J Cell Biochem, 2003 Jul 1, 89(4), 674 - 87 Cell cycle-dependent transduction of cell-permeant Cre recombinase proteins; Jo D et al.; Protein transduction has been widely used to analyze biochemical processes in living cells quantitatively and under non-steady-state conditions . The present study analyzed the effects of cell cycle on the uptake and activity of cell-permeant Cre recombinase proteins . Previous studies had suggested that the efficiency of recombination and/or protein transduction varied among individual cells, even within a clonal population . We report here that cells in the G1 phase of the cell cycle undergo recombination at a lower rate than cells at other phases of the cell cycle, and that this variation results largely from differences in protein uptake, associated with differences in cell size . These results have implications regarding the mechanism of protein transduction and identify a source of heterogeneity that can influence the response of individual cells to cell-permeant proteins . J Cell Biochem, 2003 Jul 1, 89(4), 647 - 52 Monoclonal antibody to fibulin-1 generated by genetic immunization; Pupa SM et al.; Fibulin-1 (Fbln-1) is an extracellular matrix (ECM) and plasma glycoprotein . Considering the growing evidence indicating that Fbln-1 plays a role in cancer we sought to develop monospecific antibodies to better facilitate further studies of the function of Fbln-1 in breast cancer . Using a plasmid expression vector encoding full-length human Fbln-1D as an immunogen and CpG oligodeoxyribonucleotides as adjuvant a monoclonal antibody (MAb) against Fbln-1 was produced . This MAb, designated MEM-2 was of IgM isotype and reacted with bacterially expressed Fbln-1 . Furthermore, MEM-2 reacted with Fbln-1 expressed in the ECM released by cultured human breast carcinoma SKBR-3 cells in ELISA, and also with Fbln-1 present in SKBR-3 cell extract in immunoprecipitation and Western blotting . MEM-2 also reacted with Fbln-1 in human breast carcinoma specimens . These findings illustrate the utility of genetic immunization as a means of generating monoclonal antibodies to tumor-related ECM proteins . MEM-2 represents a useful new tool for the study of Fbln-1 in breast cancer . Nat Biotechnol, 2003 Aug, 21(8), 927 - 31 Epub 2003 Jul 13. Metabolic labeling of C . elegans and D . melanogaster for quantitative proteomics; Krijgsveld J et al.; A crucial issue in comparative proteomics is the accurate quantification of differences in protein expression levels . To achieve this, several methods have been developed in which proteins are labeled with stable isotopes either in vivo via metabolic labeling or in vitro by protein derivatization . Although metabolic labeling is the only way to obtain labeling of all proteins, it has thus far only been applied to single- celled organisms and cells in culture . Here we describe quantitative 15N metabolic labeling of the multicellular organisms Caenorhabditis elegans, a nematode, and Drosophila melanogaster, the common fruit fly, achieved by feeding them on 15N-labeled Escherichia coli and yeast, respectively . The relative abundance of individual proteins obtained from different samples can then be determined by mass spectrometry (MS) . The applicability of the method is exemplified by the comparison of protein expression levels in two C . elegans strains, one with and one without a germ line . The methodology described provides tools for accurate quantitative proteomic studies in these model organisms. Proc Natl Acad Sci U S A, 2003 Jul 22, 100(15), 8654 - 9 Epub 2003 Jul 11. Chirality sensing by Escherichia coli topoisomerase IV and the mechanism of type II topoisomerases; Stone MD et al.; Escherichia coli topoisomerase (Topo) IV is an essential type II Topo that removes DNA entanglements created during DNA replication . Topo IV relaxes (+) supercoils much faster than (-) supercoils, promoting replication while sparing the essential (-) supercoils . Here, we investigate the mechanism underlying this chiral preference . Using DNA binding assays and a single-molecule DNA braiding system, we show that Topo IV recognizes the chiral crossings imposed by the left-handed superhelix of a (+) supercoiled DNA, rather than global topology, twist deformation, or local writhe . Monte Carlo simulations of braid, supercoil, and catenane configurations demonstrate how a preference for a single-crossing geometry during strand passage can allow Topo IV to perform its physiological functions . Single-enzyme braid relaxation experiments also provide a direct measure of the processivity of the enzyme and offer insight into its mechanochemical cycle. J Virol, 2003 Aug, 77(15), 8216 - 26 Human immunodeficiency virus (HIV) type 1 transframe protein can restore activity to a dimerization-deficient HIV protease variant; Dautin N et al.; The protease (PR) from human immunodeficiency virus (HIV) is essential for viral replication: this aspartyl protease, active only as a dimer, is responsible for cleavage of the viral polyprotein precursors (Gag and Gag-Pol), to release the functional mature proteins . In this work, we have studied the structure-function relationships of the HIV PR by combining a genetic test to detect proteolytic activity in Escherichia coli and a bacterial two-hybrid assay to analyze PR dimerization . We showed that a drug-resistant PR variant isolated from a patient receiving highly active antiretroviral therapy is impaired in its dimerization capability and, as a consequence, is proteolytically inactive . We further showed that the polypeptide regions adjacent to the PR coding sequence in the Gag-Pol polyprotein precursor, and in particular, the transframe polypeptide (TF), located at the N terminus of PR, can facilitate the dimerization of this variant PR and restore its enzymatic activity . We propose that the TF protein could help to compensate for folding and/or dimerization defects in PR arising from certain mutations within the PR coding sequence and might therefore function to buffer genetic variations in PR. Plant Physiol, 2003 Jul, 132(3), 1228 - 40 Cytokinin oxidase gene expression in maize is localized to the vasculature, and is induced by cytokinins, abscisic acid, and abiotic stress; Brugiere N et al.; Cytokinins are hormones that play an essential role in plant growth and development . The irreversible degradation of cytokinins, catalyzed by cytokinin oxidase, is an important mechanism by which plants modulate their cytokinin levels . Cytokinin oxidase has been well characterized biochemically, but its regulation at the molecular level is not well understood . We isolated a cytokinin oxidase open reading frame from maize (Zea mays), called Ckx1, and we used it as a probe in northern and in situ hybridization experiments . We found that the gene is expressed in a developmental manner in the kernel, which correlates with cytokinin levels and cytokinin oxidase activity . In situ hybridization with Ckx1 and transgenic expression of a transcriptional fusion of the Ckx1 promoter to the Escherichia coli beta-glucuronidase reporter gene revealed that the gene is expressed in the vascular bundles of kernels, seedling roots, and coleoptiles . We show that Ckx1 gene expression is inducible in various organs by synthetic and natural cytokinins . Ckx1 is also induced by abscisic acid, which may control cytokinin oxidase expression in the kernel under abiotic stress . We hypothesize that under non-stress conditions, cytokinin oxidase in maize plays a role in controlling growth and development via regulation of cytokinin levels transiting in the xylem . In addition, we suggest that under environmental stress conditions, cytokinin oxidase gene induction by abscisic acid results in aberrant degradation of cytokinins therefore impairing normal development. J Biol Chem, 2003 Sep 19, 278(38), 36959 - 65 Epub 2003 Jul 11. CCAAT/enhancer-binding protein family members recruit the coactivator CREB-binding protein and trigger its phosphorylation; Kovacs KA et al.; CCAAT/enhancer-binding protein (C/EBP) family members are transcription factors involved in important physiological processes, such as cellular proliferation and differentiation, regulation of energy homeostasis, inflammation, and hematopoiesis . Transcriptional activation by C/EBPalpha and C/EBPbeta involves the coactivators CREB-binding protein (CBP) and p300, which promote transcription by acetylating histones and recruiting basal transcription factors . In this study, we show that C/EBPdelta is also using CBP as a coactivator . Based on sequence homology with C/EBPalpha and -beta, we identify in C/EBPdelta two conserved amino acid segments that are necessary for the physical interaction with CBP . Using reporter gene assays, we demonstrate that mutation of these residues prevents CBP recruitment and diminishes the transactivating potential of C/EBPdelta . In addition, our results indicate that C/EBP family members not only recruit CBP but specifically induce its phosphorylation . We provide evidence that CBP phosphorylation depends on its interaction with C/EBPdelta and define point mutations within one of the two conserved amino acid segments of C/EBPdelta that abolish CBP phosphorylation as well as transcriptional activation, suggesting that this new mechanism could be important for C/EBP-mediated transcription. J Biol Chem, 2003 Sep 26, 278(39), 37965 - 73 Epub 2003 Jul 10. An oxidized purine nucleoside triphosphatase, MTH1, suppresses cell death caused by oxidative stress; Yoshimura D et al.; MTH1 hydrolyzes oxidized purine nucleoside triphosphates such as 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) and 2-hydroxy-2'-deoxyadenosine 5'-triphosphate (2-OH-dATP) and thus protects cells from damage caused by their misincorporation into DNA . In the present study, we established MTH1-null mouse embryo fibroblasts that were highly susceptible to cell dysfunction and death caused by exposure to H2O2, with morphological features of pyknosis and electron-dense deposits accumulated in mitochondria . The cell death observed was independent of both poly(ADP-ribose) polymerase and caspases . A high performance liquid chromatography tandem mass spectrometry analysis and immunofluorescence microscopy revealed a continuous accumulation of 8-oxo-guanine both in nuclear and mitochondrial DNA after exposure to H2O2 . All of the H2O2-induced alterations observed in MTH1-null mouse embryo fibroblasts were effectively suppressed by the expression of wild type human MTH1 (hMTH1), whereas they were only partially suppressed by the expression of mutant hMTH1 defective in either 8-oxo-dGTPase or 2-OH-dATPase activity . Human MTH1 thus protects cells from H2O2-induced cell dysfunction and death by hydrolyzing oxidized purine nucleotides including 8-oxo-dGTP and 2-OH-dATP, and these alterations may be partly attributed to a mitochondrial dysfunction. J Biol Chem, 2003 Sep 19, 278(38), 36242 - 9 Epub 2003 Jul 10. Modulation of the 3'-->5'-exonuclease activity of human apurinic endonuclease (Ape1) by its 5'-incised Abasic DNA product; Wong D et al.; The major abasic endonuclease of human cells, Ape1 protein, is a multifunctional enzyme with critical roles in base excision repair (BER) of DNA . In addition to its primary activity as an apurinic/apyrimidinic endonuclease in BER, Ape1 also possesses 3'-phosphodiesterase, 3'-phosphatase, and 3'-->5'-exonuclease functions specific for the 3' termini of internal nicks and gaps in DNA . The exonuclease activity is enhanced at 3' mismatches, which suggests a possible role in BER for Ape1 as a proofreading activity for the relatively inaccurate DNA polymerase beta . To elucidate this role more precisely, we investigated the ability of Ape1 to degrade DNA substrates that mimic BER intermediates . We found that the Ape1 exonuclease is active at both mismatched and correctly matched 3' termini, with preference for mismatches . In our hands, the exonuclease activity of Ape1 was more active at one-nucleotide gaps than at nicks in DNA, even though the latter should represent the product of repair synthesis by polymerase beta . However, the exonuclease activity was inhibited by the presence of nearby 5'-incised abasic residues, which result from the apurinic/apyrimidinic endonuclease activity of Ape1 . The same was true for the recently described exonuclease activity of Escherichia coli endonuclease IV . Exonuclease III, the E . coli homolog of Ape1, did not discriminate among the different substrates . Removal of the 5' abasic residue by polymerase beta alleviated the inhibition of the Ape1 exonuclease activity . These results suggest roles for the Ape1 exonuclease during BER after both DNA repair synthesis and excision of the abasic deoxyribose-5-phosphate by polymerase beta. Exp Parasitol, 2002 Nov-Dec, 102(3-4), 194 - 200 Paragonimus westermani: molecular cloning, expression, and characterization of a recombinant yolk ferritin; Kim TY et al.; Ferritin is an intracellular protein involved in iron metabolism . A cDNA PwYF-1 cloned from the adult Paragonimus westermani cDNA library encoded a putative polypeptide of 216 amino acids homologous with ferritins of vertebrates and invertebrates . Febinding motifs identified in PwYF-1 polypeptide were conserved and predicted to form a ferroxidase center . PwYF-1 polypeptide contained an extended peptide of 45 amino acids at its C-terminus . Recombinant PwYF-1 protein, expressed and purified from Escherichia coli, showed iron-uptake ability and ferroxidase activity . Ferroxidase activity of recombinant PwYF-1 protein was reactivated by secondary addition of apotransferrin to assay mixture . Mouse immune serum raised against the recombinant PwYF-1 protein recognized specifically 24 kDa protein from adult P . westermani lysate . PwYF-1 protein was localized to vitelline follicles and the eggs of P . westermani . Collectively, PwYF-1 protein was identified as a P . westermani yolk ferritin. Exp Parasitol, 2002 Nov-Dec, 102(3-4), 178 - 86 A potentially important excretory-secretory product of Giardia lamblia; Shant J et al.; In this study we have reported the detailed characterization of a 58 kDa excretory-secretory product (ESP) of Giardia lamblia . The method of purification has been simplified which has improved the purification fold as well as the yield of the ESP . The binding efficacy of disialoganglioside (GD2) to the purified ESP was found to be maximum among all other gangliosides used . The N-terminal sequence of the immunoreactive 29 kDa peptide obtained from partial tryptic digest of the ESP was found to be AD-FVPQVST . The IgG against the purified ESP (IgGES) showed cross-reactivity with the binding subunit of the commercially available cholera toxin and also with two protein bands of western cottonmouth moccasin snake toxin . The ESP could accumulate fluid in the intestine of sealed adult mice and also induce morphological changes in HEp-2 cells . The crude extract of G . lamblia trophozoites preincubated with Escherichia coli revealed 8-fold augmentation in the cytopathic activity on HEp-2 cells as compared to that of crude preparation from trophozoites only. Science, 2003 Jul 11, 301(5630), 213 - 5 A B cell-based sensor for rapid identification of pathogens; Rider TH et al.; We report the use of genetically engineered cells in a pathogen identification sensor . This sensor uses B lymphocytes that have been engineered to emit light within seconds of exposure to specific bacteria and viruses . We demonstrated rapid screening of relevant samples and identification of a variety of pathogens at very low levels . Because of its speed, sensitivity, and specificity, this pathogen identification technology could prove useful for medical diagnostics, biowarfare defense, food- and water-quality monitoring, and other applications. Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9256 - 61 Epub 2003 Jul 10. The 1.1-A resolution crystal structure of DJ-1, the protein mutated in autosomal recessive early onset Parkinson's disease; Wilson MA et al.; Mutations in DJ-1, a human gene with homologues in organisms from all kingdoms of life, have been shown to be associated with autosomal recessive, early onset Parkinson's disease (PARK7) . We report here the three-dimensional structure of the DJ-1 protein, determined at a resolution of 1.1 A by x-ray crystallography . The chain fold of DJ-1 resembles those of a bacterial protein, PfpI, that has been annotated as a cysteine protease, and of a domain of a bacterial catalase whose role in the activity of that enzyme is uncertain . In contrast to PfpI, a hexameric protein whose oligomeric structure is essential for its putative proteolytic activity, DJ-1 is a dimer with completely different intersubunit contacts . The proposed catalytic triad of PfpI is absent from the corresponding region of the structure of DJ-1, and biochemical assays fail to detect any protease activity for purified DJ-1 . A highly conserved cysteine residue, which is catalytically essential in homologues of DJ-1, shows an extreme sensitivity to radiation damage and may be subject to other forms of oxidative modification as well . The structure suggests that the loss of function caused by the Parkinson's-associated mutation L166P in DJ-1 is due to destabilization of the dimer interface . Taken together, the crystal structure of human DJ-1 plus other observations suggest the possible involvement of this protein in the cellular oxidative stress response and a general etiology of neurodegenerative diseases. Microbiology, 2003 Jul, 149(Pt 7), 1871 - 82 The Escherichia coli AIDA autotransporter adhesin recognizes an integral membrane glycoprotein as receptor; Laarmann S et al.; The AIDA-I autotransporter adhesin, as a prototype of the AIDA adhesin family, represents a tripartite antigen consisting of the functional adhesin AIDA-I (alpha-domain), which mediates the specific attachment of bacteria to target cells, and a two-domain translocator (AIDA(c)) organized in the beta(1)- and beta(2)-domains . Cellular receptor moieties for the adhesin AIDA-I have not been identified . Here, it is demonstrated that the purified adhesin binds specifically to a high-affinity class of receptors on HeLa cells . Additionally, the adhesin was found to bind to a variety of mammalian cell types, indicating a broad tissue distribution of the receptor moiety . By using complementary techniques, including co-immunoprecipitation and one- and two-dimensional gel electrophoresis, the AIDA-I binding protein on HeLa cells was identified as a surface glycoprotein of about 119 kDa (gp119) . The gp119 AIDA-I cellular receptor protein was characterized biochemically and found to be an integral N-glycosylated membrane protein with a pI of 5.2. Microbiology, 2003 Jul, 149(Pt 7), 1797 - 805 Identification of genes in the tomato big bud phytoplasma and comparison to those in sweet potato little leaf-V4 phytoplasma; Streten C et al.; Genetic relatedness of phytoplasmas is commonly defined on the basis of differences in the highly conserved 16S rRNA gene, which may not resolve closely related phytoplasmas . An example of this is the closely related tomato big bud (TBB) and sweet potato little leaf strain V4 (SPLL-V4) phytoplasmas, which cannot easily be differentiated by their 16S rRNA gene sequences . This study aimed to identify genes on the TBB phytoplasma chromosome which could be used to examine genetic variation between these two closely related phytoplasmas . Random clones generated from TBB phytoplasma genomic DNA were sequenced and characterized by database analysis . Twenty-three genes were identified within 19 random clones, which contained approximately 18.0 kbp of TBB phytoplasma genomic DNA . Half of the TBB phytoplasma genes identified were involved in DNA replication, transcription and translation . The remaining TBB phytoplasma genes were involved in protein secretion, cellular processes and energy metabolism . Phylogenetic analysis of representative genes showed that the TBB phytoplasma grouped with the mycoplasmas with the exception of the TBB phytoplasma secA gene, which grouped with the onion yellows phytoplasma . PCR primers were designed based on the new genes and tested on isolates of the TBB and SPLL-V4 phytoplasmas . Most primers amplified a product from TBB and SPLL-V4 phytoplasma samples . When amplified products were subjected to RFLP analysis, the restriction patterns were the same as the respective original clones . This result confirmed that the same sequence had been amplified by PCR and showed that these isolates were indistinguishable using the new genes . This study showed that in fact the TBB and SPLL-V4 phytoplasmas are closely related even with the analysis of new genes . These new genes have, however, provided insight into the biology of the TBB and SPLL-V4 phytoplasmas. J Biol Chem, 2003 Sep 19, 278(38), 36953 - 8 Epub 2003 Jul 10. Heat shock protein 90 as an endogenous protein enhancer of inducible nitric-oxide synthase; Yoshida M et al.; Nitric oxide (NO) generated by inducible NO synthase (iNOS) plays crucial roles in inflammation and host defense . With an intrinsically bound calmodulin, iNOS is fully active once expressed in cells . Thus, regulation of NO production from iNOS was thought to primarily occur at the enzyme transcriptional level . Here we show that NO synthesis from iNOS can be profoundly modulated by heat shock protein 90 (hsp90) through protein-protein interaction . To study whether hsp90 affects iNOS function, recombinant murine iNOS was purified from an Escherichia coli expression system by affinity chromatography . Hsp90, at physiological concentrations (10-500 nm), dose-dependently increased iNOS activity . This was a specific effect because neither denatured hsp90 nor irrelevant bovine serum albumin affected iNOS function . Overexpression of hsp90 enhanced NO production in iNOS-transfected cells . On the contrary, hsp90 inhibition dramatically decreased NO formation from iNOS in macrophages . Co-immunoprecipitation studies showed that hsp90 and iNOS associated with each other in cells . Overexpression of iNOS resulted in NO-mediated cellular injury . Hsp90 inhibition markedly attenuated NO formation and prevented cellular injury . These results demonstrated that hsp90 is an allosteric enhancer of iNOS . iNOS is coupled with hsp90 in cells, and this coupling facilitates NO synthesis . In light of the critical role of hsp90 in iNOS-mediated cytotoxic action, modulating the interaction between hsp90 and iNOS may be a new approach to intervene inflammation and immune response. Blood, 2003 Oct 15, 102(8), 2856 - 61 Epub 2003 Jul 10. The murine platelet and plasma factor V pools are biosynthetically distinct and sufficient for minimal hemostasis; Sun H et al.; Coagulation factor V (FV) is a central regulator of the coagulation cascade . Circulating FV is found in plasma and within platelet alpha granules . The specific functions of these distinct FV pools are uncertain . We now report the generation of transgenic mice with FV gene expression restricted to either the liver or megakaryocyte/platelet lineage using bacterial artificial chromosome (BAC) constructs . Six of 6 independent albumin BAC transgenes rescue the neonatal lethal hemorrhage of FV deficiency . Rescued mice all exhibit liver-specific Fv expression at levels ranging from 6% to 46% of the endogenous Fv gene, with no detectable FV activity within the platelet pool . One of the 3 Pf4 BAC transgenes available for analysis also rescues the lethal FV null phenotype, with FV activity restricted to only the platelet pool (approximately 3% of the wild-type FV level) . FV-null mice rescued by either the albumin or Pf4 BAC exhibit nearly normal tail bleeding times . These results demonstrate that Fv expression in either the platelet or plasma FV pool is sufficient for basal hemostasis . In addition, these findings indicate that the murine platelet and plasma FV pools are biosynthetically distinct, in contrast to a previous report demonstrating a plasma origin for platelet FV in humans. Bioinformatics, 2003, 19 Suppl 1, i323 - 30 Extraction of correlated gene clusters from multiple genomic data by generalized kernel canonical correlation analysis; Yamanishi Y et al.; MOTIVATION: A major issue in computational biology is the reconstruction of pathways from several genomic datasets, such as expression data, protein interaction data and phylogenetic profiles . As a first step toward this goal, it is important to investigate the amount of correlation which exists between these data . RESULTS: These methods are successfully tested on their ability to recognize operons in the Escherichia coli genome, from the comparison of three datasets corresponding to functional relationships between genes in metabolic pathways, geometrical relationships along the chromosome, and co-expression relationships as observed by gene expression data. FEMS Microbiol Lett, 2003 Jul 15, 224(1), 113 - 8 Purification and characterization of KpsT, the ATP-binding component of the ABC-capsule exporter of Escherichia coli K1; Nsahlai CJ et al.; The K1 capsule, an alpha(2,8)-linked polymer of sialic acid, is an important virulence determinant of invasive Escherichia coli . The 17-kb kps gene cluster of E . coli K1 encodes the information necessary for capsule expression at the cell surface . Two proteins, KpsM and KpsT, play a role in the transport of capsular polysaccharide across the cytoplasmic membrane, utilizing the energy from ATP hydrolysis . They belong to the ATP-binding cassette superfamily of transport proteins . In this study, we purified KpsT in its native form and show that the purified protein is able to bind ATP, undergo an ATP-dependent conformational change and hydrolyze ATP . Protease accessibility studies demonstrate the in vivo interaction between KpsM and KpsT. FEMS Microbiol Lett, 2003 Jul 15, 224(1), 91 - 5 Francisella strains express hemolysins of distinct characteristics; Lai XH et al.; Historically, Francisella strains have been described as nonhemolytic . In this study, we show by use of solid and liquid hemolysis assays that some Francisella strains have hemolytic properties . The Francisella novicida type strain U112 is hemolytic to horse erythrocytes and Francisella philomiragia type strain FSC144 is hemolytic towards both human and horse erythrocytes . The F . novicida strain U112 released a protein (novilysin A) into the culture supernatant which cross-reacted with antiserum against Escherichia coli HlyA whereas there was no similar protein detectable with this cross-reactive property from the supernatant of the F . philomiragia strain. FEMS Microbiol Lett, 2003 Jul 15, 224(1), 1 - 15 F factor conjugation is a true type IV secretion system; Lawley TD et al.; The F sex factor of Escherichia coli is a paradigm for bacterial conjugation and its transfer (tra) region represents a subset of the type IV secretion system (T4SS) family . The F tra region encodes eight of the 10 highly conserved (core) gene products of T4SS including TraAF (pilin), the TraBF, -KF (secretin-like), -VF (lipoprotein) and TraCF (NTPase), -EF, -LF and TraGF (N-terminal region) which correspond to TrbCP, -IP, -GP, -HP, -EP, -JP, DP and TrbLP, respectively, of the P-type T4SS exemplified by the IncP plasmid RP4 . F lacks homologs of TrbBP (NTPase) and TrbFP but contains a cluster of genes encoding proteins essential for F conjugation (TraFF, -HF, -UF, -WF, the C-terminal region of TraGF, and TrbCF) that are hallmarks of F-like T4SS . These extra genes have been implicated in phenotypes that are characteristic of F-like systems including pilus retraction and mating pair stabilization . F-like T4SS systems have been found on many conjugative plasmids and in genetic islands on bacterial chromosomes . Although few systems have been studied in detail, F-like T4SS appear to be involved in the transfer of DNA only whereas P- and I-type systems appear to transport protein or nucleoprotein complexes . This review examines the similarities and differences among the T4SS, especially F- and P-like systems, and summarizes the properties of the F transfer region gene products. Birth Defects Res A Clin Mol Teratol, 2003 Apr, 67(4), 240 - 5 Zinc treatment prevents lipopolysaccharide-induced teratogenicity in mice; Carey LC et al.; BACKGROUND: During pregnancy, exposure to lipopolysaccharide (LPS) can lead to abortion, preterm delivery, and teratogenicity . The mechanisms underlying these effects are unclear . Both LPS and ethanol are potent inducers of liver metallothionein (MT), a key Zn binding protein . The teratogenic effects of ethanol have been linked to MT-induced changes in maternal-fetal Zn homeostasis, leading tofetal deficiency . This study was designed to assess whether the teratogenic effects of LPS are also related to MT induction and changes in Zn homeostasis . METHODS: Non-pregnant normal (MT +/+) and MT-null (MT -/-) mice were injected subcutaneously with 0.5 microg/gm LPS and killed over 48 hr . In MT +/+ mice, liver MT concentrations were elevated from 6 hr, and were maximal at 24 hr (30-fold basal), whereas liver Zn levels were also increased from 6 hr . Plasma Zn concentrations decreased by 80% at 6 hr, and were below normal between 6 and 24 hr . In MT -/- mice, plasma Zn levels were increased from basal between 6 and 16 hr . Dams were injected with LPS, saline, or LPS and ZnSO4 (2 microg/gm, MT +/+ only) on Day 8 of gestation (GDS), killed on GD18, and the fetuses examined for malformations . RESULTS: External abnormalities were most prevalent in fetuses from MT +/+ dams exposed to LPS, where 34% of fetuses in each litterwere affected . MT +/+ dams treated with LPS and ZnSO4, and MT -/- dams treated with LPS had litters in which 5.4 and 4.8% of fetuses were abnormal respectively . CONCLUSIONS: The findings of this study strongly support the hypothesis that LPS teratogenicity is mediated at least in part by MT-induced changes in maternal Zn homeostasis,which compromises fetal Zn supply. Masui, 2003 Jun, 52(6), 656 - 61 {Evaluation of the animal model for endotoxin-induced shock}; Taniguchi T et al.; BACKGROUND: We evaluated our animal model for endotoxin-induced shock and compared its characteristics with those of clinical endotoxic shock . METHODS AND RESULTS: <Model 1> Male Japanese rabbits anesthetized with urethane and ventilated mechanically were assigned to one of two groups: the endotoxin group, receiving intravenous E . coli endotoxin (0.5 mg.kg-1) via the mesenteric vein; control group, receiving 0.9% saline . After the endotoxin injection, the mean arterial pressure, cardiac output, and systemic vascular resistance decreased progressively . Four hours after the endotoxin injection, the levels of both interleukin (IL)-6 and IL-8 in the endotoxin group were higher than those in the control group . <Model 2> Male Wistar rats anesthetized with pentobarbital and ventilated mechanically were assigned to one of two groups: endotoxin group, receiving intravenous E . coli endotoxin (15 mg.kg-1); and control group, receiving 0.9% saline . After the endotoxin injection, the systolic arterial pressure and heart rate decreased progressively . The plasma tumor necrosis factor alpha and IL-6 increased in the endotoxin group . CONCLUSIONS: We could not observe the hyperdynamic state and multiple organ dysfunctions and did not administer vasopressors and antibiotics in our endotoxin shock models, compared with the characteristics of clinical endotoxin shock . Further refinement of our animal models for endotoxin shock is challenging. World J Gastroenterol, 2003 Jul, 9(7), 1521 - 4 Aggregate formation of hepatitis B virus X protein affects cell cycle and apoptosis; Song CZ et al.; AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses . METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography . Anti-HBx monoclonal antibody was developed for immunocytochemical detection . Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 cells . Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells . The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry . RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules . Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours . Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time . EGFP fluorescence in HBx expression cells was significantly decreased . CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase . Our data may provide helpful information to understand the biological effects of HBx aggregates on cells. World J Gastroenterol, 2003 Jul, 9(7), 1455 - 9 Preparation and characterization of polyclonal antibodies against ARL-1 protein; Jin JF et al.; AIM: To prepare and characterize polyclonal antibodies against aldose reductase-like (ARL-1) protein . METHODS: ARL-1 gene was inserted into the E . coli expression vector pGEX-4T-1(His)(6)C and vector pQE-30 . Recombinant ARL-1 proteins named ARL-(His)(6) and ARL-GST were expressed . They were purified by affinity chromatography . Sera from domestic rabbits immunized with ARL-(His) (6) were purified by CNBr-activated sepharose 4B coupled ARL-GST . Polyclonal antibodies were detected by Western blotting . RESULTS: Recombinant proteins of ARL-(His)(6) with molecular weight of 35.7 KD and ARL-GST with molecular weight of 60.8 KD were highly expressed . The expression levels of ARL-GST and ARL-(His)(6) were 15.1 % and 27.7 % among total bacteria proteins, respectively . They were soluble, predominantly in supernatant . After purification by non-denatured way, SDS-PAGE showed one band . In the course of polyclonal antibodies purification, only one elution peak could be seen . Western blotting showed positive signals in the two purified proteins and the bacteria transformed with pGEX-4T-1(His) (6) C-ARL and pQE-30-ARL individually . CONCLUSION: Polyclonal antibodies are purified and highly specific against ARL-1 protein . ARL-GST and ARL-(His) (6) are highly expressed and purified. J Infect Dis, 2003 Jul 15, 188(2), 286 - 9 Epub 2003 Jul 01. Murein lipoprotein, peptidoglycan-associated lipoprotein, and outer membrane protein A are present in purified rough and smooth lipopolysaccharides; Hellman J et al.; Purified lipopolysaccharides (LPSs) have been used for many decades to gain insight into processes that occur during sepsis . Previous studies indicate that purified LPSs often contain trace protein contaminants . To identify protein contaminants of LPSs, we performed immunoblotting using, as antigen, purified LPS from various species of bacteria and, as primary antibodies, anti-murein lipoprotein (MLP), peptidoglycan-associated lipoprotein (PAL), and outer membrane protein A (OmpA) . MLP, PAL, and/or OmpA were detected in 10 of the 13 LPS preparations and were present in LPS from rough and smooth bacteria . PAL and MLP have been reported to stimulate inflammation . The studies indicate that PAL and MLP are common contaminants of purified LPS and raise the possibility that these contaminants may influence results of studies performed using purified LPS. Nucleic Acids Res, 2003 Jul 15, 31(14), 4017 - 23 Hfq affects the length and the frequency of short oligo(A) tails at the 3' end of Escherichia coli rpsO mRNAs; Le Derout J et al.; Polyadenylation plays an important role in RNA degradation in bacterial cells . In Escherichia coli, exoribonucleases, mostly RNase II and polynucleotide phosphorylase, antagonize the synthesis of poly(A) tails by poly(A) polymerase I (PAP I) . In accordance with earlier observations showing that only a small fraction of bacterial RNA is polyadenylated, we demonstrate here that approximately 10% of rpsO mRNA harbors short oligo(A) tails ranging from one to five A residues in wild-type cells . We also compared the length, frequency and distribution of poly(A) tails at the 3'-end of rpsO transcripts in vivo in the presence and absence of Hfq, a host factor that in vitro stimulates the activity of PAP I, and found that Hfq affects all three parameters . In the hfq(+) strain the average length of oligo(A) tails and frequency of polyadenylated transcripts was higher than in the hfq(-) strain and a smaller proportion of tails was found at the 3' end of transcripts terminated at the Rho- independent terminator . Our data led us to the conclusion that Hfq is involved in the recognition of 3' RNA extremities by PAP I. Nucleic Acids Res, 2003 Jul 15, 31(14), 3971 - 81 Histone deacetylases in fungi: novel members, new facts; Trojer P et al.; Acetylation is the most prominent modification on core histones that strongly affects nuclear processes such as DNA replication, DNA repair and transcription . Enzymes responsible for the dynamic equilibrium of histone acetylation are histone acetyltransferases (HATs) and histone deacetylases (HDACs) . In this paper we describe the identification of novel HDACs from the filamentous fungi Aspergillus nidulans and the maize pathogen Cochliobolus carbonum . Two of the enzymes are homologs of Saccharomyces cerevisiae HOS3, an enzyme that has not been identified outside of the established yeast systems until now . One of these homologs, HosB, showed intrinsic HDAC activity and remarkable resistance against HDAC inhibitors like trichostatin A (TSA) when recombinant expressed in an Escherichia coli host system . Phylo genetic analysis revealed that HosB, together with other fungal HOS3 orthologs, is a member of a separate group within the classical HDACs . Immunological investigations with partially purified HDAC activities of Aspergillus showed that all classical enzymes are part of high molecular weight complexes and that a TSA sensitive class 2 HDAC constitutes the major part of total HDAC activity of the fungus . However, further biochemical analysis also revealed an NAD(+)-dependent activity that could be separated from the other activities by different types of chromatography and obviously represents an enzyme of the sirtuin class. Nucleic Acids Res, 2003 Jul 15, 31(14), 3946 - 53 Importance of the reverse Hoogsteen base pair 54-58 for tRNA function; Zagryadskaya EI et al.; To elucidate the general constraints imposed on the structure of the D- and T-loops in functional tRNAs, active suppressor tRNAs were selected in vivo from a combinatorial tRNA gene library in which several nucleotide positions of these loops were randomized . Analysis of the nucleotide sequences of the selected clones demonstrates that among the randomized nucleotides, the most conservative are nucleotides 54 and 58 in the T-loop . In most cases, they make the combination U54-A58, which allows the formation of the normal reverse Hoogsteen base pair . Surprisingly, other clones have either the combination G54-A58 or G54-G58 . However, molecular modeling shows that these purine-purine base pairs can very closely mimic the reverse Hoogsteen base pair U-A and thus can replace it in the T-loop of a functional tRNA . This places the reverse Hoogsteen base pair 54-58 as one of the most important structural aspects of tRNA functionality . We suggest that the major role of this base pair is to preserve the conformation of dinucleotide 59-60 and, through this, to maintain the general architecture of the tRNA L-form. Nucleic Acids Res, 2003 Jul 15, 31(14), 3893 - 900 Incision at hypoxanthine residues in DNA by a mammalian homologue of the Escherichia coli antimutator enzyme endonuclease V; Moe A et al.; Deamination of DNA bases can occur spontaneously, generating highly mutagenic lesions such as uracil and hypoxanthine . In Escherichia coli two enzymes initiate repair at hypoxanthine residues in DNA . The alkylbase DNA glycosylase, AlkA, initiates repair by removal of the damaged base, whereas endonuclease V, Endo V, hydrolyses the second phosphodiester bond 3' to the lesion . We have identified and characterised a mouse cDNA with striking homology to the E.coli nfi gene, which also has significant similarities to motifs required for catalytic activity of the UvrC endonuclease . The 37-kDa mouse enzyme (mEndo V) incises the DNA strand at the second phosphodiester bond 3' to hypoxanthine- and uracil-containing nucleotides . The activity of mEndo V is elevated on single-stranded DNA substrate in vitro . Expression of the mouse protein in a DNA repair-deficient E.coli alkA nfi strain suppresses its spontaneous mutator phenotype . We suggest that mEndo V initiates an alternative excision repair pathway for hypoxanthine removal . It thus appears that mEndo V has properties overlapping the function of alkylbase DNA glycosylase (Aag) in repair of deaminated adenine, which to some extent could explain the absence of phenotypic abnormalities associated with Aag knockout in mice. Proc Natl Acad Sci U S A, 2003 Jul 22, 100(15), 8682 - 7 Epub 2003 Jul 09. X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli; Vila-Sanjurjo A et al.; Protein biosynthesis on the ribosome requires accurate reading of the genetic code in mRNA . Two conformational rearrangements in the small ribosomal subunit, a closing of the head and body around the incoming tRNA and an RNA helical switch near the mRNA decoding site, have been proposed to select for complementary base-pairing between mRNA codons and tRNA anticodons . We determined x-ray crystal structures of the WT and a hyper-accurate variant of the Escherichia coli ribosome at resolutions of 10 and 9 A, respectively, revealing that formation of the intact 70S ribosome from its two subunits closes the conformation of the head of the small subunit independent of mRNA decoding . Moreover, no change in the conformation of the switch helix is observed in two steps of tRNA discrimination . These 70S ribosome structures indicate that mRNA decoding is coupled primarily to movement of the small subunit body, consistent with previous proposals, whereas closing of the head and the helical switch may function in other steps of protein synthesis. EMBO J, 2003 Jul 15, 22(14), 3503 - 13 Mechanism of the electron transfer catalyst DsbB from Escherichia coli; Grauschopf U et al.; The membrane protein DsbB from Escherichia coli is essential for disulfide bond formation and catalyses the oxidation of the periplasmic dithiol oxidase DsbA by ubiquinone . DsbB contains two catalytic disulfide bonds, Cys41-Cys44 and Cys104-Cys130 . We show that DsbB directly oxidizes one molar equivalent of DsbA in the absence of ubiquinone via disulfide exchange with the 104-130 disulfide bond, with a rate constant of 2.7 x 10 M(-1) x s(-1) . This reaction occurs although the 104-130 disulfide is less oxidizing than the catalytic disulfide bond of DsbA (E(o)' = -186 and -122 mV, respectively) . This is because the 41-44 disulfide, which is only accessible to ubiquinone but not to DsbA, is the most oxidizing disulfide bond in a protein described so far, with a redox potential of -69 mV . Rapid intramolecular disulfide exchange in partially reduced DsbB converts the enzyme into a state in which Cys41 and Cys44 are reduced and thus accessible for reoxidation by ubiquinone . This demonstrates that the high catalytic efficiency of DsbB results from the extreme intrinsic oxidative force of the enzyme. Clin Diagn Lab Immunol, 2003 Jul, 10(4), 680 - 5 Production and characterization of a human recombinant monoclonal Fab fragment specific for influenza A viruses; Desogus A et al.; A human recombinant monoclonal Fab fragment that specifically recognizes all the influenza A virus strains tested was produced in transformed Escherichia coli using the phage display technique . No strain of influenza B virus reacted with it . It was purified after four cycles of panning and by a single passage through an immunoaffinity column . About 1 mg of pure monoclonal antibody was obtained from 1 liter of culture medium in 3 working days . The Fab fragment reacted with a viral 27-kDa protein, which could reasonably be a matrix protein . Indirect immunofluorescence tests performed on virus-infected MDCK cells showed that this Fab fragment was at least equally efficient as other commercial monoclonal antibody-based systems in detecting influenza A viral infections . The potential advantages of human recombinant Fabs on murine monoclonal antibodies are discussed. Immunol Lett, 2003 Jul 3, 88(1), 43 - 6 Selective induction of CD8+CD4- thymocyte apoptosis mediated by the B-subunit of Escherichia coli heat-labile enterotoxin; Salmond RJ et al.; Receptor-binding by the B-subunit of Escherichia coli heat-labile enterotoxin (EtxB) induces apoptosis of peripheral CD8(+), but not CD4(+) T-cells . Given that peripheral CD8(+) and CD4(+) T cells arise from a common developmental pathway in the thymus, we investigated the effects of EtxB on different thymocyte populations . We show that the acquisition of sensitivity to EtxB-mediated cell death arises following transition of CD4(+)CD8(+) double positive cells into the CD4(-)CD8(+) pathway . Maturation of T cells into CD4(-)CD8(+) single positive cells is associated with upregulated expression of receptors for EtxB. Gene, 2003 Jun 5, 311, 153 - 63 Gene replacement without selection: regulated suppression of amber mutations in Escherichia coli; Herring CD et al.; We have developed a method called "gene gorging" to make precise mutations in the Escherichia coli genome at frequencies high enough (1-15%) to allow direct identification of mutants by PCR or other screen rather than by selection . Gene gorging begins by establishing a donor plasmid carrying the desired mutation in the target cell . This plasmid is linearized by in vivo expression of the meganuclease I-SceI, providing a DNA substrate for lambda Red mediated recombination . This results in efficient replacement of the wild type allele on the chromosome with the modified sequence . We demonstrate gene gorging by introducing amber stop codons into the genes xylA, melA, galK, fucI, citA, ybdO, and lacZ . To compliment this approach we developed an arabinose inducible amber suppressor tRNA . Controlled expression mediated by the suppressor was demonstrated for the lacZ and xylA amber mutants. Bioorg Med Chem Lett, 2003 Aug 4, 13(15), 2493 - 6 High throughput screening identifies novel inhibitors of Escherichia coli dihydrofolate reductase that are competitive with dihydrofolate; Zolli-Juran M et al.; This communication describes the high-throughput screen of a diverse library of 50,000 small molecules against Escherichia coli dihydrofolate reductase to detect inhibitors . Sixty-two compounds were identified as having significant inhibitory activity against the enzyme . Secondary screening of these revealed twelve molecules that were competitive with dihydrofolate, nine of which have not been previously characterized as inhibitors of dihydrofolate reductase . These novel molecules ranged in potency (K(i)) from 26 nM to 11 microM and may represent fresh starting points for new small molecule therapeutics directed against dihydrofolate reductase. Zhongguo Wei Zhong Bing Ji Jiu Yi Xue, 2003 Jan, 15(1), 26 - 8 {Effects of efferent vagus nerve excitation on inflammatory response in heart tissue in rats with endotoxemia}; Shi DG et al.; OBJECTIVE: To study the effects of electrical stimulation of efferent vagus nerve on the inflammatory response in heart tissue in rats with endotoxemia . METHODS: Two hundred and ten Wistar rats were randomly divided into seven groups . Animals were subjected to bilateral cervical vagotomy, or a comparable sham surgical procedure in which the vagus nerves were isolated but not transected . The distal end of a vagus nerve trunk was placed across bipolar electrodes connected to a stimulation module and controlled by an acquisition system . Stimuli with constant voltage (5 V, 2 ms, 1 Hz) were applied to the nerve for 20 minutes immediately after administration of lipopolysaccharide (LPS, 10 mg/kg, E coli O111: B4, Sigma), and then were repeated 2 times after each of 10 minutes interval . Blood and tissue samples of these rats were collected at 1, 1.5 and 2 hours after LPS administration in all groups . Tumor necrosis factor-alpha (TNF-alpha) and mycloperoxidase (MPO) in heart tissue and serum MB isoenzyme of creatine kinase(CK-MB) were determined . RESULTS: The electrical stimulation of the efferent vagus nerve significantly decreased the contents of TNF-alpha and MPO in heart tissue and CK-MB in serum, and alleviated myocardial inflammation in heart tissue at all time points, especially at 1.5 hours after endotoxin challenge . CONCLUSION: The results suggested that excitation of the efferent vagus nerve can significantly alleviate the inflammatory response in the heart tissue, thus it might produce a potential protective effect on the heart during endotoxemia in rats. J Biol Chem, 2003 Sep 19, 278(38), 36285 - 95 Epub 2003 Jul 08. Identification of the required acyltransferase step in the biosynthesis of the phosphatidylinositol mannosides of mycobacterium species; Kordulakova J et al.; Fatty acyl functions of the glycosylated phosphatidylinositol (GPI) anchors of the phosphatidylinositol mannosides (PIM), lipomannan (LM), and lipoarabinomannan (LAM) of mycobacteria play a critical role in both the physical properties and biological activities of these molecules . In a search for the acyltransferases that acylate the GPI anchors of PIM, LM, and LAM, we examined the function of the mycobacterial Rv2611c gene that encodes a putative acyltransferase involved in the early steps of phosphatidylinositol mannoside synthesis . A Rv2611c mutant of Mycobacterium smegmatis was constructed which exhibited severe growth defects and contained an increased amount of phosphatidylinositol mono- and di-mannosides and a decreased amount of acylated phosphatidylinositol di-mannosides compared with the wild-type parental strain . In cell-free assays, extracts from M . smegmatis overexpressing the M . tuberculosis Rv2611c gene incorporated {14C}palmitate into acylated phosphatidylinositol mono- and di-mannosides, and transferred cold endogenous fatty acids onto 14C-labeled phosphatidylinositol mono- and di-mannosides more efficiently than extracts from the wild-type strain . Cell-free extracts from the Rv2611c mutant of M . smegmatis were greatly impaired in these respects . This work provides evidence that Rv2611c is the acyltransferase that catalyzes the acylation of the 6-position of the mannose residue linked to position 2 of myo-inositol in phosphatidylinositol mono- and di-mannosides, with the mono-mannosylated lipid acceptor being the primary substrate of the enzyme . We also provide the first evidence that two distinct pathways lead to the formation of acylated PIM2 from PIM1 in mycobacteria. J Biol Chem, 2003 Sep 19, 278(38), 36128 - 38 Epub 2003 Jul 08. Efficient Hsp90-independent in vitro activation by Hsc70 and Hsp40 of duck hepatitis B virus reverse transcriptase, an assumed Hsp90 client protein; Beck J et al.; Hsp90 is a specialized chaperone that controls the activity of many key regulator proteins such as steroid hormone receptors (SHRs) . Hormone binding, and therefore SHR activation, requires Hsp90, which is loaded onto the receptors by a series of events involving Hsp70, Hsp40, Hop, and p23 . The reverse transcriptase (RT) of hepatitis B viruses, small DNA-containing viruses that replicate via an RNA intermediate, has been reported to depend similarly on Hsp90 for enzymatic activity . Using an in vitro reconstitution system consisting of recombinant duck hepatitis B virus RT, purified chaperones, and the authentic RNA template Depsilon, we demonstrate here that this RT can be activated efficiently by just Hsp40 and Hsc70 plus energy, without the need for Hsp90 or other cofactors . The reaction appears to proceed selectively with the Hdj1 variant of Hsp40 but not Hdj2 or its yeast homolog Ydj1 . The primary reaction product is a metastable, RNA binding-competent intermediate that decays quickly in the absence of its cognate RNA but, in its presence, accumulates in an initiation-competent form over several hours . Because deletion of the RNase H domain rendered the protein partly chaperone-independent, the chaperones may be needed indirectly to relieve occlusion of the RNA binding site by this domain . Our results do not exclude that other factors contribute to RT activation in vivo, but they challenge a fundamental SHR-like dependence on Hsp90 . Thus Hsc70, mostly known for its role in general protein folding, is able to effect activation of a highly specialized target protein. J Biol Chem, 2003 Oct 10, 278(41), 40272 - 81 Epub 2003 Jul 08. Mechanism of the delta wrench in opening the beta sliding clamp; Indiani C et al.; The beta sliding clamp encircles DNA and tethers DNA polymerase III holoenzyme to the template for high processivity . The clamp loader, gamma complex (gamma 3 delta delta'chi psi), assembles beta around DNA in an ATP-fueled reaction . The delta subunit of the clamp loader opens the beta ring and is referred to as the wrench; ATP modulates contact between beta and delta among other functions . Crystal structures of delta.beta and the gamma 3 delta delta' minimal clamp loader make predictions of the clamp loader mechanism, which are tested in this report by mutagenesis . The delta wrench contacts beta at two sites . One site is at the beta dimer interface, where delta appears to distort the interface by via a steric clash between a helix on delta and a loop near the beta interface . The energy for this steric clash is thought to derive from the other site of interaction, in which delta binds to a hydrophobic pocket in beta . The current study demonstrates that rather than a simple steric clash with beta, delta specifically contacts beta at this site, but not through amino acid side chains, and thus is presumably mediated by peptide backbone atoms . The results also imply that the interaction of delta at the hydrophobic site on beta contributes to destabilization of the beta dimer interface rather than acting solely as a grip of delta on beta . Within the gamma complex, delta' is proposed to prevent delta from binding to beta in the absence of ATP . This report demonstrates that one or more gamma subunits also contribute to this role . The results also indicate that delta' acts as a backboard upon which the gamma subunits push to attain the ATP induced change needed for the delta wrench to bind and open the beta ring. Am J Physiol Gastrointest Liver Physiol, 2003 Aug, 285(2), G332 - 43 A novel mitogenic protein that is highly expressed in cells of the gastric antrum mucosa; Martin TE et al.; Human and pig cDNAs for a novel stomach protein, the product of a gene expressed at high levels specifically in cells of the antrum mucosa, have been characterized . The general exon/intron structure of the genomic DNA is conserved in humans and mice . The predicted protein sequences of the human and mouse mRNAs contain 185 and 184 amino acids, respectively . The protein isolated from pig antral extracts has an NH2 terminus consistent with cleavage of a 20-amino acid signal peptide . Human cDNA was expressed in E . coli to generate a protein antigen for antibody production . The antibodies detected polypeptides of approximately 18 kDa in antrum extracts from all mammalian species tested . Immunocytochemistry located antrum mucosal protein (AMP)-18 to surface mucosal cells of the mouse antrum and, specifically, to secretion granules, suggesting that it is cosecreted with mucins . Antrum extracts and recombinant human AMP-18 exhibit growth-promoting activity on epithelial cells that can be blocked by the specific antisera . We suggest that AMP-18 is a "gastrokine" that maintains the integrity of the gastric mucosal epithelium. Xenobiotica, 2003 Jun, 33(6), 603 - 13 Diterpene quinone tanshinone IIA selectively inhibits mouse and human cytochrome p4501A2; Ueng YF et al.; 1 . Tanshinone IIA is the main active diterpene quinone in the herbal medicine Salvia miltiorrhiza . In untreated mouse liver microsomes, tanshinone IIA selectively inhibited 7-ethoxyresorufin O-deethylation (EROD) and 7-methoxyresorufin O-demethylation (MROD) activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, N-nitrosodimethylamine and nifedipine . Tanshinone IIA was a competitive inhibitor of MROD activity with a K(i) of 7.2 +/- 0.7 nM . 2 . In 3-methylcholanthrene-treated mouse liver microsomes, tanshinone IIA and two minor tanshinones, tanshinone I and cryptotanshinone, inhibited liver microsomal MROD activity without affecting EROD and benzo(a)pyrene hydroxylation activities at the concentrations up to 1 microM . Tanshinone IIA induced a type I binding spectrum with a spectral dissociation constant K(s) of 2.3 +/-0.8 microM without cooperativity . 3 . In human liver microsomes, tanshinone IIA decreased EROD and MROD activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, chlorzoxazone and nifedipine . 4 . In Escherichia coli membranes expressing bicistronic human CYP1A enzymes, tanshinone IIA inhibited EROD activity of CYP1A1 with an IC(50) 48 times higher than that for CYP1A2 . Tanshinone I and cryptotanshinone had the same IC(50) ratio (1A1/1A2) of 4 . 5 . The results indicate that tanshinone represents a new group of CYP1A inhibitors, and tanshinone IIA had the highest selectivity in inhibition of CYP1A2. Vaccine, 2003 Jul 28, 21(24), 3374 - 6 Transfer of antibody via mother's milk; Van de Perre P; Differing from humans, IgG from breast milk in many animal species (rodents, bovines, cats, ferrets, etc.) are transported across the intestinal epithelium into the neonatal circulation . This transport is located at the duodenal and jejunal level where enterocytes express a surface membrane receptor able to bind Fc of IgG and to facilitate transcytosis of these immunoglobulins . Fcgamma-R, which is very similar to the placenta receptor responsible for active transplacental transfer of IgG in humans, binds IgG but not other isotypes . Maternal milk antibodies represent an important part of circulating IgG in these animals, as they are involved in the negative feedback of endogenous IgG synthesis . This phenomenon stops abruptly as soon as weaning takes place . Neonatal calves that have a defect in such transfer of maternal immunoglobulins are at high risk of systemic infectious diseases . In humans, in whom gut closure occurs precociously, breast milk antibodies do not enter neonatal/infant circulation . A large part of immunoglobulins excreted in milk are IgA that protect mainly against enteric infections . The specificity of maternal milk IgA is driven by an entero-mammary cell circulation . Human milk also contains anti-idiotypic antibodies capable of enhancing infant antibody response . Maternal milk antibodies coat infant mucosal surfaces and some have a clear protective role . This has been studied extensively in infectious disease models such as rotavirus, E . coli, poliovirus, and retroviruses . In the rotavirus model, antirotaviral IgA can be detected in stools of breast-fed but not bottle-fed neonates . In a large cohort of lactating women infected with HIV-1 in Rwanda, anti-HIV milk antibodies of the IgG isotype were more frequently detected followed by secretory IgM . Surprisingly, anti-HIV-1 SIgA were less frequently found . The presence of milk SIgA at 15 days as well as the persistence of a SIgM response during the whole lactation period was associated with lower risk of HIV transmission from the mother to the infant . Recently, HIV-1 antibodies from maternal milk have been shown to block transcytosis in vitro in a monolayer enterocyte model . Among these antibodies, those directed against the ELDKWA epitope had higher neutralising activity than serum antibodies.In humans, milk excreted antibodies play a major role in protecting infants from infection by pathogens having a mucosal portal of entry. Biochim Biophys Acta, 2003 Jul 9, 1628(1), 71 - 7 Genomic structure and expression analysis of the gene encoding a silkworm basic Kunitz-type chymotrypsin inhibitor; He N et al.; Kunitz-type chymotrypsin inhibitor CIb1 of silkworm Bombyx mori is a basic peptide consisting of 62 amino acid residues . To elucidate the mechanisms of transcriptional regulation of CIb1 gene expression, we cloned it for genomic structure analysis . CIb1 cDNA was used as a probe to screen a BAC sub-library . One positive clone containing the upstream sequences was isolated and the sequence result showed that CIb1 gene consists of three exons spaced by two introns . In the 5'-flanking region, consensus TATA and CCAAT boxes were identified . Other binding sites for transcription factors such as NF-kappaB, GATA, C/EBP, COUP-TF/HNF-4, RORalpha1, SRY, and HOXA3 were also detected . Southern blot analysis suggested a single copy of CIb1 gene in the silkworm genome . Northern blot analysis indicated that the expression of CIb1 gene is transcriptionally regulated during development and is apparently tissue-specific . The CIb1 mRNA was detected in fat body, ovary, trachea, and skin . We furthermore investigated the CIb1 expression profiles after LPS and E . coli injection . The fluctuations of CIb1 transcript in challenged larvae confirm our proposal that CIb1 is an immune responsible gene . According to our data, we discussed the transcriptional factors putatively responsible for the physiological role of CIb1 in the silkworm hemolymph. Biochim Biophys Acta, 2003 Jul 9, 1628(1), 30 - 9 Cel6A, a major exoglucanase from the cellulosome of the anaerobic fungi Piromyces sp . E2 and Piromyces equi; Harhangi HR et al.; Anaerobic fungi possess high cellulolytic activities, which are organised in high molecular mass (HMM) complexes . Besides catalytic modules, the cellulolytic enzyme components of these complexes contain non-catalytic modules, known as dockerins, that play a key role in complex assembly . Screening of a genomic and a cDNA library of two Piromyces species resulted in the isolation of two clones containing inserts of 5.5 kb (Piromyces sp . E2) and 1.5 kb (Piromyces equi) . Both clones contained the complete coding region of a glycoside hydrolase (GH) from family 6, consisting of a 20 amino acid signal peptide, a 76 (sp . E2)/81 (P . equi) amino acid stretch comprising two fungal non-catalytic docking domains (NCDDs), a 24 (sp . E2)/16 (P . equi) amino acid linker, and a 369 amino acid catalytic module . Homology modelling of the catalytic module strongly suggests that the Piromyces enzymes will be processive cellobiohydrolases . The catalytic residues and all nearby residues are conserved . The reaction is thus expected to proceed via a classical single-displacement (inverting) mechanism that is characteristic of this family of GHs . The enzyme, defined as Cel6A, encoded by the full-length Piromyces E2 sequence was expressed in Escherichia coli . The recombinant protein expressed had a molecular mass of 55 kDa and showed activity against Avicel, supporting the observed relationship of the sequence to those of known cellobiohydrolases . Affinity-purified cellulosomes of Piromyces sp . E2 were analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis . A major band was detected with the molecular weight of Cel6A . A tryptic fingerprint of this protein confirmed its identity. J Mol Biol, 2003 Jul 18, 330(4), 851 - 66 Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization; Russo AT et al.; Unfolding and refolding kinetics of human FKBP12 C22A were monitored by fluorescence emission over a wide range of urea concentration in the presence and absence of protecting osmolytes glycerol, proline, sarcosine and trimethylamine-N-oxide (TMAO) . Unfolding is well described by a mono-exponential process, while refolding required a minimum of two exponentials for an adequate fit throughout the urea concentration range considered . The bi-exponential behavior resulted from complex coupling between protein folding, and prolyl isomerization in the denatured state in which the urea-dependent rate constant for folding was greater than, equal to, and less than the rate constants for prolyl isomerization within the urea concentration range of zero to five molar . Amplitudes and the observed folding and unfolding rate constants were fitted to a reversible three-state model composed of two sequential steps involving the native state and a folding-competent denatured species thermodynamically linked to a folding-incompetent denatured species . Excellent agreement between thermodynamic parameters for FKBP12 C22A folding calculated from the kinetic parameters and those obtained directly from equilibrium denaturation assays provides strong support for the applicability of the mechanism, and provides evidence that FKBP12 C22A folding/unfolding is two-state, with prolyl isomer heterogeneity in the denatured ensemble . Despite the chemical diversity of the protecting osmolytes, they all exhibit the same kinetic behavior of increasing the rate constant of folding and decreasing the rate constant for unfolding . Osmolyte effects on folding/unfolding kinetics are readily explained in terms of principles established in understanding osmolyte effects on protein stability . These principles involve the osmophobic effect, which raises the Gibbs energy of the denatured state due to exposure of peptide backbone, thereby increasing the folding rate . This effect also plays a key role in decreasing the unfolding rate when, as is often the case, the activated complex exposes more backbone than is exposed in the native state. J Mol Biol, 2003 Jul 18, 330(4), 813 - 9 NMR and temperature-jump measurements of de novo designed proteins demonstrate rapid folding in the absence of explicit selection for kinetics; Gillespie B et al.; We address the importance of natural selection in the origin and maintenance of rapid protein folding by experimentally characterizing the folding kinetics of two de novo designed proteins, NC3-NCAP and ENH-FSM1 . These 51 residue proteins, which adopt the helix-turn-helix homeodomain fold, share as few as 12 residues in common with their most closely related natural analog . Despite the replacement of up to 3/4 of their residues by a computer algorithm optimizing only thermodynamic properties, the designed proteins fold as fast or faster than the 35,000 s(-1) observed for the closest natural analog . Thus these de novo designed proteins, which were produced in the complete absence of selective pressures or design constraints explicitly aimed at ensuring rapid folding, are among the most rapidly folding proteins reported to date. J Mol Biol, 2003 Jul 18, 330(4), 675 - 85 Multiple interactions within the hepatitis C virus RNA polymerase repress primer-dependent RNA synthesis; Ranjith-Kumar CT et al.; The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) initiates RNA synthesis in vivo by a de novo mechanism . In vitro, however, the HCV RdRp can initiate de novo or extend from a primed template . A novel beta-loop near the RdRp active site was previously found to prevent the use of primed templates . We found that, in addition to the beta-loop, the C-terminal tail of the HCV RdRp and the de novo initiation GTP are required to exclude the use of primed-templates . GTP binding to the NTPi site of the HCV RdRp orchestrates the participation of other structures . The interactions of the beta-loop, C-terminal tail, and GTP provide an elegant solution to ensure de novo initiation of HCV RNA synthesis. J Mol Biol, 2003 Jul 18, 330(4), 651 - 6 Structural plasticity and the evolution of antibody affinity and specificity; Yin J et al.; The germline precursor to the ferrochelatase antibody 7G12 was found to bind the polyether jeffamine in addition to its cognate hapten N-methylmesoporphyrin . A comparison of the X-ray crystal structures of the ligand-free germline Fab and its complex with either hapten or jeffamine reveals that the germline antibody undergoes significant conformational changes upon the binding of these two structurally distinct ligands, which lead to increased antibody-ligand complementarity . The five somatic mutations introduced during affinity maturation lead to enhanced binding affinity for hapten and a loss in affinity for jeffamine . Moreover, a comparison of the crystal structures of the germline and affinity-matured antibodies reveals that somatic mutations not only fix the optimal binding site conformation for the hapten, but also introduce interactions that interfere with the binding of non-hapten molecules . The structural plasticity of this germline antibody and the structural effects of the somatic mutations that result in enhanced affinity and specificity for hapten likely represent general mechanisms used by the immune response, and perhaps primitive proteins, to evolve high affinity, selective receptors for so many distinct chemical structures. Metab Eng, 2003 Apr, 5(2), 74 - 85 A metabolic network analysis & NMR experiment design tool with user interface-driven model construction for depth-first search analysis; Zhu T et al.; A Windows program for metabolic engineering analysis and experimental design has been developed . A graphical user interface enables the pictorial, "on-screen" construction of a metabolic network . Once a model is composed, balance equations are automatically generated . Model construction, modification and information exchange between different users is thus considerably simplified . For a given model, the program can then be used to predict all the extreme point flux distributions that optimize an objective function while satisfying balances and constraints by using a depth-first search strategy . One can also find the minimum reaction set that satisfies different conditions . Based on the identified flux distributions or linear combinations, the user can simulate the NMR and GC/MS spectra of selected signal molecules . Alternately, spectra vectorization allows for the automated optimization of labeling experiments that are intended to distinguish between different, yet plausible flux extreme point distributions . The example provided entails predicting the flux distributions associated with deleting pyruvate kinase and designing 13C NMR experiments that can maximally discriminate between the flux distributions. Biochem Biophys Res Commun, 2003 Jul 18, 307(1), 172 - 9 Specificity for inhibitors of metal-substituted methionine aminopeptidase; Li JY et al.; Methionine aminopeptidases (MetAPs) have been studied in vitro as Co(II) enzymes, but their in vivo metal remains to be defined . While activation of Escherichia coli MetAP (EcMetAP1) by Co(II), Mn(II), and Zn(II) was detectable by a colorimetric Met-S-Gly-Phe assay, significant activation by Ni(II) was shown in a fluorescence Met-AMC assay, in addition to Co(II) and Mn(II) activation . When tested on the metal-substituted EcMetAP1s, a few inhibitors that we obtained recently from a random screening on Co-EcMetAP1 either became much weak or lost activity on Mn- or Zn-EcMetAP1, although they kept inhibitory activity on Ni-EcMetAP1 . A couple of peptidic inhibitors and the methionine mimetic (3R)-amino-(2S)-hydroxyheptanoic acid (AHHpA, 6) maintained moderate activities on Co-, Mn-, Zn-, and Ni-EcMetAP1s . Our results clearly demonstrate that the metal-substitution has changed the enzyme specificity for substrates and inhibitors . Therapeutic applications call for inhibitors specific for MetAP with a physiologically relevant metal at its active site. Autoimmun Rev, 2003 Jan, 2(1), 19 - 24 T cell immunity and primary biliary cirrhosis; Ishibashi H et al.; T lymphocytes play a pivotal role in the autoimmune response in primary biliary cirrhosis (PBC) . Recent studies have shown that there is overlapping in the PDC-E2-specific T and B cell epitopes . In addition, helper T and cytotoxic T cell epitopes all contain a shared peptide sequence . In addition, recognition of exogenous antigens including bacterial antigens by autoantigen-specific T cell and the mechanism of molecular mimicry provide a clue to clarifying the pathogenesis of PBC . Furthermore, the findings that autoantigen-immune complexes cross present and also that the presentation of autoantigen is of a higher relative efficiency, define a unique role of autoantibodies in the pathogenesis of the autoimmune disease . The mechanism of immune-mediated bile duct damage in PBC, including the possible role of T cell-mediated cytotoxicity and molecular mimicry is discussed. Plant J, 2003 Jul, 35(2), 164 - 76 Functional expression of AtHMA4, a P1B-type ATPase of the Zn/Co/Cd/Pb subclass; Mills RF et al.; Mechanisms are required by all organisms to maintain the concentration of essential heavy metals (e.g . Zn and Cu) within physiological limits and to minimise the detrimental effects of non-essential heavy metals (e.g . Cd) . Heavy-metal P-type ATPases (HMAs) are a subgroup of the P-type ATPase superfamily that may contribute to metal homeostasis in plants . We cloned and characterised a member of this family, AtHMA4, from Arabidopsis thaliana that clusters with the Zn/Co/Cd/Pb subclass of HMAs on phylogenetic analysis . Sequencing of the AtHMA4 cDNA showed that it contained the conserved motifs found in all P-type ATPases and also motifs that are characteristic of heavy-metal ATPases . Escherichia coli mutants defective in the HMAs, CopA and ZntA, were used in functional complementation studies . AtHMA4 was able to restore growth at high {Zn} in the zntA mutant but not at high {Cu} in the copA mutant, suggesting a role in zinc transport . Heterologous expression of AtHMA4 in Saccharomyces cerevisiae made the yeast more resistant to Cd but did not affect sensitivity to other metals compared with vector-transformed controls . The organ specificity of AtHMA4 was analysed in Arabidopsis and showed that AtHMA4 was expressed in a range of tissues with highest expression in roots . AtHMA4 was upregulated in roots exposed to elevated levels of Zn and Mn but downregulated by Cd . Possible physiological roles of this transporter in Arabidopsis are discussed. J Bioenerg Biomembr, 2003 Feb, 35(1), 31 - 40 Multiple biochemical activities of NM23/NDP kinase in gene regulation; Postel EH; NM23/NDPk proteins play critical roles in cancer and development; however, our understanding of the underlying biochemical mechanisms is still limited . This large family of highly conserved proteins are known to participate in many events related to DNA metabolism, including nucleotide binding and nucleoside triphosphate synthesis, DNA binding and transcription, and cleavage of DNA strands via covalent protein-DNA complexes . The chemistry of the DNA-cleavage reaction of NM23-H2/NDPk is characteristic of DNA repair enzymes . Both the DNA cleavage and the NDPk reactions are conserved between E . coli and the human enzymes, and several conserved amino acid side chains involved in catalysis are shared by these reactions . It is proposed here that NM23/NDP kinases are important regulators of gene expression during development and cancer via previously unrecognized roles in DNA repair and recombination, and via previously unrecognized pathways and mechanisms of genetic control. J Allergy Clin Immunol, 2003 Jul, 112(1), 159 - 67 Persistent protective effect of heat-killed Escherichia coli producing "engineered," recombinant peanut proteins in a murine model of peanut allergy; Li XM et al.; BACKGROUND: Peanut allergy (PNA) is a life-threatening food allergy for which there is no definitive treatment . OBJECTIVE: We investigated the long-term immunomodulatory effect of heat-killed Escherichia coli producing engineered (mutated) Ara h1, 2, and 3 (HKE-MP123) administered rectally (pr) in a murine model of PNA . METHODS: Peanut-allergic C3H/HeJ mice received 0.9 (low dose), 9 (medium dose), or 90 (high dose) microg HKE-MP123 pr, HKE-containing vector (HKE-V) alone, or vehicle alone (sham) weekly for 3 weeks . Mice were challenged 2 weeks later . A second and third challenge were performed at 4-week intervals . RESULTS: After the first challenge, all 3 HKE-MP123 and HKE-V-treated groups exhibited reduced symptom scores (P <.01,.01,.05,.05, respectively) compared with the sham-treated group . Interestingly, only the medium- and high-dose HKE-MP123-treated mice remained protected for up to 10 weeks after treatment accompanied by a significant reduction of plasma histamine levels compared with sham-treated mice (P <.05 and.01, respectively) . IgE levels were significantly lower in all HKE-MP123-treated groups (P <.001), being most reduced in the high-dose HKE-MP123-treated group at the time of each challenge . IL-4, IL-13, IL-5, and IL-10 production by splenocytes of high-dose HKE-MP123-treated mice were significantly decreased (P <.01;.001,.001, and.001, respectively), and IFN-gamma and TGF-beta production were significantly increased (P <.001 and.01, respectively) compared with sham-treated mice at the time of the last challenge . CONCLUSIONS: Treatment with pr HKE-MP123 can induce long-term "downregulation" of peanut hypersensitivity, which might be secondary to decreased antigen-specific T(H)2 and increased T(H)1 and T regulatory cytokine production. J Immunol, 2003 Jul 15, 171(2), 812 - 20 Modular organization of the carboxyl-terminal, globular head region of human C1q A, B, and C chains; Kishore U et al.; The first step in the activation of the classical complement pathway, by immune complexes, involves the binding of the globular heads of C1q to the Fc regions of aggregated IgG or IgM . Located C-terminal to the collagen region, each globular head is composed of the C-terminal halves of one A (ghA), one B (ghB), and one C chain (ghC) . To dissect their structural and functional autonomy, we have expressed ghA, ghB, and ghC in Escherichia coli as soluble proteins linked to maltose-binding protein (MBP) . The affinity-purified fusion proteins (MBP-ghA, -ghB, and -ghC) bound differentially to heat-aggregated IgG and IgM, and also to three known C1q-binding peptides, derived from HIV-1, HTLV-I, and beta-amyloid . In the ELISAs, the MBP-ghA bound to heat-aggregated IgG and IgM as well as to the HIV-1 gp41 peptide; the MBP-ghB bound preferentially to IgG rather than IgM, in addition to binding beta-amyloid peptide, whereas the MBP-ghC showed a preference for IgM and the HTLV-I gp21 peptide . Both MBP-ghA and MBP-ghB also inhibited C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes . However, for IgM-coated erythrocytes, MBP-ghC was a better inhibitor of C1q than MBP-ghB . The recombinant forms of ghA, ghB, and ghC also bound specifically to apoptotic PBMCs . We conclude that the C1q globular head region is likely to have a modular organization, being composed of three structurally and functionally independent modules, which retains multivalency in the form of a heterotrimer . The heterotrimeric organization thus offers functional flexibility and versatility to the whole C1q molecule. J Biol Chem, 2003 Sep 19, 278(38), 35903 - 13 Epub 2003 Jul 07. Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD; Taxis C et al.; The endoplasmic reticulum (ER) harbors a protein quality control system, which monitors protein folding in the ER . Elimination of malfolded proteins is an important function of this protein quality control . Earlier studies with various soluble and transmembrane ER-associated degradation (ERAD) substrates revealed differences in the ER degradation machinery used . To unravel the nature of these differences we generated two type I membrane ERAD substrates carrying malfolded carboxypeptidase yscY (CPY*) as the ER-luminal ERAD recognition motif . Whereas the first, CT* (CPY*-TM), has no cytoplasmic domain, the second, CTG*, has the green fluorescent protein present in the cytosol . Together with CPY*, these three substrates represent topologically diverse malfolded proteins, degraded via ERAD . Our data show that degradation of all three proteins is dependent on the ubiquitin-proteasome system involving the ubiquitin-protein ligase complex Der3/Hrd1p-Hrd3p, the ubiquitin conjugating enzymes Ubc1p and Ubc7p, as well as the AAA-ATPase complex Cdc48-Ufd1-Npl4 and the 26S proteasome . In contrast to soluble CPY*, degradation of the membrane proteins CT* and CTG* does not require the ER proteins Kar2p (BiP) and Der1p . Instead, CTG* degradation requires cytosolic Hsp70, Hsp40, and Hsp104p chaperones. Eur J Biochem, 2003 Jul, 270(14), 3083 - 91 Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca; Irwin DC et al.; Thermobifida fusca xyloglucan-specific endo-beta-1,4-glucanase (Xeg)74 and the Xeg74 catalytic domain (CD) were cloned, expressed in Escherichia coli, purified and characterized . This enzyme has a glycohydrolase family-74 CD that is a specific xyloglucanase followed by a family-2 carbohydrate binding module at the C terminus . The Michaelis constant (Km) and maximal rate (Vmax) values for hydrolysis of tamarind seed xyloglucan (tamXG) are 2.4 micro m and 966 micro mol xyloglucan oligosaccharides (XGOs) min-1 . micro mol protein-1 . More than 75% of the activity was retained after a 16-h incubation at temperatures up to 60 degrees C . The enzyme was most active at pH 6.0-9.4 . NMR analysis showed that its catalytic mechanism is inverting . The oligosaccharide products from hydrolysis of tamXG were determined by MS analysis . Cel9B, an active carboxymethylcellulose (CMC)ase from T . fusca, was also found to have activity on xyloglucan (XG) at 49 micro mol.min-1 . micro mol protein-1, but it could not hydrolyze XG units containing galactose . An XG/cellulose composite was prepared by growing Gluconacetobacterxylinus on glucose with tamXG in the medium . Although a mixture of purified cellulases was unable to degrade this material, the composite material was fully hydrolyzed when Xeg74 was added . T . fusca was not able to grow on tamXG, but Xeg74 was found in the culture supernatant at the same level as was found in cultures grown on Solka Floc . The function of this enzyme appears to be to break down the XG surrounding cellulose fibrils found in biomass so that T . fusca can utilize the cellulose as a carbon source. Eur J Biochem, 2003 Jul, 270(14), 2945 - 9 Photochemical cross-linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups; Taranenko M et al.; Formamidopyrimidine-DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7-hydro-8-oxoguanine, from damaged DNA . In order to identify specific contacts between nucleobases of DNA and amino acids from the E . coli Fpg protein, photochemical cross-linking was employed using new reactive DNA duplexes containing 5-{4-{3-(trifluoromethyl)-3H-diazirin-3-yl}phenyl}-2'-deoxyuridine dU* residues near the 7-hydro-8-oxoguanosine (oxoG) lesion . The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them . The nicking efficiency of the DNA duplex containing a dU* residue 5' to the oxoG was higher as compared to oxidized native DNA . The conditions for the photochemical cross-linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross-linked product . Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5' adjacent to oxoG and the other 5' adjacent to the cytidine residue pairing with oxoG in the other strand . The approaches developed may be applicable to pro- and eukaryotic homologues of the E . coli Fpg protein as well as to other repair enzymes. Eur J Biochem, 2003 Jul, 270(14), 2937 - 44 The calcium-induced switch in the troponin complex probed by fluorescent mutants of troponin I; Oliveira DC et al.; The Ca2+-induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction . Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5-hydroxytryptophan (5HW) . Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N-domain of TnC are the inhibitory region (residues 96-116) and a neighboring region that includes position 121 . Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC . We also discuss the possibility that the regulatory sites in the N-terminal domain of TnC might be the high affinity Ca2+-binding sites in the troponin complex. Biochemistry, 2003 Jul 15, 42(27), 8369 - 76 2H, 13C, and 15N kinetic isotope effects on the reaction of the ammonia-rescued K258A mutant of aspartate aminotransferase; Wright SK et al.; Deuterium isotope effects at C2 of aspartate and heavy atom isotope effects at C2, C3, and the amino group of aspartate were determined for the reaction of the lysine-258 to alanine mutant of Escherichia coli rescued with exogenous ammonia . We were able to calculate an (15)N intrinsic isotope effect of 1.034 . The intrinsic (13)C isotope effect at C3 is 1.0060, and the (13)C isotope effect at C2 is 1.0016 . These isotope effects reveal that collapse of the carbinolamine (or gem-diamine) to give the final product is the rate-determining step in this system . Furthermore, these results indicate that lysine-258 is critical to the catalysis of the final breakdown to give product, and in fact this step is more strongly affected by mutation of lysine-258 than the deprotonation of the external aldimine. Biochemistry, 2003 Jul 15, 42(27), 8289 - 97 Sequential structural changes of Escherichia coli thioesterase/protease I in the serial formation of Michaelis and tetrahedral complexes with diethyl p-nitrophenyl phosphate; Tyukhtenko SI et al.; Escherichia coli thioesterase/protease I (TEP-I) belongs to a new subclass of lipolytic enzymes of the serine hydrolase superfamily . Here we report the first direct NMR observation of the formation of the Michaelis complex (MC) between TEP-I and diethyl p-nitrophenyl phosphate (DENP), an active site directed inhibitor of serine protease, and its subsequent conversion to the tetrahedral complex (TC) . NMR, ESI-MS, and kinetic data showed that DENP binds to TEP-I in a two-step process, a fast formation of MC followed by a slow conversion to TC . NMR chemical shift perturbation further revealed that perturbations were confined mainly to four conserved segments comprising the active site . Comparable magnitudes of chemical shift perturbations were detected in both steps . The largest chemical shift perturbation occurred around the catalytic Ser(10) . In MC, the conformation of the mobile Ser(10) was stabilized, and its amide resonance became observable . From the large chemical shift perturbation upon conversion from MC to TC, we propose that the amide protons of Ser(10) and Gly(44) serve as the oxyanion hole proton donors that stabilize the tetrahedral adduct . The pattern of residues perturbed in both steps suggests a sequential, stepwise structural change upon binding of DENP . The present study also demonstrates the important catalytic roles of conserved residues in the SGNH family of proteins. Biochemistry, 2003 Jul 15, 42(27), 8163 - 70 The small protein CP12: a protein linker for supramolecular complex assembly; Graciet E et al.; CP12 is an 8.5-kDa nuclear-encoded chloroplast protein, isolated from higher plants . It forms part of a core complex of two dimers of phosphoribulokinase (PRK), two tetramers of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and CP12 . The role of CP12 in this complex assembly has not been determined . To address this question, we cloned a cDNA encoding the mature CP12 from the green alga Chlamydomonas reinhardtii and expressed it in Escherichia coli . Sequence alignments show that it is very similar to other CP12s, with four conserved cysteine residues forming two disulfide bridges in the oxidized CP12 . On the basis of reconstitution assays and surface plasmon resonance binding studies, we show that oxidized, but not reduced, CP12 acts as a linker in the assembly of the complex, and we propose a model in which CP12 associates with GAPDH, causing its conformation to change . This GAPDH/CP12 complex binds PRK to form a half-complex (one unit) . This unit probably dimerizes due partially to interactions between the enzymes of each unit . Reduced CP12 being unable to reconstitute the complex, we studied the structures of oxidized and reduced CP12 by NMR and circular dichroism to determine whether reduction induced structural transitions . Oxidized CP12 is mainly composed of alpha helix and coil segments, and is extremely flexible, while reduced CP12 is mainly unstructured . Remarkably, CP12 has similar physicochemical properties to those of "intrinsically unstructured proteins" that are also involved in regulating macromolecular complexes, or in their assembly . CP12s are thus one of the few protein families of intrinsically unstructured proteins specific to plants. Bioorg Khim, 2003 May-Jun, 29(3), 310 - 5 {A composite polyaniline-containing silica sorbent for DNA isolation}; Kapustin DV et al.; A composite sorbent based on porous glass beads modified with thin polyaniline coating was prepared by precipitating aniline polymerization in the presence of carrier particles . It was shown that the modification ensures the uniform coating of the inner surface of the carrier pores with the polymer layer approximately 70 A thick . It was shown that the resulting material retains the initial porosity of the carrier and is selective in the separation of nucleic acids and proteins . The polyaniline-coated sorbents were shown to be efficient for both the preparative DNA isolation from bacterial lysates and for analytical purposes, in particular, for studying DNA fragmentation during apoptosis proceeding under UV irradiation of cell lysates of colon carcinoma . The morphological and chromatographic characteristics of the new sorbent were demonstrated to be similar to those of the polyfluorobutadiene sorbent. Rapid Commun Mass Spectrom, 2003, 17(14), 1579 - 84 Stable isotope labeling of phosphopeptides for multiparallel kinase target analysis and identification of phosphorylation sites; Glinski M et al.; An approach for multiparallel target identification and relative quantification of in vitro kinase activities in two different biological samples, using liquid chromatography/mass spectrometry (LC/MS), is described . Synthetic target peptides, containing the putative regulatory phosphorylation sites of sucrose-phosphate synthase (SPS) isoenzymes from Arabidopsis thaliana, were simultaneously in vitro phosphorylated and their phosphorylation states determined . Quantification was achieved by stable isotope labeling of the phosphoserine moiety with ethanethiol and {(2)D(5)}-ethanethiol . This revealed different kinase activities in extracts of wild-type (WT) plants and mutant plants lacking plastidic phosphoglucomutase (PGM) . The multiparallel assay allowed the determination of favored substrate specificities among the putative phosphorylation sites in SPS . Additionally, we extended the method to unambiguously identify phosphorylation sites in peptides via differential labeling . Braz J Med Biol Res, 2003 Jul, 36(7), 829 - 37 Epub 2003 Jun 26. Shared control of maltose and trehalose utilization in Candida utilis; Rolim MF et al.; Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose . Here we report the catabolism of exogenous trehalose by Candida utilis . In contrast to the biphasic growth in glucose, the growth of C . utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect . Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 mol trehalose min-1 mg cell (dry weight)-1 . The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol . Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively . Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70% of the total activity . In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter . These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli . We show here for the first time that trehalose induces the catabolism of maltose in yeast. Sci Aging Knowledge Environ . 2003 Feb 05;2003(5):PE3. Structure-(Dys)function relationships in mitochondrial electron transport chain complex II? Kristal BS, Krasnikov BF. It has been postulated that mitochondrially derived reactive oxygen species (ROS) play a major causative role in aging processes . The primary sources of these oxidants are believed to be complexes I and III of the electron transport chain, with little evidence supporting oxidant formation at complex II (succinate dehydrogenase) . Mutation of a complex II protein has, however, been shown to cause increased oxidative stress and decreased life expectancy in the Caenorhabditis elegans mutant mev-1 . A recent study by Yankovskaya and colleagues, in which the structure of Escherichia coli succinate dehydrogenase was determined, provides an explanation for these observations . Furthermore, these results suggest possible mechanisms by which electron leakage might occur at this site in the aged organism. J Biol Chem, 2003 Sep 19, 278(38), 36077 - 84 Epub 2003 Jul 05. Glutamate counteracts the denaturing effect of urea through its effect on the denatured state; Mandal AK et al.; The urea induced equilibrium denaturation behavior of glutaminyl-tRNA synthetase from Escherichia coli (GlnRS) in 0.25 m potassium l-glutamate, a naturally occurring osmolyte in E . coli, has been studied . Both the native to molten globule and molten globule to unfolded state transitions are shifted significantly toward higher urea concentrations in the presence of l-glutamate, suggesting that l-glutamate has the ability to counteract the denaturing effect of urea . d-Glutamate has a similar effect on the equilibrium denaturation of glutaminyl-tRNA synthetase, indicating that the effect of l-glutamate may not be due to substrate-like binding to the native state . The activation energy of unfolding is not significantly affected in the presence of 0.25 m potassium l-glutamate, indicating that the native state is not preferentially stabilized by the osmolyte . Dramatic increase of coefficient of urea concentration dependence (m) values of both the transitions in the presence of glutamate suggests destabilization and increased solvent exposure of the denatured states . Four other osmolytes, sorbitol, trimethylamine oxide, inositol, and triethylene glycol, show either a modest effect or no effect on native to molten globule transition of glutaminyl-tRNA synthetase . However, glycine betaine significantly shifts the transition to higher urea concentrations . The effect of these osmolytes on other proteins is mixed . For example, glycine betaine counteracts urea denaturation of tubulin but promotes denaturation of S228N lambda-repressor and carbonic anhydrase . Osmolyte counteraction of urea denaturation depends on osmolyte-protein pair. Eur J Med Res, 2003 May 30, 8(5), 208 - 11 Comparison of ability of apoptosis induction by lipopolysaccharide of Porphyromonas gingivalis with Escherichia coli; Hirai K et al.; We compared effects of Porphyromonas gingivalis LPS with Escherichia coli LPS to the murine peritoneal macrophage . E . coli LPS possessed a threshold dose between 100 micro g and 10 micro g, the higher dose induced apoptosis at the murine peritoneal macrophage while the lower dose did not . The ability of apoptosis induction at the murine peritoneal macrophage of P . gingivalis LPS was weaker than E . coli LPS . P . gingivalis LPS did not induce significant apoptosis in all tested dose . However, the morphology of the peritoneal macrophage treated by P . gingivalis LPS was obviously different from that of the unstimulated cells. Zhongguo Shi Yan Xue Ye Xue Za Zhi, 2003 Jun, 11(3), 238 - 42 {A new method for construction of EGFP-labled recombinant adenovirus containing hVEGF(165) and its property in vitro}; Zhong ZD et al.; By using AdEasy system, which is based on the homologous recombination in bacteria, an EGFP labled recombinant adenovirus vector containing hVEGF(165) was generated quickly and its property was studied in vitro . First, hVEGF(165) coding sequence was subcloned into the shuttle plasmid pAdTrack-CMV, then linearized and cotransferred with adenoviral backbone vector pAdEasy-1 into E . coli strain BJ(5183) . After positive kanamycin-resistant colony was picked up, the recombinant adenoviral plasmid was identified by restriction analysis with PacI and transfected into HEK 293 cells to assembly replication-defective adenovirus Ad-EGFP/hVEGF(165) . The further amplified recombinant adenoviruses were purified by CsCl banding at 32,000 rpm for 18 to 24 hours . Electron microscopy and PCR were performed for testing the recombinant adenovirus . The results showed that the purified particles were homogenous hexagon with a high titer up to 2 x 10(12)pfu/ml . An amplified band of 540 bp fragment demonstrated the successful insert of hVEGF(165) . Under fluorescence microscopy, the expression of EGFP was easily detected in HEK 293 and other target cells . The maximal stimulating effect on the proliferation of hUVEC was obtained when the given multiplicity of infection (MOI) of Ad-EGFP/hVEGF(165) was 100 . The rate of EGFP positive mouse bone marrow mononuclear cells analysed by flow cytometry was 27.3% after 24 hour-incubation with Ad-EGFP/hVEGF(165) (MOI = 100), and the expression of hVEGF(165) protein in the conditioned medium was 1385 +/- 332 pg/10(6) cells . It is concluded that the construction of adenovirus vector by homologous recombination in bacteria using AdEasy system can be quickly and easily performed, and the prepared high titer of Ad-EGFP/hVEGF(165) is an efficient helpful vector to transfer genes into target cells, all of which make the further in vivo experiments with VEGF(165) possible. Biosci Biotechnol Biochem, 2003 Jun, 67(6), 1368 - 75 Preparation and characterization of recombinant murine p65/L-plastin expressed in Escherichia coli and high-titer antibodies against the protein; Shinomiya H et al.; We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages . It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells . p65/L-plastin is characterized by a series of Ca(2+)-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells . We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to beta-actin and prepared high-titer antibodies using large amounts of the protein as immunogen . Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components . We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes. Biosci Biotechnol Biochem, 2003 Jun, 67(6), 1358 - 67 Molecular and immunochemical characteristics of monoclonal and recombinant antibodies specific to bisphenol A; Nishi K et al.; Four anti-bisphenol A monoclonal antibodies (mabs) were obtained and each characterized by an enzyme-linked immunosorbent assay (ELISA) . Among these mabs, BBA-2187 was the most reactive towards bisphenol A . The quantitation limit of the ELISA assay for bisphenol A was 0.13 ng/ml, which is more sensitive than the other immunoassays reported . Then, the cDNA clones encoding variable heavy and variable light chains of these four mabs were isolated, and used for construction of four single-chain Fv (scFv) antibody genes, which were expressed in Escherichia coli cells . The reactivity of four scFv antibodies towards bisphenol A in ELISA was comparable to those of the parent mabs . The most sensitive assay was achieved with BBA-2187scFv . Its cross-reactivity to the related compounds was similar to that of the parent mab . Based on the reactivity of heterologous combinations of VH and VL fragments, it was found that the unique structure of the framework region 2 in the VL of BBA-2187 appeared to be important for specific assembly together with the VH. Biosci Biotechnol Biochem, 2003 Jun, 67(6), 1335 - 41 Identification by the phage-display technique of peptides that bind to H7 flagellin of Escherichia coli; Ide T et al.; The four peptides interacting with H7 flagellin of Escherichia coli were selected from a phage display library . The library was selected four times, and the interacting phage peptides were competitively eluted with H7 flagellin . An enzyme-linked immunosorbent assay (ELISA) showed that these peptides were reactive with the H7 flagellin in a dose-dependent manner . Among them, a D1 phage clone showed the highest binding affinity to the H7 flagellin . We synthesized the D1 peptide (LHIHRPTLSIQG) corresponding to the peptide-encoding region of the D1 phage clone . The synthetic peptide showed micro-molar affinity (EC(50) value=1.9 microM) for the H7 flagellin . Furthermore, this D1 peptide interacted more specifically with the H7 flagellin than with the other flagellins (H1, H5, H12, or H23) of E . coli . In situ hybridization clearly showed that the peptide only detected those cells harboring the H7 flagellin gene (fliC) . The peptide may specifically bind to the H7 flagellin on the cell surface . These results suggest that the phage-display technique could be used as a tool for identifying peptides as an alternative to using a ligand as a diagnostic reagent in food products or in clinical testing. Methods Mol Biol, 2001, 181, 67 - 81 A Transgenic Approach to Studying Imprinted Genes: Modified BACs and PACs; John RM et al.; The advantages of using large genomic clones in the analysis of imprinted genes is described in Chapter 4 with particular reference to yeast artificial chromosomes (YACs) . These contain on average 500-600 kb of DNA but can be much larger (>1 Mb) . YACS are propagated in yeast and are therefore amenable to genetic modification by homologous recombination, and there are now many examples of their use to generate transgenic mice . This chapter describes a relatively new strategy for using large genomic clones that relies on Escherichia coli-based systems. Science, 2003 Jul 4, 301(5629), 102 - 5 Inferring genetic networks and identifying compound mode of action via expression profiling; Gardner TS et al.; The complexity of cellular gene, protein, and metabolite networks can hinder attempts to elucidate their structure and function . To address this problem, we used systematic transcriptional perturbations to construct a first-order model of regulatory interactions in a nine-gene subnetwork of the SOS pathway in Escherichia coli . The model correctly identified the major regulatory genes and the transcriptional targets of mitomycin C activity in the subnetwork . This approach, which is experimentally and computationally scalable, provides a framework for elucidating the functional properties of genetic networks and identifying molecular targets of pharmacological compounds. J Nucl Med, 2003 Jul, 44(7), 1087 - 91 A bivalent leukotriene B(4) antagonist for scintigraphic imaging of infectious foci; van Eerd JE et al.; Several radiolabeled chemotactic peptides have been tested for their suitability to show infection and inflammation . Leukotriene B(4) (LTB(4)) receptor-binding ligands could be useful agents for revealing neutrophilic infiltrations because the LTB(4) receptor is abundantly expressed on neutrophils after an inflammatory stimulus . In this study, we investigated the in vivo and in vitro characteristics of a new hydrophilic (111)In-labeled LTB(4) antagonist . METHODS: The LTB(4) antagonist DPC11870-11 was labeled with (111)In and intravenously injected into New Zealand White rabbits with Escherichia coli infection in the left thigh muscle . The pharmacokinetics and biodistribution were studied by serial scintigraphic imaging (0-24 h after injection) and by ex vivo counting of dissected tissues (6 and 24 h after injection) . The receptor-mediated in vivo localization of the compound was investigated in 3 rabbits that received an excess of nonradioactive indium-labeled agent 2 min before the administration of the (111)In-labeled LTB(4) antagonist . RESULTS: In rabbits with intramuscular E . coli infection, the abscess was visualized as early as 2 h after injection . Accumulation in the abscess increased with time, resulting in excellent images at 6 h after injection . Blood clearance was rapid in the first hours after injection (alpha-half-life = 30 +/- 6 min, 85%; beta-half-life = 25.7 +/- 0.8 h, 15%) . Abscess-to-background ratios, as derived from the region-of-interest analysis, increased to 34 +/- 7 at 24 h after injection . The images of both groups showed moderate uptake in the liver, spleen, kidneys, and bone marrow . No activity was seen in the bladder, indicating almost complete retention in the kidneys . The uptake in the abscess could be blocked completely by injection of an excess of nonradioactive agent, indicating a specific receptor-ligand interaction of the radiolabeled agent in the infected tissue . Biodistribution data showed that after saturation of the LTB(4) receptor, the abscess uptake, in percentage injected dose per gram, was significantly reduced (0.03 +/- 0.02 vs . 0.24 +/- 0.06, P = 0.008) . CONCLUSION: The modified LTB(4) antagonist showed infectious foci rapidly after injection because of specific receptor-ligand interaction . Because of the high abscess-to-background ratios that were obtained and the fact that no accumulation of radioactivity was observed in the gastrointestinal tract, this compound has excellent characteristics for revealing infectious and inflammatory foci. Blood, 2003 Oct 15, 102(8), 2835 - 42 Epub 2003 Jul 03. The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation; Isordia-Salas I et al.; Crohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation . In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility . Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma . A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis) . The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation . We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation . We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK . The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F . When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased . This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats. J Biol Chem, 2003 Sep 12, 278(37), 34751 - 6 Epub 2003 Jul 02. Integration of b subunits of unequal lengths into F1F0-ATP synthase; Grabar TB et al.; In Escherichia coli the peripheral stalk of F1F0-ATP synthase consists of a parallel dimer of identical b subunits . However, the length of the two b subunits need not be fixed . This led us to ask whether it is possible for two b subunits of unequal length to dimerize in a functional enzyme complex . A two-plasmid expression system has been developed that directs production of b subunits of unequal lengths in the same cell . Two b subunits differing in length have been expressed with either a histidine or V5 epitope tag to facilitate nickel-affinity resin purification (Ni-resin) and Western blot analysis . The epitope tags did not materially affect enzyme function . The system allowed us to determine whether the different b subunits segregate to form homodimers or, conversely, whether a heterodimer consisting of both the shortened and lengthened b subunits can occur in an intact enzyme complex . Experiments expressing different b subunits lengthened and shortened by up to 7 amino acids were detected in the same enzyme complex . The V5-tagged b subunit shortened by 7 amino acids (b Delta 7-V5) was detected in Ni-resin-purified membrane preparations only when coexpressed with a histidine-tagged b subunit in the same cell . The results demonstrate that the enzyme complex can tolerate a size difference between the two b subunits of up to 14 amino acids . Moreover, the experiments demonstrated the feasibility of constructing enzyme complexes with non-identical b subunits that will be valuable for research requiring specific chemical modification of a single b subunit. J Biol Chem, 2003 Oct 3, 278(40), 38194 - 205 Epub 2003 Jul 03. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3; Rauchenberger R et al.; The human combinatorial antibody library Fab 1 (HuCAL-Fab 1) was generated by transferring the heavy and light chain variable regions from the previously constructed single-chain Fv library (Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., and Virnekas, B . (2000) J . Mol . Biol . 296, 57-86), diversified in both complementarity-determining regions 3 into a novel Fab display vector, yielding 2.1 x 10(10) different antibody fragments . The modularity has been retained in the Fab display and screening plasmids, ensuring rapid conversion into various antibody formats as well as antibody optimization using prebuilt maturation cassettes . HuCAL-Fab 1 was challenged against the human fibroblast growth factor receptor 3, a potential therapeutic antibody target, against which, to the best of our knowledge, no functional antibodies could be generated so far . A unique screening mode was designed utilizing recombinant functional proteins and cell lines differentially expressing fibroblast growth factor receptor isoforms diversified in expression and receptor dependence . Specific Fab fragments with subnanomolar affinities were isolated by selection without any maturation steps as determined by fluorescence flow cytometry . Some of the selected Fab fragments completely inhibit target-mediated cell proliferation, rendering them the first monoclonal antibodies against fibroblast growth factor receptors having significant function blocking activity . This study validates HuCAL-Fab 1 as a valuable source for the generation of target-specific antibodies for therapeutic applications. Arterioscler Thromb Vasc Biol, 2003 Sep 1, 23(9), 1677 - 83 Epub 2003 Jul 03. Chlamydia pneumoniae binds to platelets and triggers P-selectin expression and aggregation: a causal role in cardiovascular disease? Kalvegren H, Majeed M, Bengtsson T. OBJECTIVE: Evidence linking Chlamydia pneumoniae to atherosclerotic cardiovascular disease is expanding . Platelets are considered to play an essential role in cardiovascular diseases; however, so far platelets have not been associated with an infectious cause of atherosclerosis . This study aims to clarify the interaction between C pneumoniae and platelets and possibly present a novel mechanism in the pathogenesis of atherosclerosis . METHODS AND RESULTS: The effects of C pneumoniae on platelet aggregation and secretion were assessed with lumiaggregometry, and the ability of C pneumoniae to bind to platelets and stimulate expression of P-selectin was analyzed with flow cytometry . We found that C pneumoniae, at a chlamydia:platelet ratio of 1:15, adheres