J Cell Sci, 1999 Aug, 112 ( Pt 16), 2705 - 14 Quantitative measurement of mammalian chromosome mitotic loss rates using the green fluorescent protein; Burns EM et al.; We have measured the mitotic loss rates of mammalian chromosomes in cultured cells . The green fluorescent protein (GFP) gene was incorporated into a non-essential chromosome so that cells containing the chromosome fluoresced green, while those lacking it did not . The proportions of fluorescent and non-fluorescent cells were measured by fluorescence activated cell sorter (FACS) analysis . Loss rates ranged from 0.005% to 0.20% per cell division in mouse LA-9 cells, and from 0.02% to 0.40% in human HeLa cells . The rate of loss was elevated by treatment with aneugens, demonstrating that the system rapidly identifies agents which induce chromosome loss in mammalian cells. J Cell Sci, 1999 Aug, 112 ( Pt 16), 2631 - 8 The nuclear envelope serves as an intermediary between the ER and Golgi complex in the intracellular parasite Toxoplasma gondii; Hager KM et al.; Morphological examination of the highly polarized protozoan parasite Toxoplasma gondii suggests that secretory traffic in this organism progresses from the endoplasmic reticulum to the Golgi apparatus using the nuclear envelope as an intermediate compartment . While the endoplasmic reticulum is predominantly located near the basal end of the parasite, the Golgi is invariably adjacent to the apical end of the nucleus, and the space between the Golgi and nuclear envelope is filled with numerous coatomer-coated vesicles . Staining with antiserum raised against recombinant T . gondii beta-COP confirms its association with the apical juxtanuclear region . Perturbation of protein secretion using brefeldin A, microtubule inhibitors or dithiothreitol disrupts the Golgi, causing swelling of the nuclear envelope, particularly at its basal end . Prolonged drug treatment leads to gross distention of the endoplasmic reticulum, filling the basal end of the parasite . Cloning and sequencing of the T . gondii homolog of the chaperonin protein BiP identifies the carboxy-terminal amino acid sequence HDEL as this organism's endoplasmic reticulum-retention signal . Appending the HDEL motif to a recombinant secretory protein (a chimera between the parasite's major surface protein fusion, P30, and the Green Fluorescent Protein) causes this secretory reporter to be retained intracellularly . P30-GFP-HDEL fluorescence was most intense within the nuclear envelope, particularly at the apical end . These data support a model of secretion in which protein traffic from the endoplasmic reticulum to Golgi occurs via the apical end of the nuclear envelope. Exp Cell Res, 1999 Aug 1, 250(2), 313 - 28 The HMG protein T160 colocalizes with DNA replication foci and is down-regulated during cell differentiation; Hertel L et al.; The high mobility group protein T160, the murine homolog of the human structure-specific recognition protein 1, was first supposed to be involved in the process of V-(D)-J recombination, since it could bind to recombination signal sequence probes . We have recently cloned T160 by using an unrelated DNA probe and shown that it binds to either cruciform or linear DNA with no sequence specificity . In this work, we performed a detailed analysis of T160 expression and immunolocalization . We show that T160 is a phosphoprotein broadly conserved from yeast to mammals, with a high level of expression in all the cell lines tested and in tissues containing a high degree of proliferating cells . Indirect immunofluorescence analysis by confocal laser microscopy revealed that T160 distribution in the cell nucleus is not uniform, and focus-like staining was observed . Cell cycle studies by BrdU incorporation suggest that the appearance of T160 nuclear foci is specific of mid to late S phase . Furthermore, while T160 expression does not change during the cell cycle, it is dramatically down-regulated when cells begin to differentiate, as highlighted in C2C12 myoblasts and myotubes . The disappearance of T160 nuclear staining in multinucleated myotubes is shown . Taken together, these data suggest that its function may be less specific than V-(D)-J recombination and more related to some cellular basic process, such as DNA replication or repair . Mol Biochem Parasitol, 1999 Jun 25, 101(1-2), 161 - 72 A protein linked to mitochondrion development in Trypanosoma brucei; Perez-Morga D et al.; The use of the two-hybrid system in yeast allowed us to isolate a new mitochondrial protein of Trypanosoma brucei, termed PIE8, for putative protein interacting with ESAG8 . This protein was found to localize progressively in the single mitochondrion of the parasite during the mitochondrial reactivation needed to adapt the parasite from the glycolysis-based metabolism in the mammalian host, to the cytochrome-mediated respiration in the fly vector . Once this reactivation is established, PIE8 is lost from the mitochondrion . Thus, the temporary presence of PIE8 in the mitochondrion is linked to mitochondrial reactivation. Braz J Med Biol Res, 1999 May, 32(5), 651 - 7 Adhesion of the human pathogen Sporothrix schenckii to several extracellular matrix proteins; Lima OC et al.; The pathogenic fungus Sporothrix schenckii is the causative agent of sporotrichosis . This subcutaneous mycosis may disseminate in immunocompromised individuals and also affect several internal organs and tissues, most commonly the bone, joints and lung . Since adhesion is the first step involved with the dissemination of pathogens in the host, we have studied the interaction between S . schenckii and several extracellular matrix (ECM) proteins . The binding of two morphological phases of S . schenckii, yeast cells and conidia, to immobilized type II collagen, laminin, fibronectin, fibrinogen and thrombospondin was investigated . Poly (2-hydroxyethyl methacrylate) (poly-HEMA) was used as the negative control . Cell adhesion was assessed by ELISA with a rabbit anti-S . schenckii antiserum . The results indicate that both morphological phases of this fungus can bind significantly to type II collagen, fibronectin and laminin in comparison to the binding observed with BSA (used as blocking agent) . The adhesion rate observed with the ECM proteins (type II collagen, fibronectin and laminin) was statistically significant (P < 0.05) when compared to the adhesion obtained with BSA . No significant binding of conidia was observed to either fibrinogen or thrombospondin, but yeast cells did bind to the fibrinogen . Our results indicate that S . schenckii can bind to fibronectin, laminin and type II collagen and also show differences in binding capacity according to the morphological form of the fungus. An Acad Bras Cienc, 1999, 71(2), 273 - 7 Action of chlorogenic acid on the complement system; Ejzemberg R et al.; Previous research on plants used in folk medicine as antidotes against snake-bite revealed some constituents responsible for such protection . Chlorogenic acid (3-0-caffeoyl quinic acid) was one of these substances, studied with more attention . It has been shown that this substance binds to proteins through hydrophobic interactions and hydrogen bonds . This paper shows the preliminary results about the anti-complementary action of chlorogenic acid . Human and guinea pig sera, treated with chlorogenic acid, were added to the hemolytic system (sheep erythrocyte sensitized with hemolysin) to study its effect on the activation of the classical complement pathway . The action on the alternative pathway was studied with human serum treated with chlorogenic acid and zymosan . Our results show that chlorogenic acid presents anti-complementary action at the classical pathway, since the sera are not able to lysis the indicator system . The presence of C3b fragments on the surface of the yeast cells demonstrates that the alternative pathway was not affected. Mol Cell Endocrinol, 1999 May 25, 151(1-2), 5 - 16 The ubiquitin system in gametogenesis; Baarends WM et al.; Ubiquitin is a ubiquitous and highly conserved protein of 76 amino acid residues, that can be covalently attached to cellular acceptor proteins . The attachment of ubiquitin to target proteins is achieved through a multi-step enzymatic pathway, which involves activities of ubiquitin-activating E1 enzymes, ubiquitin-conjugating E2 enzymes, and ligating E3 enzymes . Mono- or poly-ubiquitination of proteins can lead to protein degradation or modification of protein activity . Many components of the complex ubiquitin system show remarkable evolutionary conservation, from yeast to mammalian species . The ubiquitin system is essential to all eukaryotic cells . Among others, several signal transduction cascades show involvement of the ubiquitin system, but there are currently little data supporting a specific role of the ubiquitin system in hormonal control of reproduction . Interestingly, during gametogenesis, many specialized and important aspects of the ubiquitin system become apparent . Components of the ubiquitin system appear to be involved in different steps and processes during gametogenesis, including control of meiosis, and reorganization of chromatin structure. J Biol Chem, 1999 Jul 23, 274(30), 21478 - 84 Fiz1, a novel zinc finger protein interacting with the receptor tyrosine kinase Flt3; Wolf I et al.; The receptor tyrosine kinase Flt3 has been shown to play a role in proliferation and survival of hematopoietic progenitor cells as well as differentiation of early B lymphoid progenitors . However, the signaling events that control growth or differentiation are not completely understood . In order to identify new signaling molecules interacting with the cytoplasmic domain of Flt3, we performed a yeast two-hybrid screen . In addition to several SH2 domain-containing proteins, we have isolated a novel Flt3 interacting zinc finger protein (Fiz1) with 11 C(2)H(2)-type zinc fingers . Fiz1 binds to the catalytic domain of Flt3 but not to the structurally related receptor tyrosine kinases Kit, Fms, and platelet-derived growth factor receptor . This association is independent of kinase activity . The interaction between Flt3 and Fiz1 detected in yeast was confirmed by in vitro and in vivo coprecipitation assays . Fiz1 mRNA is expressed in all murine cell lines and tissues tested . Anti-Fiz1 antibodies recognize a 60-kDa protein, which is localized in the nucleus as well as in the cytoplasm . Together, these results identified a novel class of interaction between a receptor tyrosine kinase and a signaling molecule which is independent of the well established SH2 domain/phosphotyrosine binding. J Biol Chem, 1999 Jul 23, 274(30), 21425 - 9 Selective association of G protein beta(4) with gamma(5) and gamma(12) subunits in bovine tissues; Asano T et al.; The beta and gamma subunits of G proteins are tightly bound under physiological conditions, and so far, seven beta and 11 gamma subunit isoforms have been found . The relative abilities of the beta and gamma subunits to associate with each other have been studied using transfected cell assays, in vitro translation and the yeast two-hybrid system, but have not been fully characterized in various tissues . In the present study, we demonstrated the selectivity of association of the beta with gamma isoforms in bovine tissues . Immunoprecipitation of betagamma complexes from tissue extracts with antibodies against various gamma subunits and subsequent analyses revealed that beta(4) associated with the gamma subunits with the following rank order of selectivity: gamma(5) > gamma(12) > gamma(2) > gamma(3), while beta(2) bound to gamma(2), gamma(3), and gamma(12) more selectively than to gamma(5) . By contrast, beta(1) associated with all gamma subunits without significant selectivity . Analyses of purified betagamma complexes containing various gamma isoforms revealed beta subunit compositions similar to those found in the immunoprecipitates . Particular combinations of beta and gamma subunit isoforms may contribute to maintaining efficient and specific signal transduction mediated by G proteins. J Biol Chem, 1999 Jul 23, 274(30), 21375 - 86 Identification, expression, and characterization of a cDNA encoding human endoplasmic reticulum mannosidase I, the enzyme that catalyzes the first mannose trimming step in mammalian Asn-linked oligosaccharide biosynthesis; Gonzalez DS et al.; We have isolated a full-length cDNA clone encoding a human alpha1, 2-mannosidase that catalyzes the first mannose trimming step in the processing of mammalian Asn-linked oligosaccharides . This enzyme has been proposed to regulate the timing of quality control glycoprotein degradation in the endoplasmic reticulum (ER) of eukaryotic cells . Human expressed sequence tag clones were identified by sequence similarity to mammalian and yeast oligosaccharide-processing mannosidases, and the full-length coding region of the putative mannosidase homolog was isolated by a combination of 5'-rapid amplification of cDNA ends and direct polymerase chain reaction from human placental cDNA . The open reading frame predicted a 663-amino acid type II transmembrane polypeptide with a short cytoplasmic tail (47 amino acids), a single transmembrane domain (22 amino acids), and a large COOH-terminal catalytic domain (594 amino acids) . Northern blots detected a transcript of approximately 2.8 kilobase pairs that was ubiquitously expressed in human tissues . Expression of an epitope-tagged full-length form of the human mannosidase homolog in normal rat kidney cells resulted in an ER pattern of localization . When a recombinant protein, consisting of protein A fused to the COOH-terminal luminal domain of the human mannosidase homolog, was expressed in COS cells, the fusion protein was found to cleave only a single alpha1,2-mannose residue from Man(9)GlcNAc(2) to produce a unique Man(8)GlcNAc(2) isomer (Man8B) . The mannose cleavage reaction required divalent cations as indicated by inhibition with EDTA or EGTA and reversal of the inhibition by the addition of Ca(2+) . The enzyme was also sensitive to inhibition by deoxymannojirimycin and kifunensine, but not swainsonine . The results on the localization, substrate specificity, and inhibitor profiles indicate that the cDNA reported here encodes an enzyme previously designated ER mannosidase I . Enzyme reactions using a combination of human ER mannosidase I and recombinant Golgi mannosidase IA indicated that that these two enzymes are complementary in their cleavage of Man(9)GlcNAc(2) oligosaccharides to Man(5)GlcNAc(2). Chem Res Toxicol, 1999 Jul, 12(7), 555 - 9 Design, synthesis, and characterization of 7-methoxy-4-(aminomethyl)coumarin as a novel and selective cytochrome P450 2D6 substrate suitable for high-throughput screening; Onderwater RC et al.; In this study, a selective substrate for cytochrome P450 2D6 was designed using a small molecule model developed by M . J . De Groot et al . {(1997) Chem . Res . Toxicol . 10, 41-48} . The substrate, 7-methoxy-4-(aminomethyl)coumarin (MAMC), and its putative O-demethylated metabolite 7-hydroxy-4-(aminomethyl)coumarin (HAMC) were synthesized, and their respective fluorescence properties were characterized . The selectivity of MAMC for P450 2D6 was characterized using microsomes containing single human P450 isoenzymes and human liver microsomes . Formation of the metabolic product HAMC was easily assessed in real time with fluorescence spectroscopy, since MAMC and HAMC excitation and emission wavelengths differed significantly . HPLC analysis confirmed that HAMC was the single metabolic product of MAMC and that HAMC formation accounts for the total increase in fluorescence . It was found that, in microsomes from yeast or lymphoblastoid cells selectively expressing P450 isoenzymes, MAMC was selective for P450 2D6 at a concentration of 25 microM with only P450 1A2 contributing significantly to the formation of HAMC . P450s 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 3A4, and 3A5 were shown not to metabolize MAMC at a concentration of 25 microM . K(m) and v(max) values of MAMC for P450 2D6 were found to be 26.2 +/- 2.8 microM and 2.9 +/- 0.07 min(-)(1), respectively . For P450 1A2, MAMC was found to have a K(m) value of 29.7 +/- 6.2 microM and a v(max) of 0.57 +/- 0.07 min(-)(1) . Formation of HAMC in human liver microsomes could be completely inhibited by quinidine, at a concentration of 0.5 microM selective for P450 2D6, and furafylline, at a concentration of 30 microM selective for P450 1A2 . In conclusion, O-demethylation of 7-methoxy-4-(aminomethyl)coumarin is a rapid and easily determined parameter for P450 2D6 activity and, due to the fluorescent properties of the metabolite formed, may be a valuable new tool for high-throughput screening purposes. Am J Physiol, 1999 Jul, 277(1 Pt 1), C163 - 73 Phagocytic and macropinocytic activity in MARCKS-deficient macrophages and fibroblasts; Carballo E et al.; Macrophages express high levels of the myristoylated, alanine-rich, C kinase substrate (MARCKS), an actin cross-linking protein . To investigate a possible role of MARCKS in macrophage function, fetal liver-derived macrophages were generated from wild-type and MARCKS knockout mouse embryos . No differences between the wild-type and MARCKS-deficient macrophages with respect to morphology (Wright's stain) or actin distribution (staining with rhodamine-phalloidin, under basal conditions or after treatment with phorbol esters, lipopolysaccharide, or both) were observed . We then evaluated phagocytosis mediated by different receptors: Fc receptors tested with IgG-coated sheep red blood cells, complement C3b receptors tested with C3b-coated yeast, mannose receptors tested with unopsonized zymosan, and nonspecific phagocytosis tested with latex beads . We also studied fluid phase endocytosis in macrophages and mouse embryo fibroblasts by using FITC-dextran to quantitate this process . In most cases, there were no differences between the cells derived from wild-type and MARCKS-deficient mice . However, a minor but significant and reproducible difference in rates of zymosan phagocytosis at 45-60 min was observed, with lower rates of phagocytosis in the MARCKS-deficient cells . Our data indicate that MARCKS deficiency may lead to slightly decreased rates of zymosan phagocytosis. Trends Cell Biol, 1999 Aug, 9(8), 323 - 8 An exegesis of IAPs: salvation and surprises from BIR motifs; Miller LK; The BIR (baculovirus IAP repeat) motif is a conserved sequence of approximately 70 amino acids that was identified originally in the 'inhibitor of apoptosis' (IAP) family of proteins . BIR-containing proteins (BIRPs) are found in viruses, yeast and metazoans . Recent genetic analysis of a nematode BIRP demonstrated an essential role in cytokinesis instead of apoptosis . It is likely that BIRs originated in eukaryotes to serve a role in cytokinesis and/or mitotic spindle function during cell division and that, with gene duplication, the more recent adaptation of some BIRPs to the regulation of apoptosis was possible . IAPs interact with a variety of proteins, including members of the caspase protease family . This article discusses current research on the structure and function of the BIR motifs and how it could provide insight into the function of BIRPs in cell division. Biochim Biophys Acta, 1999 Jul 13, 1432(2), 222 - 33 Conformational structure and binding mode of glyceraldehyde-3-phosphate dehydrogenase to tRNA studied by Raman and CD spectroscopy; Carmona P et al.; Recently it has been suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play a role in nuclear tRNA export . As the structural basis of binding of GAPDH to tRNA is as yet unknown, we have employed Raman and CD spectroscopy as probes of the solution structures of GAPDH from rabbit and tRNA(Phe) from brewers yeast . Additionally, we have obtained the Raman and CD spectra of GAPDH when bound to tRNA(Phe) . In the complex we find the following results: (a) The most part of the tRNA(Phe) structure is conserved, but with a slight perturbation toward a B-like form . (b) No significant changes in the secondary structure of the protein upon binding are observed . (c) The surface enhanced Raman spectra are consistent with a GAPDH-tRNA(Phe) complex molecular model that involves the insertion of TRNA(Phe) into the GAPDH tetramer groove containing the R and P axes . (d) The specific interactions that occur between GAPDH and the tRNA(Phe) involve, mainly, stacking between nucleobases and aromatic amino-acid residues, and ionic interactions of basic amino-acid residues with phosphate groups of the ribose-phosphate backbone . The above stacking interactions are also supported by the significant relatedness that we have found between an amino-acid sequence (residues 303-308) of GAPDH and RNP2 binding motifs of some RNA binding proteins. Anal Chem, 1999 Jul 1, 71(13), 2270 - 8 Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry; Tong W et al.; A method to directly identify proteins in complex mixtures by solid-phase microextraction (micro-SPE)/multistep elution/capillary electrophoresis (CE)/tandem mass spectrometry (MS/MS) is described . A sheathless liquid-metal junction interface is used to interface CE and electrospray ionization MS/MS . A subfemtomole detection limit is achieved for protein identification through database searching using MS/MS data . The SPE serves as a semiseparation dimension using an organic-phase step-elution gradient in combination with the second separation dimension for increased resolving power of complex peptide mixtures . This approach improves the concentration detection limit for CE and allows more proteins in complex mixtures to be identified . A 75-protein complex from yeast ribosome is analyzed using this method and 80-90% of the proteins in the complex can be identified by searching the database using the MS/MS data from a complete analysis . This multidimensional CE/MS/MS methodology provides an alternative to multidimensional liquid chromatography/MS/MS for direct identification of small amounts of protein in mixtures. Mol Endocrinol, 1999 Jul, 13(7), 1169 - 82 Molecular modeling of human P450c17 (17alpha-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations; Auchus RJ et al.; P450c17 (17alpha-hydroxylase/17,20-lyase) catalyzes steroid 17alpha-hydroxylase and 17,20-lyase activities in the biosynthesis of androgens and estrogens . These two activities are differentially regulated in a tissue-specific and developmentally programmed manner . To visualize the active site topology of human P450c17 and to study the structural basis of its substrate specificity and catalytic selectivity, we constructed a second-generation computer-graphic model of human P450c17 . The energetics of the model are comparable to those of the principal template of the model, P450BMP, as determined from its crystallographic coordinates . The protein structure analysis programs PROCHECK, WHATIF, and SurVol indicate that the predicted P450c17 structure is reasonable . The hydrophobic active site accommodates both delta4 and delta5 steroid substrates in a catalytically favorable orientation . The predicted contributions of positively charged residues to the redox-partner binding site were confirmed by site-directed mutagenesis . Molecular dynamic simulations with pregnenolone, 17-OH-pregnenolone, progesterone, and 17-OH-progesterone docked into the substrate-binding pocket demonstrated that regioselectivity of the hydroxylation reactions is determined both by proximity of hydrogens to the iron-oxo complex and by the stability of the carbon radicals generated after hydrogen abstraction . The model explains the activities of all known naturally occurring and synthetic human P450c17 mutants . The model predicted that mutation of lysine 89 would disrupt 17,20-lyase activity to a greater extent than 17alpha-hydroxylase activity; expression of a test mutant, K89N, in yeast confirmed this prediction . Hydrogen peroxide did not support catalysis of the 17,20-lyase reaction, as would be predicted by mechanisms involving a ferryl peroxide . Our present model and biochemical data suggest that both the hydroxylase and lyase activities proceed from a common steroid-binding geometry by an iron oxene mechanism . This model will facilitate studies of sex steroid synthesis and its disorders and the design of specific inhibitors useful in chemotherapy of sex steroid-dependent cancers. Anticancer Drug Des, 1999 Apr, 14(2), 107 - 14 Influence of p53 mutation on pathological grade, but not prognosis of non-Hodgkin's lymphoma; Osada M et al.; Mutations in the p53 gene were detected in 27 of the 107 (25%) cases of non-Hodgkin's lymphoma (NHL), examined by assaying the transcriptional activity of p53 in yeast . A relatively high mutation rate of p53 was observed in B-cell intermediate-grade NHL and in T-cell high-grade immunoblastic NHL, in contrast to the relatively low mutation rate observed in other pathological classifications . However, retrospective analyses of all 76 cases revealed that the survival profile and therapeutic responses were very similar in NHL patients bearing lymphomas with a mutant p53 or with the wild-type p53 even within the subclasses characterized by frequent p53 mutation . In patients with high-intermediate grade tumors, the median survival period was 24 months in mutated p53 cases and 14 months in wild-type cases . Complete remission (CR) was observed in 9 of the 17 patients (53%) with mutated forms of p53 and 18 of the 35 patients (51%) with wild-type p53 genes . Our analyses of NHL patients revealed that the presence of p53 mutations may influence pathological grades of NHL, but did not strongly correlate with poor prognosis or reduced chemo/radiosensitivity in NHL . Hence, mutations of p53 do not serve as a prognostic, or chemo/radiosensitivity marker in NHL. J Clin Microbiol, 1999 Aug, 37(8), 2699 - 702 Indigenous disseminated Penicillium marneffei infection in the state of Manipur, India: report of four autochthonous cases; Singh PN et al.; We describe four cases of disseminated infection caused by endemic Penicillium marneffei in human immunodeficiency virus (HIV)-infected patients from the Manipur state of India . The most common clinical features observed were fever, anorexia, weight loss, hepatosplenomegaly, and, more importantly, skin lesions resembling molluscum contagiosum . The diagnosis in each of the four cases was achieved by direct examination of smears, observance of intracellular yeast-like cells multiplying by fission in biopsied tissue from skin lesions, and isolation of the dimorphic P . marneffei in pure culture in each case . In one case, fluorescent antibody studies allowed specific diagnosis . This report documents a new area in which P . marneffei is endemic, located in eastern India, and describes the first occurrence in India of P . marneffei in HIV-infected patients as well as the extension of the areas of P . marneffei endemicity westward to the northeastern state of Manipur. Comput Chem, 1999 Jun 15, 23(3-4), 209 - 17 Protein-coding region discovery in organisms underrepresented in databases; Quentin Y et al.; The prediction of coding sequences has received a lot of attention during the last decade . We can distinguish two kinds of methods, those that rely on training with sets of example and counter-example sequences, and those that exploit the intrinsic properties of the DNA sequences to be analyzed . The former are generally more powerful but their domains of application are limited by the availability of a training set . The latter avoid this drawback but can only be applied to sequences that are long enough to allow computation of the statistics . Here, we present a method that fills the gap between the two approaches . A learning step is applied using a set of sequences that are assumed to contain coding and non-coding regions, but with the boundaries of these regions unknown . A test step then uses the discriminant function obtained during the learning to predict coding regions in sequences from the same organism . The learning relies upon a correspondence analysis and prediction is presented on a graphical display . The method has been evaluated on a sample of yeast sequences, and the analysis of a set of expressed sequence tags from the Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza illustrates the relevance of the approach in its biological context. Biochem Biophys Res Commun, 1999 Jul 5, 260(2), 309 - 12 BCR binds to the xeroderma pigmentosum group B protein; Maru Y et al.; The BCR gene is involved in the formation of the BCR-ABL oncogene responsible for the pathogenesis of Philadelphia chromosome-positive human leukemias . We have previously shown that P210 BCR-ABL binds to the xeroderma pigmentosum group B protein (XPB) through the portion of BCR that is homologous to the catalytic domain of GDP-GTP exchangers such as yeast CDC24 and Dbl . In the baculovirus overexpression system which facilitates binding of coexpressed proteins, we now show that XPB binds to the intact BCR protein efficiently but not to CDC24 or Dbl, suggesting specificity of this interaction . The binding of endogenous BCR and XPB proteins was also detected in Hela cells, and this was inhibited by a blocking peptide . Full-length (1-782) XPB and its truncated form (203-782), which does not contain the nuclear localization signal, were tagged with glutathione S-transferase (GST) and were expressed in Rat1 fibroblasts . GST-XPB(203-782) was localized predominantly in the cytoplasm and bound to BCR but not to p62, one of the other components in TFIIH . GST-XPB(1-782) was largely in the nucleus and bound to p62 and BCR . Although the biological significance of the binding remains to be uncovered, BCR binds to the XPB/p62 complex . Cell Biochem Biophys, 1999, 30(3), 413 - 35 A potentially exhaustive screening strategy reveals two novel divergent myosins in Dictyostelium; Schwarz EC et al.; In recent years, the myosin superfamily has kept expanding at an explosive rate, but the understanding of their complex functions has been lagging . Therefore, Dictyostelium discoideum, a genetically and biochemically tractable eukaryotic amoeba, appears as a powerful model organism to investigate the involvement of the actomyosin cytoskeleton in a variety of cellular tasks . Because of the relatively high degree of functional redundancy, such studies would be greatly facilitated by the prior knowledge of the whole myosin repertoire in this organism . Here, we present a strategy based on PCR amplification using degenerate primers and followed by negative hybridization screening which led to the potentially exhaustive identification of members of the myosin family in D . discoideum . Two novel myosins were identified and their genetic loci mapped by hybridization to an ordered YAC library . Preliminary inspection of myoK and myoM sequences revealed that, despite carrying most of the hallmarks of myosin motors, both molecules harbor features surprisingly divergent from most known myosins. Cell Motil Cytoskeleton, 1999, 43(3), 243 - 54 Regulated expression of p14 (cofactor A) during spermatogenesis; Fanarraga ML et al.; The correct folding of tubulins and the generation of functional alpha beta-tubulin heterodimers require the participation of a series of recently described molecular chaperones and CCT (or TRiC), the cytosolic chaperonin containing TCP-1 . p14 (cofactor A) is a highly conserved protein that forms stable complexes with beta-tubulin which are not apparently indispensable along the in vitro beta-tubulin folding route . Consequently, the precise role of p14 is still unknown, though findings on Rb12p (its yeast homologue) suggest p14 might play a role in meiosis and/or perhaps to serve as an excess beta-tubulin reservoir in the cell . This paper investigates the in vivo possible role of p14 in testis where mitosis, meiosis, and intense microtubular remodeling processes occur . Our results confirm that p14 is more abundantly expressed in testis than in other adult mammalian tissues . Northern blot, Western blot, in situ hybridization, and immunocytochemical analyses have all demonstrated that p14 is progressively upregulated from the onset of meiosis through spermiogenesis, being more abundant in differentiating spermatids . The close correlation observed between the mRNA expression waves for p14 and testis specific tubulin isotypes beta 3 and alpha 3/7, together with the above results, suggest that p14 role in testis would presumably be associated to beta-tubulin processing rather than meiosis itself . Additional in vitro beta 3-tubulin synthesis experiments have shown that p14 plays a double role in beta-tubulin folding, enhancing the dimerization of newly synthesized beta-tubulin isotypes as well as capturing excess beta-tubulin monomers . The above evidence suggests that p14 is a chaperone required for the actual beta-tubulin folding process in vivo and storage of excess beta-tubulin in situations, such as in testis, where excessive microtubule remodeling could lead to a disruption of the alpha-beta balance . As seen for other chaperones, p14 could also serve as a route to lead excess beta-tubulin or replaced isotypes towards degradation. Plant Cell, 1999 Jul, 11(7), 1267 - 76 Nuclear export in plants . Use of geminivirus movement proteins for a cell-based export assay; Ward BM et al.; The nuclear export of proteins and RNAs has been studied in heterokaryons or by microinjecting test substrates into nuclei of HeLa cells or Xenopus oocytes . We have previously shown that the two movement proteins BR1 and BL1 encoded by the plant pathogenic squash leaf curl virus act in a coordinated manner to facilitate virus cell-to-cell movement and that one of these (BR1) is a nuclear shuttle protein . By using a novel in vivo cell-based assay for nuclear export in which nuclear-localized BR1 is trapped by BL1 and redirected to the cortical cytoplasm, we demonstrate that residues 177 to 198 of BR1 contain a leucine-rich nuclear export signal (NES) of the type found in the Rev protein encoded by the human immunodeficiency virus and in Xenopus TFIIIA . We further show that the TFIIIA NES can functionally replace the NES of BR1 in both nuclear export and viral infectivity . These findings suggest that this basic pathway for nuclear export is highly conserved among plant and animal cells and in yeast. Plant Cell, 1999 Jul, 11(7), 1227 - 38 A maize homolog of mammalian CENPC is a constitutive component of the inner kinetochore; Dawe RK et al.; Genes for three maize homologs (CenpcA, CenpcB, and CenpcC) of the conserved kinetochore assembly protein known as centromere protein C (CENPC) have been identified . The C-terminal portion of maize CENPC shares similarity with mammalian CENPC and its yeast homolog Mif2p over a 23-amino acid region known as region I . Immunolocalization experiments combined with three-dimensional light microscopy demonstrated that CENPC is a component of the kinetochore throughout interphase, mitosis, and meiosis . It is shown that sister kinetochore separation occurs in two discrete phases during meiosis . A partial separation of sister kinetochores occurs in prometaphase I, and a complete separation occurs in prometaphase II . CENPC is absent on structures known as neocentromeres that, in maize, demonstrate poleward movement but lack other important features of centromeres/kinetochores . CENPC and a previously identified centromeric DNA sequence interact closely but do not strictly colocalize on meiotic chromosomes . These and other data indicate that CENPC occupies an inner domain of the maize kinetochore. Neuron, 1999 May, 23(1), 181 - 92 A YAC mouse model for Huntington's disease with full-length mutant huntingtin, cytoplasmic toxicity, and selective striatal neurodegeneration; Hodgson JG et al.; We have produced yeast artificial chromosome (YAC) transgenic mice expressing normal (YAC18) and mutant (YAC46 and YAC72) huntingtin (htt) in a developmental and tissue-specific manner identical to that observed in Huntington's disease (HD) . YAC46 and YAC72 mice show early electrophysiological abnormalities, indicating cytoplasmic dysfunction prior to observed nuclear inclusions or neurodegeneration . By 12 months of age, YAC72 mice have a selective degeneration of medium spiny neurons in the lateral striatum associated with the translocation of N-terminal htt fragments to the nucleus . Neurodegeneration can be present in the absence of macro- or microaggregates, clearly showing that aggregates are not essential to initiation of neuronal death . These mice demonstrate that initial neuronal cytoplasmic toxicity is followed by cleavage of htt, nuclear translocation of htt N-terminal fragments, and selective neurodegeneration. Neuron, 1999 May, 23(1), 45 - 54 latheo encodes a subunit of the origin recognition complex and disrupts neuronal proliferation and adult olfactory memory when mutant; Pinto S et al.; The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants . The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain . Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae . latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene . lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC) . Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation. J Virol, 1999 Aug, 73(8), 6282 - 92 The gag domains required for avian retroviral RNA encapsidation determined by using two independent assays; Lee E et al.; The Rous sarcoma virus (RSV) Gag precursor polyprotein is the only viral protein which is necessary for specific packaging of genomic RNA . To map domains within Gag which are important for packaging, we constructed a series of Gag mutations in conjunction with a protease (PR) active-site point mutation in a full-length viral construct . We found that deletion of either the matrix (MA), the capsid (CA), or the protease (PR) domain did not abrogate packaging, although the MA domain is likely to be required for proper assembly . A previously characterized deletion of both Cys-His motifs in RSV nucleocapsid protein (NC) reduced both the efficiency of particle release and specific RNA packaging by 6- to 10-fold, consistent with previous observations that the NC Cys-His motifs played a role in assembly and RNA packaging . Most strikingly, when amino acid changes at Arg 549 and 551 immediately downstream of the distal NC Cys-His box were made, RNA packaging was reduced by more than 25-fold with no defect in particle release, demonstrating the importance of this basic amino acid region in packaging . We also used the yeast three-hybrid system to study avian retroviral RNA-Gag interactions . Using this assay, we found that the interactions of the minimal packaging region (Mpsi) with Gag are of high affinity and specificity . Using a number of Mpsi and Gag mutants, we have found a clear correlation between a reporter gene activation in a yeast three-hybrid binding system and an in vivo packaging assay . Our results showed that the binding assay provides a rapid genetic assay of both RNA and protein components for specific encapsidation. J Biol Chem, 1999 Jul 16, 274(29), 20679 - 87 ZRP-1, a zyxin-related protein, interacts with the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E; Murthy KK et al.; Protein-protein interactions play an important role in the specificity of cellular signaling cascades . By using the yeast two-hybrid system, a specific interaction was identified between the second PDZ domain of the cytosolic protein tyrosine phosphatase hPTP1E and a novel protein, which was termed ZRP-1 to indicate its sequence similarity to the Zyxin protein family . The mRNA encoding this protein is distributed widely in human tissues and contains an open reading frame of 1428 base pairs, predicting a polypeptide of 476 amino acid residues . The deduced protein displays a proline-rich amino-terminal region and three double zinc finger LIM domains at its carboxyl terminus . The specific interaction of this novel protein with the second PDZ domain of hPTP1E was demonstrated both in vitro, using bacterially expressed proteins, and in vivo, by co-immunoprecipitation studies . Deletion analysis indicated that an intact carboxyl terminus is required for its interaction with the second PDZ domain of hPTP1E in the yeast two-hybrid system and suggested that other sequences, including the LIM domains, also participate in the interaction . The genomic organization of the ZRP-1 coding sequence is identical to that of the lipoma preferred partner gene, another Zyxin-related protein, suggesting that the two genes have evolved from a recent gene duplication event. J Biol Chem, 1999 Jul 16, 274(29), 20619 - 27 Functional staging of ADP/ATP carrier translocation across the outer mitochondrial membrane; Ryan MT et al.; The ADP/ATP carrier (AAC) is the major representative of the inner membrane carrier proteins of mitochondria that are synthesized without cleavable presequences . The characterization of the import pathway of AAC into mitochondria has mainly depended on an operational staging system . Here, we introduce two approaches for analyzing the import of AAC, blue native electrophoresis and folding-induced translocation arrest, that allow a functional staging of AAC transport across the outer membrane . (i) Blue native electrophoresis permits a direct monitoring of the receptor stage of AAC and its chase into mitochondria . Binding to this stage requires the receptor protein Tom70 but not Tom37 or Tom20 . (ii) A fusion protein between AAC and dihydrofolate reductase can be selectively arrested in the general import pore complex of the outer membrane by ligand induced folding of the passenger protein . Cross-linking demonstrates that the arrested preprotein is in close contact not only with several receptors and Tim10 but also with the channel protein Tom40, providing the first direct evidence that cleavable preproteins and carrier preproteins interact with the same outer membrane channel . The staging system presented here permits a molecular dissection of AAC transport across the outer mitochondrial membrane, relates it to functional units of the translocases, and indicates a coordinated and successive cooperation of distinct translocase subcomplexes during transfer of the preprotein. J Biol Chem, 1999 Jul 16, 274(29), 20505 - 12 p125 is a novel mammalian Sec23p-interacting protein with structural similarity to phospholipid-modifying proteins; Tani K et al.; COPII-coated vesicles are involved in protein transport from the endoplasmic reticulum to the Golgi apparatus . COPII consists of three parts: Sar1p and the two protein complexes, Sec23p-Sec24p and Sec13p-Sec31p . Using a glutathione S-transferase fusion protein with mouse Sec23p, we identified a novel mammalian Sec23p-interacting protein, p125, which is clearly distinct from Sec24p . The N-terminal region of p125 is rich in proline residues, and the central and C-terminal regions exhibit significant homology to phospholipid-modifying proteins, especially phosphatidic acid preferring-phospholipase A1 . We transiently expressed p125 and mouse Sec23p in mammalian cells and examined their interaction . The results showed that the N-terminal region of p125 is important for the interaction with Sec23p . We confirmed the interaction between the two proteins by a yeast two-hybrid assay . Overexpression of p125, like that of mammalian Sec23p, caused disorganization of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus, suggesting its role in the early secretory pathway. J Biol Chem, 1999 Jul 16, 274(29), 20489 - 98 SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5'-CACCT sequences in candidate target genes; Verschueren K et al.; Activation of transforming growth factor beta receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes . We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system . Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation . SIP1 is a new member of the deltaEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins . Like deltaEF1, SIP1 binds to 5'-CACCT sequences in different promoters, including the Xenopus brachyury promoter . Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2 promoter in vitro, prevented expression of the endogenous Xbra gene in early Xenopus embryos . Therefore, SIP1, like deltaEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways . The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor beta members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo. J Biol Chem, 1999 Jul 16, 274(29), 20432 - 7 DRBP76, a double-stranded RNA-binding nuclear protein, is phosphorylated by the interferon-induced protein kinase, PKR; Patel RC et al.; The interferon-induced double-stranded RNA-activated protein kinase PKR is the prototype of a class of double-stranded (dsRNA)-binding proteins (DRBPs) which share a dsRNA-binding motif conserved from Drosophila to humans . Here we report the purification of DRBP76, a new human member of this class of proteins . Sequence from the amino terminus of DRBP76 matched that of the M phase-specific protein, MPP4 . DRBP76 was also cloned by the yeast two-hybrid screening of a cDNA library using a mutant PKR as bait . Analysis of the cDNA sequence revealed that it is the full-length version of MPP4, has a bipartite nuclear localization signal, two motifs that can mediate interactions with both dsRNA and PKR, five epitopes for potential M phase-specific phosphorylation, two potential sites for phosphorylation by cyclin-dependent kinases, a RG2 motif present in many RNA-binding proteins and predicts a protein of 76 kDa . DsRNA and PKR interactions of DRBP76 were confirmed by analysis of in vitro translated and purified native proteins . Cellular expression of an epitope-tagged DRBP76 demonstrated its nuclear localization, and its co-immunoprecipitation with PKR demonstrated that the two proteins interact in vivo . Finally, purified DRBP76 was shown to be a substrate of PKR in vitro, indicating that this protein's cellular activities may be regulated by PKR-mediated phosphorylation. J Biol Chem, 1999 Jul 16, 274(29), 20229 - 34 The linkage of Kennedy's neuron disease to ARA24, the first identified androgen receptor polyglutamine region-associated coactivator; Hsiao PW et al.; Although the linkage of polyglutamine (poly-Q) repeat expansion in the androgen receptor (AR) to Kennedy's disease (X-linked spinal and bulbar muscular atrophy) was a major step forward, the detailed molecular mechanism of how the change in poly-Q length contributes to the disease remains unclear . Here we report the identification of a nuclear G-protein, Ras-related nuclear protein/ARA24, as the first AR coactivator that can bind differentially with different lengths of poly-Q within AR . In the yeast and mammalian reciprocal interacting assays, our data suggested the interaction of AR N-terminal domain with ARA24 diminishes as the poly-Q length increases . The coactivation of ARA24 also diminishes with the poly-Q expansion within AR . Deletion of the acidic hexapeptide (DEDDDL) at the C terminus of ARA24 further enhances its AR coactivation . Together, our data suggest that poor interaction and weaker coactivation of ARA24 to the longer poly-Q AR in the X-linked spinal and bulbar muscular atrophied AR could contribute to the weaker transactivation of AR . The consequence of poor interaction and weak coactivation may eventually lead to the partial androgen insensitivity during the development of Kennedy's disease. J Biol Chem, 1999 Jul 16, 274(29), 20215 - 22 Peptide specificity determinants at P-7 and P-6 enhance the catalytic efficiency of Ca2+/calmodulin-dependent protein kinase I in the absence of activation loop phosphorylation; Hook SS et al.; Phosphorylation of Ca2+/calmodulin-dependent protein kinase I (CaM KI) at Thr-177 by recombinant rat Ca2+/calmodulin-dependent kinase kinase B (CaM KKB) modulates the kinetics of synapsin-(4-13) peptide phosphorylation by reducing the Km 44-fold and decreasing the KCaM 4-fold . There is also a slight decrease in Km for ATP and increase in enzyme Vmax . A synthetic peptide substrate from the yeast transcription factor, ADR1-(222-234)G233 is a 15-fold better substrate for the Thr-177 dephospho-form of CaM KI than synapsin-(4-13) . The Thr-177 dephospho-enzyme has a Km and Vmax for ADR1-(222-234)G233 similar to the values with synapsin-(4-13) using the Thr-177 phosphorylated enzyme . Likewise, with ADR1-(222-234)G233 as substrate, phosphorylation of Thr-177 or substitution of T177A had very little effect on the kinetic values . Using chimeric peptides between synapsin-(4-13) and ADR1-(222-234)G233 we found that N-terminal basic residues at P-7 and P-6 positions were sufficient to allow efficient phosphorylation by the Thr-177 dephospho-form of CaM KI . Phosphorylation of Thr-177 expands the substrate specificity of CaM KI and is not merely an "on-off" switch for kinase activity. J Bacteriol, 1999 Jul, 181(14), 4285 - 91 Glucose transport in the extremely thermoacidophilic Sulfolobus solfataricus involves a high-affinity membrane-integrated binding protein; Albers SV et al.; The archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 2.5 to 3.5 on carbon sources such as yeast extracts, tryptone, and various sugars . Cells rapidly accumulate glucose . This transport activity involves a membrane-bound glucose-binding protein that interacts with its substrate with very high affinity (Kd of 0 . 43 microM) and retains high glucose affinity at very low pH values (as low as pH 0.6) . The binding protein was extracted with detergent and purified to homogeneity as a 65-kDa glycoprotein . The gene coding for the binding protein was identified in the S . solfataricus P2 genome by means of the amino-terminal amino acid sequence of the purified protein . Sequence analysis suggests that the protein is anchored to the membrane via an amino-terminal transmembrane segment . Neighboring genes encode two membrane proteins and an ATP-binding subunit that are transcribed in the reverse direction, whereas a homologous gene cluster in Pyrococcus horikoshii OT3 was found to be organized in an operon . These data indicate that S . solfataricus utilizes a binding-protein-dependent ATP-binding cassette transporter for the uptake of glucose. Genes Dev, 1999 Jul 1, 13(13), 1678 - 91 Identification of an SCF ubiquitin-ligase complex required for auxin response in Arabidopsis thaliana; Gray WM et al.; The plant hormone auxin regulates diverse aspects of plant growth and development . We report that in Arabidopsis, auxin response is dependent on a ubiquitin-ligase (E3) complex called SCFTIR1 . The complex consists of proteins related to yeast Skp1p and Cdc53p called ASK and AtCUL1, respectively, as well as the F-box protein TIR1 . Mutations in either ASK1 or TIR1 result in decreased auxin response . Further, overexpression of TIR1 promotes auxin response suggesting that SCFTIR1 is limiting for the response . These results provide new support for a model in which auxin action depends on the regulated proteolysis of repressor proteins. J Infect Dis, 1999 Aug, 180(2), 534 - 7 Association of plasma levels of human immunodeficiency virus type 1 RNA and oropharyngeal Candida colonization; Gottfredsson M et al.; The pathophysiology of oropharyngeal candidiasis in patients infected with human immunodeficiency virus (HIV) type 1 is poorly understood . Association between oropharyngeal yeast carriage and various clinical factors in HIV-1-infected patients was studied in 83 patients with no clinical evidence of thrush and no recent antifungal use . Of the clinical factors measured, the only correlate of yeast colonization was with plasma HIV-1 RNA levels (P=.001), whereas the correlation with CD4 cell count was poor (P=.36) . By multivariable regression modeling, plasma HIV-1 RNA was the only parameter that correlated with the extent of colonization with Candida infection (P=.003) . These data indicate that the presence and amount of asymptomatic oropharyngeal yeast carriage in persons with HIV-1 infection is more significantly correlated with plasma HIV-1 RNA levels than with CD4 cell count . Further studies on the effect of HIV-1 on oropharyngeal yeast colonization, infection, and local immunity are warranted. Mol Biol Cell, 1999 Jul, 10(7), 2251 - 64 The plant vesicle-associated SNARE AtVTI1a likely mediates vesicle transport from the trans-Golgi network to the prevacuolar compartment; Zheng H et al.; Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes . Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast . AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively . By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I . Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis . Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC . AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC . In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts . We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process. Blood, 1999 Jul 15, 94(2), 724 - 32 Detection of t(4;14)(p16.3;q32) chromosomal translocation in multiple myeloma by double-color fluorescent in situ hybridization; Finelli P et al.; Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies . In multiple myeloma (MM), variable chromosome partners have been identified by conventional cytogenetics, including the 11q13, 8q24, 18q21, and 6p21 loci . We and others have recently reported a novel, karyotypically undetectable chromosomal translocation t(4;14)(p16 . 3;q32) in MM-derived cell lines, as well as in primary tumors . The 4p16.3 breakpoints are relatively scattered and located less than 100 kb centromeric of the fibroblast growth factor receptor 3 (FGFR3) gene or within the recently identified WHSC1 gene, both of which are apparently deregulated by the translocation . To assess the frequency of the t(4;14)(p16.3;q32) translocation in MM, we performed a double-color fluorescent in situ hybridization (FISH) analysis of interphase nuclei with differently labeled probes specific for the IGH locus (a pool of plasmid clones specific for the IGH constant regions) or 4p16.3 (yeast artificial chromosome (YAC) 764-H1 spanning the region involved in breakpoints) . Thirty MM patients, the MM-derived cell lines KMS-11 and OPM2, and six normal controls were examined . The identification of a t(4;14) translocation, evaluated as the presence of a der(14) chromosome, was based on the colocalization of signals specific for the two probes; a cutoff value of 15% (mean + 3 standard deviation {SD}) derived from the interphase FISH of the normal controls (range, 5% to 11%; mean +/- SD, 8.16 +/- 2.2) was used for the quantification analysis . In interphase FISH, five patients (one in clinical stage I, two in stage II, one in stage III, and a plasma cell leukemia) were found to be positive (approximately 15%) . FISH metaphases with split or colocalized signals were detected in only two of the translocated cases and confirmed the pattern found in the interphase nuclei . Furthermore, in three of the five cases with the translocation, FISH analysis with the IGH joining probe (JH) showed the presence of the reciprocal product of the translocation {der(4) chromosome} . Overall, our study indicates that the t(4;14)(p16 . 3;q32) chromosomal translocation is a recurrent event in MM tumors and may contribute towards the detection of this lesion and our understanding of its pathogenetic and clinical implications in MM. Acta Biochim Pol, 1998, 45(4), 949 - 76 Oligonucleotide synthesis using the 2-(levulinyloxymethyl)-5-nitrobenzoyl group for the 5'-position of nucleoside 3'-phosphoramidite derivatives; Kamaike K et al.; A comparative study on the utility of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) and 2-(levulinyloxymethyl)benzoyl (LMBz) protecting groups for the 5'-positions of nucleoside 3'-phosphoramidite derivatives in the oligonucleotide synthesis is presented in terms of the syntheses of TpTpT, TpTpTpT, and UpCpApGpUpUpGpG . In addition we describe the synthesis, using the LMNBz protecting group, of the CpCpA terminus triplet of tRNAs bearing exocyclic amino groups with 15N-labeling, and the trimer Gp{A*}pG containing 2'-O-(beta-D-ribofuranosyl)adenosine ({A*}), the latter of which is found at position 64 in the yeast initiator tRNA(Met). Gene, 1999 Jul 8, 234(2), 353 - 60 The Drosophila ortholog of the human XPG gene; Houle JF et al.; Xeroderma pigmentosum complementation group G (XPG) protein is a junction-specific endonuclease which is indispensable for nucleotide excision repair (NER) of DNA in eukaryotes . Recent studies have hinted at a second, essential function for the XPG protein in higher eukaryotes . We undertook a comparison of the amino acid sequences of multiple XPG orthologs to determine if a motif or domain could be identified that is conserved uniquely in higher eukaryotes . A search of current databases allowed us to retrieve complete amino acid sequences for the human, mouse and Xenopus XPG proteins, and for two yeast orthologs . We also identified an incomplete Drosophila open reading frame (ORF) that was a good candidate for the XPG protein . We cloned a complete Drosophila cDNA for this ORF and examination of the primary amino acid sequence suggests that this cDNA encodes the Drosophila ortholog of XPG . A comparison of all six orthologous polypeptides reveals the presence of two previously unidentified conserved domains . One of these is unique to all four higher eukaryotic sequences . Conceivably this domain evolved to support the essential function of XPG protein. Genomics, 1999 Jul 1, 59(1), 51 - 8 Identification and characterization of AFG3L2, a novel paraplegin-related gene; Banfi S et al.; We recently identified a gene responsible for an autosomal recessive form of hereditary spastic paraplegia (HSP) . This gene encodes paraplegin, a mitochondrial protein highly homologous to the yeast mitochondrial ATPases Afg3p and Rcalp, which have both proteolytic and chaperone-like activities at the inner mitochondrial membrane . By screening the Expressed Sequence Tag database, we identified and characterized a novel human cDNA, ATPase family gene 3-like 2 (AFG3L2, Human Gene Nomenclature Committee-approved symbol), which is closely related to paraplegin . This cDNA encodes a 797-amino-acid predicted protein highly similar to paraplegin as well as to yeast Afg3p and Rca1p . Immunofluorescence studies revealed that AFG3L2 and paraplegin share a similar expression pattern and the same subcellular localization, the mitochondrial compartment . We subsequently mapped AFG3L2 to chromosome 18p11 by radiation hybrid analysis . AFG3L2 may represent a candidate gene for other forms of HSPs and possibly for other neurodegenerative disorders . Curr Opin Cell Biol, 1999 Jun, 11(3), 342 - 6 Mechanism and regulation of transcriptional elongation by RNA polymerase II; Reines D et al.; Over the past few years, biochemical and genetic studies have shed considerable light on the structure and function of the RNA polymerase II (pol II) elongation complex and the transcription factors that control it . Novel elongation factors have been identified and their mechanisms of action characterized in increasing detail; new insights into the biological roles of elongation factors have been gained from genetic studies of the regulation of mRNA synthesis in yeast; and intriguing links between the pol II elongation machinery and the pathways of DNA repair and recombination have emerged. Curr Opin Cell Biol, 1999 Jun, 11(3), 330 - 5 Activation of RNA polymerase II transcription; Berk AJ; Multiple alternative interactions between activators and co-activators stimulate transcription by RNA polymerase II . In the past two years, multiprotein co-activator complexes have been characterized and their subunits defined . TATA-box binding protein associated factor (TAF) subunits of yeast TFIID were found to be generally required for transcription in vivo . Mammalian multisubunit coactivator complexes with homologs of the yeast SRB/Mediator subunits have been characterized . Structures of nuclear receptor-coactivator complexes have been determined. Curr Opin Cell Biol, 1999 Jun, 11(3), 391 - 401 The nuclear pore complex: from molecular architecture to functional dynamics; Stoffler D et al.; Toward dissecting the molecular composition and architecture of the nuclear pore complex (NPC), over the past 18 months novel nucleoporins and NPC subcomplexes were identified and characterized . The three-dimensional structure of isolated yeast NPCs was determined by electron cryomicroscopy . New specimen preparation and labeling protocols localized a number of nucleoporins and NPC subcomplexes within the three-dimensional architecture of the yeast NPC . Structural changes of native NPCs mediated by physiological effectors such as calcium or ATP were monitored by time-lapse atomic force microscopy, thus revealing a first glimpse of the NPC's functional dynamics. J Protein Chem, 1999 Apr, 18(3), 315 - 23 Soybean beta-conglycinin alpha subunit is phosphorylated on two distinct serines by protein kinase CK2 in vitro; Ralet MC et al.; Protein kinase CK2 purified from the yeast Yarrowia lipolytica was used to phosphorylate soybean beta-conglycinin alpha subunit . CK2 is known to phosphorylate serines and threonines in the consensus sequence Ser/Thr-X-X-Glu/Asp/SerP/TyrP . Beta-conglycinin alpha subunit (68 kDa) presents seven consensus sequences, but only 0.5-1 mol P/mol alpha subunit was incorporated by CK2 . {32P}Phosphorylated beta-conglycinin alpha subunit was cleaved either by cyanogen bromide or by trypsin . 32P was incorporated into the largest cyanogen bromide fragment only (50 kDa, N-terminal) and only two radiolabeled zones were detected after HPLC of the trypsic digest . The corresponding phosphorylated zones were collected and further analyzed by RP-HPLC coupled to electrospray ionization mass spectrometry (LC-ESMS) . Two phosphorylated sites, Ser 75 and Ser 117, were determined after MS-MS analysis of three phosphopeptides identified as 70-89, 116-126, and 116-127 sequences . Over the seven consensus sequences of beta-conglycinin alpha subunit, Ser 75 is the only one which was phosphorylated . Ser 117 was phosphorylated although it is not an expected phosphorylation site according to the canonical consensus sequence criteria as there is no acidic determinant at the +3 position . Both Ser 75 and Ser 117 are located inside very acidic sequences, by contrast with the other unphosphorylated potential sites. J Protein Chem, 1999 Apr, 18(3), 259 - 68 Structure of pyridoxal kinase from sheep brain and role of the tryptophanyl residues; Maras B et al.; The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods . The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da . The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy . The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers . Chemical modification with NBS abolishes the catalytic activity of the kinase . The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported {Hanna, Turner, and Kirkness, (1997), J . Biol . Chem . 272, 10756-10760} . Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity . In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not . Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution. Mol Gen Genet, 1999 Jun, 261(4-5), 820 - 30 Molecular cloning and characterisation of a maize cDNA for a homologue of the large subunit of the eukaryotic initiation factor 3 (eIF3); Sabelli PA et al.; In order to identify genes that are specifically expressed in distinct cell populations of the maize root apex, we have constructed PCR-directed cDNA libraries from microdissected populations of cells, and screened them by differential hybridisation . A meristem-specific cDNA was isolated and characterised . This cDNA, termed ZmeIF3A, encodes a protein homologous to the large subunit of the eukaryotic translation Initiation Factor 3 (eIF3), which is an essential multi-protein complex for the initiation of protein synthesis . The ZmeIF3A protein is most similar to the yeast homologue RPG1, lacking the repeated C-terminal domain characteristic of its mammalian counterparts . However, despite this similarity, it fails to replace the RPG1 protein in complementation experiments on yeast mutants . Analysis of gene expression in situ showed that the ZmeIF3A transcript is expressed in the region of the root meristem surrounding the central stele . ZmeIF3A mRNA is also expressed in the young root, the male inflorescence, and the developing cob and seed . In maize, ZmeIF3A is encoded by one or two genomic sequences . This is the first report on the isolation and characterisation of a cDNA from higher plants that encodes a product homologous to a component of the eIF3 complex. Mycoses, 1999 Apr, 42(1-2), 111 - 5 Case report . Cutaneous phaeohyphomycosis caused by Cladosporium oxysporum; Romano C et al.; The case of a 66-year-old woman with Cushing syndrome and a 1-year history of papulo-nodular lesions on the right leg is reported . Biopsy revealed septate hyphae and yeast-like cells in granulomatous dermo-hypodermal lesions . Culture of biopsy fragments on Sabouraud glucose agar without cycloheximide produced colonies that were olive green on top and greenish black underneath . On the basis of microscope findings and scanning electron microscopy observation of fragments of colonies, a diagnosis of cutaneous phaeohyphomycosis due to Cladosporium oxysporum was made . The patient was initially treated with itraconazole, which led to clinical improvement, but mycological recovery was obtained after a course of ketoconazole, made necessary by the presence of pituitary adenoma . To our knowledge, this is the first reported case of subcutaneous phaeohyphomycosis due to Cl . oxysporum. Int Arch Allergy Immunol, 1999 Jun, 119(2), 95 - 100 Activation of Fc epsilon RI inhibits the pyruvate kinase through direct interaction with the gamma-chain; Oak MH et al.; The downstream signaling components of high-affinity IgE receptor (FcepsilonRI) were studied using yeast two-hybrid screening of the cDNA library constructed from RBL-2H3 cells . The cytoplasmic part of the gamma-chain but not that of the beta-chain was found to interact with pyruvate kinase in the yeast . The in-vitro-translated pyruvate kinase also specifically interacted with the bacterially expressed glutathione-S transferase fusion protein of the cytoplasmic part of the gamma-chain . When RBL-2H3 cells were challenged with antigen, the activity of pyruvate kinase gradually decreased, reaching the minimum activity around 5 min after the activation, and then slowly returned to the normal level . The dose-response curve (antigen vs . pyruvate kinase activity) plotted at 5 min after stimulation showed that the pyruvate kinase was dose-dependently inhibited and the maximum inhibition was reached at the concentration of 0.1 microgram/ml of antigen . Direct interaction between FcepsilonRI and pyruvate kinase was also demonstrated by co-immunoprecipitation in RBL-2H3 cells . These data suggest that pyruvate kinase is functionally linked with FcepsilonRI and might exert an important role in controlling cellular functions following the activation of FcepsilonRI. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 8265 - 70 High gene density is conserved at syntenic loci of small and large grass genomes; Feuillet C et al.; Comparative genomic analysis at the genetic-map level has shown extensive conservation of the gene order between the different grass genomes in many chromosomal regions . However, little is known about the gene organization in grass genomes at the microlevel . Comparison of gene-coding regions between maize, rice, and sorghum showed that the distance between the genes is correlated with the genome size . We have investigated the microcolinearity at Lrk gene loci in the genomes of four grass species: wheat, barley, maize, and rice . The Lrk genes, which encode receptor-like kinases, were found to be consistently associated with another type of receptor-like kinase (Tak) on chromosome groups 1 and 3 in Triticeae and on chromosomes homoeologous to Triticeae group 3 in the other grass genomes . On Triticeae chromosome group 1, Tak and Lrk together with genes putatively encoding NBS/LRR proteins form a cluster of genes possibly involved in signal transduction . Comparison of the gene composition at orthologous Lrk loci in wheat, barley, and rice revealed a maximal gene density of one gene per 4-5 kb, very similar to the gene density in Arabidopsis thaliana . We conclude that small and large grass genomes contain regions that are highly enriched in genes with very little or no repetitive DNA . The comparison of the gene organization suggested various genome rearrangements during the evolution of the different grass species. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 8184 - 9 A single amino acid substitution in the cyclin D binding domain of the infected cell protein no . 0 abrogates the neuroinvasiveness of herpes simplex virus without affecting its ability to replicate; Van Sant C et al.; The infected cell protein no . 0 (ICP0) of herpes simplex virus 1 is a promiscuous transactivator shown to enhance the expression of genes introduced into cells by infection or transfection . The protein interacts with several viral and cellular proteins . Earlier studies have shown that ICP0 binds and stabilizes cyclin D3 but does interfere with the phosphorylation of retinoblastoma protein, its major function . Cyclin D3 plays a key role in the transition from G1 to S phase . To define the role of cyclin D3 in productive infection, the ICP0 binding site for cyclin D3 was mapped and mutagenized by substitution of aspartic acid codon 199 with the alanine codon . We report that the substitution precluded the interaction of this protein with cyclin D3 in the yeast two-hybrid system and the stabilization of cyclin D3 in infected cells . A recombinant virus carrying this mutation could not be differentiated from wild-type parent with respect to replication in dividing cells but yielded 10-fold less progeny from infected resting cells and serum-deprived or contact-inhibited human fibroblasts . In mice, the mutant was only slightly less pathogenic than the wild-type parent by intracerebral route but was significantly less neuroinvasive after peripheral inoculation . Replacement of the mutated amino acid with aspartic acid restored wild-type phenotype . Stabilization of cyclin D3 therefore is linked to higher virus yields in nondividing cells and potentially higher virulence in experimental and natural hosts . One function of ICP0 is to scavenge the cell for proteins that could bolster viral replication. Proc Natl Acad Sci U S A, 1999 Jul 6, 96(14), 8040 - 5 A human DAZ transgene confers partial rescue of the mouse Dazl null phenotype; Slee R et al.; In a subset of infertile men, a spectrum of spermatogenic defects ranging from a complete absence of germ cells (sertoli cell only) to oligozoospermia is associated with microdeletions of the DAZ (deleted in azoospermia) gene cluster on human distal Yq . DAZ encodes a testis-specific protein with RNA-binding potential recently derived from a single-copy gene DAZL1 (DAZ-like) on chromosome 3 . Y chromosomal DAZ homologues are confined to humans and higher primates . It remains unclear which function unique to higher primate spermatogenesis DAZ may serve, and the functional status of the gene recently has been questioned . To assess the extent of functional conservation we have tested the capacity of a human DAZ gene contained in a 225-kb yeast artificial chromosome to complement the sterile phenotype of the Dazl null mouse (Dazl-/-), which is characterized by severe germ-cell depletion and meiotic failure . Although Dazl-/- mice remained infertile when the DAZ transgene was introduced, histological examination revealed a partial and variable rescue of the mutant phenotype, manifest as a pronounced increase in the germ cell population of the seminiferous tubules and survival to the pachytene stage of meiosis . As well as constituting definitive proof of the spermatogenic role of the DAZ gene product, these findings confirm the high degree of functional conservation between the DAZ and DAZL1 genes, suggesting they may constitute a single target for contraceptive intervention and raising the possibility of therapeutic up-regulation of the DAZL1 gene in infertile men. Cytogenet Cell Genet, 1999, 84(3-4), 225 - 9 The application of AFLP fingerprinting to construct a YAC contig containing ADH2 and MTP on sheep chromosome 6; Lumsden JM et al.; The low density of genetic markers on livestock maps limits progress in positional cloning projects . We demonstrate a strategy of combining comparative mapping with AFLP fingerprinting to develop physical maps in a defined region of the sheep genome . Sequence tagged sites for alcohol dehydrogenase 2 (ADH2) and microsomal triglyceride transfer protein (MTP) were developed and used to screen a sheep yeast artificial chromosome (YAC) library . Nine YACs were identified containing the microsatellite marker BM1329 and either ADH2 or MTP . Additional markers in the region were not available, and AFLP analysis was developed to identify sheep-specific bands within the YACs to determine their degree of overlap . Fourteen bands common to more than one YAC were analysed and provided the markers necessary to develop a YAC contig containing the three STS markers . One YAC (yac260B5) containing all three markers (ADH2, MTP, and BM1329) was mapped to sheep chromosome 6q1.6-->q1.8 by FISH analysis. Cytogenet Cell Genet, 1999, 84(3-4), 220 - 4 Assignment of human teratocarcinoma derived growth factor (TDGF) sequences to chromosomes 2q37, 3q22, 6p25 and 19q13.1; Scognamiglio B et al.; The human teratocarcinoma derived growth factor 1 (TDGF1) gene maps on chromosome (Chr) 3p21.3 . One pseudogene (TDGF3) maps on Chr Xq21-->q22 . We now report the nucleotide sequence and chromosome location of three additional TDGF pseudogenes . The three new sequences (TDGF2, TDGF4 and TDGF5) are truncated at the 5' end and have accumulated several point mutations, deletions and insertions . TDGF2, TDGF4 and TDGF6 map on Chrs 2q37, 6p25 and 3q22, respectively . Finally, Southern blot analysis of DNA from normal individuals shows a highly variable restriction pattern of the TDGF sequences. Cytogenet Cell Genet, 1999, 84(3-4), 173 - 8 Characterization of the human laminin beta2 chain locus (LAMB2): linkage to a gene containing a nonprocessed, transcribed LAMB2-like pseudogene (LAMB2L) and to the gene encoding glutaminyl tRNA synthetase (QARS); Durkin ME et al.; The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes . We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1) . Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified . The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS) . About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L) . The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene . The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1). Cytogenet Cell Genet, 1999, 84(3-4), 167 - 72 Cloning and characterization of ASH2L and Ash2l, human and mouse homologs of the Drosophila ash2 gene; Ikegawa S et al.; Drosophila ash2 belongs to the trithorax (trx) gene family . The ash2 product positively regulates expression of homeotic selector genes, and is implicated in early development and formation of various disc patterns in the fruit fly . Through large-scale sequencing of human genomic DNA coupled with in silico gene trapping, we identified a gene (ASH2L) on chromosome 8p11.2 whose predicted product was highly homologous to ash2, characterized it, and identified its mouse counterpart . The human ash2 cDNA is 2368 bp long, encoding 628 amino acids . The 16-exon gene spans more than 34 kb of genomic DNA between STS markers WI-9207 (centromere) and AFMA295ZD5 (telomere) on chromosome 8, with transcription oriented telomere to centromere . The ash2 genes are highly conserved among different species, including C . elegans and yeast . The presence of a conserved bipartite nuclear localization signal and a PHD finger motif in the human ash2 gene suggests that the gene product would function as a transcriptional regulator in humans, as its homologue does in Drosophila. J Biochem (Tokyo), 1999 Jul, 126(1), 68 - 77 Molecular cloning and functional expression of the human Golgi UDP-N-acetylglucosamine transporter; Ishida N et al.; We have cloned the human UDP-N-acetylglucosamine (UDP-GlcNAc) transporter cDNA, which was recognized through a homology search in the expressed sequence tags database (dbEST) based on its similarity to the human UDP-galactose transporter . The chromosomal location of the UDP-GlcNAc transporter gene was assigned to chromosome 1p21 by fluorescence in situ hybridization (FISH) . The transporter was expressed ubiquitously in every tissue so far examined . Expression of the transporter cDNA in CHO-K1 cells in its native and in a C-terminally HA-tagged form indicated that the human UDP-GlcNAc transporter was localized in the Golgi apparatus . The membrane vesicles prepared from yeast cells expressing the cDNA product exhibited UDP-GlcNAc-specific transporting activity . Comparison among UDP-galactose, CMP-sialic acid, and UDP-GlcNAc transporters from several organisms enabled us to identify residues highly conserved among the transporters and residues specific for each group of transporters. J Biochem (Tokyo), 1999 Jul, 126(1), 1 - 6 Carboxypeptidase Y: structural basis for protein sorting and catalytic triad; Jung G et al.; A yeast vacuolar protease, carboxypeptidase Y (CPY), is known to be involved in the C-terminal processing of peptides and proteins; however, its real function remains unclear . The CPY biosynthetic pathway has been used as a model system for protein sorting in eukaryotes . CPY is synthesized as a prepro-form that travels through the ER and Golgi to its final destination in vacuoles . In the course of studies on the transport mechanism of CPY, various post-translational events have been identified, e.g . carbohydrate modification and cleavage of the pre-segments . In addition, sorting signals and various sorting vehicles, similar to those found in higher eukaryotic cells, have been found . The catalytic triad in the active site of CPY makes this enzyme a serine protease . A unique feature distinguishing CPY from other serine proteases is its wide pH optimum, in particular its high activity at acidic pH . Several structural properties which might contribute to this unique feature exist such as a conserved free cysteine residue in the S1 substrate binding pocket, a recognition site for a C-terminal carboxyl group, and a disulfide zipper motif . The structural bases in CPY functions are discussed in this article. Gene, 1999 Jun 24, 234(1), 61 - 9 Interaction between the two ubiquitously expressed transcription factors NF-Y and Sp1; Roder K et al.; The regulation of the rat fatty acid synthase gene by mediators such as diet, hormones, cAMP, sterols or retinoic acid is controlled by three NF-Y binding sites . All three sites have a neighbouring Sp1-binding GC-box . This NF-Y/Sp1 motif is conserved in the FAS promoters of rat, human, goose and chicken . We have previously shown cooperative binding of NF-Y and Sp1 to the promoter region at -500 coincident with a diet-induced DNAse I-hypersensitive site . Here, we show an in-vivo interaction of NF-YA with Sp1 using the yeast two-hybrid system . The interacting domains are located between amino acids 55 and 139 of the NF-Y subunit NF-YA and between amino acids 139 and 344 of Sp1 . In addition, we show by co-immunoprecipitation direct interaction of NF-Y subunit NF-YA with Sp1 in extracts of rat hepatoma cells H4IIE . Furthermore, we demonstrate by the GST pull-down assay that NF-YA interacts physically with Sp1 in-vitro in the absence of DNA . Therefore, NF-Y can be added to the list of transcription factors interacting with Sp1. EMBO J, 1999 Jul 1, 18(13), 3676 - 87 An adipogenic cofactor bound by the differentiation domain of PPARgamma; Castillo G et al.; Ligand activation of the nuclear receptor PPARgamma induces adipogenesis and increases insulin sensitivity, while activation of other PPAR isoforms (-alpha and -delta) induces little or no fat cell differentiation . Expression and activation of chimeras formed between PPARgamma and PPARdelta in fibroblasts has allowed us to localize a major domain of PPARgamma responsible for adipogenesis to the N-terminal 138 amino acids, a region with AF-1 transcriptional activity . Using this region of PPARgamma as bait, we have used a yeast two-hybrid screen to clone a novel protein, termed PGC-2, containing a partial SCAN domain . PGC-2 binds to and increases the transcriptional activity of PPARgamma but does not interact with other PPARs or most other nuclear receptors . Ectopic expression of PGC-2 in preadipocytes containing endogenous PPARgamma causes a dramatic increase in fat cell differentiation at both the morphological and molecular levels . These results suggest that interactions between PGC-2, a receptor isoform-selective cofactor and PPARgamma contribute to the adipogenic action of this receptor. EMBO J, 1999 Jul 1, 18(13), 3604 - 15 A Caenorhabditis elegans JNK signal transduction pathway regulates coordinated movement via type-D GABAergic motor neurons; Kawasaki M et al.; The c-Jun N-terminal kinase (JNK) of the MAP kinase superfamily is activated in response to a variety of cellular stresses and is involved in apoptosis in neurons . However, the roles of the JNK signaling pathway in the nervous system are unknown . The genes for the Caenorhabditis elegans homolog of JNK, JNK-1, and its direct activator, JKK-1, were isolated based on their abilities to function in the Hog1 MAP kinase pathway in yeast . JKK-1 is a member of the MAP kinase kinase superfamily and functions as a specific activator of JNK . Both jnk-1 and jkk-1 are expressed in most neurons . jkk-1 null mutant animals exhibit defects in locomotion that can be rescued by the conditional expression of JKK-1 in mutant adults, suggesting that the defect is not due to a developmental error . Furthermore, ectopic expression of JKK-1 in type-D motor neurons is sufficient to rescue the movement defect . Thus, the C.elegans JNK pathway functions in type-D GABAergic motor neurons and thereby modulates coordinated locomotion. Front Biosci, 1999 Jul 01, 4, C1 - 3 Multiprobe RNase protection assay with internally labeled radioactive probes, generated by RT-PCR and nested PCR; von Wolff M et al.; RNase Protection Assay (RPAs) is a highly sensitive and reproducible method of quantitating the levels of specific mRNA transcripts . The introduction of the commercially available Multiprobe RPAs allow comparing and quantifying the expression of up to different mRNA species in a single sample of 1-20 micrograms of total RNA . To generate probes which are not commercially available, we prepared highly specific probes by RT-PCR and nested PCR . Then, after ligation of a T7 promoter, another PCR was performed with a primer set consisting of a specific sense primer and antisense T7 primer . Only the antisense strand of the double stranded PCR-product contained the T7-promoter sequence on its 5' end, allowing in vitro transcription and internal labeling with {alpha-32}UTP . Probe concentration was determined in a scintillation counter and equal counts were introduced in the assay . In vitro transcription of the PCR generated probes resulted in radioactive probes with a very high specific activity, allowing simultaneous analysis of 70 different RNA samples . RPA could be performed under the same conditions as recommended for the commercially available probe sets, avoiding time consuming optimization of reaction conditions . Negative controls consisted of yeast RNA and sense RNA probes . Positive controls were single stranded templates, generated by asymmetric PCR . Dilution series revealed a high reproducibility and the potential of this technique to semi-quantitate mRNA in different RNA samples . In conclusion, probes may be generated by RT-PCR and nested PCR that will work with the commercially available Multiprobe RPAs . The high probe yield allows analysis of a great number of samples using the same set of probes with a high reproducibility. Biochem J, 1999 Jul 15, 341 ( Pt 2), 257 - 63 Talin contains three similar vinculin-binding sites predicted to form an amphipathic helix; Bass MD et al.; Using recombinant talin polypeptides and an SDS/PAGE-blot overlay assay, we have previously identified three regions of talin that are involved in binding to vinculin {Gilmore, Wood, Ohanian, Jackson, Patel, Rees, Hynes and Critchley (1993) J . Cell Biol . 122, 337-347} . We have confirmed these observations by using a yeast two-hybrid assay and shown that talin residues 498-656, 852-950 and 1929-2029 are each capable of binding to vinculin residues 1-258 . We have further defined the three vinculin-binding sites in talin to residues 607-636, 852-876 and 1944-1969; alignment of these sequences shows 59% similarity, although there are only two identical residues . Predictions of secondary structure indicate that this vinculin-binding motif forms an amphipathic alpha-helix . The hydrophobic face of helix 607-636 contains three aligned leucines (residues 608, 615 and 622), which show conservative substitutions in the other two sites . To test the possibility that this might constitute a leucine zipper involved in vinculin binding, we mutated each leucine residue to an alanine . The results showed that this leucine repeat is not essential to the interaction between talin and vinculin . We also used the yeast two-hybrid system to define further the talin-binding site within vinculin residues 1-258 . C-terminal deletions made in accordance with exon boundaries showed that vinculin residues 1-167 are capable of interacting with each of the three vinculin-binding sites in talin . However, all N-terminal deletions abolished binding . The results suggest that the talin-binding site in vinculin has a relatively complex fold, whereas the vinculin-binding motif in talin is contained within a short linear peptide sequence that is repeated three times in the talin rod domain. Cell Growth Differ, 1999 Jun, 10(6), 387 - 96 Nuclear c-Abl is a COOH-terminal repeated domain (CTD)-tyrosine (CTD)-tyrosine kinase-specific for the mammalian RNA polymerase II: possible role in transcription elongation; Baskaran R et al.; The c-Abl tyrosine kinase has been shown to interact with the COOH-terminal repeated domain (CTD) of mammalian RNA polymerase II and can phosphorylate the tyrosine residues in the CTD . Interestingly, the Drosophila or the yeast CTD were not efficiently phosphorylated by the mammalian c-Abl . This species-specificity was found to be determined by the extreme COOH-terminal CTD sequences that are not conserved through evolution . In vitro, COOH-terminal-truncated CTD could neither bind to, nor be phosphorylated by, c-Abl . In vivo, coexpression of a full length CTD prevents c-Abl from inducing the tyrosine phosphorylation of endogenous RNA polymerase II, and such inhibitory effect was not observed with the coexpression of COOH-terminal-truncated CTD . Serine/threonine phosphorylation of the CTD has been linked to the regulation of transcription elongation . Transcription from the human immunodeficiency virus type 1 (HIV-1) promoter requires CTD-phosphorylation, which is stimulated by the viral Tat protein through the recruitment of cellular Ser/Thr CTD kinases . In transient cotransfection experiments, the c-Abl kinase was found to activate the HIV promoter in the absence of Tat . The activation of the HIV promoter required the nuclear localization of c-Abl and could be correlated with increased tyrosine phosphorylation of RNA polymerase II . These observations suggest that tyrosine phosphorylation of the CTD may be functionally equivalent to its serine/threonine phosphorylation in stimulating transcription elongation. J Biol Chem, 1999 Jul 9, 274(28), 19894 - 900 Association of the D2 dopamine receptor third cytoplasmic loop with spinophilin, a protein phosphatase-1-interacting protein; Smith FD et al.; Signaling through D2 class dopamine receptors is crucial to correct brain development and function, and dysfunction of this system is implicated in major neurological disorders such as Parkinson's disease and schizophrenia . To investigate potential novel mechanisms of D2 receptor regulation, the third cytoplasmic loop of the D2 dopamine receptor was used to screen a rat hippocampal yeast two-hybrid library . Spinophilin, a recently characterized F-actin and protein phosphatase-1-binding protein with a single PDZ domain was identified as a protein that specifically associates with this region of D2 receptors . A direct interaction between spinophilin and the D2 receptor was confirmed in vitro using recombinant fusion proteins . The portion of spinophilin responsible for interacting with the D2 third cytoplasmic loop was narrowed to a region that does not include the actin-binding domain, the PDZ domain, or the coiled-coil . This region is distinct from the site of interaction with protein phosphatase-1, and both D2 receptors and protein phosphatase-1 may bind spinophilin at the same time . The interaction is not mediated via the unique 29-amino acid insert in D2long; both D2long and D2short third cytoplasmic loops interact with spinophilin in vitro and in yeast two-hybrid assays . Expression of D2 receptors containing an extracellular hemagglutinin epitope in Madin-Darby canine kidney cells results in co-localization of receptor and endogenous spinophilin as determined by immunocytochemistry using antibodies directed against spinophilin and the HA tag . We hypothesize that spinophilin is important for establishing a signaling complex for dopaminergic neurotransmission through D2 receptors by linking receptors to downstream signaling molecules and the actin cytoskeleton. Nat Genet, 1999 Jul, 22(3), 305 - 8 The gene mutated in thiamine-responsive anaemia with diabetes and deafness (TRMA) encodes a functional thiamine transporter; Fleming JC et al.; Thiamine-responsive megaloblastic anaemia with diabetes and deafness (TRMA; MIM 249270) is an autosomal recessive disease thought to be due to a defect in thiamine (vitamin B1) transport . Pharmacological doses of thiamine correct the anaemia, and in some cases improve the diabetes, although progressive sensorineural deafness is irreversible . Previous studies localized the TRMA gene to a 4-cM region on chromosome 1q23.3 (ref . 5), and fine-mapping has recently narrowed that region further . We have previously demonstrated that fibroblasts from people with TRMA lack high-affinity thiamine transport . Expression of a gene encoding a known yeast thiamine transporter, THI10 (refs 8-10), in TRMA mutant cells prevents apoptotic cell death in thiamine-depleted medium . On the basis of these studies, we hypothesized that a defective thiamine transporter causes TRMA . We undertook a candidate gene approach to identify putative thiamine transporters in the 1q23.3 critical region . Here we present evidence that the gene SLC19A2 (for solute carrier family 19 (thiamine transporter), member 2) encodes the first known mammalian thiamine transporter, which we designate thiamine transporter-1 (THTR-1). Nat Genet, 1999 Jul, 22(3), 291 - 4 Mutations in the gene encoding 3 beta-hydroxysteroid-delta 8, delta 7-isomerase cause X-linked dominant Conradi-Hünermann syndrome; Braverman N et al.; X-linked dominant Conradi-Hunermann syndrome (CDPX2; MIM 302960) is one of a group of disorders with aberrant punctate calcification in cartilage, or chondrodysplasia punctata (CDP) . This is most prominent around the vertebral column, pelvis and long bones in CPDX2 . Additionally, CDPX2 patients may have asymmetric rhizomesomelia, sectorial cataracts, patchy alopecia, ichthyosis and atrophoderma . The phenotype in CDPX2 females ranges from stillborn to mildly affected individuals identified in adulthood . CDPX2 is presumed lethal in males, although a few affected males have been reported . We found increased 8(9)-cholestenol and 8-dehydrocholesterol in tissue samples from seven female probands with CDPX2 (ref . 4) . This pattern of accumulated cholesterol intermediates suggested a deficiency of 3beta-hydroxysteroid-delta8,delta7-isomerase (sterol-delta8-isomerase), which catalyses an intermediate step in the conversion of lanosterol to cholesterol . A candidate gene encoding a sterol-delta8-isomerase (EBP) has been identified and mapped to Xp11.22-p11.23 (refs 5,6) . Using SSCP analysis and sequencing of genomic DNA, we found EBP mutations in all probands . We confirmed the functional significance of two missense alleles by expressing them in a sterol-delta8-isomerase-deficient yeast strain . Our results indicate that defects in sterol-delta8-isomerase cause CDPX2 and suggest a role for sterols in bone development. J Biol Chem, 1999 Jul 9, 274(28), 19771 - 7 Cloning and characterization of a novel RING finger protein that interacts with class V myosins; El-Husseini AE et al.; We have identified a novel protein (BERP) that is a specific partner for the tail domain of myosin V . Class V myosins are a family of molecular motors thought to interact via their unique C-terminal tails with specific proteins for the targeted transport of organelles . BERP is highly expressed in brain and contains an N-terminal RING finger, followed by a B-box zinc finger, a coiled-coil (RBCC domain), and a unique C-terminal beta-propeller domain . A yeast two-hybrid screening indicated that the C-terminal beta-propeller domain mediates binding to the tail of the class V myosin myr6 (myosin Vb) . This interaction was confirmed by immunoprecipitation, which also demonstrated that BERP could associate with myosin Va, the product of the dilute gene . Like myosin Va, BERP is expressed in a punctate pattern in the cytoplasm as well as in the neurites and growth cones of PC12 cells . We also found that the RBCC domain of BERP is involved in protein dimerization . Stable expression of a mutant form of BERP lacking the myosin-binding domain but containing the dimerization domain resulted in defective PC12 cell spreading and prevented neurite outgrowth in response to nerve growth factor . Our studies present a novel interaction for the beta-propeller domain and provide evidence for a role for BERP in myosin V-mediated cargo transport. Trends Biochem Sci, 1999 Jul, 24(7), 271 - 5 DNA recombination: the replication connection; Haber JE; Chromosomal double-strand breaks (DSBs) arise after exposure to ionizing radiation or enzymatic cleavage, but especially during the process of DNA replication itself . Homologous recombination plays a critical role in repair of such DSBs . There has been significant progress in our understanding of two processes that occur in DSB repair: gene conversion and recombination-dependent DNA replication . Recent evidence suggests that gene conversion and break-induced replication are related processes that both begin with the establishment of a replication fork in which both leading- and lagging-strand synthesis occur . There has also been much progress in characterization of the biochemical roles of recombination proteins that are highly conserved from yeast to humans. Appl Environ Microbiol, 1999 Jul, 65(7), 2926 - 33 Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus trichoderma harzianum rifai 1295-22 Altomare C, Norvell WA, Bjorkman T, Harman GE. We investigated the capability of the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of chelating metabolites, and redox activity . T-22 was able to solubilize MnO2, metallic zinc, and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium, as determined by inductively coupled plasma emission spectroscopy . Acidification was not the major mechanism of solubilization since the pH of cultures never fell below 5.0 and in cultures containing MnO2 the pH rose from 6.8 to 7.4 . Organic acids were not detected by high-performance thin-layer chromatography in the culture filtrates . Fe2O3, MnO2, Zn, and rock phosphate were also solubilized by cell-free culture filtrates . The chelating activity of T-22 culture filtrates was determined by a method based on measurement of the equilibrium concentration of the chrome azurol S complex in the presence of other chelating substances . A size exclusion chromatographic separation of the components of the culture filtrates indicated the presence of a complexed form of Fe but no chelation of Mn . In liquid culture, T . harzianum T-22 also produced diffusible metabolites capable of reducing Fe(III) and Cu(II), as determined by the formation of Fe(II)-Na2-bathophenanthrolinedisulfonic acid and Cu(I)-Na2-2, 9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid complexes . This is the first report of the ability of a Trichoderma strain to solubilize insoluble or sparingly soluble minerals . This activity may explain, at least partially, the ability of T-22 to increase plant growth . Solubilization of metal oxides by Trichoderma involves both chelation and reduction . Both of these mechanisms also play a role in biocontrol of plant pathogens, and they may be part of a multiple-component action exerted by T-22 to achieve effective biocontrol under a variety of environmental conditions. Antimicrob Agents Chemother, 1999 Jul, 43(7), 1716 - 8 Activities of sordarins in murine histoplasmosis; Graybill JR et al.; Sordarins are new antifungals which inhibit fungal protein synthesis by blocking elongation factor 2 . Three compounds were evaluated in a murine model of histoplasmosis . Immune-competent mice were infected intravenously with 10(6) to 10(8) CFU of Histoplasma capsulatum yeast cells . Mice were treated either orally with sordarins or fluconazole from day 2 through 8 after infection or intraperitoneally with amphotericin B during the same period . Protection was measured by increased rates of survival for 30 days after infection or reduction of lung or kidney tissue counts 9 days after infection . All three of the antifungal drugs tested were protective compared with controls . Sordarins were effective at doses as low as 2 mg/kg of body weight/day . This novel class of drugs compared favorably with amphotericin B and fluconazole for the treatment of histoplasmosis. Exp Cell Res, 1999 Jul 10, 250(1), 142 - 54 Characterization of NF-L and betaIISigma1-spectrin interaction in live cells; Macioce P et al.; Neurofilaments (NFs) are neuron-specific intermediate filaments (IFs) composed of three different subunits, NF-L, NF-M, and NF-H . NFs move down the axon with the slow component of axonal transport, together with microtubules, microfilaments, and alphaII/betaII-spectrin (nonerythroid spectrin or fodrin) . It has been shown that alphaII/betaII-spectrin is closely associated with NFs in vivo and that betaII-spectrin subunit binds to NF-L filaments in vitro . In the present study we seek to elucidate the relationship between NF-L and betaII-spectrin in vivo . We transiently transfected full-length NF-L and carboxyl-terminal deleted NF-L mutants in SW13 Cl.2 Vim- cells, which lack an endogenous IF network and express alphaII/betaIISigma1-spectrin . Double-immunofluorescence and electron microscopy studies showed that a large portion of betaIISigma1-spectrin colocalizes with the structures formed by NF-L proteins . We found a similar association between NF-L proteins and actin . However, coimmunoprecipitation experiments in transfected cells and the yeast two-hybrid system results failed to demonstrate a direct interaction of NF-L with betaIISigma1-spectrin in vivo . The presence of another protein that acts as a bridge between the membrane skeleton and neurofilaments or modulating their association may therefore be required . Biochemistry, 1999 Jun 29, 38(26), 8204 - 16 An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha; Plastino J et al.; The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis . The D319E mutant catalyzes the oxidation of methylamine and phenethylamine, but not that of benzylamine . kcat/Km for methylamine is found to be 80-fold reduced compared to that of the wild type . Viscosogen and substrate and solvent deuteration have no effect on this parameter for D319E, which is suggestive of limitation of kcat/Km by a conformational change . This conformational change is proposed to be the movement of the cofactor into a productive orientation upon the binding of substrate . In the absence of substrate, a flipped cofactor orientation is likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen . kcat for D319E methylamine oxidase is reduced 200-fold compared to that of the wild type and is unaffected by substrate deuteration, but displays a substantial solvent isotope effect . A 428 nm absorbance is evident under conditions of saturating methylamine and oxygen with D319E . The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity . Resonance Raman spectroscopy indicates the formation of a covalent ammonia adduct and identifies it as the deprotonated iminoquinone . In contrast, when the D319E mutant is reacted with ammonia, it gives predominantly a 340-350 nm species . This absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct formation . Resonance Raman spectroscopic examination of the steady state species of D319E methylamine oxidation, in combination with the kinetic data, indicates that the 428 nm species is the deprotonated iminoquinone produced upon reoxidation of the reduced cofactor . A model is proposed in which a central role of the active site base is to position the free cofactor and several enzyme intermediates for optimal activity. Biochemistry, 1999 Jun 15, 38(24), 7828 - 36 Comparison of the substrate specificities of human liver cytochrome P450s 2C9 and 2C18: application to the design of a specific substrate of CYP 2C18; Minoletti C et al.; A series of 2-aroylthiophenes derived from tienilic acid by replacement of its OCH2COOH substituent with groups bearing various functions have been synthesized and studied as possible substrates of recombinant human liver cytochrome P450s 2C9 and 2C18 expressed in yeast . Whereas only compounds bearing a negative charge acted as substrates of CYP 2C9 and were hydroxylated at position 5 of their thiophene ring at a significant rate, many neutral 2-aroylthiophenes were 5-hydroxylated by CYP 2C18 with kcat values of >2 min-1 . Among the various compounds that were studied, those bearing an alcohol function were the best CYP 2C18 substrates . One of them, compound 3, which bears a terminal O(CH2)3OH function, appeared to be a particularly good substrate of CYP 2C18 . It was regioselectively hydroxylated by CYP 2C18 at position 5 of its thiophene ring with a KM value of 9 +/- 1 microM and a kcat value of 125 +/- 25 min-1, which are the highest described so far for a CYP 2C . A comparison of the oxidations of 3, by yeast-expressed CYP 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5, showed that only CYP 2C8, 2C18, and 2C19 were able to catalyze the 5-hydroxylation of 3 . However, the catalytic efficiency of CYP 2C18 for that reaction was considerably higher (kcat/KM value being 3-4 orders of magnitude larger than those found for CYP 2C8 and 2C19) . Several human P450s exhibited small activities for the oxidative O-dealkylation of 3 . The four recombinant CYP 2Cs were the best catalysts for that reaction (kcat between 1 and 5 min-1) when compared to all the P450s that were tested, even though it is a minor reaction in the case of CYP 2C18 . All these results show that compound 3 is a new, selective, and highly efficient substrate for CYP 2C18 that should be useful for the study of this P450 in various organs and tissues . They also suggest some key differences between the active sites of CYP 2C9 and CYP 2C18 for substrate recognition. FASEB J, 1999 Jul, 13(10), 1249 - 57 A Sec7-related protein in Paramecium; Nair S et al.; We have cloned and sequenced a SEC7-related gene in Paramecium tetraurelia that contains an open reading frame for 1135 amino acids encoding a 133 kDa protein, PSec7 . Sec7, first identified in vesicular trafficking mutants in yeast, and its phylogenetic homologues function as guanine-nucleotide exchange factors for small G-proteins such as ARF (ADP-ribosylation factor) . The deduced amino acid sequence in PSec7 for the motifs that form the ARF binding site are more than 70% identical to yeast Sec7 and similarly identical to ARNO, the human ARF exchange factor, with correct positioning of the critical glutamic acid residue within the motif region . Overall, the identity of PSec7 to yeast Sec7 is 32% . The deduced amino acid sequence also has five sequences that resemble IQ motifs, EF hand binding domains found in all myosins, and two pleckstrin homology domains . Similar sequences are present in yeast Sec7 and other Sec7-related molecules . A protein kinase A phosphorylation site may also be present . Southern blots suggest that a single gene encodes PSec7 . Northern blots show that the message encoding PSec7 is induced on deciliation, followed by ciliogenesis, which suggests a role for PSec7 in cilia such as transport or targeting of ciliary membrane components. J Cell Biol, 1999 Jun 28, 145(7), 1471 - 82 The zinc finger protein A20 inhibits TNF-induced NF-kappaB-dependent gene expression by interfering with an RIP- or TRAF2-mediated transactivation signal and directly binds to a novel NF-kappaB-inhibiting protein ABIN; Heyninck K et al.; The zinc finger protein A20 is a tumor necrosis factor (TNF)- and interleukin 1 (IL-1)-inducible protein that negatively regulates nuclear factor-kappa B (NF-kappaB)-dependent gene expression . However, the molecular mechanism by which A20 exerts this effect is still unclear . We show that A20 does not inhibit TNF- induced nuclear translocation and DNA binding of NF-kappaB, although it completely prevents the TNF- induced activation of an NF-kappaB-dependent reporter gene, as well as TNF-induced IL-6 and granulocyte macrophage-colony stimulating factor gene expression . Moreover, NF-kappaB activation induced by overexpression of the TNF receptor-associated proteins TNF receptor-associated death domain protein (TRADD), receptor interacting protein (RIP), and TNF recep- tor-associated factor 2 (TRAF2) was also inhibited by expression of A20, whereas NF-kappaB activation induced by overexpression of NF-kappaB-inducing kinase (NIK) or the human T cell leukemia virus type 1 (HTLV-1) Tax was unaffected . These results demonstrate that A20 inhibits NF-kappaB-dependent gene expression by interfering with a novel TNF-induced and RIP- or TRAF2-mediated pathway that is different from the NIK-IkappaB kinase pathway and that is specifically involved in the transactivation of NF-kappaB . Via yeast two-hybrid screening, we found that A20 binds to a novel protein, ABIN, which mimics the NF-kappaB inhibiting effects of A20 upon overexpression, suggesting that the effect of A20 is mediated by its interaction with this NF-kappaB inhibiting protein, ABIN. Endocrinology, 1999 Jul, 140(7), 3318 - 27 P34H sperm protein is preferentially expressed by the human corpus epididymidis; Legare C et al.; During epididymal transit, mammalian spermatozoa acquire new surface proteins that are necessary for gamete interaction . We have previously described a 34-kDa human epididymal sperm protein, P34H, that has been shown to be involved in sperm-zona pellucida interaction . In the present study, we report the cloning and characterization of the full-length complementary DNA encoding human P34H . The predicted amino acid sequence revealed 65% identity with P26h, the hamster counterpart of the P34H . The deduced P34H amino acid sequence revealed a 71% similarity with a pig lung tetrameric carbonyl reductase, a member of the short chain dehydrogenase/ reductase family proteins . Northern blot analysis revealed that P34H messenger RNA (mRNA) was highly expressed in the human epididymis, principally in the corpus region . A single 912-bp P34H transcript was detected . In situ hybridization experiments showed that the P34H mRNA was predominantly expressed in the proximal and distal sections of the corpus epididymidis . The staining was restricted to the principal cells of the epididymal epithelium . The localization of P34H mRNA was in agreement with the appearance of P34H protein along the male reproductive tract . Western blot analysis revealed that recombinant P34H expressed by a