Allergy, 2002 Sep, 57(9), 776 - 84 Assessment of profilin as an allergen for latex-sensitized patients; Nieto A et al.; BACKGROUND: The presence of the actin-binding protein, profilin, has been demonstrated in natural latex extracts; but the clinical significance of this molecule as an allergen for latex-allergic patients is not clear . We studied the allergenic relevance of isolated latex natural and recombinant profilin, by in vivo and in vitro techniques, in two populations of spina bifida children (SB) and adults allergic to latex (AL) . METHODS: Profilin is present in small amounts in latex extracts obtained from low ammoniated (LA) natural latex . Its purification by affinity chromatography resulted difficult due to Heb v 1 unspecific binding . Therefore a method was developed to obtain natural profilin from natural latex, combining affinity chromatography (PLP, poly-L-proline Sepharose column) and previous ammonium sulfate fractionation . Alternatively, latex c-serum containing a low amount of Hev b 1 and a relatively higher profilin content could be used . Recombinant latex profilin isoform (rHev b 8) was cloned by PCR amplification . The entire coding region of Hev b 8 was subcloned into the expression vector pKN172 and a non-fusion form of Hev b 8 was expressed in Escherichia coli BL21 (DE3) . Purified recombinant protein was obtained after a single passage through PLP-Sepharose column . RESULTS: Natural and recombinant purified Hev b 8 were tested cutaneously by intradermoreaction (ID) in 17 SB and 14 AL patients . They were positive in 15 SB and 14 AL patients . No wheals were produced when tested in nonatopic control patients . Only 42% of sera from latex-allergic patients revealed specific IgE titers of class 1 or higher by enzyme immunoassay and only 39% of them exhibited IgE binding by SDS-PAGE immunoblotting with any natural or recombinant Hev b 8 forms . CONCLUSION: It seems that profilin is a relevant allergen for both groups of patients from a frequency point of view, but with scarce presence in natural latex extracts and raw sources, with a subsequent low IgE induction capacity. Biochem J, 2002 Nov 15, 368(Pt 1), 273 - 81 Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites; Singh A et al.; Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets . As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P . vinckei (vinckepain-2) and P . berghei (berghepain-2) were characterized . These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system) . Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme . The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin . However, key differences between the plasmodial proteases were identified . In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly . Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity ( k cat/ K m increased approx . 100-fold) and more rapid autohydrolysis . The present study identifies key animal-model parasite targets . It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases. DNA Res, 2002 Jun 30, 9(3), 71 - 8 Biochemical examination of the potential eukaryotic-type protein kinase genes in the complete genome of the unicellular Cyanobacterium synechocystis sp . PCC 6803; Kamei A et al.; The complete genome of the unicellular motile cyanobacterium Synechocystis sp . PCC 6803 harbors seven putative genes for a subfamily Pkn2 of the eukaryotic-type (or "Hanks-type") protein kinase . Previously, SpkA and SpkB were shown to have protein kinase activity and to be required for cell motility . Here, the other five genes were examined . These genes, except for spkG (slr0152), were successfully expressed in Escherichia coli . Eukaryotic-type protein kinase activity of the expressed SpkC (Slr0599), SpkD (S110776) and SpkF (Slr1225) was demonstrated as autophosphorylation and phosphorylation of the general substrate proteins . SpkE (Slr1443) did not show any activity, a finding consistent with its lack of several key amino acid residues in its kinase motif . Gene-disrupted mutants showed no discernible defect in phenotype except that spkD was apparently essential for survival. Anticancer Res, 2002 Mar-Apr, 22(2B), 1217 - 23 Tumor-specificity and radical scavenging activity of poly-herbal formula; Miyamoto M et al.; A total of 14 poly-herbal formula extracts were compared for their biological activities both in vivo and in vitro . Pretreatment of mice with the extracts protected them from E . coli infection to various extents . Among the extracts, the HD-12 and DLH-3073 extracts showed the highest cytotoxicity against both HIV-infected and mock-infected MT4 cells, without induction of any apparent anti-HIV activity . The extracts showed significantly higher cytotoxic activity against five human tumor cell lines (HSC-2, HSC-3, HSG, MT-4, HL-60) than against three normal human cell lines (HGF, HPC, HPLF) . Agarose gel electrophoresis demonstrated that the HD-12 and DLH-3073 extracts induced intemucleosomal DNA fragmentation in HL-60 cells . ESR spectroscopy showed that all the extracts produced radicals and this was paralleled by their ability to scavenge the superoxide anion (produced by hypoxanthine-xanthine oxidase reaction), the hydroxyl radical (produced by Fenton reaction) and nitric oxide (produced by NOC- 7) in the presence of radical trapping agents . Higher and lower concentrations of extracts enhanced or reduced respectively, the radical intensity of sodium ascorbate, suggesting their bimodal actions . The tumor specificity and antioxidant properties of the herb extracts further suggest their medicinal efficacy. Anticancer Res, 2002 May-Jun, 22(3), 1361 - 8 The combination regimen of idarubicin and taxotere is effective against human drug-resistant leukemic cell lines; Majlessipour F et al.; Up-regulation of Bcl-2 protein may contribute to drug resistance, by decreasing apoptosis after treatment, in pre-B and B-cell leukemias in pediatric patients . By contrast, augmented caspase-3 activity, an effector caspase, may be indicative of drug sensitivity due to increased cellular apoptosis . We have reported the development of an in vitro human T-lymphoblastic leukemia model resistant to ara-C and/or native E . coli L-asparaginase (ASNase), mimicking the drug resistance to the Capizzi II regimen . We have investigated the potential drug synergism between Idarubicin (IDA) and Taxotere (TXR) that may be active in the ara-C and ASNase double drug-resistant cell lines . The additive or synergistic activity between IDA and TXR is drug concentration-dependent in inducing caspase-3 activation and cellular apoptosis . We exposed two human drug-resistant cell lines that do not express the MDRI phenotype, one resistant to ASNase alone (CEM/ASNase-1-3) and the other resistant to both ara-C and ASNase (CEM/ara-C/I/ASNase-0.5-2), to physiologically achievable concentrations of IDA, TXR, or their combination . Both lines showed either synergistic drug activity to the combination regimen in comparison to either drug used alone, as determined by MTT assay, or additivity as demonstrated by flow cytometry after Annexin V and propidium iodide (PI) staining . After exposure of the ASNase-resistant line to various concentrations, the intracellular levels of Bcl-2 protein decreased to near zero relative to untreated control cells . The Bcl-2 protein reductions in these cells ranged from 30% to <1%, 49% to <1%, and 27% to 3% when treated with IDA or TXR as a single drug or IDA + TXR combination, respectively . Similarly, intracellular Bcl-2 levels in the double-resistant cell line decreased with reductions ranging from 24% to <1%, 87% to <1%, and 46% to <1% of the untreated control after treatment with IDA, TXR, or their combination, respectively . Conversely, the caspase-3 activity increased in a dose-dependent manner and inversely-correlated with loss of cell viability (r= 0.91) after exposure to IDA + TXR combination in the double drug-resistant line to both ara-C and ASNase . We conclude that the combination of the IDA + TXR regimen is highly synergistic or additive in drug resistant human leukemic cell clones . The molecular mechanism of action is due to the down-regulation of Bcl-2 protein and up-regulation of caspase-3 activity . This drug combination warrants further investigation for use in the treatment of patients with ara-C and/or ASNase refractory leukemias. J Orthop Res, 2002 Jul, 20(4), 747 - 55 Collagen-targeted BMP3 fusion proteins arrayed on collagen matrices or porous ceramics impregnated with Type I collagen enhance osteogenesis in a rat cranial defect model; Han B et al.; Bone morphogenetic protein 3 (BMP3) is a potent osteoinductive growth factor belonging to the TGF-beta superfamily . In this study, we engineered a recombinant BMP3 protein to include an auxiliary collagen-targeting domain derived from von Willebrand coagulation factor (vWF) . The collagen-targeted BMP3 fusion protein (rhBMP3-C) was expressed in E . coli, purified from bacterial inclusion bodies, renatured under controlled redox conditions, and assayed for biological activity in vitro and in vivo . The renatured rhBMP3-C fusion protein bound tightly to collagen matrices and inhibited DNA synthesis in normal rat calvaria cells and in two out of three human osteosarcoma cell lines tested . Alkaline phosphatase activity was increased in rat calvarial cells and was decreased in osteosarcoma cells in vitro in a dose-dependent manner . Collagen sponges impregnated with rhBMP3-C and implanted subcutaneously in Fischer-344 rats induced dose-dependent dystrophic calcification of the collagen matrix, with no evidence of ectopic bone formation . However, local injection of rhBMP3-C infused in a collagen suspension induced new bone formation on the periosteal surface of rat calvaria . Finally, in a rat cranial defect model, surgical implantation of rhBMP3-C arrayed on either collagen sponges or on porous ceramics coated with Type I collagen exhibited marked osteoinductive properties . Taken together, these results demonstrate the feasibility of engineering and manufacturing targeted-BMPs which exhibit an integral gain-of-function that may be exploited to therapeutic advantage in (i) the enhancement of effective local concentrations, (ii) the prevention of systemic biodistribution and side effects, and (iii) the design of improved osteoinductive matrices. Int J Oncol, 2002 Sep, 21(3), 661 - 6 Cancer-specific killing by the CD suicide gene using the human telomerase reverse transcriptase promoter; Liu J et al.; Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase, is transcriptionally upregulated in more than 90% of tumor cells . It may be used as a tool for driving a gene to kill tumors specifically . To test this idea, luciferase reporter gene was used and the results showed that hTERT promoter could restrict the gene expression in the telomerase-positive tumor cells . A tumor-specific expression plasmid phTERT-CD was constructed, in which the E . coli cytosine deaminase (CD) gene was controlled by the hTERT promoter . A colorectal cancer cell line (LoVo) and a normal amnion cell line (WISH) were transfected by this plasmid . It was shown that the expression of the CD gene increased the sensitivity of LoVo cells to the prodrug, 5-fluorocytosine (5FC), over 800-fold, while the sensitivity of WISH cells to 5FC was increased only 6-fold . Mixed cell experiments showed a strong "bystander effect" on CD-negative cells . Furthermore, a significant anti-tumor effect of the phTERT-CD/5FC system was observed in nude mice bearing mammalian carcinoma induced by s.c . inoculation of LoVo cells when the mice were given 250 mg/kg 5FC twice a day for 10 consecutive days . These results indicated that hTERT promoter could target the suicidal effect of CD gene to tumor cells, and therefore, may be a novel and promising targeting approach to the treatment of cancer. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(4), 397 - 401 Expression of the APLA(2) Gene from Agkistrodon halys Pallas; Liu XL et al.; The APLA(2) gene from Agkistrodon halys Pallas has been cloned into the expression plasmid pBLMVL2 and expressed in E . coli RR1 . The molecular weight of the expressed product is approximately 14 kD as shown by SDS-PAGE, its expression level is about 30% of the total cellular proteins . The protein was produced as insoluble inclusion bodies . After partially purified by washing the inclusion bodies, the product was denatured and refolded into active form . Then, the expressed APLA(2) was purified by FPLC Superose (TM) 12 and was a single band as shown by SDS-PAGE . The purified expressed protein had specific activity as the native enzyme and cross-reacted with antisera prepared against the native enzyme . The successful expression of the APLA(2) gene from Agkistrodon halys Pallas provides a good basis for further structure-function studies. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(4), 382 - 386 Expression of Fusion Protein Containing Androgen Receptor Segment and Preparation of Polyclonal Antibodies to Androgen Receptor; Xie F et al.; The cDNA segment (1105-2224) of the androgen receptor was cloned into the pGEX-3X expression vector and expressed in E . coli . The soluble fusion proteins GST-AR were used to immunize rabbits to obtain polyclonal antibodies to androgen receptors . The antibodies were purified by GST-Sepharose 4B and indicated the specificity to androgen receptors . The purified antibodies can be used in study the function of AR. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(5), 520 - 524 Cloning, Expression and Identification of Matrix Protein Gene of Wheat Rosette Stunt Virus (WRSV); Ye YJ et al.; cDNA fragment located downstream of the WRSV NS protein gene was sequenced . Following an 80 nt untranslated region at the 5 'terminus, there is a 453 nt open reading frame(ORF) encoding a 17 kD protein, which was allowed to be expressed in E . coli using bacterial expression vector pGEX-3X and produced a 43 kD fusion protein . The result of Western blot showed that the fusion protein was able to react strongly with antibody raised against the purified WRSV particles . According to the similarities between the gene organizations of VSV and WRSV and between the molecular weights of deduced and expressed proteins, as well as the viral structural M protein, and to the result of Western blot, the 453 nt ORF was identified as WRSV M protein gene. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(5), 515 - 519 Cloning and Sequencing of the Gene Encoding NS Protein of Wheat Rosette Stunt Virus; Shen JL et al.; The cDNA fragment located downstream of the WRSV N protein gene was obtained from the cDNA library of WRSV by hybridization with a 24 nucleotides sequence fragment of the 3' end of mRNA of the N protein . The cDNA fragment was sequenced and on ORF encoding a 40 kD protein was found . The gene was expressed in E . coli DE3 and was identified by Western blot experiment as the NS gene of WRSV. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(5), 477 - 482 Cloning, Sequencing and Expression of the atpE Gene of Broad Bean Chloroplast; Gu Y et al.; The ninth KpnI fragment (about 4.0 kb) of broad bean chloroplast DNA contains the entire chloroplast atpE gene and atpB gene . This fragment was inserted into the pUC18 polylinker region, yielding plasmid pUK1 . Southern hybridization using atpE gene from maize chloroplast as probe revealed that atpE gene encoding epsilon subunit of broad bean chloroplast was localized in 0.9 kb ClaI fragment of pUK1 . The whole sequence of atpE gene of broad bean chloroplast was determined by using primer F and primer R of the polylinker region of pBluescript KS (+) DNA . The entire atpE gene of broad bean chloroplast was inserted into the polylinker region of vector pJLA505 to form recombinant plasmids pJLA505-atpE . The expression plasmid was transformed into E . coli DH5alpha cells which were then induced at 42 degrees . By analysis with SDS-PAGE, the product of interest accounted for more than 38% of total E . coli cell proteins . Identification of the expressed product by Western blot analysis demonstrated that it can react specifically with anti-epsilon serum of maize chloroplast CF(1) . The expressed product aggregated to form insoluble inclusion bodies and was purified . The purified product had the same function as that of the native epsilon subunit. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(5), 454 - 458 Cloning of Schistosoma japonicum Chinese Strain TPI Gene and Characterization of Its Expression Product in Escherichia coli; Cai XZ et al.; Triose-phosphate isomerase is an important candidate for schistosoma antigens . An 800 bp DNA fragment was amplified by RT-PCR from adult Schistosoma japonicum mRNA . Sequence analysis revealed that this fragment contained S . japonicum (Chinese strain) triose-phosphate isomerase gene . Then this gene was cloned into the expression vector pGEX-4T and subsequently expressed in E . coli . The recombinant GST-fusion protein was purified by glutathione agarose affinity chromatography . Its molecular weight was determined to be 54 kD . The yield of expression was around 30 mg/L E . coli culture . Western blotting showed that the recombinant protein had good antigenicity which could be helpful for the making of anti-S . japonicum multi-valent recombinant vaccine. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(6), 618 - 622 A prostate-specific Protein Factor Inhibits the Interaction of Androgen Receptors with Hormone Response Elements; Xie F et al.; The fragments of the androgen receptor (amino acids: 359-732) and of the glucocorticoid receptor (amino acids: 396-548) were expressed in E . coli as fusion proteins with GST . Both fusion proteins, denoted GST-AR and GST-GR, contained the DNA-binding domain and some flanking amino acids . In gel retardation assay both fusion proteins could bind the androgen/glucocorticoid response element (ARE/GRE) . We found that both cytosol and nuclear extracts from rat ventral prostate (v.p), but not from other source tested could abolish the interaction of GST-AR and GST-GR with ARE/GRE (from C3 (1) gene and MMTV LTR) . The inhibition was androgen-dependent and sensitive to temperature and trypsin treatment . It implies that a protein inhibitor was present in the rat ventral prostate. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 1998, 30(6), 607 - 610 Mutation of G138P Enhanced the Thermostability of D-glucose Isomerase; Zhu GP et al.; In order to enhance the thermostability of D-glucose isomerase (GI), Gly 138 was decided to be the target to be replaced by molecular design . The mutant G138P was obtained by in vitro site-directed mutagenesis of GI gene . The recombinant plasmid pTKD-GI containing mutant site was expressed in E . coli K38 strain . The comparison experiments of GIG138P with wild-type GI showed that: (1) The half time of GIG138P was as about two times as that of the wild type . (2) The optimum temperature of GIG138P was increased by 10-12 degrees . (3) The specific-activity of GIG138P was similar to the wild-type GI . We supposed, based on the above facts, that the substitution of Pro for Gly at position 138 introduced a pyrrolidine ring, which could just fill perfectly the empty hole leaved by Gly-138 which has no side chain and could make the protein structure more rigid, therefore the mutant G138P enhanced the thermostability of SM33GI. Mol Cell Biol, 2002 Sep, 22(17), 6111 - 21 Targeted deletion of mNth1 reveals a novel DNA repair enzyme activity; Ocampo MT et al.; DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues . We generated a null mouse (mNth1(-/-)) by gene targeting . After almost 2 years, such mice exhibited no overt abnormalities . Tissues of mNth1(-/-) mice contained an enzymatic activity which cleaved DNA at sites of oxidized thymine residues (thymine glycol {Tg}) . The activity was greater when Tg was paired with G than with A . This is in contrast to Nth1, which is more active against Tg:A pairs than Tg:G pairs . We suggest that there is a back-up mammalian repair activity which attacks Tg:G pairs with much greater efficiency than Tg:A pairs . The significance of this activity may relate to repair of oxidized 5-methyl cytosine residues (5meCyt) . It was shown previously (S . Zuo, R . J . Boorstein, and G . W . Teebor, Nucleic Acids Res . 23:3239-3243, 1995) that both ionizing radiation and chemical oxidation yielded Tg from 5meCyt residues in DNA . Thus, this previously undescribed, and hence novel, back-up enzyme activity may function to repair oxidized 5meCyt residues in DNA while also being sufficient to compensate for the loss of Nth1 in the mutant mice, thereby explaining the noninformative phenotype. Proc Natl Acad Sci U S A, 2002 Sep 17, 99(19), 12126 - 31 Epub 2002 Aug 07. The mechanism of type IA topoisomerases; Dekker NH et al.; The topology of cellular DNA is carefully controlled by enzymes called topoisomerases . By using single-molecule techniques, we monitored the activity of two type IA topoisomerases in real time under conditions in which single relaxation events were detected . The strict one-at-a-time removal of supercoils we observed establishes that these enzymes use an enzyme-bridged strand-passage mechanism that is well suited to their physiological roles and demonstrates a mechanistic unity with type II topoisomerases. J Biol Chem, 2002 Oct 11, 277(41), 39035 - 44 Epub 2002 Aug 06. Differential partitioning of lipids metabolized by separate yeast glycerol-3-phosphate acyltransferases reveals that phospholipase D generation of phosphatidic acid mediates sensitivity to choline-containing lysolipids and drugs; Zaremberg V et al.; In this study we demonstrate that the GAT1 and GAT2 genes encode the major glycerol-3-phosphate acyltransferase activities in Saccharomyces cerevisiae . Genetic inactivation of either GAT1 or GAT2 did not alter cell growth but inactivation of both resulted in growth cessation . Metabolic analyses of gat1 and gat2 yeast detected that the major differences were: (i) a 50% increase in the rate of triacylglycerol synthesis in gat1 yeast and a corresponding 50% decrease in gat2 yeast, and (ii) a 5-fold increase in glycerophosphocholine production through deacylation of phosphatidylcholine synthesized through the CDP-choline pathway in gat1 yeast, whereas gat2 yeast displayed a 10-fold decrease . To address why we observed alterations in phospholipid turnover specific to phosphatidylcholine produced through the CDP-choline pathway in gat1 and gat2 yeast we tested their sensitivity to various cytotoxic lysolipids and observed that gat2 cells were more sensitive to lysophosphatidylcholine, but not other lysolipids . To pursue the mechanism we analyzed their sensitivity to choline-containing lysolipids or drugs that could not be deacylated and/or reacylated . Our data showed that gat1 and gat2 yeast were resistant and sensitive to lysoplatelet activating factor, platelet activating factor, and the anti-tumor lipid edelfosine, respectively, indicating that their sensitivity to these compounds was not because of differences in rates of phosphatidylcholine deacylation . As growth of gat2 cells was impaired in the presence of ethanol, a phospholipase D (Spo14p) inhibitor, we inferred that phospholipase D may play important biologic and metabolic roles in phenotypes observed in gat yeast . Genetic inactivation of the SPO14 gene resulted in increased susceptibility, whereas expression of Escherichia coli diacylglycerol kinase relieved growth inhibition, to choline-containing lysolipids and drugs . Our results are consistent with a model whereby phosphatidic acid generated from phosphatidylcholine hydrolysis by Spo14p regulates susceptibility to choline-containing lysolipid analogs and drugs. J Biol Chem, 2002 Oct 18, 277(42), 39642 - 8 Epub 2002 Aug 06. Novel {2Fe-2S}-type redox center C in SdhC of archaeal respiratory complex II from Sulfolobus tokodaii strain 7; Iwasaki T et al.; The SdhC subunit of the archaeal respiratory complex II (succinate:quinone oxidoreductase) from Sulfolobus tokodaii strain 7 has a novel cysteine rich motif and is also related to archaeal and bacterial heterodisulfide reductase subunits . We overexpressed the sdhC gene heterologously in Escherichia coli and characterized the gene product in greater detail . Low temperature resonance Raman and x-ray absorption spectroscopic investigation collectively demonstrate the presence of a {2Fe-2S} cluster core with complete cysteinyl ligation (Center C) and an isolated zinc site in the recombinant SdhC . The {2Fe-2S}2+ cluster core is sensitive to dithionite, resulting in irreversible breakdown of the Fe-Fe interaction . EPR analysis confirmed that the novel Center C is an inherent redox center in the archaeal complex II, showing unique EPR signals in the succinate-reduced state . Distinct subunit and cofactor arrangements in the S . tokodaii respiratory complex II, as compared with those in mitochondrial and some mesophilic bacterial enzymes, indicate modular evolution of this ubiquitous electron entry site in the respiratory chains of aerobic organisms. J Biol Chem, 2002 Oct 18, 277(42), 39296 - 303 Epub 2002 Aug 06. Inhibition of bovine phenol sulfotransferase (bSULT1A1) by CoA thioesters . Evidence for positive cooperativity and inhibition by interaction with both the nucleotide and phenol binding sites; Tulik GR et al.; Previous work with the bovine phenol sulfotransferase (bSULT1A1, EC ) demonstrated inhibition by CoA that was competitive with respect to the sulfuryl donor substrate, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) (Leach, M., Cameron, E., Fite, N., Stassinopoulos, J., Palmreuter, N., and Beckmann, J . D . (1999) Biochem . Biophys . Res . Commun . 261, 815-819) . Here we report that long chain acyl-CoAs are more potent inhibitors of bSULT1A1 and also of human dopamine sulfotransferase (SULT1A3) when compared with unesterified CoA and short chain-length acyl-CoAs . A complex pattern of inhibition was revealed by systematic variation of palmitoyl-CoA, PAPS, and 7-hydroxycoumarin, the acceptor substrate . Convex plots of apparent K(m)/V(max) versus {palmitoyl-CoA} were adequately modeled using an ordered rapid equilibrium scheme with PAPS as the leading substrate and by accounting for the possible binding of two equivalents of inhibitor to the dimeric enzyme . Interestingly, the first K(i) of 2-3 microm was followed by a second K(i) of only 0.01-0.05 microm, suggesting that positive subunit cooperativity enhances binding of long chain acyl-CoAs to this sulfotransferase . Simultaneous interaction of palmitoyl-CoA with both the nucleotide and phenol binding sites is suggested by two experiments . First, the acyl-CoA displaced 7-hydroxycoumarin from the highly fluorescent bSULT1A1.PAP.7-HC complex in a cooperative manner . Second, palmitoyl-CoA prevented the quenching of bSULT1A1 fluorescence observed with pentachlorophenol . Finally, titrations of bSULT1A1-pentachlorophenol complex with palmitoyl-CoA caused the return of protein fluorescence, and the binding of palmitoyl-CoA was highly cooperative (Hill constant of 1.9) . Overall, these results suggest a model of sulfotransferase inhibition in which the 3'-phosphoadenosine-5'-diphosphate moiety of CoA docks to the PAPS domain, and the acyl-pantetheine group docks to the hydrophobic phenol binding domain. J Biol Chem, 2002 Oct 18, 277(42), 39809 - 14 Epub 2002 Aug 06. Function of DcuS from Escherichia coli as a fumarate-stimulated histidine protein kinase in vitro; Janausch IG et al.; The two-component regulatory system DcuSR of Escherichia coli controls the expression of genes of C(4)-dicarboxylate metabolism in response to extracellular C(4)- dicarboxylates such as fumarate or succinate . DcuS is a membrane-integral sensor kinase, and the sensory and kinase domains are located on opposite sides of the cytoplasmic membrane . The intact DcuS protein (His(6)-DcuS) was overproduced and isolated in detergent containing buffer . His(6)-DcuS was reconstituted into liposomes made from E . coli phospholipids . Reconstituted His(6)-DcuS catalyzed, in contrast to the detergent-solubilized sensor, autophosphorylation by {gamma-(33)P}ATP with an approximate K(D) of 0.16 mm for ATP . Up to 7% of the reconstituted DcuS was phosphorylated . Phosphorylation was stimulated up to 5.9-fold by C(4)-dicarboxylates, but not by other carboxylates . The phosphoryl group of DcuS was rapidly transferred to the response regulator DcuR . Upon phosphorylation, DcuR bound specifically to dcuB promoter DNA . The reconstituted DcuSR system therefore represents a defined in vitro system, which is capable of the complete transmembrane signal transduction by the DcuSR two-component system from the stimulus (fumarate) to the DNA, including signal transfer across the phospholipid membrane. J Biol Chem, 2002 Oct 4, 277(40), 36978 - 86 Epub 2002 Aug 06. Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase; Failer BU et al.; We have isolated and characterized the cDNA encoding a Ca(2+)-dependent nucleoside diphosphatase (EC ) related to two secreted ATP- and ADP-hydrolyzing apyrases of the bloodsucking insects, Cimex lectularius and Phlebotomus papatasi . The rat brain-derived cDNA has an open reading frame of 1209 bp encoding a protein of 403 amino acids and a calculated molecular mass of 45.7 kDa . The mRNA was expressed in all tissues investigated, revealing two major transcripts with varying preponderance . The immunohistochemical analysis of the Myc-His-tagged enzyme expressed in Chinese hamster ovary cells revealed its association with the endoplasmic reticulum and also with pre-Golgi intermediates . Ca(2+)-dependent nucleoside diphosphatase is a membrane protein with its catalytic site facing the organelle lumen . It hydrolyzes nucleoside 5'-diphosphates in the order UDP >GDP = IDP >>>CDP but not ADP . Nucleoside 5'-triphosphates were hydrolyzed to a minor extent, and no hydrolysis of nucleoside 5'-monophosphates was observed . The enzyme was strongly activated by Ca(2+), insensitive to Mg(2+), and had a K(m) for UDP of 216 microm . Ca(2+)-dependent nucleoside diphosphatase may support glycosylation reactions related to quality control in the endoplasmic reticulum. J Biol Chem, 2002 Oct 18, 277(42), 39102 - 11 Epub 2002 Aug 07. Nopp140 is a mediator of the protein kinase A signaling pathway that activates the acute phase response alpha1-acid glycoprotein gene; Chiu CM et al.; The acute phase response (APR) in liver during inflammation is one of the well known examples for elucidating the signaling pathways that lead to the combinatorial regulation of gene expression . The APR is exemplified by alpha(1)-acid glycoprotein gene (agp) expression . A number of transcription factors, including CCAAT/enhancer-binding protein beta (C/EBPbeta), glucocorticoid receptor, cAMP-response element-binding protein (CREB), and Nopp140, are known to participate in its induction . The underlying mechanism of Nopp140 and other factors for regulating agp expression remains unclear . Here we demonstrate that protein kinase A (PKA)-dependent phosphorylation of Nopp140, together with C/EBPbeta, induces agp gene expression synergistically . The cooperative activation of the agp gene by Nopp140 and forskolin is sensitive to inhibition by PKI . Results from biochemical and functional characterizations of Nopp140 mutants defective in PKA phosphorylation sites suggest that PKA-dependent Nopp140 phosphorylation is important for its role in agp gene activation . Furthermore, maximal activation of the agp gene by PKA-phosphorylated Nopp140 depends on the presence of CREB and C/EBPbeta . The participation of CREB in the activation is, however, independent of its PKA-mediated phosphorylation . In summary, we demonstrate the existence of a novel Nopp140-mediated PKA signaling pathway that leads to the activation of agp, one of the major acute phase response genes. FEMS Microbiol Lett, 2002 Aug 6, 213(2), 225 - 30 Recombinant expression and in vitro folding of proinsulin are stimulated by the synthetic dithiol Vectrase-P; Winter J et al.; Recombinant production of native proinsulin in the periplasm of Escherichia coli is limited by formation of the correct disulfide bonds and inclusion body formation . These limitations can be overcome during in vitro folding of proinsulin by using a redox system and also protein disulfide isomerase . Here, we added a redox active substance, Vectrase-P, to the cultivation medium of E . coli cells producing proinsulin . We show that this synthetic dithiol partially mimicking the redox activity of protein disulfide isomerase provides an improved redox situation in the periplasm and, therefore, provides optimum conditions for folding of proinsulin in that cell compartment resulting in an increase in yield of 60% . The in vivo results were confirmed by analyzing in vitro folding of proinsulin in the presence of the dithiol Vectrase-P. Virology, 2002 Jul 20, 299(1), 122 - 32 Expression, purification, and characterization of the RNA 5'-triphosphatase activity of dengue virus type 2 nonstructural protein 3; Bartelma G et al.; Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5' end . The viral RNA is translated to produce a single polyprotein precursor that is processed to yield three virion proteins and at least seven nonstructural proteins (NS) in the infected host . NS3 is a multifunctional protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity . The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside triphosphatases (NTPases)/RNA helicases . In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5'-RNA triphosphatase activity, suggesting its role in the synthesis of the 5'-cap structure . In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form . The purified protein has 5'-RNA triphosphatase activity that cleaves the gamma-phosphate moiety of the 5'-triphosphorylated RNA substrate . Biochemical and mutational analyses of the NS3 protein indicate that the nucleoside triphosphatase and 5'-RNA triphosphatase activities of NS3 share a common active site. J Agric Food Chem, 2002 Aug 14, 50(17), 4803 - 11 Differential scanning calorimetry and circular dichroism spectrometry of walleye pollack myosin and light meromyosin; Togashi M et al.; The thermodynamic properties of myosin and its C-terminal fragment, light meromyosin (LMM), from walleye pollack, a typical cold-water fish efficiently utilized on an industrial scale, were analyzed by using differential scanning calorimetry (DSC) and circular dichroism (CD) spectrometry . Recombinant walleye pollack LMM expressed in Escherichia coli was also subjected to DSC and CD measurements for reference . The two proteins prepared from frozen surimi showed three endothermic peaks, the transition temperatures (T(m)) of which were quite similar, although overall DSC patterns differed considerably from one another . Their alpha-helical contents determined by CD were low compared to values reported before for other species . On the other hand, recombinant LMM gave four endothermic peaks at 27.4, 30.8, 36.5, and 43.4 degrees C in DSC and showed an alpha-helical content of approximately 80% . The peak at 27.4 degrees C could not be observed in walleye pollack LMM prepared from frozen surimi and thus was possibly attributed to its C terminus, because this extreme C-terminal region is supposedly truncated during preparation of LMM by tryptic digestion. Shock, 2002 Aug, 18(2), 158 - 62 A 4-amino analogue of tetrahydrobiopterin attenuates endotoxin-induced hemodynamic alterations and organ injury in rats; Fitzal F et al.; Most recently we have shown that 4-aminotetrahydrobiopterin (4-ABH4), an analogue of tetrahydrobiopterin (cofactor of NO synthase), even administered 2 h after endotoxin challenge, improves survival rate in rats . The following experiment was performed to examine the effects of 4-ABH4 with respect to endotoxin-induced hemodynamic alterations and organ failure . At 2 h after endotoxic challenge (10 mg kg(-1) body weight) animals received 4-ABH4 at a dose of 1, 10, or 100 mg kg(-1) body weight . The controls were treated similarly but received saline at the same volume . Eight hours after endotoxin challenge cardiac index and stroke volume were significantly increased in animals treated with 10 mg 4-ABH4 compared to controls (0.23 +/- 0.06 vs . 0.16 +/- 0.04 mL min(-1) kg(-1) and 0.29 +/- 0.05 vs . 0.22 +/- 0.03 mL beat(-1)) while mean arterial pressure and peripheral vascular resistance index did not significantly differ among the groups . Plasma alanine aminotransferase (ALT) and creatinine levels were significantly increased in endotoxin controls compared with laboratory controls (ALT: 1643 +/- 1436 vs . 74 +/- 17 U L(-1); Creatinine: 91 +/- 29 vs . 42 +/- 3 micromol L(-1)) which was attenuated in animals treated with 10 mg kg(-1) 4-ABH4 (ALT: 417 +/- 318 U L(-1); Creatinine: 78 +/- 26 micromol L(-1)) . Moreover, endotoxin-induced lung edema and intestinal necrosis were significantly reduced by 4-ABH4 . Our study provides information that tetrahydrobiopterin analogue, 4-ABH4, improves LPS induced hemodynamic conditions and organ injury . This may, at least in part, account for the previously observed protection of rats by 4-ABH4 against endotoxin-induced mortality in the same endotoxic shock model. RNA, 2002 Jul, 8(7), 904 - 12 Trans-acting RNA inhibits tRNA suppressor activity in vivo; Attardi DG et al.; We constructed two aptamers, each of which contains a 7-nt-long loop complementary to the anticodon loop of a suppressor tRNA . One of these aptamers can form a stable bimolecular complex with the suppressor tRNA in vitro and protects the 7 nt in the suppressor's anticodon loop from RNase S1 . An Escherichia coli strain, carrying an amber mutation in the lac Z gene, produces beta-galactosidase only if the suppressor is present; the aptamer's coexpression in the cell inhibits the activity of the suppressor tRNA . Moreover, in E . coli extract, the aptamer partially inhibits the read-through of the stop codon on the part of the suppressor tRNA . These results point to a novel strategy that need not be limited to the suppressor tRNA . By constructing appropriate inducible aptamers, it may well be possible to effectively control translation in vivo. RNA, 2002 Jul, 8(7), 878 - 89 Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: a possible case of ribosomal RNA-messenger RNA molecular mimicry; Guillier M et al.; In addition to being a component of the large ribosomal subunit, ribosomal protein L20 of Escherichia coli also acts as a translational repressor . L20 is synthesized from the IF3 operon that contains three cistrons coding for IF3, and ribosomal proteins L35 and L20 . L20 directly represses the expression of the gene encoding L35 and the expression of its own gene by translational coupling . All of the cis-acting sequences required for repression by L20, called the operator, are found on an mRNA segment extending from the middle of the IF3 gene to the start of the L35 gene . L20-mediated repression requires a long-range base-pairing interaction between nucleotide residues within the IF3 gene and residues just upstream of the L35 gene . This interaction results in the formation of a pseudoknot . Here we show that L20 causes protection of nucleotide residues in two regions of the operator in vitro . The first region is the pseudoknot itself and the second lies in an irregular stem located upstream of the L35 gene . By primer extension analysis, we show that L20 specifically induces reverse transcriptase stops in both regions . Therefore, these two regions define two L20-binding sites in the operator . Using mutations and deletions of rpml'-'lacZ fusions, we show that both sites are essential for repression in vivo . However L20 can bind to each site independently in vitro . One site is similar to the L20-binding site on 23S rRNA . Here we propose that L20 recognizes its mRNA and its rRNA in similar way. J Am Coll Nutr, 2002 Aug, 21(4), 351 - 6 Endotoxin-mediated hepatic lipid accumulation during parenteral nutrition in rats; Dickerson RN et al.; OBJECTIVE: To assess the effect of endotoxemia on hepatic lipid content during parenteral nutrition (PN) in rats . METHODS: Twenty male Sprague-Dawley rats (185-230 gm) were randomized to receive PN (n=9) or PN plus a continuous infusion of E . coli 026:B6 lipopolysaccharide (LPS; n= 11) . All animals received isocaloric (170 kcal/kg/day), isonitrogenous (1.1 g N/kg/day), glucose-based PN for the next 78 hours . After 30 hours of adaptation to TPN, the animals were randomized to receive PN or PN plus LPS at 6 mg/kg/day for the remaining 48 hours of study . The animals were euthanized and the livers were harvested . RESULTS: Liver weight increased significantly (by 60%) from 7.5+/-0.6 g to 12.1+/-2.4 g (p < or = 0.01) in the animals who received PN versus LPS, respectively . The proportion of liver water remained the same for PN and LPS groups (72.9+/-3.2% versus 72.3+/-3.8%, respectively, p = N.S.) . However, liver fat increased disproportionately (by about 130%) from 0.20+/-0.05 g to 0.46+/-0.20 g (p < or = 0.01) total fat weight or from 9.6+/-1.8% to 13.6+/-4.1% (p < or = 0.02) lipid content (g/g) of the dry liver weight for the PN and LPS groups, respectively . CONCLUSION: Endotoxin, when given concomitantly with parenteral nutrition, increases hepatic lipid accumulation and thus augments the development of parenteral nutrition-associated fatty liver in rats. J Immunol, 2002 Aug 15, 169(4), 1978 - 83 Accelerated clearance of Escherichia coli in experimental peritonitis of histamine-deficient mice; Hori Y et al.; We prepared a model of experimental peritonitis by introducing Escherichia coli into the peritoneal cavity of the histamine-deficient mice generated by a disruption of the gene for histidine decarboxylase (HDC), the unique histamine-synthesizing enzyme . When we inoculated E . coli into the peritoneal cavities of the HDC(-/-) (histamine-deficient) mice, they eliminated E . coli more efficiently than did the wild-type mice . Histamine was released efficiently from the peritoneal cells after E . coli inoculation in HDC(+/+) mice, although only trace amounts were detected in the peritoneal cells of HDC(-/-) mice . Two histamine agonists (6-{2-(4-imidazolyl)ethylamino}-N-(4-trifluoromethylphenyl)hepatanecarboxamide (H(1)) and dimaprit (H(2))) impaired the clearance of E . coli from the peritoneal cavity in HDC(-/-) mice, suggesting that the activation of both H(1) and H(2) receptors suppresses the clearance . In contrast, two kinds of H(1) and H(2) receptor antagonists, cimetidine and pyrilamine, promoted the clearance of E . coli in HDC(+/+) mice . Phagocytosis appeared to be enhanced in HDC(-/-) mice, since the number of neutrophils in the peritoneal cavity of HDC(-/-) mice was markedly increased . This enhanced recruitment of neutrophils was suppressed in the presence of the histamine agonists, 6-{2-(4-imidazolyl)ethylamino}-N-(4-trifluoromethylphenyl)hepatanecarboxamide and dimaprit . In this report histamine was first shown to be an important mediator in an E . coli infectious peritonitis model, causing a delay in the elimination of bacteria . This also raised the possibility of the use of antihistamine drugs for bacterial infection. J Immunol, 2002 Aug 15, 169(4), 1744 - 52 Role of B7 costimulatory molecules in the adjuvant activity of the heat-labile enterotoxin of Escherichia coli; Martin M et al.; Much interest has been directed at understanding the adjuvant properties of the heat-labile enterotoxin of Escherichia coli (LT) . In this study, we have assessed how LT compared with the nonenzymatic mutant LT (E112K) affect the level of B7-1 and B7-2 expression on APCs, and we determined how these costimulatory molecules influence their adjuvant properties . Analysis of B7-1 and B7-2 expression on B cells revealed that LT enhanced B7-2 but not B7-1, while LT (E112K) had no effect on the expression of either costimulatory molecule . Treatment of macrophage or dendritic cells with LT resulted in a predominant enhancement of B7-2, while LT (E112K) induced mainly B7-1 expression . Analysis of LT- and LT (E112K)-treated B cells, macrophage, and dendritic cells also revealed significant differences in their ability to enhance anti-CD3-stimulated CD4(+) T cell proliferative responses via B7-1 and B7-2 . Furthermore, the ability of LT to enhance both Ab and CD4(+) T cell responses to a coadministered Ag was severely abrogated in B7-2- but not B7-1-deficient mice . In contrast, the in vivo adjuvant properties of LT (E112K) appeared to be mediated by both B7-1 and B7-2 for optimal CD4(+) T cell responses, while B7-1 appeared to be the predominant B7 molecule involved in the ability of LT (E112K) to augment Ab responses to a coadministered Ag . These findings demonstrate distinct differences in the ability of LT and LT (E112K) to enhance B7-1 and B7-2 on APC, as well as a dependence upon these costimulatory molecules for their adjuvant properties. J Immunol Methods, 2002 Sep 15, 267(2), 213 - 26 Fab-scFv fusion protein: an efficient approach to production of bispecific antibody fragments; Lu D et al.; The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods . Here, we describe an efficient approach for the production of a novel bispecific antibody fragment by genetically fusing a single-chain Fv (scFv) to the C-terminus of either the light chain or the heavy chain of a Fab fragment of different antigen-binding specificity . The bispecific Fab-scFv fragments were expressed in a single Escherichia coli host and purified to homogeneity by a one-step affinity chromatography . Two different versions of the bispecific Fab-scFv fragments were constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor . These bispecific antibody fragments not only retained the antigen-binding capacity of each of the parent antibodies, but also are capable of binding to both targets simultaneously as demonstrated by a cross-linking ELISA . Further, the bispecific antibodies were comparable to their parent antibodies in their potency in blocking ligand binding to the receptors and in inhibiting ligand-induced biological activities . This design for BsAb fragments should be applicable to any pair of antigen specificities. Neurosci Lett, 2002 Aug 30, 329(2), 185 - 8 Establishment of a novel enzyme-linked immunosorbent assay for Thy-1; quantitative assessment of neuronal degeneration; Seki M et al.; In the central nervous system (CNS), Thy-1 is expressed predominantly on neurons and serves as a specific marker for neurons . In the present study, we established a two-site enzyme-linked immunosorbent assay (ELISA) that detects trace amounts of Thy-1 protein . Recombinant Thy-1 protein expressed in Escherichia coli was purified and used as a standard . Of the regions of the nervous system examined, the highest Thy-1 concentration was found in the striatum followed by the hippocampus, neocortex, cerebellum, spinal cord, retina and optic nerve . We found that injection of a neurotoxin, N-methyl-D-aspartate, into the vitreous cavity reduced the Thy-1 level in the retina . Thy-1 ELISA will be useful for quantitative assessment of neurodegeneration in the CNS . Mol Biochem Parasitol, 2002 Aug 7, 123(1), 35 - 45 Identification, expression, and functional characterization of MAEBL, a sporozoite and asexual blood stage chimeric erythrocyte-binding protein of Plasmodium falciparum; Ghai M et al.; MAEBL is a chimeric erythrocyte binding protein reported in rodent malaria parasites Plasmodium yoelii and Plasmodium berghei, that has the gene structure similar to erythrocyte binding proteins, but N-terminal homology to subdomains I and II of Apical membrane antigen-1 . We report here the sequence analysis and gene structure of the Plasmodium falciparum maebl gene . We have cloned and expressed a putative red cell binding domain, M2, of this gene in Escherichia coli, purified the recombinant protein (r-PfM2) and studied its in vitro binding specificity to human red cells . Binding of r-PfM2 protein to red cells was abolished by pretreatment with papain, while increased binding was observed to neuraminidase-treated red cells . Polyclonal antibodies to r-PfM2 recognized native MAEBL protein in blood stage schizont extracts of the parasite on Western blots and within the apical complex of free merozoites, by indirect immunofluorescent assay (IFA) . MAEBL expression in P . falciparum sporozoites was also detected by reverse transcriptase polymerase chain reaction (RT-PCR) and IFA . High titer antibodies to r-PfM2 were observed in human sera obtained from a malaria endemic region some of which inhibited r-PfM2 binding to red cells . Individuals immunized with irradiated sporozoites tested positive for anti-MAEBL antibodies by ELISA . The dual stage expression of MAEBL makes it an excellent pre-erythrocytic and erythrocytic stage vaccine target antigen . Mol Biochem Parasitol, 2002 Aug 7, 123(1), 23 - 33 Bacterially expressed and refolded receptor binding domain of Plasmodium falciparum EBA-175 elicits invasion inhibitory antibodies; Pandey KC et al.; Malaria parasites make specific receptor-ligand interactions to invade erythrocytes . A 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A during invasion of human erythrocytes . The receptor-binding domain of EBA-175 lies in a conserved, amino-terminal, cysteine-rich region, region F2 of EBA-175 (PfF2), that is homologous to the binding domains of other erythrocyte binding proteins such as Plasmodium vivax Duffy binding protein . We have developed methods to produce recombinant PfF2 in its functional form . Recombinant PfF2 was expressed in Escherichia coli, purified from inclusion bodies, renatured by oxidative refolding and purified to homogeneity by ion-exchange and gel filtration chromatography . Refolded PfF2 has been characterized using biochemical and biophysical methods and shown to be pure, homogenous and functional in that it binds human erythrocytes with specificity . Immunization with refolded PfF2 yields high titre antibodies that efficiently inhibit P . falciparum invasion of erythrocytes in vitro . Importantly, antibodies raised against PfF2 block invasion by a P . falciparum field isolate that invades erythrocytes using multiple pathways . These observations support the development of recombinant PfF2 as a vaccine candidate for P . falciparum malaria . Hybrid Hybridomics, 2002 Jun, 21(3), 169 - 78 Development of a second generation monoclonal single chain variable fragment antibody against Venezuelan equine encephalitis virus: expression and functional analysis; Alvi AZ et al.; We have generated a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (MAb) against Venezuelan equine encephalitis virus (VEE), by cloning variable regions of the heavy (V(H)) and the light (V(L)) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E . MAb 1A4A1 was successfully cloned as a ScFv in Escherichia coli strain TG-1 and expressed as a approximately 30 kDa ScFv protein which was functional in recognizing VEE by ELISA . Results were reproduced in Escherichia coli strain HB2151 where the same clone, designated A116, was expressed primarily as soluble periplasmic protein . The 30 kDa A116 antibody displayed weak binding specificity to VEE antigen . Sequence analysis revealed a frame shift in the N-terminal region of the V(L) domain, upstream to the complementarity-determining region 1 (CDR1), as the probable cause of reduced activity . The protein sequence of A116 was highly homologous to published murine ScFv protein sequences except in the region of the identified frame shift. Helicobacter, 2002 Aug, 7(4), 237 - 44 The role of the Ferric Uptake Regulator (Fur) in regulation of Helicobacter pylori iron uptake; van Vliet AH et al.; BACKGROUND: Availability of the essential nutrient iron is thought to vary greatly in the gastric mucosa, and thus the human gastric pathogen Helicobacter pylori requires regulatory responses to these environmental changes . Bacterial iron-responsive regulation is often mediated by Ferric Uptake Regulator (Fur) homologs, and in this study we have determined the role of H . pylori Fur in regulation of H . pylori iron uptake . METHODS: Wild-type H . pylori and fur mutant derivatives were compared after growth in iron-restricted and iron-replete conditions . Iron-uptake was measured using 55Fe-labeled iron, whereas gene expression was monitored at the transcriptional level using Northern hybridization and lacZ reporter gene fusions . RESULTS: Iron-uptake and total cellular iron content were approximately five-fold increased in the fur mutant compared with the wild-type strain, which indicated that in the fur mutant iron-uptake is not repressed by excess iron . A comprehensive screening of all H . pylori genes encoding putative iron-uptake proteins indicated that some of these H . pylori genes are constitutively expressed, while others are iron- and Fur-regulated . CONCLUSIONS: Iron uptake in H . pylori is in part differently regulated compared with other bacteria, since in H . pylori some iron-uptake systems are constitutively expressed . However, other iron uptake systems of H . pylori display the iron- and Fur-mediated repression that is common in bacteria . Taken together, this Fur-mediated modulation of iron-uptake capacity may be a specific adaptation to the conditions in the human stomach, where iron starvation and iron overload can be encountered in relatively short time intervals. Kidney Int, 2002 Sep, 62(3), 846 - 56 Shiga toxin-2 triggers endothelial leukocyte adhesion and transmigration via NF-kappaB dependent up-regulation of IL-8 and MCP-1; Zoja C et al.; BACKGROUND: Shiga toxin (Stx)-producing E . coli is a causative agent of the epidemic form of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children . Endothelial injury and leukocyte activation are instrumental to the development of microangiopathic lesions . To obtain more insight into the mechanisms favoring endothelium-leukocyte interaction, we studied (1) the effect of Stx-2 on leukocyte adhesion and transmigration in human endothelial cells under flow; (2) the effect of Stx-2 on endothelial expression of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) and their functional role in the adhesive phenomena; and (3) the role of nuclear factor-kappaB (NF-kappaB) in endothelial chemokine expression . METHODS: For adhesion experiments, human umbilical vein endothelial cells (HUVEC) and human glomerular endothelial cells (GEC) were incubated for 24 hours with Stx-2 (25 pmol/L), with or without anti-IL-8 or MCP-1 antibodies, and then exposed to leukocyte suspension under flow (1.5 dynes/cm2) . IL-8 and MCP-1 expression was evaluated in Stx-2 treated endothelial cells (6 hours) by Northern blot . NF-kappaB activity was assessed by electrophoretic mobility shift assay . The role of NF-kappaB in Stx-induced chemokines was evaluated by transfecting HUVEC with an adenovirus coding for IkappaBalpha . RESULTS: Stx-2 significantly enhanced the number of leukocytes that adhered and then migrated across the endothelium . Stx-2 increased the expression of IL-8 and MCP-1, which was preceded by NF-kappaB activation . Blocking of endothelial IL-8 and MCP-1 with corresponding antibodies significantly inhibited Stx-induced leukocyte adhesion and migration either in HUVEC or GEC . Adenovirus-mediated gene transfer of IkappaBalpha down-regulated IL-8 and MCP-1 mRNA and also inhibited the adhesion and transmigration of leukocytes in Stx-treated HUVEC . CONCLUSIONS: Stx-2 via a transcriptional activation mechanism specifically mediated by NF-kappaB up-regulates endothelial MCP-1 and IL-8 . Both chemokines are important modulators of leukocyte adhesion and transmigration under flow . These findings might be relevant to understand the nature of microvascular lesions in HUS and open future perspectives for better treatment of microvascular thrombosis. Kidney Int, 2002 Sep, 62(3), 757 - 62 Decreased sulfotransferase SULT1C2 gene expression in DPT-induced polycystic kidney; Sugimura K et al.; BACKGROUND: The pathogenesis of polycystic kidney disease (PKD) remains unclear despite the identification of the genes responsible for hereditary PKD . In this study, we investigated the alteration of gene expressions in an acquired PKD model induced by 2-amino-4,5-diphenylthiazole (DPT) using the differential display method . METHODS: Kidney mRNA from a Sprague-Dawley rat fed with 1% DPT for 4 days and from a control rat was compared by the RT-PCR differential display method . Differentially expressed bands were re-amplified and subcloned . Using these subclones as probes, the changes in gene expressions were confirmed by Northern blot analysis . Subsequently, mouse kidney cDNA library was screened . RESULTS: The isolated 1.5-kb cDNA contained an open reading frame encoding 296 amino acids, which shared 94.3% identity with rat SULT1C2 sulfotransferase, and was considered to be its mouse ortholog (GenBank Accession No . AY005469) . Mouse SULT1C2 mRNA was abundant in the kidney and stomach among normal mouse tissues . The expression of SULT1C2 mRNA was decreased in the rat kidney after DPT feeding but not in the stomach . Mouse SULT1C2 was expressed successfully using pET plasmid vector and E . coli . The recombinant 34-kD protein was capable of catalyzing the sulfation of p-nitrophenol at a Km of 3.1 mmol/L, by utilizing 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor . CONCLUSIONS: Although the physiological substrate and function of SULT1C2 have yet to be elucidated, its down-regulation could be involved in the cystic changes of tubules by decreasing the sulfation of the tubular basement membrane components. Parasite Immunol, 2002 Jul, 24(7), 355 - 61 Effect of salivary gland extracts from the tick, Boophilus microplus, on leucocytes from Brahman and Hereford cattle; Turni C et al.; The effect of salivary gland extract (SGE) from Boophilus microplus on peripheral blood lymphocytes, neutrophils and monocytes from Brahman (Bos indicus) and Hereford (Bos taurus) cattle was investigated . SGE (8 micro g) significantly inhibited the proliferation response of lymphocytes to concanavalin A from both Brahman and Hereford cattle by 89% and 41%, respectively . The difference in inhibition between the two breeds was highly significant (P < 0.01), whilst at 1 micro g of SGE, significant inhibition of lymphocytes occurred only in Hereford cattle (34%) . Flow cytometric analysis of monocytes and neutrophils showed that SGE (40 micro g) significantly reduced both the proportion of cells actively phagocytosing Escherichia coli labelled with fluorescein isothiocyanate (E . coli-FITC) and the uptake of E . coli-FITC in Brahman cattle . However, in Hereford cattle, a significant depression in uptake was only observed in neutrophils . The proportion of monocytes and neutrophils with oxidative activity was significantly suppressed in the presence of SGE in both breeds of cattle . These results indicate that peripheral blood leucocytes from different breeds of cattle respond differently to SGE. Plant J, 2002 Aug, 31(3), 243 - 54 Two distantly related genes encoding 1-deoxy-d-xylulose 5-phosphate synthases: differential regulation in shoots and apocarotenoid-accumulating mycorrhizal roots; Walter MH et al.; Isopentenyl diphosphate, the universal precursor of isoprenoids, is synthesized by two separate routes, one in the cytosol and the other in plastids . The initial step of the plastidial pathway is catalysed by 1-deoxy-d-xylulose 5-phosphate synthase (DXS), which was previously thought to be encoded by a single-copy gene . We have identified two distinct classes of DXS-like cDNAs from the model legume Medicago truncatula . The deduced mature MtDXS1 and MtDXS2 proteins, excluding the predicted plastid-targeting peptides, are similar in size (72.7 and 71.2 kDa) yet share only 70% identity in their amino acid sequences, and both encode functional DXS proteins as shown by heterologous expression in Escherichia coli . Available DXS sequences from other plants can easily be assigned to either class 1 or class 2 . Partial sequences of multiple DXS genes in a single genome may be found in the databases of several monocot and dicot plants . Blot analyses of RNA from M . truncatula, maize, tomato and tobacco demonstrate preferential expression of DXS1 genes in many developing plant tissues except roots . By contrast, DXS2 transcript levels are low in most tissues but are strongly stimulated in roots upon colonization by mycorrhizal fungi, correlated with accumulation of carotenoids and apocarotenoids . Monoterpene-synthesizing gland cells of leaf trichomes appear to be another site of DXS2 gene activity . The potential importance of DXS1 in many housekeeping functions and a still hypothetical role of DXS2 in the biosynthesis of secondary isoprenoids is discussed. J Drug Target, 2002 Jun, 10(4), 345 - 52 Transluminal gene transfer into brain capillary endothelial cells in vivo with HVJ-liposomes; Jiang C et al.; Bioactive proteins or peptides cannot be effectively delivered into brain capillary endothelial cells (BCECs) or brain parenchyma . In this study, we selectively transferred Escherichia coli beta-galactosidase gene (beta-gal) as a model gene into BCECs by using the hemagglutination virus of Japan (HVJ)-liposomes . HVJ-liposomes encapsulating a beta-gal plasmid were used to transfect MBEC4 cells in vitro, and were administrated via the internal carotid artery to rat in vivo . Success of the procedure was confirmed by the detection of 116 kDa beta-gal protein in transfected MBEC4 cells and in brain capillaries isolated from transfected rats, by Western blot analysis and histological staining . The enzymatic activities of beta-galactosidase were 5- to 10-fold and 20-fold higher than when beta-gal-containing liposomes without fusogenic activity (uncoated liposomes) or plasmid alone were employed in vitro and in vivo, respectively . Thus, HVJ-liposomes were demonstrated to be a useful vector to transfer a foreign gene into the brain capillary endothelium in vivo via the transluminal route. J Membr Biol, 2002 Jun 1, 187(3), 239 - 53 Site-directed mutagenesis within the central constriction site of ScrY (sucroseporin): effect on ion transport and comparison of maltooligosaccharide binding to LamB of Escherichia coli; Kim BH et al.; The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography . The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids . The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places . The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end . The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes . In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates . The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels . The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels . The results suggest that both on- and off-rate constants were affected by the mutations . Most of them showed a substantial effect on carbohydrate binding kinetics . Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties. Crit Care Med, 2002 Aug, 30(8), 1826 - 33 Adenosine triphosphate-magnesium dichloride during hyperdynamic porcine endotoxemia: effects on hepatosplanchnic oxygen exchange and metabolism; Asfar P et al.; OBJECTIVE: To assess the effects of adenosine triphosphate-magnesium dichloride (ATP-MgCl2) on systemic and hepatosplanchnic hemodynamics, oxygen exchange, and energy metabolism over 24 hrs of hyperdynamic normotensive porcine endotoxemia . DESIGN: Prospective, randomized, controlled experimental study with repeated measures . SETTING: Investigational animal laboratory . SUBJECTS: Seventeen pigs were divided into two groups: eight animals receiving endotoxin served as a control group and nine animals received endotoxin (lipopolysaccharide) and ATP-MgCl2 . INTERVENTIONS: Pigs were anesthetized, mechanically ventilated, and instrumented . Endotoxemia was achieved by continuous intravenous infusion of Escherichia coli lipopolysaccharide . Animals were resuscitated by hetastarch targeted to maintain mean arterial pressure of >75 mm Hg . Twelve hours after the start of the endotoxin infusion, ATP-MgCl2, or its vehicle, were administered for 12 hrs . MEASUREMENTS AND MAIN RESULTS: Mean arterial pressure was maintained in the control group because of a sustained increase in cardiac output achieved by fluid resuscitation, whereas ATP-MgCl2 significantly decreased mean arterial pressure because of further systemic vasodilatation . ATP-MgCl2 markedly increased portal venous flow . In contrast to the controls, hepatic arterial flow remained unchanged until the end of the experiment, despite the further increase in cardiac output . The ileal mucosal-arterial PCO2 gap (Delta PCO2) progressively increased (p <.05) in control animals, whereas it was restored to prelipopolysaccharide levels during ATP-MgCl2 infusion . Changes in Delta PCO2 correlated with those of portal vein blood flow in these animals (r = -.68, p <.05) . Moreover, ATP-MgCl2 blunted the lipopolysaccharide-induced decrease in hepatic lactate balance but did not affect portal venous pH, hepatosplanchnic oxygen exchange, splanchnic lactate/pyruvate ratios, isoprostane, NO2- + NO3-, cytokine concentrations, or tissue nucleotide content . CONCLUSION: During long-term hyperdynamic porcine endotoxemia, ATP-MgCl2 normalized the otherwise progressive rise of the ileal mucosal-arterial Delta PCO2 . Furthermore, it allowed blunting of the continuous decrease in hepatic lactate clearance, thus preserving the metabolic coupling between lactate release from the intestine and lactate utilization by the liver. Crit Care Med, 2002 Aug, 30(8), 1820 - 5 Effect of pretreatment with interleukin-1 beta on inflammatory infiltrates and tissue damage after experimental endotoxic challenge; Tellez-Gil L et al.; OBJECTIVE: To evaluate the effect of treatment with murine recombinant interleukin-1 beta on inflammatory infiltrate and tissue damage after experimental endotoxic challenge . DESIGN: Randomized, controlled study . SETTING: Experimental Unit, Virgen de las Nieves University Hospital . SUBJECTS: Seventy-two female CBA/H mice, 20-21 g, supplied by the animal center of the Experimental Unit . INTERVENTION: The mice were randomized into three groups of 24 . Group 1 (sham) received two intraperitoneal doses of 0.1 mL of phosphate-buffered saline; group 2 (lipopolysaccharide) was injected with 125 mg/kg lipopolysaccharide (Escherichia coli, intraperitoneally) 24 hrs after 0.1 mL of phosphate-buffered saline; group 3 was pretreated with 80 ng of recombinant interleukin-1 beta per mouse (intraperitoneally) 24 hrs before the endotoxic challenge . MEASUREMENTS AND MAIN RESULTS: At 1, 2, 4, and 24 hrs after the endotoxic challenge, we studied inflammatory infiltrate and tissue damage in lung, liver, and intestine by determining myeloperoxidase and malondialdehyde tissue concentrations . When we compared the pretreated group with the lipopolysaccharide group, myeloperoxidase concentrations decreased significantly in lung (p <.001) and liver (p <.001) at all study times, and in intestine (p <.001) at 2, 4, and 24 hrs; malondialdehyde concentrations significantly decreased in lung at 1 (p <.05), 2 (p <.01), and 24 (p <.001) hrs, in liver at 2 (p <.001), 4 (p <.01), and 24 (p <.001) hrs, and in intestine at 1 (p <.001), 2, 4 (p <.05), and 24 (p <.001) hrs . CONCLUSION: Pretreatment with recombinant interleukin-1 beta significantly reduces inflammatory infiltrate and tissue damage in mouse lung, liver, and intestine after an experimental endotoxic challenge. Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11103 - 8 Epub 2002 Aug 05. Transition to the open state of the TolC periplasmic tunnel entrance; Andersen C et al.; The TolC channel-tunnel spans the bacterial outer membrane and periplasm, providing a large exit duct for protein export and multidrug efflux when recruited by substrate-engaged inner membrane complexes . The sole constriction in the single pore of the homotrimeric TolC is the periplasmic tunnel entrance, which in its resting configuration is closed by dense packing of the 12 tunnel-forming alpha-helices . Recruitment of TolC must trigger opening for substrate transit to occur, but the mechanism underlying transition from the closed to the open state is not known . The high resolution structure of TolC indicates that the tunnel helices are constrained at the entrance by a circular network of intra- and intermonomer hydrogen bonds and salt bridges . To assess how opening is achieved, we disrupted these connections and monitored changes in the aperture size by measuring the single channel conductance of TolC derivatives in black lipid bilayers . Elimination of individual connections caused incremental weakening of the circular network, accompanied by gradual relaxation from the closed state and increased flexibility of the entrance . Simultaneous abolition of the key links caused a substantial increase in conductance, generating an aperture that corresponds to the modeled open state, with the capacity to allow access and passage of diverse substrates . The results support a model in which transition to the open state of TolC is achieved by an iris-like realignment of the tunnel entrance helices. J Biol Chem, 2002 Oct 18, 277(42), 40125 - 31 Epub 2002 Aug 05. Projection structure of P-glycoprotein by electron microscopy . Evidence for a closed conformation of the nucleotide binding domains; Lee JY et al.; The structure of P-glycoprotein (Pgp) from mouse has been studied by electron microscopy and image analysis . Two-dimensional crystals of Pgp in a lipid bilayer were generated by reconstituting pure, detergent-solubilized protein containing a C-terminal six-histidine tag using the lipid monolayer technique . The crystals belong to plane group P1 with a = b = 104 +/- 2 A and gamma = 90 +/- 4 degrees . The projection structure of Pgp calculated at a resolution of 22 A shows two closely interacting protein domains that can be interpreted as the N- and C-terminal halves of the protein . The projection structure of Pgp is consistent with the recently published x-ray structure of MsbA, a lipid A flippase from Escherichia coli with high sequence homology to Pgp but only when the two MsbA subunits are rotated to bring their nucleotide binding domains together. J Biol Chem, 2002 Oct 18, 277(42), 39251 - 8 Epub 2002 Aug 05. CaiT of Escherichia coli, a new transporter catalyzing L-carnitine/gamma -butyrobetaine exchange; Jung H et al.; l-Carnitine is essential for beta-oxidation of fatty acids in mitochondria . Bacterial metabolic pathways are used for the production of this medically important compound . Here, we report the first detailed functional characterization of the caiT gene product, a putative transport protein whose function is required for l-carnitine conversion in Escherichia coli . The caiT gene was overexpressed in E . coli, and the gene product was purified by affinity chromatography and reconstituted into proteoliposomes . Functional analyses with intact cells and proteoliposomes demonstrated that CaiT is able to catalyze the exchange of l-carnitine for gamma-butyrobetaine, the excreted end product of l-carnitine conversion in E . coli, and related betaines . Electrochemical ion gradients did not significantly stimulate l-carnitine uptake . Analysis of l-carnitine counterflow yielded an apparent external K(m) of 105 microm and a turnover number of 5.5 s(-1) . Contrary to related proteins, CaiT activity was not modulated by osmotic stress . l-Carnitine binding to CaiT increased the protein fluorescence and caused a red shift in the emission maximum, an observation explained by ligand-induced conformational alterations . The fluorescence effect was specific for betaine structures, for which the distance between trimethylammonium and carboxyl groups proved to be crucial for affinity . Taken together, the results suggest that CaiT functions as an exchanger (antiporter) for l-carnitine and gamma-butyrobetaine according to the substrate/product antiport principle. J Biol Chem, 2002 Oct 18, 277(42), 40036 - 42 Epub 2002 Aug 05. X-ray structure of pyruvate formate-lyase in complex with pyruvate and CoA . How the enzyme uses the Cys-418 thiyl radical for pyruvate cleavage; Becker A et al.; The glycyl radical enzyme pyruvate formate-lyase (PFL) synthesizes acetyl-CoA and formate from pyruvate and CoA . With the crystal structure of the non-radical form of PFL in complex with its two substrates, we have trapped the moment prior to pyruvate cleavage . The structure reveals how the active site aligns the scissile bond of pyruvate for radical attack, prevents non-radical side reactions of the pyruvate, and confines radical migration . The structure shows CoA in a syn conformation awaiting pyruvate cleavage . By changing to an anti conformation, without affecting the adenine binding mode of CoA, the thiol of CoA could pick up the acetyl group resulting from pyruvate cleavage. FEBS Lett, 2002 Aug 14, 525(1-3), 111 - 5 Biophysical study of a molecular intermediate preceding collapse of tight couple and Kaltschmidt-Wittmann ribosomes; Bonincontro A et al.; In previous works we evidenced, by different biophysical approaches, two levels of structural organization in Escherichia coli ribosomal particles . Thermal treatment up to a defined and non-denaturing temperature causes demolition of only one level of structural complexity . By consequence the ribosomal particle exists in an intermediate state between the native form and the completely collapsed one . In this communication we report on a structural comparison of this intermediate state in Kaltschmidt-Wittmann (LC) and 'tight couple' (TC) ribosomes . Three different biophysical approaches were adopted: dielectric spectroscopy, fluorescence and light scattering . Differential responses to thermal treatment are evidenced in the two ribosomal species . In particular TC show a more compact structure and the overall particle population is more homogeneous than LC in the native state . On the other hand, LC particles after thermal treatment undergo major alterations of geometry and/or phenomena of supra-particle aggregation. FEBS Lett, 2002 Aug 14, 525(1-3), 65 - 70 Topology determination and functional analysis of the Escherichia coli TatC protein; Gouffi K et al.; The TatC protein is an essential component of the bacterial Tat system . By using alkaline phosphatase and beta-glucuronidase fusions we found that TatC contains four transmembrane helices . Three insertions of Ala-Ser dipeptide at the cytoplasmic N- and C-termini and in the cytoplasmic loop had no or only partial effect on the TatC function . In contrast, five of seven insertions in the two periplasmic loops abolished the Tat function . Four insertions analyzed had no effect on the stability of the altered TatC proteins or on membrane assembly of the TatA and TatB proteins . These data provide a novel base for more detailed studies of the mechanism of the Tat system. FEBS Lett, 2002 Aug 14, 525(1-3), 39 - 42 The hppA gene of Helicobacter pylori encodes the class C acid phosphatase precursor; Godlewska R et al.; Screening of the Helicobacter pylori genomic library with sera from infected humans and from immunized rabbits resulted in identification of the 25 kDa protein cell envelope (HppA) which exhibits acid phosphatase activity . Enzyme activity was demonstrated by specific enzymatic assays with whole-cell protein preparations of H . pylori strain N6 and from Escherichia coli carrying the hppA gene (pUWM192) . HppA showed optimum activity at pH 5.6 and was resistant to inhibition by EDTA . Bioinformatics analysis and site-directed mutagenesis of two putative active site residues (D73 and D192) provide further insight into the sequence-structure-function relationships of HppA as a member of the DDDD phosphohydrolase superfamily. FEBS Lett, 2002 Aug 14, 525(1-3), 33 - 8 Influence of lipids on membrane assembly and stability of the potassium channel KcsA; van Dalen A et al.; Recently we observed in an in vitro system that newly synthesized KcsA assembles efficiently into a tetramer in lipid vesicles {van Dalen et al . (2002) FEBS Lett . 511, 51-58} . Here we used this system to get insight into the importance of the lipid composition for KcsA membrane association and tetramerization and we compared this to the lipid dependency of the thermo-stability of the KcsA tetramer . It was found that a large amount of phosphatidylethanolamine (>40 mol%) and a lower amount of phosphatidylglycerol (approximately 20-30 mol%) were optimal for efficient KcsA membrane association and tetramerization . Strikingly, vesicles of the abundant and commonly used membrane lipid phosphatidylcholine did not support assembly, further demonstrating the importance of membrane lipid composition for KcsA assembly . The in vitro assembled KcsA tetramer showed similar thermo-stability in biological and pure lipid membranes, demonstrating that both tetramers are alike . In addition, we show that solubilization of the membrane with detergent reduces the thermo-stability of the tetramer . The highest KcsA tetramer stability was observed in intact bilayers in the presence of anionic lipids. FEBS Lett, 2002 Aug 14, 525(1-3), 25 - 8 Mutagenesis identifies a conserved tyrosine residue important for the activity of uroporphyrinogen III synthase from Anacystis nidulans; Roessner CA et al.; Uroporphyrinogen III synthase from the cyanobacterium Anacystis nidulans was overproduced in Escherichia coli and analyzed by site specific mutagenesis . Of the nine conserved amino acids altered, only a single tyrosine mutant (Y166F) showed any significant decrease in activity suggesting this residue is critical for proper substrate binding and/or catalysis. FEBS Lett, 2002 Aug 14, 525(1-3), 13 - 9 Oligomerization of DsRed is required for the generation of a functional red fluorescent chromophore; Sacchetti A et al.; The coral red fluorescent protein (DsRed) absorbs and emits light at much higher wavelengths than the structurally homologous green fluorescent protein, raising questions about the properties of its chromophore . We have analyzed the relationship between the aggregation state and fluorescence of native, 6-histidine-tagged, or maltose-binding protein-fused DsRed . In all cases, newly synthesized DsRed molecules were largely monomeric and devoid of covalently closed chromophores . Maturation in vitro induces the expression of red fluorescent chromophores but only in oligomeric forms of the protein, whereas monomers are essentially devoid of fluorescence . NaOH-denatured samples demonstrated a generalized breakdown of the DsRed oligomers to monomers, which refolded after neutralization into weakly green fluorescent and still monomeric species . Red fluorescent chromophores were regenerated only upon oligomerization . These findings demonstrate that 'red' chromophores form and are functional only as oligomers, and suggest that the smallest red fluorescent functional unit is a dimer . A comparison of alkali-, acid- and guanidinium-denatured DsRed indicates that stabilization of the DsRed chromophore by concerted steps of folding and oligomerization may play a critical role in its maturation process. J Steroid Biochem Mol Biol, 2002 Jul, 81(3), 255 - 62 Membrane reconstitution of recombinant guinea pig cytochrome P45017alpha and the effects of site-directed mutagenesis on androgen formation; Owaki A et al.; Although cytochrome P45017alpha catalyzes the formation of androgen from both pregnenolone and progesterone, the production of androstenedione from progesterone is a major pathway in the guinea pig, rat, mouse, and hamster . In contrast, human, bovine and sheep P45017alpha produce dehydroepiandrosterone from pregnenolone . Cytochrome P45017alphas from all of these animals have high homology in the amino acid sequence around the 340-370 region . To investigate the substrate preferences for androgen production, we replaced a few amino acids in the 340-370 region of guinea pig P45017alpha with those found in the other animals . The recombinant P45017alphas were expressed in E . coli DH5alpha, purified by column chromatography and incorporated into liposome membranes . The (His)(4) tag in the recombinant P45017alphas had little effect on the interaction with NADPH-P450 reductase in the membranes . The recombinant P45017alphas with a single-position mutation of F344I, H349R or M352L and with double-position mutations of F344I and H349R and triple-position mutations showed decreases in the production of 17alpha-hydroxypregnenolone, androstenedione and dehydroepiandrosterone . The activity for 17alpha-hydroxyprogeterone production was increased significantly by the F344I mutation . The addition of cytochrome b5 did not have much of an effect on the 17alpha-hydroxylation but had a significant effect on androgen production in both the nonmutated and mutated P45017alphas. J Steroid Biochem Mol Biol, 2002 Jul, 81(3), 217 - 25 Ligand and coactivator recruitment preferences of peroxisome proliferator activated receptor alpha; Mukherjee R et al.; The mechanism by which ligands of nuclear receptors show differential effects on gene transcription is not fully understood, but is believed to result in part from the preferential recruitment and/or displacement of coactivators and corepressors . We have explored the interaction of several known ligands and the nuclear receptor (peroxisome proliferator activated receptor alpha, PPARalpha) using scintillation proximity assay (SPA) and the interaction of LXXLL containing peptides derived from three coactivators (SRC-1, CBP and PGC-1) with PPARalpha in the presence of PPARalpha agonist ligands using fluorescence resonance energy transfer (FRET) . The EC(50)s of the individual ligands for recruitment showed the same rank order regardless of the coactivator peptide used, with GW2331<WY14643=ciprofibrate<L165041<gemfibrozil . Similarly, for all ligands tested, the rank order of EC(50) for peptide recruitment was CBP<PGC-1<SRC-1 . These data suggest that for these LXXLL coactivator peptides, the ligands do not substantially differ in their preferences . Partial agonism was observed with ciprofibrate and PGC-1 and gemfibrozil and CBP giving a lower FRET at saturation than with the other ligands . This suggests that ciprofibrate and gemfibrozil induce a different conformation to the receptor-PGC-1 and receptor-CBP complex, respectively . In cotransfection assays, unexpected differences in potencies and efficacies were observed and the rank order of EC(50)s for activation differed from that predicted by FRET assays . In most cases, the presence of a coactivator peptide led to decrease in the EC(50)s seen in FRET assays compared to the K(i)s observed in binding to receptor only, consistent with the lower EC(50)s obtained in the transfection assays . Our data demonstrate that ligand induced coactivator preferences of PPARalpha contribute to transcription potency and efficacy. J Mol Biol, 2002 Aug 16, 321(3), 397 - 409 Mutational analysis of polynucleotide phosphorylase from Escherichia coli; Jarrige A et al.; Polynucleotide phosphorylase (PNPase), a homotrimeric exoribonuclease present in bacteria, is involved in mRNA degradation . In Escherichia coli, expression of this enzyme is autocontrolled at the translational level . We introduced about 30 mutations in the pnp gene by site-directed mutagenesis, most of them in phylogenetically conserved residues, and determined their effects on the three catalytic activities of PNPase, phosphorolysis, polymerisation and phosphate exchange, as well as on the efficiency of translational repression . The data are presented and discussed in the light of the crystallographic structure of PNPase from Streptomyces antibioticus . The results show that both PNPase activity and the presence of the KH and S1 RNA-binding domains are required for autocontrol . Deletions of these RNA-binding domains do not abolish any of the three catalytic activities, indicating that they are contained in a domain independent of the catalytic centre . Moreover, the catalytic centre was located around the tungsten-binding site identified by crystallography . Some mutations affect the three catalytic activities differently, an observation consistent with the presence of different subsites. Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1423 - 6 Transglutaminase activity of human ER-60; Okudo H et al.; Human recombinant ER-60 was confirmed to have transglutaminase activity by a microtiter plate assay . Transglutaminase activity of ER-60 did not require calcium and was inhibited by cystamine, a substrate analogue . In addition, the transglutaminase activity of ER-60 was not inhibited by SH-blocking reagents . These results suggest that the properties of the transglutaminase activity of ER-60 are different from those in the cases of known mammalian transglutaminases of which the active site includes a cysteine residue. Biosci Biotechnol Biochem, 2002 Jun, 66(6), 1378 - 81 Cloning and expression of an endo-1,6-beta-D-glucanase gene (neg1) from Neurospora crassa; Oyama S et al.; A gene (neg1) encoding an endo-1,6-beta-D-glucanase from Neurospora crassa was cloned . The putative neg1 was 1443-bp long and encoded a mature endo-1,6-beta-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids . The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-beta-D-glucanase activity . No genes similar in sequence were found in yeasts and fungi. Mol Cell Biochem, 2002 May-Jun, 234-235(1-2), 11 - 8 Signal transduction by nitric oxide in cellular stress responses; Demple B; Nitric oxide (NO) has received wide attention as a biological signaling molecule that uses cyclic GMP as a cellular second messenger . Other work has supported roles for cysteine oxidation or nitrosylation as signaling events . Recent studies in bacteria and mammalian cells now point to the existence of at least two other pathways independent of cGMP . For the E . coli SoxR protein, signaling occurs by nitrosylation of its binuclear iron-sulfur clusters, a reaction that is unprecedented in gene activation . In intact cells, these nitrosylated centers are very rapidly replaced by unmodified iron-sulfur clusters, a result that points to the existence of an active repair pathway for this type of protein damage . Exposure of mammalian cells to NO elicits an adaptive resistance that confers elevated resistance of the cells to higher levels of NO . This resistance in many cell types involves the important defense protein heme oxygenase 1, although the mechanism by which this enzyme mediates NO resistance remains unknown . Induction of heme oxygenase in some cell types occurs through the stabilization of its mRNA . NO-induced stabilization of mRNA is mediated by pre-existing proteins and points to the existence of an important new signaling pathway that counteracts the damage and stress exerted by this free radical. Poult Sci, 2002 Jul, 81(7), 958 - 65 Effect of dietary supplementation with vitamin D metabolites in an experimental model of turkey osteomyelitis complex; Huff GR et al.; Supplementation with vitamin D3 was previously shown to protect Escherichia coli challenged birds that underwent two dexamethasone (DEX) treatments at 5 and 12 wk of age in an experimental model of turkey osteomyelitis complex (TOC) . The purpose of the present study was to determine the effects of dietary supplementation with 10 microg of 1,25 dihydroxyvitamin D3 (1,25D)/ kg feed or 99 microg of 25-hydroxyvitamin D3 (25D)/kg feed on disease resistance in the same model . Birds were fed the supplemented diets continuously and ad libitum . Seven hundred twenty turkey poults were placed into 24 floor pens in a 3 x 2 x 2 design (three vitamin D treatments, two DEX treatments, two E . coli treatments, with two replicate pens per treatment) . At 5 wk of age, half of the birds were treated with DEX, and half of the DEX-treated birds and half of the nontreated birds were challenged with E . coli . All mortalities and lame birds were necropsied . At 9 wk, all of the DEX- or E . coli-treated birds were given another series of DEX injections; 2 wk later 10 birds per pen were necropsied . At 12 wk, survivors of the previous challenges were given a third DEX treatment, and all birds were necropsied 2 wk later . After the first series of DEX injections, mortality was increased in the 25D-supplemented birds that were given the DEX treatment and the E . coli challenge . After the second series of DEX injections, the main effect mean BW was significantly lower in birds given 1,25D as compared to controls and 25D-supplemented birds . Mortality was higher in 1,25D-supplemented birds that were challenged with E . coli at 5 wk and treated with DEX at 9 wk as compared to 25D-supplemented birds . The 1,25D-treated birds that were treated with DEX at 5 and 9 wk and challenged with E . coli at 5 wk had higher mortality and air sacculitis scores as compared to controls and 25D-treated birds . The main effect mean mortality was significantly higher in birds given 1,25D as compared to controls and 25D-treated birds . The percentage of birds with TOC lesions was decreased from 27% to 0 by 25D and 1,25D in the groups given two DEX treatments and E . coli challenge . After the third DEX treatment, BW of 1,25D-suppplemented birds was decreased, and mortality and air sacculitis scores were increased . Bone strength was generally increased by supplementation with 1,25D, whereas 25D supplementation increased bone strength only in birds challenged at 5 wk and treated with DEX at Weeks 9 and 12 . In this study, supplementation with vitamin D metabolites decreased TOC incidence in E . coli-challenged birds given two DEX treatments . However, toxic effects were observed in most supplemented DEX-treated birds and may be attributed to an additive effect of DEX treatment, E . coli septicemia, and vitamin D supplementation. Poult Sci, 2002 Jul, 81(7), 951 - 7 Influence of a propionic acid feed additive on performance of turkey poults with experimentally induced poult enteritis and mortality syndrome; Roy RD et al.; Poult enteritis and mortality syndrome (PEMS) has multiple etiological agents associated with its occurrence, including two viruses and at least three Escherichia coli isolates . Myco Curb (MC) contains organic acids and is used as a feed additive to inhibit growth of many bacteria and toxin-producing molds but not viruses . Studies evaluating the influence of MC on BW, feed conversion, and mortality indicate that turkey poults tolerate MC at 1.25% but not 2.50%, but higher MC content in feed provides greater suppression of growth of bacterial isolates commonly associated with PEMS . In two PEMS experiments, 1.25% MC was blended into poult starter feed and was maintained in the feed for the duration of the 3-wk experiments . In these experiments, 1-d-old commercial poults were placed into battery brooders and were given turkey starter feed and water ad libitum . At 6 d posthatch, PEMS-designated poults were given a 1-mL oral gavage of a 10% suspension of feces from PEMS-infected poults . BW depression due to PEMS was not alleviated by MC, although there was less variation in mean BW of the MC-fed poults, and there was a highly significant reduction in mortality (68% in PEMS-exposed with MC vs . 32.5% in PEMS-exposed without MC) . The reduction in mortality in the MC-fed poults was attributed to decreased bacterial content of the gut and to maintenance of packed cell volume and hemoglobin content . It was concluded that MC might be a potential nutritional intervention during PEMS. Nat Biotechnol, 2002 Sep, 20(9), 908 - 13 Epub 2002 Aug 05. Rational cytokine design for increased lifetime and enhanced potency using pH-activated "histidine switching"; Sarkar CA et al.; We describe a method for the rational design of more effective therapeutic proteins using amino acid substitutions that reduce receptor binding affinity in intracellular endosomal compartments, thereby leading to increased recycling in the ligand-sorting process and consequently resulting in longer half-life in extracellular medium . We demonstrate this approach for granulocyte colony-stimulating factor by using computationally predicted histidine substitutions that switch protonation states between cell-surface and endosomal pH . Molecular modeling of binding electrostatics indicates two different single-histidine mutants that fulfill our design requirements; experimental assays demonstrate that each mutant indeed exhibits an order-of-magnitude increase in medium half-life along with enhanced potency due to increased endocytic recycling. Nat Struct Biol, 2002 Sep, 9(9), 659 - 64 3-Methyladenine DNA glycosylase I is an unexpected helix-hairpin-helix superfamily member; Drohat AC et al.; The Escherichia coli enzyme 3-methyladenine DNA glycosylase I (TAG) hydrolyzes the glycosidic bond of 3-methyladenine (3-MeA) in DNA and is found in many bacteria and some higher eukaryotes . TAG shows little primary sequence identity with members of the well-known helix-hairpin-helix (HhH) superfamily of DNA repair glycosylases, which consists of AlkA, EndoIII, MutY and hOGG1 . Unexpectedly, the three-dimensional solution structure reported here reveals TAG as member of this superfamily . The restricted specificity of TAG for 3-MeA bases probably arises from its unique aromatic rich 3-MeA binding pocket and the absence of a catalytic aspartate that is present in all other HhH family members. J Biol Chem, 2002 Oct 18, 277(42), 39228 - 34 Epub 2002 Aug 02. Sticky DNA: effect of the polypurine.polypyrimidine sequence; Vetcher AA et al.; The polypurine.polypyrimidine sequence requirements for the formation of sticky DNA were evaluated in Escherichia coli plasmid systems to determine the potential occurrence of this conformation throughout biological systems . A mirror repeat, dinucleotide tract of (GA.TC)(37), which is ubiquitous in eukaryotes, formed sticky DNA, but shorter sequences of 10 or 20 repeats were inert . (GGA.TCC)(n) inserts (where n = 126, 159, and 222 bp) also formed sticky DNA . As shown previously, the control sequence (GAA.TTC)(150) (450 bp) readily adopted the X-shaped sticky structure; however, this structure has never been found for the nonpathogenic (GAAGGA.TCCTTC)(65) of the same approximate length (390 bp) . A sequence that is replete with polypurine.polypyrimidine tracts that can form triplexes and slipped structures but lacks long repeating motifs (the 2.5-kbp intron 21 sequence from the polycystic kidney disease gene 1) was also inert . Interestingly, tracts of (GAA.TTC)(n) (where n = 176 or 80) readily formed sticky DNA with (GAAGGA.TCCTTC)(65) cloned into the same plasmid when the pair of inserts was in the direct, but not in the indirect (inverted), orientation . The stabilities of the triple base (Watson-Crick and Hoogsteen) interactions in the DNA/DNA associated triplex region of the sticky conformations account for these observations . Our results have significant chemical and biological implications for the structure and function of this unusual DNA conformation in Friedreich's ataxia. J Biol Chem, 2002 Oct 18, 277(42), 39815 - 22 Epub 2002 Aug 02. Escherichia coli DnaA protein loads a single DnaB helicase at a DnaA box hairpin; Carr KM et al.; The molecular engine that drives bidirectional replication fork movement from the Escherichia coli replication origin (oriC) is the replicative helicase, DnaB . At oriC, two and only two helicase molecules are loaded, one for each replication fork . DnaA participates in helicase loading; DnaC is also involved, because it must be in a complex with DnaB for delivery of the helicase . Since DnaA induces a local unwinding of oriC, one model is that the limited availability of single-stranded DNA at oriC restricts the number of DnaB molecules that can bind . In this report, we determined that one DnaB helicase or one DnaB-DnaC complex is bound to a single-stranded DNA in a biologically relevant DNA replication system . These results indicate that the availability of single-stranded DNA is not a limiting factor and support a model in which the site of entry for DnaB is altered so that it cannot be reused . We also show that 2-4 DnaA monomers are bound on the single-stranded DNA at a specific site that carries a DnaA box sequence in a hairpin structure. Bioorg Med Chem Lett, 2002 Sep 2, 12(17), 2281 - 5 Quantum chemical- and 3-D-QSAR (CoMFA) studies of benzalacetones and 1,1,1-trifluoro-4-phenyl-3-buten-2-ones; Yamagami C et al.; The inhibitory effect (IC(50)) of the title compounds on UV-induced mutagenesis in Escherichia coli WP2uvrA was analyzed quantitatively by using various quantum chemical descriptors and also by the CoMFA method: both approaches provided results of similar quality . The activity was shown to be increased by electron-withdrawing substituents and also by hydrogen-bonding between 2-hydroxy group and the bio-component. J Struct Biol, 2002 Apr-May, 138(1-2), 34 - 46 Three-dimensional spectral signal-to-noise ratio for a class of reconstruction algorithms; Penczek PA; A three-dimensional (3D) version of the spectral signal-to-noise ratio (SSNR)-based resolution measure is introduced . The measure is defined for a class of 3D reconstruction algorithms that use interpolation in Fourier space . The statistical properties of the SSNR are discussed and related to the properties of another resolution measure, the Fourier shell correlation (FSC) . The new measure was tested on 3D structures calculated from a simulated set of quasi-evenly spaced 2D projections using a nearest-neighbor interpolation and a gridding algorithm . In the latter case, the results agree very well with the FSC-based estimate, with the exception of very high SSNR values . The main applicability of the 3D SSNR is tomography, where due to the small number of projections collected, FSC cannot be used . The new measure was applied to three sets of tomographic data . It was demonstrated that the measure is sufficiently sensitive to yield theoretically expected results . Therefore, the 3D SSNR opens up the possibility of evaluating the quality of tomographic reconstructions in an objective manner . The 3D distribution of SSNR is of major interest in single-particle analysis . It is shown that the new measure can be used to evaluate the anisotropy of 3D reconstructions . The distribution of SSNR is characterized by three anisotropy indices derived from principal axes of the 3D inertia covariance matrix of the SSNR . These indices are used to construct a 3D Fourier filter which, when applied to a 3D reconstruction of a macromolecule, maximizes the SNR in real space and minimizes real-space artifacts caused by uneven distribution of 2D projections. Res Microbiol, 2002 Jun, 153(5), 289 - 300 Evaluation of PCR and PCR-RFLP protocols for identifying Shiga toxins; Ziebell KA et al.; This study evaluated two generic polymerase chain reaction (PCR) protocols, and nine subtyping protocols and three PCR-restriction fragment length polymorphism (RFLP) protocols for detection of stx genes . The PCR protocols were evaluated by testing 12 reference isolates and 496 field strains of Shiga toxin-producing Escherichia coli (STEC) . Both generic methods detected all stx genes . In tests with the reference isolates, all methods detected stx1 and stx2, seven subtyping methods detected stx2v(EH250), seven detected stx2e and only two detected stx2f . Four of the subtyping protocols identified stx genes in all of the field isolates . The PCR-RFLP protocols gave contradictory results for approximately 20% of the strains tested . The observed limitations of the protocols were shown to be due to nucleotide sequence variation in the region of the PCR primers . One subtyping protocol that detected the virulence-related genes, eae and ehxA, and all stx except for the stx2f gene, was modified by newly designed primers so that it identified all stx genes . This modified protocol provides comprehensive characterization of STEC in a single multiplex reaction. Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11020 - 4 Epub 2002 Aug 01. Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli; Chin JW et al.; Benzophenones are among the most useful photocrosslinking agents in biology . We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-l-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG . This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase . Irradiation resulted in efficient crosslinking (>50%) of the protein subunits . This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo. Proc Natl Acad Sci U S A, 2002 Aug 20, 99(17), 11109 - 14 Epub 2002 Aug 01. Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains; Kurek I et al.; Cellulose synthase (CesA) proteins are components of CesA complexes (rosettes) and are thought to catalyze the chain elongation step in glucan polymerization . Little is understood about rosette assembly, including how CesAs interact with each other or with other components within the complexes . The first conserved region at the N terminus of plant CesA proteins contains two putative zinc fingers that show high homology to the RING-finger motif . We show that this domain in GhCesA1 can bind two atoms of Zn2+, as predicted by its structure . Analysis in the yeast two-hybrid system indicates that the N-terminal portions of cotton fiber GhCesA1 and GhCesA2 containing these domains can interact to form homo- or heterodimers . Although Zn(2+) binding occurs only when the protein is in the reduced form, biochemical analyses show that under oxidative conditions, the GhCesA1 zinc-finger domain and also the full-length protein dimerize via intermolecular disulfide bonds, indicating CesA dimerization can be regulated by redox state . We also provide evidence that the herbicide CGA 325'615 (Syngenta, Basel), which inhibits synthesis of crystalline cellulose and leads to a disruption of rosette architecture, may affect the oxidative state of the zinc-finger domain that is necessary for rosette stability . Taken together, these results support a model in which at least part of the process of rosette assembly and function may involve oxidative dimerization between CesA subunits. Plant Cell Physiol, 2002 Jul, 43(7), 810 - 5 Spatial expression and characterization of a putative ethylene receptor protein NTHK1 in tobacco; Xie C et al.; A putative ethylene receptor gene NTHK1 encodes a protein with a putative signal peptide, three transmembrane segments, a putative histidine kinase domain and a putative receiver domain . The receiver domain was expressed in an Escherichia coli expression system, purified and used to generate polyclonal antibodies for immunohistochemistry analysis . The spatial expression of the NTHK1 protein was then investigated . We found that NTHK1 was abundant during flower and ovule development . It was also expressed in glandular hairs, stem, and in leaves that had been wounded . The NTHK1 gene was further introduced into the tobacco plant and we found that, in different transgenic lines, the NTHK1 gene was transcribed to various degrees . Upon ACC treatment, the etiolated transgenic seedlings showed reduced ethylene sensitivity when compared with the control, indicating that NTHK1 is a functional ethylene receptor in plants. J Cell Sci, 2002 Sep 1, 115(Pt 17), 3509 - 15 Leukotriene D(4) induces stress-fibre formation in intestinal epithelial cells via activation of RhoA and PKCdelta; Massoumi R et al.; The intestinal epithelial barrier, which is regulated by the actin cytoskeleton, exhibits permeability changes during inflammation . Here we show that activation of the CysLT(1) receptor by the inflammatory mediator leukotriene D(4) (LTD(4)) causes a rapid increase in stress-fibre formation in intestinal epithelial cells . This effect was mimicked by cytotoxic necrotising factor-1 (CNF-1)-induced activation of RhoA, overexpression of constitutively active RhoA (L63-RhoA) and phorbol-ester-induced activation of protein kinase C (PKC) . In accordance, inhibition of RhoA, by C3 exoenzyme or by dominant-negative RhoA (N19-RhoA), as well as GF109203X-induced inhibition of PKC, suppressed the LTD(4)-induced stress-fibre formation . Introduction of the dominant-negative regulatory domain of PKCdelta, but not the corresponding structures from PKCalpha, betaII or epsilon, blocked the LTD(4)-induced stress-fibre formation . Evaluating the relationship between PKCdelta and RhoA in LTD(4)-induced stress-fibre formation, we found that C3 exoenzyme inhibited the rapid LTD(4)-elicited translocation of PKCdelta to the plasma membrane . Furthermore, CNF-1-induced stress-fibre formation was blocked by GF109203X and by overexpression of the regulatory domain of PKC-delta, whereas PKC-induced stress-fibre production was not affected by N19-RhoA . We conclude that PKC-delta is located downstream of RhoA and that active RhoA and PKCdelta are both necessary for LTD(4)-induced stress-fibre formation. Am J Respir Crit Care Med, 2002 Aug 1, 166(3), 268 - 73 The role of CC chemokine receptor 2 in alveolar monocyte and neutrophil immigration in intact mice; Maus U et al.; The CC chemokine ligand 2 (CCL2) (JE, monocyte chemotactic protein-1 {MCP-1}) and its CC chemokine receptor 2 (CCR2) are critical regulators of monocyte/macrophage trafficking . Recently, we demonstrated that application of exogenous CCL2 in the lungs of mice induced monocyte accumulation in the airspace, whereas combined bronchoalveolar instillation of CCL2 and Escherichia coli endotoxin provoked both enhanced monocyte accumulation and extensive neutrophil influx associated with loss of pulmonary endothelial/epithelial barrier function . In this study, we investigated the role of the CCL2 receptor CCR2 in alveolar leukocyte traffic . In CCR2 knockout mice or wild-type mice treated with the anti-CCR2-blocking monoclonal antibody MC21, monocyte accumulation in response to alveolar CCL2 or CCL2 plus endotoxin was inhibited by more than 90% . Unexpectedly, alveolar neutrophil accumulation in the CCL2/lipopolysaccharide (LPS) model was also drastically reduced by both approaches of CCR2 function interference . When wild-type mice treated with anti-Gr-1 monoclonal antibody to deplete neutrophils selectively or treated with antileukinate, a CXC receptor inhibitor, were challenged with alveolar CCL2 plus LPS, alveolar monocyte accumulation was markedly decreased . Wild-type mice treated with MC21 to block CCR2 function or with anti-Gr-1 to deplete neutrophils did not exhibit the vascular leakage that typically accompanies inflammation triggered by CCL2 and LPS in wild-type mice . These findings confirm a central role for CCR2 in the process of alveolar monocyte recruitment in response to CCL2 alone and combined CCL2 plus LPS and reveal a previously unobserved interdependence between monocyte and neutrophil trafficking that has important implications for the concomitant increase in vascular permeability. J Biochem (Tokyo), 2002 Aug, 132(2), 291 - 300 Molecular cloning and characterization of a group II chaperonin delta-subunit from soybean; Nong VH et al.; Molecular characterization of plant group II chaperonin (CCT, c-cpn, or TriC) still remains elusive . By PCR-based cloning techniques using soybeans, we have made a successful attempt to clone a delta-subunit homologue of CCT (CCTdelta) . This subunit is responsible for the binding of an in vivo substrate, alpha-actin, by assisting the correct folding of the cytoskeletal protein in mouse, and the occurrence of the subunit homologue in plant CCT was unclear . As the cloning strategy, a putative amino acid segment, NH(2)-Gly-Gly-Gly-Ala-Pro-Glu-COOH, which is tightly conserved in all known animal and yeast CCTdelta subunits, was chosen for designing a degenerate primer of the PCR-cloning . The resultant 1881-bp cDNA was found to have an open-reading frame of 533 amino acids with a calculated molecular mass of 57,677 Da and to share about 58-65% identity overall at the amino acid level with the corresponding subunits known to date . Using antibodies raised against Escherichia coli-produced soybean insoluble CCTdelta as a monitoring tool, we purified soybean CCT from the extract of its immature seeds . STEM images demonstrated that the molecular shape of soybean CCT is a double eight-membered ring, which resembles the known group II chaperonins . The CCT also reactivated a denatured firefly luciferase with a significant, but limited level of the native enzymic activity in an in vitro system . Northern blot analysis showed that soybean CCTdelta gene, which is intronless and composed of a small family, was only expressed at a very early stage of seed development of soybean. J Biochem (Tokyo), 2002 Aug, 132(2), 217 - 21 Interactions of arsenic with human metallothionein-2; Toyama M et al.; Arsenic is a toxic element that is found in the atmosphere, as well as in aquatic and terrestrial environments . We have demonstrated that As(3+) binds to human metallothionein-2 (hMT-2) by UV absorption spectroscopy, inductively coupled plasma-atomic emission spectrometry (ICP-AES), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) . MALDI-TOF-MS revealed that the structure of the adduct formed by arsenic and hMT-2 (As-hMT-2) was not homogeneous . The maximum molar ratio of arsenic to hMT-2 was found to be more than 6:1 on ICP-AES, UV absorption spectroscopy and MALDI-TOF-MS . The ratio of the number of sulfhydryl groups in hMT-2 that bound arsenic was 3:1, which is the same as the ratios reported previously for arsenic-glutathione and arsenic-phytochelatin complexes. Eur J Biochem, 2002 Aug, 269(15), 3697 - 704 Kinetic studies of human tyrosyl-DNA phosphodiesterase, an enzyme in the topoisomerase I DNA repair pathway; Cheng TJ et al.; Tyrosyl-DNA phosphodiesterase (TDP) cleaves the phosphodiester bond linking the active site tyrosine residue of topoisomerase I with the 3' terminus of DNA in topoisomerase I-DNA complexes which accumulate during treatment of cancer with camptothecin . In yeast, TDP mutation confers a 1000-fold hypersensitivity to camptothecin in the presence of an additional mutation of RAD9 gene {Pouliot, J.J., Yao, K.C., Robertson, C.A . & Nash, H.A . (1999) Science 286, 552-555} . Based on the recently solved crystal structure, human TDP belongs to a distinct class within the phospholipase D superfamily in spite of very low sequence homology {Interthal, H., Pouliot, J.J . & Champoux, J.J . (2001) Proc . Natl Acad .