Prikl Biokhim Mikrobiol, 2001 May-Jun, 37(3), 289 - 91 {Study of the lytic activity of actinomycetes isolated from various soils in Georgia}; Kudukhashvili PG et al.; The lytic activities of 310 cultures from the Collection of Actinomycetes of the Institute of Biochemistry and Biotechnology, National Academy of Sciences of Georgia, were studied; 18% of these strains appeared capable of lysing the yeast cell wall . The active producer of the enzyme was selected . This culture was isolated from chestnut soil in Gardabani raion (Central Georgia) . Its cultural-morphological, biochemical, and antagonistic properties allowed the culture to be ascribed to the species Geodermatophilus obseurus Luedemann, 1968 . The maximal lytic activity under deep cultivation conditions, exceeding twofold the activity of Actinomyces griseinus, was observed during the logarithmic growth phase. Am J Hematol, 2001 Aug, 67(4), 247 - 51 Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization; Ranganathan S et al.; We have developed a laboratory method combining a random mutagenesis method and a yeast two-hybrid system to study effects of mutation on human erythrocyte spectrin tetramerization . A PCR-based procedure was used to generate random mutations in DNA fragments of the first 55 residues of alpha-spectrin . Each of the DNA fragments from random mutagenesis was fused with a DNA fragment of native spectrin consisting of residues 56 to 368 to give a DNA fragment of the first 368 residues in alpha-spectrin . The alpha-spectrin DNA fragment and a DNA fragment containing the last 449 residues in beta-spectrin were introduced into the yeast two-hybrid system for rapid screening of alpha- and beta-spectrin interaction . Yeast colonies with interacting alpha- and beta-peptides were blue, and those with non-interacting alpha- and beta-peptides were white . Six single amino acid mutations (R27G, Y35N, F38S, L49H, Y53N, and Y53C) and a double amino acid mutation (K16M, I24N) were identified from 8 white colonies, but no mutations were found in the DNA fragments of 14 blue colonies . Thus this simple laboratory method allows us to study effects of mutation on interactions of alpha- and beta-spectrin at the tetramerization site. Glycoconj J, 2000 Nov, 17(11), 745 - 8 Filamentous fungus Aspergillus oryzae has two types of alpha-1,2-mannosidases, one of which is a microsomal enzyme that removes a single mannose residue from Man9GlcNAc2; Yoshida T et al.; alpha-Mannosidase activities towards high-mannose oligosaccharides were examined with a detergent-solubilized microsomal preparation from a filamentous fungus, Aspergillus oryzae . In the enzymatic reaction, the pyridylaminated substrate Man(9)GlcNAc(2)-PA was trimmed to Man(8)GlcNAc(2)-PA which lacked one alpha-1,2-mannose residue at the nonreducing terminus of the middle branch (Man8B isomer), and this mannooligosaccharide remained predominant through the overall reaction . Trimming was optimal at pH 7.0 in PIPES buffer in the presence of calcium ion and kifunensine was inhibitory with IC(50) below 0.1 microM . These results suggest that the activity is the same type as was previously observed with human and yeast endoplasmic reticulum (ER) alpha-mannosidases . Considering these results together with previous data on a fungal alpha-1,2-mannosidase that trimmed Man(9)GlcNAc(2) to Man(5)GlcNAc(2) (Ichishima, E., et al . (1999) Biochem J, 339: 589-597), the filamentous fungi appear to have two types of alpha-1,2-mannosidases, each of which acts differently on N-linked mannooligosaccharides. J Biol Chem, 2001 Sep 7, 276(36), 33375 - 83 Epub 2001 Jul 06. Identification and characterization of RRM-containing coactivator activator (CoAA) as TRBP-interacting protein, and its splice variant as a coactivator modulator (CoAM); Iwasaki T et al.; We previously cloned and characterized thyroid hormone receptor-binding protein (TRBP) as an LXXLL-containing general coactivator that associates with coactivator complexes through its C terminus . To identify protein cofactors for TRBP action, a Sos-Ras yeast two-hybrid cDNA library was screened using TRBP C terminus as bait . A novel coactivator was isolated, coactivator activator (CoAA), that specifically associates with TRBP . Human CoAA is composed of 669 amino acids with a TRBP-interacting domain and two highly conserved RNA recognition motifs (RRM) commonly found in ribonucleoproteins . A splice variant lacking the entire TRBP-interacting domain was also isolated as a coactivator modulator (CoAM), a 156-amino acid protein containing only the RRM region . Human CoAA and CoAM mRNAs are encoded by a single gene located on chromosome 11q13; alternative splicing in exon 2 of CoAA yields CoAM . CoAA interacts with both TRBP and p300 in vitro . In addition, CoAA potently coactivates transcription mediated by multiple hormone-response elements and acts synergistically with TRBP and CREB-binding protein (CBP) . Furthermore, CoAA is associated with the DNA-dependent protein kinase-poly(ADP-ribose) polymerase complex . Strikingly, CoAM, which lacks a TRBP-interacting domain, strongly represses both TRBP and CBP action suggesting that CoAM may modulate endogenous CoAA function . These data suggest that CoAA may serve as a mediator of coactivators such as TRBP in gene activation. Lett Appl Microbiol, 2001 Jul, 33(1), 89 - 93 Ethanol-induced dimorphism and lipid composition changes in Mucor fragilis CCMI 142; Serrano I et al.; AIMS: To study the effect of ethanol on morphology, lipid production and fatty acid profile of Mucor fragilis CCMI 142 cultures . METHODS AND RESULTS: Cell inhibition in shake flask cultures due to alcohol toxicity grew linearly from 0.418 mol x 1(-1) to 0.816 mol x 1(-1) ethanol corresponding to a decrease of specific growth rate . The growth inhibition constant took the value of 2.27 mol x 1(-1) . The germination of fungal spores into hyphae is inhibited by concentrations from 0.418 mol x 1(-1) to 0.816 mol x 1(-1) ethanol . In this range, M . fragilis CCMI 142 spores form, exclusively, budding yeast-like cells instead of filaments . Below 0.418 mol 1-1 ethanol the formation of yeast-like cells was stimulated and there was a spore germination delay . CONCLUSION: The lipid content decreased as the concentration of ethanol increased, and was associated with an increase of unsaturated/saturated fatty acid ratio . SIGNIFICANCE AND IMPACT OF THE STUDY: The major conclusion of the study is the production of an enriched unsaturated fatty acids final product with particular emphasis to the presence of gamma-linolenic acid (18:3omega6) a biologically active compound with a useful impact in nutraceutical science. Plant Mol Biol, 2001 May, 46(2), 229 - 39 Functional characterization of beta-ketoacyl-CoA synthase genes from Brassica napus L; Han J et al.; Seed-specifically expressed beta-ketoacyl-CoA synthase genes of Brassica napus (Bn-FAE1.1 genes) were cloned from two cultivars, namely Askari, a high-erucic-acid type, and Drakkar, a low-erucic-acid type . The genes from the two cultivars were found to be nearly identical . They encode proteins of 507 amino acids, the sequences of which differ only at position 282 . The Bn-FAE1.1 gene of Askari, unlike that of Drakkar, was functionally expressed in yeast cells suggesting that the single amino acid exchange effects the low erucic acid phenotype at the E1 gene locus . In yeast cells the beta-ketoacyl-CoA synthase of Askari elongated not only oleoyl but also palmitoleoyl groups as well as saturated acyl groups in such a way that monounsaturated acyl groups of 22 carbons and saturated ones of 26 carbons were formed as main products . A reporter gene fused to the promoter region of the Bn-FAE1.1 gene from Askari showed seed-specific expression in transgenic rapeseed plants . Over-expression of the coding region of the Askari gene in developing seeds of transgenic Drakkar plants resulted in a significant increase in the levels of eicosenoic acid and erucic acid esterified in the seed oil . On the other hand, in transgenic high-erucic-acid rapeseed plants the increase in erucic acid level was at most 60% although the chimeric Bn-FAE1.1 gene was co-expressed with an erucoyl-CoA-specific lysophosphatidate acyltransferase gene enabling trierucoyl glycerol to accumulate in the seed oil. Plant Mol Biol, 2001 May, 46(2), 143 - 60 NIMIN-1, NIMIN-2 and NIMIN-3, members of a novel family of proteins from Arabidopsis that interact with NPR1/NIM1, a key regulator of systemic acquired resistance in plants; Weigel RR et al.; NPR1/NIM1 is a key regulator of systemic acquired resistance (SAR) in Arabidopsis . Using the yeast two-hybrid system, we have identified three novel genes, NIMIN-1, NIMIN-2 and NIMIN-3 (NIMIN for NIM1-interacting) that encode structurally related proteins interacting physically with NPR1/NIM1 . NIMIN-1 and NIMIN-2 both bind strongly to NPR1/NIM1 via a common binding motif interacting with the C-terminal moiety of NPR1/NIM1, whereas NIMIN-3 interacts with NPR1/NIM1 via the N-terminal part of NPR1/NIM1 . In addition, NIMIN-1, NIMIN-2, and NIMIN-3 are able to interact via NPR1/NIM1 with basic leucine zipper transcription factors of the TGA family in a yeast tri-hybrid system . A mutant protein of NPR1/NIM1, npr1-2, which has been shown to be severely impaired in induction of SAR gene expression, failed to bind the NIMIN proteins . The NIMIN genes are expressed in Arabidopsis plants in response to SAR-inducing treatments, and the NIMIN proteins, like NPR1/NIM1, carry functional nuclear localization signals as revealed by expression of fusion proteins in yeast and in transgenic plants . Taken together, these data indicate that the NIMIN proteins, via physical interaction with NPR1/NIM1, are part of the signal transduction pathway leading to SAR gene expression in Arabidopsis. J Basic Microbiol, 2001, 41(2), 131 - 7 Glutathione metabolism and dimorphism in Aureobasidium pullulans; Jurgensen CW et al.; Yeast<-->mycelium morphological transitions of Aureobasidium pullulans are influenced by numerous environmental factors . In general, changes in the glutathione (GSH) metabolism of dimorphic fungi may lead to alterations in the reduced thiol status of the cells that are hypothesised to initialise morphological transitions . In accordance with this hypothesis, the specific GSH levels found in A . pullulans yeast cells were always significantly higher than those in mycelia . One the other hand, there was no significant difference between the GSH/GSSG redox status of the cells with either yeast or mycelial morphology . The cascade of events leading to morphological transitions was therefore unlikely to proceed via redox modulation of protein thiols . Although there were morphology-dependent differences in the specific activities of some GSH metabolic enzymes, e.g . glutathione reductase (GR), gamma-glutamyltranspeptidase (gamma GT), glucose-6-phosphate dehydrogenase (G6PD), they were not satisfactory to explain the observed alterations in the intracellular GSH levels . It is noteworthy that very similar specific gamma GT and G6PD activities were found in cells separated from mixed morphology cultures independently of the actual cell morphology . On the other hand, the specific gamma GT and G6PD activities of A . pullulans cells sharing the same morphology but separated from pure and mixed morphology cultures showed marked differences. J Biol Chem, 2001 Sep 21, 276(38), 35768 - 77 Epub 2001 Jul 05. PDZ domain protein GIPC interacts with the cytoplasmic tail of melanosomal membrane protein gp75 (tyrosinase-related protein-1); Liu TF et al.; Tyrosinase and tyrosinase-related proteins (TRPs) are a family of melanosomal membrane proteins involved in mammalian pigmentation . Whereas the melanogenic functions of TRPs are localized in their amino-terminal domains that reside within the lumen of melanosomes, the sorting and targeting of these proteins to melanosomes is mediated by signals in their cytoplasmic domains . To identify proteins that interact with the cytoplasmic tail of gp75 (TRP-1), the most abundant melanosomal membrane protein, we performed yeast two-hybrid screening of a melanocyte cDNA library . Here, we show that the cytoplasmic domain of gp75 interacts with a PDZ domain-containing protein . The gp75-interacting protein is identical to GIPC, an RGS (regulator of G protein signaling)/GAIP-interacting protein, and to SEMCAP-1, a transmembrane semaphorin-binding protein . Carboxyl-terminal amino acid residues, Ser-Val-Val, of gp75 are necessary and sufficient for interaction of gp75 with the single PDZ domain in GIPC . Although endogenous and transfected GIPCs bind efficiently to transiently expressed gp75, only a small amount of GIPC is found associated with gp75 at steady state . Using a strategy to selectively synchronize the biosynthesis of endogenous gp75, we demonstrate that only newly synthesized gp75 associates with GIPC, primarily in the juxtanuclear Golgi region . Our data suggest that GIPC/SEMCAP-1 plays a role in biosynthetic sorting of proteins, specifically gp75, to melanosomes. J Biol Chem, 2001 Aug 31, 276(35), 33079 - 85 Epub 2001 Jul 05. Four subunit a isoforms of Caenorhabditis elegans vacuolar H+-ATPase . Cell-specific expression during development; Oka T et al.; We have identified four genes (vha-5, vha-6, vha-7, and unc-32) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit a in Caenorhabditis elegans, the first example of four distinct isoforms in eukaryotes . Their products had nine putative transmembrane regions, exhibited 43-60% identity and 62-84% similarity with the bovine subunit a1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activity (Leng, X . H., Manolson, M . F., and Forgac, M . (1998) J . Biol . Chem . 273, 6717-6723) . The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase . Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells; vha-5 was strongly expressed in an H-shaped excretory cell, vha-6 was strongly expressed in intestine, vha-7 was strongly expressed in hypodermis, and unc-32 was strongly expressed in nerve cells . Furthermore, the vha-7 and unc-32 genes were also expressed in the uteri of hermaphrodites . RNA interference analysis showed that the double-stranded RNA for unc-32 caused embryonic lethality similar to that seen with other subunit genes (vha-1, vha-4, and vha-11) (Oka, T., and Futai, M . (2000) J . Biol . Chem . 275, 29556-29561) . The progenies of worms injected with the vha-5 or vha-6 double-stranded RNA became died at a specific larval stage, whereas the vha-7 double-stranded RNA showed no effect on development . These results suggest that V-ATPases with these isoforms generate acidic compartments essential for worm development in a cell-specific manner. Oncogene, 2001 Jun 28, 20(29), 3869 - 79 Ras-GAP SH3 domain binding protein (G3BP) is a modulator of USP10, a novel human ubiquitin specific protease; Soncini C et al.; Degradation of cellular proteins through ubiquitination is a fundamental strategy for regulating biological pathways . De-ubiquitination, i.e . the removal of ubiquitin from proteins and peptides to which ubiquitin is attached, is catalyzed by processing proteases known as de-ubiquitinating enzymes . We are studying the biology of a family of de-ubiquitinating enzymes, the mammalian ubiquitin-specific proteases (USPs), some of which appear to play a role in growth control . Given the fact that the modes of regulation of USPs and of their substrate specificity are poorly understood, we decided to attempt the identification of USP interacting proteins . Using the yeast two-hybrid system (2HS), we have isolated a cDNA clone whose product specifically interacts with USP10 but not with other USP baits tested . The isolated clone encodes a protein known to interact with the Ras-GTPase activating protein (G3BP) . This interaction was further confirmed by performing a 2HS with G3BP, which led to the isolation of USP10 encoding cDNAs . We validated the interaction between the two proteins by performing in vitro binding assays and immunoprecipitations in human cells . G3BP does not appear to be a substrate of USP10; it rather inhibits the ability of USP10 to disassemble ubiquitin chains . The USP10/G3BP complex appears to co-immunoprecipitate with ubiquitinated species that could be substrates of USP10. Plant J, 2001 May, 26(4), 385 - 94 APETALA1 and SEPALLATA3 interact to promote flower development; Pelaz S et al.; In Arabidopsis, the closely related APETALA1 (AP1) and CAULIFLOWER (CAL) MADS-box genes share overlapping roles in promoting flower meristem identity . Later in flower development, the AP1 gene is required for normal development of sepals and petals . Studies of MADS-domain proteins in diverse species have shown that they often function as heterodimers or in larger ternary complexes, suggesting that additional proteins may interact with AP1 and CAL during flower development . To identify proteins that may interact with AP1 and CAL, we used the yeast two-hybrid assay . Among the five MADS-box genes identified in this screen, the SEPALLATA3 (SEP3) gene was chosen for further study . Mutations in the SEP3 gene, as well as SEP3 antisense plants that have a reduction in SEP3 RNA, display phenotypes that closely resemble intermediate alleles of AP1 . Furthermore, the early flowering phenotype of plants constitutively expressing AP1 is significantly enhanced by constitutive SEP3 expression . Taken together, these studies suggest that SEP3 interacts with AP1 to promote normal flower development. J Neurobiol, 2001 Aug, 48(2), 120 - 30 Dendritic growth induced by BMP-7 requires Smad1 and proteasome activity; Guo X et al.; Bone morphogenetic proteins (BMPs) induce dendritic growth in cultured sympathetic neurons; however, the signaling pathways that mediate this dendrite-promoting activity have not been previously characterized . Here we report studies of the signaling events that regulate the growth of these afferent processes . We find that Smad1 is expressed in sympathetic neurons and that BMPs rapidly induce its phosphorylation and translocation from the cytoplasm to the nucleus . Furthermore, a dominant negative form of Smad1 inhibits BMP-7-induced dendritic growth, suggesting a requirement for Smad1 activation in this biological activity of BMP-7 . A physical interaction between Smad1 and components involved in the proteasome-mediated degradation system was detected with a yeast two-hybrid screen, thereby prompting an examination of the effects of proteasome inhibitors on dendritic growth . Lactacystin and ALLN (N-acetyl-Leu-Leu-norleucinal) selectively blocked BMP-7-induced dendritic growth without adversely affecting either cell viability or axonal growth . Moreover, studies of transfected P19 cells suggest that the proteasome inhibitors directly block the effects of Smad1 on the transcriptional activity of the Tlx-2 promoter . These data indicate that BMP-induced dendritic growth requires Smad1 activation and involves proteasome-mediated degradation events . Mol Cell Biol, 2001 Aug, 21(15), 4996 - 5007 NoBP, a nuclear fibroblast growth factor 3 binding protein, is cell cycle regulated and promotes cell growth; Reimers K et al.; Secreted and nuclear forms of fibroblast growth factor 3 (FGF3) have opposing effects on cells . The secreted form stimulates cell growth and transformation, while the nuclear form inhibits DNA synthesis and cell proliferation . By using the yeast two-hybrid system we have identified a nucleolar FGF3 binding protein (NoBP) which coimmunoprecipitated and colocalized with FGF3 in transfected COS-1 cells . Characterization of the NoBP binding domain of FGF3 exactly matched the sequence requirements of FGF3 for its translocation into the nucleoli, suggesting that NoBP might be the nucleolar binding partner of FGF3 essential for its nucleolus localization . Carboxyl-terminal domains of NoBP contain linear nuclear and nucleolar targeting motifs which are capable of directing a heterologous protein beta-galactosidase to the nucleus and the nucleoli . While NoBP expression was detected in all analyzed proliferating established cell lines, NoBP transcription was rapidly downregulated in the promyelocytic leukemia cell line HL60 when induced to differentiate . Analysis on the expression pattern of NoBP mRNA throughout the cell cycle in HeLa cells synchronized by lovastatin demonstrated a substantial upregulation during the late G(1)/early S phase . NoBP overexpression conferred a proliferating effect onto NIH 3T3 cells and can counteract the inhibitory effect of nuclear FGF3, suggesting a role of NoBP in controlling proliferation in cells . We propose that NoBP is the functional target of nuclear FGF3 action. J Biol Chem, 2001 Sep 7, 276(36), 33569 - 75 Epub 2001 Jul 03. The DNA glycosylase T:G mismatch-specific thymine DNA glycosylase represses thyroid transcription factor-1-activated transcription; Missero C et al.; The transcription factor thyroid transcription factor-1 (TTF-1) is a homeodomain-containing protein that belongs to the NK2 family of genes involved in organogenesis . TTF-1 is required for normal development of the forebrain, lung, and thyroid . In a search for factors that regulate TTF-1 transcriptional activity, we isolated three genes (T:G mismatch-specific thymine DNA glycosylase (TDG), homeodomain-interacting protein kinase 2 (HIPK2), and Ajuba), whose products can interact with TTF-1 in yeast and in mammalian cells . TDG is an enzyme involved in base excision repair . In the present paper, we show that TDG acts as a strong repressor of TTF-1 transcriptional activity in a dose-dependent manner, while HIPK2 and Ajuba display no effect on TTF-1 activity, at least under the tested conditions . TDG-mediated inhibition occurs specifically on TTF-1-responsive promoters in thyroid and non thyroid cells . TDG associates with TTF-1 in mammalian cells through the TTF-1 carboxyl-terminal activation domain and is independent of the homeodomain . These findings reveal a previously unsuspected role for the repair enzyme TDG as a transcriptional repressor and open new routes toward the understanding of the regulation of TTF-1 transcriptional activity. J Biol Chem, 2001 Sep 7, 276(36), 33899 - 905 Epub 2001 Jul 03. Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression; Daher A et al.; Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA . It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding . TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity . We used the yeast two-hybrid assay to map the interaction sites between the two proteins . We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay . We characterized two independent PKR-binding sites in TRBP . These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2 . TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression . In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain . These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners. J Biol Chem, 2001 Sep 14, 276(37), 34941 - 7 Epub 2001 Jul 03. Definition of the extended substrate specificity determinants for beta-tryptases I and II; Harris JL et al.; Tryptases betaI and betaII were heterologously expressed and purified in yeast to functionally characterize the substrate specificity of each enzyme . Three positional scanning combinatorial tetrapeptide substrate libraries were used to determine the primary and extended substrate specificity of the proteases . Both enzymes have a strict primary preference for cleavage after the basic amino acids, lysine and arginine, with only a slight preference for lysine over arginine . betaI and betaII tryptase share similar extended substrate specificity, with preference for proline at P4, preference for arginine or lysine at P3, and P2 showing a slight preference for asparagine . Measurement of kinetic constants with multiple substrates designed for beta-tryptases reveal that selectivity is highly dependent on ground state substrate binding . Coupled with the functional determinants, structural determinants of tryptase substrate specificity were identified . Molecular docking of the preferred substrate sequence to the three-dimensional tetrameric tryptase structure reveals a novel extended substrate binding mode that involves interactions from two adjacent protomers, including P4 Thr-96', P3 Asp-60B' and Glu-217, and P1 Asp-189 . Based on the determined substrate information, a mechanism-based tetrapeptide-chloromethylketone inhibitor was designed and shown to be a potent tryptase inhibitor . Finally, the cleavage sites of several physiologically relevant substrates of beta-tryptases show consistency with the specificity data presented here. Cell Microbiol, 2001 Jul, 3(7), 439 - 47 Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCalpha and defective phagosome maturation; Holm A et al.; Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes . The repeating disaccharide-phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection . LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines . LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F-actin . In the present study, we have characterized the dynamics of F-actin during the phagocytosis of L . donovani promastigotes in J774 macrophages . We observed that F-actin accumulated progressively around phagosomes containing wild-type L . donovani promastigotes during the first hour of phagocytosis . Using LPG-defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG . LPG also disturbed cortical actin turnover during phagocytosis . The LPG-dependent accumulation of periphagosomal F-actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCalpha to the phagosome . Accumulation of periphagosomal F-actin during phagocytosis of L . donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome. Biochem Biophys Res Commun, 2001 Jul 6, 285(1), 98 - 104 Obtusifoliol 14alpha-demethylase (CYP51) antisense Arabidopsis shows slow growth and long life; Kushiro M et al.; Obtusifoliol 14alpha-demethylase is a plant orthologue of sterol 14alpha-demethylase (CYP51) essential in sterol biosynthesis . We have prepared CYP51 antisense Arabidopsis in order to shed light on the sterol and steroid hormone biosynthesis in plants . Arabidopsis putative CYP51 cDNA (AtCYP51) was obtained from Arabidopsis expressed sequence tag (EST) library and its function was examined in a yeast lanosterol 14alpha-demethylase (Erg11) deficient mutant . A recombinant AtCYP51 protein fused with a yeast Erg11 signal-anchor peptide was able to complement the erg11 mutation, which confirmed AtCYP51 to be a functional sterol 14alpha-demethylase . AtCYP51 was then used to generate transgenic Arabidopsis by transforming with pBI vector harboring AtCYP51 in the antisense direction under CaMV35S promoter . The resulting transgenic plants were decreased in accumulation of AtCYP51 mRNA and increased in the amount of endogenous obtusifoliol . They showed a semidwarf phenotype in the early growth stage and a longer life span than control plants . This newly found phenotype is different from previously characterized brassinosteroid (BR)-deficient campesterol biosynthesis mutants . J Biol Chem, 2001 Sep 7, 276(36), 33554 - 60 Epub 2001 Jul 02. Ligand-dependent interaction of estrogen receptor-alpha with members of the forkhead transcription factor family; Schuur ER et al.; Estrogen acting through the estrogen receptor (ER) is able to regulate cell growth and differentiation of a variety of normal tissues and hormone-responsive tumors . Ligand-activated ER binds DNA and transactivates the promoters of estrogen target genes . In addition, ligand-activated ER can interact with other factors to alter the physiology and growth of cells . Using a yeast two-hybrid screen, we have identified an interaction between ER alpha and the proapoptotic forkhead transcription factor FKHR . The ER alpha-FKHR interaction depends on beta-estradiol and is reduced significantly in the absence of hormone or the presence of Tamoxifen . A glutathione S-transferase pull-down assay was used to confirm the interaction and localized two interaction sites, one in the forkhead domain and a second in the carboxyl terminus . The FKHR interaction was specific to ER alpha and was not detected with other ligand-activated steroid receptors . The related family members, FKHRL1 and AFX, also bound to ER alpha in the presence of beta-estradiol . FKHR augmented ER alpha transactivation through an estrogen response element . Conversely, ER alpha repressed FKHR-mediated transactivation through an insulin response sequence, and cell cycle arrest induced by FKHRL1 in MCF7 cells was abrogated by estradiol . These results suggest a novel mechanism of estrogen action that involves regulation of the proapoptotic forkhead transcription factors. FEBS Lett, 2001 Jun 29, 500(1-2), 41 - 4 Rat protein tyrosine phosphatase eta physically interacts with the PDZ domains of syntenin; Iuliano R et al.; The tyrosine phosphatase r-PTPeta is able to suppress the malignant phenotype of rat thyroid tumorigenic cell lines . To identify r-PTPeta interacting proteins, a yeast two-hybrid screening was performed and an insert corresponding to the full-length syntenin cDNA was isolated . It encodes a protein containing two PDZ domains that mediates the binding of syntenin to proteins such as syndecan, proTGF-alpha, beta-ephrins and neurofascin . We show that r-PTPeta is able to interact with syntenin also in mammalian cells, and although syntenin is a tyrosine-phosphorylated protein it is not a substrate of r-PTPeta . The integrity of both PDZ domains of syntenin and the carboxy-terminal region of r-PTPeta are required for the interaction between syntenin and r-PTPeta. Genes Chromosomes Cancer, 2001 Aug, 31(4), 333 - 44 Defining a common region of deletion at 13q21 in human cancers; Chen C et al.; Previous molecular genetic analyses identified a region of deletion at 13q21 in a variety of human cancers, suggesting the existence of a tumor suppressor gene(s) at this locus . In our earlier study on prostate cancer, the region of deletion was confined to a 3.1 cM interval between D13S152 and D13S162 . At present, however, no known gene located in this interval has been firmly implicated in cancer, and the region remains too large for gene identification . To fine-map the area of interest, we established a contig of bacterial artificial chromosome (BAC) clones, narrowed the region of deletion by loss of heterozygosity (LOH) and homozygosity-mapping-of-deletion (HOMOD) analyses in different types of cancers, and tested a candidate gene from the region for mutation and alteration of expression in prostate cancers . The contig consisted of 75 overlapping BAC clones . In addition to the generation of 47 new sequence-tagged-site (STS) markers from the ends of BAC inserts, 76 known STS and expressed sequence tag markers were mapped to the contig (25 kb per marker on average) . The minimal region of deletion was further defined to be about 700 kb between markers D13S791 and D13S166 by LOH analysis of 42 cases of prostate cancer, and by HOMOD analysis of eight prostate cancer cell lines/xenografts and 49 cell lines from cancers of the breast, ovary, endometrium, and cervix, using 18 microsatellite markers encompassing the deletion region . A gene that is homologous to the WT1 tumor suppressor gene, AP-2rep (KLF12), was mapped in this region and was analyzed for its expression and genetic mutation . In addition to low levels of expression in both normal and neoplastic cells of the prostate, this gene did not have any mutations in a group of aggressive prostate cancers and cell lines/xenografts, as assessed by the methods of polymerase chain reaction-single strand conformational polymorphism analysis and direct sequencing . These studies suggest that a 700 kb interval at 13q21 harbors a tumor suppressor gene(s) that seems to be involved in multiple types of cancer, and that the AP-2rep gene is unlikely to be an important tumor suppressor gene in prostate cancer . The BAC contig and high-resolution physical map of the defined region of deletion should facilitate the cloning of a tumor suppressor gene(s) at 13q21 . Nucleic Acids Res, 2001 Jul 1, 29(13), 2836 - 42 14-3-3tau associates with and activates the MEF2D transcription factor during muscle cell differentiation; Choi SJ et al.; Myocyte enhancer binding factor 2 (MEF2) proteins belong to the MADS box family of transcription factors and four MEF2 proteins, MEF2A, MEF2B, MEF2C and MEF2D, have been found . MEF2 proteins have been shown to play critical roles in differentiation of muscles and neuronal tissues . How transactivational activity of MEF2 proteins is regulated is not fully understood . MEF2 proteins are activated by several kinases, including Erk5 and calcium/calmodulin-dependent kinase, and interact with repressors, including histone deacetylases 4 and 5 (HDAC4 and HDAC5) and Cabin1 . During the effort to understand regulation of MEF2 activity, we identified 14-3-3tau as a MEF2D-interacting molecule by yeast two-hybrid screening . We found that 14-3-3tau forms a complex with MEF2D in vivo and specifically enhances MEF2 transactivational activity . The results from transient transfection and co-precipitation experiments suggest that 14-3-3tau activates MEF2D by competitively inhibiting HDAC4 from binding to MEF2D and thereby affects muscle cell differentiation. Nucleic Acids Res, 2001 Jul 1, 29(13), 2691 - 8 A sensitive, single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis; Hooker L et al.; We describe a fast and robust new assay format to measure poly(A) polymerase (PAP) activity in a microtiter plate format . The new assay principle uses only natural nucleotide triphosphates and avoids a labour-intensive filtration step . A coupled enzymatic system combining PAP and reverse transcriptase forms the basis of the assay . The PAP generates a poly(A) tail on a RNA substrate and the reverse transcriptase is used to quantify the polyadenylated RNA by extension of a biotinylated oligo-dT primer . We demonstrate the principle of the assay using influenza virus RNA polymerase and yeast PAP as examples . A specific increase in the K(m) value for ATP and the observation of burst kinetics in the polyadenylation dependent, but not in the polyadenylation independent, assay suggest that a rate limiting step of influenza polymerase activity occurs after transcription elongation . Yeast PAP was used to validate the assay as an example of a template independent PAP . The new yeast PAP assay was approximately 100-fold more sensitive than the conventional TCA precipitation assay for yeast PAP, but the kinetic analysis of the PAP reaction gave similar results in both assays . The two enzymes show important differences with respect to inhibition by 3'-deoxy-ATP . Whereas the K(i) value for 3'-deoxy-ATP (105-117 microM) is similar to the K(m) value for ATP (186 microM) in the case of influenza RNA polymerase, the K(i) value for 3'-deoxy-ATP (0.4-0.6 microM) is approximately 100-fold lower than the K(m) value for ATP (50 microM) in the case of yeast PAP. J Biochem (Tokyo), 2001 Jul, 130(1), 73 - 8 Association of phosphatidylinositol 3-kinase composed of p110beta-catalytic and p85-regulatory subunits with the small GTPase Rab5; Kurosu H et al.; A family of phosphatidylinositol 3-kinases (PI 3-kinase), comprising three major classes (I-III) in terms of substrate specificity and regulation, play important roles in a variety of cell functions . We previously reported that the class-I heterodimeric PI 3-kinase consisting of p110beta-catalytic and p85-regulatory subunits is synergistically activated by two different types of membrane receptors, one possessing tyrosine kinase activity and the other activating trimeric G proteins . Here we report an additional unique feature of the p110beta/p85 PI 3-kinase . The small GTPase Rab5 was identified as a binding protein for the p110beta-catalytic subunit in a yeast two-hybrid screening system . The interaction appears to require at least two separated amino-acid sequences present specifically in the beta isoform of p110 and the GTP-bound form of Rab5 . The expressions of constitutively active and dominant negative mutants of Rab5 in THP-1 cells induce the stimulation and inhibition, respectively, of protein kinase B activity, which is dependent on the PI 3-kinase product phosphatidylinositol 3,4,5-triphosphate . These results suggest that there is a specific interaction between GTP-bound Rab5 and the p110beta/p85 PI 3-kinase, leading to efficient coupling of the lipid kinase product to its downstream target, protein kinase B. Med Pregl, 2001 Jan-Feb, 54(1-2), 45 - 51 {Etiopathogenesis, clinical picture and diagnosis of onychomycoses}; Tasic S et al.; ETIOLOGY OF ONYCHOMYCOSES: Onychomycoses can be caused by dermatophytes, molds and yeasts . However, dermatophytes appear to be the chief organisms capable of a primary attack on the nail . By far the most frequent dermatophytes isolated from nails are Trichophyton rubrum, T . mentagrophytes var . interdigitale and Epidermophyton floccosum . Molds virtually only invade toenails, but their role as a primary pathogen is still debated . Yeasts have been isolated from diseased nails at highly different rates . Nails may be infected by two different dermatophytes, two dermatophytes and a yeast, a dermatophyte, a yeast and a mold, etc . PATHOGENESIS OF ONYCHOMYCOSES: The mode of infection is still under debate . In many cases palmar and/or plantar tinea, exists but can often remain asymptomatic for years . After spreading to the nail, the fungus invades the hyponychium or lateral nail sulcus to finally reach the nail bed where it moves proximally to the matrix . Proximal subungual onychomycosis probably starts with a fungal skin infection, whereas white superficial onychomycosis seems to be a culture of T . mentagrophytes on a softened nail surface . Total dystrophic onychomycosis may result from both distal and proximal subungual onychomycosis or from C . albicans in chronic mucocutaneous candidiasis . Candida infections occur most often due to previous Candida paronychia, but it appears that a number of cases of so called idiopathic onycholysis are also caused by C . albicans with damage to the hyponychium being the portal-of entry . CLINICAL PICTURE OF ONYCHOMYCOSES: Onychomycoses can be divided into four different types . Distal subungual onychomycosis is the most common . The most frequent presenting clinical features are thickening and opaci-fication of the nail plate along the distal and lateral borders . The discoloration ranges from white to brown . The edge of the affected nail is usually uneven and often one or more streaks of dystrophic discoloured nail extend towards the distal border . Proximal subungual onychomycosis is uncommon . A white spot appears beneath the proximal nail fold and may extend distally to involve the deeper layers of the whole nail . Superficial white onychomycosis is also uncommon . The surface is the initial site of invasion . The causative organisms produce small superficial white and powdery patches over the nail . The surface becomes rough and the texture softer than normal . Total dystrophic onychomycosis represents the most advanced from all the previous three types, especially the distal subungual onychomycosis . The nail matrix has become permanently scarred by chronic infection . The nail is thick, elevated, denser and opaque . Candidomycotic onychomycosis shows erythematous and swollen proximal and lateral nail folds . Consequently, the nail plate becomes detached from the eponychium . Mycotic onycholysis is characterized by detachment of the nail plate from the bed, distal nail erosions, and grayish-yellow paste-like material under the nail . DIAGNOSIS OF ONYCHOMYCOSES: The diagnosis of onychomycoses cannot be made on the basis of clinical observation alone . Direct microscopy plays an important role in diagnosing nail fungal infections . However, fungal cultures are the only definitive test that can be used to identify the genus and the species of the infectious organism . Histological examination is a routine technique useful for defining the nature and localization of fungi in the nail plate . Immunohistochemistry applied to onychomycosis is an experimental approach bringing prominent information about identification of fungi . In vivo confocal microscopy represents a technique of the future. Nat Genet, 2001 Jul, 28(3), 276 - 80 Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation; Hiroi Y et al.; The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases . Using the yeast two-hybrid system with Nkx2-5 as the 'bait', we isolated the T-box-containing transcription factor Tbx5; mutations in TBX5 cause heart and limb malformations in Holt-Oram syndrome (HOS) . Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells showed that they also associate with each other in mammalian cells . Glutathione S-transferase (GST) 'pull-down' assays indicated that the N-terminal domain and N-terminal part of the T-box of Tbx5 and the homeodomain of Nkx2-5 were necessary for their interaction . Tbx5 and Nkx2-5 directly bound to the promoter of the gene for cardiac-specific natriuretic peptide precursor type A (Nppa) in tandem, and both transcription factors showed synergistic activation . Deletion analysis showed that both the N-terminal domain and T-box of Tbx5 were important for this transactivation . A G80R mutation of Tbx5, which causes substantial cardiac defects with minor skeletal abnormalities in HOS, did not activate Nppa or show synergistic activation, whereas R237Q, which causes upper-limb malformations without cardiac abnormalities, activated the Nppa promoter to a similar extent to that of wildtype Tbx5 . P19CL6 cell lines overexpressing wildtype Tbx5 started to beat earlier and expressed cardiac-specific genes more abundantly than did parental P19CL6 cells, whereas cell lines expressing the G80R mutant did not differentiate into beating cardiomyocytes . These results indicate that two different types of cardiac transcription factors synergistically induce cardiac development. Nat Genet, 2001 Jul, 28(3), 272 - 5 Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria; Ono T et al.; Extensive complementation between fused mitochondria is indicated by recombination of 'parental' mitochondrial (mt) DNA (ref . 1,2) of yeast and plant cells . It has been difficult, however, to demonstrate the occurrence of complementation between fused mitochondria in mammalian species through the presence of recombinant mtDNA molecules, because sequence of mtDNA throughout an individual tends to be uniform owing to its strictly maternal inheritance . We isolated two types of respiration-deficient cell lines, with pathogenic mutations in mitochondrial tRNAIle or tRNALeu(UUR) genes from patients with mitochondrial diseases . The coexistence of their mitochondria within hybrids restored their normal morphology and respiratory enzyme activity by 10-14 days after fusion, indicating the presence of an extensive and continuous exchange of genetic contents between the mitochondria . This complementation between fused mitochondria may represent a defence of highly oxidative organelles against mitochondrial dysfunction caused by the accumulation of mtDNA lesions with age. J Biol Chem, 2001 Sep 7, 276(36), 33512 - 7 Epub 2001 Jun 28. Molecular and Biochemical Characterization of Rat epsilon -N-Trimethyllysine Hydroxylase, the First Enzyme of Carnitine Biosynthesis; Vaz FM et al.; epsilon-N-Trimethyllysine hydroxylase (EC ) is the first enzyme in the biosynthetic pathway of l-carnitine and catalyzes the formation of beta-hydroxy-N-epsilon-trimethyllysine from epsilon-N-trimethyllysine, a reaction dependent on alpha-ketoglutarate, Fe(2+), and oxygen . We purified the enzyme from rat kidney and sequenced two internal peptides by quadrupole-time-of-flight mass spectroscopy . The peptide sequences were used to search the Expressed Sequence Tag data base, which led to the identification of a rat cDNA of 1218 base pairs encoding a polypeptide of 405 amino acids with a calculated molecular mass of 47.5 kDa . Using the rat sequence we also identified the homologous cDNAs from human and mouse . Heterologous expression of both the rat and human cDNAs in COS cells confirmed that they encode epsilon-N-trimethyllysine hydroxylase . Subcellular fractionation studies revealed that the rat enzyme is localized exclusively in mitochondria . Expression studies in yeast indicated that the rat enzyme is synthesized as a 47.5-kDa precursor and subsequently processed to a mature protein of 43 kDa, presumably upon import in mitochondria . The Michaelis-Menten constants of the purified rat enzyme for trimethyllysine, alpha-ketoglutarate, and Fe(2+) were 1.1 mm, 109 microm, and 54 microm, respectively . Both gel filtration and blue native polyacrylamide gel electrophoresis analysis showed that the native enzyme has a mass of approximately 87 kDa, indicating that in rat epsilon-N-trimethyllysine hydroxylase is a homodimer. J Biol Chem, 2001 Sep 14, 276(37), 35060 - 70 Epub 2001 Jun 28. Rich, a rho GTPase-activating protein domain-containing protein involved in signaling by Cdc42 and Rac1; Richnau N et al.; A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4) . The protein was named RICH-1 (RhoGAP interacting with CIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain . Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays . In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA . Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1. Mol Cell, 2001 Jun, 7(6), 1201 - 11 Analysis of telomerase processivity: mechanistic similarity to HIV-1 reverse transcriptase and role in telomere maintenance; Peng Y et al.; The key protein subunit of the telomerase complex, known as TERT, possesses a reverse transcriptase (RT)-like domain that is conserved in enzymes encoded by retroviruses and retroelements . Structural and functional analysis of HIV-1 RT suggests that RT processivity is governed, in part, by the conserved motif C, motif E, and a C-terminal domain . Mutations in analogous regions of the yeast TERT were found to have anticipated effects on telomerase processivity in vitro, suggesting a great deal of mechanistic and structural similarity between TERT and retroviral RTs, and a similarity that goes beyond the homologous domain . A close correlation was uncovered between telomerase processivity and telomere length in vivo, suggesting that enzyme processivity is a limiting factor for telomere maintenance. Mol Cell, 2001 Jun, 7(6), 1143 - 52 The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release; Kohler A et al.; Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP) . Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP . To test whether degradation products exit through this channel, we analyzed their size distribution . Their median length from an open-channel CP mutant was 40% greater than that from the wild-type . Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation . These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP. J Org Chem, 2001 Feb 9, 66(3), 997 - 1001 Chemoenzymatic synthesis of pyrrolo{2,1-b}quinazolinones: lipase-catalyzed resolution of vasicinone; Kamal A et al.; A facile synthesis of bronchodilatory pyrrolo{2,1-b}quinazoline alkaloids by azidoreductive cyclization strategy employing TMSCl-NaI and bakers' yeast is described . Both the chemical and enzymatic methods are mild and take place at room temperature in good yields . Further, synthesis and resolution of vasicinone has been carried out by employing different lipases . It has been observed that lipase PS provides acetate of (S)-vasicinone in 98% ee. J Biol Chem, 2001 Sep 28, 276(39), 36566 - 74 Epub 2001 Jun 27. CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway; Schoch G et al.; The 4- and 5-hydroxylations of phenolic compounds in plants are catalyzed by cytochrome P450 enzymes . The 3-hydroxylation step leading to the formation of caffeic acid from p-coumaric acid remained elusive, however, alternatively described as a phenol oxidase, a dioxygenase, or a P450 enzyme, with no decisive evidence for the involvement of any in the reaction in planta . In this study, we show that the gene encoding CYP98A3, which was the best possible P450 candidate for a 3-hydroxylase in the Arabidopsis genome, is highly expressed in inflorescence stems and wounded tissues . Recombinant CYP98A3 expressed in yeast did not metabolize free p-coumaric acid or its glucose or CoA esters, p-coumaraldehyde, or p-coumaryl alcohol, but very actively converted the 5-O-shikimate and 5-O-d-quinate esters of trans-p-coumaric acid into the corresponding caffeic acid conjugates . The shikimate ester was converted four times faster than the quinate derivative . Antibodies directed against recombinant CYP98A3 specifically revealed differentiating vascular tissues in stem and root . Taken together, these data show that CYP98A3 catalyzes the synthesis of chlorogenic acid and very likely also the 3-hydroxylation of lignin monomers . This hydroxylation occurs on depsides, the function of which was so far not understood, revealing an additional and unexpected level of networking in lignin biosynthesis. Jpn J Cancer Res, 2001 Jun, 92(6), 638 - 44 Ectopic expression of MAFB gene in human myeloma cells carrying (14;20)(q32;q11) chromosomal translocations; Hanamura I et al.; Chromosome 14q +, which represents a chromosomal rearrangement involving the immunoglobulin heavy chain gene (IgH) locus, is a genetic hallmark of human multiple myeloma (MM) . Here, we report the identification of (14;20)(q32;q11) chromosomal translocations found in MM cells . Double color fluorescence in situ hybridization analyses pinpointed the breakpoints at the 20q11 locus in two MM cell lines within a length of at most 680 kb between the KIAA0823 and MAFB gene loci . Among the transcribed sequences in the vicinity of the breakpoints, an ectopic expression of the MAFB gene, which is located at 450 - 680 kb telomeric to one of the breakpoints and encodes a member of the MAF family basic region / leucine zipper transcription factor, was demonstrated to be associated with t(14;20) . This finding, together with that of a previous study describing its transforming activity, suggests that the MAFB gene may be one of the targets deregulated by regulatory elements of the IgH gene as a result of t(14;20). Proc Natl Acad Sci U S A, 2001 Jul 3, 98(14), 7724 - 9 Epub 2001 Jun 26. Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55(Gag); VerPlank L et al.; Ubiquitination appears to be involved in virus particle release from infected cells . Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles . Here we report that the p6 region in the Pr55(Gag) structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins . Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55(Gag) and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag . The PTAPP motif {or late (L) domain} within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55(Gag) . The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6 . Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein . The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus. Physiol Rev, 2001 Jul, 81(3), 1353 - 92 Spectrin and ankyrin-based pathways: metazoan inventions for integrating cells into tissues; Bennett V et al.; The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms . This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1 . The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes . However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins . These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions . These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life . Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum . Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets. J Clin Microbiol, 2001 Jul, 39(7), 2640 - 1 Rapid diagnosis of Histoplasma capsulatum endocarditis using the AccuProbe on an excised valve; Chemaly RF et al.; Histoplasma capsulatum is an infrequent but serious cause of endocarditis . The definitive diagnosis requires culture, which may require a long incubation . We demonstrated the ability of the Histoplasma capsulatum AccuProbe to accurately identify this organism when applied directly on an excised valve that contained abundant yeast forms consistent with H . capsulatum. Enzyme Microb Technol, 2001 Jul 5, 29(1), 62 - 69 Dechlorination of chlorophenols using extracellular peroxidases produced by streptomyces albus ATCC 3005; Antonopoulos VT et al.; Streptomyces albus ATCC 3005 was found to produce higher levels of extracellular peroxidase activity (3.420 U mg(-1)) than previously reported for any other actinomycete . Maximum peroxidase activity was obtained after 72 h of incubation at a temperature of 30 degrees C in a liquid medium (pH 7.6) containing (in w/v) 0.8% to 0.9% oat spelts xylan and 0.6% yeast extract, corresponding to a C:N ratio of around 8.4:1 . Characterization of the peroxidases revealed that the optimal temperature for peroxidase activity, using the standard 2,4-dichlorophenol (2,4-DCP) assay was 53 degrees C, when the enzyme reaction was performed at pH 7.2 . A study of the effect of temperature on the stability of peroxidase over time, showed that the enzyme was stable at 40 degrees C, with a half-life of 224 min, while at higher temperatures the stability and activity was reduced such that at 50 degrees C and 70 degrees C the half-life of the enzyme was 50 min and 9 min respectively . The optimum pH for the activity of the enzyme occurred between pH 8.1 and 10.4 . In terms of substrate specificity, the peroxidase was able to catalyze a broad range of substrates including 2,4-DCP, L-3,4-dihydroxyphenylalanine (L-DOPA), 2,4,5-trichlorophenol and other chlorophenols in the presence of hydrogen peroxide . Ion exchange chromatography was used to confirm that the enzyme was able to release chloride ions from a range of chlorophenols. Enzyme Microb Technol, 2001 Jul 5, 29(1), 28 - 33 Effect of contact time and inhibitor concentration on the affinity mediated adsorption of cells to surfaces; Lam A et al.; Cell detachment by shear stress under conditions of laminar flow was used to investigate the effect of incubation time and soluble binding competitors on affinity mediated cell/surface interactions . Fractional attachment between yeast and a Concanavalin A (Con A) coated surface was studied as a function of adhesion time prior to exposure to shear in a parallel plate flow chamber . Two, four and sixteen hours adhesion times gave rise to significantly different fractional attachment profiles, with four hours giving greater cell retention.The effect of dextran as a competitive displacer of pre-attached cells was also examined using a number of exposure regimes . While the presence of dextran in the displacement buffer led to higher fractional displacement of pre-attached cells, this effect was magnified if an equilibration period between dextran solution and pre-attached cells was allowed before detachment was attempted . The decline in fractional attachment increased with incubation time up to 30 min, with longer periods resulting in a smaller effect . Pre-incubation of the Con A surface with dextran prior to the introduction of cells led to a 60% reduction in attachment.Attempts to determine critical shear values were complicated by the presence of a tightly bound cell fraction of approximately 15% that was not removed at the highest shear values used. Oncogene, 2001 May 31, 20(25), 3281 - 9 The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10; de Bruijn DR et al.; As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome . Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function . We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait . Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL . Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells . Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments . The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs . The C-terminal interaction domain of AF10 is located outside known functional domains . Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors . This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias. Oncogene, 2001 May 31, 20(25), 3185 - 92 Regulation of microtubule assembly by human EB1 family proteins; Bu W et al.; The EB1 family proteins are highly conserved microtubule-associated proteins . The EB1 protein in yeast has been shown to play an important role in regulating microtubule dynamics and chromosome segregation . Human EB1 family proteins include EB1, RP1 and EBF3 . Although EB1 and RP1 have been shown to associate with microtubules, the subcellular localization of endogenous EBF3 had not been characterized . The function of human EB1 family proteins was also not clear . We therefore investigated the cellular localization of EBF3 and the regulation of microtubule organization by EB1 family proteins . As do EB1 and RP1, EBF3 was found to colocalize with microtubules, preferentially at their plus ends, throughout the cell cycle . Moreover, there was a very strong EBF3 signal at the centrosome in interphase cells and at the spindle poles in mitotic cells . When EB1 family proteins were overexpressed, they associated with the entire microtubule cytoskeleton . In addition, EB1 and EBF3 induced microtubule bundling in some cells overexpressing these proteins . These microtubule bundles were more resistant to nocodazole and were more acetylated than regular microtubules . Our results demonstrate for the first time that human EB1 family proteins could regulate microtubule assembly and stability. J Biol Chem, 2001 Sep 7, 276(36), 33328 - 35 Epub 2001 Jun 22. Analysis of the alpha-actinin/zyxin interaction; Li B et al.; The yeast two-hybrid system was used to search for interaction partners of human zyxin . Screening of two different cDNA libraries, one prepared from human placenta, the other from human heart, yielded several positive clones that occurred in both searches, including clones coding for cyclophilin, nebulette, and alpha-actinin . The zyxin/alpha-actinin interaction was analyzed in detail . By site-directed mutagenesis, a linear motif of 6 amino acids (Phe-Gly-Pro-Val-Val-Ala) present at the N terminus of zyxin was found to play a critical role . Replacement of a single amino acid within this motif abolished binding to alpha-actinin in blot overlays as well as in living cells . On the other hand, the interaction site in alpha-actinin was mapped to a conformational determinant present in the center of the protein as demonstrated by a fragment deletion analysis . This binding site involved a tandem array of two complete spectrin-like domains . Only fragments that were able to dimerize in yeast also bound to zyxin, suggesting that dimerization of alpha-actinin is essential for zyxin binding. J Biol Chem, 2001 Aug 17, 276(33), 30995 - 1003 Epub 2001 Jun 22. Characterization of a general stabilizer element that blocks deadenylation-dependent mRNA decay; Ruiz-Echevarria MJ et al.; mRNA degradation is a regulated process that can play an important role in determining the level of expression of specific genes . The rate at which a specific mRNA is degraded depends largely on specific cis-acting sequences located throughout the transcript . cis-Acting destabilizer sequences that promote increased rates of decay have been identified in several short-lived mRNAs . However, little is known about elements that promote stability, known as stabilizer elements (STEs), and how they function . The work presented here describes the characterization of a STE in the PGK1 transcript . The PGK1 stabilizer element (P-STE) has been delineated to a 64-nucleotide sequence from the coding region that can stabilize a chimeric transcript containing the instability elements from the 3'-untranslated region of the MFA2 transcript . The P-STE is located within the PGK1 coding region and functions when located in the translated portion of the transcript and at a minimum distance from the 3'-untranslated region . These results further support the link between translation and mRNA degradation . A conserved sequence in the TEF1/2 transcript has been identified that also functions as a STE, suggesting that this sequence element maybe a general stability determinant found in other yeast mRNAs. J Biol Chem, 2001 Aug 24, 276(34), 31709 - 12 Epub 2001 Jun 21. Adenosine 5'-monophosphate inhibits the association of 14-3-3 proteins with the plant plasma membrane H(+)-ATPase; Camoni L et al.; Although a well ascertained evidence proves that the activity of the plant plasma membrane H(+)-ATPase is regulated by 14-3-3 proteins, information about physiological factors modulating the phosphorylation-dependent association between 14-3-3 proteins and the proton pump is largely incomplete . In this paper we show that the 5'-AMP-mimetic, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), inhibits the fusicoccin-promoted proton extrusion in maize roots . We also demonstrate that 5'-AMP inhibits the association of 14-3-3 proteins with the C-terminal domain of the H(+)-ATPase in an overlay assay as well as the 14-3-3-dependent stimulation of the Arabidopsis thaliana H(+)-ATPase AHA1 isoform expressed in yeast membranes . Finally, by means of affinity chromatography with immobilized 5'-AMP and trinitrophenyl-AMP fluorescence analysis, we demonstrate that the 14-3-3 isoform GF14-6 from maize is able to bind 5'-AMP . The possible role of 5'-AMP as a general regulator of 14-3-3 functions in the plant cell is discussed. Diabetes, 2001 Jul, 50(7), 1531 - 8 Novel arguments in favor of the substrate-transport model of glucose-6-phosphatase; Gerin I et al.; The purpose of this work was to discriminate between two models for glucose-6-phosphatase: one in which the enzyme has its catalytic site oriented toward the lumen of the endoplasmic reticulum, requiring transporters for glucose-6-phosphate, inorganic phosphate (Pi), and glucose (substrate-transport model), and a second one in which the hydrolysis of glucose-6-phosphate occurs inside the membrane (conformational model) . We show that microsomes preloaded with yeast phosphoglucose isomerase catalyzed the detritiation of {2-(3)H}glucose-6-phosphate and that this reaction was inhibited by up to 90% by S3483, a compound known to inhibit glucose-6-phosphate hydrolysis in intact but not in detergent-treated microsomes . These results indicate that glucose-6-phosphate is transported to the lumen of the microsomes in an S3483-sensitive manner . Detritiation by intramicrosomal phosphoglucose isomerase was stimulated twofold by 1 mmol/l vanadate, a phosphatase inhibitor, indicating that glucose-6-phosphatase and the isomerase compete for the same intravesicular pool of glucose-6-phosphate . To investigate the site of release of Pi from glucose-6-phosphate, we incubated microsomes with Pb(2+), which forms an insoluble complex with Pi, preventing its rapid exit from the microsomes . Under these conditions, approximately 80% of the Pi that was formed after 5 min was intramicrosomal, compared with <10% in the absence of Pb(2+) . We also show that, when incubated with glucose-6-phosphate and mannitol, glucose-6-phosphatase formed mannitol-1-phosphate and that this nonphysiological product was initially present within the microsomes before being released to the medium . These results indicate that the primary site of product release by glucose-6-phosphatase is the lumen of the endoplasmic reticulum. Comp Biochem Physiol A Mol Integr Physiol, 2001 Jun, 129(2-3), 615 - 30 Mannose-receptor-mediated clearance of lysosomal alpha-mannosidase in scavenger endothelium of cod endocardium; Sorensen KK et al.; Mannose-receptor-mediated clearance of circulating glycoproteins was studied in Atlantic cod (Gadus morhua) . Distribution studies with radioiodinated and fluorescently labelled ligands showed that cod liver lysosomal alpha-mannosidase and yeast invertase were rapidly eliminated from blood via a mannose specific pathway in liver parenchymal cells and endocardial endothelial cells of atrium and ventricle . Asialo-orosomucoid, a galactose-terminated glycoprotein, was cleared by liver only . In vitro studies were performed with primary cultures of atrial-endocardial endothelial cells (AEC), incubated at 12 degrees C in a serum free medium . Cod AEC endocytosed mannose-terminated glycoproteins (125I-alpha-mannosidase, 125I-invertase, 125I-mannan, 125I-ovalbumin and unlabelled lysosomal alpha-mannosidase), whereas 125I-asialo-orosomucoid was not recognised . Uptake of radiolabelled mannose-terminated ligands was inhibited 80-100% in the presence of excess amounts of mannan, invertase, D-mannose, L-fucose or EGTA . Our results suggest that the cod endocardial endothelial cells express a specific Ca(2+)-dependent mannose receptor, analogous to the mannose receptor on mammalian macrophages and liver sinusoidal endothelial cells. Eur J Biochem, 2001 Jun, 268(12), 3407 - 15 Generation and epitope mapping of high-affinity scFv to eukaryotic elongation factor 1A by dual application of phage display; Kjaer S et al.; To generate specific tools for, in particular, localization studies of the eukaryotic elongation factor 1A (eEF1A), we have applied phage display in various formats to affinity-improve and map epitopes of two previously isolated, low-affinity single-chain Fv (scFv) G3 and D1 . The scFv differ in their reactivity toward the eEF1A isoforms, eEF1A-1 and eEF1A-2 . By PCR-based randomization of six residues within the variable light chain CDR3 (LCDR3), and subsequent phage-based affinity-selection, two 'families' of affinity-improved scFv were obtained . The scFv of highest affinity, A8, has a Kd of 9 nM to eEF1A-1 . Interestingly, two affinity-improved scFvs have abnormally short LCDR3 consisting of two and four residues compared to 11 in the parental scFv . Hence, the LCDR3 of the parental clones may play a modulating rather than a direct role in antigen-binding . Despite different preferences for the eEF1A isoforms, both families of scFv recognize antigenic determinant(s), which was mapped to residues 413-450 of eEF1A-1/2 by Western blot analysis of recombinant human eEF1A (hEF1A) fragments . Prior to the Western blotting analysis, the epitope location had been suggested using a novel approach where phage-antibody repertoire derived scFv were used to select phage-displayed peptides . Hereby, peptides containing a SFXD motif, matching the SFSD(414-418) sequence found in hEF1A-1 were isolated . The structure of eukaryotic EF1A from yeast indicates a discontinuous nature of the epitope with distal functional elements juxtaposed by the protein fold . Finally, the scFv A8 was applied for immunofluorescence studies of transformed human amnion cells and MCF-7 fibroblasts . In both cases a perinuclear localization of hEF1A was observed . No evidence for the reported nuclear localization of hEF1A was obtained. Biochemistry (Mosc), 2001 Jun, 66(6), 623 - 7 Effect of two conserved amino acid residues on DREB1A function; Cao ZF et al.; Transcription factors of the DREBP subgroup and the EREBP subgroup contain conserved DNA-binding domains called AP2/EREBP domains, which specifically bind to DRE cis-element and GCC-box, respectively . The 14th and 19th amino acid residues of AP2/EREBP domains are absolutely conserved in the transcription factors of the DREBP subgroup as well as in the EREBP subgroup . However, these two residues of transcription factors of the DREBP subgroup are different from those of the EREBP subgroup . To assess the functional significance of these two residues in binding to the target sequence, the Val (14th residue) and Glu (19th residue) of the AP2/EREBP domain of DREB1A (a transcription factor of the DREBP subgroup) were mutated individually or doubly to Ala and Asp, respectively . This made the 14th and 19th amino acid residues of mutant DREB1A identical to the corresponding residues of transcription factors of the EREBP subgroup . Yeast in vivo analysis showed that: 1) on a selective medium plate of SD/His- Ura- Trp- + 30 mM approximately 60 mM 3-AT, the growth of yeast cells containing HIS and lacZ double reporter genes was normal in the transformation of the 19th singly mutated DREB1A, obviously inhibited in the transformation of the 14th singly mutated DREB1A, and seriously inhibited in the transformation of the 14th/19th doubly mutated DREB1A; 2) quantitative assay of beta-galactosidase activity showed that the intensities of lacZ expression decreased in the transformations of the 14th singly mutated and 14th/19th doubly mutated types . The experimental results revealed that the 19th site mutation did not affect the binding of the DREB1A transcription factor to the DRE cis-element; the 14th site mutation obviously inhibited their binding; and the double mutation of the 14th/19th sites seriously inhibited their binding . This suggests that the conserved Val (14th) and Glu (19th) residues are crucial in the regulation of the binding activity of DREB1A to the DRE cis-element. RNA, 2001 Jun, 7(6), 904 - 19 An unexpected, conserved element of the U3 snoRNA is required for Mpp10p association; Wormsley S et al.; The U3 small nucleolar ribonucleoprotein (snoRNP) is composed of a small nucleolar RNA (snoRNA) and at least 10 proteins . The U3 snoRNA base pairs with the pre-rRNA to carry out the A0, A1, and A2 processing reactions that lead to the release of the 18S rRNA from the nascent pre-rRNA transcript . The yeast U3 snoRNA can be divided into a short 5' domain (nt 1-39) and a larger 3' domain (73 to the 3' end) separated by a stretch of nucleotides called the hinge region (nt 40-72) . The sequences required for pre-rRNA base pairing are found in the 5' domain and hinge region whereas the 3' domain is largely covered with proteins . Mpp10p, one of the protein components unique to the U3 snoRNP, plays a role in processing at the A1 and A2 sites . Because of its critical role in U3 snoRNP function, we determined which sequences in the U3 snoRNA are required for Mpp10p association . Unlike fibrillarin and all the previous U3 snoRNP components studied in this manner, sequences in the 3' domain are not sufficient for Mpp10p association . Instead, a conserved sequence element in the U3 snoRNA hinge region is required, placing Mpp10p near the 5' domain that carries out the pre-rRNA base-pairing interactions in the functional center of the U3 snoRNP. Neoplasia, 2001 Mar-Apr, 3(2), 99 - 104 Loss of expression of human spectrin src homology domain binding protein 1 is associated with 10p loss in human prostatic adenocarcinoma; Macoska JA et al.; The gene encoding human spectrin Src homology domain binding protein 1, or Hssh3bp1, which is a marker of macropinocytic vesicles and a potential regulator of macropinocytosis, co-localizes to a YAC containing chromosome 10p sequences at loci D10S89 and D10S111 that are frequently deleted in prostate tumors . Expression of Hssh3bp1 was evaluated at the protein level in 17 paired normal and malignant prostate tumor samples using the monoclonal antibody 2G8 to Hssh3bp1 . These experiments demonstrated that 4/6 tumors (67%) with 10p deletion failed to express Hssh3bp1 protein compared to 5/11 (46%) tumors with intact 10p . Thus, loss of Hssh3bp1 expression is concordant with allelic loss of adjacent 10p sequences in human prostate tumors . In addition, two prostate tumor cell lines contain an exon skipping mutation in the Hssh3bp1 gene that leads to the abnormal splicing of the mRNA and loss of a portion of Abl tyrosine kinase SH3 domain binding site in the protein . These data are consistent with a role for Hssh3bp1 as a candidate tumor suppressor gene inactivated during prostate tumorigenesis. Oncogene, 2001 May 28, 20(24), 3076 - 85 ATP-dependent chromatin remodeling factors: nucleosome shufflers with many missions; Varga-Weisz P; This review addresses recent developments in the field of ATP-dependent chromatin remodeling factors . These factors use the energy of ATP hydrolysis to introduce superhelical torsion into DNA, which suggests a common mechanistic basis of action . Chromatin remodeling factors function both in transcriptional activation and repression, but they may have roles outside of transcriptional regulation such as DNA repair . A study of the nucleosome dependent ATPase ISWI in yeast illustrates the involvement of ATP-dependent chromatin remodeling in transcriptional repression by setting up inaccessible chromatin structures at promoters . However, factors such as ISWI are also involved in the restructuring of large chromatin domains and even whole chromosomes . Transcriptional regulation by ATP-dependent chromatin remodeling factors occurs in concert with histone modifying enzymes such as histone acetyltransferases and histone deacetylases: In yeast, SWI/SNF targeting is a requirement for histone acetyltransferases activity at promoters that are active at late stages of mitosis, when the chromatin is still condensed . This demonstrates that ATP-dependent remodeling factors facilitate covalent histone modifications . However, they are also regulated by histone modifications and in some circumstances they function in parallel with histone modifications towards the same goal. Oncogene, 2001 May 10, 20(21), 2720 - 5 p53-interacting protein 53BP2 inhibits clonogenic survival and sensitizes cells to doxorubicin but not paclitaxel-induced apoptosis; Ao Y et al.; 53BP2 was initially identified as a protein interacting with p53 in a yeast two-hybrid screen and subsequently shown to enhance p53 transcriptional transactivation and induce apoptosis when transiently overexpressed in cell lines . In order to further study the biologically relevant effects of 53BP2, we have constructed HEK293 stable cell lines where 53BP2 expression can be regulated using an ecdysone inducible expression system . Our results indicate that the response of cells is dependent on the amount of 53BP2 that is expressed . High levels of 53BP2 expression (>or=140-fold above endogenous) impede cell cycle progression and induce apoptosis . Lower levels of 53BP2 expression (6-11-fold above endogenous) suppress colony formation but do not lead to detectable perturbations in the cell cycle or apoptosis . Lower levels of 53BP2 expression sensitized cells to apoptosis induced by DNA damaging chemotherapy agents doxorubicin, ara-C and VP16, but not microtubule active agents paclitaxel and vinblastine . Our results demonstrate that high levels of 53BP2 expression have profound biological effects ultimately leading to apoptosis, whereas lower levels of 53BP2 expression have more subtle effects on growth and sensitize cells to some chemotherapy agents. Mamm Genome, 2001 Jul, 12(7), 561 - 5 Comparative mapping of BTA15 and HSA11 including a region containing a QTL for meat tenderness; Rexroad CE 3rd et al.; The starting point of the present study was the reported identification of a chromosomal region on bovine Chromosome (Chr) 15 (BTA15) carrying loci affecting meat tenderness . A comparative linkage map of BTA15 and human Chr 11 (HSA11) was constructed to identify potential positional candidate genes and to provide a resource of genetic markers to support marker-assisted selection (MAS) . Relative rearrangements between the bovine and human genomes for these chromosomes are the most complex observed in comparative mapping between the two species, with nine alternating blocks of conserved synteny between HSA11 and bovine Chrs 15 and 29 . The results of this study were the addition of nine genes to the HSA11/BTA15 comparative linkage map, and development of five microsatellite markers within the quantitative trait locus (QTL) interval . One gene with known effects on muscle development (MYOD1) was mapped to the interval . A second gene (CALCA) involved in regulation of calcium levels, a key factor in postmortem tenderization, also mapped within the interval . Refinement of the comparative map and QTL position will reduce the interval on the human transcription map to be scanned in search of candidates, reducing the effort and resources required to identify the allelic variation responsible for the genetic effect. Nature, 2001 Jun 21, 411(6840), 969 - 74 ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses; Bao S et al.; Genotoxic stress triggers the activation of checkpoints that delay cell-cycle progression to allow for DNA repair . Studies in fission yeast implicate members of the Rad family of checkpoint proteins, which includes Rad17, Rad1, Rad9 and Hus1, as key early-response elements during the activation of both the DNA damage and replication checkpoints . Here we demonstrate a direct regulatory linkage between the human Rad17 homologue (hRad17) and the checkpoint kinases, ATM and ATR . Treatment of human cells with genotoxic agents induced ATM/ATR-dependent phosphorylation of hRad17 at Ser 635 and Ser 645 . Overexpression of a hRad17 mutant (hRad17AA) bearing Ala substitutions at both phosphorylation sites abrogated the DNA-damage-induced G2 checkpoint, and sensitized human fibroblasts to genotoxic stress . In contrast to wild-type hRad17, the hRad17AA mutant showed no ionizing-radiation-inducible association with hRad1, a component of the hRad1-hRad9-hHus1 checkpoint complex . These findings demonstrate that ATR/ATM-dependent phosphorylation of hRad17 is a critical early event during checkpoint signalling in DNA-damaged cells. J Biol Chem, 2001 Aug 24, 276(34), 32330 - 7 Epub 2001 Jun 20. Synemin may function to directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres; Bellin RM et al.; Synemin is a large intermediate filament (IF) protein that has been identified in all types of muscle cells in association with desmin- and/or vimentin-containing IFs . Our previous studies (Bellin, R . M., Sernett, S . W., Becker, B., Ip, W., Huiatt, T . W., and Robson, R . M . (1999) J . Biol . Chem . 274, 29493-29499) demonstrated that synemin forms heteropolymeric IFs with major IF proteins and contains a binding site for the myofibrillar Z-line protein alpha-actinin . By utilizing blot overlay assays, we show herein that synemin also interacts with the costameric protein vinculin . Furthermore, extensive assays utilizing the Gal4 yeast two-hybrid system demonstrate interactions of synemin with desmin and vimentin and additionally define more precisely the protein subdomains involved in the synemin/alpha-actinin and synemin/vinculin interactions . The C-terminal approximately 300-amino acid region of synemin binds to the N-terminal head and central rod domains of alpha-actinin and the approximately 150-amino acid C-terminal tail of vinculin . Overall, these interactions indicate that synemin may anchor IFs to myofibrillar Z-lines via interactions with alpha-actinin and to costameres at the sarcolemma via interactions with vinculin and/or alpha-actinin . These linkages would enable the IFs to directly link all cellular myofibrils and to anchor the peripheral layer of myofibrils to the costameres. J Biol Chem, 2001 Sep 21, 276(38), 35305 - 11 Epub 2001 Jun 19. Acute phase protein alpha 1-acid glycoprotein interacts with plasminogen activator inhibitor type 1 and stabilizes its inhibitory activity; Boncela J et al.; alpha(1)-Acid glycoprotein, one of the major acute phase proteins, was found to interact with plasminogen activator inhibitor type 1 (PAI-1) and to stabilize its inhibitory activity toward plasminogen activators . This conclusion is based on the following observations: (a) alpha(1)-acid glycoprotein was identified to bind PAI-1 by a yeast two-hybrid system . Three of 10 positive clones identified by this method to interact with PAI-1 contained almost the entire sequence of alpha(1)-acid glycoprotein; (b) this protein formed complexes with PAI-1 that could be immunoprecipitated from both the incubation mixtures and blood plasma by specific antibodies to either PAI-1 or alpha(1)-acid glycoprotein . Such complexes could be also detected by a solid phase binding assay; and (c) the real-time bimolecular interactions monitored by surface plasmon resonance indicated that the complex of alpha(1)-acid glycoprotein with PAI-1 is less stable than that formed by vitronectin with PAI-1, but in both cases, the apparent K(D) values were in the range of strong interactions (4.51 + 1.33 and 0.58 + 0.07 nm, respectively) . The on rate for binding of PAI-1 to alpha(1)-glycoprotein or vitronectin differed by 2-fold, indicating much faster complex formation by vitronectin than by alpha(1)-acid glycoprotein . On the other hand, dissociation of PAI-1 bound to vitronectin was much slower than that from the alpha(1)-acid glycoprotein, as indicated by 4-fold lower k(off) values . Furthermore, the PAI-1 activity toward urokinase-type plasminogen activator and tissue-type plasminogen activator was significantly prolonged in the presence of alpha(1)-acid glycoprotein . These observations suggest that the complex of PAI-1 with alpha(1)-acid glycoprotein can play a role as an alternative reservoir of the physiologically active form of the inhibitor, particularly during inflammation or other acute phase reactions. Blood, 2001 Jul 1, 98(1), 201 - 9 EAF1, a novel ELL-associated factor that is delocalized by expression of the MLL-ELL fusion protein; Simone F et al.; The (11;19)(q23;p13.1) translocation in acute leukemia leads to the generation of a chimeric protein that fuses MLL to the transcriptional elongation factor ELL . A novel protein was isolated from a yeast 2-hybrid screen with ELL that was named EAF1 for ELL-associated factor 1 . Using specific antibodies, the endogenous EAF1 and ELL proteins were coimmunoprecipitated from multiple cell lines . In addition, endogenous EAF1 also exhibited the capacity to interact with ELL2 . Database comparisons with EAF1 identified a region with a high content of serine, aspartic acid, and glutamic acid residues that exhibited homology with the transcriptional activation domains of several translocation partner proteins of MLL, including AF4, LAF4, and AF5q31 . A similar transcriptional activation domain has been identified in this region of EAF1 . By confocal microscopy, endogenous EAF1 and ELL colocalized in a distinct nuclear speckled pattern . Transfection of the MLL-ELL fusion gene delocalized EAF1 from its nuclear speckled distribution to a diffuse nucleoplasmic pattern . In leukemic cell lines derived from mice transplanted with MLL-ELL-transduced bone marrow, EAF1 speckles were not detected . Taken together, these data suggest that expression of the MLL-ELL fusion protein may have a dominant effect on the normal protein-protein interactions of ELL. Trends Genet, 2001 Jul, 17(7), 370 - 3 Evolutionary expansion of CRIB-containing Cdc42 effector proteins; Pirone DM et al.; Cdc42, a small GTPase, regulates actin polymerization and other signaling pathways through interaction with many different downstream effector proteins . Most of these effector proteins contain a Cdc42-binding domain, called a CRIB domain . Here, we describe the evolutionary analysis of these CRIB-containing proteins in yeast, worms, flies and humans . The number of CRIB-containing effector proteins increases from yeast to humans, involving both an increase within families and the emergence of new families . These evolutionary changes correlate with the development of the more complex signaling pathways present in higher organisms. Biochim Biophys Acta, 2001 Jun 28, 1519(3), 199 - 208 Wheat mitochondria ccmB encodes the membrane domain of a putative ABC transporter involved in cytochrome c biogenesis; Faivre-Nitschke SE et al.; Assembly of cytochromes c is mediated by different proteins depending on the organism and organelle considered . In land plants, mitochondria follow a pathway distinct from that of yeast and animal mitochondria, more similar to that described for alpha- and gamma-proteobacteria . Indeed, in plant mitochondria, four genes were identified based on the similarities of their products with bacterial proteins involved in c-type cytochrome maturation . We report the characterisation of one of these mitochondrial genes in Triticum aestivum, TaccmB, which is proposed to encode a subunit of an ABC transporter . The transcript extremities were mapped and cDNA sequencing revealed 42 C to U editing positions in the 618 nucleotide long coding region . This high editing rate affects the identity of 32 amino acids out of 206 . Antibodies directed against wheat CcmB recognise a 28 kDa protein in an enriched inner mitochondrial membrane protein fraction, a location which is in agreement with the high hydrophobicity of the protein and its function as a putative transmembrane domain of an ABC transporter involved in cytochrome c and c1 biogenesis in plant mitochondria. FEBS Lett, 2001 Jun 15, 499(1-2), 133 - 6 CSN3 interacts with IKKgamma and inhibits TNF- but not IL-1-induced NF-kappaB activation; Hong X et al.; The transcription factor nuclear factor kappaB (NF-kappaB) plays a pivotal role in immune and inflammatory responses . Activation of NF-kappaB requires the activity of IKK, a kinase complex that contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit IKKgamma . To understand how IKK activity is regulated, we searched for IKKgamma-interacting proteins by the yeast two-hybrid system . These screenings identified CSN3, a component of the COP9 signalsome, as a protein specifically interacting with IKKgamma . Overexpression of CSN3 inhibits NF-kappaB activation triggered by tumor necrosis factor (TNF), but not interleukin-1 (IL-1) . Moreover, overexpression of CSN3 also inhibits NF-kappaB activation triggered by proteins involved in TNF signaling, including TNF-R1, TRAF2, RIP, and NIK, but not by TRAF6, a protein involved in IL-1 signaling . These data suggest that CSN3 is a specific negative regulator of TNF- but not IL-1-induced NF-kappaB activation pathways. Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7188 - 93 Nonnucleoside reverse transcriptase inhibitors are chemical enhancers of dimerization of the HIV type 1 reverse transcriptase; Tachedjian G et al.; Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are allosteric inhibitors of the HIV type 1 (HIV-1) reverse transcriptase (RT) . Yeast grown in the presence of many of these drugs exhibited dramatically increased association of the p66 and p51 subunits of the HIV-1 RT as reported by a yeast two-hybrid assay . The enhancement required drug binding by RT; introduction of a drug-resistance mutation into the p66 construct negated the enhancement effect . The drugs could also induce heterodimerization of dimerization defective mutants . Coimmunoprecipitation of RT subunits from yeast lysates confirmed the induction of heterodimer formation by the drugs . In vitro-binding studies indicate that NNRTIs can bind tightly to p66 but not p51 and then mediate subsequent heterodimerization . This study demonstrates an unexpected effect of NNRTIs on the assembly of RT subunits. Mol Cell Biol, 2001 Jul, 21(14), 4818 - 28 The cell cycle-regulatory CDC25A phosphatase inhibits apoptosis signal-regulating kinase 1; Zou X et al.; CDC25A phosphatase promotes cell cycle progression by activating G(1) cyclin-dependent kinases and has been postulated to be an oncogene because of its ability to cooperate with RAS to transform rodent fibroblasts . In this study, we have identified apoptosis signal-regulating kinase 1 (ASK1) as a CDC25A-interacting protein by yeast two-hybrid screening . ASK1 activates the p38 mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal protein kinase-stress-activated protein kinase (JNK/SAPK) pathways upon various cellular stresses . Coimmunoprecipitation studies demonstrated that CDC25A physically associates with ASK1 in mammalian cells, and immunocytochemistry with confocal laser-scanning microscopy showed that these two proteins colocalize in the cytoplasm . The carboxyl terminus of CDC25A binds to a domain of ASK1 adjacent to its kinase domain and inhibits the kinase activity of ASK1, independent of and without effect on the phosphatase activity of CDC25A . This inhibitory action of CDC25A on ASK1 activity involves diminished homo-oligomerization of ASK1 . Increased cellular expression of wild-type or phosphatase-inactive CDC25A from inducible transgenes suppresses oxidant-dependent activation of ASK1, p38, and JNK1 and reduces specific sensitivity to cell death triggered by oxidative stress, but not other apoptotic stimuli . Thus, increased expression of CDC25A, frequently observed in human cancers, could contribute to reduced cellular responsiveness to oxidative stress under mitogenic or oncogenic conditions, while it promotes cell cycle progression . These observations propose a mechanism of oncogenic transformation by the dual function of CDC25A on cell cycle progression and stress responses. Mol Cell Biol, 2001 Jul, 21(14), 4614 - 25 Inhibition of androgen receptor-mediated transcription by amino-terminal enhancer of split; Yu X et al.; A yeast two-hybrid assay has identified an androgen-dependent interaction of androgen receptor (AR) with amino-terminal enhancer of split (AES), a member of the highly conserved Groucho/TLE family of corepressors . Full-length AR, as well as the N-terminal fragment of AR, showed direct interactions with AES in in vitro protein-protein interaction assays . AES specifically inhibited AR-mediated transcription in a well-defined cell-free transcription system and interacted specifically with the basal transcription factor (TFIIE) in HeLa nuclear extract . These observations implicate AES as a selective repressor of ligand-dependent AR-mediated transcription that acts by directly interacting with AR and by targeting the basal transcription machinery. Biochem J, 2001 Jul 1, 357(Pt 1), 269 - 74 Conformational stability and warfarin-binding properties of human serum albumin studied by recombinant mutants; Watanabe H et al.; Correctly folded recombinant wild-type human serum albumin and the single-residue mutants K199A, W214A, R218H and H242Q were produced with the use of a yeast expression system . The changes in R218H resulted in a pronounced decrease in intrinsic fluorescence . Thermodynamic parameters for thermal denaturation of the present mutants and of five additional mutants have been determined, showing small increases in stability for two mutants (R218H and H242Q) and a larger decrease in stability for one (W214A) . In the last of these, denaturation was a heterogeneous process starting at physiological temperature . The high-affinity binding constant for warfarin at pH 7.4 was determined by fluorescence spectroscopy: there was a significant increase in affinity for binding of warfarin to H242Q and K199A and a smaller decrease in affinity for W214A and R218H . The findings show that Trp-214 is not as essential for the high-affinity binding of warfarin as has previously been thought. Biochem J, 2001 Jul 1, 357(Pt 1), 225 - 32 Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells; Myles T et al.; Members of the phosphoprotein phosphatase (PPP) family of protein serine/threonine phosphatases, including protein phosphatase (PP)1, PP2A and PP2B, share invariant active-site residues that are critical for catalytic function {Zhuo, Clemens, Stone and Dixon (1994) J . Biol . Chem . 269, 26234-26238} . Mutation of the active-site residues Asp(88) or His(118) within the human PP2A catalytic subunit (PP2Ac)alpha impaired catalytic activity in vitro; the D88N and H118N substitutions caused a 9- and 23-fold reduction in specific activity respectively, when compared with wild-type recombinant PP2Ac, indicating an important role for these residues in catalysis . Consistent with this, the D88N and H118N substituted forms failed to provide PP2A function in vivo, because, unlike wild-type human PP2Acalpha, neither substituted for the endogenous PP2Ac enzyme of budding yeast . Relative to wild-type PP2Ac, the active-site mutants were dramatically overexpressed in High Five insect cells using the baculovirus system . Milligram quantities of PP2Ac were purified from 1x10(9) High Five cells and the kinetic constants for dephosphorylation of the peptide RRA(pT)VA (single-letter amino-acid notation) by PP2Ac (K(m)=337.5 microM; k(cat)=170 s(-1)) and D88N (K(m)=58.4 microM; k(cat)=2 s(-1)) were determined . The results show that the substitution impairs catalysis severely without a significant effect on substrate binding, consistent with the PPP catalytic mechanism . Combination of the baculovirus and yeast systems provides a strategy whereby the structure-function of PP2Ac may be fully explored, a goal which has previously proven difficult, owing to the stringent auto-regulatory control of PP2Ac protein levels in vivo. Appl Microbiol Biotechnol, 2001 May, 55(5), 585 - 9 Pre-termination in aflR of Aspergillus sojae inhibits aflatoxin biosynthesis; Matsushima K et al.; The aflR gene product is the main transcriptional regulator of aflatoxin biosynthesis in Aspergillus parasiticus and Aspergillus flavus . Although A . sojae strains do not produce aflatoxins, they do have an aflR homologue . When compared with the aflR of A . parasiticus, the A . sojae gene contains two mutations: an HAHA motif and a premature stop codon . To investigate the functionality of the A . sojae aflR gene product, we used a GAL4 one-hybrid system in yeast . The transcription-activating activity of AflR from A . sojae was 15% of that from A . parasiticus . The introduction of an additional aflR from A . sojae into an A . parasiticus strain did not affect aflatoxin productivity . A hybrid aflR comprising the amino-terminal region of A . sojae aflR and the carboxy-terminal region of A . parasiticus aflR suppressed the effect associated with pre-termination of the A . sojae AflR . We conclude that the premature stop codon of the A . sojae aflR is the key to its functionality and leads to prevention of aflatoxin biosynthesis through loss of the transcription of aflatoxin biosynthesis-related genes. Environ Sci Technol, 2001 Jun 1, 35(11), 2365 - 8 Degradation of bisphenol A in water by TiO2 photocatalyst; Ohko Y et al.; The photocatalytic degradation of bisphenol A (BPA), a representative endocrine disruptor, was carried out in TiO2 aqueous suspension . The main purposes were to confirm the total mineralization of BPA and to evaluate the estrogenic activity in the treated water during the photocatalytic reaction . An initial BPA concentration of 175 microM in water was totally degraded to carbon dioxide by TiO2-photocatalyzed reactions under UV irradiation of 10 mW cm-2 for 20 h . Four HPLC peaks indicating intermediate products appeared in chromatograms monitored at 275 nm, but the heights relative to that of the initial BPA were very low, at most 0.04 in the time period 5-10 h after the start of UV irradiation . All of the peaks finally disappeared after 20 h . For the treated water, the transcriptional estrogenic activity in response to human estrogen receptor in a yeast hybrid assay decreased drastically to less than 1% of the initial BPA's activity within 4 h . On the basis of these results, we conclude that TiO2 photocatalysis could be a useful technology for the purification of water containing BPA without generating any serious secondary pollution. Curr Genet, 2001 May, 39(3), 183 - 9 Primary structure and transcription patterns of RPL36, a ribosomal protein-encoding gene of the mycoparasitic fungus, Trichoderma hamatum; Fekete C et al.; We report the isolation and expression profiles of a single-copy gene from the mycoparasitic fungus Trichoderma hamatum encoding a 60 S cytoplasmic ribosomal protein . The gene, named RPL36, was cloned through its nutrient-mediated expression, using mRNA differential screening . Its predicted ORF, interrupted by two introns, encoded a 105-aa polypeptide . The deduced rpL36 protein showed high overall homologies with other L36-type ribosomal proteins isolated from yeast, rat and human . Analysis of the promoter region of RPL36 revealed the presence of two ribosomal protein gene (RPG) boxes and a T-rich region known to be involved in the regulation of most ribosomal protein genes . Expression of RPL36 was tightly regulated by carbon and nitrogen availability . The mRNA levels of this gene decreased upon exposure of the mycelium to different stresses, whereas the addition of cycloheximide resulted in a super-induction . Levels of RPL36 transcripts also increased during mycoparasitic interaction between T . hamatum and Botrytis cinerea. J Virol, 2001 Jul, 75(14), 6337 - 47 A Ty1 reverse transcriptase active-site aspartate mutation blocks transposition but not polymerization; Uzun O et al.; Reverse transcriptases (RTs) are found in a wide variety of mobile genetic elements including viruses, retrotransposons, and infectious organellar introns . An invariant triad of aspartates is thought to be required for the catalytic function of RTs . We generated RT mutants in the yeast retrotransposon Ty1, changing each of these active-site aspartates to asparagine or glutamate . All but one of the mutants lacked detectable polymerase activity . The novel exception, D(211)N, retained near wild-type in vitro polymerase activity within virus-like particles but failed to carry out in vivo transposition . For this mutant, minus-strand synthesis is impaired and formation of the plus-strand strong-stop intermediate is eliminated . Intragenic second-site suppressor mutations of the transposition defect map to the RNase H domain of the enzyme . Our results demonstrate that one of the three active-site aspartates in a retrotransposon RT is not catalytically critical . This implies a basic difference in the polymerase active-site geometry of Ty1 and human immunodeficiency virus RT and shows that subtle mutations in one domain can cause dramatic functional effects on a distant domain of the same enzyme. J Biol Chem, 2001 Aug 24, 276(34), 31635 - 41 Epub 2001 Jun 18. hPop5, a protein subunit of the human RNase MRP and RNase P endoribonucleases; van Eenennaam H et al.; The RNase MRP and RNase P particles both function as endoribonucleases . RNase MRP has been implicated in the processing of precursor-rRNA, whereas RNase P has been shown to function in the processing of pre-tRNA . Both ribonucleoprotein particles have an RNA component that can be folded into a similar secondary structure and share several protein components . We have identified human, rat, mouse, cow, and Drosophila homologues of the Pop5p protein subunit of the yeast RNase MRP and RNase P complexes . The human Pop5 cDNA encodes a protein of 163 amino acids with a predicted molecular mass of 18.8 kDa . Polyclonal antibodies raised against recombinant hPop5 identified a 19-kDa polypeptide in HeLa cells and showed that hPop5 is associated with both RNase MRP and RNase P . Using affinity-purified anti-hPop5 antibodies, we demonstrated that the endogenous hPop5 protein is localized in the nucleus and accumulates in the nucleolus, which is consistent with its association with RNase MRP and RNase P . Catalytically active RNase P was partially purified from HeLa cells, and hPop5 was shown to be associated with it . Finally, the evolutionarily conserved acidic C-terminal tail of hPop5 appeared to be required neither for complex formation nor for RNase P activity. Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1215 - 9 Candida sorbosivorans sp . nov., a new member of the genus Candida Berkhout; James SA et al.; A yeast, strain NCYC 2938T, was isolated from contaminated industrial material . This material was involved in a cascade continuous process for oxidizing sorbitol (D-glucitol) to L-sorbose . The isolate is similar, although not identical, to Candida geochares and Candida magnoliae in its physiological characteristics . Sequence analysis of the 26S rDNA D1/D2 variable domain showed that it was similar to those of both Candida species, but differed sufficiently to be considered as a separate species . Both the physiological characteristics and the unique 26S rDNA D1/D2 sequence of NCYC 2938T are described here, and the yeast has been named Candida sorbosivorans sp . nov . The type strain is NCYC 2938T (= CBS 8768T). Biol Pharm Bull, 2001 Jun, 24(6), 727 - 8 2,2-Diphenyl-1-picrylhydrazyl hydrate, a stable free radical, is an alpha-glucosidase inhibitor; Lee DS et al.; Glycosidases play a pivotal role in processing of various glycoproteins and glycolipids . It is well known that glycosidases are also involved in a variety of degenerative metabolic disorders such as cancer and AIDS . In order to develop potent alpha-glucosidase inhibitors, we first screened 2,2-diphenyl-1-picrylhydrazyl hydrate as a candidate . 2,2-Diphenyl-1-picrylhydrazyl hydrate was found to inhibit alpha- and beta-glucosidases as well as alpha- and beta-mannosidases . It was also shown to be a non-competitive inhibitor of yeast alpha-glucosidase with a Ki value of 1.1 x 10(-6) M . Taken together, we anticipate that 2,2-diphenyl-1-picrylhydrazyl hydrate may be a potent inhibitor for some incurable metabolic disorders including AIDS. Sheng Wu Gong Cheng Xue Bao, 2001 Mar, 17(2), 221 - 3 {Studies on eicosapentaenoic acid production by submersion culture of Cryphecodinium cohnii}; Yang G et al.; The effects of the incubation temperature, initial pH of the medium, carbon source and nitrogen source on the production of Crypthecodinium cohnii C98 were studied . Through orthogonal experiments, the optimum culture medium was obtained (g/L): Glucose, 65; Yeast extract, 2.0; KNO3 3.0, KH2PO4 1.0, MgSO4.7H2O 0.6, NaCl 5.8, CaCl2.2H2O 0.1, ZnSO4.7H2O 0.0067, FeCl3.6H2O 0.014, CuSO4.5H2O 0.0004, MnSO4.H2O 0.0002 . Under the optimum culture conditions, the microalgae dry cell weight and eicosapentaenoic acid was 18.3 g/L and 0.891 g/L, respectively . The submersion culture process was analysed. Nucleic Acids Res, 2001 Jun 15, 29(12), 2558 - 66 A novel fluorometric oligonucleotide assay to measure O( 6)-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase; Kreklau EL et al.; DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents . Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures . We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O(6)-methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract . This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein . This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample . In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling . In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities. Gene, 2001 Jun 13, 271(1), 81 - 6 Molecular characterization of homologues of both subunits A (SPO11) and B of the archaebacterial topoisomerase 6 in plants; Hartung F et al.; The Spo11 protein is an eukaryotic homologue of the topoisomerase 6 subunit A from archaebacteria . In yeast Spo11p has been found to bind covalently to double-strand breaks (DSBs) during meiosis . Single homologues of the SPO11 gene exist in various eukaryotes, except plants . Previously, we found in the Arabidopsis thaliana genome two ancient paralogs, AtSPO11-1 and 2 . Here we report on the molecular characterization of a third one, AtSPO11-3 . This puzzling finding might be explained by the fact that we detected additionally--for the first time outside of the archaebacterial kingdom--a homologue of the subunit B of topoisomerase 6, AtTOP6B . Both AtSPO11-3 and AtTOP6B are abundantly expressed in Arabidopsis and EST comparisons indicate the presence of both genes in various plant species . Via two hybrid studies we could demonstrate that full length AtTop6B is able to interact with AtSpo11-2 and 3 but not with AtSpo11-1 . Our data suggest that plants possess in contrast to other eukaryotes an additional archaebacterial kind of topoisomerase. EMBO J, 2001 Jun 15, 20(12), 3018 - 28 The B-box dominates SAP-1-SRF interactions in the structure of the ternary complex; Hassler M et al.; The serum response element (SRE) is found in several immediate-early gene promoters . This DNA sequence is necessary and sufficient for rapid transcriptional induction of the human c-fos proto-oncogene in response to stimuli external to the cell . Full activation of the SRE requires the cooperative binding of a ternary complex factor (TCF) and serum response factor (SRF) to their specific DNA sites . The X-ray structure of the human SAP-1-SRF-SRE DNA ternary complex was determined (Protein Data Bank code 1hbx) . It shows SAP-1 TCF bound to SRF through interactions between the SAP-1 B-box and SRF MADS domain in addition to contacts between their respective DNA-binding motifs . The SAP-1 B-box is part of a flexible linker of which 21 amino acids become ordered upon ternary complex formation . Comparison with a similar region from the yeast MATalpha2-MCM1-DNA complex suggests a common binding motif through which MADS-box proteins may interact with additional factors such as Fli-1. Biochim Biophys Acta, 2001 Jun 6, 1512(2), 357 - 66 Iron-induced oxidative damage of corn root plasma membrane H(+)-ATPase; Souza-Santos P et al.; The effect of iron on the activity of the plasma membrane H(+)-ATPase (PMA) from corn root microsomal fraction (CRMF) was investigated . In the presence of either Fe(2+) or Fe(3+) (100-200 microM of FeSO(4) or FeCl(3), respectively), 80-90% inhibition of ATP hydrolysis by PMA was observed . Half-maximal inhibition was attained at 25 microM and 50 microM for Fe(2+) and Fe(3+), respectively . Inhibition of the ATPase activity was prevented in the presence of metal ion chel