Plant Cell, 2002 May, 14(5), 993 - 1003 Arabidopsis SGT1b is required for defense signaling conferred by several downy mildew resistance genes; Tor M et al.; We describe the identification of a mutant in the Arabidopsis accession Columbia (Col-0) that exhibits enhanced downy mildew (edm1) susceptibility to several Peronospora parasitica isolates, including the RPP7-diagnostic isolate Hiks1 . The mutation was mapped to chromosome IV and characterized physically as a 35-kb deletion spanning seven genes . One of these genes complemented the mutant to full wild-type resistance against all of the Peronospora isolates tested . This gene (AtSGT1b) encodes a predicted protein of 39.8 kD and is an Arabidopsis ortholog of yeast SGT1, which was described originally as a key regulatory protein in centromere function and ubiquitin-mediated proteolysis . AtSGT1b contains three tetratricopeptide repeats at the N terminus followed by a bipartite chord-containing SGT domain and an SGT-specific domain at the C terminus . We discuss the role of AtSGT1b in disease resistance and its possible involvement in ubiquitin-mediated proteolysis in plants. Nucleic Acids Res, 2002 Jun 1, 30(11), 2270 - 9 Synergistic activation of the rat laminin gamma1 chain promoter by the gut-enriched Kruppel-like factor (GKLF/KLF4) and Sp1; Higaki Y et al.; Laminin is a multifunctional heterotrimeric protein present in extracellular matrix where it regulates processes that compose tissue architecture including cell differentiation . Laminin gamma1 is the most widely expressed laminin chain and its absence causes early lethality in mouse embryos . Laminin gamma1 chain gene (LAMC1) promoter contains several GC/GT-rich motifs including the bcn-1 element . Using the bcn-1 element as a bait in the yeast one-hybrid screen, we cloned the gut-enriched Kruppel-like factor (GKLF or KLF4) from a rat mesangial cell library . We show that GKLF binds bcn-1, but this binding is not required for the GKLF-mediated activation of the LAMC1 promoter . The activity of GKLF is dependent on a synergism with another Kruppel-like factor, Sp1 . The LAMC1 promoter appears to have multiple GKLF- and Sp1-responsive elements which may account for the synergistic activation . We provide evidence that the synergistic action of GKLF and Sp1 is dependent on the promoter context and the integrity of GKLF activation and DNA-binding domain . GKLF is thought to participate in the switch from cell proliferation to differentiation . Thus, the Sp1-GKLF synergistic activation of the LAMC1 promoter may be one of the avenues for expression of laminin gamma1 chain when laminin is needed to regulate cell differentiation. J Biol Chem, 2002 Aug 9, 277(32), 29108 - 15 Epub 2002 May 28. Alix (ALG-2-interacting protein X), a protein involved in apoptosis, binds to endophilins and induces cytoplasmic vacuolization; Chatellard-Causse C et al.; ALG-2-interacting protein X (Alix), also known as AIP1, is a cytoplasmic protein ubiquitously expressed and concentrated in phagosomes and exosomes . Alix may regulate apoptosis since it binds apoptosis-linked gene 2 (ALG-2), a Ca2+-binding protein necessary for cell death, and also overexpression of its C-terminal half (Alix-CT) blocks death induced by several stimuli . This part of Alix contains a long proline-rich domain containing several potential SH3-binding sites . Using Alix as bait in a yeast two-hybrid system to screen a mouse brain library, we have found that SH3p4, SH3p8, and SH3p13, collectively known as endophilins, bind to Alix . Co-immunoprecipitations and overlay experiments allowed us to demonstrate that endophilins bind to Alix-CT through an SH3/proline-rich domain interaction . We have narrowed the region of Alix interacting with endophilins down to 14 amino acids containing a PXRPPPP consensus sequence, also present in synaptojanin and germinal center kinase-like kinase, allowing their interaction to endophilins . We further show that overexpression of Alix-CT, which blocks cell death, leads to cytoplasmic vacuolization into tubulo-vesicular structures delineated by Alix-CT . This vacuolization phenomenon is greatly enhanced upon co-expression with endophilins and may be part of the protecting mechanism afforded by Alix-CT. J Biol Chem, 2002 Aug 9, 277(32), 28972 - 80 Epub 2002 May 28. Calmodulin binds RalA and RalB and is required for the thrombin-induced activation of Ral in human platelets; Clough RR et al.; Ral GTPases may be involved in calcium/calmodulin-mediated intracellular signaling pathways . RalA and RalB are activated by calcium, and RalA binds calmodulin in vitro . It was examined whether RalA can bind calmodulin in vivo, whether RalB can bind calmodulin, and whether calmodulin is functionally involved in Ral activation . Yeast two-hybrid analyses demonstrated both Rals interact directly but differentially with calmodulin . Coimmunoprecipitation experiments determined that calmodulin and RalB form complexes in human platelets . In vitro pull-down experiments in platelets and in vitro binding assays showed endogenous Ral and calmodulin interact in a calcium-dependent manner . Truncated Ral constructs determined in vitro and in vivo that RalA has an additional calmodulin binding domain to that previously described, that although RalB binds calmodulin, its C-terminal region is involved in partially inhibiting this interaction, and that in vitro RalA and RalB have an N-terminal calcium-independent and a C-terminal calcium-dependent calmodulin binding domain . Functionally, in vitro Ral-GTP pull-down experiments determined that calmodulin is required for the thrombin-induced activation of Ral in human platelets . We propose that differential binding of calmodulin by RalA and RalB underlies possible functional differences between the two proteins and that calmodulin is involved in the regulation of the activation of Ral-GTPases. J Biol Chem, 2002 Aug 16, 277(33), 29634 - 42 Epub 2002 May 28. Different roles for the cyclic nucleotide binding domain and amino terminus in assembly and expression of hyperpolarization-activated, cyclic nucleotide-gated channels; Proenza C et al.; In mammalian heart and brain, pacemaker currents are produced by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, which probably exist as heteromeric assemblies of different subunit isoforms . To investigate the molecular domains that participate in assembly and membrane trafficking of HCN channels, we have used the yeast two-hybrid system, patch clamp electrophysiology, and confocal microscopy . We show here that the N termini of the HCN1 and HCN2 isoforms interacted and were essential for expression of functional homo- or heteromeric channels on the plasma membrane of Chinese hamster ovary cells . We also show that the cyclic nucleotide binding domain (CNBD) of HCN2 was required for the expression of functional homomeric channels . This expression was dependent on a 12-amino acid domain corresponding to the B-helix in the CNBD of the catabolite activator protein . However, co-expression with HCN1 of an HCN2 deletion mutant lacking the CNBD rescued surface immunofluorescence and currents, indicating that a CNBD need not be present in each subunit of a heteromeric HCN channel . Furthermore, neither CNBDs nor other COOH-terminal domains of HCN1 and HCN2 interacted in yeast two-hybrid assays . Thus, interaction between NH(2)-terminal domains is important for HCN subunit assembly, whereas the CNBD is important for functional expression, but its absence from some subunits will still allow for the assembly of functional channels. J Biol Chem, 2002 Aug 16, 277(33), 30079 - 90 Epub 2002 May 28. Identification, molecular cloning, and characterization of a novel GABAA receptor-associated protein, GRIF-1; Beck M et al.; A novel 913-amino acid protein, gamma-aminobutyric acid type A (GABA(A)) receptor interacting factor-1 (GRIF-1), has been cloned and identified as a GABA(A) receptor-associated protein by virtue of its specific interaction with the GABA(A) receptor beta 2 subunit intracellular loop in a yeast two-hybrid assay . GRIF-1 has no homology with proteins of known function, but it is the rat orthologue of the human ALS2CR3/KIAA0549 gene . GRIF-1 is expressed as two alternative splice forms, GRIF-1a and a C-terminally truncated form, GRIF-1b . GRIF-1 mRNA has a wide distribution with a major transcript size of 6.2 kb . GRIF-1a protein is only expressed in excitable tissues, i.e . brain, heart, and skeletal muscle major immunoreactive bands of M(r) approximately 115 and 106 kDa and, in muscle and heart only, an additional 88-kDa species . When expressed in human embryonic kidney 293 cells, GRIF-1a yielded three immunoreactive bands with M(r) approximately 115, 106, and 98 kDa . Co-expression of GRIF-1a and alpha 1 beta 2 gamma 2 GABA(A) receptors in mammalian cells revealed some co-localization in the cell cytoplasm . Anti-FLAG-agarose specifically precipitated GRIF-1(FLAG) and GABA(A) receptor beta 2 subunits from human embryonic kidney 293 cells co-transfected with GRIF-1a(FLAG) and beta 2 subunit clones . Further, immobilized GRIF-1-(8-633) specifically precipitated in vitro GABA(A) receptor alpha 1 and beta 2 subunit immunoreactivities from detergent extracts of adult rat brain . The respective GABA(A) receptor beta 2 subunit/GRIF-1 binding domains were mapped using the yeast two-hybrid reporter gene assays . A possible role for GRIF-1 as a GABA(A) receptor beta 2 subunit trafficking factor is proposed. Biochim Biophys Acta, 2002 Apr 22, 1554(1-2), 48 - 56 The quantitation of ADP diffusion gradients across the outer membrane of heart mitochondria in the presence of macromolecules; Gellerich FN et al.; We have previously provided evidence that diffusion of metabolites across the porin pores of mitochondrial outer membrane is hindered . A functional consequence of this diffusion limitation is the dynamic compartmentation of ADP in the intermembrane space . These earlier studies were done on isolated mitochondria suspended in isotonic media without macromolecules, in which intermembrane space of mitochondria is enlarged . The present study was undertaken to assess the diffusion limitation of outer membrane in the presence of 10% (w/v) dextran M20, in order to mimic the action of cytosolic macromolecules on mitochondria . Under these conditions, mitochondria have a more native, condensed configuration.Flux-dependent concentration gradients of ADP were estimated by measuring the ADP diffusion fluxes across the porin pores of isolated rat heart mitochondria incubated together with pyruvate kinase (PK), both of which compete for ADP regenerated by mitochondrial creatine kinase (mtCK) within the intermembrane space or by yeast hexokinase (HK) extramitochondrially . From diffusion fluxes and bulk phase concentrations of ADP, its concentrations in the intermembrane space were calculated using Fick's law of diffusion . Flux-dependent gradients up to 23 microM ADP (for a diffusion rate of J(Dif)=1.9 micromol ADP/min/mg mitochondrial protein) were observed . These gradients are about twice those estimated in the absence of dextran and in the same order of magnitude as the cytosolic ADP concentration (30 microM), but they are negligibly low for cytosolic ATP (5 mM) . Therefore, it is concluded that the dynamic ADP compartmentation is of biological importance for intact heart cells.If mtCK generates ADP within the intermembrane space, the local ADP concentration can be clearly higher than in the cytosol resulting in higher extramitochondrial phosphorylation potentials . In this way, mtCK contributes to ensure optimal kinetic conditions for ATP-splitting reactions in the extramitochondrial compartment. Biophys Chem, 2002 May 2, 96(2-3), 259 - 71 Folding and stability of different oligomeric states of thiamin diphosphate dependent homomeric pyruvate decarboxylase; Killenberg-Jabs M et al.; The folding and stability of recombinant homomeric (alpha-only) pyruvate decarboxylase from yeast was investigated . Different oligomeric states (tetramers, dimers and monomers) of the enzyme occur under defined conditions . The enzymatic activity is used as a sensitive probe for structural differences between the active and inactive form (mis-assembled forms, aggregates) of the folded protein . Unfolding kinetics starting from the native protein comprise both the dissociation of the oligomers into monomers and their subsequent denaturation, which could be monitored by stopped-flow kinetics . In the course of unfolding, the tetramers do not directly dissociate into monomers, but via a stable dimeric state . Starting from the unfolded state, a reactivation of homomeric pyruvate decarboxylase requires both refolding to monomers and their correct association to enzymatically active dimers or tetramers . The reactivation yield under the in vitro conditions used follows an optimum behavior. Virology, 2002 Apr 10, 295(2), 307 - 19 Silencing of a gene encoding a protein component of the oxygen-evolving complex of photosystem II enhances virus replication in plants; Abbink TE et al.; It has been suggested that, in addition to viral proteins, host proteins are involved in RNA virus replication . In this study the RNA helicase domain of the Tobacco mosaic virus (TMV) replicase proteins was used as bait in the yeast two-hybrid system to identify tobacco proteins with a putative role in TMV replication . Two host proteins were characterized . One protein (designated #3) belongs to a protein family of ATPases associated with various activities (AAA), while the second host protein (designated #13) is the 33K subunit of the oxygen-evolving complex of photosystem II . Using Tobacco rattle virus vectors, genes #3 and #13 were silenced in Nicotiana benthamiana, after which the plants were challenged by TMV infection . Silencing of gene #13 resulted in a 10-fold increase of TMV accumulation, whereas silencing of gene #3 caused a twofold reduction of TMV accumulation . Additionally, silencing of genes #3 and #13 decreased and increased, respectively, the accumulation of two other viruses . Similar to silencing of gene #13, inhibition of photosystem II by application of an herbicide increased TMV accumulation several fold . Infection of N . benthamiana with TMV resulted in a decrease of #13 mRNA levels . Silencing of gene #13 may reflect a novel strategy of TMV to suppress basal host defense mechanisms . The two-hybrid screenings did not identify tobacco proteins involved in helicase domain-induced N-mediated resistance . (c) 2002 Elsevier Science (USA). Oncogene, 2002 May 16, 21(22), 3507 - 16 The TRC8 hereditary kidney cancer gene suppresses growth and functions with VHL in a common pathway; Gemmill RM et al.; VHL is part of an SCF related E3-ubiquitin ligase complex with 'gatekeeper' function in renal carcinoma . However, no mutations have been identified in VHL interacting proteins in wild type VHL tumors . We previously reported that the TRC8 gene was interrupted by a t(3;8) translocation in a family with hereditary renal and non-medullary thyroid cancer . TRC8 encodes a multi-membrane spanning protein containing a RING-H2 finger with in vitro ubiquitin ligase activity . We isolated the Drosophila homologue, DTrc8, and studied its function by genetic manipulations and a yeast 2-hybrid screen . Human and Drosophila TRC8 proteins localize to the endoplasmic reticulum . Loss of either DTrc8 or DVhl resulted in an identical ventral midline defect . Direct interaction between DTrc8 and DVhl was confirmed by GST-pulldown and co-immunoprecipitation experiments . CSN-5/JAB1 is a component of the COP9 signalosome, recently shown to regulate SCF function . We found that DTrc8 physically interacts with CSN-5 and that human JAB1 localization is dependent on VHL mutant status . Lastly, overexpression of DTrc8 inhibited growth consistent with its presumed role as a tumor suppressor gene . Thus, VHL, TRC8, and JAB1 appear to be linked both physically and functionally and all three may participate in the development of kidney cancer. Proc Natl Acad Sci U S A, 2002 May 28, 99(11), 7774 - 9 Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome; Preisig-Muller R et al.; Andersen's syndrome, an autosomal dominant disorder related to mutations of the potassium channel Kir2.1, is characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic bone structure . The aim of our study was to find out whether heteromerization of Kir2.1 channels with wild-type Kir2.2 and Kir2.3 channels contributes to the phenotype of Andersen's syndrome . The following results show that Kir2.x channels can form functional heteromers: (i) HEK293 cells transfected with Kir2.x-Kir2.y concatemers expressed inwardly rectifying K(+) channels with a conductance of 28-30 pS . (ii) Expression of Kir2.x-Kir2.y concatemers in Xenopus oocytes produced inwardly rectifying, Ba(2+) sensitive currents . (iii) When Kir2.1 and Kir2.2 channels were coexpressed in Xenopus oocytes the IC(50) for Ba(2+) block of the inward rectifier current differed substantially from the value expected for independent expression of homomeric channels . (iv) Coexpression of nonfunctional Kir2.x constructs, in which the GYG region of the pore region was replaced by AAA, with wild-type Kir2.x channels produced both homomeric and heteromeric dominant-negative effects . (v) Kir2.1 and Kir2.3 channels could be coimmunoprecipitated in membrane extracts from isolated guinea pig cardiomyocytes . (vi) Yeast two-hybrid analysis showed interaction between the N- and C-terminal intracellular domains of different Kir2.x subunits . Coexpression of Kir2.1 mutants related to Andersen's syndrome with wild-type Kir2.x channels showed a dominant negative effect, the extent of which varied between different mutants . Our results suggest that differential tetramerization of the mutant allele of Kir2.1 with wild-type Kir2.1, Kir2.2, and Kir2.3 channels represents the molecular basis of the extraordinary pleiotropy of Andersen's syndrome. J Lipid Res, 2002 Jun, 43(6), 911 - 20 Cloning and characterization of a mammalian fatty acyl-CoA elongase as a lipogenic enzyme regulated by SREBPs; Matsuzaka T et al.; The mammalian enzyme involved in the final elongation of de novo fatty acid biosynthesis following the building of fatty acids to 16 carbons by fatty acid synthase has yet to be identified . In the process of searching for genes activated by sterol regulatory element-binding protein 1 (SREBP-1) by using DNA microarray, we identified and characterized a murine cDNA clone that is highly similar to a fatty acyl-CoA elongase gene family such as Cig30, Sscs, and yeast ELOs . Studies on the cells overexpressing the full-length cDNA indicate that the encoded protein, designated fatty acyl-CoA elongase (FACE), has a FACE activity specific for long-chains; C12-C16 saturated- and monosaturated-fatty acids . Hepatic expression of this identified gene was consistently activated in the livers of transgenic mice overexpressing nuclear SREBP-1a, -1c, or -2 . FACE mRNA levels are markedly induced in a refed state after fasting in the liver and adipose tissue . This refeeding response is significantly reduced in SREBP-1 deficient mice . Dietary PUFAs caused a profound suppression of this gene expression, which could be restored by SREBP-1c overexpression . Hepatic FACE expression was also highly up-regulated in leptin-deficient ob/ob mice . Hepatic FACE mRNA was markedly increased by administration of a pharmacological agonist of liver X-activated receptor (LXR), a dominant activator for SREBP-1c expression . These data indicated that this elongase is a new member of mammalian lipogenic enzymes regulated by SREBP-1, playing an important role in de novo synthesis of long-chain saturated and monosaturated fatty acids in conjunction with fatty acid synthase and stearoyl-CoA desaturase. J Biol Chem, 2002 Aug 9, 277(32), 28545 - 53 Epub 2002 May 24. The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels; Yus-Najera E et al.; We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels . CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+ . Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two alpha-helices (A and B) . These two helices encompass approximately 85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of approximately 130 amino acids . Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1-5-10 CaM-binding motifs in helix B . Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay . Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct . Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur . These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the {Ca2+} is raised . Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels. Diabetes, 2002 Jun, 51(6), 1859 - 66 Tanis: a link between type 2 diabetes and inflammation? Walder K, Kantham L, McMillan JS, Trevaskis J, Kerr L, De Silva A, Sunderland T, Godde N, Gao Y, Bishara N, Windmill K, Tenne-Brown J, Augert G, Zimmet PZ, Collier GR. Here we describe a novel protein, which we have named Tanis, that is implicated in type 2 diabetes and inflammation . In Psammomys obesus, a unique polygenic animal model of type 2 diabetes and the metabolic syndrome, Tanis is expressed in the liver in inverse proportion to circulating glucose (P = 0.010) and insulin levels (P = 0.004) and in direct proportion with plasma triglyceride concentrations (P = 0.007) . Hepatic Tanis gene expression was markedly increased (3.1-fold) after a 24-h fast in diabetic but not in nondiabetic P . obesus . In addition, glucose inhibited Tanis gene expression in cultured hepatocytes (P = 0.006) as well as in several other cell types (P = 0.001-0.011) . Thus, Tanis seems to be regulated by glucose and is dysregulated in the diabetic state . Yeast-2 hybrid screening identified serum amyloid A (SAA), an acute-phase inflammatory response protein, as an interacting protein of Tanis, and this was confirmed by Biacore experiments . SAA and other acute-phase proteins have been the focus of recent attention as risk factors for cardiovascular disease, and we contend that Tanis and its interaction with SAA may provide a mechanistic link among type 2 diabetes, inflammation, and cardiovascular disease. J Microbiol Methods, 2002 Aug, 50(3), 319 - 23 Evaluation of rapid DNA extraction methods for the quantitative detection of fungi using real-time PCR analysis; Haugland RA et al.; Three comparatively rapid methods for the extraction of DNA from fungal conidia and yeast cells in environmental (air, water and dust) samples were evaluated for use in real-time PCR (TaqMan) analyses . A simple bead milling method was developed to provide sensitive, accurate and precise quantification of target organisms in air and water (tap and surface) samples . However, quantitative analysis of dust samples required further purification of the extracted DNA by a streamlined silica adsorption procedure. Biochim Biophys Acta, 2002 Jun 7, 1576(1-2), 127 - 35 High affinity copper transport protein in the lizard Podarcis sicula: molecular cloning, functional characterization and expression in somatic tissues, follicular oocytes and eggs; Riggio M et al.; Copper (Cu) is an essential element required in many biological processes including cellular growth and development . The molecular mechanisms involved in copper homeostasis include proteins that play a role in Cu uptake . Genes encoding high affinity copper transporters (Ctr) have been identified in yeast, plant and mammalian cells.Analysis of copper and zinc content in growing ovarian follicles and ovulated eggs of the reptilian Podarcis sicula demonstrated that the levels of both metals rise during oocyte growth, reaching the maximum in ovulated eggs . By exploiting the remarkable evolutionary conservation of the primary structure of Ctr proteins, cDNA encoding a Ctr was isolated from the liver of the lizard P . sicula by reverse transcriptase PCR and RACE strategy by using primers designed based on consensus motifs present in mammalian Ctr . The predicted protein sequence contains three transmembrane domains and a putative hydrophilic extracellular amino-terminal domain . Besides complementing the respiratory deficiency of yeast cells defective in high affinity Cu transport, expression of lizard Ctr1(1) in Hek293 cells stimulates Cu uptake.Gene expression assessed by Northern blot hybridization of RNA from different tissues of P . sicula shows the highest levels of transcript in both intestine and liver . The profile of Ctr1 mRNA in growing ovarian follicles and eggs demonstrates that the transcript accumulates during the oocyte growth and reaches the highest levels in ovulated eggs . These results suggest that lizard Ctr1 protein may function in Cu acquisition in growing oocytes and eggs. Oral Microbiol Immunol, 2002 Jun, 17(3), 181 - 5 The isolation, identification and molecular analysis of Candida spp . isolated from the oral cavities of patients with diabetes mellitus; Manfredi M et al.; Previous studies have shown a high incidence (77%) of isolation of Candida spp . from the oral cavities of patients with type 1 diabetes mellitus . The aim of the present study was to assess the prevalence of yeast in the oral cavities of patients suffering from type 1 and type 2 diabetes mellitus . The patients were classified according to the level of diabetic control (HbA1c), and further stratified on the presence or absence of dental prosthesis . Oral rinse samples were assessed for the growth of yeast and the degree of colonization . Oral isolates were defined to the species level by both phenotypic and novel molecular methods . The overall proportion (60%) of diabetic patients who had Candida spp . isolated from the oral cavity was similar to that previously reported . Local oral factors, such as the presence of dentures, seemed to have a greater influence than diabetic status on the amount and species of Candida isolated from the oral cavities of diabetic patients . Diabetic patients with dentures had more non-albicans Candida isolated from their mouths than dentate diabetic patients . Candida dubliniensis was isolated from diabetic patients and may have a predilection for dentate patients. Mol Cell Biochem, 2002 Mar, 232(1-2), 163 - 7 The transcription co-repressor TLE1 interacted with the intracellular region of gpl30 through its Q domain; Liu F et al.; As the common signal transducer for IL-6 family cytokines, gp 130 interacts with various signal molecules . Our previous work found the amino-terminal enhancer of split (AES) molecule interacts with gp130 intracellular region through its conserved glutamine-rich (Q) domain . The Q domain in AES shares high homology with those in the transcription co-repressor transducin-like enhancer of split (TLE) proteins . The yeast two-hybrid assay, gluthione S-transferase fusion protein pull-down assay and immuno-co-precipitation assay indicated that the Q domain of TLE1 is capable of binding gp130 intracellular domain, and the intracellular membrane proximal region of gp 130 containing conserved Box1 and Box2 motifs seemed essential for this interaction . The interaction between gp130 and TLE1 indicated that molecules of TLE family might play a role in gp 130 signaling. J Food Prot, 2002 May, 65(5), 840 - 4 Polymerase chain reaction-mediated characterization of molds belonging to the Aspergillus flavus group and detection of Aspergillus parasiticus in peanut kernels by a multiplex polymerase chain reaction; Chen RS et al.; The Aspergillus flavus group covers species of A . flavus and Aspergillus parasiticus as aflatoxin producers and Aspergillus oryzae and Aspergillus sojae as koji molds . Genetic similarity among these species is high, and aflatoxin production of a culture may be affected by cultivation conditions and substrate composition . Therefore, a polymerase chain reaction (PCR)-mediated method of detecting the aflatoxin-synthesizing genes to indicate the degree of risk a genotype has of being a phenotypic producer was demonstrated . In this study, 19 strains of the A . flavus group, including A . flavus, A . parasiticus, A . oryzae, A . sojae, and one Aspergillus niger, were subjected to PCR testing in an attempt to detect four genes, encoding for norsolorinic acid reductase (nor-1), versicolorin A dehydrogenase (ver-1), sterigmatocystin O-methyltransferase (omt-1), and a regulatory protein (apa-2), involved in aflatoxin biosynthesis . Concurrently, the strains were cultivated in yeast-malt (YM) broth for aflatoxin detection . Fifteen strains were shown to possess the four target DNA fragments . With regard to aflatoxigenicity, all seven aflatoxigenic strains possessed the four DNA fragments, and five strains bearing less than the four DNA fragments did not produce aflatoxin . When peanut kernels were artificially contaminated with A . parasiticus and A . niger for 7 days, the contaminant DNA was extractable from a piece of cotyledon (ca . 100 mg), and when subjected to multiplex PCR testing using the four pairs of primers coding for the above genes, they were successfully detected . The target DNA fragments were detected in the kernels infected with A . parasiticus, and none was detected in the sound (uninoculated) kernels or in the kernels infected with A . niger. Eur J Biochem, 2002 May, 269(10), 2546 - 56 Human sprouty 4, a new ras antagonist on 5q31, interacts with the dual specificity kinase TESK1; Leeksma OC et al.; The Drosophila melanogaster protein sprouty is induced upon fibroblast growth factor (FGF)- and epidermal growth factor (EGF)-receptor tyrosine kinase activation and acts as an inhibitor of the ras/MAP kinase pathway downstream of these receptors . By differential display RT-PCR of activated vs . resting umbilical artery smooth muscle cells (SMCs) we detected a new human sprouty gene, which we designated human sprouty 4 (hspry4) based on its homology with murine sprouty 4 . Hspry4 is widely expressed and Northern blots indicate that different isoforms of hspry4 are induced upon cellular activation . The hspry4 gene maps to 5q31.3 . It encodes a protein of 322 amino acids, which, in support of a modulating role in signal transduction, contains a prototypic cysteine-rich region, three, potentially Src homology 3 (SH3) binding, proline-rich regions and a PEST sequence . This new sprouty orthologue can suppress the insulin- and EGF-receptor transduced MAP kinase signaling pathway, but fails to inhibit MAP kinase activation by constitutively active V12 ras . Hspry4 appears to impair the formation of active GTP-ras and exert its activity at the level of wild-type ras or upstream thereof . In a yeast two-hybrid screen, using hspry4 as bait, testicular protein kinase 1 (TESK1) was identified from a human fetal liver cDNA library as a partner of hspry4 . The hspry4-TESK1 interaction was confirmed by coimmunoprecipitation experiments and increases by growth factor stimulation . The two proteins colocalize in apparent cytoplasmic vesicles and do not show substantial translocation to the plasma membrane upon receptor tyrosine kinase stimulation. Novartis Found Symp, 2002, 245, 207 - 18; discussion 218-22, 261-4 A protein chemical approach to channel structure and function: the proton channel of the vacuolar H(+)-ATPase; Findlay JB et al.; The vacuolar H(+)-ATPase provides a channel through which protons can be pumped across the bilayer . It is a complex assembly of about 20 subunits made up from 13 different polypeptide chains . The proton channel is located in the bilayer and therefore must be formed from one or both of the two intramembraneous subunits, called in yeast Vphlp (100 kDa) and Vma3p (16 kDa) . Electron microscopy and the use of water soluble and hydrophobic chemical probes in conjunction with mutagenesis to cysteine or glutamic acid residues, suggest that the membrane sector consists of a single Vphlp subunit in association with a hexameric complex of the four-helical bundle Vma3p subunit . This hexamer encloses a large central pore lined by the first transmembrane helix . This pore appears to be impermeable, however; instead, a glutamic acid residue critical to transport function is located on the outside of the hexamer, deeply buried in the membrane and accessible to probes and inhibitors resident in the hydrophobic phase of the bilayer . The stoichiometry and chemistry of inhibitor binding supports the postulate that the mechanism of action involves rotation of the hexamer in the plane of the bilayer . Mutagenesis data on the Vphlp subunit indicate that it is vital to proton transport . It is likely, therefore, that the proton channel is formed at the interface of the Vphlp and Vma3p subunits, the protons moving via a network of interacting charged amino acid side-chains. Environ Sci Technol, 2002 May 1, 36(9), 1980 - 7 Products of aqueous chlorination of bisphenol A and their estrogenic activity; Hu JY et al.; To assess the estrogenic activity potentially stemming from bisphenol A (BPA) in drinking water, APCI/LC/MS and NMR were used to identify the products of its aqueous chlorination under the following conditions: 500 microg/L bisphenol A and 1.46 mg/L sodium hypochlorite (pH 7.5) at 25 degrees C . The 13 products (4-chloro-BPA; 2,6'-dichloro-BPA; 2,6-dichloro-BPA; 2,2',6'-trichloro-BPA; 2,2',6,6'-tetrachloro-BPA; trichlorophenol; 4-isopropyl-2'-hydroxylphenol; and six kinds of polychlorinated phenoxyphenols (PCPPs)) were found in the chlorinated BPA solution . Three main pathways are proposed: (1) chlorine-substitution reactions on the aromatic ring, followed by dehydration to form the chlorine-substituted BPA, (2) chlorine substitution reactions followed by cleavage of the alpha-C on the isopropyl moiety with positive partial charge and beta'-C on the benzene moiety with a negative partial charge to form trichlorophenol and 4-isopropyl-2'-hydroxylphenol, and (3) the formation of PCPPs . Especially for pathway 2, the reaction mechanism was clarified based on semiempirical quantum mechanical calculations . The reaction proceeded by attack of the OH and Cl (from HOCl) on the alpha-C on the isopropyl moiety with a positive partial charge and on the beta'-C with a negative partial charge on the benzene moiety . The activation energies forthe HOCl/4-chloro-BPA and 2,2',6,6'-tetrachloro-BPA reactions were 0.14 and 0.15 kcal/mol, respectively . Finally, the estrogenic activity of the aqueous chlorinated BPA solution was assessed by an estrogen receptor binding assay and a yeast two-hybrid system . It was found that the binding affinity of the chlorinated aqueous BPA at 60 min was 24 times that before chlorination . The transcriptional activation-induced by products were detected by a yeast two-hybrid system based on the ligand-dependent interaction of two proteins, a human ER and a coactivator, suggesting that the chlorinated BPA solution elicits an ability to mimic the effect of the estrogen hormone. Infect Control Hosp Epidemiol, 2002 May, 23(5), 279 - 81 Environmental legionellosis and oropharyngeal colonization by Legionella in immunosuppressed patients; Pedro-Botet ML et al.; Inhalation of contaminated aerosols is considered the most common route of Legionella transmission . The aim of this study was to determine whether contact with water contaminated by Legionella was related to oropharyngeal colonization in immunosuppressed patients . Eighty-five oropharyngeal swabs (April 1996 to June 1996) were seeded on selective and nonselective buffered charcoal-yeast extract media . Legionella was not isolated. Mol Cell Biol, 2002 Jun, 22(12), 4390 - 401 The DNA architectural protein HMGB1 displays two distinct modes of action that promote enhanceosome assembly; Mitsouras K et al.; HMGB1 (also called HMG-1) is a DNA-bending protein that augments the affinity of diverse regulatory proteins for their DNA sites . Previous studies have argued for a specific interaction between HMGB1 and target proteins, which leads to cooperative binding of the complex to DNA . Here we propose a different model that emerged from studying how HMGB1 stimulates enhanceosome formation by the Epstein-Barr viral activator Rta on a target gene, BHLF-1 . HMGB1 stimulates binding of individual Rta dimers to multiple sites in the enhancer . DNase I and hydroxyl radical footprinting, electrophoretic mobility shift assays, and immobilized template assays failed to reveal stable binding of HMGB1 within the complex . Furthermore, mutational analysis failed to identify a specific HMGB1 target sequence . The effect of HMGB1 on Rta could be reproduced by individual HMG domains, yeast HMO1, or bacterial HU . These results, combined with the effects of single-amino-acid substitutions within the DNA-binding surface of HMGB1 domain A, argue for a mechanism whereby DNA-binding and bending by HMGB1 stimulate Rta-DNA complex formation in the absence of direct interaction with Rta or a specific HMGB1 target sequence . The data contrast with our analysis of HMGB1 action on another BHLF-1 regulatory protein called ZEBRA . We discuss the two distinct modes of HMGB1 action on a single regulatory region and propose how HMGB1 can function in diverse contexts. Mol Cell Biol, 2002 Jun, 22(12), 4346 - 57 UBA1 and UBA2, two proteins that interact with UBP1, a multifunctional effector of pre-mRNA maturation in plants; Lambermon MH et al.; Nicotiana plumbaginifolia UBP1 is an hnRNP-like protein associated with the poly(A)(+) RNA in the cell nucleus . Consistent with a role in pre-mRNA processing, overexpression of UBP1 in N . plumabaginifolia protoplasts enhances the splicing of suboptimal introns and increases the steady-state levels of reporter mRNAs, even intronless ones . The latter effect of UBP1 is promoter specific and appears to be due to UBP1 binding to the 3' untranslated region (3'-UTR) and protecting the mRNA from exonucleolytic degradation (M . H . L . Lambermon, G . G . Simpson, D . A . Kirk, M . Hemmings-Mieszczak, U . Klahre, and W . Filipowicz, EMBO J . 19:1638-1649, 2000) . To gain more insight into UBP1 function in pre-mRNA maturation, we characterized proteins interacting with N . plumbaginifolia UBP1 and one of its Arabidopsis thaliana counterparts, AtUBP1b, by using yeast two-hybrid screens and in vitro pull-down assays . Two proteins, UBP1-associated proteins 1a and 2a (UBA1a and UBA2a, respectively), were identified in A . thaliana . They are members of two novel families of plant-specific proteins containing RNA recognition motif-type RNA-binding domains . UBA1a and UBA2a are nuclear proteins, and their recombinant forms bind RNA with a specificity for oligouridylates in vitro . As with UBP1, transient overexpression of UBA1a in protoplasts increases the steady-state levels of reporter mRNAs in a promoter-dependent manner . Similarly, overexpression of UBA2a increases the levels of reporter mRNAs, but this effect is promoter independent . Unlike UBP1, neither UBA1a nor UBA2a stimulates pre-mRNA splicing . These and other data suggest that UBP1, UBA1a, and UBA2a may act as components of a complex recognizing U-rich sequences in plant 3'-UTRs and contributing to the stabilization of mRNAs in the nucleus. Mol Cell Biol, 2002 Jun, 22(12), 4319 - 33 Recruitment of the NCoA/SRC-1/p160 family of transcriptional coactivators by the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator complex; Beischlag TV et al.; The aryl hydrocarbon receptor complex heterodimeric transcription factor, comprising the basic helix-loop-helix-Per-ARNT-Sim (bHLH-PAS) domain aryl hydrocarbon receptor (AHR) and aryl hydrocarbon receptor nuclear translocator (ARNT) proteins, mediates the toxic effects of TCDD (2,3,7,8 tetrachlorodibenzo-p-dioxin) . The molecular events underlying TCDD-inducible gene activation, beyond the activation of the AHRC, are poorly understood . The SRC-1/NCoA-1, NCoA-2/GRIP-1/TIF-2, and p/CIP/AIB/ACTR proteins have been shown to act as mediators of transcriptional activation . In this report, we demonstrate that SRC-1, NCoA-2, and p/CIP are capable of independently enhancing TCDD-dependent induction of a luciferase reporter gene by the AHR/ARNT dimer . Furthermore, injection of anti-SRC-1 or anti-p/CIP immunoglobulin G into mammalian cells abolishes the transcriptional activity of a TCDD-dependent reporter gene . We demonstrate by coimmunoprecipitation and by a reporter gene assay that SRC-1 and NCoA-2 but not p/CIP are capable of interacting with ARNT in vivo after transient transfection into mammalian cells, while AHR is capable of interacting with all three coactivators . We confirm the interactions of ARNT and AHR with SRC-1 with immunocytochemical techniques . Furthermore, SRC-1, NCoA-2, and p/CIP all associate with the CYP1A1 enhancer region in a TCDD-dependent fashion, as demonstrated by chromatin immunoprecipitation assays . We demonstrate by yeast two-hybrid, glutathione S-transferase pulldown, and mammalian reporter gene assays that ARNT requires its helix 2 domain but not its transactivation domain to interact with SRC-1 . This indicates a novel mechanism of action for SRC-1 . SRC-1 does not require its bHLH-PAS domain to interact with ARNT or AHR, but utilizes distinct domains proximal to its p300/CBP interaction domain . Taken together, these data support a role for the SRC family of transcriptional coactivators in TCDD-dependent gene regulation. Mol Cell Biol, 2002 Jun, 22(12), 4256 - 67 A Krüppel-associated box-zinc finger protein, NT2, represses cell-type-specific promoter activity of the alpha 2(XI) collagen gene; Tanaka K et al.; Type XI collagen is composed of three chains, alpha 1(XI), alpha 2(XI), and alpha 3(XI), and plays a critical role in the formation of cartilage collagen fibrils and in skeletal morphogenesis . It was previously reported that the -530-bp promoter segment of the alpha 2(XI) collagen gene (Col11a2) was sufficient for cartilage-specific expression and that a 24-bp sequence from this segment was able to switch promoter activity from neural tissues to cartilage in transgenic mice when this sequence was placed in the heterologous neurofilament light gene (NFL) promoter . To identify a protein factor that bound to the 24-bp sequence of the Col11a2 promoter, we screened a mouse limb bud cDNA expression library in the yeast one-hybrid screening system and obtained the cDNA clone NT2 . Sequence analysis revealed that NT2 is a zinc finger protein consisting of a Kruppel-associated box (KRAB) and is a homologue of human FPM315, which was previously isolated by random cloning and sequencing . The KRAB domain has been found in a number of zinc finger proteins and implicated as a transcriptional repression domain, although few target genes for KRAB-containing zinc finger proteins has been identified . Here, we demonstrate that NT2 functions as a negative regulator of Col11a2 . In situ hybridization analysis of developing mouse cartilage showed that NT2 mRNA is highly expressed by hypertrophic chondrocytes but is minimally expressed by resting and proliferating chondrocytes, in an inverse correlation with the expression patterns of Col11a2 . Gel shift assays showed that NT2 bound a specific sequence within the 24-bp site of the Col11a2 promoter . We found that Col11a2 promoter activity was inhibited by transfection of the NT2 expression vector in RSC cells, a chondrosarcoma cell line . The expression vector for mutant NT2 lacking the KRAB domain failed to inhibit Col11a2 promoter activity . These results demonstrate that KRAB-zinc finger protein NT2 inhibits transcription of its physiological target gene, suggesting a novel regulatory mechanism of cartilage-specific expression of Col11a2. Biochem J, 2002 Jun 1, 364(Pt 2), 571 - 7 Repression of Smad2 and Smad3 transactivating activity by association with a novel splice variant of CCAAT-binding factor C subunit; Chen F et al.; Activation by transforming growth factor-beta (TGF-beta)/activin receptors leads to phosphorylation of Smad2 (Sma- and Mad-related protein 2) and Smad3, which function as transcription factors to regulate gene expression . Using the MH2 domain (Mad homologue domain of Smad proteins 2) of Smad3 in a yeast two-hybrid screening, we isolated a novel splice variant of CAATT-binding factor subunit C (CBF-C), designated CBF-Cb, that associated with Smad3 . CBF-C is one of the subunits that form a heterotrimeric CBF complex capable of binding and activating the CAATT motif found in the promoters of many eukaryotic genes . CBF-Cb is 62 amino acids shorter than the wild-type CBF-C in the N-terminal region . In addition, CBF-Cb is expressed ubiquitously in various mouse tissues . By an immunoprecipitation assay, we detected an in vivo association of CBF-Cb with Smad2 and Smad3, independent of signalling by activated TGF-beta type I receptors . In transient transfection experiments, overexpression of CBF-Cb was able to repress the transactivating activity of Smad2 and Smad3, mediated either by direct binding to the Smad-responsive element or through their association with the Smad-interacting transcription factor FAST-2 (forkhead activin signal transducer-2) . The Smad-mediated transcriptional response after TGF-beta receptor activation was also inhibited by overexpression of unspliced CBF-C . In addition, the repressive activity of CBF-Cb on Smad2- and Smad3-mediated transcriptional regulation was abrogated by co-expression of the general transcription activator p300 . The association of CBF-Cb with Smad2 was competitively inhibited by overexpression of p300 . These data indicate a novel mechanism for modulation of the transcriptional activity of Smad proteins, whereby the interaction of CBF-Cb, as well as canonical CBF-C, with the MH2 domain of Smads may prevent the association of Smads with transcriptional co-activators. Biochem J, 2002 Jun 1, 364(Pt 2), 497 - 505 Biochemical characterization and subcellular localization of human copper transporter 1 (hCTR1); Klomp AE et al.; The human copper transporter 1 gene (hCTR1) was previously identified by functional complementation in ctr1-deficient yeast . Overexpression of hCTR1 in wild-type yeast leads to increased sensitivity to copper toxicity, and mice with a homozygous disruption at the Ctr1 locus die early during embryogenesis . It is proposed that hCTR1 is responsible for high-affinity copper uptake into human cells, but the underlying molecular mechanisms are unknown . To begin to investigate the biochemical characteristics of hCTR1, a polyclonal antiserum was raised against recombinant hCTR1-fusion peptides . Biosynthetic studies using this antiserum revealed that hCTR1 was synthesized as a precursor protein of 28 kDa containing N-linked oligosaccharides, and is then converted to a mature protein of approx . 35 kDa, which is ubiquitously expressed . Immunofluorescence studies showed that subcellular hCTR1 localization differed markedly between cell types . In some cell lines, hCTR1 was located predominantly in an intracellular vesicular perinuclear compartment, and in others hCTR1 was located predominantly at the plasma membrane . In contrast with the copper export P-type ATPases mutated in Wilson disease and Menkes disease, the localization of hCTR1 was not influenced by copper concentrations . Inhibition of endocytosis by methyl-beta-cyclodextrin caused a partial redistribution of hCTR1 to the cell surface of HeLa cells . Taken together, the results in this study suggest a cell-specific control of copper uptake, which involves subcellular localization of the hCTR1 protein. Biochem Soc Trans, 2002 Apr, 30(2), 146 - 9 Splicing signals and factors in plant intron removal; Brown JW et al.; Constitutive splicing of the potato invertase mini-exon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the adjacent intron and a U(11) element found just downstream of the branchpoint in the upstream intron {Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422-433} . The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences . Plant introns differ from their vertebrate and yeast counterparts in being UA- or U-rich (up to 85% UA) . One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins . We are adopting three approaches to studying the RNA-protein interactions in plant splicing . First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants . Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts . Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened. J Immunol, 2002 Jun 1, 168(11), 5746 - 55 BAD1, an essential virulence factor of Blastomyces dermatitidis, suppresses host TNF-alpha production through TGF-beta-dependent and -independent mechanisms; Finkel-Jimenez B et al.; We investigated how BAD1, an adhesin and virulence factor of Blastomyces dermatitidis, suppresses phagocyte proinflammatory responses . Wild-type yeast cocultured with murine neutrophils or macrophages prompted release of a soluble factor into conditioned supernatant that abolished TNF-alpha production in response to the fungus; isogenic, attenuated BAD1 knockout yeast did not have this effect . Phagocytes released 4- to 5-fold more TGF-beta in vitro in response to wild-type yeast vs BAD1 knockout yeast . Treatment of inhibitory, conditioned supernatant with anti-TGF-beta mAb neutralized detectable TGF-beta and restored phagocyte TNF-alpha production . Similarly, addition of anti-TGF-beta mAb into cultures of phagocytes and wild-type yeast reversed BAD1 inhibition of TNF-alpha production . Conversely, TGF-beta treatment of phagocytes cultured with knockout yeast suppressed TNF-alpha production . Hence, TGF-beta mediates BAD1 suppression of TNF-alpha by wild-type B . dermatitidis cultured in vitro with phagocytes . In contrast to these findings, neutralization of elevated TGF-beta levels during experimental pulmonary blastomycosis did not restore BAD1-suppressed TNF-alpha levels in the lung or ameliorate disease . Soluble BAD1 was found to accumulate in the alveoli of infected mice at levels that suppressed TNF-alpha production by phagocytes . However, in contrast to yeast cell surface BAD1, which induced TGF-beta, soluble BAD1 failed to do so and TNF-alpha suppression mediated by soluble BAD1 was unaffected by neutralization of TGF-beta . Thus, BAD1 of B . dermatitidis induces suppression of TNF-alpha and progressive infection by both TGF-beta-dependent and -independent mechanisms. J Biol Chem, 2002 Aug 16, 277(33), 29908 - 18 Epub 2002 May 21. Vesicle-associated membrane protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to modify export from the endoplasmic reticulum; Wyles JP et al.; Oxysterol-binding protein (OSBP) is 1 of 12 related proteins implicated in the regulation of vesicle transport and sterol homeostasis . A yeast two-hybrid screen using full-length OSBP as bait was undertaken to identify partner proteins that would provide clues to the function of OSBP . This resulted in the cloning of vesicle-associated membrane protein-associated protein-A (VAP-A), a syntaxin-like protein implicated in endoplasmic reticulum (ER)/Golgi vesicle transport, and phospholipid regulation in mammalian cells and yeast, respectively . By using a combination of yeast two-hybrid, glutathione S-transferase pull-down and immunoprecipitation experiments, the VAP-A-binding region in OSBP was localized to amino acids 351-442 . This region did not include the pleckstrin homology (PH) domain but overlapped with the N terminus of the oxysterol binding and OSBP homology domains . C- and N-terminal truncations or deletions of VAP prevented interaction with OSBP but did not affect VAP multimerization . Although the OSBP PH domain was not necessary for VAP-A binding in vitro, interaction with VAP-A was enhanced in cells by mutation of the conserved PH domain tryptophan (OSBP W174A) or deletion of the C-terminal half of the PH domain (OSBP Delta 132-182) . OSBP W174A retained oxysterol binding activity, association with phospholipid vesicles via the PH domain, and localized with VAP in unusual ER-associated structures . At 40 degrees C, misfolded ts045-vesicular stomatitis virus G protein fused to green fluorescent protein was co-localized with VAP-A/OSBP W174A structures on the ER but was exported to the Golgi when folded normally at 32 degrees C . A fluorescent ceramide analogue also accumulated in these ER inclusions, and export to the Golgi was partially inhibited as indicated by decreased Golgi staining and a 30% reduction in sphingomyelin synthesis . These studies show that OSBP binding to the ER and Golgi apparatus is regulated by its PH domain and VAP interactions, and the complex is involved at a stage of protein and ceramide transport from the ER. FEBS Lett, 2002 May 22, 519(1-3), 169 - 72 Human septin-septin interaction: CDCrel-1 partners with KIAA0202; Blaser S et al.; Septins are evolutionary conserved cytoskeletal GTPases forming heteropolymer complexes involved in cytokinesis and other cellular processes . CDCrel-1 (cell division cycle related-1) is a recently cloned and characterized human septin which is highly expressed in non-dividing cells, such as neurons . Using a yeast two-hybrid system we demonstrate that CDCrel-1 partners with another uncharacterized human septin, KIAA0202 . The interaction of CDCrel-1 and KIAA0202 was confirmed in the human leukemia cell line K-562 using pull-down assays with a KIAA0202-glutathione S-transferase fusion protein and by immunoprecipitation of the CDCrel-1-KIAA0202 complex with an anti-KIAA0202 antibody . Expression studies of the two human septins revealed a concomitant expression of both proteins in certain cells. Biochimie, 2002 Feb-Mar, 84(2-3), 131 - 41 Mitochondrial reactive oxygen species in cell death signaling; Fleury C et al.; During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype . Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death . These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs . However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways . ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms . In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state . Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2. RNA, 2002 May, 8(5), 698 - 704 Interaction of RNA with phage display selected peptides analyzed by capillary electrophoresis mobility shift assay; Mucha P et al.; A sensitive capillary electrophoresis mobility shift assay (CEMSA) to analyze RNA/peptide interactions has been developed . Capillary electrophoresis (CE) has been adapted for investigating the interaction between variously methylated 17-nt analogs of the yeast tRNAPhe anticodon stem and loop domain (ASL(Phe)) and 15-amino-acid peptides selected from a random phage display library (RPL) . A peptide-concentration-dependent formation of RNA/peptide complex was clearly visible during CEMSA . In the presence of peptide, the UV-monitored CE peak for ASLPhe with three of the five naturally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5C40) shifted from 18.16 to 20.90 min . The mobility shift was observed only for methylated RNA . The negative effects of diffusion, electroosmotic flow and adhesion of molecules to the capillary internal wall were suppressed by using a buffer containing a sieving polymer and a polyacrylamide-coated capillary . Under these conditions, well-shaped peaks and resolution of RNA free and bound to peptide were achieved . Peptide tF2, the most populated ligand in the RPL, specifically bound triply methylated ASLPhe in a methylated nucleoside-dependent manner . CE was found to be an efficient and sensitive method for the qualitative analysis of RNA-peptide interaction and should be generally applicable to the study of RNA-peptide (protein) interactions. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 May, 34(3), 353 - 7 {Cloning and analysis of a novel gene encoding N-terminal acetyltransferase subunit}; He YG et al.; N-terminal acetylation is the most common modification in eukaryotic proteins, affecting stability and activity of proteins . NatA is one of the N-terminal acetytransferases in yeast . It is composed of two subunits, NAT1 and ARD1 . Defect in one of them leads to loss of activity of NatA . Null mutant of NAT1 in yeast exhibits a variety of phenotypes, including depression of a silent mating type locus (HML), failing to enter G(0) in poor nutrient situations and chromosomes instability . Based on homology of NAT1 between yeast and other organisms, the full-length CDS (coding sequence) of HNAT1 was cloned and sequenced . Result of in situ hybridization in testis of rat showed that expression of NAT1 was high and its expression was different in different phases of spermatogenesis . The gene may play an important role in spermatogenesis. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2002 May, 34(3), 279 - 84 {TFAR19 enhances the opening of permeability transition pore in the mitochondrial membrane of mice liver}; Tian HK et al.; TFAR19 TF-1 cell apoptosis related gene 19 is a novel apoptosis-related gene cloned from human leukemia cell line TF-1 cells undergoing apoptosis in 1999 (accession number AF014955 in GenBank) . The human TFAR19 encodes a protein which shares significant homology to the corresponding proteins of species ranging from yeast to mice . TFAR19 exhibits a ubiquitous expression pattern and its expression is upregulated in tumor cells undergoing apoptosis . Overexpression of TFAR19 could enhance apoptosis of some tumor cells induced by growth factor withdrawal or serum deprivation . But the exact mechanism of TFAR19 is unclear . Mitochondria not only provides energy for the cell, but also plays a critical role on cell death or survival . The release of apoptosis promoting factor, such as cytochrome c from mitochondria, resulted by the damage of mitochondrial membrane integrity, is the key factor controlling apoptosis . The permeability transition pore (PTP) of mitochondria is a protein complex located between the mitochondrial membranes, and it plays an important role in regulating the integrity of mitochondrial membrane . In this study, the effect of recombinant TFAR19 on isolated mitochondrial PTP, membrane potential, and release of cytochrome c was investigated in vitro . The results indicated that recombinant TFAR19 facilitated the isolated mitochondrial PTP opening, decreased the membrane potential, and promoted the release of cytochrome c . The effect of TFAR19 on mitochondria is implemented by opening the mitochondrial PTP . Experimental results implicate that TFAR19 may positively feedback apoptosis signal of mitochondria, forming a positive loop to promote apoptosis. J Biol Chem, 2002 Aug 2, 277(31), 28191 - 9 Epub 2002 May 17. BLOC-1, a novel complex containing the pallidin and muted proteins involved in the biogenesis of melanosomes and platelet-dense granules; Falcon-Perez JM et al.; Recent studies have led to the identification of a group of genes required for normal biogenesis of lysosome-related organelles such as melanosomes and platelet-dense granules . Two of these genes, which are defective in the pallid and muted mutant mouse strains, encode small, coiled-coil-forming proteins that display no homology to each other or to any known protein . We report that these two proteins, pallidin and muted, are components of a novel protein complex . We raised antibodies that allow for detection of pallidin from a wide variety of mammalian cells . Endogenous pallidin was distributed in both soluble and peripheral membrane protein fractions . Size-exclusion chromatography and sedimentation velocity analyses indicated that the bulk of cytosolic pallidin is a component of an asymmetric protein complex with a molecular mass of approximately 200 kDa . We named this complex BLOC-1 (for biogenesis of lysosome-related organelles complex 1) . Steady-state pallidin protein levels were reduced in fibroblasts derived from muted and reduced pigmentation mice, suggesting that the genes defective in these two mutant strains could encode components of BLOC-1 that are required for pallidin stability . Co-immunoprecipitation and immunodepletion experiments using an antibody to muted confirmed that this protein is a subunit of BLOC-1 . Yeast two-hybrid analyses revealed that pallidin is capable of self-association through a region that contains its two coiled-coil forming domains . Unlike AP-3-deficient pearl fibroblasts, which display defects in intracellular zinc storage, zinc distribution was not noticeably affected in pallid or muted fibroblasts . Interestingly, immunofluorescence and in vitro binding experiments demonstrated that pallidin/BLOC-1 is able to associate with actin filaments . We propose that BLOC-1 mediates the biogenesis of lysosome-related organelles by a mechanism that may involve self-assembly and interaction with the actin cytoskeleton. Cancer Res, 2002 May 15, 62(10), 2897 - 905 Initiation of human astrocytoma by clonal evolution of cells with progressive loss of p53 functions in a patient with a 283H TP53 germ-line mutation: evidence for a precursor lesion; Fulci G et al.; Little is known about the genetic and molecular events leading to the early stages of human astrocytoma formation . To examine this issue, we analyzed the significance of sequential accumulation of two somatic point mutations (R267W and E258D) in the TP53 gene during the initiation of astrocytoma in a patient born with a single germ-line p53 point mutation (R283H) . We adapted a p53 transcriptional assay in yeast to establish the temporal occurrence and allelic distribution of the p53 mutations present in the patient and characterized these mutations through functional assays and structural modeling . Our results show that the first somatic mutation occurred at codon 267 on the p53 allele harboring the germ-line mutation R283H, whereas the second somatic mutation occurred in the remaining wild-type (wt) allele at codon 258 . These two mutations induced the formation of tumor cells with the genotype p53(267W+283H/258D), which comprised 70% of the cells in the primary WHO grade II astrocytoma . Another 8% of cells within the tumor had the partially mutated genotype p53(267W+283H/WT) and represented the remnants of a clinically undetectable intermediate stage of astrocytic neoplastic transformation . The remaining 22% of cells had the constitutive p53(283H/WT) genotype and likely consisted of nontumor cells . Functional analysis of the p53 alleles present in the patient's tumor indicated that the germ-line p53(R283H) could transactivate the CDKN1A((p21, WAF1, cip1, SDI1)) but not the BAX gene and retained the ability to induce growth arrest of human glioblastoma cells . The p53(R267W+R283H) and p53(E258D) were incapable of transactivating either promoter or inducing growth arrest . Modeling of p53 interaction with DNA suggests that R283H mutation may weaken the sequence-specific interaction of p53 lysine 120 with the BAX gene but not the CDKN1A p53-responsive elements . Taken together, these results have characterized, for the first time, the genetic events defining a clinically undetectable precursor lesion leading to a grade II astrocytoma . They also suggest that astrocytoma initiation in this patient resulted from monoclonal evolution driven by a sequential loss of proapoptotic and growth arrest functions of p53. Antimicrob Agents Chemother, 2002 Jun, 46(6), 2029 - 31 In vitro activities of pentamidine, pyrimethamine, trimethoprim, and sulfonamides against Aspergillus species; Afeltra J et al.; The susceptibilities of 70 strains of Aspergillus species were tested against seven different sulfa drugs and pentamidine by a microdilution method with RPMI 1640 and yeast nitrogen base media . Sulfamethoxazole, sulfadiazine, and pentamidine were active in vitro . The MICs obtained with RPMI 1640 were significantly higher than those with yeast nitrogen base . More studies are needed to further elucidate the action of these drugs. Recent Prog Horm Res, 2002, 57, 103 - 28 Male germ cell gene expression; Eddy EM; Formation of the male gamete occurs in sequential mitotic, meiotic, and postmeiotic phases . Many germ cell-specific transcripts are produced during this process . Their expression is developmentally regulated and stage specific . Some of these transcripts are product of genes that are male germ cell-specific homologs of genes expressed in somatic cells, while some are expressed from unique genes unlike any others in the genome . Others are alternate transcripts derived from the same gene as transcripts in somatic cells but differing from them in size and/or overall sequence . They are generated during gene expression by using promoters and transcription factors that activate transcription at different start sites upstream or downstream of the usual site, by incorporation of alternate exons, by germ cell-specific splicing events, and by using alternate initiation sites for polyadenylation . Male germ cell development consists of an assortment of unique processes, including meiosis, genetic recombination, haploid gene expression, formation of the acrosome and flagellum, and remodeling and condensation of the chromatin . These processes are intricate, highly ordered, and require novel gene products and a precise and well-coordinated program of gene expression to occur . The regulation of gene expression in male germ cells occurs at three levels: intrinsic, interactive, and extrinsic . A highly conserved genetic program "intrinsic" to germ cells determines the sequence of events that underlies germ cell development . This has been underscored by recent studies showing that meiosis involves many genes that have been conserved during evolution from yeast to man . During meiosis and other processes unique to germ cells, the intrinsic program determines which genes are utilized and when they are expressed . In the postmeiotic phase, it coordinates the expression of genes whose products are responsible for constructing the sperm . The process of spermatogenesis occurs in overlapping waves, with cohorts of germ cells developing in synchrony . The intrinsic program operating within a particular germ cell requires information from and provides information to neighboring cells to achieve this coordination . Sertoli cells are crucial for this "interactive" process as well as for providing essential support for germ cell proliferation and progression through the phases of development . The interactive level of regulation is dependent on "extrinsic" influences, primarily testosterone and follicle-stimulating hormone (FSH) . Studies during the last 4 years have established that FSH is not essential for germ cell development but instead serves an important supportive role for this process . While testosterone is essential for maintenance of spermatogenesis, it acts on Sertoli cells and peritubular cells and has indirect effects on germ cells . The extrinsic and interactive processes are extremely important for establishing and maintaining an optimum environment within which gametogenesis occurs . Nevertheless, an intrinsic evolutionarily conserved genetic program regulates male germ cell gene expression and development. Mamm Genome, 2002 May, 13(5), 239 - 44 Mapping of the mouse hyh gene to a YAC/BAC contig on proximal Chromosome 7; Chae TH et al.; Mice that are homozygous for the autosomal recessive hydrocephaly with hop gait (hyh) mutation on Chromosome (Chr) 7 have congenital hydrocephalus characterized by an interhemispheric cyst arising from the third ventricle and agenesis of the corpus callosum . Analysis of more than 500 backcross and intercross progeny maps the hyh locus to proximal Chr 7, approximately 13 cM centromeric to its originally reported map position . Analysis of recombinants at several MIT microsatellite markers localized the hyh locus between D7Mit75 and D7Mit56 . Development of several new SSLP markers allowed us to refine the hyh candidate interval to a region defined by the cone-rod homeobox ( Crx) gene proximally and D7Mit56 distally . A contig of yeast artificial chromosome (YAC) clones and bacterial artificial chromosome (BAC) clones spanning this entire region has been developed, and a number of potential candidate genes for hyh within this interval have been identified . Gene content is conserved between this region of mouse Chr 7 and human Chr 19q13.3 . Physical mapping of the regions around D7Mit75 and D7Mit56 has also determined the order of a number of MIT markers that remain unresolved on the Mouse Genome Database (MGD) map . Our physical map and transcript map may be useful for positional cloning of genes in this unusually gene-rich region of the genome. Mem Inst Oswaldo Cruz, 2002 Mar, 97(2), 253 - 7 Multicenter Brazilian study of oral Candida species isolated from AIDS patients; Sant'Ana Pde L et al.; Oropharyngeal candidiasis continues to be considered the most common opportunistic disease in Aids patients . This study was designed to investigate species distribution, serotype and antifungal susceptibility profile among Candida spp . isolated from the oral cavity of Aids patients recruited from six Brazilian university centers . Oral swabs from 130 Aids patients were plated onto CHROMagar Candida medium and 142 isolates were recovered . Yeast isolates were identified by classical methods and serotyped using the Candida Check or target system-Iatron . Antifungal susceptibility testing was performed according to the NCCLS microbroth assay . C . albicans was the most frequently isolated species (91%), and 70% of the isolates belonged to serotype A . We detected 12 episodes of co-infection (9%), including co-infection with both serotypes of C . albicans . Non-albicans species were isolated from 12 episodes, 50% of them exhibited DDS or resistance to azoles . Otherwise, only 8 out 130 isolates of C . albicans exhibited DDS or resistance to azoles . Brazilian Aids patients are infected mainly by C . albicans serotype A, most of them susceptible to all antifungal drugs. Bioinformatics, 2002 Apr, 18(4), 585 - 96 Analysis of mRNA expression and protein abundance data: an approach for the comparison of the enrichment of features in the cellular population of proteins and transcripts; Greenbaum D et al.; MOTIVATION: Protein abundance is related to mRNA expression through many different cellular processes . Up to now, there have been conflicting results on how correlated the levels of these two quantities are . Given that expression and abundance data are significantly more complex and noisy than the underlying genomic sequence information, it is reasonable to simplify and average them in terms of broad proteomic categories and features (e.g . functions or secondary structures), for understanding their relationship . Furthermore, it will be essential to integrate, within a common framework, the results of many varied experiments by different investigators . This will allow one to survey the characteristics of highly expressed genes and proteins . RESULTS: To this end, we outline a formalism for merging and scaling many different gene expression and protein abundance data sets into a comprehensive reference set, and we develop an approach for analyzing this in terms of broad categories, such as composition, function, structure and localization . As the various experiments are not always done using the same set of genes, sampling bias becomes a central issue, and our formalism is designed to explicitly show this and correct for it . We apply our formalism to the currently available gene expression and protein abundance data for yeast . Overall, we found substantial agreement between gene expression and protein abundance, in terms of the enrichment of structural and functional categories . This agreement, which was considerably greater than the simple correlation between these quantities for individual genes, reflects the way broad categories collect many individual measurements into simple, robust averages . In particular, we found that in comparison to the population of genes in the yeast genome, the cellular populations of transcripts and proteins (weighted by their respective abundances, the transcriptome and what we dub the translatome) were both enriched in: (i) the small amino acids Val, Gly, and Ala; (ii) low molecular weight proteins; (iii) helices and sheets relative to coils; (iv) cytoplasmic proteins relative to nuclear ones; and (v) proteins involved in 'protein synthesis,' 'cell structure,' and 'energy production.' SUPPLEMENTARY INFORMATION: http://genecensus.org/expression/translatome Cell, 2002 May 3, 109(3), 297 - 306 TFIIH plays an essential role in RNA polymerase I transcription; Iben S et al.; TFIIH is a multisubunit protein complex that plays an essential role in nucleotide excision repair and transcription of protein-coding genes . Here, we report that TFIIH is also required for ribosomal RNA synthesis in vivo and in vitro . In yeast, pre-rRNA synthesis is impaired in TFIIH ts strains . In a mouse, part of cellular TFIIH is localized within the nucleolus and is associated with subpopulations of both RNA polymerase I and the basal factor TIF-IB . Transcription systems lacking TFIIH are inactive and exogenous TFIIH restores transcriptional activity . TFIIH is required for productive but not abortive rDNA transcription, implying a postinitiation role in transcription . The results provide a molecular link between RNA polymerase I transcription and transcription-coupled repair of active ribosomal RNA genes. J Biol Chem, 2002 Jul 26, 277(30), 27154 - 61 Epub 2002 May 20. Novel Mediator proteins of the small Mediator complex in Drosophila SL2 cells; Gu JY et al.; The Mediator complex is generally required for transcriptional regulation in species ranging from yeast to human . Throughout evolution, the functional diversity of the Mediator complex has been enhanced to meet the increasing requirements for sophisticated gene regulation . It is likely that greater structural complexity is thus required to accomplish these new, complex regulatory functions . In this study, we took systematic steps to examine various types of Mediator complexes in Drosophila melanogaster . Such efforts led to the identification of three distinct forms of Mediator complexes . In exploring their compositional and functional heterogeneity, we found that the smallest complex (C1) is highly enriched in a certain type of Drosophila cells and possesses novel Mediator proteins . The subunits shared among the three Mediator complexes (C1, C2, and C3) appear to form a stable modular structure that serves as a binding surface for transcriptional activator proteins . However, only C2 and C3 were able to support activated transcription in vitro . These findings suggest that different cell types may require distinct Mediator complexes, some of which may participate in nuclear processes other than the previously identified functions. Biol Reprod, 2002 Jun, 66(6), 1813 - 9 Early growth response gene-1 regulates the expression of the rat luteinizing hormone receptor gene; Yoshino M et al.; LH receptor gene expression is primarily regulated via specific interactions of trans-acting proteins and cis-acting DNA sequences in the upstream region of the gene . In this study, we report, using luciferase assays, that the region between -171 and -137 base pairs (bp) is essential for basal expression of the rat LH receptor gene . To identify factors that interact with the region between -171 and -137 bp and regulate expression of the gene, a rat granulosa cell cDNA library was screened using a yeast one-hybrid system . A positive clone, isolated by the screening, encodes a transcription factor early growth response gene-1 (Egr-1) . To determine the sequence to which Egr-1 protein binds, electrophoretic mobility shift assay (EMSA) was employed . The Egr-1 protein was produced by an in vitro transcription/translation system using a full-length rat Egr-1 cDNA . The upstream region between -171 and -137 bp contains 2 overlapping Egr-1 consensus sequences . The EMSA revealed that Egr-1 binds independently to both sites . The overexpression of Egr-1 in MA-10 cells caused an approximately 2-fold increase in reporter luciferase activity . However, no induction of the luciferase activity was observed when luciferase constructs that lacked or had mutations in either or both of the Egr-1 sites were used, indicating that Egr-1 positively regulates LH receptor gene expression . In differentiated granulosa cells that had been pretreated with FSH for 48 h, the levels of both mRNA and Egr-1 protein were induced by hCG or cAMP, reaching maximal levels approximately 1.5 h after treatment and then returning to basal levels 8 h thereafter . No Egr-1 mRNA or protein was detected in undifferentiated granulosa cells, even after stimulation with 8-bromoadenosine-cAMP . These results suggest that Egr-1 functions only in luteinized granulosa cells after stimulation with hCG or cAMP . In conclusion, the findings demonstrate that Egr-1 actually binds to the regulatory upstream region of the LH receptor gene and positively regulates receptor gene expression . In addition, Egr-1 expression was observed only in luteinized granulosa cells after stimulation with hCG or cAMP . The present study provides further support to the hypothesis that Egr-1 plays important roles in the pituitary-gonadal axis. Insect Biochem Mol Biol, 2002 Jun, 32(6), 591 - 7 A deleted portion of one of the two xanthine dehydrogenase genes causes translucent larval skin in the oq mutant of the silkworm (Bombyx mori); Komoto N; A silkworm mutant, oq, has translucent larval skin because it is deficient in xanthine dehydrogenase (XDH) activity and is unable to synthesize uric acid, which is normally accumulated in the larval epidermis and makes the skin white and opaque . Two XDH bands were found in zymograms of the silkworm fat body: an intense band (XDHalpha) and a faint one (XDHbeta) . The oq mutant lacks only XDHalpha, which seemed to be the major source of XDH activity in the fat body . An 8-bp deletion found in BmXDH1, a silkworm XDH gene, generates a premature stop codon . The resulting truncated BmXDH1 protein lacks three molybdenum cofactor-binding domains necessary for enzyme activity . BmXDH2, the other XDH gene, does not show any apparent deficiencies . BmXDH1 expressed in yeast cells yielded an activity band with the same mobility as that of XDHalpha in zymograms . BmXDH1 of the oq mutant did not yield active XDH in yeast, while the activity was restored by filling in the deleted sequence . These results showed that BmXDH1 deletion in the oq mutant is responsible for the absence of significant XDH activity, resulting in the translucent larval skin of the mutant phenotype. Biochem J, 2002 Sep 1, 366(Pt 2), 471 - 80 Stereochemical features of the hydrolysis of 9,10-epoxystearic acid catalysed by plant and mammalian epoxide hydrolases; Summerer S et al.; cis-9,10-epoxystearic acid was used as a tool to probe the active sites of epoxide hydrolases (EHs) of mammalian and plant origin . We have compared the stereochemical features of the hydrolysis of this substrate catalysed by soluble and membrane-bound rat liver EHs, by soluble EH (purified to apparent homogeneity) obtained from maize seedlings or celeriac roots, and by recombinant soybean EH expressed in yeast . Plant EHs were found to differ in their enantioselectivity, i.e . their ability to discriminate between the two enantiomers of 9,10-epoxystearic acid . For example, while the maize enzyme hydrated both enantiomers at the same rate, the EH from soybean exhibited very high enantioselectivity in favour of 9R,10S-epoxystearic acid . This latter enzyme also exhibited a strict stereoselectivity, i.e . it hydrolysed the racemic substrate with a very high enantioconvergence, yielding a single chiral diol product, threo-9R,10R-dihydroxystearic acid . Soybean EH shared these distinctive stereochemical features with the membrane-bound rat liver EH . The stereochemical outcome of these enzymes probably results from a stereoselective attack by the nucleophilic residue on the oxirane ring carbon having the (S)-configuration, leading to the presumed (in plant EH) covalent acyl-enzyme intermediate . In sharp contrast, the reactions catalysed by cytosolic rat liver EH exhibited a complete absence of enantioselectivity and enantioconvergence; this latter effect might be ascribed to a regioselective formation of the acyl-enzyme intermediate involving C-10 of 9,10-epoxystearic acid, independent of its configuration . Thus, compared with soybean EH, the active site of rat liver soluble EH displays a very distinct means of anchoring the oxirane ring of the fatty acid epoxides, and therefore appears to be a poor model for mapping the catalytic domain of plant EHs. Biochem J, 2002 Aug 15, 366(Pt 1), 79 - 86 Polyamine-modulated factor 1 binds to the human homologue of the 7a subunit of the Arabidopsis COP9 signalosome: implications in gene expression; Wang Y et al.; Polyamines have been identified to play a role in the transcription of various growth-related genes . The recently discovered polyamine responsive element and the associated trans-acting proteins involved in polyamine-regulated transcription have provided a model system for the study of the role of polyamines in transcription . Polyamine-modulated factor 1 (PMF-1) was identified as one of the transacting factors that binds to NF-E2 related factor-2 (Nrf-2) to regulate the transcription of spermidine/spermine N(1)-acetyltransferase (SSAT) . The possibility that PMF-1 also binds to other proteins involved in transcriptional regulation cannot be ruled out . Using a yeast two-hybrid strategy, it was found that PMF-1 binds to a human homologue of the Arabidopsis COP9 signalosome subunit 7a (CSN 7) protein . In the present study, we describe human CSN 7, a 275-amino-acid- containing protein that may have a direct role in regulating gene expression . CSN 7 and PMF-1 bind to each other, as well as compete with each other for binding to Nrf-2 . This competition for Nrf-2 binding and interaction with each other is implicated in the regulation of SSAT transcription . CSN 7 possesses a C-terminal coiled-coil domain similar to the domain that mediates the interaction between PMF-1 and Nrf-2, suggesting that coiled-coil domains also mediate the interaction between CSN 7 and PMF-1 . Since CSN 7 does not contain a DNA-binding domain, its effects on transcription must occur in conjunction with binding to other proteins . The results presented here demonstrate that PMF-1 and Nrf-2 can act as protein partners of CSN 7. J Appl Toxicol, 2002 May-Jun, 22(3), 161 - 5 Effects of trivalent chromium on hepatic CYP-linked monooxygenases in laying hens; Guerra MC et al.; Chromium is an essential nutrient required for the metabolism of carbohydrates and lipids in humans and many animal species . We evaluated whether feeding laying hens with high levels of different chemical forms of trivalent chromium may affect hepatic metabolizing cytochrome P-450 (CYP)-linked enzymes . Modulation of CYP-dependent monooxygenases (which play a pivotal role in the endogenous metabolism) by Cr(III) was tested using either specific substrates as probes of different CYPs or testosterone as a multi-bioprobe . The CYP-supported oxidases were studied in liver microsomes from laying hens fed with diet supplemented with either 25 or 50 ppm chromium chloride as a yeast or as aminoniacinate . Although at 25 ppm no appreciable effects were observed, at 50 ppm a general inactivation of the recorded monooxygenases (ranging from 34% loss for ethoxycoumarin O-deethylase with chromium chloride to 85% loss for O-deethylation of ethoxyresorufin with either chromium yeast or aminoniacinate) were achieved in the supplemented groups vs controls . The only exception was the O-dealkylation of pentoxyresorufin, which was significantly boosted by both chromium yeast (up to 65% increase) and chromium aminoniacinate (up to 141%) . Measurements of the regio- and stereoselective hydroxylation of testosterone used as a multi-bioprobe corroborated the inactivating properties of Cr(III) at the higher dose . Taken as a whole, these findings seem to indicate that high levels of Cr(III) supplementation can substantially impair CYP-catalysed drug metabolism in laying hens . J Biol Chem, 2002 Aug 16, 277(33), 30165 - 76 Epub 2002 May 15. Transforming acidic coiled-coil-containing protein 4 interacts with centrosomal AKAP350 and the mitotic spindle apparatus; Steadman BT et al.; AKAP350 is a multiply spliced family of 350-450-kDa protein kinase A-anchoring proteins localized to the centrosomes and the Golgi apparatus . Using AKAP350A as bait in a yeast two-hybrid screen of a rabbit parietal cell library, we have identified a novel AKAP350-interacting protein, transforming acidic coiled-coil-containing protein 4 (TACC4) . Two-hybrid binary assays demonstrate interaction of both TACC3 and TACC4 with AKAP350A and AKAP350B . Antibodies raised to a TACC4-specific peptide sequence colocalize TACC4 with AKAP350 at the centrosome in interphase Jurkat cells . Mitotic cell staining reveals translocation of TACC4 from the centrosome to the spindle apparatus with the majority of TACC4 at the spindle poles . Truncated TACC4 proteins lacking the AKAP350 minimal binding domain found in the carboxyl coiled-coil region of TACC4 could no longer target to the centrosome . Amino-truncated TACC4 proteins could no longer target to the spindle apparatus . Further, overexpression of TACC4 fusion proteins that retained spindle localization in mitotic cells resulted in an increased proportion of cells present in prometaphase . We propose that AKAP350 is responsible for sequestration of TACC4 to the centrosome in interphase, whereas a separate TACC4 domain results in functional localization of TACC4 to the spindle apparatus in mitotic cells. J Biol Chem, 2002 Jul 26, 277(30), 26821 - 30 Epub 2002 May 15. The gene encoding the Acyl-CoA-binding protein is activated by peroxisome proliferator-activated receptor gamma through an intronic response element functionally conserved between humans and rodents; Helledie T et al.; The acyl-CoA-binding protein (ACBP) is a 10-kDa intracellular protein that specifically binds acyl-CoA esters with high affinity and is structurally and functionally conserved from yeast to mammals . In vitro studies indicate that ACBP may regulate the availability of acyl-CoA esters for various metabolic and regulatory purposes . The protein is particularly abundant in cells with a high level of lipogenesis and de novo fatty acid synthesis and is significantly induced during adipocyte differentiation . However, the molecular mechanisms underlying the regulation of ACBP expression in mammalian cells have remained largely unknown . Here we report that ACBP is a novel peroxisome proliferator-activated receptor (PPAR)gamma target gene . The rat ACBP gene is directly activated by PPARgamma/retinoid X receptor alpha (RXRalpha) and PPARalpha/RXRalpha, but not by PPARdelta/RXRalpha, through a PPAR-response element in intron 1, which is functionally conserved in the human ACBP gene . The intronic PPAR-response element (PPRE) mediates induction by endogenous PPARgamma in murine adipocytes and confers responsiveness to the PPARgamma-selective ligand BRL49653 . Finally, we have used chromatin immunoprecipitation to demonstrate that the intronic PPRE efficiently binds PPARgamma/RXR in its natural chromatin context in adipocytes . Thus, the PPRE in intron 1 of the ACBP gene is a bona fide PPARgamma-response element. Structure (Camb), 2002 May, 10(5), 609 - 18 The structure of the mammalian 20S proteasome at 2.75 A resolution; Unno M et al.; The 20S proteasome is the catalytic portion of the 26S proteasome . Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively . Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution . The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits . A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit . We also determined the site of the nuclear localization signals in the molecule . A model of the immunoproteasome was predicted from this constitutive structure. Curr Biol, 2002 May 14, 12(10), 854 - 8 Transitions regulating the timing of cytokinesis in embryonic cells; Shuster CB et al.; Anaphase, mitotic exit, and cytokinesis proceed in rapid succession, and while mitotic exit is a requirement for cytokinesis in yeast, it may not be a direct requirement for furrow initiation in animal cells . In this report, we physically manipulated the proximity of the mitotic apparatus (MA) to the cell cortex in combination with microinjection of effectors of the spindle checkpoint and CDK1 activity to determine how the initiation of cytokinesis is coupled to the onset of anaphase and mitotic exit . Whereas precocious contact between the MA and the cell surface advanced the onset of cytokinesis into early anaphase A, furrowing could not be advanced prior to the metaphase-anaphase transition . Additionally, while cells arrested in anaphase could be induced to initiate cleavage furrows, cells arrested in metaphase could not . Finally, activation of the mitotic checkpoint in one spindle of a binucleate cell failed to arrest cytokinesis induced by the control spindle but did inhibit the formation of furrows between the arrested MA and the control, nonarrested MA . Our experiments suggest that the competence of the mitotic apparatus to initiate cytokinesis is not dependent on cyclin degradation but does require anaphase-promoting complex (APC) activity and, thus, inactivation of the mitotic checkpoint. Curr Biol, 2002 May 14, 12(10), 829 - 33 Sister chromatids fail to separate during an induced endoreplication cycle in Drosophila embryos; Vidwans SJ et al.; When mitosis is bypassed, as in some cancer cells or in natural endocycles, sister chromosomes remain paired and produce four-stranded diplochromosomes or polytene chromosomes . Cyclin/Cdk1 inactivation blocks entry into mitosis and can reset G2 cells to G1, allowing another round of replication . Reciprocally, persistent expression of Cyclin A/Cdk1 or Cyclin E/Cdk2 blocks Drosophila endocycles . Inactivation of Cyclin A/Cdk1 by mutation or overexpression of the Cyclin/Cdk1 inhibitor, Roughex (Rux), converts the 16(th) embryonic mitotic cycle to an endocycle; however, we show that Rux expression fails to convert earlier cell cycles unless Cyclin E is also downregulated . Following induction of a Rux transgene in Cyclin E mutant embryos during G2 of cell cycle 14 (G2(14)), Cyclins A, B, and B3 disappeared and cells reentered S phase . This rereplication produced diplochromosomes that segregated abnormally at a subsequent mitosis . Thus, like the yeast CKIs Rum1 and Sic1, Drosophila Rux can reset G2 cells to G1 . The observed cyclin destruction suggests that cell cycle resetting by Rux was associated with activation of the anaphase-promoting complex (APC), while the presence of diplochromosomes implies that this activation of APC outside of mitosis was not sufficient to trigger sister disjunction. Curr Biol, 2002 May 14, 12(10), 787 - 97 C . elegans PAT-4/ILK functions as an adaptor protein within integrin adhesion complexes; Mackinnon AC et al.; BACKGROUND: Mammalian integrin-linked kinase (ILK) was identified in a yeast two-hybrid screen for proteins binding the integrin beta(1) subunit cytoplasmic domain . ILK has been implicated in integrin-mediated signaling and is also an adaptor within integrin-associated cytoskeletal complexes . RESULTS: We identified the C . elegans pat-4 gene in previous genetic screens for mutants unable to assemble integrin-mediated muscle cell attachments . Here, we report that pat-4 encodes the sole C . elegans homolog of ILK . In pat-4 null mutants, embryonic muscle cells form integrin foci, but the subsequent recruitment of vinculin and UNC-89 as well as actin and myosin filaments to these in vivo focal adhesion analogs is blocked . Conversely, PAT-4/ILK requires the ECM component UNC-52/perlecan, the transmembrane protein integrin, and the novel cytoplasmic attachment protein UNC-112 to be properly recruited to nascent attachments . Transgenically expressed "kinase-dead" ILK fully rescues pat-4 loss-of-function mutants . We also identify UNC-112 as a new binding partner for ILK . CONCLUSIONS: Our data strengthens the emerging view that ILK functions primarily as an adaptor protein within integrin adhesion complexes and identifies UNC-112 as a new ILK binding partner. Zhonghua Xue Ye Xue Za Zhi, 2002 Jan, 23(1), 30 - 2 {Detection of inv (16) in acute myelomonocytic leukemia by interphase fluorescence in situ hybridization}; Li J et al.; OBJECTIVE: To evaluate fluorescence in situ hybridization (FISH) in the detection of inv (16) (p13; q22) . METHODS: Spectrum red labeled yeast artificial chromosome (YAC) clone 854E2 which spans the breakpoint cluster region in MYH11 in band 16p13 and single color interphase FISH were used to detect inv (16) in 26 cases of acute myelomonocytic leukemias (AML-M(4)), and the results were compared with that of conventional cytogenetic analysis . RESULTS: R banding karyotyping test revealed no inv (16) in 25 cases, one AML M(4Eo) case showed inv (16) by G banding . Nine cases including all three M(4Eo) had inv (16) by FISH analysis, among whom the characteristic fluorescence signal pattern of the inv (16) was seen in 13.3% to 32.1% (median, 21.3%) of the tested cells . CONCLUSION: YAC 854E2 and interphase FISH provide a powerful technique in the detection of inv (16) (p13q22). Biochem J, 2002 Sep 1, 366(Pt 2), 585 - 94 Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response; Okada T et al.; In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), a homoeostatic response, termed the unfolded protein response (UPR), is activated in all eukaryotic cells . The UPR involves only transcriptional regulation in yeast, and approx . 6% of all yeast genes, encoding not only proteins to augment the folding capacity in the ER, but also proteins working at various stages of secretion, are induced by ER stress {Travers, Patil, Wodicka, Lockhart, Weissman and Walter (2000) Cell (Cambridge, Mass.) 101, 249-258} . In the present study, we conducted microarray analysis of HeLa cells, although our analysis covered only a small fraction of the human genome . A great majority of human ER stress-inducible genes (approx . 1% of 1800 genes examined) were classified into two groups . One group consisted of genes encoding ER-resident molecular chaperones and folding enzymes, and these genes were directly regulated by the ER-membrane-bound transcription factor activating transcription factor (ATF) 6 . The ER-membrane-bound protein kinase double-stranded RNA-activated protein kinase-like ER kinase (PERK)-mediated signalling pathway appeared to be responsible for induction of the remaining genes, which are not involved in secretion, but may be important after cellular recovery from ER stress . In higher eukaryotes, the PERK-mediated translational-attenuation system is known to operate in concert with the transcriptional-induction system . Thus we propose that mammalian cells have evolved a strategy to cope with ER stress different from that of yeast cells. Proc Natl Acad Sci U S A, 2002 May 14, 99(10), 6925 - 30 Siva-1 binds to and inhibits BCL-X(L)-mediated protection against UV radiation-induced apoptosis; Xue L et al.; We previously cloned Siva-1 by using the cytoplasmic tail of CD27, a member of the tumor necrosis factor receptor family, as the bait in the yeast two-hybrid system . The Siva gene is organized into four exons that code for the predominant full-length Siva-1 transcript, whereas its alternate splice form, Siva-2, lacks exon 2 coding sequence . Various groups have demonstrated a role for Siva-1 in several apoptotic pathways . Interestingly, the proapoptotic properties of Siva-1 are lacking in Siva-2 . The fact that Siva-1 is partly localized to mitochondria despite the absence of any mitochondrial targeting signal, it harbors a 20-aa-long putative amphipathic helical structure that is absent in Siva-2, and that its expression is restricted to double-positive (CD3(+), CD4(+), CD8(+)) thymocytes like BCL-X(L), prompted us to test for a potential interaction between Siva-1 and BCL-X(L) . Here, we show that Siva-1 binds to and inhibits BCL-X(L)-mediated protection against UV radiation-induced apoptosis . Indeed, the unique amphipathic helical region (SAH) present in Siva-1 is required for its binding to BCL-X(L) and sensitizing cells to UV radiation . Natural complexes of Siva-1/BCL-X(L) are detected in HUT78 and murine thymocyte, suggesting a potential role for Siva-1 in regulating T cell homeostasis. Blood, 2002 Jun 1, 99(11), 3931 - 8 Rescue of the lethal scl(-/-) phenotype by the human SCL locus; Sinclair AM et al.; The stem cell leukemia (SCL) gene encodes a basic helix-loop-helix transcription factor with a critical role in the development of both blood and endothelium . Loss-of-function studies have shown that SCL is essential for the formation of hematopoietic stem cells, for subsequent erythroid development and for yolk sac angiogenesis . SCL exhibits a highly conserved pattern of expression from mammals to teleost fish . Several murine SCL enhancers have been identified, each of which directs reporter gene expression in vivo to a subdomain of the normal SCL expression pattern . However, regulatory elements necessary for SCL expression in erythroid cells remain to be identified and the size of the chromosomal domain needed to support appropriate SCL transcription is unknown . Here we demonstrate that a 130-kilobase (kb) yeast artificial chromosome (YAC) containing the human SCL locus completely rescued the embryonic lethal phenotype of scl(-/-) mice . Rescued YAC(+) scl(-/-) mice were born in appropriate Mendelian ratios, were healthy and fertile, and exhibited no detectable abnormality of yolk sac, fetal liver, or adult hematopoiesis . The human SCL protein can therefore substitute for its murine homologue . In addition, our results demonstrate that the human SCL YAC contains the chromosomal domain necessary to direct expression to the erythroid lineage and to all other tissues in which SCL performs a nonredundant essential function. Eur J Cancer, 2002 May, 38(8), 1126 - 32 Novel expressed sequences obtained by means of a suppression subtractive hybridisation analysis from the 6q21 region that is frequently deleted in gastric cancer; Carvalho B et al.; In our search for genomic regions that are involved in the development of gastric cancer, we recently identified a 2-cM minimal region of overlapping heterozygous deletions in 6q16.3-q23.1 . Here, we describe an application of the suppression subtraction method (SSH) to search for genes in this small region of the genome, taking advantage of the fact that many human genes present on yeast artificial chromosomes (YACs) are expressed in yeast . Subtraction was performed with two virtually contiguous YACs that cover a region of approximately 2.5 Mb . Combined forward and reversed subtractions resulted in the identification of 12 clones of human origin, all of which could be confirmed by sequence analysis as originating from the 6q21 region . Expression in human tissues could be confirmed by Northern analysis for two of the clones, one of them showing a high level of expression in stomach tissue. Curr Biol, 2002 Apr 30, 12(9), R334 - 6 Cytokinesis: myosin spots the ring; Hou MC et al.; Faithful actomyosin ring assembly is pivotal for successful cell division . The mechanisms by which the actomyosin ring is assembled at the correct time and place remain unclear . Recent studies in fission yeast have shown that a myosin II-containing spot may be a novel progenitor structure essential for actomyosin ring assembly. Curr Biol, 2002 Apr 30, 12(9), 695 - 704 Targeting of Golgi-specific pleckstrin homology domains involves both PtdIns 4-kinase-dependent and -independent components; Levine TP et al.; BACKGROUND: Phosphoinositides are required for the recruitment of many proteins to both the plasma membrane and the endosome; however, their role in protein targeting to other organelles is less clear . The pleckstrin homology (PH) domains of oxysterol binding protein (OSBP) and its relatives have been shown to bind to the Golgi apparatus in yeast and mammalian cells . Previous in vitro binding studies identified phosphatidylinositol (PtdIns) (4)P and PtdIns(4,5)P(2) as candidate ligands, but it is not known which is recognized in vivo and whether phosphoinositide specificity can account for Golgi-specific targeting . RESULTS: We have examined the distribution of GFP fusions to the PH domain of OSBP and to related PH domains in yeast strains carrying mutations in individual phosphoinositide kinases . We find that Golgi targeting requires the activity of the PtdIns 4-kinase Pik1p but not phosphorylation of PtdIns at the 3 or 5 positions and that a PH domain specific for PtdIns(4,5)P(2) is targeted exclusively to the plasma membrane . However, a mutant version of the OSBP PH domain that does not bind phosphoinositides in vitro still shows some targeting in vivo . This targeting is independent of Pik1p but dependent on the Golgi GTPase Arf1p . CONCLUSIONS: Phosphorylation of PtdIns at the 4 position but not conversion to PtdIns(4,5)P(2) contributes to recruitment of PH domains to the Golgi apparatus . However, potential phosphoinositide ligands for these PH domains are not restricted to the Golgi, and the OSBP PH domain also recognizes a second determinant that is ARF dependent, indicating that organelle specificity reflects a combinatorial interaction. Mol Biol Cell, 2002 May, 13(5), 1439 - 48 Competition of spontaneous protein folding and mitochondrial import causes dual subcellular location of major adenylate kinase; Strobel G et al.; Sorting of cytoplasmically synthesized proteins to their target compartments usually is highly efficient so that cytoplasmic precursor pools are negligible and a particular gene product occurs at one subcellular location only . Yeast major adenylate kinase (Adk1p/Aky2p) is one prominent exception to this rule . In contrast to most mitochondrial proteins, only a minor fraction (6-8%) is taken up into the mitochondrial intermembrane space, whereas the bulk of the protein remains in the cytosol in sequence-identical form . We demonstrate that Adk1p/Aky2p uses a novel mechanism for subcellular partitioning between cytoplasm and mitochondria, which is based on competition between spontaneous protein folding and mitochondrial targeting and import . Folding is spontaneous and rapid and can dispense with molecular chaperons . After denaturation, enzymatic activity of Adk1p/Aky2p returns within a few minutes and, once folded, the protein is thermally and proteolytically very stable . In an uncoupled cell-free organellar import system, uptake of Adk1p/Aky2p is negligible, but can be improved by previous chaotropic denaturation . Import ensues independently of Hsp70 or membrane potential . Thus, nascent Adk1p/Aky2p has two options: either it is synthesized to completion and folds into an enzymatically active import-incompetent conformation that remains in the cytosol; or, during synthesis and before commencement of significant tertiary structure formation, it reaches a mitochondrial surface receptor and is internalized. J Cell Sci, 2002 Jun 1, 115(Pt 11), 2423 - 31 The plant Spc98p homologue colocalizes with gamma-tubulin at microtubule nucleation sites and is required for microtubule nucleation; Erhardt M et al.; The molecular basis of microtubule nucleation is still not known in higher plant cells . This process is better understood in yeast and animals cells . In the yeast spindle pole body and the centrosome in animal cells, gamma-tubulin small complexes and gamma-tubulin ring complexes, respectively, nucleate all microtubules . In addition to gamma-tubulin, Spc98p or its homologues plays an essential role . We report here the characterization of rice and Arabidopsis homologues of SPC98 . Spc98p colocalizes with gamma-tubulin at the nuclear surface where microtubules are nucleated on isolated tobacco nuclei and in living cells . AtSpc98p-GFP also localizes at the cell cortex . Spc98p is not associated with gamma-tubulin along microtubules . These data suggest that multiple microtubule-nucleating sites are active in plant cells . Microtubule nucleation involving Spc98p-containing gamma-tubulin complexes could then be conserved among all eukaryotes, despite differences in structure and spatial distribution of microtubule organizing centers. J Biol Chem, 2002 Jul 19, 277(29), 26452 - 9 Epub 2002 May 10. Mechanism of N-terminal autoinhibition in the Arabidopsis Ca(2+)/H(+) antiporter CAX1; Pittman JK et al.; Regulation of Ca(2+)/H(+) antiporters may be an important function in determining the duration and amplitude of cytosolic Ca(2+) oscillations . Previously the Arabidopsis Ca(2+)/H(+) transporter, CAX1 (cation exchanger 1), was identified by its ability to suppress yeast mutants defective in vacuolar Ca(2+) transport . Recently, a 36-amino acid N-terminal regulatory region on CAX1 has been identified that inhibits CAX1-mediated Ca(2+)/H(+) antiport . Here we show that a synthetic peptide designed against the CAX1 36 amino acids inhibited Ca(2+)/H(+) transport mediated by an N-terminal-truncated CAX1 but did not inhibit Ca(2+) transport by other Ca(2+)/H(+) antiporters . Ca(2+)/H(+) antiport activity measured from vacuolar-enriched membranes of Arabidopsis root was also inhibited by the CAX1 peptide . Through analyzing CAX chimeric constructs the region of interaction of the N-terminal regulatory region was mapped to include 7 amino acids (residues 56-62) within CAX1 . The CAX1 N-terminal regulatory region was shown to physically interact with this 7-amino acid region by yeast two-hybrid analysis . Mutagenesis of amino acids within the N-terminal regulatory region implicated several residues as being essential for regulation . These findings describe a unique mode of antiporter autoinhibition and demonstrate the first detailed mechanisms for the regulation of a Ca(2+)/H(+) antiporter from any organism. Cloning Stem Cells, 2002, 4(1), 39 - 46 Gene transfer strategies in animal transgenesis; Montoliu L; Position effects in animal transgenesis have prevented the reproducible success and limited the initial expectations of this technique in many biotechnological projects . Historically, several strategies have been devised to overcome such position effects, including the progressive addition of regulatory elements belonging to the same or to a heterologous expression domain . An expression domain is thought to contain all regulatory elements that are needed to specifically control the expression of a given gene in time and space . The lack of profound knowledge on the chromatin structure of expression domains of biotechnological interest, such as mammary gland-specific genes, explains why most standard expression vectors have failed to drive high-level, position-independent, and copy-number-dependent expression of transgenes in a reproducible manner . In contrast, the application of artificial chromosome-type constructs to animal transgenesis usually ensures optimal expression levels . YACs, BACs, and PACs have become crucial tools in animal transgenesis, allowing the inclusion of distant key regulatory sequences, previously unknown, that are characteristic for each expression domain . These elements contribute to insulating the artificial chromosome-type constructs from chromosomal position effects and are fundamental in order to guarantee the correct expression of transgenes. Toxicol Ind Health, 2001 Feb, 17(1), 31 - 9 Effects of butylparaben on the male reproductive system in rats; Oishi S; Parabens are a group of compounds widely used as preservatives in foodstuffs, cosmetics, toiletries and pharmaceuticals . These compounds are known to exert a weak estrogenic activity, with butylparaben showing the most potent activity among methyl-, ethyl- and propyl esters in in vitro recombinant yeast assay and in in vivo uterotrophic assay . To account for potential reproductive effects in male animals, butylparaben was administered to 3-week-old Wistar rats divided in groups of eight subjects, at doses of 0.00%, 0.01%, 0.10% and 1.00% with the animal's diet . After 8 weeks, the rats were killed by decapitation and the weights of the testes, epididymides, prostates, seminal vesicles and preputial glands were recorded . The absolute and relative weights of epididymides were decreased in a dose-dependent manner and the decrease was statistically significant at 0.10% and above . The cauda epididymal sperm reserve of all treated groups was significantly decreased . The sperm count of the group receiving the highest dose was 58.2% of control values . The daily sperm production (DSP) in the testis was also significantly lower in all treated groups when compared to controls . Serum testosterone concentration was lowered dose-dependently and was significant at 0.1% or more . The daily intake of butylparaben that caused these disruptions is similar to the lower level of acceptable daily intake (ADI) for parabens in the European Community (EC) and in Japan . The results of the present experiments show for the first time that exposure of a postweaning mammal to butylparaben had an adverse effect on the secretion of testosterone and in the functions of the male reproductive system. Environ Health Perspect, 2002 May, 110(5), 533 - 6 Confirmation of uterotrophic activity of 3-(4-methylbenzylidine)camphor in the immature rat; Tinwell H et al.; In this study we found that the ultraviolet sunscreen component 3-(4-methylbenzylidine)camphor (4MBC) is uterotrophic in immature rats when administered by either subcutaneous injection or oral gavage . These data confirm