Escherichia coli

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 coli LPS (5 mg/kg of BW) or distilled saline (control) . Sickness responses were measured at 6, 12, 24, 48, and 72 h following injection (n = 10 at each point in time for each line) . Although LPS induced widespread sickness symptoms in all of the treated chicks, the reactions were in a genotypic- and phenotypic-specific manner . Compared with LGPS and DXL chicks, HGPS chicks had acute, transient behavioral and physical changes with less effect on BW gain, organ development, and core temperature, which were in the order HGPS < DXL < LGPS . The effects of heritable factors and LPS challenge on the differential responses among the present lines may reflect each line's unique adaptability to stress and resistance to infection and inflammation . The results suggested that the present chicken lines may provide a valuable animal model for investigating the effects of genetic-environmental interactions on the behavioral and physiological homeostasis in response to stress and disease.

Environ Mol Mutagen, 2004, 43(4), 226 - 34
Effect of deletion of SOS-induced polymerases, pol II, IV, and V, on spontaneous mutagenesis in Escherichia coli mutD5; Nowosielska A et al.; The E . coli dnaQ gene encodes the epsilon subunit of DNA polymerase III (pol III) responsible for the proofreading activity of this polymerase . The mutD5 mutant of dnaQ chronically expresses the SOS response and exhibits a mutator phenotype . In this study we have constructed a set of E . coli AB1157 mutD5 derivatives deleted in genes encoding SOS-induced DNA polymerases, pol II, pol IV, and pol V, and estimated the frequency and specificity of spontaneous argE3-->Arg(+) reversion in exponentially growing and stationary-phase cells of these strains . We found that pol II exerts a profound effect on the specificity of spontaneous mutation in exponentially growing cells . Analysis of growth-dependent Arg(+) revertants in mutD5 polB(+) strains revealed that Arg(+) revertants were due to tRNA suppressor formation, whereas those in mutD5 DeltapolB strains arose by back mutation at the argE3 ochre site . In stationary-phase bacteria, Arg(+)revertants arose mainly by back mutation, regardless of whether they were proficient or deficient in pol II . Our results also indicate that in a mutD5 background, the absence of pol II led to increased frequency of Arg(+) growth-dependent revertants, whereas the lack of pol V caused its dramatic decrease, especially in mutD5 DeltaumuDC and mutD5 DeltaumuDC DeltapolB strains . In contrast, the rate of stationary-phase Arg(+)revertants increased in the absence of pol IV in the mutD5 DeltadinB strain . We postulate that the proofreading activity of pol II excises DNA lesions in exponentially growing cells, whereas pol V and pol IV are more active in stationary-phase cultures .

Arch Microbiol . 2004 May 12; {Epub ahead of print}
Use of Tn KPK2 for sequencing a 10.6-kb PstI DNA fragment of Bradyrhizobium japonicum and for the construction of aspA and ndvA mutants; Wiedemann G et al.; Transposon Tn KPK2 was used to saturate a randomly cloned Bradyrhizobium japonicum PstI fragment and the insertions were used as starting points for the sequence determination . The first gene of the 10.6-kb DNA insert encodes a homologue to ndvA, the product of which is known to be involved in the formation of periplasmic cyclic glucans . Selected Tn KPK2 insertions were introduced into the B . japonicum wild-type strain . The resulting mutants were subsequently tested for their symbiotic interactions with soybeans . As in Sinorhizobium meliloti, a B . japonicum ndvA mutant was affected in salt-stress tolerance and exhibited symbiotic defects in that it induced the formation of ineffective soybean nodules . The central nodule tissue was infected by bacteroids, but within the infected cells the mutant was not properly maintained . Another gene was found to be highly similar to bacterial aspartases and thus was named aspA . The putative function of the product of this gene was confirmed by genetic complementation of aspartase-less Escherichia coli strain TK237 . The symbiotic phenotype of a B . japonicum aspA::Tn KPK2 mutant consisted of enlarged symbiosomes that made the system ineffective . In general, Tn KPK2 is a suitable means for fast sequencing . In combination with pJQ200SK, the resulting recombinant plasmids can be directly used to create genetically defined mutants.

J Biol Chem, 2004 Aug 6, 279(32), 33684 - 95 Epub 2004 May 12.
Reduced pain hypersensitivity and inflammation in mice lacking microsomal prostaglandin e synthase-1; Kamei D et al.; We examined the in vivo role of membrane-bound prostaglandin E synthase (mPGES)-1, a terminal enzyme in the PGE2-biosynthetic pathway, using mPGES-1 knockout (KO) mice . Comparison of PGES activity in the membrane fraction of tissues from mPGES-1 KO and wild-type (WT) mice indicated that mPGES-1 accounted for the majority of lipopolysaccharide (LPS)-inducible PGES in WT mice . LPS-stimulated production of PGE2, but not other PGs, was impaired markedly in mPGES-1-null macrophages, although a low level of cyclooxygenase-2-dependent PGE2 production still remained . Pain nociception, as assessed by the acetic acid writhing response, was reduced significantly in KO mice relative to WT mice . This phenotype was particularly evident when these mice were primed with LPS, where the stretching behavior and the peritoneal PGE2 level of KO mice were far less than those of WT mice . Formation of inflammatory granulation tissue and attendant angiogenesis in the dorsum induced by subcutaneous implantation of a cotton thread were reduced significantly in KO mice compared with WT mice . Moreover, collagen antibody-induced arthritis, a model for human rheumatoid arthritis, was milder in KO mice than in WT mice . Collectively, our present results provide unequivocal evidence that mPGES-1 contributes to the formation of PGE2 involved in pain hypersensitivity and inflammation.

J Biol Chem, 2004 Jul 23, 279(30), 31026 - 32 Epub 2004 May 12.
Targeting and translocation of two lipoproteins in Escherichia coli via the SRP/Sec/YidC pathway; Froderberg L et al.; In Escherichia coli, two main protein targeting pathways to the inner membrane exist: the SecB pathway for the essentially posttranslational targeting of secretory proteins and the SRP pathway for cotranslational targeting of inner membrane proteins (IMPs) . At the inner membrane both pathways converge at the Sec translocase, which is capable of both linear transport into the periplasm and lateral transport into the lipid bilayer . The Sec-associated YidC appears to assist the lateral transport of IMPs from the Sec translocase into the lipid bilayer . It should be noted that targeting and translocation of only a handful of secretory proteins and IMPs have been studied . These model proteins do not include lipoproteins . Here, we have studied the targeting and translocation of two secretory lipoproteins, the murein lipoprotein and the bacteriocin release protein, using a combined in vivo and in vitro approach . The data indicate that both murein lipoprotein and bacteriocin release protein require the SRP pathway for efficient targeting to the Sec translocase . Furthermore, we show that YidC plays an important role in the targeting/translocation of both lipoproteins.

Annu Rev Biophys Biomol Struct, 2004, 33, 119 - 40
Structure, dynamics, and catalytic function of dihydrofolate reductase; Schnell JR et al.; Molecular motions are widely regarded as contributing factors in many aspects of protein function . The enzyme dihydrofolate reductase (DHFR), and particularly that from Escherichia coli, has become an important system for investigating the linkage between protein dynamics and catalytic function, both because of the location and timescales of the motions observed and because of the availability of a large amount of structural and mechanistic data that provides a detailed context within which the motions can be interpreted . Changes in protein dynamics in response to ligand binding, conformational change, and mutagenesis have been probed using numerous experimental and theoretical approaches, including X-ray crystallography, fluorescence, nuclear magnetic resonance (NMR), molecular dynamics simulations, and hybrid quantum/classical dynamics methods . These studies provide a detailed map of changes in conformation and dynamics throughout the catalytic cycle of DHFR and give new insights into the role of protein motions in the catalytic activity of this enzyme.

Neuropathology, 2004 Jun, 24(2), 136 - 43
Expression of glial fibrillary acidic protein in developing rat brain after intrauterine infection; Yu HM et al.; In order to investigate the neuropathological effects on the developing rat brain after intrauterine infection, identification of GFAP was observed . Escherichia coli (E . coli) was inoculated into uterine horn of pregnant rats when gestation was 70% complete (15 days) and the control group was inoculated with normal saline . Immunohistochemistry was used for evaluation of GFAP expression in pup brains at postnatal day 1 (P1), P3, P7, P14 and P21, and RT-PCR was used to analyze GFAP mRNA, interleukin-1beta, mRNA (IL-1beta mRNA) and tumor necrosis factor-alpha mRNA (TNF-alpha mRNA) expression in pup brains at P1, P3 and P7 . At P1 and P3, GFAP was expressed very scarcely in periventricular white matter but not in other brain regions between the two groups . Compared with the control group, at P7 GFAP expression of the E . coli-treated pups was remarkably increased in periventricular white matter and hippocampus . The E . coli-treated pups at P14 showed a marked increase of GFAP expression in periventricular white matter, corpus callosum and cortex . However, no significant difference in levels of GFAP expression in any brain regions were found at P21 between the two groups . GFAP mRNA expression of the E . coli-treated pups was higher than the control at P1 and P3, but there was no significant difference between the two groups at P7 . IL-1beta mRNA and TNF-alpha mRNA expressions of the E . coli-treated pups were higher than the control at P1 but there was no significant difference between the two groups at P3 and P7 . These present results suggest that intrauterine infection could increase GFAP expression in the pup brain and indicate that intrauterine infection might damage the developing white matter and IL-1beta, TNF-alpha might be a mechanism mediating between the two events.

Extremophiles, 2004 Aug, 8(4), 301 - 8 Epub 2004 May 12.
Molecular and biochemical characterization of a fructose-6-phosphate-forming and ATP-dependent fructokinase of the hyperthermophilic archaeon Thermococcus litoralis; Qu Q et al.; Close to an operon encoding an ABC transporter for maltose and trehalose, Thermococcus litoralis contains a gene whose encoded sequence showed similarity to sugar kinases . We cloned this gene, now called frk, and expressed it as a C-terminal His-tag version in Escherichia coli . We purified the recombinant protein, identified it as an ATP-dependent and fructose-6-phosphate-forming fructokinase (Frk) and determined its biochemical properties . At its optimal temperature of 80 degrees C, the apparent Km and Vmax values of Frk were 2.3 mM and 730 U/mg protein for fructose at saturating ATP concentration, and 0.81 mM and 920 U/mg protein for ATP at saturating fructose concentration . The enzyme did not lose activity at 80 degrees C for 4 h . Under denaturating conditions in SDS-PAGE, it exhibited a molecular mass of 35 kDa . Gel-filtration chromatography revealed a molecular mass of 58 kDa, indicating a dimer under nondenaturating, in vitro conditions.

Curr Genet, 2004 Jul, 46(1), 37 - 46 Epub 2004 Apr 27.
Cloning, functional characterization, and near-ultraviolet radiation-enhanced expression of a photolyase gene (PHR1) from the phytopathogenic fungus Bipolaris oryzae; Kihara J et al.; Photolyase is a DNA repair enzyme that can absorb blue/ultraviolet A light as energy and split a pyrimidine dimer induced by ultraviolet radiation . We isolated and characterized PHR1, a gene encoding photolyase, from the phytopathogenic fungus Bipolaris oryzae . Sequence analysis showed that PHR1 encodes a putative protein that has 634 amino acids, a molecular mass of 72.6 kDa, and 51.3-55.5% sequence identity to other fungal photolyases . Complementation of the photoreactivation-deficient Escherichia coli mutant by PHR1 cDNA demonstrated that the PHR1 gene from B . oryzae encodes a functional photolyase . Real-time PCR analysis showed that the PHR1 transcripts were specifically enhanced by near-ultraviolet radiation (300-400 nm) and by sunlight.

Proc Natl Acad Sci U S A, 2004 May 18, 101(20), 7566 - 71 Epub 2004 May 11.
An expanded genetic code with a functional quadruplet codon; Anderson JC et al.; With few exceptions the genetic codes of all known organisms encode the same 20 amino acids, yet all that is required to add a new building block are a unique tRNA/aminoacyl-tRNA synthetase pair, a source of the amino acid, and a unique codon that specifies the amino acid . For example, the amber nonsense codon, TAG, together with orthogonal Methanococcus jannaschii or Escherichia coli tRNA/synthetase pairs have been used to genetically encode a variety of unnatural amino acids in E . coli and yeast, respectively . However, the availability of noncoding triplet codons ultimately limits the number of amino acids encoded by any organism . Here, we report the design and generation of an orthogonal synthetase/tRNA pair derived from archaeal tRNA(Lys) sequences that efficiently and selectively incorporates an unnatural amino acid into proteins in response to the quadruplet codon, AGGA . Frameshift suppression with L-homoglutamine (hGln) does not significantly affect protein yields or cell growth rates and is mutually orthogonal with amber suppression, permitting the simultaneous incorporation of two unnatural amino acids, hGln and O-methyl-L-tyrosine, at distinct positions within myoglobin . This work suggests that neither the number of available triplet codons nor the translational machinery itself represents a significant barrier to further expansion of the genetic code.

J Biol Chem, 2004 Aug 6, 279(32), 33547 - 57 Epub 2004 May 11.
Differential activation of nitric-oxide synthase isozymes by calmodulin-troponin C chimeras; Newman E et al.; The interactions of neuronal nitric-oxide synthase (nNOS) with calmodulin (CaM) and mutant forms of CaM, including CaM-troponin C chimeras, have been previously reported, but there has been no comparable investigation of CaM interactions with the other constitutively expressed NOS (cNOS), endothelial NOS (eNOS), or the inducible isoform (iNOS) . The present study was designed to evaluate the role of the four CaM EF hands in the activation of eNOS and iNOS . To assess the role of CaM regions on aspects of enzymatic function, three distinct activities associated with NOS were measured: NADPH oxidation, cytochrome c reduction, and nitric oxide (*NO) generation as assessed by the oxyhemoglobin capture assay . CaM activates the cNOS enzymes by a mechanism other than stimulating electron transfer into the oxygenase domain . Interactions with the reductase moiety are dominant in cNOS activation, and EF hand 1 is critical for activation of both nNOS and eNOS . Although the activation patterns for nNOS and eNOS are clearly related, effects of the chimeras on all the reactions are not equivalent . We propose that cytochrome c reduction is a measure of the release of the FMN domain from the reductase complex . In contrast, cytochrome c reduction by iNOS is readily activated by each of the chimeras examined here and may be constitutive . Each of the chimeras were co-expressed with the human iNOS enzyme in Escherichia coli and subsequently purified . Domains 2 and 3 of CaM contain important elements required for the Ca2+/CaM independence of *NO production by the iNOS enzyme . The disparity between cytochrome c reduction and *NO production at low calcium can be attributed to poor association of heme and FMN domains when the bound CaM constructs are depleted of Ca2+ . In general cNOSs are much more difficult to activate than iNOS, which can be attributed to their extra sequence elements, which are adjacent to the CaM-binding site and associated with CaM control.

J Biol Chem, 2004 Jul 16, 279(29), 29879 - 88 Epub 2004 May 10.
La protein binding at the GCAC site near the initiator AUG facilitates the ribosomal assembly on the hepatitis C virus RNA to influence internal ribosome entry site-mediated translation; Pudi R et al.; Human La autoantigen has been shown to influence internal initiation of translation of hepatitis C virus (HCV) RNA . Previously, we have demonstrated that, among the three RRMs of La protein, the RRM2 interacts with HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG present in the stem region of stem-loop IV (SL IV) (Pudi, R., Abhiman, S., Srinivasan, N., and Das S . (2003) J . Biol . Chem . 278, 12231-12240) . Here, we have demonstrated that the mutations in the GCAC motif, which altered the binding to RRM2, had drastic effect on HCV IRES-mediated translation, both in vitro and in vivo . The results indicated that the primary sequence of the stem region of SL IV plays an important role in mediating internal initiation . Furthermore, we have shown that the mutations also altered the ability to bind to ribosomal protein S5 (p25), through which 40 S ribosomal subunit is known to contact the HCV IRES RNA . Interestingly, binding of La protein to SL IV region induced significant changes in the circular dichroism spectra of the HCV RNA indicating conformational alterations that might assist correct positioning of the initiation complex . Finally, the ribosome assembly analysis using sucrose gradient centrifugation implied that the mutations within SL IV of HCV IRES impair the formation of functional ribosomal complexes . These observations strongly support the hypothesis that La protein binding near the initiator AUG facilitates the interactions with ribosomal protein S5 and 48 S ribosomal assembly and influences the formation of functional initiation complex on the HCV IRES RNA to mediate efficient internal initiation of translation.

J Biol Chem, 2004 Jul 16, 279(29), 30037 - 46 Epub 2004 May 10.
The DinI protein stabilizes RecA protein filaments; Lusetti SL et al.; When DinI is present at concentrations that are stoichiometric with those of RecA or somewhat greater, DinI has a substantial stabilizing effect on RecA filaments bound to DNA . Exchange of RecA between free and bound forms was almost entirely suppressed, and highly stable filaments were documented with several different experimental methods . DinI-mediated stabilization did not affect RecA-mediated ATP hydrolysis and LexA co-protease activities . Initiation of DNA strand exchange was affected in a DNA structure-dependent manner, whereas ongoing strand exchange was not affected . Destabilization of RecA filaments occurred as reported in earlier work but only when DinI protein was present at very high concentrations, generally superstoichiometric, relative to the RecA protein concentration . DinI did not facilitate RecA filament formation but stabilized the filaments only after they were formed . The interaction between the RecA protein and DinI was modulated by the C terminus of RecA . We discuss these results in the context of a new hypothesis for the role of DinI in the regulation of recombination and the SOS response.

Hepatobiliary Pancreat Dis Int, 2004 May, 3(2), 219 - 22
Construction of eukaryotic expression plasmids of hepatitis B surface antigen and helper T lymphocyte epitope; Piao YF et al.; BACKGROUND: DNA immunization provides a promising approach to elicit protective humoral and cellular immune responses against HBV . This study was to construct an eukaryotic expression plasmid containing helper T lymphocyte epitope, which will enhance the immunogenicity of a novel hepatitis B virus (HBV) fusion protein DNA vaccine . METHODS: The target gene containing pan-DR helper T cell epitopes (PADRE) and HBsAg was amplified by polymerase chain reaction (PCR) . The PCR products were linked with PMD-18T vector . Plasmid DNA was purified from transformed E.coli competent cell JM109 and digested with Hind III and EcoR I . Then, the target gene was cloned in pcDNA3.1(+) digested by Hind III and EcoR I . Finally, the identity of DNA was verified by digestion and DNA sequencing . RESULTS: The recombinant expression vectors of pcDNA3.1(+)-PADRE/HBs were identified by restriction enzyme digestion and DNA sequencing . The insert DNA fragment was consistent with the expected sequence . CONCLUSIONS: The constructed eukaryotic expression plasmid of pcDNA3.1(+)-PADRE/HBs is convenient for further study of eukaryotic transfection and response for cellular and humoral immunity against HBV.

Mol Biochem Parasitol, 2004 Jul, 136(1), 101 - 7
Identification and characterization of serine proteinase inhibitors from Neospora caninum; Bruno S et al.; Two cDNA clones obtained from the Neospora caninum Expressed Sequence Tag project were selected by their homology with the Toxoplasma gondii serine proteinase inhibitor (serpin) gene, TgPI-1 and TgPI-2 . One of them, named NcPI-H, showed several premature stop codons . The other cDNA, named NcPI-S, encoded a 79 amino acid protein containing a putative signal peptide and only one non-classical Kazal domain . Two other N . caninum EST sequences (NcEST1 and NcEST2) and one from Eimeria tenella (EtPI-S) were retrieved from the database . Amino acid sequence analysis suggested that NcEST1 and NcEST2 might be the N . caninum counterparts of TgPI-1 and TgPI-2, respectively . EtEST-S, as NcPI-S, is a single domain serpin . The open reading frame encoding the mature version of NcPI-S was expressed as recombinant protein, fused to a 6 histidine tag in Escherichia coli . Specific rabbit antiserum generated against the recombinant NcPI-S was used in immunoblot assays . Bands of 20, 30, 40, and 66-kDa were detected by SDS-PAGE of whole parasite homogenate . In addition, when an anti-TgPI-1 serum was used, bands of 25 and 35-kDa were detected indicating that there is no cross-reactivity between both serpins, and showing as well, the presence of another putative serpin in N . caninum . The recombinant protein NcPI-S, inhibited bacterial subtilisin completely, and showed lower inhibitory capacity on human neutrophil elastase, animal trypsin, and chymotrypsin, suggesting differences in effectiveness.

Biochem J, 2004 Aug 15, 382(Pt 1), 247 - 52
Mycobacterium tuberculosis DnaA initiator protein: purification and DNA-binding requirements; Zawilak A et al.; The Mycobacterium tuberculosis oriC (the origin of chromosomal replication) region contains 13 non-perfect DnaA boxes . The M . tuberculosis initiator protein, DnaA, was overexpressed in Escherichia coli as a soluble His-tagged fusion protein . The purified protein His6MtDnaA was investigated for its binding properties to DnaA boxes from the oriC region . Gel retardation demonstrated that the DnaA from M . tuberculosis requires two DnaA boxes for efficient binding . Electron microscopy as well as DNase I footprinting showed that the His6MtDnaA protein binds to four specific regions, which correspond to the location of 11 out of 13 previously identified DnaA boxes within the M . tuberculosis oriC . Probably, in M . tuberculosis, DnaA molecules by co-operative binding of numerous 'non-perfect' DnaA boxes assemble along the oriC region and subsequently form a massive nucleoprotein complex.

Biopolymers, 2004 May-Jun 5, 74(1-2), 69 - 72
Fourier transform infrared spectroscopic study on the conformational reorganization in Escherichia coli complex I due to redox-driven proton translocation; Hellwig P et al.; The proton-pumping NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane . Electron transfer is accomplished by flavin mononucleotide (FMN) and a series of iron-sulfur (Fe/S) clusters . A novel mechanism has been proposed wherein the electron transfer reaction induces conformational changes that subsequently lead to the translocation of protons . Redox-induced Fourier transform infrared difference spectra have been obtained, showing strong conformational changes in the amide I region . The amplitude of the signal is pH dependent, as expected for an energy coupling step in the enzymes reaction . Furthermore, pH-dependent protonation events and quinone binding were detected .

Biopolymers, 2004 May-Jun 5, 74(1-2), 60 - 3
Changes of near-UV CD spectrum of human hemoglobin upon oxygen binding: a study of mutants at alpha 42, alpha 140, beta 145 tyrosine or beta 37 tryptophan; Jin Y et al.; The deoxy-form of human adult hemoglobin (Hb A) exhibits a distinct negative CD band at 287 nm that disappears in the oxy-form . It has been suggested that the negative CD band is due to the environmental alteration of Tyr-alpha 42 or Trp-beta 37 at the alpha(1)beta(2) contact upon deoxygenation . To evaluate the contributions of the aromatic residues at the alpha(1)beta(2) contact and the penultimate tyrosine residues of the alpha and beta subunits (alpha 140 and beta 145) to the negative CD band, three recombinant (r) Hbs (rHb Ser-alpha 42, rHb His-beta 37, and rHb Thr-beta 145) were produced in Escherichia coli, and we compared the near-uv CD spectra of these three rHbs and Hb Rouen (Tyr-alpha 140-->His) with the spectra of Hb A under the condition in which all mutant Hbs were able to undergo the T-->R transition (Hill's n > 2.0) . The contributions of Tyr-alpha 42, Trp-beta 37, Tyr-alpha 140, and Tyr-beta 145 to the negative CD band were estimated from changes in the ellipticity of the negative CD band at 287 nm to be 4, 18, 32, and 27%, respectively . These results indicate that environmental alteration of the penultimate tyrosine residues caused by the formation of salt bridges upon the R-->T transition is primarily responsible for the negative CD band .

Biotechnol Bioeng, 2004 Jun 20, 86(6), 687 - 97
Engineering green fluorescent protein as a dual functional tag; Paramban RI et al.; A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins . Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus . The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC) . The purification efficiencies by IMAC for all variants were found to be comparable . Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis . The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants . Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants . Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157 . The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner . The MBP-GFP172 fusion protein remained fluorescent and was purified from E . coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC . For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1 . The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion .

J Nucl Med, 2004 May, 45(5), 867 - 77
Single-chain Fv-streptavidin substantially improved therapeutic index in multistep targeting directed at disialoganglioside GD2; Cheung NK et al.; Multistep targeting can improve the therapeutic index of antibody-based targeting, particularly relevant to pediatric tumors where acute toxicity and late effects of treatment are major concerns . Neuroblastoma is uniquely suited for such investigations because of its abundance of surface ganglioside GD2 . METHODS: 5F11scFv (scFv = single-chain variable fragment) was constructed from the variable regions of the heavy (V(H)) and kappa-light (V(L)) chain complementary DNA (cDNA) of anti-GD2 IgM hybridoma 5F11 and ligated to full-length streptavidin cDNA for expression in Escherichia coli . Purified 5F11-scFv-streptavidin (5F11-scFv-SA) was a homotetramer and showed comparable avidity to 5F11 IgM and a 30-fold improvement over monomeric scFv . Biodistribution of 5F11-scFv-SA was studied in nude mice xenografted with neuroblastoma LAN-1 . Twenty-four hours after intravenous injection of 300-900 microg 5F11-scFv-SA, 150-450 microg of a thiogalactoside-containing clearing agent, (Gal-NAc)(16)-alpha-S-C(5)H(10)-NH-LC-N-Me-biotin (molecular weight, 8652), were administered intravenously, followed by approximately 2.5 microg (1.85-3.7 MBq) (111)In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin ((111)In-DOTA-biotin) intravenously 4 h later and clocked as time 0 . RESULTS: Tumor uptake (percentage of injected dose per gram {%ID/g}) at 2 h was 7 %ID/g and decayed with a half-life of 72 h, whereas blood %ID/g rapidly decreased to <1/500 of that of tumor after the first 24 h . The tumor-to-nontumor (T/NT) ratio at 72 h was high (median, 106; range, 3.4 {kidney} to 1660 {blood}) . When the area under the radioactivity curve was computed, the T/NT organ ratio was favorable (4.8 for kidney and 162 for blood) . When human and murine tumors were surveyed, the T/NT ratio of (111)In-DOTA-biotin uptake correlated with their levels of GD2 expression as assayed by flow cytometry . Biotinylated polypeptides (bovine serum albumin and vasointestinal peptides) achieved selective tumor targeting when the multistep strategy was applied . CONCLUSION: Improvement in the T/NT ratio using pretargeting strategy may increase the efficacy and safety of scFv-based approaches in cancer therapy . Additionally, since biotinylated polypeptides can be rendered tumor selective, a large repertoire of agents can potentially be explored.

J Biol Chem, 2004 Aug 20, 279(34), 35622 - 9 Epub 2004 May 10.
The structure of the pantothenate kinase.ADP.pantothenate ternary complex reveals the relationship between the binding sites for substrate, allosteric regulator, and antimetabolites; Ivey RA et al.; Pantothenate kinase catalyzes the first step in the biosynthesis of coenzyme A, the major acyl group carrier in biology . In bacteria, regulation of pantothenate kinase activity is a major factor in controlling intracellular coenzyme A levels, and pantothenate analogs are growth-inhibiting antimetabolites . We have extended the structural information on Escherichia coli pantothenate kinase by determining the structure of the enzyme.ADP . pantothenate ternary complex . Pantothenate binding induces a significant conformational change in amino acids 243-263, which form a "lid" that folds over the open pantothenate binding groove . The positioning of the substrates suggests the reaction proceeds by a concerted mechanism that involves a dissociative transition state, although the negative charge neutralization of the gamma-phosphate by Arg-243, Lys-101, and Mg(2+) coupled with hydrogen bonding of the C1 of pantothenate to Asp-127 suggests different interpretations of the phosphoryl transfer mechanism of pantothenate kinase . N-alkylpantothenamides are substrates for pantothenate kinase . Modeling these antimetabolites into the pantothenate active site predicts that they bind in the same orientation as pantothenate with their alkyl chains interacting with the hydrophobic dome over the pantothenate pocket, which is also accessed by the beta-mercaptoethylamine moiety of the allosteric regulator, coenzyme A . These structural/biochemical studies illustrate the intimate relationship between the substrate, allosteric regulator, and antimetabolite binding sites on pantothenate kinase and provide a framework for studies of its catalysis and feedback regulation.

J Biol Chem, 2004 Jul 30, 279(31), 32569 - 77 Epub 2004 May 10.
Localization of human Mcm10 is spatially and temporally regulated during the S phase; Izumi M et al.; Mcm10 (Dna43) is an essential protein for the initiation of DNA replication in Saccharomyces cerevisiae . Recently, we identified a human Mcm10 homolog and found that it is regulated by proteolysis and phosphorylation in a cell cycle-dependent manner and that it binds chromatin exclusively during the S phase of the cell cycle . However, the precise roles that Mcm10 plays are still unknown . To study the localization dynamics of human Mcm10, we established HeLa cell lines expressing green fluorescent protein (GFP)-tagged Mcm10 . From early to mid-S phase, GFP-Mcm10 appeared in discrete nuclear foci . In early S phase, several hundred foci appeared throughout the nucleus . In mid-S phase, the foci appeared at the nuclear periphery and nucleolar regions . In the late S and G phases, GFP-Mcm10 was localized to nucleoli . Although (2)the distributions of GFP-Mcm10 during the S phase resembled those of replication foci, GFP-Mcm10 foci did not colocalize with sites of DNA synthesis in most cases . Furthermore, the transition of GFP-Mcm10 distribution patterns preceded changes in replication foci patterns or proliferating cell nuclear antigen foci patterns by 30-60 min . These results suggest that human Mcm10 is temporarily recruited to the replication sites 30-60 min before they replicate and that it dissociates from chromatin after the activation of the prereplication complex.

Anal Biochem, 2004 Jun 1, 329(1), 11 - 20
Toward pyrosequencing on surface-attached genetic material by use of DNA-binding luciferase fusion proteins; Ehn M et al.; Mutation detection and single-nucleotide polymorphism genotyping require screening of large samples of materials and therefore the importance of high-throughput DNA analysis techniques is significant . Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technology based on the sequencing-by-synthesis principle . Currently, the technique is limited to simultaneous analysis of 96 or 384 samples . Earlier, attempts to increase the sample capacity were made using micromachined filter chamber arrays where parallel analyses of nanoliter samples could be monitored in real time . We have developed a strategy for specific immobilization of the light-producing enzyme luciferase to the DNA template within a reaction chamber . By this approach, luciferase is genetically fused to a DNA-binding protein (Klenow polymerase or Escherichia coli single-stranded DNA-binding (SSB) protein) and to a purification handle (Z(basic)) . The proteins are produced in E . coli and purified using cation and anion exchange chromatography with removal of Z(basic) . The produced proteins have been analyzed using an assay for complete primer extension of DNA templates immobilized on magnetic beads detected by pyrosequencing chemistry . Results from these experiments show that the proteins bind selectively to the immobilized DNA and that their enzymatic domains were active . Z(basic)-SSB-luciferase produced the highest signal in this assay and was further exploited as enzymatic reagent for DNA sequencing.

Biochim Biophys Acta, 2004 May 12, 1656(1), 1 - 31
Microsecond freeze-hyperquenching: development of a new ultrafast micro-mixing and sampling technology and application to enzyme catalysis; Cherepanov AV et al.; A novel freeze-quench instrument with a characteristic <<dead-time>> of 137 +/- 18 micros is reported . The prototype has several key features that distinguish it from conventional freeze-quench devices and provide a significant improvement in time resolution: (a) high operating pressures (up to 400 bar) result in a sample flow with high linear rates (up to 200 m s(-1)); (b) tangential micro-mixer with an operating volume of approximately 1 nl yields short mixing times (up to 20 micros); (c) fast transport between the mixer and the cryomedium results in short reaction times: the ageing solution exits the mixer as a free-flowing jet, and the chemical reaction occurs "in-flight" on the way to the cryomedium; (d) a small jet diameter (approximately 20 microm) and a high jet velocity (approximately 200 m s(-1)) provide high sample-cooling rates, resulting in a short cryofixation time (up to 30 micros) . The dynamic range of the freeze-quench device is between 130 micros and 15 ms . The novel tangential micro-mixer efficiently mixes viscous aqueous solutions, showing more than 95% mixing at eta < or = 4 (equivalent to protein concentrations up to 250 mg ml(-1)), which makes it an excellent tool for the preparation of pre-steady state samples of concentrated protein solutions for spectroscopic structure analysis . The novel freeze-quench device is characterized using the reaction of binding of azide to metmyoglobin from horse heart . Reaction samples are analyzed using 77 K optical absorbance spectroscopy, and X-band EPR spectroscopy . A simple procedure of spectral analysis is reported that allows (a) to perform a quantitative analysis of the reaction kinetics and (b) to identify and characterize novel reaction intermediates . The reduction of dioxygen by the bo3-type quinol oxidase from Escherichia coli is assayed using the MHQ technique . In these pilot experiments, low-temperature optical absorbance measurements show the rapid oxidation of heme o3 in the first 137 micros of the reaction, accompanied by the formation of an oxo-ferryl species . X-band EPR spectroscopy shows that a short-living radical intermediate is formed during the oxidation of heme o3 . The radical decays within approximately 1 ms concomitant with the oxidation of heme b, and can be attributed to the PM reaction intermediate converting to the oxoferryl intermediate F . The general field of application of the freeze-quench methodology is discussed .

DNA Repair (Amst), 2004 Jun 3, 3(6), 639 - 47
Functional interactions between the MutL and Vsr proteins of Escherichia coli are dependent on the N-terminus of Vsr; Monastiriakos SK et al.; The crystal structure of the Escherichia coli Vsr endonuclease bound to a C(T/G)AGG substrate revealed that the DNA is held by a pincer composed of a trio of aromatic residues which intercalate into the major groove, and an N-terminus alpha helix which lies across the minor groove . We have constructed an N-terminus truncation (Delta14) which removes most of the alpha helix . The mutant is still fairly proficient in mediating very short patch repair . However, its endonuclease activity is considerably reduced and, in contrast to that of the wild type protein, cannot be stimulated by MutL . We had shown previously that excess Vsr in vivo causes mutagenesis, probably by inhibiting the participation of MutL in mismatch repair . The Delta14 mutant has diminished mutagenicity . In contrast, four enzymatically inactive mutants, with intact N-termini, are as mutagenic as the wild type protein . On the basis of these results we suggest that MutL causes a conformational change in the N-terminus of Vsr which enhances Vsr activity, and that this functional interaction between Vsr and MutL decreases the ability of MutL to carry out mismatch repair.

DNA Repair (Amst), 2004 Jun 3, 3(6), 603 - 15
Disruption of Drosophila Rad50 causes pupal lethality, the accumulation of DNA double-strand breaks and the induction of apoptosis in third instar larvae; Gorski MM et al.; The Rad50/Mre11/Nbs1 protein complex has a crucial role in DNA metabolism, in particular in double-strand break (DSB) repair through homologous recombination (HR) . To elucidate the role of the Rad50 protein complex in DSB repair in a multicellular eukaryote, we generated a Rad50 deficient Drosophila strain by P-element mediated mutagenesis . Disruption of Rad50 causes retarded development and pupal lethality . To investigate the mechanism of pupal death, brains and wing imaginal discs from third instar larvae were studied in more detail . Wing imaginal discs from Rad50 mutant larvae displayed a 3.5-fold increase in the induction of spontaneous apoptotic cells in comparison to their heterozygous siblings . This finding correlates with increased levels of phosphorylated histone H2Av, indicating an accumulation of DSBs in Rad50 mutant larvae . A 45-fold increase in the frequency of anaphase bridges was detected in the brains of Rad50 deficient larvae, consistent with a role for Rad50 in telomere maintenance and/or replication of DNA . The induction of DSBs and defects in chromosome segregation are in agreement with a role of Drosophila Rad50 in repairing the DSBs that arise during replication.

Mutat Res, 2004 Jun 4, 550(1-2), 33 - 48
Long-chain adducts of trans-4-hydroxy-2-nonenal to DNA bases cause recombination, base substitutions and frameshift mutations in M13 phage; Kowalczyk P et al.; Oxidative stress enhances lipid peroxidation (LPO) implicated in the promotion and progression of carcinogenesis . One of the major LPO products is trans-4-hydroxy-2-nonenal (HNE), which was shown to react with guanosine and under peroxidizing conditions also with adenosine . We show here that all four DNA bases are targets for HNE, although displaying different reactivity: dG > dC > dA approximately equal to dT . HPLC and mass spectrometry analyses of HNE reactions with deoxynucleosides showed in each case the formation of several products, with mass peaks corresponding to HNE-dN adducts at a 1:1 and also 2:1 and 3:1 ratios . In the dA, dC and dG reactions, mass peaks corresponding to heptyl-substituted etheno-adducts were also detected, indicating HNE oxidation to its epoxide by air oxygen . In DNA pretreated with HNE, DNA synthesis by T7 DNA polymerase was stopped in a sequence-dependent manner at G > or = C > A and T sites . HNE increased the mutation rates in the lac Z gene of M13 phage transfected into wild type Escherichia coli . The most frequent event was the recombination between lacZ gene sequences in M13 and the E . coli F' factor DNA . Base substitutions and frameshifts were also observed in approximately similar numbers . Over 50% of base substitutions were the C-->T transitions, followed by the G-->C and A-->C transversions . In the E . coli recA strain recombination was not observed, although one mutational G-->T hot-spot appeared within the DNA fragment undergoing recombination in the wild type E . coli . We conclude that long chain HNE adducts to DNA bases arrest DNA synthesis and cause recombination, base substitutions and frameshift mutations in ssDNA.

Mutat Res, 2004 Jun 4, 550(1-2), 25 - 32
Mechanism of 2-aminopurine-stimulated mutagenesis in Escherichia coli; Pitsikas P et al.; 2-Aminopurine (2AP), a base analog, causes both transition and frameshift mutations in Escherichia coli . The analog is thought to cause mutations by two mechanisms: directly, by mispairing with cytosine, and indirectly, by saturation of mismatch repair (MMR) . The goal of this work was to measure the relative contribution of these two mechanisms to the occurrence of transition mutations . Our data suggest that, in contrast to 2-aminopurine-stimulated frameshift mutations, the majority of transition mutations are a direct effect of base mispairing.

FEMS Microbiol Lett, 2004 May 15, 234(2), 325 - 31
An improved method for deleting large regions of Escherichia coli K-12 chromosome using a combination of Cre/loxP and lambda Red; Fukiya S et al.; We have established an improved large deletion method in Escherichia coli genome using a combination of two different recombination systems, lambda Red and Cre/loxP . The loxP site could be rapidly and efficiently integrated in the genome by lambda Red and large deletions of both 117- and 165-kbp regions could be generated in 100% efficiency by Cre/loxP . Comparative genomic hybridization microarray experiments of deletion strains indicated that deletions were generated only in expected regions of the genome . These results have demonstrated that the method is useful for genome engineering in E . coli .

FEMS Microbiol Lett, 2004 May 15, 234(2), 303 - 8
Topological studies on the twin-arginine translocase component TatC; Behrendt J et al.; The twin-arginine translocation (Tat) system can translocate folded proteins across biological membranes . Among the known Tat-system components in Escherichia coli, TatC is the only protein with multiple trans-membrane domains . TatC is important for translocon interactions with Tat substrates . The knowledge of its membrane topology is therefore crucial for the understanding of substrate binding and translocon function . Recently, based on active PhoA reporter fusions to the second predicted cytoplasmic loop of TatC, a topology with four trans-membrane domains has been suggested, calling in silico predictions of six trans-membrane domains into question . Here we report studies with translational fusions of TatC to the topological marker enzymes PhoA and LacZ which provide strong evidence for a six-trans-membrane domain topology . The stop transfer capacity of the fourth trans-membrane domain was found to be strongly influenced by the succeeding cytoplasmic domain . The presence of linker sequences at PhoA-fusion sites of the cytoplasmic domain induced PhoA leakage . In the case of one tested fusion (S185-PhoA), the stop-transfer efficiency was already low due to the negative charge in the center of the fourth trans-membrane domain (E170) . The results point to the importance of cytoplasmic loops for the stabilization of stop-transfer sequences and revoke evidence for only four trans-membrane domains of TatC .

FEMS Microbiol Lett, 2004 May 15, 234(2), 297 - 301
Use of a novel allele of the Escherichia coli aacC4 aminoglycoside resistance gene as a genetic marker in mycobacteria; Consaul SA et al.; The aacC4 gene from Escherichia coli can be expressed in mycobacteria and confers resistance to apramycin . However, the major limitation of the aacC4 gene as a genetic tool is that the gene also confers resistance to kanamycin and gentamicin, two antibiotics commonly used for selection in mycobacterial genetics, thus reducing the utility of the aacC4 gene in the mycobacterial field . To overcome this problem we constructed, by chemical mutagenesis, a mutant allele of the E . coli aacC4 gene that still confers resistance to apramycin but has a reduced ability to confer resistance to kanamycin and gentamicin . We then constructed a variety of E . coli-mycobacteria shuttle plasmids containing this mutant allele .

Vet Microbiol, 2004 May 20, 100(1-2), 55 - 64
Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A . centrale vaccine strain; Molad T et al.; Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed . The identity between Anaplasma marginale and A . centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences . A . centrale msp5 nucleotide sequences shared 86.8% identity with A . marginale msp5, and there was 92.9% homology between A . centrale and A . marginale encoded amino acids of the MSP5 protein . Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A . centrale are encoded by single copy genes . Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A . marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A . centrale and A . marginale . These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A . centrale and A . marginale . This conservation is consistent with the cross-protective immunity between A . marginale and A . centrale and supports the development of improved vaccines based upon common outer membrane proteins.

Protein Expr Purif, 2004 Jun, 35(2), 360 - 5
Purification and refolding of human alpha5-subunit (PSMA5) of the 20S proteasome, expressed as inclusion bodies in Escherichia coli; Han YG et al.; The 20S proteasome is the central enzyme of nonlysosomal protein degradation in both the cytosol and nucleus . It is composed of 28 protein subunits which are arranged into four staggered heptameric rings . The outer rings consist of alpha-subunits which are responsible for binding of proteasome activators, inhibitors, and regulators . To better characterize human alpha5-subunit (PSMA5) of the 20S proteasome, we have established a high-efficiency Escherichia coli expression system . The DNA-coding sequence for the human PSMA5, which was subcloned into the vector pET-22b (+), has been expressed as inclusion bodies in E . coli BL21 (DE3) . To produce the native PSMA5, straightforward protocols have been developed for refolding the human PSMA5 in the presence of surfactants using dilution refolding and size-exclusion chromatography matrix refolding methods . After refolding, recovery yields of about 20% were obtained, respectively, with purity above 95% . The human PSMA5 was detected by dynamic light scattering in refolding process, and the molecular weight of the final refolded product was measured using gel filtration chromatography, which indicates that the human PSMA5 exists mainly as tetramer.

Protein Expr Purif, 2004 Jun, 35(2), 353 - 9
Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli; Neves FO et al.; Interferons (IFNs) represent an important defense mechanism in vertebrates . In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences . The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein . DNA sequences were cloned into pAE, an E . coli vector that allows heterologous protein expression with or without a histidine tag . Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies . Purification of the recombinant His-tagged proteins was achieved in a single step by Ni(2+)-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being approximately 210 and 75mg/L, respectively . The rhIFNs expressed within this system were biologically active ( approximately 1,1x10(8)IU/mg) based on antiviral assay . The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest.

Protein Expr Purif, 2004 Jun, 35(2), 344 - 52
Cloning, expression, and structural analysis of recombinant BJcuL, a c-type lectin from the Bothrops jararacussu snake venom; Kassab BH et al.; The lactose-binding lectin from Bothrops jararacussu venom (BJcuL) is a homodimer belonging to group VII of the c-type animal lectins . BJcuL has also been shown to serve as an interesting tool for combating tumor progression by inhibiting cancer and endothelial cell growth . However, detailed structural studies of BJcuL and its biological mechanisms of cytotoxicity are yet to be reported, perhaps because of the non-availability of recombinant proteins in necessary quantities . Intending to increase the present information about structural and consequently the understating of biological studies, the cDNA coding for BJcuL from a venom gland has been cloned and sequenced . The mature protein-coding region was amplified by PCR with specific oligonucleotides, and subcloned into the pET-15b vector to express the recombinant BJcuL in Escherichia coli BL21 (DE3) . The deduced amino acid sequence exhibits a high degree of sequence identity with c-type lectins (CTLs) and c-type lectin-like domains (CTLDs) . An insoluble and inactive 18.5-kDa protein was overexpressed after 1.0mM IPTG induction . The recombinant BJcuL was recovered and denatured in a buffer with 6M urea and purified on a nickel-affinity column . Protein refolding was carried out on this column, during procedure purification, followed by dialysis against CTBS and then by gel filtration for separation of the active dimmer . The refolding process of rBJcuL and the analysis of its structure were confirmed by biological assay, circular dichroism, and MALDI-TOF.

Protein Expr Purif, 2004 Jun, 35(2), 334 - 43
Expression, purification, and in vitro characterization of recombinant salmon insulin-like growth factor-II; Wilkinson RJ et al.; The insulin-like growth factors, IGF-I and IGF-II, are single chain polypeptides, which are structurally related to proinsulin and promote proliferation and differentiation of cells in many vertebrate species . Previous attempts to produce recombinant salmon IGF-II (rsIGF-II) were compromised by low expression levels and co-purification of incorrectly cleaved protein with the authentic recombinant product . In this study, a gene containing the coding region for Atlantic salmon (Salmo salar) IGF-II was cloned into a modified pET32a expression vector and transformed into Escherichia coli BL21 trxB (DE3) cells . Upon growth and induction (with IPTG) of the transformant, recombinant salmon IGF-II (rsIGF-II) was expressed as an insoluble, 28kDa thioredoxin.sIGF-II fusion protein linked by a protease cleavage motif (trx.FAHY.sIGF-II) in inclusion bodies . The inclusion bodies were subsequently solubilized and the fusion protein was purified by Ni-affinity chromatography . Recombinant IGF-II (7.8kDa) was then released from the fusion partner using H64A subtilisin BPN' protease and purified by reversed-phase HPLC . Homogeneity of the final recombinant product was confirmed by N-terminal amino acid sequencing, ion-spray mass spectrometry, SDS-polyacrylamide gel electrophoresis, and analytical reversed-phase HPLC . The biological activity of rsIGF-II was demonstrated in cultured rat L6 myoblasts and was found to be approximately 9- and 5-fold less potent than recombinant human IGF-I and recombinant salmon IGF-I, respectively, a result similar to that demonstrated previously with other recombinant fish IGF-II's in non-homologous cell lines.

Protein Expr Purif, 2004 Jun, 35(2), 327 - 33
Bacterially expressed recombinant p68 RNA helicase is phosphorylated on serine, threonine, and tyrosine residues; Yang L et al.; We previously reported the expression and purification of recombinant p68 RNA helicase in a bacterial expression system . The recombinant p68 is an RNA-dependent ATPase and ATP-dependent RNA helicase . In the process of characterizing the ATPase and RNA unwinding activities of the recombinant p68, we observed that the bacterially expressed p68 RNA helicase is phosphorylated on tyrosine, serine, and threonine residues . Our data demonstrated that phosphorylations on the recombinant p68 RNA helicase affect the enzymatic activities of the protein . This is the first observation that recombinant protein expressed in bacteria Escherichia coli is phosphorylated at multiple residues by bacterial endogenous protein kinases . Our observations suggest an important mechanism in controlling the function of p68 RNA helicase by signal transduction pathways.

Protein Expr Purif, 2004 Jun, 35(2), 320 - 6
Expression and purification of His-tagged rat mitochondrial 3-ketoacyl-CoA thiolase wild-type and His352 mutant proteins; Zeng J et al.; Mitochondrial 3-ketoacyl-CoA thiolase is a key enzyme for the beta-oxidation of fatty acids, and the deficiency of this enzyme in patients has been previously reported . We cloned a cDNA of rat mitochondrial 3-ketoacyl-CoA thiolase into a bacterial expression vector pLM1 with six continuous histidine codons attached to the 5' end of the gene . The cloned cDNA was overexpressed in Escherichia coli and the soluble protein was purified with a nickel Hi-Trap chelating metal affinity column in 92% yield to apparent homogeneity . The specific activity of the purified His-tagged rat mitochondrial 3-ketoacyl-CoA thiolase was 25U/mg . It has been proposed that His352 is a catalytic residue responsible for activation of coenzyme A by deprotonation of a sulfhydryl group . We constructed four mutant expression plasmids of the enzyme using site-directed mutagenesis . Mutant proteins were overexpressed in E . coli and purified with a nickel metal affinity column . Kinetic studies of wild-type and mutant proteins were carried out, and the result confirmed that His352 is a catalytic residue of rat mitochondrial 3-ketoacyl-CoA thiolase . Our overexpression in E . coli and one-step purification of the highly active rat mitochondrial 3-ketoacyl-CoA thiolase greatly facilitated our further investigation of this enzyme, and our result from site-directed mutagenesis increased our understanding of the mechanism for the reaction catalyzed by 3-ketoacyl-CoA thiolase.

Protein Expr Purif, 2004 Jun, 35(2), 298 - 303
Expression, purification, and DNA-binding activity of the Herbaspirillum seropedicae RecX protein; Galvao CW et al.; The Herbaspirillum seropedicae RecX protein participates in the SOS response: a process in which the RecA protein plays a central role . The RecX protein of the H . seropedicae, fused to a His-tag sequence (RecX His-tagged), was over-expressed in Escherichia coli and purified by metal-affinity chromatography to yield a highly purified and active protein . DNA band-shift assays showed that the RecX His-tagged protein bound to both circular and linear double-stranded DNA and also to circular single-stranded DNA . The apparent affinity of RecX for DNA decreased in the presence of Mg(2+) ions . The ability of RecX to bind DNA may be relevant to its function in the SOS response.

Protein Expr Purif, 2004 Jun, 35(2), 293 - 7
In vivo increase of solubility of overexpressed Hha protein by tandem expression with interacting protein H-NS; Pons JI et al.; Gene cloning in appropriate vectors followed by protein overexpression in Escherichia coli is the common means for protein purification . This approach has many advantages but also some drawbacks; one of these is that many proteins fail to achieve a soluble conformation when overexpressed in E . coli . Hha protein belongs to a family of nucleoid-associated proteins functionally related to the H-NS family of proteins . Hha-like proteins and H-NS-like proteins are able to semidirectly bind to each other . We show in this work that overexpressed Hha or HisHha protein (a functional derivative of Hha containing a 6x His tag at the amino end) from a T7-polymerase promoter in BL21 DE3 E . coli strains results in the vast majority of the protein accumulated in insoluble aggregates (inclusion bodies) . We also show that tandem overexpression of HisHha and H-NS increases the solubility of HisHha and prevents the formation of inclusion bodies . Single amino acid substitutions in the HisHha protein, which impair interaction with H-NS, render insoluble protein even when tandem-expressed with H-NS, tandem expression of an insoluble protein and an interacting partner is an experimental strategy which could be useful to increase the solubility of other overexpressed proteins in E . coli.

Protein Expr Purif, 2004 Jun, 35(2), 276 - 83
Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus; Anguera MC et al.; Folate metabolism is necessary for the biosyntheses of purine nucleotides and thymidylate and for the synthesis of S-adenosylmethionine, a cofactor required for cellular methylation reactions and a precursor of spermidine and spermine syntheses . Disruption of folate metabolism is associated with several pathologies and developmental anomalies including cancer and neural tube defects . The enzyme 5,10-methenyltetrahydrofolate synthetase (MTHFS, EC 6.3.3.2) catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, and has been shown to affect intracellular folate concentrations by accelerating folate degradation . Mammalian MTHFS proteins described to date are not stable and no recombinant mammalian MTHFS protein has been successfully expressed in Escherichia coli . The three-dimensional structure of MTHFS has not been solved . The cDNA coding for Mus musculus MTHFS was isolated and expressed in E . coli with a hexa-histidine tag . Milligram quantities of recombinant mouse MTHFS were purified using metal affinity chromatography and the protein was stabilized with Tween 20 . Mouse MTHFS has a molecular mass of 23kDa and is 84% identical in amino acid sequence to the human enzyme . Activity assays confirmed the functionality of the recombinant protein, with Km =5 microM for (6S)-5-formyltetrahydrofolate and Km=769 microM for Mg-ATP . This is the first example of a mammalian form of MTHFS expressed in E . coli that yielded sufficient quantities of stable purified protein to allow for detailed characterization of its three-dimensional structure and kinetic properties.

Protein Expr Purif, 2004 Jun, 35(2), 257 - 63
Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants; Zhang DQ et al.; Antifreeze proteins (AFPs) enable organisms to survive under freezing or sub-freezing conditions . AFPs have a great potential in the low temperature storage of cells, tissues, organs, and foods . This process will require a large number of recombinant AFPs . In the present study, the recombinant carrot AFP was highly expressed in Escherichia coli strain BL21 (DE3) . The activity of the purified and refolded recombinant proteins was analyzed by measurement of thermal hysteresis (TH) activity and detection of in vitro antifreeze activity by measuring enhanced cold resistance of bacteria . Two carrot AFP mutants generated by site-directed mutagenesis were also expressed and purified under these conditions for use in parallel experiments . Recombinant DcAFP displayed a TH activity equivalent to that of native DcAFP, while mutants DcAFP-N130Q and rDcAFP-N130V showed 32 and 43% decreases in TH activity, respectively . Both the recombinant DcAFP and its mutants were able to enhance the cold resistance of bacteria, to degrees consistent with their respective TH activities.

Protein Expr Purif, 2004 Jun, 35(2), 225 - 36
Expression, refolding, and purification of recombinant human phosphodiesterase 3B: definition of the N-terminus of the catalytic core; Varnerin JP et al.; We have developed an expression, refolding, and purification protocol for the catalytic domain of human Phosphodiesterase 3B (PDE3B) . High level expression in Escherichia coli has been achieved with yields of up to 20mg/L . The catalytic domain of the enzyme was purified by affinity chromatography utilizing a novel affinity ligand . PDE3B, purified by affinity chromatography, with no single impurity #10878;1% as determined by SDS-PAGE, has a specific activity of 2210+/-442nmol/min/mg and a KM for cAMP of 44+/-4.5nM . Reducing the size of the expressed catalytic domain from residues 387-1112 to residues 654-1086 greatly reduced the aggregation phenomena observed with the affinity purified PDE3B . The definition of the N-terminus of the catalytic core was examined through the generation of several truncation mutants spanning amino acid residues 636-674 . Constructs starting at E665 and M674 were fully active and devoid of activity, respectively . A construct starting at D668 had a Vmax reduced by approximately 10-fold relative to the longer constructs, yet the KM was not affected . This indicates the minimal N-terminus of the catalytic core lies between E665 and Y667 . Refolding and affinity purification of the 654-1073 catalytic core of PDE3B has been employed to produce large quantities of highly pure enzyme for structural studies.

Protein Expr Purif, 2004 Jun, 35(2), 199 - 205
A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1; Wang B et al.; An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL . The recombinant protein was purified by Ni-chelation affinity chromatography . It showed a single band with a molecular mass of 18kDa in SDS-PAGE . The purified enzyme exhibited both phospholipase A(2) and esterase activities with the optimal catalytic temperature at 90 degrees C . The enzyme activity was Ca(2+)-independent . Kinetic analysis revealed its Km, k cat, and Vm for the p-nitrophenyl propionate substrate were 103microM, 39s(-1), and 249micromol/min/mg, respectively . The recombinant protein was thermostable and its half-life at 100 degrees C was about 1h.

Protein Expr Purif, 2004 Jun, 35(2), 190 - 8
Purification of functional full-length liver X receptor beta produced in Escherichia coli; Toresson G et al.; Liver X receptor beta (LXRbeta) is a ligand dependent transcription factor that is a member of the nuclear receptor superfamily . LXRbeta and its isoform LXRalpha have recently been recognized as important regulators of lipid homeostasis in vertebrates . N-terminally hexahistidine-tagged rat LXRbeta was expressed in Escherichia coli as a full-length protein and purified in two chromatographic steps, immobilized metal affinity chromatography and gel filtration . From 10g of bacterial cells, 2.5mg of protein was recovered . The purified LXRbeta is functional with respect to ligand-, DNA-, and coactivator-binding . The synthetic ligand T0901317 bound to LXRbeta with high affinity yielding a K(d) of 2.7nM . Specific interaction with DR4 response elements, in the presence of RXR, was demonstrated with electrophoretic mobility shift assay . Furthermore, surface plasmon resonance analysis of LXRbeta binding to coactivator peptides revealed a ligand dependent interaction with the C-terminal nuclear receptor binding site of the coactivator RAP250 . The purified LXRbeta constitutes an important tool for further functional and structural studies.

Protein Expr Purif, 2004 Jun, 35(2), 171 - 80
Cloning, expression, purification, and characterization of Nocardia sp . GTP cyclohydrolase I; He A et al.; The sequence of the gene from Nocardia sp . NRRL 5646 encoding GTP cyclohydrolase I (GCH), gch, and its adjacent regions was determined . The open reading frame of Nocardia gch contains 684 nucleotides, and the deduced amino acid sequence represents a protein of 227 amino acid residues with a calculated molecular mass of 24,563Da . The uncommon start codon TTG was identified by matching the N-terminal amino acid sequence of purified Nocardia GCH with the deduced amino acid sequence . A likely ribosomal binding site was identified 9bp upstream of the translational start site . The 3' end flank region encodes a peptide that shares high homology with dihydropteroate synthases . Nocardia GCH has 73 and 60% identity to the proteins encoded by the putative gch of Mycobacterium tuberculosis and Streptomyces coelicolor, respectively . Nocardia GCH was highly expressed in Escherichia coli cells carrying a pHAT10 based expression vector, and moderately expressed in Mycobacterium smegmatis cells carrying a pSMT3 based expression vector . Enterokinase digestion of recombinant Nocardia GCH, and in-gel digestion of Nocardia GCH and recombinant GCH followed by MALDI-TOF-MS analysis, confirmed that the actual subunit size of the enzyme was 24.5kDa . Thus, we conclude that the active form of native Nocardia GCH is a decamer . Our earlier incorrect conclusion was that the native enzyme was an octamer derived from the anomalous SDS-PAGE migration of the subunit.

FEBS Lett, 2004 May 7, 565(1-3), 203 - 6
Stimulation of Fe-S cluster insertion into apoFNR by Escherichia coli glutaredoxins 1, 2 and 3 in vitro; Achebach S et al.; The oxygen sensor fumarate nitrate reductase regulator (FNR) of Escherichia coli contains in the active (anaerobic) state a {4Fe-4S}(2+) cluster which is lost after exposure to O(2) . In aerobically prepared apoFNR, or in FNR obtained by treatment of {4Fe-4S} . FNR with O(2) in vitro, intramolecular cysteine disulfides are found, including the cysteine residues which serve as ligands for the Fe-S cluster . It is shown here that the reconstitution of {4Fe-4S} . FNR from this form of aerobic apoFNR was preceded by a long lag phase when glutathione was used as the reducing agent . Addition of E . coli glutaredoxins (Grx) 1, 2 or 3 decreased the lag phase greatly and stimulated the reconstitution rate slightly (about twofold) . Reconstitution of anaerobically prepared apoFNR, which has a lower cysteine disulfide content, showed only a short lag phase, which further decreased in the presence of Grx . It is concluded that in the lag phase the cysteine disulfides of apoFNR become reduced for the incorporation of the {4Fe-4S} cluster and that this reaction is stimulated by Grx . Thioredoxin (Trx) 1 showed no stimulation of FNR reconstitution in vitro . It is suggested that the function of Grx might be of significance for the insertion of FeS cluster in proteins containing disulfides.

FEBS Lett, 2004 May 7, 565(1-3), 111 - 6
Identification of a novel protein 3a from severe acute respiratory syndrome coronavirus; Yu CJ et al.; The open reading frame 3 of the severe acute respiratory syndrome coronavirus (SARS-CoV) genome encodes a predicted protein 3a, consisting of 274 amino acids, that lacks any significant similarities to any known protein . We generated specific antibodies against SARS protein 3a by using a synthetic peptide (P2) corresponding to amino acids 261-274 of the putative protein . Anti-P2 antibodies and the sera from SARS patients could specifically detect the recombinant SARS protein 3a expressed in Escherichia coli and in Vero E6 cells . Expression of SARS protein 3a was detected at 8-12 h after infection and reached a higher level after approximately 24 h in SARS-CoV-infected Vero E6 cells . Protein 3a was also detected in the alveolar lining pneumocytes and some intra-alveolar cells of a SARS-CoV-infected patient's lung specimen . Recombinant protein 3a expressed in Vero E6 cells and protein 3a in the SARS-CoV-infected cells was distributed over the cytoplasm in a fine punctate pattern with partly concentrated staining in the Golgi apparatus . Our study demonstrates that SARS-CoV indeed expresses a novel protein 3a, which is present only in SARS-CoV and not in other known CoVs.

FEBS Lett, 2004 May 7, 565(1-3), 101 - 5
Identification of an Arabidopsis inorganic pyrophosphatase capable of being imported into chloroplasts; Schulze S et al.; An Arabidopsis cDNA coding for a previously uncharacterized isoform of inorganic pyrophosphatase was isolated . It was used to complement an E . coli mutant, demonstrating that it coded for an active enzyme . MgCl(2) was necessary for the protein's activity, whilst NaF inhibited it . The K(m) for pyrophosphate and the pH optimum of the protein was determined . The gene coding for this protein was expressed in all tissues, and its expression in rosette leaves was induced by incubation on metabolizable sugars . In vitro import experiments demonstrated that the protein could be imported into chloroplasts and localized to the stromal compartment.

FEBS Lett, 2004 May 7, 565(1-3), 75 - 80
Cross talk between DevS sensor kinase homologue, Rv2027c, and DevR response regulator of Mycobacterium tuberculosis; Saini DK et al.; Rv2027c is a putative orphan histidine sensor kinase that bears strong homology to DevS of the hypoxia-responsive DevR-DevS two-component system in M . tuberculosis . The cytosolic C-terminal domain of Rv2027c protein (Rv2027c(194)) was overexpressed in E . coli and biochemically characterized . Rv2027c(194) underwent autophosphorylation at a conserved His(392) residue and engaged in phosphotransfer with DevR response regulator . The rates of autophosphorylation and the stabilities of the phosphorylated species were broadly similar in Rv2027c and DevS . However, unlike DevS, Rv2027c utilized Ca(2+) as an alternative divalent ion during autophosphorylation . In contrast to DevS which completed phosphotransfer to DevR in 5-10 min, phosphotransfer from Rv2027c approximately P was only partial at 30 min . Unlike devS transcription that was hypoxia-responsive, Rv2027c transcript levels were not upregulated from basal levels during hypoxia . The differential regulation of devS and Rv2027c genes, the ability of Rv2027c to utilize Ca(2+) as a divalent cation in autophosphorylation at physiological concentrations and to engage in phosphotransfer with DevR suggests that the DevR regulon could be modulated by more than one environmental cue relayed through DevS and Rv2027c.

FEBS Lett, 2004 May 7, 565(1-3), 59 - 64
X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold; Ericsson UB et al.; Pseudouridine synthases catalyse the isomerisation of uridine to pseudouridine in structural RNA . The pseudouridine synthase TruD, that modifies U13 in tRNA, belongs to a recently identified and large family of pseudouridine synthases present in all kingdoms of life . We report here the crystal structure of Escherichia coli TruD at 2.0 A resolution . The structure reveals an overall V-shaped molecule with an RNA-binding cleft formed between two domains: a catalytic domain and an insertion domain . The catalytic domain has a fold similar to that of the catalytic domains of previously characterised pseudouridine synthases, whereas the insertion domain displays a novel fold.

FEBS Lett, 2004 May 7, 565(1-3), 39 - 42
Recombinant ATPases of the yeast 26S proteasome activate protein degradation by the 20S proteasome; Takeuchi J et al.; The 26S proteasome contains a proteolytic core, 20S proteasome, and its regulatory particle, 19S complex . That regulatory particle contains six ATPases that are involved in unfolding and translocation of substrates to the 20S proteasome's catalytic chamber . We expressed ATPase-encoding genes of the regulatory particle of Saccharomyces cerevisiae and found that some recombinant ATPases can self-assemble into a high-molecular-weight protein complex in Escherichia coli . Purification of the Rpt1Rpt2 hetero-complex and the Rpt4 homo-complex for functional characterization demonstrated their contribution to energy-dependent protein degradation . Our finding, production of a functional subunit of the 19S regulatory particle in bacteria, is a simpler and technically advanced system to functionally characterize individual subunits.

FEBS Lett, 2004 May 7, 565(1-3), 11 - 8
Molecular and functional analysis of Caenorhabditis elegans CHIP, a homologue of Mammalian CHIP; Khan LA et al.; A recently identified molecule C-terminus of Hsc70 interacting protein (CHIP) has been reported to be an E3 ubiquitin ligase collaborating with molecular chaperones for the degradation of misfolded or unfolded proteins . The physiological roles of CHIP in animal and plant development remain largely unknown . Here, we show that the knockdown of CeCHIP by RNAi and knockout by a deletion mutation arrests the development of the animal at the larval stage . CeCHIP expresses ubiquitously in all tissues but there are tissue specific variations of expression level . CeCHIP produces dose dependent phenotypes in vivo . Over expression of CHIP causes embryonic lethality, while a comparatively lower level of over expression causes locomotion and egg laying defects, and the CHIP over expressed animals form dauers at a higher temperature.

J Inorg Biochem, 2004 May, 98(5), 856 - 61
Aberrant activity of the DNA repair enzyme AlkB; Henshaw TF et al.; Escherichia coli AlkB is a DNA/RNA repair enzyme containing a mononuclear Fe(II) site that couples the oxidative decomposition of alpha-ketoglutarate (alphaKG) to the hydroxylation of 1-methyladenine or 3-methylcytosine lesions in DNA or RNA, resulting in release of formaldehyde and restoration of the normal bases . In the presence of Fe(II), alphaKG, and oxygen, but the absence of methylated DNA, AlkB was found to catalyze an aberrant reaction that generates a blue chromophore . The color is proposed to derive from Fe(III) coordinated by a hydroxytryptophan at position 178 as revealed by mass spectrometric analysis . Protein structural modeling confirms that Trp 178 is reasonably positioned to react with the Fe(IV)-oxo intermediate proposed to form at the active site.

J Inorg Biochem, 2004 May, 98(5), 775 - 85
Zn-, Cd-, and Pb-transcription factor IIIA: properties, DNA binding, and comparison with TFIIIA-finger 3 metal complexes; Huang M et al.; Properties of the metal ion binding sites of Zn-transcription factor IIIA (TFIIIA) were investigated to understand the potential of this type of zinc finger to undergo reactions that remove Zn(2+) from the protein . Zn-TFIIIA was purified from E . coli containing the cloned sequence for Xenopus laevis oocyte TFIIIA and its stoichiometry of bound Zn(2+) was shown to depend on the details of the isolation process . The average dissociation constant of Zn(2+) in Zn-TFIIIIA was 10(-7) . The dissociation constant for Zn-F3, the third finger from the N-terminus of TFIIIA, was 1.0 x 10(-8) . The reactivity of Zn-TFIIIA with a series of metal binding ligands, including 2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene (zincon), 4-(2-pyridylazo)-resorcinol (PAR), and 3-ethoxy-2-oxo-butyraldehyde-bis-(N(4)-dimethylthiosemicarbazone) (H(2)KTSM(2)) revealed similar kinetics . The reactivity of PAR with Zn-TFIIIA declined substantially when the protein was bound to the internal control region (ICR) of the 5S ribosomal DNA . Both Cd(2+) and Pb(2+) disrupt TFIIIA binding to its cognate DNA sequence . The Pb(2+) dissociation constant of Pb-F3 was measured as 2.5 x 10(-8) . According to NMR spectroscopy, F3 does not fold into a regular conformation in the presence of Pb(2+).

Biochim Biophys Acta, 2004 May 6, 1698(2), 251 - 4
Crystallization and preliminary crystallographic studies of a bifunctional restriction endonuclease Eco57I; Tamulaitiene G et al.; Restriction endonuclease Eco57I from Escherichia coli recognizes asymmetric DNA sequence 5'-CTGAAG and has both restriction (DNA cleavage a short distance away from the recognition site) and modification (methylation) activities residing in a single polypeptide chain . Single crystals of wild-type Eco57I ternary complexes with double-stranded DNA and sinefungin, a stimulator of endonuclease activity, were obtained by the vapor diffusion technique and characterized crystallographically for different variants of the DNA component . The best data for the complex with 25-mer DNA were collected to 4.2-A resolution at 100 K using synchrotron radiation . The crystals are orthorhombic, space group P2(1)2(1)2, with a=164.3, b=293.0, c=71.1 A, and contain two to four copies of the protein in the asymmetric unit.

Mini Rev Med Chem, 2004 May, 4(4), 351 - 9
Structural requirements for efficient phosphorylation of nucleotide analogs by human thymidylate kinase; Lavie A et al.; Successive phosphorylation of nucleoside analog prodrugs to their triphosphate forms is required for the pharmacological activity of these compounds in the chemotherapeutic treatment of viral infections and cancer . Human thymidylate kinase (TMPK), apart from its essential physiological role in the biosynthesis of TTP, is also required for the activation of thymidine analogs, such as the clinically used anti-HIV prodrugs AZT and d4T . This enzyme is rate determining in the three-step cascade of AZT phosphorylation . Our structural work on human, yeast and E . coli TMPKs, in conjunction with sequence homology analyses and biochemical data, has demonstrated that three loops are crucial for the function of this enzyme: the first is the highly conserved P-loop motif, which binds and positions the phosphoryl groups of ATP, the second critical loop contains the DR(Y/H) motif that supplies a catalytic arginine and is also important for the binding and positioning of the magnesium ion complexed to ATP, and the third loop is the so-called Lid-region that is a flexible stretch which closes on ATP when it binds . Modifications of the sugar moieties of nucleoside monophosphates are shown to exert drastic effects on the enzyme's conformation and, thus, reduced activity . Our structural work on several TMPKs has formed the basis for generating mutants of human TMPK that are about 100 times more efficient in phosphorylating AZTMP . These enzyme variants could potentially be introduced into HIV-targeted cells in order to significantly improve AZT's antiviral activity.

Biochemistry, 2004 May 18, 43(19), 5902 - 11
Full-length influenza hemagglutinin HA2 refolds into the trimeric low-pH-induced conformation; Swalley SE et al.; The influenza virus uses hemagglutinin (HA) to fuse the viral and cellular membranes . As part of an effort to study the membrane-interacting elements of HA, the fusion peptide, and the C-terminal transmembrane anchor, we have expressed in Escherichia coli the full-length HA(2) chain with maltose-binding protein fused at its N-terminus . The chimeric protein can be refolded in vitro in the presence of specific detergents to yield stable, homogeneous trimers, as determined by analytical ultracentrifugation . The trimers have the so-called "low pH" conformation-the rearranged HA(2) conformation obtained when intact HA(1)/HA(2) is induced to refold by exposure to low pH-as detected by electron microscopy and monoclonalantibody reactivity . These results provide further evidence for the notion that the neutral-pH structure of intact HA is metastable and that binding of protons lowers the kinetic barriers that prevent rearrangement to the minimum-free-energy conformation . The refolded chimeric protein described here is a suitable species for undertaking studies of how the fusion peptide inserts into membranes and assessing the nature of possible intermediates in the fusion process.

Biochemistry, 2004 May 18, 43(19), 5716 - 27
Evolution of enzymatic activity in the enolase superfamily: structural studies of the promiscuous o-succinylbenzoate synthase from Amycolatopsis; Thoden JB et al.; Divergent evolution of enzyme function is commonly explained by a gene duplication event followed by mutational changes that allow the protein encoded by the copy to acquire a new function . An alternate hypothesis is that this process is facilitated when the progenitor enzyme acquires a second function while maintaining the original activity . This phenomenon has been suggested to occur in the o-succinylbenzoate synthase (OSBS) from a species of Amycolatopsis that catalyzes not only the physiological syn-dehydration reaction of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate but also an accidental racemization of N-acylamino acids {Palmer, D . R., Garrett, J . B., Sharma, V., Meganathan, R., Babbitt, P . C., and Gerlt, J . A . (1999) Biochemistry 38, 4252-4258} . To understand the molecular basis of this promiscuity, three-dimensional structures of liganded complexes of this enzyme have been determined, including the product of the OSBS reaction and three N-acylamino acid substrates for the N-acylamino acid racemase (NAAAR) reaction, N-acetylmethionine, N-succinylmethionine, and N-succinylphenylglycine, to 2.2, 2.3, 2.1, and 1.9 A resolution, respectively . These structures show how the active-site cavity can accommodate both the hydrophobic substrate for the OSBS reaction and the substrates for the accidental NAAAR reaction . As expected, the N-acylamino acid is sandwiched between lysines 163 and 263, which function as the catalytic bases for the abstraction of the alpha-proton in the (R)- and (S)-racemization reactions, respectively {Taylor Ringia, E . A., Garrett, J . B, Thoden, J . B., Holden, H . M., Rayment, I., and Gerlt, J . A . (2004) Biochemistry 42, 224-229} . Importantly, the protein forms specific favorable interactions with the hydrophobic amino acid side chain, alpha-carbon, carboxylate, and the polar components of the N-acyl linkage . Accommodation of the components of the N-acyl linkage appears to be the reason that this enzyme is capable of a racemization reaction on these substrates, whereas the orthologous OSBS from Escherichia coli lacks this functionality.

Biochemistry, 2004 May 18, 43(19), 5661 - 71
Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins; Wijffels G et al.; The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair . A universal interaction motif is proposed to be one mechanism by which those proteins bind the E . coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer . The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence . To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro . A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence . The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Lipids, 2004 Feb, 39(2), 111 - 6
Lactational changes in the fatty acid composition of human milk gangliosides; Martin-Sosa S et al.; The objectives of this work were to study the FA composition of milk gangliosides, as well as to gain further insight into the characterization of human milk gangliosides . The potential capacity of human milk gangliosides to adhere to human enterotoxigenic Escherichia coli (ETEC-strains) was also studied . Human milk gangliosides were isolated and identified by high-performance TLC or immunoassay . The latter also was used to assay bacterial adhesion . The FA composition of gangliosides was studied by GC . The presence of O-acetyl GD3 (Neu5,9Ac2alpha2-8 NeuAcalpha2-3Galbeta1-4GlcCer) and trace amounts of GM1 {Galgamma1}3-3GalNAcgamma1,-3(Neualpha2-3)Galbeta1-4GlcCerl in human milk was confirmed . Medium-chain FA were almost absent in colostrum, whereas in the subsequent stages they rose to 20% . The levels of long-chain FA decreased after colostrum . With respect to the degree of saturation, gangliosides from colostrum were richer in monounsaturated FA than gangliosides synthesized during the rest of the lactation period, opposite to the pattern for PUFA . A human-ETEC colonization factor antigen II-expressing strain showed binding capacity to human milk GM3 (NeuAcalpha2-3Gal{1-4GlcCer) . New data on human milk gangliosides have been gathered . A thorough knowledge of their composition is needed since they may have important biological implications in regard to newborns' defense against infection.

Biomed Sci Instrum, 2004, 40, 232 - 7
Probing the elastic nature of spider silk in pursuit of the next designer fiber; Brooks AE et al.; Spider silk, one of nature's greatest accomplishments, has a combination of strength and elasticity that is unrivaled . Spiders produce up to 7 different silks; each one with a unique combination of tensile strength and elasticity that allows the spiders' web to hold prey while being resilient enough not to break upon impact . In an attempt to determine the sequences responsible for these enviable mechanical properties, several different amino acid motifs have been defined . Much of the recent work is now concentrated on correlating amino acid motifs with a specific mechanical property . The current hypothesis is that the strength property of spider silk is conferred by a poly-alanine or alanine rich (An or GAn) motif whereas the elastic nature of spider silk is conferred by the amino acid motif, GPGXX, where X is Q, S, A, G, or Y . Despite the fact that these different motifs are now known, the combination of strength and elasticity are yet to be duplicated ex vivo . In an attempt to verify that the GPGXX motif imparts elasticity to the spider silk, the number of repeats and/or the amino acid composition of the Argiope aurantia "elastic motif" were varied and expressed in various strains of E . coli to change the elastic properties of the resulting film and/or fiber . Concurrent with work on the elasticity motif is ongoing work on the strength module . Understanding these two different motifs will aide efforts to produce a designer biomaterial for medical, commercial, and military applications.

Shanghai Kou Qiang Yi Xue, 2004 Apr, 13(2), 126 - 9
{Cloning of human amelogenin gene encoding mature peptide}; Cheng L et al.; PURPOSE: The purpose of this study was to clone human amelogenin gene encoding mature protein, which provides a basis for expressing the recombinant human amelogenin in Escherichia coli . in the future . METHODS: In this study, total RNA was extracted from the dental germ of a legally aborted embryo by Trizol . Using RT-PCR technique we obtained synthesis of cDNA from the total RNA, and the desired DNA products were conducted with PCR from cDNA . The segment was inserted into expression vector PQE30 and the interesting plasmid was transformed into Escherichia coli . host DH5alpha . The double-stranded DNA of positive clone was analyzed by PCR, restriction endonuclease mapping and DNA sequence analysis . RESULTS: The sequence analysis of recombinant plasmid showed that the human amelogenin encoding mature protein was inserted into vector PQE30 accurately . CONCLUSIONS: We conducted human amelogenin encoding mature protein from dental germ of a legally aborted embryo and got the recombinant plasmid which may express amelogenin gene for further research.

Protein Sci, 2004 Jun, 13(6), 1566 - 71 Epub 2004 May 07.
Modification of halogen specificity of a vanadium-dependent bromoperoxidase; Ohshiro T et al.; The halide specificity of vanadium-dependent bromoperoxidase (BPO) from the marine algae, Corallina pilulifera, has been changed by a single amino acid substitution . The residue R397 has been substituted by the other 19 amino acids . The mutant enzymes R397W and R397F showed significant chloroperoxidase (CPO) activity as well as BPO activity . These mutant enzymes were purified and their properties were investigated . The maximal velocities of CPO activities of the R397W and R397F enzymes were 31.2 and 39.2 units/mg, and the K(m) values for Cl(-) were 780 mM and 670 mM, respectively . Unlike the native enzyme, both mutant enzymes were inhibited by NaN(3) . In the case of the R397W enzyme, the incorporation rate of vanadate into the active site was low, compared with the R397F and the wild-type enzyme . These results supported the existence of a specific halogen binding site within the catalytic cleft of vanadium haloperoxidases.

Protein Sci, 2004 Jun, 13(6), 1684 - 92 Epub 2004 May 07.
Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer; Pokkuluri PR et al.; The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution . This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome . Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively . Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli . The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7) . The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8% . Unlike the two c(7) molecules with known structures, one from G . sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue . As a result, the corresponding heme could have a higher potential than the other two hemes . The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.

Protein Sci, 2004 Jun, 13(6), 1594 - 602 Epub 2004 May 07.
Sequence-structure mapping errors in the PDB: OB-fold domains; Venclovas C et al.; The Protein Data Bank (PDB) is the single most important repository of structural data for proteins and other biologically relevant molecules . Therefore, it is critically important to keep the PDB data, as much as possible, error-free . In this study, we have analyzed PDB crystal structures possessing oligonucleotide/oligosaccharide binding (OB)-fold, one of the highly populated folds, for the presence of sequence-structure mapping errors . Using energy-based structure quality assessment coupled with sequence analyses, we have found that there are at least five OB-structures in the PDB that have regions where sequences have been incorrectly mapped onto the structure . We have demonstrated that the combination of these computation techniques is effective not only in detecting sequence-structure mapping errors, but also in providing guidance to correct them . Namely, we have used results of computational analysis to direct a revision of X-ray data for one of the PDB entries containing a fairly inconspicuous sequence-structure mapping error . The revised structure has been deposited with the PDB . We suggest use of computational energy assessment and sequence analysis techniques to facilitate structure determination when homologs having known structure are available to use as a reference . Such computational analysis may be useful in either guiding the sequence-structure assignment process or verifying the sequence mapping within poorly defined regions.

Protein Sci, 2004 Jun, 13(6), 1698 - 703 Epub 2004 May 07.
Two-promoter vector is highly efficient for overproduction of protein complexes; Kim KJ et al.; The use of bicistronic vectors, which contain two target genes under one promoter, has been the most common practice for the heterologous production of binary protein complexes . The major problem of this method is the much lower expression of the second gene compared with that of the first gene next to the promoter . We tested a simple idea of whether inclusion of an additional promoter in front of the second gene may remove the problem . Compared with bicistronic vectors, corresponding two-promoter vectors yielded four to nine times larger amounts of the complexes between BCL-2 family proteins, BCL-X(L):BAD, BCL-X(L):BIM-S, and CED-9:EGL-1 in bacterial cells as a result of significantly increased expression of the second genes in a manner independent of the order of the target genes . With the two-promoter system, we produced two other complexes in large quantity suitable for extensive crystallization trial . The method does not accompany any technical disadvantages, and represents a significant improvement from the conventional method, which should enjoy wide application for the coexpression of binary or higher order protein complexes by extension.

Protein Sci, 2004 Jun, 13(6), 1466 - 75 Epub 2004 May 07.
Accessibility of introduced cysteines in chemoreceptor transmembrane helices reveals boundaries interior to bracketing charged residues; Boldog T et al.; Two hydrophobic sequences, 24 and 30 residues long, identify the membrane-spanning segments of chemoreceptor Trg from Escherichia coli . As in other related chemoreceptors, these helical sequences are longer than the minimum necessary for an alpha-helix to span the hydrocarbon region of a biological membrane . Thus, the specific positioning of the segments relative to the hydrophobic part of the membrane cannot be deduced from sequence alone . With the aim of defining the positioning for Trg experimentally, we determined accessibility of a hydrophilic sulfhydryl reagent to cysteines introduced at each position within and immediately outside the two hydrophobic sequences . For both sequences, there was a specific region of uniformly low accessibility, bracketed by regions of substantial accessibility . The two low-accessibility regions were each 19 residues long and were in register in the three-dimensional organization of the transmembrane domain deduced from independent data . None of the four hydrophobic-hydrophilic boundaries for these two membrane-embedded sequences occurred at a charged residue . Instead, they were displaced one to seven residues internal to the charged side chains bracketing the extended hydrophobic sequences . Many hydrophobic sequences, known or predicted to be membrane-spanning, are longer than the minimum necessary helical length, but precise membrane boundaries are known for very few . The cysteine-accessibility approach provides an experimental strategy for determining those boundaries that could be widely applicable.

Microbiology, 2004 May, 150(Pt 5), 1495 - 505
Properties of haemolysin E (HlyE) from a pathogenic Escherichia coli avian isolate and studies of HlyE export; Wyborn NR et al.; Haemolysin E (HlyE) is a novel pore-forming toxin first identified in Escherichia coli K-12 . Analysis of the 3-D structure of HlyE led to the proposal that a unique hydrophobic beta-hairpin structure (the beta-tongue, residues 177-203) interacts with the lipid bilayer in target membranes . In seeming contradiction to this, the hlyE sequence from a pathogenic E . coli strain (JM4660) that lacks all other haemolysins has been reported to encode an Arg residue at position 188 that was difficult to reconcile with the proposed role of the beta-tongue . Here it is shown that the JM4660 hlyE sequence encodes Gly, not Arg, at position 188 and that substitution of Gly188 by Arg in E . coli K-12 HlyE abolishes activity, emphasizing the importance of the head domain in HlyE function . Nevertheless, 76 other amino acid substitutions were confirmed compared to the HlyE protein of E . coli K-12 . The JM4660 HlyE protein was dimeric, suggesting a mechanism for improving toxin solubility, and it lysed red blood cells from many species by forming 36-41 A diameter pores . However, the haemolytic phenotype of JM4660 was found to be unstable due to defects in HlyE export, indicating that export of active HlyE is not an intrinsic property of the protein but requires additional components . TnphoA mutagenesis of hlyE shows that secretion from the cytoplasm to the periplasm does not require the carboxyl-terminal region of HlyE . Finally, disruption of genes associated with cell envelope function, including tatC, impairs HlyE export, indicating that outer membrane integrity is important for effective HlyE secretion.

Microbiology, 2004 May, 150(Pt 5), 1457 - 66
Increased transcription rates correlate with increased reversion rates in leuB and argH Escherichia coli auxotrophs; Reimers JM et al.; Escherichia coli auxotrophs of leuB and argH were examined to determine if higher rates of transcription in derepressed genes were correlated with increased reversion rates . Rates of leuB and argH mRNA synthesis were determined using half-lives and concentrations, during exponential growth and at several time points during 30 min of amino acid starvation . Changes in mRNA concentration were primarily due to increased mRNA synthesis and not to increased stability . Four strains of E . coli amino acid auxotrophs, isogenic except for relA and argR, were examined . In both the leuB and argH genes, rates of transcription and mutation were compared . In general, strains able to activate transcription with guanosine tetraphosphate (ppGpp) had higher rates of mRNA synthesis and mutation than those lacking ppGpp (relA2 mutants) . argR knockout strains were constructed in relA(+) and relA mutant strains, and rates of both argH reversion and mRNA synthesis were significantly higher in the argR knockouts than in the regulated strains . A statistically significant linear correlation between increased rates of transcription and mutation was found for data from both genes . In general, changes in mRNA half-lives were less than threefold, whereas changes in rates of mRNA synthesis were often two orders of magnitude . The results suggest that specific starvation conditions target the biosynthetic genes for derepression and increased rates of transcription and mutation.

Microbiology, 2004 May, 150(Pt 5), 1413 - 26
Quantitative relationships for specific growth rates and macromolecular compositions of Mycobacterium tuberculosis, Streptomyces coelicolor A3(2) and Escherichia coli B/r: an integrative theoretical approach; Cox RA; Further understanding of the physiological states of Mycobacterium tuberculosis and other mycobacteria was sought through comparisons with the genomic properties and macromolecular compositions of Streptomyces coelicolor A3(2), grown at 30 degrees C, and Escherichia coli B/r, grown at 37 degrees C . A frame of reference was established based on quantitative relationships observed between specific growth rates ( micro ) of cells and their macromolecular compositions . The concept of a schematic cell based on transcription/translation coupling, average genes and average proteins was developed to provide an instantaneous view of macromolecular synthesis carried out by cells growing at their maximum rate . It was inferred that the ultra-fast growth of E . coli results from its ability to increase the average number of rRNA (rrn) operons per cell through polyploidy, thereby increasing its capacity for ribosome synthesis . The maximum growth rate of E . coli was deduced to be limited by the rate of uptake and consumption of nutrients providing energy . Three characteristic properties of S . coelicolor A3(2) growing optimally ( micro =0.30 h(-1)) were identified . First, the rate of DNA replication was found to approach the rate reported for E . coli ( micro =1.73 h(-1)); secondly, all rrn operons were calculated to be fully engaged in precursor-rRNA synthesis; thirdly, compared with E . coli, protein synthesis was found to depend on higher concentrations of ribosomes and lower concentrations of aminoacyl-tRNA and EF-Tu . An equation was derived for E . coli B/r relating micro to the number of rrn operons per genome . Values of micro =0.69 h(-1) and micro =1.00 h(-1) were obtained respectively for cells with one or two rrn operons per genome . Using the author's equation relating the number of rrn operons per genome to maximum growth rate, it is expected that M . tuberculosis with one rrn operon should be capable of growing much faster than it actually does . Therefore, it is suggested that the high number of insertion sequences in this species attenuates growth rate to still lower values.

J Biol Chem, 2004 Jul 9, 279(28), 29211 - 7 Epub 2004 May 08.
Functional properties of the herpes simplex virus type I origin-binding protein are controlled by precise interactions with the activated form of the origin of DNA replication; Macao B et al.; The herpes simplex virus, type I origin-binding protein, OBP, is a superfamily II DNA helicase encoded by the UL9 gene . OBP binds in a sequence-specific and cooperative way to the viral origin of replication oriS . OBP may unwind partially and introduce a hairpin into the double-stranded origin of replication . The formation of the novel conformation referred to as oriS* also requires the single-stranded DNA-binding protein, ICP8, and ATP hydrolysis . OBP forms a stable complex with oriS* . The hairpin in oriS* provides a site for sequence-specific attachment, and a single-stranded region triggers ATP hydrolysis . Here we use Escherichia coli exonuclease I to map the binding of the C-terminal domain of OBP to the hairpin and the helicase domains to the single-stranded tail . The helicase domains cover a stretch of 23 nucleotides of single-stranded DNA . Using streptavidin-coated magnetic beads, we show that OBP may bind two copies of double-stranded DNA (one biotin-labeled and the other one radioactively labeled) but only one copy of oriS* . It is the length of the single-stranded tail that determines the stoichiometry of OBP.DNA complexes . OBP interacts with the bases of the single-stranded tail, and ATP hydrolysis is triggered by position-specific interactions between OBP and bases in the single-stranded tail of oriS*.

J Biol Chem, 2004 Aug 6, 279(32), 33806 - 15 Epub 2004 May 07.
Structure of human MTH1, a Nudix family hydrolase that selectively degrades oxidized purine nucleoside triphosphates; Mishima M et al.; Oxygen radicals generated through normal cellular respiration processes can cause mutations in genomic and mitochondrial DNA . Human MTH1 hydrolyzes oxidized purine nucleoside triphosphates, such as 8-oxo-dGTP and 2-hydroxy-dATP, to monophosphates, thereby preventing the misincorporation of these oxidized nucleotides during replication . Here we present the solution structure of MTH1 solved by multidimensional heteronuclear NMR spectroscopy . The protein adopts a fold similar to that of Escherichia coli MutT, despite the low sequence similarity between these proteins outside the conserved Nudix motif . The substrate-binding pocket of MTH1, deduced from chemical shift perturbation experiments, is located at essentially the same position as in MutT; however, a pocket-forming helix is largely displaced in MTH1 (approximately 9 A) such that the shape of the pocket differs between the two proteins . Detailed analysis of the pocket-forming residues enabled us to identify Asn33 as one of the key residues in MTH1 for discriminating the oxidized form of purine, and mutation of this residue modifies the substrate specificity . We also show that MTH1 catalyzes hydrolysis of 8-oxo-dGTP through nucleophilic substitution of water at the beta-phosphate.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Jan, 19(1), 71 - 3
{Construction and expression of mouse-human chimeric Fab fragment gene of monoclonal antibody SZ-2 against platelet}; Dai KS et al.; AIM: To reduce immunogenicity of a monoclonal antibody SZ-2 specific for human platelet . METHODS: Reverse transcription and polymerize chain reaction were used to amplify the variable region genes of monoclonal antibody SZ-2 . The cloned V(H) and V(L) genes were sequenced and fused to human IgG1 constant region gene CH1 and Ckappa in plasmid pSW1 . The recombinant plasmid were transformed into E . coli . The expressed recombinant proteins were analysed . RESULTS: The V(H) and V(L) genes were homologous with the published gene sequences of mouse antibody variable region . The concentration of chimeric Fab fragment in expression supernatant was about 180 microg/L detected by ELISA . Western blot analysis showed that SZ-2 Fab/Hu maintained the binding activity to human platelet GPIb . The recombinant proteins could suppress platelet aggregation induced by Ristocatin . CONCLUSION: The variable region genes of SZ-2 are cloned and the mouse-human chimeric Fab fragment is expressed successfully in E . coli.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2003 Jan, 19(1), 68 - 70
{Immunopotency of the recombinant urease B subunit vaccine of Helicobacter pylori after intranasal administration to mice}; Gao ZG et al.; AIM: To observe the immunopotency of the recombinant urease B subunit (rUreB) of Helicobacter pylori after intranasal administration to mice . MDTHODS: BALB/c mice were immunized intranasally with rUreB of 20 microg,10 microg and rUreB plus different adjuvants, such as cholera toxin B subunit (CTB), Escherichia coli heat labile enterotoxin B subunit (LTB) and carbopol respectively, four times at an intervals of 7 days . The serum and washing solution from gastric, intestinal, nasal and tracheal mucosas were collected in 7 days after final immunization . IgG and IgA antibodies specific for rUreB were detected by ELISA . RESULTS: The levels of IgA and IgG antibodies in sera every groups of mice immunized intranasally were significantly increased compared with control group (P<0.01) . Only the levels of serum IgG of mice immunized with 20 microg dose were higher than those of mice immunized with 10 microg dose . Carbopol could enhance the level of IgA antibodies in washing solution from mouse gastric mucosa after intranasal immunization.The efficacy of LTB as a nasal mucosal immuno-adjuvant was stronger than that of CTB . CONCLUSION: CTB, LTB and carbopol can play the role of adjuvant in nasal mucosal immunization . Intranasal immunization with rUreB can induce not only serum IgG antibody production but also antibody responses of different mucosa . Thus intranasal inoculation is a convenient, effective and cheap immunization way.

Cancer Sci, 2004 May, 95(5), 442 - 7
Conditional gene silencing utilizing the lac repressor reveals a role of SHP-2 in cagA-positive Helicobacter pylori pathogenicity; Higuchi M et al.; RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems . The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells . We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor . Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-1-thio-beta-D-galactopyranoside (IPTG) . Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence . This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells . Our results indicate that SHP-2 is a critical downstream effector of H . pylori CagA . The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach.

Biomacromolecules, 2004 May-Jun, 5(3), 1015 - 20
Dynamics of the Escherichia coli O91 O-antigen polysaccharide in solution as studied by carbon-13 NMR relaxation; Lycknert K et al.; The dynamics of the O-antigen part of the lipopolysaccharide from the enterohemorrhagic Escherichia coli O91 has been determined in solution using (13)C NMR relaxation measurements at two magnetic field strengths, 9.4 and 14.1 T, thereby facilitating the testing of several dynamical models . The biological repeating unit, consisting of five sugar residues and substituents, could be determined by spectral analysis of different (1)H,(13)C correlations and corroborated by the relaxation data . The site specifically (13)C-labeled material was shown to have approximately 10 repeating units with a narrow distribution . A model-free analysis of the relaxation data revealed a complex dynamical behavior where the sugar residues could be described by a global correlation time (tau(m) = 5.4 ns), generalized order parameters (S(2) approximately 0.63), and different correlation times for internal motions related to their position in the repeating unit along the polymer (tau(e) approximately 360-520 ps) . One of the sugar residues showed, in addition, a chemical exchange contribution . Furthermore, a substituent on another sugar residue was described by two order parameters (S(f)(2) = 0.51 and S(s)(2) = 0.21) . The solution dynamics of the polysaccharide are thus described by highly intricate motions, both in amplitude and time scales . These results are of significance in the general description of polysaccharides surrounding bacterial cell surfaces and in the presentation of antigenic epitopes to the immune system of an invaded host.

Methods Mol Med, 2004, 99, 215 - 23
Single-cell laser-capture microdissection and RNA amplification; Kamme F et al.; Generating gene-expression profiles from laser-captured cells requires the successful combination of laser-capture microdissection, RNA extraction, RNA amplification, and microarray analysis . To permit single-cell gene-expression profiling, the RNA amplification method has to be sufficiently powerful to bridge the gap between the amount of RNA available from a single cell to what is required by the microarray, a gap that spans 5 to 6 orders of magnitude . This chapter focuses on the amplification of RNA using a two-round T7 RNA amplification method . The protocols described are adapted for laser-captured material and have been used to generate gene expression profiles from single laser-captured cells.

Biol Trace Elem Res, 2004 Jun, 98(3), 209 - 18
Effects of zinc on cell-mediated immunity in chronic hemodialysis patients; Ribeiro RC et al.; Thirteen healthy subjects and 20 hemodialysis patients were studied to observe the delayed hypersensitivity skin tests (DHSTs) and phytohemagglutinin (PHA)-stimulating lymphocyte blastogenesis . Significant differences were observed between the groups . Controls had a higher proportion of positive skin reaction than hemodialysis patients in relation to Escherichia coli (p<0.01) and tuberculin (PPD) (p<0.05) . Regarding lymphocyte blastogenesis stimulated by phytohemagglutinin (PHA), cell proliferation was more accentuated in controls than hemodialysis patients (p<0.05) . On the other hand, serum zinc was elevated in controls (78 +/- 8 microg/dL) in comparison to hemodialysis patients (71 +/- 33 microg/dL) (p<0.05) . Of the 20 hemodialysis patients, 8 patients were maintained on long-term hemodialysis before and after zinc therapy, with the aim of studying DHST and PHA-stimulating lymphocyte blastogenesis . There was a significant improvement of DHST response to E . coli antigen after 100 d of zinc treatment (p<0.01), and with the discontinuation of therapy, the DHST responses decreased back to the initial values (p<0.05) . Zinc administration also increased the lymphocyte proliferation induced by PHA from 31386 +/- 3974 to 42480 +/- 5242 cpm (mean +/- SD) (p<0.05) . These results indicated that zinc therapy improved in vivo and in vitro DHST and lymphocyte function of hemodialysis patients and that its discontinuation suppressed all of the benefits observed .

J Biol Chem, 2004 Jul 16, 279(29), 30546 - 53 Epub 2004 May 05.
Lysine beta311 of protein geranylgeranyltransferase type I partially replaces magnesium; Hartman HL et al.; Protein geranylgeranyltransferase type I (GGTase I) catalyzes the attachment of a geranylgeranyl lipid group near the carboxyl terminus of protein substrates . Unlike protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type II, which require both Zn(II) and Mg(II) for maximal turnover, GGTase I turnover is dependent only on Zn(II) . In FTase, the magnesium ion is coordinated by aspartate beta352 and the diphosphate of farnesyl diphosphate to stabilize the developing charge in the transition state (Pickett, J . S., Bowers, K . E., and Fierke, C . A . (2003) J . Biol . Chem . 278, 51243-51250) . In GGTase I, lysine beta311 is substituted for this aspartate and is proposed to replace the catalytic function of Mg(II) (Taylor, J . S., Reid, T . S., Terry, K . L., Casey, P . J., and Beese, L . S . (2003) EMBO J . 22, 5963-5974) . Here we demonstrate that the prenylation rate constant catalyzed by wild type GGTase I (k(chem) = 0.18 +/- 0.02 s(-1)) is not dependent on Mg(II), is approximately 20-fold slower than the maximal rate constant catalyzed by FTase, and has a single pKa of 6.4 +/- 0.1, likely reflecting deprotonation of the peptide thiol . Mutation of lysine beta311 in GGTase I to alanine (Kbeta311A) or aspartate (Kbeta311D) decreases the k(chem) in the absence of magnesium 9-41-fold without significantly affecting the binding affinity of either substrate . Furthermore, the geranylgeranylation rate constant is enhanced by the addition of Mg(II) for Kbeta311A and Kbeta311D GGTase I 2-5-fold compared with wild type GGTase I with K(Mg) of 140 +/- 10 mm and 6.4 +/- 0.8 mm, respectively . These results demonstrate that lysine beta311 of GGTase I partially replaces the catalytic function of Mg(II) observed in FTase.

J Biol Chem, 2004 Jul 16, 279(29), 30836 - 43 Epub 2004 May 05.
Structure and function of recombinant cobra venom factor; Kock MA et al.; Cobra venom factor (CVF) is the complement-activating protein from cobra venom . It is a structural and functional analog of complement component C3 . CVF functionally resembles C3b, the activated form of C3 . Like C3b, CVF binds factor B, which is subsequently cleaved by factor D to form the bimolecular complex CVF,Bb . CVF,Bb is a C3/C5 convertase that cleaves both complement components C3 and C5 . CVF is a three-chain protein that structurally resembles the C3b degradation product C3c, which is unable to form a C3/C5 convertase . Both C3 and CVF are synthesized as single-chain prepro-proteins . This study reports the recombinant expression of pro-CVF in two insect cell expression systems (baculovirus-infected Sf9 Spodoptera frugiperda cells and stably transfected S2 Drosophila melanogaster cells) . In both expression systems pro-CVF is synthesized initially as a single-chain pro-CVF molecule that is subsequently proteolytically processed into a two-chain form of pro-CVF that structurally resembles C3 . The C3-like form of pro-CVF can be further proteolytically processed into another two-chain form of pro-CVF that structurally resembles C3b . Unexpectedly, all three forms of pro-CVF exhibit functional activity of mature, natural CVF . Recombinant pro-CVF supports the activation of factor B in the presence of factor D and Mg2+ and depletes serum complement activity like natural CVF . The bimolecular convertase pro-CVF,Bb exhibits both C3 cleaving and C5 cleaving activity . The activity of pro-CVF and the resulting C3/C5 convertase is indistinguishable from CVF and the CVF,Bb convertase . The ability to produce active forms of pro-CVF recombinantly ensures the continued availability of an important research reagent for complement depletion because cobra venom as the source for natural CVF will be increasingly difficult to obtain as the Indian cobra is on the list of endangered species . Experimental systems to express pro-CVF recombinantly will also be invaluable for studies to delineate the structure and function relationship of CVF and its differences from C3 as well as to generate human C3 derivatives with CVF-like function for therapeutic complement depletion ("humanized CVF").

Appl Bioinformatics, 2002, 1(3), 111 - 9
Consensus sequence Zen; Schneider TD; Consensus sequences are widely used in molecular biology but they have many flaws . As a result, binding sites of proteins and other molecules are missed during studies of genetic sequences and important biological effects cannot be seen . Information theory provides a mathematically robust way to avoid consensus sequences . Instead of using consensus sequences, sequence conservation can be quantitatively presented in bits of information by using sequence logo graphics to represent the average of a set of sites, and sequence walker graphics to represent individual sites.

Appl Bioinformatics, 2003, 2(3), 185 - 8
Genomic Object Net: II . Modelling biopathways by hybrid functional Petri net with extension; Doi A et al.; This paper demonstrates how to create an HFPNe (hybrid functional Petri net with extension) model, using the lac operon gene regulatory mechanism and glycolytic pathway as an example . Using this example, readers can then model other biopathways of interest . Simulations of the HFPNe model were performed using the software package Genomic Object Net.

Arch Biochem Biophys, 2004 Jun 1, 426(1), 43 - 54
The role of cysteine 160 in thiamine diphosphate binding of the Calvin-Benson-Bassham cycle transketolase of Rhodobacter sphaeroides; Bobst CE et al.; The transketolase gene (cbbT) that encodes the Calvin-Benson-Bassham pathway transketolase (CbbT) of Rhodobacter sphaeroides was overexpressed in Escherichia coli and the recombinant protein purified to homogeneity . Like other transketolases, R . sphaeroides CbbT was found to be inactivated in the presence of oxygen . At its optimal pH of 7.8, CbbT displays a specific activity of 37 U/mg, a KR5P of 949 microM, a KXu5P of 11 microM, and a KThDP of 1.8 microM . Cysteine 160, equivalent to Cys159 of the yeast enzyme, is found within the active site and is loosely conserved amongst several sources of transketolase . To investigate the role of cysteine 160 found in the active site of R . sphaeroides CbbT, this residue was targeted for mutagenesis . Cys160 was changed to alanine, serine, aspartate, and glutamate . To compare the effect of these mutations on ThDP binding, spectral techniques were employed in addition to analysis by enzymatic activity . Fluorescence quenching was used to measure both equilibrium binding constants as well as first order rates of binding . The results of these studies indicated that Cys160 played an important and substantial role in cofactor binding, revealing the importance of this loosely conserved residue . In addition, the Cys160 mutants did not appear to alter oxygen-mediated inactivation.

Arch Biochem Biophys, 2004 Jun 1, 426(1), 32 - 42
Oligomerization is required for betaine-homocysteine S-methyltransferase function; Szegedi SS et al.; Betaine-homocysteine methyltransferase (BHMT) is a member of a family (Pfam 02574) of zinc- and thiol/selenol-dependent methyltransferases . All family members purified to date are monomers, except BHMT, which is an oligomer . We have studied how C-terminal truncation or mutagenic replacement of residues within or associated with the unique dimerization arm of this enzyme affects oligomerization and function . Two C-terminal truncation mutants, S325 and D371, do not express well in Escherichia coli and are inactive . Residues within the dimerization arm (H338, R346, W352, R361, P362, Y363, N364, and P365) and one that forms a hydrogen bond to the arm (E266) were changed to alanine . All mutants maintained a normal or near-normal ability to bind zinc . E266A, R361A, P362A, Y363A, N364A, and P365A displayed near-normal catalytic activity, but H338A had only 10% of the wild-type enzyme activity . Like the wild-type enzyme, most mutants eluted as tetramers from gel filtration columns and formed discrete bands on SDS-PAGE gels following glutaraldehyde crosslinking . Mutants R346A and W352A had negligible activity, eluted as dimers, and displayed aberrant crosslinking properties . These data indicate that unlike other Pfam 02574 members, oligomerization of BHMT is required for function.

Biomaterials, 2004 Oct, 25(23), 5433 - 40
In vivo studies of endotoxin removal by lysine-cellulose adsorbents; Fang H et al.; A new type adsorbent for removal of bacterial endotoxins was prepared by immobilizing lysine covalently onto cellulose beads . Endotoxins (Escherichia coli O55: B5) were injected into 13 healthy New Zealand white rabbits to induce infectious symptoms . Hemoperfusion using the adsorbent column removed endotoxins in the blood of eight rabbits during 2h while other five rabbits were used as control . The mean blood endotoxin concentration was reduced significantly from 5.56 +/- 0.54 EU/ml (1 EU = 100 pg) before treatment to 0.41 +/- 0.26 EU/ml after perfusion as measured by the limulus amebocyte lysate test (Chromogenix) . Liver function and renal function tests showed significant improvement of septic symptoms in contrast to the control group . Other parameters such as superoxide dismutase and malondialdehyde were ameliorated markedly after the treatment . Moreover, the adsorbent showed good results in mechanical strength, blood compatibility and cytotoxicity, which suggested that lysine-cellulose adsorbent was of high ET-binding efficacy without significant side effect . It has a high potential of clinical application for treatment of patients with severe sepsis .

Mol Cell Endocrinol, 2004 Apr 15, 218(1-2), 21 - 38
Crustacean retinoid-X receptor isoforms: distinctive DNA binding and receptor-receptor interaction with a cognate ecdysteroid receptor; Wu X et al.; We have identified cDNA clones that encode homologs of the ecdysteroid receptor (EcR) and retinoid-X receptor (RXR)/USP classes of nuclear receptors from the fiddler crab Uca pugilator (UpEcR and UpRXR) . Several UpRXR cDNA splicing variants were found in coding regions that could potentially influence function . A five-amino acid (aa) insertion/deletion is located in the "T" box in the hinge region . Another 33-aa insertion/deletion is found inside the ligand-binding domain (LBD), between helix 1 and helix 3 . Ribonuclease protection assays (RPA) showed that four UpRXR transcripts {UpRXR(+5+33), UpRXR(-5+33), UpRXR(+5-33) and UpRXR(-5-33)} were present in regenerating limb buds . UpRXR(-5+33) was the most abundant transcript present in regenerating limb buds in both early blastema and late premolt growth stages . Expression vectors for these UpRXR variants and UpEcR were constructed, and the proteins expressed in E . coli and in vitro expression systems . The expressed crab nuclear receptors were then characterized by electrophoretic mobility shift assay (EMSA) and glutathione S-transferase (GST) pull down experiments . EMSA results showed that UpEcR/UpRXR(-5+33) heterocomplexes bound with a series of hormone response elements (HREs) including eip28/29, IRper-1, DR-4, and IRhsp-1 with appreciable affinity . Competition EMSA also showed that the affinity decreased as sequence composition deviated from a perfect consensus element . Binding to IRper-1 HREs occurred only if the heterodimer partner UpRXR contained the 33-aa LBD insertion . UpRXR lacking both the 5-aa and 33-aa insertion bound to a DR-1G HRE in the absence of UpEcR . The results of GST-pull down experiments showed that UpEcR interacted only with UpRXR variants containing the 33-aa insertion, and not with those lacking the 33-aa insertion . These in vitro receptor protein-DNA and receptor protein-protein interactions occurred in the absence of hormone (20-hydroxyecdysone and 9-cis retinoid acid, 9-cis RA) . Transactivation studies using a hybrid UpEcR ligand-binding domain construct and UpRXR (+/-33) ligand-binding domain constructs also showed that the 33-aa insertion was indispensable in mediating ecdysteroid stimulated transactivation.

Structure (Camb), 2004 May, 12(5), 861 - 9
Structure of the n-terminal domain of Escherichia coli glutamine synthetase adenylyltransferase; Xu Y et al.; We report the crystal structure of the N-terminal domain of Escherichia coli adenylyltransferase that catalyzes the reversible nucleotidylation of glutamine synthetase (GS), a key enzyme in nitrogen assimilation . This domain (AT-N440) catalyzes the deadenylylation and subsequent activation of GS . The structure has been divided into three subdomains, two of which bear some similarity to kanamycin nucleotidyltransferase (KNT) . However, the orientation of the two domains in AT-N440 differs from that in KNT . The active site of AT-N440 has been identified on the basis of structural comparisons with KNT, DNA polymerase beta, and polyadenylate polymerase . AT-N440 has a cluster of metal binding residues that are conserved in polbeta-like nucleotidyl transferases . The location of residues conserved in all ATase sequences was found to cluster around the active site . Many of these residues are very likely to play a role in catalysis, substrate binding, or effector binding.

Zhonghua Jie He He Hu Xi Za Zhi, 2004 Mar, 27(3), 188 - 90
{Molecular cloning, purification, and serological characterization of four specific antigens of Mycobacterium tuberculosis}; Xu L et al.; OBJECTIVE: To express the 14,000, 37,000, and 6,000 early secretory antigenic target (ESAT-6) and mtb81 antigen genes in bacteria, and to purify the product and determine their activity . METHODS: The 14,000, 37,000 , ESAT-6, and mtb81 antigen genes were amplified from Mycobacterium tuberculosis genomic DNA by polymerase chain reactions and cloned into pGEX 4T-1 expression vector . BL21 strain of Escherichia coli was transformed with the recombinant vectors and induced to express recombinant proteins . The proteins were purified by affinity chromatography . The biological activity of purified proteins were estimated by enzyme-linked immunoabsorbant assay (ELISA) . RESULTS: The BL21 strains of Escherichia coli with recombinant vectors showed high level of 14,000, 37,000, ESAT-6, and mtb81 gene expressions after induction . The products were purified successfully and showed high antigenicity and specificity . The sensitivity of 38,000, 14,000, ESAT-6, and mtb81 were 54%, 60%, 44%, and 36%, respectively . CONCLUSION: The expressions and purifications of recombinant 14,000, 37,000, ESAT-6, and mtb81 antigens with natural activity facilitate their research and application.

Mol Microbiol, 2004 May, 52(4), 1055 - 67
The effect of host-encoded nucleoid proteins on transposition: H-NS influences targeting of both IS903 and Tn10; Swingle B et al.; Nucleoid proteins are small, abundant, DNA-binding proteins that profoundly affect the local and global structure of the chromosome, and play a major role in gene regulation . Although several of these proteins have been shown to enhance assembly of transpososomes before initiating transposition, no systematic survey has been carried out examining the in vivo role(s) of these proteins in transposition . We have examined the requirement of the six most abundant nucleoid proteins in transposition for three different transposons, IS903, Tn10 and Tn552 . Most notably, H-NS was required for efficient transposition of all three elements in a papillation assay, suggesting a general role for H-NS in bacterial transposition . Further studies indicated that H-NS was exerting its effect on target capture . Targeting preferences for IS903 into the Escherichia coli chromosome were dramatically altered in the absence of H-NS . In addition, the alterations observed in the IS903 target profile emphasized the important role that H-NS plays in chromosome organization . A defect in target capture was also inferred for Tn10, as an excised transposon fragment, a precursor to target capture, accumulated in in vivo induction assays . Furthermore, a transposase mutant that is known to increase target DNA bending and to relax target specificity eliminated this block to target capture . Together, these results imply a role for H-NS in target capture, either by providing regions of DNA more accessible to transposition or by stabilizing transpososome binding to captured targets immediately before strand transfer.

Mol Microbiol, 2004 May, 52(4), 1029 - 44
Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli; Lai EM et al.; Bacteria show asymmetric subcellular distribution of many proteins involved in diverse cellular processes such as chemotaxis, motility, actin polymerization, chromosome partitioning and cell division . In many cases, the specific subcellular localization of these proteins is critical for proper regulation and function . Although cellular organization of the bacterial cell clearly plays an important role in cell physiology, systematic studies to uncover asymmetrically distributed proteins have not been reported previously . In this study, we undertook a proteomics approach to uncover polar membrane proteins in Escherichia coli . We identified membrane proteins enriched in E . coli minicells using a combination of two-dimensional electrophoresis and mass spectrometry . Among a total of 173 membrane protein spots that were consistently detected, 36 spots were enriched in minicell membranes, whereas 15 spots were more abundant in rod cell membranes . The minicell-enriched proteins included the inner membrane proteins MCPs, AtpA, AtpB, YiaF and AcrA, the membrane-associated FtsZ protein and the outer membrane proteins YbhC, OmpW, Tsx, Pal, FadL, OmpT and BtuB . We immunolocalized two of the minicell-enriched proteins, OmpW and YiaF, and showed that OmpW is a bona fide polar protein whereas YiaF displays a patchy membrane distribution with a polar and septal bias.

Mol Microbiol, 2004 May, 52(4), 1013 - 28
Phage T4 early promoters are resistant to inhibition by the anti-sigma factor AsiA; Orsini G et al.; Phage T4 early promoters are transcribed in vivo and in vitro by the Escherichia coli RNA polymerase holoenzyme Esigma(70) . We studied in vitro the effects of the T4 anti-sigma(70) factor AsiA on the activity of several T4 early promoters . In single-round transcription, promoters motB, denV, mrh.2, motA wild type and UP element-deleted motA are strongly resistant to inhibition by AsiA . The alpha-C-terminal domain of Esigma(70) is crucial to this resistance . DNase I footprinting of Esigma(70) and Esigma(70)AsiA on motA and mrh.2 shows extended contacts between the holoenzyme with or without AsiA and upstream regions of these promoters . A TG --> TC mutation of the extended -10 motif in the motA UP element-deleted promoter strongly increases susceptibility to inhibition by AsiA, but has no effect on the motA wild-type promoter: either the UP element or the extended -10 site confers resistance to AsiA . Potassium permanganate reactivity shows that the two structure elements are not equivalent: with AsiA, the motA UP element-deleted promoter opens more slowly whereas the motA TC promoter opens like the wild type . Changes in UV laser photoreactivity at position +4 on variants of motA reveal an analogous distinction in the roles of the extended -10 and UP promoter elements.

Mol Microbiol, 2004 May, 52(4), 963 - 83
Differential recognition of members of the carcinoembryonic antigen family by Afa/Dr adhesins of diffusely adhering Escherichia coli (Afa/Dr DAEC); Berger CN et al.; Little is known about the molecular bases underlying the virulence of diffusely adhering Escherichia coli (DAEC) harbouring the Afa/Dr family of adhesins . These adhesins recognize as receptors the GPI-anchored proteins CD55 (decay-accelerating factor, DAF) and CD66e (carcinoembryonic antigen, CEA) . CD66e is a member of the CEA-related cell adhesion molecules (CEACAM) family, comprising seven members . We analysed the interactions of Afa/Dr DAEC with the CEACAMs using CEACAM-expressing CHO and HeLa cells . The results demonstrate that only E . coli expressing a subfamily of Afa/Dr adhesins, named here Afa/Dr-I, including Dr, F1845 and AfaE-III adhesins, bound onto CHO cells expressing CEACAM1, CEA or CEACAM6 . Whereas all the Afa/Dr adhesins elicit recruitment of CD55 around adhering bacteria, only the Afa/Dr-I subfamily elicits the recruitment of CEACAM1, CEA and CEACAM6 . In addition, although CEACAM3 is not recognized as a receptor by the subfamily of Afa/Dr adhesins, it is recruited around bacteria in HeLa cells . The recruited CEACAM1, CEA and CEACAM6 around adhering bacteria resist totally or in part a detergent extraction, whereas the recruited CEACAM3 does not . Finally, the results show that recognition of CEA and CEACAM6, but not CEACAM1, is accompanied by tight attachment to bacteria of cell surface microvilli-like extensions, which are elongated . Moreover, recognition of CEA is accompanied by an activation of the Rho GTPase Cdc42 and by a phosphorylation of ERM, which in turn elicit the observed cell surface microvilli-like extensions.

Proc Natl Acad Sci U S A, 2004 May 18, 101(20), 7583 - 8 Epub 2004 May 05.
SecB is a bona fide generalized chaperone in Escherichia coli; Ullers RS et al.; It is known that the DnaK and Trigger Factor (TF) chaperones cooperate in the folding of newly synthesized cytosolic proteins in Escherichia coli . We recently showed that despite a very narrow temperature range of growth and high levels of aggregated cytosolic proteins, E . coli can tolerate deletion of both chaperones, suggesting that other chaperones might be involved in this process . Here, we show that the secretion-dedicated chaperone SecB efficiently suppresses both the temperature sensitivity and the aggregation-prone phenotypes of a strain lacking both TF and DnaK . SecB suppression is independent of a productive interaction with the SecA subunit of the translocon . Furthermore, in vitro cross-linking experiments demonstrate that SecB can interact both co- and posttranslationally with short nascent chains of both secretory and cytosolic proteins . Finally, we show that such cotranslational substrate recognition by SecB is greatly suppressed in the presence of ribosome-bound TF, but not by DnaK . Taken together, our data demonstrate that SecB acts as a bona fide generalized chaperone.

J Immunol, 2004 May 15, 172(10), 6490 - 500
Molecular characterization of polygalacturonases as grass pollen-specific marker allergens: expulsion from pollen via submicronic respirable particles; Swoboda I et al.; Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma . We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases) . The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5 . rPhl p 13 was expressed in Escherichia coli and purified to homogeneity . Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens . The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls . rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release . Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains . The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen . Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.

J Biol Chem, 2004 Jun 25, 279(26), 26807 - 10 Epub 2004 May 05.
Chemokines generally exhibit scavenger receptor activity through their receptor-binding domain; Shimaoka T et al.; Chemokines are a family of cytokines that induce directed migration of various types of leukocytes through specific interactions with a group of seven transmembrane receptors . Scavenger receptors are a heterogenous family of transmembrane molecules that commonly bind and uptake oxidized low density lipoprotein and bacteria . Here, we show that not only CXC chemokine 16 (CXCL16)/SR-PSOX, a transmembrane chemokine with scavenger receptor activity, but also 12 out of 15 chemokines examined efficiently bound scavenger receptor ligands in competition with cells expressing their specific chemokine receptors . Furthermore both the chemotactic and scavenger receptor activities of SR-PSOX/CXCL16 were similarly impaired in a series of mutants altered in the chemokine domain, indicating that SR-PSOX/CXCL16 binds scavenger receptor ligands as well as CXCR6 using highly overlapping binding motifs . Taken together, chemokines generally have scavenger receptor-like activity through their receptor-binding domain, suggesting a close evolutionary relationship between chemokines and scavenger receptors.

J Biol Chem, 2004 Jul 30, 279(31), 32008 - 17 Epub 2004 May 05.
Refolding processes of cytochrome P450cam from ferric and ferrous acid forms to the native conformation . Formations of folding intermediates with non-native heme coordination state; Egawa T et al.; Changes in heme coordination state and protein conformation of cytochrome P450(cam) (P450(cam)), a b-type heme protein, were investigated by employing pH jump experiments coupled with time-resolved optical absorption, fluorescence, circular dichroism, and resonance Raman techniques . We found a partially unfolded form (acid form) of ferric P450(cam) at pH 2.5, in which a Cys(-)-heme coordination bond in the native conformation was ruptured . When the pH was raised to pH 7.5, the acid form refolded to the native conformation through a distinctive intermediate . Formations of similar acid and intermediate forms were also observed for ferrous P450(cam) . Both the ferric and ferrous forms of the intermediate were found to have an unidentified axial ligand of the heme at the 6th coordination sphere, which is vacant in the high spin ferric and ferrous forms at the native conformation . For the ferrous form, it was also indicated that the 5th axial ligand is different from the native cysteinate . The folding intermediates identified in this study demonstrate occurrences of non-native coordination state of heme during the refolding processes of the large b-type heme protein, being akin to the well known folding intermediates of cytochromes c, in which c-type heme is covalently attached to a smaller protein.

Appl Environ Microbiol, 2004 May, 70(5), 2660 - 6
Heat shock protein-mediated resistance to high hydrostatic pressure in Escherichia coli; Aertsen A et al.; A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress . This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure . Several further observations provide additional support for this hypothesis: (i) . the expression of rpoH, encoding the heat shock-specific sigma factor sigma(32), was also induced by high pressure; (ii) . heat shock rendered E . coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii) . basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E . coli compared to wild-type levels.

J Carcinog . 2004 May 5;3(1):7.
Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay; Boerrigter ME; BACKGROUND: Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators . The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect . METHODS: Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP), 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period . Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls).The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids . RESULTS: Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice . Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied . CONCLUSION: The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.

Eur J Biochem, 2004 May, 271(10), 1952 - 62
Purification and characterization of Helicobacter pylori arginase, RocF: unique features among the arginase superfamily; McGee DJ et al.; The urea cycle enzyme arginase (EC 3.5.3.1) hydrolyzes l-arginine to l-ornithine and urea . Mammalian arginases require manganese, have a highly alkaline pH optimum and are resistant to reducing agents . The gastric human pathogen, Helicobacter pylori, also has a complete urea cycle and contains the rocF gene encoding arginase (RocF), which is involved in the pathogenesis of H . pylori infection . Its arginase is specifically involved in acid resistance and inhibits host nitric oxide production . The rocF gene was found to confer arginase activity to Escherichia coli; disruption of plasmid-borne rocF abolished arginase activity . A translationally fused His(6)-RocF was purified from E . coli under nondenaturing conditions and had catalytic activity . Remarkably, the purified enzyme had an acidic pH optimum of 6.1 . Both purified arginase and arginase-containing H . pylori extracts exhibited optimal catalytic activity with cobalt as a metal cofactor; manganese and nickel were significantly less efficient in catalyzing the hydrolysis of arginine . Viable H . pylori or E . coli containing rocF had significantly more arginase activity when grown with cobalt in the culture medium than when grown with manganese or no divalent metal . His(6)-RocF arginase activity was inhibited by low concentrations of reducing agents . Antibodies raised to purified His(6)-RocF reacted with both H . pylori and E . coli extracts containing arginase, but not with extracts from rocF mutants of H . pylori or E . coli lacking the rocF gene . The results indicate that H . pylori RocF is necessary and sufficient for arginase activity and has unparalleled features among the arginase superfamily, which may reflect the unique gastric ecological niche of this organism.

Eur J Biochem, 2004 May, 271(10), 1885 - 94
Cloning, expression and characterization of two new IgE-binding proteins from the yeast Malassezia sympodialis with sequence similarities to heat shock proteins and manganese superoxide dismutase; Andersson A et al.; Malassezia sympodialis is an opportunistic yeast that colonizes human skin and may induce IgE and T cell reactivity in patients with atopic eczema/dermatitis syndrome (AEDS) . Previously, we have cloned and expressed six recombinant allergens (rMala s 1 and rMala s 5 to rMala s 9) from this yeast . By combining high throughput screening and phage surface display techniques, 27 complete and partial IgE-binding clones of M . sympodialis have been identified . Here we enlarged the panel of recombinant M . sympodialis allergens by RACE-PCR, cloning and nucleotide sequencing to obtain the coding sequences of two new IgE-binding clones . The coding sequences of one of the clones showed similarity to the heat shock protein (HSP) family and the other to manganese superoxide dismutase (MnSOD), and both had a high degree of homology to human counterparts . The coding sequences were expressed in Escherichia coli as six-histidine tagged recombinant proteins and generated products with molecular masses of 86.1 kDa for HSP and 22.4 kDa for MnSOD . Their IgE-binding frequencies were shown to be 69% and 75%, respectively, to 28 sera from AEDS patients with serum IgE to M . sympodialis extract, indicating that HSP and MnSOD are major M . sympodialis allergens . In inhibition immunoblotting, M . sympodialis extract could inhibit the binding of serum IgE from AEDS patients to rHSP and rMnSOD in a concentration-dependent manner . The high frequency of sera from AEDS patients, showing IgE binding to both HSP and MnSOD, indicates that these allergens, designated Mala s 10 and Mala s 11, could play a role in AEDS.

Eur J Biochem, 2004 May, 271(10), 1864 - 72
The metabolic role and evolution of L-arabinitol 4-dehydrogenase of Hypocrea jecorina; Pail M et al.; L-Arabinitol 4-dehydrogenase (Lad1) of the cellulolytic and hemicellulolytic fungus Hypocrea jecorina (anamorph: Trichoderma reesei) has been implicated in the catabolism of L-arabinose, and genetic evidence also shows that it is involved in the catabolism of D-xylose in xylitol dehydrogenase (xdh1) mutants and of D-galactose in galactokinase (gal1) mutants of H . jecorina . In order to identify the substrate specificity of Lad1, we have recombinantly produced the enzyme in Escherichia coli and purified it to physical homogeneity . The resulting enzyme preparation catalyzed the oxidation of pentitols (L-arabinitol) and hexitols (D-allitol, D-sorbitol, L-iditol, L-mannitol) to the same corresponding ketoses as mammalian sorbitol dehydrogenase (SDH), albeit with different catalytic efficacies, showing highest k(cat)/K(m) for L-arabinitol . However, it oxidized galactitol and D-talitol at C4 exclusively, yielding L-xylo-3-hexulose and D-arabino-3-hexulose, respectively . Phylogenetic analysis of Lad1 showed that it is a member of a terminal clade of putative fungal arabinitol dehydrogenase orthologues which separated during evolution of SDHs . Juxtapositioning of the Lad1 3D structure over that of SDH revealed major amino acid exchanges at topologies flanking the binding pocket for d-sorbitol . A lad1 gene disruptant was almost unable to grow on L-arabinose, grew extremely weakly on L-arabinitol, D-talitol and galactitol, showed reduced growth on D-sorbitol and D-galactose and a slightly reduced growth on D-glucose . The weak growth on L-arabinitol was completely eliminated in a mutant in which the xdh1 gene had also been disrupted . These data show not only that Lad1 is indeed essential for the catabolism of L-arabinose, but also that it constitutes an essential step in the catabolism of several hexoses; this emphasizes the importance of such reductive pathways of catabolism in fungi.

C R Biol, 2004 Mar, 327(3), 211 - 24
Modeling operon dynamics: the tryptophan and lactose operons as paradigms; Mackey MC et al.; Understanding the regulation of gene control networks and their ensuing dynamics will be a critical component in the understanding of the mountain of genomic data being currently collected . This paper reviews recent mathematical modeling work on the tryptophan and lactose operons which are, respectively, the classical paradigms for repressible and inducible operons.

Plant Cell Rep, 2004 Jul, 22(12), 931 - 8 Epub 2004 May 04.
Heritable transgene expression pattern imposed onto maize ubiquitin promoter by maize adh-1 matrix attachment regions: tissue and developmental specificity in maize transgenic plants; Torney F et al.; Matrix attachment regions (MARs) have been used to enhance transgene expression and to reduce transgene expression instability in various organisms . In plants, contradictory data question the role of MAR sequences . To assess the use of MAR sequences in maize, we have used two well-characterized MARs from the maize adh-1 region . The MARs have been cloned either 5' to or at both sides of a reporter gene expression cassette to reconstitute a MAR-based domain . Histochemical staining revealed a new transgene expression pattern in roots of regenerated plants and their progeny . Furthermore, MARs systematically induced variegation . We show here that maize adh-1 MARs are able to modify transgene expression patterns as a heritable trait, giving a new and complementary outcome following use of MARs in genetic transformation.

Int Arch Occup Environ Health, 2004 Jun, 77(5), 335 - 40 Epub 2004 May 01.
Flow cytometric determination of neutrophil respiratory burst activity in workers exposed to formaldehyde; Lyapina M et al.; AIM: The aim of the study was to investigate neutrophil respiratory burst activity (NRBA) in workers who were occupationally exposed to formaldehyde . METHODS: NRBA, spontaneous and stimulated with E . coli, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA), was studied by means of quantitative flow cytometric determination in 29 workers who were occupationally exposed to formaldehyde; 21 healthy subjects, not exposed to formaldehyde, served as controls . All subjects underwent clinical assessment, including a review of a summary of their medical history and a physical examination . Routine haematological tests were performed . RESULTS: A statistically significant predominance of subjective symptoms and objective clinical findings of chronic upper respiratory tract inflammation, as well as decreased resistance to infections, was observed in the 29 workers exposed to formaldehyde, compared with the controls (chi2 = 9.28, P = 0.02) . No statistically significant difference in the spontaneous and stimulated NRBA between the exposed workers and the control group was observed . The spontaneous NRBA (percentage oxidizing cells) was significantly lower in the group of exposed workers with upper respiratory tract findings and frequent and long-lasting infectious inflammatory relapses (median and range 0.45 (0.02-2.03), mean values 0.65 +/- 0.74) than in the healthy controls (median and range 1.35 (0.07-8.69), mean values 2.42 +/- 2.47; P < 0.05), and in the group of exposed workers with rare and short, acute, inflammation of the upper respiratory tract or without any inflammations (median and range 1.00 (0.02-8.67), mean values 1.67 +/- 2.08; P < 0.05) . A significant negative correlation between the duration of occupational exposure to formaldehyde and erythrocyte count and haematocrit was found . CONCLUSIONS: The observed decrease of spontaneous NRBA in workers with a history and clinical findings of frequent and long-lasting relapses of chronic inflammation of the upper respiratory tract could be due to formaldehyde exposure and individual susceptibility . The results obtained suggest that functional changes in polymorphonuclear neutrophil granulocytes could serve as an early indicator of an impact of formaldehyde on NRBA . The applied method might be used for identifying groups at increased toxicological risk .

Proc Natl Acad Sci U S A, 2004 May 18, 101(20), 7594 - 9 Epub 2004 May 04.
Automated structure prediction of weakly homologous proteins on a genomic scale; Zhang Y et al.; We have developed TASSER, a hierarchical approach to protein structure prediction that consists of template identification by threading, followed by tertiary structure assembly via the rearrangement of continuous template fragments guided by an optimized C(alpha) and side-chain-based potential driven by threading-based, predicted tertiary restraints . TASSER was applied to a comprehensive benchmark set of 1,489 medium-sized proteins in the Protein Data Bank . With homologues excluded, in 927 cases, the templates identified by our threading algorithm PROSPECTOR_3 have a rms deviation from native <6.5 A with approximately 80% alignment coverage . After template reassembly, this number increases to 1,172 . This shows significant and systematic improvement of the final models with respect to the initial template alignments . Furthermore, significant improvements in loop modeling are demonstrated . We then apply TASSER to the 1,360 medium-sized ORFs in the Escherichia coli genome; approximately 920 can be predicted with high accuracy based on confidence criteria established in the Protein Data Bank benchmark . These results from our unprecedented comprehensive folding benchmark on all protein categories provide a reliable basis for the application of TASSER to structural genomics, especially to proteins of low sequence identity to solved protein structures.

J Biol Chem, 2004 Jun 25, 279(26), 26876 - 84 Epub 2004 May 04.
The Escherichia coli 3-methyladenine DNA glycosylase AlkA has a remarkably versatile active site; O'Brien PJ et al.; 3-Methyladenine DNA glycosylase II (AlkA) from Escherichia coli is induced in response to DNA alkylation, and it protects cells from alkylated nucleobases by catalyzing their excision . In contrast to the highly specific 3-methyladenine DNA glycosylase I (E . coli TAG) that catalyzes the excision of 3-methyl adducts of adenosine and guanosine from DNA, AlkA catalyzes the excision of a wide variety of alkylated bases including N-3 and N-7 adducts of adenosine and guanosine and O(2) adducts of thymidine and cytidine . We have investigated how AlkA can recognize a diverse set of damaged bases by characterizing its discrimination between oligonucleotide substrates in vitro . Similar rate enhancements are observed for the excision of a structurally diverse set of substituted purine bases and of the normal purines adenine and guanine . These results are consistent with a remarkably indiscriminate active site and suggest that the rate of AlkA-catalyzed excision is dictated not by the catalytic recognition of a specific substrate but instead by the reactivity of the N-glycosidic bond of each substrate . Damaged bases with altered base pairing have a modest advantage, as mismatches are processed up to 400-fold faster than stable Watson-Crick base pairs . Nevertheless, AlkA does not effectively exclude undamaged DNA from its active site . The resulting deleterious excision of normal bases is expected to have a substantial cost associated with the expression of AlkA.

J Bacteriol, 2004 May, 186(10), 3262 - 5
The nucleotide transporter of Caedibacter caryophilus exhibits an extended substrate spectrum compared to the analogous ATP/ADP translocase of Rickettsia prowazekii; Daugherty RM et al.; The two obligate intracellular alphaproteobacteria Rickettsia prowazekii and Caedibacter caryophilus, a human pathogen and a paramecium endosymbiont, respectively, possess transport systems to facilitate ATP uptake from the host cell cytosol . These transport proteins, which have 65% identity at the amino acid level, were heterologously expressed in Escherichia coli, and their properties were compared . The results presented here demonstrate that the caedibacter transporter had a broader substrate than the more selective rickettsial transporter . ATP analogs with modified sugar moieties, dATP and ddATP, inhibited the transport of ATP by the caedibacter transporter but not by the rickettsial transporter . Both transporters were specific for di- and trinucleotides with an adenine base in that adenosine tetraphosphate, AMP, UTP, CTP, and GTP were not competitive inhibitors . Furthermore, the antiporter nature of both transport systems was shown by the dependence of the efflux of {alpha-32P}ATP on the influx of substrate (ATP but not dATP for rickettsiae, ATP or dATP for caedibacter).

J Bacteriol, 2004 May, 186(10), 3230 - 7
Horizontal transfer of CS1 pilin genes of enterotoxigenic Escherichia coli; Froehlich B et al.; CS1 is one of a limited number of serologically distinct pili found in enterotoxigenic Escherichia coli (ETEC) strains associated with disease in people . The genes for the CS1 pilus are on a large plasmid, pCoo . We show that pCoo is not self-transmissible, although our sequence determination for part of pCoo shows regions almost identical to those in the conjugative drug resistance plasmid R64 . When we introduced R64 into a strain containing pCoo, we found that pCoo was transferred to a recipient strain in mating . Most of the transconjugant pCoo plasmids result from recombination with R64, leading to acquisition of functional copies of all of the R64 transfer genes . Temporary coresidence of the drug resistance plasmid R64 with pCoo leads to a permanent change in pCoo so that it is now self-transmissible . We conclude that when R64-like plasmids are transmitted to an ETEC strain containing pCoo, their recombination may allow for spread of the pCoo plasmid to other enteric bacteria.

J Bacteriol, 2004 May, 186(10), 3173 - 81
Rusty, jammed, and well-oiled hinges: Mutations affecting the interdomain region of FliG, a rotor element of the Escherichia coli flagellar motor; Van Way SM et al.; The FliG protein is a central component of the bacterial flagellar motor . It is one of the first proteins added during assembly of the flagellar basal body, and there are 26 copies per motor . FliG interacts directly with the Mot protein complex of the stator to generate torque, and it is a crucial player in switching the direction of flagellar rotation from clockwise (CW) to counterclockwise and vice versa . A primarily helical linker joins the N-terminal assembly domain of FliG, which is firmly attached to the FliF protein of the MS ring of the basal body, to the motility domain that interacts with MotA/MotB . We report here the results of a mutagenic analysis focused on what has been called the hinge region of the linker . Residue substitutions in this region generate a diversity of phenotypes, including motors that are strongly CW biased, infrequent switchers, rapid switchers, and transiently or permanently paused . Isolation of these mutants was facilitated by a "sensitizing" mutation (E232G) outside of the hinge region that was accidentally introduced during cloning of the chromosomal fliG gene into our vector plasmid . This mutation partially interferes with flagellar assembly and accentuates the defects associated with mutations that by themselves have little phenotypic consequence . The effects of these mutations are analyzed in the context of a conformational-coupling model for motor switching and with respect to the structure of the C-terminal 70% of FliG from Thermotoga maritima.

J Bacteriol, 2004 May, 186(10), 3097 - 107
Distinctive protein signatures provide molecular markers and evidence for the monophyletic nature of the deinococcus-thermus phylum; Griffiths E et al.; The Deinococcus-Thermus group of species is currently recognized as a distinct phylum solely on the basis of their branching in 16S rRNA trees . No unique biochemical or molecular characteristics that can distinguish this group from all other bacteria are known at present . In this work, we describe eight conserved indels (viz., inserts or deletions) in seven widely distributed proteins that are distinctive characteristics of the Deinococcus-Thermus phylum but are not found in any other group of bacteria . The identified signatures include a 7-amino-acid (aa) insert in threonyl-tRNA synthetase, 1- and 3-aa inserts in the RNA polymerase beta' subunit, a 5-aa deletion in signal recognition particle (Ffh/SR54), a 2-aa insert in major sigma factor 70 (sigma70), a 2-aa insert in seryl-tRNA synthetase (SerRS), a 1-aa insert in ribosomal protein L1, and a 2-aa insert in UvrA homologs . By using PCR primers for conserved regions, fragments of these genes were amplified from a number of Deinococcus-Thermus species, and all such fragments (except SerRS in Deinococcus proteolyticus) were found to contain the indicated signatures . The presence of these signatures in various species from all three known genera within this phylum, viz., Deinococcus, Thermus, and Meiothermus, provide evidence that they are likely distinctive characteristics of the entire phylum which were introduced in a common ancestor of this group . The signature in SerRS, which is absent in D . proteolyticus, was likely introduced after the branching of this species . Phylogenetic studies as well as the nature of the inserts in some of these proteins (viz., sigma70 and SerRS) also support a sister group relationship between the Thermus and the Meiothermus genera . The identified signatures provide strong evidence for the monophyletic nature of the Deinococcus-Thermus phylum . These molecular markers should prove very useful in the identification of new species related to this group.

J Bacteriol, 2004 May, 186(10), 3086 - 96
Instability of pathogenicity islands in uropathogenic Escherichia coli 536; Middendorf B et al.; The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs) . One main feature of these distinct DNA regions is their instability . We applied the so-called island-probing approach and individually labeled all five PAIs of E . coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions . Furthermore, we investigated the boundaries of these PAIs . According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable . In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli . Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences . In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates . Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay . Our data indicate that the genome content of uropathogenic E . coli can be modulated by deletion of PAIs.

J Bacteriol, 2004 May, 186(10), 3006 - 14
Signal transduction cascade between EvgA/EvgS and PhoP/PhoQ two-component systems of Escherichia coli; Eguchi Y et al.; Transcriptional analysis of a constitutively active mutant of the EvgA/EvgS two-component system of Escherichia coli resulted in enhanced expression of 13 PhoP/PhoQ-regulated genes, crcA, hemL, mgtA, ompT, phoP, phoQ, proP, rstA, rstB, slyB, ybjG, yrbL, and mgrB . This regulatory network between the two systems also occurred as a result of overproduction of the EvgA regulator; however, enhanced transcription of the phoPQ genes did not further activate expression of the PhoP/PhoQ-regulated genes . These results demonstrated signal transduction from the EvgA/EvgS system to the PhoP/PhoQ system in E . coli and also identified the genes that required the two systems for enhanced expression . This is one example of the intricate signal transduction networks that are posited to exist in E . coli.

Genetics, 2004 Apr, 166(4), 1775 - 82
Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31; Groth AC et al.; The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites . Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos . Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47% . In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells . Two lines of Drosophila were created by integrating an attP site into the genome with a P element . phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring . A total of 100% of these progeny carried a precise integration event at the genomic attP site . These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.

Genetics, 2004 Apr, 166(4), 1631 - 40
RuvAB and RecG are not essential for the recovery of DNA synthesis following UV-induced DNA damage in Escherichia coli; Donaldson JR et al.; Ultraviolet light induces DNA lesions that block the progression of the replication machinery . Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur . Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo . To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants . We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells . The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo.

Cancer Res, 2004 May 1, 64(9), 3096 - 102
Deficiencies in mouse Myh and Ogg1 result in tumor predisposition and G to T mutations in codon 12 of the K-ras oncogene in lung tumors; Xie Y et al.; Oxidative DNA damage is unavoidably and continuously generated by oxidant byproducts of normal cellular metabolism . The DNA damage repair genes, mutY and mutM, prevent G to T mutations caused by reactive oxygen species in Escherichia coli, but it has remained debatable whether deficiencies in their mammalian counterparts, Myh and Ogg1, are directly involved in tumorigenesis . Here, we demonstrate that deficiencies in Myh and Ogg1 predispose 65.7% of mice to tumors, predominantly lung and ovarian tumors, and lymphomas . Remarkably, subsequent analyses identified G to T mutations in 75% of the lung tumors at an activating hot spot, codon 12, of the K-ras oncogene, but none in their adjacent normal tissues . Moreover, malignant lung tumors were increased with combined heterozygosity of Msh2, a mismatch repair gene involved in oxidative DNA damage repair as well . Thus, oxidative DNA damage appears to play a causal role in tumorigenesis, and codon 12 of K-ras is likely to be an important downstream target in lung tumorigenesis . The multiple oxidative repair genes are required to prevent mutagenesis and tumor formation . The mice described here provide a valuable model for studying the mechanisms of oxidative DNA damage in tumorigenesis and investigating preventive or therapeutic approaches.

J Oral Rehabil, 2004 Jan, 31(1), 85 - 9
The effect of washing water on bonding to etched enamel; Schneider DJ et al.; There is current concern about bacterial contamination of dental unit waterlines . This research hypothesized that the presence of increasing concentrations of bacteria in water used to wash etched enamel would result in a corresponding decrease in both shear bond strength (SBS) and critical surface tension (gammaC) of enamel . A further hypothesis was made that there is a correlation between SBS and gammaC . The effect of 3.5 ppm iodine in the water as a bacteriostatic agent was also assessed . Five groups of 10 samples of bovine enamel were etched, washed, and a resin composite bonded to them . The control group was washed with distilled water . Another group was washed with the dilute iodine solution . The remaining three groups used a different concentration of Escherichia coli DH5alpha as follows (in cfu mL(-1)): group 1: 10(2); group 2: 10(4); group 3: 10(6) . Shear bond strength data were measured on an Instron testing machine at a crosshead speed of 1 mm min(-1) . Adhesion data were (MPa): control: 24.6 +/- 6.0; with iodine: 20.8 +/- 2.7; group 1: 19.8 +/- 2.7; group 2: 13.5 +/- 3.0; group 3: 13.9 +/- 3.6 . The F-test yielded a highly significant difference between control group, iodine group and group 1, compared with groups 2 and 3 (P < 0.0001) . Tukey's Studentized Range Test was used for pairwise comparison testing between groups . Using a Cahn dynamic contact angle analyzer and linear regression analysis, the plots of surface tension versus costheta were extrapolated to costheta = 1 to give gammaC data for the control group and groups 1-3 . In all cases reasonable linearity was observed (r2 > or = 0.87) . Data (mN m(-1)) were: control group: 50.8; group 1: 45.1; group 2: 43.2; group 3: 39.5 . The SBS and gammaC were then plotted against each other and linear regression analysis performed . It was concluded that increasing concentrations of bacteria in wash water decreased both SBS and gammaC and that a linear correlation (R2 = 0.84) was found between the values of these two parameters.

Mol Biol (Mosk), 2004 Mar-Apr, 38(2), 250 - 5
{Creation of a new construct for cloning DNA and modeling the structure of Drosophila polytene chromosomes}; Zimin PI et al.; Modification of P element-based transformation vector pCaSpeR3New yielded a new construct, pICon, which contains the structural region of the Escherichia coli lacZ, the adjacent 5' and 3' regulatory regions of hsp70, pUC19, and two tandem FRTs . Owing to the hsp70 promoter, the pICon insertion site may be localized on polytene chromosomes after heat shock by light or electron microscopy . The pUC19 sequence with a polylinker allows cloning of the genomic sequence adjacent to the 3' end of pICon by the rescue of the P-element target . Functional FRTs allow insertion or deletion of various DNA fragments . The construct is large (22046 bp), forms easily detectable structures in polytene chromosomes, and may be used to study the structural and functional organization of the Drosophila melanogaster genome, in particular, to elucidate the causes of banding pattern formation . To map the molecular boundaries of interband 3C6/C7, the DNA sequence of this region was cloned between the two FRTs.

Proc Natl Acad Sci U S A, 2004 May 11, 101(19), 7439 - 44 Epub 2004 Apr 27.
Interactions of glutaredoxins, ribonucleotide reductase, and components of the DNA replication system of Escherichia coli; Ortenberg R et al.; A strain of Escherichia coli missing three members of the thioredoxin superfamily, thioredoxins 1 and 2 and glutaredoxin 1, is unable to grow, a phenotype presumed to be due to the inability of cells to reduce the essential enzyme ribonucleotide reductase . Two classes of mutations can restore growth to such a strain . First, we have isolated a collection of mutations in the gene for the protein glutaredoxin 3 that suppress the growth defect . Remarkably, all eight independent mutations alter the same amino acid, methionine-43, changing it to valine, isoleucine, or leucine . From the position of the amino acid changes and their effects, we propose that these alterations change the protein so that its properties are closer to those of glutaredoxin 1 . The second means of suppressing the growth defects of the multiply mutant strain was by mutations in the DNA replication genes, dnaA and dnaN . These mutations substantially increase the expression of ribonucleotide reductase, most likely by altering the interaction of the regulatory protein DnaA with the ribonucleotide reductase promoter . Our results suggest that this increase in the concentration of ribonucleotide reductase in the cell allows more effective interaction with glutaredoxin 3, thus restoring an effective pool of deoxyribonucleotides . Our studies present direct evidence that ribonucleotide reductase is the only essential enzyme that requires the three reductive proteins missing in our strains . Our results also suggest an unexpected regulatory interaction between the DnaA and DnaN proteins.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 6882 - 7 Epub 2004 Apr 27.
Turning on ribonucleotide reductase by light-initiated amino acid radical generation; Chang MC et al.; Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms, providing the monomeric precursors required for DNA replication and repair . The class I RNRs are composed of two subunits; the R1 subunit contains the active site for nucleotide reduction and allosteric effector binding sites, whereas the R2 subunit houses the essential diirontyrosyl (Y.) radical cofactor . A major unresolved issue is the mechanism by which the tyrosyl radical on R2 (Y122, Escherichia coli numbering) reversibly generates the transient thiyl radical (S.) on R1 that initiates nucleotide reduction . This intersubunit radical initiation is postulated to occur through a defined pathway involving conserved aromatic amino acids (R2: Y122, W48, Y356; R1: Y731, Y730) over a long distance of 35 A . A 20-mer peptide identical to the C-terminal tail of R2 (356-375) and containing Y356 is a competitive inhibitor with respect to R2, and it effectively blocks nucleotide reduction . We now report that a 21-mer peptide, in which a tryptophan has been incorporated at the N terminus of the 20th mer, can replace the R2 subunit and initiate nucleotide reduction by photoinitiated radical generation . The deoxynucleotide generated depends on the presence of allosteric effector and is pathway-dependent . Replacement of Y731 of R2 with phenylalanine prevents deoxynucleotide formation . These results provide direct evidence for the chemical competence of aromatic amino acid radicals and the importance of Y356 in R2 in the radical initiation process of the class I RNRs.

Proc Natl Acad Sci U S A, 2004 May 11, 101(19), 7293 - 8 Epub 2004 May 03.
The C-terminal domain of DNA gyrase A adopts a DNA-bending beta-pinwheel fold; Corbett KD et al.; DNA gyrase is unique among enzymes for its ability to actively introduce negative supercoils into DNA . This function is mediated in part by the C-terminal domain of its A subunit (GyrA CTD) . Here, we report the crystal structure of this approximately 35-kDa domain determined to 1.75-A resolution . The GyrA CTD unexpectedly adopts an unusual fold, which we term a beta-pinwheel, that is globally reminiscent of a beta-propeller but is built of blades with a previously unobserved topology . A large, conserved basic patch on the outer edge of this domain suggests a likely site for binding and bending DNA; fluorescence resonance energy transfer-based assays show that the GyrA CTD is capable of bending DNA by > or =180 degrees over a 40-bp region . Surprisingly, we find that the CTD of the topoisomerase IV A subunit, which shares limited sequence homology with the GyrA CTD, also bends DNA . Together, these data provide a physical explanation for the ability of DNA gyrase to constrain a positive superhelical DNA wrap, and also suggest that the particular substrate preferences of topoisomerase IV might be dictated in part by the function of this domain.

Proc Natl Acad Sci U S A, 2004 May 11, 101(19), 7427 - 32 Epub 2004 May 03.
Carbon nutrition of Escherichia coli in the mouse intestine; Chang DE et al.; Whole-genome expression profiling revealed Escherichia coli MG1655 genes induced by growth on mucus, conditions designed to mimic nutrient availability in the mammalian intestine . Most were nutritional genes corresponding to catabolic pathways for nutrients found in mucus . We knocked out several pathways and tested the relative fitness of the mutants for colonization of the mouse intestine in competition with their wild-type parent . We found that only mutations in sugar pathways affected colonization, not phospholipid and amino acid catabolism, not gluconeogenesis, not the tricarboxylic acid cycle, and not the pentose phosphate pathway . Gluconate appeared to be a major carbon source used by E . coli MG1655 to colonize, having an impact on both the initiation and maintenance stages . N-acetylglucosamine and N-acetylneuraminic acid appeared to be involved in initiation, but not maintenance . Glucuronate, mannose, fucose, and ribose appeared to be involved in maintenance, but not initiation . The in vitro order of preference for these seven sugars paralleled the relative impact of the corresponding metabolic lesions on colonization: gluconate > N-acetylglucosamine > N-acetylneuraminic acid = glucuronate > mannose > fucose > ribose . The results of this systematic analysis of nutrients used by E . coli MG1655 to colonize the mouse intestine are intriguing in light of the nutrient-niche hypothesis, which states that the ecological niches within the intestine are defined by nutrient availability . Because humans are presumably colonized with different commensal strains, differences in nutrient availability may provide an open niche for infecting E . coli pathogens in some individuals and a barrier to infection in others.

Proc Natl Acad Sci U S A, 2004 May 11, 101(19), 7451 - 6 Epub 2004 May 03.
Chlamydial histone-DNA interactions are disrupted by a metabolite in the methylerythritol phosphate pathway of isoprenoid biosynthesis; Grieshaber NA et al.; The chlamydial developmental cycle is characterized by an intracellular replicative form, termed the reticulate body, and an extracellular form called the elementary body . Elementary bodies are characterized by a condensed chromatin, which is maintained by a histone H1-like protein, Hc1 . Differentiation of elementary bodies to reticulate bodies is accompanied by dispersal of the chromatin as chlamydiae become transcriptionally active, although the mechanisms of Hc1 release from DNA have remained unknown . Dissociation of the nucleoid requires chlamydial transcription and translation with negligible loss of Hc1 . A genetic screen was therefore designed to identify chlamydial genes rescuing Escherichia coli from the lethal effects of Hc1 overexpression . CT804, a gene homologous to ispE, which encodes an intermediate enzyme of the non-mevalonate methylerythritol phosphate (MEP) pathway of isoprenoid biosynthesis, was selected . E . coli coexpressing CT804 and Hc1 grew normally, although they expressed Hc1 to a level equivalent to that which condensed the chromatin of parent Hc1-expressing controls . Inhibition of the MEP pathway with fosmidomycin abolished IspE rescue of Hc1-expressing E . coli . Deproteinated extract from IspE-expressing bacteria caused dispersal of purified chlamydial nucleoids, suggesting that chlamydial histone-DNA interactions are disrupted by a small metabolite within the MEP pathway rather than by direct action of IspE . By partial reconstruction of the MEP pathway, we determined that 2-C-methylerythritol 2,4-cyclodiphosphate dissociated Hc1 from chlamydial chromatin . These results suggest that chlamydial histone-DNA interactions are disrupted upon germination by a small metabolite in the MEP pathway of isoprenoid biosynthesis.

J Biol Chem, 2004 Jul 2, 279(27), 28515 - 21 Epub 2004 Apr 27.
The residue 129 polymorphism in human prion protein does not confer susceptibility to Creutzfeldt-Jakob disease by altering the structure or global stability of PrPC; Hosszu LL et al.; There are two common forms of prion protein (PrP) in humans, with either methionine or valine at position 129 . This polymorphism is a powerful determinant of the genetic susceptibility of humans toward both sporadic and acquired forms of prion disease and restricts propagation of particular prion strains . Despite its key role, we have no information on the effect of this mutation on the structure, stability, folding, and dynamics of the cellular form of PrP (PrP(C)) . Here, we show that the mutation has no measurable effect on the folding, dynamics, and stability of PrP(C) . Our data indicate that the 129M/V polymorphism does not affect prion propagation through its effect on PrP(C); rather, its influence is likely to be downstream in the disease mechanism . We infer that the M/V effect is mediated through the conformation or stability of disease-related PrP (PrP(Sc)) or intermediates or on the kinetics of their formation.

J Biol Chem, 2004 Jul 16, 279(29), 30514 - 22 Epub 2004 Apr 27.
Structural basis for coronavirus-mediated membrane fusion . Crystal structure of mouse hepatitis virus spike protein fusion core; Xu Y et al.; The surface transmembrane glycoprotein is responsible for mediating virion attachment to cell and subsequent virus-cell membrane fusion . However, the molecular mechanisms for the viral entry of coronaviruses remain poorly understood . The crystal structure of the fusion core of mouse hepatitis virus S protein, which represents the first fusion core structure of any coronavirus, reveals a central hydrophobic coiled coil trimer surrounded by three helices in an oblique, antiparallel manner . This structure shares significant similarity with both the low pH-induced conformation of influenza hemagglutinin and fusion core of HIV gp41, indicating that the structure represents a fusion-active state formed after several conformational changes . Our results also indicate that the mechanisms for the viral fusion of coronaviruses are similar to those of influenza virus and HIV . The coiled coil structure has unique features, which are different from other viral fusion cores . Highly conserved heptad repeat 1 (HR1) and HR2 regions in coronavirus spike proteins indicate a similar three-dimensional structure among these fusion cores and common mechanisms for the viral fusion . We have proposed the binding regions of HR1 and HR2 of other coronaviruses and a structure model of their fusion core based on our mouse hepatitis virus fusion core structure and sequence alignment . Drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation may be applied to the inhibition of a number of emerging infectious diseases, including severe acute respiratory syndrome.

J Biol Chem, 2004 Jul 2, 279(27), 28136 - 42 Epub 2004 May 03.
Antagonistic cross-talk between Rac and Cdc42 GTPases regulates generation of reactive oxygen species; Diebold BA et al.; Cross-talk between Rho GTPase family members (Rho, Rac, and Cdc42) plays important roles in modulating and coordinating downstream cellular responses resulting from Rho GTPase signaling . The NADPH oxidase of phagocytes and nonphagocytic cells is a Rac GTPase-regulated system that generates reactive oxygen species (ROS) for the purposes of innate immunity and intracellular signaling . We recently demonstrated that NADPH oxidase activation involves sequential interactions between Rac and the flavocytochrome b(558) and p67(phox) oxidase components to regulate electron transfer from NADPH to molecular oxygen . Here we identify an antagonistic interaction between Rac and the closely related GTPase Cdc42 at the level of flavocytochrome b(558) that regulates the formation of ROS . Cdc42 is unable to stimulate ROS formation by NADPH oxidase, but Cdc42, like Rac1 and Rac2, was able to specifically bind to flavocytochrome b(558) in vitro . Cdc42 acted as a competitive inhibitor of Rac1- and Rac2-mediated ROS formation in a recombinant cell-free oxidase system . Inhibition was dependent on the Cdc42 insert domain but not the Switch I region . Transient expression of Cdc42Q61L inhibited ROS formation induced by constitutively active Rac1 in an NADPH oxidase-expressing Cos7 cell line . Inhibition of Cdc42 activity by transduction of the Cdc42-binding domain of Wiscott-Aldrich syndrome protein into human neutrophils resulted in an enhanced fMetLeuPhe-induced oxidative response, consistent with inhibitory cross-talk between Rac and Cdc42 in activated neutrophils . We propose here a novel antagonism between Rac and Cdc42 GTPases at the level of the Nox proteins that modulates the generation of ROS used for host defense, cell signaling, and transformation.

J Biol Chem, 2004 Jul 16, 279(29), 30634 - 42 Epub 2004 May 03.
Crystal structure of PapA5, a phthiocerol dimycocerosyl transferase from Mycobacterium tuberculosis; Buglino J et al.; Polyketide-associated protein A5 (PapA5) is an acyltransferase that is involved in production of phthiocerol and phthiodiolone dimycocerosate esters, a class of virulence-enhancing lipids produced by Mycobacterium tuberculosis . Structural analysis of PapA5 at 2.75-A resolution reveals a two-domain structure that shares unexpected similarity to structures of chloramphenicol acetyltransferase, dihydrolipoyl transacetylase, carnitine acetyltransferase, and VibH, a non-ribosomal peptide synthesis condensation enzyme . The PapA5 active site includes conserved histidine and aspartic acid residues that are critical to PapA5 acyltransferase activity . PapA5 catalyzes acyl transfer reactions on model substrates that contain long aliphatic carbon chains, and two hydrophobic channels were observed linking the PapA5 surface to the active site with properties consistent with these biochemical activities and substrate preferences . An additional alpha helix not observed in other acyltransferase structures blocks the putative entrance into the PapA5 active site, indicating that conformational changes may be associated with PapA5 activity . PapA5 represents the first structure solved for a protein involved in polyketide synthesis in Mycobacteria.

J Biol Chem, 2004 Jul 2, 279(27), 28106 - 12 Epub 2004 Apr 29.
The hepatitis B virus X protein inhibits secretion of apolipoprotein B by enhancing the expression of N-acetylglucosaminyltransferase III; Kang SK et al.; The X protein of hepatitis B virus (HBx) plays a major role on hepatocellular carcinoma (HCC) . Apolipoprotein B (apoB) in the liver is an important glycoprotein for transportation of very low density lipoproteins and low density lipoproteins . Although lipid accumulation in the liver is known as one of the factors for the HCC, the relationship between HBx and apoB during the HCC development is poorly understood . To better understand the biological significance of HBx in HCC, liver Chang cells that specifically express HBx were established and characterized . In this study we demonstrate that overexpression of HBx significantly up-regulates the expression of UDP-N-acetylglucosamine:beta-d-mannoside-1,4-N-acetylglucosaminyltransferase-III (GnT-III), an enzyme that functions as a bisecting-N-acetylglucosamine (GlcNAc) transferase in apoB, and increases GnT-III promoter activity in a chloramphenicol acetyltransferase assay . GnT-III expression levels of HBx-transfected cells appeared to be higher than that of hepatocarcinoma cells as well as GnT-III-transfected cells, indicating that HBx may has a strong GnT-III promotor-enhancing activity . Intracellular levels of apoBs, which contained the increased bisecting GlcNAc, were accumulated in HBx-transfected liver cells . These cells as well as GnT-III-transfected liver cells revealed the inhibition of apoB secretion and the increased accumulation of intracellular triglyceride and cholesterol compared with vector-transfected cells . Moreover, overexpression of GnT-III and HBx in liver cells was shown to down-regulate the transcriptional level of microsomal triglyceride transfer protein, which regulates the assembly and secretion of apoB . Therefore, our study strongly suggested that the HBx increase in intracellular accumulation of aberrantly glycosylated apoB resulted in inhibition of secretion of apoB as well as intracellular lipid accumulation by elevating the expression of GnT-III.

J Mol Biol, 2004 May 21, 339(1), 243 - 50
Structural adaptation of the glycophorin A transmembrane homodimer to D-amino acid modifications; Gerber D et al.; Protein-protein recognition is an essential process in life . The chemistry of these kind of interactions is predominantly stereospecific (i.e . receptor-ligand, antibody-hapten binding) . Here, we investigated whether the hydrophobic nature of the membrane affects this stereospecificity . To this end, we synthesized a diastereomer analogue (2D-GPA) of the glycophorin A transmembrane helix, with two l-valine residues replaced by their d-enantiomer . This ensures a disruption of the secondary structure . We investigated the ability of the diastereomer peptide to recognize the GPA chimera in the ToxR homodimer reporting system, in vivo . The peptide demonstrated a dose-dependent dominant negative effect on the GPA transmembrane in the bacterial ToxR system, suggesting a wild-type like interaction . This result was corroborated in vitro by fluorescence energy transfer between 2D-GPA and all-l GPA . Peptide binding to the bacteria was confirmed through confocal imaging, and Western blot confirmed that ToxR GPA receptor levels are not affected by the presence of the exogenous peptide . In order to understand the structural basis for heterodimer formation, homodimer and heterodimer structures, based on the NMR 3D structure of GPA, were subjected to a molecular dynamics simulation . The resulting heterodimer structure maintained most of the original inter-helical interactions, and its structure is similar to that of the homodimer . We postulate that the need to satisfy all H-bonds can compensate for the structural strain induced by the presence of the d-amino acid residues.

J Mol Biol, 2004 May 21, 339(1), 199 - 205
Phi value analysis of an allosteric transition of GroEL based on a single-pathway model; Inobe T et al.; There are currently two contradictory models for the kinetics of the ATP-induced GroEL allosteric transition occurring around 20 microM ATP . One model, proposed by Horovitz et al . demonstrates the existence of two parallel pathways for the allosteric transition and an abrupt ATP-dependent switch from one pathway to the other . The other model, which was proposed by the present authors, shows no need to assume the parallel pathways, and a combination of the transition-state theory and the Monod-Wyman-Changeux model of allostery can explain the kinetics as well as the equilibrium of the transition . The discrepancy appears to be due to whether we regard the transition as reversible or irreversible . Thus, here we have investigated the reversibility of the allosteric transition between 0 microM and 70 microM ATP by the use of a stopped-flow double-jump technique, which has allowed us to monitor the kinetics of the reverse reaction from the relaxed state at a high ATP concentration to the tense state at a low ATP concentration . The tryptophan fluorescence of a tryptophan-inserted variant of GroEL was used to follow the kinetics . As a result, the allosteric transition was shown to be a reversible process, supporting the validity of our model . We also show that the structural environment around the ATP-binding site of GroEL in the transition state is very similar to that in the relaxed state (Phi=0.9) by using a Phi value analysis in the kinetic Monod-Wyman-Changeux model, which is analogous to the mutational Phi value analysis in protein folding.

J Mol Biol, 2004 May 21, 339(1), 89 - 102
Loop relaxation, a mechanism that explains the reduced specificity of rabbit 20alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily; Couture JF et al.; The aldo-keto reductase rabbit 20alpha-hydroxysteroid dehydrogenase (rb20alpha-HSD; AKR1C5) is less selective than other HSDs, since it exerts its activity both on androgens (C19 steroids) and progestins (C21 steroids) . In order to identify the molecular determinants responsible for this reduced selectivity, binary (NADPH) and ternary (NADP(+)/testosterone) complex structures were solved to 1.32A and 2.08A resolution, respectively . Inspection of the cofactor-binding cavity led to the identification of a new interaction between side-chains of residues His222 and Lys270, which cover the central phosphate chain of the cofactor, reminiscent of the "safety-belt" found in other aldo-keto reductases . Testosterone is stabilized by a phenol/benzene tunnel composed of side-chains of numerous residues, among which Phe54, which forces the steroid to take up an orientation markedly contrasting with that found in HSD ternary complexes reported . Combining structural, site-directed mutagenesis, kinetic and fluorescence titration studies, we found that the selectivity of rb20alpha-HSD is mediated by (i) the relaxation of loop B (residues 223-230), partly controlled by the nature of residue 230, (ii) the nature of the residue found at position 54, and (iii) the residues found in the C-terminal tail of the protein especially the side-chain of the amino acid 306.

J Mol Biol, 2004 May 21, 339(1), 53 - 66
Dynamics of DNA loop capture by the SfiI restriction endonuclease on supercoiled and relaxed DNA; Embleton ML et al.; The SfiI endonuclease is a prototype for DNA looping . It binds two copies of its recognition sequence and, if Mg(2+) is present, cuts both concertedly . Looping was examined here on supercoiled and relaxed forms of a 5.5 kb plasmid with three SfiI sites: sites 1 and 2 were separated by 0.4 kb, and sites 2 and 3 by 2.0 kb . SfiI converted this plasmid directly to the products cut at all three sites, though DNA species cleaved at one or two sites were formed transiently during a burst phase . The burst revealed three sets of doubly cut products, corresponding to the three possible pairings of sites . The equilibrium distribution between the different loops was evaluated from the burst phases of reactions initiated by adding MgCl(2) to SfiI bound to the plasmid . The short loop was favored over the longer loops, particularly on supercoiled DNA . The relative rates for loop capture were assessed after adding SfiI to solutions containing the plasmid and MgCl(2) . On both supercoiled and relaxed DNA, the rate of loop capture across 0.4 kb was only marginally faster than over 2.0 kb or 2.4 kb . The relative strengths and rates of looping were compared to computer simulations of conformational fluctuations in DNA . The simulations concurred broadly with the experimental data, though they predicted that increasing site separations should cause a shallower decline in the equilibrium constants than was observed but a slightly steeper decline in the rates for loop capture . Possible reasons for these discrepancies are discussed.

J Mol Biol, 2004 May 21, 339(1), 31 - 9
Polarity effects in the lactose operon of Escherichia coli; Li Y et al.; An intergenic RNA segment between lacY and lacA of the lactose operon in Escherichia coli is cleaved by RNase P, an endoribonuclease . The cleavage of the intergenic RNA was ten times less efficient than cleavage of a tRNA precursor in vitro . Fragments of the RNase P cleavage product are detectable in vivo in the wild-type strain but not in a mutant strain at the restrictive temperature . The cleavage product that contains lacA in the wild-type strain was quickly degraded . When this intergenic segment was cloned upstream of a reporter gene, the expression of the reporter gene was also inhibited substantially in wild-type E.coli, but not in a temperature sensitive mutant strain in RNase P at the restrictive temperature . These results support data regarding the natural polarity between lacZ versus lacA, the downstream gene.

Fish Shellfish Immunol, 2004 Feb, 16(2), 139 - 49
A complement C3 fragment equivalent to mammalian C3d from the common carp (Cyprinus carpio): generation in serum after activation of the alternative pathway and detection of its receptor on the lymphocyte surface; Nakao M et al.; A terminal degradation product (C3d) of mammalian complement component C3 plays an important role in modulation of the adaptive immune response through the interaction with complement receptor type 2 (CR2) on B cells . The present study is aimed at determining whether this is a functional bridge between the innate and adaptive immune systems in bony fish . The fragmentation of the complement component C3 in carp (Cyprinus carpio) serum, activated with zymosan, was analysed to ascertain if carp C3 also generates a mammalian C3d-like fragment under physiological conditions . A 35 kDa peptide reactive to the anti-carp C3 alpha-chain was detected on the zymosan particles and in the activated serum . Its N-terminal amino acid sequence identified it as carp C3d derived from the C3-H1 isoform . Another C3 isoform, C3-S, of carp was found to yield a C3d fragment at lower efficiency than C3-H1 . Recombinant C3d fragments derived from C3-H1 and C3-S were produced in Escherichia coli as fusion proteins with glutathione-S-transferase (GST), and used for ligands to examine the presence of a possible CR2-like C3d receptor on carp lymphocytes . An enzyme-linked immunoadsorbent assay system, using the recombinant C3d proteins and anti-GST on a microplate to which was attached carp peripheral lymphocytes, detected a significant binding of carp C3d to the lymphocyte . The degree of binding of C3-H1-derived C3d was higher than that of C3-S-derived C3d . In addition, the binding of both ligands was inhibited by anti-C3 alpha-chain, but not by EDTA or EGTA, indicating that the putative C3d receptor does not require divalent cation . These properties agree well with those reported for mammalian CR2.

Fish Shellfish Immunol, 2004 Mar, 16(3), 453 - 9
Analysis and characterisation of IL-1beta processing in rainbow trout, Oncorhynchus mykiss; Hong S et al.; Mammalian IL-1beta is produced as a biologically inactive 31 kDa precursor, which is converted to the active 18 kDa form by proteolytic processing . Synthesis and processing of native piscine IL-1beta is poorly understood . In the present study, the native IL-1beta precursor or mature peptides were detected at sizes of approx . 29 kDa and 24 kDa in cell lysates of a rainbow trout macrophage cell line RTS-11, with or without LPS stimulation, by Western blot analysis using a polyclonal antibody against the putative trout mature IL-1beta (rmIL-1beta) produced in Escherichia coli . Processing of the 29 kDa precursor into a 24 kDa mature peptide was confirmed by analysis of such proteins using a monoclonal conjugate (Ni-NTA-HRP) against 6 histidines in lysates of the RTS-11 cells transfected with an expression plasmid containing the IL-1beta precursor molecule tagged with 6 histidines at its C terminus . Only the recombinant mature 24 kDa) IL-1beta/HIS protein was purified from the culture supernatants of the transfected cells, indicating the molecule is cleaved to be secreted . These findings strongly suggest that the trout IL-1beta molecule is processed in trout macrophages in an analogous way to the situation with mammalian IL-1beta despite the lack of a clear ICE cut site.

Chem Biol, 2004 Mar, 11(3), 327 - 35
Mass spectrometric interrogation of thioester-bound intermediates in the initial stages of epothilone biosynthesis; Hicks LM et al.; Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS) . Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC . Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.0 Da mass shift consistent with the formation of the methylthiazolyl-methacrylyl product by incorporation of acetyl-CoA, cysteine, and methylmalonyl-CoA . Thioester-templated reaction intermediates and products are typically characterized by quantifying radioactive substrates, either enzyme bound or chemically hydrolyzed . In contrast, the MS-based methodology described here provides semiquantifiable ratios of free enzyme, intermediate, and product occupancy and reveals that certain substrates result in a >50% formation of nonproductive intermediates.

Biochemistry, 2004 May 11, 43(18), 5334 - 40
Perforation of the tunnel wall in carbamoyl phosphate synthetase derails the passage of ammonia between sequential active sites; Kim J et al.; Carbamoyl phosphate synthetase (CPS) from Escherichia coli consists of a small subunit (approximately 42 kDa) and a large subunit (approximately 118 kDa) and catalyzes the biosynthesis of carbamoyl phosphate from MgATP, bicarbonate, and glutamine . The enzyme is able to utilize external ammonia as an alternative nitrogen source when glutamine is absent . CPS contains an internal molecular tunnel, which has been proposed to facilitate the translocation of reaction intermediates from one active site to another . Ammonia, the product from the hydrolysis of glutamine in the small subunit, is apparently transported to the next active site in the large subunit of CPS over a distance of about 45 A . The ammonia tunnel that connects these two active sites provides a direct path for the guided diffusion of ammonia and protection from protonation . Molecular damage to the ammonia tunnel was conducted in an attempt to induce leakage of ammonia directly to the protein exterior by the creation of a perforation in the tunnel wall . A hole in the tunnel wall was made by mutation of integral amino acid residues with alanine residues . The triple mutant alphaP360A/alphaH361A/betaR265A was unable to utilize glutamine for the synthesis of carbamoyl phosphate . However, the mutant enzyme retained full catalytic activity when external ammonia was used as the nitrogen source . The synchronization of the partial reactions occurring at the three active sites observed with the wild-type CPS was seriously disrupted with the mutant enzyme when glutamine was used as a nitrogen source . Overall, the catalytic constants of the mutant were consistent with the model where the channeling of ammonia has been disrupted due to the leakage from the ammonia tunnel to the protein exterior.

Biochemistry, 2004 May 11, 43(18), 5324 - 33
The catalytic subunit of Escherichia coli nitrate reductase A contains a novel {4Fe-4S} cluster with a high-spin ground state; Rothery RA et al.; We have used EPR spectroscopy, redox potentiometry, and protein crystallography to characterize the {4Fe-4S} cluster (FS0) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG) . FS0 is clearly visible in the crystal structure of NarGHI {Bertero, M . G., et al . (2003) Nat . Struct . Biol . 10, 681-687} but has novel coordination comprising one His residue and three Cys residues . At low temperatures (<15 K), reduced NarGHI exhibits a previously unobserved EPR signal comprising peaks at g = 5.023 and g = 5.556 . We have assigned these features to a {4Fe-4S}(+) cluster with an S = (3)/(2) ground state, with the g = 5.023 and g = 5.556 peaks corresponding to subpopulations exhibiting DeltaS = (1)/(2) and DeltaS = (3)/(2) transitions, respectively . Both peaks exhibit midpoint potentials of approximately -55 mV at pH 8.0 and are eliminated in the EPR spectrum of apomolybdo-NarGHI . The structure of apomolybdo-NarGHI reveals that FS0 is still present but that there is significant conformational disorder in a segment of residues that includes one of the Cys ligands . On the basis of these observations, we have assigned the high-spin EPR features of reduced NarGHI to FS0.

Biochemistry, 2004 May 11, 43(18), 5296 - 303
Conformational change of the dimeric DsbC molecule induced by GdnHCl . A study by intrinsic fluorescence; Stepanenko OV et al.; Unfolding-refolding of Escherichia coli DsbC, a homodimeric molecule, induced by GdnHCl was studied by intrinsic fluorescence . Interpretation of experimental fluorescence data was done together with the analysis of protein 3D structure . It is shown that although Cys 141 is the next neighbor of the single tryptophan residue (Trp 140), the sulfur atoms of the disulfide bond Cys 141-Cys 163 are far apart from the indole ring and cannot quench its fluorescence, while the potential quenchers are Met 136 and His 170 . It was revealed that though each subunit of DsbC contains eight tyrosine residues, only three tyrosine residues (Tyr 171, Tyr 38, and Tyr 52) contribute to the bulk fluorescence of the molecule . The character of intrinsic fluorescence intensity changes induced by GdnHCl (equilibrium and kinetic data) and its parametric representation, the existence of an isosbestic point of fluorescence spectra at different GdnHCl concentrations, allowed suggesting a one-step character of DsbC denaturation and its reversibility.

Hepatology, 2004 May, 39(5), 1297 - 302
Murine alanine aminotransferase: cDNA cloning, functional expression, and differential gene regulation in mouse fatty liver; Jadhao SB et al.; Alanine aminotransferase (ALT) is a widely used index of liver integrity or hepatocellular damage in clinics as well as a key enzyme in intermediatary metabolism . In this study, we have cloned the complementary DNAs of murine homologues of human alanine aminotransferase 1 and 2 (ALT1 and ALT2) . The deduced peptides of murine ALT1 (mALT1) and ALT2 (mALT2) share 87% and 93% identity, respectively, with their human counterparts at the amino acid level . Murine ALT genes localize to separate chromosomes, with mALT1 gene (gpt1) on chromosome 15 and mALT2 gene (gpt2) on chromosome 8 . The murine gpt1 and gpt2 differ in messenger RNA expression: gpt1 is mainly expressed in liver, bowel, and white adipose tissue and gpt2 is highly expressed in muscle, liver, and white adipose tissue . Expression of recombinant mALT1 and mALT2 proteins in Escherichia coli (E . coli) produced functional enzymes that catalyze alanine transamination . The potential diagnostic value of ALT isoenzymes in liver disease was evaluated in an obese animal model . In fatty livers of obese mice, ALT2 gene expression is induced 2-fold, but ALT1 remains the same . Furthermore, in fatty liver, total hepatic ALT activity is elevated significantly by 30% whereas aspartate aminotransferase (AST) activity remains unchanged . In conclusion, these results indicate that ALT2 may be responsible for the increased ALT activity in hepatic steatosis and provide evidence that an ALT isoenzyme-specific assay may have more diagnostic value than the total ALT activity assay currently in clinical use.

Nat Struct Mol Biol, 2004 Jun, 11(6), 551 - 7 Epub 2004 May 02.
The sigma 70 subunit of RNA polymerase induces lacUV5 promoter-proximal pausing of transcription; Brodolin K et al.; The sigma(70) subunit of Escherichia coli RNA polymerase (RNAP) is a transcription initiation factor that can also be associated with RNAP during elongation . We provide biochemical evidence that sigma(70) induces a transcription pause at the lacUV5 promoter after RNAP has synthesized a 17-nucleotide transcript . The sigma(70)-dependent pausing requires an interaction between sigma(70) and a part of the lac repressor operator sequence resembling a promoter -10 consensus . The polysaccharide heparin triggers the release of sigma(70) from the paused complexes, supporting the view that during the transition from initiation to elongation the interactions between sigma(70) and core RNAP are weakened . We propose that the binding and retention of sigma(70) in elongation complexes are stabilized by its ability to form contacts with DNA of the transcription bubble . In addition, we suggest that the sigma(70) subunit in the elongation complex may provide a target for regulation of gene expression.

Mol Biotechnol, 2004 May, 27(1), 7 - 14
Separate control of Rep and Cap expression using mutant and wild-type loxP sequences and improved packaging system for adeno-associated virus vector production; Mizukami H et al.; Adeno-associated virus (AAV) vectors are a practical choice for gene transfer, and demand for them is increasing . To cope with the necessity in the near future, we have developed a number of approaches to establish packaging cell lines for the production of AAV vectors . In our previous study, a highly regulated expression of large Rep proteins was obtained by using the Cre-loxP switching system . Therefore, in the present study, to regulate Cap expression as well, we developed an inducible expression system for both Rep and Cap proteins by using an additional set of mutant loxP sequences . The mutants possess two base alterations in the spacer region of loxP and recombine specifically with the same counterpart in the presence of Cre . By using two separate plasmids, one with mutant and the other with wild-type loxP sequences, the expression of two different proteins can be induced simultaneously by Cre recombinase . When the LacZ-encoding plasmid vector was used as a packaging model, a significant packaging titer of 2.1 x 1010 genome copies per 10-cm dish was obtained . These results indicate the importance of controlling Cap expression, in addition to Rep, to achieve an optimum production rate for AAV vectors.

Plant Physiol, 2004 May, 135(1), 95 - 102 Epub 2004 Apr 30.
The key role of phloroglucinol O-methyltransferase in the biosynthesis of Rosa chinensis volatile 1,3,5-trimethoxybenzene; Wu S et al.; 1,3,5-Trimethoxybenzene is a key component of the Chinese rose odor . This compound is synthesized in three successive methylation steps from phloroglucinol, the initial precursor . A novel, to our knowledge, phloroglucinol O-methyltransferase (POMT) characterized here methylates the first step to produce the intermediate 3,5-dihydroxyanisole, while two previously described orcinol O-methyltransferases catalyze the subsequent steps . We isolated POMT from rose petals and determined partial amino acid sequences of the purified enzyme . The full-length POMT cDNA was isolated and expressed in Escherichia coli . Both the native and recombinant POMT exhibited substrate specificity for phloroglucinol . POMT was expressed specifically in floral organs, in accordance with its role as a key enzyme in the synthesis of rose floral scent compounds.

Plant Physiol, 2004 May, 135(1), 137 - 44 Epub 2004 Apr 30.
Both subunits of ADP-glucose pyrophosphorylase are regulatory; Cross JM et al.; The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis . Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate . The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation . We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective . Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes . Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties . Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation . The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase . Alterations in the small subunit condition drastically different allosteric properties . In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.

Nucleic Acids Res, 2004 Apr 30, 32(8), 2430 - 40 Print 2004.
Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D; Jokela M et al.; The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related . The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively . Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily . Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3'-5' proofreading exonuclease activity of these enzymes . To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn2+-dependent 3'-5' exonuclease . The putative polymerase subunit DP2 is not required . The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity . MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.

J Biotechnol, 2004 May 27, 110(2), 137 - 42
Rational affinity purification of native Streptomyces family 10 xylanase; Ito S et al.; Xylanase SoXyn10A from Streptomyces olivaceoviridis E-86 comprises a family 10 catalytic module linked to a family 13 carbohydrate-binding module (SoCBM13) . The SoCBM13 has a beta-trefoil structure, with binding sites in each subdomain (alpha, beta and gamma) . Subdomain alpha, but not subdomains beta and gamma, binds tightly to lactose . It was, therefore, thought that immobilized lactose could be used for the affinity purification of SoXyn10A . Lactosyl-Sepharose was prepared and tested as an affinity matrix . SoXyn10A produced from the cloned xyn10A gene by Escherichia coli, and native SoXyn10A in culture supernatants from S . olivaceoviridis, were purified to homogeneity in a single step by affinity chromatography using this matrix . This simple purification of SoXyn10A makes the enzyme an attractive candidate for applications requiring xylanase . The CBM also has the potential for use as an affinity tag for the purification of other proteins.

J Am Soc Mass Spectrom, 2004 May, 15(5), 647 - 53
An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins; Hegeman AD et al.; A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins . The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phosphatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d(4)), is described in detail . Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chromatography-mass spectrometry (microLC-MS) using a Micromass Q-TOF2 mass spectrometer . Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy . The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorylated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within approximately 20%).

Res Vet Sci, 2004 Aug, 77(1), 89 - 91
Plasma intestinal fatty acid binding protein (I-FABP) concentrations increase following intestinal ischemia in pigs; Niewold TA et al.; Intestinal fatty acid binding protein (I-FABP) is an intracellular epithelial protein in the intestinal mucosa of many animals . I-FABP appears in the circulation following epithelial damage, and in humans, is proven to be a parameter for damage to the mucosa . In this paper, an ELISA test designed for human I-FABP analysis was used to assay pig blood samples . The test recognized I-FABP cloned from pig small intestine and expressed in Escherichia coli . Furthermore, in our experimental model of (low flow) intestinal ischemia and reperfusion a significant rise in plasma I-FABP concentrations 15-30 min after clamping of the mesenteric artery was demonstrated . This is the first report that in pigs circulating I-FABP is a useful marker for (mild) intestinal injury, and could possibly be used to monitor (intestinal) health in clinical practice.

Res Vet Sci, 2004 Aug, 77(1), 17 - 21
traT and CNF2 genes of Escherichia coli isolated from milk of healthy cows and sheep; Acik MN et al.; The objectives of the present study were to isolate Escherichia coli from milk of apparently healthy cows and sheep and to investigate the presence of traT and cytotoxic necrotising factor-2 (CNF2) virulence genes by multiplex polymerase chain reaction (PCR) . Milk samples collected from a total of 1028 apparently healthy ruminants (737 cows and 291 sheep) in eastern Turkey were streaked onto 5% sheep-blood agar . E . coli was isolated and identified by biochemical tests in 5.9% (61/1028) of milk samples . Correct amplification with the molecular length of 232 bp was obtained from all E . coli isolates by the species-specific PCR . The isolation rates of the agent were calculated to be 7.6% (56/737) in cows and 1.7% (5/291) in sheep . The difference between these proportions was statistically significant (p < 0.001) . Multiplex PCR showed that traT and CNF2 genes were present in 62.3% and 6.6% of all isolates, respectively . Both genes were present in 16.4% of the isolates, with only 14.7% having neither gene.

Biochem Biophys Res Commun, 2004 May 28, 318(2), 601 - 14
Analyzing and enhancing mRNA translational efficiency in an Escherichia coli in vitro expression system; Voges D et al.; The dependence of efficiency of translation initiation on mRNA sequence parameters was investigated in an Escherichia coli in vitro expression system . We designed a large-scale expression experiment focussing on the influence of sequence variations in the translated region (TR) of the mRNA without changing the 5'-untranslated region (5'-UTR) . The level of translated protein from 756 expression constructs was measured and the influence of a large number of possible effector attributes was statistically analyzed . Base exchanges immediately adjacent to the start codon up to nucleotide (+)25 had a profound effect on translational efficiency . Correlation analysis revealed a significant dependence on base pair probability and G+C content on the expression level, indicating that mRNA secondary structure in this region hampers translation . Using our training data, we developed a methodology to predict and improve the translation efficiency of open reading frames (ORFs).

Biochem Biophys Res Commun, 2004 May 28, 318(2), 507 - 13
The effect of variable domain orientation and arrangement on the antigen-binding activity of a recombinant human bispecific diabody; Lu D et al.; In recent years a variety of recombinant methods have been developed for efficient production of bispecific antibodies (BsAb) in various formats . Bispecific diabody (bDAb), a 55-60 kDa molecule comprising two non-covalently associated cross-over single chain Fv (scFv) polypeptides, represents one of the most promising as well the most straightforward approaches to BsAb production . Here we constructed a bDAb, using two human scFv, 11F8 and A12, directed against the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR), respectively, as the building blocks . A total of 8 scFv and diabody constructs were prepared comprising the same two variable heavy (V(H)) and variable light (V(L)) chain domains but arranged in different orientations . V(H)/V(L) orientation, i.e., V(H)-linker-V(L) or V(L)-linker-V(H), showed significant effects on the expression and antigen-binding activity of scFv and monospecific diabody of both 11F8 and A12 . Further, only 2 out of the 4 possible V(H)/V(L) orientations/arrangements in bDAb construction yielded active products that retain binding activity to both EGFR and IGFR . Both active bDAb preparations retained their original antigen-binding activity after incubation at 37 degrees C in mouse serum for up to 7 days, indicating excellent stability of the constructs . Taken together, our results underscore the importance of identifying/selecting optimal V(H)/V(L) orientation/arrangement for efficient production of active bDAb.

Biochem Biophys Res Commun, 2004 May 28, 318(2), 470 - 6
Crystal structure of the glutaredoxin-like protein SH3BGRL3 at 1.6 Angstrom resolution; Nardini M et al.; We report the 1.6 Angstrom resolution crystal structure of SH3BGRL3, a member of a new mammalian protein family of unknown function . The observed "thioredoxin fold" of SH3BGRL3 matches the tertiary structure of glutaredoxins, even in the N-terminal region where the sequence similarity between the two protein families is negligible . In particular, SH3BGRL3 displays structural modifications at the N-terminal Cys-x-x-Cys loop, responsible for glutathione binding and catalysis in glutaredoxins . The loop hosts a six residue insertion, yielding an extra N-terminal-capped helical turn, first observed here for the thioredoxin fold . This, together with deletion of both Cys residues, results in a substantial reshaping of the neighboring cleft, where glutathione is hosted in glutaredoxins . While not active in redox reaction and glutathione binding, SH3BGRL3 may act as an endogenous modulator of glutaredoxin activities by competing, with its fully conserved thioredoxin fold, for binding to yet unknown target proteins.

Biochem Biophys Res Commun, 2004 May 28, 318(2), 453 - 60
Human neuroglobin interacts with flotillin-1, a lipid raft microdomain-associated protein; Wakasugi K et al.; Neuroglobin (Ngb) is a newly discovered vertebrate globin that is expressed in the brain and that can reversibly bind oxygen . It has been reported that Ngb levels increase in neurons in response to oxygen deprivation, and that it protects neurons from hypoxia . However, the mechanism of this neuroprotection remains unclear . Recently, we found that oxidized human Ngb bound to the alpha-subunits of heterotrimeric G proteins (Galpha) and acted as a guanine nucleotide dissociation inhibitor for Galpha . To identify other Ngb-binding proteins, we herein screened a human brain cDNA library by using a yeast two-hybrid system . Among the plasmids isolated from positive clones, one contained an insert with 100% sequence identity to human flotillin-1 . The interaction of Ngb with flotillin-1 was confirmed by glutathione S-transferase pull-down experiments . Since Galpha exists within lipid rafts critical for signal transduction and flotillin-1 recruits signaling proteins to lipid rafts, flotillin-1 might recruit Ngb to lipid rafts as a means of preventing neuronal death.

Biochem Biophys Res Commun, 2004 May 28, 318(2), 391 - 6
Human programmed cell death 5 protein has a helical-core and two dissociated structural regions; Liu D et al.; Programmed cell death 5 (PDCD5) protein is phylogenetically conserved in both the nucleus and cytoplasm . The human PDCD5 protein is expressed in tumor cells during apoptosis independent of the apoptosis-inducing stimuli, and recently it was found that PDCD5 is an important regulator in both apoptotic and non-apoptotic programmed cell death . In this study, human PDCD5 was expressed in Escherichia coli cell and studied using heteronuclear NMR method . The NMR results indicate that PDCD5 protein can be divided into three structural regions, a core region and two dissociated terminal regions . The core region (41-101) represents a rigid sub-domain consisting mainly of a triple-helix bundle . The N-terminal 38 residues (3-40) are ordered, but not a rigid structural region which contains abundant secondary structure, and packs very loosely against the core . The C-terminal 17 residues (102-118) represent a mobile unstructured region, which may be capable of interaction with nucleic acid.

J Steroid Biochem Mol Biol, 2004 Mar, 88(3), 235 - 45
Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex; Lala P et al.; Human cytochrome P450 aromatase (P450arom) is responsible for biosynthesis of estrogens from androgens . Monoclonal antibody MAb3-2C2 to P450arom specifically binds to a conformational epitope and suppresses the enzyme activity in a dose-dependent manner . The crystal structure of the Fab fragment of MAb3-2C2 has been used to engineer a recombinant single chain antibody fragment (scFv) and a homodimeric variable domain of the light chain (VL(2)) . These recombinant antibody fragments have been expressed in Escherichia coli and purified . Here, we show that the recombinant scFv suppresses P450arom activity with an IC(50) value similar to that of natural MAb3-2C2 F(ab')(2) . The recombinant VL(2) also exhibits dose-dependent suppression of the P450arom activity, but at a reduced level, demonstrating that the homodimer is unable to fully mimic the complementarity determining region (CDR) of a variable heavy chain (VH)-VL heterodimer . We prepare and purify a stable complex of P450arom with MAb3-2C2 F(ab')(2) and show that the complex migrates and precipitates as a single molecular assembly . Efforts to crystallize P450arom for structure-function studies have yielded small single crystals . Our results suggest that formation of stable complexes with fragments of the monoclonal antibody could provide an alternative method for crystallization of P450arom.

Microb Pathog, 2004 Jun, 36(6), 319 - 25
Loss of lipopolysaccharide receptor CD14 from the surface of human macrophage-like cells mediated by Porphyromonas gingivalis outer membrane vesicles; Duncan L et al.; Porphyromonas gingivalis, the major etiologic agent of chronic periodontitis, produces a broad spectrum of virulence factors, including outer membrane vesicles . In this study, we investigated the capacity of P . gingivalis vesicles to promote the shedding or cleavage of the lipopolysaccharide (LPS) receptor CD14 from the surface of human U937 macrophage-like cells . SDS-PAGE/Western immunoblotting analysis of gingival crevicular fluid samples from patients affected by moderate or advanced periodontitis revealed the presence of soluble CD14 and CD14 fragments, thus supporting the hypothesis of an in vivo shedding and cleavage of CD14 receptors . Flow cytometry analysis of macrophage-like cells treated with a vesicle-containing culture supernatant of P . gingivalis showed a significant decrease in the binding of anti-human CD14 to the cell surface . However, no accumulation of soluble CD14 or immunoreactive CD14 fragments in the assay supernatant could be demonstrated by ELISA . Treatment of macrophage-like cells with various concentrations of P . gingivalis vesicles substantially suppressed TNF-alpha production triggered by Escherichia coli LPS . This suppressive effect was much less important using heat-treated vesicles or in the presence of leupeptin, a gingipain inhibitor, during the treatment . Recombinant human CD14 receptors were found to be susceptible to proteolytic degradation by P . gingivalis vesicles . A purified Arg-gingipain preparation produced much more degradation than a Lys-gingipain preparation . This study provides evidence that P . gingivalis outer membrane vesicles contribute to the loss of membrane-bound CD14 receptors and that gingipains degrade this LPS receptor . Such a phenomenon, which results in an hyporesponsiveness of macrophages to LPS stimulation, may contribute to an increased capacity of P . gingivalis, and other periodontopathogens, to evade the host immune system mechanisms.

Plant Physiol Biochem, 2004 Apr, 42(4), 291 - 7
Cloning and over-expression of a cDNA encoding a polyketide synthase from Cannabis sativa; Raharjo TJ et al.; A polyketide synthase has been suggested to play an important role in cannabinoid biosynthesis in Cannabis sativa L . This enzyme catalyzes the biosynthesis of olivetolic acid, one of the precursors for cannabinoid biosynthesis . Using a reverse transcriptase-polymerase chain reaction (RT-PCR) based on the DNA homology of chalcone synthase (EC 2.3.1.156) and valerophenone synthase (EC 2.3.1.156) of hop (Humulus lupulus), a cDNA encoding a polyketide synthase in C . sativa was identified . The coding region of the gene is 1170 bp long encoding a 389 amino acid protein of a predicted 42.7 kDa molecular mass and with a pI of 6.04 . The gene shares a high homology with a chalcone synthase gene of H . lupulus, 85% and 94% homology on the level of DNA and protein, respectively . Over-expression of the construct in Escherichia coli M15 resulted in a 45 kDa protein . The protein has chalcone synthase activity as well as valerophenone synthase activity, a chalcone synthase-like activity . Using n-hexanoyl-CoA and malonyl-CoA as substrates did not give olivetol or olivetolic acid as a product.

J Nanobiotechnology . 2004 Apr 30;2(1):4.
Simultaneous measurement of sensor-protein dynamics and motility of a single cell by on-chip microcultivation system; Inoue I et al.; Measurement of the correlation between sensor-protein expression, motility and environmental change is important for understanding the adaptation process of cells during their change of generation . We have developed a novel assay exploiting the on-chip cultivation system, which enabled us to observe the change of the localization of expressed sensor-protein and the motility for generations . Localization of the aspartate sensitive sensor protein at two poles in Escherichia coli decreased quickly after the aspartate was added into the cultivation medium . However, it took more than three generations for recovering the localization after the removal of aspartate from the medium . Moreover, the tumbling frequency was strongly related to the localization of the sensor protein in a cell . The results indicate that the change of the spatial localization of sensor protein, which was inherited for more than three generations, may contribute to cells, motility as the inheritable information.

Biosci Biotechnol Biochem, 2004 Apr, 68(4), 939 - 41
In vivo bioconversion of tetrahydroisoquinoline by recombinant coclaurine N-methyltransferase; Morishige T et al.; Coclaurine N-methyltransferase from Coptis japonica catalyzes the N-methylation of coclaurine as well as simple tetrahydroisoquinoline . We examined the possibility of converting 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline into its N-methylated product using transgenic Escherichia coli, which expressed recombinant coclaurine N-methyltransferase, without the addition of a methyl-group donor . Transgenic E . coli successfully N-methylated the substrate added to the medium and excreted the product . Limitation of bioconversion by the supply of methyl-group donor is discussed.

Biosci Biotechnol Biochem, 2004 Apr, 68(4), 841 - 7
Involvement of tyrosines at fucose-binding sites of Aleuria aurantia lectin: non-equal response to site-directed mutagenesis among five sites; Amano K et al.; Since the involvement of Tyr residues in the fucose-binding of Aleuria aurantia lectin (AAL) was proved by chemical modification using the Tyr-specific reagent tetranitromethane, site-directed mutagenesis was attempted . Since the tertiary structure of AAL was determined recently to be a six-bladed beta-propeller fold, and five fucose-binding sites per subunit were found, based on positions of Tyr residues in the tertiary structure, three classes of mutants were constructed: 1) Tyr on the 2nd beta-strand of each blade (beta-2 mutants), 2) Tyr or Trp on the 3rd beta-strand (beta-3 mutants), and 3) Tyr outside of binding sites (other-Y mutants) . The mutagenized cDNA was expressed in Escherichia coli as His-tag-AAL, and the hemagglutinating activity was assayed . Among 14 mutants, three beta-2 mutants (Y26A, Y79A, and Y181A), and three beta-3 mutants (Y92A, W149A, and Y241A) showed decreased activity . These mutated residues resided at Sites 1, 2, and 4, at the same locations relatively in the binding sites . Mutagenesis of Tyr or Trp at the corresponding locations in Sites 3 and 5 did not lead to a reduction in activity . Results indicate that the properties of Sites 1, 2, and 4 are different from those of Sites 3 and 5, and that the contribution of these two sites to the hemagglutination reaction was minor.

Biosci Biotechnol Biochem, 2004 Apr, 68(4), 835 - 40
In vivo and in vitro interactions of the Bombyx mori chymotrypsin inhibitor b1 with Escherichia coli; He N et al.; Various chymotrypsin inhibitors occur in the hemolymph of silkworm larvae . Interaction of chymotrypsin inhibitor b1 (CI-b1) with Escherichia coli was examined from the viewpoint of action against invading bacteria . Injection of dead E . coli cells into larva reduced the CI-b1 content of the hemolymph, suggesting in vivo binding of CI-b1 to the outer membrane of the cell . Results from incubation of E . coli in cell-free hemolymph in the presence or absence of lipopolysaccharide indicated that CI-b1 is the only CI bound to E . coli and that it interacts with lipopolysaccharide . CI-b1 formed a complex with lipopolysaccharide in vitro; the value of the dissociation constant was relatively large . Inhibitory activity of CI-b1 changed insignificantly in mixture with lipopolysaccharide . CI-b1 affected the growth of E . coli but never worked lethally . CI-b1 is speculated to be a mediator that scavenges intruding bacteria rather than a direct anti-bacterial factor . This is the first report confirming that CI-b1 is a lipopolysaccharide binding protein.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 6969 - 74 Epub 2004 Apr 26.
Dual architectural roles of HU: formation of flexible hinges and rigid filaments; van Noort J et al.; The nucleoid-associated protein HU is one of the most abundant proteins in Escherichia coli and has been suggested to play an important role in bacterial nucleoid organization and regulation . Although the regulatory aspects of HU have been firmly established, much less is understood about the role of HU in shaping the bacterial nucleoid . In both functions (local) modulation of DNA architecture seems an essential feature, but information on the mechanical properties of this type of sequence-independent nucleoprotein complex is scarce . In this study we used magnetic tweezers and atomic force microscopy to quantify HU-induced DNA bending and condensation . Both techniques revealed that HU can have two opposing mechanical effects depending on the protein concentration . At concentrations <100 nM, individual HU dimers induce very flexible bends in DNA that are responsible for DNA compaction up to 50% . At higher HU concentrations, a rigid nucleoprotein filament is formed in which HU appears to arrange helically around the DNA without inducing significant condensation.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 7011 - 6 Epub 2004 Apr 26.
Directed evolution of protein enzymes using nonhomologous random recombination; Bittker JA et al.; We recently reported the development of nonhomologous random recombination (NRR) as a method for nucleic acid diversification and applied NRR to the evolution of DNA aptamers . Here, we describe a modified method, protein NRR, that enables proteins to access diversity previously difficult or impossible to generate . We investigated the structural plasticity of protein folds and the ability of helical motifs to function in different contexts by applying protein NRR and in vivo selection to the evolution of chorismate mutase (CM) enzymes . Functional CM mutants evolved using protein NRR contained many insertions, deletions, and rearrangements . The distribution of these changes was not random but clustered in certain regions of the protein . Topologically rearranged but functional enzymes also emerged from these studies, indicating that multiple connectivities can accommodate a functional CM active site and demonstrating the ability to generate new domain connectivities through protein NRR . Protein NRR was also used to randomly recombine CM and fumarase, an unrelated but also alpha-helical protein . Whereas the resulting library contained fumarase fragments in many contexts before functional selection, library members surviving selection for CM activity invariably contained a CM core with fumarase sequences found only at the termini or in one loop . These results imply that internal helical fragments cannot be swapped between these proteins without the loss of nearly all CM activity . Our findings suggest that protein NRR will be useful in probing the functional requirements of enzymes and in the creation of new protein topologies.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 6911 - 6 Epub 2004 Apr 26.
A mutant spacer sequence between -35 and -10 elements makes the Plac promoter hyperactive and cAMP receptor protein-independent; Liu M et al.; To determine whether the spacer region between the -35 and -10 elements plays any sequence-specific role, we randomized the GC-rich sequence ((-20)CCGGCTCG(-13)) within the spacer region of the cAMP-dependent lac promoter and selected an activator-independent mutant, which showed extraordinarily high intrinsic activity . The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the -15 sequence, which must be juxtaposed to the upstream end of the -10 sequence for the hyperactivity . The transcription enhancement functions only in the presence of a -35 element . The spacer sequence enhanced both RNA polymerase binding and open complex formation . Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters . Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the -18/-9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.

J Biol Chem, 2004 Jul 2, 279(27), 28243 - 50 Epub 2004 Apr 26.
Maltose-binding protein is open in the catalytic transition state for ATP hydrolysis during maltose transport; Austermuhle MI et al.; The maltose transport complex of Escherichia coli, a member of the ATP-binding cassette superfamily, mediates the high affinity uptake of maltose at the expense of ATP . The membrane-associated transporter consists of two transmembrane subunits, MalF and MalG, and two copies of the cytoplasmic ATP-binding cassette subunit, MalK . Maltose-binding protein (MBP), a soluble periplasmic protein, delivers maltose to the MalFGK(2) transporter and stimulates hydrolysis by the transporter . Site-directed spin labeling electron paramagnetic resonance spectroscopy is used to monitor binding of MBP to MalFGK(2) and conformational changes in MBP as it interacts with MalFGK(2) . Cysteine residues and spin labels have been introduced into the two lobes of MBP so that spin-spin interaction will report on ligand-induced closure of the protein (Hall, J . A., Thorgeirsson, T . E., Liu, J., Shin, Y . K., and Nikaido, H . (1997) J . Biol . Chem . 272, 17610-17614) . At least two different modes of interaction between MBP and MalFGK(2) were detected . Binding of MBP to MalFGK(2) in the absence of ATP resulted in a decrease in motion of spin label at position 41 in the C-terminal domain of MBP . In a vanadate-trapped transition state intermediate, all free MBP became tightly bound to MalFGK(2), spin label in both lobes became completely immobilized, and spin-spin interactions were lost, suggesting that MBP was in an open conformation . Binding of non-hydrolyzable MgATP analogs or ATP in the absence of Mg is sufficient to stabilize a complex of open MBP and MalFGK(2) . Taken together, these data suggest that closure of the MalK dimer interface coincides with opening of MBP and maltose release to the transporter.

J Chromatogr A, 2004 Apr 30, 1035(1), 83 - 6
Purification of recombinant green fluorescent protein by three-phase partitioning; Jain S et al.; The technique of three-phase partitioning (TPP) was used to purify the green fluorescent protein (GFP) in a single step . TPP uses a combination of ammonium sulphate and tert-butanol to precipitate proteins from their crude extracts . In the first round of TPP with 20% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:1 (v/v), most of the GFP remains in the lower aqueous phase . When subjected to a second round of TPP with 60% ammonium sulphate saturation at the ratio of crude to tert-butanol 1:2 (v/v) gives 78% recovery of GFP with a 20-fold purification . The sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of purified preparation shows single band . The fluorescence excitation and emission spectra agreed with values reported in literature.

J Chromatogr A, 2004 Apr 23, 1034(1-2), 155 - 9
Ion exchange using poorly activated supports, an easy way for purification of large proteins; Pessela BC et al.; Ion-exchange chromatography using commercial ionic supports is a commonly used technique for protein purification . However, selective adsorption of a target protein from a given extract onto commercial ion exchangers seems to be quite complex since they are designed to adsorb the maximum percentage of proteins with the opposite charge . In this paper, ion-exchanger supports with different activation degrees (from 1 to 40 micromol of amino groups per g of agarose) have been prepared and used for the purification of large proteins . These kinds of proteins have large surfaces to interact by many points with the support . Therefore, it was possible to purify large proteins as beta-galactosidase from Thermus sp . strain T2 from a crude extract from Escherichia coli or bovine liver catalase from a commercial preparation, with tailor-made ion-exchanger supports . A simple step of adsorption/desorption on lowly activated supports rendered both enzymes rather pure as confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis . Moreover, this strategy makes also easy the desorption step that requires rather low NaCl concentrations, which may become a serious problem for desorption of large proteins when using conventional supports, due to their ability of generating a very strong adsorption.

J Infect Dis, 2004 May 1, 189(9), 1556 - 64
Ability of blood group A-active glycosphingolipids to act as Escherichia coli heat-labile enterotoxin receptors in HT-29 cells; Galvan EM et al.; We examined the ability of blood group A-active glycoconjugates to act as receptors for Escherichia coli heat-labile type I enterotoxin (LT-I) in HT-29 cells . These cells contained ~4 times more specific binding sites for LT-I than for cholera toxin (CT) . Binding of LT-I could not be blocked by the B subunit of CT (CT-B), indicating the existence of LT-I receptors in addition to the glycosphingolipid GM1 . LT-I was able to increase levels of cyclic adenosine monophosphate (AMP), even in the presence of CT-B . Helix pomatia and anti-blood group A antibody caused a dose-dependent inhibition of binding of LT-I to cells and production of cyclic AMP . LT-I recognized several complex blood group A-active glycosphingolipids from cells, and this interaction was also interfered with by H . pomatia . Treatment of cells with D,L-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol diminished surface expression of blood group A-active glycosphingolipids and binding of LT-I to non-GM1 receptors . These observations suggest that blood group A-active glycosphingolipids can function as alternative receptors for LT-I in HT-29 cells.

Protein Eng Des Sel, 2004 Mar, 17(3), 261 - 6 Epub 2004 Apr 28.
Thermal stabilization of penicillolysin, a thermolabile 19 kDa Zn2+-protease, obtained by site-directed mutagenesis; Doi Y et al.; Penicillolysin is a member of the clan MX and the family of M35 proteases . The enzyme is a thermolabile Zn(2+)- protease from Penicillium citrinum with a unique substrate profile . We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein . We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the edible mushroom Grifora frondsa . The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.

Mol Cell Proteomics, 2004 Aug, 3(8), 780 - 7 Epub 2004 Apr 28.
Only a small subset of the horizontally transferred chromosomal genes in Escherichia coli are translated into proteins; Taoka M et al.; Horizontally transferred genes are believed to play a critical role in the divergence of bacterial strains from a common ancestor, but whether all of these genes express functional proteins in the cell remains unknown . Here, we used an integrated LC-based protein identification technology to analyze the proteome of Escherichia coli strain K12 (JM109) and identified 1,480 expressed proteins, which are equivalent to approximately 35% of the total open reading frames predicted in the genome . This subset contained proteins with cellular abundance of several dozens to hundreds of thousands of copies, and included nearly all types of proteins in terms of chemical characteristics, subcellular distribution, and function . Interestingly, the subset also contained 138 of 164 gene products that are currently known to be essential for bacterial viability (84% coverage) . However, the subset contained only a very small population (10%) of protein products from genes mapped within K-loops, which are "hot spots" for the integration of foreign DNAs within the K12 genome . On the other hand, these genes in K-loops appeared to be transcribed to RNAs almost as efficiently as the native genes in the bacterial cell as monitored by DNA microarray analysis, raising the possibility that most of the recently acquired foreign genes are inadequate for the translational machinery for the native genes and do not generate functional proteins within the cell.

J Biochem (Tokyo), 2004 Apr, 135(4), 555 - 65
A new recombinant single chain trispecific antibody recruits T lymphocytes to kill CEA (carcinoma embryonic antigen) positive tumor cells in vitro efficiently; Wang XB et al.; Anti-tumor associated antigen (TAA).CD3.CD28 trispecific antibody(TsAb) is able to provide two signals for fully and continuously activation of T lymphocytes and recruit them around tumor cells, presenting an attractive concept in tumor immunotherapy . Here, a new single chain trispecific antibody (scTsAb), named CEA-scTsAb, was constructed by fusion of anti-CEA (Carcinoma Embryonic Antigen) single chain antibody (scFv), anti-CD3 scFv and anti-CD28 VH, spaced by polypeptide interlinkers taken from the fragment of constant region (FC) of human IgG and human serum albumin (HSA) . It was expressed in Escherichia coli at low temperature (30 degrees C) with up to 50% of the antibody being present in soluble form . After one step of DEAE anion chromatography, the soluble product was sufficiently pure for further in vitro activity assays . First, it was proved that CEA-scTsAb could recognize three antigens (CEA, CD28 and Jurkat cell membrane antigen) specifically and could distinguish antigen positive cells from antigen negative cells in vitro . Then fresh PBMC (peripheral blood mononuclear cells), without being pre-treated by co-stimulatory reagents, such as IL-2 or CD28 mAb, were used as effector cells to test their ability to mediate tumor specific cytolysis of CEA-positive tumor cells, SW1116 . It was found by photomicrography that T lymphocytes were attracted to SW1116 cells in the presence of CEA-scTsAb, which resulted in effective cytolysis of tumor cells . As shown by MTT assay, the efficacy of tumor specific cytolysis mediated by CEA-scTsAb related to both the quantity and activation of PBMC . At an effector cells/target cells ratio (E/T) of 5, it was proved by dual-color FACS with propidium iodide (PI) and FITC-annexin V that both necrosis and apoptosis of tumor cells were causes of tumor specific cytolysis . In summary, a new single chain trispecific (CEA x CD3 x CD28) antibody was constructed and characterized carefully in this paper and was found to possess functions: (i) to activate T lymphocytes independently of additional co-stimulatory signal, (ii) to attract activated T lymphocytes around CEA-positive tumor cells, (iii) to attack CEA-positive tumor cells with recruited T lymphocytes . Because it recognizes a widely distributed tumor antigen (CEA), with moderate molecular weight (about 75 kDa) and a simple production procedure, and is able to mediate a high level of tumor specific cytolysis without any additional co-stimulating reagents, CEA-scTsAb is very promising for the task of immunotherapy in future.

J Biochem (Tokyo), 2004 Apr, 135(4), 495 - 9
Characterization of Escherichia coli uridine phosphorylase by single-site mutagenesis; Oliva I et al.; The Escherichia coli udp gene encodes uridine phosphorylase (UP), which catalyzes the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate . The X-ray structure of E . coli UP resolved by two different groups produced conflicting results . In order to cast some light on the E . coli UP catalytic site, we mutagenized several residues in UP and measured by RP-HPLC the phosphorolytic activity of the mutant UP proteins in vitro . Mutations Thr94Ala, Phe162Ala, and Tyr195Gly caused a drastic decrease in UP activity . These three residues were suggested to be involved in the nucleoside binding site . However, surprisingly, Tyr195Ala caused a relative increase in enzymatic activity . Both Met197Ala and Met197Ser conserved low activity, suggesting a minor role for this residue in the UP active site . Glu196Ala completely lost UP activity, whereas the more conservative Glu196Asp mutation was still partially active, confirming the importance of maintaining the correct charge in the surroundings of this position . Glu198 was mutated to either Gly, Asp and Gln . All three substitutions caused complete loss of enzymatic activity suggesting an important role of Glu198 both in ribose binding and in interaction with phosphate ions . Arg30Ala and Arg91Ala eliminated UP activity, whereas Arg30Lys and Arg91Lys presented a very low activity, confirming that these residues might interact with and stabilize the phosphate ions . Ile69Ala did not decrease UP activity, whereas His8Ala lowered the activity to about 20% . Both amino acids were suggested to take part in subunit interactions . Our results confirm the structural similarity between E . coli UP and E . coli purine nucleoside phosphorylase (PNP).

J Biochem (Tokyo), 2004 Apr, 135(4), 487 - 94
Mass spectrometry of hydrogen/deuterium exchange of Escherichia coli dihydrofolate reductase: effects of loop mutations; Yamamoto T et al.; To address the effects of single amino acid substitutions on the structural fluctuation of Escherichia coli dihydrofolate reductase (DHFR), hydrogen/deuterium exchange kinetics were investigated at 15 degrees C with wild-type and mutant DHFRs at Gly67 (six mutants) and Gly121 (eight mutants) located in two flexible loops, by means of electrospray ionization mass spectrometry . These mutations induced significant changes in the first-order rate constant of proton exchange, k(ex) (0.10-0.27 min(-1)), the number of fast-exchangeable protons, Delta M(o) (164-222 Da), and the number of protons protected from exchange, Delta M(infinity) (15-56 Da), relative to the corresponding values for the wild-type enzyme (k(ex) = 0.18 min(-1), Delta M(o) = 164 Da, and Delta M(infinity) = 50.5 Da) . These kinetic parameters were strongly correlated with the volume of introduced amino acids, but partly correlated with adiabatic compressibility (volume fluctuation), stability, and enzymatic activity . These results indicate that the local structure change due to a single amino acid substitution in loop regions is dramatically magnified to affect the structural fluctuation of the whole DHFR molecule, resulting in complicated changes in its stability and function.

J Biochem (Tokyo), 2004 Apr, 135(4), 479 - 85
In vitro phosphorylation of initiation factor 2 alpha (aIF2 alpha) from hyperthermophilic archaeon Pyrococcus horikoshii OT3; Tahara M et al.; Eukaryotic initiation factor 2 (eIF2) is a heterotrimeric protein composed of alpha, beta, and gamma subunits, of which the alpha subunit (eIF2 alpha) plays a crucial role in regulation of protein synthesis through phosphorylation at Ser51 . All three subunit genes are conserved in Archaea . To examine the properties of archaeal initiation factor 2 alpha (aIF2 alpha), three genes encoding alpha, beta, and gamma subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 were expressed in Escherichia coli cells, and the resulting proteins, aIF2 alpha, aIF2 beta, and aIF2 gamma, were characterized with reference to the properties of eIF2 . aIF2 alpha preferentially interacts with aIF2 gamma, but does not interact with aIF2 beta, which is consistent with data obtained with eIF2, of which eIF2 gamma serves as a core subunit, interacting with eIF2 alpha and eIF2 beta . It was found that aIF2 alpha was, albeit to a lower degree, phosphorylated by double-stranded RNA-dependent protein kinase (hPKR) from human, and a primary target site was suggested to be Ser48 within aIF2 alpha . This finding led us to the search for a putative aIF2 specific kinase gene (PH0512) in the P . horikoshii genome . The gene product Ph0512p unambiguously phosphorylated aIF2 alpha, and Ser48, as in the phosphorylation by hPKR, was suggested to be a target amino acid residue for the PKR homologue Ph0152p in P . horikoshii . These findings suggest that aIF2 alpha, like eIF2 alpha in eukaryotes, plays a role in regulation of the protein synthesis in Archaea through phosphorylation and dephosphorylation.

Dig Liver Dis, 2004 Apr, 36(4), 265 - 70
Gastric effects of the selective cyclooxygenase-2 inhibitor, celecoxib, in the rat; Coppelli G et al.; BACKGROUND: Recent studies have revealed that cyclooxygenase-2 is involved in the protection of the damaged gastric mucosa, mediating, in particular, the acceleration of ulcer healing and angiogenesis; therein, it has been suggested that selective cyclooxygenase-2 inhibitors, although safe in healthy stomach, may have deleterious effects on the injured gastric mucosa . Moreover, no information is available about direct effects of these drugs on gastric surface epithelium . AIMS: To investigate the gastric effects of the selective cyclooxygenase-2 inhibitor, celecoxib, in healthy and damaged rat gastric mucosa . METHODS: Gastric toxicity was studied in the rat by measuring gastric potential difference and mucosal lesions . Celecoxib was administered intragastrically, either in basal conditions or in combination with damaging (acetylsalicylic acid and ethanol) or protective (sodium nitroprusside and lipopolysaccharides from Escherichia coli) agents . The anti-inflammatory activity was evaluated in the carrageenan-induced paw oedema assay . The non-selective inhibitors indomethacin and acetylsalicylic acid were used for comparison . RESULTS: In conscious rats celecoxib, indomethacin and acetylsalicylic acid significantly reduced the paw oedema induced by carrageenan . While acetylsalicylic acid and indomethacin significantly reduced basal gastric potential difference and caused gastric mucosal lesions, celecoxib was ineffective; moreover, it did not aggravate the direct damaging effect of intragastric ethanol or aspirin . Pretreatment with the non-selective nitric oxide synthase inhibitor N-nitro-L-argynine methyl ester did not significantly change the gastric effects of celecoxib . Both celecoxib and indomethacin prevented the gastroprotective effects induced by sodium nitroprusside (nitric oxide donor) or by bacterial lipopolysaccharides (inducer of nitric oxide synthesis) . CONCLUSIONS . These data indicate that the selective cyclooxygenase-2 inhibitor celecoxib did not alter gastric mucosal barrier nor induced mucosal lesions in the healthy or nitric oxide-deficient rat gastric mucosa . However, cyclooxygenase-2 inhibition impaired nitric oxide-dependent gastroprotection, indicating that cyclooxygenase-2 derived prostaglandins may be involved in the gastric mucosal defence.

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