Am J Physiol Lung Cell Mol Physiol, 2004 Sep, 287(3), L552 - 8 Epub 2004 May 21. CpG DNA-mediated immune response in pulmonary endothelial cells; Li J et al.; Although the CpG DNA immune response mediated by Toll-like receptor 9 (TLR9) has been extensively studied in a number of immune cells, the response to CpG DNA in endothelial cells (EC) is not well understood . In this study, we show that both mouse and rat lung EC display constitutive expression of TLR9 mRNA . Exposure to CpG DNA induced a potent proinflammatory response as manifested by an increased expression of IL-8 and ICAM-1 in mouse pulmonary EC . The proinflammatory response was sensitive to chloroquine, consistent with a role of endosomal contribution . A role for p38 MAPK and NF-kappaB pathway was apparent as the response was sensitive to inhibitors of p38 MAPK and NF-kappaB but was not affected by inhibitors of ERK1/2 . A synergistic effect of CpG DNA and LPS on the inflammatory response is consistent with multiple TLR interaction in EC . This study suggests a possible role for CpG DNA-mediated EC immune response in the host defense system . It also has important implications in plasmid DNA-mediated pulmonary endothelium gene transfer. Antimicrob Agents Chemother, 2004 Jun, 48(6), 2308 - 13 High-level resistance to ceftazidime conferred by a novel enzyme, CTX-M-32, derived from CTX-M-1 through a single Asp240-Gly substitution; Cartelle M et al.; A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (bla(CTX-M-32)) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution . Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis Expert Opin Biol Ther, 2004 May, 4(5), 719 - 28 Plant-derived vaccines against diarrhoeal diseases; Tacket CO; Transgenic plant-derived vaccines offer a new strategy for the development of safe, inexpensive vaccines against diarrhoeal diseases . In animal and Phase I clinical studies, these vaccines have been safe and immunogenic without the need for a buffer or vehicle other than the plant cell . This review examines some early attempts to develop oral transgenic plant vaccines against enteric infections such as enterotoxigenic Escherichia coli infection, cholera and norovirus infection. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 May, 19(3), 248 - 51 {Study on the preparing of polyclonal antibodies against plant-selected maker gene hpt expression protein by DNA immunization}; Yang LC et al.; AIM: To prepare the polyclonal antibodies (pAbs) against HPT, a kind of plant-selected maker gene encoding protein, by DNA immunization technique and explore the influencing factors for this gene immunization . METHODS: The coding sequence of hpt was cloned into eukaryotic expression vector pCDNA3 . The sequence of the plasmid pCDNA3-HPT was demonstrated by restricting enzyme digestion analysis and DNA sequencing . The sequence-correct recombinant plasmids were purified and used to immunize BALB/c mice . The titer and specificity of antisera were detected by ELISA and Western blot, respecitively . RESULTS: No pAb against HPT was detected following three immunization with hpt genes . Then mice were divided into three groups when the forth booster immunization was carried out: 1st group (immunization with the endotoxin-free recombinant plasmids), 2nd group (immunization with (His)(6)-HPT fusion protein expressed in E.coli) and 3rd group (immunization with the same plasmids as before mentioned) . As a result, the pAb titer of the 1st group mice increased to 1:200, and the that of 2nd group was up to 1:2 000 . Yet the 3rd group detected no anti-HPT antibody . Western blot analysis had proved that antisera of the first two groups could produce specific binding reaction to the purified GST-HPT, (His)(6)-HPT protein and their expressed product (bacterial protein) . CONCLUSION: We have got successfully the specific pAb against HPT by DNA immunization, but its titer is yet unsatisfactory, inferring that the character of the hpt gene self and its expression level may play an important role . In order to raise level of serum antibody prepared by DNA immunization, the farther study on various influencing factors still need to be performed. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 May, 19(3), 215 - 7 {Expression of amino terminal of nucleoprotein and glycoprotein G2 of Hantaan virus in insect cells in the form of fusion protein}; Liu Y et al.; AIM: To express the glycoprotein G2 and amino terminal of nucleoprotein (NP) of Hantaan virus in Bac-to-Bac baculovirus expression system in the form of fusion protein . METHODS: The recombinant baculovirus expression vector pFBDHTa-G2S 0.7 was constructed . The chimeric gene was inserted into bacmid in E.coli DH10Bac with the help of Tn7 transposition system . Then the recombinant baculovirus was screened and the fusion protein was expressed in insect cells . The expression product was detected by ELISA, immunofluorescence assay and Western blot analysis . RESULTS: The recombinant baculovirus containing the chimeric gene G2S 0.7 had been constructed successfully and the fusion protein could be expressed in insect cells . The expressed protein could be recognized by the Hantaan virus NP-specific mAb and glycoprotein G2-specific mAb . CONCLUSION: The successful expression of fusion protein G2S 0.7 with biological activity in insect cells lays the foundation for further research on its immunological characteristic. Biochem J, 2004 Aug 1, 381(Pt 3), 685 - 91 The CBP/p300 TAZ1 domain in its native state is not a binding partner of MDM2; Matt T et al.; The transcriptional co-activator CBP {CREB (cAMP-response-element-binding protein)-binding protein} and its paralogue p300 play a key role in the regulation of both activity and stability of the tumour suppressor p53 . Degradation of p53 is mediated by the ubiquitin ligase MDM2 (mouse double minute protein) and is also reported to be regulated by CBP/p300 . Direct protein-protein interaction between a central domain of MDM2 and the TAZ1 (transcriptional adaptor zinc-binding domain) {C/H1 (cysteine/histidine-rich region 1)} domain of p300 and subsequent formation of a ternary complex including p53 have been reported previously . We expressed and purified the proposed binding domains of HDM2 (human homologue of MDM2) and CBP, and examined their interactions using CD spectroscopy . The binding studies were extended by using natively purified GST (glutathione S-transferase)-p300 TAZ1 and GST-p53 fusion proteins, together with in vitro translated HDM2 fragments, under similar solution conditions to those in previous studies, but omitting added EDTA, which causes unfolding and aggregation of the zinc-binding TAZ1 domain . Comparing the binding properties of the known TAZ1 interaction partners HIF-1alpha (hypoxia-inducible factor 1), CITED2 (CBP/p300-interacting transactivator with glutamic- and aspartic-rich tail) and STAT2 (signal transducer and activator of transcription 2) with HDM2, our data suggest that TAZ1 in its native state does not serve as a specific recognition domain of HDM2 . Rather, unfolded TAZ1 and HDM2 proteins have a high tendency to aggregate, and non-specific protein complexes are formed under certain conditions. J Mol Microbiol Biotechnol, 2003, 6(3-4), 133 - 44 Enhancement of translation initiation by A/T-rich sequences downstream of the initiation codon in Escherichia coli; Qing G et al.; The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E . coli . In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene . A total of 119 random clones showing higher beta-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced . Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them . Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation . Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E . coli gene expression . Methods Mol Biol, 2004, 270, 319 - 34 Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments; Chusacultanachai S et al.; The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy . This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP) . These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity . The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step . After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication . The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations . This strategy is suitable for generation of random mutations with low frequency in a large target DNA . Error-prone PCR is one of the most widely used random mutagenesis . During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction . We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained . Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries . DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides . This strategy uses two chemically synthesized degenerate oligonucleotides as primers . By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to control both the position and rate of mutation in degenerated region of the primers . The primers are integrated into newly synthesized plasmid DNA by primer extension reaction using Pfu DNA polymerase . After plasmid DNA template encoding wild-type sequence is eliminated from the reaction by DpnI digestion, the pool of mutagenized plasmids can then be used directly in screening procedures. Methods Mol Biol, 2004, 270, 299 - 318 In vitro shuttle mutagenesis using engineered mariner transposons; Robinson KA et al.; Advances in our understanding of the protozoan parasite Leishmania have been facilitated by the development of molecular and genetic tools . One powerful approach for gene identification and analysis is transposon mutagenesis . This can be performed directly in vivo, but often it is more convenient to generate transpositions in vitro for subsequent analysis in vivo, in a process termed "shuttle mutagenesis." The Drosophila element mariner is well suited for application by either route . Minimal mariner elements containing cis-acting elements required for transposition have been generated, which can be further modified to suit the needs of the experimenter . Additional genetic markers and/or reporters can be introduced, which are useful for procedures such as insertional mutagenesis, shotgun sequencing, or the generation of protein and transcriptional fusions for subsequent analysis . Active transposase can readily be generated following expression in Escherichia coli, and efficiencies of 10-3/target can be obtained, allow-ing the generation of large transposon insertion libraries suitable for subsequent screening in vivo . This chapter explains the steps necessary to purify active Mos1 transposase and conduct an in vitro transposition reaction . We also discuss some of the considerations relevant to the design and application of functional mariner elements (donor plasmids) relevant to studies in Leishmania and other organisms. Artif Organs, 2004 Jun, 28(6), 529 - 36 Maintenance of glucose-sensitive insulin secretion of cryopreserved human islets with University of Wisconsin solution and ascorbic acid-2 glucoside; Arata T et al.; Normal human islet cells are an ideal source for pancreas-targeted cell therapies, but the availability of human donor pancreata for islet isolation is severely limited . To effectively utilize such scarce donor organs for cell therapies, it is crucial to develop an excellent isolation, effective cryopreservation, and efficient gene transfer techniques for the transportation of isolated cells . In the present study, we investigate the effect of University of Wisconsin (UW) solution and ascorbic acid-2 glucoside (AA2G) on the cryopreservation of human islets . We also evaluate the gene transfer efficiency of a lentiviral vector expressing the E . coli LacZ gene, Lt-NLS/LacZ, in human islets . Human islets were isolated with a standard digestion method at the University of Alberta . Isolated islets were transported to Japan for 40 h and then subjected to cryopreservation experiments . The following preservation solutions were tested: UW solution with 100 micro g/mL of AA2G, UW solution, 100% fetal bovine serum (FBS), and CMRL supplemented with 10% FBS . Following three months of cryopreservation, the islets were thawed and analyzed for viability, glucose-sensitive insulin secretion, proinsulin gene expression profile, and in vivo engraftment . The islets were also subjected to monolayer formation with 804G-cell-line-derived extracellular matrix (ECM), followed by Lt-NLS/LacZ transduction . The viability, morphology, glucose-sensitive insulin secretion, proinsulin gene expression, and monolayer formation efficiency of the thawed cryopreserved islets are significantly better maintained by the use of UW solution . When AA2G (100 microg/mL) is combined with UW, such parameters are further improved . The adequate engraftment of UW + AA2G-cryopreserved human islets is achieved in the liver of nude mice . Efficient Lt-NLS/LacZ transduction is identified in monolayered islets cryopreserved with UW solution with AA2G . The present work demonstrates that the combination of UW solution with AA2G (100 microg/mL) would be a useful cryopreservation means for human islets . Human islets monolayer-cultured with 804G-derived ECM are efficiently transduced with a lentiviral vector Lt-NLS/LacZ. J Vet Diagn Invest, 2004 May, 16(3), 191 - 6 Development of quantitative competitive-reverse transcriptase-polymerase chain reaction for detection and quantitation of avian leukosis virus subgroup J; Kim Y et al.; Infection with avian leukosis virus subgroup J (ALV-J) causes severe economic losses in the broiler industry by increasing mortality, producing tumors, and decreasing weight gain in chickens . The quantitation of ALV-J is difficult because of its failure to produce a cytopathic effect in cell culture systems and the nonspecificity of antigen-capture enzyme-linked immunosorbent assay (ELISA) tests . This study was performed to develop a quantitative competitive-reverse transcriptase-polymerase chain reaction (QC-RT-PCR) method based on coamplification of ALV-J genomic RNA and a known amount of a synthesized RNA competitor . The 369 bp RNA competitor was constructed by restriction enzyme treatment of an ALV-J specific 545 bp PCR product, ligation, transformation into Escherichia coli, and in vitro transcription . The competitor contained the same amplification primer annealing sites and sequence as the original viral RNA, except that it had a 176 bp internal deletion . Coamplified RT-PCR products were visualized by electrophoresis and ethidium bromide staining, and fluorescences were quantified using computer-assisted image analysis . The sensitivity of this new QC-RT-PCR method was 25 fg of viral RNA, and 10-fold dilutions were differentiable . This method allowed absolute and relative quantification of ALV-J RNA copy numbers and was simpler than previously published methods for ALV-J quantification. Biotechniques, 2004 May, 36(5), 878 - 85 Evaluation of the CFP-substrate-YFP system for protease studies: advantages and limitations; Felber LM et al.; A protease can be defined as an enzyme capable of hydrolyzing peptide bonds . Thus, characterization of a protease involves identification of target peptide sequences, measurement of activities toward these sequences, and determination of kinetic parameters . Biological protease substrates based on fluorescent protein pairs, which allow for use of fluorescence resonance energy transfer (FRET), have been recently developed for in vivo protease activity detection and represent a very interesting alternative to chemical substrates for in vitro protease characterization . Here, we analyze a FRET system consisting of cyan and yellow fluorescent proteins (CFP and YFP, respectively), which are fused by a peptide linker serving as protease substrate . Conditions for CFP-YFP fusion protein production in Escherichia coli and purification of proteins were optimized . FRET between CFP and YFP was found to be optimum at a pH between 5.5 and 10.0, at low concentrations of salt and a temperature superior to 25 degrees C . For efficient FRET to occur, the peptide linker between CFP and YFP can measure up to 25 amino acids . The CFP-substrate-YFP system demonstrated a high degree of resistance to nonspecific proteolysis, making it suitable for enzyme kinetic analysis . As with chemical substrates, substrate specificity of CFP-substrate-YFP proteins was tested towards different proteases and kcat/Km values were calculated. Biotechniques, 2004 May, 36(5), 864 - 70 Linker peptide and affinity tag for detection and purification of single-chain Fv fragments; Kuttner G et al.; The peptide tag GATPQDLNTML, corresponding to amino acids 46-56 of the human immunodeficiency virus type 1 (HIV-1) capsid protein p24, is the linear epitope of the murine monoclonal antibody CB4-1 . This antibody shows high affinity (KD = 1.8 x 10(-8) M) to the free epitope peptide in solution . The original p24 peptide tag and mutant derivatives were fused to the C terminus of a single-chain antibody (scFv) and characterized with respect to sensitivity in Western blot analyses and behavior in purification procedures using affinity chromatography . The p24 tag also proved to be a suitable alternative to the (Gly4Ser)3 linker commonly used to connect single-chain antibody variable regions derived from a heavy (VH) and light chain (VL) . Binding of CB4-1 antibody to the p24 tag was not hampered when the tag was located internally in the protein sequence, and the specific antigen affinity of the scFv was only slightly reduced . All scFv variants were solubly expressed in Escherichia coli and could be purified from the periplasm . Our results highlight the p24 tag as a useful tool for purifying and detecting recombinantly expressed scFvs. Analyst, 2004 Jun, 129(6), 529 - 34 Epub 2004 Apr 26. Respiration activity of Escherichia coli entrapped in a cone-shaped microwell and cylindrical micropore monitored by scanning electrochemical microscopy (SECM); Kaya T et al.; The metabolic activity of E . coli cells embedded in collagen gel microstructures in a cone-shaped well and in a cylindrical micropore was investigated using scanning electrochemical microscopy (SECM), based on the oxygen consumption rate and the conversion rate from ferrocyanide to ferricyanide . The analysis of the concentration profiles for oxygen and ferrocyanide afforded the oxygen consumption rate and the ferrocyanide production rate . A comparison indicated that the ferrocyanide production rates were larger than the oxygen consumption rate, and also that the rates observed in the cylindrical micropore were larger than those observed in the cone-shaped well . The ferrocyanide production rate of a single E . coli cell was calculated to be (5.4 +/- 2.6) x 10(-19) mol s(-1), using a cylindrical micropore system. Protein Sci, 2004 Jun, 13(6), 1458 - 65 Solution structure of the hypothetical protein Mth677 from Methanobacterium thermoautotrophicum: a novel alpha+beta fold; Blanco FJ et al.; The structure of Mth677, a hypothetical protein from Methanobacterium thermoautotrophicum (Mth), has been determined by using heteronuclear nuclear magnetic resonance (NMR) methods on a double-labeled (15)N-(13)C sample . Mth677 adopts a novel alpha+beta fold, consisting of two alpha-helices (one N terminal and one C terminal) packed on the same side of a central beta-hairpin . This structure is likely shared by its three orthologs, detected in three other Archaebacteria . There are no clear features in the sequences of these proteins or in the genome organization of Mth to make a reliable functional assignment to this protein . However, the structural similarity to Escherichia coli MinE, the protein which controls that division occurs at the midcell site, lends support to the proposal that Mth677 might be, in Mth, the counterpart of the topological specificity domain of MinE in E . coli. J Biol Chem, 2004 Jul 30, 279(31), 32674 - 83 Epub 2004 May 19. Mutants in a small heat shock protein that affect the oligomeric state . Analysis and allele-specific suppression; Giese KC et al.; Oligomerization is an essential property of small heat shock proteins (sHSPs) that appears to regulate their chaperone activity . We have examined the role of conserved hydrophobic residues that are postulated to stabilize sHSP oligomers . We identified a mutation of Synechocystis Hsp16.6 that impairs function in vivo and in vitro . The V143A mutation is in the C-terminal extension, a region predicted to form an oligomeric interaction with a hydrophobic region that includes the site of a previously characterized mutation, L66A . Both mutants were dimeric, but V143A had a stronger oligomerization defect than L66A . However, V143A protected a model substrate better than L66A . This suggests that although the two regions both play a role in oligomerization, they are not equivalent . Nevertheless, the addition of either dimeric sHSP enhanced the in vitro chaperone activity of wild type Hsp16.6, consistent with models that the sHSP dimers initiate interactions with substrates . Suppressor analysis of V143A identified mutations in the N terminus that restored activity by restabilizing the oligomer . These mutants were allele-specific and unable to suppress L66A, although they suppressed a dimeric C-terminal truncation of Hsp16.6 . Conversely, suppressors of L66A were unable to suppress either V143A or the truncation, although they, like suppressors of V143A, stabilize the Hsp16.6 oligomer . We interpret these data as evidence that the mutations V143A and L66A stabilize two different dimeric structures and as further support that sHSP dimers are active species. J Biol Chem, 2004 Jul 23, 279(30), 31964 - 72 Epub 2004 May 19. Human immunodeficiency virus type 1 Gag assembly through assembly intermediates; Morikawa Y et al.; Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process . To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction . When E . coli expression was performed at 37 degrees C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S . Electron microscopy confirmed the presence of spherical particles in the E . coli cells . In contrast, expression at 30 degrees C resulted in the production of only the 60 S form of Gag multimer, and crescent-shaped structures or small patches with double electron-dense layers were accumulated, but no complete particles . In vitro assembly reactions using purified Gag protein, when performed at 37 degrees C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30 degrees C reaction produced only the 60 S multimers . However, when the 60 S multimers were cross-linked so as not to allow conformational changes, in vitro assembly reactions at 37 degrees C did not produce any higher order of multimers . ATP depletion did not halt Gag assembly in the E . coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37 degrees C, were solely responsible for the Gag assembly observed here . We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag. J Biol Chem, 2004 Aug 20, 279(34), 35849 - 57 Epub 2004 May 19. Activation and proteasomal degradation of rho GTPases by cytotoxic necrotizing factor-1 elicit a controlled inflammatory response; Munro P et al.; The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli . CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells . CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation . Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin . The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response . Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3alpha (MIP-3alpha) and E-selectin . Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3alpha, MCP-1, and IL-8 . These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially . Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert . Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses. J Biol Chem, 2004 Jul 16, 279(29), 30236 - 43 Epub 2004 May 18. Binding of SeqA protein to hemi-methylated GATC sequences enhances their interaction and aggregation properties; Han JS et al.; The SeqA protein regulates chromosome initiation and is involved in segregation in Escherichia coli . One SeqA protein binds to two hemi-methylated GATC sequences to form a stable SeqA-DNA complex . We found that binding induced DNA bending, which was pronounced when the two sequences were on the same face of the DNA . Two SeqA molecules bound cooperatively to each pair of hemi-methylated sites when the spacing between the sites was < or = 30 bp . This cooperative binding was able to stabilize the binding of a wild type to a single hemi-methylated site, or mutant form of SeqA protein to hemi-methylated sites, although such binding did not occur without cooperative interaction . Two cooperatively bound SeqA molecules interacted with another SeqA bound up to 185 bp away from the two bound SeqA proteins, and this was followed by aggregation of free SeqA proteins onto the bound proteins . These results suggest that the stepwise interaction of SeqA proteins with hemi-methylated GATC sites enhances their interaction and leads to the formation of SeqA aggregates . Cooperative interaction followed by aggregation may be the driving force for formation of the SeqA foci that appear to be located behind replication forks. J Med Microbiol, 2004 Jun, 53(Pt 6), 573 - 9 Phenotypic and functional characterization of intraepithelial lymphocytes in a bovine ligated intestinal loop model of enterohaemorrhagic Escherichia coli infection; Menge C et al.; Ruminants are a major reservoir of enterohaemorrhagic Escherichia coli (EHEC), which cause acute gastroenteritis in humans with potentially life-threatening sequelae . The mechanisms underlying EHEC persistence in ruminant hosts are poorly understood . EHEC produce several cytotoxins that inhibit the proliferation of bovine lymphocytes in vitro and influence EHEC persistence in calves, suggesting that bacterial suppression of mucosal inflammation may be important in vivo . In order to address this hypothesis, intraepithelial lymphocytes (IEL) obtained from ligated intestinal loops of five 9-14 day old calves were characterized 12 h after inoculation with E . coli strains . Loops were inoculated with an EHEC O103 : H2 strain, an isogenic Deltastx1 mutant incapable of producing Shiga toxin 1 (Stx1) and a porcine non-pathogenic E . coli strain . The IEL mainly comprised activated CD2(+) CD3(+) CD6(+) CD8alpha(+) T cells and resembled IEL obtained from the intestinal mucosa of orally challenged calves . Forty per cent of all IEL were potentially sensitive to Stx1 in that they expressed the receptor for Stx1 . Nevertheless, analysis of IEL from inoculated loops failed to detect a significant effect of the different E . coli strains on proliferative capacity, natural killer cell activity or the cytokine mRNA profile . However, the EHEC wild-type strain reduced the percentage of CD8alpha(+) T cells in the ileal mucosa compared with loops inoculated with the Deltastx1 mutant . This shift in IEL composition was not associated with inhibition of IEL proliferation in situ, since the majority of the IEL from all loops were in the G(0)/G(1) phase of the cell cycle . These studies indicate that the ligated ileal loop model will be a useful tool to dissect the mechanisms underlying suppression of mucosal inflammation by EHEC in the reservoir host. J Cell Sci, 2004 Jun 1, 117(Pt 13), 2757 - 67 Epub 2004 May 18. Two classic cadherin-related molecules with no cadherin extracellular repeats in the cephalochordate amphioxus: distinct adhesive specificities and possible involvement in the development of multicell-layered structures; Oda H et al.; We previously reported the existence of Bb-cadherin, a molecule related to classic cadherin, in the cephalochordate amphioxus (Branchiostoma belcheri) . The structure of Bb-cadherin is unique in that it lacks the cadherin extracellular repeats, although its cytoplasmic domain shows close similarities to those of typical classic cadherins . The extracellular region of Bb-cadherin consists of laminin globular domains and a cysteine-rich EGF-like domain that are similar to domains in nonchordate classic cadherins . In this study, we identified a second amphioxus cadherin . It was designated Bb2-cadherin (Bb2C) while the previously reported cadherin has been renamed Bb1-cadherin (Bb1C) . Bb2C is very similar to Bb1C in its overall structure and amino acid sequence . Genomic BLAST searches and phylogenetic analyses suggested that these two amphioxus genes have been generated through a gene duplication that occurred after separation of the cephalochordates from the other animals . They also bear distinct adhesive specificities . Immunohistochemical analyses showed that Bb1C and Bb2C, together with beta-catenin, appear to function as adherens junction constituents in the epithelia of different germ layers of the amphioxus embryo . Differential expression of the two cadherins was also observed in the developing, multicell-layered notochord . These observations suggest that, despite their unique structures, the functions and developmental roles of Bb1C and Bb2C are comparable to those of the classic cadherins characterized to date in other animal groups, such as the vertebrate E- and N-cadherins and the Drosophila DE- and DN-cadherins . The possible involvement of Bb1C and Bb2C in the development of multicell-layered structures characteristic of the cephalochordate body plan is presented. J Biol Chem, 2004 Jul 23, 279(30), 31505 - 13 Epub 2004 May 18. Mutagenesis of residue betaArg-246 in the phosphate-binding subdomain of catalytic sites of Escherichia coli F1-ATPase; Ahmad Z et al.; Residues responsible for phosphate binding in F(1)F(0)-ATP synthase catalytic sites are of significant interest because phosphate binding is believed linked to proton gradient-driven subunit rotation . From x-ray structures, a phosphate-binding subdomain is evident in catalytic sites, with conserved betaArg-246 in a suitable position to bind phosphate . Mutations betaR246Q, betaR246K, and betaR246A in Escherichia coli were found to impair oxidative phosphorylation and to reduce ATPase activity of purified F(1) by 100-fold . In contrast to wild type, ATPase of mutants was not inhibited by MgADP-fluoroaluminate or MgADP-fluoroscandium, showing the Arg side chain is required for wild-type transition state formation . Whereas 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by approximately 50%, although reaction still occurred at residue betaTyr-297, proximal to betaArg-246 in the phosphate-binding pocket . Inhibition characteristics supported the conclusion that NBD-Cl reacts in betaE (empty) catalytic sites, as shown previously by x-ray structure analysis . Phosphate protected against NBD-Cl inhibition in wild type but not in mutants . The results show that phosphate can bind in the betaE catalytic site of E . coli F(1) and that betaArg-246 is an important phosphate-binding residue. J Bacteriol, 2004 Jun, 186(11), 3663 - 9 Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions; Hazan R et al.; mazEF is an Escherichia coli suicide module specific for a stable toxin and a labile antitoxin . Inhibiting mazEF expression appeared to activate the module to cause cell death . Here we show that several stressful conditions, including high temperatures, DNA damage, and oxidative stress, also induce mazEF-mediated cell death . We also show that this process takes place only during logarithmic growth and requires an intact relA gene. J Bacteriol, 2004 Jun, 186(11), 3578 - 89 Recognition of ferric catecholates by FepA; Annamalai R et al.; Escherichia coli FepA transports certain catecholate ferric siderophores, but not others, nor any noncatecholate compounds . Direct binding and competition experiments demonstrated that this selectivity originates during the adsorption stage . The synthetic tricatecholate Fe-TRENCAM bound to FepA with 50- to 100-fold-lower affinity than Fe-enterobactin (FeEnt), despite an identical metal center, and Fe-corynebactin only bound at much higher concentrations . Neither Fe-agrobactin nor ferrichrome bound at all, even at concentrations 10(6)-fold above the Kd . Thus, FepA only adsorbs catecholate iron complexes, and it selects FeEnt among even its close homologs . We used alanine scanning mutagenesis to study the contributions of surface aromatic residues to FeEnt recognition . Although not apparent from crystallography, aromatic residues in L3, L5, L7, L8, and L10 affected FepA's interaction with FeEnt . Among 10 substitutions that eliminated aromatic residues, Kd increased as much as 20-fold (Y481A and Y638A) and Km increased as much as 400-fold (Y478), showing the importance of aromaticity around the pore entrance . Although many mutations equally reduced binding and transport, others caused greater deficiencies in the latter . Y638A and Y478A increased Km 10- and 200-fold more, respectively, than Kd . N-domain loop deletions created the same phenotype: Delta60-67 (in NL1) and Delta98-105 (in NL2) increased Kd 10- to 20-fold but raised Km 500- to 700-fold . W101A (in NL2) had little effect on Kd but increased Km 1,000-fold . These data suggested that the primary role of the N terminus is in ligand uptake . Fluorescence and radioisotopic experiments showed biphasic release of FeEnt from FepA . In spectroscopic determinations, k(off1) was 0.03/s and k(off2) was 0.003/s . However, FepAY272AF329A did not manifest the rapid dissociation phase, corroborating the role of aromatic residues in the initial binding of FeEnt . Thus, the beta-barrel loops contain the principal ligand recognition determinants, and the N-domain loops perform a role in ligand transport. J Bacteriol, 2004 Jun, 186(11), 3516 - 24 Transcriptome analysis of Crp-dependent catabolite control of gene expression in Escherichia coli; Gosset G et al.; We report here the transcriptome analyses of highly expressed genes that are subject to catabolite repression or activation mediated by the cyclic AMP receptor protein (Crp) . The results reveal that many operons encoding enzymes of central carbon metabolic pathways (e.g., Krebs cycle enzymes), as well as transporters and enzymes that initiate carbon metabolism, are subject to direct Crp-mediated catabolite repression . By contrast, few enzyme-encoding genes (direct regulation) but many ribosomal protein- and tRNA-encoding genes (indirect regulation) are subject to Crp-dependent glucose activation . Additionally, Crp mediates strong indirect catabolite repression of many cytoplasmic stress response proteins, including the major chaperone proteins, five ATP-dependent protease complexes, and several cold and heat shock proteins . These results were confirmed by (i) phenotypic analyses, (ii) real-time PCR studies, (iii) reporter gene fusion assays, and (iv) previously published reports about representative genes . The results serve to define and extend our appreciation of the Crp regulon. J Bacteriol, 2004 Jun, 186(11), 3508 - 15 Interaction of the sliding clamp beta-subunit and Hda, a DnaA-related protein; Kurz M et al.; In Escherichia coli, interactions between the replication initiation protein DnaA, the beta subunit of DNA polymerase III (the sliding clamp protein), and Hda, the recently identified DnaA-related protein, are required to convert the active ATP-bound form of DnaA to an inactive ADP-bound form through the accelerated hydrolysis of ATP . This rapid hydrolysis of ATP is proposed to be the main mechanism that blocks multiple initiations during cell cycle and acts as a molecular switch from initiation to replication . However, the biochemical mechanism for this crucial step in DNA synthesis has not been resolved . Using purified Hda and beta proteins in a plate binding assay and Ni-nitrilotriacetic acid pulldown analysis, we show for the first time that Hda directly interacts with beta in vitro . A new beta-binding motif, a hexapeptide with the consensus sequence QL{SP}LPL, related to the previously identified beta-binding pentapeptide motif (QL{SD}LF) was found in the amino terminus of the Hda protein . Mutants of Hda with amino acid changes in the hexapeptide motif are severely defective in their ability to bind beta . A 10-amino-acid peptide containing the E . coli Hda beta-binding motif was shown to compete with Hda for binding to beta in an Hda-beta interaction assay . These results establish that the interaction of Hda with beta is mediated through the hexapeptide sequence . We propose that this interaction may be crucial to the events that lead to the inactivation of DnaA and the prevention of excess initiation of rounds of replication. J Bacteriol, 2004 Jun, 186(11), 3423 - 30 Cloning and functional analysis by gene disruption of a gene encoding a gamma-butyrolactone autoregulator receptor from Kitasatospora setae; Choi SU et al.; Gamma-butyrolactone autoregulator receptors of the genus Streptomyces have a common activity as DNA-binding transcriptional repressors, controlling secondary metabolism and/or morphological differentiation . A gene encoding a gamma-butyrolactone autoregulator receptor was cloned from a bafilomycin B1 producer, Kitasatospora setae, for the first time from a non-Streptomyces genus of actinomycetes, and its function was evaluated by in vitro and in vivo analyses . The gene fragment was initially cloned by PCR with primers designed from two highly conserved regions of Streptomyces autoregulator receptors (BarA, FarA, ScbR, and ArpA), followed by genomic Southern hybridization yielding a 7-kb BamHI fragment on which a 654-bp receptor gene (ksbA) was identified . The recombinant KsbA protein demonstrated clear binding activity toward 3H-labeled autoregulators, especially toward {3H}SCB1, confirming that ksbA encodes a real autoregulator receptor of K . setae . To clarify the in vivo function of ksbA, a ksbA-disrupted strain was constructed by means of homologous recombination after introducing a ksbA disruption construct via transconjugation from Escherichia coli . No difference in morphology was found between the wild-type strain and the ksbA disruptants . However, the ksbA disruptants started producing bafilomycin 18 h earlier than the wild-type strain and showed a 2.4-fold-higher accumulation of bafilomycin . The phenotype was restored to the original wild-type phenotype by complementation with intact ksbA, indicating that the autoregulator receptor protein of K . setae acts as a primary negative regulator of the biosynthesis of bafilomycin but plays no role in cytodifferentiation of K . setae . This indicates that, unlike the A-factor receptor of Streptomyces griseus, the autoregulator receptor (ksbA) of K . setae belongs to a family of autoregulator receptors which control secondary metabolism but play no role in morphological differentiation. J Inorg Biochem, 2004 Jun, 98(6), 1032 - 6 Studies on the interaction of Escherichia coli agmatinase with manganese ions: structural and kinetic studies of the H126N and H151N variants; Salas M et al.; The H126N and H151N variants of Escherichia coli agmatinase (EC 3.5.3.11) were produced by site-directed mutagenesis, and their kinetic and structural properties were examined . About 51% and 30% of wild-type activity were expressed by fully manganese activated species of the H126N and H151N variants, respectively . Mutations were not accompanied by changes in the K(m) value for arginine (1.2+/-0.3 mM), K(i) value for putrescine inhibition (3.2+/-0.4 mM), molecular weight (M(r) 67,000+/-2000), tryptophan fluorescence properties (lambda(max) = 342 nm) or CD spectra of the enzyme . However, the interaction with the required manganese ions was significantly altered, as indicated by the effects of dialysis of the enzymes against metal-free buffer . We conclude that replacement of His151 with asparagine results in the loss of a catalytically essential Mn(2+) upon dialysis and concomitant reversible inactivation of the H151N mutant, and that the affinity of a more weakly bound Mn(2+) is decreased in the H126N variant. J Inorg Biochem, 2004 Jun, 98(6), 925 - 30 Mannose-Escherichia coli interaction in the presence of metal cations studied in vitro by colorimetric polydiacetylene/glycolipid liposomes; Sun C et al.; Supramolecular assemblies of liposomes (vesicles) made of diacetylenic lipids and synthetic mannoside derivative glycolipid receptors were successfully used to mimic the molecular recognition occurring between mannose and Escherichia coli . This specific molecular recognition was translated into visible blue-to-red color transition (biochromism) of the polymerized liposomes, readily quantified by UV-visible spectroscopy . Some transition metal cations (Cd(2+), Ag(+), Cu(2+), Fe(3+), Zn(2+) and Ni(2+)) and alkali earth metal cations (Ca(2+), Mg(2+) and Ba(2+)) were introduced into the system to analyze their effects on specific biochromism . Results showed that the presence of Cd(2+), Ag(+), Ca(2+), Mg(2+) and Ba(2+) enhanced biochromism . A possible enhancement mechanism was proposed in the process of bacterial adhesion to host cells . However, Cu(2+), Fe(3+), Zn(2+) and Ni(2+) exhibited inhibitory effects that cooperated with diacetylene lipid with a carboxylic group and increased the rigidity of the liposomal outer leaflet, blocking changes in the side chain conformation and electrical structure of polydiacetylene polymer during biochromism. Reprod Biomed Online, 2004 Apr, 8(4), 398 - 407 Flow cytometry and microscopic acridine orange test: relationship with standard semen analysis; Apedaile AE et al.; Improved prediction of male fertility requires advances in semen analysis . This study examined the reproducibility and independence of the flow cytometry acridine orange test (FCM-AOT) of sperm chromatin integrity as an assessment of semen quality . The study found that FCM-AOT results are not significantly affected by up to 6 h delay in semen preparation (n = 9) or contamination of semen with moderate concentrations of bacteria (<10(8)/ml E . coli or Staph . epidermidis, n = 14) . The variation of replicate measurements within samples was low (%Abnormal alpha(t): SD = 1.4, 95%CI = 4.6, n = 25) and different samples from the same men were mostly within the range of measurement error (n = 35) . FCM-AOT variables, in particular %Abnormal alpha(t), displayed significant correlations with motility (r = -0.557), vitality (r = -0.469) and morphology (r = -0.464, n = 201), which are similar in magnitude to those existing between the standard semen variables . Surprisingly, no correlation was found between %Abnormal alpha(t) and the microscopic acridine orange test (M-AOT) (n = 185), suggesting the FCM results are sensitive to a different aspect of sperm quality . In summary, this study confirms that although not totally independent of standard semen analysis or the M-AOT, it is found to be a robust, sensitive and reproducible measure of semen quality, representative of the individual. Kidney Int, 2004 Jun, 65(6), 2184 - 92 In vitro study of the potential role of guanidines in leukocyte functions related to atherogenesis and infection; Glorieux GL et al.; BACKGROUND: The blunted immune response upon stimulation in chronic renal failure (CRF) is often coupled to a baseline inflammatory status which has been related to atherogenesis . Uremic biologic fluids and several specific uremic retention solutes alter cell-mediated immune responses, as well as the interaction of calcitriol with the immune system . METHODS: The present study evaluated the influence of different guanidino compounds on DNA synthesis, chemiluminescence production, and CD14 expression of undifferentiated and calcitriol-differentiated HL-60 cells . In a second setup, these guanidino compounds were evaluated for their specific effect on normal human leukocyte oxidative burst activity and tumor necrosis factor-alpha (TNF-alpha) expression . RESULTS: First, several guanidino compounds elicited proinflammatory effects on leukocytes . Methylguanidine and guanidine stimulated the proliferation of undifferentiated HL-60 cells and the antiproliferative effect of calcitriol (P < 0.05) was neutralized in the presence of methylguanidine (P < 0.05) and guanidinosuccinic acid (P < 0.05) . The phorbol-myristate-acetate (PMA)-stimulated chemiluminescence production of the calcitriol differentiated HL-60 cells was enhanced in the presence of guanidine (P < 0.05) . Methylguanidine and guanidinoacetic acid enhanced the lipopolysaccharide (LPS)-stimulated intracellular production of TNF-alpha by normal human monocytes (P < 0.05) . Second, several guanidino compounds inhibited the function of leukocytes if they were activated . The PMA-stimulated chemiluminescence production of the calcitriol differentiated HL-60 cells was inhibited by the presence of methylguanidine (P < 0.05), guanidinoacetic acid (P < 0.05) and guanidinosuccinic acid (P < 0.05) . After incubation of whole blood in the presence of methylguanidine, the Escherichia coli stimulated oxidative burst activity of the granulocyte population was significantly inhibited (P < 0.05) . In addition, guanidinosuccinic acid had an inhibitory effect on the LPS-stimulated intracellular production of TNF-alpha by human monocytes (P < 0.01) . CONCLUSION: Guanidino compounds exert proinflammatory as well as anti-inflammatory effects on monocyte/macrophage function . This could contribute to the altered prevalence of cardiovascular disease and propensity to infection in patients with CRF. Biochem J, 2004 Sep 1, 382(Pt 2), 759 - 67 Transketolase from Leishmania mexicana has a dual subcellular localization; Veitch NJ et al.; Transketolase has been characterized in Leishmania mexicana . A gene encoding this enzyme was identified and cloned . The gene was expressed in Escherichia coli and the protein was purified and characterized . An apparent K(m) of 2.75 mM for ribose 5-phosphate was determined . X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 A (1 A identical with 0.1 nm) . The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization . Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes . Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle. Bioconjug Chem, 2004 May-Jun, 15(3), 658 - 63 Direct production of proteins with N-terminal cysteine for site-specific conjugation; Gentle IE et al.; Proteins with N-terminal cysteine can undergo native chemical ligation and are useful for site-specific N-terminal labeling or protein semisynthesis . Recombinant production of these has usually been by site-specific cleavage of a precursor fusion protein at an internal cysteine residue . Here we describe a simpler route to producing these proteins . Overexpression in E . coli of several proteins containing cysteine as the second amino acid residue yielded products in which the initiating methionine residue had been completely cleaved by endogenous methionine aminopeptidase . While secondary modification of the terminal cysteine was a complicating factor, conditions were identified to eliminate or minimize this problem . Recombinant proteins produced in this way were suitable for site-specific modification of the amino terminus via native chemical ligation technology, as demonstrated by conjugation of a thioester-containing derivative of fluorescein to one such protein . The ability to directly produce proteins with N-terminal cysteine should simplify the application of native chemical ligation technology to recombinant proteins and make the technique more amenable to researchers with limited expertise in protein chemistry. Bioconjug Chem, 2004 May-Jun, 15(3), 576 - 82 Synthesis and in vitro evaluation of PNA-peptide-DETA conjugates as potential cell penetrating artificial ribonucleases; Petersen L et al.; We report the synthesis of novel artificial ribonucleases with potentially improved cellular uptake . The design of trifunctional conjugates 1a and 1b is based on the specific RNA-recognizing properties of PNA, the RNA-cleaving abilities of diethylenetriamine (DETA), and the peptide (KFF)(3)K for potential uptake into E . coli . The conjugates were assembled in a convergent synthetic route involving native chemical ligation of a PNA, containing an N-terminal cysteine, with the C-terminal thioester of the cell-penetrating (KFF)(3)K peptide to give 12a and 12b . These hybrids contained a free cysteine side-chain, which was further functionalized with an RNA-hydrolyzing diethylenetriamine (DETA) moiety . The trifunctional conjugates (1a, 1b) were evaluated for RNA-cleaving properties in vitro and showed efficient degradation of the target RNA at two major cleavage sites . It was also established that the cleavage efficiency strongly depended on the type of spacer connecting the PNA and the peptide. Methods Mol Biol, 2004, 266, 47 - 69 Comparative genomics: digging for data; Avison MB; Comparative genomics is a science in its infancy . It has been driven by a huge increase in freely available genome-sequence data, and the development of computer techniques to allow whole-genome sequence analyses . Other approaches, which use hybridization as a method for comparing the gene content of related organisms, are rising alongside these more bioinformatic methods . All these approaches have been pioneered using bacterial genomes because of their simplicity and the large number of complete genome sequences available . The aim of bacterial comparative genomics is to determine what genotypic differences are important for the expression of particular traits . The benefits of such studies will be a deeper understanding of these phenomena; the possibility of exposing novel drug targets, including those for antivirulence drugs; and the development of molecular techniques that reveal patients who are infected with virulent organisms so that health care resources can be allocated appropriately . With more and more genome sequences becoming available, the rise of comparative genomics continues apace. Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 7943 - 8 Epub 2004 May 17. Activity-based probes for protein tyrosine phosphatases; Kumar S et al.; Protein tyrosine phosphatases (PTPs) are involved in the regulation of many aspects of cellular activity including proliferation, differentiation, metabolism, migration, and survival . Given the large number and complexity of PTPs in cell signaling, new strategies are needed for the integrated analysis of PTPs in the whole proteome . Unfortunately, the activities of many PTPs are tightly regulated by posttranslational mechanisms, limiting the utility of standard genomics and proteomics methods for functional characterization of these enzymes . To facilitate the global analysis of PTPs, we designed and synthesized two activity-based probes that consist of alpha-bromobenzylphosphonate as a PTP-specific trapping device and a linker that connects the trapping device with a biotin tag for visualization and purification . We showed that these probes are active site-directed irreversible inactivators of PTPs and form a covalent adduct with PTPs involving the active site Cys residue . Additionally, we demonstrated that the probes are extremely specific toward PTPs while remaining inert to other proteins, including the whole proteome from Escherichia coli . Consequently, these activity-based PTP probes can be used to profile PTP activity in complex proteomes . The ability to interrogate the entire PTP family on the basis of changes in their activity should greatly accelerate both the assignment of PTP function and the identification of potential therapeutic targets. Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 7902 - 6 Epub 2004 May 17. Trigger factor binds to ribosome-signal-recognition particle (SRP) complexes and is excluded by binding of the SRP receptor; Buskiewicz I et al.; Trigger factor (TF) and signal recognition particle (SRP) bind to the bacterial ribosome and are both crosslinked to protein L23 at the peptide exit, where they interact with emerging nascent peptide chains . It is unclear whether TF and SRP exclude one another from their ribosomal binding site(s) . Here we show that SRP and TF can bind simultaneously to ribosomes or ribosome nascent-chain complexes exposing a SRP-specific signal sequence . Based on changes of the crosslinking pattern and on results obtained by fluorescence measurements using fluorescence-labeled SRP, TF binding induces structural changes in the ribosome-SRP complex . Furthermore, we show that binding of the SRP receptor, FtsY, to ribosome-bound SRP excludes TF from the ribosome . These results suggest that TF and SRP sample nascent chains on the ribosome in a nonexclusive fashion . The decision for ribosome nascent-chain complexes exposing a signal sequence to enter SRP-dependent membrane targeting seems to be determined by the binding of SRP, which is stabilized by signal sequence recognition, and promoted by the exclusion of TF due to the binding of the SRP receptor to ribosome-bound SRP. Proc Natl Acad Sci U S A, 2004 May 25, 101(21), 7925 - 30 Epub 2004 May 17. Design of temperature-sensitive mutants solely from amino acid sequence; Chakshusmathi G et al.; Temperature-sensitive (Ts) mutants are a powerful tool with which to study gene function in vivo . Ts mutants are typically generated by random mutagenesis followed by laborious screening procedures . By using the Escherichia coli cytotoxin CcdB as a model system, simple procedures for generating Ts mutants at high frequency through site-directed mutagenesis were developed . Putative buried, hydrophobic residues are selected through analysis of the protein sequence . Residue burial is confirmed by ensuring that substitution of the residue by Asp leads to protein inactivation . At such sites, a Ts phenotype can typically be generated either by (i) substitution of two predicted, buried residues with the 18 remaining amino acids or (ii) introduction of Lys, Ser, Ala, and Trp at three to four predicted buried sites . By using these design strategies, 17 tight Ts mutants of CcdB were isolated at four predicted buried sites . The rules were further verified by making several Ts mutants of yeast Gal4 at residues 68, 69, and 70 . No Ts mutants of either protein have been previously reported . Such Ts mutants of Gal4 can be used for conditional expression of a variety of genes by using the well characterized upstream-activating-sequence-Gal4 system. Nucleic Acids Res, 2004 May 17, 32(9), 2751 - 9 Print 2004. CsdA, a cold-shock RNA helicase from Escherichia coli, is involved in the biogenesis of 50S ribosomal subunit; Charollais J et al.; CsdA, a DEAD-box protein from Escherichia coli, has been proposed to participate in a variety of processes, such as translation initiation, gene regulation after cold-shock, mRNA decay and biogenesis of the small ribosomal subunit . Whether the protein really plays a direct role in these multiple processes is however, not clear . Here, we show that CsdA is involved in the biogenesis of the large rather than the small ribosomal subunit . Deletion of the csdA gene leads to a deficit in free 50S subunits at low temperatures and to the accumulation of a new particle sedimenting around 40S . Analysis of the RNA and protein contents of this particle indicates that it corresponds to a mis-assembled large subunit . Sucrose gradient fractionation shows that in wild-type cells CsdA associates mainly with a pre50S particle . Presumably the RNA helicase activity of CsdA permits a structural rearrangement during 50S biogenesis at low temperature . We showed previously that SrmB, another DEAD-box RNA helicase, is also involved in 50S assembly in E.coli . Our results suggest that CsdA is required at a later step than SrmB . However, over-expression of CsdA corrects the ribosome defect of the srmB-deleted strain, indicating that some functional overlap exists between the two proteins. J Biol Chem, 2004 Jul 16, 279(29), 30210 - 8 Epub 2004 May 17. Essential role of methionine residues in calmodulin binding to Bordetella pertussis adenylate cyclase, as probed by selective oxidation and repair by the peptide methionine sulfoxide reductases; Vougier S et al.; Bordetella pertussis, the causative agent of whooping cough, secretes among other virulence factors an adenylate cyclase (AC) toxin that is able to enter into eukaryotic cells where it is activated upon binding to endogenous calmodulin (CaM) and synthesizes supraphysiological cAMP levels . In vivo, the AC toxin, through its specific interaction with the CD11b/CD18 integrin, primarily targets phagocytic cells such as neutrophils and macrophages . Because neutrophil priming and activation result in the production of reactive oxygen species that may cause intracellular oxidation, we have examined the biological consequences of the oxidation of CaM methionines upon its interaction with AC . We show here that the interaction of CaM with AC is dependent on the reduced state of methionines, because oxidation of all methionine residues of CaM dramatically decreases its affinity for AC . Peptide methionine sulfoxide reductases A (MsrA) and B (MsrB) were able to partially reduce the oxidized CaM, and these partially "repaired" forms could interact with AC nearly as efficiently as the native protein . We further showed that the CaM.AC complex is resistant to oxidation with tert-butylhydroperoxide, and we identified methionine residues 109, 124, and 145 as critical for binding to AC . The resistance of the AC.CaM complex to oxidation and the ability of AC to be efficiently activated by partially oxidized CaM molecules should allow the toxin to exert its cytotoxic effects on activated neutrophils and contribute to the host colonization. J Biol Chem, 2004 Sep 10, 279(37), 38683 - 92 Epub 2004 May 17. Recognition of fold and sugar linkage for glycosyltransferases by multivariate sequence analysis; Rosen ML et al.; Glycosyltransferases (GTs) are among the largest groups of enzymes found and are usually classified on the basis of sequence comparisons into many families of varying similarity (CAZy systematics) . Only two different Rossman-like folds have been detected (GT-A and GT-B) within the small number of established crystal structures . A third uncharacterized fold has been indicated with transmembrane organization (GT-C) . We here use a method based on multivariate data analyses (MVDAs) of property patterns in amino acid sequences and can with high accuracy recognize the correct fold in a large data set of GTs . Likewise, a retaining or inverting enzymatic mechanism for attachment of the donor sugar could be properly revealed in the GT-A and GT-B fold group sequences by such analyses . Sequence alignments could be correlated to important variables in MVDA, and the separating amino acid positions could be mapped over the active sites . These seem to be localized to similar positions in space for the alpha/beta/alpha binding motifs in the GT-B fold group structures . Analogous, active-site sequence positions were found for the GT-A fold group . Multivariate property patterns could also easily group most GTs annotated in the genomes of Escherichia coli and Synechocystis to proper fold or organization group, according to benchmarking comparisons at the MetaServer . We conclude that the sequence property patterns revealed by the multivariate analyses seem more conserved than amino acid types for these GT groups, and these patterns are also conserved in the structures . Such patterns may also potentially define substrate preferences. Biochem Biophys Res Commun, 2004 Jun 11, 318(4), 970 - 6 Vital roles of an interhelical insertion in catalase-peroxidase bifunctionality; Li Y et al.; The loop connecting the F and G helices of catalase-peroxidases contains a approximately 35 amino acid structure (the FG insertion) that is absent from monofunctional peroxidases . These two groups of enzymes share highly similar active sites, yet the monofunctional peroxidases lack appreciable catalase activity . Thus, the FG insertion may serve a role in catalase-peroxidase bifunctionality, despite its peripheral location relative to the active site . We produced a variant of Escherichia coli catalase-peroxidase (KatG) lacking its FG insertion (KatG(DeltaFG)) . Absorption spectra indicated the heme environment of KatG(DeltaFG) was highly similar to wild-type KatG, but the variant retained only 0.2% catalase activity . In contrast, the deletion reduced peroxidase activity by only 50% . Kinetic parameters for the peroxidase and residual catalase activities of KatG(DeltaFG) as well as pH dependence studies suggested that the FG insertion supports hydrogen-bonded networks critical for reactions involving H2O2 . The structure also appears to regulate access of electron donors to the active site. Biochem Biophys Res Commun, 2004 Jun 11, 318(4), 862 - 7 Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate; Kuo CJ et al.; SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs . Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome . This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM . In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration . This makes finding the tight inhibitor (nanomolar range IC50) impossible . In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors . The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease . Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters . Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM) . This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease. FEBS Lett, 2004 May 21, 566(1-3), 311 - 5 Replacement of domain b of human protein disulfide isomerase-related protein with domain b' of human protein disulfide isomerase dramatically increases its chaperone activity; Horibe T et al.; We have reported that human protein disulfide isomerase-related protein (hPDIR) has isomerase and chaperone activities that are lower than those of the human protein disulfide isomerase (hPDI), and that the b domain of hPDIR is critical for its chaperone activity {J . Biol . Chem . 279 (2004) 4604} . To investigate the basis of the differences between hPDI and hPDIR, and to determine the functions of each hPDIR domain in detail, we constructed several hPDIR domain mutants . Interestingly, when the b domain of hPDIR was replaced with the b' domain of hPDI, a dramatic increase in chaperone activity that was close to that of hPDI itself was observed . However, this mutant showed decreased oxidative refolding of alpha1-antitrypsin . The replacement of the b domain of hPDIR with the c domain of hPDI also increased its chaperone activity . These observations suggest that putative peptide-binding sites of hPDI determine both its chaperone activity and its substrate specificity. FEBS Lett, 2004 May 21, 566(1-3), 207 - 12 The mature part of proNGF induces the structure of its pro-peptide; Kliemannel M et al.; Human nerve growth factor (NGF) belongs to the structural family of cystine knot proteins, characterized by a disulfide pattern in which one disulfide bond threads through a ring formed by a pair of two other disulfides connecting two adjacent beta-strands . Oxidative folding of NGF revealed that the pro-peptide of NGF stimulates in vitro structure formation . In order to learn more about this folding assisting protein fragment, a biophysical analysis of the pro-peptide structure has been performed . While proNGF is a non-covalent homodimer, the isolated pro-peptide is monomeric . No tertiary contacts stabilize the pro-peptide in its isolated form . In contrast, the pro-peptide appears to be structured when bound to the mature part . The results presented here demonstrate that the mature part stabilizes the structure in the pro-peptide region . This is the first report that provides a biophysical analysis of a pro-peptide of the cystine knot protein family. FEBS Lett, 2004 May 21, 566(1-3), 201 - 6 Neocarzinostatin naphthoate synthase: an unique iterative type I PKS from neocarzinostatin producer Streptomyces carzinostaticus; Sthapit B et al.; Enediyne antibiotics are known for their potent antitumor activities . One such enediyne, neocarzinostatin (NCS), consists of a 1:1 complex of non-peptide chromophore (1a), and peptide apoprotein . The structurally diverse non-peptide chromophore is responsible for its biological activity . One of its structural components, the naphthoic acid moiety (2,7-dihydroxy-5-methyl-1-naphthoic acid, 1d) is synthesized by a polyketide synthase (PKS) pathway through condensing six intact acetate units . The 5.45 kb iterative type I PKS, neocarzinostatin naphthoate synthase (NNS), responsible for naphthoic acid moiety biosynthesis, shares sequence homology with 6-methyl salicylic acid synthase of fungi and orsellinic acid synthases (AviM and CalO5) of Streptomyces origin . Cultures of S . lividans TK24 and S . coelicolor YU105 containing plasmids with NNS were able to produce 2-hydroxy-5-methyl-1-naphthoic acid (2a), a key intermediate of naphthoic acid moiety in NCS . In addition to 2a, a novel product, 2-hydroxy-5-hydroxymethyl-1-naphthoic acid (2d) was isolated . This is the first report of a bacterial iterative type I PKS from an enediyne producer which enables the biosynthesis of bicyclic aromatic compounds. FEBS Lett, 2004 May 21, 566(1-3), 162 - 8 A novel ninein-interaction protein, CGI-99, blocks ninein phosphorylation by GSK3beta and is highly expressed in brain tumors; Howng SL et al.; To explore more hNinein interacting proteins, the yeast two-hybrid screening using ninein C-terminal domain as bait protein was performed . One novel gene, CGI-99, was demonstrated to associate with hNinein in the yeast two-hybrid method and in vitro GST pull-down assay . Molecular characterization also showed that CGI-99 possessed a transcriptional activity at the N-terminal . In addition, CGI-99 formed a dimer with the C-terminal, which overlapped with hNinein binding site . In kinase assay, CGI-99 binds to hNinein and completely blocks the phosphorylation of hNinein by GSK3beta . Moreover, CGI-99 was highly expressed in all brain tumors which is in agreement with the Northern blot analysis . Taken together, we have isolated a novel protein CGI-99, which may be involved in the functional regulation of human ninein in the centrosome structure and may also be important in brain development and tumorigenesis. FEBS Lett, 2004 May 21, 566(1-3), 105 - 9 The homozygous M712T mutation of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase results in reduced enzyme activities but not in altered overall cellular sialylation in hereditary inclusion body myopathy; Hinderlich S et al.; Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder, caused by mutations in UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid biosynthesis . In Middle Eastern patients a single homozygous mutation occurs, converting methionine-712 to threonine . Recombinant expression of the mutated enzyme revealed slightly reduced N-acetylmannosamine kinase activity, in agreement with the localization of the mutation within the kinase domain . B lymphoblastoid cell lines derived from patients expressing the mutated enzyme also display reduced UDP-N-acetylglucosamine 2-epimerase activity . Nevertheless, no reduced cellular sialylation was found in those cells by colorimetric assays and lectin analysis, indicating that HIBM is not directly caused by an altered overall expression of sialic acids. FEBS Lett, 2004 May 21, 566(1-3), 95 - 9 Reshaping the folding energy landscape by chloride salt: impact on molten-globule formation and aggregation behavior of carbonic anhydrase; Boren K et al.; During chemical denaturation different intermediate states are populated or suppressed due to the nature of the denaturant used . Chemical denaturation by guanidine-HCl (GuHCl) of human carbonic anhydrase II (HCA II) leads to a three-state unfolding process (Cm,NI=1.0 and Cm,IU=1.9 M GuHCl) with formation of an equilibrium molten-globule intermediate that is stable at moderate concentrations of the denaturant (1-2 M) with a maximum at 1.5 M GuHCl . On the contrary, urea denaturation gives rise to an apparent two-state unfolding transition (Cm=4.4 M urea) . However, 8-anilino-1-naphthalene sulfonate (ANS) binding and decreased refolding capacity revealed the presence of the molten globule in the middle of the unfolding transition zone, although to a lesser extent than in GuHCl . Cross-linking studies showed the formation of moderate oligomer sized (300 kDa) and large soluble aggregates (>1000 kDa) . Inclusion of 1.5 M NaCl to the urea denaturant to mimic the ionic character of GuHCl leads to a three-state unfolding behavior (Cm,NI=3.0 and Cm,IU=6.4 M urea) with a significantly stabilized molten-globule intermediate by the chloride salt . Comparisons between NaCl and LiCl of the impact on the stability of the various states of HCA II in urea showed that the effects followed what could be expected from the Hofmeister series, where Li+ is a chaotropic ion leading to decreased stability of the native state . Salt addition to the completely urea unfolded HCA II also led to an aggregation prone unfolded state, that has not been observed before for carbonic anhydrase . Refolding from this state only provided low recoveries of native enzyme. FEBS Lett, 2004 May 21, 566(1-3), 48 - 54 A tradeoff between protein stability and conformational mobility in homotrimeric dUTPases; Takacs E et al.; Oligomerization directs active site formation in homotrimeric 2'-deoxyuridine triphosphate pyrophosphatases (dUTPases) . Stability of the homotrimer is a central determinant in enzyme function . The present comparative studies of bacterial and fruitfly dUTPases with homologous 3D structures by differential scanning microcalorimetry; fluorescence, circular dichorism and infrared spectroscopies, demonstrate that unfolding is a two-state highly cooperative transition in both dUTPases excluding a significantly populated intermediate state of dissociated and folded monomers . The eukaryotic protein is much less resistant against either thermal or guanidine hydrochloride-induced denaturation . Results suggest that hydrophobic packing of the inner threefold channel of the dUTPase homotrimer greatly contributes to stability. Int J Biochem Cell Biol, 2004 Aug, 36(8), 1585 - 98 A Trypanosoma cruzi heat shock protein 40 is able to stimulate the adenosine triphosphate hydrolysis activity of heat shock protein 70 and can substitute for a yeast heat shock protein 40; Edkins AL et al.; The process of assisted protein folding, characteristic of members of the heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) molecular chaperone families, is important for maintaining the structural integrity of cellular protein machinery under normal and stressful conditions . Hsp70 and Hsp40 cooperate to bind non-native protein conformations in a process of adenosine triphosphate (ATP)-regulated assisted protein folding . We have analysed the molecular chaperone activity of the cytoplasmic inducible Hsp70 from Trypanosoma cruzi (TcHsp70) and its interactions with its potential partner Hsp40s (T . cruzi DnaJ protein 1 {Tcj1} and T . cruzi DnaJ protein 2 {Tcj2}) . Histidine-tagged TcHsp70 (His-TcHsp70), Tcj1 (Tcj1-His) and Tcj2 (His-Tcj2) were over-produced in Escherichia coli and purified by nickel affinity chromatography . The in vitro basal specific ATP hydrolysis activity (ATPase activity) of His-TcHsp70 was determined as 40 nmol phosphate/min/mg protein, significantly higher than that reported for other Hsp70s . The basal specific ATPase activity was stimulated to a maximal level of 60 nmol phosphate/min/mg protein in the presence of His-Tcj2 and a model substrate, reduced carboxymethylated alpha-lactalbumin . In vivo complementation assays showed that Tcj2 was able to overcome the temperature sensitivity of the ydj1 mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 homologue (yeast DnaJ protein 1, Ydj1) . These data suggest that Tcj2 is involved in cytoprotection in a similar fashion to Ydj1, and that TcHsp70 and Tcj2 may interact in a nucleotide-regulated process of chaperone-assisted protein folding. Biochem J, 2004 Aug 15, 382(Pt 1), 191 - 8 Topology of the substrate-binding site of a Lys49-phospholipase A2 influences Ca2+-independent membrane-damaging activity; Sa JM et al.; BthTx-I (bothropstoxin-I) is a myotoxic Lys49-PLA2 (phospholipase A2 with Lys49) isolated from Bothrops jararacussu venom, which damages liposome membranes by a Ca2+-independent mechanism . The highly conserved Phe5/Ala102/Phe106 motif in the hydrophobic substrate-binding site of the Asp49-PLA2s is substituted by Leu5/Val102/Leu106 in the Lys49-PLA2s . The Leu5/Val102/Leu106 triad in BthTx-I was sequentially mutated via all single- and double-mutant combinations to the Phe5/Ala102/Phe106 mutant . All mutants were expressed as inclusion bodies in Escherichia coli, and the thermal stability (Tm), together with the myotoxic and Ca2+-independent membrane-damaging activities of the recombinant proteins, were evaluated . The far-UV CD profiles of the native, wild-type recombinant and the L106F (Leu106-->Phe) and L5F/F102A/L106F mutant proteins were identical . The L5F, V102A, L5F/V102A and V102A/L106F mutants showed distorted far-UV CD profiles; however, only the L5F and L5F/V102A mutants showed significant decreases in Tm . Alterations in the far-UV CD spectra correlated with decreased myotoxicity and protein-induced release of a liposome-entrapped marker . However, the V102A/L106F and L5F/V102A/L106F mutants, which presented high myotoxic activities, showed significantly reduced membrane-damaging activity . This demonstrates that the topology of the substrate-binding region of BthTx-I has a direct effect on the Ca2+-independent membrane damage, and implies that substrate binding retains an important role in this process. Biochem J, 2004 Aug 15, 382(Pt 1), 51 - 7 Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase; Yang W et al.; CTP:phosphoethanolamine cytidylyltransferase (ECT) is considered to be the regulatory enzyme in the CDP-ethanolamine pathway of phosphatidylethanolamine (PE) biosynthesis . The ECT cDNA of Chlamydomonas reinhardtii encodes a protein of 443 amino acid residues, which is longer than the same protein in yeast, rat or human . The translated product of cloned cDNA was expressed as a fusion protein in Escherichia coli, and was shown to have ECT activity . The deduced amino acid sequence has 41% identity with that of human or rat, and 30% with yeast . The ECT protein has a repetitive internal sequence in its N- and C-terminal halves and a signature peptide sequence, RTXGVSTT, typical of the cytidylyltransferase family . The first 70 amino acid residues do not match the N-terminal part of the cytidylyltransferases from other organisms, and we hypothesize that it is a subcellular targeting signal to mitochondria . ECT and organelle marker enzyme assays showed that the total activity of ECT correlates well with that of fumarase, a marker enzyme for mitochondria . Northern blots showed an increase in mRNA abundance during reflagellation, indicating a possibility of transcriptional regulation . A notable change in the enzyme activity in C . reinhardtii cells was observed during the cell cycle, increasing during the dark and then decreasing during the light period, while the mRNA level did not alter, providing evidence for post-translational regulation. Biochemistry, 2004 May 25, 43(20), 6293 - 303 Engineering a thermostable human prolyl endopeptidase for antibody-directed enzyme prodrug therapy; Heinis C et al.; We present a new antibody-directed enzyme prodrug therapy strategy (ADEPT) based on a post-proline cleaving endopeptidase and prodrugs, in which cytotoxic moieties are linked to a proline-containing peptide . Human prolyl endopeptidase was expressed in Escherichia coli and purified to homogeneity . The enzyme was active in buffer and in human serum but was rapidly thermally inactivated by incubation at 37 degrees C, thus preventing applications in vivo . While prolyl endopeptidase display on filamentous phage abolished viral infectivity and prevented directed evolution strategies based on phage display, we robotically screened 10752 individual colonies of mutant enzymes using a fluorogenic assay to improve enzyme stability . A single amino acid mutation (Glu289 --> Gly) improved protein stability, resulting in a half-life of 16 h at 37 degrees C in phosphate buffer . Two prodrugs were synthesized, in which an N-protected glycine-proline dipeptide was covalently coupled to doxorubicin and melphalan . (Benzyloxycarbonyl)glycylprolylmelphalan, but not the more sterically hindered doxorubicin prodrug, could be efficiently activated by prolyl endopeptidase {specific activity = 813.3 nmol min(-1) (mg of enzyme)(-1) at 25 degrees C} . The melphalan prodrug was essentially nontoxic to CHO, F9 teratocarcinoma, MCF7 breast adenocarcinoma, and p3U1 mouse myeloma cells up to millimolar concentrations, while prodrug incubation with the engineered prolyl endopeptidase mutant led to a cell killing profile superimposable to the one of melphalan . The prolyl endopeptidase mutant was then chemically coupled to the human antibody L19, specific to the EDB domain of fibronectin, a marker of angiogenesis . The resulting immunoconjugate retains antigen binding and enzymatic activity, thus opening the way to anticancer ADEPT applications. Biochemistry, 2004 May 25, 43(20), 5953 - 64 Use of a chemical trigger for electron transfer to characterize a precursor to cluster X in assembly of the iron-radical cofactor of Escherichia coli ribonucleotide reductase; Saleh L et al.; A key step in generation of the catalytically essential tyrosyl radical (Y122(*)) in protein R2 of Escherichia coli ribonucleotide reductase is electron transfer (ET) from the near-surface residue, tryptophan 48 (W48), to a (Fe(2)O(2))(4+) complex formed by addition of O(2) to the carboxylate-bridged diiron(II) cluster . Because this step is rapid, the (Fe(2)O(2))(4+) complex does not accumulate and, therefore, has not been characterized . The product of the ET step is a "diradical" intermediate state containing the well-characterized Fe(IV)Fe(III) cluster, X, and a W48 cation radical (W48(+)(*)) . The latter may be reduced from solution to complete the two-step transfer of an electron to the buried diiron site . In this study, a (Fe(2)O(2))(4+) state that is probably the precursor to the X-W48(+)(*) diradical state in the reaction of the wild-type protein (R2-wt) has been characterized by exploitation of the observation that in R2 variants with W48 replaced with alanine (A), the otherwise disabled ET step can be mediated by indole compounds . Mixing of the Fe(II) complex of R2-W48A/Y122F with O(2) results in accumulation of an intermediate state that rapidly converts to X upon mixing with 3-methylindole (3-MI) . The state comprises at least two species, of which each exhibits an apparent Mossbauer quadrupole doublet with parameters characteristic of high-spin Fe(III) ions . The isomer shifts of these complexes and absence of magnetic hyperfine coupling in their Mossbauer spectra suggest that both are antiferromagnetically coupled diiron(III) clusters . The fact that both rapidly convert to X upon treatment with a molecule (3-MI) shown in the preceding paper to mediate ET in W48A R2 variants indicates that they are more oxidized than X by one electron, which suggests that they have a bound peroxide equivalent . Their failure to exhibit either the long-wavelength absorption (at 650-750 nm) or Mossbauer doublet with high isomer shift (>0.6 mm/s) that are characteristic of the putatively mu-1,2-peroxo-bridged diiron(III) intermediates that have been detected in the reactions of methane monooxygenase (P or H(peroxo)) and variants of R2 with the D84E ligand substitution suggests that they have geometries and electronic structures different from those of the previously characterized complexes . Supporting this deduction, the peroxodiiron(III) complex that accumulates in R2-W48A/D84E is much less reactive toward 3-MI-mediated reduction than the (Fe(2)O(2))(4+) state in R2-W48A/Y122F . It is postulated that the new (Fe(2)O(2))(4+) state is either an early adduct in an orthogonal pathway for oxygen activation or, more likely, the successor to a (mu-1,2-peroxo)diiron(III) complex that is extremely fleeting in R2 proteins with the wild-type ligand set but longer lived in D84E-containing variants. Biochemistry, 2004 May 25, 43(20), 5943 - 52 Mediation by indole analogues of electron transfer during oxygen activation in variants of Escherichia coli ribonucleotide reductase R2 lacking the electron-shuttling tryptophan 48; Saleh L et al.; Activation of dioxygen by the carboxylate-bridged diiron(II) cluster in the R2 subunit of class I ribonucleotide reductase from Escherichia coli results in the one-electron oxidation of tyrosine 122 (Y122) to a stable radical (Y122*) . A key step in this reaction is the rapid transfer of a single electron from a near-surface residue, tryptophan 48 (W48), to an adduct between O(2) and diiron(II) cluster to generate a readily reducible cation radical (W48(+)(*)) and the formally Fe(IV)Fe(III) intermediate known as cluster X . Previous work showed that this electron injection step is blocked in the R2 variant with W48 replaced by phenylalanine {Krebs, C., Chen, S., Baldwin, J., Ley, B . A., Patel, U., Edmondson, D . E., Huynh, B . H., and Bollinger, J . M., Jr . (2000) J . Am . Chem . Soc . 122, 12207-12219} . In this study, we show that substitution of W48 with alanine similarly disables the electron transfer (ET) but also permits its chemical mediation by indole compounds . In the presence of an indole mediator, O(2) activation in the R2-W48A variant produces approximately 1 equiv of stable Y122* and more than 1 equiv of the normal (micro-oxo)diiron(III) product . In the absence of a mediator, the variant protein generates primarily altered Fe(III) products and only one-fourth as much stable Y122* because, as previously reported for R2-W48F, most of the Y122* that is produced decays as a consequence of the inability of the protein to mediate reductive quenching of one of the two oxidizing equivalents of the initial diiron(II)-O(2) complex . Mediation of ET is effective in W48A variants containing additional substitutions that also impact the reaction mechanism or outcome . In the reaction of R2-W48A/F208Y, the presence of mediator suppresses formation of the Y208-derived diiron(III)-catecholate product (which is predominant in R2-F208Y in the absence of reductants) in favor of Y122* . In the reaction of R2-W48A/D84E, the presence of mediator affects the outcome of decay of the peroxodiiron(III) intermediate known to accumulate in D84E variants, increasing the yield of Y122* by as much as 2.2-fold to a final value of 0.75 equiv and suppressing formation of a 490 nm absorbing product that results from decay of the two-electron oxidized intermediate in the absence of a functional ET apparatus. Equine Vet J, 2004 Apr, 36(3), 273 - 8 Endotoxin-induced digital vasoconstriction in horses: associated changes in plasma concentrations of vasoconstrictor mediators; Menzies-Gow NJ et al.; REASONS FOR PERFORMING STUDY: Lipopolysaccharide (LPS) infusion reduces digital perfusion, but the mediators responsible remain undetermined . OBJECTIVES: To identify vasoconstrictor mediators released following LPS infusion and relate their appearance in plasma to digital blood flow alterations . METHODS: Blood flow in the lateral digital vessels of 6 Thoroughbred horses, following a 30 min infusion of LPS (E . coli 055:B5; 30 ng/kg), was measured using Doppler ultrasonography . Concomitant measurements of hoof wall and coronary band surface temperatures (HWST and CBST) were made . Serial blood samples were collected and plasma LPS, tumour necrosis factor alpha (TNFalpha), 5-HT, thromboxane B2 (TxB2) and endothelin measured . RESULTS: Plasma LPS concentrations reached a maximum of 13.2 pg/ml during the infusion, followed by an increase in plasma TNFalpha concentration . Digital arterial and venous blood flow decreased by 43 and 63%, respectively; HWST and CBST similarly decreased . Systemic blood pressure remained unaltered . Plasma concentrations of TxB2 and 5-HT increased, coinciding with the onset of digital hypoperfusion . Plasma endothelin concentrations remained unchanged . CONCLUSIONS: The temporal relationship between the onset of digital hypoperfusion and increases in plasma 5-HT and TxB2 concentrations is consistent with these platelet-derived mediators being associated with LPS-induced laminitis . POTENTIAL RELEVANCE: These experimental data support the use of anti-platelet therapy in the prevention of laminitis associated with endotoxaemic conditions. Arzneimittelforschung, 2004, 54(4), 242 - 9 A new gene encoding the ribosome-inactivating protein from mistletoe extracts; Tonevitsky AG et al.; Extracts from mistletoe (Viscum album L.) contain three main toxic proteins--the lectins MLI (also known as viscumin), MLII and MLIII . A catalytic subunit of the mistletoe plant toxic lectin MLIII has been cloned and expressed in Escherichia coli cells . The structure and immunochemical properties of recombinant MLIII A-subunit were investigated using a panel of monoclonal antibodies against ML-toxins . Ribosome-inactivating activity of the recombinant MLIII A-subunit was determined in a cell-free system exhibiting inhibition of endogenous protein synthesis . The comparative analysis of nucleotide and deduced amino acid sequences of the cloned MLIII A and the native MLI A-subunits was performed, revealing the main differences in the primary structure of these proteins . Antigenicity analysis of the MLIII A-subunit has revealed a new epitope D179-E184 that is not present in viscumin . The role of toxic lectins with respect to the immunological properties of mistletoe extracts is discussed. Hum Mutat . 2004 Jun;23(6):631. Identification and functional analysis of two novel mutations in the CBS gene in Polish patients with homocystinuria; Orendae M et al.; Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is an inherited disorder of homocysteine transsulfuration, which manifests by neurological, vascular and connective tissue involvement . So far, 130 pathogenic mutations have been recognized in the CBS gene . We examined 10 independent alleles in Polish patients suffering from CBS deficiency, and we detected four already described mutations (c.1224-2A>C, c.684C>A, c.833T>C, and c.442G>A) and two novel mutations (c.429C>G and c.1039+1G>T) . The pathogenicity of the novel mutations was demonstrated by expression in E.coli . This is the first published communication on mutations leading to CBS deficiency in Poland . RNA, 2004 Jun, 10(6), 954 - 64 The structure of a ribosomal protein S8/spc operon mRNA complex; Merianos HJ et al.; In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron . The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward . A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site . Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs . S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron. RNA, 2004 Jun, 10(6), 907 - 13 A novel partial modification at C2501 in Escherichia coli 23S ribosomal RNA; Andersen TE et al.; Escherichia coli is the best-characterized organism with respect to posttranscriptional modifications of its ribosomal RNA (rRNA) . It is presently believed that all the modified nucleotides have been identified, primarily on the basis of two detection methods; modification-induced inhibition of the enzyme reverse transcriptase or analysis by combined HPLC and electrospray ionization mass spectrometry . Comparison of data from these different approaches reveals a disagreement regarding modification of C2501 in E . coli 23S rRNA . A . Bakin and J . Ofengand previously reported the detection of a modification at this site based on a reverse transcriptase assay . J.A . McCloskey and coworkers could not confirm the existence of such a modification using an electrospray ionization mass spectrometry approach . C2501 is therefore generally considered unmodified . We have used a strategy involving isolation of a specific rRNA fragment from E . coli 23S rRNA followed by Matrix Assisted Laser Desorption/Ionization mass spectrometry and tandem mass spectrometry to investigate this controversy . Our data reveal a novel 16-Da partial modification at C2501 . We believe that the data reported here clarify the above discrepancy, because a minor partial modification detected in a reverse transcriptase assay would not necessarily be detected by the original mass spectrometry approach . The level of modification was furthermore monitored in different growth situations, and we found a significant positive regulation in stationary phase cells . C2501 is universally conserved and implicated in structure folds very close to the catalytic center of the ribosome . Moreover, several antibiotics bind to nucleotides in this region, which altogether make a modification at this site interesting. J Biol Chem, 2004 Jul 23, 279(30), 31050 - 7 Epub 2004 May 15. Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase; Kasrayan A et al.; Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis . The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related . With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs) . We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure . The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis . Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2 . Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments . The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction . Based on the experimental setup, we propose that the inhibition of enzyme activity in the E . coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2 . Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold. Trends Genet, 2004 Jun, 20(6), 232 - 4 Evidence against the selfish operon theory; Pal C et al.; According to the selfish operon hypothesis, the clustering of genes and their subsequent organization into operons is beneficial for the constituent genes because it enables the horizontal gene transfer of weakly selected, functionally coupled genes . The majority of these are expected to be non-essential genes . From our analysis of the Escherichia coli genome, we conclude that the selfish operon hypothesis is unlikely to provide a general explanation for clustering nor can it account for the gene composition of operons . Contrary to expectations, essential genes with related functions have an especially strong tendency to cluster, even if they are not in operons . Moreover, essential genes are particularly abundant in operons. Trends Genet, 2004 Jun, 20(6), 224 - 7 Activation induced deaminase: the importance of being specific; Smith HC et al.; Activation-induced deaminase (AID) is required for class switch recombination and somatic hypermutation in immunoglobulin genes . Although the preponderance of evidence suggests that AID functions by deaminating deoxycytidine in DNA, the question remains whether it can also deaminate cytidine in mRNA, as originally proposed based on its homology to RNA-editing enzymes . Recently, the biological relevance of assaying mammalian enzymes for DNA deaminase activity using Escherichia coli DNA as a reporter has been questioned, representing another round in the ongoing debate. Vet Microbiol, 2004 Jun 3, 100(3-4), 241 - 6 Inhibition of adhesion of F18+ Escherichia coli to piglet intestinal villous enterocytes by monoclonal antibody against blood group H-2 antigen; Snoeck V et al.; Enterotoxigenic and verotoxigenic F18+ Escherichia coli colonising the pig small intestine, adhere to receptors on intestinal villous enterocytes by F18 fimbriae . The aim of the present study was to define the F18R nature . The knowledge on the nature of this receptor could be important for the development of receptor-based treatments against F18+ E . coli-induced disease . The adhesion of F18+ E . coli to pig intestinal villous enterocytes was analysed in an in vitro assay . The adhesion of F18+ E . coli but not of F4ac+ E . coli was strongly inhibited by monoclonal antibodies (mAb) with blood group H-2 specificity . Conversely, blood group H-1 specific mAb could not inhibit the adhesion of F18+ E . coli nor F4ac+ E . coli . Moreover, the blood group H-2 trisaccharide strongly inhibited the adhesion of F18+ E . coli, but only partially the adhesion of F4ac+ E . coli . These data demonstrate that the F18 receptor contains the blood group antigen H-2 (alpha-fuc-(1-2)-beta-Gal-(1-4)-GlcNAc) as major carbohydrate. Gene, 2004 May 12, 332, 97 - 106 Isolation, expression and bioactivity of feline granulocyte-macrophage colony-stimulating factor; Dunham SP et al.; A cDNA encoding feline granulocyte-macrophage colony stimulating factor was cloned from alveolar macrophages using the reverse transcriptase-polymerase chain reaction (RT-PCR) . The cDNA is 426 bp in length and encodes a predicted mature protein of 127 amino acids and the majority of the signal peptide . The recombinant protein (rfGM-CSF) was expressed in both Escherichia coli, as a calmodulin fusion protein, and mammalian cells . Biological activity of both recombinant proteins was demonstrated using the human erythroleukaemic cell line, TF-1 . In a soft agar clonogenic assay, rfGM-CSF supported the development of granulocyte, macrophage and granulocyte-macrophage colonies . In combination with phytohaemagglutin (PHA) lymphocyte-conditioned medium, the number and size of such colonies were increased . Culture of feline bone marrow cells with rfGM-CSF was an efficient method for producing cells with morphology typical of dendritic cells (DC) . The availability of the recombinant cytokine will permit further studies, in particular, the evaluation of the role of dendritic cells in feline immunopathology and its potential as a vaccine adjuvant. Zhonghua Jie He He Hu Xi Za Zhi, 2004 Apr, 27(4), 249 - 52 {Constructing shuttle plasmid fusion expressing Ag85B-ESAT-6 on the surface of Mycobacterium}; Shi CH et al.; OBJECTIVE: To construct the E . coli.-BCG (Bacille Calmette-Guerin) shuttle vector expressing Mycobacterium tuberculosis secreted protein Ag85B-ESAT-6 on the surface of Mycobacterium vaccae . METHODS: The gene fragment containing 19 000 antigen (19-ss) were amplified by polymerase chain reaction (PCR) from the Mycobacterium tuberculosis H(37)Ra . We cloned the 19ss gene into the E . coli.-BCG shuttle vector pOLYG and named the pCW, which can shuttle and express exogenous antigen gene on cell wall of Mycobacterium . Then Mycobacterium tuberculosis secret protein Ag85B and ESAT-6 gene were cloned into the vector and determined by indirect immunofluorescence . RESULTS: The sequence of 19-ss gene was identified with Genbank reported by sequencing . The constructed E . coli.-BCG shuttle vector using 19ss gene had the function of shuttle between E . coli . and Mycobacteria . By indirect immunofluorescence technique the secreted protein Ag85B-ESAT-6 can be fused and expressed on surface of Mycobacterium vaccae . CONCLUSION: The E . coli.-BCG shuttle vector is constructed successfully which could express exogenous antigen gene as a chimeric exported membrane. Zhonghua Jie He He Hu Xi Za Zhi, 2004 Apr, 27(4), 244 - 8 {Preparation and application of recombinant Mycobacterium tuberculosis CFP10-ESAT-6 fusion protein}; Chen Q et al.; OBJECTIVE: To prepare the recombinant CFP10-ESAT-6 fusion protein, and to study its immunological characteristics, and its potential for serodiagnosis of tuberculosis . METHODS: The lhp-ESAT-6 fusion gene was amplified by Gene SOEing, and then cloned into pQE30 plasmid . The recombinant CFP10-ESAT-6 fusion protein was expressed and purified . Its antigenicity was confirmed by Western blot . Animal models infected with M . tuberculosis H(37)Rv strain and M . bovis BCG respectively were made to evaluate the potential value of the fusion protein in the serodiagnosis of tuberculosis . RESULTS: The sequence of recombinant plasmid pQE30-CFP10-ESAT-6 was identical to the predicted sequence . The recombinant protein (rCFP10-ESAT-6), about 26 000, existed in the cytoplasm of DH5alpha in soluble form and represented 40% of the total bacterial protein . The purity and concentration of the final product was 98% and 1.2 g/L, respectively . Western blot showed that the rCFP10-ESAT-6 had good immunoreactivity with sera from patients with active tuberculosis and rabbits immunized with CFP10 and ESAT-6 respectively . The positive cutoff value was A(490) plus 2 standard deviation from negative guinea pig sera detected by ELISA . Serological reactivity to rCFP10-ESAT-6 was observed in 11 of the serum samples from guinea pigs with tuberculosis and 1 of sera from guinea pigs infected with BCG, while the serological reactivity to PPD was observed in 11 of sera from guinea pigs with tuberculosis and in 11 of sera from guinea pigs infected with BCG . CONCLUSIONS: The rCFP10-ESAT-6 fusion protein was highly expressed in soluble form in E . coli . It had antigenicity of both CFP10 and ESAT-6, and could be used to differentiate infection with M . tuberculosis H(37)Rv strain from immunization with M . bovis BCG . The study provided experimental data for potential application of rCFP10-ESAT-6 in the diagnosis of tuberculosis. Rev Invest Clin, 2004 Jan-Feb, 56(1), 43 - 50 Production of recombinant rat hepatic histidase; Ortiz V et al.; Rat liver histidase was expressed in E . coli by using a PCR product of the coding sequence obtained from the rat liver cDNA of histidase cloned in the expression vector pRSET . The construct (pRSET-HAL) produced a fusion protein containing a tail of polyhistidine . The expression product was purified with a resin containing Ni+ that retains proteins with polyhistidine fragments . pRSET-HAL was analyzed by restriction enzyme mapping and by sequencing confirming the correct orientation and nucleotide sequence . Native rat liver histidase was also purified and it had a Mr of 72 kDa . An antiserum against native histidase was obtained in rabbit . Western blot analysis revealed one band of 72 kDa observed in membranes containing purified histidase or rat liver high speed supernatants . The same antiserum also detected in cell lysates of E . coli transformed with the pRSET-HAL plasmid a single band of 74 kDa of the recombinant histidase before cleavage with enterokinase . After the proteolysis, the Western blot analysis showed a single band of approximately 72 kDa . Kinetic analysis of the recombinant histidase showed similar Km and Vmax compared with native histidase. J Biol Chem, 2004 Aug 20, 279(34), 35840 - 8 Epub 2004 May 13. Isotype-specific degradation of Rac activated by the cytotoxic necrotizing factor 1; Pop M et al.; The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates members of the Rho family by deamidation of glutamine 61/63 . Because this amino acid is crucial for GTP hydrolysis, deamidation of glutamine 61/63 results in constitutively active Rho proteins . Recently, it was shown that the level of CNF1-activated Rac is rapidly diminished in CNF1-treated cells by proteolytic degradation . Here, we studied the requirements for CNF1-induced Rac degradation . By overexpressing His-tagged activated Rac mutants we show that constitutive activation is necessary for degradation of Rac . However, permanent activation is not sufficient for degradation, because Rac that is constitutively activated by transamidation at glutamine 61 by the Bordetella dermonecrotic toxin is not degraded . Overexpression of His-tagged Rac mutants deficient in interaction with GTPase-activating protein (Rac(N92D) and Rac(Y64H)) and guanosine nucleotide dissociation inhibitor (Rac(H103E)) were degraded after activation by CNF1, whereas Rac(Y40C), which is not able to interact with CRIB domain effectors or plenty of SH3, was not degraded . Isoprenylation and the presence of a putative mitotic destruction box are essential for CNF-induced degradation . In contrast to Rac1, Rac2, and Rac3 were not degraded following constitutive activation by CNF1 . Using site-directed mutagenesis, we defined the polybasic region and amino acids 90, 107, 147, and 151 as responsible for isotype-specific degradation. Am J Respir Crit Care Med, 2004 Sep 15, 170(6), 613 - 20 Epub 2004 May 13. Inhaled carbon monoxide confers antiinflammatory effects against ventilator-induced lung injury; Dolinay T et al.; Ventilator-induced lung injury (VILI) is a major cause of morbidity and mortality in intensive care units . The stress-inducible gene product, heme oxygenase-1, and carbon monoxide (CO), a major by-product of heme oxygenase catalysis of heme, have been shown to confer potent antiinflammatory effects in models of tissue and cellular injury . In this study, we observed increased expression of heme oxygenase-1 mRNA and protein in a rat model of VILI . To assess the physiologic function of heme oxygenase-1 induction in VILI, we determined whether low concentration of inhaled CO could serve to protect the lung against VILI . Low concentration of inhaled CO significantly reduced tumor necrosis factor-alpha levels and total cell count in lavage fluid, while simultaneously elevating levels of antiinflammatory interleukin-10 levels . To better characterize the mechanism of CO-mediated antiinflammatory effects, we examined key signaling pathways, which may mediate CO-induced antiinflammatory effects . We demonstrate that inhaled CO exerts antiinflammatory effects in VILI via the p38 mitogen-activated protein kinase pathway but independent of activator protein-1 and nuclear factor-kappaB pathways . Our data lead to a tempting speculation that inhaled CO might be useful in minimizing VILI. Prog Biophys Mol Biol, 2004 Jun-Jul, 85(2-3), 217 - 34 A multi-scaled approach for simulating chemical reaction systems; Burrage K et al.; In this paper we give an overview of some very recent work, as well as presenting a new approach, on the stochastic simulation of multi-scaled systems involving chemical reactions . In many biological systems (such as genetic regulation and cellular dynamics) there is a mix between small numbers of key regulatory proteins, and medium and large numbers of molecules . In addition, it is important to be able to follow the trajectories of individual molecules by taking proper account of the randomness inherent in such a system . We describe different types of simulation techniques (including the stochastic simulation algorithm, Poisson Runge-Kutta methods and the balanced Euler method) for treating simulations in the three different reaction regimes: slow, medium and fast . We then review some recent techniques on the treatment of coupled slow and fast reactions for stochastic chemical kinetics and present a new approach which couples the three regimes mentioned above . We then apply this approach to a biologically inspired problem involving the expression and activity of LacZ and LacY proteins in E . coli, and conclude with a discussion on the significance of this work . Res Microbiol, 2004 May, 155(4), 231 - 7 When replication travels on damaged templates: bumps and blocks in the road; Courcelle J et al.; Escherichia coli can accurately replicate their genome even when it contains hundreds of damaged bases . In this situation, processes such as DNA repair, translesion DNA synthesis, and recombination all contribute to the cell's ability to successfully complete this task . However, under conditions when these reactions go awry, these same processes can result in cell lethality, mutagenesis, or genetic instability . In order to understand the molecular events that can lead this normally faithful duplication of the genome to become less than perfect, it is essential to define the substrates and conditions when each of these processes are recruited to the replication fork. Bioorg Med Chem, 2004 Jun 1, 12(11), 2965 - 72 Slow-binding inhibition of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase; Braga R et al.; 2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase is a key enzyme in the Entner-Doudoroff pathway of bacteria . It catalyzes the reversible production of KDPG from pyruvate and D-glyceraldehyde 3-phosphate through a class I Schiff base mechanism . On the basis of aldolase mechanistic pathway, various pyruvate analogues bearing beta-diketo structures were designed and synthesized as potential inhibitors . Their capacity to inhibit aldolase catalyzed reaction by forming stabilized iminium ion or conjugated enamine were investigated by enzymatic kinetics and UV-vis difference spectroscopy . Depending of the substituent R (methyl or aromatic ring), a competitive or a slow-binding inhibition takes place . These results were examined on the basis of the three-dimensional structure of the enzyme. Bioorg Med Chem, 2004 Jun 1, 12(11), 2853 - 61 Design and synthesis of chromogenic thiopeptolide substrates as MetAPs active site probes; Cui YM et al.; Twenty one chromogenic thiopeptolide substrates were designed and synthesized as the active site probes and analyzed with each S1 site of mutant residues and enzymes of wild-type MetAP1s . The preliminary enzymatic experiments indicate that cysteine 70 or 202, at either Escherichia coli or human MetAP1, played a crucial role in the methionine hydrolysis. Mol Imaging, 2004 Jan, 3(1), 33 - 42 Visualization of the dynamics of gene expression in the living mouse; Ryan A et al.; Reporter genes can monitor the status and activity of recombinant genomes in a diverse array of organisms, from bacteria and yeast to plants and animals . We have combined luciferase reporter genes with a conditional gene expression system based on regulatory elements from the lac operon of Escherichia coli to visualize the dynamics of gene expression in realtime in the living mouse . Using this technology, we have determined the rate of gene induction and repression, the level of target gene activity in response to different doses of inducer, and the schedule of induction during early embryogenesis of both the endogenous and the experimentally manipulated programs of mammalian gene expression associated with the HD/Hdh locus . The combination of in vivo imaging and lac regulation is a powerful tool for generating conditional transgenic mice that can be screened rapidly for optimal regulation and expression patterns, and for monitoring the induction and repression of regulated genes noninvasively in the living animal. Poult Sci, 2004 May, 83(5), 707 - 15 Endotoxin stress responses in chickens from different genetic lines . 1 . Sickness, behavioral, and physical responses; Cheng HW et al.; Genetic variation in response to lipopolysaccharide (LPS) challenge was studied in chicken lines divergently selected for high (HGPS) and low (LGPS) group productivity and survivability resulting from cannibalism and flightiness in colony cages and in a Dekalb XL (DXL) commercial line . Six-week-old chicks were randomly assigned to control or experimental groups and were injected intravenously with Escherichia