Methods Mol Biol, 2004, 270, 379 - 94 FISH mapping for helminth genome; Hirai H et al.; Basic techniques for fluorescence in situ hybridization (FISH) mapping that have been used in genome projects on schistosomes and filariae are introduced . The chapter shows techniques specific for bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) clones and includes experiences of chromosome preparation, DNA labeling, hybridization, microscopy, and localization of BAC clones. Methods Mol Biol, 2004, 270, 353 - 78 Chromosome fragmentation in leishmania; Dubessay P et al.; Chromosome fragmentation (CF) constitutes one means of manipulating eukaryotic genomes and provides a powerful tool for examining both the structure and function of chromosomes . During the past 15 yr, CF, which is based on the use of transfection, has been widely used in yeast and mammals to elucidate the functional elements required for normal chromosome maintenance . However, in view of the relatively late development of parasite genome projects, this strategy has only been used recently in parasites . Here, we describe basic methods for CF (except telomere-mediated fragmentation) experiments and analysis in Leishmania . Current limitations of this methodology are precisely the lack of knowledge of the nature of centromeres and autonomously replicating sequences in this and other protozoa, the poor understanding of precise recombination mechanisms, as well as the fact that the deletion of unknown genes essential to parasite survival may interfere with recombination events and chromosomal rearrangements . Still, this powerful method has enriched our basic knowledge of chromosomal structure and maintenance. J Vet Diagn Invest, 2004 May, 16(3), 248 - 50 Granulomatous lymphadenitis and splenitis associated with Monocillium indicum infection in a dog; Mackie JT et al.; This report describes severe generalized granulomatous lymphadenitis and splenitis in a 5-year-old, spayed female, Rottweiler dog with anorexia and diarrhea . There was replacement or effacement of much of the parenchyma of the lymph nodes and spleen by sheets of macrophages, multinucleated giant cells, and myriad nonpigmented fungal organisms, most of which appeared to be intracellular . These organisms were very pleomorphic, including large chlamydospore-like cells, small round yeast-like cells, and septate hyphae . A fungus identified as Monocillium indicum was isolated from lymph node tissue . To the authors' knowledge, this is the first report of infection with Monocillium in either humans or other animals. EMBO J, 2004 Jun 16, 23(12), 2369 - 80 Epub 2004 May 20. Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase; Yeung F et al.; NF-kappaB is responsible for upregulating gene products that control cell survival . In this study, we demonstrate that SIRT1, a nicotinamide adenosine dinucleotide-dependent histone deacetylase, regulates the transcriptional activity of NF-kappaB . SIRT1, the mammalian ortholog of the yeast SIR2 (Silencing Information Regulator) and a member of the Sirtuin family, has been implicated in modulating transcriptional silencing and cell survival . SIRT1 physically interacts with the RelA/p65 subunit of NF-kappaB and inhibits transcription by deacetylating RelA/p65 at lysine 310 . Treatment of cells with resveratrol, a small-molecule agonist of Sirtuin activity, potentiates chromatin-associated SIRT1 protein on the cIAP-2 promoter region, an effect that correlates with a loss of NF-kappaB-regulated gene expression and sensitization of cells to TNFalpha-induced apoptosis . While SIRT1 is capable of protecting cells from p53-induced apoptosis, our work provides evidence that SIRT1 activity augments apoptosis in response to TNFalpha by the ability of the deacetylase to inhibit the transactivation potential of the RelA/p65 protein. EMBO J, 2004 Jun 2, 23(11), 2206 - 15 Epub 2004 May 20. Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein; Lee RJ et al.; Aberrant polypeptides in the endoplasmic reticulum (ER) are retro-translocated to the cytoplasm and degraded by the 26S proteasome via ER-associated degradation (ERAD) . To begin to resolve the requirements for the retro-translocation and degradation steps during ERAD, a cell-free assay was used to investigate the contributions of specific factors in the yeast cytosol and in ER-derived microsomes during the ERAD of a model, soluble polypeptide . As ERAD was unaffected when cytoplasmic chaperone activity was compromised, we asked whether proteasomes on their own supported both export and degradation in this system . Proficient ERAD was observed if wild-type cytosol was substituted with either purified yeast or mammalian proteasomes . Moreover, addition of only the 19S cap of the proteasome catalyzed ATP-dependent export of the polypeptide substrate, which was degraded upon subsequent addition of the 20S particle. Development, 2004 Jun, 131(12), 2971 - 81 Epub 2004 May 19. Identification of new members of Fertilisation Independent Seed Polycomb Group pathway involved in the control of seed development in Arabidopsis thaliana; Guitton AE et al.; In higher plants, double fertilisation initiates seed development . One sperm cell fuses with the egg cell and gives rise to the embryo, the second sperm cell fuses with the central cell and gives rise to the endosperm . The endosperm develops as a syncytium with the gradual organisation of domains along an anteroposterior axis defined by the position of the embryo at the anterior pole and by the attachment to the placenta at the posterior pole . We report that ontogenesis of the posterior pole in Arabidopsis thaliana involves oriented migration of nuclei in the syncytium . We show that this migration is impaired in mutants of the three founding members of the FERTILIZATION INDEPENDENT SEED (FIS) class, MEDEA (MEA), FIS2 and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) . A screen based on a green fluorescent protein (GFP) reporter line allowed us to identify two new loci in the FIS pathway, medicis and borgia . We have cloned the MEDICIS gene and show that it encodes the Arabidopsis homologue of the yeast WD40 domain protein MULTICOPY SUPRESSOR OF IRA (MSI1) . The mutations at the new fis loci cause the same cellular defects in endosperm development as other fis mutations, including parthenogenetic development, absence of cellularisation, ectopic development of posterior structures and overexpression of the GFP marker. J Biol Chem, 2004 Jul 23, 279(30), 31164 - 70 Epub 2004 May 18. Human SAD1 kinase is involved in UV-induced DNA damage checkpoint function; Lu R et al.; Checkpoint activation by DNA damage during G(2) prevents activation of cyclin B/Cdc2 complexes, and as a consequence, mitotic entry is blocked . Although initiation and maintenance of G(2) arrest are known to be regulated by at least two distinct signaling pathways, including those of p38MAPK and ataxia-telangiectasia-mutated (ATM)- and Rad3-related (ATR)-Chk1 in higher eukaryotes, the actual number of signaling pathways involved in this regulation is still elusive . In the present study, we identified human SAD1 (hsSAD1) by searching a sequence data base . The predicted hsSAD1 protein comprises 778 amino acids and shares significant homology with the fission yeast Cdr2, a mitosis-regulatory kinase, and Caenorhabditis elegans SAD1, a neuronal cell polarity regulator . HsSAD1 transcript was expressed ubiquitously with the highest levels of expression in brain and testis . HsSAD1 specifically phosphorylated Wee1A, Cdc25-C, and -B on Ser-642, Ser-216, and Ser-361 in vitro, respectively . Overexpression of hsSAD1 resulted in an increased phosphorylation of Cdc25C on Ser-216 in vivo . DNA damage induced by UV or methyl methane sulfonate but not by IR enhanced endogenous hsSAD1 kinase activity in a caffeine-sensitive manner and caused translocation of its protein from cytoplasm to nucleus . Overexpression of wild-type hsSAD1 induced G(2)/M arrest in HeLa S2 cells . Furthermore, UV-induced G(2)/M arrest was partially abrogated by the reduced expression of hsSAD1 using small interfering RNA . These results suggest that hsSAD1 acts as checkpoint kinase upon DNA damage induced by UV or methyl methane sulfonate . The identification of this new kinase suggests the existence of an alternative checkpoint pathway other than those of ATR-Chk1 and p38MAPK. Mol Cell, 2004 May 21, 14(4), 420 - 1 Telomeres are double-strand DNA breaks hidden from DNA damage responses; Shay JW et al.; A network of ATM/ATR-mediated events regulates cell cycle checkpoints and genomic integrity and contributes to the processing of DNA double-strand breaks in both genomic DNA and at telomeres . In yeast and in human cells, investigators, including, and Herbig et al., published in this issue of Molecular Cell, are beginning to decipher the signaling pathways involved at the telomeres. Biochem Biophys Res Commun, 2004 Jun 11, 318(4), 920 - 6 Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach; Arora A et al.; Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function . Cholesterol is the most representative sterol present in higher eukaryotes . It is often found distributed non-randomly in domains or pools in biological and model membranes . Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic . Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component . Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear . We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH . Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization . These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes. FEBS Lett, 2004 May 21, 566(1-3), 60 - 4 Involvement of GSK-3beta in TWEAK-mediated NF-kappaB activation; De Ketelaere A et al.; Glycogen synthase kinase-3beta (GSK-3beta) is a key component of several signaling pathways . We found that a short variant of 'TNF-like weak inducer of apoptosis' (shortTWEAK) formed a complex with GSK-3beta in a yeast two-hybrid system . We demonstrate that shortTWEAK and GSK-3beta colocalize in the nucleus of human neuroblastoma cells . We also show that TWEAK is internalized in different cell lines and that it translocates to the nucleus . This event causes the degradation of IkappaBalpha, the nuclear translocation of both GSK-3beta and p65, and the induction of NF-kappaB-driven gene expression . We demonstrate that the induction of IL-8 expression by TWEAK can be counteracted by LiCl . Taken together, these data suggest that GSK-3beta plays an important role in the signal transduction pathway between TWEAK and NF-kappaB. J Mol Biol, 2004 Jun 4, 339(3), 495 - 504 Identification of a new antizyme mRNA +1 frameshifting stimulatory pseudoknot in a subset of diverse invertebrates and its apparent absence in intermediate species; Ivanov IP et al.; The expression of eukaryotic antizyme genes requires +1 translational frameshifting . The frameshift in decoding most vertebrate antizyme mRNAs is stimulated by an RNA pseudoknot 3' of the frameshift site . Although the frameshifting event itself is conserved in a wide variety of organisms from yeast to mammals, until recently no corresponding 3' RNA pseudoknot was known in invertebrate antizyme mRNAs . A pseudoknot, different in structure and origin from its vertebrate counterparts, is now shown to be encoded by the antizyme genes of distantly related invertebrates . Identification of the 3' frameshifting stimulator in intermediate species or other invertebrates remains unresolved. RNA, 2004 Jun, 10(6), 942 - 53 Xenopus U3 snoRNA docks on pre-rRNA through a novel base-pairing interaction; Borovjagin AV et al.; U3 small nucleolar RNA (snoRNA) is essential for rRNA processing to form 18S ribosomal RNA (rRNA) . Previously, it has been shown that nucleolin is needed to load U3 snoRNA on pre-rRNA . However, as documented here, this is not sufficient . We present data that base-pairing between the U3 hinges and the external transcribed spacer (ETS) is critical for functional alignment of U3 on its pre-rRNA substrate . Additionally, the interaction between the U3 hinges and the ETS is proposed to serve as an anchor to hold U3 on the pre-rRNA substrate, while box A at the 5' end of U3 snoRNA swivels from ETS contacts to 18S rRNA contacts . Compensatory base changes revealed base-pairing between the 3' hinge of U3 snoRNA and region E1 of the ETS in Xenopus pre-rRNA; this novel interaction is required for 18S rRNA production . In contrast, base-pairing between the 5' hinge of U3 snoRNA and region E2 of the ETS is auxiliary, unlike the case in yeast where it is required . Thus, higher and lower eukaryotes use different interactions for functional association of U3 with pre-rRNA . The U3 hinge sequence varies between species, but covariation in the ETS retains complementarity . This species-specific U3-pre-rRNA interaction offers a potential target for a new class of antibiotics to prevent ribosome biogenesis in eukaryotic pathogens. Bioinformatics, 2004 Nov 1, 20(16), 2711 - 8 Epub 2004 May 14. Mining gene expression data for positive and negative co-regulated gene clusters; Ji L et al.; MOTIVATION: Analysis of gene expression data can provide insights into the positive and negative co-regulation of genes . However, existing methods such as association rule mining are computationally expensive and the quality and quantities of the rules are sensitive to the support and confidence values . In this paper, we introduce the concept of positive and negative co-regulated gene cluster (PNCGC) that more accurately reflects the co-regulation of genes, and propose an efficient algorithm to extract PNCGCs . RESULTS: We experimented with the Yeast dataset and compared our resulting PNCGCs with the association rules generated by the Apriori mining algorithm . Our results show that our PNCGCs identify some missing co-regulations of association rules, and our algorithm greatly reduces the large number of rules involving uncorrelated genes generated by the Apriori scheme . AVAILABILITY: The software is available upon request. Biol Cell, 2004 May, 96(4), 251 - 6 VAMP subfamilies identified by specific R-SNARE motifs; Rossi V et al.; In eukaryotes, interactions among the alpha-helical coiled-coil domains (CCDs) of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in mediating the fusion among vesicles and target membranes . Surface residues of such CCDs are major candidates to regulate the specificity of membrane fusion, as they may alter local charge at the interaction layers and surface of the fusion complex, possibly modulating its formation and/or the binding of non-SNARE regulatory factors . Based on alternate patterns in surface residues, we have identified two motifs which group vesicular SNAREs in two novel subfamilies: RG-SNAREs and RD-SNAREs . The RG-SNARE CCD is common to all members of the widely conserved family of long VAMPs or longins and to yeast and non-neuronal VAMPs, possibly mediating "basic" fusion mechanisms; instead, only synaptobrevins from Bilateria share an RD-SNARE CCD, which is likely to mediate interactions to specific, yet unknown, regulatory factors and/or be the landmark of rapid fusion reactions like that mediating the release of neurotransmitters. Curr Opin Cell Biol, 2004 Jun, 16(3), 285 - 92 mRNA export: an assembly line from genes to nuclear pores; Vinciguerra P et al.; mRNAs are transported from the nucleus to the cytoplasm by a machinery conserved from yeast to humans . Previous studies showed that mRNA export factors are loaded on nascent mRNAs during elongation, coupling transcription to export . More recently identified mRNA export factors connect transcription initiation to the export machinery associated with nuclear pores, and potentially tether active genes to the nuclear periphery . Furthermore, a newly identified link between the nuclear exosome and the transcription, 3'-end formation and export machineries suggests that early messenger ribonucleoprotein complex (mRNP) assembly is co-transcriptionally monitored . Moreover, inefficient mRNP assembly impairs transcription elongation, indicating tight interdependence of these processes . Finally, nuclear retention of unspliced mRNAs by the perinuclear Mlp proteins reveals a novel mechanism of mRNP surveillance prior to export. Gene, 2004 May 12, 332, 119 - 27 cDNA cloning and characterization of the human THRAP2 gene which maps to chromosome 12q24, and its mouse ortholog Thrap2; Musante L et al.; Characterization of a balanced t(2;12)(q37;q24) translocation in a patient with suspicion of Noonan syndrome revealed that the chromosome 12 breakpoint lies in the vicinity of a novel human gene, thyroid hormone receptor-associated protein 2 (THRAP2) . We therefore characterized this gene and its mouse counterpart in more detail . Human and mouse THRAP2/Thrap2 span a genomic region of about 310 and >170 kilobases (kb), and both contain 31 exons . Corresponding transcripts are approximately 9.5 kb long . Their open reading frames code for proteins of 2210 and 2203 amino acids, which are 93% identical . By northern blot analysis, human and mouse THRAP2/Thrap2 genes showed ubiquitous expression . Transcripts were most abundant in human skeletal muscle and in mouse heart . THRAP2 protein is 56% identical to human TRAP240, which belongs to the thyroid hormone receptor associated protein (TRAP) complex and is evolutionary conserved up to yeast . This complex is involved in transcriptional regulation and is believed to serve as adapting interface between regulatory proteins bound to specific DNA sequences and RNA polymerase II. Biochem Biophys Res Commun, 2004 Jun 4, 318(3), 714 - 8 NuRD complex component Mi-2beta binds to and represses RORgamma-mediated transcriptional activation; Johnson DR et al.; RORgamma is a nuclear receptor that binds to DNA motifs as a monomer to constitutively activate target genes . RORgamma plays an important role in thymocyte development and lymph node organogenesis, while the regulation of RORgamma-mediated transcriptional activation is currently unclear . The purpose of this study was to identify other nuclear proteins that interact with RORgamma . A yeast two-hybrid screen with Y190 yeast cells under stringent conditions resulted in the identification of CHD4, also known as Mi-2beta, as a RORgamma-interacting protein . This interaction was confirmed by GST pull-down assays . This interaction occurred within the middle regulatory region (amino acids 719-1164) of Mi-2beta . Transfection of Gal4-RORgamma into HeLa cells resulted in constitutive transactivation of the MH100-tk-luc reporter . The addition of Mi-2beta resulted in a dramatic 50% decrease in Gal4-RORgamma-mediated transactivation . These data demonstrate that RORgamma forms a protein-protein interaction with the regulatory region of Mi-2beta, resulting in inhibition of RORgamma transcriptional activity . These results may provide evidence as to how RORgamma-mediated transactivation is regulated by other nuclear proteins. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Apr, 21(2), 188 - 92 {DNA microarray reveals changes in gene expression of endothelial cells under shear stress}; Cheng M et al.; cDNA microarray technology is used as a powerful tool for rapid, comprehensive, and quantitative analysis of gene profiles of cultured human umbilical vein endothelial cells(HUVECs) in the normal static group and the shear stressed (4.20 dyne/cm2, 2 h) group . The total RNA from normal static cultured HUVECs was labeled by Cy3-dCTP, and total RNA of HUVECs from the paired shear stressed experiment was labeled by Cy5-dCTP . The expression ratios reported are the average from the two separate experiments . After bioinformatics analysis, we identified a total of 108 genes (approximately 0.026%) revealing differential expression . Of these 53 genes expressions were up-regulated, the most enhanced ones being human homolog of yeast IPP isomerase, human low density lipoprotein receptor gene, Squalene epoxidase gene, 7-dehydrocholesterol reductase, and 55 were down-regulated, the most decreased ones being heat shock 70 kD protein 1, TCB gene encoding cytosolic thyroid hormone-binding protein in HUVECs exposed to low shear stress . These results indicate that the cDNA microarray technique is effective in screening the differentially expressed genes in endothelial cells induced by various experimental conditions and the data may serve as stimuli to further researches. Mol Cell Biol, 2004 Jun, 24(11), 5016 - 27 Multiple genetic pathways involving the Caenorhabditis elegans Bloom's syndrome genes him-6, rad-51, and top-3 are needed to maintain genome stability in the germ line; Wicky C et al.; Bloom's syndrome (BS) is an autosomal-recessive human disorder caused by mutations in the BS RecQ helicase and is associated with loss of genomic integrity and an increased incidence of cancer . We analyzed the mitotic and the meiotic roles of Caenorhabditis elegans him-6, which we show to encode the ortholog of the human BS gene . Mutations in him-6 result in an enhanced irradiation sensitivity, a partially defective S-phase checkpoint, and in reduced levels of DNA-damage induced apoptosis . Furthermore, him-6 mutants exhibit a decreased frequency of meiotic recombination that is probably due to a defect in the progression of crossover recombination . In mitotically proliferating germ cells, our genetic interaction studies, as well as the assessment of the number of double-strand breaks via RAD-51 foci, reveal a complex regulatory network that is different from the situation in yeast . Although the number of double-strand breaks in him-6 and top-3 single mutants is elevated, the combined depletion of him-6 and top-3 leads to mitotic catastrophe concomitant with a massive increase in the level of double-strand breaks, a phenotype that is completely suppressed by rad-51 . him-6 and top-3 are thus needed to maintain low levels of double-strand breaks in normally proliferating germ cells, and both act in partial redundant pathways downstream of rad-51 to prevent mitotic catastrophy . Finally, we show that topoisomerase IIIalpha acts independently during a late stage of meiotic recombination. Mol Cell Biol, 2004 Jun, 24(11), 4824 - 34 Paired-type homeodomain transcription factors are imported into the nucleus by karyopherin 13; Ploski JE et al.; We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin beta family member Karyopherin 13 (Kap13) . Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen . Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation . Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay . Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain . Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted . The paired-type homeodomain transcription factor family includes more than 20 members . All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13 . We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13. J Exp Biol, 2004 May, 207(Pt 12), 2147 - 55 The sea urchin complement homologue, SpC3, functions as an opsonin; Clow LA et al.; The purple sea urchin Strongylocentrotus purpuratus expresses a homologue of complement component C3 (SpC3), which acts as a humoral opsonin . Significantly increased phagocytic activity was evident when yeast target cells were opsonized after incubation with coelomic fluid containing SpC3 . SpC3 could be detected on the surface of yeast, and phagocytic activity could be inhibited by an anti-SpC3 antibody . This indicates that SpC3 promotes phagocytosis by physically tagging target cells for ingestion . Confocal microscopy showed that opsonized yeast were phagocytosed by a single coelomocyte type (polygonal phagocytes), presumably because these cells express SpC3 receptors . Overall, these data indicate that SpC3 is a major humoral opsonin in S . purpuratus coelomic fluid. Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 987 - 93 Auriculibuller fuscus gen . nov., sp . nov . and Bullera japonica sp . nov., novel taxa in the Tremellales; Sampaio JP et al.; Seven phylloplane yeast strains that were collected in the Arrabida Natural Park, Portugal, and identified preliminarily as Bullera alba, the anamorphic stage of Bulleromyces albus, were investigated . In contrast to Bulleromyces albus, these isolates produced a brownish pigment when grown on potato dextrose agar . The pigment caused darkening of the cultures and diffused into the culture medium . Mating studies revealed that the Arrabida isolates did not react with the different mating types of Bulleromyces albus, but were sexually compatible with them and produced mycelium with clamp connections, haustoria and transversally septate basidia that ejected the basidiospores . Various taxonomic criteria that were evaluated during the present study and comparison with other sexual taxa of the Tremellales indicated that this teleomorph should be classified in a novel genus . Therefore, Auriculibuller fuscus gen . nov., sp . nov . (type strain, PYCC 5690(T)=CBS 9648(T)) is proposed . In addition, during the course of this investigation, a member of a novel Bullera species, Bullera japonica sp . nov . (type strain, PYCC 4534(T)=CBS 2013(T)), was found among collection isolates that were identified formerly as Bullera alba . In molecular phylogenetic analysis of the D1/D2 domains of the 26S rDNA and the internal transcribed spacer region, the two taxa were found to be closely related, but distinct at the species level. J Hosp Infect, 2004 May, 57(1), 8 - 13 Candidosis in the intensive care unit: a 20-year survey; Tortorano AM et al.; Deep-seated candidosis is a major problem in critically ill patients . Colonization with candida has been identified as an important independent risk factor for the development of candidaemia . Since the 1980s routine surveillance cultures have been performed on patients admitted for six or more days to the 'E . Vecla' intensive care unit (ICU) of the IRCCS Ospedale Maggiore di Milano . Colonization was observed on admission to the ICU in 59 of 117 (50%) patients in 2000 and 10 others developed colonization during their stay on the unit . A similar colonization rate was found in a survey performed 16 years earlier . The incidence of non-albicans Candida species, however, increased in 2000 . In particular, 24 patients were culture positive for Candida glabrata at some point during their hospital stay, whereas this species was isolated from only one patient in 1983-1984 . Antifungal susceptibility testing performed by Sensititre Yeast One revealed no resistance among 19 C . albicans strains tested . In contrast, fluconazole resistance was observed in two of 39 (5%) C . glabrata isolates from 23 patients . In the period 1983-2002, 28 candida bloodstream infections were identified and 12 were considered to be ICU-acquired (2.6/1000 hospitalized patients; 0.33/1000 patient days) . The low rate of ICU-acquired candidaemia despite the inclusion of severely compromised patients in this study confirms the usefulness of routine mycological surveillance in preventing deep-seated candidosis. DNA Cell Biol, 2004 Apr, 23(4), 223 - 5 Heat-shock proteins reverse the G2 arrest caused by HIV-1 viral protein R; Bukrinsky M et al.; HIV-1 Vpr is an important contributor to viral pathogenesis . Vpr displays several highly conserved pathogenic activities, including induction of cell cycle G(2) arrest and cell death . The host immune system, in turn, preferentially targets Vpr in an attempt to reduce its pathogenic effects . To identify innate anti-Vpr factors, we performed a genetic search for multicopy suppressors of Vpr-induced G(2) arrest in fission yeast . Several heat-shock proteins were identified in these experiments . Analyses in mammalian cells demonstrated that heatshock proteins HSP27 and HSP70 suppress Vpr-induced G2 arrest . This effect appears to be mediated by an interaction between heat shock proteins and Vpr . These results illustrate another example of antagonistic interactions between the viral and cellular proteins. Biochem J, 2004 Aug 15, 382(Pt 1), 307 - 14 Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase; Palumaa P et al.; Cox17, a copper chaperone for cytochrome c oxidase, is an essential and highly conserved protein . The structure and mechanism of functioning of Cox17 are unknown, and even its metalbinding stoichiometry is elusive . In the present study, we demonstrate, using electrospray ionization-MS, that porcine Cox17 binds co-operatively four Cu+ ions . Cu4Cox17 is stable at pH values above 3 and fluorescence spectra indicate the presence of a solvent-shielded multinuclear Cu(I) cluster . Combining our results with earlier EXAFS results on yeast CuCox17, we suggest that Cu4Cox17 contains a Cu4S6-type cluster . At supramillimolar concentrations, dithiothreitol extracts metals from Cu4Cox17, and an apparent copper dissociation constant KCu=13 fM was calculated from these results . Charge-state distributions of different Cox17 forms suggest that binding of the first Cu+ ion to Cox17 causes a conformational change from an open to a compact state, which may be the rate-limiting step in the formation of Cu4Cox17 . Cox17 binds non-co-operatively two Zn2+ ions, but does not bind Ag+ ions, which highlights its extremely high metal-binding specificity . We further demonstrate that porcine Cox17 can also exist in partly oxidized (two disulphide bridges) and fully oxidized (three disulphide bridges) forms . Partly oxidized Cox17 can bind one Cu+ or Zn2+ ion, whereas fully oxidized Cox17 does not bind metals . The metal-binding properties of Cox17 imply that, in contrast with other copper chaperones, Cox17 is designed for the simultaneous transfer of up to four copper ions to partner proteins . Metals can be released from Cox17 by non-oxidative as well as oxidative mechanisms. Poult Sci, 2004 May, 83(5), 776 - 82 Nitric oxide inhibition after Toxoplasma gondii infection of chicken macrophage cell lines; Guillermo LV et al.; Toxoplasma gondii infects many warm-blooded animals, including chickens . However, little is known about how this protozoan behaves within chicken macrophages . Thus, the microbicidal biology of HD11 and MQ-NCSU (available chicken macrophage cell lines) and the escaping mechanism of T . gondii were investigated . After infection, both cell lines were activated with lipopolysaccharide (LPS) and nitric oxide (NO), and reactive oxygen intermediates (ROI) were evaluated . T . gondii infected both cell lines, and 30 and 60% inhibition of NO production was detected in MQ-NCSU and HD11, respectively . In HD11, NO inhibition was not dependent on cyclooxygenase products . Although NO was partially inhibited, it did control T . gondii multiplication, showing the importance of this microbicidal molecule . Production of ROI was not detected in either cell line after T . gondii or yeast interaction . NADPH diaphorase (NADPH-d) activity, a histochemical marker of inducible NO synthase (iNOS), was detected at various levels in the HD11 population activated with LPS . The HD11 population infected with T . gondii showed a decrease in NADPH-d, indicating that NO production inhibition was related to iNOS disappearance in infected macrophages . These results demonstrate that in chicken macrophages T . gondii can also inhibit NO production, which suggests that an iNOS suppression mechanism might be used for better survival in macrophages. Mol Biol Evol, 2004 Aug, 21(8), 1534 - 7 Epub 2004 May 12. An assessment of accuracy, error, and conflict with support values from genome-scale phylogenetic data; Taylor DJ et al.; Despite the importance of molecular phylogenetics, few of its assumptions have been tested with real data . It is commonly assumed that nonparametric bootstrap values are an underestimate of the actual support, Bayesian posterior probabilities are an overestimate of the actual support, and among-gene phylogenetic conflict is low . We directly tested these assumptions by using a well-supported yeast reference tree . We found that bootstrap values were not significantly different from accuracy . Bayesian support values were, however, significant overestimates of accuracy but still had low false-positive error rates (0% to 2.8%) at the highest values (>99%) . Although we found evidence for a branch-length bias contributing to conflict, there was little evidence for widespread, strongly supported among-gene conflict from bootstraps . The results demonstrate that caution is warranted concerning conclusions of conflict based on the assumption of underestimation for support values in real data. J Biol Chem, 2004 Jun 4, 279(23), 24873 - 80 Epub 2004 Mar 26. PIAS3 suppresses NF-kappaB-mediated transcription by interacting with the p65/RelA subunit; Jang HD et al.; Nuclear factor-kappaB (NF-kappaB) is a transcription factor critical for key cellular processes, including immune response, apoptosis, and cell cycle progression . A yeast two-hybrid screening, using the Rel homology domain (RHD) of the p65 subunit (RelA) of NF-kappaB as bait, led to the isolation of PIAS3, previously identified as a specific inhibitor of STAT3 . We show that PIAS3 can directly associate with p65 using an in vitro pull-down and in vivo coimmunoprecipitation assays . When overexpressed, PIAS3 inhibits NF-kappaB-dependent transcription induced by treatment with tumor necrosis factor alpha (TNF-alpha) or interleukin-1beta or by overexpression of TNF family receptors such as RANK, TNFR1, and CD30 or signal transducers of TNF receptor-associated factors (TRAFs), including TRAF2, TRAF5, and TRAF6 . Downregulation of PIAS3 by RNA interference reverses its effect on TNF-alpha-mediated NF-kappaB activation . We found that an N-terminal region of PIAS3 is necessary for both the interaction with p65 and the transcriptional suppression activity . In addition, we found that an LXXLL coregulator signature motif located within the N-terminal region of PIAS3 is the minimal requirement for the interaction with p65 . Furthermore, we demonstrate that PIAS3 interferes with p65 binding to the CBP coactivator, thereby resulting in a decreased NF-kappaB-dependent transcription . Taken together, these data suggest that PIAS3 may function in vivo as a modulator in suppressing the transcriptional activity of p65. J Biol Chem, 2004 Jun 4, 279(23), 24834 - 43 Epub 2004 Mar 31. Transcriptional regulation by the repressor of estrogen receptor activity via recruitment of histone deacetylases; Kurtev V et al.; Histone acetyltransferases and deacetylases are recruited by transcription factors and adapter proteins to regulate specific subsets of target genes . We were interested in identifying interaction partners of histone deacetylase 1 (HDAC1) that might be involved in conferring target or substrate specificity . Using the yeast two-hybrid system, we isolated the repressor of estrogen receptor activity (REA) as a novel HDAC1-associated protein . We demonstrated the in vivo interaction of REA with HDAC1 and characterized the respective domains required for their interaction in vitro . In addition, we found that REA also associates with the class II histone deacetylase HDAC5 . In luciferase reporter assays, REA decreased transcription, and this repression was sensitive to the deacetylase inhibitor trichostatin A . Finally, we showed that REA specifically interacts with the chicken ovalbumin upstream binding transcription factors and II . The nuclear receptor chicken ovalbumin upstream binding transcription factor I was found to cooperate with REA and histone deacetylases in the repression of target genes . We, therefore, propose a novel function for REA as a mediator of transcriptional repression by nuclear hormone receptors via recruitment of histone deacetylases. J Neurosci Res, 2004 Jun 1, 76(5), 613 - 22 JAB1 enhances HAND2 transcriptional activity by regulating HAND2 DNA binding; Dai YS et al.; HAND2 (also known as dHAND) is a basic helix-loop-helix (bHLH) transcription factor essential for development of the heart, limbs, and neural crest-derived lineages . HAND2 expression is observed in a number of tissues derived from the neural crest, including components of the peripheral nervous system, where it has been shown to regulate sympathetic nervous system development . Here we show that HAND2 is expressed in both the sympathetic and the parasympathetic divisions of the autonomic nervous system (ANS) . How HAND2 functions during development of these neuronal lineages is uncertain . An important mechanism involved in HAND2's function is its interactions with other proteins . To understand better the molecular interactions regulating HAND2 during ANS development, we employed a yeast two-hybrid screen to identify HAND2-interacting proteins . One protein identified in this screen, Jun activation domain-binding protein (JAB1), is involved in numerous cell processes, including regulation of transcription and protein turnover . We show that JAB1 binds directly to the HLH domain of HAND2 and increases HAND2 transcription-stimulating activity . However, JAB1 does not contain a transcriptional activation domain, nor does it recruit an activation domain to HAND2 . Our data indicate that JAB1 augments HAND2 transcriptional activity by enhancing HAND2 DNA binding . We further show that enhanced HAND2 DNA binding is mediated through the HLH domain and not through the DNA binding domain . These results show that JAB1 regulates the transcriptional activity of HAND2 in a unique manner that may account, in part, for the apparent ability of this bHLH factor to regulate gene expression through numerous mechanisms . Mol Genet Genomics, 2004 Jun, 271(5), 532 - 44 Epub 2004 May 12. Identification of critical domains and putative partners for the Caenorhabditis elegans spindle component LIN-5; Fisk Green R et al.; Successful cell division requires proper assembly, placement and functioning of the spindle apparatus that segregates the chromosomes . The Caenorhabditis elegans gene lin-5 encodes a novel coiled-coil component of the spindle required for spindle positioning and chromosome segregation . To gain further insights into lin-5 function, we screened for dominant suppressors of the partial loss-of-function phenotype associated with the mutation lin-5(ev571ts ), and isolated 68 suppressing mutations . Eight out of the ten suppressors sequenced contained intragenic missense mutations immediately upstream of the lesion in lin-5(ev571ts ) . These probably help to stabilize protein-protein interactions mediated by the coiled-coil domain . This domain was found to be required for binding to several putative LIN-5 interacting (LFI) proteins identified in yeast two-hybrid screens . Interestingly, interaction with the coiled-coil protein LFI-1 was specifically reduced by the lin-5(ev571ts ) mutation and restored by a representative intragenic suppressor mutation . Immunostaining experiments showed that LIN-5 and LFI-1 may co-localize around the kinetochore microtubules during metaphase, indicating potential interaction in vivo . The coiled-coil domain of LIN-5 was also found to mediate homodimerization, while the C-terminal region of LIN-5 was sufficient for interaction with GPR-1, a recently identified component of a LIN-5 spindle-regulatory complex . A single amino-acid substitution in the N-terminal region of LIN-5, encoded by the e1457 allele, abolished all LIN-5 interactions . Taken together, our results indicate that the spindle functions of LIN-5 depend on interactions with multiple protein partners, and that these interactions are mediated through several different domains of LIN-5. PLoS Biol . 2004 May;2(5):E129 . Epub 2004 May 11. Early myocardial function affects endocardial cushion development in zebrafish; Bartman T et al.; Function of the heart begins long before its formation is complete . Analyses in mouse and zebrafish have shown that myocardial function is not required for early steps of organogenesis, such as formation of the heart tube or chamber specification . However, whether myocardial function is required for later steps of cardiac development, such as endocardial cushion (EC) formation, has not been established . Recent technical advances and approaches have provided novel inroads toward the study of organogenesis, allowing us to examine the effects of both genetic and pharmacological perturbations of myocardial function on EC formation in zebrafish . To address whether myocardial function is required for EC formation, we examined silent heart (sih(-/-)) embryos, which lack a heartbeat due to mutation of cardiac troponin T (tnnt2), and observed that atrioventricular (AV) ECs do not form . Likewise, we determined that cushion formation is blocked in cardiofunk (cfk(-/-)) embryos, which exhibit cardiac dilation and no early blood flow . In order to further analyze the heart defects in cfk(-/-) embryos, we positionally cloned cfk and show that it encodes a novel sarcomeric actin expressed in the embryonic myocardium . The Cfk(s11) variant exhibits a change in a universally conserved residue (R177H) . We show that in yeast this mutation negatively affects actin polymerization . Because the lack of cushion formation in sih- and cfk-mutant embryos could be due to reduced myocardial function and/or lack of blood flow, we approached this question pharmacologically and provide evidence that reduction in myocardial function is primarily responsible for the defect in cushion development . Our data demonstrate that early myocardial function is required for later steps of organogenesis and suggest that myocardial function, not endothelial shear stress, is the major epigenetic factor controlling late heart development . Based on these observations, we postulate that defects in cardiac morphogenesis may be secondary to mutations affecting early myocardial function, and that, in humans, mutations affecting embryonic myocardial function may be responsible for structural congenital heart disease. J Biol Chem, 2004 Jul 16, 279(29), 30287 - 97 Epub 2004 May 11. TALE homeodomain proteins regulate gonadotropin-releasing hormone gene expression independently and via interactions with Oct-1; Rave-Harel N et al.; Gonadotropin-releasing hormone (GnRH) is the central regulator of reproductive function . Expression of the GnRH gene is confined to a rare population of neurons scattered throughout the hypothalamus . Restricted expression of the rat GnRH gene is driven by a multicomponent enhancer and an evolutionarily conserved promoter . Oct-1, a ubiquitous POU homeodomain transcription factor, was identified as an essential factor regulating GnRH transcription in the GT1-7 hypothalamic neuronal cell line . In this study, we conducted a two-hybrid interaction screen in yeast using a GT1-7 cDNA library to search for specific Oct-1 cofactors . Using this approach, we isolated Pbx1b, a TALE homeodomain transcription factor that specifically associates with Oct-1 . We show that heterodimers containing Pbx/Prep1 or Pbx/Meis1 TALE homeodomain proteins bind to four functional elements within the GnRH regulatory region, each in close proximity to an Oct-1-binding site . Cotransfection experiments indicate that TALE proteins are essential for GnRH promoter activity in the GT1-7 cells . Moreover, Pbx1 and Oct-1, as well as Prep1 and Oct-1, form functional complexes that enhance GnRH gene expression . Finally, Pbx1 is expressed in GnRH neurons in embryonic as well as mature mice, suggesting that the associations between TALE homeodomain proteins and Oct-1 regulate neuron-specific expression of the GnRH gene in vivo. Biochem J, 2004 Aug 15, 382(Pt 1), 169 - 76 Functional analysis of the CXXC motif using phage antibodies that cross-react with protein disulphide-isomerase family proteins; Kimura T et al.; Polyclonal antibodies that had been raised against particular PDI (protein disulphide-isomerase) family proteins did not cross-react with other PDI family proteins . To evade immune tolerance to the important self-motif Cys-Xaa-Xaa-Cys, which is present in PDI family proteins, we used the phage display library {established by Griffiths, Williams, Hartley, Tomlinson, Waterhouse, Crosby, Kontermann, Jones, Low, Allison et al . (1994) EMBO J . 13, 3245-3260} to isolate successfully the phage antibodies that can cross-react with human and bovine PDIs, human P5, human PDI-related protein and yeast PDI . By measuring the binding of scFv (single-chain antibody fragment of variable region) to synthetic peptides and to mutants of PDI family proteins in a surface plasmon resonance apparatus, we identified clones that recognized sequences containing the CGHC motif or the CGHCK sequence . By using the isolated phage antibodies, we demonstrated for the first time that a lysine residue following the CXXC motif significantly increases the isomerase activities of PDI family proteins . Moreover, we demonstrated that the affinity of isolated scFvs for mutant PDI family proteins is proportional to the isomerase activities of their active sites. Biochem J, 2004 Aug 15, 382(Pt 1), 111 - 9 Analysis of two CBP (cAMP-response-element-binding protein-binding protein) interacting sites in GRIP1 (glucocorticoid-receptor-interacting protein), and their importance for the function of GRIP1; Huang SM et al.; The p160 co-activators, SRC1 (steroid receptor co-activator 1), GRIP1 (glucocorticoid-receptor-interacting protein 1) and ACTR (activator for thyroid hormone and retinoid receptors), have two ADs (activation domains), AD1 and AD2 . AD1 is a binding site for the related co-activators, CBP (cAMP-response-element-binding protein-binding protein) and p300, whereas AD2 binds to another co-activator, co-activator-associated arginine methyltransferase 1 (CARM1) . Here, we identified two CBP-interacting sites {amino acids 1075-1083 (site I) and 1095-1106 (site II)} in a so-called CBP-dependent transactivation domain (AD1; amino acids 1057-1109) of GRIP1 . Site I was the major site for CBP-dependent AD1 transactivation activity of GRIP1 whereas, following the deletion of site II, full or partial transactivation activity was retained without the recruitment of CBP in yeast, HeLa, human embryonic kidney 293 and CV-1 cells . GRIP1 (with a deletion of site II) expressed stronger co-activator activity than that of wild-type GRIP1 in the TR (thyroid receptor) and the AR (androgen receptor), but not the ER (oestrogen receptor), systems in HeLa cells . We also demonstrated that these CBP-binding sites of GRIP1 are not the only functional domains for its AD1 function in TR, AR and ER systems in HeLa cells by the exogenous overexpression of one E1A mutant, which led to a lack of CBP-binding ability . Our results suggest that these two CBP-interacting sites in the GRIP AD1 domain not only determine its AD1 activity, but are also involved in its co-activator functions in some nuclear receptors. J Biol Chem, 2004 Jul 23, 279(30), 31002 - 9 Epub 2004 May 10. Microtubule-associated protein light chain 2 is a stargazin-AMPA receptor complex-interacting protein in vivo; Ives JH et al.; The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors . The extreme C terminus of stargazin (sequence, -TTPV), confers high affinity for PDZ domain-containing proteins e.g . PSD-95 . Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface . Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin . Positive interactors included PDZ domain-containing proteins e.g . SAP97, SAP102, and PIST . Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor . This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin . Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2 . Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2 . Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2 . Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse. J Biol Chem, 2004 Jul 16, 279(29), 30490 - 7 Epub 2004 May 10. CGI-58 interacts with perilipin and is localized to lipid droplets . Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome; Yamaguchi T et al.; Lipid droplets (LDs) are a class of ubiquitous cellular organelles that are involved in lipid storage and metabolism . Although the mechanisms of the biogenesis of LDs are still unclear, a set of proteins called the PAT domain family have been characterized as factors associating with LDs . Perilipin, a member of this family, is expressed exclusively in the adipose tissue and regulates the breakdown of triacylglycerol in LDs via its phosphorylation . In this study, we used a yeast two-hybrid system to examine the potential function of perilipin . We found direct interaction between perilipin and CGI-58, a deficiency of which correlated with the pathogenesis of Chanarin-Dorfman syndrome (CDS) . Endogenous CGI-58 was distributed predominantly on the surface of LDs in differentiated 3T3-L1 cells, and its expression increased during adipocyte differentiation . Overexpressed CGI-58 tagged with GFP gathered at the surface of LDs and colocalized with perilipin . This interaction seems physiologically important because CGI-58 mutants carrying an amino acid substitution identical to that found in CDS lost the ability to be recruited to LDs . These mutations significantly weakened the binding of CGI-58 with perilipin, indicating that the loss of this interaction is involved in the etiology of CDS . Furthermore, we identified CGI-58 as a binding partner of ADRP, another PAT domain protein expressed ubiquitously, by yeast two-hybrid assay . GFP-CGI-58 expressed in non-differentiated 3T3-L1 or CHO-K1 cells was colocalized with ADRP, and the CGI-58 mutants were not recruited to LDs carrying ADRP, indicating that CGI-58 may also cooperate with ADRP. Anal Biochem, 2004 Jun 1, 329(1), 58 - 67 Increased sample capacity for genotyping and expression profiling by kinetic polymerase chain reaction; Watson RM et al.; We fabricated and evaluated high-throughput kinetic thermal cyclers with 768-reaction capacity for kinetic polymerase chain reaction (kPCR)-based genotyping and kinetic reverse transcription (kRT)-PCR-based transcript quantitation . The system uses dye-based detection with ethidium bromide and a single DNA polymerase-based PCR or RT-PCR assay . Allele-specific detection of the two most common hereditary hemochromotosis mutant alleles, C282Y and H63D, was reliably measured by kPCR using human DNA templates as low as 10 genome equivalents per assay . Transcript profiling was performed for 16 yeast transcripts ranging in intracellular abundance over four orders of magnitude . Standard deviations of the PCR cycle threshold values determined from multiple kRT-PCR assays in three different instruments ranged from 0.11 to 0.97 PCR cycles and were reproducible, transcript specific, and instrument independent . The effects of the sin3, gal11, and snf2 knockout mutations on expression of 385 yeast genes were evaluated by kRT-PCR and compared to published values determined by high-density oligonucleotide array and/or microarray analysis for snf2 and sin3 . The 768-reaction kinetic thermalcyclers, each with a capacity for more than a half million assays per year, are well suited to genomics applications such as single nucleotide polymorphism/disease association studies and genomewide transcription profiling where high sensitivity and accuracy are required. Dev Biol, 2004 Jun 1, 270(1), 19 - 30 Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis; Amthor H et al.; Follistatin is known to antagonise the function of several members of the TGF-beta family of secreted signalling factors, including Myostatin, the most powerful inhibitor of muscle growth characterised to date . In this study, we compare the expression of Myostatin and Follistatin during chick development and show that they are expressed in the vicinity or in overlapping domains to suggest possible interaction during muscle development . We performed yeast and mammalian two-hybrid studies and show that Myostatin and Follistatin interact directly . We further show that single modules of the Follistatin protein cannot associate with Myostatin suggesting that the entire protein is required for the interaction . We analysed the interaction kinetics of the two proteins and found that Follistatin binds Myostatin with a high affinity of 5.84 x 10(-10) M . We next tested whether Follistatin suppresses Myostatin activity during muscle development . We confirmed our previous observation that treatment of chick limb buds with Myostatin results in a severe decrease in the expression of two key myogenic regulatory genes Pax-3 and MyoD . However, in the presence of Follistatin, the Myostatin-mediated inhibition of Pax-3 and MyoD expression is blocked . We additionally show that Myostatin inhibits terminal differentiation of muscle cells in high-density cell cultures of limb mesenchyme (micromass) and that Follistatin rescues muscle differentiation in a concentration-dependent manner . In summary, our data suggest that Follistatin antagonises Myostatin by direct protein interaction, which prevents Myostatin from executing its inhibitory effect on muscle development. FEBS Lett, 2004 May 7, 565(1-3), 81 - 8 Reconstitution and functional characterization of the unusual bi-subunit type I DNA topoisomerase from Leishmania donovani; Das BB et al.; Leishmania donovani topoisomerase I is an unusual bi-subunit enzyme . The activity of the enzyme has been detected when the genes of the individual subunits were co-expressed in yeast {J . Biol . Chem . 278 (2003) 3521} . Here, we report for the first time, the in vitro reconstitution of the two recombinant proteins, LdTOP1L and LdTOP1S, corresponding to the large and small subunits and localization of the active enzyme in both the nucleus and kinetoplast . The proteins were purified from bacterial extract and the activity was measured by plasmid DNA relaxation assay . LdTOP1L and LdTOP1S form a direct 1:1 heterodimer complex through protein-protein interaction . Under standard relaxation assay condition (50 mM KCl and 10 mM Mg(2+)), reconstituted enzyme (LdTOP1LS) showed reduced processivity as well as 2-fold reduced affinity for DNA compared to eukaryotic monomeric rat liver topoisomerase I (RLTOP1) . Cleavage assay at various salt concentrations reveals that Camptothecin (CPT) enhanced the formation of "cleavable complex" at low salt . Interaction between the two subunits leading to the formation of an active complex could be explored as an insight for development of new therapeutic agents with specific selectivity. FEBS Lett, 2004 May 7, 565(1-3), 23 - 7 Low-density lipoprotein receptor-related protein interacts with MafB, a regulator of hindbrain development; Petersen HH et al.; The intracellular domain (ICD) of the low-density lipoprotein receptor-related protein (LRP) functionally interacts with adaptor proteins both as an integral part of the receptor polypeptide and after proteolytic release . Identification of such adaptors has been difficult because the ICD is self-activating in conventional transcription factor-based yeast two-hybrid screens . We adopted an alternative screen for the ICD that depends on the activation of the Ras-signaling pathway and uncovered the transcription factor MafB as novel ICD interacting protein . MafB is a regulator of hindbrain segmentation and interacts with the ICD through a leucine zipper domain . The ICD co-localizes with MafB to the nucleus and negatively regulates its transcriptional activity, suggesting a possible role for LRP in brain development. J Theor Biol, 2004 Jun 7, 228(3), 293 - 301 Effects of stochasticity in models of the cell cycle: from quantized cycle times to noise-induced oscillations; Steuer R; Noise and fluctuations are ubiquitous in living systems . Still, the interaction between complex biochemical regulatory systems and the inherent fluctuations ('noise') is only poorly understood . As a paradigmatic example, we study the implications of noise on a recently proposed model of the eukaryotic cell cycle, representing a complex network of interactions between several genes and proteins . The purpose of this work is twofold: First, we show that the inclusion of noise into the description of the system accounts for several recent experimental findings, as e.g . the existence of quantized cycle times in wee1- cdc25delta double-mutant cells of fission yeast . In the main part, we then focus on more general aspects of the interplay between noise and the dynamics of the system . In particular, we demonstrate that a stochastic description leads to qualitative changes in the dynamics, such as the emergence of noise-induced oscillations . These findings will be discussed in the light of an ongoing debate on models of cell division as limit-cycle oscillators versus checkpoint mechanisms. Urology, 2004 May, 63(5), 989 - 93 Clinical evaluation of p53 mutations in urothelial carcinoma by IHC and FASAY; Watanabe J et al.; OBJECTIVES: To assess the clinical benefit of the methods of detection of p53 mutations in human urothelial carcinoma . METHODS: A total of 75 surgical specimens of urothelial carcinoma were analyzed using a yeast functional assay (FASAY) and immunohistochemistry (IHC), in combination with sequencing analysis . RESULTS: Of the 75 specimens, 24 (32.0%) were positive (mutant) by FASAY and 23 (30.7%) were positive by IHC . The sequencing analysis confirmed that all 24 FASAY-positive tumors harbored mutations, and no mutations were detected in any FASAY-negative tumors . In contrast, nuclear accumulation of p53 protein was detected in 9 (17.6%) of 51 tumors with no mutation, and 10 (41.7%) of 24 tumors with mutation showed no positive staining on IHC . The mutations detected by FASAY and IHC were both associated with stage and grade, but null mutations of p53 were not associated with stage . Concerning chemosensitivity, 6 (85.7%) of 7 responders harbored p53 missense mutations in at least one allele (P = 0.01), and only 4 (57.1%) were judged positive by IHC (P = 0.13) . CONCLUSIONS: FASAY is more accurate than IHC in detecting the various types of p53 mutations, suggesting that a comprehensive approach for the detection of p53 mutations may be essential to elucidate their clinical significance. J Inorg Biochem, 2004 May, 98(5), 814 - 23 The stability of the cytochrome c scaffold as revealed by NMR spectroscopy; Berners-Price SJ et al.; NMR spectroscopy was used to study the effect of guanidinium chloride on the unfolding of horse heart and yeast iso-1 cytochrome c under mild alkaline conditions . The structural changes on the horse heart protein were detected through NOESY (Nuclear Overhauser Effect SpectroscopY) experiments whereas (15)N-(1)H heteronuclear NMR was used to monitor the behavior of the yeast protein . The latter represents the first characterization through (15)N-(1)H heteronuclear NMR spectroscopy of the guanidinium chloride induced unfolding of mitochondrial cytochrome c . The presence of denaturants decreases the temperature at which the native Met80 axial ligand is displaced from the iron center under the present mild alkaline conditions . The process can be described in terms of protein fragments behaving as unfolding units of different stability . The comparison between the two proteins indicates that the loop+helix connecting the proximal and distal sites, as well as the long Met80-containing loop immediately after a short helix, are structural characteristics of mitochondrial cytochrome c that appear to be responsible for the Met80-iron(III) bond fragility. Mol Cell Endocrinol, 2004 Mar 31, 217(1-2), 221 - 7 The myosin binding protein is a novel mineralocorticoid receptor binding partner; Bratton MR et al.; The mineralocorticoid receptor (MR) plays a role in congestive heart failure; however, the molecular mechanism(s) remains undefined . We hypothesized that interaction of the MR with a cardiac protein modulates the transcriptional activation function of the MR within the heart . We used the yeast two-hybrid technique to screen a human heart library and found an aldosterone-dependent interaction between the hMR and the cardiac myosin binding protein (cMBP-c) . The EC(50) of the hMR-MBP-c interaction was approximately 80nM, and the cMBP-c did not interact with the glucocorticoid receptor (GR) . The GST pull-down technique was used to confirm an interaction between the MR and the cMBP-c as well as the lack of interaction with the GR . Spironolactone partially blocked this interaction, further suggesting MR specificity . We also determined the cMBP-c binding site lies within the C-terminus of the MR . We propose that interaction of the MR with cMBP-c may play a role in cardiac remodeling. Chromosoma, 2004 May, 112(7), 360 - 71 Epub 2004 May 07. The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionarily conserved; Barber CM et al.; Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood . Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation . However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes . In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis . We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals . Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis . We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase . The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase. Nat Struct Mol Biol, 2004 Jun, 11(6), 558 - 66 Epub 2004 May 09. Genome-wide analysis of mRNAs regulated by the THO complex in Drosophila melanogaster; Rehwinkel J et al.; In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export . The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p . Whether a complex with similar functions assembles in metazoa has not yet been established . Here we report that Drosophila melanogaster THO consists of THO2, HPR1 and three proteins, THOC5-THOC7, which have no orthologs in budding yeast . Gene expression profiling in cells depleted of THO components revealed that <20% of the transcriptome was regulated by THO . Nonetheless, export of heat-shock mRNAs under heat stress was strictly dependent on THO function . Notably, 8% of upregulated genes encode proteins involved in DNA repair . Thus, although THO function seems to be conserved, the vast majority of mRNAs are transcribed and exported independently of THO in D . melanogaster. Oncogene, 2004 Jul 22, 23(33), 5654 - 63 An activated mTOR mutant supports growth factor-independent, nutrient-dependent cell survival; Edinger AL et al.; In yeast, TOR couples cellular growth and metabolism to the availability of extracellular nutrients . In contrast, mammalian TOR kinase activity has been reported to be regulated by growth factor stimulation via the PI3K/Akt pathway . Consistent with this, growth factor deprivation results in dephosphorylation of the mTOR target proteins p70S6k and 4EBP1 in the face of abundant extracellular nutrients . To determine whether the activation of mTOR was sufficient to support cell survival in the absence of other growth factor-mediated signal transduction, we evaluated the ability of a growth factor-independent mTOR mutant, DeltaTOR, to protect cells from growth factor deprivation . DeltaTOR- but not wild-type mTOR-expressing cells were protected from many of the sequelae of growth factor deprivation including amino-acid transporter degradation, reduction of the glycolytic rate, cellular atrophy, decreased mitochondrial membrane potential, and Bax activation . Furthermore, DeltaTOR expression increased growth factor-independent, nutrient-dependent cell survival and enhanced the ability of p53-/- MEFs to form colonies in soft agar . These results suggest that activating mutations of mTOR can contribute to apoptotic resistance and might contribute to cellular transformation . EMBO Rep, 2004 Jun, 5(6), 626 - 31 Epub 2004 May 07. Sgt1 is required for human kinetochore assembly; Steensgaard P et al.; Budding yeast Sgt1 is required for kinetochore assembly, and its homologues have a role in cAMP signalling in fungi and pathogen resistance in plants . The function of mammalian Sgt1 is unknown . We report that RNA interference-mediated depletion of Sgt1 from HeLa cells causes dramatic alterations of the mitotic spindle and problems in chromosome alignment . Cells lacking Sgt1 undergo a mitotic delay due to activation of the spindle checkpoint . The checkpoint response, however, is significantly weakened in Sgt1-depleted cells, and this correlates with a dramatic reduction in kinetochore levels of Mad1, Mad2 and BubR1 . These effects are explained by a problem in kinetochore assembly that prevents the localization of Hec1, CENP-E, CENP-F, CENP-I, but not CENP-C, to mitotic kinetochores . Our studies implicate Sgt1 as an essential protein and a critical assembly factor for the mammalian kinetochore, and lend credit to the hypothesis of a kinetochore assembly pathway that is conserved from yeast to man. Nat Cell Biol, 2004 Jun, 6(6), 540 - 6 Epub 2004 May 09. The endogenous ligand Stunted of the GPCR Methuselah extends lifespan in Drosophila; Cvejic S et al.; Many extracellular signals are transmitted to the interior of the cell by receptors with seven membrane-spanning helices that trigger their effects by means of heterotrimeric guanine-nucleotide-binding regulatory proteins (G proteins) . These G-protein-coupled receptors (GPCRs) control various physiological functions in evolution from pheromone-induced mating in yeast to cognition in humans . The potential role of the G-protein signalling system in the control of animal ageing has been highlighted by the genetic revelation that mutation of a GPCR encoded by methuselah extends the lifespan of adult Drosophila flies . How methuselah functions in controlling ageing is not clear . A first essential step towards the understanding of methuselah function is to determine the ligands of Methuselah . Here we report the identification and characterization of two endogenous peptide ligands of Methuselah, designated Stunted A and B . Flies with mutations in the gene encoding these ligands show an increase in lifespan and resistance to oxidative stress . We conclude that the Stunted-Methuselah system is involved in the control of animal ageing. Biol Pharm Bull, 2004 May, 27(5), 628 - 33 Association of N-myc downregulated gene 1 with heat-shock cognate protein 70 in mast cells; Sugiki T et al.; N-Myc downregulated gene (NDRG) 1 is markedly induced during in vitro maturation of mouse immature bone marrow-derived mast cells (BMMCs) into a mature connective tissue mast cell (CTMC)-like phenotype . However, cellular function of this unique cytosolic protein is currently obscure . In this study, we sought potential NDRG1-binding proteins using yeast two-hybrid analysis and found that NDRG1 is capable of binding to heat-shock cognate protein 70 (Hsc70) both in vitro and in mast cells . The expression of Hsc70 was markedly elevated during the in vitro maturation of BMMCs into CTMC-like cells in accordance with the increased expression of NDRG1 . Deletion of the C-terminal hydrophilic tandem repeats from NDRG1 facilitated the interaction with Hsc70 in vitro . Interaction between NDRG1 and Hsc70 was constitutive in mast cells and was not altered following cell activation . Although NDRG1 undergoes phosphorylation (accompanying paper), the binding of NDRG1 to Hsc70 was not affected by this event . Interestingly, the NDRG1-Hsc70 complex transiently appeared in the nuclear fraction of activated mast cells. Mol Biol Cell, 2004 Jul, 15(7), 3073 - 82 Epub 2004 May 07. IRI-1, a LIN-15B homologue, interacts with inositol-1,4,5-triphosphate receptors and regulates gonadogenesis, defecation, and pharyngeal pumping in Caenorhabditis elegans; Walker DS et al.; Inositol-1,4,5-triphosphate receptors (IP(3)Rs) are ligand-gated Ca(2+) channels that control Ca(2+) release from intracellular stores . They are central to a wide range of cellular responses . IP(3)Rs in Caenorhabditis elegans are encoded by a single gene, itr-1, and are widely expressed . Signaling through IP(3) and IP(3)Rs is important in ovulation, control of the defecation cycle, modulation of pharyngeal pumping rate, and embryogenesis . To further elucidate the molecular basis of the diversity of IP(3)R function, we used a yeast two-hybrid screen to search for proteins that interact with ITR-1 . We identified an interaction between ITR-1 and IRI-1, a previously uncharacterized protein with homology to LIN-15B . Iri-1 is widely expressed, and its expression overlaps significantly with that of itr-1 . In agreement with this observation, iri-1 functions in known itr-1-mediated processes, namely, upregulation of pharyngeal pumping in response to food and control of the defecation cycle . Knockdown of iri-1 in an itr-1 loss-of-function mutant potentiates some of these effects and sheds light on the signaling pathways that control pharyngeal pumping rate . Knockdown of iri-1 expression also results in a sterile, evl phenotype, as a consequence of failures in early Z1/Z4 lineage divisions, such that gonadogenesis is severely disrupted. Microbiology, 2004 May, 150(Pt 5), 1571 - 80 Linear versus circular mitochondrial genomes: intraspecies variability of mitochondrial genome architecture in Candida parapsilosis; Rycovska A et al.; The yeast species Candida parapsilosis, an opportunistic pathogen, exhibits genetic and genomic heterogeneity . To assess the polymorphism at the level of mitochondrial DNA (mtDNA), the organization of the mitochondrial genome in strains belonging to the three variant groups of this species was investigated . Although these analyses revealed a group-specific restriction fragment pattern of mtDNA, strains belonging to different groups appear to have similar genes in the same gene order . An extensive survey of C . parapsilosis isolates uncovered surprising alterations in the molecular architecture of their mitochondrial genome . A screening strategy for strains harbouring mtDNA with rearranged architecture showed that nearly all strains from groups I and III possess linear mtDNA molecules terminating with arrays of tandem repeat units, while most of the group II strains have a circular mitochondrial genome . In addition, it was found that linear genophores in mitochondria of strains from different groups differ in the sequence of the mitochondrial telomeric repeat unit . The occurrence of altered forms of mtDNA among C . parapsilosis strains opens up the unique possibility to address questions concerning the evolutionary origin and replication strategy of linear and circular genomes in mitochondria. Mol Cell Proteomics, 2004 Aug, 3(8), 809 - 19 Epub 2004 May 06. Biochemical characterization of protein complexes from the Helicobacter pylori protein interaction map: strategies for complex formation and evidence for novel interactions within type IV secretion systems; Terradot L et al.; We have investigated a large set of interactions from the Helicobacter pylori protein interaction map previously identified by high-throughput yeast two-hybrid (htY2H)-based methods . This study had two aims: i) to validate htY2H as a source of protein-protein interaction complexes for high-throughput biochemical and structural studies of the H . pylori interactome; and ii) to validate biochemically interactions shown by htY2H to involve components of the H . pylori type IV secretion systems . Thus, 17 interactions involving 31 proteins and protein fragments were studied, and a general strategy was designed to produce protein-interacting partners for biochemical and structural characterization . We show that htY2H is a valid source of protein-protein complexes for high-throughput proteome-scale characterization of the H . pylori interactome, because 76% of the interactions tested were confirmed biochemically . Of the interactions involving type IV secretion proteins, three could be confirmed . One interaction is between two components of the type IV secretion apparatus, ComB10 and ComB4, which are VirB10 and VirB4 homologs, respectively . Another interaction is between a type IV component (HP0525, a VirB11 homolog) and a non-type IV secretion protein (HP01451), indicating that proteins other than the core VirB (1-11)-VirD4 proteins may play a role in type IV secretion . Finally, a third interaction was biochemically confirmed between CagA, a virulence factor secreted by the type IV secretion system encoded by the Cag pathogenicity island, and a non-type IV secretion protein, HP0496. J Biol Chem, 2004 Jul 2, 279(27), 27845 - 8 Epub 2004 May 07. A novel role for the immunophilin FKBP52 in copper transport; Sanokawa-Akakura R et al.; FK506-binding protein 52 (FKBP52) is an immunophilin that possesses peptidylprolyl cis/trans-isomerase (PPIase) activity and is a component of a subclass of steroid hormone receptor complexes . Several recent studies indicate that immunophilins can regulate neuronal survival and nerve regeneration although the molecular mechanisms are poorly understood . To investigate the function of FKBP52 in the nervous system, we employed a yeast two-hybrid strategy using the PPIase domain (domain I) as bait to screen a neonatal rat dorsal root ganglia cDNA expression library . We identified an interaction between FKBP52 domain I and Atox1, a copper-binding metallochaperone . Atox1 interacts with Menkes disease protein and Wilson disease protein (WD) and functions in copper efflux . The interaction between FKBP52 and Atox1 was observed in both glutathione S-transferase pull-down experiments and when proteins were ectopically expressed in human embryonic kidney (HEK) 293T cells and was sensitive to FK506 . Interestingly, the FKBP52/Atox1 interaction was enhanced when HEK 293T cells were cultured in copper-supplemented medium and decreased in the presence of the copper chelator, bathocuproine disulfate, suggesting that the interaction is regulated in part by intracellular copper . Overexpression of FKBP52 increased rapid copper efflux in (64)Cu-loaded cells, as did the overexpression of WD transporter . Taken together, our present findings suggest that FKBP52 is a component of the copper efflux machinery, and in so, may also promote neuroprotection from copper toxicity. Cancer Sci, 2004 May, 95(5), 424 - 9 Targeted gene delivery using humanized single-chain antibody with negatively charged oligopeptide tail; Suzuki M et al.; We have recently developed the so-called recombinant immunoporter as a non-viral vector based on a single-chain antibody (scFv) derived from a monoclonal antibody B4G7 against epidermal growth factor (EGF) receptor . This immunoporter (mBBD20) was composed of single-chain antibody and negatively charged oligopeptide tail {5 units of Asn4Ser (D20)}, and was expressed in yeast as a secreted protein . The purified mBBD20 was converted to an immunogene by mixing it with DNA and a cationic polymer, polyethyleneimine (PEI) . The resulting complex, namely recombinant immunogene, exhibited gene transfer activity with EGFR-specificity in vitro (Suzuki et al., Gene Ther . 2003) . In this paper, we further improved various conditions necessary for the formation of the proper recombinant immunogene, retaining receptor specificity of its binding, intracellular processing of the receptor-bound gene, and efficient gene expression . Moreover, we provided evidence that the recombinant immunoporter made with humanized scFv could be used as a potent gene transfer vehicle to target particular tumor cells . This approach seems worthy of clinical trial. EMBO J, 2004 May 19, 23(10), 2156 - 65 Epub 2004 May 06. CITRX thioredoxin interacts with the tomato Cf-9 resistance protein and negatively regulates defence; Rivas S et al.; To identify proteins involved in tomato Cf-9 resistance protein function, a yeast two-hybrid screen was undertaken using the cytoplasmic C-terminus of Cf-9 as bait . A thioredoxin-homologous clone, interacting specifically with Cf-9, was identified and called CITRX (Cf-9-interacting thioredoxin) . Virus-induced gene silencing (VIGS) of CITRX resulted in an accelerated Cf-9/Avr9-triggered hypersensitive response in both tomato and Nicotiana benthamiana, accompanied by enhanced accumulation of reactive oxygen species, alteration of protein kinase activity and induction of defence-related genes . VIGS of CITRX also conferred increased resistance to the fungal pathogen Cladosporium fulvum in the otherwise susceptible Cf0 tomato . CITRX acts as a negative regulator of the cell death and defence responses induced through Cf-9, but not Cf-2 . Recognition of the Cf-9 C-terminus by CITRX is necessary and sufficient for this negative regulation . This is the first study that implicates thioredoxin activity in the regulation of plant disease resistance. Methods Mol Med, 2004, 99, 239 - 53 Functional genomic analysis in pain research using hybridization arrays; Walker SJ et al.; Hybridization array technology makes it possible to compare global gene-expression patterns in any experimental context for which good-quality RNA can be generated . To date, DNA arrays have been used as a tool to compare functional genomic changes (differences in wholesale gene expression) in studies that cover an impressive variety of research disciplines including cancer, yeast genomics, and, more recently, neuroscience and behavior . The basic premise of the array experiment is that one interrogates a panel of probes (gene-specific cDNA fragments or gene-specific oligonucleotides that have been immobilized on a solid support) with RNAs (targets) from control and treated experimental samples that have been either radioactively or fluorescently labeled . Signal derived from either competitive (both samples on a single chip) or differential (one sample/one chip) hybridization is used to calculate relative gene expression . There are three widely used platforms available to perform array experiments (Affymetrix GeneChips, oligonucleotide arrays, and membrane-based cDNA arrays) and each platform offers advantages and limitations . The experimental description in this chapter explains, in detail, how to perform a hybridization array using the macroarray platform. Bioinformatics, 2004 Nov 1, 20(16), 2605 - 17 Epub 2004 May 06. A dynamically growing self-organizing tree (DGSOT) for hierarchical clustering gene expression profiles; Luo F et al.; MOTIVATION: The increasing use of microarray technologies is generating large amounts of data that must be processed in order to extract useful and rational fundamental patterns of gene expression . Hierarchical clustering technology is one method used to analyze gene expression data, but traditional hierarchical clustering algorithms suffer from several drawbacks (e.g . fixed topology structure; mis-clustered data which cannot be reevaluated) . In this paper, we introduce a new hierarchical clustering algorithm that overcomes some of these drawbacks . RESULT: We propose a new tree-structure self-organizing neural network, called dynamically growing self-organizing tree (DGSOT) algorithm for hierarchical clustering . The DGSOT constructs a hierarchy from top to bottom by division . At each hierarchical level, the DGSOT optimizes the number of clusters, from which the proper hierarchical structure of the underlying dataset can be found . In addition, we propose a new cluster validation criterion based on the geometric property of the Voronoi partition of the dataset in order to find the proper number of clusters at each hierarchical level . This criterion uses the Minimum Spanning Tree (MST) concept of graph theory and is computationally inexpensive for large datasets . A K-level up distribution (KLD) mechanism, which increases the scope of data distribution in the hierarchy construction, was used to improve the clustering accuracy . The KLD mechanism allows the data misclustered in the early stages to be reevaluated at a later stage and increases the accuracy of the final clustering result . The clustering result of the DGSOT is easily displayed as a dendrogram for visualization . Based on a yeast cell cycle microarray expression dataset, we found that our algorithm extracts gene expression patterns at different levels . Furthermore, the biological functionality enrichment in the clusters is considerably high and the hierarchical structure of the clusters is more reasonable . AVAILABILITY: DGSOT is available upon request from the authors. Bioinformatics, 2004 Nov 1, 20(16), 2626 - 35 Epub 2004 May 06. A statistical framework for genomic data fusion; Lanckriet GR et al.; MOTIVATION: During the past decade, the new focus on genomics has highlighted a particular challenge: to integrate the different views of the genome that are provided by various types of experimental data . RESULTS: This paper describes a computational framework for integrating and drawing inferences from a collection of genome-wide measurements . Each dataset is represented via a kernel function, which defines generalized similarity relationships between pairs of entities, such as genes or proteins . The kernel representation is both flexible and efficient, and can be applied to many different types of data . Furthermore, kernel functions derived from different types of data can be combined in a straightforward fashion . Recent advances in the theory of kernel methods have provided efficient algorithms to perform such combinations in a way that minimizes a statistical loss function . These methods exploit semidefinite programming techniques to reduce the problem of finding optimizing kernel combinations to a convex optimization problem . Computational experiments performed using yeast genome-wide datasets, including amino acid sequences, hydropathy profiles, gene expression data and known protein-protein interactions, demonstrate the utility of this approach . A statistical learning algorithm trained from all of these data to recognize particular classes of proteins--membrane proteins and ribosomal proteins--performs significantly better than the same algorithm trained on any single type of data . AVAILABILITY: Supplementary data at http://noble.gs.washington.edu/proj/sdp-svm Mech Ageing Dev, 2004 May, 125(5), 341 - 9 Effects of simultaneous over-expression of Cu/ZnSOD and MnSOD on Drosophila melanogaster life span; Sun J et al.; The FLP-out technique, based on yeast FLP recombinase, allows induced over-expression of transgenes in Drosophila adults . With FLP-out control and over-expressing flies have identical genetic backgrounds and therefore differences in life span must result from transgene induction . The amount of over-expression achieved varies between independent transgenic lines, and previously for both Cu/ZnSOD and MnSOD life span was found to be increased in proportion to the increase in enzyme activity . To determine if greater increases in enzyme and life span could be achieved with FLP-out, enzyme over-expression and life span were analyzed in eight lines containing two MnSOD transgenes, three lines containing three MnSOD transgenes, and three lines containing a MnSOD transgene plus a Cu/ZnSOD transgene . Life span was again found to be increased in proportion to the increase in MnSOD enzyme activity, with increases of up to 40% in mean and maximum life span . However the increases in enzyme activity and life span conferred per transgene were reduced when more than one transgene was present at the same time . When the reduced efficiency of enzyme over-expression per transgene was taken into account, simultaneous over-expression of MnSOD and Cu/ZnSOD was found to have partially additive effects on life span. Trends Cell Biol, 2004 May, 14(5), 215 - 8 Mitochondrial building blocks; Jensen RE et al.; Despite many genomic and proteomic attempts, approximately half of all mitochondrial proteins remain unidentified . Moreover, the composition of mitochondria varies in different mammalian cell types and the details of this tissue specificity are unclear . Two recent reports provide a major advance in our understanding of mitochondrial function . Sickmann et al . used an exhaustive proteomic approach and came very close to identifying the complete set of yeast mitochondrial proteins . Mootha et al . examined mitochondria from mouse brain, heart, kidney and liver cells, finding that a surprising fraction of the proteins are expressed in only a subset of tissues. Dev Cell, 2004 May, 6(5), 607 - 8 Cyclin C makes an entry into the cell cycle; Sage J; From yeast to humans, cell cycle progression is orchestrated by the oscillation of kinase activities associated with cyclins . In an article published recently in Cell, Ren and Rollins investigate mechanisms controlling the G0/G1 transition in quiescent cells and identify new cyclin C/Cdk3 complexes as key regulators of cell cycle reentry in human cells. J Neurosci, 2004 May 5, 24(18), 4421 - 31 p35/cyclin-dependent kinase 5 phosphorylation of ras guanine nucleotide releasing factor 2 (RasGRF2) mediates Rac-dependent Extracellular Signal-regulated kinase 1/2 activity, altering RasGRF2 and microtubule-associated protein 1b distribution in neurons; Kesavapany S et al.; Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase the activity of which is dependent on association with its neuron-specific activators, p35 and p39 . Cdk5 activity is critical for the proper formation of cortical structures and lamination during development . In the adult nervous system, Cdk5 function is implicated in cellular adhesion, dopamine signaling, neurotransmitter release, and synaptic activity . In addition, Cdk5 is also involved in "cross-talk" with other signal transduction pathways . To further examine its involvement in cross-talk with other pathways, we identified proteins that interacted with p35 using the yeast two-hybrid system . We report here that p35 associates with Ras guanine nucleotide releasing factor 2 (RasGRF2) in coimmunoprecipitation and colocalization studies using transfected cell lines as well as primary cortical neurons . Additionally, Cdk5 phosphorylates RasGRF2 both in vitro and in vivo, leading to a decrease in Rac-guanidine exchange factor activity and a subsequent reduction in extracellular signal-regulated kinase 1/2 activity . We show that p35/Cdk5 phosphorylates RasGRF2 on serine737, which leads to an accumulation of RasGRF2 in the neuronal cell bodies coinciding with an accumulation of microtubule-associated protein 1b . The membrane association of p35 and subsequent localization of Cdk5 activity toward RasGRF2 and Rac provide insights into important cellular signaling processes that occur at the membrane, resulting in downstream effects on signal transduction cascades. J Biol Chem, 2004 Jul 9, 279(28), 29101 - 8 Epub 2004 May 05. The heme synthesis defect of mutants impaired in mitochondrial iron-sulfur protein biogenesis is caused by reversible inhibition of ferrochelatase; Lange H et al.; Mitochondria are responsible for the synthesis of both iron-sulfur clusters and heme, but the potential connection between the two major iron-consuming pathways is unknown . Here, we have shown that mutants in the yeast mitochondrial iron-sulfur cluster (ISC) assembly machinery displayed reduced cytochrome levels and diminished activity of the heme-containing cytochrome c oxidase, in addition to iron-sulfur protein defects . In contrast, mutants in components of the mitochondrial ISC export machinery, which are specifically required for maturation of cytosolic iron-sulfur proteins, were not decreased in heme synthesis or cytochrome levels . Heme synthesis does not involve the function of mitochondrial ISC components, because immunological depletion of various ISC proteins from mitochondrial extracts did not affect the formation and amounts of heme . The heme synthesis defects of ISC mutants were found in vivo in isolated mitochondria and in mitochondrial detergent extracts and were confined to an inhibition of ferrochelatase, the enzyme catalyzing the insertion of iron into protoporphyrin IX . In support of these findings, immunopurification of ferrochelatase from ISC mutants restored its activity to wild-type levels . We conclude that the reversible inhibition of ferrochelatase is the molecular reason for the heme deficiency in ISC assembly mutants . This inhibitory mechanism may be used for regulation of iron distribution between the two iron-consuming processes. Genome Biol . 2004;5(5):222 . Epub 2004 Apr 22. The mechanism of prion strain propagation; Telling GC; Studies of mammalian prion diseases such as bovine spongiform encephalopathy have suggested that different strains consist of prion proteins with different conformations . Two recent studies of yeast prions have now formally demonstrated that multiple stable protein conformations are the basis of strain variation. BMC Cell Biol . 2004 May 05;5(1):18. TMF is a golgin that binds Rab6 and influences Golgi morphology; Fridmann-Sirkis Y et al.; BACKGROUND: Golgins are coiled-coil proteins associated with the Golgi apparatus, that are believed to be involved in the tethering of vesicles and the stacking of cisternae, as well as other functions such as cytoskeletal association . Many are peripheral membrane proteins recruited by GTPases . Several have been described in animal cells, and some in yeast, but the relationships between golgins from different species can be hard to define because although they share structural features, their sequences are not well conserved . RESULTS: We show here that the yeast protein Sgm1, previously shown to be recruited to the Golgi by the GTPase Ypt6, binds to Ypt6:GTP via a conserved 100-residue coiled-coil motif that can be identified in a wide range of eukaryotes . The mammalian equivalent of Sgm1 is TMF/ARA160, a protein previously identified in various screens as a putative transcription or chromatin remodelling factor . We show that it is a Golgi protein, and that it binds to the three known isoforms of the Ypt6 homologue Rab6 . Depletion of the protein by RNA interference in rat NRK cells results in a modest dispersal of Golgi membranes around the cell, suggesting a role for TMF in the movement or adherence of Golgi stacks . CONCLUSION: We have identified TMF as an evolutionarily conserved golgin that binds Rab6 and contributes to Golgi organisation in animal cells. Mol Immunol, 2004 Mar, 40(17), 1257 - 61 The Filamin-A is a partner of Tc-mip, a new adapter protein involved in c-maf-dependent Th2 signaling pathway; Grimbert P et al.; Using a yeast two-hybrid screen, we identified Filamin-A as a binding partner of the new adapter protein c-mip (c-maf inducing protein) and it's splice variant Tc-mip (truncated c-maf inducing protein) . We have previously shown that Tc-mip is involved in Th2 signaling pathway and cytoskeletal reorganization in patients with minimal change nephrotic syndrome (MCNS), the most frequent glomerular disease in children . We showed that Filamin-A and c-mip or Tc-mip co-immunoprecipitate from c-mip or Tc-mip Jurkat transfected cells using antibodies directed against both types of proteins . In co-immunoprecipitate Jurkat cells, Filamin-A and c-mip were distributed evenly in the cytoplasm, whereas in Tc-mip-transfected Jurkat cells, Filamin-A was expressed in zones facing the cell contact . Moreover, we found that Filamin-A was upregulated in T lymphocytes of MCNS patients, as compared to normal subjects . These findings suggest that Filamin-A interacts with c-mip/Tc-mip in this new T-cell signaling pathway. Biotechnol Lett, 2004 Mar, 26(6), 517 - 9 Biotransformation of lignin polymers derived from beech wood pulping by Sporobolomyces roseus isolated from leafy material; Kosikova B et al.; The ability of the yeast, Sporobolomyces roseus, isolated from leafy material, to modify lignin derived from beechwood pulping was examined by FTIR and 13C NMR spectroscopy, which revealed oxidative cleavage of the Calpha-Cbeta linkages between lignin units . Using veratryl alcohol as a model substrate confirmed that Sp . roseus could oxidize veratryl alcohol into veratric acid . This yeast might be suitable for the pretreatment of lignocellulosic materials and/or for biotransformation of technical lignins. J Mol Neurosci, 2004, 23(1-2), 23 - 34 Interactions among alpha-synuclein, dopamine, and biomembranes: some clues for understanding neurodegeneration in Parkinson's disease; Rochet JC et al.; Parkinson's disease (PD) is a neurologic disorder resulting from the loss of dopaminergic neurons in the brain . Two lines of evidence suggest that the protein alpha-synuclein plays a role in the pathogenesis of PD: Fibrillar alpha-synuclein is a major component of Lewy bodies in diseased neurons, and two mutations in alpha-synuclein are linked to early-onset disease . Accordingly, the fibrillization of alpha-synuclein is proposed to contribute to neurodegeneration in PD . In this report, we provide evidence that oligomeric intermediates of the alpha-synuclein fibrillization pathway, termed protofibrils, might be neurotoxic . Analyses of protofibrillar alpha-synuclein by atomic force microscopy and electron microscopy indicate that the oligomers consist of spheres, chains, and rings . alpha-Synuclein protofibrils permeabilize synthetic vesicles and form pore-like assemblies on the surface of brain-derived vesicles . Dopamine reacts with alpha-synuclein to form a covalent adduct that slows the conversion of protofibrils to fibrils . This finding suggests that cytosolic dopamine in dopaminergic neurons promotes the accumulation of toxic alpha-synuclein protofibrils, which might explain why these neurons are most vulnerable to degeneration in PD . Finally, we note that aggregation of alpha-synuclein likely occurs via different mechanisms in the cell versus the test tube . For example, the binding of alpha-synuclein to cellular membranes might influence its self-assembly . To address this point, we have developed a yeast model that might enable the selection of random alpha-synuclein mutants with different membrane-binding affinities . These variants might be useful to test whether membrane binding by alpha-synuclein is necessary for neurodegeneration in transgenic animal models of PD. J Clin Endocrinol Metab, 2004 May, 89(5), 2498 - 500 Tetrahydrogestrinone is a potent androgen and progestin; Death AK et al.; Tetrahydrogestrinone (THG) was recently identified as a novel steroid used illicitly to improve athletic performance . Although its structure is closely related to gestrinone, a 19-nor progestin, and resembles that of trenbolone, THG was never marketed, so information on its hormonal properties is not known . In this study, we demonstrate that THG is a highly potent androgen and progestin in a yeast-based in vitro bioassay system expressing human androgen and progesterone receptors . It has no estrogenic activity and no antagonism for any of the three steroid receptor classes. Genetics, 2004 Apr, 166(4), 1715 - 25 The selective values of alleles in a molecular network model are context dependent; Peccoud J et al.; Classical quantitative genetics has applied linear modeling to the problem of mapping genotypic to phenotypic variation . Much of this theory was developed prior to the availability of molecular biology . The current understanding of the mechanisms of gene expression indicates the importance of nonlinear effects resulting from gene interactions . We provide a bridge between genetics and gene network theories by relating key concepts from quantitative genetics to the parameters, variables, and performance functions of genetic networks . We illustrate this methodology by simulating the genetic switch controlling galactose metabolism in yeast and its response to selection for a population of individuals . Results indicate that genes have heterogeneous contributions to phenotypes and that additive and nonadditive effects are context dependent . Early cycles of selection suggest strong additive effects attributed to some genes . Later cycles suggest the presence of strong context-dependent nonadditive effects that are conditional on the outcomes of earlier selection cycles . A single favorable allele cannot be consistently identified for most loci . These results highlight the complications that can arise with the presence of nonlinear effects associated with genes acting in networks when selection is conducted on a population of individuals segregating for the genes contributing to the network. Plant J, 2004 May, 38(4), 685 - 98 Sorting signals in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer; Contreras I et al.; In mammals and yeast, a cytosolic dilysine motif is critical for endoplasmic reticulum (ER) localization of type I membrane proteins . Retrograde transport of type I membrane proteins containing dilysine motifs at their cytoplasmic carboxy (C)-terminal tail involves the interaction of these motifs with the COPI coat . The C-terminal dilysine motif has also been shown to confer ER localization to type I membrane proteins in plant cells . Using in vitro binding assays, we have analyzed sorting motifs in the cytosolic tail of membrane proteins, which may be involved in the interaction with components of the COPI coat in plant cells . We show that a dilysine motif in the -3,-4 position (relative to the cytosolic C-terminus) recruits in a very specific manner all the subunits of the plant coatomer complex . Lysines cannot be replaced by arginines or histidines to bind plant coatomer . A diphenylalanine motif in the -7,-8 position, which by itself has a low ability to bind plant coatomer, shows a clear cooperativity with the dilysine motif . Both dilysine and diphenylalanine motifs are present in the cytosolic tail of several proteins of the p24 family of putative cargo receptors, which has several members in plant cells . The cytosolic tail of a plant p24 protein is shown to recruit not only coatomer but also ADP ribosylation factor 1 (ARF1), a process which depends on both dilysine and diphenylalanine motifs . ARF1 binding increases twofold upon treatment with brefeldin A (BFA) and is completely abolished upon treatment with GTPgammaS, suggesting that ARF1 can only interact with the cytosolic tail of p24 proteins in its GDP-bound form. Arch Latinoam Nutr, 2003 Dec, 53(4), 400 - 7 {Functional properties of Sphagnum magellanicum fiber and its direct use in formulation of bakery products}; Villarroel M et al.; Characterization of functional properties of Sphagnum magellanicum fiber were investigated . Water absortion (WAC) and water retention (WRC) capacities, swelling capacity (SC); organic molecule absortion capacity (OMAC) and cationic interchange capacity (CIC) were evaluated, as well as its incorporation as fiber source to bakery produts . Diferent particles sizes were selected to evaluate their effects on the functional properties of moss fiber: T1(1.4 mm);T2(1.0 mm); T3(0.42 mm); T4(0.18 mm) . Best results of CAA, CRA; SC and OMRC were obtained with T3, whereas best values of CIC were attained with T1 . An optimized formulation of fiber enriched bread was developed analizing simultaneously the effect of four independent variables (yeast, moss fiber, fluffy agent and shortening) on the sensory quality of products . Shelf life studies were carried out by storing samples of fiber enriched breads at 20 degrees C and 6 degrees C . At the end of the study, refrigerated samples showed better sensory quality stability. Med Mycol, 2004 Apr, 42(2), 165 - 75 Clinical, histopathological and immunological effects of exposure of canine skin to Malassezia pachydermatis; Bond R et al.; The effects of the daily application for 7 days of suspensions of Malassezia pachydermatis to normal canine skin were evaluated in 10 beagle dogs . Four out of six dogs challenged without occlusion developed transient lesions generally characterized clinically by mild erythema with papules and histologically by mild epidermal hyperplasia and a superficial perivascular dermatitis . Saline-treated control sites showed no clinical signs . In four dogs challenged with occlusion, skin lesions occurred at both yeast and saline-treated sites; erythema and papules were more severe at the yeast-treated sites in three dogs . Occlusion induced more persistent lesions, which resolved within 24 days . Population densities of the yeast were highest at day 8 and declined rapidly following cessation of application . Peripheral blood mononuclear cell proliferation indices following M . pachydermatis exposure in vitro and serum concentrations of M . pachydermatis-specific IgG antibodies did not vary significantly during the study . Delayed (24 h) intradermal test reactivity to M . pachydermatis antigens developed in all eight dogs with clinical signs following yeast exposure . This study suggests that the resistance of healthy canine skin to infection by M . pachydermatis is mediated by local delayed hypersensitivity responses and, or innate epidermal immune mechanisms. J Biol Chem, 2004 Jul 9, 279(28), 29728 - 39 Epub 2004 Apr 28. PKD2 interacts and co-localizes with mDia1 to mitotic spindles of dividing cells: role of mDia1 IN PKD2 localization to mitotic spindles; Rundle DR et al.; Mutations in pkd2 result in the type 2 form of autosomal dominant polycystic kidney disease, which accounts for approximately 15% of all cases of the disease . PKD2, the protein product of pkd2, belongs to the transient receptor potential superfamily of cation channels, and it can function as a mechanosensitive channel in the primary cilium of kidney cells, an intracellular Ca(2+) release channel in the endoplasmic reticulum, and/or a nonselective cation channel in the plasma membrane . We have identified mDia1/Drf1 (mammalian Diaphanous or Diaphanous-related formin 1 protein) as a PKD2-interacting protein by yeast two-hybrid screen . mDia1 is a member of the RhoA GTPase-binding formin homology protein family that participates in cytoskeletal organization, cytokinesis, and signal transduction . We show that mDia1 and PKD2 interact in native and in transfected cells, and binding is mediated by the cytoplasmic C terminus of PKD2 binding to the mDia1 N terminus . The interaction is more prevalent in dividing cells in which endogenous PKD2 and mDia1 co-localize to the mitotic spindles . RNA interference experiments reveal that endogenous mDia1 knockdown in HeLa cells results in the loss of PKD2 from mitotic spindles and alters intracellular Ca(2+) release . Our results suggest that mDia1 facilitates the movement of PKD2 to a centralized position during cell division and has a positive effect on intracellular Ca(2+) release during mitosis . This may be important to ensure equal segregation of PKD2 to the daughter cell to maintain a necessary level of channel activity . Alternatively, PKD2 channel activity may be important in the cell division process or in cell fate decisions after division. J Biol Chem, 2004 Jul 16, 279(29), 30265 - 73 Epub 2004 Apr 27. Merlin, a tumor suppressor, interacts with transactivation-responsive RNA-binding protein and inhibits its oncogenic activity; Lee JY et al.; The neurofibromatosis type 2 gene-encoded protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of membrane-cytoskeleton-associated proteins . Recent studies suggest that the loss of neurofibromatosis type 2 function contributes to tumor development and metastasis . Although the cellular functions of merlin as a tumor suppressor are relatively well characterized, the cellular mechanism whereby merlin controls cell proliferation from membrane locations is still poorly understood . During our efforts to find potential merlin modulators through protein-protein interactions, we identified transactivation-responsive RNA-binding protein (TRBP) as a merlin-binding protein in a yeast two-hybrid screen . The interaction between TRBP and merlin was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation, and co-localization experiments . The carboxyl-terminal regions of each protein were responsible for their interaction . Cells overexpressing TRBP showed enhanced cell growth in cell proliferation assays and also exhibited transformed phenotypes, such as anchorage-independent cell growth and tumor development in mouse xenografts . Merlin efficiently inhibited these oncogenic activities of TRBP in our experiments . These results provide the first clue to the functional interaction between TRBP and merlin and suggest a novel mechanism for the tumor suppressor function of merlin both in vitro and in vivo. J Biol Chem, 2004 Jul 9, 279(28), 28936 - 44 Epub 2004 Apr 27. Mutational analysis of the archaeal tyrosine recombinase SSV1 integrase suggests a mechanism of DNA cleavage in trans; Letzelter C et al.; The only tyrosine recombinase so far studied in archaea, the SSV1 integrase, harbors several changes in the canonical residues forming the catalytic pocket of this family of recombinases . This raised the possibility of a different mechanism for archaeal tyrosine recombinase . The residues of Int(SSV) tentatively involved in catalysis were modified by site-directed mutagenesis, and the properties of the corresponding mutants were studied . The results show that all of the targeted residues are important for activity, suggesting that the archaeal integrase uses a mechanism similar to that of bacterial or eukaryotic tyrosine recombinases . In addition, we show that Int(SSV) exhibits a type IB topoisomerase activity because it is able to relax both positive and negative supercoils . Interestingly, in vitro complementation experiments between the inactive integrase mutant Y314F and all other inactive mutants restore in all cases enzymatic activity . This suggests that, as for the yeast Flp recombinase, the active site is assembled by the interaction of the tyrosine from one monomer with the other residues from another monomer . The shared active site paradigm of the eukaryotic Flp protein may therefore be extended to the archaeal tyrosine recombinase Int(SSV). J Biol Chem, 2004 Jul 16, 279(29), 29944 - 51 Epub 2004 Apr 29. An Hsp27-related, dominant-negative-acting intracellular estradiol-binding protein; Chen H et al.; New World primates (NWPs) exhibit a compensated form of resistance to gonadal steroid hormones . We demonstrated recently that estrogen resistance in NWP cells was associated with the overexpression of two proteins, a nonreceptor-related, dominant-negative-acting estrogen response element (ERE)-binding protein (ERE-BP) and an intracellular estradiol-binding protein (IEBP) . Based on the N-terminal sequences of tryptic fragments of IEBP isolated from a 17beta-estradiol (E2) affinity column we cloned a full-length cDNA for IEBP from the estrogen-resistant NWP cell line, B95-8 . Subsequent sequence analysis revealed 87% sequence identity between the deduced peptide for IEBP and human Hsp27 . When hormone-responsive, wild-type Old World primate (OWP) cells were transiently transfected with IEBP cDNA, E2-directed ERE reporter luciferase activity was reduced by 50% compared with vector only-transfected OWP cells (p < 0.0018) . When IEBP and ERE-BP were cotransfected, ERE promoter-reporter activity was reduced by a further 60% (p < 0.0001) . Electrophoresis mobility shift analyses showed that IEBP neither bound to ERE nor competed with the estrogen receptor (ER) for binding to ERE . However, there was evidence of protein-protein interaction of IEBP and ERalpha; IEBP was coimmunoprecipitated with anti-ERalpha antibody in wild-type cells stably transfected with IEBP . A specific interaction between ERalpha and IEBP was confirmed in glutathione S-transferase pull-down and yeast two-hybrid assays . Data indicate that the Hsp27-related IEBP interacts with the ligand binding domain of the ERalpha . In summary, by inhibiting the ERalpha-E2 interaction, IEBP acts to squelch ERalpha-directed ERE-regulated transactivation and promote estrogen resistance in NWP cells. J Mol Biol, 2004 May 21, 339(1), 173 - 83 Structural analysis of the human Golgi-associated plant pathogenesis related protein GAPR-1 implicates dimerization as a regulatory mechanism; Serrano RL et al.; The plant pathogenesis related proteins group 1 (PR-1) and a variety of related mammalian proteins constitute a PR-1 protein family that share sequence and structural similarities . GAPR-1 is a unique family member as thus far it is the only PR-1 family member that is not co-translationally targeted to the lumen of the endoplasmic reticulum before trafficking to either vacuoles or secretion . Here we report that GAPR-1 may form dimers in vitro and in vivo, as determined by yeast two-hybrid screening, biochemical and biophysical assays . The 1.55A crystal structure demonstrates that GAPR-1 is structurally homologous to the other PR-1 family members previously solved (p14a and Ves V 5) . Through an examination of inter-molecular interactions between GAPR-1 molecules in the crystal lattice, we propose a number of the highly conserved amino acid residues of the PR-1 family to be involved in the regulation of dimer formation of GAPR-1 with potential implications for other PR-1 family members . We show that mutagenesis of these conserved amino acid residues leads to a greatly increased dimer population . A recent report suggests that PR-1 family members may exhibit serine protease activity and further examination of the dimer interface of GAPR-1 indicates that a catalytic triad similar to that of serine proteases may be formed across the dimer interface by residues from both molecules within the dimer. Fish Shellfish