Yeasts

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 ubiquitin E3 ligase, designated Smurf2, that negatively regulates Smad2 signaling . In both yeast two-hybrid and in vitro binding assays, we found that Smurf2 could interact with receptor-activated Smads (R-Smads), including Smad1, Smad2, and Smad3 but not Smad4 . Ectopic expression of Smurf2 was sufficient to reduce the steady-state levels of Smad1 and Smad2 but not Smad3 or Smad4 . Significantly, Smurf2 displayed preference to Smad2 as its target for degradation . Furthermore, Smurf2 exhibited higher binding affinity to activated Smad2 upon TGF-beta stimulation . The ability of Smurf2 to promote Smad2 destruction required the HECT catalytic activity of Smurf2 and depended on the proteasome-dependent pathway . Consistent with these results, Smurf2 potently reduced the transcriptional activity of Smad2 . These data suggest that a ubiquitin/proteasome-dependent mechanism is important for proper regulation of TGF-beta signaling.

J Pept Sci, 2000 Sep, 6(9), 453 - 8
Assessing the protease and protease inhibitor content of the human genome; Southan C; The revealing of the entire complement of protease and protease inhibitor sequences by the Human Genome Project will be of great importance to both academic and pharmaceutical research . Although the finishing phase is not yet complete, a selection of secondary annotation sources and comparisons with completed model organism genomes already allow useful estimates to be made . Conservative extrapolation suggests a total of approximately 1.8% for human proteases . This is close to the figures for yeast (1.7%) and worm (1.8%) but lower than the fly (3.4%) which has a large trypsin-like protease content . Using estimates for the human proteome of between 40,000 and 60,000 genes would extrapolate to 700-1,100 proteases, compared with approximately 360 currently represented as GenBank mRNAs . Preliminary comparisons between domain annotations for predicted human gene products and completed proteins suggest the genomic protease family and mechanistic class distributions will broadly reflect those in the current transcript data . The protease:inhibitor ratio at the mRNA level is currently approximately 9:1, but genome annotation data indicate that inhibitory domains are more widespread than this ratio would indicate.

Mol Gen Genet, 2000 Sep, 264(1-2), 119 - 26
Characterization of Ce-atl-1, an ATM-like gene from Caenorhabditis elegans; Aoki H et al.; An ATM-like gene was identified in the genome of Caenorhabditis elegans . The putative product of the gene, termed Ce-atl-1 (C . elegans ATM-like 1) consists of 2514 amino acid residues . The C-terminal sequence, which contains a PI-3 kinase-like domain, showed good homology with the products of the gene MEC1/ESR1 from budding yeast, the rad3+ gene of fission yeast and mammalian ATM (ataxia-telangiectasia and rad3+ related) genes . The results of RNA-mediated interference indicated that the major phenotype associated with repression of Ce-atl-1 was lethality (approximately 50-80%) during early embryogenesis . Among the surviving progeny, males (XO animals) arose at a high frequency (2-30%) . In addition, 5% of oocyte chromosomes demonstrated aneuploidy due to a defect in pre-meiotic chromosomal segregation . Gene expression analyses indicated that Ce-atl-1 mRNA was expressed in all larval stages and that its level increased about fivefold in the adult stage . The adult expression level was decreased in the glp-4 mutant, which is defective in germ line proliferation . Ce-atl-1 was strongly expressed in both the mitotic and meiotic cells of adult gonads . In summary, Ce-atl-1 appears to be important for early embryogenesis, and loss of its function results in a defect in chromosome segregation, similar to what has been observed for AT-related proteins.

Physiol Rev, 2000 Oct, 80(4), 1483 - 521
Calcineurin: form and function; Rusnak F et al.; Calcineurin is a eukaryotic Ca(2+)- and calmodulin-dependent serine/threonine protein phosphatase . It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an active site dinuclear metal center, and a tightly associated, myristoylated, Ca(2+)-binding subunit, calcineurin B . The primary sequence of both subunits and heterodimeric quaternary structure is highly conserved from yeast to mammals . As a serine/threonine protein phosphatase, calcineurin participates in a number of cellular processes and Ca(2+)-dependent signal transduction pathways . Calcineurin is potently inhibited by immunosuppressant drugs, cyclosporin A and FK506, in the presence of their respective cytoplasmic immunophilin proteins, cyclophilin and FK506-binding protein . Many studies have used these immunosuppressant drugs and/or modern genetic techniques to disrupt calcineurin in model organisms such as yeast, filamentous fungi, plants, vertebrates, and mammals to explore its biological function . Recent advances regarding calcineurin structure include the determination of its three-dimensional structure . In addition, biochemical and spectroscopic studies are beginning to unravel aspects of the mechanism of phosphate ester hydrolysis including the importance of the dinuclear metal ion cofactor and metal ion redox chemistry, studies which may lead to new calcineurin inhibitors . This review provides a comprehensive examination of the biological roles of calcineurin and reviews aspects related to its structure and catalytic mechanism.

Physiol Rev, 2000 Oct, 80(4), 1291 - 335
Structure, function, and control of phosphoinositide-specific phospholipase C; Rebecchi MJ et al.; Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids . Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol . The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs . New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies . Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response . Clues to their true biological roles were also obtained . Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development . In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function.

J Med Genet, 2000 Oct, 37(10), 771 - 5
Two translocations of chromosome 15q associated with dyslexia; Nopola-Hemmi J et al.; Developmental dyslexia is characterised by difficulties in learning to read . As reading is a complex cognitive process, multiple genes are expected to contribute to the pathogenesis of dyslexia . The genetics of dyslexia has been a target of molecular studies during recent years, but so far no genes have been identified . However, a locus for dyslexia on chromosome 15q21 (DYX1) has been established in previous linkage studies . We have identified two families with balanced translocations involving the 15q21-q22 region . In one family, the translocation segregates with specific dyslexia in three family members . In the other family, the translocation is associated with dyslexia in one family member . We have performed fluorescence in situ hybridisation (FISH) studies to refine the position of the putative dyslexia locus further . Our results indicate that both translocation breakpoints on 15q map within an interval of approximately 6-8 Mb between markers D15S143 and D15S1029, further supporting the presence of a locus for specific dyslexia on 15q21.

Genetics, 2000 Oct, 156(2), 711 - 21
The Drosophila mus101 gene, which links DNA repair, replication and condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1 C-terminus domains; Yamamoto RR et al.; The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents . Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically . The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101(lcd) . To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy . We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control . Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIbeta . Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.

Endocrinology, 2000 Oct, 141(10), 3534 - 45
Conformational changes and coactivator recruitment by novel ligands for estrogen receptor-alpha and estrogen receptor-beta: correlations with biological character and distinct differences among SRC coactivator family members; Kraichely DM et al.; Ligands for the estrogen receptor (ER) that have the capacity to selectively bind to or activate the ER subtypes ERalpha or ERbeta would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity . In this study, we examine three compounds of novel structure that act as ER subtype-selective ligands . These are a propyl pyrazole triol (PPT), which is a potent agonist on ERalpha but is inactive on ERbeta, and a pair of substituted tetrahydrochrysenes (THC), one enantiomer of which (S,S-THC) is an agonist on both ERalpha and ERbeta, the other (R,R-THC) being an agonist on ERalpha but an antagonist on ERbeta . To investigate the molecular mechanisms underlying the ER subtype-selective actions of these compounds, we have determined the conformational changes induced in ERalpha and ERbeta by these ligands using protease digestion sensitivity, and we have tested the ability of these ligands to promote the recruitment of representatives of the three SRC/p160 coactivator protein family members (SRC-1, GRIP-1, ACTR, respectively) to ERalpha and ERbeta using yeast two-hybrid and glutathione-S-transferase (GST) pull-down assays . We find that the ligand-ER protease digestion pattern is distinctly different for stimulatory and inhibitory ligands, and that this assay, as well as coactivator recruitment, are excellent indicators of their agonist/antagonist character . Interestingly however, compared with estradiol, the novel agonist ligands show some quantitative differences in their ability to recruit SRC-1, -2, and -3 . This implies that while generally similar to estradiol, these ligands induce ER conformations that differ somewhat from that induced by estradiol, differences that are illustrative of the nature of their biological character . The application of methods to characterize the conformations induced in ER subtypes by novel ligands, as done in this study, enables a greater understanding of how ligand-receptor conformations relate to estrogen agonist or antagonist behavior.

Nature, 2000 Sep 21, 407(6802), 401 - 5
The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans; Zhou Z et al.; In metazoans, most pre-messenger RNAs contain introns that are removed by splicing . The spliced mRNAs are then exported to the cytoplasm . Recent studies showed that splicing promotes efficient mRNA export, but the mechanism for coupling these two processes is not known . Here we show that Aly, the metazoan homologue of the yeast mRNA export factor Yralp (ref . 2), is recruited to messenger ribonucleoprotein (mRNP) complexes generated by splicing . In contrast, Aly does not associate with mRNPs assembled on identical mRNAs that already have no introns or with heterogenous nuclear RNP (hnRNP) complexes . Aly is recruited during spliceosome assembly, and then becomes tightly associated with the spliced mRNP . Aly shuttles between the nucleus and cytoplasm, and excess recombinant Aly increases both the rate and efficiency of mRNA export in vivo . Consistent with its splicing-dependent recruitment, Aly co-localizes with splicing factors in the nucleus . We conclude that splicing is required for efficient mRNA export as a result of coupling between the splicing and the mRNA export machineries.

J Vet Med B Infect Dis Vet Public Health, 2000 Aug, 47(6), 433 - 9
Isolation of Candida rugosa from turkeys; Moretti A et al.; The present study describes the isolation of Candida rugosa from young turkeys that died 10 days after the end of a therapeutic treatment for a recent outbreak of coccidiosis . Candida rugosa was isolated from the digestive tract of all the birds examined . This isolation is the first in turkeys and corroborates the fact that C . rugosa is an opportunistic yeast which circumvents host defences when other predisposing factors are present.

J Biol Chem, 2000 Dec 29, 275(52), 41124 - 32
Protein-tyrosine phosphatase D1, a potential regulator and effector for Tec family kinases; Jui HY et al.; Etk, also named Bmx, is a member of the Tec tyrosine kinase family, which is characterized by a multimodular structure including a pleckstrin homology (PH) domain, an SH3 domain, an SH2 domain, and a catalytic domain . The signaling mechanisms regulating Etk kinase activity remain largely unknown . To identify factor(s) regulating Etk activity, we used the PH domain and a linker region of Etk as a bait for a yeast two-hybrid screen . Three independent clones encoding protein-tyrosine phosphatase D1 (PTPD1) fragments were isolated . The binding of PTPD1 to Etk is specific since PTPD1 cannot associate with either the Akt PH domain or lamin . In vitro and in vivo binding studies demonstrated that PTPD1 can interact with Etk and that residues 726-848 of PTPD1 are essential for this interaction . Deletion analysis of Etk indicated that the PH domain is essential for PTPD1 interaction . Furthermore, the Etk-PTPD1 interaction stimulated the kinase activity of Etk, resulting in an increased phosphotyrosine content in both factors . The Etk-PTPD1 interaction also increased Stat3 activation . The effect of PTPD1 on Etk activation is specific since PTPD1 cannot potentiate Jak2 activity upon Stat3 activation . In addition, Tec (but not Btk) kinase can also be activated by PTPD1 . Taken together, these findings indicate that PTPD1 can selectively associate with and stimulate Tec family kinases and modulate Stat3 activation.

J Biol Chem, 2001 Jan 12, 276(2), 1434 - 8
p13(SUC1) and the WW domain of PIN1 bind to the same phosphothreonine-proline epitope; Landrieu I et al.; The WW domain of the human PIN1 and p13(SUC1), a subunit of the cyclin-dependent kinase complex, were previously shown to be involved in the regulation of the cyclin-dependent kinase complex activity at the entry into mitosis, by an unresolved molecular mechanism . We report here experimental evidence for the direct interaction of p13(SUC1) with a model CDC25 peptide, dependent on the phosphorylation state of its threonine . Chemical shift perturbation of backbone (1)H(N), (15)N, and (13)Calpha resonances during NMR titration experiments allows accurate identification of the binding site, primarily localized around the anion-binding site, occupied in the crystal structure of the homologous p9(CKSHs2) by a sulfate molecule . The epitope recognized by p13(SUC1) includes the proline at position +1 of the phosphothreonine, as was shown by the decrease in affinity for a mutated CDC25 phosphopeptide, containing an alanine/proline substitution . No direct interaction between the PIN1 WW domain or its catalytic proline cis/trans-isomerase domain and p13(SUC1) was detected, but our study showed that in vitro the WW domain of the human PIN1 antagonizes the binding of the p13(SUC1) to the CDC25 phosphopeptide, by binding to the same phosphoepitope . We thus propose that the full cyclin-dependent kinase complex stimulates the phosphorylation of CDC25 through binding of its p13(SUC1) module to the phosphoepitope of the substrate and that the reported WW antagonism of p13(SUC1)-stimulated CDC25 phosphorylation is caused by competitive binding of both protein modules to the same phosphoepitope.

Genomics, 2000 Oct 1, 69(1), 54 - 62
Identification and structural analysis of human RBM8A and RBM8B: two highly conserved RNA-binding motif proteins that interact with OVCA1, a candidate tumor suppressor; Salicioni AM et al.; The OVCA1 gene is a candidate for the breast and ovarian tumor suppressor gene at chromosome 17p13.3 . To help determine the function(s) of OVCA1, we used a yeast two-hybrid screening approach to identify OVCA1-associating proteins . One such protein, which we initially referred to as BOV-1 (binder of OVCA1-1) is 173 or 174 amino acids in length and appears to be a new member of a highly conserved RNA-binding motif (RBM) protein family that is highly conserved evolutionarily . Northern blot analysis revealed that BOV-1 is ubiquitously expressed and that three distinct messenger RNA species are expressed, 1-, 3.2-, and 5.8-kb transcripts . The 1-kb transcript is the most abundant and is expressed at high levels in the testis, heart, placenta, spleen, thymus, and lymphocytes . Using fluorescence in situ hybridization and the 5.8-kb complementary DNA probe, we determined that BOV-1 maps to both chromosome 5q13-q14 and chromosome 14q22-q23 . Further sequence analysis determined that the gene coding the 1- and the 3.2-kb transcripts (HGMW-approved gene symbol RBM8A) maps to 14q22-q23, whereas a second highly related gene coding for the 5.8-kb transcript resides at chromosome 5q13-q14 (HGMW-approved gene symbol RBM8B) . The predicted proteins encoded by RBM8A and RBM8B are identical except that RBM8B is 16 amino acids shorter at its N-terminus . Molecular modeling of the RNA-binding domain of RBM8A and RBM8B, based on homology to the sex-lethal protein of Drosophila, identifies conserved residues in the RBM8 protein family that are likely to contact RNA in a protein-RNA complex . The conservation of sequence and structure through such an evolutionarily divergent group of organisms suggests an important function for the RBM8 family of proteins .

Genomics, 2000 Oct 1, 69(1), 1 - 13
Fine-scale comparative mapping of the human 7q11.23 region and the orthologous region on mouse chromosome 5G: the low-copy repeats that flank the Williams-Beuren syndrome deletion arose at breakpoint sites of an evolutionary inversion(s); Valero MC et al.; Williams-Beuren syndrome (WBS) is a developmental disorder caused by haploinsufficiency for genes deleted in chromosome band 7q11.23 . A common deletion including at least 16-17 genes has been defined in the great majority of patients . We have completed a physical and transcription map of the WBS region based on analysis of high-throughput genome sequence data and assembly of a BAC/PAC/YAC contig, including the characterization of large blocks of gene-containing low-copy-number repeat elements that flank the commonly deleted interval . The WBS deletions arise as a consequence of unequal crossing over between these highly homologous sequences, which confer susceptibility to local chromosome rearrangements . We have also completed a clone contig, genetic, and long-range restriction map of the mouse homologous region, including the orthologues of all identified genes in the human map . The order of the intradeletion genes appears to be conserved in mouse, and no low-copy-number repeats are found in the region . However, the deletion region is inverted relative to the human map, exactly at the flanking regions . Thus, we have identified an evolutionary inversion with chromosomal breakpoints at the sites where the human 7q11.23 low-copy-number repeats are located . Additional comparative mapping suggests a model for human chromosome 7 evolution due to serial inversions leading to genomic duplications . This high-resolution mouse map provides the framework required for the generation of mouse models for WBS mimicking the human molecular defect .

Stem Cells, 1996, 14 Suppl 1, 38 - 47
The regulated expression of a TATA-less, platelet-specific gene, alphaIIb; Block KL et al.; The megakaryocyte (MK)-specific integrin, alphaIIb, is the alpha-subunit of the alphaIIb/beta3 complex found on the surface of platelets . This complex is a receptor for fibrinogen and other ligands when platelets are activated . Because the alphaIIb gene is specifically expressed in MKs, this gene was studied as a potential model for MK-specific gene expression . Previous studies have defined some of the important regulatory elements in 912 bp of the immediate 5'-flanking region of this gene . These studies defined several important elements including two GATA-binding elements and an Ets-binding element . Using a primary rat marrow expression system, we demonstrated that one of the GATA-binding elements, -454 bp upstream of the transcriptional start site (GATA454), is critical for expression of the alphaIIb gene . A potential negative regulatory element was found between -100 and -200 bp upstream of both the rat and human alphaIIb genes . The biological basis by which this negative regulatory region effects expression is not well understood . Recent studies have focused on the issue of the molecular basis by which this TATA-less gene is properly transcribed . We found that a GA-rich region approximately 14 bp upstream from the transcriptional start site appears to be a nonconsensus Sp1-binding site that interacts with an Ets-consensus site approximately 20 bp further upstream . These studies provide further evidence of the role of interactions between Ets-like proteins and Sp1 in transcriptional activation when a TATA box is not present in the promoter region of a gene . Based on the presented studies and previous results, a model is proposed for the regulation of expression of the alphaIIb gene . In studies looking at more distal regulatory elements, we have found, using the primary rat marrow expression system, that 2.9 kb of 5'-flanking alphaIIb sequence has as high a level of expression as the 912 bp construct . Whether either of these lengths of 5'-flanking region can result in tissue-specific expression in transgenic models is presently being investigated . In addition, while a published report suggests that the two genes alphaIIb and beta3 are physically linked within a 250 kb region of genomic DNA, analysis of yeast artificial chromosome clones and genomic pulsed field gel electrophoresis analysis are consistent with these two genes not being tightly linked and being >1 mb apart, suggesting that these two genes do not form a single, tissue-specific locus.

Appl Environ Microbiol, 2000 Oct, 66(10), 4253 - 7
A methylotrophic pathway participates in pectin utilization by Candida boidinii; Nakagawa T et al.; The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source . When cells were grown on 1% (wt/vol) pectin, C . boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg) . The numbers of peroxisomes also increased ca . two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell) . The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant . Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

Exp Cell Res, 2000 Oct 10, 260(1), 96 - 104
14-3-3 protein family members have a regulatory role in retinoic acid-mediated induction of cytokeratins in F9 cells; Takihara Y et al.; We have found that the expression of five 14-3-3 protein isoforms is induced during the retinoic acid (RA)-mediated differentiation of mouse embryonal carcinoma F9 cells . The induced expression of the 14-3-3 proteins is presumed to have a role in enhancing the mitogen-activated protein kinase (MAPK) activity during RA-mediated F9 cell differentiation, because using genetically engineered budding yeast we showed that these isoforms enhanced the signaling in the MAPK cascade mainly through the interaction with Raf-1 . Then we assessed the role of increased MAPK activity in F9 cell differentiation by interfering with signaling in the MAPK cascade in F9 cells . The exogenous expression of dominant-negative MEK1 efficiently abrogated RA-mediated induction of the cytokeratins EndoA and EndoC in the F9 cells . These results suggest that the 14-3-3 proteins play a role in the efficient induction of the cytokeratins during F9 cell differentiation through their signal enhancing activity in the MAPK cascade .

J Neurosci, 2000 Oct 1, 20(19), 7252 - 7
PICK1 interacts with and regulates PKC phosphorylation of mGLUR7; Dev KK et al.; The G-protein-coupled metabotropic glutamate receptor subtype 7a (mGluR7a) is a member of group III metabotropic glutamate receptors that plays an important role as a presynaptic receptor in regulating transmitter release at glutamatergic synapses . Here we report that the protein interacting with C-kinase (PICK1) binds to the C terminus (ct) of mGluR7a . In the yeast two-hybrid system, the extreme ct of mGluR7a was shown to interact with the PSD-95/Discs large/ZO-1 (PDZ) domain of PICK1 . Pull-down assays indicated that PICK1 was retained by a glutathione S-transferase fusion of ct-mGluR7a . Furthermore, recombinant and native PICK1/mGluR7a complexes were coimmunoprecipitated from COS-7 cells and rat brain tissue, respectively . Confocal microscopy showed that both PICK1 and mGluR7a displayed synaptic colocalization in cultured hippocampal neurons . PICK1 has previously been shown to bind protein kinase C alpha-subunit (PKCalpha), and mGluR7a is known to be phosphorylated by PKC . We show a relationship between these three proteins using recombinant PICK1, mGluR7, and PKCalpha, where they were co-immunoprecipitated as a complex from COS-7 cells . In addition, PICK1 caused a reduction in PKCalpha-evoked phosphorylation of mGluR7a in in vitro phosphorylation assays . These results suggest a role for PICK1 in modulating PKCalpha-evoked phosphorylation of mGluR7a.

Chromosoma, 2000, 109(5), 318 - 27
A neocentromere in the DAZ region of the human Y chromosome; Floridia G et al.; We describe a novel rearranged human Y chromosome consisting of an inverted duplication of the long arm heterochromatin and a small amount of euchromatin: rea(Y)(qter-q11.2::q11.2-qter) . The normal centromere has been deleted and a neocentromere containing CENP-A, -C, -E and Mad2 but not CENP-B has formed close to the breakpoint . A 2.7 Mb yeast artificial chromosome contig spanning the breakpoint was constructed and the breakpoint was localised to a region of <120 kb close to the DAZ gene cluster . Combined immunofluorescence and fluorescence in situ hybridisation showed that the centromeric protein-binding domain of the neocentromere was located near the breakpoint and within the DAZ cluster.

Parasitol Today, 2000 Oct, 16(10), 421 - 7
Traffic jams: protein transport in Plasmodium falciparum; van Dooren GG et al.; Protein targeting in malaria parasites is a complex process, involving several cellular compartments that distinguish these cells from more familiar systems, such as yeast or mammals . At least a dozen distinct protein destinations are known . The best studied of these is the vestigial chloroplast (the apicoplast), but new tools promise rapid progress in understanding how Plasmodium falciparum and related apicomplexan parasites traffic proteins to their invasion-related organelles, and how they modify the host by trafficking proteins into its cytoplasm and plasma membrane . Here, Giel van Dooren and colleagues discuss recent insights into protein targeting via the secretory pathway in this fascinating and important system . This topic emerged as a major theme at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000.

Biochem Biophys Res Commun, 2000 Sep 16, 276(1), 350 - 4
Receptor isoform-specific interaction of prostaglandin EP3 receptor with muskelin; Hasegawa H et al.; By using the yeast two-hybrid system, muskelin was found to bind with the carboxy-terminal tail of the prostaglandin EP3 receptor alpha isoform but not with either the beta or gamma isoform . A direct interaction between the carboxy-terminal tail of the alpha isoform and muskelin was confirmed in vitro using recombinant fusion proteins . Analysis by confocal microscopy indicated that the isoform and muskelin were distributed at the plasma membrane in transfected cells . When the isoform was stimulated by agonist, the receptor was internalized in the cells expressing the receptor alone, but this internalization was partially inhibited by the cotransfection with muskelin . Furthermore, muskelin enhanced the Gi activity of the isoform . Thus, muskelin appears to be an isoform-specific anchoring protein for the EP3 receptor .

Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11286 - 91
Intracellular delivery of phosphoinositides and inositol phosphates using polyamine carriers; Ozaki S et al.; Phosphoinositide signaling regulates events in endocytosis and exocytosis, vesicular trafficking of proteins, transduction of extracellular signals, remodeling of the actin cytoskeleton, regulation of calcium flux, and apoptosis . Obtaining mechanistic insights in living cells is impeded by the membrane impermeability of these anionic lipids . We describe a carrier system for intracellular delivery of phosphoinositide polyphosphates (PIP(n)s) and fluorescently labeled PIP(n)s into living cells, such that intracellular localization can be directly observed . Preincubation of PIP(n)s or inositol phosphates with carrier polyamines produced complexes that entered mammalian, plant, yeast, bacterial, and protozoal cells in seconds to minutes via a nonendocytic mechanism . Time-dependent transit of both PIP(n)s and the carrier to specific cytosolic and nuclear compartments was readily visualized by fluorescence microscopy . Platelet-derived growth factor treatment of NIH 3T3 fibroblasts containing carrier-delivered phosphatidylinositol 4,5-bisphosphate {PtdIns(4, 5)P(2)}-7-nitrobenz-2-oxa-1,3-diazole resulted in the redistribution of the fluorescent signal, suggesting that fluorescent PtdIns(4, 5)P(2) was a substrate for phospholipase C . We also observed a calcium flux in NIH 3T3 cells when complexes of carrier and PtdIns(4, 5)P(2) or inositol 1,4,5-trisphosphate were added extracellularly . This simple intracellular delivery system allows for the efficient translocation of biologically active PIP(n)s, inositol phosphates, and their fluorescent derivatives into living cells in a physiologically relevant context.

Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11632 - 7
Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions; Uno Y et al.; The induction of the dehydration-responsive Arabidopsis gene, rd29B, is mediated mainly by abscisic acid (ABA) . Promoter analysis of rd29B indicated that two ABA-responsive elements (ABREs) are required for the dehydration-responsive expression of rd29B as cis-acting elements . Three cDNAs encoding basic leucine zipper (bZIP)-type ABRE-binding proteins were isolated by using the yeast one-hybrid system and were designated AREB1, AREB2, and AREB3 (ABA-responsive element binding protein) . Transcription of the AREB1 and AREB2 genes is up-regulated by drought, NaCl, and ABA treatment in vegetative tissues . In a transient transactivation experiment using Arabidopsis leaf protoplasts, both the AREB1 and AREB2 proteins activated transcription of a reporter gene driven by ABRE . AREB1 and AREB2 required ABA for their activation, because their transactivation activities were repressed in aba2 and abi1 mutants and enhanced in an era1 mutant . Activation of AREBs by ABA was suppressed by protein kinase inhibitors . These results suggest that both AREB1 and AREB2 function as transcriptional activators in the ABA-inducible expression of rd29B, and further that ABA-dependent posttranscriptional activation of AREB1 and AREB2, probably by phosphorylation, is necessary for their maximum activation by ABA . Using cultured Arabidopsis cells, we demonstrated that a specific ABA-activated protein kinase of 42-kDa phosphorylated conserved N-terminal regions in the AREB proteins.

Histol Histopathol, 2000 Oct, 15(4), 1271 - 84
Identification and characterization of genes responsive to apoptosis: application of DNA chip technology and mRNA differential display; Sun Y; Apoptosis (programmed cell death) is a genetically programmed active cell death process for maintaining homeostasis under physiological conditions and for responding to various stimuli . Many human diseases have been associated with either increased apoptosis (such as AIDS and neurodegenerative disorders) or decreased apoptosis (such as cancer and autoimmune disorders) . In an attempt to understand apoptosis signaling pathway and genes associated with apoptosis, we established two cell model systems on which apoptosis is induced either by DNA damaging agent, etoposide or by redox agent, 1,10-phenanthroline (OP) . DNA chip profiling or mRNA differential display (DD) was utilized to identify genes responsive to apoptosis induced by these two agents . In etoposide model with chip hybridization, we defined signaling pathways that mediate apoptosis in p53 dependent manner (through activation of p53 target genes such as Waf-1/p21, PCNA, GPX, S100A2 and PTGF-beta) as well as in p53-independent manner (through activation of ODC and TGF-beta receptor, among others) . In OP model with DD screening, we cloned and characterized two genes: glutathione synthetase, encoding an enzyme involved in glutathione synthesis and Sensitive to Apoptosis Gene (SAG), a novel evolutionarily conserved gene encoding a zinc RING finger protein . Both genes appear to protect cells from apoptosis induced by redox agents . Further characterization of SAG revealed that it is a growth essential gene in yeast and belongs to a newly identified gene family that promotes protein ubiquitination and degradation . Through this activity, SAG regulates cell cycle progression and many other key biological processes . Thus, SAG could be a valid drug target for anti-cancer and anti-inflammation therapies.

Curr Top Dev Biol, 2000, 49, 251 - 66
Centrosome replication in somatic cells: the significance of G1 phase; Balczon R; Proper cell division requires that the cell be able to form a bipolar spindle during mitosis . To achieve this, the centrosome must be replicated accurately during interphase . Our understanding of the mechanisms that allow centrosome doubling to be coordinated with other cell cycle progression processes is advancing at a rapid pace . Several different experimental systems have been developed that are allowing detailed studies of centrosome replication . For example, the identification of mutants in yeast that are unable to duplicate the SPB accurately during interphase has provided important insights concerning centrosome duplication . In addition, intact embryonic cells and extracts prepared from unfertilized eggs are powerful tools for investigating the molecular regulation of centrosome doubling during the cell cycle . Many of the observations from these embryonic systems are directly applicable to understanding centrosome doubling in somatic cells . Finally, transgenic mouse models and cultured mammalian cell systems have been developed for analyzing the regulation of centrosome doubling in cells with more complex cell cycles . As our knowledge of the cell cycle advances, particularly our understanding of the intricate series of events that must occur for somatic cells to traverse G1 phase, it should be possible to use the systems that have been developed to determine how the replication of the centrosome is coordinated with other cell cycle progression processes . The next few years should see rapid advances in our understanding of this critical cell biological process.

Biochim Biophys Acta, 2000 Jun 15, 1479(1-2), 1 - 14
Posttranslational processing and differential glycosylation of Tractin, an Ig-superfamily member involved in regulation of axonal outgrowth; Jie C et al.; Tractin is a novel member of the Ig-superfamily which has a highly unusual structure . It contains six Ig domains, four FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine-rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ domain binding motif . By generating domain-specific antibodies, we show that Tractin is proteolytically processed at two cleavage sites, one located in the third FNIII domain, and a second located just proximal to the transmembrane domain resulting in the formation of four fragments . The most NH(2)-terminal fragment which is glycosylated with the Lan3-2, Lan4-2, and Laz2-369 glycoepitopes is secreted, and we present evidence which supports a model in which the remaining fragments combine to form a secreted homodimer as well as a transmembrane heterodimer . The extracellular domain of the dimers is mostly made up of the collagen-like PG/YG-repeat domain but also contains 11/2 FNIII domain and the acidic domain . The collagen-like PG/YG-repeat domain could be selectively digested by collagenase and we show by yeast two-hybrid analysis that the intracellular domain of Tractin can interact with ankyrin . Thus, the transmembrane heterodimer of Tractin constitutes a novel protein domain configuration where sequence that has properties similar to that of extracellular matrix molecules is directly linked to the cytoskeleton through interactions with ankyrin.

Biochim Biophys Acta, 2000 Jul 24, 1492(2-3), 513 - 6
Cloning and characterization of a novel human ninein protein that interacts with the glycogen synthase kinase 3beta; Hong YR et al.; Using human glycogen synthase kinase 3beta (GSK-3beta) as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, hNinein . When the full length cDNA of hNinein was sequenced, it showed that an open reading frame encoded a protein consisting of 2047 amino acids with a predicted molecular mass of 239 kDa . The features of this protein include a potential GTP binding site, a large coiled-coil domain together with four leucine zipper domains and a GSK-3beta binding site . Fluorescence microscopy experiment showed that hNinein is localized in the pericentriolar matrix of the centrosome . In addition, hNinein also showed to react with centrosomal autoantibody sera . Our findings suggest that hNinein may be involved in the formation of centrosome matrix and interacts with the GSK-3beta, implying that it may also be regulated by GSK-3beta phosphorylation signaling.

Biochim Biophys Acta, 2000 Jun 21, 1492(1), 242 - 6
New isoforms of cytochrome c oxidase subunit IV in tuna fish; Huttemann M; In the present study, the cDNA sequences of cytochrome c oxidase subunit IV isoforms from tuna fish are reported . The cDNAs share 57% identity among each other and the deduced amino acid sequences of the mature proteins 56% identity . Until now, only in yeast are two isoforms of the corresponding subunit V known, which are expressed in response to the oxygen supply . The hypothetical function of the new isoforms in fish for adaptation to different oxygen partial pressures in tissues of higher organisms is discussed.

Development, 2000 Oct, 127(20), 4455 - 67
Modular long-range regulation of Myf5 reveals unexpected heterogeneity between skeletal muscles in the mouse embryo; Hadchouel J et al.; The myogenic factor Myf5 plays a key role in muscle cell determination, in response to signalling cascades that lead to the specification of muscle progenitor cells . We have adopted a YAC transgenic approach to identify regulatory sequences that direct the complex spatiotemporal expression of this gene during myogenesis in the mouse embryo . Important regulatory regions with distinct properties are distributed over 96 kb upstream of the Myf5 gene . The proximal 23 kb region directs early expression in the branchial arches, epaxial dermomyotome and in a central part of the myotome, the epaxial intercalated domain . Robust expression at most sites in the embryo where skeletal muscle forms depends on an enhancer-like sequence located between -58 and -48 kb from the Myf5 gene . This element is active in the epaxial and hypaxial myotome, in limb muscles, in the hypoglossal chord and also at the sites of Myf5 transcription in prosomeres p1 and p4 of the brain . However later expression of Myf5 depends on a more distal region between -96 and -63 kb, which does not behave as an enhancer . This element is necessary for expression in head muscles but strikingly only plays a role in a subset of trunk muscles, notably the hypaxially derived ventral body muscles and also those of the diaphragm and tongue . Transgene expression in limb muscle masses is not affected by removal of the -96/-63 region . Epaxially derived muscles and some hypaxial muscles, such as the intercostals and those of the limb girdles, are also unaffected . This region therefore reveals unexpected heterogeneity between muscle masses, which may be related to different facets of myogenesis at these sites . Such regulatory heterogeneity may underlie the observed restriction of myopathies to particular muscle subgroups.

J Nutr Biochem, 2000 Jun 1, 11(6), 341 - 347
Selenium supplementation of Chinese women with habitually low selenium intake increases plasma selenium, plasma glutathione peroxidase activity, and milk selenium, but not milk glutathione peroxidase activity(1); Moore MA et al.; Twenty-one pregnant women living in Xichang County, China, a selenium-deficient area, were divided into two groups and given either a placebo (n = 10) as yeast or selenium-enriched yeast tablets (n = 11) to provide 100 microg selenium per day . This supplementation was begun the last trimester of pregnancy and continued for 3 months after parturition . Plasma selenium levels and glutathione peroxidase (GPX) activity steadily declined in supplemented women, but a curvilinear response occurred in milk selenium and GPX activity in both supplemented and deficient women and in plasma selenium and GPX activity in deficient women . The milk selenium levels were higher in supplemented women but there were no differences in the milk GPX activity between the two groups of women . The plasma alpha-tocopherol concentrations declined after parturition in both groups but no differences were found between the two groups of women . Plasma thiobarbituric acid reactive substances declined in supplemented women but showed a curvilinear response in unsupplemented women, suggesting peroxidative stress in these women . GPX, selenium, and peroxidative responses in plasma and milk following parturition is advocated as a new method to assess selenium status of lactating women.

Mamm Genome, 2000 Oct, 11(10), 862 - 5
Genetic linkage analysis of X-ray hypersensitivity in the LEC mutant rat; Agui T et al.; The LEC rat has been reported to exhibit X-ray hypersensitivity and deficiency in DNA double-strand break (DSB) repair . The present study was performed to map the locus responsible for this phenotype, the xhs (X-ray hypersensitivity), as the first step in identifying the responsible gene . Analysis of the progeny of (BN x LEC)F(1) x LEC backcrosses indicated that the X-ray hypersensitive phenotype was controlled by multiple genetic loci in contrast to the results reported previously . Quantitative trait loci (QTL) linkage analysis revealed two responsible loci located on Chromosomes (Chr) 4 and 1 . QTL on Chr 4 exhibited very strong linkage to the X-ray hypersensitive phenotype, while QTL on Chr 1 showed weak linkage . The Rad52 locus, mutation of which results in hypersensitivity to ionizing radiation and impairment of DNA DSB repair in yeast, was reported to be located on the synteneic regions of mouse Chr 6 and human Chr 12 . However, mapping of the rat Rad52 locus indicated that it was located 23 cM distal to the QTL on Chr 4 . Furthermore, none of the radio-sensitivity-related loci mapped previously in the rat chromosome were identical to the QTL on Chrs 4 and 1 in the LEC rat . Thus, it seems that X-ray hypersensitivity in the LEC rat is caused by mutation(s) in as-yet-undefined genes.

Mol Cell Biol, 2000 Oct, 20(20), 7813 - 25
Protein 4.1 R-135 interacts with a novel centrosomal protein (CPAP) which is associated with the gamma-tubulin complex; Hung LY et al.; Using a yeast two-hybrid system, we isolated a novel human centrosomal protein, CPAP (centrosomal P4.1-associated protein), which specifically interacts with the head domain of the 135-kDa protein 4.1R isoform (4.1R-135) . Sequence analysis revealed that the carboxyl terminus of CPAP has 31.3% amino acid identity with human Tcp-10 (a t-complex responder gene product) . Interestingly, most of the sequence identity is restricted to two conserved regions . One carries a leucine zipper, which may form a series of heptad repeats involved in coiled-coil formation; the other contains unusual glycine repeats with unknown function . Immunofluorescence analysis revealed that CPAP and gamma-tubulin are localized within the centrosome throughout the cell cycle . CPAP cosediments with gamma-tubulin in sucrose gradients and coimmunoprecipitates with gamma-tubulin, indicating that CPAP is a part of the gamma-tubulin complex . Furthermore, functional analysis revealed that CPAP is localized within the center of microtubule asters and may participate in microtubule nucleation . The formation of microtubule asters was significantly inhibited by anti-CPAP antibody . Together, these observations indicate that CPAP may play an important role in cell division and centrosome function.

Mol Cell Biol, 2000 Oct, 20(20), 7662 - 72
Induction of human fetal globin gene expression by a novel erythroid factor, NF-E4; Zhou W et al.; The stage selector protein (SSP) is a heteromeric complex involved in preferential expression of the human gamma-globin genes in fetal-erythroid cells . We have previously identified the ubiquitous transcription factor CP2 as a component of this complex . Using the protein dimerization domain of CP2 in a yeast two-hybrid screen, we have cloned a novel gene, NF-E4, encoding the tissue-restricted component of the SSP . NF-E4 and CP2 coimmunoprecipitate from extract derived from a fetal-erythroid cell line, and antiserum to NF-E4 ablates binding of the SSP to the gamma promoter . NF-E4 is expressed in fetal liver, cord blood, and bone marrow and in the K562 and HEL cell lines, which constitutively express the fetal globin genes . Enforced expression of NF-E4 in K562 cells and primary erythroid progenitors induces endogenous fetal globin gene expression, suggesting a possible strategy for therapeutic intervention in the hemoglobinopathies.

Mol Cell Biol, 2000 Oct, 20(20), 7527 - 40
Repression of virus-induced interferon A promoters by homeodomain transcription factor Ptx1; Lopez S et al.; Interferon A (IFN-A) genes are differentially expressed after virus induction . The differential expression of individual IFN-A genes is modulated by substitutions in the proximal positive virus responsive element A (VRE-A) of their promoters and by the presence or absence of a distal negative regulatory element (DNRE) . The functional feature of the DNRE is to specifically act by repression of VRE-A activity . With the use of the yeast one-hybrid system, we describe here the identification of a specific DNRE-binding protein, the pituitary homeobox 1 (Ptx1 or Pitx1) . Ptx1 is detectable in different cell types that differentially express IFN-A genes, and the endogenous Ptx1 protein binds specifically to the DNRE . Upon virus induction, Ptx1 negatively regulates the transcription of DNRE-containing IFN-A promoters, and the C-terminal region, as well as the homeodomain of the Ptx1 protein, is required for this repression . After virus induction, the expression of the Ptx1 antisense RNA leads to a significant increase of endogenous IFN-A gene transcription and is able to modify the pattern of differential expression of individual IFN-A genes . These studies suggest that Ptx1 contributes to the differential transcriptional strength of the promoters of different IFN-A genes and that these genes may provide new targets for transcriptional regulation by a homeodomain transcription factor.

Mol Cell Biol, 2000 Oct, 20(20), 7480 - 9
SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA; Kipp M et al.; SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix . The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood . We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins . This domain was first identified in human scaffold attachment factor A (SAF-A) and was thus designated SAF-Box . The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain . We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity . Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments . This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.

Biochemistry (Mosc), 2000 Aug, 65(8), 933 - 9
Isolation and properties of a mannan-binding lectin from the coelomic fluid of the holothurian Cucumaria japonica; Bulgakov AA et al.; A new 44-kD, C-type mannan-binding lectin (MBL-C) consisting of two identical subunits was isolated from the coelomic fluid of the holothurian Cucumaria japonica . In the direct hemagglutination assay, the lectin was effectively inhibited by highly branched mannans similar in structure to yeast mannans and composed of alpha-(1-->2)- and alpha-(1-->6)-bound D-mannopyranose residues . Hemagglutination was not inhibited by mannosaccharides, common constituents of the hydrocarbon chains of "normal" glycoproteins . The lectin reaction depends on Ca2+ concentration: maximum activity of MBL-C is observed at 10 mM Ca2+ . The activity of MBL-C increases in the pH range from 5 to 7 and reaches maximum at pH 7.0 . The lectin is sensitive to temperature . Heating of the lectin solution at temperatures above 40 degrees C decreases activity, while incubation at 90 degrees C for 1 h leads to complete irreversible inactivation . Carbohydrate specificity, Ca2+-dependence, and amino acid composition indicate that MBL-C belongs to the C-type mannan-binding lectins . Polyclonal antibodies against MBL-C revealed its immunochemical similarity to a mannan-binding lectin from another holothurian species, Stichopus japonicus; this provides evidence for structural homology between these proteins.

Hum Mol Genet, 2000 Sep 22, 9(15), 2305 - 12
Identification and characterization of an ataxin-1-interacting protein: A1Up, a ubiquitin-like nuclear protein; Davidson JD et al.; Expansion of a polyglutamine tract within ataxin-1 causes spinocerebellar ataxia type 1 (SCA1) . In this study, we used the yeast two-hybrid system to identify an ataxin-1-interacting protein, A1Up . A1Up localized to the nucleus and cytoplasm of transfected COS-1 cells . In the nucleus, A1Up co-localized with mutant ataxin-1, further demonstrating that A1Up interacts with ataxin-1 . Expression analyses demonstrated that A1U mRNA is widely expressed as an approximately 4.0 kb transcript and is present in Purkinje cells, the primary site of SCA1 cerebellar pathology . Sequence comparisons revealed that A1Up contains an N-terminal ubiquitin-like (UbL) region, placing it within a large family of similar proteins . In addition, A1Up has substantial homology to human Chap1/Dsk2, a protein that binds the ATPase domain of the HSP70-like Stch protein . These results suggest that A1Up may link ataxin-1 with the chaperone and ubiquitin-proteasome pathways . In addition, these data support the concept that ataxin-1 may function in the formation and regulation of multimeric protein complexes within the nucleus.

Hum Mol Genet, 2000 Sep 22, 9(15), 2281 - 9
Alzheimer's disease-associated presenilin 2 interacts with DRAL, an LIM-domain protein; Tanahashi H et al.; Using the yeast two-hybrid system, we screened for proteins interacting with presenilin 2 (PS2) and cloned DRAL . DRAL is an LIM-only protein containing four LIM domains and an N-terminal half LIM domain . Previously DRAL has been cloned as a co-activator of the androgen receptor and as a protein interacting with a DNA replication regulatory protein, hCDC47 . Our yeast two-hybrid assay showed that DRAL interacted with a hydrophilic loop region (amino acids 269-298) in the endoproteolytic N-terminal fragment of PS2, but not that of PS1, although the region 269-298 of PS2 and the corresponding PS1 sequence differ by only three amino acids . Each point mutation within this region, R275A, T280A, Q282A, R284A, N285A, P287T, I288L, F289A and S296A, in PS2 abolished the binding . This suggests that DRAL recognizes the PS2 structure specifically . The in vitro interaction was confirmed by affinity column assay and the physiological interactions between endogenous PS2 and DRAL by co-immunoprecipitation from human lung fibroblast MRC5 cells . Furthermore, in PS2-overexpressing HEK293 cells, we found an increase in the amount of DRAL in the membrane fraction and an increase in the amount of DRAL that was co-immunoprecipitated with PS2 . The potential role of DRAL in the cellular signaling suggests that DRAL functions as an adaptor protein that links PS2 to an intracellular signaling.

Hum Mol Genet, 2000 Sep 22, 9(15), 2231 - 9
Identification of WTAP, a novel Wilms' tumour 1-associating protein; Little NA et al.; The Wilms' tumour suppressor gene WT1 is essential for the normal development of the genitourinary system . It appears to play a role in both transcriptional and post-transcriptional regulation of certain cellular genes . However, the mechanisms behind WT1 function are not clearly understood despite the identification of numerous potential target genes and the isolation of several WT1-binding proteins . This study therefore sets out to identify other WT1-associating proteins to help to unravel how WT1 interacts with the cellular machinery . We report the identification of a novel human WT1-associating protein, WTAP, which was isolated using the yeast two-hybrid system . Both in vitro and in vivo assays have shown that the interaction between WTAP and WT1 is specific and occurs endogenously in cells . The mouse homologue of WTAP was isolated and found to be >90% conserved at the nucleotide and protein levels . The human and mouse genes were mapped using fluorescence in situ hybridization to regions in chromosomes 6 (which is thought to harbour a tumour suppressor gene) and 17, respectively . The expression pattern of WTAP was investigated and shown to be ubiquitous, perhaps reflecting a housekeeping role . WTAP is a nuclear protein, which like WT1 localizes throughout the nucleoplasm as well as in speckles and partially co-localizes with splicing factors . Although the significance of this interaction is not yet known, WTAP promises to be an interesting WT1-binding partner.

Nature, 2000 Sep 14, 407(6801), 202 - 7
CAP defines a second signalling pathway required for insulin-stimulated glucose transport; Baumann CA et al.; Insulin stimulates the transport of glucose into fat and muscle cells . Although the precise molecular mechanisms involved in this process remain uncertain, insulin initiates its actions by binding to its tyrosine kinase receptor, leading to the phosphorylation of intracellular substrates . One such substrate is the Cbl proto-oncogene product . Cbl is recruited to the insulin receptor by interaction with the adapter protein CAP, through one of three adjacent SH3 domains in the carboxy terminus of CAP . Upon phosphorylation of Cbl, the CAP-Cbl complex dissociates from the insulin receptor and moves to a caveolin-enriched, triton-insoluble membrane fraction . Here, to identify a molecular mechanism underlying this subcellular redistribution, we screened a yeast two-hybrid library using the amino-terminal region of CAP and identified the caveolar protein flotillin . Flotillin forms a ternary complex with CAP and Cbl, directing the localization of the CAP-Cbl complex to a lipid raft subdomain of the plasma membrane . Expression of the N-terminal domain of CAP in 3T3-L1 adipocytes blocks the stimulation of glucose transport by insulin, without affecting signalling events that depend on phosphatidylinositol-3-OH kinase . Thus, localization of the Cbl-CAP complex to lipid rafts generates a pathway that is crucial in the regulation of glucose uptake.

Mol Cell Endocrinol, 2000 Sep 25, 167(1-2), 139 - 50
Interactions between androgen and estrogen receptors and the effects on their transactivational properties; Panet-Raymond V et al.; The physiological interplay of androgen and estrogen action in endocrine tissues is well recognized . The biochemical processes responsible for this interplay have yet to be fully defined . We have demonstrated that the androgen receptor (AR) and estrogen receptor-alpha (ERalpha) can interact directly using the yeast and mammalian two-hybrid systems . These interactions occurred between the C-terminal ERalpha ligand-binding domain and either the N-terminal AR transactivational domain or the full-length AR . Estrogen receptor-beta (ERbeta) did not interact with the AR . DNA cotransfection studies employing AR, ERalpha and ERbeta expression vectors and AR- or ER-reporter gene constructs were used to identify and measure potential functional effects of AR-ER interaction . Coexpression of ERalpha with AR decreased AR transactivation by 35%; coexpression of AR with ERalpha decreased ERalpha transactivation by 74% . Coexpression of AR and ERbeta did not significantly modulate AR or ERbeta transactivation . In summary, we have shown that specific domains of AR and ERalpha physically interact and have demonstrated the functional consequences of such interaction . These results may help explain the nature of the physiological interplay between androgens and estrogens.

Nucleic Acids Res, 2000 Oct 1, 28(19), 3771 - 8
Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells; Sargent RG et al.; Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates . Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1(-) cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3' tails . To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3' tails after treatment with the rare-cutting endonuclease I-SCE:I . Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells . Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3' tails, like its Rad1/Rad10 counterpart in yeast . Thus, it is likely that misprocessing of non-homologous 3' tails is the primary source of recombination-dependent rearrangements in mammalian cells . We also find an unexpected effect of ERCC1 deficiency on I-SCE:I-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SCE:I-induced double-strand breaks.

J Virol, 2000 Oct, 74(20), 9637 - 45
Human herpesvirus 8 LANA interacts with proteins of the mSin3 corepressor complex and negatively regulates Epstein-Barr virus gene expression in dually infected PEL cells; Krithivas A et al.; The human herpesvirus 8 (HHV-8) latency-associated nuclear antigen (LANA) is expressed in all latently HHV-8 infected cells and in HHV-8-associated tumors, including primary effusion lymphoma (PEL) . To better understand the contribution of LANA to tumorigenesis and to the PEL phenotype, we performed a yeast two-hybrid screen which identified the corepressor protein SAP30 as a LANA binding protein . SAP30 is a constituent of a large multicomponent complex that brings histone deacetylases to the promoter . Glutathione S-transferase affinity assays confirmed interaction between LANA and SAP30 and also demonstrated interactions between LANA and two other members of the corepressor complex, mSin3A and CIR . The corepressors bound to the amino-terminal 340-amino-acid domain of LANA . In transient expression assays, this same domain of LANA mediated repression when targeted to a 5xGal4tk-CAT reporter as a GAL4-LANA fusion . PEL cells have the unusual feature that they are frequently dually infected with both HHV-8 and Epstein-Barr virus (EBV) . We found that EBV EBNA-1 expression is downregulated in PEL cells at both the RNA and protein levels . In transient expression assays, LANA repressed activated expression from the EBV Qp and Cp latency promoters . Reduction of endogenous Qp activity could also be demonstrated in EBV-infected Rael cells transfected with a LANA expression plasmid . In contrast to the effect of LANA on EBV latency promoters, LANA activated expression from its own promoter . The data indicate that LANA can mediate transcriptional repression through recruitment of an mSin3 corepressor complex and further that LANA-mediated repression is likely to contribute to the low level of EBV latency gene expression seen in dually infected PEL cells.

J Nat Prod, 2000 Sep, 63(9), 1273 - 6
Use of COMPARE analysis to discover new natural product drugs: isolation of camptothecin and 9-methoxycamptothecin from a new source; Zhou BN et al.; Analysis of cytotoxicity data of extracts from the National Cancer Institute's Active Repository by the COMPARE protocol was carried out using camptothecin as a reference point . Extracts identified by this process were further characterized by a selective yeast bioassay for inhibitors of topoisomerase I and by a biochemical assay for compounds that stabilize the topoisomerase I-DNA covalent binary complex . Five of the extracts were positive in the yeast bioassay, and eight extracts showed activity on the assay that monitors stabilization of the topoisomerase I-DNA complex . Four of the latter extracts were inactive in the yeast bioassay, and thus would not have been identified as hits without the COMPARE preselection process . One of the extracts, from Pyrenacantha klaineana, was selected for detailed investigation, and fractionation of this extract yielded camptothecin and 9-methoxycamptothecin as the bioactive constituents.

RNA, 2000 Sep, 6(9), 1226 - 35
Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay; Bhattacharya A et al.; The Upf1 protein in yeast has been implicated in the modulation of efficient translation termination as well as in the accelerated turnover of mRNAs containing premature stop codons, a phenomenon called nonsense-mediated mRNA decay (NMD) . A human homolog of the yeast UPF1, termed HUpf1/RENT1, has also been identified . The HUpf1 has also been shown to play a role in NMD in mammalian cells . Comparison of the yeast and human UPF1 proteins demonstrated that the amino terminal cysteine/histidine-rich region and the region comprising the domains that define this protein as a superfamily group I helicase have been conserved . The yeast Upf1p demonstrates RNA-dependent ATPase and 5' --> 3' helicase activities . In this paper, we report the expression, purification, and characterization of the activities of the human Upf1 protein . We demonstrate that human Upf1 protein displays a nucleic-acid-dependent ATPase activity and a 5'--> 3' helicase activity . Furthermore, human Upf1 is an RNA-binding protein whose RNA-binding activity is modulated by ATP . Taken together, these results indicate that the activities of the Upf1 protein are conserved across species, reflecting the conservation of function of this protein throughout evolution.

Plant Mol Biol, 2000 Jun, 43(2-3), 221 - 34
Transcriptional transgene silencing and chromatin components; Meyer P; Contrary to simplistic views that have long prevailed in genetics textbooks, gene transcription in higher organisms cannot be fully understood by analysing binding of transcription factors to DNA target sites within the promoter regions, just as it would be inappropriate to picture the genetic information within a nucleus as a simple string of DNA . Instead, DNA is embedded in a highly complex chromatin structure that controls the location and accessibility of individual genetic regions in a way we are still far from understanding in detail . What has become obvious, mainly due to ground-breaking research in yeast and animal systems, is that the packaging of certain genes into a chromosomal matrix is regulated via sophisticated chromatin remodelling mechanisms that define whether and when a gene becomes accessible to the transcription machinery . In plants, especially the analysis of transgenes and transposable elements has reminded us of the presence of epigenetic control mechanisms, which can affect the reliable expression of transgenes . There is increasing evidence that chromatin components play an important part in plant epigenetics . The purpose of this review is to describe the main general principles of chromatin remodelling as they have been elucidated in non-plant systems and to discuss their relevance for the control of gene expression in plants.

Nucleosides Nucleotides Nucleic Acids, 2000 Jul, 19(7), 1145 - 58
Invasion of strongly binding oligonucleotides into tRNA structure; Petyuk V et al.; Interaction of yeast tRNA(Phe) with oligodeoxyribonucleotides containing 5-methylcytosine, 2-aminoadenine, and 5-propynyl-2'-deoxyuridine was investigated . The modified oligonucleotides show increased binding capacity although the association rates are similar for the modified and natural oligonucleotides . The most pronounced increase in association constant (70 times) due to the incorporation of the strongly binding units was achieved in the case of oligonucleotide complementary to the sequence 65-76 of the tRNA(Phe).

Biochem J, 2000 Oct 1, 351(Pt 1), 215 - 20
The catabolic function of the alpha-aminoadipic acid pathway in plants is associated with unidirectional activity of lysine-oxoglutarate reductase, but not saccharopine dehydrogenase; Zhu X et al.; Whereas plants and animals use the alpha-aminoadipic acid pathway to catabolize lysine, yeast and fungi use the very same pathway to synthesize lysine . These two groups of organisms also possess structurally distinct forms of two enzymes in this pathway, namely lysine-oxoglutarate reductase (lysine-ketoglutarate reductase; LKR) and saccharopine dehydrogenase (SDH): in plants and animals these enzymes are linked on to a single bifunctional polypeptide, while in yeast and fungi they exist as separate entities . In addition, yeast LKR and SDH possess bi-directional activities, and their anabolic function is regulated by complex transcriptional and post-transcriptional controls, which apparently ascertain differential accumulation of intermediate metabolites; in plants, the regulation of the catabolic function of these two enzymes is not known . To elucidate the regulation of the catabolic function of plant bifunctional LKR/SDH enzymes, we have used yeast as an expression system to test whether a plant LKR/SDH also possesses bi-directional LKR and SDH activities, similar to the yeast enzymes . The Arabidopsis enzyme complemented a yeast SDH, but not LKR, null mutant . Identical results were obtained when deletion mutants encoding only the LKR or SDH domains of this bifunctional polypeptide were expressed individually in the yeast cells . Moreover, activity assays showed that the Arabidopsis LKR possessed catabolic, but not anabolic, activity, and its uni-directional activity stems from its structure rather than its linkage to SDH . Our results suggest that the uni-directional activity of LKR plays an important role in regulating the catabolic function of the alpha-amino adipic acid pathway in plants.

Biochem J, 2000 Oct 1, 351(Pt 1), 1 - 12
Amino acid regulation of gene expression; Fafournoux P et al.; The impact of nutrients on gene expression in mammals has become an important area of research . Nevertheless, the current understanding of the amino acid-dependent control of gene expression is limited . Because amino acids have multiple and important functions, their homoeostasis has to be finely maintained . However, amino-acidaemia can be affected by certain nutritional conditions or various forms of stress . It follows that mammals have to adjust several of their physiological functions involved in the adaptation to amino acid availability by regulating the expression of numerous genes . The aim of the present review is to examine the role of amino acids in regulating mammalian gene expression and protein turnover . It has been reported that some genes involved in the control of growth or amino acid metabolism are regulated by amino acid availability . For instance, limitation of several amino acids greatly increases the expression of the genes encoding insulin-like growth factor binding protein-1, CHOP (C/EBP homologous protein, where C/EBP is CCAAT/enhancer binding protein) and asparagine synthetase . Elevated mRNA levels result from both an increase in the rate of transcription and an increase in mRNA stability . Several observations suggest that the amino acid regulation of gene expression observed in mammalian cells and the general control process described in yeast share common features . Moreover, amino acid response elements have been characterized in the promoters of the CHOP and asparagine synthetase genes . Taken together, the results discussed in the present review demonstrate that amino acids, by themselves, can, in concert with hormones, play an important role in the control of gene expression.

Biochemistry, 2000 Sep 12, 39(36), 11065 - 73
ASD4, a new GATA factor of Neurospora crassa, displays sequence-specific DNA binding and functions in ascus and ascospore development; Feng B et al.; A new gene encoding a novel GATA factor, ASD4, of Neurospora crassa was isolated and demonstrated to possess one intron and to specify an open reading frame encoding a protein with 427 amino acid residues . The ASD4 protein contains a single GATA-type zinc finger and a putative coiled-coil domain . Unlike related proteins, DAL80 in yeast and NREB in Penicillium, ASD4 does not appear to be involved in regulation of nitrogen metabolism . An Asd-4 null mutant obtained by the rip procedure did not show any effect upon nitrogen control, but instead resulted in severe defects in ascus and ascospore genesis . The Asd-4 rip mutant is dominant to Asd-4+ . A cross of the Asd-4 mutant with wild-type resulted in fruiting bodies that appeared to be normal macroscopically but which were complete devoid of asci and ascospores . Introduction of the Asd-4+ gene into the Asd-4 rip mutant corrected the defect in ascus and ascospore development in crosses with wild-type . Mobility shift assays demonstrated that ASD4 acts as a sequence-specific DNA binding protein and recognizes DNA fragments that contain GATA core elements . Gel filtration and cross-linking experiments revealed that the ASD4 protein exists as a tetramer in solution . These results suggest that the ASD4 protein functions positively as a transcriptional regulator of sexual development in Neurospora.

Eur J Biochem, 2000 Oct, 267(19), 6025 - 43
Insect chitin synthase cDNA sequence, gene organization and expression; Tellam RL et al.; Chitin is a major component of the cuticle of arthropods . However, the synthesis of chitin is poorly understood . Feeding larvae of the insect Lucilia cuprina on the fungal chitin synthase competitive inhibitor, nikkomycin Z resulted in strong concentration-dependent mortality of the larvae (LD50 = 280 nM) . This result demonstrates that chitin is an essential component of this insect . The complete cDNA and deduced amino-acid sequences of the first arthropod chitin synthase-like protein, LcCS-1, from the larvae of the insect L . cuprina have been determined . The cDNA sequence is 5757 bp in length and codes for a large complex protein containing 1592 amino acids (Mr = 180 717) . Analysis of the whole protein sequence reveals low, but significant, similarity to yeast chitin synthases with stronger areas of conservation centred on local regions implicated in the active sites of the yeast enzymes . Strikingly, LcCS-1 contains 15-18 potential transmembrane segments, indicating that the protein is an integral membrane protein . Two alternative topographical models of LcCS-1 are described, which involve its association with either the plasma membrane or the membrane of intracellular vesicles . LcCS-1 mRNA is produced in all life stages of the insect with expression in the larval stage limited to the integument and trachea . In a third instar larva the mRNA was localized to a single layer of epidermal cells immediately underlying the procuticle region of the integument . cDNA or genomic sequences that are highly related to fragments of LcCS-1 were demonstrated in three insect orders, one arachnid and Caenorhabditis elegans, thereby attesting to the importance of this enzyme in these chitin-producing organisms . Bioinformatics has been used to deduce the gene sequence and organization of the highly homologous Drosophila melanogaster orthologue of LcCS-1, DmCS-1.

DNA Res, 2000 Aug 31, 7(4), 273 - 81
Prediction of the coding sequences of unidentified human genes . XVIII . The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro; Nagase T et al.; In our series of human cDNA projects for accumulating sequence information on the coding sequences of unidentified genes, we herein present the entire sequences of 100 cDNA clones of unidentified genes, named KIAA1544 to KIAA1643, from two sets of size-fractionated human adult and fetal brain cDNA libraries . The average sizes of the inserts and corresponding open reading frames of cDNA clones analyzed here reached 4.6 kb and 2.8 kb (930 amino acid residues), respectively . By computer-assisted database search of the deduced amino acid sequences, 48 predicted gene products were classified into the five functional categories of proteins relating to cell signaling/communication, nucleic acid management, cell structure/motility, protein management and metabolism . Homology search against the databases for proteins deduced from yeast, nematode and fly full genome sequences revealed only one gene (KIAA1630) was entirely conserved among human and these three organisms in the 100 genes reported here . Additionally, their chromosomal loci were determined by using human-rodent hybrid panels unless they were already assigned in the public databases . Furthermore, the expression profiles of the genes were also studied in 10 human tissues, 8 brain regions, spinal cord, fetal brain and fetal liver by reverse transcription-coupled polymerase chain reaction, products of which were quantified by enzyme-linked immunosorbent assay.

Curr Biol, 2000 Sep 21, 10(18), 1147 - 50
Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation; Wakiyama M et al.; The poly(A)-binding protein Pab1p interacts directly with the eukaryotic translation initiation factor 4G (eIF4G) to facilitate translation initiation of polyadenylated mRNAs in yeast {1,2} . Although the eIF4G-PABP interaction has also been demonstrated in a mammalian system {3,4}, its biological significance in vertebrates is unknown . In Xenopus oocytes, cytoplasmic polyadenylation of several mRNAs coincides with their translational activation and is critical for maturation {5-7} . Because the amount of PABP is very low in oocytes {8}, it has been argued that the eIF4G-PABP interaction does not play a major role in translational activation during oocyte maturation . Also, overexpression of PABP in Xenopus oocytes has only a modest stimulatory effect on translation of polyadenylated mRNA and does not alter either the efficiency or the kinetics of progesterone-induced maturation {9} . Here, we report that the expression of an eIF4GI mutant defective in PABP binding in Xenopus oocytes reduces translation of polyadenylated mRNA and dramatically inhibits progesterone-induced maturation . Our results show that the eIF4G-PABP interaction is critical for translational control of maternal mRNAs during Xenopus development.

FEBS Lett, 2000 Sep 15, 481(2), 147 - 51
Characterization of Solt, a novel SoxLZ/Sox6 binding protein expressed in adult mouse testis; Yamashita A et al.; SoxLZ/Sox6, a member of the Sox protein family, contains a leucine zipper motif in addition to an HMG box, which is its DNA binding domain . Here we have identified a novel SoxLZ/Sox6 binding protein, termed Solt, which we obtained independently using both a far-Western blot and a yeast two-hybrid screen . Like SoxLZ/Sox6 mRNA, Solt mRNA was exclusively expressed in the testis in mouse . Solt contains an unusual leucine zipper, which bound to the leucine zipper region of SoxLZ/Sox6 in vitro . In transient transfection assays in CHO cells with SoxLZ/Sox6 containing the transactivational region of herpes simplex virus VP16, expression of a reporter gene that carries a cis binding region for Sox proteins was significantly enhanced by the co-expression of Solt and Ca(2+)/calmodulin-dependent protein kinase IV.

Curr Biol, 2000 Sep 7, 10(17), 1075 - 8
INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow; Adams RR et al.; Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements . The 'chromosomal passenger' proteins {1}, which include the inner centromere protein (INCENP {2}), the Aurora-related serine-threonine protein kinase AIRK2 {3,4} and the unidentified human autoantigen TD-60 {5}, have been suggested to integrate mitotic events . These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase . Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis {6,7}, whereas interference with AIRK2 function primarily affects cytokinesis {3,8} . Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro . This association was found to be evolutionarily conserved . Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box . This interaction between INCENP and Aurora kinase was found to be biologically relevant . INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.

J Biol Chem, 2000 Dec 29, 275(52), 41287 - 98
Structural basis for catalysis and inhibition of N-glycan processing class I alpha 1,2-mannosidases; Vallee F et al.; Endoplasmic reticulum (ER) class I alpha1,2-mannosidase (also known as ER alpha-mannosidase I) is a critical enzyme in the maturation of N-linked oligosaccharides and ER-associated degradation . Trimming of a single mannose residue acts as a signal to target misfolded glycoproteins for degradation by the proteasome . Crystal structures of the catalytic domain of human ER class I alpha1,2-mannosidase have been determined both in the presence and absence of the potent inhibitors kifunensine and 1-deoxymannojirimycin . Both inhibitors bind to the protein at the bottom of the active-site cavity, with the essential calcium ion coordinating the O-2' and O-3' hydroxyls and stabilizing the six-membered rings of both inhibitors in a (1)C(4) conformation . This is the first direct evidence of the role of the calcium ion . The lack of major conformational changes upon inhibitor binding and structural comparisons with the yeast alpha1, 2-mannosidase enzyme-product complex suggest that this class of inverting enzymes has a novel catalytic mechanism . The structures also provide insight into the specificity of this class of enzymes and provide a blueprint for the future design of novel inhibitors that prevent degradation of misfolded proteins in genetic diseases.

J Biol Chem, 2000 Dec 29, 275(52), 40765 - 76
Identification and characterization of a SUMO-1 conjugation system that modifies neuronal calcium/calmodulin-dependent protein kinase II in Drosophila melanogaster; Long X et al.; Drosophila Uba2 and Ubc9 SUMO-1 conjugation enzyme homologs (DmUba2 and DmUbc9) were isolated as calcium/calmodulin-dependent kinase II (CaMKII) interacting proteins by yeast two-hybrid screening of an adult head cDNA library . We found that at least one isoform of Drosophila neuronal CaMKII is conjugated to DmSUMO-1 in vivo . The interactions observed in the two-hybrid screen may therefore reflect catalytic events . To understand the role of SUMO conjugation in the brain, we undertook a characterization of the system . The other required components of the system, Drosophila Aos1 and SUMO-1 (DmAos1 and DmSUMO-1), were identified in expressed sequence tag data base searches . Purified recombinant DmUba2/DmAos1 dimer can activate DmSUMO-1 in vitro and transfer DmSUMO-1 to recombinant DmUbc9 . DmSUMO-1 conjugation occurs in all developmental stages of Drosophila and in the adult central nervous system . Overexpression of a putative dominant negative DmUba2(C175S) mutant protein in the Drosophila central nervous system resulted in an increase in overall DmSUMO-1 conjugates and a base-sensitive p120 species, which is likely to be DmUba2(C175S) linked to endogenous DmSUMO-1 through an oxygen ester bond . Overexpression of DmUba2(wt) protein in vivo also led to increased levels of DmSUMO-1 conjugates . High level overexpression of either DmUba2(wt) or DmUba2(C175S) in the Drosophila central nervous system caused pupal and earlier stage lethality . Expression in the developing eye led to a rough eye phenotype with retinal degeneration . These results suggest that normal SUMO conjugation is essential in the differentiated nervous system and reveal a potential novel mechanism that regulates neuronal calcium/calmodulin-dependent protein kinase II function.

Genomics, 2000 Sep 15, 68(3), 322 - 9
Physical localization of the mesoderm development (mesd) functional region; Wines ME et al.; The mesoderm development (mesd) functional interval is essential for primitive streak formation and mesoderm induction . Mesd is defined by overlapping albino (c) deletions on chromosome 7 . We have constructed a bacterial artificial chromosome (BAC) contig that spans the mesd functional region . BAC end-sequence identifies three segments that recognize novel expressed sequences . Localization of the proximal breakpoints from Del(7)Tyr(c-3YPSd) and Del(7)Tyr(c-112K) within the contig defines a deletion interval of 310-350 kb that is essential for mesd function . Importantly, using BAC transgene rescue, we define a 75-kb mesd critical region containing at least one expressed sequence .

Genomics, 2000 Sep 15, 68(3), 273 - 82
A 2-Mb YAC/BAC-based physical map of the ovum mutant (Om) locus region on mouse chromosome 11; Cohen-Tannoudji M et al.; The embryonic lethal phenotype observed when DDK females are crossed with males from other strains results from a deleterious interaction between the egg cytoplasm and the paternal pronucleus soon after fertilization . We have previously mapped the Om locus responsible for this phenotype, called the DDK syndrome, to an approximately 2-cM region of chromosome 11 . Here, we report the generation of a physical map of 28 yeast and bacterial artificial chromosome clones encompassing the entire genetic interval containing the Om locus . This contig, spanning approximately 2 Mb, was used to map precisely genes and genetic markers of the region . We determined the maximum physical interval for Om to be 1400 kb . In addition, 11 members of the Scya gene family were found to be organized into two clusters at the borders of the Om region . Two other genes (Rad51l3 and Schlafen 2) and one EST (D11Wsu78e) were also mapped in the Om region . This integrated map provides support for the identification of additional candidate genes for the DDK syndrome .

Genomics, 2000 Sep 15, 68(3), 264 - 72
Childhood absence epilepsy in 8q24: refinement of candidate region and construction of physical map; Sugimoto Y et al.; Childhood absence epilepsy (CAE), one of the common idiopathic generalized epilepsies, accounts for 8 to 15% of all childhood epilepsies . Inherited as an autosomal dominant trait, frequent absence attacks start in early or midchildhood and disappear by 30 years of age or may persist through life . Recently, we mapped the locus for CAE persisting with tonic-clonic seizures to chromosome 8q24 (ECA1) by genetic linkage analysis . As a further step in the identification of the ECA1 gene, we constructed a bacterial artificial chromosome- and yeast artificial chromosome-based physical map for the 8q24 region, spanning about 3 Mb between D8S1710 and D8S523 . Accurately ordered STS markers within the physical map aided in the analysis of haplotypes and recombinations and reduced the ECA1 region to 1.5 Mb flanked by D8S554 and D8S502 . Pairwise analysis in six families confirmed linkage with a pooled lod score of 4.10 (θ = 0) at D8S534 . The sequence-ready physical map as well as the narrowed candidate region described here should contribute to the identification of the ECA1 gene .

J Agric Food Chem, 2000 Sep, 48(9), 3898 - 905
Quantitative study of the formation of endoproteolytic activities during malting and their stabilities to kilning; Jones BL et al.; The proteinases of germinating barley (Hordeum vulgare L.) hydrolyze storage proteins into amino acids and small peptides that can be used by the growing plant or, during brewing, by yeast . They are critical for the malting and brewing processes because several aspects of brewing are affected by the amounts of protein, peptide, and amino acids that are in the wort . This study was carried out to quantitatively measure when endoproteinases form in green malt and whether they are inactivated at the high temperatures that occur during malt kilning . Little endoproteolytic activity was present in ungerminated barley, but the activities began forming 1 day into the "germination" phase of malting, and they were nearly maximal by the third germination day . Quantitative studies with azogelatin "in solution" assays showed that the green malt endoproteolytic activities were not inactivated under commercial kilning conditions that use temperatures as high as 85 degrees C but that some actually increased during the final kilning step . Qualitative (2-D, IEF x PAGE) analyses, which allow the study of individual proteases, showed that some of the enzymes were affected by heating at 68 and 85 degrees C, during the final stages of kilning . These changes obviously did not, however, decrease the overall proteolytic activity.

Cell Tissue Res, 2000 Sep, 301(3), 329 - 40
Subcellular localization of protein kinase CK2 . A key to its function?
Faust M, Montenarh M.
More than 46 years ago, Burnett and Kennedy first described protein kinase CK2 (formerly known as casein kinase 2) in liver extracts . Since then, protein kinase CK2 has been investigated in many organisms from yeast to man . It is now well established that protein kinase CK2 is a pleiotropic and ubiquitous serine or threonine kinase, which is highly conserved during evolution . A great number of studies deal with substrates of CK2, but the fact that over 160 substrates exist is more confusing than elucidatory . The holoenzyme is composed of two regulatory beta-subunits and two catalytic alpha- or alpha'-subunits . There is now increasing evidence for individual functions of the subunits that are different from their functions in the holoenzyme . Furthermore, more and more studies describe interacting partners of the kinase that may be decisive in the regulation of this enzyme . A big step forward has been the determination of the crystal structure of the two subunits of protein kinase CK2 . Now the interactions of the catalytic subunit of CK2 with ATP as well as GTP and the interaction between the regulatory subunits can be explained . However, cellular functions of protein kinase CK2 still remain unclear . In the present review we will focus our interest on the subcellular localization of protein kinase CK2 . Protein kinase CK2 is found in many organisms and tissues and nearly every subcellular compartment . There is ample evidence that protein kinase CK2 has different functions in these compartments and that the subcellular localization of protein kinase CK2 is tightly regulated . Therefore studying the subcellular localization of protein kinase CK2 may be a key to its function.

J Biol Chem, 2000 Dec 29, 275(52), 41192 - 200
hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions; Nix SL et al.; Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane . All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK) . We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes . We now report that hCASK binds another MAGUK, human discs large (hDlg) . Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine . These proteins co-precipitate from lysates of an intestinal cell line, Caco-2 . The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells . In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding . The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK . Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo . These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.

J Mol Biol, 2000 Sep 29, 302(4), 991 - 1004
The tRNA-dependent activation of arginine by arginyl-tRNA synthetase requires inter-domain communication; Lazard M et al.; The tRNA-dependent amino acid activation catalyzed by mammalian arginyl-tRNA synthetase has been characterized . A conditional lethal mutant of Chinese hamster ovary cells that exhibits reduced arginyl-tRNA synthetase activity (Arg-1), and two of its derived revertants (Arg-1R4 and Arg-1R5) were analyzed at the structural and functional levels . A single nucleotide change, resulting in a Cys to Tyr substitution at position 599 of arginyl-tRNA synthetase, is responsible for the defective phenotype of the thermosensitive and arginine hyper-auxotroph Arg-1 cell line . The two revertants have a single additional mutation resulting in a Met222 to Ile change for Arg-1R4 or a Tyr506 to Ser change for Arg-1R5 . The corresponding mutant enzymes were expressed in yeast and purified . The Cys599 to Tyr mutation affects both the thermal stability of arginyl-tRNA synthetase and the kinetic parameters for arginine in the ATP-PP(i) exchange and tRNA aminoacylation reactions . This mutation is located underneath the floor of the Rossmann fold catalytic domain characteristic of class 1 aminoacyl-tRNA synthetases, near the end of a long helix belonging to the alpha-helix bundle C-terminal domain distinctive of class 1a synthetases . For the Met222 to Ile revertant, there is very little effect of the mutation on the interaction of arginyl-tRNA synthetase with either of its substrates . However, this mutation increases the thermal stability of arginyl-tRNA synthetase, thereby leading to reversion of the thermosensitive phenotype by increasing the steady-state level of the enzyme in vivo . In contrast, for the Arg-1R5 cell line, reversion of the phenotype is due to an increased catalytic efficiency of the C599Y/Y506S double mutant as compared to the initial C599Y enzyme . In light of the location of the mutations in the 3D structure of the enzyme modeled using the crystal structure of the closely related yeast arginyl-tRNA synthetase, the kinetic analysis of these mutants suggests that the obligatory tRNA-induced activation of the catalytic site of arginyl-tRNA synthetase involves interdomain signal transduction via the long helices that build the tRNA-binding domain of the enzyme and link the site of interaction of the anticodon domain of tRNA to the floor of the active site .

Dev Biol, 2000 Oct 1, 226(1), 45 - 56
The Caenorhabditis elegans Ldb/NLI/Clim orthologue ldb-1 is required for neuronal function; Cassata G et al.; LIM homeodomain (LIM-HD) and nuclear LIM-only proteins play important roles in a variety of developmental processes in animals . In some cases their activities are modulated by a nuclear LIM binding protein family called Ldb/NLI/Clim . Here we characterize the Ldb/NLI/Clim orthologue ldb-1 of the nematode Caenorhabditis elegans . Two alternatively spliced variants exist, which differ in their amino-termini . The ldb-1 orthologue of Caenorhabditis briggsae has the same structure as that of C . elegans and is highly conserved throughout the open reading frame, while conservation to fly and vertebrate proteins is restricted to specific domains: the dimerization domain, the nuclear localization sequence, and the LIM interaction domain . C . elegans ldb-1 is expressed in neurogenic tissues in embryos, in all neurons in larval and adult stages, and in vulval cells, gonadal sheath cells, and some body muscle cells . C . elegans LDB-1 is able to specifically bind LIM domains in yeast two-hybrid assays . RNA inactivation studies suggest that C . elegans ldb-1 is not required for the differentiation of neurons that express the respective LIM-HD genes or for LIM-HD gene autoregulation . In contrast, ldb-1 is necessary for several neuronal functions mediated by LIM-HD proteins, including the transcriptional activation of mec-2, the mechanosensory neuron-specific stomatin .

Nature, 2000 Sep 7, 407(6800), 102 - 6
TFIIH is negatively regulated by cdk8-containing mediator complexes; Akoulitchev S et al.; The mammalian cyclin-dependent kinase 8 (cdk8) gene has been linked with a subset of acute lymphoblastic leukaemias, and its corresponding protein has been functionally implicated in regulation of transcription . Mammalian cdk8 and cyclin C, and their respective yeast homologues, Srb10 and Srb11, are components of the RNA polymerase II holoenzyme complex where they function as a protein kinase that phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (ref . 7) . The yeast SRB10 and SRB11 genes have been implicated in the negative regulation of transcription . The cdk8/cyclin C protein complex is also found in a number of mammalian Mediator-like protein complexes, which repress activated transcription independently of the CTD in vitro . Here we show that cdk8/cyclin C can regulate transcription by targeting the cdk7/cyclin H subunits of the general transcription initiation factor IIH (TFIIH) . cdk8 phosphorylates mammalian cyclin H in the vicinity of its functionally unique amino-terminal and carboxy-terminal alpha-helical domains . This phosphorylation represses both the ability of TFIIH to activate transcription and its CTD kinase activity . In addition, mimicking cdk8 phosphorylation of cyclin H in vivo has a dominant-negative effect on cell growth . Our results link the Mediator complex and the basal transcription machinery by a regulatory pathway involving two cyclin-dependent kinases . This pathway appears to be unique to higher organisms.

Infect Immun, 2000 Oct, 68(10), 5530 - 8
A region of Plasmodium falciparum antigen Pfs25 that is the target of highly potent transmission-blocking antibodies; Stowers AW et al.; Each of the four epidermal growth factor (EGF)-like domains of the Plasmodium falciparum sexual-stage antigen Pfs25 has been individually expressed as a yeast-secreted recombinant protein (yEGF1 through yEGF4) . All four are recognized by the immune sera of animals and humans vaccinated with TBV25H (the corresponding yeast-secreted full-length recombinant form of Pfs25), with antibody titers to yEGF1 and yEGF2 weakly correlating with the ability of the sera to block the transmission of parasites to the mosquito host . All four proteins are poorly immunogenic in mice vaccinated with aluminum hydroxide-absorbed formulations . However, all four successfully primed the mice to mount an effective secondary antibody response after a single boost with TBV25H . Sera from mice vaccinated with yEGF2-TBV25H completely block the development of oocysts in mosquito midguts in membrane-feeding assays . Further, of the four proteins, only the depletion of antibodies to yEGF2 from the sera of rabbits vaccinated with TBV25H consistently abolished the ability of those sera to block oocyst development . Thus, antibodies to the second EGF-like domain of Pfs25 appear to mediate a very potent blocking activity, even at low titers . Vaccination strategies that target antibody response towards this domain may improve the efficacy of future transmission-blocking vaccines.

J Biol Chem, 2000 Dec 15, 275(50), 39497 - 506
Domain interactions in the gelatinase A.TIMP-2.MT1-MMP activation complex . The ectodomain of the 44-kDa form of membrane type-1 matrix metalloproteinase does not modulate gelatinase A activation; Overall CM et al.; On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2 . A 44-kDa remnant of MT1-MMP, with an N terminus at Gly(285), is also present on the cell after autolytic shedding of the catalytic domain from the hemopexin carboxyl (C) domain, but its role in gelatinase A activation is unknown . We investigated intermolecular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and solution-phase assays, cell culture, and immunocytochemistry . A strong interaction between the TIMP-2 C domain (Glu(153)-Pro(221)) and the gelatinase A hemopexin C domain (Gly(446)-Cys(660)) was demonstrated by the yeast two-hybrid system . Epitope masking studies showed that the anionic TIMP-2 C tail lost immunoreactivity after binding, indicating that the tail was buried in the complex . Using recombinant MT1-MMP hemopexin C domain (Gly(285)-Cys(508)), no direct role for the 44-kDa form of MT1-MMP in cell surface activation of progelatinase A was found . Exogenous hemopexin C domain of gelatinase A, but not that of MT1-MMP, blocked the cleavage of the 68-kDa gelatinase A activation intermediate to the fully active 66-kDa enzyme by concanavalin A-stimulated cells . The MT1-MMP hemopexin C domain did not form homodimers nor did it bind the gelatinase A hemopexin C domain, the C tail of TIMP-2, or full-length TIMP-2 . Hence, the ectodomain of the remnant 44-kDa form of MT1-MMP appears to play little if any role in the activation of gelatinase A favoring the hypothesis that it accumulates on the cell surface as an inactive, stable degradation product.

J Biol Chem, 2000 Dec 15, 275(50), 39081 - 9
Interaction between interferon consensus sequence-binding protein and COP9/signalosome subunit CSN2 (Trip15) . A possible link between interferon regulatory factor signaling and the COP9/signalosome; Cohen H et al.; Interferon consensus sequence-binding protein (ICSBP) is a member of the interferon regulatory factors (IRF) that has a pivotal role in mediating resistance to pathogenic infections in mice and in promoting the differentiation of myeloid cells . ICSBP exerts some of its transcriptional activities via association with other factors that enable its binding to a variety of promoters containing DNA composite elements . These interactions are mediated through a specific COOH-terminal domain termed IAD (IRF association domain) . To gain a broader insight of the capacity of ICSBP to interact with other factors, yeast two-hybrid screens were performed using ICSBP-IAD as a bait against a B-cell cDNA library . Trip15 was identified as a specific interacting factor with ICSBP in yeast cells, which was also confirmed by in vitro glutathione S-transferase pull-down assays and by coimmunoprecipitation studies in COS7 cells . Trip15 was recently identified as a component of the COP9/signalosome (CSN) complex composed of eight evolutionary conserved subunits and thus termed CSN2 . This complex has a role in cell-signaling processes, which is manifested by its associated novel kinase activity and by the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression . We show that in vitro association of ICSBP with the CSN leads to phosphorylation of ICSBP at a unique serine residue within its IAD . The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity of ICSBP exerted on IRF-1 . This suggests that the CSN has a role in integrating incoming signals that affect the transcriptional activity of ICSBP.

Hum Genet, 1999 Sep, 105(3), 217 - 25
Isolation of CAG/CTG repeats from within the chromosome 2p21-p24 locus for autosomal dominant spastic paraplegia (SPG4) by YAC fragmentation; Del-Favero J et al.; Pure autosomal dominant spastic paraplegia (SPG) is a genetically heterogeneous neurodegenerative disorder of the central nervous system clinically characterized by progressive spasticity mainly affecting the lower limbs . Three distinct loci have been mapped to chromosomes 14q (SPG3), 2p (SPG4) and 15q (SPG6) . In particular, SPG4 families show striking intrafamilial variability suggestive of anticipation and evidence has been provided that CAG/CTG repeat expansions may be involved . To isolate CAG/CTG repeat containing sequences from within the SPG4 candidate region, a novel approach was developed . Fragmentation vectors were assembled allowing direct fragmentation of yeast artificial chromosomes (YACs) with a short (> or = 21 bp) CAG/CTG sequence as the target site for homologous recombination . We used the CAG/CTG YAC fragmentation vectors to isolate CAG/CTG containing sequences from four YACs spanning the SPG4 candidate region between D2S400 and D2S367 . A total of four CAG/CTG containing sequences were isolated of which three were novel . However, none of the four CAG/CTG repeats showed expanded alleles in two Belgian SPG4 families . In addition, we showed that the CAG/CTG alleles detected by the repeat expansion detection (RED) method could be fully explained by two polymorphic nonpathogenic CAG/CTG repeats on chromosomes 17 and 18, respectively . Also, the RED expansions in six SPG families could not be explained by amplification of the CAG/CTG repeats at the SPG4 locus . Together, our data do not support the hypothesis of a CAG/CTG repeat expansion as the molecular mechanism underlying SPG4 pathology.

Cancer Res, 2000 Sep 1, 60(17), 4729 - 34
Isolation and mapping of a human septin gene to a region on chromosome 17q, commonly deleted in sporadic epithelial ovarian tumors; Russell SE et al.; Allele losses from chromosome 17 are common in sporadic ovarian tumors . Previously, we reported high rates of LOH (up to 70%) from 17q25 at the marker THH59 in a bank of malignant ovarian tumors . We have extended this study to 70 tumors with 17 markers from the long arm of chromosome 17 . In most cases, the data are consistent with whole chromosome loss, but we have identified a minimal region of deletion that is centered around 4 microsatellites with zero recombination at map position 106.9 cM . A P1/BAC contig across the region (approximately 200 kb) was constructed and used to determine the precise position and order of the microsatellites . The contig was shown to hybridize to 17q25 by fluorescence in situ hybridization analysis . The DNA sequence of the entire contig was determined and analyzed by BLAST searches . A 4-kb cDNA was subsequently identified with homology to the yeast, Drosophila and mammalian septin family of genes . We have designated this gene Ovarian/Breast (Ov/Br) septin . Two splice variants were demonstrated within the 200-kb contig, which differ only at exon 1 . Within the contig, approximately 45% of the septin alpha transcript was identified and 38% of the septin beta transcript . The septins are a family of genes involved in cytokinesis and cell cycle control . Their known functions are consistent with the hypothesis that the human 17q25 septin gene is a candidate for the ovarian tumor suppressor gene.

Cancer Res, 2000 Sep 1, 60(17), 4697 - 700
Aberrant transcripts of the cyclin-dependent kinase-associated protein phosphatase in hepatocellular carcinoma; Yeh CT et al.; The cyclin-dependent kinase (Cdk)-associated protein phosphatase (KAP) is a human dual specificity protein phosphatase that dephosphorylates Cdk2 on threonine 160 in a cyclin-dependent manner . To investigate whether mutations of this enzyme occur in hepatocellular carcinoma (HCC), KAP mRNA was analyzed by reverse transcription-PCR (RT-PCR), followed by cloning and sequencing . Eight of 14 biopsy tissues obtained from advanced HCC, 6 of 13 surgically removed HCC tissues, and 2 of the adjacent noncancerous tissues contained aberrant KAP transcripts . Using the yeast two-hybrid system, five of seven representative KAP mutants were shown to be defective in interacting with Cdk2 . These data suggest a possible role of KAP mutations in multiple-step hepatocarcinogenesis.

J Reprod Med, 2000 Aug, 45(8), 679 - 84
Clinical profile of vulvodynia patients . A prospective study of 300 patients; Sadownik LA; OBJECTIVE: To define the demographic and clinical characteristics of women presenting with vulvodynia . STUDY DESIGN: Vulvodynia patients seen by the author between September 1996 and June 1999 were included in the study . Patients completed a standardized questionnaire and were interviewed and clinically examined . RESULTS: Three hundred one patients completed the questionnaire . The average age was 38 years old, 72% reported postsecondary education, 54% were nulligravid, and 55% were married . Average duration of symptoms was 38 months . Patients reported dyspareunia (71%), vulvar burning (57%) and vulvar itching (46%) . One-third reported problems with sexual response . The majority (64%) reported a "history" of yeast infections . Over 64% of the time all therapeutic interventions tried by patients made the vulvar symptoms no better or worse . Approximately 55% reported another chronic health condition . Positive physical findings were often limited to inflammation in the vestibule (25%) and pain on palpation of the posterior vestibule (69%) . Patients reported that their vulvodynia limited their physical activities . CONCLUSION: Physicians should approach management of vulvodynia using a chronic pain model that emphasizes multidisciplinary health care and "improvement" in health, rather than single interventions and cure of disease.

J Basic Microbiol, 2000, 40(4), 289 - 92
Purification of a soluble cytochrome P450 from Trichosporon montevideense; Stundl UM et al.; The yeast Trichosporon montevideense CBS 6721 expressed large amounts of cytochrome P450 after cultivation in a glucose-peptone medium . The P450, which could be detected in the cytosolic fraction after cell breakage and ultracentrifugation, was purified to electrophoretic homogeneity and migrated in SDS-PAGE with a M(r) of 43,000 . As indicated by IEF, the preparation consisted of two different P450 isoforms with pI-values of 5.9 and 6.2, which were named P450MS1 and P450MS2 respectively . Both isoforms had a characteristic maximum at 446 nm in the reduced carbon monoxide difference spectra . Partial N-terminal sequencing of P450MS1 and P450MS2 demonstrated a high degree of sequence homology between the soluble P450 enzymes of T . montevideense CBS 6721 and their close relationship to the soluble P450 forms of Trichosporon spec . SBUG 752, T . cutaneum ATCC 58094 and to the P450s of the CYP55 family of Fusarium oxysporum and Cylindrocarpon tonkinense.

Curr Biol, 2000 Aug 24, 10(16), 997 - 1000
Sister-chromatid cohesion via MEI-S332 and kinetochore assembly are separable functions of the Drosophila centromere; Lopez JM et al.; Attachment, or cohesion, between sister chromatids is essential for their proper segregation in mitosis and meiosis {1,2} . Sister chromatids are tightly apposed at their centromeric regions, but it is not known whether this is due to cohesion at the functional centromere or at flanking centric heterochromatin . The Drosophila MEI-S332 protein maintains sister-chromatid cohesion at the centromeric region {3} . By analyzing MEI-S332's localization requirements at the centromere on a set of minichromosome derivatives {4}, we tested the role of heterochromatin and the relationship between cohesion and kinetochore formation in a complex centromere of a higher eukaryote . The frequency of MEI-S332 localization is decreased on minichromosomes with compromised inheritance, despite the consistent presence of two kinetochore proteins . Furthermore, MEI-S332 localization is not coincident with kinetochore outer-plate proteins, suggesting that it is located near the DNA . We conclude that MEI-S332 localization is driven by the functional centromeric chromatin, and binding of MEI-S332 is regulated independently of kinetochore formation . These results suggest that in higher eukaryotes cohesion is controlled by the functional centromere, and that, in contrast to yeast {5}, the requirements for cohesion are separable from those for kinetochore assembly.

Curr Biol, 2000 Aug 24, 10(16), R586 - 8
Cyclin transcription: Timing is everything; Breeden LL; The stage-specific activation of cyclin-dependent kinases controls the order and timing of cell-cycle transitions . Recent studies offer insight into the mechanism of cell-cycle-regulated transcription of the mitotic cyclins of budding yeast.

Proc Natl Acad Sci U S A, 2000 Oct 10, 97(21), 11557 - 62
The gamma-aminobutyric acid type A (GABAA) receptor-associated protein (GABARAP) promotes GABAA receptor clustering and modulates the channel kinetics; Chen L et al.; A microtubule-associated protein, gamma-aminobutyric acid type A (GABA(A)) receptor-associated protein (GABARAP), was previously identified as binding to the intracellular domain of GABA(A) receptors by using the yeast two-hybrid screen . In the present work, immunofluorescent staining and green fluorescent protein-tagged receptor subunits showed that GABARAP is associated with and promotes the clustering of GABA(A) receptors in QT-6 quail fibroblasts . The tubulin-binding motif of GABARAP and the gamma2 subunit of the receptor are required . Disruption of microtubules prevents the clustering in a time-dependent manner . When green fluorescent protein-tagged alpha1 or gamma2 subunit coexpressed with beta2, gamma2L, and GABARAP was used, recordings from visually identified cells revealed that clustered GABA(A) receptor had an EC(50) of about 20 microM, vs . 5.7 microM for the diffuse receptor . Clustered receptors deactivated faster and desensitized slower than the diffuse receptors, because of decrease in the apparent affinity of GABA binding . Different properties for clustered receptors relative to unclustered receptors in heterologous cells suggest that homologous differences between extrasynaptic and synaptic clustered receptors in neurons may be due to the organization of the postsynaptic machinery.

J Biol Chem, 2000 Dec 8, 275(49), 38863 - 9
ZIC2 and Sp3 repress Sp1-induced activation of the human D1A dopamine receptor gene; Yang Y et al.; The human D(1A) dopamine receptor is transcribed from a tissue-specific regulated gene under the control of two promoters . An activator region (AR1) located between nucleotides -1154 and -1136 (relative to the first ATG) enhances transcription from the upstream promoter that is active in the brain . In this investigation, we sought to identify the nuclear factors that regulate the D(1A) gene through their binding to AR1 using yeast one-hybrid screening . Sp3 and Zic2 were among the positive clones isolated . Although Sp1 was not isolated from this screening and purified Sp1 alone does not bind to AR1 in gel shift experiments, this general transcription factor binds to AR1 in the presence of D(1A) expressing NS20Y nuclear extract and activates the D(1A) promoter . Thus, Sp1 appears to require an unknown factor(s) or post-translational modification to interact with AR1 . On the other hand, Zic2 and Sp3 inhibit Sp1-induced activation of the D(1A) gene in an AR1-dependent manner . Zic2 and D(1A) genes have reciprocal brain regional distributions; Zic2 is expressed primarily in the cerebellum, and D(1A) is highly expressed in corpus striatum . These observations collectively suggest that one of the physiologic functions of Zic2 is repression of D(1A) gene transcription and that the intracellular balance among Sp1, Sp3 and Zic2 is important for regulating the tissue-specific expression of this dopamine receptor.

J Biol Chem, 2000 Dec 29, 275(52), 41234 - 42
FATZ, a filamin-, actinin-, and telethonin-binding protein of the Z-disc of skeletal muscle; Faulkner G et al.; We report the identification and characterization of a novel 32-kDa protein expressed in skeletal muscle and located in the Z-disc of the sarcomere . We found that this protein binds to three other Z-disc proteins; therefore, we have named it FATZ, gamma-filamin/ABP-L, alpha-actinin and telethonin binding protein of the Z-disc . From yeast two-hybrid experiments we are able to show that the SR3-SR4 domains of alpha-actinin 2 are required to bind the COOH-terminal region of the FATZ as does gamma-filamin/ABP-L . Furthermore, by using a glutathione S-transferase overlay assay we find that FATZ also binds telethonin . The level of FATZ protein in muscle cells increases during differentiation, being clearly detectable before the onset of myosin . Although FATZ has no known interaction domains, it would appear to be involved in a complex network of interactions with other Z-band components . On the basis of the information known about its binding partners, we could envisage a central role for FATZ in the myofibrillogenesis . After screening our muscle expressed sequence tag data base and the public expressed sequence tag data bases, we were able to assemble two other muscle transcripts that show a high level of identity with FATZ in two different domains . Therefore, FATZ may be the first member of a small family of novel muscle proteins.

J Cell Sci, 2000 Oct, 113 Pt 19, 3419 - 26
Distinct roles for pbs21 and pbs25 in the in vitro ookinete to oocyst transformation of Plasmodium berghei; Siden-Kiamos I et al.; We have developed an in vitro culture system for early sporogonic stages of Plasmodium berghei, which can be used to study developmental events normally taking place in the midgut of an infected mosquito . These include penetration of insect cells by the mature ookinete, transformation into oocysts and the early development of the latter, sustained through several rounds of nuclear division . The system, based upon co-culture of enriched ookinetes with several established insect cell lines, was used to study the development of mutant ookinetes lacking both the Pbs21 and Pbs25 surface proteins . Motility and entry of double knockout and Pbs21 single knockout ookinetes into the insect cells are normal, but the number of ookinetes successfully transforming into oocysts expressing the CSP protein are substantially reduced . Finally, using the yeast two-hybrid system we also show that Pbs25 has the capacity to homodimerise as well as to form heterodimers with Pbs21.

Mol Cell, 2000 Aug, 6(2), 395 - 407
The protooncogene TCL1 is an Akt kinase coactivator; Laine J et al.; Human T cell prolymphocytic leukemia can result from chromosomal translocations involving 14q32.1 or Xq28 regions . The regions encode a family of protooncogenes (TCL1, MTCP1, and TCL1b) of unknown function . In yeast two-hybrid screening, we found that TCL1 interacts with Akt . All TCL1 isoforms bind to the Akt pleckstrin homology domain . Both in vitro and in vivo TCL1 increases Akt kinase activity and as a consequence enhances substrate phosphorylation . In vivo, TCL1 stabilizes the mitochondrial transmembrane potential and enhances cell proliferation and survival . In vivo, TCL1 forms trimers, which associate with Akt . TCL1 facilitates the oligomerization and activation of Akt . Our data show that TCL1 is a novel Akt kinase coactivator, which promotes Akt-induced cell survival and proliferation.

Mol Cell, 2000 Aug, 6(2), 339 - 48
Control of transfer RNA maturation by phosphorylation of the human La antigen on serine 366; Intine RV et al.; Conversion of a nascent precursor tRNA to a mature functional species is a multipartite process that involves the sequential actions of several processing and modifying enzymes . La is the first protein to interact with pre-tRNAs in eukaryotes . An opal suppressor tRNA served as a functional probe to examine the activities of yeast and human (h)La proteins in this process in fission yeast . An RNA recognition motif and Walker motif in the metazoan-specific C-terminal domain (CTD) of hLa maintain pre-tRNA in an unprocessed state by blocking the 5'-processing site, impeding an early step in the pathway . Faithful phosphorylation of hLa on serine 366 reverses this block and promotes tRNA maturation . The results suggest that regulation of tRNA maturation at the level of RNase P cleavage may occur via phosphorylation of serine 366 of hLa.

J Food Prot, 2000 Sep, 63(9), 1295 - 8
Reduction of aflatoxins by Korean soybean paste and its effect on cytotoxicity and reproductive toxicity--part 1 . Inhibition of growth and aflatoxin production of Aspergillus parasiticus by Korean soybean paste (Doen-jang) and identification of the active component; Kim JG et al.; The inhibitory effect of methanol extract of Korean soybean paste on the mold growth and aflatoxin production of a toxigenic strain of Aspergillus parasiticus ATCC 15517 was studied using different concentrations of the extract in yeast-extract sucrose broth . While inhibition in mold growth due to increasing the concentration of the extract was observed, the more remarkable effect was the inhibition of aflatoxin production . Reduction of mycelial weight as a result of addition of the extract was observed to range between 1.5 to 12.9% while reduction of aflatoxin production quantified by high-performance liquid chromatography ranged from 14.3 to 41.7% . Five percent of the extract significantly reduced aflatoxin production at the end of the incubation period (P < 0.05), although the effect on mycelial growth was less pronounced . This study indicates that soybean paste could also be an effective inhibitor of aflatoxin production even though mycelial growth may be permitted . The main active component identified by gas chromatography-mass spectroscopy was linoleic acid.

Mol Cell Biol, 2000 Oct, 20(19), 7319 - 31
Transcriptional regulation of the CLC-K1 promoter by myc-associated zinc finger protein and kidney-enriched Krüppel-like factor, a novel zinc finger repressor; Uchida S et al.; The expression of CLC-K1 and CLC-K2, two kidney-specific CLC chloride channels, is transcriptionally regulated on a tissue-specific basis . Previous studies have shown that a GA element near their transcriptional start sites is important for basal and cell-specific activities of the CLC-K1 and CLC-K2 gene promoters . To identify the GA-binding proteins, the human kidney cDNA library was screened by a yeast one-hybrid system . A novel member of the Cys2-His2 zinc finger gene designated KKLF (for "kidney-enriched Kruppel-like factor") and the previously isolated MAZ (for "myc-associated zinc finger protein") were cloned . KKLF was found to be abundantly expressed in the liver, kidneys, heart, and skeletal muscle, and immunohistochemistry revealed the nuclear localization of KKLF protein in interstitial cells in heart and skeletal muscle, stellate cells, and fibroblasts in the liver . In the kidneys, KKLF protein was localized in interstitial cells, mesangial cells, and nephron segments, where CLC-K1 and CLC-K2 were not expressed . A gel mobility shift assay revealed sequence-specific binding of recombinant KKLF and MAZ proteins to the CLC-K1 GA element, and the fine-mutation assay clarified that the consensus sequence for the KKLF binding site was GGGGNGGNG . In a transient-transfection experiment, MAZ had a strong activating effect on transcription of the CLC-K1-luciferase reporter gene . On the other hand, KKLF coexpression with MAZ appeared to block the activating effect of MAZ . These results suggest that a novel set of zinc finger proteins may help regulate the strict tissue- and nephron segment-specific expression of the CLC-K1 and CLC-K2 channel genes through their GA cis element.

J Biol Chem, 2000 Dec 1, 275(48), 37588 - 95
Importance of conserved alpha -subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase; Pedersen PA et al.; The segment (708)TGDGVNDSPALKK(720) in the alpha-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg(2+) or ATP and energy transduction . Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr(708) --> Ser mutation and alterations of Asp(714) abolish all catalytic reactions . In mutations of Asp(710) and Asn(713), ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced . Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp(710) contributes to coordination of Mg(2+) during transfer of gamma-phosphate to Asp(369) in the high energy Mg.E(1)P{3Na} intermediate and that Asn(713) is involved in these processes . In contrast, Asp(710) and Asp(713) do not contribute to Mg(2+) binding in the E(2)P.ouabain complex . Transition to E(2)P thus involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713), and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369) . The Asp(710) --> Ala mutation blocks interaction with vanadate, whereas Asn(713) --> Ala interferes with phosphorylation from P(i) of the E(2).ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction . The Asp(710) --> Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl(+) 2- to 3-fold, suggesting a role in transmission of K(+) stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.

Nucleic Acids Res, 2000 Sep 15, 28(18), 3636 - 41
Anchoring of rice BAC clones to the rice genetic map in silico; Yuan Q et al.; A wealth of molecular resources have been developed for rice genomics, including dense genetic maps, expressed sequence tags (ESTs), yeast artificial chromosome maps, bacterial artificial chromosome (BAC) libraries and BAC end sequence databases . Integration of genetic and physical maps involves labor-intensive empirical experiments . To accelerate the integration of the bacterial clone resources with the genetic map for the International Rice Genome Sequencing Project, we cleaned and filtered the available EST and BAC end sequences for repetitive sequences and then searched all available rice genetic markers with our filtered databases . We identified 418 genetic markers that aligned with at least one BAC end sequence with >95% sequence identity, providing a set of large insert clones with an average separation of 1 Mb that can serve as nucleation points for the sequencing phase of the International Rice Genome Sequencing Project.

Nucleic Acids Res, 2000 Sep 15, 28(18), 3581 - 6
MDM2 interacts with the C-terminus of the catalytic subunit of DNA polymerase epsilon; Vlatkovic N et al.; MDM2 is induced by p53 in response to cellular insults such as DNA damage and can have effects upon the cell cycle that are independent or downstream of p53 . We used a yeast two-hybrid screen to identify proteins that bind to MDM2 and which therefore might be involved in these effects . We found that MDM2 can bind to the C-terminus of the catalytic subunit of DNA polymerase epsilon (DNA pol epsilon), to a region that is known to be essential in yeast . In an in vitro system we confirmed that MDM2 could bind to the homologous regions of both mouse and human DNA pol epsilon and to full-length human DNA pol epsilon . DNA pol epsilon co-immunoprecipitated with MDM2 from transfected H1299 cells and also from a HeLa cell nuclear extract . We show here that the DNA pol epsilon-interacting domain of MDM2 is located between amino acids 50 and 166 . Our studies provide evidence that MDM2 interacts with a region of DNA pol epsilon that plays a critical role in the function of DNA pol epsilon.

Nucleic Acids Res, 2000 Sep 15, 28(18), 3478 - 85
RBT1, a novel transcriptional co-activator, binds the second subunit of replication protein A; Cho JM et al.; Replication Protein A (RPA) is required for DNA recombination, repair and replication in all eukaryotes . RPA participation in these pathways is mediated by single-stranded DNA binding and protein interactions . We herein identify a novel protein, Replication Protein Binding Trans-Activator (RBT1), in a yeast two-hybrid assay employing the second subunit of human RPA (RPA32) as bait . RBT1-RPA32 binding was confirmed by glutathione S:-transferase pull-down and co-immunoprecipitation . Fluorescence microscopy indicates that green fluorescence protein-tagged RBT1 is localized to the nucleus in vivo . RBT1 mRNA expression, determined by semi-quantitative RT-PCR, is significantly higher in cancer cell lines MCF-7, ZR-75, SaOS-2 and H661, compared to the cell lines normal non-immortalized human mammary epithelial cells and normal non-immortalized human bronchial epithelial cells . Further, yeast and mammalian one-hybrid analysis shows that RBT1 is a strong transcriptional co-activator . Interestingly, mammalian transactivation data is indicative of significant variance between cell lines; the GAL4-RBT1 fusion protein has significantly higher transcriptional activity in human cancer cells compared to human normal primary non-immortalized epithelial cells . We propose that RBT1 is a novel transcriptional co-activator that interacts with RPA, and has significantly higher activity in transformed cells.

Plant Physiol, 2000 Sep, 124(1), 125 - 33
Expression of arabidopsis CAX2 in tobacco . Altered metal accumulation and increased manganese tolerance; Hirschi KD et al.; Metal transport from the cytosol to the vacuole is thought to be an important component of ion tolerance and of a plant's potential for use in phytoremediation . The Arabidopsis antiporter CAX2 (calcium exchanger 2) may be a key mediator of this process . CAX2 expression in yeast suppressed both Ca(2+) and Mn(2+) growth defects . A peptide-specific antibody to the antiporter reacted with a 39-kD protein from plant vacuolar membranes . Tobacco (Nicotiana tabacum) plants expressing CAX2 accumulated more Ca(2+), Cd(2+), and Mn(2+) and were more tolerant to elevated Mn(2+) levels . Expression of CAX2 in tobacco increased Cd(2+) and Mn(2+) transport in isolated root tonoplast vesicles . These results suggest that CAX2 has a broad substrate range and modulation of this transporter may be an important component of future strategies to improve plant ion tolerance.

Hum Genet, 2000 Jan, 106(1), 127 - 9
Physical mapping of the human ATX1 homologue (HAH1) to the critical region of the 5q- syndrome within 5q32, and immediately adjacent to the SPARC gene; Boultwood J et al.; The 5q- syndrome is a myelodysplastic syndrome with the 5q deletion as the sole karyotypic abnormality . The human ATX1 homologue (HAH1), encodes a copper-binding protein with a role in antioxidant defence . We have mapped this gene to the 3 Mb critical region of gene loss of the 5q- syndrome within 5q32, flanked by the genes for ADRB2 and IL12B, using gene dosage analysis . Fine physical mapping of the HAH1 gene within this genomic interval was then performed by screening YAC and BAC contigs spanning the critical region of the 5q- syndrome using PCR amplification . The HAH1 gene maps immediately adjacent to the SPARC gene at 5q32, and is flanked by the genetic markers D5S1838 and D5S1419 . The HAH1 gene is expressed in haematological tissues and plays a role in antioxidant defence . Antioxidant levels are low in most cancers and the importance of antioxidant enzymes in cancer genesis is well recognised . Genomic localisation, function and expression would suggest that the HAH1 gene represents a candidate gene for the 5q-syndrome.

Hum Genet, 2000 Jan, 106(1), 29 - 35
Incidence of mosaic cell lines in vivo and malsegregation of chromosome 21 in lymphocytes in vitro of trisomy 21 patients: detection by fluorescence in situ hybridization on binucleated lymphocytes; Shi Q et al.; In order to detect aneuploidy in interphase human lymphocytes, both in vivo and in vitro, fluorescence in situ hybridization (FISH) was carried out on binucleated cells cytokinesis-blocked by cytochalasin B at the first mitosis after phytohemagglutinin stimulation . A pericentric chromosome-21-specific DNA probe prepared from yeast artificial chromosome clone 881D2 by the polymerase chain reaction was employed . One thousand binucleated cells per individual were scored from cultures from twelve trisomy 21 patients aged 0.01-8.9 years (mean 4.3 years) and 20 normal children of similar age . Of trisomy 21 patients, increased frequencies of disomic cells in vivo (1.690+/-1.070%) and cells containing six signals with nondisjunction (0.822+/-0.554%) were found, compared with those of monosomic 21 cells in vivo (0.265+/-0.130%) and cells containing four signals with nondisjunction in normal children (0.369+/-0.250%; P=0.000 and P=0.000, respectively) . These results show that malsegregation of chromosome 21 occurs more often in trisomic 21 cells than in disomic cells from normal children . The frequency of nondisjunction was significantly higher than the loss of chromosome 21 in both cultured trisomic (0.822+/-0.554% vs 0.043+/-0.049%, P=0.000) and disomic (0.369+/-0.250% vs 0.010+/-0.30%, P=0.000) cells . Comparisons of in vivo and in vitro data on aneuploidy indicate that a cell selection mechanism may exist in vivo . All these results show that FISH, with a chromosome-specific probe, on binucleated lymphocytes is a powerful tool for simultaneously detecting mosaic cell lines in vivo and malsegregation (loss and nondisjunction) of a corresponding chromosome in vitro in the same cell population.

Nat Cell Biol, 2000 Sep, 2(9), 672 - 6
Polo-like kinase-1 is a target of the DNA damage checkpoint; Smits VA et al.; Polo-like kinases (PLKs) have an important role in several stages of mitosis . They contribute to the activation of cyclin B/Cdc2 and are involved in centrosome maturation and bipolar spindle formation at the onset of mitosis . PLKs also control mitotic exit by regulating the anaphase-promoting complex (APC) and have been implicated in the temporal and spatial coordination of cytokinesis . Experiments in budding yeast have shown that the PLK Cdc5 may be controlled by the DNA damage checkpoint . Here we report the effects of DNA damage on Polo-like kinase-1 (Plk1) in a variety of human cell lines . We show that Plk1 is inhibited by DNA damage in G2 and in mitosis . In line with this, we show that DNA damage blocks mitotic exit . DNA damage does not inhibit the kinase activity of Plk1 mutants in which the conserved threonine residue in the T-loop has been changed to aspartic acid, suggesting that DNA damage interferes with the activation of Plk1 . Significantly, expression of these mutants can override the G2 arrest induced by DNA damage . On the basis of these data we propose that Plk1 is an important target of the DNA damage checkpoint, enabling cell-cycle arrests at multiple points in G2 and mitosis.

Haematologica, 2000 Sep, 85(9), 908 - 12
Deletions at 11q23 in different lymphoma subtypes; Zhu Y et al.; BACKGROUND AND OBJECTIVES: Chromosome band 11q23 is frequently deleted in various types of neoplasm . The region represented by yeast artificial chromosome (YAC) clone 755b11 at 11q23 has been shown to be the minimal common region of deletion in mantle cell lymphoma (MCL) and B-cell chronic lymphocytic leukemia (B-CLL) . The aim of the study was to determine the frequencies of 11q23 deletion in different lymphoma subtypes . DESIGN AND METHODS: We performed fluorescence in situ hybridization (FISH) analysis with YAC755b11 on either peripheral blood or lymph node biopsy (LN) specimens of patients diagnosed as having MCL (47), CLL/small lymphocytic lymphoma (SLL) (62), diffuse large cell lymphoma (DLCL) (17), follicular lymphoma (FL) (9), and Hodgkin's disease (HD) (11) . Fifteen cases of reactive or normal lymph node biopsies were studied as controls . RESULTS: Forty of the 161 (25%) samples exhibited deletions in the region represented by YAC755b11 . The 11q23 deletion was found only in MCL (23, 49%), CLL / SLL (13, 21%) and DLCL (4, 24%) . Three cases were classified as Richter's syndrome and they all exhibited the deletion at 11q23 . The deletion frequencies in the blood specimens of typical CLL (30%) and lymph node specimens of CLL/SLL (13%) were remarkably different . INTERPRETATION AND CONCLUSIONS: Our study demonstrated that the 11q23 deletion is not common in lymphomas other than MCL, CLL and DLCL . It also showed the possible correlation of the 11q23 deletion with the transformation of localized lymphoma to CLL, and with the development of Richter's syndrome.

Oncogene, 2000 Sep 7, 19(38), 4328 - 36
Human endogenous retrovirus protein cORF supports cell transformation and associates with the promyelocytic leukemia zinc finger protein; Boese A et al.; Human endogenous retrovirus sequences (HERVs) reside in the genomes of primates and humans for several million years . The majority of HERVs is non-coding but a limited set is intact and can express proteins . We have recently identified an almost intact HERV-K(HML-2) provirus on chromosome 7 and have documented that most patients with germ cell tumors (GCTs) display antibodies directed against proteins of HERV-K(HML-2) . To address whether these proteins merely represent tumor markers or contribute to neoplastic transformation, we examined the transforming potential of various HERV sequences and studied physical interactions between HERV and cellular proteins by yeast two-hybrid and biochemical assays . cORF, a protein encoded by the C-terminal open reading frame within the env gene, supports tumor growth in nude mice and associates with the promyelocytic leukemia zinc finger protein (PLZF) . The interaction domains map between amino acid residues 21 and 87 of cORF, and between residues 245 and 543 of PLZF . PLZF is critical for spermatogenesis in mice . Abnormal spermatogenesis or maturation of gonocytes is thought to predispose humans to the development of germ cell tumors . Thus, cORF of human endogenous retroviruses may contribute to tumor development by interfering with processes during spermatogenesis that involve PLZF.

Oncogene, 2000 Aug 31, 19(37), 4273 - 82
A novel adaptor-like protein which is a substrate for the non-receptor tyrosine kinase, BRK; Mitchell PJ et al.; The brk gene encodes a non-receptor tyrosine kinase that has been found to be overexpressed in approximately two thirds of breast tumours . Using a yeast two-hybrid based screen, we have cloned cDNAs encoding a novel protein, BKS, that is a substrate for the kinase activity of BRK and has the characteristics of an adaptor protein . BKS possesses an N-terminal PH-like domain followed by an SH2-like domain . In co-transfection experiments, high levels of phosphotyrosine were observed on BKS and BRK was found to be associated with BKS, both of which were dependent on the catalytic activity of BRK . The phosphorylation of and association with BKS by BRK was also dependent on the SH2-like domain present within BKS . In addition, BKS recruited an unidentified 100 kDa protein that was also phosphorylated on tyrosine residues in the presence of BRK . We have determined that the BKS protein is expressed in most adult human tissues . Oncogene (2000) 19, 4273 - 4282

Curr Opin Genet Dev, 2000 Oct, 10(5), 529 - 35
Genetic control of cell size; Stocker H et al.; Over the past 25 years, the genetic control of cell size has mainly been addressed in yeast, a single-celled organism . Recent insights from Drosophila have shed light on the signalling pathways responsible for adjusting and maintaining cell size in metazoans . Evidence is emerging for a signalling cascade conserved in evolution that links external nutrient sources to cell size.

Curr Opin Genet Dev, 2000 Oct, 10(5), 489 - 96
Molecular motors and developmental asymmetry; Fischer JA; The generation of distinct cell fates can require movement of specific molecules or organelles to particular locations within the cell . These subcellular movements are often the jobs of motor proteins . Seemingly disparate developmental processes--determination of right and left in vertebrates, setting up the axes of polarity in insect embryos, mating-type switching in yeast, and coordinated organelle movements in Drosophila--converge in their dependence on motor proteins . The extent of possible regulatory complexity is only beginning to emerge.

Brain Res, 2000 Sep 15, 877(1), 110 - 23
UNCL, the mammalian homologue of UNC-50, is an inner nuclear membrane RNA-binding protein; Fitzgerald J et al.; We isolated a mammalian homologue of the C . elegans gene unc-50 that we have named UNCL . The 777 kb rat UNCL cDNA encodes a 259 amino acid protein that is expressed in a wide variety of tissues with highest mRNA levels in brain, kidney and testis . Hydropathy plot analysis and in vitro translation experiments with microsomal membranes indicate that UNCL is a transmembrane protein . Hemagglutinin tagged UNCL was stably transfected into SaOS-2 osteosarcoma cells and exhibited a nuclear rim staining pattern which was retained following extraction with 1% Triton X-100, suggesting that UNCL localizes to the inner nuclear membrane . UNCL-HA was extractable in 350 mM NaCl, suggesting that UNCL is not associated with the nuclear matrix . Homopolymer RNA-binding assays performed on in vitro translated UNCL protein and 'structural modeling by homology' suggest that UNCL binds RNA via an amino-terminal RNA Recognition-like Motif . Since unc-50 is required for expression of assembled muscle-type nicotinic receptors in the nematode we investigated whether UNCL had a similar function for mammalian nicotinic receptors . When UNCL was co-expressed with neural nicotinic receptors in Xenopus oocytes or COS cells it increased expression of functional cell surface receptors up to 1 . 6-fold . We conclude that UNCL is a novel inner nuclear membrane protein that associates with RNA and is involved in the cell-surface expression of neuronal nicotinic receptors . UNCL plays a broader role because UNCL homologues are present in two yeast and a plant species, none of which express nicotinic receptors and it is also found in tissues that lack nicotinic receptors.

J Biol Chem, 2000 Dec 1, 275(48), 37815 - 23
ARL4, an ARF-like protein that is developmentally regulated and localized to nuclei and nucleoli; Lin CY et al.; ADP-ribosylation factors (ARFs) are highly conserved approximately 20-kDa guanine nucleotide-binding proteins that participate in both exocytic and endocytic vesicular transport pathways via mechanisms that are only partially understood . Although several ARF-like proteins (ARLs) are known, their biological functions remain unclear . To characterize its molecular properties, we cloned mouse and human ARL4 (mARL4 and hARL4) cDNA . The appearance of mouse ARL4 mRNA during embryonic development coincided temporally with the sequential formation of somites and the establishment of brain compartmentation . Using ARL4-specific antibody for immunofluorescence microscopy, we observed that endogenous mARL4 in cultured Sertoli and neuroblastoma cells was mainly concentrated in nuclei . When expressed in COS7 cells, ARL4-T34N mutant, predicted to exist with GDP bound, was concentrated in nucleoli . Yeast two-hybrid screening and in vitro protein-interaction assays showed that hARL4 interacted with importin-alpha through its C-terminal NLS region and that the interaction was not nucleotide-dependent . Like ARL2 and -3, recombinant hARL4 did not enhance cholera toxin-catalyzed auto-ADP-ribosylation . Its binding of guanosine 5'-O-(thiotriphosphate) was modified by phospholipid and detergent, and the N terminus of hARL4, like that of ARF, was myristoylated . Our findings suggest that ARL4, with its distinctive nuclear/nucleolar localization and pattern of developmental expression, may play a unique role(s) in neurogenesis and somitogenesis during embryonic development and in the early stages of spermatogenesis in adults.

Biocell, 2000 Aug, 24(2), 145 - 50
A quantitative approach to correlate astrocyte differentiation and phagocytic activity; Iacono RF et al.; A triple staining procedure (PAP labeling for GFAP, PAS reaction for added yeast cells and hematoxylin for nuclear staining of the whole cell monolayer) had disclosed that Junin virus infection enhanced phagocytic activity by inducing greater astrocyte differentiation . Here, we resorted to a mathematical approach for simultaneous evaluation of astrocyte differentiation and potential phagocytosis . At light microscopy level, the total number of: a) PAS-stained yeast cells, b) PAS-stained yeast cells associated to GFAP-positive astrocytes, c) GFAP-positive astrocytes, and d) total number of GFAP-labeled and non-labeled astrocytes, were counted within the monolayer area delimited by a grid with a total area of 0.01 mm2 . As the percentage of PAS-stained yeast cells associated to GFAP-positive astrocytes correlated significantly with the percentage of GFAP-positive astrocytes for the three yeast cell incubation times (24, 48 and 72 h), a mathematical approach involving a so-called beta parameter representing the percentage of differentiated astrocytes capable of taking up 50% of added yeast cells, was developed . Since beta value dropped along yeast cell incubation time, and more markedly in Junin-virus infected samples, a numerical value was thus available to assess enhanced phagocytic activity in astrocytes undergoing differentiation . Therefore, the application of a mathematical approach to cell monolayers subjected to current staining techniques, allows more objective analysis of data provided by cursory visualization at light microscopy level.

Exp Gerontol, 2000 Aug, 35(5), 533 - 41
Nonhuman primate models in biogerontology; Lane MA; A variety of animal models are utilized in biogerontological studies including yeast, nematodes, fruit flies, hamsters, mice, rats, and nonhuman primates . Species selection for research is based on many factors including economic feasibility, husbandry, generalizability of findings, available background information, adaptability to experimentation, and often, relevance to human aging . Each model offers its own strengths and limitations; however, nonhuman primates offer the unique advantage of phylogenetic proximity to humans . Among others, costs to purchase and maintain research subjects represent major limitations of nonhuman primate models . Although several nonhuman primate species have been utilized in aging research, rhesus monkeys (Macaca mulatta) are the best characterized and most extensively studied in biomedical gerontology . Nonhuman primate models have been employed as models for human aging in many research areas including neurobiology, skeletal, and reproductive aging and age-related diseases such as cardiovascular disease and diabetes . Primate models are now also being utilized to study interventions into aging such as caloric restriction . It will be several more years until definitive conclusions regarding lifespan effects can be made . However, existing data strongly suggest that many of the beneficial effects reported in rodents on CR also occur in primate models thereby strengthening the possibility that this nutritional paradigm may also impact favorably upon human aging.

Biochim Biophys Acta, 2000 Sep 7, 1493(1-2), 255 - 8
BSPRY, a novel protein of the Ro-Ret family; Schenker T et al.; Utilizing the yeast two-hybrid system we have identified a novel protein of the Ro-Ret family that was termed BSPRY . This protein is composed of a B-box, an alpha-helical coiled coil and a SPRY domain . BSPRY from human beings shares 80% sequence identity with the homologous protein from mice . The gene for BSPRY resides on human chromosome 9 and is specifically expressed in testis . It comprises six exons and five introns and possesses a GC rich promoter forming a typical CpG island . The function of BSPRY is not known, but several related proteins of the RBCC family have been implicated in cell transformation.

Biochim Biophys Acta, 2000 Sep 7, 1493(1-2), 249 - 54
A novel protein (Fbf-1) that binds to CD95/APO-1/FAS and shows sequence similarity to trichohyalin and plectin; Schmidt T et al.; The Fas/Apo-1/CD95 cell surface receptor belongs to the TNF receptor family of cell death inducing molecules . A number of cytosolic adapter proteins that mediate signal transduction of CD95 have been characterized, but some features of the molecular mechanisms of CD95-induced cell death remain elusive . We describe here a novel protein that can interact with the cytosolic domain of the murine CD95 receptor in a yeast two-hybrid assay . This novel protein was termed Fbf-1 for Fas binding factor and bears no sequence similarity to the known CD95 adapter proteins . Fbf-1 is 1173 aa long and has a theoretical molecular weight of around 130 kDa . The protein is expressed in a wide variety of tissues and is localized in the cytoplasm . Fbf-1 is a very hydrophilic protein, highly conserved between mouse and human and bears a carboxyterminal leucine heptad repeat reminiscent of leucine zipper protein interaction domains . In addition, it shows sequence similarity to trichohyalin and plectin pointing to a function as a structural protein.

Proc Int Conf Intell Syst Mol Biol, 2000, 8, 317 - 28
Genes, themes and microarrays: using information retrieval for large-scale gene analysis; Shatkay H et al.; The immense volume of data resulting from DNA microarray experiments, accompanied by an increase in the number of publications discussing gene-related discoveries, presents a major data analysis challenge . Current methods for genome-wide analysis of expression data typically rely on cluster analysis of gene expression patterns . Clustering indeed reveals potentially meaningful relationships among genes, but can not explain the underlying biological mechanisms . In an attempt to address this problem, we have developed a new approach for utilizing the literature in order to establish functional relationships among genes on a genome-wide scale . Our method is based on revealing coherent themes within the literature, using a similarity-based search in document space . Content-based relationships among abstracts are then translated into functional connections among genes . We describe preliminary experiments applying our algorithm to a database of documents discussing yeast genes . A comparison of the produced results with well-established yeast gene functions demonstrates the effectiveness of our approach.

Proc Int Conf Intell Syst Mol Biol, 2000, 8, 93 - 103
Biclustering of expression data; Cheng Y et al.; An efficient node-deletion algorithm is introduced to find submatrices in expression data that have low mean squared residue scores and it is shown to perform well in finding co-regulation patterns in yeast and human . This introduces "biclustering", or simultaneous clustering of both genes and conditions, to knowledge discovery from expression data . This approach overcomes some problems associated with traditional clustering methods, by allowing automatic discovery of similarity based on a subset of attributes, simultaneous clustering of genes and conditions, and overlapped grouping that provides a better representation for genes with multiple functions or regulated by many factors.

Proc Int Conf Intell Syst Mol Biol, 2000, 8, 67 - 74
Regulatory element detection using a probabilistic segmentation model; Bussemaker HJ et al.; The availability of genome-wide mRNA expression data for organisms whose genome is fully sequenced provides a unique data set from which to decipher how transcription is regulated by the upstream control region of a gene . A new algorithm is presented which decomposes DNA sequence into the most probable "dictionary" of motifs or words . Identification of words is based on a probabilistic segmentation model in which the significance of longer words is deduced from the frequency of shorter words of various length . This eliminates the need for a separate set of reference data to define probabilities, and genome-wide applications are therefore possible . For the 6,000 upstream regulatory regions in the yeast genome, the 500 strongest motifs from a dictionary of size 1,200 match at a significance level of 15 standard deviations to a database of cis-regulatory elements . Analysis of sets of genes such as those up-regulated during sporulation reveals many new putative regulatory sites in addition to identifying previously known sites.

Hokkaido Igaku Zasshi, 2000 Jul, 75(4), 265 - 74
{Functional restoration of tumor suppressor p53 alters susceptibility of glioblastoma cells to irradiation--analysis using a cell line containing a temperature-sensitive mutant}; Tsuchiya K; To examine the functional role of tumor suppressor p53 in radiosensitivity of glioblastoma multiforme cells, I analyzed radiosensitivity of three glioblastoma cell lines with different p53 statuses: LN444 (wild-type p53), U251MG (mutant R273Q), and LN382 (mutant V197L) . The mutant V197L is a temperature-sensitive (ts) mutant, of which transcriptional activity is almost normal at 34 degrees C but lost at 37 degrees C in yeast p53 functional assay . Transcriptional activity V197L p53 on mdm2, p21/WAF1, and Bax promoters determined by a luciferase assay increased respectively by 6.5, 33.4, and 4.9 folds at 34 degrees C, compared to those at 37 degrees C . Semiquantitative multiplex RT-PCR showed a slight increase of Bax mRNA expression at 34 degrees C but a marked increase of p21 mRNA expression, indicating that this ts mutant restored transcriptional activity predominantly on p21 promoter at the permissible temperature . Corresponding to the p21 transactivation, growth of LN382 cells was completely arrested without apoptosis when cultured at 34 degrees C but this arrest was reversed at 37 degrees C with a delay time subsequently to 24 or 48 h of culture at 34 degrees C . Clonogenic assay showed that dose-dependent radiosensitivity of LN382 cells to gamma-irradiation was markedly enhanced at 34 degrees C compared to that at 37 degrees C, whereas those of LN444 and U251MG were not changed between 34 and 37 degrees C . This sensitization was not attributable to apoptosis induction, since no DNA ladder formation nor sub-G1/0 phase cells were observed . Instead, cell cycle analysis demonstrated G1 and G2M arrest of the LN382 cells cultured at 34 degrees C after irradiation . In agreement to this, an increase of p21 expression was further enhanced by irradiation . In conclusion, these findings altogether suggest that p21 expression by restoration of p53 function can increase the radiosensitivity of glioblastoma cells by arresting the cells at G1 and G2M phases.

DNA Cell Biol, 2000 Aug, 19(8), 475 - 85
NFBD1/KIAA0170 is a novel nuclear transcriptional transactivator with BRCT domain; Ozaki T et al.; The BRCT (BRCA1 C-terminus) superfamily includes a large number of nuclear proteins closely involved in DNA repair, recombination, and cell-cycle control . The human cDNA clone NFBD1 (previously designated KIAA0170) encodes a novel protein (2089 amino acids in length; calculated molecular mass 226,440 D) with possible BRCT domains at its carboxy terminus (amino acid residues 1894-2089) . This gene product has been described as one of the BRCT superfamily proteins . However, its biological significance has been unclarified . Expression of green fluorescent protein (GFP)-tagged full-length NFBD1 or a series of deletion mutants indicated that NFBD1 was localized to the nucleus in various mammalian cells, and a 197-amino acid segment near the amino terminus (amino acid residues 142-338) contained a nuclear targeting signal . In vitro DNA-binding experiments showed that the highly basic region of NFBD1 (amino acid residues 1841-1893) possessed DNA-binding activity . The region encoding amino acids 508-995 of NFBD1 fused inframe with GAL4 DNA-binding domain activated transcription in both yeast and mammalian cells, while the possible BRCT domains of NFBD1 failed to induce transcription in mammalian cells . Overexpression of antisense NFBD1 RNA in a p53-deficient human osteogenic sarcoma cell line (SAOS-2) resulted in remarkable suppression of SAOS-2 colony formation . These results suggest that NFBD1 is a nuclear transcriptional transactivator with possible BRCT domains and may contribute to cell growth control.

J Cell Biol, 2000 Sep 4, 150(5), 939 - 48
Atrophin-1, the dentato-rubral and pallido-luysian atrophy gene product, interacts with ETO/MTG8 in the nuclear matrix and represses transcription; Wood JD et al.; Dentato-rubral and pallido-luysian atrophy (DRPLA) is one of the family of neurodegenerative diseases caused by expansion of a polyglutamine tract . The drpla gene product, atrophin-1, is widely expressed, has no known function or activity, and is found in both the nuclear and cytoplasmic compartments of neurons . Truncated fragments of atrophin-1 accumulate in neuronal nuclei in a transgenic mouse model of DRPLA, and may underlie the disease phenotype.Using the yeast two-hybrid system, we identified ETO/MTG8, a component of nuclear receptor corepressor complexes, as an atrophin-1-interacting protein . When cotransfected into Neuro-2a cells, atrophin-1 and ETO/MTG8 colocalize in discrete nuclear structures that contain endogenous mSin3A and histone deacetylases . These structures are sodium dodecyl sulfate-soluble and associated with the nuclear matrix . Cotransfection of ETO/MTG8 with atrophin-1 recruits atrophin-1 to the nuclear matrix, while atrophin-1 and ETO/MTG8 cofractionate in nuclear matrix preparations from brains of DRPLA transgenic mice . Furthermore, in a cell transfection-based assay, atrophin-1 represses transcription . Together, these results suggest that atrophin-1 associates with nuclear receptor corepressor complexes and is involved in transcriptional regulation.Emerging links between disease-associated polyglutamine proteins, nuclear receptors, translocation-leukemia proteins, and the nuclear matrix may have important repercussions for the pathobiology of this family of neurodegenerative disorders.

Biochem Biophys Res Commun, 2000 Sep 7, 275(3), 764 - 7
Oxidative stress regulates the interaction of p16 with Cdk4; Martin EA et al.; Oxidative stress can have a myriad of effects on many different cell types . The mechanisms by which these effects occur are not completely known . Chimeric proteins of the GAL4 DNA binding domain and Cdk4, or the GAL4 activation domain with p16, were expressed in the yeast two-hybrid system . Cells expressing these chimeric proteins were cultured with hydrogen peroxide and decreases in beta-galactosidase activity were observed when compared to cells incubated without hydrogen peroxide . When cells, which expressed the intact GAL4 binding protein, were cultured in the presence of hydrogen peroxide the opposite was observed . Incubation of cells with buthionine sulfoximine augmented these responses to hydrogen peroxide . These data suggest that one of the mechanisms by which oxidative stress acts is via the modulation of protein-protein interactions and demonstrate that the yeast two-hybrid system may be a model by which to study protein interactions due to oxidative stress .

Brain Res, 2000 Sep 8, 876(1-2), 55 - 61
Developmental profile of kainate receptor subunit KA1 revealed by Cre expression in YAC transgenic mice; Kask K et al.; To determine the spatio-temporal expression in brain of the high-affinity kainate receptor subunit KA1, we generated transgenic mice expressing Cre recombinase from the KA1 gene on a chromosomally integrated 550 kb yeast artificial chromosome (YAC) . Activity of the KA1 gene promoter during brain development was visualized by Cre immunohistochemistry, and by X-gal staining of beta-galactosidase induced by Cre recombinase in double transgenic KA1-Cre/lacZ indicator mice . During early brain development, expression from the YAC-carried KA1-Cre transgene was observed in all major brain areas, predicting a function for KA1 in the developing central nervous system . In the adult brain, KA1-Cre transgene expression was restricted mainly to hippocampal CA3 pyramidal and dentate gyrus granule cells, an adult expression pattern characteristic for the endogenous KA1 alleles . KA1-Cre transgenic mice may help in elucidating the role of floxed genes ablated in vivo in KA1 expressing neurons.

Proc Natl Acad Sci U S A, 2000 Sep 12, 97(19), 10389 - 94
Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro; Matsuoka S et al.; The protein kinase Chk2, the mammalian homolog of the budding yeast Rad53 and fission yeast Cds1 checkpoint kinases, is phosphorylated and activated in response to DNA damage by ionizing radiation (IR), UV irradiation, and replication blocks by hydroxyurea (HU) . Phosphorylation and activation of Chk2 are ataxia telangiectasia-mutated (ATM) dependent in response to IR, whereas Chk2 phosphorylation is ATM-independent when cells are exposed to UV or HU . Here we show that in vitro, ATM phosphorylates the Ser-Gln/Thr-Gln (SQ/TQ) cluster domain (SCD) on Chk2, which contains seven SQ/TQ motifs, and Thr68 is the major in vitro phosphorylation site by ATM . ATM- and Rad3-related also phosphorylates Thr68 in addition to Thr26 and Ser50, which are not phosphorylated to a significant extent by ATM in vitro . In vivo, Thr68 is phosphorylated in an ATM-dependent manner in response to IR, but not in response to UV or HU . Substitution of Thr68 with Ala reduced the extent of phosphorylation and activation of Chk2 in response to IR, and mutation of all seven SQ/TQ motifs blocked all phosphorylation and activation of Chk2 after IR . These results suggest that in vivo, Chk2 is directly phosphorylated by ATM in response to IR and that Chk2 is regulated by phosphorylation of the SCD.

Nat Genet, 2000 Sep, 26(1), 85 - 8
Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation; Wong KK et al.; Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes . Loss of telomere function is associated with rampant genetic instability and loss of cellular viability and renewal potential . The telomere also participates in processes of chromosomal repair, as evidenced by the 'capture' or de novo synthesis of telomere repeats at double-stranded breaks and by the capacity of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in non-homologous end-joining (NHEJ) . Here we used the telomerase-deficient mouse, null for the essential telomerase RNA gene (Terc), to assess the role of telomerase and telomere function on the cellular and organismal response to ionizing radiation . Although the loss of telomerase activity per se had no discernable impact on the response to ionizing radiation, the emergence of telomere dysfunction in late-generation Terc-/- mice imparted a radiosensitivity syndrome associated with accelerated mortality . On the cellular level, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts (MEFs) showed diminished dose-dependent clonogenic survival . The radiosensitivity of telomere dysfunctional cells correlated with delayed DNA break repair kinetics, persistent chromosomal breaks and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation . Our findings establish a intimate relationship between functionally intact telomeres and the genomic, cellular and organismal response to ionizing radiation.

Biotechnol Bioeng, 2000 Oct 20, 70(2), 197 - 207
Growth characteristics and metabolic flux analysis of Candida milleri; Granstrom TB et al.; The growth characteristics of the sourdough yeast Candida milleri was studied in a carbon-limited aerobic chemostat culture on defined medium . The effect of glucose, xylose, and glucose-xylose mixture on metabolite production and on key enzyme activities was evaluated . Xylose as a sole carbon source was not metabolized by C . milleri . Glucose as a sole carbon source produced only biomass and carbon dioxide . When a glucose-xylose mixture (125:125 C-mM) was used as a carbon source, a small amount of xylose was consumed and a low concentration of xylitol was produced (7.20 C-mM) . Enzymatic assays indicated that C . milleri does not possess xylitol dehydrogenase activity and its xylose reductase is exclusively NADPH-dependent . In glucose medium both NAD(+)- and NADP(+)-dependent aldehyde dehydrogenase activities were found, whereas in a glucose-xylose medium only NADP(+)-dependent aldehyde dehydrogenase activity was detected . The developed metabolic flux analysis corresponded well with the experimentally measured values of metabolite production, oxygen consumption (OUR), and carbon dioxide production (CER) . Turnover number in generation and consumption of ATP, mitochondrial and cytosolic NADH, and cytosolic NADPH could be calculated and redox balance was achieved . Constraints were imposed on the flux estimates such that the directionality of irreversible reactions is not violated, and cofactor dependence of the measured enzyme activities were taken into account in constructing the metabolic flux network .

Plant J, 2000 Sep, 23(5), 633 - 41
Analysis of the G-overhang structures on plant telomeres: evidence for two distinct telomere architectures; Riha K et al.; Telomeres are highly conserved structures essential for maintaining the integrity of eukaryotic genomes . In yeast, ciliates and mammals, the G-rich strand of the telomere forms a 3' overhang on the chromosome terminus . Here we investigate the architecture of telomeres in the dicot plants Silene latifolia and Arabidopsis thaliana using the PENT (primer extension/nick translation) assay . We show that both Arabidopsis and Silene telomeres carry G-overhangs longer than 20-30 nucleotides . However, in contrast to yeast and ciliate telomeres, only half of the telomeres in Silene seedlings possess detectable G-overhangs . PENT reactions using a variety of primers and reaction conditions revealed that the remaining fraction of Silene telomeres carries either no overhangs or overhangs less than 12 nucleotides in length . G-overhangs were observed in Silene seeds and leaves, tissues that lack telomerase activity . These findings suggest that incomplete DNA replication of the lagging strand, rather than synthesis by telomerase, is the primary mechanism for G-overhang synthesis in plants . Unexpectedly, we found that the fraction of telomeres with detectable G-overhangs decreased from 50% in seedlings to 35% in leaves . The difference may reflect increased susceptibility of the G-overhangs to nuclease attack in adult leaves, an event that could act as a precursor for the catabolic processes accompanying leaf senescence

Plant J, 2000 Aug, 23(4), 527 - 38
Biochemical characterization of the Arabidopsis K+ channels KAT1 and AKT1 expressed or co-expressed in insect cells; Urbach S et al.; KAT1 and AKT1 belong to the multigenic family of the inwardly rectifying Shaker-like plant K+ channels . They were biochemically characterized after expression in insect cells using recombinant baculoviruses . The channels were solubilized from microsomal fractions prepared from infected cells (among eight different detergents only one, L-alpha-lysophosphatidylcholine, was efficient for solubilization), and purified to homogeneity using immunoaffinity (KAT1) or ion-exchange and size exclusion (AKT1) techniques . The following results were obtained with the purified polypeptides: (i) neither KAT1 nor AKT1 was found to be glycosylated; (ii) both polypeptides were mainly present as homotetrameric structures, supporting the hypothesis of a tetrameric structure for the functional channels; (iii) no heteromeric KAT1/AKT1 assembly was detected when the two polypeptides were co-expressed in insect cells . The use of the two-hybrid system in yeast also failed to detect any interaction between KAT1 and AKT1 polypeptides . Because of these negative results, the hypothesis that plant K+-channel subunits are able to co-assemble without any discrimination, previously put forward based on co-expression in Xenopus oocytes of various K+-channel subunits (including KAT1 and AKT1), has still to be supported by independent approaches . Co-localization of channel subunits within the same plant tissue/cell does not allow us to conclude that the subunits form heteromultimeric channels.

Plant J, 2000 Aug, 23(4), 517 - 25
A dominant negative mutant of sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells; Takeuchi M et al.; Protein secretion plays an important role in plant cells as it does in animal and yeast cells, but the tools to study molecular events of plant secretion are very limited . We have focused on the Sar1 GTPase, which is essential for the vesicle formation from the endoplasmic reticulum (ER) in yeast, and have previously shown that tobacco and Arabidopsis SAR1 complement yeast sar1 mutants . In this study, we have established a transient expression system of GFP-fusion proteins in tobacco and Arabidopsis cultured cells . By utilizing confocal laser scanning microscopy, we demonstrate that a dominant negative mutant of Arabidopsis Sar1 inhibits the ER-to-Golgi transport of Golgi membrane proteins, AtErd2 and AtRer1B, and locates them to the ER . The same mutant Sar1 also blocks the exit from the ER of a vacuolar storage protein, sporamin . These results not only provide the first evidence that the Sar1 GTPase functions in the ER-to-Golgi transport in plant cells, but also prove that conditional expression of dominant mutants of secretory machinery can be a useful tool in manipulating vesicular trafficking.

Curr Opin Microbiol, 2000 Aug, 3(4), 339 - 43
Molecular basis of pathogenicity in Blastomyces dermatitidis: the importance of adhesion; Klein BS; An understanding of the molecular bases of pathogenicity in Blastomyces dermatitidis and related systemic dimorphic fungi has been limited until recent years . Yeast cells of B . dermatitidis display an adhesion promoting protein termed WI-1 . Recent studies entailing homologous gene targeting and mutation of WI-1 have provided null mutants at this locus and demonstrated the crucial role of the WI-1 adhesin in pathogenesis of blastomycosis . Ongoing studies are pointing to a link between phase-specific expression of WI-1 and the observation that transition to yeast cells is essential for the acquisition of pathogenicity by B . dermatitidis . Recombinant attenuated yeast that lack WI-1 are serving as invaluable tools for induction of vaccine resistance and are pointing to new insights about adaptive immunity to B . dermatitidis.

Genes Cells, 2000 Sep, 5(9), 739 - 47
Transactivation mechanisms of mouse clock transcription factors, mClock and mArnt3; Takahata S et al.; BACKGROUND: The Arnt3 (also termed as BMAL1 or MOP3)/Clock heterodimer is a positive regulator of circadian rhythm and activates the transcription of target genes such as per1 and vasopressin . RESULTS: We investigated the transcriptional mechanism of mArnt3/mClock heterodimer . While mClock did not possess any distinct activation domain, mArnt3 contained a transcriptional activation domain at the most C-terminal end, the activity of which was not expressed, even in the one hybrid system, until it was bound by mClock . It has been suggested that mClock plays a regulatory or structural role in exerting a transcription enhancing effect of the mArnt3/mClock heterodimer . Deletion proceeding from amino acids 559-492 of mClock markedly reduced the transactivation activity of mArnt3/mClock heterodimer, in consistence with the results of the Clock-delta 19 mutant . Yeast and mammalian two-hybrid systems revealed that CBP and p300 interacted with mArnt3 via the CREB binding domain . The In vivo interaction between mArnt3 and CBP was confirmed by the GST pull down assay . CONCLUSION: Taken together, these results suggest that the mArnt3/mClock heterodimer exerted its transactivation activity via CBP or p300 interacting with mArnt3 in the heterodimer with mClock playing a structural or regulatory role in the transactivation process.

Eur J Biochem, 2000 Sep, 267(18), 5805 - 9
Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome c; Kotlyar A et al.; A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported . The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6, 8-trisulfonate in complex with cytochrome oxidase . The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue . Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6, 8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase . The maximum efficiency by a single laser pulse resulted in the reduction of approximately 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase . At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.

EMBO J, 2000 Sep 1, 19(17), 4493 - 502
The CXXCXXC motif determines the folding, structure and stability of human Ero1-Lalpha; Benham AM et al.; The presence of correctly formed disulfide bonds is crucial to the structure and function of proteins that are synthesized in the endoplasmic reticulum (ER) . Disulfide bond formation occurs in the ER owing to the presence of several specialized catalysts and a suitable redox potential . Work in yeast has indicated that the ER resident glycoprotein Ero1p provides oxidizing equivalents to newly synthesized proteins via protein disulfide isomerase (PDI) . Here we show that Ero1-Lalpha, the human homolog of Ero1p, exists as a collection of oxidized and reduced forms and covalently binds PDI . We analyzed Ero1-Lalpha cysteine mutants in the presumed active site C(391)VGCFKC(397) . Our results demonstrate that this motif is important for protein folding, structural integrity, protein half-life and the stability of the Ero1-Lalpha-PDI complex.

J Biol Chem, 2000 Dec 1, 275(48), 37317 - 23
Kinetic characterization of glutathione reductase from the malarial parasite Plasmodium falciparum . Comparison with the human enzyme; Bohme CC et al.; The homodimeric flavoenzyme glutathione reductase (GR) maintains high intracellular concentrations of the antioxidant glutathione (GSSG + NADPH + H(+) <--> 2 GSH + NADP(+)) . Due to its central function in cellular redox metabolism, inhibition of GR from the malarial parasite Plasmodium falciparum represents an important approach to antimalarial drug development; therefore, the catalytic mechanism of GR from P . falciparum has been analyzed and compared with the human host enzyme . The reductive half-reaction is similar to the analogous reaction with GR from other species . The oxidative half-reaction is biphasic, reflecting formation and breakdown of a mixed disulfide between the interchange thiol and GSH . The equilibrium between the E(ox)-EH(2) and GSSG-GSH couples has been modeled showing that the Michaelis complex, mixed disulfide-GSH, is the predominant enzyme form as the oxidative half-reaction progresses; rate constants used in modeling allow calculation of an K(eq) from the Haldane relationship, 0.075, very similar to the K(eq) of the same reaction for the yeast enzyme (0.085) (Arscott, L . D., Veine, D . M., and Williams, C . H., Jr . (2000) Biochemistry 39, 4711-4721) . Enzyme-monitored turnover indicates that E(FADH(-))(S-S) . NADP(+) and E(FAD)(SH)(2).NADPH are dominant enzyme species in turnover . Since the individual forms of the enzyme differ in their susceptibility to inhibitors, the prevailing states of GR in the cell are of practical relevance.

Bioorg Med Chem, 2000 Jan, 8(1), 223 - 32
Stereospecific syntheses of trans-vinyldioxidosqualene and 3-hydroxysulfide derivatives, as potent and time-dependent 2,3-oxidosqualene cyclase inhibitors; Viola F et al.; trans-Vinyldioxidosqualene and beta-hydroxysulfide derivatives were synthesized stereospecifically and evaluated as inhibitors of animal and yeast oxidosqualene cyclases . Only trans-vinyldioxidosqualene and 2,3-epoxy-vinyl-beta-hydroxysulfides, having the reactive function at crucial positions 14,15 and 18,19, were active as inhibitors of animal and yeast cyclases . (14-trans)-28-Methylidene-2,3: 14,15-dioxidoundecanorsqualene 27 was the most potent inhibitor of the series of pig liver cyclase, with an IC50 of 0.4 microM, and it behaved also as the most active time-dependent inhibitor of the animal enzyme.

Mycopathologia, 1999, 147(2), 89 - 96
Production of T-2 toxin by a Fusarium resembling Fusarium poae; Torp M et al.; A Fusarium species with a micro morphology similar to F . poae and a metabolite profile resembling that of F . sporotrichioides has been identified . Like typical F . poae, the microconidia have a globose to pyriform shape, but the powdery appearance, especially on Czapek-Dox Iprodione Dichloran agar (CZID), less aerial mycelium and the lack of fruity odour on Potato Sucrose Agar (PSA) make it different from F . poae . The lack of macroconidia, polyphialides and chlamydospores differentiates it from F . sporotrichioides . All 18 isolates investigated, 15 Norwegian, two Austrian and one Dutch, produced T-2 toxin (25-400 micrograms/g) on PSA or Yeast Extract Sucrose agar (YES) . In addition, neosolaniol, iso-neosolaniol, HT-2 toxin, 4- and 15-acetyl T-2 tetraol, T-2 triol and T-2 tetraol and 4,15-diacetoxyscirpenol were formed in variable amounts . Neither nivalenol, 4- or 15-acetylnivalenol or 4,15-diacetylnivalenol were detected in any of the cultures, while these toxins were produced at least in small amounts by all the 12 typical F . poae isolates studied . The question of whether this Fusarium should be classified as F . poae or F . sporotrichioides or a separate taxon should be addressed.

Mycopathologia, 1999, 147(2), 63 - 5
The antifungal action of dandruff shampoos; Bulmer AC et al.; The disease commonly known as "dandruff" is caused by numerous host factors in conjunction with the normal flora yeast Malassezia furfur (Pityrosporum ovale) . Indeed, clinical studies have shown that administration of antifungal agents correlates with an improved clinical condition . Almost all commercially available hair shampoos publicize that they contain some form of antifungal agent(s) . However, few studies have been published in which antifungal activity of commercially available hair shampoos have been contrasted experimentally . In this study six commercially available shampoos (in the Philippines) were assessed for antifungal activity against a human (dandruff) isolate of M . furfur: (a) Head & Shoulders (Proctor & Gamble); (b) Gard Violet (Colgate-Palmolive); (c) Nizoral 1% (Janssen); (d) Nizoral 2% (Janssen); (e) Pantene Blue (Proctor & Gamble); and (f) Selsun Blue (Abbott) . The results demonstrated that all six of the assayed hair shampoos have some antifungal effect on the test yeast . However, there was consider variation in potency of antifungal activity . Nizoral 1% and Nizoral 2% shampoo preparations were the most effective . The 1% Nizoral shampoo was consistently 10X better at killing yeast cells than the next closest rival shampoo . The 2% Nizoral shampoo was 10X better than the Nizoral 1% product and 100 times better than any of the other products assayed . The study demonstrated that shampoos containing a proven antifungal compound were the most effective in controlling the causative yeast.

J Cell Biochem, 2000 Aug 2, 79(2), 213 - 24
The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2; Barritt DS et al.; A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan . Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1 . Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein . NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan . The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1 . The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules . MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm .

Int J Pharm, 2000 Aug 10, 203(1-2), 169 - 77
Polysaccharide coated liposomes for oral immunization--development and characterization; Venkatesan N et al.; Polysaccharide coated liposomes were prepared, characterized and evaluated for their potential use in oral immunization . Liposomes were prepared by reverse phase evaporation method . Bovine serum albumin (BSA) was chosen as the model antigen . Pulluan, a naturally occurring polysaccharide produced by a yeast like fungus, was chemically modified into its palmitoyl derivative (O-palmitoylpullulan; OPP) and was used for coating of the liposomes . The synthesized OPP was characterized by IR and NMR spectroscopy . The liposomes prepared were characterized for their size, shape, surface charge, encapsulation efficiency and stability in simulated gastric fluid . The immune stimulating activity was studied by measuring the serum IgA and IgG following oral administration of the prepared polysaccharide coated liposomes . Similarly, other formulations were studied and the results were compared . BSA loaded liposomes coated with OPP and plain polysaccharide could produce better IgG and IgA titre levels as compared to plain alum adsorbed BSA . The plain liposomes containing BSA could however produce significantly higher IgG and IgA levels as compared to equivalent BSA-alum based oral immunization . The results indicate that chemically modified polysaccharide coated liposomes can be used as a potential adjuvants for effective oral immunization.

Mamm Genome, 2000 Sep, 11(9), 774 - 8
Fine linkage and physical mapping suggests cross-over suppression with a retroposon insertion at the npc1 mutation; Hsu SJ et al.; Mouse Niemann-Pick disease type C1 (npc1), formerly designated spm (sphingomyelinosis), is an autosomal recessive lipid storage disorder . We generated a high-resolution linkage map in the 2.24-cM npc1 critical region by typing eight polymorphic markers in 2322 meioses (948 of these were previously reported) . A minimal set of overlapping yeast artificial chromosomes (YACs) had previously been assembled (Hsu and Erickson 2000) . The YAC 313-B-8, which covered this whole region, has been used to construct cosmid libraries . Three cosmid contigs were built, and one of them contained the npc1 locus . Two (CA)(n) microsatellites were identified, and the one new one was characterized, from the YAC-derived cosmids . The most proximal cosmid contig overlaps with markers near twirler (Tw) . Both the physical map and genetic linkage map have been integrated to study the recombination frequencies in this particular region of the mouse genome, and recombination suppression due to the heterozygous insertion of DNA was suggested.

Mamm Genome, 2000 Sep, 11(9), 755 - 62
Complete nucleotide sequence and genomic structure of the human NRAMP1 gene region on chromosome region 2q35; Marquet S et al.; Several lines of independent evidence suggest that human Natural Resistance Associated Macrophage Protein 1 gene (NRAMP1) is an important regulator of susceptibility to infectious diseases caused by certain intracellular pathogens . Here, we report the nucleotide sequence of 32198 bp of genomic DNA overlapping NRAMP1 on chromosomal region 2q35 . The NRAMP1 gene spans 13604 bp . The gene and its 5' genomic region are highly enriched for DNA repeat sequences . A second gene was identified in the immediate vicinity of NRAMP1 and was tentatively named Nuclear LIM Interactor-Interacting Factor (NLI-IF) by analogy to its closest ortholog . The human NLI-IF gene begins 4721 bp downstream of the NRAMP1 stop codon and is composed of seven exons varying in size from 57 bp to 1644 bp . The gene gives rise to a 2655-bp mRNA transcript that contains a 783-bp open reading frame . The predicted molecular weight of the 261-amino acid NLI-IF protein is 29.2 kDa . Several putative gene regulatory elements were identified in the 5' upstream region of NLI-IF, including consensus binding sequences for Sp1, AP-2, NF-kappa B, and PU 1 . The NLI-IF amino acid sequence has homology to proteins that have a high degree of homology with the NLI-interacting factor from Gallus gallus and are found in divergent species ranging from yeast to plants . NLI-IF is part of a human gene family encoding four related proteins of unknown function . Northern blot analysis of 15 different human tissues revealed a 2.6-kb NLI-IF mRNA that was ubiquitously expressed, but at varying levels . A second transcript with estimated size of 7 kb was expressed only in the placenta . Our data provide new sequence information about the NRAMP1 gene region that will be useful in the search for genetic variants causally involved in altered susceptibility to infectious diseases.

J Biol Chem, 2000 Nov 17, 275(46), 36457 - 64
Characterization of TCL, a new GTPase of the rho family related to TC10 andCcdc42; Vignal E et al.; GTPases of the Rho family control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation, and apoptosis . We report here the characterization of a new Rho member, which shares 85% and 78% amino acid similarity to TC10 and Cdc42, respectively . This GTPase, termed as TC10-like (TCL) is encoded by an unexpectedly large locus, made of five exons spanning over 85 kilobases on human chromosome 14 . TCL mRNA is 2.5 kilobases long and is mainly expressed in heart . In vitro, TCL shows rapid GDP/GTP exchange and displays higher GTP dissociation and hydolysis rates than TC10 . Using the yeast two-hybrid system and GST pull-down assays, we show that GTP-bound but not GDP-bound TCL protein directly interacts with Cdc42/Rac interacting binding domains, such as those found in PAK and WASP . Despite its overall similarity to TC10 and Cdc42, the constitutively active TCL mutant displays distinct morphogenic activity in REF-52 fibroblasts, producing large and dynamic F-actin-rich ruffles on the dorsal cell membrane . Interestingly, TCL morphogenic activity is blocked by dominant negative Rac1 and Cdc42 mutants, suggesting a cross-talk between these three Rho GTPases.

Toxicol Sci, 2000 Sep, 57(1), 54 - 60
Lack of significant estrogenic or antiestrogenic activity of pyrethroid insecticides in three in vitro assays based on classic estrogen receptor alpha-mediated mechanisms; Saito K et al.; Estrogenic and antiestrogenic activity of pyrethroid insecticides (d-trans-allethrin, cypermethrin, empenthrin, fenvalerate, imiprothrin, permethrin, d-phenothrin and prallethrin) was evaluated using a suite of three in vitro assays based on classic human estrogen receptor alpha (hER alpha)-mediated mechanisms . A mammalian cell-based luciferase reporter gene assay was developed for examining effects on hER alpha-mediated gene activation . hER alpha-independent effects on the gene activation were examined using control cells with constitutive luciferase activation by a herpes simplex virus thymidine kinase (HSV-TK) promoter for determining appropriate dose levels of test chemicals . Moreover, the test chemical-dependent interaction between hER alpha and a coactivator (transcriptional intermediary factor 2: TIF2) was analyzed by a yeast two-hybrid method, competitive binding to hER alpha being assayed by a fluorescence polarization method . Significant (p < 0.05) positive effects of estrogenic substances (E2/estradiol, diethylstilbestrol, and p-nonylphenol) were detected in all assays . An antiestrogen, 4-hydroxytamoxifen, significantly inhibited E2-mediated transactivation and interaction between hER alpha and TIF2 through hER alpha binding (p < 0.05) . However, none of the pyrethroids tested showed significant (p < 0.05) estrogenic or antiestrogenic effects (100 nM-10 microM), indicating that they do not impact on the classic hER alpha-mediated activation pathway in vitro.

Endocrinology, 2000 Sep, 141(9), 3440 - 50
Thyroid receptor activator molecule, TRAM-1, is an androgen receptor coactivator; Tan JA et al.; An androgen receptor (AR) interacting protein was isolated from a HeLa cell complementary DNA library by two-hybrid screening in yeast using the AR DNA and ligand binding domains {amino acids (aa) 481-919} as bait . AR binding of the protein in yeast was dependent on the presence of testosterone or dihydrotestosterone (DHT) . The isolated protein is identical to thyroid receptor activator molecule TRAM-1 but lacking aa 1-458 . TRAM-1 is a steroid receptor coactivator-3 (SRC-3) subtype . In affinity matrix assays, 35S-labeled TRAM-1 bound the GST-AR ligand binding domain (aa 624-919) and GST-AR N-terminal and DNA binding domains (aa 1-660), but not the GST-AR DNA binding domain (aa 544-634) alone . Coexpression of TRAM-1 increased DHT-dependent AR transactivation 5-fold and constitutive activity of AR (aa 1-660) N-terminal and DNA-binding domains increased 9-fold . Full-length TRAM-1 (aa 1-1424) and the partial (aa 459-1424) were AR and GR coactivators as was SRC-1 . In human testis, immunostaining of SRC-3 colocalized with AR in nuclei of Sertoli cells and peritubular myoid cells, indicating it could function as an AR coactivator in these cells . SRC-3 was also present in nuclei of spermatogenic cells where AR was not expressed, suggesting it might also be a coactivator with other nuclear receptors that regulate spermatogenesis.

Cytogenet Cell Genet, 2000, 89(3-4), 168 - 70
An anchored YAC-STS framework for the rat genome; Cai L et al.; We report here the first YAC-STS framework for the rat genome . A total of 417 anchor microsatellite markers were used to screen a 10-fold redundant YAC library . One or more unambiguous YACs were identified for 372 markers . Assuming the genetic length of the rat genome to be 2,000 cM (Bihoreau et al . 1997b), the YAC-STS framework will provide, on average, one informative YAC clone every 5.4 cM . A total of 111 anchor markers used in this study were derived from known gene regions . We also demonstrated one of the important and immediate uses of this YAC-STS framework, which is to establish a correlation between the genetic and cytogenetic maps in the rat through FISH analysis .

J Biochem (Tokyo), 2000 Sep, 128(3), 493 - 8
Protein N-arginine methylation in subcellular fractions of lymphoblastoid cells; Lin CH et al.; Arginine methylation in RNA-binding proteins containing arginine- and glycine-rich RGG motifs is catalyzed by specific protein arginine N-methyltransferase in cells . We previously showed that lymphoblastoid cells grown in the presence of an indirect methyltransferase inhibitor, adenosine dialdehyde (AdOx), accumulated high level of hypomethylated protein substrates for the endogenous protein methyltransferases or recombinant yeast arginine methyltransferase {Li, C . et al . (1998) Arch . Biochem . Biophys . 351, 53-59} . In this study we fractionated the lymphoblastoid cells to locate the methyltransferases and the substrates in cells . Different sets of hypomethylated methyl-accepting polypeptides with wide range of molecular masses were present in cytosolic, ribosomal, and nucleus fractions . The methylated amino acid residues of the methyl-accepting proteins in these fractions were determined . In all three fractions, dimethylarginine was the most abundant methylated amino acid . The protein-arginine methyltransferase activities in the three fractions were analyzed using recombinant fibrillarin (a nucleolar RGG protein) as the methyl-accepting substrate . Fibrillarin methylation was strongest in the presence of the cytosolic fraction, followed by the ribosomal and then the nucleus fractions . The results demonstrated that protein-arginine methyltransferases as well as their methyl-accepting substrates were widely distributed in different subcellular fractions of lymphoblastoid cells.

J Biol Chem, 2000 Nov 17, 275(46), 35840 - 7
The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase; Ravid T et al.; 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA . This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome . Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase . Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA . Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme . Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR . Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR . Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.

J Biol Chem, 2000 Oct 20, 275(42), 32387 - 90
The calcium-independent receptor for alpha-latrotoxin from human and rodent brains interacts with members of the ProSAP/SSTRIP/Shank family of multidomain proteins; Kreienkamp HJ et al.; Subtypes of the calcium-independent receptors for alpha-latrotoxin (CIRL1-3) define a distinct subgroup within the large family of the seven-transmembrane region cell surface receptors . The physiological function of CIRLs is unknown because neither extracellular ligands nor intracellular coupling proteins (G-proteins) have been identified . Using yeast two-hybrid screening, we identified a novel interaction between the C termini of CIRL1 and -2 and the PSD-95/discs large/ZO-1 (PDZ) domain of a recently discovered multidomain protein family (ProSAP/SSTRIP/Shank) present in human and rat brain . In vitro, CIRL1 and CIRL2 interacted strongly with the PDZ domain of ProSAP1 . The specificity of this interaction has been verified by in vivo experiments using solubilized rat brain membrane fractions and ProSAP1 antibodies; only CIRL1, but not CIRL2, was co-immunoprecipitated with ProSAP1 . In situ hybridization revealed that ProSAP1 and CIRL1 are co-expressed in the cortex, hippocampus, and cerebellum . Colocalization was also observed at the subcellular level, as both CIRL1 and ProSAP1 are enriched in the postsynaptic density fraction from rat brain . Expression of all three CIRL isoforms is highly regulated during postnatal brain development, with CIRL3 exhibiting its highest expression levels immediately after birth, followed by CIRL2 and finally CIRL1 in aged rats.

Environ Health Perspect, 2000 Aug, 108(8), 723 - 9
Quantitative comparisons of in vitro assays for estrogenic activities; Fang H et al.; Substances that may act as estrogens show a broad chemical structural diversity . To thoroughly address the question of possible adverse estrogenic effects, reliable methods are needed to detect and identify the chemicals of these diverse structural classes . We compared three assays--in vitro estrogen receptor competitive binding assays (ER binding assays), yeast-based reporter gene assays (yeast assays), and the MCF-7 cell proliferation assay (E-SCREEN assay)--to determine their quantitative agreement in identifying structurally diverse estrogens . We examined assay performance for relative sensitivity, detection of active/inactive chemicals, and estrogen/antiestrogen activities . In this examination, we combined individual data sets in a specific, quantitative data mining exercise . Data sets for at least 29 chemicals from five laboratories were analyzed pair-wise by X-Y plots . The ER binding assay was a good predictor for the other two assay results when the antiestrogens were excluded (r(2) is 0.78 for the yeast assays and 0.85 for the E-SCREEN assays) . Additionally, the examination strongly suggests that biologic information that is not apparent from any of the individual assays can be discovered by quantitative pair-wise comparisons among assays . Antiestrogens are identified as outliers in the ER binding/yeast assay, while complete antagonists are identified in the ER binding and E-SCREEN assays . Furthermore, the presence of outliers may be explained by different mechanisms that induce an endocrine response, different impurities in different batches of chemicals, different species sensitivity, or limitations of the assay techniques . Although these assays involve different levels of biologic complexity, the major conclusion is that they generally provided consistent information in quantitatively determining estrogenic activity for the five data sets examined . The results should provide guidance for expanded data mining examinations and the selection of appropriate assays to screen estrogenic endocrine disruptors.

Virology, 2000 Sep 1, 274(2), 246 - 54
The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies; Gowda S et al.; Citrus tristeza virus (CTV) has 10 3' open reading frames (ORFs) of unknown function except for the two coat proteins . The highest produced subgenomic RNAs are those of the major coat protein gene (p25) and the 3' most genes, p20 and p23 . The proteins from three ORFs, p25, p27, and p20, were examined in the yeast two-hybrid assay for the interactions between themselves and to one another . The p20 protein exhibited a high affinity for itself, suggesting that it might aggregate in infected cells . The cytopathology of CTV infections includes characteristic paracrystalline and amorphous inclusions in the phloem elements of infected citrus . Polyclonal antiserum raised against the bacterial expressed p20 gene product detected a protein of approximately 22-23 kDa, which accumulated to relatively high levels in CTV-infected citrus, but not in healthy citrus . Immunogold localization using antibodies to p20 protein showed strong and specific labeling of the amorphous inclusion bodies present in CTV-infected cells . Mesophyll protoplasts of Nicotiana benthamiana transfected with a CTV mutant containing the green fluorescent protein (GFP) ORF fused in-frame to the 3' end of p20 protein ORF expressed high levels of GFP . The fusion protein was concentrated in one specific area in the cytoplasm and lacked an organized shape . Accumulation of high levels of p20 protein in infected tissue, specific localization of the p20-GFP fusion protein, immunolocalization of p20 protein into amorphous inclusions, and strong homologous p20 protein-p20 protein interactions in the yeast-two-hybrid assay suggest that the p20 protein of CTV is a major component of the amorphous inclusion bodies present in CTV-infected cells .

Biochem Biophys Res Commun, 2000 Aug 28, 275(2), 509 - 16
Mouse prenylated Rab acceptor is a novel Golgi membrane protein; Liang Z et al.; We have cloned a mouse prenylated Rab acceptor (mPRA), which interacts with various Rab proteins in the yeast two-hybrid system . This study investigated its intracellular localization and characterized the localization signal . The mPRA was found to be an integral membrane protein that was localized to the Golgi complex at steady state as determined by confocal fluorescence microscopy . With green fluorescent protein attached to the N-terminus of mPRA, the fusion protein was expressed in BHK cells and was shown to exhibit the same Golgi localization as the native mPRA . Systematic truncations from the N- and C-termini of mPRA revealed that the entire N-terminal half (91 residues) of the protein was dispensable for the Golgi localization . In contrast, deletion of only 5 residues from the C-terminus diminished the Golgi localization of mPRA, leading to its accumulation in the ER . The data indicate that the C-terminal half (94 residues) of mPRA is necessary and sufficient for proper folding, ER export, and Golgi localization . The Golgi localization of mPRA suggests that it may play a role in the structural organization and function of the Golgi complex .

Biochem Biophys Res Commun, 2000 Aug 28, 275(2), 394 - 400
Molecular cloning and characterization of a copper chaperone for copper/zinc superoxide dismutase from the rat; Hiromura M et al.; Copper chaperone is an essential cytosolic factor that maintains copper homeostasis in living cells . Cytosolic metallochaperones have been recently identified in plant, yeast, rodents, and human cells . During our investigation, we found a new member of the copper chaperone family for copper/zinc superoxide dismutase, which was cloned from rats . The new copper chaperone was named rCCS (rat Copper Chaperone for Superoxide dismutase) . The cDNA of rCCS was found to have a length of 1094 bp, and the protein analyzed from the cDNA was deduced to contain 274 amino acids . The amino acid sequence of rCCS consists of three domains: A metal binding domain, which has a MXCXXC motif in domain I, a homolog of the Cu/Zn SOD in domain II, and a CXC motif in domain III . The binding of rCCS to Cu/Zn SOD was analyzed by GST column binding assay, and the domain II of rCCS was found to be essential for binding to Cu/Zn SOD, which in turn activates Cu/Zn SOD .

Genomics, 2000 Sep 1, 68(2), 220 - 8
A physical and transcript map based upon refinement of the critical interval for PPH1, a gene for familial primary pulmonary hypertension . The International PPH Consortium; Machado RD et al.; Primary pulmonary hypertension (PPH), an often fatal disorder, is characterized by sustained elevation of pulmonary artery pressure of unknown cause . In its familial form (FPPH), the disorder segregates as an autosomal dominant and displays markedly reduced penetrance . A gene for FPPH was previously localized to a 25-cM interval on the long arm of chromosome 2 (2q31-q33) . We now report a complete yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC)/P1 artificial chromosome contig (PAC), assembled by STS content mapping, across a newly identified minimum nonrecombinant interval containing the gene designated PPH1 . The physical map has served to establish polymorphic marker order unequivocally, enabling the establishment of detailed haplotypes for the region . Together with the identification of novel recombination events in affected individuals from six newly ascertained kindreds, these data have allowed the significant reduction of the minimum PPH1 critical interval to a 4.8-cM region . The region, flanked by the polymorphic markers D2S115 (centromeric) and D2S1384 (telomeric), corresponds to a minimum physical distance of 5.8 Mb at 2q33 . Numerous expressed sequence tags and known genes were placed on the YAC/BAC contig spanning the PPH1 gene critical region .

Genomics, 2000 Sep 1, 68(2), 127 - 35
Molecular cloning of the critical region for glomerulopathy with fibronectin deposits (GFND) and evaluation of candidate genes; Vollmer M et al.; Glomerulopathy with fibronectin deposits (GFND, MIM 601894) is an autosomal dominant kidney disease that leads to terminal renal failure at a median age of 47 years . It represents a distinct entity of membranoproliferative glomerulonephritis (MPGN) type III and is characterized by the unique feature of massive glomerular deposits of fibronectin . We have recently localized a gene locus for GFND to human chromosome 1q32 by total genome linkage analysis in a large kindred, within a 4.1-cM critical interval between markers D1S2872 and D1S2891 . This interval contains a cluster of genes for "regulators of complement activation" (RCA), which represent strong candidates for GFND . To identify positional candidate genes for GFND within the critical genetic interval, we here report the cloning of the entire critical GFND region in a complete YAC and partial PAC contig . We constructed a high-resolution transcriptional map, thereby defining positional and functional candidate genes for the disease . To evaluate their role in GFND, we performed functional studies on RCA proteins in GFND patients from the large kindred, as well as mutational analysis of the genes for complement receptor-2 (CR2), membrane cofactor protein (MCP), and decay accelerating factor (DAF) . Although no loss-of-function mutation has been identified as yet, these data provide a basis for the examination of candidate genes for GFND and other genes for MPGN, which localize to the vicinity of the GFND region .

Dev Biol, 2000 Sep 1, 225(1), 124 - 34
A role for a TIMP-3-sensitive, Zn(2+)-dependent metalloprotease in mammalian gamete membrane fusion; Correa LM et al.; During fertilization, sperm and egg plasma membranes adhere and then fuse by a mechanism that is not well understood . Zinc metalloproteases are necessary for some intercellular fusion events, for instance, cell-cell fusion in yeast . In this study we tested the effects of class-specific and family-specific protease inhibitors on mouse gamete fusion . Capacitated, acrosome-reacted sperm and zona-free eggs were used in assays designed to define the effects of inhibitors on sperm-egg plasma membrane binding or fusion . Inhibitors of the aspartic, cysteine, and serine protease classes had no effect on sperm-egg binding or fusion . Both a synthetic metalloprotease substrate (succinyl-Ala-Ala-Phe-amidomethylcoumarin) and the zinc chelator 1,10-phenanthroline inhibited sperm-egg fusion but did not decrease sperm-egg binding . The fusion-inhibition effect of phenanthroline was reversible and activity of the inhibitable zinc metalloprotease was shown to be required during a short time window, the first 15 min after insemination . Tissue inhibitor of metalloprotease-3 and Ro 31-9790, specific inhibitors of zinc metalloproteases in the matrixin and adamalysin families, also inhibited sperm-egg fusion but not sperm-egg binding . These data indicate a role in gamete fusion for one or more zinc metalloproteases of the matrixin and/or adamalysin families that act after plasma membrane binding and before sperm-egg membrane fusion .

Matrix Biol, 2000 Aug, 19(4), 289 - 301
The EMILIN protein family; Colombatti A et al.; The EMILINs are a new family of glycoproteins of the extracellular matrix . The prototype of this family is the chicken EMILIN that was originally identified in extracts of aortas; it was then found to be widely distributed in several tissues associated with elastin and localized at the interface between amorphous elastin and microfibrils . Based on peptide sequences, chicken and human cDNAs coding for EMILIN were isolated by RT/PCR by screening kidney and heart cDNA libraries . By using a C-terminal fragment of human EMILIN-1 as a bait in the yeast two-hybrid system, a second family member, EMILIN-2, has also been isolated . EMILINs are characterized by a C-terminal gC1q globular domain, a short collagenous sequence, a long coiled-coil region and a new cysteine-rich N-terminal domain that can be considered a hallmark of the family being present also in multimerin . The gene for EMILIN-1 was mapped on chromosome 2p23 overlapping with the promoter region of the ketohexokinase gene . The gC1q domain of EMILIN-1 can form relatively stable and compact homotrimers and this association is then followed by a multimeric assembly of disulfide-bonded protomers . Recombinant EMILIN-1 purified from the supernatant of 293 cells represents a very efficient ligand for cell adhesion of several cell types.

Proc Natl Acad Sci U S A, 2000 Sep 12, 97(19), 10637 - 42
Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds; Luo M et al.; The promoters of MEA (FIS1), FIS2, and FIE (FIS3), genes that repress seed development in the absence of pollination, were fused to beta-glucuronidase (GUS) to study their activity pattern . The FIS2GUS product is found in the embryo sac, in each of the polar cell nuclei, and in the central cell nucleus . After pollination, the maternally derived FIS2GUS protein occurs in the nuclei of the cenocytic endosperm . Before cellularization of the endosperm, activity is terminated in the micropylar and central nuclei of the endosperm and subsequently in the nuclei of the chalazal cyst . MEAGUS has a pattern of activity similar to that of FIS2GUS, but FIEGUS protein is found in many tissues, including the prepollination embryo sac, and in embryo and endosperm postpollination . The similarity in mutant phenotypes; the activity of FIE, MEA, and FIS2 in the same cells in the embryo sac; and the fact that MEA and FIE proteins interact in a yeast two-hybrid system suggest that these proteins operate in the same system of control of seed development . Maternal and not paternal FIS2GUS, MEAGUS, and FIEGUS show activity in early endosperm, so these genes may be imprinted . When fis2, mea, and fie mutants are pollinated, seed development is arrested at the heart embryo stage . The seed arrest of mea and fis2 is avoided when they are fertilized by a low methylation parent . The wild-type alleles of MEA or FIS2 are not required . The parent-of-origin-determined differential activity of MEA, FIS2, and FIE is not dependent on DNA methylation, but methylation does control some gene(s) that have key roles in seed development.

J Biol Chem, 2000 Nov 24, 275(47), 37055 - 61
The inositol polyphosphate 5-phosphatases and the apurinic/apyrimidinic base excision repair endonucleases share a common mechanism for catalysis; Whisstock JC et al.; Inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from the inositol ring of phosphatidylinositol-derived signaling molecules; however, the mechanism of catalysis is only partially characterized . These enzymes play critical roles in regulating cell growth, apoptosis, intracellular calcium oscillations, and post-synaptic vesicular trafficking . The UCLA fold recognition server (threader) predicted that the conserved 300-amino acid catalytic domain, common to all 5-phosphatases, adopts the fold of the apurinic/apyrimidinic (AP) base excision repair endonucleases . PSI-BLAST searches of GENPEPT, using the amino acid sequence of AP endonuclease exonuclease III, identified all members of the 5-phosphatase family with highly significant scores . A sequence alignment between exonuclease III and all known 5-phosphatases revealed six highly conserved motifs containing residues that corresponded to the catalytic residues in the AP endonucleases . Mutation of each of these residues to alanine in the mammalian 43-kDa, or yeast Inp52p 5-phosphatase, resulted in complete loss of enzyme activity . We predict the 5-phosphatase enzymes share a similar mechanism of catalysis to the AP endonucleases, consistent with other common functional similarities such as an absolute requirement for magnesium for activity . Based on this analysis, functional roles have been assigned to conserved residues in all 5-phosphatase enzymes.

J Biol Chem, 2000 Nov 17, 275(46), 36316 - 23
Covalent modification of p73alpha by SUMO-1 . Two-hybrid screening with p73 identifies novel SUMO-1-interacting proteins and a SUMO-1 interaction motif; Minty A et al.; Two-hybrid screening in yeast with p73alpha isolated SUMO-1 (small ubiquitin-like modifier 1), the enzyme responsible for its conjugation, Ubc-9, and a number of novel SUMO-1-interacting proteins, including thymine DNA glycosylase, PM-Scl75, PIASx, PKY, and CHD3/ZFH . A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with SUMO-1 in the two-hybrid system . We show here that p73alpha, but not p73beta, can be covalently modified by SUMO-1 . The major SUMO-1-modified residue in p73alpha is the C-terminal lysine (Lys(627)) . The sequence surrounding this lysine conforms to a consensus SUMO-1 modification site b(X)XXhKXE, where b is a basic amino acid . SUMO-1-modified p73 is more rapidly degraded by the proteasome than unmodified p73, although SUMO-1 modification is not required for p73 degradation . SUMO-1 modification does not affect the transcriptional activity of p73alpha on an RGC-luciferase reporter gene in SK-N-AS cells . Instead, SUMO-1 modification may alter the subcellular localization of p73, because SUMO-1-modified p73 is preferentially found in detergent-insoluble fractions . Alternatively, it may modulate the interaction of p73 with other proteins that are substrates for SUMO-1 modification or which interact with SUMO-1, such as those identified here.

Biochem Soc Trans, 2000, 28(4), 481 - 5
Auxin transport: providing a sense of direction during plant development; Swarup R et al.; Auxins are key regulators of plant development . Plants employ a specialized delivery system termed polar auxin transport to convey indole-3-acetic acid from source to target tissues . Auxin transport is mediated by the combined activities of specialized influx and efflux carriers . Mutational approaches in the model plant, Arabidopsis thaliana, have led to the molecular genetic characterization of putative auxin influx and efflux carrier components, AUX1 and AtPIN1 . Both genes belong to distinct gene families that are being functionally characterized by using a reverse genetic approach in Arabidopsis . AtPIN proteins are asymmetrically localized within plant plasma membranes, providing a molecular mechanism for the characteristic polarity of auxin transport . We outline the epitope tagging strategy being used in our laboratory to immunolocalize AUX1 and discuss the implications of its subcellular localization for auxin redistribution within root apical tissues . Lastly, we describe a novel carrier-based mechanism that plant cells might use to determine their relative position(s) within an auxin gradient, drawing parallels with the mechanism of glucose perception in yeast.

Biochem Soc Trans, 2000, 28(4), 376 - 9
Mechanisms for ATP-dependent chromatin remodelling; Whitehouse I et al.; Gene regulation involves the generation of a local chromatin topology that is conducive to transcription . Several classes of chromatin remodelling activity have been shown to play a role in this process . ATP-dependent chromatin-remodelling activities use energy derived from the hydrolysis of ATP to alter the structure of chromatin, making it more accessible for transcription factor binding . The yeast SWI-SWF complex is the founding member of this family of ATP-dependent chromatin-remodelling activities . We have developed a model system to study the ability of the SWI-SWF complex to alter chromatin structure . Using this system, we find that SWI-SWF is able to alter the position of nucleosomes along the DNA . This is consistent with recent reports that other ATP-dependent chromatin-remodelling activities can alter the positions of nucleosomes along DNA . This suggests that nucleosome mobilization may be a general feature of the activity of ATP-dependent chromatin-remodelling activities . Some of the mechanisms by which nucleosomes may be moved along DNA are discussed.

Sud Med Ekspert, 2000 Jul-Aug, 43(4), 22 - 4
{Some aspects in preparation and use of wide spectrum antiglobulin sera in forensic medical studies}; Kolokolva GP; Wide spectrum (polyspecific) antiglobulin sera were obtained by rabbit immunization with 10% solution of zymosan-charged globulin (product of yeast digestion with trypsin) with complete Freund's adjuvant . The resultant sera meet the international standards and can be used for forensic medical identification of serum G1m(1) group and for detecting incomplete antierythrocytic complement-fixing anti-Daffi, anti-Kell, and other antibodies in blood transfusion service.

Cancer Lett, 2000 Oct 1, 158(2), 141 - 50
Mutation analysis of mitotic checkpoint genes (hBUB1 and hBUBR1) and microsatellite instability in adult T-cell leukemia/lymphoma; Ohshima K et al.; Adult T-cell leukemia/lymphoma (ATLL) is a neoplasm of T-lymphocytes, and human T-cell lymphotropic virus type-I (HTLV-I) is etiologically considered as the causative virus of ATLL . The karyotypes of ATLL are very complex in both number and structure, although no specific karyotype abnormalities have been identified . HTLV-I is thought to integrate its provirus into random sites in host chromosomal DNA and induces chromosomal instability . The BUB gene is a component of the mitotic checkpoint in budding yeast . Recently, human homologues of the BUB were identified and mutant alleles of hBUB1 and hBUBR1 were detected in two colorectal tumor cell lines, which showed microsatellite instability (MIN) . In vitro, BUB proteins form a complex of monomers . These proteins interact with the human MAD1 gene product, a target of the HTLV-1 tax oncogene . We examined the role of checkpoint gene in the chromosomal abnormalities of ATLL by investigating mutations of hBUB1 and hBUBR1, and MIN of replication errors of BAX, insulin-like growth factor, and transforming growth factor beta type II . We analyzed ten cases with ATLL and eight B-cell lymphomas (five diffuse large cell lymphomas, three follicular lymphomas) . Complex chromosomal abnormalities were detected in ATLL, while B-cell lymphomas showed only simple or minimal chromosomal abnormalities . Significant mutations/deletion of hBUB1 or hBUBR1 were detected in four of ten cases with ATLL, including two heterozygous point mutations, one homozygous point mutation, and one with a 47 bp deletion . In contrast, only one of eight B-cell lymphomas showed nonsense mutation of hBUBR1 . None of the ATLL and B-cell lymphomas showed MIN . In the multistage process of leukemogenesis of ATLL, our findings indicate that mutations of mitotic checkpoint genes may play an important role in the induction of complex chromosomal abnormalities.

J Nutr, 2000 Sep, 130(9), 2384 - 9
Selenium from high selenium broccoli protects rats from colon cancer; Finley JW et al.; Colon cancer is the third most common newly diagnosed cancer in the United States and the third most common cause of cancer-related deaths . Previous supplementation studies have demonstrated the efficacy of selenium (Se) for prevention of colon cancer in humans . The metabolism of Se depends on its chemical form, and studies have shown that the chemical form of Se in broccoli does not accumulate in the body as fast as other forms of Se and may be especially beneficial for prevention of cancer . In the first experiment of the present study, Fisher F-344 rats (n = 45) were allotted randomly to torula yeast-based diets supplemented with the following: 1) no Se; 2) 0.1 microg Se/g diet as selenate; 3) 1.0 microg Se/g diet as selenate; 4) 0.1 microg Se/g diet as selenized broccoli (Se concentration of approximately 500 microg/g); or 5) 1.0 microg Se/g diet as selenized broccoli . In Experiment 2, rats (n = 80) were allotted randomly to the same basal diet supplemented with the following: 1) no added Se; 2) 2.0 microg Se/g diet as selenite; 3) 2 . 0 microg Se/g diet as selenite + low Se broccoli; and 4) 2.0 microg Se/g diet as selenized broccoli . Rats were fed the diets for 2 wk and injected with a chemical carcinogen (3,2 dimethyl 4-amino biphenyl or dimethyl-hydrazine in Experiment 1 or dimethyl hydrazine in Experiment 2; 2 rats/treatment were used as vehicle controls) . Supranutritional amounts of Se supplied as high Se broccoli significantly decreased (P: < 0.05) the incidence of aberrant crypts (AC) and aberrant crypt foci (ACF; preneoplastic lesions indicative of colon cancer) compared with other dietary treatments . Diets were controlled for the presence or absence of broccoli and for the total amount of Se . The reduction in AC and ACF was a function of Se in high Se broccoli and not a result of broccoli alone or Se alone . Adequate dietary Se supplied as high Se broccoli did not accumulate in tissues or increase glutathione peroxidase activity as well as other forms and amounts of Se . Thus, Se from high Se broccoli may be metabolized in a manner that diverts much of the Se into a pool that provides protection against colon cancer.

J Biol Chem, 2000 Nov 17, 275(46), 36204 - 10
The G protein-coupled receptor CL1 interacts directly with proteins of the Shank family; Tobaben S et al.; PDZ domains play a pivotal role in the synaptic localization of ion channels, receptors, signaling enzymes, and cell adhesion molecules . These domains mediate protein-protein interactions via the recognition of a conserved sequence motif at the extreme C terminus of their target proteins . By means of a yeast two-hybrid screen using the C terminus of the G protein-coupled alpha-latrotoxin receptor CL1 as bait, three PDZ domain proteins of the Shank family were identified . These proteins belong to a single protein family characterized by a common domain organization . The PDZ domain is highly conserved among the family members, significantly different from other known PDZ domains, and specifically binds to the C terminus of CL1 . Shank1 and CL1 are expressed primarily in brain, and both proteins co-enrich in the postsynaptic density . Furthermore, Shank1 induces a clustering of CL1 in transfected cells, strongly supporting an interaction of both proteins in vivo.

Mol Cell Biol, 2000 Sep, 20(18), 6984 - 95
p50(Cdc37) can buffer the temperature-sensitive properties of a mutant of Hck; Scholz G et al.; Genetic studies have previously revealed that Cdc37p is required for the catalytic competence of v-Src in yeast . We have reasoned that temperature-sensitive mutants of Src family kinases might be more sensitive to the cellular level of p50(Cdc37), the mammalian homolog of Cdc37p, than their wild-type counterpart, thus potentially providing a unique opportunity to elucidate the involvement of p50(Cdc37) in the folding and stabilization of Src family kinases . A temperature-sensitive mutant of a constitutively active form of Hck (i.e., tsHck499F) was created by mutating two amino acids within the kinase domain of Hck499F . Significantly, overexpression of p50(Cdc37) rescues the catalytic activity of tsHck499F at 33 degrees C, while partially buffering it against inactivation at higher temperatures (e.g., 37 and 39 degrees C) . Hsp90 function is required for tsHck499F activity and its stabilization by p50(Cdc37), but overexpression of Hsp90 is not sufficient to stabilize tsHck499F . Overexpression of p50(Cdc37) promotes the association of tsHck499F with Hsp90, suggesting that the cellular level of p50(Cdc37) might be the rate-limiting step in the association of tsHck499F with Hsp90 . A truncation mutant of p50(Cdc37) that cannot bind Hsp90 still has a limited capacity to rescue the catalytic activity of tsHck499F and promote its association with Hsp90 . This is a particularly important observation, since it argues that rather than solely acting as a passive adapter protein to tether tsHck499F to Hsp90, p50(Cdc37) may also act allosterically to enhance the association of tsHck499F with Hsp90 . The findings presented here might also have implications for our understanding of the evolution of protein kinases and tumor development.

Hum Mol Genet, 2000 Sep 1, 9(14), 2107 - 16
Mutations in the CNGB3 gene encoding the beta-subunit of the cone photoreceptor cGMP-gated channel are responsible for achromatopsia (ACHM3) linked to chromosome 8q21; Kohl S et al.; Achromatopsia is an autosomal recessive disorder featuring total colour blindness, photophobia, reduced visual acuity and nystagmus . While mutations in the CNGA3 gene on chromosome 2q11 are responsible for achromatopsia in a subset of patients, previous linkage studies have localized another achromatopsia locus, ACHM3, on chromosome 8q21 . Using achromatopsia families in which CNGA3 mutations have been excluded, we refined the ACHM3 locus to a 3.7 cM region enclosed by markers D8S1838 and D8S273 . Two yeast artificial chromosome (YAC) contigs covering nearly the entire ACHM3 interval were constructed . Database searches with YAC content sequences identified two overlapping high throughput genomic sequencing phase (HTGS) entries which contained sequences homologous to the murine cng6 gene encoding the putative beta-subunit of the cone photoreceptor cGMP-gated channel . Using RT-PCR and RACE, we identified and cloned the human cDNA homologue, designated CNGB3, which encodes an 809 amino acid polypeptide . Northern blot analysis revealed a major transcript of approximately 4.4 kb specifically expressed in the retina . The human CNGB3 gene consists of 18 exons distributed over approximately 200 kb of genomic sequence . Analysis of the CNGB3 gene in achromats revealed six different mutations including a missense mutation (S435F), two stop codon mutations (R203X and E336X), a 1 bp and an 8 bp deletion (1148delC and 819-826del) and a putative splice site mutation of intron 13 . The 1148delC mutation was identified recurrently in several families, and in total was present on 11 of 22 disease chromosomes segregating in our families.

Hum Mol Genet, 2000 Sep 1, 9(14), 2095 - 105
The retinitis pigmentosa GTPase regulator (RPGR) interacts with novel transport-like proteins in the outer segments of rod photoreceptors; Roepman R et al.; Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene cause X-linked retinitis pigmentosa type 3 (RP3), a severe, progressive and degenerative retinal dystrophy eventually leading to complete blindness . RPGR is ubiquitously expressed, yet mutations in the RPGR gene lead to a retina-restricted phenotype . To date, all RP3 associated missense mutations that have been identified are located in the RCC1-homologous domain (RHD) of RPGR . To investigate the molecular pathogenesis of RP3, we screened retinal yeast two-hybrid libraries with the RHD of RPGR . We identified several alternatively spliced gene products, some with retina-restricted expression, that interact specifically with RPGR in vivo and in vitro . Thus, these proteins were named RPGR-interacting protein 1 (RPGRIP1) isoforms . They contain a C-terminal RPGR-interacting domain and stretches of variable coiled-coil domains homologous to proteins involved in vesicular trafficking . The interaction between RPGR and RPGRIP1 isoforms was impaired in vivo by RP3-associated mutations in RPGR . Moreover, RPGR and RPGRIP1 co-localize in the outer segment of rod photoreceptors, which is in full agreement with the retinitis pigmentosa phenotype observed in RP3 patients . The localization of RPGRIP1 at 14q11 makes it a strong candidate gene for RP16 . These results provide a clue for the retina-specific pathogenesis in RP3, and hint towards the involvement of RPGR and RPGRIP1 in mediating vesicular transport-associated processes.

Hum Mol Genet, 2000 Sep 1, 9(14), 2085 - 93
Identification of a novel protein interacting with RPGR; Boylan JP et al.; A novel protein, called RPGRIP, has been identified as interacting with the RPGR protein, which is mutated in a severe form of human retinal degeneration, X-linked retinitis pigmentosa (RP3 type) . The bovine RPGRIP was identified initially by screening for RPGR-interacting proteins with a bovine retina cDNA library using the yeast two-hybrid system . The specificity of the interaction was confirmed by co-immunoprecipitation of in vitro translated protein and using RPGR mutants . The human RPGRIP gene was isolated and shown to be expressed in retina and testis . Human RPGRIP spans a genomic interval of 34 kb, and consists of 15 exons, some of which are alternatively spliced . It was mapped using monochromosomal and radiation hybrid cell lines to chromosomal region 14q11 . The function of RPGRIP is unknown; it shows no homology to proteins of known function, although it is predicted to form two coiled-coil domains at the N-terminus . RPGRIP is a strong candidate gene for causing human retinal degeneration.

Genome Res, 2000 Aug, 10(8), 1138 - 47
Discovery of a novel, paternally expressed ubiquitin-specific processing protease gene through comparative analysis of an imprinted region of mouse chromosome 7 and human chromosome 19q13.4; Kim J et al.; Using mouse BAC clones spanning an imprinted interval of proximal mouse chromosome 7 and the genomic sequence of the related interval of human chromosome 19q13.4, we have identified a novel mouse gene, Usp29 (ubiquitin-specific processing protease 29), near two known imprinted genes, Peg3 and Zim1 . Gene Usp29 is located directly adjacent to Peg3 in a "head-to-head" orientation, and comprises exons distributed over a genomic distance of at least 400 kb . A similar human gene is also found in the homologous location in human chromosome 19q13.4 . The mouse Usp29 gene is also imprinted and is transcribed mainly from the paternal allele with highest expression levels in adult brain, especially in the cerebral cortex and hippocampus, and in the forebrain, face, and limb buds of midgestation mouse embryos . Analysis of a full-length 7.6-kb cDNA clone revealed that Usp29 encodes an 869-amino-acid protein that displays significant homology with yeast and nematode ubiquitin carboxyl-terminal hydrolases . These data suggest that, like the candidate Angelman syndrome gene Ube3a (ubiquitin ligase), Usp29 may represent another imprinted gene involved in the ubiquitination pathway . This identification of a third imprinted gene, Usp29, from the Peg3/Zim1-region confirms the presence of a conserved imprinted domain spanning at least 500 kb in the proximal portion of mouse chromosome 7 (Mmu7).

Genome Res, 2000 Aug, 10(8), 1103 - 7
Gene survey of the pathogenic protozoan Trypanosoma cruzi; Porcel BM et al.; We have performed a survey of the active genes in the important human pathogen Trypanosoma cruzi by analyzing 5013 expressed sequence tags (ESTs) generated from a normalized epimastigote cDNA library . Clustering of all sequences resulted in 771 clusters, comprising 54% of the ESTs . In total, the ESTs corresponded to 3054 transcripts that might represent one-fourth of the total gene repertoire in T . cruzi . About 33% of the T . cruzi transcripts showed similarity to sequences in the public databases, and a large number of hitherto undiscovered genes predicted to be involved in transcription, cell cycle control, cell division, signal transduction, secretion, and metabolism were identified . More than 140 full-length gene sequences were derived from the ESTs . Comparisons with all open reading frames in yeast and in Caenorhabditis elegans showed that only 12% of the T . cruzi transcripts were shared among diverse eukaryotic organisms . Comparison with other kinetoplastid sequences identified 237 orthologous genes that are shared between these evolutionarily divergent organisms . The generated data are a useful resource for further studies of the biology of the parasite and for development of new means to combat Chagas' disease.

J Biol Chem, 2000 Dec 1, 275(48), 37957 - 65
Molecular characterization of CRMP5, a novel member of the collapsin response mediator protein family; Fukada M et al.; The CRMP (collapsin response mediator protein) family is thought to play key roles in growth cone guidance during neural development . The four members (CRMP1-4) identified to date have been demonstrated to form hetero-multimeric structures through mutual associations . In this study, we cloned a novel member of this family, which we call CRMP5, by the yeast two-hybrid method . This protein shares relatively low amino acid identity with the other CRMP members (49-50%) and also with dihydropyrimidinase (51%), whereas CRMP1-4 exhibit higher identity with each other (68-75%), suggesting that CRMP5 might be categorized into a third subfamily . The mouse CRMP5 gene was located at chromosome 5 B1 . Northern blot and in situ hybridization analyses indicated that CRMP5 is expressed throughout the nervous system similarly to the other members (especially CRMP1 and CRMP4) with the expression peak in the first postnatal week . Association experiments using the yeast two-hybrid method and co-immunoprecipitation showed that CRMP5 interacts with dihydropyrimidinase and all the CRMPs including itself, except for CRMP1, although the expression profile almost overlaps with that of CRMP1 during development . These results suggest that CRMP complexes in the developing nervous system are classifiable into two populations that contain either CRMP1 or CRMP5 . This indicates that different complexes may have distinct functions in shaping the neural networks.

J Biol Chem, 2000 Dec 1, 275(48), 37798 - 806
Positive- and negative-acting Kruppel-like transcription factors bind a transforming growth factor beta control element required for expression of the smooth muscle cell differentiation marker SM22alpha in vivo; Adam PJ et al.; Transforming growth factor beta (TGF-beta) is implicated in the regulation of smooth muscle cell (SMC) differentiation . We previously identified a novel TGF-beta control element (TCE) in the promoters of SMC differentiation marker genes, including alpha-smooth muscle actin and SM22alpha . In this study, the importance of the TCE in regulation of SM22alpha gene expression in vivo was investigated by mutating it within the context of a mouse SM22alpha promoter-lacZ transgenic construct . Mutation of the TCE completely abolished SM22alpha promoter activity in arterial SMCs as well as in developing heart and skeletal muscle . To identify the transcription factor(s) binding to the TCE, we performed yeast one-hybrid cloning analysis and identified gut-enriched Kruppel-like factor (GKLF) . However, cotransfection studies in cultured cells showed that GKLF repressed the TGF-beta-dependent increases in SM22alpha and alpha-smooth muscle actin promoter activities . Furthermore, GKLF was not highly expressed in differentiated SMCs in vivo, and TGF-beta down-regulated GKLF expression in dedifferentiated cultured SMCs . In contrast, overexpression of a related factor (BTEB2) transactivated SM22alpha promoter activity . Thus, our findings suggest a reciprocal role for related Kruppel-like transcription factors in the regulation of SMC differentiation through a TCE-dependent mechanism.

J Biol Chem, 2000 Nov 10, 275(45), 35233 - 41
Subsets of human origin recognition complex (ORC) subunits are expressed in non-proliferating cells and associate with non-ORC proteins; Thome KC et al.; The origin recognition complex (ORC) in yeast is a complex of six tightly associated subunits essential for the initiation of DNA replication . Human ORC subunits are nuclear in proliferating cells and in proliferative tissues like the testis, consistent with a role of human ORC in DNA replication . Orc2, Orc3, and Orc5 also are detected in non-proliferating cells like cardiac myocytes, adrenal cortical cells, and neurons, suggesting an additional role of these proteins in non-proliferating cells . Although Orc2-5 co-immunoprecipitate with each other under mild extraction conditions, a holo complex of the subunits is difficult to detect . When extracted under more stringent extraction conditions, several of the subunits co-immunoprecipitate with stoichiometric amounts of other unidentified proteins but not with any of the known ORC subunits . The variation in abundance of individual ORC subunits (relative to each other) in several tissues, expression of some subunits in non-proliferating tissues, and the absence of a stoichiometric complex of all the subunits in cell extracts indicate that subunits of human ORC in somatic cells might have activities independent of their role as a six subunit complex involved in replication initiation . Finally, all ORC subunits remain consistently nuclear, and Orc2 is consistently phosphorylated through all stages of the cell cycle, whereas Orc1 is selectively phosphorylated in mitosis.

J Biol Chem, 2000 Dec 1, 275(48), 37742 - 51
The BNIP-2 and Cdc42GAP homology domain of BNIP-2 mediates its homophilic association and heterophilic interaction with Cdc42GAP; Low BC et al.; We recently showed that BNIP-2 is a putative substrate of the fibroblast growth factor receptor tyrosine kinase and it possesses GTPase-activating activity toward the small GTPase, Cdc42 . The carboxyl terminus of BNIP-2 shares high homology to the non-catalytic domain of Cdc42GAP, termed BCH (for BNIP-2 and Cdc42GAP homology) domain . Despite the lack of obvious homology to any known catalytic domains of GTPase-activating proteins (GAPs), the BCH domain of BNIP-2 bound Cdc42 and stimulated the GTPase activity via a novel arginine-patch motif similar to that employed by one contributing partner in a Cdc42 homodimer . In contrast, the BCH domain of Cdc42GAP, although it can bind Cdc42, is catalytically inactive . This raises the possibility that these domains might have other roles in the cell . Using glutathione S-transferase recombinant proteins, immunoprecipitation studies, and yeast two-hybrid assays, it was found that BNIP-2 and Cdc42GAP could form homo and hetero complexes via their conserved BCH domains . Molecular modeling of the BNIP-2 BCH homodimer complex and subsequent deletion mutagenesis helped to identify the region (217)RRKMP(221) as the major BCH interaction site within BNIP-2 . In comparison, deletion of either the arginine-patch (235)RRLRK(239) (necessary for GAP activity) or region (288)EYV(290) (a Cdc42 binding sequence) had no effect on BCH-BCH interaction . Extensive data base searches showed that the BCH domain is highly conserved across species . The results suggest that BCH domains of BNIP-2 and Cdc42GAP represent a novel protein-protein interaction domain that could potentially determine and/or modify the physiological roles of these molecules.

J Biol Chem, 2000 Nov 3, 275(44), 34521 - 7
Identification of Mrj, a DnaJ/Hsp40 family protein, as a keratin 8/18 filament regulatory protein; Izawa I et al.; To elucidate the function of keratins 8 and 18 (K8/18), major components of the intermediate filaments of simple epithelia, we searched for K8/18-binding proteins by screening a yeast two-hybrid library . We report here that human Mrj, a DnaJ/Hsp40 family protein, directly binds to K18 . Among the interactions between DnaJ/Hsp40 family proteins and various intermediate filament proteins that we tested using two-hybrid methods, Mrj specifically interacted with K18 . Immunostaining with anti-Mrj antibody showed that Mrj colocalized with K8/18 filaments in HeLa cells . Mrj was immunoprecipitated not only with K18, but also with the stress-induced and constitutively expressed heat shock protein Hsp/c70 . Mrj bound to K18 through its C terminus and interacted with Hsp/c70 via its N terminus, which contains the J domain . Microinjection of anti-Mrj antibody resulted in the disorganization of K8/18 filaments, without effects on the organization of actin filaments and microtubules . Taken together, these results suggest that Mrj may play an important role in the regulation of K8/18 filament organization as a K18-specific co-chaperone working together with Hsp/c70.

J Cell Sci, 2000 Sep, 113 ( Pt 18), 3267 - 75
The mammalian homologue of the Caenorhabditis elegans polarity protein PAR-6 is a binding partner for the Rho GTPases Cdc42 and Rac1; Johansson A et al.; A mammalian homologue of the PDZ domain containing Caenorhabditis elegans protein PAR-6 was found in a yeast two-hybrid system screen as binding to the Rho family member Cdc42 . PAR-6 contains a PDZ domain and in C . elegans it has been shown to be crucial for the asymmetric cleavage and establishment of cell polarity during the first cell divisions in the growing embryo . Mammalian PAR-6 interacted with Cdc42 and Rac1 both in the yeast two-hybrid system and in in vitro binding assays . Co-immunoprecipitation experiments, employing transiently transfected Cos-1 cells, further confirmed that Cdc42 and Rac1 are physiological binding partners for PAR-6 . We found that, in epithelial Madin-Darby canine kidney cells (MDCK), endogenous PAR-6 was present in the tight junctions, as judged from its co-localisation with the tight junction protein ZO-1, however, PAR-6 was also detected in the cell nucleus . Stimulation of MDCK cells with scatter factor/hepatocyte growth factor induced a loss of PAR-6 from the areas of cell-cell contacts in conformity with their progressive breakdown . In C . elegans PAR-6 co-localises with PAR-3 and has been suggested to form a direct complex . In agreement with earlier studies, mammalian PAR-3 was found to be present in tight junctions of MDCK cells but, in contrast to PAR-6, the protein could not be detected in the nucleus . Furthermore, co-immunoprecipitation experiments, employing Cos-1 cells, demonstrated that mammalian PAR-6 and PAR-3 formed a direct complex . These findings, together with the reported roles of PAR-6 and PAR-3 in C . elegans, suggest that Cdc42 and Rac1 and PAR-6/PAR-3 are involved in the establishment of cell polarity in epithelial cells.

Mol Gen Genet, 2000 Jul, 263(6), 908 - 15
Identification and chromosomal localization of the monkey retrotransposon in Musa sp; Balint-Kurti PJ et al.; Retroelements are ubiquitous features of eukaryotic genomes, often accounting for a substantial fraction of their total DNA content . One major group of retroelements, which includes the gypsy and copia-like elements, is distinguished by the presence of long terminal repeats (LTRs) . We have identified and partially characterized a sequence from banana (Musa acuminata cv . Grand Nain) which shows significant homology to gypsy-like LTR retroelements from other species . The element, named monkey, shows a high degree of homology to the reverse transcriptase, RNase H and integrase genes of retroelements from plants, fungi and yeast . However, several stop codons are present in the major ORF of this element, suggesting that this copy of monkey, if functional, is non-autonomous . Southern analysis indicated that monkey is present in both the A and B genomes of Musa, and that it is found in 200-500 copies per haploid genome in cv . Grand Nain . Chromosomal localization by fluorescent in-situ hybridization indicates that copies of monkey are concentrated in the nucleolar organizer regions and colocalize with rRNA genes . Other copies of monkey appear to be dispersed throughout the genome.

Tsitologiia, 2000, 42(6), 573 - 7
{Effect of polyamine synthesis inhibitors separately and in combination with epidermal growth factor on fusion of lysosomes with phagosomes and F-actin level in mouse peritoneal macrophages}; Mozhenok TP et al.; Effects of polyamine (PA) synthesis inhibitors--alpha-difluoromethylornithinchloride (DFMO) and alpha-methylornithinchloride (MO)--separately or in combination with the epidermal growth factor (EGF)--on lysosome-phagosome fusion (P-LF) and F-actin content in murine peritoneal macrophages were studied using fluorescent dye Acridine orange for lysosome labelling, FITC-phalloidin for F-actin, and yeast cells as a target . DFMO and MO significantly inhibited P-LF and decreased F-actin content in murine peritoneal macrophages . A combination of DFMO and MO with EGF failed to inhibit P-LF or to decrease F-actin content in these cells . The results obtained with DFMO and MO suggested new cellular targets of their effects . These results may be extended to cancer research to provide a rationale for clinical trials using combinations of EGF with DFMO or MO.

Anticancer Res, 2000 Jul-Aug, 20(4), 2653 - 8
Modulation of bcl-2 and cytotoxicity by licochalcone-A, a novel estrogenic flavonoid; Rafi MM et al.; Herbal therapies are commonly used by patients with cancer, despite little understanding about their clinical and biological activity . We recently demonstrated that the herbal combination PC-SPES, which contains licorice root, had potent estrogenic activity in vitro, in animals, and in patients with prostate cancer . Licochalcone-A (LA) is one flavonoid extracted from licorice root with antiparasitic and anti-tumor activity, but the effect on the human estrogen receptor and mechanism of anti-tumor activity is unknown . Recent studies demonstrated that the mechanism of cytotoxic effect by some estrogens may involve modulation of the anti-apoptotic protein bcl-2 . In the present study, we determined if LA had estrogenic activity, anti-tumor activity, and modulated the apoptotic protein bcl-2 in human cell lines derived from acute leukemia, breast cancer, and prostate cancer . A yeast growth-based assay under the control of the human estrogen receptor (hER) demonstrated that LA was a phytoestrogen . A cell viability assay demonstrated that LA had anti-tumor activity in all cell lines tested and enhanced the effect of paclitaxel and vinblastine chemotherapy . LA induced apoptosis in MCF-7 and HL-60 cell lines, as demonstrated by cleavage of PARP, the substrate of ICE-like proteases . Immunoblot analysis demonstrated that LA decreased the anti-apoptotic protein bcl-2 and altered the bcl-2/bax ratio in favor of apoptosis . In contrast, the parent compound chalcone or estradiol did not decrease bc1-2 expression . Therefore, these data demonstrate that LA is a phytoestrogen with anti-tumor activity and is capable of modulating bcl-2 protein expression . The modulation of bcl-2 may be dependent on specific structural differences between LA and the parent compound chalcone and independent of LA estrogenicity.

Mol Pharmacol, 2000 Sep, 58(3), 584 - 90
Functional differences between the amino-terminal domains of estrogen receptors alpha and beta; Delaunay F et al.; Human estrogen receptors alpha (ERalpha) and beta (ERbeta) are ligand-inducible transcription factors that are highly homologous in their central DNA-binding and carboxyl-terminal ligand-binding domains . In contrast, there is very little conservation between ERalpha and ERbeta in the amino-terminal domain . Using different human cell lines, we show that wild-type ERbeta transcriptional activity is lower or similar to that of ERalpha, depending on the cell type . Deletion of the amino-terminal domain in both ER subtypes resulted in no or a lower decrease of transcriptional activity of ERbeta compared with ERalpha, suggesting that the ERbeta amino-terminal domain contains a weaker transcriptional activation function-1 . Using ERalpha and ERbeta deletion mutants, we showed that the amino-terminal transcriptional activity of ERbeta maps to amino acids 1-31 . Interestingly, this domain contains a six amino-acid motif (amino acids 5-10 in human ERbeta) that is part of the ERalpha-activation function-1 region (amino acids 49-54 in human ERalpha) and highly conserved among all mammalian ERalpha amino-terminal domains . Despite this similarity between the two ER subtypes, no autonomous and ligand-independent activity of the ERbeta-amino-terminal domain was observed in yeast and mammalian cells in contrast to ERalpha . This study provides a molecular basis for the difference in transcriptional activity between ERalpha and ERbeta and establishes that ERbeta contains a structurally and functionally restricted amino-terminal transcriptional activity.

J Cell Biol, 2000 Aug 21, 150(4), 881 - 6
Peroxisomal membrane fusion requires two AAA family ATPases, Pex1p and Pex6p; Titorenko VI et al.; Two AAA family ATPases, NSF and p97, have been implicated in membrane fusion during assembly and inheritance of organelles of the secretory pathway . We have now investigated the roles of AAA ATPases in membrane fusion during assembly of the peroxisome, an organelle outside the classical secretory system . Here, we show that peroxisomal membrane fusion in the yeast Yarrowia lipolytica requires two AAA ATPases, Pex1p and Pex6p . Release of membrane- associated Pex1p and Pex6p drives the asymmetric priming of two fusion partners . The next step, peroxisome docking, requires release of Pex1p from one partner . Subsequent fusion of the peroxisomal membranes is independent of both Pex1p and Pex6p.

J Biol Chem, 2000 Nov 10, 275(45), 35393 - 401
Genetic mapping of the human C5a receptor . Identification of transmembrane amino acids critical for receptor function; Geva A et al.; Many hormones and sensory stimuli signal through a superfamily of seven transmembrane-spanning receptors to activate heterotrimeric G proteins . How the seven transmembrane segments of the receptors (a molecular architecture of bundled alpha-helices conserved from yeast to man) work as "on/off" switches remains unknown . Previously, we used random saturation mutagenesis coupled with a genetic selection in yeast to determine the relative importance of amino acids in four of the seven transmembrane segments of the human C5a receptor (Baranski, T . J., Herzmark, P., Lichtarge, O., Gerber, B . O., Trueheart, J., Meng, E . C., Iiri, T., Sheikh, S . P., and Bourne, H . R . (1999) J . Biol . Chem . 274, 15757-15765) . In this study, we evaluate helices I, II, and IV, thereby furnishing a complete mutational map of the seven transmembrane helices of the human C5a receptor . Our analysis identified 19 amino acid positions resistant to non-conservative substitutions . When combined with the 25 essential residues previously identified in helices III and V-VII, they delineate two distinct components of the receptor switch: a ligand-binding surface at or near the extracellular surface of the helix bundle and a core cluster in the cytoplasmic half of the bundle . In addition, we found critical amino acids in the first and second helices that are predicted to face the lipid membrane . These residues form an extended surface that might mediate interactions with lipids and other membrane proteins or function as an oligomerization domain with other receptors.

Br J Cancer, 2000 Sep, 83(6), 725 - 8
Serial analysis of gene expression identifies putative metastasis-associated transcripts in colon tumour cell lines; Parle-McDermott A et al.; We have used serial analysis of gene expression (SAGE) to identify gene expression differences between a primary colon tumour cell line (SW480) and an isogenic lymph-node metastasis cell line (SW620) . Differential expression was confirmed for the following genes: keratin K5, cystatin S, serum amyloid A, the human homologue of yeast ribosomal S28 and the p32 subunit of human pre-mRNA splicing factor SF2 . Expression of confirmed differences were also analysed in other metastatic cell lines .

Oncogene, 2000 Aug 10, 19(34), 3925 - 30
The Src/Csk regulatory circuit arose early in metazoan evolution; Miller MA et al.; We have identified a gene encoding a member of the Csk family of non-receptor protein-tyrosine kinases (PTKs) in the early-diverging metazoan Hydra . In situ hybridization analysis of the distribution of RNA from the Hydra Csk gene indicates that it is expressed in most of the epithelial cells of the adult polyp and in gametogenic cells . Comparison of the expression pattern of Hydra Csk with that of STK, the Hydra Src gene orthologue, reveals that the two genes are largely co-expressed . Such co-expression is consistent with a role for Hydra Csk in regulation of STK activity . This possibility was tested directly by coexpressing Hydra Csk with STK in yeast . Co-expression suppressed the growth inhibition seen when STK alone is expressed in yeast . Suppression was dependent on the presence of the putative regulatory tyrosine in the carboxyl-terminal tail of STK . Phosphotyrosine immunoblot analysis confirmed that expression of Csk resulted in suppression of STK kinase activity . Taken together these data indicate that the regulatory circuit involving Src and Csk PTKs was established prior to the divergence of the phylum Cnidaria from the rest of the metazoans.

Oncogene, 2000 Aug 10, 19(34), 3894 - 901
The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain; Erdmann KS et al.; Mutations of the tumor suppressor protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer . Here we describe a novel interaction between the APC protein and the protein tyrosine phosphatase PTP-BL carrying five PDZ protein-protein interaction domains . Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC protein, as determined by yeast two-hybrid studies . Using surface plasmon resonance analysis we established a dissociation constant (K(D)) of 8.1 x 10(-9) M . We find that a naturally occurring splice insertion of five amino acids (PDZ2b) abolishes its binding affinity to the APC protein . The in vivo interaction between PTP-BL and the APC protein was shown by coprecipitation experiments in transfected COS cells . Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions . The interaction of the APC protein with a protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated proteins, such as beta-catenin playing a major role in the regulation of cell division, migration and cell adhesion.

Eur J Hum Genet, 2000 Aug, 8(8), 613 - 20
Linkage disequilibrium in inbred North African families allows fine genetic and physical mapping of triple A syndrome; Hadj-Rabia S et al.; Triple A syndrome (Allgrove syndrome, MIM No . 231550) is a rare autosomal recessive disorder characterised by ACTH-resistant adrenal insufficiency, achalasia of the cardia, and alacrimia . The triple A gene has been previously mapped to chromosome 12q13 in a maximum interval of 6 cM between loci D12S1629 and D12S312 . Using linkage analysis in 12 triple A families, mostly originating from North Africa, we confirm that the disease locus maps to the 12q13 region (Zmax = 10.89 at theta = 0 for D12S1604) and suggest that triple A is a genetically homogeneous disorder . Recombination events as well as homozygosity for polymorphic markers enabled us to reduce the genetic interval to a 3.9 cM region . Moreover, total linkage disequilibrium was found at the D12S1604 locus between a rare allele and the mutant chromosomes in North African patients . Analysis of markers at five contiguous loci showed that most of the triple A chromosomes are derived from a single founder chromosome . As all markers are located in a 0 cM genetic interval and only allele 5 at the D12S1604 locus was conserved in mutant chromosomes, we speculate that the triple A mutation is due to an ancient Arabian founder effect that occurred before migration to North Africa . Since we also found linkage disequilibrium at D12S1604 in two patients from Southern Europe (France and Spain), the founder effect might well extend to other Mediterranean countries . Taking advantage of a YAC contig encompassing the triple A minimal physical region, the triple A gene was mapped to a 1.7 Mb DNA fragment accessible to gene cloning.

Eur J Hum Genet, 2000 Aug, 8(8), 561 - 70
Physical map of a 1.5 mb region on 12p11.2 harbouring a synpolydactyly associated chromosomal breakpoint; Debeer P et al.; Synpolydactyly (SPD) is a rare malformation of the distal limbs known to be caused by mutations in HOXD13 . We have previously described a complex form of SPD associated with synostoses in three members of a Belgian family, which co-segregates with a t(12;22)(p11.2;q13.3) chromosomal translocation . The chromosome 12 breakpoint of this translocation maps to 12p11.2 between markers D12S1034 and D12S1596 . Here we show that a mutation in the HOXD13 gene is not responsible for the phenotype, and present a physical map of the region around the 12p11.2 breakpoint . Starting from D12S1034 and D12S1596, we have established a contig approximately 1.5 Mb in length, containing 13 YAC clones, 16 BAC clones, and 11 cosmid clones . FISH analysis shows that cosmid LL12NCO1-149H4 maps across the breakpoint, and Southern blot experiments using fragments of this cosmid as probes identify a rearranged BamHI fragment in the patients carrying the translocation . A search for expressed sequences within the contig have so far revealed one CpG island, seven anonymous ESTs and three previously characterised genes, DAD-R, KRAG and HT21, all of which were found not to be directly disrupted by the translocation . The gene represented by EST R72964 was found to be disrupted by the translocation . These findings lay the groundwork for further efforts to characterise a gene critical for normal distal limb development that is perturbed by this translocation.

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