11304692 Matullo G, Guarrera S, Carturan S, Peluso M, Malaveille C, Davico L, Piazza A, Vineis P: DNA repair gene polymorphisms, bulky DNA adducts in white blood cells and bladder cancer in a case-control study. Int J Cancer. 2001 May 15;92(4):562-7. Individuals differ widely in their ability to repair DNA damage, and DNA-repair deficiency may be involved in modulating cancer risk. In a case-control study of 124 bladder-cancer patients and 85 hospital controls (urological and non-urological), 3 DNA polymorphisms localized in 3 genes of different repair pathways (XRCC1-Arg399Gln, exon 10; XRCC3-Thr241Met, exon 7; XPD-Lys751Gln, exon 23) have been analyzed. Results were correlated with DNA damage measured as (32)P-post-labeling bulky DNA adducts in white blood cells from peripheral blood. Genotyping was performed by PCR-RFLP analysis, and allele frequencies in cases/controls were as follows: XRCC1-399Gln = 0.34/0.39, XRCC3-241Met = 0.48/0.35 and XPD-751Gln = 0.42/0.42. Odds ratios (ORs) were significantly greater than 1 only for the XRCC3 (exon 7) variant, and they were consistent across the 2 control groups. XPD and XRCC1 appear to have no impact on the risk of bladder cancer. Indeed, the effect of XRCC3 was more evident in non-smokers [OR = 4.8, 95% confidence interval (CI) 1.1-21.2]. XRCC3 apparently interacted with the N-acetyltransferase type 2 (NAT-2) genotype. The effect of XRCC3 was limited to the NAT-2 slow genotype (OR = 3.4, 95% CI 1.5-7.9), suggesting that XRCC3 might be involved in a common repair pathway of bulky DNA adducts. In addition, the risk of having DNA adduct levels above the median was higher in NAT-2 slow acetylators, homozygotes for the XRCC3-241Met variant allele (OR = 14.6, 95% CI 1.5-138). However, any discussion of interactions should be considered preliminary because of the small numbers involved. Our results suggest that bladder-cancer risk can be genetically modulated by XRCC3, which may repair DNA cross-link lesions produced by aromatic amines and other environmental chemicals. XRCC3 apparently interacted with the N-acetyltransferase type 2 (NAT-2) genotype. 8901578 Hintermann E, Grieder NC, Amherd R, Brodbeck D, Meyer UA: Cloning of an arylalkylamine N-acetyltransferase (aaNAT1) from Drosophila melanogaster expressed in the nervous system and the gut. Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12315-20. In insects, neurotransmitter catabolism, melatonin precursor formation, and sclerotization involve arylalkylamine N-acetyltransferase (aaNAT, EC 2.3.1.87) activity. It is not known if one or multiple aaNAT enzymes are responsible for these activities. We recently have purified an aaNAT from Drosophila melanogaster. Here, we report the cloning of the corresponding aaNAT cDNA (aaNAT1) that upon COS cell expression acetylates dopamine, tryptamine, and the immediate melatonin precursor serotonin. aaNAT1 represents a novel gene family unrelated to known acetyl-transferases, except in two weakly conserved amino acid motifs. In situ hybridization studies of aaNAT1 mRNA in embryos reveal hybridization signals in the brain, the ventral cord, the gut, and probably in oenocytes, indicating a broad tissue distribution of aaNAT1 transcripts. Moreover, in day/ night studies we demonstrate a diurnal rhythm of melatonin concentration without a clear-cut change in aaNAT1 mRNA levels. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes. The data suggest that tissue-specific regulation of aaNAT1 may be associated with different enzymatic functions and do not exclude the possibility of additional aaNAT genes. 11309290 Vulevic B, Chen Z, Boyd JT, Davis W Jr, Walsh ES, Belinsky MG, Tew KD: Cloning and characterization of human adenosine 5'-triphosphate-binding cassette, sub-family A, transporter 2 (ABCA2). Cancer Res. 2001 Apr 15;61(8):3339-47. We have isolated the full-length cDNA for human ATP-binding cassette, sub-family A, member 2 transporter (ABCA2). The ORF of this cDNA encodes a protein consisting of 2436 amino acids with apparent molecular weight of M(r) 270,000. Accordingly, ABCA2 is the largest known mammalian ABC transporter described thus far. Analysis of mRNA expression levels indicated that ABCA2 is highest in human brain and has a broad expression pattern in a panel of tumor cell lines. Using specific antibodies to ABCA2 and various organelle marker proteins, ABCA2 was found to colocalize with the lysosomal/endosomal marker LAMP1, forming discrete, punctate intracellular vesicles. In ABCA2-transfected cells, the transporter also colocalized with a fluorescently labeled steroid analogue, estramustine. The sequestration of the steroid into the lysosomal/endosomal compartment indicates a potential substrate specificity for ABCA2. Furthermore, the presence of a lipocalin signature motif in the ABCA2 sequence suggests a possible broad role for this protein in the transport of steroids, lipids, and related molecules. Using specific antibodies to ABCA2 and various organelle marker proteins, ABCA2 was found to colocalize with the lysosomal/endosomal marker LAMP1, forming discrete, punctate intracellular vesicles. 11368359 Shteper PJ, Siegfried Z, Asimakopoulos FA, Palumbo GA, Rachmilewitz EA, Ben-Neriah Y, Ben-Yehuda D: ABL1 methylation in Ph-positive ALL is exclusively associated with the P210 form of BCR-ABL. Leukemia. 2001 Apr;15(4):575-82. In human Ph-positive leukemia there is a clear association of different forms of the BCR-ABL oncogene with distinct types of leukemia. The P190 form of BCR-ABL is rarely observed in chronic myeloid leukemia (CML) but is present in 50% of Ph-positive acute lymphoblastic leukemia (ALL). In contrast, the P210 form is observed both in CML and 50% of Ph-positive ALL. Methylation of the proximal promoter of the ABL1 gene has been shown to be a nearly universal event associated with clinical progression of CML. This raises the question of whether methylation of the ABL1 promoter is an epigenetic modification also associated with Ph-positive ALL. To study this issue, we used methylation-specific PCR and bisulfite sequencing to determine the methylation status of the ABL1 promoter in 18 Ph-positive ALL samples. We report here that gene-specific ABL1 promoter methylation is associated mainly with the P210 form of BCR-ABL and not the P190 form. While six out of the seven P210-positive ALL samples had ABL1 promoter methylation, none of the 11 P190-positive ALL samples demonstrated ABL1 promoter methylation. In addition, we estimated the extent and relative abundance of ABL1 promoter methylation in several Ph-positive ALL samples and compared it to the methylation pattern in chronic, accelerated and blastic crisis phases of CML. We put forth a model that correlates the different types of leukemias with the different levels of ABL1 promoter methylation. We report here that gene-specific ABL1 promoter methylation is associated mainly with the P210 form of BCR-ABL and not the P190 form. 10231028 Daigo Y, Isomura M, Nishiwaki T, Tamari M, Ishikawa S, Kai M, Murata Y, Takeuchi K, Yamane Y, Hayashi R, Minami M, Fujino MA, Hojo Y, Uchiyama I, Takagi T, Nakamura Y: Characterization of a 1200-kb genomic segment of chromosome 3p22-p21.3. DNA Res. 1999 Feb 26;6(1):37-44. We previously determined the nucleotide sequence and characterized the 685-kb proximal half of CEPH YAC936c1, which corresponds to a portion of human chromosome 3p21.3. In the study reported here, we characterized the remaining 515-kb of this YAC clone corresponding to the telomeric half of its human insert. The newly sequenced region contained a total of ten genes including six reported previously: phospholipase C delta 1 (PLCD1), human activin receptor type IIB (hActR-IIB), organic cation transporter-like 1 (OCTL1), organic cation transporter-like 2 (OCTL2), oxidative stress response 1 (OSR1), and human xylulokinase-like protein (XYLB). The remaining four genes present in the telomeric region included two known genes, MyD88 and ACAA, and two novel genes. One (designated ENGL) of the novel sequences was found to encode an amino-acid sequence homologous to the family of DNA/RNA endonucleases, especially endonuclease G. The other gene F56 revealed no significant homology to any known genes. These results disclosed complete physical and transcriptional maps of the 1200-kb region of 3p present in YAC 936c1. The remaining four genes present in the telomeric region included two known genes, MyD88 and ACAA, and two novel genes. 11872884 Pezzini A, Del Zotto E, Archetti S, Negrini R, Bani P, Albertini A, Grassi M, Assanelli D, Gasparotti R, Vignolo LA, Magoni M, Padovani A: Plasma homocysteine concentration, C677T MTHFR genotype, and 844ins68bp CBS genotype in young adults with spontaneous cervical artery dissection and atherothrombotic stroke. Stroke. 2002 Mar;33(3):664-9. BACKGROUND AND PURPOSE: The role of mild hyperhomocysteinemia as a risk factor for cerebral ischemia may depend on stroke subtype. To test this hypothesis, we undertook a prospective case-control study of a group of patients with spontaneous cervical artery dissection (sCAD), a group of patients with atherothrombotic stroke (non-CAD), and a group of control subjects. METHODS: Fasting total plasma homocysteine (tHcy) concentration, C677T MTHFR genotype, and 844ins68bp CBS genotype were determined in 25 patients with sCAD, 31 patients <45 years of age with non-CAD ischemic stroke, and 36 control subjects. Biochemical data in the patient groups were obtained within the first 72 hours of stroke onset. RESULTS: Median tHcy levels were significantly higher in patients with sCAD (13.2 micromol/L; range, 7 to 32.8 micromol/L) compared with control subjects (8.9 micromol/L; range, 5 to 17.3 micromol/L; 95% CI, 1.05 to 1.52; P=0.006). Cases with tHcy concentration above the cutoff level of 12 micromol/L were significantly more represented in the group of patients with sCAD compared with control subjects (64% versus 13.9%; 95% CI, 2.25 to 44.23; P=0.003); a significant association between the MTHFR TT genotype and sCAD was also observed (36% versus 11.1%; 95% CI, 1.10 to 19.23; P=0.045). No significant difference in tHcy levels and in the prevalence of thermolabile MTHFR was found between patients with non-CAD ischemic stroke and control subjects and between patients with sCAD and non-CAD ischemic stroke. The distribution of the 844ins68bp CBS genotype and the prevalence of subjects carrying both the TT MTHFR and 844ins68bp CBS genotypes were not significantly different among the 3 groups. CONCLUSIONS: Our results are consistent with the hypothesis that increased plasma homocysteine levels and the TT MTHFR genotype may represent risk factors for sCAD. In contrast, their role in atherothrombotic strokes remains a contentious issue. No significant difference in tHcy levels and in the prevalence of thermolabile MTHFR was found between patients with non-CAD ischemic stroke and control subjects and between patients with sCAD and non-CAD ischemic stroke. 11739374 Duggan A, Garcia-Anoveros J, Corey DP: The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J Biol Chem. 2002 Feb 15;277(7):5203-8. Epub 2001 Dec 5. Members of the BNaC/ASIC family of ion channels have been implicated in mechanotransduction and nociception mediated by dorsal root ganglion (DRG) neurons. These ion channels are also expressed in the CNS. We identified the PDZ domain protein PICK1 as an interactor of BNaC1(ASIC2) in a yeast two-hybrid screen. We show by two-hybrid assays, glutathione S-transferase pull-down assays, and coimmunoprecipitations that the BNaC1-PICK1 interaction is specific, and that coexpression of both proteins leads to their clustering in intracellular compartments. The interaction between BNaC1 and PICK1 requires the PDZ domain of PICK1 and the last four amino acids of BNaC1. BNaC1 is similar to two other BNaC/ASIC family members, BNaC2 (ASIC1) and ASIC4, at its extreme C terminus, and we show that PICK1 also interacts with BNaC2. We found that PICK1, like BNaC1 and BNaC2, is expressed by DRG neurons and, like the BNaC1alpha isoform, is present at their peripheral mechanosensory endings. Both PICK1 and BNaC1alpha are also coexpressed by some pyramidal neurons of the cortex, by pyramidal neurons of the CA3 region of hippocampus, and by cerebellar Purkinje neurons, localizing to their dendrites and cell bodies. Therefore, PICK1 interacts with BNaC/ASIC channels and may regulate their subcellular distribution or function in both peripheral and central neurons. BNaC1 is similar to two other BNaC/ASIC family members, BNaC2 (ASIC1) and ASIC4, at its extreme C terminus, and we show that PICK1 also interacts with BNaC2. 8180167 Hermans JM, Jones R, Stone SR: Rapid inhibition of the sperm protease acrosin by protein C inhibitor. Biochemistry. 1994 May 10;33(18):5440-4. Heparin was found to be an allosteric modulator of the amidolytic activity of the protease acrosin. In the presence of saturating concentrations of heparin, there was a 4.9-fold decrease in the value of the Michaelis constant for the substrate D-Ile-Pro-Arg-p-nitroanilide and the value of kcat was 2.5-fold lower. Analysis of the data yielded a dissociation constant of 0.22 +/- 0.04 microM for the heparin-acrosin complex. The presence of relatively high concentrations of protein C inhibitor in seminal plasma [Laurell, M., Christensson, A., Abrahamson, P., Stenflo, J., & Lilja, H. (1992) J. Clin. Invest. 89, 1094-1101] suggests that this serpin may be involved in the control of the activity of acrosin. Acrosin was found to be rapidly inhibited by protein C inhibitor with the association rate constant (kass) for the formation of the complex being (2.41 +/- 0.03) x 10(5) M-1 s-1. The value of kass showed a bell-shaped dependence on the concentration of heparin; it was maximal at concentrations of heparin between 0.08 and 3 microM and decreased at lower and higher concentrations. At the optimal heparin concentration, the value of kass for the acrosin-protein C inhibitor reaction was 230-fold higher ((5.6 +/- 0.1) x 10(7) M-1 s-1) than in the absence of heparin. The results suggest that protein C inhibitor may be important in the physiological control of acrosin activity, particularly where the presence of heparin-like glycosaminoglycans would stimulate the acrosin-protein C inhibitor reaction. Acrosin was found to be rapidly inhibited by protein C inhibitor with the association rate constant (kass) for the formation of the complex being (2.41 +/- 0.03) x 10 (5) M-1 s-1. 8980351 O'Briain DS, Sheils O, McElwaine S, McCann SR, Lawler M: Sorting out mix-ups. The provenance of tissue sections may be confirmed by PCR using microsatellite markers. Am J Clin Pathol. 1996 Dec;106(6):758-64. Standard identification systems usually ensure that biopsy material is correctly associated with a given patient. Sometimes, as when a tumor is unexpectedly found, the provenance (proof of origin) of a tissue sample may be questioned; the tissue may have been mislabelled or contaminated with tissue from another patient. Techniques used to confirm tissue provenance include comparing either tissue markers of gender or ABO blood groups; however, these methods have weak confirmatory power. Recently, the use of DNA-based polymerase chain reaction (PCR) techniques has been reported. Paired, formalin-fixed, paraffin-embedded, 10 microns tissue sections were selected from 17 patients, 8 of whom had carcinoma, either by dividing a biopsy section, using sequential biopsies, or sequential biopsy and autopsy tissue. The resulting 36 samples were coded before analysis. In two additional cases, 1-mm fragments of tumor from one patient were included in the tissue block of benign tissue from another patient, the tumor fragments were identified on hematoxylin-and-eosin-stained sections, separately scraped off the glass slide, and analyzed. Tissue from two clinical cases, one of suspected mislabelling and one with a suspected carry-over of malignant tissue were also investigated. Short tandem repeat sequences (STR) or microsatellites, are 2-5 base pair repeats that vary in their repeat number between individuals. This variation (polymorphism) can be assessed using a PCR. A panel of markers of 3 STRs; ACPP, INT 2, and CYP 19 (on chromosomes 3, 11, and 15, respectively) were used. DNA was isolated from the samples after xylene deparaffinization and proteinase digestion, and was then amplified in a radioactive PCR using primers selected to give a product size ranging from 136-178 bases. Amplified products were electrophoresed on denaturing polyacrylamide gels, dried, and autoradiographed. DNA segments were successfully extracted from all samples but one, which was fixed in Bouin's fluid. By comparing allele sizes from the panel, all tissue pairs (other than the Bouin's pair) were successfully matched, the 1-mm tumor fragments were correctly assigned, and the two clinical problems were solved. STRs are highly informative and robust markers, well suited to PCR of small portions of tissue sections, and are an effective method to confirm the provenance of benign and malignant biopsy and autopsy material. A panel of markers of 3 STRs; ACPP, INT 2, and CYP 19 (on chromosomes 3, 11, and 15, respectively) were used. 8941379 Ueyama H, Inazawa J, Nishino H, Ohkubo I, Miwa T: FISH localization of human cytoplasmic actin genes ACTB to 7p22 and ACTG1 to 17q25 and characterization of related pseudogenes. Cytogenet Cell Genet. 1996;74(3):221-4. Human beta- and gamma-cytoplasmic actin genes (ACTB and ACTG1) were mapped to chromosomes 7p22 and 17q25, respectively, by fluorescence in situ hybridization (FISH). Four processed pseudogenes, beta-actin-related ACTBP9 and gamma-actin-related ACTGP1, ACTGP3, and ACTGP9, were isolated from human libraries. By PCR of somatic cell hybrid DNAs, ACTBP9 and two beta-actin-related pseudogenes (ACTBP7 and ACTBP8) were mapped to human chromosomes 18, 15, and 6, respectively. The gamma-actin-related pseudogenes were mapped by FISH to chromosomes 3q23 (ACTGP1), 20p13 (ACTGP3), and 6p21.1 (ACTGP9). By PCR of somatic cell hybrid DNAs, ACTBP9 and two beta-actin-related pseudogenes (ACTBP7 and ACTBP8) were mapped to human chromosomes 18, 15, and 6, respectively. 9544840 Egeo A, Mazzocco M, Sotgia F, Arrigo P, Oliva R, Bergonon S, Nizetic D, Rasore-Quartino A, Scartezzini P: Identification and characterization of a new human cDNA from chromosome 21q22.3 encoding a basic nuclear protein. Hum Genet. 1998 Mar;102(3):289-93. Congenital heart disease (CHD) affects over 40% of Down syndrome (DS) patients. The region proposed to contain the gene(s) for DS CHD has been restricted to 21q22.2-22.3, from D21S55 to MX1. The identification and functional characterization of the genes mapping to this region is a necessary step to understand the pathogenesis of CHD in DS. In an effort to contribute to the construction of a transcriptional map of the DS CHD region we have performed direct cDNA selection using a YAC contig that maps between ETS2 and D21S15 and cDNAs synthesised from fetal heart structures. Here we describe the identification and characterization of a new gene, WRB, that maps to 21q22.3 between ACTL5 and HMG 14 and appears to be widely expressed in adult and fetal tissues. The new gene encodes a basic protein of unknown function containing a tryptophan-rich carboxyl-terminal region and a potential nuclear localization signal. Immunofluorescence analysis shows a predominant localization in the cell nucleus. The understanding of the biological function of the protein product should clarify the potential role of WRB in the pathogenesis of DS CHD. Here we describe the identification and characterization of a new gene, WRB, that maps to 21q22.3 between ACTL5 and HMG 14 and appears to be widely expressed in adult and fetal tissues. 11864567 Nayak S, Santiago FE, Jin H, Lin D, Schedl T, Kipreos ET: The Caenorhabditis elegans Skp1-related gene family: diverse functions in cell proliferation, morphogenesis, and meiosis. Curr Biol. 2002 Feb 19;12(4):277-87. BACKGROUND: The SCF ubiquitin-ligase complex targets the ubiquitin-mediated degradation of proteins in multiple dynamic cellular processes. A key SCF component is the Skp1 protein that functions within the complex to link the substrate-recognition subunit to a cullin that in turn binds the ubiquitin-conjugating enzyme. In contrast to yeast and humans, Caenorhabditis elegans contains multiple expressed Skp1-related (skr) genes. RESULTS: The 21 Skp1-related (skr) genes in C. elegans form one phylogenetic clade, suggesting that a single ancestral Skp1 gene underwent independent expansion in C. elegans. The cellular and developmental functions of the 21 C. elegans skr genes were probed by dsRNA-mediated gene inactivation (RNAi). The RNAi phenotypes of the skr genes fall into two classes. First, the highly similar skr-7, -8, -9, and -10 genes are required for posterior body morphogenesis, embryonic and larval development, and cell proliferation. Second, the related skr-1 and -2 genes are required for the restraint of cell proliferation, progression through the pachytene stage of meiosis, and the formation of bivalent chromosomes at diakinesis. CUL-1 was found to interact with SKR-1, -2, -3, -7, -8, and -10 in the yeast two-hybrid system. Interestingly, SKR-3 could interact with both CUL-1 and its close paralog CUL-6. CONCLUSIONS: Members of the expanded skr gene family in C. elegans perform critical functions in regulating cell proliferation, meiosis, and morphogenesis. The finding that multiple SKRs are able to bind cullins suggests an extensive set of combinatorial SCF complexes. CUL-1 was found to interact with SKR-1, -2, -3, -7, -8, and -10 in the yeast two-hybrid system. 2542383 Miller YE, Minna JD, Gazdar AF: Lack of expression of aminoacylase-1 in small cell lung cancer. Evidence for inactivation of genes encoded by chromosome 3p. J Clin Invest. 1989 Jun;83(6):2120-4. A deletion involving chromosome 3p (14-23) characteristically occurs in small cell lung cancer (SCLC). Reduction to homozygosity, rather than complete loss, is typically observed for genes in the deleted region. Lack of expression for genes encoded by this region, implying inactivation of all alleles, has not been previously described. We have examined the expression of aminoacylase-1 (ACY-1), encoded by chromosome 3p21, using both an electrophoretic activity assay and a monoclonal antibody-based ELISA. A variety of human tissues, including lung, brain, liver, kidney, heart, adrenal medulla, and erythrocytes have previously been tested for ACY-1 activity and antigen; all but erythrocytes are positive. Thus, ACY-1 is expressed in all nucleated human cells examined to date. ACY-1 was undetectable in a significant number of SCLC cell lines (4/29) and tumors (1/8), but not in non-small cell lung cancer (NSCLC) cell lines (0/19) or tumors (0/9), nor in a variety of other human cell lines (0/15) or colon tumors (0/8). In addition, reduced (approximately 10% of normal) ACY-1 expression was common in SCLC cell lines (14/29) and tumors (3/8), but not in NSCLC cell lines (1/19) or tumors (0/9), nor in other human cell lines (0/15) or colon tumors (0/8). Thus, low or undetectable ACY-1 expression is highly specific for SCLC and occurs in both cell lines and tumor tissue. The finding of undetectable ACY-1 expression in SCLC supports the hypothesis that inactivation of all alleles of specific chromosome 3p genes occurs in a SCLC in a fashion analogous to Rb gene inactivation in retinoblastoma, and suggests that the structural gene for ACY-1 may be closely linked to a putative SCLC tumor suppressor gene. The finding of undetectable ACY-1 expression in SCLC supports the hypothesis that inactivation of all alleles of specific chromosome 3p genes occurs in a SCLC in a fashion analogous to Rb gene inactivation in retinoblastoma, and suggests that the structural gene for ACY-1 may be closely linked to a putative SCLC tumor suppressor gene. 12176995 Lim R, Winteringham LN, Williams JH, McCulloch RK, Ingley E, Tiao JY, Lalonde JP, Tsai S, Tilbrook PA, Sun Y, Wu X, Morris SW, Klinken SP: MADM, a novel adaptor protein that mediates phosphorylation of the 14-3-3 binding site of myeloid leukemia factor 1. J Biol Chem. 2002 Oct 25;277(43):40997-1008. Epub 2002 Aug 9. A yeast two-hybrid screen was conducted to identify binding partners of Mlf1, an oncoprotein recently identified in a translocation with nucleophosmin that causes acute myeloid leukemia. Two proteins isolated in this screen were 14-3-3zeta and a novel adaptor, Madm. Mlf1 contains a classic RSXSXP sequence for 14-3-3 binding and is associated with 14-3-3zeta via this phosphorylated motif. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. In addition, Madm recruited a serine kinase, which phosphorylated both Madm and Mlf1 including the RSXSXP motif. In contrast to wild-type Mlf1, the oncogenic fusion protein nucleophosmin (NPM)-MLF1 did not bind 14-3-3zeta, had altered Madm binding, and localized exclusively in the nucleus. Ectopic expression of Madm in M1 myeloid cells suppressed cytokine-induced differentiation unlike Mlf1, which promotes maturation. Because the Mlf1 binding region of Madm and its own dimerization domain overlapped, the levels of Madm and Mlf1 may affect complex formation and regulate differentiation. In summary, this study has identified two partner proteins of Mlf1 that may influence its subcellular localization and biological function. Madm co-immunoprecipitated with Mlf1 and co-localized in the cytoplasm. 7766992 Edelhoff S, Villacres EC, Storm DR, Disteche CM: Mapping of adenylyl cyclase genes type I, II, III, IV, V, and VI in mouse. Mamm Genome. 1995 Feb;6(2):111-3. The adenylyl cyclases (AC) act as second messengers in regulatory processes in the central nervous system. They might be involved in the pathophysiology of diseases, but their biological function is unknown, except for AC type I, which has been implicated in learning and memory. We previously mapped the gene encoding AC I to human Chromosome (Chr) 7p12. In this study we report the mapping of the adenylyl cyclase genes type I-VI to mouse chromosomes by fluorescence in situ hybridization (FISH): Adcy1 to Chr 11A2, Adcy2 to 13C1, Adcy3 to 12A-B, Adcy4 to 14D3, Adcy5 to 16B5, and Adcy6 to 15F. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11.2. The mapping data confirm known areas of conservation between mouse and human chromosomes. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11. 10650952 Park HJ, Lee J, Wang L, Park JH, Kwon HB, Arimura A, Chun SY: Stage-specific expression of pituitary adenylate cyclase-activating polypeptide type I receptor messenger ribonucleic acid during ovarian follicle development in the rat. Endocrinology. 2000 Feb;141(2):702-9. Expression of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide with considerable homology to vasoactive intestinal peptide, has been shown to be stimulated by gonadotropins in the ovary. The present studies further evaluated the cell-type specific expression and gonadotropin regulation of PACAP type I receptor (PACAPR) messenger RNA in immature rat ovaries and in cultured preovulatory follicles. Northern blot analysis of ovaries obtained from prepubertal rats revealed the increased expression of PACAPR during prepubertal development. The major cell types expressing PACAPR messenger RNA were granulosa cells of large preantral follicles. Treatment of immature rats with PMSG caused a decrease in ovarian PACAPR expression. In contrast, treatment with human (h) CG at 2 days after PMSG treatment stimulated ovarian PACAPR messenger RNA within 3-6 h in granulosa cells of preovulatory follicles. Treatment of cultured preovulatory follicles in vitro with LH further confirmed the time- and dose-dependent stimulation of PACAPR by gonadotropins in granulosa cells of preovulatory follicles. Moreover, RNase protection assay revealed that the short variant of ovarian PACAPR was the predominant form stimulated during prepubertal development and by gonadotropins. These results demonstrate the expression of PACAPR messenger RNA in granulosa cells of growing follicles and of preovulatory follicles stimulated by gonadotropins, and suggest that PACAP may play a role in the growth of developing follicles and in ovulation as an autocrine/paracrine factor. In contrast, treatment with human (h) CG at 2 days after PMSG treatment stimulated ovarian PACAPR messenger RNA within 3-6 h in granulosa cells of preovulatory follicles. 1996113 Duester G, Shean ML, McBride MS, Stewart MJ: Retinoic acid response element in the human alcohol dehydrogenase gene ADH3: implications for regulation of retinoic acid synthesis. Mol Cell Biol. 1991 Mar;11(3):1638-46. Retinoic acid regulation of one member of the human class I alcohol dehydrogenase (ADH) gene family was demonstrated, suggesting that the retinol dehydrogenase function of ADH may play a regulatory role in the biosynthetic pathway for retinoic acid. Promoter activity of human ADH3, but not ADH1 or ADH2, was shown to be activated by retinoic acid in transient transfection assays of Hep3B human hepatoma cells. Deletion mapping experiments identified a region in the ADH3 promoter located between -328 and -272 bp which confers retinoic acid activation. This region was also demonstrated to confer retinoic acid responsiveness on the ADH1 and ADH2 genes in heterologous promoter fusions. Within a 34-bp stretch, the ADH3 retinoic acid response element (RARE) contains two TGACC motifs and one TGAAC motif, both of which exist in RAREs controlling other genes. A block mutation of the TGACC sequence located at -289 to -285 bp eliminated the retinoic acid response. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. Since ADH catalyzes the conversion of retinol to retinal, which can be further converted to retinoic acid by aldehyde dehydrogenase, these results suggest that retinoic acid activation of ADH3 constitutes a positive feedback loop regulating retinoic acid synthesis. As assayed by gel shift DNA binding studies, the RARE region (-328 to -272 bp) of ADH3 bound the human retinoic acid receptor beta (RAR beta) and was competed for by DNA containing a RARE present in the gene encoding RAR beta. 12379507 Evans JJ, Youssef AH, Yandle TG, Lewis LK, Nicholls MG: Effects of endothelin-1 on release of adrenomedullin and C-type natriuretic peptide from individual human vascular endothelial cells. J Endocrinol. 2002 Oct;175(1):225-32. Regulation of cardiovascular system activity involves complex interactions amongst numerous factors. Three of these vasoactive factors are adrenomedullin, C-type natriuretic peptide (CNP) and endothelin-1 (ET-1), each of which is claimed to have important local effects. To investigate paracrine/autocrine regulation of the secretion of these peptides we used a cell immunoblot method. We postulated that basal release of adrenomedullin and CNP by endothelial cells is modulated by ET-1. Dispersed human aortic endothelial cells were attached to a protein binding membrane and incubated for 1 or 4 h with control medium or with ET-1, endothelin receptor antagonists or antibody to ET-1, and then submitted to immunohistochemical staining. Peptides (adrenomedullin, CNP and ET-1) within individual cells were stained, as was peptide secreted and adjacent to the cell. It was demonstrated that adrenomedullin, CNP and ET-1 can be contained within the same cell. In addition, we observed that individual endothelial cells can secrete all three peptides. The endothelin ET-A/ET-B receptor antagonist, bosentan, the ET-B receptor antagonist, BQ-788, and anti-ET-1 serum decreased the percentage of endothelial cells that secreted adrenomedullin and CNP relative to control. Conversely, the addition of ET-1 induced an increase in the number of endothelial cells that secreted adrenomedullin and CNP. These results provide strong evidence that endogenous ET-1, from human vascular endothelial cells, acts in a paracrine/autocrine manner to modulate the basal release of adrenomedullin and CNP. Our observations of this modulation suggest that vascular endothelial cells of humans constitute an important component of a self-responsive vasoregulatory system. Conversely, the addition of ET-1 induced an increase in the number of endothelial cells that secreted adrenomedullin and CNP. 8039425 Loftus SK, Shiang R, Warrington JA, Bengtsson U, McPherson JD, Wasmuth JJ: Genes encoding adrenergic receptors are not clustered on the long arm of human chromosome 5. Cytogenet Cell Genet. 1994;67(2):69-74. The distal portion of the long arm of chromosome 5 (5q) contains a large number of genes encoding membrane receptors belonging to various gene families, including G protein-coupled adrenergic receptors. Previous reports indicated that the genes for two of the adrenergic receptors, ADRB2 and ADRA1B, were within 300 kb of one another on 5q. In an effort to determine if a third adrenergic receptor assigned to 5q, ADRA1A, was physically close to the genes encoding the other adrenergic receptors, we attempted to place all three loci on a radiation hybrid map of 5q. The results conflicted with previous mapping results in two ways. First, ADRA1B is on 5q but is several million bases, rather than a few hundred thousand bases, from ADRB2. Second, ADRA1A is not on chromosome 5, but rather on chromosome 20. Thus, even though 5q contains an extraordinary number of genes encoding receptors for various hormones, growth factors, and neurotransmitters, there is no particular clustering of genes encoding adrenergic receptors in this region. First, ADRA1B is on 5q but is several million bases, rather than a few hundred thousand bases, from ADRB2. 1676978 Oakey RJ, Caron MG, Lefkowitz RJ, Seldin MF: Genomic organization of adrenergic and serotonin receptors in the mouse: linkage mapping of sequence-related genes provides a method for examining mammalian chromosome evolution. Genomics. 1991 Jun;10(2):338-44. Five sequence-related genes encoding four adrenergic receptors and a serotonin receptor were localized to specific regions of four mouse chromosomes with respect to 11 other genetic markers. Linkage was established by the analysis of the haplotypes of 114 interspecific backcross mice. Adra2r (alpha 2-C10) and Adrb1r (beta 1) receptors mapped to the distal region of mouse chromosome 19. These genes were separated by 2.6 +/- 1.5 cM in a segment of mouse chromosome 19 that has a similar organization of these genes on the long arm of human chromosome 10. The Adra1r (alpha 1B), Adrb2r (beta 2), and Htra1 (5HT1A) genes mapped to proximal mouse chromosome 11, proximal mouse chromosome 18, and distal mouse chromosome 13, respectively. The organization of genes linked to these loci on regions of the three mouse chromosomes is consistent with the organization of homologous human genes on human chromosome 5. These findings further define the relationship of linkage groups conserved during the evolution of the mouse and human genomes. We have identified a region that may have been translocated during evolution and suggest that the human genomic organization of adrenergic receptors more closely resembles that of a putative primordial ancestor. Adra2r (alpha 2-C10) and Adrb1r (beta 1) receptors mapped to the distal region of mouse chromosome 19. 9165520 Valik D, Miner PT, Jones JD: First U.S. case of adenylosuccinate lyase deficiency with severe hypotonia. Pediatr Neurol. 1997 Apr;16(3):252-5. Adenylosuccinate lyase (ASL) deficiency is a defect in purine de novo synthesis pathway. The disease has variable clinical presentation involving psychomotor retardation, seizures, hypotonia, and autism. The presence of succinyladenosine and succinylaminoimidazole carboxamide riboside (SAICA riboside) in body fluids characterizes the biochemical phenotype. All cases of ASL deficiency described to date have been diagnosed in Europe. Using a high-resolution thin-layer chromatography (TLC) technique combining screening for ASL deficiency and disorders of saccharide metabolism, we found the first case of this disease in the US. The patient presented with delayed motor development and profound hypotonia. The family history and routine laboratory tests were negative. Screening for metabolic disorders detected the presence of succinyladenosine and SAICA riboside in urine. The activity of ASL in the patient's skin fibroblasts was 43% of controls (patient, mean = 1.20 nmol/min/mg of protein, s = 0.21, n = 3; controls, mean = 2.78 nmol/min/mig of protein, s = 0.61, n = 7). In a 15-month-old girl with profound hypotonia, we established the diagnosis of ASL deficiency by demonstrating succinyladenosine and SAICA riboside in urine and decreased residual activity of ASL in skin fibroblasts. Adenylosuccinate lyase (ASL) deficiency is a defect in purine de novo synthesis pathway. 9570147 Xin X, Day R, Dong W, Lei Y, Fricker LD: Cloning, sequence analysis, and distribution of rat metallocarboxypeptidase Z. DNA Cell Biol. 1998 Apr;17(4):311-9. A cDNA encoding human carboxypeptidase Z (CPZ), a novel metallocarboxypeptidase, was recently cloned (Song and Fricker, J. Biol. Chem., 272, 1054, 1997). In the present study, a cDNA encoding the rat homolog of CPZ was identified. As with the human form, rat CPZ contains an N-terminal domain of 120 amino acids that has 20% to 30% amino acid identity with the "frizzled" domain found on proteins that interact with Wnt, a protein involved in tissue polarity in early embryogenesis. Sequence analysis showed rat and human CPZ to be highly conserved within the frizzled domain (77% amino acid identity), the carboxypeptidase domain (91%), and the C-terminal 28 residues (78%). The entire rat CPZ protein has high sequence similarity with human CPZ (81% amino acid identity), moderate sequence similarity to human carboxypeptidase N (45%), human carboxypeptidase E (41%), and human carboxypeptidase M (33%), and less sequence similarity with other metallocarboxypeptidases. Northern blot analysis showed rat CPZ mRNA to be abundant in the placenta, with low to moderate levels in the brain, lung, thymus, and kidney. The BRL3A rat liver cell line and the PC12 rat adrenal cell line express high levels of CPZ mRNA. In situ hybridization analysis indicated that CPZ is expressed only in specific cell types. For example, in the brain, CPZ mRNA is present in leptomeningeal cells, but not in the majority of other cell types. This distribution in leptomeningeal cells is shared by AEBP1, a recently reported member of the metallocarboxypeptidase gene family. However, the distribution of CPZ and AEBP1 differ in pituitary and thyroid. Taken together, these studies suggest that CPZ functions in a range of cell types. However, the distribution of CPZ and AEBP1 differ in pituitary and thyroid. 11920564 Islam TC, Lindvall J, Wennborg A, Branden LJ, Rabbani H, Smith CI: Expression profiling in transformed human B cells: influence of Btk mutations and comparison to B cell lymphomas using filter and oligonucleotide arrays. Eur J Immunol. 2002 Apr;32(4):982-93. We have used both Clontech Atlas Human Hematology/Immunology cDNA microarrays, containing 588 genes, and Affymetrix oligonucleotide U95Av2 human array complementary to more than 12,500 genes to get a global view of genes expressed in Epstein-Barr virus (EBV)-transformed B cells and genes regulated by Bruton's tyrosine kinase (Btk). We compared EBV-transformed wild-type (WT) B cells from a healthy individual, WT1 and an X-linked agammaglobulinemia (XLA) patient cell line, XLA1, using the Clontech filters arrays. Eleven genes were > or =1.9-fold induced in absence of functional Btk. Furthermore, we analyzed a second patient cell line, XLA2, and compared this to two WT cell lines using oligonucleotide arrays. A total of 391 genes were found to be differentially expressed, including kinases and transcriptions factors. Furthermore, one expressed sequence tag and eight complementary DNA clones with unknown function were down-regulated in XLA2, indicating their biological role. Higher-fold inductions, Fyn (39.5), Hck (15.5) and Cyp1B1 (5.8), were observed using oligonucleotide array and were confirmed using real-time PCR for Fyn (20.8), Hck (6.7) and Cyp1B1 (10). Two genes, B cell translocation gene1 (BTG1) and B cell-specific OCT binding factor-1 (OBF-1) were induced > or =1.9-fold in both XLA1 and XLA2 analyzed by Atlas filter arrays andAffymetrix chips, respectively. Data from both filter and oligonucleotide arrays were compared to the gene clusters of a previously published lymphoma expression profile by linking to the UniGene transcript database. Our findings demonstrate for the first time the use of microarray to study the influence of Btk mutations and the use of functional annotation and validation of expression data by comparison of microarray analyses. Two genes, B cell translocation gene1 (BTG1) and B cell-specific OCT binding factor-1 (OBF-1) were induced > or =1.9-fold in both XLA1 and XLA2 analyzed by Atlas filter arrays andAffymetrix chips, respectively. 7486253 Zhang W, Haley CS, Moran C: Mapping the soluble angiotensin binding protein (ABP1) locus to porcine chromosome 16. Anim Genet. 1995 Oct;26(5):337-9. A highly polymorphic (AT)nTm microsatellite located in a PRE1 SINE element in the 3'UTR of the soluble angiotensin binding protein (ABP1) gene has enabled the linkage mapping on the PiGMaP reference families of the ABP1 gene to porcine chromosome 16, to a region homologous with the short arm of human chromosome 5. Mapping the soluble angiotensin binding protein (ABP1) locus to porcine chromosome 16. 3263401 Dang-Vu AP, Olsen EA, Vollmer RT, Greenberg ML, Hershfield MS: Treatment of cutaneous T cell lymphoma with 2'-deoxycoformycin (pentostatin). J Am Acad Dermatol. 1988 Oct;19(4):692-8. 2'-Deoxycoformycin, a potent inhibitor of adenosine deaminase, was administered to three patients with cutaneous T cell lymphoma refractory to multiple treatment modalities. Patient 1, who received 5 mg/m2/day for 3 days at 35- to 71-day intervals, has achieved a complete remission greater than 16 months in duration. Patient 2 had progressive disease despite two courses of 2'-deoxycoformycin at a dose of 5 mg/m2/day for 3 days at 28-day intervals. The third patient, who was treated with 4 mg/m2 2'-deoxycoformycin weekly to biweekly, had an initial response, but the disease progressed after eight treatments. Only one patient had any side effects: Patient 1 developed reversible episcleritis, mild elevation of liver enzymes, and persistent nausea and vomiting. In red blood cells of all patients, there was near complete inhibition of adenosine deaminase (91% to 96%) and S-adenosylhomocysteine hydrolase (89% to 95%) activities with treatment. In peripheral blood lymphocytes, adenosine deaminase was inhibited by 85% to 98% and S-adenosylhomocysteine hydrolase by 51% to 88%. The deoxyadenosine triphosphate level, reflected by the total cellular adenine deoxyribonucleotide measurement in erythrocytes, was noted to be modestly elevated during treatment, with the highest level in the patient who demonstrated the only complete response and the only toxic effects. Low-dose 2'-deoxycoformycin appears to be safe but may be an insufficiently intensive regimen to treat refractory cutaneous T cell lymphoma. With proper biochemical monitoring, higher doses may be both safe and more effective. In peripheral blood lymphocytes, adenosine deaminase was inhibited by 85% to 98% and S-adenosylhomocysteine hydrolase by 51% to 88%. 2848721 Daveau M, Christian-Davrinche, Julen N, Hiron M, Arnaud P, Lebreton JP: The synthesis of human alpha-2-HS glycoprotein is down-regulated by cytokines in hepatoma HepG2 cells. FEBS Lett. 1988 Dec 5;241(1-2):191-4. The regulation of the synthesis of alpha-2-HS glycoprotein (AHSG) by inflammatory mediators from activated monocytes was studied on the human hepatoma cell line HepG2 and compared to that of albumin. Monocyte-conditioned medium, recombinant human interleukin-6 (rhIL6) and interleukin-1 beta (rhIL1 beta) all down-regulated the synthesis of AHSG. This decrease was found both at the protein and the mRNA level. The most efficient mediator was the monocyte-conditioned medium, when rhIL1 beta was found to be less efficient than rhIL6. The combination of rhIL6 and rhIL1 beta resulted in an additive down-regulation of the AHSG mRNA levels. Similar results were obtained with albumin. These data indicate that AHSG is a negative acute-phase protein whose synthesis is regulated by cytokines in a manner similar to that of albumin. Monocyte-conditioned medium, recombinant human interleukin-6 (rhIL6) and interleukin-1 beta (rhIL1 beta) all down-regulated the synthesis of AHSG. 292306 Mohandas T, Sparkes RS, Sparkes MC, Shulkin JD, Toomey KE, Funderburk SJ: Regional localization of human gene loci on chromosome 9: studies of somatic cell hybrids containing human translocations. Am J Hum Genet. 1979 Sep;31(5):586-600. Somatic cell hybrids were derived from the fusion of (1) Chinese hamster cells deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) and human cells carrying an X/9 translocation and (2) Chinese hamster cells deficient in thymidine kinase (TK) and human cells carrying a 17/9 translocation. Several independent primary hybrid clones from these two series of cell hybrids were analyzed cytogenitically for human chromosome content and electrophoretically for the expression of human markers known to be on human chromosome 9. The results allow the assignment of the loci for the enzymes galactose-1-phosphate uridyltransferase (GALT), soluble aconitase (ACONs), and adenylate kinase-3 (AK3) to the short arm of chromosome 9 (p11 to pter) and the locus for the enzyme adenylate kinase-1 (AK1) to the distal end of the long arm of human chromosome 9 (hand q34). Earlier family studies have shown that the locus for AK1 is closely linked to the ABO blood group locus and to the locus of the nail-patella (Np) syndrome. Thus the regional localization of AK1 locus permits the localization of the AK1-Np-ABO linkage group. Earlier family studies have shown that the locus for AK1 is closely linked to the ABO blood group locus and to the locus of the nail-patella (Np) syndrome. 11049814 Schwartz BS, Lee BK, Lee GS, Stewart WF, Simon D, Kelsey K, Todd AC: Associations of blood lead, dimercaptosuccinic acid-chelatable lead, and tibia lead with polymorphisms in the vitamin D receptor and [delta]-aminolevulinic acid dehydratase genes. Environ Health Perspect. 2000 Oct;108(10):949-54. A cross-sectional study was performed to evaluate the influence of polymorphisms in the [delta]-aminolevulinic acid dehydratase (ALAD) and vitamin D receptor (VDR) genes on blood lead, tibia lead, and dimercaptosuccinic acid (DMSA)-chelatable lead levels in 798 lead workers and 135 controls without occupational lead exposure in the Republic of Korea. Tibia lead was assessed with a 30-min measurement by (109)Cd-induced K-shell X-ray fluorescence, and DMSA-chelatable lead was estimated as 4-hr urinary lead excretion after oral administration of 10 mg/kg DMSA. The primary goals of the analysis were to examine blood lead, tibia lead, and DMSA-chelatable lead levels by ALAD and VDR genotypes, controlling for covariates; and to evaluate whether ALAD and VDR genotype modified relations among the different lead biomarkers. There was a wide range of blood lead (4-86 microg/dL), tibia lead (-7-338 microg Pb/g bone mineral), and DMSA-chelatable lead (4.8-2,103 microg) levels among lead workers. Among lead workers, 9.9% (n = 79) were heterozygous for the ALAD(2) allele and there were no homozygotes. For VDR, 10.7% (n = 85) had the Bb genotype, and 0.5% (n = 4) had the BB genotype. Although the ALAD and VDR genes are located on different chromosomes, lead workers homozygous for the ALAD(1) allele were much less likely to have the VDR bb genotype (crude odds ratio = 0.29, 95% exact confidence interval = 0.06-0.91). In adjusted analyses, subjects with the ALAD(2) allele had higher blood lead levels (on average, 2.9 microg/dL, p = 0.07) but no difference in tibia lead levels compared with subjects without the allele. In adjusted analyses, lead workers with the VDR B allele had significantly (p < 0.05) higher blood lead levels (on average, 4.2 microg/dL), chelatable lead levels (on average, 37.3 microg), and tibia lead levels (on average, 6.4 microg/g) than did workers with the VDR bb genotype. The current data confirm past observations that the ALAD gene modifies the toxicokinetics of lead and also provides new evidence that the VDR gene does so as well. The current data confirm past observations that the ALAD gene modifies the toxicokinetics of lead and also provides new evidence that the VDR gene does so as well. 1671851 van Oost BA, van Zandvoort PM, Tunte W, Brunner HG, Hoogeboom AJ, Maaswinkel-Mooy PD, Bakkeren J, Hamel B, Ropers HH: Linkage analysis in X-linked adrenoleukodystrophy and application in post- and prenatal diagnosis. Hum Genet. 1991 Feb;86(4):404-7. We have performed linkage analysis with the DNA markers DXS52 and the clotting factor VIII gene (F8C), in several large families with X-linked adrenoleukodystrophy (ALD). The tight linkage to DXS52 could be extended giving a maximal LOD score of 22.5 at 1 cM. F8C was also tightly linked to ALD with a maximal LOD score of 7.8 without recombination. Multipoint linkage analysis with the markers DXS304, DXS52, and F8C indicated that both the gene for ALD and for F8C are distal to DXS52. In four patients with ALD, no major structural rearrangement in the Xqter region was observed; in particular, there were no abnormalities in the vision blindness genes. DNA analysis appeared to be of use in determination of the carrier status of females at risk, for the determination of the origin of the mutation in a particular family, and for prenatal diagnosis. F8C was also tightly linked to ALD with a maximal LOD score of 7.8 without recombination. 11044606 Suzuki R, Shintani T, Sakuta H, Kato A, Ohkawara T, Osumi N, Noda M: Identification of RALDH-3, a novel retinaldehyde dehydrogenase, expressed in the ventral region of the retina. Mech Dev. 2000 Nov;98(1-2):37-50. In the developing retina, a retinoic acid (RA) gradient along the dorso-ventral axis is believed to be a prerequisite for the establishment of dorso-ventral asymmetry. This RA gradient is thought to result from the asymmetrical distribution of RA-generating aldehyde dehydrogenases along the dorso-ventral axis. Here, we identified a novel aldehyde dehydrogenase specifically expressed in the chick ventral retina, using restriction landmark cDNA scanning (RLCS). Since this molecule showed enzymatic activity to produce RA from retinaldehyde, we designated it retinaldehyde dehydrogenase 3 (RALDH-3). Structural similarity suggested that RALDH-3 is the orthologue of human aldehyde dehydrogenase 6. We also isolated RALDH-1 which is expressed in the chick dorsal retina and implicated in RA formation. Raldh-3 was preferentially expressed first in the surface ectoderm overlying the ventral portion of the prospective eye region and then in the ventral retina, earlier than Raldh-1 in chick and mouse embryos. High level expression of Raldh-3 was also observed in the nasal region. In addition, we found that Pax6 mutants are devoid of Raldh-3 expression. These results suggested that Raldh-3 is the key enzyme in the formation of an RA gradient along the dorso-ventral axis during the early eye development, and also in the development of the olfactory system. In addition, we found that Pax6 mutants are devoid of Raldh-3 expression. 12374760 Weinhofer I, Forss-Petter S, Zigman M, Berger J: Cholesterol regulates ABCD2 expression: implications for the therapy of X-linked adrenoleukodystrophy. Hum Mol Genet. 2002 Oct 15;11(22):2701-8. X-linked adrenoleukodystrophy (X-ALD) is a severe neurodegenerative disorder with impaired very long-chain fatty acid (VLCFA) metabolism. The disease-associated ABCD1 (ALD) gene encodes a peroxisomal membrane protein, which belongs to the superfamily of ATP-binding cassette transporters. Several treatment regimes have been tried without satisfactory clinical benefit. Recently, the cholesterol-lowering drug lovastatin was reported to normalize VLCFA levels in two out of three clinical studies. This investigation aimed to disclose the molecular mechanism of successful reduction of VLCFA accumulation in order to fill in the gap in the understanding how dietary cholesterol lowering affects the levels of VLCFA in patients with X-ALD and to allow more efficacious treatment. Overexpression of ABCD2 (ALDR), the closest relative of ABCD1, restores VLCFA accumulation in cultured ABCD1-deficient cells. Here we show by real-time PCR that the ABCD2 gene is induced in cultured human fibroblasts and monocytes upon sterol depletion via a mechanism requiring the activation of sterol regulatory element-binding proteins (SREBPs), a family of transcription factors that control the metabolism of cholesterol and fatty acids. This is unexpected and the first report that extends the mechanism of transcriptional regulation by SREBPs to a peroxisomal protein, thus providing a closer link between peroxisomes, cholesterol and fatty acid biosynthesis. Using reporter gene studies, site-directed mutagenesis and gel shift assays, we identified a functional sterol regulatory element in the proximal promoter region of ABCD2. Finally, we demonstrated that ABCD2 induction by sterol depletion significantly reduced the accumulation of VLCFA in X-ALD fibroblasts. Thus, lowering cholesterol leads to SREBP maturation, increased ABCD2 expression and reduced VLCFA accumulation. Cholesterol regulates ABCD2 expression: implications for the therapy of X-linked adrenoleukodystrophy. 11350124 Krieg P, Marks F, Furstenberger G: A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression. Genomics. 2001 May 1;73(3):323-30. Epidermis-type lipoxygenases, a distinct subclass within the multigene family of mammalian lipoxygenases (LOX), comprise recently discovered novel isoenzymes isolated from human and mouse skin including human 15-LOX-2, human and mouse 12R-LOX, mouse 8S-LOX, and mouse e-LOX-3. We have isolated the human homologue of mouse e-LOX-3. The cDNA of 3362 bp encodes a 711-amino-acid protein displaying 89% sequence identity with the mouse protein and exhibiting the same unusual structural feature, i.e., an extra segment of 41 amino acids, which can be located beyond the N-terminal beta-barrel domain at the surface of the C-terminal catalytic domain. The gene encoding e-LOX-3, ALOXE3, was found to be part of a gene cluster of approximately 100 kb on human chromosome 17p13.1 containing in addition the 12R-LOX gene, ALOX12B, the 15-LOX-2 gene, ALOX15B, and a novel 15-LOX pseudogene, ALOX15P. ALOXE3 and ALOX12B are arranged in a head-to-tail fashion separated by 8.5 kb. The genes are split into 15 exons and 14 introns spanning 22 and 15 kb, respectively. ALOX15P was found on the opposite DNA strand directly adjacent to the 3'-untranslated region of ALOX12B. ALOX15B is located in the same orientation 25 kb downstream of ALOX12B, and is composed of 14 exons and 13 introns spanning a total of 9.7 kb of genomic sequence. RT-PCR analysis demonstrated a predominant expression of ALOXE3, ALOX12B, and ALOX15B in skin. ALOXE3 and ALOX12B are arranged in a head-to-tail fashion separated by 8.5 kb. 11350124 Krieg P, Marks F, Furstenberger G: A gene cluster encoding human epidermis-type lipoxygenases at chromosome 17p13.1: cloning, physical mapping, and expression. Genomics. 2001 May 1;73(3):323-30. Epidermis-type lipoxygenases, a distinct subclass within the multigene family of mammalian lipoxygenases (LOX), comprise recently discovered novel isoenzymes isolated from human and mouse skin including human 15-LOX-2, human and mouse 12R-LOX, mouse 8S-LOX, and mouse e-LOX-3. We have isolated the human homologue of mouse e-LOX-3. The cDNA of 3362 bp encodes a 711-amino-acid protein displaying 89% sequence identity with the mouse protein and exhibiting the same unusual structural feature, i.e., an extra segment of 41 amino acids, which can be located beyond the N-terminal beta-barrel domain at the surface of the C-terminal catalytic domain. The gene encoding e-LOX-3, ALOXE3, was found to be part of a gene cluster of approximately 100 kb on human chromosome 17p13.1 containing in addition the 12R-LOX gene, ALOX12B, the 15-LOX-2 gene, ALOX15B, and a novel 15-LOX pseudogene, ALOX15P. ALOXE3 and ALOX12B are arranged in a head-to-tail fashion separated by 8.5 kb. The genes are split into 15 exons and 14 introns spanning 22 and 15 kb, respectively. ALOX15P was found on the opposite DNA strand directly adjacent to the 3'-untranslated region of ALOX12B. ALOX15B is located in the same orientation 25 kb downstream of ALOX12B, and is composed of 14 exons and 13 introns spanning a total of 9.7 kb of genomic sequence. RT-PCR analysis demonstrated a predominant expression of ALOXE3, ALOX12B, and ALOX15B in skin. ALOX15B is located in the same orientation 25 kb downstream of ALOX12B, and is composed of 14 exons and 13 introns spanning a total of 9.7 kb of genomic sequence. 7920663 Hentati A, Bejaoui K, Pericak-Vance MA, Hentati F, Speer MC, Hung WY, Figlewicz DA, Haines J, Rimmler J, Ben Hamida C, et al.: Linkage of recessive familial amyotrophic lateral sclerosis to chromosome 2q33-q35. Nat Genet. 1994 Jul;7(3):425-8. Amyotrophic lateral sclerosis (ALS) usually presents as a sporadic disorder of motor neurons. However, familial forms of ALS have been described--autosomal dominant forms (ALS1, ALS3), clinically indistinguishable from the sporadic form, and autosomal recessive forms with early onset and slower progression of symptoms (ALS2). To localize the gene for one of the autosomal recessive forms of ALS, we applied linkage analysis to a large inbred family from Tunisia. A lod score maximum of Zmax = 8.2 at theta = 0.00 was obtained with marker D2S72 located on chromosome 2q33-q35. The fine mapping of this region suggested that the ALS2 locus lies in the 8 cM segment flanked by D2S155 and D2S115. However, familial forms of ALS have been described--autosomal dominant forms (ALS1, ALS3), clinically indistinguishable from the sporadic form, and autosomal recessive forms with early onset and slower progression of symptoms (ALS2). 7533900 Rouet P, Raguenez G, Tronche F, Mfou'ou V, Salier JP: Hierarchy and positive/negative interplays of the hepatocyte nuclear factors HNF-1, -3 and -4 in the liver-specific enhancer for the human alpha-1-microglobulin/bikunin precursor. Nucleic Acids Res. 1995 Feb 11;23(3):395-404. Alpha-1-microglobulin and bikunin are two plasma glycoproteins encoded by an alpha-1-microglobulin/bikunin precursor (AMBP) gene. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. In the present study, electromobility shift assays of wild-type or mutated probes demonstrated that the boxes 1-5 have a binding capacity for their cognate HNF protein. Box 5 is also a target for another, as yet unidentified, factor. A functional analysis of the wild-type or mutated enhancer, driving its homologous promoter and a reporter CAT gene in the HepG2 hepatoma cell line, demonstrated that all six boxes participate in the enhancer activity, with the primary influence of box 4 (HNF-1) and box 2 (HNF-4). A similar analysis in the HNF-free CHO cell line co-transfected with one or several HNF factors further demonstrated various interplays between boxes: box 3 (HNF-3 alpha and beta) has a negative influence over the major HNF-4 box 2 as well as a positive influence over the major HNF-1 box 4. The strict liver-specific expression of the AMBP gene is controlled by a potent enhancer made of six clustered boxes numbered 1-6 that have been reported to be proven or potential binding sites for the hepatocyte-enriched nuclear factors HNF-1, -4, -3, -1, -3, -4, respectively. 9634515 Kant SG, Polinkovsky A, Mundlos S, Zabel B, Thomeer RT, Zonderland HM, Shih L, van Haeringen A, Warman ML: Acromesomelic dysplasia Maroteaux type maps to human chromosome 9. Am J Hum Genet. 1998 Jul;63(1):155-62. Acromesomelic dysplasias are skeletal disorders that disproportionately affect the middle and distal segments of the appendicular skeleton. We report genetic mapping studies in four families with acromesomelic dysplasia Maroteaux type (AMDM), an autosomal recessive osteochondrodysplasia. A peak LOD score of 5.1 at recombination fraction 0 was obtained with fully informative markers on human chromosome 9. In three of the four families, the affected offspring are products of consanguineous marriages; if it is assumed that these affected offspring are homozygous by descent for the region containing the AMDM locus, a 6.9-cM AMDM candidate interval can be defined by markers D9S1853 and D9S1874. The mapping of the AMDM locus to human chromosome 9 indicates that AMDM is genetically distinct from the two other mapped acromesomelic dysplasias, Hunter-Thompson type and Grebe type, which are caused by mutations in CDMP1 on human chromosome 20. The mapping of the AMDM locus to human chromosome 9 indicates that AMDM is genetically distinct from the two other mapped acromesomelic dysplasias, Hunter-Thompson type and Grebe type, which are caused by mutations in CDMP1 on human chromosome 20. 9398738 Sayegh RA, Tao XJ, Leykin L, Isaacson KB: Endometrial alpha-2 macroglobulin; localization by in situ hybridization and effect on mouse embryo development in vitro. J Clin Endocrinol Metab. 1997 Dec;82(12):4189-95. alpha-2 macroglobulin (A2M) is a 718,000-kDA broad spectrum plasma protease inhibitor whose production by the human endometrium was recently reported. The multifunctional A2M receptor, also known as low-density lipoprotein receptor-related protein, was also recently immunolocalized to the endometrial stroma. The objective of this study was to further characterize the endometrial site of expression of A2M, and to study its effects on mouse embryo development in vitro, to gain some insight into the functional significance of its endometrial production. Formalin-fixed, paraffin-embedded human endometrium from hysterectomy and endometrial biopsy specimen was used for in situ hybridization analysis, with 35S-labeled riboprobes representing subcloned A2M complementary DNA (cDNA) fragments. Duplicate sections of human endometrium were hybridized with sense and antisense probe and coated with photographic emulsion. Resultant autoradiograms were analyzed qualitatively by light- and darkfield microscopy and quantitatively by a computerized analysis of the signal intensity. Immunohistochemistry and immunoblotting for endometrial tissues were performed using an affinity-purified polyclonal antibody to human A2M. The effect of A2M on mouse embryo development was studied by exposure of one cell mouse embryo in culture to physiological concentrations of biologically active and inactive A2M. Expression signals for A2M were more numerous and intense in the secretory endometrium, compared with proliferative endometrium. Endothelial cells lining the endometrial blood vessels seemed to be the main source of A2M expression. The A2M expression signals in secretory endothelium were 2- to 3-fold stronger than the proliferative endothelium, suggesting transcriptional activation of A2M expression in the secretory endothelium. Glandular expression was observed in secretory endometrium from two patients with endometriosis. Ectopic endometrial tissues also produced A2M. A2M at concentrations of 400-500 mumol/L significantly inhibited blastocyst development of mouse embryos in vitro. A2M is expressed predominantly by the endometrial endothelial cells and may be involved in endometrial physiology. Physiological concentrations of A2M inhibit mouse embryo development in vitro, suggesting that endometrial production of A2M may play a role in regulating preimplantation embryo development. Ectopic endometrial tissues also produced A2M. 10894947 Leeb T, Breen M, Brenig B: Genomic structures and sequences of two closely linked genes (AMT, TCTA) on dog chromosome 20q15.1-->q15.2. Cytogenet Cell Genet. 2000;89(1-2):98-100. Analysis of genomic sequence from canine chromosome 20q15.1-->q15.2 revealed the presence of two closely linked genes. The two genes represent the corresponding canine orthologs of human aminomethyltransferase (AMT) and the human T-cell leukemia translocation associated (TCTA) gene. Aminomethyltransferase or glycine cleavage system T-protein is an important enzyme in glycine metabolism. The reported canine AMT gene spans 5 kb and consists of nine exons. It encodes a protein of 403 amino acids with 88% identity to human aminomethyltransferase. Human TCTA is located on 3p21 near the breakpoint of a t(1;3) translocation observed in some cancer cell lines. The 4-kb canine TCTA gene consists of three exons and probably represents a pseudogene. It is located adjacent to AMT and very close to DAG1 and BSN. The two genes represent the corresponding canine orthologs of human aminomethyltransferase (AMT) and the human T-cell leukemia translocation associated (TCTA) gene. 2424823 Davis MM, Hodes ME, Munsick RA, Ulbright TM, Goldstein DJ: Pancreatic amylase expression in human pancreatic development. Hybridoma. 1986 Summer;5(2):137-45. Enzymatic and immunologic methods were used to study amylase expression in amniotic fluid and human pancreatic tissue from fetuses of various gestational ages. A starch-coated slide assay was used to quantitate amylase activity in amniotic fluids, and samples with activity were studied by electrophoresis to determine the presence of salivary amylase (Amy1) and pancreatic amylase (Amy2) isozymes. Immunohistochemical studies were performed with both a rabbit anti-human Amy1 antibody and a murine anti-human Amy2 antibody (Amy2/SP2/1). Amy2/SP2/1 was used to discriminate between Amy1 and Amy2. Both Amy1 and Amy2 activities were present in amniotic fluid from 14 weeks' gestation, and immunologic activity was present in pancreatic tissue sections from 15.9 weeks' gestation. Amy2/SP2/1 was used to discriminate between Amy1 and Amy2. 10922985 Carter WB, Ward MD: HER2 regulatory control of angiopoietin-2 in breast cancer. Surgery. 2000 Aug;128(2):153-8. BACKGROUND: HER2 overexpression is a marker of aggressive breast cancer. Tumors that overexpress HER2 induce endothelial cell retraction and endothelial destabilization. Because angiopoietin-2 (Ang-2) also destabilizes microvessels, we postulated that HER2 signaling upregulates Ang-2 as a mechanism of angioinvasion. METHODS: We tested human breast cancers and breast cancer cell lines for coexpression of HER2 and Ang-2 with Northern blot, reverse transcriptase-polymerase chain reaction, and enzyme-linked immunosorbent assay. Further, we manipulated HER2 signaling with 100 ng/mL MAbHu HER2 (Herceptin; Genentech, San Francisco, Calif) and Heregulin beta1 (100 ng/mL; R&D Systems, Inc, Minneapolis, Minn) to test for HER2 regulation of Ang-2 production. RESULTS: Three of 4 breast cancer cell lines expressed HER2 protein and Ang-2 mRNA. HER cells, a stably transfected cell line that overexpresses HER2 6-fold, showed a 430% increase in Ang-2 mRNA compared to parental MCF-7 cells. Heregulin beta1 stimulation of HER2 signaling in MCF-7 cells increased Ang-2 by 20% (P <.05). HER2 signaling blockade with 100 ng/mL Herceptin reduced Ang-2 mRNA 90% (P <.001). Five of 11 cancers expressed both HER2 and Ang-2; 2 cancers expressed only Ang-2. CONCLUSIONS: We conclude that human breast cancers express Ang-2. HER2 signaling appears to regulate Ang-2 expression, although other signaling pathways may also regulate Ang-2. Ang-2 may be a therapeutic target in these cancers and may define which patients would benefit from Herceptin therapy. HER2 regulatory control of angiopoietin-2 in breast cancer. 1328390 Kilpatrick L, McCawley L, Nachiappan V, Greer W, Majumdar S, Korchak HM, Douglas SD: Alpha-1-antichymotrypsin inhibits the NADPH oxidase-enzyme complex in phorbol ester-stimulated neutrophil membranes. J Immunol. 1992 Nov 1;149(9):3059-65. The generation of superoxide anion and release of granule contents are essential to the bactericidal function of neutrophils, but may also contribute to host tissue damage during inflammation. In previous studies (J. Immunol. 146:2388), we have demonstrated that the acute phase reactant alpha-1-antichymotrypsin (ACT), a potent inhibitor of the serine protease cathepsin G, also suppresses superoxide anion generation. The inhibitory effect of ACT was not directly linked to its antiproteolytic activity and may reflect interaction at a site other than its reactive loop. To further characterize the mechanism of inhibition, we investigated the direct effects of ACT on the NADPH oxidase enzyme complex and the signaling pathways that regulate motivation of the respiratory burst. We present evidence that ACT does not intefer with agonist-stimulated calcium mobilization or translocation and activity of protein kinase C. ACT was an effective inhibitor of superoxide anion generation in membrane preparations isolated from PMA-activated cells. These results support the notion that ACT is acting on a component of the active assembled NADPH oxidase complex. Thus, ACT may have an important role in regulation of specific aspects of the inflammatory processes and the modulation of toxic oxygen-based host tissue damage. Alpha-1-antichymotrypsin inhibits the NADPH oxidase-enzyme complex in phorbol ester-stimulated neutrophil membranes. 9419419 Golay J, Broccoli V, Borleri GM, Erba E, Faretta M, Basilico L, Ying GG, Piccinini G, Shapiro LH, Lovric J, Nawrath M, Molling K, Rambaldi A, Introna M: Redundant functions of B-Myb and c-Myb in differentiating myeloid cells. Cell Growth Differ. 1997 Dec;8(12):1305-16. We show in this report that the human myeloid leukemia cell line GFD8 is a useful model to compare the biological function of the structurally related c-Myb and B-Myb proto-oncogenes and to investigate the c-myb domains required for this function. GFD8 cells are dependent for growth on granulocyte-macrophage colony-stimulating factor and differentiate in response to phorbol myristate acetate (PMA). We have stably transfected this cell line with constructs constitutively expressing c-Myb or B-Myb. Deregulated expression of both c-Myb and B-Myb inhibited the differentiation observed in response to PMA and, in particular, the induction of the CD11b and CD11c antigens on the cell surface, and the induction of adherence. Furthermore, c-Myb and B-Myb enhanced expression of CD13 upon PMA treatment. Although deregulated Myb expression did not alter the growth factor dependence of the cells, it led to an increase in G2 relative to G1 arrest in cells induced to differentiate in response to PMA, whereas control vector-transfected cells were blocked mostly in G1. This decrease in G1 block took place despite normal induction of the cyclin-dependent kinase inhibitor protein p21 (CIP1/WAF1). Thus, GFD8 cells stably expressing the human B-Myb protein behaved in a manner indistinguishable from those stably expressing C-Myb for both differentiation and cell cycle parameters. In agreement with these findings and differently from most previous reports, transactivation assays show that B-myb can indeed act as a strong activator of transcription. Finally, we demonstrated that although the DNA-binding domain of c-myb is required for both the differentiation block and the shift in cell cycle after PMA treatment, phosphorylation by casein kinase II and mitogen-activated protein kinase at positions 11 and 12 or 532 of c-myb, respectively, are not. We conclude that c-Myb and B-Myb may activate a common cellular program in the GFD8 cell line involved in both differentiation and cell cycle control. Furthermore, c-Myb and B-Myb enhanced expression of CD13 upon PMA treatment. 12730231 MacLeod TJ, Kwon M, Filipenko NR, Waisman DM: Phospholipid-associated annexin A2-S100A10 heterotetramer and its subunits: characterization of the interaction with tissue plasminogen activator, plasminogen, and plasmin. J Biol Chem. 2003 Jul 11;278(28):25577-84. Epub 2003 Apr 30. Annexin A2 (p36) is a highly alpha-helical molecule that consists of two opposing sides, a convex side that contains the phospholipid-binding sites and a concave side, which faces the extracellular milieu and contains multiple ligand-binding sites. The amino-terminal region of annexin A2 extends along the concave side of the protein and contains the binding site for the S100A10 (p11) subunit. The interaction of these subunits results in the formation of the heterotetrameric form of the protein, annexin A2-S100A10 heterotetramer (AIIt). To simulate the orientation of AIIt on the plasma membrane we bound AIIt to a phospholipid bilayer that was immobilized on a BIAcore biosensor chip. Surface plasmon resonance was used to observe in real time the molecular interactions between phospholipid-associated AIIt or its annexin A2 subunit and the ligands, tissue-type plasminogen activator (t-PA), plasminogen, and plasmin. AIIt bound t-PA (Kd = 0.68 microm), plasminogen (Kd = 0.11 microm), and plasmin (Kd = 75 nm) with moderate affinity. Contrary to previous reports, the phospholipid-associated annexin A2 subunit failed to bind t-PA or plasminogen but bound plasmin (Kd = 0.78 microm). The S100A10 subunit bound t-PA (Kd = 0.45 microm), plasminogen (Kd = 1.81 microm), and plasmin (Kd = 0.36 microm). Removal of the carboxyl-terminal lysines from the S100A10 subunit attenuated t-PA and plasminogen binding to AIIt. These results show that the carboxyl-terminal lysines of S100A10 form t-PA and plasminogen-binding sites. In contrast, annexin A2 and S100A10 contain distinct binding sites for plasmin. The amino-terminal region of annexin A2 extends along the concave side of the protein and contains the binding site for the S100A10 (p11) subunit. 8938449 Fernandez MP, Jenkins NA, Gilbert DJ, Copeland NG, Morgan RO: Sequence and chromosomal localization of mouse annexin XI. Genomics. 1996 Nov 1;37(3):366-74. Mouse annexin XI (anx11)2 was cloned from a macrophage cDNA library and characterized by genetic linkage mapping, DNA sequencing, and structural comparison with other annexins. The Anx11 gene localized to mouse chromosome 14 in close linkage with the Rarb, Plau, and Wnt5a genes near the centromere and 1.8 cM distal from the Anx7 gene. The open reading frame was flanked by long, untranslated regions and encoded a 503-amino-acid protein with 93.1% identity to its human orthologue. Its 189-aa amino terminus corresponded to the widely expressed variant 1 of two possible, alternatively spliced forms. A previously described peptide from Aplysia brasiliana was identified as a closely related invertebrate homologue. Since annexin XI is known to be localized in the nucleus at certain stages of development, the identification of a region in tetrad repeats 3 and 4 resembling the "chromo box" domain may be relevant to a nuclear regulatory function of annexin XI. Knowledge of the mouse cDNA sequence and genetic map location will assist in the analysis of genomic organization and expression and provide a useful animal model to investigate gene function and hereditary phenotype for annexin XI. The Anx11 gene localized to mouse chromosome 14 in close linkage with the Rarb, Plau, and Wnt5a genes near the centromere and 1.8 cM distal from the Anx7 gene. 12162552 Murasugi A, Tohma-Aiba Y: Characterization of partially truncated human midkine expressed in Pichia pastoris. Biosci Biotechnol Biochem. 2002 Jun;66(6):1295-300. Recombinant human midkine (rh-midkine) was expressed under the control of the AOX1 gene promoter in Pichiapastoris. Approximately 640 mg of rh-midkine was secreted into one liter of medium of the high cell-density fermentation. The protein processing of the rh-midkine was done efficiently and correctly in P. pastoris, and O-mannosylation was not detected in the purified rh-midkine. However, only about 30% of the purified rh-midkine was intact. The other ones lost 5-12 amino acid residues from the amino-termini, provably by proteolysis. Even the mixture of these truncated midkines could promote CHO cell proliferation as well as the authentic rh-midkine. Recombinant human midkine (rh-midkine) was expressed under the control of the AOX1 gene promoter in Pichiapastoris. 10833507 Lee DS, Tomita S, Kirino Y, Suzuki T: Regulation of X11L-dependent amyloid precursor protein metabolism by XB51, a novel X11L-binding protein. J Biol Chem. 2000 Jul 28;275(30):23134-8. We isolated a cDNA encoding a novel protein, XB51, that interacts with the amino-terminal domain of the neuron-specific X11-like protein (X11L). The protein XB51 inhibited the association of X11L with amyloid precursor protein through a non-competitive mechanism and abolished the suppression of beta-amyloid production by X11L. The majority of XB51 is localized around the nucleus and recovered in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) buffer-insoluble fraction when XB51 is expressed in cells. Association of XB51 with X11L changed the intracellular distribution of XB51 and resulted in redistribution of XB51 into the CHAPS buffer-soluble fraction. These observations suggest that XB51, together with X11L, plays an important role in the regulatory system of amyloid precursor protein metabolism and beta-amyloid generation. The protein XB51 inhibited the association of X11L with amyloid precursor protein through a non-competitive mechanism and abolished the suppression of beta-amyloid production by X11L. 10446967 Batchu RB, Shammas MA, Wang JY, Munshi NC: Interaction of adeno-associated virus Rep78 with p53: implications in growth inhibition. Cancer Res. 1999 Aug 1;59(15):3592-5. Adeno-associated virus (AAV) is a nonpathogenic, single-stranded DNA virus belonging to the parvoviridae family. Onco-suppressive properties of AAV against adenovirus, a DNA tumor virus, have been well documented. Rep78, a major regulatory protein of AAV, is believed to be responsible for its antioncogenic properties. Most DNA tumor viruses disturb the cell cycle pathways by essentially abrogating the functions of p53. Here we present evidence that AAV acts as an antiproliferative agent against adenovirus by protecting the adenoviral-mediated degradation of p53 as confirmed by both Western blot analysis and immunoprecipitation analysis with anti-p53 antibody. Coimmunoprecipitation experiments revealed that the AAV Rep78 is physically bound to p53 in vivo. Furthermore, the binding of purified p53 to the AAV Rep78 affinity column confirms their interaction. These results document for the first time that the antiproliferative effects of AAV against adenovirus are mediated, at least in part, by the interaction of AAV Rep78 with p53. Coimmunoprecipitation experiments revealed that the AAV Rep78 is physically bound to p53 in vivo. 12480705 Taketani S, Kakimoto K, Ueta H, Masaki R, Furukawa T: Involvement of ABC7 in the biosynthesis of heme in erythroid cells: interaction of ABC7 with ferrochelatase. Blood. 2003 Apr 15;101(8):3274-80. Epub 2002 Dec 12. A mitochondrial half-type ATP-binding cassette (ABC) protein, ABC7, plays a role in iron homeostasis in mitochondria, and defects in human ABC7 were shown to be responsible for the inherited disease X-linked sideroblastic anemia/ataxia. We examined the role of ABC7 in the biosynthesis of heme in erythroid cells where hemoglobin is a major product of iron-containing compounds. RNA blots showed that the amount of ABC7 mRNA in dimethylsulfoxide (Me(2)SO)-treated mouse erythroleukemia (MEL) cells increased markedly in parallel with the induction of the mRNA expression of ferrochelatase, the last enzyme in the pathway to synthesize heme. The transfection of the antisense oligonucleotide to mouse ABC7 mRNA into Me(2)SO-treated MEL cells led to a decrease of heme production, as compared with sense oligonucleotide-transfected cells. ABC7 protein was shown to be colocalized with ferrochelatase in mitochondria, as assessed by immunostaining. Furthermore, in vitro and in vivo pull-down assays revealed that ABC7 protein is interacted with the carboxy-terminal region containing the iron-sulfur cluster of ferrochelatase. The transient expression of ABC7 in mouse embryo liver BNL-CL2 cells resulted in an increase in the activity and level of ferrochelatase and thioredoxin, a cytosolic protein containing iron-sulfur. These increases were also observed in MEL cells stably expressing ABC7. When ABC7 transfectants were treated with Me(2)SO, an increase in cellular heme concomitant with a marked induction of the expression of ferrochelatase was observed. The extent of these increases was 3-fold greater than in control cells. The results indicated that ABC7 positively regulates not only the expression of extramitochondrial thioredoxin but also that of an intramitochondrial iron-sulfur-containing protein, ferrochelatase. Then, the expression of ABC7 contributes to the production of heme during the differentiation of erythroid cells. ABC7 protein was shown to be colocalized with ferrochelatase in mitochondria, as assessed by immunostaining. 7965771 Lacal PM, Amici C, Bonmassar E, D'Onofrio C: Effects of cyclopentenone prostaglandins on myeloid cells during early infection with HTLV-I. II. Regulation of synthesis of inducible p72 heat shock protein. J Pharmacol Exp Ther. 1994 Nov;271(2):1096-102. Differentiation of cells of myelomonocitic lineage influences both cellular permissivity to infection with human T-cell leukemia virus type I after cell-to-cell virus transmission and sensitivity to the antiproliferative effect of cyclopentenone prostaglandins (PG)A1 and PGJ2. Growth inhibition and control of infection were found to be associated with high intracellular levels of inducible p72 heat shock protein (HSP70). Pluripotent K562 cells produced higher HSP70 base-line levels than promyelocytic HL60 or monoblastoid U937 cells. Treatment with PGA1 and especially with PGJ2 enhanced the synthesis of HSP70 in all these cells. Notably, HSP70 accumulated in virus-exposed U937 cells (but not in K562 or HL60 cells). Because in lethally irradiated virus-donor cells HSP70 production was barely detectable, expression of this protein in cocultured U937 cells can be prevalently attributed to virus-recipient cells. Treatment with PGA1 and even more with PGJ2 remarkably enhanced the synthesis of HSP70 in virus-exposed U937 cells, thus resulting in persistently high levels of HSP70 protein in the cells. As shown previously, in U937 cells treatment with PGs was associated with reduced percentages of virus p19gag positive cells and enhanced specific lysis of virus-donor cells at early time points after cell-to-cell transmission. Because the HSP70 protein family is involved in the control of cell proliferation as well as in antigen processing function during the immune response to pathogens, it is possible that persistent high expression levels of HSP70 in PG-treated cells play a critical role in regulating both cell cycling and antiviral cellular responses. Treatment with PGA1 and especially with PGJ2 enhanced the synthesis of HSP70 in all these cells. 9211900 Wang B, Golemis EA, Kruh GD: ArgBP2, a multiple Src homology 3 domain-containing, Arg/Abl-interacting protein, is phosphorylated in v-Abl-transformed cells and localized in stress fibers and cardiocyte Z-disks. J Biol Chem. 1997 Jul 11;272(28):17542-50. Arg and c-Abl represent the mammalian members of the Abelson family of protein-tyrosine kinases. A novel Arg/Abl-binding protein, ArgBP2, was isolated using a segment of the Arg COOH-terminal domain as bait in the yeast two-hybrid system. ArgBP2 contains three COOH-terminal Src homology 3 domains, a serine/threonine-rich domain, and several potential Abl phosphorylation sites. ArgBP2 associates with and is a substrate of Arg and v-Abl, and is phosphorylated on tyrosine in v-Abl-transformed cells. ArgBP2 is widely expressed in human tissues and extremely abundant in heart. In epithelial cells ArgBP2 is located in stress fibers and the nucleus, similar to the reported localization of c-Abl. In cardiac muscle cells ArgBP2 is located in the Z-disks of sarcomeres. These observations suggest that ArgBP2 functions as an adapter protein to assemble signaling complexes in stress fibers, and that ArgBP2 is a potential link between Abl family kinases and the actin cytoskeleton. In addition, the localization of ArgBP2 to Z-disks suggests that ArgBP2 may influence the contractile or elastic properties of cardiac sarcomeres and that the Z-disk is a target of signal transduction cascades. ArgBP2 associates with and is a substrate of Arg and v-Abl, and is phosphorylated on tyrosine in v-Abl-transformed cells. 12856127 Mensink M, Blaak EE, Vidal H, De Bruin TW, Glatz JF, Saris WH: Lifestyle changes and lipid metabolism gene expression and protein content in skeletal muscle of subjects with impaired glucose tolerance. Diabetologia. 2003 Aug;46(8):1082-9. Epub 2003 Jul 11. AIMS/HYPOTHESIS. Skeletal muscle of pre-diabetic patients is characterised by a diminished capacity to handle fatty acids. A diminished content of several enzymes involved in fatty-acid transport and oxidation have been suggested to underlie these defects. The aim of this study was to investigate whether the combination of dietary advice, increased physical activity and weight loss improves lipid metabolic gene and protein expression in skeletal muscle of subjects with impaired glucose tolerance. METHODS. Before and after 1 year of a lifestyle-intervention programme, expression of several genes and proteins involved in lipid metabolism were measured in vastus lateralis muscle biopsies from subjects in the intervention ( n=7) and control group ( n=6). RESULTS. After 1 year the intervention group had an improved glycaemic control and reduced body fat compared to the control group. Significant differences were observed for acetyl CoA-carboxylase 2 and uncoupling protein 2 expression (ACC2: -16.8+/-12.4% vs +51.5+/-32.3% for the intervention and control group respectively; p<0.05) (UCP2: -26.9+/-10.3% vs +10.5+/-6.2% for the intervention and control group respectively; p<0.05). Change in 3-hydroxyacyl-CoA dehydrogenase protein content tended to be different between groups (+3.2+/-1.1 vs -0.9+/-1.9 U/mg.ww for the intervention and control group, p=0.07). CONCLUSIONS/INTERPRETATION. Lifestyle changes leading to an improved glycaemic control and reduced adiposity, resulted in a down-regulation of ACC-2 and UCP2 expression and in an increase in HAD protein content, reflecting a better capacity to utilise fatty acids. Significant differences were observed for acetyl CoA-carboxylase 2 and uncoupling protein 2 expression (ACC2: -16.8+/-12.4% vs +51.5+/-32.3% for the intervention and control group respectively; p <0.05) (UCP2: -26.9+/-10.3% vs +10.5+/-6.2% for the intervention and control group respectively; p <0.05). 2655995 Seiffert UB, Siede WH, Oremek G: Endoprotease in human liver transforming multiple forms of alkaline phosphatase. Clin Biochem. 1989 Apr;22(2):103-7. Two forms of alkaline phosphatase, extracted from human liver and named API1 and API3, are of high molecular mass, but API3 is the larger molecule and is membrane-bound while API1 is smaller and soluble. Enzyme kinetics are identical. It is suggested that API1 is produced from API3 by an endoprotease. We demonstrated the action of an endoprotease in human liver homogenate converting API3 into API1. In the absence of this enzyme no conversion occurred. This enzyme is active at an acidic pH (less than 6.5) in the presence of Ca.. or Mg.. -ions. It is inhibited by traces of EDTA. It is insensitive to diisopropyl fluoro-phosphate, to leupeptin and to reducing or oxidizing chemicals. At alkaline pH (8.6) its activity is rapidly destroyed. The enzyme is stable in acidic buffer. We conclude that API1 is indeed formed from API3 in the living cell by enzymatic conversion. It is suggested that API1 is produced from API3 by an endoprotease. 12609736 Zhou C, Blumberg B: Overlapping gene structure of human VLCAD and DLG4. Gene. 2003 Feb 27;305(2):161-6. Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a major enzyme catalysing the first step in mitochondrial beta-oxidation of long-chain fatty acids. During analysis of the VLCAD promoter, we discovered that another gene, discs-large-related 4 (DLG4), overlaps VLCAD and is transcribed in the opposite direction. DLG4 encodes postsynaptic density-95 (PSD95) protein, which plays critical roles in the formation and maintenance of synaptic junctions. The transcription start site of the VLCAD gene was determined by primer extension analysis and the overlapping structure of VLCAD and DLG4 was clarified. VLCAD and DLG4 are arranged in a head-to-head orientation on chromosome 17p13, and share a 245 bp overlapping region that contains part of DLG4 exon 1 and the entire exon 1 of VLCAD including 62 bp of protein coding sequence. Despite the overlap of their 5' ends, DLG4 and VLCAD exhibit peak mRNA expression in different tissues, suggesting that they are independently regulated at the transcriptional level. Interestingly, VLCAD and DLG4 genes do not overlap in the mouse or Drosophila genomes. Overlapping gene structure of human VLCAD and DLG4. 10625210 Ieko M, Sawada KI, Koike T, Notoya A, Mukai M, Kohno M, Wada N, Itoh T, Yoshioka N: The putative mechanism of thrombosis in antiphospholipid syndrome: impairment of the protein C and the fibrinolytic systems by monoclonal anticardiolipin antibodies. Semin Thromb Hemost. 1999;25(5):503-7. The mechanism of thrombosis in patients with antiphospholipid syndrome is not clear. To investigate it, we examined the effect of monoclonal anticardiolipin (aCL) antibodies and beta2-glycoprotein I (beta2-GPI), which is required for formation of the aCL epitopes, on activated protein C (APC) and on fibrinolytic activity. First, APC activities were measured in the presence and absence of beta2-GPI or gamma M immunoglobulin (IgM) monoclonal aCLs (EY1C8 and EY2C9), or both, established from peripheral blood lymphocytes obtained from a patient with aCL. beta2-GPI exhibited a procoagulant activity by inhibiting APC activity as well as an anticoagulant activity by inhibiting thrombin generation. Any further inhibition of APC activity was caused by monoclonal aCL, and then only in the presence of beta2-GPI. The remaining tissue plasminogen activator (t-PA) of the sample consisting of beta2-GPI, two-chain recombinant t-PA, and plasminogen activator inhibitor (PAI)-1 was measured by a chromogenic assay using the synthetic substrate S-2251, Glu-plasminogen, and soluble fibrin monomer. beta2-GPI protected t-PA activity from inhibition by PAI-1. However, monoclonal aCLs (EY1C8 and EY2C9) inhibited the effect of beta2-GPI on fibrinolytic activity; that is, monoclonal aCLs inhibited fibrinolytic activity by elevating PAI-1 activity. Thrombosis in patients with aCL can be explained in part by both the inhibition of APC anticoagulant activity and the impairment of fibrinolytic activity by aCL. However, monoclonal aCLs (EY1C8 and EY2C9) inhibited the effect of beta2-GPI on fibrinolytic activity; that is, monoclonal aCLs inhibited fibrinolytic activity by elevating PAI-1 activity. 3100097 Koren E, Puchois P, Alaupovic P, Fesmire J, Kandoussi A, Fruchart JC: Quantification of two different types of apolipoprotein A-I containing lipoprotein particles in plasma by enzyme-linked differential-antibody immunosorbent assay. Clin Chem. 1987 Jan;33(1):38-43. We describe a method for measuring apolipoprotein A-I (ApoA-I) associated and unassociated with apolipoprotein A-II (ApoA-II) in plasma. To directly determine associated ApoA-I, we coated microtiter plates with antibody to ApoA-II, blocked the nonspecific binding sites, and incubated the plate with plasma, immobilizing the lipoprotein particles containing both ApoA-II and ApoA-I. The unbound constituents of plasma were washed away, peroxidase-labeled antibody to ApoA-I was added, the plate rewashed, peroxidase substrate added, and the resulting color measured. ApoA-I unassociated with ApoA-II was evaluated by subtracting the concentration of associated ApoA-I from the total ApoA-I concentration. The method is specific, rapid, and precise. Within- and between-assay CVs were 5.6 and 9.8%, respectively. Analytical recovery of ApoA-I was 94%. The average normolipidemic concentration of ApoA-I associated with ApoA-II in 50 women was 790 mg/L; in 50 men, it was 788 mg/L. The corresponding values for unassociated ApoA-I were 644, 577 mg/L. Both lipoprotein forms of ApoA-I were detected in all major density classes, but were most abundant in high-density lipoproteins. The technique is applicable to measurement of any two apolipoproteins that occur in both associated and unassociated forms in plasma. We describe a method for measuring apolipoprotein A-I (ApoA-I) associated and unassociated with apolipoprotein A-II (ApoA-II) in plasma. 10393433 Viggiano L, Rocchi M, Svelto M, Calamita G: Assignment of the aquaporin-8 water channel gene (AQP8) to human chromosome 16p12. Cytogenet Cell Genet. 1999;84(3-4):208-10. Assignment of the aquaporin-8 water channel gene (AQP8) to human chromosome 16p12. 3987576 Bottini E, Carapella E, Scacchi R, Lucarini N, Gloria-Bottini F, Pascone R, Bonci E, Maggioni G: Serum haptoglobin appearance during neonatal period is associated with acid phosphatase (ACP1) phenotype. Early Hum Dev. 1985 Jan;10(3-4):237-43. Erythrocyte acid phosphatase (ACP1) is a polymorphic enzyme found in many tissues and acts in vivo as a flavin-mononucleotide phosphatase. We have recently observed a relation between this enzyme and length of gestation. The present study shows that the pattern of appearance of serum haptoglobin during the neonatal period is associated with ACP1 phenotype suggesting some important function of this polymorphic enzyme in human development. Serum haptoglobin appearance during neonatal period is associated with acid phosphatase (ACP1) phenotype. 12354724 Hoerstrup SP, Kadner A, Melnitchouk S, Trojan A, Eid K, Tracy J, Sodian R, Visjager JF, Kolb SA, Grunenfelder J, Zund G, Turina MI: Tissue engineering of functional trileaflet heart valves from human marrow stromal cells. Circulation. 2002 Sep 24;106(12 Suppl 1):I143-50. BACKGROUND: We previously demonstrated the successful tissue engineering and implantation of functioning autologous heart valves based on vascular-derived cells. Human marrow stromal cells (MSC) exhibit the potential to differentiate into multiple cell-lineages and can be easily obtained clinically. The feasibility of creating tissue engineered heart valves (TEHV) from MSC as an alternative cell source, and the impact of a biomimetic in vitro environment on tissue differentiation was investigated. METHODS AND RESULTS: Human MSC were isolated, expanded in culture, and characterized by flow-cytometry and immunohistochemistry. Trileaflet heart valves fabricated from rapidly bioabsorbable polymers were seeded with MSC and grown in vitro in a pulsatile-flow-bioreactor. Morphological characterization included histology and electron microscopy (EM). Extracellular matrix (ECM)-formation was analyzed by immunohistochemistry, ECM protein content (collagen, glycosaminoglycan) and cell proliferation (DNA) were biochemically quantified. Biomechanical evaluation was performed using Instron(TM). In all valves synchronous opening and closing was observed in the bioreactor. Flow-cytometry of MSC pre-seeding was positive for ASMA, vimentin, negative for CD 31, LDL, CD 14. Histology of the TEHV-leaflets demonstrated viable tissue and ECM formation. EM demonstrated cell elements typical of viable, secretionally active myofibroblasts (actin/myosin filaments, collagen fibrils, elastin) and confluent, homogenous tissue surfaces. Collagen types I, III, ASMA, and vimentin were detected in the TEHV-leaflets. Mechanical properties of the TEHV-leaflets were comparable to native tissue. CONCLUSION: Generation of functional TEHV from human MSC was feasible utilizing a biomimetic in vitro environment. The neo-tissue showed morphological features and mechanical properties of human native-heart-valve tissue. The human MSC demonstrated characteristics of myofibroblast differentiation. Collagen types I, III, ASMA, and vimentin were detected in the TEHV-leaflets. 8941379 Ueyama H, Inazawa J, Nishino H, Ohkubo I, Miwa T: FISH localization of human cytoplasmic actin genes ACTB to 7p22 and ACTG1 to 17q25 and characterization of related pseudogenes. Cytogenet Cell Genet. 1996;74(3):221-4. Human beta- and gamma-cytoplasmic actin genes (ACTB and ACTG1) were mapped to chromosomes 7p22 and 17q25, respectively, by fluorescence in situ hybridization (FISH). Four processed pseudogenes, beta-actin-related ACTBP9 and gamma-actin-related ACTGP1, ACTGP3, and ACTGP9, were isolated from human libraries. By PCR of somatic cell hybrid DNAs, ACTBP9 and two beta-actin-related pseudogenes (ACTBP7 and ACTBP8) were mapped to human chromosomes 18, 15, and 6, respectively. The gamma-actin-related pseudogenes were mapped by FISH to chromosomes 3q23 (ACTGP1), 20p13 (ACTGP3), and 6p21.1 (ACTGP9). By PCR of somatic cell hybrid DNAs, ACTBP9 and two beta-actin-related pseudogenes (ACTBP7 and ACTBP8) were mapped to human chromosomes 18, 15, and 6, respectively. 8941379 Ueyama H, Inazawa J, Nishino H, Ohkubo I, Miwa T: FISH localization of human cytoplasmic actin genes ACTB to 7p22 and ACTG1 to 17q25 and characterization of related pseudogenes. Cytogenet Cell Genet. 1996;74(3):221-4. Human beta- and gamma-cytoplasmic actin genes (ACTB and ACTG1) were mapped to chromosomes 7p22 and 17q25, respectively, by fluorescence in situ hybridization (FISH). Four processed pseudogenes, beta-actin-related ACTBP9 and gamma-actin-related ACTGP1, ACTGP3, and ACTGP9, were isolated from human libraries. By PCR of somatic cell hybrid DNAs, ACTBP9 and two beta-actin-related pseudogenes (ACTBP7 and ACTBP8) were mapped to human chromosomes 18, 15, and 6, respectively. The gamma-actin-related pseudogenes were mapped by FISH to chromosomes 3q23 (ACTGP1), 20p13 (ACTGP3), and 6p21.1 (ACTGP9). The gamma-actin-related pseudogenes were mapped by FISH to chromosomes 3q23 (ACTGP1), 20p13 (ACTGP3), and 6p21.1 (ACTGP9). 10656685 Nikolopoulos SN, Spengler BA, Kisselbach K, Evans AE, Biedler JL, Ross RA: The human non-muscle alpha-actinin protein encoded by the ACTN4 gene suppresses tumorigenicity of human neuroblastoma cells. Oncogene. 2000 Jan 20;19(3):380-6. alpha-Actinins are actin-binding proteins important in organization of the cytoskeleton and in cell adhesion. We have cloned and characterized a cDNA from human neuroblastoma cell variants which encodes the second non-muscle alpha-actinin isoform designated ACTN4 (actinin-4). mRNA encoded by the ACTN4 gene, mapped to chromosome 4, is abundant in non-tumorigenic, substrate-adherent human neuroblastoma cell variants but absent or only weakly expressed in malignant, poorly substrate-adherent neuroblasts. It is also present in many adherent tumor cell lines of diverse tissue origins. Cell lines typically co-express ACTN4 and ACTN1, a second non-muscle alpha-actinin gene. Expression is correlated with substrate adhesivity. Analysis of deduced amino acid sequences suggests that the two isoforms may differ in function and in regulation by calcium. Moreover, ACTN4 exhibits tumor suppressor activity. Stable clones containing increased levels of alpha-actinin, isolated from highly malignant neuroblastoma stem cells [BE(2)-C] after transfection with a full-length ACTN4 cDNA, show decreased anchorage-independent growth ability, loss of tumorigenicity in nude mice, and decreased expression of the N-myc proto-oncogene. Cell lines typically co-express ACTN4 and ACTN1, a second non-muscle alpha-actinin gene. 7890768 Xu J, McKeehan K, Matsuzaki K, McKeehan WL: Inhibin antagonizes inhibition of liver cell growth by activin by a dominant-negative mechanism. J Biol Chem. 1995 Mar 17;270(11):6308-13. The beta:beta activin homodimer and alpha:beta inhibin heterodimer are mutual antagonists which share a common beta subunit. Recently, it has been shown that, similar to transforming growth factor-beta 1, activin is an inhibitor of hepatocyte DNA synthesis. The activin receptor appears to be an obligatory complex of genetically distinct type I and II transmembrane serine/threonine kinases. Activin type I receptors, SKR1 and SKR2, were first cloned from well differentiated human hepatoma cells (HepG2). This prompted us to investigate the binding of activin and inhibin to receptors from HepG2 cells and the effect of the two ligands on DNA synthesis. Here we show that beta:beta activin binds to the activin type II receptor kinase (ActRII) which induces activin binding to the type I receptor kinase SKR2 to form ActRII.beta:beta.SKR2 complexes in which an activin beta chain occupies each receptor subunit. Inhibin also binds to ActRII through its beta subunit, competes with the binding of activin to ActRII, but fails to form the ActRII.SKR2 complex. No specific binding site for inhibin could be demonstrated in HepG2 cells. Inhibin, which had no activity of its own, antagonized the inhibitory effect of activin on DNA synthesis. The results suggest that inhibin may be a natural antagonist of assembly of the heterodimeric activin receptor complex through a dominant-negative mechanism. Here we show that beta:beta activin binds to the activin type II receptor kinase (ActRII) which induces activin binding to the type I receptor kinase SKR2 to form ActRII.beta:beta.SKR2 complexes in which an activin beta chain occupies each receptor subunit. 2334420 Yu XB, Burke C, Zhang J, Marchitelli LJ, Davis EA, Ackerman S: Transcription factor IIA of wheat and human interacts similarly with the adenovirus-2 major late promoter. Biochem Biophys Res Commun. 1990 Apr 30;168(2):498-505. Transcription factor IIA (TFIIA) is a necessary component of many RNA polymerase II transcription complexes. Assembly of the transcription complex begins when TFIIA interacts with the promoter. We have previously purified wheat germ TFIIA to homogeneity and demonstrated that it substitutes for human TFIIA in a human in vitro transcription system which utilizes the adenovirus-2 major late promoter (Ad-2 MLP). We now show, by gel retardation assays, that wheat TFIIA interacts with the Ad-2 MLP. Extensively purified human (HeLa) TFIIA interacts with the Ad-2 MLP similarly. Both wheat and human TFIIA interact with a DNA fragment comprising the minimal promoter region (-51/+32) but not with upstream or downstream regions. With both TFIIAs multiple complexes form; the fastest wheat TFIIA/DNA complex appears to be larger than the corresponding human TFIIA/DNA complex. Limited point mutation analysis of the Ad-2 MLP demonstrates that changes at -30 (TATAA region), +1, and -1 diminish TFIIA binding, but a change at -40 does not. DNA footprint analysis of this region is not definitive, but does indicate that following TFIIA binding there are changes in the pattern of hypersensitive sites. Extensively purified human (HeLa) TFIIA interacts with the Ad-2 MLP similarly. 12589427 Tazi-Ahnini R, Cork MJ, Wengraf D, Wilson AG, Gawkrodger DJ, Birch MP, Messenger AG, McDonagh AJ: Notch4, a non-HLA gene in the MHC is strongly associated with the most severe form of alopecia areata. Hum Genet. 2003 Apr;112(4):400-3. Epub 2003 Feb 14. Alopecia areata (AA) is a disorder primarily affecting the hair and nails in which associated autoimmune or atopic disease is common. Genetically, it is a complex trait with evidence of a role for genes of the major histocompatibility complex (MHC), the interleukin-1 cluster and chromosome 21 in the pathogenesis. The strongest association is with HLA class II alleles, although whether this indicates a direct contribution to the pathogenesis or results merely from linkage disequilibrium with nearby disease genes is unknown. Notch4 is a recently defined gene in the HLA class III region. Notch signalling is a direct determinant of keratinocyte growth arrest and entry into differentiation. A possible role for Notch in hair growth has been indicated by transgenic mouse findings that activation of the Notch pathway in the hair cortex leads to aberrant differentiation of adjacent hair-shaft layers. Notch4 is therefore a plausible candidate gene for AA. We have examined two polymorphisms in the coding sequence of the Notch4 gene at positions +1297 and +3063 in a case-control study of 116 AA patients and 142 ethnically matched, healthy control subjects. The initial analysis showed a significant association of AA in the overall data set with the Notch4(T+1297C) polymorphism (P<0.001) but not with Notch4(A+3063G). To confirm this association, we genotyped an additional 62 patients and found that the risk for disease was higher in Notch4(+1297C) homozygotes [odds ratio (OR) 3.43 (1.63, 7.19)] than in heterozygotes [OR 2.58 (1.57, 4.24)]. On classifying the patients by severity of disease, the association appeared to be confined to the severest form (alopecia universalis) [OR 4.02 (1.64, 9.88), P=0.0014]. These results support previous findings showing that different HLA susceptibility alleles are associated with mild and severe AA. We have examined two polymorphisms in the coding sequence of the Notch4 gene at positions +1297 and +3063 in a case-control study of 116 AA patients and 142 ethnically matched, healthy control subjects. 9819405 Gelmetti V, Zhang J, Fanelli M, Minucci S, Pelicci PG, Lazar MA: Aberrant recruitment of the nuclear receptor corepressor-histone deacetylase complex by the acute myeloid leukemia fusion partner ETO. Mol Cell Biol. 1998 Dec;18(12):7185-91. Nuclear receptor corepressor (CoR)-histone deacetylase (HDAC) complex recruitment is indispensable for the biological activities of the retinoic acid receptor fusion proteins of acute promyelocytic leukemias. We report here that ETO (eight-twenty-one or MTG8), which is fused to the acute myelogenous leukemia 1 (AML1) transcription factor in t(8;21) AML, interacts via its zinc finger region with a conserved domain of the corepressors N-CoR and SMRT and recruits HDAC in vivo. The fusion protein AML1-ETO retains the ability of ETO to form stable complexes with N-CoR/SMRT and HDAC. Deletion of the ETO C terminus abolishes CoR binding and HDAC recruitment and severely impairs the ability of AML1-ETO to inhibit differentiation of hematopoietic precursors. These data indicate that formation of a stable complex with CoR-HDAC is crucial to the activation of the leukemogenic potential of AML1 by ETO and suggest that aberrant recruitment of corepressor complexes is a general mechanism of leukemogenesis. Deletion of the ETO C terminus abolishes CoR binding and HDAC recruitment and severely impairs the ability of AML1-ETO to inhibit differentiation of hematopoietic precursors. 12859334 Kawahara Y, Ito K, Sun H, Kanazawa I, Kwak S: Low editing efficiency of GluR2 mRNA is associated with a low relative abundance of ADAR2 mRNA in white matter of normal human brain. Eur J Neurosci. 2003 Jul;18(1):23-33. The ionotropic glutamate receptor (GluR) subunits GluR2, GluR5 and GluR6 are subject to RNA editing at their Q/R sites, resulting in significant alterations in the channel properties of the receptors. RNA editing at the Q/R site of GluRs is both developmentally and regionally regulated. Here we provide the first quantitative measurements of both mRNAs of the GluR subunits and mRNAs of the RNA editing enzymes ADAR1-ADAR3 in a comparison of the efficiency of editing at the Q/R site with the expression levels of ADAR mRNA in human brain. We demonstrate that the Q/R site of GluRs in white matter is edited significantly less than in grey matter. In addition, by means of quantitative reverse transcription-polymerase chain reaction methods, we demonstrate that the relative abundance of ADAR2 mRNA to GluR2 mRNA is significantly lower in white matter than in grey matter and that the GluR2 Q/R site editing decreased only when the ratio of ADAR2 mRNA (not that of ADAR1 mRNA) to GluR2 mRNA dropped below a threshold (20 x 10(-3)). These results suggest that Q/R site of GluRs editing is regulated in a regional, and hence presumably cell-specific, manner and that the GluR2 Q/R site editing is critically regulated by ADAR2 in human brain. Low editing efficiency of GluR2 mRNA is associated with a low relative abundance of ADAR2 mRNA in white matter of normal human brain. 10931828 Tyzack JK, Wang X, Belsham GJ, Proud CG: ABC50 interacts with eukaryotic initiation factor 2 and associates with the ribosome in an ATP-dependent manner. J Biol Chem. 2000 Nov 3;275(44):34131-9. Eukaryotic initiation factor 2 (eIF2) plays a key role in the process of translation initiation and in its control. Here we demonstrate that highly purified mammalian eIF2 contains an additional polypeptide of apparent molecular mass of 110 kDa. This polypeptide co-purified with eIF2 through five different chromatography procedures. A cDNA clone encoding the polypeptide was isolated, and its sequence closely matched that of a protein previously termed ABC50, a member of the ATP-binding cassette (ABC) family of proteins. Antibodies to ABC50 co-immunoprecipitated eIF2 and vice versa, indicating that the two proteins interact. The presence of ABC50 had no effect upon the ability of eIF2 to bind GDP but markedly enhanced the association of methionyl-tRNA with the factor. Unlike the majority of ABC proteins, which are membrane-associated transporters, ABC50 associates with the ribosome and co-sediments in sucrose gradients with the 40 and 60 S ribosomal subunits. The association of ABC50 with ribosomal subunits was increased by ATP and decreased by ADP. ABC50 is related to GCN20 and eEF3, two yeast ABC proteins that are not membrane-associated transporters and are instead implicated in mRNA translation and/or its control. Thus, these data identify ABC50 as a third ABC protein with a likely function in mRNA translation, which associates with eIF2 and with ribosomes. ABC50 is related to GCN20 and eEF3, two yeast ABC proteins that are not membrane-associated transporters and are instead implicated in mRNA translation and/or its control. 1352876 You M, Wang Y, Stoner G, You L, Maronpot R, Reynolds SH, Anderson M: Parental bias of Ki-ras oncogenes detected in lung tumors from mouse hybrids. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5804-8. A mouse strain with low lung tumor susceptibility (C3H) and a strain with high lung tumor susceptibility (A/J) were reciprocally crossed to produce C3A and AC3 F1 hybrid mice. Ki-ras oncogenes were detected in spontaneous and chemically induced lung tumors obtained from the C3A and AC3 mice. To further explore the genetics of the Ki-ras gene in mouse lung tumor susceptibility, the parental origin of Ki-ras oncogenes detected in lung tumors from the F1 hybrids was determined by a strategy based on a 37-base-pair deletion in the second intron of the A/J Ki-ras allele. Ki-ras oncogenes were derived from the A/J parent in 38 of 40 tumors obtained from C3A mice and 30 of 30 tumors from AC3 mice. The observation that the activated oncogene in hybrids originates from the susceptible parent suggests that the Ki-ras gene is directly linked to mouse lung tumor susceptibility. This finding may have implications for pulmonary adenocarcinoma development in humans, since Ki-ras oncogenes are detected in 35% of this human tumor type. Ki-ras oncogenes were detected in spontaneous and chemically induced lung tumors obtained from the C3A and AC3 mice. 11584144 Takeshita T, Yang X, Morimoto K: Association of the ADH2 genotypes with skin responses after ethanol exposure in Japanese male university students. Alcohol Clin Exp Res. 2001 Sep;25(9):1264-9. BACKGROUND: A contribution of the alcohol dehydrogenase-2 (ADH2) polymorphism to alcohol sensitivity and alcohol drinking behavior is still controversial. In this study, we examined the effects of the ADH2 genotypes on skin reactions to ethanol and habitual alcohol intake among Japanese male university students, controlling for the effects of the low Km aldehyde dehydrogenase (ALDH2) genotype, as an extension of our previous study. METHODS: The study subjects were 357 Japanese male students [average age (mean +/- SD) was 23.7 +/- 3.0 years] in a medical university. The subjects completed a questionnaire regarding self-reported alcohol-associated symptoms and alcohol-drinking behavior. The ADH2 and ALDH2 genotypes were determined through digestion of polymerase chain reaction products by restriction enzymes. All subjects participated in the ethanol patch test. We observed skin responses at 0, 5, 15, and 20 min after removal of the tape. RESULTS: Among the ALDH2*1/*1 genotypes, only some subjects with ADH2*1/*2 or ADH2*2/*2 exhibited a positive response, which increased with increasing time after the removal. However, none of comparisons between the different ADH2 genotypes reached statistical significance. Among the ALDH2*1/*2 genotypes, those with ADH2*1/*2 or ADH2*2/*2 showed a significant increase in response with increasing time after the removal and revealed a significantly higher positivity rate at 15 min than those with ADH2*1/*1. In those with the ALDH2*1/*2 genotype, the positive rate of facial flushing with one glass of beer was higher in those with ADH2*1/*2 and ADH2*2/*2 than those with ADH2*1/*1, although this was not significant. However, the ADH2 genotype did not seem to influence drinking frequency or amounts of alcohol intake in each ALDH2 genotype. CONCLUSIONS: This study finds further evidence for a contribution of the ADH2 polymorphism to skin reactions after either local or systemic ethanol exposure in Asian people. However, the effects of the ADH2 polymorphism may be mild because this polymorphism does not seem to influence alcohol drinking behavior in these study subjects. However, the ADH2 genotype did not seem to influence drinking frequency or amounts of alcohol intake in each ALDH2 genotype. 11306061 Hoog J, Brandt M, Hedberg JJ, Stromberg P: Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6. Chem Biol Interact. 2001 Jan 30;130-132(1-3):395-404. Alcohol dehydrogenases (ADH) of classes V and VI, ADH5 and ADH6, have been defined in man and rodents, respectively. Sequence data have been obtained at cDNA and genomic levels, but limited data are available for functionality and substrate repertoire. The low positional identity (65%) between the two ADHs, place them into separate classes. We have shown that the ADH5 gene yields two differently processed mRNAs and harbors a gene organization identical to other mammalian ADHs. This is probably due to an alternative splicing in the eighth intron that results in a shorter message missing the ninth exon or a normal message with the expected number of codons. The isolated rat ADH6 cDNA was found to be fused to ADH2 at the 5'-end. The resulting main open reading frame translates into an N-terminally extended polypeptide. In vitro translation results in a polypeptide of about 42 kDa and further, protein was possible to express in COS cells as a fusion product with Green Fluorescent Protein. Both ADH5 and ADH6 show genes and gene products that are processed comparably to other mammalian ADHs and the deduced amino acid sequences indicate a lack of ethanol dehydrogenase activity that probably explains why no corresponding proteins have been isolated. The functionality of these ADHs is therefore still an enigma. Mammalian alcohol dehydrogenase of higher classes: analyses of human ADH5 and rat ADH6. 8020991 MacCollin M, Peterfreund R, MacDonald M, Fink JS, Gusella J: Mapping of a human A2a adenosine receptor (ADORA2) to chromosome 22. Genomics. 1994 Mar 15;20(2):332-3. Mapping of a human A2a adenosine receptor (ADORA2) to chromosome 22. 11834538 O'Donnell J, Boulton FE, Manning RA, Laffan MA: Amount of H antigen expressed on circulating von Willebrand factor is modified by ABO blood group genotype and is a major determinant of plasma von Willebrand factor antigen levels. Arterioscler Thromb Vasc Biol. 2002 Feb 1;22(2):335-41. To investigate whether the effect of ABO blood group on plasma von Willebrand factor (vWF) levels is mediated by the ABH antigenic determinants carried on N-linked glycans of vWF, we studied 158 group A and group O healthy volunteers. vWF antigen (vWF:Ag) and factor VIII antigen (FVIII:Ag) levels were highest in A(1)A(1) individuals and higher in A(1)O(1) than in A(2)O(1) or O(1)O(1) individuals. Plasma A transferase activity and the amount of A antigen expressed per unit vWF (AvWF) were significantly higher in A(1)A(1) than in A(1)O(1) individuals and higher in A(1)O(1) than in A(2)O(1) individuals. AvWF was correlated strongly with plasma levels of A transferase activity. Thus, we have clearly demonstrated a direct relationship between ABO genotype, A transferase expression, and the amount of A antigen expressed on circulating vWF. H antigen expression per unit vWF (HvWF) was highest in group O individuals. Among group A individuals, the pattern of HvWF expression was A(2)O(1)>A(1)O(1)>A(1)A(1). In group O and group A(2)O(1) individuals, HvWF was inversely correlated with plasma vWF levels. In contrast, among group A(1)A(1) and A(1)O(1) individuals, there was no relationship between AvWF and plasma vWF levels. These findings suggest that it is H antigen expression that mediates the ABO effect on plasma vWF concentration. These findings suggest that it is H antigen expression that mediates the ABO effect on plasma vWF concentration. 9344667 Manca A, Volpi EV, Laficara F, Muresu R, Gray IC, Spurr NK, Nobile C: Detailed physical analysis of a 1.5-megabase YAC contig containing the MXI1 and ADRA2A genes. Genomics. 1997 Oct 15;45(2):407-11. The distal long arm of chromosome 10 harbors genes of biomedical interest such as MXI1, a putative tumor suppressor gene, and those encoding the adrenergic receptors alpha2A (ADRA2A) and beta1 (ADRB1). As part of a physical and genetic study of this genomic region, we constructed a 1.5-Mb YAC contig mapping to 10q25 that contains MXI1 and ADRA2A as well as a number of STSs. Rare cutting restriction site analysis of overlapping YACs allowed fine mapping of these genes and markers along the contig and revealed the presence of four CpG islands. MXI1 and ADRA2A appear to be about 600 kb apart, whereas ADRB1 is separated from ADRA2A by a distance larger than previously reported. Detailed physical analysis of a 1.5-megabase YAC contig containing the MXI1 and ADRA2A genes. 8530022 Hinsdale ME, Farmer SC, Johnson KR, Davisson MT, Hamm DA, Tolwani RJ, Wood PA: RNA expression and chromosomal location of the mouse long-chain acyl-CoA dehydrogenase gene. Genomics. 1995 Jul 20;28(2):163-70. The cDNA for mouse long-chain acyl-CoA dehydrogenase (Acadl, gene symbol; LCAD, enzyme) was cloned and characterized. The cDNA was obtained by library screening and reverse transcription-polymerase chain reaction (RT-PCR). The deduced amino acid sequence showed a high degree of homology to both the rat and the human LCAD sequence. Northern analysis of multiple tissues using the mouse Acadl cDNA as a probe showed two bands in all tissues examined. We found a total of three distinct mRNAs for Acadl. These three mRNAs were encoded by a single gene that we mapped to mouse chromosome 1. The three transcripts differed in the 3' untranslated region due to use of alternative polyadenylation sites. Quantitative evaluation of a multitissue Northern blot showed a varied ratio of the larger transcript as compared with the smaller transcripts. The cDNA for mouse long-chain acyl-CoA dehydrogenase (Acadl, gene symbol; LCAD, enzyme) was cloned and characterized. 12124994 Nakai K, Habano W, Fujita T, Nakai K, Schnackenberg J, Kawazoe K, Suwabe A, Itoh C: Highly multiplexed genotyping of coronary artery disease-associated SNPs using MALDI-TOF mass spectrometry. Hum Mutat. 2002 Aug;20(2):133-8. Highly multiplexed genotyping methods are needed to support a comprehensive analysis of single nucleotide polymorphisms (SNPs) in coronary artery disease (CAD)-related genes. In this study we evaluated chip-based MALDI-TOF mass spectrometry for multiplexed genotyping of SNPs associated with CAD. Our analysis included 14 healthy Japanese individuals and 19 Japanese patients with myocardial infarction whose first attack occurred before age 50. We selected 29 candidate genes involved in 1) the renin-angiotensin system, 2) lipid metabolism, 3) cytokines and adhesion molecules, 4) growth factors, and 5) the coagulation-fibrinolysis system. Genotyping of candidate SNPs was performed by MALDI-TOF MS using a MassARRAY system, and 4-plex analysis was achieved at a maximum. All 39 SNPs determined by the fluorescent dye-terminator cycle sequencing method from four randomly selected patients were found to be in complete agreement with the results obtained from MassARRAY system. Significant differences were observed in the -1965delG of PAI1 (SERPINE1) with respect to allelic frequency, the G>A in the promoter region SNP in SM22 (TAGLN) for dominant genotype, and in two other SNPs (C>T in intron 1 of HGF, and -1965delG of PAI1) for recessive genotype. Three SNPs (803T>C of AGT, 677CT of MTHFR, 190T>C of ADRB3) showed weak differences in allelic frequency. MALDI-TOF-MS provided high performance with a multiplex assay design for analysis of CAD-related SNPs by increasing the throughput while maintaining a high level of accuracy. Three SNPs (803T> C of AGT, 677CT of MTHFR, 190T> C of ADRB3) showed weak differences in allelic frequency. 11399774 Yang JB, Duan ZJ, Yao W, Lee O, Yang L, Yang XY, Sun X, Chang CC, Chang TY, Li BL: Synergistic transcriptional activation of human Acyl-coenzyme A: cholesterol acyltransterase-1 gene by interferon-gamma and all-trans-retinoic acid THP-1 cells. J Biol Chem. 2001 Jun 15;276(24):20989-98. Epub 2001 Feb 28. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme involved in cellular cholesterol homeostasis and in atherosclerotic foam cell formation. Human ACAT-1 gene contains two promoters (P1 and P7), each located in a different chromosome (1 and 7) (Li, B. L., Li, X. L., Duan, Z. J., Lee, O., Lin, S., Ma, Z. M., Chang, C. C., Yang, X. Y., Park, J. P., Mohandas, T. K., Noll, W., Chan, L., and Chang, T. Y. (1999) J. Biol Chem. 274, 11060-11071). Interferon-gamma (IFN-gamma), a cytokine that exerts many pro-atherosclerotic effects in vivo, causes up-regulation of ACAT-1 mRNA in human blood monocyte-derived macrophages and macrophage-like cells but not in other cell types. To examine the molecular nature of this observation, we identified within the ACAT-1 P1 promoter a 159-base pair core region. This region contains 4 Sp1 elements and an IFN-gamma activated sequence (GAS) that overlaps with the second Sp1 element. In the monocytic cell line THP-1 cell, the combination of IFN-gamma and all-trans-retinoic acid (a known differentiation agent) enhances the ACAT-1 P1 promoter but not the P7 promoter. Additional experiments showed that all-trans-retinoic acid causes large induction of the transcription factor STAT1, while IFN-gamma causes activation of STAT1 such that it binds to the GAS/Sp1 site in the ACAT-1 P1 promoter. Our work provides a molecular mechanism to account for the effect of IFN-gamma in causing transcriptional activation of ACAT-1 in macrophage-like cells. Additional experiments showed that all-trans-retinoic acid causes large induction of the transcription factor STAT1, while IFN-gamma causes activation of STAT1 such that it binds to the GAS/Sp1 site in the ACAT-1 P1 promoter. 11850634 Lembo PM, Grazzini E, Groblewski T, O'Donnell D, Roy MO, Zhang J, Hoffert C, Cao J, Schmidt R, Pelletier M, Labarre M, Gosselin M, Fortin Y, Banville D, Shen SH, Strom P, Payza K, Dray A, Walker P, Ahmad S: Proenkephalin A gene products activate a new family of sensory neuron--specific GPCRs. Nat Neurosci. 2002 Mar;5(3):201-9. Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception. Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). 10386818 Sivashanmugam P, Richardson RT, Hall S, Hamil KG, French FS, O'Rand MG: Cloning and characterization of an androgen-dependent acidic epididymal glycoprotein/CRISP1-like protein from the monkey. J Androl. 1999 May-Jun;20(3):384-93. A cDNA encoding an acidic epididymal glycoprotein (AEG)-like, CRISP1 (cysteine-rich secretory protein) protein from the monkey (Macaca mullata) epididymis has been cloned and sequenced. The monkey AEG (mAEG) has an open reading frame that encodes a protein containing 249 amino acids with a deduced molecular mass of 28 kDa. The mAEG protein sequence is 85% identical to human and 44% identical to mouse CRISP1, including all 16 conserved cysteine residues. mAEG also shows a significant amino acid homology with other CRISP proteins, rat AEG/DE, human TPX1/CRISP2, and guinea pig acrosomal autoantigen 1 (AA1). In addition, mAEG shows somewhat less homology to a toxin from the Mexican beaded lizard and to a human glioma pathogenesis-related protein. Northern blot analysis shows that the mRNA for mAEG is expressed in all the regions of the epididymis except the caput and was not detected in the testis, prostate, seminal vesicle, and brain. In castrated animals, mAEG gene expression in the epididymis is significantly diminished; however, testosterone enanthate replacement restored the normal level of expression, demonstrating that expression of mAEG is androgen dependent. Western blot analysis of monkey epididymal regions using mouse antirecombinant human AEG identified a 28-kDa protein only in the caudal region. Immunohistochemical analysis identified mAEG only in the principal cells of the cauda epididymal epithelium. Immunofluorescence analysis identified mAEG on the principal piece of the sperm tail and as small patches over the middle piece and head regions. The results described in the present study suggest that mAEG (CRISP1) is secreted in the monkey epididymis, regulated by androgens and present on epididymal spermatozoa. The results described in the present study suggest that mAEG (CRISP1) is secreted in the monkey epididymis, regulated by androgens and present on epididymal spermatozoa. 8244345 Just W, Klett C, Vetter U, Vogel W: Assignment of the human aggrecan gene AGC1 to 15q25-->q26.2 by in situ hybridization. Hum Genet. 1993 Nov;92(5):516-8. The human aggrecan gene (AGC1) has been localized to 15q25-->q26.2 by in situ hybridization. Although no genetic diseases of connective tissue map to this location, the malignant melanoma-associated surface antigen mel-CSPG is located here; mel-CSPG is a chondroitin sulfate proteoglycan. This raises the possibility that AGC1 and mel-CSPG may be the same gene. This raises the possibility that AGC1 and mel-CSPG may be the same gene. 11007781 Ki SW, Ishigami K, Kitahara T, Kasahara K, Yoshida M, Horinouchi S: Radicicol binds and inhibits mammalian ATP citrate lyase. J Biol Chem. 2000 Dec 15;275(50):39231-6. Six different biotinylated radicicol derivatives were synthesized as affinity probes for identification of cellular radicicol-binding proteins. Derivatives biotinylated at the C-17 (BR-1) and C-11 (BR-6) positions retained the activity of morphological reversion in v-src-transformed 3Y1 fibroblasts. Two radicicol-binding proteins, 120 and 90-kDa in size, were detected in HeLa cell extracts by employing BR-1 and BR-6, respectively. The 90-kDa protein bound to BR-6 was identified to be Hsp90 by immunoblotting. The 120-kDa protein bound to BR-1 was purified from rabbit reticulocyte lysate, and its internal amino acid sequence was identical to that of human and rat ATP citrate lyase. The identity of the 120-kDa protein as ATP citrate lyase was confirmed by immunoblotting. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. These results suggest that radicicol binds the two proteins through different molecular portions of its structure. BR-1-bound ATP citrate lyase isolated from rabbit reticulocyte lysate showed no enzymatic activity. The activity of rat liver ATP citrate lyase was inhibited by radicicol and BR-1 but not by BR-6. Kinetic analysis demonstrated that radicicol was a non-competitive inhibitor of ATP citrate lyase with K(i) values for citrate and ATP of 13 and 7 microm, respectively. Interaction between BR-1 and ATP citrate lyase was blocked by radicicol but not by herbimycin A that interacts with Hsp90. 2843104 Saha AK, Dowling JN, Pasculle AW, Glew RH: Legionella micdadei phosphatase catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate in human neutrophils. Arch Biochem Biophys. 1988 Aug 15;265(1):94-104. The legionellae are facultative intracellular bacterial pathogens which multiply in host phagocytes. Legionella micdadei cells contain an acid phosphatase (ACP2) which blocks superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe (fMLP) [A. K. Saha, et al. (1985) Arch. Biochem. Biophys. 243, 150-160]. In the present study, we have purified the Legionella phosphatase to homogeneity as indicated by the finding of a single 68,000-Da band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We explored the possibility that ACP2 acts by interfering with polyphosphoinositide hydrolysis and the production of the intracellular second messengers, inositol trisphosphate (IP3) and diacylglycerol, following neutrophil stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) was hydrolyzed rapidly by ACP2 in vitro. The rate of hydrolysis of PIP2 was higher at pH 7.0 (Km 2.0 microM; 4 X 10(3) units/mg protein; 1 unit equals 1 nmol of Pi released/h) than at lower pH. IP3 was also a good substrate for ACP2 in vitro. When human neutrophil phosphoinositides were prelabeled with 32Pi, subsequent incubation with ACP2 resulted in an 85% loss of the labeled PIP2 over 2 h. Following fMLP stimulation of [3H]inositol-labeled neutrophils, the quantity of IP3 produced by ACP2-treated cells was reduced by 44%. Prior treatment of neutrophils with ACP2 also reduced by 45% the amount of diacylglycerol they produced when stimulated by fMLP. These results indicate that the Legionella phosphatase may compromise the neutrophils' microbicidal response to the organism by hydrolyzing PIP2, the progenitor of IP3 and diacylglycerol, and by hydrolyzing IP3 itself. Prior treatment of neutrophils with ACP2 also reduced by 45% the amount of diacylglycerol they produced when stimulated by fMLP. 11823199 Jacquet C, Gouin E, Jeannel D, Cossart P, Rocourt J: Expression of ActA, Ami, InlB, and listeriolysin O in Listeria monocytogenes of human and food origin. Appl Environ Microbiol. 2002 Feb;68(2):616-22. Expression of proteins involved in the adhesion of Listeria monocytogenes to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of L. monocytogenes strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90; P = 1 x 10(-4)). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89; P = 1 x 10(-2)) and central nervous system infections were associated with group ActA2 strains (OR = 3.04; P = 1 x 10(-3)) and group ActA3 strains (OR = 3.91; P = 1 x 10(-3)). Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 and Ami2) and into four groups with ActA (groups ActA1, ActA2, ActA3, and ActA4). 10361383 Wang J, Killinger DW, Hegele RA: A microdeletion within DAX-1 in X-linked adrenal hypoplasia congenita and hypogonadotrophic hypogonadism. J Investig Med. 1999 May;47(5):232-5. BACKGROUND: X-linked adrenal hypoplasia congenita (AHC) is a developmental disorder characterized by primary adrenal gland failure, which produces extreme and potentially fatal endocrine deficiencies. Hypogonadotrophic hypogonadism (HHG) also may be associated with AHC. AHC has been shown to result from a variety of mutations in the DAX-1 gene, which encodes a member of the nuclear hormone receptor superfamily. METHODS: The proband, one of the world's oldest living patients with AHC and HHG, was diagnosed in 1955. He was on corticosteroid replacement therapy since that time and androgen replacement therapy since puberty. We sequenced his DAX-1 gene. RESULTS: We found a 4 bp ACTC deletion between nucleotides 1464 and 1467 in the second exon of the normal DAX-1 sequence. This mutation caused a shift in the reading frame and predicted a premature stop codon at amino acid position 416. The mutation abolished a recognition site for DdeI, allowing for confirmation by restriction analysis. CONCLUSIONS: The position of the mutation confirms the functional importance of the COOH-terminal 10% of the DAX-1 sequence. The clinical history also reinforces the importance of early diagnosis in AHC, which can be associated with longevity and no obvious morbidity after more than 40 years of hormone replacement therapy. RESULTS: We found a 4 bp ACTC deletion between nucleotides 1464 and 1467 in the second exon of the normal DAX-1 sequence. 10876051 Haskell-Luevano C, Monck EK, Wan YP, Schentrup AM: The agouti-related protein decapeptide (Yc[CRFFNAFC]Y) possesses agonist activity at the murine melanocortin-1 receptor. Peptides. 2000 May;21(5):683-9. Agouti-related protein (AGRP) is a naturally occurring antagonist of the brain melanocortin receptors (MC3R and MC4R) and is physiologically implicated as participating in feeding behavior and energy homeostasis. The human AGRP decapeptide Yc[CRFFNAFC]Y has been previously reported as binding to the human MC3 and MC4 receptors and antagonizing the MC4 receptor. We have synthesized this decapeptide and pharmacologically characterized it at the murine melanocortin receptors and found it to possess MC4R antagonist activity (pA(2) = 6.8) and, unexpectedly, MC1R agonist activity (EC(50) = 2.89 microM). This study characterizes the first AGRP-based peptide agonist at the melanocortin receptors. The human AGRP decapeptide Yc [CRFFNAFC] Y has been previously reported as binding to the human MC3 and MC4 receptors and antagonizing the MC4 receptor. 10656685 Nikolopoulos SN, Spengler BA, Kisselbach K, Evans AE, Biedler JL, Ross RA: The human non-muscle alpha-actinin protein encoded by the ACTN4 gene suppresses tumorigenicity of human neuroblastoma cells. Oncogene. 2000 Jan 20;19(3):380-6. alpha-Actinins are actin-binding proteins important in organization of the cytoskeleton and in cell adhesion. We have cloned and characterized a cDNA from human neuroblastoma cell variants which encodes the second non-muscle alpha-actinin isoform designated ACTN4 (actinin-4). mRNA encoded by the ACTN4 gene, mapped to chromosome 4, is abundant in non-tumorigenic, substrate-adherent human neuroblastoma cell variants but absent or only weakly expressed in malignant, poorly substrate-adherent neuroblasts. It is also present in many adherent tumor cell lines of diverse tissue origins. Cell lines typically co-express ACTN4 and ACTN1, a second non-muscle alpha-actinin gene. Expression is correlated with substrate adhesivity. Analysis of deduced amino acid sequences suggests that the two isoforms may differ in function and in regulation by calcium. Moreover, ACTN4 exhibits tumor suppressor activity. Stable clones containing increased levels of alpha-actinin, isolated from highly malignant neuroblastoma stem cells [BE(2)-C] after transfection with a full-length ACTN4 cDNA, show decreased anchorage-independent growth ability, loss of tumorigenicity in nude mice, and decreased expression of the N-myc proto-oncogene. Cell lines typically co-express ACTN4 and ACTN1, a second non-muscle alpha-actinin gene. 1339456 Beggs AH, Byers TJ, Knoll JH, Boyce FM, Bruns GA, Kunkel LM: Cloning and characterization of two human skeletal muscle alpha-actinin genes located on chromosomes 1 and 11. J Biol Chem. 1992 May 5;267(13):9281-8. Conserved sequences of dystrophin, beta-spectrin, and alpha-actinin were used to plan a set of degenerate oligonucleotide primers with which we amplified a portion of a human alpha-actinin gene transcript. Using this short clone as a probe, we isolated and characterized full-length cDNA clones for two human alpha-actinin genes (ACTN2 and ACTN3). These genes encode proteins that are structurally similar to known alpha-actinins with approximately 80% amino acid identity to each other and to the previously characterized human nonmuscle gene. ACTN2 is the human homolog of a previously characterized chicken gene while ACTN3 represents a novel gene product. Northern blot analysis demonstrated that ACTN2 is expressed in both skeletal and cardiac muscle, but ACTN3 expression is limited to skeletal muscle. As with other muscle-specific isoforms, the EF-hand domains in ACTN2 and ACTN3 are predicted to be incapable of binding calcium, suggesting that actin binding is not calcium sensitive. ACTN2 was mapped to human chromosome 1q42-q43 and ACTN3 to 11q13-q14 by somatic cell hybrid panels and fluorescent in situ hybridization. These results demonstrate that some of the isoform diversity of alpha-actinins is the result of transcription from different genetic loci. ACTN2 is the human homolog of a previously characterized chicken gene while ACTN3 represents a novel gene product. 10869574 Schreiber R, Pavenstadt H, Greger R, Kunzelmann K: Aquaporin 3 cloned from Xenopus laevis is regulated by the cystic fibrosis transmembrane conductance regulator. FEBS Lett. 2000 Jun 23;475(3):291-5. The cystic fibrosis transmembrane conductance regulator (CFTR) is essential for epithelial electrolyte transport and has been shown to be a regulator of epithelial Na(+), K(+), and Cl(-) channels. CFTR also enhances osmotic water permeability when activated by cAMP. This was detected initially in Xenopus oocytes and is also present in human airway epithelial cells, however, the mechanisms remain obscure. Here, we show that CFTR activates aquaporin 3 expressed endogenously and exogenously in oocytes of Xenopus laevis. The interaction requires stimulation of wild type CFTR by cAMP and an intact first nucleotide binding domain as demonstrated for other CFTR-protein interactions. Here, we show that CFTR activates aquaporin 3 expressed endogenously and exogenously in oocytes of Xenopus laevis. 7782067 Radice P, Pensotti V, Jones C, Perry H, Pierotti MA, Tunnacliffe A: The human archain gene, ARCN1, has highly conserved homologs in rice and Drosophila. Genomics. 1995 Mar 1;26(1):101-6. A novel human gene, ARCN1, has been identified in chromosome band 11q23.3. It maps approximately 50 kb telomeric to MLL, a gene that is disrupted in a number of leukemia-associated translocation chromosomes. cDNA clones representing ARCN1 hybridize to 4-kb mRNA species present in all tissues tested. Sequencing of cDNAs suggests that at least two forms of mRNA with alternative 5' ends are present within the cell. The mRNA with the longest open reading frame gives rise to a protein of 57 kDa. Although the sequence reported is novel, remarkable similarity is observed with two predicted protein sequences from partial DNA sequences generated by rice (Oryza sativa) and fruit fly (Drosophila melanogaster) genome projects. The degree of sequence conservation is comparable to that observed for highly conserved structural proteins, such as heat shock protein HSP70, and is greater than that of gamma-tubulin and heat shock protein HSP60. A more distant relationship to the group of clathrin-associated proteins suggests a possible role in vesicle structure or trafficking. In view of its ancient pedigree and a potential involvement in cellular architecture, we propose that the ARCN1 protein be named archain. In view of its ancient pedigree and a potential involvement in cellular architecture, we propose that the ARCN1 protein be named archain. 8668142 Deitz SB, Wu C, Silve S, Howell KE, Melancon P, Kahn RA, Franzusoff A: Human ARF4 expression rescues sec7 mutant yeast cells. Mol Cell Biol. 1996 Jul;16(7):3275-84. Vesicle-mediated traffic between compartments of the yeast secretory pathway involves recruitment of multiple cytosolic proteins for budding, targeting, and membrane fusion events. The SEC7 gene product (Sec7p) is a constituent of coat structures on transport vesicles en route to the Golgi complex in the yeast Saccharomyces cerevisiae. To identify mammalian homologs of Sec7p and its interacting proteins, we used a genetic selection strategy in which a human HepG2 cDNA library was transformed into conditional-lethal yeast sec7 mutants. We isolated several clones capable of rescuing sec7 mutant growth at the restrictive temperature. The cDNA encoding the most effective suppressor was identified as human ADP ribosylation factor 4 (hARF4), a member of the GTPase family proposed to regulate recruitment of vesicle coat proteins in mammalian cells. Having identified a Sec7p-interacting protein rather than the mammalian Sec7p homolog, we provide evidence that hARF4 suppressed the sec7 mutation by restoring secretory pathway function. Shifting sec7 strains to the restrictive temperature results in the disappearance of the mutant Sec7p cytosolic pool without apparent changes in the membrane-associated fraction. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. These results suggest a requirement for Sec7p cycling on and off of the membranes for cell growth and vesicular traffic. In addition, overexpression of the yeast GTPase-encoding genes ARF1 and ARF2, but not that of YPT1, suppressed the sec7 mutant growth phenotype in an allele-specific manner. This allele specificity indicates that individual ARFs are recruited to perform two different Sec7p-related functions in vesicle coat dynamics. The introduction of hARF4 to the cells maintained the balance between cytosolic and membrane-associated Sec7p pools. 12810105 Bronte V, Serafini P, Mazzoni A, Segal DM, Zanovello P: L-arginine metabolism in myeloid cells controls T-lymphocyte functions. Trends Immunol. 2003 Jun;24(6):302-6. Although current attention has focused on regulatory T lymphocytes as suppressors of autoimmune responses, powerful immunosuppression is also mediated by a subset of myeloid cells that enter the lymphoid organs and peripheral tissues during times of immune stress. If these myeloid suppressor cells (MSCs) receive signals from activated T lymphocytes in the lymphoid organs, they block T-cell proliferation. MSCs use two enzymes involved in arginine metabolism to control T-cell responses: inducible nitric oxide synthase (NOS2), which generates nitric oxide (NO) and arginase 1 (Arg1), which depletes the milieu of arginine. Th1 cytokines induce NOS2, whereas Th2 cytokines upregulate Arg1. Induction of either enzyme alone results in a reversible block in T-cell proliferation. When both enzymes are induced together, peroxynitrites, generated by NOS2 under conditions of limiting arginine, cause activated T lymphocytes to undergo apoptosis. Thus, NOS2 and Arg1 might act separately or synergistically in vivo to control specific types of T-cell responses, and selective antagonists of these enzymes might prove beneficial in fighting diseases in which T-cell responses are inappropriately suppressed. This Opinion is the second in a series on the regulation of the immune system by metabolic pathways. Thus, NOS2 and Arg1 might act separately or synergistically in vivo to control specific types of T-cell responses, and selective antagonists of these enzymes might prove beneficial in fighting diseases in which T-cell responses are inappropriately suppressed. 11279102 Parks WT, Frank DB, Huff C, Renfrew Haft C, Martin J, Meng X, de Caestecker MP, McNally JG, Reddi A, Taylor SI, Roberts AB, Wang T, Lechleider RJ: Sorting nexin 6, a novel SNX, interacts with the transforming growth factor-beta family of receptor serine-threonine kinases. J Biol Chem. 2001 Jun 1;276(22):19332-9. Epub 2001 Mar 8. Sorting nexins (SNX) comprise a family of proteins with homology to several yeast proteins, including Vps5p and Mvp1p, that are required for the sorting of proteins to the yeast vacuole. Human SNX1, -2, and -4 have been proposed to play a role in receptor trafficking and have been shown to bind to several receptor tyrosine kinases, including receptors for epidermal growth factor, platelet-derived growth factor, and insulin as well as the long form of the leptin receptor, a glycoprotein 130-associated receptor. We now describe a novel member of this family, SNX6, which interacts with members of the transforming growth factor-beta family of receptor serine-threonine kinases. These receptors belong to two classes: type II receptors that bind ligand, and type I receptors that are subsequently recruited to transduce the signal. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. Of the type I receptors, SNX6 was found to interact only with inactivated TbetaRI. SNXs 1-4 also interacted with the transforming growth factor-beta receptor family, showing different receptor preferences. Conversely, SNX6 behaved similarly to the other SNX proteins in its interactions with receptor tyrosine kinases. Strong heteromeric interactions were also seen among SNX1, -2, -4, and -6, suggesting the formation in vivo of oligomeric complexes. These findings are the first evidence for the association of the SNX family of molecules with receptor serine-threonine kinases. Of the type II receptors, SNX6 was found to interact strongly with ActRIIB and more moderately with wild type and kinase-defective mutants of TbetaRII. 9671486 Guasch RM, Scambler P, Jones GE, Ridley AJ: RhoE regulates actin cytoskeleton organization and cell migration. Mol Cell Biol. 1998 Aug;18(8):4761-71. The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation. 10699171 Prakash SK, Paylor R, Jenna S, Lamarche-Vane N, Armstrong DL, Xu B, Mancini MA, Zoghbi HY: Functional analysis of ARHGAP6, a novel GTPase-activating protein for RhoA. Hum Mol Genet. 2000 Mar 1;9(4):477-88. Microphthalmia with linear skin defects (MLS) is an X-linked dominant, male-lethal syndrome characterized by microphthalmia, aplastic skin and agenesis of the corpus callosum, and is caused by the deletion of a 500 kb critical region in Xp22.3. Our laboratory isolated a novel rho GTPase-activating protein (rhoGAP) gene named ARHGAP6 from the MLS region. ARHGAP6 contains 14 exons encoding a 974 amino acid protein with three putative SH3-binding domains. Because exons 2-14 are deleted in all MLS patients, we hypothesized that ARHGAP6 may be responsible for some of the phenotypic features of MLS. We pursued two approaches to study the function of ARHGAP6 and its role in the pathogenesis of MLS: gene targeting of the rhoGAP domain in mouse embryonic stem cells and in vitro expression studies. Surprisingly, loss of the rhoGAP function of Arhgap6 does not cause any detectable phenotypic or behavioral abnormalities in the mutant mice. Transfected mammalian cells expressing ARHGAP6 lose their actin stress fibers, retract from the growth surface and extend thin, branching processes resembling filopodia. The ARHGAP6 protein co-localizes with actin filaments through an N-terminal domain and recruits F-actin into the growing processes. Mutation of a conserved arginine residue in the rhoGAP domain prevents the loss of stress fibers but has little effect on process outgrowth. These results suggest that ARHGAP6 has two independent functions: one as a GAP with specificity for RhoA and the other as a cytoskeletal protein that promotes actin remodeling. These results suggest that ARHGAP6 has two independent functions: one as a GAP with specificity for RhoA and the other as a cytoskeletal protein that promotes actin remodeling. 11303027 Van Valkenburgh H, Shern JF, Sharer JD, Zhu X, Kahn RA: ADP-ribosylation factors (ARFs) and ARF-like 1 (ARL1) have both specific and shared effectors: characterizing ARL1-binding proteins. J Biol Chem. 2001 Jun 22;276(25):22826-37. Epub 2001 Apr 12. Despite the 40-60% identity between ADP-ribosylation factors (ARFs) and ARF-like (ARL) proteins, distinct functional roles have been inferred from findings that ARLs lack the biochemical or genetic activities characteristic of ARFs. The potential for functional overlap between ARFs and ARLs was examined by comparing effects of expression on intact cells and the ability to bind effectors. Expression of [Q71L]ARL1 in mammalian cells led to altered Golgi structure similar to, but less dramatic than, that reported previously for [Q71L]ARF1. Two previously identified partners of ARFs, MKLP1 and Arfaptin2/POR1, also bind ARL1 but not ARL2 or ARL3. Two-hybrid screens of human cDNA libraries with dominant active mutants of human ARL1, ARL2, and ARL3 identified eight different but overlapping sets of binding partners. Specific interactions between ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and characterized in more detail. Like ARFs and ARL1, the binding of SCOCO to Golgi membranes is rapidly reversed by brefeldin A, suggesting the presence of a brefeldin A-sensitive ARL1 exchange factor. These data reveal a complex network of interactions between GTPases in the ARF family and their effectors and reveal a potential for cross-talk not demonstrated previously. Specific interactions between ARL1 and two binding proteins, SCOCO and Golgin-245, are defined and characterized in more detail. 8589438 Abbott BD, Probst MR: Developmental expression of two members of a new class of transcription factors: II. Expression of aryl hydrocarbon receptor nuclear translocator in the C57BL/6N mouse embryo. Dev Dyn. 1995 Oct;204(2):144-55. The aryl hydrocarbon receptor (AhR) and the AhR nuclear translocator protein (ARNT) are basic-helix-loop-helix (bHLH) proteins involved in transcriptional regulation. The AhR is a ligand-activated partner of the ARNT protein. Both proteins are required to transcriptionally regulate gene expression. ARNT must be complexed to AhR to permit binding to the regulatory DNA sequence. The AhR-ligand complex is known to mediate a range of biological responses, such as developmental toxicity, induction of cleft palate, and hydronephrosis. AhR and ARNT are expressed in human embryonic palatal cells and AhR was recently shown to have a specific developmental pattern of expression in the mouse embryo. In the present study, expression of ARNT is characterized in C57Bl/6N mouse embryos from gestation day (GD) 10-16 using immunohistochemistry and in situ hybridization. Af affinity purified antibody against human ARNT (1.1 micrograms/ml) was detected with an avidin-biotin-peroxidase complex. ARNT mRNA was localized with a 35S-RNA probe from pBM5/NEO-M1-1. Specific spatial and temporal patterns of ARNT expression emerged and mRNA and protein expression correlated. The GD 10-11 embryos showed highest levels of ARNT in neuroepithelial cells of the neural tube, visceral arches, otic and optic placodes, and preganglionic complexes. The heart also had significant expression of ARNT with strong nuclear localization. After GD11, expression in heart and brain declined. In GD 12-13 embryos expression was highest in the liver where expression increased from GD 12-16. At GD 15-16 the highest levels of ARNT occurred in adrenal gland and liver, although ARNT was also detected in submandibular gland, ectoderm, tongue, bone, and muscle. In all of these tissues ARNT was cytoplasmic as well as nuclear, except in some of the cortical adrenal cells in which ARNT was strongly cytoplasmic with little or no nuclear localization. These specific patterns of ARNT expression, which differ in certain tissues from the expression of AhR, suggest that ARNT may have additional roles in normal embryonic development. The AhR is a ligand-activated partner of the ARNT protein. 9720496 Khan A, Krishna M, Baker SP, Banner BF: Cathepsin B and tumor-associated laminin expression in the progression of colorectal adenoma to carcinoma. Mod Pathol. 1998 Aug;11(8):704-8. Cathepsin B is a lysosomal cysteine proteinase associated with degradation of laminin. It is increased in colorectal carcinoma (CA). Laminin is a major component of basement membrane involved in cell-matrix interactions and tumor progression. The aim of this study was to correlate cathepsin B and tumor-associated laminin in colorectal adenomas (ADs) with increasing grades of dysplasia and in invasive CAs. Forty-five ADs (8 tubular, 16 tubulovillous, 21 villous), 13 adenomas with high-grade dysplasia/carcinoma in situ (AHDs), and 17 invasive CAs were immunostained with polyclonal antibodies to cathepsin B and laminin. Statistical analysis was performed using exact linear by linear association test and Spearman rank correlation coefficient. Cathepsin B-positive tumor cells were seen in 30 (67%) ADs, 13 (100%) AHDs, and 17 (100%) CAs. The grade of cathepsin B staining was significantly increased in AHDs and CAs, compared with ADs (P < .0001). Laminin was continuous in all of the ADs and discontinuous or fragmented in the AHDs and CAs (P < .0001). The degree of cathepsin B staining in tumor cells also correlated with breakdown of laminin. Increased cathepsin B expression and decrease in tumor-associated laminin might suggest a mechanism for progression of ADs to CAs. The grade of cathepsin B staining was significantly increased in AHDs and CAs, compared with ADs (P < .0001). 12714565 Autieri MV, Kelemen SE, Wendt KW: AIF-1 is an actin-polymerizing and Rac1-activating protein that promotes vascular smooth muscle cell migration. Circ Res. 2003 May 30;92(10):1107-14. Epub 2003 Apr 24. Development of vascular restenosis is a multifaceted process characterized by migration and proliferation of vascular smooth muscle cells (VSMCs), resulting in loss of lumen diameter. Characterization of proteins that mediate this process is essential in our understanding of the pathogenesis of arterial injury. Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding protein that is expressed in VSMCs by allograft and balloon angioplasty injury. AIF-1 is not present in cultured human VSMCs but is induced by cytokines, and overexpression of AIF-1 results in increased VSMC growth and cell-cycle gene expression. To characterize AIF-1 modulatory effects in primary human VSMCs, AIF-1-interacting proteins were identified by an AIF-1/glutathione S transferase fusion protein affinity assay. MALDI-TOF mass spectrophotometric amino analysis identified actin as an AIF-1 interacting protein. This interaction was verified by coimmunoprecipitation. This is a functional interaction, because AIF-1 binds to and polymerizes F-actin in vitro. In unstimulated VSMCs, AIF-1 colocalizes with F-actin but translocates to lamellipodia on stimulation with platelet-derived growth factor. VSMCs stably transduced with AIF-1 retrovirus migrate 2.6-fold more rapidly (85.1+/-2.9 versus 225.5+/-16.6; P<0.001) in response to platelet-derived growth factor versus control cells. AIF-1 colocalizes with Rac1, and AIF-1-transduced VSMCs show a constitutive and enhanced activation of Rac1, providing a mechanism for the increased migration. These data indicate that AIF-1 binds and polymerizes F actin and also regulates Rac1 activity and VSMC migration. Considering the AIF-1 expression pattern in injured arteries, this suggests that AIF-1 may be involved in the cytoskeletal signaling network leading to vascular remodeling. AIF-1 colocalizes with Rac1, and AIF-1-transduced VSMCs show a constitutive and enhanced activation of Rac1, providing a mechanism for the increased migration. 5940 Wilson DE, Povey S, Harris H: Adenylate kinases in man: evidence for a third locus. Ann Hum Genet. 1976 Jan;39(3):305-13. The tissue distribution of the adenylate kinase isozymes in man has been examined using various substrates. The isozymes attributable to the AK1 and AK2 loci were identified, and an additional set of isozymes probably attributable to a third locus was also found. This locus has been provisionally designated AK3. The AK3 isozymes show activity with either GTP + AMP or ITP + AMP but do not show activity with ATP + AMP. They also differ from the AK1 and AK2 isozymes in electrophoretic mobility and from the AK1 isozymes in being resistant to silver inhibition. They are similar in molecular size to the AK1 isozymes whereas the AK2 isozymes are apparently larger. The AK3 isozymes evidently correspond to the enzyme nucleosidetriphosphate-adenylate kinase (2.7.4.10). Somatic cell hybrid studies indicate that the AK3 locus is not syntenic with that of AK2 (chromosome 1). The AK3 locus is however, probably syntenic with the AK1 locus, on chromosome 9. Genetically determined variation of AK3 has not been seen in a survey of about 80 individuals. The AK3 locus is however, probably syntenic with the AK1 locus, on chromosome 9. 10029606 Cotter PD, May A, Li L, Al-Sabah AI, Fitzsimons EJ, Cazzola M, Bishop DF: Four new mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causing X-linked sideroblastic anemia: increased pyridoxine responsiveness after removal of iron overload by phlebotomy and coinheritance of hereditary hemochromatosis. Blood. 1999 Mar 1;93(5):1757-69. X-linked sideroblastic anemia (XLSA) in four unrelated male probands was caused by missense mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). All were new mutations: T647C, C1283T, G1395A, and C1406T predicting amino acid substitutions Y199H, R411C, R448Q, and R452C. All probands were clinically pyridoxine-responsive. The mutation Y199H was shown to be the first de novo XLSA mutation and occurred in a gamete of the proband's maternal grandfather. There was a significantly higher frequency of coinheritance of the hereditary hemochromatosis (HH) HFE mutant allele C282Y in 18 unrelated XLSA hemizygotes than found in the normal population, indicating a role for coinheritance of HFE alleles in the expression of this disorder. One proband (Y199H) with severe and early iron loading coinherited HH as a C282Y homozygote. The clinical and hematologic histories of two XLSA probands suggest that iron overload suppresses pyridoxine responsiveness. Notably, reversal of the iron overload in the Y199H proband by phlebotomy resulted in higher hemoglobin concentrations during pyridoxine supplementation. The proband with the R452C mutation was symptom-free on occasional phlebotomy and daily pyridoxine. These studies indicate the value of combined phlebotomy and pyridoxine supplementation in the management of XLSA probands in order to prevent a downward spiral of iron toxicity and refractory anemia. Four new mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causing X-linked sideroblastic anemia: increased pyridoxine responsiveness after removal of iron overload by phlebotomy and coinheritance of hereditary hemochromatosis. 10854777 Slavov D, Clark M, Gardiner K: Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish. Gene. 2000 May 30;250(1-2):41-51. One type of RNA editing involves the deamination of adenosine (A) residues to inosines (I) at specific sites in specific pre-mRNAs. These inosines are subsequently read as guanosines by the ribosome, with potentially significant consequences for protein sequence. In mammals, two such A-to-I RNA editases are RED1, which edits some serotonin and glutamate receptors, and RED2, with unidentified substrates. To study the evolutionary conservation among these editases, we have isolated homologous genes from the Japanese pufferfish, Fugu rubripes. Fugu has two genes homologous to Red1 that are similar in size and organization and that show a fivefold compaction relative to the human gene; they differ, however, in their base compositional features. The Fugu gene for RED2 is unusually large, spanning more than 50kb; within the largest intron, there is evidence for a novel gene on the opposite strand. Because of these unusual features, the partial genomic structure was determined for the mouse RED2 gene. A partial cDNA for RED1 was also isolated from zebrafish. Comparisons between fish and between fish and mammals of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA-binding domains vary within a single protein in their levels of conservation. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation. Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish. 7766992 Edelhoff S, Villacres EC, Storm DR, Disteche CM: Mapping of adenylyl cyclase genes type I, II, III, IV, V, and VI in mouse. Mamm Genome. 1995 Feb;6(2):111-3. The adenylyl cyclases (AC) act as second messengers in regulatory processes in the central nervous system. They might be involved in the pathophysiology of diseases, but their biological function is unknown, except for AC type I, which has been implicated in learning and memory. We previously mapped the gene encoding AC I to human Chromosome (Chr) 7p12. In this study we report the mapping of the adenylyl cyclase genes type I-VI to mouse chromosomes by fluorescence in situ hybridization (FISH): Adcy1 to Chr 11A2, Adcy2 to 13C1, Adcy3 to 12A-B, Adcy4 to 14D3, Adcy5 to 16B5, and Adcy6 to 15F. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11.2. The mapping data confirm known areas of conservation between mouse and human chromosomes. We also confirmed previously reported mapping results of the corresponding human loci ADCY2, ADCY3, ADCY5, and ADCY6 to human chromosomes and, in addition, determined the chromosomal location of ADCY4 to human Chr 14q11. 12810632 Lahortiga I, Vizmanos JL, Agirre X, Vazquez I, Cigudosa JC, Larrayoz MJ, Sala F, Gorosquieta A, Perez-Equiza K, Calasanz MJ, Odero MD: NUP98 is fused to adducin 3 in a patient with T-cell acute lymphoblastic leukemia and myeloid markers, with a new translocation t(10;11)(q25;p15). Cancer Res. 2003 Jun 15;63(12):3079-83. The nucleoporin 98 gene (NUP98) has been reported to be fused to 13 partner genes in hematological malignancies with 11p15 translocations. Twelve of them have been identified in patients with myeloid neoplasias and only 1, RAP1GDS1 (4q21), is fused with NUP98 in five patients with T-cell acute lymphoblastic leukemia (T-ALL). Three of these patients coexpressed T and myeloid markers, suggesting the specific association of t(4;11)(q21;p15) with a subset of T-ALL originating from an early progenitor, which has the potential to express mature T-cell antigens as well as myeloid markers. We describe here a new NUP98 partner involved in a t(10;11)(q25;p15) in a patient with acute biphenotypic leukemia, showing coexpression of mature T and myeloid markers. The gene involved, located in 10q25, was identified as ADD3 using 3'-RACE. ADD3 codes for the ubiquitous expressed subunit gamma of the adducin protein, and it seems to play an important role in the skeletal organization of the cell membrane. Both NUP98-ADD3 and ADD3-NUP98 fusion transcripts are expressed in the patient. This is the second partner of NUP98 described in T-ALL. Adducin shares with the product of RAP1GDS1, and with all of the nonhomeobox NUP98 partners, the presence of a region with significant probability of adopting a coiled-coil conformation. This region is always retained in the fusion transcript with the NH(2) terminus FG repeats of NUP98, suggesting an important role in the mechanism of leukemogenesis. NUP98 is fused to adducin 3 in a patient with T-cell acute lymphoblastic leukemia and myeloid markers, with a new translocation t (10;11)(q25;p15). 2893758 Ceci JD, Zheng YW, Felder MR: Molecular analysis of mouse alcohol dehydrogenase: nucleotide sequence of the Adh-1 gene and genetic mapping of a related nucleotide sequence to chromosome 3. Gene. 1987;59(2-3):171-82. The mouse has three genes (Adh) encoding alcohol dehydrogenase (ADH) enzymes of different tissue specificity and catalytic properties. Identified regulatory loci are known to affect the expression of Adh-1 and Adh-3, which are closely linked on chromosome 3. The Adh-1 gene product is expressed predominantly in liver, and its mRNA product is androgen-inducible in kidney. In this study, genomic clones of Adh-1 were obtained from a Balb/cJ DNA library. The nucleotide sequences of all exons, intron/exon boundaries and 5'- and 3'-flanking regions were obtained. The gene spans nearly 13 kb and is divided into nine exons and eight introns. The transcription start point of this gene was determined by S1 nuclease mapping studies and presumptive regulatory regions in the 5'-flanking regions were identified, including a TATA box and a glucocorticoid-responsive element. A restriction fragment length polymorphism in the Adh-1 gene was identified among inbred strains and mapped at the [Adh-1, Adh-3] complex on chromosome 3. An additional 'Adh-like' sequence in the genome was also mapped to chromosome 3 approx. 9 centiMorgans from Adh-1. Identified regulatory loci are known to affect the expression of Adh-1 and Adh-3, which are closely linked on chromosome 3. 6202736 Chang PL, Moudgil G: A specific ultrastructural stain for arylsulfatase A activity in human cultured fibroblasts. J Histochem Cytochem. 1984 Jun;32(6):617-24. A staining reaction was developed to specifically detect arylsulfatase A activity in the presence of arylsulfatases B and C. Nitrocatechol, generated by all arylsulfatases from the substrate p-nitrocatechol sulfate, can be coupled to produce Hatchett 's brown which reacts with 3,3'-diaminobenzidine to yield an osmiophilic polymer visible under the electron microscope. The reaction was made specific for arylsulfatase A by inhibiting arylsulfatase C activity with low pH and arylsulfatase B activity with pyrophosphate. The specificity was confirmed both by electrophoretic analysis and by patient fibroblasts deficient only in arylsulfatase A activity. Under optimal conditions for preserving structural integrity and enzyme activity, enzyme reaction deposits were found mainly around vesicles. Some of these vesicles were large and heterogeneous (48-330 nm in diameter), distributed randomly within the cytoplasm, but most of the positive-reacting vesicles were uniform in size (86 +/- 18 nm in diameter) and distributed in a peripheral zone about 0.1-0.5 micron wide. These periplasmic vesicles might be partly fused with each other or with the plasma membrane. In conclusion, a specific stain for arylsulfatase A activity suitable for light and electron microscopy and the optimal conditions for structural and enzymatic preservations were developed. Although this enzyme has been considered to be lysosomal in origin, most of the activity was detected in periplasmic vesicles near the cell surface. The reaction was made specific for arylsulfatase A by inhibiting arylsulfatase C activity with low pH and arylsulfatase B activity with pyrophosphate. 9192838 Puca AA, Zollo M, Repetto M, Andolfi G, Guffanti A, Simon G, Ballabio A, Franco B: Identification by shotgun sequencing, genomic organization, and functional analysis of a fourth arylsulfatase gene (ARSF) from the Xp22.3 region. Genomics. 1997 Jun 1;42(2):192-9. We recently reported the isolation of two new members of the sulfatase gene family, arylsulfatase D (ARSD) and E (ARSE), located approximately 50 kb from each other in the Xp22.3 region. Mutation analysis indicated ARSE as the gene responsible for X-linked recessive chondrodysplasia punctata. Expression of the ARSE gene in COS cells resulted in a heat-labile arylsulfatase activity that was inhibited by warfarin. At the same time, we detected the presence of a 1.2-kb fragment located at approximately 60 kb from ARSD and ARSE with significant homology to these two genes, suggesting the existence of another sulfatase gene, arylsulfatase F (ARSF), in Xp22.3. We have used a combined approach of long-range genomic sequencing and screening of cDNA libraries to isolate the ARSF gene. Expression of the ARSF cDNA in COS cells resulted in a heat-labile arylsulfatase activity that is not inhibited by warfarin, supporting our hypothesis that only ARSE is specifically inhibited by warfarin and is most likely involved in warfarin embryopathy. Genomic analysis revealed that ARSF has an intron/exon organization highly similar to those of ARSD and ARSE, which is also shared by another Xp22.3 sulfatase gene, ARSC (arylsulfatase C, also known as steroid sulfatase), with the splice sites occurring at the same position in all four genes. The data obtained from sequence analysis and presented in this paper indicate that the ARSC, ARSD, ARSE, and ARSF genes are more similar to each other than to other members of the sulfatase gene family, supporting our hypothesis that they represent a subfamily of related proteins created through duplication events that occurred in an ancestral pseudoautosomal region. Expression of the ARSF cDNA in COS cells resulted in a heat-labile arylsulfatase activity that is not inhibited by warfarin, supporting our hypothesis that only ARSE is specifically inhibited by warfarin and is most likely involved in warfarin embryopathy. 12919952 George CH, Higgs GV, Lai FA: Ryanodine receptor mutations associated with stress-induced ventricular tachycardia mediate increased calcium release in stimulated cardiomyocytes. Circ Res. 2003 Sep 19;93(6):531-40. Epub 2003 Aug 14. Ca2+ release from the sarcoplasmic reticulum mediated by the cardiac ryanodine receptor (RyR2) is a fundamental event in cardiac muscle contraction. RyR2 mutations suggested to cause defective Ca2+ channel function have recently been identified in catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD) affected individuals. We report expression of three CPVT-linked human RyR2 (hRyR2) mutations (S2246L, N4104K, and R4497C) in HL-1 cardiomyocytes displaying correct targeting to the endoplasmic reticulum. N4104K also localized to the Golgi apparatus. Phenotypic characteristics including intracellular Ca2+ handling, proliferation, viability, RyR2:FKBP12.6 interaction, and beat rate in resting HL-1 cells expressing mutant hRyR2 were indistinguishable from wild-type (WT) hRyR2. However, Ca2+ release was augmented in cells expressing mutant hRyR2 after RyR activation (caffeine and 4-chloro-m-cresol) or beta-adrenergic stimulation (isoproterenol). RyR2:FKBP12.6 interaction remained intact after caffeine or 4-CMC activation, but was dramatically disrupted by isoproterenol or forskolin, an activator of adenylate cyclase. Isoproterenol and forskolin elevated cyclic-AMP to similar magnitudes in all cells and were associated with equivalent hyperphosphorylation of mutant and WT hRyR2. CPVT-linked mutations in hRyR2 did not alter resting cardiomyocyte phenotype but mediated augmented Ca2+ release on RyR-agonist or beta-AR stimulation. Furthermore, equivalent interaction between mutant and WT hRyR2 and FKBP12.6 was demonstrated. RyR2 mutations suggested to cause defective Ca2+ channel function have recently been identified in catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVD) affected individuals. 1362387 Edman K, Maret W: Alcohol dehydrogenase genes: restriction fragment length polymorphisms for ADH4 (pi-ADH) and ADH5 (chi-ADH) and construction of haplotypes among different ADH classes. Hum Genet. 1992 Dec;90(4):395-401. Of the five human alcohol dehydrogenase (ADH) genes located in the region q21-25 of chromosome 4, genetic markers have been reported previously only for class I enzymes, ADH1-3. Here, new restriction fragment length polymorphisms (RFLPs) are described for the genes of two other classes, ADH4 (pi) and ADH5 (chi or formaldehyde dehydrogenase, FDH). The frequencies and modes of inheritance of these RFLPs were determined with DNA both from unrelated individuals and from families. A polymorphic PstI site is assigned to the fourth intron of the ADH4 gene. Pairwise linkage disequilibrium calculations for these new RFLPs and already known RFLPs at the ADH2 and ADH3 loci establish strong linkage disequilibria between polymorphic MspI and BstXI sites in the ADH5 gene as well as between XbaI and MspI sites in the ADH3 gene. Furthermore, linkage disequilibria were detected between RFLPs of the ADH2 and ADH3 genes as well as between those of the ADH4 and ADH5 genes. The latter disequilibrium implies a hitherto unknown physical proximity of two genes belonging to different ADH classes. The RFLPs were used to construct chromosomal haplotypes that include three ADH classes. Of the 16 possible haplotypes for four RFLP markers used here, 10 were experimentally detected. The potential application of the ADH RFLPs and haplotypes in linkage or association studies of inherited diseases such as familial "alcoholism" is discussed. A polymorphic PstI site is assigned to the fourth intron of the ADH4 gene. 9888993 Woods MO, Young TL, Parfrey PS, Hefferton D, Green JS, Davidson WS: Genetic heterogeneity of Bardet-Biedl syndrome in a distinct Canadian population: evidence for a fifth locus. Genomics. 1999 Jan 1;55(1):2-9. Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive disease characterized by retinal dystrophy, renal structural abnormalities, obesity, dysmorphic extremities, and hypogenitalism in males. BBS is genetically heterogeneous with four known loci: BBS1 (11q), BBS2 (16q), BBS3 (3p), and BBS4 (15q). The prevalence of BBS in Newfoundland is approximately 10-fold greater than in Switzerland (1:160,000) and similar to the prevalence among the Bedouin of Kuwait (1:13,500). A population-based genetic survey was performed on 17 BBS families from the island portion of the province of Newfoundland, a comparatively isolated region of Canada. The families in the study had a total of 36 well-documented, affected individuals with 12 families having 2 or more affected individuals. Linkage at each of the four known loci was tested with two-point linkage and haplotype analysis. Three of the 17 kindreds showed linkage to 11q, 1 to 16q, and 1 to 3p. The latter is the first BBS3 family identified in a population of northern European descent. Six families remain undetermined because of poor pedigree structure or inconclusive haplotype analyses. Six families were excluded from all four known BBS loci, indicating that there is at least a fifth BBS locus (BBS5). Six families were excluded from all four known BBS loci, indicating that there is at least a fifth BBS locus (BBS5). 7950978 Fredholm BB, Altiok N: Adenosine A2B receptor signalling is altered by stimulation of bradykinin or interleukin receptors in astroglioma cells. Neurochem Int. 1994 Jul;25(1):99-102. The human astroglioma cell D384 possesses adenosine A2B receptors coupled to the formation of cyclic AMP. These cells also possess bradykinin B2 receptors coupled to phospholipase C and consequent increases in intracellular calcium and protein kinase C. Interleukin 1 beta causes an increase in c-fos, AP-1 transcriptional activity and an increased expression of several genes including NGF, but the initial signalling events are unknown. Bradykinin causes a rapid decrease in A2B receptor mediated cAMP formation, via a mechanism that involves calcium, but not cGMP, and appears to depend upon a direct decrease in adenylyl cyclase. Il-1 beta causes a slowly developing (18-24 h) increase in A2B receptor signalling. The results indicate that adenosine effects in glial cells, believed to be important in neuroprotection, are modified in the short and long-term by inflammatory mediators. Adenosine A2B receptor signalling is altered by stimulation of bradykinin or interleukin receptors in astroglioma cells. 8177505 Chang NC, Jenkins NA, Gilbert DJ, Copeland NG, Chang YH, Chen WM, Chang AC: Assignment of two alpha 2 adrenoceptor subtype genes to murine chromosomes. Neurosci Lett. 1994 Feb 14;167(1-2):105-8. Subtype-specific probes to the murine homologs of the human ADRA2B and ADRA2C genes were prepared by PCR amplification and used to map these two genes to mouse chromosomes 2 and 5, respectively. Both genes mapped in regions of mouse chromosomes consistent with their map location in humans. These mapping results provide additional insights into the linkage relationships among members of this important gene family. Subtype-specific probes to the murine homologs of the human ADRA2B and ADRA2C genes were prepared by PCR amplification and used to map these two genes to mouse chromosomes 2 and 5, respectively. 10701773 Wang C, Valtavaara M, Myllyla R: Lack of collagen type specificity for lysyl hydroxylase isoforms. DNA Cell Biol. 2000 Feb;19(2):71-7. Lysyl hydroxylase is the enzyme catalyzing the formation of hydroxylysyl residues in collagens. Large differences in the extent of hydroxylysyl residues are found among collagen types. Three lysyl hydroxylase isoenzymes (LH1, LH2, LH3) have recently been characterized from human and mouse tissues. Nothing is known about the distribution of these isoforms within cells or whether they exhibit collagen type specificity. We measured mRNA levels of the three isoforms, as well as the mRNAs of the main collagen types I, III, IV, and V and the alpha subunit of prolyl 4-hydroxylase, another enzyme involved in collagen biosynthesis, in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. Immunoblotting was utilized to confirm the results of Northern hybridization. The levels of mRNA of LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase showed significant correlations with each other. The LH3 mRNA levels did not correlate with those of LH1, LH2, or the alpa subunit of prolyl 4-hydroxylase, clearly indicating a difference in the regulation of LH3. No correlation was observed between LH isoforms and individual collagen types, indicating a lack of collagen type specificity for lysyl hydroxylase isoforms. Our observations suggest that LH1, LH2, and the alpha subunit of prolyl 4-hydroxylase are coregulated together with total collagen synthesis but not with the specific collagen types and indicate that LH3 behaves differently from LH1 and LH2, implying a difference in their substrates. These observations set the basis for further studies to define the functions of lysyl hydroxylase isoforms. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. 8884272 Kurdi-Haidar B, Aebi S, Heath D, Enns RE, Naredi P, Hom DK, Howell SB: Isolation of the ATP-binding human homolog of the arsA component of the bacterial arsenite transporter. Genomics. 1996 Sep 15;36(3):486-91. Arsenite resistance in bacteria is mediated by an efflux pump composed of the arsA and arsB gene products. We have isolated the human homolog of the bacterial arsA (hARSA-I), a member of the ATPase superfamily with no transmembrane domain. Southern and Northern analyses indicated the presence of two cross-hybridizing genes in the human genome and expression of hARSA-I in many tissues. A rabbit antiserum raised against a glutathione-S-transferase (GST)/hARSA-I fusion protein identified two cross-reacting proteins of 37 and 42 kDa by Western analysis in two different human cell lines. Overexpression of hARSA-I in the embryonal human kidney 293 cell line was accompanied by overproduction of the 37-kDa protein Biochemical analysis using the GST/hARSA-I fusion protein indicated that hARSA-I is an ATPase analogous to the bacterial ArsA. Thus, hARSA-I is a new eukaryotic member of a highly conserved ATP-binding superfamily of proteins. We have isolated the human homolog of the bacterial arsA (hARSA-I), a member of the ATPase superfamily with no transmembrane domain. 1440058 Todd S, Naylor SL: New chromosomal mapping assignments for argininosuccinate synthetase pseudogene 1, interferon-beta 3 gene, and the diazepam binding inhibitor gene. Somat Cell Mol Genet. 1992 Jul;18(4):381-5. Argininosuccinate synthetase pseudogene 1 (ASSP1), interferon-beta 3 (IFNB3) gene, and diazepam binding inhibitor (DBI) gene have previously been mapped to human chromosome 2. Their nucleotide sequences, recorded in the GENBANK data base, were used to generate DNA primers to amplify specific sequences using the polymerase chain reaction (PCR). These primers failed to amplify DNA sequences when used to analyze microcell hybrid clones containing human chromosome 2. In order to map these genes, a panel of somatic cell hybrids was analyzed by PCR with these primer sets. The results of these experiments place ASSP1 sequences on human chromosome 6, IFNB3 on human chromosome 8, and DBI on human chromosome 6. The results of these experiments place ASSP1 sequences on human chromosome 6, IFNB3 on human chromosome 8, and DBI on human chromosome 6. 12738776 Elorza A, Penela P, Sarnago S, Mayor F Jr: MAPK-dependent degradation of G protein-coupled receptor kinase 2. J Biol Chem. 2003 Aug 1;278(31):29164-73. Epub 2003 May 8. G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). Altered expression of GRK2 has been described to occur during pathological conditions characterized by impaired GPCR signaling. We have reported recently that GRK2 is rapidly degraded by the proteasome pathway and that beta-arrestin function and Src-mediated phosphorylation are involved in targeting GRK2 for proteolysis. In this report, we show that phosphorylation of GRK2 by MAPK also triggers GRK2 turnover by the proteasome pathway. Modulation of MAPK activation alters the degradation of transfected or endogenous GRK2, and a GRK2 mutant that mimics phosphorylation by MAPK shows an enhanced degradation rate, thus indicating a direct effect of MAPK on GRK2 turnover. Interestingly, MAPK-mediated modulation of wild-type GRK2 stability requires beta-arrestin function and is facilitated by previous phosphorylation of GRK2 on tyrosine residues by c-Src. Consistent with an important physiological role, interfering with this GRK2 degradation process results in altered GPCR responsiveness. Our data suggest that both c-Src and MAPK-mediated phosphorylation would contribute to modulate GRK2 degradation, and put forward the existence of new feedback mechanisms connecting MAPK cascades and GPCR signaling. G protein-coupled receptor kinase 2 (GRK2) is a key modulator of G protein-coupled receptors (GPCR). 8595912 Druck T, Gu Y, Prabhala G, Cannizzaro LA, Park SH, Huebner K, Keen JH: Chromosome localization of human genes for clathrin adaptor polypeptides AP2 beta and AP50 and the clathrin-binding protein, VCP. Genomics. 1995 Nov 1;30(1):94-7. Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed "assembly proteins," or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2 alpha and AP2 beta, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2 beta (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with APs. Chromosomal in situ hybridization of a human genomic clone demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2 beta fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2 beta fragment from a panel of rodent-human hybrid DNAs. 9545647 Shah ZH, Migliosi V, Miller SC, Wang A, Friedman TB, Jacobs HT: Chromosomal locations of three human nuclear genes (RPSM12, TUFM, and AFG3L1) specifying putative components of the mitochondrial gene expression apparatus. Genomics. 1998 Mar 15;48(3):384-8. We have mapped the chromosomal locations of three human nuclear genes for putative components of the apparatus of mitochondrial gene expression, using a combination of in situ hybridization and interspecies hybrid mapping. The genes RPMS12 (mitoribosomal protein S12, a conserved protein component of the mitoribosomal accuracy center), TUFM (mitochondrial elongation factor EF-Tu), and AFG3L1 (similar to the yeast genes Afg3 and Rca1 involved in the turnover of mistranslated or misfolded mtDNA-encoded polypeptides) were initially characterized by a combination of database sequence analysis, PCR, cloning, and DNA sequencing. RPMS12 maps to chromosome 19q13.1, close to the previously mapped gene for autosomal dominant hearing loss DFNA4. The TUFM gene is located on chromosome 16p11.2, with a putative pseudogene or variant (TUFML) located very close to the centromere of chromosome 17. AFG3L1 is located on chromosome 16q24, very close to the telomere. By virtue of their inferred functions in mitochondria, these genes should be regarded as candidates of disorders sharing features with mitochondrial disease syndromes, such as sensorineural deafness, diabetes, and retinopathy. Chromosomal locations of three human nuclear genes (RPSM12, TUFM, and AFG3L1) specifying putative components of the mitochondrial gene expression apparatus. 2061309 Wellstein A, Dobrenski AF, Radonovich MN, Brady JF, Riegel AT: Purification of PO-B, a protein that has increased affinity for the pro-opiomelanocortin gene promoter after dephosphorylation. J Biol Chem. 1991 Jul 5;266(19):12234-41. The region -15 to -3 of the pro-opiomelanocortin (POMC) gene promoter specifically binds a transcription factor previously designated PO-B. This region of the POMC gene is involved in the control of constitutive POMC gene expression since mutation of the PO-B DNA-binding site severely reduces transcription from the POMC promoter both in vivo and in vitro (Riegel, A. T., Remenick, J., Wolford, R., Berard, D., and Hager, G. (1990) Nucleic Acids Res. 18, 4513-4521). We have now purified PO-B from HeLa cells approximately 25,000-fold to greater than 90% homogeneity by a combination of ion exchange and reversed phase chromatography. In addition we have studied post-translational modifications that alter the affinity of purified PO-B for its cognate DNA binding site. In Southwestern analysis of column fractions, two bands of apparent molecular masses of 54 and 56 kDa bound specifically to the PO-B recognition sequence. The two copurified components have indistinguishable amino acid composition, are highly hydrophobic, and are heat and acid stable. DNA-binding specificity studies suggest that PO-B does not represent any previously described transcription factor. In addition, dephosphorylation of both species with acid phosphatase induced an about 30-fold increase in DNA binding but failed to produce any significant change in electrophoretic mobility. We conclude that the purified PO-B species represent products of the same gene and suggest that the in vivo function of PO-B may be regulated by its phosphorylation status. This region of the POMC gene is involved in the control of constitutive POMC gene expression since mutation of the PO-B DNA-binding site severely reduces transcription from the POMC promoter both in vivo and in vitro (Riegel, A. T., Remenick, J., Wolford, R., Berard, D., and Hager, G. (1990) Nucleic Acids Res. 18, 4513-4521). 11584144 Takeshita T, Yang X, Morimoto K: Association of the ADH2 genotypes with skin responses after ethanol exposure in Japanese male university students. Alcohol Clin Exp Res. 2001 Sep;25(9):1264-9. BACKGROUND: A contribution of the alcohol dehydrogenase-2 (ADH2) polymorphism to alcohol sensitivity and alcohol drinking behavior is still controversial. In this study, we examined the effects of the ADH2 genotypes on skin reactions to ethanol and habitual alcohol intake among Japanese male university students, controlling for the effects of the low Km aldehyde dehydrogenase (ALDH2) genotype, as an extension of our previous study. METHODS: The study subjects were 357 Japanese male students [average age (mean +/- SD) was 23.7 +/- 3.0 years] in a medical university. The subjects completed a questionnaire regarding self-reported alcohol-associated symptoms and alcohol-drinking behavior. The ADH2 and ALDH2 genotypes were determined through digestion of polymerase chain reaction products by restriction enzymes. All subjects participated in the ethanol patch test. We observed skin responses at 0, 5, 15, and 20 min after removal of the tape. RESULTS: Among the ALDH2*1/*1 genotypes, only some subjects with ADH2*1/*2 or ADH2*2/*2 exhibited a positive response, which increased with increasing time after the removal. However, none of comparisons between the different ADH2 genotypes reached statistical significance. Among the ALDH2*1/*2 genotypes, those with ADH2*1/*2 or ADH2*2/*2 showed a significant increase in response with increasing time after the removal and revealed a significantly higher positivity rate at 15 min than those with ADH2*1/*1. In those with the ALDH2*1/*2 genotype, the positive rate of facial flushing with one glass of beer was higher in those with ADH2*1/*2 and ADH2*2/*2 than those with ADH2*1/*1, although this was not significant. However, the ADH2 genotype did not seem to influence drinking frequency or amounts of alcohol intake in each ALDH2 genotype. CONCLUSIONS: This study finds further evidence for a contribution of the ADH2 polymorphism to skin reactions after either local or systemic ethanol exposure in Asian people. However, the effects of the ADH2 polymorphism may be mild because this polymorphism does not seem to influence alcohol drinking behavior in these study subjects. However, the ADH2 genotype did not seem to influence drinking frequency or amounts of alcohol intake in each ALDH2 genotype. 9161417 Hsu LC, Chang WC, Yoshida A: Human aldehyde dehydrogenase genes, ALDH7 and ALDH8: genomic organization and gene structure comparison. Gene. 1997 Apr 11;189(1):89-94. The structure of two human aldehyde dehydrogenase genes, ALDH7 and ALDH8, have been determined. The ALDH7 gene spans about 20 kb of the human genomic DNA and is composed of 9 coding exons. The ALDH8 gene is over 10 kb in length and consists of at least 10 exons. The ALDH8 gene contains an in-frame stop codon at the 17th codon position from the first initiator Met. The coding region of the ALDH7 gene shows about 86% nucleotide identity with the corresponding region of the ALDH8 gene. The numbers and positions of the introns of the two genes are conserved, suggesting that gene duplication is involved in the expansion of the ALDH gene family. The human ALDH7 and -8 genes have a closer evolutionary relationship with the human ALDH3. The structure of two human aldehyde dehydrogenase genes, ALDH7 and ALDH8, have been determined. 10640806 Suzuki T, Kansaku N, Kurosaki T, Shimada K, Zadworny D, Koide M, Mano T, Namikawa T, Matsuda Y: Comparative FISH mapping on Z chromosomes of chicken and Japanese quail. Cytogenet Cell Genet. 1999;87(1-2):22-6. Using direct R-banding fluorescence in situ hybridization, we assigned five functional genes-growth hormone receptor (GHR), prolactin receptor (PRLR), spleen tyrosine kinase (SYK), aldolase B (ALDOB), and muscle skeletal receptor tyrosine kinase (MUSK)-to the chicken Z chromosome. SYK and MUSK were newly localized to the chicken Z chromosome in this study. GHR and PRLR were situated close to each other on the short arm of the chicken Z chromosome, as are their counterparts on human chromosome 5. SYK, MUSK, and ALDOB, which have been mapped to human chromosome 9, were localized to the long arm of the chicken Z chromosome. Thus, the present results indicate the presence of conserved synteny between the chicken Z chromosome and human chromosomes 5 and 9. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. The locations of these four Z-linked genes were conserved between chicken and Japanese quail. The results support the notion that the avian Z chromosome and the mammalian X chromosome did not evolve from a common ancestral linkage group. Using the same method, four of the genes (GHR, PRLR, ALDOB, and MUSK) were assigned to the Japanese quail Z chromosome. 11960987 Siu F, Bain PJ, LeBlanc-Chaffin R, Chen H, Kilberg MS: ATF4 is a mediator of the nutrient-sensing response pathway that activates the human asparagine synthetase gene. J Biol Chem. 2002 Jul 5;277(27):24120-7. Epub 2002 Apr 17. Transcription from the asparagine synthetase (A.S.) gene is increased in response to either amino acid (amino acid response) or glucose (endoplasmic reticulum stress response) deprivation. These two independent pathways converge on the same set of genomic cis-elements within the A.S. promoter referred to as nutrient-sensing response elements (NSRE) 1 and 2, both of which are necessary for gene activation. The NSRE-1 sequence was used to screen ATF/CREB family members by electrophoresis mobility shift assays and supershift by specific antibodies. The results indicated that ATF4 binds to the NSRE-1 sequence and that the amount of the ATF4 complex was increased when extracts from amino acid-deprived or glucose-deprived cells were tested. Using electrophoresis mobility shift assay experiments and a probe that contained both NSRE-1 and NSRE-2, mutation of the NSRE-1 sequence completely prevented formation of the ATF4-containing complexes, whereas mutation of the NSRE-2 sequence did not. Overexpression of ATF4 increased A.S. promoter-driven transcription, whereas an inhibitory dominant negative ATF4 mutant blocked both basal and starvation-enhanced transcription. Collectively, the results provide both in vitro and in vivo evidence for a role of ATF4 in the transcriptional activation of the A.S. gene in response to nutrient deprivation. ATF4 is a mediator of the nutrient-sensing response pathway that activates the human asparagine synthetase gene. 10814707 Yanagisawa H, Bundo M, Miyashita T, Okamura-Oho Y, Tadokoro K, Tokunaga K, Yamada M: Protein binding of a DRPLA family through arginine-glutamic acid dipeptide repeats is enhanced by extended polyglutamine. Hum Mol Genet. 2000 May 22;9(9):1433-42. Dentatorubral-pallidoluysian atrophy (DRPLA) is one of the hereditary neurodegenerative disorders caused by expansion of CAG/glutamine repeats. To investigate the normal function of the DRPLA gene and the pathogenic mechanism of neuron death in specific areas of the brain, we isolated and analyzed a gene that shares a notable motif with DRPLA, arginine-glutamic acid (RE) dipeptide repeats. The gene isolated, designated RERE, has an open reading frame of 1566 amino acids, of which the C-terminal portion has 67% homology to DRPLA, whereas the N-terminal portion is distinctive. RERE also contains arginine-aspartic acid (RD) dipeptide repeats and putative nuclear localization signal sequences, but no polyglutamine tracts. RERE is expressed at a low level in most tissues examined. Immunoprecipitation and in vitro binding assays demonstrate that the DRPLA and RERE proteins bind each other, for which one of the RE repeats has a primary role, and extended polyglutamine enhances the binding. With engineered constructs fused with a tag, the RERE protein localized predominantly in the nucleus. Moreover, when RERE is overexpressed, the distribution of endogenous DRPLA protein alters from the diffused to the speckled pattern in the nucleus so as to co-localize with RERE. More RERE protein is recruited into nuclear aggregates of the DRPLA protein with extended polyglutamine than into those of pure polyglutamine. These results reveal a function for the DRPLA protein in the nucleus and the RE repeat in the protein-protein interaction. More RERE protein is recruited into nuclear aggregates of the DRPLA protein with extended polyglutamine than into those of pure polyglutamine. 9022753 Liu YW, Chen BK, Chen CJ, Arakawa T, Yoshimoto T, Yamamoto S, Chang WC: Epidermal growth factor enhances transcription of human arachidonate 12-lipoxygenase in A431 cells. Biochim Biophys Acta. 1997 Jan 7;1344(1):38-46. Epidermal growth factor (EGF), determined by immunoprecipitation and Western blot analysis, increased both enzyme activity and protein level of 12-lipoxygenase in the solubilized microsomes of human epidermoid carcinoma A431 cells, respectively. The EGF-induced expression of 12-lipoxygenase mRNA was inhibited by transcription inhibitors such as actinomycin D and 5,6-dichlorobenzimidazole riboside. Promoters of different lengths for human 12-lipoxygenase gene were used to prepare the luciferase fusion vectors. These construct plasmids were transiently transfected into A431 cells, and the induction of luciferase expression by EGF was examined. A 4- to 6-fold increase in luciferase reporter activity stimulated by EGF for 18 h treatment was observed in plasmids with the 5'-flanking region length of -951 bp and that of -224 bp upstream from translation starting site. The time-dependent induction of luciferase activity by EGF paralleled the EGF-induced enzyme activity and expression of 12-lipoxygenase protein. Taken together, the results of this study indicate that EGF enhanced the transcription of the human 12-lipoxygenase gene, resulting in an increase in the amount and activity of 12-lipoxygenase. Epidermal growth factor enhances transcription of human arachidonate 12-lipoxygenase in A431 cells. 11353452 Philibert RA, Cheung D, Welsh N, Damschroder-Williams P, Thiel B, Ginns EI, Gershenfeld HK: Absence of a significant linkage between Na(+),K(+)-ATPase subunit (ATP1A3 and ATP1B3) genotypes and bipolar affective disorder in the old-order Amish. Am J Med Genet. 2001 Apr 8;105(3):291-4. Previous studies provide evidence for a genetic component for susceptibility to bipolar affective disorder (BPAD) in the old-order Amish population. El-Mallakh and Wyatt [1995: Biol Psychiatry 37:235-244] have suggested that the Na(+),K(+)-ATPase may be a candidate gene for BPAD. This study examines the relationship between BPAD in the old-order Amish cohort and the Na(+),K(+)-ATPase alpha1 and beta3 subunit genes (ATP1A3, ATP1B3). A total of 166 sibling pairs were analyzed for linkage via nonparametric methods. Suggestive levels of statistical significance were not reached in any stratification model for affective illness. Overall, the results do not support linkage of bipolar disorder to the Na(+),K(+)-ATPase alpha subunit gene (ATP1A3) and beta subunit gene (ATP1B3) in these old-order Amish families and they show that these Na(+),K(+)-ATPase subunit genes are not major effect genes (>or=fourfold increased genetic risk of disease) for BPAD in the old-order Amish pedigrees. We cannot exclude other genetic variants of the Na(+),K(+)-ATPase hypothesis for BPAD, whereby other loci may modifying Na(+),K(+)-ATPase activity. Overall, the results do not support linkage of bipolar disorder to the Na (+),K (+)-ATPase alpha subunit gene (ATP1A3) and beta subunit gene (ATP1B3) in these old-order Amish families and they show that these Na (+),K (+)-ATPase subunit genes are not major effect genes (> or=fourfold increased genetic risk of disease) for BPAD in the old-order Amish pedigrees. 11353452 Philibert RA, Cheung D, Welsh N, Damschroder-Williams P, Thiel B, Ginns EI, Gershenfeld HK: Absence of a significant linkage between Na(+),K(+)-ATPase subunit (ATP1A3 and ATP1B3) genotypes and bipolar affective disorder in the old-order Amish. Am J Med Genet. 2001 Apr 8;105(3):291-4. Previous studies provide evidence for a genetic component for susceptibility to bipolar affective disorder (BPAD) in the old-order Amish population. El-Mallakh and Wyatt [1995: Biol Psychiatry 37:235-244] have suggested that the Na(+),K(+)-ATPase may be a candidate gene for BPAD. This study examines the relationship between BPAD in the old-order Amish cohort and the Na(+),K(+)-ATPase alpha1 and beta3 subunit genes (ATP1A3, ATP1B3). A total of 166 sibling pairs were analyzed for linkage via nonparametric methods. Suggestive levels of statistical significance were not reached in any stratification model for affective illness. Overall, the results do not support linkage of bipolar disorder to the Na(+),K(+)-ATPase alpha subunit gene (ATP1A3) and beta subunit gene (ATP1B3) in these old-order Amish families and they show that these Na(+),K(+)-ATPase subunit genes are not major effect genes (>or=fourfold increased genetic risk of disease) for BPAD in the old-order Amish pedigrees. We cannot exclude other genetic variants of the Na(+),K(+)-ATPase hypothesis for BPAD, whereby other loci may modifying Na(+),K(+)-ATPase activity. Overall, the results do not support linkage of bipolar disorder to the Na (+),K (+)-ATPase alpha subunit gene (ATP1A3) and beta subunit gene (ATP1B3) in these old-order Amish families and they show that these Na (+),K (+)-ATPase subunit genes are not major effect genes (> or=fourfold increased genetic risk of disease) for BPAD in the old-order Amish pedigrees. 1364010 Chambers JA, Gardner E, Hauptmann R, Ponder BA, Mulligan LM: TaqI polymorphisms at the annexin VIII locus (ANX8). Hum Mol Genet. 1992 Oct;1(7):550. TaqI polymorphisms at the annexin VIII locus (ANX8). 10329626 Bash J, Zong WX, Banga S, Rivera A, Ballard DW, Ron Y, Gelinas C: Rel/NF-kappaB can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors. EMBO J. 1999 May 17;18(10):2803-11. Jagged1 belongs to the DSL family of ligands for Notch receptors that control the proliferation and differentiation of various cell lineages. However, little is known about the transcription factors that regulate its expression. Here, we show that Jagged1 is a Rel/NF-kappaB-responsive gene. Both c-Rel and RelA induced jagged1 gene expression, whereas a mutant defective for transactivation did not. Importantly, jagged1 transcripts were also upregulated by endogenous NF-kappaB activation and this effect was inhibited by a dominant mutant of IkappaBalpha, a physiological inhibitor of NF-kappaB. Cell surface expression of Jagged1 in c-Rel-expressing cell monolayers led to a functional interaction with lymphocytes expressing the Notch1/TAN-1 receptor. This correlated with the initiation of signaling downstream of Notch, as evidenced by increased levels of HES-1 transcripts in co-cultivated T cells and of CD23 transcripts in co-cultivated B cells. Consistent with its Rel/NF-kappaB-dependent induction, Jagged1 was found to be highly expressed in splenic B cells where c-Rel is expressed constitutively. These results demonstrate that c-Rel can trigger the Notch signaling pathway in neighboring cells by inducing jagged1 gene expression, and suggest a role for Jagged1 in B-cell activation, differentiation or function. These findings also highlight the potential for an interplay between the Notch and NF-kappaB signaling pathways in the immune system. Both c-Rel and RelA induced jagged1 gene expression, whereas a mutant defective for transactivation did not. 8825033 Verboomen H, Mertens L, Eggermont J, Wuytack F, Van Den Bosch L: Modulation of SERCA2 activity: regulated splicing and interaction with phospholamban. Biosci Rep. 1995 Oct;15(5):307. Ca(2+)-uptake into intracellular stores is mediated by the sarco/endoplasmic reticulum Ca(2+)ATPases (SERCAs). This review deals first with the gene structural and the characterization of the tissue-specific SERCA2 transcript processing. Secondly, the two different protein isoforms and their regulation are described. Finally, this review ends with a discussion on the possible physiological role of the SERCA2 isoform diversity. Modulation of SERCA2 activity: regulated splicing and interaction with phospholamban. 11802776 Ito M, Amizuka N, Ozawa H, Oda K: Retention at the cis-Golgi and delayed degradation of tissue-non-specific alkaline phosphatase with an Asn153-->Asp substitution, a cause of perinatal hypophosphatasia. Biochem J. 2002 Feb 1;361(Pt 3):473-80. Tissue-non-specific alkaline phosphatase (TNSALP) is an ectoenzyme anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). A TNSALP mutant with an Asn(153)-->Asp (N153D) substitution was reported in a foetus diagnosed with perinatal hypophosphatasia (Mornet, Taillandier, Peyramaure, Kaper, Muller, Brenner, Bussiere, Freisinger, Godard, Merrer et al. (1998) Eur. J. Hum. Genet. 6, 308-314). When expressed ectopically in COS-1 cells, the wild-type TNSALP formed active non-covalently associated dimers, whereas TNSALP (N153D) formed aberrant disulphide-bonded high-molecular-mass aggregates devoid of enzyme activity. Cell-surface biotinylation and digestion with phosphatidylinositol-specific phospholipase C showed that TNSALP (N153D) failed to reach the cell surface. Instead, double immunofluorescence demonstrated that TNSALP (N153D) partially co-localized with a cis-Golgi marker (GM-130) at the steady-state. Upon treatment with brefeldin A, TNSALP (N153D) was still co-localized with GM-130, further supporting the finding that this mutant is localized in the cis-Golgi. Consistent with morphological results, pulse-chase experiments showed that newly synthesized TNSALP (N153D) remained endo-beta-N-acetylglucosaminidase H-sensitive throughout the chase. Eventually, after a prolonged chase time, the mutant was found to be partly degraded in a proteasome-dependent manner. Since the mutant TNSALP was significantly labelled with [3H]ethanolamine, a component of GPI, comparable with the wild-type enzyme, it is unlikely that the abortive synthesis of the mutant is due to a defect in GPI-attachment. Interestingly, when asparagine was replaced by glutamine at position 153 (N153D), TNSALP (N153Q) was indistinguishable from the wild-type enzyme in terms of its molecular properties, suggesting the possible importance of amino acids with a polar amide group at position 153. Taken together, these findings indicate that replacing asparagine with aspartic acid at position 153 causes misfolding and incorrect assembly of TNSALP, which results in its retention at the cis-Golgi en route to the cell surface, followed by a delayed degradation, presumably as part of a quality-control process. We postulate that the molecular basis of the perinatal hypophosphatasia associated with TNSALP (N153D) is due to the absence of mature TNSALP at the cell surface. Instead, double immunofluorescence demonstrated that TNSALP (N153D) partially co-localized with a cis-Golgi marker (GM-130) at the steady-state. 10662549 Cox PR, Zoghbi HY: Sequencing, expression analysis, and mapping of three unique human tropomodulin genes and their mouse orthologs. Genomics. 2000 Jan 1;63(1):97-107. Tropomodulin (TMOD) is the actin-capping protein for the slow-growing end of filamentous actin, and a neuronal-specific isoform, neuronal tropomodulin (NTMOD), is the major binding protein to brain tropomyosin in rat. The Drosophila TMOD homolog, Sanpodo, alters sibling cell fate determination, so we used a cross-species approach to identify additional TMOD family members that may play a critical role in this process. We characterized the human and mouse orthologs to rat NTMOD (TMOD2 and Tmod2, respectively) as well as two novel tropomodulin family members (TMOD3, Tmod3 and TMOD4, Tmod4). Their expression patterns vary extensively, from ubiquitous (TMOD3 and Tmod3) to muscle (TMOD4) or neuronal tissues only (TMOD2 and Tmod2). TMOD2 and TMOD3 map next to one another on chromosome 15q21.1-q21.2, and their mouse orthologs map to a homologous region on mouse chromosome 9; TMOD4 maps to the telomeric end of 1q12 and Tmod4 to a homologous region of mouse chromosome 3. Their location and expression patterns make TMOD2 and TMOD3 candidate genes for amyotrophic lateral sclerosis 5 (ALS5) and dyslexia-1 (DYX1) and TMOD4 a candidate gene for limb girdle muscular dystrophy 1B (LGMD1B). Our mapping efforts revealed new regions of paralogy among chromosomes 1q, 9q, 15q, and 19p. Their location and expression patterns make TMOD2 and TMOD3 candidate genes for amyotrophic lateral sclerosis 5 (ALS5) and dyslexia-1 (DYX1) and TMOD4 a candidate gene for limb girdle muscular dystrophy 1B (LGMD1B). 8787675 Burglen L, Amiel J, Viollet L, Lefebvre S, Burlet P, Clermont O, Raclin V, Landrieu P, Verloes A, Munnich A, Melki J: Survival motor neuron gene deletion in the arthrogryposis multiplex congenita-spinal muscular atrophy association. J Clin Invest. 1996 Sep 1;98(5):1130-2. The survival motor neuron (SMN) gene was lacking in 6/12 patients with arthrogryposis multiplex congenita (AMC) associated with spinal muscular atrophy (SMA). Neither point mutation in the SMN gene nor evidence for linkage to chromosome 5q13 were found in the other patients. Hitherto, arthrogryposis was regarded as an exclusion criterion in SMA. Our data strongly suggest that AMC of neurogenic origin is genetically heterogeneous, with a subgroup being allelic to SMA. Absence or interruption of the SMN gene in the AMC-SMA association will make the diagnosis easier and genetic counselling will now become feasible. The survival motor neuron (SMN) gene was lacking in 6/12 patients with arthrogryposis multiplex congenita (AMC) associated with spinal muscular atrophy (SMA). 8700162 Santiago-Garcia J, Mas-Oliva J, Saavedra D, Zarain-Herzberg A: Analysis of mRNA expression and cloning of a novel plasma membrane Ca(2+)-ATPase splice variant in human heart. Mol Cell Biochem. 1996 Feb 23;155(2):173-82. Four different plasma membrane Ca(2+)-ATPase (PMCA) genes and three sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) genes have been previously cloned and characterized. In this study we have investigated the expression of the mRNA encoding the various PMCA and SERCA proteins in fetal and adult human heart and placenta by the reverse-transcriptase-polymerase-chain-reaction (RT-PCR) and cDNA cloning. We have found that PMCA1 and PMCA4 genes were expressed in 8-, 12- and 20-week fetal heart and in adult heart. PMCA2 gene was expressed at low levels in adult heart but was not detected in fetal heart. PMCA3 mRNA was not detected in the heart nor placenta. In contrast, the mRNA encoding SERCA2a, SERCA2b and SERCA3 were expressed in all cardiac developmental stages. Multiple alternatively spliced mRNA transcripts which differ at splice site A and B/C of the PMCA1, PMCA2 and PMCA4 genes were detected in the human heart. Interestingly, a novel tissue specific variant of the PMCA4 gene was detected in both fetal and adult human heart but not in placenta that accounts for about 30% of the total PMCA4 mRNA variant expression. DNA sequence analysis of this novel variant revealed that it corresponds to the equivalent of the PMCA1d variant and accordingly we have named it PMCA4d. We cloned and sequenced eight cDNA inserts encoding for the PMCA1 and PMCA4 variants from a fetal human heart cDNA library confirming that these are the two main PMCA genes expressed in cardiac muscle. We have found that PMCA1 and PMCA4 genes were expressed in 8-, 12- and 20-week fetal heart and in adult heart. 9233643 Ferrari D, Chiozzi P, Falzoni S, Dal Susino M, Melchiorri L, Baricordi OR, Di Virgilio F: Extracellular ATP triggers IL-1 beta release by activating the purinergic P2Z receptor of human macrophages. J Immunol. 1997 Aug 1;159(3):1451-8. Extracellular ATP (ATPe) is known to cause release of processed IL-1 beta from LPS-treated macrophages and microglial cells. IL-1 beta release is fast and thought to be associated with cell death. We have reinvestigated this process to identify 1) the purinergic receptor involved; 2) the relationship to cell death; and 3) pharmacologic agonists or antagonists able to modulate IL-1 beta release. Our data confirm that ATPe is a powerful stimulus for IL-1 beta release from LPS-treated human macrophages; however, we also show that IL-1 beta release is not necessarily associated with cell death, as it occurs at lower ATP concentrations and much earlier than leakage of cytoplasmic markers. The selective purinergic P2Z receptor agonist benzoylbenzoyl ATP was at least one order of magnitude more powerful than ATP, but also had a strong cytotoxic effect. 2-Methylthio-ATP was equipotent as ATPe at the optimal concentration of 1 mM, but markedly inhibitory at higher concentrations. The irreversible P2Z blocker-oxidized ATP completely inhibited ATPe-induced IL-1 beta release. IL-1 beta release also was inhibited by increasing the K+ concentration of the incubation medium. These data suggest that ATPe triggers IL-1 beta via the purinergic P2Z receptor recently shown to be expressed by human macrophages and identified as a new member of the P2X family (P2X7), and provide pharmacologic tools for the modulation of IL-1 beta release in vitro and, possibly, in vivo. These data suggest that ATPe triggers IL-1 beta via the purinergic P2Z receptor recently shown to be expressed by human macrophages and identified as a new member of the P2X family (P2X7), and provide pharmacologic tools for the modulation of IL-1 beta release in vitro and, possibly, in vivo. 11085934 Knott TG, Birdsey GM, Sinclair KE, Gallagher IM, Purdue PE, Danpure CJ: The peroxisomal targeting sequence type 1 receptor, Pex5p, and the peroxisomal import efficiency of alanine:glyoxylate aminotransferase. Biochem J. 2000 Dec 1;352 Pt 2:409-18. Unlike most organellar proteins, some peroxisomal proteins are often found in significant amounts in the cytosol. Such apparent import inefficiency is very marked in guinea pig (Cavia porcellus) hepatocytes in which the cytosolic levels of two peroxisomal proteins, catalase and alanine:glyoxylate aminotransferase (AGT), are much higher than those found in human (Homo sapiens) hepatocytes, for example. In an attempt to provide an explanation for this phenomenon, we have cloned the guinea pig CpPEX5 gene, which encodes the peroxisomal targeting sequence type 1 (PTS1) import receptor Pex5p, and functionally compared it with its human homologue, HsPex5p. Our results showed the following: (1) CpPEX5, like its human homologue, encodes two splice variants differing by the presence or absence of an internal region of 37 amino acids; (2) both variants were expressed in all guinea pig tissues studied; (3) both variants were equally able to complement peroxisomal import of PTS1 proteins in microinjected Deltapex5 human fibroblasts; (4) CpPex5p was as efficient as HsPex5p in mediating the peroxisomal import of proteins possessing the consensus PTS1, Ser-Lys-Leu, but much less efficient in mediating the import of proteins possessing non-consensus PTS1s (i.e. Lys-Lys-Leu of human AGT and Ala-Asn-Leu of human catalase); (5) reporter proteins with the consensus PTS1, Ser-Lys-Leu, inhibited the peroxisomal import of endogenous catalase, whereas AGT with the non-consensus Lys-Lys-Leu did not; (6) high concentrations of HsPex5p, but not CpPex5p, markedly inhibited the import of AGT, but not catalase or proteins ending in Ser-Lys-Leu; and (7) in the yeast two-hybrid system, AGT-Ser-Lys-Leu interacted with the tetratricopeptide repeat domain of HsPex5p, but AGT-Lys-Lys-Leu did not. In addition, AGT-Ser-Lys-Leu was targeted to peroxisomes in Saccharomyces cerevisiae, whereas AGT-Lys-Lys-Leu was not. These data suggest that the inefficient peroxisomal import of AGT and catalase in guinea pig cells is due to the inefficiency with which CpPex5p mediates the peroxisomal import of proteins containing non-consensus PTS1s. They also suggest that the non-consensus PTS1 of human AGT might interact with HsPex5p very differently compared with the consensus PTS1, Ser-Lys-Leu. The peroxisomal targeting sequence type 1 receptor, Pex5p, and the peroxisomal import efficiency of alanine:glyoxylate aminotransferase. 11866458 Skoldberg F, Ronnblom L, Thornemo M, Lindahl A, Bird PI, Rorsman F, Kampe O, Landgren E: Identification of AHNAK as a novel autoantigen in systemic lupus erythematosus. Biochem Biophys Res Commun. 2002 Mar 8;291(4):951-8. To identify candidate autoantigens associated with arthritis, a rat chondrocyte cDNA library was immunoscreened with serum from a patient with rheumatoid arthritis. One isolated cDNA encoded part of AHNAK, a 700-kDa phosphoprotein with DNA binding properties, that appears to be involved in several signal transduction pathways. Immunoreactivity against an in vitro translated human AHNAK fragment was detected in 4.6% (5/109) of patients with rheumatoid arthritis, 29.5% (18/61) of patients with systemic lupus erythematosus (SLE), and 1.2% (2/172) of blood donors. Anti-AHNAK antibodies reacted with a recombinant human AHNAK fragment and with native AHNAK from C32 cell lysates. In vitro translated AHNAK fragment could be cleaved by granzyme B and caspase-3. Anti-AHNAK positive SLE patients had a higher frequency of homogeneous antinuclear antibody staining patterns and a lower frequency of recent mucosal ulcerations. This is the first report that AHNAK can be targeted by the immune system in autoimmune disease. In vitro translated AHNAK fragment could be cleaved by granzyme B and caspase-3. 12082568 Kim SJ, Young LJ, Gonen D, Veenstra-VanderWeele J, Courchesne R, Courchesne E, Lord C, Leventhal BL, Cook EH Jr, Insel TR: Transmission disequilibrium testing of arginine vasopressin receptor 1A (AVPR1A) polymorphisms in autism. Mol Psychiatry. 2002;7(5):503-7. Impairment in social reciprocity is a central component of autism. In preclinical studies, arginine vasopressin (AVP) has been shown to increase a range of social behaviors, including affiliation and attachment, via the V(1a) receptor (AVPR1A) in the brain. Both the behavioral effects of AVP and the neural distribution of the V1a receptor vary greatly across mammalian species. This difference in regional receptor expression as well as differences in social behavior may result from a highly variable repetitive sequence in the 5' flanking region of the V1a gene (AVPR1A). Given this comparative evidence for a role in inter-species variation in social behavior, we explored whether within our own species, variation in the human AVPR1A may contribute to individual variations in social behavior, with autism representing an extreme form of social impairment. We genotyped two microsatellite polymorphisms from the 5' flanking region of AVPR1A for 115 autism trios and found nominally significant transmission disequilibrium between autism and one of the microsatellite markers by Multiallelic Transmission/Disequilibrium test (MTDT) that was not significant after Bonferroni correction. We also screened approximately 2 kb of the 5' flanking region and the coding region and identified 10 single nucleotide polymorphisms. In preclinical studies, arginine vasopressin (AVP) has been shown to increase a range of social behaviors, including affiliation and attachment, via the V (1a) receptor (AVPR1A) in the brain. 10190716 Dalekos GN, Wedemeyer H, Obermayer-Straub P, Kayser A, Barut A, Frank H, Manns MP: Epitope mapping of cytochrome P4502D6 autoantigen in patients with chronic hepatitis C during alpha-interferon treatment. J Hepatol. 1999 Mar;30(3):366-75. BACKGROUND/AIMS: Cytochrome P450 2D6 (CYP2D6) has been documented as the major target antigen of liver kidney microsomal autoantibodies type-1 (anti-LKM-1) in both autoimmune hepatitis type-2 (AIH-2) and hepatitis C (HCV). In HCV/anti-LKM-1-positive patients, the choice between alpha-interferon (alpha-IFN) or immunosuppression may be difficult. This study was conducted to evaluate the course and outcome of alpha-IFN therapy in HCV/anti-LKM-1-positive and -negative patients and the alterations in these autoantibody titers by the indirect immunofluorescence and a novel radioligand assay. Epitope mapping was also performed to screen for a potential shift in anti-LKM-1 binding towards small linear epitopes, which are more often detected in AIH-2 patients. METHODS: Twenty-one patients with HCV infection received alpha-IFN. Seven patients were anti-LKM-1 positive (study group) and 14 patients were anti-LKM-1 negative (disease control group). Anti-CYP2D6 detection was based on immunoprecipitation of [35S]-methionine-labeled CYP2D6 recombinant protein (rCYP2D6) produced by in vitro transcription/translation. RESULTS: Four out of seven (57%) patients in the study group and 5/14 (36%) in the disease control group initially responded, but subsequently relapsed. During follow-up, alanine aminotransferase significantly increased in the study group compared to the disease control group (p<0.01). A slight increase, followed by a plateau of autoantibody titers was recorded by the radioligand assay and by indirect immunofluorescence during therapy and follow-up in most cases. In one patient, however, gamma-globulins and anti-LKM-1 titers increased, reaching very high levels (1:40 960). alpha-IFN was interrupted and immunosuppression was started. HCV/anti-CYP2D6 positive sera recognized CYP2D6 expressed in E. coli and two truncated proteins (aa 250-494 and 321-494). Two out of seven sera, in addition reacted with a small linear epitope of aa 257-269 (one of which also reacted with a C-terminal domain of aa 350-494). CONCLUSIONS: A rather mild deterioration in liver disease was observed in only 1/7 HCV/anti-LKM-1-positive patients during alpha-IFN treatment. This patient showed high anti-CYP2D6 titers before the initiation of therapy, a sharp increase in anti-LKM-1 titers during treatment, and reactivities to a small linear epitope and an infrequently recognized C-terminal domain of CYP2D6. After switching to immunosuppressive treatment, a complete and sustained response was recorded. Further prospective studies from many centers are needed to define whether these features have general, clinical significance or not. BACKGROUND/AIMS: Cytochrome P450 2D6 (CYP2D6) has been documented as the major target antigen of liver kidney microsomal autoantibodies type-1 (anti-LKM-1) in both autoimmune hepatitis type-2 (AIH-2) and hepatitis C (HCV). 10233855 O'Donnell K, Harkes IC, Dougherty L, Wicks IP: Expression of receptor tyrosine kinase Axl and its ligand Gas6 in rheumatoid arthritis: evidence for a novel endothelial cell survival pathway. Am J Pathol. 1999 Apr;154(4):1171-80. Angiogenesis and synovial cell hyperplasia are characteristic features of rheumatoid arthritis (RA). Many growth and survival factors use receptors belonging to the tyrosine kinase family that share conserved motifs within the intracellular catalytic domains. To understand further the molecular basis of cellular hyperplasia in RA, we have used degenerate primers based on these motifs and RNA obtained from the synovium of a patient with RA to perform reverse transcriptase-polymerase chain reaction. We report detection of the receptor tyrosine kinase (RTK) Axl in RA synovium and we document the expression pattern of Axl in capillary endothelium, in vascular smooth muscle cells of arterioles and veins, and in a subset of synovial cells in RA synovial tissue. Gas6 (for growth arrest-specific gene 6), which is a ligand for Axl and is related to the coagulation factor protein S, was found in synovial fluid and tissue from patients with RA and osteoarthritis. Axl expression and function was studied in human umbilical vein endothelial cells (HUVECs). Gas6 bound to HUVECs; soluble Axl inhibited this binding. Exogenous Gas6 protected HUVECs from apoptosis in response to growth factor withdrawal and from TNFalpha-mediated cytotoxicity. These findings may reveal a new aspect of vascular physiology, which may also be relevant to formation and maintenance of the abnormal vasculature in the rheumatoid synovium. Gas6 bound to HUVECs; soluble Axl inhibited this binding. 8621683 Chertov O, Michiel DF, Xu L, Wang JM, Tani K, Murphy WJ, Longo DL, Taub DD, Oppenheim JJ: Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils. J Biol Chem. 1996 Feb 9;271(6):2935-40. Reports that interleukin-8 (IL-8) induces the infiltration of neutrophils followed by T-cells into injection sites led us to postulate that by stimulation of neutrophil degranulation IL-8 may cause the release of factors with chemoattractant activity for T-lymphocytes. Extracts of human neutrophil granules were chromatographed to isolate and purify T-lymphocyte chemoattractant factors. Two major peaks of T-cell chemotactic activity were purified by C18 reversed phase high pressure liquid chromatography (HPLC). The first peak was resolved further by C4 reversed phase HPLC and yielded an active fraction shown by NH2-terminal amino acid sequence analysis to contain defensins HNP-1, HNP-2, and HNP-3. Purified defensins HNP-1 and HNP-2 (kindly provided by Dr. R. I. Lehrer, UCLA) were also potent chemoattractants for human T-cells, while HNP-3 was inactive. The second peak of T-cell chemoattractant activity was also further purified to homogeneity by C4 reversed phase HPLC and identified by NH2-terminal sequence analysis as CAP37/azurocidin, a protein with sequence homology to serine proteases. 0.1 100 ng of defensins and 1.0 100 ng/ml CAP37 were able to stimulate in vitro T-cell chemotaxis. Neutrophil activating factors, i.e. IL-8, phorbol 12-myristate 13-acetate/ionomycin, and formylmethionylleucylphenylalanine each induced the release of CAP37 and defensins from neutrophil granules. Subcutaneous administration of defensins or CAP37/azurocidin into BALB/c mice resulted in a moderate neutrophil and mononuclear cell infiltrate by 4 h, which was greater by 24 h at the site of injection. Additionally, subcutaneous injection of defensins into chimeric huPBL-SCID mice resulted in significant infiltration by human CD3+ cells within 4 h. These results identify the antimicrobial proteins, CAP37/azurocidin and defensins HNP-1 and HNP-2, as potent neutrophil-derived chemoattractants for T-cells. These proteins represent primordial antimicrobial peptides which may have evolved into acute inflammatory cell-derived signals that mobilize immunocompetent T-cells and other inflammatory cells. 0.1 100 ng of defensins and 1.0 100 ng/ml CAP37 were able to stimulate in vitro T-cell chemotaxis. 12458207 Barthwal MK, Sathyanarayana P, Kundu CN, Rana B, Pradeep A, Sharma C, Woodgett JR, Rana A: Negative regulation of mixed lineage kinase 3 by protein kinase B/AKT leads to cell survival. J Biol Chem. 2003 Feb 7;278(6):3897-902. Epub 2002 Nov 27. Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that activates c-jun N-terminal kinase (JNK) and can induce cell death in neurons. By contrast, the activation of phosphatidylinositol 3-kinase and AKT/protein kinase B (PKB) acts to suppress neuronal apoptosis. Here, we report a functional interaction between MLK3 and AKT1/PKBalpha. Endogenous MLK3 and AKT1 interact in HepG2 cells, and this interaction is regulated by insulin. The interaction domain maps to the C-terminal half of MLK3 (amino acids 511-847), and this region also contains a putative AKT phosphorylation consensus sequence. Endogenous JNK, MKK7, and MLK3 kinase activities in HepG2 cells are significantly attenuated by insulin treatment, whereas the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin reversed the effect. Finally, MLK3-mediated JNK activation is inhibited by AKT1. AKT phosphorylates MLK3 on serine 674 both in vitro and in vivo. Furthermore, the expression of activated AKT1 inhibits MLK3-mediated cell death in a manner dependent on serine 674 phosphorylation. Thus, these data provide the first direct link between MLK3-mediated cell death and its regulation by a cell survival signaling protein, AKT1. Finally, MLK3-mediated JNK activation is inhibited by AKT1. 2416779 Cooke NE, David EV: Serum vitamin D-binding protein is a third member of the albumin and alpha fetoprotein gene family. J Clin Invest. 1985 Dec;76(6):2420-4. A near full-length cDNA encoding the human vitamin D-binding protein (hDBP) was isolated from a human liver mRNA expression library. Complete sequence analysis of this clone predicts the full-length amino acid sequence of the pre-hDBP. Comparison of the sequence of the hDBP mRNA and protein to existing protein and nucleic acid data banks demonstrates a strong and highly characteristic homology of the hDBP with human albumin (hALB) and human alpha-fetoprotein (hAFP). Based upon this structural comparison, we establish that DBP is a member of the ALB and AFP gene family. Based upon this structural comparison, we establish that DBP is a member of the ALB and AFP gene family. 10086964 Loh E, Rebbeck TR, Mahoney PD, DeNofrio D, Swain JL, Holmes EW: Common variant in AMPD1 gene predicts improved clinical outcome in patients with heart failure. Circulation. 1999 Mar 23;99(11):1422-5. BACKGROUND: This study was undertaken to identify gene(s) that may be associated with improved clinical outcome in patients with congestive heart failure (CHF). The adenosine monophosphate deaminase locus (AMPD1) was selected for study. We hypothesized that inheritance of the mutant AMPD1 allele is associated with increased probability of survival without cardiac transplantation in patients with CHF. METHODS AND RESULTS: AMPD1 genotype was determined in 132 patients with advanced CHF and 91 control reference subjects by use of a polymerase chain reaction-based, allele-specific oligonucleotide detection assay. In patients with CHF, those heterozygous (n=20) or homozygous (n=1) for the mutant AMPD1 allele (AMPD1 +/- or -/-, respectively) experienced a significantly longer duration of heart failure symptoms before referral for transplantation evaluation than CHF patients homozygous for the wild-type allele (AMPD1 +/+; n=111; 7.6+/-6.5 versus 3.2+/-3.6 years; P<0.001). The OR of surviving without cardiac transplantation >/=5 years after initial hospitalization for CHF symptoms was 8.6 times greater (95% CI: 3.05, 23.87) in those patients carrying >/=1 mutant AMPD1 allele than in those carrying 2 wild-type AMPD1 +/+ alleles. CONCLUSIONS: After the onset of CHF symptoms, the mutant AMPD1 allele is associated with prolonged probability of survival without cardiac transplantation. The mechanism by which the presence of the mutant AMPD1 allele may modify the clinical phenotype of heart failure remains to be determined. The adenosine monophosphate deaminase locus (AMPD1) was selected for study. 2788608 Groot PC, Bleeker MJ, Pronk JC, Arwert F, Mager WH, Planta RJ, Eriksson AW, Frants RR: The human alpha-amylase multigene family consists of haplotypes with variable numbers of genes. Genomics. 1989 Jul;5(1):29-42. Polymorphic amylase protein patterns have suggested the presence in the human genome of various haplotypes encoding these allozymes. To investigate the genomic organization of the human alpha-amylase genes, we isolated the pertinent genes from a cosmid library constructed of DNA from an individual expressing three different salivary amylase allozymes. From the restriction maps of the overlapping cosmids and a comparison of these maps with the restriction enzyme patterns of DNA from the donor and family members, we were able to identify two haplotypes consisting of very different numbers of salivary amylase genes. The short haplotype contains two pancreatic genes (AMY2A and AMY2B) and one salivary amylase gene (AMY1C), arranged in the order 2B-2A-1C, encompassing a total length of approximately 100 kb. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes (AMY1A and AMY1B) and a pseudogene lacking the first three exons (AMYP1). The order of the amylase genes within the repeat is 1A-1B-P1. All genes are in a head-to-tail orientation except AMY1B, which has the reverse orientation with respect to the other genes. Analysis of somatic cell hybrids confirmed the presence of these short and long haplotypes. Furthermore, we present evidence for the existence of additional haplotypes in the human population and propose a general model for the evolution of the human alpha-amylase multigene family. A general designation 2B-2A-(1A-1B-P)n-1C can describe these haplotypes, n being 0 and 2 for the short and the long haplotypes presented in this paper, respectively. The long haplotype spans about 300 kb and contains six additional genes arranged in two repeats, each one consisting of two salivary amylase genes (AMY1A and AMY1B) and a pseudogene lacking the first three exons (AMYP1). 9894799 Riordan D, Dawson AJ: The evaluation of 15q proximal duplications by FISH. Clin Genet. 1998 Dec;54(6):517-21. Six patients from the clinical cytogenetics laboratory identified as having the normal variant dup(15)(q12) were further evaluated using fluorescence in situ hybridization (FISH). The purpose of this study was to ascertain whether any of the Prader Willi Angelman Chromosome Region (PWCR and ANCR, respectively) loci were duplicated in these patients. The results indicated that the patients could be categorized into two groups. The first group showed no duplication of the PWCR ANCR loci and appeared to belong to the dup(15)(q12) class which is phenotypically silent and is therefore called the normal variant. The second group also showed no duplication of the PWCR/ANCR loci. but had a large alpha satellite variant with D15Z. It is hypothesized that these patients do not have a duplication of 15q12 but rather a centromeric variant which mimics the dup(15)(q12). This study confirms the importance of evaluating apparent variations in the proximal region of chromosome 15 with FISH. The purpose of this study was to ascertain whether any of the Prader Willi Angelman Chromosome Region (PWCR and ANCR, respectively) loci were duplicated in these patients. 8943845 Adachi M, Sekiya M, Torigoe T, Takayama S, Reed JC, Miyazaki T, Minami Y, Taniguchi T, Imai K: Interleukin-2 (IL-2) upregulates BAG-1 gene expression through serine-rich region within IL-2 receptor beta c chain. Blood. 1996 Dec 1;88(11):4118-23. BAG-1 is a Bci-2-binding protein which functions in protection from apoptotic cell death. Here we provide evidence for interleukin-2 (IL-2)-mediated upregulation of BAG-1 expression. In hematopoietic cell line BAF-B03 F7 cells, gene transfer mediated expression of the IL-2R beta c chain is sufficient to confer proliferation and cell survival responses to IL-2. In these IL-2R beta c-expressing cells, BAG-1 mRNA was dramatically induced by IL-2. The IL-2-mediated induction of BAG-1 expression required the activation of tyrosine kinase(s) and was sensitive to rapamycin as the induction of bcl-2 expression was. Analysis of the transfectants which express mutant IL-2R beta c chains or mutant Janus family protein tyrosine kinase Jak3 lacking the kinase domain showed that the IL-2-mediated BAG-1 gene expression required the serinerich region within the IL-2R beta c chain, but Jak3 activation was dispensable. The signaling pathway for BAG-1 gene expression thus highly resembles that for bcl-2 gene expression, strongly suggesting that their induction shares the same signaling pathway. In addition, deletion of the serine-rich region led to loss of IL-2-mediated protection from apoptotic cell death. Taken together, these studies demonstrate that the serine-rich region of the IL-2R beta c chain mediates the coordinated expression of bcl-2 and BAG-1 genes, thereby contributing to suppression of apoptosis. In these IL-2R beta c-expressing cells, BAG-1 mRNA was dramatically induced by IL-2. 11454518 Barzon L, Chilosi M, Fallo F, Martignoni G, Montagna L, Palu G, Boscaro M: Molecular analysis of CDKN1C and TP53 in sporadic adrenal tumors. Eur J Endocrinol. 2001 Aug;145(2):207-12. OBJECTIVE: To evaluate the roles of the CDKN1C (P57KIP2) gene, which encodes for the cyclin-dependent kinase inhibitor CDNC, and the TP53 tumor suppressor gene in adrenal tumorigenesis, as a means of investigating the molecular basis of sporadic adrenal tumors, which is unknown. DESIGN: Screening for the presence CDKN1C and TP53 mutations and analyzing the expression pattern of CDNC, P53 and its downstream effector CDN1 (P21WAF1/CIP1) in a series of 79 sporadic adrenal tumors. METHODS: Single-strand conformation polymorphism and sequencing were used for mutation analysis of CDKN1C and TP53 in blood and adrenal tissue samples. In a subgroup of 48 tissues, CDKN1C expression was evaluated by RT-PCR and immunohistochemistry. Immunohistochemical analysis of P53 and CDN1 was performed. RESULTS: No somatic mutations of CDKN1C were found in the tumors analyzed, in spite of low/absent CDNC expression in adrenocortical adenomas and carcinomas. Mutations in the TP53 gene were present in 70% of adrenocortical carcinomas, associated with abnormal P53 and CDN1 expression, but not in benign neoplasms. In the normal adrenal cortex, CDNC expression was strictly nuclear and confined to the cortical zone (i.e. zona glomerulosa and reticularis), with no staining in the medulla. CONCLUSIONS: Mutations in the TP53 gene are frequent in adrenocortical carcinomas and might be used as a marker of malignancy. In the normal adrenal cortex, the zone-specific pattern of expression of CDNC suggests a role in adrenal differentiation. Immunohistochemical analysis of P53 and CDN1 was performed. 9039982 Beales PL, Warner AM, Hitman GA, Thakker R, Flinter FA: Bardet-Biedl syndrome: a molecular and phenotypic study of 18 families. J Med Genet. 1997 Feb;34(2):92-8. The autosomal recessive disorder Bardet-Biedl syndrome is characterised by retinal degeneration, polydactyly, obesity, mental retardation, hypogenitalism, renal dysplasia, and short stature. It is heterogeneous with at least four gene loci (BBS1-4) having been mapped to date. We have studied 18 multiply affected families noting the presence of both major and minor manifestations. Using a fluorescently based PCR technique, we genotyped each family member and assigned linkage to one of the four loci. Given this degree of heterogeneity we hoped to find phenotypic differences between linkage categories. We found 44% of families linked to 11q13 (BBS1) and 17% linked to 16q21 (BBS2). Only one family was linked to 15q22 (BBS4) and none to 3p12. We conclude that BBS1 is the major locus among white Bardet-Biedl patients and that BBS3 is extremely rare. Only subtle phenotypic differences were observed, the most striking of which was a finding of taller affected offspring compared with their parents in the BBS1 category. Affected subjects in the BBS2 and 4 groups were significantly shorter than their parents. Twenty eight percent of pedigrees did not show linkage to any known locus, evidence for at least a fifth gene. We conclude that the different genes responsible for Bardet-Biedl syndrome may influence growth characteristics such as height. We found 44% of families linked to 11q13 (BBS1) and 17% linked to 16q21 (BBS2). 10352685 King G, Hirst L, Holmes R: Human corneal and lens aldehyde dehydrogenases. Localization and function(s) of ocular ALDH1 and ALDH3 isozymes. Adv Exp Med Biol. 1999;463:189-98. Localization and function (s) of ocular ALDH1 and ALDH3 isozymes. 11790142 Izaguirre G, Pietruszko R, Cho S, MacKerell A Jr: Human aldehyde dehydrogenase catalytic activity and structural interactions with coenzyme analogs. J Biomol Struct Dyn. 2001 Dec;19(3):429-47. K(m) and V(max) values for 10 coenzyme analogs never previously studied with any aldehyde dehydrogenase and NADP(+) were compared with those for NAD(+) for three human aldehyde dehydrogenases (EC 1.2.1.3); the cytoplasmic E1 (the product of the aldh1 gene), the mitochondrial E2 (the product of the aldh2 gene) and the cytoplasmic E3 (the product of the aldh9 gene) isozymes. Structural information on changes in coenzyme-protein interactions were obtained via molecular dynamics (MD) studies with the E2 isozyme and quantum mechanical (QM) calculations were used to study changes in charge distribution of the pyridine ring and relative free energies of solvation of the purine ring in the analogs. E1 showed the broadest substrate specificity and was the only isozyme subject to substrate inhibition, both of which are suggested to be due to the two coenzyme conformations observed previously in the sheep crystal structure. NADP(+) selectivity is indicated to be influenced by Glu195 in E1 and E2. Substitutions in the purine ring affected K(m) but not V(max), with the changes in K(m) being dominated by the hydrophobicity of the purine ring as indicted by the QM calculations. Substitutions in the pyridine ring sometimes rendered the coenzymes inactive, with no consistent pattern observed for the three coenzymes. Structural analysis of the coenzyme analog-E2 MD simulations revealed different structural perturbations of the surrounding active site, though interactions with Asn169 and Glu399 were preserved in all cases. K (m) and V (max) values for 10 coenzyme analogs never previously studied with any aldehyde dehydrogenase and NADP (+) were compared with those for NAD (+) for three human aldehyde dehydrogenases (EC 1.2.1.3); the cytoplasmic E1 (the product of the aldh1 gene), the mitochondrial E2 (the product of the aldh2 gene) and the cytoplasmic E3 (the product of the aldh9 gene) isozymes. 10510318 O'connor T, Ireland LS, Harrison DJ, Hayes JD: Major differences exist in the function and tissue-specific expression of human aflatoxin B1 aldehyde reductase and the principal human aldo-keto reductase AKR1 family members. Biochem J. 1999 Oct 15;343 Pt 2:487-504. Complementary DNA clones encoding human aflatoxin B(1) aldehyde reductase (AKR7A2), aldehyde reductase (AKR1A1), aldose reductase (AKR1B1), dihydrodiol dehydrogenase 1 (AKR1C1) and chlordecone reductase (AKR1C4) have been expressed in Escherichia coli. These members of the aldo-keto reductase (AKR) superfamily have been purified from E. coli as recombinant proteins. The recently identified AKR7A2 was shown to differ from the AKR1 isoenzymes in being able to catalyse the reduction of 2-carboxybenzaldehyde. Also, AKR7A2 was found to exhibit a narrow substrate specificity, with activity being restricted to succinic semialdehyde (SSA), 2-nitrobenzaldehyde, pyridine-2-aldehyde, isatin, 1,2-naphthoquinone (1,2-NQ) and 9,10-phenanthrenequinone. In contrast, AKR1A1 reduces a broad spectrum of carbonyl-containing compounds, displaying highest specific activity for SSA, 4-carboxybenzaldehyde, 4-nitrobenzaldehyde, pyridine-3-aldehyde, pyridine-4-aldehyde, 4-hydroxynonenal, phenylglyoxal, methylglyoxal, 2,3-hexanedione, 1, 2-NQ, 16-ketoestrone and d-glucuronic acid. Comparison between the kinetic properties of AKR7A2 and AKR1A1 showed that both recombinant enzymes exhibited roughly similar k(cat)/K(m) values for SSA, 1,2-NQ and 16-ketoestrone. Many of the compounds which are substrates for AKR1A1 also serve as substrates for AKR1B1, though the latter enzyme was shown to display a specific activity significantly less than that of AKR1A1 for most of the aromatic and aliphatic aldehydes studied. Neither AKR1C1 nor AKR1C4 was found to possess high reductase activity towards aliphatic aldehydes, aromatic aldehydes, aldoses or dicarbonyls. However, unlike AKR1A1 and AKR1B1, both AKR1C1 and AKR1C4 were able to catalyse the oxidation of 1-acenaphthenol and, in addition, AKR1C4 could oxidize di- and tri-hydroxylated bile acids. Specific antibodies raised against AKR7A2, AKR1A1, AKR1B1, AKR1C1 and AKR1C4 have been used to show the presence of all of the reductases in human hepatic cytosol; the levels of AKR1B1 and AKR1C1 were markedly elevated in livers with alcohol-associated injury, and indeed AKR1B1 was only detectable in livers with evidence of alcoholic liver disease. Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues. These experiments revealed however, that the expression of AKR1A1 is restricted primarily to brain, kidney, liver and small intestine. The AKR1C family members proved not to be as widely expressed as the other reductases, with AKR1C1 being observed in only kidney, liver and testis, and AKR1C4 being found in liver alone. As human kidney is a rich source of AKR, the isoenzymes in this organ have been studied further. Anion-exchange chromatography of human renal cytosol on Q-Sepharose allowed resolution of AKR1A1, AKR1B1, AKR1C1 and AKR7A2, as identified by substrate specificity and Western blotting. Immunohistochemistry of human kidney demonstrated that AKR7A2 is expressed in a similar fashion to the AKR1 family members in proximal and distal convoluted renal tubules. Furthermore, both AKR7A2 and AKR1 members were expressed in renal carcinoma cells, suggesting that these groups of isoenzymes may be engaged in related physiological functions. Western blotting of extracts from brain, heart, kidney, liver, lung, prostate, skeletal muscle, small intestine, spleen and testis showed that AKR7A2 is present in all of the organs examined, and AKR1B1 is similarly widely distributed in human tissues. 12911785 Sayers I, Barton S, Rorke S, Sawyer J, Peng Q, Beghe B, Ye S, Keith T, Clough JB, Holloway JW, Sampson AP, Holgate ST: Promoter polymorphism in the 5-lipoxygenase (ALOX5) and 5-lipoxygenase-activating protein (ALOX5AP) genes and asthma susceptibility in a Caucasian population. Clin Exp Allergy. 2003 Aug;33(8):1103-10. BACKGROUND: 5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are essential for cysteinyl-leukotriene (cys-LT) production, critical mediators in asthma. OBJECTIVE: We sought to identify novel promoter polymorphisms within the FLAP (ALOX5AP) gene promoter and test the role of these and the previously identified 5-LO (ALOX5) Sp1 promoter polymorphism in asthma susceptibility. METHODS: To assess genetic association with asthma phenotypes, we genotyped 341 Caucasian families (containing two asthmatic siblings) and non-asthmatic control subjects (n=184). Genetic association was determined by case-control and transmission disequilibrium test (TDT) analyses. To determine the functional role of polymorphisms on basal transcription, we generated ALOX5AP-promoter-luciferase constructs and transiently transfected human HeLa cells. RESULTS: A novel G/A substitution at -336 bp and a poly(A) repeat (n=19 or 23) at position -169 to -146 bp were identified in the ALOX5AP promoter. Genotyping found the -336 A and poly(A19) alleles at frequencies of q=0.06 and 0.12, respectively. No ALOX5AP allele was associated with asthma or asthma-related phenotypes in case-control or TDT analyses. ALOX5AP-promoter-luciferase analyses did not support a functional role of the -336 or poly(A) polymorphism in determining basal transcription. The ALOX5 Sp1 polymorphism was predominantly homozygous wild-type 5/5 (frequency q=0.70) and heterozygous 4/5 (q=0.23) genotypes and no allele was associated with asthma or asthma-related phenotypes. CONCLUSION: Taken together, these data do not support a significant role for these polymorphisms in genetic susceptibility to asthma in the Caucasian population. Promoter polymorphism in the 5-lipoxygenase (ALOX5) and 5-lipoxygenase-activating protein (ALOX5AP) genes and asthma susceptibility in a Caucasian population. 1670751 Asher JH Jr, Morell R, Friedman TB: Waardenburg syndrome (WS): the analysis of a single family with a WS1 mutation showing linkage to RFLP markers on human chromosome 2q. Am J Hum Genet. 1991 Jan;48(1):43-52. Waardenburg syndrome type I (WS1; MIM 19350) is caused by a pleiotropic, autosomal dominant mutation with variable penetrance and expressivity. Of individuals with this mutation, 20%-25% are hearing impaired. A multilocus linkage analysis of RFLP data from a single WS1 family with 11 affected individuals indicates that the WS1 mutation in this family is linked to the following four marker loci located on the long arm of chromosome 2: ALPP (alkaline phosphatase, placental), FN1 (fibronectin 1), D2S3 (a unique-copy DNA segment), and COL6A3 (collagen VI, alpha 3). For the RFLP marker loci, a multilocus linkage analysis using MLINK produced a peak lod (Z) of 3.23 for the following linkage relationships and recombination fractions (theta i): (ALPP----.000----FN1)----.122----D2S3----.267----CO L6A3. A similar analysis produced a Z of 6.67 for the following linkage relationships and theta i values among the markers and WS1: (FN1----.000----WS1----.000----ALPP)----.123----D2S 3----.246----COL6A3. The data confirm the conclusion of Foy et al. that at least some WS1 mutations map to chromosome 2q. A multilocus linkage analysis of RFLP data from a single WS1 family with 11 affected individuals indicates that the WS1 mutation in this family is linked to the following four marker loci located on the long arm of chromosome 2: ALPP (alkaline phosphatase, placental), FN1 (fibronectin 1), D2S3 (a unique-copy DNA segment), and COL6A3 (collagen VI, alpha 3). 11807986 Wimmer K, Zhu Xx XX, Rouillard JM, Ambros PF, Lamb BJ, Kuick R, Eckart M, Weinhausl A, Fonatsch C, Hanash SM: Combined restriction landmark genomic scanning and virtual genome scans identify a novel human homeobox gene, ALX3, that is hypermethylated in neuroblastoma. Genes Chromosomes Cancer. 2002 Mar;33(3):285-94. Restriction landmark genome scanning (RLGS) allows comparative analysis of several thousand DNA fragments in the genome and provides a means to identify CpG islands that are altered in tumor cells as a result of amplification, deletion, or methylation changes. We have developed a novel informatics tool, designated virtual genome scan (VGS), that makes it possible to predict automatically the sequence of fragments in RLGS patterns by matching to the human genome sequence. A combination of RLGS and VGS was utilized to identify changes of chromosome 1-derived fragments in neuroblastoma. A NotI-EcoRV fragment was found to be absent frequently in neuroblastoma cell line RLGS patterns. Sequence prediction by VGS as well as cloning of the fragment showed that it contained a CpG island that is part of the human orthologue of the hamster homeobox gene Alx3. Expression analysis in a panel of human and mouse tissues showed predominant expression of ALX3 in brain tissue. Methylation-sensitive sequence analysis of the promoter region in neuroblastoma cell lines indicated that methylation of specific sequences correlated with repression of the ALX3 gene. Expression was re-induced after treatment with the methylation inhibitor 5-aza-2'-deoxycytidine. Promoter methylation analysis of ALX3 in primary neuroblastoma tumors, using methylation-sensitive polymerase chain reaction, found preferential ALX3 methylation in advanced-stage tumors. The VGS approach we have implemented in combination with RLGS is useful for the identification of genomic CpG island-related methylation changes or deletions in cancer. Combined restriction landmark genomic scanning and virtual genome scans identify a novel human homeobox gene, ALX3, that is hypermethylated in neuroblastoma. 12777383 Luciakova K, Barath P, Poliakova D, Persson A, Nelson BD: Repression of the human adenine nucleotide translocase-2 gene in growth-arrested human diploid cells: the role of nuclear factor-1. J Biol Chem. 2003 Aug 15;278(33):30624-33. Epub 2003 May 30. Adenine nucleotide translocase-2 (ANT2) catalyzes the exchange of ATP for ADP across the mitochondrial membrane, thus playing an important role in maintaining the cytosolic phosphorylation potential required for cell growth. Expression of ANT2 is activated by growth stimulation of quiescent cells and is down-regulated when cells become growth-arrested. In this study, we address the mechanism of growth arrest repression. Using a combination of transfection, in vivo dimethyl sulfate mapping, and in vitro DNase I mapping experiments, we identified two protein-binding elements (Go-1 and Go-2) that are responsible for growth arrest of ANT2 expression in human diploid fibroblasts. Proteins that bound the Go elements were purified and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as members of the NF1 family of transcription factors. Chromatin immunoprecipitation analysis showed that NF1 was bound to both Go-1 and Go-2 in quiescent human diploid cells in vivo, but not in the same cells stimulated to growth by serum. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. Furthermore, overexpression of NF1-A, -C, and -X in NIH3T3 cells repressed expression of an ANT2-driven reporter gene construct. Two additional putative repressor elements in the ANT2 promoter, an Sp1 element juxtaposed to the transcription start site and a silencer centered at nucleotide -332, did not appear to contribute to growth arrest repression. Thus, enhanced binding of NF1 is a key step in the growth arrest repression of ANT2 transcription. To our knowledge, this is the first report showing a role for NF1 in growth arrest. NF1 binding correlated with the disappearance of ANT2 transcripts in quiescent cells. 9503022 Morgan RO, Bell DW, Testa JR, Fernandez MP: Genomic locations of ANX11 and ANX13 and the evolutionary genetics of human annexins. Genomics. 1998 Feb 15;48(1):100-10. We have reconstructed a molecular genetic history of human annexins to chronicle their origins and dispersal throughout the genome. This involved the completion of chromosomal mapping, determination of ancestral relationships, and estimation of gene duplication dates. Fluorescence in situ hybridization localized human annexin XI (ANX11) to 10q22.3-q23.1 and annexin XIII (ANX13) to 8q24.1-q24.2. Orthologous annexins showed minor rate variation when calibrated to species separation times given by the fossil record, but paralogous subfamilies have diverged at fivefold variable rates. The rates and extents of sequence divergence were used to predict a mean separation time of 450 million years between vertebrate annexins, although their common ancestor may have emanated from invertebrate stock. Annexins XIII and VII formed a phylogenetically early clade, and annexins II and VIa were the most divergent members of two distinct clades. ANX6 may have been created by tandem duplication about 500 million years ago (Mya) and duplicated again to form ANX5 400 Mya, whereas ANX4 and ANX8 are proposed to be sequential duplication products from annexin XI. Vertebrate annexins thus proliferated via a cascade of gene duplications in higher metazoa to form at least three diverging groups of ubiquitous and structurally related genes. These can be distinguished by their dispersed genomic locations as well as their individual patterns of expression and partially differentiated functions. Genomic locations of ANX11 and ANX13 and the evolutionary genetics of human annexins. 9479504 McLennan AG, Flannery AV, Morten JE, Ridanpaa M: Chromosomal localization of the human diadenosine 5',5"'-P1,P4-tetraphosphate pyrophosphohydrolase (Ap4A hydrolase) gene (APAH1) to 9p13. Genomics. 1998 Jan 15;47(2):307-9. Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) hydrolase is the enzyme responsible for maintaining the intracellular level of the dinucleotide Ap4A, the function of which has yet to be established. The APAH1 gene encoding this Ap4A hydrolase has been mapped by fluorescence in situ hybridization and PCR to human chromosome 9p13. Radiation hybrid panel mapping further located APAH1 between the IL11RA and the GALT genes, thus excluding it as a candidate gene for cartilage-hair hypoplasia, which maps proximal to GALT. Several tumor suppressor genes have previously been mapped within the 9p13-p21 region. Given that the FHIT gene at 3p14.2, which encodes a diadenosine 5',5"'-P1,P3-triphosphate (AP3A) hydrolase, is a candidate tumor suppressor, APAH1 should also be considered a potential tumor suppressor. Given that the FHIT gene at 3p14.2, which encodes a diadenosine 5',5"'-P1,P3-triphosphate (AP3A) hydrolase, is a candidate tumor suppressor, APAH1 should also be considered a potential tumor suppressor. 8855266 Guenette SY, Chen J, Jondro PD, Tanzi RE: Association of a novel human FE65-like protein with the cytoplasmic domain of the beta-amyloid precursor protein. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10832-7. We identified a novel human homologue of the rat FE65 gene, hFE65L, by screening the cytoplasmic domain of beta-amyloid precursor protein (beta PP) with the "interaction trap." The cytoplasmic domains of the beta PP homologues, APLP1 and APLP2 (amyloid precursor-like proteins), were also tested for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. We confirmed these interactions in vivo by successfully coimmunoprecipatating endogenous beta PP and APLP2 from mammalian cells overexpressing a hemagglutinin-tagged fusion of the C-terminal region of hFE65L. We report the existence of a human FE65 gene family and evidence supporting specific interactions between members of the beta PP and FE65 protein families. Sequence analysis of the FE65 human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. Our data show that a single PI domain is sufficient for binding of hFE65L to the cytoplasmic domain of beta PP and APLP2. The PI domain of the protein, Shc, is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2. Thus, it is likely that the PI domains present in the C-terminal moiety of the hFE65L protein bind the NPXY motif located in the cytoplasmic domain of beta PP and APLP2. 7789170 Maric SC, Crozat A, Louhimo J, Knuutila S, Janne OA: The human S-adenosylmethionine decarboxylase gene: nucleotide sequence of a pseudogene and chromosomal localization of the active gene (AMD1) and the pseudogene (AMD2). Cytogenet Cell Genet. 1995;70(3-4):195-9. S-adenosylmethionine decarboxylase (AdoMet-DC) is a key enzyme in polyamine biosynthesis. The human genome contains at least two loci for the AdoMetDC gene (AMD), one of which (AMD1) has previously been mapped to chromosome 6 and the other (AMD2) to the X chromosome. The locus on chromosome 6 is the transcriptionally active gene. We now report characterization of the AMD2 locus (GenBank Accession No. U02035) on the X chromosome, which contains sequences that cross-hybridize with human AdoMetDC cDNA. This DNA lacks all of the introns present in AMD1 and has numerous mutations in the protein-coding region. Its overall nucleotide sequence identity with AdoMetDC cDNA is about 90%. AMD2 is therefore a processed pseudogene, which, because of multiple mutations, cannot be translated to an active AdoMetDC enzyme, even if it were transcribed. Chromosomal loci for human AMD sequences were determined by in situ hybridization to metaphase chromosomes, with genomic DNAs from the active gene and the pseudogene loci as probes. AMD1 was localized to chromosome region 6q21-->q22 and AMD2 to band Xq28. AMD1 was localized to chromosome region 6q21--> q22 and AMD2 to band Xq28. 2982946 Suzuki T, Butler JL, Cooper MD: Human B cell responsiveness to B cell growth factor after activation by phorbol ester and monoclonal anti-mu antibody. J Immunol. 1985 Apr;134(4):2470-6. The effect of phorbol ester on human B cell activation was examined. Picomolar to nanomolar concentrations of phorbol ester induced a high level of proliferation in small IgM-positive B cells isolated from peripheral blood by fluorescence-activated cell sorting. The addition of optimal doses of anti-mu antibody resulted in enhanced proliferation of phorbol ester-activated B cells. The addition of B cell growth factor (BCGF) to phorbol ester-activated B cells also resulted in a dose-dependent synergistic effect and maximal enhancement on day 3. BCGF activity could be absorbed with either phorbol ester- or anti-mu-activated B cells, but not with resting B cells, thus confirming the induction of functional BCGF receptor expression. Cell proliferation was not necessary for the induction of functional BCGF receptors. Phorbol ester was a more efficient inducer of BCGF receptor expression than was anti-mu antibody; gamma-interferon treatment had no effect. BCGF enhanced transferrin receptor expression by phorbol ester-activated B cells. The results suggest that phorbol ester-activated small B cells can be used to monitor BCGF activity, and this synergistic combination may be useful in establishing BCGF-dependent B cell clones in culture. BCGF enhanced transferrin receptor expression by phorbol ester-activated B cells. 12524539 Fan Z, Beresford PJ, Zhang D, Xu Z, Novina CD, Yoshida A, Pommier Y, Lieberman J: Cleaving the oxidative repair protein Ape1 enhances cell death mediated by granzyme A. Nat Immunol. 2003 Feb;4(2):145-53. Epub 2003 Jan 13. The cytolytic T lymphocyte protease granzyme A (GzmA) initiates a caspase-independent cell death pathway. Here we report that the rate-limiting enzyme of DNA base excision repair, apurinic endonuclease-1 (Ape1), which is also known as redox factor-1 (Ref-1), binds to GzmA and is contained in the SET complex, a macromolecular complex of 270-420 kDa that is associated with the endoplasmic reticulum and is targeted by GzmA during cell-mediated death. GzmA cleaves Ape1 after Lys31 and destroys its known oxidative repair functions. In so doing, GzmA may block cellular repair and force apoptosis. In support of this, cells with silenced Ape1 expression are more sensitive, whereas cells overexpressing noncleavable Ape1 are more resistant, to GzmA-mediated death. GzmA cleaves Ape1 after Lys31 and destroys its known oxidative repair functions. 11269478 Kara M, Ohta Y, Tanaka Y, Oda M, Watanabe Y: Autocrine motility factor receptor expression in patients with stage I non-small cell lung cancer. Ann Thorac Surg. 2001 Mar;71(3):944-8. BACKGROUND: Expression of autocrine motility factor receptor (AMFR) associates with increased cell migration and poor survival in certain types of human cancers. We assessed the possible correlation between AMFR, clinicopathologic features, and survival in stage I non-small cell lung cancer (NSCLC). METHODS: AMFR expression was analyzed immunohistochemically, using a monoclonal antibody (3F3A) in tumor specimens from 97 patients with curative resection. Vascular endothelial growth factor (VEGF) expression was also examined after accounting for AMFR expression. RESULTS: Out of 97 tumors, 38 (39.2%) were positively stained with AMFR. The AMFR expression was significantly associated with histologic type of tumor, mainly in adenocarcinoma. Overall survival of patients with AMFR-positive tumors was significantly worse than that of AMFR-negative tumors (p = 0.0050). The AMFR expression appears to be associated with VEGF expression. Patients who were AMFR positive and had high VEGF expression had a worse prognosis compared with the AMFR-negative and low VEGF-expression group (p < 0.0001). Multivariate analysis revealed an independent prognostic impact of AMFR on survival (p = 0.0039). CONCLUSIONS: These results indicate that evaluation of AMFR expression may provide useful guidance in follow-up of patients with NSCLC. The AMFR expression appears to be associated with VEGF expression. 14517957 Henneke M, Flaschker N, Helbling C, Muller M, Schadewaldt P, Gartner J, Wendel U: Identification of twelve novel mutations in patients with classic and variant forms of maple syrup urine disease. Hum Mutat. 2003 Nov;22(5):417. Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder of panethnic distribution caused by a deficiency of the activity of branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. Mutations in the human BCKD genes E1alpha (BCKDHA), E1beta (BCKDHB) and E2 (DBT) are known to result in MSUD, referred to as type Ia, Ib and II mutations respectively. In this study 16 patients with the classic severe form of MSUD and three patients with milder variant forms of the disease were investigated for mutations in the E1alpha-, E1beta- and E2-gene by single-strand conformation polymorphism (SSCP) analysis and DNA sequencing. The patients' clinical and biochemical phenotypes were well characterized. One novel type Ia missense mutation, eight novel type Ib (three missense, two nonsense, two small deletions, one small duplication) and three novel type II (two missense, one splice site) mutations were identified in patients. Moreover, eleven previously described mutations were detected: five type Ia (four missense, one nonsense), three type Ib mutations (two missense, one nonsense) and three type II mutations (two missense, one small deletion). Fourteen patients are homozygous for one single mutation, five patients are compound-heterozygous for two different mutations affecting one of the three genes. Thus, in all 19 patients the identified mutations can most probably be considered the molecular basis of the disease. Mutations in the human BCKD genes E1alpha (BCKDHA), E1beta (BCKDHB) and E2 (DBT) are known to result in MSUD, referred to as type Ia, Ib and II mutations respectively. 10460257 Homayouni R, Rice DS, Sheldon M, Curran T: Disabled-1 binds to the cytoplasmic domain of amyloid precursor-like protein 1. J Neurosci. 1999 Sep 1;19(17):7507-15. Disruption of the disabled-1 gene (Dab1) results in aberrant migration of neurons during development and disorganization of laminar structures throughout the brain. Dab1 is thought to function as an adapter molecule in signal transduction processes. It contains a protein-interaction (PI) domain similar to the phosphotyrosine-binding domain of the Shc oncoprotein, it is phosphorylated by the Src protein tyrosine kinase, and it binds to SH2 domains in a phosphotyrosine-dependent manner. To investigate the function of Dab1, we searched for binding proteins using the yeast two-hybrid system. We found that the PI domain of Dab1 interacts with the amyloid precursor-like protein 1 (APLP1). The association of Dab1 with APLP1 was confirmed in biochemical assays, and the site of interaction was localized to a cytoplasmic region of APLP1 containing the amino acid sequence motif Asn-Pro-x-Tyr (NPxY). NPxY motifs are involved in clathrin-mediated endocytosis, and they have been shown to bind to PI domains present in several proteins. This region of APLP1 is conserved among all members of the amyloid precursor family of proteins. Indeed, we found that Dab1 also interacts with amyloid precursor protein (APP) and APLP2 in biochemical association experiments. In transiently transfected cells, Dab1 and APLP1 colocalized in membrane ruffles and vesicular structures. Cotransfection assays in cultured cells indicated that APP family members increased serine phosphorylation of Dab1. Dab1 and APLP1 are expressed in similar cell populations in developing and adult brain tissue. These results suggest that Dab1 may function, at least in part, through association with APLP1 in the brain. We found that the PI domain of Dab1 interacts with the amyloid precursor-like protein 1 (APLP1). 10748223 Slepnev VI, Ochoa GC, Butler MH, De Camilli P: Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites. J Biol Chem. 2000 Jun 9;275(23):17583-9. Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. 649160 Teng YS, Tan SG, Lopez CG, Ng T, Lie-Injo LE: Genetic markers in Malaysians: variants of soluble and mitochondrial glutamic oxaloacetic transaminase and salivary and pancreatic amylase, phosphoglucomutase III and saliva esterase polymorphisms. Hum Genet. 1978 Apr 24;41(3):347-54. Malaysians of Malay, Chinese, and Indian ancestries were electrophoretically phenotyped for Amy1 and saliva esterase region 1 (Set-1) from saliva, Amy2 from plasma, soluble and mitochondrial GOT and PGM3 from leukocyte and placenta. Kadazans and Bajaus, the indigenous people of Sabah, East Malaysia were surveyed for Amy2. Three types of variants were observed for Amy1, one type for Amy2. Only Indians were found to be polymorphic for Amy1. Two GOTs 2-1 and three GOTm 2-1 variants were found among 281 Chinese while three GOTm 2-1 variants were found among 311 Malays. Malaysian Malays, Chinese, and Indians were found to be polymorphic for Set-1 and PGM3. The gene frequencies in Malays are Set-1F=0.601 +/- 0.021, Set-1S = 0.399 +/- 0.021; PGM13 = 0.788 +/- 0.020, PGM23 = 0.212 +/- 0.020; in Chinese Set-1F = 0.497 +/- 0.028, Set-1S = 0.503 +/- 0.028; PGM13 = 0.745 +/- 0.24, PGM23 = 0.255 +/- 0.024; in Indians, Set-1F = 0.449 +/- 0.031, Set-1S = 0.551 +/- 0.031; PGM13 = 0.755 +/- 0.029, PGM23 = 0.245 +/- 0.029. Three types of variants were observed for Amy1, one type for Amy2. 8633065 Nobile V, Vallee BL, Shapiro R: Characterization of mouse angiogenin-related protein: implications for functional studies on angiogenin. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4331-5. Angiogenin-related protein (Angrp), the putative product of a recently discovered mouse gene, shares 78% sequence identity with mouse angiogenin (Ang). In the present study, the relationship of Angrp to Ang has been investigated by producing both proteins in bacteria and comparing their functional properties. We find that mouse Ang is potently angiogenic, but Angrp is not, even when assayed at relatively high doses. A deficiency in catalytic capacity, which is essential for the biological activity of Ang, does not appear to underlie Angrp's lack of angiogenicity. In fact, Angrp has somewhat greater ribonucleolytic activity toward tRNA and dinucleotide substrates than does Ang. Instead, an inability to bind cellular receptors is implicated since Angrp does not inhibit Ang-induced angiogenesis. Poor conservation of the Ang receptor recognition sequence 58-69 in Angrp most likely contributes to this defect. However, other substitutions must also influence receptor binding since an Angrp quadruple mutant that is identical to Ang in this segment still lacks both angiogenic activity and the capacity to inhibit Ang. The functional differences between Ang and Angrp, together with evidence presented herein that Angrp is regulated differently than Ang, suggest that the roles of the two proteins in vivo may be quite distinct. The functional differences between Ang and Angrp, together with evidence presented herein that Angrp is regulated differently than Ang, suggest that the roles of the two proteins in vivo may be quite distinct. 8159688 Devarajan P, Scaramuzzino DA, Morrow JS: Ankyrin binds to two distinct cytoplasmic domains of Na,K-ATPase alpha subunit. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2965-9. Ankyrin has emerged as a ubiquitous protein linking integral membrane transport proteins such as Na,K-ATPase to an underlying spectrin cytoskeleton. This interaction is mediated by the alpha subunit of Na,K-ATPase; however, the nature of the ankyrin binding site in Na,K-ATPase is unknown. As a step to determine the mechanism of this interaction, the ankyrin binding region of human erythrocyte spectrin and each of five putative cytoplasmic domains of the Na,K-ATPase alpha subunit have been prepared as recombinant fusion proteins in bacteria and analyzed for their interaction with erythrocyte and kidney ankyrin (Ank1 and Ank3, respectively) in vitro. Spectrin binds both Ank1 and Ank3 avidly, as expected. Two of the Na,K-ATPase domains, immobilized on a bioaffinity column, also interact specifically with both of these ankyrins. These ATPase domains are encoded by codons 140-290 (domain II) and 345-784 (domain III), with domain II displaying the greatest apparent affinity. Sequences in domain II are highly conserved between species and isoforms of Na,K-ATPase and are homologous to a cytoplasmic domain in H,K-ATPase and to a limited region of sequence in Ca-ATPase. Conversely, domain II shares no significant homology with other ankyrin binding proteins such as band 3 and Na(+)-channel proteins. These results identify a clear function for a conserved but previously not understood region of the alpha subunit of Na,K-ATPase and suggest that the interaction of ankyrin with membrane transport proteins may involve complex tertiary structural determinants not easily deduced from the primary sequence. Spectrin binds both Ank1 and Ank3 avidly, as expected. 8490661 Schiebel K, Weiss B, Wohrle D, Rappold G: A human pseudoautosomal gene, ADP/ATP translocase, escapes X-inactivation whereas a homologue on Xq is subject to X-inactivation. Nat Genet. 1993 Jan;3(1):82-7. We report the cloning of a highly conserved pseudoautosomal gene on the human sex chromosomes. A cDNA clone was selected by crosshybridization with a microdissected clone from the chromosomal subregion Xp22.3. It encodes a previously characterized member of the ADP/ATP translocase family and plays a fundamental role in cellular energy metabolism. This gene, ANT3, is located approximately 1,300 kilobases from the telomere, proximal to the pseudoautosomal gene CSF2RA, and escapes X-inactivation. Interestingly, a homologue of ANT3, ANT2, maps to Xq and is subject to X-inactivation. These genes provide the first evidence of two closely related X-chromosomal genes, which show striking differences in their X-inactivation behaviour. Interestingly, a homologue of ANT3, ANT2, maps to Xq and is subject to X-inactivation. 12600746 Ogawa M, Hiraoka Y, Aiso S: The Polycomb-group protein ENX-2 interacts with ZAP-70. Immunol Lett. 2003 Mar 3;86(1):57-61. Human ENX-2 is a homologue of Drosophila Enhancer of zeste, which is a member of Polycomb-group proteins regulating the expression of homeotic genes as chromatin-associated proteins. In this study, we demonstrate that ENX-2 plays an important role as a signaling molecule involved in T cell receptor-mediated signaling pathway. In immunoprecipitation experiments, ENX-2 and zeta associated protein-70 (ZAP-70) were co-precipitated from T cell lysate. When probed with an anti-phospho-tyrosine antibody, ENX-2 was found to be phosphorylated on tyrosine. On the other hand, ENX-2 was not phosphorylated on tyrosine in the mutant Jurkat cell, J.Cam1.6 lacking the activity of lymphocyte protein tyrosine kinase p56(lck). The interaction between ENX-2 and ZAP-70 was abolished in the mutant cell. Furthermore, in-vitro kinase assay using purified p56(lck) demonstrated that ENX-2 became tyrosine phosphorylated by this kinase. These findings show that the phosphorylation of ENX-2 is responsible for the interaction between ENX-2 and ZAP-70. The Polycomb-group protein ENX-2 interacts with ZAP-70. 2193101 Kovach NL, Yee E, Munford RS, Raetz CR, Harlan JM: Lipid IVA inhibits synthesis and release of tumor necrosis factor induced by lipopolysaccharide in human whole blood ex vivo. J Exp Med. 1990 Jul 1;172(1):77-84. Tumor necrosis factor (TNF) released by lipopolysaccharide (LPS)-stimulated mononuclear phagocytes is a critical mediator of sepsis. We examined the capacities of rough mutant Salmonella typhimurium LPS (Rc) and LPS partial structures lipid A, monophosphoryl lipid A (MPLA), lipid IVA, and lipid X to induce production of TNF in whole blood. Rc LPS (0.0001-10 ng/ml) produced a dose-dependent release of TNF as determined by cytotoxicity of actinomycin D-sensitized L929 murine fibroblasts. Lipid A, MPLA, lipid IVA, and lipid X exhibited decreasing capacities to stimulate production of TNF in whole blood, respectively. Fractional deacylation of LPS by incubation with acyloxyacyl hydrolase isolated from human leukocytes produced a reduction in the capacity of LPS to induce TNF release in whole blood. Maximal enzymatic deacylation reduced activity of LPS by greater than 100-fold. Coincubation with lipid IVA inhibited TNF release induced by Rc LPS or lipid A, but not by phorbol ester. In contrast, MPLA, lipid X, and deacylated LPS failed to inhibit LPS-stimulated release of TNF. Corresponding to the inhibition of the release of TNF protein, lipid IVA also inhibited the accumulation of TNF mRNA in LPS-stimulated mononuclear cells. These results suggest that lipid IVA may act as a competitive antagonist of LPS, perhaps at the receptor level. Fractional deacylation of LPS by incubation with acyloxyacyl hydrolase isolated from human leukocytes produced a reduction in the capacity of LPS to induce TNF release in whole blood. 9880564 Wang X, Driscoll DM, Morton RE: Molecular cloning and expression of lipid transfer inhibitor protein reveals its identity with apolipoprotein F. J Biol Chem. 1999 Jan 15;274(3):1814-20. Published studies demonstrate that lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein (CETP) activity. Although LTIP inhibits CETP activity among different lipoprotein classes, it preferentially suppresses transfer events involving low density lipoprotein (LDL), whereas transfers involving high density lipoprotein as donor are less affected. In this study, we report the purification of LTIP and the expression of its cDNA in cultured cells. Purification of LTIP, in contrast to other published protocols, took advantage of the tight association of this protein with LDL. Ultracentrifugally isolated LDL was further purified on anti-apoE and apoA-I affinity columns. Affinity purified LDL was delipidated by tetramethylurea, and the tetramethylurea-soluble proteins were separated by SDS-polyacrylamide gel electrophoresis. The protein migrating at a molecular mass of approximately 33 kDa was excised from the gel and its N-terminal amino acid sequence determined. The 14-amino acid sequence obtained showed complete homology with the sequence deduced for apolipoprotein F (apoF) cDNA isolated from Hep G2 cells. On Western blots, peptide-specific antibodies raised against synthetic fragments of apoF reacted with the same 33-kDa protein in LTIP-containing fractions purified from LDL and from lipoprotein-deficient plasma. In contrast to that previously reported, apoF was shown to be associated almost exclusively with LDL, identical to the distribution of LTIP activity. The cDNA for apoF was cloned from a human liver cDNA library, ligated into a mammalian expression vector, and transiently transfected into COS-7 cells. Conditioned media containing secreted apoF demonstrated CETP inhibitor activity, whereas cells transfected with vector alone did not. This CETP inhibitor activity was efficiently removed from the media by nickel-Sepharose, consistent with the 6-His tag incorporated into recombinant apoF. By Western blot, the 6-His-tagged protein had a molecular weight slightly larger than native apoF. The CETP inhibitor activity of recombinant apoF possessed the same LDL specificity, oleate sensitivity, and dependence on lipoprotein concentration as previously noted for LTIP. We conclude that LTIP and apoF are identical. Conditioned media containing secreted apoF demonstrated CETP inhibitor activity, whereas cells transfected with vector alone did not. 12618593 Hein DW, Doll MA, Xiao GH, Feng Y: Prostate expression of N-acetyltransferase 1 (NAT1) and 2 (NAT2) in rapid and slow acetylator congenic Syrian hamster. Pharmacogenetics. 2003 Mar;13(3):159-67. Arylamine carcinogens induce prostate tumours in rodent models and may contribute to the aetiology of human prostate cancers. N-acetylation and O-acetylation, catalysed by N-acetyltransferase 1 (NAT1) and 2 (NAT2), activate and/or deactivate arylamines to electrophilic intermediates that bind DNA and initiate tumours in target organs. NAT1 and NAT2 are both subject to genetic polymorphism in humans, and molecular epidemiological investigations suggest that NAT1 and/or NAT2 acetylator genotype modifies risk for prostate cancers. A Syrian hamster model congenic at the NAT2 locus was used to investigate the role of acetylator genotype in N- and O-acetylation of aromatic and heterocyclic amine carcinogens in the liver and prostate. A gene dose-response (NAT2*15/*15>NAT2*15/*16A>NAT2*16A/*16A) relationship was observed in liver and prostate cytosol towards the N-acetylation of p-aminobenzoic acid, 2-aminofluorene, beta-napthylamine, 4-aminobiphenyl, and 3,2'-dimethyl-4-aminobiphenyl. NAT1 and NAT2 were separated and partially purified from liver and prostate cytosol. NAT1 and NAT2 in liver and prostate catalysed -acetylation of the arylamines above and O-acetylation of N-hydroxy derivatives of 2-aminofluorene, 4-aminobiphenyl and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. Rates were higher in rapid versus slow acetylators when catalysed by NAT2 but not when catalysed by NAT1. Partially purified prostate NAT2 exhibited higher apparent K(m) and V(max) than NAT1. Prostate NAT1 mRNA levels were higher than NAT2 and neither NAT1 nor NAT2 mRNA level differed with NAT2 acetylator genotype. The results provide mechanistic support for a role of NAT1 and/or NAT2 acetylator polymorphism(s) in human prostate cancer risk related to aromatic and/or heterocyclic amine carcinogens. Rates were higher in rapid versus slow acetylators when catalysed by NAT2 but not when catalysed by NAT1. 8871610 Zhang X, Li L, Choe J, Krajewski S, Reed JC, Thompson C, Choi YS: Up-regulation of Bcl-xL expression protects CD40-activated human B cells from Fas-mediated apoptosis. Cell Immunol. 1996 Oct 10;173(1):149-54. CD40--CD40L interactions between resting B cells and activated T cells are essential for germinal center formation. It has been shown that CD40L can induce both Fas expression and susceptibility to Fas-mediated killing in B cells, while anti-Ig can partially rescue B cells from Fas-mediated killing. However, the intracellular mechanism for this phenomenon is not known. We examined the expression of Fas and bcl-2 family gene products, such as Bcl-2, Bcl-x, Bax, and Mcl-1, in human tonsillar B cells. The activation of naive B cells by CD40L induced transient expression of Bcl-xL. As the Bcl-xL level decreased in CD40-activated B cells, the cells became susceptible to apoptosis by anti-Fas antibodies. Though anti-Ig did not change the Fas expression, it protected CD40-activated B cells from Fas-mediated killing by up-regulating Bcl-xL expression. The addition of anti-Ig did not significantly change Bcl-2, Bax, and Mcl-1 levels compared to those of B cells activated by CD40L alone. The activation of naive B cells by CD40L induced transient expression of Bcl-xL. 12160729 Dyomin VG, Chaganti SR, Dyomina K, Palanisamy N, Murty VV, Dalla-Favera R, Chaganti RS: BCL8 is a novel, evolutionarily conserved human gene family encoding proteins with presumptive protein kinase A anchoring function. Genomics. 2002 Aug;80(2):158-65. BCL8 is a novel human gene family initially identified through cloning of BCL8A, located at the t(14;15)(q32;q11-q13) translocation breakpoint, in a case of diffuse large B-cell lymphoma. Multiple copies of BCL8A map to the 1-Mb proximal duplicated region at 15q. We identified additional copies on human chromosomes 13 (BCL8B), 22 (BCL8C), 2 (BCL8D), and 10 (BCL8E) by cDNA cloning and sequence analysis. BCL8A, BCL8C, BCL8D, and BCL8E are truncated at the genomic level and are presumably pseudogenes or sterile transcripts. BCL8B is expressed predominantly in human brain and encodes a 327-kDa protein with extensive homology to the Drosophila melanogaster protein kinase A anchoring protein RG. LRBA, a human gene with a ubiquitous expression pattern mapping to 4q32, encodes a protein closely related to BCL8. The phylogenetically conserved BCL8 gene family evolved by transchromosomal and intrachromosomal duplications within the human genome. LRBA, a human gene with a ubiquitous expression pattern mapping to 4q32, encodes a protein closely related to BCL8. 10326698 Noguchi S, Kasugai T, Miki Y, Fukutomi T, Emi M, Nomizu T: Clinicopathologic analysis of BRCA1- or BRCA2-associated hereditary breast carcinoma in Japanese women. Cancer. 1999 May 15;85(10):2200-5. BACKGROUND: The purpose of this investigation was to elucidate the clinicopathologic characteristics of BRCA1- and BRCA2-associated hereditary breast carcinomas (HBCs) in Japanese women. METHODS: Various clinicopathologic characteristics of HBCs arising in patients with BRCA1 or BRCA2 germline mutations were compared with those of the control group (sporadic breast carcinomas). RESULTS: The mean age at the time of diagnosis of BRCA1-associated HBCs and that of BRCA2-associated HBCs (44 years for both) were significantly younger than that of the control group (54 years) and the incidence of bilateral tumors was significantly higher in the BRCA1-associated HBCs (32%) and BRCA2-associated HBCs (29%) than in the control group (6%). BRCA1-associated HBCs showed a tendency (P = 0.06) toward an increase in solid-tubular type tumors and a significant increase in histologic grade 3 tumors (P < 0.01) and lymphatic invasion positive tumors (P < 0.05) compared with the control group. BRCA1-associated HBCs were significantly more estrogen receptor negative (P < 0.01), c-erb B-2 negative (P < 0.05), and p53 positive (P < 0.01), and they also showed a significant increase in MIB-1 staining grades (P < 0.01) as well as microvessel counts (P < 0.05) compared with the control group. However, there was no significant difference in these parameters between the BRCA2-associated HBCs and the control group. CONCLUSIONS: BRCA1-associated HBCs in Japanese women have biologically aggressive phenotypes. However, BRCA2-associated HBCs are without distinctive clinicopathologic features compared with sporadic breast carcinomas. BACKGROUND: The purpose of this investigation was to elucidate the clinicopathologic characteristics of BRCA1- and BRCA2-associated hereditary breast carcinomas (HBCs) in Japanese women. 11563861 Tsukamoto K, Hirano K, Tsujii K, Ikegami C, Zhongyan Z, Nishida Y, Ohama T, Matsuura F, Yamashita S, Matsuzawa Y: ATP-binding cassette transporter-1 induces rearrangement of actin cytoskeletons possibly through Cdc42/N-WASP. Biochem Biophys Res Commun. 2001 Sep 28;287(3):757-65. Positional cloning approaches revealed that Tangier disease (TD), a genetic high density lipoprotein deficiency, is associated with mutations in the ATP-binding cassette transporter-1 (ABCA1) gene. However, the biological function of ABCA1 is still not fully investigated. Recently, we have reported that the cells from the patients with TD had abnormal actin cytoskeletons in association with decreased expression of Cdc42, a member of RhoGTPases family. In the present study, we have found that actin cytoskeletons were altered in HEK293 cells transfected with human ABCA1 (hABCA1) cDNA. Cells expressing hABCA1 were divided into the following two groups by the distinct morphology with altered actin cytoskeletons: one had increased formation of filopodia (designated as Type I) and the other had long protrusions (designated as Type II). Type I cells had morphology similar to that of cells transfected with dominant active form of Cdc42 (Cdc42-DA, V12Cdc42Hs-DA). Type II cells had morphology similar to that of cells transfected with neural Wiskott-Aldrich Syndrome Protein (N-WASP),one of the established downstream effector molecules of Cdc42. We have obtained the data showing a possible pathway of ABCA1/Cdc42/N-WASP by the following experiments. Introduction of mutant of Cdc42 (dominant negative form of Cdc42, N17Cdc42Hs-DN) and N-WASP (N-WASP lacking verprolin homology domain, N-WASPDeltaVPH), both of which are supposed to have potential to inhibit rearrangement of actin cytoskeletons, significantly inhibited the morphological changes induced by expression of hABCA1. Immunoprecipitation study with FLAG-tagged ABCA1 (hABCA1-FLAG) revealed that Cdc42 was coimmunoprecipitated with hABCA1-FLAG. In addition, we have demonstrated possible intracellular colocalization of these two molecules in the overexpressing cells by the confocal laser microscopy. These results may suggest that hABCA1 regulates actin organization through the possible interaction with Cdc42Hs. These results may suggest that hABCA1 regulates actin organization through the possible interaction with Cdc42Hs. 11687513 Shroyer NF, Lewis RA, Yatsenko AN, Lupski JR: Null missense ABCR (ABCA4) mutations in a family with stargardt disease and retinitis pigmentosa. Invest Ophthalmol Vis Sci. 2001 Nov;42(12):2757-61. PURPOSE: To determine the type of ABCR mutations that segregate in a family that manifests both Stargardt disease (STGD) and retinitis pigmentosa (RP), and the functional consequences of the underlying mutations. METHODS: Direct sequencing of all 50 exons and flanking intronic regions of ABCR was performed for the STGD- and RP-affected relatives. RNA hybridization, Western blot analysis, and azido-adenosine triphosphate (ATP) labeling was used to determine the effect of disease-associated ABCR mutations in an in vitro assay system. RESULTS: Compound heterozygous missense mutations were identified in patients with STGD and RP. STGD-affected individual AR682-03 was compound heterozygous for the mutation 2588G-->C and a complex allele, [W1408R; R1640W]. RP-affected individuals AR682-04 and-05 were compound heterozygous for the complex allele [W1408R; R1640W] and the missense mutation V767D. Functional analysis of the mutation V767D by Western blot and ATP binding revealed a severe reduction in protein expression. In vitro analysis of ABCR protein with the mutations W1408R and R1640W showed a moderate effect of these individual mutations on expression and ATP-binding; the complex allele [W1408R; R1640W] caused a severe reduction in protein expression. CONCLUSIONS: These data reveal that missense ABCR mutations may be associated with RP. Functional analysis reveals that the RP-associated missense ABCR mutations are likely to be functionally null. These studies of the complex allele W1408R; R1640W suggest a synergistic effect of the individual mutations. These data are congruent with a model in which RP is associated with homozygous null mutations and with the notion that severity of retinal disease is inversely related to residual ABCR activity. CONCLUSIONS: These data reveal that missense ABCR mutations may be associated with RP. 8262969 Tan EC, Leung T, Manser E, Lim L: The human active breakpoint cluster region-related gene encodes a brain protein with homology to guanine nucleotide exchange proteins and GTPase-activating proteins. J Biol Chem. 1993 Dec 25;268(36):27291-8. GTPase-activating proteins (GAPs) modulate the activity of the ras superfamily of proteins by converting active GTP-bound to inactive GDP-bound p21s. Employing a novel GAP overlay assay (Manser, E., Leung, T., Monfries, C., Teo, M., Hall, C., and Lim, L. (1992) J. Biol. Chem. 267, 16025-16028), we demonstrated a diversity of proteins with GAP activities in different tissues. Using a polymerase chain reaction strategy exploiting conserved residues in the GAP domains of n-chimaerin and the product of the breakpoint cluster region gene (BCR), we isolated a human brain 5.3-kilobase cDNA containing a 486-base pair region with complete identity to a previously reported active BCR-related (ABR) gene sequence on human chromosome 17. The brain cDNA encoded a 98-kDa protein (ABR) resembling BCR (68% identity), containing both the oncogene dbl-related domain at the N terminus and the GAP domain at the C terminus; however, it lacks the N-terminal BCR protein kinase domain. The ABR GAP domain expressed as an Escherichia coli fusion protein was active against Rac1 and Cdc42 of the rho subfamily. The ABR mRNA is highly enriched in the brain. ABR probably corresponds to the brain-enriched 100-kDa GAP for Rac and Cdc42Hs previously detected. The relationship of ABR to Miller-Dieker syndrome, a neurological disorder co-mapping to 17p13.3, is discussed. ABR probably corresponds to the brain-enriched 100-kDa GAP for Rac and Cdc42Hs previously detected. 12914524 Maehara K, Hida T, Abe Y, Koga A, Ota K, Kutoh E: Functional interference between estrogen-related receptor alpha and peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha heterodimer complex in the nuclear receptor response element-1 of the medium chain acyl-coenzyme A dehydrogenase gene. J Mol Endocrinol. 2003 Aug;31(1):47-60. We undertook a study of molecular interference of nuclear orphan receptors. Nuclear receptor response element-1 (NRRE-1) from the human medium-chain acyl coenzyme A dehydrogenase (MCAD) gene promoter was shown to contain three hexamer elements (site 1 through 3) that are known to interact with a number of nuclear receptors including chicken ovalbumin upstream promoter transcription factor (COUP-TF) and estrogen-related receptor alpha (ERRalpha). We demonstrated that the peroxisome proliferator-activated receptor alpha/9-cis-retinoic acid receptor alpha (PPARalpha/RXRalpha) heterodimer complex can also bind to the two hexamer repeat sequences (between site 1 and site 3) arranged as an everted imperfect repeat separated by 14 bp (ER14). Mutations of the putative core elements have shown that these three sites are differentially involved in ERRalpha and PPARalpha/RXRalpha binding. Homodimer of ERRalpha was shown to interact between site 1 and site 3 (ER14). To date, no nuclear receptor is known to bind to response elements over such long intervals. Interestingly, site 1 was shown to be essential for ERRalpha binding while site 3 supports its binding only in the presence of site 1. Furthermore, it was shown that the binding profile of ERRalpha and PPARalpha/RXRalpha are competitive rather than making a high order complex within NRRE-1. At the cellular level, transcriptional activation driven by the PPARalpha/RXRalpha complex was counteracted by the expression of ERRalpha in HeLa cells. These results suggest that ERRalpha and PPARalpha/RXRalpha could interfere with each other's function through binding to similar DNA elements, thereby finetuning the transcriptional outcome of the target gene. Our findings suggest a mechanism whereby multiple nuclear receptors can activate or repress DNA binding or transcription via a single pleiotropic regulatory element. Nuclear receptor response element-1 (NRRE-1) from the human medium-chain acyl coenzyme A dehydrogenase (MCAD) gene promoter was shown to contain three hexamer elements (site 1 through 3) that are known to interact with a number of nuclear receptors including chicken ovalbumin upstream promoter transcription factor (COUP-TF) and estrogen-related receptor alpha (ERRalpha). 11739374 Duggan A, Garcia-Anoveros J, Corey DP: The PDZ domain protein PICK1 and the sodium channel BNaC1 interact and localize at mechanosensory terminals of dorsal root ganglion neurons and dendrites of central neurons. J Biol Chem. 2002 Feb 15;277(7):5203-8. Epub 2001 Dec 5. Members of the BNaC/ASIC family of ion channels have been implicated in mechanotransduction and nociception mediated by dorsal root ganglion (DRG) neurons. These ion channels are also expressed in the CNS. We identified the PDZ domain protein PICK1 as an interactor of BNaC1(ASIC2) in a yeast two-hybrid screen. We show by two-hybrid assays, glutathione S-transferase pull-down assays, and coimmunoprecipitations that the BNaC1-PICK1 interaction is specific, and that coexpression of both proteins leads to their clustering in intracellular compartments. The interaction between BNaC1 and PICK1 requires the PDZ domain of PICK1 and the last four amino acids of BNaC1. BNaC1 is similar to two other BNaC/ASIC family members, BNaC2 (ASIC1) and ASIC4, at its extreme C terminus, and we show that PICK1 also interacts with BNaC2. We found that PICK1, like BNaC1 and BNaC2, is expressed by DRG neurons and, like the BNaC1alpha isoform, is present at their peripheral mechanosensory endings. Both PICK1 and BNaC1alpha are also coexpressed by some pyramidal neurons of the cortex, by pyramidal neurons of the CA3 region of hippocampus, and by cerebellar Purkinje neurons, localizing to their dendrites and cell bodies. Therefore, PICK1 interacts with BNaC/ASIC channels and may regulate their subcellular distribution or function in both peripheral and central neurons. BNaC1 is similar to two other BNaC/ASIC family members, BNaC2 (ASIC1) and ASIC4, at its extreme C terminus, and we show that PICK1 also interacts with BNaC2. 8643505 Gray NK, Pantopoulos K, Dandekar T, Ackrell BA, Hentze MW: Translational regulation of mammalian and Drosophila citric acid cycle enzymes via iron-responsive elements. Proc Natl Acad Sci U S A. 1996 May 14;93(10):4925-30. The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H2O2), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation.