Yeasts

Biochem Soc Trans, 2001 Nov, 29(Pt 6), 768 - 73
UCP3 and its putative function: consistencies and controversies; Harper ME et al.; The physiological function of uncoupling protein 3 (UCP3) is as yet unknown . Based on its 57% homology to UCP1 whose physiologic function is uncoupling and thermogenesis, UCP3 was attributed with the function of mitochondrial uncoupling through proton-leak reactions . UCP3 is expressed selectively in muscle, a tissue in which it has been estimated that proton leak accounts for approx . 50% of resting energy metabolism . Genetic linkage, association and variant studies suggest a role for UCP3 in obesity and/or diabetes . Studies of the heterologous expression of UCP3 in yeast provide support for the idea that UCP3 can uncouple mitochondrial oxidative phosphorylation, but the physiological relevance of these results is questionable . In vitro studies of mitochondria from Ucp3(-/-) mice provide support, but there are no changes in resting metabolic rate (RMR) of mice . In vivo studies demonstrate increased ATP synthesis, but estimates of substrate oxidation rate indicate no change . Mice that greatly overexpress Ucp3 in muscle have increased RMR . Inconsistent with the function of uncoupling are the observations that fasting results in increased expression of UCP3, but no change in muscle proton leak . Moreover, fasting decreases energy expenditure in muscle . Expression patterns for Ucp3 and lipid-metabolism genes support a physiological role in fatty acid oxidation . Overall, findings support a role for Ucp3 in fatty acid metabolism that may have implications for obesity and/or Type II diabetes.

Biochem Soc Trans, 2001 Nov, 29(Pt 6), 722 - 8
Regulation of the serotonin transporter by interacting proteins; Haase J et al.; The serotonin transporter (SERT) plays a critical role in the maintenance of normal neurotransmission by serotonin {5-hydroxytryptamine (5-HT)} . Recent evidence suggests that SERT and other neurotransmitter transporters are tightly regulated . Activation of protein kinase C results in a decrease in SERT-mediated 5-HT uptake, which is due to an internalization of the transporter . However, to date little is known about the mechanism and proteins involved in the down-regulation of the transporter . One candidate SERT-regulatory protein is the SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) protein, syntaxin 1A (Syn1A), which has recently been implicated in the regulation of ion channels as well as the SERT-related gamma-aminobutyric acid- and glycine-transporters . Using 5-HT uptake assays, confocal microscopy and glutathione S-transferase (GST) pull-down assays we showed that Syn1A also interacts with SERT and alters the subcellular localization of the transporter, resulting in a reduction of 5-HT transport . In addition, we have used the yeast two-hybrid system to search for novel regulatory proteins that interact with the cytoplasmic N-terminal domain of SERT . By screening rat brain cDNA library we have identified six potential SERT-binding proteins . Here we also present progress towards the elucidation of the biological relevance of these proteins and their potential role for the regulation of the serotonin transporter.

Biochem Biophys Res Commun, 2001 Nov 23, 289(1), 240 - 4
Analysis of the expression pattern of Ebp1, an ErbB-3-binding protein; Xia X et al.; Ebp1, a member of the PA2G4 family, was isolated as an ErbB-3-binding protein in our laboratory using yeast two hybrid analysis . Although Ebp1 mRNA is ubiquitously expressed, little is known about either the expression of Ebp1 protein in vivo or its translation initiation site . Western blotting analysis of a wide range of cell lines and primary tissue indicated that in the majority of cases Ebp1 is expressed as a single protein which migrates at 48 kDa in SDS-polyacrylamide gels . We show using epitope-tagged expression constructs that the second, not the first, in-frame ATG is used for the initiation of translation of the endogenous protein, encoding a protein predicted to be 41.5 kDa . The molecular mass of endogenous Ebp1 protein derived from mouse liver and brain was determined by mass spectrometry and the data confirm that translation of endogenous Ebp1 in tissues is initiated from the second in-frame ATG .

Biogerontology, 2000, 1(1), 47 - 54
Deletion and dosage modulation of the eEF1A gene in Podospora anserina: effect on the life cycle; Silar P et al.; eEF1A is encoded by a unique gene in the filamentous fungus Podospora anserina . We show here that (1) this gene is essential for vegetative growth, (2) readthrough at UGA stop codon level is positively correlated with eEF1A level, (3) eEF1A level is regulated in P . anserina . (4) Increasing eEF1A gene dosage does not modify P . anserina life cycle parameters, especially longevity is not changed . These data confirm and extend those previously obtained in yeast and Drosophila.

Mamm Genome, 2001 Dec, 12(12), 925 - 9
Genomic characterization of human SEC14L1 splice variants within a 17q25 candidate tumor suppressor gene region and identification of an unrelated embedded expressed sequence tag; Kalikin LM et al.; Human SEC14L1 shows partial sequence homology to the budding yeast SEC14 protein and the Japanese flying squid retinal-binding protein and was previously generally localized to 17q25 . We more precisely mapped SEC14L1 within a discrete region of 17q25 that likely harbors at least one putative breast and ovarian tumor suppressor gene . We determined that this gene consists of 18 exons ranging in size from 70 bp (exon 11) to 3088 bp (exon 17) and spanning at least 58 kb of DNA . Exon 17 contained a highly polymorphic variable number of tandem repeats (VNTR) and was present only in the larger ubiquitously expressed 5.5-kb transcript . The 3.0-kb ubiquitously expressed transcript included sequences at the beginning of exon 17 (designated exon 17a) and the end of exon 17 (designated exon 18), but lacked the internal 2439 bp of exon 17, including the VNTR . This alternative splicing resulted in a predicted protein of 719 residues from the smaller transcript with four more terminal amino acids than the 715 residue protein predicted from the larger transcript . EST H49244 spanned exon 11 of SEC14L1 and was specifically expressed in human peripheral blood leukocytes . One intragenic single nucleotide polymorphism (SNP) was confirmed . SEC14L1 contained the CRAL/TRIO domain also found in alpha-tocopherol transfer protein (TTPA) and cellular retinaldehyde-binding protein (CRALBP) . As retinoids have been shown to inhibit the growth of breast cancer cells, loss of the proposed SEC14L1 retinal-binding function may contribute to breast tumorigenesis . As TTPA and CRALBP have been implicated in retinitis pigmentosa (RP), altered SEC14L1 expression may contribute to RP in previously unlinked families . Coding exon-specific PCR primers were designed to aid in future expression and mutational analyses.

Mamm Genome, 2001 Dec, 12(12), 887 - 92
Physical and transcriptional map of the mouse Chromosome 10 proximal region syntenic to human 6q16-q21; Chalhoub N et al.; Toward the isolation of the grey-lethal (gl) gene, we have genetically localized this locus on mouse Chromosome (Chr) 10 between the Fyn gene and the D10Mit148 microsatellite marker . Here, we have screened five yeast artificial chromosome (YAC) libraries and isolated more than 100 YAC clones mapping to this region . Forty-two clones were characterized and assembled in an approximately 8.5 megabases (Mb) contig showing high linkage conservation with the human 6q16-q21 interval . During this study, 24 specific novel sequence-tagged sites (STSs) were derived from YAC insert ends, and 15 mouse genes were precisely mapped to the contig . The physical and transcriptional map presented here will provide novel resources to isolate the gl locus associated with osteopetrosis, and will also provide candidate loci for other defects mapped on human Chr 6q.

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13595 - 600 Epub 2001 Nov 13.
Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase; Jennewein S et al.; A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases . A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast . The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates . This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product . The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism . Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 14038 - 43 Epub 2001 Nov 13.
SNAP-29: a general SNARE protein that inhibits SNARE disassembly and is implicated in synaptic transmission; Su Q et al.; Using the yeast two-hybrid system with syntaxin-1A as bait, we isolated soluble NSF attachment protein (SNAP)-29 from a human brain cDNA library . Synaptosomal fractionation and immunocytochemical staining of hippocampal neurons in culture showed that SNAP-29 is present at synapses and is predominantly associated with synaptic vesicles . The interaction of SNAP-29 with syntaxin-1 was further confirmed with immunoprecipitation analysis . Binding competition studies with SNAP-29 demonstrated that it could compete with alpha-SNAP for binding to synaptic SNAP receptors (SNAREs) and consequently inhibit disassembly of the SNARE complex . Introduction of SNAP-29 into presynaptic superior cervical ganglion neurons in culture significantly inhibited synaptic transmission in an activity-dependent manner . Although SNAP-29 has been suggested to be a general SNARE component in membrane trafficking, our findings suggest that it may function as a regulator of SNARE complex disassembly and modulate the process of postfusion recycling of the SNARE components.

J Cell Sci, 2001 Oct, 114(Pt 20), 3619 - 29
A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation; Munafo DB et al.; Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome . These vesicles fuse with lysosomes and the sequestered material is degraded . Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells . We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process . We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC . With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway . Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles . We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles . Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles . Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation . Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.

J Biol Chem, 2002 Jan 25, 277(4), 2413 - 8 Epub 2001 Nov 13.
The arabidopsis Na+/H+ exchanger AtNHX1 catalyzes low affinity Na+ and K+ transport in reconstituted liposomes; Venema K et al.; In saline environments, plants accumulate Na(+) in vacuoles through the activity of tonoplast Na(+)/H(+) antiporters . The first gene for a putative plant vacuolar Na(+)/H(+) antiporter, AtNHX1, was isolated from Arabidopsis and shown to increase plant tolerance to NaCl . However, AtNHX1 mRNA was up-regulated by Na(+) or K(+) salts in plants and substituted for the homologous protein of yeast to restore tolerance to several toxic cations . To study the ion selectivity of the AtNHX1 protein, we have purified a histidine-tagged version of the protein from yeast microsomes by Ni(2+) affinity chromatography, reconstituted the protein into lipid vesicles, and measured cation-dependent H(+) exchange with the fluorescent pH indicator pyranine . The protein catalyzed Na(+) and K(+) transport with similar affinity in the presence of a pH gradient . Li(+) and Cs(+) ions were also transported with lower affinity . Ion exchange by AtNHX1 was inhibited 70% by the amiloride analog ethylisopropyl-amiloride . Our data indicate a role for intracellular antiporters in organelle pH control and osmoregulation.

J Biol Chem, 2002 Jan 4, 277(1), 9 - 12 Epub 2001 Nov 13.
Jab1 interacts directly with HIF-1alpha and regulates its stability; Bae MK et al.; Hypoxia-inducible factor-1 (HIF-1) is a master transcription factor that controls transcriptional activation of a number of genes responsive to the low cellular oxygen tension, including vascular endothelial growth factor (VEGF), erythropoietin, and glycolytic enzymes . The stability and activity of HIF-1alpha are regulated by binding to various proteins such as pVHL, p53, and p300/CBP . Here, using the yeast two-hybrid screening system, we found that HIF-1alpha interacts with Jab1 (Jun activation domain-binding protein-1), which is a coactivator of AP-1 transcription factor and fifth subunit of COP9 signalosome complex . The interaction of Jab1 with HIF-1alpha was confirmed by GST pull-down assay and also reproduced in vivo in HEK 293 cells, where endogenous Jab1 was coimmunoprecipitated with the overexpressed HIF-1alpha . Moreover, Jab1-enhanced transcriptional activity of HIF-1 under hypoxia led to increase the expression of VEGF, a major HIF-1 target gene . Furthermore, Jab1 increased HIF-1alpha protein levels, which was due to the enhanced HIF-1alpha stability . The binding of HIF-1alpha and p53 tumor suppressor protein, negative regulator of HIF-1alpha stability, was interfered in a Jab1-dependent manner . Taken together, these results indicate that Jab1 should be considered as a novel regulator of HIF-1alpha stability via direct interaction.

EMBO J, 2001 Nov 15, 20(22), 6475 - 84
Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps; Saveanu C et al.; Eukaryotic ribosome maturation depends on a set of well ordered processing steps . Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein . Nog2p contains a putative GTP-binding site, which is essential in vivo . Kinetic and steady-state measurements of the levels of pre-rRNAs in Nog2p-depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SB(S) and 7S(S) precursors . We found Nog2p physically associated with large pre-60S complexes highly enriched in the 27SB and 7S rRNA precursors . These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP-binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins . In the absence of Nog2p, the pre-60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm . These results suggest that transient, possibly GTP-dependent association of Nog2p with the pre-ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.

FEBS Lett, 2001 Nov 9, 508(1), 75 - 9
Cyclic AMP affinity purification and ESI-QTOF MS-MS identification of cytosolic glyceraldehyde 3-phosphate dehydrogenase and two nucleoside diphosphate kinase isoforms from tobacco BY-2 cells; Laukens K et al.; The soluble protein fraction of tobacco bright yellow 2 cells contained adenosine 3',5'-cyclic monophosphate (cAMP)-binding activity, detected with both a conventional binding assay and a surface plasmon resonance biosensor . A cAMP-agarose-based affinity purification procedure yielded three proteins which were identified by mass spectrometry as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and two nucleoside diphosphate kinases (NDPKs) . This is the first report describing an interaction between cAMP and these proteins in higher plants . Our findings are discussed in view of the reported role of the interaction of cAMP with GAPDH and NDPK in animals and yeast . In addition, we provide a rapid method to isolate both proteins from higher plants.

Genomics, 2001 Nov, 78(1-2), 73 - 82
Comparative genomics of the SOX9 region in human and Fugu rubripes: conservation of short regulatory sequence elements within large intergenic regions; Bagheri-Fam S et al.; Campomelic dysplasia (CD), a human skeletal malformation syndrome with XY sex reversal, is caused by heterozygous mutations in and around the gene SOX9 . SOX9 has an extended 5' control region, as indicated by CD translocation breakpoints scattered over 1 Mb proximal to SOX9 and by expression data from mice transgenic for human SOX9-spanning yeast artificial chromosomes . To identify long-range regulatory elements within the SOX9 5' control region, we compared approximately 3.7 Mb and 195 kb of sequence around human and Fugu rubripes SOX9, respectively . We identified only seven and five protein-coding genes in the human and F . rubripes sequences, respectively . Four of the F . rubripes genes have been mapped in humans; all reside on chromosome 17 but show extensive intrachromosomal gene shuffling compared with the gene order in F . rubripes . In both species, very large intergenic distances separate SOX9 from its directly flanking genes: 2 Mb and 500 kb on either side of SOX9 in humans, and 68 and 97 kb on either side of SOX9 in F . rubripes . Comparative sequence analysis of the intergenic regions revealed five conserved elements, E1-E5, up to 290 kb 5' to human SOX9 and up to 18 kb 5' to F . rubripes SOX9, and three such elements, E6-E8, 3' to SOX9 . Where available, mouse sequences confirm conservation of the elements . From the yeast artificial chromosome transgenic data, elements E3-E5 are candidate enhancers for SOX9 expression in limb and vertebral column, and 8 of 10 CD translocation breakpoints separate these elements from SOX9.

Braz J Biol, 2001 Aug, 61(3), 405 - 8 Epub 2002 Jan 28.
Development of Lutzomyia intermedia and Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) larvae in different diets; Wermelinger ED et al.; The objective of this research was to evaluate, in laboratory, the development of Lutzomyia intermedia and Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) larvae, vectors of leishmaniasis in Brazil, in the following diets: industrialized food for rabbits, dogs, hamsters and aquarium fishes, besides liver powder, cooked lettuce, wheat germ, beer yeast, oat, wheat bran and a diet denominated aged food . Except wheat bran for L . intermedia, all diets provided adequate development for both species, which showed that any of them can be used in laboratory insectaries for these insects . L . intermedia showed better development with most nutritious diets and both species presented better development with aged food . Fungi as an additional nutrient source for L . intermedia and L . longipalpis is suggested.

Eur J Cancer B Oral Oncol, 1993 Oct, 29B(4), 291 - 4
Lesions of the oral mucosa in lymphoma patients receiving cytostatic drugs; Laine PO et al.; The 1-year incidence of oral mucosal lesions during cytostatic therapy was investigated in 67 patients {34 men and 33 women (mean age 49 years)} out of 79 original patients, being treated for non-Hodgkin lymphoma or Hodgkin's disease . The incidence of lesions during examinations was 43.4% . Recurrent lesions were observed in 19.4% of cases . Mean leukocyte counts were statistically significantly lower (P < 0.01) during lesion periods than before cytostatic therapy in all lesion groups . Leukocytopenia was found in 85.4% of patients with hairy leukoplakia-like lesions (HLL), and in 81.8% of the patients with angular cheilitis . 5 out of 14 patients with oral ulcers (35.7%) had episodes of septicaemia . Mean thrombocyte counts of patients in various lesion groups were normal (< 140 x 10/1) . However, low thrombocyte counts were more statistically significant (P < 0.05), when haemorrhages or HLL were present . Clinical candidiasis was diagnosed in 28.4% of patients during the treatment . However, cultivation revealed that 62.3% of salivary yeast cultures were positive . The study reported here shows a correlation between mucosal ulcers and septicemia, and between leukocytopenia, angular cheilitis and HLL . The disparity between clinically diagnosed candidiasis and the occurrence of salivary yeast counts suggests that antifungal drugs might be of prophylactic value during cytostatic therapy.

Plant Physiol, 2001 Nov, 127(3), 842 - 51
Diversity of Arabidopsis genes encoding precursors for phytosulfokine, a peptide growth factor; Yang H et al.; Phytosulfokine-alpha (PSK-alpha), a unique plant peptide growth factor, was originally isolated from conditioned medium of asparagus (Asparagus officinalis) mesophyll cell cultures . PSK-alpha has several biological activities including promoting plant cell proliferation . Four genes that encode precursors of PSK-alpha have been identified from Arabidopsis . Analysis of cDNAs for two of these, AtPSK2 and AtPSK3, shows that both of these genes consist of two exons and one intron . The predicted precursors have N-terminal signal peptides and only a single PSK-alpha sequence located close to their carboxyl termini . Both precursors contain dibasic processing sites flanking PSK, analogous to animal and yeast prohormones . Although the PSK domain including the sequence of PSK-alpha and three amino acids preceding it are perfectly conserved, the precursors bear very limited similarity among Arabidopsis and rice (Oryza sativa), suggesting a new level of diversity among polypeptides that are processed into the same signaling molecule in plants, a scenario not found in animals and yeast . Unnatural {serine-4}PSK-beta was found to be secreted by transgenic Arabidopsis cells expressing a mutant of either AtPSK2 or AtPSK3 cDNAs, suggesting that both AtPSK2 and AtPSK3 encode PSK-alpha precursors . AtPSK2 and AtPSK3 were expressed demonstrably not only in cultured cells but also in intact plants, suggesting that PSK-alpha may be essential for plant cell proliferation in vivo as well as in vitro . Overexpression of either precursor gene allowed the transgenic calli to grow twice as large as the controls . However, the transgenic cells expressing either antisense cDNA did not dramatically decrease mitogenic activity, suggesting that these two genes may act redundantly.

J Biol Chem, 2002 Feb 15, 277(7), 4738 - 46 Epub 2001 Nov 12.
GmZIP1 encodes a symbiosis-specific zinc transporter in soybean; Moreau S et al.; The importance of zinc in organisms is clearly established, and mechanisms involved in zinc acquisition by plants have recently received increased interest . In this report, the identification, characterization and location of GmZIP1, the first soybean member of the ZIP family of metal transporters, are described . GmZIP1 was found to possess eight putative transmembrane domains together with a histidine-rich extra-membrane loop . By functional complementation of zrt1zrt2 yeast cells no longer able to take up zinc, GmZIP1 was found to be highly selective for zinc, with an estimated K(m) value of 13.8 microm . Cadmium was the only other metal tested able to inhibit zinc uptake in yeast . An antibody raised against GmZIP1 specifically localized the protein to the peribacteroid membrane, an endosymbiotic membrane in nodules resulting from the interaction of the plant with its microsymbiont . The specific expression of GmZIP1 in nodules was confirmed by Northern blot, with no expression in roots, stems, or leaves of nodulated soybean plants . Antibodies to GmZIP1 inhibited zinc uptake by symbiosomes, indicating that at least some of the zinc uptake observed in isolated symbiosomes could be attributed to GmZIP1 . The orientation of the protein in the membrane and its possible role in the symbiosis are discussed.

J Biol Chem, 2002 Jan 18, 277(3), 1739 - 48 Epub 2001 Nov 08.
Differential regulation of the orphan nuclear receptor small heterodimer partner (SHP) gene promoter by orphan nuclear receptor ERR isoforms; Sanyal S et al.; The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) interacts with a wide array of nuclear receptors and represses their transcriptional activity . SHP expression is regulated by several other members of the nuclear receptor superfamily, including the orphan receptors SF-1 and LRH-1, and the bile acid receptor FXR . We have found that the SHP promoter is also activated by the estrogen receptor-related receptor gamma (ERRgamma) but not the related ERRalpha and ERRbeta isoforms . SHP and ERRgamma mRNAs are coexpressed in several tissues, including pancreas, kidney, and heart, confirming the potential relevance of this transactivation . ERRgamma transactivation is dependent on only one of five previously characterized DNA-binding sites for SF-1, and this element differs from previously reported ERR response elements . However, treatment with the histone deacetylase inhibitor trichostatin A significantly increased ERRalpha and ERRbeta activity on this element indicating that the lack of activity of ERRalpha and -beta may depend on their association with co-repressor in vivo . Furthermore, using protease sensitivity assays on DNA bound receptors it was demonstrated that DNA sequence of different response elements may cause allosteric modulation of ERR proteins, which in turn may be responsible for the differential activities of these receptors on different response elements . SHP inhibits ERRgamma transactivation and physically interacts with all three members of ERR subfamily, as demonstrated by both yeast two-hybrid and biochemical assays . As with other SHP targets, this interaction is dependent on the AF-2 coactivator-binding site of ERRgamma and the previously described N-terminal receptor interaction domain of SHP . Several recently described SHP mutations associated with moderate obesity in humans block the inhibition of ERRgamma activity . Overall, these results identify a new autoregulatory loop controlling SHP gene expression and significantly extend the potential functional roles of the three ERRs.

Infect Immun, 2001 Dec, 69(12), 7671 - 8
Potential role for extracellular glutathione-dependent ferric reductase in utilization of environmental and host ferric compounds by Histoplasma capsulatum; Timmerman MM et al.; The mammalian host specifically limits iron during Histoplasma capsulatum infection, and fungal acquisition of iron is essential for productive infection . H . capsulatum expresses several iron acquisition mechanisms under iron-limited conditions in vitro . These components include hydroxamate siderophores, extracellular glutathione-dependent ferric reductase enzyme, extracellular nonproteinaceous ferric reductant(s), and cell surface ferric reducing agent(s) . We examined the relationship between these mechanisms and a potential role for the extracellular ferric reductase in utilization of environmental and host ferric compounds through the production of free, soluble Fe(II) . Siderophores and ferric reducing agents were coproduced under conditions of iron limitation . The H . capsulatum siderophore dimerum acid and the structurally similar basidiomycete siderophore rhodotorulic acid acted as substrates for the ferric reductase, and rhodotorulic acid removed Fe(III) bound by transferrin . The mammalian Fe(III)-binding compounds hemin and transferrin served both as substrates for the ferric reductase and as iron sources for yeast-phase growth at neutral pH . In the case of transferrin, there was a correlation between the level of iron saturation and efficacy for both of these functions . Our data are not consistent with an entirely pH-dependent mechanism of iron acquisition from transferrin, as has been suggested to occur in the macrophage phagolysosome . The foreign siderophore ferrioxamine B also acted as a substrate for the ferric reductase, while the foreign siderophore ferrichrome did not . Both ferrioxamine and ferrichrome served as iron sources for yeast- and mold-phase growth, the latter presumably by some other acquisition mechanism(s).

Clin Cancer Res, 2001 Nov, 7(11), 3404 - 9
Chromosome 6 abnormalities in ovarian surface epithelial tumors of borderline malignancy suggest a genetic continuum in the progression model of ovarian neoplasms; Tibiletti MG et al.; PURPOSE: We used conventional cytogenetics, molecular cytogenetics, and molecular genetic analyses to study the pattern of allelic loss on chromosome 6q in a cohort of borderline epithelial ovarian tumors . EXPERIMENTAL DESIGN: Fifteen tumor samples were collected from patients undergoing surgery for ovarian tumors . The tumors of borderline malignancy, classified according to the standard criteria, included 4 mucinous and 11 serous tumors . Cytogenetic and molecular cytogenetic (with yeast artificial chromosome clones from 6q26-27) studies were performed on tumor areas contiguous to those used for histological examination ensuring the appropriate sampling . Moreover loss of heterozygosity analysis was performed using PCR amplification of eight microsatellite markers mapping on 6q27 (D6S193, D6S297), 6q26 (D6S305, D6S415, D6S441), 6q21 (D6S287), 6q16 (D6S311), and 6q14 (D6S300) . RESULTS: Deletions of this chromosome arm, in particular of 6q24-27, were the most frequent lesions found in this set of tumors . In a tumor with a normal karyotype the only detectable alteration was a deletion of approximately 300 kb within the D6S149-D6S193 interval at band 6q27 . This is, to date, the smallest deletion described for borderline tumors . CONCLUSION: Alterations in the above-mentioned interval are a common finding in advanced ovarian carcinomas but also in benign ovarian cysts, implying that some tumors of borderline malignancy may arise from benign tumors and that malignant ones may evolve from tumors of borderline malignancy . Genes located in 6q27 seem to be crucial for this mechanism of early events in ovarian tumorigenesis.

J Biol Chem, 2002 Jan 18, 277(3), 2012 - 8 Epub 2001 Nov 09.
Multiple roles for phosphatidylinositol 4-kinase in biosynthetic transport in polarized Madin-Darby canine kidney cells; Bruns JR et al.; Phosphatidylinositols (PI) play important roles in regulating numerous cellular processes including cytoskeletal organization and membrane trafficking . The control of PI metabolism by phosphatidylinositol kinases has been the subject of extensive investigation; however, little is known about how phosphatidylinositol kinases regulate traffic in polarized epithelial cells . Because phosphatidylinositol 4-kinase (PI4K)-mediated phosphatidylinositol 4-phosphate (PI(4)P) production has been suggested to regulate biosynthetic traffic in yeast and mammalian cells, we have examined the role of PI4Kbeta in protein delivery in polarized MDCK cells, at different levels of the biosynthetic pathway . Expression of wild type PI4Kbeta had no effect on the rate of transport of influenza hemagglutinin (HA) through the Golgi complex, but inhibited the rate of trans-Golgi network (TGN)-to-cell surface delivery of this protein . By contrast, expression of dominant-negative, kinase-dead PI4Kbeta (PI4Kbeta(D656A)) inhibited intra-Golgi transport but stimulated TGN-to-cell surface delivery of HA . Moreover, expression of PI4Kbeta(D656A) significantly increased the solubility in cold Triton X-100 of HA staged in the TGN, suggesting that altered association of HA with lipid rafts may be responsible for the enhanced transport rate . Both wild type and kinase-dead PI4Kbeta inhibited basolateral delivery of vesicular stomatitis virus G protein, suggesting an effector function for PI4Kbeta in the regulation of basolateral traffic . Thus, by contrast with the observed requirement for PI4Kbeta activity and PI(4)P for efficient transport in yeast, our data suggest that changes in PI(4)P levels can stimulate and inhibit Golgi to cell surface delivery in mammalian cells.

Am J Clin Dermatol, 2000 Mar-Apr, 1(2), 75 - 80
Management of seborrheic dermatitis and pityriasis versicolor; Faergemann J; Pityriasis (tinea) versicolor and seborrheic dermatitis are two very common skin diseases . Pityriasis versicolor is a chronic superficial fungal disease usually located on the upper trunk, neck, or upper arms . In pityriasis versicolor, the lipophilic yeast Malassezia (also know as Pityrosporum ovale or P . orbiculare) changes from the blastospore form to the mycelial form under the influence of predisposing factors . The most important exogenous factors are high temperatures and a high relative humidity which probably explain why pityriasis versicolor is more common in the tropics . The most important endogenous factors are greasy skin, hyperhidrosis, hereditary factors, corticosteroid treatment and immunodeficiency . There are many ways of treating pityriasis versicolor topically . Options include propylene glycol, ketoconazole shampoo, zinc pyrithione shampoo, ciclopiroxamine, selenium sulfide, and topical antifungals . In difficult cases, short term treatment with fluconazole or itraconazole is effective and well tolerated . To avoid recurrence a prophylactic treatment regimen is mandatory . Seborrheic dermatitis is characterized by red scaly lesions predominantly located on the scalp, face and upper trunk . There are now many studies indicating that Malassezia plays an important role in this condition . Even a normal number of Malassezia will start an inflammatory reaction . Mild corticosteroids are effective in the treatment of seborrheic dermatitis . However, the disease recurs quickly, often within just a few days . Antifungal therapy is effective in the treatment of seborrheic dermatitis and, because it reduces the number of Malassezia, the time to recurrence is increased compared with treatment with corticosteroids . Antifungal therapy should be the primary treatment of this disease.

Cytogenet Cell Genet, 2001, 94(1-2), 55 - 61
An integrated genetic and physical map of the 650-kb region containing the congenital polycystic kidney (cpk) locus on mouse chromosome 12; Mrug M et al.; Mice homozygous for the congenital polycystic kidney (cpk) mutation develop a rapidly progressive form of polycystic kidney disease . We report an integrated genetic and physical map of the 650-kb region containing the cpk locus and the exclusion of Rrm2 and Idb2 as candidate cpk genes . Our study establishes the requisite foundation for positional cloning of the cpk gene .

Cytogenet Cell Genet, 2001, 94(1-2), 49 - 54
Conserved synteny and gene order difference between human chromosome 12 and pig chromosome 5; Goureau A et al.; A comparative map of human chromosome 12 (HSA 12) and pig chromosome 5 (SSC 5) was constructed using ten pig expressed sequence tags (ESTs) . These ESTs were isolated from primary granulosa cell cultures by differential display (EST b10b), or from a granulosa cDNA library (VIIIE1, DRIM, N*9, RIIID2 and RVIC1) or from a small intestine cDNA library (ATPSB, ITGB7, MYH9, and STAT2) . Also used were two Traced Orthologous Amplified Sequence Tags (TOASTs) (LALBA, TRA1), one microsatellite-associated gene (IGF1) and finally five human YACs selected for their cytogenetic position, with a view to increasing the number of informative markers for the comparison . Large-insert clones were obtained by screening a pig bacterial artificial chromosome (BAC) library with specific primers for each EST and TOAST and for IGF1 . These BACs were used as probes for fluorescent in situ hybridisation (FISH) both on porcine and human metaphases . In addition, the human YACs were FISH mapped on pig chromosomes . This allowed us to refine and, in some cases, to correct the previous mapping obtained with a somatic cell hybrid panel . While these data confirm chromosome painting results showing that the distal part of SSC 5p arm is conserved on HSA 22, while the rest of the chromosome corresponds to HSA 12, they also demonstrate gene-order differences between human and pig . In addition, it was also possible to determine the position of the synteny breakpoint .

Annu Rev Genomics Hum Genet, 2001, 2, 435 - 62
The genetics of aging; Finch CE et al.; The genetic analysis of life span has only begun in mammals, invertebrates, such as Caenorhabditis elegans and Drosophila, and yeast . Even at this primitive stage of the genetic analysis of aging, the physiological observations that rate of metabolism is intimately tied to life span is supported . In many examples from mice to worms to flies to yeast, genetic variants that affect life span also modify metabolism . Insulin signaling regulates life span coordinately with reproduction, metabolism, and free radical protective gene regulation in C . elegans . This may be related to the findings that caloric restriction also regulates mammalian aging, perhaps via the modulation of insulin-like signaling pathways . The nervous system has been implicated as a key tissue where insulin-like signaling and free radical protective pathways regulate life span in C . elegans and Drosophila . Genes that determine the life span could act in neuroendocrine cells in diverse animals . The involvement of insulin-like hormones suggests that the plasticity in life spans evident in animal phylogeny may be due to variation in the timing of release of hormones that control vitality and mortality as well as variation in the response to those hormones . Pedigree analysis of human aging may reveal variations in the orthologs of the insulin pathway genes and coupled pathways that regulate invertebrate aging . Thus, genetic approaches may identify a set of circuits that was established in ancestral metazoans to regulate their longevity.

Annu Rev Genomics Hum Genet, 2001, 2, 129 - 51
Congenital disorders of glycosylation; Jaeken J et al.; Congenital disorders of glycosylation (CDG) are a rapidly growing group of genetic diseases that are due to defects in the synthesis of glycans and in the attachment of glycans to other compounds . Most CDG are multisystem diseases that include severe brain involvement . The CDG causing sialic acid deficiency of N-glycans can be diagnosed by isoelectrofocusing of serum sialotransferrins . An efficient treatment, namely oral D-mannose, is available for only one CDG (CDG-Ib) . In many patients with CDG, the basic defect is unknown (CDG-x) . Glycan structural analysis, yeast genetics, and knockout animal models are essential tools in the elucidation of novel CDG . Eleven primary genetic glycosylation diseases have been discovered and their basic defects identified: six in the N-glycan assembly, three in the N-glycan processing, and two in the O-glycan (glycosaminoglycan) assembly . This review summarizes their clinical, biochemical, and genetic characteristics and speculates on further developments in this field.

Toxicol Lett, 2001 Dec 15, 125(1-3), 75 - 81
Enantiomer-specific activity of o,p'-DDT with the human estrogen receptor; Hoekstra PF et al.; There is a growing concern that environmental xenobiotics may be affecting human and wildlife health by disrupting normal endocrine function via interaction with steroid hormone receptors . Several of these persistent contaminants are chiral and may have enantiomer-specific biological properties . Previous experiments have demonstrated that (-)-o,p'-DDT enantiomer is a more active estrogen-mimic than the (+)-enantiomer in rats . However, these results have not been extrapolated to other biological systems . This study used a yeast-based assay to assess the enantiomer-specific transcriptional activity of DDT with the human estrogen receptor (hER) . (+)-17beta-estradiol, racemic DDT and individual DDT enantiomers were added to yeast cultures and hER activity was measured by quantification of beta-galactosidase . The relative activity of o,p'-DDT was weak compared to estradiol . For o,p'-DDT, the (-)-enantiomer was the active estrogen mimic whereas the hER activity of (+)-o,p'-DDT was negligible . The presence of the (+)-enantiomer at relatively greater concentration decreased the transcriptional activity of (-)-o,p'-DDT . This data demonstrates the need to consider stereochemistry of environmental contaminants and their potential influence on biological responses.

Biochem Biophys Res Commun, 2001 Nov 16, 288(5), 1078 - 86
HSH2: a novel SH2 domain-containing adapter protein involved in tyrosine kinase signaling in hematopoietic cells; Oda T et al.; We isolated a cDNA clone encoding a novel Src homology (SH)2 domain-containing protein of 47 kDa from a human cDNA library . As its transcript was predominantly expressed in hematopoietic cells, this gene was termed HSH2 for hematopoietic SH2 protein . This protein contains several putative protein-binding motifs, SH3-binding proline-rich regions, and phosphotyrosine sites, but lacks enzymatic motifs . In a yeast two-hybrid screen, we identified a cytokine-regulated tyrosine kinase c-FES and an activated Cdc42-associated tyrosine kinase ACK1 as HSH2 interactors . HSH2 bound c-FES via its C-terminal region as well as its N-terminal region including the SH2 domain, whereas it bound ACK1 via its N-terminal proline-rich region . Furthermore, these two kinases bound and tyrosine-phosphorylated HSH2 in mammalian cells . Hence, we postulate that HSH2 functions as an adapter protein involved in tyrosine kinase signaling, and possibly regulates cytokine signaling and cytoskeletal reorganization, in hematopoietic cells .

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 14174 - 9 Epub 2001 Nov 06.
Regulation of cyclic peptide biosynthesis in a plant pathogenic fungus by a novel transcription factor; Pedley KF et al.; Strains of the filamentous fungus Cochliobolus carbonum that produce the host-selective compound HC-toxin, a cyclic tetrapeptide, are highly virulent on certain genotypes of maize (Zea mays L.) . Production of HC-toxin is under the control of a complex locus, TOX2, which is composed of at least seven linked and duplicated genes that are present only in toxin-producing strains of C . carbonum . One of these genes, TOXE, was earlier shown to be required for the expression of the other TOX2 genes . TOXE has four ankyrin repeats and a basic region similar to those found in basic leucine zipper (bZIP) proteins, but lacks any apparent leucine zipper . Here we show that TOXE is a DNA-binding protein that recognizes a ten-base motif (the "tox-box") without dyad symmetry that is present in the promoters of all of the known TOX2 genes . Both the basic region and the ankyrin repeats are involved in DNA binding . A region of TOXE that includes the first ankyrin repeat is necessary and sufficient for transcriptional activation in yeast . The data indicate that TOXE is the prototype of a new family of transcription factor, so far found only in plant-pathogenic fungi . TOXE plays a specific regulatory role in HC-toxin production and, therefore, pathogenicity by C . carbonum.

Blood, 2001 Nov 15, 98(10), 3082 - 6
Rearrangements of the c-myc oncogene are present in 15% of primary human multiple myeloma tumors; Avet-Loiseau H et al.; Rearrangements of the c-myc oncogene have been found in most plasmacytomas induced in mice and human myeloma cell lines (HMCLs) analyzed so far . However, neither induced mouse plasmacytomas nor HMCLs represent relevant models for human multiple myeloma (MM) . To evaluate the incidence of c-myc rearrangements in human plasma cell dyscrasias, sets of probes were generated to allow direct assessment of c-myc translocations on interphase plasma cells by using fluorescence in situ hybridization . After validation of these probes, a large cohort of patients with either newly diagnosed MM (n = 529), relapsed MM (n = 58), primary plasma cell leukemia (PCL; n = 23), monoclonal gammopathy of undetermined significance (n = 65), or smoldering MM (n = 24) were analyzed . C-myc rearrangements were identified in 15% of patients with MM or primary PCL, independently of the stage of the disease (ie, diagnosis or relapse and MM or primary PCL) . Analysis of the 2 main translocations observed on karyotyping, ie, t(8;14) and t(8;22), revealed that these specific translocations represented only 25% (23 of 91) of c-myc rearrangements . c-myc rearrangements were then correlated with several other patients' characteristics: illegitimate IgH recombinations, chromosome 13 deletions, and serum beta2-microglobulin levels . The only significant correlation was with a high beta2-microglobulin level (P =.002), although a trend for association with t(4;14) was observed (P =.08) . Thus, c-myc rearrangement analysis in patients with MM revealed a strikingly lower incidence than that in HMCLs and plasmacytomas induced in mice, indicating that data obtained with these models cannot be directly extrapolated to human MM.

Curr Opin Cell Biol, 2001 Dec, 13(6), 731 - 7
The coupling of cell growth to the cell cycle; Tapon N et al.; The development of a complex multicellular organism requires a coordination of growth and cell division under the control of patterning mechanisms . Studies in yeast have pioneered our understanding of the relationship between growth and cell division . In recent years, many of the pathways that regulate growth in multicellular eukaryotes have been identified . This work has revealed interesting and unexpected relationships between mechanisms that regulate growth and the cell cycle machinery.

J Mol Biol, 2001 Nov 2, 313(4), 733 - 49
Trypanosoma brucei 5'ETS A'-cleavage is directed by 3'-adjacent sequences, but not two U3 snoRNA-binding elements, which are all required for subsequent pre-small subunit rRNA processing events; Hartshorne T et al.; Trypanosoma brucei pre-rRNA processing commences by cleavage near the 5' end of 5.8 S sequences . The 5' external transcribed spacer (5'ETS) is removed from pre-small subunit (SSU) rRNAs by sequential cleavages at internal A' and A0 sites, and A1 at the 5' end of SSU rRNA . The A' and A0 sites positionally resemble the U3 small nucleolar RNA-dependent, primary pre-rRNA cleavages of vertebrates and yeast, respectively . Uniquely in T . brucei, two U3-crosslinkable 5'ETS sites are essential for SSU rRNA production: site1b is novel in its 3' location to the A' site, and site3 lies upstream of A0 in a position analogous to the yeast U3-binding site . Here, in vivo analysis of mutated 5'ETS sequences shows that sequences 5' to the A' site are not needed for A' cleavage or SSU rRNA production . A' cleavage is linked to, but is not sufficient to trigger, downstream pre-SSU rRNA processing events . These events require an intact 11 nt sequence, 3'-adjacent to A', which directs efficient and accurate A' cleavage . Neither the A' nearby site1b nor the site3 U3-binding elements affect A' processing, yet each is required for A0 and A1 cleavage, and SSU rRNA production . The same U3 3' hinge bases evidently bind a core element, UGUu/gGGU, within site1a and site3; the U3-site1b interaction is less reliant on base-pairing than the U3-site3 interaction . As yeast U3 5' hinge bases pair to 5'ETS sequences, it is clear that distinct U3 hinge regions can interact at both novel and related 5'ETS sites to promote 3'-proximal 5'ETS processing events in diverse organisms . The T . brucei data fit a model wherein processing factors assemble at the 5'ETS site1a to affect A' cleavage and stabilize a U3-site1b complex, which may work in concert with the downstream U3-site3 complex to assist processing events leading to ribosomal SSU production .

Exp Cell Res, 2001 Nov 15, 271(1), 28 - 35
Endocytosis in Drosophila: progress, possibilities, prognostications; Narayanan R et al.; By many outside the field, endocytosis is often perceived as a "house-keeping" function performed via identical mechanisms in yeast and man . Recent discoveries have done much to reduce this misperception . (1) Endocytosis occurs via different mechanisms and different pathways in different cellular contexts . (2) Molecular mechanisms that regulate homologous pathways in unicellular and multicellular organisms show considerable variance . (3) Temporally controlled endocytosis of specific regulatory molecules underlies several important and intricate biological processes including synapse formation, synaptic plasticity, cell fate determination, and morphogen gradient formation . Interactions between endocytosis and cytoskeletal and signaling pathways have been particularly revealing . In this intellectual context, Drosophila has become invaluable as a metazoan genetic model in which to understand the many faces of endocytosis . This review discusses two aspects of work in Drosophila: (a) its contributions toward understanding fundamental mechanisms that underlie the operation of endocytic pathways; (b) how analyses in Drosophila provide insights into varied biological processes regulated by endocytosis . In addition, while offering our commentary on merits and limitations of Drosophila work, we speculate on likely areas for contributions and future research on endocytosis in Drosophila .

DNA Seq, 2001 Jul, 12(1), 59 - 65
Characterisation of a cDNA encoding chick eukaryotic translation initiation factor-2 beta; Sneesby KJ et al.; A full length cDNA for the beta subunit of chick (Gallus gallus) eukaryotic translation initiation factor-2 is described . This cDNA was isolated by screening a chick cDNA library with a probe derived via differential display of developing chick heart tissue . Up-regulated expression of eIF-2 beta mRNA was confirmed by reverse Northern dot blot analysis . eIF-2 beta, together with eIF-2 alpha and eIF-2 gamma, comprise subunits of a complex that promotes the binding of methionyl-tRNA to ribosomes during the initiation of protein translation . The nucleotide sequence of the chick eIF-2 beta cDNA predicts a protein of 334 amino acids that has 95%, 93%, 56% and 37% sequence identity with rabbit, human, drosophila and yeast eIF-2 beta, respectively . The deduced eIF-2 beta protein contains a number of functional motifs and domains consistent with the putative function of this protein; these include a potential C2-C2 zinc-finger binding domain, three polylysine regions, and three acidic regions.

J Biol Chem, 2002 Jan 18, 277(3), 1712 - 8 Epub 2001 Nov 05.
Olf-1/early B cell factor is a regulator of glut4 gene expression in 3T3-L1 adipocytes; Dowell P et al.; A negative regulatory element in the 5'-flanking region of the murine glut4 gene mediates chronic insulin- and cAMP-induced repression in 3T3-L1 adipocytes . Previous work demonstrated that members of the nuclear factor 1 (NF1) family of transcription factors and an unidentified factor bind to and mediate repression from this regulatory element . By using a yeast one-hybrid screen, Olf-1/Early B cell factor (O/E-1) was isolated as a candidate for this unidentified factor . A protein complex from 3T3-L1 adipocyte nuclear extract that bound the negative regulatory element was recognized by O/E-specific antiserum, and binding activity was competed effectively by distinct O/E-binding sequences . O/E binding activity was also detected in nuclear extracts from insulin-responsive, GLUT4-expressing tissues including adipose, skeletal muscle, and heart . Mutations within the negative regulatory element that abolish binding of O/E proteins concomitantly blocked insulin-induced repression in reporter gene assays . These results suggest that one or more members of the O/E transcription factor family function as important regulators of glut4 gene expression and therefore may play a heretofore unanticipated role in glucose homeostasis and insulin signaling.

FEBS Lett, 2001 Nov 2, 507(3), 331 - 5
Human homolog of mouse tescalcin associates with Na(+)/H(+) exchanger type-1; Mailander J et al.; A novel regulatory protein, tescalcin (TSC), recently isolated from mouse embryonic testes, has been implicated in gonadal differentiation . Employing the yeast two-hybrid system with the Na(+)/H(+) exchanger type-1 (NHE1) carboxyterminal domain as a bait we have identified a novel NHE1-associated protein of 214 amino acid residues representing the human homolog of mouse TSC (96.7% identity) . Co-precipitation experiments demonstrated the interaction of human TSC with NHE1 in vitro and in vivo, and 45Ca(2+) overlay assay revealed that TSC binds Ca(2+) . Immunofluorescence studies indicated that TSC is prominent in cellular lamellipodia where it colocalizes with NHE1 . Abundant expression of TSC mRNA in the heart suggests that TSC may play important role(s) in concert with NHE1 in cardiac tissues.

Curr Biol, 2001 Oct 30, 11(21), 1716 - 21
The DExH/D box protein HEL/UAP56 is essential for mRNA nuclear export in Drosophila; Gatfield D et al.; Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells . Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC) . In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56 . This suggested a role for these proteins in nuclear transport . Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells . In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus . Consequently, incorporation of {35S}methionine into newly synthesized proteins is inhibited . This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs . In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC) . We conclude that HEL is essential for the export of bulk mRNA in Drosophila . The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.

Curr Biol, 2001 Oct 30, 11(21), 1695 - 9
Cell cycle controlling the silencing and functioning of mammalian activators; Mullen AC et al.; Naive CD4(+) helper T (T(H)) cells respond to stimulation by terminally differentiating into two mature classes, T(H)1 cells, which express interferon gamma (IFN-gamma), and T(H)2 cells, which express interleukin 4 (IL-4) . The transcriptional activators T-bet and Gata-3 mediate commitment to the T(H)1 and T(H)2 fates, respectively, including chromatin remodeling of signature genes . The cytokine IL-12 fosters growth of committed T(H)1 cells, while IL-4 fosters growth of committed T(H)2 cells . IL-12 and IL-4 also play critical roles in commitment by promoting transcriptional silencing of Gata-3 and T-bet, respectively . We now show that both T-bet and Gata-3 are induced in a cell cycle-independent manner in bipotent progenitor cells . In contrast, both lineage-restricted gene induction by the activator proteins and heritable silencing of the transcription of each activator, the hallmarks of terminal differentiation, are cell cycle dependent . We found that cells that cannot cycle remain uncommitted and bipotent in response to the most polarizing signals for maturation . These results provide mechanistic insight into a mammalian model of terminal differentiation by illustrating that cell cycle-coupled epigenetic effects, as originally described in yeast, may represent an evolutionarily conserved strategy for organizing signaling and cell fate.

Curr Biol, 2001 Oct 30, 11(21), 1686 - 90
Interaction of heterotrimeric G13 protein with an A-kinase-anchoring protein 110 (AKAP110) mediates cAMP-independent PKA activation; Niu J et al.; Heterotrimeric G proteins and protein kinase A (PKA) are two important transmitters that transfer signals from a wide variety of cell surface receptors to generate physiological responses . The established mechanism of PKA activation involves the activation of the Gs-cAMP pathway . Binding of cAMP to the regulatory subunit of PKA (rPKA) leads to a release and subsequent activation of a catalytic subunit of PKA (cPKA) . Here, we report a novel mechanism of PKA stimulation that does not require cAMP . Using yeast two-hybrid screening, we found that the alpha subunit of G13 protein interacted with a member of the PKA-anchoring protein family, AKAP110 . Using in vitro binding and coimmunoprecipitation assays, we have shown that only activated G alpha 13 binds to AKAP110, suggesting a potential role for AKAP110 as a G alpha subunit effector protein . Importantly, G alpha 13, AKAP110, rPKA, and cPKA can form a complex, as shown by coimmunoprecipitation . By characterizing the functional significance of the G alpha 13-AKAP110 interaction, we have found that G alpha 13 induced release of the cPKA from the AKAP110-rPKA complex, resulting in a cAMP-independent PKA activation . Finally, AKAP110 significantly potentiated G alpha 13-induced activation of PKA . Thus, AKAP110 provides a link between heterotrimeric G proteins and cAMP-independent activation of PKA.

Plant J, 2001 Oct, 28(1), 61 - 71
Amino acid permeases in developing seeds of Vicia faba L.: expression precedes storage protein synthesis and is regulated by amino acid supply; Miranda M et al.; Full length cDNAs encoding three amino acid permeases were isolated from seed-specific libraries of Vicia faba . The predicted proteins VfAAP1, VfAAP3 and VfAAP4 share up to 66% identity among themselves . Functional characterization of VfAAP1 and VfAAP3 in a yeast mutant showed that these permeases transport a broad range of amino acids . However, VfAAP1 had a preference for cysteine and VfAAP3 for lysine and arginine . VfAAP1 was highly expressed in cotyledons at early developmental stages and moderately in other sink tissues . Its peak of expression in cotyledons corresponded to the appearance of storage protein transcripts, suggesting that this transporter fulfills an important role in providing amino acids for storage protein biosynthesis . VfAAP3 was expressed most abundantly in maternal tissues, that is in roots, stems, gynoecia, pods and seed coats at different developmental stages . VfAAP4 transcripts could not be detected by northern hybridization . In situ hybridization showed that VfAAP1 mRNA is distributed throughout cotyledon storage parenchyma cells, but could not be detected in the abaxial epidermal cell layer . It also accumulate in the chlorenchyma and thin-walled parenchyma cells of seed coats . VfAAP1 mRNA levels were lower in cotyledons cultured in the presence of glutamine, whereas expression of a vicilin storage protein gene was up-regulated under similar conditions . Cysteine repressed the expression of the GUS reporter gene under control of the VfAAP1 promoter, suggesting that this transporter is modulated at the transcriptional level . Regulation of amino acid transport in relation to storage protein accumulation is discussed.

Plant J, 2001 Oct, 28(1), 27 - 39
The Arabidopsis MALE STERILITY1 (MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors; Wilson ZA et al.; We report here the molecular characterisation of the Arabidopsis MALE STERILITY1 gene, which is a critical sporophytic controlling factor for anther and pollen development . Homozygous ms1 mutants do not produce viable pollen, but are otherwise phenotypically normal . Degeneration of pollen occurs soon after microspore release from the tetrads, at which time the tapetum also appears abnormally vacuolated . The MS1 gene is expressed at low levels in anthers from closed buds, with expression in the tapetum at the stage of microspore release . No expression is seen in open flowers . The deduced MS1 protein sequence shows strong homology to the PHD-finger motif found in known transcription factors from humans, yeast and higher plants . Six alleles of ms1 have been identified; all result in premature termination of the MS1 protein and loss of the PHD-finger motif . MS1 is likely to play a key role in regulating transcription during specific stages of male gametogenesis and anther development . As such, MS1 provides a valuable tool for the manipulation of male sterility in higher plants.

Lett Appl Microbiol, 2001 Nov, 33(5), 367 - 70
Production of gamma-linolenic acid by Mortierella isabellina grown on hexadecanol; Xian M et al.; AIMS: To optimize the production of linolenic acid by Mortierella isabellina grown on hexadecanol . METHODS AND RESULTS: Effects of culture conditions such as culture time, pH of medium, hexadecanol concentration, incubation temperature and ageing of mycelia on production of linolenic acid were studied . The production of gamma-linolenic acid reached 2.44 mg ml-1 (271 mg g-1 dry cells) when Mortierella isabellina was cultivated in a medium consisting of 2% hexadecanol and 1% yeast extract at 23 degrees C for 120 h and then the mycelia, after removal of medium by suction filtration, were allowed to stand for a further 15 d at 5 degrees C . CONCLUSION: Ageing of mycelia and incubation temperature showed predominant effects on the increased linolenic acid production . SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights effective conditions for increasing linolenic acid production by Mortierella isabellina grown on hexadecanol.

Phytomedicine, 2001 Sep, 8(5), 331 - 7
CNS active potentials of some Hypericum species of India; Mukherjee PK et al.; Hypericum is a large genus comprising 200 species, wide spread on temperate region and tropical mountains . Several different species are available in Indian subcontinent . Psychopharmacological profiles of the two different Hypericum species e.g., H . hookerianum and H . patulum available in Nilgiris, India were investigated at two different doses (200 and 400 mg/kg, p.o.) in different animal models viz . Spontaneous motor activity (SMA) test in mice; Exploratory behaviour test by Head dip test in mice and Y-maze test in rats; Effects on pentobarbitone induced sleeping time in mice and study of the effects on body temperature in rats . All the extracts tested showed enhancement in spontaneous motor activity (SMA) in mice and exploratory behavior by head dip test in mice and Y-maze test in rats . The extracts reduce significantly the pentobarbitone induced sleeping time in mice . When tested for their effect on body temperature in rats, the extract of H . hookerianum showed significant reduction in yeast induced pyrexia with no effect on normal body temperature, while H . patulum showed no activity in this experimental model.

Pest Manag Sci, 2001 Oct, 57(10), 946 - 50
Chitin synthesis and inhibition: a revisit; Cohen E; Chitin is an abundant biologically important aminopolysaccharide composed of N-acetyl-D-glucosamine units . Individual polymers, which are synthesized intracellularly by chitin synthase (CS), a membrane-bound glycosyl transferase, are translocated across the plasma membrane and coalesce to form rigid crystallites . These crystallites, inter alia, are integral parts of septa and cell walls in yeast and filamentous fungi, respectively, and of cuticles in invertebrates, notably crustaceans and insects . Despite decades of intensive research, many events associated with the complexity of chitin formation and deposition are still obscure, or only partially understood . The list includes the hormonal control of CS at the transcriptional and translational levels as well as the post-translational CS packaging; trafficking and guidance of CS clusters to proper sites in the cells and their intricate insertion into the plasma membranes; activation of the catalytic step and its control or modulation; and translocation of chitin chains across cell membranes, their orientation, fibrillogenesis and association with other extracellular structural components such as polysaccharides (fungi) and cuticular proteins (insects) . Also the precise biochemical lesions inflicted by CS inhibitors, such as the acylurea insect growth regulators, are largely unclear . The recent isolation and sequencing of insect CS genes should help in elucidating various aspects of chitin biochemistry and inhibition . In particular, the large number of transmembrane segments, characteristic of the insect CS, are speculated to be involved in chitin translocation and are expected to shed light on the mode of action of acylurea insecticides.

J Nutr, 2001 Nov, 131(11), 2988S - 93S
Regulation of translation via TOR signaling: insights from Drosophila melanogaster; Miron M et al.; The target of rapamycin (TOR) proteins are large protein kinases evolutionarily conserved from yeast to human . A large body of evidence demonstrates that TOR proteins function in a nutrient-sensing checkpoint whose role is to restrict growth under conditions of low nutrient availability . Under such conditions, TOR blocks the transmission of growth-promoting signals from extracellular stimuli . Recent data obtained by genetic studies in the fruit fly Drosophila melanogaster demonstrate the importance of both insulin-like signaling and TOR signaling in promoting growth . Importantly, these studies identified a major downstream target of TOR and insulin-like signaling as the translational machinery.

Mol Biol Cell, 2001 Nov, 12(11), 3353 - 64
Yarrowia lipolytica cells mutant for the peroxisomal peroxin Pex19p contain structures resembling wild-type peroxisomes; Lambkin GR et al.; PEX genes encode peroxins, which are proteins required for peroxisome assembly . The PEX19 gene of the yeast Yarrowia lipolytica was isolated by functional complementation of the oleic acid-nonutilizing strain pex19-1 and encodes Pex19p, a protein of 324 amino acids (34,822 Da) . Subcellular fractionation and immunofluorescence microscopy showed Pex19p to be localized primarily to peroxisomes . Pex19p is detected in cells grown in glucose-containing medium, and its levels are not increased by incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes . pex19 cells preferentially mislocalize peroxisomal matrix proteins and the peripheral intraperoxisomal membrane peroxin Pex16p to the cytosol, although small amounts of these proteins could be reproducibly localized to a subcellular fraction enriched for peroxisomes . In contrast, the peroxisomal integral membrane protein Pex2p exhibits greatly reduced levels in pex19 cells compared with its levels in wild-type cells . Importantly, pex19 cells were shown by electron microscopy to contain structures that resemble wild-type peroxisomes in regards to size, shape, number, and electron density . Subcellular fractionation and isopycnic density gradient centrifugation confirmed the presence of vesicular structures in pex19 mutant strains that were similar in density to wild-type peroxisomes and that contained profiles of peroxisomal matrix and membrane proteins that are similar to, yet distinct from, those of wild-type peroxisomes . Because peroxisomal structures form in pex19 cells, Pex19p apparently does not function as a peroxisomal membrane protein receptor in Y . lipolytica . Our results are consistent with a role for Y . lipolytica Pex19p in stabilizing the peroxisomal membrane.

J Comput Biol, 2001, 8(5), 523 - 47
Constrained global optimization for estimating molecular structure from atomic distances; Williams GA et al.; Finding optimal three-dimensional molecular configurations based on a limited amount of experimental and/or theoretical data requires efficient nonlinear optimization algorithms . Optimization methods must be able to find atomic configurations that are close to the absolute, or global, minimum error and also satisfy known physical constraints such as minimum separation distances between atoms (based on van der Waals interactions) . The most difficult obstacles in these types of problems are that 1) using a limited amount of input data leads to many possible local optima and 2) introducing physical constraints, such as minimum separation distances, helps to limit the search space but often makes convergence to a global minimum more difficult . We introduce a constrained global optimization algorithm that is robust and efficient in yielding near-optimal three-dimensional configurations that are guaranteed to satisfy known separation constraints . The algorithm uses an atom-based approach that reduces the dimensionality and allows for tractable enforcement of constraints while maintaining good global convergence properties . We evaluate the new optimization algorithm using synthetic data from the yeast phenylalanine tRNA and several proteins, all with known crystal structure taken from the Protein Data Bank . We compare the results to commonly applied optimization methods, such as distance geometry, simulated annealing, continuation, and smoothing . We show that compared to other optimization approaches, our algorithm is able combine sparse input data with physical constraints in an efficient manner to yield structures with lower root mean squared deviation.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 92 - 7
The use of extracellular enzymes from Streptomyces albus ATCC 3005 for the bleaching of eucalyptus kraft pulp; Antonopoulos VT et al.; The suitability of culture supernatant from Streptomyces albus ATCC 3005 for use in the biobleaching of eucalyptus kraft pulp was investigated . S . albus was found to grow on a minimal salts medium containing oat spelts xylan and yeast extract as the main carbon and nitrogen sources, respectively . Maximal extracellular xylanase and peroxidase production was detected after 120 h (11.97 U ml(-1)) and 72 h (0.58 U ml(-1)), respectively . Importantly, no cellulase activity could be detected . When the effect of pH on enzyme activity was examined, maximal xylanase and peroxidase activity was obtained at pH 6.5 and pH 9.9, respectively . The optimum hydrogen peroxide (H2O2) concentration for peroxidase activity was found to occur at 20 mM, with peroxidase remaining active at 100 mM H2O2 after 1 h incubation at 53 degrees C; the half-life of the enzyme at that temperature was estimated to be 33 min . Short-term (1 h) biobleaching of eucalyptus kraft pulp with culture supernatant from S . albus in the presence of H2O2 resulted in a significant reduction of kappa number (2.85 units) with no change in viscosity . These results suggest a potential application of cellulase-free culture supernatants from S . albus in biobleaching.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 117 - 23
Purification and properties of a beta-1,6-glucanase from Streptomyces sp . EF-14, an actinomycete antagonistic to Phytophthora spp; Fayad KP et al.; Extracellular enzymes with glucanase activities are an important component of actinomycete-fungus antagonism . Streptomyces sp . EF-14 has been previously identified as one of the most potent antagonists of Phytophthora spp . A beta-1,6-glucanase (EC 3.2.1.75; glucan endo-1,6-beta-glucosidase) was purified by four chromatographic steps from the culture supernatant of strain EF-14 grown on a medium with lyophilized cells of Candida utilis as main nutrient source . The glucanase level in this medium followed a characteristic pattern in which the rise of beta-1,6-glucanase activity always preceded that of beta-1,3-glucanase . The molecular mass of the enzyme was estimated to be 65 kDa and the pI approximately 5.5 . It hydrolyzed pustulan by an endo-mechanism generating gentiobiose and glucose as final products . Laminarin was not hydrolyzed indicating that the enzyme does not recognize beta-1,6-links flanked by beta-1,3-links . No significant clearing of yeast cell walls in liquid suspensions or in agar plates was observed indicating that this beta-1,6-glucanase is a non-lytic enzyme . This is the first beta-1,6-glucanase characterized from an actinomycete.

Pharmacogenetics, 2001 Nov, 11(8), 739 - 41
In-vitro analysis of the contribution of CYP2D6.35 to ultra-rapid metabolism; Allorge D et al.; From 10 to 30% of CYP2D6 ultra-rapid metabolizers of Caucasian origin harbor alleles with duplicated or amplified functional CYP2D6 genes . Recently, the CYP2D6*35 allele has been reported to be more frequent in ultra-rapid metabolizing subjects than in extensive metabolizers, suggesting a possible role of this variant in CYP2D6 duplication-negative ultra-rapid metabolizing subjects . In this study, we examined the functional consequences of the Val11Met, Arg296Cys and Ser486Thr amino acid substitutions associated with the CYP2D6*35 on the expression and catalytic activity of the variant enzyme, heterologously expressed in yeast . Our results indicate that the functional activity and level of expression of recombinant CYP2D6.35 are comparable with those of the wild-type enzyme, thus precluding the hypothesis that the high level of enzyme activity in CYP2D6 duplication-negative ultra-rapid metabolizing subjects is a consequence of the expression of a more catalytically effective CYP2D6.35 enzyme.

Science, 2001 Nov 2, 294(5544), 1108 - 11
Interaction of the response regulator ARR4 with phytochrome B in modulating red light signaling; Sweere U et al.; The Arabidopsis thaliana response regulator 4, expressed in response to phytochrome B action, specifically interacts with the extreme amino-terminus of the photoreceptor . The response regulator 4 stabilizes the active Pfr form of phytochrome B in yeast and in planta, thus elevates the level of the active photoreceptor in vivo . Accordingly, transgenic Arabidopsis plants overexpressing the response regulator 4 display hypersensitivity to red light but not to light of other wavelengths . We propose that the response regulator 4 acts as an output element of a two-component system that modulates red light signaling on the level of the phytochrome B photoreceptor.

Nucleic Acids Res, 2001 Nov 1, 29(21), 4433 - 40
Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype; Chennathukuzhi VM et al.; The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved . To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes . GTP, but not GDP, reduces RNA binding by approximately 50% and the poorly hydrolyzed GTP analog, GTPgammaS, reduces binding by >90% in gel shift and immunoprecipitation assays . No similar reduction of DNA binding is seen . When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP(GTP) and TB-RBP(GTP) no longer binds to RNA, although DNA binding is not affected . Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP(GTP) will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax) . Transfection of TB-RBP(GTP) into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP(GTP) in cells . These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.

Nucleic Acids Res, 2001 Nov 1, 29(21), 4319 - 33
The Arabidopsis thaliana genome contains at least 29 active genes encoding SET domain proteins that can be assigned to four evolutionarily conserved classes; Baumbusch LO et al.; SET domains are conserved amino acid motifs present in chromosomal proteins that function in epigenetic control of gene expression . These proteins can be divided into four classes as typified by their Drosophila members E(Z), TRX, ASH1 and SU(VAR)3-9 . Homologs of all four classes have been identified in yeast and mammals, but not in plants . A BLASTP screening of the Arabidopsis genome identified 37 genes: three E(z) homologs, five trx homologs, four ash1 homologs and 15 genes similar to Su(var)3-9 . Seven genes were assigned as trx-related and three as ash1-related . Only four genes have been described previously . Our classification is based on the characteristics of the SET domains, cysteine-rich regions and additional conserved domains, including a novel YGD domain . RT-PCR analysis, cDNA cloning and matching ESTs show that at least 29 of the genes are active in diverse tissues . The high number of SET domain genes, possibly involved in epigenetic control of gene activity during plant development, can partly be explained by extensive genome duplication in Arabidopsis . Additionally, the lack of introns in the coding region of eight SU(VAR)3-9 class genes indicates evolution of new genes by retrotransposition . The identification of putative nuclear localization signals and AT-hooks in many of the proteins supports an anticipated nuclear localization, which was demonstrated for selected proteins.

Genome Res, 2001 Nov, 11(11), 1826 - 32
A short pseudoautosomal region in laboratory mice; Perry J et al.; The pseudoautosomal region (PAR) of mammalian sex chromosomes is a small region of sequence identity that is the site of an obligatory pairing and recombination event between the X and Y chromosomes during male meiosis . During female meiosis, X chromosomes can pair and recombine along their entire length; recombination in the PAR is therefore approximately 10x greater in male meiosis compared with female meiosis . A consequence of the presence of the PAR in two copies in males and females is that genes in the region escape the process of X-inactivation . Although the structure and gene content of the human PAR at Xq/Yq is well understood, the mouse PAR, which appears to be of independent evolutionary origin, is poorly characterized . Here we describe a yeast artificial chromosome (YAC) contig covering the distal part of the mouse X chromosome, which we have used to define the pseudoautosomal boundary, that is, the point of divergence of X-specific and X-Y-identical sequences . In addition, we have investigated the size of the mouse PAR by integrating a unique restriction endonuclease recognition site just proximal to the pseudoautosomal boundary by homologous recombination . Restriction digestion of this modified DNA and pulsed field gel electrophoresis reveal that the PAR in these cells is approximately 700 kb . Thus, the mouse PAR, although small in size, has retained essential sex chromosome pairing functions despite its rapid rate of evolution.

Genes Dev, 2001 Nov 1, 15(21), 2886 - 99
The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis; Mohr SE et al.; In Drosophila melanogaster, formation of the axes and the primordial germ cells is regulated by interactions between the germ line-derived oocyte and the surrounding somatic follicle cells . This reciprocal signaling results in the asymmetric localization of mRNAs and proteins critical for these oogenic processes . Mago Nashi protein interprets the posterior follicle cell-to-oocyte signal to establish the major axes and to determine the fate of the primordial germ cells . Using the yeast two-hybrid system we have identified an RNA-binding protein, Tsunagi, that interacts with Mago Nashi protein . The proteins coimmunoprecipitate and colocalize, indicating that they form a complex in vivo . Immunolocalization reveals that Tsunagi protein is localized within the posterior oocyte cytoplasm during stages 1-5 and 8-9, and that this localization is dependent on wild-type mago nashi function . When tsunagi function is removed from the germ line, egg chambers develop in which the oocyte nucleus fails to migrate, oskar mRNA is not localized within the posterior pole, and dorsal-ventral pattern abnormalities are observed . These results show that a Mago Nashi-Tsunagi protein complex is required for interpreting the posterior follicle cell-to-oocyte signal to define the major body axes and to localize components necessary for determination of the primordial germ cells.

Cancer Res, 2001 Nov 1, 61(21), 7943 - 9
Inactivation of human SRBC, located within the 11p15.5-p15.4 tumor suppressor region, in breast and lung cancers; Xu XL et al.; A cDNA clone encoding human SRBC {serum deprivation response factor (sdr)-related gene product that binds to c-kinase} was isolated in a yeast two-hybrid screening, with amino acids 1-304 of BRCA1 as the probe . The human SRBC gene (hSRBC) was mapped to chromosome region 11p15.5-p15.4, close to marker D11S1323, at which frequent loss of heterozygosity (LOH) has been observed in sporadic breast, lung, ovarian, and other types of adult cancers as well as childhood tumors . hSRBC-coding region mutations including frame shift and truncation mutations were detected in a few ovarian and lung cancer cell lines . More significantly, the expression of hSRBC protein was down-regulated in a large fraction {30 (70%) of 43} of breast, lung, and ovarian cancer cell lines, whereas strong expression of hSRBC protein was detected in normal mammary and lung epithelial cells . The down-regulation of hSRBC expression in cancer cells was associated with hypermethylation of CpG dinucleotides in its promoter region, and 3 (60%) of 5 primary breast tumors and 11 (79%) of 14 primary lung tumors were also found to be hypermethylated . Treatment of breast cancer MCF7 cells with 5'azacytidine and Trichostatin A resulted in expression of hSRBC, confirming DNA methylation as the mode of inactivation . Our results suggest that epigenetic or mutational inactivation of hSRBC may contribute to the pathogenesis of several types of human cancers, marking hSRBC as a candidate tumor suppressor gene.

Mol Cell Biol, 2001 Dec, 21(23), 8035 - 44
Identification of components of the murine histone deacetylase 6 complex: link between acetylation and ubiquitination signaling pathways; Seigneurin-Berny D et al.; The immunopurification of the endogenous cytoplasmic murine histone deacetylase 6 (mHDAC6), a member of the class II HDACs, from mouse testis cytosolic extracts allowed the identification of two associated proteins . Both were mammalian homologues of yeast proteins known to interact with each other and involved in the ubiquitin signaling pathway: p97/VCP/Cdc48p, a homologue of yeast Cdc48p, and phospholipase A2-activating protein, a homologue of yeast UFD3 (ubiquitin fusion degradation protein 3) . Moreover, in the C-terminal region of mHDAC6, a conserved zinc finger-containing domain named ZnF-UBP, also present in several ubiquitin-specific proteases, was discovered and was shown to mediate the specific binding of ubiquitin by mHDAC6 . By using a ubiquitin pull-down approach, nine major ubiquitin-binding proteins were identified in mouse testis cytosolic extracts, and mHDAC6 was found to be one of them . All of these findings strongly suggest that mHDAC6 could be involved in the control of protein ubiquitination . The investigation of biochemical properties of the mHDAC6 complex in vitro further supported this hypothesis and clearly established a link between protein acetylation and protein ubiquitination.

Mol Cell Biol, 2001 Dec, 21(23), 8022 - 34
Kinectin is a key effector of RhoG microtubule-dependent cellular activity; Vignal E et al.; RhoG is a member of the Rho family of GTPases that activates Rac1 and Cdc42 through a microtubule-dependent pathway . To gain understanding of RhoG downstream signaling, we performed a yeast two-hybrid screen from which we identified kinectin, a 156-kDa protein that binds in vitro to conventional kinesin and enhances microtubule-dependent kinesin ATPase activity . We show that RhoG(GTP) specifically interacts with the central domain of kinectin, which also contains a RhoA binding domain in its C terminus . Interaction was confirmed by coprecipitation of kinectin with active RhoG(G12V) in COS-7 cells . RhoG, kinectin, and kinesin colocalize in REF-52 and COS-7 cells, mainly in the endoplasmic reticulum but also in lysosomes . Kinectin distribution in REF-52 cells is modulated according to endogenous RhoG activity . In addition, by using injection of anti-kinectin antibodies that challenge RhoG-kinectin interaction or by blocking anti-kinesin antibodies, we show that RhoG morphogenic activity relies on kinectin interaction and kinesin activity . Finally, kinectin overexpression elicits Rac1- and Cdc42-dependent cytoskeletal effects and switches cells to a RhoA phenotype when RhoG activity is inhibited or microtubules are disrupted . The functional links among RhoG, kinectin, and kinesin are further supported by time-lapse videomicroscopy of COS-7 cells, which showed that the microtubule-dependent lysosomal transport is facilitated by RhoG activation or kinectin overexpression and is severely stemmed upon RhoG inhibition . These data establish that kinectin is a key mediator of microtubule-dependent RhoG activity and suggest that kinectin also mediates RhoG- and RhoA-dependent antagonistic pathways.

J Virol, 2001 Dec, 75(23), 11791 - 802
Interaction of zyxin, a focal adhesion protein, with the e6 protein from human papillomavirus type 6 results in its nuclear translocation; Degenhardt YY et al.; Zyxin, a focal adhesion molecule, interacts specifically with the E6 protein from human papillomavirus (HPV) type 6 in a yeast two-hybrid screen of a cDNA library prepared from human keratinocytes . Zyxin does not interact significantly with E6 proteins from HPV types 11, 16, or 18 . The interaction was confirmed by in vitro and in vivo analyses and it requires the LIM domains (Lin-11, Isl-1, and Mec-3 {G . Freyd, S . K . Kim, and H . R . Horvitz, Nature 344:876-879, 1990}) found at the carboxyl terminus of zyxin . Cotransfection of E6 from HPV ((6)E6) and zyxin results in the accumulation of zyxin in the nucleus where it can function as a transcriptional activator . (6)E6 can also mobilize endogenous zyxin to the nucleus.

J Virol, 2001 Dec, 75(23), 11664 - 76
Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells; Miller DJ et al.; The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents . The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells . To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication . We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses . Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis . Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes . Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures . We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.

J Biol Chem, 2002 Jan 4, 277(1), 455 - 61 Epub 2001 Oct 31.
Multi-PDZ domain protein 1 (MUPP1) is concentrated at tight junctions through its possible interaction with claudin-1 and junctional adhesion molecule; Hamazaki Y et al.; Claudins, most of which end in valine at their COOH termini, constitute tight junction (TJ) strands, suggesting that TJ strands strongly attract PDZ-containing proteins . Indeed, ZO-1, -2, and -3, each of which contains three PDZ domains, were shown to directly bind to claudins . Using the yeast two-hybrid system, we identified ZO-1 and MUPP1 (multi-PDZ domain protein 1) as binding partners for the COOH terminus of claudin-1 . MUPP1 has been identified as a protein that contains 13 PDZ domains, but it has not been well characterized . In vitro binding assays with recombinant MUPP1 confirmed the interaction between MUPP1 and claudin-1 and identified PDZ10 as the responsible domain for this interaction . A polyclonal antibody specific for MUPP1 was then generated . Immunofluorescence confocal microscopy as well as immunoelectron microscopy with this antibody revealed that in polarized epithelial cells MUPP1 was exclusively concentrated at TJs . Furthermore, in vitro binding and transfection experiments showed that junctional adhesion molecule, another TJ adhesion molecule, also bound to the PDZ9 domain of MUPP1 . These findings suggested that MUPP1 is concentrated at TJs in epithelial cells through its binding to claudin and junctional adhesion molecule and that it may function as a multivalent scaffold protein that recruits various proteins to TJs.

EMBO J, 2001 Nov 1, 20(21), 6028 - 36
Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress; Morita K et al.; Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that activates the JNK and p38 MAP kinase cascades and is activated in response to oxidative stress such as hydrogen peroxide (H(2)O(2)) . A yeast two-hybrid screening identified a serine/threonine protein phosphatase 5 (PP5) as a binding partner of ASK1 . PP5 directly dephosphorylated an essential phospho-threonine residue within the kinase domain of ASK1 and thereby inactivated ASK1 activity in vitro and in vivo . The interaction between PP5 and ASK1 was induced by H(2)O(2) treatment and was followed by the decrease in ASK1 activity . PP5 inhibited not only H(2)O(2)-induced sustained activation of ASK1 but also ASK1-dependent apoptosis . Thus, PP5 appears to act as a physiological inhibitor of ASK1-JNK/p38 pathways by negative feedback.

EMBO J, 2001 Nov 1, 20(21), 5876 - 86
Identification of interaction domains of the prion protein with its 37-kDa/67-kDa laminin receptor; Hundt C et al.; Cell-binding and internalization studies on neuronal and non-neuronal cells have demonstrated that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor for the cellular prion protein (PrP) . Here we identify direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of the cellular PrP to its receptor, which we demonstrated in vitro on recombinant proteins . Mapping analyses in the yeast two-hybrid system and cell-binding assays identified PrPLRPbd1 {amino acids (aa) 144-179} as a direct and PrPLRPbd2 (aa 53-93) as an indirect HSPG-dependent laminin receptor precursor (LRP)-binding site on PrP . The yeast two-hybrid system localized the direct PrP-binding domain on LRP between aa 161 and 179 . Expression of an LRP mutant lacking the direct PrP-binding domain in wild-type and mutant HSPG-deficient Chinese hamster ovary cells by the Semliki Forest virus system demonstrates a second HSPG-dependent PrP-binding site on LRP . Considering the absence of LRP homodimerization and the direct and indirect LRP-PrP interaction sites, we propose a comprehensive model for the LRP-PrP-HSPG complex.

Front Biosci, 2001 Nov 01, 6, A25 - 32
Specific regions of the extracellular domain of dlk, an EGF-like homeotic protein involved in differentiation, participate in intramolecular interactions; Baladron V et al.; The level of expression of dlk, an EGF-like protein possessing six EGF-like repeats in its extracellular region, is critical for 3T3-L1 fibroblasts to differentiate into adipocytes in response to IGF1 . The mechanism of action of dlk is not well understood, but its localization on the cell membrane suggests that dlk may function as a receptor, as a ligand or as a regulatory protein modulating the binding, the signaling, or the expression of other molecules involved in cell differentiation and growth . In this work, we demonstrate, by using the Yeast Two-Hybrid system, that dlk interacts with itself through specific regions of its extracellular domain . The strongest interactions were observed between specific EGF-like repeats and between a non EFG-like region where unknown proteases act to generate soluble forms of dlk . These observations suggest that the interaction between two membrane dlk molecules belonging to the same or to different cells, or the interaction between soluble and membrane dlk variants, may be important to regulate dlk function.

Virology, 2001 Oct 25, 289(2), 269 - 82
Characterization and template properties of RNA dimers generated during flock house virus RNA replication; Albarino CG et al.; Flock house virus (FHV) is the best studied member of the Nodaviridae, a family of small, nonenveloped, isometric RNA viruses of insects and fish . Nodavirus genomes comprise two single-stranded positive-sense RNA segments (RNAs 1 and 2) that encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively . The RdRp replicates both genomic RNAs and also generates a subgenomic RNA (RNA3) that is not encapsidated . Although genomic RNAs replicate through negative-sense intermediates, little is known about these RNAs or the details of the replication mechanism . Negative-sense RNAs 1, 2, and 3, as well as putative dimers of RNAs 2 and 3, have been detected in previous studies . In this study we detected dimers of RNAs 1, 2, and 3 by Northern blot analyses of RNA samples from FHV-infected Drosophila cells, as well as from mammalian and yeast cells supporting FHV RNA replication . Characterization of these RNA species by RT-PCR and sequence determination showed that they contained head-to-tail junctions of FHV RNAs . RNAs containing the complete sequence of RNA2 joined to RNA3 were also detected during replication . To examine the template properties of these dimeric RNAs, we made corresponding cDNAs and transcribed them from a T7 promoter in mammalian cells constitutively expressing T7 RNA polymerase, together with RNA1 to provide the RdRp . Although heterologous terminal extensions inhibit FHV RNA replication, monomeric RNA2 was resolved and replicated from complete or partial homodimer templates and from an RNA2-RNA3 heterodimer .

Biochem Biophys Res Commun, 2001 Nov 9, 288(4), 1018 - 26
Floral transcription factor AGAMOUS interacts in vitro with a leucine-rich repeat and an acid phosphatase protein complex; Gamboa A et al.; We are interested in identifying potential protein interactors of MADS domain transcription factors during Arabidopsis thaliana flower development . We based our biochemical search on a conserved motif in the MADS domain that includes putative phosphatase and phosphorylation sites that may mediate protein interactions . An affinity column with this motif and a few surrounding hypervariable amino acids derived from the AGAMOUS sequence was prepared and used to isolate potential interactors from floral crude extracts . Only two proteins were specifically bound to the affinity column . The first corresponds to a carpel specific storage protein, VSP1, that presents acid phosphatase activity, and the second is a novel leucine-rich repeat protein that we have named FLOR1 . Coimmunoprecipitation, two-hybrid yeast, and affinity column assays show that the FLOR1-VSP1 complex interacts with AGAMOUS and that this transcription factor directly interacts with FLOR1 . This is the first assay to show an interaction between plant MADS domain factors and non-MADS proteins .

Biol Chem, 2001 Sep, 382(9), 1379 - 85
ERH (enhancer of rudimentary homologue), a conserved factor identical between frog and human, is a transcriptional repressor; Pogge von Strandmann E et al.; Drosophila enhancer of rudimentary {e(r)} interacts genetically with the rudimentary gene, which encodes a protein possessing the first three enzymatic activities of the pyrimidine biosynthesis pathway . A regulatory or enzymatic activity of e(r) in pyrimidine biosynthesis and the cell cycle has been suggested, but nothing is known about its molecular function . The factor is evolutionarily highly conserved since homologues exist in plants and mammals . We cloned the Xenopus enhancer of rudimentary homologue (XERH) as an interaction partner of DCoH/PCD (dimerisation cofactor of HNF1/pterin-4alpha-carbinolamine dehydratase) in the yeast two-hybrid assay . DCoH/PCD is a multifunctional factor originally identified as a positive cofactor of the HNF1 homeobox transcription factors . XERH is a 104 amino acid protein that is identical to its mammalian homologues . The mRNA is expressed maternally, enriched in ectodermal derivatives during development and ubiquitously detectable in the adult . Fused to the DNA binding region of the GAL4 transcription factor domain, XERH represses the activity of a GAL4 responsive reporter in HeLa, but not in NIH3T3 cells . Furthermore, the DCoH/PCD coactivation of a HNF1 responsive reporter is inhibited by XERH . We propose that XERH is a cell type-specific transcriptional repressor, probably interfering with HNF1-dependent gene regulation via DCoH/PCD.

Proc Natl Acad Sci U S A, 2001 Nov 6, 98(23), 13078 - 83 Epub 2001 Oct 30.
The coactivator dTAF(II)110/hTAF(II)135 is sufficient to recruit a polymerase complex and activate basal transcription mediated by CREB; Felinski EA et al.; A specific TATA binding protein-associated factor (TAF), dTAF(II)110/hTAF(II)135, interacts with cAMP response element binding protein (CREB) through its constitutive activation domain (CAD), which recruits a polymerase complex and activates transcription . The simplest explanation is that the TAF is a coactivator, but several studies have questioned this role of TAFs . Using a reverse two-hybrid analysis in yeast, we previously mapped the interaction between dTAF(II)110 (amino acid 1-308) and CREB to conserved hydrophobic amino acid residues in the CAD . That mapping was possible only because CREB fails to activate transcription in yeast, where all TAFs are conserved, except for the TAF recognizing CREB . To test whether CREB fails to activate transcription in yeast because it lacks a coactivator, we fused dTAF(II)110 (amino acid 1-308) to the TATA binding protein domain of the yeast scaffolding TAF, yTAF(II)130 . Transformation of yeast with this hybrid TAF conferred activation by the CAD, indicating that interaction with yTFIID is sufficient to recruit a polymerase complex and activate transcription . The hybrid TAF did not mediate activation by VP16 or vitamin D receptor, each of which interacts with TFIIB, but not with dTAF(II)110 (amino acid 1-308) . Enhancement of transcription activation by dTAF(II)110 in mammalian cells required interaction with both the CAD and TFIID and was inhibited by mutation of core hydrophobic residues in the CAD . These data demonstrate that dTAF(II)110/hTAF(II)135 acts as a coactivator to recruit TFIID and polymerase and that this mechanism of activation is conserved in eukaryotes.

J Biol Chem, 2001 Dec 28, 276(52), 48863 - 70 Epub 2001 Oct 30.
Cloning and characterization of liver-specific isoform of Chk1 gene from rat; Shann YJ et al.; We have isolated and characterized an isoform of protein kinase Chk1 gene from rat liver and a rat liver cDNA library by 5'-rapid amplification of cDNA ends . The gene (Cil) contains the C-terminal region of the Chk1 gene, but the 5'-end is derived from a sequence in the intron of Chk1 preceding the C-terminal domain by differential RNA splicing . The kinase domain of Chk1 gene is absent in this isoform . Tissue RNA and protein blot analyses indicated that Cil was specifically expressed only in rat liver, and its expression increased with liver development . Expression of Cil was found to be reduced in three rat hepatoma cell lines examined . A promoter trap experiment suggested that a promoter was located in the intron preceding the C-terminal domain of Chk1, and transcription from this novel promoter generated the new 5' noncoding exon of Cil . Thus Cil was generated by both alternate promoter usage and differential RNA splicing . UV irradiation induced caffeine-sensitive phosphorylation of both Chk1 and Cil at Ser-345 in Chk1 and its equivalent site in Cil, implying a role for ATR kinase in the phosphorylation of both proteins . We demonstrated the interaction between the kinase domain of Chk1 and Cil using a yeast two-hybrid assay and pull-down technique . In contrast to the effect of Chk1, Cil was found to decrease the transactivating function of p53, and the S63A mutation of Cil abolished this effect . These results suggest that Cil may serve as a dominant negative competitor of Chk1 as suggested previously.

Cochrane Database Syst Rev . 2001;(4):CD002845.
Oral versus intra-vaginal imidazole and triazole anti-fungal treatment of uncomplicated vulvovaginal candidiasis (thrush); Watson MC et al.; BACKGROUND: Anti-fungals are available for oral and intra-vaginal treatment of uncomplicated vulvovaginal candidiasis (thrush) . OBJECTIVES: The primary objective of this review was to assess the relative effectiveness of oral versus intra-vaginal anti-fungals for the treatment of uncomplicated vulvovaginal candidiasis . The secondary objectives of the review were to assess the cost-effectiveness, safety and patient preference of oral versus intra-vaginal anti-fungals . SEARCH STRATEGY: The following sources were searched: The Cochrane Library (Issue 4, 1999), MEDLINE (January 1985 to May 2000), EMBASE (January 1980 to January 2000) and the Cochrane Collaboration Sexually Transmitted Disease Group Specialised Register of Controlled Trials . The reference lists of retrieved articles were reviewed manually . The manufacturers of anti-fungals available in the UK were contacted . SELECTION CRITERIA: ~bullet~Randomised controlled trials published in any language . ~bullet~Trials had to compare at least one oral anti-fungal with one intra-vaginal anti-fungal . ~bullet~Women (aged 16 years or over) with uncomplicated vulvovaginal candidiasis . ~bullet~The diagnosis of vulvovaginal candidiasis to be made mycologically (i.e . a positive culture and / or microscopy for yeast) . ~bullet~Trials were excluded if they solely involved subjects who were HIV positive, immunocompromised, pregnant, breastfeeding or diabetic . ~bullet~The primary outcome measure was clinical cure . DATA COLLECTION AND ANALYSIS: Duplicate scrutiny was performed of the titles and abstracts of the electronic search results . Full article formats of all selected abstracts were retrieved and independently assessed by two reviewers . Independent duplicate abstraction was performed by four reviewers . Disagreements regarding trial inclusion or data abstraction were resolved by discussion between the reviewers . Odds ratios were pooled using the random effects model . Chi-squared tests with a p-value of less than 0.1 indicated heterogeneity in the results . MAIN RESULTS: Seventeen trials are included in the review, reporting 19 oral versus intra-vaginal anti-fungal comparisons . No statistically significant differences were shown between oral and intra-vaginal anti-fungal treatment for clinical cure at short term (OR 1.00 (95% CI, 0.72 to 1.40)) and long term (OR 1.03 (95% CI, 0.72 to 1.49)) follow-up . No statistically significant differences for mycological cure were observed between oral and intra-vaginal treatment at short term (OR 1.20 (95% CI, 0.87 to 1.65)) or long term follow-up (OR 1.30 (95% CI, 0.99 to 1.71)) . Two trials each reported one withdrawal from treatment due to an adverse reaction . Treatment preference data were poorly reported . REVIEWER'S CONCLUSIONS: No differences exist in terms of the relative effectiveness (measured as clinical and mycological cure) of anti-fungals administered by the oral and intra-vaginal routes for the treatment of uncomplicated vaginal candidiasis . No definitive conclusion can be made regarding the relative safety of oral and intra-vaginal anti-fungals for uncomplicated vaginal candidiasis . The oral route of administration is the preferred route for anti-fungals for the treatment of vulvovaginal candidiasis . The decision to prescribe or recommend the purchase of an anti-fungal for oral or intra-vaginal administration should take into consideration: safety, cost and treatment preference . Unless there is a previous history of adverse reaction to one route of administration or contraindications, women who are purchasing their own treatment should be given full information about the characteristics and costs of treatment to make their own decision . If health services are paying the treatment cost, decision-makers should consider whether the higher cost of oral anti-fungal administration is worth the gain in convenience, if this is the patient's preference.

J Biol Chem, 2002 Jan 18, 277(3), 2050 - 8 Epub 2001 Oct 29.
Regulation of internal ribosome entry site-mediated translation by eukaryotic initiation factor-2alpha phosphorylation and translation of a small upstream open reading frame; Fernandez J et al.; Adaptation to amino acid deficiency is critical for cell survival . In yeast, this adaptation involves phosphorylation of the translation eukaryotic initiation factor (eIF) 2alpha by the kinase GCN2 . This leads to the increased translation of the transcription factor GCN4, which in turn increases transcription of amino acid biosynthetic genes, at a time when expression of most genes decreases . Here it is shown that translation of the arginine/lysine transporter cat-1 mRNA increases during amino acid starvation of mammalian cells . This increase requires both GCN2 phosphorylation of eIF2alpha and the translation of a 48-amino acid upstream open reading frame (uORF) present within the 5'-leader of the transporter mRNA . When this 5'-leader was placed in a bicistronic mRNA expression vector, it functioned as an internal ribosomal entry sequence and its regulated activity was dependent on uORF translation . Amino acid starvation also induced translation of monocistronic mRNAs containing the cat-1 5'-leader, in a manner dependent on eIF2alpha phosphorylation and translation of the 48-amino acid uORF . This is the first example of mammalian regulation of internal ribosomal entry sequence-mediated translation by eIF2alpha phosphorylation during amino acid starvation, suggesting that the mechanism of induced Cat-1 protein synthesis is part of the adaptive response of cells to amino acid limitation.

FEBS Lett, 2001 Oct 26, 507(2), 133 - 6
Interaction between two adapter proteins, PAG and EBP50: a possible link between membrane rafts and actin cytoskeleton; Brdickova N et al.; Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains) . PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk . In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG . We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na(+)/H(+) exchanger regulatory factor), as a specific PAG-binding partner . The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50 . As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG-EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

Mol Cell, 2001 Oct, 8(4), 729 - 30
Vesicle tethering factors united; Pfeffer S; In the October 2001 issue of Developmental Cell, Whyte and Munro elucidate the composition of a novel vesicle tethering complex and in the process uncover previously undetected homology between tethering complexes that catalyze a variety of different transport events in yeast and mammalian cells.

Genes Cells, 2001 Oct, 6(10), 913 - 21
Cofilin-2, a novel type of cofilin, is expressed specifically at aggregation stage of Dictyostelium discoideum development; Aizawa H et al.; BACKGROUND: A conventional cofilin, cofilin-1 in Dictyostelium discoideum plays significant roles in cell proliferation, phagocytosis, chemotactic movement and macropinocytosis . RESULTS: We identified a new member of the cofilin family, named cofilin-2 in D . discoideum . Cofilin-2 shows significant homology to a conventional Dictyostelium cofilin, cofilin-1, through its entire sequence, and contains residues conserved among the cofilin family that are responsible for actin-binding . On the other hand, several residues that are conserved among the cofilin family are missing from cofilin-2 . Purified cofilin-2 depolymerized actin filaments in a dose- and pH-dependent manner and reduced the apparent viscosity of an actin solution, although they did not co-sediment with actin filaments at all . Cofilin-2 was not expressed in vegetative cells, but was transiently induced during the aggregation stage of development, whereas cofilin-1 was predominantly expressed in vegetative cells . Immunocytochemistry revealed that cofilin-2 localizes at substrate adhesion sites, where cofilin-1 is almost completely excluded . Disruption of the cofilin-2 gene caused an increase in actin accumulation at the substrate adhesion sites . We also found that cofilin-2 did not rescue Deltacof1 yeast cells, whereas cofilin-1 did . CONCLUSIONS: Cofilin-2 may play a distinct role from that of cofilin-1 in destabilization of the actin cytoskeleton during Dictyostelium development.

Mol Genet Genomics, 2001 Oct, 266(2), 180 - 9
A transcript encoding a nucleic acid-binding protein specifically expressed in maize seeds; Heyl A et al.; A cDNA clone has been obtained for a low-abundance, seed-specific mRNA that encodes a polypeptide which defines a novel family of plant proteins with some similarities to the DnaJ class of molecular chaperones . The MEM1 (Maize Endosperm Motif binding protein) protein is capable of binding to the endosperm motif and activating transcription in the yeast one-hybrid system . Recombinant MEM1 was shown to bind in vitro to nucleic acids, with a preference for RNA over DNA . MEM1 is capable of forming homodimers, a property that is dependent on a domain close to the C-terminus of the protein . The protein is expressed in mid- to late-term endosperm cells . Subcellular fractionation and size fractionation under non-denaturing conditions indicate that the protein is present in the cytosol of endosperm cells . Possible roles of MEM1 in endosperm and protein body development are discussed.

Mol Endocrinol, 2001 Nov, 15(11), 1870 - 9
Mice lacking pituitary tumor transforming gene show testicular and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant cell cycle progression, and premature centromere division; Wang Z et al.; Tumorigenic pituitary tumor transforming gene (PTTG) is a mammalian homolog of Xenopus securin that inhibits chromatid separation, is overexpressed in many human tumor types, and mediates transcriptional activation . Loss of yeast securin Pds1p or Drosophila securin pimples is lethal . Here we show that mice lacking PTTG (PTTG -/-) are, surprisingly, viable and fertile; but they have testicular and splenic hypoplasia, thymic hyperplasia, and thrombocytopenia . PTTG -/- mouse embryo fibroblasts exhibited aberrant cell cycle progression with prolonged G2-M phase and binucleated and multinucleated nuclei with increased aneuploidy . PTTG -/- mouse embryo fibroblast metaphases contained quadriradial, triradial, and chromosome breaks, as well as premature centromere division . The results show that PTTG functions to maintain chromosome stability, cell cycle progression, and appropriate cell division . Moreover, mammalian sister chromatid separation, an important transition in the cell cycle, is likely regulated by mechanisms in addition to securin.

J Cell Sci, 2001 Oct, 114(Pt 19), 3479 - 85
Signal recognition particle protein 19 is imported into the nucleus by importin 8 (RanBP8) and transportin; Dean KA et al.; The signal recognition particle (SRP) is a cytoplasmic RNA-protein complex that targets proteins to the rough endoplasmic reticulum . Although SRP functions in the cytoplasm, RNA microinjection and cDNA transfection experiments in animal cells, as well as genetic analyses in yeast, have indicated that SRP assembles in the nucleus . Nonetheless, the mechanisms responsible for nuclear-cytoplasmic transport of SRP RNA and SRP proteins are largely unknown . Here we show that the 19 kDa protein subunit of mammalian SRP, SRP19, was efficiently imported into the nucleus in vitro by two members of the importin beta superfamily of transport receptors, importin 8 and transportin; SRP19 was also imported less efficiently by several other members of the importin beta family . Although transportin is known to import a variety of proteins, SRP19 import is the first function assigned to importin 8 . Furthermore, we show that a significant pool of endogenous SRP19 is located in the nucleus, as well as the nucleolus . Our results show that at least one mammalian SRP protein is specifically imported into the nucleus, by members of the importin beta family of transport receptors, and the findings add additional evidence for nuclear assembly of SRP.

J Cell Sci, 2001 Oct, 114(Pt 19), 3413 - 8
GGA proteins: new players in the sorting game; Boman AL; The GGA proteins are a novel family of proteins that were discovered nearly simultaneously by several labs studying very different aspects of membrane trafficking . Since then, several studies have described the GGA proteins and their functions in yeast and mammalian cells . Four protein domains are present in all GGA proteins, as defined by sequence homology and function . These different domains interact directly with ARF proteins, cargo and clathrin . Alteration of the levels of GGA proteins by gene knockout or overexpression affects specific trafficking events between the trans-Golgi network and endosomes . These data suggest that GGAs function as ARF-dependent, monomeric clathrin adaptors to facilitate cargo sorting and vesicle formation at the trans-Golgi network.

J Biol Chem, 2002 Jan 4, 277(1), 679 - 85 Epub 2001 Oct 26.
Rab5 association with the angiotensin II type 1A receptor promotes Rab5 GTP binding and vesicular fusion; Seachrist JL et al.; Previous studies have demonstrated that the internalization of the angiotensin II type 1A receptor (AT(1A)R) may be mediated by both beta-arrestin-sensitive and -insensitive mechanisms . Therefore, we have used the AT(1A)R carboxyl-terminal tail to screen a rat brain yeast two-hybrid expression library for novel AT(1A)R-interacting proteins that might contribute to the regulation of AT(1A)R internalization . We have identified Rab5a as an AT(1A)R-binding protein that selectively associates with the AT(1A)R and not with the beta2-adrenergic receptor . A Rab5a-S34N mutant defective in GTP binding does not prevent the internalization of the AT(1A)R but does prevent the trafficking of the AT(1A)R into larger hollow cored vesicular structures . Agonist activation of the AT(1A)R promotes both the formation of Rab5a.AT(1A)R protein complexes and Rab5a GTP binding . Rab5a interactions with the AT(1A)R are mediated in part by the last 10 amino acid residues of the AT(1A)R carboxyl-terminal tail, and although a mutant receptor lacking these residues internalizes normally, it does not redistribute into larger hollow vesicles . Our data suggest that AT(1A)R activation modulates Rab5a activity leading to the homotypic fusion of endocytic vesicles . These observations suggest that vesicular cargo proteins, such as the AT(1A)R, may control their targeting between intracellular compartments by directly regulating the activity of components of the intracellular trafficking machinery such as Rab5a.

Curr Opin Genet Dev, 2001 Dec, 11(6), 681 - 4
Gene and genome duplication; Sankoff D; Genomic sequencing projects have revealed the productivity of processes duplicating genes or entire chromosome segments . Substantial proportions of the yeast, Arabidopsis and human gene complements are made up of duplicates . This has prompted much interest in the processes of duplication, functional divergence and loss of genes, has renewed the debate on whether an early vertebrate genome was tetraploid, and has inspired mathematical models and algorithms in computational biology.

FEBS Lett, 2001 Oct 19, 507(1), 1 - 5
Cleavage of translation initiation factor 4AI (eIF4AI) but not eIF4AII by foot-and-mouth disease virus 3C protease: identification of the eIF4AI cleavage site; Li W et al.; The translation initiation factor eIF4A is cleaved within mammalian cells infected by foot-and-mouth disease virus (FMDV) . The FMDV 3C protease cleaves eIF4AI (between residues E143 and V144), but not the closely related eIF4AII . Modification of eIF4AI, to produce a sequence identical to eIF4AII around the cleavage site, blocked proteolysis . Alignment of mammalian eIF4AI onto the three-dimensional structure of yeast eIF4A located the scissile bond within an exposed, flexible portion of the molecule . The N- and C-terminal cleavage products of eIF4AI generated by FMDV 3C dissociate . Cleavage of eIF4AI by FMDV 3C is thus expected to inactivate it.

Vet J, 2001 Nov, 162(3), 219 - 25
Evaluation in vitro of canine neutrophil function; Comazzi S et al.; Polymorphonuclear granulocyte (PMN) phagocytosis may be affected by many pathological changes . A panel of tests requiring relatively small volumes of blood was applied to 16 healthy dogs in order to obtain normal values and to standardize techniques . PMNs were isolated by discontinuous Percoll gradients; chemotaxis was tested in a modified Boyden chamber using the leading front method; fluorescinated yeast uptake was evaluated on a slide and superoxide (SO) production and adherence was carried out on a microtitre plate . The different aspects of phagocytosis showed no correlation with one another . Better results were obtained using a 60 min incubation period using interleukin-8 (25 ng/mL) as an activator for chemotaxis, and incubating plates for 30 min with phorbol myristate acetate (10(-6)mol/L) to assess SO production .

J Biol Chem, 2002 Jan 4, 277(1), 735 - 45 Epub 2001 Oct 25.
Structure-function analysis of the heat shock factor-binding protein reveals a protein composed solely of a highly conserved and dynamic coiled-coil trimerization domain; Tai LJ et al.; Heat shock factor-binding protein (HSBP) 1 is a small, evolutionarily conserved protein originally identified in a yeast two-hybrid screen using the trimerization domain of heat shock factor (HSF) 1 as the bait . Similar in size to HSF1 trimerization domain, human HSBP1 contains two arrays of hydrophobic heptad repeats (designated HR-N and HR-C) characteristic of coiled-coil proteins . Proteins of the HSBP family are relatively small (<100 residues), comprising solely a putative coiled-coil oligomerization domain without any other readily recognizable structural or functional motif . Our biophysical and biochemical characterization of human HSBP1 reveals a cooperatively folded protein with high alpha-helical content and moderate stability . NMR analyses reveal a single continuous helix encompassing both HR-N and HR-C in the highly conserved central region, whereas the less conserved carboxyl terminus is unstructured and accessible to proteases . Unlike previously characterized coiled-coils, backbone 15N relaxation measurements implicate motional processes on the millisecond time scale in the coiled-coil region . Analytical ultracentrifugation and native PAGE studies indicate that HSBP1 is predominantly trimeric over a wide concentration range . NMR analyses suggest a rotationally symmetric trimer . Because the highly conserved hydrophobic heptad repeats extend over 60% of HSBP1, we propose that HSBP most likely regulates the function of other proteins through coiled-coil interactions.

J Biol Chem, 2001 Dec 28, 276(52), 49034 - 42 Epub 2001 Oct 25.
hTid-1, a human DnaJ protein, modulates the interferon signaling pathway; Sarkar S et al.; The Jak family of protein-tyrosine kinases are crucial for the signaling of a large number of different polypeptide ligands, including the interferons, many cytokines, erythropoietin, and growth factors . Through their interaction with receptors, the Jaks initiate a signaling cascade resulting in the activation of gene transcription and ultimately a cellular response to various ligands . In addition to their role in cellular signaling, alteration of Jak activity has been implicated in several disease states . In identifying Jak2-interacting proteins with the yeast two-hybrid system, we cloned the human homologue of the Drosophila melanogaster tumor suppressor gene lethal () tumorous imaginal discs, which encodes the protein Tid56 . Drosophila Tid56 and its human homologue hTid-1 represent members of the DnaJ family of molecular chaperones . The TID1 gene encodes two splice variants hTid-1(S) and hTid-1(L) . We confirmed the interaction between Jak2 and hTid-1(S) or hTid-1(L) by immunoprecipitation from COS-1 cells expressing these proteins . The interaction between endogenous hTid-1 and Jak2 was shown in HEp2 cells . We further showed that hTid-1 interacts with the human interferon-gamma (Hu-IFN-gamma) receptor subunit IFN-gamma R2 . In addition, using a chimeric construct where the extracellular domain of IFN-gamma R2 was fused to the kinase domain of Jak2, we showed that hTid-1 binds more efficiently to the chimera with an active kinase domain than to a similar construct with an inactive kinase domain . Additionally, the data demonstrate that hTid-1 isoforms as well as Jak2 interact with Hsp70/Hsc70 in vivo, and the interaction between Hsp70/Hsc70 and hTid-1 is reduced after IFN-gamma treatment . Furthermore, both hTid-1(S) and hTid-1(L) can modulate IFN-gamma-mediated transcriptional activity.

Mol Microbiol, 2001 Oct, 42(1), 3 - 12
SEPH, a Cdc7p orthologue from Aspergillus nidulans, functions upstream of actin ring formation during cytokinesis; Bruno KS et al.; In the filamentous fungus, Aspergillus nidulans, multiple rounds of nuclear division occur before cytokinesis, allowing an unambiguous identification of genes required specifically for cytokinesis . As in animal cells, both an intact microtubule cytoskeleton and progression through mitosis are required for actin ring formation and contraction . The sepH gene from A . nidulans was discovered in a screen for temperature-sensitive cytokinesis mutants . Sequence analysis showed that SEPH is 42% identical to the serine-threonine kinase Cdc7p from fission yeast . Signalling through the Septation Initiation Network (SIN), which includes Cdc7p and the GTPase Spg1p, is emerging as a primary regulatory pathway used by fission yeast to control cytokinesis . A similar group of proteins comprise the Mitotic Exit Network (MEN) in budding yeast . This is the first direct evidence for the existence of a functional SIN-MEN pathway outside budding and fission yeast . In addition to SEPH, potential homologues were also identified in other fungi and plants but not in animal cells . Deletion of sepH resulted in a viable strain that failed to septate at any temperature . Interestingly, quantitative analysis of the actin cytoskeleton revealed that sepH is required for construction of the actin ring . Therefore, SEPH is distinct from its counterpart in fission yeast, in which SIN components operate downstream of actin ring formation and are necessary for ring contraction and later events of septation . We conclude that A . nidulans has components of a SIN-MEN pathway, one of which, SEPH, is required for early events during cytokinesis.

Mol Cell Biochem, 2001 Jun, 222(1-2), 205 - 11
Molecular biology of nickel carcinogenesis; Costa M et al.; A review of the molecular mechanisms of nickel carcinogenesis has been compiled . This work is based upon approximately 20 years of research conducted in my laboratory . Molecular mechanisms of nickel carcinogenesis are considered from the point-of-view of the uptake of nickel, both soluble and insoluble particles in cells, its dissolution and its effects on heterochromatin . Molecular mechanisms by which nickel induces gene silencing in cells by DNA hypermethylation in mammalian cells and by inhibiting histone acetylation in yeast cells are also discussed.

Semin Radiat Oncol, 2001 Oct, 11(4), 352 - 72
When X-ray-inducible proteins meet DNA double strand break repair; Leskov KS et al.; Cellular responses to ionizing radiation (IR) include (a) activation of signal transduction enzymes; (b) stimulation of DNA repair, most notably DNA double strand break (DSB) repair by homologous or nonhomologous recombinatorial pathways; (c) activation of transcription factors and subsequent IR-inducible transcript and protein changes; (d) cell cycle checkpoint delays in G(1), S, and G(2) required for repair or for programmed cell death of severely damaged cells; (e) activation of zymogens needed for programmed cell death (although IR is a poor inducer of such responses in epithelial cells); and (f) stimulation of IR-inducible proteins that may mediate bystander effects influencing signal transduction, DNA repair, angiogenesis, the immune response, late responses to IR, and possibly adaptive survival responses . The overall response to IR depends on the cell's inherent genetic background, as well as its ability to biochemically and genetically respond to IR-induced damage . To improve the anti-tumor efficacy of IR, our knowledge of these pleiotropic responses must improve . The most important process for the survival of a tumor cell following IR is the repair of DNA double strand breaks (DSBs) . Using yeast two-hybrid analyses along with other molecular and cellular biology techniques, we cloned transcripts/proteins that are involved in, or presumably affect, nonhomologous DNA double strand break end-joining (NHEJ) repair mediated by the DNA-PK complex . Using Ku70 as bait, we isolated a number of Ku-binding proteins (KUBs) . We identified the first X-ray-inducible transcript/protein (xip8, Clusterin (CLU)) that associates with DNA-PK . A nuclear form of CLU (nCLU) prevented DNA-PK-mediated end joining, and stimulated cell death in response to IR or when overexpressed in the absence of IR . Structure-function analyses using molecular and cellular (including green fluorescence-tagged protein trafficking) biology techniques showed that nCLU appears to be an inactive protein residing in the cytoplasm of epithelial cells . Following IR injury, nCLU levels increase and an as yet undefined posttranslational modification appears to alter the protein, exposing nuclear localization sequences (NLSs) and coiled-coil domains . The modified protein translocates to the nucleus and triggers cell death, presumably through its interaction specifically with Ku70 . Understanding nCLU responses, as well as the functions of the KUBs, will be important for understanding DSB repair . Knowledge of DSB repair may be used to improve the antitumor efficacy of IR, as well as other chemotherapeutic agents .

J Biol Chem, 2001 Dec 21, 276(51), 48100 - 7 Epub 2001 Oct 24.
CCAAT/enhancer-binding protein-beta is a mediator of the nutrient-sensing response pathway that activates the human asparagine synthetase gene; Siu F et al.; Transcription from the human asparagine synthetase (AS) gene is increased in response to either amino acid (amino acid response) or glucose (unfolded protein response) deprivation . These two independent pathways converge on the same set of genomic cis-elements within the AS promoter, which are referred to as nutrient-sensing response element (NSRE)-1 and -2, both of which are absolutely necessary for gene activation . The NSRE-1 sequence was used to identify the corresponding transcription factor by yeast one-hybrid screening . Based on those results, electrophoretic mobility shift assays for individual CCAAT/enhancer-binding protein-beta (C/EBP) family members were performed to test for supershifting of complexes by specific antibodies . The results indicated that of all the family members, C/EBPbeta bound to the NSRE-1 sequence to the greatest extent and that the absolute amount of this complex was increased when extracts from amino acid- or glucose-deprived cells were tested . Using electrophoretic mobility shift assays, mutation of the NSRE-1 sequence completely prevented formation of the C/EBPbeta-containing complexes . In contrast, mutation of the NSRE-2 sequence did not block C/EBPbeta binding . Overexpression in HepG2 hepatoma cells of the activating isoform of C/EBPbeta increased AS promoter-driven transcription, whereas the inhibitory dominant-negative isoform of C/EBPbeta blocked enhanced transcription following amino acid or glucose deprivation . Collectively, the results provide both in vitro and in vivo evidence for a role of C/EBPbeta in the transcriptional activation of the AS gene in response to nutrient deprivation.

Curr Biol, 2001 Oct 16, 11(20), 1595 - 9
mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos; Garner M et al.; Drosophila double park encodes a homolog of Cdt1 that functions in initiation of DNA replication in fission yeast and Xenopus . dup mutants complete the first 15 embryonic cell cycles, presumably via maternal dup products, and show defects in the 16(th) S phase (S16) . Cells carrying dup(a1) allele forgo S16 altogether but enter mitosis 16 (M16) . We find that the timing of entry into M16 is similar in dup(a1) and heterozygous or wild-type (wt) controls . In contrast, we find that mutant cells carrying another allele, dup(a3), undergo a partial S16 and delay the entry into M16 . Thus, initiation of S16 appears necessary for delaying M16 . This delay is absent in double mutants of dup(a3) and mei-41 (Drosophila ATR), indicating that a mei-41-dependent checkpoint acts to delay the entry into mitosis in response to incomplete DNA replication . dup(a3) and dup(a1) mutant cells that enter M16 become arrested in M16 . We find that mitotic cyclins are stabilized and that a spindle checkpoint protein, Bub1, localizes onto chromosomes during mitotic arrest in dup mutants . These features suggest an arrest prior to metaphase-anaphase transition . dup(a3) bub1 double mutant cells exit M16, indicating that a bub1-mediated checkpoint acts to block mitotic exit in dup mutants . To our knowledge, this is the first report of (1) incomplete DNA replication affecting both the entry into and the exit from mitosis in a single cell cycle via different mechanisms and (2) the role of bub1 in regulating mitotic exit in response to incomplete DNA replication.

J Invest Dermatol, 2001 Oct, 117(4), 914 - 9
Induction of apoptosis in melanoma cell lines by p53 and its related proteins; Yamashita T et al.; Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53 . By using recombinant adenoviruses that express p53 or p53-related p51A or p73beta, we tested their apoptotic activities in melanoma cells . Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA {K} instead of GAA {E}) in one cell line, 70W, out of six human melanoma cell lines analyzed (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W, and G361) . Adenovirus-mediated transfer of p53, p51A, and/or p73beta suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells . Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73beta . By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members . Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73beta, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells . Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells . We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73beta . It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members.

Proc Natl Acad Sci U S A, 2001 Oct 23, 98(22), 12602 - 7
P{Switch}, a system for spatial and temporal control of gene expression in Drosophila melanogaster; Roman G et al.; We have developed a method for turning on and off the expression of transgenes within Drosophila in both time and space . Two different enhancer detector elements carrying an RU486-inducible form of the yeast transcription factor GAL4 were constructed and used to generate enhancer detector lines . These lines were screened for RU486-inducible reporter gene expression in the adult head . We identified lines that exhibit inducible expression in many cell and tissue types, verifying that the elements respond to nearby enhancers . No expression was detected in the absence of the ligand . The P{Switch1} element responded to genomic enhancers less efficiently than P{Switch2} but produced more specific patterns of expression . Two P{Switch} lines were used to ablate fat body tissue in adult females through the induced expression of diphtheria toxin . These females were sterile, which correlates with fat body loss, and they died prematurely.

Cancer Genet Cytogenet, 2001 Oct 15, 130(2), 111 - 7
Cytogenetic and fluorescence in situ hybridization characterization of chromosome 8 rearrangements in head and neck squamous cell carcinomas; Jin Y et al.; Structural rearrangements of chromosome 8 are frequently encountered in squamous cell carcinomas of the head and neck (HNSCC) . These aberrations often affect the centromeric region, resulting in the formation of isochromosome i(8q) and whole arm translocations . Some tumors may display structural rearrangements of 8p23 . To characterize further the localization of the breakpoints in such rearrangements, 12 HNSCC known to carry pericentromeric rearrangements of chromosome 8 and 8p23 abnormalities were investigated with fluorescence in situ hybridization (FISH) by the use of 15 YAC clones spanning 8p23 and 8p11 to 8q11 . FISH confirmed that all, except one, aberrations cytogenetically interpreted to be i(8q) were true, monocentric i(8q) . Similarly, all whole-arm translocations appeared as centric fusions . It could thus be concluded that the essential outcome of these rearrangements is genomic imbalances and not rearrangement of genes in the pericentromeric region . By the use of five YAC clones mapping to 8p23, different breakpoints at the molecular level were disclosed in cases with cytogenetically identical 8p23 rearrangements . An evaluation of the genomic imbalances detected in the present series revealed that overrepresentation of 8q material was present in 11 of the 12 tumors . The most commonly gained segment was 8q22 approximately qter, found in all cases with 8q overrepresentation . Loss of parts of or the entire 8p was seen in 10 tumors . The smallest overlapping deleted region was localized to the subtelomeric region of 8p.

J Org Chem, 1999 Jun 25, 64(13), 4914 - 4919
2-Piperidone Type of Chiral Building Block for 3-Piperidinol Alkaloid Synthesis; Toyooka N et al.; An enantiomeric pair of a new 2-piperidone type of chiral building block (1) has been prepared by bakers' yeast reduction of beta-keto ester (2) or lipase-mediated transesterification of hydroxy ester (+/-)-(1), derived from NaBH(4) reduction of 2, in enantiopure form . The absolute stereochemistry of (-)-1 was verified by its conversion to known piperidine (-)-3, an intermediate for the synthesis of (-)-spectaline . The 2-piperidone (-)-1 was converted to all four diastereomers of 2,6-disubstituted 3-piperidinol chiral building blocks on the basis of homologation of (-)-1 at the lactam carbonyl using the Eschenmoser method via corresponding thiolactams (-)-9, (-)-20, (-)-25, (-)-27, and (-)-34, followed by stereocontrolled reduction of the resulting vinylogous urethanes (+)-10, (+)-15, (+)-23, (+)-28, and (+)-32, respectively, and epimerization of the hydroxyls at the 3-position {(-)-16 via (+)-17 to (-)-18 and (+)-29 via (+)-30 to (+)-31} . The versatility of these chiral buliding blocks has been demonstrated by the chiral synthesis of the 3-piperidinol alkaloids (+)-prosafrinine, (-)-iso-6-cassine, (-)-prosophylline, and (-)-prosopinine from (-)-37, (-)-14, (+)-36, and (-)-26, respectively.

Arch Biochem Biophys, 2001 Nov 1, 395(1), 78 - 84
Human sterol 14alpha-demethylase activity is enhanced by the membrane-bound state of cytochrome b(5); Lamb DC et al.; Human sterol 14alpha-demethylase (P45051; CYP51) catalyzes the oxidative removal of the C32 methyl group of dihydrolanosterol, an essential step in the cholesterol biosynthetic pathway . The reaction is dependent upon NADPH cytochrome P450 reductase (CPR) that donates the electrons for the catalytic cycle . Here we used a recombinant yeast CPR to investigate the abilities of four different forms of cytochrome b(5) to support sterol demethylation activity of CYP51 . The cytochrome b(5) derivatives were genetically engineered forms of the native rat cytochrome b(5) core-tail: the soluble globular b(5) core (core), the core linked at its N-terminus with the secretory signal sequence of alkaline phosphatase (signal-core), and the signal sequence linked to the native b(5) (signal-core-tail) . The rat core-tail enzyme greatly stimulated sterol demethylation, whereas the signal-core-tail was only marginally active . In contrast, the core and signal-core constructs were completely inactive in stimulating the demethylation reaction . Additionally, cytochrome b(5) enhanced sterol demethylation by more than threefold by accepting electrons from soluble yeast CPR and in its ability to reduce P450 . We show that the nature of transient linkage between the hemoproteins and the redox partners is most likely brought about electrostatically, although productive interaction between cytochrome b(5) and CYP51 is governed by the membrane-insertable hydrophobic region in the cytochrome b(5) which in turn determines the correct spatial orientation of the core . This is the first report showing the stimulation of CYP51 by cytochrome b(5) .

Plant Cell Physiol, 2001 Oct, 42(10), 1156 - 68
A MADS box gene from lily (Lilium Longiflorum) is sufficient to generate dominant negative mutation by interacting with PISTILLATA (PI) in Arabidopsis thaliana; Tzeng TY et al.; Lily MADS box gene 1 (LMADS1), with sequence homology to the AP3 family of genes, was cloned and characterized from lily (Lilium longiflorum) . LMADS1 protein contains almost complete consensus sequence of the PISTILLATA (PI)-derived motif (YEFRVQPSQPNLH) found in the AP3 family of genes and paleoAP3 motif (YGSHDLRLA) found in the AP3 family of genes from the low eudicot, magnolid dicot and monocot species . LMADS1 mRNA was expressed in all four whorls of the flower and absent in the vegetative leaves . The LMADS1 protein was only detected in the petals and stamens, indicating that LMADS1 is possibly post-transcriptionally regulated in lily . Arabidopsis plants transformed with 35S::LMADS1 produced flowers with short petals and stamens, however, no floral organ conversion was observed . Ectopic expression of LMADS1 cDNA truncated with the MADS box domain in Arabidopsis generated the ap3-like dominant negative mutation in which the petals were converted into sepal-like structures and the stamens were converted into carpel-like structures . Yeast two-hybrid analysis indicated that LMADS1 truncated with the MADS box domain is able to sufficiently interact with the Arabidopsis PI protein . This result supports that LMADS1 is the functional counterpart of the AP3 gene in lily . Interestingly, in contrast to other B functional genes, LMADS1 truncated with the MADS box domain is able to strongly form homodimers . LMADS1 may represent an ancestral form of the B function gene, which retains the ability to form homodimers in regulating petal and stamen development in lily.

Neurology, 2001 Oct 23, 57(8), 1440 - 6
Homozygosity (E140K) in SCO2 causes delayed infantile onset of cardiomyopathy and neuropathy; Jaksch M et al.; OBJECTIVE: To report three unrelated infants with a distinctive phenotype of Leigh-like syndrome, neurogenic muscular atrophy, and hypertrophic obstructive cardiomyopathy . The patients all had a homozygous missense mutation in SCO2 . BACKGROUND: SCO2 encodes a mitochondrial inner membrane protein, thought to function as a copper transporter to cytochrome c oxidase (COX), the terminal enzyme of the respiratory chain . Mutations in SCO2 have been described in patients with severe COX deficiency and early onset fatal infantile hypertrophic cardioencephalomyopathy . All patients so far reported are compound heterozygotes for a missense mutation (E140K) near the predicted CxxxC metal binding motif; however, recent functional studies of the homologous mutation in yeast failed to demonstrate an effect on respiration . METHODS: Here we present clinical, biochemical, morphologic, functional, MRI, and MRS data in two infants, and a short report in an additional patient, all carrying a homozygous G1541A transition (E140K) . RESULTS: The disease onset and symptoms differed significantly from those in compound heterozygotes . MRI and muscle morphology demonstrated an age-dependent progression of disease with predominant involvement of white matter, late appearance of basal ganglia lesions, and neurogenic muscular atrophy in addition to the relatively late onset of hypertrophic cardiomyopathy . The copper uptake of cultured fibroblasts was significantly increased . CONCLUSIONS: The clinical spectrum of SCO2 deficiency includes the delayed development of hypertrophic obstructive cardiomyopathy and severe neurogenic muscular atrophy . There is increased copper uptake in patients' fibroblasts indicating that the G1541A mutation effects cellular copper metabolism.

J Biol Chem, 2002 Jan 4, 277(1), 535 - 43 Epub 2001 Oct 22.
The EWS/NOR1 fusion gene product gains a novel activity affecting pre-mRNA splicing; Ohkura N et al.; In extraskeletal myxoid chondrosarcoma, chromosomal translocation creates a gene fusion between EWS and the orphan nuclear receptor NOR1 . The resulting fusion gene product, EWS/NOR1, has been believed to lead to malignant transformation by functioning as a transcriptional activator, but an alternative mechanism may also be involved . Here, using a newly developed functional complementation screening in yeast, we found that EWS/NOR1, but not EWS or NOR1, complemented the loss of function of the small nuclear ribonucleoprotein Snu23p, an essential factor for pre-mRNA splicing in yeast . To verify the potential function of EWS/NOR1 in mammalian cells, we next showed that overexpression of EWS/NOR1 caused increased usage of the distal 5'-splice site of pre-mRNA splicing and that EWS/NOR1 interacted with the human splicing protein U1C; neither EWS nor NOR1 had the same activity or interaction as EWS/NOR1 . Altogether, our findings reveal that EWS/NOR1 gains a novel activity affecting pre-mRNA splicing.

J Biol Chem, 2002 Feb 15, 277(7), 4609 - 17 Epub 2001 Oct 22.
A dominant-negative mutant of androgen receptor coregulator ARA54 inhibits androgen receptor-mediated prostate cancer growth; Miyamoto H et al.; The ligand-bound androgen receptor (AR) regulates target genes via a mechanism involving coregulators such as androgen receptor-associated 54 (ARA54) . We investigated whether the interruption of the AR coregulator function could lead to down-regulation of AR activity . Using in vitro mutagenesis and a yeast two-hybrid screening assay, we have isolated a mutant ARA54 (mt-ARA54) carrying a point mutation at amino acid 472 changing a glutamic acid to lysine, which acts as a dominant-negative inhibitor of AR transactivation . In transient transfection assays of prostate cancer cell lines, the mt-ARA54 suppressed endogenous mutated AR-mediated and exogenous wild-type AR-mediated transactivation in LNCaP and PC-3 cells, respectively . In DU145 cells, the mt-ARA54 suppressed exogenous ARA54 but not other coregulators, such as ARA55-enhanced or SRC-1-enhanced AR transactivation . In the LNCaP cells stably transfected with the plasmids encoding the mt-ARA54 under the doxycycline inducible system, the overexpression of the mt-ARA54 inhibited cell growth and endogenous expression of prostate-specific antigen . Mammalian two-hybrid assays further demonstrated that the mt-ARA54 can disrupt the interaction between wild-type ARA54 molecules, suggesting that ARA54 dimerization or oligomerization may play an essential role in the enhancement of AR transactivation . Together, our results demonstrate that a dominant-negative AR coregulator can suppress AR transactivation and cell proliferation in prostate cancer cells . Further studies may provide a new therapeutic approach for blocking AR-mediated prostate cancer growth.

J Biol Chem, 2002 Jan 11, 277(2), 896 - 906 Epub 2001 Oct 22.
A Trypanosoma brucei protein complex that binds G-overhangs and co-purifies with telomerase activity; Cano MI et al.; The chromosomal ends of Trypanosoma brucei, like those of most eukaryotes, contain conserved 5'-TTAGGG-3' repeated sequences and are maintained by the action of telomerase . Fractionated T . brucei cell extracts with telomerase activity were used as a source of potential regulatory factors or telomerase-associated components that might interact with T . brucei telomeres . Electrophoretic mobility shift assays and UV cross-linking were used to detect possible single-stranded telomeric protein.DNA complexes and to estimate the approximate size of the protein constituents . Three single-stranded telomeric protein.DNA complexes were observed . Complex C3 was highly specific for the G-strand telomeric repeat sequence and shares biochemical characteristics with G-rich, single-stranded telomeric binding proteins and with components of the telomerase holoenzyme described in yeast, ciliates, and humans . Susceptibility to RNase A or chemical nuclease (hydroxyl radical) pre-treatment showed that complex C3 was tightly associated with an RNA component . Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to estimate the molecular mass of the peptides obtained by in-gel Lys-C digestion of low abundance C3-associated proteins . The molecular masses of the peptides showed no homologies with other proteins from trypanosomes or with any protein in the data bases screened.

Biol Reprod, 2001 Nov, 65(5), 1437 - 43
A functional composite cis-element for NF kappa b and RBJ kappa in the rat pregnancy-specific glycoprotein gene; Wang CD et al.; The rat pregnancy-specific glycoprotein gene rnCGM3 is primarily expressed in the placenta . Previously, three DNase I footprinting sites (FPI, FPII, and FPIII) were identified in the rnCGM3 promoter region, a yeast one-hybrid screen was performed to identify the nuclear factors binding to the FPIII (5'-GCCTGGGAAAAAACTC-3') element, and RBPJ kappa, a downstream effector of the Notch signaling pathway, was identified as one of the FPIII-binding factors . In the present study, the NF kappa B member p65 was identified as another FPIII-binding factor . Electrophoretic mobility shift assays showed that NF kappa B members, including p50 and p65, bound to the FPIII site . The core binding sequence in the FPIII element for p50 and p65 is GGGAAA, which overlaps with that for RBPJ kappa . Competition exists between p50 and RBPJ kappa for binding to the FPIII element . Transient expression analyses revealed that p65 significantly stimulated the expression of a reporter gene directed by the NF kappa B core sequence in the FPIII element . However, RBPJ kappa could block this stimulation . These results suggest that the regulation of rnCGM3 expression involves both NF kappa B and RBPJ kappa, and they are mutually exclusive in the FPIII element.

Cancer Genet Cytogenet, 2001 Oct 1, 130(1), 75 - 8
FISH characterization of t(8;12)(q12;p13) observed as the sole karyotypic anomaly in a myelodysplastic syndrome patient; Finelli P et al.; We report a t(8;12)(q12; p13) as the sole cytogenetic anomaly in a patient with a myelodysplastic syndrome (MDS) . By means of FISH, we mapped the genomic region involved in the breakpoint (bkp) on both chromosomes . The 12p13 bkp mapped between markers WI-664 and WI-9218, immediately distal to the breakpoint cluster region frequently involved in hematological neoplasms targeted by y964C10 . The 8q12 bkp (not yet investigated by FISH) was characterized and found to occur between markers WI-3263 and D8S524 within the region recognized by y874E10.

Biochem J, 2001 Nov 1, 359(Pt 3), 591 - 7
N-terminal truncation affects the kinetics and structure of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana; Villadsen D et al.; The enzyme fructose-6-phosphate 2-kinase (F6P,2K; 6-phosphofructo-2-kinase)/fructose-2,6-bisphosphatase (F26BPase) catalyses the formation and degradation of the regulatory metabolite fructose 2,6-bisphosphate . A cDNA encoding the bifunctional plant enzyme isolated from Arabidopsis thaliana (AtF2KP) was expressed in yeast, and the substrate affinities and allosteric properties of the affinity-purified enzyme were characterized . In addition to the known regulators 3-phosphoglycerate, dihydroxyacetone phosphate, fructose 6-phosphate and P(i), several metabolites were identified as important new effectors . PP(i), phosphoenolpyruvate and 2-phosphoglycerate strongly inhibited F6P,2K activity, whereas fructose 1,6-bisphosphate and 6-phosphogluconate inhibited F26BPase activity . Furthermore, pyruvate was an activator of F6P,2K and an inhibitor of F26BPase . Both kinase and phosphatase activities were rapidly inactivated by mild heat treatment (42 degrees C, 10 min), but the presence of phosphate protected both enzyme activities from inactivation . In addition to the catalytic regions, the Arabidopsis enzyme comprises a 345-amino-acid N-terminus of unknown function . The role of this region was examined by the expression of a series of N-terminally truncated enzymes . The full-length and truncated enzymes were analysed by gel-filtration chromatography . The full-length enzyme was eluted as a homotetramer, whereas the truncated enzymes were eluted as monomers . Deletion of the N-terminus decreased the kinase/phosphatase activity ratio by 4-fold, and decreased the affinity for the substrate fructose 6-phosphate . The data show that the N-terminus is important both for subunit assembly and for defining the kinetic properties of the enzyme.

Biochem J, 2001 Nov 1, 359(Pt 3), 485 - 96
LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1; Weiskirchen R et al.; Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts . The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis . In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells . The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2 . During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo . The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex . To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen . The protein inhibitor of activated STAT1 ('PIAS1') was shown to associate selectively with the C-terminal LIM domain of CRP2 . Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis . These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells.

Plant Mol Biol, 2001 Nov, 47(4), 461 - 74
Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development; Hei U et al.; A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized . The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP) . VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family . We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL . During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells . At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles . However, mRNA localization in stage VI cotyledons during the prestorage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization . The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene . The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation . Our data suggest different functions of VfSBPL during seed development.

Int J Cancer, 2001 Oct 1, 94(1), 35 - 43
Restoration of endogenous wild-type p53 activity in a glioblastoma cell line with intrinsic temperature-sensitive p53 induces growth arrest but not apoptosis; Ikeda J et al.; p53 protein is a transcription factor involved in multiple tumor-suppressor activities including cell cycle control and apoptosis . TP53 gene is frequently mutated in glioblastoma, suggesting the importance of inactivation of this gene product in gliomagenesis . Restoration of p53 function in glioblastoma cell lines deficient for p53 has shown that p53 induces growth arrest or apoptosis depending on the cell line and vector used to transduce wild-type TP53 alleles . Considering that astrocytes grow and express p53, it is not clear whether these results reflect physiologic responses or the result of p53 overexpression in combination with cellular responses to viral vector infection . Here, we reassessed this issue using a glioblastoma cell line (LN382) that expresses an endogenous temperature-sensitive mutant p53 . This cell line expresses TP53 alleles (100% as determined by a p53 transcriptional assay in yeast) mutated at codon 197 GTG (Val) > CTG (Leu) . We found that the p53 protein in these cells acted as an inactive mutant at 37 degrees C and as a functional wild-type p53 below 34 degrees C as demonstrated by several lines of evidence, including (i) restoration of transactivating ability in yeast, (ii) induction of p53-modulated genes such as CDKN1(p21) and transforming growth factor-alpha, (iii) disappearance of accumulated p53 protein in the nucleus and (iv) decrease in steady state p53 protein levels . This temperature switch allowed p53 levels, which were close to physiological levels to dramatically reduce LN382 cell proliferation by inducing a G(1)/S cell cycle block, but not to induce apoptosis . The lack of apoptosis was considered to be a result of the low level p53 expression, because increasing wild-type p53 levels by adenoviral-mediated gene transfer caused apoptosis in these cells . The LN382 cell line will be extremely useful for investigations into the roles of p53 in cellular responses to a variety of stimuli or damages .

J Org Chem, 1996 Nov 15, 61(23), 8010 - 8015
Cuprate-Mediated Synthesis and Biological Evaluation of Cyclopropyl- and tert-Butylfarnesyl Diphosphate Analogs; Mu Y et al.; The novel farnesyl diphosphate (FPP) analog 3-cyclopropyl-3-desmethylfarnesyl diphosphate (3-cpFPP, 1) was designed as a potential mechanism-based inhibitor of the FPP-utilizing enzyme protein-farnesyl transferase (PFTase) . The key step in the synthesis of 1 involved the stereoselective coupling of vinyl triflate 8 with a lower order cyclopropyl cyanocuprate to afford the desired cyclopropyl ester 13 . The sterically encumbered analog 3-desmethyl-3-tert-butylfarnesyl diphosphate (3-tbFPP, 7) was synthesized via a similar route . The use of the more reactive higher order tert-butyl cyanocuprate led to lower yields of ester 11, the key intermediate in the synthesis of 7 . Biological evaluation of 3-cpFPP demonstrates that it is not a time-dependent inhibitor of recombinant yeast PFTase . Instead, 3-cpFPP is an alternative substrate for this enzyme that exhibits a K(m) comparable to FPP and a k(cat) only 5-fold lower than the natural substrate . In contrast, 3-tbFPP is an exceptionally poor substrate for yeast PFTase and acts as an inhibitor of this enzyme.

J Org Chem, 1996 Sep 6, 61(18), 6296 - 6301
Synthesis of Protein Farnesyltransferase and Protein Geranylgeranyltransferase Inhibitors: Rapid Access to Chaetomellic Acid A and Its Analogues; Ratemi ES et al.; A facile two-step stereospecific synthesis of the protein farnesyltransferase inhibitor chaetomellic acid A (1) and its analogues was developed . Addition of organocuprates derived from Grignard reagents (e.g . tetradecylmagnesium chloride and CuBr.Me(2)S) to dimethyl acetylenedicarboxylate (DMAD) in tetrahydrofuran containing hexamethylphosphoramide was followed by capture of the resulting copper enolates with a variety of electrophiles (e.g . methyl iodide) to give dimethyl cis-butenedioate derivatives 4-11 . Hydrolysis with lithium hydroxide generated the corresponding lithium carboxylates, which readily closed to 2,3-disubstituted maleic anhydrides 17-20 upon acid treatment . Compound 16, an analogue wherein the tetradecyl group of 1 is replaced by a farnesyl moiety, is 7-fold more potent than 1 as an inhibitor of protein farnesyltransferase from yeast and displays a 100:1 selectivity for this enzyme relative to yeast protein geranylgeranyltransferase . In contrast, analogue 15, which contains a geranylgeranyl side chain, shows ca . 10:1 selectivity for the latter enzyme.

Science . 1984 Sep 14;225(4667):1134.
Cohen-Boyer patent finally issued; Norman C; KIE: On 28 August 1984, the Patent Office finally approved a second Cohen-Boyer patent which covers hybrid bacterial plasmids . This patent, together with an earlier one covering methods, provides the University of California, San Francisco, and Stanford University with proprietary control over the basic techniques and tools used in gene splicing . The Patent Office rejected a claim by an alleged third co-inventor because of doubts about whether information in the patent application would enable the plasmids to be duplicated . The universities accepted the patent's restriction to bacterial plasmids and will pursue a separate application for yeast plasmids .

Mamm Genome, 2001 Sep, 12(9), 734 - 40
Genetic analysis of the organic cation transporter genes Orct2/Slc22a2 and Orct3/Slc22a3 reduces the critical region for the t haplotype mutant t(w73) to 200 kb; Zwart R et al.; Here we report an analysis of two candidate genes for the t(w73) implantation mutation . The t(w73) gene maps to a 20-cM region of mouse Chromosome (Chr) 17 known as the t-complex, which exists in a wild-type and t haplotype form in present-day mice . The t haplotype variants contain several mutant alleles affecting male fertility and embryonic viability and offer the opportunity to identify genes critical for these processes . t(w73) homozygous embryos are defective in trophoblast production and fail to implant adequately, with death occurring at approximately 7.5 days post coitum (pc) . Two recently described organic cation transporter genes, Slc22a2 (Orct2) and Slc22a3 (Orct3), fulfill criteria predicted for t(w73) candidate genes, since both map to the previously defined 500-kb t(w73) minimal region and both are also expressed in 7.5 days pc post-implantation embryos . The genomic locus of the Orct2 gene appears similar in wild-type and t(w73) chromosomes . In contrast, the genomic locus of Orct3 is amplified and displays an altered expression profile in all t haplotype variant chromosomes tested . In addition, Orct3 shows a t(w73) specific polymorphism . To test whether either Orct2 or Orct3 is involved in the t(w73) phenotype, we have performed a genetic rescue experiment using YAC transgenes overexpressing Orct2, and genetic complementation with an allele in which the Orct3 gene was inactivated by homologous recombination . The results eliminate both Orct2 and Orct3 as candidates and further reduce the critical region containing the t(w73) mutant from 500 kb to 200 kb.

Mamm Genome, 2001 Sep, 12(9), 719 - 23
Generation and exploration of a dense genetic map in a region of a QTL affecting corpora lutea in a Meishan x Yorkshire cross; Braunschweig MH et al.; Previously genomic scans revealed quantitative trait loci (QTL) on porcine Chromosome 8 (SSC8) as significantly affecting the number of corpora lutea (CL) in swine . In one study, statistical evidence for the putative QTL was found in the chromosomal region defined by the microsatellites (MS) SW205, SW444, SW206, and SW29 . A Yeast Artificial Chromosome library was screened by using the corresponding primers for clones containing these MS by PCR . From five positive YAC clones, 10 additional MS were isolated and mapped to SSC8 with the INRA-University of Minnesota porcine Radiation Hybrid (IMpRH) panel . The genetic map position of the QTL has been refined by addition of these 10 markers . The QTL evaluation included pedigrees of F2-intercross Meishan x Yorkshire design, with phenotypic data of 108 F2 female offspring and genotypic data for 29 MS markers on SSC8 . The analysis was performed by using the least squares regression method . The calculated QTL effect for CL obtained by the multilocus least squares method showed a maximum test statistic (F value = 13.98) at position 99 cM between three MS derived from YACs containing SW205 and SW1843 spanning an interval of 7.1 cM . The point-wise (nominal) P-value was 5.21 x 10-6 corresponding to a genome-wide P-value of 0.009 . The additive QTL effect explained 17.4% of the phenotypic variance.

J Biol Chem, 2002 Jan 4, 277(1), 816 - 22 Epub 2001 Oct 18.
Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation; D'Alonzo RC et al.; Previously, we determined that the activator protein-1 (AP-1)-binding site and the runt domain (RD)-binding site and their binding proteins, c-Fos.c-Jun and Cbfa, regulate the collagenase-3 promoter in parathyroid hormone-treated and differentiating osteoblasts . Here we show that Cbfa1 and c-Fos.c-Jun appear to cooperatively bind the RD- and AP-1-binding sites and form ternary structures in vitro . Both in vitro and in vivo co-immunoprecipitation and yeast two-hybrid studies further demonstrate interaction between Cbfa1 with c-Fos and c-Jun in the absence of phosphorylation and without binding to DNA . Additionally, only the runt domain of Cbfa1 was required for interaction with c-Jun and c-Fos . In mammalian cells, overexpression of Cbfa1 enhanced c-Jun activation of AP-1-binding site promoter activity, demonstrating functional interaction . Finally, insertion of base pairs that disrupted the helical phasing between the AP-1- and RD-binding sites also inhibited collagenase-3 promoter activation . Thus, we provide direct evidence that Cbfa1 and c-Fos.c-Jun physically interact and cooperatively bind the AP-1- and RD-binding sites in the collagenase-3 promoter . Moreover, the AP-1- and RD-binding sites appear to be organized in a specific required helical arrangement that facilitates transcription factor interaction and enables promoter activation.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3373 - 6
Analysis of metabolic pathways by the growth of cells in the presence of organic solvents; Spinnler HE et al.; A new approach to the analysis of metabolic pathways involving poorly water-soluble intermediates is proposed . It relies upon the ability of the hydrophobic intermediates formed by a sequence of intracellular reactions to cross the membrane(s) and partition between aqueous and organic phases, when cells are incubated in the presence of a nonpolar and nontoxic organic solvent . As a result of this thermodynamically driven efflux of the formed intermediates from the cell, they accumulate in the organic medium in sufficient quantities for GC-MS analysis and identification . This enables direct determination of the sequence of chemical reactions involved with no requirement for the isolation of each individual metabolite from a cell-free extract . The feasibility of the proposed methodology has been demonstrated by the elucidation of the biosynthesis of (R)-gamma-decalactone from (R)-ricinoleic acid catalyzed by the yeast Sporidiobolus ruinenii grown in the presence of decane . The corresponding 4-hydroxy-acid intermediates, formed in the course of beta-oxidation of (R)-ricinoleic acid, were simultaneously observed in a single experiment on the same chromatogram . Potential applications of this proposed methodology are briefly discussed.

Proc Natl Acad Sci U S A, 1991 Oct 15, 88(20), 9320 - 4
Nuclear scaffolds and scaffold-attachment regions in higher plants; Hall G Jr et al.; DNA in the nuclei of eukaryotic organisms undergoes a hierarchy of folding to be packaged into interphase and metaphase chromosomes . The first level of packaging is the 11-nm nucleosome fiber, which is further coiled into a 30-nm fiber . Evidence from fungal and animal systems reveals the existence of higher order packaging consisting of loops of the 30-nm fibers attached to a proteinaceous nuclear scaffold by an interaction between the scaffold and specific DNA sequences called scaffold-attachment regions (SARs) . Support for the ubiquitous nature of such higher order packaging of DNA is presented here by our work with plants . We have isolated scaffolds from tobacco nuclei using buffers containing lithium diiodosalicylate to remove histones and then using restriction enzymes to remove the DNA not closely associated with the scaffold . We have used Southern hybridization to show that the DNA remaining bound to the scaffolds after nuclease digestion includes SARs flanking three root-specific tobacco genes . This assay for SARs is termed the endogenous assay because it identifies genomic sequences as SARs by their endogenous association with the scaffold . Another assay, the exogenous assay, depends upon the ability of scaffolds to specifically bind exogenously added DNA fragments containing SARs . The tobacco scaffolds specifically bind a well-characterized yeast SAR, and cloned DNA fragments derived from the 3'-flanking regions of the root-specific genes are confirmed to contain SARs by this exogenous assay.

J Biol Chem, 2002 Jan 11, 277(2), 1531 - 7 Epub 2001 Oct 17.
Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action; Aguirre V et al.; Serine phosphorylation of insulin receptor substrate-1 (IRS-1) inhibits insulin signal transduction in a variety of cell backgrounds, which might contribute to peripheral insulin resistance . However, because of the large number of potential phosphorylation sites, the mechanism of inhibition has been difficult to determine . One serine residue located near the phosphotyrosine-binding (PTB) domain in IRS-1 (Ser(307) in rat IRS-1 or Ser(312) in human IRS-1) is phosphorylated via several mechanisms, including insulin-stimulated kinases or stress-activated kinases like JNK1 . During a yeast tri-hybrid assay, phosphorylation of Ser(307) by JNK1 disrupted the interaction between the catalytic domain of the insulin receptor and the PTB domain of IRS-1 . In 32D myeloid progenitor cells, phosphorylation of Ser(307) inhibited insulin stimulation of the phosphatidylinositol 3-kinase and MAPK cascades . These results suggest that inhibition of PTB domain function in IRS-1 by phosphorylation of Ser(307) (Ser(312) in human IRS-1) might be a general mechanism to regulate insulin signaling.

J Biol Chem, 2001 Dec 14, 276(50), 46693 - 6 Epub 2001 Oct 17.
Identification of a conserved archaeal RNA polymerase subunit contacted by the basal transcription factor TFB; Magill CP et al.; Archaea possess two general transcription factors that are required to recruit RNA polymerase (RNAP) to promoters in vitro . These are TBP, the TATA-box-binding protein and TFB, the archaeal homologue of TFIIB . Thus, the archaeal and eucaryal transcription machineries are fundamentally related . In both RNAP II and archaeal transcription systems, direct contacts between TFB/TFIIB and the RNAP have been demonstrated to mediate recruitment of the polymerase to the promoter . However the subunit(s) directly contacted by these factors has not been identified . Using systematic yeast two-hybrid and biochemical analyses we have identified an interaction between the N-terminal domain of TFB and an evolutionarily conserved subunit of the RNA polymerase, RpoK . Intriguingly, homologues of RpoK are found in all three nuclear RNA polymerases (Rpb6) and also in the bacterial RNA polymerase (omega-subunit).

J Med Chem, 2001 Oct 25, 44(22), 3622 - 31
Synthesis of sulfaphenazole derivatives and their use as inhibitors and tools for comparing the active sites of human liver cytochromes P450 of the 2C subfamily; Ha-Duong NT et al.; Twenty-three new derivatives of sulfaphenazole (SPA) were synthesized to further explore the topology of the active sites of human liver cytochromes P450 of the 2C subfamily and to find new selective inhibitors of these cytochromes . These compounds are derived from SPA by replacement of the NH(2) and H (of the SO(2)NH function) substituents of SPA with various R(1) and R(2) groups, respectively . Their inhibitory effects were studied on recombinant CYP 2C8, 2C9, 2C18, and 2C19 expressed in yeast . High affinities for CYP 2C9 (IC(50) < 1 microM) were only observed for SPA derivatives having the SO(2)NH function and a relatively small R(1) substituent (R(1) = NH(2), CH(3)) . Any increase in the size of R(1) led to a moderate decrease of the affinity, and the N-alkylation of the SO(2)NH function of SPA to a greater decrease of this affinity . The same structural changes led to opposite effects on molecular recognition by CYP 2C8 and 2C18, which generally exhibited similar behaviors . Thus, contrary to CYP 2C9, CYP 2C8 and 2C18 generally prefer neutral compounds with relatively large R(1) and R(2) substituents . CYP 2C19 showed an even lower affinity for anionic compounds than CYP 2C8 and 2C18 . However, as CYP 2C8 and 2C18, CYP 2C19 showed a much better affinity for neutral compounds derived from N-alkylation of SPA and for anionic compounds bearing a larger R(1) substituent . One of the new compounds (R(1) = methyl, R(2) = propyl) inhibited all human CYP 2Cs with IC(50) values between 10 and 20 microM, while another one (R(1) = allyl, R(2) = methyl) inhibited all CYP 2Cs except CYP 2C9, and a third one (R(1) = R(2) = methyl) inhibited all CYP 2Cs except CYP 2C8 . Only 2 compounds of the 25 tested derivatives were highly selective toward one human CYP 2C; these are SPA and compound 1 (R(1) = CH(3), R(2) = H), which acted as selective CYP 2C9 inhibitors . However, some SPA derivatives selectively inhibited CYP 2C8 and 2C18 . Since CYP 2C18 is hardly detectable in human liver, these derivatives could be interesting molecules to selectively inhibit CYP 2C8 in human liver microsomes . Thus, compound 11 (R(1) = NH(2), R(2) = (CH(2))(2)CH(CH(3))(2)) appears to be particularly interesting for that purpose as its IC(50) value for CYP 2C8 is low (3 microM) and 20-fold smaller than those found for CYP 2C9 and 2C19.

Biochem Biophys Res Commun, 2001 Oct 26, 288(2), 420 - 6
Apoptosis-linked gene 2 binds to the death domain of Fas and dissociates from Fas during Fas-mediated apoptosis in Jurkat cells; Jung YS et al.; Apoptosis-linked gene 2 (ALG-2) is a member of the family of Ca(2+)-binding proteins with penta-EF-hand and is essential for the execution of apoptosis by various signals including Fas activation . We studied the regulation of ALG-2 during Fas-mediated apoptosis in Jurkat cells . The 22-kDa ALG-2 protein is cleaved and becomes a 19-kDa protein after Fas activation . The appearance of 19-kDa ALG-2 protein increases for 4 h after treatment with 200 ng/ml of anti-Fas Ab treatment and gradually degrades afterward . Confocal microscopic analysis showed that ALG-2 translocated from the plasma membrane to the cytosol during Fas-mediated apoptosis . Therefore, we examined if ALG-2 interacts with Fas . The protein-protein interaction of ALG-2 with Fas was demonstrated using yeast two-hybrid assays as well as in vitro GST pull-down assay . Endogenous ALG-2 was immunoprecipitated with anti-Fas Ab in Jurkat cells without Fas activation . However, the endogenous ALG-2 was no longer immunoprecipitated with anti-Fas Ab 2 h after anti-Fas Ab treatment . This study, for the first time, presents a direct molecular connection of ALG-2 to apoptosis by its direct interaction with Fas, and enlists ALG-2 as a new member of posttranslationally modified proteins during Fas-mediated apoptotic process .

J Microbiol Immunol Infect, 2001 Sep, 34(3), 171 - 7
Factors accounting for misidentification of Candida species; Lo HJ et al.; From April 15 through June 15, 1999, a total of 660 yeast isolates were collected from 22 hospitals in Taiwan to investigate factors determining the accuracy of yeast identification . The germ tube test was the method most frequently used by hospitals for yeast identification, followed by the API-32C, cornmeal agar window test, and assimilation method . All of the submitted isolates were re-speciated in the National Health Research Institutes laboratory . The frequencies of inconsistent identification of isolates between hospitals and the National Health Research Institutes laboratory varied with the location and the type of hospital . The sensitivity and specificity of the germ tube test were 95% and 98.6%, respectively . This study showed that hospitals using the germ tube test as the first step in yeast identification had fewer inconsistent identifications of isolates than those using other methods . The VITEK Yeast Biochemical Card and API-32C had a sensitivity of 92.6% and 98.3%, respectively . No single method consistently identified all yeast isolates . Thus, every laboratory should have at least 2 methods available for yeast identification.

Prikl Biokhim Mikrobiol, 2001 Sep-Oct, 37(5), 573 - 7
{Formation of xylitol in Candida guilliermondii 2581 culture}; Zagustina NA et al.; The yeast strain Candida guilliermondii 2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose . The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed . The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm . It was shown that the growth under conditions of limited aeration favors the reduction of xylose.

Int J Mol Med, 2001 Nov, 8(5), 513 - 20
Molecular cloning and sequencing of the cDNA coding for a novel regucalcin gene promoter region-related protein in rat, mouse and human liver; Misawa H et al.; The molecular cloning and sequencing of the cDNA coding for a novel regucalcin gene promoter region-related protein (RGPR) was investigated using rat, mouse and human liver cDNA library with a yeast one-hybrid system and a rapid amplification of cDNA ends (RACE) method . The clone coding an unknown protein was isolated, and a novel protein was identified . This protein was termed as RGPR-p117 . RGPR-p117 in rat, mouse and human liver consisted of 1058, 1051 and 1060 amino acid residues with calculated molecular mass of 117, 115 and 117 kDa and estimated pI of 5.69, 5.70 and 5.71, respectively . The homologies of amino acids among rat, mouse and human RGPR-p117 were at least 70% . RGPR-p117 had a leucine zipper motif . The expression of RGPR-p117 mRNA was found in the liver, kidney, heart, spleen, and brain of rats . The database search of the human RGPR-p117 showed that its gene consisted of at least 26 exons spanning approximately 4.1 kbp and localized on human chromosome 1q25.2 . Furthermore, we found a cDNA clone which was highly identical to a front half part of the human RGPR-p117 cDNA, using the BLAST search of human RGPR-p117 . This cDNA clone was a splicing variant of human RGPR-p117, which derived from human placental choriocarcinoma . Our study demonstrates that a novel gene coding RGPR-p117 is present in rat, mouse and human.

Mol Cell Biol, 2001 Nov, 21(22), 7775 - 86
N-terminal domains of the human telomerase catalytic subunit required for enzyme activity in vivo; Armbruster BN et al.; Most tumor cells depend upon activation of the ribonucleoprotein enzyme telomerase for telomere maintenance and continual proliferation . The catalytic activity of this enzyme can be reconstituted in vitro with the RNA (hTR) and catalytic (hTERT) subunits . However, catalytic activity alone is insufficient for the full in vivo function of the enzyme . In addition, the enzyme must localize to the nucleus, recognize chromosome ends, and orchestrate telomere elongation in a highly regulated fashion . To identify domains of hTERT involved in these biological functions, we introduced a panel of 90 N-terminal hTERT substitution mutants into telomerase-negative cells and assayed the resulting cells for catalytic activity and, as a marker of in vivo function, for cellular proliferation . We found four domains to be essential for in vitro and in vivo enzyme activity, two of which were required for hTR binding . These domains map to regions defined by sequence alignments and mutational analysis in yeast, indicating that the N terminus has also been functionally conserved throughout evolution . Additionally, we discovered a novel domain, DAT, that "dissociates activities of telomerase," where mutations left the enzyme catalytically active, but was unable to function in vivo . Since mutations in this domain had no measurable effect on hTERT homomultimerization, hTR binding, or nuclear targeting, we propose that this domain is involved in other aspects of in vivo telomere elongation . The discovery of these domains provides the first step in dissecting the biological functions of human telomerase, with the ultimate goal of targeting this enzyme for the treatment of human cancers.

Neuron, 2001 Oct 11, 32(1), 13 - 23
Identification of a novel tetramerization domain in large conductance K(ca) channels; Quirk JC et al.; More than 50 genes are known to encode K(+) channel monomers and can coassemble to form hetero-tetrameric K(+) channels . However, only a subset of possible monomer combinations come together to form functional ion channels . The assembly and tetramerization of appropriate channel monomers is mediated by association domains (ADs) . To identify such domains in human large-conductance Ca(2+)-activated K(+) channels (hSlo1), we screened hSlo1 domains for self-association using yeast two-hybrid assays . Putative ADs were subjected to functional assays in Xenopus oocytes and further characterized by coprecipitation, native gel electrophoresis, and sucrose density gradient centrifugation assays . This led to the identification of a single intracellular association domain localized near the channel pore and required for channel function . We conclude that this novel tetramerization domain, referred to as BK-T1, promotes the assembly of hSlo1 monomers into functional K(Ca) channels.

BMC Genomics . 2001;2(1):6 . Epub 2001 Sep 26.
Characterization of the mouse Dazap1 gene encoding an RNA-binding protein that interacts with infertility factors DAZ and DAZL; Dai T et al.; BACKGROUND: DAZAP1 (DAZ Associated Protein 1) was originally identified by a yeast two-hybrid system through its interaction with a putative male infertility factor, DAZ (Deleted in Azoospermia) . In vitro, DAZAP1 interacts with both the Y chromosome-encoded DAZ and an autosome-encoded DAZ-like protein, DAZL . DAZAP1 contains two RNA-binding domains (RBDs) and a proline-rich C-terminal portion, and is expressed most abundantly in the testis . To understand the biological function of DAZAP1 and the significance of its interaction with DAZ and DAZL, we isolated and characterized the mouse Dazap1 gene, and studied its expression and the subcellular localization of its protein product . RESULTS: The human and mouse genes have similar genomic structures and map to syntenic chromosomal regions . The mouse and human DAZAP1 proteins share 98% identity and their sequences are highly similar to the Xenopus orthologue Prrp, especially in the RBDs . Dazap1 is expressed throughout testis development . Western blot detects a single 45 kD DAZAP1 protein that is most abundant in the testis . Although a majority of DAZAP1 is present in the cytoplasmic fraction, they are not associated with polyribosomes . CONCLUSIONS: DAZAP1 is evolutionarily highly conserved . Its predominant expression in testes suggests a role in spermatogenesis . Its subcellular localization indicates that it is not directly involved in mRNA translation.

Radiat Res, 2001 Nov, 156(5 Pt 2), 642 - 7
The role of ATM in telomere structure and function; Pandita TK; Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder with a wide variety of phenotypic manifestations . AT patients are cancer prone and hypersensitive to ionizing radiation . Cells derived from AT patients require higher levels of serum factors, exhibit cytoskeletal defects, and undergo premature senescence in culture . The gene responsible for AT is ATM (ataxia-telangiectasia mutated), and its product has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, and cell cycle control . Because of the homology of the human ATM gene to the TEL1 and rad3 genes of yeast, it has been suggested that mutations in ATM could lead to defective telomere maintenance . The ATM gene product influences chromosome end associations, telomere length, and telomere clustering . The defective telomere metabolism in AT cells could be due to altered interactions between the telomeres and the nuclear matrix . These interactions were studied in nuclear matrix halos before and after irradiation . Altered telomere-nuclear matrix interactions were observed in cells derived from individuals with AT . AT cells also had different nucleosomal periodicity in their telomeres from normal cells . Both telomere-nuclear matrix interactions and nucleosomal periodicity were altered by treatment of primary AT fibroblasts with ionizing radiation . This effect was not observed in cells derived from normal individuals . A link was also found between altered telomere-nuclear matrix interactions, aberrant telomere clustering, and gonadal atrophy . The telomere defect was not corrected by the ectopic expression of the catalytic subunit of telomerase (TERT) . Since alteration of the yeast telomere chromatin structure is known to influence gene expression, we compared expressed sequence tags (ESTs) of Atm-null mouse cells and normal mouse cells . Several ESTs were found to be aberrantly expressed in Atm-null mouse cells . This paper summarizes our recent publications and presents some new data on the influence of ATM on telomere metabolism.

J Virol, 2001 Nov, 75(22), 10683 - 95
Proteasome-independent disruption of PML oncogenic domains (PODs), but not covalent modification by SUMO-1, is required for human cytomegalovirus immediate-early protein IE1 to inhibit PML-mediated transcriptional repression; Xu Y et al.; Human cytomegalovirus (HCMV) major immediate-early protein IE1 is an abundant 72-kDa nuclear phosphoprotein that is thought to play an important role in efficient triggering of the lytic cycle, especially at low multiplicity of infection . The best-known properties of IE1 at present are its transient targeting to punctate promyelocytic leukemia protein (PML)-associated nuclear bodies (PML oncogenic domains {PODs} or nuclear domain 10 {ND10}), with associated displacement of the cellular PML tumor suppressor protein into a diffuse nucleoplasmic form and its association with metaphase chromosomes . Recent studies have shown that the targeting of PML (and associated proteins such as hDaxx) to PODs is dependent on modification of PML by ubiquitin-like protein SUMO-1 . In this study, we provide direct evidence that IE1 is also covalently modified by SUMO-1 in both infected and cotransfected cells, as well as in in vitro assays, with up to 30% of the protein representing the covalently conjugated 90-kDa form in stable U373/IE1 cell lines . Lysine 450 was mapped as the major SUMO-1 conjugation site, but a point mutation of this lysine residue in IE1 did not interfere with its targeting to and disruption of the PODs . Surprisingly, unlike PML or IE2, IE1 did not interact with either Ubc9 or SUMO-1 in yeast two-hybrid assays, suggesting that some additional unknown intranuclear cofactors must play a role in IE1 sumoylation . Interestingly, stable expression of either exogenous PML or exogenous Flag-SUMO-1 in U373 cell lines greatly enhanced both the levels and rate of in vivo IE1 sumoylation during HCMV infection . Unlike the disruption of PODs by the herpes simplex virus type 1 IE110(ICP0) protein, the disruption of PODs by HCMV IE1 proved not to involve proteasome-dependent degradation of PML . We also demonstrate here that the 560-amino-acid PML1 isoform functions as a transcriptional repressor when fused to the GAL4 DNA-binding domain and that wild-type IE1 inhibits the repressor function of PML1 in transient cotransfection assays . Furthermore, both IE1(1-346) and IE1(L174P) mutants, which are defective in displacing PML from PODs, failed to inhibit the repression activity of PML1, whereas the sumoylation-negative IE1(K450R) mutant derepressed as efficiently as wild-type IE1 . Taken together, our results suggest that proteasome-independent disruption of PODs, but not IE1 sumoylation, is required for efficient IE1 inhibition of PML-mediated transcriptional repression.

J Biol Chem, 2001 Dec 21, 276(51), 48318 - 24 Epub 2001 Oct 15.
Interaction with BRCA2 suggests a role for filamin-1 (hsFLNa) in DNA damage response; Yuan Y et al.; The BRCA2 tumor suppressor plays significant roles in DNA damage response . The human actin binding protein filamin-1 (hsFLNa, also known as ABP-280) participates in orthogonal actin network, cellular stress responses, signal transduction, and cell migration . Through a yeast two-hybrid system, an in vitro binding assay, and in vivo co-immunoprecipitations, we identified an interaction between BRCA2 and hsFLNa . The hsFLNa binding domain of BRCA2 was mapped to an internal conserved region, and the BRCA2-interacting domain of hsFLNa was mapped to its C terminus . Although hsFLNa is known for its cytoplasmic functions in cell migration and signal transduction, some hsFLNa resides in the nucleus, raising the possibility that it participates in DNA damage response through a nuclear interaction with BRCA2 . Lack of hsFLNa renders a human melanoma cell line (M2) more sensitive to several genotoxic agents including gamma irradiation, bleomycin, and ultraviolet-c light . These results suggest that BRCA2/hsFLNa interaction may serve to connect cytoskeletal signal transduction to DNA damage response pathways.

Exp Gerontol, 2001 Aug, 36(8), 1349 - 59
A cost of reproduction: oxidative stress susceptibility is associated with increased egg production in Drosophila melanogaster; Wang Y et al.; The present study tests the hypothesis that reproduction is correlated with decreased oxidative stress resistance . In numerous species, it has been observed that longevity is negatively correlated with reproduction but the physiological basis of this cost is not well understood . In the present study, female egg production was stimulated by adding live yeast to the surface of Drosophila food . After females were held on yeast-supplemented and unmodified medium for 6-12 days, susceptibility to oxidative stress was measured by exposure to methyl viologen . Added yeast was associated with stress susceptibility of fertile females but not of sterile females . The results of the present study suggest that oxidative stress susceptibility is a physiological cost of reproduction.

EMBO Rep, 2001 Oct, 2(10), 899 - 904
RNA and sex determination in Caenorhabditis elegans . Post-transcriptional regulation of the sex-determining tra-2 and fem-3 mRNAs in the Caenorhabditis elegans hermaphrodite; Puoti A et al.; The Caenorhabditis elegans hermaphrodite sequentially produces sperm and oocytes from a single pool of precursors . Therefore, the hermaphrodite's germ line is the site of two major cell fate decisions: a germ cell precursor first undergoes a mitosis/meiosis decision and then a sperm/oocyte decision . While the mitosis/meiosis decision is governed by Notch/GLP-1 signalling, the sperm/oocyte decision relies on post-transcriptional regulation of two key mRNAs, tra-2 and fem-3 . This review focuses on factors that are required for the silencing of these mRNAs, which results in the sequential production of sperm and oocytes . Most factors that regulate the expression of tra-2 and fem-3 are homologous to proteins involved in RNA regulation in yeast, mammals or Drosophila, suggesting that at least some of the molecular mechanisms regulating the two worm mRNAs have been conserved throughout evolution.

Cell Stress Chaperones, 2001 Jul, 6(3), 238 - 46
The Hsp90 family of proteins in Arabidopsis thaliana; Krishna P et al.; The 90-kDa heat shock protein (Hsp90) is an essential molecular chaperone in eukaryotic cells, with key roles in the folding and activation of proteins involved in signal transduction and control of the cell cycle . A search for Hsp90 sequences in the Arabidopsis thaliana genome revealed that this family includes 7 members . The AtHsp90-1 through AtHsp90-4 proteins constitute the cytoplasmic subfamily, whereas the AtHsp90-5, AtHsp90-6, and AtHsp90-7 proteins are predicted to be within the plastidial, mitochondrial, and endoplasmic reticulum compartments, respectively . The deduced amino acid sequences of each of the cytoplasmic proteins contains the highly conserved C-terminal pentapeptide MEEVD . All of the AtHsp90 sequences include a conserved adenosine triphosphate-binding domain, whereas only the cytoplasmic and endoplasmic reticulum-resident sequences include an adjacent charged linker domain that is common in mammalian and yeast sequences . The occurrence of multiple AtHsp90 proteins in the cytoplasm and of family members in other subcellular compartments suggests a range of specific functions and target polypeptides.

Zhongguo Zhong Yao Za Zhi, 1998 Nov, 23(11), 687 - 90, 704-inside back cover
{Experimental studies on anti-inflammatory, antipyretic and diuretic effects of several species of tongcao and xiao-tongcao}; Shen Y et al.; OBJECTIVE: To observe and compare the anti-inflammatory, antipyretic and diuretic effects of three kinds of Tongcao(Tongtuomu, Panyezhangyeshu and Luosan) and seven kinds of Xiao-tongcao(Ximashanjinjiehua, Xinanxiuqiu1 . 2., Ditanghua, Suixuezhangcai, Qingjiaye and Zhongguo jinjiehua) . METHOD: Decoctions prepared from the above kinds of Tongcao and Xiao-tongcao crude drugs were given to rats at dosages of 8 g/kg and 4 g/kg by ig . Pharmacological actions were observed by means of carrageenan-induced swelling paws, fever models induced by beer-yeast or carrageenan and metabolic cage method in rats . RESULT: All the experimental decoctions could inhibit carrageenan-induced swelling of rat paws in different degrees and exerted anti-pyretic effect on rat fever models induced by beer-yeast or carrageenan . Three kinds of Xiaotongcao(Ximashanjingjiehua, Xinanxiuqui2., Zhongguojinjiehua) had obvious diuretic effect on rats . CONCLUSION: Decoctions of different species of Tongcao and Xiao-tongcao all have anti-inflammatory, antipyretic and diuretic effects, thus providing some pharmacologic basis for the efficacy assay, clinical application, species collation and quality study of Tongcao and Xiao-tongcao.

Mol Biol Cell, 2001 Oct, 12(10), 3060 - 73
Characterization of human palladin, a microfilament-associated protein; Mykkanen OM et al.; Actin-containing microfilaments control cell shape, adhesion, and contraction . In striated muscle, alpha-actinin and other Z-disk proteins coordinate the organization and functions of actin filaments . In smooth muscle and nonmuscle cells, periodic structures termed dense bodies and dense regions, respectively, are thought to serve functions analogous to Z-discs . We describe here identification and characterization of human palladin, a protein expressed mainly in smooth muscle and nonmuscle and distributed along microfilaments in a periodic manner consistent with dense regions/bodies . Palladin contains three Ig-domains most homologous to the sarcomeric Z-disk protein myotilin . The N terminus includes an FPPPP motif recognized by the Ena-Vasp homology domain 1 domain in Ena/vasodilatator-stimulated phosphoprotein (VASP)/Wiscott-Aldrich syndrome protein (WASP) protein family . Cytoskeletal proteins with FPPPP motif target Ena/VASP/WASP proteins to sites of actin modulation . We identified palladin in a yeast two-hybrid search as an ezrin-associated protein . An interaction between palladin and ezrin was further verified by affinity precipitation and blot overlay assays . The interaction was mediated by the alpha-helical domain of ezrin and by Ig-domains 2-3 of palladin . Ezrin is typically a component of the cortical cytoskeleton, but in smooth muscle cells it is localized along microfilaments . These cells express palladin abundantly and thus palladin may be involved in the microfilament localization of ezrin . Palladin expression was up-regulated in differentiating dendritic cells (DCs), coinciding with major cytoskeletal and morphological alterations . In immature DCs, palladin localized in actin-containing podosomes and in mature DCs along actin filaments . The regulated expression and localization suggest a role for palladin in the assembly of DC cytoskeleton.

J Biol Chem, 2001 Dec 7, 276(49), 46073 - 8
Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damaging agents; Tsirigotis M et al.; There is convincing evidence from studies in yeast that a functional ubiquitin/proteasome pathway is required to degrade misfolded or oxidatively damaged proteins but for technical reasons, it has been difficult to perform comparable studies in mammalian cells . To investigate the possibility that the ubiquitin/proteasome pathway is cytoprotective for mammalian cells, we have introduced epitope-tagged wild-type ubiquitin or dominant-negative mutant versions of ubiquitin into mouse HT4 neuroblastoma cells . Cells expressing mutant versions of ubiquitin were found to be sensitive to cadmium, an agent that causes oxidative damage to cellular components, and to canavanine, an amino acid analog that generates misfolded proteins . The greatest sensitivity to canavanine was observed in cells expressing a mutant version of ubiquitin unable to support the formation of Lys(48) linkages . Substrates of the proteasome were found to accumulate in these cells, suggesting a general deficit in proteolysis . Our data suggest that defects in the ubiquitin-mediated proteolytic system predispose mammalian cells to the toxic effects of abnormal protein.

J Biol Chem, 2001 Dec 14, 276(50), 47542 - 9 Epub 2001 Oct 11.
Characterization of fortilin, a novel antiapoptotic protein; Li F et al.; Apoptosis is meticulously controlled in living organisms . Its dysregulation has been shown to play a key role in a number of human diseases, including neoplastic, cardiovascular, and degenerative disorders . Bcl-2 family member proteins and inhibitors of apoptosis proteins are two major negative regulators of apoptosis . We report here the characterization of novel antiapoptotic protein, fortilin, which we identified through yeast two-hybrid library screening . Sequence analysis of fortilin revealed it to be a 172-amino acid polypeptide highly conserved from mammals to plants . Fortilin is structurally unrelated to either Bcl-2 family member proteins or inhibitors of apoptosis proteins . Northern blot analysis showed the fortilin message to be ubiquitous in normal tissue but especially abundant in the liver, kidney, and small intestine . Western blot analysis using anti-fortilin antibody showed more extensive expression in cancerous cell lines (H1299, MCF-7, and A549) than in cell lines derived from normal tissue (HEK293) . Immunocytochemistry using HeLa cells transiently expressing FLAG-tagged fortilin and immunohistochemistry using human breast ductal carcinoma tissue and anti-fortilin antibody both showed that fortilin is predominantly localized in the nucleus . Functionally, the transient overexpression of fortilin in HeLa cells prevented them, in a dose-dependent fashion, from undergoing etoposide-induced apoptosis . Consistently, U2OS cells stably expressing fortilin protected the cells from cell death induced by etoposide over various concentrations and durations of exposure . In addition, fortilin overexpression inhibited caspase-3-like activity as assessed by the cleavage of fluorogenic substrate benzyloxycarbonyl-DEVD-7-amido-4-(trifluoromethyl)coumarin . Furthermore, the antisense depletion of fortilin from breast cancer cell line MCF-7 was associated with massive cell death . These data suggest that fortilin represents a novel antiapoptotic protein involved in cell survival and apoptosis regulation.

J Biol Chem, 2001 Dec 7, 276(49), 46088 - 93 Epub 2001 Oct 11.
Interaction between the G alpha subunit of heterotrimeric G(12) protein and Hsp90 is required for G alpha(12) signaling; Vaiskunaite R et al.; The G alpha subunit of G(12) protein, one of the heterotrimeric G proteins, regulates diverse and complex cellular responses by transducing signals from the cell surface, presumably involving more than one downstream effector . Yeast two-hybrid screening of a human testis cDNA library identified a large fragment of Hsp90 as a protein that interacted with G alpha(12) . The interaction between G alpha(12) and Hsp90 was further substantiated by a co-immunoprecipitation technique . We have determined that Hsp90 is not required for the interaction of G alpha(12) with its binding partners, p115(RhoGEF) and the G beta subunit . Importantly, Hsp90 is required for G alpha(12)-induced serum response element activation, cytoskeletal changes, and mitogenic response . Closely related to G alpha(12), the G alpha(13) subunit did not interact with Hsp90 and did not require functional Hsp90 for serum response element activation . Thus, our results identify a novel signaling module of G alpha(12) and Hsp90.

J Biol Chem, 2001 Dec 21, 276(51), 48237 - 42 Epub 2001 Oct 11.
Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdc14a; Bembenek J et al.; Two forms of the anaphase-promoting complex (APC) mediate the degradation of critical cell cycle regulators . APC(Cdc20) promotes sister-chromatid separation by ubiquitinating securin, whereas APC(Cdh1) ubiquitinates mitotic cyclins, allowing the exit from mitosis . Here we show that phosphorylation of human Cdh1 (hCdh1) by cyclin B-Cdc2 alters the conformation of hCdh1 and prevents it from activating APC . A human homologue of yeast Cdc14, human Cdc14a (hCdc14a), dephosphorylates hCdh1 and activates APC(Cdh1) . In contrast, hCdc14a does not affect the activity of APC(Cdc20) . hCdc14a is a major phosphatase for hCdh1 and localizes to centrosomes in HeLa cells . Therefore, hCdc14a may promote the activation of APC(Cdh1) and exit from mitosis in mammalian cells.

Infect Immun, 2001 Nov, 69(11), 6874 - 80
Involvement of fungal cell wall components in adhesion of Sporothrix schenckii to human fibronectin; Lima OC et al.; Systemic sporotrichosis is an emerging infection potentially fatal for immunocompromised patients . Adhesion to extracellular matrix proteins is thought to play a crucial role in invasive fungal diseases . Here we report studies of the adhesion of Sporothrix schenckii to the extracellular protein fibronectin (Fn) . Both yeast cells and conidia of S . schenckii were able to adhere to Fn as detected by enzyme-linked immunosorbent binding assays . Adhesion of yeast cells to Fn is dose dependent and saturable . S . schenckii adheres equally well to 40-kDa and 120-kDa Fn proteolytic fragments . While adhesion to Fn was increased by Ca(2+), inhibition assays demonstrated that it was not RGD dependent . A carbohydrate-containing cell wall neutral fraction blocked up to 30% of the observed adherence for the yeast cells . The biochemical nature of this fraction suggests the participation of cell surface glycoconjugates in binding by their carbohydrate or peptide moieties . These results provide new data concerning S . schenckii adhesion mechanisms, which could be important in host-fungus interactions and the establishment of sporotrichosis.

Brain Res, 2001 Oct 19, 916(1-2), 1 - 10
A novel protein interacts with a clock-related protein, rPer1; Matsuki T et al.; Mammalian Per proteins are thought to be important in the mechanism of circadian rhythm . We identified a novel protein PIPS (Per1 interacting protein of the suprachiasmatic nucleus) with the yeast two-hybrid system using PAS domain of rat Per1 (rPer1) as a bait . PIPS is about a 180-kDa protein and expressed mainly in the brain, especially in the hypothalamus including the suprachiasmatic nuclei (SCN) . PIPS interacts with mouse Per1 (mPer1) in vitro and in cultured cells transfected with both molecules . Furthermore, it was found that mPer1 translocated PIPS into the nuclei in the cultured cells . Thus, these findings suggest a possibility that PIPS is involved in the feedback loop or output mechanism of circadian rhythm through interacting with Per1 in the SCN.

Genomics, 2001 Oct, 77(3), 127 - 34
Skipping of exon 9 of human CFTR in YAC-transgenic mice; Manson A et al.; Exon 9 of the human gene CFTR is skipped in some mRNA transcripts in human tissues . The level of skipping correlates with the number of TG's and T's in the 5' splice acceptor of exon 9 . Poorly spliced alleles are associated with mild cystic fibrosis related phenotypes . Here we describe transgenic mice carrying a yeast artificial chromosome (YAC) with the intact human gene CFTR . When the YAC carries 10 TG's and 7 T's at the splice acceptor, there is about 50% skipping of exon 9 in most tissues, whereas 12 TG's and 5 T's give about 90% skipping . The level of skipping is quite uniform over many tissues, except the testis, in which there is a much higher level of correct splicing . These mice confirm that the TG(m)T(n) polymorphism has an effect on splicing and should be valuable for studying this phenomenon.

Plant Cell, 2001 Oct, 13(10), 2283 - 95
Identification and characterization of GONST1, a golgi-localized GDP-mannose transporter in Arabidopsis; Baldwin TC et al.; Transport of nucleotide sugars across the Golgi apparatus membrane is required for the luminal synthesis of a variety of plant cell surface components . We identified an Arabidopsis gene encoding a nucleotide sugar transporter (designated GONST1) that we have shown by transient gene expression to be localized to the Golgi . GONST1 complemented a GDP-mannose transport-defective yeast mutant (vrg4-2), and Golgi-rich vesicles from the complemented strain displayed increased GDP-mannose transport activity . GONST1 promoter::beta-glucuronidase studies suggested that this gene is expressed ubiquitously . The identification of a Golgi-localized nucleotide sugar transporter from plants will allow the study of the importance of this class of proteins in the synthesis of plant cell surface components such as cell wall polysaccharides.

Plant Cell, 2001 Oct, 13(10), 2269 - 81
HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein; Li J et al.; Stamen and carpel identities are specified by the combinatorial activities of several floral homeotic genes, APETALA3, PISTILLATA, AGAMOUS (AG), SEPALLATA1 (SEP1), SEPALLATA2 (SEP2), and SEPALLATA3 (SEP3), all of which code for MADS domain DNA binding proteins . AG and the SEP genes also control floral determinacy . HUA1 and HUA2 were identified previously as regulators of stamen and carpel identities and floral determinacy because the recessive hua1-1 or hua2-1 allele affected these processes in plants with a lower dosage of functional AG (either homozygous for the weak ag-4 allele or heterozygous for the strong ag-1 allele) . HUA2 was cloned previously and shown to code for a novel protein . We isolated the HUA1 gene using a map-based approach and show that it encodes a protein with six CCCH-type zinc finger motifs that is also found in yeast, Caenorhabditis elegans, Drosophila melanogaster, and mammalian proteins . Several such genes from invertebrates and mammals are known to play key regulatory roles in development . Therefore, HUA1 are another example of non-MADS domain proteins involved in organ identity specification . We demonstrated that HUA1 binds ribohomopolymers, preferentially poly rU and poly rG, but not double-stranded DNA in vitro . This finding suggests that HUA1, like several mammalian CCCH zinc finger proteins, is an RNA binding protein . Therefore, HUA1 likely participates in a new regulatory mechanism governing flower development.

Gene, 2001 Aug 8, 273(2), 285 - 93
The sedlin gene for spondyloepiphyseal dysplasia tarda escapes X-inactivation and contains a non-canonical splice site; Mumm S et al.; Mutations in the sedlin gene cause spondyloepiphyseal dysplasia tarda (SEDT), a rare X-linked chondrodysplasia . Affected males suffer short stature, deformation of the spine and hips, and deterioration of intervertebral discs with characteristic radiographic changes in the vertebrae . We have sequenced two full-length cDNA clones corresponding to the human sedlin gene . The longest cDNA is 2836 bp, containing a 218 bp 5' untranslated region, a 423 bp coding region, and a 2195 bp 3' untranslated region . The second cDNA does not contain exon 2, suggesting alternative splicing . Sedlin was finely mapped in Xp22.2 by Southern blot analysis on a yeast artificial chromosome/bacterial artificial chromosome map . Comparison of the cDNA sequence and genomic sequence identified six sedlin exons of 67, 142, 112, 147, 84, and 2259 bp . The corresponding introns vary in size from 339 to 14,061 bp . Splice site sequences for four of the five introns conform to the GT/AG consensus sequences, however, the splice site between exons 4 and 5 displays a rare non-canonical splice site sequence, AT/AC . Northern blot analysis showed expression of the sedlin gene in all human adult and fetal tissues tested, with the highest levels in kidney, heart, skeletal muscle, liver, and placenta . Four mRNA sizes were detected with the major band being 3 kb and minor bands of 5, 1.6, and 0.9 kb (the smallest product may reflect a sedlin pseudogene) . Sedlin is expressed from both the active and the inactive human X chromosomes helping to explain the recessive nature and consistent presentation of the disease . Human sedlin shows homology to a yeast gene, which conditions endoplasmic reticulum/golgi transport . Characterization of the human sedlin cDNA and determination of the sedlin gene structure enable functional studies of sedlin and elucidation of the pathogenesis of SEDT.

Gene, 2001 Aug 8, 273(2), 227 - 37
The iodocyanopindolol and SM-11044 binding protein belongs to the TM9SF multispanning membrane protein superfamily; Sugasawa T et al.; SM-11044 is the only beta-adrenergic agonist that inhibits guinea pig eosinophil chemotaxis and induces relaxation of depolarized rat colon tonus . We have previously reported the purification of a 34 kDa photoaffinity-labeled SM-11044 binding protein (SMBP) from rat colon that may mediate the biological effects of the ligand and that differs from all known monoamine receptors (Sugasawa et al., J . Biol . Chem . 272 (1997) 21244) . The present report describes partial amino acid sequence of rat SMBP and molecular cloning of corresponding human SMBP (hSMBP) cDNA . This cDNA encodes a 588 amino acid residue polypeptide comprising a signal peptide, a long hydrophilic amino-terminal region, and a highly hydrophobic C-terminal portion organized into nine putative transmembrane domains . The sequence and structure of hSMBP shows homology to members of a new transmembrane protein 9 superfamily (TM9SF) . Comparison of hSMBP with related protein sequences from yeast, plant and human revealed two subgroups within TM9SF . The members of these groups differ in length and have characteristic amino acid sequence motifs in their amino-terminal portion . Northern blot analysis revealed two major SMBP mRNAs, at 3.4 and 3.8 kb, that were present in all the human tissues examined . Western blot experiments detected SMBP as a 70 kDa protein that may be further cleaved into an active 34 kDa N-terminal polypeptide . Stable Chinese Hamster Ovary cell transfectants expressing hSMBP cDNA displayed specific binding of {(125)I}iodocyanopindolol that was displaced by SM-11044 in a dose-dependent manner . Thus, SMBP is the first member of TM9SF with functional ligand binding properties, suggesting that some of these integral membrane proteins may function as channels, small molecule transporters or receptors.

Gene, 2001 Aug 8, 273(2), 181 - 9
Identification of tumor suppressor candidate genes by physical and sequence mapping of the TSLC1 region of human chromosome 11q23; Pletcher MT et al.; Loss of heterozygosity for a locus on human chromosome 11q22-23 is observed at high frequency in non-small cell lung carcinoma (NSCLC) . Introduction of a 1.1 Mb fragmented yeast artificial chromosome (YAC) mapping to this region completely suppresses the tumorigenic properties of a human NSCLC cell line, A549 . Smaller fragmented YACs give partial but not complete suppression . To further localize the gene(s) responsible for this partial suppression, a bacterial artificial chromosome (BAC) and P1-based artificial chromosome (PAC) contig was constructed, completely spanning the candidate region . End sequence generated in the construction of the BAC/PAC contig identified a previously unmapped EST and served to order genomic sequence contigs from the publicly available Celera Genomics (CG) and Human Genome Project (HGP) efforts . Comparison showed that CG provided larger contigs, while HGP provided more coverage . Neither CG nor HGP provided complete sequence coverage, alone or in combination . The sequence was used to map 110 ESTs and to predict new genes, including two GenScan gene predictions that overlapped ESTs and were shown to be differentially expressed in tumorigenic and suppressed A549 cell lines.

Biochem Biophys Res Commun, 2001 Oct 19, 288(1), 56 - 61
The role of the coiled-coil motif in interactions mediated by TPD52; Sathasivam P et al.; TPD52 (D52)-like proteins are small coiled-coil motif-bearing proteins first identified through their expression in human breast carcinoma that mutually interact in hetero- and homomeric fashions . However, it has been unclear whether the coiled-coil motif is sufficient, or even necessary, for these interactions to occur . We have therefore examined the binding activities of a panel of C-terminally deleted D52 proteins in both the yeast two-hybrid system and pull-down assays . In the yeast two-hybrid system, interactions were only detected when regions C-terminal to the coiled-coil motif were also present . However, using pull-down assays, interactions were detected for all deletion mutants which included the coiled-coil motif . This suggests that the coiled-coil motif is indeed necessary for interactions mediated by D52 proteins, but that C-terminal protein regions facilitate and/or stabilize these interactions .

Oncogene, 2001 Sep 27, 20(43), 6245 - 9
Loss of heterozygosity analysis defines a 3-cM region of 15q commonly deleted in human malignant mesothelioma; De Rienzo A et al.; Previous comparative genomic hybridization and allelic loss analyses demonstrated frequent deletions from 15q11.1-15 in malignant mesothelioma . Recurrent losses of 15q11-22 have also been reported in several other tumor types such as breast and colorectal cancers . To more precisely map the commonly deleted region, we have performed a high density loss of heterozygosity analysis of 46 malignant mesotheliomas, using 26 polymorphic microsatellite markers spanning the entire long arm of chromosome 15 . Allelic loss from 15q was observed in 22 of 46 (48%) cases . These analyses have defined a minimally deleted region of approximately 3-cM, which was confirmed to reside at 15q15 by fluorescence in situ hybridization analysis with yeast artificial chromosome probes . No tumor suppressor genes have been reported to map to this site . The minimally deleted region identified in this investigation overlaps those observed in other kinds of cancer, and is the smallest site of recurrent 15q loss identified to date in human tumors . The identification of this commonly deleted site implicates a putative tumor suppressor gene(s) at 15q15 involved in diverse forms of human neoplasia.

Oncogene, 2001 Sep 27, 20(43), 6132 - 41
High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth; Pierantoni GM et al.; The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors . The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours . In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait . This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase . HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells . The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y) . We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay . In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells . Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle . Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.

Oncogene, 2001 Sep 13, 20(41), 5920 - 9
Chromosomal localization and promoter analysis of the adenomatous polyposis coli binding protein RP1; Wadle A et al.; The EB1/RP1 family is a new protein family that is characterized by the ability of its members to serve as interacting partners for the adenomatous polyposis coli (APC) tumour suppressor protein and tubulin . Data obtained with highly conserved yeast homologues suggest that the EB1/RP1 protein family promotes cytoplasmic microtubule dynamics and contributes to the sensor mechanism controlling the cytokinesis checkpoint during mitosis . However, the precise function of this protein family in mammalian cells has not been elucidated so far and remains unclear . Here, we report on the genomic localization of the RP1 gene and the characterization of the corresponding promoter . The RP1 gene was found to be encoded on chromosome 18q21, a locus which is altered or deleted in up to 50% of all patients with colorectal cancer . Promoter analysis revealed that the RP1 gene is under the control of a strong promoter that was 10 times more active in mammalian cells when compared to SV40 promoter . Members of the cyclic AMP response element binding protein family (CREB1 and CREB2) could be identified as transcription factors binding specifically within the RP1 promoter sequence.

Oncogene, 2001 Sep 13, 20(41), 5913 - 9
Functional interaction of Yaf2 with the central region of MycN; Bannasch D et al.; MYCN is often amplified in advanced-stage neuroblastomas with the consequence of enhanced MycN protein expression . By employing the yeast two-hybrid system we found that Yaf2 binds to the central region of MycN . Binding was also seen in vitro and in vivo . Ectopically expressed Yaf2, like MycN, is localized in the nuclei of neuroblastoma cells . Endogenous Yaf2 is expressed in all three tested neuroblastoma cell lines, all of which also express MycN . Yaf2 was able to enhance MycN-mediated transactivation from an E-box promoter, deletion of the Yaf2 binding region in MycN abrogates this effect . Thus, the binding of Yaf2 to the central region of MycN is functional in mammalian cells.

Oncogene, 2001 Sep 13, 20(41), 5903 - 7
Identification of a prostate-specific G-protein coupled receptor in prostate cancer; Xia C et al.; Membrane receptors coupled to heterotrimeric G-proteins play an essential role in the transmission of signals from the extracellular environment to the cytoplasm of the cell . A wide variety of external stimuli, including neurotransmitters, hormones, phospholipids, photons, odorants, taste ligands, and growth factors, can activate specific members of the G-protein coupled receptors (GPCRs) . Besides essential functions in fully differentiated cells and tissues, GPCRs are also involved in embryogenesis, tissue regeneration, cell growth stimulation, and cell proliferation . In this study, we identified a novel prostate-specific G-protein coupled receptor that interacts with Galpha(12) in our yeast two-hybrid assays . The expression of the receptor protein is highly restricted to human prostate tissues using multiple-tissue Northern blot analysis, and tissue expression array . Furthermore, the expression of prostate-specific receptor is increased significantly in prostate tumors in comparison with the matched normal prostate tissues using PCR and Southern blot analysis, suggesting a potential role of this tissue-specific G-protein coupled receptor in prostate cancer development.

Oncogene, 2001 Sep 13, 20(41), 5846 - 55
Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitor of apoptosis; Aouacheria A et al.; In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans . Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes . The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2 . RT-PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys . Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end . Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized . Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope . Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.

Braz J Med Biol Res, 2001 Oct, 34(10), 1237 - 45
Molecular characterization of DDX26, a human DEAD-box RNA helicase, located on chromosome 7p12; Camargo AA et al.; DEAD-box proteins comprise a family of ATP-dependent RNA helicases involved in several aspects of RNA metabolism . Here we report the characterization of the human DEAD-box RNA helicase DDX26 . The gene is composed of 14 exons distributed over an extension of 8,123 bp of genomic sequence and encodes a transcript of 1.8 kb that is expressed in all tissues evaluated . The predicted amino acid sequence shows a high similarity to a yeast DEAD-box RNA helicase (Dbp9b) involved in ribosome biogenesis . The new helicase maps to 7p12, a region of frequent chromosome amplifications in glioblastomas involving the epidermal growth factor receptor (EGFR) gene . Nevertheless, co-amplification of DDX26 with EGFR was not detected in nine tumors analyzed.

Proc Natl Acad Sci U S A, 2001 Oct 9, 98(21), 11997 - 2002 Epub 2001 Oct 02.
Functional analysis of mutant and wild-type Drosophila origin recognition complex; Chesnokov I et al.; The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes . We have reconstituted a functional recombinant Drosophila ORC and compared activities of the wild-type and several mutant ORC variants . Drosophila ORC is an ATPase, and our studies show that the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding . Moreover, DNA binding by ORC reduces its ATP hydrolysis activity . In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA(+) nucleotide-binding domain of ORC1 . Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication . However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions . We also provide evidence that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane . Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features among Drosophila and budding yeast ORC complexes.

J Biol Chem, 2001 Dec 14, 276(50), 47411 - 20 Epub 2001 Oct 09.
The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis; Kawasaki-Nishi S et al.; The 100-kDa "a" subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1 . The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase . We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi . V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion . To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform . Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion . Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal . Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxyl-terminal domain of Stv1p . Our results suggest that whereas targeting and in vivo dissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.

Biol Chem, 2001 Aug, 382(8), 1103 - 7
The eukaryotic gene transcription machinery; Kornberg RD; Seven purified proteins may be combined to reconstitute regulated, promoter-dependent RNA polymerase II transcription: five general transcription factors, Mediator, and RNA polymerase II . The entire system has been conserved across species from yeast to humans . The structure of RNA polymerase II, consisting of 10 polypeptides with a mass of about 500 kDa, has been determined at atomic resolution . On the basis of this structure, that of an actively transcribing RNA polymerase II complex has been determined as well.

Eur J Immunol, 2001 Oct, 31(10), 2885 - 91
LAP, a lymphocyte activation gene-3 (LAG-3)-associated protein that binds to a repeated EP motif in the intracellular region of LAG-3, may participate in the down-regulation of the CD3/TCR activation pathway; Iouzalen N et al.; The threshold, extent and termination of TCR activation is controlled in part by inhibitory co-receptors expressed on activated T cells . The lymphocyte activation gene product (LAG-3), a ligand for MHC class II molecules co-caps with the CD3/TCR complex and inhibits cell proliferation and cytokine secretion in response to CD3 signaling . We first investigated whether LAG-3 is localized in activated T cells in detergent-resistant membrane rafts enriched in glycosphingolipids and cholesterol . We showed that both LAG-3 and MHC class II are present in the cell fraction of glycosphingolipid-rich complexes (GSL complexes) before the assembly of the immunological synapse by CD3/TCR complex cross-linking . Using the LAG-3 intracytoplasmic region as bait in the yeast two-hybrid cloning system, we next identified a novel protein termed LAP for LAG-3-associated protein . LAP is encoded by a 1.8-kb RNA message in lymphocytes and encodes a 45-kDa protein that is expressed in most tissues . We showed that LAP binds specifically in vitro and in vivo to the Glu-Pro (EP) repeated motif present in the LAG-3 intracytoplasmic region . LAP also binds to the EP motif of another functionally important receptor, the PDGFR . Thus, LAP is a candidate molecule for a new type of signal transduction and/or coupling of clustered rafts to the microtubule networks that could explain how negative signaling of co-receptors may occur through molecules devoid of any immunoreceptor tyrosine-based inhibitory motif consensus sequence.

J Immunol, 2001 Oct 15, 167(8), 4504 - 10
An ancient lectin-dependent complement system in an ascidian: novel lectin isolated from the plasma of the solitary ascidian, Halocynthia roretzi; Sekine H et al.; Mannose-binding lectin (MBL) is a C-type lectin involved in the first line of host defense against pathogens and it requires MBL-associated serine protease (MASP) for activation of the complement lectin pathway . To elucidate the origin and evolution of MBL, MBL-like lectin was isolated from the plasma of a urochordate, the solitary ascidian Halocynthia roretzi, using affinity chromatography on a yeast mannan-Sepharose . SDS-PAGE of the eluted proteins revealed a major band of approximately 36 kDa (p36) . p36 cDNA was cloned from an ascidian hepatopancreas cDNA library . Sequence analysis revealed that the carboxy-terminal half of the ascidian lectin contains a carbohydrate recognition domain (CRD) that is homologous to C-type lectin, but it lacks a collagen-like domain that is present in mammalian MBLs . Purified p36 binds specifically to glucose but not to mannose or N-acetylglucosamine, and it was designated glucose-binding lectin (GBL) . The two ascidian MASPs associated with GBL activate ascidian C3, which had been reported to act as an opsonin . The removal of GBL-MASPs complex from ascidian plasma using Ab against GBL inhibits C3-dependent phagocytosis . These observations strongly suggest that GBL acts as a recognition molecule and that the primitive complement system, consisting of the lectin-proteases complex and C3, played a major role in innate immunity before the evolution of an adaptive immune system in vertebrates.

J Cell Biol, 2001 Oct 15, 155(2), 239 - 49 Epub 2001 Oct 08.
Functional characterization of the KNOLLE-interacting t-SNARE AtSNAP33 and its role in plant cytokinesis; Heese M et al.; Cytokinesis requires membrane fusion during cleavage-furrow ingression in animals and cell plate formation in plants . In Arabidopsis, the Sec1 homologue KEULE (KEU) and the cytokinesis-specific syntaxin KNOLLE (KN) cooperate to promote vesicle fusion in the cell division plane . Here, we characterize AtSNAP33, an Arabidopsis homologue of the t-SNARE SNAP25, that was identified as a KN interactor in a yeast two-hybrid screen . AtSNAP33 is a ubiquitously expressed membrane-associated protein that accumulated at the plasma membrane and during cell division colocalized with KN at the forming cell plate . A T-DNA insertion in the AtSNAP33 gene caused loss of AtSNAP33 function, resulting in a lethal dwarf phenotype . atsnap33 plantlets gradually developed large necrotic lesions on cotyledons and rosette leaves, resembling pathogen-induced cellular responses, and eventually died before flowering . In addition, mutant seedlings displayed cytokinetic defects, and atsnap33 in combination with the cytokinesis mutant keu was embryo lethal . Analysis of the Arabidopsis genome revealed two further SNAP25-like proteins that also interacted with KN in the yeast two-hybrid assay . Our results suggest that AtSNAP33, the first SNAP25 homologue characterized in plants, is involved in diverse membrane fusion processes, including cell plate formation, and that AtSNAP33 function in cytokinesis may be replaced partially by other SNAP25 homologues.

J Biol Chem, 2001 Dec 21, 276(51), 48596 - 607 Epub 2001 Oct 08.
Intracellular localization of the Ret finger protein depends on a functional nuclear export signal and protein kinase C activation; Harbers M et al.; The Ret finger protein (RFP) was identified initially as an oncogene product and belongs to a family of proteins that contain a tripartite motif consisting of a RING finger, a B box, and a coiled-coil domain . RFP represses transcription by interacting with Enhancer of Polycomb and is localized to the cytoplasm or nucleus depending on the cell type . Here, we have identified the nuclear export signal (NES) located in the coiled-coil region of RFP . Mutation of this NES or treatment with leptomycin B abrogated the nuclear export of RFP in NIH3T3 cells . In addition, fusion of this NES to other nuclear proteins, such as yeast transcription factor Gal4, resulted in their release into the cytoplasm of NIH3T3 cells . Although the NES function of RFP in HepG2 cells is masked by another domain in RFP or by another protein, 12-O-tetradecanoylphorbol-13-acetate treatment or overexpression of constitutively active protein kinase Calpha (PKCalpha) abrogated masking, leading to the cytoplasmic localization of RFP . Furthermore, treatment of NIH3T3 cells with PKC inhibitors blocked the function of NES, resulting in nuclear localization of RFP . Thus, the nuclear export of RFP is regulated positively by PKC activation . However, RFP was not a direct substrate of PKC, and additional signaling pathways may be involved in the regulation of nuclear export of RFP.

J Cell Sci, 2001 Sep, 114(Pt 17), 3207 - 12
Alterations in an IRE1-RNA complex in the mammalian unfolded protein response; Bertolotti A et al.; IRE1 proteins mediate cellular responses to accumulation of malfolded proteins in the endoplasmic reticulum in the yeast and mammalian unfolded protein responses . A sensitive in vivo u.v . crosslinking assay showed that IRE1 proteins are intimately associated with RNA in mammalian cells . The IRE1-associated RNA fragments recovered by this assay were different in stressed and unstressed cells . The amount of RNA associated with IRE1 that could be revealed by end-labeling with T4 kinase was greater in IRE1-containing complexes isolated from stressed cells . Furthermore, the RNA fragments recovered from complexes found in stressed cells were shorter than those from unstressed cells, revealing a dynamic change in the IRE1-RNA complex during the UPR . Formation of the complex between IRE1 and RNA was dependent on both the kinase and endonuclease domains of IRE1, and involved pre-existing RNA species . When viewed in the context of the known importance of Ire1p-HAC1 mRNA interactions to the yeast unfolded protein response, these findings suggest that full-length mammalian IRE1s also engage RNA molecules as downstream effectors.

J Biol Chem, 2001 Nov 30, 276(48), 45298 - 306 Epub 2001 Oct 05.
Identification and characterization of a novel Golgi protein, GCP60, that interacts with the integral membrane protein giantin; Sohda M et al.; We demonstrated previously that the integral membrane protein giantin has the Golgi localization signal at the COOH-terminal cytoplasmic domain (Misumi, Y., Sohda, M., Tashiro, A., Sato, H., and Ikehara, Y . (2001) J . Biol . Chem . 276, 6867-6873) . In the present study, using this domain as bait in the yeast two-hybrid screening system, we identified a novel protein interacting with giantin . The 3.6-kilobase mRNA encoding a 528-amino acid protein of 60 kDa designated GCP60 was ubiquitously expressed and was especially abundant in the testis and ovary . Immunofluorescence and immunoelectron microscopy confirmed that GCP60 was co-localized with giantin in the Golgi complex . GCP60 was found to be a peripheral protein associated with the Golgi membrane, where a COOH-terminal domain of GCP60 interacts with the COOH-terminal cytoplasmic domain of giantin . Overexpression of the COOH-terminal domain of GCP60 caused disassembly of the Golgi structure and blocked protein transport from the endoplasmic reticulum to the Golgi . Taken together, these results suggest that GCP60 is involved in the maintenance of the Golgi structure by interacting with giantin, affecting protein transport between the endoplasmic reticulum and the Golgi.

J Biol Chem, 2001 Dec 14, 276(50), 47583 - 9 Epub 2001 Oct 04.
Protein associated with Myc (PAM) is a potent inhibitor of adenylyl cyclases; Scholich K et al.; Using the yeast two-hybrid assay and the second of the two large cytosolic domains of type V adenylyl cyclase (ACV) as bait, we identified a small region (amino acids 1028-1231) in the protein associated with Myc (PAM) as an interaction site for ACV . This small region of PAM as well as purified full-length PAM inhibited the activity of ACV . Additionally, full-length PAM was a very potent inhibitor of ACI and AC activities in S49 cyc(-) cells and HeLa cells with IC(50) values in the pm and low nm range . Moreover, the regulator of chromatin condensation 1-like domain of PAM (amino acids 446-1062) was sufficient and as potent as full-length PAM at inhibiting the activity of ACV . Interestingly, full-length PAM did not inhibit ACII activity that was stimulated by either forskolin of Galpha(s) . When endogenous levels of PAM in HeLa cells were decreased using antisense oligodeoxynucleotides, the basal cAMP content was elevated, and the dose-response curve for vasoactive intestinal peptide-elicited cAMP accumulation in HeLa cells was shifted to the left . Therefore, we conclude that PAM is a very potent, novel inhibitor of specific isoforms of AC . Furthermore, the regulator of chromatin condensation 1-like domain of PAM is sufficient to exert the effects of the full-length protein on AC and decreases in endogenous PAM levels in HeLa cells can modulate both basal and agonist stimulated cAMP accumulation.

Hum Mol Genet, 2001 Sep 15, 10(19), 2049 - 59
Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6; Kleinjan DA et al.; The transcription factor PAX6 plays a critical, evolutionarily conserved role in eye, brain and olfactory development . Homozygous loss of PAX6 function affects all expressing tissues and is neonatally lethal; heterozygous null mutations cause aniridia in humans and the Small eye (Sey) phenotype in mice . Several upstream and intragenic PAX6 control elements have been defined, generally through transgenesis . However, aniridia cases with chromosomal rearrangements far downstream of an intact PAX6 gene suggested a requirement for additional cis-acting control for correct gene expression . The likely location of such elements is pinpointed through YAC transgenic studies . A 420 kb yeast artificial chromosome (YAC) clone, extending well beyond the most distant patient breakpoint, was previously shown to rescue homozygous Small eye lethality and correct the heterozygous eye phenotype . We now show that a 310 kb YAC clone, terminating just 5' of the breakpoint, fails to influence the Sey phenotypes . Using evolutionary sequence comparison, DNaseI hypersensitivity analysis and transgenic reporter studies, we have identified a region, >150 kb distal to the major PAX6 promoter P1, containing regulatory elements . Components of this downstream regulatory region drive reporter expression in distinct partial PAX6 patterns, indicating that the functional PAX6 gene domain extends far beyond the transcription unit.

Eur J Biochem, 2001 Oct, 268(19), 5167 - 75
B"-associated factor(s) involved in RNA polymerase III preinitiation complex formation and start-site selection; Andrau JC et al.; The TFIIIB transcription factor is the central component of the RNA polymerase III transcriptional machinery . In yeast, this factor is composed of three essential polypeptides TBP, TFIIIB70 and TFIIIB90, that are sufficient as recombinant proteins, together with TFIIIC, to promote accurate transcription in vitro . Here we show that a partially purified fraction, named B", that contains the TFIIIB90 subunit, displays properties distinct from recombinant TFIIIB90 . This fraction contains at least a component that interacts with DNA*TFIIIC complexes, either alone or in combination with TFIIIB90, and increases the resistance of the complexes to heparin treatment . In addition, primer extension and single round transcriptions experiment reveal a different start-site selection pattern directed by B" or rTFIIIB90 . In mixing experiments, we show that an activity in B", distinct from TFIIIB90, can promote transcription initiation at the +1 site without affecting the rate of preinitiation complex formation . Our data suggest the existence of at least one new component that participates in preinitiation complex formation and influences start-site selection by RNA polymerase III.

Mol Genet Genomics, 2001 Sep, 266(1), 133 - 41
Transcriptional activation of the human Cu/Zn superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through the xenobiotic-responsive element; Cho JS et al.; Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress . Here we have investigated the effect of the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the promoter of the Cu/Zn superoxide dismutase (SOD1) gene in HepG2 and HeLa cells using the chloramphenicol acetyltransferase gene as a reporter . The SOD1 promoter was activated 4- to 5-fold by TCDD treatment, in a concentration-dependent manner . In addition, the level of SOD1 mRNA and the enzymatic activity of the SOD1 protein were also enhanced on exposure of the cells to TCDD . Functional analysis of the regulatory region of the SOD1 gene by deletion and point mutation, and the use of a heterologous promoter system, showed that the SOD1 gene was transactivated by TCDD via the xenobiotic-responsive element (XRE) . Gel mobility shift assays also confirmed the induction and the inducible binding of a receptor-ligand complex to XRE . Yeast cells that overexpress hSOD1 appeared to be more resistant to TCDD than the wild type . These results demonstrate that SOD1 is induced by TCDD via the XRE . The induced SOD1 may accelerate the neutralization of the superoxide anion and thus reduce the oxidative damage associated with dioxin toxicity.

Int J Food Microbiol, 2001 Sep 19, 69(1-2), 141 - 6
Survey on mycoflora of cow and buffalo dairy products from Southern Italy; Minervini F et al.; Economic losses of dairy products due to spoilage by yeasts have been increasing in European companies because of the reduced use of preservatives, packaging in modified atmospheres, or new formulations that do not strictly control the growth of these organisms . This study reports the results of a survey of yeast species and populations in 145 samples of cow and buffalo dairy products collected in some regions of Southern Italy . Yeasts were isolated from 74% and 57% of cow and buffalo products, respectively . Candida inconspicua was the predominant species in unripened products from cow's milk, while C . famata was detected in medium and long-term ripened dairy products, mostly in association with other yeasts and with moulds belonging to the genus Penicillium . For dairy products produced from buffalo milk, C . inconspicua was the most important yeast frequently isolated from dairy products . Total yeast populations ranged from 5 x 10(2) to 5 x 10(5) cfu/g, indicating a good hygienic quality of the products . The isolation of C . albicans from one stracciatella sample is noteworthy, as this yeast represents a potential contamination by human . Even though yeasts are considered as environmental contaminants, the occurrence of some of them in dairy products at high levels could represent a risk for human health, in particular for immunocompromised patients.

J Chromatogr A, 2001 Aug 31, 928(1), 77 - 90
Determination of folates in foods by high-performance liquid chromatography with fluorescence detection after precolumn conversion to 5-methyltetrahydrofolates; Ndaw S et al.; A liquid chromatographic-fluorimetric determination of folates in foodstuffs including their extraction, without or with deconjugation, chemical conversion to 5-CH3-H4PteGlu(n) and purification of the extract by affinity chromatography is reported . The conversion enables the analysis of total folates and also of the contents of the different mono- and polyglutamate forms of the folates . The method has a satisfactory day-to-day repeatability (never,more than 10%) and a very low detection limit (0.02 pmol per injection) . Depending on the folate studied, the recovery rates varied from 78% (10-CHO-PteGlu) to 98% (5-CHO-H4PteGlu) . Furthermore it has been possible to show that the deconjugation of the folates by rat plasma conjugase was incomplete in foodstuffs whereas chicken pancreas conjugase effectively converted the different folate polyglutamates into folate diglutamates . It could not be demonstrated that prior hydrolysis with a protease and amylase was useful for the analysis of the different foodstuffs studied (yeast, spinach, beef liver, beef fillet and peas) when deconjugation was performed with the chicken pancreas conjugase.

Eur J Paediatr Neurol, 2001, 5 Suppl A, 33 - 5
Fine mapping of ovine ceroid lipofuscinosis confirms orthology with CLN6; Broom MF et al.; The neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases with severe neurodegenerative pathology . An ovine model (OCL) has well defined parallels with the human disease at a biochemical and pathological level . The gene for OCL is located in the chromosomal region OAR 7q13-15 . This region is syntenic with HSA 15q21-23 suggesting that OCL and CLN6 represent mutations in orthologous genes . New microsatellite markers were used for refinement of the OCL critical region . YAC clones that span the critical region have been isolated and comparative gene mapping confirms that the regions for CLN6 and OCL are equivalent.

J Neurosci, 2001 Oct 15, 21(20), 7985 - 92
An NMDA receptor signaling complex with protein phosphatase 2A; Chan SF et al.; Regulation of protein phosphatase 2A (PP2A) activity and NMDA receptor (NMDAR) phosphorylation state contribute to the modulation of synaptic plasticity, yet these two mechanisms have not been functionally linked . The NMDAR subunit NR3A is equipped with a unique carboxyl domain that is different from other NMDAR subunits . We hypothesized that the NR3A C-terminal intracellular domain might serve as synaptic anchor for the phosphatase in the developing CNS . A cDNA library was screened by the yeast two-hybrid method using the NR3A carboxyl domain as the bait . The catalytic subunit of the serine-threonine PP2A was found to be associated with the NR3A carboxyl domain . Immunoprecipitation studies indicated that the NR3A subunit formed a stable complex with PP2A in the rat brain in vivo . Association of PP2A with NMDARs led to an increase in the phosphatase activity of PP2A and the dephosphorylation of serine 897 of the NMDAR subunit NR1 . Stimulation of NMDARs led to the dissociation of PP2A from the complex and the reduction of PP2A activity . A peptide corresponding to the PP2A-NR3A binding domain functioned as a negative regulator of PP2A activity . These data suggest that NMDARs are allosteric modulators of PP2A, which in turn controls their phosphorylation state . The data delineate a mechanistic model of the dynamic regulation of a PP2A-NMDAR signaling complex, mediated by the interaction of NR3A and PP2A, and suggest a novel NMDAR-mediated signaling mechanism in addition to the traditional ionotropic functions of NMDARs.

J Biol Chem, 2001 Nov 23, 276(47), 43499 - 502 Epub 2001 Oct 03.
Effects of acetylation of histone H4 at lysines 8 and 16 on activity of the Hat1 histone acetyltransferase; Makowski AM et al.; During nucleosome assembly in vivo, newly synthesized histone H4 is specifically diacetylated on lysines 5 and 12 within the H4 NH(2)-terminal tail domain . The highly conserved "K5/K12" deposition pattern of acetylation is thought to be generated by the Hat1 histone acetyltransferase, which in vivo is found in the HAT-B complex . In the following report, the activity and substrate specificity of the human HAT-B complex and of recombinant yeast Hat1p have been examined, using synthetic H4 NH(2)-terminal peptides as substrates . As expected, the unacetylated H4 peptide was a good substrate for acetylation by yeast Hat1p and human HAT-B, while the K5/K12-diacetylated peptide was not significantly acetylated . Notably, an H4 peptide previously diacetylated on lysines 8 and 16 was a very poor substrate for acetylation by either yeast Hat1p or human HAT-B . Treating the K8/K16-diacetylated peptide with histone deacetylase prior to the HAT-B reaction raised acetylation at K5/K12 to 70-80% of control levels . These results present strong support for the model of H4-Hat1p interaction proposed by Dutnall et al . (Dutnall, R . N., Tafrov, S . T., Sternglanz, R., and Ramakrishnan, V . (1998) Cell 94, 427-438) and provide evidence for the first time that site-specific acetylation of histones can regulate the acetylation of other substrate sites.

Nat Cell Biol, 2001 Oct, 3(10), 933 - 8
Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin; McCartney BM et al.; Proper positioning of mitotic spindles ensures equal allocation of chromosomes to daughter cells . This often involves interactions between spindle and astral microtubules and cortical actin . In yeast and Caenorhabditis elegans, some of the protein machinery that connects spindles and cortex has been identified but, in most animal cells, this process remains mysterious . Here, we report that the tumour suppressor homologue APC2 and its binding partner Armadillo both play roles in spindle anchoring during the syncytial mitoses of early Drosophila embryos . Armadillo, alpha-catenin and APC2 all localize to sites of cortical spindle attachment . APC2-Armadillo complexes often localize with interphase microtubules . Zeste-white 3 kinase, which can phosphorylate Armadillo and APC, is also crucial for spindle positioning and regulates the localization of APC2-Armadillo complexes . Together, these data suggest that APC2, Armadillo and alpha-catenin provide an important link between spindles and cortical actin, and that this link is regulated by Zeste-white 3 kinase.

J Biol Chem, 2001 Dec 7, 276(49), 46445 - 52 Epub 2001 Oct 02.
A DnaJ protein, apobec-1-binding protein-2, modulates apolipoprotein B mRNA editing; Lau PP et al.; Mammalian homologues of DnaJ proteins, also known as Hsp40 proteins, are co-chaperonins that complement Hsp70 chaperone function . Using the yeast two-hybrid system, we cloned an apolipoprotein (apo) B mRNA editing complementation protein, called apobec-1-binding protein-2 (ABBP-2), and found that it is a Class II DnaJ homologue . ABBP-2 binds to apobec-1, the mammalian apoB mRNA editase, via its J domain and neighboring G/F domain . It is a ubiquitously expressed protein, and, by transfection analysis of GFP-ABBP-2, we found that the protein is located in both the nucleus and cytosol of transfected cells, with predominance in the nucleus . Down-regulation of ABBP-2 expression in cultured cells inhibits endogenous apobec-1-mediated apoB mRNA editing . Like other Hsp40 proteins, ABBP-2 binds to Hsp70 and has ATPase-stimulating activity . Apobec-1-mediated apoB mRNA editing activity of in vitro tissue extracts requires the presence of Hsp70/ABBP-2 . Although exogenously added ATP is not required for editing activity, removal of the endogenous ATP present in these extracts, which disrupts ABBP-2-Hsp70 interaction, completely inhibits editing . ABBP-2 differs from previously described auxiliary proteins (ABBP-1, ACF, and GRY-RBP) in that it does not contain any RNA recognition motifs . Not only is ABBP-2 required for efficient apoB mRNA editing, this newly discovered apobec-1-binding protein may help determine the subcellular distribution and trafficking of apobec-1 via its interaction with the chaperonin Hsp70.

J Biol Chem, 2001 Nov 30, 276(48), 45270 - 5 Epub 2001 Oct 02.
Topology of a human equilibrative, nitrobenzylthioinosine (NBMPR)-sensitive nucleoside transporter (hENT1) implicated in the cellular uptake of adenosine and anti-cancer drugs; Sundaram M et al.; The human equilibrative nucleoside transporter hENT1, the first identified member of the ENT family of integral membrane proteins, is the primary mechanism for the cellular uptake of physiologic nucleosides, including adenosine, and many anti-cancer nucleoside drugs . We have produced recombinant hENT1 in Xenopus oocytes and used native and engineered N-glycosylation sites in combination with immunological approaches to experimentally define the membrane architecture of this prototypic nucleoside transporter . hENT1 (456 amino acid residues) is shown to contain 11 transmembrane helical segments with an amino terminus that is intracellular and a carboxyl terminus that is extracellular . Transmembrane helices are linked by short hydrophilic regions, except for a large glycosylated extracellular loop between transmembrane helices 1 and 2 and a large central cytoplasmic loop between transmembrane helices 6 and 7 . Sequence analyses suggest that this membrane topology is common to all mammalian, insect, nematode, protozoan, yeast, and plant members of the ENT protein family.

Insect Biochem Mol Biol, 2001 Nov 1, 31(12), 1165 - 71
Cloning of two hexokinase isoenzyme sequences from Drosophila melanogaster; Jayakumar PC et al.; Hexokinase coding DM1 and DM2 sequences were obtained from genomic DNA of a Drosophila melanogaster cell line by PCR amplification strategy . Both the sequences were found to encode an enzyme with a molecular weight of 50,000 Da . Amino acid sequence alignment of DM1 and DM2 shows approximately 45% homology with yeast and human hexokinases . The sequences also indicated the presence of conserved amino acid residues and motifs that are present in mammalian hexokinases and are involved in the binding of different substrates . Southern blot analysis suggests that the D . melanogaster genome contain a single copy of DM1 and DM2 sequences . Northern analysis indicates DM1 is expressed as more than one transcript in adult as well as in the D.Mel2 cell line . DM2 is expressed as a single transcript in adult flies . Expression levels for DM1 and DM2 encoded message were found to be similar in different stages of development as seen by RT-PCR . The biotechnological significance of these sequences in metabolic engineering of cells is discussed.

Biochem J, 2001 Oct 15, 359(Pt 2), 255 - 63
Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75; Provost P et al.; Coactosin-like protein (CLP) was recently identified in a yeast two-hybrid screen using 5-lipoxygenase as bait . In the present study, we report the functional characterization of CLP as a human filamentous actin (F-actin)-binding protein . CLP mRNA shows a wide tissue distribution and is predominantly expressed in placenta, lung, kidney and peripheral-blood leucocytes . Endogenous CLP is localized in the cytosol of myeloid cells . Using a two-hybrid approach, actin was identified as a CLP-interacting protein . Binding experiments indicated that CLP associates with F-actin, but does not form a stable complex with globular actin . In transfected mammalian cells, CLP co-localized with actin stress fibres . CLP bound to actin filaments with a stoichiometry of 1:2 (CLP: actin subunits), but could be cross-linked to only one subunit of actin . Site-directed mutagenesis revealed the involvement of Lys(75) of CLP in actin binding, a residue highly conserved in related proteins and supposed to be exposed on the surface of the CLP protein . Our results identify CLP as a new human protein that binds F-actin in vitro and in vivo, and indicate that Lys(75) is essential for this interaction.

J Biol Chem, 2001 Dec 7, 276(49), 46544 - 52 Epub 2001 Oct 01.
Polycystin-1 interacts with intermediate filaments; Xu GM et al.; Polycystin-1, the protein defective in a majority of patients with autosomal dominant polycystic kidney disease, is a ubiquitously expressed multi-span transmembrane protein of unknown function . Subcellular localization studies found this protein to be a component of various cell junctional complexes and to be associated with the cytoskeleton, but the specificity and nature of such associations are not known . To identify proteins that interact with the polycystin-1 C-tail (P1CT), this segment was used as bait in a yeast two-hybrid screening of a kidney epithelial cell library . The intermediate filament (IF) protein vimentin was identified as a strong polycystin-1-interacting partner . Cytokeratins K8 and K18 and desmin were also found to interact with P1CT . These interactions were mediated by coiled-coil motifs in polycystin-1 and IF proteins . Vimentin, cytokeratins K8 and K18, and desmin also bound directly to P1CT in GST pull-down and in in vitro filament assembly assays . Two observations confirmed these interactions in vivo: (i) a cell membrane-anchored form of recombinant P1CT decorated the IF network and was found to associate with the cytoskeleton in detergent-solubilized cells and (ii) endogenous polycystin-1 distributed with IF at desmosomal junctions . Polycystin-1 may utilize this association for structural, storage, or signaling functions.

Invest Ophthalmol Vis Sci, 2001 Oct, 42(11), 2652 - 63
A novel gene for autosomal dominant Stargardt-like macular dystrophy with homology to the SUR4 protein family; Edwards AO et al.; PURPOSE: To describe a novel gene causing a Stargardt-like phenotype in a family with dominant macular dystrophy and the exclusion of all known genes within the disease locus . METHODS: Meiotic breakpoint mapping in a family of 2314 individuals enabled refinement of the location of the disease gene . The genomic organization and expression profile of known and putative genes within the critical region were determined using bioinformatics, cDNA cloning, and RT-PCR . The coding sequence of genes expressed within the retina was scanned for mutations, by using DNA sequencing . RESULTS: The disease-causing gene (STGD3) was further localized to 562 kb on chromosome 6 between D6S460 and a new polymorphic marker centromeric to D6S1707 . Of the four genes identified within this region, all were expressed in the retina or retinal pigment epithelium . The only coding DNA sequence variant identified in these four genes was a 5-bp deletion in exon 6 of ELOVL4 . The deletion is predicted to lead to a truncated protein with a net loss of 44 amino acids, including a dilysine endoplasmic reticulum retention motif . The ELOVL4 gene is the fourth known example of a predicted human protein with homology to mammalian and yeast enzymes involved in the membrane-bound fatty acid chain elongation system . The genomic organization of ELOVL4 and primer sets for exon amplification are presented . CONCLUSIONS: ELOVL4 causes macular dystrophy in this large family distributed throughout North America and implicates fatty acid biosynthesis in the pathogenesis of macular degeneration . The PCR-based assay for the 5-bp deletion will facilitate more accurate genetic counseling and identification of other branches of the family.

Genes Dev, 2001 Oct 1, 15(19), 2613 - 25
LAF1, a MYB transcription activator for phytochrome A signaling; Ballesteros ML et al.; The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals . Here, we describe a new Arabidopsis mutant, laf1 (long after far-red light 1) that has an elongated hypocotyl specifically under far-red light . Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths . As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction . Further analyses revealed that laf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates . LAF1 encodes a nuclear protein with strong homology with the R2R3-MYB family of DNA-binding proteins . Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein . LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles . This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO).

Neuron, 2001 Sep 27, 31(6), 913 - 27
Polyglutamine-expanded ataxin-7 antagonizes CRX function and induces cone-rod dystrophy in a mouse model of SCA7; La Spada AR et al.; Spinocerebellar ataxia type 7 (SCA7) is an autosomal dominant disorder caused by a CAG repeat expansion . To determine the mechanism of neurotoxicity, we produced transgenic mice and observed a cone-rod dystrophy . Nuclear inclusions were present, suggesting that the disease pathway involves the nucleus . When yeast two-hybrid assays indicated that cone-rod homeobox protein (CRX) interacts with ataxin-7, we performed further studies to assess this interaction . We found that ataxin-7 and CRX colocalize and coimmunoprecipitate . We observed that polyglutamine-expanded ataxin-7 can dramatically suppress CRX transactivation . In SCA7 transgenic mice, electrophoretic mobility shift assays indicated reduced CRX binding activity, while RT-PCR analysis detected reductions in CRX-regulated genes . Our results suggest that CRX transcription interference accounts for the retinal degeneration in SCA7 and thus may provide an explanation for how cell-type specificity is achieved in this polyglutamine repeat disease.

Biochemistry, 2001 Oct 9, 40(40), 12112 - 22
Ticlopidine as a selective mechanism-based inhibitor of human cytochrome P450 2C19; Ha-Duong NT et al.; Experiments using recombinant yeast-expressed human liver cytochromes P450 confirmed previous literature data indicating that ticlopidine is an inhibitor of CYP 2C19 . The present studies demonstrated that ticlopidine is selective for CYP 2C19 within the CYP 2C subfamily . UV-visible studies on the interaction of a series of ticlopidine derivatives with CYP 2C19 showed that ticlopidine binds to the CYP 2C19 active site with a K(s) value of 2.8 +/- 1 microM . Derivatives that do not involve either the o-chlorophenyl substituent, the free tertiary amine function, or the thiophene ring of ticlopidine did not lead to such spectral interactions and failed to inhibit CYP 2C19 . Ticlopidine is oxidized by CYP 2C19 with formation of two major metabolites, the keto tautomer of 2-hydroxyticlopidine (1) and the dimers of ticlopidine S-oxide (TSOD) (V(max) = 13 +/- 2 and 0.4 +/- 0.1 min(-1)) . During this oxidation, CYP 2C19 was inactivated; the rate of its inactivation was time and ticlopidine concentration dependent . This process meets the chemical and kinetic criteria generally accepted for mechanism-based enzyme inactivation . It occurs in parralel with CYP 2C19-catalyzed oxidation of ticlopidine, is inhibited by an alternative well-known substrate of CYP 2C19, omeprazole, and correlates with the covalent binding of ticlopidine metabolite(s) to proteins . Moreover, CYP 2C19 inactivation is not inhibited by the presence of 5 mM glutathione, suggesting that it is due to an alkylation occurring inside the CYP 2C19 active site . The effects of ticlopidine on CYP 2C19 are very analogous with those previously described for the inactivation of CYP 2C9 by tienilic acid . This suggests that a similar electrophilic intermediate, possibly a thiophene S-oxide, is involved in the inactivation of CYP 2C19 and CYP 2C9 by ticlopidine and tienilic acid, respectively . The kinetic parameters calculated for ticlopidine-dependent inactivation of CYP 2C19, i.e., t(1/2max) = 3.4 min, k(inact) = 3.2 10(-3) s(-1), K(I) = 87 microM, k(inact)/K(I) = 37 L.mol(-1).s(-1), and r (partition ratio) = 26 (in relation with formation of 1 + TSOD), classify ticlopidine as an efficient mechanism-based inhibitor although somewhat less efficient than tienilic acid for CYP 2C9 . Importantly, ticlopidine is the first selective mechanism-based inhibitor of human liver CYP 2C19 and should be a new interesting tool for studying the topology of the active site of CYP 2C19.

Sci STKE . 2001 Sep 18;2001(100):RE11.
Ypt/rab gtpases: regulators of protein trafficking; Segev N; Ypt/Rab guanosine triphosphatases (GTPases) have emerged in the last decade as key regulators of protein transport in all eukaryotic cells . They seem to be involved in all aspects of vesicle trafficking: vesicle formation, motility, and docking, and membrane remodeling and fusion . The functions of Ypt/Rabs are themselves controlled by upstream regulators that stimulate both their nucleotide cycling and their cycling between membranes . Ypt/Rabs transmit signals to downstream effectors in a guanosine triphosphate (GTP)-dependent manner . The identity of upstream regulators and downstream effectors is known for a number of Ypt/Rabs, and models for their mechanisms of action are emerging . In at least two cases, Ypt/Rab upstream regulators and downstream effectors are found together in a single complex . In agreement with the idea that Ypt/Rabs function in all aspects of vesicular transport, their diverse effectors have recently been shown to function in all identified aspects of vesicle transport . Activators and effectors for individual Ypt/Rabs share no similarity, but are conserved between yeast and mammalian cells . Finally, cross talk demonstrated among the various Ypt/Rabs, and between Ypt/Rabs and other signaling factors, suggests possible coordination among secretory steps, as well as between protein transport and other cellular processes.

J Comp Pathol, 2001 Aug-Oct, 125(2-3), 219 - 23
Disseminated histoplasmosis in a sea otter (Enhydra lutris); Morita T et al.; Disseminated histoplasmosis was diagnosed in a 4.75-year-old, captive female sea otter (Enhydra lutris) . At necropsy, the liver was found to be markedly swollen, with many nodules (4-12 mm in diameter) . Histologically, macrophages containing numerous intracellular yeast-like organisms were noted in the liver, spleen, lung and kidney . These organisms were labelled immunohistochemically with anti-histoplasma yeast antibody . Ultrastructurally, the yeast-like organisms, 2-4 microm in diameter, were found within membranous structures in the cytoplasm of macrophages . This is the first confirmed report of disseminated histoplasmosis in sea otters . Copyright Harcourt Publishers Ltd.

Scand J Rheumatol, 2001, 30(4), 208 - 12
Selenium supplementation in rheumatoid arthritis investigated in a double blind, placebo-controlled trial; Peretz A et al.; INTRODUCTION: Selenium is an essential trace element with antioxidant properties . Trials with selenium have been conducted in rheumatoid arthritis (RA) to correct impaired selenium status and increase defences against deleterious oxidant species . AIM OF THE STUDY: To investigate in a double blind multi-centric placebo-controlled study the effects of selenium supplementation in RA . METHODS: Fifty five patients with moderate RA received during 90 days either capsules containing selenium-enriched yeast (200 microg/d) or a placebo . RESULTS: The visual analog scale, the Ritchie index, the number of swollen and painful joints, and morning stiffness significantly decreased with time in both groups (p<0.001), but no difference between groups could be identified . When examining the quality of life a significant (p<0.01) improvement in arm movements and health feeling was evidenced in selenium-treated patients . CONCLUSION: Selenium treatment did not show clinical benefit on RA . Interestingly, the improval in both groups demonstrated a placebo effect of the intervention trial.

Plant Cell Physiol, 2001 Sep, 42(9), 976 - 81
An Arabidopsis SNF1-related protein kinase, AtSR1, interacts with a calcium-binding protein, AtCBL2, of which transcripts respond to light; Nozawa A et al.; AtSR1 is a protein kinase of Arabidopsis thaliana, which belongs to the SNF1-related protein kinase subfamily 3 . We previously showed accumulation of its transcripts to be responsive to light . In this study, we examined the interaction between AtSR1 and six calcineurin B like proteins of Arabidopsis and found that AtSR1 prominently interacts with one of them, AtCBL2, by yeast two-hybrid assay . Interaction between AtSR1 and AtCBL2 could also be directly confirmed in vitro by pull down assay . RNA blot and reverse transcription-polymerase chain reaction analyses showed that transcripts of AtCBL2, and also of AtCBL1, another CBL, increased upon illumination of leaves . The physiological meaning of the interaction of AtSR1and AtCBL2 is not clear, but they presumably function in signal transduction of light.

Plant Cell Physiol, 2001 Sep, 42(9), 900 - 5
A putative two pore channel AtTPC1 mediates Ca(2+) flux in Arabidopsis leaf cells; Furuichi T et al.; The gene encoding voltage-gated channel with high affinity for Ca(2+) permeation has not been cloned from plants . In the present study, we isolated a full-length cDNA encoding a putative Ca(2+ )channel (AtTPC1) from Arabidopsis . AtTPC1 has two conserved homologous domains, both of which contain six transmembrane segments (S1-S6) and a pore loop (P) between S5 and S6 in each domain, and has the highest homology with the two pore channel TPC1 recently cloned from rat . The overall structure is similar to the half of the general structure of alpha-subunits of voltage-activated Ca(2+) channels from animals . AtTPC1 rescued the Ca(2+) uptake activity of a yeast mutant cch1 . Sucrose-induced luminescence, which reflects a cytosolic free Ca(2+) increase in aequorin-expressing Arabidopsis leaves, was enhanced by overexpression of AtTPC1 and suppressed by antisense expression of it . Sucrose-H(+) symporters AtSUC1 and 2, which depolarize membrane potential of cells receiving sucrose, also depressed a Ca(2+) increase by their antisense expression . These results suggest that AtTPC1 mediates a voltage-activated Ca(2+ )influx in Arabidopsis leaf cells.

Obstet Gynecol, 2001 Oct, 98(4), 588 - 91
Clobetasol propionate in the treatment of premenarchal vulvar lichen sclerosus; Smith YR et al.; OBJECTIVE: To assess the effectiveness of treating premenarchal vulvar lichen sclerosus with clobetasol propionate . METHODS: A retrospective chart review was performed of girls presenting to the University of Michigan Pediatric and Adolescent Gynecology Clinic from January, 1995, to July, 2000, with premenarchal lichen sclerosus . Subjects in the study were treated with topical clobetasol propionate ointment 0.05% for 2-4 weeks, and then tapered to a less potent steroid . Information was extracted concerning age at onset, symptoms, vulvar examination, previous treatments, effectiveness of clobetasol, follow-up, and complications . The parents were contacted for a follow-up telephone survey . RESULTS: Fifteen girls averaging 5.7 years at the start of symptoms met criteria . The diagnosis of lichen sclerosus was made visually in 11 and by biopsy in four . Follow-up ranged from 2 months to 6 years . Fourteen girls had good improvement within 4-7 weeks . One girl developed a yeast superinfection and one developed transient erythema . At least 1 year of follow-up by clinic visit or telephone interview was available in 11 girls . Of these 11, two girls had no further vulvar symptoms after the initial treatment, five had one or two total flares, three reported three to eight flares per year, and one girl continues to be unresponsive to therapy . CONCLUSION: Clobetasol propionate was an effective treatment of premenarchal vulvar lichen sclerosus in this small group; however, recurrences were common and required additional steroid treatment . Furthermore, complications of treatment were infrequent, minor, and easily treatable.

Plant J, 2001 Sep, 27(5), 393 - 405
The ubiquitin-specific protease UBP14 is essential for early embryo development in Arabidopsis thaliana; Doelling JH et al.; The ubiquitin/26S proteasome pathway is a major route for selectively degrading cytoplasmic and nuclear proteins in eukaryotes . In this pathway, chains of ubiquitins become attached to short-lived proteins, signalling recognition and breakdown of the modified protein by the 26S proteasome . During or following target degradation, the attached multi-ubiquitin chains are released and subsequently disassembled by ubiquitin-specific proteases (UBPs) to regenerate free ubiquitin monomers for re-use . Here, we describe Arabidopsis thaliana UBP14 that may participate in this recycling process . Its amino acid sequence is most similar to yeast UBP14 and its orthologues, human IsoT1-3 and Dictyostelium UbpA, and it can functionally replace yeast UBP14 in a ubp14Delta mutant . Like its orthologues, AtUBP14 can disassemble multi-ubiquitin chains linked internally via epsilon-amino isopeptide bonds using Lys48 and can process some, but not all, translational fusions of ubiquitin linked via alpha-amino peptide bonds . However, unlike its yeast and Dictyostelium orthologues, AtUBP14 is essential in Arabidopsis . T-DNA insertion mutations in the single gene that encodes AtUBP14 cause an embryonic lethal phenotype, with the homozygous embryos arresting at the globular stage . The arrested seeds have substantially increased levels of multi-ubiquitin chains, indicative of a defect in ubiquitin recycling . Taken together, the data demonstrate an essential role for the ubiquitin/26S proteasome pathway in general and for AtUBP14 in particular during early plant development.

Plant Mol Biol, 2001 Aug, 46(6), 639 - 50
Dual targeting properties of the N-terminal signal sequence of Arabidopsis thaliana THI1 protein to mitochondria and chloroplasts; Chabregas SM et al.; thi1 has been recently isolated from Arabidopsis thaliana and is probably involved in both thiamine biosynthesis and as protection of organellar DNA from damage . Studies of thiamine biosynthesis in plants suggests a plastid location for the pathway, which is in agreement with the predicted THI1 N-terminal chloroplastic transit peptide (TP) . On the other hand, thiamine is synthesized in mitochondria in yeast cells . Interestingly, A . thaliana thi1 cDNA complements a yeast strain disrupted for the homologous gene . Analysis of THI1 amino acid sequence revealed the presence of a putative amphiphilic alpha-helix, which is typical for mitochondrial presequences, located downstream of the chloroplast transit peptide . To define the putative role of the two predicted targeting sequences in tandem, we produced two chimeric genes encompassing the chloroplastic THI1 TP and either 4 or 27 (including the putative mitochondrial presequence) N-terminal residues of the mature THI1, both linked to the reporter (gusA) gene . Analysis of GUS distribution in subcellular fractions of transgenic plants revealed that in the construct retaining only 4 residues of mature THI1, GUS was found in the chloroplastic fraction . Extension of the THI1 transit peptide to 27 residues of the mature protein allowed import and processing of GUS into both mitochondria and chloroplasts . Direct analysis by immunogold-labeling with an anti-THI1 polyclonal antibody identified THI1 in both organelles in Arabidopsis . We also provide evidence that the precursors of both organellar isoforms are encoded by a single nuclear transcript . Thus, THI1 is targeted simultaneously to mitochondria and chloroplasts by a post transcriptional mechanism.

Nucleic Acids Res, 2001 Oct 1, 29(19), 3975 - 81
Promiscuous patching of broken chromosomes in mammalian cells with extrachromosomal DNA; Lin Y et al.; To study double-strand break (DSB)-induced mutations in mammalian chromosomes, we stably transfected thymidine kinase (tk)-deficient mouse fibroblasts with a DNA substrate containing a recognition site for yeast endonuclease I-SceI embedded within a functional tk gene . Cells were then electroporated with a plasmid expressing endonuclease I-SceI to induce a DSB, and clones that had lost tk function were selected . In a previous study of DSB-induced tk-deficient clones, we found that approximately 8% of recovered tk mutations involved the capture of one or more DNA fragments at the DSB site . Almost half of the DNA capture events involved the I-SceI expression plasmid, and several events involved retrotransposable elements . To learn whether only certain DNA sequences or motifs are efficiently captured, in the current work we electroporated an I-SceI expression plasmid along with HaeIII fragments of φX174 genomic DNA . We report that 18 out of 132 tk-deficient clones recovered had captured DNA fragments, and 14 DNA capture events involved one or more fragments of φX174 DNA . Microhomology existed at most junctions between φX174 DNA and genomic sequences . Our work suggests that virtually any extrachromosomal DNA molecule may be recruited for the patching of DSBs in a mammalian genome.

Nucleic Acids Res, 2001 Oct 1, 29(19), 3939 - 48
PNRC2 is a 16 kDa coactivator that interacts with nuclear receptors through an SH3-binding motif; Zhou D et al.; PNRC2 (proline-rich nuclear receptor co-regulatory protein 2) was identified using mouse steroidogenic factor 1 (SF1) as bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library . PNRC2 is an unusual coactivator in that it is the smallest coactivator identified so far, with a molecular weight of 16 kDa, and interacts with nuclear receptors using a proline-rich sequence . In yeast two-hybrid assays PNRC2 interacted with orphan receptors SF1 and estrogen receptor-related receptor alpha1 in a ligand-independent manner . PNRC2 was also found to interact with the ligand-binding domains of estrogen receptor, glucocorticoid receptor, progesterone receptor, thyroid receptor, retinoic acid receptor and retinoid X receptor in a ligand-dependent manner . A functional activation function 2 domain is required for nuclear receptors to interact with PNRC2 . Using the yeast two-hybrid assay, the region amino acids 85-139 was found to be responsible for the interaction with nuclear receptors . This region contains an SH3 domain-binding motif (SEPPSPS) and an NR box-like sequence (LKTLL) . A mutagenesis study has shown that the SH3 domain-binding motif is important for PNRC2 to interact with all the nuclear receptors tested . Our results reveal that PNRC2 has a structure and function similar to PNRC, a previously characterized coactivator . These two proteins represent a new type of nuclear receptor co-regulatory proteins.

J Biol Chem, 2001 Dec 7, 276(49), 46340 - 6
Roles of phytanoyl-CoA alpha-hydroxylase in mediating the expression of human coagulation factor VIII; Chen C et al.; The coagulation factor VIII (FVIII) is the coagulation factor deficient in the X-chromosome-linked bleeding disorder hemophilia A . Previous transfection studies demonstrated that factor VIII was 10-100-fold less efficiently expressed than the homologous coagulation factor, factor V . To investigate the regulatory mechanisms of FVIII synthesis and secretion, we used the yeast two-hybrid system as an approach to search for proteins that associated with FVIII . The A2 domain (337-740 amino acids) of factor VIII (FVIII-A2) was used as a bait and phytanoyl-CoA alpha-hydroxylase (PAHX) was identified as a binding protein of FVIII-A2 . PAHX had potential to interact with the residues 373-508 within the A2 domain, but not with A1 and A3 (the homologous domains of A2) . The interaction between the A2 domain and PAHX was independent of the type 2 peroxisomal targeting signal (PTS2) of PAHX . Overexpression of PAHX in FVIII-produced cells decreased the expression of FVIII by about 70% . The elevated expression of von Willebrand factor had no effect on the suppression of FVIII secretion by PAHX . Expression of the green fluorescent PAHX fusion protein in SMMC-7721 cells affected the intracellular trafficking of FVIII-A2 . These results suggested that the interaction between PAHX and FVIII-A2 was in part responsible for the low-level expression of factor VIII.

J Biol Chem, 2001 Dec 21, 276(51), 48196 - 205 Epub 2001 Sep 26.
An isoform of branched-chain aminotransferase is a novel co-repressor for thyroid hormone nuclear receptors; Lin HM et al.; The functions of thyroid hormone receptors (TRs) are regulated by a host of co-regulatory proteins . Tissue-specific expression of these co-regulators leads to distinct expression patterns and regulation of thyroid hormone (T3) target genes in tissues . Previously we have found that human colon carcinoma RKO cells exhibit strong T3-independent transcriptional activity . We therefore searched for co-regulatory proteins in RKO cells using a yeast two-hybrid system with the intact TRbeta1 as bait . One of the three positive clones, designated as P3, was identified to be an isoform of human mitochondria branched-chain aminotransferase (BCATm) . P3 was a spliced variant of BCATm with an internal 12-amino acid deletion near the carboxyl-terminal region and was abundantly expressed in RKO cells . The expressed protein localized both to the mitochondria and the nucleus of transfected CV1 cells . P3 physically interacted with TRbeta1 in a T3-independent manner that led to the inhibition in binding of TRbeta1 to thyroid hormone-responsive element . P3 not only enhanced the repressor activity of the unliganded TR but also repressed the ligand-dependent activation of TR . This repression was reversed by treatment of cells with trichostatin A, suggesting that in addition to the inhibition of DNA binding, the repression activity of P3 on TR may also be mediated by histone deacetylase activity . Thus, unlike the currently known co-repressors, P3 is a novel ligand-independent co-repressor for TR.

EMBO J, 2001 Oct 1, 20(19), 5513 - 20
Rad52 partially substitutes for the Rad51 paralog XRCC3 in maintaining chromosomal integrity in vertebrate cells; Fujimori A et al.; Yeast Rad52 DNA-repair mutants exhibit pronounced radiation sensitivity and a defect in homologous re combination (HR), whereas vertebrate cells lacking Rad52 exhibit a nearly normal phenotype . Bio chemical studies show that both yeast Rad52 and Rad55-57 (Rad51 paralogs) stimulate DNA-strand exchange mediated by Rad51 . These findings raise the possibility that Rad51 paralogs may compensate for lack of Rad52 in vertebrate cells, explaining the absence of prominent phenotypes for Rad52-deficient cells . To test this hypothesis, using chicken DT40 cells, we generated conditional mutants deficient in both RAD52 and XRCC3, which is one of the five vertebrate RAD51 paralogs . Surprisingly, the rad52 xrcc3 double-mutant cells were non-viable and exhibited extensive chromosomal breaks, whereas rad52 and xrcc3 single mutants grew well . Our data reveal an overlapping (but non-reciprocal) role for Rad52 and XRCC3 in repairing DNA double-strand breaks . The present study shows that Rad52 can play an important role in HR repair by partially substituting for a Rad51 paralog.

EMBO J, 2001 Oct 1, 20(19), 5400 - 11
Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4; Feys BJ et al.; The Arabidopsis EDS1 and PAD4 genes encode lipase-like proteins that function in resistance (R) gene-mediated and basal plant disease resistance . Phenotypic analysis of eds1 and pad4 null mutants shows that EDS1 and PAD4 are required for resistance conditioned by the same spectrum of R genes but fulfil distinct roles within the defence pathway . EDS1 is essential for elaboration of the plant hypersensitive response, whereas EDS1 and PAD4 are both required for accumulation of the plant defence-potentiating molecule, salicylic acid . EDS1 is necessary for pathogen-induced PAD4 mRNA accumulation, whereas mutations in PAD4 or depletion of salicylic acid only partially compromise EDS1 expression . Yeast two-hybrid analysis reveals that EDS1 can dimerize and interact with PAD4 . However, EDS1 dimerization is mediated by different domains to those involved in EDS1-PAD4 association . Co-immunoprecipitation experiments show that EDS1 and PAD4 proteins interact in healthy and pathogen-challenged plant cells . We propose two functions for EDS1 . The first is required early in plant defence, independently of PAD4 . The second recruits PAD4 in the amplification of defences, possibly by direct EDS1-PAD4 association.

Gene, 2001 Sep 5, 275(1), 107 - 14
Cloning and analysis of mold-specific genes in the dimorphic fungus Histoplasma capsulatum; Tian X et al.; A critical feature in the pathogenesis of the respiratory pathogen Histoplasma capsulatum is the conversion from the mold form (found in soil) to the yeast form in the lungs of the host . Little is known about the molecular biology of Histoplasma dimorphism . In particular, the possible roles of genes which are transcriptionally silent in yeast (i.e . mold-specific) have not been studied . We have produced a cDNA library highly enriched for mold-upregulated clones by fragmenting cDNA and removing yeast-specific and common sequences with a highly efficient enzyme degrading subtraction method . Screening of randomly selected clones identified cDNA fragments representing 16 different mold-upregulated genes . Because multiple cDNA fragments can be treated as alleles in a genetic screen, we were able to apply probability analysis to estimate the total number of mold-upregulated genes . We estimate that there are 27 upregulated genes; cDNA fragments of 16 have been isolated . Here we report the first isolation and analysis of cDNA from two mold-specific genes, MS8 (GenBank AF292398) and MS88 (GenBank AF357882) . The MS8 transcript was very strongly expressed in mold but not detected on Northern blots with yeast RNA . The putative MS8 protein was predicted to be 21.3 kDa (203 aa), very rich in glutamine and glycine and had a calculated pI of 6.76 . The MS88 transcript was weakly expressed in mold and not detected in yeast . The putative MS88 protein was predicted to be 22.5 kDa (219 aa) with a pI of 4.46 . GenBank similarity searches revealed that the putative MS8 protein was similar to a glutamine-rich protein, of unknown function, from the fungus Colletotrichum gloeosporioides (GenBank U94186) . No significant matches were found for the putative MS88 protein.

Genome Biol . 2001;2(9):REVIEWS0007 . Epub 2001 Aug 31.
The nuclear pore complex; Adam SA; Nuclear pore complexes, the conduits for information exchange between the nucleus and cytoplasm, appear broadly similar in eukaryotes from yeast to human . Precisely how nuclear pore complexes regulate macromolecular and ionic traffic remains unknown, but recent advances in the identification and characterization of components of the complex by proteomics and genomics have provided new insights.

Biochem Biophys Res Commun, 2001 Oct 5, 287(4), 941 - 8
Basolateral sorting of human poliovirus receptor alpha involves an interaction with the mu1B subunit of the clathrin adaptor complex in polarized epithelial cells; Ohka S et al.; Poliovirus receptor (hPVR/CD155) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily but its natural function remains unknown . Two membrane-bound isoforms, hPVRalpha and hPVRdelta, are known to date, and they differ only in the amino acid sequence of their cytoplasmic domains . To gain an insight into the possible function of the cytoplasmic domains, we examined the localization of introduced hPVRalpha and hPVRdelta in polarized epithelial cells deficient of native hPVRs . Basolateral sorting of hPVRalpha was observed in Madine-Darby canine kidney cells expressing mu1B, but not in LLC-PK1 porcine kidney cells deficient in mu1B . Distribution of hPVRdelta, however, occurred both on the apical and basolateral plasma membranes of these two cell lines . Basolateral sorting of hPVRalpha was also seen in LLC-PK1 cells that expressed an intact exogenous mu1B, but not in the cells that expressed a mutant mu1B lacking binding ability to tyrosine-containing signals . These results indicate that mu1B is involved in the distribution of hPVRalpha to the basolateral membrane . Comparative distribution analysis of hPVRalpha using a series of mutants with truncations and substitutions in the cytoplasmic tail demonstrated that determinant for the basolateral sorting resided in the tyrosine-containing motif of the cytoplasmic tail . Furthermore, yeast two hybrid analysis strongly suggested that the tyrosine motif directly interacted with mu1B protein . Thus, basolateral sorting of hPVRalpha appears to involve the interaction with mu1B through a tyrosine motif existing in the cytoplasmic domain .

Proc Natl Acad Sci U S A, 2001 Sep 25, 98(20), 11016 - 23
Controlling potassium channel activities: Interplay between the membrane and intracellular factors; Yi BA et al.; Neural signaling is based on the regulated timing and extent of channel opening; therefore, it is important to understand how ion channels open and close in response to neurotransmitters and intracellular messengers . Here, we examine this question for potassium channels, an extraordinarily diverse group of ion channels . Voltage-gated potassium (Kv) channels control action-potential waveforms and neuronal firing patterns by opening and closing in response to membrane-potential changes . These effects can be strongly modulated by cytoplasmic factors such as kinases, phosphatases, and small GTPases . A Kv alpha subunit contains six transmembrane segments, including an intrinsic voltage sensor . In contrast, inwardly rectifying potassium (Kir) channels have just two transmembrane segments in each of its four pore-lining alpha subunits . A variety of intracellular second messengers mediate transmitter and metabolic regulation of Kir channels . For example, Kir3 (GIRK) channels open on binding to the G protein betagamma subunits, thereby mediating slow inhibitory postsynaptic potentials in the brain . Our structure-based functional analysis on the cytoplasmic N-terminal tetramerization domain T1 of the voltage-gated channel, Kv1.2, uncovered a new function for this domain, modulation of voltage gating, and suggested a possible means of communication between second messenger pathways and Kv channels . A yeast screen for active Kir3.2 channels subjected to random mutagenesis has identified residues in the transmembrane segments that are crucial for controlling the opening of Kir3.2 channels . The identification of structural elements involved in potassium channel gating in these systems highlights principles that may be important in the regulation of other types of channels.

DNA Res, 2001 Aug 31, 8(4), 163 - 71
Mapping of quantitative trait locus related to submergence tolerance in rice with aid of chromosome walking; Kamolsukyunyong W et al.; The major QTL for submergence tolerance was locate in the 5.9 cM interval between flanking RFLP markers . To narrow down this region, a physical map was constructed using YAC and BAC clones . A 400-kb YAC was identified in this region and later its end fragments were used to screen a rice BAC library . Through chromosome walking, 24 positive BAC clones formed two contigs around linked-RFLP markers, R1164 and RZ698 . Using one YAC end, six BAC ends and three RFLP markers, a fine-scale map was constructed of the 6.8-cM interval of S10709-RZ698 on rice chromosome 9 . The submergence tolerance and related trait were located in a small, well-defined region around BAC-end marker 180D1R and RFLP marker R1164 . The physical-to-map distance ratio in this region is as small as 172.5 kb/cM, showing that this region is a hot spot for recombination in the rice genome.

Cell Tissue Res, 2001 Sep, 305(3), 285 - 98
Function and interactions of integrins; van der Flier A et al.; Integrins are heterodimeric cell adhesion molecules that link the extracellular matrix to the cytoskeleton . The integrin family in man comprises 24 members, which are the result of different combinations of 1 of 18 alpha- and 1 of 8 beta-subunits . Alternative splicing of mRNA of some alpha- and beta-subunits and postranslational modifications of integrin subunits further increase the diversity of the integrin family . In their capacity as adhesion receptors that organize the cytoskeleton, integrins play an important role in controlling various steps in the signaling pathways that regulate processes as diverse as proliferation, differentiation, apoptosis, and cell migration . The intracellular signals that lead to these effects may be transduced via cytoplasmic components, which have been identified as integrin-binding proteins in yeast two-hybrid screens and which could mediate the coupling of integrins to intracellular signaling pathways . In this review an overview is given of the function and ligand-binding properties of integrins as well as of proteins that associate with integrins and may play a role in their signaling function.

J Biol Chem, 2001 Dec 7, 276(49), 46632 - 8 Epub 2001 Sep 24.
Ligand-regulated binding of FAP68 to the hepatocyte growth factor receptor; Grisendi S et al.; We have used the yeast two-hybrid system to identify proteins that interact with the intracellular portion of the hepatocyte growth factor (HGF) receptor (Met) . We isolated a human cDNA encoding a novel protein of 68 kDa, which we termed FAP68 . This protein is homologous to a previously described FK506-binding protein-associated protein, FAP48, which derives from an alternative spliced form of the same cDNA, lacking an 85-nucleotide exon and leading to an early stop codon . Here we show that epithelial cells, in which the HGF receptor is naturally expressed, contain FAP68 and not FAP48 proteins . FAP68 binding to Met requires the last 30 amino acids of the C-terminal tail, which are unique to the HGF receptor . Indeed, FAP68 does not interact with related tyrosine kinases of the Met and insulin receptor families . FAP68 interacts specifically with the inactive form of HGF receptor, such as a kinase-defective receptor or a dephosphorylated wild type receptor . In vivo, endogenous FAP68 can be coimmunoprecipitated with the HGF receptor in the absence of stimuli and not upon HGF stimulation . Thus, FAP68 represents a novel type of effector that interacts with the inactive HGF receptor and is released upon receptor phosphorylation . Free FAP68 exerts a specific stimulatory activity toward the downstream target p70 S6 protein kinase (p70S6K) . Significantly, nonphosphorylated HGF receptor prevents FAP68 from stimulating p70S6K . These data suggest a role for FAP68 in coupling HGF receptor signaling to the p70S6K pathway.

J Biol Chem, 2001 Nov 16, 276(46), 43065 - 73 Epub 2001 Sep 24.
Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein; O'Connell S et al.; The products of Polycomb group (PcG) genes are required for the epigenetic repression of a number of important developmental regulatory genes, including homeotic genes . Enhancer of zeste (E(Z)) is a Drosophila PcG protein that previously has been shown to bind directly to another PcG protein, Extra Sex Combs (ESC), and is present along with ESC in a 600-kDa complex in Drosophila embryos . Using yeast two-hybrid and in vitro binding assays, we show that E(Z) binds directly to another PcG protein, Polycomblike (PCL) . PCL.E(Z) interaction is shown to be mediated by the plant homeodomain (PHD) fingers domain of PCL, providing evidence that this motif can act as an independent protein interaction domain . An association was also observed between PHF1 and EZH2, human homologs of PCL and E(Z), respectively, demonstrating the evolutionary conservation of this interaction . E(Z) was found to not interact with the PHD domains of three Drosophila trithorax group (trxG) proteins, which function to maintain the transcriptional activity of homeotic genes, providing evidence for the specificity of the interaction of E(Z) with the PCL PHD domain . Coimmunoprecipitation and gel filtration experiments demonstrate in vivo association of PCL with E(Z) and ESC in Drosophila embryos . We discuss the implications of PCL association with ESC.E(Z) complexes and the possibility that PCL may either be a subunit of a subset of ESC.E(Z) complexes or a subunit of a separate complex that interacts with ESC.E(Z) complexes.

J Biol Chem, 2001 Nov 30, 276(48), 44604 - 12 Epub 2001 Sep 24.
PrPC directly interacts with proteins involved in signaling pathways; Spielhaupter C et al.; The cellular prion protein (PrP(C)) is a conserved glycoprotein predominantly expressed in neuronal cells . Its purpose in living cells is still enigmatic . To elucidate on its cellular function, we performed a yeast two-hybrid screen for interactors . We used murine PrP(C) (amino acids 23-231) as bait to search a mouse brain cDNA expression library . Several interaction partners were identified . Three of them with a high homology to known sequences were further characterized . These candidates were the neuronal phosphoprotein synapsin Ib, the adaptor protein Grb2, and the still uncharacterized prion interactor Pint1 . The in vivo interaction of the three proteins with PrP(C) was confirmed by co-immunoprecipitation assays with recombinant and authentic proteins in mammalian cells . The binding regions were mapped using truncated PrP constructs . As both synapsin Ib and Grb2 are implicated in neuronal signaling processes, our findings further strengthen the putative role of the prion protein in signal transduction.

EMBO Rep, 2001 Oct, 2(10), 905 - 9 Epub 2001 Sep 24.
Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing; Van Dyck E et al.; Double-strand breaks (DSBs) occur frequently during DNA replication . They are also caused by ionizing radiation, chemical damage or as part of the series of programmed events that occur during meiosis . In yeast, DSB repair requires RAD52, a protein that plays a critical role in homologous recombination . Here we describe the actions of human RAD52 protein in a model system for single-strand annealing (SSA) using tailed (i.e . exonuclease resected) duplex DNA molecules . Purified human RAD52 protein binds resected DSBs and promotes associations between complementary DNA termini . Heteroduplex intermediates of these recombination reactions have been visualized by electron microscopy, revealing the specific binding of multiple rings of RAD52 to the resected termini and the formation of large protein complexes at heteroduplex joints formed by RAD52-mediated annealing.

Appl Environ Microbiol, 2001 Oct, 67(10), 4752 - 9
Diversity of Geotrichum candidum strains isolated from traditional cheesemaking fabrications in France; Marcellino N et al.; The diversity of French fungus-ripened cheeses is due partly to the succession of fungi that colonize the cheese during ripening . Geotrichum candidum appears in the early stages of ripening on soft cheeses such as Camembert and semihard cheeses such as St . Nectaire and Reblochon . Its lipases and proteases promote flavor development, and its aminopeptidases reduce bitterness imparted by low-molecular-weight peptides in cheese . We assessed the genetic diversity of G . candidum strains by using random amplification of polymorphic DNA (RAPD)-PCR correlated with phenotypic tests for carbon assimilation and salt tolerance . Strains were isolated from milk, curd, and cheese collected in seven major cheesemaking regions of France . Sixty-four isolates were characterized . We found high genetic diversity of G . candidum even within the same cheesemaking regions . Strains did not group according to region . All of the strains from the Haute-Savoie were able to assimilate lactate as the sole source of carbon, while lactate assimilation varied among strains from the Auvergne . Strains varied in D-mannitol assimilation, and none used citrate as the sole source of carbon . Yeast-like colony morphology predominated in Reblochon, while all of the strains isolated from St . Nectaire were filamentous . The RAPD-PCR technique readily differentiated Geotrichum fragrans isolated from milk and curd in a St . Nectaire cheesemaking facility . This study reveals an enormous diversity of G . candidum that has been empirically selected through the centuries by the cheesemakers of France.

Appl Environ Microbiol, 2001 Oct, 67(10), 4701 - 7
Expression, gene cloning, and characterization of five novel phytases from four basidiomycete fungi: Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens; Lassen SF et al.; Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate . In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast . One full-length phytase-encoding cDNA was isolated from each library, except for the Ceriporia sp . library where two different phytase-encoding cDNAs were found . All five phytases were expressed in Aspergillus oryzae, purified, and characterized . The phytases revealed temperature optima between 40 and 60 degrees C and pH optima at 5.0 to 6.0, except for the P . lycii phytase, which has a pH optimum at 4.0 to 5.0 . They exhibited specific activities in the range of 400 to 1,200 U . mg, of protein(-1) and were capable of hydrolyzing phytate down to myo-inositol monophosphate . Surprisingly, (1)H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases . Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26).

Exp Cell Res, 2001 Oct 1, 269(2), 312 - 21
Transcription activating property of autoantigen SG2NA and modulating effect of WD-40 repeats; Zhu W et al.; Autoantibodies to intracellular proteins have been detected in sera of patients with various forms of cancer . Nuclear autoantigen SG2NA (S, G2 phase nuclear antigen) was isolated using autoantibodies from a patient with bladder and lung cancers and its expression is enhanced in the S and G2 phases of the cell cycle . Molecular cloning revealed that the C-terminal region of SG2NA contains six WD-40 repeats, motifs that are present in a large family of proteins with diverse functions . We show that the N-terminal region of SG2NA (aa 1-391) acted as a strong transcriptional activator in both yeast and mammalian cells . In contrast, the C-terminal WD-40 repeats had an inhibitory effect on transcription activation . We performed molecular swapping experiments by substituting the WD-40 repeats of SG2NA with those of yeast Met30 and Cdc4 and showed that the WD-40 regions from either Met30 or Cdc4 were capable of reproducing transcription repression function . The SG2NA WD-40 repeats were also able to repress basal level transcription and transactivation function of a GAL4-VP16 chimera . These observations suggest that some WD-40 repeats may have, as one of their functions, a negative regulatory role in the biological activities of their own and perhaps other proteins .

Biofactors, 2001, 14(1-4), 153 - 9
An analysis of cancer prevention by selenium; Combs GF Jr et al.; The nutritional functions of selenium (Se) are recognized as being due to a number of Se-containing proteins . It is not clear, however, whether any of these function in the anti-tumorigenic effects of Se most of which have been demonstrated for Se exposures greater than those required for selenoprotein expression . Indeed, other anti-tumorigenic mechanisms have been demonstrated for certain Se-metabolites . The Nutritional Prevention of Cancer Trial found supplemental Se (200 microg/day, as Se-enriched yeast) to be associated with significant reductions in cancer risks in subjects with pre-treatment plasma Se concentrations below ca . 120 ng/ml (1.5 nmoles/ml), which level would appear to require food-Se intakes of ca . 1.5 microg/kg body weight/day . However, the putative anti-carcinogenic Se-metabolite(s) should be more relevant than total plasma Se as a supplementation target for cancer prevention . These may be components of the non-protein-bound fraction of Se in plasma, which constitutes 2-4% of total plasma Se.

J Biol Chem, 2001 Dec 7, 276(49), 45677 - 85
A transcriptionally inactive E2F-1 targets the MDM family of proteins for proteolytic degradation; Strachan GD et al.; E2F-1-activated transcription promotes cell cycle progression and apoptosis . These functions are regulated by several factors including the E2F-1-binding protein MDM2 and the retinoblastoma protein pRb . Using a yeast two-hybrid screen we have identified the MDM2-related protein, MDMX, as an E2F-1-binding protein . In these studies we find that coexpression of MDMX with E2F-1 results in degradation of the MDMX protein . Although this proteolytic degradation can be blocked by the protease inhibitors bafilomycin A(1), N-acetyl-Leu-Leu-Norleu-AL, and N-acetyl-Leu-Leu-Met-AL, MDMX degradation is not inhibited by lactacystin, suggesting that degradation occurs by a proteasome-independent mechanism . Using an E2F-1 deletion mutant (E2F-1(180-437)) we show that E2F-1-targeted degradation of MDMX does not require the E2F-1 DNA binding domain and therefore is independent of E2F-1-driven transcription . We also find that this transcriptionally inactive E2F-1 mutant is capable of degrading the MDMX-related protein MDM2 and the MDMX isoform MDMX-S . Mapping of the E2F-1 C terminus reveals that neither a previously characterized C-terminal MDM2 binding domain nor the pRb binding domain on E2F-1 is required for MDMX and MDM2 degradation.

J Biol Chem, 2001 Nov 23, 276(47), 43818 - 23 Epub 2001 Sep 20.
Characterization of the human beta -glucan receptor and its alternatively spliced isoforms; Willment JA et al.; beta-1,3-d-Glucans are biological response modifiers with potent effects on the immune system . A number of receptors are thought to play a role in mediating these responses, including murine Dectin-1, which we recently identified as a beta-glucan receptor . In this study we describe the characterization of the human homologue of this receptor and show that it is structurally and functionally similar to the mouse receptor . The human beta-glucan receptor is a type II transmembrane receptor with a single extracellular carbohydrate recognition domain and an immunoreceptor tyrosine activation motif in its cytoplasmic tail . The human beta-glucan receptor is widely expressed and functions as a pattern recognition receptor, recognizing a variety of beta-1,3- and/or beta-1,6-linked glucans as well as intact yeast . In contrast to the murine receptor, the human receptor mRNA is alternatively spliced, resulting in two major (A and B) and six minor isoforms . The two major isoforms differ by the presence of a stalk region separating the carbohydrate recognition domain from the transmembrane region and are the only isoforms that are functional for beta-glucan binding . The human receptor also binds T-lymphocytes at a site distinct from the beta-glucan binding site, indicating that this receptor can recognize both endogenous and exogenous ligands.

EMBO J, 2001 Sep 17, 20(18), 5232 - 41
Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3; Jacobs SA et al.; Recent studies show that heterochromatin-associated protein-1 (HP1) recognizes a 'histone code' involving methylated Lys9 (methyl-K9) in histone H3 . Using in situ immunofluorescence, we demonstrate that methyl-K9 H3 and HP1 co-localize to the heterochromatic regions of Drosophila polytene chromosomes . NMR spectra show that methyl-K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain . This interaction requires methyl-K9 to reside within the proper context of H3 sequence . NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues . Using fluorescence anisotropy and isothermal titration calorimetry, we determined that this interaction occurs with a K(D) of approximately 100 microM, with the binding enthalpically driven . A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3-binding interface, and abolishes methyl-K9 H3 tail binding . Finally, we note that sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation . For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl- K9 H3, but instead shows preference for unmodified H3 tail.

Toxicol In Vitro, 2001 Aug-Oct, 15(4-5), 477 - 88
Fish cell lines as versatile tools in ecotoxicology: assessment of cytotoxicity, cytochrome P4501A induction potential and estrogenic activity of chemicals and environmental samples; Fent K; In vitro systems such as primary cells and cell lines are of growing importance in ecotoxicology . Cells from different tissues and species of fish are used for the assessment of toxic action of chemicals and evaluation of environmental samples . For organotins and substituted phenols, we have found that the in vitro cytotoxicity is positively correlated with the acute toxicity in vivo, and therefore cytotoxicity assays may serve as an alternative for acute fish toxicity testing . We have been using the hepatocellular carcinoma (PLHC-1) cell line for the assessment of the cytochrome P4501A (CYP1A) induction potential of polyaromatic hydrocarbons (PAHs), nitro-PAHs and azaarenes . For these compounds, the CYP1A induction potential is found to be related to the molecular structure and lipophilicity . In mixtures, CYP1A induction of individual compounds is additive . Based on the comparative investigation of the induction potential we derived an induction equivalency (IEQ) concept that can be applied for the evaluation of environmental samples such as landfill leachates, sediments and motorway runoffs . Fish cell lines are also valuable, rapid and cost-effective tools for the assessment of estrogenic activity of chemicals and environmental samples . We have developed an estrogen-responsive reporter gene system using the rainbow trout gonad cell line RTG-2, in which an estrogen receptor beta form is expressed at very low levels, but is not inducible . As the estrogenic activity is dependent on the cellular level of estrogen receptor (ER), ER has to be co-transfected in transient transfections in addition to an estrogen-responsive reporter gene . Using a dual luciferase system, the estrogenic activity of 12 compounds including alkylphenols, DDT-isomers and its metabolites have been assessed . Our system shows a high sensitivity with a detection limit of 0.05 nM estradiol and is therefore more sensitive than many other mammalian or yeast systems . The relative estrogenic activity (e.g . o,p'-DDT) and other toxicological effects may differ from those in mammalian systems, indicating that a risk evaluation for fish could only be meaningfully assessed in fish-specific systems . This paper illustrates the versatility and high potential of fish cell lines in ecotoxicology.

Toxicol In Vitro, 2001 Aug-Oct, 15(4-5), 421 - 5
Measurement of estrogenic activity of chemicals for the development of new dental polymers; Hashimoto Y et al.; The estrogenic activities of 13 Bisphenol-A (BPA)-related chemicals for development of new polymers by three in vitro bioassay have been examined in the presence and absence of a post-mitochondrial metabolizing system (S9 mix) . BPA, Bisphenol-B (BPB), Bisphenol-F (BPF), Bisphenol-S (BPS), 4,4-ethylidenebisphenol (BP1), 4,4-dihydroxybenzophenone (BP2), 2,2-bis (4-hydroxyphenyl)-hexafluoropropane (BP3), 4,4-(1,4-phenylenediisopropylidene) bisphenol (BP4), 4,4-cyclohexylidenebisphenol (BP5), 4,4-dihydroxydiphenyl ether (BP6), 4-hydroxydiphenylmethane (BP7), 4-cumylphenol (BP8) and 4,4-dihydroxydiphenyl sulfide (BP9) were each diluted with dimethyl sulfoxide to final concentrations ranging from 10(-7) to 10(-3) M in both the yeast two-hybrid system and in a fluorescence polarization system . Dilutions of 10(-9) to 10(-4) M were assayed in the E-screen, respectively . Except for BPS and BP4, the chemicals tested showed estrogenic activity in the absence of cut S9 mix preparation and the activity was enhanced with S9 mix . BPS, which was initially negative, was active with S9 mix in the yeast two-hybrid system . BP2 was weakly estrogenic with or without S9 mix . Chemicals other than BP2 were positive in the competition binding assay . All chemicals tested showed estrogenic activity in the E-screen, the concentration level of which was 10(4) times lower than those of the other two assays.

Toxicol In Vitro, 2001 Aug-Oct, 15(4-5), 413 - 9
Endocrine disrupters--testing strategies to assess human hazard; Baker VA; During the last decade an hypothesis has been developed linking certain chemicals (natural and synthetic) to observed and suspected adverse effects on reproduction in both wildlife and humans . The issue of 'endocrine disruption' originally focused on chemicals that mimic the action of the natural hormone oestrogen . However, the concern is now encompassing effects on the whole endocrine system . In response to public awareness, regulatory agencies (including the US EPA) and the OECD are formulating potential testing strategies and have begun the process of validating defined tests to systematically assess chemicals for their endocrine-disrupting activities . In order to investigate chemicals that have the potential to cause endocrine disruption, a large number of in vitro and in vivo assays have been identified . In vitro test systems (particularly when used in combination) offer the possibility of providing an early screen for large numbers of chemicals and can be useful in characterising the mechanism of action and potency . In vitro assays in widespread use for the screening/characterisation of endocrine disrupting potential include hormone receptor ligand binding assays (determination of the ability of a chemical to bind to the hormone receptor), cell proliferation assays (analysis of the ability of a chemical to stimulate growth of oestrogen sensitive cells), reporter gene assays in yeast or mammalian cells (analysis of the ability of a chemical to stimulate the transcription of a reporter gene construct in cell culture), and the analysis of the regulation of endogenous oestrogen sensitive genes in cell lines . However, in vitro assays do not always reliably predict the outcome in vivo due to differences in metabolic capabilities of the test systems used and the diverse range of mechanisms by which endocrine disrupting chemicals may act . Therefore a complementary battery of short- and long-term in vitro and in vivo assays (that assess both receptor and non-receptor mediated mechanisms of action) seems the most appropriate way at present of assessing the potential endocrine disrupting activities of chemicals . At Unilever we have used a combination of in vitro assays (receptor binding, reporter gene and cell proliferation assays) together with short-term in vivo tests (uterotrophic assay in immature rodents) to examine the oestrogenic potential of a large number of chemicals . An evaluation of the advantages and limitations of these methods is provided . Finally, any potential test system needs to be validated and standardized before the information generated can be for the identification of hazard, and possibly for risk assessment purposes.

Cancer Genet Cytogenet, 2001 Sep, 129(2), 112 - 9
Molecular characterization of the breakpoint region associated with a constitutional t(2;15)(q34;q26) in a patient with multiple myeloma; Kitamura E et al.; The molecular cloning of the translocation breakpoints from constitutional chromosome rearrangements in patients with a variety of human diseases has consistently led to the isolation of genes important in the development of the phenotype . We used fluorescence in situ hybridization (FISH) to analyze the breakpoint region of a constitutional chromosome translocation involving regions 2q34 and 15q26 observed in a patient with multiple myeloma (MM), a malignant disorder of plasma cells secreting monoclonal immunoglobulin . FISH analysis of this rearrangement showed that the chromosome 2-specific yeast artificial chromosome (YAC) 914E7 and the chromosome 15-specific YAC 757H6 span the translocation breakpoints, respectively . In order to characterize the location of the breakpoints further, somatic cell hybrids were constructed between mouse NIH3T3 cells and t(2;15)-bearing lymphoblastoid cells . Using these somatic cell hybrids, we have shown that the breakpoint on chromosome 2 lies between D2S3007 and D2S3004 and the chromosome 15 breakpoint lies between D15S107 and WI5967 (D15S836) . YAC fragmentation has been used to define a 350 kb region containing the 15q26 breakpoint.

Int J Parasitol, 2001 Oct, 31(12), 1381 - 91
Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes; Taraschi TF et al.; During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium . Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane . Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system . It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations . The identification of: (1) a P . falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P . falciparum erythrocyte membrane protein 1 and P . falciparum erythrocyte membrane protein 3 in the host cell cytosol of P . falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane . The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings . These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible . It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.

Int J Parasitol, 2001 Oct, 31(12), 1343 - 53
Endocytosis in different lifestyles of protozoan parasitism: role in nutrient uptake with special reference to Toxoplasma gondii; Robibaro B et al.; A fundamental property of any eukaryotic cell is endocytosis, that is the ability to take up external fluid, solutes and particulate matter into membrane-bound intracellular vesicles by various mechanisms . Toxoplasma gondii is an intracellular protozoan parasite of the phylum Apicomplexa with a wide geographical and host range distribution . Significant progress in studying the cell biology of this parasite has been accomplished over the last few years . Only recently endocytic compartments and endocytic trafficking have come to a closer dissection in T . gondii . In this review, we discuss the evidence for an endocytic compartment and present a model for an endocytic pathway in Toxoplasma against a background of endocytosis in kinetoplastida and the extensive insights gained from mammalian and yeast cells.

Physiol Behav, 2001 Aug, 73(5), 859 - 71
Murine models for Down syndrome; Dierssen M et al.; The availability of the recently published DNA sequence of human chromosome 21 (HSA21) is a landmark contribution that will have an immediate impact on the study of the role of specific genes to Down syndrome (DS) . Trisomy 21 or DS is the only autosomal aneuploidy that is not lethal in the fetal or early postnatal period . DS phenotypes show variable penetrance, affecting many different organs, including brain (mental retardation, early onset of Alzheimer's disease, AD), muscle (hypotonia), skeleton, and blood . DS phenotypes may stem directly from the cumulative effect of overexpression of specific HSA21 gene products or indirectly through the interaction of these gene products with the whole genome, transcriptome, or proteome . Mouse genetic models have played an important role in the elucidation of the contribution of specific genes to the DS phenotype . To date, the strategies used for modeling DS in mice have been three: (1) to assess single-gene contributions to DS phenotype, using transgenic techniques to create models overexpressing single or combinations of genes, (2) to assess the effects of overexpressing large foreign DNA pieces, introduced on yeast artificial chromosomes (YACs) or bacterial artificial chromosomes (BACs) into transgenic mice, and (3) mouse trisomies that carry all or part of MMU16, which has regions of conserved homology with HSA21 . Here we review the existing murine models and the relevance of their contribution to DS research.

Structure (Camb), 2001 Sep, 9(9), 759 - 64
DNA lesion bypass polymerases open up; Beard WA et al.; Structures of catalytic fragments of two DNA lesion bypass DNA polymerases, yeast DNA polymerase eta and an archeon DinB homolog, have recently been solved . These structures share several common architectural and structural features observed in other DNA polymerases, including a hand-like architecture with fingers, palm, and thumb subdomains . The new structures provide the first structural insights into DNA lesion bypass . The fingers and thumb are smaller than those in other DNA polymerases . Modeled substrates suggest that the fingers in the vicinity of the incoming nucleotide is closed, a conformation not previously observed for an unliganded polymerase . However, the template binding pocket appears to be more open, indicating that for DNA polymerase eta, a covalently linked thymine-thymine dimer could be accommodated.

RNA, 2001 Sep, 7(9), 1192 - 212
Computational modeling of eukaryotic mRNA turnover; Cao D et al.; The process of eukaryotic gene expression involves a diverse number of steps including transcription, RNA processing, transport, translation, and mRNA turnover . A critical step in understanding this process will be the development of mathematical models that quantitatively describe and predict the behavior of this complex system . We have simulated eukaryotic mRNA turnover in a linear multicomponent model based on the known mRNA decay pathways in yeast . Using rate constants based on experimental data for the yeast unstable MFA2 and stable PGK1 transcripts, the computational modeling reproduces experimental observations after minor adjustments . Subsequent analysis and a series of in silico experiments led to several conclusions . First, we demonstrate that mRNA half-life as commonly measured underestimates the average life span of an mRNA . Second, due to the properties of the pathways, the measurement of a half-life can predominantly measure different steps in the decay network . A corollary of this fact is that different mRNAs will be affected differentially by changes in specific rate constants . Third, the way to obtain the largest change of levels of mRNA for the smallest changes in rate is by changing the rate of deadenylation, where a large amount of regulation of mRNA decay occurs . Fourth, the 3'-to-5' degradation of mRNA shows mRNA-specific rates of degradation that are dependent on the 5' structure of the mRNA . These programs can be run over the Web, are adaptable to other eukaryotes, and provide outputs as graphs and virtual northern gels, which can be directly compared to experimental data . Therefore, this model constitutes a useful tool for the quantitative analysis of the process and control of mRNA degradation in eukaryotic cells.

Nature, 2001 Sep 20, 413(6853), 327 - 31
RNA-binding protein Nrd1 directs poly(A)-independent 3'-end formation of RNA polymerase II transcripts; Steinmetz EJ et al.; A eukaryotic chromosome contains many genes, each transcribed separately by RNA polymerase (pol) I, II or III . Transcription termination between genes prevents the formation of polycistronic RNAs and anti-sense RNAs, which are generally detrimental to the correct expression of genes . Terminating the transcription of protein-coding genes by pol II requires a group of proteins that also direct cleavage and polyadenylation of the messenger RNA in response to a specific sequence element, and are associated with the carboxyl-terminal domain of the largest subunit of pol II (refs 1, 2, 3, 4, 5, 6) . By contrast, the cis-acting elements and trans-acting factors that direct termination of non-polyadenylated transcripts made by pol II, including small nucleolar and small nuclear RNAs, are not known . Here we show that read-through transcription from yeast small nucleolar RNA and small nuclear RNA genes into adjacent genes is prevented by a cis-acting element that is recognized, in part, by the essential RNA-binding protein Nrd1 . The RNA-binding protein Nab3, the putative RNA helicase Sen1, and the intact C-terminal domain of pol II are also required for efficient response to the element . The same proteins are required for maintaining normal levels of Nrd1 mRNA, indicating that these proteins may control elongation of a subset of mRNA transcripts.

Nature, 2001 Sep 20, 413(6853), 316 - 22
Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line; Strohmaier H et al.; Cyclin E, one of the activators of the cyclin-dependent kinase Cdk2, is expressed near the G1-S phase transition and is thought to be critical for the initiation of DNA replication and other S-phase functions . Accumulation of cyclin E at the G1-S boundary is achieved by periodic transcription coupled with regulated proteolysis linked to autophosphorylation of cyclin E . The proper timing and amplitude of cyclin E expression seem to be important, because elevated levels of cyclin E have been associated with a variety of malignancies and constitutive expression of cyclin E leads to genomic instability . Here we show that turnover of phosphorylated cyclin E depends on an SCF-type protein-ubiquitin ligase that contains the human homologue of yeast Cdc4, which is an F-box protein containing repeated sequences of WD40 (a unit containing about 40 residues with tryptophan (W) and aspartic acid (D) at defined positions) . The gene encoding hCdc4 was found to be mutated in a cell line derived from breast cancer that expressed extremely high levels of cyclin E.

Mol Cell Biol, 2001 Oct, 21(20), 7010 - 9
MSY2 and MSY4 bind a conserved sequence in the 3' untranslated region of protamine 1 mRNA in vitro and in vivo; Giorgini F et al.; Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells . We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4 . We show here that MSY2 and MSY4 bind a sequence, 5'-UCCAUCA-3', present in the 3' untranslated region of the translationally repressed protamine 1 (Prm1) mRNA . Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site . Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay . A consensus site of U(AC)C(A)CAU(C)CA(CU) (subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis . These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system . Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene . The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.

Mol Cell Biol, 2001 Oct, 21(20), 6984 - 98
Novel meiosis-specific isoform of mammalian SMC1; Revenkova E et al.; Structural maintenance of chromosomes (SMC) proteins fulfill pivotal roles in chromosome dynamics . In yeast, the SMC1-SMC3 heterodimer is required for meiotic sister chromatid cohesion and DNA recombination . Little is known, however, about mammalian SMC proteins in meiotic cells . We have identified a novel SMC protein (SMC1beta), which-except for a unique, basic, DNA binding C-terminal motif-is highly homologous to SMC1 (which may now be called SMC1alpha) and is not present in the yeast genome . SMC1beta is specifically expressed in testes and coimmunoprecipitates with SMC3 from testis nuclear extracts, but not from a variety of somatic cells . This establishes for mammalian cells the concept of cell-type- and tissue-specific SMC protein isoforms . Analysis of testis sections and chromosome spreads of various stages of meiosis revealed localization of SMC1beta along the axial elements of synaptonemal complexes in prophase I . Most SMC1beta dissociates from the chromosome arms in late-pachytene-diplotene cells . However, SMC1beta, but not SMC1alpha, remains chromatin associated at the centromeres up to metaphase II . Thus, SMC1beta and not SMC1alpha is likely involved in maintaining cohesion between sister centromeres until anaphase II.

Mol Cell Biol, 2001 Oct, 21(20), 6841 - 50
Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1; Connor JH et al.; The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library . Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated I-1(1-171) . Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding . In contrast, PKA phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment . Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483) . By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus . While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase . In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated . In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited . These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.

J Cell Biol, 2001 Sep 17, 154(6), 1209 - 23
The actin-binding protein Hip1R associates with clathrin during early stages of endocytosis and promotes clathrin assembly in vitro; Engqvist-Goldstein AE et al.; Huntingtin-interacting protein 1 related (Hip1R) is a novel component of clathrin-coated pits and vesicles and is a mammalian homologue of Sla2p, an actin-binding protein important for both actin organization and endocytosis in yeast . Here, we demonstrate that Hip1R binds via its putative central coiled-coil domain to clathrin, and provide evidence that Hip1R and clathrin are associated in vivo at sites of endocytosis . First, real-time analysis of Hip1R-YFP and DsRed-clathrin light chain (LC) in live cells revealed that these proteins show almost identical temporal and spatial regulation at the cell cortex . Second, at the ultrastructure level, immunogold labeling of 'unroofed' cells showed that Hip1R localizes to clathrin-coated pits . Third, overexpression of Hip1R affected the subcellular distribution of clathrin LC . Consistent with a functional role for Hip1R in endocytosis, we also demonstrated that it promotes clathrin cage assembly in vitro . Finally, we showed that Hip1R is a rod-shaped apparent dimer with globular heads at either end, and that it can assemble clathrin-coated vesicles and F-actin into higher order structures . In total, Hip1R's properties suggest an early endocytic function at the interface between clathrin, F-actin, and lipids.

Biochem Biophys Res Commun, 2001 Sep 28, 287(3), 733 - 8
The PX domain as a novel phosphoinositide- binding module; Ago T et al.; The phox (phagocyte oxidase) homology (PX) domain occurs in the mammalian phox proteins p40(phox) and p47(phox), the polarity establishment protein Bem1p in budding yeast, and a variety of proteins involved in membrane trafficking . Here we show that the PX domains of p40(phox) and p47(phox) directly bind to phosphoinositides: p40(phox) prefers Ptdlns(3)P, while p47(phox) does Ptdlns(4)P and Ptdlns(3,4)P(2) . In addition, the Bem1p PX domain also interacts with Ptdlns(4)P . When the p40(phox) PX domain is expressed as a fusion to green fluorescent protein in HeLa cells, it exists at early endosomes where Ptdlns(3)P is enriched . Furthermore, a mutant p40(phox) PX carrying the substitution of Lys for Arg105 only weakly binds to phosphoinositides in vitro, and fails to locate to early endosomes . Thus the PX domain functions as a novel phosphoinositide-binding module and likely participates in targeting of proteins to membranes .

Mol Pharmacol, 2001 Oct, 60(4), 725 - 31
Site-directed mutagenesis of m1-toxin1: two amino acids responsible for stable toxin binding to M(1) muscarinic receptors; Krajewski JL et al.; m1-Toxin1 binds specifically and irreversibly to M(1) muscarinic receptors and can slow the dissociation of {(3)H}N-methylscopolamine ({(3)H}NMS) from these receptors . Yet only 7 of its 65 amino acids are not conserved in six other mamba toxins that bind reversibly to M(2)-M(5) muscarinic receptors . Two of these seven residues (Phe(38), Lys(65)) were mutated to corresponding residues of the other toxins (Ile(38), Glu(65)), to evaluate amino acids in m1-toxin1 that confer its remarkable affinity and specificity . The cDNA for m1-toxin1 was cloned from venom gland mRNA using polymerase chain reaction (PCR)-based techniques . Its nucleotide sequence is remarkably similar to those of other short-chain neurotoxins . The cDNAs for mutant toxins Phe(38) to Ile(38) (F38I) and Lys(65) to Glu(65) (K65E) were constructed by PCR-based techniques . Each cDNA was expressed in yeast, and the toxins were purified from yeast media by cation-exchange and reversed phase chromatography . Recoveries were 40 to 152 microg/l . Recombinant m1-toxin1 was identical to the native toxin (observed mass: 7471 Da; irreversible blockade of {(3)H}NMS binding to cloned M(1) receptors at 25 degrees C; no blockade of M(2)-M(5) receptors; 6-fold slowing of {(3)H}NMS dissociation at 37 degrees C) . F38I also bound specifically to M(1) receptors, but reversibly and without effect on NMS dissociation . Thus, Phe(38) contributes to the stability of toxin-receptor complexes, but not to M(1)-selectivity . K65E bound selectively and irreversibly to unliganded M(1) receptors but did not slow NMS dissociation . It is suggested that the C-terminal Lys(65) of m1-toxin1 may contact an outer loop of the M(1) receptor.

Comb Chem High Throughput Screen, 2001 Nov, 4(7), 585 - 91
Predicting in vivo protein peptide interactions with random phage display; Smothers JF et al.; Binding sites in protein complexes occasionally map to small peptides within one or more proteins . Random peptide display methods simulate binding interactions by providing all possible peptide combinations with an equal opportunity to bind a protein of interest . The natural substrates for the protein are typically known in advance . However, it is often the case that such substrates are identified as putative partner proteins by using in vivo methods such as yeast two hybrid screening . Unfortunately, such methods often produce lengthy datasets of protein sequences and offer little mechanistic insight into how such interactions might take place in vivo . Here, we review an approach that addresses this problem . First, sequence alignment tools identify and characterize blocks of conserved sequences among peptides recovered during random peptide display . Next, searching programs detect similar blocks of conserved sequences within naturally occurring proteins to predict partner proteins . Finally, the significance of an interaction is tested using site specific mutagenesis, binding competition or co-immunoprecipitation experiments . This strategy should become increasingly powerful with the growing popularity of interaction studies, sequencing projects and microarray analyses in modern biology.

Yeast, 2001 Sep 30, 18(13), 1197 - 205
Genetic analysis of TAF68/61 reveals links to cell cycle regulators; Reese JC et al.; In yeast, inactivation of certain TBP-associated factors (TAF(II)s) results in arrest at specific stages of the cell cycle . In some cases, cell cycle arrest is not observed because overlapping defects in other cellular processes precludes the manifestation of an arrest phenotype . In the latter situation, genetic analysis has the potential to reveal the involvement of TAF(II)s in cell cycle regulation . In this report, a temperature-sensitive mutant of TAF68/61 was used to screen for high-copy dosage suppressors of its growth defect . Ten genes were isolated: TAF suppressor genes, TSGs 1-10 . Remarkably, most TSGs have either a genetic or a direct link to control of the G(2)/M transition . Moreover, eight of the 10 TSGs can suppress a CDC28 mutant specifically defective for mitosis (cdc28-1N) but not an allele defective for passage through start . The identification of these genes as suppressors of cdc28-1N has identified four unreported suppressors of this allele . Moreover, synthetic lethality is observed between taf68-9 and cdc28-1N . The isolation of multiple genes involved in the control of a specific phase of the cell cycle argue that the arrest phenotypes of certain TAF(II) mutants reflect their role in specifically regulating cell cycle functions .

J Biol Chem, 2001 Dec 7, 276(49), 46276 - 83 Epub 2001 Sep 17.
The Sos1-Rac1 signaling . Possible involvement of a vacuolar H(+)-ATPase E subunit; Miura K et al.; We have purified and identified a 32-kDa protein interacting with the Dbl oncogene homology domain of mSos1(Sos-DH) from rat brains by glutathione S-transferase-Sos-DH affinity chromatography . Peptide sequencing revealed that the protein is identical to a positive regulatory E subunit (V-ATPase E) of a vacuolar H(+)-ATPase, which is responsible for acidification of endosome and alkalinization of intracellular pH . The interaction between V-ATPase E and Sos-DH was confirmed by yeast two-hybrid assay . A coimmunoprecipitation assay demonstrated that a V-ATPase E protein physiologically bound to mSos1, and the protein was colocalized with mSos1 in the cytoplasm, as determined by immunohistochemistry . mSos1 was found in the early endosome fraction together with V-ATPase E and Rac1, suggesting the functional involvement of mSos1/V-ATPase E complexes in the Rac1 activity at endosomes . Overexpression of V-ATPase E in COS cells enhanced the ability of mSos1 to promote the guanine nucleotide exchange activity for Rac1 and stimulated the kinase activity of Jun kinase, a downstream target of Rac1 . Thus, the data indicate that V-ATPase E may participate in the regulation of the mSos1-dependent Rac1 signaling pathway involved in growth factor receptor-mediated cell growth control.

Genetics, 2001 Sep, 159(1), 159 - 72
The divergent Caenorhabditis elegans beta-catenin proteins BAR-1, WRM-1 and HMP-2 make distinct protein interactions but retain functional redundancy in vivo; Natarajan L et al.; beta-Catenins function both in cell adhesion as part of the cadherin/catenin complex and in Wnt signal transduction as transcription factors . Vertebrates express two related proteins, beta-catenin and plakoglobin, while Drosophila has a single family member, Armadillo . Caenorhabditis elegans expresses three beta-catenin-related proteins, BAR-1, HMP-2, and WRM-1, which are quite diverged in sequence from each other and other beta-catenins . While BAR-1 and WRM-1 are known to act in Wnt-mediated processes, and HMP-2 acts in a complex with cadherin/alpha-catenin homologs, it is unclear whether all three proteins retain the other functions of beta-catenin . Here we show that BAR-1, like vertebrate beta-catenin, has redundant transcription activation domains in its amino- and carboxyl-terminal regions but that HMP-2 and WRM-1 also possess the ability to activate transcription . We show via yeast two-hybrid analysis that these three proteins display distinct patterns of protein interactions . Surprisingly, we find that both WRM-1 and HMP-2 can substitute for BAR-1 in C . elegans when expressed from the bar-1 promoter . Therefore, although their mutant phenotypes and protein interaction patterns strongly suggest that the functions of beta-catenin in other species have been segregated among three diverged proteins in C . elegans, these proteins still retain sufficient similarity to display functional redundancy in vivo.

Genetics, 2001 Sep, 159(1), 147 - 57
Regulation of physiological rates in Caenorhabditis elegans by a tRNA-modifying enzyme in the mitochondria; Lemieux J et al.; We show that the phenotype associated with gro-1(e2400) comprises the whole suite of features that characterize the phenotype of the clk mutants in Caenorhabditis elegans, including deregulated developmental, behavioral, and reproductive rates, as well as increased life span and a maternal effect . We cloned gro-1 and found that it encodes a highly conserved cellular enzyme, isopentenylpyrophosphate:tRNA transferase (IPT), which modifies a subset of tRNAs . In yeast, two forms of the enzyme are produced by alternative translation initiation, one of which is mitochondrial . In the gro-1 transcript there are also two possible initiator ATGs, between which there is a sequence predicted to encode a mitochondrial localization signal . A functional GRO-1::GFP fusion protein is localized diffusely throughout the cytoplasm and nucleus . A GRO-1::GFP initiated from the first methionine is localized exclusively to the mitochondria and rescues the mutant phenotype . In contrast, a protein initiated from the second methionine is localized diffusely throughout the cell and does not rescue the mutant phenotype . As oxygen consumption and ATP concentration have been reported to be unaffected in gro-1 mutants, our observations suggest that GRO-1 acts in mitochondria and regulates global physiology by unknown mechanisms.

Genomics, 2001 Aug, 76(1-3), 30 - 6
X/autosomal translocations in the Xq critical region associated with premature ovarian failure fall within and outside genes; Mumm S et al.; Premature ovarian failure curtails female reproductive life and is often linked to balanced Xq/autosomal translocations in a critical region . We mapped regions around translocations at the edges of this zone (one in Xq13.3, two in Xq26) in large-insert clones and analyzed their sequence . One Xq26 region is extensively transcribed and, in agreement with a recent independent analysis, the breakpoint interrupts a gene that encodes a widely expressed peptidase . In contrast 430 kb around the second Xq26 breakpoint has no putative or detected gene content . In 260 kb around the Xq13 translocation, the breakpoint falls among a cluster of repetitive elements at least 59 kb from the only detected gene (a rarely expressed T-box family transcription factor) . We discuss our results in relation to models that ascribe premature ovarian failure to interruption of ovarian genes or to a failure of interactions involving DNA of the critical region during follicle development.

J Cell Sci, 2001 Jul, 114(Pt 13), 2449 - 60
Rab7 regulates phagosome maturation in Dictyostelium; Rupper A et al.; A Dictyostelium Rab7 homolog has been demonstrated to regulate fluid-phase influx, efflux, retention of lysosomal hydrolases and phagocytosis . Since Rab7 function appeared to be required for efficient phagocytosis, we sought to further characterize the role of Rab7 in phagosomal maturation . Expression of GFP-Rab7 resulted in labeling of both early and late phagosomes containing yeast, but not forming phagocytic cups . In order to determine if Rab7 played a role in regulating membrane traffic between the endo/lysosomal system and maturing phagosomes, latex bead containing (LBC) phagosomes were purified from wild-type cells at various times after internalization . Glycosidases, cysteine proteinases, Rab7 and lysosomally associated membrane proteins were delivered rapidly to nascent phagosomes in control cells . LBC phagosomes isolated from cells overexpressing dominant negative (DN) Rab7 contained very low levels of LmpA (lysosomal integral membrane protein) and alpha-mannosidase was not detectable . Interestingly, cysteine proteinases were delivered to phagosomes as apparent pro-forms in cells overexpressing DN Rab7 . Despite these defects, phagosomes in cells overexpressing DN Rab7 matured to form multi-particle spacious phagosomes, except that these phagosomes remained significantly more acidic than control phagosomes . These results suggested that Rab7 regulates both an early and late steps of phagosomal maturation, similar to its role in the endo/lysosomal system.

J Org Chem, 2001 Sep 21, 66(19), 6217 - 28
Substrate specificity of the glycosyl donor for oligosaccharyl transferase; Tai VW et al.; Oligosaccharyl transferase (OT) catalyzes the co-translational transfer of a dolichol-linked tetradecasaccharide (Dol-PP-GlcNAc(2)Man(9)Glc(3), 1a) to an asparagine side chain of a nascent polypeptide inside the lumen of the endoplasmic reticulum (ER) . The glycosyl acceptor requires an Asn-Xaa-Thr/Ser sequon, where Xaa can be any natural amino acid except proline, for N-linked glycosylation to occur . To address the substrate specificity of the glycosyl donor, three unnatural dolichol-linked disaccharide analogues (Dol-PP-GlcNTFA-GlcNAc 1c, Dol-PP-2DFGlc-GlcNAc 1d, and Dol-PP-GlcNAc-Glc 1e) were synthesized and evaluated as substrates or inhibitors for OT from yeast . The synthetic analogue Dol-PP-GlcNAc-Glc 1e, with substitution in the distal sugar, was found to be a substrate (K(m)(app)() = 26 microM) for OT . On the other hand, the analogues Dol-PP-GlcNTFA-GlcNAc 1c (K(i) = 154 microM) and Dol-PP-2DFGlc-GlcNAc 1d (K(i) = 252 microM), with variations in the proximal sugar, were inhibitors for OT . The dolichol-linked monosaccharide Dol-PP-GlcNAc 3 was found to be the minimum unit for glycosylation to occur.

FEBS Lett, 2001 Sep 7, 505(1), 92 - 6
Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells; Kausalya PJ et al.; Zonula occludens protein 1 (ZO-1) is a cytosolic tight junction protein that tethers transmembrane proteins such as occludin, claudin and junctional adhesion molecule to the actin cytoskeleton . The interaction between ZO-1 and claudin or junctional adhesion molecule occurs via the amino-terminal PSD95/Dlg/ZO-1 (PDZ) domains in ZO-1 . A yeast two-hybrid screen to search for proteins that interact with the PDZ domains of ZO-1 identified connexin (Cx) 45 . Cx45 interacts with the PDZ domains of ZO-1 and ZO-3, but not ZO-2, via a short C-terminal PDZ binding motif (SVWI) . In transfected epithelial Madin-Darby canine kidney cells, Cx45 co-localizes with endogenous ZO-1 at or near tight junctions and co-precipitation experiments show that Cx45 and ZO-1 directly interact . Inactivating the C-terminal PDZ-binding motif in Cx45 affects its co-precipitation and co-localization with ZO-1 . The growing number of connexins (i.e . Cx43 and Cx45) that can associate with ZO proteins indicate that ZO proteins may play a more general role in organizing gap junctions and/or in recruiting signaling molecules that regulate intercellular communication.

FEBS Lett, 2001 Sep 7, 505(1), 81 - 6
Homodimerization of presenilin N-terminal fragments is affected by mutations linked to Alzheimer's disease; Cervantes S et al.; Mutations on human presenilins 1 and 2 cause dominant early-onset familial Alzheimer's disease (FAD) . Presenilins are polytopic transmembrane proteins endoproteolytically processed in vivo to N- and C-terminal fragments (NTFs and CTFs) . The functional presenilin unit consists of a high molecular weight complex that contains both fragments . Here we show NTF:NTF, CTF:CTF and NTF:CTF interactions by yeast two-hybrid and in vivo endoplasmic reticulum split-ubiquitin assays . Our results also highlight the involvement of HL1--the hydrophilic loop between TMI and TMII--in the NTF:NTF binding site . Besides, nine FAD-linked presenilin mutations substantially affected HL1:HL1 binding . From the evidence of NTF and CTF homodimerization, we propose the contribution of two NTFs and two CTFs, instead of a single NTF:CTF heterodimer, to the functional presenilin-gamma-secretase complex and that FAD mutations affect the assembly or stability of this complex.

FEBS Lett, 2001 Sep 7, 505(1), 7 - 12
Antisense inhibition of Chk2/hCds1 expression attenuates DNA damage-induced S and G2 checkpoints and enhances apoptotic activity in HEK-293 cells; Yu Q et al.; The cellular response to DNA damage involves checkpoint controls that delay cell cycle progression in order to provide time for repair of damaged DNA . Chk2/hCds1 is a recently identified homolog of the yeast Cds1 kinase that is involved in cell cycle checkpoint response to DNA damage . To investigate the functions of Chk2/hCds1 in response to DNA damage in mammalian cells, we established a stable human kidney embryonic cell line (HEK-293) that expresses antisense Chk2/hCds1 (Chk2AS) under the control of an inducible promoter . Cells that express Chk2AS display defective S-phase delay in response to DNA replication-mediated DNA damage induced by the topoisomerase I inhibitor camptothecin . The defective G2 checkpoint was also observed in Chk2AS cells exposed to the DNA damaging agent VP-16 or gamma-radiation . Enhanced apoptosis was observed in Chk2AS cells after exposure to gamma-radiation or camptothecin . No p53 activation was observed after DNA damage in HEK-293 or Chk2AS cells . Our results indicate that perturbation of Chk2/hCds1 expression adversely affects the S- and G2-phase checkpoints following DNA damage or DNA replication block, and suggest that reduced expression of Chk2/hCds1 might promote a p53-independent apoptotic response.

Arch Biochem Biophys, 2001 Sep 15, 393(2), 271 - 80
First simultaneous isolation of a ribosome inactivating protein and an antifungal protein from a mushroom (Lyophyllum shimeji) together with evidence for synergism of their antifungal effects; Lam SK et al.; From the fruiting bodies of the mushroom Lyophyllum shimeji, a novel ribosome inactivating protein with a molecular weight of 20 kDa and exhibiting antifungal activity against Physalospora piricola (IC(50) = 2.5 microM) and Coprinus comatus was isolated . The protein, designated lyophyllin, was purified by ion exchange chromatography on CM-cellulose, affinity chromatography on Affi-gel Blue Gel, and then ion exchange chromatography on Mono S . Lyophyllin possessed an N-terminal sequence with some similarity to those of plant ribosome-inactivating proteins . It inhibited translation in rabbit reticulocyte lysate with an IC(50) of 1 nM, thymidine uptake by murine splenocytes with an IC(50) of 1 microM and HIV-1 reverse transcriptase activity with an IC(50) of 7.9 nM . Lyophyllin did not manifest ribonuclease or hemagglutinating activity . An antifungal protein, designated Lyophyllum antifungal protein (LAP), with a molecular weight of 14 kDa, and an N-terminal sequence somewhat analogous to those of angiosperm thaumatin-like proteins and thaumatins and an inactive variant of the ubiquitin-conjugating enzyme, was first isolated from Lyophyllum shimeji . LAP was adsorbed on CM-cellulose, Affi-gel blue gel, and Mono S . LAP exerted antifungal activity against P . piricola (IC(50) = 70 nM) and Mycosphaerella arachidicola but not against Rhizoctonia solani, Colletotrichum gossypii, and Coprinus comatus . It exerted very low translation inhibitory activity in a rabbit reticulocyte lysate system (IC(50) = 70 microM) and negligible ribonuclease activity toward yeast transfer RNA and hemagglutinating activity toward rabbit erythrocytes . It inhibited HIV-1 reverse transcriptase with an IC(50) of about 5.2 nM . A synergism in antifungal activities of LAP and lyophyllin against P . piricola was demonstrable .

Arch Virol, 2001 Jul, 146(7), 1415 - 26
Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 ORF50 gene product contains a potent C-terminal activation domain which activates gene expression via a specific target sequence; Wang S et al.; The ART (Activator of Replication and Transcription) protein of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus-8 (HHV-8), is encoded by the ORF50 gene . It is expressed as an immediate-early gene and plays a crucial role in the transition between latency and productive infection . HHV-8 ART is a transcriptional transactivator which can up-regulate viral gene expression . Transient expression assays showed that ART strongly activated ORF57 and K8 promoter-directed gene expression in both CV-1 and BJAB cells . The ART target site was mapped to a 40-bp region compassing nt 81904 to 81943 on the ORF57 promoter . When linked upstream to a heterologous SV40 promoter, this region by itself was able to confer ART responsiveness . This 40-bp segment contains a 16-bp consensus sequence which is also found in the K8 promoter region located between nt 74769 to 74784 . Deletion of the fragment including this 16-bp consensus abrogated the ART responsiveness of the K8 promoter . The role of this 16-bp consensus in ART transactivation was further supported by site-directed mutagenesis . Mutations of the conserved nucleotides within the 16-bp consensus in the ORF57 promoter dramatically impaired its responsiveness to ART . Fusion protein analysis with chimeric proteins containing the DNA binding domain of yeast transactivator Gal4 (residues 1 to 147) and different ART segments defined an acidic C-terminal region (amino acids {aa} 527 to 634) as a potent activator . Deletions of this activation domain in the ART protein resulted in a decrease or loss of its ability to activate ORF57 and K8 promoters containing the ART responsive element in transfected cells . How the ART activation domain activates ORF57 and K8 gene expression through the 16-bp consensus sequence remains to be determined.

J Biol Chem, 2001 Nov 30, 276(48), 44905 - 11 Epub 2001 Sep 12.
The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase; Nishitani H et al.; S-phase onset is controlled, so that it occurs only once every cell cycle . DNA is licensed for replication after mitosis in G(1), and passage through S-phase removes the license to replicate . In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replication occurs once per cell cycle . We show that the human Cdt1 homologue (hCdt1), a nuclear protein, is present only during G(1) . After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G(2) . hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages . hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1 . Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase . In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis . These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells.

J Biol Chem, 2001 Nov 16, 276(46), 43471 - 81 Epub 2001 Sep 12.
Binding of 14-3-3beta regulates the kinase activity and subcellular localization of testicular protein kinase 1; Toshima JY et al.; Testicular protein kinase 1 (TESK1) is a serine/threonine kinase that phosphorylates cofilin and induces actin cytoskeletal reorganization . The kinase activity of TESK1 is stimulated by integrin-mediated signaling pathways, but the mechanism of regulation has remained unknown . By using the yeast two-hybrid system, we identified 14-3-3beta to be the binding protein of TESK1 . Specific interaction between TESK1 and 14-3-3beta became evident in in vitro and in vivo co-precipitation assays . 14-3-3beta interacts with TESK1 through the C-terminal region of TESK1 and in a manner dependent on the phosphorylation of Ser-439 within an RXXSXP motif . Binding of 14-3-3beta inhibited the kinase activity of TESK1 . During cell spreading on fibronectin, the TESK1/14-3-3beta interaction significantly decreased, in a time course that inversely correlated with increase in TESK1 kinase activity . Thus, the dissociation of 14-3-3beta from a TESK1/14-3-3beta complex is likely to be involved in the integrin-mediated TESK1 activation . In HeLa cells, TESK1, together with 14-3-3beta, accumulated at the cell periphery when cells were plated on fibronectin, whereas they were diffusely distributed in the cytoplasm in the case of non-stimulated cells . We propose that 14-3-3beta plays important roles in regulating the kinase activity of TESK1 and localizing TESK1 to cell adhesion sites following integrin stimulation.

Hum Mol Genet, 2001 Sep 1, 10(18), 1945 - 52
Telomere maintenance by telomerase and by recombination can coexist in human cells; Cerone MA et al.; Immortal human cells maintain their telomeres by two independent mechanisms, a prevalent one dependent on de novo synthesis of telomeric DNA by telomerase, and a rarer one based on telomere recombination {alternative lengthening of telomeres (ALT)} . Studies with yeast have indicated that expression of telomerase inhibits telomere recombination . In the present study, we have investigated whether expression of telomerase in cells that use ALT would similarly reveal dominance of telomere elongation by telomerase over telomere recombination . Telomerase-negative WI38 VA13/2RA ALT cells were reconstituted for telomerase activity through ectopic expression of the enzyme subunits, hTERT and hTR, and the presence and function of telomerase and ALT were monitored during long term cell growth by enzymatic assays, detection of the ALT-associated PML bodies (APBs) and analysis of telomere dynamics . Our results indicate that telomerase activity and APBs persisted in the cells over at least 90 population doublings . The activity of both pathways on telomeres was determined by analysis of telomere length versus time by gel electrophoresis and in situ hybridization . ALT cells are characterized by very heterogeneous telomeres with a much longer average size than the telomeres of telomerase-positive cells . Telomere dynamics in our cells were compatible with both ALT and telomerase being biologically active since the long telomeres typical of ALT were maintained, while short telomeres, thought to be the preferential substrate of telomerase, were elongated . These findings, indicating that human cells may be capable of concomitantly utilizing both mechanisms of telomere maintenance without effects on their growth and viability, have implications for cancer therapy.

Traffic, 2001 Sep, 2(9), 622 - 30
Ubiquitin sorts proteins into the intralumenal degradative compartment of the late-endosome/vacuole; Urbanowski JL et al.; Many studies have demonstrated a role for ubiquitin (Ub) in the down-regulation of cell surface proteins . In yeast, down-regulation is marked by the internalization of proteins, followed by their delivery to the lumen of the vacuole where both the cytosolic and lumenal domains are degraded . It is generally believed that the regulatory step of this process is internalization from the plasma membrane and that protein delivery to the lysosome or vacuole is by default . By separating the process of internalization from degradation, we demonstrate that incorporation of proteins into intralumenal vesicles represents a distinct sorting step along the endocytic pathway that is controlled by recognition of ubiquitin . We show that attachment of a single ubiquitin can serve as a specific sorting signal for the degradative pathway by redirecting recycling Golgi proteins and resident vacuolar proteins into intralumenal vesicles of the yeast vacuole . This pathway is independent of PtdIns(3,5) P2 and does not rely on the specific composition of transmembrane domain segments . These data provide a physiological basis for how ubiquitination of cell surface proteins guides their degradation and removal from the recycling pathway.

Traffic, 2001 Sep, 2(9), 612 - 21
Late endosomes: sorting and partitioning in multivesicular bodies; Piper RC et al.; Late endosomes, which have the morphological characteristics of multivesicular bodies, have received relatively little attention in comparison with early endosomes and lysosomes . Recent work in mammalian and yeast cells has given insights into their structure and function, including the generation of their multivesicular morphology . Lipid partitioning to create microdomains enriched in specific lipids is observed in late endosomes, with some lumenal vesicles enriched in lysobisphosphatidic acid and others in phosphatidylinositol 3-phosphate . Sorting of membrane proteins into the lumenal vesicles may occur because of the properties of their trans-membrane domains, or as a result of tagging with ubiquitin . Yeast class E Vps proteins and their mammalian orthologs are the best candidates to make up the protein machinery that controls inward budding, a process that starts in early endosomes . Late endosomes are able to undergo homotypic fusion events and also heterotypic fusion with lysosomes, a process that delivers endocytosed macromolecules for proteolytic degradation.

Oral Microbiol Immunol, 2001 Oct, 16(5), 270 - 8
Salivary anticandidal activity and saliva composition in an HIV-infected cohort; Lin AL et al.; This study investigated salivary anticandidal activity and salivary composition in stimulated whole saliva of 18 advanced HIV-infected patients and compared these values to healthy controls . Stimulated whole saliva from HIV-infected patients showed decreased anticandidal activity . The flow rate was reduced by 40% as compared with controls . The saliva flow rate for HIV-infected patients who had recoverable yeast in their saliva was reduced as compared to HIV-infected patients without recoverable yeast . For HIV-infected patients, the saliva concentrations of lactoferrin, secretory IgA and Cl- were increased while the secretion rate of lysozyme, total protein and K+ were reduced . There was no difference in any parameter as a function of taking the antifungal drug fluconazole . There was no association between salivary anticandidal activity and any salivary component . This study shows reduced anticandidal activity and salivary flow rate in HIV-infected patients . These alterations may contribute to their increased incidence of oral candidal infections.

J Microsc, 2001 Sep, 203(Pt 3), 285 - 94
A new approach for cryofixation by high-pressure freezing; Studer D et al.; A newly designed high-pressure freezing machine for cryofixation was established and tested (Leica EMPACT), based on ideas originally proposed by Moor & Riehle in 1968 . The new machine, essentially an improved version of our prototype, pressurizes the sample to 2000 bar in a small container (using methylcyclohexane as hydraulic fluid) and at the same time cools the outer surface of the container with a jet of liquid nitrogen . The advantage of this approach is that the machine uses little liquid nitrogen and can be built small and light . The machine is able to vitrify and freeze well a variety of specimens, for example, plant leaves, yeast cells, liver or nerve tissue (more samples are shown at: Cooling efficiency is the same as in the traditional machines that use liquid nitrogen to pressurize and simultaneously cool the sample.

Genes Cells, 2001 Sep, 6(9), 815 - 24
Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen; Ibe S et al.; BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells . Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity . Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus . The N region would be synthesized with the large protein complex . RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT . The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo . TdT binds directly to a PCNA trimer, as shown by gel filtration . TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain . TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation . CONCLUSION: TdT interacts directly with PCNA through its DPD . A functional consequence of this interaction is the negative regulation of TdT activity . These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.

Genes Cells, 2001 Sep, 6(9), 743 - 63
Bir1/Cut17 moving from chromosome to spindle upon the loss of cohesion is required for condensation, spindle elongation and repair; Morishita J et al.; BACKGROUND: In mammals, proteins containing BIR domains (IAPs and survivin) are implicated in inhibiting apoptosis and sister chromatid separation . In the nematode, Bir1 is required for a proper localization of aurora kinase, which moves from the mitotic chromosome in metaphase to the spindle midzone in anaphase as a passenger . Fission yeast Bir1/Pbh1 is essential for normal mitosis . RESULTS: A temperature sensitive mutant cut17-275 exhibits the defect in condensation and spindle elongation at 36 degrees C, while securin is degraded . Gene cloning shows that the cut17+ gene is identical to bir1+/pbh1+ . At 26 degrees C, cut17-275 is UV sensitive as the repair of DNA damage is severely compromised . Bir1/Cut17 is a nuclear protein in interphase, which is then required for recruiting condensin to the mitotic nucleus, and concentrates to form a discrete number of dots from prometaphase to metaphase . Once the chromatids are separated, Bir1/Cut17 no longer binds to kinetochores and instead moves to the middle of spindle . Chromatin immunoprecipitation suggested that Bir1/Cut17 associates with the outer repetitious centromere region in metaphase . Following the initiation of anaphase the protein switches from being a chromosomal protein to a spindle protein . This transit is stringently regulated by the state of sister chromatid cohesion proteins Mis4 and Rad21 . Ark1, is an aurora kinase homologue whose mitotic distribution is identical to, and under the control of Bir1/Cut17 . CONCLUSIONS: Bir1/Cut17 and Ark1 act as "passengers" but they may play a main role as a recruitment factor, essential for condensation, spindle elongation and DNA repair . Bir1/Cut17 should have roles both in mitotic and in interphase chromosome . The proper location of Ark1 requires Bir1/Cut17, and the mitotic localization of Bir1/Cut17 requires sister cohesion.

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