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 sh