Yeasts

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 Immunol, 2004 Apr, 16(4), 527 - 37
Enhancement of innate immunity in rainbow trout (Oncorhynchus mykiss Walbaum) associated with dietary intake of carotenoids from natural products; Amar EC et al.; The effects of orally administered carotenoids from natural sources on the non-specific defense mechanisms of rainbow trout were evaluated in a nine-week feeding trial . Fish were fed four diets containing either beta-carotene or astaxanthin at 100 and 200 mg kg-1 from the marine algae Dunaliella salina and red yeast Phaffia rhodozyma, respectively, and a control diet containing no supplemented carotenoids . Specific growth rate and feed:gain ratio were not affected by dietary carotenoid supplementation . Among the humoral factors, serum alternative complement activity increased significantly in all carotenoid supplemented groups when compared to the control . On the other hand, serum lysozyme activity increased in the Dunaliella group but not in the Phaffia group, whereas plasma total immunoglobulin levels were not altered by the feeding treatments . As for the cellular responses, the superoxide anion production from the head kidney remained unchanged while the phagocytic rate and index in all supplemented groups were significantly higher than those of the control . These findings demonstrate that dietary carotenoids from both D . salina and P . rhodozyma can modulate some of the innate defense mechanisms in rainbow trout.

Chem Biol, 2004 Feb, 11(2), 151 - 3
Finding Cinderella after the ball: a three-hybrid approach to drug target identification; Lefurgy S et al.; A major bottleneck in drug discovery is identifying the targets of small molecules . The yeast three-hybrid assay extends the two-hybrid approach to screen for protein-small molecule interactions . In this issue of Chemistry & Biology, GPC Biotech reports the first application of this promising assay.

Oncogene, 2004 Jul 8, 23(31), 5394 - 404
A novel crosstalk mechanism between nuclear receptor-mediated and growth factor/Ras-mediated pathways through PNRC-Grb2 interaction; Zhou D et al.; It has been demonstrated that proline-rich nuclear receptor coregulatory protein (PNRC) is a nuclear receptor coactivator that interacts with nuclear receptors through an SH3-binding motif located in its C-terminus . In the present report, a physical interaction between PNRC and Grb2 (an adapter protein involved in growth factor/Ras-mediated pathways) has been demonstrated using the GST pull-down assay, the yeast two-hybrid assay, as well as by coimmunoprecipitation . Cotransfection and fluorescence imaging have also confirmed the colocalization of PNRC and Grb2 in mammalian cells . Transient transfection experiments have demonstrated that, by interacting with each other, Grb2 decreases the coactivator activity of PNRC for nuclear receptors, and that PNRC suppresses Grb2-mediated Ras/MAP-kinase activation . Furthermore, it was discovered that HeLa cells overexpressing PNRC grew more slowly when compared to matched controls . Additionally, using a RT-PCR analysis of mRNA on six pairs of cancer/noncancer tissues, PNRC expression was found to be significantly lower in breast cancer tissue than in noncancer tissue . Based on these findings, we believe that PNRC and Grb2, by interacting with each other, can suppress nuclear receptor-mediated regulation and growth factor-mediated regulation in human breast tissue . This is a newly identified crosstalk mechanism for modulating these two important types of regulatory pathways.

Mol Biotechnol, 2004 May, 27(1), 33 - 57
Calmodulin's flexibility allows for promiscuity in its interactions with target proteins and peptides; Yamniuk AP et al.; The small bilobal calcium regulatory protein calmodulin (CaM) activates numerous target enzymes in response to transient changes in intracellular calcium concentrations . Binding of calcium to the two helix-loop-helix calcium-binding motifs in each of the globular domains induces conformational changes that expose a methionine-rich hydrophobic patch on the surface of each domain of the protein, which it uses to bind to peptide sequences in its target enzymes . Although these CaM-binding domains typically have little sequence identity, the positions of several bulky hydrophobic residues are often conserved, allowing for classification of CaM-binding domains into recognition motifs, such as the 1-14 and 1-10 motifs . For calcium-independent binding of CaM, a third motif known as the IQ motif is also common . Many CaM-peptide complexes have globular conformations, where CaM's central linker connecting the two domains unwinds, allowing the protein to wrap around a single predominantly alpha-helical target peptide sequence . However, novel structures have recently been reported where the conformation of CaM is highly dissimilar to these globular complexes, in some instances with less than a full compliment of bound calcium ions, as well as novel stoichiometries . Furthermore, many divergent CaM isoforms from yeast and plant species have been discovered with unique calcium-binding and enzymatic activation characteristics compared to the single CaM isoform found in mammals.

Plant Physiol, 2004 May, 135(1), 71 - 84
Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the cyanogenic glucosides lotaustralin and linamarin in Lotus japonicus; Forslund K et al.; Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin . The content of cyanogenic and nitrile glucosides in L . japonicus depends on plant developmental stage and tissue . The cyanide potential is highest in young seedlings and in apical leaves of mature plants . Roots and seeds are acyanogenic . Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid l-Ile, whereas linamarin is derived from Val . In silico homology searches identified two cytochromes P450 designated CYP79D3 and CYP79D4 in L . japonicus . The two cytochromes P450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L . japonicus . CYP79D3 and CYP79D4 are differentially expressed . CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots . Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with l-Ile as substrate than with l-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L . japonicus . Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L . japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amounts of lotaustralin and rhodiocyanoside A/D were unaltered.

Mol Biol Cell, 2004 Jul, 15(7), 3244 - 56 Epub 2004 Apr 30.
A human telomerase-associated nuclease; Oulton R et al.; Ciliate and yeast telomerase possess a nucleolytic activity capable of removing DNA from the 3' end of a single-stranded oligonucleotide substrate . The nuclease activity is thought to assist in enzyme proofreading and/or processivity . Herein, we report a previously uncharacterized human telomerase-associated nuclease activity that shares several properties with ciliate and yeast telomerases . Partially purified human telomerase, either from cell extracts or recombinantly produced, demonstrated an ability to remove 3' nontelomeric nucleotides from a substrate containing 5' telomeric DNA, followed by extension of the newly exposed telomeric sequence . This cleavage/extension activity was apparent at more than one position within the telomeric DNA and was influenced by sequences 5' to the telomeric/nontelomeric boundary and by substitution with a methylphosphonate moiety at the telomeric/nontelomeric DNA boundary . Our data suggest that human telomerase is associated with an evolutionarily conserved nucleolytic activity and support a model in which telomerase-substrate interactions can occur distal from the 3' primer end.

Mol Biol Cell, 2004 Jul, 15(7), 3095 - 105 Epub 2004 Apr 30.
Sorting nexin 17 accelerates internalization yet retards degradation of P-selectin; Williams R et al.; The transient appearance of P-selectin on the surface of endothelial cells helps recruit leukocytes into sites of inflammation . The tight control of cell surface P-selectin on these cells depends on regulated exocytosis of Weibel-Palade bodies where the protein is stored and on its rapid endocytosis . After endocytosis, P-selectin is either sorted via endosomes and the Golgi apparatus for storage in Weibel-Palade bodies or targeted to lysosomes for degradation . A potential player in this complex endocytic itinerary is SNX17, a member of the sorting nexin family, which has been shown in a yeast two-hybrid assay to bind P-selectin . Here, we show that overexpression of SNX17 in mammalian cells can influence two key steps in the endocytic trafficking of P-selectin . First, it promotes the endocytosis of P-selectin from the plasma membrane . Second, it inhibits the movement of P-selectin into lysosomes, thereby reducing its degradation.

Exp Cell Res, 2004 May 15, 296(1), 12 - 20
Nuclear export of mRNA: from the site of transcription to the cytoplasm; Erkmann JA et al.; Cellular mRNAs are produced in the nucleus and must be exported to the cytoplasm to allow for their translation into proteins . Recruitment of export factors to nascent mRNA starts cotranscriptionally and involves elaborate systems of quality control . Correctly processed mRNAs are committed for export in the form of large ribonucleoprotein complexes (mRNPs) . Translocation of mRNPs through the nuclear pore complex (NPC) is mediated by a conserved heterodimeric transport receptor (NXF1/p15 in metazoa and Mex67p/Mtr2p in yeast) that bridges the interaction between the mRNP and the NPC . In this review, we describe the cis- and trans-requirements for mRNA export as well as the different mechanisms of recruiting export factors to mRNPs . We also discuss the significance of linking mRNA export with both downstream and upstream events in gene expression.

Biochem Biophys Res Commun, 2004 May 28, 318(2), 571 - 8
Aryl hydrocarbon receptor-mediated induction of microsomal drug-metabolizing enzyme activity by indirubin and indigo; Sugihara K et al.; Indirubin and indigo, which are thought to be natural ligands for aryl hydrocarbon receptor (AhR), showed marked AhR ligand activities in a reporter gene assay using recombinant yeast . Their activities were comparable with or more potent than that of 2,3,7,8-tetrachlorodibenzo-p-dioxin . When indirubin and indigo were administered to mice, ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in the liver were increased, but subsequently decreased within 2 days . Indirubin was more potent than indigo . Levels of cytochrome P450 1A1/2 proteins and mRNAs in the liver of mice dosed with indirubin were also enhanced . These enhancing effects of indirubin and indigo were not observed in AhR knock-out mice . Ethoxyresorufin-O-dealkylase and methoxyresorufin-O-dealkylase activities in rat hepatocytes and HepG2 cells were enhanced by the addition of indirubin or indigo, but less potently than by 2,3,7,8-tetrachlorodibenzo-p-dioxin . Indigocarmine, a sulfate derivative of indigo, which is used as food additive, did not show these inducing effects on drug-metabolizing enzymes . Our results suggest that indirubin and indigo act as inducers for cytochrome P450 1A1/2 mediated by AhR in mammals in vivo.

J Ethnopharmacol, 2004 Apr, 91(2-3), 237 - 42
Anti-inflammatory activity of methanolic extracts from Ventilago harmandiana Pierre; Panthong A et al.; Methanolic extracts from the heart wood, stem bark, and stem wood of Ventilago harmandiana Pierre (Family Rhamnaceae) were assessed for anti-inflammatory effects using both acute and chronic inflammatory models . Analgesic and antipyretic activities of the extracts were also evaluated . It was found that all extracts possessed strong inhibitory effects on the acute phase of inflammation as seen in ethyl phenylpropiolate (EPP)- and arachidonic acid (AA)-induced ear edema as well as in carrageenin-induced paw edema in rats . The extracts elicited only weak inhibitory activity on cotton pellet-induced granuloma formation, a subchronic inflammatory model . In the analgesic test, all extracts exerted pronounced inhibitory activity in acetic acid-induced writhing response but showed only weak effects in the tail-flick test . The extracts also showed excellent antipyretic activity on yeast-induced hyperthermia in rats.

Curr Biol, 2004 May 4, 14(9), R355 - 6
Molecular motors: Dynein's gearbox; Cross RA; A new optical trapping study shows that the stepsize of cytoplasmic dynein varies according to the applied force, suggesting that this motor can change gear . Complementary biochemical kinetic work on yeast dynein mutants hints at the allosteric mechanisms involved.

Curr Biol, 2004 May 4, 14(9), 824 - 8
The Ubx2 and Ubx3 cofactors direct Cdc48 activity to proteolytic and nonproteolytic ubiquitin-dependent processes; Hartmann-Petersen R et al.; Valosin-containing protein, VCP/p97 or Cdc48, is a eukaryotic ATPase involved in membrane fusion, protein transport, and protein degradation . We describe two proteins, Ubx2 and Ubx3, which interact with Cdc48 in fission yeast . Ubx3 is the ortholog of p47/Shp1, a previously described Cdc48 cofactor involved in membrane fusion, whereas Ubx2 is a novel protein . Cdc48 binds the UBX domains present in both Ubx2 and Ubx3, indicating that this domain is a general Cdc48-interacting module . Ubx2 and Ubx3 also interact with ubiquitin chains . Disruption of the ubx3(+)-gene causes both temperature and canavanine sensitivity and stabilizes some ubiquitin-protein conjugates including the CDK inhibitor Rum1, but not a model substrate of the ER-degradation pathway . Moreover the ubx3 null displays synthetic lethality with a pus1 null mutant, a multiubiquitin binding subunit of the 26S proteasome . In contrast, the ubx2 null mutant did not display any obvious protein-degradation phenotype . In conclusion Ubx3/p47 is not, as previously thought, only important for membrane fusion; it's also important for the specific degradation of a subset of cell proteins . Our genetic analyses revealed that Ubx3/p47 functionally parallels a substrate receptor of the 26S proteasome, Pus1/Rpn10, indicating that the Cdc48-Ubx3 complex is involved in delivering substrates to the 26S proteasome.

Eur J Cancer, 2004 Apr, 40(6), 785 - 93
Update on NCI in vitro drug screen utilities; Holbeck SL; Development of new anti-cancer drugs is a costly and risky proposition . The Developmental Therapeutics Program (DTP) of the National Cancer Institutes of the United States (U.S.) facilitates the drug development process by providing access to preclinical screening services . Since the early 1990's, DTP has screened tens of thousands of compounds against a panel of 60 human tumour cell lines representing nine tissue sites . At the same time, DTP began to accumulate information on the expression of molecular entities in the same 60 cell line panel . Many of these data are freely available to the public at . More recently, additional, more focused screens have entered the picture, with data also available through the web site . These include screening of roughly 100000 compounds against a panel of yeast mutants, and screening of the NCI Diversity Set in assays designed to detect effects on Molecular Targets of interest.

BMC Dermatol . 2004 May 01;4(1):5.
Study of the distribution of Malassezia species in patients with pityriasis versicolor and healthy individuals in Tehran, Iran; Tarazooie B et al.; BACKGROUND: Pityriasis versicolor is a superficial infection of the stratum corneum which caused by a group of yeasts formerly named pityrosporium . The taxonomy of these lipophilic yeasts has recently been modified and includes seven species referred as Malassezia . The aim of this study is to compare the distribution of Malassezia species isolated from pityriasis versicolor lesions and those isolated from healthy skins . METHODS: Differentiation of all malassezia species performed using morphological features and physiological test including catalase reaction, Tween assimilation test and splitting of esculin . RESULTS: In pityriasis versicolor lesions, the most frequently isolated species was M . globosa (53.3%), followed by M . furfur (25.3%), M . sympodialis(9.3%), M . obtusa (8.1%) and M . slooffiae (4.0%) . The most frequently isolated species in the skin of healthy individuals were M . globosa, M . sympodialis, M . furfur, M . sloofiae and M . restricta which respectively made up 41.7%, 25.0%, 23.3%, 6.7% and 3.3% of the isolated species . CONCLUSIONS: According to our data, M . globosa was the most prevalent species in the skin of healthy individuals which recovered only in the yeast form . However, the Mycelial form of M . globosa was isolated as the dominant species from pityriasis versicolor lesions . Therefore, the role of predisposing factors in the conversion of this yeast to mycelium and its subsequent involvement in pityriasis versicolor pathogenicity should be considered.

Mycopathologia, 2004 Feb, 157(2), 233 - 41
Aflatoxin binders II: reduction of aflatoxin M1 in milk by sequestering agents of cows consuming aflatoxin in feed; Diaz DE et al.; Sequestering agents bind dietary aflatoxin B1 (AFB1) and reduce absorption from an animal's gastrointestinal tract . As a result, they protect an animal from the toxic effects of AFB1 and reduce transfer of the metabolite, aflatoxin M1 (AFM1), into milk . Three experiments, using late-lactation Holstein cows fed AFB1-contaminated feed, were conducted to evaluate several potential sequestering agents for their abilities to prevent or reduce the transmission of AFM1 into milk . Six agents previously tested in our laboratory for AFB1 binding in vitro were evaluated in these experiments . These were: SA-20, an activated carbon (AC-A); Astra-Ben-20, a sodium bentonite (AB-20); MTB-100, an esterified glucomannan (MTB-100); Red Crown, a calcium bentonite (RC); Flow Guard, a sodium bentonite (FG); and Mycrosorb, a sodium bentonite (MS) . Five of the six sequestering agents significantly (P < 0.01) reduced AFM1 contamination of milk (AB-20, 61%; FG, 65%; MS, 50%; MTB-100, 59%; and RC, 31%); whereas, AC-A, activated carbon, had no effect on AFM1 transmission at 0.25% of feed . By the first milking (1 day after cows consumed contaminated feed), AFM1 appeared in milk, then reached maximum levels after three days, and was absent from milk within four days after AFB1 was removed from the feed . Sodium bentonites at 1.2% of feed showed good potential as AFB1 binders; MTB-100, a yeast cell wall product, was equally effective at 0.05% in feed . Potential AFB1 binding agents should be evaluated experimentally to demonstrate efficacy . Our data show that sequestering agents can reduce AFM1 in milk of cows fed AFB1-contaminated feed.

Am J Obstet Gynecol, 2004 Apr, 190(4), 1004 - 10
Predictive value of the clinical diagnosis of lower genital tract infection in women; Landers DV et al.; OBJECTIVE: We hypothesized that diagnostic approaches to lower genital tract infections are inaccurate and proposed this study to evaluate typical approaches . STUDY DESIGN: Clinical diagnoses were made with symptoms, direct observation, wet mount, vaginal pH, and amines in 598 women with genital complaints . Laboratory testing for N gonorrhoeae, yeast, T vaginalis, C trachomatis, and bacterial vaginosis by Gram stain . RESULTS: The most frequent symptoms were vaginal discharge (64%), change in discharge (53%), malodor (48%), and pruritis (32%) . The infection rates were 46% bacterial vaginosis, 29% yeast, 12% trichomoniasis, 11% chlamydia or gonorrhea; 21% of the patients had no infection . The symptoms did not predict laboratory diagnosis . Clinical signs and symptoms with office-based tests and microscopy improved the accuracy of diagnoses . Amsel's clinical diagnosis of bacterial vaginosis was the most sensitive at 92% . The sensitivity of wet mount diagnosis of trichomoniasis was 62%, of yeast by microscopy was 22%, and of mucopus for the prediction of gonorrhea and/or chlamydia was 30% . CONCLUSION: Symptoms alone should not be used to direct treatment in instances in which resources permit more complete evaluation with office-based testing that includes microscopy . Treatment failures or diagnostic uncertainty should prompt specific laboratory testing.

J Biol Chem, 2004 Jul 2, 279(27), 28522 - 30 Epub 2004 Apr 26.
Herpes simplex virus type 1 capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and plays a role in retrograde cellular transport; Douglas MW et al.; Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules . There is increasing evidence that the retrograde transport of herpes simplex virus type 1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known . Here we report that the herpes simplex virus outer capsid protein VP26 interacts with dynein light chains RP3 and Tctex1 and is sufficient to mediate retrograde transport of viral capsids in a cellular model . A library of herpes simplex virus capsid and tegument structural genes was constructed and tested for interactions with dynein subunits in a yeast two-hybrid system . A strong interaction was detected between VP26 and the homologous 14-kDa dynein light chains RP3 and Tctex1 . In vitro pull-down assays confirmed binding of VP26 to RP3, Tctex1, and intact cytoplasmic dynein complexes . Recombinant herpes simplex virus capsids were constructed either with or without VP26 . In pull-down assays VP26+ capsids bound to RP3; VP26-capsids did not . To investigate intracellular transport, the recombinant viral capsids were microinjected into living cells and incubated at 37 degrees C . After 1 h VP26+ capsids were observed to co-localize with RP3, Tctex1, and microtubules . After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, whereas VP26-capsids remained in a random distribution . We propose that VP26 mediates binding of incoming herpes simplex virus capsids to cytoplasmic dynein during cellular infection, through interactions with dynein light chains.

J Biol Chem, 2004 Jul 9, 279(28), 29325 - 35 Epub 2004 Apr 26.
90-kDa ribosomal S6 kinase is a direct target for the nuclear fibroblast growth factor receptor 1 (FGFR1): role in FGFR1 signaling; Hu Y et al.; Fibroblast growth factor receptor 1 (FGFR1) is a transmembrane protein capable of transducing stimulation by secreted FGFs . In addition, newly synthesized FGFR1 enters the nucleus in response to cellular stimulation and during development . Nuclear FGFR1 can transactivate CRE (cAMP responsive element), activate CRE-binding protein (CREB)-binding protein (CBP) and gene activities causing cellular growth and differentiation . Here, a yeast two-hybrid assay was performed to identify FGFR1-binding proteins and the mechanism of nuclear FGFR1 action . Ten FGFR1-binding proteins were identified . Among the proteins detected with the intracellular FGFR1 domain was a 90-kDa ribosomal S6 kinase (RSK1), a regulator of CREB, CBP, and histone phosphorylation . FGFR1 bound to the N-terminal region of RSK1 . The FGFR1-RSK1 interaction was confirmed by co-immunoprecipitation and colocalization in the nucleus and cytoplasm of mammalian cells . Predominantly nuclear FGFR1-RSK1 interaction was observed in the rat brain during neurogenesis and in cAMP-stimulated cultured neural cells . In TE671 cells, transfected FGFR1 colocalized and coimmunoprecipitated, almost exclusively, with nuclear RSK1 . Nuclear RSK1 kinase activity and RSK1 activation of CREB were enhanced by transfected FGFR1 . In contrast, kinase-deleted FGFR1 (TK-), which did not bind to RSK1 failed to stimulate nuclear RSK1 activity or RSK1 activation of CREB . Kinase inactive FGFR1 (K514A) bound effectively to nuclear RSK1, but it failed to stimulate RSK1 . Thus, active FGFR1 kinase regulates the functions of nuclear RSK1 . The interaction of nuclear FGFR1 with pluripotent RSK1 offers a new mechanism through which FGFR1 may control fundamental cellular processes.

J Biol Chem, 2004 Jul 2, 279(27), 28653 - 61 Epub 2004 Apr 26.
The Human hydroxyacylglutathione hydrolase (HAGH) gene encodes both cytosolic and mitochondrial forms of glyoxalase II; Cordell PA et al.; In yeast and higher plants, separate genes encode the cytosolic and mitochondrial forms of glyoxalase II . In contrast, although glyoxalase II activity has been detected both in the cytosol and mitochondria of mammals, only a single gene encoding glyoxalase II has been identified . Previously it was thought that this gene (the hydroxyacylglutathione hydrolase gene), comprised 8 exons that are transcribed into mRNA and that the resulting mRNA species encoded a single cytosolic form of glyoxalase II . Here we show that this gene gives rise to two distinct mRNA species transcribed from 9 and 10 exons, respectively . The 9-exon-derived transcript encodes two protein species: mitochondrially targeted glyoxylase II, which is initiated from an AUG codon in a previously uncharacterized part of the mRNA sequence, and cytosolic glyoxalase II, which is initiated by internal ribosome entry at a downstream AUG codon . The transcript deriving from 10 exons has an in-frame termination codon between the two initiating AUG codons and hence only encodes the cytosolic form of the protein . Confocal fluorescence microscopy indicates that the mitochondrially targeted form of glyoxalase II is directed to the mitochondrial matrix . Analysis of glyoxalase II mRNA sequences from a number of species indicates that dual initiation from alternative AUG codons is conserved throughout vertebrates.

Bioinformatics, 2004 Oct 12, 20(15), 2466 - 70 Epub 2004 Apr 29.
HPID: the Human Protein Interaction Database; Han K et al.; The Human Protein Interaction Database was designed (1) to provide human protein interaction information pre-computed from existing structural and experimental data, (2) to predict potential interactions between proteins submitted by users and (3) to provide a depository for new human protein interaction data from users . Two types of interaction are available from the pre-computed data: (1) interactions at the protein superfamily level and (2) those transferred from the interactions of yeast proteins . Interactions at the superfamily level were obtained by locating known structural interactions of the PDB in the SCOP domains and identifying homologs of the domains in the human proteins . Interactions transferred from yeast proteins were obtained by identifying homologs of the yeast proteins in the human proteins . For each human protein in the database and each query submitted by users, the protein superfamilies and yeast proteins assigned to the protein are shown, along with their interacting partners . We have also developed a set of web-based programs so that users can visualize and analyze protein interaction networks in order to explore the networks further . AVAILABILITY: http://www.hpid.org.

BMC Neurosci . 2004 Apr 29;5(1):16.
Targeted mutagenesis of the Sap47 gene of Drosophila: flies lacking the synapse associated protein of 47 kDa are viable and fertile; Funk N et al.; BACKGROUND: Conserved proteins preferentially expressed in synaptic terminals of the nervous system are likely to play a significant role in brain function . We have previously identified and molecularly characterized the Sap47 gene which codes for a novel synapse associated protein of 47 kDa in Drosophila . Sequence comparison identifies homologous proteins in numerous species including C . elegans, fish, mouse and human . First hints as to the function of this novel protein family can be obtained by generating mutants for the Sap47 gene in Drosophila . RESULTS: Attempts to eliminate the Sap47 gene through targeted mutagenesis by homologous recombination were unsuccessful . However, several mutants were generated by transposon remobilization after an appropriate insertion line had become available from the Drosophila P-element screen of the Bellen/Hoskins/Rubin/Spradling labs . Characterization of various deletions in the Sap47 gene due to imprecise excision of the P-element identified three null mutants and three hypomorphic mutants . Null mutants are viable and fertile and show no gross structural or obvious behavioural deficits . For cell-specific over-expression and "rescue" of the knock-out flies a transgenic line was generated which expresses the most abundant transcript under the control of the yeast enhancer UAS . In addition, knock-down of the Sap47 gene was achieved by generating 31 transgenic lines expressing Sap47 RNAi constructs, again under UAS control . When driven by a ubiquitously expressed yeast transcription factor (GAL4), Sap47 gene suppression in several of these lines is highly efficient resulting in residual SAP47 protein concentrations in heads as low as 6% of wild type levels . CONCLUSION: The conserved synaptic protein SAP47 of Drosophila is not essential for basic synaptic function . The Sap47 gene region may be refractory to targeted mutagenesis by homologous recombination . RNAi using a construct linking genomic DNA to anti-sense cDNA in our hands is not more effective than using a cDNA-anti-sense cDNA construct . The tools developed in this study will now allow a detailed analysis of the molecular, cellular and systemic function of the SAP47 protein in Drosophila.

Traffic, 2004 Jun, 5(6), 393 - 9
Rab GTPases and myosin motors in organelle motility; Seabra MC et al.; The actin cytoskeleton is essential to ensure the proper location of, and communication between, intracellular organelles . Some actin-based myosin motors have been implicated in this process, particularly members of the class V myosins . We discuss here the emerging role of the Ras-like GTPases of the Rab family as regulators of myosin function in organelle transport . Evidence from yeast secretory vesicles and mitochondria, and mammalian melanosomes and endosomes suggests that Rab GTPases are crucial components of the myosin organelle receptor machinery . Better understood is the case of the melanosome where Rab27a recruits a specific effector called melanophilin, which in turn binds myosin Va . The presence of a linker protein between a Rab and a myosin may represent a general mechanism . We argue that Rabs are ideally suited to perform this role as they are exquisite organelle markers . Furthermore, the molecular switch property of Rabs may enable them to regulate the timing of the myosin association with the target organelle.

Thromb Haemost, 2004 May, 91(5), 959 - 66
The novel human platelet septin SEPT8 is an interaction partner of SEPT4; Blaser S et al.; Septins are a family of GTP-binding proteins, which are essential for active membrane movement such as cytokinesis and vesicle trafficking . In non-dividing cells (such as platelets and neurons) septins are implicated in exocytosis . Platelets from a SEPT5 knockout mouse showed an altered serotonin secretion and platelet aggregation, suggesting that SEPT5 is involved in secretion in platelets . Septins form complexes consisting of multiple septin polypeptides . Using the yeast two-hybrid system we had demonstrated that SEPT5 partners with SEPT8 . The aim of this study was to identify other interaction partners of the human platelet septin SEPT8 . Using the yeast two-hybrid system with SEPT8 as bait protein we identified the human septin SEPT4 as an interaction partner of SEPT8 . The interaction between SEPT4 and SEPT8 was confirmed by immunoprecipitation . Expression analysis revealed that SEPT4 is also expressed in human platelets . Thus, SEPT4 is the third described platelet septin besides SEPT5 and SEPT8 . Transmission electron microscopy of platelets revealed that SEPT8 and SEPT4 are localized surrounding alpha-granules (as it had been shown for the septin SEPT5) suggesting that the three septins may be components of the septin complex in platelets and contribute in such a way to platelet biology . Activation of platelets by agonists resulted in the translocation of SEPT4 and SEPT8 to the platelet surface indicating a possible functional role of these proteins in platelet granular secretion.

Oncogene, 2004 Apr 29, 23(20), 3700 - 7
cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP; Hanissian SH et al.; Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1) . We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells . The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA . The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites . MLF1IP transcripts are expressed in a variety of tissues (e.g . fetal liver, bone marrow, thymus and testis) . MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes . These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

Protein Eng Des Sel, 2004 Apr, 17(4), 293 - 304 Epub 2004 Apr 28.
Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 degrees C; Graff CP et al.; An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37 degrees C . Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry . Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level . This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.

Hum Mol Genet, 2004 Jun 15, 13(12), 1257 - 65 Epub 2004 Apr 28.
Ataxin-7 is a subunit of GCN5 histone acetyltransferase-containing complexes; Helmlinger D et al.; Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder caused by a CAG repeat expansion in the SCA7 gene leading to elongation of a polyglutamine tract in ataxin-7, a protein of unknown function . A putative ataxin-7 yeast orthologue (SGF73) has been identified recently as a new component of the SAGA (Spt/Ada/Gcn5 acetylase) multisubunit complex, a coactivator required for transcription of a subset of RNA polymerase II-dependent genes . We show here that ataxin-7 is an integral component of the mammalian SAGA-like complexes, the TATA-binding protein-free TAF-containing complex (TFTC) and the SPT3/TAF9/GCN5 acetyltransferase complex (STAGA) . In agreement, immunoprecipitation of ataxin-7 retained a histone acetyltransferase activity, characteristic for TFTC-like complexes . We further identified a minimal domain in ataxin-7 that is required for interaction with TFTC/STAGA subunits and is conserved highly through evolution, allowing the identification of a SCA7 gene family . We showed that this domain contains a conserved Cys(3)His motif that binds zinc, forming a new zinc-binding domain . Finally, polyglutamine expansion in ataxin-7 did not affect its incorporation into TFTC/STAGA complexes purified from SCA7 patient cells . We demonstrate here that ataxin-7 is the human orthologue of the yeast SAGA SGF73 subunit and is a bona fide subunit of the human TFTC-like transcriptional complexes.

Hum Mol Genet, 2004 Jun 15, 13(12), 1241 - 8 Epub 2004 Apr 28.
Direct interaction of FANCD2 with BRCA2 in DNA damage response pathways; Hussain S et al.; Fanconi anaemia (FA) is a chromosomal instability disorder characterized by cellular sensitivity to DNA interstrand crosslinking agents and a high risk of cancer . Six of the eight proteins encoded by the known FA genes form a nuclear complex which is required for the monoubiquitination of the FANCD2 protein . FANCD2 complexes and colocalizes with BRCA1, but its presumptive role in DNA repair has not yet been clearly defined . We used yeast two-hybrid analysis to test for interaction between FANCD2 and 10 proteins involved in homologous recombination repair . FANCD2 did not interact with RAD51, the five RAD51 paralogs, RAD52, RAD54 or DMC1 . However, it bound to a highly conserved C-terminal site in BRCA2 that also binds FANCG/XRCC9 . FANCD2 and BRCA2 can be coimmunoprecipitated from cell extracts of both human and Chinese hamster wild-type cells, thus confirming that the interaction occurs in vivo . Formation of nuclear foci of FANCD2 was normal in the BRCA2 mutant CAPAN-1 cells, which indicates that the recruitment of FANCD2 to sites of DNA-repair is independent of wild-type BRCA2 function . FANCD2 colocalized with RAD51 in foci following treatment with mitomycin C or hydroxyurea, and colocalized very tightly with PCNA after treatment with hydroxyurea . These findings suggest that FANCD2 may have a role in the cellular response to stalled replication forks or in the repair of replication-associated double-strand breaks, irrespective of the type of primary DNA lesion.

Cell Stress Chaperones, 2003 Winter, 8(4), 381 - 94
Tomato heat stress protein Hsp16.1-CIII represents a member of a new class of nucleocytoplasmic small heat stress proteins in plants; Siddique M et al.; We describe a new class of plant small heat stress proteins (sHsps) with dominant nuclear localization (Hsp17-CIII) . The corresponding proteins in tomato, Arabidopsis, and rice are encoded by unique genes containing a short intron in the beta4-encoding region of the alpha-crystallin domain (ACD) . The strong nuclear localization results from a cluster of basic amino acid residues in the loop between beta5 and beta6 of the ACD . Using yeast 2-hybrid tests, analyses of native complexes of the sHsps, and immunofluorescence data, we demonstrate that, in contrast to earlier observations (Kirschner et al 2000), proteins of the sHsp classes CI, CII, and CIII interact with each other, thereby influencing oligomerization state and intracellular localization.

Srp Arh Celok Lek, 2003 Nov-Dec, 131(11-12), 454 - 7
{First case of Malassezia globosa isolation in Serbia}; Arsic-Arsenijevic V et al.; Today is known that genus Malassezia includes seven species: M . furfur, M . sympodialis, M . obtusa, M . globosa, M . restricta, M . sloofflae and M . pachydermatis, but role of each of the species in the pathogenesis of disease has not been elucidated yet, so further laboratory isolation and identification are necessary . We report the first case of isolation of Malassezia globosa in Serbia (Belgrade), in a patient suffering from Pityriasis versicolor . Identification of M . globosa was based on macroscopic, microscopic and biochemical characteristics . Isolation was done on Leeming and Notman medium and on mDixona agar, at 350C, during 7 days in aerobic conditions . Also the yeast's biochemical phenotype was determined as catalase (+), lipase (+), esculin degradation (-), Tween (20, 40, 60 and 80) assimilation (-) . M . globosa is a lipophilic yeast of the genus Malassezia and the common member of the skin flora . In concordance with some predisponing factors M . globosa is implicated in the pathogenesis of several skin diseases (pityriasis versicolor, malassezia foliculitis, seborheic dermatitis and some forms of atopic dermatitis) . In immunocompromised patients and neonates this yeast can even cause fatal systemic infections . Because the role of Malassezia spp . In pathogenesis of skin disease is not still determined, we suggest laboratory diagnosis and identification of these species as a routine diagnostic procedure.

Bioessays, 2004 May, 26(5), 523 - 32
Insulator dynamics and the setting of chromatin domains; Fourel G et al.; The early discovery of cis-regulatory elements able to promote transcription of genes over large distances led to the postulate that elements, termed insulators, should also exist that would limit the action of enhancers, LCRs and silencers to defined domains . Such insulators were indeed found during the past fifteen years in a wide range of organisms, from yeast to humans . Recent advances point to an important role of transcription factors in insulator activity and demonstrate that the operational observation of an insulator effect relies on a delicate balance between the "efficiency" of the insulator and that of the element to be counteracted . In addition, genuine insulator elements now appear less common than initially envisaged, and they are only found at loci displaying a high density of coding or regulatory information . Where this is not the case, chromatin domains of opposing properties are thought to confront each other at "fuzzy" boundaries . In this article, we propose models for both fixed and fuzzy boundaries that incorporate probabilistic and dynamic parameters .

J Ind Microbiol Biotechnol, 2004 May, 31(4), 155 - 60 Epub 2004 Apr 27.
A general method for the analysis of random bisubstrate enzyme mechanisms; Leskovac V et al.; In the present communication, a general method for the kinetic analysis of random bisubstrate mechanisms is described . The method comprises a stepwise application of the following kinetic and ligand-binding experiments: determination of steady-state kinetic constants, product inhibition patterns, maximum rate relationships, application of alternate substrates, application of dead-end inhibitors, direct binding of substrates, kinetic isotope effects, and isotope exchange studies . This general method was applied to a practical example: a yeast alcohol dehydrogenase-catalyzed oxidation of 2-propanol by NAD(+) at pH 7.0, 25 degrees C . It was found that this fully reversible reaction proceeds by a steady-state random Bi-Bi mechanism, whereby both dead-end complexes are formed.

Invest Ophthalmol Vis Sci, 2004 May, 45(5), 1297 - 305
Prosaposin gene expression in normal and dystrophic RCS rat retina; Van Den Berghe L et al.; PURPOSE: To identify proteins secreted by the retinal pigment epithelium (RPE) and to analyze their cellular distribution in normal and pathologic rat retinas at various stages of eye development . METHODS: A cDNA library was constructed with RNA isolated from porcine RPE sheets and screened by using the yeast signal sequence trap system . In situ hybridization, immunohistochemistry, and semiquantitative RT-PCR analysis were performed on rat retinas . RESULTS: The cDNA encoding prosaposin was isolated . This is the first time this gene has been shown to be expressed in the retina . Prosaposin mRNA was detected in the rat RPE cell monolayer and in ganglion cells 14, 21, and 45 days after birth . The amount of prosaposin mRNA increased between days 14 and 45 after birth in normal retinas (rdy+), but not in the pathologic retinas (rdy-) of RCS rats . CONCLUSIONS: Several techniques were used to determine the localization of prosaposin in rat retinas . The increase in the amount of prosaposin mRNA in normal retinas coincided with the maturation of photoreceptor cells and the beginning of the phagocytosis process . In addition, the RCS rdy- RPE cells, characterized by the abrogation of the ingestion phase of the photoreceptor outer segments, are deficient in prosaposin expression.

FEBS Lett, 2004 Apr 30, 564(3), 239 - 44
The role and structure of mitochondrial carriers; Kunji ER; Members of the mitochondrial carrier family transport compounds over the inner mitochondrial membrane to link the biochemical pathways in the cytosol with those in the mitochondrial matrix . X-ray crystallography has recently provided us with the first atomic model of the bovine ADP/ATP carrier, which is a member of this family . The structure explains the typical three-fold sequence repeats and signature motif of mitochondrial carriers . However, the carrier was crystallised as a monomer in detergent, which is inconsistent with the consensus that mitochondrial carriers exist as homo-dimers . The projection structure of the yeast ADP/ATP carrier by electron crystallography shows that carriers could form homo-dimers in the membrane.

Int J Food Microbiol, 2004 Apr 15, 92(2), 171 - 9
Dormancy, activation and viability of Rhizopus oligosporus sporangiospores; Thanh NV et al.; Interruption of dormancy to improve viability of Rhizopus oligosporus sporangiospores is crucial for the application of stored starter cultures for fungal (tempe) production . We aimed to assess the extent of dormancy and factors that could result in activation . Whereas heat treatments were unsuccessful, Malt Extract Broth (MEB) showed to be a good activation medium, with 80% of dormant spores being activated as measured by fluorescence microscopy using a fluorescent marker, compared with 11% with the control . Peptone and yeast extract but not glucose played an important role in activating dormant spores . Metabolically active (fluorescent) and swollen spores, followed by germ tubes were obtained after activation in MEB for 25 min., 2 and 4 h, respectively, at 37 degrees C . Simultaneously, some interesting transitions took place . Dormant spores represent 85-90% of the total spores at harvest and after drying . Their number decreased to 21-32% after activation with MEB with a concomitant increase of metabolically active spores . As a result of storage, some dormancy was lost, yielding an increase of active spores from 11.2% at harvest to 28.8% after 3 months storage . Levels of active spores were well correlated with their viability . By activation of dormant spores, their viability increased; levels of viable and active spores were maximum in 1 month old starter (61.7% and 75.9% of total spores, respectively) but gradually decreased with concomitant increase of the number of dead spores.

Leuk Res, 2004 Apr, 28(4), 409 - 14
Domains involved in ETO and human N-CoR interaction and ETO transcription repression; Wang J et al.; The (8;21) translocation between the AML1 and ETO genes is seen in approximately 12-15% of all acute myeloid leukemia (AML) and is a frequently observed nonrandom genetic alteration associated with AML . The ETO moiety was shown to interact with the nuclear receptor co-repressor (N-CoR) complex, which includes mSin3A and the histone deacetylase, HDAC1 . Repression of AML1-responsive hematopoietic genes by AML1-ETO and the N-CoR complex may play a mechanistic role in t(8;21) leukemogenesis . In order to characterize the interaction between ETO and N-CoR, mutants of either protein were constructed and tested for binding in both yeast two-hybrid and immunoprecipitation assays . We found that two domains of human N-CoR, amino acid residues 988-1126 and 1551-1803, were necessary for interaction with ETO . Previously, we and other investigators had reported that two unusual zinc finger motifs at the C-terminus of ETO mediated binding to N-CoR . Here, using mammalian two-hybrid assays, we found that transcription repression by ETO was substantially decreased when either zinc finger motif of ETO is deleted or mutated . In addition, we identified a second transcription repression domain located between residues 275 and 487 . Characterization of the ETO interaction domains within human N-CoR and of the transcription domains of ETO is a first step in designing targeted molecular therapy for t(8;21) AML.

BMC Genomics . 2004 Apr 26;5(1):25.
Rapid prefractionation of complex protein lysates with centrifugal membrane adsorber units improves the resolving power of 2D-PAGE-based proteome analysis; Doud MK et al.; BACKGROUND: Two-dimensional gel electrophoresis (2D-PAGE) has proven over the years to be a reliable and efficient method for separation of hundreds of proteins based on charge and mass . Nevertheless, the complexity of even the simplest proteomes limits the resolving power of 2D-PAGE . This limitation can be partially alleviated by sample prefractionation using a variety of techniques . RESULTS: Here, we have used Vivapure Ion Exchange centrifugal adsorber units to rapidly prefractionate total fission yeast protein lysate based on protein charge . Three fractions were prepared by stepwise elution with increasing sodium chloride concentrations . Each of the fractions, as well as the total lysate, were analyzed by 2D-PAGE . This simple prefractionation procedure considerably increased the resolving power of 2D-PAGE . Whereas 308 spots could be detected by analysing total protein lysate, 910 spots were observed upon prefractionation . Thorough gel image analysis demonstrated that prefractionation visualizes an additional set of 458 unique fission yeast proteins not detected in whole cell lysate . CONCLUSIONS: Prefractionation with Vivapure Q spin columns proved to be a simple, fast, reproducible, and cost-effective means of increasing the resolving power of 2D-PAGE using standard laboratory equipment.

Biochem J, 2004 Jun 15, 380(Pt 3), 605 - 10
Alternative reading frame protein (ARF)-independent function of CARF (collaborator of ARF) involves its interactions with p53: evidence for a novel p53-activation pathway and its negative feedback control; Hasan MK et al.; CARF, a collaborator of ARF (alternative reading frame protein), was cloned as a novel ARF-binding protein from a yeast-interaction screen . It potentiated ARF-mediated p53 function, and also caused a moderate increase in p53 activity in the absence of ARF . We herein report the molecular mechanism of ARF-independent function of CARF . By employing a variety of approaches, including overexpression of CARF, its suppression by small interfering RNA and use of protease inhibitors, we demonstrate that: (i) CARF directly interacts with wild-type p53, causing its stabilization and functional activation; and (ii) CARF and p53 levels show an inverse relationship that is instigated by a negative-feedback control via a proteasome-mediated degradation pathway.

Biochemistry, 2004 May 4, 43(17), 4944 - 54
Studies of the enzymic mechanism of Candida tenuis xylose reductase (AKR 2B5): X-ray structure and catalytic reaction profile for the H113A mutant; Kratzer R et al.; Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldo-keto reductase (AKR) superfamily of proteins and enzymes . Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding . We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 A structure of H113A mutant bound to NADP(+), and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant . Deuterium kinetic isotope effects (KIEs) on k(cat) and k(cat)/K(m xylose) show that, relative to the wild type, the hydride transfer rate constant (k(7) approximately 0.16 s(-1)) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected . No solvent isotope effect was seen on k(cat) and k(cat)/K(m xylose) for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation . The pH profiles of log(k(cat)/K(m xylose)) for the wild type and H113A decreased above apparent pK(a) values of 8.85 and 7.63, respectively . The DeltapK(a) of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50 . A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on k(cat) and k(cat)/K(m aldehyde) into an electronic effect and bonding effects (which are lacking in the wild type) . Using the Hammett sigma scale, electronic parameter coefficients (rho) of +0.64 (k(cat)) and +0.78 (k(cat)/K(m aldehyde)) were calculated and clearly differ from rho(k(cat)/K(aldehyde)) and rho(k(cat)) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme . Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants . An enzymic mechanism is proposed in which His-113, through a hydrogen bond from Nepsilon2 to aldehyde O1, assists in catalysis by optimizing the C=O bond charge separation and orbital alignment in the ternary complex.

Nat Cell Biol, 2004 May, 6(5), 393 - 404 Epub 2004 Apr 25.
FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P; Godi A et al.; The molecular mechanisms underlying the formation of carriers trafficking from the Golgi complex to the cell surface are still ill-defined; nevertheless, the involvement of a lipid-based machinery is well established . This includes phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) . In yeast, PtdIns(4)P exerts a direct role, however, its mechanism of action and its targets in mammalian cells remain uncharacterized . We have identified two effectors of PtdIns(4)P, the four-phosphate-adaptor protein 1 and 2 (FAPP1 and FAPP2) . Both proteins localize to the trans-Golgi network (TGN) on nascent carriers, and interact with PtdIns(4)P and the small GTPase ADP-ribosylation factor (ARF) through their plekstrin homology (PH) domain . Displacement or knockdown of FAPPs inhibits cargo transfer to the plasma membrane . Moreover, overexpression of FAPP-PH impairs carrier fission . Therefore, FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers.

Oncogene, 2004 Jul 8, 23(31), 5316 - 29
BRCA1 cooperates with NUFIP and P-TEFb to activate transcription by RNA polymerase II; Cabart P et al.; The tumor suppressor gene product BRCA1 is a component of the RNA polymerase II (pol II) holoenzyme that is involved, through binding to various regulatory proteins, in either activation or repression of transcription . Using a yeast two-hybrid screen, we have identified a human zinc-finger-containing protein NUFIP that interacts with BRCA1 . The ubiquitous, stably expressed, nuclear protein NUFIP specifically stimulates activator-independent pol II transcription in vitro and in vivo . Immunodepletion of the endogenous NUFIP causes a marked decrease of pol II transcription, which is then shown to be restored by stable complex of ectopically produced NUFIP and associated factors . NUFIP not only interacts with BRCA1 but also associates with the positive elongation factor P-TEFb through interaction with the regulatory Cyclin T1 subunit . Cyclin T1 is required for BRCA1- and NUFIP-dependent synergistic activation of pol II transcription in 293 cells . Mutation of the zinc-finger domain abolishes the NUFIP-mediated transcriptional activation . We show that NUFIP is associated with preinitiation complexes, open transcription complexes, and elongation complexes . In addition, NUFIP facilitates ATP-dependent dissociation of hyperphosphorylated pol II from open transcription complexes in vitro.

Mol Biol Cell, 2004 Jul, 15(7), 3106 - 13 Epub 2004 Apr 23.
Ribosomal S6 kinase as a mediator of keratinocyte growth factor-induced activation of Akt in epithelial cells; Pan ZZ et al.; The keratinocyte growth factor receptor (KGFR) is a member of the fibroblast growth factor receptor (FGFR) superfamily . The proximal signaling molecules of FGFRs are much less characterized compared with other growth factor receptors . Using the yeast two-hybrid assay, we have identified ribosomal S6 kinase (RSK) to be a protein that associates with the cytoplasmic domain of the KGFR . The RSK family of kinases controls multiple cellular processes, and our studies for the first time show association between the KGFR and RSK . Using a lung-specific inducible transgenic system we have recently demonstrated protective effects of KGF on the lung epithelium and have demonstrated KGF-induced activation of the prosurvival Akt pathway both in vivo and in vitro . Here we show that a kinase inactive RSK mutant blocks KGF-induced Akt activation and KGF-mediated inhibition of caspase 3 activation in epithelial cells subjected to oxidative stress . It was recently shown that RSK2 recruits PDK1, the kinase responsible for both Akt and RSK activation . When viewed collectively, it appears that the association between the KGFR and RSK plays an important role in KGF-induced Akt activation and consequently in the protective effects of KGF on epithelial cells.

Mol Biol Cell, 2004 Jul, 15(7), 3053 - 60 Epub 2004 Apr 23.
Adaptor protein complex 1 mediates the transport of lysosomal proteins from a Golgi-like organelle to peripheral vacuoles in the primitive eukaryote Giardia lamblia; Touz MC et al.; Giardia lamblia is an early branching protist that possesses peripheral vacuoles (PVs) with characteristics of lysosome-like organelles, located underneath the plasma membrane . In more evolved cells, lysosomal protein trafficking is achieved by cargo recognition involving adaptor protein (AP) complexes that recognize specific amino acid sequences (tyrosine and/or dileucine motifs) within the cytoplasmic tail of membrane proteins . Previously, we reported that Giardia has a tyrosine-based sorting system, which mediates the targeting of a membrane-associated cysteine protease (encystation-specific cysteine protease, ESCP) to the PVs . Here, we show that Giardia AP1 mediates the transport of ESCP and the soluble acid phosphatase (AcPh) to the PVs . By using the yeast two-hybrid assay we found that the ESCP tyrosine-based motif interacts specifically with the medium subunit of AP1 (Gimicroa) . Hemagglutinin-tagged Gimicroa colocalizes with ESCP and AcPh and coimmunoprecipitates with clathrin, suggesting that protein trafficking toward the PVs is clathrin-adaptin dependent . Targeted disruption of Gimicroa results in mislocalization of ESCP and AcPh but not of variant-specific surface proteins . Our results suggest that, unlike mammalian cells, only AP1 is involved in anterograde protein trafficking to the PVs in Giardia . Moreover, even though Giardia trophozoites lack a morphologically discernible Golgi apparatus, the presence of a clathrin-adaptor system suggests that this parasite possess a primitive secretory organelle capable of sorting proteins similar to that of more evolved cells.

Mol Biol Cell, 2004 Jul, 15(7), 3031 - 41 Epub 2004 Apr 23.
Ent5p is required with Ent3p and Vps27p for ubiquitin-dependent protein sorting into the multivesicular body; Eugster A et al.; At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body . Both PtdIns(3,5)P(2) and ubiquitin are necessary for proper sorting of some of these cargoes . We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P(2) via its ENTH domain . In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected . Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body . Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P(2)- and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body.

J Leukoc Biol, 2004 Jul, 76(1), 86 - 94 Epub 2004 Apr 23.
Expression of the beta-glucan receptor, Dectin-1, on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions; Reid DM et al.; Dectin-1 is a pathogen-recognition receptor on macrophages (MPhis), neutrophils, and dendritic cells (DCs) . On MPhis and bone marrow-derived DCs, it has been shown to mediate the nonopsonic recognition of and response to soluble and particulate yeast beta-glucans . We have optimized the immunohistochemical detection of Dectin-1 and demonstrated its expression on neutrophils, subpopulations of MPhis in splenic red and white pulp, alveolar MPhis, Kupffer cells, and MPhis and DCs in the lamina propria of gut villi . This is consistent with its role in pathogen surveillance . A significant proportion of CD11c(+) splenic DCs expressed Dectin-1, but expression was not restricted to any one subset . Dectin-1 expression was low on resident MPhis and DCs of skin and was not detected on resident MPhis or DCs in kidney, heart, brain, or eye . The proposed, additional role of Dectin-1 as a coreceptor for T cell activation is supported by its expression on DCs in the T cell areas of the spleen and lymph nodes . Strong expression of Dectin-1 on subpopulations of MPhis and DCs in the medullary and corticomedullary regions of the thymus suggests a role distinct from pathogen recognition . Tissue localization thus revealed potential roles of Dectin-1 in leukocyte interactions during innate immune responses and T cell development.

J Biol Chem, 2004 Jul 16, 279(29), 30643 - 53 Epub 2004 Apr 23.
An androgen receptor NH2-terminal conserved motif interacts with the COOH terminus of the Hsp70-interacting protein (CHIP); He B et al.; The NH2-terminal sequence of steroid receptors is highly variable between different receptors and in the same receptor from different species . In this study, a primary sequence homology comparison identified a 14-amino acid NH2-terminal motif of the human androgen receptor (AR) that is common to AR from all species reported, including the lower vertebrates . The evolutionarily conserved motif is unique to AR, with the exception of a partial sequence in the glucocorticoid receptor of higher species . The presence of the conserved motif in AR and the glucocorticoid receptor and its absence in other steroid receptors suggests convergent evolution . The function of the AR NH2-terminal conserved motif was suggested from a yeast two-hybrid screen that identified the COOH terminus of the Hsp70-interacting protein (CHIP) as a binding partner . We found that CHIP functions as a negative regulator of AR transcriptional activity by promoting AR degradation . In support of this, two mutations in the AR NH2-terminal conserved motif previously identified in the transgenic adenocarcinoma of mouse prostate model reduced the interaction between CHIP and AR . Our results suggest that the AR NH2-terminal domain contains an evolutionarily conserved motif that functions to limit AR transcriptional activity . Moreover, we demonstrate that the combination of comparative sequence alignment and yeast two-hybrid screening using short conserved peptides as bait provides an effective strategy to probe the structure-function relationships of steroid receptor NH2-terminal domains and other intrinsically unstructured transcriptional regulatory proteins.

EMBO Rep, 2004 Apr, 5(4), 385 - 91
DNA double-strand breaks, recombination and synapsis: the timing of meiosis differs in grasshoppers and flies; Viera A et al.; The temporal and functional relationships between DNA events of meiotic recombination and synaptonemal complex formation are a matter of discussion within the meiotic field . To analyse this subject in grasshoppers, organisms that have been considered as models for meiotic studies for many years, we have studied the localization of phosphorylated histone H2AX (gamma-H2AX), which marks the sites of double-strand breaks (DSBs), in combination with localization of cohesin SMC3 and recombinase Rad51 . We show that the loss of gamma-H2AX staining is spatially and temporally linked to synapsis, and that in grasshoppers the initiation of recombination, produced as a consequence of DSB formation, precedes synapsis . This result supports the idea that grasshoppers display a pairing pathway that is not present in other insects such as Drosophila melanogaster, but is similar to those reported in yeast, mouse and Arabidopsis . In addition, we have observed the presence of gamma-H2AX in the X chromosome from zygotene to late pachytene, indicating that the function of H2AX phosphorylation during grasshopper spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA DSBs.

Mikrobiol Z, 2004 Jan-Feb, 66(1), 3 - 9
{Keratinolytic activity of Streptomyces sp . 1382}; Ivanko OV et al.; Research in the growth dynamics of Streptomyces sp . 1382--a producer of keratinolytic enzyme--has shown that maximum of its biosynthesis in the culture medium is achieved on the 5th day, that correlates with maximum of biomass accumulation . The enzyme complex, obtained from culture liquid, is active in the wide range of pH (4-11) and temperature (10-70 degrees C) . Under such conditions it is stable and does not lose its activity during several hours . The preparation possesses different pH-optima of keratinase and total proteolytic activities: in neutral (pH 7.0) and weakly alkali (pH 8.0) regions, as well as different thermal optima at 37-40 and 50-60 degrees C, respectively . Four types of colonies whose level of keratinase activity was considerably different have been found, when analyzing heterogeneity of the strain-producer population . Study of the effect of various sources of carbon and nitrogen on proteases synthesis by the culture has found the activating effect of arabinose, maltose, peptone and yeast autolysate . Under such conditions synthesis of keratinolytic enzyme increased 6-7 time, only the sheep wool and chicken feather being added as the only source of carbon and nitrogen . The induction with substrate is discussed as a possible mechanism of keratinase synthesis regulation.

Eur J Mass Spectrom (Chichester, Eng), 2004, 10(2), 289 - 94
Optimization of conditions for studies of protein unfolding by hydrogen exchange/mass spectrometry; Raza AS et al.; Understanding the forces driving protein folding and aggregation is an essential step in developing means for controlling these important processes . Amide hydrogen exchange, coupled with mass spectrometry, has become an important method for studying protein unfolding and refolding . To extend procedures developed to study unfolding of relatively soluble proteins to less soluble, aggregation-prone proteins requires special considerations . This publication describes a general strategy developed using yeast transaldolase, which aggregates easily under conditions required to study its unfolding . Results presented here show that reducing the protein concentration to the nanomolar range is essential for managing aggregation of transaldolase . In addition, the present results point to use of relatively high concentrations of denaturants and short incubation times to minimize aggregation . These results also show how amide hydrogen exchange, coupled with mass spectrometry, can be used to study soluble aggregates.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 6934 - 9 Epub 2004 Apr 21.
Probing TBP interactions in transcription initiation and reinitiation with RNA aptamers that act in distinct modes; Fan X et al.; The TATA-binding protein (TBP) is a critical general transcription factor that associates with the core promoter and acts as a nexus for gene regulation through its interactions with other factors . A large number of proteins recognize the relatively small yet highly conserved C-terminal domain of TBP . One subset of these proteins (general transcription factors) interacts with the TBP.TATA complex and RNA polymerase II to create the preinitiation complex . To study TBP functions in preinitiation complex and other complexes, we generated a set of RNA aptamers with high affinity to yeast TBP . These aptamers act on TBP in different ways: all of them bind TBP competitively with DNA bearing the TATA element, and some can actively disrupt the TBP.TATA interaction in preformed, higher-order complexes containing the additional general transcription factors TFIIB and TFIIA . In crude cell extracts, the aptamers inhibit transcription in ways that reveal the dynamic nature of TBP interactions during initiation and reinitiation.

J Biol Chem, 2004 Jul 2, 279(27), 28393 - 401 Epub 2004 Apr 21.
Identification of snapin and three novel proteins (BLOS1, BLOS2, and BLOS3/reduced pigmentation) as subunits of biogenesis of lysosome-related organelles complex-1 (BLOC-1); Starcevic M et al.; Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a ubiquitously expressed multisubunit protein complex required for the normal biogenesis of specialized organelles of the endosomal-lysosomal system, such as melanosomes and platelet dense granules . The complex is known to contain the coiled-coil-forming proteins, Pallidin, Muted, Cappuccino, and Dysbindin . The genes encoding these proteins are defective in inbred mouse strains that serve as models of Hermansky-Pudlak syndrome (HPS), a genetic disorder characterized by hypopigmentation and platelet storage pool deficiency . In addition, mutation of human Dysbindin causes HPS type 7 . Here, we report the identification of another four subunits of the complex . One is Snapin, a coiled-coil-forming protein previously characterized as a binding partner of synaptosomal-associated proteins 25 and 23 and implicated in the regulation of membrane fusion events . The other three are previously uncharacterized proteins, which we named BLOC subunits 1, 2, and 3 (BLOS1, -2, and -3) . Using specific antibodies to detect endogenous proteins from human and mouse cells, we found that Snapin, BLOS1, BLOS2, and BLOS3 co-immunoprecipitate, and co-fractionate upon size exclusion chromatography, with previously known BLOC-1 subunits . Furthermore, steady-state levels of the four proteins are significantly reduced in cells from pallid mice, which carry a mutation in Pallidin and display secondary loss of other BLOC-1 subunits . Yeast two-hybrid analyses suggest a network of binary interactions involving all of the previously known and newly identified subunits . Interestingly, the HPS mouse model strain, reduced pigmentation, carries a nonsense mutation in the gene encoding BLOS3 . As judged from size exclusion chromatographic analyses, the reduced pigmentation mutation affects BLOC-1 assembly less severely than the pallid mutation . Mutations in the human genes encoding Snapin and the BLOS proteins could underlie novel forms of HPS.

Eur J Pain, 1997, 1(1), 43 - 52
Intraplantar zymosan as a reliable, quantifiable model of thermal and mechanical hyperalgesia in the rat; Meller ST et al.; The study of the mechanisms of thermal and mechanical hyperalgesia produced in human inflammatory conditions is dependent on a reliable, consistent model . The present investigation shows that the intraplantar administration of zymosan in the rat hindpaw produces a reliable and quantifiable thermal and mechanical hyperalgesia accompanied by oedema that closely mimics the symptoms of inflammation in man . Prior to the intraplantar injection of zymosan, there was no significant difference in withdrawal latencies, mechanical withdrawal thresholds or paw thickness between the left and right hindpaws . The intraplantar injection of zymosan (0.313-6.25 mg), an extract from yeast, produced a dose- and time-dependent thermal and mechanical hyperalgesia, a robust oedema and, at the greatest doses, produced evidence of spontaneous pain . At the least dose of zymosan tested (0.313 mg), there was a slight oedema; oedema was greatest at dosages > or =2.5 mg and was always maximal by 30 min postinjection, irrespective of the dose . On the other hand, thermal and mechanical hyperalgesia showed a more complex dose- and time-dependence . Mechanical hyperalgesia did not appear until dosages > or =1.25 mg and was maximal by 5 mg . In addition, mechanical hyperalgesia showed a time-dependent progressive onset so that hyperalgesia was maximal by the 4-h testing time-point . In contrast, thermal hyperalgesia showed a biphasic nature with two apparent maximal time-points (30 min and 4 h) . There was an early-phase thermal hyperalgesia (maximal by 30 min) that was dose-dependent at dosages > or =2.5 mg (not apparent at lower dosages) and a late-phase (maximal by 4 h) that was dose-dependent at dosages > or =0.0625 mg . At the greatest doses administered (5 and 6.25 mg), there was evidence of spontaneous pain from the time of injection for up to 4 h that was characterized by occasional spontaneous flicking of the hindpaw, but more usually by holding the paw in an elevated position for extended periods of time . In addition, at the greatest dose tested (6.25 mg), all rats showed evidence of licking, biting and shaking of the injected hindpaw for up to 30-45 min after injection . These data demonstrate that the intraplantar injection of zymosan is a reliable and quantifiable model of tonic pain characterized by a dose- and time-dependent thermal and mechanical hyperalgesia accompanied by a robust oedema . This model is likely to be a useful, reliable model in which to study further the central and peripheral mechanisms of hyperalgesia.

Biochem J, 2004 Aug 1, 381(Pt 3), 895 - 904
Binding of proteins to the PDZ domain regulates proteolytic activity of HtrA1 serine protease; Murwantoko et al.; HtrA1, a member of the mammalian HtrA (high temperature requirement A) serine protease family, has a highly conserved protease domain followed by a PDZ domain . Accumulating evidence has indicated that PDZ domains regulate protease activity of HtrA proteins . We searched for binding partners of the PDZ domain of mouse HtrA1 by yeast two-hybrid screening, and isolated proteins that were recognized by the HtrA1 PDZ domain through their C-terminal ends with a core consensus Phi-X-Phi-{V/L/F/A}-COOH sequence (where Phi is a hydrophobic/non-polar amino acid) . C-propeptides of fibrillar collagens were most frequently isolated . Type III procollagen alpha1 C-propeptide, which was used as a model protein, was digested by HtrA1 . HtrA1 cleavage of the collagen C-propeptide was enhanced by reductive denaturation of the C-propeptide and partly inhibited by removal of the C-terminal four amino acids from the C-propeptide, suggesting that the substrate recognition was facilitated by the binding of the free C-terminal ends of substrates to the PDZ domain of HtrA1 . The synthetic oligopeptide (GM130Pep) that fitted the consensus recognition sequence bound to HtrA1 with a high affinity (K(d)=6.0 nM) . GM130Pep stimulated HtrA1 protease activity 3- to 4-fold, but did not efficiently stimulate the activity of an HtrA1 mutant lacking the PDZ domain, supporting the notion that the PDZ domain enhances protease activity upon ligand binding . The peptide derived from Type III collagen alpha1 C-propeptide specifically stimulated protease activity of HtrA1, but did not stimulate nor significantly bind to HtrA2, suggesting that the collagen C-propeptide is a specific physiological regulator of HtrA1.

Environ Toxicol, 2004 Jun, 19(3), 216 - 25
Survey of hormone activities in municipal biosolids and animal manures; Lorenzen A et al.; The potential exists for natural or synthetic hormonal chemicals present in agricultural fertilizers to be transferred to adjacent aquatic environments in order to alter endocrine function in exposed wildlife . Recombinant yeast and mammalian cell line (BG1Luc4E2) assays were used to screen crude organic extracts of municipal biosolids and animal manures for estrogen-, androgen-, and progesterone receptor gene transcription activities . Of the biosolid extracts, those samples that had undergone aerobic digestion had no or minimal estrogen- and no androgen receptor gene transcription activities . In contrast, those biosolid samples that had undergone anaerobic digestion had much higher estrogen- and, for all but one site, androgen receptor gene transcription activities . Extracts prepared from animal manure samples had variable levels of androgen- and estrogen receptor gene transcription activities, which may be related to the type, sex, age, and reproductive status of the animals . The diet and treatment of animals with hormone implants also appeared to be factors influencing hormone activity in animal manure . Progesterone receptor gene transcription activity was observed for only one chicken litter sample . Overall, results of this study suggest that in vitro bioassays can be used to survey and detect hormone activity in municipal biosolids and animal manures . Furthermore, results of these assays can be used to develop practices that will minimize the potential environmental endocrine-disrupting effects of these substances .

Proc Natl Acad Sci U S A, 2004 Apr 27, 101(17), 6484 - 9 Epub 2004 Apr 19.
Human Claspin works with BRCA1 to both positively and negatively regulate cell proliferation; Lin SY et al.; Claspin is a homolog of Mrc1, a checkpoint protein required for the DNA replication checkpoint in yeast . In Xenopus, phosphorylated Claspin binds to xChk1 and regulates xChk1 activation in response to replication stress . In this study, we have shown that the human homolog of Claspin is required for resistance to multiple forms of genotoxic stress including UV, IR, and hydroxyurea . Phosphorylation of Claspin was found to depend on the ataxia telangiectasia mutated-Rad3 related (ATR) pathway . DNA damage induces the formation of a complex between Claspin and BRCA1, a second regulator of Chk1 activation . Claspin was found to control BRCA1 phosphorylation on serine 1524, a site whose phosphorylation is controlled by the ATR pathway . These results are consistent with a model in which ATR regulates Claspin phosphorylation in response to DNA damage and replication stress resulting in recruitment and phosphorylation of BRCA1 . BRCA1 and Claspin then function to activate the tumor suppressor Chk1 . Unexpectedly, we found that Claspin has a second, positive role in control of the cell cycle as Claspin overexpression increased cell proliferation . These results suggest that Claspin has properties of both a tumor suppressor and an oncogene.

J Biol Chem, 2004 Jul 2, 279(27), 28345 - 57 Epub 2004 Apr 19.
The hepatitis E virus open reading frame 3 protein activates ERK through binding and inhibition of the MAPK phosphatase; Kar-Roy A et al.; The hepatitis E virus causes acute viral hepatitis endemic in much of the developing world and is a serious public health problem . However, due to the lack of an in vitro culture system or a small animal model, its biology and pathogenesis are poorly understood . We have shown earlier that the ORF3 protein (pORF3) of hepatitis E virus activates ERK, a member of the MAPK superfamily . Here we have explored the mechanism of pORF3-mediated ERK activation and demonstrated it to be independent of the Raf/MEK pathway . Using biochemical assays, yeast two-hybrid analysis, and intracellular fluorescence resonance energy transfer we showed that pORF3 binds Pyst1, a prototypic member of the ERK-specific MAPK phosphatase . The binding regions in the two proteins were mapped to the N terminus of pORF3 and a central portion of Pyst1 . Expression of pORF3 protected ERK from the inhibitory effects of ectopically expressed Pyst1 . This is the first example of a viral protein regulating ERK activation by inhibition of its cognate dual specificity phosphatase.

Int Immunol, 2004 May, 16(5), 727 - 36 Epub 2004 Apr 13.
SAP increases FynT kinase activity and is required for phosphorylation of SLAM and Ly9; Simarro M et al.; The free Src homology 2 (SH2) domain protein SAP, encoded by the X-linked lymphoproliferative disease gene SH2D1A, controls signal transduction initiated by engagement of the SLAM-related receptors in T and NK cells . Here we demonstrate that SAP is required for phosphorylation of both SLAM and Ly9 in thymocytes and peripheral T cells . Furthermore, in vitro protein interaction studies and yeast two-hybrid analyses indicated that SAP binds directly to FynT and Lck . While SAP bound to both the SH3 domain and to the kinase domain of FynT, SAP bound solely to the kinase domain of Lck . The existence of a strong interaction between SAP and the SH3 domain of FynT prompted us to study the role of SAP in modulating the activity of FynT . In vitro addition of SAP to the autoinhibited form of FynT caused a large increase in FynT catalytic activity . By contrast, the SAP mutant R78E, which is unable to bind to the FynT SH3 domain, did not increase FynT activity and also displayed a reduced adaptor function upon transfection into T cells . Our results demonstrate that SAP is an adaptor that bridges SLAM and Ly9 with Src-like protein tyrosine kinases (PTKs), and has the ability to activate FynT.

J Cell Biochem, 2004 May 1, 92(1), 65 - 76
Induction of G1 cell cycle arrest and P15INK4b expression by ECRG1 through interaction with Miz-1; Zhao N et al.; ECRG1 is a novel candidate of tumor suppressor gene identified from human esophagus . To study the biological role of ECRG1 gene, we performed a GAL4-based yeast two-hybrid screen of a human fetal liver cDNA library . Using the ECRG1 cDNA as bait, we identified two putative clones as associated proteins, Miz-1 and FLNA (Filamin A) . The interaction of ECRG1 and Miz-1 was confirmed by glutathione-S-transferase (GST)-pull-down assays in vitro and co-immunoprecipitation experiments in vivo . ECRG1 was co-localized with Miz-1 in nucleus, as shown by confocal microscopy . Transfection of ECRG1 gene into the esophageal cancer (EC) cells inhibited cell proliferation and induced G1 phase arrest of cell cycle . In the co-transfection of ECRG1 and Miz-1 assays, we found inhibition of cell proliferation and G1/S phase in EC cells, but the levels of cell proliferation inhibition and G1/S phase arrest were more strongly compared with the transfection of ECRG1 or Miz-1 alone . In addition, the interaction of ECRG1 and Miz-1 could induce expression of P15(INK4b) gene in esophageal cancer 9706 (EC9706) cells . However, the transfection of ECRG1 or Miz-1 alone was not revealed the expressions of P15(INK4b) gene . When antisense ECRG1 interdicted expression of endogenous ECRG1 in Balb/c-3T3 cells, Transfection of Miz-1 couldn't induce P15(INK4b) expression . The results provide evidences that ECRG1 and Miz-1 in EC cells may be acting as a co-functional protein associated with regulation of cell cycle and induction of P15(INK4b) expression . It suggests that ECRG1 may inhibit tumor cell growth by affecting cell cycle, and that expression of P15(INK4b) may be likely to enhance G1 cell cycle arrest during the interaction of ECRG1 and Miz-1 . The physical interaction of ECRG1 and Miz-1 may play an important role in carcinogenesis of EC .

J Cell Biochem, 2004 May 1, 92(1), 29 - 41
Identification of FHOD1-binding proteins and mechanisms of FHOD1-regulated actin dynamics; Westendorf JJ et al.; Formin homology-2-domain containing protein 1 (FHOD1) regulates gene transcription, actin-cytoskeleton structure, and cell migration . To gain insight into the mechanisms by which FHOD1 mediates these diverse activities, a yeast-two-hybrid screen was performed to identify FHOD1-binding proteins . Three proteins specifically interacted with the carboxy-terminal two-thirds of FHOD1, which includes the FH1, FH2, and diaphanous activating domains (DAD) . The newly identified FHOD1-binding proteins are protein kinase C binding protein 1 (PRKCBP1), cyclophilin B, and an isoform of WASP-interacting SH3-domain protein/diaphanous-interacting protein 1 (WISH/DIP1), named WISH-B . The proline-rich FH1 domain of FHOD1 was sufficient to interact with the central portion of PRKCP1 and full-length cyclophilin B . The FH1 domain also interacted with full-length WISH-B, but the extreme amino-terminus was sufficient to associate with WISH-B as well . WISH-B altered the solubility of FHOD1 in vitro and a truncation mutant containing the amino-terminal 227 residues of WISH-B disrupted FHOD1-induced stress fibers . WISH-B did not affect FHOD1-induced gene transcription through the serum response factor (SRF) recognition site on the skeletal alpha actin promoter (SkA) . However, stabilization of F-actin prevented FHOD1 dependent activation of this promoter in presence of high, but not low serum concentrations . Thus, the identification of a new FHOD1-binding protein provides insight into the mechanisms by which FHOD1 regulates actin polymerization and transcription .

Biochem Biophys Res Commun, 2004 May 14, 317(4), 1195 - 9
DISC1 localizes to the centrosome by binding to kendrin; Miyoshi K et al.; Disrupted-In-Schizophrenia 1 (DISC1) was identified as a novel gene disrupted by a (1;11)(q42.1;q14.3) translocation that segregated with major mental disorders in a Scottish family . Using the yeast two-hybrid system, we screened a human brain cDNA library for interactors of the DISC1 protein . One of the positive clones encoded kendrin/pericentrin-B, a giant protein known to localize specifically to the centrosome . The interaction between DISC1 and kendrin in mammalian cells was demonstrated by an immunoprecipitation assay . Residues 446-533 of DISC1 were essential for the interaction with kendrin . Immunocytochemical analysis revealed the colocalization of DISC1 and kendrin to the centrosome . These data indicate that DISC1 localizes to the centrosome by binding to kendrin . Kendrin has been reported to anchor the gamma-tubulin complex to the centrosome, providing microtubule nucleation sites . The present study suggests the possible involvement of DISC1 in the pathophysiology of mental disorders due to its putative effect on centrosomal function.

Gen Comp Endocrinol, 2004 May 15, 137(1), 89 - 98
Effects of high doses of cortisol on innate cellular immune response of seabream (Sparus aurata L.); Esteban MA et al.; The effects of high doses of cortisol, biologically the most active corticosteroid in the circulating blood of teleost fish, on gilthead seabream (Sparus aurata L.) leucocytes were determined . Leucocytes isolated from the head-kidney (the principal haemopoietic organ of this fish species) were incubated with five different concentrations (ranging from 0 to 10(-1) mM) of cortisol for 30, 120, 240, 360, and 480 min and their effects on leucocyte viability and some of the main innate cellular immune responses were evaluated . The viability and the cytotoxic activity of leucocytes against tumor cells were not significantly affected by in vitro incubation with cortisol, at any of the assayed concentration or incubation times . With cortisol concentrations of 10(-9), 10(-7) or 10(-5) mM, the respiratory burst activity of head-kidney leucocytes were significantly depressed after 30 min of incubation . On the other hand, with cortisol concentrations of 10(-1) mM, the phagocytosis of yeast cells and the total peroxidase content of leucocytes were significantly depressed at incubation times longer than 240 min . To corroborate that the observed effects were due to the cortisol treatment, assays were developed using both cortisol and cycloheximide, which blocked the inhibitory effects of cortisol . The present results demonstrate that cortisol plays an important role in the down-regulation of phagocytic but not of cytotoxic activity in seabream leucocytes.

Gene, 2004 Apr 28, 331, 107 - 14
Heterogeneous nuclear ribonucleoprotein E2 binds to tau exon 10 and moderately activates its splicing; Broderick J et al.; Tau is a microtubule-associated protein whose transcript undergoes complex-regulated splicing in the mammalian nervous system . Exon 10 of the gene is an alternatively spliced cassette, which is adult-specific and codes for a microtubule-binding domain . Mutations that affect splicing of exon 10 have been shown to cause frontotemporal dementia with parkinsonism (FTDP) . Using tau exon 10 as a bait in a yeast three-hybrid screen, we discovered that it interacts with hnRNPE2 . Cotransfection assays show that hnRNPE2 isoforms moderately activate the splicing of exon 10.

FEBS Lett, 2004 Apr 23, 564(1-2), 205 - 11
P66(ShcA) interacts with MAPKAP kinase 2 and regulates its activity; Yannoni YM et al.; Three mitogen activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2, MK2) interacting proteins were identified using a yeast two-hybrid approach . ShcA, a signaling phospho-protein, human polyhomeotic 2 (HPH2), a transcriptional regulator, and highly similar to smoothelin (HSTS), which is related to the cytoskeletal associated protein smoothelin, interact specifically with MK2 . The interaction of MK2 with the 66 kDa isoform of ShcA, p66(ShcA), and HPH2 was confirmed using co-immunoprecipitation . MK2 is activated with p66(ShcA) co-expression and p66(ShcA) is an in vitro substrate for MK2, further demonstrating their association and suggesting a biological role for p66(Shc) in MK2 activation.

FEBS Lett, 2004 Apr 23, 564(1-2), 131 - 5
Interaction of neuron-specific K+-Cl- cotransporter, KCC2, with brain-type creatine kinase; Inoue K et al.; gamma-Aminobutyric acid, a major inhibitory neurotransmitter within the adult central nervous system, is also known to be excitatory at early developmental stages due to the elevated intracellular Cl(-) concentration . This functional change is primarily attributable to a K(+)-Cl(-) cotransporter, KCC2, the expression of which is developmentally regulated in neurons . However, little detail information is available concerning the intracellular regulation of KCC2 function . Here, we identify an interaction between KCC2 and brain-type creatine kinase by means of yeast two-hybrid screening . This interaction, which was also detected in cultured cells and brain extracts, might contribute to KCC2-mediated modulation of Cl(-) homeostasis.

Curr Opin Struct Biol, 2004 Apr, 14(2), 147 - 53
A structural view of the COPII vesicle coat; Bickford LC et al.; The COPII vesicle coat coordinates the budding of transport vesicles from the endoplasmic reticulum in the initial step of the secretory pathway . The coat orchestrates a sequence of events including self-assembly on the membrane, cargo and SNARE molecule selection, and deformation of the membrane into a bud to drive vesicle fission . Recent molecular-level studies have helped to explain how the three components of yeast COPII - Sar1 GTPase, the Sec23/24 subcomplex and the Sec13/31 subcomplex - combine to organize this complex process.

Biosens Bioelectron, 2004 Jun 15, 19(11), 1439 - 44
Dynamic formation of optically trapped microstructure arrays for biosensor applications; Daria VR et al.; We explore the dynamic properties of multiple-beam optical traps to manipulate arrays of microstructures for biosensor applications . Multiple optical traps are generated by a virtually loss-less transformation of input phase patterns into high-intensity trapping-beams . A direct image projection of the phase patterns enables an adjustable number of optical traps in addition to instantaneous control of the position, size, shape and intensity of each trapping-beam . We present experimental results showing various colloidal formations through dynamic optical manipulation of polystyrene microspheres and yeast cells in aqueous media . The experimental configurations are geared towards the use of multiple-beam optical traps for biosensor applications.

BMC Neurosci . 2004 Apr 16;5(1):14.
Parkin counteracts symptoms in a Drosophila model of Parkinson's disease; Haywood AF et al.; BACKGROUND: Parkinson's disease, a prevalent neurodegenerative disease, is characterized by the reduction of dopaminergic neurons resulting in the loss of motor control, resting tremor, the formation of neuronal inclusions and ultimately premature death . Two inherited forms of PD have been linked to mutations in the alpha-synuclein and parkin genes . The parkin protein functions as an ubiquitin ligase targeting specific proteins for degradation . Expression of human alpha-synuclein in Drosophila neurons recapitulates the loss of motor control, the development of neuronal inclusions, degeneration of dopaminergic neurons and the ommatidial array to provide an excellent genetic model of PD . RESULTS: To investigate the role of parkin, we have generated transgenic Drosophila that conditionally express parkin under the control of the yeast UAS enhancer . While expression of parkin has little consequence, co-expression of parkin with alpha-synuclein in the dopaminergic neurons suppresses the alpha-synuclein-induced premature loss of climbing ability . In addition directed expression of parkin in the eye counteracts the alpha-synuclein-induced degeneration of the ommatidial array . These results show that parkin suppresses the PD-like symptoms observed in the alpha-synuclein-dependent Drosophila model of PD . CONCLUSION: The highly conserved parkin E3 ubiquitin ligase can suppress the damaging effects of human alpha-synuclein . These results are consistent with a role for parkin in targeting alpha-synuclein to the proteasome . If this relationship is conserved in humans, this suggests that up-regulation of parkin should suppress alpha-synucleinopathic PD . The development of therapies that regulate parkin activity may be crucial in the treatment of PD.

EMBO Rep, 2004 May, 5(5), 515 - 20 Epub 2004 Apr 16.
gurke and pasticcino3 mutants affected in embryo development are impaired in acetyl-CoA carboxylase; Baud S et al.; Normal embryo development is required for correct seedling formation . The Arabidopsis gurke and pasticcino3 mutants were isolated from different developmental screens and the corresponding embryos exhibit severe defects in their apical region, affecting bilateral symmetry . We have recently identified lethal acc1 mutants affected in acetyl-CoA carboxylase 1 (ACCase 1) that display a similar embryo phenotype . A series of crosses showed that gk and pas3 are allelic to acc1 mutants, and direct sequencing of the ACC1 gene revealed point mutations in these new alleles . The isolation of leaky acc1 alleles demonstrated that ACCase 1 is essential for correct plant development and that mutations in ACCase affect cellular division in plants, as is the case in yeast . Interestingly, significant metabolic complementation of the mutant phenotype was obtained by exogenous supply of malonate, suggesting that the lack of cytosolic malonyl-CoA is likely to be the initial factor leading to abnormal development in the acc1 mutants.

J Biol Chem, 2004 Jun 25, 279(26), 26948 - 58 Epub 2004 Apr 15.
The zinc finger transcription factor transforming growth factor beta-inducible early gene-1 confers myeloid-specific activation of the leukocyte integrin CD11d promoter; Noti JD et al.; CD11d encodes the alpha(D) subunit for a leukocyte integrin that is expressed on myeloid cells . In this study we show that the -100 to -20 region of the CD11d promoter confers myeloid-specific activation of the CD11d promoter . Transforming growth factor beta-inducible early gene-1 (TIEG1) was isolated in a yeast one-hybrid screen using the -100 to -20 region of the CD11d promoter as bait . Purified GST.TIEG1 protein was able to bind within the -61 to -45 region that overlaps a shorter binding site for Sp1 . Transient overexpression of TIEG1 activated the CD11d promoter specifically in myeloid cells, whereas, down-regulation of TIEG1 with small interfering TIEG1 RNA also down-regulated expression of CD11d . In vivo, TIEG1 does not physically interact with Sp1 . Cotransfection and electrophoretic mobility shift analyses of TIEG1, Sp1, and Sp3 revealed that TIEG1 competes with these Sp proteins for binding to overlapping sites in the CD11d promoter . Although TIEG1 and Sp1 are ubiquitously expressed in myeloid and non-myeloid cells, chromatin immunoprecipitation assays revealed differential occupancy of the CD11d promoter by these factors . In undifferentiated myeloid and non-myeloid cells, occupancy of the CD11d promoter by TIEG1 is similar . Upon differentiation of myeloid cells and subsequent up-regulation of CD11d expression, TIEG1 occupancy increases . In contrast, occupancy by TIEG1 remains low in non-myeloid cells exposed to phorbol ester . We propose that up-regulation of CD11d expression following differentiation of myeloid cells is mediated through increased binding of TIEG1 binding to the CD11d promoter.

J Biol Chem, 2004 Jun 18, 279(25), 26425 - 32 Epub 2004 Apr 15.
Identification and characterization of a novel BASH N terminus-associated protein, BNAS2; Imamura Y et al.; A B cell-specific adaptor protein, BASH (also known as BLNK or SLP-65), is crucial for B cell receptor (BCR) signaling . BASH binds to various signaling intermediates, such as Btk, PLCgamma2, Vav, and Grb2, through its well defined motifs . Although functional significance of such interactions has been documented, BASH-mediated signal transduction mechanism is not fully understood . Using the yeast two-hybrid system, we have identified a novel protein that binds to a conserved N-terminal domain of BASH, which we named BNAS2 (BASH N terminus associated protein 2) . From its deduced amino acid sequence, BNAS2 is presumed to contain four transmembrane domains, which are included in a central MARVEL domain, and to localize to endoplasmic reticulum . BNAS2 was co-precipitated with BASH as well as Btk and ERK2 from a lysate of mouse B cell line . In the transfected cells, the exogenous BNAS2 was localized in a mesh-like structure in the cytoplasm resembling that of endoplasmic reticulum (ER) and nuclear membrane . BASH was co-localized with BNAS2 in a manner dependent on its N-terminal domain . RT-PCR analysis indicated that BNAS2 mRNA is expressed ubiquitously except for plasma cells . In chicken B cell line DT40, overexpression of BNAS2 resulted in an enhancement of BCR ligation-mediated transcriptional activation of Elk1, but not of NF-kappaB, in a manner dependent on the dose of BNAS2 . Thus BNAS2 may serve as a scaffold for signaling proteins such as BASH, Btk, and ERK at the ER and nuclear membrane and may facilitate ERK activation by signaling from cell-surface receptors.

Gene, 2004 Apr 14, 330, 9 - 18
Identification of the TBX5 transactivating domain and the nuclear localization signal; Zaragoza MV et al.; TBX5 is a member of the T-box gene family and encodes a transcription factor involved in cardiac and limb development . Mutations of TBX5 cause Holt-Oram syndrome (HOS), an autosomal-dominant condition with congenital cardiac defects and forelimb anomalies . Here, we used a GAL4-TBX5 fusion protein in a modified yeast-one hybrid system to elucidate the TBX5 transactivating domain . Using a series of deletion mutations of TBX5, we narrowed down its functional domain to amino acids 339-379 of its C-terminal half; point mutagenesis analysis then showed that the loss of amino acids 349-351 abolished transactivation . This result was confirmed in mammalian cells . Furthermore, wild-type TBX5, but not TBX5 with mutations at the amino acids 349-351, has ability to inhibit NCI-H1299 cell growth also suggesting that these amino acids are crucial for the TBX5 function in mammalian cells . In addition, to identify the nuclear localization signal of TBX5, we searched for cluster of basic amino acids . We found that the deletion of the KRK sequence at amino acids 325-327 mislocalizes TBX5 to cytoplasm, suggesting that these amino acids serve as a nuclear localization signal . These studies enhance our understanding of the structure-function relationship of TBX5 and suggest that truncation mutations of TBX5 could cause HOS through the loss of its transactivating domain and/or the nuclear localization signal.

Plant J, 2004 May, 38(3), 526 - 38
DISTORTED2 encodes an ARPC2 subunit of the putative Arabidopsis ARP2/3 complex; El-Din El-Assal S et al.; Arabidopsis trichomes are unicellular, branched structures that have highly constrained requirements for the cytoskeleton . The 'distorted group' genes function downstream from microtubule-based branch initiation, and are required during the actin-dependent phase of polarized stalk and branch expansion . Of the eight known 'distorted group' genes, a subset encode homologs of ARP2/3 complex subunits . In eukaryotic cells, the seven-protein ARP2/3 complex nucleates actin filament networks that push on the plasma membrane and organelles . In plants cells, the existence and function of an ARP2/3 complex is unclear . In this paper, we report that DISTORTED2 (DIS2) encodes a paralogue of the ARP2/3 complex subunit ARPC2 . DIS2 has ARPC2 activity, based on its ability to rescue the growth defects of arpc2 (arc35Delta) null yeast cells . Like known ARPC2s, DIS2 physically interacts with ARPC4 . Mutations in DIS2 cause a distorted trichome phenotype, defects in cell-cell adhesion, and a modest reduction in shoot FW . The actin cytoskeleton in dis2 trichomes is extensive, but developing branches fail to generate and maintain highly organized cytoplasmic actin bundles.

Mol Biol Evol, 2004 Jul, 21(7), 1459 - 61 Epub 2004 Apr 14.
Using consensus networks to visualize contradictory evidence for species phylogeny; Holland BR et al.; Building species phylogenies from genome data requires the evaluation of phylogenetic evidence from independent gene loci . We propose an approach to do this using consensus networks . We compare gene trees for eight yeast genomes and show that consensus networks have potential for helping to visualize contradictory evidence for species phylogenies.

Mol Biol Evol, 2004 Jul, 21(7), 1455 - 8 Epub 2004 Apr 14.
Genome-scale phylogeny and the detection of systematic biases; Phillips MJ et al.; Phylogenetic inference from sequences can be misled by both sampling (stochastic) error and systematic error (nonhistorical signals where reality differs from our simplified models) . A recent study of eight yeast species using 106 concatenated genes from complete genomes showed that even small internal edges of a tree received 100% bootstrap support . This effective negation of stochastic error from large data sets is important, but longer sequences exacerbate the potential for biases (systematic error) to be positively misleading . Indeed, when we analyzed the same data set using minimum evolution optimality criteria, an alternative tree received 100% bootstrap support . We identified a compositional bias as responsible for this inconsistency and showed that it is reduced effectively by coding the nucleotides as purines and pyrimidines (RY-coding), reinforcing the original tree . Thus, a comprehensive exploration of potential systematic biases is still required, even though genome-scale data sets greatly reduce sampling error.

J Biol Chem, 2004 Jun 18, 279(25), 26227 - 32 Epub 2004 Apr 14.
The role of p22 NF-E4 in human globin gene switching; Zhou W et al.; The human stage selector protein, a complex containing the ubiquitous transcription factor CP2 and the erythroid-specific factor p22 NF-E4, facilitates the interaction of the gamma-globin genes with the locus control region in fetal erythroid cells . Enforced expression of p22 NF-E4 in K562 cells and human cord blood progenitors increases fetal globin gene expression, and in progenitors, reduces beta-globin expression . To examine the role of NF-E4 in an in vivo model of hemoglobin switching, we enforced the expression of p22 NF-E4 in transgenic mice carrying the human beta-globin locus yeast artificial chromosome . Although murine erythropoiesis and globin gene expression is unaffected in these mice, the expression profile of the human globin genes is altered . All three transgenic lines displayed an increased gamma:beta-globin ratio in E12.5-14.5 fetal liver, resulting in a delay in the fetal/adult switch . At E12.5, this is primarily due to a reduction of beta-gene expression, whereas at E14.5, the increased gamma:beta ratio is due to enhanced gamma-gene expression . Despite this, the switch in globin subtype is fully completed in the adult bone marrow . These findings indicate that p22 NF-E4 is capable of influencing human globin gene expression in vivo but is incapable of overriding the intrinsic mechanisms governing gamma-gene silencing in this context.

Virus Res, 2004 Jun 15, 102(2), 151 - 63
The non-structural 3 (NS3) protein of dengue virus type 2 interacts with human nuclear receptor binding protein and is associated with alterations in membrane structure; Chua JJ et al.; Flaviviral infections produce a distinct array of virus-induced intracellular membrane alterations that are associated with the flaviviral replication machinery . Currently, it is still unknown which flaviviral protein(s) is/are responsible for this induction . Using yeast two-hybrid and co-immunoprecipitation analyses, we demonstrated that the NS3 protein of dengue virus type 2 interacted specifically with nuclear receptor binding protein (NRBP), a host cellular protein that influences trafficking between the endoplasmic reticulum (ER) and Golgi, and that interacts with Rac3, a member of the Rho-GTPase family . Co-expression of NS3 and NRBP in baby hamster kidney cells exhibited significant subcellular co-localization, and revealed the redistribution of NRBP from the cytoplasm to the perinuclear region . Furthermore, a set of membrane structures affiliated with the rough ER at the perinuclear region was induced in cells transfected with NS3 . These structures are reminiscent of the virus-induced convoluted membranes previously observed in flavivirus-infected cells . This interaction between dengue viral and host cell proteins as well as the formation of the NS3-induced membrane structures suggest that NS3 may subvert the role of NRBP in ER-Golgi trafficking.

Mol Cell Biol, 2004 May, 24(9), 3782 - 93
Normal development and fertility of knockout mice lacking the tumor suppressor gene LRP1b suggest functional compensation by LRP1; Marschang P et al.; LRP1b and the closely related LRP1 are large members of the low-density lipoprotein receptor family . At the protein level LRP1b is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cargo transport and cellular signaling . Somatic LRP1b mutations frequently occur in non-small cell lung cancer and urothelial cancers, suggesting a role in the modulation of cellular growth . In contrast to LRP1, LRP1b-deficient mice develop normally, most likely due to its restricted expression pattern and functional compensation by LRP1 or other receptors . LRP1b is expressed predominantly in the brain, and a differentially spliced form is present in the adrenal gland and in the testis . Despite the presence of a potential furin cleavage site and in contrast to LRP1, immunoblotting for LRP1b reveals the presence of a single 600-kDa polypeptide species . Using a yeast two-hybrid approach, we have identified two intracellular proteins, the postsynaptic density protein 95 and the aryl hydrocarbon receptor-interacting protein, that bind to the intracellular domain of LRP1b . In addition, we have found several potential ligands that bind to the extracellular domain . Analysis of LRP1b knockout mice may provide further insights into the role of LRP1b as a tumor suppressor and into the mechanisms of cancer development.

Mol Cell Biol, 2004 May, 24(9), 3734 - 46
Suppressor of sable, a putative RNA-processing protein, functions at the level of transcription; Kuan YS et al.; The Drosophila melanogaster su(s) gene product negatively regulates the expression of mutant alleles with transposon insertions in the 5'-transcribed region by an unknown mechanism . We have investigated here su(s) function through in vivo structure-function analysis, heterologous reporter gene assays, and in vivo transcriptional induction experiments . We have shown that mutations of two arginine-rich motifs (ARMs), an acidic region, or two CCCH zinc fingers affect the ability of Su(s) to downregulate the expression of an insertion mutant allele and to autoregulate genomic su(s) transgenes . Using yeast and HeLa cell assays, we found that, when tethered to the promoter region, the N- and C-terminal regions of Su(s) can repress reporter gene expression, and all three motifs, but most significantly the ARMs, contribute to the repression activity . Finally, we showed that, in vivo, Su(s) inhibits the transcriptional induction of a transgene with an insertion in the first exon but does not affect induction of a similar transgene with a consensus 5' splice site near the upstream boundary of the insertion . Together, these results reveal a link between Su(s), transcription, and pre-mRNA processing.

J Biol Chem, 2004 Jun 18, 279(25), 26469 - 74 Epub 2004 Apr 13.
Molecular cloning and characterization of a human multisubstrate specific nucleotide-sugar transporter homologous to Drosophila fringe connection; Suda T et al.; Nucleotide-sugar transporters are crucial components in the synthesis of glycoconjugates . We identified a novel human nucleotide-sugar transporter gene, hfrc1, which is homologous to Drosophila melanogaster fringe connection, Caenorhabditis elegans sqv-7, and human UGTrel7 . HFRC1 was localized within the Golgi apparatus following its transient expression in HCT116 cells . In human tissues, hfrc1 and UGTrel7 exhibited similar tissue distributions, although hfrc1 transcripts showed a 10 times greater abundance than those of UGTrel7 . The heterologous expression of HFRC1 in the yeast revealed the multisubstrate specific transport activity of HFRC1 (for UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-glucose (UDP-Glc), and GDP-mannose (GDP-Man), with apparent K(m) values of 8.0, 2.1, and 0.14 microm, respectively) . In the mammalian cells, HFRC1 transported UDP-GlcNAc and UDP-Glc, but not GDP-Man . Overexpression of the hfrc1 gene in HCT116 cells modulated the cell surface heparan sulfate expression status . These results suggest that HFRC1 takes part in the synthesis of heparan sulfate by regulating the level of UDP-GlcNAc, a donor substrate for the heparan sulfate synthases.

J Biol Chem, 2004 Jun 11, 279(24), 25805 - 12 Epub 2004 Apr 12.
Sun2 is a novel mammalian inner nuclear membrane protein; Hodzic DM et al.; Sun protein (Sun1 and Sun2) cDNAs were previously cloned based on the homology of their C-terminal regions (SUN (Sad1 and UNC) domain) with the Caenorhabditis elegans protein UNC-84 whose mutation disrupts nuclear migration/positioning . In this study, we raised an anti-Sun2 serum and identified Sun2 in mammalian cells . In HeLa cells, Sun2 displays a nuclear rim-like pattern typical for a nuclear envelope protein . The Sun2 antibody signal co-localizes with nuclear pore and INM markers signals . The rim-like pattern was also observed with the recombinant full-length Sun2 protein fused to either EGFP or V5 epitopes . In addition, we found that a recombinant truncated form of Sun2, extending from amino acids 26 to 339, is sufficient to specify the nuclear envelope localization . Biochemical analyses show that Sun2 is an 85-kDa protein that is partially insoluble in detergent with high salt concentration and in chaotropic agents . Furthermore, Sun2 is enriched in purified HeLa cell nuclei . Electron microscopy analysis shows that Sun2 localizes in the nuclear envelope with a sub-population present in small clusters . Additionally, we show that the SUN domain of Sun2 is localized to the periplasmic space between the inner and the outer nuclear membranes . From our data, we conclude that Sun2 is a new mammalian inner nuclear membrane protein . Because the SUN domain is conserved from fission yeast to mammals, we suggest that Sun2 belongs to a new class of nuclear envelope proteins with potential relevance to nuclear membrane function in the context of the involvement of its components in an increasing spectrum of human diseases.

Alcohol Alcohol, 2004 May-Jun, 39(3), 178 - 82
New simple method for purification of class I alcohol dehydrogenase; Negoro M et al.; AIMS: The purpose of this study was to develop a new simple method for purification of rat class I alcohol dehydrogenase (ADH, EC 1.1.1.1) . METHODS AND RESULTS: Immobilized p-hydroxyacetophenone was used as a ligand for affinity chromatography for the initial purification step after ammonium sulfate precipitation of the cytosolic fraction of rat liver . Then the eluant was separated by using ion-exchange chromatography, and homogenous class I ADH, as judged by the results of SDS-PAGE and confirmed by the results of the amino-acid sequence of peptides degraded from a 39 kDa protein, was obtained with a high yield (57%) . The purified ADH showed kinetic constants of 1.3 mmol/l for Km and 62.4 per min for Kcat with ethanol as a substrate . ADH was also successfully purified from yeast by a similar method using p-hydroxyacetophenone affinity chromatography . CONCLUSIONS: This simple method involving only two chromatographic procedures may be very useful for purification of ADH.

Anal Biochem, 2004 May 1, 328(1), 29 - 34
A rapid filtration apparatus for harvesting cells under controlled conditions for use in genome-wide temporal profiling studies; Burke PV et al.; Gene expression can respond rapidly to changes in environmental conditions . To effectively monitor these responses, we built a filtration apparatus that allows for the rapid harvesting and processing of moderate volumes of yeast cells under controlled atmospheric conditions (e.g., anaerobic conditions) . Harvesting by filtration offers several advantages over that by centrifugation, especially when rapid, repeated sampling of dilute cultures is required . A number of different filter membranes, including cellulose acetate, mixed esters of cellulose, regenerated cellulose, polycarbonate, and polyvinylidene fluoride, were assayed for harvest efficiency and the quality of RNA obtained by hot-phenol extraction from cells directly adhering to the membranes . To determine the suitability of the RNA for microarray analyses, we quantified both cDNA yield from reverse transcription and the indirect coupling of Cyan dyes . In general, filtration times, cell yields, and RNA quality were similar among the filters examined, although some media components (e.g., antifoam) can cause fouling of smaller-pore-sized filters . Thus, choice of a membrane will depend on the particular medium, ease of filter handling, or on other experimental considerations . We routinely use this filtration apparatus with Osmonics 1.2 microm cellulose acetate filters for isolating RNA for genome-wide temporal profiling analyses.

Immunol Lett, 2004 Apr 15, 92(3), 221 - 6
Specific recruitment of SPA-1 to the immunological synapse: involvement of actin-bundling protein actinin; Harazaki M et al.; SPA-1 is involved in the regulation of T cell activation in response to antigens through the control of Rap1 GTPase signaling . In this study, the subcellular localization of SPA-1 in the T cells was examined by using anti-SPA-1 antibody and GFP-SPA-1 . While SPA-1 was detected diffusely at the surface cortical region in the floating unpolarized T cells, it was concentrated at the matrix-adhesion region with dense actin-cytoskeleton . Upon interaction with specific antigen-presenting cells, SPA-1 was highly concentrated at the immunological synapse closely co-localizing with actin . By yeast two-hybrid system, SPA-1 was shown to interact with an actin-bundling protein alpha-actinin, and it was indicated that SPA-1 co-localized with alpha-actinin at the immunological synapse . The results have suggested that SPA-1 in the T cells is selectively recruited to the immunological synapse with dense actin-cytoskeletal reorganization and keeps restraining the levels of Rap1GTP at the local TCR-signaling complex for the T cell activation.

Biochem Biophys Res Commun, 2004 May 7, 317(3), 930 - 8
Homology modeling of the structure of tobacco acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis; Le DT et al.; A reliable model of tobacco acetohydroxy acid synthase (AHAS) was obtained by homology modeling based on a yeast AHAS X-ray structure using the Swiss-Model server . Conserved residues at the dimer interface were identified, of which the functional roles of four residues, namely H142, E143, M489, and M542, were determined by site-directed mutagenesis . Eight mutants were successfully generated and purified, five of which (H142T, M489V, M542C, M542I, and M542V) were found to be inactive under various assay conditions . The H142K mutant was moderately altered in all kinetic parameters to a similar extent . In addition, the mutant was more thermo-labile than wild type enzyme . The E143A mutant increased the Km value more than 20-fold while other parameters were not significantly changed . All mutations carried out on residue M542 inactivated the enzyme . Though showing a single band on SDS-PAGE, the M542C mutant lost its native tertiary structure and was aggregated . Except M542C, each of the other mutants showed a secondary structure similar to that of wild type enzyme . Although all the inactive mutants were able to bind FAD, the mutants M489V and M542C showed a very low affinity for FAD . None of the active mutants constructed was strongly resistant to three tested herbicides . Taken together, the results suggest that the residues of H142, E143, M489, and M542 are essential for catalytic activity . Furthermore, it seems that H142 residue is involved in stabilizing the dimer interaction, while E143 residue may be involved in binding with substrate pyruvate . The data from the site-directed mutagenesis imply that the constructed homology model of tobacco AHAS is realistic.

Bioorg Med Chem Lett, 2004 May 3, 14(9), 2137 - 40
Didehydrofarnesyl diphosphate: an intrinsically fluorescent inhibitor of protein farnesyltransferase; Liu XH et al.; Didehydrofarnesyl diphosphate (delta delta FPP), a fluorescent pentaene analogue of farnesyl diphosphate (FPP), was synthesized using stereoselective Wittig reactions . Although delta delta FPP was not an alternative substrate for yeast protein farnesyltransferase (FTase), the fluorescent analogue was a potent competitive inhibitor with a K(i) value of 8.8 microM (K (m) (FPP) = 27 microM).

Anal Chem, 2004 Apr 15, 76(8), 2196 - 202
Integrated and ultrasensitive gel protein identification; Cooper JW et al.; An integrated gel protein identification technology is developed and demonstrated for the effective ( approximately 90% recovery), rapid (less than 5 min), and sensitive identification (as low as 1 ng gel protein loading) of gel-resolved proteins using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) . This integrated technology involves on-line combination of electronic protein transfer with nanoscale proteolytic digestion in a capillary platform, enabling electrokinetic-based protein extraction and stacking, real-time proteolytic cleavage of extracted proteins, and direct deposition of protein digests onto MALDI targets . By revisiting the yeast two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) in similar isoelectric point and molecular mass ranges as studied by Gygi and co-workers (Gygi, S . P.; Corthals, G . L.; Zhang, Y.; Rochon, Y.; Aebersold, R . Proc . Natl . Acad . Sci . U.S.A . 2000, 97, 9390-9395), we are additionally able to identify a large number of low abundance proteins with codon adaptation index (CAI) values of <0.2 and increase the proteome coverage to nearly 50% . The CAI value distribution for identified yeast proteins now more closely approximates that predicted for the entire yeast proteome . We further note that the current single-capillary methodology can be easily expanded to a multiplexed capillary platform as a ultrahigh throughput and greatly effective tool for linking 2-D PAGE with MS, particularly for the analysis of low-abundance proteins.

Clin Chem Lab Med, 2004 Mar, 42(3), 279 - 82
Acid ribonuclease and alkaline ribonuclease isoenzymes in plasma of patients with decreased glomerular filtration rate; Naskalski J et al.; Removal of low molecular weight proteins from plasma by kidneys depends on glomerular filtration rate (GFR), protein-glomerular membrane electric charge, steric interactions and a number of functionally active nephrons present in the kidneys . There is a well documented relationship between the concentration of low molecular weight proteins in plasma and GFR value in patients with impaired renal function . Accumulation of low molecular weight proteins in plasma along with a decrease in GFR value may in the long run enhance formation of protein tissue deposits known as various forms of amyloidosis . In this paper we present studies on plasma concentrations of acid leukocyte-type ribonuclease (RNase) and alkaline pancreatic-type RNase and GFR value in 54 patients with renal failure . RNase isoenzymes' activities were assayed by measuring their enzyme activities manifested as ability to decompose yeast RNA and assay of digestion products' concentration by spectrophotometry . The studies show that decreasing filtration rate produces an increase in serum activities of both acid and alkaline RNases, which is proportional to the logarithm of GFR value . However, the increase rate vs . GFR value is by four times higher for acid RNase than for alkaline RNase . Acid RNase in human plasma is mostly of leukocytic origin and differs from pancreatic-type alkaline RNase, which is of pancreatic origin . The obtained results may suggest that leukocyte originating proteins essentially contribute to low molecular weight protein accumulation in plasma of patients with chronic renal insufficiency.

Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 5910 - 5 Epub 2004 Apr 12.
Molecular basis for the inhibition of the carboxyltransferase domain of acetyl-coenzyme-A carboxylase by haloxyfop and diclofop; Zhang H et al.; Acetyl-CoA carboxylases (ACCs) are crucial for the metabolism of fatty acids, making these enzymes important targets for the development of therapeutics against obesity, diabetes, and other diseases . The carboxyltransferase (CT) domain of ACC is the site of action of commercial herbicides, such as haloxyfop, diclofop, and sethoxydim . We have determined the crystal structures at up to 2.5-A resolution of the CT domain of yeast ACC in complex with the herbicide haloxyfop or diclofop . The inhibitors are bound in the active site, at the interface of the dimer of the CT domain . Unexpectedly, inhibitor binding requires large conformational changes for several residues in this interface, which create a highly conserved hydrophobic pocket that extends deeply into the core of the dimer . Two residues that affect herbicide sensitivity are located in this binding site, and mutation of these residues disrupts the structure of the domain . Other residues in the binding site are strictly conserved among the CT domains.

J Virol, 2004 May, 78(9), 4817 - 26
A novel motif in geminivirus replication proteins interacts with the plant retinoblastoma-related protein; Arguello-Astorga G et al.; The geminivirus replication factor AL1 interacts with the plant retinoblastoma-related protein (pRBR) to modulate host gene expression . The AL1 protein of tomato golden mosaic virus (TGMV) binds to pRBR through an 80-amino-acid region that contains two highly predicted alpha-helices designated 3 and 4 . Earlier studies suggested that the helix 4 motif, whose amino acid sequence is strongly conserved across geminivirus replication proteins, plays a role in pRBR binding . We generated a series of alanine substitutions across helix 4 of TGMV AL1 and examined their impact on pRBR binding using yeast two-hybrid assays . These experiments showed that several helix 4 residues are essential for efficient pRBR binding, with a critical residue being a leucine at position 148 in the middle of the motif . Various amino acid substitutions at leucine-148 indicated that both structural and side chain components contribute to pRBR binding . The replication proteins of the geminiviruses tomato yellow leaf curl virus and cabbage leaf curl virus (CaLCuV) also bound to pRBR in yeast dihybrid assays . Mutation of the leucine residue in helix 4 of CaLCuV AL1 reduced binding . Together, these results suggest that helix 4 and the conserved leucine residue are part of a pRBR-binding interface in begomovirus replication proteins.

J Cell Biol, 2004 Apr, 165(1), 111 - 22
Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer; Seaman MN; fEndosome-to-Golgi retrieval of the mannose 6-phosphate receptor (MPR) is required for lysosome biogenesis . Currently, this pathway is poorly understood . Analyses in yeast identified a complex of proteins called "retromer" that is essential for endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p . Retromer comprises five distinct proteins: Vps35p, 29p, 26p, 17p, and 5p, which are conserved in mammals . Here, we show that retromer is required for the efficient retrieval of the cation-independent MPR (CI-MPR) . Cells lacking mammalian VPS26 fail to retrieve the CI-MPR, resulting in either rapid degradation of or mislocalization to the plasma membrane . We have localized mVPS26 to multivesicular body endosomes by electron microscopy, and through the use of CD8 reporter protein constructs have examined the effect of loss of mVPS26 upon the trafficking of membrane proteins that cycle between the endosome and the Golgi . The data presented here support the hypothesis that retromer performs a selective function in endosome-to-Golgi transport, mediating retrieval of the CI-MPR, but not furin.

Basic Clin Pharmacol Toxicol, 2004 Apr, 94(4), 177 - 83
Expression of CYP4F12 in gastrointestinal and urogenital epithelia; Stark K et al.; Cytochrome P450 4F12 (CYP4F12) was originally cloned from human liver and small intestine . CYP4F12 can oxidize arachidonic acid, two stable prostaglandin H2 analogues, and an antihistamine, ebastine, but the tissue distribution and catalytic properties of CYP4F12 have not been fully investigated . An antipeptide polyclonal antibody was raised against the C-terminal of CYP4F12 (PLNVGLQ), evaluated by Western blot analysis and used for immunohistological analysis of 50 human tissues . Western blot analysis of recombinant CYP4F12, expressed in yeast, and microsomal proteins from adult and foetal liver, kidney, placenta at term, seminal vesicles, the prostate gland and purified prostasomes showed that the polyclonal antibody detected a protein of the expected size, approximately 60 kDa . CYP4F12 mRNA could be detected in seminal vesicles and prostate gland by reverse transcription-PCR . Prominent CYP4F12 immunoreactivity occurred, inter alia, in the epithelial cells of the gastrointestinal tract (stomach, small intestine, and colon), collecting tubules, transitional epithelium, ovarian follicles, the endothelium of microvessels of placental villi (first trimester), and epidermis . We screened recombinant CYP4F12 for catalytic activity . Arachidonic acid (20:4n-6) was hydroxylated at C18 and laurate at C11, but significant amounts of metabolites of 18:2n-6, 20:3n-9, 20:5n-3, 22:5n-6, and some prostaglandins could not be detected . We conclude that CYP4F12 is widely distributed in gastrointestinal and urogenital epithelia and exhibits a narrow substrate specificity.

Plant J, 2004 Apr, 38(2), 348 - 57
Development of an efficient method for the isolation of factors involved in gene transcription during rice embryo development; Ye R et al.; Summary An efficient yeast-based system was developed for the isolation of plant cDNAs encoding transcription factors (TFs) and proteins with transcription activation functions (co-activators) . The system consists of two vectors: (i) a reporter vector (pG221) harboring the iso-1-cytochrome c (CYC1) core promoter and the beta-galactosidase (lacZ) gene; and (ii) a cDNA library construction vector (pYF503), which yields a library of plant peptides fused to the GAL4-binding domain (GAL4-BD) . Expression of a peptide harboring the characteristics of a transcriptional activator leads to expression of lacZ, allowing for selection of relevant colonies . TFs during rice embryo development were isolated through this system . Approximately 200 confirmed positive colonies were obtained from screening 10(6) yeast colonies, and sequence analysis of conserved domains identified 75 independent cDNAs, 20 of which encoded plant TFs or co-activators, including members of the APETALA2 (AP2)/ethylene-responsive element-binding protein (EREBP), MYB and growth-regulating factor (GRF) families . Peptides encoded by 13 of the isolated cDNAs were classified as potential TFs or co-activators because of the presence of conserved TF-like domains . Additionally, 2, 11, and 13 clones encoded kinases, chromosome-related proteins, and unknown proteins, respectively, while the remaining 16 cDNAs were associated with specific functions seemingly unrelated to TFs . Expression pattern analysis of selected TF-encoding genes via RT-PCR revealed that these genes were expressed during seed development, with differential transcription observed during various stages . This work provides informative hints for further study of the regulatory mechanism of rice seed development and illustrates an identification strategy that will be of practical value for the isolation of TFs and co-activators associated with specific plant developmental processes.

Plant J, 2004 Apr, 38(2), 320 - 31
Characterization of heterotrimeric G protein complexes in rice plasma membrane; Kato C et al.; Two genes in the rice genome were identified as those encoding the gamma subunits, gamma1 and gamma2, of heterotrimeric G proteins . Using antibodies against the recombinant proteins for the alpha, beta, gamma1, and gamma2 subunits of the G protein complexes, all of the subunits were proven to be localized in the plasma membrane in rice . Gel filtration of solubilized plasma membrane proteins showed that all of the alpha subunits were present in large protein complexes (about 400 kDa) containing the other subunits, beta, gamma1, and gamma2, and probably also some other proteins, whereas large amounts of the beta and gamma (gamma1 and gamma2) subunits were freed from the large complexes and took a 60-kDa form . A yeast two-hybrid assay and co-immunoprecipitation experiments showed that the beta subunit interacted tightly with the gamma1 and gamma2 subunits, and so the beta and gamma subunits appeared to form dimers in rice cells . Some dimers were associated with the alpha subunit, because few beta, gamma1, and gamma2 subunits were present in the 400-kDa complexes in a rice mutant, d1, which was lacking in the alpha subunit . When a constitutively active form of the alpha subunit was prepared by the exchange of one amino acid residue and introduced into d1, the mutagenized subunit was localized in the plasma membrane of the transformants and took a free, and not the 400-kDa, form.

Curr Med Chem, 2004 Apr, 11(8), 981 - 6
Biology of heme in health and disease; Wijayanti N et al.; Heme is an essential molecule with contradictory biological functions . In hemoproteins such as hemoglobin and cytochromes protein-bound heme is a prosthetic group serving physiological functions as a transporter for oxygen and electrons . On the other hand free heme can have deleterious effects by generating reactive oxygen species that cause oxidative stress . Consequently, heme homeostasis of the cell must be tightly controlled . The biosynthesis of heme is catalyzed by eight enzymes that are differentially regulated in liver and erythroid cells . Recent findings on proinflammatory functions of heme and its role in the pathogenesis of diseases, such as rhabdomyolysis or atherosclerosis are summarized . The regulation of gene expression by heme in yeast and mammalian cells and the underlying molecular mechanisms are presented . Finally, we discuss the functional significance of the heme-degrading enzyme heme oxygenase and heme-binding proteins for the regulation of heme homeostasis.

Oncogene, 2004 May 27, 23(25), 4422 - 9
Metastatic tumor antigen 1 short form (MTA1s) associates with casein kinase I-gamma2, an estrogen-responsive kinase; Mishra SK et al.; Recent studies have shown that metastasis-associated protein-1 short form (MTA1s) - metastatic tumor antigen 1 short form sequesters estrogen receptor-alpha (ER-alpha) in the cytoplasm of breast cancer cells . Using a yeast two-hybrid screening to clone MTA1s-interacting proteins, we identified casein kinase I-gamma 2 (CKI-gamma2, a ubiquitously expressed cytoplasmic kinase) as an MTA1s-binding protein . We show that MTA1s interacts with CKI-gamma2 both in vitro and in vivo and colocalizes in the cytoplasm . In addition, we found that CKI-gamma2 can phosphorylate MTA1s, but not ER, in an antiestrogen-dependent manner and that estrogen stimulates CKI-gamma2 activity that could be effectively blocked by a specific inhibitor of CKI . CKI-gamma2 could further potentiate the ER corepressive function of MTA1s . Kinase dead CK1-gamma2 could not repress estrogen-induced ER transactivation functions . Results from mutagenesis studies suggest that substitution of the serine residue at 321 to alanine, which is a possible CKI-gamma2 phopshorylation site in MTA1s, results in a significant reduction in the ability of MTA1s to repress ER transactivation . These findings identified MTA1s as a target of CKI-gamma2, and provided new evidence to suggest that CKI-gamma2 phosphorylates and modulates the functions of MTA1s, and that these extranuclear effects of estrogen might have important implications in regulating the functions of MTA1s in human mammary epithelial and cancer cells.

Oncogene, 2004 Apr 12, 23(16), 2891 - 906
Autophagy as a cell death and tumor suppressor mechanism; Gozuacik D et al.; Autophagy is characterized by sequestration of bulk cytoplasm and organelles in double or multimembrane autophagic vesicles, and their delivery to and subsequent degradation by the cell's own lysosomal system . Autophagy has multiple physiological functions in multicellular organisms, including protein degradation and organelle turnover . Genes and proteins that constitute the basic machinery of the autophagic process were first identified in the yeast system and some of their mammalian orthologues have been characterized as well . Increasing lines of evidence indicate that these molecular mechanisms may be recruited by an alternative, caspase-independent form of programmed cell death, named autophagic type II cell death . In some settings, autophagy and apoptosis seem to be interconnected positively or negatively, introducing the concept of 'molecular switches' between them . Additionally, mitochondria may be central organelles integrating the two types of cell death . Malignant transformation is frequently associated with suppression of autophagy . The recent implication of tumor suppressors like Beclin 1, DAP-kinase and PTEN in autophagic pathways indicates a causative role for autophagy deficiencies in cancer formation . Autophagic cell death induction by some anticancer agents underlines the potential utility of its induction as a new cancer treatment modality.

J Biol Chem, 2004 Jul 9, 279(28), 29247 - 54 Epub 2004 Apr 09.
The scaffold protein CNK1 interacts with the tumor suppressor RASSF1A and augments RASSF1A-induced cell death; Rabizadeh S et al.; The connector enhancer of KSR (CNK) is a multidomain scaffold protein discovered in Drosophila, where it is necessary for Ras activation of the Raf kinase . Recent studies have shown that CNK1 also interacts with RalA and Rho and participates in some aspects of signaling by these GTPases . Herein we demonstrate a novel aspect of CNK1 function, i.e . reexpression of CNK1 suppresses tumor cell growth and promotes apoptosis . As shown previously for apoptosis induced by Ki-Ras(G12V), CNK1-induced apoptosis is suppressed by a dominant inhibitor of the mammalian sterile 20 kinases 1 and (MST1/MST2) . Immunoprecipitates of MST1 endogenous to LoVo colon cancer cells contain endogenous CNK1; however, no association of these two polypeptides can be detected in a yeast two-hybrid assay . CNK1 does, however, bind directly to the RASSF1A and RASSF1C polypeptides, constitutive binding partners of the MST1/2 kinases . Deletion of the MST1 carboxyl-terminal segment that mediates its binding to RASSF1A/C eliminates the association of MST1 with CNK1 . Coexpression of CNK1 with the tumor suppressive isoform, RASSF1A, greatly augments CNK1-induced apoptosis, whereas the nonsuppressive RASSF1C isoform is without effect on CNK1-induced apoptosis . Overexpression of CNK1-(1-282), a fragment that binds RASSF1A but is not proapoptotic, blocks the apoptosis induced by CNK1 and by Ki-Ras(G12V) . Thus, in addition to its positive role in the proliferative outputs of active Ras, the CNK1 scaffold protein, through its binding of a RASSF1A.MST complex, also participates in the proapoptotic signaling initiated by active Ras.

J Biol Chem, 2004 Jul 16, 279(29), 30375 - 84 Epub 2004 Apr 09.
Identification of the epitope for the epidermal growth factor receptor-specific monoclonal antibody 806 reveals that it preferentially recognizes an untethered form of the receptor; Johns TG et al.; The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial cancers, an observation often correlated with poor clinical outcome . Overexpression of the EGFR is commonly caused by EGFR gene amplification and is sometimes associated with expression of a variant EGFR (de2-7 EGFR or EGFRvIII) bearing an internal deletion in its extracellular domain . Monoclonal antibody (mAb) 806 is a novel EGFR antibody with significant antitumor activity that recognizes both the de2-7 EGFR and a subset of the wild type (wt) EGFR when overexpressed but does not bind the wt EGFR expressed in normal tissues . Despite only binding to a low proportion of the wt EGFR expressed in A431 tumor cells (approximately 10%), mAb 806 displays robust antitumor activity against A431 xenografts grown in nude mice . To elucidate the mechanism leading to its unique specificity and mode of antitumor activity, we have determined the EGFR binding epitope of mAb 806 . Analysis of mAb 806 binding to EGFR fragments expressed either on the surface of yeast or in an immunoblot format identified a disulfide-bonded loop (amino acids 287-302) that contains the mAb 806 epitope . Indeed, mAb 806 binds with apparent high affinity (approximately 30 nm) to a synthetic EGFR peptide corresponding to these amino acids . Analysis of EGFR structures indicates that the epitope is fully exposed only in the transitional form of the receptor that occurs because EGFR changes from the inactive tethered conformation to a ligand-bound active form . It would seem that mAb 806 binds this small proportion of transient receptors, preventing their activation, which in turn generates a strong antitumor effect . Finally, our observations suggest that the generation of antibodies to transitional forms of growth factor receptors may represent a novel way of reducing normal tissue targeting yet retaining antitumor activity.

J Cell Sci, 2004 Apr 1, 117(Pt 9), 1699 - 708 Epub 2004 Mar 09.
ATPase-deficient hVPS4 impairs formation of internal endosomal vesicles and stabilizes bilayered clathrin coats on endosomal vacuoles; Sachse M et al.; Epidermal growth factor receptors (EGFRs) destined for lysosomal degradation are sorted in the early endosomal vacuole into small, lumenal vesicles that arise by inward budding of the limiting membrane . We have previously shown that, before their incorporation into internal vesicles, EGFRs are concentrated in flat bilayered-clathrin coats on the endosomal vacuole . Here, we show that an ATPase-deficient mutant of hVPS4 (hVPS4(EQ)) increases the association of bilayered coats with endosomal vacuoles . In addition, hVPS4(EQ) leads to a reduction in the number of internal vesicles in early and late endosomal vacuoles, and retention of EGFRs at the limiting membrane . Interestingly, hVPS4(EQ) was predominantly found on non-coated regions of endosomal vacuoles, often at the rim of a coated area . In line with published data on Vps4p function in yeast, these results suggest that hVPS4 is involved in the release of components of the bilayered coat from the endosomal membrane . Moreover, our data suggest that disassembly of the coat is required for the formation of internal vesicles.

J Biol Chem, 2004 Jul 9, 279(28), 29270 - 7 Epub 2004 Apr 08.
The Caenorhabditis elegans ortholog of TRAP240, CeTRAP240/let-19, selectively modulates gene expression and is essential for embryogenesis; Wang JC et al.; Mediator complexes are large multiprotein assemblies that function in the regulation of eukaryotic gene transcription . In yeast, certain mediator subunits appear to comprise a subcomplex that acts in the regulation of a specific subset of genes . We investigated in a metazoan, Caenorhabditis elegans, the roles and interactions of two of those subunits, CeTRAP240/let-19 and CeTRAP230/dpy-22 . We found that CeTRAP240/let-19 contains four domains that are conserved in the human TRAP240 protein and that one of those domains displays intrinsic transcriptional repression activity . Using RNA interference, we found that reduced expression of CeTRAP240/let-19 displayed a high penetrance of embryonic lethality in F1 progeny; animals that escaped embryonic arrest showed mutant phenotypes such as burst vulva and molting defects . CeTRAP240/let-19 appeared to affect specific genes, as CeTRAP240/let-19(RNAi) led to selectively reduced expression of a subset of reporter genes examined . Genetic experiments supported the view that CeTRAP240/let-19 and CeTRAP230/dpy-22, like their Drosophila and yeast counterparts, can operate on common pathways . Thus, a male tail phenotype caused by the pal-1(e2091) mutation was suppressed not only by CeTRAP230/dpy-22 mutants, as reported previously, but also by reduced expression of CeTRAP240/let-19 . Additionally, CeTRAP240/let-19(RNAi) in a CeTRAP230/dpy-22 mutant background produced a strong synthetic lethal phenotype . Overall, our results establish specific roles of CeTRAP240/let-19 in C . elegans embryonic development and a functional interaction between CeTRAP240/let-19 and CeTRAP230/dpy-22 . Interestingly, whereas this interaction has been conserved from yeast to mammals, the subcomplex modulates metazoan-specific genetic pathways, likely in addition to those also controlled in yeast.

J Mol Endocrinol, 2004 Apr, 32(2), 425 - 36
Cinnamic acid based thiazolidinediones inhibit human P450c17 and 3beta-hydroxysteroid dehydrogenase and improve insulin sensitivity independent of PPARgamma agonist activity; Arlt W et al.; Thiazolidinediones improve insulin sensitivity in type 2 diabetes mellitus by acting as peroxisome proliferator-associated receptor gamma (PPARgamma) agonists, and decrease circulating androgen concentrations in polycystic ovary syndrome by unknown mechanisms . Some thiazolidinediones directly inhibit the steroidogenic enzymes P450c17 and 3beta-hydroxysteroid dehydrogenase type II (3betaHSDII) by distinct mechanisms . We synthesized five novel thiazolidinediones, CLX-M1 to -M5 by linking a 2,4-thiazolidinedione moiety to a substituted alpha-phenyl cinnamic acid previously shown to have glucose-lowering effects . Using yeast microsomes expressing human P450c17 and 3betaHSDII we found that cinnamic acid methyl esters with a double bond in the thiazolidinedione core structure (M3, M5) were stronger inhibitors of P450c17 than methyl esters with the conventional core (M1, M4) . These four compounds inhibited 3betaHSDII equally well, while the free cinnamic acid analog (M2) did not inhibit either enzyme . Thus, the inhibition of P450c17 and 3betaHSDII by these novel thiazolidinediones reveals structure-activity relationships independent of PPARgamma transactivation . PPARgamma transactivation was moderate (M1), weak (M2, M3) or even absent (M4, M5) . While the PPARgamma agonist activity of M1 was only 3% of that of rosiglitazone, both increased glucose uptake by 3T3-L1 adipocytes and reduced serum glucose levels in ob/ob and db/db mice to a similar extent . The similar glucose-lowering effects of M1 and rosiglitazone, despite their vast differences in PPARgamma agonist activity, suggests these two actions may occur by separate mechanisms.

EMBO J, 2004 May 5, 23(9), 1977 - 86 Epub 2004 Apr 08.
A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier; Komatsu M et al.; Several studies have addressed the importance of various ubiquitin-like (UBL) post-translational modifiers . These UBLs are covalently linked to most, if not all, target protein(s) through an enzymatic cascade analogous to ubiquitylation, consisting of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes . In this report, we describe the identification of a novel ubiquitin-fold modifier 1 (Ufm1) with a molecular mass of 9.1 kDa, displaying apparently similar tertiary structure, although lacking obvious sequence identity, to ubiquitin . Ufm1 is first cleaved at the C-terminus to expose its conserved Gly residue . This Gly residue is essential for its subsequent conjugating reactions . The C-terminally processed Ufm1 is activated by a novel E1-like enzyme, Uba5, by forming a high-energy thioester bond . Activated Ufm1 is then transferred to its cognate E2-like enzyme, Ufc1, in a similar thioester linkage . Ufm1 forms several complexes in HEK293 cells and mouse tissues, revealing that it conjugates to the target proteins . Ufm1, Uba5, and Ufc1 are all conserved in metazoa and plants but not in yeast, suggesting its potential roles in various multicellular organisms.

EMBO Rep, 2004 May, 5(5), 484 - 9 Epub 2004 Apr 08.
Dystroglycan, a scaffold for the ERK-MAP kinase cascade; Spence HJ et al.; Dystroglycan is an important cell adhesion receptor linking the actin cytoskeleton, via utrophin and dystrophin, to laminin in the extracellular matrix . To identify adhesion-related signalling molecules associated with dystroglycan, we conducted a yeast two-hybrid screen and identified mitogen-activated protein (MAP) kinase kinase 2 (MEK2) as a beta-dystroglycan interactor . Pull-down experiments and localization studies substantiated a physiological link between beta-dystroglycan and MEK and localized MEK with dystroglycan in membrane ruffles . Moreover, we also identified active extracellular signal-regulated kinase (ERK), the downstream kinase from MEK, as another interacting partner for beta-dystroglycan and localized both active ERK and dystroglycan to focal adhesions in fibroblast cells . These studies suggest a role for dystroglycan as a multifunctional adaptor or scaffold capable of interacting with components of the ERK-MAP kinase cascade including MEK and ERK . These findings have important implications for our understanding of the role of dystroglycan in normal cellular processes and in disease states such as muscular dystrophy.

Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 5940 - 5 Epub 2004 Apr 07.
Crystal structure and characterization of a cytochrome c peroxidase-cytochrome c site-specific cross-link; Guo M et al.; A specific covalently cross-linked complex between redox partners yeast cytochrome c peroxidase (CCP) and cytochrome c (cyt . c) has been made by engineering cysteines into CCP and cyt . c that form an intermolecular disulfide bond in high yield . The crystal structure of the cross-linked complex has been solved to 1.88-A resolution and closely resembles the structure of the noncovalent complex {Pellitier, H . & Kraut, J . (1992) Science 258, 1748-1755} . The higher resolution of the covalent complex has enabled the location of ordered water molecules at the peroxidase-cytochrome c interface that serve to bridge between the two proteins by hydrogen bonding . As in the noncovalent complex, direct electrostatic interactions between protein groups appear not to be critical in complex formation . UV-visible spectroscopic and stopped-flow studies indicate that CCP in the covalent complex reacts normally with H(2)O(2) to give compound I . Stopped-flow kinetic studies also show that intramolecular electron transfer between the cross-linked ferrocytochrome c and the Trp-191 cation radical site in CCP compound I occurs fast and is nearly complete within the dead time ( approximately 2 ms) of the instrument . These results indicate that the structure of the covalent complex closely mimics the physiological electron transfer complex . In addition, single-turnover and steady-state experiments reveal that CCP compound I in the covalent complex oxidizes exogenously added ferrocytochrome c at a slow rate (t(1/2) approximately 2 min), indicating that CCP does not have a second independent site for physiologically relevant electron transfer.

J Clin Microbiol, 2004 Apr, 42(4), 1790 - 3
Modified colorimetric assay for susceptibility testing of azole antifungal drugs against Candida species; Chen J et al.; We modified a rapid susceptibility assay (RSA) for antifungal susceptibility testing of azoles based upon glucose utilization . This modified RSA method provides quantitative endpoint readings in 6 h . In this study, the modified RSA and the National Committee for Clinical Laboratory Standards M27-A methods were used to determine the MICs of fluconazole and itraconazole for 118 Candida isolates . Yeast nitrogen base containing 0.12 g of glucose per liter was used for the modified RSA method . For fluconazole, agreement among assays within one or two twofold dilutions was 72.9 and 88.1%, respectively; for itraconazole, agreement within one or two twofold dilutions was 82.2 and 89.8%, respectively . These data suggest that the modified RSA method is a reliable and rapid method for azole antifungal susceptibility testing against Candida species.

Biol Reprod, 2004 Aug, 71(2), 629 - 36 Epub 2004 Apr 07.
Human follicle-stimulating hormone (FSH) receptor interacts with the adaptor protein APPL1 in HEK 293 cells: potential involvement of the PI3K pathway in FSH signaling; Nechamen CA et al.; Selection of a dominant follicle that will ovulate likely occurs by activation of cell survival pathways and suppression of death-promoting pathways in a mechanism involving FSH and its cognate receptor (FSHR) . A yeast two-hybrid screen of an ovarian cDNA library was employed to identify potential interacting partners with human FSHR intracellular loops 1 and 2 . Among eight cDNA clones identified in the screen, APPL1 (adaptor protein containing PH domain, PTB domain, and leucine zipper motif; also known as APPL or DIP13alpha) was chosen for further analysis . APPL1 appears to coimmunoprecipitate with FSHR in HEK 293 cells stably expressing FSHR (293/FSHR cells), confirming APPL1 as a potential FSHR-interacting partner . The phosphorylation status of members of the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway was also examined because of the proposed role of APPL1 in the antiapoptotic PI3K/Akt pathway . FOXO1a, also referred to as forkhead homologue in rhabdomyosarcoma, is a downstream effector in the pathway and tightly linked to expression of proapoptotic genes . FOXO1a, but not the upstream kinase Akt, is rapidly phosphorylated, and FOXO1a is thereby inactivated when 293/FSHR cells are treated with FSH . In addition, FSHR coimmunoprecipitates with Akt . The identification of APPL1 as a potential interactor with FSHR and the finding that FOXO1a is phosphorylated in response to FSH provide a possible link between FSH and PI3K/Akt signaling, which may help to delineate a survival mechanism whereby FSH selects the dominant follicle to survive.

Proc Natl Acad Sci U S A, 2004 Mar 30, 101(13), 4507 - 12 Epub 2004 Mar 19.
Body plan evolution of ascomycetes, as inferred from an RNA polymerase II phylogeny; Liu YJ et al.; The mode of evolution of the biologically diverse forms of ascomycetes is not well understood, largely because the descent relationships remain unresolved . By using sequences of the nuclear gene RPB2, we have inferred with considerable resolution the phylogenetic relationships between major groups within the phylum Ascomycota . These relationships allow us to deduce a historical pattern of body plan evolution . Within Taphrinomycotina, the most basal group, two simple body plans exist: uncovered asci with unicellular growth, or rudimentary ascoma with hyphal growth . Ancestral ascomycetes were filamentous; hyphal growth was lost independently in the yeast forms of Taphrinomycotina and Saccharomycotina . Pezizomycotina, the sister group to Saccharomycotina, retained mycelial growth while elaborating two basic ontogenetic pathways for ascoma formation and centrum development . The RPB2 phylogeny shows with significant statistical support that taxa in Pezizomycotina with ascohymenial ontogeny (ascoma generally forms after nuclear pairing) are ancestral and paraphyletic, whereas ascolocular fungi with fissitunicate asci are a clade derived from them . Ascolocular lichens are polyphyletic, whereas ascohymenial lichens comprise a monophyletic group that includes the Lecanorales . Our data are not consistent with a derived origin of Eurotiomycetes including Aspergillus and Trichophyton from within a lichen-forming ancestral group . For these reasons, the results of this study are considerably at variance with the conclusion that major fungal lineages are derived from lichensymbiotic ancestors . Interpretation of our results in the context of early work suggests that ascoma ontogeny and centrum characters are not in conflict with the molecular data.

Proc Natl Acad Sci U S A, 2004 Mar 30, 101(13), 4361 - 6 Epub 2004 Mar 02.
Mitochondrial DNA ligase in Crithidia fasciculata; Sinha KM et al.; Kinetoplast DNA (kDNA), the form of mitochondrial DNA in trypanosomatids, consists of thousands of interlocked circular DNAs organized into a compact disk structure . A type II DNA topoisomerase, a DNA polymerase beta, and a structure-specific endonuclease have been localized to antipodal sites flanking the kDNA disk along with nascent DNA minicircles . We have cloned a gene (LIG k) encoding a mitochondrial DNA ligase in the trypanosomatid Crithidia fasciculata, and we show that an epitope-tagged form of the ligase colocalizes with the other replication proteins at the antipodal sites and also at the two faces of the kDNA disk . DNA LIG k becomes adenylated in reactions with ATP, and the adenylate moiety is removed by incubation with pyrophosphate or nicked DNA . The ligase interacts physically with the beta polymerase and is proposed to be involved in the repair of gaps in the newly synthesized minicircles . In yeast and mammals, a single gene encodes both nuclear and mitochondrial forms of DNA ligase . The LIG K protein sequence has low similarity to mitochondrial DNA ligases in other eukaryotes and is distinct from the C . fasciculata nuclear DNA ligase (LIG I).

BMC Cell Biol . 2004 Feb 29;5(1):9.
Mig12, a novel Opitz syndrome gene product partner, is expressed in the embryonic ventral midline and co-operates with Mid1 to bundle and stabilize microtubules; Berti C et al.; BACKGROUND: Opitz G/BBB syndrome is a genetic disorder characterized by developmental midline abnormalities, such as hypertelorism, cleft palate, and hypospadias . The gene responsible for the X-linked form of this disease, MID1, encodes a TRIM/RBCC protein that is anchored to the microtubules . The association of Mid1 with the cytoskeleton is regulated by dynamic phosphorylation, through the interaction with the alpha4 subunit of phosphatase 2A (PP2A) . Mid1 acts as an E3 ubiquitin ligase, regulating PP2A degradation on microtubules . RESULTS: In spite of these findings, the biological role exerted by the Opitz syndrome gene product is still unclear and the presence of other potential interacting moieties in the Mid1 structure prompted us to search for additional cellular partners . Through a yeast two-hybrid screening approach, we identified a novel gene, MIG12, whose protein product interacts with Mid1 . We confirmed by immunoprecipitation that this interaction occurs in vivo and that it is mediated by the Mid1 coiled-coil domain . We found that Mig12 is mainly expressed in the neuroepithelial midline, urogenital apparatus, and digits during embryonic development . Transiently expressed Mig12 is found diffusely in both nucleus and cytoplasm, although it is enriched in the microtubule-organizing center region . Consistently with this, endogenous Mig12 protein is partially detected in the polymerized tubulin fraction after microtubule stabilization . When co-transfected with Mid1, Mig12 is massively recruited to thick filamentous structures composed of tubulin . These microtubule bundles are resistant to high doses of depolymerizing agents and are composed of acetylated tubulin, thus representing stabilized microtubule arrays . CONCLUSIONS: Our findings suggest that Mig12 co-operates with Mid1 to stabilize microtubules . Mid1-Mig12 complexes might be implicated in cellular processes that require microtubule stabilization, such as cell division and migration . Impairment in Mig12/Mid1-mediated microtubule dynamic regulation, during the development of embryonic midline, may cause the pathological signs observed in Opitz syndrome patients.

J Biol Chem, 2004 Jul 2, 279(27), 28817 - 25 Epub 2004 Apr 06.
Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase; Xie M et al.; CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids . Previous work demonstrated that CCT is a homodimer . In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT . Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions . The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles . Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent . Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes . The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines . We found that the N-terminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236) . We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface.

Virus Res, 2004 Jun 1, 102(1), 109 - 15
RNA silencing: a conserved antiviral immunity of plants and animals; Ding SW et al.; RNA silencing is a novel RNA-guided gene regulatory mechanism operational in a wide range of eukaryotic organisms from fission yeast, plants, to mammals . This article reviews the recent progress on aspects of RNA silencing that are related to its biological function as a conserved antiviral immunity of plants and animals, and highlights features of this novel antiviral response in invertebrate animals as compared to the known innate and adaptive immunities . Finally, we discuss evidence that suggests a natural antiviral role for RNA silencing in vertebrates as well as experimental approaches that may facilitate the identification of first mammalian viral suppressors of RNA silencing.

Commun Dis Public Health, 2003 Dec, 6(4), 320 - 4
Rapid and frequent induction of protective immunity exceeding UK recommendations for healthcare settings by MF59-adjuvated hepatitis B vaccine; Lewis DJ et al.; The ability of three doses of a novel MF59-adjuvanted hepatitis B virus (HBV) vaccine containing surface and pre-S2 antigens given at 0, 1, and 6 months to induce levels of HBV surface antibody (sAb) > or = 100 mIU/ml was compared with a UK licensed alum-adjuvanted yeast-derived HBV vaccine in HBV-naive healthcare workers (HCWs) . One month after second immunization with HBV/MF59, 100% of HCWs had sAb > or = 100 mIU/mL, compared with only 11% and 85% after two or three immunisations with Engerix-B . The sAb GMT of the Engerix B immunised group remained below 100 mIU/mL until month seven, (compared with month one for HBV/MF59), and was 123-fold lower at this time (208,561 vs . 1,686 mIU/mL) . In our subjects HBV/MF59 vaccine rapidly induced sAb to levels far in excess of those recommended by the Department of Health for high-risk situations (e.g . HCWs and patients on dialysis) . It has the potential for shorter schedules and reduced need for serology and boosters.

J Biomed Sci, 2004 May-Jun, 11(3), 391 - 7
Effect of experimental oxidative stress on steroidogenesis and DNA damage in mouse testis; Kaur P et al.; The objective of the present study was to evaluate the effect of oxidative stress induced by feeding various levels of selenium on steroidogenesis and DNA damage in mouse testes . To create various levels of oxidative stress in mice, diets with three different Se levels were fed to separate groups for 8 weeks . Group 1 animals were fed a yeast-based diet, which was considered a Se-deficient diet (0.02 ppm) . Group 2 and 3 animals were fed a Se-deficient diet supplemented with 0.2 and 1 ppm Se as sodium selenite, respectively . After completion of the diet feeding, estimations were carried out, and results were compared with those of group 2 . A significant decrease in Se levels was observed in group 1 animals, whereas they were greatly enhanced in group 3 . Glutathione peroxidase (GSH-Px) activity was greatly reduced in both the liver and testes in group 1, whereas no significant changes were found in GSH-Px activity in group 3 . Serum luteinizing hormone, follicle-stimulating hormone (FSH), and testosterone levels were reduced in group 1 . Significant decreases of sperm number and motility were observed in group 1 when compared to group 2 male mice . No changes in these parameters were observed in group 3 . DNA fragmentation was observed in both groups 1 and 3; however, the damage was more prevalent in group 1 . The results clearly demonstrate the effect of oxidative stress generated by feeding various Se levels on the steroidogenesis and DNA fragmentation in mice testes .

J Biol Chem, 2004 Jun 18, 279(25), 26074 - 81 Epub 2004 Apr 05.
Structure-function analysis of the estrogen receptor alpha corepressor scaffold attachment factor-B1: identification of a potent transcriptional repression domain; Townson SM et al.; Scaffold attachment factor-B1 (SAFB1) is a nuclear matrix protein that has been proposed to couple chromatin structure, transcription, and RNA processing . We have previously shown that SAFB1 can repress estrogen receptor (ERalpha)-mediated transactivation . Here we present a structure-function study showing that transactivation is mediated via an intrinsic and transferable C-terminal repression domain (RD) . A similar C-terminal RD was found in the family member SAFB2 . Removal of the RD from SAFB1 resulted in a dominant-negative SAFB1 protein that increased ligand-dependent and -independent ERalpha activity . SAFB1RD-mediated repression was partly blocked by histone deacetylase inhibitors; however, no histone deacetylase inhibitors were identified in a yeast two-hybrid screen using the RD as bait . Instead, SAFB1RD was found to interact with TAFII68, a member of the basal transcription machinery . We propose a model in which SAFB1 represses ERalpha activity via indirect association with histone deacetylation and interaction with the basal transcription machinery.

Cancer Genet Cytogenet, 2004 Apr 15, 150(2), 136 - 43
Heterogeneity of structural abnormalities in the 7q31.3 approximately q34 region in myeloid malignancies; Gonzalez MB et al.; Abnormalities in the long arm of chromosome 7 are a frequent chromosomal aberration in myeloid disorders . Most studies have focused on the analysis of del(7q), demonstrating the presence of several minimal deleted regions in 7q22 approximately q31 . By contrast, few studies in myeloid disorders have been devoted to the analysis of translocations, either balanced or unbalanced, involving 7q . In this study, we used fluorescence in situ hybridization (FISH) to characterize the 7q31.3 approximately q34 region (markers D7S480-D7S2227) in patients with deletion or translocation of 7q . A total of 910 cases of myeloid disorders were studied by conventional cytogenetics . Fifty-eight (6%) patients had structural aberrations of 7q . FISH studies were carried out in the 27 patients with involvement of 7q31 approximately q34: 14 cases had an acute myelogenous leukemia and 13 cases had a myelodysplastic syndrome . FISH analysis revealed the existence of high complexity in the 7q31.3 approximately q34 region in patients with unbalanced translocations . No breakpoints in 7q31.3 approximately q34 were found in the cases with deletion or balanced translocation . Nevertheless, studies of unbalanced translocations showed several breakpoints in markers D7S480-D7S2227, which delineate a commonly altered region . The complexity of 7q rearrangements suggests that a synergy of different genetic factors, rather than the alteration of a single tumor suppressor gene, could be involved in the pathogenesis of del(7q) in myeloid disorders.

Methods Mol Biol, 2004, 261, 327 - 36
Mammalian two-hybrid assay for detecting protein-protein interactions in vivo; Lee JW et al.; Mammalian two-hybrid assay is a convenient, powerful tool to investigate protein-protein interactions in vivo . In particular, this method has a major advantage over the better known yeast version in that one can study interactions between mammalian proteins that may not fold correctly in yeast or that require post-translational modification or external stimulation that are not present in yeast.

Plant Physiol, 2004 Apr, 134(4), 1632 - 41 Epub 2004 Apr 02.
Identification and characterization of four chrysanthemum MADS-box genes, belonging to the APETALA1/FRUITFULL and SEPALLATA3 subfamilies; Shchennikova AV et al.; Four full-length MADS-box cDNAs from chrysanthemum, designated Chrysanthemum Dendrathema grandiflorum MADS (CDM) 8, CDM41, CDM111, and CDM44, have been isolated and further functionally characterized . Protein sequence alignment and expression patterns of the corresponding genes suggest that CDM8 and CDM41 belong to the FRUITFULL (FUL) clade, CDM111 is a member of the APETALA1 (AP1) subfamily, and CDM44 is a member of the SEPALLATA3 (SEP3) subfamily of MADS-box transcription factors . Overexpression of CDM111 in Arabidopsis plants resulted in an aberrant phenotype that is reminiscent of the phenotype obtained by ectopic expression of the AP1 gene . In addition, CDM111 was able to partially complement the ap1-1 mutant from Arabidopsis, illustrating that CDM111 is the functional equivalent to AP1 . Yeast two- and three-hybrid studies were performed to investigate the potential protein interactions and complexes in which these chrysanthemum MADS-box proteins are involved . Based on these studies, we conclude that CDM44 is most likely the SEP3 functional equivalent, because the CDM44 protein interacts with CDM proteins of the AP1/FUL and AG subfamilies, and as a higher order complex with the heterodimer between the presumed B-type CDM proteins.

FEBS Lett, 2004 Apr 9, 563(1-3), 93 - 7
Identification of the human sphingolipid C4-hydroxylase, hDES2, and its up-regulation during keratinocyte differentiation; Mizutani Y et al.; The C4-hydroxylation of dihydrosphingosine or dihydroceramide is a key reaction in the biosynthesis of phytosphingolipids, both in yeasts and in mammalian cells . Mouse DES2 (mDES2) was recently cloned and shown to work as a Delta4-desaturase/C4-hydroxylase, when expressed in yeast cells . Here, we cloned a human homologue of mDES2, hDES2, by homology search utilizing a BLAST program . When expressed in HEK 293 cells, hDES2 exhibited hydroxylase activity for dihydroceramide . Northern blot analyses of hDES2 revealed high expression in skin, intestines, and kidney, sites reportedly possessing high levels of phytosphingolipids . Furthermore, up-regulation of hDES2 mRNA expression and subsequent phytoceramide production were observed during vitamin C/serum-induced differentiation of human keratinocytes . These results suggest that the newly cloned hDES2 plays an essential role in phytosphingolipid synthesis in human skin and other phytosphingolipid-containing tissues.

Biochim Biophys Acta, 2004 Apr 5, 1688(3), 232 - 6
Identification of the interaction between the human recombinant histamine releasing factor/translationally controlled tumor protein and elongation factor-1 delta (also known as eElongation factor-1B beta); Langdon JM et al.; The human recombinant histamine releasing factor (HrHRF), also known as translationally controlled tumor protein (TCTP), p23 and fortilin, has been described to have both extra- and intracellular functions . To elucidate an extra- or intracellular role for HrHRF, we used the yeast two-hybrid system with HrHRF as the bait and a Jurkat T cell library . We isolated a partial cDNA clone of the human elongation factor-1 delta (EF-1delta) encoding for amino acids 12 to 281 . This interaction was confirmed by co-immunoprecipitation experiments . Previously, both HrHRF and EF-1delta have been isolated and identified in association with malignancy in numerous studies . EF-1delta is part of the EF-1 complex responsible for kinetic proofreading in protein synthesis . Additionally, DNA microarray data classifies TCTP (HrHRF) as co-regulated with ribosomal proteins and recent structural analysis of TCTP (HrHRF) relates it to a guanine nucleotide-free chaperone . Our findings of an interaction between HrHRF and EF-1delta taken with some of the recently published information concerning the TCTP (HrHRF) mentioned above suggest a possible intracellular role for TCTP/HrHRF.

Mol Cell Endocrinol, 2003 Dec 31, 213(1), 71 - 8
Mixed lineage kinase 2 enhances trans-repression of Alien and nuclear receptors; Eckey M et al.; Alien was previously identified as a corepressor for the thyroid hormone receptor (TR) and DAX-1 which belong both to the superfamily of nuclear receptors . Here, we isolated the mixed lineage kinase 2 (MLK2) as an interacting partner for the corepressor Alien using a yeast two hybrid screen . MLK2 is an upstream activator of JNKs and activation of MLK2-mediated signaling cascades play roles in neurodegenerative and apoptotic mechanisms in the central nervous system . MLK2 has been shown to be localized both in the cytoplasm and cell nucleus . We confirmed the Alien-MLK2 interaction using GST pull-down experiments and also show that MLK2 is able to phosphorylate Alien in immune-kinase assays . Functional analyses revealed that Alien, DAX-1 and thyroid hormone receptor mediated transcriptional silencing is strongly enhanced in the presence of active MLK2 . Since MAP kinase signaling pathways are important mediators of cellular responses to a wide variety of stimuli, our data suggest that signaling pathways not only regulate transactivation but also enhancement of transcriptional silencing . This novel cross-talk may represent a link between MLK2-mediated signaling and transcriptional repression of target genes during neuronal differentiation processes.

Curr Biol, 2004 Apr 6, 14(7), 548 - 59
The microtubule plus end-tracking proteins mal3p and tip1p cooperate for cell-end targeting of interphase microtubules; Busch KE et al.; BACKGROUND: CLIP-170 and EB1 protein family members localize to growing microtubule tips and link spatial information with the control of microtubule dynamics . It is unknown whether these proteins operate independently or whether their actions are coordinated . In fission yeast the CLIP-170 homolog tip1p is required for targeting of microtubules to cell ends, whereas the role of the EB1 homolog mal3p in microtubule organization has not been investigated . RESULTS: We show that mal3p promotes the initiation of microtubule growth and inhibits catastrophes . Premature catastrophes occur randomly throughout the cell in the absence of mal3p . mal3p decorates the entire microtubule lattice and localizes to particles along the microtubules and at their growing tips . Particles move in two directions, outbound toward the cell ends or inbound toward the cell center . At cell ends, the microtubule tip-associated mal3p particles disappear followed by a catastrophe . mal3p localizes normally in tip1-deleted cells and disappears from microtubule tips preceding the premature catastrophes . In contrast, tip1p requires mal3p to localize at microtubule tips . mal3p and tip1p directly interact in vitro . CONCLUSIONS: mal3p and tip1p form a system allowing microtubules to target cell ends . We propose that mal3p stimulates growth initiation and maintains growth by suppressing catastrophes . At cell ends, mal3p disappears from microtubule tips followed by a catastrophe . mal3p is involved in recruiting tip1p to microtubule tips . This becomes important when microtubules contact the cell cortex outside the cell ends because mal3p dissociates prematurely without tip1p, which is followed by a premature catastrophe.

Mol Cell Biol, 2004 Apr, 24(8), 3077 - 88
Differential targeting of two distinct SWI/SNF-related Drosophila chromatin-remodeling complexes; Mohrmann L et al.; The SWI/SNF family of ATP-dependent chromatin-remodeling factors plays a central role in eukaryotic transcriptional regulation . In yeast and human cells, two subclasses have been recognized: one comprises yeast SWI/SNF and human BAF, and the other includes yeast RSC and human PBAF . Therefore, it was puzzling that Drosophila appeared to contain only a single SWI/SNF-type remodeler, the Brahma (BRM) complex . Here, we report the identification of two novel BRM complex-associated proteins: Drosophila Polybromo and BAP170, a conserved protein not described previously . Biochemical analysis established that Drosophila contains two distinct BRM complexes: (i) the BAP complex, defined by the presence of OSA and the absence of Polybromo and BAP170, and (ii) the PBAP complex, containing Polybromo and BAP170 but lacking OSA . Determination of the genome-wide distributions of OSA and Polybromo on larval salivary gland polytene chromosomes revealed that BAP and PBAP display overlapping but distinct distribution patterns . Both complexes associate predominantly with regions of open, hyperacetylated chromatin but are largely excluded from Polycomb-bound repressive chromatin . We conclude that, like yeast and human cells, Drosophila cells express two distinct subclasses of the SWI/SNF family . Our results support a close reciprocity of chromatin regulation by ATP-dependent remodelers and histone-modifying enzymes.

Genome Res, 2004 Apr, 14(4), 766 - 79
Integrated and sequence-ordered BAC- and YAC-based physical maps for the rat genome; Krzywinski M et al.; As part of the effort to sequence the genome of Rattus norvegicus, we constructed a physical map comprised of fingerprinted bacterial artificial chromosome (BAC) clones from the CHORI-230 BAC library . These BAC clones provide approximately 13-fold redundant coverage of the genome and have been assembled into 376 fingerprint contigs . A yeast artificial chromosome (YAC) map was also constructed and aligned with the BAC map via fingerprinted BAC and P1 artificial chromosome clones (PACs) sharing interspersed repetitive sequence markers with the YAC-based physical map . We have annotated 95% of the fingerprint map clones in contigs with coordinates on the version 3.1 rat genome sequence assembly, using BAC-end sequences and in silico mapping methods . These coordinates have allowed anchoring 358 of the 376 fingerprint map contigs onto the sequence assembly . Of these, 324 contigs are anchored to rat genome sequences localized to chromosomes, and 34 contigs are anchored to unlocalized portions of the rat sequence assembly . The remaining 18 contigs, containing 54 clones, still require placement . The fingerprint map is a high-resolution integrative data resource that provides genome-ordered associations among BAC, YAC, and PAC clones and the assembled sequence of the rat genome.

Circ Res, 2004 Apr 2, 94(6), 724 - 34
Point-counterpoint of sphingosine 1-phosphate metabolism; Saba JD et al.; Sphingosine 1-phosphate (S1P), an evolutionarily conserved bioactive lipid mediator, is now recognized as a potent modulator of cell regulation . In vertebrates, S1P interacts with cell surface G protein-coupled receptors of the EDG family and induces profound effects in a variety of organ systems . Indeed, an S1P receptor agonist is undergoing clinical trials to combat immune-mediated transplant rejection . Recent information on S1P receptor biology suggests potential utility in the control of cardiovascular processes, including angiogenesis, vascular permeability, arteriogenesis, and vasospasm . However, studies from diverse invertebrates, such as yeast, Dictyostelium, Drosophila, and Caenorhabditis elegans have shown that S1P is involved in important regulatory functions in the apparent absence of EDG S1P receptor homologues . Metabolic pathways of S1P synthesis, degradation, and release have recently been described at the molecular level . Genetic and biochemical studies of these enzymes have illuminated the importance of S1P signaling systems both inside and outside of cells . The revelation of receptor-dependent pathways, as well as novel metabolic/intracellular pathways has provided new biological insights and may ultimately pave the way for the development of novel therapeutic approaches for cardiovascular diseases.

Gastroenterology, 2004 Apr, 126(4), 1157 - 66
Involvement of chloride channels in hepatic copper metabolism: ClC-4 promotes copper incorporation into ceruloplasmin; Wang T et al.; BACKGROUND & AIMS: Copper transport in hepatocytes is regulated by the interaction of multiple pumps, chaperones, and accessory proteins . Intracellular chloride channels are essential for copper metabolism in yeast but their role in Cu transport in hepatocytes is unknown . The aim of this study was to determine whether chloride channels are modulators of copper incorporation into ceruloplasmin (CP) . METHODS: The effects of chloride concentration and chloride channel expression on secretion of holoCp and apoCp was measured by gel electrophoresis and immunoblotting . ClC family chloride channel expression in hepatocytes was determined by Western blotting . The association of ClC-4 and the Wilson's disease protein (ATP7B) was determined by co-immunoprecipitation . RESULTS: Chloride substitution reduced total Cp secretion and the ratio of secreted holoCp to apoCp (P = 0.038) . The role of specific chloride channels was examined by cotransfection of ceruloplasmin and the chloride channel . Overexpression of ClC-4 doubled copper incorporation into ceruloplasmin (P = 0.011), whereas identical overexpression of ClC-3 had no effect . The effect of ClC-4 was most pronounced under copper-limiting conditions in which it increased copper incorporation more than 4-fold (P = 0.037) . ClC-4 protein was abundant in hepatocyte membranes and was localized in intracellular vesicles containing ATP7B.CONCLUSIONS: ClC-4 is an intracellular chloride channel that stimulates copper incorporation into ceruloplasmin, probably by improving the efficiency of the ATP7B copper pump . It is thus an important component of the regulation of hepatic copper transport and may modulate Cu transport rates during copper deficiency, Wilson's disease, and other copper toxicosis syndromes.

Curr Microbiol, 2004 Feb, 48(2), 118 - 23
Specificity of DNA methylation changes during fungal dimorphism and its relationship to polyamines; Reyna-Lopez GE et al.; We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis . To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M . rouxii and Y . lipolytica . In an odc null mutant of U . maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine . Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism . Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition.

EMBO J, 2004 Apr 21, 23(8), 1900 - 10 Epub 2004 Apr 01.
Snf1-related protein kinase 1 is needed for growth in a normal day-night light cycle; Thelander M et al.; The yeast Snf1 protein kinase and its animal homologue, the AMP-activated protein kinase, play important roles in metabolic regulation, by serving as energy gauges that turn off energy-consuming processes and mobilize energy reserves during low-energy conditions . The closest homologue of these kinases in plants is Snf1-related protein kinase 1 (SnRK1) . We have cloned two SnRK1-encoding genes, PpSNF1a and PpSNF1b, in the moss Physcomitrella patens, where gene function can be studied directly by gene targeting in the haploid gametophyte . A snf1a snf1b double knockout mutant is viable, but lacks all Snf1-like protein kinase activity . The mutant has a complex phenotype that includes developmental abnormalities, premature senescence and altered sensitivities to plant hormones . Remarkably, the double knockout mutant also requires continuous light, and is unable to grow in a normal day-night light cycle . This suggests that SnRK1 is needed for metabolic changes that help the plant cope with the dark hours of the night.

Biol Pharm Bull, 2004 Apr, 27(4), 548 - 53
Estrogenic and antiestrogenic activities of the roots of Moghania philippinensis and their constituents; Ahn EM et al.; In the course of our search for natural estrogenic compounds from medicinal plants, we found that the methanolic extract from the roots of Moghania philippinensis (Fabaceae) showed significant effects on the proliferation of MCF-7 cells (human breast cancer) and induction of beta-galactosidase activity in a yeast two-hybrid assay . Through estrogenic activity-guided fractionation, we isolated several active flavonoids including prenylated ones . The CHCl(3) fraction and its new constituent, 8-(1,1-dimethylallyl)genistein (9), appreciably increased the uterine weight in ovariectomized rats when administered orally for 14 consecutive days, in which compound 9 showed stronger estrogenic activity than genistein . Antiestrogenic activities were also examined based on the inhibition of MCF-7 cell proliferation and beta-galactosidase activity in the yeast two-hybrid assay, mediated by 17beta-estradiol . 5,7,3',4'-Tetrahydroxy-6,8-diprenylisoflavone (6) showed the strongest antiestrogenic activity.

IEEE Trans Inf Technol Biomed, 2004 Mar, 8(1), 5 - 15
Cluster analysis of gene expression data based on self-splitting and merging competitive learning; Wu S et al.; Cluster analysis of gene expression data from a cDNA microarray is useful for identifying biologically relevant groups of genes . However, finding the natural clusters in the data and estimating the correct number of clusters are still two largely unsolved problems . In this paper, we propose a new clustering framework that is able to address both these problems . By using the one-prototype-take-one-cluster (OPTOC) competitive learning paradigm, the proposed algorithm can find natural clusters in the input data, and the clustering solution is not sensitive to initialization . In order to estimate the number of distinct clusters in the data, we propose a cluster splitting and merging strategy . We have applied the new algorithm to simulated gene expression data for which the correct distribution of genes over clusters is known a priori . The results show that the proposed algorithm can find natural clusters and give the correct number of clusters . The algorithm has also been tested on real gene expression changes during yeast cell cycle, for which the fundamental patterns of gene expression and assignment of genes to clusters are well understood from numerous previous studies . Comparative studies with several clustering algorithms illustrate the effectiveness of our method.

Gen Dent, 2003 Jul-Aug, 51(4), 361 - 6; quiz 367
Seizure disorders: update of medical and dental considerations; Stoopler ET et al.; Seizure disorders and epilepsy represent neurologic conditions that commonly are seen among patients requiring dental treatment . When dentists possess a working knowledge of seizures, in addition to an understanding of updated therapies for seizure management and oral complications associated with pharmacological therapy, they are able to treat patients with these disorders more effectively . Neurologic consultations and selecting an appropriate venue for treatment may need to be addressed prior to treatment, depending on the level of seizure control . Laboratory tests designed to evaluate medication levels, leukocyte counts, and clotting ability also may be required . Frequent recall visits may be necessary for seizure disorder patients who display adverse oral complications from medication, such as gingival hypertrophy, xerostomia, and oral yeast infections.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 417 - 32
Secondary membranes for flux optimization in membrane filtration of biologic suspensions; Nemade PR et al.; We employ in situ deposited secondary membranes of yeast (SMYs) to optimize permeate flux during microfiltration and ultrafiltration of protein solutions . The deposited secondary membrane was periodically removed by backflushing, and a new cake layer was deposited at the start of the next cycle . The effects of backflushing time, backflushing strength, wall shear rate, and amount of secondary membrane deposited on the permeate flux were examined . Secondary membranes were found to increase the permeate flux in microfiltration by severalfold . Protein transmission was also enhanced owing to the presence of the secondary membrane, and the amount of protein recovered was more than twice that obtained during filtration of protein-only solutions under otherwise identical conditions . In ultrafiltration, the flux enhancement owing to the secondary membrane was only 50% or less . In addition, the flux for ultrafiltration was relatively insensitive to changes in the concentration of yeast used during deposition of SMY and to the backflushing strength used to periodically remove the secondary membrane.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 843 - 55
Production of fumaric acid using rice bran and subsequent conversion to succinic acid through a two-step process; Moon SK et al.; The fungal production of fumaric acid using rice bran and subsequent bacterial conversion of succinic acid using fungal culture broth were investigated . Since the rice bran contains abundant proteins, amino acids, vitamins, and minerals, it is suitable material that fungi use as a nitrogen source . The effective concentration of rice bran to produce fumaric acid was 5 g/L . A large amount of rice bran caused excessive fungal growth rather than enhance fumaric acid production . In addition, we could produce fumaric acid without the addition of zinc and iron . Fungal culture broth containing approx 25 g/L of fumaric acid was directly employed for succinic acid conversion . The amount of glycerol and yeast extract required for succinic acid conversion was reduced to 70 and 30%, respectively, compared with the amounts cited in previous studies.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 145 - 59
Screening of Dowex anion-exchange resins for invertase immobilization; Tomotani EJ et al.; Commercial yeast invertase (Bioinvert) was immobilized by adsorption on anion-exchange resins, collectively named Dowex(R) (1x8:50-400, 1x4:50-400, and 1x2:100-400) . Optimal binding was obtained at pH 5.5 and 32 degrees C . Among different polystyrene beads, the complex Dowex-1x4-200/invertase showed a yield coupling and an immobilization coefficient equal to 100% . The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated . The complex Dowex/invertase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form . The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0 . In both cases, the optimal pH values were found in the range of the stability interval . The Km and Vmax for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL.

J Biol Chem, 2004 Jun 11, 279(24), 25374 - 80 Epub 2004 Mar 30.
Growth factor receptor-bound protein 2 interaction with the tyrosine-phosphorylated tail of amyloid beta precursor protein is mediated by its Src homology 2 domain; Zhou D et al.; The sequential processing of the familial disease gene product amyloid beta precursor protein (AbetaPP) by beta- and gamma-secretases generates amyloid beta, which is considered to be the pathogenic factor of Alzheimer's disease, and the AID peptide (AbetaPP intracellular domain) . The AID peptide acts as a positive regulator of apoptosis and modulates transcription and calcium release . To gain clues about the molecular mechanisms regulating the function of AbetaPP and AID, proteins interacting with the AID region of AbetaPP have been isolated using the yeast two-hybrid system . Recent evidence indicates that AbetaPP undergoes post-translational modification events in the AID region and that phosphorylation might regulate its affinity for interacting proteins . To test this possibility and to uncover AbetaPP-binding partners whose interaction depends on AbetaPP phosphorylation, we used a proteomic approach . Here we describe a protein, growth factor receptor-bound protein 2 (Grb2), that specifically binds AbetaPP, phosphorylated in Tyr(682) . Furthermore, we show that this interaction is direct and that Grb2 binds to phospho-AbetaPP via its Src homology 2 region . Together with the evidence that Grb2 is in complex with AbetaPP in human brains and that these complexes are augmented in brains from Alzheimer's cases, our data indicate that Grb2 may mediate some biological and possibly pathological AbetaPP-AID function.

Mol Cell, 2004 Mar 26, 13(6), 766 - 8
Homolog pairing in S . pombe: the ends are the means; Burgess SM; The pairing of homologous chromosomes is a universal feature of meiosis and is important for the accurate segregation of chromosomes in the first of two meiotic divisions . In the March issue of Developmental Cell, report findings in fission yeast which point to telomere clustering and movement as being important determinants of homolog pairing.

Anal Chem, 2004 Apr 1, 76(7), 2071 - 82
Parallel, quantitative measurement of protein binding to a 120-element double-stranded DNA array in real time using surface plasmon resonance microscopy; Shumaker-Parry JS et al.; Quantitative, real-time measurement of kinetics of sequence-specific binding of DNA-binding proteins to double-stranded DNA (dsDNA) immobilized in a 10 x 12 array on a planar gold surface is demonstrated using surface plasmon resonance (SPR) microscopy . This binding of the yeast transcription factor Gal4 to a 120-spot dsDNA array made with alternating 200-microm spots of its dsDNA operator sequence and an unrelated DNA sequence proves that this method could be used to simultaneously monitor the kinetics of binding of proteins to 120 different dsDNA sequences with a sensitivity to approximately 0.5 pg (<2 x 10(7) molecules) of bound protein in each array spot at a time resolution of 1 s . The method is label free and also allows absolute quantitative determination of the binding stoichiometry (i.e., the number of proteins bound per dsDNA) at each time . The dsDNA array was fabricated using a robotic microspotting system to deliver nanoliter droplets of biotinylated dsDNA solutions onto a streptavidin linker layer immobilized with biotinylated alkylthiols on a thin gold film . Simultaneous monitoring of binding to the many array elements allows the use of reference spots (i.e., array elements with unrelated dsDNA sequences) to correct the signal for nonspecific protein-DNA binding and changes in bulk refractive index of the solutions in the SPR microscope's flow cell . This allows high-throughput analyses of the kinetics and equilibrium of protein-dsDNA binding.

Cell Mol Life Sci, 2004 Mar, 61(6), 629 - 40
Transcriptional coregulator SNW/SKIP: the concealed tie of dissimilar pathways; Folk P et al.; Eukaryotic gene expression requires that all the steps of messenger RNA production are regulated in concert to integrate the diverse inputs cells receive . We discuss the functioning of SNW/SKIP, an essential spliceosomal component and transcriptional coregulator, which may provide regulatory coupling of transcription initiation and splicing . SNW/SKIP potentiates the activity of important transcription factors, such as vitamin D receptor, CBF1 (RBP-Jkappa), Smad2/3, and MyoD . It synergizes with Ski in overcoming pRb-mediated cell cycle arrest, and it is targeted by the viral transactivators EBNA2 and E7 . SNW/SKIP may aid in conformational transition of the gene expression machine through its avidity to nuclear matrix fractions or by recruiting foldases such as the prolyl isomerase PPIL1 . The extensive list of SNW/SKIP partners, its unique primary structure, conserved from yeast to humans, and its essential character suggest a distinct function of general importance.

Nat Genet, 2004 Apr, 36(4), 400 - 4 Epub 2004 Mar 28.
Mutations in VPS33B, encoding a regulator of SNARE-dependent membrane fusion, cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome; Gissen P et al.; ARC syndrome (OMIM 208085) is an autosomal recessive multisystem disorder characterized by neurogenic arthrogryposis multiplex congenita, renal tubular dysfunction and neonatal cholestasis with bile duct hypoplasia and low gamma glutamyl transpeptidase (gGT) activity . Platelet dysfunction is common . Affected infants do not thrive and usually die in the first year of life . To elucidate the molecular basis of ARC, we mapped the disease to a 7-cM interval on 15q26.1 and then identified germline mutations in the gene VPS33B in 14 kindreds with ARC . VPS33B encodes a homolog of the class C yeast vacuolar protein sorting gene, Vps33, that contains a Sec1-like domain important in the regulation of vesicle-to-target SNARE complex formation and subsequent membrane fusion.

J Nutr, 2004 Apr, 134(4), 717 - 23
Phosphorylation of eIF2alpha is involved in the signaling of indispensable amino acid deficiency in the anterior piriform cortex of the brain in rats; Gietzen DW et al.; Sensing of indispensable amino acid (IAA) deficiency, an acute challenge to protein homeostasis, is demonstrated by rats as rejection of IAA-deficient diets within 20 min . The anterior piriform cortex (APC) of the brain in rats and birds is essential for this nutrient sensing, and is activated by IAA deficiency . Yet the mechanisms that sense and transduce IAA reduction to signaling in the APC, or indeed in any animal cells, are unknown . Because rejection of a deficient diet within 20 min is too rapid to be explained by transcription-derived signals, brain tissue was taken from rats after 20 min access to either a threonine-basal, -devoid, or -corrected diet and examined for proteins associated with early signaling of IAA deficiency in the yeast model . Western blots and immunohistochemistry showed that the phosphorylation of eukaryotic initiation factor 2-alpha (p-eIF2alpha{Ser51}) and translation of its downstream product, c-Jun, were increased (47%, P < 0.005, and 55%, P < 0.025, respectively) in APC from rats offered devoid, but not corrected diets, compared with those offered basal diets . This was not seen in other brain areas . In cells intensely labeled for cytoplasmic p-eIF2alpha, there was intense fluorescence for c-Jun in the nucleus . Thus, p-eIF2alpha, which is pivotal in the initiation of global protein translation, and its downstream product, the leucine zipper protein, c-Jun, are increased in the mammalian APC within the time frame necessary for the behavioral response . We suggest that p-eIF2alpha and c-Jun participate in signaling nutrient deficiency in the IAA-sensitive neurons of the APC.

J Biol Chem, 2004 Jun 4, 279(23), 24420 - 6 Epub 2004 Mar 29.
Formin homology domain protein (FHOD1) is a cyclic GMP-dependent protein kinase I-binding protein and substrate in vascular smooth muscle cells; Wang Y et al.; Cyclic GMP-dependent protein kinase I (PKGI) mediates vascular relaxation by nitric oxide and related nitrovasodilators and inhibits vascular smooth muscle cell (VSMC) migration . To identify VSMC proteins that interact with PKGI, the N-terminal protein interaction domain of PKGIalpha was used to screen a yeast two-hybrid human aortic cDNA library . The formin homology (FH) domain-containing protein, FHOD1, was found to interact with PKGIalpha in this screen . FH domain-containing proteins bind Rho-family GTPases and regulate actin cytoskeletal dynamics, cell migration, and gene expression . Antisera to FHOD1 were raised and used to characterize FHOD1 expression and distribution in vascular cells . FHOD1 is highly expressed in human coronary artery, aortic smooth muscle cells, and in human arterial and venous endothelial cells . In glutathione S-transferase pull-down experiments, the FHOD1 C terminus (amino acids 964-1165) binds full-length PKGI . Both in vitro and intact cell studies demonstrate that the interaction between FHOD1 and PKGI is decreased 3- to 5-fold in the presence of the PKG activator, 8Br-cGMP . Immunofluorescence studies of human VSMC show that FHOD1 is cytoplasmic and is concentrated in the perinuclear region . PKGI also directly phosphorylates FHOD1, and studies with wild-type and mutant FHOD1-derived peptides identify Ser-1131 in the FHOD1 C terminus as the unique PKGI phosphorylation site in FHOD1 . These studies demonstrate that FHOD1 is a PKGI-interacting protein and substrate in VSMCs and show that cyclic GMP negatively regulates the FHOD1-PKGI interaction . Based on the known functions of FHOD1, the data are consistent with a role for FHOD1 in cyclic GMP-dependent inhibition of VSMC stress fiber formation and/or migration.

Anal Biochem, 2004 Apr 15, 327(2), 176 - 83
High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity; Rowlands MG et al.; The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic . The intrinsic ATPase activity of HSP90 is essential to this activity . HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy . Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described . A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties . A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90 . The Km for ATP determined in the assay was 510+/-70 microM . The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay . The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.

J Invertebr Pathol, 2004 Feb, 85(2), 80 - 8
The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests; Nahar P et al.; The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated . Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer . When grown in a yeast extract-peptone medium, M . anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz . chitin deacetylase (CDA) and chitosanase . Chitinase activity was induced in chitin-containing medium . Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands . SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively . Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected . Following germination of M . anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone . Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan . Hydrolysis of chitin with constitutively produced enzymes of M . anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis . Thus, chitin deacetylase was important in initiating pathogenesis of M . anisopliae softening the insect cuticle to aid mycelial penetration . Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity . Chemical assays of M . anisopliae cell wall composition revealed the presence of chitosan . CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.

Oncogene, 2004 May 13, 23(22), 3915 - 31
Bcl-xES, a BH4- and BH2-containing antiapoptotic protein, delays Bax oligomer formation and binds Apaf-1, blocking procaspase-9 activation; Schmitt E et al.; Bcl-2 family members either negatively or positively regulate the apoptotic threshold of cells . Bcl-xES (extra short), a novel Bcl-x member, possesses a unique combination of BH4 and BH2 domains as well as a COOH-terminal hydrophobic transmembrane anchor domain . Bcl-xES contains sequences of hydrophobic alpha-6 helices but lacks sequences of alpha-5 helices, suggesting that it does not have pore channel-forming activity but functions uniquely as a trapping protein . mRNA expression analysis by reverse transcriptase-polymerase chain reaction and RNase protection assay reveal that Bcl-xES is expressed in a variety of human cancer cell lines and human tumors, including bone marrow from patients with acute lymphoblastic leukemia . Bcl-xES expression is much less pronounced in some specimens of normal human tissues, including the breast, ovary, testis and lung . Stable, transfected human B lymphoma Namalwa variant cells expressing Bcl-xES were derived to investigate its role in apoptosis . Bcl-xES had a preventive effect on cell death induced by tumor necrosis factor-alpha and various concentrations of anticancer drugs, including camptothecin, etoposide and cisplatin . Its protective action on cell death was correlated with the inhibition of mitochondrial cytochrome c release and caspase activation . In a yeast two-hybrid system, Bcl-xES interacted with most Bcl-2 family members, including those containing only a BH3 domain, and with the Ced-4 homolog Apaf-1 . Co-immunoprecipitation and gel filtration chromatography experiments suggest that Bcl-xES delays drug-induced apoptosis by disturbing the formation of Bax oligomers and preventing cytochrome c release, but also by interacting with Apaf-1 and inhibiting procaspase-9 activation, thus averting the apoptogenic proteolytic caspase cascade and cell death.

Protein Eng Des Sel, 2004 Feb, 17(2), 175 - 82 Epub 2004 Feb 20.
Distribution of proline-rich (PxxP) motifs in distinct proteomes: functional and therapeutic implications for malaria and tuberculosis; Ravi Chandra B et al.; We have conducted a survey of proline-rich (PxxP) motifs in the proteomes of human, mouse, yeast, Mycobacterium tuberculosis and Plasmodium falciparum . Our analyses reveal a strikingly high occurrence of these motifs in each organism, suggesting a wide dependence on protein-protein interaction networks in cellular systems . All proteomes considered have an abundance of PxxP motifs which can potentially participate in binding to SH3 domain-containing proteins . A large fraction of these motifs can be assigned to structurally conserved types of class I and class II sequences . We propose that while maintaining the primary biochemical function, many proteins are likely to participate in additional interactions involving molecular cross-talk with other proteins using proline-rich and other motifs . We have also identified PxxP-containing motifs that are unique to P.falciparum and M.tuberculosis . These sequences may serve as leads for the development of peptidomimics that specifically target these organisms . We propose a novel drug target selection strategy where shared PxxP-containing motifs can be used to direct the development of inhibitors that focus on multiple targets in the cell . Screening for such unique PxxP-containing motifs in the P.falciparum proteome yielded highly conserved sequences in the variant surface antigen family that can be used to initiate design of peptidomimics that may potentially abrogate parasite cytoadherence during malaria infections.

Mol Biol Cell, 2004 Jun, 15(6), 2771 - 81 Epub 2004 Mar 26.
AKAP350 interaction with cdc42 interacting protein 4 at the Golgi apparatus; Larocca MC et al.; The A kinase anchoring protein 350 (AKAP350) is a multiply spliced type II protein kinase A anchoring protein that localizes to the centrosomes in most cells and to the Golgi apparatus in epithelial cells . In the present study, we sought to identify AKAP350 interacting proteins that could yield insights into AKAP350 function at the Golgi apparatus . Using yeast two-hybrid and pull-down assays, we found that AKAP350 interacts with a family of structurally related proteins, including FBP17, FBP17b, and cdc42 interacting protein 4 (CIP4) . CIP4 interacts with the GTP-bound form of cdc42, with the Wiscott Aldrich Syndrome group of proteins, and with microtubules, and exerts regulatory effects on cytoskeleton and membrane trafficking . CIP4 is phosphorylated by protein kinase A in vitro, and elevation of intracellular cyclic AMP with forskolin stimulates in situ phosphorylation of CIP4 . Our results indicate that CIP4 interacts with AKAP350 at the Golgi apparatus and that either disruption of this interaction by expressing the CIP4 binding domain in AKAP350, or reduction of AKAP350 expression by RNA interference leads to changes in Golgi structure . The results suggest that AKAP350 and CIP4 influence the maintenance of normal Golgi apparatus structure.

J Virol, 2004 Apr, 78(8), 3851 - 62
RoXaN, a novel cellular protein containing TPR, LD, and zinc finger motifs, forms a ternary complex with eukaryotic initiation factor 4G and rotavirus NSP3; Vitour D et al.; Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery . This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3' consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I . To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein . Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3 . This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions . A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region . In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids . Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions . RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection . Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341) . The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation.

J Virol, 2004 Apr, 78(8), 3805 - 10
Transactivator protein BICP0 of bovine herpesvirus 1 (BHV-1) is blocked by prostaglandin D2 (PGD2), which points to a mechanism for PGD2-mediated inhibition of BHV-1 replication; Saydam O et al.; The immediate-early protein, BICP0, of bovine herpesvirus 1 (BHV-1) transactivates a variety of viral and cellular genes . In a yeast two-hybrid cDNA library screening, we found that lipocalin-type prostaglandin D synthase, which catalyzes the production of prostaglandin D(2) (PGD(2)), is a cellular target of BICP0 . We observed that, during wild-type BHV-1 infection, PGD(2) levels were increased intracellularly and decreased in the medium . These effects were absent upon infection with recombinant BHV-1 expressing beta-galactosidase instead of BICP0 (A2G2) . Transient-expression assays showed that BICP0 alone caused a significant increase in PGD(2) levels in the cell . PGD(2) repressed BHV-1 replication in cultured cells . Antiviral activities of prostaglandins have been documented long ago, but their mode of action remains to be clarified . Here we provide evidence that PGD(2) impairs the transactivation ability of BICP0 that is necessary for efficient virus replication.

J Biol Chem, 2004 Jun 11, 279(24), 25823 - 9 Epub 2004 Mar 26.
Molecular determinants of the interaction of Mad with the PAH2 domain of mSin3; Le Guezennec X et al.; The Sin3 co-repressor acts as a protein scaffold to recruit transcription factors via its four highly homologous paired amphipathic helix (PAH) domains . PAH2 has been shown to interact strongly with the Sin3 interacting domain (SID) of the tumor suppressor Mad . This PAH2/Mad complex has been studied extensively by NMR, but the molecular determinants that dictate the specificity of interaction remain to be elucidated . To uncover residues that convey the specificity of interaction between PAH2 and Mad, PAH2 residues contacted by the Mad-SID were introduced into the PAH1 domain of mSin3b and tested for gain-of-interaction in vivo in a yeast two-hybrid setting and further confirmed in a cell-free system . This approach led to the identification of PAH2-Phe-7 as a critical residue . Stabilization of the interaction between PAH1-Phe-7 and the Mad-SID was achieved by introducing Val-14 and Gln-39 into PAH1 . Substitution of PAH2 residues contacted by the Mad-SID with their respective residues in PAH1 corroborated and extended the critical role of Phe-7 and the stabilizing role of Val-14 and Gln-39 . We conclude that Phe-7 is the critical determinant and provides the molecular specificity for the association between Sin3 and Mad in regulating cell growth and differentiation.

J Biol Chem, 2004 Jun 11, 279(24), 25783 - 8 Epub 2004 Mar 27.
The path of the DNA along the dimer interface of topoisomerase II; Roca J; The eukaryotic DNA topoisomerase II is a dyadic enzyme that, upon ATP binding, transports one duplex DNA (T-segment) through a transient double-stranded break in another (G-segment) . The path of the T-segment involves the sequential crossing of three gates along the dimer interface: the entrance or N-gate, the DNA gate, and the exit or C-gate . Coordination among these gates is critical for dimer stability and the prevention of chromosome damage . This study examines DNA transactions by yeast topoisomerase II derivatives defective in gate function . The results indicate that, although the N-gate is not required for G-segment cleavage, the DNA gate per se is not able to widen unless ATP binds to the N-gate . Next, a captured T-segment cannot be held in the interdomainal region between the N-gate and the DNA gate . Finally, the G-segment can be religated while a T-segment is held in the central cavity of the enzyme between the DNA gate and the C-gate . These quaternary couplings for gate opening and closing suggest that topoisomerase II ensures a transient DNA gating state, during which dimer interface contacts are maximized and backtracking of the transported DNA is minimized.

Biochem Biophys Res Commun, 2004 Apr 23, 317(1), 54 - 9
ERBP, a novel estrogen receptor binding protein enhancing the activity of estrogen receptor; Bu H et al.; To understand the mechanism by which estrogen receptor (ER) activates transcription in a tissue specific fashion, we isolated ERalpha binding protein (ERBP) by performing yeast two-hybrid screening with human mammary gland cDNA library . ERBP is a nuclear protein and its mRNA is ubiquitously expressed . The in vitro interaction of ERBP with ERalpha was demonstrated by GST pull-down assay and this interaction was enhanced by estrogen . In addition, ERBP also bound to PPARgamma, RXRalpha, and ERbeta . ERBP interacted with the DNA binding domain and the hinge region of ERalpha . There are two ERalpha binding regions on ERBP . The binding of ERBP region at C-terminus to ERalpha is increased by estrogen while the binding of ERBP region at N-terminus is not affected by estrogen . The interaction of ERBP with ERalpha was further confirmed in vivo by immunoprecipitation . Transient transfection experiment demonstrated that ERBP enhanced the transcriptional activity of ERalpha.

Syst Appl Microbiol, 2004 Mar, 27(2), 261 - 7
Bee pollen, a substrate that stimulates ochratoxin A production by Aspergillus ochraceus Wilh; Medina A et al.; The capacity of bee pollen as a substrate for production of ochratoxin A (OTA) by a strain of Aspergillus ochraceus was studied . For control purposes corn, wheat and rice grains, and eleven liquid media were assayed . They were Yeast Extract Sucrose broth (YES), YES supplemented with 0.05, 0.1, 0.5, 1 and 5% bee pollen, YES supplemented with 0.5% peptone, 50% must, Wickerham medium, Aflatoxin Production medium and Coconut Broth Medium . Cultures were maintained at 28 degrees C for 4 weeks and were analyzed every seven days for OTA by liquid chromatography with fluorescence detection . OTA production in bee pollen was significantly (P < 0.01) higher than production in corn, wheat and rice grains regardless of incubation time . With regard to liquid cultures, OTA accumulation in YES supplemented with 5% bee pollen was significantly higher than in pollen-free liquid cultures . A positive correlation between the proportion of pollen added to YES medium and OTA level was observed . This is the first report concerning the use of bee pollen as a substrate to stimulate OTA production . On the basis of the preliminary results obtained in this study it can be hypothesized that bee pollen may constitute an important risk factor concerning the presence of OTA in the diet of consumers of that nutritious food.

Arch Virol, 2004 Apr, 149(4), 799 - 807 Epub 2004 Jan 05.
Cloning and sequencing of full-length cDNAs of RNA1 and RNA2 of a Tomato black ring virus isolate from Poland; Jonczyk M et al.; Full-length cDNA clones corresponding to the RNA1 and RNA2 of the Polish isolate MJ of Tomato black ring virus (TBRV, genus Nepovirus) were obtained using a direct recombination strategy in yeast, and their complete nucleotide sequences were established . RNA1 is 7358 nucleotides and RNA2 is 4633 nucleotides in length, excluding the poly(A) tails . Both RNAs contain a single open reading frame encoding polyproteins of 254 kDa and 149 kDa for RNA1 and RNA2 respectively . Putative cleavage sites were identified, and the relationships between TBRV and related nepoviruses were studied by sequence comparison.

PLoS Biol . 2004 May;2(5):E126 . Epub 2004 Mar 23.
Accelerated evolution of the ASPM gene controlling brain size begins prior to human brain expansion; Kouprina N et al.; Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume . The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates . Mutations in ASPM, which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH . Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast . We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change . The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years . Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM . Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals . We report the first direct application of TAR cloning technology to the study of human evolution . Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.

EMBO J, 2004 Apr 21, 23(8), 1857 - 67 Epub 2004 Mar 25.
Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins; Wang C et al.; Mammalian H/ACA small nucleolar RNAs and telomerase RNA share common sequence and secondary structure motifs that form ribonucleoprotein particles (RNPs) with the same four core proteins, NAP57 (also dyskerin or in yeast Cbf5p), GAR1, NHP2, and NOP10 . The assembly and molecular interactions of the components of H/ACA RNPs are unknown . Using in vitro transcription/translation in combination with immunoprecipitation of core proteins, UV-crosslinking, and electrophoretic mobility shift assays, we demonstrate the following . NOP10 associates with NAP57 as a prerequisite for NHP2 binding . Although NHP2 on its own binds RNA nonspecifically, this NAP57-NOP10-NHP2 core trimer specifically recognizes H/ACA RNAs . GAR1 associates independently with NAP57 near the pseudouridylase core of mature H/ACA RNPs . In contrast to other RNPs whose assembly is initiated by protein-RNA interactions, the four H/ACA core proteins form a protein-only particle that associates with H/ACA RNAs . Nonetheless, functional H/ACA snoRNPs assembled in cytosolic extracts are stable and do not exchange their RNA components, suggesting that new particle formation requires de novo synthesis.

EMBO J, 2004 Apr 7, 23(7), 1647 - 56 Epub 2004 Mar 25.
The Arabidopsis cytochrome P450 CYP707A encodes ABA 8'-hydroxylases: key enzymes in ABA catabolism; Kushiro T et al.; The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism . In plants, ABA 8'-hydroxylation is thought to play a predominant role in ABA catabolism . ABA 8'-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified . Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes . These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1-CYP707A4, encode ABA 8'-hydroxylases . Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition . During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed . Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type . These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.

Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 4810 - 4 Epub 2004 Mar 24.
Dynamics of coilin in Cajal bodies of the Xenopus germinal vesicle; Deryusheva S et al.; Cajal bodies (CBs) are complex organelles found in the nuclei of a wide variety of organisms, including vertebrates, invertebrates, plants, and yeast . In most cell types CBs are <1 microm in diameter, severely limiting the range of experimental observations that can be made on them . By contrast, CBs in the amphibian oocyte nucleus (also called the germinal vesicle) are 2-10 microm in diameter . We have taken advantage of this large size to carry out kinetic studies on coilin, a protein that is specifically enriched in CBs . We labeled coilin with photoactivatable green fluorescent protein and analyzed the movement of the protein by confocal microscopy in unfixed germinal vesicles isolated in oil . We showed that coilin leaves the CB relatively slowly (minutes rather than seconds) with kinetics similar to earlier measurements on its entrance . We also showed that coilin diffuses very slowly within the CB, consistent with its being in a large macromolecular complex . Finally, we found that the movement of coilin is not directly affected by the transcriptional state of the nucleus or ongoing nucleocytoplasmic exchange . These data on the kinetics of coilin reinforce the conclusion that CB components are in a constant state of flux, consistent with models that postulate an active role for CBs in nuclear physiology.

J Biol Chem, 2004 May 28, 279(22), 23045 - 51 Epub 2004 Mar 24.
Identification of a major microfibril-associated glycoprotein-1-binding domain in fibrillin-2; Werneck CC et al.; Using yeast two-hybrid, ligand blotting, and solid phase binding assays, we have shown that microfibril-associated glycoprotein-1 (MAGP-1) interacts with the 8-cysteine motif of fibrillin-2 encoded by exon 24 . Binding to this sequence was demonstrated for full-length MAGP-1 as well as for the MAGP-1 matrix-binding domain encoded by exons 7 and 8 . The matrix-binding domain, but not the full-length protein, also bound to regions of fibrillin-2 defined by exons 16 and 17, exon 20, and exons 23 and 24 . Interestingly, no binding was detected to sequences near the N or C terminus where MAGP-1 and MAGP-2, respectively, were shown to interact with fibrillin-1 . The localization of MAGP-1 binding to the 8-Cys domain encoded by exon 24 suggests that the bead structure of microfibrils consists of exon 24 and portions of the central region of fibrillin-2 . Exon 24 in fibrillin lies in the region of the molecule where mutations produce the most severe phenotypes associated with Marfan syndrome (fibrillin-1) and congenital contractural arachnodactyly (fibrillin-2) . It is possible that these mutations alter the ability of fibrillin to bind MAGP-1, which may contribute to the severity of the disease.

Biochem Biophys Res Commun, 2004 Apr 16, 316(4), 1116 - 23
Human MCRS2, a cell-cycle-dependent protein, associates with LPTS/PinX1 and reduces the telomere length; Song H et al.; Human LPTS/PinX1 is a telomerase-inhibitory protein, which binds to the telomere protein Pin2/TRF1 and the catalytic subunit hTERT of telomerase . To explore the proteins that might be involved in the telomerase pathway, we performed a yeast two-hybrid screening with LPTS/PinX1 as the bait . A novel gene, MCRS2, encoding for an isoform of MCRS1/p78 and MSP58 was isolated . The expression of MCRS2 protein is cell-cycle dependent, accumulating in the very early S phase . MCRS2 interacts with LPTS/PinX1 in vitro, in vivo and colocalizes with LPTS/PinX1 in cells . MCRS2 and its amino terminus inhibit telomerase activity in vitro and long-term overexpression of MCRS2 in SMMC-7721 cells results in a gradual and progressive shortening of telomeres . Our findings suggest that MCRS2 might be a linker between telomere maintenance and cell-cycle regulation.

FEBS Lett, 2004 Mar 26, 562(1-3), 118 - 24
Gaining insight into the role of serine 282 in B . napus FAE1 condensing enzyme; Katavic V et al.; To gain some insight whether there is an absolute requirement for the serine 282 to yield a functional fatty acid elongase 1 condensing enzyme we have introduced point mutations in the FAE1 coding sequence which led to the substitution of serine 282 with several aliphatic or aromatic amino acids . The mutated FAE1 polypeptides were expressed in yeast . Gas chromatography analyses of the fatty acid methyl esters from yeast lysates and fatty acid elongase activity assays demonstrated that there is not an absolute requirement for serine at position 282 to yield a functional FAE1 condensing enzyme.

Curr Biol, 2004 Mar 23, 14(6), 458 - 66
A complex of Armadillo, Legless, and Pygopus coactivates dTCF to activate wingless target genes; Thompson BJ; BACKGROUND: Upon receiving a Wnt signal, cells accumulate beta-catenin (Armadillo in Drosophila), which binds directly to TCF transcription factors, leading to the transcription of Wnt target genes . It is generally thought that beta-catenin/Armadillo is a transcriptional coactivator when bound to TCF in the nucleus and that this function is mediated by its C terminus . However, recent findings in Drosophila indicated that Armadillo may activate dTCF in the cytoplasm . RESULTS: Here, I reexamine the mechanism of Armadillo's signaling function in light of Legless and Pygopus, two nuclear factors recently discovered to be essential for this function . I show that Armadillo, in order to activate dTCF, must enter the nucleus and form a complex with Legless and Pygopus . The ability of this complex to stimulate TCF-mediated transcription can be altered by linkage of a strong transcriptional activator or repressor to Armadillo . Furthermore, Armadillo is a strong transcriptional activator when fused to the yeast GAL4 DNA binding domain-an activity that depends on regions of the Armadillo repeat domain that mediate binding to Legless and to chromatin modifying and remodeling factors . Finally, linkage of the N-terminal region of Pygopus, but not the C terminus of Armadillo, to dominant-negative dTCF can restore its signaling activity in transgenic flies . CONCLUSIONS: My evidence argues in favor of a revised coactivator factor model in which Armadillo's coactivator function depends on regions within its Armadillo repeat domain to which Legless/Pygopus and other transcriptional coactivators can bind . In contrast, the C terminus of Armadillo plays a less direct role in this function.

Anal Bioanal Chem, 2004 May, 379(1), 156 - 62 Epub 2004 Mar 24.
Modification of the Limulus amebocyte lysate assay for the analysis of glucan in indoor environments; Foto M et al.; beta-1,3- D-Glucan is a biologically active component mainly from fungi that has been shown in several studies to be related to respiratory health outcomes from damp building exposures . Here, we report the development and application of a method for the analysis of the glucan extracted in 0.5 N NaOH solution making use of an available preparation of Limulus amebocyte lysate (LAL) . The method yields reproducible beta-1,3- D-glucan measurements from samples of outdoor air, yeast cells, fungal spore preparations and ragweed pollen, and is more sensitive than competing measurements . The LAL-based measurement compared favourably to that based on size-exclusion chromatography using UV and refractive index detection . Growth conditions of the fungi did not materially change the concentrations of glucan in spores indicating that this is a stable property . Glucan content was proportional to spore surface area; however, some species contain higher relative spore glucan contents.

Am J Obstet Gynecol, 2004 Mar, 190(3), 663 - 7
Immunoglobulin E antibodies to seminal fluid in women with vulvar vestibulitis syndrome: relation to onset and timing of symptoms; Babula O et al.; OBJECTIVE: Patients with vulvar vestibulitis syndrome and control subjects were tested for evidence of allergy to seminal fluid to differentiate women with a clinical diagnosis of vulvar vestibulitis syndrome into discrete categories . STUDY DESIGN: Plasma samples from 52 women with vulvar vestibulitis syndrome and 43 control subjects were tested for immunoglobulin E antibodies to seminal fluid, total immunoglobulin E, interleukin-4, and interleukin-12 by enzyme-linked immunosorbent assay . Demographic and medical histories were obtained by questionnaire and interview . RESULTS: Sixteen of the patients (30.8%) with vulvar vestibulitis syndrome and 2 control subjects (4.7%) tested positive for immunoglobulin E antiseminal fluid . Symptoms began after sexual intercourse in 43.8% of the women who tested immunoglobulin E positive and 11.1% of the women who tested immunoglobulin E negative (P=.02) . Symptom initiation after a yeast infection was reported by 31.3% of the women who tested immunoglobulin E positive and by 2.8% of the women who tested immunoglobulin E negative (P=.008) . Other symptom-initiating events were reported by 47.2% of the women who tested immunoglobulin E negative and by none of the women who tested immunoglobulin E positive (P=.0008) . Fifty percent of the women who tested immunoglobulin E positive, as opposed to 22.2% of the women who tested immunoglobulin E negative, reported pain only after intercourse (P=.05) . Pain at other times occurred in 50% of the women who tested immunoglobulin E positive and in 72.2% of the women who tested immunoglobulin E negative (P=.001) . There was no relation between immunoglobulin E antiseminal fluid and total immunoglobulin E, interleukin-4,or interleukin-12 . CONCLUSION: A subset of women with vulvar vestibulitis syndrome are sensitized to seminal fluid, and an allergic reaction to seminal fluid may be associated with the initiation and persistence of their symptoms.

Vaccine, 2004 Feb 17, 22(7), 865 - 71
Recombinant antibodies: a natural partner in combinatorial antifungal therapy; Matthews RC et al.; Monotherapy, in the form of amphotericin B or one of its liposomal derivatives, is the usual treatment for invasive fungal infections, due to lack of a safe, effective combination of antifungal drugs . Combination therapy is not necessarily beneficial-there may be mutual antagonism or indifference, increased toxicity or interference with concomitant medication . But the benefits of a well-tolerated, synergistic combination would be great-the enhanced efficacy would improve clinical outcome, reduce the need for prolonged courses of treatment and prevent the emergence of antifungal drug resistance . Antifungal antibodies would be a natural partner in a combinatorial approach to antifungal therapy . Analysis of the antibody response which occurs in patients with invasive candidiasis, being treated with amphotericin B, showed a close correlation between recovery and antibody to the immunodominant heat shock protein 90 (hsp90) . The molecular chaperone hsp90 is essential for yeast viability . Mycograb is a human recombinant antibody to hsp90 which shows intrinsic antifungal activity and synergy with amphotericin B both in vitro and in vivo . It is now the subject of a multinational, double-blind, placebo-controlled trial, in patients with culture-confirmed invasive candidiasis on liposomal amphotericin B.

Nat Rev Mol Cell Biol, 2004 Feb, 5(2), 133 - 47
The dynamin superfamily: universal membrane tubulation and fission molecules?
Praefcke GJ, McMahon HT.
Dynamins are large GTPases that belong to a protein superfamily that, in eukaryotic cells, includes classical dynamins, dynamin-like proteins, OPA1, Mx proteins, mitofusins and guanylate-binding proteins/atlastins . They are involved in many processes including budding of transport vesicles, division of organelles, cytokinesis and pathogen resistance . With sequenced genomes from Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans, yeast species and Arabidopsis thaliana, we now have a complete picture of the members of the dynamin superfamily from different organisms . Here, we review the superfamily of dynamins and their related proteins, and propose that a common mechanism leading to membrane tubulation and/or fission could encompass their many varied functions.

Acta Crystallogr D Biol Crystallogr, 2004 Apr, 60(Pt 4), 770 - 2 Epub 2004 Mar 23.
Crystallization and X-ray analysis of the Y75N mutant of Mucor pusillus pepsin complexed with inhibitor; Badasso MO et al.; Y75N mutant Mucor pusillus pepsin has been overexpressed in yeast, purified and cocrystallized with the iodine-containing human renin inhibitor CP-113972 {(2R,3S}-isopropyl 3-{(L-prolyl-p-iodo-L-phenylalanyl-S-methyl-cysteinyl)amino-4}-cyclohexyl-2-hydroxybutanoate} for X-ray crystallography . Tetragonal complex crystals with space group P4(3)2(1)2 were produced by the hanging-drop vapour-diffusion method and diffracted to 3.0 A . The crystals exhibited unit-cell parameters a = b = 182.5, c = 99.1 A and contained four molecules in the asymmetric unit . A 96% complete data set was collected at 298 K using Cu Kalpha X-rays from a rotating-anode generator . Solution of the crystal structure of Y75N mutant M . pusillus pepsin is under way by molecular replacement using the molecular coordinates of wild-type M . pusillus pepsin as a model.

J Biol Chem, 2004 Jun 18, 279(25), 26433 - 44 Epub 2004 Mar 23.
Hox transcription factor ultrabithorax Ib physically and genetically interacts with disconnected interacting protein 1, a double-stranded RNA-binding protein; Bondos SE et al.; The Hox protein family consists of homeodomain-containing transcription factors that are primary determinants of cell fate during animal development . Specific Hox function appears to rely on protein-protein interactions; however, the partners involved in these interactions and their function are largely unknown . Disconnected Interacting Protein 1 (DIP1) was isolated in a yeast two-hybrid screen of a 0-12-h Drosophila embryo library designed to identify proteins that interact with Ultrabithorax (Ubx), a Drosophila Hox protein . The Ubx.DIP1 physical interaction was confirmed using phage display, immunoprecipitation, pull-down assays, and gel retardation analysis . Ectopic expression of DIP1 in wing and haltere imaginal discs malforms the adult structures and enhances a decreased Ubx expression phenotype, establishing a genetic interaction . Ubx can generate a ternary complex by simultaneously binding its target DNA and DIP1 . A large region of Ubx, including the repression domain, is required for interaction with DIP1 . These more variable sequences may be key to the differential Hox function observed in vivo . The Ubx.DIP1 interaction prevents transcriptional activation by Ubx in a modified yeast one-hybrid assay, suggesting that DIP1 may modulate transcriptional regulation by Ubx . The DIP1 sequence contains two dsRNA-binding domains, and DIP1 binds double-stranded RNA with a 1000-fold higher affinity than either single-stranded RNA or double-stranded DNA . The strong interaction of Ubx with an RNA-binding protein suggests a wider range of proteins may influence Ubx function than previously appreciated.

J Am Chem Soc, 2004 Mar 31, 126(12), 3769 - 76
Probing the transition states of four glucoside hydrolyses with 13C kinetic isotope effects measured at natural abundance by NMR spectroscopy; Lee JK et al.; Kinetic isotope effects (KIEs) were measured for methyl glucoside (4) hydrolysis on unlabeled material by NMR . Twenty-eight (13)C KIEs were measured on the acid-catalyzed hydrolysis of alpha-4 and beta-4, as well as enzymatic hydrolyses with yeast alpha-glucosidase and almond beta-glucosidase . The 1-(13)C KIEs on the acid-catalyzed reactions of alpha-4 and beta-4, 1.007(2) and 1.010(6), respectively, were in excellent agreement with the previously reported values (1.007(1), 1.011(2): Bennet and Sinnott, J . Am . Chem . Soc . 1986, 108, 7287) . Transition state analysis of the acid-catalyzed reactions using the (13)C KIEs, along with the previously reported (2)H KIEs, confirmed that both reactions proceed with a stepwise D(N)A(N) mechanism and showed that the glucosyl oxocarbenium ion intermediate exists in an E(3) sofa or (4)H(3) half-chair conformation . (13)C KIEs showed that the alpha-glucosidase reaction also proceeded through a D(N)*A(N) mechanism, with a 1-(13)C KIE of 1.010(4) . The secondary (13)C KIEs showed evidence of distortions in the glucosyl ring at the transition state . For the beta-glucosidase-catalyzed reaction, the 1-(13)C KIE of 1.032(1) demonstrated a concerted A(N)D(N) mechanism . The pattern of secondary (13)C KIEs was similar to the acid-catalyzed reaction, showing no signs of distortion . KIE measurement at natural abundance makes it possible to determine KIEs much more quickly than previously, both by increasing the speed of KIE measurement and by obviating the need for synthesis of isotopically labeled compounds.

Proc Natl Acad Sci U S A, 2004 Apr 6, 101(14), 4930 - 5 Epub 2004 Mar 22.
A global suppressor motif for p53 cancer mutants; Baroni TE et al.; The transcription factor and tumor suppressor protein p53 is frequently inactivated in human cancers . In many cases, p53 gene mutations result in high levels of inactive, full-length p53 protein with one amino acid change in the core domain that recognizes p53 DNA-binding sites . The ability to endow function to mutated p53 proteins would dramatically improve cancer therapy, because it would reactivate a central apoptotic pathway . By using genetic strategies and p53 assays in yeast and mammalian cells, we identified a global suppressor motif involving codons 235, 239, and 240 . These intragenic suppressor mutations, either alone or in combination, restored function to 16 of 30 of the most common p53 cancer mutants tested . The 235-239-240 suppressor motif establishes that manipulation of a small region of the core domain is sufficient to activate a large number of p53 cancer mutants . Understanding the structural basis of the rescue mechanism will allow the pursuit of small compounds able to achieve a similar stabilization of p53 cancer mutants.

J Biol Chem, 2004 Jul 9, 279(28), 29308 - 19 Epub 2004 Mar 22.
The ORF3 protein of hepatitis E virus interacts with liver-specific alpha1-microglobulin and its precursor alpha1-microglobulin/bikunin precursor (AMBP) and expedites their export from the hepatocyte; Tyagi S et al.; Hepatitis E virus (HEV), a plus-stranded RNA virus contains three open reading frames . Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein and ORF3 codes for a phosphoprotein of undefined function . Using the yeast two-hybrid system to screen a human cDNA liver library we have isolated, an N-terminal deleted protein, alpha(1) -microglobulin/bikunin precursor (AMBP) that specifically interacts with the ORF3 protein of HEV . Independently cloned, full-length AMBP was obtained and tested positive for interaction with ORF3 using a variety of in vivo and in vitro techniques . AMBP, a liver-specific precursor protein codes for two different unrelated proteins alpha(1)-microglobulin (alpha(1)m) and bikunin . alpha(1) m individually interacted with ORF3 . The above findings were validated by COS-1 cell immunoprecipitation, His(6) pull-down experiments, and co-localization experiments followed by fluorescence resonance energy transfer analysis . Human liver cells showing co-localization of ORF3 with endogenously expressing alpha(1) m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of alpha(1)m out of the hepatocyte . Using drugs to block the secretory pathway, we showed that alpha m was not degraded in the presence of ORF3 . Finally, (1)pulse labeling of alpha(1)m showed that its secretion was expedited out of the liver cell at faster rates in the presence of the ORF3 protein . Hence, ORF3 has a direct biological role in enhancing alpha(1)m export from the hepatocyte.

Biochem Pharmacol, 2004 Feb 1, 67(3), 453 - 8
Glycine 154 of the equilibrative nucleoside transporter, hENT1, is important for nucleoside transport and for conferring sensitivity to the inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep; SenGupta DJ et al.; hENT1 and hENT2 are members of the human equilibrative nucleoside transporter family . hENT1 is ubiquitously expressed and plays an important role in the disposition and pharmacological activity of nucleoside drugs and nucleosides, such as adenosine . hENT2 is expressed in only a few tissues (e.g . muscle) . hENT1 and hENT2 differ in their affinity for nucleoside substrates and in their sensitivity to inhibitors, such as nitrobenzylthioinosine (NBMPR) . hENT1 has higher (or equal) affinity to hENT2 for all natural nucleosides except inosine . hENT1 is also more sensitive to NBMPR inhibition (IC50 approximately 0.4-8 nM) when compared with hENT2 (IC50 approximately 2.8 microM) . This difference in inhibition potency is substantially dependent on the difference in amino acid at position 154 in hENT1 (glycine) and hENT2 (serine) . Since NBMPR competitively inhibits nucleoside transporter activity, we hypothesized that G154 may also play a role in the transport of natural nucleosides and in the inhibition by other hENT1 inhibitors, dipyridamole (DP), and dilazep (DZ) . Our results, using a yeast expression system, demonstrate that substituting glycine 154 of hENT1 with serine of hENT2 converts hENT1 to a transporter that exhibits partial characteristics of hENT2 . For example, this conversion reduces sensitivity of hENT1 to the inhibitors NBMPR, DP, and DZ and reduces its transport affinity for the natural nucleosides cytidine and adenosine . However, this conversion renders hENT1 less sensitive to inhibition by anti-HIV drugs azidothymidine, dideoxyinosine, and the nucleobase, hypoxanthine . Collectively, these results suggest that glycine 154 plays an important role in the transport of nucleosides and in sensitivity to the inhibitors NBMPR, DP, and DZ.

Neurosci Lett, 2004 Feb 26, 357(1), 63 - 7
Alzheimer's disease-associated amyloid beta interacts with the human serine protease HtrA2/Omi; Park HJ et al.; Amyloid beta (Abeta), a principle component of the cerebral plaques found in the brains of patients with Alzheimer's disease (AD), is a pivotal factor implicated in the pathogenesis of AD . Recent reports show that not only extracellular Abeta but also intracellular Abeta induces neuronal apoptosis; however, the mechanism remains to be elucidated . Using yeast two-hybrid assays, we found that Abeta interacts with HtrA2/Omi, an essential human serine protease with proapoptotic activity . Additionally, we mapped the C-terminal region containing the PDZ domain of HtrA2/Omi as the binding determinant for Abeta? The interaction of Abeta with HtrA2/Omi was further confirmed through in vivo co-immunoprecipitation assay in HEK293 cells . This study suggests the possibility that the accumulation of intracellular Abeta and a function of proapoptotic protease, HtrA2/Omi are correlated.

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