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