Histochem Cell Biol, 2003 Jan, 119(1), 1 - 13 Epub 2002 Dec 21. Colocalisation of the protein tyrosine phosphatases PTP-SL and PTPBR7 with beta4-adaptin in neuronal cells; Dilaver G et al.; The mouse gene Ptprr encodes the neuronal protein tyrosine phosphatases PTP-SL and PTPBR7 . These proteins differ in their N-terminal domains, with PTP-SL being a cytosolic, membrane-associated phosphatase and PTPBR7 a type I transmembrane protein . In this study, we further explored the nature of the PTP-SL-associated vesicles in neuronal cells using a panel of organelle markers and noted a comparable subcellular distribution for PTP-SL and the beta4-adaptin subunit of the AP4 complex . PTP-SL, PTPBR7 and beta4-adaptin are localised at the Golgi apparatus and at vesicles throughout the cytoplasm . Immunohistochemical analysis demonstrated that PTP-SL, PTPBR7 and beta4-adaptin are all endogenously expressed in brain . Interestingly, coexpression of PTP-SL and beta4-adaptin leads to an altered subcellular localisation for PTP-SL . Instead of the Golgi and vesicle-type staining pattern, still observable for beta4-adaptin, PTP-SL is now distributed throughout the cytoplasm . Although beta4-adaptin was found to interact with the phosphatase domain of PTP-SL and PTPBR7 in the yeast two-hybrid system, it failed to do so in transfected neuronal cells . Our data suggest that the tyrosine phosphatases PTP-SL and PTPBR7 may be involved in the formation and transport of AP4-coated vesicles or in the dephosphorylation of their transmembrane cargo molecules at or near the Golgi apparatus. Am J Pathol, 2003 Feb, 162(2), 373 - 9 Multicolor deconvolution microscopy of thick biological specimens; Maierhofer C et al.; One limitation in understanding disease at the cellular level has been the inability to efficiently analyze DNA on a cell-to-cell basis within the natural tissue context . However, DNA analyses at a single-cell resolution should be instrumental for the understanding of cancer cell biology, cancer evolution, for chromosomal mosaic analysis and rare cell events, and should provide otherwise inaccessible information on essential biological processes . Here we present a fluorescence in situ hybridization-based multicolor deconvolution technique for three-dimensional microscopy . We use up to seven different color channels for probe detection, which allows the simultaneous high-resolution localization of multiple point-like sources within a biological specimen with a thickness of up to 30 micro m . In addition, a DNA counterstain is used for volume labeling of the nuclei offering the opportunity for a simultaneous segmentation of nuclei . Furthermore, as the instrumentation consists of a standard fluorescence microscope it represents a low-cost method as compared to confocal microscopy. Trends Genet, 2003 Feb, 19(2), 60 - 2 Searching for nuclear-mitochondrial genes; Chinnery PF; Recently, a novel strategy has been developed to identify yeast genes that are important for mitochondrial respiratory chain function . This approach found a large number of genes that were not previously thought to be involved, providing new candidate disease genes for mitochondrial disorders . These genes could cast light on the intricate relationship between genotype and phenotype in a wide range of inherited human diseases. Curr Opin Chem Biol, 2003 Feb, 7(1), 103 - 9 Genome-wide analysis of signaling domain function; Yu JW et al.; Approximately 2.5% of human gene products contain one or more small domains that drive interactions between proteins and other cellular components in cell signaling processes . The many interactions driven by these relatively simple domains are thought to cooperate with one another to yield complex signaling networks that allow very fine control of cell function . In principle, if we can understand all domain-mediated interactions it should be possible to model these networks . Genome-wide analysis of signaling domain interactions represents a first step in this direction, and several advances of this sort in yeast have been reported over the past year . These reports suggest, for some domains at least, that the prospect of generating 'wiring diagrams' with this simple approach is feasible. Blood Cells Mol Dis, 2002 Nov-Dec, 29(3), 536 - 47; discussion 548-52 Iron metabolism and mitochondrial abnormalities in Friedreich ataxia; Pandolfo M; Friedreich ataxia is an autosomal recessive disease causing degeneration in the central and peripheral nervous system, cardiomyopathy, skeletal abnormalities and increased risk of diabetes . It is caused by deficiency of frataxin, a highly conserved nuclear-encoded mitochondrial protein . The genetic mutation found in 98% of Friedreich ataxia chromosomes is the unstable hyperexpansion of a GAA triplet repeat in the first intron of the gene . The expanded GAA repeat, by adopting an abnormal triple helical structure, impairs frataxin transcription . Longer repeats cause a more profound frataxin deficiency and are associated with earlier onset and increased severity of the disease . Yeast cells deficient in the frataxin homologue (Deltayfh1) become unable to carry out oxidative phosphorylation, lose mitochondrial DNA, accumulate iron in mitochondria, show unregulated high expression of high affinity iron uptake, and have an increased sensitivity to oxidative stress . Loss of respiratory competence in Deltayfh1 is iron-dependent . Additional properties of these cells include a deficiency of iron-sulfur cluster containing proteins (ISPs) and impaired iron efflux out of mitochondria . Evidence of oxidative stress, mitochondrial dysfunction, deficiency of multiple ISPs and iron deposits are also found in the human disease and in mouse models . The primary function of frataxin is still unknown, however much recent evidence suggests that it enhances iron-sulfur cluster synthesis and protects iron from free radical-generating reactions . The search for frataxin function stimulated more investigations on the role of mitochondria in cellular iron homeostasis . Their results suggest that these organelles may play a central role in controlling iron homeostasis, which is not surprising considering that they are the major cellular site where this metal is utilized . I propose a model, valid in yeast as well as in higher eukaryotes, in which iron transport into mitochondria is directly coupled to its uptake at the cell membrane and iron transport out of mitochondria depends on adequate iron-sulfur cluster synthesis . Regulatory mechanisms in the cytosol would then sense a post-mitochondrial iron pool . Much circumstantial evidence from genetically manipulated yeast and from human diseases supports this model. Zhonghua Gan Zang Bing Za Zhi, 2003 Jan, 11(1), 8 - 10 {Screening of the genes of hepatitis B virus PreS2 interacting proteins}; Lu YY et al.; OBJECTIVE: To screen and clone the genes of proteins in hepatocytes interacting with hepatitis B virus (HBV) PreS2 by yeast-two hybridization technique . METHODS: The HBV PreS2 gene was amplified by polymerase chain reaction (PCR) and HBV PreS2 bait plasmid was constructed by using yeast-two hybridization system 3, then transformed into yeast AH109, followed by mating with yeast Y187 containing liver cDNA library plasmid in 2 YPDA medium . Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) and synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) containing X-alpha-gal for selecting positive blue clones, then amplified by PCR, sequenced, and performed bioinformatics analysis . RESULTS: HBV PreS2 gene was cloned successfully and expressed in yeast AH109.Twenty-six positive colonies were selected, among them, twelve containing metallothionein 2A, one cytochrome C oxidase II, two cytochrome P450 subfamily IV4F, two cytochrome c oxidase subunit 4 isoform 1, three albumin (ALB), one Na(+)K(+) transporting ATPase beta-1 polypeptide, two prealbumin, one lectin galactoside-binding subunit, and Two new genes with unknown function . CONCLUSION: Genes of HBV PreS2 interacting proteins have been successfully cloned, which brings some new clues for studying the biological functions of HBV PreS2 and related proteins. Biochem Soc Trans, 2003 Feb, 31(Pt 1), 263 - 5 Sister chromatid cohesion and genome stability in vertebrate cells; Morrison C et al.; For successful eukaryotic mitosis, sister chromatid pairs remain linked after replication until their kinetochores have been attached to opposite spindle poles by microtubules . This linkage is broken at the metaphase-anaphase transition and the sisters separate . In budding yeast, this sister chromatid cohesion requires a multi-protein complex called cohesin . A key component of cohesin is Scc1/Mcd1 (Rad21 in fission yeast) . Disruption of the chicken orthologue of Scc1 by gene targeting in DT40 cells causes premature sister chromatid separation . Cohesion between sister chromatids is likely to provide a substrate for post-replicative DNA repair by homologous recombination . In keeping with this role of cohesion, Scc1 mutants also show defects in the repair of spontaneous and induced DNA damage . Scc1-deficient cells frequently fail to complete metaphase chromosome alignment and show chromosome segregation defects, suggesting aberrant kinetochore function . Consistent with this, the chromosomal passenger protein, INCENP (inner centromere protein) fails to localize to centromeres . Survivin, another passenger protein and one which interacts with INCENP, also fails to localize to centromeres in Scc1-deficient cells . These results show that cohesin maintains genomic stability by ensuring appropriate DNA repair and equal chromosome segregation at mitosis. Sheng Li Ke Xue Jin Zhan, 2001 Jul, 32(3), 229 - 32 {New type of two-hybrid systems in protein-protein interaction studies}; Xue YN; Some new type of two-hybrid systems, such as split-ubiquitin system, protein-fragment complementation assay, repressor reconstitution assay and SOS recruitment system, have been developed recently . Similar to the original transcription-based yeast two-hybrid system, these systems are to establish an assay for protein-protein interactions, in which some functional proteins can be split into two parts in structure and their activities can be recovered by reconstitution . Due to their non-transcriptional properties, these new types of two-hybrid systems have become useful extension of the yeast two-hybrid system and powerful tools for the study of protein interactions. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 138 - 42 {Expression and characterization of Kringle 1-4.5 domains of human plasminogen}; Zhou QW et al.; The cDNA encoding Kringle 1-4 and part of Kringle 5 domains of human plasminogen (K1-4.5), obtained from HepG2 by RT-PCR, was cloned into expression vector pHIL-S1 . The recombinant plasmid pHIL-K1-4.5 was transformed into Pichi pastoris GS115 and the recombinant yeast was induced to express the recombinant proteins by methanol . The expressed proteins were purified by lysine affinity chromatography to a purity of 95% . The recombinant K1-4.5 inhibited the growth of bovine capillary endothelial cells (BAEC) stimulated by the basic fibroblast growth factor (bFGF), in a dosage-dependent manner with a half maximal concentration of 2 mg/L . rhK1-4.5 also inhibited 40% of the BAEC migration stimulated by bFGF in the concentration of 1 mg/L. Science, 2003 Jan 24, 299(5606), 572 - 4 Extended longevity in mice lacking the insulin receptor in adipose tissue; Bluher M et al.; Caloric restriction has been shown to increase longevity in organisms ranging from yeast to mammals . In some organisms, this has been associated with a decreased fat mass and alterations in insulin/insulin-like growth factor 1 (IGF-1) pathways . To further explore these associations with enhanced longevity, we studied mice with a fat-specific insulin receptor knockout (FIRKO) . These animals have reduced fat mass and are protected against age-related obesity and its subsequent metabolic abnormalities, although their food intake is normal . Both male and female FIRKO mice were found to have an increase in mean life-span of approximately 134 days (18%), with parallel increases in median and maximum life-spans . Thus, a reduction of fat mass without caloric restriction can be associated with increased longevity in mice, possibly through effects on insulin signaling. Cancer Res, 2003 Jan 15, 63(2), 404 - 12 An integrated physical and gene map of the 3.5-Mb chromosome 3p21.3 (AP20) region implicated in major human epithelial malignancies; Protopopov A et al.; To facilitate the identification of tumor suppressor genes in the chromosome 3p21.3-p22 AP20 subregion, we constructed a 3.5-Mb physical and gene map of this segment (between markers D3S4285 and D3S3873) that spans the distance from 124.4cR3000 to 133.5 cR3000 of the GB4 genetic map . We used NotI-linking and -jumping clones, sequence-tagged site PCR marker analysis, and multicolor and fiber fluorescence in situ hybridization to confirm the sequence order and map orientation . An integrated clone contig composed of 5 yeast artificial chromosome, 15 bacterial artificial chromosome, 5 P1 artificial chromosome, and 8 NotI-linking clones provided the physical base of the map . We unequivocally established the order of 28 sequence-tagged sites and 35 genes in the region . Gaps between published bacterial artificial chromosome contigs were determined and covered by our own sequence data . Furthermore, three new genes were isolated, namely the human homologue to the rat Golgi peripheral membrane protein p65, GOLPH5 (GORASP1), the gene for stress-inducible protein, STI2, and the AP20-region gene 1, APRG1 . The tumor suppressor gene candidate APRG1 was positioned close to the border of the homozygous deletion in a small cell lung cancer cell line ACC-LC5 . Expression analysis with a tissue-specific panel of cDNA revealed seven distinct tissue-specific splice variants (A-G) of the message (size range, 1.0-1.8 kb) . Although the gene was expressed at a low level in all tested tissues, comparatively higher expression was detected in pancreas (splice forms B and D), kidney (A) and placenta (B and C) . The APRG1 gene encoded a predicted protein of 170 amino acids (isoform B), which had an NH2-terminal part conserved among members of the eukaryotic translation factor 6 gene family . A Prosite pattern corresponding to the cell attachment sequence Arg-Gly-Asp was also found . The presence of this domain raised the intriguing possibility that APRG1B may be directly involved in membrane interactions and cell adhesion . We showed that the AP20 region was duplicated during mammalian evolution and homologous gene clusters were present in human chromosome 2 and syntenic mouse regions on chromosomes 1, 2, and 9 . Interestingly, the HYA22 gene (human ortholog of the yeast YA22 gene) was located at the borders of both breakpoints, evolutionarily conserved gene cluster and homozygous deletions detected in lung, kidney and other cancers . NotI digestion revealed that the AP20 region was frequently and extensively methylated in renal carcinoma cell lines and tumor biopsies. Parasitol Int, 2003 Mar, 52(1), 1 - 11 Transmission-blocking vaccine of vivax malaria; Tsuboi T et al.; Malaria remains one of the leading causes of both morbidity and mortality of humans residing in tropical countries . For many malarious regions outside of Africa, development of effective transmission-blocking vaccines will require coverage against both Plasmodium falciparum and Plasmodium vivax . The genes coding for two potential P . vivax transmission-blocking antigens, Pvs25 and Pvs28, have been cloned . Mice vaccinated with yeast-produced recombinant proteins Pvs25 and Pvs28 adsorbed to aluminum hydroxide developed strong antibody responses against the immunogens . The development of oocysts in mosquitoes was completely inhibited when these antisera were ingested with the P . vivax Salvador (Sal) I strain-infected chimpanzee blood . In a large collection of P . vivax field isolates, we found only 5 nucleotide changes that would result in amino acid substitutions in Pvs25 . In contrast, the Pvs28 gene had 22 nucleotide changes that would result in conservative amino acid substitutions . How the antigenic polymorphism of Pvs25 and Pvs28 would affect the efficacy of Sal I based vaccine remains to be elucidated . Clinical trials with Pvs25 and the P . falciparum ortholog Pfs25 are in preparation . J Anim Sci, 2002 Dec, 80(12), 3257 - 67 Effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on swine performance, brain regional neurochemistry, and serum chemistry and the efficacy of a polymeric glucomannan mycotoxin adsorbent; Swamy HV et al.; The co-occurrence of Fusarium mycotoxins in contaminated swine diets has been shown to result in synergistic toxicity beyond that observed for individual toxins . An experiment was conducted, therefore, to investigate the effects of feeding a blend of grains naturally contaminated with Fusarium mycotoxins on growth, brain regional neurochemistry, serum immunoglobulin (Ig) concentrations, serum chemistry, hematology, and organ weights of starter pigs . Three levels of glucomannan polymer (GM polymer, extract of yeast cell wall, Alltech Inc.) were also tested for its efficacy to overcome Fusarium mycotoxicoses . A total of 175 starter pigs (initial weight of 10 +/- 1.1 kg) were fed five diets (seven pens of five pigs per diet) for 21 d . Diets included (1) control, (2) blend of contaminated grains, (3) contaminated grains + 0.05% GM polymer (4) contaminated grains + 0.10% GM polymer and (5) contaminated grains + 0.20% GM polymer . Diets containing contaminated grains averaged 5.5 ppm deoxynivalenol, 0.5 ppm 15-acetyldeoxynivalenol, 26.8 ppm fuuric acid, and 0.4 ppm zearalenone . Feed intake and weight gain of all pigs fed contaminated grains was significantly reduced compared to controls throughout the experiment . The weights of liver and kidney, expressed as a percentage of body weight, were lower in pigs fed the contaminated diet than in those fed the control diet . The feeding of contaminated grains significantly reduced concentrations of dopamine in the hypothalamus and pons and concentrations of dihydroxyphenylacetic acid and norepinephrine in the pons . The ratios of 5-hydroxyindoleacetic acid to serotonin, however, were elevated in the hypothalamus and pons . The feeding of contaminated grains increased serum IgM and IgA concentrations, while serum IgG concentrations were not altered . The supplementation of GM polymer prevented some of the mycotoxin-induced alterations in brain neurotransmitter and serum Ig concentrations . In summary, the feeding of grains naturally contaminated with Fusarium mycotoxins reduced growth, altered brain neurochemistry, increased serum Ig concentrations, and decreased organ weights in starter pigs . Some of the Fusarium mycotoxin-induced changes in neurochemistry and serum Ig concentrations can be prevented by the feeding of yeast cell wall polymer at appropriate concentrations, although this was not reflected in increased growth rate under these experimental conditions. Se Pu, 2000 Nov, 18(6), 500 - 2 {Separation and determination of purine bases and pyrimidine bases from nucleic acid hydrolysis by HPLC on BDS column}; Huang XL et al.; The hydrolysates of nucleic acid, six purine bases and pyrimidine bases (cytosine, uracil, guanine, hypoxanthine, adenine and thymine) were separated and determined by using HPLC . It is discussed how the column and mobile phase affect the separation . The peaks of cytosine and adenine are tailed on ordinary C18 column, and they are very good on BDS-C18 column . The KH2PO4-H3PO4 buffer can be used in separation the hydrolysates of RNA and DNA, and the NaAc-HAc buffer is only used in DNA . In addition, pH value is a very important factor for separation . With pH value of mobile phase increasing, the retention times of guanine, hypoxanthine and thymine were first increased and then decreased, adenine was increased, and cytosine and uracil were almost constant . The chosen mobile phase was 0.1 mol/L KH2PO4-H3PO4 buffer, with a pH value of 4.05 . It was detected at UV 260 nm . The determination was completed within 10 min . The RSDs were all less than 3% and the recoveries were in the range of 82%-114% . The method has been applied to the detection of yeast hydrolysates. J Biol Chem, 2003 Apr 4, 278(14), 12231 - 40 Epub 2003 Jan 21. Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by specific interaction of independent regions of human La autoantigen; Pudi R et al.; The human La autoantigen has been shown to interact with the internal ribosome entry site (IRES) of hepatitis C virus (HCV) in vitro . Using a yeast three-hybrid system, we demonstrated that, in addition to full-length La protein, both N- and C-terminal halves were able to interact with HCV IRES in vivo . The exogenous addition of purified full-length and truncated La proteins in rabbit reticulocyte lysate showed dose-dependent stimulation of HCV IRES-mediated translation . However, an additive effect was achieved adding the terminal halves together in the reaction, suggesting that both might play critical roles in achieving full stimulatory activity of the full-length La protein . Using computational analysis, three-dimensional structures of the RNA recognition motifs (RRM) of the La protein were independently modeled . Of the three putative RRMs, RRM2 was predicted to have a good binding pocket for the interaction with the HCV IRES around the GCAC motif near the initiator AUG and RRM3 binds perhaps in a different location . This observation was further investigated by the filter-binding and toe-printing assays . The results presented here strongly suggest that both the N- and C-terminal halves can interact independently with the HCV IRES and are involved in stimulating internal initiation of translation. Bioessays, 2003 Feb, 25(2), 152 - 62 The CRP/MLP/TLP family of LIM domain proteins: acting by connecting; Weiskirchen R et al.; In vertebrates, members of the cysteine-rich protein (CRP) family are characterized by the presence of two LIM domains linked to short glycine-rich repeats . These proteins mediate protein-protein interactions and are of fundamental importance for cell differentiation, cytoskeletal remodeling, and transcriptional regulation . To date, a vast amount of information about vertebrate CRPs has become available, including their biological functions, interacting partners, and three-dimensional structures . Compatible with a molecular adapter role, structural data reveal that the LIM domains within these proteins represent completely independent folded units bridged by flexible linker regions . The physiological roles for individual CRPs was determined by targeted gene disruption analysis and by identification of common and specific binding partners by means of yeast and mammalian two-hybrid screens . Several CRP-like LIM domain proteins with close structural and sequence similarity were identified in arthropods, protozoas and plants, supporting the notion that this subset of LIM domain proteins has been highly conserved over the span of evolution thereby emphasizing the importance of their function . Prostate, 2003 Mar 1, 54(4), 315 - 21 Selenomethionine does not affect PSA secretion independent of its effect on LNCaP cell growth; Bhamre S et al.; BACKGROUND: Individuals supplemented with selenium have reduced incidence of prostate cancer . This study determines whether selenomethionine specifically affects the secretion of prostate specific antigen (PSA) in vitro . METHODS: LNCaP cells were supplemented with selenomethionine for 7 days . PSA secretion was determined by ELISA . Cell proliferation was assessed by enumeration of trypan blue excluding cells . Colony formation was determined in soft agar . Cell cycle distribution was determined by FACS analysis of propidium iodide stained cells . RESULTS: Selenomethionine at > or = 70 microM inhibited LNCaP cell growth and colony formation . 0-100 microM selenomethionine did not affect the secretion of PSA by LNCaP cells in cell culture supernatants when normalized to the number of cells in culture . At supra-nutritional concentrations of selenomethionine, LNCaP cells had longer G(0)/G(1) phase in agreement with the inhibitory effects on cell growth . CONCLUSIONS: PSA secretion is not specifically inhibited by concentrations of selenomethionine corresponding to plasma selenium concentrations found in individuals supplemented with chemopreventive concentrations of selenized yeast . These data suggest that changes in serum PSA levels in individual patients during selenium supplementation is not an effect specific for PSA secretion, but rather may be a useful indicator for changes in disease progression in individual patients . Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1128 - 33 Epub 2003 Jan 21. Modular organization of cellular networks; Rives AW et al.; We investigated the organization of interacting proteins and protein complexes into networks of modules . A network-clustering method was developed to identify modules . This method of network-structure determination was validated by clustering known signaling-protein modules and by identifying module rudiments in exclusively high-throughput protein-interaction data with high error frequencies and low coverage . The signaling network controlling the yeast developmental transition to a filamentous form was clustered . Abstraction of a modular network-structure model identified module-organizer proteins and module-connector proteins . The functions of these proteins suggest that they are important for module function and intermodule communication. J Biol Chem, 2003 Mar 28, 278(13), 11696 - 704 Epub 2003 Jan 21. A developmentally regulated splice variant from the complex lola locus encoding multiple different zinc finger domain proteins interacts with the chromosomal kinase JIL-1; Zhang W et al.; Using a yeast two-hybrid screen we have identified a novel isoform of the lola locus, Lola zf5, that interacts with the chromosomal kinase JIL-1 . We characterized the lola locus and provide evidence that it is a complex locus from which at least 17 different splice variants are likely to be generated . Fifteen of these each have a different zinc finger domain, whereas two are without . This potential for expression of multiple gene products suggests that they serve diverse functional roles in different developmental contexts . By Northern and Western blot analyses we demonstrate that the expression of Lola zf5 is developmentally regulated and that it is restricted to early embryogenesis . Immunocytochemical labeling with a Lola zf5-specific antibody of Drosophila embryos indicates that Lola zf5 is localized to nuclei . Furthermore, by creating double-mutant flies we show that a reduction of Lola protein levels resulting from mutations in the lola locus acts as a dominant modifier of a hypomorphic JIL-1 allele leading to an increase in embryonic viability . Thus, genetic interaction assays provide direct evidence that gene products from the lola locus function within the same pathway as the chromosomal kinase JIL-1. Prostaglandins Leukot Essent Fatty Acids, 2003 Feb, 68(2), 107 - 12 Mechanism of fatty acid desaturation: a bioorganic perspective; Behrouzian B et al.; The desaturation of long chain fatty acids is a ubiquitous transformation which plays a critical role in the biosynthesis of lipids . Of particular interest to the bioorganic chemist is the unique ability of desaturases to oxidize unactivated hydrocarbon chains in a chemo-, regio- and stereoselective manner . The mechanism of membrane-bound desaturases has been examined using regiospecifically labelled analogues bearing deuterium, sulfur or fluorine-substituted methylene isosteres . These probes have been applied in the study of several biomedically important desaturase systems including a prototypical yeast stearoyl CoA delta(9) desaturase . In all cases, it has been found that the dehydrogenation (desaturation) process is initiated by a kinetically important hydrogen activation step at the carbon of the incipient double bond which is closest to the acyl terminus of the fatty acid chain . These results point to a common active site architecture which is highly conserved among a wide range of membranous desaturases. Radiat Res, 2003 Feb, 159(2), 139 - 48 Molecular anatomy of the DNA damage and replication checkpoints; Qin J et al.; Cell cycle checkpoints are signal transduction pathways that enforce the orderly execution of the cell division cycle and arrest the cell cycle upon the occurrence of undesirable events, such as DNA damage, replication stress, and spindle disruption . The primary function of the cell cycle checkpoint is to ensure that the integrity of chromosomal DNA is maintained . DNA lesions and disrupted replication forks are thought to be recognized by the DNA damage checkpoint and replication checkpoint, respectively . Both checkpoints initiate protein kinase-based signal transduction cascade to activate downstream effectors that elicit cell cycle arrest, DNA repair, or apoptosis that is often dependent on dose and cell type . These actions prevent the conversion of aberrant DNA structures into inheritable mutations and minimize the survival of cells with unrepairable damage . Genetic components of the damage and replication checkpoints have been identified in yeast and humans, and a working model is beginning to emerge . We summarize recent advances in the DNA damage and replication checkpoints and discuss the essential functions of the proteins involved in the checkpoint responses. Arch Virol, 2003 Jan, 148(1), 99 - 113 Interaction of Bombyx mori nucleopolyhedrovirus BRO-A and host cell protein laminin; Kang WK et al.; The Bombyx mori nucleopolyhedrovirus (BmNPV) contains five baculovirus repeated ORF ( bro) genes, all of which are expressed as delayed early genes . We have recently reported that BmNPV BRO proteins, specially BRO-A and BRO-C, contain a nucleic acid binding activity and are involved in nucleosome structures in nuclei of infected cells . To further understand the function of bro-a gene, we looked for factors interacting with BmNPV BRO-A using the yeast two-hybrid system . Fifteen clones obtained from a cDNA library of mock-infected cells and one from a library prepared at 2 h postinfection (p.i.) were found to comprise one distinct gene, which was identified as the Bombyx homolog (bLaminin) of Drosophila laminin beta1 . A direct interaction between BRO-A and N-terminal region of bLaminin was demonstrated by in vitro pull-down experiments . Further pull-down assays using BmN cell extracts and anti-laminin antibodies also showed interaction of both proteins . In addition, two more clones were obtained from cDNA library of 12 h p.i . and were found to encode BRO-A itself, indicating that BRO-A forms an oligomer . Taken together, we propose that BRO-A may function as a laminin binding protein. Acta Neuropathol (Berl), 2003 Feb, 105(2), 177 - 84 Epub 2002 Nov 12. Synphilin in normal human brains and in synucleinopathies: studies with new antibodies; Murray IJ et al.; Mutations in the gene encoding alpha-synuclein (alpha-syn) have recently been linked to rare hereditary forms of Parkinson's disease . A yeast two-hybrid screen with alpha-synuclein (alpha-syn) identified synphilin as an alpha-syn-interacting protein, potentially implicating synphilin in the pathogenesis of synucleinopathies . Co-transfection of synphilin and the central (NAC) region of alpha-syn in HEH293 cells resulted in synuclein inclusions . Furthermore, synphilin immunoreactivity has been observed in Lewy bodies (LBs) and glial cytoplasmic inclusions of synucleinopathies . To further characterize synphilin, we utilized two new anti-synphilin antibodies for biochemical and immunohistochemical studies in normal and disease brain tissues . In normal brain tissue, synphilin localized predominantly to large neurons, such as substantia nigra neurons, hippocampal pyramidal and cerebellar Purkinje cells . However, in a few pathological cases synphilin immunoreactivity was present in glial cells and a small percentage of cortical and nigral LBs . In brain extracts, synphilin was observed primarily as a 90-kDa band but protein bands of 50 and 65 kDa were also present in both soluble (high salt) and lipid (Triton X-100) fractions . Additionally, less abundant higher molecular mass species, including a 120-kDa band of similar size to that of synphilin expressed in transiently transfected cells were recovered in 8 M urea-solubilized pellets after sequential extraction of brain tissue with buffers of increasing strengths . The presence of the synphilin of higher molecular mass was detected regardless of alpha-syn pathology and may represent an immature form of synphilin . Thus, although synphilin may be an alpha-syn-interacting protein present in some alpha-syn lesions, it still remains to be determined whether synphilin plays a critical role in mechanisms of brain degeneration in human synucleinopathies. Biochem Biophys Res Commun, 2003 Jan 31, 301(1), 71 - 7 A novel zinc finger protein, ZZaPK, interacts with ZAK and stimulates the ZAK-expressing cells re-entering the cell cycle; Yang JJ; ZAK has been implicated in cell cycle arrest regulation through its function on decreasing cyclin E expression . To explore the mechanistic basis for this regulation, the yeast two-hybrid system was used with a novel Kruppel-type C2H2 zinc finger member cloned . This cloned cDNA encodes a novel protein with Kruppel-type zinc fingers designed as ZZaPK (zinc finger and ZAK associated protein with KRAB domain) and is widely expressed . ZZaPK, when it is expressed in cells, is growth promoted and might lead to increasing E2F expression and induce cyclin E/CDK2 activity, which counteracts the ZAK function . The model proposed here is that ZAK might play a role as an upstream signal to suppress the ZZaPK function and decrease E2F expression. Mol Cell, 2003 Jan, 11(1), 151 - 61 A shared surface of TBP directs RNA polymerase II and III transcription via association with different TFIIB family members; Zhao X et al.; The TATA box binding protein TBP is highly conserved and the only known basal factor that is involved in transcription by all three eukaryotic nuclear RNA polymerases from promoters with or without a TATA box . By mutagenesis and analysis on a selected set of four model pol II and pol III TATA box-containing and TATA-less promoters, we demonstrate that human TBP utilizes two modes to achieve its versatile functions . First, it uses a different set of surfaces on the conserved and structured TBP core domain to direct transcription from each of the four model promoters . Second, unlike yeast TBP, human TBP can use a shared surface to interact with two different TFIIB family members--TFIIB and Brf2--to initiate transcription by different RNA polymerases. Mol Cell, 2003 Jan, 11(1), 1 - 3 A new connection: chaperones meet a mitochondrial receptor; Voos W; Cytosolic chaperones stabilize cellular proteins under stress conditions and protect nascent protein chains during normal growth . Recent data from Young et al . (2003) extend the function of chaperones by demonstrating that Hsp90 and Hsp70 specifically interact with the mitochondrial protein import receptor Tom70 at the outer membrane and are required for translocation of precursor proteins. Plant J, 2003 Jan, 33(2), 305 - 17 GeBP, the first member of a new gene family in Arabidopsis, encodes a nuclear protein with DNA-binding activity and is regulated by KNAT1; Curaba J et al.; Trichomes of Arabidopsis are single-celled epidermal hair that are a useful model for studying plant cell fate determination . Trichome initiation requires the activity of the GLABROUS1 (GL1) gene whose expression in epidermal and trichome cells is dependent on the presence of a 3'-cis-regulatory element . Using a one-hybrid screen, we have isolated a cDNA, which encodes for a protein, GL1 enhancer binding protein (GeBP), that binds this regulatory element in yeast and in vitro . GeBP and its three homologues in Arabidopsis share two regions: a central region with no known motifs and a C-terminal region with a putative leucine-zipper motif . We show that both regions are necessary for trans-activation in yeast . A translational fusion with the Yellow Fluorescent Protein (YFP) indicates that GeBP is a nuclear protein whose localization is restricted to, on average, 3-5 subnuclear foci that might correspond to nucleoli . Transcriptional fusion with the GUS reporter indicates that GeBP is mainly expressed in vegetative meristematic tissues and in very young leaf primordia . We looked at GeBP expression in plants mutated in or misexpressing KNAT1, a KNOX gene, expressed in the shoot apical meristem and downregulated in leaf founder cells, and found that GeBP transcript level is regulated by KNAT1 suggesting that KNAT1 is a transcriptional activator of GeBP . This regulation suggests that GeBP is acting as a repressor of leaf cell fate. Lett Appl Microbiol, 2003, 36(2), 69 - 72 Rapid reduction of Legionella pneumophila on stainless steel with zeolite coatings containing silver and zinc ions; Rusin P et al.; AIMS: To determine the rate of reduction of Legionella pneumophila by stainless steel surfaces with zeolite ceramic coatings containing 2.5% (w/w) silver (Ag) and 14% zinc (Zn) ions . METHODS AND RESULTS: Stainless steel pans with and without Ag/Zn coatings were inoculated with solutions of Leg . pneumophila ATCC 33155 and incubated at 37 degrees C . Survival was monitored using the spread-plate technique on selective buffered charcoal yeast extract agar . Significant reductions of Leg . pneumophila were effected by the Ag/Zn zeolite coatings within 2 h of exposure . CONCLUSIONS, Significance and Impact of the Study: Zeolite ceramic Ag/Zn coatings impart significant anti-Legionella properties to stainless steel surfaces . Coated stainless steel could be used in the manufacture of air ducts, condensation pans and intake and exhaust vents . These products have the potential to reduce numbers of Legionella in air-handling systems. J Appl Microbiol, 2003, 94(2), 330 - 9 Liquid formulation of the biocontrol agent Candida sake by modifying water activity or adding protectants; Torres R et al.; AIMS: To evaluate the effect of modification of water activity (aw) and the addition of protective substances in the preservation medium of liquid formulations of the biocontrol agent Candida sake stored at 4 and 20 degrees C . METHODS AND RESULTS: The aw of the preservation medium of C . sake was modified from 0.72 to 0.95 by adding glycerol or polyethylene glycol (PEG) . Moreover, several protectant substances at different concentrations were evaluated . Modification of lower aw-levels (0.721-0.901) with glycerol did not maintain the viability of the yeast cells . Higher aw-levels (0.93-0.95) with either glycerol or PEG improved the viability but not at acceptable viability levels . C . sake cells maintained viabilities >60% when sugars, such as trehalose, and polyols, such as glycerol and PEG were used as protectants in liquid formulations . Moreover, liquid formulations of C . sake stored at 4 degrees C showed higher number of viable counts than at 20 degrees C . When different sugars were tested, all of them, except 10% fructose, resulted in a viability higher than 50% of the C . sake formulations . Biocontrol of liquid formulation treatments was similar to fresh cells in controlling Penicillium expansum on wounded apples . CONCLUSIONS: Sugars such as lactose and trehalose could be considered as good protectants in order to obtain liquid formulations of C . sake cells as they maintain the viability >70% for 4 months at 4 degrees C . SIGNIFICANCE AND IMPACT OF STUDY: This study shows that a suitable liquid formulation for commercial application can be produced with high viability and conservation of biocontrol efficacy . Moreover, if 10% lactose is the protectant used in the formulation, the economic costs would not be limiting for industrial production. Tsitologiia, 2002, 44(9), 830 - 8 {A new human cellular protein AUP1 . I . In vitro interaction of AUP1 with adenoviral proteins E4ORF3 and E1A}; Karpisheva KV et al.; The 11-kDa product of adenovirus early region 4 (E4) open reading frame (ORF) 3 participates in many processes occurring in infected cell, including post-transcriptional steps in late viral gene expression and viral DNA synthesis . In addition, E4ORF3 from adenovirus type 5 (Ad5) displays the features of a viral oncoprotein . It initiates focal transformation of primary rat cells in cooperation with Ad5 El genes and confers multiple additional transformed properties on E1-expressing cells . Biochemical details of E4ORF3 activities in these processes are not well understood . A large body of evidence indicates that its lytic and transforming functions are mediated by physical interactions with viral and cellular components involved in DNA transcription and repair, as well as by host cell factors that regulate the integrity of nuclear multiprotein complexes known as PML oncogenic domains (PODs) . In this study we have employed the two-hybrid screen in yeast to isolate human cDNAs encoding for E4ORF3-interacting proteins . Among 15 positive clones five cDNAs encode for a cellular protein called AUP1 . In vitro-binding assays demonstrated that AUP1 fused to glutathione S-transferase (GST) specifically binds to E4ORF3 from Ad5, Ad9 and Ad40 generated in a coupled transcription-translation system, whereas no interactions was observed with ORF3 from Ad12 . Interestingly, GST-AUP1 interacted also specifically with in vitro translated Ad5 E1A proteins . Regions involved in the Ad5 E4ORF3/AUP1 interaction in vitro map to the central part of E4 protein and the carboxy-terminal region of AUP1, while E1A binds to an amino-terminal segment of the cell protein . Taken together, these studies indicate that AUP1 may represent a cellular target of both adenovirus E4ORF3 and E1A proteins . Additional studies are currently under way to confirm the significance of these interactions in living cells in vivo. Bull Exp Biol Med, 2002 Oct, 134(4), 366 - 9 Photoprotective activity of melanin preparations in human skin exposed to UV irradiation: dependence on previous photoexposure; Paramonov BA et al.; Photoprotective effects of three melanin preparations (from black yeast fungi and Sepia sp.) were studied . These preparations in aqueous solutions (5 g/ml, dark exposure for 7 days) demonstrated high photomodification capacity upon exposure to visible light in doses of up to 1.8 kJ/m2 . Preliminary exposure of these solutions to visible light in a dose of 360 kJ/m2 notably decreased the photoprotective effect of melanins during UV exposure of the skin treated with these solutions (at UV dose of 3.4 kJ/m2) . This necessitates empirical selection of the dose and storage condition of melanin preparations for attaining the optimal photoprotective effect. J Gen Virol, 2003 Jan, 84(Pt 1), 193 - 202 Virus-cell interactions in the induction of type 1 interferon by influenza virus in mouse spleen cells; Miller JL et al.; Inactivated influenza A virus and fixed, virus-infected cells induce type 1 interferon (IFN-alpha/beta) production in murine splenocytes . In this study, we have explored the nature of the virus-spleen cell interaction that leads to IFN-alpha/beta induction and the reason for the poor response to some virus strains . IFN-alpha/beta induction by horse serum-sensitive, but not -resistant, strains of influenza virus was inhibited in the presence of horse serum, indicating that binding of the virus to sialylated cell receptors is a necessary step in the induction process . Furthermore, influenza viruses A/PR/8/34 (H1N1) and A/WS/33 (H1N1), which were poor inducers of IFN-alpha/beta in spleen cells, were shown to have a more active neuraminidase than strains that induced higher IFN levels, and IFN-alpha/beta induction by A/PR/8/34 (H1N1) and A/WS/33 (H1N1) was restored in the presence of a neuraminidase inhibitor . Growth of virus in different cell types altered the level of IFN-alpha/beta induced in spleen cells by particular virus strains, suggesting that the nature of the carbohydrate moieties on the viral glycoproteins may also influence IFN-alpha/beta induction in this system . Consistent with this notion, treatment of egg-grown virus with periodate to oxidize viral carbohydrate greatly reduced its capacity for IFN-alpha/beta induction . Furthermore, induction of IFN-alpha/beta was inhibited in the presence of the saccharides yeast mannan and laminarin . Together these findings indicate: (i) a requirement for interaction of the virus with sialylated receptors on the IFN-producing cell; (ii) an influence of viral carbohydrate on the response; and (iii) possible involvement of a lectin-like receptor on the IFN-producing cell in the induction of IFN-alpha/beta or in regulation of this response. J Neurosci, 2003 Jan 15, 23(2), 403 - 15 Modulation of type 1 inositol (1,4,5)-trisphosphate receptor function by protein kinase a and protein phosphatase 1alpha; Tang TS et al.; Type 1 inositol (1,4,5)-trisphosphate receptors (InsP3R1s) play a major role in neuronal calcium (Ca2+) signaling . The InsP3R1s are phosphorylated by protein kinase A (PKA), but the functional consequences of InsP3R1 phosphorylation and the mechanisms that control the phosphorylated state of neuronal InsP3R1s are poorly understood . In a yeast two-hybrid screen of rat brain cDNA library with the InsP3R1-specific bait, we isolated the protein phosphatase 1alpha (PP1alpha) . In biochemical experiments, we confirmed the specificity of the InsP3R1-PP1alpha association and immunoprecipitated the InsP3R1-PP1 complex from rat brain synaptosomes and from the neostriatal lysate . We also established that the association with PP1 facilitates dephosphorylation of PKA-phosphorylated InsP3R1 by the endogenous neostriatal PP1 and by the recombinant PP1alpaha . We demonstrated that exposure of neostriatal slices to 8-bromo-cAMP, dopamine, calyculin A, or cyclosporine A, but not to 10 nM okadaic acid, promotes the phosphorylation of neostriatal InsP3R1 by PKA in vivo . We discovered that PKA activates and PP1alpha inhibits the activity of recombinant InsP3R1 reconstituted into planar lipid bilayers . We found that phosphorylation of InsP3R1 by PKA induces at least a fourfold increase in the sensitivity of InsP3R1 to activation by InsP3 without shifting the peak of InsP3R1 bell-shaped Ca2+ dependence . Based on these data, we suggest that InsP3R1 may participate in cross talk between cAMP and Ca2+ signaling in the neostriatum and possibly in other regions of the brain. Biol Reprod, 2003 Feb, 68(2), 543 - 52 Novel RING finger protein OIP1 binds to conserved amino acid repeats in sperm tail protein ODF1; Zarsky HA et al.; Outer dense fibers (ODFs) and the fibrous sheath (FS) are unique structures of the mammalian sperm tail . Recently, progress has been made in the molecular cloning of ODF and FS proteins, and because of this, questions addressing the morphogenesis and underlying protein network that make up sperm tail structures and their function can now be addressed . Using the N-terminal leucine zipper motif of the major ODF protein ODF1, we had previously isolated interacting proteins Odf2, Spag4, and Spag5 . We report here a yeast two-hybrid strategy to isolate a novel rat testicular protein, OIP1, that binds to the evolutionarily conserved Cys-Gly-Pro repeats in the C-terminus of ODF1 . OIP1 is expressed in round spermatids as well as in spermatocytes and several somatic tissues, albeit at a lower level . No expression was detectable in epididymis, heart, and smooth muscle . OIP1 protein localizes to the sperm tail in a pattern expected for an ODF1-interacting protein . OIP1 belongs to the family of RING finger proteins of the H2 subclass . Deletion of the putative RING motif significantly decreased binding to ODF1 . Genomic analysis of rat Oip1 and Oip1 homologs indicates that Oip1 is highly conserved . Oip1 is subject to differential splicing and alternative polyadenylation events . It is interesting that Oip1 mRNAs have been reported that lack the exon encoding the putative RING finger. Sheng Li Ke Xue Jin Zhan, 2000 Jan, 31(1), 35 - 42 {A brief introduction to the methods for novel gene cloning}; Sun CX et al.; There are a lot of methods for novel gene cloning, but how to clone candidate gene(s) quickly and correctly? This is a brief introduction to methods of novel gene cloning, these methods includes: differential display reverse transcriptase polymerase chain reaction(DD RT-PCR), suppression subtractive hybridization(SSH), RNA arbitrarily primed PCR(RAP-PCR), representational difference analysis(RDA), yeast two-hybrid system, cDNA capturation, et al . We not only introduced these methods, but also discussed the advantages and disadvantages of them . However, no single method is omnipotent, one should pick up the method most suitable for a special purpose. World J Gastroenterol, 2003 Feb, 9(2), 300 - 3 Interaction between hepatitis C virus core protein and translin protein--a possible molecular mechanism for hepatocellular carcinoma and lymphoma caused by hepatitis C virus; Li K et al.; AIM: To investigate the interaction between hepatitis C virus core protein and translin protein and its role in the pathogenensis of hepatocellular carcinoma and lymphoma . METHODS: With the components of the yeast two hybrid system 3, "bait" plasmids of HCV core the gene was constructed . After proving that hepatitis C virus core protein could be firmly expressed in AH109 yeast strains, yeast two- hybrid screening was performed by mating AH109 with Y187 that transformed with liver cDNA library plasmids-pACT2 and then plated on quadruple dropout (QDO) medium and then assayed for alpha-gal activity . Sequencing analysis of the genes of library plasmids in yeast colonies that could grow on QDO with alpha-gal activity was performed . The interaction between HCV core protein and the protein we obtained from positive colony was further confirmed by repeating yeast two - hybrid analysis and coimmunoprecipitation in vitro . RESULTS: A gene from a positive colony was the gene of translin, a recombination hotspot binding protein . The interaction between HCV core protein and translin protein could be proved not only in yeast, but also in vitro . CONCLUSION: The core protein of HCV can interact with translin protein . This can partly explain the molecular mechanism for hepatocellular carcinoma and lymphoma caused by HCV. J Cell Biochem, 2003 Feb 15, 88(3), 557 - 68 The E2F-1 transcription factor is negatively regulated by its interaction with the MDMX protein; Strachan GD et al.; Several proteins with important roles in oncogenesis have been shown to regulate the function of the E2F-1 transcription factor, which is known to activate the expression of genes required for proliferation and apoptosis . Here we identify the MDMX oncoprotein as an E2F-1-binding factor, from a yeast-two hybrid screen using a portion of the E2F-1 protein as "bait." We demonstrate that the region within MDMX needed for the E2F-1:MDMX interaction is located in the central part of the protein, C-terminal of the p53-binding domain . The region within E2F-1 needed for this association is adjacent to the DNA binding domain . Further, when expressed in vivo or in vitro the MDMX protein migrates as two isoforms on SDS-PAGE, the faster migrating isoform having the stronger affinity for the E2F-1 proteins . It appears that this interaction reduces the ability of E2F-1 to bind DNA . Expression of MDMX along with E2F-1 and Dp-1 in Saos2 cells reduces the ability of E2F-1 to bind to its consensus DNA sequence, without altering E2F-1 protein levels . These data indicate that the MDMX protein is capable of associating with E2F-1 and negatively regulating its DNA binding ability . Mamm Genome, 2003 Jan, 14(1), 1 - 6 Characterization of the mouse genes for mitochondrial transcription factors B1 and B2; Rantanen A et al.; We have recently fully reconstituted the basal human mitochondrial transcription machinery in a pure in vitro system . Surprisingly, we found two different transcription factors (TFB1M and TFB2M) that each interact with mitochondrial RNA polymerase in human mitochondria, whereas there is only one such factor in budding yeast mitochondria . This unexpected finding raised important questions concerning the regulation of mitochondrial transcription in mammals in general and in other metazoans . We have now further analyzed putative homologs to TFB1M and TFB2M in different species . We mapped the mouse homologs, Tfb1m and Tfb2m, by linkage analysis to mouse Chr 17 and Chr 1, respectively . These regions display conserved linkage synteny with human Chr 6 and Chr 1, where TFB1M and TFB2M map . The intron-exon arrangements of Tfb1m and TFB1M and of Tfb2m and TFB2M were identical, and the promoter regions had similar predicted recognition elements for transcriptional factors NRF2 and Sp1 . Northern blot analyses showed that Tfb1m and Tfb2m were ubiquitously expressed and had expression patterns that were very similar to the previously reported expression patterns for TFB1M and TFB2M . These findings show that Tfb1m and Tfb2m indeed are orthologs to TFB1M and TFB2M . Bioinformatic analyses indicated that most metazoans have two TFBM genes, since putative homologs to both TFB1M and TFB2M were found in D . melanogaster . Our data thus suggest that a duplication event of the TFBM gene in early metazoan evolution has permitted a more flexible regulation of mtDNA transcription, possibly in response to the complex physiological demands of multicellular organisms. J Dermatol, 2002 Dec, 29(12), 797 - 802 A case of primary cutaneous histoplasmosis in a patient with diabetes and multi-infarct dementia; Krunic AL et al.; We report a case of primary cutaneous histoplasmosis in a fifty-year-old African-American woman with diabetes and multi-infarct dementia . The patient developed fever and crusted, nodulo-ulcerative lesions of the skin after accidental superficial trauma to the forehead . The biopsy revealed suppurative granulomatous inflammation with intracellular and extracellular yeast-like cells with associated clear halo measuring 3-4 mm in size . Systemic involvement was not found . The lesions cleared after treatment with itraconazole 200 mg twice a day for 3 weeks . The medication was continued for a total period of 3 months, with no signs of recurrence after one-year of follow-up. Anal Chem, 2003 Jan 1, 75(1), 42 - 8 Affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase for the multiplex analysis of congenital disorders of glycosylation types Ia and Ib; Li Y et al.; We report a new application of affinity capture-elution electrospray mass spectrometry (ACESI-MS) to assay the enzymes phosphomannomutase (PMM) and phosphomannose isomerase (PMI), which when deficient cause congenital disorders of glycosylation CDG-type Ia and type Ib, respectively . The novel feature of this mass-spectrometry-based assay is that it allows one to distinguish and quantify enzymatic products that are isomeric with their substrates that are present simultaneously in complex mixtures, such as cultured human cell homogenates . This is achieved by coupled assays in which the PMM and PMI primary products are in vitro subjected to another enzymatic reaction with yeast transketolase that changes the mass of the products to be detected by mass spectrometry . The affinity purification procedure is fully automated, and the mass spectrometric analysis is multiplexed in a fashion that is suitable for high-throughput applications. Endocr Res, 2002 Nov, 28(4), 505 - 13 Differential regulation of SF-1-cofactor interactions; Lund J et al.; The orphan nuclear receptor steroidogenic factor-1 (SF-1) plays pivotal roles in the development and function of steroidogenic organs . Here we describe the differential effect of protein kinase A (PKA) on coregulation of SF-1 dependent transcription by two p160 family members, p300/CBP co-integrator-associated protein (p/CIP) and transcription intermediary factor-2 (TIF2) . Thus, whereas p/CIP-stimulated SF-1 dependent transcription is further potentiated by PKA, we show that activation of PKA leads to selective downregulation of TIF2 protein and a subsequent repression of TIF2 coactivator function . Using a yeast two-hybrid screen we also identified a novel zinc finger containing protein, which interacts with SF-1 via the AF-2 domain. Insect Biochem Mol Biol, 2002 Nov, 32(11), 1507 - 16 Expression and characterization of a novel teratocyte protein of the braconid, Microplitis croceipes (cresson); Rana RL et al.; Microplitis croceipes wasps overcome host immunity by inducing changes in host physiology using factors derived from the embryo and/or larva . Teratocytes of some parasitic wasps circulate in the host hemolymph after egg hatch and synthesize proteins (TSPs), some of which are secreted to alter host physiology in support of endoparasitoid development . TSPs appear to alter host physiology, at least in part, by inhibiting synthesis of certain proteins . M . croceipes teratocytes synthesize a 13.9 kDa protein (TSP14), which inhibits synthesis of host proteins that are linked to larval growth and development . A cDNA encoding TSP14 was generated by RT-PCR from teratocyte RNA, and cloned into yeast expression vectors to produce sufficient recombinant protein for functional analyses . RecTSP14 was produced using the yeast expression system at a concentration of more than 300 micrograms/L . The recTSP14 inhibited in vitro translation of larval Heliothis virescens RNA, with the activity sensitive to boiling, protein concentration, incubation time, and storage temperatures . Although recTSP14 inhibited translation of some cellular RNAs in vitro, the in vivo incorporation of {35S}-methionine into proteins of selected insect and mammalian cell lines was not inhibited . These findings suggest that recTSP14 entry is cell type-specific and required to inhibit synthesis of target protein(s). Plant Physiol, 2003 Jan, 131(1), 27 - 40 Identification and characterization of the ARIADNE gene family in Arabidopsis . A group of putative E3 ligases; Mladek C et al.; ARIADNE (ARI) proteins were recently identified in fruitfly (Drosophila melanogaster), mouse, and man because of their specific interaction with the ubiquitin-conjugating (E2) enzymes UbcD10, UbcM4, UbcH7, and UbcH8 . They are characterized by specific motifs and protein structures that they share with PARKIN, and there is increasing evidence that ARI/PARKIN proteins function as E2-dependent ubiquitin-protein ligases . On the basis of homology and motif searches, 16 AtARI genes were identified in Arabidopsis . Analysis of the position of exons/introns and their chromosomal localization indicates that the AtARI gene family expanded via larger and smaller genome duplications . We present evidence that retroposition of processed mRNA may have also contributed to enlarging this gene family . Phylogenetic analyses divides the AtARI proteins into three subgroups . Two groups are absent in yeast, invertebrates, and vertebrates and may therefore represent new plant-specific subfamilies . Examination of the predicted protein sequences revealed that the ARI proteins share an additional leucine-rich region at the N terminus that is highly conserved in all phyla analyzed . Furthermore, conserved consensus signals for casein kinase II-dependent phosphorylation and for nuclear localization were identified . The in silico-based analyses were complemented with experimental data to quantify expression levels . Using real-time polymerase chain reaction, we show that the ARI genes are differentially transcribed . AtARI1 is highly expressed in all organs, whereas no transcripts could be detected for AtARI11, AtARI13, and AtARI14 . AtARI12 and AtARI16 are expressed in an organ-specific manner in the roots and siliques, respectively. Mol Biol Cell, 2003 Jan, 14(1), 262 - 73 SVIP is a novel VCP/p97-interacting protein whose expression causes cell vacuolation; Nagahama M et al.; VCP/p97 is involved in a variety of cellular processes, including membrane fusion and ubiquitin-dependent protein degradation . It has been suggested that adaptor proteins such as p47 and Ufd1p confer functional versatility to VCP/p97 . To identify novel adaptors, we searched for proteins that interact specifically with VCP/p97 by using the yeast two-hybrid system, and discovered a novel VCP/p97-interacting protein named small VCP/p97-interacting protein (SVIP) . Rat SVIP is a 76-amino acid protein that contains two putative coiled-coil regions, and potential myristoylation and palmitoylation sites at the N terminus . Binding experiments revealed that the N-terminal coiled-coil region of SVIP, and the N-terminal and subsequent ATP-binding regions (ND1 domain) of VCP/p97, interact with each other . SVIP and previously identified adaptors p47 and ufd1p interact with VCP/p97 in a mutually exclusive manner . Overexpression of full-length SVIP or a truncated mutant did not markedly affect the structure of the Golgi apparatus, but caused extensive cell vacuolation reminiscent of that seen upon the expression of VCP/p97 mutants or polyglutamine proteins in neuronal cells . The vacuoles seemed to be derived from endoplasmic reticulum membranes . These results together suggest that SVIP is a novel VCP/p97 adaptor whose function is related to the integrity of the endoplasmic reticulum. Mol Biol Cell, 2003 Jan, 14(1), 190 - 200 The Drosophila Cog5 homologue is required for cytokinesis, cell elongation, and assembly of specialized Golgi architecture during spermatogenesis; Farkas RM et al.; The multisubunit conserved oligomeric Golgi (COG) complex has been shown previously to be involved in Golgi function in yeast and mammalian tissue culture cells . Despite this broad conservation, several subunits, including Cog5, were not essential for growth and showed only mild effects on secretion when mutated in yeast, raising questions about what functions these COG complex subunits play in the life of the cell . Here, we show that function of the gene four way stop (fws), which encodes the Drosophila Cog5 homologue, is necessary for dramatic changes in cellular and subcellular morphology during spermatogenesis . Loss-of-function mutations in fws caused failure of cleavage furrow ingression in dividing spermatocytes and failure of cell elongation in differentiating spermatids and disrupted the formation and/or stability of the Golgi-based spermatid acroblast . Consistent with the lack of a growth defect in yeast lacking Cog5, animals lacking fws function were viable, although males were sterile . Fws protein localized to Golgi structures throughout spermatogenesis . We propose that Fws may directly or indirectly facilitate efficient vesicle traffic through the Golgi to support rapid and extensive increases in cell surface area during spermatocyte cytokinesis and polarized elongation of differentiating spermatids . Our study suggests that Drosophila spermatogenesis can be an effective sensitized genetic system to uncover in vivo functions for proteins involved in Golgi architecture and/or vesicle transport. Mol Cell Biol, 2003 Feb, 23(3), 1004 - 13 Transcriptional coactivation of bone-specific transcription factor Cbfa1 by TAZ; Cui CB et al.; Core-binding factor 1 (Cbfa1; also called Runx2) is a transcription factor belonging to the Runt family of transcription factors that binds to an osteoblast-specific cis-acting element (OSE2) activating the expression of osteocalcin, an osteoblast-specific gene . Using the yeast two-hybrid system, we identified a transcriptional coactivator, TAZ (transcriptional coactivator with PDZ-binding motif), that binds to Cbfa1 . A functional relationship between Cbfa1 and TAZ is demonstrated by the coimmunoprecipitation of TAZ by Cbfa1 and by the fact that TAZ induces a dose-dependent increase in the activity of osteocalcin promoter-luciferase constructs by Cbfa1 . A dominant-negative construct of TAZ in which the coactivation domains have been deleted reduces osteocalcin gene expression down to basal levels . NIH 3T3, MC 3T3, and ROS 17/2.8 cells showed the expected nuclear localization of Cbfa1, whereas TAZ was distributed throughout the cytoplasm with some nuclear localization when transfected with either Cbfa1 or TAZ . Upon cotransfection by both Cbfa1 and TAZ, the transfected TAZ shows predominant nuclear localization . The dominant-negative construct of TAZ shows minimal nuclear localization upon cotransfection with Cbfa1 . These data indicate that TAZ is a transcription coactivator for Cbfa1 and may be involved in the regulation of osteoblast differentiation. Mol Cell Biol, 2003 Feb, 23(3), 908 - 15 Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality; Buchou T et al.; Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival . Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases . In this work, we show that knockout of the CK2beta gene in mice leads to postimplantation lethality . Mutant embryos were reduced in size at embryonic day 6.5 (E6.5) . They did not exhibit signs of apoptosis but did show reduced cell proliferation . Mutant embryos were resorbed at E7.5 . In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage . Attempts to generate homozygous embryonic stem (ES) cells failed . By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts . This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta . Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution. J Biol Chem, 2003 Apr 18, 278(16), 14461 - 8 Epub 2003 Jan 15. Uncleaved BAP31 in association with A4 protein at the endoplasmic reticulum is an inhibitor of Fas-initiated release of cytochrome c from mitochondria; Wang B et al.; BAP31 is a polytopic integral protein of the endoplasmic reticulum membrane and, like BID, is a preferred substrate of caspase-8 . Upon Fas/CD95 stimulation, BAP31 is cleaved within its cytosolic domain, generating proapoptotic p20 BAP31 . In human KB epithelial cells expressing the caspase-resistant mutant crBAP31, Fas stimulation resulted in cleavage of BID and insertion of BAX into mitochondrial membrane, but subsequent oligomerization of BAX and BAK, egress of cytochrome c to the cytosol, and apoptosis were impaired . Bap31-null mouse cells expressing crBAP31 cannot generate the endogenous p20 BAP31 cleavage product, yet crBAP31 conferred resistance to cellular condensation and cytochrome c release in response to activation of ectopic FKBPcasp8 by FK1012z . Full-length BAP31, therefore, is a direct inhibitor of these caspase-8-initiated events, acting independently of its ability to sequester p20, with which it interacts . Employing a novel split ubiquitin yeast two-hybrid screen for BAP31-interacting membrane proteins, the putative ion channel protein of the endoplasmic reticulum, A4, was detected and identified as a constitutive binding partner of BAP31 in human cells . Ectopic A4 that was introduced into A4-deficient cells cooperated with crBAP31 to resist Fas-induced egress of cytochrome c from mitochondria and cytoplasmic apoptosis. J Biol Chem, 2003 Mar 28, 278(13), 11633 - 41 Epub 2003 Jan 15. The N-terminal end of Bax contains a mitochondrial-targeting signal; Cartron PF et al.; The translocation of Bax alpha, a pro-apoptotic member of the BCL-2 family from the cytosol to mitochondria, is a central event of the apoptotic program . We report here that the N-terminal (NT) end of Bax alpha, which contains its first alpha helix (Eta alpha 1), is a functional mitochondrial-addressing signal both in mammals and in yeast . Similar results were obtained with a newly described variant of Bax called Bax psi, which lacks the first 20 amino acids of Bax alpha and is constitutively associated with mitochondria . Deletion of Eta alpha 1 impairs the binding of Bax psi to mitochondria, whereas a fusion of the N terminus of Bax alpha, which contains Eta alpha 1 with a cytosolic protein, results in the binding of the chimeric proteins to mitochondria both in a cell-free assay and in vitro . More importantly, the mitochondria-bound chimeric proteins inhibit the interaction of Bax psi with mitochondria as well as Bax-apoptogenic properties . The mutations of the Eta alpha 1, which inhibit Bax alpha and Bax psi translocation to mitochondria, also block the subsequent activation of the execution phase of apoptosis . Conversely, a deletion of the C terminus does not appear to influence Bax alpha and Bax psi mitochondrial addressing . Taken together, our results suggest that Bax is targeted to mitochondria by its NT and thus through a pathway that is unique for a member of the BCL-2 family. J Biol Chem, 2003 Mar 21, 278(12), 10374 - 80 Epub 2003 Jan 15. p21-activated protein kinase 4 (PAK4) interacts with the keratinocyte growth factor receptor and participates in keratinocyte growth factor-mediated inhibition of oxidant-induced cell death; Lu Y et al.; Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family (also known as FGF-7), is an important protective factor for epithelial cells . The receptor for KGF (also called FGFR2-IIIb), which has intrinsic tyrosine kinase activity, is expressed specifically on epithelial cells and in the lung epithelium . Administration of KGF has been shown to protect the lung from various insults, but the mechanism of protection is not well understood . To understand the mechanism by which KGF exerts protective functions on epithelial cells, we used the yeast two-hybrid assay to identify proteins that interact with the KGF receptor (KGFR) . Here we show that the cytoplasmic domain of KGFR interacts with p21-activated protein kinase (PAK) 4, which is a new member of the PAK family . The PAKs are regulated by the Rho-family GTPases Rac and Cdc42 . PAK4 is the most divergent member of the PAK family of proteins and may have distinct functions . However, stimuli that regulate PAK4 activity are not known . Our data show that PAK4 can associate with the KGFR, which is dependent on KGFR tyrosine kinase activity . We show that a dominant negative mutant of PAK4 blocks KGF-mediated inhibition of caspase-3 activation in epithelial cells subjected to oxidant stress . Our data demonstrate that PAK4 is an important mediator of the anti-apoptotic effects of KGF on epithelial cells. Lupus, 2002, 11(12), 776 - 9 Anti-DNA antibodies--structure and function; Rahman A et al.; Expression of monoclonal anti-DNA antibodies in vitro can be used to study the relationships between molecular structure, binding properties and pathogenicity . Bacterial and yeast systems can be used to produce antibody fragments such as Fab . The yields are potentially sufficient to allow structural studies such as crystallization, but purification of the anti-DNA Fab from the bacterial periplasm may be challenging . Mammalian cell expression systems produce lower yields, but the products are whole antibodies, which can be used in assays of pathogenicity . This article describes some recent experiments in which bacterial and mammalian systems were used to study human monoclonal anti-DNA antibodies . Light chain sequence motifs were found to be important both in binding to antigens and in determining pathogenicity of the antibodies in severe combined immunodeficiency mice . The distribution of B cell subpopulations is disturbed in patients with systemic lupus erythematosus (SLE) . These patients, like those with infectious mononucleosis, have an overall B cell lymphopenia but an increased frequency of plasmablasts/early plasma cells in their blood . Some of these early plasma cells belong to clones that have rearranged the V(H) gene V4-34 . There is a selective rise in immunoglobulins encoded by this gene in both infectious mononucleosis and SLE. Can J Vet Res, 2003 Jan, 67(1), 56 - 9 Evaluation of the effect of pH on in vitro growth of Malassezia pachydermatis; Matousek JL et al.; The purpose of this study was to evaluate the effects of pH on the growth of canine Malassezia pachydermatis isolates in vitro . Yeast growth was monitored by measuring the optical density with a spectrophotometer . The growth of American Type Culture Collection and field strains of M . pachydermatis was optimal between the pH values of 4.0 and 8.0, and inhibited at the ranges of 1.0 to 3.0 and 9.0 to 10.0 . An analysis of covariance showed no significant differences among the growth curves at pH levels 5.0 to 8.0 . Although specific contrast tests showed that the growth slope at pH 4.0 was significantly different from that at pH 5.0 to 8.0, only small, random differences were found when the growth slope at pH 4.0 was compared to the individual slopes at pH 5.0, 6.0, 7.0, and 8.0 . The findings of this study suggest that topical acidifying products could be beneficial therapeutic options for cutaneous yeast infections in dogs. Oncogene, 2003 Jan 9, 22(1), 28 - 33 Interaction between BRCA2 and replication protein A is compromised by a cancer-predisposing mutation in BRCA2; Wong JM et al.; Mutations in the BRCA1 and BRCA2 genes predispose women to familial, early-onset breast cancer . Both the BRCA1 and BRCA2 proteins appear to function in the homologous recombination pathway of DNA double-strand break repair . Both BRCA1 and BRCA2 have also been implicated in transcription by RNA polymerase II, for both proteins have domains which, when tethered adjacent to a promoter, can activate transcription . In experiments reported here, we have used protein affinity chromatography and coimmunoprecipitation techniques to show that the putative N-terminal acidic transcriptional activation domain of BRCA2 interacts with replication protein A (RPA), a protein essential for DNA repair, replication and recombination . This interaction was not mediated by DNA and was specific for human RPA but not yeast RPA . Since the cancer-predisposing mutation Y42C in BRCA2 significantly compromised the interaction between RPA and BRCA2, this interaction may be biologically important . That BRCA2 protein in HeLa cell extract also coimmunoprecipitated with RPA suggested that this interaction occurs in vivo . Therefore, the transcriptional activation domains within BRCA2, and perhaps BRCA1, may provide links to RPA and DNA repair processes rather than transcription. J Biol Chem, 2003 Mar 28, 278(13), 11676 - 85 Epub 2003 Jan 13. MTA1 interacts with MAT1, a cyclin-dependent kinase-activating kinase complex ring finger factor, and regulates estrogen receptor transactivation functions; Talukder AH et al.; The transcriptional activity of estrogen receptor-alpha is controlled by coregulators . MTA1 (metastasis-associated protein 1) represses estrogen receptor-alpha-driven transcription by recruiting histone deacetylases (HDACs) to the estrogen response element containing target gene chromatin in breast cancer cells . Using a yeast two-hybrid screen with the MTA1 C-terminal domain as bait, we identified MAT1 (menage a trois 1) as an MTA1-binding protein . MAT1 is an assembly/targeting factor for cyclin-dependent kinase-activating kinase (CAK), which has been shown to functionally interact with general transcriptional factor TFIIH, a known inducer of ER transactivation . We show that estrogen signaling promotes nuclear translocation of MAT1 and that MTA1 interacts with MAT1 both in vitro and in vivo . MAT1 binds to the C-terminal 389-441 amino acids GATA domain and N-terminal 1-164 amino acids bromo-domain of MTA1, whereas MTA1 binds to the N-terminal ring finger domain of the MAT1 . In addition, MAT1 interacts with the activation function 2 domain of ER and colocalizes with ER in activated cells . MTA1 deregulation in breast cancer cells led to its interactions with the CAK complex components, ER, and HDAC2 . Accordingly, MTA1 inhibited CAK stimulation of ER transactivation that was partially relieved by HDAC inhibitor trichostatin A, suggesting that MTA1 might inhibit CAK-induced transactivation function of ER by recruiting HDAC . Furthermore, MTA1 overexpression inhibited the ability of CAK complex to phosphorylate ER . Together, these findings identified MAT1 as a target of MTA1 and provided new evidence to suggest that the transactivation functions of ER might be influenced by the regulatory interactions between CAK and MTA1 in breast cancer cells. J Cell Biol, 2003 Jan 20, 160(2), 245 - 53 Epub 2003 Jan 13. Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles; Bagnato P et al.; Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells . The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known . We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin . Obscurin is a giant sarcomeric protein of approximately 800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures . The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells . In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin . The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells . The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils.In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum . Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin . Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum. Di Yi Jun Yi Da Xue Xue Bao, 2003 Jan, 23(1), 30 - 3 {Inhibitory effect of recombinant human endostatin on angiogenesis and lung metastasis of mouse lung adenocarcinoma LA795}; Xia H et al.; OBJECTIVE: To study the inhibitory effect of recombinant human endostatin (rhES) on the angiogenesis and lung metastasis of mouse lung adenocarcinoma LA795 . METHODS: The recombinant yeast strain containing the gene sequence encoding highly soluble rhES was induced by methanol for rhES production, which was purified with heparin affinity chromatography . T739 mice with subcutaneous inoculation of LA795 cells were randomized into 2 groups (10 in each group) to receive injection of either rhES (20 mg/kg x b x w x per day) or PBS in the same volume for 14 consecutive days starting from the sixth day after the inoculation . The angiogenesis and lung metastasis of the implanted tumors were subsequently observed . RESULTS: Purified rhES was successfully obtained . As shown by immunohistochemistry, the tumors in the mice receiving rhES treatment exhibited less density of the microvessels than those in the PBS-treated mice did (P<0.01) . Pathological examination of the lung tissue of the mice in rhES group found no visible signs of tumor metastasis, which, in contrast, was widespread in PBS group . The weight of the lungs was also significantly different (P<0.01) . CONCLUSION: rhES possesses good biological properties and can potently inhibit the angiogenesis and lung metastasis of mouse lung adenocarcinoma LA795. FEBS Lett, 2003 Jan 16, 534(1-3), 133 - 8 Distinct roles of the Src family kinases, SRC-1 and KIN-22, that are negatively regulated by CSK-1 in C . elegans; Hirose T et al.; To elucidate the primitive roles of the Src family kinases (SFKs), here we characterized Caenorhabditis elegans orthologues of SFKs (src-1 and kin-22) and their regulator kinase Csk (csk-1) . SRC-1 and KIN-22 possess the C-terminal regulatory tyrosines characteristic of SFKs, and their activities are negatively regulated by CSK-1 in a yeast expression system . The src-1 and csk-1 genes are co-expressed in some head neurons, the anchor cell and the tail region, while kin-22 and csk-1 genes are co-expressed in pharyngeal muscles and tail region . Expression of KIN-22 induced morphological defects in the pharynx, whereas expression of SRC-1 did not show any overt phenotype in adult . RNA interference of src-1, but not that of kin-22, caused a developmental arrest in early development . These results suggest that SRC-1 and KIN-22 play distinct roles under the control of CSK-1. FEBS Lett, 2003 Jan 16, 534(1-3), 26 - 32 Photoreceptor synaptic protein HRG4 (UNC119) interacts with ARL2 via a putative conserved domain; Kobayashi A et al.; Human retinal gene 4 (HRG4) (UNC119) is a photoreceptor synaptic protein of unknown function, shown when mutated to cause retinal degeneration in a patient and in a confirmatory transgenic model . ADP-ribosylation factor-like protein 2 (ARL2) was identified as an interactor of HRG4 by the yeast two-hybrid strategy . The presence of ARL2 in the retina and co-localization with HRG4 was confirmed by Western blot and double immunofluorescence analysis, respectively . The interaction of ARL2 with HRG4 was further confirmed by co-immunoprecipitation and direct binding analysis . Phosphodiesterase delta (PDEdelta) is an ARL2-binding protein homologous to HRG4 . Amino acid residues of PDEdelta involved in binding ARL2 and forming a hydrophobic pocket were shown to be highly conserved in HRG4, suggesting similarity in binding mechanism and function. J Mol Biol, 2003 Jan 31, 325(5), 963 - 77 Reconstructing the binding site of factor Xa in trypsin reveals ligand-induced structural plasticity; Reyda S et al.; In order to investigate issues of selectivity and specificity in protein-ligand interactions, we have undertaken the reconstruction of the binding pocket of human factor Xa in the structurally related rat trypsin by site-directed mutagenesis . Three sequential regions (the "99"-, the "175"- and the "190"- loops) were selected as representing the major structural differences between the ligand binding sites of the two enzymes . Wild-type rat trypsin and variants X99rT and X(99/175/190)rT were expressed in yeast, and analysed for their interaction with factor Xa and trypsin inhibitors . For most of the inhibitors studied, progressive loop replacement at the trypsin surface resulted in inhibitory profiles akin to factor Xa . Crystals of the variants were obtained in the presence of benzamidine (3), and could be soaked with the highly specific factor Xa inhibitor (1) . Binding of the latter to X99rT results in a series of structural adaptations to the ligand, including the establishment of an "aromatic box" characteristic of factor Xa . In X(99/175/190)rT, introduction of the 175-loop results in a surprising re-orientation of the "intermediate helix", otherwise common to trypsin and factor Xa . The re-orientation is accompanied by an isomerisation of the Cys168-Cys182 disulphide bond, and burial of the critical Phe174 side-chain . In the presence of (1), a major re-organisation of the binding site takes place to yield a geometry identical to that of factor Xa . In all, binding of (1) to trypsin and its variants results in significant structural rearrangements, inducing a binding surface strongly reminiscent of factor Xa, against which the inhibitor was optimised . The structural data reveal a plasticity of the intermediate helix, which has been implicated in the functional cofactor dependency of many trypsin-like serine proteinases . This approach of grafting loops onto scaffolds of known related structures may serve to bridge the gap between structural genomics and drug design. J Mol Biol, 2003 Jan 31, 325(5), 949 - 62 Polycystin-2 associates with tropomyosin-1, an actin microfilament component; Li Q et al.; Polycystin-2 (PC2) is the product of the second cloned gene (PKD2) responsible for autosomal dominant polycystic kidney disease and has recently been shown to be a calcium-permeable cation channel . PC2 has been shown to connect indirectly with the actin microfilament . Here, we report a direct association between PC2 and the actin microfilament . Using a yeast two-hybrid screen, we identified a specific interaction between the PC2 cytoplasmic C-terminal domain and tropomyosin-1 (TM-1), a component of the actin microfilament complex . Tropomyosins constitute a protein family of more than 20 isoforms arising mainly from alternative splicing and are present in muscle as well as non-muscle cells . We identified a new TM-1 splicing isoform in kidney and heart (TM-1a) that differs from TM-1 in the C terminus and interacted with PC2 . In vitro biochemical methods, including GST pull-down, blot overlay and microtiter binding assays, confirmed the interaction between PC2 and the two TM-1 isoforms . Further experiments targeted the interacting domains to G821-R878 of PC2 and A152-E196, a common segment of TM-1 and TM-1a . Indirect double immunofluorescence experiments showed partial co-localization of PC2 and TM-1 in transfected mouse fibroblast NIH 3T3 cells . Co-immunoprecipitation (co-IP) studies using 3T3 cells and Xenopus oocytes co-expressing PC2 and TM-1 (or TM-1a) revealed in vivo association between the protein pairs . Furthermore, the in vivo interaction between the endogenous PC2 and TM-1 was demonstrated also by reciprocal co-IP using native human embryonic kidney cells and human adult kidney . Considering previous reports that TM-1 acts as a suppressor of neoplastic growth of transformed cells, it is possible that TM-1 contributes to cyst formation/growth when the anchorage of PC2 to the actin microfilament via TM-1 is altered. Mikrobiologiia, 2002 Nov-Dec, 71(6), 809 - 18 {Phenotypic features of Ferroplasma acidiphilum strains Yt and Y-2}; Pivovarova TA et al.; Earlier, we described a new family of mesophilic, strictly autotrophic Fe(2+)-oxidizing archaebacteria, Ferroplasmaceae, which belongs to the order Thermoplasmales and includes the genus Ferroplasma and species F . acidiphilum (strain YT) {1} . The present work is concerned with a comparative study of phenotypic characteristics of the type strain YT and a new strain, F . acidiphilum Y-2, isolated from dense pulps produced during oxidation of arsenogold concentrates from the Bakyrchikskoe (Kazakhstan) and Olimpiadinskoe (Krasnoyarsk Krai) ore deposits, respectively . The G + C content of DNA from strains YT and Y-2 comprised 35.1 and 35.2 mol%, respectively; the level of DNA-DNA homology between the strains was 84% . Restriction profiles of chromosomal DNA from both strains exhibited a similarity coefficient of 0.87 . Genotypic characteristics of these strains indicate their affiliation to the same species . The cells of both strains are polymorphic and lack cell walls . Strains of F . acidiphilum oxidized ferrous oxide and pyrite as the sole source of energy and fixed carbon dioxide as the sole carbon source . Strains required yeast extract as a growth factor . Optimum pH for cell growth ranged from 1.7 to 1.8; the temperature optima for the growth of strains YT and Y-2 were 34-36 and 40-42 degrees C, respectively . Comparative analysis of total lipids revealed their close similarity in the strains; two glycophospholipids comprised 90% of total lipids: lipid I, beta-D-glucopyranosylcaldarchaetidylglycerol (about 55%), and lipid II, trihexosylcaldarchaetidylglycerol (26%), whose isopranyl chains contained no cyclopentane rings . The carbohydrate fraction of lipid I hydrolysate contained only D-glucose, whereas hydrolysate of lipid II contained both D-glucose and D-galactose in a molar ratio of 2:1 . Thus, it was established that the intraspecific phylogenetic divergence within F . acidiphilum is manifested in two the strains by different temperature optima against the background of similarity in other phenotypic properties. Exp Mol Med, 2002 Nov 30, 34(5), 367 - 73 Inhibition of BETA2/NeuroD by Id2; Ghil SH et al.; Id (Inhibitor of Differentiation) proteins belong to a family of transcriptional modulators that are characterized by a helix loop helix (HLH) region but lack the basic amino acid domain . Id proteins are known to interact with basic helix-loop-helix (bHLH) transcription factors and function as their negative regulators . The negative role of Id proteins has been well demonstrated in muscle development and some in neuronal cells . In this study, we investigated the effect of Id on the function of BETA2/NeuroD, a bHLH transcription factor responsible for neuron and endocrine cell specific gene expression . cDNAs of several Id isoforms were isolated by yeast two-hybrid system using the bHLH domain of E47, a ubiquitous bHLH partner as a bait . Id proteins expressed in COS M6 cells, were found in both cytosolic and nuclear fractions . Electrophoretic mobility shift assay showed that coexpression of Id2 proteins inhibited BETA2/ NeuroD binding to its target sequence, E-box . Id2 inhibited E-box mediated gene expression in a dose dependent manner in BETA2/NeuroD expressing HIT cells . Id coexpressed with BETA2/NeuroD in HeLa cells, inhibited the stimulatory activity of BETA2/NeuroD . These results suggest that Id proteins may negatively regulate tissue specific gene expression induced by BETA2/NeuroD in neuroendocrine cells and the inhibitory role of Id proteins during differentiation may be conserved in various tissues. Exp Mol Med, 2002 Dec 31, 34(6), 489 - 95 Deoxyhypusine synthase is phosphorylated by protein kinase C in vivo as well as in vitro; Kang KR et al.; Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein . We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca(2+), suggesting a possible involvement of PKC . We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell . Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells . The highest degree of phosphorylation was found in CHO cells . Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca(2+)/DAG . Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca(2+) plus DAG . Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca(2+)/phospholipid dependent . These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis. Proc Natl Acad Sci U S A, 2003 Jan 21, 100(2), 550 - 5 Epub 2003 Jan 13. RFPL4 interacts with oocyte proteins of the ubiquitin-proteasome degradation pathway; Suzumori N et al.; Oocyte meiosis and early mitotic divisions in developing embryos rely on the timely production of cell cycle regulators and their clearance via proteasomal degradation . Ret Finger Protein-Like 4 (Rfpl4), encoding a RING finger-like protein with a B30.2 domain, was discovered during an in silico search for germ cell-specific genes . To study the expression and functions of RFPL4 protein, we performed immunolocalizations and used yeast two-hybrid and other protein-protein interaction assays . Immunohistochemistry and immunofluorescence showed that RFPL4 accumulates in all growing oocytes and quickly disappears during early embryonic cleavage . We used a yeast two-hybrid model to demonstrate that RFPL4 interacts with the E2 ubiquitin-conjugating enzyme HR6A, proteasome subunit beta type 1, ubiquitin B, as well as a degradation target protein, cyclin B1 . Coimmunoprecipitation analyses of in vitro translated proteins and extracts of transiently cotransfected Chinese hamster ovary (CHO)-K1 cells confirmed these findings . We conclude that, like many RING-finger containing proteins, RFPL4 is an E3 ubiquitin ligase . The specificity of its expression and these interactions suggest that RFPL4 targets cyclin B1 for proteasomal degradation, a key aspect of oocyte cell cycle control during meiosis and the crucial oocyte-to-embryo transition to mitosis. J Biol Chem, 2003 Mar 21, 278(12), 10668 - 74 Epub 2003 Jan 13. MAGE-A4 interacts with the liver oncoprotein gankyrin and suppresses its tumorigenic activity; Nagao T et al.; Hepatocellular carcinoma ranks among the most common malignancies in Southeast Asia and South Africa . Although there are many modalities of treatment, the recurrence and metastasis rates are high, and the prognosis is unsatisfactory . Gankyrin, a recently found oncoprotein, is a promising target for drug therapy because it is overexpressed in all studied hepatocellular carcinomas . Gankyrin contains six ankyrin repeats and interacts with Rb, Cdk4, and the S6 ATPase of the 26 S proteasome . In this study, a yeast two-hybrid screen with gankyrin has identified MAGE-A4 as another interacting protein . The interaction, mediated by the C-terminal half of MAGE-A4, was reproduced in mammalian cells . The interaction was specific to MAGE-A4, because other MAGE family proteins structurally similar to MAGE-A4, i.e . MAGE-A1, MAGE-A2, and MAGE-A12, did not bind to gankyrin . MAGE-A4 partially suppressed both anchorage-independent growth in vitro and tumor formation in athymic mice of gankyrin-overexpressing cells . The ability of mutant MAGE-A4 to interact with gankyrin correlated with the ability to suppress the anchorage-independent growth . These results demonstrate that MAGE-A4 binds to gankyrin and suppresses its oncogenic activity . So far, the major focus of studies on the MAGE proteins has been on their potential for cancer immunotherapy . Our results may also shed light on novel functions for MAGE-A proteins. EMBO Rep, 2003 Jan, 4(1), 59 - 63 Trithorax interacts with type 1 serine/threonine protein phosphatase in Drosophila; Rudenko A et al.; The catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c) was shown to bind trithorax (TRX) in the yeast two-hybrid system . Interaction between PP1c and TRX was confirmed in vivo by co-immunoprecipitation from Drosophila extracts . An amino-terminal fragment of TRX, containing a putative PP1c-binding motif, was shown to be sufficient for binding to PP1c by in vitro glutathione S-transferase pull-down assays using recombinant protein and fly extracts expressing epitope tagged PP1c . Disruption of the PP1c-binding motif abolished binding, indicating that this motif is necessary for interaction with PP1 . On polytene chromosomes, PP1c is found at many discrete bands, which are widely distributed along the chromosomes . Many of the sites that stain strongly for PP1c correspond to sites of TRX, consistent with a physical association of PP1c with chromatin-bound TRX . Homeotic transformations of haltere to wing in flies mutant for trx are dominantly suppressed by PP1c mutants, indicating that PP1c not only binds TRX, but is a physiologically relevant regulator of TRX function in vivo. J Protein Chem, 2002 Oct, 21(7), 447 - 53 Arginine methylation of recombinant murine fibrillarin by protein arginine methyltransferase; Lin CH et al.; Fibrillarin is a conserved nucleolar SnoRNP with a diverse N-terminal glycine- and arginine-rich (GAR) domain in most eukaryotes . This region in human fibrillarin is known to contain modified dimethylarginines . In this report we demonstrate that recombinant murine fibrillarin is a substrate for protein arginine methyltransferase, including the purified recombinant enzyme (rat PRMT1 and yeast RMT1) and the protein methyltransferases present in lymphoblastoid cell extracts . Our results of protease digestion, methylation competition reactions, and immunoblotting with a methylarginine-specific antibody all indicate that the methylation of fibrillarin is in the N-terminal GAR domain and arginyl residues are modified . Finally, amino acid analyses revealed that the modification of recombinant murine fibrillarin forms methylarginines, mostly as dimethylarginines. J Hum Genet, 2002, 47(12), 681 - 3 Identification of a novel human DDX40gene, a new member of the DEAH-box protein family; Xu J et al.; The DExH/D-box superfamily of RNA helicases seems to play key roles during RNA metabolism, such as pre-mRNA splicing, ribosome biogenesis, and others . We have cloned a new gene of the DEAH-box protein subgroup, designated DDX40 (DEAD/H-box polypeptide 40 gene) . DDX40 contains 3656 nucleotides and codes for a putative 779-amino-acid protein . Sequence analysis of the cDNA product revealed that it contained a DEAH (Asp-Glu-Ala-His) sequence motif and other conserved motifs . The DDX40 protein shared 53% and 43% amino acid identity with human DDX8 and yeast Drh1, respectively, in the conserved region . Northern blot analysis showed that DDX40 was expressed ubiquitously in the eight tissues examined, implying a general physiological function of the protein . We speculate that, like other members of the DExH/D-box superfamily, DDX40 may play roles in pre-mRNA splicing, ribosome biogenesis and other RNA processing functions. J Am Acad Dermatol, 2003 Jan, 48(1), 123 - 7 Disseminated blastomycosis; Assaly RA et al.; A 26-year-old veiled Saudi-Arabian woman presented with hemoptysis, and multiple nodules and abscesses . A skin biopsy specimen revealed yeast forms consistent with Blastomyces dermatitidis . Fungal cultures from bronchoscopy and skin specimens also grew B dermatitidis . She was treated with oral itraconazole (200 mg twice a day) . Both lung and skin lesions showed improvement within 6 weeks. J Biomol NMR, 2002 Nov, 24(3), 171 - 89 Protein NMR structure determination with automated NOE-identification in the NOESY spectra using the new software ATNOS; Herrmann T et al.; Novel algorithms are presented for automated NOESY peak picking and NOE signal identification in homonuclear 2D and heteronuclear-resolved 3D {(1)H,(1)H}-NOESY spectra during de novo protein structure determination by NMR, which have been implemented in the new software ATNOS (automated NOESY peak picking) . The input for ATNOS consists of the amino acid sequence of the protein, chemical shift lists from the sequence-specific resonance assignment, and one or several 2D or 3D NOESY spectra . In the present implementation, ATNOS performs multiple cycles of NOE peak identification in concert with automated NOE assignment with the software CANDID and protein structure calculation with the program DYANA . In the second and subsequent cycles, the intermediate protein structures are used as an additional guide for the interpretation of the NOESY spectra . By incorporating the analysis of the raw NMR data into the process of automated de novo protein NMR structure determination, ATNOS enables direct feedback between the protein structure, the NOE assignments and the experimental NOESY spectra . The main elements of the algorithms for NOESY spectral analysis are techniques for local baseline correction and evaluation of local noise level amplitudes, automated determination of spectrum-specific threshold parameters, the use of symmetry relations, and the inclusion of the chemical shift information and the intermediate protein structures in the process of distinguishing between NOE peaks and artifacts . The ATNOS procedure has been validated with experimental NMR data sets of three proteins, for which high-quality NMR structures had previously been obtained by interactive interpretation of the NOESY spectra . The ATNOS-based structures coincide closely with those obtained with interactive peak picking . Overall, we present the algorithms used in this paper as a further important step towards objective and efficient de novo protein structure determination by NMR. Science, 2003 Jan 31, 299(5607), 716 - 9 Epub 2003 Jan 09. ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation; Zilberman D et al.; Proteins of the ARGONAUTE family are important in diverse posttranscriptional RNA-mediated gene-silencing systems as well as in transcriptional gene silencing in Drosophila and fission yeast and in programmed DNA elimination in Tetrahymena . We cloned ARGONAUTE4 (AGO4) from a screen for mutants that suppress silencing of the Arabidopsis SUPERMAN (SUP) gene . The ago4-1 mutant reactivated silent SUP alleles and decreased CpNpG and asymmetric DNA methylation as well as histone H3 lysine-9 methylation . In addition, ago4-1 blocked histone and DNA methylation and the accumulation of 25-nucleotide small interfering RNAs (siRNAs) that correspond to the retroelement AtSN1 . These results suggest that AGO4 and long siRNAs direct chromatin modifications, including histone methylation and non-CpG DNA methylation. J Biol Chem, 2003 Mar 21, 278(12), 10041 - 7 Epub 2003 Jan 09. Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase delta and proliferating cell nuclear antigen; Liu L et al.; The yeast two-hybrid screening method was used to identify novel proteins that associate with human DNA polymerase delta (pol delta) . Two baits were used in this study . These were the large (p125) and small (p50) subunits of the core pol delta heterodimer . p50 was the only positive isolated with p125 as the bait . Two novel protein partners, named PDIP38 and PDIP46, were identified from the p50 screen . In this study, the interaction of PDIP38 with pol delta was further characterized . PDIP38 encodes a protein of 368 amino acids whose C terminus is conserved with the bacterial APAG protein and with the F box A protein . It was found that PDIP38 also interacts with proliferating cell nuclear antigen (PCNA) . The ability of PDIP38 to interact with both the p50 subunit of pol delta and with PCNA was confirmed by pull-down assays using glutathione S-transferase (GST)-PDIP38 fusion proteins . The PCNA-PDIP38 interaction was also demonstrated by PCNA overlay experiments . The association of PDIP38 with pol delta was shown to occur in calf thymus tissue and mammalian cell extracts by GST-PDIP38 pull-down and coimmunoprecipitation experiments . PDIP38 was associated with pol delta isolated by immunoaffinity chromatography . The association of PDIP38 with pol delta could also be demonstrated by native gel electrophoresis. J Biol Chem, 2003 Mar 21, 278(12), 10048 - 54 Epub 2003 Jan 08. Identification of targets for calcium signaling through the copine family of proteins . Characterization of a coiled-coil copine-binding motif; Tomsig JL et al.; We provide evidence that copines, members of a ubiquitous family of calcium-dependent, membrane-binding proteins, may represent a universal transduction pathway for calcium signaling because we find copines are capable of interacting with a wide variety of "target" proteins including MEK1, protein phosphatase 5, and the CDC42-regulated kinase, that are themselves components of intracellular signaling pathways . The copine target proteins were identified by yeast two-hybrid screening and the interactions were verified in vitro using purified proteins . In the majority of cases the copine binds to a domain of the target protein that is predicted to form a characteristic coiled-coil . A consensus sequence for the coiled-coil copine-binding site was derived and found to have predictive value for identifying new copine targets . We also show that interaction with copines may result in recruitment of target proteins to membrane surfaces and regulation of the enzymatic activities of target proteins. Wei Sheng Yan Jiu, 2000 Nov, 29(6), 400 - 1 {Study on the revision of hygiene standard for fresh-cream cake}; Wang S et al.; In order to revise the hygiene standard for fresh-cream cake, 44 samples were examined . With regard to the specific selling condition and the results inspected, a reference for acid value, peroxide value, bacterial colony forming efficiency and mould count was proposed . Moreover the yeast count was suggested as one of the hygienic standard for GB7099 too . The result of this study could be used as a reference for developing the law on the hygienic inspection and control of fresh-cream cake. Nucleic Acids Res, 2003 Jan 1, 31(1), 432 - 5 Plant snoRNA database; Brown JW et al.; The Plant snoRNA database provides information on small nucleolar RNAs from Arabidopsis and eighteen other plant species . Information includes sequences, expression data, methylation and pseudouridylation target modification sites, initial gene organization (polycistronic, single gene and intronic) and the number of gene variants . The Arabidopsis information is divided into box C/D and box H/ACA snoRNAs, and within each of these groups, by target sites in rRNA, snRNA or unknown . Alignments of orthologous genes and gene variants from different plant species are available for many snoRNA genes . Plant snoRNA genes have been given a standard nomenclature, designed wherever possible, to provide a consistent identity with yeast and human orthologues. Nucleic Acids Res, 2003 Jan 1, 31(1), 248 - 50 BIND: the Biomolecular Interaction Network Database; Bader GD et al.; The Biomolecular Interaction Network Database (BIND: archives biomolecular interaction, complex and pathway information . A web-based system is available to query, view and submit records . BIND continues to grow with the addition of individual submissions as well as interaction data from the PDB and a number of large-scale interaction and complex mapping experiments using yeast two hybrid, mass spectrometry, genetic interactions and phage display . We have developed a new graphical analysis tool that provides users with a view of the domain composition of proteins in interaction and complex records to help relate functional domains to protein interactions . An interaction network clustering tool has also been developed to help focus on regions of interest . Continued input from users has helped further mature the BIND data specification, which now includes the ability to store detailed information about genetic interactions . The BIND data specification is available as ASN.1 and XML DTD. J Biol Chem, 2003 Mar 14, 278(11), 9979 - 85 Epub 2003 Jan 07. ADP-ribosylation factor-dependent phospholipase D2 activation is required for agonist-induced mu-opioid receptor endocytosis; Koch T et al.; Agonist exposure of many G protein-coupled receptors induces a rapid receptor phosphorylation and uncoupling from G proteins . Resensitization of these desensitized receptors requires endocytosis and subsequent dephosphorylation . Using a yeast two-hybrid screen, the rat mu-opioid receptor (MOR1, also termed MOP) was found to be associated with phospholipase D2 (PLD2), a phospholipid-specific phosphodiesterase located in the plasma membrane, which has been implicated in the formation of endocytotic vesicles . Coimmunoprecipitation experiments in HEK293 cells coexpressing MOR1 and PLD2 confirmed that MOR1 constitutively interacts with PLD2 . Treatment with the mu receptor agonist DAMGO ({d-Ala(2), Me Phe(4), Glyol(5)}enkephalin) led to an increase in PLD2 activity, whereas morphine, which does not induce MOR1 receptor internalization, failed to induce PLD2 activation . The DAMGO-mediated PLD2 activation was inhibited by brefeldin A, an inhibitor of ADP-ribosylation factor (ARF) but not by the protein kinase C (PKC) inhibitor calphostin C indicating that opioid receptor-mediated activation of PLD2 is ARF- but not PKC-dependent . Furthermore, heterologous stimulation of PLD2 by phorbol ester led to an accelerated internalization of the mu-opioid receptor after both DAMGO and morphine exposure . Conversely the inhibition of PLD2-mediated phosphatidic acid formation by 1-butanol or overexpression of a negative mutant of PLD2 prevented agonist-mediated endocytosis of MOR1 . Together, these data suggest that PLD2 play a key role in the regulation of agonist-induced endocytosis of the mu-opioid receptor. Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 307 - 12 Epub 2002 Dec 23. Direct interaction with a nuclear protein and regulation of gene silencing by a variant of the Ca2+-channel beta 4 subunit; Hibino H et al.; The beta subunits of voltage-gated Ca(2+) channels are known to be regulators of the channels' gating properties . Here we report a striking additional function of a beta subunit . Screening of chicken cochlear and brain cDNA libraries identified beta(4c), a short splice variant of the beta(4) subunit . Although beta(4c) occurs together with the longer isoforms beta(4a) or beta(4b) in the brain, eye, heart, and lung, the cochlea expresses exclusively beta(4c) . The association of beta(4c) with the Ca(2+)-channel alpha(1) subunit has slight but significant effects on the kinetics of channel activation and inactivation . Yeast two-hybrid and biochemical assays revealed that beta(4c) interacts directly with the chromo shadow domain of chromobox protein 2heterochromatin protein 1gamma (CHCB2HP1gamma), a nuclear protein involved in gene silencing and transcriptional regulation . Coexpression of this protein specifically recruits beta(4c) to the nuclei of mammalian cells . Furthermore, beta(4c) but not beta(4a) dramatically attenuates the gene-silencing activity of chromobox protein 2heterochromatin protein 1gamma . The beta(4c) subunit is therefore a multifunctional protein that not only constitutes a portion of the Ca(2+) channel but also regulates gene transcription. J Clin Microbiol, 2003 Jan, 41(1), 479 - 82 Trichosporon loubieri infection in a patient with adult polycystic kidney disease; Padhye AA et al.; A 45-year-old man from Nepal with a 13-year history of polycystic kidney disease was diagnosed as suffering from chronic renal failure with end-stage renal disease . After receiving empirical antituberculosis treatment, he was treated with broad-spectrum antibiotics . A left nephrectomy was performed, and after 4 months, he received a kidney transplant . The left kidney was grossly enlarged, with multiple cystic spaces filled with blackish material . Histologic examination of the excised left kidney tissue stained with hematoxylin and eosin and Gomori's methenamine silver stains showed numerous hyaline, septate, fungal hyphae of various lengths, many broken into rectangular arthroconidia in the cysti