Yeasts

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 cystic spaces . Culture of the kidney tissue yielded white, glabrous, yeast-like colonies . Based on its micromorphology, growth at 42 degrees C, and ribosomal DNA (rDNA) sequence analysis, and also sequence analysis of the internal-transcribed-spacer and D1/D2 rDNA regions, the yeast was identified as Trichosporon loubieri . Postsurgically, the patient was treated with amphotericin B and oral itraconazole, followed by maintenance therapy with fluconazole . He remained afebrile and asymptomatic . At the final follow-up, all parameters were found normal and the patient was doing well, with normal renal function reports . This paper presents the first known case of human infection caused by T . loubieri.

J Clin Microbiol, 2003 Jan, 41(1), 205 - 8
Nested PCR assays for detection of Blastomyces dermatitidis DNA in paraffin-embedded canine tissue; Bialek R et al.; A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family ONYGENACEAE: We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively . The B . dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy . Sequencing revealed that all PCR products were homologous to the B . dermatitidis WI-1 adhesin gene . No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma . The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively . Only six products were identified as being identical to B . dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B . dermatitidis nested PCR . For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H . capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B . dermatitidis PCR assay . In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B . dermatitidis in canine tissue.

Trends Biochem Sci, 2003 Jan, 28(1), 41 - 8
Linking chromatin function with metabolic networks: Sir2 family of NAD(+)-dependent deacetylases; Denu JM; Chromatin remodeling enzymes rely on coenzymes derived from metabolic pathways, suggesting a tight synchronization among apparently diverse cellular processes . A unique example of this link is the recently described NAD(+)-dependent protein and/or histone deacetylases . The founding member of this family - yeast silent information regulator 2 (ySir2) - is involved in gene silencing, chromosomal stability and ageing . Sir2-like enzymes catalyze a reaction in which the cleavage of NAD(+)and histone and/or protein deacetylation are coupled to the formation of O-acetyl-ADP-ribose, a novel metabolite . The dependence of the reaction on both NAD(+) and the generation of this potential second messenger offers new clues to understanding the function and regulation of nuclear, cytoplasmic and mitochondrial Sir2-like enzymes.

Zhongguo Zhong Yao Za Zhi, 2000 Oct, 25(10), 619 - 21
{Effect and mechanism of active fraction A guizhi decoction on dual-directional thermoregulation: effect on heat shock protein in hypothalamus of rats}; Huo HR et al.; OBJECTIVE: To investigate the effect of active fraction A in Guizhi Decoction (Fr.A) on dual-directional thermoregulation and its mechanism of influencing heat shock protein (HSP) in hypothalamus . METHOD: Using Western blot method to measure HSP of hypothalamus in febrile and hypothermal rats . RESULTS: Regulating the body temperature in dual-direction, Fr.A could antagonize the decrease of HSP contents of hypothalamus in hypothermal rats induced by aminopyrine, and abate the HSP content in febrile rats induced by yeast . CONCLUSION: Fr.A adjusts the body temperature through regulating the contents of HSP of hypothalamus in febrile and hypothermal rats.

Exp Mol Med, 2002 Sep 30, 34(4), 313 - 7
Human FEN-1 can process the 5'-flap DNA of CTG/CAG triplet repeat derived from human genetic diseases by length and sequence dependent manner; Lee S et al.; Trinucleotide repeat (TNR) instability can cause a variety of human genetic diseases including myotonic dystrophy and Huntington's disease . Recent genetic data show that instability of the CAG/CTG repeat DNA is dependent on its length and replication origin . In yeast, the RAD27 (human FEN-1 homologue) null mutant has a high expansion frequency at the TNR loci . We demonstrate here that FEN-1 processes the 5'-flap DNA of CTG/CAG repeats, which is dependent on the length in vitro . FEN-1 protein can cleave the 5'-flap DNA containing triplet repeating sequence up to 21 repeats, but the activity decreases with increasing size of flap above 11 repeats . In addition, FEN-1 processing of 5'-flap DNA depends on sequence, which play a role in the replication origin-dependent TNR instability . Interestingly, FEN-1 can cleave the 5'-flap DNA of CTG repeats better than CAG repeats possibly through the flap-structure . Our biochemical data of FEN-1's activity with triplet repeat DNA clearly shows length dependence, and aids our understanding on the mechanism of TNR instability.

Neurochem Res, 2002 Dec, 27(12), 1719 - 24
Allosteric regulation of mouse brain serine racemase; Neidle A et al.; Serine racemase, purified from mouse brain, consisted of two isoforms . They had similar enzymatic properties and had molecular weights of about 55 kDa based on size exclusion chromatography . This is about twice that reported from its electrophoretic mobility on SDS gels or from the amino acid sequence of the recombinant enzyme . In addition to the previously reported requirements for pyridoxal phosphate and reducing agents, we found that both forms of the enzyme required Mg2+ and were strongly stimulated by yeast extract . The yeast extract could be replaced by ATP, GTP, or ADP and, to a lesser extent, by other nucleotides . In the presence of 1 mM ATP, the Km for L-serine decreased from 13 mM to 1.8 mM with little change in Vmax, indicating an allosteric mechanism for nucleotide activation . In addition to acting as a serine racemase, the enzyme has been reported to act on L-serine O-sulfate as a dehydratase . When measured by HPLC, after derivatization with 2,4 dinitrophenylhydrazine, we found, as expected, a very rapid formation of pyruvate from this substrate . L-serine was also converted to pyruvate at about twice the racemization rate . L-serine O-sulfate dehydration was inhibited by ATP, while L-serine dehydration, like racemization, was activated by nucleotides, indicating that, for L-serine, dehydration and racemization take place at the same site.

Plant Cell Physiol, 2002 Dec, 43(12), 1558 - 67
Molecular properties of a matrix attachment region-binding protein located in the nucleoli of tobacco cells; Fujiwara S et al.; We cloned a cDNA for matrix-attachment region (MAR)-binding protein from Nicotiana tabacum cells to elucidate the structure and function of the nuclear matrix . The cDNA encodes a protein of 555 amino acids (61,050 Da) with an isoelectric point of 9.4 . We named the protein NtMARBP61 . The sequence is 45% identical to yeast Nop58p, which is involved in rRNA processing . The C-terminal part is unique and rich in lysine residues . The recombinant C-terminal part had the ability to bind double-stranded DNAs of 12 tobacco MARs . The intracellular localization was determined to be in the nucleolus by fluorescent microscopy using the antibody to the recombinant NtMARBP61 . The mRNA level was high in the lag and early-log phases of cultured cells but low in the stationary phase . The protein was accumulated only in the middle- and late-log phases, suggesting that NtMARBP61 is essential for growing cells . The results suggest at least the structural and regulatory function of NtMARBP61 in the nucleolus as a MAR-binding protein in a growth-stage specific manner.

J Biol Chem, 2003 Mar 14, 278(11), 9195 - 202 Epub 2003 Jan 06.
JNK1 physically interacts with WW domain-containing oxidoreductase (WOX1) and inhibits WOX1-mediated apoptosis; Chang NS et al.; Transient activation of c-Jun N-terminal kinase (JNK) promotes cell survival, whereas persistent JNK activation induces apoptosis . Bovine testicular hyaluronidase PH-20 activates JNK1 and protects L929 fibroblasts from staurosporine-mediated cell death . PH-20 also induces the expression of a p53-interacting WW domain-containing oxidoreductase (WOX1, also known as WWOX or FOR) in these cells . WOX1 enhances the cytotoxic function of tumor necrosis factor and mediates apoptosis synergistically with p53 . Thus, the activated JNK1 is likely to counteract WOX1 in mediating apoptosis . Here it is demonstrated that ectopic JNK1 inhibited WOX1-mediated apoptosis of L929 fibroblasts, monocytic U937 cells, and other cell types . Also, JNK1 blocked WOX1 prevention of cell cycle progression . By stimulating cells with anisomycin or UV light, JNK1 became activated, and WOX1 was phosphorylated at Tyr(33) . The activated JNK1 physically interacted with the phosphorylated WOX1, as determined by co-immunoprecipitation . Alteration of Tyr(33) to Arg(33) in WOX1 abrogated its binding interaction with JNK1 and its activity in mediating cell death, indicating that Tyr(33) phosphorylation is needed to activate WOX1 . A dominant negative WOX1 was developed and shown to block p53-mediated apoptosis and anisomycin-mediated WOX1 phosphorylation but could not inhibit JNK1 activation . This mutant protein bound p53 but could not interact with JNK1, as determined in yeast two-hybrid analysis . Taken together, phosphorylation of JNK1 and WOX1 is necessary for their physical interaction and functional antagonism.

J Interferon Cytokine Res, 2002 Nov, 22(11), 1113 - 21
The mouse interferon-inducible gene Ifi204 product interacts with the Tpr protein, a component of the nuclear pore complex; De Andrea M et al.; We have used yeast two-hybrid screening to isolate cDNA-encoding proteins interacting with the protein encoded by the interferon (IFN)-inducible gene Ifi204 . Four independent overlapping clones were isolated from an NIH3T3 cDNA library . The largest clone encoded a protein (1203 amino acids in length) sharing 94% identity with the C-terminal portion of the human translocated promoter region (Tpr) protein . Northern blot analysis revealed a 7.5-kilobase mRNA present in both mouse and human cell lines . In addition, in vivo interaction was demonstrated by coimmunoprecipitation experiments . Anti-Tpr polyclonal monospecific antibodies (Ab) used for immunofluorescence staining labeled the nuclear envelope (NE) in a punctate pattern characteristic of nucleoporins and also yielded staining throughout the nuclear interior . The intranuclear Tpr occurred in apparently discrete foci . When superimposed on optical sections obtained with anti-p204 Abs, these colocalized, with the sole exception of the nucleolar compartment stained by the anti-p204 Abs only . Although the specific function of Tpr is not defined, it appears to mediate p204 translocation from the cytoplasmic to the nuclear compartment following IFN treatment.

J Physiol Pharmacol, 2002 Dec, 53(4 Pt 1), 675 - 88
In vitro studies of activation of phagocytic cells by bioactive peptides; Stoika RS et al.; The effect of formyl chemotactic peptide (fCTP, fMet-Leu-Phe), beta-amyloid peptides (beta-AP, 1-42, 1-16 and 25-35), and bradykinin (BK) on functional activity of phagocytic cells has been investigated . Wheat germ agglutinin (WGA) was also used as a model membrane binding agent of polypeptide nature . Murine monocyte-macrophage cell line J774.2 and normal human blood polymorphonuclear (PMN) cells were used as target phagocytic cells . Their activity was quantitatively estimated by measuring phagocytosis of killed yeast cells . Beta-AP (1-41) maximally stimulated phagocytosis at 0.1 microg/ml, BK--at 1.0 microg/ml, and fCTP--at 2.0 microg/ml . Beta-AP (1-16) and beta-AP (25-35) were inactive in used test-systems . Phagocytosis-inducing activity of beta-AP (1-42) and BK reached maximal levels in 2 h and decreased after 4-6 h of incubation . Phagocytosis numbers were compared with the indicators of phagocytic cell activation, such as absorption of neutral red dye, glucose utilization, production of super-oxide anion (NBT-test) and nitrite accumulation (indicator of NO production) . NBT-test, which may be related to the killing ability of phagocytic cells towards the ingested objects, was positive only in stimulated PMN leukocytes, while the nitrite accumulation was detected only in stimulated macrophages . Nitrite accumulation in macrophages was markedly induced by lipopolysaccharide and to a lower extent by 0.5 microg/ml beta-AP (1-42) . In high dose (5.0 microg/ml) beta-AP suppressed nitrite accumulation in macrophages stimulated by lipopolysaccharide . Other studied peptides were inactive in inducing nitrite accumulation . Transforming growth factor type beta suppressed phagocytic activity of PMN cells activated by beta-AP or WGA . The anti-inflammatory drugs (indomethacin and L-lysine aescinate) inhibited beta-AP (1-42)-induced phagocytosis . The interrelations between the regulatory pathways of BK, beta-AP and fCTP are discussed.

Zhongguo Zhong Yao Za Zhi, 2000 May, 25(5), 297 - 9
{A pharmacodynamic research on chromone glucosides of fangfeng}; Xue BY et al.; OBJECTIVE: To investigate the pharmacological activity of prim-O-glucosylcimifugin and 4'-O-beta-D-glucosyl-5-O-methylvisamminol con tained in Fangfeng . METHODS: Analgesic, antipyretic and antiinflammatory effects were assessed using the models of yeast-induced rat fever, acetic acidinduced peritoneal pain thermalpain, and dimethylbenzol-induced mouse ear edema . RESULTS: Prim-O-glucosylcimifugin and 4'-O-beta-D-glucosyl-5-O-methylvisamminol could reduce the body temperature of rats in a model of yeast-induced fever . Both components could decrease the spasm action induced by peritoneal injection of acetic acid, increase the pain-threshold in a thermal-pain model, relieve the mouse ear edema caused by dimethybenzol and significantly inhibit ADP-induced platelet aggregation . CONCLUSION: Prim-O-glucosylcimifugin and 4'-O-beta-D-glucosyl-5-O-methylvisamminol have significant analgesic, antipyretic, anti-inflammatory and anti-platelet aggregation actions.

Apoptosis, 2003 Jan, 8(1), 39 - 44
CSE1L/CAS: its role in proliferation and apoptosis; Behrens P et al.; CAS/CSE1L is the human homologue of the yeast gene CSE1 . It was first cloned while searching for genes that rendered breast cancer cells resistant towards toxin induced apoptosis . Since depletion of CSE1 leads to cell-cycle arrest, CAS is thought to be involved in proliferation . CAS functions in the mitotic spindle checkpoint . CAS is located on chromosome 20q13, a locus often amplified in cancers of various origin, e.g . colonic or breast cancer . Since genetic instability is a hallmark of cancer, amplification or over expression of the CAS gene might interfere with or override its role in the mitotic spindle checkpoint . CAS is also implicated in the nuclear to cytoplasmic reshuffling of importin alpha, which itself is necessary for the nuclear transport of several proliferation activating proteins, transcription factors, oncogene and tumor suppressor gene products such as p53 and BRCA1 . Inhibition of MEK1 mediated phosphorylation has been shown to enhance paclitaxel (Taxol) induced apoptosis in breast, ovarian, and lung tumor cell lines in-vitro . Since CAS is also phosphorylated (activated) by MEK1, and since the anti-cancer drug Taxol alters the microtubule assembly and activates pro-apoptotic signaling pathways, altering the activity/phosphorylation status of CAS via MEK1 inhibition may present a potential strategy in experimental cancer therapy.

Oral Oncol, 2003 Feb, 39(2), 163 - 9
Specific p53 mutations predict poor prognosis in oral squamous cell carcinoma; Yamazaki Y et al.; In this study, we focused on p53 mutations in specific regions, including DNA-binding surface regions, to clarify the correlation between mutations within the specific regions of p53 and clinical outcomes of patients with oral cancers . We analyzed p53 mutations in 121 fresh primary oral squamous cell carcinomas (SCCs) by polymerase chain reaction-single-strand conformation polymorphism or a yeast functional assay . p53 mutations were detected in 51/121 (42%) cases . Mutation of p53 was not associated with any clinicopathological parameters; however, tumors containing specific p53 mutations, e.g . DNA-binding surface regions (L2, L3 and the LSH motif) and conserved regions (II-V), had significantly poorer prognoses than tumors with mutations outside of those regions . Moreover, locoregional failure, lymph node metastasis and the occurrence of subsequent distant metastasis were also significantly associated with mutations within DNA-binding surface regions . These data indicate that specific mutations of p53 could be important prognostic factors in oral SCCs .

Plant Cell, 2003 Jan, 15(1), 119 - 32
AtATM is essential for meiosis and the somatic response to DNA damage in plants; Garcia V et al.; In contrast to yeast or mammalian cells, little is known about the signaling responses to DNA damage in plants . We previously characterized AtATM, an Arabidopsis homolog of the human ATM gene, which is mutated in ataxia telangiectasia, a chromosome instability disorder . The Atm protein is a protein kinase whose activity is induced by DNA damage, particularly DNA double-strand breaks . The phosphorylation targets of Atm include proteins involved in DNA repair, cell cycle control, and apoptosis . Here, we describe the isolation and functional characterization of two Arabidopsis mutants carrying a T-DNA insertion in AtATM . Arabidopsis atm mutants are hypersensitive to gamma-radiation and methylmethane sulfonate but not to UV-B light . In correlation with the radiation sensitivity, atm mutants failed to induce the transcription of genes involved in the repair and/or detection of DNA breaks upon irradiation . In addition, atm mutants are partially sterile, and we show that this effect is attributable to abundant chromosomal fragmentation during meiosis . Interestingly, the transcription of DNA recombination genes during meiosis was not dependent on AtATM, and meiotic recombination occurred at the same rate as in wild-type plants, raising questions about the function of AtAtm during meiosis in plants . Our results demonstrate that AtATM plays a central role in the response to both stress-induced and developmentally programmed DNA damage.

Mol Cell Biol, 2003 Jan, 23(2), 450 - 61
Mcm1p-induced DNA bending regulates the formation of ternary transcription factor complexes; Lim FL et al.; The yeast MADS-box transcription factor Mcm1p plays an important regulatory role in several diverse cellular processes . In common with a subset of other MADS-box transcription factors, Mcm1p elicits substantial DNA bending . However, the role of protein-induced bending by MADS-box proteins in eukaryotic gene regulation is not understood . Here, we demonstrate an important role for Mcm1p-mediated DNA bending in determining local promoter architecture and permitting the formation of ternary transcription factor complexes . We constructed mutant mcm1 alleles that are defective in protein-induced bending . Defects in nuclear division, cell growth or viability, transcription, and gene expression were observed in these mutants . We identified one likely cause of the cell growth defects as the aberrant formation of the cell cycle-regulatory Fkh2p-Mcm1p complex . Microarray analysis confirmed the importance of Mcm1p-mediated DNA bending in maintaining correct gene expression profiles and revealed defects in Mcm1p-mediated repression of Ty elements and in the expression of the cell cycle-regulated YFR and CHS1 genes . Thus, we discovered an important role for DNA bending by MADS-box proteins in the formation and function of eukaryotic transcription factor complexes.

J Biol Chem, 2003 Mar 7, 278(10), 8356 - 62 Epub 2002 Dec 30.
Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor; Tall GG et al.; The activation of heterotrimeric G proteins is accomplished primarily by the guanine nucleotide exchange activity of ligand-bound G protein-coupled receptors . The existence of nonreceptor guanine nucleotide exchange factors for G proteins has also been postulated . Yeast two-hybrid screens with Galpha(o) and Galpha(s) as baits were performed to identify binding partners of these proteins . Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B . Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for a subset of Galpha proteins including Galpha(q), Galpha(i1), and Galpha(o), but not Galpha(s) . The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated . Ric-8A interacts with GDP-bound Galpha proteins, stimulates release of GDP, and forms a stable nucleotide-free transition state complex with the Galpha protein; this complex dissociates upon binding of GTP to Galpha.

J Biol Chem, 2003 Mar 7, 278(10), 7875 - 83 Epub 2002 Dec 31.
The Clf1p splicing factor promotes spliceosome assembly through N-terminal tetratricopeptide repeat contacts; Wang Q et al.; Spliceosome assembly follows a well conserved pathway of subunit addition that includes both small nuclear ribonucleoprotein (snRNP) particles and non-snRNP splicing factors . Clf1p is an unusual splicing factor composed almost entirely of direct repeats of the tetratricopeptide repeat (TPR) protein-binding motif . Here we show that the Clf1p protein resides in at least two multisubunit protein complexes, a small nuclear RNA-free structure similar to what was reported as the Prp19p complex (nineteen complex; NTC) and an RNP structure that contains the U2, U5, and U6 small nuclear RNAs . Thirty Ccf (Clf1p complex factor) proteins have been identified by mass spectroscopy or immune detection as known or suspected components of the yeast spliceosome . Deletion of TPR1 or TPR2 from an epitope-tagged Clf1p protein (i.e . Clf1Delta2-TAP) destabilizes Clf1p complexes assembled in vivo, causing the release of the Cef1p and Prp19p NTC factors and decreased association of the Rse1p, Snu114p, and Hsh155p snRNP proteins . In vitro, temperature inactivation of Clf1Delta2p impairs the prespliceosome to spliceosome transition and prevents Prp19p recruitment to the splicing complex . These and related data support the view that the poly-TPR Clf1p splicing factor promotes the functional integration of the U4/U6.U5 tri-snRNP particle into the U1-, U2-dependent prespliceosome.

DNA Repair (Amst), 2003 Jan 2, 2(1), 1 - 11
Suppression of high-fidelity double-strand break repair in mammalian chromosomes by pifithrin-alpha, a chemical inhibitor of p53; Lin Y et al.; We investigated the effect of pifithrin-alpha (PFTalpha), a chemical inhibitor of p53, on DNA double-strand break (DSB) repair in mammalian chromosomes . Thymidine kinase-deficient mouse fibroblasts were stably transfected with DNA substrates containing one or two recognition sites for yeast endonuclease I-SceI embedded within a herpes simplex virus thymidine kinase gene . Genomic DSBs were induced by introducing an I-SceI expression plasmid into cells in the presence or absence of 20 microM PFTalpha . From cells containing the DNA substrate with a single I-SceI site we recovered low-fidelity nonhomologous end-joining (NHEJ) events in which one or more nucleotides were deleted or inserted at the DSB . From cells containing the substrate with two I-SceI sites we recovered high-fidelity DNA end-joining (precise ligation (PL)) events . We found that treatment of cells with PFTalpha caused a 5-10-fold decrease in recovery of PL but decreased recovery of NHEJ by less than two-fold . Deletion sizes associated with NHEJ were unaffected by treatment with PFTalpha . Our work suggests the possibility that p53 facilitates high-fidelity DSB repair while playing little or no role in mutagenic NHEJ.

DNA Repair (Amst), 2002 Jun 21, 1(6), 449 - 61
The carboxy-terminal domain of the XPC protein plays a crucial role in nucleotide excision repair through interactions with transcription factor IIH; Uchida A et al.; The xeroderma pigmentosum group C (XPC) protein specifically involved in genome-wide damage recognition for nucleotide excision repair (NER) was purified as a tight complex with HR23B, one of the two mammalian homologs of RAD23 in budding yeast . This XPC-HR23B complex exhibits strong binding affinity for single-stranded DNA, as well as preferential binding to various types of damaged DNA . To examine the structure-function relationship of XPC, a series of truncated mutant proteins were generated and assayed for various binding activities . The two domains participating in binding to HR23B and damaged DNA, respectively, were mapped within the carboxy-terminal half of XPC, which also contains an evolutionary conserved amino acid sequence homologous to the yeast RAD4 protein . We established that the carboxy-terminal 125 amino acids are dispensable for both HR23B and damaged DNA binding, while interactions with transcription factor IIH (TFIIH) are significantly impaired by truncation of this domain . Furthermore, deletion of the extreme carboxy-terminal domain totally abolished XPC activity in the cell-free NER reaction . These results suggest that following initial damage recognition, the carboxy terminus of XPC may be essential for the recruitment of TFIIH, and that most truncation mutations identified in XP-C patients result in non-functional proteins.

DNA Repair (Amst), 2002 Jul 17, 1(7), 559 - 69
Activities of human DNA polymerase kappa in response to the major benzo{a}pyrene DNA adduct: error-free lesion bypass and extension synthesis from opposite the lesion; Zhang Y et al.; In cells, the major benzo{a}pyrene DNA adduct is the highly mutagenic (+)-trans-anti-BPDE-N(2)-dG . In eukaryotes, little is known about lesion bypass of this DNA adduct during replication . Here, we show that purified human Polkappa can effectively bypass a template (+)-trans-anti-BPDE-N(2)-dG adduct in an error-free manner . Kinetic parameters indicate that Polkappa bypass of the (-)-trans-anti-BPDE-N(2)-dG adduct was approximately 41-fold more efficient compared to the (+)-trans-anti-BPDE-N(2)-dG adduct . Furthermore, we have found another activity of human Polkappa in response to the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts: extension synthesis from mispaired primer 3' ends opposite the lesion . In contrast, the two adducts strongly blocked DNA synthesis by the purified human Polbeta and the purified catalytic subunits of yeast Polalpha, Poldelta, and Pol epsilon right before the lesion . Extension by human Polkappa from the primer 3' G opposite the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts was mediated by a -1 deletion mechanism, probably resulting from re-aligning the primer G to pair with the next template C by Polkappa prior to DNA synthesis . Thus, sequence contexts 5' to the lesion strongly affect the fidelity and mechanism of the Polkappa-catalyzed extension synthesis . These results support a dual-function model of human Polkappa in bypass of BPDE DNA adducts: it may function both as an error-free bypass polymerase alone and an extension synthesis polymerase in combination with another polymerase .

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2309 - 14
Reclassification of the Sporobolomyces roseus and Sporidiobolus pararoseus complexes, with the description of Sporobolomyces phaffii sp . nov; Bai FY et al.; More than 50 ballistoconidium-forming yeast strains, isolated from plant leaves collected in Yunnan, China, were identified as Sporobolomyces roseus Kluyver & van Niel by conventional methods . However, comparison of the internal transcribed spacer (ITS) region and 265 rDNA D1/D2 domain sequences indicated that these strains represented more than one species . Type or authentic strains of the synonyms of Sporobolomyces roseus and the closely related species Sporidiobolus pararoseus Fell & Tallman were employed in the rDNA sequence comparison . Sporobolomyces boleticola Ramirez, Sporobolomyces pollaccii Verona & Ciferri, Sporobolomyces roseus var . madurae Janke and Torulopsis somala Verona were confirmed to be conspecific with Sporobolomyces roseus . Another synonym of this species, Sporobolomyces salmoneus Derx, was located together with Sporobolomyces marcillae Santa Maria in a separate clade . Two synonyms of Sporidiobolus pararoseus, Sporobolomyces carnicolor Yamasaki & Fujii (nom . inval.) and Sporobolomyces japonicus Iizuka & Goto, were revealed to represent two distinct species . The name Sporobolomyces carnicolor is validated, with strain CBS 4215(T) as the type strain . A novel species represented by five of the selected Yunnan strains was confirmed, for which the name Sporobolomyces phaffii sp . nov . is proposed (type strain CH 2.052(T) = AS 2.2137(T) = JCM 11491(T) = CBS 9129(T)) . This study also indicates that yeast species with similar ITS sequences may have quite different D1/D2 sequences.

J Cell Sci, 2003 Feb 1, 116(Pt 3), 453 - 61
Functional interaction of megalin with the megalinbinding protein (MegBP), a novel tetratrico peptide repeat-containing adaptor molecule; Petersen HH et al.; Megalin is a member of the LDL receptor gene family that plays an important role in forebrain development and in cellular vitamin D metabolism through endocytic uptake of vitamin D metabolites . Similar to other receptors in this gene family, megalin is believed to functionally interact with intracellular proteins through adaptors that bind to the receptor tail and regulate its endocytic and signal transducing activities . Using yeast two-hybrid screens, we identified a novel scaffold protein with tetratrico peptide repeats, the megalin-binding protein (MegBP) that associates with the receptor . The binding site of MegBP was mapped to an N-terminal region on the receptor tail harboring a proline-rich peptide element . MegBP binding did not block the endocytic activity of the receptor; however, overexpression resulted in cellular lethality . In further screens, we identified proteins that bound to MegBP and thus might be recruited to the megalin tail . MegBP-interacting partners included several transcriptional regulators such as the SKI-interacting protein (SKIP), a co-activator of the vitamin D receptor . These finding suggest a model whereby megalin directly participates in transcriptional regulation through controlled sequestration or release of transcription factors via MegBP.

J Cell Sci, 2003 Feb 1, 116(Pt 3), 447 - 51
Rescue of arrested RNA polymerase II complexes; Svejstrup JQ; In the past few months, several discoveries relating to the mechanism underlying transcription-coupled DNA repair (TCR) have been reported . These results make it timely to propose a hypothesis for how eukaryotic cells might deal with arrested RNA polymerase II (Pol II) complexes . In this model, the transcription-repair coupling factor Cockayne Syndrome B (or the yeast equivalent Rad26) uses DNA translocase activity to remodel the Pol II-DNA interface, possibly to push the polymerase past the obstruction or to remove it from the DNA so that repair can take place if the obstacle is a DNA lesion . However, when this action is not possible and Pol II is left irreversibly trapped on DNA, the polymerase is instead ubiquitylated and eventually removed by proteolysis.

J Exp Bot, 2003 Jan, 54(382), 477 - 88
Decreased sucrose content triggers starch breakdown and respiration in stored potato tubers (Solanum tuberosum); Hajirezaei MR et al.; To change the hexose-to-sucrose ratio within phloem cells, yeast-derived cytosolic invertase was expressed in transgenic potato (Solanum tuberosum cv . Desiree) plants under control of the rolC promoter . Vascular tissue specific expression of the transgene was verified by histochemical detection of invertase activity in tuber cross-sections . Vegetative growth and tuber yield of transgenic plants was unaltered as compared to wild-type plants . However, the sprout growth of stored tubers was much delayed, indicating impaired phloem-transport of sucrose towards the developing bud . Biochemical analysis of growing tubers revealed that, in contrast to sucrose levels, which rapidly declined in growing invertase-expressing tubers, hexose and starch levels remained unchanged as compared to wild-type controls . During storage, sucrose and starch content declined in wild-type tubers, whereas glucose and fructose levels remained unchanged . A similar response was found in transgenic tubers with the exception that starch degradation was accelerated and fructose levels increased slightly . Furthermore, changes in carbohydrate metabolism were accompanied by an elevated level of phosphorylated intermediates, and a stimulated rate of respiration . Considering that sucrose breakdown was restricted to phloem cells it is concluded that, in response to phloem-associated sucrose depletion or hexose elevation, starch degradation and respiration is triggered in parenchyma cells . To study further whether elevated hexose and/or hexose-phosphates or decreased sucrose levels are responsible for the metabolic changes observed, sucrose content was decreased by tuber-specific expression of a bacterial sucrose isomerase . Sucrose isomerase catalyses the reversible conversion of sucrose into palatinose, which is not further metabolizable by plant cells . Tubers harvested from these plants were found to accumulate high levels of palatinose at the expense of sucrose . In addition, starch content decreased slightly, while hexose levels remained unaltered, compared with the wild-type controls . Similar to low sucrose-containing invertase tubers, respiration and starch breakdown were found to be accelerated during storage in palatinose-accumulating potato tubers . In contrast to invertase transgenics, however, no accumulation of phosphorylated intermediates was observed . Therefore, it is concluded that sucrose depletion rather than increased hexose metabolism triggers reserve mobilization and respiration in stored potato tubers.

Bioresour Technol, 2003 May, 87(3), 359 - 62
Effect of media supplements and culture conditions on inulinase production by an actinomycete strain; Gill PK et al.; Streptomyces sp . GNDU 1 produced high levels of extra-cellular inulinase (0.552 IU/ml) after 24 h at pH 7.5, temperature 46 degrees C in the presence of 1% inulin . The optimum temperature and pH for enzyme activity were 60 degrees C and 5.5 respectively . Yeast extract as a nitrogen source was found to be most suitable one for inulinase production whereas ammonium ion was inhibitory to the enzymatic production . All these conditions make Streptomyces sp . GNDU 1, a potential candidate for industrial enzymatic production of fructose from inulin.

Biochem Biophys Res Commun, 2003 Jan 17, 300(3), 637 - 44
GATE-16 and GABARAP are authentic modifiers mediated by Apg7 and Apg3; Tanida I et al.; GATE-16, GABARAP, and LC3 are three mammalian counterparts of yeast Apg8p/Aut7p . Here, we show that GATE-16 and GABARAP are authentic modifiers, as is the case of LC3 modification . The C-terminal Phe(117) of proGATE-16 and the C-terminal Leu(117) of proGABARAP are post-translationally cleaved to expose an essential Gly(116) within GATE-16 and GABARAP, with the products designated GATE-16-I and GABARAP-I, respectively . The Gly(116) within GATE-16 and GABARAP are essential for further formation of the intermediates between them and Apg7p(C572S) and Apg3p(C264S) . When Apg7p and Apg3p are expressed, GATE-16-I and GABARAP-I are modified to a secondary ubiquitin-like modified form, GATE-16-II and GABARAP-II, respectively . GATE-16-I and GABARAP-I, but not LC3-I, localize to membrane compartments before their modification . These results indicate that GATE-16 and GABARAP are authentic modifiers, but that they have different biochemical characteristics from those of LC3.

J Mol Biol, 2003 Jan 24, 325(4), 785 - 94
The crystal structure at 2A resolution of the Ca2+ -binding protein S100P; Zhang H et al.; S100P is a small calcium-binding protein of the S100 EF-hand-containing family of proteins . Elevated levels of its mRNA are reported to be associated with the progression to hormone independence and metastasis of prostate cancer and to be associated with loss of senescence in human breast epithelial cells in vitro . The first structure of human recombinant S100P in calcium-bound form is now reported at 2.0A resolution by X-ray diffraction . A flexible linker connects the two EF-hand motifs . The protein exists as a homodimer formed by non-covalent interactions between large hydrophobic areas on monomeric S100P . Experiments with an optical biosensor to study binding parameters of the S100P monomer interaction showed that the association rate constant was faster in the presence of calcium than in their absence, whereas the dissociation rate constant was independent of calcium . The K(d) values were 64(+/-24)nM and 2.5(+/-0.8) microM in the presence and in the absence of calcium ions, respectively . Dimerization of S100P is demonstrated in vivo using the yeast two-hybrid system . The effect of mutation of specific amino acids suggests that dimerization in vivo can be affected by amino acids on the dimer interface and in the hydrophobic core.

Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 358 - 63 Epub 2002 Dec 27.
NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants; Moon H et al.; NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation . However, much less is known about the functional significance of NDPKs in plants . We show here that NDPK is associated with H(2)O(2)-mediated mitogen-activated protein kinase signaling in plants . H(2)O(2) stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2) . Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants . Mutants lacking AtNDPK2 had higher levels of ROS than wild type . H(2)O(2) treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H(2)O(2)-activated A . thaliana mitogen-activated protein kinases . In the absence of H(2)O(2) treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant . Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6 . Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3 in vitro . Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ . Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H(2)O(2)-mediated MAPK signaling in plants.

Proc Natl Acad Sci U S A, 2003 Jan 7, 100(1), 289 - 94 Epub 2002 Dec 27.
Disrupted-in-Schizophrenia-1 (DISC-1): mutant truncation prevents binding to NudE-like (NUDEL) and inhibits neurite outgrowth; Ozeki Y et al.; Disrupted-in-Schizophrenia-1 (DISC-1) is a gene whose mutant truncation is associated with major psychiatric illness with a predominance of schizophrenic symptomatology . We have cloned and characterized rodent DISC-1 . DISC-1 expression displays pronounced developmental regulation with the highest levels in late embryonic life when the cerebral cortex develops . In yeast two-hybrid analyses, DISC-1 interacts with a variety of cytoskeletal proteins . One of these, NudE-like (NUDEL), is associated with cortical development and is linked to LIS-1, the disease gene for a form of lissencephaly, a disorder of cortical development . The disease mutant form of DISC-1 fails to bind NUDEL . Expression of mutant, but not wild-type, DISC-1 in PC12 cells reduces neurite extension . As schizophrenia is thought to reflect defects in cortical development that are determined by cytoskeletal protein activities, the cellular disturbances we observe with mutant DISC-1 may be relevant to psychopathologic mechanisms.

J Biol Chem, 2003 Mar 7, 278(10), 8118 - 25 Epub 2002 Dec 27.
Negative regulation of MAPKK by phosphorylation of a conserved serine residue equivalent to Ser212 of MEK1; Gopalbhai K et al.; The MAPKKs MEK1 and MEK2 are activated by phosphorylation, but little is known about how these enzymes are inactivated . Here, we show that MEK1 is phosphorylated in vivo at Ser(212), a residue conserved among all MAPKK family members . Mutation of Ser(212) to alanine enhanced the basal activity of MEK1, whereas the phosphomimetic aspartate mutation completely suppressed the activation of both wild-type MEK1 and the constitutively activated MEK1(S218D/S222D) mutant . Phosphorylation of Ser(212) did not interfere with activating phosphorylation of MEK1 at Ser(218)/Ser(222) or with binding to ERK2 substrate . Importantly, mimicking phosphorylation of the equivalent Ser(212) residue of the yeast MAPKKs Pbs2p and Ste7p similarly abrogated their biological function . Our findings suggest that Ser(212) phosphorylation represents an evolutionarily conserved mechanism involved in the negative regulation of MAPKKs.

FEBS Lett, 2003 Jan 2, 533(1-3), 14 - 20
Characterization of a new family of proteins that interact with the C-terminal region of the chromatin-remodeling factor CHD-3; Lemos TA et al.; The two human proteins Ki-1/57 and CGI-55 have highly similar amino acid sequences but their functions are unknown . We analyzed them by yeast two-hybrid screens and found that they interact with the C-terminal region of the human chromatin-remodeling factor CHD-3 (chromo-helicase-DNA-binding domain protein-3) . The interaction of CGI-55 and CHD-3 could be confirmed in vitro and in vivo by co-immunoprecipitations from Sf9 insect cells . Mapping showed that CGI-55 interacts with CHD-3 via two regions at its N- and C-terminals . The CGI-55 and Ki-1/57 mRNAs show highest expression in muscle, colon and kidney . A CGI55-GFP fusion protein was localized in the cytoplasm, nucleus and perinuclear regions of HeLa cells . These data suggest the possibility that CGI-55 and Ki-1/57 might be involved in nuclear functions like the remodeling of chromatin.

Mol Cell Neurosci, 2002 Dec, 21(4), 575 - 83
The insulin receptor substrate IRSp53 links postsynaptic shank1 to the small G-protein cdc42; Soltau M et al.; The multidomain shank/ProSAP/SSTRIP proteins are major scaffold proteins in glutamatergic synapses in the mammalian brain; expression of shank1/SSTRIP in hippocampal neurons induces morphological changes in dendritic spines, suggesting that shank1 is involved in synapse formation and activity-dependent changes of synaptic structure . Using part of the proline-rich region of shank1 in a yeast two hybrid screen, we identified the insulin receptor substrate IRSp53 as an interaction partner . Overlay assays verified a strong interaction between a proline-rich sequence (residues 911-940) in shank1 and the SH3 domain of IRSp53 . When coexpressed in HEK cells, shank1 colocalizes with IRSp53 in intracellular structures, preventing targeting of IRSp53 to filopodia which are induced by IRSp53 expression in the absence of shank1 . IRSp53 also binds to the activated form of the small G-protein cdc42 . Interestingly, IRSp53 coprecipitates with shank1 from transfected HEK cells in a small G-protein-regulated manner . Thus, IRSp53 constitutes a cdc42-regulated ligand for shank1 which may provide a molecular basis for small G-protein mediated effects on the structure of the postsynaptic complex.

Biochem Biophys Res Commun, 2003 Jan 10, 300(2), 494 - 500
The c-Cbl-associated protein and c-Cbl are two new partners of the SH2-containing inositol polyphosphate 5-phosphatase SHIP2; Vandenbroere I et al.; SHIP2 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains motifs susceptible to mediate protein-protein interaction . Using yeast two-hybrid, GST-pulldown, and coimmunoprecipitation studies, we isolated the CAP cDNA as a specific partner of SHIP2 proline-rich domain and showed by GST-pulldown experiments that the interaction took place with the SH3C of CAP . The interaction was not modulated in COS-7 cells stimulated by EGF neither in CHO cells overexpressing the insulin receptor in the presence or absence of insulin stimulation . We also showed that SHIP2 was able to coimmunoprecipitate with endogenous c-Cbl protein in the absence of CAP and with the insulin receptor in CHO-IR cell extracts . The presence of SHIP2 in a complex around the insulin receptor could account for the very specific increase in insulin sensitivity of SHIP2 knock-out mice.

Int Rev Cytol, 2003, 222, 237 - 93
Comparative analysis of spore coat formation, structure, and function in Dictyostelium; West CM; Dictyostelium produces spores at the end of its developmental cycle to propagate the lineage . The spore coat is an essential feature of spore biology contributing a semipermeable chemical and physical barrier to protect the enclosed amoeba . The coat is assembled from secreted proteins and a polysaccharide, and from cellulose produced at the cell surface . They are organized into a polarized molecular sandwich with proteins forming layers surrounding the microfibrillar cellulose core . Genetic and biochemical studies are beginning to provide insight into how the deliveries of protein and cellulose to the cell surface are coordinated and how cysteine-rich domains of the proteins interact to form the layers . A multidomain inner layer protein, SP85/PsB, seems to have a central role in regulating coat assembly and contributing to a core structural module that bridges proteins to cellulose . Coat formation and structure have many parallels in walls from plant, algal, yeast, protist, and animal cells.

J Virol, 2003 Jan, 77(2), 943 - 52
The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes; Yoshimatsu K et al.; Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA) . Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers . Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers . The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization . The NP-NP interaction was also detected by using a yeast two-hybrid assay . Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast . The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast . However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA . Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA . Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.

Microbiol Res, 2002, 157(4), 283 - 92
Colony variation in Sinorhizobium meliloti inoculant strain U 45; Bloem JF et al.; A culture of Sinorhizobium meliloti strain U 45, maintained on yeast extract-mannitol (YM) agar, produced a mixture of Congo red-absorbing (R1) and non-absorbing (W1) colonies when grown on YM medium containing Congo red . The original freeze-dried (FD) culture formed gummy (G), white (W2) and small red (R2) colony types on the above medium . All colonies were stable except G, which segregated into G and W2-like types . Immune diffusion patterns of all colony types were identical . The W1 colony type dominated R1 when a 1:1 combination was sub-cultured on YM agar . The parent cultures and their variants exhibited a range of N2-fixing effectiveness and competitiveness when inoculated onto two cultivars of Medicago sativa . Variant R2 from the FD culture was ineffective on both cultivars . Genomic DNA fingerprinting with insertion elements ISRm3 and ISRm2011-2 suggested that transposition of these elements was not a cause of variation, but a DNA band was absent in the profiles of two out of three W2-like colonies . Protein profile comparisons showed high similarity (r = 0.98) between the colony types when grown in YM broth . When grown on Tryptone-Yeast extract medium, variants from the FD and agar-maintained cultures formed separate clusters with r = 0.79 . Polymerase chain reaction fingerprinting using repetitive, site-directed and arbitrary primers failed to differentiate the variants . The results emphasize the need to monitor culture variability to maintain the quality of legume inoculants.

J Biol Chem, 2003 Feb 28, 278(9), 7146 - 53 Epub 2002 Dec 24.
The chaperone protein 14-3-3 interacts with 3BP2/SH3BP2 and regulates its adapter function; Foucault I et al.; Lymphocyte stimulation by immunoreceptors is achieved through the activation of multiple signaling pathways leading to cytokine gene transcription . Adapter proteins are critical signaling components that can integrate multiple pathways by allowing the assembly of multimolecular signaling complexes . We previously showed that the cytoplasmic adapter 3BP2 (also known as SH3BP2) promotes NFAT/AP-1 transcriptional activities in T cells through the activation of Ras- and calcineurin-dependent pathways . However, the molecular mechanisms by which 3BP2/SH3BP2 regulates cell signaling and activation remain poorly documented . In this study, using a combination of yeast two-hybrid analysis and biochemical approaches, we present evidence for a physical interaction between 3BP2 and the chaperone protein 14-3-3 . This interaction was direct and constitutively detected in yeast and in mammalian cells . Phorbol ester, pervanadate, and forskolin/isobutylmethylxanthine stimulations enhanced this interaction, as well as co-expression of constitutive active mutants of serine/threonine kinases, including protein kinase C . We found that dephosphorylation of 3BP2 by alkaline phosphatase disrupted its interaction with 14-3-3 and that 3BP2 was a substrate of purified protein kinase C in vitro, suggesting that the phosphorylation of 3BP2 by upstream kinases was required for 14-3-3 binding . Using deletion mutants of 3BP2, two 14-3-3 binding domains were mapped to two proline-rich (residues 201-240 and 270-310) domains of 3BP2 . These domains were shown to contain two 14-3-3 consensus binding motifs . We identified residues Ser(225) and Ser(277) of 3BP2 as being essential for interaction with 14-3-3 family proteins, optimal 3BP2 serine phosphorylation, and then for 3BP2-dependent function . Indeed, a 3BP2 mutant protein incapable of binding 14-3-3 showed increased capacity to stimulate NFAT transcriptional activities, suggesting that 14-3-3 binding to 3BP2 negatively regulates 3BP2 adapter function in lymphocytes.

Indian J Physiol Pharmacol, 2002 Apr, 46(2), 235 - 40
Evaluation of analgesic, antipyretic and anti-inflammatory activity of spirobarbitunylphenothiazines in rodents; Sharma S et al.; Analgesic, antipyretic and anti-inflammatory activities of newly synthesized spirobarbitunylphenothiazines viz 10-{7, 11-Di(4-4' dimethoxphenyl)-3-oxo-9-methylaminoimino-2, 4-diazaspiro {5.5} undecane 1, 5 dione} acetylphenothiazine (test drug A) and 10-{7, 11-Di (N.N-dimethylaminophenyl)-3-oxo-9-methylaminoimino-2, 4-diazaspiro {5, 5} undecane-1, 5 dione} acetylphenothiazine (test drug B) have been screened in Swiss mice and Wistar rats . The peripheral analgesic activity of test drugs A and B was investigated by acetic acid induced writhing test in Swiss mice while the central analgesic action was assessed by hot-wire (tail flick test) of the analgesiometer and tail-clip test in Wistar rats . Antipyretic activity was assessed on Brewer's yeast induced pyrexic model while antiinflammatory activity was seen on carrageenan induced hind paw oedema . Analgesic activity was found to be only of peripheral type as there was reduction of 66% in writhing responses by test drugs A and B in dose of 80 mg/kg in mice . No change in the tail flick responses was observed on analgesiometer or by tail clip by both the test drugs . Reduction of 1.5 to 2.0 degrees C in rectal temperature was observed in pyretic rats by test drugs A and B in dose of 80 mg/kg . 80% reduction in paw volume was noted in 80 mg/kg dose of both the test drugs which was comparable to the anti-inflammatory activity of 300 mg/kg, p.o . of phenylbutazone.

Biol Pharm Bull, 2002 Dec, 25(12), 1639 - 41
Determination of ethanol in biological samples by gas chromatography with an electron-capture detection; Otsuka M; A simple and sensitive procedure for determination of ethanol in biological samples was established . In this procedure, ethanol in biological samples was first converted to acetaldehyde by yeast alcohol dehydrogenase . Then, acetaldehyde formed was derivatized to 2,4-dinitrophenylhydrazone, which was determined by gas chromatography with an electron-capture detection . Ethanol concentration in 1 ml of rat blood plasma can be measured as 19.5 nmol/ml with 98.0% recovery . Since this procedure enable to determine minute amount of ethanol in biological samples, the method is useful to study the metabolism of ethanol.

Hum Mol Genet, 2003 Jan 15, 12(2), 155 - 67
Interaction of alphaPIX (ARHGEF6) with beta-parvin (PARVB) suggests an involvement of alphaPIX in integrin-mediated signaling; Rosenberger G et al.; Members of the Rho GTPase family are key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton . It is now well established that these small GTPases are also crucial for neuronal morphogenesis and connectivity . Moreover, mutations in ARHGEF6 (also known as alphaPIX or Cool-2 ), encoding a Rac1/Cdc42-specific guanine nucleotide exchange factor, have been implicated in X-linked mental retardation . In an attempt to get insight into the biological function of ARHGEF6 and the upstream signaling cascades leading to its activation, we used the full-length coding region of ARHGEF6 as bait in yeast-two hybrid screens and identified PARVB (beta-parvin or affixin) as a novel binding partner . The interaction was confirmed by co-immunoprecipitation and GST pull-down . We showed by immunofluorescence that ARHGEF6 and PARVB co-localize at the cell periphery to lamellipodia and ruffles in well-spread and actively spreading cells adhered to fibronectin . In addition, interaction of ARHGEF6 to ARHGEF7 (betaPIX or Cool-1), a close homolog of ARHGEF6, was confirmed . In in vivo assays, two ARHGEF6 mutations identified previously in patients with X-linked non-specific mental retardation, ARHGEF6 deltaaa56-83 and deltaaa396-776, abolished interaction of ARHGEF6 to PARVB . Binding between ARHGEF6 and ARHGEF7 was not affected by ARHGEF6 deltaaa56-83 but did not occur with ARHGEF6 deltaaa396-776 . These data suggest that both the N-terminal calponin homology (CH) and C-terminal coiled-coil domains are necessary for the ARHGEF6-PARVB binding . In contrast, it seems that only the coiled-coil domain is required for the interaction and heterodimerization of ARHGEF6 and ARHGEF7 . PARVB is known to interact with integrin-linked kinase (ILK) and is involved in the early stage of cell-substrate interaction through integrins . The identification of PARVB as an ARHGEF6 interacting partner together with the co-localization of ARHGEF6 and ILK in spreading cells suggest that ARHGEF6 is involved in integrin-mediated signaling leading to activation of the GTPases Rac1 and/or Cdc42.

J Biol Chem, 2003 Mar 7, 278(10), 8786 - 94 Epub 2002 Dec 23.
The alpha-helical D1 domain of the tobacco bZIP transcription factor BZI-1 interacts with the ankyrin-repeat protein ANK1 and is important for BZI-1 function, both in auxin signaling and pathogen response; Kuhlmann M et al.; The tobacco (Nicotiana tabacum) bZIP transcription factor BZI-1 is involved in auxin-mediated growth responses and in establishing pathogen defenses . Transgenic plants expressing a dominant-negative BZI-1-DeltaN derivative, which lacks the N-terminal activation domain, showed altered vegetative growth . In particular auxin-induced rooting and formation of tobacco mosaic virus-induced hypersensitive response lesions are affected . BZI-1-related proteins described in various plant species share the conserved domains D1, D2, BD, and D4 . To define those BZI-1 domains involved in transcription factor function, BZI-1 deletion derivatives were expressed in transgenic plants . The domains D1 or BD are crucial for BZI-1-DeltaN function in planta . The basic BD domain is mediating DNA binding of BZI-1 . Yeast two-hybrid and in vitro binding studies reveal the ankyrin-repeat protein ANK1, which specifically interacts with a part of the BZI-1 protein (amino acids 73-222) encoding the D1 domain . ANK1 does not bind DNA or act as a co-activator of BZI-1-mediated transcription . Moreover, green fluorescence protein localization studies propose that ANK1 is acting mainly inside the cytosol . Transcription analysis reveals that ANK1 is ubiquitously expressed, but after pathogen attack transcription is transiently down-regulated . Along these lines, ANK1 homologous proteins in Arabidopsis thaliana have been reported to function in pathogen defense . We therefore propose that the D1 domain serves as an interaction surface for ANK1, which appears to regulate BZI-1 function in auxin signaling as well as pathogen response.

J Ethnopharmacol, 2003 Jan, 84(1), 31 - 5
Anti-inflammatory, antipyretic and antinociceptive activities of Tabernaemontana pandacaqui Poir; Taesotikul T et al.; Studies on carrageenin-induced rat paw edema, yeast-induced hyperthermia in rat and writhing response induced by acetic acid in mice showed that the alcoholic extract of stems of Tabernaemontana pandacaqui (T . pandacaqui) has significant anti-inflammatory, antipyretic and antinociceptive activities . These activities are due to alkaloidal components since they were also observed when the crude alkaloidal (CA) fraction separated from alcoholic extract was tested in the same models .

Mol Cell Neurosci, 2002 Nov, 21(3), 454 - 62
Interaction of synaptophysin with the AP-1 adaptor protein gamma-adaptin; Horikawa HP et al.; Synaptophysin is one of the most abundant proteins of the synaptic vesicle membrane . Here, we selected the cytoplasmic carboxyterminal region of synaptophysin to search for interacting proteins by using the yeast two-hybrid system . This identified gamma-adaptin, a component of the AP-1 adaptor complex, as a synaptophysin binding protein . An anti-synaptophysin antibody coimmunoprecipitated gamma-adaptin from brain extracts, and immunocytochemistry disclosed a partial colocalization of synaptophysin and gamma-adaptin in the perinuclear region of cultured hippocampal neurons . Our results are consistent with synaptophysin serving as a docking site for AP-1 during clathrin-dependent vesicle budding and/or kinesin-based transport reactions.

Curr Biol, 2002 Dec 23, 12(24), 2142 - 6
The cell cycle regulatory factor TAF1 stimulates ribosomal DNA transcription by binding to the activator UBF; Lin CY et al.; Control of ribosome biogenesis is a potential mechanism for the regulation of cell size during growth, and a key step in regulating ribosome production is ribosomal RNA synthesis by RNA polymerase I (Pol I) . In humans, Pol I transcription requires the upstream binding factor UBF and the selectivity factor SL1 to assemble coordinately on the promoter . UBF is an HMG box-containing factor that binds to the rDNA promoter and activates Pol I transcription through its acidic carboxy-terminal tail . Using UBF (284-670) as bait in a yeast two-hybrid screen, we have identified an interaction between UBF and TAF1, a factor involved in the transcription of cell cycle and growth regulatory genes . Coimmunoprecipitation and protein-protein interaction assays confirmed that TAF1 binds to UBF . Confocal microscopy showed that TAF1 colocalizes with UBF in Hela cells, and cell fractionation experiments provided further evidence that a portion of TAF1 is localized in the nucleolus, the organelle devoted to ribosomal DNA transcription . Cotransfection and in vitro transcription assays showed that TAF1 stimulates Pol I transcription in a dosage-dependent manner . Thus, TAF1 may be involved in the coordinate expression of Pol I- and Pol II-transcribed genes required for protein biosynthesis and cell cycle progression.

Curr Biol, 2002 Dec 23, 12(24), 2066 - 75
A Formin Homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C . elegans; Severson AF et al.; BACKGROUND: F-actin is enriched at the cortex of embryonic cells in the nematode Caenorhabditis elegans and is required for multiple processes that include the establishment of an anterior-posterior (A-P) axis and cytokinesis . However, the mechanisms that regulate cortical microfilament (MF) assembly remain poorly understood . RESULTS: We show here that a profilin called PFN-1 accumulates at the cortex independent of the actin cytoskeleton and is required for the assembly or maintenance of cortical MFs and myosin . Reducing PFN-1 levels by RNAi results in cytokinesis and A-P polarity defects . PFN-1 binds to the Formin Homology (FH) protein CYK-1, which also is required for cortical MFs . In contrast to PFN-1 and CYK-1, the Arp2/3 complex appears to be dispensable for the assembly of cortical MFs, for A-P polarity, and for cytokinesis . Instead, the Arp2/3 complex is required for cell migrations that occur during gastrulation and may also be involved in cellular rearrangements required for epidermal enclosure prior to elongation of ovoid embryos into vermiform larvae . CONCLUSIONS: We conclude that the FH protein CYK-1 and the profilin PFN-1 mediate the Arp2/3-independent assembly of MFs and are required for cytokinesis in the early embryo . These data suggest that CYK-1 and PFN-1 may nucleate MFs, as has recently been shown for an FH protein and a profilin in yeast.

Tohoku J Exp Med, 2002 Feb, 196(2), 65 - 70
Investigation of intracellular factors involved in methylmercury toxicity; Naganuma A et al.; Methylmercury is a known pollutant that causes severe central nervous system disorders . It is capable of passing through the blood-brain barrier and accumulates in cerebral cells . However, little is known regarding the mechanism of its toxicity at the molecular level . Using yeast cells, we searched for the genes involved in the expression of methylmercury toxicity, and found that genes encoding L-glutamine.D-fructose-6-phosphate amidotransferase (GFAT) and ubiquitin transferase (Ubc3) confer methylmercury resistance on the cells . It has also been shown that GFAT is the target molecule of methylmercury in yeast cells . These findings provide important clues about the mechanism underlying methylmercury toxicity in mammals.

Eur J Cell Biol, 2002 Nov, 81(11), 577 - 84
A novel family of nuclear transport receptors mediates the export of messenger RNA to the cytoplasm; Izaurralde E; Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis . A general feature of mRNA export is that it is an active, receptor-mediated process . The mRNA export receptors are thought to recognize and bind to the mRNA-export cargoes either directly or indirectly (via adaptor proteins) and facilitate their translocation across the central channel of the nuclear pore complex (NPC) . On the cytoplasmic side of the NPC, the exported mRNA is released and the receptor returns to the nucleoplasm, without the cargo, to initiate additional rounds of export . Recent, studies in yeast and in higher eukaryotes have led to the elucidation of an evolutionarily conserved pathway for the export of bulk mRNA to the cytoplasm.

J Immunol, 2003 Jan 1, 170(1), 487 - 94
Identification of heat shock protein 60 as the ligand on Histoplasma capsulatum that mediates binding to CD18 receptors on human macrophages; Long KH et al.; Histoplasma capsulatum (Hc), is a facultative intracellular fungus that binds to CD11/CD18 receptors on macrophages (Mphi) . To identify the ligand(s) on Hc yeasts that is recognized by Mphi, purified human complement receptor type 3 (CR3, CD11b/CD18) was used to probe a Far Western blot of a detergent extract of Hc cell wall and cell membrane . CR3 recognized a single 60-kDa protein, which was identified as heat shock protein 60 (hsp60) . Biotinylation of viable yeasts, followed by precipitation with streptavidin-coated beads, and Western blotting with anti-hsp60 demonstrated that hsp60 was on the surface of Hc yeasts . Electron and confocal microscopy revealed that hsp60 resided on the yeast cell wall in discrete clusters . Recombinant hsp60 (rhsp60) inhibited attachment of Hc yeasts to Mphi . Recombinant hsp60 and Abs to CD11b and CD18 inhibited binding of yeasts to Chinese hamster ovary cells transfected with CR3 (CHO3) . Polystyrene beads coated with rhsp60 bound to Mphi, and attachment was inhibited by Abs to CD11 and CD18 . Freeze/thaw extract (F/TE), a preparation of Hc yeast surface proteins that contained hsp60, inhibited the attachment of Hc yeasts to Mphi . Depletion of hsp60 from F/TE removed the capacity of F/TE to block binding of Hc to Mphi . Interestingly, rhsp60 did not inhibit binding of Hc yeasts to dendritic cells (DC), which recognize Hc via very late Ag 5 . Moreover, F/TE inhibited attachment of Hc to DC even when depleted of hsp60 . Thus, Hc hsp60 appears to be a major ligand that mediates attachment of Hc to Mphi CD11/CD18, whereas DC recognize Hc via a different ligand(s).

J Biol Chem, 2003 Feb 21, 278(8), 6610 - 7 Epub 2002 Dec 22.
Manganese specificity determinants in the Arabidopsis metal/H+ antiporter CAX2; Shigaki T et al.; In plants and fungi, vacuolar transporters help remove potentially toxic cations from the cytosol . Metal/H(+) antiporters are involved in metal sequestration into the vacuole . However, the specific transport properties and the ability to manipulate these transporters to alter substrate specificity are poorly understood . The Arabidopsis thaliana cation exchangers, CAX1 and CAX2, can both transport Ca(2+) into the vacuole . There are 11 CAX-like transporters in Arabidopsis; however, CAX2 was the only characterized CAX transporter capable of vacuolar Mn(2+) transport when expressed in yeast . To determine the domains within CAX2 that mediate Mn(2+) specificity, six CAX2 mutants were constructed that contained different regions of the CAX1 transporter . One class displayed no alterations in Mn(2+) or Ca(2+) transport, the second class showed a reduction in Ca(2+) transport and no measurable Mn(2+) transport, and the third mutant, which contained a 10-amino acid domain from CAX1 (CAX2-C), showed no reduction in Ca(2+) transport and a complete loss of Mn(2+) transport . The subdomain analysis of CAX2-C identified a 3-amino acid region that is responsible for Mn(2+) specificity of CAX2 . This study provides evidence for the feasibility of altering substrate specificity in a metal/H(+) antiporter, an important family of transporters found in a variety of organisms.

J Biol Chem, 2003 Mar 28, 278(13), 11312 - 9 Epub 2002 Dec 19.
The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids; Brown AJ et al.; GPR41 and GPR43 are related members of a homologous family of orphan G protein-coupled receptors that are tandemly encoded at a single chromosomal locus in both humans and mice . We identified the acetate anion as an agonist of human GPR43 during routine ligand bank screening in yeast . This activity was confirmed after transient transfection of GPR43 into mammalian cells using Ca(2+) mobilization and {(35)S}guanosine 5'-O-(3-thiotriphosphate) binding assays and by coexpression with GIRK G protein-regulated potassium channels in Xenopus laevis oocytes . Other short chain carboxylic acid anions such as formate, propionate, butyrate, and pentanoate also had agonist activity . GPR41 is related to GPR43 (52% similarity; 43% identity) and was activated by similar ligands but with differing specificity for carbon chain length, with pentanoate being the most potent agonist . A third family member, GPR42, is most likely a recent gene duplication of GPR41 and may be a pseudogene . GPR41 was expressed primarily in adipose tissue, whereas the highest levels of GPR43 were found in immune cells . The identity of the cognate physiological ligands for these receptors is not clear, although propionate is known to occur in vivo at high concentrations under certain pathophysiological conditions.

J Biol Chem, 2003 Mar 7, 278(10), 8837 - 45 Epub 2002 Dec 19.
Identification of UNC119 as a novel activator of SRC-type tyrosine kinases; Cen O et al.; Lyn, an Src-type tyrosine kinase, is associated with the interleukin (IL)-5 receptor in eosinophils . The mechanism of its activation is unknown . Through yeast two-hybrid screening we have cloned and characterized a new signaling molecule, Unc119, that associates with IL-5Ralpha and Src family tyrosine kinases . Unc119 induces the catalytic activity of these kinases through interaction with Src homology 2 and 3 domains . IL-5 stimulation of eosinophils increases Unc119 association with Lyn and induces its catalytic activity . Lyn is important for eosinophil survival . Eosinophils that are transduced with Unc119 have increased Lyn activity and demonstrate prolonged survival in the absence of IL-5 . Inhibition of Unc119 down-regulates eosinophil survival . To our knowledge Unc119 is the first receptor-associated activator of Src family tyrosine kinases.

J Biol Chem, 2003 Feb 28, 278(9), 7639 - 44 Epub 2002 Dec 20.
Molecular basis of sulfonylurea herbicide inhibition of acetohydroxyacid synthase; Pang SS et al.; Acetohydroxyacid synthase (AHAS) (acetolactate synthase, EC ) catalyzes the first step in branched-chain amino acid biosynthesis and is the target for sulfonylurea and imidazolinone herbicides . These compounds are potent and selective inhibitors, but their binding site on AHAS has not been elucidated . Here we report the 2.8 A resolution crystal structure of yeast AHAS in complex with a sulfonylurea herbicide, chlorimuron ethyl . The inhibitor, which has a K(i) of 3.3 nm, blocks access to the active site and contacts multiple residues where mutation results in herbicide resistance . The structure provides a starting point for the rational design of further herbicidal compounds.

J Biol Chem, 2003 Apr 4, 278(14), 12175 - 81 Epub 2002 Dec 20.
A new member of the LIM protein family binds to filamin B and localizes at stress fibers; Takafuta T et al.; Human filamins are 280-kDa proteins containing an N-terminal actin-binding domain followed by 24 characteristic repeats . They also interact with a number of other cellular proteins . All of those identified to date, with the exception of actin, bind to the C-terminal third of a filamin . In a yeast two-hybrid search of a human placental library, using as bait repeats 10-18 of filamin B, we isolated a cDNA coding for a novel 374 amino acid protein containing a proline-rich domain near its N terminus and two LIM domains at its C terminus . We term this protein filamin-binding LIM protein-1, FBLP-1 . Yeast two-hybrid studies with deletion mutants localized the areas of interaction in FBLP-1 to its N-terminal domain and in filamin B to repeats 10-13 . FBLP-1 mRNA was detected in a variety of tissues and cells including platelets and endothelial cells . We also have identified two FBLP-1 variants . Both contain three C-terminal LIM domains, but one lacks the N-terminal proline-rich domain . Transfection of FBLP-1 into 293A cells promoted stress fiber formation, and both FBLP-1 and filamin B localized to stress fibers in the transfected cells . The association between filamin B and FBLP-1 may play a hitherto unknown role in cytoskeletal function, cell adhesion, and cell motility.

Infect Immun, 2003 Jan, 71(1), 465 - 73
Differential cytokine pattern in the spleens and livers of BALB/c mice infected with Penicillium marneffei: protective role of gamma interferon; Sisto F et al.; Penicillium marneffei is an intracellular opportunistic fungus causing invasive mycosis in AIDS patients . T cells and macrophages are important for protection in vivo . However, the role of T-cell cytokines in the immune response against P . marneffei is still unknown . We studied by semiquantitative reverse transcription-PCR and biological assays the patterns of expression of Th1 and Th2 cytokines in the organs of wild-type (wt) and gamma interferon (IFN-gamma) knockout (GKO) mice infected intravenously with P . marneffei conidia . At 3 x 10(5) conidia/mouse, a self-limiting infection developed in wt BALB/c mice, whereas all GKO mice died at day 18 postinoculation . Splenic and hepatic granulomas were present in wt mice, whereas disorganized masses of macrophages and yeast cells were detected in GKO mice . The infection resolved faster in the spleens than in the livers of wt mice and was associated with the local expression of type 1 cytokines (high levels of interleukin-12 {IL-12} and IFN-gamma) but not type 2 cytokines (low levels of IL-4 and IL-10) . Conversely, both type 1 and type 2 cytokines were detected in the livers of wt animals . Disregulation of the cytokine profile was seen in the spleens but not in the livers of GKO mice . The inducible nitric oxide synthase mRNA level was low and the TNF-alpha level was high in both spleens and livers of GKO mice compared to wt mice . These data suggest that the polarization of a protective type 1 immune response against P . marneffei is regulated at the level of individual organs and that the absence of IFN-gamma is crucial for the activation of fungicidal macrophages and the development of granulomas.

Int J Cancer, 2003 Feb 20, 103(5), 616 - 23
Clustering of deletions on chromosome 13 in benign and low-malignant lipomatous tumors; Dahlen A et al.; Deletions and structural rearrangements of the long arm of chromosome 13 are frequently observed in benign and low-malignant lipomatous tumors, but nothing is known about their molecular genetic consequences . We assessed the karyotypes of 40 new and 22 previously published cases (35 ordinary lipomas, 15 spindle cell/pleomorphic lipomas, 2 myxolipomas, 1 angiomyxolipoma and 9 atypical lipomatous tumors) with chromosome 13-abnormalities, and found bands 13q12-22 to be frequently affected . Twenty-seven cases with structural abnormalities within this region were selected for breakpoint and deletion mapping by metaphase fluorescence in situ hybridization (FISH), using a set of 20 probes . Deletions were found in 23 of 27 cases . The remaining 4 cases had seemingly balanced rearrangements . The breakpoints were scattered but clustered to band 13q14, and in all cases with unbalanced abnormalities, a limited region within band 13q14 was partially or completely deleted . A deletion within band 13q14 was found together with a breakpoint on the other homologue in 5 cases, 4 of which could be tested further with regard to the status of the retinoblastoma (RB1)-gene . In all 4 cases, only 1 copy of the gene was deleted . In addition to the breaks and deletions in the vicinity of the RB1-locus, several other regions of 13q were recurrently affected, e.g., in the vicinity of the hereditary breast cancer (BRCA2; 13q12)- and lipoma HMGIC fusion partner (LHFP; 13q13)- genes . Our findings strongly indicate that deletion of a limited region (approximately 2.5 Mbp) within 13q14, distal to the RB1-locus, is of importance in the development of a subset of lipomatous tumors .

J Exp Bot, 2003 Jan, 54(381), 213 - 21
Seed germination is blocked in Arabidopsis putative vacuolar sorting receptor (atbp80) antisense transformants; Laval V et al.; The membrane receptor protein from pea, peabp80, has been shown to function by in vitro binding studies, and in vivo in yeast mutant, as a vacuolar sorting receptor (VSR) . Families of proteins with homology to peabp80 have been identified in many plants including ARABIDOPSIS: The family of membrane receptors, atbp80a-f (Arabidopsis thaliana binding protein 80 kDa) is highly homologous to peabp80 and may also function as vacuolar sorting receptors . Interactions with vacuolar sorting determinants have been shown only in vitro for atbp80b . In this paper, atbp80b was over- and under-expressed in Arabidopsis . Transgenic plants that over-expressed atbp80b showed no visible phenotype . However, antisense transformants were defective in germination . In non-germinating antisense transformants the embryo appeared to be normal, but, using several methods, it was not possible to rescue the non-germinating seeds, indicating that the mechanisms were probably independent of a seed-coat-imposed inhibition . To make a correlation between the lack of germination and gene expression, transcription analysis of all atbp80 genes was performed in the non-germinating antisense seeds indicating that all the normally transcribed genes were not detected . Then, a gene expression study of atbp80s genes was carried-out following seed imbibition and in various organs during wild-type plant development showing that all the genes from the family were transcribed and differentially expressed.

J Biol Chem, 2003 Mar 7, 278(10), 8460 - 7 Epub 2002 Dec 18.
Grb10 inhibits insulin-stimulated insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase/Akt signaling pathway by disrupting the association of IRS-1/IRS-2 with the insulin receptor; Wick KR et al.; Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context . We have investigated the mechanism by which full-length hGrb10gamma inhibits signaling through the insulin receptor substrate (IRS) proteins . Overexpression of hGrb10gamma in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 . Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation . In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10gamma to both delay and reduce phosphorylation of Akt at Thr(308) and Ser(473) in response to insulin stimulation . Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10gamma directly associates with the IR kinase regulatory loop . However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10 . In cells, hGrb10gamma inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr(972) in the juxtamembrane region and Tyr(1158/1162/1163) in the regulatory domain . We conclude that binding of hGrb10gamma to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.

J Biol Chem, 2003 Feb 28, 278(9), 7486 - 93 Epub 2002 Dec 19.
Basic helix-loop-helix transcription factor epicardin/capsulin/Pod-1 suppresses differentiation by negative regulation of transcription; Funato N et al.; Epicardin/capsulin/Pod-1, expressed in skeletal myoblasts within brachial arches and in the condensing mesenchyme, is a member of the basic helix-loop-helix (bHLH) transcription factor family that is involved in various cell differentiation processes . In this study, we examined the functional properties of epicardin/capsulin/Pod-1 in differentiation . The yeast and mammalian two-hybrid systems showed physical associations between epicardin/capsulin/Pod-1 and E2A, both of which were present in the nuclei . The bHLH domains mediated this association . Ectopic expression of epicardin/capsulin/Pod-1 inhibited E2A-dependent activation of the exogenous and endogenous expression of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) gene, and the muscle creatine kinase gene that encodes the predominant creatine kinase isoform expressed in mammalian skeletal muscle . Transfection with epicardin/capsulin/Pod-1 small interfering RNA abolished the epicardin/capsulin/Pod-1-mediated suppression of E12-dependent activation of the p21 promoter . Chromatin immunoprecipitation assay showed that epicardin/capsulin/Pod-1 was physically associated with the muscle creatine kinase promoter in vivo . Moreover, terminal differentiation of C2C12 myoblasts was inhibited by exogenous introduction of epicardin/capsulin/Pod-1 . These inhibitory functions of epicardin/capsulin/Pod-1 closely resemble those of the bHLH inhibitor Twist protein . These results indicate that epicardin/capsulin/Pod-1 functions as a negative regulator of differentiation of myoblasts through transcription in at least two distinct steps, cell growth arrest and lineage-specific differentiation.

J Biol Chem, 2003 Mar 7, 278(10), 8494 - 500 Epub 2002 Dec 19.
Determinants of Rab5 interaction with the N terminus of early endosome antigen 1; Merithew E et al.; The Rab5 effector early endosome antigen 1 (EEA1) is a parallel coiled coil homodimer with an N-terminal C(2)H(2) Zn(2+) finger and a C-terminal FYVE domain . Rab5 binds to independent sites at the N and C terminus of EEA1 . To gain further insight into the structural determinants for endosome tethering and fusion, we have characterized the interaction of Rab5C with truncation and site-specific mutants of EEA1 using quantitative binding measurements . The results demonstrate that the C(2)H(2) Zn(2+) finger is both essential and sufficient for the N-terminal interaction with Rab5 . Although the heptad repeat C-terminal to the C(2)H(2) Zn(2+) finger provides the driving force for stable homodimerization, it does not influence either the affinity or stoichiometry of Rab5 binding . Hydrophobic residues predicted to cluster on a common face of the C(2)H(2) Zn(2+) finger play a critical role in the interaction with Rab5 . Although the homologous C(2)H(2) Zn(2+) finger of the Rab5 effector Rabenosyn binds to Rab5 with comparable affinity, the analogous C(2)H(2) Zn(2+) finger of the yeast homologue Vac1 shows no detectable interaction with Rab5, reflecting non-conservative substitutions of critical residues . Large changes in the intrinsic tryptophan fluorescence of Rab5 accompany binding to the C(2)H(2) Zn(2+) finger of EEA1 . These observations can be explained by a mode of interaction in which a partially exposed tryptophan residue located at the interface between the switch I and II regions of Rab5 lies within a hydrophobic interface with a cluster of non-polar residues in the C(2)H(2) Zn(2+) finger of EEA1.

Am J Respir Crit Care Med, 2003 Apr 1, 167(7), 991 - 8 Epub 2002 Dec 18.
TA-19, a novel protein antigen of Trichosporon asahii, in summer-type hypersensitivity pneumonitis; Matsunaga Y et al.; The most common form of hypersensitivity pneumonitis in Japan is summer-type hypersensitivity pneumonitis (SHP), which is caused by the inhalation of Trichosporon asahii or Trichosporon mucoides . To seek protein antigens relevant to the immunopathogenesis of SHP, we constructed a cDNA expression library of T . asahii, a major causative yeast species of SHP . Using the immunoscreening method, we identified and cloned a novel gene encoding a 19-kD protein, named TA-19, which proved to be specifically recognized in the bronchoalveolar lavage (BAL) fluids and sera of patients with SHP . IgG, IgA, and IgM antibodies to the recombinant TA-19 protein were significantly elevated in the sera as well as in the BAL fluids from SHP patients compared with those from non-SHP groups . This protein also induced SHP-specific proliferation of the mononuclear cells from both the peripheral blood and BAL . These results reveal that TA-19 derived from T . asahii may play a relevant role in specific cellular and humoral immune responses in patients with SHP.

Sci Total Environ, 2003 Jan 1, 301(1-3), 87 - 96
The potential for oestrogenic effects of pesticides in headwater streams in the UK; Hurst MR et al.; Twenty-six pesticidal compounds and samples of stormwater from two different agricultural catchments were screened for oestrogenic activity using an in vitro recombinant yeast-based assay . Only six fungicides showed an oestrogenic response with low comparative biological activity of 5000 to 2.5 million times less potent than 17beta-estradiol (E2), a natural steroidal oestrogen . Concentrations of biological activity expressed as E(2) equivalents for the headwater stream stormwater samples ranged from <0.01 to 0.11 ng E2/l . These values are at least one order of magnitude below levels that have been documented to produce oestrogenic effects in fish and are therefore considered to represent a low risk to associated headwater stream communities . The potential sources of the oestrogenic activity measured in the headwater streams are discussed .

Plant J, 2002 Dec, 32(6), 949 - 60
The cellulose-deficient Arabidopsis mutant rsw3 is defective in a gene encoding a putative glucosidase II, an enzyme processing N-glycans during ER quality control; Burn JE et al.; rsw3 is a temperature-sensitive mutant of Arabidopsis thaliana showing radially swollen roots and a deficiency in cellulose . The rsw3 gene was identified by a map-based strategy, and shows high similarity to the catalytic alpha-subunits of glucosidase II from mouse, yeast and potato . These enzymes process N-linked glycans in the ER, so that they bind and then release chaperones as part of the quality control pathway, ensuring correct protein folding . Putative beta-subunits for the glucosidase II holoenzyme identified in the Arabidopsis and rice genomes share characteristic motifs (including an HDEL ER-retention signal) with beta-subunits in mammals and yeast . The genes encoding the putative alpha- and beta-subunits are single copy and, like the rsw3 phenotype, widely expressed . rsw3 reduces cell number more strongly than cell size in stamen filaments and probably stems . Most features of the rsw3 phenotype are shared with other cellulose-deficient mutants, but some--notably, production of multiple rosettes and a lack of secreted seed mucilage--are not and may reflect glucosidase II affecting processes other than cellulose synthesis . The rsw3 root phenotype develops more slowly than the rsw1 and rsw2 phenotypes when seedlings are transferred to the restrictive temperature . This is consistent with rsw3 reducing glycoprotein delivery from the ER to the plasma membrane whereas rsw1 and rsw2 act more rapidly by affecting the properties of already delivered enzymes.

Eur J Biochem, 2003 Jan, 270(1), 76 - 83
Mixed lineage kinase LZK and antioxidant protein-1 activate NF-kappaB synergistically; Masaki M et al.; Leucine zipper-bearing kinase (LZK) is a novel member of the mixed lineage kinase (MLK) family {Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J . P., and Kawasaki, T . (1997) J . Biol . Chem.272, 28622-28629} . We have previously shown that LZK activates the c-Jun-NH2 terminal kinase (JNK) pathway, but not the extracellular signal-related kinase (ERK) pathway, by acting as a mitogen-activated protein kinase kinase kinase (MAPKKK) {Ikeda, A., Hasegawa, K., Masaki, M., Moriguchi, T., Nishida, E., Kozutsumi, Y., Oka, S., and Kawasaki, T . (2001) J . Biochem.130, 773-781} . However, the mode of activation of LZK remains largely unknown . By means of a yeast two-hybrid screening system, we have identified a molecule localized to mitochondria, antioxidant protein-1 (AOP-1), that binds to LZK and which acts as a modulator of LZK activity . Recently, several MAPKKKs involved in the JNK pathway, such as MEKK1, TAK1 and MLK3, were shown, using over-expression assay systems, to activate a transcription factor, NF-kappaB, through activation of the IKK complex . Using similar assay systems, we demonstrated that LZK activated NF-kappaB-dependent transcription through IKK activation only weakly, but this was reproducible, and that AOP-1 enhanced the LZK-induced NF-kappaB activation . We also provided evidence that LZK was associated directly with the IKK complex through the kinase domain, and that AOP-1 was recruited to the IKK complex through the binding to LZK.

Virology, 2002 Nov 25, 303(2), 232 - 9
Mapping interaction sites of the A20R protein component of the vaccinia virus DNA replication complex; Ishii K et al.; The vaccinia virus A20R protein is required for DNA replication, is associated with the processive form of the viral DNA polymerase, and directly interacts with the viral proteins encoded by the D4R, D5R, and H5R open reading frames as determined by a genome-wide yeast two-hybrid analysis . The purpose of the present study was to further analyze the latter protein-protein interactions . Association of an epitope-tagged A20R protein with an epitope-tagged D4R or H5R protein, expressed in vaccinia virus-infected cells, was demonstrated by binding the complex to one mAb followed by Western blotting with another . Interaction between the A20R and D5R proteins, which was weakest in the yeast two-hybrid analysis, could not be demonstrated by this method . A panel of N- and C-terminal truncated forms of the A20R protein was tested for interaction with the D4R, H5R, and D5R proteins using the yeast two-hybrid system . These studies revealed that nonoverlapping regions of A20R comprising amino acids 1 to 25, 26 to 76, and 201 to 251 were required for binding of D4R, H5R, and D5R, respectively . By contrast, no interaction of A20R with D4R could be detected after deletion of only 25 codons from either end of the latter open reading frame . A fusion protein containing either full-length A20R or only the N-terminal 25 amino acids of A20R was sufficient to capture the D4R protein, whereas the fusion protein containing A20R amino acids 26 to 426 was not, confirming the results of the yeast two-hybrid analysis . The distinct protein binding domains of the A20R protein may contribute to the assembly or stability of the multiprotein DNA replication complex.

J Struct Biol, 2002 Oct-Dec, 140(1-3), 232 - 40
Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos; Cohen M et al.; Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants . However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates . In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM) . We show that the NPCs are bigger in C . elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes . We also localized the C . elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy . Both proteins are present at or near the inner nuclear membrane . A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior . Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin . Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus . Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C . elegans nuclear envelope.

DNA Cell Biol, 2002 Nov, 21(11), 819 - 25
Hox proteins activate the IGFBP-1 promoter and suppress the function of hPR in human endometrial cells; Gao J et al.; Previous studies have shown that progestin activates the transcription of IGFBP-1 (insulin-like growth factor binding protein-1) . Four regions in the IGFBP-1 promotor have been identified to enhance the transcription . Two of the regions, located at -73 to -65 bp and -319 to -311 bp formed identical DNA-protein complexes with the nuclear extracts of endometrial stromal/decidual cells . To identify the binding protein(s) in endometrial cells that interact with these two regions, we have used the TGTCAATTA repeats (-319 to -11 bp of the IGFBP-1 promoter) to screen the human decidual cDNA library by yeast one-hybrid system . We found that Hox A10, HoxA11, HoxB2, HoxB4, and HoxD11 interacted with the TGTCAATTA repeats in yeast cells . Among these hox genes, the full-length coding region of HoxA10, HoxA11, and HoxB4 were used for functional analysis in three types of endometrial cells, undifferentiated endometrial stromal cells, decidual cells (differentiated stromal cells) and endometrial adenocarcinoma cell line (HEC1-B) . All these endometrial cells produce IGFBP-1 . Transient transfection assay showed that HoxA10 expression vector increased the promoter activity (the IGFBP-1 proximal promoter containing TGC/TCAATTA and two functional PRE sites) in endometrial stromal cells and in HEC-1B cells, but not in decidual cells . HoxB4 enhanced the promoter activity only in decidual cells, while HoxA11 had no apparent effect in all three types of cells . To evaluate whether Hox proteins would interact with progesterone receptor (hPR), cells were transfected with the promoter construct, Hox and hPR expression vectors . hPR alone activated the IGFBP-1 promoter activity, but expression of Hox gene suppressed the activation . Hox proteins also suppressed the hPR enhanced promoter activities of MMTV (containing consensus-PRE sites) and glycodelin (GdA, containing Sp1 site which mediates the hPR function) . These data showed that Hox genes selectively activate the transcription of the IGFBP-1 and GdA genes in different types of endometrial cells . Hox genes, however, suppress the hPR enhanced activities . In addition, we found that HoxB4 expression was induced by estrogen and progestin . Other investigators have shown that HoxA10 and 11 were stimulated by progestin . These findings show that Hox proteins are molecular mediators of the steroid hormones during endometrial cell development.

Biochem J, 2003 Apr 15, 371(Pt 2), 385 - 93
Regulation of transcription by the heterogeneous nuclear ribonucleoprotein E1B-AP5 is mediated by complex formation with the novel bromodomain-containing protein BRD7; Kzhyshkowska J et al.; E1B-AP5 was initially identified as a target of the early adenovirus E1B-55 kDa protein during the course of lytic infection . E1B-AP5 belongs to the heterogeneous nuclear ribonucleoprotein family and was demonstrated to be involved in mRNA processing and transport {Gabler, Schutt, Groitl, Wolf, Shenk and Dobner (1998) J . Virol . 72, 7960-7971} . In the present paper, we demonstrate that E1B-AP5 differentially regulates basic and ligand-dependent transcription . We found that E1B-AP5 represses basic transcription driven by several virus and cellular promoters, and mapped the repression activity to the N-terminal part of the protein . In contrast with basic repression, E1B-AP5 activated the glucocorticoid-dependent promoter in the absence of dexamethasone, but did not contribute to the dexamethasone-induced activation . Mutant analysis indicated the presence of an additional cellular factor that modulates E1B-AP5 transcriptional activity . Using yeast two-hybrid screening, we identified a novel chromatin-associated bromodomain-containing protein, BRD7, as an E1B-AP5 interaction partner . We confirmed E1B-AP5-BRD7 complex formation in vivo and in vitro . We found that, although BRD7 binds to histones H2A, H2B, H3 and H4 through its bromodomain, this domain was not necessary for the interaction with E1B-AP5 . Indeed, the triple complex formation of E1B-AP5, BRD7 and histones was demonstrated . Disruption of the E1B-AP5-BRD7 complex increased E1B-AP5 repression activity for basic transcription and converted it from being an activator of the hormone-dependent promoter into being a strong repressor . We conclude that complex formation between BRD7 and E1B-AP5 links chromatin events with mRNA processing at the level of transcriptional regulation.

Connect Tissue Res, 2002, 43(2-3), 308 - 19
Novel functions of the matricellular proteins osteopontin and osteonectin/SPARC; Sodek J et al.; Osteopontin (OPN) and osteonectin/SPARC (ON/SPARC) are prominent matricellular components of the extracellular matrix of mineralized tissues of bones and teeth in which they can regulate the formation and growth of hydroxyapatite crystals and influence a variety of cell activities . OPN regulates cell responses through several integrin receptors and is also a ligand for the CD44 receptor, through which it acts as a chemoattractant . Although a cell-surface receptor for SPARC has not been identified it can block cell-cell and cell-matrix interactions and inhibit cell migration and chemotaxis . OPN and SPARC also appear to function inside cells . Thus, OPN appears to exist in association with the CD44 receptor inside migratory cells, while intracellular SPARC is associated with axonemal tubulin in ciliated epithelial cells . Analyses of fibroblasts and peritoneal macrophages from OPN-null and CD44-null cells show impaired functionality involving migration and cell fusion required for osteoclast formation, while disruption of SPARC expression leads to developmental defects in Xenopus . To gain further insights into the intracellular functions of OPN and SPARC, we have used the yeast two-hybrid system to identify potential interacting molecules . Using full-length SPARC as bait the carboxy-terminal domain, which contains two EF-hand, high-affinity binding sites, was found to have transcriptional activity, while several novel proteins that interact with the amino-terminal domains of SPARC and full-length OPN have been identified . The identification of OPN and SPARC inside specialized cells introduces a novel concept in cellular regulation by matricellular proteins.

Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16666 - 71 Epub 2002 Dec 16.
Z-DNA-binding proteins can act as potent effectors of gene expression in vivo; Oh DB et al.; The role of Z-DNA-binding proteins in vivo is explored in yeast . A conformation-specific yeast one-hybrid system is made in which formation of Z-DNA is studied near a minimal promoter site where it can be stabilized by negative supercoiling in addition to protein binding . Experiments were carried out with a Z-DNA-binding protein domain from the editing enzyme, double-stranded RNA adenosine deaminase 1 . In the one-hybrid system, the reporter gene is activated when a Z-DNA-specific binding domain is fused with an activation domain and expressed in vivo . Significantly, it was found that even in the absence of the activation domain there is substantial transcription of the reporter gene if the Z-DNA-binding protein is expressed in the cell . This result suggests that Z-DNA formation in the promoter region induced or stabilized by a Z-DNA-binding protein can act as a cis-element in gene regulation . Related results have been found recently when the human chromatin-remodeling system converts a segment of DNA in the promoter region of the human colony-stimulating factor 1 gene into the left-handed Z-conformation.

Proc Natl Acad Sci U S A, 2002 Dec 24, 99(26), 16788 - 93 Epub 2002 Dec 16.
Dynamics of microtubule asters in microfabricated chambers: the role of catastrophes; Faivre-Moskalenko C et al.; Recent in vivo as well as in vitro experiments have indicated that microtubule pushing alone is sufficient to position a microtubule-organizing center within a cell . Here, we investigate the effect of catastrophes on the dynamics of microtubule asters within microfabricated chambers that mimic the confining geometry of living cells . The use of a glass bead as the microtubule-organizing center allows us to manipulate the aster by using optical tweezers . In the case in which microtubules preexist, we show that because of microtubule buckling, repositioning almost never occurs after relocation with the optical tweezers, although initial microtubule growth always leads the aster to the geometrical center of the chamber . When a catastrophe promoter is added, we find instead that the aster is able to efficiently explore the chamber geometry even after being relocated with the optical tweezers . As predicted by theoretical calculations, the results of our in vitro experiments clearly demonstrate the need for catastrophes for proper positioning in a confining geometry . These findings correlate with recent observations of nuclear positioning in fission yeast cells.

EMBO J, 2002 Dec 16, 21(24), 6915 - 24
Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures; van Dijk E et al.; We have cloned cDNAs for the human homologues of the yeast Dcp1 and Dcp2 factors involved in the major (5'-3') and NMD mRNA decay pathways . While yeast Dcp1 has been reported to be the decapping enzyme, we show that recombinant human Dcp2 (hDcp2) is enzymatically active . Dcp2 activity appears evolutionarily conserved . Mutational and biochemical analyses indicate that the hDcp2 MutT/Nudix domain mediates this activity . hDcp2 generates m7GDP and 5'-phosphorylated mRNAs that are 5'-3' exonuclease substrates . Corresponding decay intermediates are present in human cells showing the relevance of this activity . hDcp1 and hDcp2 co-localize in cell cytoplasm, consistent with a role in mRNA decay . Interestingly, these two proteins show a non-uniform distribution, accumulating in specific foci.

Genes Cells, 2002 Dec, 7(12), 1231 - 42
Interaction of the chromatin compaction-inducing domain (LR domain) of Ki-67 antigen with HP1 proteins; Kametaka A et al.; BACKGROUND: The LR domain of marsupial chmadrin is defined by its C-terminal amino acid sequence, which contains several pairs of leucine (L) and arginine (R) residues . The LR domain of chmadrin causes a significant compaction of chromatin over the entire length of chromosomes when it is overproduced . The possible human homologue of chmadrin, Ki-67 antigen (pKi-67), also has a stretch of LR pairs, but with no obvious overall similarity, at its C-terminus . RESULTS: The LR domain of human pKi-67 also induced chromatin compaction, both in human and marsupial cells . A yeast two-hybrid assay and an in vitro binding assay demonstrated that the human LR domain binds to heterochromatin protein 1 (HP1), a well-characterized molecule as a mediator of heterochromatin formation . In fixed cells stained with specific antibodies, the pKi-67 was found to be co-localized partially with HP1 at foci on chromosomes in an early G1 phase . Time-lapse observation in living cells co-expressing the fluorescently tagged proteins showed that the LR domain formed foci on chromosomes over a limited period of the cell cycle from the telophase to early G1 phase and that HP1 subsequently accumulated at these foci of the LR domain . CONCLUSIONS: Marsupial chmadrin and human pKi-67 induce chromatin compaction across species, possibly via the interaction of its LR domain with HP1.

Genetica, 2002 Sep, 116(1), 137 - 49
Prospects for using genetic transformation for improved SIT and new biocontrol methods; Handler AM; The genetic manipulation of non-drosophilid insect species is possible by the creation of recombinant DNA constructs that can be integrated into host genomes by several transposon-based vector systems . This technology will allow the development and testing of a variety of systems that can improve existing biological control methods, and the development of new highly efficient methods . For programs such as sterile insect technique (SIT), transgenic strains may include fluorescent protein marker genes for detection of released insects, and conditional gene expression systems that will result in male sterility and female lethality for genetic sexing . Conditional expression systems include the yeast GAL4 system and the bacterial Tet-off and Tet-on systems that can, respectively, negatively or positively regulate expression of genes for lethality or sterility depending on a dietary source of tetracycline . Importantly, strains for male sterility must also incorporate an effective system for genetic sexing, since typically, surviving females would remain fertile . Models for the use of these expression systems and associated genetic material come from studies in Drosophila and, while many of these systems should be transferable to other insects, continued research will be necessary in insects of interest to clone genes, optimize germ-line transformation, and perform vector stability studies and risk assessment for their release as transgenic strains.

J Gen Appl Microbiol, 2000 Dec, 46(6), 297 - 310
Four new species of Kockovaella isolated from plant leaves collected in Vietnam; Luong DT et al.; Five ballistoconidiogenous yeast strains, isolated from plant leaves at Cuc Phuong National Forest of Ninh Binh, Vietnam, were assigned to the genus Kockovaella based on morphological and chemotaxonomical characteristics . They represent four new species based on analyses of 18S rDNA sequence, sequences of internal transcribed spacer regions, and DNA-DNA reassociation experiments . Four new species, Kockovaella calophylli (1 strain), Kockovaella cucphuongensis (2 strains), Kockovaella litseae (1 strain), and Kockovaella vietnamensis (1 strain) are proposed for these strains.

Nat Biotechnol, 2003 Jan, 21(1), 89 - 92 Epub 2002 Dec 16.
An efficient protein complex purification method for functional proteomics in higher eukaryotes; Forler D et al.; The ensemble of expressed proteins in a given cell is organized in multiprotein complexes . The identification of the individual components of these complexes is essential for their functional characterization . The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast . The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes . Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells . This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.

Curr Opin Hematol, 2003 Jan, 10(1), 2 - 7
PX domain takes shape; Wientjes FB et al.; In recent years, a number of protein domains have been identified that bind phosphoinositides and direct proteins to membrane targets . A recent addition to this group is the Phox homology or PX domain, a 120-amino acid domain conserved from yeast to humans, which is present in proteins involved in cell signaling, protein sorting, vesicle fusion, and the assembly of components of the superoxide generating system of neutrophils . These domains have varying affinities for phosphatidylinositol-3-phosphate (PI(3)P), and PI(3,4) and (4,5) bisphosphates, which couple the PI kinase and phosphatase signaling networks to the assembly of proteins at membrane surfaces . These PX domains also contain a PXXP motif, allowing them to bind to proteins containing Src homology 3 (SH3) domains.

Mol Cell Biol, 2003 Jan, 23(1), 238 - 49
LXXLL-related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1; Suzuki T et al.; The orphan receptor Ad4BP/SF-1 (NR5A1) is a constitutive activator, and its activity is repressed by another orphan receptor, Dax-1 (NR0B1) . In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1 . Yeast two-hybrid and transient-transfection assays confirmed the necessity of three LXXLL-related motifs in Dax-1 for interaction with and repression of Ad4BP/SF-1 . In vitro pull-down experiments confirmed that Dax-1 interacts with Ad4BP/SF-1 and also with LRH-1 (NR5A2) . The target specificity of the LXXLL-related motifs was indicated by the observations that Ad4BP/SF-1, ERalpha (NR3A1), LRH-1, ERR2 (NR3B2), and fly FTZ-F1 (NR5A3) interacted through their ligand binding domains with all the LXXLL-related motifs in Dax-1 whereas HNF4 (NR2A1) and RORalpha (NR1F1) did not . Transcriptional activities of the receptors whose DNA binding domains (DBDs) were replaced by the GAL4 DBD were repressed by Dax-1 to various levels, which correlated with the strength of interaction . Amino acid substitutions revealed that Ad4BP/SF-1 and LRH-1 preferentially interact with L(+1)XXLL-related motifs containing serine, tyrosine, serine, and threonine at positions -2, +2, +3, and +6, respectively . Taken together, our results indicate that the specificities of LXXLL-related motifs in Dax-1 based on their amino acid sequences play an important role in regulation of orphan receptors.

Mol Cell Biol, 2003 Jan, 23(1), 26 - 37
The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation; Yang HS et al.; Pdcd4 is a novel transformation suppressor that inhibits tumor promoter-induced neoplastic transformation and the activation of AP-1-dependent transcription required for transformation . A yeast two-hybrid analysis revealed that Pdcd4 associates with the eukaryotic translation initiation factors eIF4AI and eIF4AII . Immunofluorescent confocal microscopy showed that Pdcd4 colocalizes with eIF4A in the cytoplasm . eIF4A is an ATP-dependent RNA helicase needed to unwind 5' mRNA secondary structure . Recombinant Pdcd4 specifically inhibited the helicase activity of eIF4A and eIF4F . In vivo translation assays showed that Pdcd4 inhibited cap-dependent but not internal ribosome entry site (IRES)-dependent translation . In contrast, Pdcd4(D418A), a mutant inactivated for binding to eIF4A, failed to inhibit cap-dependent or IRES-dependent translation or AP-1 transactivation . Recombinant Pdcd4 prevented eIF4A from binding to the C-terminal region of eIF4G (amino acids 1040 to 1560) but not to the middle region of eIF4G(amino acids 635 to 1039) . In addition, both Pdcd4 and Pdcd4(D418A) bound to the middle region of eIF4G . The mechanism by which Pdcd4 inhibits translation thus appears to involve inhibition of eIF4A helicase, interference with eIF4A association-dissociation from eIF4G, and inhibition of eIF4A binding to the C-terminal domain of eIF4G . Pdcd4 binding to eIF4A is linked to its transformation-suppressing activity, as Pdcd4-eIF4A binding and consequent inhibition of translation are required for Pdcd4 transrepression of AP-1.

J Cell Sci, 2003 Jan 15, 116(Pt 2), 401 - 14
A dominant negative form of the AAA ATPase SKD1/VPS4 impairs membrane trafficking out of endosomal/lysosomal compartments: class E vps phenotype in mammalian cells; Fujita H et al.; SKD1 is a member of the family of ATPases associated with cellular activities whose yeast homologue Vps4p has been implicated in endosomal/vacuolar membrane transports . When a mutant of SKD1 that lacks ATPase activity {SKD1(E235Q)} was overexpressed in mammalian cells, it induced a dominant negative phenotype characterized by aberrant endosomal structures (denoted as E235Q compartments) . Expression of SKD1(E235Q) caused an accumulation of basolateral recycling receptors, such as asialoglycoprotein receptor and low-density lipoprotein in polarized hepatocytes and Madin-Darby canine kidney cells, respectively, in E235Q compartments . In addition, SKD1(E235Q) also abrogated, via endosomes, transport to the trans-Golgi network, as indicated by an accumulation of TGN38 in E235Q compartments . Three lines of evidence further demonstrated that SKD1 participates in the membrane transport from early endosomes to late endosomes/lysosomes: (1) a redistribution of a late endosomal and lysosomal membrane protein endolyn in E235Q compartments; (2) an inhibition of epidermal growth factor receptor degradation, due to an accumulation of the receptors in E235Q compartments; and (3) a mis-sorting of and defect in the proteolytic processing of newly synthesized cathepsin D . An intriguing finding was that the expression of SKD1(E235Q) caused the number of lysosomes to decrease (to one-sixth of control numbers) but their size to increase (2.4-fold larger in diameter than control lysosomes) . Indeed, an ultrastructural analysis revealed that the expression of SKD1(E235Q) causes an accumulation of hybrid organelles formed by direct fusion between late endosomes and lysosomes . We conclude that SKD1 regulates multiple steps of membrane transport out of early endosomes and the reformation of lysosomes from a hybrid organelle.

J Cell Sci, 2003 Jan 15, 116(Pt 2), 387 - 99
Analysis of the interactions between BP180, BP230, plectin and the integrin alpha6beta4 important for hemidesmosome assembly; Koster J et al.; Hemidesmosomes (HDs) are multi-protein complexes that promote stable adhesion of epithelial cells to the underlying extracellular matrix . We assessed the interactions between different hemidesmosomal components with each other, mapped the binding sites and studied the importance of these interactions for HD assembly in yeast two-hybrid and cell-transfection assays . The results show that: (1) bullous pemphigoid antigen (BP) 180 binds not only to BP230, but also to plectin . The interactions between these proteins are facilitated by the Y subdomain in the N-terminal plakin domain of BP230 and plectin, and residues 145-230 of the cytoplasmic domain of BP180; (2) different, but overlapping, sequences on BP180 mediate binding to beta4, which, in turn associates with BP180 via its third fibronectin type III repeat; (3) sequences in the N-terminal extremity of BP230 mediate its binding to beta4, which requires the C-terminal end of the connecting segment up to the fourth FNIII repeat of the beta4 subunit . (4) Finally, cell-transfection studies showed that the localization of BP230 into hemidesmosome-like structures depends on its Z-Y subdomains as well as on the availability of BP180 . By having further uncovered interactions between various hemidesmosomal components, mapped the involved binding sites and dissected a hierarchy of interactions relevant for their topogenic fate, our findings give novel insights into the molecular organization of hemidesmosomes.

J Cell Sci, 2003 Jan 15, 116(Pt 2), 345 - 52
RNAi reveals anti-apoptotic and transcriptionally repressive activities of DAXX; Michaelson JS et al.; The function of DAXX, a highly conserved mammalian gene, has remained controversial; this is due, in part, to its identification in a variety of yeast two-hybrid screens . Targeted deletion in the mouse revealed that DAXX is essential for embryonic development . Furthermore, the increased levels of apoptosis observed in Daxx-knockout embryos and embryonic stem cell lines suggested that DAXX functions in an anti-apoptotic capacity . In contrast, overexpression studies showed that DAXX may promote apoptosis . Additional studies showed that, when overexpressed, DAXX could function as a transcriptional repressor . To clarify these matters, we have used RNAi to deplete endogenous DAXX and thereby assess DAXX function in cell lines previously tested in overexpression studies . Increased apoptosis was observed in DAXX-depleted cells, showing DAXX to be anti-apoptotic . The apoptosis induced by the absence of DAXX was rescued by Bcl-2 overexpression . In addition, transcriptional derepression was observed in RNAi-treated cells, indicating the ability of endogenous DAXX to repress gene expression and allowing for the identification of novel targets of DAXX repression, including nuclear factor kappaB (NF-kappaB)- and E2F1- regulated targets . Thus, depletion of DAXX by RNAi has verified the crucial role of endogenous DAXX as an anti-apoptotic regulator, and has allowed the identification of probable physiological targets of DAXX transcriptional repression.

J Cell Sci, 2003 Jan 15, 116(Pt 2), 211 - 6
ANChors away: an actin based mechanism of nuclear positioning; Starr DA et al.; Mechanisms for nuclear migration and nuclear anchorage function together to control nuclear positioning . Both tubulin and actin networks play important roles in nuclear positioning . The actin cytoskeleton has been shown to position nuclei in a variety of systems from yeast to plants and animals . It can either act as a stable skeleton to anchor nuclei or supply the active force to move nuclei . Two C . elegans genes and their homologues play important roles in these processes . Syne/ANC-1 anchors nuclei by directly tethering the nuclear envelope to the actin cytoskeleton, and UNC-84/SUN functions at the nuclear envelope to recruit Syne/ANC-1.

J Biol Chem, 2003 Feb 28, 278(9), 6873 - 8 Epub 2002 Dec 12.
Functional analysis of the nucleotide binding domain of membrane-associated guanylate kinases; Olsen O et al.; Membrane-associated guanylate kinases (MAGUKs) regulate cellular adhesion and signal transduction at sites of cell-cell contact . MAGUKs are composed of modular protein-protein interaction motifs including L27, PDZ, Src homology (SH) 3, and guanylate kinase domains that aggregate adhesion molecules and receptors . Genetic analyses reveal that lethal mutations of MAGUKs often occur in the guanylate kinase domain, indicating a critical role for this domain . Here, we explored whether GMP binding to the guanylate kinase domain regulates MAGUK function . Surprisingly, and in contrast to previously published studies, we failed to detect GMP binding to the MAGUKs postsynaptic density-95 (PSD-95) and CASK . Two amino acid residues in the GMP binding pocket that differ between MAGUKs and authentic guanylate kinase explain this lack of binding, as swapping these residues largely prevent GMP binding to yeast guanylate kinase . Conversely, these mutations restore GMP binding but not catalytic activity to PSD-95 . Protein ligands for the PSD-95 guanylate kinase domain, guanylate kinase-associated protein (GKAP) and MAP1A, appear not to interact with the canonical GMP binding pocket, and GMP binding does not influence the intramolecular SH3/guanylate kinase (GK) interaction within PSD-95 . These studies indicate that MAGUK proteins have lost affinity for GMP but may have retained the guanylate kinase structure to accommodate a related regulatory ligand.

Virology, 2002 Nov 10, 303(1), 69 - 78
Interaction of human papillomavirus type 16 L2 with cellular proteins: identification of novel nuclear body-associated proteins; Gornemann J et al.; Two structural proteins form the Papillomavirus (PV) capsids . While the functions of the major structural protein L1 are well established, the exact functions for the minor structural protein L2 are much less well defined, except for some information on a role in viral entry and maturation of infectious virions . To gain more insight in the function of L2 we used the yeast two hybrid system with the Human Papillomavirus (HPV) 11 L2 and HPV16 L2 as bait proteins to isolate putative cellular interaction partners . We identified four proteins interacting with L2 proteins of at least two different HPV types and this interaction was confirmed in vitro by pull-down assays . Further evidence for this interaction was obtained by in vivo localization studies . Two of the proteins, the previously described PATZ and a novel protein, designated PLINP, were localized in discrete nuclear domains and colocalized with L2 . The third protein, designated PMSP, is a newly identified cytoplasmic protein which was recruited to nuclear dots when coexpressed with L2 . The fourth protein interacting with HPV16, 11 and 1 L2, the tubular-nephritis antigen related protein (TIN-Ag-RP), shows a cytoplasmic as well as a membrane bound subcellular distribution . Taken together, our data indicate that L2 of HPVs with different phenotypes interacts with several cellular host proteins, recruits one of them to the nucleus, and is complexed with at least three cellular proteins in specific nuclear domains . These findings suggest an HPV type-independent modulatory function of L2 on host-cell functions that involves discrete nuclear domains and alteration of the subcellular distribution of cellular proteins . The interacting cellular proteins identified may play a role in the viral life cycle and establishment of viral persistence.

Cell Stress Chaperones, 2002 Jul, 7(3), 258 - 68
Small glutamine-rich protein/viral protein U-binding protein is a novel cochaperone that affects heat shock protein 70 activity; Angeletti PC et al.; Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together . These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex . Here we provide evidence that small glutamine-rich protein/viral protein U-binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70 . By "Far-Western" and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90 . The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95-195) that are flanked by charged residues . Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro . This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP . A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (delta SGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent . Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex.

Ecotoxicol Environ Saf, 2002 Sep, 53(1), 65 - 9
Estrogenic effects from household stoves; Wu WZ et al.; With the application of a genetically modified yeast, estrogen receptor-activating compounds were detected in the soot and emission gas of a wood-burning household stove . The EC50 value of 17beta-estradiol was divided by the EC50 value of soot, and the obtained relative estrogenic value for raw soot was 2.37E-5, indicating that soot was about 100,000 times less estrogenic than 17beta-estradiol . Chemical analysis revealed that alkyl phenol, benzonic acid, and PAHs represented the major constituents in the most potent fractions of the soot . Along with PAHs, other constituents might also contribute to the estrogenicity of soot.

Ann N Y Acad Sci, 2002 Nov, 977, 45 - 64
Atherosclerotic lesions and mitochondria DNA deletions in brain microvessels as a central target for the development of human AD and AD-like pathology in aged transgenic mice; Aliev G et al.; We have studied the ultrastructural features of vascular lesions and mitochondria in brain vascular wall cells from human AD brain biopsy, human short postmortem brain tissues, and yeast artificial chromosome (YAC) and C57B6/SJL transgenic positive (Tg+) mice overexpressing amyloid beta precursor protein (AbetaPP) . In situ hybridization using mitochondrial DNA (mtDNA) probes for human wild type, 5 kb deleted, and mouse mtDNA was performed, along with immunocytochemistry using antibodies against amyloid precursor protein (APP), 8-hydroxy-2'-guanosine (8-OHG), and cytochrome c oxidase (COX) . There was a higher degree of amyloid deposition in the vascular walls of the human AD, YAC, and C57B6/SJL Tg (+) mice compared to age-matched controls . In addition, vessels with more severe lesions showed immunopositive staining for APP and possessed large, lipid-laden vacuoles in the cytoplasm of endothelial cells (EC) . Significantly more mitochondrial abnormalities were seen in human AD, YAC, and C57B6/SJL Tg (+) mouse microvessels where lesions occurred . In situ hybridization using wild and chimera (5 kb) mtDNA probes revealed positive signals in damaged mitochondria from the vascular endothelium and in perivascular cells of lesioned microvessels close to regions of large amyloid deposition . These features were absent in undamaged regions of human AD tissues, YAC and C57B6/SJL Tg (+) mouse tissues, and in age-matched control subjects . In addition, vessels with atherosclerotic lesions revealed endothelium and perivascular cells possessing clusters of wild and deleted mtDNA positive probes . These mtDNA deletions were accompanied by increased amounts of immunoreactive APP, 8-OHG, and COX in the same cellular compartment . Our observations demonstrate that vascular wall cells, especially their mitochondria, appear to be a central target for oxidative stress-induced damage.

Biochim Biophys Acta, 2002 Sep 23, 1599(1-2), 152 - 5
SSDP1 gene encodes a protein with a conserved N-terminal FORWARD domain; Bayarsaihan D; I describe the characterization of mouse, human and chicken SSDP1 orthologs that encode a highly conserved protein with over 90% identity at the amino acid level . Structurally, the protein consists of a well-preserved FWD (FORWARD)-domain at the N-terminal end and a proline-, glycine-, methionine- and serine-rich sequence in the central and C-terminal regions . The FORWARD domain, comprised of three alpha-helices, is characterized by the presence of a FWD-box of unknown function conserved not only in vertebrates, but also in nematode, plants, fly and yeast . Human SSDP1 spans about 200 kb on the chromosome 1p31-p32 region and consists of 17 exons . The SSDP1 mRNA transcripts are distributed ubiquitously in adult human and mouse tissues.

Basic Res Cardiol, 2002, 97 Suppl 1, I25 - 30
Sodium pump isoform expression in heart failure: implication for treatment; Muller-Ehmsen J et al.; In the human heart several isoforms of the sodium pump (Na,K-ATPase, the cardiac glycoside receptor) are expressed (alpha1beta1, alpha2beta1, and alpha3beta1) . Their expression is regulated in a highly specific manner, so that there are region specific differences in the expression pattern . The isoform expression pattern is also known to be organ specific in many cases (e.g., kidney, skeletal muscle), suggesting isoform specific functions . In human heart, we have demonstrated that the isoform composition of the left ventricle is altered during heart failure in man and postulate a role of Na,K-ATPase isoforms in the compensatory mechanisms of this disease . When Na,K-ATPase isoforms were expressed separately in yeast cells, we found that the affinities of K and ouabain were lower for alpha2beta1 than for alpha1beta1 or alpha3beta1 . In addition, alpha3beta1 had a lower turnover rate than alpha1beta1 . Similar results were found in a study, where Na,K-ATPase isoforms were expressed in Xenopus oocytes . Thus, there is evidence for specific biochemical properties of the Na,K-ATPase isoforms . In heterozygous knock-out mice, in which either alpha1 or alpha2 isoforms were selectively reduced, only the lower expression and activity of alpha2 led to a hypercontractile response as seen with cardiac glycosides . Therefore in mice, the effect of cardiac glycosides seems to be mediated specifically by alpha2 . In summary, there is a tissue-specific regulation of Na,K-ATPase isoform expression in humans, as well as a highly specific regulation of the isoforms during disease, e.g., heart failure . There is also evidence for specific biochemical properties of different isoforms of the human Na,K-ATPase as well as for a specific functional impact on cardiac contractility in mice . Therefore, the isoforms of human Na,K-ATPase are not exchangeable and targeting specific isoforms by drugs or gene therapy may promise therapeutic benefit in diseases like heart failure or atrial fibrillation.

Gynecol Obstet Fertil, 2002 Oct, 30(10), 817 - 21
{Human infertility: meiotic genes as potential candidates}; Mandon-Pepin B et al.; Up to now, the identification of gene mutations causing infertility in humans remains poorly investigated . Temporal progression through meiosis and meiosis specific genes had been extensively characterized in yeast . Recently some mammalian homologous were found . The molecular mechanisms regulating entry into and progression through meiosis in mammals are still unknown . However, disruption of some meiotic genes in mouse showed an essential role of them in meiotic chromosome synapsis and gametogenesis . Moreover, the phenotype of gonads in null mutant mice for some meiotic genes (failure to initiate or blockage in meiosis, lack of gametes or small size of gonads...) could be strikingly similar to clinical observations found in human infertility . The aim of this study was to identify putative mutations in 5 meiotic genes of several clinically well-characterized patients who present unexplained infertility (normal karyotype, women with premature ovarian failure, men with azospermia and without Y micro-deletion) . For this purpose, the exons of these 5 genes (DMC1, SPO11, MSH4, MSH5, CCNA1) were all amplified by PCR with specific primers and each amplified-exon was sequenced . Sequences were aligned in comparison to the human corresponding gene available in Genbank . Many heterozygous mutations were found in different genes . Two homozygous mutations were found in MSH4 and DMC1 genes in a young man presenting a testis vanishing syndrome and a woman presenting a premature ovarian failure, respectively . Consequences of such mutations will be examined and verified in model organisms (yeast, mouse) to check the relevance of the mutations in clinical setting.

J Virol, 2003 Jan, 77(1), 159 - 66
Vaccinia virus uracil DNA glycosylase has an essential role in DNA synthesis that is independent of its glycosylase activity: catalytic site mutations reduce virulence but not virus replication in cultured cells; De Silva FS et al.; Previous findings that the vaccinia virus uracil DNA glycosylase is required for virus DNA replication, coupled with an inability to isolate a mutant with an active site substitution in the glycosylase gene, were surprising, as such enzymes function in DNA repair and bacterial, yeast, and mammalian null mutants are viable . To further study the role of the viral protein, we constructed recombinant vaccinia viruses with single or double mutations (D68N and H181L) in the uracil DNA glycosylase conserved catalytic site by using a complementing cell line that constitutively expresses the viral enzyme . Although these mutations abolished uracil DNA glycosylase activity, they did not prevent viral DNA replication or propagation on a variety of noncomplementing cell lines or human primary skin fibroblasts . In contrast, replication of a uracil DNA glycosylase deletion mutant occurred only in the complementing cell line . Therefore, the uracil DNA glycosylase has an essential role in DNA replication that is independent of its glycosylase activity . Nevertheless, the conservation of the catalytic site in all poxvirus orthologs suggested an important role in vivo . This idea was confirmed by the decreased virulence of catalytic-site mutants when administered by the intranasal route to mice.

Eukaryot Cell, 2002 Dec, 1(6), 875 - 83
Cloning and characterization of scon-3+, a new member of the Neurospora crassa sulfur regulatory system; Sizemore ST et al.; The sulfur regulatory system of Neurospora crassa consists of a group of sulfur-regulated structural genes (e.g., arylsulfatase) that are under coordinate control of the CYS3 positive regulator and sulfur controller (SCON) negative regulators . Here we report on the cloning of scon-3(+), which encodes a polypeptide of 171 amino acids and is a Skp1 family homolog . Repeat-induced point mutation of scon-3(+) resulted in a phenotype of constitutive expression of arylsulfatase, a phenotype consistent with other sulfur controller mutants . Northern analysis indicated that, unlike other members of the sulfur regulatory system, expression of scon-3(+) is not under the direct control of the CYS3 transcriptional activator . In particular, scon-3(+) mRNA was detectable under sulfur repressing or derepressing conditions in a Deltacys-3 mutant . In yeast, Skp1p and an F-box protein binding partner are core constituents of a class of E3 ubiquitin ligases known as SCF complexes . The N . crassa negative regulator SCON2 contains an F-box motif essential for the operation of the sulfur regulatory system and suggests a role for an SCF complex in the N . crassa sulfur regulatory system . A crucial set of experiments, by using a yeast two-hybrid approach with confirming coimmunoprecipitation assays, demonstrated that SCON3 interacts with SCON2 in a manner dependent upon the F-box motif of SCON2 . The protein-protein interaction detected between SCON2 and SCON3 represents the initial demonstration in a filamentous fungus of functional interaction between putative core components of a SCF complex.

Scand J Infect Dis, 2002, 34(10), 753 - 5
Prevalence of dermatomycoses in Mal de Meleda patients: a field study; Ergin C et al.; Mal de Meleda is a rare autosomal recessive form of palmoplantar keratoderma characterized by hyperkeratosis of the palms and soles . The presence of yeast and dermatophytes was investigated in 29 mal de Meleda patients from Koprucay canyon, Turkey, a newer geographical focus, and was found in 62.0% and 20.7% of cases, respectively . Antifungal resistance of isolates was not detected.

Sci STKE . 2002 Dec 10;2002(162):PE49.
Hitting the target: emerging technologies in the search for kinase substrates; Manning BD et al.; Through phosphorylation, protein kinases can alter the activity, localization, protein association, and stability of their targets . Despite the importance to our understanding of all aspects of cell biology, progress toward identifying bona fide substrates of specific protein kinases has been slow . Traditionally used techniques to identify true kinase substrates, such as genetics, yeast two-hybrid screens, and biochemical purification, are often laborious and unreliable . However, several new approaches have recently been developed and used successfully to identify genuine in vivo substrates of certain protein kinases . These methods include screening for phosphorylation of proteins from phage expression libraries, peptide library screens to determine optimal motifs favored by specific kinases, the use of phospho-motif antibodies, and an approach that uses structurally altered kinases and allele-specific adenosine triphosphate analogs and kinase inhibitors . We describe these approaches and discuss their utility and inherent caveats.

J Biol Chem, 2003 Feb 14, 278(7), 5195 - 204 Epub 2002 Dec 09.
Tankyrase 1 interacts with Mcl-1 proteins and inhibits their regulation of apoptosis; Bae J et al.; Mcl-1L (myeloid cell leukemia-1 long) is an antiapoptotic Bcl-2 family protein discovered as an early induction gene during leukemia cell differentiation . Previously, we identified Mcl-1S (short) as a short splicing variant of the Mcl-1 gene with proapoptotic activity . To identify Mcl-1-interacting proteins, we performed yeast two-hybrid screening and found cDNAs encoding tankyrase 1 . This protein possesses poly(ADP-ribose) polymerase activity and presumably facilitates the turnover of substrates following ADP-ribosylation . In yeast and mammalian cells, tankyrase 1 interacts with both Mcl-1L and Mcl-1S, but does not bind to other Bcl-2 family proteins tested . Analysis of truncated tankyrase 1 mutants indicated that the first 10 ankyrin repeats are involved in interaction with Mcl-1 . In the N terminus of Mcl-1, a stretch of 25 amino acids is sufficient for binding to tankyrase 1 . Overexpression of tankyrase 1 antagonizes both Mcl-1L-mediated cell survival and Mcl-1S-induced cell death . Furthermore, coexpression of tankyrase 1 with Mcl-1L or Mcl-1S decreased the levels of Mcl-1 proteins . Although tankyrase 1 down-regulates Mcl-1 protein expression, no ADP-ribosylation of Mcl-1 was detected . In contrast, overexpression of Mcl-1 proteins suppressed the ADP-ribosylation of the telomeric repeat binding factor 1, another tankyrase 1-interacting protein . Thus, interaction of Mcl-1L and Mcl-1S with tankyrase 1 could serve as a unique mechanism to decrease the expression of these Bcl-2 family proteins, thereby leading to the modulation of the apoptosis pathway.

J Biol Chem, 2003 Feb 21, 278(8), 6323 - 9 Epub 2002 Dec 09.
NFBD1, a novel nuclear protein with signature motifs of FHA and BRCT, and an internal 41-amino acid repeat sequence, is an early participant in DNA damage response; Shang YL et al.; Efficient repair of DNA double-strand breaks depends on the intact signaling cascade, comprising molecules involved in DNA damage signal pathways and checkpoints . Budding yeast Rad9 (scRad9) is required for activation of scRad53 (mammalian homolog Chk2) and transduction of the signal further downstream in this pathway . In the search for a mammalian homolog, three proteins in the human data base, including BRCA1, 53BP1, and nuclear factor with BRCT domains protein 1 (NFBD1), were found to share significant homology with the BRCT motifs of scRad9 . Because BRCA1 and 53BP1 are involved in DNA damage responses, a similar role for NFBD1 was tested . We show that NFBD1 is a 250-kDa nuclear protein containing a forkhead-associated motif at its N terminus, two BRCT motifs at its C terminus, and 13 internal repetitions of a 41-amino acid sequence . Five minutes after gamma-irradiation, NFBD1 formed nuclear foci that colocalized with the phosphorylated form of H2AX and Chk2, two phosphorylation events known to be involved in early DNA damage response . NFBD1 foci are also detected in response to camptothecin, etoposide, and methylmethanesulfonate treatments . Deletion of the forkhead-associated motif or the internal repeats of NFBD1 has no effect on DNA damage-induced NFBD1 foci formation . Conversely, deletion of the BRCT motifs abrogates damage-induced NFBD1 foci . Ectopic expression of the BRCT motifs reduced damage-induced NFBD1 foci and compromised phosphorylated Chk2- and phosphorylated H2AX-containing foci . These results suggest that NFBD1, like BRCA1 and 53BP1, participates in the early response to DNA damage.

Mol Biol Cell, 2002 Dec, 13(12), 4167 - 78
Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor; Posern G et al.; Signal-induced activation of the transcription factor serum response factor (SRF) requires alterations in actin dynamics . SRF activity can be inhibited by ectopic expression of beta-actin, either because actin itself participates in SRF regulation or as a consequence of cytoskeletal perturbations . To distinguish between these possibilities, we studied actin mutants . Three mutant actins, G13R, R62D, and a C-terminal VP16 fusion protein, were shown not to polymerize in vivo, as judged by two-hybrid, immunofluorescence, and cell fractionation studies . These actins effectively inhibited SRF activation, as did wild-type actin, which increased the G-actin level without altering the F:G-actin ratio . Physical interaction between SRF and actin was not detectable by mammalian or yeast two-hybrid assays, suggesting that SRF regulation involves an unidentified cofactor . SRF activity was not blocked upon inhibition of CRM1-mediated nuclear export by leptomycin B . Two actin mutants were identified, V159N and S14C, whose expression favored F-actin formation and which strongly activated SRF in the absence of external signals . These mutants seemed unable to inhibit SRF activity, because their expression did not reduce the absolute level of G-actin as assessed by DNase I binding . Taken together, these results provide strong evidence that G-actin, or a subpopulation of it, plays a direct role in signal transduction to SRF.

Mol Biol Cell, 2002 Dec, 13(12), 4111 - 3
Mammalian septins nomenclature; Macara IG et al.; There are 10 known mammalian septin genes, some of which produce multiple splice variants . The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene . Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues . Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee . The human and mouse septin genes will be named SEPT1-SEPT10 and Sept1-Sept10, respectively . Splice variants will be designated by an underscore followed by a lowercase "v" and a number, e.g., SEPT4_v1.

Phytochemistry, 2003 Jan, 62(1), 71 - 5
6H-dibenzo{b,d}pyran-6-one derivatives from the cultured lichen mycobionts of Graphis spp . and their biosynthetic origin; Tanahashi T et al.; The spore-derived mycobionts of the lichen Graphis prunicola, G . cognata and G . scripta were cultivated on a malt-yeast extract medium supplemented with 10% sucrose and their metabolites were investigated . Graphislactones A-D were isolated from the cultures of G . prunicola, while alternariol and graphislactones A and C were isolated from those of G . cognata . From the cultured mycobionts of G . scripta, a new 6H-dibenzo{b,d}pyran-6-one derivative, graphislactone E with graphislactones A and C was obtained . On the other hand, cultivation of the mycobionts of G . prunicola on a malt-yeast extract medium supplemented with 2.5% sucrose and 0.25% sodium acetate produced two new metabolites, graphislactones E and F . Their structures were determined by spectroscopic methods . The biogenetic origin of the carbon skeleton in both compounds was verified by administering sodium {1-13C}-acetate and sodium {1,2-13C(2)}-acetate.

J Agric Food Chem, 2002 Dec 18, 50(26), 7593 - 9
Production of trichothecenes and other secondary metabolites by Fusarium culmorum and Fusarium equiseti on common laboratory media and a soil organic matter agar: an ecological interpretation; Hestbjerg H et al.; Fusarium culmorum and F . equiseti were characterized with regard to production of trichothecenes and other secondary metabolites . Results following growth on laboratory media are interpreted with the aim of increasing the understanding of fungal metabolism in the field environment . While trichothecene production was detected for 94 of 102 F . culmorum isolates, only 8 of 57 F . equiseti isolates were positive . Profiles of secondary metabolites were compared by following growth on yeast extract sucrose agar (YES), potato sucrose agar (PSA), and an agar medium, prepared from soil organic matter (SOM), which was included to simulate growth conditions in soil . SOM supported the production of chrysogine by F . culmorum . The two species utilized the media differently . F . culmorumproduced zearalenone (ZEA) on YES, whereas some F . equiseti isolates produced ZEA on PSA . Other F . equiseti isolates produced equisetin . These differences may reflect that F . culmorum depends on a pathogenic life style while F . equiseti has a more saprotrophic mode of existence.

Cell Mol Life Sci, 2002 Oct, 59(10), 1632 - 9
Nascent-polypeptide-associated complex; Rospert S et al.; Nascent-polypeptide-associated complex (NAC) is a heterodimeric complex which can reversibly bind to eukaryotic ribosomes . NAC is located in direct proximity to newly synthesized polypeptide chains as they emerge from the ribosome . Although its function is thought to be conserved from yeast to humans our current knowledge about what NAC actually does in a living cell is incomplete . It has been suggested that NAC is a (i) dynamic component of the ribosomal exit tunnel, providing a shield for nascent polypeptides, (ii) negative regulator of translocation into the endoplasmic reticulum and (iii) positive regulator of translocation into the mitochondria . However, none of these hypotheses is generally accepted . Moreover, the individual subunits of NAC have been implicated in processes related to transcription rather than translation, and it is currently under debate whether NAC might be a protein of dual function . This review attempts to summarize the data from different fields and to discuss the partly controversial results in a common context.

J Pathol, 2003 Jan, 199(1), 18 - 27
The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B; Schmidt MH et al.; The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression . Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function . This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system . A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions . Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified . In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found . Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13 . Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed . To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells . Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression . It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase .

Eur J Biochem, 2002 Dec, 269(24), 6241 - 9
Identification and characterization of a novel activated RhoB binding protein containing a PDZ domain whose expression is specifically modulated in thyroid cells by cAMP; Mircescu H et al.; In a search for genes regulated in response to cAMP we have identified a new protein, p76RBE, whose mRNA and protein expression is enhanced in thyrocytes following thyrotropin stimulation of the cAMP transduction cascade . This protein presents important similarities with Rhophilin and contains different protein-protein interaction motifs . The presence of HR1 and PDZ motifs as well as a potential PDZ binding domain motif suggests that p76RBE could be implicated in targeting or scaffolding processes . By yeast two-hybrid screenings and coimmunoprecipitation, we show here that p76RBE is a specific binding protein of RhoB and binds selectively to the GTP-bound form of this small GTPase . p76RBE also binds in vitro to components of the cytoskeleton, including cytokeratin 18 . p76RBE is essentially cytoplasmic in transfected COS-7 mammalian cells and seems to be recruited to an endosomal compartment when coexpressed with the activated form of RhoB . p76RBE was shown to be mainly expressed in tissues with high secretion activity . Our data suggest that p76RBE could play a key role between RhoB and potential downstream elements needed under stimulation of the thyrotropin/cAMP pathway in thyrocytes and responsible for intracellular motile phenomena such as the endocytosis involved in the thyroid secretory process.

Eur J Biochem, 2002 Dec, 269(24), 6233 - 40
Reconstructing the replication complex of AcMNPV; Hefferon KL et al.; Baculoviruses are well known for their large, circular, double-stranded DNA genomes . The type member, AcMNPV, is the best characterized and undergoes a succession of early, late and very late gene expression during its infection cycle . The viral genes involved in DNA replication have previously been identified and their products are required for the activation of late gene expression . In this study, we FLAG- and HA-tagged the replication late expression factors of AcMNPV, examined their expression and functional activities by CAT assay and Western blot analysis, and determined their subcellular localization in transfected cells by subcellular fractionation and immunofluorescent microscopy . We found that all replication LEFs with the exception of P143 and P35 resided in the nucleus of transfected cells . We further investigated the interactions among various replication LEFs using both yeast two-hybrid and coprecipitation strategies . A summary of the interactive properties of the replication LEFs is presented and a model for a putative AcMNPV replication complex is offered.

Mycoses, 2002 Dec, 45(11-12), 488 - 91
Fungal flora of human toe webs; Oyeka CA et al.; A total of 100 young adults (67 males and 33 females) participated in the study . Clinical evaluation showed that only 10 of the volunteers showed some scaling, fissuring and peeling of the toe webs . Four of these complained of occasional itching . Fourteen different genera of fungi were recovered from 78 of the 100 youths screened . Yeasts were recovered from 21 (27%) of the positive cases, nondermatophytes from 38 (49%) and dermatophytes from 19 (24%) . Microsporum gypseum was the most commonly recovered dermatophyte . Rhizopus stolonifer and Trichosporon cutanueum were the most frequently recovered nondermatophytic mould and yeast, respectively . More males (62.8%) harboured these organisms than females (37.2%) . The study further showed that human toe webs that are apparently healthy harbour a variety of fungi, that may be potential pathogens.

Mycoses, 2002 Dec, 45(11-12), 465 - 9
Adherence of Candida strains isolated from the human gastrointestinal tract; Biasoli MS et al.; The adherence of different Candida strains isolated from the human gastrointestinal tract was studied . The 23 Candida strains isolated from faeces were C . albicans (12), C . glabrata (2), C . krusei (2), C . parapsilosis (2), C . tropicalis (2), C . colliculosa (1), C . kefyr (1) and C . lusitaniae (1) . Buccal epithelial cells from different healthy donors were used . Adherence values were maximal for C . albicans and minimal for C . krusei . A relation exists between yeast adherence capacity and the ability to colonize mucosal surfaces.

Mycoses, 2002 Dec, 45(11-12), 449 - 54
Physiological characters of Sporothrix schenckii isolates; Ghosh A et al.; Sporotrichosis is endemic in three regions (east, north and south) in India . The colony morphology and physiological characteristics of 49 clinical isolates from these three regions (25 from north India, 17 from east India and seven from south India) were analysed in both mycelial and yeast forms . No difference in colony character was seen among the 49 isolates on three different media . Growth of all isolates was inhibited at 40 degrees C . The yeast forms were found to be more tolerant to osmotic pressure and salt concentrations . Most mycelial forms grew well between pH 3-12.0 whereas most yeast forms could tolerate a pH range of 2.4 to 9.5 . Variations in assimilation of arabinose, dextrin, raffinose, rhamnose and starch was observed among strains from different geographical regions . The yeast forms did not show any urease activity but the mycelial forms of all isolates could split urea . Phenol oxidase and potassium nitrate assimilation were positive and gelatinase activity and casein hydrolysis were negative for all isolates.

Mycoses, 2002 Dec, 45(11-12), 443 - 8
Exopolysaccharides and capsules in human pathogenic Exophiala species; Yurlova NA et al.; The black yeasts Exophiala spinifera and E . dermatitidis produce extracellular slimes, which may be either in the form of a well-delimited capsule or of diffusely exuded exopolysaccharides (EPS) . The optimal conditions for their production were studied . The presence or absence of polysaccharide material can be used for recognition of the two species . Five-day-old cultures grown on potato glucose agar at 24 degrees C were observed in India ink, and positive identification for E . spinifera was obtained when significant halos were seen around yeast cells . In contrast, E . dermatitidis had irregular EPS with a fibrillar substructure made visible by alcian blue staining . Other Exophiala species produce insignificant amounts of extracellular mucus or none at all . The diagnostic method is particularly useful with yeast-like primary cultures, which often consist entirely of budding cells and lack the characteristic structures of the filamentous Exophiala synanamorph.

Mycoses, 2002 Dec, 45(11-12), 437 - 42
Serological differences in three Blastomyces dermatitidis strains; Axtell RC et al.; Yeast phase lysate antigens, prepared from three isolates of Blastomyces dermatitidis (T-58, Tennessee dog; 48089, Zaire human; ERC-2, Wisconsin dog) were assayed for their ability to detect antibodies in human sera, dog sera and sera from rabbits immunized with each of the lysate antigens . The dog sera were from animals diagnosed with blastomycosis from various endemic regions in North America . T-58 and ERC-2 lysate antigens exhibited a high reactivity with the serum from dogs infected with blastomycosis; however, 48089 lysate showed low reactivity with the same sera . With the immunized rabbit sera, 48089 lysate was the only lysate with a high reactivity with the 48089 serum and it exhibited little reactivity with the heterologous sera . The T-58 and ERC-2 lysate antigens reacted minimally with the 48089 serum but reacted highly with both the T-58 and ERC-2 sera . The human sera were from individuals potentially exposed to B . dermatitidis while working on a prairie dog relocation project in Colorado . Remarkably, all three lysate antigens could detect antibodies in the individuals diagnosed with blastomycosis . This study indicated that there were serological differences in the 48089 Zaire lysate compared with the other lysate antigens and it may be designated serotype 2.

Essays Biochem, 2000, 36, 27 - 35
Glycosylation and protein transport; Scheiffele P et al.; Transport along the secretory pathway is largely signal-mediated . Proteins in the secretory pathway can be covalently modified with various carbohydrate structures, most commonly O-glycans, N-glycans and/or proteoglycans . Carbohydrate modifications can change the physical properties of proteins or can function as specific recognition epitopes . Glycosylation can act as an apical sorting signal in polarized epithelial cells and provide a signal for surface transport in non-polarized fibroblasts . Homologues of leguminous plant lectins have been identified in yeast, fruitflies, worms and humans . Intracellular lectins are candidate receptors in the secretory pathway to mediate concentration of cargo in carrier vesicles.

Essays Biochem, 2000, 35, 33 - 42
Unconventional myosins; Kalhammer G et al.; Myosins constitute a large superfamily of F-actin-based motor proteins found in many organisms from yeast to humans . A phylogenetic comparison of their head sequences has allowed them to be grouped into 15 different classes . Unconventional myosins can be monomeric or dimeric, but are thought not to form filaments, unlike conventional myosin . The double-headed class-V myosins are good candidates for transporting vesicles, organelles and (mRNA) particles along actin filaments . Class-I myosins are involved in membrane dynamics and actin organization at the cell cortex, thus affecting cell migration, endocytosis, pinocytosis and phagocytosis . A class-III myosin from Drosophila is required for phototransduction and maintenance of the rhabdomere . Class-IX myosins negatively regulate the small G-protein Rho, a signalling molecule that regulates the organization of the actin cytoskeleton . Protein kinases that are regulated by members of the Rho small G-protein family regulate the motor activities of different myosins.

J Immunol, 2002 Dec 15, 169(12), 6883 - 9
TNFR-associated factor-3 is associated with BAFF-R and negatively regulates BAFF-R-mediated NF-kappa B activation and IL-10 production; Xu LG et al.; TALL-1 is a member of the TNF family that is critically involved in B cell survival, maturation, and progression of lupus-like autoimmune diseases . TALL-1 has three receptors, including BCMA, TACI, and BAFF-R, which are mostly expressed by B lymphocytes . Gene knockout studies have indicated that BAFF-R is the major stimulatory receptor for TALL-1 signaling and is required for normal B cell development . The intracellular signaling mechanisms of BAFF-R are not known . In this report, we attempted to identify BAFF-R-associated downstream proteins by yeast two-hybrid screening . This effort identified TNFR-associated factor (TRAF)3 as a protein specifically interacting with BAFF-R in yeast two-hybrid assays . Coimmunoprecipitation experiments indicated that BAFF-R interacts with TRAF3 in B lymphoma cells and this interaction is stimulated by TALL-1 treatment . Domain mapping experiments indicated that both a 6-aa membrane proximal region and the C-terminal 35 aa of BAFF-R are required for its interaction with TRAF3 . Moreover, overexpression of TRAF3 inhibits BAFF-R-mediated NF-kappaB activation and IL-10 production . Taken together, our findings suggest that TRAF3 is a negative regulator of BAFF-R-mediated NF-kappaB activation and IL-10 production.

Biochem Biophys Res Commun, 2002 Dec 20, 299(5), 793 - 800
Interaction of Axl receptor tyrosine kinase with C1-TEN, a novel C1 domain-containing protein with homology to tensin; Hafizi S et al.; Axl receptor tyrosine kinase is implicated in several malignancies and is the receptor for the vitamin K-dependent growth factor Gas6 . From a yeast two-hybrid screen of protein-protein interactions with the Axl cytoplasmic domain, we detected a previously uncharacterised SH2 domain-containing protein . We cloned two novel splice variants of this protein that give rise to 1409- and 1419-amino acid proteins, differing only in their N-terminal residues and yielding a 150-kDa protein product by in vitro translation . The Axl-interacting C-terminus contains a tandem SH2 and PTB domain combination homologous to the focal adhesion protein tensin . We detected interaction of Axl with both domains in mammalian cells by co-immunoprecipitation and two-hybrid analyses . In addition, the protein possesses an N-terminal putative phorbol ester-binding C1 domain as well as a central tyrosine phosphatase motif . Thus, we have named the protein C1 domain-containing phosphatase and TENsin homologue (C1-TEN) . Northern blot analysis of C1-TEN in human tissues revealed highest expression in heart, kidney, and liver . In summary, we have identified a novel multi-domain intracellular protein that interacts with Axl and which may furthermore be involved in other signal transduction pathways.

J Biochem Mol Biol, 2002 Nov 30, 35(6), 629 - 36
The Ring-H2 finger motif of CKBBP1/SAG is necessary for interaction with protein kinase CKII and optimal cell proliferation; Kim YS et al.; Protein kinase CKII (CKII) is required for progression through the cell division cycle . We recently reported that the beta subunit of protein kinase CKII (CKIIbeta) associates with CKBBP1 that contains the Ring-H2 finger motif in the yeast two-hybrid system . We demonstrate here that the Ring-H2 finger-disrupted mutant of CKBBP1 does not interact with purified CKIIbeta in vitro, which shows that the Ring-H2 finger motif is critical for direct interaction with CKIIbeta . The CKII holoenzyme is efficiently co-precipitated with the wild-type CKBBP1, but not with the Ring-H2 finger-disrupted CKBBP1, from whole cell extracts when epitope-tagged CKBBP1 is transiently expressed in HeLa cells . Disruption of the Ring-H2 finger motif does not affect the cellular localization of CKBBP1 in HeLa cells . The increased expression of either the wild-type CKBBP1 or Ring-H2 finger-disrupted CKBBP1 does not modulate the protein or the activity levels of CKII in HeLa cells . However, the stable expression of Ring-H2 finger-disrupted CKBBP1 in HeLa cells suppresses cell proliferation and causes the accumulation of the G1/G0 peak of the cell cycle . The Ring-H2 finger motif is required for maximal CKBBP1 phosphorylation by CKII, suggesting that the stable binding of CKBBP1 to CKII is necessary for its efficient phosphorylation . Taken together, these results suggest that the complex formation of CKIIbeta with CKBBP1 and/or CKII-mediated CKBBP1 phosphorylation is important for the G1/S phase transition of the cell cycle.

J Biochem Mol Biol, 2002 Nov 30, 35(6), 562 - 7
Interaction of Heliothis armigera nuclear polyhedrosis viral capsid protein with its host actin; Lu SY et al.; In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed . Then VP39 was used as bait . The host actin gene was isolated from the cDNA library with the yeast two-hybrid system . This demonstrated that VP39 could interact with its host actin in yeast . In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39 . The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter . The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin . Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39 . Red actin cables were also formed in the cytoplasm at the same time . Actin was aggregated in the nucleus 9 h after the transfection . The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin . Such a combination would result in the actin skeleton rearrangement.

Int J Oncol, 2003 Jan, 22(1), 51 - 7
Role of p53 in G2/M cell cycle arrest and apoptosis in response to gamma-irradiation in ovarian carcinoma cell lines; Concin N et al.; We investigated the cell cycle and apoptotic response to irradiation in 4 human ovarian carcinoma cell lines, i.e., PA-1, Caov-3, SK-OV-3, and ES-2 . Cell lines were also analysed for their p53 and Bax expression to address the relationship with cell cycle and apoptotic response . Apoptosis was examined by flow cytometric measurement of annexin V binding and by determination of cytoplasmic histone-associated DNA fragments with a photometric enzyme immunoassay . Cell cycle analyses were performed on the basis of flow cytometry . p53 and Bax protein expression was examined by immunocytochemistry in untreated cells and after irradiation . p53 cDNA sequencing and a functional yeast-based assay (FASAY) were performed to determine the p53 mutational status . All cell lines exhibited a dose-dependent G2/M arrest . No arrest in G1 was seen . A strong correlation was found between the G2/M arrest and the induction of apoptosis . PA-1, the only cell line found to express wild-type p53, showed the highest susceptibility to accumulate in G2/M and the strongest apoptotic response after irradiation . In this cell line irradiation resulted in an unequivocal accumulation of p53 protein and in an increased expression of Bax protein . Caov-3, lacking wild-type p53, showed upregulation of Bax expression after irradiation . Caov-3 proved to be relative sensitive to apoptosis compared to SK-OV-3 and ES-2 . These two cell lines were found to be p53 mutated in sequence analysis and irradiation had no effect on the expression of p53 . No change in Bax expression was seen in ES-2, while SK-OV-3 exhibited decreased Bax protein levels after irradiation . Our data suggest that the G2/M arrest is an important component of the pathway leading from irradiation-induced DNA damage to apoptosis in the examined cell lines . The G2/M arrest and associated apoptosis found in the examined cell lines does not necessarily require wild-type p53, although wild-type p53 and possibly Bax may contribute to a maximum response to irradiation . Two independent mechanisms, p53-dependent and p53-independent, are suggested in the examined cell lines.

Nat Immunol, 2003 Jan, 4(1), 55 - 62 Epub 2002 Dec 09.
TCRs with high affinity for foreign pMHC show self-reactivity; Holler PD et al.; T cells with high-affinity T cell receptors (TCRs) for a foreign peptide-major histocompatibility complex (pMHC) appear to be negatively selected, even though they have never seen the foreign antigen . To examine how this process operates, we used in vitro yeast display to isolate high-affinity TCRs from the T cell clone 2C . The TCRs showed fast on-rates, which were consistent with reduced CDR (complementarity determining region) flexibility, and cross-reactivity with other cognate pMHCs . T cell hybridomas transfected with a high-affinity TCR were stimulated by endogenous self-pMHC, which suggested that T cells bearing the TCR would be negatively selected . The immune system appears to maintain a repertoire of flexible, low-affinity TCRs at the expense of more effective high-affinity TCRs.

Protein Eng, 2002 Oct, 15(10), 805 - 9
Robustness of hen lysozyme monitored by random mutations; Kunichika K et al.; We investigated the robustness of hen lysozyme by using random mutant libraries . Six random mutant libraries containing 1, 1.5, 2, 3, 5 and 14 amino acid mutations per hen lysozyme were systematically constructed by varying the concentrations of Mg(2+) and Mn(2+) on polymerase chain reaction . The mutated genes from the six libraries were cloned to a yeast expression vector and a total of 4000 clones were screened on the basis of lysis activity and ELISA employing monoclonal antibody that recognized only lysozyme with native conformation . About 80% of the clones with an average of two amino acid mutations retained active structure . Almost all clones with an average of five mutations lost active structure . On the other hand, 80% of the clones with an average of two amino acid mutations retained both gross conformation and active structure and 24% of the clones with an average of 14 amino acid mutations retained gross conformation . These results show that gross conformation is robust against mutations and so is active structure to a lesser extent.

J Biol Chem, 2003 Feb 28, 278(9), 7439 - 44 Epub 2002 Dec 04.
The Coxsackievirus and adenovirus receptor (CAR) forms a complex with the PDZ domain-containing protein ligand-of-numb protein-X (LNX); Sollerbrant K et al.; The Coxsackievirus and adenovirus receptor (CAR) functions as a virus receptor, but its primary biological function is unknown . A yeast two-hybrid screen was used to identify Ligand-of-Numb protein-X (LNX) as a binding partner to the intracellular tail of CAR . LNX harbors several protein-protein interacting domains, including four PDZ domains, and was previously shown to bind to and regulate the expression level of the cell-fate determinant Numb . CAR was able to bind LNX both in vivo and in vitro . Efficient binding to LNX required not only the consensus PDZ domain binding motif in the C terminus of CAR but also upstream sequences . The CAR binding region in LNX was mapped to the second PDZ domain . CAR and LNX were also shown to colocalize in vivo in mammalian cells . We speculate that CAR and LNX are part of a larger protein complex that might have important functions at discrete subcellular localizations in the cell.

Lung Cancer, 2002 Dec, 38 Suppl 3, S9 - 17
Molecular approaches to lung cancer evaluation; Niklinski J et al.; The stagnation of therapeutic results in lung cancer over the last decade(s) is a matter of great concern, also due to the constantly increasing incidence of the disease . Among the reasons for this failing therapeutic progress is the lack of understanding of the molecular mechanisms underlying the disease . Presently molecular biology techniques contribute to the deciphering of these mechanisms . This review article gives an overview of the actual situation . The genetic changes leading to malignancy are successively considered at the DNA, RNA and protein levels . Alterations at the DNA level represent the bulk of the available data, being related to p53 mutations, alterations in the beta-tubulin gene, microsatellite alterations, methods for identifying individual and isolated aberrant cells, identification of epigenetic mechanisms such as methylation of the promoter region of tumor suppressor genes; alterations in pre-neoplastic lesions are also evoked . In all cases, the techniques are described and results presented . RNA based methods are critically considered, and the yeast functional assay described . Protein based methods are also considered . The use of cDNA microarrays opens new perspectives and brings the simultaneous identification of numerous DNA alterations at a grip, with hopefully significant improvements in treatment results and increased efficiency for early detection and prevention .

Mol Biochem Parasitol, 2002 Nov-Dec, 125(1-2), 135 - 46
Role of dihydrolipoyl dehydrogenase (E3) and a novel E3-binding protein in the NADH sensitivity of the pyruvate dehydrogenase complex from anaerobic mitochondria of the parasitic nematode, Ascaris suum; Harmych S et al.; The pyruvate dehydrogenase complex (PDC) plays changing roles during the aerobic-anaerobic transition in the life cycle of the parasitic nematode, Ascaris suum . However, the dihydrolipoyl dehydrogenase (E3) subunit appears to be identical in all stages, despite the fact that the PDC is less sensitive to NADH inhibition in anaerobic muscle . Therefore, we have cloned cDNAs encoding E3 and a novel anaerobic-specific E3-binding protein (E3BP) that lacks the terminal lipoyl domain found in E3BPs from yeast and mammals, and functionally expressed E3 and E3 mutants designed to have decreased dimer stability on the assumption that the binding of E3 to an anaerobic-specific E3BP might stabilize the E3 dimer interface and decrease E3 sensitivity to NADH inhibition . As predicted, the mutants exhibited decreased thermal stability, increased sensitivity to NADH and the binding of E3(Y18F) to the E3-depleted core of the pig heart PDC increased E3 activity and decreased E3 sensitivity to NADH inhibition . However, although the free A . suum E3 was less sensitive to NADH inhibition than the pig heart E3, both E3s were significantly more sensitive to NADH inhibition when assayed with dihydrolipoamide than their corresponding PDCs assayed with pyruvate . More importantly, the binding of rE3 to its core complex had little effect on its apparent K(m) for NAD(+), K(i) for NADH inhibition, or the NADH/NAD(+) ratio yielding 50% inhibition . These data suggest that although binding to the core stabilizes the E3 dimer interface, it does not play a significant role in reducing the sensitivity of the A . suum PDC to NADH inhibition during anaerobiosis.

Mol Biochem Parasitol, 2002 Nov-Dec, 125(1-2), 11 - 21
Trypanosoma brucei MRE11 is non-essential but influences growth, homologous recombination and DNA double-strand break repair; Tan KS et al.; MRE11 is a conserved multi-functional protein that is important for maintaining genomic integrity in yeast and mammalian cells . By database searching, we identified a full-length candidate MRE11 on Trypanosoma brucei chromosome II . We subsequently cloned and sequenced the corresponding gene from the Lister 427 strain . MRE11 is a single copy gene that encodes an 83 kDa protein of 763 amino acids . GFP-MRE11 and Ty1-MRE11 fusion proteins localized to the nucleus of bloodstream and procyclic T . brucei . Interestingly, Ty1-MRE11 associated, to some extent, with telomeres of procyclic but not bloodstream forms . This association appears cell-cycle dependent, with the highest co-localization in G1 cells . We were able to generate an MRE11 null mutant in bloodstream forms, indicating that it is non-essential . However, the null mutant was impaired in homologous recombination, as evidenced by the reduced integration efficiency of transfected DNA . A conditional null mutant, containing a tetracycline-inducible ectopic Ty1-MRE11, exhibited reduced growth and plating efficiency and increased sensitivity to DNA double-strand breaks, induced by methyl methanesulphonate or ionizing radiation, in the absence of tetracycline.

An Esp Pediatr, 2002 Nov, 57(5), 452 - 6
{Cutaneous colonization by Malassezia spp . in neonates}; Juncosa Morros T et al.; BACKGROUND: Malassezia spp . is a lipophilic yeast considered to be a normal component of the human skin flora . It has been associated with sepsis in patients receiving intravenous infusion of lipid emulsions through central venous catheters (CVC) . Current evidence indicates a high rate of skin colonization in healthy adults, in contrast with the low rate of colonization in prepubertal children . Of note is the high prevalence of colonized infants in the neonatal intensive care unit (NICU) . METHODS: We performed a prospective open observational study of colonization in all infants admitted to the NICU during a nine-month period (October 1997-June 1998) . Length of stay in the unit, birthweight and the use of CVC for parenteral fat infusion were evaluated . RESULTS: Seventy-seven neonates were included in the study . The mean length of stay in the NICU was 24 days . A total of 63.6 % weighed less than 2,500 g at birth and 72 % were given parenteral nutrition supplemented with fat emulsion through a CVC . The overall rate of colonization in the unit was 41.5 and 75 % of the patients became colonized within the first two weeks of admission . CONCLUSIONS: These data emphasize the need for preventive measures to reduce the transmission of these yeasts in the NICU and to prevent the occurrence of neonatal sepsis due to Malassezia spp . in immunologically immature infants.

Microsc Res Tech, 2002 Dec 15, 59(6), 536 - 41
Confocal laser scanning microscopy to study formation and properties of polyelectrolyte nanocapsules derived from CdCO3 templates; Silvano D et al.; Three-dimensional confocal laser scanning microscopy (CLSM) was used as an essential investigation method to obtain information about the formation and morphological characteristics of nanocapsules . Nanocapsules are built by layer-by-layer deposition of alternatively charged polyelectrolytes on templates forming nanostructured hollow shells . CLSM is unique in allowing for monitoring of the core dissolution process in real time and for studying nanocapsule functioning in hydrated conditions within a three-dimensional and temporal framework . Since we are also interested in the identification of other possible templates, we briefly report on the use of yeast cells as biocolloidal cores monitored by means of two-photon microscopy . Here we focus our attention on the use of CdCO(3) crystals as template candidates for the preparation of stable capsules . Both cubic and spherical CdCO(3) cores have been produced . Cubic cores exhibit higher monodispersity and smaller size compared to spherical ones . Capsules templated on these cores have a higher surface-to-volume ratio that is valuable for applications related to drug delivery, functional properties of the shells and adsorption of proteins, and other biologically relevant molecules . Microsc . Res . Tech . 59:536-541, 2002 .

Nucleic Acids Res, 2002 Dec 1, 30(23), 5301 - 9
PSKH1, a novel splice factor compartment-associated serine kinase; Brede G et al.; Small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP splicing factors containing a serine/arginine-rich domain (SR proteins) concentrate in splicing factor compartments (SFCs) within the nucleus of interphase cells . Nuclear SFCs are considered mainly as storage sites for splicing factors, supplying splicing factors to active genes . The mechanisms controlling the interaction of the various spliceosome constituents, and the dynamic nature of the SFCs, are still poorly understood . We show here that endogenous PSKH1, a previously cloned kinase, is located in SFCs . Migration of PSKH1-FLAG into SFCs is enhanced during co-expression of T7-tagged ASF/SF2 as well as other members of the SR protein family, but not by two other non-SR nuclear proteins serving as controls . Similar to the SR protein kinase family, overexpression of PSKH1 led to reorganization of co-expressed T7-SC35 and T7-ASF/SF2 into a more diffuse nuclear pattern . This redistribution was not dependent on PSKH1 kinase activity . Different from the SR protein kinases, the SFC-associating features of PSKH1 were located within its catalytic kinase domain and within its C-terminus . Although no direct interaction was observed between PSKH1 and any of the SR proteins tested in pull-down or yeast two-hybrid assays, forced expression of PSKH1-FLAG was shown to stimulate distal splicing of an E1A minigene in HeLa cells . Moreover, a GST-ASF/SF2 fusion was not phosphorylated by PSKH1, suggesting an indirect mechanism of action on SR proteins . Our data suggest a mutual relationship between PSKH1 and SR proteins, as they are able to target PSKH1 into SFCs, while forced PSKH1 expression modulates nuclear dynamics and the function of co-expressed splicing factors.

Nucleic Acids Res, 2002 Dec 1, 30(23), 5205 - 12
Functional domains involved in the interaction between Orc1 and transcriptional repressor AlF-C that bind to an origin/promoter of the rat aldolase B gene; Saitoh Y et al.; The promoter of the rat aldolase B (AldB) gene functions in vivo as an origin of DNA replication in the cells in which transcription of the gene is repressed . Previously, we identified two closely related DNA-binding proteins, AlF-C1 and AlF-C2, which repressed the AldB gene promoter . We also reported that the binding site of these proteins, site C, is one of the required DNA elements of the AldB gene origin/promoter for autonomously replicating activity in transfected cells . In the present study, we show that AlF-C1 and AlF-C2 bind directly to Orc1, a subunit of the origin recognition complex (ORC) . Deletion analyses revealed a functional domain in AlF-C2 for binding to Orc1, which is located separately from the DNA-binding domain . In addition, we found a novel protein-interacting domain in Orc1 required for the binding of AlF-C2, which was conserved in human, mouse and Chinese hamster, but not in Drosophila, frog and yeast . Thus, it is assumed that in mammalian cells, sequence- specific DNA-binding proteins are involved in recruiting ORC to regulate replication initiation and/or transcription repression.

Nucleic Acids Res, 2002 Dec 1, 30(23), 5182 - 92
Methylation of Xenopus CIRP2 regulates its arginine- and glycine-rich region-mediated nucleocytoplasmic distribution; Aoki K et al.; Cold-inducible RNA-binding protein (CIRP) was originally found in mammalian cells as a protein that is overexpressed upon a temperature downshift . Recently, we identified a Xenopus homolog of CIRP, termed xCIRP2, as a major cytoplasmic RNA-binding protein in oocytes . In this study we found by yeast two-hybrid screening that the Xenopus homolog of protein arginine N-methyltransferase 1 (xPRMT1) interacted with xCIRP2 . We found that an arginine- and glycine-rich region of xCIRP2, termed the RG4 domain, was a target of xPRMT1 for methylation in vitro . xCIRP2 expressed in cultured cells accumulated in the nucleus as does mammalian CIRP . Interestingly, the RG4 domain was necessary for nuclear localization of xCIRP2 . RG4-mediated nuclear accumulation of xCIRP2 was diminished in the presence of transcription inhibitors, suggesting that nuclear localization of xCIRP2 was dependent on ongoing transcription with RNA polymerase II . Analysis of interspecies heterokaryons revealed that xCIRP2 was capable of nucleocytoplasmic shuttling and the RG4 domain functioned as a nucleocytoplasmic shuttling signal . Methylation by overexpressed xPRMT1 caused cytoplasmic accumulation of xCIRP2 . Possible implications of the relationship between regulation of intracellular localization and multiple functions of xCIRP2 will be discussed.

Nucleic Acids Res, 2002 Dec 1, 30(23), 5017 - 28
The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1; Al-Maghrebi M et al.; Previously, we have shown that the vimentin 3' untranslated region (3'UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA . This region was shown to specifically bind protein(s) by band shift analyses . UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass . Here, we have used this sequence as 'bait' to isolate RNA binding proteins using the yeast three-hybrid method . This technique relies on a functional assay detecting bona fide RNA-protein interaction in vivo . Three cDNA isolates, HAX-1, eEF-1gamma and hRIP, code for proteins of a size consistent with in vitro cross- linking studies . In all cases, recombinant proteins were capable of binding RNA in vitro . Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-1gamma and HAX-1 . Moreover, HAX-1 binding appears to be specific to vimentin's 3'UTR . Both in vivo synthesized eEF-1gamma and HAX-1 proteins were 'pulled out' of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin's 3'UTR . Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1gamma or HAX-1 . Thus, in addition to their known functions, both eEF-1gamma and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.

J Biol Chem, 2003 Feb 14, 278(7), 5188 - 94 Epub 2002 Dec 03.
Identification of the LIM protein FHL2 as a coactivator of beta-catenin; Wei Y et al.; Beta-catenin is a key mediator of the Wnt pathway, which plays a critical role in embryogenesis and oncogenesis . As a transcriptional activator, beta-catenin binds the transcription factors, T-cell factor and lymphoid enhancer factor, and regulates gene expression in response to Wnt signaling . Abnormal activation of beta-catenin has been linked to various types of cancer . In a yeast two-hybrid screen, we identified the four and a half of LIM-only protein 2 (FHL2) as a novel beta-catenin-interacting protein . Here we show specific interaction of FHL2 with beta-catenin, which requires the intact structure of FHL2 and armadillo repeats 1-9 of beta-catenin . FHL2 cooperated with beta-catenin to activate T-cell factor/lymphoid enhancer factor-dependent transcription from a synthetic reporter and the cyclin D1 and interleukin-8 promoters in kidney and colon cell lines . In contrast, coexpression of beta-catenin and FHL2 had no synergistic effect on androgen receptor-mediated transcription, whereas each of these two coactivators independently stimulated AR transcriptional activity . Thus, the ability of FHL2 to stimulate the trans-activating function of beta-catenin might be dependent on the promoter context . The detection of increased FHL2 expression in hepatoblastoma, a liver tumor harboring frequent beta-catenin mutations, suggests that FHL2 might enforce beta-catenin transactivation activity in cancer cells . These findings reveal a new function of the LIM coactivator FHL2 in transcriptional activation of Wnt-responsive genes.

J Biol Chem, 2003 Feb 21, 278(8), 6420 - 6 Epub 2002 Dec 03.
Sorting of encystation-specific cysteine protease to lysosome-like peripheral vacuoles in Giardia lamblia requires a conserved tyrosine-based motif; Touz MC et al.; Encystation-specific cysteine protease (ESCP) was the first membrane-associated protein described to be part of the lysosome-like peripheral vacuoles in the intestinal parasite Giardia lamblia . ESCP is homologous to cathepsin C enzymes of higher eukaryotes, but is distinguished from other lysosomal cysteine proteases because it possesses a transmembrane domain and a short cytoplasmic tail . Tyrosine-based motifs within tails of membrane proteins are known to participate in endosomal/lysosomal protein sorting in higher eukaryotes . In this study, we show that a YRPI motif within the ESCP cytoplasmic tail is necessary and sufficient to mediate ESCP sorting to peripheral vacuoles in Giardia . Deletion and point mutation analysis demonstrated that the tyrosine residue is critical for ESCP sorting, whereas amino acids located at the Y+1 (Arg), Y+2 (Pro), and Y+3 (Ile) positions show minimal effect . Loss of the motif resulted in surface localization, whereas addition of the motif to a variant-specific surface protein resulted in lysosomal localization . Although Giardia trophozoites lack a morphologically discernible Golgi apparatus, our findings indicate that this parasite directs proteins to the lysosomes using a conserved sorting signal similar to that used by yeast and mammalian cells . Because Giardia is one of the earliest branching protist, these results demonstrate that sorting motifs for specific protein traffic developed very early during eukaryotic evolution.

Development, 2003 Jan, 130(2), 343 - 54
The Drosophila trithorax group gene tonalli (tna) interacts genetically with the Brahma remodeling complex and encodes an SP-RING finger protein; Gutierrez L et al.; The trithorax group genes are required for positive regulation of homeotic gene function . The trithorax group gene brahma encodes a SWI2/SNF2 family ATPase that is a catalytic subunit of the Brm chromatin-remodeling complex . We identified the tonalli (tna) gene in Drosophila by genetic interactions with brahma . tna mutations suppress Polycomb phenotypes and tna is required for the proper expressions of the Antennapedia, Ultrabithorax and Sex combs reduced homeotic genes . The tna gene encodes at least two proteins, a large isoform (TnaA) and a short isoform (TnaB) . The TnaA protein has an SP-RING Zn finger, conserved in proteins from organisms ranging from yeast to human and thought to be involved in the sumoylation of protein substrates . Besides the SP-RING finger, the TnaA protein also has extended homology with other eukaryotic proteins, including human proteins . We show that tna mutations also interact with mutations in additional subunits of the Brm complex, with mutations in subunits of the Mediator complex, and with mutations of the SWI2/SNF2 family ATPase gene kismet . We propose that Tna is involved in postranslational modification of transcription complexes.

Org Lett, 2002 Dec 12, 4(25), 4459 - 62
Directed evolution experiments reveal mutations at cycloartenol synthase residue His477 that dramatically alter catalysis; Segura MJ et al.; {reaction: see text} Cycloartenol synthase cyclizes and rearranges oxidosqualene to the protosteryl cation and then specifically deprotonates from C-19 . To identify mutants that deprotonate differently, randomly generated mutant cycloartenol synthases were selected in a yeast lanosterol synthase mutant . A novel His477Asn mutant was uncovered that produces 88% lanosterol and 12% parkeol . The His477Gln mutant produces 73% parkeol, 22% lanosterol, and 5% Delta(7)-lanosterol . These are the most accurate lanosterol synthase and parkeol synthase that have been generated by mutagenesis.

J Biol Chem, 2003 Feb 14, 278(7), 4603 - 10 Epub 2002 Dec 02.
Delta 12-oleate desaturase-related enzymes associated with formation of conjugated trans-delta 11, cis-delta 13 double bonds; Iwabuchi M et al.; Conjugated linolenic acids are present as major seed oils in several plant species . Punicic acid (or trichosanic acid) is a conjugated linolenic acid isomer containing cis-delta9, trans-delta11, cis-delta13 double bonds in the C(18) carbon chain . Here we report cDNAs, TkFac and PgFac, isolated from Trichosanthes kirilowii and Punica granatum, that encode a class of conjugases associated with the formation of trans-delta11, cis-delta13 double bonds . Expression of TkFac and PgFac in Arabidopsis seeds under transcriptional control of the seed-specific napin promoter resulted in accumulation of punicic acid up to approximately 10% (w/w) of the total seed oils . In contrast, no punicic acid was found in lipids from leaves even when the conjugases were driven under control of the cauliflower mosaic virus 35S promoter . In yeast cells grown without exogenous fatty acids in the culture medium, TkFac and PgFac expression resulted in punicic acid accumulation accompanied by 16:2delta(9cis, 12cis) and 18:2delta(9cis, 12cis) production . Thus, TkFac and PgFac are defined as bifunctional enzymes having both conjugase and delta12-oleate desaturase activity . Furthermore, we demonstrate that 16:2delta(9cis, 12cis) and 18:3delta(9cis, 12cis, 15cis) as well as 18:2delta(9cis, 12cis) are potential substrates for the conjugases to form trans-delta11 and cis-delta13 double bonds.

Mol Biol Rep, 2002 Sep, 29(3), 317 - 23
Screen and identification of proteins interacting with ADAM19 cytoplasmic tail; Huang L et al.; ADAM family plays important roles in neurogenesis . The cytoplasmic tail of ADAM19 (ADAM19-CT) contains 193 residues . The presence of two putative SH3 ligand-binding sites suggests potential interactions with cytosolic proteins, which could be possibly linked to the functions of ADAM19 . To address these issues, a yeast two-hybrid screen was performed in human fetal brain cDNA library to isolate proteins that interact with the cytoplasmic tail of ADAM19 . Four proteins were obtained, ArgBP1, beta-cop, ubiquitin and a novel protein . GST-Pulldown assay has confirmed the interaction between AdAM19 and ArgBP1 . By constructing series of deletion mutants of ADAM19-CT and ArgBP1 respectively, the interaction regions have been identified . They are the SH3 binding sites in ADAM19-CT and the P4 region in ArgBP1 . And the interaction is specific . ArgBP1 does not bind to ADAM22, ADAM29 or ADAM9 (mouse) . ArgBP1 may be the key protein, which accounts for the physiological function of ADAM19.

Biochem Genet, 2002 Dec, 40(11-12), 359 - 78
Genetic selection for modulators of the MAP kinase and beta-catenin growth-control pathways in mammalian cells; Wheatley W et al.; Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways . In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway . We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade . Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases . In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered . In neither selection were peptide inhibitors observed . We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.

Med Mycol, 2002 Oct, 40(5), 501 - 5
Effect of CD40 ligand on the course of murine histoplasmosis; Wheat LJ et al.; CD40 ligand-CD40 ligation is important in the development of T-cell-mediated immune responses . The purpose of this study was to examine the role of CD40L in recovery from histoplasmosis using a murine model of intratracheally induced infection . B6C3F1 mice were infected intratracheally with Histoplasma capsulatum yeast and monitored for clearance of the organism from the lungs and spleen . CD40L treatment was begun on either day -2 or +2 post inoculation and continued until day 14 in CD4-depleted animals and from day -2 to day +4 in non-immunosuppressed animals . Amphotericin B treatment was begun four days following inoculation and given every other day for 10 days . CD40L reduced fungal burden by less than one log when started two days before infection but did not act synergistically with low-dosage amphotericin B (0.2 mg kg(-1) qod) in CD4 depleted mice . Low-dose amphotericin B, CD40L, and the combination of the two failed to lower the fungal burden in a second experiment using a more virulent isolate of the same strain of H . capsulatum in CD4-depleted mice . Furthermore, CD40L did not increase the concentrations of IFN-gamma, IL-12 or IL-10 in the lungs or spleens of infected animals . In summary, CD40L had minimal or no effect on the course of infection in this murine model of histoplasmosis.

J Cell Biochem, 2003 Jan 1, 88(1), 95 - 103
Establishment of dependence relationships between genome replication and mitosis; Clarke DJ; Although budding yeast cell biology and genetics provided a powerful system to isolate S-phase checkpoint mutants, initial studies relied on a defect not likely to be relevant in higher eukaryotes . The first mutants were isolated for their inability to restrain mitotic spindle elongation in S-phase . Since most eukaryotes do not assemble spindles until prometaphase the validity of this approach might have been questioned . However, these early studies were designed with a highly valid assumption in mind; that checkpoints have a variety of targets, but comprise conserved kinase cascades that make up these signaling pathways . The task that lies ahead is to determine targets of the S-phase checkpoint relevant to mammals . One step forward might be the realization that the budding yeast S-phase checkpoint prevents loss of sister chromatid cohesion while DNA replication is ongoing . If this mechanism is conserved in mammals, it could prove vital for chromosome segregation fidelity .

Genes Chromosomes Cancer, 2003 Jan, 36(1), 57 - 69
Deletion mapping on the long arm of chromosome 7 in splenic lymphoma with villous lymphocytes; Gruszka-Westwood AM et al.; Splenic lymphoma with villous lymphocytes (SLVL) is a low-grade lymphoproliferative disorder characterized by splenomegaly and circulating villous lymphocytes in the peripheral blood . It is considered to be the leukemic form of splenic marginal zone lymphoma (SMZL) . The genetic basis of this lymphoma type remains unknown . Conventional cytogenetic studies have identified frequent structural abnormalities of chromosome 7, in the form of translocations, mainly unbalanced, and 7q deletions . In this current study, we undertook deletion mapping of the long arm of chromosome 7 in a series of cases with SLVL . Metaphase fluorescence in situ hybridization (FISH) was used in the first instance, followed by a study of loss of heterozygosity (LOH) . The common area of deletion identified by FISH spanned from the YAC clone HSC7E1289 (mapping to 7q32.1) to in between YACs HSC7E195 and HSC7E648 (7q32-3) . By application of 50 microsatellite markers mapping to the FISH-CDR and to areas of deletion reported in other studies, four distinct hotspot loci were identified, with abnormalities present in 29-55% cases . In three of them, both LOH and biallelic deletions were found . The LOH in the majority of patients was noncontiguous . The presence of a high incidence of abnormalities in the established hotspot areas and in particular the finding of biallelic deletions is indicative of the existence of genes important for the pathogenesis of SLVL in these areas .

Glycobiology, 2002 Nov, 12(11), 771 - 83
Characterization of POMT2, a novel member of the PMT protein O-mannosyltransferase family specifically localized to the acrosome of mammalian spermatids; Willer T et al.; Over the past few years it has emerged that O-mannosyl glycans are not restricted to yeasts and fungi but are also present in higher eukaryotes, including humans . They play a substantial role in the onset of muscular dystrophy and neuronal migration disorders, like muscle-eye-brain disease . Protein O-mannosyltransferase genes (PMTs) are evolutionarily conserved from yeast to human; however, little is known about these enzymes in higher eukaryotes . In this study, we cloned the first PMT2 subfamily members from human (hPOMT2), mouse (mPomt2), and Drosophila (DmPOMT2) . A detailed characterization of the mammalian POMT2, with emphasis on mouse Pomt2, shows that mammalian POMT2 is predominantly expressed in testis tissue . Due to differential transcription initiation of the mPomt2 gene, two distinct mRNA species that vary in length are formed . The shorter transcript is present in all somatic tissues examined . Expression of the corresponding hPOMT2 cDNA in mammalian cells identified POMT2 as an integral membrane protein of the endoplasmic reticulum with an apparent molecular weight of 83 kDa . The longer mPomt2 transcript is restricted to testis and encodes a testis-specific mPOMT2 protein isoform . Using in situ hybridization and immunolocalization, we demonstrate that in testis tissue mPOMT2 localizes to maturing spermatids and is abundant within the acrosome, a sperm-specific organelle essential for fertilization . Our data suggest a novel and specific role for the putative protein O-mannosyltransferase POMT2 in the maturation and/or function of sperm in mammals.

Jpn J Cancer Res, 2002 Nov, 93(11), 1183 - 6
Identification of the CAB2/hCOS16 gene required for the repair of DNA double-strand breaks on a core amplified region of the 17q12 locus in breast and gastric cancers; Nezu M et al.; We previously reported that CAB1 and c-ERBB-2 genes were found to be located in a core amplified region of the 17q12 locus, which is frequently amplified in various cancers . During identification of this core region, CAB2, a human homologue of the yeast COS16 required for the repair of DNA double-strand breaks was cloned . Autofluorescence analysis of cells transfected with its GFP fusion protein demonstrated that CAB2 translocates into vesicles, suggesting that overexpression of CAB2 may decrease intercellular Mn2+ by accumulating it in the vesicles, in the same way as yeast COS16 . This is the first report identifying all of the genes on the core amplified region of the 17q12 locus in breast and gastric cancers.

Biochemistry (Mosc), 2002 Oct, 67(10), 1197 - 202
The regulatory alpha subunit of phosphorylase kinase may directly participate in the binding of glycogen phosphorylase; Andreeva IE et al.; The yeast two-hybrid screen has been used to identify potential regions of interaction of the largest regulatory subunit, alpha, of phosphorylase kinase (PhK) with two fragments of its protein substrate, glycogen phosphorylase b (Phb) . One fragment, corresponding to residues 17-484 (PhbN'), contained the regulatory domain of the protein, but in missing the first 16 residues was devoid of the sole phosphorylation site of Phb, Ser14; the second fragment corresponded to residues 485-843 (PhbC) and contained the catalytic domain of Phb . Truncation fragments of the alpha subunit were screened for interactions against these two substrate fragments . PhbC was not found to interact with any alpha constructs; however, PhbN' interacted with a region of alpha (residues 864-1014) that is near the phosphorylatable region of that subunit . PhbN' was also screened for interactions against a variety of fragments of the catalytic gamma subunit of PhK; however, no interactions were detected, even with full-length gamma . Our results support the idea that amino acid residues proximal to the convertible serine of Phb are important for its specific interaction with the catalytic subunit of PhK, but that regions distinct from the convertible serine residue of Phb and from the catalytic domain of PhK may also be involved in the interaction of these two proteins.

FEBS Lett, 2002 Dec 4, 532(1-2), 211 - 5
DelGEF, a homologue of the Ran guanine nucleotide exchange factor RanGEF, binds to the exocyst component Sec5 and modulates secretion; Sjolinder M et al.; In order to identify the function of deafness locus putative guanine nucleotide exchange factor (DelGEF), a protein homologous to the nucleotide exchange factor for the small GTPase Ran, a cDNA library was screened for interacting proteins using a yeast two-hybrid system . The human homologue of Sec5, a protein involved in vesicle transport and secretion, was identified as a binding partner . The interaction between DelGEF and Sec5 was found to be dependent on Mg2+ and stimulated by guanosine triphosphate (GTP) or deoxycytidine triphosphate (dCTP) . Downregulation of endogenous DelGEF in HeLa cells induced increased extracellular secretion of proteoglycans indicating a possible role for DelGEF in the secretion process.

FEBS Lett, 2002 Dec 4, 532(1-2), 27 - 30
Interaction of FLU, a negative regulator of tetrapyrrole biosynthesis, with the glutamyl-tRNA reductase requires the tetratricopeptide repeat domain of FLU; Meskauskiene R et al.; Regulation of tetrapyrrole biosynthesis in plants has been attributed to feedback control of glutamyl-tRNA reductase (GLU-TR) by heme . Recently, another negative regulator, the FLU protein, has been discovered that operates independently of heme . A truncated form of FLU that contains two domains implicated in protein-protein interaction was co-expressed in yeast with either GLU-TR or glutamate-1-semialdehyde-2-1-aminotransferase (GSA-AT), the second enzyme involved in delta-aminolevulinic acid (ALA) biosynthesis . FLU interacts strongly with GLU-TR but not with GSA-AT . Two variants of FLU that carry single amino acid exchanges within their coiled coil and tetratricopeptide repeat (TPR) domains, respectively, were also tested . Only the FLU variant with the mutated TPR motif lost the capacity to interact with GLU-TR.

Gene, 2002 Oct 16, 299(1-2), 195 - 204
Cloning and comparative sequence analysis of PUM1 and PUM2 genes, human members of the Pumilio family of RNA-binding proteins; Spassov DS et al.; Drosophila gene Pumilio (Pum) is a founder member of an evolutionarily conserved family of RNA-binding proteins that are present from yeast to mammals, and act as translational repressors during embryo development and cell differentiation . The human genome contains two Pumilio related genes, PUM1 and PUM2, that encode 127 and 114 kDa proteins with evolutionarily highly conserved Pum RNA-binding domain (86 and 88% homology with the fly Pum protein) . PUM1 and PUM2 proteins share 83% overall similarity, with RNA-binding domain being 91% identical . Both PUM1 and PUM2 show relatively widespread and mostly overlapping expression in human tissues, and are very large genes with highly conserved gene structure . PUM1 consists of 22 exons, spanning about 150 kb on chromosome 1p35.2, whereas PUM2 consists of 20 exons and spans at least 80 kb on chromosome 2p23-24 . Extremely high evolutionary conservation of the RNA-binding domain from yeast to humans, and conserved function of Pumilio proteins in invertebrates and lower vertebrates suggest that mammalian Pumilio proteins could also play an important role in translational regulation of embryogenesis and cell development and differentiation.

J Microbiol Methods, 2003 Feb, 52(2), 221 - 9
Visual clone identification of Penicillium commune isolates; Hansen ME et al.; A method for visual clone identification of Penicillium commune isolates was developed . The method is based on images of fungal colonies acquired after growth on a standard medium and involves a high degree of objectivity, which in future studies will make it possible for non-experts to perform a qualified identification of different species as well as clones within a species . A total of 77 P . commune isolates from a cheese dairy were 3-point inoculated on Yeast Extract Sucrose (YES) agar and incubated for 7 days at 25 degrees C . After incubation, the isolates were classified into groups containing the same genotype determined by DNA fingerprinting (AFLP) . Each genotype also has a specific phenotype such as different colony colours . By careful image acquisition, colours were measured in a reproducible way . Prior to image analysis, each image was corrected with respect to colour, geometry and self-illumination, thereby gaining a set of directly comparable images . A method for automatic extraction of a given number of concentric regions was used . Using the positions of the regions, a number of relevant features--capturing colour and colour-texture from the surface of the fungal colonies--was extracted for further analysis . We introduced the Jeffreys-Matusitas (JM) distance between the feature distributions to express the similarity between regions in two colonies, and to evaluate the overall (weighted) similarity . The nearest neighbour (NN) classification rule was used . On a dataset from 137 isolates, we obtained a "leave-one-out" cross-validation identification rate of approximately 93-98% compared with the result of DNA fingerprinting .

Biochem Biophys Res Commun, 2002 Dec 13, 299(4), 594 - 8
The binding of the RyR2 calcium channel to its gating protein FKBP12.6 is oppositely affected by ARVD2 and VTSIP mutations; Tiso N et al.; Arrhythmogenic right ventricular dysplasia/cardiomyopathy type 2 (ARVD2, OMIM 600996) and stress-induced polymorphic ventricular tachycardia (VTSIP, OMIM 604772) are two cardiac diseases causing juvenile sudden death, both associated with mutations in the RyR2 calcium channel . By using a quantitative yeast two-hybrid system, we show that VTSIP- and ARVD2-associated point mutations influence positively and negatively, respectively, the binding of RyR2 to its gating protein FKBP12.6 . These findings suggest that ARVD2 mutations increase RyR2-mediated calcium release to cytoplasm, while VTSIP mutations do not affect significantly cytosolic calcium levels, thereby explaining the clinical differences between the two diseases . The present two-hybrid system appears to be an efficient molecular tool to assay the binding of FKBP12s proteins to both cardiac RyR2 and skeletal muscle RyR1 isoforms, circumventing the full-length expression of this class of giant channels . We also provide evidence of the suitability of this system to test new drugs that target RyRs-FKBP12s interactions and do not affect yeast growth.

J Biol Chem, 2003 Feb 7, 278(6), 3590 - 8 Epub 2002 Nov 27.
The proteoglycan NG2 is complexed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamate receptor interaction protein (GRIP) in glial progenitor cells . Implications for glial-neuronal signaling; Stegmuller J et al.; The proteoglycan NG2 is expressed by immature glial cells in the developing and adult central nervous system . Using the COOH-terminal region of NG2 as bait in a yeast two-hybrid screen, we identified the glutamate receptor interaction protein GRIP1, a multi-PDZ domain protein, as an interacting partner . NG2 exhibits a PDZ binding motif at the extreme COOH terminus which binds to the seventh PDZ domain of GRIP1 . In addition to the published expression in neurons, GRIP1 is expressed by immature glial cells . GRIP1 is known to bind to the GluRB subunit of the AMPA glutamate receptor expressed by subpopulations of neurons and immature glial cells . In cultures of primary oligodendrocytes, cells coexpress GluRB and NG2 . A complex of NG2, GRIP1, and GluRB can be precipitated from transfected mammalian cells and from cultures of primary oligodendrocytes . Furthermore, NG2 and GRIP can be coprecipitated from developing brain tissue . These data suggest that GRIP1 acts as a scaffolding molecule clustering NG2 and AMPA receptors in immature glia . In view of the presence of synaptic contacts between neurons and NG2-positive glial cells in the hippocampus and the close association of NG2-expressing glial cells with axons, we suggest a role for the NG2.AMPA receptor complex in glial-neuronal recognition and signaling.

J Biol Chem, 2003 Feb 21, 278(8), 6181 - 6 Epub 2002 Nov 27.
A broad but restricted requirement for TAF-5 (human TAFII100) for embryonic transcription in Caenorhabditis elegans; Walker AK et al.; As conserved components of the transcription factor (TF) IID- and TFTC/SAGA-related complexes, TATA-binding protein-associated factors (TAF(II)s) are important for eukaryotic mRNA transcription . In yeast, genetic analyses suggest that, although some individual TAF(II)s are required for transcription of most genes, others have highly specialized functions . Much less is known about the functions of TAF(II)s in metazoans, which have more complex genomes that include many tissue-specific genes . TAF-5 (human (h) TAF(II)100) is of particular interest because it is predicted to have an important structural role . Here we describe the first genetics-based analysis of TAF-5 in a metazoan . By performing RNA interference in Caenorhabditis elegans embryos, which can survive for several cell generations without transcription, we found that taf-5 is important for a significant fraction of transcription . However, TAF-5 is apparently not essential for the expression of multiple developmental and other metazoan-specific genes . This phenotype remarkably resembles the previously described effects of similarly depleting two C . elegans histone fold TAF(II)s, TAF-9 (hTAF(II)31/32) and TAF-10 (hTAF(II)30), but is distinct from the widespread transcription block caused by TAF-4 (hTAF(II)130) depletion . Our findings suggest that TAF-5, TAF-9, and TAF-10 are part of a functional module of TFIID- and TFTC/SAGA-related complexes that can be bypassed in many metazoan-specific genes.

J Biol Chem, 2003 Feb 7, 278(6), 3882 - 90 Epub 2002 Nov 27.
Conserved N-terminal motifs of telomerase reverse transcriptase required for ribonucleoprotein assembly in vivo; Bosoy D et al.; Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats . The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase (RT)-like motifs that directly mediate nucleotide addition . The RT motifs are located in the C-terminal region of the polypeptide . Sequence alignments also revealed the existence of four conserved motifs (named GQ, CP, QFP, and T) in the N-terminal region of TERT . The GQ motif of yeast TERT has been demonstrated previously to be essential for telomerase catalysis and may participate in RNP formation . In this report, we show that substitution of conserved residues in the CP, QFP, and T motifs of yeast TERT also impairs both telomere maintenance and telomerase activity, thus confirming the validity of the sequence alignment . The extent of telomere shortening correlates with the extent of reduction in the level of telomerase activity, TERT protein, and TERT-associated TLC1 RNA . Overexpression of the mutant proteins does not result in telomere shortening, implying that assembly rather than catalytic function was affected . This notion was further supported by comparing the efficiency of RNP formation in the wild type and the overexpression strains . Taken together, our results show that three of the four N-terminal motifs are required for efficient telomerase RNP formation in vivo but not for the enzymatic function of telomerase . We also show that the majority of telomerase-associated TLC1 RNA has a more upstream 3' end than previously reported, consistent with additional processing events during RNP maturation.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Dec 25, 782(1-2), 267 - 89
Multidimensional chromatography coupled to electrospray ionization time-of-flight mass spectrometry as an alternative to two-dimensional gels for the identification and analysis of complex mixtures of intact proteins; Liu H et al.; The limitations of 2-D gels for global proteomics have encouraged the development of alternative approaches for identifying proteins in complicated mixtures, and determining their modification state . In this work, we describe the application of multidimensional liquid chromatography (SCX-RPLC) coupled with electrospray time-of-flight mass spectrometry and off-line fraction collection to analyze complex intact protein mixtures . Methods were developed using both standard proteins and an enriched yeast ribosomal fraction sample containing approximately 100 proteins, which permitted assessment of the effectiveness of the individual separation dimensions, as well as investigation of the interplay between separation capacity and electrospray MS performance.

Virus Res, 2002 Dec, 90(1-2), 113 - 8
Novel cellular interacting partners of the human papillomavirus 16 transcription/replication factor E2; Boner W et al.; Human papillomaviruses (HPVs) are causative agents in a number of human diseases . HPV can be divided into two groups: low risk that cause diseases such as genital warts, and high risk that cause ano-genital cancers . Of the high-risk group, HPV16 is the most commonly found in cervical cancer . All HPV encode an E2 protein and this protein regulates transcription from, and replication of, the viral genome making it essential for the viral life cycle . In order to function E2 must interact with cellular proteins; identification of these cellular partners will provide targets for disruption of the viral life cycle and will also provide insights into the processes of transcription and replication . To identify the cellular interacting partners for HPV16 E2, we carried out a yeast two-hybrid screen with the amino-terminus of E2 that is essential for mediating transcription and replication . Here we describe how this screen was carried out and detail the interacting partners that were identified; these include the proteins TopBP1, RACK1, POMP, p27(BBP), ODC antizyme, and Delta-adaptin . Several of these partners have characteristics that make them ideal candidates for mediating E2 function.

Curr Opin Microbiol, 2002 Dec, 5(6), 580 - 5
Polarity in filamentous fungi: establishment, maintenance and new axes; Momany M; Germ tube emergence in filamentous fungi appears to be similar to bud emergence in yeast . Several key proteins (e.g . Cdc42, septins, Bni1 formin, Rho1 and Rho3) play common roles in polarity establishment and early polarity maintenance in both processes . Although germ tube extension, which can be thought of as extreme polarity maintenance, uses some of the same genes, they are likely to be regulated differently . Mutations in polarity maintenance genes often lead to a split tip in filamentous fungi, a phenotype without an analogue in yeast . Cell cycle regulation differs between tip splitting and subapical branching, but in both processes filamentous fungi maintain several axes of polar growth simultaneously.

Curr Opin Microbiol, 2002 Dec, 5(6), 575 - 9
Chemotaxis and cell differentiation in Dictyostelium; Kay RR; Dictyostelium genome sequencing predicts an unexpectedly large number of genes . Many are absent from yeast but present in animals and presumably support cellular abilities not found in yeast . Prominent amongst these abilities is chemotaxis, where great strides are being made in understanding how cells orient in a gradient and mobilise their cytoskeleton for movement . In multicellular development, a regulatory scheme for proportioning prespore and prestalk-O cells has emerged.

Mol Endocrinol, 2002 Dec, 16(12), 2764 - 79
Dual mechanism of signal transducer and activator of transcription 5 activation by the insulin receptor; Le MN et al.; Insulin stimulates signal transducer and activator of transcription 5 (Stat5) activation in insulin receptor (IR)-overexpressing cell lines and in insulin target tissues of mice . Stat5b and insulin receptor substrate 1 (IRS-1) interact with the same autophosphorylation site in the IR {phosphotyrosine (pY) 972} in yeast two-hybrid assays, and the IR phosphorylates Stat5b in vitro . These data suggest that Stat5 proteins might be recruited to, and phosphorylated by, the activated IR in vivo . Nevertheless, insulin activates Janus kinases (JAKs) in IR-overexpressing cell lines and in insulin target tissues . To determine whether Stat5 proteins must be recruited to the pY972LSA motif in the IR for insulin-stimulated activation in mammalian cells, we generated and tested a series of IR mutants . The L973R/A975D mutation abolishes the ability of the IR to induce Stat5 activation, whereas IRS-1 phosphorylation is unaffected . In contrast, the N969A/P970A mutation in the IR has no effect on Stat5 activation but significantly reduces IRS-1 phosphorylation . In coimmunoprecipitation assays, insulin-stimulated Stat5 activation correlates with Stat5 recruitment to the IR . We also find that insulin stimulates tyrosine phosphorylation of JAKs that are constitutively associated with the IR . Expression of dominant-negative (DN) JAKs, the JAK inhibitor suppressor of cytokine signaling 1, or pretreatment with the JAK inhibitor, AG490, reduces, but does not eliminate, insulin-induced Stat5 activation . Expression of the appropriate pair of DN JAKs in each of the singly JAK-deficient cell lines further establishes a component of insulin-stimulated Stat5 activation that is JAK independent . This likely represents phosphorylation of Stat5 proteins by the IR, as we find that IR kinase domain phosphorylates Stat5b in vitro on Y699 as efficiently as JAK2 . Increasing the concentration of Stat5 proteins in cells favors the direct phosphorylation of Stat5 by the IR kinase where the DN-JAK inhibition of insulin-stimulated Stat5 activation becomes insignificant . At physiological levels of Stat5 however, we propose that JAKs and the IR both contribute to the insulin-induced phosphorylation of Stat5.

J Exp Bot, 2003 Jan, 54(380), 11 - 23
Meiosis, recombination and chromosomes: a review of gene isolation and fluorescent in situ hybridization data in plants; Schwarzacher T; Evidence is now increasing that many functions and processes of meiotic genes are similar in yeast and higher eukaryotes . However, there are significant differences and, most notably, yeast has considerably higher recombination frequencies than higher eukaryotes, different cross-over interference and possibly more than one pathway for recombination, one late and one early . Other significant events are the timing of double-strand breaks (induced by Spo11) that could be either cause or consequence of homologous chromosome synapsis and SC formation depending on the organisms, yeast plants and mammals versus Drosophila melanogaster and Caenorhabditis elegans . Many plant homologues and heterologues to meiotic genes of yeast and other organisms have now been isolated, in particular in Arabidopsis thaliana, showing that overall recombination genes are very conserved while synaptonemal complex and cohesion proteins are not . In addition to the importance of unravelling the meiotic processes by gene discovery, this review discusses the significance of chromatin packaging, genome organization, and distribution of specific repeated DNA sequences for homologous chromosome cognition and pairing, and the distribution of recombination events along the chromosomes.

J Cell Sci, 2003 Jan 1, 116(Pt 1), 39 - 49
Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites; Bergman LW et al.; Apicomplexan host cell invasion and gliding motility depend on the parasite's actomyosin system located beneath the plasma membrane of invasive stages . Myosin A (MyoA), a class XIV unconventional myosin, is the motor protein . A model has been proposed to explain how the actomyosin motor operates but little is known about the components, topology and connectivity of the motor complex . Using the MyoA neck and tail domain as bait in a yeast two-hybrid screen we identified MTIP, a novel 24 kDa protein that interacts with MyoA . Deletion analysis shows that the 15 amino-acid C-terminal tail domain of MyoA, rather than the neck domain, specifically interacts with MTIP . In Plasmodium sporozoites MTIP localizes to the inner membrane complex (IMC), where it is found clustered with MyoA . The data support a model for apicomplexan motility and invasion in which the MyoA motor protein is associated via its tail domain with MTIP, immobilizing it at the outer IMC membrane . The head domain of the immobilized MyoA moves actin filaments that, directly or via a bridging protein, connect to the cytoplasmic domain of a transmembrane protein of the TRAP family . The actin/TRAP complex is then redistributed by the stationary MyoA from the anterior to the posterior end of the zoite, leading to its forward movement on a substrate or to penetration of a host cell.

EMBO J, 2002 Dec 2, 21(23), 6440 - 51
Lack of mitochondrial citrate synthase discloses a new meiotic checkpoint in a strict aerobe; Ruprich-Robert G et al.; Mitochondrial citrate synthase (mCS) is the initial enzyme of the tricarboxylic acid (TCA) cycle . Despite the key position of this protein in respiratory metabolism, very few studies have addressed the question of the effects of the absence of mCS in development . Here we report on the characterization of 15 point mutations and a complete deletion of the cit1 gene, which encodes mCS in the filamentous fungus Podospora anserina . This gene was identified genetically through a systematic search for suppressors of the metabolic defect of the peroxisomal pex2 mutants . The cit1 mutant strains exhibit no visible vegetative defects . However, they display an unexpected developmental phenotype: in homozygous crosses, cit1 mutations impair meiosis progression beyond the diffuse stage, a key stage of meiotic prophase . Enzyme assays, immunofluorescence and western blotting experiments show that the presence of the mCS protein is more important for completion of meiosis than its well-known enzyme activity . Combined with observations made in budding yeast, our data suggest that there is a general metabolic checkpoint at the diffuse stage in eukaryotes.

Blood, 2003 Apr 1, 101(7), 2804 - 9 Epub 2002 Nov 27.
Felic (CIP4b), a novel binding partner with the Src kinase Lyn and Cdc42, localizes to the phagocytic cup; Dombrosky-Ferlan P et al.; Through its Src homology 3 (SH3) and SH2 domains, the Src kinase Lyn interacts with a small number of phosphoproteins, such as Shc, Cbl, and Vav, which regulate cell cycle and the cytoskeleton . Using Lyn's Unique, SH3, and SH2 domains as bait in a yeast 2-hybrid screen, we isolated a novel gene product with features of a scaffolding protein . We named it Felic because it contains a domain homologous to the tyrosine kinase Fes and the cytoskeletal protein ezrin and forms a Lyn interaction with the GTPase Cdc42 (Felic) . Felic was expressed in both hematopoietic and nonhematopoietic tissues . Because it represents an alternative splice product related to the Cdc42-interacting protein 4, CIP4, we also refer to Felic as CIP4b . Felic contains an SH3 recognition site RXPXXP and multiple tyrosine residues . In insulin or serum-stimulated HEK293 cells, Felic became tyrosine phosphorylated . Like CIP4, Felic associated with Cdc42 in its activated form only . Unlike CIP4, Felic does not possess a C-terminal SH3 domain . Coprecipitation studies show that Felic bound to Lyn or activated forms of Cdc42 . Overexpression of Felic or CIP4 inhibited NIH 3T3 cell invasiveness in a Matrigel assay . Because Lyn and Cdc42 are involved in phagocytosis, we examined the distribution of Felic in RAW macrophages during particle ingestion . Felic was recruited more efficiently than CIP4 to the phagocytic cups . Altogether, these data suggest that CIP4/Felic constitute a novel family of cytoskeletal scaffolding proteins, integrating Src and Cdc42 pathways . The absence of an SH3 domain in Felic provides a structural basis for functional differences.

Eukaryot Cell, 2002 Apr, 1(2), 249 - 56
The mold-specific MS8 gene is required for normal hypha formation in the dimorphic pathogenic fungus Histoplasma capsulatum; Tian X et al.; The dimorphic fungus Histoplasma capsulatum is the etiologic agent of one of the most common systemic mycoses of humans, histoplasmosis . In the environment, H . capsulatum grows in a differentiated mold form and shifts to an undifferentiated yeast form after mold fragments or spores are inhaled . This mold-to-yeast shift is required for disease . Little is known about the molecular biology of dimorphism in Histoplasma, and most studies have been directed toward yeast-specific genes . While it is important to examine the role of genes upregulated in the yeast morphotype, genes which are silenced in the yeast (i.e., mold-specific genes) may also play a critical role in dimorphism . To begin to examine this hypothesis, we report here the first misexpression and knockout analysis of a mold-specific gene in Histoplasma . The strongly expressed MS8 gene encodes a predicted 21-kDa protein extremely rich in glycine and glutamine . Forced expression of MS8 driven by the TEF1 promoter in yeast did not alter the yeast morphology at 37 degrees C or mold formation at 25 degrees C . Yeast expressing MS8 did exhibit clumping in liquid medium and formed "sticky" colonies on agar plates . Allelic replacement of MS8 was accomplished by a positive-negative selection procedure . ms8 knockout mutants formed apparently normal yeast at 37 degrees C but gave rise to aberrant mycelia at 25 degrees C . The mold colonies of the knockouts were less than half as large as normal, had a granular surface, produced a dark-red pigment, and formed short hyphae which were 40% wider with a distinctive twisted "zig-zag" shape.

Invest Ophthalmol Vis Sci, 2002 Dec, 43(12), 3609 - 12
A peptidase gene in chromosome 8q is disrupted by a balanced translocation in a duane syndrome patient; Pizzuti A et al.; PURPOSE: To identify the gene disrupted by a de novo reciprocal balanced translocation t(6;8)(q26;q13) in a patient with Duane retraction syndrome (DURS) . The break point in chromosome arm 8q is positioned within the DURS1 critical region . METHODS: Fluorescence in situ hybridization (FISH) analysis using cosmid and BAC clones covering the DURS1 locus was performed to define the break point position and its relationship with expressed sequence tags (ESTs) in the region . Once the interrupted gene was identified, the full-length cDNA was sequenced and the genomic organization defined . Eighteen patients with sporadic DURS without cytogenetic abnormalities involving the DURS1 region were screened for point mutations in the candidate DURS1 gene . RESULTS: A carboxypeptidase gene (CPAH) was directly interrupted between the first and second exons in a patient with DURS who carried a de novo reciprocal balanced translocation t(6;8)(q26;q13) involving the DURS1 region on chromosome arm 8q13 . The gene was transcribed in at least two alternative mRNA forms, with different start and stop codons . CONCLUSIONS: The CPAH gene was interrupted in a patient with DURS carrying a translocation break point in the DURS1 region on chromosome 8q13 . CPAH is therefore a likely candidate for this abnormality, even if the possibility that other genes are involved, either by direct effects on transcription units present in the first CPAH intron or by position effects, cannot be ruled out . Functional studies of the influence of this gene on the morphogenesis of eye muscles and their innervation may clarify this question.

Diabetes, 2002 Dec, 51(12), 3362 - 7
Identification of the insulin-regulated interaction of phosphodiesterase 3B with 14-3-3 beta protein; Onuma H et al.; Phosphodiesterase (PDE)-3B, a major PDE isoform in adipocytes, plays a pivotal role in the antilipolytic action of insulin . Insulin-induced phosphorylation and activation of PDE3B is phosphatidylinositol 3-kinase (PI3-K) and Akt dependent, but the precise mechanism of PDE3B activation is not fully understood . We have identified 14-3-3 beta, a critical scaffolding molecule in signal transduction, as a protein that interacts with PDE3B using the yeast two-hybrid system . The interaction between PDE3B and 14-3-3 beta was then confirmed in vitro . The glutathione S-transferase (GST)-tagged 14-3-3 beta interacts with endogenous PDE3B of rat adipocytes, and this interaction is enhanced when adipocytes are treated with insulin . Coimmunoprecipitation experiments reveal that endogenous PDE3B also associates with endogenous 14-3-3 beta in rat adipocytes, and this interaction is enhanced by insulin . Two different PI3-K inhibitors, wortmannin and Ly294002, block this induction, suggesting that PI3-K is required . Synthetic 15 amino acid peptides of rat PDE3B containing phosphorylated Ser-279 or -302 inhibit this interaction, indicating that the insulin-regulated phosphorylation of these serine residues is involved . Because insulin receptor substrate-1 also associates with 14-3-3, the dimeric 14-3-3 beta could function as a scaffolding protein in the activation of PDE3B by insulin.

Mol Cell, 2002 Nov, 10(5), 1189 - 99
Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination; Frank-Vaillant M et al.; The stability of DNA ends generated by the HO endonuclease in yeast is surprisingly high with a half-life of more than an hour . This transient stability is unaffected by mutations that abolish nonhomologous end joining (NHEJ) . The unprocessed ends interact with Yku70p and Yku80p, two proteins required for NHEJ, but not significantly with Rad52p, a protein involved in homologous recombination (HR) . Repair of a double-strand break by NHEJ is unaffected by the possibility of HR, although the use of HR is increased in NHEJ-defective cells . Partial in vitro 5' strand processing suppresses NHEJ but not HR . These results show that NHEJ precedes HR temporally, and that the availability of substrate dictates the particular pathway used . We propose that transient stability of DNA ends is a foundation for the permanent stability of telomeres.

Traffic, 2002 Dec, 3(12), 906 - 21
Drosophila sec10 is required for hormone secretion but not general exocytosis or neurotransmission; Andrews HK et al.; The sec6/8, or exocyst, complex is implicated in trafficking of secretory vesicles to fusion sites in the plasma membrane . Genetic analyses have been done primarily in yeast, where mutation of the eight protein subunits similarly disrupts polarized vesicle fusion . The goal of this study was to assay the sec6/8 complex in Drosophila, and specifically to test its widely hypothesized functions in synaptogenesis and neurotransmission . We used a transgenic RNAi approach to remove the most highly conserved complex component, Drosophila sec10 (dSec10) . Ubiquitous dSec10 RNAi resulted in early postembryonic lethality, demonstrating that dSec10 is essential . Surprisingly, tissue-specific dSec10 RNAi revealed no essential requirement in nervous system, musculature, gut or epidermis . Assays of polarized secretion in all these tissues failed to reveal any role for dSec10 . In particular, the neuromuscular synapse showed no defects in morphogenesis or vesicle trafficking/fusion underlying neurotransmission . The essential requirement for dSec10 was restricted to the ring gland, the Drosophila organ specialized for endocrine function . The developmental arrest of dSec10 RNAi animals was partially rescued by feeding ecdysone, suggesting dSec10 mediates steroid hormone secretion . We conclude that dSec10 has no detectable role in most forms of polarized trafficking/exocytosis, including neurotransmission, but rather is essential for endocrine secretion.

Int J Dermatol, 2002 Nov, 41(11), 771 - 2
Nargile (Hubble-Bubble) smoking-induced hand eczema; Onder M et al.; A 65-year-old retired man with hand eczema presented to the Dermatology clinic in October of 2001 . He complained of scaly, fissured plaque-type lesions over the radial margin of his right index finger and thumb (Fig . 1) . He first noticed these changes 2 years ago.There was no history of irritation from his occupation . None of the other family members were affected . There was no history of atopy or psoriasis . The physical examination was remarkable for scaly, fissured, hyperkeratotic patches on the palms and palmar surfaces of the finger tips of the right hand . No nail changes were noted . The other fingers were free from lesions.There were no changes on the feet or soles . A diagnosis of eczema was suspected . Hobbies and repeated trauma to the hands were investigated . He had a habit of "nargile" smoking,starting at 35 years of age and he was using this apparatus more than 2 h a day . We performed patch tests with European standart test serial and they were negative . Yeast examination using KOH was negative . The diagnosis of Nargile (Hubble-bubble) eczema was made . It was advised that he stop smoking . Mild topical corticosteroids and emollient with urea were started . Clinical evaluation demonstrated resolution of the lesions after 2 weeks of therapy.

J Appl Microbiol, 1997 Feb, 82(2), 149 - 56
Purification and characterization of the protease enzymes of Streptomyces thermovulgaris grown in rapemeal-derived media; Yeoman KH et al.; When grown in a particulate-free, protein-rich medium derived from rapemeal (termed medium B), Streptomyces thermovulgaris produced multiple protease enzymes . The main protease activity was attributed to two types of serine protease, denoted as SV1 and SV2 . A metallo protease component (SV3) and an azocaseinase component (SV4) were also present . Protease SV1 had a molecular weight of 30 kDa and a pI of 5.8 . Protease SV2 was characterized by a high thermostability in the presence of calcium ions and had a pI of 8.4 . This enzyme had a molecular weight of 60 kDa, but we suggest that this is the dimeric form, with 30 kDa being the monomer unit . The method chosen for initial downstream processing influenced both the yield and type of protease purified . When cell-free supernatant fluid was concentrated using ultrafiltration, rather than acetone precipitation, a higher percentage and a greater range of proteases were recovered . The medium used for the growth of Strep . thermovulgaris also appeared to affect the type of protease produced . A more diverse range of proteases were produced on rapemeal-derived medium when compared to yeast extract medium.

Int J Med Microbiol, 2002 Oct, 292(5-6), 381 - 90
Molecular genetic studies of the model dematiaceous pathogen Wangiella dermatitidis; Szaniszlo PJ; The rapidly improving molecular genetic tractability of Wangiella (Exophiala) dermatitidis significantly enhances its usefulness as a model for the more than 100 other dematiaceous (melanized) agents of human disease . Previously this model was based almost exclusively on its vegetative polymorphism, which at the simplest level is expressed as three well-characterized modes of growth (e.g., blastic, apical and isotropic) that produce myriad yeast, hyphal and sclerotic phenotypes . This cellular plasticity is important for a dematiaceous model pathogen because some are hyphal in nature but exist almost exclusively as sclerotic bodies in infected tissue, whereas others are hyphal both in nature and in tissue, and still others exist in nature predominantly as yeast, but as mixtures of yeast, hyphae and sclerotic bodies in tissue . By exploiting the polymorphism of W . dermatitidis, any phenotype of another dematiaceous pathogen can be produced for study of the regulation of its development and its contribution to pathogenicity and virulence . The coupling of this asset with the recent finding that its haploid, uninucleate yeast cell is easily transformed molecularly, and the even more recent development of systems for both random and targeted gene disruptions and for site-specific, integrative gene overexpression studies suggest that it will continue as the preferred model for the dematiaceous fungi and irrespective of the mycosis involved . The results reviewed here aim to confirm this contention, stimulate others to study this fungus, and demonstrate that W . dermatitidis is exceptionally useful for discovering by molecular genetic techniques cell wall-associated virulence factors in fungi, and in particular in the dematiaceous fungi.

Int J Med Microbiol, 2002 Oct, 292(5-6), 349 - 61
Molecular cell biology and molecular genetics of Histoplasma capsulatum; Ignatov A et al.; Histoplasma capsulatum is a dimorphic ascomycete which is capable of producing a broad spectrum of disease ranging from mild asymptomatic, pulmonary illness to severe, life-threatening systemic mycosis . Regulatory mechanisms that use temperature and other environmental cues are paramount to the successful adaptation of the organism as an effective intracellular pathogenic yeast . Although the biochemistry and phenomenology of reversible morphogenesis have been well examined in Histoplasma, the identification and functional characterization of genes and their products that are required for early establishment or maintenance of the parasitic yeast phase in intracellular host compartments have only recently been fruitful . Advances in the molecular biology of Histoplasma, including approaches to introduce telomeric plasmids, reporter fusion constructs, and gene disruption cassettes into the fungus are poised to solidify the pre-eminence of this fungus as a model system which can be applied to other dimorphic fungal pathogens that exhibit similar cellular and immunological complexities . This review centers on recent developments in the molecular cell biology and molecular genetics of Histoplasma capsulatum that provide important new avenues for examining the mold-to-yeast phase transition beyond the historical, binary view of dimorphism and the implications that these successful approaches may have on seminal issues in fungal pathogenesis.

Int J Med Microbiol, 2002 Oct, 292(5-6), 331 - 47
Control of morphogenesis in the human fungal pathogen Penicillium marneffei; Andrianopoulos A; Fungal pathogens are an increasing threat to human health due to the increasing population of immunocompromised individuals and the increased incidence of treatment-derived infections . Penicillium marneffei is an emerging fungal pathogen endemic to South-east Asia, where it is AIDS defining . Like many other fungal pathogens, P . marneffei is capable of alternating between a filamentous and a yeast growth form, known as dimorphic switching, in response to environmental stimuli . P . marneffei grows in the filamentous form at 25 degrees C and in the yeast form at 37 degrees C . During filamentous growth and in response to environmental cues, P . marneffei undergoes asexual development to form complex multicellular structures from which the infectious agents, the conidia, are produced . At 37 degrees C, P . marneffei undergoes the dimorphic switching program to produce the pathogenic yeast cells . These yeast cells are found intracellularly in the mononuclear phagocyte system of the host and divide by fission, in contrast to the budding mode of division exhibited by most other fungal pathogens . In addition, P . marneffei is evolutionarily distinct from most other dimorphic fungal pathogens and is the only known Penicillium species which exhibits dimorphic growth . The unique evolutionary history of P . marneffei and the rapidly increasing incidence of infection, coupled with the presence of both complex asexual development and dimorphic switching programs in one organism, makes this system a valuable one for the study of morphogenesis and pathogenicity . Recent development of molecular genetic techniques for P . marneffei, including DNA-mediated transformation, have greatly facilitated the study of these two important morphogenetic programs, asexual development and dimorphic switching, and we are beginning to uncover important determinants which control these events . Understand these programs is providing insights into the biology of P . marneffei and its pathogenic capacity.

Cell Motil Cytoskeleton, 2003 Jan, 54(1), 29 - 40
Dynamic localization of myosin-I to endocytic structures in Acanthamoeba; Ostap EM et al.; We used fluorescence microscopy of live Acanthamoeba to follow the time course of the concentration of myosin-I next to the plasma membrane at sites of macropinocytosis and phagocytosis . We marked myosin-I with a fluorescently labeled monoclonal antibody (Cy3-M1.7) introduced into the cytoplasm by syringe loading . M1.7 binds myosin-IA and -IC without affecting their activities, but does not bind myosin-IB . Cy3-M1.7 concentrates at two different macropinocytic structures: large circular membrane ruffles that fuse to create macropinosomes, and smaller endocytic structures that occur at the end of stalk-like pseudopodia . These dynamic structures enclose macropinosomes every 30-60 s . Cy3-M1.7 accumulates rapidly as these endocytic structures form and dissipate rapidly after they internalize . Double labeling fixed cells with Cy3-M1.7 and polyclonal antibodies specific for myosin-IA, -IB, or -IC revealed that all three myosin-I isoforms associate with macropinocytic structures, but individual structures vary in their myosin-I isoform composition . Myosin-I and actin also concentrate transiently at sites where amoebae ingest yeast or the pseudopodia of neighboring cells (heterophagy) by the process of phagocytosis . Within 3 min of yeast attachment to the amoeba, myosin-I concentrates around the phagocytic cup, yeast are internalized, and myosin-I de-localizes . Despite known differences in the regulation of macropinocytosis and phagocytosis, the morphology, protein composition, and dynamics of phagocytosis and macropinocytosis are similar, indicating that they share common structural properties and contractile mechanisms .

J Am Acad Dermatol, 2002 Dec, 47(6), 852 - 5
Treatment of dandruff with 5% tea tree oil shampoo; Satchell AC et al.; BACKGROUND: Dandruff appears to be related to the yeast Pityrosporum ovale . Tea tree oil has antifungal properties with activity against P ovale and may be useful in the treatment of dandruff . OBJECTIVE: We conducted a randomized, single-blind, parallel-group study to investigate the efficacy and tolerability of 5% tea tree oil and placebo in patients with mild to moderate dandruff . METHODS: One hundred twenty-six male and female patients, aged 14 years and older, were randomly assigned to receive either 5% tea tree oil shampoo or placebo, which was used daily for 4 weeks . The dandruff was scored on a quadrant-area-severity scale and by patient self-assessment scores of scaliness, itchiness, and greasiness . RESULTS: The 5% tea tree oil shampoo group showed a 41% improvement in the quadrant-area-severity score compared with 11% in the placebo group (P <.001) . Statistically significant improvements were also observed in the total area of involvement score, the total severity score, and the itchiness and greasiness components of the patients' self-assessments . The scaliness component of patient self-assessment improved but was not statistically significant . There were no adverse effects . CONCLUSION: Five percent tea tree oil appears to effective and well tolerated in the treatment of dandruff.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 263 - 70
Methanogenium marinum sp . nov., a H2-using methanogen from Skan Bay, Alaska, and kinetics of H2 utilization; Chong SC et al.; A methanogen, strain AK-1, was isolated from permanently cold marine sediments, 38- to 45-cm below the sediment surface at Skan Bay, Alaska . The cells were highly irregular, nonmotile coccoids (diameter, 1 to 1.2 microm), occurring singly . Cells grew by reducing CO2 with H2 or formate as electron donor . Growth on formate was much slower than that on H2 . Acetate, methanol, ethanol, 1- or 2-propanol, 1- or 2-butanol and trimethylamine were not catabolized . The cells required acetate, thiamine, riboflavin, a high concentration of vitamin B12, and peptones for growth; yeast extract stimulated growth but was not required . The cells grew fastest at 25 degrees C (range 5 degrees C to 25 degrees C), at a pH of 6.0-6.6 (growth range, pH 5.5-7.5), and at a salinity of 0.25-1.25 M Na+ . Cells of this and other H2-using methanogens from saline environments metabolized H2 to a very low threshold pressure (less than 1 Pa) that was dependent on the methane partial pressure . We propose that the threshold pressure may be limited by the energetics of catabolism . The sequence of the 16S rDNA gene of strain AK-1 was most similar (98%) to the sequences of Methanogenium cariaci JR-1 and Methanogeniumfrigidum Ace-2 . DNA-DNA hybridization between strain AK-1 and these two strains showed only 34.9% similarity to strain JR-1 and 56.5% similarity to strain Ace-2 . These analyses indicated strain AK-1 should be classified as a new species within the genus Methanogenium . Phenotypic differences between strain AK-1 and these strains (including growth temperature, salinity range, pH range, and nutrient requirements) support this . Therefore, a new species, Methanogenium marinum, is proposed with strain AK-1 as type strain.

Bioessays, 2002 Dec, 24(12), 1095 - 109
The dynamics of cell cycle regulation; Tyson JJ et al.; Major events of the cell cycle--DNA synthesis, mitosis and cell division-are regulated by a complex network of protein interactions that control the activities of cyclin-dependent kinases . The network can be modeled by a set of nonlinear differential equations and its behavior predicted by numerical simulation . Computer simulations are necessary for detailed quantitative comparisons between theory and experiment, but they give little insight into the qualitative dynamics of the control system and how molecular interactions determine the fundamental physiological properties of cell replication . To that end, bifurcation diagrams are a useful analytical tool, providing new views of the dynamical organization of the cell cycle, the role of checkpoints in assuring the integrity of the genome, and the abnormal regulation of cell cycle events in mutants . These claims are demonstrated by an analysis of cell cycle regulation in fission yeast .

J Mass Spectrom, 2002 Nov, 37(11), 1118 - 30
Molecular mass determination of plasma-derived glycoproteins by ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with internal calibration; Belgacem O et al.; Human plasma-derived antithrombin III (AT-III), factor IX (FIX) and vitronectin (VN) were characterized as native glycoproteins and in their de-N-glycosylated form by means of MALDI mass spectrometry . The average molecular masses of the three complex glycoproteins were determined applying internal calibration with high-mass, well-defined protein calibrants . Internal calibration generated for the 47 kDa yeast protein enolase a mass precision in the continuous and delayed extraction mode of +/-0.12 and +/-0.022%, respectively . The achievable mass accuracy for such a high-mass, unmodified protein was in the range of 0.02% in the continuous mode, which turned out to be better than in the delayed extraction mode . Purification of all (glyco) proteins (even the calibration proteins) by means of ZipTip technology and direct elution with a solvent system containing the appropriate MALDI matrix turned out to be a prerequisite to measure the exact molecular masses with an internal calibration . The average molecular masses of the two different forms of AT-III, namely AT-III(alpha) and AT-III(beta), were shown to be 57.26 and 55.04 kDa, respectively . The 2.22 kDa mass difference is attributed to the known difference in carbohydrate content at one specific site (Asn-135) . After exhaustive de-N-glycosylation (by means of PNGase F) of the alpha- and beta-form and subsequent MALDI-MS analysis, average molecular masses of 48.96 and 48.97 kDa, respectively, were obtained . These values are in good agreement (-0.15%) with the calculated molecular mass (49.039 kDa) of the protein part based on SwissProt data . The molecular mass of the heavily post-translational modified glycoprotein FIX was found to be 53.75 kDa with a peak width at 10% peak height of 4.5 kDa, because of the presence of many different posttranslational modifications (N- and O-glycosylation at multiple sites, sulfation, phosphorylation, hydroxylation and numerous gamma-carboxyglutamic acids) . MALDI-MS molecular mass determination of the native, size-exclusion chromatography-purified, VN sample revealed that the glycoprotein was present as dimer with molecular mass of 117.74 kDa, which could be corroborated by non-reducing SDS-PAGE . After sample treatment with guanidine hydrochloride and mass spectrometric analysis, a single, new main component was detected . The molecular mass turned out to be 59.45 kDa, representing the monomeric form of VN, known as V75 . The determined molecular mass value was shown to be on one hand lower than from SDS-PAGE and on the other higher than the calculated amino acid sequence molecular mass (52 277 Da), pointing to the well-known SDS-PAGE bias and to considerable post-translational modifications . Further treatment of the sample with a reducing agent and subsequent MALDI-MS revealed two new components with molecular masses of 49.85 and 9.41 kDa, corresponding to V65 and V10 subunits of VN . PNGase F digest of the V75 and V65 units and MS analysis, exhibiting a molecular mass reduction of 6.37 kDa in both cases, verified the presence of a considerable amount of N-glycans . Copyright 2002 John Wiley & Sons, Ltd.

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