J Neurosci, 2000 Nov 1, 20(21), 7932 - 40 Regulation of AMPA receptor GluR1 subunit surface expression by a 4 . 1N-linked actin cytoskeletal association; Shen L et al.; The synaptic localization, clustering, and immobilization of neurotransmitter receptors and ion channels play important roles in synapse formation and synaptic transmission . Although several proteins have been identified that interact with AMPA receptors and that may regulate their synaptic targeting, little is known about the interaction of AMPA receptors with the cytoskeleton . In studies examining the interaction of the AMPA receptor GluR1 subunit with neuronal proteins, we determined that GluR1 interacts with the 4.1G and 4.1N proteins, homologs of the erythrocyte membrane cytoskeletal protein 4.1 . Using the yeast two-hybrid system and a heterologous cell system, we demonstrated that both 4.1G and 4.1N bind to a membrane proximal region of the GluR1 C terminus, and that a region within the C-terminal domain of 4.1G or 4.1N is sufficient to mediate the interaction . We also found that 4.1N can associate with GluR1 in vivo and colocalizes with AMPA receptors at excitatory synapses . Disruption of the interaction of GluR1 with 4.1N or disruption of actin filaments decreased the surface expression of GluR1 in heterologous cells . Moreover, disruption of actin filaments in cultured cortical neurons dramatically reduced the level of surface AMPA receptors . These results suggest that protein 4.1N may link AMPA receptors to the actin cytoskeleton. J Biol Chem, 2000 Dec 15, 275(50), 39262 - 6 Filamin (280-kDa actin-binding protein) is a caspase substrate and is also cleaved directly by the cytotoxic T lymphocyte protease granzyme B during apoptosis; Browne KA et al.; We used yeast two-hybrid screening to identify the cytoskeletal protein filamin as a ligand for the proapoptotic protease granzyme B, produced by cytotoxic T lymphocytes . Filamin was directly cleaved by granzyme B when target cells were exposed to granzyme B and the lytic protein perforin, but it was also cleaved in a caspase-dependent manner following the ligation of Fas receptors . A similar pattern of filamin cleavage to polypeptides of approximately 110 and 95 kDa was observed in Jurkat cells killed by either mechanism . However, filamin cleavage in response to granzyme B was not inhibited by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone at concentrations that abolished DNA fragmentation . Filamin staining was redistributed from the cell membrane into the cytoplasm of Jurkat cells exposed to granzyme B and perforin and following ligation of Fas receptors, coincident with the morphological changes of apoptosis . Filamin-deficient human melanoma cells were significantly (although not completely) protected from granzyme B-mediated death compared with isogenic filamin-expressing cells, both in clonogenic survival and (51)Cr release assays, whereas death from multiple other stimuli was not affected by filamin deficiency . Thus, filamin is a functionally important substrate for granzyme B, as its cleavage may account at least partly for caspase-independent cell death mediated by the granzyme. Environ Health Perspect, 2000 Oct, 108(10), 983 - 7 Prediction and assessment of the effects of mixtures of four xenoestrogens; Payne J et al.; The assessment of mixture effects of estrogenic agents is regarded as an issue of high priority by many governmental agencies and expert decision-making bodies all over the world . However, the few mixture studies published so far have suffered from conceptual and experimental problems and are considered to be inconclusive . Here, we report the results of assessments of two-, three- and four-component mixtures of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol, all compounds with well-documented estrogenic activity . Extensive concentration-response analyses with the single agents were carried out using a recombinant yeast screen (yeast estrogen screen, YES) . Based on the activity of the single agents in the YES assay we calculated predictions of entire concentration-response curves for mixtures of our chosen test agents assuming additive combination effects . For this purpose we employed the models of concentration addition and independent action, both well-established models for the calculation of mixture effects . Experimental concentration-response analyses revealed good agreement between predicted and observed mixture effects in all cases . Our results show that the combined effect of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol in the YES assay does not deviate from expected additivity . We consider both reference models as useful tools for the assessment of combination effects of multiple mixtures of xenoestrogens. Neuropeptides, 2000 Oct, 34(5), 292 - 302 Molecular models to analyse preprotachykinin-A expression and function; Quinn JP et al.; Towards an understanding of the mechanisms controlling Preprotachykinin A (PPT) expression we have generated a variety of molecular models to determine the mechanisms regulating both the tissue-specific and stimulus-inducible expression of the PPT gene . The approaches used include transgenic and virus vector models complementing biochemical analysis of promoter interactions with transcription factors . We have identified and characterised a yeast artificial chromosome (YAC) containing the human PPT gene and generated transgenic mouse lines containing multiple copies of this chromosome on a normal mouse genetic background . This resulted in a pattern of expression in the nervous system remarkably similar to that reported for PPT mRNA in rodents . In addition, this transgenic model has been constructed in such a manner to allow for over expression of tachykinins based on the number of extra alleles in the transgenic mouse . These animals allow us to further examine the function of the tachykinins and acts as a useful complement to existing PPT ablated mice . In vitro we have introduced the proximal PPT promoter in reporter gene constructs into adult neurones in both DRG and the CNS by an adenoassociated virus (AAV) vector or by biolistic transfection respectively . Using the AAV vector we have demonstrated that the proximal promoter can mediate the effects of NGF in adult rat DRG . These models allow us to delineate transcriptional domains involved in the physiological and pathological expression of the PPT gene . Biol Trace Elem Res, 2000 Feb, 73(2), 113 - 25 Selenium supplementation of children in a selenium-deficient area in China: blood selenium levels and glutathione peroxidase activities; Alfthan G et al.; Keshan disease is a cardiomyopathy restricted to the endemic areas of China and seen in residents having an extremely low selenium (Se) status . Prophylactic administration of sodium selenite has been shown to decrease significantly the incidence of acute and subacute cases . The aim offthe study was to assess the relative bioavailability of selenite versus organic Se-yeast in a Se-deficient area in China with a randomized double-blind double-dummy design . Healthy children (n=30) between 14 and 16 yr of age were randomized into three equal groups receiving either 200 microg/d selenite Se or 200 microg/d Se-yeast or placebo for 12 wk . Blood was drawn at baseline, 4, 8, and 12 wk and 4 wk postsupplementation . The plasma Se concentration (mean +/- SD) was 0.16+/-0.03 micromol/L at baseline . Selenite and Se-yeast supplementation increased plasma Se to plateau values, 1.0+/-0.2 and 1.3+/-0.2 micromol/L, respectively . In red cells, Se-yeast increased the selenium level sixfold and selenite threefold compared to placebo . The relative bioavailability of Se-yeast versus selenite measured as glutathione peroxidase (GSHPx) activity was similar in plasma, red blood cells, and platelets . GSHPx activity reached maximal levels in plasma and platelets of 300% and 200%, respectively, after 8 wk compared to the placebo group, but continued to increase in red cells for 16 wk . Our study showed that although both forms of Se were equally effective in raising GSHPx activity, Se-yeast provided a longer lasting body pool of Se . Se-yeast may be a better alternative to selenite in the prophylaxis of Keshan disease with respect to building up of body stores. Biol Trace Elem Res, 2000 Apr, 74(1), 55 - 70 Selenium regulates expression in rat liver of genes for proteins involved in iron metabolism; Christensen MJ et al.; Suppression subtractive hybridization analysis in our laboratory recently revealed that transferrin mRNA may be elevated in Sedeficient rat liver . In this work, we compared expression in rat liver of genes for transferrin, transferrin receptor, ferritin light and heavy chains, and iron-regulatory proteins 1 and 2 in Se adequacy and deficiency . Weanling male Sprague-Dawley rats were fed Torula yeast diets supplemented with 0 or 0.15 microg Se/kg diet as sodium selenite for 15 wk . Activity of cellular glutathione peroxidase was virtually abolished in Se-deficient rat liver, whereas activity of glutathione S-transferase was 43% higher than in Se-adequate liver . There were no differences in hematocrit, hemoglobin, or liver iron content . To examine differential gene expression, we used a multiplex relative reverse transcriptase-polymerase chain reaction method . Three of the six genes examined showed modest but consistent upregulation in Se deficiency . Transferrin mRNA was 30% more abundant in Se-deficient than in Se-adequate liver . For the transferrin receptor, the difference was 32%, and for iron regulatory protein 1, it was 63% . No consistent differences were observed for iron regulatory protein 2 or for ferritin light or heavy chain . These findings suggest a possible role for dietary Se in moderating iron metabolism. Ceska Gynekol, 1999 Sep, 64(5), 316 - 22 {Diagnosis and treatment of chronic vaginal candidiasis}; Spott J et al.; OBJECTIVE OF STUDY: The objective of the study is to determine the optimum diagnostics of vaginal yeast infections and to compare the effects of treatment of these infections by Natamycin with those by Clotrimazol . TYPE OF STUDY: Prospective comparison of both option of treatment of vaginal infections . NAME AND PLACE OF RESEARCH: Obstetrics and gynaecology department, Brno-Bohunice . METHODS: 30 patients treated with hydrophobe Natamycin and 20 patients treated with hydrophyll Clotrimazol formed a sample of 50 women . Regular checks were made on the 10th and 30th day after the beginning of treatment . Diagnosis was performed by means of native microscopy supplemented by an examination for cultivated yeasts in the culture medium "FUNGI-QUICK" . At the same time a microscopic examination of slides stained by Gram and Giems was made . RESULTS: A correlation between the evaluated native slide and the culture examined thereafter was 96% . Statistical evaluation of the difference of the rate of success of treatment between the two groups by means of the t-test revealed a value of 0.29, the level of probability was 0.05 at N1 = 0.1795 and N2 = 0.2179 . CONCLUSION: Native microscopy is irreplaceable in the diagnosis of vaginal candidosis . No significant differences in the effects of treatment with Natamycin and Clotrimazol were found . On the basis of these results we made some recommendations on the principles of optimum treatment. Mol Cell Biol, 2000 Nov, 20(22), 8613 - 22 A family of LIM-only transcriptional coactivators: tissue-specific expression and selective activation of CREB and CREM; Fimia GM et al.; Transcription factors of the CREB family control the expression of a large number of genes in response to various signaling pathways . Regulation mediated by members of the CREB family has been linked to various physiological functions . Classically, activation by CREB is known to occur upon phosphorylation at an essential regulatory site (Ser133 in CREB) and the subsequent interaction with the ubiquitous coactivator CREB-binding protein (CBP) . However, the mechanism by which selectivity is achieved in the identification of target genes, as well as the routes adopted to ensure tissue-specific activation, remains unrecognized . We have recently described the first tissue-specific coactivator of CREB family transcription factors, ACT (activator of CREM in testis) . ACT is a LIM-only protein which associates with CREM in male germ cells and provides an activation function which is independent of phosphorylation and CBP . Here we characterize a family of LIM-only proteins which share common structural organization with ACT . These are referred to as four-and-a-half-LIM-domain (FHL) proteins and display tissue-specific and developmentally regulated expression . FHL proteins display different degrees of intrinsic activation potential . They provide powerful activation function to both CREB and CREM when coexpressed either in yeast or in mammalian cells, specific combinations eliciting selective activation . Deletion analysis of the ACT protein shows that the activation function depends on specific arrangements of the LIM domains, which are essential for both transactivation and interaction properties . This study uncovers the existence of a family of tissue-specific coactivators that operate through novel, CBP-independent routes to elicit transcriptional activation by CREB and CREM . The future identification of additional partners of FHL proteins is likely to reveal unappreciated aspects of tissue-specific transcriptional regulation. Mol Cell Biol, 2000 Nov, 20(22), 8382 - 9 ERK5 is a novel type of mitogen-activated protein kinase containing a transcriptional activation domain; Kasler HG et al.; Previous studies have shown that upregulation of the orphan steroid receptor Nur77 is required for the apoptosis of immature T cells in response to antigen receptor signals . Transcriptional upregulation of Nur77 in response to antigen receptor signaling involves two binding sites for the MEF2 family of transcription factors located in the Nur77 promoter . Calcium signals greatly increase the activity of MEF2D in T cells via a posttranslational mechanism . The mitogen-activated protein (MAP) kinase ERK5 was isolated in a yeast two-hybrid screen using the MADS-MEF2 domain of MEF2D as bait . ERK5 resembles the other MAP kinase family members in its N-terminal half, but it also contains a 400-amino-acid C-terminal domain of previously uncharacterized function . We report here that the C-terminal region of ERK5 contains a MEF2-interacting domain and, surprisingly, also a potent transcriptional activation domain . These domains are both required for coactivation of MEF2D by ERK5 . The MEF2-ERK5 interaction was found to be activation dependent in vivo and inhibitable in vitro by the calcium-sensitive MEF2 repressor Cabin 1 . The transcriptional activation domain of ERK5 is required for maximal MEF2 activity in response to calcium flux in T cells, and it can activate the endogenous Nur77 gene when constitutively recruited to the Nur77 promoter via MEF2 sites . These studies provide insights into a mechanism whereby MEF2 activity can respond to calcium signaling and suggest a novel, unexpected mechanism of MAP kinase function. Protein Sci, 2000 Sep, 9(9), 1743 - 52 Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription; Chen HT et al.; Transcription factor IIB (TFIIB) is an essential component in the formation of the transcription initiation complex in eucaryal and archaeal transcription . TFIIB interacts with a promoter complex containing the TATA-binding protein (TBP) to facilitate interaction with RNA polymerase II (RNA pol II) and the associated transcription factor IIF (TFIIF) . TFIIB contains a zinc-binding motif near the N-terminus that is directly involved in the interaction with RNA pol II/TFIIF and plays a crucial role in selecting the transcription initiation site . The solution structure of the N-terminal residues 2-59 of human TFIIB was determined by multidimensional NMR spectroscopy . The structure consists of a nearly tetrahedral Zn(Cys)3(His)1 site confined by type I and "rubredoxin" turns, three antiparallel beta-strands, and disordered loops . The structure is similar to the reported zinc-ribbon motifs in several transcription-related proteins from archaea and eucarya, including Pyrococcus furiosus transcription factor B (PfTFB), human and yeast transcription factor IIS (TFIIS), and Thermococcus celer RNA polymerase II subunit M (TcRPOM) . The zinc-ribbon structure of TFIIB, in conjunction with the biochemical analyses, suggests that residues on the beta-sheet are involved in the interaction with RNA pol II/TFIIF, while the zinc-binding site may increase the stability of the beta-sheet. J Virol, 2000 Nov, 74(22), 10819 - 21 The genomic RNA in Ty1 virus-like particles is dimeric; Feng YX et al.; The yeast retrotransposon Ty1 resembles retroviruses in a number of important respects but also shows several fundamental differences from them . We now report that, as in retroviruses, the genomic RNA in Ty1 virus-like particles is dimeric . The Ty1 dimers also resemble retroviral dimers in that they are stabilized during the proteolytic maturation of the particle . The stabilization of the dimer suggests that one of the cleavage products of TyA1 possesses nucleic acid chaperone activity. J Biol Chem, 2001 Jan 12, 276(2), 1585 - 93 Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins; Bernard ML et al.; AGS3 (activator of G-protein signaling 3) was isolated in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins . As an initial approach to define the role of AGS3 in mammalian signal processing, we defined the AGS3 subdomains involved in G-protein interaction, its selectivity for G-proteins, and its influence on the activation state of G-protein . Immunoblot analysis with AGS3 antisera indicated expression in rat brain, the neuronal-like cell lines PC12 and NG108-15, as well as the smooth muscle cell line DDT(1)-MF2 . Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell . AGS3 coimmunoprecipitated with Galpha(i3) from cell and tissue lysates, indicating that a subpopulation of AGS3 and Galpha(i) exist as a complex in the cell . The coimmunoprecipitation of AGS3 and Galpha(i) was dependent upon the conformation of Galpha(i3) (GDP GTPgammaS (guanosine 5'-3-O-(thio)triphosphate)) . The regions of AGS3 that bound Galpha(i) were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro(463)-Ser(650)), each of which were capable of binding Galpha(i) . AGS3-GPR domains selectively interacted with Galpha(i) in tissue and cell lysates and with purified Galpha(i)/Galpha(t) . Subsequent experiments with purified Galpha(i2) and Galpha(i3) indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one Galpha(i) subunit at the same time . The AGS3-GPR domains effectively competed with Gbetagamma for binding to Galpha(t(GDP)) and blocked GTPgammaS binding to Galpha(i1) . AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways. Genome Res, 2000 Oct, 10(10), 1445 - 52 HEAT repeats associated with condensins, cohesins, and other complexes involved in chromosome-related functions; Neuwald AF et al.; HEAT repeats correspond to tandemly arranged curlicue-like structures that appear to serve as flexible scaffolding on which other components can assemble . Using sensitive sequence analysis techniques we detected HEAT repeats in various chromosome-associated proteins, including four families of proteins associated with condensins and cohesins, which are nuclear complexes that contain structural maintenance of chromosome (SMC) proteins . Among the proteins detected were the XCAP-D2 and XCAP-G subunits of the Xenopus laevis 13S condensin complex, the Aspergillus BimD and Sordaria macrospora Spo76p proteins, the budding yeast Scc2p protein, and the related Drosophila transcriptional activator Nipped-B . Clathrin adaptor and COP-I coatomer subunits, which function in vesicle coat assembly and were previously noted to share weak sequence similarity to condensin subunits, also contain HEAT repeats . HEAT repeats were also found in the TBP-associated TIP120 protein, a global enhancer of transcription, and in the budding yeast Mot1p protein, which is a member of the SWI2/SNF2 family . SWI2/SNF2 proteins, some of which are helicases, perform diverse roles in transcription control, DNA repair, and chromosome segregation and form chromatin-remodeling complexes . HEAT repeats also were found in dis1-TOG family and cofactor D family microtubule-associated proteins, which, owing to their roles in microtubule dynamics, perform functions related to mitotic progression and chromosome segregation . Hence, our analysis predicts structural features of these proteins and suggests that HEAT repeats may play important roles in chromosome dynamics. J Biol Chem, 2001 Feb 16, 276(7), 5152 - 65 Epub 2000 Oct 19. Proteomics characterization of abundant Golgi membrane proteins; Bell AW et al.; A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34) . The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins . Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs . Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin . Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking. Plant Cell, 2000 Oct, 12(10), 1893 - 902 GRCD1, an AGL2-like MADS box gene, participates in the C function during stamen development in Gerbera hybrida; Kotilainen M et al.; Despite the differences in flower form, the underlying mechanism in determining the identity of floral organs is largely conserved among different angiosperms, but the details of how the functions of A, B, and C are specified varies greatly among plant species . Here, we report functional analysis of a Gerbera MADS box gene, GRCD1, which is orthologous to AGL2-like MADS box genes . Members of this group of genes are being reported in various species in growing numbers, but their functions remained largely unsettled . GRCD1 expression is detected in all four whorls, but the strongest signal is seen in the developing stamen and carpel . Downregulating GRCD1 expression by antisense transformation revealed that lack of GRCD1 caused homeotic changes in one whorl only: sterile staminodes, which normally develop in whorl 3 of marginal female florets, were changed into petals . This indicates that the GRCD1 gene product is active in determining stamen identity . Transgenic downregulation of GRCD1 causes a homeotic change similar to that in the downregulation of the Gerbera C function genes GAGA1 and GAGA2, but one that is limited to whorl 3 . Downregulation of GRCD1 expression does not reduce expression of GAGA1 or GAGA2, or vice versa; and in yeast two-hybrid analysis, GRCD1 is able to interact with GAGA1 and GAGA2 . We propose that a heterodimer between the GRCD1 and GAGA1/2 gene products is needed to fulfill the C function in whorl 3 in Gerbera. Biochemistry, 2000 Oct 24, 39(42), 12939 - 52 Kinetic characterization of the second step of group II intron splicing: role of metal ions and the cleavage site 2'-OH in catalysis; Gordon PM et al.; The ai5gamma group II intron from yeast excises itself from precursor transcripts in the absence of proteins . When a shortened form of the intron containing all but the 3'-terminal six nucleotides is incubated with an exon 1 oligonucleotide and a 3' splice site oligonucleotide, a nucleotidyl transfer reaction occurs that mimics the second step of splicing . As this tripartite reaction provides a means to identify important functional groups in 3' splice site recognition and catalysis, we establish here a minimal kinetic framework and demonstrate that the chemical step is rate-limiting . We use this framework to characterize the metal ion specificity switch observed previously upon sulfur substitution of the 3'-oxygen leaving group and to elucidate by atomic mutagenesis the role of the neighboring 2'-OH in catalysis . The results suggest that both the 3'-oxygen leaving group and the neighboring 2'-OH are important ligands for metal ions in the transition state but not in the ground state and that the 2'-OH may play an additional role in transition state stabilization by donating a hydrogen bond . Metal specificity switch experiments combined with quantitative analysis show that the Mn(2+) that interacts with the leaving group binds to the ribozyme with the same affinity as the metal ion that interacts with the neighboring 2'-OH, raising the possibility that a single metal ion mediates interactions with the 2'- and 3'-oxygen atoms at the 3' splice site. Dev Growth Differ, 2000 Oct, 42(5), 507 - 17 Direct molecular interaction of a conserved yolk granule protein in sea urchins; Wessel GM et al.; The regulation of yolk storage in oocytes and subsequent utilization in embryos is critical for embryogenesis . In sea urchins, the major yolk protein is made in the intestines, transported to the ovaries and accumulated in developing oocytes within membrane-bound vesicles comprising approximately 10% of the mass of an egg . Here, a non-yolk protein that accumulates specifically in yolk granules is reported . This protein was identified by cDNA cloning and, by use of antibodies to the recombinant protein, it was shown that this molecule is stored selectively in yolk granules of oocytes and embryos . No accumulation was seen in the accessory cells, testis, or intestines . In situ ribonucleic acid (RNA) hybridizations showed that the transcript accumulated only in oocytes, and was more highly concentrated in young oocytes . However, later in oogenesis, the messenger ribonucleic acid (mRNA) levels decreased significantly so that no signal was detectable in mature haploid eggs or at any later stage in development . However, by immunofluorescence and western blot analysis, the 30 kDa band was present throughout development . The predicted sequence of this protein shows that it is a member of the bep, HLC-32, EBP family of sea urchin proteins, but as it does not accumulate at the cell surface, nor in the hyaline layer in the two species studied here, as do other members of the family, it has been referred to as YP30 (30 kDa protein of the yolk platelet) . To address its potential function, yeast two-hybrid analysis was performed to screen for proteins that potentially interact with YP30 . It was found that it binds itself, and forms strongly interacting dimers . It is hypothesized that YP30 participates in the packaging and storage of major yolk protein during oogenesis, or in the utilization of the major yolk protein in development. Mycopathologia, 1999, 147(3), 139 - 48 Effect of climatic conditions on natural mycoflora and fumonisins in freshly harvested corn of the State of Paraná, Brazil; Ono EY et al.; Natural mycoflora associated with fumonisins were analyzed in 150 samples of freshly harvested corn from Central-Southern, Central-Western and Northern regions of the State of Parana, Brazil and correlated to climatic conditions . The corn samples were frequently contaminated with Fusarium sp . (98.7 to 100%) and Penicillium sp . (93 to 100%), when compared to Aspergillus sp . (not detected to 27.7%) . The highest contamination with potentially mycotoxigenic fungi occurred in corn harvested in the Central-Western region, where total mould and yeast counts ranged from 5.5 x 10(3) to 5.2 x 10(6) CFU/g, with 98.7% contaminated by Fusarium sp . and 93% by Penicillium sp . In this region F . moniliforme (F . verticillioides) was the predominant Fusarium sp., and was isolated in 85.9% of the samples . Aspergillus sp . was isolated from 27.7% samples . FB1 was detected in 100% of the samples (mean of 2.39 micrograms/g) and FB2 in 97.7% (mean of 1.09 micrograms/g) . Fumonisins were also detected in all samples from Northern region, with mean of 4.56 micrograms/g (FB1) and 2.20 micrograms/g (FB2) . Considering 1.0 microgram/g as the threshold, 72% of the corn samples from the Central-West and 92% from the North were contaminated with concentrations above this value, in contrast to a 18.5% contamination rate from Central-Southern samples . Between corn planting to harvesting season, the average maximum temperature and relative humidity were 26 degrees C and 77.1% (Central-Southern), 27 degrees C and 69% (Northern) and 29.9 degrees C and 89.1% (Central-Western) . Therefore, the higher fumonisins contamination of corn from Northern region when compared to the Central-South were due to the differences in rainfall levels (92.8 mm in Central-Southern, 202 mm in Northern) during the month preceding harvest. Virology, 2000 Oct 25, 276(2), 424 - 34 Pseudosubstrate inhibition of protein kinase PKR by swine pox virus C8L gene product; Kawagishi-Kobayashi M et al.; The interferon-induced protein kinase PKR is activated upon binding double-stranded RNA and phosphorylates the translation initiation factor eIF2alpha on Ser-51 to inhibit protein synthesis in virally infected cells . Swinepox virus C8L and vaccinia virus K3L gene products structurally resemble the amino-terminal third of eIF2alpha . We demonstrate that the C8L protein, like the K3L protein, can reverse the toxic effects caused by high level expression of human PKR in yeast cells . In addition, expression of either the K3L or C8L gene product was found to reverse the inhibition of reporter gene translation caused by PKR expression in mammalian cells . The inhibitory function of the K3L and C8L gene products in these assays was found to be critically dependent on residues near the carboxyl-termini of the proteins including a sequence motif shared among eIF2alpha and the C8L and K3L gene products . Thus, despite significant sequence differences both the C8L and K3L proteins function as pseudosubstrate inhibitors of PKR . Prenat Diagn, 2000 Oct, 20(10), 842 - 6 Incidental prenatal detection of an Xp deletion using an anonymous primer pair for fetal sexing; Jakubiczka S et al.; We report on the incidental prenatal detection of an interstitial X-chromosomal deletion in a male fetus and his mother by fetal sexing with a primer pair recognizing an X-Y homologous locus (DXYS19), formerly unassigned on the X chromosome . The proband asked for prenatal diagnosis because of her elevated age and risk of Duchenne muscular dystrophy (DMD) . Prior to molecular genetic testing for DMD, fetal sexing was carried out on DNA prepared from cultured amniocytes . PCR analysis revealed the expected Y-chromosomal product, but did not show the constitutive X-chromosomal fragment . The absence of the X-chromosomal fragment in the fetus and on one X chromosome of the mother was confirmed by Southern hybridization of HindIII restricted DNA with probe pJA1165 (DXYS19) . DXYS19X was mapped to Xp22.3 by combining several approaches, including: (1) analysis of somatic cell hybrid lines containing different fragments of the human X chromosome; (2) Southern hybridization of a yeast artificial chromosome (YAC)-filter panel provided by the Resource Center/Primary Database (RZPD); (3) FISH analysis; and (4) re-evaluation of two patients with interstitial deletions in Xp22.3 . The extent of the deletion in the fetus was estimated by further markers from Xp22.3 and found to include the STS gene . Mental retardation could not be excluded since some mentally retarded patients exhibit overlapping deletions . Exp Parasitol, 2000 Sep, 96(1), 23 - 31 Trypanosoma cruzi: cloning and characterization of a RAB7 gene; Leal ST et al.; The small monomeric GTP-binding proteins of the RAB subfamily are key regulatory elements of the machinery that controls membrane traffic in eukaryotic cells . These proteins have been localized to many different intracellular organelles, on both endocytic and exocytic compartments, suggesting that each step of vesicular traffic can involve a specific RAB protein . The presence of conserved amino acid domains in these proteins has allowed the cloning of their genes from several organisms, including yeast, plants, humans, and parasites . In this work we describe the identification, cloning, and characterization of a RAB7 gene homologue in Trypanosoma cruzi (TcRAB7) . Our data indicate that this gene is present as a single copy in the T . cruzi genome, located on a 2.25-Mb chromosomal DNA . TcRAB7 is expressed in T . cruzi epimastigotes, metacyclic trypomastigotes, and spheromastigotes . We established transformed cell lines that express two versions of an epitope-tagged TcRAB7 protein: one wild type (pTAG) and one deleted at the C-terminal cysteines (pDeltaCXC) . Wild-type TcRAB7 protein (pTAG) appears to be localized exclusively in the membrane fraction, while the mutated TcRAB7 protein (pDeltaCXC) loses the ability to associate with the membrane, showing only cytosolic localization . Also, we produced the recombinant TcRAB7 protein and demonstrated that it binds GTP . The identification of exo- and endocytic machinery components in T . cruzi and their function would provide specific markers of these subcellular compartments, thereby unveiling important aspects of vesicular traffic in this parasite . J Biol Chem, 2001 Jan 19, 276(3), 1881 - 8 Epub 2000 Oct 18. Transcription factors Zic1 and Zic2 bind and transactivate the apolipoprotein E gene promoter; Salero E et al.; We have used the yeast one-hybrid system to identify transcription factors that bind to specific sequences in proximal regions of the apolipoprotein E gene promoter . The sequence between -163 and -124, that has been previously defined as a functional promoter element, was used as a bait to screen a human brain cDNA library . Ten cDNA clones that encoded portions of the human Zic1 (five clones) and Zic2 (five clones) transcription factors were isolated . Electrophoretic mobility shift assays confirmed the presence of a binding site for Zic1 and Zic2 in the -136/-125 region . Displacement of binding with oligonucleotides derived from adjacent sequences within the APOE promoter revealed the existence of two additional Zic-binding sequences in this promoter . These sequences were identified by electrophoretic mobility shift assays and mutational analysis in regions -65/-54 and -185/-174 . Cotransfection of Zic1 and Zic2 expression vector and different APOE promoter-luciferase reporter constructs in U87 glioblastoma cell line showed that the three binding sites partially contributed to the trans-stimulation of the luciferase reporter . Ectopic expression of Zic1 and Zic2 in U87 cells also trans-stimulated the expression of the endogenous gene, increasing the amount of apolipoprotein E produced by glial cells . These data indicate that Zic proteins might contribute to the transcriptional activity of the apolipoprotein E gene and suggest that apolipoprotein E could mediate some of the developmental processes in which Zic proteins are involved. J Cell Biol, 2000 Oct 16, 151(2), 235 - 48 Indications for a novel muscular dystrophy pathway . gamma-filamin, the muscle-specific filamin isoform, interacts with myotilin; van der Ven PF et al.; gamma-Filamin, also called ABP-L, is a filamin isoform that is specifically expressed in striated muscles, where it is predominantly localized in myofibrillar Z-discs . A minor fraction of the protein shows subsarcolemmal localization . Although gamma-filamin has the same overall structure as the two other known isoforms, it is the only isoform that carries a unique insertion in its immunoglobulin (Ig)-like domain 20 . Sequencing of the genomic region encoding this part of the molecule shows that this insert is encoded by an extra exon . Transient transfections of the insert-bearing domain in skeletal muscle cells and cardiomyocytes show that this single domain is sufficient for targeting to developing and mature Z-discs . The yeast two-hybrid method was used to identify possible binding partners for the insert-bearing Ig-like domain 20 of gamma-filamin . The two Ig-like domains of the recently described alpha-actinin-binding Z-disc protein myotilin were found to interact directly with this filamin domain, indicating that the amino-terminal end of gamma-filamin may be indirectly anchored to alpha-actinin in the Z-disc via myotilin . Since defects in the myotilin gene were recently reported to cause a form of autosomal dominant limb-girdle muscular dystrophy, our findings provide a further contribution to the molecular understanding of this disease. J Biol Chem, 2001 Jan 12, 276(2), 910 - 4 Identification of an inhibitor of hsc70-mediated protein translocation and ATP hydrolysis; Fewell SW et al.; Members of the hsc70 family of molecular chaperones are critical players in the folding and quality control of cellular proteins . Because several human diseases arise from defects in protein folding, the activity of hsc70 chaperones is a potential therapeutic target for these disorders . By using a known hsc70 modulator, 15-deoxyspergualin, as a seed, we identified a novel inhibitor of hsc70 activity . This compound, R/1, inhibits the endogenous and DnaJ-stimulated ATPase activity of hsc70 by 48 and 51%, respectively, and blocks the hsc70-mediated translocation of a preprotein into yeast endoplasmic reticulum-derived microsomal vesicles . Biochemical studies demonstrate that R/1 most likely exerts these effects by altering the oligomeric state of hsc70. Proc Natl Acad Sci U S A, 2000 Oct 24, 97(22), 12239 - 43 Frataxin activates mitochondrial energy conversion and oxidative phosphorylation; Ristow M et al.; Friedreich's ataxia (FA) is an autosomal recessive disease caused by decreased expression of the mitochondrial protein frataxin . The biological function of frataxin is unclear . The homologue of frataxin in yeast, YFH1, is required for cellular respiration and was suggested to regulate mitochondrial iron homeostasis . Patients suffering from FA exhibit decreased ATP production in skeletal muscle . We now demonstrate that overexpression of frataxin in mammalian cells causes a Ca(2+)-induced up-regulation of tricarboxylic acid cycle flux and respiration, which, in turn, leads to an increased mitochondrial membrane potential (delta psi(m)) and results in an elevated cellular ATP content . Thus, frataxin appears to be a key activator of mitochondrial energy conversion and oxidative phosphorylation. J Biol Chem, 2001 Jan 5, 276(1), 172 - 8 Regulation of DNA binding and trans-activation by a xenobiotic stress-activated plant transcription factor; Johnson C et al.; As-1-type cis-elements augment transcription of both nuclear and pathogen genes in response to stress and defense cues in plants . Basic/leucine zipper proteins termed "TGA factors" that specifically bind as-1 elements are likely candidates for mediating these transcription activities . Our earlier work has shown that 2, 4-dichlorophenoxyacetic acid-induced xenobiotic stress enhances trans-activation by a chimeric fusion protein of the yeast Gal4 binding domain and TGA1a, a TGA factor of tobacco . Here we demonstrate that xenobiotic stress also enhances the ability of native TGA1a to bind as-1 and activate transcription of a known target gene . In addition, the previously identified xenobiotic stress-responsive domain of TGA1a was found to inhibit this factor's trans-activation potential by a mechanism that appears to involve stimulus-reversible interactions with a nuclear repressor protein . Results from these and other studies can now be placed in the context of a working model to explain basal and xenobiotic stress-induced activities of TGA1a through its cognate cis-acting element. J Cell Sci, 2000 Nov, 113 Pt 21, 3825 - 37 The C . elegans septin genes, unc-59 and unc-61, are required for normal postembryonic cytokineses and morphogenesis but have no essential function in embryogenesis; Nguyen TQ et al.; Septins have been shown to play important roles in cytokinesis in diverse organisms ranging from yeast to mammals . In this study, we show that both the unc-59 and unc-61 loci encode Caenorhabditis elegans septins . Genomic database searches indicate that unc-59 and unc-61 are probably the only septin genes in the C . elegans genome . UNC-59 and UNC-61 localize to the leading edge of cleavage furrows and eventually reside at the midbody . Analysis of unc-59 and unc-61 mutants revealed that each septin requires the presence of the other for localization to the cytokinetic furrow . Surprisingly, unc-59 and unc-61 mutants generally have normal embryonic development; however, defects were observed in post-embryonic development affecting the morphogenesis of the vulva, male tail, gonad, and sensory neurons . These defects can be at least partially attributed to failures in post-embryonic cytokineses although our data also suggest other possible roles for septins . unc-59 and unc-61 double mutants show similar defects to each of the single mutants. FEBS Lett, 2000 Sep 1, 480(2-3), 283 - 6 Ulip6, a novel unc-33 and dihydropyrimidinase related protein highly expressed in developing rat brain; Horiuchi M et al.; Here, we report the identification of Ulip6, a novel unc-33 and dihydropyrimidinase related protein that belongs to the Ulip/CRMP protein family . Ulip6 was found in a yeast two-hybrid screen using the neuronal glycine transporter GlyT2 as bait . The rat and human Ulip6 sequences are highly homologous and most closely related to the liver enzyme dihydropyrimidinase (Ulip5) . Northern and Western analysis of rat tissues revealed that the distribution of the Ulip6 mRNA and protein resembles those of brain-type Ulip proteins . Like Ulip1-4, Ulip6 is highly expressed in embryonic and early postnatal brain and spinal cord . These findings are consistent with Ulip6 having a function in neuronal differentiation and/or axon growth. FEBS Lett, 2000 Sep 1, 480(2-3), 201 - 7 N-terminal and core-domain random mutations in human topoisomerase II alpha conferring bisdioxopiperazine resistance; Jensen LH et al.; Random mutagenesis of human topoisomerase II alpha cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance . The mutations L169F, G551S, P592L, D645N, and T996L confer > 37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively . Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not . This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs . In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds. Mol Cell Biol Res Commun, 2000 Jun, 3(6), 367 - 73 Repression of transcription by HoxC11 upon phorbol ester stimulation; Sur IP et al.; Hox genes encode transcription factors with a conserved DNA-binding domain and exhibit similar DNA-binding preferences . The in vivo specificity required for their biological function is brought about by combinatorial interactions with other factors . Such interactions also modulate their activation state . Here we show that HoxC11 can either activate or repress transcription in a signal-specific manner . We report the isolation of HoxC11 in a yeast one-hybrid screen for factors binding to a phorbol-ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) response element (VLTRE), which is also a target for TPA-induced binding of Rel factors in gel-shift experiments . Although we detect no binding of in vitro translated HoxC11 to the TPA response element in EMSA, overexpression of HoxC11 in the HepG2 cell line leads to a complete block of TPA-induced transcription from a VLTRE-luciferase reporter . There is, however, no repression of the basal levels . The repression is furthermore not dependent on homeo-domain DNA binding . Our data suggest an interaction of HoxC11 with the basal-transcription machinery . We propose that HoxC11 is capable of mediating transcriptional activation or repression in a signal-specific manner and that its activation of the DNA target sequence in yeast might reflect in vivo recruitment to the promoter complex . Oncogene, 2000 Sep 28, 19(41), 4706 - 12 Identification and characterization of JunD missense mutants that lack menin binding; Knapp JI et al.; Menin, the product of the MEN1 tumor suppressor gene, binds to the AP1 transcription factor JunD and represses JunD transcriptional activity . The effects of human or mouse JunD missense mutations upon menin interaction were studied by random and alanine scanning mutagenesis of the menin binding region of JunD (amino acids 1-70) . JunD mutant proteins were tested for menin binding in a reverse yeast two-hybrid assay, and for transcriptional regulation by menin in AP1-reporter assays . Random mutagenesis identified two different mutations that disrupted menin interaction at mouse JunD amino acid 42 (G42E and G42R) . Mutation G42A generated by alanine scanning did not affect menin binding, likely reflecting the conserved nature of this amino acid substitution . Furthermore, by size exclusion chromatography menin co-migrated with wild type JunD but not with the JunD mutant tested (G42E) . Alanine scanning mutagenesis of residues 30-55 revealed two different amino acids, P41 and P44, of mouse JunD that were critical for interaction with menin . Mouse JunD missense mutants P41A, G42R, G42E and P44A failed to bind menin and also escaped menin's control over their transcriptional activity . At lower amounts of transfected menin, the transcriptional effect of menin on the mutants P41A, G42R and G42E was changed from repression to activation, similar to that with c-jun . In conclusion, a small N-terminal region of JunD mediates a key difference between JunD and c-jun, and a component of this difference is dependent on JunD binding to menin. Genomics, 2000 Oct 15, 69(2), 242 - 51 Gene structure and expression study of the SEDL gene for spondyloepiphyseal dysplasia tarda; Gecz J et al.; Spondyloepiphyseal dysplasia tarda (SEDL) is an X-linked recessive disorder of endochondral bone formation caused by mutations in the SEDL gene . Here we present the structural analysis and subcellular localization of human SEDL . The SEDL gene is composed of six exons and spans a genomic region of approximately 20 kb in Xp22 . It contains four Alu sequences in its 3' UTR and an alternatively spliced MER20 sequence in its 5' UTR (exon 2) . Complex alternative splicing was detected for exon 4 . Altogether seven SEDL pseudogenes were detected in the human genome: SEDLP1, a transcribed retropseudogene (or retro-xaptonuon) on chromosome 19q13.4 with potential to encode a protein identical to that of the SEDL gene; SEDLP2, another retropseudogene (not transcribed) on chromosome 8; and five truncated pseudogenes, SEDLP3-SEDLP7, on chromosome Yq11.23 . Based on the knowledge of the yeast SEDL ortholog we speculated that the SEDL protein may participate along the ER-to-Golgi transport compartments . To test this hypothesis we performed transient transfection studies with tagged recombinant mammalian SEDL proteins in Cos-7 cells . The tagged SEDL proteins localized to perinuclear structures that partly overlapped with the intermediate ER-Golgi compartment (ERGIC; or vesicular tubular complex, VTC) . Two human SEDL mutations (157-158delAT and C271T(STOP)) introduced into SEDL FLAG and GFP constructs led to the misplacement of the SEDL protein primarily to the cell nucleus and partially to the cytoplasm . Based on these experiments we suggest that the COOH end of the SEDL protein might be responsible for proper targeting of SEDL along the ER-Golgi membrane compartments (including Golgi and ERGIC/VTC) . Genomics, 2000 Oct 15, 69(2), 174 - 81 Identification and characterization of an Xq26-q27 duplication in a family with spina bifida and panhypopituitarism suggests the involvement of two distinct genes; Hol FA et al.; We investigated a family with a duplication, dup(X)q26-q27, that was present in two brothers, their mother, and their maternal grandmother . The brothers carrying the duplication displayed spina bifida and panhypopituitarism, whereas a third healthy brother inherited the normal X chromosome . Preferential inactivation of the X chromosome containing the duplication was evident in healthy carrier females . We determined the boundaries of the Xq26-q27 duplication . Via interphase FISH analysis we narrowed down each of the two breakpoint regions to approximately 300-kb intervals . The proximal breakpoint is located in Xq26.1 between DXS1114 and HPRT and is contained in YAC yWXD599, while the distal breakpoint is located in Xq27.3 between DXS369 and DXS1200 and contained in YAC yWXD758 . The duplication comprises about 13 Mb . Evidence from the literature points to a predisposing gene for spina bifida in Xq27 . We hypothesize that the spina bifida in the two brothers may be due to interruption of a critical gene in the Xq27 breakpoint region . Several candidate genes were mapped to the Xq27 critical region but none was shown to be disrupted by the duplication event . Recently, M . Lagerstrom-Fermer et al . (1997, Am . J . Hum . Genet . 60, 910-916) reported on a family with X-linked recessive panhypopituitarism associated with a duplication in Xq26; however, no details were reported on the extent of the duplication . Our study corroborates their hypothesis that X-linked recessive panhypopituitarism is likely to be caused by a gene encoding a dosage-sensitive protein involved in pituitary development . We place the putative gene between DXS1114 and DXS1200, corresponding to the interval defined by the duplication in the present family . Oncogene, 2000 Sep 21, 19(40), 4557 - 62 p55CDC/hCDC20 is associated with BUBR1 and may be a downstream target of the spindle checkpoint kinase; Wu H et al.; Eukaryotic cells have evolved a mechanism that delays the progression of mitosis until condensed chromosomes are properly positioned on the mitotic spindle . We have been studying genes that regulated the spindle checkpoint in human cells . Enforced expression of human BUBR1, but not a BUBR1 mutant allele, enhances accumulation of mitotic cells . Yeast two-hybrid system and GST-pulldown analyses show that p55CDC/hCdc20, a protein known to link spindle checkpoint components such as MAD2 to anaphase promoting complex (APC), interacts with BUBR1 . In addition, p55CDC is capable of pulling down BUBR1 in sf-9 cells infected with both p55CDC and His6-BUBR1 recombinant baculoviruses but not in the cells infected with p55CDC baculoviruses or with the baculoviral vector alone . Moreover, immunoprecipitation followed by Western blot analyses confirmed that native p55CDC is associated with BUBR1 in HeLa cells . Spindle checkpoint activation by nocodazole treatment enhances the association between p55CDC and His6-BUBR1 . In nocodazole-arrested mitotic cells, both CDC16 and hyperphosphorylated CDC27, two APC components, preferentially associate with His6-BUBR1 resins, but not the control resins . Furthermore, BUBR1 phosphorylates p55CDC in vitro, and the phosphorylation of p55CDC by BUBR1 appears to be correlated with spindle checkpoint activation . Together, our studies strongly suggest that BUBR1 may target APC via p55CDC. J Enzyme Inhib, 2000, 15(5), 509 - 15 Competitive inhibition of Trypanosoma brucei phosphoglucose isomerase by D-arabinose-5-phosphate derivatives; Hardre R et al.; We report four new strong high energy intermediate analog competitive inhibitors of fructose-6-phosphate isomerization catalyzed by purified Trypanosoma brucei phosphoglucose isomerase: D-arabinonhydroxamic acid-5-phosphate, D-arabinonate-5-phosphate, D-arabinonamide-5-phosphate and D-arabinonhydrazide-5-phosphate . For comparison, the inhibitory properties of the corresponding non-phosphorylated analogues D-arabinonhydroxamic acid, D-arabinonate, D-arabinonamide and D-arabinonhydrazide were also evaluated . D-Arabinonhydroxamic acid-5-phosphate appears as the most potent competitive inhibitor ever evaluated on a phosphoglucose isomerase with an inhibition constant value of 50 nM and a Michaelis constant over inhibition constant ratio of about 2000 . Our results show that anionic high energy intermediate analogues, and more particularly D-arabinonhydroxamic acid-5-phosphate, display a weak but significant specificity for Trypanosoma brucei phosphoglucose isomerase versus yeast phosphoglucose isomerase, while neutral high energy intermediate analogues are not selective at all . This would indicate the presence of more positively charged residues in the active site for Trypanosoma brucei phosphoglucose isomerase as compared to that of yeast phosphoglucose isomerase. Genes Cells, 2000 Oct, 5(10), 839 - 47 Identification and characterization of human Wee1B, a new member of the Wee1 family of Cdk-inhibitory kinases; Nakanishi M et al.; BACKGROUND: In eukaryotic cells, the kinase activity of the mitosis-promoting complex composed of cyclin B and Cdc2 (Cdk1) is negatively regulated by the phosphorylation of Cdk1 on threonine or tyrosine residues within its ATP binding domain . RESULTS: We identified human Wee1B by searching a sequence database . The predicted human Wee1B protein comprises 561 amino acids . Northern blot analysis revealed that human Wee1B mRNA is particularly abundant in testis . Interestingly, RT-PCR using early embryos revealed that the Wee1B product was readily detectable at the mature oocyte, but abruptly disappeared at embryonic day 2.5, suggesting that the amount of Wee1B mRNA is dependent on the maternal expression . GFP-Wee1B showed a predominantly nuclear localization in HeLa cells . Human Wee1B was able to rescue the lethal phenotype of the fission yeast wee1-50Deltamik1 mutant, and over-expression of the human protein in these cells resulted in cell elongation as a result of arrest of the cell cycle at the G2-M transition . Recombinant Wee1B effectively phosphorylated cyclin B-associated Cdk1 on tyrosine-15, resulting in an inactivation of the kinase activity of Cdk1 . CONCLUSION: We identified human Wee1B as a novel Cdk1-inhibitory kinase . The identification of this new member of the Wee1 family suggests that inhibition of Cdk1 is mediated at multiple levels in mammals. J Mol Evol, 2000 Sep, 51(3), 265 - 77 Evolutionary studies on uricases of fungal endosymbionts of aphids and planthoppers; Hongoh Y et al.; Aphids belonging to the three genera Tuberaphis, Glyphinaphis, and Cerataphis contain extracellular fungal symbionts that resemble endocellular yeast-like symbionts of planthoppers . Whereas the symbiont of planthoppers has a uricase (urate oxidase; EC 1.7.3.3) and recycles uric acid that the host stores, no uric acid was found in Tuberaphis styraci, and its fungal symbiont did not exhibit the uricase activity . However, the fungal symbionts of these aphids, including that of T . styraci, were shown to have putative uricase genes, or pseudogenes, for the uricase . Sequence analysis of these genes revealed that deleterious mutations occurred independently on each lineage of Glyphinaphis and Tuberaphis, while no such mutation was found in the lineage of Cerataphis . These genes were almost identical to those cloned from the symbionts of planthoppers, though the host aphids and planthoppers are phylogenetically distant . To estimate the phylogenetic relationship in detail between the fungal symbionts of aphids and those of planthoppers, a gene tree was constructed based on the sequences of the uricase genes including their flanking regions . As a result, the symbionts of planthoppers and Tuberaphis aphids formed a sister group against those of Glyphinaphis and Cerataphis aphids with high bootstrap confidence levels, which strongly suggests that symbionts have been horizontally transferred from the aphids' lineage to the planthoppers'. Mol Biol Cell, 2000 Oct, 11(10), 3411 - 24 A role for the START gene-specific transcription factor complex in the inactivation of cyclin B and Cut2 destruction; Tournier S et al.; Hyperactivation of Cdc2 in fission yeast causes cells to undergo a lethal premature mitosis called mitotic catastrophe . This phenotype is observed in cdc2-3w wee1-50 cells at high temperature . Eleven of 17 mutants that suppress this phenotype define a single complementation group, mcs1 . The mcs1-77 mutant also suppresses lethal inactivation of the Wee1 and Mik1 tyrosine kinases and thus delays mitosis independently of Cdc2 tyrosine phosphorylation . We have cloned mcs1 by isolating suppressors of the cell cycle arrest phenotype of mcs1-77 cdc25-22 cells and found that it encodes Res2, a component of the START gene-specific transcription factor complex MBF (also known as DSC-1) . The mcs1-77 mutant bears a single point mutation in the DNA-binding domain of Res2 that causes glycine 68 to be replaced by a serine residue . Importantly, two substrates of the anaphase-promoting complex (APC), the major B-type cyclin, Cdc13, and the anaphase inhibitor, Cut2, are unstable in G2-phase mcs1-77 cells . Consistent with this, we observe abnormal sister chromatid separation in mcs1-77 cdc25-22 cells at the restrictive temperature . Mutation of either Cdc10 or Res1 also deregulates MBF-dependent transcription and causes a G2 delay . We find that this cell cycle delay is abolished in the absence of the APC regulator Ste9/Srw1 and that the periodic expression of Ste9/Srw1 is controlled by the MBF complex . These data suggest that in fission yeast the MBF complex plays a key role in the inactivation of cyclin B and Cut2 destruction by controlling the periodic production of APC regulators. Cell Signal, 2000 Aug, 12(8), 515 - 24 Small G-protein networks: their crosstalk and signal cascades; Matozaki T et al.; Small GTP-binding proteins (G-proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members . This superfamily is structurally classified into at least five families: the Ras, Rho/Rac/Cdc42, Rab, Sar1/Arf, and Ran families . They play key roles not only in temporal but also in spatial determination of specific cell functions . It has become clear that multiple small G-proteins form signalling cascades that are involved in various cellular functions, such as budding processes of the yeast and regulation of the actin cytoskeleton in fibroblasts . In addition, two distinct small G-proteins regulate specific cellular functions in a cooperative or antagonistic manner . A single small G-protein exerts various biological responses through different downstream effectors . Moreover, some of these downstream effectors sequentially activate further downstream effector proteins . Thus, small G-proteins appear to exert their functions through their mutual crosstalk and multiple downstream effectors in a variety of cellular functions. Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 817 - 22 Gene structure and promoter function of murine Munc18-2, a nonneuronal exocytic Sec1 homolog; Agrawal A et al.; Sec1 family proteins are regulators of diverse exocytic processes, from yeast to man . Three mammalian homologues, Munc18-1, -2, and -3 have been described . We have studied the structure and expression of the mouse Munc18-2 gene . The Munc18-2 gene comprises 19 exons whose sizes range from 50 to 158 bp, with a total gene size of approximately 11 kb . A single transcript of 2.1 kb is expressed in multiple non-neuronal murine tissues . Munc18-2 has a striking resemblance to Munc18-1 in structure despite only 60% sequence identity, suggesting a recent gene duplication event . Analysis of the region upstream of the transcription start site shows that Munc18-2 has a TATA-less promoter, with a consensus initiator (Inr) sequence at the start of transcription, several Sp1 binding sites, and strong promoter activity in RBL-2H3 mast cells . The region from +5 to -430 is more active than +5 to -800, suggesting upstream repressor elements . Biochem Biophys Res Commun, 2000 Sep 24, 276(2), 642 - 8 Chimeric proteins between UCP1 and UCP3: the middle third of UCP1 is necessary and sufficient for activation by fatty acids; Hagen T et al.; Uncoupling protein (UCP) 1 and UCP3 are mitochondrial inner membrane proteins which both mediate proton leak and thus decrease the mitochondrial transmembrane proton gradient . However, UCP1 and UCP3 differ in their biochemical regulation . UCP1 is activated by free fatty acids and inhibited by purine nucleotides . Using heterologous expression studies in yeast, UCP3 was found to lack both fatty acid activation and purine nucleotide inhibition . To assess which domains are responsible for the regulation of UCP1 by free fatty acids and by purine nucleotides and the absence of such regulation in UCP3, chimeric proteins were generated . Given that uncoupling proteins, like all members of the mitochondrial carrier family, possess a tripartite structure and consist of three repeated domains of approximately 100 residues, swaps in the three repeated domains were made between UCP1 and UCP3 . Regulation of the resulting six different chimeric proteins by free fatty acids and purine nucleotides was studied after heterologous expression in yeast mitochondria . In this study, it is shown that activation of UCP1 by free fatty acids is mediated by the second repeated domain, since substitution of the second repeat of UCP1 by the equivalent repeat of UCP3 abolishes fatty acid activation . In contrast, replacing the second repeat of UCP3 by the corresponding repeated domain of UCP1 results in fatty acid activation similar to wild type UCP1 . The lack of free fatty acid activation of UCP3 is not due to the absence of the histidine pair H145 and H147 found in the second repeated domain of UCP1 . Furthermore, the findings with respect to purine nucleotide inhibition are consistent with a significant role of the C-terminal repeated domain of UCP1 in mediating purine nucleotide inhibition . Biochem Biophys Res Commun, 2000 Sep 24, 276(2), 587 - 93 Isolation of a novel ubiquitin-like protein from Pleurotus ostreatus mushroom with anti-human immunodeficiency virus, translation-inhibitory, and ribonuclease activities; Wang HX et al.; A glycoprotein, with a ubiquitin-like N-terminal sequence, has been prepared from an extract of fruiting bodies of the mushroom Pleurotus ostreatus, using a procedure which included ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and Mono Q and gel filtration on Superdex 75 . It exhibited a molecular weight of 12.5 kDa and was unadsorbed on DEAE-cellulose and Mono Q, but adsorbed on Affi-blue gel and SP-Sepharose . It inhibited translation in a rabbit reticulocyte lysate system (IC(50) = 160 nM) and exhibited low ribonucleolytic activity (14 micro/mg) toward yeast tRNA . It also expressed an inhibitory activity toward human immunodeficiency virus-1 reverse transcriptase, which could be enhanced by succinylation . Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11575 - 80 Overexpression of the human VPAC2 receptor in the suprachiasmatic nucleus alters the circadian phenotype of mice; Shen S et al.; The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) belong to a superfamily of structurally related peptide hormones that includes glucagon, glucagon-like peptides, secretin, and growth hormone-releasing hormone . Microinjection of VIP or PACAP into the rodent suprachiasmatic nucleus (SCN) phase shifts the circadian pacemaker and VIP antagonists, and antisense oligodeoxynucleotides have been shown to disrupt circadian function . VIP and PACAP have equal potency as agonists of the VPAC(2) receptor (VPAC(2)R), which is expressed abundantly in the SCN, in a circadian manner . To determine whether manipulating the level of expression of the VPAC(2)R can influence the control of the circadian clock, we have created transgenic mice overexpressing the human VPAC(2)R gene from a yeast artificial chromosome (YAC) construct . The YAC was modified by a strategy using homologous recombination to introduce (i) the HA epitope tag sequence (from influenza virus hemagglutinin) at the carboxyl terminus of the VPAC(2)R protein, (ii) the lacZ reporter gene, and (iii) a conditional centromere, enabling YAC DNA to be amplified in culture in the presence of galactose . High levels of lacZ expression were detected in the SCN, habenula, pancreas, and testis of the transgenic mice, with lower levels in the olfactory bulb and various hypothalamic areas . Transgenic mice resynchronized more quickly than wild-type controls to an advance of 8 h in the light-dark (LD) cycle and exhibited a significantly shorter circadian period in constant darkness (DD) . These data suggest that the VPAC(2)R can influence the rhythmicity and photic entrainment of the circadian clock. Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11365 - 70 Werner protein recruits DNA polymerase delta to the nucleolus; Szekely AM et al.; Werner syndrome is a Mendelian disorder of man that produces a number of manifestations resembling human aging . This disorder is caused by inactivation of the wrn gene, a member of the RecQ family of DNA helicases . The helicase and exonuclease activities of the Werner protein (WRN) suggest that it functions in DNA transactions, but the physiological function of WRN remains elusive . We present several lines of evidence that WRN interacts specifically with the p50 subunit of polymerase delta, the major DNA polymerase required for chromosomal DNA replication . P50, identified by yeast two-hybrid screening, interacts physically with the C terminus of WRN . Native WRN protein coimmunoprecipitates with p50 in a cellular fraction enriched in nucleolar proteins, and this immunocomplex also includes p125, the catalytic subunit of polymerase delta . In subcellular localization studies of cells transfected with WRN, p50 and p125 redistribute to the nucleolus and colocalize with WRN . These results suggest that one of the functions of WRN protein is to directly modify DNA replication via its interaction with p50 and abet dynamic relocalization of the DNA polymerase delta complexes within the nucleus. Mol Cell Biol, 2000 Nov, 20(21), 8124 - 33 Multiple mechanisms of suppression circumvent transcription defects in an RNA polymerase mutant; Tan Q et al.; Using a high-copy-number suppressor screen to obtain clues about the role of the yeast RNA polymerase II subunit RPB4 in transcription, we identified three suppressors of the temperature sensitivity resulting from deletion of the RPB4 gene (DeltaRPB4) . One suppressor is Sro9p, a protein related to La protein, another is the nucleosporin Nsp1p, and the third is the RNA polymerase II subunit RPB7 . Suppression by RPB7 was anticipated since its interaction with RPB4 is well established both in vitro and in vivo . We examined the effect of overexpression of each suppressor gene on transcription . Interestingly, suppression of the temperature-sensitive phenotype correlates with the correction of a characteristic transcription defect of this mutant: each suppressor restored the level of promoter-specific, basal transcription to wild-type levels . Examination of the effects of the suppressors on other in vivo transcription aberrations in DeltaRPB4 cells revealed significant amelioration of defects in certain inducible genes in Sro9p and RPB7, but not in Nsp1p, suppressor cells . Analysis of mRNA levels demonstrated that overexpression of each of the three suppressors minimally doubled the mRNA levels during stationary phase . However, the elevated mRNA levels in Sro9p suppressor cells appear to result from a combination of enhanced transcription and message stability . Taken together, these results demonstrate that these three proteins influence transcription and implicate Sro9p in both transcription and posttranscription events. Mol Cell Biol, 2000 Nov, 20(21), 8112 - 23 Target selectivity of bicoid is dependent on nonconsensus site recognition and protein-protein interaction; Zhao C et al.; We describe experiments to compare the activities of two Drosophila homeodomain proteins, Bicoid (Bcd) and an altered-specificity mutant of Fushi tarazu, Ftz(Q50K) . Although the homeodomains of these proteins share a virtually indistinguishable ability to recognize a consensus Bcd site, only Bcd can activate transcription from natural enhancer elements when assayed in both yeast and Drosophila Schneider S2 cells . Our analysis of chimeric proteins suggests that both the homeodomain of Bcd and sequences outside the homeodomain contribute to its ability to recognize natural enhancer elements . We further show that, unlike the Bcd homeodomain, the Ftz(Q50K) homeodomain fails to recognize nonconsensus sites found in natural enhancer elements . The defect of a chimeric protein containing the homeodomain of Ftz(Q50K) in place of that of Bcd can be preferentially restored by converting the nonconsensus sites in natural enhancer elements to consensus sites . Our experiments suggest that the biological specificity of Bcd is determined by combinatorial contributions of two important mechanisms: the nonconsensus site recognition function conferred by the homeodomain and the cooperativity function conferred primarily by sequences outside the homeodomain . A systematic comparison of different assay methods and enhancer elements further suggests a fluid nature of the requirements for these two Bcd functions in target selection. Biotechnol Prog, 2000 Sep-Oct, 16(5), 736 - 43 Effect of PDI overexpression on recombinant protein secretion in CHO cells; Davis R et al.; In eukaryotic cells, protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins . PDI also functions as a molecular chaperone and has been found associated with proteins in the ER . In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins . Increasing PDI activity in bacterial, yeast, and insect cell expression systems can lead to increased secretion of heterologous proteins containing disulfide bridges . Since Chinese hamster ovary (CHO) cells are widely used for the expression of recombinant proteins, we expressed recombinant human PDI (rhu PDI) in CHO cells to increase cellular PDI levels and examined its effect on the secretion of two different recombinant proteins: interleukin 15 (IL-15) and a tumor necrosis factor receptor:Fc fusion protein (TNFR:Fc) . Secretion of TNFR:Fc (a disulfide-rich protein) is decreased in cells overexpressing PDI; the TNFR:Fc protein is retained inside these cells and colocalizes with the overexpressed rhu PDI protein in the endoplasmic reticulum . PDI overexpression did not result in intracellular retention of IL15 . The nature of the interaction between PDI and TNFR:Fc was further investigated by expressing a disulfide isomerase mutant PDI in CHO cells to determine if the functional activity of PDI is involved in the cellular retention of TNFR:Fc protein. Biochemistry, 2000 Oct 17, 39(41), 12723 - 30 Pseudouridine synthase 3 from mouse modifies the anticodon loop of tRNA; Chen J et al.; A cDNA encoding mouse pseudouridine synthase 3 (mPus3p) has been cloned . The predicted protein has 34% identity with yeast pseudouridine synthase 3 (Pus3), an enzyme known to form pseudouridine at positions 38 and 39 in yeast tRNA . The cDNA is 1.7 kb, and when used as a probe on a Northern blot of total RNA from mouse tissues or cells in culture, a band at 1.8 kb was observed . The open reading frame codes for a protein of 481 amino acids with a predicted molecular mass of 55 552 Da . When mPus3p was in vitro translated and used in reactions with tRNA substrates from both yeast and humans, uridines at position 39 were modified to pseudouridine . In a tRNA substrate with a uridine at position 38 (human tRNA(Leu)), there was very slight formation of pseudouridine at that position after incubation with mPus3p. Biophys Chem, 2000 Aug 30, 86(2-3), 231 - 7 Transcription through nucleosomes; Felsenfeld G et al.; Transcriptionally active genes in eukaryotes still retain most of the Chromatin packaging that is characteristic of eukaryotic DNA . Nucleosomes and even some higher order structure are present, although the histones may be chemically modified, for example by acetylation or phosphorylation, as part of the activation process . The presence of nucleosomes on the coding region of active genes raises the question: How does an RNA polymerase transcribe such a template? We have attempted to answer this question with relatively simple model systems involving a template carrying a single positioned nucleosome . We have shown that when a phage polymerase, SP6, transcribes such a template, the histone octamer of the nucleosome is not released into solution . Instead it is retained on the same DNA molecule, but displaced from its original binding site . Further studies have allowed us to propose a detailed model, which appears to hold not only for SP6 but also for transcription by the much larger RNA polymerase III from yeast . Our most recent results, obtained by electron cryomicroscopy, confirm and refine this model. Biophys Chem, 2000 Aug 30, 86(2-3), 191 - 201 Intermolecular dynamics and function in actin filaments; Kim E et al.; Structural models of F-actin suggest that three segments in actin, the DNase I binding loop (residues 38-52), the hydrophobic plug (residues 262-274) and the C-terminus, contribute to the formation of an intermolecular interface between three monomers in F-actin . To test these predictions and also to assess the dynamic properties of intermolecular contacts in F-actin, Cys-374 pyrene-labeled skeletal alpha-actin and pyrene-labeled yeast actin mutants, with Gln-41 or Ser-265 replaced with cysteine, were used in fluorescence experiments . Large differences in Cys-374 pyrene fluorescence among copolymers of subtilisin-cleaved (between Met-47 and Gly-48) and uncleaved alpha-actin showed both intra- and intermolecular interactions between the C-terminus and loop 38-52 in F-actin . Excimer band formation due to intermolecular stacking of pyrene probes attached to Cys-41 and Cys-265, and Cys-41 and Cys-374, in mutant yeast F-actin confirmed the proximity of these residues on the paired sites (to within 18 A) in accordance with the models of F-actin structure . The dynamic properties of the intermolecular interface in F-actin formed by loop 38-52, plug 262-274 and the C-terminus may account for the observed cross-linking of these sites with reagents < 18 A . The functional importance of actin filament dynamics was demonstrated by the inhibition of the in vitro motility in the Gln-41-Cys-374 cross-linked actin filaments. J Cell Physiol, 2000 Nov, 185(2), 269 - 79 Molecular characterization of celtix-1, a bromodomain protein interacting with the transcription factor interferon regulatory factor 2; Staal A et al.; Transcriptional control at the G1/S-phase transition of the cell cycle requires functional interactions of multimeric promoter regulatory complexes that contain DNA binding proteins, transcriptional cofactors, and/or chromatin modifying enzymes . Transcriptional regulation of the human histone H4/n gene (FO108) is mediated by Interferon Regulatory Factor-2 (IRF-2), as well as other histone gene promoter factors . To identify proteins that interact with cell-cycle regulatory factors, we performed yeast two-hybrid analysis with IRF-2 and identified a novel human protein termed Celtix-1 which binds to IRF-2 . Celtix-1 contains several phylogenetically conserved domains, including a bromodomain, which is found in a number of transcriptional cofactors . Using a panel of IRF-2 deletion mutants in yeast two-hybrid assays, we established that Celtix-1 contacts the C-terminus of IRF-2 . Celtix-1 directly interacts with IRF-2 based on binding studies with glutathione S-transferase (GST)/IRF-2 fusion proteins, and immunofluorescence studies suggest that Celtix-1 and IRF-2 associate in situ . Celtix-1 is distributed throughout the nucleus in a heterodisperse pattern . A subset of Celtix-1 colocalizes with the hyperacetylated forms of histones H3 and H4, as well as with the hyperphosphorylated, transcriptionally active form of RNA polymerase II . We conclude that the bromodomain protein Celtix-1 is a novel IRF-2 interacting protein that associates with transcriptionally active chromatin in situ . Nucleic Acids Res, 2000 Oct 15, 28(20), 3853 - 63 Structure of HAP1-PC7 bound to DNA: implications for DNA recognition and allosteric effects of DNA-binding on transcriptional activation; Lukens AK et al.; HAP1 is a transcription factor in yeast whose DNA-binding domain has been implicated in directly affecting transcriptional activation . Two separate mutations in the DNA-binding domain, S63G (HAP1-PC7) and S63R (HAP1-18), retain wild-type binding affinity . However, HAP1-PC7 is transcriptionally silent while HAP1-18 shows highly elevated levels of transcription . We have determined the X-ray crystal structure of the DNA-binding domain of HAP1-PC7 bound to its DNA target, UAS(CYC7), and compared it to the previously solved HAP1-wt and HAP1-18 complexes to UAS(CYC7) . Additionally, we have quantitatively compared the DNA-binding affinity and specificity of the HAP1-PC7, HAP1-18 and HAP1-wt DNA-binding domains . We show that, although the DNA-binding domains of these three proteins bind UAS(CYC7) with comparable affinity and specificity, the protein-DNA interactions are dramatically different between the three complexes . Conserved protein-DNA interactions are largely restricted to an internal DNA sequence that excludes one of the two conserved DNA half-sites of UAS(CYC7) suggesting a mode of recognition distinct from other HAP1 family members . Alternative protein-DNA interactions result in divergent DNA configurations between the three complexes . These results suggest that the differential transcriptional activities of the HAP1, HAP1-18 and HAP1-PC7 proteins are due, at least in part, to alternative protein-DNA contacts, and implies that HAP1-DNA interactions have direct allosteric effects on transcriptional activation. J Virol, 2000 Nov, 74(21), 10217 - 22 Cytoplasmic dynein LC8 interacts with lyssavirus phosphoprotein; Jacob Y et al.; Using a yeast two-hybrid human brain cDNA library screen, the cytoplasmic dynein light chain (LC8), a 10-kDa protein, was found to interact strongly with the phosphoprotein (P) of two lyssaviruses: rabies virus (genotype 1) and Mokola virus (genotype 3) . The high degree of sequence divergence between these P proteins (only 46% amino acid identity) favors the hypothesis that this interaction is a common property shared by all lyssaviruses . The P protein-dynein LC8 interaction was confirmed by colocalization with laser confocal microscopy in infected cells and by coimmunoprecipitation . The dynein-interacting P protein domain was mapped to the 186 amino acid residues of the N-terminal half of the protein . Dynein LC8 is a component of both cytoplasmic dynein and myosin V, which are involved in a wide range of intracellular motile events, such as microtubule minus-end directed organelle transport in axon "retrograde transport" and actin-based vesicle transport, respectively . Our results provide support for a model of viral nucleocapsid axoplasmic transport . Furthermore, the role of LC8 in cellular mechanisms other than transport, e.g., inhibition of neuronal nitric oxide synthase, suggests that the P protein interactions could be involved in physiopathological mechanisms of rabies virus-induced pathogenesis. J Virol, 2000 Nov, 74(21), 10104 - 11 Interaction of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) with HS1-associated protein X-1: implication of cytoplasmic function of EBNA-LP; Kawaguchi Y et al.; Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) consists of W1W2 repeats and a unique C-terminal Y1Y2 domain and has been suggested to play an important role in EBV-induced transformation . To identify the cellular factors interacting with EBNA-LP, we performed a yeast two-hybrid screen, using EBNA-LP cDNA containing four W1W2 repeats as bait and an EBV-transformed human peripheral blood lymphocyte cDNA library as the source of cellular genes . Our results were as follows . (i) All three cDNAs in positive yeast colonies were found to encode the same cellular protein, HS1-associated protein X-1 (HAX-1), which is localized mainly in the cytoplasm and has been suggested to be involved in the regulation of B-cell signal transduction and apoptosis . (ii) Mutational analysis of EBNA-LP revealed that the association with HAX-1 is mediated by the W1W2 repeat domain . (iii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with HAX-1 transiently expressed in COS-7 cells . (iv) When EBNA-LP and HAX-1 were coexpressed in COS-7 cells, EBNA-LP was specifically coimmunoprecipitated with HAX-1 . (v) Careful cell fractionation experiments of an EBV-infected lymphoblastoid cell line revealed that EBNA-LP is localized in the cytoplasm as well as in the nucleus . (vi) When EBNA-LP containing four W1W2 repeats was expressed in COS-7 cells, EBNA-LP was detected mainly in the nucleus by immunofluorescence assay . Interestingly, when EBNA-LP containing a single W1W2 repeat was expressed in COS-7 cells, EBNA-LP was localized predominantly in the cytoplasm and was colocalized with HAX-1 . These results indicate that EBNA-LP is in fact present and may have a significant function in the cytoplasm, possibly by interacting with and affecting the function of HAX-1. J Biol Chem, 2001 Jan 5, 276(1), 738 - 41 Mutations in the Kv beta 2 binding site for NADPH and their effects on Kv1.4; Peri R et al.; Kv beta 2 enhances the rate of inactivation and level of expression of Kv1.4 currents . The crystal structure of Kv beta 2 binds NADP(+), and it has been suggested that Kv beta 2 is an oxidoreductase enzyme () . To investigate how this function might relate to channel modulation, we made point mutations in Kv beta 2 in either the NADPH docking or putative catalytic sites . Using the yeast two-hybrid system, we found that these mutations did not disrupt the interaction of Kv beta 2 with Kv alpha 1 channels . To characterize the Kv beta 2 mutants functionally, we coinjected wild-type or mutant Kv beta 2 cRNAs and Kv1.4 cRNA in Xenopus laevis oocytes . Kv beta 2 increased both the amplitude and rate of inactivation of Kv1.4 currents . The cellular content of Kv1.4 protein was unchanged on Western blot, but the amount in the plasmalemma was increased . Mutations in either the orientation or putative catalytic sites for NADPH abolished the expression-enhancing effect on Kv1.4 current . Western blots showed that both types of mutation reduced Kv1.4 protein . Like the wild-type Kv beta 2, both types of mutation increased the rate of inactivation of Kv1.4, confirming the physical association of mutant Kv beta 2 subunits with Kv1.4 . Thus, mutations that should interfere with NADPH function uncouple the expression-enhancing effect of Kv beta 2 on Kv1.4 currents from its effect on the rate of inactivation . These results suggest that the binding of NADPH and the putative oxidoreductase activity of Kv beta 2 may play a role in the processing of Kv1.4. J Biol Chem, 2000 Dec 29, 275(52), 40981 - 5 AGS3 inhibits GDP dissociation from galpha subunits of the Gi family and rhodopsin-dependent activation of transducin; Natochin M et al.; A number of recently discovered proteins that interact with the alpha subunits of G(i)-like G proteins contain homologous repeated sequences named G protein regulatory (GPR) motifs . Activator of G protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence of the pheromone receptor, has a domain with four such repeats . To elucidate the potential mechanisms of regulation of G protein signaling by proteins containing GPR motifs, we examined the effects of the AGS3 GPR domain on the kinetics of guanine nucleotide exchange and GTP hydrolysis by G(i)alpha(1) and transducin-alpha (G(t)alpha) . The AGS3 GPR domain markedly inhibited the rates of spontaneous guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding to G(i)alpha and rhodopsin-stimulated GTPgammaS binding to G(t)alpha . The full-length AGS3 GPR domain, AGS3-(463-650), was approximately 30-fold more potent than AGS3-(572-629), containing two AGS3 GPR motifs . The IC(50) values for the AGS3-(463-650) inhibitory effects on G(i)alpha and transducin were 0.12 and 0.15 microm, respectively . Furthermore, AGS3-(463-650) and AGS3-(572-629) effectively blocked the GDP release from G(i)alpha and rhodopsin-induced dissociation of GDP from G(t)alpha . The potencies of AGS3-(572-629) and AGS3-(463-650) to suppress the GDP dissociation rates correlated with their ability to inhibit the rates of GTPgammaS binding . Consistent with the inhibition of nucleotide exchange, the AGS3 GPR domain slowed the rate of steady-state GTP hydrolysis by G(i)alpha . The catalytic rate of G(t)alpha GTP hydrolysis, measured under single turnover conditions, remained unchanged with the addition of AGS3-(463-650) . Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G protein-coupled receptor-independent activators of Gbetagamma signaling pathways, act as GDP dissociation inhibitors and negatively regulate the activation of a G protein by a G protein-coupled receptor. FASEB J, 2000 Oct, 14(13), 1876 - 88 Co-repressors 2000; Burke LJ et al.; In the last 5 years, many co-repressors have been identified in eukaryotes that function in a wide range of species, from yeast to Drosophila and humans . Co-repressors are coregulators that are recruited by DNA-bound transcriptional silencers and play essential roles in many pathways including differentiation, proliferation, programmed cell death, and cell cycle . Accordingly, it has been shown that aberrant interactions of co-repressors with transcriptional silencers provide the molecular basis of a variety of human diseases . Co-repressors mediate transcriptional silencing by mechanisms that include direct inhibition of the basal transcription machinery and recruitment of chromatin-modifying enzymes . Chromatin modification includes histone deacetylation, which is thought to lead to a compact chromatin structure to which the accessibility of transcriptional activators is impaired . In a general mechanistic view, the overall picture suggests that transcriptional silencers and co-repressors act in analogy to transcriptional activators and coactivators, but with the opposite effect leading to gene silencing . We provide a comprehensive overview of the currently known higher eukaryotic co-repressors, their mechanism of action, and their involvement in biological and pathophysiological pathways . We also show the different pathways that lead to the regulation of co-repressor-silencer complex formation. Development, 2000 Nov, 127(21), 4519 - 29 A human YAC transgene rescues craniofacial and neural tube development in PDGFRalpha knockout mice and uncovers a role for PDGFRalpha in prenatal lung growth; Sun T et al.; The platelet-derived growth factor alpha-receptor (PDGFRalpha) plays a vital role in the development of vertebrate embryos, since mice lacking PDGFRalpha die in mid-gestation . PDGFRalpha is expressed in several types of migratory progenitor cells in the embryo including cranial neural crest cells, lung smooth muscle progenitors and oligodendrocyte progenitors . To study PDGFRalpha gene regulation and function during development, we generated transgenic mice by pronuclear injection of a 380 kb yeast artificial chromosome (YAC) containing the human PDGFRalpha gene . The YAC transgene was expressed in neural crest cells, rescued the profound craniofacial abnormalities and spina bifida observed in PDGFRalpha knockout mice and prolonged survival until birth . The ultimate cause of death was respiratory failure due to a defect in lung growth, stemming from failure of the transgene to be expressed correctly in lung smooth muscle progenitors . However, the YAC transgene was expressed faithfully in oligodendrocyte progenitors, which was not previously observed with plasmid-based transgenes containing only upstream PDGFRalpha control sequences . Our data illustrate the complexity of PDGFRalpha genetic control, provide clues to the location of critical regulatory elements and reveal a requirement for PDGF signalling in prenatal lung growth, which is distinct from the known requirement in postnatal alveogenesis . In addition, we found that the YAC transgene did not prolong survival of Patch mutant mice, indicating that genetic defects outside the PDGFRalpha locus contribute to the early embryonic lethality of Patch mice. J Biol Chem, 2001 Jan 19, 276(3), 1688 - 95 Epub 2000 Oct 10. Flavonoid 6-hydroxylase from soybean (Glycine max L.), a novel plant P-450 monooxygenase; Latunde-Dada AO et al.; Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons . We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity . When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 microm) and showed typical type I absorption spectra . These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings . Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones . Isoflavones were hardly hydroxylated . As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated . Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate . For its structural identification, the chemically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment . The structures of the final reaction products for both enzymes were confirmed by NMR and mass spectrometry . Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation . This is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species. J Infect Dis, 2000 Nov, 182(5), 1531 - 5 Epub 2000 Oct 09. A randomized, placebo-controlled trial of granulocyte-macrophage colony-stimulating factor and nucleoside analogue therapy in AIDS; Brites C et al.; Preliminary preclinical and clinical data suggest that granulocyte-macrophage colony-stimulating factor (GM-CSF) may decrease viral replication . Therefore, 105 individuals with AIDS who were receiving nucleoside analogue therapy were enrolled in a placebo-controlled, double-blind study and were randomized to receive either 125 microgram/m(2) of yeast-derived, GM-CSF (sargramostim) or placebo subcutaneously twice weekly for 6 months . Subjects were evaluated for toxicity and disease progression . A significant decrease in mean virus load (VL) was observed for the GM-CSF treatment group at 6 months (-0.07 log(10) vs . -0.60 log(10); P=.02) . More subjects achieved human immunodeficiency virus (HIV)-RNA levels <500 copies/mL at >/=2 evaluations (2% on placebo vs . 11% on GM-CSF; P=.04) . Genotypic analysis of 46 subjects demonstrated a lower frequency of zidovudine-resistant mutations among those receiving GM-CSF (80% vs . 50%; P=.04) . No difference was observed in the incidence of opportunistic infections (OIs) through 6 months or survival, despite a higher risk for OI among GM-CSF recipients . GM-CSF reduced VL and limited the evolution of zidovudine-resistant genotypes, potentially providing adjunctive therapy in HIV disease. Bull Exp Biol Med, 2000 Jun, 129(6), 524 - 6 Liver resistance to CCl(4)-induced injury after stimulation of macrophages with various preparations; Kutina SN et al.; Acute toxic hepatitis in male Wistar rats was produced by single injection of 40% CCl(4) (0.2 ml per 100 g body weight in oil) . Pretreatment with various immunostimulators (bacterial polysaccharides prodigiozan and salmozan; yeast polysaccharides zymosan, peptidoglycan, and mannan; and hydrolytic enzyme egg lysozyme) produced a hepatoprotective effect correlating which the stimulatory influence on macrophages and increasing in the following order: mannan<peptidoglycan<zymosan<lysozyme<salmozan<prodigiozan. Science, 2000 Oct 6, 290(5489), 144 - 7 Regulation of STAT3 by direct binding to the Rac1 GTPase; Simon AR et al.; The signal transducers and activators of transcription (STAT) transcription factors become phosphorylated on tyrosine and translocate to the nucleus after stimulation of cells with growth factors or cytokines . We show that the Rac1 guanosine triphosphatase can bind to and regulate STAT3 activity . Dominant negative Rac1 inhibited STAT3 activation by growth factors, whereas activated Rac1 stimulated STAT3 phosphorylation on both tyrosine and serine residues . Moreover, activated Rac1 formed a complex with STAT3 in mammalian cells . Yeast two-hybrid analysis indicated that STAT3 binds directly to active but not inactive Rac1 and that the interaction occurs via the effector domain . Rac1 may serve as an alternate mechanism for targeting STAT3 to tyrosine kinase signaling complexes. Science, 2000 Oct 6, 290(5489), 142 - 4 A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants; Anandalakshmi R et al.; Posttranscriptional gene silencing (PTGS) is an ancient eukaryotic regulatory mechanism in which a particular RNA sequence is targeted and destroyed . The helper component-proteinase (HC-Pro) of plant potyviruses suppresses PTGS in plants . Using a yeast two-hybrid system, we identified a calmodulin-related protein (termed rgs-CaM) that interacts with HC-Pro . Here we report that rgs-CaM, like HC-Pro itself, suppresses gene silencing . Our work is the first report identifying a cellular suppressor of PTGS. Microbiol Immunol, 2000, 44(8), 637 - 41 Blood lysate staining, a new microscopic method for diagnosis of fungemia using peripheral blood; Makimura K et al.; We developed a microscopy method for the detection of fungal cells in peripheral blood, termed blood lysate staining, using an approximately 5x5 mm dotted blood lysate . This method was able to detect the emerging fungal pathogen Trichosporon asahii in murine models of systemic fungal infection and fungemia in patients quickly and at minimal cost . Pathogenic yeasts were successfully detected in 6 of 8 blood samples which were taken from feverish immunocompromised patients who were clinically suspected of having fungal infections . Fungal cells were observed as ovoid to elongated, 3x3 to 7x10 microm, and occurred singly, budding, and in short chains and clusters in a periodic acid-Schiff-stained blood smear . The yeast cells were easily distinguished from blood-cell debris by their size, shape and smooth yet rigid outline. J Biol Chem, 2000 Dec 29, 275(52), 41469 - 75 Two-step processing of human frataxin by mitochondrial processing peptidase . Precursor and intermediate forms are cleaved at different rates; Cavadini P et al.; We showed previously that maturation of the human frataxin precursor (p-fxn) involves two cleavages by the mitochondrial processing peptidase (MPP) . This observation was not confirmed by another group, however, who reported only one cleavage . Here, we demonstrate conclusively that MPP cleaves p-fxn in two sequential steps, yielding a 18,826-Da intermediate (i-fxn) and a 17,255-Da mature (m-fxn) form, the latter corresponding to endogenous frataxin in human tissues . The two cleavages occur between residues 41-42 and 55-56, and both match the MPP consensus sequence RX downward arrow (X/S) . Recombinant rat and yeast MPP catalyze the p --> i step 4 and 40 times faster, respectively, than the i --> m step . In isolated rat mitochondria, p-fxn undergoes a sequence of cleavages, p --> i --> m --> d(1) --> d(2), with d(1) and d(2) representing two C-terminal fragments of m-fxn produced by an unknown protease . The i --> m step is limiting, and the overall rate of p --> i --> m does not exceed the rate of m --> d(1) --> d(2), such that the levels of m-fxn do not change during incubations as long as 3 h . Inhibition of the i --> m step by a disease-causing frataxin mutation (W173G) leads to nonspecific degradation of i-fxn . Thus, the second of the two processing steps catalyzed by MPP limits the levels of mature frataxin within mitochondria. Yakugaku Zasshi, 2000 Sep, 120(9), 749 - 65 {Pharmacognosical study on secondary metabolites}; Shoyama Y; Clonal micropropagation on various medicinal plants was set up resulting in the regenerated plants which possessed a homogeneous quality . The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization of mass spectrometry . A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigene-BSA conjugate with mouse myeloma cells . Competitive enzyme-linked immunosorbent assay (ELISA) using MAb was set up as a high sensitive, specific and reproducible qualitative method . A method of determination for ginsenosides by using a unique western blotting was established . Immunoaffinity column chromatography using an anti-ginsenoside Rb1MAb has made possible a single-step separation of ginsenoside Rb1 from a crude ginseng extract . Single chain Fv gene of anti-forskolin MAb was prepared from mRNA of hybridoma secreting anti-forskolin MAb and cloned . Gene was constructed into a pET-28a(+) vector producing a scFv protein . Modeling of forskolin and scFV was investigated . THCA synthase was purified from the homogenate of Cannabis sativa leaves on successive column chromatographies . THCA synthase was confirmed to be homogeneity having 75 kDa . To obtain the corresponding cDNA clone of THCA synthase, a set of degenerate promers was constructed based on N-terinal and internal amino acid sequences of THCA synthase . The 5' and 3' ends of cDNA were amplified by RACE . A full sequencing has been determined to be corded a polypeptide having 545 amino acid residues . The cDNA clone was expressed in yeast system via PUC19 vector resulting in THCA synthase activity. Biochim Biophys Acta, 2000 Oct 2, 1493(3), 289 - 301 The human transcriptional repressor protein NAB1: expression and biological activity; Thiel G et al.; The zinc finger protein early growth response 1 (Egr-1) is a transcriptional activator involved in the regulation of growth and differentiation . Egr-1 has a large activating domain and three zinc finger motifs that function as a DNA binding region . We show here that a third functional domain of the Egr-1 protein, localized between the extended activation domain and the zinc finger DNA binding region, acts as a transcriptional repressor domain when fused to a heterologous DNA binding domain (DBD) . Through protein-protein interaction this inhibitory domain of Egr-1 brings the transcriptional corepressor NAB1 in close proximity to the transcription unit . NAB1 is expressed ubiquitously in human cell lines as shown by RNase protection mapping . Overexpression studies revealed that NAB1 is able to completely block transcription mediated by Egr-1 . In addition, the transcriptional repression activity of a fusion protein containing the inhibitory domain of Egr-1 and the DBD of the yeast transcription factor GAL4 was increased by overexpression of NAB1 . A fusion protein consisting of the DBD of GAL4 and the coding region of human NAB1 repressed transcription from model promoters with engineered upstream GAL4 binding sites . The GAL4-NAB1 fusion protein functioned from proximal and distal positions indicating that NAB1 displays transcriptional repressor activity at any position within the transcription unit . Thus, the biological function of the inhibitory domain of Egr-1 is solely to provide a docking site for NAB1 via protein-protein interaction. J Cell Biol, 2000 Oct 2, 151(1), 53 - 68 Synbindin, A novel syndecan-2-binding protein in neuronal dendritic spines; Ethell IM et al.; Dendritic spines are small protrusions on the surface of dendrites that receive the vast majority of excitatory synapses . We previously showed that the cell-surface heparan sulfate proteoglycan syndecan-2 induces spine formation upon transfection into hippocampal neurons . This effect requires the COOH-terminal EFYA sequence of syndecan-2, suggesting that cytoplasmic molecules interacting with this sequence play a critical role in spine morphogenesis . Here, we report a novel protein that binds to the EFYA motif of syndecan-2 . This protein, named synbindin, is expressed by neurons in a pattern similar to that of syndecan-2, and colocalizes with syndecan-2 in the spines of cultured hippocampal neurons . In transfected hippocampal neurons, synbindin undergoes syndecan-2-dependent clustering . Synbindin is structurally related to yeast proteins known to be involved in vesicle transport . Immunoelectron microscopy localized synbindin on postsynaptic membranes and intracellular vesicles within dendrites, suggesting a role in postsynaptic membrane trafficking . Synbindin coimmunoprecipitates with syndecan-2 from synaptic membrane fractions . Our results show that synbindin is a physiological syndecan-2 ligand on dendritic spines . We suggest that syndecan-2 induces spine formation by recruiting intracellular vesicles toward postsynaptic sites through the interaction with synbindin. Genes Dev, 2000 Oct 1, 14(19), 2534 - 46 A multifactor complex of eukaryotic initiation factors, eIF1, eIF2, eIF3, eIF5, and initiator tRNA(Met) is an important translation initiation intermediate in vivo; Asano K et al.; Translation initiation factor 2 (eIF2) bound to GTP transfers the initiator methionyl tRNA to the 40S ribosomal subunit . The eIF5 stimulates GTP hydrolysis by the eIF2/GTP/Met-tRNA(i)(Met) ternary complex on base-pairing between Met-tRNA(i)(Met) and the start codon . The eIF2, eIF5, and eIF1 all have been implicated in stringent selection of AUG as the start codon . The eIF3 binds to the 40S ribosome and promotes recruitment of the ternary complex; however, physical contact between eIF3 and eIF2 has not been observed . We show that yeast eIF5 can bridge interaction in vitro between eIF3 and eIF2 by binding simultaneously to the amino terminus of eIF3 subunit NIP1 and the amino-terminal half of eIF2beta, dependent on a conserved bipartite motif in the carboxyl terminus of eIF5 . Additionally, the amino terminus of NIP1 can bind concurrently to eIF5 and eIF1 . These findings suggest the occurrence of an eIF3/eIF1/eIF5/eIF2 multifactor complex, which was observed in cell extracts free of 40S ribosomes and found to contain stoichiometric amounts of tRNA(i)(Met) . The multifactor complex was disrupted by the tif5-7A mutation in the bipartite motif of eIF5 . Importantly, the tif5-7A mutant is temperature sensitive and displayed a substantial reduction in translation initiation at the restrictive temperature . We propose that the multifactor complex is an important intermediate in translation initiation in vivo. Genes Dev, 2000 Oct 1, 14(19), 2441 - 51 Functional selectivity of recombinant mammalian SWI/SNF subunits; Kadam S et al.; The SWI/SNF family of chromatin-remodeling complexes plays a key role in facilitating the binding of specific transcription factors to nucleosomal DNA in diverse organisms from yeast to man . Yet the process by which SWI/SNF and other chromatin-remodeling complexes activate specific subsets of genes is poorly understood . We show that mammalian SWI/SNF regulates transcription from chromatin-assembled genes in a factor-specific manner in vitro . The DNA-binding domains (DBDs) of several zinc finger proteins, including EKLF, interact directly with SWI/SNF to generate DNase I hypersensitivity within the chromatin-assembled beta-globin promoter . Interestingly, we find that two SWI/SNF subunits (BRG1 and BAF155) are necessary and sufficient for targeted chromatin remodeling and transcriptional activation by EKLF in vitro . Remodeling is achieved with only the BRG1-BAF155 minimal complex and the EKLF zinc finger DBD, whereas transcription requires, in addition, an activation domain . In contrast, the BRG1-BAF155 complex does not interact or function with two unrelated transcription factors, TFE3 and NF-kappaB . We conclude that specific domains of certain transcription factors differentially target SWI/SNF complexes to chromatin in a gene-selective manner and that individual SWI/SNF subunits play unique roles in transcription factor-directed nucleosome remodeling. J Biol Chem, 2001 Feb 16, 276(7), 5166 - 76 Epub 2000 Oct 03. Mechanism of Cu,Zn-superoxide dismutase activation by the human metallochaperone hCCS; Rae TD et al.; The mechanism for copper loading of the antioxidant enzyme copper, zinc superoxide dismutase (SOD1) by its partner metallochaperone protein is not well understood . Here we show the human copper chaperone for Cu,Zn-SOD1 (hCCS) activates either human or yeast enzymes in vitro by direct protein to protein transfer of the copper cofactor . Interestingly, when denatured with organic solvents, the apo-form of human SOD1 cannot be reactivated by added copper ion alone, suggesting an additional function of hCCS such as facilitation of an active folded state of the enzyme . While hCCS can bind several copper ions, metal binding studies in the presence of excess copper scavengers that mimic the intracellular chelation capacity indicate a limiting stoichiometry of one copper and one zinc per hCCS monomer . This protein is active and unlike the yeast protein, is a homodimer regardless of copper occupancy . Matrix-assisted laser desorption ionization-mass spectrometry and metal binding studies suggest that Cu(I) is bound by residues from the first and third domains and no bound copper is detected for the second domain of hCCS in either the full-length or truncated forms of the protein . Copper-induced conformational changes in the essential C-terminal peptide of hCCS are consistent with a "pivot, insert, and release" mechanism that is similar to one proposed for the well characterized metal handling enzyme, mercuric ion reductase. Nat Struct Biol, 2000 Oct, 7(10), 889 - 93 Structural basis for the diversity of DNA recognition by bZIP transcription factors; Fujii Y et al.; The basic region leucine zipper (bZIP) proteins form one of the largest families of transcription factors in eukaryotic cells . Despite relatively high homology between the amino acid sequences of the bZIP motifs, these proteins recognize diverse DNA sequences . Here we report the 2.0 A resolution crystal structure of the bZIP motif of one such transcription factor, PAP1, a fission yeast AP-1-like transcription factor that binds DNA containing the novel consensus sequence TTACGTAA . The structure reveals how the Pap1-specific residues of the bZIP basic region recognize the target sequence and shows that the side chain of the invariant Asn in the bZIP motif adopts an alternative conformation in Pap1 . This conformation, which is stabilized by a Pap1-specific residue and its associated water molecule, recognizes a different base in the target sequence from that in other bZIP subfamilies. Nat Genet, 2000 Oct, 26(2), 225 - 8 Human-mouse genome comparisons to locate regulatory sites; Wasserman WW et al.; Elucidating the human transcriptional regulatory network is a challenge of the post-genomic era . Technical progress so far is impressive, including detailed understanding of regulatory mechanisms for at least a few genes in multicellular organisms, rapid and precise localization of regulatory regions within extensive regions of DNA by means of cross-species comparison, and de novo determination of transcription-factor binding specificities from large-scale yeast expression data . Here we address two problems involved in extending these results to the human genome: first, it has been unclear how many model organism genomes will be needed to delineate most regulatory regions; and second, the discovery of transcription-factor binding sites (response elements) from expression data has not yet been generalized from single-celled organisms to multicellular organisms . We found that 98% (74/75) of experimentally defined sequence-specific binding sites of skeletal-muscle-specific transcription factors are confined to the 19% of human sequences that are most conserved in the orthologous rodent sequences . Also we found that in using this restriction, the binding specificities of all three major muscle-specific transcription factors (MYF, SRF and MEF2) can be computationally identified. J Biol Chem, 2000 Dec 29, 275(52), 41107 - 13 A novel nuclear localization signal in human DNA topoisomerase I; Mo YY et al.; DNA topoisomerase (topo) I is a nuclear enzyme that plays an important role in DNA metabolism . Based on conserved nuclear targeting sequences, four classic nuclear localization signals (NLSs) have been proposed at the N terminus of human topo I, but studies with yeast have suggested that only one of them (amino acids (aa) 150-156) is sufficient to direct the enzyme to the nucleus . In this study, we expressed human topo I fused to enhanced green fluorescent protein (EGFP) in mammalian cells and demonstrated that whereas aa 150-156 are sufficient for nuclear localization, the nucleolar localization requires aa 157-199 . More importantly, we identified a novel NLS within aa 117-146 . In contrast to the classic NLSs that are rich in basic amino acids, the novel NLS identified in this study is rich in acidic amino acids . Furthermore, this novel NLS alone is sufficient to direct not only EGFP into the nucleus but also topo I; and the EGFP.topo I fusion driven by the novel NLS is as active in vivo as the wild-type topo I in response to the topo I inhibitor topotecan . Together, our results suggest that human topo I carries two independent NLSs that have opposite amino acid compositions. J Biol Chem, 2000 Nov 24, 275(47), 36818 - 22 Smurf2 is a ubiquitin E3 ligase mediating proteasome-dependent degradation of Smad2 in transforming growth factor-beta signaling; Lin X et al.; Smads are important intracellular signaling effectors for transforming growth factor-beta (TGF-beta) and related factors . Proper TGF-beta signaling requires precise control of Smad functions . In this study, we have identified a novel HECT class ubiquit