Yeasts

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 yeast expression system, is antigenically related to the native P34H sperm protein . Based on its pattern of expression and its function in one of the key steps leading to fertilization, P34H can be considered as a marker of epididymal sperm maturation in humans.

Microbiol Immunol, 1999, 43(4), 323 - 30
Role of superoxide anion in the fungicidal activity of murine peritoneal exudate macrophages against Penicillium marneffei; Kudeken N et al.; Penicillium marneffei is an important opportunistic fungal pathogen . The mechanisms of host defense against P . marneffei are not fully understood . In the present study, we, for the first time, investigated the role of superoxide anion (O2-) in the killing of two forms of P . marneffei, yeast cells and conidia, and the role of this killing mediator in the fungicidal activity of IFN-gamma-stimulated murine peritoneal macrophages . P . marneffei yeast cells were susceptible to the killing effect of activated macrophages and chemically generated O2, while conidia were not . These results suggested that O2- played some role in the fungicidal activity of macrophages . However, an oxygen radical scavenger, superoxide dismutase (SOD), did not suppress, but rather enhanced the fungicidal activity of IFN-gamma-stimulated macrophages against P . marneffei yeast cells . This inconsistency was explained by the release of insufficient concentrations of O2- by activated macrophages as compared with the amount of O2- necessary for the killing of yeast cells, which was predicted in a chemical generating system . On the other hand, SOD enhanced the production of nitric oxide (NO) by IFN-gamma-activated macrophages, and their increased fungicidal activity was significantly inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of NO synthase . Our results suggested that O2- does not function as the killing mediator of macrophages against P . marneffei, but rather plays an important role in the regulation of the NO-mediated killing system by suppressing NO production.

Eur J Clin Microbiol Infect Dis, 1999 Apr, 18(4), 302 - 4
In vitro activity of the new echinocandin antifungal, MK-0991, against common and uncommon clinical isolates of Candida species; Barchiesi F et al.; A broth macrodilution method, performed as recommended by the National Committee for Clinical Laboratory Standards, was used for comparative testing of the new echinocandin antifungal agent MK-0991 and fluconazole against 50 yeast isolates belonging to 12 species of Candida . MK-0991 was shown to be highly effective against both fluconazole-susceptible and -resistant Candida spp., yielding minimum inhibitory concentrations ranging from < or = 0.19 to 0.78 microg/ml . Fungicidal activity was exerted at < or = 1.5 microg/ml for 73% of the isolates tested . This study suggests that MK-0991 has significant potential for clinical development.

Eur J Cell Biol, 1999 May, 78(5), 305 - 10
The ADP-ribosylation factor GTPase-activating protein Glo3p is involved in ER retrieval; Dogic D et al.; Retrograde transport of proteins from the Golgi to the endoplasmic reticulum (ER) has been the subject of some interest in the recent past . Here a new thermosensitive yeast mutant defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum was characterized . The ret4-1 mutant also exhibited a selective defect in forward ER-to-Golgi transport of some secreted proteins at the non-permissive temperature . The corresponding RET4 gene was found to encode Glo3p, a GTPase-activating protein (GAP) specific for ADP-ribosylation factor (ARF) . In vitro, the Glo3 thermosensitive mutant showed a reduced ARF1-GAP activity . The Glo3 protein belongs to a family of zinc finger proteins that may include additional ARF-GAPs . Gene deletion experiments of other family members showed that only GLO3 deletion resulted in impaired retrieval of dilysine-tagged proteins back to the ER . These results demonstrate that Glo3p is the main ARF-GAP specifically involved in ER retrieval.

Glia, 1999 Apr, 26(2), 176 - 85
Edg-2 in myelin-forming cells: isoforms, genomic mapping, and exclusion in Charcot-Marie-Tooth disease; Allard J et al.; Edg-2 is an heptahelical receptor whose spatio-temporal distribution during rat brain development is consistent with a role in the control of myelination . We have now identified two splice variants of Edg-2 mRNA in rat brain that encode two receptor isoforms differing by a stretch of 18 amino acids in the NH2-terminal extracellular tail of the receptor . Prenatally (i.e., before oligodendrocyte myelination), the two variants detected by selective in situ hybridization are equally abundant, vary in parallel, and remain restricted to proliferative zones in the brain . Postnatally, the long isoform becomes predominant in myelinating structures, where its abundance increases sharply during the period of myelination . In the adult human brain, only the long variant was detected, while in situ hybridization showed it selectively expressed in the white matter and in clusters of cells showing features of oligodendrocytes of the temporal cerebral cortex . Consequently, the human Edg-2 gene was studied to assess its possible contribution in inherited neuropathies . The coding sequence was found to be contained in three exons and to map to chromosome 9q31.3-32 by using radiation hybrid panel and Yeast-Artificial Chromosomes . Two intragenic bi-allelic polymorphisms and a rare mutation were identified . As a first application to molecular genetic studies, they were used to exclude the Edg-2 gene in six families with phenotype of demyelinating Charcot-Marie-Tooth disease of unknown origin.

Pediatr Clin North Am, 1999 Jun, 46(3), 631 - 42, ix
Gynecologic care of medically complicated adolescents; Owens K et al.; Gynecologic care of adolescents presents a challenge under the best of circumstances, but when the patient has significant medical processes that interact with the process of puberty, the care of these patients may become extremely difficult . A review of the more common medical illnesses of adolescents and the interaction on the events of puberty and normal menstrual function is presented with emphasis on contraception and future fertility . Although many of the contraceptive options present a possible increased risk to these patients, it must be kept in mind that these adolescent patients will develop emotionally and become sexually active at some point in their lives, and the potential risk of the resultant pregnancy must be weighed carefully . The various options of management for gynecologic problems are discussedPIP: This paper discusses issues on gynecologic care of medically complicated adolescents . Gynecologists and health personnel should give special attention to the chronic medical illness or disabilities of adolescents throughout their development . They must understand the interactions of the normal endocrinology of development and the pathophysiology of the medical illness . Chronically ill and disabled adolescents are least likely to receive information and guidance on sexual issues . They need to know the potential impact of their illness on pregnancy; genetic risk of their illness, treatment, and disability pose to their unborn children; and their limitations on both sexual function and fertility . In addition, it inverted question marks important that these adolescents be given counseling addressing social and judgmental skills in cases of social isolation . Illnesses related to pubertal developments include: diabetes mellitus; cystic fibrosis; renal insufficiency and failure; gynecologic issues with various other chronic illnesses; sickle cell disease; seizure disorders; asthma; tuberculosis; inflammatory bowel disease; chronic yeast infections; menstrual abnormalities and coagulopathies or thrombocytopenia; adolescent and childhood malignancies; and developmental delayed and physical disability .

Mamm Genome, 1999 Jul, 10(7), 662 - 9
Genomic organization and expression analysis of the mouse qkI locus; Kondo T et al.; qkI, encoding a KH domain-containing RNA binding protein, has been isolated as a candidate gene for the mouse neurological mutation quaking . Here, we describe detailed studies on its genomic structure and expression pattern . We isolated approximately 1 Mb of genomic region containing the quaking locus and determined its genomic organization . The qkI locus contains at least 9 exons spanning approximately 65 kb of DNA . It gives rise to six distinct transcripts encoding, theoretically, five different protein isoforms . Exons 1 through 4 are shared by all the transcripts, whereas coding exons and two distinct 3'-UTRs downstream to the exon 4 are differentially utilized . One isoform has a truncated KH domain and may act as an antagonist to the others . These findings and identification of a single transcription initiation site suggest that differential expression of each transcript is regulated by alternative splicing . Expression of each alternative transcript and protein product was also examined . Two types of transcripts, 5 kb-A and B, are most abundant in the brain of newborn mice and are gradually downregulated thereafter . In contrast, the other three messages, 6 kb, 7 kb-A and B, increase as myelination proceeds and peak at 2 weeks of age, corresponding to the most active stage of myelination . Although the qkI messages and their products are abundant in brain and heart, a lower level of expression was found in various other tissues tested . Alternative transcripts that share the same 3'-UTR showed very similar expression patterns, suggesting a regulatory role of the 3'-UTRs in qkI gene expression.

Mamm Genome, 1999 Jul, 10(7), 657 - 61
Genetic and physical maps of the mouse rd3 locus; exclusion of the ortholog of USH2A; Danciger JS et al.; The rd3 retinal degeneration gene was previously mapped 10 +/- 2.5 cM distal to Akp1 on mouse Chromosome (Chr) 1 (Chang et al., 1993), a region that may be homologous to the locus of the human USH2A gene, which carries mutations responsible for Usher IIa retinal degeneration/hearing loss syndrome . An intercross from an Rb(11, 13)4Bnr(rd3/rd3) x C57BL/6J mating was set up, 428 F2 meioses were analyzed, and the rd3 gene was placed between the markers D1MIT292/D1MIT209 and D1MIT510, a distance of 1.40 +/- 0.57 cM . These flanking markers and the mouse ortholog of USH2A (Mush2a) were mapped in the T31 mouse radiation hybrid (RH) panel, with the result that D1MIT292/D1MIT209 and D1MIT510 were 7.9 cR3000 apart ( approximately 800 kb), and Mush2a was > 30 cR3000 proximal to the pair, excluding it from the rd3 locus . A contig spanning the rd3 locus and consisting of 2 YACs and one BAC was generated, and Mush2a was absent from it, confirming its exclusion from the locus . Comparison of adjacent marker pairs in the Whitehead genetic map and our genetic map showed some discrepancies in order of markers and genetic distances . Comparison of our genetic map and the RH map showed some highly skewed relationships between genetic and physical distances.

Carcinogenesis, 1999 Jul, 20(7), 1161 - 8
Taxol, vincristine or nocodazole induces lethality in G1-checkpoint-defective human astrocytoma U373MG cells by triggering hyperploid progression; Hong FD et al.; In this report, we describe a novel lytic mechanism exploited by antimicrotubule drugs (AMDs) such as Taxol which are frequently used to treat multiple human cancers including breast and ovarian cancers . In cells lacking the G1-arresting capacity due to the defect in retinoblastoma or p53 gene function, AMDs trigger hyperploid progression and death . The hyperploid progression occurs via continued cell-cycle progression without cell division . Blocking hyperploid progression through hydroxyurea or ectopically expressed p27(Kip1), a G1-specific Cdk inhibitor, abrogates AMD cytotoxicity . Thus, AMDs induce lethality in G1-checkpoint-defective cells by triggering hyperploid progression . The phenomenon is reminiscent of that observed previously with bub-1 yeast mutant . The potential significance of this finding lies in that hyperploid progression-mediated death may be exploited to develop a therapy with tumor-specificity at the genetic level . As a large fraction of human cancers are mutated in p53 gene, it may have a wide therapeutic applicability.

J Biol Chem, 1999 Jul 2, 274(27), 19025 - 34
Association of atypical protein kinase C isotypes with the docker protein FRS2 in fibroblast growth factor signaling; Lim YP et al.; FRS2 is a docker protein that recruits signaling proteins to the plasma membrane in fibroblast growth factor signal transduction . We report here that FRS2 was associated with PKC lambda when Swiss 3T3 cells were stimulated with basic fibroblast growth factor . PKC zeta, the other member of the atypical PKC subfamily, could also bind FRS2 . The association between FRS2 and PKC lambda is likely to be direct as shown by yeast two-hybrid analysis . The C-terminal fragments of FRS2 (amino acid residues 300-508) and SNT2 (amino acids 281-492), an isoform bearing 50% identity to FRS2, interacted with PKC lambda at a region (amino acids 240-562) that encompasses the catalytic domain . In vitro kinase assays revealed neither FRS2 nor SNT2 was a substrate of PKC lambda or zeta . Mutation of the alanine residue (Ala-120) to glutamate in the pseudo-substrate region of PKC lambda results in a constitutively active kinase that exhibited more than 2-fold greater binding to FRS2 in vitro than its "closed" wild-type counterpart . Tyrosine phosphorylation of FRS2 did not affect its binding to the constitutively active PKC lambda mutant, suggesting that the activation of PKC lambda is necessary and sufficient for its association with FRS2 . It is likely that FRS2 serves as an anchoring protein for targeting activated atypical PKCs to the cell plasma membrane in signaling pathways.

J Biol Chem, 1999 Jul 2, 274(27), 19003 - 10
Direct interaction in T-cells between thetaPKC and the tyrosine kinase p59fyn; Ron D et al.; The protein kinase C (PKC) family has been clearly implicated in T-cell activation as have several nonreceptor protein-tyrosine kinases associated with the T-cell receptor, including p59fyn . This report demonstrates that thetaPKC and p59fyn specifically interact in vitro, in the yeast two-hybrid system, and in T-cells . Further indications of direct interaction are that p59fyn potentiates thetaPKC catalytic activity and that thetaPKC is a substrate for tyrosine phosphorylation by p59fyn . This interaction may account for the localization of thetaPKC following T-cell activation, pharmacological disruption of which results in specific cell-signaling defects . The demonstration of a physical interaction between a PKC and a protein-tyrosine kinase expands the class of PKC-anchoring proteins (receptors for activated C kinases (RACKs)) and demonstrates a direct connection between these two major T-cell-signaling pathways.

DNA Res, 1999 Apr 30, 6(2), 131 - 6
Complete DNA sequence and characterization of a 330-kb VNTR-rich region on chromosome 6q27 that is commonly deleted in ovarian cancer; Minaguchi T et al.; We report the complete genomic DNA sequence and the characterization of a 330-kb region on chromosome 6q27 that is often deleted in ovarian cancers . Using computer programs to predict exonic sequences, we isolated four novel genes, HGC6.1-4, as well as the known AF-6 gene . None of the deduced products of the novel genes exhibited significant homology to previously known proteins . We also identified ten microsatellites and 12 different VNTR sequences within the target region . HGC6.3 contained a VNTR within a coding exon, each repeat consisting of 42 nucleotides; the predicted 14-amino-acid consensus unit is MTPTVFSSQHTAGG . At least nine different sizes of this VNTR locus were detected among 20 unrelated DNA samples from caucasians . The polymorphic markers and the transcript map documented here may contribute to identification of novel genes or allelic aberrations associated with the development of ovarian cancers.

J Food Prot, 1999 Jun, 62(6), 657 - 61
Medium optimization for the production of antioxidants from Aspergillus candidus; Yen GC et al.; The objective of this study was to optimize the factors for the production of antioxidant from Aspergillus candidus CCRC 31543 . Extracts of broth filtrate had higher antioxidant activity (inhibition of peroxidation {IP} >98%) when sucrose or lactose was used as a carbon source . Sucrose in the medium also resulted in a higher yield of extracts . Ethyl acetate extracts had the highest yield and antioxidant activity compared with the other two solvents . For the production of antioxidant, inorganic nitrogen sources were found to be more suitable than organic nitrogen sources, and ammonium sulfate was better than sodium nitrate . Yeast extract had a strong influence on the yield of antioxidant extracts . Both mycelium and broth filtrate of A . candidus CCRC 31543 showed similar antioxidant activity (IP = 95%), and they also had similar extraction yields.

Electrophoresis, 1999 Jun, 20(6), 1233 - 8
Horizontal two-dimensional electrophoresis of complex DNA samples using precast gel systems; Schickle H et al.; We have developed a simplified procedure for the separation of enzyme-digested genomic DNA, or of complex mixtures of cloned DNA, into two dimensions . The procedure relies on the use of precast gels for horizontal electrophoretic separations . Precast agarose-type gels are used for the first-dimensional separation of fragments based on size . Precast polyacrylamide gels are used for the second-dimensional separation of fragments . The separated fragments are subjected to enzymatic digestion in situ prior to their transfer to the second-dimensional gel . Applications of this procedure include the analysis of DNA libraries, analysis of yeast artificial chromosomes (YACs) as well as other similar preparations, and the screening of genomic DNA for the occurrence of multi-copy DNA fragments as in the case of genomic amplifications in cancer.

Cell Death Differ, 1999 Apr, 6(4), 314 - 25
A novel adenovirus E1B19K-binding protein B5 inhibits apoptosis induced by Nip3 by forming a heterodimer through the C-terminal hydrophobic region; Ohi N et al.; The adenovirus E1B19K protein inhibits apoptosis induced by E1A and other divergent signals . The cellular proteins that interact with E1B19K have been analyzed by isolating cDNA clones by the yeast two hybrid system . One of these clones encodes B5 which consists of 219 amino acid residues and contains the putative BH3 and transmembrane regions . B5 binds strongly to Nip3 and itself, weakly to E1B19K, but not to Bcl-2 and localizes in nuclear envelope, endoplasmic reticulum and mitochondria . B5 has sequence homology with Nip3 in the middle and C-terminal regions, but not in the N-terminal region . Unlike other E1B19K binding BH3 proteins so far characterized, B5 does not induce apoptosis, but inhibits apoptosis induced by Nip3 . However the deletion mutant B5Delta1-31 lacking the N-terminus does induce apoptosis, although weaker than does Nip3, suggesting that the N-terminal region is masking the apoptosis-inducing capacity of B5.

Mech Dev, 1999 May, 83(1-2), 95 - 105
A double interaction screen identifies positive and negative ftz gene regulators and ftz-interacting proteins; Yu Y et al.; Regulatory genes directing embryonic development are expressed in complex patterns . The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a striped pattern that is controlled by several discrete and large cis- regulatory elements . One key cis-element is the ftz proximal enhancer which is required for stripe establishment and which mediates autoregulation by direct binding of Ftz protein . To identify the trans-acting factors that regulate ftz expression and autoregulation, we developed a modified yeast two hybrid screen, the Double Interaction Screen (DIS) . The DIS was designed to isolate both DNA binding transcriptional regulators that interact with the proximal enhancer and proteins that interact with Ftz itself when it is bound to the enhancer . The screen identified two candidate Ftz protein cofactors as well as activators and repressors of ftz transcription that bind directly to the enhancer . One of these (Tramtrack (Ttk)) was previously shown to bind to at least five sites in the proximal enhancer; genetic studies suggested that Ttk acts as a repressor of ftz in the embryo . Here we show that, in yeast cells, Ttk protein strongly activates transcription, suggesting that yeast may be missing a necessary co-repressor which is present in Drosophila embryos . Further, we have characterized the activity of a second candidate ftz repressor isolated in the screen - the product of the pair-rule gene sloppy paired - a member of the forkhead family . We show that Slp1 is a DNA binding protein . We have identified a high affinity binding site for Slp1 in the ftz proximal enhancer . Slp1 represses transcription via this binding site in yeast cells, consistent with its role as a direct repressor of ftz stripes in interstripe regions during late stages of embryogenesis . The DIS should be a generally useful method to identify DNA binding transcriptional regulators and protein partners of previously characterized DNA binding proteins .

Brain Res Mol Brain Res, 1999 Jun 18, 70(1), 66 - 73
Identification of a novel lithium regulated gene in rat brain; Wang JF et al.; Differential display PCR was used to identify genes regulated by mood stabilizer lithium in rat cerebral cortex . A differentially displayed lithium regulated gene fragment was isolated in rat cerebral cortex after chronic treatment with lithium (1.69 g/kg, p.o . ) for three weeks . A 1216-nucleotide cDNA for a novel lithium regulated gene (NLRG) was isolated from a rat brain cDNA library with RACE (rapid amplification of 5' cDNA end) PCR using a prime from the differentially displayed NLRG gene fragment . The deduced protein sequence was 321 amino acids long, and shows a significant homology with yeast nitrogen permease regulator 2 (NPR2) . NLRG expression induced by lithium was confirmed by Northern and slot blot analysis in rat cerebral cortex and neuroblastomaxglioma NG108-15 cells, respectively . In situ hybridization revealed that chronic treatment with lithium increased NLRG gene expression in frontal cortex and hippocampus, but not in striatum, hypothalamus and thalamus regions of rat brain . These results suggest a novel target for lithium which may be relevant to its mechanism of action .

Blood, 1999 Jul 1, 94(1), 225 - 32
Isochromosome 17q in blast crisis of chronic myeloid leukemia and in other hematologic malignancies is the result of clustered breakpoints in 17p11 and is not associated with coding TP53 mutations; Fioretos T et al.; An isochromosome of the long arm of chromosome 17, i(17q), is the most frequent genetic abnormality observed during the disease progression of Philadelphia chromosome-positive chronic myeloid leukemia (CML), and has been described as the sole anomaly in various other hematologic malignancies . The i(17q) hence plays a presumably important pathogenetic role both in leukemia development and progression . This notwithstanding, the molecular consequences of this abnormality have not been investigated in detail . We have analyzed 21 hematologic malignancies (8 CML in blast crisis, 8 myelodysplastic syndromes {MDS}, 2 acute myeloid leukemias, 2 chronic lymphocytic leukemias, and 1 acute lymphoblastic leukemia) with i(17q) by fluorescence in situ hybridization (FISH) . Using a yeast artificial chromosome (YAC) contig, derived from the short arm of chromosome 17, all cases were shown to have a breakpoint in 17p . In 12 cases, the breaks occurred within the Smith-Magenis Syndrome (SMS) common deletion region in 17p11, a gene-rich region which is genetically unstable . In 10 of these 12 cases, we were able to further map the breakpoints to specific markers localized within a single YAC clone . Six other cases showed breakpoints located proximally to the SMS common deletion region, but still within 17p11, and yet another case had a breakpoint distal to this region . Furthermore, using chromosome 17 centromere-specific probes, it could be shown that the majority of the i(17q) chromosomes (11 of 15 investigated cases) were dicentric, ie, they contained two centromeres, strongly suggesting that i(17q) is formed through an intrachromosomal recombination event, and also implicating that the i(17q), in a formal sense, should be designated idic(17)(p11) . Because i(17q) formation results in loss of 17p material, potentially uncovering the effect of a tumor suppressor on the remaining 17p, the occurrence of TP53 mutations was studied in 17 cases by sequencing the entire coding region . In 16 cases, no TP53 mutations were found, whereas one MDS displayed a homozygous deletion of TP53 . Thus, our data suggest that there is no association between i(17q) and coding TP53 mutations, and that another tumor suppressor gene(s), located in proximity of the SMS common deletion region, or in a more distal location, is of pathogenetic importance in i(17q)-associated leukemia.

Mol Genet Metab, 1999 Jul, 67(3), 227 - 35
Isolation and characterization of a novel transcript embedded within HIRA, a gene deleted in DiGeorge syndrome; Pizzuti A et al.; We have isolated a few cDNAs from different human tissues, transcribed from the first intron of HIRA, a gene deleted in the DiGeorge syndrome . These cDNAs are produced by an intronic gene (22k48) which is transcribed by the HIRA opposite strand and is itself arranged in exons and subjected to alternative splicing . The longest continuum cDNA sequence we obtained is 3.6 kb long and contains 3 different exons and 2 introns . 22k48 cDNA is composed of several tandemly arranged repeated elements (Alu, LINEs, CAn) surrounding a unique sequence . In situ hybridization showed the presence of 22k48 RNA in the cytoplasm of CNS and PNS neurons . 22k48 RNA is able to bind cytoplasmic proteins in the range of 45 to 60 kDa . 22k48 is a new member of the small group of genes that are transcribed but not translated, and its haploinsufficiency could contribute to the pathogenesis of the DiGeorge syndrome .

J Virol Methods, 1999 May, 79(2), 205 - 17
Purification and characterization of hepatitis B virus surface antigen particles produced in Drosophila Schneider-2 cells; Deml L et al.; The small surface antigen of hepatitis B virus (HBV) was produced in Drosophila melanogaster Schneider-2 (DS-2) cells transfected stably using an inducible Drosophila metallothionein promoter . Selected clonal DS-2 cell-lines expressed and secreted large quantities of HBsAg particles consisting exclusively of non-glycosylated 25 kDa proteins . HBsAg produced by DS-2 cells had physical and biochemical properties very similar to 22 nm particles derived from the human hepatoma cell-line PLC/PRF/5 . DS-2 cell-derived HBsAg particles were purified near homogeneity by a strategy based on protein concentration, precipitation and ultracentrifugation . The resulting HBsAg product was < 98% pure . A single immunisation of BALB/c mice with both DS-2 and yeast-cell derived purified HBsAg particles without adjuvants elicited a substantial humoral antibody and class-I restricted cytotoxic T lymphocyte (CTL) response . Adsorption of HBsAg particles to aluminium hydroxide resulted in increased levels of HBsAg-specific antibodies . However, CTLs were not elicited by HBsAg/Alum combinations . Thus, stably transfected DS-2 cells provide a useful source for the production of HBV subviral particles for diagnostic and research purposes as well as for novel vaccine development.

J Leukoc Biol, 1999 Jun, 65(6), 808 - 14
Mycoplasma arginini enhances cytotoxicity of thioglycollate-elicited murine macrophages toward YAC-1 tumor cells through production of NO; Ribeiro-Dias F et al.; Bacterial products stimulate macrophage tumoricidal activity through release of tumor necrosis factor (TNF) and nitric oxide (NO) . We show here that thioglycollate-elicited macrophages acquire cytotoxic activity when cocultured with Mycoplasma arginini-infected YAC-1 tumor cells and release TNF and NO . Fixed mycoplasma-infected cells, supernatants from infected-cell cultures, or purified heat-killed mycoplasma obtained from cell-free cultures were all able to induce TNF and NO production . Thus, the mycoplasma per se and not a product of infected cells induce the release of these molecules . Addition of prostaglandin E2 (PGE2) to the cocultures, which reduced TNF release, or antibodies to TNF, did not affect macrophage cytotoxicity nor NO release . Inhibition of NO production by L-NAME or aminoguanidine reduced the cytotoxicity, and treatment with a NO donor was toxic to YAC-1 cells . These results indicate that M . arginini activates thioglycollate-elicited murine macrophages for NO and TNF release increasing their cytotoxic activity toward YAC-1 cells and that this activity is dependent on NO but not TNF release.

J Leukoc Biol, 1999 Jun, 65(6), 771 - 7
The synthetic non-toxic drug 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline inhibits neutrophil production of reactive oxygen species; Harbecke O et al.; The effects of the non-toxic drug 2,3-dimethyl-6(2-dimethylaminoethyl)-6H-indolo-(2,3-b)quinoxaline (B220) on the generation of reactive oxygen species froin human neutrophils were investigated . The data show that B220 inhibits neutrophil release of reactive oxygen species, as well as intracellular generation of reactive oxygen species . The inhibition is not achieved through direct oxygen radical scavenger activity of B220, and the drug has no immediate effects on the activity of the assembled oxidase . Radical production and release were inhibited by all agonists tested {i.e . the protein kinase C-activating phorbol ester phobol myristate acetate; the receptor-specific agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP); and serum-opsonized yeast particles} in the presence of B220 . The drug also inhibits phagocytosis and fMLP-induced mobilization of granules . However, based on the fact that the effects of B220 on phagocytosis and granule mobilization are much less significant than its effect on radical production, we suggest that the signal(s) affected by B220 is (are) located mainly downstream of the point at which the signals are generated to promote oxidase activation and phagocytosis or granule secretion, respectively.

Oncogene, 1999 Jun 17, 18(24), 3608 - 16
MBP1: a novel mutant p53-specific protein partner with oncogenic properties; Gallagher WM et al.; Using a yeast two-hybrid screening strategy with a common tumour-derived p53 mutant as bait, we identified several mutant p53-interacting partners including the known proteins wild-type (wt) p53, hUBC9 and GBP/PIAS1 . In addition, a novel protein partner was identified which we have termed MBP1, for Mutant p53-Binding Protein 1 . MBP1 is a new member of the emerging fibulin gene family, which currently comprises fibulin-1, fibulin-2 and S1-5 . Expression of MBP1 mRNA is differentially regulated both temporally during development of the mouse embryo and in a tissue-specific manner within the adult . Specific interaction between MBP1 and mutant p53 was illustrated by both two-hybrid analysis in yeast and co-immunoprecipitation in mammalian cells . MBP1 displayed the following order of binding specificity towards different p53 forms: H175 > G281 > H273 > or = W248>wt p53 . Thus, MBP1 appears to bind preferentially to p53 mutants of the 'structural' rather than 'contact' class, reflecting a potential bias towards those mutants having a significant alteration in conformation from that assumed by wt p53 . We propose that MBP1 is the product of a candidate oncogene as rates of both neoplastic transformation and tumour cell growth were shown to be significantly enhanced when the protein is ectopically overexpressed . Furthermore, MBP1 may play a role in determining if a 'gain of function' effect is seen with certain p53 mutants.

Plant Mol Biol, 1999 Apr, 39(6), 1113 - 26
Characterization of ATDRG1, a member of a new class of GTP-binding proteins in plants; Etheridge N et al.; We report the initial characterization of an Arabidopsis thaliana cDNA (atdrg1), a member of a new class of GTP-binding proteins (G-proteins) in plants . The predicted ATDRG1 protein contains all five structural motifs characteristic of the G-protein superfamily . Apart from these motifs, the amino acid sequence differs substantially from all known G-proteins except for a recently discovered new family named developmentally regulated G-proteins (DRGs) . Sequences closely related to atdrg1 are found in species as distant as human (80% amino acid conservation), Drosophila (74%), yeast (77%) and Caenorhabditis elegans (77%) . The remarkable evolutionary conservation of these proteins suggests an important, but as yet unclear role . Phylogenetic analysis of the available homologous sequences strongly suggests a diphyletic origin of the eukaryotic DRG proteins . Northern analysis shows high levels of atdrg1 mRNA in all Arabidopsis tissues studied, and homologues of atdrg1 are present throughout the plant kingdom . In situ hybridization reveals that atdrg1 is highly expressed in actively growing tissues and reproductive organs . Southern analysis indicates the presence of either one or two copies of atdrg1 in the Arabidopsis genome . Immunolocalization studies show that the protein is present in cytoplasmic vesicles found mainly in actively growing tissues suggesting a putative role for ATDRG1 in either the regulation of vesicle transport or the regulation of enzymes involved in storage protein processing.

Plant Mol Biol, 1999 Apr, 39(6), 1091 - 100
Splicing-independent processing of plant box C/D and box H/ACA small nucleolar RNAs; Leader DJ et al.; Small nucleolar RNAs (snoRNAs) are involved in various aspects of ribosome biogenesis and rRNA maturation . Plants have a unique organisation of snoRNA genes where multiple, different genes are tightly clustered at a number of different loci . The maize gene clusters studied here include genes from both of the two major classes of snoRNAs (box C/D and box H/ACA) and are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter . In contrast to vertebrate and yeast intron-encoded snoRNAs, which are processed from debranched introns by exonuclease activity, the particular organisation of plant snoRNA genes suggests a different mode of expression and processing . Here we show that single and multiple plant snoRNAs can be processed from both non-intronic and intronic transcripts such that processing is splicing-independent and requires endonucleolytic activity . Processing of these different snoRNAs from the same polycistronic transcript suggests that the processing machineries needed by each class are not spatially separated in the nucleolus/nucleus.

Insect Biochem Mol Biol, 1999 May, 29(5), 435 - 43
Cloning and functional expression of a cDNA encoding a metabolic acyl-CoA delta 9-desaturase of the cabbage looper moth, Trichoplusia ni; Liu W et al.; Acyl-CoA delta 9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity . In this research, a cDNA sequence was obtained from Trichoplusia ni adult fat body mRNA by using RT-PCR with degenerate primers based on other characterized delta 9-desaturase sequences . The remainder of the sequence was amplified using 3'- and 5'-RACE . A 1439 bp cDNA reconstructed from three overlapping PCR products contains an ORF encoding a 353-amino acids (aa) protein that shows clear homology (greater than 50% aa identity and greater than 65% aa similarity to characterized insect and vertebrate desaturases) . The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transformation . The newly characterized desaturase from T . ni produced fatty acids delta 9-16 and delta 9-18 in a 1:6 ratio, compared to a 5:1 ratio, respectively, with the yeast delta 9 desaturase . A Northern blot hybridization and a RT-PCR experiment showed that temporal and tissue-specific patterns of expression of the corresponding mRNA are distinct from those of the delta 11-desaturase mRNA present in the pheromone glands of adult females . Based on its homology to other desaturases, the widespread distribution of its corresponding mRNA in various tissues, and its functional assay, we conclude that this cDNA encodes the apoprotein corresponding to the desaturase component of the metabolic delta 9-desaturase complex of T . ni.

Genes Chromosomes Cancer, 1999 Jul, 25(3), 277 - 83
Identification and mapping of novel tumor suppressor loci on 6p in diffuse large B-cell non-Hodgkin's lymphoma; Nagai H et al.; Allelic deletions have been thought to be indicators of the presence of tumor suppressor genes (TSGs) . As indicated by this allelotype study using 39 highly informative microsatellite markers distributed among all autosomal chromosomes, frequent loss of heterozygosity (LOH) has been found at 6p in B-cell non-Hodgkin's lymphoma . To identify the commonly deleted regions (CDRs), we performed fine deletion mapping using 26 highly polymorphic microsatellite markers on 6p . The most frequent LOH occurred at D651721, where 9 of 18 of the informative cases (50%) had allelic losses . Seventeen of 32 cases (53%) exhibited LOH at least at one locus on 6p . Ten of these 17 cases showed interstitial deletions, and their LOH patterns indicated two CDRs on 6p; one between D6S1721 and D6S260 (at 6p23-24), and the other between D6S265 and D6S291 (at 6p21) . The genetic distance of both CDRs was 6 cM . The CDKN1A (p21) gene is reported to be located within the interval of the CDR at 6p21, but no mutation of the gene was found in these 32 patients . These data suggested that these two loci might harbor novel putative TSGs responsible for the pathogenesis of malignant lymphoma . We have constructed a contig of yeast artificial chromosome (YAC) clones spanning the most frequent CDR at 6p23-24 . This YAC contig can be used for fine physical mapping of the region and cloning of candidate TSGs.

Genes Chromosomes Cancer, 1999 Jul, 25(3), 270 - 6
Deletion mapping at 12p12-13 in metastatic prostate cancer; Kibel AS et al.; The identification of homozygous deletions in malignant tissue is a powerful tool for the localization of tumor suppressor genes . Representational difference analysis (RDA) uses selective hybridization and the polymerase chain reaction (PCR) to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes, such as BRCA2 and PTEN . We have recently identified a 1-5-cM homozygous deletion on 12p12-13 in a prostate cancer xenograft and found that 47% of patients who died of prostate carcinoma demonstrate focal loss of heterozygosity (LOH) in this region in metastatic deposits . We have now characterized the region of interest by assembling a yeast artificial chromosome (YAC) contig spanning the homozygous deletion and identifying which known genes and expressed sequence tags (EST) lie within the homozygous deletion . A rib metastasis was harvested at autopsy and placed subcutaneously in a male SCID mouse . Genomic DNA from this xenograft and from the patient's normal renal tissue was extracted . Multiplex PCR, with the xenograft and normal DNA used as template, was performed using primers for loci on the Whitehead contig 12.1 believed to be near our region of interest . We found that our deletion lay in a 1-2-Mb interval between WI-664 and D12S358 . We then used the same primers to construct a YAC contig across the homozygous deletion . PCR amplification of YAC DNA, using primers for the genomic sequences of known genes and ESTs reported to lie on 12p12-13, was used to identify candidate genes that lay within the deletion . Duplex PCR, with control primers known not to be deleted in the xenograft, was used to confirm that both the CDKN1B and ETV6 genes were homozygously deleted in the xenograft . Mutations in either or both of these genes may play an important role in metastatic prostate carcinoma.

Genes Chromosomes Cancer, 1999 Jul, 25(3), 230 - 40
Mapping of the breakpoints on the short arm of chromosome 17 in neoplasms with an i(17q); Scheurlen WG et al.; Isochromosomes are monocentric or dicentric chromosomes with homologous arms that are attached in a reverse configuration as mirror images . With an incidence of 3-4%, the i(17q) represents the most frequent isochromosome in human cancer . It is found in a variety of tumors, particularly in blast crisis of chronic myeloid leukemia (CML-BC), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), and medulloblastoma (MB), and indicates a poor prognosis . To determine the breakpoints on the molecular genetic level, we analyzed 18 neoplasms (six CML, four AML, one NHL, and seven MB) with an i(17q) and two MB with a pure del(17p) applying fluorescence in situ hybridization (FISH) with yeast artificial chromosome (YAC) clones, P1-artificial chromosome (PAC) clones, and cosmids from a well-characterized contig covering more than 6 Mb of genomic DNA . We identified four different breakpoint cluster regions . One is located close to or within the centromere of chromosome 17 and a second in the Charcot-Marie-Tooth (CMT1A) region at 17(p11.2) . A third breakpoint was found telomeric to the CMT1A region . The fourth, most common breakpoint was detected in MB, AML, and in CML-BC specimens and was bordered by two adjacent cosmid clones (clones D14149 and M0140) within the Smith-Magenis syndrome (SMS) region . These results indicate that the low copy number repeat gene clusters which are present in the CMT and SMS regions may be one of the factors for the increased instability that may trigger the formation of an i(17q).

Plant J, 1999 May, 18(3), 243 - 52
Tobacco retinoblastoma-related protein phosphorylated by a distinct cyclin-dependent kinase complex with Cdc2/cyclin D in vitro; Nakagami H et al.; The retinoblastoma (Rb) protein was originally identified as a product of a tumour suppressor gene that plays a pivotal role in regulating both the cell cycle and differentiation in mammals . The growth-suppressive activity of Rb is regulated by phosphorylation with cyclin-dependent kinase (CDK), and inactivation of the Rb function is one of the critical steps for transition from the G1 to the S phase . We report here the cloning of a cDNA (NtRb1) from Nicotiana tabacum which encodes a Rb-related protein, and show that this gene is expressed in all the organs examined at the mRNA level . We have demonstrated that NtRb1 interacts with tobacco cyclin D by using yeast two-hybrid and in vitro binding assays . In mammals, cyclin D can assemble with CDK4 and CDK6, but not with Cdc2, to form active complexes . Surprisingly, tobacco cyclin D and Cdc2 proteins can form a complex in insect cells, which is able to phosphorylate tobacco Rb-related protein in vitro . Using immunoprecipitation with the anti-cyclin D anti-body, cyclin D can be found in a complex with Cdc2 in suspension-cultured tobacco BY-2 cells . These results suggest that the cdc2 gene modulates the cell cycle through the phosphorylation of Rb-related protein by forming an active complex with cyclin D in plants.

DNA Seq, 1999, 10(2), 109 - 13
Caenorhabditis elegans contains structural homologs of human prk and plk; Ouyang B et al.; We and others have recently reported cloning and characterization of human prk and plk, members of the polo family of protein serine/threonine kinases that includes the budding yeast cdc5 and Drosophila melanoganster polo . The cdc5 gene is essential for cell cycle progression through mitosis and controls adaptation to the yeast DNA damage checkpoint . Here we report the identification of two new cdc5 homologs from Ceanorhabditis elegans, named plc1 and plc2 . The deduced amino acid sequences of Plc1 and Plc2 share strong homology with both human Prk and Plk . plc1 and plc2 genes are closely linked on chromosome III and share 40% residue identity, suggesting that gene duplication followed by independent evolution gives rise to multiple polo homologous genes within a species . Similar to polo family members in other species, two distinct domains are present in Plc1 and Plc2 with the N-terminal half being the putative kinase domain . Interestingly, Plc2, unlike Plc1, contains a less conserved polo box within the C-terminal half of the protein, suggesting a functional division between these two kinases.

Bioessays, 1999 May, 21(5), 412 - 21
Protein glycosylation in development and disease; Dennis JW et al.; N- and O-linked glycan structures of cell surface and secreted glycoproteins serve a variety of functions related to cell-cell communication in systems affecting development and disease . The more sophisticated N-glycan biosynthesis pathway of metazoans diverges from that of yeast with the appearance of the medial-Golgi beta-N-acetylglucosaminyltransferases (GlcNAc-Ts) . Tissue-specific regulation of medial- and trans-Golgi glycosyltransferases contribute structural diversity to glycoproteins in metazoans, and this can affect their molecular properties including localization, half-life, and biological activity . Null mutations in glycosyltransferase genes positioned later in the biosynthetic pathway disrupt expression of smaller subsets of glycan structures and are progressively milder in phenotype . In this review, we examine data on targeted mutations affecting glycosylation in mice and congenital mutations in man, with a view to understanding the molecular functions of glycan structures as modulators of glycoprotein activity . Finally, pathology associated with the expression of GlcNAc-Ts in cancer and diabetes-induced cardiac hypertrophy suggest that inhibitors of these enzymes may have therapeutic value.

FEBS Lett, 1999 Jun 4, 452(1-2), 87 - 91
Initiation of DNA replication in eukaryotes: questioning the origin; Francon P et al.; Although proteins involved in DNA replication in yeast have counterparts in multicellular organisms, the definition of an origin of DNA replication and its control in higher eukaryotes might obey to different rules . Origins of DNA replication that are site-specific have been found, supporting the notion that specific DNA regions are used to initiate DNA synthesis along metazoan chromosomes . However, the notion that specific sequences will define origins is still being debated . The variety and complexity of transcriptional programs that have to be regulated in multicellular organisms may impose a plasticity that would not be compatible with a fixed origin simply defined at the sequence level . Such a plasticity would be essential to developmental programs where the control of DNA replication could be more integrated to the control of gene expression than in unicellular eukaryotes.

Biotechniques, 1999 Jun, 26(6), 1150 - 6, 1158, 1160
Construction of gene targeting vectors from lambda KOS genomic libraries; Wattler S et al.; We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors . The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones . The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones . The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes . The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert . We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

Gene, 1999 Jun 11, 233(1-2), 189 - 95
Cloning, mapping and expression of UBL3, a novel ubiquitin-like gene; Chadwick BP et al.; A novel Drosophila melanogaster gene UBL3 was characterized and shown to be highly conserved in man and Caenorhabditis elegans (C . elegans) . The human and mouse homologues were cloned and sequenced . UBL3 is a ubiquitin-like protein of unknown function with no conserved homologues in yeast . Mapping of the human and mouse UBL3 genes places them within a region of shared gene order between human and mouse chromosomes on human chromosome 13q12-13 and telomeric mouse chromosome 5 (MMU5).

Gene, 1999 Jun 11, 233(1-2), 13 - 9
Cloning of the cDNA encoding phenylalanyl tRNA synthetase regulatory alpha-subunit-like protein whose expression is down-regulated during differentiation; Zhou X et al.; Hybrid polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA), induce differentiation of transformed cells . Differential display of RNA was used to identify genes whose expression is changed during SAHA-induced differentiation of murine erythroleukemia (MEL) cells . One such cDNA was identified whose mRNA level decreased by 50% after 8h of SAHA treatment as determined by Northern blot analysis . The full-length cDNA (1944bp in length) was cloned by sequencing of an EST clone and rapid amplification of 5' cDNA ends (5'-RACE) . The predicted amino acid sequence is 589 amino acids and shares 45% identity with the yeast cytoplasmic phenylalanyl tRNA synthetase (PheRS) regulatory alpha-subunit . Human EST clones which share over 90% identity of predicted amino acid sequence with this cDNA map to chromosome 2 near the paired box homeotic gene 3 (PAX3) locus, a region syngenic to mouse chromosome 1 . This is the first report of the cloning of the full-length cDNA for the murine PheRS regulatory alpha-subunit-like protein . The level of PheRS alpha-subunit-like mRNA is regulated during differentiation but not during cell cycle progression.

Curr Opin Plant Biol, 1999 Jun, 2(3), 198 - 206
Source-sink regulation by sugar and stress; Roitsch T; The regulation of carbon partitioning between source and sink tissues in higher plants is not only important for plant growth and development, but insight into the underlying regulatory mechanism is also a prerequisite to modulating assimilate partitioning in transgenic plants . Hexoses, as well as sucrose, have been recognised as important signal molecules in source-sink regulation . Components of the underlying signal transduction pathways have been identified and parallels, as well as distinct differences, to known pathways in yeast and animals have become apparent . There is accumulating evidence for crosstalk, modulation and integration between signalling pathways responding to phytohormones, phosphate, light, sugars, and biotic and abiotic stress-related stimuli . These complex interactions at the signal transduction levels and co-ordinated regulation of gene expression seem to play a central role in source-sink regulation.

Leukemia, 1999 May, 13(5), 708 - 12
Discontinuous deletions at 11q23 in B cell chronic lymphocytic leukemia; Zhu Y et al.; Loss of genomic material in 11q is one of the most common structural chromosome aberrations in B cell chronic lymphocytic leukemia (B-CLL) . In order to characterize the deletions of 11q23 in B-CLL, we performed fluorescence in situ hybridization (FISH) with eight YAC (yeast artificial chromosome) probes on peripheral leukocytes of 30 patients . These YACs form a contig spanning 7.8 Mb at 11q23.1-q23.3 . We found deletions in nine out of 30 cases (30%) and five of them had discontinuous deletions in this region . The region represented by YAC 755b11 (1.6 Mb in size) was involved in all cases with deletions, supporting the hypothesis that this region might contain a novel gene of pathogenic importance to B-CLL . A more distal region represented by YAC 785e12 (760 kb in size) was deleted frequently and specifically . Whether there is another novel gene of pathogenic importance to B-CLL and what is its potential relationship to the deletions in the region represented by YAC 755b11, are issues that require further studies.

Mar Biotechnol (NY), 1999 Mar, 1(2), 200 - 206
Identification and Expression Pattern of DNA Polymerase alpha Gene in a Marine Diatom, Skeletonema costatum; Hwang SP et al.; : To investigate the potential of DNA polymerase alpha as a marker for DNA replication in phytoplankton, two gene fragments that showed a high degree of similarity with eukaryotic DNA polymerase alpha were cloned from two strains of a diatom, Skeletonema costatum (Greville) Cleve . The gene fragments amplified with the polymerase chain reaction were 397 and 396 bp in length, respectively . The deduced amino acid sequences showed 44% to 61% similarity to the corresponding regions of DNA polymerase alpha sequences of eukaryotic organisms ranging from yeast to humans . The similarity was especially high in three evolutionarily conserved regions within the amplified fragments . Further, hybridization patterns from Southern blotting confirmed that the amplified fragments were an integral part on the genome of S . costatum . In batch cultures abundant messenger of DNA polymerase alpha appeared in the late exponential phase and the early stationary phase . This pattern suggests that DNA polymerase alpha expression is associated with actively dividing cells.

Mar Biotechnol (NY), 1999 Mar, 1(2), 191 - 199
Prolidase in the Marine Sponge Suberites domuncula: Enzyme Activity, Molecular Cloning, and Phylogenetic Relationship; Wiens M et al.; : The enzyme prolidase hydrolyzes the peptide bond that involves the imino nitrogen of proline or hydroxyproline; hence, it catalyzes the final step in collagen degradation . From mammals it is known that this enzyme plays a major role in the recycling of proline for collagen synthesis and can be considered to be essential for the control of cell growth . The dominant organic exoskeleton in sponges, especially in Demospongiae, is collagen and the collagen-related spongin . Here we demonstrate that crude extracts of the demosponge Suberites domuncula contain prolidase or prolidase-like activity . The complementary DNA encoding the putative prolidase was cloned from a library of the same animal . Two different forms of cDNAs, termed SDPEPD1 and SDPEPD2, were identified, coding for the putative polypeptides PEPD_SD-1 with a molecular mass of 55,805 Da and PEPD_SD-2 with 51,684 . Evidence is presented suggesting that the two different transcripts originate from the same gene but are formed by an alternative splicing event . We conclude that demosponges contain the activity as well as the gene for prolidase, a major enzyme involved in collagen metabolism, spicule formation, and cell motility . Phylogenetic analysis revealed that the sponge prolidase branches off first from the common ancestor of metazoan prolidases and later than the yeast prolidase; only distantly related are the bacterial enzymes.

Nucleic Acids Res . 1999 Jul 1;27(13):e4.
A novel in vivo assay for the analysis of protein-protein interaction; Maroun M et al.; The Ras Recruitment System (RRS) is a method for identification and isolation of protein-protein interaction . The method is based on translocation of cytoplasmic mammalian Ras protein to the inner leaflet of the plasma membrane through protein-protein interaction . The system is studied in a temperature-sensitive yeast strain where the yeast Ras guanyl nucleotide exchange factor is inactive at 36 degrees C . Protein-protein interaction results in cell growth at the restrictive temperature . We developed a gene reporter assay for the analysis of protein-protein interaction in mammalian cells . Ras activation in mammalian cells induces the mitogen-activated kinase cascade (MAPK), which can be monitored using Ras-dependent reporter genes . This greatly extends the usefulness of the system and provides a novel assay for protein-protein interaction in mammalian cells.

Mol Cell Biol, 1999 Jul, 19(7), 5001 - 13
Human Cdc34 and Rad6B ubiquitin-conjugating enzymes target repressors of cyclic AMP-induced transcription for proteolysis; Pati D et al.; Ubiquitin-mediated proteolysis controls diverse physiological processes in eukaryotes . However, few in vivo targets of the mammalian Cdc34 and Rad6 ubiquitin-conjugating enzymes are known . A yeast-based genetic assay to identify proteins that interact with human Cdc34 resulted in three cDNAs encoding bZIP DNA binding motifs . Two of these interactants are repressors of cyclic AMP (cAMP)-induced transcription: hICERIIgamma, a product of the CREM gene, and hATF5, a novel ATF homolog . Transfection assays with mammalian cells demonstrate both hCdc34- and hRad6B-dependent ubiquitin-mediated proteolysis of hICERIIgamma and hATF5 . This degradation requires an active ubiquitin-conjugating enzyme and results in abrogation of ICERIIgamma- and ATF5-mediated repression of cAMP-induced transcription . Consistent with these results, the endogenous ICER protein is elevated in cells which are null for murine Rad6B (mHR6B-/-) or transfected with dominant negative and antisense constructs of human CDC34 . Based on the requirement for CREM/ICER and Rad6B proteins in spermatogenesis, we determined expression of Cdc34, Rad6B, CREM/ICER isoforms, and the Skp1-Cullin-F-box ubiquitin protein ligase subunits Cul-1 and Cul-2, which are associated with Cdc34 activity during murine testicular development . Cdc34, Rad6B, and the Cullin proteins are expressed in a developmentally regulated manner, with distinctly different patterns for Cdc34 and the Cullin proteins in germ cells . The Cdc34 and Rad6B proteins are significantly elevated in meiotic and postmeiotic haploid germ cells when chromatin modifications occur . Thus, the stability of specific mammalian transcription factors is the result of complex targeting by multiple ubiquitin-conjugating enzymes and may have an impact on cAMP-inducible gene regulation during both meiotic and mitotic cell cycles.

Mol Cell Biol, 1999 Jul, 19(7), 4944 - 52
Cloning and characterization of two evolutionarily conserved subunits (TFIIIC102 and TFIIIC63) of human TFIIIC and their involvement in functional interactions with TFIIIB and RNA polymerase III; Hsieh YJ et al.; Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that mediates transcription of class III genes through direct recognition of promoters (for tRNA and virus-associated RNA genes) or promoter-TFIIIA complexes (for the 5S RNA gene) and subsequent recruitment of TFIIIB and RNA polymerase III . We describe the cognate cDNA cloning and characterization of two subunits (hTFIIIC63 and hTFIIIC102) that are present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC and are related in structure and function to two yeast TFIIIC subunits (yTFIIIC95 and yTFIIIC131) previously shown to interact, respectively, with the promoter (A box) and with a subunit of yeast TFIIIB . hTFIIIC63 and hTFIIIC102 show parallel in vitro interactions with the homologous human TFIIIB and RNA polymerase III components, as well as additional interactions that may facilitate both TFIIIB and RNA polymerase III recruitment . These include novel interactions of hTFIIIC63 with hTFIIIC102, with hTFIIIB90, and with hRPC62, in addition to the hTFIIIC102-hTFIIIB90 and hTFIIIB90-hRPC39 interactions that parallel the previously described interactions in yeast . As reported for yTFIIIC131, hTFIIIC102 contains acidic and basic regions, tetratricopeptide repeats (TPRs), and a helix-loop-helix domain, and mutagenesis studies have implicated the TPRs in interactions both with hTFIIIC63 and with hTFIIIB90 . These observations further document conservation from yeast to human of the structure and function of the RNA polymerase III transcription machinery, but in addition, they provide new insights into the function of hTFIIIC and suggest direct involvement in recruitment of both TFIIIB and RNA polymerase III.

Mol Cell Biol, 1999 Jul, 19(7), 4611 - 22
Ras-specific exchange factor GRF: oligomerization through its Dbl homology domain and calcium-dependent activation of Raf; Anborgh PH et al.; The full-length versions of the Ras-specific exchange factors Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2), which are expressed in brain and a restricted number of other organs, possess an ionomycin-dependent activation of Erk mitogen-activated protein kinase activity in 293T cells (C . L . Farnsworth et al., Nature 376:524-527, 1995; N . P . Fam et al., Mol . Cell . Biol . 17:1396-1406, 1996) . Each GRF protein contains a Dbl homology (DH) domain . A yeast two-hybrid screen was used to identify polypeptides that associate with the DH domain of GRF1 . In this screen, a positive cDNA clone from a human brain cDNA library was isolated which consisted of the GRF2 DH domain and its adjacent ilimaquinone domain . Deletion analysis verified that the two-hybrid interaction required only the DH domains, and mutation of Leu-263 to Gln (L263Q) in the N terminus of the GRF1 DH domain abolished the two-hybrid interaction, while a cluster of more C-terminally located mutations in the DH domain did not eliminate the interaction . Oligomers between GRF1 and GRF2 were detected in a rat brain extract, and forced expression of GRF1 and GRF2 in cultured mammalian cells formed homo- and hetero-oligomers . Introduction of the L263Q mutation in GRF1 led to a protein that was deficient in oligomer formation, while GRF1 containing the DH cluster mutations formed homo-oligomers with an efficiency similar to that of wild type . Compared to wild-type GRF1, the focus-forming activity on NIH 3T3 cells of the GRF1 DH cluster mutant was reduced, while the L263Q mutant was inactive . Both mutants were impaired in their ability to mediate ionomycin-dependent Erk activity in 293T cells . In the absence of ionomycin, 293T cells expressing wild-type GRF1 contained much higher levels of Ras-GTP than control cells; the increase in Erk activity induced by ionomycin in the GRF1-expressing cells also induced a concomitant increase in Raf kinase activity, but without a further increase in the level Ras-GTP . We conclude that GRF1 and GRF2 can form homo- and hetero-oligomers via their DH domains, that mutational inactivation of oligomer formation by GRF1 is associated with impaired biological and signaling activities, and that in 293T cells GRF1 mediates at least two pathways for Raf activation: one a constitutive signal that is mainly Ras-dependent, and one an ionomycin-induced signal that cooperates with the constitutive signal without further augmenting the level of GTP-Ras.

J Biol Chem, 1999 Jun 25, 274(26), 18785 - 92
Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination; Kumar S et al.; The human papilloma virus E6-associated protein (E6AP) functions as a ubiquitin protein ligase (E3) in the E6-mediated ubiquitination of p53 . E6AP is also an E3 in the absence of E6, but its normal cellular substrates have not yet been identified . Here we report the identification of HHR23A, one of the human homologues of the yeast DNA repair protein Rad23, as an E6-independent target of E6AP . HHR23A binds E6AP and is ubiquitinated in vitro in an E6AP-dependent manner . Ubiquitinated forms of endogenous HHR23A are detectable in mammalian cells . Overexpression of wild-type E6AP in vivo enhances the ubiquitination of HHR23A, whereas a dominant negative E6AP mutant inhibits HHR23A ubiquitination . Although HHR23A is a stable protein in non-synchronized cells, its levels are regulated in a cell cycle-dependent manner, with specific degradation occurring during S phase . The S phase degradation of HHR23A could be blocked in vivo by dominant negative E6AP, providing direct evidence for the involvement of E6AP in the regulation of HHR23A . Consistent with a role of the HHR23 proteins in DNA repair, UV-induced DNA damage inhibited HHR23A degradation . Although the precise role of HHR23 proteins in DNA repair and cell cycle progression remains to be elucidated, our data suggest that E6AP-mediated ubiquitination of HHR23A may have important implications in DNA repair and cell cycle progression.

J Biol Chem, 1999 Jun 25, 274(26), 18515 - 23
Valine 571 functions as a regional organizer in programming the glucocorticoid receptor for differential binding of glucocorticoids and mineralocorticoids; Lind U et al.; The glucocorticoid receptor (GR) interacts specifically with glucocorticoids, whereas its closest relative, the mineralocorticoid receptor (MR), interacts with both glucocorticoids and mineralocorticoids, such as aldosterone . To investigate the mechanism underlying the glucocorticoid/mineralocorticoid specificity of the GR, we used a yeast model system to screen for GR ligand-binding domain mutants, substituted with MR residues in the segment 565-574, that can be efficiently activated by aldosterone . In all such increased activity mutants, valine 571 was replaced by methionine, even though most mutants also contained substitutions of other residues with their MR counterparts . Further analysis in yeast and COS-7 cells has revealed that the identity of residue 571 determines the behavior of other MR substituted residues in the 565-574 segment . Generally, MR substitutions in this region are only consistent with aldosterone binding if residue 571 is also replaced with methionine (MR conformation) . If residue 571 is valine (GR conformation), most other MR substitution mutants drastically reduce interaction with both mineralocorticoid and glucocorticoid hormones . Based on these functional data, we hypothesize that residue 571 functions as a regional organizer involved in discriminating between glucocorticoid and mineralocorticoid hormones . We have used a molecular model of the GR ligand-binding domain in an attempt to interpret our functional data in structural terms.

J Biol Chem, 1999 Jun 25, 274(26), 18446 - 54
EHSH1/intersectin, a protein that contains EH and SH3 domains and binds to dynamin and SNAP-25 . A protein connection between exocytosis and endocytosis?
Okamoto M, Schoch S, Sudhof TC.
In yeast two-hybrid screens for proteins that bind to SNAP-25 and may be involved in exocytosis, we isolated a protein called EHSH1 (for EH domain/SH3 domain-containing protein) . Cloning of full-length cDNAs revealed that EHSH1 is composed of an N-terminal region with two EH domains, a central region that is enriched in lysine, leucine, glutamate, arginine, and glutamine (KLERQ domain), and a C-terminal region comprised of five SH3 domains . The third SH3 domain is alternatively spliced . Data bank searches demonstrated that EHSH1 is very similar to Xenopus and human intersectins and to human SH3P17 . In addition, we identified expressed sequence tags that encode a second isoform of EHSH1, called EHSH2 . EHSH1 is abundantly expressed in brain and at lower levels in all other tissues tested . In binding studies, we found that the central KLERQ domain of EHSH1 binds to recombinant or native brain SNAP-25 and SNAP-23 . The C-terminal SH3 domains, by contrast, quantitatively interact with dynamin, a protein involved in endocytosis . Dynamin strongly binds to the alternatively spliced central SH3 domain (SH3C) and the two C-terminal SH3 domains (SH3D and SH3E) but not to the N-terminal SH3 domains (SH3A and SH3B) . Immunoprecipitations confirmed that both dynamin and SNAP-25 are complexed to EHSH1 in brain . Our data suggest that EHSH1/intersectin may be a novel adaptor protein that couples endocytic membrane traffic to exocytosis . The ability of multiple SH3 domains in EHSH1 to bind to dynamin suggests that EHSH1 can cluster several dynamin molecules in a manner that is regulated by alternative splicing.

J Biol Chem, 1999 Jun 25, 274(26), 18407 - 13
The identification of phosphatidylinositol 3,5-bisphosphate in T-lymphocytes and its regulation by interleukin-2; Jones DR et al.; In recent times 3-phosphoinositides have emerged as important regulators of cell metabolism, survival, and proliferation . During the last year, the phospholipid phosphatidylinositol 3, 5-bisphosphate (PtdIns3,5P2) was identified in yeast, fibroblasts, SV40-transformed kidney (COS-7) cells, and platelets . The discovery of this novel phospholipid has increased the complexity of the metabolism relating to the generation of biologically active inositol-containing lipids . We describe here the identification of PtdIns3,5P2 in the CTLL-2 mouse T-lymphocyte cell line using two in vivo radiolabeling protocols . Treatment of the cells with UV radiation led to an increase in the cellular content of PtdIns3,5P2 . In contrast, preincubation of the cells with wortmannin or treatment with hypertonic medium (high concentration sorbitol) led to the opposite effect . Herein we demonstrate that interleukin-2 (IL-2), the growth factor required for CTLL-2 cell proliferation, was able to increase the level of PtdIns3,5P2 with similar kinetics to that of the formation of phosphatidylinositol 3,4-bisphosphate (PtdIns3, 4P2) . An increase in this novel 3-phosphorylated lipid in response to IL-2 seems to be a general property of this cytokine because a similar result was obtained when the pre-B cell line BaF/3 expressing the high affinity IL-2 receptor was used . Using a constitutively active regulatory subunit of type I phosphatidylinositol 3-kinase and cells expressing a deletion of the serine-rich domain of the IL-2 receptor beta chain, which is required for IL-2-stimulated type I phosphatidylinositol 3-kinase activation, we demonstrate that IL-2-induced generation of PtdIns3, 5P2 is related to the activation of this enzyme . The results show for the first time the identification of PtdIns3,5P2 in both T- and B-lymphocytes and indicate its positive regulation by the mitogen IL-2.

J Biol Chem, 1999 Jun 25, 274(26), 18327 - 34
Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent; Kelly JD et al.; Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions . In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis . In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter . The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --> TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p < 10(-6), Poisson's normal distribution) . A hypothetical model for the interpretation of mutational events at this site is proposed . The present work is the first report on mutational properties of Me-lex . Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner.

J Biol Chem, 1999 Jun 25, 274(26), 18145 - 8
The head domain of plakophilin-1 binds to desmoplakin and enhances its recruitment to desmosomes . Implications for cutaneous disease; Kowalczyk AP et al.; The contribution of desmosomes to epidermal integrity is evident in the inherited blistering disorder associated with the absence of a functional gene for plakophilin-1 . To define the function of plakophilin-1 in desmosome assembly, interactions among the desmosomal cadherins, desmoplakin, and the armadillo family members plakoglobin and plakophilin-1 were examined . In transient expression assays, plakophilin-1 formed complexes with a desmoplakin amino-terminal domain and enhanced its recruitment to cell-cell borders; this recruitment was not dependent on the equimolar expression of desmosomal cadherins . In contrast to desmoplakin-plakoglobin interactions, the interaction between desmoplakin and plakophilin-1 was not mediated by the armadillo repeat domain of plakophilin-1 but by the non-armadillo head domain, as assessed by yeast two-hybrid and recruitment assays . We propose a model whereby plakoglobin serves as a linker between the cadherins and desmoplakin, whereas plakophilin-1 enhances lateral interactions between desmoplakin molecules . This model suggests that epidermal lesions in patients lacking plakophilin-1 are a consequence of the loss of integrity resulting from a decrease in binding sites for desmoplakin and intermediate filaments at desmosomes.

Genomics, 1999 Jun 15, 58(3), 318 - 22
An integrated somatic cell hybrid, YAC, and BAC map of the Rmc1 region of mouse chromosome 1; Hunter K et al.; Rmc1, the cellular receptor for the polytropic class of murine retroviruses, determines the tissue tropism of the virus and therefore plays a critical role in the pathogenesis of polytropic virus-induced leukemia . Previously we reported the physical mapping of this gene to a 5-cM region of mouse chromosome 1 and the construction of a yeast artificial chromosome (YAC) contig across this region . In this report we describe the refinement of the Rmc1 candidate region to approximately 600 kb and the generation of an integrated somatic cell hybrid, YAC, and bacterial artificial chromosome contig spanning the region . A number of genes and loci were physically ordered along the chromosome, including a recently identified candidate for Rmc1 .

Curr Eye Res, 1999 Apr, 18(4), 283 - 91
Elements regulating the transcription of human interstitial retinoid-binding protein (IRBP) gene in cultured retinoblastoma cells; Fong SL et al.; PURPOSE: To identify cis-acting elements and trans-acting factors involved in the expression of human IRBP gene . METHODS: Transient transfection of WERI-Rb1 and HeLa cells, DNase 1 footprinting, gel mobility-shift assay and yeast one-hybrid system were used to study the regulatory elements that are involved in the expression of human IRBP gene . RESULTS: A region between -1620 and -1411 was shown to have enhancer properties . Using nuclear extracts from WERI-Rb1 and HeLa cells, four footprints were identified in the proximal promoter region (-206 to +68) . The core promoter element IP1 binds to OTX2 in the yeast one-hybrid system . By cotransfecting HeLa cells, OTX2 could transactivate the irbp promoter . The functions of IP2 (from -119 to -86) and IP3 (from -183 to -147) remain to be determined . The region containing the HeLa cell-specific footprint IP4 (from -202 to -180) could silence the OTX2 transactivation of the irbp promoter . CONCLUSION: The 5'-flanking region of irbp contains an enhancer sequence . The possible silencer upstream from the core promoter may serve to suppress expression of irbp in HeLa cells . When the proximal promoter is used to identify binding proteins in a human retina library by the yeast one hybrid system, nine of the identified clones contained the cDNA sequence for the homeodomain protein OTX2 . Since no clones for the homeodomain protein CRX were found, and since OTX2 can transcriptionally activate irbp in normally non-expressing HeLa cells, it is possible that OTX2 rather than CRX is the transcriptional activator for irbp in human photoreceptors.

J Clin Endocrinol Metab, 1999 Jun, 84(6), 2104 - 10
Medroxyprogesterone acetate and dexamethasone are competitive inhibitors of different human steroidogenic enzymes; Lee TC et al.; Medroxyprogesterone acetate (MPA), a widely used progestin, can suppress the hypothalamic-pituitary-gonadal axis but can also directly inhibit gonadal steroidogenesis; the success of MPA as a treatment for gonadotropin-independent sexual precocity derives from its direct action on steroidogenic tissues . Dexamethasone, a widely used glucocorticoid, can suppress the hypothalamic-pituitary-adrenal axis, but its potential effect directly on the adrenal is unclear . Previous reports suggested that these two drugs may act on the initial steps in the rodent steroidogenic pathway; therefore, we investigated their abilities to inhibit the first three human enzymes in steroidogenesis: the cholesterol side-chain cleavage enzyme (P450scc), the 17alpha-hydroxylase/17,20-lyase (P450c17), and type II 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSDII) . We found no effect of either drug on P450scc in intact human choriocarcinoma JEG-3 cells . Using microsomes from yeast expressing human P450c17 or microsomes from human adrenals, we found that dexamethasone inhibited P450c17 with a Ki of 87 micromol/L, which is about 1000 times higher than typical therapeutic concentrations, but that MPA has no detectable action on P450c17 . Using microsomes from yeast expressing human 3betaHSDII, we found that this enzyme has indistinguishable apparent Km values of 5.2-5.5 micromol/L and similar maximum velocities of 0.34-0.56 pmol steroid/min x microg microsomal protein for the three principal endogenous substrates, pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone . In this system, MPA inhibited 3betaHSDII with a Ki of 3.0 micromol/L, which is near concentrations achieved by high therapeutic doses of 5-20 mg MPA/kg x day . These data establish the mechanism of action of MPA as an inhibitor of human steroidogenesis, and are in contrast with the results of earlier studies indicating that MPA inhibited both P450c17 and 3betaHSD in rat Leydig cells . These studies establish the "humanized yeast" system as a model for studying the actions of drugs on human steroidogenic enzymes and suggest that 3betaHSDII may be an appropriate target for pharmacological interventions in human disorders characterized by androgen excess or sex steroid dependency.

Arch Med Res, 1999 Mar-Apr, 30(2), 106 - 15
Congo red effect on cyst viability and cell wall structure of encysting . Entamoeba invadens; Garcia-Zapien AG et al.; BACKGROUND: The cell wall of Entamoeba invadens cysts is composed of chitin microfibrils as the main structural component . It has been demonstrated in yeast that the chitin cell wall assembly is altered by dyes such as Congo red (CR) and Calcofluor . METHODS: The purpose of this work was to study the cell wall assembly under the effect of CR dye on encysting E . invadens by means of light and electron microscopy, after the amebas were subjected to the effect of 100-2,000 micrograms CR/mL . Experiments were performed either in BI-S-33 or in mLG media . RESULTS: Trophozoite growth was not inhibited by 100-1,000 micrograms/mL CR after 8 days of incubation in BI-S-33 medium . However, low levels of growth were observed with 2,000 micrograms/mL of dye . No significant differences in morphologically viable (hyaline) cyst production occurred after 24-48 h, when 100 micrograms CR/mL was used, while the highest concentration of CR (2,000 micrograms/mL) resulted in a significant decrease of hyaline cyst yield; dead cysts prevailed in cultures, particularly at 72 h of CR treatment . Differentiation of amebas incubated in the presence of 500-2,000 micrograms/mL CR produced abnormal chitin deposits, rendering irregularly thick or double cell walls, as shown by transmission and scanning electron microscopy . Cyst cultures obtained under 100 micrograms/mL CR produced as many trophozoites as did the control when they were incubated in BI-S-33, but only low numbers of trophozoites were found in culture cysts obtained under higher CR doses . CONCLUSION: Our results suggest that CR affects E . invadens encystment, alters the cell wall formation, and also affects the cyst viability.

Ann N Y Acad Sci, 1999 Apr 20, 873, 239 - 44
From concept to market in industrial impedance applications; Davey C et al.; This paper discusses some of the technical aspects of converting a laboratory idea into a commercial product . The example used is the development of the Aber Instruments Biomass Monitor, which is now used worldwide in industry for the pitching of yeast in breweries and for biomass measurements in the pharmaceutical industry . Although the issues raised will relate to instrumentation in a production environment, many of the themes will be equally applicable to medical instruments.

FEBS Lett, 1999 May 21, 451(2), 203 - 8
Identification of a novel alternatively spliced septin; Fung ET et al.; Septins are a family of cytoskeletal proteins involved in cytokinesis, targeting of proteins to specific sites on the plasma membrane, and cellular morphogenesis . While many aspects of their function in cytokinesis in yeast cells have been investigated, the function of septins in mammalian cells is less well understood . For example, septins are present in post-mitotic neurons, suggesting they have other roles in, for example, establishing cell polarity . The full extent of the septin gene family is not known in mammalian cells . To better understand the septin gene family, we have cloned and characterized a novel mammalian septin.

FEBS Lett, 1999 May 21, 451(2), 142 - 6
Analysis of gene expression data using self-organizing maps; Toronen P et al.; DNA microarray technologies together with rapidly increasing genomic sequence information is leading to an explosion in available gene expression data . Currently there is a great need for efficient methods to analyze and visualize these massive data sets . A self-organizing map (SOM) is an unsupervised neural network learning algorithm which has been successfully used for the analysis and organization of large data files . We have here applied the SOM algorithm to analyze published data of yeast gene expression and show that SOM is an excellent tool for the analysis and visualization of gene expression profiles.

Clin Obstet Gynecol, 1999 Jun, 42(2), 221 - 33
Clinical management of vulvodynia; Davis GD et al.; Vulvodynia represents a group of vulvar disorders that can be clinically perplexing . Unfortunately, there are no simple tests for its diagnosis . Patients often are reluctant to report vulvar pain . Some are embarrassed to reveal their perceived "sexual dysfunction" caused by dyspareunia . Others have been told in the past "It's all in you head," or "It's just a yeast infection." Most patients with vulvodynia are seen by multiple practitioners and are placed on a variety of treatments before a correct diagnosis is made . All too often, these treatments are not only ineffective but also damaging . Careful clinical investigation is required for the correct diagnosis and treatment of vulvodynia (Figure 2).

Mol Cell Probes, 1999 Jun, 13(3), 199 - 202
Mapping of human WHN gene in a 17q11.2 YAC contig and identification of an intragenic STR; Corrado L et al.; The recently isolated human WHN gene has been previously assigned to chromosome 17q11-12 using radiation hybrids . In this study, we constructed a YAC contig covering 17q11.2 between crystallinBA1 and neurofibromatosis 1 genes . By ordering known and novel markers, we determined the position of the WHN gene, and of the closely linked retinal 4 and sodium/dicarboxylate cotransporter genes . We also identified a new mononucleotide polymorphism contained within the untranslated exon 1 of the WHN gene, which may be useful for linkage and LOH studies .

Nat Genet, 1999 Jun, 22(2), 199 - 202
A single EFEMP1 mutation associated with both Malattia Leventinese and Doyne honeycomb retinal dystrophy; Stone EM et al.; Malattia Leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) refer to two autosomal dominant diseases characterized by yellow-white deposits known as drusen that accumulate beneath the retinal pigment epithelium (RPE) . Both loci were mapped to chromosome 2p16-21 (refs 5,6) and this genetic interval has been subsequently narrowed . The importance of these diseases is due in large part to their close phenotypic similarity to age-related macular degeneration (AMD), a disorder with a strong genetic component that accounts for approximately 50% of registered blindness in the Western world . Just as in ML and DHRD, the early hallmark of AMD is the presence of drusen . Here we use a combination of positional and candidate gene methods to identify a single non-conservative mutation (Arg345Trp) in the gene EFEMP1 (for EGF-containing fibrillin-like extracellular matrix protein 1) in all families studied . This change was not present in 477 control individuals or in 494 patients with age-related macular degeneration . Identification of this mutation may aid in the development of an animal model for drusen, as well as in the identification of other genes involved in human macular degeneration.

EMBO J, 1999 Jun 15, 18(12), 3392 - 403
Net, a negative Ras-switchable TCF, contains a second inhibition domain, the CID, that mediates repression through interactions with CtBP and de-acetylation; Criqui-Filipe P et al.; Signalling cascades are integrated at the transcriptional level by the interplay between factors such as the ternary complex factors (TCFs) that interact with serum response factor (SRF) and the serum response element (SRE) of the fos promoter . Net is a negative TCF that is switched to a positive regulator by the Ras signal . To understand the mechanisms of repression by Net, we used a yeast two-hybrid screen to identify factors that interact with its inhibitory domain . We isolated mCtBP1, the murine homologue of huCtBP1, a factor implicated in negative regulation of transformation by E1A plus Ras . We show that mCtBP1 interacts strongly with Net both in vitro and in vivo . The CtBP interaction domain of Net, the CID, mediates repression independently of the previously identified negative element, the NID . The CID inhibits by recruiting the co-repressor mCtBP1 . The CID and mCtBP1 need to use de-acetylase activity for repression, whereas the NID apparently represses by other mechanisms . Finally, we provide evidence that CtBP and de-acetylation repress the c-fos SRE in low serum when it is inactive, but not in high serum when it is active . These results provide insights into the cross-talk between pathways that inhibit and stimulate transformation at the level of Net, a regulator of gene expression.

EMBO J, 1999 Jun 15, 18(12), 3305 - 16
Drainin required for membrane fusion of the contractile vacuole in Dictyostelium is the prototype of a protein family also represented in man; Becker M et al.; The contractile vacuole expels water by forming a channel with the plasma membrane and thus enables cells to survive in a hypo-osmotic environment . Here we characterize drainin, a Dictyostelium protein involved in this process, as the first member of a protein family represented in fission yeast, Caenorhabditis elegans and man . Gene replacement in Dictyostelium shows that drainin acts at a checkpoint of channel formation between the contractile vacuole and the plasma membrane . A green fluorescent protein fusion of drainin localizes specifically to the contractile vacuole and rescues its periodic discharge in drainin-null cells . Drainin is a peripheral membrane protein, requiring a short hydrophobic stretch in its C-terminal region for localization and function . We suggest that drainin acts in a signaling cascade that couples a volume-sensing device in the vacuolar membrane to the membrane fusion machinery.

Hum Genet, 1999 Apr, 104(4), 326 - 32
The human neuregulin-2 (NRG2) gene: cloning, mapping and evaluation as a candidate for the autosomal recessive form of Charcot-Marie-Tooth disease linked to 5q; Ring HZ et al.; Neuregulin-2 (NRG2) is a novel member of the neuregulin family of growth and differentiation factors . Through interaction with the ErbB family of receptors, neuregulin-2 induces the growth and differentiation of epithelial, neuronal, glial and other types of cells . In this study, we have cloned the human neuregulin-2 gene, and determined its genomic structure and alternative splicing patterns . By using radiation hybrid mapping panels, the human NRG2 gene was mapped to the D5S658-D5S402 region within 5q23-q33, close to an autosomal recessive form of demyelinating Charcot-Marie-Tooth (CMT) disease . The NRG2 gene was found to be on two yeast artificial chromosomes overlapping the candidate interval and was, thus, considered a good positional candidate for this form of CMT . When the entire neuregulin-2 coding sequence and splice junctions were explored, however, no mutation was identified in one CMT family linked to 5q23-q33 . In addition, three intronic single nucleotide polymorphisms were identified in the NRG2 gene . Genotyping in two families localized the NRG2 gene outside of the revised candidate interval between D5S402-D5S210 and excluded NRG2 as the gene responsible for this form of CMT disease.

Bioinformatics, 1999 May, 15(5), 370 - 5
Statistical mechanical simulation of polymeric DNA melting with MELTSIM; Blake RD et al.; MOTIVATION: MELTSIM is a windows-based statistical mechanical program for simulating melting curves of DNAs of known sequence and genomic dimensions under different conditions of ionic strength with great accuracy . The program is useful for mapping variations of base compositions of sequences, conducting studies of denaturation, establishing appropriate conditions for hybridization and renaturation, determinations of sequence complexity, and sequence divergence . RESULTS: Good agreement is achieved between experimental and calculated melting curves of plasmid, bacterial, yeast and human DNAs . Denaturation maps that accompany the calculated curves indicate non-coding regions have a significantly lower (G+C) composition than coding regions in all species examined . Curves of partially sequenced human DNA suggest the current database may be heavily biased with coding regions, and excluding large (A+T)-rich elements . AVAILABILITY: MELTSIM 1.0 is available at: //TSIM/MELTSIM-1.0-Win/meltsim . zip . Melting curve plots in this paper were made with GNUPLOT 3.5, available at: o.html Contact : blake@maine.maine.edu;

Plant Cell, 1999 Jun, 11(6), 1179 - 90
Sac3, an Snf1-like serine/threonine kinase that positively and negatively regulates the responses of Chlamydomonas to sulfur limitation; Davies JP et al.; The Sac3 gene product of Chlamydomonas positively and negatively regulates the responses of the cell to sulfur limitation . In wild-type cells, arylsulfatase activity is detected only during sulfur limitation . The sac3 mutant expresses arylsulfatase activity even when grown in nutrient-replete medium, which suggests that the Sac3 protein has a negative effect on the induction of arylsulfatase activity . In contrast to its effect on arylsulfatase activity, Sac3 positively regulates the high-affinity sulfate transport system-the sac3 mutant is unable to fully induce high-affinity sulfate transport during sulfur limitation . We have complemented the sac3 mutant and cloned a cDNA copy of the Sac3 gene . The deduced amino acid sequence of the Sac3 gene product is similar to the catalytic domain of the yeast Snf1 family of serine/threonine kinases and is therefore classified as a Snf1-related kinase (SnRK) . Specifically, Sac3 falls within the SnRK2 subfamily of kinases from vascular plants . In addition to the 11 subdomains common to Snf1-like serine/threonine kinases, Sac3 and the plant kinases have two additional subdomains and a highly acidic C-terminal region . The role of Sac3 in the signal transduction system that regulates the responses of Chlamydomonas to sulfur limitation is discussed.

Biosystems, 1999 May, 50(2), 83 - 97
Diversity of temporal self-organized behaviors in a biochemical system; De la Fuente IM; The numerical study of a glycolytic model formed by a system of three delay-differential equations revealed a notable richness of temporal structures which included the three main routes to chaos, as well as a multiplicity of stable coexisting states . The Feigenbaum, intermitency and quasiperiodicity routes to chaos can emerge in the biochemical oscillator . Moreover, different types of birhythmicity, trirhythmicity and hard excitation emerge in the phase space . For a single range of the control parameter it can be observed the coexistence of two quasiperiodicity routes to chaos, the coexistence of a stable steady state with a stable torus, and the coexistence of a strange attractor with different stable regimes such as chaos with different periodic regimes, chaos with bursting behavior, and chaos with torus . In most of the numerical studies, the biochemical oscillator has been considered under periodic input flux being the mean input flux rate 6 mM/h . On the other hand, several investigators have observed quasiperiodic time patterns and chaotic oscillations by monitoring the fluorescence of NADH in glycolyzing yeast under sinusoidal glucose input flux . Our numerical results match well with these experimental studies.

Matrix Biol, 1999 Feb, 18(1), 5 - 17
Biology and function of hemidesmosomes; Nievers MG et al.; Hemidesmosomes are cell-substratum adhesion sites that connect the extracellular matrix to the keratin cytoskeleton . Our knowledge of the function of these structures has greatly increased as a result of studies on patients with aberrant expression of hemidesmosome components and studies using targeted inactivation of mouse genes encoding these components . Insight into the formation of hemidesmosomes, as well as into protein-protein interactions that occur in these junctional complexes, has recently been gained by in vitro cell transfections, blot overlay and yeast two-hybrid assays . In addition, recent results indicate that the alpha6 beta4 integrin is involved in the transduction of signals that are induced by the extracellular matrix and which modulate processes as diverse as cell proliferation, differentiation, apoptosis, migration and tissue morphogenesis . Thus it seems that hemidesmosomes do not merely maintain dermo-epidermal adhesion and tissue integrity, but that they are also implicated in intracellular signaling . Here we discuss recently published data on the biology and function of hemidesmosomes.

Biochim Biophys Acta, 1999 Jun 15, 1432(1), 137 - 41
Nucleotide sequences of genes for ribosomal protein L41 and tRNAThr(AGU) from Coprinus cinereus; Aimi T et al.; The nucleotide sequences of genes for the homolog in Coprinus cinereus of the eukaryotic ribosomal protein L41 and for tRNAThr(AGU) are reported . The gene for tRNAThr(AGU) was located upstream of the gene for the L41 ribosomal protein, and these genes were adjacent to each other but in opposite orientations . The deduced amino acid sequence of ribosomal protein L41 exhibited strong homology to those of L41 proteins of several yeasts . The 56th amino acid of the deduced protein was proline, as it is in the L41 protein of a cycloheximide-sensitive strain of yeast . The putative secondary structure of the tRNA gene resembled the characteristic cloverleaf structure of tRNAs . Elements resembling an A-box and a B-box were found in the gene for tRNAThr(AGU) . These boxes are known as internal promoter elements in genes for eukaryotic tRNAs.

J Cell Biol, 1999 Jun 14, 145(6), 1277 - 92
A myristoylated calcium-binding protein that preferentially interacts with the Alzheimer's disease presenilin 2 protein; Stabler SM et al.; It is well established that mutations in the presenilin 1 and 2 genes cause the majority of early onset Alzheimer's disease (AD) . However, our understanding of the cellular functions of the proteins they encode remains rudimentary . Knowledge of proteins with which the presenilins interact should lead to a better understanding of presenilin function in normal and disease states . We report here the identification of a calcium-binding protein, calmyrin, that interacts preferentially with presenilin 2 (PS2) . Calmyrin is myristoylated, membrane-associated, and colocalizes with PS2 when the two proteins are overexpressed in HeLa cells . Yeast two-hybrid liquid assays, affinity chromatography, and coimmunoprecipitation experiments confirm binding between PS2 and calmyrin . Functionally, calmyrin and PS2 increase cell death when cotransfected into HeLa cells . These results allude to several provocative possibilities for a dynamic role of calmyrin in signaling, cell death, and AD.

Genomics, 1999 Jun 1, 58(2), 207 - 10
A sequence-ready map of the human chromosome 17p telomere; Xiang Z et al.; A half-YAC clone derived from human chromosome 17p was mapped at high resolution using cosmid subclone fingerprint analysis . Colinearity of the half-YAC with the telomeric human genomic DNA fragment was ascertained by RecA-assisted restriction endonuclease cleavage mapping . Previously isolated and radiation hybrid-mapped markers TEL17P37, TEL17P49, and TEL17P80 mapped 30-60 kb from the 17p terminus . This sequence-ready map permits high-resolution integration of genetic maps with the DNA sequences directly adjacent to the tip of human chromosome 17p, and will provide the cloned DNA required for ascertaining the nucleotide sequence of this subtelomeric region .

Genomics, 1999 Jun 1, 58(2), 188 - 201
Integrated STS/YAC physical, genetic, and transcript map of human Xq21.3 to q23/q24 (DXS1203-DXS1059); Srivastava AK et al.; A map has been assembled that extends from the XY homology region in Xq21.3 to proximal Xq24, approximately 20 Mb, formatted with 200 STSs that include 25 dinucleotide repeat polymorphic markers and more than 80 expressed sequences including 30 genes . New genes HTRP5, CAPN6, STPK, 14-3-3PKR, and CALM1 and previously known genes including BTK, DDP, GLA, PLP, COL4A5, COL4A6, PAK3, and DCX are localized; candidate loci for other disorders for which genes have not yet been identified, including DFN-2, POF, megalocornea, and syndromic and nonsyndromic mental retardation, are also mapped in the region . The telomeric end of the contig overlaps a yeast artificial chromosome (YAC) contig from Xq24-q26 and with other previously published contigs provides complete sequence-tagged site (STS)/YAC-based coverage of the long arm of the X chromosome . The order of published landmark loci in genetic and radiation hybrid maps is in general agreement . Combined with high-density STS landmarks, the multiple YAC clone coverage and integrated genetic, radiation hybrid, and transcript map provide resources to further disease gene searches and sequencing .

Genomics, 1999 Jun 1, 58(2), 171 - 80
Identification of a human homolog of the Drosophila rotated abdomen gene (POMT1) encoding a putative protein O-mannosyl-transferase, and assignment to human chromosome 9q34.1; Jurado LA et al.; We have isolated a human gene homologous to Drosophila melanogaster rotated abdomen, rt, a poorly viable recessive mutation causing a clockwise twisted abdomen in affected flies due to defects in embryonic muscle development . The human gene, like rt, encodes a protein with high homology to the yeast mannosyl-transferases (Pmts) and has been named POMT1 . POMT1 is expressed as a 3.1-kb transcript in all tissues tested, with highest levels in testis and fetal brain . Alternative splicing of several exons in all tissues predicts the generation of several protein isoforms . The most common mRNA variant encodes a 725-aa protein with 40% identity and 62.5% similarity to rt, as well as 30.5% identity and 54% similarity to yeast Pmts . Computer prediction of protein sorting suggests that the POMT1 product could be an integral protein of the endoplasmic reticulum membrane . Given the strong conservation of protein motifs between POMT1 and the yeast Pmts, POMT1 may function as a mannosyl-transferase involved in O-mannosylation of proteins, being the first of such a class found in mammals . The POMT1 locus has been assigned to human chromosome 9q34.1 by somatic cell hybrids, radiation hybrids, and linkage analysis . On the basis of the rt phenotype, POMT1 could be a candidate for uncharacterized genetic disorders of the muscular system, such as some forms of congenital muscular dystrophy or congenital myopathy .

Genomics, 1999 Jun 1, 58(2), 138 - 45
A complete physical contig and partial transcript map of the Williams syndrome critical region; Hockenhull EL et al.; Williams syndrome (WS) is a contiguous gene syndrome caused by hemizygosity for a chromosomal deletion at 7q11.23 . The range of phenotypes includes mental retardation, dysmorphic facies, heart abnormalities, short stature, a specific cognitive profile, hyperacusis, and infantile hypercalcaemia . To identify all the deleted genes, we have constructed a detailed physical map and complete BAC/PAC contig of the critical region, extending a distance of approximately 2 Mb and delimited by the nondeleted markers D7S1816 and D7S489A . Somatic cell hybrids of WS patients were made and used to define the centromeric and telomeric deletion breakpoints, enabling the size of the WS deletion to be defined as approximately 1.4 Mb . Genes previously mapped to the region have been located on the contig, and we have isolated eight transcripts, two of which have been characterized as the genes CPETR1 and CPETR2 . This contig and expressed sequence map will form the basis for the construction of a complete transcription map of the deleted region and will enable genotype-phenotype correlations to be attempted to identify the individual components of WS .

Nature, 1999 Jun 3, 399(6735), 483 - 7
Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC; Shimizu S et al.; During transduction of an apoptotic (death) signal into the cell, there is an alteration in the permeability of the membranes of the cell's mitochondria, which causes the translocation of the apoptogenic protein cytochrome c into the cytoplasm, which in turn activates death-driving proteolytic proteins known as caspases . The Bcl-2 family of proteins, whose members may be anti-apoptotic or pro-apoptotic, regulates cell death by controlling this mitochondrial membrane permeability during apoptosis, but how that is achieved is unclear . Here we create liposomes that carry the mitochondrial porin channel (also called the voltage-dependent anion channel, or VDAC) to show that the recombinant pro-apoptotic proteins Bax and Bak accelerate the opening of VDAC, whereas the anti-apoptotic protein Bcl-x(L) closes VDAC by binding to it directly . Bax and Bak allow cytochrome c to pass through VDAC out of liposomes, but passage is prevented by Bcl-x(L) . In agreement with this, VDAC1-deficient mitochondria from a mutant yeast did not exhibit a Bax/Bak-induced loss in membrane potential and cytochrome c release, both of which were inhibited by Bcl-x(L) . Our results indicate that the Bcl-2 family of proteins bind to the VDAC in order to regulate the mitochondrial membrane potential and the release of cytochrome c during apoptosis.

Nature, 1999 Jun 3, 399(6735), 479 - 83
The MAPK kinase Pek1 acts as a phosphorylation-dependent molecular switch; Sugiura R et al.; The mitogen-activated protein kinase (MAPK) pathway is a highly conserved eukaryotic signalling cascade that converts extracellular signals into various outputs, such as cell growth and differentiation . MAPK is phosphorylated and activated by a specific MAPK kinase (MAPKK): MAPKK is therefore considered to be an activating regulator of MAPK . Pmk1 is a MAPK that regulates cell integrity and which, with calcineurin phosphatase, antagonizes chloride homeostasis in fission yeast . We have now identified Pek1, a MAPKK for Pmk1 MAPK . We show here that Pek1, in its unphosphorylated form, acts as a potent negative regulator of Pmk1 MAPK signalling . Mkh1, an upstream MAPKK kinase (MAPKKK), converts Pek1 from being an inhibitor to an activator . Our results indicate that Pek1 has a dual stimulatory and inhibitory function which depends on its phosphorylation state . This switch-like mechanism could contribute to the all-or-none physiological response mediated by the MAPK signalling pathway.

J Virol, 1999 Jul, 73(7), 5388 - 401
Translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 Gag polyprotein; Cimarelli A et al.; Human immunodeficiency virus type 1 (HIV-1) gag-encoded proteins play key functions at almost all stages of the viral life cycle . Since these functions may require association with cellular factors, the HIV-1 matrix protein (MA) was used as bait in a yeast two-hybrid screen to identify MA-interacting proteins . MA was found to interact with elongation factor 1-alpha (EF1alpha), an essential component of the translation machinery that delivers aminoacyl-tRNA to ribosomes . EF1alpha was then shown to bind the entire HIV-1 Gag polyprotein . This interaction is mediated not only by MA, but also by the nucleocapsid domain, which provides a second, independent EF1alpha-binding site on the Gag polyprotein . EF1alpha is incorporated within HIV-1 virion membranes, where it is cleaved by the viral protease and protected from digestion by exogenously added subtilisin . The specificity of the interaction is demonstrated by the fact that EF1alpha does not bind to nonlentiviral MAs and does not associate with Moloney murine leukemia virus virions . The Gag-EF1alpha interaction appears to be mediated by RNA, in that basic residues in MA and NC are required for binding to EF1alpha, RNase disrupts the interaction, and a Gag mutant with undetectable EF1alpha-binding activity is impaired in its ability to associate with tRNA in cells . Finally, the interaction between MA and EF1alpha impairs translation in vitro, a result consistent with a previously proposed model in which inhibition of translation by the accumulation of Gag serves to release viral RNA from polysomes, permitting the RNA to be packaged into nascent virions.

J Biol Chem, 1999 Jun 18, 274(25), 17806 - 12
Identification of the cell cycle regulator VCP (p97/CDC48) as a substrate of the band 4.1-related protein-tyrosine phosphatase PTPH1; Zhang SH et al.; The human band 4.1-related protein-tyrosine phosphatase PTPH1 was introduced into NIH3T3 cells under the control of a tetracycline-repressible promoter . Ectopic expression of wild type PTPH1 dramatically inhibited cell growth, whereas a catalytically impaired mutant showed no effect . To identify the direct target of PTPH1 in the cell, we generated a substrate-trapping mutant, in which an invariant aspartate residue was changed to alanine (D811A in PTPH1) . The PTPH1-D811A mutant trapped primarily a 97-kDa tyrosine-phosphorylated protein, which was determined to be VCP (also named p97 or yeast CDC48), from various cell lysates in vitro . However, when expressed in mammalian cells, the D811A mutant was observed to contain high levels of phosphotyrosine and did not trap substrates . Mutation of tyrosine 676 to phenylalanine (Y676F) in the PTPH1-D811A mutant led to a marked reduction in phosphotyrosine content . Furthermore, this double mutant specifically trapped VCP in vivo and recognized the C-terminal tyrosines of VCP, whose phosphorylation is important for cell cycle progression in yeast . Like wild type PTPH1, this double mutant also inhibited cell proliferation . Moreover, induction of wild type PTPH1 resulted in specific dephosphorylation of VCP without changing the overall phosphotyrosine profile of the cells . VCP has been implicated in control of a variety of membrane functions, including membrane fusions, and is a regulator of the cell cycle . Our results suggest that PTPH1 may exert its effects on cell growth through dephosphorylation of VCP, thus implicating tyrosine phosphorylation as an important regulator of VCP function.

J Biol Chem, 1999 Jun 18, 274(25), 17599 - 604
Tip60 is a nuclear hormone receptor coactivator; Brady ME et al.; The androgen receptor (AR) is a member of the nuclear hormone receptor superfamily . Recent work in this field has been focused upon defining the mechanisms of transcriptional control exacted by members of this superfamily . Using a COOH-terminal region of the human AR in a yeast two-hybrid screen, we have identified Tip60 as an AR-interacting protein . In this report, we show that Tip60, which was originally identified as a coactivator for the human immunodeficiency virus TAT protein, can enhance AR-mediated transactivation in a ligand-dependent manner in LNCaP and COS-1 cell lines . In addition, our experiments show that Tip60 can also enhance transactivation through the estrogen receptor and progesterone receptor in a ligand-dependent manner; thus identifying Tip60 as a nuclear hormone receptor coactivator . Our studies also demonstrate that Tip60 co-immunoprecipitates with the full-length AR in vitro and that, in our system, Tip60 enhances transactivation to levels observed with the coactivators steroid receptor coactivator 1, p300, and CREB-binding protein . The importance of such proteins in enhancing nuclear hormone receptor-mediated transcriptional activation is widely accepted, and this work suggests that Tip60 may have an equally important role to play.

J Biol Chem, 1999 Jun 18, 274(25), 17417 - 23
Brefeldin A inhibited activity of the sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors; Morinaga N et al.; A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cytosol . Cloning and expression of the cDNA confirmed that the recombinant protein (p200) is a BFA-sensitive ARF GEP . p200 contains a domain that is 50% identical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain . Sec7 domains have been identified also in other proteins with ARF GEP activity, some of which are not inhibited by BFA . To identify structural elements that influence GEP activity and its BFA sensitivity, several truncated mutants of p200 were made . Deletion of sequence C-terminal to the Sec7 domain did not affect GEP activity . A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had high activity . The mutant lacking 630 N-terminal amino acids was, however, only 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-terminal residues) . It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 595 and 630) modifies activity dramatically . Myristoylated recombinant ARFs were better than non-myristoylated as substrates; ARFs 1 and 3 were better than ARF5, and no activity was detected with ARF6 . Physical interaction of the Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein . Effects of BFA on p200 and all mutants with high activity were similar with approximately 50% inhibition at </=50 microM . The inactive BFA analogue B36 did not inhibit the Sec7 domain or p200 . Thus, the Sec7 domain of p200, like that of Sec7 itself (Sata, M., Donaldson, J . G., Moss, J., and Vaughan, M . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 4204-4208), plays a role in BFA inhibition as well as in GEP activity, although the latter is markedly modified by other structural elements.

Genes Dev, 1999 Jun 1, 13(11), 1475 - 85
Hrs, a FYVE finger protein localized to early endosomes, is implicated in vesicular traffic and required for ventral folding morphogenesis; Komada M et al.; Hrs is an early endosomal protein homologous to Vps27p, a yeast protein required for vesicular trafficking . Hrs has a FYVE double zinc finger domain, which specifically binds phosphatidylinositol(3)-phosphate and is conserved in several proteins involved in vesicular traffic . To understand the physiological role of Hrs, we generated mice carrying a null mutation of the gene . Hrs homozygous mutant embryos developed with their ventral region outside of the yolk sac, had two independent bilateral heart tubes (cardia bifida), lacked a foregut, and died around embryonic day 11 (E11) . These phenotypes arise from a defect in ventral folding morphogenesis that occurs normally around E8.0 . Significant apoptosis was detected in the ventral region of mutant embryos within the definitive endoderm, suggesting an important role of this germ layer in ventral folding morphogenesis . Abnormally enlarged early endosomes were detected in the mutants in several tissues including definitive endoderm, suggesting that a deficiency in vesicular transport via early endosomes underlies the mutant phenotype . The vesicular localization of Hrs was disrupted in cells treated with wortmannin, implicating Hrs in the phosphatidylinositol 3-kinase pathway of membrane trafficking.

Exp Gerontol, 1999 Apr, 34(2), 173 - 84
Extended longevity lines of Drosophila melanogaster: abundance of yolk protein gene mRNA in fat body and ovary; Carlson KA et al.; Lines of Drosophila melanogaster selected for late-life female reproduction typically exhibit correlated responses of reduced early fecundity and increased longevity . This relationship suggests a tradeoff between reproductive effort and somatic maintenance, which in turn, underlies some evolutionary theories of senescence . The mechanistic basis of the apparent tradeoff between increased longevity and reduced early-age fecundity has remained obscure . The present manuscript addresses the issues of whether the reduced early-age fecundity in selected lines corresponds to reduced yolk-protein mRNA production, and whether long-lived flies exhibit somatic maintenance in terms of relatively reduced yolk-protein mRNA production in the fat body . Yolk protein is one of the most abundant proteins used for female reproduction . By comparing a set of lines selected for late life reproduction with the corresponding control lines, we show that that yolk-protein gene mRNA relative abundance during the first four days posteclosion did not correspond to reduced early-life fecundity in the selected lines . In D . melanogaster, yolk protein is produced in the fat body and ovarian follicle cells . On the fourth day posteclosion, relatively more yolk-protein gene mRNA was present in the fat body . On day 1 posteclosion, supplemental yeast did not alter relative yolk-protein gene mRNA abundance . However, on day 4 posteclosion, supplemental yeast stimulated yolk-protein gene mRNA production in the fat body, which suggests an underlying mechanism for the nutrition-based phenotypic plasticity of fecundity previously documented in these lines . On medium without supplemental yeast, the relatively low abundance of fat body yolk-protein gene mRNA in the selected lines on day 4 posteclosion corresponds to a prediction derived from the disposable soma theory.

Mol Biol Rep, 1999 Apr, 26(1-2), 137 - 46
Structure and functional analysis of the 26S proteasome subunits from plants; Fu H et al.; As initial steps to define how the 26S proteasome degrades ubiquitinated proteins in plants, we have characterized many of the subunits that comprise the proteolytic complex from Arabidopsis thaliana . A set of 23 Arabidopsis genes encoding the full complement of core particle (CP) subunits and a collection encoding 12 out of 18 known eukaryotic regulatory particle (RP) subunits, including six AAA-ATPase subunits, were identified . Several of these 26S proteasome genes could complement yeast strains missing the corresponding orthologs . Using this ability of plant subunits to functionally replace yeast counterparts, a parallel structure/function analysis was performed with the RP subunit RPN 10/MCB1, a putative receptor for ubiquitin conjugates . RPN10 is not essential for yeast viability but is required for amino acid analog tolerance and degradation of proteins via the ubiquitin-fusion degradation pathway, a subpathway within the ubiquitin system . Surprisingly, we found that the C-terminal motif required for conjugate recognition by RPN10 is not essential for in vivo functions . Instead, a domain near the N-terminus is required . We have begun to exploit the moss Physcomitrella patens as a model to characterize the plant 26S proteasome using reverse genetics . By homologous recombination, we have successfully disrupted the RPN10 gene . Unlike yeast rpn10delta strains which grow normally, Physcomitrella rpn10delta strains are developmentally arrested, being unable to initiate gametophorogenesis . Further analysis of these mutants revealed that RPN10 is likely required for a developmental program triggered by plant hormones.

Mol Biol Rep, 1999 Apr, 26(1-2), 53 - 7
Proteasome-dependent degradation of human CDC25B phosphatase; Cans C et al.; The CDC25 dual specificity phosphatase is a universal cell cycle regulator . The evolutionary conservation of this enzyme from yeast to man bears witness to its major role in the control of cyclin-dependent kinases (CDK) activity that are central regulators of the cell cycle machinery . CDC25 phosphatase both dephosphorylates and activates CDKs . Three human CDC25s have been identified . CDC25A is involved in the control of G1/S, and CDC25C at G2/M throught the activation of CDK 1-cyclin B . The exact function of CDC25B however remains elusive . We have found that CDC25B is degraded by the proteasome pathway in vitro and in vivo . This degradation is dependent upon phosphorylation by the CDK1-cyclin A complex, but not by CDK1-cyclin B . Together with the observations of others made in yeast and mammals, our results suggest that CDC25B might act as a 'mitotic starter' triggering the activation of an auto-amplification loop before being degraded.

Mol Biol Rep, 1999 Apr, 26(1-2), 15 - 9
Assembly of the regulatory complex of the 26S proteasome; Gorbea C et al.; The 19S regulatory complex (RC) of 26S proteasomes is a 900-1000 kDa particle composed of 18 distinct subunits (S1-S15) ranging in molecular mass from 25 to 110 kDa . This particle confers ATP-dependence and polyubiquitin (polyUb) recognition to the 26S proteasome . The symmetry and homogenous structure of the proteasome contrasts sharply with the remarkable complexity of the RC . Despite the fact that the primary sequences of all the subunits are now known, insight has been gained into the function of only eight subunits . The six ATPases within the RC constitute a subfamily (S4-like ATPases) within the AAA superfamily and we have shown that they form specific pairs in vitro . We have now determined that putative coiled-coils within the variable N-terminal regions of these proteins are likely to function as recognition elements that direct the proper placement of the ATPases within the RC . We have also begun mapping putative interactions between non-ATPase subunits and S4-like ATPases . These studies have allowed us to build a model for the specific arrangement of 9 subunits within the human regulatory complex . This model agrees with recent findings by Glickman et al . who have reported that two subcomplexes, termed the base and the lid, form the RC of budding yeast 26S proteasomes.

J Cell Sci, 1999 Jul, 112 ( Pt 13), 2167 - 75
Receptor mediated and fluid phase pathways for internalization of the ER Hsp90 chaperone GRP94 in murine macrophages; Wassenberg JJ et al.; Immunization of mice with GRP94, the endoplasmic reticulum (ER) Hsp90, elicits cytotoxic T lymphocyte (CTL) responses to chaperone-bound, source cell-derived peptides . Elicitation of a CTL response requires that GRP94-associated peptides be transferred onto major histocompatability complex (MHC) class I molecules, a process that is postulated to accompany GRP94 internalization by antigen presenting cells, such as macrophages (Mphi) and dendritic cells (DC) . In studies of GRP94 uptake in elicited Mphi, we report that Mphi display specific cell surface binding of GRP94, and that surface-bound GRP94 can be internalized via receptor mediated endocytosis . GRP94 internalized by this pathway co-localized predominately with transferrin-positive early endosomes . At time periods of up to 20 minutes, little trafficking of GRP94 to the lysosomal compartment was observed . When GRP94 was present in the medium, and thus accessible to both receptor-mediated and fluid phase internalization pathways, internalization was modestly inhibited in the presence of yeast mannan, a competitive inhibitor of mannose/fucose receptor activity, and substantially inhibited by dimethylamiloride, an inhibitor of macropinocytosis . GRP94 internalized via macropinocytosis did not display prominent co-staining with the lysosomal marker LAMP-2 . These data identify multiple pathways of GRP94 internalization and indicate that receptor-dependent uptake of GRP94 is not dependent upon its high mannose oligosaccharide moiety . Most significantly, these data demonstrate the existence of cell surface receptor(s), apparently unique to antigen presenting cells, that function in the binding and internalization of the ER chaperone GRP94.

Oncogene, 1999 Jun 3, 18(22), 3316 - 23
The Bcl-3 oncoprotein acts as a bridging factor between NF-kappaB/Rel and nuclear co-regulators; Dechend R et al.; The proto-oncoprotein Bcl-3 is a member of the IkappaB family and is present predominantly in the nucleus . To gain insight into specific nuclear functions of Bcl-3 we have isolated proteins that interact with its ankyrin repeat domain . Using the yeast two-hybrid-system we identified four novel binding partners of Bcl-3 in addition to NF-kappaB p50 and p52, previously known to associate with Bcl-3 . The novel Bcl-3 interactors Jab1, Pirin, Tip60 and Bard1 are nuclear proteins which also bind to other transcription factors including c-Jun, nuclear factor I (NFI), HIV-1 Tat or the tumor suppressor and PolII holoenzyme component Brca1, respectively . Bcl-3, p50, and either Bard1, Tip60 or Pirin are sequestered into quarternary complexes on NF-kappaB DNA binding sites, whereas Jab1 enhances p50-Bcl-3-DNA complex formation . Furthermore, the histone acetylase Tip60 enhances Bcl-3-p50 activated transcription through an NF-kappaB binding site, indicating that quarternary complexes containing Bcl-3 interactors modulate NF-kappaB driven gene expression . These data implicate Bcl-3 as an adaptor between NF-kappaB p50/p52 and other transcription regulators and suggest that its gene activation function may at least in part be due to recruitment of the Tip60 histone actetylase.

Clin Genet, 1999 Apr, 55(4), 269 - 76
Partial DiGeorge syndrome in two patients with a 10p rearrangement; Van Esch H et al.; We describe 2 patients with a partial DiGeorge syndrome (facial dysmorphism, hypoparathyroidism, renal agenesis, mental retardation) and a rearrangement of chromosome 10p . The first patient carries a complex chromosomal rearrangement, with a reciprocal insertional translocation between the short arm of chromosome 10 and the long arm of chromosome 8, with karyotype 46, XY ins(8;10) (8pter 8q13::10p15-->10p14::8q24.1-->8qter) ins(10:8) (10pter--> 10p15::8q24.1-->8q13::10p14-->10qter) . The karyotype of the second patient shows a terminal deletion of the short arm of chromosome 10 . In both patients, the breakpoints on chromosome 10p reside outside the previously determined DiGeorge critical region II (DGCRII) . This is in agreement with previous reports of patients with a terminal deletion of 10p with breakpoints distal to the DGCRII and renal malformations/hypoparathyroidism, and thus adds to evidence that these features may be caused by haploinsufficiency of one or more genes distal to the DGCRII.

Clin Genet, 1999 Apr, 55(4), 248 - 55
Linkage disequilibrium mapping of the Nova Scotia variant of Niemann-Pick disease; Greer WL et al.; Niemann-Pick type D (NPD) disease is a severe degenerative disorder of the nervous system characterized by the accumulation of tissue cholesterol and sphingomyelin . Because of a founder effect, it is unusually common in southwestern Nova Scotia, Canada . We have confirmed that almost all patients from 20 affected sibships descended on both sides from a small group of Acadians who settled in this region in about the year 1767 . Previously using classic linkage analysis of this large kindred, we defined the critical gene region to a 13-cM chromosome segment between D18S869 and D18S66 . Seven ESTs have been positioned within this interval . Carstea et al . (Niemann Pick C disease gene: homology to mediators of cholesterol homeostasis . Science 1997: 277: 232-235) recently demonstrated that one of these ESTs is the Niemann-Pick type C (NPCI) gene, the gene disrupted in most patients with NPC disease, and we have shown that a G3097-->T mutation in the NPC1 gene is also responsible for NPD . Here we report the development of five new polymorphic microsatellite markers and the testing for complete linkage disequilibrium in our single large NPD kindred that allowed us to reduce the NPD critical region to a 1-cM (1.3-1.6 Mb) interval between D18S1398 and D18S1108 . In contrast, Carstea et al., using classic linkage analysis, required more than 18 unrelated NPC families to reduce the NPC1 critical region to a 5-cM interval . Our work supports the finding that NPD is an allelic variant of NPC1, and illustrates the power of large kindreds, which are common in Atlantic Canada and other relatively isolated areas, for gene mapping and identification.

Curr Opin Struct Biol, 1999 Jun, 9(3), 408 - 15
Protein families in multicellular organisms; Copley RR et al.; The complete sequence of the nematode worm Caenorhabditis elegans contains the genetic machinery that is required to undertake the core biological processes of single cells . However, the genome also encodes proteins that are associated with multicellularity, as well as others that are lineage-specific expansions of phylogenetically widespread families and yet more that are absent in non-nematodes . Ongoing analysis is beginning to illuminate the similarities and differences among human proteins and proteins that are encoded by the genomes of the multicellular worm and the unicellular yeast, and will be essential in determining the reliability of transferring experimental data among phylogenetically distant species.

Fungal Genet Biol, 1999 Apr, 26(3), 236 - 52
sodVIC is an alpha-COP-related gene which is essential for establishing and maintaining polarized growth in Aspergillus nidulans; Whittaker SL et al.; Strains of Aspergillus nidulans carrying the conditional-lethal mutation sodVIC1 (stabilization of disomy) are defective in nuclear division and hyphal extension . The mutation affects both the establishment and maintenance of polar growth, since mutant spores do not germinate at restrictive temperature and preexisting hyphae stop growing upon upshift . The defect is reversible within the first 3-4 h at restrictive temperature but longer periods of incubation are lethal due to cell lysis and morphological abnormalities . There is no evidence for a specific cell cycle lesion, suggesting the existence of a feedback mechanism whereby hyphal extension is coordinated with nuclear partitioning . The sodVIC gene has been cloned from a chromosome VI-specific cosmid library and its product exhibits strong homology to the alpha-COP subunit of the coatomer complex involved in the secretory pathway in yeast and higher organisms . Molecular disruption of the gene is lethal, indicating that SodVIC is essential for growth in A . nidulans .

Mol Cell, 1999 May, 3(5), 679 - 85
DNA polymerase epsilon catalytic domains are dispensable for DNA replication, DNA repair, and cell viability; Kesti T et al.; DNA polymerase epsilon (Pol epsilon) is believed to play an essential catalytic role during eukaryotic DNA replication and is thought to participate in recombination and DNA repair . That Pol epsilon is essential for progression through S phase and for viability in budding and fission yeasts is a central element of support for that view . We show that the amino-terminal portion of budding yeast Pol epsilon (Pol2) containing all known DNA polymerase and exonuclease motifs is dispensable for DNA replication, DNA repair, and viability . However, the carboxy-terminal portion of Pol2 is both necessary and sufficient for viability . Finally, the viability of cells lacking Pol2 catalytic function does not require intact DNA replication or damage checkpoints.

Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 7098 - 103
Map-based cloning of chloronerva, a gene involved in iron uptake of higher plants encoding nicotianamine synthase; Ling HQ et al.; The uptake of iron in plants is a highly regulated process that is induced on iron starvation . In tomato, the mutant chloronerva exhibits constitutive expression of iron uptake responses and intercostal chlorosis . Biochemically, chloronerva is an auxotroph for nicotianamine, a key polyamine in plant iron uptake metabolism . The chloronerva gene has been fine-mapped onto the long arm of chromosome 1 in a large segregating tomato population and yeast artificial chromosome clones encompassing the region were isolated by using flanking markers . A cosmid contig containing the chloronerva gene was established, and complementing cosmids were identified by transformation into the mutant . The chloronerva transcript was identified by cDNA isolation using the complementing cosmids . The gene encodes a unique protein of 35 kDa . The mutant harbors a single base change compared with the wild type . Based on enzyme activity and sequence similarity to the coding DNA sequence of the purified barley enzyme the chloronerva gene encodes the enzyme nicotianamine synthase.

Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6797 - 801
The Rpd3 histone deacetylase is required for segmentation of the Drosophila embryo; Mannervik M et al.; Previous studies have implicated histone deacetylation and chromatin condensation as critical mechanisms of transcription repression in yeast and mammals . A specific histone deacetylase, Rpd3, interacts with a variety of sequence-specific transcriptional repressors, including Mad-Max heterodimers and members of the nuclear receptor superfamily . Here, we present evidence that a strong hypomorphic mutation in the Drosophila Rpd3 gene causes embryonic lethality and a specific pair-rule segmentation phenotype . The analysis of a number of segmentation genes suggests that the repressor function of Even-skipped (Eve) may be diminished, causing an indirect loss of Ftz-mediated activation of engrailed . The relatively mild defects observed in Rpd3 mutants suggest that the recently identified Groucho and dCtBP corepressor proteins do not function solely through the recruitment of histone deacetylases . We discuss the possibility that Eve mediates multiple mechanisms of repression, so that Rpd3 mutants disrupt the regulation of just a subset of Eve target genes.

Proc Natl Acad Sci U S A, 1999 Jun 8, 96(12), 6694 - 9
Regulation of eukaryotic protein synthesis: selective influenza viral mRNA translation is mediated by the cellular RNA-binding protein GRSF-1; Park YW et al.; To better understand regulation of eukaryotic protein synthesis, we studied cellular and viral mRNA translation in influenza virus-infected cells . Influenza virus infection results in a dramatic shut-off of cellular protein synthesis that is concomitant with selective viral mRNA translation . Earlier work showed that these events are mediated by viral and/or cellular factors binding to the 5' untranslated region (5' UTR) of viral mRNAs . To identify trans-acting cellular proteins responsible for selective viral protein synthesis, we employed the yeast three-hybrid system . Using the 5' UTR of the influenza virus nucleocapsid protein (NP) mRNA as bait, we identified the cellular RNA-recognition motif containing RNA-binding protein G-rich sequence factor 1 (GRSF-1) as a positive-acting translational regulatory factor . The in vivo yeast assay revealed GRSF-1 specifically bound to the NP 5' UTR but not select NP 5' UTR mutants or cellular RNA 5' UTRs . These data were confirmed by gel shift assays using recombinant GRSF-1 . Importantly, recombinant GRSF-1 specifically stimulated translation of a NP 5' UTR-driven template in cell-free translation systems . Furthermore, translation efficiency of NP 5' UTR-driven templates was reduced markedly in GRSF-1-depleted HeLa cell extracts, but restored in GRSF-1-reconstituted extracts . GRSF-1 also stimulated translation of an NP 5' UTR-driven template in HeLa cell extracts that were depleted of essential factors by addition of RNA oligonucleotides representing the viral 5' UTR RNA . Taken together, these data document the functional demonstration of a cellular protein binding to influenza virus RNAs and, importantly, suggest that influenza virus may recruit GRSF-1 to the 5' UTR to ensure preferential translation of viral mRNAs in infected cells.

Mol Biol Cell, 1999 Jun, 10(6), 1985 - 95
The human G2 checkpoint control protein hRAD9 is a nuclear phosphoprotein that forms complexes with hRAD1 and hHUS1; St Onge RP et al.; Eukaryotic cells actively block entry into mitosis in the presence of DNA damage or incompletely replicated DNA . This response is mediated by signal transduction cascades called cell cycle checkpoints . We show here that the human checkpoint control protein hRAD9 physically associates with two other checkpoint control proteins, hRAD1 and hHUS1 . Furthermore, hRAD1 and hHUS1 themselves interact, analogously to their fission yeast homologues Rad1 and Hus1 . We also show that hRAD9 is present in multiple phosphorylation forms in vivo . These phosphorylated forms are present in tissue culture cells that have not been exposed to exogenous sources of DNA damage, but it remains possible that endogenous damage or naturally occurring replication intermediates cause the observed phosphorylation . Finally, we show that hRAD9 is a nuclear protein, indicating that in this signal transduction pathway, hRAD9 is physically proximal to the upstream (DNA damage) signal rather than to the downstream, cytoplasmic, cell cycle machinery.

J Clin Invest, 1999 Jun, 103(11), 1517 - 25
Oligospermic infertility associated with an androgen receptor mutation that disrupts interdomain and coactivator (TIF2) interactions; Ghadessy FJ et al.; Structural changes in the androgen receptor (AR) are one of the causes of defective spermatogenesis . We screened the AR gene of 173 infertile men with impaired spermatogenesis and identified 3 of them, unrelated, who each had a single adenine-->guanine transition that changed codon 886 in exon 8 from methionine to valine . This mutation was significantly associated with the severely oligospermic phenotype and was not detected in 400 control AR alleles . Despite the location of this substitution in the ligand-binding domain (LBD) of the AR, neither the genital skin fibroblasts of the subjects nor transfected cell types expressing the mutant receptor had any androgen-binding abnormality . However, the mutant receptor had a consistently (approximately 50%) reduced capacity to transactivate each of 2 different androgen-inducible reporter genes in 3 different cell lines . Deficient transactivation correlated with reduced binding of mutant AR complexes to androgen response elements . Coexpression of AR domain fragments in mammalian and yeast two-hybrid studies suggests that the mutation disrupts interactions of the LBD with another LBD, with the NH2-terminal transactivation domain, and with the transcriptional intermediary factor TIF2 . These data suggest that a functional element centered around M886 has a role, not for ligand binding, but for interdomain and coactivator interactions culminating in the formation of a normal transcription complex.

FEBS Lett, 1999 May 7, 450(3), 280 - 4
Identification and characterization of GABA, proline and quaternary ammonium compound transporters from Arabidopsis thaliana; Breitkreuz KE et al.; Arabidopsis thaliana grows efficiently on GABA as the sole nitrogen source, thereby providing evidence for the existence of GABA transporters in plants . Heterologous complementation of a GABA uptake-deficient yeast mutant identified two previously known plant amino acid transporters, AAP3 and ProT2, as GABA transporters with Michaelis constants of 12.9 +/- 1.7 and 1.7 +/- 0.3 mM at pH 4, respectively . The simultaneous transport of {1-14C}GABA and {2,3-3H}proline by ProT2 as a function of pH, provided evidence that the zwitterionic state of GABA is an important parameter in substrate recognition . ProT2-mediated {1-14C}GABA transport was inhibited by proline and quaternary ammonium compounds.

Chem Phys Lipids, 1999 Apr, 98(1-2), 87 - 94
FYVE finger proteins as effectors of phosphatidylinositol 3-phosphate; Gaullier JM et al.; Phosphatidylinositol 3-phosphate (PtdIns(3)P), generated via the phosphorylation of phosphatidylinositol by phosphatidylinositol 3-kinase (PI 3-kinase), plays an essential role in intracellular membrane traffic . The underlying mechanism is still not understood in detail, but the recent identification of the FYVE finger as a protein domain that binds specifically to PtdIns(3)P provides a number of potential effectors for PtdIns(3)P . The FYVE finger (named after the first letter of the four proteins containing it; Fab1p, YOTB, Vac1p and EEA1) is a double-zinc binding domain that is conserved in more than 30 proteins from yeast to mammals . It is found in several proteins involved in intracellular traffic, and FYVE finger mutations that affect zinc binding are associated with the loss of function of several of these proteins . The interaction of FYVE fingers with PtdIns(3)P may serve three alternative functions: First, to recruit cytosolic FYVE finger proteins to PtdIns(3)P-containing membranes (in concert with accessory molecules); second, to enrich for membrane bound FYVE finger proteins into PtdIns(3)P containing microdomains within the membrane; and third, to modulate the activity of membrane bound FYVE finger proteins.

J Biol Chem, 1999 Jun 11, 274(24), 17372 - 8
A novel insect V-ATPase subunit M9.7 is glycosylated extensively; Merzendorfer H et al.; Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide . Based on N-terminal protein sequencing, we cloned a corresponding cDNA . The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa . Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal anti- body to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned . The 20-kDa polypeptide is glycosylated, as deduced from lectin staining . Treatment with N-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptide-specific monoclonal antibody . Thus, extensive N-glycosylation of the novel Vo subunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis . M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the Vo complex . However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M . sexta.

J Biol Chem, 1999 Jun 11, 274(24), 17309 - 17
A new nucleoporin-like protein interacts with both HIV-1 Rev nuclear export signal and CRM-1; Farjot G et al.; The HIV-1 Rev is a shuttling protein required for the nuclear export of unspliced and partially spliced viral mRNA . In this study, we have identified a new Rev-interacting protein, that specifically interacts with the Rev nuclear export signal both in yeast and mammalian cells . This protein has features found in nucleoporins including many phenylalanine-glycine repeats, a very high serine content, a putative zinc finger, and a coiled-coil domain; we thus called it NLP-1 (nucleoporin-like protein 1) . In addition, gene expression analysis and wheat germ agglutinin chromatography experiments suggested that NLP-1 is an ubiquitous O-glycosylated nuclear protein . Recently, a cellular factor called CRM-1 has been shown to be an essential nuclear export factor interacting directly with nuclear export signals including the Rev nuclear export signal in a RanGTP-dependent manner . We show here that NLP-1, like the previously described Rev-interacting protein hRIP/Rab and several nucleoporins, also interacts with CRM-1 both in yeast and mammalian cells.

J Biol Chem, 1999 Jun 11, 274(24), 17003 - 10
Physical interaction and functional antagonism between the RNA polymerase II elongation factor ELL and p53; Shinobu N et al.; ELL was originally identified as a gene that undergoes translocation with the trithorax-like MLL gene in acute myeloid leukemia . Recent studies have shown that the gene product, ELL, functions as an RNA polymerase II elongation factor that increases the rate of transcription by RNA polymerase II by suppressing transient pausing . Using yeast two-hybrid screening with ELL as bait, we isolated the p53 tumor suppressor protein as a specific interactor of ELL . The interaction involves respectively the transcription elongation activation domain of ELL and the C-terminal tail of p53 . Through this interaction, ELL inhibits both sequence-specific transactivation and sequence-independent transrepression by p53 . Thus, ELL acts as a negative regulator of p53 in transcription . Conversely, p53 inhibits the transcription elongation activity of ELL, suggesting that p53 is capable of regulating general transcription by RNA polymerase II through controlling the ELL activity . Elevated levels of ELL in cells resulted in the inhibition of p53-dependent induction of endogenous p21 and substantially protected cells from p53-mediated apoptosis that is induced by genotoxic stress . Our observations indicate the existence of a mutually inhibitory interaction between p53 and a general transcription elongation factor ELL and raise the possibility that an aberrant interaction between p53 and ELL may play a role in the genesis of leukemias carrying MLL-ELL gene translocations.

Cell Immunol, 1999 May 25, 194(1), 47 - 53
Complement deposition on immune complexes reduces the frequencies of metabolic, proteolytic, and superoxide oscillations of migrating neutrophils; Amit A et al.; Neutrophils exhibit intrinsic sinusoidal metabolite concentration oscillations of 3 min in resting cells and an additional approximately 10- or 20-s oscillation in migrating/adhering cells . To better understand immune complex (IC)-mediated leukocyte activation, we have studied neutrophil metabolic oscillations in the presence of ICs either with or without fixed complement . Using a microscope photometer we quantitated NAD(P)H autofluorescence oscillations . Cells exposed to ICs exhibited metabolic oscillation periods of approximately 12 s in the absence of complement and approximately 22 s in the presence of complement opsonization . To determine if the effects could be associated with C3 deposition, we used ICs opsonized with only C3 or only C1 and C4 . Untreated ICs, heat-inactivated complement-treated ICs, and C1,C4-treated ICs trigger rapid metabolic oscillations, as do fMLP and yeast; in contrast, ICs treated with full complement or C3 alone did not affect NAD(P)H oscillations in comparison to controls . The induction of higher frequency (approximately 10 s) NAD(P)H oscillations by ICs could be blocked by addition of anti-FcgammaRII, but not FcgammaRIII mAb fragments, suggesting the participation of FcgammaRII in cellular metabolic responses to ICs . Parallel changes in the frequencies of oxidant release and pericellular proteolysis were found for all of these stimuli . Thus, immune complex composition affects both intracellular metabolic signals and extracellular functional oscillations . We suggest that complement attenuates the phlogistic potential of ICs by reducing the frequency of cytoplasmic NAD(P)H oscillations .

Biotechnol Prog, 1999 May, 15(3), 500 - 5
Protein adsorption onto tentacle cation-exchange hollow-fiber membranes
Camperi SA, Navarro del Canizo AA, Wolman FJ, Smolko EE, Cascone O, Grasselli M.
A sulfonic group (up to 200 &mgr;mol/mL) membrane was incorporated to epoxy-activated microporous hollow fibers to obtain high-capacity convective ion exchangers . The pure water flux through the membrane decreased exponentially with sulfonic group density and protein binding capacity increased accordingly . At sulfonic group density of 70 &mgr;mol/mL, the membrane lysozyme maximum binding capacity was 84 +/- 9 mg/mL in comparison with its theoretical monolayer maximum binding capacity of 20 mg/mL, thus evidencing tentacle formation . After a cycle of adsorption in a 30 mM sodium phosphate buffer, pH 7 . 0, adsorbed lysozyme could be quantitatively recovered following elution with 0.5 M NaCl in the same buffer . Dynamic capacity for lysozyme was 67% of maximum binding, and this value did not change at space velocities ranging from 10 to 40 min-1 as shown by the superimposition of the corresponding breakthrough curves . A cartridge assembled with 21 fibers showed a dynamic-to-static capacity ratio for lysozyme of 0.60 with 1 mg/mL pure lysozyme solution, and 0.42 with a particulate feed composed of 1 mg/mL lysozyme and 0.1 mg/mL yeast.

J Lipid Res, 1999 Jun, 40(6), 994 - 1006
Transgenic mice expressing a human apolipoprotein{a} allele; Acquati F et al.; The most important determinant of plasma levels of Lp{a} are sequence differences at the highly polymorphic apolipoprotein{a} (apo{a}) locus . To define the sequences that mediate the regulation of apo{a} expression, we cloned a 370 kb DNA fragment that included a 130 kb apo{a} gene, and 40 kb 5'- and 200 kb 3'-flanking region from an individual with high plasma levels of Lp{a} using a YAC vector . This genomic clone was used to generate transgenic mice . In the YAC-apo{a} transgenic mouse, apo{a} was only expressed in the liver, as it is in humans . The mean serum level of apo{a} in 4-week-old YAC-apo{a} transgenic mice was 20 mg/dl . In the female mice the levels of apo{a} varied over a 1.5-fold range during the 4-day estrus cycle and the levels correlated directly with serum progesterone levels . The serum levels of apo{a} decreased to almost undetectable level in male mice after puberty and this decrease was reversed by castration . Ingestion of a high-fat diet resulted in a approximately 100-fold fall in hepatic apo{a} mRNA levels and >60-fold decrease in serum apo{a} levels.To delimit the control elements that mediate tissue-specific and sex hormone-responsive apo{a} transcription, we derived a reporter YAC in which 40 kb of 5' flanking sequences from the cloned apo{a} allele were linked to a luciferase reporter gene . Analysis of four independent transgenic lines revealed no hepatic luciferase expression, suggesting that important cis -acting elements located outside the apo{a} 5'-flanking region are necessary for in vivo expression of apo{a}.

FEBS Lett, 1999 May 14, 451(1), 27 - 32
Structure of VAT, a CDC48/p97 ATPase homologue from the archaeon Thermoplasma acidophilum as studied by electron tomography; Rockel B et al.; Valosine-containing protein-like ATPase from Thermoplasma acidophilum is a member of the superfamily of ATPases associated with a diversity of cellular activities and is closely related to CDC48 from yeast and p97 from higher eukaryotes and more distantly to N-ethylmaleimide-sensitive fusion protein . We have used electron tomography to obtain low-resolution (2-2.5 nm) three-dimensional maps of both the whole 500 kDa complex and the N-terminally truncated valosine-containing protein-like ATPase from T . acidophilum complex lacking the putative substrate binding domain.

J Mol Biol, 1999 Jun 11, 289(3), 639 - 44
Partially formed native tertiary interactions in the A-state of cytochrome c; Hostetter DR et al.; Considerable insight into protein structure, stability, and folding has been obtained from studies of non-native states . We have studied the extent of native tertiary contacts in one such molecule, the A-state of yeast iso-1-ferricytochrome c . Previously, we showed that the interface between the N and C-terminal helices is completely formed in the A-state . Here, we focus on interactions essential for forming the heme pocket of eukaryotic cytochromes c . To determine the extent of these interactions, we used saturation mutagenesis at the evolutionarily invariant residue leucine 68, and measured the free energy of denaturation for the native states and the A-states of functional variants . We show that, unlike the interaction between the terminal helices, the native interactions between the 60s helix and the rest of the protein are not completely formed in the A-state .

J Gen Virol, 1999 May, 80 ( Pt 5), 1127 - 31
Self-association and mapping of interaction domains of helper component-proteinase of potato A potyvirus; Guo D et al.; Potyviral helper component-proteinase (HC-Pro) is a multifunctional protein involved in aphid transmission, long-distance movement, polyprotein processing, genome amplification and symptom expression . It has been proposed that the active form of HC-Pro is a dimer and that coat protein (CP)-HC-Pro interaction is required for aphid transmission . To test these proposed interactions between CP and HC-Pro of potato A potyvirus (PVA), the yeast two-hybrid system was used . HC-Pro was shown to interact with itself in vivo in yeast cells, as did CP . Taken together with previous observations, we conclude that the functional HC-Pro is a homodimer . Deletion analysis showed that a 24 aa domain in the N-terminal half and the C-terminal proteinase part of HC-Pro were required for the interaction between HC-Pro molecules . No interactions were found between HC-Pro and CP using the genes of aphid-transmissible as well as aphid non-transmissible strains of PVA.

Mech Dev, 1999 Apr, 82(1-2), 23 - 8
A methionine aminopeptidase and putative regulator of translation initiation is required for cell growth and patterning in Drosophila; Cutforth T et al.; We have isolated mutations in the gene Drosophila methionine aminopeptidase 2 (DMAP2), which encodes a homolog of the type 2 methionine aminopeptidase from yeast, also known as the eukaryotic initiation factor 2alpha (eIF2alpha) associated protein p67 . Weak DMAP2 mutations cause ommatidial rotation defects and loss of ventral tissue in the compound eye as well as extra wing veins, whereas stronger alleles impair tissue growth . These limited phenotypes, in conjunction with the differential accumulation of DMAP2 transcripts throughout embryonic and larval development, suggest that a subset of proteins is spatially and temporally regulated at the level of post-translational processing or translation initiation during development . These results provide genetic evidence for post-transcriptional control in the development of multicellular organisms.

Nature, 1999 May 20, 399(6733), 276 - 9
Mammalian Srb/Mediator complex is targeted by adenovirus E1A protein; Boyer TG et al.; Adenovirus E1A proteins prepare the host cell for viral replication, stimulating cell cycling and viral transcription through interactions with critical cellular regulatory proteins such as RB and CBP . Here we show that the E1A zinc-finger domain that is required to activate transcription of viral early genes binds to a host-cell multiprotein complex containing homologues of yeast Srb/Mediator proteins . This occurs through a stable interaction with the human homologue of Caenorhabditis elegans SUR-2, a protein required for many developmental processes in the nematode . This human Srb/Mediator complex stimulates transcription in vitro in response to both the E1A zinc-finger and the herpes simplex virus VP16 activation domains . Interaction with human Sur-2 is also required for transcription to be activated by the activation domain of a transcription factor of the ETS-family in response to activated mitogen-activated protein (MAP) kinase.

Eur J Hum Genet, 1999 May-Jun, 7(4), 487 - 95
Detailed transcript map of a 810-kb region at 11p14 involving identification of 10 novel human 3' exons; Guillemot F et al.; A limited number of genes, including the human brain-derived neutrotrophic factor (BDNF) gene, have been identified in the human chromosome 11p14 region . Since this area is involved in a genetic disorder (WAGR syndrome) and because of interest in studying the regulation of the human BDNF gene, we have established a detailed transcript map of a 810-kb region clone in a yeast artificial chromosome (YAC), corresponding to a portion of this genomic locus . A set of nested deletion mutants has been generated to map genes at a mean resolution of 75kb . Four genic markers from available mapping databases have been mapped on the YAC . Ten potential novel human exons have been isolated by a 3' terminal exon trapping procedure directly applied to purified YAC DNA . Most of these exons display polyadenylation signals and they all yield positive signals in RT-PCR experiments, confirming their status of transcribed sequences . The BDNF gene is now co-localised with three other genes on a 120 kb DNA fragment.

Eur J Hum Genet, 1999 May-Jun, 7(4), 427 - 34
Genetic refinement and physical mapping of a chromosome 18q candidate region for bipolar disorder; Verheyen GR et al.; Recent genetic studies have implicated chromosome 18 in bipolar disorder (BP) with putative loci in the pericentromeric region and on 18q . We reported linkage to chromosome 18q21.33-q23 in a large family . In this study we typed additional markers in the family and were able to reduce the candidate region significantly . All affected family members are sharing alleles for markers spanning a genetic distance of maximal 8.9 cM . Haplotype analysis provided a marker order in agreement with published genetic and physical maps . Using yeast artificial chromosomes, we constructed a contig map that will help to identify positional candidate genes for bipolar disorder.

Nucleic Acids Res, 1999 Jun 15, 27(12), 2465 - 72
hPop4: a new protein subunit of the human RNase MRP and RNase P ribonucleoprotein complexes; van Eenennaam H et al.; RNase MRP is a ribonucleoprotein particle involved in the processing of pre-rRNA . The RNase MRP particle is structurally highly related to the RNase P particle, which is involved in pre-tRNA processing . Their RNA components fold into a similar secondary structure and they share several protein subunits . We have identified and characterised human and mouse cDNAs that encode proteins homologous to yPop4p, a protein subunit of both the yeast RNase MRP and RNase P complexes . The human Pop4 cDNA encodes a highly basic protein of 220 amino acids . Transfection experiments with epitope-tagged hPop4 protein indicated that hPop4 is localised in the nucleus and accumulates in the nucleolus . Immunoprecipitation assays using extracts from transfected cells expressing epitope-tagged hPop4 revealed that this protein is associated with both the human RNase MRP and RNase P particles . Polyclonal rabbit antibodies raised against recombinant hPop4 recognised a 30 kDa protein in total HeLa cell extracts and specifically co-immunoprecipitated the RNA components of the RNase MRP and RNase P complexes . Finally we showed that anti-hPop4 immunoprecipitates possess RNase P enzymatic activity . Taken together, these data show that we have identified a protein that represents the human counterpart of the yeast Pop4p protein.

Adolesc Med, 1990 Jun, 1(2), 333 - 344
Tinea Infections in Adolescents; Frieden IJ; Superficial fungal and yeast infections of the skin are common during adolescence . This article describes the causes, clinical manifestations, differential diagnosis, and treatment of four infections that increase in frequency during adolescence: tinea versicolor, tinea pedis, fungal nail infections, and tinea cruris.

Eur J Cell Biol, 1999 Apr, 78(4), 278 - 87
Interactions between coiled-coil proteins: Drosophila lamin Dm0 binds to the bicaudal-D protein; Stuurman N et al.; In a yeast two-hybrid screen we identified an interaction between Drosophila lamin Dm0, a structural nuclear protein, and BICD, a protein involved in oocyte development . The interaction can be reconstituted in vitro and takes place between segments of both proteins predicted to form coiled coils . The affinity for lamin Dm0 of the minimal binding site on BICD is modulated in a complex fashion by other BICD segments . A point mutation, F684I, that causes the dominant, bicaudal, Bic-D phenotype inhibits lamin binding in the context of the minimal lamin-binding site, but not in a larger BICD fragment . The minimal lamin-binding site of BICD binds to a few other coiled-coil proteins, but binding to these proteins is not influenced by the F684I point mutation, suggesting that the interaction with lamin may play a role in Bic-D function . Our structural studies demonstrated that BICD is 60-70% alpha-helical, is a dimer, and consists of two parts: a thin rod-shaped part of about 32 nm, and a thicker rod-shaped part of about 26 nm . Likely, the thinner rod-shaped part of full-length BICD consists of the N-terminal half of the protein, and the lamin-binding site is located within the thicker rod-shaped part.

Eur J Cell Biol, 1999 Apr, 78(4), 224 - 32
Phosphorylation of p97(VCP) and p47 in vitro by p34cdc2 kinase; Mayr PS et al.; The hexameric ATPase p97/yeast Cdc48p has been implicated in a number of cellular events that are regulated during mitosis, including homotypic membrane fusion, spindle pole body function, and ubiquitin-dependent protein degradation . p97/Cdc48p contains two conserved consensus p34cdc2 kinase phosphorylation sites within its second ATP binding domain . This domain is likely to play a role in stabilising the hexameric form of the protein . We therefore investigated whether p97 could be phosphorylated by p34cdc2 kinase in vitro, and whether phosphorylation might influence the oligomeric status of p97 . Monomeric, but not hexameric, p97 was phosphorylated by p34cdc2 kinase, as was the p97-associated protein p47 . However, phosphorylation by p34cdc2 kinase did not impair subsequent re-hexamerisation of p97, implying that the phosphorylated residue(s) are not critical for interaction between p97 monomers . Moreover, p97 within both interphase and mitotic cytosols was almost exclusively hexameric, suggesting that the activity of p97 is not regulated during mitosis by influencing the extent of oligomerisation.

Plant Mol Biol, 1999 Mar, 39(4), 647 - 56
GRAB proteins, novel members of the NAC domain family, isolated by their interaction with a geminivirus protein; Xie Q et al.; Geminiviruses encode a few proteins and depend on cellular factors to complete their replicative cycle . As a way to understand geminivirus-host interactions, we have searched for cellular proteins which interact with viral proteins . By using the yeast two-hybrid technology and the wheat dwarf geminivirus (WDV) RepA protein as a bait, we have isolated a family of proteins which we termed GRAB (for Geminivirus Rep A-binding) . We report here the molecular characterization of two members, GRAB1 and GRAB2 . We have found that the 37 C-terminal amino acids of RepA are required for interaction with GRAB proteins . This region contains residues conserved in an equivalent region of the RepA proteins encoded by other viruses of the WDV subgroup . The N-terminal domain of GRAB proteins is necessary and sufficient to interact with WDV RepA . GRAB proteins contain an unique acidic C-terminal domain while their N-terminal domain, of ca . 170 amino acids, are highly conserved in all of them . Interestingly, this conserved N-terminal domain of GRAB proteins exhibits a significant amino acid homology to the NAC domain present in proteins involved in plant development and senescence . GRAB1 and GRAB2 mRNAs are present in cultured cells and roots but are barely detectable in leaves . GRAB expression inhibits WDV DNA replication in cultured wheat cells . Our studies highlight the importance that the pathway(s) mediated by GRAB proteins, as well as by other NAC domain-containing proteins, might have on geminivirus DNA replication in connection to plant growth, development and senescence pathways.

Z Naturforsch {C}, 1999 Mar-Apr, 54(3-4), 278 - 84
Fluorescence studies on association of human translation initiation factor eIF4E with mRNA cap-analogues; Wieczorek Z et al.; Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5'-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence . A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence delta Fmax, during titration of eIF4E by various cap-analogues . The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct . Biol . 4, 717) . On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap . Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E-7-methyl-GDP complexes in crystal and solution.

Mech Dev, 1998 Dec, 79(1-2), 169 - 84
YAC transgenic analysis reveals Wilms' tumour 1 gene activity in the proliferating coelomic epithelium, developing diaphragm and limb; Moore AW et al.; Wilms' Tumour 1 gene (WT1) is required for the correct development of the urogenital system . To examine its regulation and expression, we created several transgenic mouse lines containing a beta-galactosidase reporter driven by the human WT1 promoter . A 5 kb promoter weakly recapitulated a subset of the endogenous Wt1 expression pattern . In contrast, 470 and 280 kb YAC transgenes reproduced the correct pattern with high activity and highlighted new expression sites . Wt1 is expressed in the septum transversum revealing how its mutation causes diaphragmatic defects . Wt1 expression in the limb demarcates a zone between chondrogenic and apoptotic domains . Finally, Wt1 is expressed in mesenchymal cells derived from the coelomic epithelium . Based upon these and further data we discuss a Wt1 role in epithelial<-->mesenchymal transitions.

Cancer Genet Cytogenet, 1999 Jun, 111(2), 119 - 23
Frequent alterations of evolutionarily conserved regions of chromosome 1 in human malignant melanoma; Zhang J et al.; Recurring alterations of chromosome 1 represent the most frequent site of structural chromosome abnormalities across all human solid tumors, including human cutaneous malignant melanoma . In melanoma, breakpoints involving chromosome 1 appear to accumulate most frequently at the paracentromeric regions, and secondly, to cluster within 1p36 . Of interest, these three band regions (1p11-12, 1q21, and 1p36) were simultaneously recognized by a single YAC clone which was isolated from sequences mapping to 1q21 . This observation indicates the common and highly conserved nature of sequences residing within these three bands . Because of this finding, we have examined the possible association of these recurring sites of rearrangements of chromosome 1 in malignant melanoma . To elucidate genomic alterations in these regions, we have analyzed melanoma samples simultaneously by fluorescence in situ hybridization (FISH) using both the YAC clone encoding 1p11, 1q21, and 1p36 homologous sequences, and an alpha-satellite probe for the chromosome 1 centromere . Twelve of 20 (60%) randomly selected melanoma cell lines showed detectable rearrangements in one or more of the chromosome 1 band regions . These results provide support for the notion that the homology between these regions is associated with chromosomal instability, and possibly, is of biologic relevance in malignant melanoma.

Cancer Genet Cytogenet, 1999 Jun, 111(2), 105 - 10
Elucidation of the mechanism of homozygous deletion of 3p12-13 in the U2020 cell line reveals the unexpected involvement of other chromosomes; Heppell-Parton AC et al.; Homozygous deletions in tumor cells have been useful in the localization and validation of tumor suppressor genes . We have described a homozygous deletion in a lung cancer cell line (U2020) which is located within the most proximal of the three regions on the short arm of chromosome 3 believed to be lost in lung cancer development . Construction of a YAC contig map indicates that the deletion spans around 8 Mb, but no large deletion was apparent on conventional cytogenetic analysis of the cell line . To investigate this paradox, whole chromosome, arm-specific, and regional paints have been used . This analysis has revealed that genetic loss has occurred by complex rearrangements of chromosomes 3, rather than simple interstitial deletion . These studies emphasize the power of molecular cytogenetics to disclose unsuspected tumor-specific translocations within the extremely complex karyotypes characteristic of solid tumors.

J Biol Chem, 1999 Jun 4, 274(23), 16304 - 10
Activity of the human centrosomal kinase, Nek2, depends on an unusual leucine zipper dimerization motif; Fry AM et al.; Nek2 is a human cell cycle-regulated kinase that is structurally related to the mitotic regulator, NIMA, of Aspergillus nidulans . Localization studies have shown that Nek2 is a core component of the centrosome, the microtubule organizing center of the cell, and functional approaches suggest a possible role for Nek2 in centrosome separation at the G2/M transition . Here, we have investigated the importance of an unusual leucine zipper coiled-coil motif present in the C-terminal noncatalytic domain of the Nek2 kinase . Glycerol gradient centrifugation indicated that endogenous Nek2 is present in HeLa cells as a salt-resistant 6 S complex, the predicted size of a Nek2 homodimer . Recombinant Nek2 overexpressed in insect cells also formed a 6 S complex, whereas a Nek2 mutant specifically lacking the leucine zipper motif was monomeric . Using yeast two-hybrid interaction analyses and coprecipitation assays, we found that Nek2 can indeed form homodimers both in vivo and in vitro and that this dimerization specifically required the leucine zipper motif . Moreover, deletion of the leucine zipper prevented a trans-autophosphorylation reaction on the C-terminal domain of Nek2 and strongly reduced Nek2 kinase activity on exogenous substrates . Finally, we emphasize that the Nek2 leucine zipper described here differs from classical leucine zippers in that it exhibits a radically different arrangement of hydrophobic and charged amino acids . Thus, this study reveals not only an important mechanism for the regulation of the Nek2 kinase but, furthermore, highlights an unusual organization of a leucine zipper dimerization motif.

J Biol Chem, 1999 Jun 4, 274(23), 16077 - 84
Identification of amino acid residues critical for aggregation of human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES . Characterization of active disaggregated chemokine variants; Czaplewski LG et al.; Human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates . To explore the biological significance of chemokine aggregation, nonaggregating variants were sought . The phenotypes of 105 hMIP-1alpha variants generated by systematic mutagenesis and expression in yeast were determined . hMIP-1alpha residues Asp26 and Glu66 were critical to the self-association process . Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml . Substitution of identical or analogous residues in homologous positions in both hMIP-1beta and RANTES demonstrated that they were also critical to aggregation . Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present . Solution of the three-dimensional NMR structure of hMIP-1alpha has enabled comparison of these residues in hMIP-1beta and RANTES . Aggregated and disaggregated forms of hMIP-1alpha, hMIP-1beta, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies . We have therefore generated novel reagents to evaluate the role of hMIP-1alpha, hMIP-1beta, and RANTES aggregation in vitro and in vivo . The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities . Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains . This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

Electrophoresis, 1999 Apr-May, 20(4-5), 1027 - 32
Technology development at the interface of proteome research and genomics: mapping nonpolymorphic proteins on the physical map of mouse chromosomes; Nock C et al.; Data obtained from protein spots by peptide mass fingerprinting are used to identify the corresponding genes in sequence databases . The relevant cDNAs are obtained as clones from the Integrated Molecular Analysis of Genome Expression (I.M.A.G.E.) consortium . Mapping of I.M.A.G.E . clones is performed in two steps: first, cDNA clones are hybridized against a 10-hit genomic mouse bacterial artificial chromosome (BAC) library . Second, interspersed repetitive sequence polymerase chain reaction (IRS-PCR) using a single primer directed against the mouse B1 repeat element is performed on BACs . As each cDNA detects several BACs, and each individual BAC has a 50% chance to recover an IRS-PCR fragment, the majority of cDNAs produce at least a single IRS-PCR fragment . Individual IRS fragments are hybridized against high-density spotted filter grids containing the three-dimensional permutated pools of yeast artificial chromosome (YAC) library resources that are currently being used to construct a physical map of the mouse genome . IRS fragments that hybridize to YAC clones already placed into contigs immediately provide highly precise map positions . This technology therefore is able to draw links between proteins detected by 2-D gel electrophoresis and the corresponding gene loci in the mouse genome.

Electrophoresis, 1999 Apr-May, 20(4-5), 749 - 54
Method for identification and quantitative analysis of protein lysine methylation using matrix-assisted laser desorption/ionization--time-of-flight mass spectrometry and amino acid analysis; Yan JX et al.; Protein methylation is a post-translational modification that might have important functional roles in cell regulation . We present a new technique with sufficient sensitivity (sub-pmol level) for analysis of methylation of proteins in abundances typically found on proteome maps produced by two-dimensional (2-D) gel electrophoresis . The method involves the identification and quantitation of lysine (Lys) methylation using Fmoc (9-fluorenylmethyl chloroformate)-based amino acid analysis (AAA) . Tri- and monomethyl-Lys were baseline-separated from other amino acids using a modified buffer system . Trimethyl-Lys was quantitatively recovered after acid hydrolysis and AAA of two known methylated proteins - yeast cytochome c and human calmodulin . The methylated peptides from tryptic digestion of those two proteins were identified by high sensitivity matrix-assisted laser desorption/ionization - time-of-flight (MALDI-TOF) mass spectrometry (MS) . An automated mass-screening approach is proposed for the study of various post-translational modifications to understand the distribution of those protein isoforms separated by two-dimensional polyacrylamide gel electrophoresis . It is concluded that the combination of AAA and MALDI-TOF-MS provides a high sensitivity quantitative tool for the analysis of protein post-translational methylation in the context of proteome studies.

Cytogenet Cell Genet, 1999, 84(1-2), 140 - 4
Cytogenetic assignment of 53 microsatellites from the USDA-MARC porcine genetic map; Lopez-Corrales NL et al.; This study provides 53 new fluorescent in situ hybridization cytogenetic assignments for microsatellite markers linked on the swine genetic map . Forty microsatellites are physically assigned for the first time . The chromosomal locations of eight markers were either confirmed or refined, while five loci were assigned to locations different from those given in previous reports . Markers were selected to provide physical anchors based on their presumed proximity to centromeres or telomeres and at approximately 30 cM intervals across the genetic map . The number of physical anchors for pig (SSC) chromosomes 8, 15, and 18 linkage groups was significantly improved . Centromeric regions were localized to areas less than 10 cM for SSC 1, 2, 3, 6, 7, 8, and 9 . Although the recombination rate was generally higher across small biarmed chromosomes and lowest for large acrocentric chromosomes, two regions with particularly low (1q2.1-->q2.9 and 13q2.3-->q4.1) and three regions with extremely high (5p1.5-->p1.2, 6p1.4-->p1.3, and 12p1.5-->p1.4) rates of recombination were detected . These assignments represent an overall 10% increase in the number of physically assigned markers in Sus scrofa and more than a 20% increase in the number of Type II loci assigned to the pig cytogenetic map.

Cytogenet Cell Genet, 1999, 84(1-2), 125 - 7
Genomic organization of the human complex I 13-kDa subunit gene NDUFA5; Tensing K et al.; We have characterized the human gene NDUFA5 encoding a 13-kDa subunit of the mitochondrial respiratory chain complex I (NADH: ubiquinone oxidoreductase) . The gene contains 5 exons and 4 introns, and spans 14 kb of genomic DNA . In the untranscribed region we observed potential transcription factor binding sites . We determined a single nucleotide variant (C/T) at -318, and its frequency in the German population . The functional gene was localised by FISH to 7q31 and by radiation hybrid panel near marker D7S648 in YAC 883_a_2.

Cytogenet Cell Genet, 1999, 84(1-2), 75 - 82
Identification of BAIAP2 (BAI-associated protein 2), a novel human homologue of hamster IRSp53, whose SH3 domain interacts with the cytoplasmic domain of BAI1; Oda K et al.; BAI1 (brain-specific angiogenesis inhibitor 1) was originally isolated as a p53-target gene specifically expressed in brain . To clarify its function, we have been searching for cellular proteins that associate with the cytoplasmic domain of BAI1 . Using its intracellular carboxyl terminus as "bait" in a yeast two-hybrid system, we isolated a cDNA clone named BAIAP2 whose nucleotide sequence would encode a 521-amino acid protein showing significant homology to a 58/53-kDa substrate of insulin-receptor kinase in the hamster . As the expression profile of BAIAP2 examined by Northern blot analysis was almost identical to that of BAI1, BAIAP2 appears to be active mainly in neurons . In vitro binding assays confirmed that a proline-rich cytoplasmic fragment of BAI1 interacted with the Src homology 3 (SH3) domain of BAIAP2 . Double-color immunofluorescent analysis revealed that BAIAP2 was localized at the cytoplasmic membrane when it was coexpressed with BAI1 in COS-7 cells; BAIAP2 not associated with BAI1 was diffused in the cytoplasm . Predominant localization of BAI1 protein in a sub-cellular fraction enriched in growth cones indicated a possible role of BAI1 as a cell adhesion molecule inducing growth cone guidance . As a protein partner of BAI1, BAIAP2 may represent an important link between membrane and cytoskeleton in the process of neuronal growth.

Cytogenet Cell Genet, 1999, 84(1-2), 39 - 42
Physical mapping of the CXC chemokine locus on human chromosome 4; O'Donovan N et al.; A physical map of the CXC chemokine locus on chromosome 4 has been constructed by PCR analysis and PFGE mapping of YAC clones . The genes for IL8, GRO1, PPBP, PF4, SCYB5 (ENA-78) and SCYB6 (GCP-2) have been co-localized on a 335-kb genomic fragment . The GRO2 and GRO3 genes did not map within this region and based on analysis of a YAC contig overlapping IL8 we speculate that GRO2 and GRO3 map downstream of this region . We have also assigned the novel CXC chemokine gene, SCYB9B (alias H174/betaR1) to chromosome 4q21, upstream and within 12 kb of INP10 . Like INP10 and MIG, INP10 and SCYB9B are arranged in a head to tail manner . The chromosomal arrangement of these genes appears to reflect the evolution of this multigene family and supports the theory that it arose by gene duplication.

Cytogenet Cell Genet, 1999, 84(1-2), 22 - 7
Integrated genetic and physical map of the 1q31-->q32.1 region, encompassing the RP12 locus, the F13B and HF1 genes, and the EEF1AL11 and RPL30 pseudogenes; van Soest S et al.; The gene for autosomal recessive retinitis pigmentosa (RP12) with preserved para-arteriolar retinal pigment epithelium was previously mapped close to the F13B gene in region 1q31-->q32.1 . A 4-Mb yeast artificial chromosome contig spanning this interval was constructed to facilitate cloning of the RP12 gene . The contig comprises 25 sequence-tagged sites, polymorphic markers, and single-copy probes, including five newly obtained probes . The contig orders the F13B and HF1 genes, as well as five expressed sequence tags, with respect to the integrated genetic map of this region . Homozygosity mapping resulted in refinement of the candidate gene locus for RP12 to a 1 . 3-cM region . Currently, approximately 1 Mb of the contig is represented in P1-derived artificial chromosome (PAC) clones . Direct screening of a cDNA library derived from neural retina with PACs resulted in identification of the human elongation factor 1alpha pseudogene (EEF1AL11) and a human ribosomal protein L30 pseudogene (RPL30) . A physical and genetic map covering the entire RP12 candidate gene region was constructed.

J Chromatogr A, 1999 Apr 30, 840(2), 205 - 13
Boronate affinity adsorption of RNA: possible role of conformational changes; Singh N et al.; Batch equilibrium adsorption isotherm determination is used to characterize the adsorption of mixed yeast RNA on agarose-immobilized m-aminophenylboronic acid . It is shown that the affinity-enhancing influence of divalent cations depends strongly on the precise nature of the cation used, with barium being far more effective than the conventionally-used magnesium . This adsorption-promoting influence of barium is suggested to arise primarily from ionic influences on the structure and rigidity of the RNA molecule, as the adsorption of ribose-based small molecules is not similarly affected . The substitution of barium for the standard magnesium counterion does not greatly promote the adsorption of DNA, implying that the effect is specific to RNA and may be useful in boronate-based RNA separations . RNA adsorption isotherms exhibit sharp transitions as functions of temperature, and these transitions occur at different temperatures with Mg2+ and with Ba2+ . Adsorption affinity and capacity were found to increase markedly at lower temperatures, suggestive of an enthalpically favored interaction process . The stoichiometric displacement parameter, Z, in Ba2+ buffer is three times the value in Mg2+ buffer, and is close to unity.

Cell Motil Cytoskeleton, 1999, 43(1), 52 - 62
Characterization of the mammalian septin H5: distinct patterns of cytoskeletal and membrane association from other septin proteins; Xie H et al.; The mechanisms controlling cytokinesis during yeast budding and animal cell fission appear quite different, yet both require members of the septin protein family . Mammalian homologs of this novel family of GTPases have been identified but little is known about their properties or functions . Using an antibody specific for the mammalian septin H5, we show that this protein is expressed at distinct levels in a variety of tissues . Tissue expression levels in different tissues did not coincide with those of the only previously characterized mammalian septin Nedd5 . H5, like Nedd5, localizes to the cleavage furrow in mitotic fibroblast cells but in non-mitotic cells these proteins associate with actin filaments in different ways . Nedd5 predominantly localizes with stress fibers, but only associates with central portions of the microfilament bundles . In contrast, H5 associates with the entire length of the stress fibers and the cortical actin network . Conditions that disrupt the actin cytoskeleton also disrupt the filamentous patterns of both Nedd5 and H5, resulting in a punctate cytoplasmic pattern . Cell fractionation revealed that H5 co-fractionated with actin, while Nedd5 was predominantly restricted to the membrane fraction . Co-immunoprecipitation experiments revealed that although H5 will co-precipitate with Nedd5, the precipitation is not quantitative . Taken together, these results not only show that H5 behaves like a septin, but also demonstrate that individual septin proteins have distinct properties, suggesting that they may play different roles in cytokinesis and in other stages of the cell cycle.

Gene Ther, 1999 Jan, 6(1), 22 - 33
Failure of wild-type p53 gene therapy in human cancer cells expressing a mutant p53 protein; Vinyals A et al.; The introduction of exogenous wild-type p53 into human cancer cells bearing p53 mutation does not necessarily result in inhibition of tumor growth . We have demonstrated this in MDA-MB468 breast cancer cells which are hemizygous for p53 mutation and also in KM12SM colorectal carcinoma cells which are heterozygous for p53 mutation . The wtp53 transfectants decreased three- to four-fold the number of colonies compared with controls . Most wtp53-expressing cells died by apoptosis at early passages, but some cells were able to form colonies and their proliferation rate was similar to control transfectants . This reversion was observed in three of the six MDA-MB-468 clones selected . When MDA-wtp53 transfectants were implanted orthotopically in nude mice only one clone showed prolonged tumor latency . No differences were found in either tumor proliferation or apoptosis in tumors . Integration and expression of exogenous wtp53 was assessed in early and late passages in vitro, and in tumors growing in vivo . Consistently, we found mutations in the exogenous wtp53 gene of MDA-MB468 transfectants . Excision of the exogenous gene was an alternative to abrogate the wtp53 function that was extremely efficient in KM12 cells, although they maintained resistance to geneticin . These results were corroborated by the functional assay in yeast . In conclusion, wtp53 is inactivated in these cancer cells by different mechanisms . The presence of mutated p53 may confer genome instability and mutator ability, which allows cells to escape the effects of the exogenous wtp53 and contributes to the failure of wtp53 gene therapy.

Heredity, 1999 Mar, 82 ( Pt 3), 267 - 75
Cannibalism facilitates the use of a novel environment in the flour beetle, Tribolium castaneum; Via S; Cannibalism is well known to affect both the population dynamics and the competitive relationships of organisms . Cannibalistic behaviour commonly increases in stressful conditions, such as when density is high or food is scarce, and cannibals often obtain a nutritional benefit . Might cannibalism also increase in a novel environment to which a population is poorly adapted physiologically? Moreover, might cannibalistic behaviour provide enough of a nutritional advantage in a nutritionally stressful environment to rescue individuals from its adverse effects and thus permit colonization and range expansion? Previous work has shown that oat flour is a particularly stressful environment for Tribolium castaneum . In the study reported here, egg cannibalism by two strains of T . castaneum was significantly enhanced in oat flour, and egg eating rescued larvae from the adverse demographic effects of this poor environment . Development time of the cannibals was accelerated almost to the level seen for individuals reared in the nutritionally superior environment (wheat plus brewer's yeast) . Their survival and fecundity also increased relative to individuals reared in oat flour without the opportunity to cannibalize . A sib analysis revealed that for larvae reared in the presence of victim eggs, the extent of cannibalism was genetically variable, so that this trait could evolve, given a selective benefit exceeding its cost . These results suggest that colonization of a marginal new environment could be facilitated by enhanced rates of cannibalism . The possible interplay between cannibalism and physiological adaptation to a new environment is discussed.

J Cell Sci, 1999 Jun, 112 ( Pt 12), 1901 - 13
Transcription, biochemistry and localization of nematode annexins; Daigle SN et al.; The transcription of three annexin genes in the nematode, Caenorhabditis elegans, was detected by reverse transcriptase/polymerase chain reaction amplification of messenger RNAs . The highest level of expression was from the nex-1 gene, with lower levels detected for the nex-2 and nex-3 genes . The expression of nex-1 was reduced in the Dauer larval stage relative to the other annexins, correlating with the absence of the spermathecal valves, a major site of nex-1 protein localization . Recombinant nex-1 protein was expressed in yeast, isolated by calcium-dependent binding to acidic phospholipids, and its membrane binding and aggregating activities characterized using bovine chromaffin granules as a representative intracellular substrate . Binding to granule membranes was promoted by calcium with half-maximal binding seen at 630 microM calcium . Chromaffin granule aggregation was similarly promoted by the nex-1 protein at 630 microM calcium . This low sensitivity to calcium suggests the annexin can only be activated in vivo near the plasma membrane or other sources of calcium . Sequences including the nex-1 promoter were fused to the gene for green fluorescent protein and this construct was introduced into nematodes by microinjection . Examination of transgenic offspring revealed specific nex-1 promoter activity in the pharynx, the hypodermal cells, the vulva, and the spermathecal valve, locations in which the annexin may function in collagen secretion/deposition and membrane-membrane interactions . A sensitive anti-nex-1 antibody labelled with rhodamine was injected into body cavities of the nematode but did not detect extracellular nex-1 protein . Therefore, this annexin is apparently cytosolic and may function on the cytoplasmic side of the plasma membrane of the spermathecal valve to chaperon the folding of this membrane during the opening and closing of the valve.

Neuropharmacology, 1999 May, 38(5), 635 - 44
The protein kinase C alpha binding protein PICK1 interacts with short but not long form alternative splice variants of AMPA receptor subunits; Dev KK et al.; Here we report an interaction between AMPA receptor subunits and a single PDZ domain-containing protein called PICK1 which is known to bind protein kinase C alpha (PKC alpha) . The interaction occurs within the last ten amino acid residues containing a novel PDZ binding motif (E S V/I K I) of the short C-terminal alternative splice variants of AMPA receptor subunits . No interaction occurs with the corresponding long splice variants which do not contain the E S V/I K I motif . The PDZ domain of PICK1 is required for the interaction and the mutation of a single amino acid in this region (Lys-27 to Glu) prevents interaction between PICK1 and GluR2 in the yeast two-hybrid assay . A similar mutation has been reported to prevent the binding of PICK1 to PKC alpha indicating that the same domain of PICK1 binds both PKC alpha and GluRs . Flag-tagged PICK1 is retained by a glutathione S-transferase (GST) fusion of the C-terminal of GluR2 (GST-ct-GluR2; short splice variant) but not by GST-ct-GluR1 (long splice variant) . Recombinant full length GluR2 is coimmunoprecipitated with flag-PICK1 using an anti-flag antibody and flag-PICK1 is coimmunoprecipitated with an N-terminal directed anti-GluR2 antibody . Transient expression of both proteins in COS cells reveals colocalization and an altered pattern of distribution for each protein from when they are expressed individually . This novel interaction provides a possible regulatory mechanism to specifically modulate distinct splice variants and may be involved in targeting the phosphorylation of short form GluRs by PKC alpha.

Curr Biol, 1999 May 20, 9(10), 539 - 42
Identification of RIP3, a RIP-like kinase that activates apoptosis and NFkappaB; Yu PW et al.; The tumor necrosis factor receptor 1 (TNFR1) and the Fas receptor recruit complexes formed by the interactions between RIP kinase, TRADD, FADD and RAIDD - adaptor proteins that contain death domains - which in turn recruit other proteins to initiate signaling {1}{2}{3}{4}{5} . To identify proteins associated with the TNF signaling pathway, we performed a yeast two-hybrid interaction screen using RIP as bait . We isolated a kinase, RIP3, which shares homology with the kinase domain of RIP and RIP2 (also known as Rick or CARDIAK) . RIP3 could be co-immunoprecipitated with RIP, TRAF2 and TNFR1 in mammalian cells . The carboxy-terminal domain of RIP3, like that of RIP, could activate the transcription factor NFkappaB and induce apoptosis when expressed in mammalian cells . Interestingly, this region shares no significant sequence homology to the death domain of RIP, the caspase-recruiting domain (CARD) of RIP2 {6}{7}{8} or any other apoptosis-inducing domain . As with RIP and RIP2, the kinase domain of RIP3 was not required for either NFkappaB activation or apoptosis induction . Overexpression of a dominant-negative mutant of RIP3 strongly inhibited the caspase activation but not the NFkappaB activation induced by TNFalpha . Therefore, RIP3 appears to function as an intermediary in TNFalpha-induced apoptosis.

FEBS Lett, 1999 Apr 23, 449(2-3), 215 - 20
Mu1B, a novel adaptor medium chain expressed in polarized epithelial cells; Ohno H et al.; The apical and basolateral plasma membrane domains of polarized epithelial cells contain distinct sets of integral membrane proteins . Biosynthetic targeting of proteins to the basolateral plasma membrane is mediated by cytosolic tail determinants, many of which resemble signals involved in the rapid endocytosis or lysosomal targeting . Since these signals are recognized by adaptor proteins, we hypothesized that there could be epithelial-specific adaptors involved in polarized sorting . Here, we report the identification of a novel member of the adaptor medium chain family, named mu1B, which is closely related to the previously described mu1A (79% amino acid sequence identity) . Northern blotting and in situ hybridization analyses reveal the specific expression of mu1B mRNA in a subset of polarized epithelial and exocrine cells . Yeast two-hybrid analyses show that mu1B is capable of interacting with generic tyrosine-based sorting signals . These observations suggest that mu1B may be involved in protein sorting events specific to polarized cells.

Mamm Genome, 1999 May, 10(5), 482 - 7
A comparative linkage and physical map of bovine chromosome 24 with human chromosome 18; Larsen NJ et al.; Polymorphic microsatellites have been developed in the vicinity of nine genes on bovine chromosome (BTA) 24, all orthologous to genes on human chromosome (HSA) 18 . The microsatellites have been isolated from bacterial and yeast artificial chromosome clones containing the genes . A linkage map was developed including these polymorphic markers and four anonymous, published microsatellites . Yeast artificial chromosomes containing six of these genes have also been mapped using fluorescent in situ hybridization (FISH), thereby tying the linkage map together with the physical map of BTA24 . Comparing gene location on HSA18 and BTA24 identifies four regions of conserved gene order, each containing at least two genes . These genes identify six regions of conserved order between human and mouse, two more than in the human-bovine comparison . The breakpoints between regions of conserved order for human-bovine are also breakpoints in the human-mouse comparison . The centromere identifies a fifth conserved region if the BTA24 centromere is orthologous with the HSA18 centromere.

Mamm Genome, 1999 May, 10(5), 438 - 43
Sequence-ready physical map of the mouse chromosome 16 region with conserved synteny to the human velocardiofacial syndrome region on 22q11.2; Lund J et al.; Proximal mouse Chromosome (Chr) 16 shows conserved synteny with human Chrs 16, 8, 22, and 3 . The mouse Chr 16/human Chr 22 conserved synteny region includes the DiGeorge/Velocardiofacial syndrome region of human Chr 22q11.2 . A physical map of the entire mouse Chr 16/human Chr 22 region of conserved synteny has been constructed to provide a substrate for gene discovery, genomic sequencing, and animal model development . A YAC contig was constructed that extends ca . 5.4 Mb from a region of conserved synteny with human Chr 8 at Prkdc through the region conserved with human Chr 3 at DVL3 . Sixty-one markers including 37 genes are mapped with average marker spacing of 90 kb . Physical distance was determined across the 2.6-Mb region from D16Mit74 to Hira with YAC fragmentation . The central region from D16Jhu28 to Igl-C1 was converted into BAC and PAC clones, further refining the physical map and providing sequence-ready template . The gene content and borders of three blocks of conserved linkage between human Chr 22q11.2 mouse Chr 16 are refined.

J Med Entomol, 1999 May, 36(3), 219 - 21
Spray-dried bovine blood: an effective laboratory diet for Ctenocephalides felis felis (Siphonaptera: Pulicidae); Richman DL et al.; Commercially available spray-dried protein sources were evaluated as replacement laboratory cat flea, Ctenocephalides felis felis (Bouche), larval diets for slaughterhouse-collected heat-dried blood . Percentage of adult emergence of fleas reared on adult flea feces (87.7%) and spray-dried bovine blood (79%) did not significantly differ, and yeast supplementation did not significantly increase adult emergence for spray-dried diets . However, yeast supplementation of heat-dried blood increased percentage adult emergence from 0 to 41.7% . Spray-dried bovine blood was found to be a satisfactory laboratory diet for cat flea larvae.

Eur J Neurosci, 1999 Jun, 11(6), 2031 - 43
Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97; Bassand P et al.; The NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor are encoded by distinct genes . In the rat brain, four C-terminal variants of the NR1 subunit (NR1-1 to NR1-4) are encoded by a single gene, and are generated by alternative splicing of the C1 and C2 exon cassettes, while four different genes encode the NR2 subunits (NR2 A-D) . Functional NMDA receptors result from the heteromultimeric assembly of NR1 variants with distinct NR2 subunits . The NR2B subunit interacts with post-synaptic density protein 95 (PSD-95), SAP97 and members of the membrane-associated guanylate-like kinase (MAGUK) family of proteins . This interaction occurs through the binding of the C-terminal tSXV intracellular motif of the NR2B subunit to the N-terminal PDZ (PSD-95, discs-large, ZO-1) domains of the PSD-95 and SAP97 proteins . Both NR1-3 and NR1-4 also display a consensus C-terminal tSXV motif . Using the two-hybrid genetic system in yeast and site-directed mutagenesis, we compared the binding of the NR2A, NR1-3 and NR1-4 tSXV motifs with the PDZ domains of PSD-95 and SAP97 . The main conclusions of the present report are that: (i) while NR2A displays a strong interaction with PSD-95 and SAP97, the NR1-3 and NR1-4 NMDA receptor subunits do not display any interaction despite the presence of tSXV motifs; (ii) the C-terminal tSXV motif of the NR2A subunit is mandatory but not sufficient for efficient interaction with the PSD-95 and SAP97 proteins; (iii) as yet unidentified upstream sequences of the receptor subunits determine whether the tSXV motifs will bind to the PSD-95 and SAP97 PDZ domains; (iv) different tSXV motifs elicit interactions of variable strengths; and (v) residues in positions -3 and -4 modulate the binding affinity of the C-terminal tSXV motifs . Using immunohistochemistry, we also compared the distribution of the PSD-95, NR2A and SAP97 proteins in adult rat brain, and we show that in the cortex, hippocampus and cerebellum, there is evidence for colocalization of these proteins.

Eur J Neurosci, 1999 Jun, 11(6), 1907 - 13
Amino-terminal region of secreted form of amyloid precursor protein stimulates proliferation of neural stem cells; Ohsawa I et al.; Beta-amyloid precursor protein (APP) has been reported to be expressed in the CNS from the early stages of development . However, the functional role of APP during early development remains unclear . In the present study, we found that the secreted form of APP (sAPP) significantly enhanced proliferation of neural stem cells . Cells were prepared from 13-day embryonic rat neocortex, which was dissected with a Pasteur pipette to make cell clusters . After 12 h of cultivation in the medium without serum, cells around the centre of the cluster were still nestin-positive proliferative cells, i.e . neural stem cells . To determine whether the proliferation of cells was regulated by sAPP, cultures were treated with recombinant sAPP695, the secreted form of human APP695 produced by yeast . Both DNA synthesis and expression of proliferating cell nuclear antigen markedly increased after 5 h of sAPP695 addition . The enhancement of DNA synthesis by sAPP695 stimulation was blocked by the 22C11 monoclonal antibody specific for the amino-terminal region of sAPP . Then, we examined the effect of the amino-terminal fragment of sAPP and the epitope peptide of 22C11 antibody, and found that both of them also promoted DNA synthesis, suggesting that the amino-terminal region of sAPP is responsible for the biological activity . Our findings indicate the possibility that sAPP enhances proliferation of neural stem cells in vivo and plays an important role during the early CNS development.

Eur J Biochem, 1999 Jun, 262(2), 426 - 34
Characterization of a chromatin remodelling activity in Xenopus oocytes; Gelius B et al.; The yeast SWI2/SNF2 protein is a component of a large protein complex which is involved in the remodelling of chromatin during transcriptional activation . Several homologous complexes have been found in Drosophila and mammals . We have examined the expression of the SWI2/SNF2 homologue BRG1 in Xenopus laevis using two antisera originally raised against the C-terminus of the rat and the human BRG1 protein . These two antisera cross-reacted with a protein found in both Xenopus liver and Xenopus oocytes . The Xenopus BRG1-like protein is expressed throughout oogenesis (stages I-VI) and embryogenesis . By injecting an expression vector containing the full-length human BRG1 cDNA into Xenopus oocytes, the relative molecular weight (Mr) of the Xenopus BRG1-like protein was shown to be slightly lower than that of the human BRG1, 190 000 and 200 000, respectively . The Xenopus BRG1-like protein elutes at a Mr of approximately 2 000 000 on Superose HR6trade mark size-exclusion chromatography, indicating that it is part of a larger complex, as are all other known SWI/SNF proteins . Nucleosome remodelling activity was co-eluted with the BRG1 immunogenic activity in both ion-exchange and size-exclusion chromatography.

J Biol Chem, 1999 May 28, 274(22), 15820 - 7
Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import . Differential role of NTF2; Hu W et al.; Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin alpha/beta . This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2 . m- and y-IMPalpha recognized both NLSs, with y-IMPalpha exhibiting higher affinity . m-IMPbeta greatly enhanced the binding of m-IMPalpha to the T-ag and N1N2 NLSs, but y-IMPbeta did not significantly affect the affinity of y-IMPalpha for the T-ag NLS . In contrast, y-IMPbeta enhanced y-IMPalpha binding to the NLS of N1N2, but to a lesser extent than the enhancement of m-IMPalpha binding by m-IMPbeta . NLS-dependent nuclear import was reconstituted in vitro using the different importin subunits together with the transport factors Ran and NTF2 . Whereas T-ag NLS-mediated nuclear import did not exhibit an absolute requirement for NTF2, N1N2 NLS-mediated transport strictly required NTF2 . High levels of NTF2 inhibited nuclear accumulation conferred by both NLSs . We conclude that different NLSs possess distinct nuclear import properties due to differences in recognition by importin and requirements for NTF2.

J Biol Chem, 1999 May 28, 274(22), 15757 - 65
C5a receptor activation . Genetic identification of critical residues in four transmembrane helices; Baranski TJ et al.; Hormones and sensory stimuli activate serpentine receptors, transmembrane switches that relay signals to heterotrimeric guanine nucleotide-binding proteins (G proteins) . To understand the switch mechanism, we subjected 93 amino acids in transmembrane helices III, V, VI, and VII of the human chemoattractant C5a receptor to random saturation mutagenesis . A yeast selection identified 121 functioning mutant receptors, containing a total of 523 amino acid substitutions . Conserved hydrophobic residues are located on helix surfaces that face other helices in a modeled seven-helix bundle (Baldwin, J . M., Schertler, G . F., and Unger, V . M . (1997) J . Mol . Biol . 272, 144-164), whereas surfaces predicted to contact the surrounding lipid tolerate many substitutions . Our analysis identified 25 amino acid positions resistant to nonconservative substitutions . These appear to comprise two distinct components of the receptor switch, a surface at or near the extracellular membrane interface and a core cluster in the cytoplasmic half of the bundle . Twenty-one of the 121 mutant receptors exhibit constitutive activity . Amino acids substitutions in these activated receptors predominate in helices III and VI; other activating mutations truncate the receptor near the extracellular end of helix VI . These results identify key elements of a general mechanism for the serpentine receptor switch.

J Biol Chem, 1999 May 28, 274(22), 15686 - 93
Cooperative transcriptional activation by serum response factor and the high mobility group protein SSRP1; Spencer JA et al.; Serum response factor (SRF) is a MADS box transcription factor that controls a wide range of genes involved in cell proliferation and differentiation . The MADS box mediates homodimerization and binding of SRF to the consensus sequence CC(A/T)6GG, known as a CArG box, which is found in the control regions of numerous serum-inducible and muscle-specific genes . Using a modified yeast one-hybrid screen to identify potential SRF cofactors, we found that SRF interacts with the high mobility group factor SSRP1 (structure-specific recognition protein) . This interaction, which occurs in yeast and mammalian cells, is mediated through the MADS box of SRF and a basic region of SSRP1 encompassing amino acids 489-542, immediately adjacent to the high mobility group domain . SSRP1 does not bind the CArG box, but interaction of SSRP1 with SRF dramatically increases the DNA binding activity of SRF, resulting in synergistic transcriptional activation of native and artificial SRF-dependent promoters . These results reveal an important role for SSRP1 as a coregulator of SRF-dependent transcription in mammalian cells.

J Biol Chem, 1999 May 28, 274(22), 15662 - 70
Identification of a novel inhibitor of nuclear factor-kappaB, RelA-associated inhibitor; Yang JP et al.; Here we report the identification and characterization of a novel protein, RelA-associated inhibitor (RAI), that binds to the NF-kappaB subunit p65 (RelA) and inhibits its transcriptional activity . RAI gene was isolated in a yeast two-hybrid screen using the central region of p65 as bait . We confirmed the physical interaction in vitro using recombinant proteins as well as in vivo by immunoprecipitation/Western blot assay . RAI gene encodes a protein with homology to the C-terminal region of 53BP2 containing four consecutive ankyrin repeats and an Src homology 3 domain . RAI mRNA was preferentially expressed in human heart, placenta, and prostate . Despite its similarity to 53BP2, RAI did not interact with p53 in a yeast two-hybrid assay . RAI inhibited the action of NF-kappaB p65 but not that of p53 in transient luciferase gene expression assays . Similarly, RAI inhibited the endogenous NF-kappaB activity induced by tumor necrosis factor-alpha . RAI specifically inhibited the DNA binding activity of p65 when co-transfected in 293 cells . RAI protein appeared to be located in the nucleus and colocalized with NF-kappaB p65 that was activated by TNF-alpha . These observations indicate that RAI is another inhibitor of NF-kappaB in addition to IkappaB proteins and may confer an alternative mechanism of regulation.

Adv Exp Med Biol, 1999, 464, 49 - 62
Monoterpenes in essential oils . Biosynthesis and properties; Loza-Tavera H; Monoterpenes are compounds found in the essential oils extracted from many plants, including fruits, vegetables, spices and herbs . These compounds contribute to the flavor and aroma of plant from which they are extracted . Monoterpenes are acyclic, monocyclic, or bicyclic C30 compounds synthesized by monoterpene synthases using geranyl pyrophosphate (GPP) as substrate . GPP is also the precursor in the synthesis of farnesyl pyrophosphate (FPP) and geranyl-geranyl pyrophosphate (GGPP), two important compounds in cell metabolism of animals, plants and yeast . Monoterpene cyclases produce cyclic monoterpenes through a multistep mechanism involving a universal intermediate, a terpinyl cation which can be transformed to several compounds . Experimental studies, using animal cancer models, have demonstrated that some monoterpenes possess anticarcinogenic properties, acting at different cellular and molecular levels . From these discoveries it seems clear that monoterpenes could be considered as effective, nontoxic dietary antitumorigenic agents that hold promise as a novel class of anticancer drugs.

Biotechnol Appl Biochem, 1999 Jun, 29 ( Pt 3), 191 - 205
Multiprobe fluorescence imaging and microspectrofluorimetry of cell transformation and differentiation: implications in terms of applied biochemistry and biotechnology; Kohen E et al.; The dichotomy of cellular transformation versus differentiation does not preclude the hypothesis of a unified underlying mechanism that can switch either way as a result of growth factors, cell-membrane receptors, secondary messengers, integrating switch kinases and/or nuclear receptors . Its study for biopharmaceutical and biotechnological applications requires a methodology capable of dealing with such pleiotropy . In the multiprobe-multiparameter approach, one must remain wary of cumulative toxic effects and misinterpretations . 'Smart' instrumentation does not mean 'smart' probes . It turns out that the cell's own endogenous probes, the fluorescent coenzymes, may be akin to 'smart' probes, open to study in situ of many-fold interrelated pathways in cell energetics and dynamics . Resolution at the micro- and even nano-compartment levels is not altogether impossible . Thus an innovative search in terms of what may be called 'intracellular reconnaissance with fluorescent probes and biopharmaceuticals' necessitates recourse to multiple tentative probings along the pleiotropic mechanisms as far in resolution as one can go . Among the characteristic findings using this approach are: (i) morphometric alterations in the mitochondria and melanosomes of melanoma cells treated with azelaic acid; (ii) deregulation of mitochondrial control and extramitochondrial metabolism in similarly treated cells; (iii) considerable acceleration of NAD(P) transient kinetics in atractylate-treated L sarcoma cells; (iv) alterations of mitochondria and Golgi in fusion-deficient myoblasts; (v) tentative recognition of beta-glucosidase deficiency in Gaucher disease cells by the use of fluorescent and fluorogenic lysosomal probes; and (vi) UVA-induced accumulation of Schiff bases (a kind of accelerated photo-aging) in yeast and kidney epithelial cells . Because these studies utilize probing at whatever points along the concerned pathways become accessible, at first glance they may look disconnected . What and where is the connecting thread, for instance, between studying melanoma metabolism, melanosome morphometry, hepatocyte organelle morphogenesis and transformation, myotube organelle morphogenesis and fusion-non-fusion, and lysosomal activity in gene-deficient cells? In the mapping of the regulatory and deregulatory mechanisms involved in the switching of differentiation or transformation, each of the above topics carries an information content towards resolution of the pleiotropic puzzle . The integration of such information with increasing resolution and access to intracellular microdomains may ultimately allow focus on the precise target, the switch from differentiation to transformation or vice versa.

Arch Biochem Biophys, 1999 Jun 1, 366(1), 107 - 15
Signal presequences increase mitochondrial permeability and open the multiple conductance channel; Kushnareva YE et al.; We have reported that the signal presequence of cytochrome oxidase subunit IV from Neurospora crassa increases the permeability of isolated rat liver mitochondria {P . M . Sokolove and K . W . Kinnally (1996) Arch . Biochem . Biophys . 336, 69} and regulates the behavior of the mutiple conductance channel (MCC) of yeast inner mitochondrial membrane {T . A . Lohret and K . W . Kinnally (1995) J . Biol . Chem . 270, 15950} . Here we examine in greater detail the action of a number of mitochondrial presequences from various sources and of several control peptides on the permeability of isolated rat liver mitochondria and on MCC activity monitored via patch-clamp techniques in both mammalian mitoplasts and a reconstituted yeast system . The data indicate that the ability to alter mitochondrial permeability is a property of most, but not all, signal peptides . Furthermore, it is clear that, although signal peptides are characterized by positive charge and the ability to form amphiphilic alpha helices, these two characteristics are not sufficient to guarantee mitochondrial effects . Finally, the results reveal a strong correlation between peptide effects on the permeability of isolated mitochondria and on MCC activity: peptides that induced swelling of mouse and rat mitochondria also activated the quiescent MCC of mouse mitoplasts and induced flickering of active MCC reconstituted from yeast mitochondrial membranes . Moreover, relative peptide efficacies were very similar for mitochondrial swelling and both types of patch-clamp experiments . We propose that patch-clamp recordings of MCC activity and the high-amplitude swelling induced by signal peptides reflect the opening of a single channel . Based on the selective responsiveness of that channel to signal peptides and the dependence of its opening in isolated mitochondria on membrane potential, we further suggest that the channel is involved in the mitochondrial protein import process .

Biochem J, 1999 Jun 1, 340 ( Pt 2), 561 - 8
Identification of peroxisomal proteins by using M13 phage protein VI phage display: molecular evidence that mammalian peroxisomes contain a 2,4-dienoyl-CoA reductase; Fransen M et al.; To elucidate unknown mammalian peroxisomal enzymes and functions, we subjected M13 phage expressing fusions between the gene encoding protein VI and a rat liver cDNA library to an immunoaffinity selection process in vitro (biopanning) with the use of antibodies raised against peroxisomal subfractions . In an initial series of biopanning experiments, four different cDNA clones were obtained . These cDNA species encoded two previously identified peroxisomal enzymes, catalase and urate oxidase, and two novel proteins that contained a C-terminal peroxisomal targeting signal (PTS1) . A primary structure analysis of these novel proteins revealed that one, ending in the tripeptide AKL, is homologous to the yeast peroxisomal 2,4-dienoyl-CoA reductase (EC 1.3.1.34; DCR), an enzyme required for the degradation of unsaturated fatty acids, and that the other, ending in the tripeptide SRL, is a putative member of the short-chain dehydrogenase/reductase (SDR) family, with three isoforms . Green fluorescent protein (GFP) fusions encoding GFP-DCR-AKL, GFP-DCR, GFP-SDR-SRL and GFP-SDR were expressed in mammalian cells . The analysis of the subcellular location of the recombinant fusion proteins confirmed the peroxisomal localization of GFP-DCR-AKL and GFP-SDR-SRL, as well as the functionality of the PTS1 . That the AKL protein is indeed an NADPH-dependent DCR was demonstrated by showing DCR activity of the bacterially expressed protein . These results demonstrate at the molecular level that mammalian peroxisomes do indeed contain a DCR . In addition, the results presented here indicate that the protein VI display system is suitable for the isolation of rare cDNA clones from cDNA libraries and that this technology facilitates the identification of novel peroxisomal proteins.

Biochem J, 1999 Jun 1, 340 ( Pt 2), 405 - 9
Glucuronidation of the environmental oestrogen bisphenol A by an isoform of UDP-glucuronosyltransferase, UGT2B1, in the rat liver; Yokota H et al.; Bisphenol A, an environmental oestrogenic chemical, was found to conjugate highly with glucuronic acid in male rat liver microsomes studied in vitro . In the various isoforms tested (1A1, 1A3, 1A5, 1A6, 1A7 and 2B1), glucuronidation of bisphenol A and of diethylstilboestrol, a synthetic crystalline compound possessing oestrogenic activity and known to be glucuronidated by liver microsomes, was catalysed by an isoform of UDP-glucuronosyltransferase (UGT), namely UGT2B1, which glucuronidates some endogenous androgens . UGT activity towards bisphenol A in liver microsomes and in UGT2B1 expressed in yeast AH22 cells (22.9 and 0.58 nmol/min per mg of microsomal proteins respectively) was higher than that towards diethylstilboestrol (75.0 and 4.66 pmol/min per mg of microsomal proteins respectively) . UGT activities towards both bisphenol A and diethylstilboestrol were distributed mainly in the liver but were also observed at substantial levels in the kidney and testis . Northern blot analysis disclosed the presence of UGT2B1 solely in the liver, and about 65% of the male rat liver microsomal UGT activities towards bisphenol A were absorbed by the anti-UGT2B1 antibody . These results indicate that bisphenol A, in male rat liver, is glucuronidated by UGT2B1, an isoform of UGT.

Genomics, 1999 May 15, 58(1), 41 - 9
Tissue-specific alternative splicing of the CSE1L/CAS (cellular apoptosis susceptibility) gene; Brinkmann U et al.; CSE1L/CAS (CAS) is a nuclear transport factor that plays a role in proliferation and apoptosis . The CAS gene consists of 25 exons . mRNA homologous over its entire length to the yeast homologue CSE1 is the predominant transcript in proliferating tissues . Additional mRNAs are generated by alternative splicing in a tissue-specific manner . An extended 3'-end is found in fetal and adult brain . A mRNA containing the 5'-end of CAS up to position 690 and an alternative 3'-end is expressed in trachea and encodes a truncated Ran-binding domain . Fetal liver expresses a mRNA with deletions of a central portion of CAS and additional sequences encoded by the last intron . SW480 colon cancer cells express another approximately 1500-base mRNA . Western blot analyses of various human tissues and immunohistology of mouse embryos show a correlation of CAS transcripts and CAS protein in different tissues . CAS isoforms may control nuclear transport of tissue-specific proteins.

J Exp Med, 1999 May 17, 189(10), 1611 - 20
A human immunoglobulin lambda locus is similarly well expressed in mice and humans; Popov AV et al.; Transgenic mice carrying a 380-kb region of the human immunoglobulin (Ig) lambda light (L) chain locus in germline configuration were created . The introduced translocus on a yeast artificial chromosome (YAC) accommodates the most proximal Iglambda variable region (V) gene cluster, including 15 Vlambda genes that contribute to >60% of lambda L chains in humans, all Jlambda-Clambda segments, and the 3' enhancer . HuIglambdaYAC mice were bred with animals in which mouse Igkappa production was silenced by gene targeting . In the kappa-/- background, human Iglambda was expressed by approximately 84% of splenic B cells . A striking result was that human Iglambda was also produced at high levels in mice with normal kappa locus . Analysis of bone marrow cells showed that human Iglambda and mouse Igkappa were expressed at similar levels throughout B cell development, suggesting that the Iglambda translocus and the endogenous kappa locus rearrange independently and with equal efficiency at the same developmental stage . This is further supported by the finding that in hybridomas expressing human Iglambda the endogenous L chain loci were in germline configuration . The presence of somatic hypermutation in the human Vlambda genes indicated that the Iglambda-expressing cells function normally . The finding that human lambda genes can be utilized with similar efficiency in mice and humans implies that L chain expression is critically dependent on the configuration of the locus.

Neurogenetics, 1998 Dec, 2(1), 34 - 42
Refined mapping and characterization of the recessive familial amyotrophic lateral sclerosis locus (ALS2) on chromosome 2q33; Hosler BA et al.; Amyotrophic lateral sclerosis (ALS) is a progressive degenerative neuromuscular disease that shows familial, autosomal dominant inheritance in 10%-15% of cases . Previous genetic analysis of one large family linked a recessive form of familial ALS (FALS-AR type 3) to the chromosome 2q33-35 region . Using additional polymorphic markers, we have narrowed the size of the linked region to approximately 1.7 cM by linkage and haplotype analysis . We have also established a yeast artificial chromosome contig across the locus that covers an approximate physical distance of 3 million bases . Based on this contig, genes and expressed sequences that map near the 2q33 region have been examined to determine whether they are located within this ALS2 candidate locus . Five identified genes and 34 expressed sequence tags map within the region defined by crossover analysis and merit further consideration as candidate genes for this disease.

Mol Cell Biol, 1999 Jun, 19(6), 4535 - 45
Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions; Ballinger CA et al.; The chaperone function of the mammalian 70-kDa heat shock proteins Hsc70 and Hsp70 is modulated by physical interactions with four previously identified chaperone cofactors: Hsp40, BAG-1, the Hsc70-interacting protein Hip, and the Hsc70-Hsp90-organizing protein Hop . Hip and Hop interact with Hsc70 via a tetratricopeptide repeat domain . In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, carboxyl terminus of Hsc70-interacting protein (CHIP) . CHIP is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture . Hsc70 and Hsp70 were identified as potential interaction partners for this protein in a yeast two-hybrid screen . In vitro binding assays demonstrated direct interactions between CHIP and both Hsc70 and Hsp70, and complexes containing CHIP and Hsc70 were identified in immunoprecipitates of human skeletal muscle cells in vivo . Using glutathione S-transferase fusions, we found that CHIP interacted with the carboxy-terminal residues 540 to 650 of Hsc70, whereas Hsc70 interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of CHIP . Recombinant CHIP inhibited Hsp40-stimulated ATPase activity of Hsc70 and Hsp70, suggesting that CHIP blocks the forward reaction of the Hsc70-Hsp70 substrate-binding cycle . Consistent with this observation, both luciferase refolding and substrate binding in the presence of Hsp40 and Hsp70 were inhibited by CHIP . Taken together, these results indicate that CHIP decreases net ATPase activity and reduces chaperone efficiency, and they implicate CHIP in the negative regulation of the forward reaction of the Hsc70-Hsp70 substrate-binding cycle.

Mol Cell Biol, 1999 Jun, 19(6), 4262 - 9
Basis for the checkpoint signal specificity that regulates Chk1 and Cds1 protein kinases; Brondello JM et al.; Six checkpoint Rad proteins (Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1) are needed to regulate checkpoint protein kinases Chk1 and Cds1 in fission yeast . Chk1 is required to prevent mitosis when DNA is damaged by ionizing radiation (IR), whereas either kinase is sufficient to prevent mitosis when DNA replication is inhibited by hydroxyurea (HU) . Checkpoint Rad proteins are required for IR-induced phosphorylation of Chk1 and HU-induced activation of Cds1 . IR activates Cds1 only during the DNA synthesis (S) phase, whereas HU induces Chk1 phosphorylation only in cds1 mutants . Here, we investigate the basis of the checkpoint signal specificity of Chk1 phosphorylation and Cds1 activation . We show that IR fails to induce Chk1 phosphorylation in HU-arrested cells . Release from the HU arrest following IR causes substantial Chk1 phosphorylation . These and other data indicate that Cds1 prevents Chk1 phosphorylation in HU-arrested cells, which suggests that Cds1 actively suppresses a repair process that leads to Chk1 phosphorylation . Cds1 becomes more highly concentrated in the nucleus only during the S phase of the cell cycle . This finding correlates with S-phase specificity of IR-induced activation of Cds1 . However, constitutive nuclear localization of Cds1 does not enhance IR-induced activation of Cds1 . This result suggests that Cds1 activation requires DNA structures or protein activities that are present only during S phase . These findings help to explain how Chk1 and Cds1 respond to different checkpoint signals.

Mol Cell Biol, 1999 Jun, 19(6), 3940 - 50
Dimeric RFX proteins contribute to the activity and lineage specificity of the interleukin-5 receptor alpha promoter through activation and repression domains; Iwama A et al.; Interleukin-5 (IL-5) plays a central role in the differentiation, proliferation, and functional activation of eosinophils . The specific action of IL-5 on eosinophils and hematopoietically related basophils is regulated by the restricted expression of IL-5 receptor alpha (IL-5Ralpha), a subunit of high-affinity IL-5R, on these cells . We have previously identified an enhancer-like cis element in the IL-5Ralpha promoter that is important for both full promoter function and lineage-specific activity . Here, we demonstrate by yeast one-hybrid screening that RFX2 protein specifically binds to this cis element . RFX2 belongs to the RFX DNA-binding protein family, the biological role of which remains obscure . Using an electrophoretic mobility shift assay, we further show that RFX1, RFX2, and RFX3 homodimers and heterodimers specifically bind to the cis element of the IL-5Ralpha promoter . The mRNA expression of RFX1, RFX2, and RFX3 was detected ubiquitously, but in transient-transfection assays, multimerized RFX binding sites in front of a basal promoter efficiently functioned in a tissue- and lineage-specific manner . To further investigate RFX functions on transcription, full-length and deletion mutants of RFX1 were targeted to DNA through fusion to the GAL4 DNA binding domain . Tissue- and lineage-specific transcriptional activation with the full-length RFX1 fusion plasmid on a reporter controlled by GAL4 binding sites was observed . Distinct activation and repression domains within the RFX1 protein were further mapped . Our findings suggest that RFX proteins are transcription factors that contribute to the activity and lineage specificity of the IL-5Ralpha promoter by directly binding to a target cis element and cooperating with other tissue- and lineage-specific cofactors.

Genome Res, 1999 May, 9(5), 417 - 27
Identification of putative programmed -1 ribosomal frameshift signals in large DNA databases; Hammell AB et al.; The cis-acting elements that promote efficient ribosomal frameshifting in the -1 (5') direction have been well characterized in several viral systems . Results from many studies have convincingly demonstrated that the basic molecular mechanisms governing programmed -1 ribosomal frameshifting are almost identical from yeast to humans . We are interested in testing the hypothesis that programmed -1 ribosomal frameshifting can be used to control cellular gene expression . Toward this end, a computer program was designed to search large DNA databases for consensus -1 ribosomal frameshift signals . The results demonstrated that consensus programmed -1 ribosomal frameshift signals can be identified in a substantial number of chromosomally encoded mRNAs and that they occur with frequencies from two- to sixfold greater than random in all of the databases searched . A preliminary survey of the databases resulting from the computer searches found that consensus frameshift signals are present in at least 21 homologous genes from different species, 2 of which are nearly identical, suggesting evolutionary conservation of function . We show that four previously described missense alleles of genes that are linked to human diseases would disrupt putative programmed -1 ribosomal frameshift signals, suggesting that the frameshift signal may be involved in the normal expression of these genes . We also demonstrate that signals found in the yeast RAS1 and the human CCR5 genes were able to promote significant levels of programmed -1 ribosomal frameshifting . The significance of these frameshifting signals in controlling gene expression is not known, however.

J Biol Chem, 1999 May 21, 274(21), 14823 - 30
Identification of the ubiquitin carrier proteins, E2s, involved in signal-induced conjugation and subsequent degradation of IkappaBalpha; Gonen H et al.; The last step in the activation of the transcription factor NF-kappaB is signal-induced, ubiquitin- and proteasome-mediated degradation of the inhibitor IkappaBalpha . Although most of the components involved in the activation and degradation pathways have been identified, the ubiquitin carrier proteins (E2) have remained elusive . Here we show that the two highly homologous members of the UBCH5 family, UBCH5b and UBCH5c, and CDC34/UBC3, the mammalian homolog of yeast Cdc34/Ubc3, are the E2 enzymes involved in the process . The conjugation reaction they catalyze in vitro is specific, as they do not recognize the S32A,S36A mutant species of IkappaBalpha that cannot be phosphorylated and conjugated following an extracellular signal . Furthermore, the reaction is specifically inhibited by a doubly phosphorylated peptide that spans the ubiquitin ligase recognition domain of the inhibitor . Cys-to-Ala mutant species of the enzymes that cannot bind ubiquitin inhibit tumor necrosis factor alpha-induced degradation of the inhibitor in vivo . Not surprisingly, they have a similar effect in a cell-free system as well . Although it is clear that the E2 enzymes are not entirely specific to IkappaBalpha, they are also not involved in the conjugation and degradation of the bulk of cellular proteins, thus exhibiting some degree of specificity that is mediated probably via their association with a defined subset of ubiquitin-protein ligases . The mechanisms that underlie the involvement of two different E2 species in IkappaBalpha conjugation are not clear at present . It is possible that different conjugating machineries operate under different physiological conditions or in different cells.

J Biol Chem, 1999 May 21, 274(21), 14624 - 31
Cholesterol biosynthesis from lanosterol . Molecular cloning, tissue distribution, expression, chromosomal localization, and regulation of rat 7-dehydrocholesterol reductase, a Smith-Lemli-Opitz syndrome-related protein; Bae SH et al.; The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and developmental abnormalities (e.g . Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced . The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization . Rat DHCR, calculated molecular mass of 54.15-kDa polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 and 87%, respectively) as compared with its other related proteins (e.g . fungal sterol Delta14-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains . Five putative sterol-sensing domains were predicted to be localized in transmembrane domains 4-8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein . The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45-kDa myc-tagged fusion protein, which was recognized with anti-myc monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors . Northern blot analysis indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain . In rat brains, the highest level of mRNA encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla . Feeding rats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increase of the enzymic activity in the liver . When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant reduction in serum total cholesterol level were observed . However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at the level of transcription of the DHCR gene under these conditions.

Toxicol Appl Pharmacol, 1999 May 15, 157(1), 36 - 42
Dietary selenium reduces the formation of aberrant crypts in rats administered 3,2'-dimethyl-4-aminobiphenyl; Feng Y et al.; Human epidemiologic studies suggest that low selenium status is associated with increased cancer risk and that selenium supplementation is associated with reduction in the incidence of several cancers, including colorectal cancer . Aromatic and heterocyclic amine carcinogens are thought to be important in the etiology of human colorectal cancer, but no information is available on the effects of selenium on aromatic amine-induced colon cancer . In order to investigate this effect, aberrant crypt foci (ACF), the putative preneoplastic lesions of colon cancer in humans and rodents, were used as a biomarker to test the hypothesis that selenium supplementation can reduce aromatic amine-induced colon carcinogenesis . Male weanling F344 inbred rats were fed a basal torula yeast selenium-deficient diet supplemented with 0, 0.1, or 2 . 0 mg selenium/kg diet as selenite, selenate, or selenomethionine (SeMet) . Animals were fed the diets for 4 weeks and then administered 1 sc injection/week for 2 weeks of 3, 2'-dimethyl-4-aminobiphenyl (DMABP; 100 mg/kg) or vehicle (peanut oil) . At 12 weeks, the rats were euthanized and the colon and rectum were removed, opened longitudinally, and fixed in 70% ethanol . Glutathione peroxidase activities in erythrocytes and liver cytosol and selenium concentrations in the colon/rectum and kidney increased significantly (p < 0.05) and in a dose-dependent manner with each of the three selenium diets . No ACF were identified in vehicle-treated rats . In DMABP-treated rats, ACF frequencies decreased significantly (p < 0.05) in groups supplemented with 0.1 or 2.0 mg selenium/kg diet as selenite and selenate but not SeMet . There were no significant differences in ACF and aberrant crypts between rats fed 0.1 vs 2.0 mg selenium/kg diet . These results suggest that dietary selenium, depending on chemical form, can reduce aromatic amine-induced colon carcinogenesis .

Biochem Biophys Res Commun, 1999 May 19, 258(3), 713 - 21
Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1; Wen Y et al.; Binding of vascular endothelial growth factor (VEGF) to its receptor, VEGFR-2 (Flk-1/KDR), induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for signaling proteins that relay the signals for cell proliferation, migration, and permeability enhancement . We explored the VEGF/receptor signaling pathway by performing a two-hybrid screen of a rat lung cDNA library in yeast using the intracellular domain of rat VEGFR-2 as bait . Two clones encoding lipocortin V were isolated . Subsequent studies with the yeast two-hybrid assay showed that the complete intracellular domain of VEGFR-2 was required for the interaction . Co-immunoprecipitation of translated proteins confirmed the interaction between the VEGF receptor and lipocortin V . VEGF induced a rapid tyrosine phosphorylation of lipocortin V in human umbilical vein endothelial cells (HUVEC) . Pretreatment of HUVEC with antisense oligodeoxyribonucleotide (ODN) for lipocortin V significantly inhibited VEGF-induced cell proliferation, which was accompanied by a decrease in protein synthesis and tyrosine phosphorylation of lipocortin V . Our results indicate that lipocortin V may function as a signaling protein for VEGFR-2 by directly interacting with the intracellular domain of the receptor and appears to be involved in regulation of vascular endothelial cell proliferation mediated by VEGFR-2 .

Biochem Biophys Res Commun, 1999 May 19, 258(3), 519 - 23
Carboxy-terminally truncated form of a coactivator UTF1 stimulates transcription from a variety of gene promoters through the TATA Box; Fukushima A et al.; We have recently isolated a novel transcriptional coactivator, UTF1, which is expressed mainly in pluripotent embryonic stem cells (Okuda, A., Fukushima, A., Nishimoto, M., Orimo, A., Yamagishi, T., Nabeshima, Y., Kuro-o, M., Nabeshima, Y., Boon, K., Keaveney, M., Stunnenberg, H . G., and Muramatsu, M . EMBO J . 17, 2019-2032, 1998) . The UTF1 does not activate transcription nonspecifically, but boosts the level of transcription strictly in a specific upstream factor, ATF-2, dependent manner in mammalian cells . However, when expressed in yeast cells, the UTF1 displays a distinct activity, being able to augment the activity of minimal promoter bearing only the TATA element . Thus, these results indicate that certain domains of UTF1 render the factor inactive in terms of stimulating transcription through the basal transcription machinery in the absence of promoter-bound ATF-2 in mammalian cells . Here we report that the region bearing the leucine zipper motif is responsible for such biochemical properties of the UTF1 . Indeed, UTF1 lacking functional leucine zipper is able to rather promiscuously stimulate transcription from a number of basal gene promoters such as those of hsp70 and E1B genes in mammalian cells . We have also shown that this activation is executed through TATA box by the experiments using a TBP allele with an altered TATA-binding specificity . Moreover, we have found that Dr1-mediated repression of transcription can be overcome by expression of this mutant UTF1, indicating that the observed stimulation of transcription is at least in part due to its action as an anti-repressor .

Biochem Biophys Res Commun, 1999 May 10, 258(2), 345 - 52
Cloning, expression, and in vitro activity of human endostatin; Dhanabal M et al.; Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis . In the present study, we have expressed human endostatin in a yeast expression system (10 mg/L) . The recombinant protein was expressed in a soluble form and purified to homogeneity . It specifically inhibited the proliferation and migration of endothelial cells . In addition, we report for the first time that endostatin caused G1 arrest of endothelial cells . Also, we show that endostatin treatment resulted in apoptosis of HUVE and HMVE cells and that all of these effects do not occur in nonendothelial cells . Collectively, these findings demonstrate the expression of a biologically active form of human endostatin in yeast and provide important mechanistic insight into endostatin action on endothelial cells .

Biochem Biophys Res Commun, 1999 May 10, 258(2), 271 - 7
Detection of DNA abnormalities by arbitrarily primed PCR fingerprinting: amplification of the MDM2 gene in a mediastinum fibrosarcoma; Kuchiki H et al.; Arbitrarily primed PCR (AP-PCR) fingerprinting method is easy and useful for analysis of genetic alterations in anonymous chromosomal regions . We applied this technology to analysis of DNA from human primary cancers and found amplification of a DNA fragment in a mediastinum fibrosarcoma . PCR-based analysis of radiation hybrid panels following cloning and nucleotide sequence determination of the fragment revealed that it was derived from a region of chromosome 12q13-q15 . In this region, the MDM2 and IFNG genes were noted as known genes that could be involved in human carcinogenesis . Southern blot analysis of genomic DNA of the tumor revealed the amplification of the MDM2 gene together with the fragment locus, but not the IFNG gene . Our results demonstrated that detection of DNA alterations by AP-PCR fingerprinting without any previous knowledge of the genes and subsequent analysis of radiation hybrid panels could lead to easy identification of candidates for genes involved in carcinogenesis .

Cell Biol Int, 1998, 22(6), 465 - 72
Effects of some biologically active compounds on phagosome-lysosome fusion in peritoneal macrophages of mice; Mozhenok T et al.; Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P-L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target . It was found that all three compounds tested enhanced P-L fusion . To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes . In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined . Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes . All three compounds tested increase F-actin content in peritoneal macrophages . Thus, the effect of BR on P-L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes . The enhancement of P-L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state .

Arch Biochem Biophys, 1999 May 15, 365(2), 216 - 22
alpha-actinin-2 is a new component of the dystrophin-glycoprotein complex; Hance JE et al.; The human skeletal muscle yeast two-hybrid cDNA library was screened with the carboxyl-terminal region (the last 200 amino acids) of dystrophin . Two interacting clones were identified corresponding to alpha-actinin-2 and actin . Interactions between alpha-actinin, actin, and dystrophin were confirmed by the ligand-blotting technique, by colocalization of dystrophin and alpha-actinin-2 to the isolated skeletal muscle sarcolemmal vesicles and to the plasma membranes isolated from C2C12 myoblasts, and by indirect immunolocalization of dystrophin and alpha-actinin-2 in skeletal muscle cells . This is the first identification of a direct interaction between alpha-actinin, actin, and the carboxyl-terminal region of dystrophin . We propose that dystrophin forms lateral, multicontact association with actin and that binding of alpha-actinin-2 to the carboxyl-terminus of dystrophin is the communication link between the integrins and the dystrophin/dystrophin-glycoprotein complex .

Chromosome Res, 1999, 7(2), 143 - 56
Structural analysis and physical mapping of a pericentromeric region of chromosome 5 of Arabidopsis thaliana; Tutois S et al.; The Arabidopsis thaliana CIC YAC 2D2, 510 kb long and containing a small block of 180 bp satellite units was subcloned after EcoR1 digestion in the pBluescript plasmid . One of these clones was mapped genetically in the pericentromeric region of chromosome 5 . The analysis of 40 subclones of this YAC showed that they all contain repeated sequences with a high proportion of transposable elements . Three new retrotransposons, two Ty-3 Gypsy-like and one Ty-1 Copia, were identified in addition to two new tandem-repeat families . A physical map of the chromosome 5 pericentromeric region was established using CIC YAC clones, spanning around 1000 kb . This contig extends from the CIC YAC 9F5 and 7A2 positioned on the left arm of chromosome 5 to a 5S rDNA genes block localized by in-situ hybridization in the pericentromeric region . Hybridization of the subclones on the CIC YAC library showed that some of them are restricted to the pericentromeric region of chromosome 5 and represent specific markers of this region.

Oncogene, 1999 Apr 8, 18(14), 2299 - 309
Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F; Ohtani K et al.; Initiation of DNA replication requires the function of MCM gene products, which participate in ensuring that DNA replication occurs only once in the cell cycle . Expression of all mammalian genes of the MCM family is induced by growth stimulation, unlike yeast, and the mRNA levels peak at G1/S boundary . In this study, we examined the transcriptional activities of isolated human MCM gene promoters . Human MCM5 and MCM6 promoters with mutation in the E2F sites failed in promoter regulation following serum stimulation and exogenous E2F expression . In addition, we identified a novel E2F-like sequence in human MCM6 promoter which cooperates with the authentic E2F sites in E2F-dependent regulation . Forced expression of E2F1 could induce expression of all members of the endogenous MCM genes in rat embryonal fibroblast REF52 cells . Our results demonstrated that the growth-regulated expression of mammalian MCM5 and MCM6 genes, and presumably other MCM members, is primarily regulated by E2F through binding to multiple E2F sites in the promoters.

J Dent Res, 1999 Apr, 78(4), 857 - 68
Natural defenses against Candida colonization breakdown in the oral cavities of the elderly; Lockhart SR et al.; Candida colonization of the oral cavity increases in the elderly . A major predisposing condition is denture use, which also increases in the elderly . To test whether the increase in colonization is age-related in a fashion independent of denture use, we analyzed the frequency (incidence) of carriage, the intensity of carriage, the multiplicity of species, and the genetic relatedness of strains in the oral cavities of 93 test subjects separated into the three age groups: 60 to 69 yr, 70 to 79 yr, and > or = 80 yr . Each age group was further subdivided into subjects with and without dentures, and into males and females . The results demonstrate that the frequency of carriage, the intensity of carriage, and multispecies carriage all increase as a function of age and differ according to gender, in both cases independent of denture use, suggesting that the natural suppression of yeast carriage in the oral cavity breaks down in the elderly . In addition, it is demonstrated that Candida glabrata colonizes the oral cavities of elderly individuals without dentures only after 80 yr of age, suggesting that there are age-related compromising conditions other than denture use in this most elderly age group.

Nucleic Acids Res, 1999 Jun 1, 27(11), 2241 - 7
Interaction of influenza virus NS1 protein and the human homologue of Staufen in vivo and in vitro; Falcon AM et al.; A screening for human proteins capable of interacting with influenza virus NS1 has been carried out using the two-hybrid genetic trap in yeast . A cDNA corresponding to the human homologue of Drosophila melanogaster Staufen protein (hStaufen) was isolated that fulfilled all genetic controls of the two-hybrid protocol . Using a hStaufen cDNA isolated from a lambda human library, the interaction of hStaufen and NS1 proteins was characterised in vivo and in vitro . Co-transfection of NS1 cDNA and a partial cDNA of hStaufen led to the relocalisation of recombinant hStaufen protein from its normal accumulation site in the cytoplasm to the nuclear location of NS1 protein . NS1 and hStaufen proteins could be co-immunoprecipitated from extracts of co-transfected cells and from mixtures of extracts containing either protein, as well as from extracts of influenza virus-infected cells . Furthermore, both proteins co-localised in the ribosomal and polysomal fractions of influenza virus-infected cells . The interaction was also detected in pull-down experiments using a resin containing purified hStaufen and NS1 protein translated in vitro . Deletion mapping of the NS1 gene indicated that a mutant protein containing the N-terminal 81 amino acids is unable to interact with hStaufen, in spite of retaining full RNA-binding capacity . These results are discussed in relation to the possible mechanisms of action of hStaufen and its relevance for influenza virus infection.

Zhonghua Yu Fang Yi Xue Za Zhi, 1998 May, 32(3), 165 - 7
{Antibody reaction to immunization with different recombinant hepatitis B vaccine in primary school children}; Liang Z et al.; OBJECTIVE: To evaluate the safety and immunogenicity of local produced hepatitis B (HB) vaccine and to lay a basis for formulating an immunization strategy . METHODS: Two batches of local produced and two batches of imported yeast derived recombinant HB vaccine (YDV), and four batches of local produced Chinese hamster ovary cell derived recombinant HB vaccine (CHOV) were administered to 550 children in primary schools to study their immunogenicity one year after immunization . RESULTS: No severe adverse reaction was found in all vaccinee . Positive seroconversion rate was more than 90 percent one month after completion of three-dose immunization with all kinds of HB vaccine mentioned above, and then declined slowly . Titer of antibody against hepatitis B surface antigen (anti-HBs) in those vaccinated with two batches of local produced YDV, except four groups, was significantly lower than that in those with imported YDV and local produced CHOV seven months after first-dose immunization, but no difference between local produced YDV and imported Merck vaccine and CHOV was found one year after immunization . Mean geometric titer (GMT) of anti-HBs reached the peak seven months after immunization in those vaccinated with all batches of HB vaccine except for the YDV imported from Amgen Co . There was no significant difference in positive seroconversion and GMT of anti-HBs between different batches of local produced YDV and CHOV . CONCLUSION: Safety and immnogenicity of local produced recombinant HB vaccine is as good as those of imported one in immunization for primary school children.

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