Yeasts

Protein Pept Lett, 2004 Dec, 11(6), 521 - 5
Interacting partners for kringle domains of plasminogen: common binding with K1 and K5 domains; Kong N et al.; We have identified MAZR and Rgl2 as specific interacting partners for kringle domains in angiostatin (K1-4) and K5 using yeast two hybrid screening . Both K1 and K1-4 have strong interaction with MAZR and Rgl2 whereas K5 only binds with Rgl2 . No interaction of K2, K3, and K4 with either of these binding proteins was detected . We suggest that a common binding motif may exist near LBS-4 that is required for binding with Rgl2 but not with MAZR.

Mol Genet Genomics . 2004 Dec 1; {Epub ahead of print}
A H(2)O(2)-producing glyoxal oxidase is required for filamentous growth and pathogenicity in Ustilago maydis; Leuthner B et al.; In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development . In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic . In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium . Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts . To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U . maydis strains . In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic . Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (<C4) and produces H(2)O(2) . The enzyme needs to be activated, presumably by auto-oxidation, to show full activity . A potential role for Glo1 during filamentous growth and pathogenic development of U . maydis is proposed.

Methods Mol Biol, 2004, 296, 345 - 54
Assaying cell cycle checkpoints: activity of the protein kinase chk1; Palermo C et al.; Eukaryotic cells regulate progression through the cell cycle in response to DNA damage . Cell cycle checkpoints are the signal transduction pathways that couple the detection of DNA damage to the proteins that control transitions in the cell cycle . The protein kinase Chk1, originally discovered in fission yeast, but conserved in humans, is essential for preventing mitotic entry in the presence of DNA damage or blocks to DNA replication that cannot be reconciled . Chk1 is phosphorylated in response to DNA damage . Phosphorylation depends on the activity of conserved components of the checkpoint pathway including Rad3, a member of the ATM/ATR family of kinases . Phosphorylation leads to activation of Chk1 kinase activity . In this chapter, we describe an assay for monitoring the activity of Chk1 isolated.

Nucleic Acids Res . 2004 Dec;32(21):e169.
In vitro selection of Jun-associated proteins using mRNA display; Horisawa K et al.; Although yeast two-hybrid assay and biochemical methods combined with mass spectrometry have been successfully employed for the analyses of protein-protein interactions in the field of proteomics, these methods encounter various difficulties arising from the usage of living cells, including inability to analyze toxic proteins and restriction of testable interaction conditions . Totally in vitro display technologies such as ribosome display and mRNA display are expected to circumvent these difficulties . In this study, we applied an mRNA display technique to screening for interactions of a basic leucine zipper domain of Jun protein in a mouse brain cDNA library . By performing iterative affinity selection and sequence analyses, we selected 16 novel Jun-associated protein candidates in addition to four known interactors . By means of real-time PCR and pull-down assay, 10 of the 16 newly discovered candidates were confirmed to be direct interactors with Jun in vitro . Furthermore, interaction of 6 of the 10 proteins with Jun was observed in cultured cells by means of co-immunoprecipitation and observation of subcellular localization . These results demonstrate that this in vitro display technology is effective for the discovery of novel protein-protein interactions and can contribute to the comprehensive mapping of protein-protein interactions.

Circ Res, 2005 Jan 7, 96(1), 73 - 81 Epub 2004 Dec 02.
Targeting to C-terminal myosin heavy chain may explain mechanotransduction involving focal adhesion kinase in cardiac myocytes; Fonseca PM et al.; Focal adhesion kinase (Fak) has been implicated as a signaling molecule involved in the early response of cardiac myocytes to mechanical stress . The mechanism of Fak activation by mechanical stimuli is not clear . In this study, we report the load-induced Fak activation and its association with myosin heavy chain in cardiac myocytes . Pressure overload lasting from 3 to 60 minutes was shown to induce Fak phosphorylation at Tyr-397, -576/7, -861, and -925 as detected by phosphospecific antibodies . This was paralleled by increases of Fak/Src association and Src activity (Tyr-418 phosphorylation) . Yeast two-hybrid screening of an adult rat cDNA library revealed an interaction between Fak and C-terminal coiled-coil region of alpha-myosin heavy chain . This was confirmed by pulldown assay with GST-C-terminal myosin fragment and native Fak from rat left ventricle . Such interaction was confirmed by coimmunoprecipitation assay with anti-Fak and anti-heavy chain cardiac myosin antibodies, confocal microscopy of double-labeled isolated cardiac myocytes and immunoelectron microscopy with anti-Fak antibody . Fak activation by mechanical stress was accompanied by a reduction of Fak/myosin heavy chain association and its relocation at subcellular sites such as costameres, Z-discs, and nuclei . Thus, our present data identify Fak interaction with C-terminal region of myosin heavy chain adding comprehensive data on Fak activation by mechanical stress and mechanotransduction in cardiac myocytes.

J Biol Chem . 2004 Dec 2; {Epub ahead of print}
Group VIA phospholipase A2 (iPLA2{beta}) forms a signaling complex with the calcium/calmodulin-dependent protein kinase II{beta} expressed in pancreatic islet beta cells; Wang Z et al.; Pancreatic islet ss-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)ss) that participates in insulin secretion and contains a calmodulin binding site and protein interaction domains . We identified the Ca(2+)/calmodulin-dependent protein kinase IIss isoform (CaMKIIss) as a potential iPLA(2)ss-interacting protein by yeast two hybrid screening of a cDNA library using iPLA(2)ss cDNA as bait . The sequence of CaMKIIss cDNA cloned from a rat islet library revealed that one dominant CaMKIIss isoform mRNA is expressed by adult islets that is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human islets . When DNA encoding that isoform was used as bait and iPLA(2)ss DNA as prey in a binary two-hybrid system, interaction between the two enzymes was confirmed, as it was when CaMKIIss was prey and iPLA(2)ss bait . Recombinant, his-tagged CaMKIIss immobilized on metal affinity matrices bound iPLA(2)ss, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA . Activities of both enzymes increased upon their association, and iPLA(2)ss reaction products reduced CaMKIIss activity . Both the iPLA(2)ss inhibitor BEL and the CaMKIIss inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both compounds inhibit insulin secretion . CaMKIIss and iPLA(2)ss can be co-immunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex . These findings suggest that iPLA(2)ss and CaMKIIss form a signaling complex in ss-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is co-induced upon differentiation of pancreatic progenitor to endocrine progenitor cells.

Nucleic Acids Res, 2004, 32(21), 6312 - 20 Print 2004.
PreSPI: a domain combination based prediction system for protein-protein interaction; Han DS et al.; With the accumulation of protein and its related data on the Internet, many domain-based computational techniques to predict protein interactions have been developed . However, most techniques still have many limitations when used in real fields . They usually suffer from low accuracy in prediction and do not provide any interaction possibility ranking method for multiple protein pairs . In this paper, we propose a probabilistic framework to predict the interaction probability of proteins and develop an interaction possibility ranking method for multiple protein pairs . Using the ranking method, one can discern the protein pairs that are more likely to interact with each other in multiple protein pairs . The validity of the prediction model was evaluated using an interacting set of protein pairs in yeast and an artificially generated non-interacting set of protein pairs . When 80% of the set of interacting protein pairs in the DIP (Database of Interacting Proteins) was used as a learning set of interacting protein pairs, high sensitivity (77%) and specificity (95%) were achieved for the test groups containing common domains with the learning set of proteins within our framework . The stability of the prediction model was also evident when tested over DIP CORE, HMS-PCI and TAP data . In the validation of the ranking method, we reveal that some correlations exist between the interacting probability and the accuracy of the prediction.

Biol Psychiatry, 2004 Dec 1, 56(11), 868 - 74
Valproate decreases inositol biosynthesis; Shaltiel G et al.; BACKGROUND: Lithium and valproate (VPA) are used for treating bipolar disorder . The mechanism of mood stabilization has not been elucidated, but the role of inositol has gained substantial support . Lithium inhibition of inositol monophosphatase, an enzyme required for inositol recycling and de novo synthesis, suggested the hypothesis that lithium depletes brain inositol and attenuates phosphoinositide signaling . Valproate also depletes inositol in yeast, Dictyostelium, and rat neurons . This raised the possibility that the effect is the result of myo-inositol-1-phosphate (MIP) synthase inhibition . METHODS: Inositol was measured by gas chromatography . Human prefrontal cortex MIP synthase activity was assayed in crude homogenate . INO1 was assessed by Northern blotting . Growth cones morphology was evaluated in cultured rat neurons . RESULTS: We found a 20% in vivo reduction of inositol in mouse frontal cortex after acute VPA administration . As hypothesized, inositol reduction resulted from decreased MIP synthase activity: .21-.28 mmol/LVPA reduced the activity by 50% . Among psychotropic drugs, the effect is specific to VPA . Accordingly, only VPA upregulates the yeast INO1 gene coding for MIP synthase . The VPA derivative N-methyl-2,2,3,3,-tetramethyl-cyclopropane carboxamide reduces MIP synthase activity and has an affect similar to that of VPA on rat neurons, whereas another VPA derivative, valpromide, poorly affects the activity and has no affect on neurons . CONCLUSIONS: The rate-limiting step of inositol biosynthesis, catalyzed by MIP synthase, is inhibited by VPA; inositol depletion is a first event shown to be common to lithium and VPA.

Genome Biol . 2004;5(12):R96 . Epub 2004.
A Drosophila protein-interaction map centered on cell-cycle regulators; Stanyon CA et al.; BACKGROUND: Maps depicting binary interactions between proteins can be powerful starting points for understanding biological systems . A proven technology for generating such maps is high-throughput yeast two-hybrid screening . In the most extensive screen to date, a Gal4-based two-hybrid system was used recently to detect over 20,000 interactions among Drosophila proteins . Although these data are a valuable resource for insights into protein networks, they cover only a fraction of the expected number of interactions . RESULTS: To complement the Gal4-based interaction data, we used the same set of Drosophila open reading frames to construct arrays for a LexA-based two-hybrid system . We screened the arrays using a novel pooled mating approach, initially focusing on proteins related to cell-cycle regulators . We detected 1,814 reproducible interactions among 488 proteins . The map includes a large number of novel interactions with potential biological significance . Informative regions of the map could be highlighted by searching for paralogous interactions and by clustering proteins on the basis of their interaction profiles . Surprisingly, only 28 interactions were found in common between the LexA- and Gal4-based screens, even though they had similar rates of true positives . CONCLUSIONS: The substantial number of new interactions discovered here supports the conclusion that previous interaction mapping studies were far from complete and that many more interactions remain to be found . Our results indicate that different two-hybrid systems and screening approaches applied to the same proteome can generate more comprehensive datasets with more cross-validated interactions . The cell-cycle map provides a guide for further defining important regulatory networks in Drosophila and other organisms.

Biotechnol Prog, 2004 Nov-Dec, 20(6), 1817 - 24
Immobilization of Candida bombicola cells on free-standing organic-gold nanoparticle membranes and their use as enzyme sources in biotransformations; Phadtare S et al.; Preparation of chemically functionalized biocompatible surfaces is of current interest, with application in the immobilization of various bioactive species such as DNA, enzymes, whole cells, etc . We report herein the one-step synthesis of a self-supporting gold nanoparticle membrane, its surface modification, and application in the immobilization of Candida bombicola (yeast) cells . The gold nanoparticle membrane is prepared by the spontaneous reduction of aqueous chloroaurate ions by a diamine at a liquid-liquid interface . The gold nanoparticles in the polymeric membrane may be capped with octadecylamine (ODA) molecules, thereby rendering the nanoparticle membrane hydrophobic . Exposure of the hydrophobized organic-gold nanoparticle membrane to C . bombicola yeast cells results in their binding to the membrane, possibly through nonspecific interactions such as hydrophobic interactions between the yeast cell walls and the ODA molecules . The enzyme cytochrome P450 present in the yeast cells immobilized on the organic-gold nanoparticle membrane was then used in the transformation of the arachidonic acid (AA) to sophorolipids followed by acid hydrolysis to form 20-hydroxyeicosatetraneoic acid (20-HETE) . The organic-gold nanoparticle membrane-C . bombicola bioconjugate could be easily separated from the reaction medium and reused a number of times.

Mol Biol Cell . 2004 Dec 1; {Epub ahead of print}
Interaction of Sla2p's ANTH Domain with PtdIns(4,5)P2 Is Important for Actin-dependent Endocytic Internalization; Sun Y et al.; Monitoring Editor: Anthony Bretscher A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known . The AP180 N-Terminal Homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis . One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization . To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain . A liposome binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain, and identified specific lysine residues required for this interaction . Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization . These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo . Strikingly, sla2 mutants defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association . In contrast, multi-color real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization . These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.

Plant Cell Physiol, 2004 Nov, 45(11), 1720 - 8
Molecular and biochemical characterization of three WD-repeat-domain-containing inositol polyphosphate 5-phosphatases in Arabidopsis thaliana; Zhong R et al.; Type II inositol polyphosphate 5-phosphatases (5PTases) in animals and yeast have been known to be important for regulating inositol and phospholipid signaling by hydrolyzing phosphate from both inositol polyphosphates and phosphoinositides . However, the molecular and biochemical properties of type II 5PTases in plants have not yet been studied . In this report, we show that three Arabidopsis genes, At5PTase12, At5PTase13 and At5PTase14, encode proteins with a 5PTase domain and a WD-repeat domain, a novel combination present only in plant 5PTases . We demonstrate that these genes are differentially expressed in Arabidopsis organs and At5PTase13 is induced in response to ABA and wounding treatments . Our biochemical studies reveal that although both At5PTase12 and At5PTase13 exhibit phosphatase activity toward only Ins(1,4,5)P3, At5PTase14 hydrolyzes phosphate from PI(4,5)P2, PI(3,4,5)P3 and Ins(1,4,5)P3 with the highest substrate affinity toward PI(4,5)P2 . All three At5PTases require Mg2+ for their phosphatase activities . Our molecular and biochemical characterization of three WD-repeat-domain-containing At5PTases provides a foundation for further elucidation of their cellular functions in Arabidopsis.

Plant Cell Physiol, 2004 Nov, 45(11), 1566 - 77
Local induction of the alc gene switch in transgenic tobacco plants by acetaldehyde; Schaarschmidt S et al.; The alc promoter system, derived from the filamentous fungi Aspergillus nidulans, allows chemically regulated gene expression in plants and thereby the study of gene function as well as metabolic and developmental processes . In addition to ethanol, this system can be activated by acetaldehyde, described as the physiological inducer in A . nidulans . Here, we show that in contrast to ethanol, acetaldehyde allows tissue-specific activation of the alc promoter in transgenic tobacco plants . Soil drenching with aqueous acetaldehyde solutions at a concentration of 0.05% (v/v) resulted in the rapid and temporary induction of the alc gene expression system exclusively in roots . In addition, the split root system allows activation to be restricted to the treated part of the root . The temporary activation of the alc system by soil drenching with acetaldehyde could be prolonged over several weeks by subsequent applications at intervals of 7 d . This effect was demonstrated for the root-specific induction of a yeast-derived apoplast-located invertase under the control of the alcohol-inducible promoter system . In leaves, which exhibit a lower responsiveness to acetaldehyde than roots, the alc system was induced in the directly treated tissue only . Thus, acetaldehyde can be used as a local inducer of the alc gene expression system in tobacco plants.

Biol Cell . 2004 Dec 1; {Epub ahead of print}
FLRG, a new ADAM 12-associated protein, modulates osteoclast differentiation; Bartholin L et al.; Background: FLRG (Follistatin Related Gene) is a secreted glycoprotein that is highly homologous to follistatin . These proteins are involved in the regulation of various biological effects mediated by their binding to TGFbeta superfamily members, activin A and BMPs (Bone Morphogenetic Proteins) . To further characterize the function of FLRG, we used a yeast two-hybrid screen to look for other possible functional partners . Results: We report a direct interaction between the cysteine-rich domain of FLRG and ADAM12 . ADAMs (A Disintegrin And Metalloproteases) are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion . Several studies have reported that ADAM12 protein, as well as activin A, are important regulators of osteoclast differentiation . We observed that the expression of ADAM12 and activin A are modulated during osteoclast formation whereas FLRG expression seems quite constant . We showed that FLRG protein inhibits osteoclast differentiation from murine primary spleen cells and macrophage RAW264.7 cells cultured in the presence of RANK-L (Receptor activator of NF-kappaB-Ligand) and M-CSF (Macrophage-Colony Stimulating Factor) . Addition of FLRG protein to precursors significantly reduces the number of osteoclasts, as well as the average number of nuclei in each osteoclast . Conclusions: Our study indicates that FLRG protein may contribute to bone formation by inhibiting osteoclast differentiation.

Environ Sci Technol, 2004 Nov 15, 38(22), 6085 - 93
Chronic effects of dietary selenium on juvenile Sacramento splittail (Pogonichthys macrolepidotus); Teh SJ et al.; The chronic effects of dietary selenium (Se) exposure in juvenile Sacramento splittail (Pogonichthys macrolepidotus) were investigated in the laboratory . A total of 960 (40 fish per tank, 3 tanks per diet) 7-month-old juvenile splittail were fed one of eight Purified-Casein diets supplemented with selenized yeast for 9 months in a flow-through system . These diets contained the following: 0.4 (control), 0.7, 1.4, 2.7, 6.6, 12.6, 26.0, and 57.6 mg of Se kg(-1) dry weight . Survival, Se tissue concentration, growth, gross morphology, and liver histopathology were assessed at 5- and 9-month of exposure . Mortalities occurred only in the two highest Se treatments and were accounted for 8.3 and 18.3% at 5-month and 10.0 and 34.3% at 9-month, respectively . Liver and muscle Se concentration were significantly correlated with dietary Se concentration . Fish exposed to 0.4-12.6 mg of Se kg(-1) diets had reached equilibrium in liver Se concentration by 5 month . Splittail fed diets at concentrations > or =26.0 mg of Se kg(-1) had not reached equilibrium in liver, and muscle Se concentrations and grew significantly slower (p < 0.05) at 5- and 9-month exposure . Se-induced deformities were observed in fish fed > or =2.7 mg of Se kg(-1) diets at 5-month and in fish fed > or =0.7 mg of Se kg(-1) diets at 9-month . Fish fed 26.0 and 57.6 mg of Se kg(-1) diets had higher liver lesion scores at 5-month while fish fed 6.6 and 57.6 mg of Se kg(-1) diet had higher liver lesion scores at 9-month . Results indicate that survivals, growth, changes of tissue Se concentrations, and histopathology of juvenile splittail were dose-dependent, but their response thresholds to dietary Se concentrations differed and depended on treatment concentrations and duration of exposure . Chronic exposure to 6.6 mg of Se kg(-1) diet induced deleterious health effects that can potentially impact survival of juvenile splittail.

Nat Rev Immunol, 2004 Dec, 4(12), 965 - 77
A BAF-centred view of the immune system; Chi T; Chromatin structure dictates whether DNA templates are accessible to nuclear proteins; therefore, it is tightly regulated . To reconfigure chromatin, cells often mobilize 'chromatin-remodelling complexes' that use energy to disrupt histone-DNA contacts . BAF complexes, which are related to the yeast SWI-SNF complex, are the prototypical mammalian chromatin-remodelling complexes . In the past few years, studies have revealed the crucial and diverse roles of BAF complexes in the regulation of the immune system - from lymphocyte development to immune responses . This review surveys these advances, highlighting the general insights these studies provide into the modes of action of BAF complexes, and it concludes with a discussion of some of the key opportunities and challenges in this field.

Mol Cell Biol, 2004 Dec, 24(24), 10894 - 904
Nonphosphorylated human La antigen interacts with nucleolin at nucleolar sites involved in rRNA biogenesis; Intine RV et al.; La is a RNA-binding protein implicated in multiple pathways related to the production of tRNAs, ribosomal proteins, and other components of the translational machinery (D . J . Kenan and J . D . Keene, Nat . Struct . Mol . Biol . 11:303-305, 2004) . While most La is phosphorylated and resides in the nucleoplasm, a fraction is in the nucleolus, the site of ribosome production, although the determinants of this localization are incompletely known . In addition to its conserved N-terminal domain, human La harbors a C-terminal domain that contains an atypical RNA recognition motif and a short basic motif (SBM) adjacent to phosphoserine-366 . We report that nonphosphorylated La (npLa) is concentrated in nucleolar sites that correspond to the dense fibrillar component that harbors nascent pol I transcripts as well as fibrillarin and nucleolin, which function in early phases of rRNA maturation . Affinity purification and native immunoprecipitation of La and fluorescence resonance energy transfer in the nucleolus reveal close association with nucleolin . Moreover, La lacking the SBM does not localize to nucleoli . Lastly, La exhibits SBM-dependent, phosphorylation-sensitive interaction with nucleolin in a yeast two-hybrid assay . The data suggest that interaction with nucleolin is, at least in part, responsible for nucleolar accumulation of La and that npLa may be involved in ribosome biogenesis.

Mol Cell Biol, 2004 Dec, 24(24), 10593 - 610
PIAS-1 is a checkpoint regulator which affects exit from G1 and G2 by sumoylation of p73; Munarriz E et al.; p73 is a recently described member of the p53 family, and, like p53, it undergoes a number of posttranslational modifications . Here we show, by yeast two-hybrid screening, pull-down assays, and coimmunoprecipitation, that p73alpha, -beta, and -gamma bind to the protein inhibitor of activated STAT-1 (PIAS-1) and that this binding stabilizes p73 . PIAS-1 also sumoylates p73alpha, although not the C-terminally truncated isoforms p73beta and -gamma, and this requires the RING finger domain of PIAS-1 . The DeltaNp73alpha isoform can also bind, and be sumoylated by, PIAS-1 . PIAS-1-mediated sumoylation decreases p73 transcriptional activity on several target promoters, such as Bax . p73 is colocalized in the nucleus with PIAS-1, and sumoylated p73 is located exclusively in the nuclear matrix . PIAS-1 is expressed predominantly during S phase, and PIAS-1 overexpression reduces p73-mediated transcription of p21, with a reduction of cells in G(1) and cell cycle reentry . Inhibition of endogenous PIAS-1 by RNA interference reduces the proportion of cells in S phase and induces G(2) arrest . These data suggest that PIAS-1, acting partly through binding and sumoylation of p73, is an important component of the cell cycle machinery.

J Clin Endocrinol Metab . 2004 Nov 30; {Epub ahead of print}
Recombinant Cell Ultra-sensitive Bioassay for Measurement of Estrogens in Post-menopausal Women; Wang S et al.; A recent analysis of data from nine studies provided convincing evidence that plasma estradiol measurements predict the risk of breast cancer in normal postmenopausal women . However, the median values detected by the various assays used in this study varied by 5 fold . These and other published data in normal postmenopausal women suggest that assays measuring low plasma estradiol concentrations suffer from problems of sensitivity, specificity and precision . Availability of a practical, low cost, specific, precise, and ultra-sensitive estrogen assay might allow enhanced prediction of the risk of breast cancer and provide an objective means of selecting post-menopausal women for breast cancer prevention . A recombinant cell ultra-sensitive bioassay (RCUB) for estrogen was recently validated for use in pre-pubertal children . We postulated that the RCUB might also prove useful for measurement of post-menopausal levels and designed the present study to examine this possibility . Thirty normal post-menopausal volunteers provided blood samples for measurement of estrogen by RCUB and, for comparison, by RIA . The estrogenic activity measured by RCUB (11.9 +/- 10.9 pmol/L mean+/-SD) <3.23 +/- 2.96 pg/ml, mean+/-SD> was significantly lower than estradiol levels measured by RIA (43.7 +/- 44.0 pmol/L) pg/ml <11.9 +/- 12.0 pg/ml> in our volunteer subjects (P < 0.00001) . Nonetheless, plasma estradiol levels measured by bioassay were significantly correlated with the estrogenic activity measured by RIA (r = 0.84 and by gas chromatography/ tandem mass spectrometry (r=0.85) . To obtain biologic evidence of the validity of the RCUB, we related plasma estrogen levels to body weight and body mass index and found highly significant correlations (r=0.54 and r=0.53 respectively) . Surprisingly, 28/30 postmenopausal women were found to have estrogen levels in the pre-pubertal range with the RCUB . The levels detected by RCUB were similar to those previously reported using an ultra-sensitive but less practical yeast bioassay . These results provide validation for the RCUB in postmenopausal women and suggest that it might prove useful for selection of women for drug therapy to prevent breast cancer.

J Cell Sci, 2004 Dec 15, 117(Pt 26), 6535 - 46 Epub 2004 Nov 30.
Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity; Ishihara N et al.; The mammalian homologues of yeast and Drosophila Fzo, mitofusin (Mfn) 1 and 2, are both essential for mitochondrial fusion and maintenance of mitochondrial morphology . Though the GTPase domain is required for Mfn protein function, the molecular mechanisms of the GTPase-dependent reaction as well as the functional division of the two Mfn proteins are unknown . To examine the function of Mfn proteins, tethering of mitochondrial membranes was measured in vitro by fluorescence microscopy using green fluorescence protein- or red fluorescent protein-tagged and Mfn1-expressing mitochondria, or by immunoprecipitation using mitochondria harboring HA- or FLAG-tagged Mfn proteins . These experiments revealed that Mfn1-harboring mitochondria were efficiently tethered in a GTP-dependent manner, whereas Mfn2-harboring mitochondria were tethered with only low efficiency . Sucrose density gradient centrifugation followed by co-immunoprecipitation revealed that Mfn1 produced oligomerized approximately 250 kDa and approximately 450 kDa complexes in a GTP-dependent manner . The approximately 450 kDa complex contained oligomerized Mfn1 from distinct apposing membranes (docking complex), whereas the approximately 250 kDa complex was composed of Mfn1 present on the same membrane or in the membrane-solubilized state (cis complex) . These results were also confirmed using blue-native PAGE . Mfn1 exhibited higher activity for this reaction than Mfn2 . Purified recombinant Mfn1 exhibited approximately eightfold higher GTPase activity than Mfn2 . These findings indicate that the two Mfn proteins have distinct activities, and suggest that Mfn1 is mainly responsible for GTP-dependent membrane tethering.

J Biol Chem . 2004 Nov 29; {Epub ahead of print}
Amino acids important for ligand specificity of the human constitutive androstane receptor; Jyrkkarinne J et al.; The human constitutive androstane receptor (CAR, NR1I3) is an important ligand-activated regulator of oxidative and conjugative enzymes and transport proteins . Due to the lack of a crystal structure of the ligand-binding domain (LBD), wide species differences in ligand specificity and the scarcity of well-characterized ligands, the factors that determine CAR ligand specificity are not clear . To address this issue, we developed highly-defined homology models of human CAR LBD to identify residues lining the ligand-binding pocket and to perform molecular dynamics simulations with known human CAR modulators . The roles of twenty-two LBD residues for basal activity, ligand selectivity, and interactions with co-regulators were studied using site-directed mutagenesis, mammalian co-transfection and yeast two-hybrid assays . These studies identified several amino acids within helices 3 (N165), 5 (V199), 11 (Y326, I330, Q331) and 12 (L343, I346) that contribute to the high basal activity of human CAR . Unique residues within helices 3 (I164, N165), 5 (C202, H203) and 7 (F234, F238) were found control the selectivity for CAR activators and inhibitors . A single residue in helix 7 (F243) appears to explain the human/mouse species difference in response of CAR to 17alpha-ethynyl-3,17beta-estradiol.

J Biol Chem . 2004 Nov 30; {Epub ahead of print}
Cell-free transport from the TGN to late endosome requires factors involved in formation and consumption of clathrin-coated vesicles; Abazeed ME et al.; Transport between the trans-Golgi network (TGN) and late endosome represents a conserved, clathrin-dependent sorting event that separates lysosomal from secretory cargo molecules and is also required for localization of integral membrane proteins to the TGN . Previously, we reported a cell-free reaction that reconstitutes transport from the yeast TGN to the late endosome/prevacuolar compartment (PVC) and requires the PVC t-SNARE Pep12p . Here, we report that factors required both for formation of clathrin-coated vesicles at the TGN (the Chc1p clathrin heavy chain and the Vps1p dynamin homolog) and for vesicle fusion at the PVC (the Vps21p rab protein and Vps45 SM (Sec1/Munc18) protein) are required for cell-free transport . The marker for TGN-PVC transport, Kex2p, is initially present in a clathrin-containing membrane compartment that is competent for delivery of Kex2p to the PVC . A Kex2p chimera containing the cytosolic tail (C-tail) of the vacuolar protein sorting receptor, Vps10p, is also efficiently transported to the PVC . Antibodies against the Kex2p and Vps10p C-tails selectively block transport of Kex2p and the Kex2-Vps10p chimera . The requirements for factors involved in vesicle formation and fusion, the identification of the donor compartment as a clathrin-containing membrane, and the need for accessibility of C-tail sequences argue that the TGN-PVC transport reaction involves selective incorporation of TGN cargo molecules into clathrin-coated vesicle intermediates . Further biochemical dissection of this reaction should help elucidate the molecular requirements and hierarchy of events in TGN-to-PVC sorting and transport.

Biochim Biophys Acta, 2004 Nov 29, 1695(1-3), 209 - 13
Productive RUPture: activation of transcription factors by proteasomal processing; Rape M et al.; Proteasomes usually degrade proteins completely into small peptides . In a few cases, however, proteasomal degradation rather results in protein processing, thereby yielding proteins of different biological activity . This process, termed "regulated ubiquitin/proteasome-dependent processing" or RUP, is essential for the function of certain transcription factors and crucial for their regulation . Examples are proteins of the mammalian NF-kappaB family and the yeast proteins SPT23 and MGA2 . In this review, we summarize the available data and suggest a mechanistic model for proteasomal processing.

Biochim Biophys Acta, 2004 Nov 29, 1695(1-3), 133 - 70
A hitchhiker's guide to the cullin ubiquitin ligases: SCF and its kin; Willems AR et al.; The SCF (Skp1-Cullin-F-box) E3 ubiquitin ligase family was discovered through genetic requirements for cell cycle progression in budding yeast . In these multisubunit enzymes, an invariant core complex, composed of the Skp1 linker protein, the Cdc53/Cul1 scaffold protein and the Rbx1/Roc1/Hrt1 RING domain protein, engages one of a suite of substrate adaptors called F-box proteins that in turn recruit substrates for ubiquitination by an associated E2 enzyme . The cullin-RING domain-adaptor architecture has diversified through evolution, such that in total many hundreds of distinct SCF and SCF-like complexes enable degradation of myriad substrates . Substrate recognition by adaptors often depends on posttranslational modification of the substrate, which thus places substrate stability under dynamic regulation by intracellular signaling events . SCF complexes control cell proliferation through degradation of critical regulators such as cyclins, CDK inhibitors and transcription factors . A plethora of other processes in development and disease are controlled by other SCF-like complexes, including those based on Cul2-SOCS-box adaptor protein and Cul3-BTB domain adaptor protein combinations . Recent structural insights into SCF-like complexes have begun to illuminate aspects of substrate recognition and catalytic reaction mechanisms.

J Mol Biol, 2005 Jan 14, 345(2), 289 - 98
Dimerisation of myomesin: implications for the structure of the sarcomeric M-band; Lange S et al.; The sarcomeric M-band is thought to provide a link between the thick and the elastic filament systems . So far, relatively little is known about its structural components and their three-dimensional organisation . Myomesin seems to be an essential component of the M-band, since it is expressed in all types of vertebrate striated muscle fibres investigated and can be found in its mature localisation pattern as soon as the first myofibrils are assembled . Previous work has shown that the N-terminal and central part of myomesin harbour binding sites for myosin, titin and muscle creatine kinase . Intrigued by the highly conserved domain layout of the C-terminal half, we screened for new interaction partners by yeast two-hybrid analysis . This revealed a strong interaction of myomesin with itself . This finding was confirmed by several biochemical assays . Our data suggest that myomesin can form antiparallel dimers via a binding site residing in its C-terminal domain 13 . We suggest that, similar to alpha-actinin in the Z-disc, the myomesin dimers cross-link the contractile filaments in the M-band . The new and the already previously identified myomesin interaction sites are integrated into the first three-dimensional model of the sarcomeric M-band on a molecular basis.

J Pediatr Endocrinol Metab, 2004 Nov, 17(11), 1545 - 9
Vulvovaginal candidiasis in children and adolescents with type 1 diabetes mellitus; Kendirci M et al.; In this prospective study we investigated the frequency of vulvovaginal candidiasis, the results of yeast cultures and detection of ketoconazole resistance in female children and adolescents with type 1 diabetes mellitus (DM1) . The study consisted of 35 patients with DM1 (age 1.7-20 years) and 22 controls (age 1.5-18 years) . Age, duration of DM1 and evidence of genital symptoms were recorded initially . After a pelvic examination, two separate swabs and samples for blood glucose and hemoglobin A1c (HbA1c) were taken . One of the swabs was used for direct examination and the second was placed on Sabouraud's dextrose agar and incubated . In vitro susceptibility of Candida species to ketoconazole was established by using Etest (AB B1ODISC) . Candida species were isolated in 32 of 61 (52.5%) swabs of patients with DM1 and five of 22 (18.2%) of the control group . The predominant Candida species isolated from patients with DM1 were C . albicans (72.7%), C . glabrata (22.7%), C . tropicalis (2.3%), and C . parapsilosis (2.3%) . The mean HbA1c in diabetic patients from whom Candida species were isolated was significantly higher than that of patients without Candida infection (p = 0.002) . Most of the C . glabrata isolates were significantly resistant to ketoconazole . During the follow-up of patients with DM1, genital candidiasis is generally overlooked . It should not be forgotten that species other than C . albicans might cause genital candidiasis.

J Biol Chem . 2004 Nov 29; {Epub ahead of print}
Noncovalent SUMO-1 binding activity of thymine DNA glycosylase (TDG) is required for its SUMO-1 modification and colocalization with the promyelocytic leukemia protein (PML); Takahashi H et al.; SUMO-1 is a member of a family of ubiquitin-like molecules that are posttranslationally conjugated to various cellular proteins in a process that is mechanistically similar to ubiquitylation . To identify molecules that bind noncovalently to SUMO-1, we performed yeast two-hybrid screening with a SUMO-1 mutant that cannot be conjugated to target proteins as the bait . This screening resulted in the isolation of cDNAs encoding the b isoform of thymine DNA glycosylase (TDGb) . A deletion mutant of TDGb {TDGb(Delta11)} that lacks a region shown to be required for noncovalent binding of SUMO-1 was also found not to be susceptible to SUMO-1 conjugation at an adjacent lysine residue, suggesting that such binding is required for covalent modification . In contrast, another mutant of TDGb {TDGb(KR)} in which the lysine residue targeted for SUMO-1 conjugation is replaced with arginine retained the ability to bind SUMO-1 noncovalently . TDGb was shown to interact with the promyelocytic leukemia protein (PML) in vitro as well as to colocalize with this protein to nuclear bodies in transfected cells . TDGb(KR) also colocalized with PML whereas TDGb(Delta11) did not, indicating that the noncovalent SUMO-1 binding activity of TDGb is required for colocalization with PML . Furthermore, SUMO-1 modification of TDGb and PML enhanced the interaction between the two proteins . These results suggest that SUMO-1 functions to tether proteins to PML-containing nuclear bodies through posttranslational modification and noncovalent protein-protein interaction.

Acta Pharmacol Sin, 2004 Dec, 25(12), 1712 - 818
Rational redesign of inhibitors of furin/kexin processing proteases by electrostatic mutations; Cai XH et al.; AIM: To model the three-dimensional structure and investigate the interaction mechanism of the proprotein convertase furin/kexin and their inhibitors (eglin c mutants) . METHODS: The three-dimensional complex structures of furin/kexin with its inhibitors, eglin c mutants, were generated by modeller program using the newly published X-ray crystallographical structures of mouse furin and yeast kexin as templates . The electrostatic interaction energy of each complex was calculated and the results were compared with the experimentally determined inhibition constants to find the correlation between them . RESULTS: High quality models of furin/kexin-eglin c mutants were obtained and used for calculation of the electrostatic interaction energies between the proteases and their inhibitors . The calculated electrostatic energies of interaction showed a linear correlation to the experimental inhibition constants . CONCLUSION: The modeled structures give good explanations of the specificity of eglin c mutants to furin/kexin . The electrostatic interactions play important roles in inhibitory activity of eglin c mutants to furin/kexin . The results presented here provided quantitative structural and functional information concerning the role of the charge-charge interactions in the binding of furin/kexin and their inhibitors.

Annu Rev Genet, 2004, 38, 203 - 32
Closing mitosis: the functions of the Cdc14 phosphatase and its regulation; Stegmeier F et al.; Completion of the cell cycle requires the temporal and spatial coordination of chromosome segregation with mitotic spindle disassembly and cytokinesis . In budding yeast, the protein phosphatase Cdc14 is a key regulator of these late mitotic events . Here, we review the functions of Cdc14 and how this phosphatase is regulated to accomplish the coupling of mitotic processes . We also discuss the function and regulation of Cdc14 in other eukaryotes, emphasizing conserved features.

Annu Rev Genet, 2004, 38, 1 - 35
Mobile group II introns; Lambowitz AM et al.; Mobile group II introns, found in bacterial and organellar genomes, are both catalytic RNAs and retrotransposable elements . They use an extraordinary mobility mechanism in which the excised intron RNA reverse splices directly into a DNA target site and is then reverse transcribed by the intron-encoded protein . After DNA insertion, the introns remove themselves by protein-assisted, autocatalytic RNA splicing, thereby minimizing host damage . Here we discuss the experimental basis for our current understanding of group II intron mobility mechanisms, beginning with genetic observations in yeast mitochondria, and culminating with a detailed understanding of molecular mechanisms shared by organellar and bacterial group II introns . We also discuss recently discovered links between group II intron mobility and DNA replication, new insights into group II intron evolution arising from bacterial genome sequencing, and the evolutionary relationship between group II introns and both eukaryotic spliceosomal introns and non-LTR-retrotransposons . Finally, we describe the development of mobile group II introns into gene-targeting vectors, "targetrons," which have programmable target specificity.

Biochemistry, 2004 Dec 7, 43(48), 15204 - 9
Twisting of the second transmembrane alpha-helix of the mitochondrial ADP/ATP carrier during the transition between two carrier conformational states; Kihira Y et al.; To investigate the structural and functional features of the second alpha-helical transmembrane segment (TM2) of the mitochondrial ADP/ATP carrier (AAC), we adopted cysteine scanning mutagenesis analysis . Single-cysteine mutations of yeast AAC were systematically introduced at residues 98-106 in TM2, and the mutants were treated with the fluorescent SH reagent eosin-5-maleimide (EMA) . EMA modified different amino acid residues of alpha-helical TM2 between the two distinct carrier conformations, called the m-state and the c-state, in which the substrate recognition site faces the matrix and cytosol, respectively . When amino acids in the helix were projected on a wheel plot, these EMA-modified amino acids were observed at distinct sides of the wheel . Since the SH reagent specifically modified cysteine in the water-accessible environment, these results indicate that distinct helical surfaces of TM2 faced the water-accessible space between the two conformations, possibly as a result of twisting of this helix . In the recently reported crystal structure of bovine AAC, several amino acids faced cocrystallized carboxyatractyloside (CATR), a specific inhibitor of the carrier . These residues correspond to those modified with EMA in the yeast carrier in the c-state . Since the binding site of CATR is known to overlap that of the transport substrate, the water-accessible space was thought to be a substrate transport pathway, and hence, the observed twisting of TM2 between the m-state and the c-state may be involved in the process of substrate translocation . On the basis of the results, the roles of TM2 in the transport function of AAC were discussed.

Biochemistry, 2004 Dec 7, 43(48), 15122 - 30
Role of s'1 loop residues in the substrate specificities of pepsin A and chymosin; Kageyama T; Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates . Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones . A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus . The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)) . Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa . A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins . The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes . An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions . In chymosin mutants, the reverse is possible . The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.

J Nat Prod, 2004 Nov, 67(11), 1829 - 32
Estrogenic and anticarcinogenic properties of kurarinone, a lavandulyl flavanone from the roots of Sophora flavescens; De Naeyer A et al.; Kurarinone, a lavandulyl flavanone, was isolated from a polyphenolic extract of the roots of Sophora flavescens using fractionation guided by estrogenic activity, which was determined by recombinant yeast and Ishikawa Var-I bioassays . Kurarinone showed weak estrogenic activity both in the yeast screen and in the Ishikawa Var-I assay with EC(50) values of 4.6 and 1.66 microM, respectively . Furthermore, kurarinone was found to have potent cytotoxic activity (IC(50) value = 22.2 microM) against human MCF-7/6 breast cancer cells in the sulforhodamine-B assay.

Virology, 2004 Dec 20, 330(2), 471 - 80
A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev; Fang J et al.; HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions . We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as "bait" . DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev- cells rescued by exogenous Rev . Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production . DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis . Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance . These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein . Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics.

J Mol Biol, 2005 Jan 7, 345(1), 141 - 51
Structure of a complex between Nedd8 and the Ulp/Senp protease family member Den1; Reverter D et al.; The Nedd8 conjugation pathway is conserved from yeast to humans and is essential in many organisms . Nedd8 is conjugated to cullin proteins in a process that alters SCF E3 ubiquitin ligase activity, and it is presumed that Nedd8 deconjugation would reverse these effects . We now report the X-ray structures of the human Nedd8-specific protease, Den1, in a complex with the inhibitor Nedd8 aldehyde, thus revealing a model for the tetrahedral transition state intermediate generated during proteolysis . Although Den1 is closely related to the SUMO-specific protease family (Ulp/Senp family), structural analysis of the interface suggests determinants involved in Nedd8 selectivity by Den1 over other ubiquitin-like family members and suggests how the Ulp/Senp architecture has been modified to interact with different ubiquitin-like modifiers.

Res Microbiol, 2004 Dec, 155(10), 861 - 6
Effects of temperature and incubation time on production of ochratoxin A by black aspergilli; Esteban A et al.; The effects of temperature (5-45 degrees C) on the growth and production of ochratoxin A (OTA) by eighteen strains of Aspergillus section Nigri, cultured on Czapek yeast autolysate agar (CYA) and on yeast extract sucrose agar (YES), were studied for an incubation period of 30 days . Isolates were selected to include different sources and different reported abilities to produce OTA . Temperature ranges for OTA production were more restrictive than those for growth and each strain tested differed in its optimum conditions for OTA production . Aspergillus niger aggregate strains achieved maximum OTA levels in YES medium mainly at 20-25 degrees C . The A . carbonarius strains produced the highest OTA levels in CYA medium at 15 or 20 degrees C . Significant amounts of OTA were produced after only five days of incubation . Due to their ability to produce OTA at a wide range of temperatures, OTA can be continuously produced in the field . This fact has to be taken into account in commodities such as grapes, raisins and wine, where A . carbonarius and members of the A . niger aggregate are considered to be the main sources of the OTA contamination.

Comput Biol Med, 2005 Feb, 35(2), 173 - 81
On exact string matching of unique oligonucleotides; Hyyro H et al.; Unique, gene-specific oligonucleotides are used for many genetic investigations such as polymerase chain reaction, gene cloning, microarray technology and antisense DNA studies . It is a computationally demanding task to extract these oligonucleotides from DNA databases . We studied the problem from the point of view of the string matching problem . We implemented and tested several exact string matching algorithms and modified the implementations to be as effective as possible . Ten different implementations were tested on yeast genomic sequence data . The run times for the best algorithms were significantly improved compared to conventional approaches, while in principle, i.e . in respect of theoretical time complexity, these algorithms do not actually differ essentially from each other.

Cell Signal, 2005 Mar, 17(3), 395 - 404
Roles of the intracellular regions of angiotensin II receptor AT2 in mediating reduction of intracellular cGMP levels; Pulakat L et al.; We have shown previously that the angiotensin II (Ang II) receptor AT2 reduces the intracellular levels of cGMP in Xenopus oocytes when activated by ligand binding, and the C-terminal cytoplasmic tail of the AT2 acts as a negative regulator of this function . Here we report the effects of mutations in the 2nd and 3rd intracellular loops of AT2 on AT2-mediated cGMP reduction . Mutating the highly conserved DRY motif (D141G-R142G-Y143A) of the 2nd ICL implicated in activating G(alpha) subunit of trimeric G-proteins did not affect AT2-mediated cGMP reduction . Moreover, anti-Gialpha antibody or phosphodiesterase inhibitor IBMX did not inhibit AT2-mediated cGMP reduction, suggesting that Gialpha activation and subsequent phosphodiesterase activation are not involved in this function . In contrast, mutations T250R-R251N and L255F-K256R located in the C-terminus of the 3rd ICL of AT2 retained ligand-binding properties of the wild-type AT2, and its ability to interact with the ErbB3 in yeast two-hybrid assay, but abolished AT2-mediated cGMP reduction . Similarities in the roles of ICLs of AT2 in AT2-mediated cGMP reduction in oocytes, and AT2-mediated SHP1 activation in COS-7 cells, (need of 3rd ICL for both functions and lack of involvement of DRY motif), suggest that the cascade of events in these two signaling mechanisms could be similar, and that an oocyte-specific SHP1-like protein may be involved in AT2-mediated cGMP reduction in these cells.

Cell Signal, 2005 Mar, 17(3), 279 - 87
cAMP-PKA signaling to the mitochondria: protein scaffolds, mRNA and phosphatases; Feliciello A et al.; Energy metabolism and, specifically, the coupling of mitochondria to growth and survival is controlled by the cAMP-PKA pathway in yeast . In higher eukaryotes, cAMP signaling originating at the plasma membrane is distributed to different subcellular districts by cAMP waves received by PKA bound to PKA anchor proteins (AKAPs) tethered to these compartments . This review focuses on the subgroup of AKAPs that anchor PKA to the mitochondrial outer membrane (mtAKAPs) . Only PKA anchored to mtAKAPs can efficiently transmit cAMP signals to mitochondria . mtAKAP complexes are remarkably heterogeneous . In addition to PKA regulatory subunits, they may include mRNAs, tyrosine phosphatase(s) and tyrosine kinase(s) . Selective regulation of these components by cAMP-PKA integrates various signal transduction pathways and can determine which subcellular compartment receives the signal . Unveiling the interactions among the components of these large complexes will shed light on how cAMP and PKA regulate vital mitochondrial processes.

Symp Soc Exp Biol, 2004, (56), 69 - 88
Plant nuclear envelope proteins; Rose A et al.; Compared to research in the animal field, the plant NE has been clearly under-investigated . The available data so far indicate similarities as well as striking differences that raise interesting questions about the function and evolution of the NE in different kingdoms . Despite a seemingly similar structure and organization of the NE, many of the proteins that are integral components of the animal NE appear to lack homologues in plant cells . The sequencing of the Arabidopsis genome has not led to the identification of homologues of animal NE components, but has indicated that the plant NE must have a distinct protein composition different from that found in metazoan cells . Besides providing a selective barrier between the nucleoplasm and the cytoplasm, the plant NE functions as a scaffold for chromatin but the scaffolding components are not identical to those found in animal cells . The NE comprises an MTOC in higher plant cells, a striking difference to the organization of microtubule nucleation in other eukaryotic cells . Nuclear pores are present in the plant NE, but identifiable orthologues of most animal and yeast nucleoporins are presently lacking . The transport pathway through the nuclear pores via the action of karyopherins and the Ran cycle is conserved in plant cells . Interestingly, RanGAP is sequestered to the NE in plant cells and animal cells, yet the targeting domains and mechanisms of attachment are different between the two kingdoms . At present, only a few proteins localized at the plant NE have been identified molecularly . Future research will have to expand the list of known protein components involved in building a functional plant NE.

Curr Genet, 2005 Jan, 47(1), 1 - 17 Epub 2004 Nov 23.
Sin3: a flexible regulator of global gene expression and genome stability; Silverstein RA et al.; SIN3 was first identified genetically as a global regulator of transcription . Sin3 is a large protein composed mainly of protein-interaction domains, whose function is to provide structural support for a heterogeneous Sin3/histone deacetylase (HDAC) complex . The core Sin3/HDAC complex is conserved from yeast to man and consists of eight proteins . In addition to HDACs, Sin3 can sequester other enzymatic functions, including nucleosome remodeling, DNA methylation, N-acetylglucoseamine transferase activity, and histone methylation . Since the Sin3/HDAC complex lacks any DNA-binding activity, it must be targeted to gene promoters by interacting with DNA-binding proteins . Although most research on Sin3 has focused on its role as a corepressor, mounting evidence suggests that Sin3 can also positively regulate transcription . Furthermore, Sin3 is key to the propagation of epigenetically silenced domains and is required for centromere function . Thus, Sin3 provides a platform to deliver multiple combinations modifications to the chromatin, using both sequence-specific and sequence-independent mechanisms.

J Neurosci, 2004 Nov 24, 24(47), 10750 - 62
Regulation of HCN channel surface expression by a novel C-terminal protein-protein interaction; Santoro B et al.; Hyperpolarization-activated cation currents (I(h)) are carried by channels encoded by a family of four genes (HCN1-4) that are differentially expressed within the brain in specific cellular and subcellular compartments . HCN1 shows a high level of expression in apical dendrites of cortical pyramidal neurons and in presynaptic terminals of cerebellar basket cells, structures with a high density of I(h) . Expression of I(h) is also regulated by neuronal activity . To isolate proteins that may control HCN channel expression or function, we performed yeast two-hybrid screens using the C-terminal cytoplasmic tails of the HCN proteins as bait . We identified a brain-specific protein, which has been previously termed TRIP8b (for TPR-containing Rab8b interacting protein) and PEX5Rp (for Pex5p-related protein), that specifically interacts with all four HCN channels through a conserved sequence in their C-terminal tails . In situ hybridization and immunohistochemistry show that TRIP8b and HCN1 are colocalized, particularly within dendritic arbors of hippocampal CA1 and neocortical layer V pyramidal neurons . The dendritic expression of TRIP8b in layer V pyramidal neurons is disrupted after deletion of HCN1 through homologous recombination, demonstrating a key in vivo interaction between HCN1 and TRIP8b . TRIP8b dramatically alters the trafficking of HCN channels heterologously expressed in Xenopus oocytes and human embryonic kidney 293 cells, causing a specific decrease in surface expression of HCN protein and I(h) density, with a pronounced intracellular accumulation of HCN protein that is colocalized in discrete cytoplasmic clusters with TRIP8b . Finally, TRIP8b expression in cultured pyramidal neurons markedly decreases native I(h) density . These data suggest a possible role for TRIP8b in regulating HCN channel density in the plasma membrane.

Exp Biol Med (Maywood), 2004 Dec, 229(11), 1111 - 9
Mammalian septin function in hemostasis and beyond; Martinez C et al.; Interest in the biology of mammalian septin proteins has undergone a birth in recent years . Originally identified as critical for yeast budding throughout the 1970s, the septin family is now recognized to extend from yeast to humans and is associated with a variety of events ranging from cytokinesis to vesicle trafficking . An emerging theme for septins is their presence at sites where active membrane or cytoplasmic partitioning is occurring . Here, we briefly review the mammalian septin protein family and focus on a prototypic human and mouse septin, termed SEPT5, that is expressed in the brain, heart, and megakaryocytes . Work from neurobiology laboratories has linked SEPT5 to the exocytic complex of neurons, with implications that SEPT5 regulates neurotransmitter release . Striking similarities exist between neurotransmitter release and the platelet-release reaction, which is a critical step in platelet response to vascular injury . Work from our laboratory has characterized the platelet phenotype from mice containing a targeted deletion of SEPT5 . Most strikingly, platelets from SEPT5(null) animals aggregate and release granular contents in response to subthreshold levels of agonists . Thus, the characterization of a SEPT5-deficient mouse has linked SEPT5 to the platelet exocytic process and, as such, illustrates it as an important protein for regulating platelet function . Recent data suggest that platelets contain a wide repertoire of different septin proteins and assemble to form macromolecular septin complexes . The mouse platelet provides an experimental framework to define septin function in hemostasis, with implications for neurobiology and beyond.

Trends Cell Biol, 2004 Dec, 14(12), 670 - 7
Flippases and vesicle-mediated protein transport; Graham TR; The best-understood mechanisms for generating transport vesicles in the secretory and endocytic pathways involve the localized assembly of cytosolic coat proteins such as clathrin, coat protein complex (COP)I and COPII onto membranes . These coat proteins can deform membranes by themselves, but accessory proteins might help to generate the tight curvature needed to form a vesicle . Enzymes that pump phospholipid from one leaflet of the bilayer to the other (flippases) can deform membranes by creating an imbalance in the phospholipid number between the two leaflets . Recent studies describe a requirement for the yeast Drs2p family of P-type ATPases in both phospholipid translocation and protein transport in the secretory and endocytic pathways . This indicates that flippases work with coat proteins to form vesicles.

Gene, 2004 Dec 8, 343, 91 - 7
Efficient somatic gene targeting in the lymphoid human cell line DG75; Feederle R et al.; Among the different approaches used to define the function of a protein of interest, alteration and/or deletion of its encoding gene is the most direct strategy . Homologous recombination between the chromosomal gene locus and an appropriately designed targeting vector results in an alteration or knockout of the gene of interest . Homologous recombination is easily performed in yeast or in murine embryonic stem cells, but is cumbersome in more differentiated and diploid somatic cell lines . Here we describe an efficient method for targeting both alleles of a complex human gene locus in DG75 cells, a cell line of lymphoid origin . The experimental approach included a conditional knockout strategy with three genotypic markers, which greatly facilitated the generation and phenotypic identification of targeted recombinant cells . The vector was designed such that it could be reused for two consecutive rounds of recombination to target both alleles . The human DG75 cell line appears similar to the chicken DT40 pre B-cell line, which supports efficient homologous recombination . Therefore, the DG75 cell line is a favorable addition to the limited number of cell lines amenable to gene targeting and should prove useful for studying gene function through targeted gene alteration or deletion in human somatic cells.

Gene, 2004 Dec 8, 343, 1 - 9
The DEK protein--an abundant and ubiquitous constituent of mammalian chromatin; Waldmann T et al.; The protein DEK is an abundant and ubiquitous chromatin protein in multicellular organisms (not in yeast) . It is expressed in more than a million copies/nucleus of rapidly proliferating mammalian cells . DEK has two DNA binding modules of which one includes a SAP box, a sequence motif that DEK shares with a number of other chromatin proteins . DEK has no apparent affinity to specific DNA sequences, but preferentially binds to superhelical and cruciform DNA, and induces positive supercoils into closed circular DNA . The available evidence strongly suggests that DEK could function as an architectural protein in chromatin comparable to the better known classic architectural chromatin proteins, the high-mobility group or HMG proteins.

J Biol Chem . 2004 Nov 24; {Epub ahead of print}
MAPKAP kinase 3pK phosphorylates and regulates chromatin-association of the polycomb-group protein Bmi1; Voncken JW et al.; Polycomb-Group (PcG) proteins form chromatin-associated, transcriptionally repressive complexes, which are critically involved in the control of cell proliferation and differentiation . Although the mechanisms involved in PcG-mediated repression are beginning to unravel, little is known about the regulation of PcG-function . We showed previously that PcG-complexes are phosphorylated in vivo, which regulates their association with chromatin . The nature of the responsible PcG-kinases remained unknown . Here we present the novel finding, that the PcG-protein Bmi1 is phosphorylated by 3pK (MAPKAP kinase 3), a convergence point downstream of activated ERK and p38 signaling pathways, and implicated in differentiation and developmental processes . We identify 3pK as an interaction partner of PcG-proteins in vitro and in vivo, by yeast two-hybrid interaction and co-immunoprecipitation respectively . Activation or overexpression of 3pK results in phosphorylation of Bmi1 and other PcG-members and their dissociation from chromatin . Phosphorylation and subsequent chromatin-dissociation of PcG-complexes is expected to result in de-repression of targets . One such reported Bmi1-target is the Cdkn2a/INK4A locus . Cells overexpressing 3pK show PcG complex/chromatin dissociation and concomitant de-repression of p14ARF, which is encoded by the Cdkn2a/INK4A locus . Thus, 3pK is a candidate regulator of phosphorylation-dependent PcG/chromatin interaction . We speculate that phosphorylation may not only affect chromatin association, but in addition the function of individual complex members . Our findings link for the first time MAPK-signaling pathways to the Polycomb transcriptional memory system . This suggests a novel mechanism by which a silenced gene status can be modulated and implicates PcG-mediated repression as a dynamically controlled process.

J Biol Chem . 2004 Nov 24; {Epub ahead of print}
Fcp1 phosphatase: Interaction with elongating RNA polymerase II holoenzyme, enzymatic mechanism of action, and genetic interaction with elongator; Kong SE et al.; Fcp1 de-phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) in vitro, and mutation of the yeast FCP1 gene results in global transcription defects and increased CTD phosphorylation levels in vivo . Here we show that the Fcp1 protein associates with elongating RNAPII holoenzyme in vitro . Our data suggest that the association of Fcp1 with elongating polymerase results in CTD de-phosphorylation when the native ternary RNAPII0/DNA/RNA complex is disrupted . Surprisingly, highly purified yeast Fcp1 de-phosphorylates serine 5, but not serine 2 of the RNAPII CTD repeat . Only free RNAPII0(ser5) and not RNAPII0/DNA/RNA ternary complexes act as a good substrate in the Fcp1 CTD de-phosphorylation reaction . In contrast, TFIIH CTD kinase has a pronounced preference for RNAPII incorporated into a ternary complex . Interestingly, the Fcp1 reaction mechanism appears to entail phosphoryl transfer from RNAPII0 directly to Fcp1 . Elongator fails to affect the phosphatase activity of Fcp1 in vitro, but genetic evidence points to a functional overlap between Elongator and Fcp1 in vivo . Genetic interactions between Elongator and a number of other transcription factors are also reported . Together, these results shed new light on mechanisms that drive the transcription cycle and point to a role for Fcp1 in the recycling of RNAPII after dissociation from active genes.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2004 Oct, 26(5), 529 - 32
{Screening, cloning, and analyzing for hSNF5 binding proteins in human fetal brain}; Zhang Y et al.; OBJECTIVE: To identify novel binding proteins of hSNF5, a subunit of chromatin remodeling complex in human fetal brain . METHODS: The yeast two-hybrid system was used for this study . Positive cDNA clones were sequenced . Sequence homology and putative functional domains were analyzed and compared with databank . RESULTS: Nine positive clones obtained were analyzed, among which the sequence of one clone was 97% homologous to the 3' mRNA of a hypothetical protein FLJ20643, while other four clones were related to protein coding sequences existed in the GenBank . The rest four clones were not in frame with any known protein coding sequence . CONCLUSIONS: Clones encoding for hSNF5 binding protein exists in cDNA library of human brain . These proteins may recruit chromatin remodeling complex via hSNF5 to modulate the transcription of their target gene and the related cell functions.

Bull Cancer, 2004 Jun, 91(6), E184 - 200
Homophilic anchorage of brain-hexokinase to mitochondria-porins revealed by specific-peptide antibody cross recognition; Oudard S et al.; In brain tumors the main source of energy is from glycolysis, which is initiated by hexokinase 1 (HK1), an enzyme bound to the mitochondrial porin . Disruption of HK binding greatly affects tumor cell survival . Little is known about the acceptor site of HK1 . Therefore, a polyclonal antibody (Pab) directed to MIAAQLLAYYFTELK (MK) peptide, corresponding to the 15-amino acids of the N-terminal sequence of brain HK1 was obtained . Anti MK antibody (aMK-Pab)bound specifically to HK as shown by ELISA . The aMK-Pab binding to MK peptide was antibody-concentration dependent and was completely abolished by its preincubation with the peptide at 6 x 10-8 M . The aMK-Pab recognized cytosolic HK (cHK) and HK solubilized (sHK)from rat-brain mitochondrial preparations, but not the yeast HK which does not have the MK sequence . An anti-brain HK Pab (aHK-Pab) directed to purified HK recognized the MK peptide; aHK-Pab bound to MK and this binding was inhibited by preincubation of the antibody with the MK peptide . It was previously demonstrated that brain HK anchors to mitochondria porins, also designated as voltage dependent-anion channels (VDAC) via the MK sequence . A specific anti-VDAC antibody (aVDAC-Pab) which specifically bound the N and C-terminal sequences of VDACwas found to bind to c-HK, sHK and MK-coated wells and this binding was abolished by aVDACPabpreincubation with MK peptide . These data suggest that the three Pabs cross-react with an epitope present in HK and VDAC, and which was presented in the MK peptide . Comparison of alignment of HK or VDAC sequences, available in the protein data bank (PDB), did not allow putative homologues responsible for the cross-reaction to be identified, suggesting that the epitope is conformational . This, added to inhibition of mitochondria-isolated HK binding by the MK peptide,suggests that there is an homophilic-type interaction between HK and porin, through a peptidic structure represented at least in part in the MK peptide.

IDrugs, 2002 Mar, 5(3), 203 - 5
Phage Display Technologies - SMi Conference; Jermutus L; This fairly small meeting, with about 50 attendees, covered all areas of antibody engineering from basic technologies to applications in both therapeutic and diagnostic areas . Despite the fact that nearly all speakers came from commercial organizations, the data presented were solid and highly informative . While new technologies, such as yeast or ribosome display, are integrating into the drug discovery process, vaccine technology looks like a promising alternative to human antibody therapy . New advances in the field, especially in technology, are primarily governed by the tight intellectual property situation.

J Biol Chem . 2004 Nov 23; {Epub ahead of print}
NDPK2 as a signal transducer in the phytochrome-mediated light signaling; Shen Y et al.; Nucleoside diphosphate kinase 2 (NDPK2) in Arabidopsis has been identified as a phytochrome interacting protein by using the C-terminal domain of phytochrome A (phyA) as the bait in yeast two-hybrid screening . The Pfr form of phyA stimulates NDPK2 -phosphate exchange activity in vitro . To better understand the multiple functions of NDPK and its role in phytochrome-mediated signaling, we characterized the interaction between phytochrome and NDPK2 . Domain studies revealed that PAS domain A in the C-terminal domain of phytochrome is the binding site for NDPK2 . Additionally, phytochrome recognizes both NDPK2 C-terminal fragment and NDPK2 hexameric structure to fulfill its binding . To illustrate the mechanism of how the Pfr form of phytochrome stimulates NDPK2, histidine-197 (H197)-surrounding residue mutants were made and tested . Results suggested that the H-bonding with H197 inside the nucleotide-binding pocket is critical for NDPK2 functioning . The pH-dependence profiles of NDPK2 indicated that mutants with different activities from the wild type have different pKa values of H197 and that NDPK2 hyperactive mutants possess lower pKa values . Since a lower pKa value of H197 accelerates NDPK2 autophosphorylation and the phospho-transfer between the phosphorylated NDPK2 and its kinase substrate, we concluded that the Pfr form of phytochrome stimulates NDPK2 by lowering the pKa value of H197.

J Biol Chem . 2004 Nov 23; {Epub ahead of print}
Inositol diphosphate signaling regulates telomere length; York SJ et al.; Activation of phospholipase C-dependent inositol polyphosphate (IP) signaling pathways generate distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export . Here we report the regulation of telomere length by production of a diphosphoryl inositol tetrakisphosphate, PP-IP(4), synthesized by the KCS1 gene product . Loss of PP-IP(4) production results in lengthening of telomeres, while overproduction leads to their shortening . This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia . Our data provide in vivo evidence of a regulatory link between IP signaling and the checkpoint kinase family, and describe a third nuclear process modulated by phospholipase C activation.

J Biol Chem . 2004 Nov 23; {Epub ahead of print}
The leukocyte integrin gene CD11d is repressed by Gut-enriched kruppel-like factor 4 in myeloid cells; Noti JD et al.; The myeloid-specific leukocyte integrin CD11d encodes the alphaD subunit for the alphaDbeta2 receptor . A yeast one-hybrid screen showed that a longer isoform of gut-enriched Kruppel-like factor 4 (GKLF) we term GKLFa interacts with the CD11d promoter . Purified GST-GKLFa protein was shown to bind within the -61 to -44 region that overlaps a binding site for the CD11d transcriptional activators Sp1 and transforming growth factor beta-inducible early gene-1 (TIEG1) . Transfection of GKLF/GKLFa in myeloid cells reduced CD11d promoter activity, whereas, downregulation of GKLF/GKLFa with small interfering RNAs led to upregulation of CD11d expression . Differentiation of myeloid cells with phorbol ester led to activation of the CD11d promoter and reduced occupancy of the promoter by GKLF/GKLFa but an increased occupancy by TIEG1 in vivo . Binding of GKLF/GKLFa, Sp1, and TIEG1 to the CD11d promoter in vivo is dependent on their zinc-finger DNA-binding domains . GKLFa physically associates with the histone deacetylases (HDAC) 1 and 2 and both HDACs are bound to the CD11d promoter in vivo but released after exposure of myeloid cells to phorbol ester suggesting that GKLF/GKLFa recruits HDACs to effect repression.

J Biol Chem . 2004 Nov 22; {Epub ahead of print}
Identification of a novel partner of Duox: EFP1, a thioredoxin-related protein; Wang D et al.; H2O2 is a crucial substrate of thyroproxidase (TPO) to iodinate thyroglobulin and synthesize thyroid hormones in thyroid . ThOX proteins (Thyroid Oxidase) also called Duox are believed to be responsible for H2O2 generating . Duoxs expressed in transfected cells do not generate an active system, nor permit their membrane localization suggesting that other proteins are required to fulfill these functions . In this study, we demonstrate interactions of Duoxs with TPO and with p22phox without any effect on Duox activity . By yeast two-hybrid method using EF-hand fragment of dog Duox1 as the bait we have isolated EFP1, one partner of Duoxs that belongs to the thioredoxin-related protein family . EFP1 shares moderate similarities with other members of thioredoxin-related proteins, but the characteristic active site and the folding structures are well conserved . EFP1 can be co-immunoprecipitated with Duoxs in transfected COS cells as well as in primary cultured human thyrocytes . It interacts also with TPO but not Tg . Immunofluorescence studies show that EFP1 and Duox proteins are co-localized inside the transfected cells, suggesting that EFP1 is not sufficient to induce either the expression of Duox at the plasma membrane or to permit H2O2 production . EFP1 and Duox mRNA share similar distribution in nine different tissues . These results suggest that EFP1 could be one of the partners in the assembly of the multi-protein complex constituting the thyroid H2O2 generating system but is certainly not sufficient to permit H2O2 generation .

Phytochemistry, 2004 Dec, 65(24), 3179 - 85
A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity; Harada E et al.; Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems . Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins . Among those are several cyanobacteria . We have studied one of the encoded proteins (alr0975 from Nostoc sp . strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity . Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine . The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine . Purified recombinant protein catalyzes the respective reaction . Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations . No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations . Expression of alr0975 was detected in Nostoc sp . cells with an antiserum raised against the protein . No indication for a responsiveness of expression to toxic metal exposure was found . Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.

Exp Cell Res, 2005 Jan 15, 302(2), 270 - 80
Common and cell type-specific responses of human cells to mitochondrial dysfunction; Miceli MV et al.; In yeast, mitochondrial dysfunction activates a specific pathway, termed retrograde regulation, which alters the expression of specific nuclear genes and results in increased replicative life span . In mammalian cells, the specific nuclear genes induced in response to loss of mitochondrial function are less well defined . This study characterizes responses in nuclear gene expression to loss of mitochondrial DNA sequences in three different human cell types: T143B, an osteosarcoma-derived cell line; ARPE19, a retinal pigment epithelium cell line; and GMO6225, a fibroblast cell population from an individual with Kearns-Sayre syndrome (KSS) . Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure gene expression of a selection of glycolysis, TCA cycle, mitochondrial, peroxisomal, extracellular matrix, stress response, and regulatory genes . Gene expression changes that were common to all three cell types included up-regulation of GCK (glucokinase), CS (citrate synthase), HOX1 (heme oxygenase 1), CKMT2 (mitochondrial creatine kinase 2), MYC (v-myc myelocytomatosis viral oncogene homolog), and WRN (Werner syndrome helicase), and down-regulation of FBP1 (fructose-1, 6-bisphosphatase 1) and COL4A1 (collagen, type IV, alpha 1) . RNA interference experiments show that induction of MYC is important in rho0 cells for the up-regulation of glycolysis . In addition, a variety of cell type-specific gene changes was detected and most likely depended upon the differentiated functions of the individual cell types . These expression changes may help explain the response of different tissues to the loss of mitochondrial function due to aging or disease.

Eur J Biochem, 2004 Nov, 271(22), 4485 - 94
Alternative initiation of transcription of the human presenilin 1 gene in SH-SY5Y and SK-N-SH cells . The role of Ets factors in the regulation of presenilin 1; Pastorcic M et al.; We have identified DNA sequences required for the expression of the presenilin 1 (PS1) gene . A promoter region has been mapped in SK-N-SH cells and includes sequences between -118 and +178 flanking the major initiation site (+1) . The PS1 gene is also efficiently transcribed in the SH-SY5Y subclone of SK-N-SH cells . However the promoter appears to be utilized in alternative ways in both cell types . Sequences both upstream as well as downstream from the initiation site mapped in SK-N-SH cells were shown by 5'- and 3'-deletion analysis to play a crucial role in both cell lines . However, in SH-SY5Y cells either upstream or downstream sequences are sufficient to direct transcription, whereas in SK-N-SH cells 5'-deletions past the +1 site eliminate over 95% of transcription . Several Ets motifs (GGAA) as well as Sp1 motifs {(G/T)GGCGGRRY} are juxtaposed both upstream and downstream from +1 . To understand how the promoter may be utilized alternatively in different cell types we have examined the effect of point mutations in these elements . Altering an Ets motif at -10 eliminates 80% of transcription in SK-N-SH cells whereas the same mutation has only a minor effect in SH-SY5Y cells . Conversely, mutation of the Ets element at +90, which eliminates 70% of transcription in SH-SY5Y cells, has a lesser effect in SK-N-SH cells . In both cell types a promoter including mutations at both -10 and +90 sites loses over 90% transcription activity indicating the crucial importance of these two Ets motifs . The effect of Sp1 mutations appears to be similar in both cell types . Hence the differential expression in each cell type may be at least partially determined by Ets factors and the -10/+90 sites . We have identified several Ets factors that recognize specifically the -10 Ets motif by the yeast one-hybrid selection including avian erythroblastosis virus E26 oncogene homologue 2, Ets-like gene 1, Ets translocation variant 1 and Ets related molecule (ERM) . We show here that ERM specifically recognizes Ets motifs on the PS1 promoter located at -10 as well as downstream at +90, +129 and +165 and activates PS1 transcription with promoter fragments containing or not the -10 Ets site.

Eur J Biochem, 2004 Nov, 271(22), 4474 - 84
Context-dependent effects of proline residues on the stability and folding pathway of ubiquitin; Crespo MD et al.; Substitution of trans-proline at three positions in ubiquitin (residues 19, 37 and 38) produces significant context-dependent effects on protein stability (both stabilizing and destabilizing) that reflect changes to a combination of parameters including backbone flexibility, hydrophobic interactions, solvent accessibility to polar groups and intrinsic backbone conformational preferences . Kinetic analysis of the wild-type yeast protein reveals a predominant fast-folding phase which conforms to an apparent two-state folding model . Temperature-dependent studies of the refolding rate reveal thermodynamic details of the nature of the transition state for folding consistent with hydrophobic collapse providing the overall driving force . Bronsted analysis of the refolding and unfolding rates of a family of mutants with a variety of side chain substitutions for P37 and P38 reveals that the two prolines, which are located in a surface loop adjacent to the C terminus of the main alpha-helix (residues 24-33), are not significantly structured in the transition state for folding and appear to be consolidated into the native structure only late in the folding process . We draw a similar conclusion regarding position 19 in the loop connecting the N-terminal beta-hairpin to the main alpha-helix . The proline residues of ubiquitin are passive spectators in the folding process, but influence protein stability in a variety of ways.

Eur J Biochem, 2004 Nov, 271(22), 4401 - 8
Regulation of phospholipid biosynthesis by phosphatidylinositol transfer protein Sec14p and its homologues . A critical role for phosphatidic acid; Holic R et al.; Transcription of yeast phospholipid biosynthesis structural genes, which contain an inositol-sensitive upstream activating sequence in their promoters, responds to the availability of the soluble precursors inositol and choline and to changes in phospholipid metabolism . The INO1 gene is deregulated (derepressed when inositol is present) under the conditions of increased phosphatidylcholine (PtdCho) turnover, as occurs in the sec14Delta cki1Delta strain (SEC14 encodes the major yeast phosphatidylinositol transfer protein; CKI1 encodes choline kinase of the cytidine diphosphate choline pathway of PtdCho biosynthesis) . Five proteins (Sfhp) share sequence homology with phosphatidylinositol transfer protein Sec14p . Two (Sfh2p and Sfh4p), when overexpressed largely complement the otherwise essential Sec14p requirement concerning growth and secretion . In this study, we analysed the ability of Sec14 homologues to correct the defect in regulation of phospholipid biosynthesis resulting from defective or missing Sec14p . We also analysed how PtdCho turnover relates to the transcriptional regulation of phospholipid biosynthesis . The results show that (a) none of the Sec14 homologues was able to substitute for Sec14p in its regulatory aspects of phospholipid biosynthesis, (b) removal of phospholipase D activity corrected the aberrant INO1 gene regulation in yeast strains with otherwise high PtdCho turnover, and (c) increased steady-state phosphatidic acid levels correlated with derepressed levels of the INO1 gene . Overall, the results support the model in which high phosphatidic acid levels lead to derepression of the genes of phospholipid biosynthesis {Henry, S.A . & Patton-Vogt, J.L . (1998) Prog . Nucleic Acid Res . Mol . Biol.61, 133-179}.

Biotechniques, 2004 Nov, 37(5), 844 - 52
Quenching accumulation of toxic galactose-1-phosphate as a system to select disruption of protein-protein interactions in vivo; Gunde T et al.; The reverse two-hybrid system has been developed to readily identify molecules or mutations that can disrupt protein-protein interactions in vivo . This system is generally based on the interaction-dependent activation of a reporter gene, whose product inhibits the growth of the engineered yeast cell . Thus, disruption of the interaction between the hybrid proteins can be positively selected because, by reducing the expression of the negative marker gene, it allows cell growth . Although several counter-selectable marker genes are currently available, their application in the reverse two-hybrid system is generally confronted with technical and practical problems such as low selectivity and relatively complex experimental procedures . Thus, the characterization of more reliable and simple counter-selection assays for the reverse two-hybrid system continues to be of interest . We have developed a novel counter-selection assay based on the toxicity of intracellular galactose-1-phosphate, which accumulates upon expression of a galactokinase-encoding GAL1 reporter gene in the absence of transferase activity . Decreased GAL1 gene expression upon dissociation of interacting proteins causes reduction of intracellular galactose-1-phosphate concentrations, thus allowing cell growth under selective conditions.

Genetika, 2004 Sep, 40(9), 1173 - 86
{Analysis of the formation and autonomous replication of an extrachromosomal mouse transgene}; Structural evidence for variable oligomerization of the N-terminal domain of cyclase-associated protein (CAP); Institute of Structural & Molecular Biology, School of Biological Sciences, The University of Edinburgh, Scotland, United KingdomCyclase-associated protein (CAP) is a highly conserved and widely distributed protein that links the nutritional response signaling to cytoskeleton remodeling . In yeast, CAP is a component of the adenylyl cyclase complex and helps to activate the Ras-mediated catalytic cycle of the cyclase . While the N-terminal domain of CAP (N-CAP) provides a binding site for adenylyl cyclase, the C-terminal domain (C-CAP) possesses actin binding activity . Our attempts to crystallize full-length recombinant CAP from Dictyostelium discoideum resulted in growth of orthorhombic crystals containing only the N-terminal domain (residues 42-227) due to auto-proteolytic cleavage . The structure was solved by molecular replacement with data at 2.2 A resolution . The present crystal structure allows the characterization of a head-to-tail N-CAP dimer in the asymmetric unit and a crystallographic side-to-side dimer . Comparison with previously published structures of N-CAP reveals variable modes of dimerization of this domain, but the presence of a common interface for the side-to-side dimer . (c) 2004 Wiley-Liss, Inc.

Oncogene, 2005 Jan 6, 24(1), 65 - 76
Cytoskeletal modification of Rho guanine nucleotide exchange factor activity: identification of a Rho guanine nucleotide exchange factor as a binding partner for Sept9b, a mammalian septin; Nagata K et al.; Small GTPase Rho and septin family proteins are thought to be related to tumorigenesis . We have identified a Rho-guanine nucleotide exchange factor (GEF) as a binding partner for a mammalian septin Sept9b using yeast two-hybrid screening . We termed this molecule septin-associated RhoGEF (SA-RhoGEF) . Molecular dissection analyses indicated that the C-terminal area of SA-RhoGEF exhibited binding to the N-terminal variable region of Sept9b . SA-RhoGEF was found by immunoprecipitation analysis to associate with septin complexes in REF52 fibroblast cells, maybe through direct interaction with Sept9b . Immunofluorescence analyses revealed the colocalization of SA-RhoGEF and Sept9b along with actin stress fibers in REF52 cells, and their colocalization along stress fibers was most likely to depend on their mutual interaction . In transient expression analyses, Sept9b inhibited SA-RhoGEF-dependent Rho activation in COS7 and HeLa cells . SA-RhoGEF and its fragments expressed in REF52 cells altered endogenous septin filament structures . To our knowledge, SA-RhoGEF is the first molecule providing a link between septins and Rho signaling.

Oncogene . 2004 Nov 22; {Epub ahead of print}
Protein stability and function of p73 are modulated by a physical interaction with RanBPM in mammalian cultured cells; Kramer S et al.; Upon a certain DNA damage including cisplatin treatment, p73 is stabilized and exerts its growth-suppressive and/or proapoptotic function . However, the precise molecular basis by which the intracellular levels of p73 are regulated remains unclear . In the present study, we have identified RanBPM as a novel binding partner of p73alpha by yeast-based two-hybrid screening, and also found that RanBPM has an ability to stabilize p73alpha . GST pull-down assays and co-immunoprecipitation experiments revealed that RanBPM directly bound to the extreme COOH-terminal region of p73alpha, whereas it failed to interact with p53 . Co-expression of RanBPM with p73alpha resulted in the nuclear translocation of RanBPM, and both proteins co-localized in cell nucleus as examined by indirect immunofluorescent staining . It is worth noting that the expression of RanBPM inhibited the ubiquitination of p73alpha, and thereby prolonged its half-life . Subsequent studies demonstrated that the proapoptotic activity of p73alpha was significantly enhanced in the presence of RanBPM . Taken together, our present findings implicate a novel role for RanBPM in the regulation of p73 stability and function.Oncogene advance online publication, 22 November 2004; doi:10.1038/sj.onc.1208257.

J Biol Chem . 2004 Nov 22; {Epub ahead of print}
Calcineurin inhibits Na+/Ca2+ exchange in phenylephrine-treated hypertrophic cardiomyocytes; Katanosaka Y et al.; The cardiac Na(+)/Ca(2+) exchanger (NCX1) is the predominant Ca(2+) extrusion mechanism from beating cardiomyocytes . The role of protein phosphorylation in the regulation of NCX1 function in normal and diseased heart remains unclear . In our search for proteins that interact with NCX1 using a yeast two-hybrid screen, we found that the C-terminus of calcineurin Abeta containing the autoinhibitory domain, binds to the beta1 repeat of the central cytoplasmic loop of NCX1 that presumably constitutes part of the allosteric Ca(2+)-regulatory site . Association of NCX1 with calcineurin was significantly increased in the BIO14.6 cardiomyopathic hamster heart compared with the normal control . In hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment, we observed a marked depression of NCX activity measured as the rate of Na(+)(i)-dependent (45)Ca(2+) uptake or the rate of Na(o)(+)-dependent (45)Ca(2+) efflux . Depressed NCX activity was partially and independently reversed by acute inhibition of calcineurin and protein kinase C (PKC) activities with little effect on myocyte hypertrophic phenotypes . Studies of NCX1 deletion mutants expressed in CCL39 cells were consistent with the view that the beta1 repeat is required for the action of endogenous calcineurin and that the large cytoplasmic loop may be required to maintain the interaction of the enzyme with its substrate . Our data suggest that NCX1 is a novel regulatory target for calcineurin, and that depressed NCX activity might contribute to the etiology of in vivo cardiac hypertrophy and dysfunction occurring under conditions in which both calcineurin and PKC are chronically activated.

Mol Pharmacol . 2004 Nov 19; {Epub ahead of print}
Residues Met89 and Ser160 in the Human Equilibrative Nucleoside Transporter 1 (hENT1) Affect hENT1's Affinity for Adenosine, Guanosine, NBMPR and Dipyridamole; Endres CJ et al.; The human equilibrative nucleoside transporter 1 (hENT1) is an important modulator of the physiological action of adenosine . We identified amino acid residues involved in adenosine transport using a yeast-based assay to rapidly screen and identify randomly generated hENT1 mutants that exhibited decreased sensitivity to inhibition of adenosine transport by various hENT1 competitive inhibitors . We identified Met89 and Ser160 as important in hENT1's affinity for various substrates and inhibitors . Mutation to Met89Cys or Ser160Cys significantly (p<0.05) increased the NBMPR IC50 by approximately 4 and 6-fold respectively (42 +/- 13 and 65 +/- 1.6 nM) when compared with the wild-type transporter (11 +/- 0.7 nM) . The double mutant, Met89Cys/Ser160Cys, synergistically increased NBMPR IC50 to approximately 19-fold of the wild-type transporter . In contrast, when compared to wild-type hENT1, the sensitivity to dipyridamole inhibition was significantly (p<0.05) increased by only the Ser160Cys (~2.6-fold) or the double Met89Cys/Ser160Cys mutant (~4.7-fold) but not by the Met89Cys mutant . Mutation to Met89Cys or Ser160Cys increased the Km of adenosine (~8 and 3-fold) and the Ki of guanosine (~6 and 2-fold) . The double mutant increased both the Km of adenosine and Ki of guanosine by ~8-fold and appeared to confer no additional reduction in adenosine or guanosine affinity than that by mutation of Met89 alone . Collectively, these data indicate that TMD2 (Met89) and TMD4 (Ser160) of hENT1 interact and are important in conferring sensitivity to NBMPR . In contrast, Ser160 and Met89 of hENT1 respectively play a dominant role in conferring sensitivity to dipyridamole and adenosine/guanosine affinity.

J Biol Chem . 2004 Nov 19; {Epub ahead of print}
Characterisation of peptides released from mitochondria: evidence for constant proteolysis and peptide efflux; Augustin S et al.; Conserved ATP-dependent proteases ensure the quality control of mitochondrial proteins and control essential steps in mitochondrial biogenesis . Recent studies demonstrated that non-assembled mitochondrially encoded proteins are degraded to peptides and amino acids which are released from mitochondria . Here, we have characterised peptides extruded from mitochondria by mass spectrometry and identified 270 peptides which are exported in an ATP- and temperature-dependent manner . The peptides originate from 51 mitochondrially and nuclearly encoded proteins localised mainly in the matrix and inner membrane indicating that peptides generated by the activity of all known mitochondrial ATP-dependent proteases can be released from the organelle . Pulse labeling experiments in logarithmically growing yeast cells revealed that ~6-12% of preexisting and newly imported proteins are degraded and contribute to this peptide pool . Under respiring conditions, we observe an increased proteolysis of newly imported proteins which suggests a higher turnover rate of respiratory chain components and thereby rationalises the predominant appearance of representatives of this functional class in the detected peptide pool . These results demonstrate a constant efflux of peptides from mitochondria and provide new insight into the stability of the mitochondrial proteome and the efficiency of mitochondrial biogenesis.

Curr Biol, 2004 Nov 23, 14(22), 2019 - 24
The mitotic arrest in response to hypoxia and of polar bodies during early embryogenesis requires Drosophila Mps1; Fischer MG et al.; Mps1 kinase plays an evolutionary conserved role in the mitotic spindle checkpoint . This system precludes anaphase onset until all chromosomes have successfully attached to spindle microtubules via their kinetochores . Mps1 overexpression in budding yeast is sufficient to trigger a mitotic arrest, which is dependent on the other mitotic checkpoint components, Bub1, Bub3, Mad1, Mad2, and Mad3 . Therefore, Mps1 might act at the top of the mitotic checkpoint cascade . Moreover, in contrast to the other mitotic checkpoint components, Mps1 is essential for spindle pole body duplication in budding yeast . Centrosome duplication in mammalian cells might also be controlled by Mps1 , but the fission yeast homolog is not required for spindle pole body duplication . Our phenotypic characterizations of Mps1 mutant embryos in Drosophila do not reveal an involvement in centrosome duplication, while the mitotic spindle checkpoint is defective in these mutants . In addition, our analyses reveal novel functions . We demonstrate that Mps1 is also required for the arrest of cell cycle progression in response to hypoxia . Finally, we show that Mps1 and the mitotic spindle checkpoint are responsible for the developmental cell cycle arrest of the three haploid products of female meiosis that are not used as the female pronucleus.

Curr Biol, 2004 Nov 23, 14(22), 1985 - 95
Guide RNAs with 5' caps and novel box C/D snoRNA-like domains for modification of snRNAs in metazoa; Tycowski KT et al.; BACKGROUND: Spliceosomal snRNAs and ribosomal RNAs in metazoans contain numerous modified residues that are functionally important . The most common modifications are site-specific 2'-O-methylation and pseudouridylation, both directed by small ribonucleoprotein particles . Each particle is composed of a short guide RNA and a set of several proteins . All previously characterized modification guide RNAs in metazoa are encoded in and processed from introns . RESULTS: We have identified and characterized three novel guide RNAs for conserved 2'-O-methylation of U2, U4, and U12 snRNAs . Two guides, termed mgU2-25/61 and mgU12-22/U4-8, appear to be independently transcribed as judged by the presence of methylated guanosine caps at their 5' ends and upstream promoters similar to those of telomerase RNA . These guide RNAs are each composed of a canonical box C/D snoRNA and a novel box C/D snoRNA-like domain, where the C'/D' motif, rather than C/D, can be folded into a conserved kink-turn structure . The snoRNA-like domains are predicted to direct 2'-O-methylation of invariant G residues that occupy analogous positions in the U2 and U12 snRNA secondary structures . A third guide, mgU2-19/30 RNA, is composed of two canonical box C/D snoRNA domains encoded within a single intron . CONCLUSIONS: This is the first description in metazoan cells of 5'-capped modification guide RNAs that appear to be independently transcribed . Since plant, yeast, and protozoan guide RNAs are mostly independently transcribed, the identification of such RNAs argues that ancestral metazoans possessed independently transcribed guide RNAs and only later, during the evolution of metazoan organisms, did the guide RNA genes shift to introns.

Anal Biochem, 2004 Dec 15, 335(2), 316 - 25
Immobilized cofactor derivatives for kinetic-based enzyme capture strategies: direct coupling of NAD(P)+; Oakey L et al.; This study reevaluates the potential for direct coupling of NAD(P)(+) to a carboxylate-terminating spacer arm using carbodiimide-promoted coupling in an attempt to develop a greatly simplified synthetic method for cofactor immobilization that would support the more widespread adoption of kinetic-based enzyme capture (KBEC) strategies for protein purification applications and protein-detecting arrays/proteomic studies . Direct coupling of NAD(+) to epoxy (1,4-butanediol diglycidyl ether)-activated Sepharose is also described . Depending on the synthetic method used, the position of attachment of cofactor is concluded to be primarily through the pyrophosphate or ribosyl hydroxyl groups . Total substitution levels varied from 0.5 to 2 micromol/g wet weight with 28-67% accessibility . Model bioaffinity chromatographic studies employing KBEC strategies are reported for bovine heart L-lactate dehydrogenase, yeast alcohol dehydrogenase, l-phenylalanine dehydrogenase from Sporosarcina, glutamate dehydrogenase (GDH) from Candida utilis, and GDH from bovine liver . The NAD(+) derivative prepared using epoxy-activated Sepharose shows most potential for further development based on total substitution levels, the apparent absence of nonbiospecific interference, reversible biospecific adsorption of some of the test enzymes using soluble KBEC/stripping ligand tactics, and the relative simplicity of the synthetic method.

Curr Opin Chem Biol, 2004 Dec, 8(6), 665 - 71
Amyloidogenic domains, prions and structural inheritance: rudiments of early life or recent acquisition?
Chernoff YO.
Amyloids are self-assembled fibre-like beta-rich protein aggregates . Amyloidogenic prion proteins propagate amyloid state in vivo and transmit it via infection or in cell divisions . While amyloid aggregation may occur in the absence of any other proteins, in vivo propagation of the amyloid state requires chaperone helpers . Yeast prion proteins contain prion domains which include distinct aggregation and propagation elements, responsible for these functions . Known aggregation and propagation elements are short in length and composed of relatively simple sequences, indicating possible ancient origin . Prion-like self-assembled structures could be involved in the initial steps of biological compartmentalization in early life.

Gene, 2004 Sep 29, 340(1), 161 - 70
Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III; Nata K et al.; Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice . Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes {Diabetes 51(Suppl . 3) (2002) S462} . Plural type III Reg genes were found in mouse and rat . On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human . In the present study, we found a novel human type III REG gene, REG III . This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP . REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine . IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis . All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins . By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12 . The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5' . These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.

Biochimie, 2004 Sep-Oct, 86(9-10), 625 - 32
Integrating a functional proteomic approach into the target discovery process; Colland F et al.; Functional proteomics is a promising technique for the rational identification of novel therapeutic targets by elucidation of the function of newly identified proteins in disease-relevant cellular pathways . Of the recently described high-throughput approaches for analyzing protein-protein interactions, the yeast two-hybrid (Y2H) system has turned out to be one of the most suitable for genome-wide analysis . However, this system presents a challenging technical problem: the high prevalence of false positives and false negatives in datasets due to intrinsic limitations of the technology and the use of a high-throughput, genetic assay . We discuss here the different experimental strategies applied to Y2H assays, their general limitations and advantages . We also address the issue of the contribution of protein interaction mapping to functional biology, especially when combined with complementary genomic and proteomic analyses . Finally, we illustrate how the combination of protein interaction maps with relevant functional assays can provide biological support to large-scale protein interaction datasets and contribute to the identification and validation of potential therapeutic targets.

Curr Opin Microbiol, 2004 Dec, 7(6), 638 - 46
Transcriptional networks: reverse-engineering gene regulation on a global scale; Chua G et al.; A major objective in post-genome research is to fully understand the transcriptional control of each gene and the targets of each transcription factor . In yeast, large-scale experimental and computational approaches have been applied to identify co-regulated genes, cis regulatory elements, and transcription factor DNA binding sites in vivo . Methods for modeling and predicting system behavior, and for reconciling discrepancies among data types, are being explored . The results indicate that a complete and comprehensive yeast transcriptional network will ultimately be achieved.

Curr Opin Microbiol, 2004 Dec, 7(6), 631 - 7
Ribosome synthesis meets the cell cycle; Dez C et al.; A large number of ribosome synthesis factors have been identified using proteomic analyses in yeast . The patterns of RNA and protein co-precipitation suggest that ribosome synthesis does not proceed via a linear progression of successive steps . Recent analyses have identified several interactions between factors clearly implicated in ribosome synthesis and specific steps in the cell division cycle . The intersections between these pathways were not anticipated, but potential explanations for their existence can be advanced.

Mol Biochem Parasitol, 2004 Dec, 138(2), 227 - 36
Cytological and biochemical evidence for a gonad-preferential interplay of SmFKBP12 and SmTbetaR-I in Schistosoma mansoni; Knobloch J et al.; In eukaryotes, FK506-binding proteins with a molecular weight of 12 kDa (FKBP12s) influence a variety of signal transduction pathways that regulate cell division, differentiation, and ion homeostasis . Amongst these, TGFbeta signaling and calcineurin (CN) phosphatase activity is modulated by FKBP12 via binding to TGFbeta-family type I receptors (TbetaR-Is) or to the CN subunit A, respectively . In this work, we demonstrate the tissue-specific expression of the Schistosoma mansoni FKBP12 homologue (SmFKBP12) in the gonads of female parasites as well as in the tegument of both genders . Components of the TGFbeta pathway have been characterized in schistosomes and their roles in mediating host-parasite or male-female interactions proposed . We show that a schistosome TGFbeta-family type I receptor (SmTbetaR-I, SmRK-1) is expressed in the female gonads, suggesting that SmFKBP12 may regulate its activity in this tissue . This hypothesis is supported by yeast two-hybrid analyses showing a direct binding of SmFKBP12 and SmTbetaR-I, which was specifically inhibited by the drug FK506 . Our data provide the first evidence for the activity of a transmembrane receptor in the vitellarium of schistosome females and indicate that FKBP12-meditated regulation of the TGFbeta pathway is evolutionarily conserved in a primitive metazoan such as Schistosoma . Furthermore, we show that the schistosome CN (SmCN) is not expressed in the female gonads, but co-localizes with SmFKBP12 only in the tegument . From these data we conclude an SmFKBP12/SmTbetaR-I, but not an SmCN/SmFKBP12 interplay in the female gonads.

Mol Biochem Parasitol, 2004 Dec, 138(2), 205 - 16
Histone acetyltransferases and deacetylase in Entamoeba histolytica; Ramakrishnan G et al.; In our efforts to understand how transcription may be regulated in Entamoeba histolytica, we have examined if this parasite has conserved enzymatic mechanisms for targeted acetylation and deacetylation of histones . Western blotting indicated that basic nuclear proteins in the size range of 16-23 kDa were acetylated in amebic trophozoites, suggesting histone acetylation . Single representatives of the GNAT and MYST family of histone acetyltransferases (HATs) were identified in the E . histolytica genome and their expression in amebic trophozoites was detected by reverse transcription of RNA followed by the polymerase chain reaction (RT-PCR) . Full-length recombinant EhMYST protein demonstrated HAT activity with calf thymus histones and showed a preference for histone H4, similar to the yeast MYST protein, Esa1 . However, ehMYST did not complement a yeast esa1 mutation . Histone deacetylase (HDAC) activity was detected in nuclear extracts from E . histolytica, and characteristically, was inhibited by trichostatin A (TSA) . Consistent with the observation of HDAC activity, RT-PCR analysis demonstrated that an amebic hdac1 homolog (ehHDAC) is expressed and appropriately spliced in E . histolytica trophozoites . Our results suggest that mechanisms for histone acetylation and deacetylation are operational in E . histolytica.

Mol Biochem Parasitol, 2004 Dec, 138(2), 171 - 82
The Schistosoma mansoni Src kinase TK3 is expressed in the gonads and likely involved in cytoskeletal organization; Kapp K et al.; Cytoplasmic protein tyrosine kinases of the Src family play a pivotal role in the regulation of cellular processes including proliferation and differentiation . Among other functions, Src kinases are involved in regulating the cell architecture . In an approach to identify protein tyrosine kinases from the medically important parasite Schistosoma mansoni, we isolated the TK3 gene by degenerate primer PCR and cDNA library screening . Sequencing of the complete cDNA and data-base analyses indicated that TK3 is a Src family kinase . Its predicted size of 71 kDa was confirmed by Western blot analysis . Southern blot analysis showed that TK3 is a single-copy gene, and Northern blot and RT-PCR experiments indicated its expression in both sexes and throughout development . Localization studies by in situ hybridization and immunolocalization revealed that TK3 is predominantly expressed in the reproductive organs such as the testes of the male and the ovary as well as the vitellarium of the female . Its enzymatic activity was confirmed by functional analyses . In transient transfection experiments with HEK293 cells, TK3 phosphorylated the well-known Src-kinase substrate p130 Cas, an intracellular scaffolding protein . Yeast two-hybrid screenings in a heterologous invertebrate system identified dAbi, vinculin and tubulin as binding partners, representing molecules that fulfill functions in the cell architecture of many organisms . These findings suggest that TK3 may play a role in signal transduction pathways organizing the cytoskeleton in the gonads of schistosomes.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 Nov, 20(6), 686 - 8
{Study on binding of HBeAg to CD81.}; Li BA et al.; AIM: To investigate the interaction between HBeAg and CD81 . METHODS: The CD81 gene was amplified by RT-PCR from HepG2 cells . The recombinant expression vector pGADT7-CD81 was constructed by routine molecular biological method . The auxotroph yeast cells were cotransfected with pGADT7-CD81 and pGBKT7-eAg and plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal . The binding of HBeAg to CD81 was also detected in vitro translation and coimmunoprecipitation test . RESULTS: The recombinant expression vector was constructed and confirmed by restriction enzyme (EcoR I and BamH I) digestion and DNA sequencing analysis . The positive yeast clones could grow and from blue colonies on SD/-Trp-Leu-His-Ade/x-alpha-gal medium . The result of immunocoprecipitation showed that HBeAg could bind to CD81 . CONCLUSION: HBeAg can bind to CD81, suggesting that CD81 plays an important role in the pathogenesis of HBV.

Pharm Res, 2004 Oct, 21(10), 1886 - 94
Screening of the interaction between xenobiotic transporters and PDZ proteins; Kato Y et al.; PURPOSE: Xenobiotic transporters have been proposed to be involved in membrane penetration of various therapeutic agents . As little information is available on molecular mechanism of their functional regulation, we have attempted to clarify the protein-protein interactions of such transporters as a first step to identify their regulators . METHODS: Yeast two-hybrid screening was performed to examine the interaction between carboxylic terminus of various xenobiotic transporters and PDZ (PSD95, D1g and ZO1) domain-containing proteins . The interaction and functional regulation were also evaluated in pull-down, immunoprecipitation and transport studies . RESULTS: Specific interaction with PDZ proteins was identified for several xenobiotic transporters including PEPT1, PEPT2, OCT3, OCTN1, OCTN2, OAT4, OATP-A, OATP-D, and OATP-F . The potent interaction was observed between PEPT2 and PDZK1, and deletion of the last four amino acids of the PEPT2 C-terminus almost completely abrogated such interaction . Recombinant PEPT2 C-terminus fusion protein can bind to purified His6-tagged PDZK1, confirming the involvement of two of four PDZ domains within PDZK1 in the interaction . Alanine-scanning mutation in PEPT2 revealed the presence of a consensus sequence (-T-X-L) that is responsible for the PDZK1 interaction . Transfection of PDZK1 increased the uptake of glycylsarcosine by PEPT2, whereas such stimulation was not observed for PEPT2 with the last four amino acids deleted . CONCLUSIONS: These results first identified the interaction between PDZ proteins and the cytosolic tail of various xenobiotic transporters . PDZK1 directly interacts with PEPT2, exerting functional regulation of its transporting activity . The current findings imply the localization of PEPT2 within a protein network constructed from PDZK1 and other transporter proteins.

J Mol Evol, 2004 Sep, 59(3), 372 - 84
Position-associated GC asymmetry of gene duplicates; Rodin SN et al.; It is well known that repositioning of a gene often exerts a strong impact on its own expression and whole development . Here we report the results of genome-wide analyses suggesting that repositioning may also radically change the evolutionary fate of gene duplicates . As an indicator of these changes, we used the GC content of gene pairs which originated by duplication . This indicator turned out to be duplicate-asymmetric, which means that genes in a pair differ significantly in GC content despite their apparent origin from a common ancestor . Such an asymmetry necessarily implies that after duplication two originally identical genes mutated in opposite directions-toward GC-rich and GC-poor content, respectively . In mammalian genomes, this trend is definitely associated with presumably methylated hypermutable CpG sites, and in a typical GC-asymmetric gene pair, its two member genes are embedded in GC-contrasting isochores . However, we unexpectedly found similar significant GC asymmetry in fish, fly, worm, and yeast . This means that neither methylation alone nor methylation in combination with isochores can be counted as a primary cause of the GC asymmetry; rather they represent specific realizations of some universal principle of genome evolution . Remarkably, genes from pairs with the greatest GC asymmetry tend to be on different chromosomes, suggesting that the mutational difference between gene duplicates is associated with translocation of a new gene to a different place in the genome, whereas GC symmetric pairs demonstrate the opposite tendency . A recently emerged extra gene copy is usually on the same chromosome as is its parent but quickly, by 0.05 substitution per synonymous site, either has perished or occupies a different chromosome . During this earliest posttranslocation period, the ratio of nonsynonymous/synonymous base substitutions is unusually high, suggesting a rapid adaptive evolution of novel functions . In a general context of evolution by gene duplication, our interpretation of this position-dependent GC asymmetry between duplicated genes is that evolution of redundant genes toward a new function has often been associated with their very early, postduplication repositioning in the genome, with a concomitant abrupt change in epigenetic control of tissue/stage-specific expression and an increase in the mutation rate . Of eight eukaryotic genomes studied, the most distinguished in this respect is the human genome.

Protist, 2004 Sep, 155(3), 323 - 30
Characterization of chitin synthases from Entamoeba; Campos-Gongora E et al.; A major component of the Entamoeba cyst wall is chitin, a homopolymer of beta-(1,4)-linked N-acetyl-D-glucosamine . Polymerization of chitin requires the presence of active chitin synthases (CHS), a group of enzymes belonging to the family of beta-glycosyl transferases . CHS have been described for fungi, insects, and nematodes; however, information is lacking about the structure and expression of this class of enzymes in protozoons such as Entamoeba . In this study, the primary structures of two putative E . histolytica CHS (EhCHS-1 and EhCHS-2) were determined by gene cloning and homologous proteins were identified in databases from E . dispar and the reptilian parasite E . invadens . The latter constitutes the widely used model organism for the study of Entamoeba cyst development . The two ameba enzymes revealed between 23% and 33% sequence similarity to CHS from other organisms with full conservation of all residues critically important for CHS activity . Interestingly, EhCHS-1 and EhCHS-2 differed substantially in their predicted molecular weights (73 kD vs . 114 kD) as well as in their isoelectric points (5.04 vs . 8.05), and homology was restricted to a central stretch of about 400 amino acid residues containing the catalytic domain . Outside the catalytic domain, EhCHS-1 was predicted to have seven transmembrane helices (TMH) of which the majority is located within the C-terminal part, resembling the situation found in yeast; whereas, EhCHS-2 is structurally related to nematode or insect chitin synthases, as it contained 17 predicted TMHs of which the majority is located within the N-terminal part of the molecule . Northern blot analysis revealed that genes corresponding to CHS-1 and CHS-2 are not expressed in Entamoeba trophozoites, but substantial amounts of CHS-1 and CHS-2 RNA were present 4 to 8 hours after induction of cyst formation by glucose deprivation of E . invadens . The time-courses of expression differed slightly between the two ameba CHS genes, as in contrast to CHS-1 RNA, expression of CHS-2 RNA was more transient and no plateau was observed between 8 and 16 hours of encystation . However, both CHS RNAs were no longer detectable after 48 hours when most of the cells had been transformed into mature cysts.

Cell, 2004 Nov 24, 119(5), 667 - 78
Mutual control of membrane fission and fusion proteins; Peters C et al.; Membrane fusion and fission are antagonistic reactions controlled by different proteins . Dynamins promote membrane fission by GTP-driven changes of conformation and polymerization state, while SNAREs fuse membranes by forming complexes between t- and v-SNAREs from apposed vesicles . Here, we describe a role of the dynamin-like GTPase Vps1p in fusion of yeast vacuoles . Vps1p forms polymers that couple several t-SNAREs together . At the onset of fusion, the SNARE-activating ATPase Sec18p/NSF and the t-SNARE depolymerize Vps1p and release it from the membrane . This activity is independent of the SNARE coactivator Sec17p/alpha-SNAP and of the v-SNARE . Vps1p release liberates the t-SNAREs for initiating fusion and at the same time disrupts fission activity . We propose that reciprocal control between fusion and fission components exists, which may prevent futile cycles of fission and fusion.

Plant Cell, 2004 Dec, 16(12), 3480 - 95 Epub 2004 Dec.
RAR1 positively controls steady state levels of barley MLA resistance proteins and enables sufficient MLA6 accumulation for effective resistance; Bieri S et al.; The polymorphic barley (Hordeum vulgare) Mla locus harbors allelic race-specific resistance (R) genes to the powdery mildew fungus Blumeria graminis f sp hordei . The highly sequence-related MLA proteins contain an N-terminal coiled-coil structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR region . Using transgenic barley lines expressing epitope-tagged MLA1 and MLA6 derivatives driven by native regulatory sequences, we show a reversible and salt concentration-dependent distribution of the intracellular MLA proteins in soluble and membrane-associated pools . A posttranscriptional process directs fourfold greater accumulation of MLA1 over MLA6 . Unexpectedly, in rar1 mutant plants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced . Furthermore, differential steady state levels of MLA1/MLA6 hybrid proteins correlate with their requirement for RAR1; the RAR1-independent hybrid protein accumulates to higher levels and the RAR1-dependent one to lower levels . Interestingly, yeast two-hybrid studies reveal that the LRR domains of RAR1-independent but not RAR1-dependent MLA isoforms interact with SGT1, a RAR1 interacting protein required for the function of many NB-LRR type R proteins . Our findings implicate the existence of a conserved mechanism to reach minimal NB-LRR R protein thresholds that are needed to trigger effective resistance responses.

Plant Cell, 2004 Dec, 16(12), 3341 - 56 Epub 2004 Dec.
Translational regulation via 5' mRNA leader sequences revealed by mutational analysis of the Arabidopsis translation initiation factor subunit eIF3h; Kim TH et al.; Eukaryotic translation initiation factor 3 (eIF3) consists of core subunits that are conserved from yeast to man as well as less conserved, noncore, subunits with potential regulatory roles . Whereas core subunits tend to be indispensable for cell growth, the roles of the noncore subunits remain poorly understood . We addressed the hypothesis that eIF3 noncore subunits have accessory functions that help to regulate translation initiation, by focusing on the Arabidopsis thaliana eIF3h subunit . Indeed, eIF3h was not essential for general protein translation . However, results from transient expression assays and polysome fractionation indicated that the translation efficiency of specific 5' mRNA leader sequences was compromised in an eif3h mutant, including the mRNA for the basic domain leucine zipper (bZip) transcription factor ATB2/AtbZip11, translation of which is regulated by sucrose . Among other pleiotropic developmental defects, the eif3h mutant required exogenous sugar to transit from seedling to vegetative development, but it was hypersensitive to elevated levels of exogenous sugars . The ATB2 mRNA was rendered sensitive to the eIF3h level by a series of upstream open reading frames . Moreover, eIF3h could physically interact with subunits of the COP9 signalosome, a protein complex implicated primarily in the regulation of protein ubiquitination, supporting a direct biochemical connection between translation initiation and protein turnover . Together, these data implicate eIF3 in mRNA-associated translation initiation events, such as scanning, start codon recognition, or reinitiation and suggest that poor translation initiation of specific mRNAs contributes to the pleiotropic spectrum of phenotypic defects in the eif3h mutant.

Cancer Res, 2004 Nov 15, 64(22), 8334 - 40
Nuclear betaII-tubulin associates with the activated notch receptor to modulate notch signaling; Yeh TS et al.; The Notch signal pathway plays important roles in proliferation, apoptosis, and differentiation . Abnormalities in Notch signaling are linked to many human diseases . After ligand binding, Notch signaling is activated through the cleavage of Notch receptors to release and translocate the Notch intracellular domain into the nucleus . The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, can modulate downstream target genes via C promoter-binding factor 1-dependent and -independent pathways . To further dissect the Notch1 signaling pathway, we screened the N1IC-associated proteins using a yeast two-hybrid system and identified nuclear beta(II)-tubulin as a candidate for the N1IC-associated proteins . It was suggested that the presence of beta(II)-tubulin in nuclei might be correlated with the cancerous state of cells . However, the function of beta(II)-tubulin locating in the nucleus still is unknown . Herein, we show that the complex of alpha- and beta(II)-tubulin is associated with N1IC in cancer cells by a coimmunoprecipitation analysis . The ankyrin domain of the Notch1 receptor alone was sufficient to associate with beta(II)-tubulin . Furthermore, alpha- and beta(II)-tubulin were localized in the nucleus and formed a complex with N1IC . Treatment with Taxol increased the amounts of nuclear alpha- and beta(II)-tubulin in K562 and HeLa cells and promoted the C promoter-binding factor 1-dependent transactivation activity of N1IC . We also show that nuclear beta(II)-tubulin was bound on the C promoter-binding factor 1 response elements via the association with N1IC . These results suggest that nuclear beta(II)-tubulin can modulate Notch signaling through interaction with N1IC in cancer cells.

Mol Biol Cell . 2004 Nov 17; {Epub ahead of print}
Analysis of Mutant Phenotypes and Splicing Defects Demonstrates Functional Collaboration between the Large and Small Subunits of the Essential Splicing Factor U2AF In Vivo; Webb CJ et al.; Monitoring Editor: Marvin P . Wickens The heterodimeric splicing factor U2AF plays an important role in 3' splice site selection, but the division of labor between the two subunits in vivo remains unclear . In vitro assays led to the proposal that the human large subunit recognizes 3' splice sites with extensive polypyrimidine tracts independently of the small subunit . We report in vivo analysis demonstrating that all five domains of spU2AF(LG) are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology . A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in S . pombe . As this is not predicted by the model for metazoan 3' splice site recognition, we sought introns for which the spU2AF(LG) and spU2AF(SM) make distinct contributions by analyzing diverse splicing events in strains harboring mutations in each partner . Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract . These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo.

Int Rev Cytol, 2004, 241, 155 - 202
Molecular and functional analysis of the dictyostelium centrosome; Graf R et al.; The centrosome is a nonmembranous, nucleus-associated organelle that functions not only as the main microtubule-organizing center but also as a cell cycle control unit . How the approximately 100 different proteins that make up a centrosome contribute to centrosome function is still largely unknown . Considerable progress in the understanding of centrosomal functions can be expected from comparative cell biology of morphologically different centrosomal structures fulfilling conserved functions . Dictyostelium is an alternative model organism for centrosome research in addition to yeast and animal cells . With the elucidation of morphological changes and dynamics of centrosome duplication, the establishment of a centrosome isolation protocol, and the identification of many centrosomal components, there is a solid basis for understanding the biogenesis and function of this fascinating organelle . Here we give an overview of the prospective protein inventory of the Dictyostelium centrosome based on database searches . Moreover, we focus on the comparative cell biology of known components of the Dictyostelium centrosome including the gamma-tubulin complex and the homologues of centrin, Nek2, XMAP215, and EB1.

J Am Chem Soc, 2004 Nov 24, 126(46), 15051 - 9
Directed evolution of a glycosynthase via chemical complementation; Lin H et al.; Recently, we reported a general assay for enzyme catalysis based on the yeast three-hybrid assay, Chemical Complementation, which is intended to expand the range of chemical reactions to which directed evolution can be applied . Here, Chemical Complementation was applied to a glycosynthase derived from a retaining glycosidase, an important class of enzymes for carbohydrate synthesis . Using the yeast three-hybrid assay, the glycosynthase activity of the E197A mutant of the Cel7B from Humicola insolens was linked to transcription of a LEU2 reporter gene, making cell growth dependent on glycosynthase activity in the absence of leucine . Then the LEU2 selection was used to isolate the most active glycosynthase from a Glu197 saturation library, yielding an E197S Cel7B variant with a 5-fold increase in glycosynthase activity . These results not only establish Chemical Complementation as a platform for the directed evolution of glycosynthases, but also show the generality of this approach and the ease with which it can be applied to new chemical reactions.

J Cell Biochem . 2004 Nov 16; {Epub ahead of print}
BMRP is a Bcl-2 binding protein that induces apoptosis; Chintharlapalli SR et al.; Members of the Bcl-2 family of proteins play important roles in the regulation of cell death by apoptosis . The yeast Two-Hybrid system was utilized to identify a protein that interacts with the anti-apoptotic protein Bcl-2, designated BMRP . This protein corresponds to a previously known mitochondrial ribosomal protein (MRPL41) . Binding experiments confirmed the interaction of BMRP to Bcl-2 in mammalian cells . Subcellular fractionation by differential centrifugation studies showed that both Bcl-2 and BMRP are localized to the same fractions (fractions that are rich in mitochondria) . Northern blot analysis revealed a major bmrp mRNA band of approximately 0.8 kb in several human tissues . Additionally, a larger 2.2 kb mRNA species was also observed in some tissues . Western blot analysis showed that endogenous BMRP runs as a band of 16-17 kDa in SDS-PAGE . Overexpression of BMRP induced cell death in primary embryonic fibroblasts and NIH/3T3 cells . Transfection of BMRP showed similar effects to those observed by overexpression of the pro-apoptotic proteins Bax or Bad . BMRP-stimulated cell death was counteracted by co-expression of Bcl-2 . The baculoviral caspase inhibitor p35 also protected cells from BMRP-induced cell death . These findings suggest that BMRP is a mitochondrial ribosomal protein involved in the regulation of cell death by apoptosis, probably affecting pathways mediated by Bcl-2 and caspases . (c) 2004 Wiley-Liss, Inc.

Mol Carcinog, 2005 Jan, 42(1), 53 - 64
Cellular ubiquitination and proteasomal functions positively modulate mammalian nucleotide excision repair; Wang QE et al.; The ubiquitin-proteasome pathway is fundamental to synchronized continuation of many cellular processes, for example, cell-cycle progression, stress response, and cell differentiation . Recent studies have shown that the ubiquitin-proteasome pathway functions in the regulation of nucleotide excision repair (NER) in yeast . In order to investigate the role of the ubiquitin-proteasome pathway in the NER of mammalian cells, global genomic repair (GGR), and transcription-coupled repair (TCR) were examined in a mouse ts20 cell line that harbors a temperature-sensitive ubiquitin-activating enzyme (E1) . We found that E1 inactivation-induced ubiquitination deficiency decreased both GGR and TCR, indicating that the ubiquitination system is involved in the optimization of entire NER machinery in mammalian cells . We specifically inhibited the function of 19S proteasome subunit by overexpressing 19S regulatory complex hSug1 or its mutant protein hSug1mk in repair competent human fibroblast, OSU-2, cells and compared their capacity for NER . The results showed that 19S regulatory complex positively modulates NER in cells . In addition, we treated OSU-2 cells with the inhibitors of 20S subunit function, MG132 and lactacystin, and demonstrated that the catalytic activity of 20S subunit is also required for efficient NER . Moreover, the UV-induced recruitment of repair factor xeroderma pigmentosum protein C (XPC) to damage sites was negatively affected by treatment of repair competent cells with MG132 . Taken together, we conclude that the ubiquitin-proteasome pathway has a positive regulatory role for optimal NER capacity in mammalian cells and appears to act through facilitating the recruitment of repair factors to DNA damage sites.

FASEB J . 2004 Nov 16; {Epub ahead of print}
Novel interaction partners of the TPR/MET tyrosine kinase; Schaaf CP et al.; A large variety of biological processes is mediated by stimulation of the receptor tyrosine kinase MET . Screening a mouse embryo cDNA library, we were able to identify several novel, putative intracellular TPR/MET-substrates: SNAPIN, DCOHM, VAV-1, Sorting nexin 2, Death associated protein kinase 3, SMC-1, Centromeric protein C, and hTID-1 . Interactions as identified by yeast two-hybrid analysis were validated in vitro and in vivo by mammalian two-hybrid studies, a far-western assay and coimmunoprecipitation . Participation in apoptosis-regulating mechanisms through interaction with DAPK-3 and cell cycle control via binding to nuclear proteins such as CENPC and SMC-1 are possible new aspects of intracellular MET signaling.

J Biol Chem . 2004 Nov 16; {Epub ahead of print}
The centrosomal protein RABP1 regulates mitotic progression by recruiting RASSF1A to spindle poles; Song MS et al.; The protein RASSF1A (RAS association domain family protein 1A), which is encoded by a gene that is frequently silenced in many types of sporadic tumor, functions in mitosis as a regulator of the anaphase-promoting complex (APC) . With the use of a yeast two-hybrid screen, we have now identified a human protein, previously designated C19ORF5, that interacts with RASSF1A . This protein, here redesignated RABP1 (RASSF1A binding protein 1), contains two MAP (microtubule-associated protein) domains, and its association with RASSF1A was confirmed in mammalian cells by immunoprecipitation and immunofluorescence analyses . RABP1 was found to be localized to the centrosome throughout the cell cycle in a manner dependent on its MAP domains . Ectopic expression of RABP1 induced both stabilization of mitotic cyclins and mitotic arrest at prometaphase in a RASSF1A-dependent manner . It also increased the extent of association between RASSF1A and Cdc20 . Conversely, depletion of RABP1 by RNA interference prevented both the localization of RASSF1A to the spindle poles as well as its binding to Cdc20, resulting in premature destruction of mitotic cyclins and acceleration of mitotic progression . These findings indicate that RABP1 is required for the recruitment of RASSF1A to the spindle poles and for its inhibition of APC-Cdc20 activity during mitosis.

J Biol Chem . 2004 Nov 16; {Epub ahead of print}
Acquisition of unprecedented PtdIns 3,5-P2 rise in hyperosmotically stressed 3T3-L1 adipocytes, mediated by ArPIKfyve -PIKfyve pathway; Sbrissa D et al.; Unlike yeast, where hyperosmotic stress induces a dramatic increase in PtdIns 3,5-P(2) synthesis, in mammalian cells, though activating a complex array of signaling events, hyperosmotic stress fails to up-regulate PtdIns 3,5-P(2), indicating the PtdIns 3,5-P{sub}2-pathway is not involved in mammalian osmoprotective responses . Here we report an unexpected and marked PtdIns 3,5-P(2) increase in response to hyperosmotic stress in differentiated 3T3-L1 adipocytes . Because this effect was not observed in the precursor preadipocytes, a specific role during acquisition of the adipocyte phenotype and transition into insulin-responsive cells could be suggested . However, acute insulin action did not result in measurable PtdIns 3,5-P(2) rise indicating the PtdIns 3,5-P(2) pathway is a specific hyperosmotically activated signaling cascade selectively operating in differentiated 3T3-L1 adipocytes . Hyperosmolarity activates different components of several kinase cascades, including p38 MAP and tyrosine kinases but these appear to be separate from the activated PtdIns 3,5-P(2) pathway . Since PtdIns 3,5-P(2) is primarily produced by PIKfyve-catalyzed synthesis and requires the upstream activator hVac14 (called herein ArPIKfyve) that physically associates with and activates PIKfyve, we examined the contribution of ArPIKfyve - PIKfyve for the hyperosmotic stress-induced rise in PtdIns 3,5-P(2) . Small interfering RNA-directed gene silencing to selectively deplete ArPIKfyve or PIKfyve in 3T3-L1 adipocytes determined the ArPIKfyve-PIKfyve axis fully accountable for the hyperosmotically activated PtdIns 3,5-P(2) . Together these results reveal a previously uncharacterized PtdIns 3,5-P(2) pathway activated selectively in hyperosmotically-stressed 3T3-L1 adipocytes and suggest a plausible role for PtdIns 3,5-P(2) in the osmoprotective response mechanism in this cell type.

Mol Cell, 2004 Nov 19, 16(4), 631 - 9
The RING domain of Mdm2 mediates histone ubiquitylation and transcriptional repression; Minsky N et al.; Histone modifications play a pivotal role in regulating transcription and other chromatin-associated processes . In yeast, histone H2B monoubiquitylation affects gene silencing . However, mammalian histone ubiquitylation remains poorly understood . We report that the Mdm2 oncoprotein, a RING domain E3 ubiquitin ligase known to ubiquitylate the p53 tumor suppressor protein, can interact directly with histones and promote in vitro monoubiquitylation of histones H2A and H2B . Moreover, Mdm2 induces H2B monoubiquitylation in vivo . Endogenous Mdm2 is tethered in vivo, presumably via p53, to chromatin comprising the p53-responsive p21(waf1) promoter, and Mdm2 overexpression enhances protein ubiquitylation in the vicinity of a p53 binding site within that promoter . Moreover, when recruited to a promoter in the absence of p53, Mdm2 can repress transcription dependently on its RING domain, suggesting that its E3 activity contributes to repression . Histone ubiquitylation may thus constitute a novel mechanism of transcriptional repression by Mdm2, possibly underlying some of its oncogenic activities.

Plant J, 2004 Dec, 40(5), 744 - 51
The effect of intron location on intron-mediated enhancement of gene expression in Arabidopsis; Rose AB; Introns are often required for full expression of genes in organisms as diverse as plants, insects, nematodes, yeast, and mammals . To explore the potential mechanisms of intron-mediated enhancement in Arabidopsis thaliana, the effect of varying the position of an intron was determined using a series of reporter gene fusions between TRYPTOPHAN BIOSYNTHESIS1 (TRP1) and GUS . Two introns that differ in the degree to which they stimulate expression were individually tested at six locations within coding sequences and two positions in the 3'-UTR . The ability of the first introns from both the TRP1 and POLYUBIQUITIN10 (UBQ10) genes to elevate mRNA accumulation in transgenic plants was found to decline with distance from the promoter, despite their being efficiently spliced from all coding sequence locations . Neither intron significantly enhanced mRNA accumulation when positioned 1.1 kb or more from the start of transcription . In addition, measurements of GUS enzyme activity revealed that both introns at all locations elevated GUS activity more than they enhanced mRNA accumulation . The stimulation mediated by two of four other introns tested at the position nearest the promoter was also greater at the level of GUS activity than mRNA accumulation . These findings support a model in which introns increase transcription and promote translation by two distinct mechanisms.

Plant J, 2004 Dec, 40(5), 699 - 711
Molecular characterization of the tobacco SET domain protein NtSET1 unravels its role in histone methylation, chromatin binding, and segregation; Yu Y et al.; Plants contain a great number of genes encoding a distinctive class of SET domain proteins which harbor a plant-specific N-terminal part together with a C-terminal part showing highest sequence similarity to the catalytic domain of the yeast CLR4, the human SUV39H1 and G9a histone-methyltransferases (HMTases) . Here we show that NtSET1, a representative member of this class from tobacco, methylated both K9 and K27 of histone H3 in vitro . Ectopic expression of NtSET1, by an inducible promoter, increased the amount of dimethylated H3K9 and induced chromosome-segregation defects in tobacco BY2 cells . Deletion analyses show that the HMTase activity, the association with specific chromatin regions and with condensed chromosomes, and the cellular effects largely depended on the C-terminal region including the SET domain of the protein . Nevertheless, the N-terminal part of NtSET1 was capable of targeting the green fluorescent protein to interphase chromatin . Finally, we show that NtSET1 bound LHP1, the Arabidopsis homolog of animal heterochromatin protein 1 (HP1), and that LHP1 co-localized with heterochromatin containing high amounts of dimethylated H3K9, suggesting a role for NtSET1 in heterochromatic function . Taken together, our results provide new insights into the molecular and global chromatin-binding activities of this particular class member of plant SET domain proteins.

Plant J, 2004 Dec, 40(5), 686 - 98
The Arabidopsis COW1 gene encodes a phosphatidylinositol transfer protein essential for root hair tip growth; Bohme K et al.; Root hairs are a major site for the uptake of water and nutrients into plants, and they form an increasingly important model system for the study of development in higher plants . We now report on the molecular genetic analysis of the srh1 mutant in Arabidopsis thaliana impaired in root hair tip growth . We show that srh1 is a new allele of cow1 (can of worms1) and we identified the COW1 gene using a positional cloning strategy . The N-terminus of the COW1 protein is 32% identical to an essential phosphatidylinositol transfer protein (PITP), the yeast Sec14 protein (sec14p) while the C-terminus is 34.5% identical to a late nodulin of Lotus japonicus, Nlj16 . We show that expression of the COW1 lipid-binding domain complements the growth defect associated with Sec14p dysfunction in yeast . In addition, we show that GFP fused to the COW1 protein specifically accumulates at the site of root hair outgrowth . We conclude that the COW1 protein is a PITP, essential for proper root hair growth.

Trends Biochem Sci, 2004 Dec, 29(12), 674 - 81
Rpb4 and Rpb7: subunits of RNA polymerase II and beyond; Choder M; RNA Polymerase II (pol II) is a large multi-subunit complex that is responsible for the synthesis of all eukaryotic mRNAs . Its correct and timely recruitment to promoter regions is a crucial step of transcription regulation, involving complicated and well-controlled networks of protein-DNA and protein-protein interactions . The best-studied pol II is the yeast complex consisting of 12 subunits (Rpb1-12) . Rpb4 and Rpb7 form a dissociable heterodimer (Rpb4/7) . The unique location of Rpb4/7 within the transcription initiation complex, and its capacity to interact with various transcription factors, suggest that it provides important links to the network of interactions that control transcription initiation . Moreover, Rpb4/7 executes some non-transcriptional activities, including mRNA transport . Hence, Rpb4/7 functions at the interface of transcriptional and post-transcriptional machinery.

Insect Biochem Mol Biol, 2004 Dec, 34(12), 1315 - 28
Expression and evolution of Delta9 and Delta11 desaturase genes in the moth Spodoptera littoralis; Rodriguez S et al.; Desaturation of fatty acids is a key reaction in the biosynthesis of moth sex pheromones . The main component of Spodoptera littoralis sex pheromone blend is produced by the action of Delta11 and Delta9 desaturases . In this article, we report on the cloning of four desaturase-like genes in this species: one from the fat body (Sls-FL1) and three (Sls-FL2, Sls-FL3 and Sls-FL4) from the pheromone gland . By means of a computational/phylogenetic method, as well as functional assays, the desaturase gene products have been characterized . The fat body gene expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1:4.5) ratio, whereas the pheromone gland Sls-FL2 expressed a Delta9 desaturase that produced (Z)-9-hexadecenoic and (Z)-9-octadecenoic acids in a (1.5:1) ratio . Although both Delta9 desaturases produced (Z)-9-tetradecenoic acid from myristic acid, transformed yeast grown in the presence of a mixture of myristic and (E)-11-tetradecenoic acids produced (Z,E)-9,11-tetradecadienoic acid, but not (Z)-9-tetradecenoic acid . The Sls-FL3 gene expressed a protein that produced a mixture of (E)-11-tetradecenoic, (Z)-11-tetradecenoic, (Z)-11-hexadecenoic and (Z)-11-octadecenoic acids in a 5:4:60:31 ratio . Despite having all the characteristics of a desaturase gene, no function could be found for Sls-FL4.

Mol Cell Biochem, 2004 Oct, 265(1-2), 71 - 8
Increased expression of H11 kinase stimulates glycogen synthesis in the heart; Wang L et al.; OBJECTIVE: H11 kinase is a serine/threonine kinase preferentially expressed in the heart, which participates in cardiac cell growth and also in cytoprotection during ischemia . A cardiac-specific transgenic mouse overexpressing H11 kinase (2- to 7-fold protein increase) has been generated, and is characterized by cardiac hypertrophy with preserved function and protection against irreversible damage during ischemia/reperfusion . In this study, we tested whether H11 kinase also participates in the metabolic adaptation to cardiac hypertrophy and ischemia . METHODS AND RESULTS: A yeast two-hybrid screen using H11 kinase as a bait in a human heart library revealed a potential interaction with phosphoglucomutase (PGM), the enzyme converting glucose 6-phosphate into glucose 1-phosphate . Interaction between H11 kinase and PGM was confirmed by co-immunoprecipitation . To test the biochemical relevance of this interaction, PGM activity was measured in the heart from wild type and transgenic mice, showing a 20% increase of Vmax in the transgenic group, without change in KM . Glycogen content was increased proportionately to the expression of the transgene, reaching a 40% increase in high-expression transgenic mice (7-fold increase in H11 kinase protein) versus wild type (p < 0.01) . Increased incorporation of glucose into glycogen was coupled to a 3-fold increase in the protein expression of the glucose transporter GLUT1 in plasma membrane of transgenic mice (p < 0.01) . CONCLUSION: H11 kinase promotes the synthesis of glycogen, an essential fuel for the stressed heart in both conditions of overload and ischemia . Therefore, H11 kinase represents an integrative sensor in the cardiac adaptation to stress by coordinating cell growth, survival and metabolism.

Nat Genet, 2004 Dec, 36(12), 1331 - 9 Epub 2004 Dec.
Rapid analysis of the DNA-binding specificities of transcription factors with DNA microarrays; Mukherjee S et al.; We developed a new DNA microarray-based technology, called protein binding microarrays (PBMs), that allows rapid, high-throughput characterization of the in vitro DNA binding-site sequence specificities of transcription factors in a single day . Using PBMs, we identified the DNA binding-site sequence specificities of the yeast transcription factors Abf1, Rap1 and Mig1 . Comparison of these proteins' in vitro binding sites with their in vivo binding sites indicates that PBM-derived sequence specificities can accurately reflect in vivo DNA sequence specificities . In addition to previously identified targets, Abf1, Rap1 and Mig1 bound to 107, 90 and 75 putative new target intergenic regions, respectively, many of which were upstream of previously uncharacterized open reading frames . Comparative sequence analysis indicated that many of these newly identified sites are highly conserved across five sequenced sensu stricto yeast species and, therefore, are probably functional in vivo binding sites that may be used in a condition-specific manner . Similar PBM experiments should be useful in identifying new cis regulatory elements and transcriptional regulatory networks in various genomes.

J Virol, 2004 Dec, 78(23), 12848 - 56
Identification of Epstein-Barr virus RK-BARF0-interacting proteins and characterization of expression pattern; Thornburg NJ et al.; The Epstein-Barr virus (EBV) BamHI A transcripts are a family of transcripts that are differentially spliced and can be detected in multiple EBV-associated malignancies . Several of the transcripts may encode proteins . One transcript of interest, RK-BARF0, is proposed to encode a 279-amino-acid protein with a possible endoplasmic reticulum-targeting sequence . In this study, the properties of RK-BARF0 were examined through identification of cellular-interacting proteins through yeast two-hybrid analysis and characterization of its expression in EBV-infected cells and tumors . In addition to the interaction previously identified with cellular Notch, it was determined that RK-BARF0 also bound cellular human I-mfa domain-containing protein (HIC), epithelin, and scramblase . An interaction between RK-BARF0 and Notch or epithelin induced proteasome-dependent degradation of Notch and epithelin but not of HIC or scramblase . Low levels of endogenous Notch expression in EBV-positive cell lines may correlate with RK-BARF0 expression . However, a screen of EBV-positive cell lines and tumors with an affinity-purified alpha-RK-BARF0 antiserum did not consistently detect RK-BARF0 . These data suggest that while RK-BARF0 may have important cellular functions during EBV infection, and while the phenotype of EBV-positive cells suggest its expression, RK-BARF0 levels may be too low to detect.

Phytochemistry, 2004 Dec, 65(23), 3119 - 23
Three isocoumarins and a benzofuran from the cultured lichen mycobionts of Pyrenula sp; Takenaka Y et al.; The spore-derived mycobionts of the lichen Pyrenula sp . were cultivated on a malt-yeast extract medium supplemented with 10% sucrose . The investigation of their metabolites resulted in isolation of four compounds, three isocoumarins and a biogenetically related benzofuran; their structures were determined by spectroscopic methods.

Exp Cell Res, 2005 Jan 1, 302(1), 61 - 8
Association of PAP-1 and Prp3p, the products of causative genes of dominant retinitis pigmentosa, in the tri-snRNP complex; Maita H et al.; PAP-1 has been identified by us as a Pim-1-binding protein and has recently been implicated as the defective gene in RP9, one type of autosomal dominant retinitis pigmentosa (adRP) . We have then shown that PAP-1 plays a role in pre-mRNA splicing . Because four causative genes for adRP, including PAP-1, Prp31, Prp8, and Prp3, encode proteins that function as splicing factors or splicing-modulating factors, we investigated the interaction of PAP-1 with Prp3p and Prp31p in this study . The results showed that PAP-1 interacted with Prp3p but not Prp31p in human cells and yeast, and that the basic region of PAP-1 and the C-terminal region of Prp3p, regions beside spots found in adRP mutations, were needed for binding . Furthermore, both Prp3p and a part of PAP-1 were found to be components of the U4/U6.U5-tri-snRNP complex, one form of the spliceosome, in Ba/F3 and K562 cells by analysis of sucrose density gradients, suggesting that PAP-1 is weakly associated with the spliceosome . These results also suggest that splicing factors implicated in adRP contribute alone or mutually to proper splicing in the retina and that loss of their functions leads to onset of adRP.

Biochem Biophys Res Commun, 2004 Dec 17, 325(3), 798 - 802
Identification and characterization of a novel gene EOLA1 stimulating ECV304 cell proliferation; Liang Z et al.; To study the changes in gene expression in endothelial cells stimulated by lipopolysaccharide (LPS) we performed subtraction hybridization on control human umbilical vein endothelial cells (HUVEC) versus HUVEC stimulated by LPS . A novel cDNA, named endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), was cloned from our differentially expressed EST database of HUVEC cDNA library (GenBank Accession No . ) . Computational analysis showed that EOLA1 is 1404bp long, encoding a 158aa, 17.8kDa protein, mapped to chromosome Xq27.4 with 5 exons, expressed in different human normal tissues and cancer cell lines . Using the EOLA1 cDNA as bait, we performed a yeast two-hybrid screening of a human liver cDNA library and identified metallothionein 2A (MT2A) as associated protein . Stable transfection of EOLA1 stimulates ECV304 cell proliferation . Our data suggest that the physical interaction of EOLA1 and MT2A may have an important role of cell protection in inflammation reaction.

Biochem Biophys Res Commun, 2004 Dec 17, 325(3), 774 - 83
MAP, a protein interacting with a tumor suppressor, merlin, through the run domain; Lee IK et al.; Merlin (or schwannomin) is a tumor suppressor encoded by the neurofibromatosis type 2 gene . Many studies have suggested that merlin is involved in the regulation of cell growth and proliferation through interactions with various cellular proteins . To better understand the function of merlin, we tried to identify the proteins that bind to merlin using the yeast two-hybrid screening . Characterization of the positive clones revealed a protein of 749 amino acids named merlin-associated protein (MAP), which showed wide tissue distribution in Northern blot analysis . Sequence analysis revealed that MAP is a potential homologue of a yeast check-point protein, BUB2, and contains TBC, SH3, and RUN domains, thereby implicating its role in the Ras-like GTPase signal pathways . MAP and merlin were directly associated in vitro and in vivo, and colocalized in NIH3T3 cells . The RUN domain of MAP and the C-terminus of merlin appeared to be responsible for their interaction . MAP decreased the AP-1-dependent promoter activity additively with merlin in NIH3T3 cells . In addition, merlin and MAP synergistically reduced the colony formation of NIH3T3 cells . These results suggest that MAP may play a cooperative role in the merlin-mediated growth suppression of cells.

Biochem Biophys Res Commun, 2004 Dec 17, 325(3), 751 - 7
The HIV-1 co-receptor CCR5 binds to alpha-catenin, a component of the cellular cytoskeleton; Schweneker M et al.; The chemokine receptors CCR5 and CXCR4 belong to the family of seven transmembrane-spanning G protein-coupled receptors, which have diverse functions in host cell defense and are associated with numerous diseases . CCR5 and CXCR4 are known as co-receptors for entry of HIV-1 . In this study the intracellular carboxy-terminus of CCR5, which is deleted in HIV-infected long-term non-progressors, was shown to interact with the carboxy-terminus of alpha-catenin, a component of the cytoskeleton, in a yeast two-hybrid screen . This interaction was verified in mammalian cells . Furthermore, the interaction of alpha-catenin with CCR5 and CXCR4 at endogenous protein levels was demonstrated in PM1 T-lymphocytes, a host cell line of HIV-1 . Our results suggest that alpha-catenin links CCR5 and CXCR4 to the cytoskeleton and is involved in the organization of these receptors at the membrane, thereby possibly affecting HIV-1 infection.

Biochem J . 2004 Nov 12; {Epub ahead of print}
Lung Kruppel-like factor (LKLF) is a transcriptional activator of the cytosolic phospholipase A 2 alpha promoter; Wick MJ et al.; Elevated expression of cytosolic phospholipase A 2 (cPLA 2) has been shown to the tumorigenesis of non-small cell lung cancer (NSCLC) . Our laboratory has previously demonstrated that oncogenic forms of Ras increase transcription of cPLA 2 in normal lung epithelial cells and NSCLC lines through activation of the ERK and JNK MAP kinase family . We have also defined a minimal region of the cPLA 2 promoter that is critical for this induction . To identify potential transcription factors which bind to this region and regulate expression, a yeast one-hybrid screen was performed with a rat lung cDNA library . Multiple members of the Kruppel family were identified, with lung Kruppel-like factor (LKLF) being isolated multiple times . Overexpression of LKLF in lung epithelial cells or drosophila SL-2 cells increased cPLA 2 promoter activity . Conversely, expression of a dominant negative form of LKLF inhibited induction of cPLA 2 promoter activity by oncogenic Ras in normal lung epithelial cells and NSCLC . By EMSA analysis, LKLF bound to a GC rich region of the cPLA 2 promoter located between -37 and -30 upstream from the transcription start site . Expression of siRNA directed against LKLF inhibited basal expression of cPLA 2 in lung epithelial cells and blocked induction by H-Ras . In NSCLC, siRNA against LKLF cooperated with siRNA against Sp1 to inhibit cPLA 2 promoter activity . Finally, recombinant LKLF was a substrate for ERKs . These data indicate that LKLF is an important regulator of cPLA 2 expression and participates in the induction of this protein which is critical for increased eicosanoid production associated with lung tumorigenesis.

Biotechnol Bioeng, 2005 Jan 5, 89(1), 53 - 77
Inferring gene regulatory relationships by combining target-target pattern recognition and regulator-specific motif examination; Wei H et al.; Although microarray data have been successfully used for gene clustering and classification, the use of time series microarray data for constructing gene regulatory networks remains a particularly difficult task . The challenge lies in reliably inferring regulatory relationships from datasets that normally possess a large number of genes and a limited number of time points . In addition to the numerical challenge, the enormous complexity and dynamic properties of gene expression regulation also impede the progress of inferring gene regulatory relationships . Based on the accepted model of the relationship between regulator and target genes, we developed a new approach for inferring gene regulatory relationships by combining target-target pattern recognition and examination of regulator-specific binding sites in the promoter regions of putative target genes . Pattern recognition was accomplished in two steps: A first algorithm was used to search for the genes that share expression profile similarities with known target genes (KTGs) of each investigated regulator . The selected genes were further filtered by examining for the presence of regulator-specific binding sites in their promoter regions . As we implemented our approach to 18 yeast regulator genes and their known target genes, we discovered 267 new regulatory relationships, among which 15% are rediscovered, experimentally validated ones . Of the discovered target genes, 36.1% have the same or similar functions to a KTG of the regulator . An even larger number of inferred genes fall in the biological context and regulatory scope of their regulators . Since the regulatory relationships are inferred from pattern recognition between target-target genes, the method we present is especially suitable for inferring gene regulatory relationships in which there is a time delay between the expression of regulating and target genes . (c) 2004 Wiley Periodicals, Inc.

Cell Cycle . 2004 Dec 7;3(12) {Epub ahead of print}
Altered Sumoylation of p63a Contributes to the Split-Hand/Foot Malformation Phenotype; Huang YP et al.; p63 mutations have been identified in several developmental abnormalities, including split-hand/foot malformation (SHFM) . In this study, we demonstrate that the C-terminal domain of p63a associates with the E2 ubiquitin conjugating enzyme, Ubc9 . A p63alpha mutation, Q634X, which naturally occurs in SHFM modulated the interaction of p63alpha with Ubc9 in yeast genetic assay . Furthermore, Ubc9 catalyzed the conjugation of p63a with small ubiquitin modifier-1 (SUMO-1), which covalently modified p63a in vitro and in vivo at two positions (K549E and K637E), each situated in a SUMO-1 modification consensus site (phiKXD/E) . In addition, p63alpha mutations (K549E and K637E) abolished sumoylation of p63alpha, dramatically activated transactivation properties of TAp63alpha, and inhibited the dominant-negative effect of DeltaNp63alpha . These p63alpha mutations also affected the transcriptional regulation of gene targets involved in bone and tooth development (e.g., RUNX2 and MINT) and therefore might contribute to the molecular mechanisms underlying the SHFM phenotype.

Arterioscler Thromb Vasc Biol, 2005 Jan, 25(1), 57 - 64 Epub 2004 Nov 11.
Regulation of transport of the angiotensin AT2 receptor by a novel membrane-associated Golgi protein; Wruck CJ et al.; OBJECTIVE: Synthesis and maturation of G protein-coupled receptors are complex events that require an intricate combination of processes including protein folding, posttranslational modifications, and transport through distinct cellular compartments . Little is known concerning the regulation of G protein-coupled receptor transport from the endoplasmic reticulum to the cell surface . METHODS AND RESULTS: Here we show that the cytoplasmatic carboxy-terminal of the angiotensin AT2 receptor (AT2R) acts independently as an endoplasmic reticulum-export signal . Using a yeast two-hybrid system, we identified a Golgi membrane-associated protein termed ATBP50 (for AT2R binding protein of 50 kDa) that binds to this motif . We also cloned ATBP60 and ATBP135 encoded by the same gene as ATBP50 that mapped to chromosomes 8p21.3 . Downregulation of ATBP50 using siRNA leads to retention of AT2R in inner compartments, reduced cell surface expression, and decreased antiproliferative effects of the receptor . CONCLUSIONS: Our results indicate that ATBP50 regulates the transport of the AT2R to cell membrane by binding to a specific motif within its cytoplasmic carboxy-terminal and thereby enabling the antiproliferative effects of the receptor.

Development, 2004 Dec, 131(23), 5897 - 907
Par-1 regulates bicoid mRNA localisation by phosphorylating Exuperantia; Riechmann V et al.; The Ser/Thr kinase Par-1 is required for cell polarisation in diverse organisms such as yeast, worms, flies and mammals . During Drosophila oogenesis, Par-1 is required for several polarisation events, including localisation of the anterior determinant bicoid . To elucidate the molecular pathways triggered by Par-1, we have performed a genome-wide, high-throughput screen for Par-1 targets . Among the targets identified in this screen was Exuperantia (Exu), a mediator of bicoid mRNA localisation . We show that Exu is a phosphoprotein whose phosphorylation is dependent on Par-1 in vitro and in vivo . We identify two motifs in Exu that are phosphorylated by Par-1, and show that their mutation abolishes bicoid mRNA localisation during mid-oogenesis . Interestingly, exu mutants in which Exu phosphorylation is specifically affected can to some extent recover from these bicoid mRNA localisation defects during late oogenesis . These results demonstrate that Par-1 establishes polarity in the oocyte by activating a mediator of bicoid mRNA localisation . Furthermore, our analysis reveals two phases of Exu-dependent bicoid mRNA localisation: an early phase that is strictly dependent on Exu phosphorylation and a late phase that is less phosphorylation dependent.

Plant Cell, 2004 Dec, 16(12), 3242 - 59 Epub 2004 Dec.
FRAGILE FIBER3, an Arabidopsis gene encoding a type II inositol polyphosphate 5-phosphatase, is required for secondary wall synthesis and actin organization in fiber cells; Zhong R et al.; Type II inositol polyphosphate 5-phosphatases (5PTases) in yeast and animals have been known to regulate the level of phosphoinositides and thereby influence various cellular activities, such as vesicle trafficking and actin organization . In plants, little is known about the phosphatases involved in hydrolysis of phosphoinositides, and roles of type II 5PTases in plant cellular functions have not yet been characterized . In this study, we demonstrate that the FRAGILE FIBER3 (FRA3) gene of Arabidopsis thaliana, which encodes a type II 5PTase, plays an essential role in the secondary wall synthesis in fiber cells and xylem vessels . The fra3 mutations caused a dramatic reduction in secondary wall thickness and a concomitant decrease in stem strength . These phenotypes were associated with an alteration in actin organization in fiber cells . Consistent with the defective fiber and vessel phenotypes, the FRA3 gene was found to be highly expressed in fiber cells and vascular tissues in stems . The FRA3 protein is composed of two domains, an N-terminal localized WD-repeat domain and a C-terminal localized 5PTase catalytic domain . In vitro activity assay demonstrated that recombinant FRA3 exhibited phosphatase activity toward PtdIns(4,5)P2, PtdIns(3,4,5)P3, and Ins(1,4,5)P3, with the highest substrate affinity toward PtdIns(4,5)P2 . The fra3 missense mutation, which caused an amino acid substitution in the conserved motif II of the 5PTase catalytic domain, completely abolished the FRA3 phosphatase activity . Moreover, the endogenous levels of PtdIns(4,5)2 and Ins(1,4,5)P3 were found to be elevated in fra3 stems . Together, our findings suggest that the FRA3 type II 5PTase is involved in phosphoinositide metabolism and influences secondary wall synthesis and actin organization.

Bioinformatics . 2004 Nov 11; {Epub ahead of print}
NetAcet: prediction of N-terminal acetylation sites; Kiemer L et al.; SUMMARY: We present here a neural network based method for prediction of amino-terminal acetylation -- by far the most abundant post-translational modification in eukaryotes . The method was developed on a yeast data set for N-acetyltransferase A (NatA) acetylation, which is the type of N-acetylation for which most examples are known and for which orthologs have been found in several eukaryotes . We obtain correlation coefficients close to 0.7 on yeast data and a sensitivity up to 74% on mammalian data, suggesting that the method is valid for other eukaryotic NatA orthologs . AVAILABILITY: The NetAcet prediction method is available as a public web server at SUPPLEMENTARY INFORMATION: http://www.cbs.dtu.dk/services/NetAcet/.

Rev Iberoam Micol, 1997 Dec, 14(4), 155 - 9
African histoplasmosis: a review; Gugnani HC et al.; African histoplasmosis caused by Histoplasma capsulatum var . duboisii is an important deep mycosis endemic in Central and West Africa and in the island of Madagascar . The disease is characterized by presence of granulomatous lesions in the skin, subcutaneous tissues and bones . Lungs and other internal organs are rarely involved . The natural reservoir of the etiological agent has only been recently discovered in a bat cave in Nigeria . The status of asymptomatic infection is not certain . Investigations on skin and serum reactivity have suggested frequent prevalence of asymptomatic infections due to H . capsulatum var . duboisii among the residents in the vicinity of the cave microfocus of the fungus . The exact portal of entry into the body is not known, but inhalation into the lungs and direct inoculation in the skin have been incriminated . Laboratory diagnosis is confirmed by in vitro conversion into large yeast forms (8-15 mum in diameter) and by the demonstration of these forms within giant cells of tissues of experimentally infected animals There are no major clean-cut physiological differences between the two varieties, viz . capsulatum and duboisii . The cell wall of H . capsulatum var duboisii contains a glucan with beta 1-4 linkages in addition to a galactomannan shared with H . capsulatum var . capsulatum . Like the var . capsulatum var . duboisii has marked proteinase and collagenase activities in both mycelial and yeast forms, suggesting a possible pathogenic role for these enzymes . Both varieties have a common exoantigen . The yeast form of H . capsulatum var . duboisii contains the antigen found in the serotype 1,4 of var . capsulatum . A monoclonal antibody test has been developed that can recognize some epitopes in H . capsulatum var . capsulatum but not in the var . duboisii . There is need to develop specific serological diagnosis for the disease . Also there should be greater international awareness about African histoplasmosis . Amphotericin B and several antimycotic azoles like ketoconazole, itraconazole and fluconazole have been successfully employed for treatment.

Rev Iberoam Micol, 1997 Dec, 14(4), 147 - 9
{Taxonomy of Malassezia furfur: state of the art.}; Aspiroz MC et al.; Malassezia furfur is a lipophilic yeast considered as a normal component of the human skin flora . Apart from pityriasis versicolor, M . furfur has been linked to several skin diseases such as seborrheic dermatitis, folliculitis or atopic dermatitis . Moreover, these yeasts have been reported as agent of invasive human diseases including pneumonia, catheter-associated sepsis and peritonitis . The existence of morphological, serological, metabolical, biochemical and karyotipical differences has been described among isolates of these yeasts . These observations gave arguments for a possible intraspecific division, and this hypothesis has been confirmed by the existence of six species within the formerly called M . furfur (lipid-dependent Malassezia strains): M . furfur, Malassezia sympodialis, Malassezia globosa, Malassezia obtusa, Malassezia restricta and Malassezia slooffiae.

Anal Chem, 2004 Nov 15, 76(22), 6618 - 27
N-terminal isotope tagging strategy for quantitative proteomics: results-driven analysis of protein abundance changes; Zappacosta F et al.; Comparing the relative abundance of each protein present in two or more complex samples can be accomplished using isotope-coded tags incorporated at the peptide level . Here we describe a chemical labeling strategy for the incorporation of a single isotope label per peptide, which is completely sequence-independent so that it potentially labels every peptide from a protein including those containing posttranslational modifications . It is based on a gentle chemical labeling strategy that specifically labels the N-terminus of all peptides in a digested sample with either a d5- or d0-propionyl group . Lysine side chains are blocked by guanidination prior to N-terminal labeling to prevent the incorporation of multiple labels . In this paper, we describe the optimization of this N-terminal isotopic tagging strategy and validate its use for peptide-based protein abundance measurements with a 10-protein standard mixture . Using a results-driven strategy, which targets proteins for identification based on MALDI TOF-MS analysis of isotopically labeled peptide pairs, we also show that this labeling strategy can detect a small number of differentially expressed proteins in a mixture as complex as a yeast cell lysate . Only peptides that show a difference in relative abundance are targeted for identification by tandem MS . Despite the fact that many peptides are quantitated, only those few showing a difference in abundance are targeted for protein identification . Proteins are identified by either targeted LC-ES MS/MS or MALDI TOF/TOF . Identifications can be accomplished equally well by either technique on the basis of multiple peptides . This increases the confidence level for both identification and quantitation . The merits of ES MS/MS or MALDI MS/MS for protein identification in a results-driven strategy are discussed.

Planta . 2004 Nov 5; {Epub ahead of print}
Isolation and functional characterization of the Ca-DREBLP1 gene encoding a dehydration-responsive element binding-factor-like protein 1 in hot pepper ( Capsicum annuum L . cv . Pukang); Hong JP et al.; Through the use of subtractive hybridization analysis, we have identified 14 partial cDNA clones (pCa-DSRs) that are rapidly induced by dehydration in hot pepper ( Capsicum annuum L.) roots . The predicted proteins encoded by Ca-DSRs are putatively involved in processes as diverse as primary and secondary metabolism, protein degradation, and stress responses, indicating the complexity of cellular responses to water deficit in hot pepper roots . Particularly, we investigated the detailed structural properties and expression profiles of Ca-DSR2 ( Ca-DREBLP1: dehydration-responsive element binding-factor-like protein 1) encoding a protein that contains a single ERF/AP2 DNA-binding domain . Based on the conserved 14th valine and 19th glutamic acid residues in the ERF/AP2 domain, a basic amino acid stretch (PKKPAGRKKFR) near its N-terminal region, and DSAW signature sequence at the end of its ERF/AP2 domain, Ca-DREBLP1 was classified as a member of a DREB1-type subfamily . Gel retardation assays revealed that Ca-DREBLP1 was able to form a specific complex with the DRE/CRT motif, but not with the GCC box . When fused to the GAL4 DNA-binding domain, the Ca-DREBLP1(190-215) mutant could effectively function as a trans-activator in yeast . This suggests that the extreme C-terminal region plays an essential role in transcription activation . In hot pepper plants, Ca-DREBLP1 was rapidly induced by dehydration, high salinity and, to a lesser extent, mechanical wounding, but not by cold stress . Thus, although the structural features of Ca-DREBLP1 resemble those of the DREB1-type proteins of Arabidopsis thaliana and rice plants, its induction patterns are reminiscent of the DREB2-type proteins, indicating that Ca-DREBLP1 is a novel class DREB subfamily in hot pepper.

J Biol Chem, 2005 Jan 14, 280(2), 1696 - 703 Epub 2004 Nov 09.
An Intermediate Form of ADP-F-actin; Bryan KE et al.; With yeast actin, contrary to other actins, filament formation, ATP hydrolysis, and P(i) release are concurrent at low actin concentrations, the condition usually employed to assess actin polymerization . This observation leads to a question concerning the conformation of the filament barbed end that might be recognized by specific actin-binding proteins . To try to detect possible new actin polymer conformations that might be intermediate in the pathway leading to mature F-actin, we monitored the change in intrinsic tryptophan fluorescence of yeast and muscle actins polymerized at pH 6 to accelerate the rate of filament formation . This allowed temporal resolution of the P(i) release process from the slower process of polymerization . With both actins, we detected a biphasic instead of the usual monophasic fluorescence change, a rapid decrease that tracks with filament formation followed by a slower rebound (the second phase) . This second phase postpolymerization conformational change requires P(i) release and occurs nearly coincident with its release . The addition of P(i) causes this second phase response to disappear, and the inclusion of P(i) during polymerization prevents its appearance . At pH 7.5, with higher yeast actin concentrations to accelerate polymerization, a two-phase fluorescence change is also observed . In this case, the second phase change lags substantially behind P(i) release . P(i) release could also be resolved from polymer formation . V159N yeast actin, hypothesized previously as remaining in a postpolymerization ATP-like state, exhibits the same two-phase intrinsic tryptophan fluorescence behavior as wild-type yeast actin . Together, these observations demonstrate the presence of an intermediate filament state between ADP-P(i) and mature ADP-F-actin.

J Biol Chem, 2005 Jan 14, 280(2), 1543 - 51 Epub 2004 Nov 09.
Molecular Basis for a Direct Interaction between the Syk Protein-tyrosine Kinase and Phosphoinositide 3-Kinase; Moon KD et al.; After engagement of the B cell receptor for antigen, the Syk protein-tyrosine kinase becomes phosphorylated on multiple tyrosines, some of which serve as docking sites for downstream effectors with SH2 or other phosphotyrosine binding domains . The most frequently identified binding partner for catalytically active Syk identified in a yeast two-hybrid screen was the p85 regulatory subunit of phosphoinositide 3-kinase . The C-terminal SH2 domain of p85 was sufficient for mediating an interaction with tyrosine-phosphorylated Syk . Interestingly, this domain interacted with Syk at phosphotyrosine 317, a site phosphorylated in trans by the Src family kinase, Lyn, and identified previously as a binding site for c-Cbl . This site interacted preferentially with the p85 C-terminal SH2 domain compared with the c-Cbl tyrosine kinase binding domain . Molecular modeling studies showed a good fit between the p85 SH2 domain and a peptide containing phosphotyrosine 317 . Tyr-317 was found to be essential for Syk to support phagocytosis mediated by FcgammaRIIA receptors expressed in a heterologous system . These studies establish a new type of p85 binding site that can exist on proteins that serve as substrates for Src family kinases and provide a molecular explanation for observations on direct interactions between Syk and phosphoinositide 3-kinase.

J Biol Chem, 2005 Jan 14, 280(2), 1199 - 208 Epub 2004 Nov 08.
A Novel Repressive E2F6 Complex Containing the Polycomb Group Protein, EPC1, That Interacts with EZH2 in a Proliferation-specific Manner; Attwooll C et al.; The transcriptional repressor E2F6 has been identified as a component of two distinct polycomb group protein (PcG)-containing complexes, suggesting a mechanism for the recruitment of repressive complexes to target sequences in DNA . Whereas one complex is involved in the repression of classic E2F target genes in G(0), a role for E2F6 within the cell cycle has yet to be defined . We searched for novel E2F6-binding proteins using a yeast two-hybrid screen and identified the PcG protein, EPC1 . We showed that, both in vitro and in vivo, E2F6, DP1, and EPC1 form a stable core complex with repressive activity . Furthermore, we identified the proliferation-specific PcG, EZH2, as an EPC1-interacting protein . Using affinity purification, we showed that E2F6, DP1, EPC1, EZH2, and Sin3B co-elute, suggesting the identification of a novel E2F6 complex that exists in vivo in both normal and transformed human cell lines . EZH2 is required for cellular proliferation and consistent with this, EZH2 elutes with the E2F6-EPC1 complex only in proliferating cells . Thus we have identified a novel E2F6-PcG complex (E2F6-EPC1) that interacts with EZH2 and may regulate genes required for cell cycle progression.

Genome Biol . 2004;5(11):R92 . Epub 2004 Oct 25.
Sparse graphical Gaussian modeling of the isoprenoid gene network in Arabidopsis thaliana; Wille A et al.; We present a novel graphical Gaussian modeling approach for reverse engineering of genetic regulatory networks with many genes and few observations . When applying our approach to infer a gene network for isoprenoid biosynthesis in Arabidopsis thaliana, we detect modules of closely connected genes and candidate genes for possible cross-talk between the isoprenoid pathways . Genes of downstream pathways also fit well into the network . We evaluate our approach in a simulation study and using the yeast galactose network.

Paediatr Perinat Epidemiol, 2004 Nov, 18(6), 441 - 7
Association of early-onset otitis media in infants and exposure to household mould; Pettigrew MM et al.; Otitis media is one of the most common infections of early childhood . Children who first experience acute otitis media at an early age (before 6 months) are at increased risk for recurrent otitis media . This prospective study investigated exposure to measured levels of airborne household mould and the risk of early otitis media in the first 6 months of life among a cohort of infants at high risk for asthma . Between September 1996 and December 1998, women were invited to participate if they had at least one other child with physician-diagnosed asthma . Mothers were given a standardised questionnaire within 4 months of their infant's birth . Airborne mould samples were also taken at this time, and culturable fungi were categorised into four levels according to the report of the Commission of European Communities: 0 (undetectable), 1-499 colony forming units (CFU)/m(3) (low), 500-999 CFU/m(3) (medium), > or =1000 CFU/m(3) (high) . Infant respiratory symptoms were collected during quarterly telephone interviews at 6, 9 and 12 months of age . Of the 806 children in the study, 27.8% experienced otitis media before six months of age . Household levels of Penicillium and Cladosporium were modestly associated with the number of otitis media episodes (P = 0.056 and 0.081 respectively) . After controlling for potential confounders, Penicillium and Cladosporium were not associated with early otitis media . High levels of 'other' mould (defined as total spore count minus counts for Penicillium, Cladosporium, and yeast) were associated with early otitis media (OR 3.49; 95% CI {1.38, 8.79}) . We also found associations between day-care outside of the home and birth during the summer or fall season with early otitis media . This study is suggestive of a relationship between otitis media and mould that warrants further study.

Curr Opin Clin Nutr Metab Care, 2004 Nov, 7(6), 615 - 22
Energy restriction and aging; Smith JV et al.; PURPOSE OF REVIEW: The focus of this review is on current research involving long-term calorie restriction and the resulting changes observed in possible biomarkers of aging . Special emphasis will be given to the basic and clinical science studies which are currently investigating the effects of controlled, high-quality energy-restricted diets on both biomarkers of longevity and on the development of chronic diseases related to age and obesity in humans . RECENT FINDINGS: Prolonged calorie restriction has been shown to extend both the median and maximal lifespan in a variety of lower species such as yeast, worms, fish, rats, and mice . Mechanisms of this lifespan extension via calorie restriction are not fully elucidated, but possibly involve significant alterations in energy metabolism, oxidative damage, insulin sensitivity, and functional changes in both the neuroendocrine and sympathetic nervous systems . Ongoing studies of prolonged energy restriction in humans are now making it possible to analyze changes in these aging biomarkers to unravel some of the mechanisms of its antiaging phenomenon . SUMMARY: With the incremental expansion of research endeavors in the area of energy or calorie restriction, data on the effects of calorie restriction in animal models and humans are becoming more accessible . Detailed analyses from controlled human trials involving long-term calorie restriction will allow investigators to link observed alterations in body composition down to changes in molecular pathways and gene expression, with their possible effects on the biomarkers of aging.

Arch Ophthalmol, 2004 Nov, 122(11), 1687 - 92
Intravitreal voriconazole: an electroretinographic and histopathologic study; Gao H et al.; BACKGROUND: Voriconazole, a novel triazole antifungal agent, presents potent activity against a broad spectrum of yeast and molds . OBJECTIVE: To determine whether voriconazole could be safely used as an intravitreal agent in the treatment of fungal endophthalmitis . METHODS: Retinal toxicity of voriconazole was examined in a rodent animal model . Voriconazole solutions were serially diluted and injected intravitreally into the eyes of normal adult Sprague-Dawley rats so that the final intravitreal concentrations were 5 microg/mL, 10 microg/mL, 25 microg/mL, 50 microg/mL, and 500 microg/mL (n = 3 for each concentration group) . Saline was injected into the fellow eyes of all animals as controls . Three weeks after injections, electroretinograms were measured, and eyes were subsequently enucleated for histologic examination . RESULTS: In electroretinographic studies, maximum scotopic b-wave, intensity needed for half saturation, and saturated a-wave amplitude were measured . There was no statistically significant difference in these parameters recorded between control eyes and voriconazole-injected eyes in any concentration groups . Histologic examination with light microscopy did not reveal any retinal abnormality in the eyes with 5 to 25 microg/mL of intravitreal voriconazole . In the eyes with 50 microg/mL and 500 microg/mL of voriconazole, small foci of retinal necrosis were occasionally observed in the outer retina, especially in the eyes with 500 microg/mL of voriconazole . CONCLUSIONS: Our results demonstrate that intravitreal voriconazole of up to 25 mg/mL causes no electroretinographic change or histologic abnormality in rat retinas . This indicates that voriconazole is a safe antifungal agent for intravitreal injection in rodents and may be used in the treatment of human fungal endophthalmitis following further study.

Med Hypotheses, 2005, 64(1), 201 - 8
Aging: gene silencing or gene activation?
Burzynski SR.
According to the author's theory of gene silencing, the key process in aging involves reduced expression of a number of genes . Silencing of genes has a complex mechanism, which involves methylation of DNA, histone modification and chromatin remodeling . In addition to deacetylation of the histones and methylation of DNA, recently described RNAi mechanism could initiate formation of silenced chromatin . Hypermethylation of the promoter will silence the gene . Genome-wide hypomethylation will induce genomic instability, amplification of oncogenes and also silencing of the genes through RNAi mechanism . Studies by different groups, conducted in yeast, worms, flies and mice, confirmed substantial changes in gene expression in aging . Among them, the most important was silencing of tumor suppressors and other genes involved in the control of cell cycle, apoptosis, detoxification, and cholesterol metabolism . There was also increased expression of the smaller group of oncogenes and other genes which are associated with typical diseases of old age . Caloric restriction normalizes expression of a substantial percentage of these genes . Animal studies confirmed importance of caloric restriction, which decreases signaling through the IGF-1/AKT pathway and expression of gene p53 . These studies, however, cannot be directly applied to human aging . It is proposed that age management therapy should attempt to normalize gene expression in the older population to the level typical for young adults . This would require activation of silenced genes and normalization of overexpressed genes . Caloric restriction and exercise are helpful in decreasing the activity of important oncogenes and activation of silenced tumor suppressors, and may have a positive impact, not only on aging, but also on prevention of cancer . Dietary supplements containing phytochemicals should normalize increased expression of oncogenes . Examples are: genistein and EGCG, which effect signaling through the IGF-1/AKT pathway and resveratrol and limonen, which do so through the RAS pathway . A group of amino acid derivatives and organic acids of animal and human origin should activate silenced tumor suppressor genes (Aminocare A10, Aminocare Extra) . Among them 3-phenylacetylamino-2, 6-piperidinedione intercalates specifically with DNA and protects sequences of tumor suppressor genes, which are vulnerable to the effects of carcinogens . Phenylacetate activates p53 and p21 through inhibition of methyltransferase and farnesylation of the RAS protein . Phenylbutyrate activates tumor suppressor genes through inhibition of histone deacetylation . Phenylacetylglutamine decreases genomic instability and expression of oncogenes and promotes apoptosis . The application of DNA microarray techniques to human studies should provide more information about differences in gene expression in different age groups and help design more effective age management regimens.

J Biol Chem, 2005 Jan 14, 280(2), 1512 - 20 Epub 2004 Nov 05.
Structure of the Ubiquitin Hydrolase UCH-L3 Complexed with a Suicide Substrate; Misaghi S et al.; Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function . Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role . The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates . We report the 1.45 A resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases . This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state . We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size . We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.

J Biol Chem, 2005 Jan 21, 280(3), 1911 - 20 Epub 2004 Nov 05.
The pathway for the production of inositol hexakisphosphate in human cells; Verbsky JW et al.; The yeast and Drosophila pathways leading to the production of inositol hexakisphosphate (InsP(6)) have been elucidated recently . The in vivo pathway in humans has been assumed to be similar . Here we show that overexpression of Ins(1,3,4)P(3) 5/6-kinase in human cell lines results in an increase of inositol tetrakisphosphate (InsP(4)) isomers, inositol pentakisphosphate (InsP(5)) and InsP(6), whereas its depletion by RNA interference decreases the amounts of these inositol phosphates . Expression of Ins(1,3,4,6)P(4) 5-kinase does not increase the amount of InsP(5) and InsP(6), although its depletion does block InsP(5) and InsP(6) production, showing that it is necessary for production of InsP(5) and InsP(6) . Expression of Ins(1,3,4,5,6)P(5) 2-kinase increases the amount of InsP(6) by depleting the InsP(5) in the cell, and depletion of 2-kinase decreases the amount of InsP(6) and causes an increase in InsP(5) . These results are consistent with a pathway that produces InsP(6) through the sequential action of Ins(1,3,4)P(3) 5/6-kinase, Ins(1,3,4,6)P(4) 5-kinase, and Ins(1,3,4,5,6)P5 2-kinase to convert Ins(1,3,4)P(3) to InsP(6) . Furthermore, the evidence implicates 5/6-kinase as the rate-limiting enzyme in this pathway.

Fungal Genet Biol, 2004 Dec, 41(12), 1132 - 40
The fungal STRE-element-binding protein Seb1 is involved but not essential for glycerol dehydrogenase (gld1) gene expression and glycerol accumulation in Trichoderma atroviride during osmotic stress; Seidl V et al.; Fungi counteract extracellular osmotic pressure by producing intracellular polyols to prevent loss of water . In yeast osmotic signaling involves a MAP-kinase pathway culminating at the STRE-binding transcription factors Msn2/4 . We investigated the role of a putative STRE-binding orthologue of Trichoderma atroviride, Seb1, in osmotic stress signaling . T . atroviride, subjected to osmotic stress (10% glucose or glycerol, 1M KCl or NaCl), responds by raising its intracellular glycerol level . In contrast to Aspergillus nidulans, no erythritol is accumulated . Accumulation of glycerol levels under osmotic stress is strongly reduced in a seb1 deletion strain . To investigate glycerol biosynthesis in T . atroviride, the genes encoding glycerol dehydrogenase (gld1) and glycerol-3-phosphate dehydrogenase (gfd1) were cloned and characterized . Although both genes contain STRE-elements in their 5'-non-coding regions, only gld1 mRNA accumulates in response to osmotic stress, whereas expression of gfd1 remains at a constitutive level . In comparison to A . nidulans gld1 transcript levels in T . atroviride rise very slowly under conditions of salt stress . Deletion of seb1 results in a delayed accumulation of the gld1 transcript, but final levels match those in the wild-type whereas gfd1 transcript accumulation remains unaffected . Assays for glycerol dehydrogenase and glycerol-3-phosphate dehydrogenase enzymatic activities reveal an increase of the former--whereas the latter remains mainly unaffected--in the wild-type and the Deltaseb1 strain under different kinds of osmotic stress . The data suggest that Seb1 is only involved in, but not essential for osmotic stress response which is in contrast to the yeast orthologues Msn2/4.

Curr Opin Genet Dev, 2004 Dec, 14(6), 680 - 5
Eukaryotic gene regulation by targeted chromatin re-modeling at dispersed, middle-repetitive sequence elements; Hodgetts R; RNA interference might have evolved to minimize the deleterious impact of transposable elements and viruses on eukaryotic genomes, because mutations in genes within the RNAi pathway cause mobilization of transposons in nematodes and flies . Although the first examples of RNAi involved post-transcriptional gene silencing, recently the pathway has been shown to act at the transcriptional level . It does so by establishing a chromatin configuration on the target DNA that has many of the hallmarks of heterochromatin, thus preventing its transcription . Members of dispersed, repeated sequence families appear to have been utilized by the RNAi machinery to regulate nearby genes in yeast . The unusual genomic distribution of three repeated element families in the chicken, fruit-fly and nematode genomes prompts speculation that some of these repeats have been co-opted to control gene expression, either locally or over extended chromosomal domains.

Curr Opin Cell Biol, 2004 Dec, 16(6), 614 - 22
New tricks for old dogs: unexpected roles for cell cycle regulators revealed using animal models; Humbert PO et al.; Studies in animal models have revealed many surprises regarding the importance of key cell cycle regulators during animal development and homeostasis, underscoring the plasticity and redundancy of cell cycle circuitry within a whole-animal context . Moreover, checkpoint regulators, which are not essential for viability in yeast and cultured cells, play important roles in cell cycle control during development.

Biochem Biophys Res Commun, 2004 Dec 10, 325(2), 626 - 31
ATFC is a novel transducer for the unfolded protein response in Bombyx mori BM5 cells; Goo TW et al.; We report the isolation of activating transcription factor of chaperone (ATFC), a novel cDNA from Bombyx mori BM5 cells that encodes a putative transducer of endoplasmic reticulum (ER) stress . The 236 amino acids of ATFC include both a basic region and a leucine zipper at the C-terminus, in contrast to Hac1p of yeast which features these structures at its N-terminus . ATFC expression was strongly up-regulated by ER stress . ATFC could specifically bind to the unfolded protein response element . BM5 cells transfected with ATFC cDNA displayed enhanced ER chaperone expression in response to ER stress . These results indicate that ATFC encodes a putative transducer of ER stress.

Curr Biol, 2004 Nov 9, 14(21), 1962 - 7
Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase; Pearson CG et al.; With a single microtubule attachment, budding-yeast kinetochores provide an excellent system for understanding the coordinated linkage to dynamic microtubule plus ends for chromosome oscillation and positioning . Fluorescent tagging of kinetochore proteins indicates that, on average, all centromeres are clustered, distinctly separated from their sisters, and positioned equidistant from their respective spindle poles during metaphase . However, individual fluorescent chromosome markers near the centromere transiently reassociate with their sisters and oscillate from one spindle half to the other . To reconcile the apparent disparity between the average centromere position and individual centromere proximal markers, we utilized fluorescence recovery after photobleaching to measure stability of the histone-H3 variant Cse4p/CENP-A . Newly synthesized Cse4p replaces old protein during DNA replication . Once assembled, Cse4-GFP is a physically stable component of centromeres during mitosis . This allowed us to follow centromere dynamics within each spindle half . Kinetochores remain stably attached to dynamic microtubules and exhibit a low incidence of switching orientation or position between the spindle halves . Switching of sister chromatid attachment may be contemporaneous with Cse4p exchange and early kinetochore assembly during S phase; this would promote mixing of chromosome attachment to each spindle pole . Once biorientation is attained, centromeres rarely make excursions beyond their proximal half spindle.

Curr Biol, 2004 Nov 9, 14(21), 1957 - 61
The Drosophila microtubule-associated protein mini spindles is required for cytoplasmic microtubules in oogenesis; Moon W et al.; The XMAP215/TOG family of proteins is a closely related set of MAPs (microtubule-associated proteins) found in animals, yeast, and plants . In yeast and animal cells, the XMAP215/TOG proteins are required for both mitosis and meiosis . Although effects of XMAP215/TOG proteins on cytoplasmic microtubules have not previously been shown in animal cells, in plants the Arabidopsis family member MOR1 is required for the organization of cortical microtubule arrays . The Drosophila family member, encoded by the mini spindles (msps) gene, is maternally expressed and loaded into the egg, where it is an essential component of meiotic and mitotic spindles . Here we show that msps is also required during oogenesis for the structure and function of cytoplasmic microtubules . Localization of bicoid (bcd) mRNA in the oocyte is a microtubule-mediated event . We show that bcd RNA localization is defective in msps mutants . We also identify defects in cytoplasmic microtubules in both the germ and follicle cells of mutant ovaries and determine the expression pattern of msps mRNA and protein in developing egg chambers . Our findings reveal a new role for msps in cell patterning and raise the possibility that other family members may perform similar functions.

Curr Biol, 2004 Nov 9, 14(21), R915 - 7
DNA replication: stalling a fork for imprinting and switching; Egel R; Mating-type switching in fission yeast has long been known to be directed by a DNA 'imprint' . This imprint has now been firmly characterized as a protected site-specific and strand-specific nick . New work also links the widely conserved Swi1-Swi3 complex to the protection of stalled replication forks in general.

J Clin Microbiol, 2004 Nov, 42(11), 5070 - 5
Role of Cannomys badius as a natural animal host of Penicillium marneffei in India; Gugnani H et al.; Infection by Penicillium marneffei in human immunodeficiency virus-positive patients in India has recently been described; the aim of our study was to survey wild rodents and their associated environment in order to identify the natural populations of this fungus . Surveys recovered P . marneffei from the internal organs of 10 (9.1%) of 110 bamboo rats (Cannomys badius) examined from Manipur state, India, an area endemic for penicilliosis marneffei . Identification of the isolates was based on a detailed study of their morphological characteristics, in vitro conversion to fission yeast form, and exoantigen tests . Multilocus microsatellite typing (MLMT) of the isolates revealed five genotypes . No genotypes were shared between sample sites, and all bamboo rats were infected with a single genotype within sample sites, demonstrating spatial genetic heterogeneity . One MLMT genotype was identical to that seen in a human isolate, suggesting that either coinfection from a common source or host-to-host transmission had occurred . This demonstrates the utility of an MLMT-based approach to elucidating the epidemiology of P . marneffei.

Circ Res, 2004 Nov 26, 95(11), 1067 - 74 Epub 2004 Nov 26.
Fibulin-5 is a novel binding protein for extracellular superoxide dismutase; Nguyen AD et al.; The extracellular superoxide dismutase (ecSOD) plays an important role in atherosclerosis and endothelial function by modulating levels of the superoxide anion (O2*-) in the extracellular space . Although heparan sulfate proteoglycan is an important ligand for ecSOD, little is known about other biological binding partners of ecSOD . The goal of this study was to identify novel proteins that interact with ecSOD . A yeast two-hybrid screening of a human aorta cDNA library using ecSOD as bait identified fibulin-5 as a predominant binding protein for ecSOD . Further analysis showed that the binding domain of ecSOD within fibulin-5 mapped to its C-terminal domain . In vitro pulldown assays and coimmunoprecipitation analysis further confirmed that ecSOD interacts with fibulin-5 in vitro and in vivo . Studies using fibulin-5-/- mice indicated that fibulin-5 is required for binding of ecSOD to vascular tissue . Importantly, the decrease in tissue-bound ecSOD levels in aortas from fibulin-5-/- mice was associated with an increase in vascular O2*- levels . Furthermore, immunohistochemical analysis using ApoE-/- mice suggested a codistribution of ecSOD and fibulin-5 in atherosclerotic vessels . In summary, we provide in this study the first evidence that the ecSOD-fibulin-5 interaction is required for ecSOD binding to vascular tissues, thereby regulating vascular O2*- levels . This interaction may represent a novel mechanism for controlling vascular redox state in the extracellular space in various cardiovascular diseases such as atherosclerosis and hypertension in which oxidative stress is increased.

Science, 2004 Nov 5, 306(5698), 990 - 5
Autophagy in health and disease: a double-edged sword; Shintani T et al.; Autophagy, the process by which cells recycle cytoplasm and dispose of excess or defective organelles, has entered the research spotlight largely owing to the discovery of the protein components that drive this process . Identifying the autophagy genes in yeast and finding orthologs in other organisms reveals the conservation of the mechanism of autophagy in eukaryotes and allows the use of molecular genetics and biology in different model systems to study this process . By mostly morphological studies, autophagy has been linked to disease processes . Whether autophagy protects from or causes disease is unclear . Here, we summarize current knowledge about the role of autophagy in disease and health.

J Biol Chem, 2005 Jan 14, 280(2), 1156 - 64 Epub 2004 Nov 04.
A Role for Rat Inositol Polyphosphate Kinases rIPK2 and rIPK1 in Inositol Pentakisphosphate and Inositol Hexakisphosphate Production in Rat-1 Cells; Fujii M et al.; Over 30 inositol polyphosphates are known to exist in mammalian cells; however, the majority of them have uncharacterized functions . In this study we investigated the molecular basis of synthesis of highly phosphorylated inositol polyphosphates (such as inositol tetrakisphosphate, inositol pentakisphosphate (IP(5)), and inositol hexakisphosphate (IP(6))) in rat cells . We report that heterologous expression of rat inositol polyphosphate kinases rIPK2, a dual specificity inositol trisphosphate/inositol tetrakisphosphate kinase, and rIPK1, an IP(5) 2-kinase, were sufficient to recapitulate IP(6) synthesis from inositol 1,4,5-trisphosphate in mutant yeast cells . Overexpression of rIPK2 in Rat-1 cells increased inositol 1,3,4,5,6-pentakisphosphate (I(1,3,4,5,6)P(5)) levels about 2-3-fold compared with control . Likewise in Rat-1 cells, overexpression of rIPK1 was capable of completely converting I(1,3,4,5,6)P(5) to IP(6) . Simultaneous overexpression of both rIPK2 and rIPK1 in Rat-1 cells increased both IP(5) and IP(6) levels . To reduce IPK2 activity in Rat-1 cells, we introduced vector-based short interference RNA against rIPK2 . Cells harboring the short interference RNA had a 90% reduction of mRNA levels and a 75% decrease of I(1,3,4,5,6)P(5) . These data confirm the involvement of IPK2 and IPK1 in the conversion of inositol 1,4,5-trisphosphate to IP(6) in rat cells . Furthermore these data suggest that rIPK2 and rIPK1 act as key determining steps in production of IP(5) and IP(6), respectively . The ability to modulate the intracellular inositol polyphosphate levels by altering IPK2 and IPK1 expression in rat cells will provide powerful tools to study the roles of I(1,3,4,5,6)P(5) and IP(6) in cell signaling.

J Biol Chem, 2004 Dec 31, 279(53), 55411 - 8 Epub 2004 Nov 03.
Rea1, a dynein-related nuclear AAA-ATPase, is involved in late rRNA processing and nuclear export of 60 S subunits; Galani K et al.; Rea1, the largest predicted protein in the yeast genome, is a member of the AAA(+) family of ATPases and is associated with pre-60 S ribosomes . Here we report that Rea1 is required for maturation and nuclear export of the pre-60 S subunit . Rea1 exhibits a predominantly nucleoplasmic localization and is present in a late pre-60 S particle together with members of the Rix1 complex . To study the role of Rea1 in ribosome biogenesis, we generated a repressible GAL::REA1 strain and temperature-sensitive rea1 alleles . In vivo depletion of Rea1 results in the significant reduction of mature 60 S subunits concomitant with defects in pre-rRNA processing and late pre-60 S ribosome stability following ITS2 cleavage and prior to the generation of mature 5.8 S rRNA . Strains depleted of the components of the Rix1 complex (Rix1, Ipi1, and Ipi3) showed similar defects . Using an in vivo 60 S subunit export assay, a strong accumulation of the large subunit reporter Rpl25-GFP (green fluorescent protein) in the nucleus and at the nuclear periphery was seen in rea1 mutants at restrictive conditions.

Gene, 2004 Nov 24, 342(2), 231 - 41
TBP-associated factors in Arabidopsis; Lago C et al.; Initiation of transcription mediated by RNA polymerase II requires a number of transcription factors among which TFIID is the major core promoter recognition factor . TFIID is composed of highly conserved factors which include the TATA-binding protein (TBP) and about 14 TBP-associated factors (TAFs) . Since TAFs play important roles in transcription they have been extensively studied in organisms like yeast, Drosophila and human . Surprisingly, TAFs have been poorly characterized in plants . With the completion of the Arabidopsis genome sequence, it is possible to search for TAFs, since many of them have conserved amino acid sequences . Mining the genome of Arabidopsis for TAFs resulted in the identification of 18 putative Arabidopsis TAFs (AtTAFs) . We have analyzed their protein structure and their genomic localisation . Expression profiling by RT-PCR showed that these TAFs are expressed in all parts of the plant which is in agreement with their general role in transcription . These analyses in combination with their evolutionary conservation with TAFs of other organisms are discussed.

Gene, 2004 Nov 10, 342(1), 57 - 66
Characterization of Oryza sativa telomerase reverse transcriptase and possible role of its phosphorylation in the control of telomerase activity; Oguchi K et al.; Telomerase reverse transcriptase (TERT) has been characterized in the dicotyledon Arabidopsis thaliana . A TERT homolog has now been identified in the monocotyledon rice (Oryza sativa L.) on the basis of its predicted homology to the A . thaliana enzyme (AtTERT) . At least five alternatively spliced transcripts of the rice TERT (OsTERT) gene were detected . The full-length OsTERT protein shares structural features with TERTs of other species, including a calculated molecular size of 144 kDa, an isoelectric point of 9.6, and conserved sequence motifs . Phylogenetic analysis showed that OsTERT clusters with AtTERT and is more related to the human and mouse enzymes than to those of yeast and ciliated protozoa, consistent with the evolutionary relations among these eukaryotes . Telomerase activity was abundant in shoot apices and cultured cells but was low or absent in leaves or roots of rice plants, whereas similarly spliced OsTERT transcripts were detected in all tissues examined and cultured cells . Similar to mouse and human TERT proteins, OsTERT contains two putative phosphorylation sites for Akt kinase . Incubation of a rice cell extract with Akt or with protein phosphatase 2A potentiated or inhibited telomerase activity, respectively, whereas Akt did not affect the activity in Arabidopsis cell extract . In addition, the kinase activated the telomerase in a leaf extract . The mechanism of telomerase regulation in rice thus appears to differ from that in Arabidopsis (which is mediated predominantly at the level of AtTERT transcription), possibly reflecting the taxonomic distance between monocotyledons and dicotyledons.

Gene, 2004 Nov 10, 342(1), 35 - 40
Functional characterization of a human histone gene cluster duplication; Braastad CD et al.; Histones are the major protein component of nucleosomes, and de novo histone synthesis is essential for packaging newly replicated DNA into chromatin . As a result, histone gene expression is exquisitely and functionally coupled with DNA replication . Vastly divergent organisms such as yeast, fly and human all demonstrate the phylogenetically conserved propensity to maintain clustering of histone genes at one or more genomic loci . Although specific mechanisms are unclear, clustering is presumed to be important for common stringent transcriptional control of these genes at the G1/S phase transition . In this study, we describe a genomic duplication of the human histone gene cluster located at chromosome 1q21, which effectively doubles the previously known size and gene number of that cluster . The duplication persists in all examined tissues and cell lines, and the duplicated genes are transcriptionally active . Levels of messenger RNAs for duplicated histone H4 genes are high relative to those for non-duplicated H4 genes . Our data suggest that transcriptionally robust histone H4 genes may have been preferentially duplicated during evolution.

Gene, 2004 Nov 10, 342(1), 1 - 11
Drosophila, an emerging model for cardiac disease; Bier E et al.; A variety of studies that are currently underway may validate the fruit fly as an in vivo model for analyzing genes involved in cardiac function . Many mutations in conserved genetic pathways have been found, including those controlling development and physiology . Because homologous genes control early developmental events as well as functional components of the Drosophila and vertebrate hearts, the fly is the simplest existing model system that can be used to assay genes involved in human congenital heart disease (CHD) . The wide variety of genetic tools available to Drosophila researchers offers many technical advantages for rapidly screening through large numbers of candidate genes . Thus, an important future and long-term direction is likely to be the use of Drosophila as a vehicle for analyzing polygenic traits as an aid in human genetics . One can anticipate a time in the not too distant future when mutant lines exist for every gene in vertebrate systems, such as mice and zebrafish . However, one of the enduring problems that will not easily be addressed by such resources will be the tracking of complex traits defined by polygenic variants . For this level of genetic analysis, simple genetic model systems including yeast, Caenorhabditis elegans, and Drosophila melanogaster will undoubtedly play a crucial ongoing role . Of them, Drosophila will be critical for examining gene networks involved in organogenesis and is clearly the system of choice for studying cardiac development, function and aging, since among the simple genetic models it is the only one with a fluid pumping heart.

Virology, 2004 Dec 5, 330(1), 284 - 94
SUMO-1 modification regulates the protein stability of the large regulatory protein Rep78 of adeno associated virus type 2 (AAV-2); Weger S et al.; The large Rep proteins Rep78 and Rep68 of the helper-dependent adeno associated virus type 2 (AAV-2) are essential for both site-specific integration of AAV DNA in the absence of helpervirus and productive AAV replication in the presence of helpervirus . We have identified UBC9, the E2 conjugating enzyme for the small ubiquitin-related polypeptide SUMO-1, as binding partner of the large Rep proteins in yeast two-hybrid analysis and in GST pulldown assays . Modification of the large Rep proteins with SUMO-1 could be demonstrated in immunoblot analysis and in immunoprecipitations, with the lysine residue at amino acid position 84 serving as the major attachment site . The largely sumolation-deficient Rep78 lysine to arginine point mutant showed a strongly reduced half-life as compared to the wild-type protein . This finding implicates a role for sumolation in the regulation of Rep78 protein stability that is assumed to be critical for the establishment and maintenance of AAV latency.

FEBS Lett, 2004 Nov 5, 577(1-2), 134 - 40
Cooperative binding of the hnRNP K three KH domains to mRNA targets; Paziewska A et al.; The heterogeneous nuclear ribonucleoprotein (hnRNP) K homology (KH) domain is an evolutionarily conserved module that binds short ribonucleotide sequences . KH domains most often are present in multiple copies per protein . In vitro studies of hnRNP K and other KH domain bearing proteins have yielded conflicting results regarding the relative contribution of each KH domain to the binding of target RNAs . To assess this RNA-binding we used full-length hnRNP K, its fragments and the yeast ortholog as baits in the yeast three-hybrid system . The results demonstrate that in this heterologous in vivo system, the three KH domains bind RNA synergistically and that a single KH domain, in comparison, binds RNA weakly.

FEBS Lett, 2004 Nov 5, 577(1-2), 9 - 16
Evidence for copper homeostasis function of metallothionein (MT3) in the hyperaccumulator Thlaspi caerulescens; Roosens NH et al.; Metallothioneins chelate metals and consequently may be a control point of metal homeostasis . Homologous to type 3 metallothioneins, TcMT3 cDNA was identified in the Cd/Zn hyperaccumulator, Thlaspi caerulescens . TcMT3 amino acid sequence showed modifications in the Cys positions when compared with its Arabidopsis orthologue . A structural model established that the MT3 carboxyterminal domain is similar to the beta domain of animal metallothioneins and predicts a smaller cavity to chelate metals for A . thaliana than for T . caerulescens . Functional testing in yeast and Northern blot analysis added further evidence for adaptative variations of MT3 for the maintenance of Cu homeostasis in a metal hyperaccumulator.

J Fam Pract, 2004 Nov, 53(11), 890 - 4
Abnormal vaginal discharge: what does and does not work in treating underlying causes; French L et al.; Antifungal medications for intravaginal use have been available in the United States for more than a decade . Women may be inclined to self-diagnose yeast infections with any vaginal discharge or other vulvovaginal symptoms that they deem abnormal . As we saw in the first part of this article, "Abnormal vaginal discharge: Using office diagnostic testing more effectively" (J Fam Pract 2004; 53{10}:805-814), abnormal discharge is more likely to be bacterial vaginosis or no pathogen at all . Potential delay in diagnosis and treatment of a sexually transmitted disease is also a concern . Increasing resistance of Candida sp . to imidazoles is associated with indiscriminate use of over-the-counter products.

Biochemistry (Mosc), 2004 Oct, 69(10), 1158 - 64
Expression and characterization of hepatitis B surface antigen in transgenic potato plants; Shulga NY et al.; Transgenic potato plants expressing the gene of hepatitis B surface antigen (HBsAg) under the control of the double promoter of 35S RNA of cauliflower mosaic virus (CaMV 35SS) and the promoter of patatin gene of potato tubers have been obtained . Biochemical analysis of the plants was performed . The amount of HBsAg in leaves, microtubers, and tubers of transgenic potatoes growing in vitro and in vivo was 0.005-0.035% of the total soluble protein . HBsAg content reached 1 microg/g in potato tubers and was maximal in plants expressing the HBsAg gene under the control of CaMV 35SS promoter . In transgenic plants expressing HBsAg gene under the control of tuber-specific patatin promoter, HBsAg was found only in microtubers and tubers and was absent in leaves . Western blot analysis of HBsAg eluted from immunoaffinity protein A-Sepharose matrix has been performed . The molecular weight of HBsAg peptide was approximately 24 kD, which is in agreement with the size of the major protein of the envelope of hepatitis B virus . Using gel filtration, it was determined that the product of HBsAg gene expression in potato plants is converted into high-molecular-weight multimeric particles . Therefore, as well as in recombinant HBsAg-yeast cells, assembling of HBsAg monomers into immunogenic aggregates takes place in HBsAg-transgenic potato, which can be used as a source of recombinant vaccine against hepatitis B virus.

J Pediatr Endocrinol Metab, 2004 Oct, 17(10), 1429 - 34
Efficacy and safety of Valtropin in the treatment of short stature in girls with Turner's syndrome; Peterkova V et al.; Valtropin (somatropin, BioPartners and LG Life Sciences {LGLS}) is a recombinant human growth hormone (GH) preparation produced using a yeast expression system . An open single-arm phase III study was conducted to evaluate efficacy and safety at a dose of 0.16 IU/kg/day (0.053 mg/kg/day) s.c . for 12 months in the treatment of short stature in girls (n = 30, aged 2-9 years) with Turner's syndrome . The primary efficacy variable was height velocity (HV) at 12 months . Secondary efficacy variables included serum GH dependent growth factors . HV increased from 3.8 +/- 1.8 cm/yr at baseline to 9.7 +/- 1.6 cm/yr (mean +/- SD) after 12 months of treatment . Marked treatment effects were also observed on other growth parameters, serum insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) . Treatment was well tolerated with no significant adverse events . It is concluded that Valtropin is as safe and effective as other human GH preparations for the treatment of growth failure in girls with Turner's syndrome.

World J Gastroenterol, 2004 Dec 1, 10(23), 3531 - 3
Effects of selenium on peripheral blood mononuclear cell membrane fluidity, interleukin-2 production and interleukin-2 receptor expression in patients with chronic hepatitis; He SX et al.; AIM: To study the effect of selenium on peripheral blood mononuclear cell (PBMC) membrane fluidity and immune function in patients with chronic hepatitis . METHODS: PBMCs were pretreated with selenium (1.156x10(-7) mol/L) for 6 h in vitro or extracted directly from patients after administration of selenium-yeast continuously for 8-12 wk (200 microg/d), and then exposed to Con-A for 48 h . The membrane fluidity, interleukin-2 (IL-2) production and interleukin-2 receptor (IL-2R) expression in PBMCs and malondialdehyde (MDA) concentration in medium and lipid peroxide (LPO) in plasma were determined . RESULTS: The PBMC membrane fluidity, IL-2 production and IL-2R expression in patients with chronic hepatitis were significantly lower than those in healthy blood donators (particle adhesive degree R, 0.17+/-0.01 vs 0.14+/-0.01, P<0.01; IL-2, 40.26+/-9.55 vs 72.96+/-11.36, P<0.01; IL-2R, 31.05+/-5.09 vs 60.58+/-10.56, P<0.01), and the MDA concentration in medium in patients with chronic hepatitis was significantly higher than that in healthy blood donators (1.44+/-0.08 vs 0.93+/-0.08, P<0.01) . Both in vitro and in vivo administration of selenium could reverse the above parameters . CONCLUSION: Supplement of selenium can suppress lipid peroxidation, and improve PBMC membrane fluidity and immune function in patients with chronic hepatitis.

Chirality, 2005 Jan, 17(1), 37 - 43
Change in enantioselectivity in bufuralol 1''-hydroxylation by the substitution of phenylalanine-120 by alanine in cytochrome P450 2D6; Masuda K et al.; The functional roles of phenylalanine at position 120 in drug oxidation by cytochrome P450 2D6 (CYP2D6) were examined using a yeast cell expression system and bufuralol (BF) enantiomers as a chiral substrate . Two mutated cDNAs, one encoding a CYP2D6 mutant having alanine instead of Phe-120 (F120A) and another encoding a mutant having alanine instead of Glu-222 (E222A), were prepared by site-directed mutagenesis and transformed into yeast cells via pGYRI vectors . The enantiomeric BF 1''-hydroxylase activities of the mutants were compared with those of the wild type . When enantiomeric BF 1''-hydroxylase activities at a substrate concentration of 100 microM were compared, the CYP2D6 wild type showed substrate enantioselectivity of (R-BF >> S-BF) and the F120A mutant exhibited substrate enantioselectivity of (R-BF < or = S-BF), whereas the product diastereoselectivity of (1''R-OH-BF << 1''-S-OH-BF) was similar between the wild type and the mutant . The activities of the other mutant (E222A) were much lower than those of the wild type and the F120A mutant, while its substrate enantioselectivity and product diastereoselectivity were the same as those of the wild type . The kinetics demonstrated that apparent K(m) values were similar among the recombinant enzymes, and V(max) values clearly reflected the selectivity described above . These results indicate that Phe-120 has a key role in the enantioselective BF 1''-hydroxylation by CYP2D6 .

Nature, 2004 Nov 18, 432(7015), 406 - 11 Epub 2004 Nov 18.
Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks; Huyen Y et al.; The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood . 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor . Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs . This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues . In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs . Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs . Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.

J Neurosci, 2004 Nov 3, 24(44), 10022 - 34
A novel epilepsy mutation in the sodium channel SCN1A identifies a cytoplasmic domain for beta subunit interaction; Spampanato J et al.; A mutation in the sodium channel SCN1A was identified in a small Italian family with dominantly inherited generalized epilepsy with febrile seizures plus (GEFS+) . The mutation, D1866Y, alters an evolutionarily conserved aspartate residue in the C-terminal cytoplasmic domain of the sodium channel alpha subunit . The mutation decreased modulation of the alpha subunit by beta1, which normally causes a negative shift in the voltage dependence of inactivation in oocytes . There was less of a shift with the mutant channel, resulting in a 10 mV difference between the wild-type and mutant channels in the presence of beta1 . This shift increased the magnitude of the window current, which resulted in more persistent current during a voltage ramp . Computational analysis suggests that neurons expressing the mutant channels will fire an action potential with a shorter onset delay in response to a threshold current injection, and that they will fire multiple action potentials with a shorter interspike interval at a higher input stimulus . These results suggest a causal relationship between a positive shift in the voltage dependence of sodium channel inactivation and spontaneous seizure activity . Direct interaction between the cytoplasmic C-terminal domain of the wild-type alpha subunit with the beta1 or beta3 subunit was first demonstrated by yeast two-hybrid analysis . The SCN1A peptide K1846-R1886 is sufficient for beta subunit interaction . Coimmunoprecipitation from transfected mammalian cells confirmed the interaction between the C-terminal domains of the alpha and beta1 subunits . The D1866Y mutation weakens this interaction, demonstrating a novel molecular mechanism leading to seizure susceptibility.

Mol Biol Evol . 2004 Nov 3; {Epub ahead of print}
Divergence Pattern of Duplicate Genes in Protein-Protein Interactions Follows the Power Law; Zhang Z et al.; The impact of the biological network structures on the divergence between the two copies of one duplicate gene pair involved in the networks has not been documented on a genome scale . Having analyzed the most recently updated Database of Interacting Proteins (DIP) by incorporating the information for duplicate genes of the same age in yeast, we find that there was a highly significantly positive correlation between the level of connectivity of ancient genes and the number of shared partners of their duplicates in the protein-protein interaction networks . This suggests that duplicate genes with a low ancestral connectivity tend to provide raw materials for functional novelty while those duplicate genes with a high ancestral connectivity tend to create functional redundancy for a genome during the same evolutionary period . Moreover, the difference in the number of partners between two copies of a duplicate pair was found to follow a power-law distribution . This suggests that loss and gain of interacting partners for most duplicate genes with a lower level of ancestral connectivity is largely symmetrical whilst the "hub duplicate genes" with a higher level of ancient connectivity display an asymmetrical divergence pattern in protein-protein interactions . Thus, it is clear that the protein-protein interaction network structures affect the divergence pattern of duplicate genes . Our findings also provide insights into the origin and development of biological networks.

J Biol Chem, 2004 Dec 31, 279(53), 54983 - 6 Epub 2004 Nov 03.
Galpha12 directly interacts with PP2A: evidence FOR Galpha12-stimulated PP2A phosphatase activity and dephosphorylation of microtubule-associated protein, tau; Zhu D et al.; The Galpha(12/13) family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton . Galpha(12/13) interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with Galpha(12), we detected an interaction with the scaffolding subunit (Aalpha) of the Ser/Thr phosphatase, protein phosphatase 2A (PP2A) . PP2A dephosphorylates multiple substrates including tau, a microtubule-associated protein that is hyperphosphorylated in neurofibrillary tangles . The interaction of Aalpha and Galpha(12) was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-Galpha(12) pull-downs from cell lysates of primary neurons . The interaction was specific for Aalpha and Galpha(12) and was independent of Galpha(12) conformation . Endogenous Aalpha and Galpha(12) colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons . In vitro reconstitution of GST-Galpha(12) or recombinant Galpha(12) with PP2A core enzyme resulted in approximately 300% stimulation of PP2A activity that was not detected with other Galpha subunits and was similar with GTPgammaS- and GDP-liganded Galpha(12) . When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation . Co-expression of wild type or QLalpha(12) with tau and the active kinase resulted in 60 +/- 15% reductions in tau phosphorylation . In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed . The Galpha(12) effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nm), in COS cells and neurons . Taken together, these findings reveal novel, direct regulation of PP2A activity by Galpha(12) and potential in vivo modulation of PP2A target proteins including tau.

J Mol Endocrinol, 2004 Oct, 33(2), 467 - 76
Thyroid hormone signaling is highly heterogeneous during pre- and postnatal brain development; Quignodon L et al.; We have generated transgenic reporter mice to analyze the spatio-temporal distribution of thyroid hormone signaling during mouse brain development . The reporter system, utilizing a chimeric yeast Gal4 DNA-binding domain-thyroid hormone alpha ligand-binding domain fusion protein to drive lacZ expression, revealed that thyroid hormone signaling starts in the midbrain roof several days before the onset of thyroid gland function, and that it remains highly heterogeneous in the central nervous system throughout pre- and postnatal development . We speculate that this heterogeneity might provide neural cells with positional information during development.

J Neurochem, 2004 Nov, 91(4), 1007 - 17
Cloning of a novel neuronally expressed orphan G-protein-coupled receptor which is up-regulated by erythropoietin, interacts with microtubule-associated protein 1b and colocalizes with the 5-hydroxytryptamine 2a receptor; Maurer MH et al.; G-protein-coupled receptors (GPCRs) are the largest group of cell surface molecules involved in signal transduction and are receptors for a wide variety of stimuli ranging from light, calcium and odourants to biogenic amines and peptides . It is assumed that systematic genomic data-mining has identified the overwhelming majority of all remaining GPCRs in the genome . Here we report the cloning of a novel orphan GPCR which was identified in a search for erythropoietin-induced genes in the brain as a strongly up-regulated gene . This unknown gene coded for a protein which had a seven-transmembrane topology and key features typical of GPCRs of the A family but a low overall identity to all known GPCRs . The protein, coded ee3, has an unusually high evolutionary conservation and is expressed in neurons in diverse areas of the CNS with relation to integrative functions or motor tasks . A yeast two-hybrid screen for interacting proteins revealed binding to the microtubule-associated protein (MAP) 1b . Coupling to MAP1a has been described for another cognate GPCR, the 5-hydroxytryptamine (5HT) 2a receptor . Surprisingly, we found complete colocalization of ee3 and the 5HT2a receptor . The interaction with MAP1b proved to be critical for the stability or folding of ee3 as in mice lacking MAP1b the ee3 protein was undetectable by immunohistochemistry, although messenger RNA levels remained unchanged . We propose that ee3 is a highly interesting new orphan GPCR with potential connections to erythropoietin and 5HT2a receptor signalling.

J Cell Biochem, 2005 Jan 1, 94(1), 168 - 77
Structural and functional analysis of domains mediating interaction between NKX-3.1 and PDEF; Chen H et al.; NKX-3.1 is a suspected prostate tumor suppressor gene that encodes a homeodomain transcription factor . NKX-3.1 has been demonstrated to interact with prostate derived Ets factor (PDEF) and to suppress the ability of PDEF to transactivate the prostate specific antigen promoter . To dissect the molecular basis of the interaction between these transcription factors, deletion analyses were preformed using the yeast two-hybrid system . The interaction of NKX-3.1 with full-length PDEF requires part of the homeodomain and a tyrosine-rich 21 amino acid sequence that lies C-terminal to the homeodomain . The interaction of PDEF with full-length NKX-3.1 requires the Ets domain and a linker region that lies between the Ets and pointed domains . Deletion of the C-terminal 21 amino acids of NKX-3.1 completely disrupts the ability to suppress the transactivation function of PDEF in prostate tumor cells, demonstrating concordance between interaction in yeast and function in mammalian cells . These studies have identified novel protein-protein interaction domains within NKX-3.1 and PDEF that operate in concert with their respective DNA binding domains to mediate functional interactions between these growth regulatory transcription factors . 2004 Wiley-Liss, Inc.

J Cell Sci, 2004 Nov 15, 117(Pt 24), 5875 - 86 Epub 2004 Nov 02.
DNA replication licensing in somatic and germ cells; Eward KL et al.; The DNA replication (or origin) licensing system ensures precise duplication of the genome in each cell cycle and is a powerful regulator of cell proliferation in metazoa . Studies in yeast, Drosophila melanogaster and Xenopus laevis have characterised the molecular machinery that constitutes the licensing system, but it remains to be determined how this important evolutionary conserved pathway is regulated in Homo sapiens . We have investigated regulation of the origin licensing factors Cdc6, Cdt1, Mcm2 and Geminin in human somatic and germ cells . Cdc6 and Cdt1 play an essential role in DNA replication initiation by loading the Mcm2-7 complex, which is required for unwinding the DNA helix, onto chromosomal origins . Geminin is a repressor of origin licensing that blocks Mcm2-7 loading onto origins . Our studies demonstrate that Cdc6, Cdt1 and Mcm2 play a central role in coordinating growth during the proliferation-differentiation switch in somatic self-renewing systems and that Cdc6 expression is rate-limiting for acquisition of replication competence in primary oocytes . In striking contrast, we show that proliferation control during male gametogenesis is not linked to Cdc6 or Mcm2, but appears to be coordinated by the negative regulator Geminin with Cdt1 becoming rate-limiting in late prophase . Our data demonstrate a striking sexual dimorphism in the mechanisms repressing origin licensing and preventing untimely DNA synthesis during meiosis I, implicating a pivotal role for Geminin in maintaining integrity of the male germline genome.

Biochem Biophys Res Commun, 2004 Dec 3, 325(1), 117 - 23
Differential regulation of phospholipase Cgamma subtypes through FcepsilonRI, high affinity IgE receptor; Yoon E et al.; The high affinity IgE receptor (FcepsilonRI) usually exists as a tetramer composed of alphabetagamma2 subunits . The COOH-tail of beta and gamma subunits contains consensus sequence termed 'immunoreceptor tyrosine-based activation motif' (ITAM) . Tyrosine phosphorylated ITAM interacts with signaling proteins that contain the Src homology domain, forming a main amplifying and signaling route for FcepsilonRI . Unlike the COOH-tail, the functional role of NH(2)-tail of beta subunit in the signaling of FcepsilonRI is not clear because it lacks the ITAM sequences . To study the roles of NH(2)-tail of beta subunit, the cDNA library of RBL-2H3 cells was screened by yeast two-hybrid assay, and the NH(2)-tail of the beta subunit was found to interact with phospholipase Cgamma2 (PLCgamma2) but not with PLCgamma1 . Since both PLCgamma1 and PLCgamma2 are expressed in RBL-2H3 cells and they possess identical cellular functions, the functional meaning of the protein-protein interaction between PLCgamma2 and NH(2)-tail of beta subunit was studied by comparing the regulatory pathways that control the FcepsilonRI-mediated tyrosine phosphorylation of the two enzymes . Our study shows that PI3-kinase and PMA-sensitive PKCs were required exclusively for the FcepsilonRI-mediated tyrosine phosphorylation of PLCgamma1 . Also the FcepsilonRI-mediated tyrosine phosphorylation of PLCgamma1 was more sensitive to the inhibitors of Src and Syk kinases . These results therefore suggest that PLCgamma1 is involved in dynamic regulation of protein kinase C activity and inositol triphosphate levels in response to cellular needs . In contrast, PLCgamma2, through continuous interaction with the NH(2)-tail of beta subunit, co-localizes with FcepsilonRI in the same signaling domain, and maintains the basal cellular PLC activity.

BMC Biol . 2004 Nov 02;2(1):23.
Acetylation of insulin receptor substrate-1 is permissive for tyrosine phosphorylation; Kaiser C et al.; BACKGROUND: Insulin receptor substrate (IRS) proteins are key moderators of insulin action . Their specific regulation determines downstream protein-protein interactions and confers specificity on growth factor signalling . Regulatory mechanisms that have been identified include phosphorylation of IRS proteins on tyrosine and serine residues and ubiquitination of lysine residues . This study investigated other potential molecular mechanisms of IRS-1 regulation . RESULTS: Using the sos recruitment yeast two-hybrid system we found that IRS-1 and histone deacetylase 2 (HDAC2) interact in the cytoplasmic compartment of yeast cells . The interaction mapped to the C-terminus of IRS-1 and was confirmed through co-immunoprecipitation in vitro of recombinant IRS-1 and HDAC2 . HDAC2 bound to IRS-1 in mammalian cells treated with phorbol ester or after prolonged treatment with insulin/IGF-1 and also in the livers of ob/ob mice but not PTP1B knockout mice . Thus, the association occurs under conditions of compromised insulin signalling . We found that IRS-1 is an acetylated protein, of which the acetylation is increased by treatment of cells with Trichostatin A (TSA), an inhibitor of HDAC activity . TSA-induced increases in acetylation of IRS-1 were concomitant with increases in tyrosine phosphorylation in response to insulin . These effects were confirmed using RNA interference against HDAC2, indicating that HDAC2 specifically prevents phosphorylation of IRS-1 by the insulin receptor . CONCLUSIONS: Our results show that IRS-1 is an acetylated protein, a post-translational modification that has not been previously described . Acetylation of IRS-1 is permissive for tyrosine phosphorylation and facilitates insulin-stimulated signal transduction . Specific inhibition of HDAC2 may increase insulin sensitivity in otherwise insulin resistant conditions.

Traffic, 2004 Dec, 5(12), 946 - 62
Differential use of two AP-3-mediated pathways by lysosomal membrane proteins; Ihrke G et al.; The adaptor protein complex AP-3 is involved in the sorting of lysosomal membrane proteins to late endosomes/lysosomes . It is unclear whether AP-3-containing vesicles form at the trans-Golgi network (TGN) or early endosomes . We have compared the trafficking routes of endolyn/CD164 and 'typical' lysosomal membrane glycoproteins (lgp120/lamp-1 and CD63/lamp-3) containing cytosolic YXXPhi-targeting motifs preceded by asparagine and glycine, respectively . Endolyn, which has a NYHTL-motif, is concentrated in lysosomes, but also occurs in endosomes and at the cell surface . We observed predominant interaction of the NYHTL-motif with the mu-subunits of AP-3 in the yeast two-hybrid system . Endolyn was mislocalized to the cell surface in AP-3-deficient pearl cells, confirming a major role of AP-3 in endolyn traffic . However, lysosomal delivery of endolyn (or a NYHTL-reporter), but not GYXXPhi-containing proteins, was practically abolished when AP-2-mediated endocytosis or traffic from early to late endosomes was inhibited in NRK and 3T3 cells . This indicates that endolyn is mostly transported along the indirect lysosomal pathway (via the cell surface), rather than directly from the TGN to late endosomes/lysosomes . Our results suggest that AP-3 mediates lysosomal sorting of some membrane proteins in early endosomes in addition to sorting of proteins with intrinsically strong AP-3-interacting lysosomal targeting motifs at the TGN.

Biochem J, 2004 Nov 15, 384(Pt 1), 59 - 68
Effects of deletions at the C-terminus of tobacco acetohydroxyacid synthase on the enzyme activity and cofactor binding; Kim J et al.; AHAS (acetohydroxyacid synthase) catalyses the first committed step in the biosynthesis of branched-chain amino acids, such as valine, leucine and isoleucine . Owing to the unique presence of these biosynthetic pathways in plants and micro-organisms, AHAS has been widely investigated as an attractive target of several classes of herbicides . Recently, the crystal structure of the catalytic subunit of yeast AHAS has been resolved at 2.8 A (1 A=0.1 nm), showing that the active site is located at the dimer interface and is near the herbicide-binding site . In this structure, the existence of two disordered regions, a 'mobile loop' and a C-terminal 'lid', is worth notice . Although these regions contain the residues that are known to be important in substrate specificity and in herbicide resistance, they are poorly folded into any distinct secondary structure and are not within contact distance of the cofactors . In the present study, we have tried to demonstrate the role of these regions of tobacco AHAS by constructing variants with serial deletions, based on the structure of yeast AHAS . In contrast with the wild-type AHAS, the truncated mutant which removes the C-terminal lid, Delta630, and the internal deletion mutant without the mobile loop, Delta567-582, impaired the binding affinity for ThDP (thiamine diphosphate), and showed different elution profiles representing a monomeric form in gel-filtration chromatography . Our results suggest that these regions are involved in the binding/stabilization of the active dimer and ThDP binding.

J Am Chem Soc, 2004 Nov 10, 126(44), 14435 - 46
Developing photoactive affinity probes for proteomic profiling: hydroxamate-based probes for metalloproteases; Chan EW et al.; The denaturing aspect of current activity-based protein profiling strategies limits the classes of chemical probes to those which irreversibly and covalently modify their targeting enzymes . Herein, we present a complimentary, affinity-based labeling approach to profile enzymes which do not possess covalently bound substrate intermediates . Using a variety of enzymes belonging to the class of metalloproteases, the feasibility of the approach was successfully demonstrated in several proof-of-concept experiments . The design template of affinity-based probes targeting metalloproteases consists of a peptidyl hydroxamate zinc-binding group (ZBG), a fluorescent reporter tag, and a photolabile diazirine group . Photolysis of the photolabile unit in the probe effectively generates a covalent, irreversible linkage between the probe and the target enzyme, rendering the enzyme distinguishable from unlabeled proteins upon separation on a SDS-PAGE gel . A variety of labeling studies were carried out to confirm that the affinity-based approach selectively labeled metalloproteases in the presence of a large excess of other proteins and that the success of the labeling reaction depends intimately upon the catalytic activity of the enzyme . Addition of competitive inhibitors proportionally diminished the extent of enzyme labeling, making the approach useful for potential in situ screening of metalloprotease inhibitors . Using different probes with varying P(1) amino acids, we were able to generate unique "fingerprint" profiles of enzymes which may be used to determine their substrate specificities . Finally, by testing against a panel of yeast metalloproteases, we demonstrated that the affinity-based approach may be used for the large-scale profiling of metalloproteases in future proteomic experiments.

Nucleic Acids Res, 2004 Nov 01, 32(19), 5851 - 60 Print 2004.
CTD kinase I is involved in RNA polymerase I transcription; Bouchoux C et al.; RNA polymerase II carboxy terminal domain (CTD) kinases are key elements in the control of mRNA synthesis . Yeast CTD kinase I (CTDK-I), is a non-essential complex involved in the regulation of mRNA synthesis at the level of transcription elongation, pre-mRNA 3' formation and nuclear export . Here, we report that CTDK-I is also involved in ribosomal RNA synthesis . We show that CTDK-I is localized in part in the nucleolus . In its absence, nucleolar structure and RNA polymerase I transcription are affected . In vitro experiments show an impairment of the Pol I transcription machinery . Remarkably, RNA polymerase I co-precipitates from cellular extracts with Ctk1, the kinase subunit of the CTDK-I complex . In vitro analysis further demonstrates a direct interaction between RNA polymerase I and Ctk1 . The results suggest that CTDK-I might participate in the regulation of distinct nuclear transcriptional machineries, thus playing a role in the adaptation of the global transcriptional response to growth signalling.

Proc Natl Acad Sci U S A, 2004 Nov 9, 101(45), 15998 - 6003 Epub 2004 Nov 01.
Sir2 mediates longevity in the fly through a pathway related to calorie restriction; Rogina B et al.; Calorie restriction can extend life span in a variety of species including mammals, flies, nematodes, and yeast . Despite the importance of this nearly universal effect, little is understood about the molecular mechanisms that mediate the life-span-extending effect of calorie restriction in metazoans . Sir2 is known to be involved in life span determination and calorie restriction in yeast mother cells . In nematodes increased Sir2 can extend life span, but a direct link to calorie restriction has not been demonstrated . We now report that Sir2 is directly involved in the calorie-restriction life-span-extending pathway in Drosophila . We demonstrate that an increase in Drosophila Sir2 (dSir2) extends life span, whereas a decrease in dSir2 blocks the life-span-extending effect of calorie reduction or rpd3 mutations . These data lead us to propose a genetic pathway by which calorie restriction extends life span and provides a framework for genetic and pharmacological studies of life span extension in metazoans.

Cancer Res, 2004 Nov 1, 64(21), 7673 - 7
A novel nuclear protein, MGC5306 interacts with DNA polymerase beta and has a potential role in cellular phenotype; Wang L et al.; A novel protein MGC5306 has been identified in yeast-two-hybrid analysis by screening a HeLa cDNA library with a truncated DNA polymerasebeta (polbetaDelta) as bait . The polbetaDelta is expressed in various types of cancers . Co-immunoprecipitation-Western blot analysis confirms not only its interaction with polbetaDelta but also with wild-type polbeta . Binding to polbeta indicates potential function of MGC5306 in repair pathway . Transfection of cells with MGC5306-GFP and Western blot analysis with anti-MGC5306 antibody reveal its nuclear localization . MGC5306 is expressed in human carcinomas and tumor cell lines but not in normal tissues, suggesting MGC5306 is most likely involved in carcinogenesis . An antigrowth activity and modulations of cell cycle events are identified in cells expressing siRNAMGC5306.

J Biol Chem, 2005 Jan 7, 280(1), 284 - 291 Epub 2004 Nov 1.
Differential Regulation of G Protein {alpha} Subunit Trafficking by Mono- and Polyubiquitination; Wang Y et al.; Previously we used mass spectrometry to show that the yeast G protein alpha subunit Gpa1 is ubiquitinated at Lys-165, located within a subdomain not present in other Galpha proteins (Marotti, L . A., Jr., Newitt, R., Wang, Y., Aebersold, R., and Dohlman, H . G . (2002) Biochemistry 41, 5067-5074) . Here we describe the functional role of Gpa1 ubiquitination . We find that Gpa1 expression is elevated in mutants deficient in either proteasomal or vacuolar protease function . Vacuolar protease pep4 mutants accumulate monoubiquitinated Gpa1, and much of the protein is localized within the vacuolar compartment . In contrast, proteasome-defective rpt6/cim3 mutants accumulate polyubiquitinated Gpa1, and in this case the protein exhibits cytoplasmic localization . Cells that lack Ubp12 ubiquitin-processing protease activity accumulate both mono- and polyubiquitinated forms of Gpa1 . In this case, Gpa1 accumulates in both the cytoplasm and vacuole . Finally, a Gpa1 mutant that lacks the ubiquitinated subdomain remains unmodified and is predominantly localized at the plasma membrane . These data reveal a strong relationship between the extent of ubiquitination and trafficking of the G protein alpha subunit to its site of degradation.

Brain Res Mol Brain Res, 2004 Nov 4, 130(1-2), 30 - 8
Induction of murine HRD1 in experimental cerebral ischemia; Qi X et al.; Hrd1p in yeast plays an important role in endoplasmic reticulum-associated degradation (ERAD) . In the present study, we used an in vivo model of hypoxia-ischemia in mice to study the expression of murine HRD1 . Hypoxia-ischemia induced a significant increase in mRNA levels of genes including GRP78, CHOP and MyD116, the expression of which are specifically activated under conditions associated with ER dysfunction . The level of mHRD1 mRNA was significantly increased after ischemia . Interestingly, induction of mHRD1 was elevated at a later time point (12-48 h) in the ischemic cortex, whereas it increased at an earlier time point (3-12 h) in the injured striatum . We also examined the changes of mHRD1 mRNA expression in neuroblastoma Neuro2a and primary glial cells exposed to hypoxia/reoxygenation . The expression of mHRD1 mRNA was remarkably up-regulated in glial cells subjected to 24 h hypoxia, whereas no significant changes were observed in Neuro2a cells under hypoxia/reoxygenation . In addition, the levels of mHRD1 mRNA were markedly elevated in glial cells exposed to treatment with tunicamycin (Tm, an ER stress inducer) . These findings suggest that hypoxia-ischemia triggers ER dysfunction and mHRD1 may play a role in ischemia-induced ER dysfunction.

Biochim Biophys Acta, 2004 Nov 3, 1666(1-2), 51 - 61
Where sterols are required for endocytosis; Pichler H et al.; Sterols are essential membrane components of eukaryotic cells . Interacting closely with sphingolipids, they provide the membrane surrounding required for membrane sorting and trafficking processes . Altering the amount and/or structure of free sterols leads to defects in endocytic pathways in mammalian cells and yeast . Plasma membrane structures functioning in the internalization step in mammalian cells, caveolae and clathrin-coated pits, are affected by cholesterol depletion . Accumulation of improper plasma membrane sterols prevents hyperphosphorylation of a plasma membrane receptor in yeast . Once internalized, sterols still interact with sphingolipids and are recycled to the plasma membrane to keep an intracellular sterol gradient with the highest amount of free sterols at the cell periphery . Interestingly, cells from patients suffering from sphingolipid storage diseases show high intracellular amounts of free cholesterol . We propose that the balanced interaction of sterols and sphingolipids is responsible for protein recruitment to specialized membrane domains and their functionality in the endocytic pathway.

Biochim Biophys Acta, 2004 Nov 3, 1666(1-2), 2 - 18
Lipids in membrane protein structures; Palsdottir H et al.; This review describes the recent knowledge about tightly bound lipids in membrane protein structures and deduces general principles of the binding interactions . Bound lipids are grouped in annular, nonannular, and integral protein lipids . The importance of lipid binding for vertical positioning and tight integration of proteins in the membrane, for assembly and stabilization of oligomeric and multisubunit complexes, for supercomplexes, as well as their functional roles are pointed out . Lipid binding is stabilized by multiple noncovalent interactions from protein residues to lipid head groups and hydrophobic tails . Based on analysis of lipids with refined head groups in membrane protein structures, distinct motifs were identified for stabilizing interactions between the phosphodiester moieties and side chains of amino acid residues . Differences between binding at the electropositive and electronegative membrane side, as well as a preferential binding to the latter, are observed . A first attempt to identify lipid head group specific binding motifs is made . A newly identified cardiolipin binding site in the yeast cytochrome bc(1) complex is described . Assignment of unsaturated lipid chains and evolutionary aspects of lipid binding are discussed.

Bioorg Med Chem, 2004 Dec 1, 12(23), 6287 - 99
Synthesis and topoisomerase I inhibitory properties of luotonin A analogues; Cagir A et al.; Luotonin A, a naturally occurring pyrroloquinazolinoquinoline alkaloid, has been previously demonstrated to be a topoisomerase I poison . A number of luotonin A derivatives have now been prepared through the condensation of anthranilic acid derivatives and 1,2-dihydropyrrolo{3,4-b}quinoline-3-one in the presence of phosphorus oxychloride . When dichloromethane was used as solvent the reaction proceeded to a single product . In contrast when the reaction was carried out in tetrahydrofuran or in phosphorus oxychloride, an additional isomeric product was obtained . The luotonin A analogues were evaluated for their ability to effect stabilization of the covalent binary complex formed between human topoisomerase I and DNA, and for cytotoxicity toward a yeast strain expressing the human topoisomerase I.

Virology, 2004 Nov 24, 329(2), 328 - 36
Protein interactions among the vaccinia virus late transcription factors; Dellis S et al.; The viral proteins A1L, A2L, G8R, and H5R positively modulate vaccinia virus late gene expression . Host-encoded proteins hnRNP A2 and RBM3 may also interact with these viral factors to influence late gene expression . In these studies, a yeast two-hybrid screen and in vitro pulldown and crosslinking experiments were used to investigate protein--protein interactions among these factors . These studies confirmed a previous observation that G8R interacts with itself and A1L . However, self-interactions of A1L and H5R, and interactions between A2L and G8R, A2L and H5R, and H5R and G8R were also observed . In addition, the proteins hnRNP A2 and RBM3 both showed some interaction with A2L . Illustration of these interactions is a step toward understanding the architecture of the late gene transcription complex as it occurs in poxviruses.

Mycopathologia, 2004 Aug, 158(2), 201 - 9
A pilot-scale expressed sequence tag analysis of Beauveria bassiana gene expression reveals a tripeptidyl peptidase that is differentially expressed in vivo; Tartar A et al.; The entomopathogen Beauveria bassiana is a dimorphic fungus that displays an in vivo-specific, yeast-like parasitic phase . In order to study the transcriptome of B . bassiana during this unique developmental phase, we developed a method to harvest in vivo B . bassiana cells from infected Manduca sexta larvae . The infected hemolymph was collected just prior to insect death and subjected to gradient centrifugation, which allowed for separation of the B . bassiana in vivo-produced cells from remaining insect hemocytes . Total RNA was extracted from the harvested fungal cells and used to construct a cDNA library that is representative of B . bassiana gene expression in vivo . Expressed Sequence Tags (ESTs) were generated and led to the cloning of two protease genes . One of these proteases was identified as a tripeptidyl peptidase (Bb TPP) . The Bb TPP protease was shown to be up-regulated during infection, and identification of a signal peptide suggested that the enzyme is secreted in the host hemolymph . Although its activity and role have yet to be characterized, the Bb TPP protease appears as a likely candidate for being involved in B . bassiana pathogenesis . The identification of this novel, up-regulated protease also suggests that random sequencing from our in vivo cDNA library may be a valuable step towards identifying biologically active metabolites produced in vivo by B . bassiana.

Planta . 2004 Oct 23; {Epub ahead of print}
Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter; Rae AL et al.; A transporter with homology to the SUT/SUC family of plant sucrose transporters was isolated from a sugarcane ( Saccharum hybrid) stem cDNA library . The gene, designated ShSUT1, encodes a protein of 517 amino acids, including 12 predicted membrane-spanning domains and a large central cytoplasmic loop . ShSUT1 was demonstrated to be a functional sucrose transporter by expression in yeast . The estimated K(m) for sucrose of the ShSUT1 transporter was 2 mM at pH 5.5 . ShSUT1 was expressed predominantly in mature leaves of sugarcane that were exporting sucrose and in stem internodes that were actively accumulating sucrose . Immunolocalization with a ShSUT1-specific antiserum identified the protein in cells at the periphery of the vascular bundles in the stem . These cells became lignified and suberized as stem development proceeded, forming a barrier to apoplasmic solute movement . However, the movement of the tracer dye, carboxyfluorescein from phloem to storage parenchyma cells suggested that symplasmic connections are present . ShSUT1 may have a role in partitioning of sucrose between the vascular tissue and sites of storage in the parenchyma cells of sugarcane stem internodes.

Nat Cell Biol, 2004 Dec, 6(12), 1189 - 94 Epub 2004 Dec.
Sorting signals can direct receptor-mediated export of soluble proteins into COPII vesicles; Otte S et al.; Soluble secretory proteins are first translocated across endoplasmic reticulum (ER) membranes and folded in a specialized ER luminal environment . Fully folded and assembled secretory cargo are then segregated from ER-resident proteins into COPII-derived vesicles or tubular elements for anterograde transport . Mechanisms of bulk-flow, ER-retention and receptor-mediated export have been suggested to operate during this transport step, although these mechanisms are poorly understood . In yeast, there is evidence to suggest that Erv29p functions as a transmembrane receptor for the export of certain soluble cargo proteins including glycopro-alpha-factor (gpalphaf), the precursor of alpha-factor mating pheromone . Here we identify a hydrophobic signal within the pro-region of gpalphaf that is necessary for efficient packaging into COPII vesicles and for binding to Erv29p . When fused to Kar2p, an ER-resident protein, the pro-region sorting signal was sufficient to direct Erv29p-dependent export of the fusion protein into COPII vesicles . These findings indicate that specific motifs within soluble secretory proteins function in receptor-mediated export from the ER . Moreover, positive sorting signals seem to predominate over potential ER-retention mechanisms that may operate in localizing ER-resident proteins such as Kar2p.

J Biol Chem, 2005 Jan 21, 280(3), 1817 - 25 Epub 2004 Oct 30.
Nucleosome Assembly Protein 1 Exchanges Histone H2A-H2B Dimers and Assists Nucleosome Sliding; Park YJ et al.; Eukaryotic chromatin is highly dynamic and turns over rapidly even in the absence of DNA replication . Here we show that the acidic histone chaperone nucleosome assembly protein 1 (NAP-1) from yeast reversibly removes and replaces histone protein dimer H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange . Transient removal of H2A-H2B dimers facilitates nucleosome sliding along the DNA to a thermodynamically favorable position . Histone exchange as well as nucleosome sliding is independent of ATP and relies on the presence of the C-terminal acidic domain of yeast NAP-1, even though this region is not required for histone binding and chromatin assembly . Our results suggest a novel role for NAP-1 (and perhaps other acidic histone chaperones) in mediating chromatin fluidity by incorporating histone variants and assisting nucleosome sliding . NAP-1 may function either untargeted (if acting alone) or may be targeted to specific regions within the genome through interactions with additional factors.

J Cell Physiol . 2004 Oct 28; {Epub ahead of print}
Cell size-dependent and independent proliferation of rodent neuroblastoma x glioma cells; Rouzaire-Dubois B et al.; For decades, the connection between cell size and division has been the subject of controversy . While in yeast, cell size checkpoints coordinate cellular growth with cell-cycle progression, it has been recently shown that large and small Schwann cells proliferate at the same rate (Conlon and Raff, 2003, J Biol 2:7) . From this point of view, it is important to know whether normal and tumoral cells are similar . During continuous culture of NG108-15 neuroblastoma x glioma cells, the rate of proliferation, cell size, and external pH changed in parallel . At constant pH, the cell size-proliferation relationship followed a bell-shaped curve, so that proliferation was optimal within a cell volume window . In contrast, external acidification decreased proliferation independently of cell size . Using electrophysiological techniques, we showed that changes in cell size were dependent on both the uptake of nutrients and the passive influx of ions . Furthermore, an increase in cell size was associated with an increase in total proteins/cell . Another way to influence cell growth and proliferation is to alter the activity of the PI-3 kinase and target of rapamycin (TOR) signaling pathway . In NG108-15 cells, pharmacological inhibition of these proteins with LY 294002 and rapamycin respectively decreased proliferation but did not modify cell size . In contrast, aphidicolin treated cells did not proliferate, but they continued to increase in size . Altogether these results indicate that the proliferation of NG108-15 cells is controlled by both cell size-dependent and independent mechanisms that include extracellular pH and PI-3 kinase activity . (c) 2004 Wiley-Liss, Inc.

Genesis, 2004 Nov, 40(3), 184 - 94
Positional cloning and characterization of mouse mei8, a disrupted allelle of the meiotic cohesin Rec8; Bannister LA et al.; A novel mutation, mei8, was isolated in a forward genetic screen for infertility mutations induced by chemical mutagenesis of ES cells . Homozygous mutant mice are sterile . Mutant females exhibit ovarian dysgenesis and lack ovarian follicles at reproductive maturity . Affected males have small testes due to arrest of spermatogenesis during meiotic prophase I . Genetic mapping and positional cloning of mei8 led to the identification of a mutation in Rec8, a homolog of the yeast meiosis-specific cohesin gene REC8 . Analysis of meiosis in Rec8(mei8)/Rec8(mei8) spermatocytes showed that, while initiation of recombination and synapsis occurs, REC8 is required for the completion and/or maintenance of synapsis, cohesion of sister chromatids, and the formation of chiasmata, as it is in other organisms . However, unlike yeast and Caenorhabditis elegans, localization of REC8 on meiotic chromosomes is not required for the assembly of axial elements .

EMBO Rep, 2004 Nov, 5(11), 1064 - 70
Dynamic interactions of a transcription factor with DNA are accelerated by a chromatin remodeller; Karpova TS et al.; Most components in the nucleus are in a state of dynamic equilibrium maintained by the rapid mobility of nuclear proteins within and between compartments . Mobility is believed to reflect transient binding, but the identity of the binding sites and the function of the transient interactions are a matter of debate . Furthermore, we know little about how these processes may be regulated . Here, we investigate the nature and regulation of transcription factor binding and mobility in the nucleus of yeast cells . Using the Ace1p transcriptional activator, we show that nonspecific DNA binding interactions seem to have a role in retarding Ace1p nuclear mobility . Surprisingly, we find that this binding is a regulated process using a chromatin remodeller to speed up Ace1p interactions at nonspecific DNA sites . Our results suggest that transcription factor mobility represents a diffusion-driven, rapid sampling of nonspecific DNA sites, and that chromatin remodellers accelerate this genomic search process.

Circ Res, 2004 Nov 26, 95(11), 1091 - 9 Epub 2004 Nov 26.
Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function; Haworth RS et al.; Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown . Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins . In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin . Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets . Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T . PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD . Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA . To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation . PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity . At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics . Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.

Bioinformatics . 2004 Oct 28; {Epub ahead of print}
Finding regulatory modules through large-scale gene-expression data analysis; Kloster M et al.; MOTIVATION: The use of gene microchips has enabled a rapid accumulation of gene-expression data . One of the major challenges of analyzing this data is the diversity, in both size and signal strength, of the various modules in the gene regulatory networks of organisms . RESULTS: Based on the Iterative Signature Algorithm {Bergmann, S., Ihmels, J . and Barkai, N . (2002) Phys . Rev . E 67, 031902}, we present an algorithm--the Progressive Iterative Signature Algorithm (PISA)--that, by sequentially eliminating modules, allows unsupervised identification of both large and small regulatory modules . We applied PISA to a large set of yeast gene-expression data, and, using the Gene Ontology database as a reference, found that the algorithm is much better able to identify regulatory modules than methods based on high-throughput transcription-factor binding experiments or on comparative genomics . Supporting material: Sections S.1-S.5, figures S1-S10 and table S1 are available at ??.

Bioinformatics . 2004 Oct 28; {Epub ahead of print}
Theoretical and practical advances in genome halving; Yin P et al.; MOTIVATION: Duplication of an organism's entire genome is a rare but spectacular event, enabling the rapid emergence of multiple new gene functions . Over time, the parallel linkage of duplicated genes across chromosomes may be disrupted by reciprocal translocations, while the intrachromosomal order of genes may be shuffled by inversions and transpositions . Some duplicate genes may evolve unrecognizably or be deleted . As a consequence, the only detectable signature of an ancient duplication event in a modern genome may be the presence of various chromosomal segments containing parallel paralogous genes, with each segment appearing exactly twice in the genome . The problem of reconstructing the linkage structure of an ancestral genome before duplication is known as genome halving with unordered chromosomes . RESULTS: In this paper, we derive a new upper bound on the genome halving distance that is tighter than the best known, and a newlower bound that is almost always tighter than the best known . We also define the notion of genome halving diameter, and obtain both upper and lower bounds for it . Our tighter bounds on genome halving distance yield a new algorithm for reconstructing an ancestral duplicated genome . We create a software package HalvingSoft based on this new algorithm and test it on the yeast genome, identifying a sequence of translocations for halving the yeast genome that is shorter than previously conjectured possible . AVAILABILITY: HalvingSoft is available from the first author upon email request.

J Biol Chem, 2004 Dec 24, 279(52), 53903 - 6 Epub 2004 Oct 27.
Cadmium inhibits the functions of eukaryotic MutS complexes; Clark AB et al.; Exposure of yeast cells to low concentrations of cadmium results in elevated mutation rates due to loss of mismatch repair (MMR), and cadmium inhibits MMR activity in extracts of human cells . Here we show that cadmium inhibits both Msh2-Msh6- and Msh2-Msh3-dependent human MMR activity in vitro . This inhibition, which occurs at a step or steps preceding repair DNA synthesis, is observed for repair directed by either a 3' or a 5' nick . In an attempt to identify the protein target(s) of cadmium inhibition, we show that cadmium inhibition of MMR is not reversed by addition of zinc to the repair reaction, suggesting that the target is not a zinc metalloprotein . We then show that cadmium inhibits ATP hydrolysis by yeast Msh2-Msh6 but has no effect on ATPase hydrolysis by yeast Mlh1-Pms1 . Steady state kinetic analysis with wild type Msh2-Msh6, and with heterodimers containing subunit-specific Glu to Ala replacements inferred to inactivate the ATPase activity of either Msh2 or Msh6, suggest that cadmium inhibits ATP hydrolysis by Msh6 but not Msh2 . Cadmium also reduces DNA binding by Msh2-Msh6 and more so for mismatched than matched duplexes . These data indicate that eukaryotic Msh2-Msh3 and Msh2-Msh6 complexes are targets for inhibition of MMR by cadmium, a human lung carcinogen that is ubiquitous in the environment.

J Biol Chem, 2005 Jan 7, 280(1), 215 - 24 Epub 2004 Oct 28.
Evidence that the plastid translocon tic40 components possess modulating capabilities; Ko K et al.; The transport of proteins into the plastid is a process that faces changing cellular needs such as the situation found in different plant organs or developing tissues . The plastid translocon must therefore be responsive to the changing cell environment to deliver efficiently different arrays of structurally diverse proteins . Although the Tic40-related envelope proteins appear to be translocon components designed to address the varying needs of protein translocation, details of their involvement remain elusive . This study was thus designed to combine plant-based experiments and yeast mitochondrion-based approaches for unveiling clues related to how the Tic40 components may behave during the protein translocation process . The main findings related to how Tic40 proteins may work are: 1) natural fluctuations are apparent in developing tissues, in different organs of the same plant, and in different species; 2) transgenic Arabidopsis seedlings can tolerate functionally a wide range of variations in Tic40 levels, from partial suppression to excessive production; 3) the Tic40 proteins themselves exhibit configurational changes in their association with yeast mitochondria in response to different carbon sources; 4) the presence of Tic40 proteins in yeast mitochondria influences regulatory aspects of the mitochondrial translocon; and 5) the Tic40 proteins associate with mitochondrial translocon components involved in regulatory-like events . The combined data provide evidence that Tic40 proteins possess modulating capabilities.

Int J Toxicol, 2004, 23 Suppl 2, 23 - 47
Final report of the amended safety assessment of PEG-5, -10, -16, -25, -30, and -40 soy sterol; A role for nucleoprotein Zap3 in the reduction of telomerase activity during embryonic stem cell differentiation; School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UKTelomerase, the enzyme which maintains the ends of linear chromosomes in eukaryotic cells is found in murine embryonic stem cells; however, its activity is downregulated during in vitro differentiation . Previous work has indicated that this is due to the transcriptional downregulation of murine reverse transcriptase unit (mTert) of telomerase . To investigate the factors that cause the transcriptional repression of mTert we defined a 300 bp region which is essential for its transcription and performed site directed mutagenesis and electrophoretic mobility shift assays . This analysis indicated that Sp1, Sp3 and c-Myc bind to the GC-boxes and E-boxes, respectively, within the promoter and help activate the transcription of mTert gene . We also identified a novel binding sequence, found repeated within the mTert core region, which when mutated caused increased mTert expression . Yeast one hybrid screening combined with electrophoretic mobility shift assays indicated that the nuclear protein Zap3 binds to this site and its overexpression leads to the downregulation of mTert during differentiation . This suggests that regulation of mTert transcription is a complex process which depends on a quantitative balance between transcription factors that cause activation or repression of this gene . Overexpression of Zap3 in murine embryonic stem cells results in reduction in telomerase activity and telomere length as well as reduced proliferative capacity and limited ability to contribute to the development of haematopoietic cells upon differentiation.

Mech Dev, 2004 Dec, 121(12), 1481 - 94
RLIP mediates downstream signalling from RalB to the actin cytoskeleton during Xenopus early development; Lebreton S et al.; The Ras protein activates at least three different pathways during early development . Two of them regulate mesodermal gene expression and the third is thought to participate in the control of actin cytoskeleton dynamics via the Ral protein . From a yeast two-hybrid screen of a Xenopus maternal cDNA library, we identified the Xenopus orthologue of the Ral interacting protein (RLIP, RIP1 or RalBP1), a putative effector of small G protein Ral . Previously, we observed that a constitutively activated form of Ral GTPase (XralB G23V) induced bleaching of the animal hemisphere and disruption of the cortical actin cytoskeleton . To demonstrate that RLIP is the effector of RalB in early development, we show that the artificial targeting of RLIP to the membrane induces a similar phenotype to that of activated RalB . We show that overexpression of the Ral binding domain (RalBD) of XRLIP, which binds to the effector site of Ral, acts in competition with the endogenous effector of Ral and protects against the destructive effect of XralB G23V on the actin cytoskeleton . In contrast, the XRLIP has a synergistic effect on the activated form of XralB, which is dependent on the RalBD of RLIP . We provide evidence for the involvement of RLIP by way of its RalBD on the dynamics of the actin cytoskeleton and propose that signalling from Ral to RLIP is required for gastrulation.

Mech Dev, 2004 Dec, 121(12), 1425 - 41
The homeodomain-containing transcription factor X-nkx-5.1 inhibits expression of the homeobox gene Xanf-1 during the Xenopus laevis forebrain development; Bayramov AV et al.; Expression of the homeobox gene Xanf-1 starts within the presumptive forebrain primordium of the Xenopus embryo at the midgastrula stage and is inhibited by the late neurula . Such stage-specific inhibition is essential for the normal development as the experimental prolongation of the Xanf-1 expression elicits severe brain abnormalities . To identify transcriptional regulators that are responsible for the Xanf-1 inhibition, we have used the yeast one-hybrid system and identified a novel Xenopus homeobox gene X-nkx-5.1 that belongs to a family of Nkx-5.1 transcription factors . In terms of gene expression, X-nkx-5.1 shares many common features with its orthologs in other species, including expression in the embryonic brain and in the ciliated cells of the otic and lateral line placodes . However, we have also observed several features specific for X-nkx-5.1, such as expression in precursors of the epidermal ciliated cells that may indicate a possible common evolutionary origin of all ciliated cells derived from the embryonic ectoderm . Another specific feature is that the X-nkx-5.1 expression in the anterior neural plate starts early, within the area overlapping the Xanf-1 expression territory at the midneurula stage, and it correlates with the beginning of the Xanf-1 inhibition . Using various loss and gain-of-function techniques, including microinjections of antisense morpholino oligonucleotides and mRNA encoding for the X-nkx-5.1 and its dominant repressor and activator versions, we have shown that X-nkx-5.1 can indeed play a role of stage-specific inhibitor of Xanf-1 in the anterior neural plate during the Xenopus development.

BMC Bioinformatics . 2004 Oct 28;5(1):172.
Incremental genetic K-means algorithm and its application in gene expression data analysis; Lu Y et al.; BACKGROUND: In recent years, clustering algorithms have been effectively applied in molecular biology for gene expression data analysis . With the help of clustering algorithms such as K-means, hierarchical clustering, SOM, etc, genes are partitioned into groups based on the similarity between their expression profiles . In this way, functionally related genes are identified . As the amount of laboratory data in molecular biology grows exponentially each year due to advanced technologies such as Microarray, new efficient and effective methods for clustering must be developed to process this growing amount of biological data . RESULTS: In this paper, we propose a new clustering algorithm, Incremental Genetic K-means Algorithm (IGKA) . IGKA is an extension to our previously proposed clustering algorithm, the Fast Genetic K-means Algorithm (FGKA) . IGKA outperforms FGKA when the mutation probability is small . The main idea of IGKA is to calculate the objective value Total Within-Cluster Variation (TWCV) and to cluster centroids incrementally whenever the mutation probability is small . IGKA inherits the salient feature of FGKA of always converging to the global optimum . C program is freely available at CONCLUSIONS: Our experiments indicate that, while the IGKA algorithm has a convergence pattern similar to FGKA, it has a better time performance when the mutation probability decreases to some point . Finally, we used IGKA to cluster a yeast dataset and found that it increased the enrichment of genes of similar function within the cluster.

BMC Bioinformatics . 2004 Oct 28;5(1):170.
PhyME: a probabilistic algorithm for finding motifs in sets of orthologous sequences; Sinha S et al.; BACKGROUND: This paper addresses the problem of discovering transcription factor binding sites in heterogeneous sequence data, which includes regulatory sequences of one or more genes, as well as their orthologs in other species . RESULTS: We propose an algorithm that integrates two important aspects of a motif's significance - overrepresentation and cross-species conservation - into one probabilistic score . The algorithm allows the input orthologous sequences to be related by any user-specified phylogenetic tree . It is based on the Expectation-Maximization technique, and scales well with the number of species and the length of input sequences . We evaluate the algorithm on synthetic data, and also present results for data sets from yeast, fly, and human . CONCLUSIONS: The results demonstrate that the new approach improves motif discovery by exploiting multiple species information.

Med Pregl, 2003, 56 Suppl 1, 97 - 102
{Phagocytic activity of peripheral blood leukocytes during acute myocardial infarction}; Durdevic PM et al.; INTRODUCTION: Coronary occlusion may cause acute myocardial infarction associated with many cellular and humoral disturbances of the immune system . The aim of this investigation was to examine phagocytic activity of peripherial blood monocytes and neutrophils as potential cellular markers of systemic immunological events in acute myocardial infarction . MATERIAL AND METHODS: This study included thirty patients following first acute myocardial infarction and thirty healthy volunteers . Immunological analyses were performed on admission and repeated on the second and seventh days after the acute event . Monocytes and neutrophils were obtained from heparinized whole blood after centrifugation and separation on density gradient and incubated with fixed number of heat inactivated and painted particles of yeast . We investigated the following parameters of phagocytic activity: percentage of phagocytosis, phagocytic index, absolute phagocytic index, phagocyte count in a fixed volume of peripherial blood and phagocytic capacity . RESULTS: Except phagocytic index, all phagocytic parameters of monocytes and neutrophils were increased in acute myocardial infarction patients on admission and on the second day of hospitalization . On the seventh day after acute event only the mononuclear phagocyte count in fixed volume of peripheral blood showed significant increase in acute myocardial infarction patients, while percentage of phagocytosis, phagocyte count in fixed volume of peripheral blood and phagocyte capacity of neutrophils were increased during the whole investigated period . CONCLUSION: These data suggest that acute myocardial infarction was followed with strong systemic inflammatory response to myocardial damage . Furthermore, activated monocytes and neutrophils could be a significant source of free radicals, which might be involved in lipid peroxidation and cause tissue damage in early postinfarction period.

Plant Cell Physiol, 2004 Sep, 45(9), 1233 - 42
Characterization of a 200 kDa microtubule-associated protein of tobacco BY-2 cells, a member of the XMAP215/MOR1 family; Hamada T et al.; A microtubule-associated protein composed of a 200 kDa polypeptide (MAP200) was isolated from tobacco-cultured BY-2 cells . Analysis of the partial amino acid sequence showed that MAP200 was identical to TMBP200, the tobacco MOR1/XMAP215 homolog . Although several homolog proteins in animal and yeast cells have been reported to promote MT dynamics in vitro, no such function has been reported for plant homologs . Turbidity measurements of tubulin solution suggested that MAP200 promoted tubulin polymerization, and analysis by dark-field microscopy revealed that this MAP increased both the number and length of microtubules (MTs) . Electron microscopy and experiments using a chemical crosslinker demonstrated that MAP200 forms a complex with tubulin . Throughout the cell cycle, some MAP200 colocalized with MT structures, including cortical MTs, the preprophase band, spindle and phragmoplast, while some MAP200 was localized in areas lacking MTs . Based on our biochemical and immunofluorescence findings, the function of MAP200 in MT polymerization is discussed.

Mol Cell Biol, 2004 Nov, 24(22), 9948 - 57
Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops; Cesare AJ et al.; A prerequisite for cellular immortalization in human cells is the elongation of telomeres through the upregulation of telomerase or by the alternative lengthening of telomeres (ALT) pathway . In this study, telomere structure in multiple ALT cell lines was examined by electron microscopy . Nuclei were isolated from GM847, GM847-Tert, and WI-38 VA13 ALT cells, psoralen photo-cross-linked in situ, and the telomere restriction fragments were purified by gel filtration chromatography . Examination of telomere-enriched fractions revealed frequent extrachromosomal circles, ranging from 0.7 to 56.8 kb . t-loops were also observed, with the loop portion ranging from 0.5 to 70.2 kb . The total length of the loop plus tail of the t-loops corresponded to the telomere restriction fragment length from the ALT cell lines as determined by pulsed-field gel electrophoresis . The presence of extrachromosomal circles containing telomeric DNA was confirmed by two-dimensional pulsed-field gel electrophoresis . These results show that extrachromosomal telomeric DNA circles are present in ALT nuclei and suggest a roll-and-spread mechanism of telomere elongation similar to that seen in previous observations of multiple yeast species . Results presented here also indicate that expression of telomerase in GM847 cells does not affect t-loop or extrachromosomal circle formation.

Mol Biol Cell, 2005 Jan, 16(1), 1 - 13 Epub 2004 Oct 27.
Phylogenetic analysis of the formin homology 2 domain; Higgs HN et al.; Formin proteins are key regulators of eukaryotic actin filament assembly and elongation, and many species possess multiple formin isoforms . A nomenclature system based on fundamental features would be desirable, to aid the rapid identification and characterization of novel formins . In this article, we attempt to systematize the formin family by performing phylogenetic analyses of the formin homology 2 (FH2) domain, an independently folding region common to all formins, which alone can influence actin dynamics . Through database searches, we identify 101 FH2 domains from 26 eukaryotic species, including 15 in mice . Sequence alignments reveal a highly conserved yeast-specific insert in the "knob loop" region of the FH2 domain, with unknown functional consequences . Phylogenetic analysis using minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML) algorithms strongly supports the existence of seven metazoan groups . Yeast FH2 domains segregate from all other eukaryotes, including metazoans, other fungi, plants, and protists . Sequence comparisons of non-FH2 regions support relationships between three metazoan groups (Dia, DAAM, and FRL) and examine previously identified coiled-coil and Diaphanous auto-regulatory domain sequences . This analysis allows for a formin nomenclature system based on sequence relationships, as well as suggesting strategies for the determination of biochemical and cellular activities of these proteins.

Bioinformatics . 2004 Oct 28; {Epub ahead of print}
Local correlation of expression profiles with gene annotations - proof of concept for a general conciliatory method; Pinto FR et al.; MOTIVATION: Integrated analysis of expression data and gene ontology annotations is a prime example of biological data that needs co-explanatory interpretation . This particular application is used to validate a new method for integrated analysis of varied biological information . RESULTS: The proposed method consists of determining local correlation coefficients and the corresponding p-values calculated per biological entity . This measure considers the combined intensity and significance of the agreement or disagreement, between two data sources about the same biological entity . The method is applied to the integrated analysis of gene expression and annotation of two gene sets, one from yeast and other from mouse . The potential of the method to generate accurate mechanistic hypothesis is also demonstrated . Specially, negative correlation results pose a new kind of biological hypothesis . Method performance was compared with annotation enrichment methods and optimal conditions for the superiority of local correlation results are discussed . AVAILABILITY: The matlab functions described in this paper are available at . SUPPLEMENTARY INFORMATION: Further information, tables and figures are available at .

Bioinformatics . 2004 Oct 27; {Epub ahead of print}
Statistical analysis of domains in interacting protein pairs; Nye TM et al.; MOTIVATION: Several methods have recently been developed to analyse large-scale sets of physical interactions between proteins in terms of physical contacts between the constituent domains, often with a view to predicting new pairwise interactions . Our aim is to combine genomic interaction data, in which domain-domain contacts are not explicitly reported, with the domain-level structure of individual proteins, in order to learn about the structure of interacting protein pairs . Our approach is driven by the need to assess the evidence for physical contacts between domains in a statistically rigorous way . RESULTS: We develop a statistical approach that assigns p-values to pairs of domain superfamilies, measuring the strength of evidence within a set of protein interactions that domains from these superfamilies form contacts . A set of p-values is calculated for SCOP superfamily pairs, based on a pooled dataset of interactions from yeast . These p-values can be used to predict which domains come into contact in an interacting protein pair . This predictive scheme is tested against protein complexes in the Protein Quaternary Structure (PQS) database, and is used to predict domain-domain contacts within 705 interacting protein pairs taken from our pooled dataset.

Hum Mol Genet, 2004 Dec 1, 13(23), 3007 - 15 Epub 2004 Oct 27.
Iron-sulfur protein maturation in human cells: evidence for a function of frataxin; Stehling O et al.; The maturation of iron-sulfur (Fe/S) proteins in eukaryotes has been intensively studied in yeast . Hardly anything is known so far about the process in higher eukaryotes, even though the high conservation of the yeast maturation components in most Eukarya suggests similar mechanisms . Here, we developed a cell culture model in which the RNA interference (RNAi) technology was used to deplete a potential component of Fe/S protein maturation, frataxin, in human HeLa cells . This protein is lowered in humans with the neuromuscular disorder Friedreich's ataxia (FRDA) . Upon frataxin depletion by RNAi, the enzyme activities of the mitochondrial Fe/S proteins, aconitase and succinate dehydrogenase, were decreased, while the activities of non-Fe/S proteins remained constant . Moreover, Fe/S cluster association with the cytosolic iron-regulatory protein 1 was diminished . In contrast, no alterations in cellular iron uptake, iron content and heme formation were found, and no mitochondrial iron deposits were observed upon frataxin depletion . Hence, iron accumulation in FRDA mitochondria appears to be a late consequence of frataxin deficiency . These results demonstrate (i) that frataxin is a component of the human Fe/S cluster assembly machinery and (ii) that it plays a role in the maturation of both mitochondrial and cytosolic Fe/S proteins.

J Biol Chem, 2005 Jan 14, 280(2), 1613 - 24 Epub 2004 Oct 26.
Mitochondria-specific RNA-modifying Enzymes Responsible for the Biosynthesis of the Wobble Base in Mitochondrial tRNAs: IMPLICATIONS FOR THE MOLECULAR PATHOGENESIS OF HUMAN MITOCHONDRIAL DISEASES; Umeda N et al.; Human mitochondrial (mt) tRNA(Lys) has a taurine-containing modified uridine, 5-taurinomethyl-2-thiouridine (taum(5)s(2)U), at its anticodon wobble position . We previously found that the mt tRNA(Lys), carrying the A8344G mutation from cells of patients with myoclonus epilepsy associated with ragged-red fibers (MERRF), lacks the taum(5)s(2)U modification . Here we describe the identification and characterization of a tRNA-modifying enzyme MTU1 (mitochondrial tRNA-specific 2-thiouridylase 1) that is responsible for the 2-thiolation of the wobble position in human and yeast mt tRNAs . Disruption of the yeast MTU1 gene eliminated the 2-thio modification of mt tRNAs and impaired mitochondrial protein synthesis, which led to reduced respiratory activity . Furthermore, when MTO1 or MSS1, which are responsible for the C5 substituent of the modified uridine, was disrupted along with MTU1, a much more severe reduction in mitochondrial activity was observed . Thus, the C5 and 2-thio modifications act synergistically in promoting efficient cognate codon decoding . Partial inactivation of MTU1 in HeLa cells by small interference RNA also reduced their oxygen consumption and resulted in mitochondria with defective membrane potentials, which are similar phenotypic features observed in MERRF.

J Biol Chem, 2005 Jan 7, 280(1), 628 - 36 Epub 2004 Oct 27.
Identification of Human VPS37C, a Component of Endosomal Sorting Complex Required for Transport-I Important for Viral Budding; Eastman SW et al.; Endosomal sorting complex required for transport-I (ESCRT-I) is one of three defined protein complexes in the class E vacuolar protein sorting (VPS) pathway required for the sorting of ubiquitinated transmembrane proteins into internal vesicles of multivesicular bodies . In yeast, ESCRT-I is composed of three proteins, VSP23, VPS28, and VPS37, whereas in mammals only Tsg101(VPS23) and VPS28 were originally identified as ESCRT-I components . Using yeast two-hybrid screens, we identified one of a family of human proteins (VPS37C) as a Tsg101-binding protein . VPS37C can form a ternary complex with Tsg101 and VPS28 by binding to a domain situated toward the carboxyl terminus of Tsg101 and binds to another class E VPS factor, namely Hrs . In addition, VPS37C is recruited to aberrant endosomes induced by overexpression of Tsg101, Hrs, or dominant negative form of the class E VPS ATPase, VPS4 . Enveloped viruses that encode PTAP motifs to facilitate budding exploit ESCRT-I as an interface with the class E VPS pathway, and accordingly, VPS37C is recruited to the plasma membrane along with Tsg101 by human immunodeficiency virus, type 1 (HIV-1) Gag . Moreover, direct fusion of VPS37C to HIV-1 Gag obviates the requirement for a PTAP motif to induce virion release . Depletion of VPS37C from cells does not inhibit murine leukemia virus budding, which is not mediated by ESCRT-I, however, if murine leukemia virus budding is engineered to be ESCRT-I-dependent, then it is inhibited by VPS37C depletion, and this inhibition is accentuated if VPS37B is simultaneously depleted . Thus, this study identifies VPS37C as a functional component of mammalian ESCRT-I.

J Biol Chem, 2004 Dec 31, 279(53), 55372 - 5 Epub 2004 Oct 27.
The ATP paradox is the expression of an economizing fuel mechanism; Aledo JC et al.; The strong negative correlation between glycolytic flux and intracellular ATP concentration observed in yeast has long been an intriguing and counterintuitive phenomenon, which has been referred to as the ATP paradox . Herein, using principles of irreversible thermodynamics it was shown that if the ATP-consuming pathways are more sensitive to extracellular glucose than glycolysis, then upon glucose addition glycolysis performance can switch from an efficient working regime to a dissipative regime, and vice versa, depending on glucose availability . The efficient regime represents a good compromise between high output power and low dissipation, whereas the dissipative working regime offers a higher output power although at a high glucose cost . The physiological and evolutionary implications of this switch strategy are discussed.

J Biol Chem, 2005 Jan 7, 280(1), 787 - 95 Epub 2004 Oct 27.
Drosophila fear of intimacy Encodes a Zrt/IRT-like Protein (ZIP) Family Zinc Transporter Functionally Related to Mammalian ZIP Proteins; Mathews WR et al.; Zinc is essential for many cellular processes, and its concentration in the cell must be tightly controlled . The Zrt/IRT-like protein (ZIP) family of zinc transporters have recently been identified as the main regulators of zinc influx into the cytoplasm (1); however, little is known about their in vivo roles . Previously, we have shown that fear of intimacy (foi) encodes a putative member of the ZIP family that is essential for development in Drosophila (2) . Here we demonstrate that FOI can act as an ion transporter in both yeast and mammalian cell assays and is specific for zinc . We also provide insight into the mechanism of action of the ZIP family through membrane topology and structure-function analyses of FOI . Our work demonstrates that Drosophila FOI is closely related to mammalian ZIP proteins at the functional level and that Drosophila represents an ideal system for understanding the in vivo roles of this family . In addition, this work indicates that the control of zinc by ZIP transporters may play a critical role in regulating developmental processes.

Oral Oncol, 2004 Nov, 40(10), 971 - 8
Cytotoxic drugs, radiotherapy and oral candidiasis; Soysa NS et al.; The increased incidence of oral candidiasis in patients with malignancies stems partly from the systemic disease itself and, partly from the therapeutic measures such as cytotoxic and other immunosuppressive drugs and radiotherapy they receive during management of such malignancies . In this review we discuss the clinical and laboratory findings on the relationship between cytotoxics, radiotherapy and oral candidiasis, possible mechanisms of pathogenicity following such therapy, as well as precautions that could be taken to minimize such recalcitrant yeast infections.

Gastroenterology, 2004 Nov, 127(5 Suppl 1), S225 - 31
Antibody-directed therapy for human hepatocellular carcinoma; Mohr L et al.; The goals of our research are to develop high-affinity and high-stability antibodies and fragments thereof for targeting tumor-specific antigens in an attempt to develop new therapeutic agents for human hepatocellular carcinoma (HCC) . Tumor-associated antigens are excellent targets for drug and gene delivery, and offer the advantage of high cellular specificity . We have explored the use of a monoclonal antibody (Mab) AF-20 raised against a human hepatoma cell line (FOCUS) as a model system . This antibody binds to a 180-kDa homodimeric cell surface glycoprotein with high affinity . The antigen is uniformly expressed in HCC-derived cell lines and human tumors, including those with distant metastasis . There is minimal expression in nontumor tissues, and none detectable in normal liver . Because the AF-20 antigen antibody interactions on the cell surface is rapidly internalized at 37 degrees C, there is an opportunity to deliver cytotoxic payloads to tumor cells . In addition, high-affinity single-chain monoclonal antibody fragments (scFv) have been created using a novel yeast display system . Drug conjugates with AF-20 monoclonal antibodies have been prepared for gene targeting of HCC both in vitro and in vivo using preclinical animal model systems . These studies show that it is possible to generate high-affinity intact scFv antibody fragments that will allow specific tumor targeting of adenoviruses containing suicide genes, chemotherapeutic agents such as methotrexate, and cytotoxic peptides to produce antitumor effects . Therefore, specific antibody targeting of antitumor agents to HCC cells has the potential for therapeutic application in this devastating disease.

Methods Mol Biol, 2004, 292, 247 - 66
Methods for measuring the replication and segregation of epstein-barr virus-based plasmids; Kapoor P et al.; Plasmids containing the Epstein-Barr virus (EBV) latent origin of replication, OriP, are stably maintained in human cells expressing the viral EBNA-1 protein . This stable maintenance is owing to the ability of EBNA-1 to activate DNA replication from OriP and to facilitate the segregation of the plasmids during cell division . Methods for quantifying the replication and stable maintenance of EBV-based plasmids in human cells are presented here, as is a reconstituted segregation system in yeast that enables the segregation activity of EBNA1 to be measured independently from its replication activity.

J Virol, 2004 Nov, 78(22), 12657 - 64
Identification of TAZ as a binding partner of the polyomavirus T antigens; Tian Y et al.; A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Delta2-4) in the common N terminus of the T antigens . To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4 . TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner . TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen {lT}:middle T antigen {mT}:sT) . The Delta2-4 mutant T antigens showed no detectable binding . The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D-->NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection . The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding . The Delta2-4 mutant was defective in viral DNA replication . Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2alpha . Wild-type polyomavirus T antigens effectively block transactivation by TAZ . The functional significance of TAZ interactions with polyomavirus T antigens is discussed.

Cell, 2004 Oct 29, 119(3), 381 - 92
Requirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RRE export function; Yedavalli VS et al.; A single transcript in its unspliced and spliced forms directs the synthesis of all HIV-1 proteins . Although nuclear export of intron-containing cellular transcripts is restricted in mammalian cells, HIV-1 has evolved the viral Rev protein to overcome this restriction for viral transcripts . Previously, CRM1 was identified as a cellular cofactor for Rev-dependent export of intron-containing HIV-1 RNA . Here, we present evidence that Rev/CRM1 activity utilizes the ATP-dependent DEAD box RNA helicase, DDX3 . We show that DDX3 is a nucleo-cytoplasmic shuttling protein, which binds CRM1 and localizes to nuclear membrane pores . Knockdown of DDX3 using either antisense vector or dominant-negative mutants suppressed Rev-RRE-function in the export of incompletely spliced HIV-1 RNAs . Plausibly, DDX3 is the human RNA helicase which functions in the CRM1 RNA export pathway analogously to the postulated role for Dbp5p in yeast mRNA export.

Genes Cells, 2004 Nov, 9(11), 1125 - 35
GRIP1tau, a novel PDZ domain-containing transcriptional activator, cooperates with the testis-specific transcription elongation factor SII-T1; Nakata A et al.; SII-T1 is a tissue-specific member of the transcription elongation factor S-II that is expressed specifically in male germ cells . In the present study, we have identified a protein named GRIP1tau interacting with SII-T1 by yeast two-hybrid screening . GRIP1tau is a novel isoform of glutamate receptor-interacting protein 1 (GRIP1) that associates with the cytoplasmic domain of the alpha-amino-3-hydroxy-5-methyl-4-isoaxazolepropionate (AMPA)-type glutamate receptor . GRIP1tau is a testis-specific nuclear protein that activates transcription when fused with a GAL4 DNA binding domain in GAL4-responsive reporter gene assays . The transactivation domain of GRIP1tau overlapped with the region essential for interaction with SII-T1, as revealed by co-immunoprecipitation assays . Also, transactivation by GRIP1tau was stimulated by SII-T1 in a dose-dependent manner . Therefore, we propose that GRIP1tau is a novel testis-specific transcriptional activator regulated by interaction with the testis-specific transcription elongation factor SII-T1.

Biochem Soc Trans, 2004 Dec, 32(Pt 6), 1021 - 4
Genes controlling the metabolic switch in hibernating mammals; Andrews MT; Hibernating mammals have the ability to decrease their metabolic rate and survive up to 6 months without food in an inactive state where body temperatures approach 0 degrees C . In hibernating 13-lined ground squirrels (Spermophilus tridecemlineatus), oxygen consumption holds at 1/30 to 1/50 of the aroused condition and heart rates are as low as 3-10 beats/min, compared with 200-300 beats/min when the animal is active . This seasonal adaptation requires a metabolic shift away from the oxidation of carbohydrates and towards the combustion of stored fatty acids as the primary source of energy . A key element in this fuel switch is the differential expression of the gene encoding pyruvate dehydrogenase kinase isoenzyme 4 . Pyruvate dehydrogenase kinase isoenzyme 4 inhibits pyruvate dehydrogenase and thus minimizes carbohydrate oxidation by preventing the flow of glycolytic products into the tricarboxylic acid cycle . Hibernators also exploit the low-temperature activity of PTL (pancreatic triacylglycerol lipase) in both heart and white adipose tissue . Lipolytic activity at body temperatures associated with hibernation was examined using recombinant ground squirrel and human PTL expressed in yeast . Enzymes from both humans and ground squirrel displayed high activity at temperatures as low as 0 degrees C and showed Q(10) = 1.2-1.5 over the temperature range 37-7 degrees C . These studies indicate that low-temperature lipolysis is a general property of PTL and does not require protein modifications unique to mammalian cells and/or the hibernating state.

Biochem J . 2004 Oct 27; {Epub ahead of print}
Is the regulation of galactose 1-phosphate tuned against gene expression noise?
de Atauri P, Orrell D, Ramsey S, Bolouri H.
The average number of mRNA molecules per active gene in yeast can be remarkably low . Consequently, the relative number of copies of each transcript per cell can vary greatly from moment to moment . When these transcripts are encoding metabolic enzymes, how do the resulting variations in enzyme concentrations affect the regulation of metabolic intermediaries? Using a kinetic model of galactose utilization in yeast, we analyze the transmission of noise from transcription and translation on metabolic intermediary regulation . In particular, the effect of the kinetic properties of the galactose 1-phosphate uridylyltransferase reaction on the transmission of noise is analyzed.

Chin Med Sci J, 2004 Sep, 19(3), 157 - 63
Inhibitory role of transcription factor COUP-TFII in expression of hTERT in HeLa cells; Wang Q et al.; OBJECTIVE: To clone and identify the proteins involved in regulating the transcription of hTERT and study the role of genes in both hTERT transcription and telomerase activity . METHODS: The full cDNA of COUP-TFII was cloned from HeLa cDNA library by hTERT promoter-based yeast one-hybrid assay and then in-frame inserted into His-tag fusion expression vector pEK318 . The His-tag COUP-TFII fusion proteins were purified by Ni-NTA chromatography . The interaction of COUP-TFII with hTERT promoter in vitro was identified by electrophoretic mobility shift assay and Footprint . The role of COUP-TFII in both hTERT transcription and telomerase activity were probed through Luciferase reporter assay, Northern blot, and TRAP-PCR ELISA . RESULTS: COUP-TFII could firmly bind to the downstream E-box and the other two binding sites in hTERT promoter . Luciferase reporter assay indicated COUP-TFII could suppress hTERT promoter activity and stable introduction of COUP-TFII into HeLa cells also decreased both endogenous hTERT transcription and telomerase activity . CONCLUSION: The human COUP-TFII can firmly bind to hTERT promoter, and inhibit telomerase activity through decreasing hTERT transcription . It will greatly facilitate understanding of telomerase regulation in normal and cancer cells.

Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15700 - 5 Epub 2004 Oct 25.
Variation in sequence and organization of splicing regulatory elements in vertebrate genes; Yeo G et al.; Although core mechanisms and machinery of premRNA splicing are conserved from yeast to human, the details of intron recognition often differ, even between closely related organisms . For example, genes from the pufferfish Fugu rubripes generally contain one or more introns that are not properly spliced in mouse cells . Exploiting available genome sequence data, a battery of sequence analysis techniques was used to reach several conclusions about the organization and evolution of splicing regulatory elements in vertebrate genes . The classical splice site and putative branch site signals are completely conserved across the vertebrates studied (human, mouse, pufferfish, and zebrafish), and exonic splicing enhancers also appear broadly conserved in vertebrates . However, another class of splicing regulatory elements, the intronic splicing enhancers, appears to differ substantially between mammals and fish, with G triples (GGG) very abundant in mammalian introns but comparatively rare in fish . Conversely, short repeats of AC and GT are predicted to function as intronic splicing enhancers in fish but are not enriched in mammalian introns . Consistent with this pattern, exonic splicing enhancer-binding SR proteins are highly conserved across all vertebrates, whereas heterogeneous nuclear ribonucleoproteins, which bind many intronic sequences, vary in domain structure and even presence/absence between mammals and fish . Exploiting differences in intronic sequence composition, a statistical model was developed to predict the splicing phenotype of Fugu introns in mammalian systems and was used to engineer the spliceability of a Fugu intron in human cells by insertion of specific sequences, thereby rescuing splicing in human cells.

Biochem Biophys Res Commun, 2004 Nov 26, 324(4), 1379 - 85
A selective requirement for copper-dependent activation of cytochrome c oxidase by Cox17p; Kako K et al.; Cox17p is cloned from yeast as a chaperone to deliver copper to the mitochondria of assembly for cytochrome c oxidase (CCO) . In mammals, CCO is a key enzyme for cellular respiration and a defect in its function is associated with severe neonatal or infantile lactic acidosis and early death . Recently, we found that Cox17p is not only required for mitochondrial oxidative phosphorylation but also is essential for embryonic growth and development in COX17 gene-deficient mice . To investigate its biochemical features, recombinant human Cox17p was overexpressed and purified without a purification tag . It specifically binds Cu(I) at a molar copper content of 3.3+/-0.04 under reduced conditions and significantly activates the mitochondrial CCO in vitro . Although the Cu-Cox17p complex was maintained between pH values from 5.0 to 7.7, Cu was completely released from Cox17p at pH 8.0 . An acute exposure of excess amount of copper ion to mouse cells resulted in a significant reduction of Cox17p mRNA expression, whereas copper starvation maintained the Cox17p transcription level . These results suggest that the stringent selectivity of Cox17p for copper is required for CCO activation, to prevent copper overload, or promote the supply of copper.

Biochem Biophys Res Commun, 2004 Nov 26, 324(4), 1324 - 32
Molecular cloning and characterization of a human gene involved in transcriptional regulation of hTERT; Tang Z et al.; Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase in human, has been identified as the rate-limiting factor in telomerase activity, but its detailed mechanism of transcription regulation remains unclear . In this investigation, a novel human gene telomerase transcriptional elements-interacting factor (TEIF) was isolated from HeLa cell based on hTERT promoter yeast one-hybrid assay . TEIF has a 2358-bp open reading frame encoding a predicted protein of 786 amino acids, which is functionally conserved in general eukaryotic species . The transcription of TEIF was detected in cells and expressed an 86-kDa native protein, distributing mainly in cellular nuclei . Its interaction with hTERT promoter was identified and the DNA binding potential was credited to C-terminus of TEIF . Transfection of TEIF into HeLa cells presented marked transactivation of hTERT promoter and stimulated both endogenous hTERT transcription and telomerase activities . These results suggest that TEIF protein might be a transcription regulator and take part in the activation of hTERT.

BMC Bioinformatics . 2004 Oct 25;5(1):158.
A computational approach for ordering signal transduction pathway components from genomics and proteomics Data; Liu Y et al.; BACKGROUND: Signal transduction is one of the most important biological processes by which cells convert an external signal into a response . Novel computational approaches to mapping proteins onto signaling pathways are needed to fully take advantage of the rapid accumulation of genomic and proteomics information . However, despite their importance, research on signaling pathways reconstruction utilizing large-scale genomics and proteomics information has been limited . RESULTS: We have developed an approach for predicting the order of signaling pathway components, assuming all the components on the pathways are known . Our method is built on a score function that integrates protein-protein interaction data and microarray gene expression data . Compared to the individual datasets, either protein interactions or gene transcript abundance measurements, the integrated approach leads to better identification of the order of the pathway components . CONCLUSIONS: As demonstrated in our study on the yeast MAPK signaling pathways, the integration analysis of high-throughput genomics and proteomics data can be a powerful means to infer the order of pathway components, enabling the transformation from molecular data into knowledge of cellular mechanisms.

J Chromatogr A, 2004 Sep 24, 1050(1), 85 - 93
Selective detection and identification of Se containing compounds--review and recent developments; Uden PC et al.; The complexity of selenium (Se) chemistry in the environment and in living organisms presents broad analytical challenges . The selective qualitative and quantitative determination of particular species of this element is vital in order to understand selenium's metabolism and significance in biology, toxicology, clinical chemistry and nutrition . This calls for state-of-the-art analytical techniques such as hyphenated methods that are reviewed with particular emphasis on interfaced separation with element-selective detection and identification of the detected selenium compounds . Atomic spectral element specific detection for monitoring chromatographic eluent enabled quantitative determination of selenium species in selenized yeast and qualitative measurement for breath samples . Gas chromatography with atomic emission detection (AED) of ethylated species and fluoroacid ion pair HPLC applied to the analysis of currently produced or archived selenized yeast and Brassica juncea have revealed the presence of a previously unrecognised Se-S amino acid, S-(methylseleno)cysteine.

Eur J Cell Biol, 2004 Aug, 83(7), 337 - 45
Testis-specific human small heat shock protein HSPB9 is a cancer/testis antigen, and potentially interacts with the dynein subunit TCTEL1; de Wit NJ et al.; Searching EST databases for new members of the human small heat shock protein family, we recently identified HSPB9, which is expressed exclusively in testis as determined by Northern blotting (Kappe et al., Biochim . Biophys . Acta 1520, 1-6, 2001) . Here we confirm this testis-specific expression pattern by RT-PCR in a larger series of normal tissues . Interestingly, while screening HSPB9 ESTs, we also noted expression in tumours, which could be verified by RT-PCR . Protein expression of HSPB9 was also detected in normal human testis and various tumour samples using immunohistochemical staining . We thus conclude that HSPB9 belongs to the steadily growing number of cancer/testis antigens . To get a better understanding of the function of HSPB9, we performed a yeast two-hybrid screen to search for HSPB9-interacting proteins . TCTEL1, a light chain component of cytoplasmic and flagellar dynein, interacted in both the yeast two-hybrid system and in immunoprecipitation experiments with HSPB9 . Additionally, immunohistochemical staining showed co-expression of HSPB9 and TCTEL1 in similar stages of spermatogenesis and in tumour cells . The possible functional significance of this interaction is discussed.

Arch Virol, 2004 Nov, 149(11), 2245 - 60 Epub 2004 Jun 30.
VP1, the RNA-dependent RNA polymerase and genome-linked protein of infectious bursal disease virus, interacts with the carboxy-terminal domain of translational eukaryotic initiation factor 4AII; Tacken MG et al.; Infectious bursal disease virus (IBDV), a member of the family Birnaviridae, is a non-enveloped, double-stranded RNA virus . Viral protein 1 (VP1), the putative RNA-dependent RNA polymerase, occurs in virions both as a free polypeptide and as a genome-linked protein, called VPg . To gain more insight in its function, we initiated a yeast two-hybrid screen . With this approach we identified the carboxy-terminal domain of eukaryotic translation initiation factor 4AII (eIF4AII) as an interactor for VP1 . The association between these molecules was confirmed by co-immunoprecipitation analyses . eIF4A plays an essential role in the initiation of translation of both capped and uncapped mRNAs . Its association with IBDV VP1 suggests an involvement of this viral protein in IBDV mRNA translation . An interaction between VP1 and full-length eIF4AII was, however, not observed . In view of the known two-domain structure of eIF4AII it is conceivable that the interaction of VP1 with full-length eIF4AII requires collaborating proteins that open up its structure and expose the VP1-binding site in the carboxy-terminal domain . The biological relevance of the potential VP1-eIF4AII interaction is discussed.

Methods Mol Biol, 2004, 289, 341 - 58
Methods to study protein-protein interactions; Meng JJ et al.; Protein-protein interactions are the underpinnings of a vast number of cellular processes . In recent years, the convergence of biochemistry, cellular, and molecular biology has made available a number of powerful techniques for studying such interactions . These techniques vary in their sensitivity, efficiency, and rapidity, but judicial deployment of a combination of them has proved to be effective and reliable . Here, we highlight a version of the yeast two-hybrid assay originally pioneered by Fields and Song (1989) and subsequently enhancements by other investigators . We also briefly describe a number of new fluorescent imaging-based biophysical techniques for studying protein-protein interactions FRET, FCS, and BiFC . Together, these constitute an impressive collection of tools for studying interactions among proteins.

J Biol Chem, 2005 Jan 7, 280(1), 104 - 11 Epub 2004 Oct 21.
Histone H2A and Spt10 Cooperate to Regulate Induction and Autoregulation of the CUP1 Metallothionein; Kuo HC et al.; Copper is an essential cellular cofactor that becomes toxic at high levels . Copper homeostasis is tightly regulated by opposing mechanisms that control copper import, export, and copper binding capacity within the cell . High levels of copper induce the expression of metallothioneins, small sulfhydryl-rich proteins with high metal binding capabilities that serve as neutralizers of toxic levels of metals . In yeast, the CUP1 gene encodes a copper metallothionein that is strongly induced in response to metals and other stress and is subsequently rapidly down-regulated . Activation of CUP1 is mediated by the copper-responsive transcriptional activator AceI, and also requires the histone acetylase Spt10 for full induction . We have examined the role of histone H2A in the normal regulation of the CUP1 gene . We have shown that specific H2A mutations in combination with spt10 deletions result in aberrant regulation of CUP1 expression . Certain lysine mutations in H2A alleviate the transcriptional defect in spt10Delta strains, though CUP1 activation is still delayed in these mutants; however, CUP1 shutdown is normal . In contrast, serine mutations in H2A prevent CUP1 shutdown when combined with spt10 deletions . In addition, swi/snf mutants exhibit both impaired CUP1 induction and failure to shut down CUP1 normally . Finally, different Spt10-dependent histone acetylation events correlate with induction and shutdown . Taken together, these data indicate that CUP1 transcriptional shutdown, like induction, is an active process controlled by the chromatin structure of the gene . These results provide new insights for the role of chromatin structure in metal homeostasis.

J Biol Chem, 2005 Jan 21, 280(3), 2213 - 9 Epub 2004 Oct 22.
Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter; Arastu-Kapur S et al.; LdNT2 is a member of the equilibrative nucleoside transporter family, which possesses several conserved residues located mainly within transmembrane domains . One of these residues, Asp(389) within LdNT2, was shown previously to be critical for transporter function without affecting ligand affinity or plasma membrane targeting . To further delineate the role of Asp(389) in LdNT2 function, second-site suppressors of the ldnt2-D389N null mutation were selected in yeast deficient in purine nucleoside transport and incapable of purine biosynthesis . A library of random mutants within the ldnt2-D389N background was screened in yeast for restoration of growth on inosine . Twelve different clones were obtained, each containing secondary mutations enabling inosine transport . One mutation, N175I, occurred in four clones and conferred augmented inosine transport capability compared with LdNT2 in yeast . N175I was subsequently introduced into an ldnt2-D389N construct tagged with green fluorescent protein and transfected into a Deltaldnt1/Deltaldnt2 Leishmania donovani knockout . GFP-N175I/D389N significantly suppressed the D389N phenotype and targeted properly to the plasma membrane and flagellum . Most interestingly, N175I increased the inosine K(m) by 10-fold within the D389N background relative to wild type GFP-LdNT2 . Additional substitutions introduced at Asn(175) established that only large, nonpolar amino acids suppressed the D389N phenotype, indicating that suppression by Asn(175) has a specific size and charge requirement . Because multiple suppressor mutations alleviate the constraint imparted by the D389N mutation, these data suggest that Asp(389) is a conformationally sensitive residue . To impart spatial information to the clustering of second-site mutations, a three-dimensional model was constructed based upon members of the major facilitator superfamily using threading analysis . The model indicates that Asn(175) and Asp(389) lie in close proximity and that the second-site suppressor mutations cluster to one region of the transporter.

J Biol Chem, 2004 Dec 31, 279(53), 55978 - 84 Epub 2004 Oct 22.
Interactor-mediated nuclear translocation and retention of protein phosphatase-1; Lesage B et al.; Protein Ser/Thr phosphatase-1 (PP1) is a ubiquitous eukaryotic enzyme that controls numerous cellular processes by the dephosphorylation of key regulatory proteins . PP1 is expressed in various cellular compartments but is most abundant in the nucleus . We have examined the determinants for the nuclear localization of enhanced green fluorescent protein-tagged PP1 in COS1 cells . Our studies show that PP1gamma(1) does not contain a functional nuclear localization signal and that its nuclear accumulation does not require Sds22, which has previously been implicated in the nuclear accumulation of PP1 in yeast (Peggie, M . W., MacKelvie, S . H., Bloecher, A., Knatko, E . V., Tatchell, K., and Stark, M . J . R . (2002) J . Cell Sci . 115, 195-206) . However, the nuclear targeting of PP1 isoforms was alleviated by the mutation of their binding sites for proteins that interact via an RVXF motif . Moreover, one of the mutants with a cytoplasmic accumulation and decreased affinity for RVXF motifs (PP1gamma(1)-F257A) could be re-targeted to the nucleus by the overexpression of nuclear interactors (NIPP1 (nuclear inhibitor of PP1) and PNUTS (PP1 nuclear targeting subunit)) with a functional RVXF motif . Also, the addition of a synthetic RVXF-containing peptide to permeabilized cells resulted in the loss of nuclear enhanced green fluorescent protein-PP1gamma(1) . Finally, NIPP1(-/-) mouse embryos showed a nuclear hyperphosphorylation on threonine, consistent with a role for NIPP1 in the nuclear targeting and/or retention of PP1 . Our data suggest that both the nuclear translocation and the nuclear retention of PP1 depend on its binding to interactors with an RVXF motif.

Exp Cell Res, 2004 Nov 15, 301(1), 16 - 22
Formins: processive cappers of growing actin filaments; Watanabe N et al.; Taking the advantage of single-molecule imaging, our recent study has revealed surprisingly long processive movement of a Formin protein, mDia1, surfing along with the growing end of actin filaments in living cells . This finding provides direct evidence for the ability of Formins to function as processive cappers that has been postulated from several lines of evidence in biochemical studies . With nucleating filaments from the profilin-actin pool, Formins may effectively generate long actin filaments, and contribute to the generation of the specific actin-based structures, that is, the contractile ring in cytokinesis, actin stress fibers in animal cells, and yeast actin cables . Furthermore, Formins have the potential to function as actin polymerization-driven molecular motors . Although much remains to be tested about the role of this novel molecular mobilization mechanism, cells might utilize actin polymerization energy for cell shape change and/or trafficking via Formin motors.

Bioorg Med Chem Lett, 2004 Dec 6, 14(23), 5743 - 5
Site-specific PEGylation of proteins containing unnatural amino acids; Deiters A et al.; Here, we report a generally applicable PEGylation methodology based on the site-specific incorporation of para-azidophenylalanine into proteins in yeast . The azido group was used in a mild {3+2} cycloaddition reaction with an alkyne derivatized PEG reagent to afford selectively PEGylated protein . This strategy should be useful for the generation of selectively PEGylated proteins for therapeutic applications.

Exp Gerontol, 2004 Oct, 39(10), 1521 - 5
Increased nuclear proteins in muscle satellite cells in aged animals as compared to young growing animals; Machida S et al.; Evidence implies that satellite cells could play some limiting role in aged muscle undergoing repair or maintenance of mass, which is of potential clinical concern as this could contribute to sarcopenia . Further, insufficient information is available concerning the cellular mechanisms responsible for the lower rat satellite cell proliferation in old animals . Thus, it was hypothesized that the following proteins would be increased in nuclei of satellite cells from old rat skeletal muscle: the cyclin-dependent kinase (CDK) inhibitors p21(WAF1/CIP1) and p27(Kip1) as well as the transcription factors p53 and Forkhead box, subgroup O1 (FOXO1) . In addition, the NAD(+)-dependent histone deacetylase SIRT1, the mammalian ortholog of the yeast SIR2 (silence information regulator 2) and a member of the Sirtuin family, was hypothesized to decrease in satellite cell nuclei of old rats . Old satellite cells (30-months old) exhibited a lesser number of BrdU-positive cells as compared to satellite cells (3-months old) from young growing animals . Western blot analysis demonstrated that nuclei of old satellite cells accumulated the cell cycle inhibitors p21(WAF1/CIP1) and p27(Kip1) . In addition, nuclear p53 and FOXO1 proteins were also higher in old satellite cells than in cells from young growing animals . These data indicated both p53/p21(WAF1/CIP1)- and FOXO1/p27(Kip1)-dependent pathways might contribute to the age-associated decrease in satellite cell proliferation . Cytoplasmic manganese superoxide dismutase (MnSOD), a gene driven by FOXO1, was higher in old satellite cells . Unexpectedly, nuclear SIRT1 was also increased in old satellite cells compared with satellite cells from young growing animals . The physiological significance of enhanced nuclear SIRT1 expression in old satellite cells remains elusive at this time . In summary, satellite cells in old rats have nuclear accumulation of proteins inhibiting the cell cycle as compared to young, growing animals.

Plant J, 2004 Nov, 40(4), 523 - 35
Potassium carrier TRH1 is required for auxin transport in Arabidopsis roots; Vicente-Agullo F et al.; Disruption of the TRH1 potassium transporter impairs root hair development in Arabidopsis, and also affects root gravitropic behaviour . Rescue of these morphological defects by exogenous auxin indicates a link between TRH1 activity and auxin transport . In agreement with this hypothesis, the rate of auxin translocation from shoots to roots and efflux of {3H}IAA in isolated root segments were reduced in the trh1 mutant, but efflux of radiolabelled auxin was accelerated in yeast cells transformed with the TRH1 gene . In roots, Pro(TRH1):GUS expression was localized to the root cap cells which are known to be the sites of gravity perception and are central for the redistribution of auxin fluxes . Consistent with these findings, auxin-dependent DR5:GUS promoter-reporter construct was misexpressed in the trh1 mutant indicating that partial block of auxin transport through the root cap is associated with upstream accumulation of the phytohormone in protoxylem cells . When {K+} in the medium was reduced from 20 to 0.1 mm, wild type roots showed mild agravitropic phenotype and DR5:GUS misexpression in stelar cells . This pattern of response to low external {K+} was also affected by trh1 mutation . We conclude that the TRH1 carrier is an important part of auxin transport system in Arabidopsis roots.

Plant J, 2004 Nov, 40(4), 462 - 73
The NAC domain mediates functional specificity of CUP-SHAPED COTYLEDON proteins; Taoka K et al.; In higher plants, although several genes involved in shoot apical meristem (SAM) formation and organ separation have been isolated, the molecular mechanisms by which they function are largely unknown . CUP-SHAPED COTYLEDON (CUC) 1 and CUC2 are examples of two such genes that encode the NAC domain proteins . This study investigated the molecular basis for their activities . Nuclear localization assays indicated that green fluorescent protein (GFP)-CUC proteins accumulate in the nucleus . Yeast one-hybrid and transient expression assays demonstrated that the C-terminal domain (CTD) of the CUC has transactivation activity . Domain-swapping experiments revealed that the functional specificity of the CUC for promoting adventitious shoot formation resides in the highly conserved NAC domain, not in the CTD in which motifs specific to the CUC subfamily are located . Taken together, these observations suggest that CUC proteins transactivate the target genes involved in SAM formation and organ separation through a specific interaction between the NAC domain and the promoter region of the target genes.

Biochem J . 2004 Oct 25; {Epub ahead of print}
Structure-based mutagenesis studies of the peptide substrate binding fragment of type I Heat shock protein 40; Li J et al.; Ydj1 is the major type I Hsp40 family member in yeast . Ydj1 can pair with yeast Hsp70 Ssa1 to facilitate protein translocation and protein folding . Ydj1 itself can also function as a molecular chaperone to bind the non-native polypeptides and suppress protein aggregations in vitro . The crystal structure of Ydj1 complexed with its peptide substrate GWLYEIS reveals that a hydrophobic pocket located on Ydj1 domain I may play a major role in mediating the interactions between Ydj1 and the peptide substrate . To understand the mechanism by which Ydj1 interacts with non-native polypeptide, we have mutated the residues forming the hydrophobic pocket based on the structural information . We have also constructed deletion mutations of the Zinc-finger motifs within Ydj1 . We have examined the functional consequences of these Ydj1 mutants by in vivo and in vitro assays . The results indicated that the hydrophobic pocket located on Ydj1 plays critical roles for its molecular chaperone activity by mediating interactions with the non-native polypeptides.

J Infect Dis, 2004 Nov 15, 190(10), 1783 - 92 Epub 2004 Oct 18.
Rare, highly pyrimethamine-resistant alleles of the Plasmodium falciparum dihydrofolate reductase gene from 5 African sites; Bates SJ et al.; In eastern and southern Africa, there has been a rapid increase in the prevalence of alleles with mutations in the Plasmodium falciparum dihydrofolate reductase gene (dhfr) associated with increased risk of clinical failure of sulfadoxine-pyrimethamine (S/P) . Molecular methods for surveillance of these mutations are now widespread, but the usual analysis detects only the most prevalent allele in a polyclonal sample . We used a yeast-expression system to identify rare, highly pyrimethamine-resistant alleles of dhfr in isolates from 5 African countries--Kenya, Tanzania, Malawi, Gabon, and Nigeria . Only the isolates from Nigeria yielded significant numbers of novel resistant alleles, and only 1 of the alleles from any location showed a >3-fold increase in resistance to S/P or to chlorproguanil-dapsone . Overall, these results suggest that dhfr alleles that confer high levels of resistance to antifolates are rare, even in eastern and southern Africa, where pyrimethamine has been intensively used.

Mol Endocrinol . 2004 Oct 21; {Epub ahead of print}
c-Src regulates AP-2 interaction with beta-arrestin and the angiotensin II type 1 receptor during clathrin-mediated internalization; Fessart D et al.; Beta-arrestins are multifunctional adaptors involved in the internalization and signaling of G protein-coupled receptors (GPCRs) . They target receptors to clathrin-coated pits (CCPs) through binding with clathrin and AP-2 complex . They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs . Here we sought to determine whether c-Src regulates the recruitment of AP-2 to betaarrestin and the Angiotensin II (Ang II) type 1 receptor (AT1R) during internalization . We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, betaarrestins and AP-2 . In vitro studies using co-immunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between betaarrestin2 and the beta-subunit of AP-2 independently of the kinase activity of c-Src . However, while c-Src expression promoted the rapid dissociation of AP-2 from both betaarrestin and AT1R after receptor stimulation, a kinase inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor . Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in HEK 293 cells using a small interfering RNA strategy . Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization . Moreover, co-immunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and betaarrestin, respectively . Together our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and betaarrestin during the clathrin-mediated internalization of receptors, and suggest a novel function for c-Src kinase in the internalization of AT1R.

FEBS Lett, 2004 Oct 22, 576(3), 375 - 80
A single in vivo-selected point mutation in the active center of Toxoplasma gondii ferredoxin-NADP+ reductase leads to an inactive enzyme with greatly enhanced affinity for ferredoxin; Thomsen-Zieger N et al.; Electron transfer between plant-type {2Fe-2S} ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) depends on the physical interaction between both proteins . We have applied a random mutagenesis approach with subsequent in vivo selection using the yeast two-hybrid system to obtain mutants of Toxoplasma gondii FNR with higher affinity for Fd . One mutant showed a 10-fold enhanced binding using affinity chromatography on immobilized Fd . A single serine-to-arginine exchange in the active site was responsible for its increased affinity . The mutant reductase was also enzymatically inactive . Homology modeling of the mutant FNR-Fd complex predicts substantial alterations of protein-FAD interactions in the active site of the enzyme with subsequent structural changes . Collectively, for the first time a point mutation in this important class of enzymes is described which leads to greatly enhanced affinity for its protein ligand.

Curr Biol, 2004 Oct 26, 14(20), 1791 - 800
Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides; Carlton J et al.; BACKGROUND: Sorting nexins (SNXs) are phox homology (PX) domain-containing proteins thought to regulate endosomal sorting of internalized receptors . The prototypical SNX is sorting nexin-1 (SNX1), a protein that through its PX domain binds phosphatidylinositol 3-monophosphate {PtdIns(3)P} and phosphatidylinositol 3,5-bisphosphate {PtdIns(3,5)P(2)} . SNX1 is associated with early endosomes, from where it has been proposed to regulate the degradation of internalized epidermal growth factor (EGF) receptors through modulating endosomal-to-lysosomal sorting . RESULTS: We show here that SNX1 contains a BAR (Bin/Amphiphysin/Rvs) domain, a membrane binding domain that endows SNX1 with the ability to form dimers and to sense membrane curvature . We present evidence that through coincidence detection, the BAR and PX domains efficiently target SNX1 to a microdomain of the early endosome defined by high curvature and the presence of 3-phosphoinositides . In addition, we show that the BAR domain endows SNX1 with an ability to tubulate membranes in-vitro and drive the tubulation of the endosomal compartment in-vivo . Using RNA interference (RNAi), we establish that SNX1 does not play a role in EGF or transferrin receptor sorting; rather it specifically perturbs endosome-to-trans Golgi network (TGN) transport of the cation-independent mannose-6-phosphate receptor (CI-MPR) . Our data support an evolutionarily conserved function for SNX1 from yeast to mammals and provide functional insight into the molecular mechanisms underlying lipid-mediated protein targeting and tubular-based protein sorting . CONCLUSIONS: We conclude that through coincidence detection SNX1 associates with a microdomain of the early endosome-characterized by high membrane curvature and the presence of 3-phosphoinositides-from where it regulates tubular-based endosome-to-TGN retrieval of the CI-MPR.

Genomics, 2004 Sep, 84(3), 536 - 49
Analysis of the human VPS13 gene family; Velayos-Baeza A et al.; The gene mutated in chorea-acanthocytosis (CHAC; approved gene symbol VPS13A) encodes chorein, a protein similar to yeast Vps13p . We detected several similar putative human proteins by BLAST analysis of chorein . We characterized the structure of three new genes encoding these CHAC-similar proteins, located on chromosomes 1p36, 8q22, and 15q21 . The most similar gene in yeast to all four human genes is Vps13, and therefore the human genes were named VPS13A (CHAC, 9q21), VPS13B (8q22), VPS13C (15q21), and VPS13D (1p36) . VPS13B has recently been reported as COH1, altered in Cohen syndrome . For each gene, we describe several alternative splicing variants; at least two transcripts per gene are major forms . The expression pattern of these genes is ubiquitous, with some tissue-specific differences between several transcript variants . Protein sequence comparisons suggest that intramolecular duplications have played an important role in the evolution of this gene family.

Water Sci Technol, 2004, 50(5), 73 - 7
The effect of nitrogen supplementation on the efficiency of colour and COD removal by Malaysian white-rot fungi in textile dyeing effluent; Lee KK et al.; White-rot fungi, namely Coriolus versicolor and Schizophyllum commune, were studied for the biodecolorization of textile dyeing effluent in shaker-flask experiments . The results showed that C . versicolor was able to achieve 68% color removal after 5 days of treatment while that of S . commune was 88% in 9 days . Both fungi achieved the above results in non-sterile condition with diammonium hydrogen phosphate as the nutrient supplement . On the other hand, the best COD removal of 80% was obtained with C . versicolor in 9 days in sterile effluent with yeast extract as nutrient supplement, while S . commune was able to remove 85% COD within 8 days in non-sterile textile effluent supplemented with diammonium hydrogen phosphate.

J Biochem (Tokyo), 2004 Aug, 136(2), 169 - 76
HTRP--an immediate-early gene product induced by HSV1 infection in human embryo fibroblasts, is involved in cellular co-repressors; Li JF et al.; The interaction between virus and receptor is a process that mimics physiological ligand binding receptors and induces signal transduction . In the investigation of the interaction between HSV1 (Herpes Simplex virus 1) and human fibroblasts via virus binding to its receptor complex on cellular membranes, the HTRP (human transcription regulator protein), a protein encoded by an immediate-early gene of cellular response against the specific stimulation of HSV1 binding, was cloned from a cDNA library established from early gene response mRNA . The localization of HTRP expressed as a fusion polypeptide with a fluorescent protein in HeLa cells was confirmed to be the nucleus . The results of a yeast two-hybrid experiment indicated that HTRP is indeed involved in the interaction with the SAP (mSin3-associate polypeptide) complex via SAP30 . A pull-down test and Western blotting in vitro, and immunoprecipitation in vivo also provided evidence in support of this result . The interaction of HTRP with SAP30 in its conserved domain implies that this protein family, as the products of immediate-early genes, comprise functional molecules involved in the transcriptional regulation of cells, which might be related to the inhibition of some cell survival genes.

Mol Biol Evol, 2005 Feb, 22(2), 367 - 77 Epub 2005 Feb.
Evolution of the AID/APOBEC Family of Polynucleotide (Deoxy)cytidine Deaminases; Conticello SG et al.; The AID/APOBEC family (comprising AID, APOBEC1, APOBEC2, and APOBEC3 subgroups) contains members that can deaminate cytidine in RNA and/or DNA and exhibit diverse physiological functions (AID and APOBEC3 deaminating DNA to trigger pathways in adaptive and innate immunity; APOBEC1 mediating apolipoprotein B RNA editing) . The founder member APOBEC1, which has been used as a paradigm, is an RNA-editing enzyme with proposed antecedents in yeast . Here, we have undertaken phylogenetic analysis to glean insight into the primary physiological function of the AID/APOBEC family . We find that although the family forms part of a larger superfamily of deaminases distributed throughout the biological world, the AID/APOBEC family itself is restricted to vertebrates with homologs of AID (a DNA deaminase that triggers antibody gene diversification) and of APOBEC2 (unknown function) identifiable in sequence databases from bony fish, birds, amphibians, and mammals . The cloning of an AID homolog from dogfish reveals that AID extends at least as far back as cartilaginous fish . Like mammalian AID, the pufferfish AID homolog can trigger deoxycytidine deamination in DNA but, consistent with its cold-blooded origin, is thermolabile . The fine specificity of its mutator activity and the biased codon usage in pufferfish IgV genes appear broadly similar to that of their mammalian counterparts, consistent with a coevolution of the antibody mutator and its substrate for the optimal targeting of somatic mutation during antibody maturation . By contrast, APOBEC1 and APOBEC3 are later evolutionary arrivals with orthologs not found in pufferfish (although synteny with mammals is maintained in respect of the flanking loci) . We conclude that AID and APOBEC2 are likely to be the ancestral members of the AID/APOBEC family (going back to the beginning of vertebrate speciation) with both APOBEC1 and APOBEC3 being mammal-specific derivatives of AID and a complex set of domain shuffling underpinning the expansion and evolution of the primate APOBEC3s.

Mol Pharmacol, 2004 Nov, 66(5), 1285 - 92
Role of mPKCI, a novel mu-opioid receptor interactive protein, in receptor desensitization, phosphorylation, and morphine-induced analgesia; Guang W et al.; The human mu-opioid receptor (HmuOR) is a G-protein coupled receptor that mediates analgesia, euphoria and other important central and peripheral neurological functions . In this study, we found in a yeast two-hybrid screen that a protein kinase C-interacting protein (PKCI) specifically interacts with the C terminus of HmuOR . The interaction of PKCI with HmuOR was recapitulated in Chinese hamster ovary cells that express the full-length HmuOR and PKCI proteins . The affinity of HmuOR for an opioid ligand and its ability to mediate the activation of a G-protein were not altered by their interaction . However, the association of PKCI with HmuOR reduced agonist-induced inhibition of adenylyl cyclase and suppressed HmuOR desensitization partially at the G protein level and completely at the adenylyl cyclase level . Furthermore, PMA-induced, but not DAMGO-induced, HmuOR phosphorylation was partially inhibited by the coexpression of PKCI, suggesting that PKCI exerts a selective regulatory effect on HmuOR signaling . This effect was specific to the mu-opioid receptor because delta-opioid receptor desensitization was unaffected by PKCI . In addition, behavioral studies revealed that both basal and morphine-induced analgesia were significantly enhanced in the mutant mice that lacked expression of PKCI gene, and these mice developed a greater extent of tolerance to morphine analgesia . Taken together, these results suggest that PKCI functions as a negative regulator in HmuOR desensitization, phosphorylation, and in mediating morphine analgesia.

Development, 2004 Nov, 131(22), 5613 - 26 Epub 2004 Oct 20.
Negative regulation of Smad2 by PIASy is required for proper Xenopus mesoderm formation; Daniels M et al.; Mesoderm induction and patterning are primarily regulated by the concentration of locally expressed morphogens such as members of the TGFbetasuperfamily . Smad2 functions as a transcription factor to regulate expression of mesodermal genes downstream of such morphogens . We have identified Xenopus PIASy (XPIASy), a member of the PIAS family, by yeast two-hybrid screening using Xenopus Smad2 (XSmad2) as a bait . During mesoderm induction, XPIASy is expressed in the animal half of embryos with a ventral high-dorsal low gradient at the marginal zone . XPIASy expression is positively and negatively regulated by activities of the XSmad2 and Wnt pathways, respectively . Interestingly, inhibition of XPIASy by morpholinos induces elongation of animal caps with induction of mesoderm genes even in the absence of their morphogen-mediated activation . In addition, their introduction into the ventral marginal zone results in a secondary axis formation . Gain-of-function analysis revealed that XPIASy inhibits mesoderm induction by specific and direct downregulation of XSmad2 transcriptional activity . These observations indicate that XPIASy functions as an essential negative regulator of the XSmad2 pathway to ensure proper mesoderm induction at the appropriate time and in the appropriate region, and suggest that both the initial step of morphogen-mediated activation of the XSmad2 pathway and regulation of the final downstream transcription step have crucial roles in mesoderm induction and patterning.

Hum Mol Genet, 2004 Dec 15, 13(24), 3171 - 9 Epub 2004 Dec 15.
Heterologous mitochondrial DNA recombination in human cells; D'Aurelio M et al.; Inter-molecular heterologous mitochondrial DNA (mtDNA) recombination is known to occur in yeast and plants . Nevertheless, its occurrence in human cells is still controversial . To address this issue we have fused two human cytoplasmic hybrid cell lines, each containing a distinct pathogenic mtDNA mutation and specific sets of genetic markers . In this hybrid model, we found direct evidence of recombination between these two mtDNA haplotypes . Recombinant mtDNA molecules in the hybrid cells were identified using three independent experimental approaches . First, recombinant molecules containing genetic markers from both parental alleles were demonstrated with restriction fragment length polymorphism of polymerase chain reaction products, by measuring the relative frequencies of each marker . Second, fragments of recombinant mtDNA were cloned and sequenced to identify the regions involved in the recombination events . Finally, recombinant molecules were demonstrated directly by Southern blot using appropriate combinations of polymorphic restriction sites and probes . This combined approach confirmed the existence of heterogeneous species of recombinant mtDNA molecules in the hybrid cells . These findings have important implications for mtDNA-related diseases, the interpretation of human evolution and population genetics and forensic analyses based on mtDNA genotyping.

Hum Mol Genet, 2004 Dec 15, 13(24), 3115 - 25 Epub 2004 Dec 15.
Huntingtin-associated protein 1 (Hap1) mutant mice bypassing the early postnatal lethality are neuroanatomically normal and fertile but display growth retardation; Dragatsis I et al.; Huntingtin-associated protein 1 (Hap1) is the first huntingtin interacting protein identified in a yeast two-hybrid screen . Although Hap1 expression has been demonstrated in neuronal and non-neuronal tissues, its molecular role is poorly understood . Recently, it has been shown that targeted disruption of Hap1 in mice results in early postnatal death as a result of depressed feeding behavior . Although this result clearly demonstrates an essential role of Hap1 in postnatal feeding, the mechanisms leading to this deficiency, as well as the role of Hap1 in adults, remain unclear . Here we show that Hap1 null mutants display suckling defects and die within the first days after birth due to starvation . Upon reduction of the litter size, some mutants survive into adulthood and display growth retardation with no apparent brain or behavioral abnormalities, suggesting that Hap1 function is essential only for early postnatal feeding behavior . Using a conditional gene repair strategy, we also show that the early lethality can be rescued if Hap1 expression is restored in neuronal cells before birth . Furthermore, no synergism was observed between Hap1 and huntingtin mutation during mouse development . Our results demonstrate that Hap1 has a fundamental role in regulating postnatal feeding in the first 2 weeks after birth and a non-essential role in the adult mouse.

J Biol Chem, 2004 Dec 24, 279(52), 54230 - 40 Epub 2004 Oct 20.
Human ADA3 binds to estrogen receptor (ER) and functions as a coactivator for ER-mediated transactivation; Meng G et al.; We have recently identified the hADA3 protein, the human homologue of yeast transcriptional coactivator yADA3, as a novel HPV16 E6 target . Using ectopic expression approaches, we further demonstrated that hADA3 directly binds to the 9-cis retinoic acid receptors alpha and beta, and functions as a coactivator for retinoid receptor-mediated transcriptional activation . Here, we examined the role of endogenous hADA3 as a coactivator for estrogen receptor (ER), an important member of the nuclear hormone receptor superfamily . We show that ADA3 directly interacts with ER alpha and ER beta . Using the chromatin immunoprecipitation assay, we also show that hADA3 is a component of the activator complexes bound to the native ER response element within the promoter of the estrogen-responsive gene pS2 . Furthermore, using an ER response element-luciferase reporter, we show that overexpression of ADA3 enhances the ER alpha- and ER beta-mediated sequence-specific transactivation . Reverse transcription-PCR analysis showed an ADA3-mediated increase in estrogen-induced expression of the endogenous pS2 gene . More importantly, using RNA interference against hADA3, we demonstrate that inhibition of endogenous hADA3 inhibited ER-mediated transactivation and the estrogen-induced increase in the expression of pS2, cathepsin D, and progesterone receptor, three widely known ER-responsive genes . The HPV E6 protein, by targeting hADA3 for degradation, inhibited the ER alpha-mediated transactivation and the protein expression of ER target genes . Thus, our results demonstrate that ADA3 directly binds to human estrogen receptor and enhances the transcription of ER-responsive genes, suggesting a broader role of mammalian hADA3 as a coactivator of nuclear hormone receptors and the potential role of these pathways in HPV oncogenesis.

J Biol Chem, 2004 Dec 24, 279(52), 54849 - 61 Epub 2004 Oct 20.
Characterization of mammalian Ecm29, a 26 S proteasome-associated protein that localizes to the nucleus and membrane vesicles; Gorbea C et al.; In addition to its thirty or so core subunits, a number of accessory proteins associate with the 26 S proteasome presumably to assist in substrate degradation or to localize the enzyme within cells . Among these proteins is ecm29p, a 200-kDa yeast protein that contains numerous HEAT repeats as well as a putative VHS domain . Higher eukaryotes possess a well conserved homolog of yeast ecm29p, and we produced antibodies to three peptides in the human Ecm29 sequence . The antibodies show that Ecm29 is present exclusively on 26 S proteasomes in HeLa cells and that Ecm29 levels vary markedly among mouse organs . Confocal immunofluorescence microscopy localizes Ecm29 to the centrosome and a subset of secretory compartments including endosomes, the ER and the ERGIC . Ecm29 is up-regulated 2-3-fold in toxinresistant mutant CHO cells exhibiting increased rates of ER-associated degradation . Based on these results we propose that Ecm29 serves to couple the 26 S proteasome to secretory compartments engaged in quality control and to other sites of enhanced proteolysis.

J Biol Chem, 2005 Jan 7, 280(1), 695 - 702 Epub 2004 Oct 20.
The Type I Hsp40 Zinc Finger-like Region Is Required for Hsp70 to Capture Non-native Polypeptides from Ydj1; Fan CY et al.; The cytosolic yeast Hsp40 Ydj1 contains a conserved zinc finger-like region (ZFLR), which has two zinc-binding domains (ZBD), that helps regulate and specify Hsp70 function . To investigate the mechanism for Ydj1 ZFLR action, ZBDI and ZBDII mutants were constructed and characterized . ZBDII mutants exhibited temperature-sensitive growth defects, but yeast tolerated mutation of ZBDI . However, ZBDI and ZBDII mutants were defective at facilitating androgen receptor (AR) folding . Defective AR folding was associated with the accumulation of complexes between AR and Ydj1 ZFLR mutants and a reduction in Hsp70.AR complex formation . Purified Ydj1 ZBDI and ZBDII mutants could bind non-native polypeptides but could not deliver luciferase to Hsp70 and were defective at luciferase refolding . Interestingly, the ability of Ydj1 to synergize with Hsp70 to suppress thermally induced protein aggregation was blocked by mutation of ZBDII, but not ZBDI . Hence, ZBDII is required for yeast to survive heat stress because it is essential for Ydj1 to cooperate with Hsp70 to suppress protein aggregation . On the other hand, protein folding is dependent upon the action of both ZBDI and ZBDII because each is required for Hsp70 to capture non-native polypeptides from Ydj1.

Biochem J . 2004 Oct 21; {Epub ahead of print}
Identification and characterization of the human ARD1-NATH protein acetyltransferase complex; Arnesen T et al.; Protein acetyltransferases and deacetylases have been implicated in oncogenesis, apoptosis and cell cycle regulation . Most of the protein acetyltransferases described acetylate epsilon-amino groups of lysine residues within proteins . Mouse ARD1 (homologue of yeast Ard1p: Arrest defective 1 protein) is the only known protein acetyltransferase catalysing acetylation of proteins at both alpha- (N-terminus) and epsilon-amino groups . Yeast Ard1p interacts with Nat1p (N-acetyltransferase 1 protein) to form a functional N-Acetyltransferase . We now describe the human homologue of Nat1p, NATH (N-Acetyl Transferase Human), as the partner of the human ARD1 protein . Included in the characterization of the NATH and hARD1 proteins is the following: 1) Endogenous NATH and hARD1 proteins are expressed in human epithelial, glioma and promyelocytic cell lines; 2) NATH and hARD1 form a stable complex as investigated by reciprocal immunoprecipitations followed by mass spectrometry analysis; 3) The NATH-hARD1 complex expresses N-terminal acetylation activity; 4) NATH and hARD1 interacts with ribosomal subunits indicating a cotranslational acetyltransferase function; 5) NATH is localised in the cytoplasm while hARD1 localises both to the cytoplasm and nucleus; 6) hARD1 partially colocalises in nuclear spots with the transcription factor HIF-1alpha, a known epsilon-amino substrate of ARD1; 7) NATH and hARD1 are cleaved during apoptosis resulting in a decreased N-acetyltransferase activity . This study identifies the human homologues of the yeast Ard1p and Nat1p proteins and presents new aspects of the NATH and hARD1 proteins relative to their yeast homologues.

Biochem J . 2004 Oct 21; {Epub ahead of print}
Neural precursor cell expressed, developmentally down-regulated 4-2 (NEDD4-2) negatively regulates transforming growth factor-beta (TGF-beta) signaling by inducing ubiquitin-mediated degradation of Smad2 and TGF-beta type I receptor; Kuratomi G et al.; Inhibitory Smad, Smad7, is a potent inhibitor of transforming growth factor (TGF)-beta superfamily signaling . By binding to activated type I receptors, it prevents the activation of receptor-regulated Smads (R-Smads) . To identify new components of the Smad pathway, we performed yeast two-hybrid screening using Smad7 as bait, and identified neural precursor cell expressed, developmentally down-regulated 4-2 (NEDD4-2) as a direct binding partner of Smad7 . NEDD4-2 is structurally similar to Smad ubiquitin regulatory factors (Smurfs) 1 and 2 which were previously identified as E3 ubiquitin ligases for R-Smads and TGF-beta superfamily receptors . NEDD4-2 functions like Smurfs 1 and 2, in that it associated with TGF-beta type I receptor via Smad7 and induces its ubiquitin-dependent degradation . Moreover, NEDD4-2 bound to TGF-beta-specific R-Smads, Smads 2 and 3, in a ligand-dependent manner, and induced degradation of Smad2, but not Smad3 . However, in contrast to Smurf2, NEDD4-2 failed to induce ubiquitination of SnoN, although NEDD4-2 bound to SnoN via Smad2 more strongly than Smurf2 . We further showed that overexpressed NEDD4-2 prevents transcriptional activity induced by TGF-beta and BMP, whereas silencing of the NEDD4-2 gene by siRNA resulted in enhancement of the responsiveness to TGF-beta superfamily cytokines . These data suggest that NEDD4-2 is a member of the Smurf-like C2-WW-HECT type E3 ubiquitin ligases, which negatively regulates TGF-beta superfamily signaling through similar, but not identical, mechanisms to those used by Smurfs.

J Pathol, 2004 Nov, 204(4), 489 - 505
The pathobiology of the septin gene family; Hall PA et al.; Septins are an evolutionarily conserved group of GTP-binding and filament-forming proteins that belong to the large superclass of P-loop GTPases . While originally discovered in yeast as cell division cycle mutants with cytokinesis defects, they are now known to have diverse cellular roles which include polarity determination, cytoskeletal reorganization, membrane dynamics, vesicle trafficking, and exocytosis . Septin proteins form homo- and hetero-oligomeric polymers which can assemble into higher-order filaments . They are also known to interact with components of the cytoskeleton, ie actin and tubulin . The precise role of GTP binding is not clear but a current model suggests that it is associated with conformational changes which alter binding to other proteins . There are at least 12 human septin genes, and although information on expression patterns is limited, most undergo complex alternative splicing with some degree of tissue specificity . Nevertheless, an increasing body of data implicates the septin family in the pathogenesis of diverse disease states including neoplasia, neurodegenerative conditions, and infections . Here the known biochemical properties of mammalian septins are reviewed in the light of the data from yeast and other model organisms . The data implicating septins in human disease are considered and a model linking these data is proposed . It is posited that septins can act as regulatable scaffolds where the stoichiometry of septin associations, modifications, GTP status, and the interactions with other proteins allow the regulation of key cellular processes including polarity determination . Derangements of such septin scaffolds thus explain the role of septins in disease states . Copyright (c) 2004 Pathological Society of Great Britain and IrelandPublication Types:

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