Escherichia coli

Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 321 - 5
{Screening strains producing inulinase and cloning of inulinase gene}; Zhang L et al.; A wild-type strain AF10 producing inulinase was screened from soil samples, the strain is identified to be a A . niger . A endoinulinase gene inuA1 from A . niger AF10 was wequenced and analyzed, after PCR amplification . The results shows inuA1 is 1551 bp in length without any intron and contains 4 potential N-glycosylation sites . The conservative sequence is WMNEPN from the N-terminus . The inuA1 was cloned to pUC118 and the recombinant vector pinuA1 was obtained and transformed into E . coli JM109 . The recombinant JM109/inuA1 included inuA1 was obtained.

Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 305 - 10
{Cloning and expression of Vitreoscilla hemoglobin in Streptomyces aureofaciens}; Meng C et al.; Vitreoscilla hemoglobin Gene was cloned in Streptomyces aureofaciens through E . coli-Streptomyces shuttle plasmids constructed by both pIJ699-pUC19(vhb) and pIJ702-pBR322(vhb) . Under low dissolved oxygen conditions, expression of hemoglobin enhanced CTC yield of engineering strain more about 40%-60% that that of control by improving oxygen transmission in cells . Hemoglobin expression by Promoter of tetracycline resistance gene was more effective for oxygen transmission than that by its native promoter regulated by dissolved oxygen concentrations in environments of locally low concentrations of dissolved oxygen.

Wei Sheng Wu Xue Bao, 2002 Jun, 42(3), 298 - 304
{Selection and characterization of peptides that specifically binding to hepatitis C virus serine protease}; Du G et al.; The HCV NS3 serine protease that plays important role in the processing of polyprotein and the replication of virus is a prime target for antiviral drugs and therapy research . Based on the crystallographic structure of HCV sreine protease, a single-chain protease was contstructed in which the central sequence of NS4A was fused to the N-terminus of NS3 serine protease domain via a flexible linker and it was expressed at high level in soluble form in E . coli . The purified protease could cleave the recombinant protein NS5ab into two parts . The purified protease was used as target to screen binding peptides from phage displayed peptide library . After three rounds of affinity screening, 37 out of 44 randomly selected phages could bind specifically with the single-chain serine protease and their specificity were verified by competitive ELISA . The 13 sequenced clones represents 6 kinds of sequences of which the amino acids composition is in bias and there is a consensus sequence: {H/F/W}-H-W-X-X-W.

Wei Sheng Wu Xue Bao, 2002 Feb, 42(1), 110 - 3
{Cloning of flocculent gene and expression in industrial yeast strain}; Guo W et al.; The partial genomic library of Saccharomyce cerevisiae FL189 possessing strong flocculation ability was constructed using Yeast-E . coli shuttle plasmid YCp50 as vector . Recombinant plasmid containing flocculation gene was obtained by screening the growth of transformants on the selective medium and measurement flocculation, designated as pCF1.pCF1 was introduced into industrial yeast strain PJ208-5-15 . Six transformants PJ208-5-15-1(pCF1)-PJ208-5-15-6(pCF1) possessing strong flocculation ability were obtained . The results of Southern blot and restriction endonuclease analysis showed that the insert is about 4.3 kb and could hybridize with the probe (2.6 kb Eco RV fragment of FLO1) . Flocculation ability assay indicated that the transformants possess strong flocculation ability . Hence, the gene controlling flocculation phenotype exists in the cloned DNA fragment . The restriction endonuclease analysis and the sequence analysis of the insert DNA fragment are in progress.

Wei Sheng Wu Xue Bao, 2002 Feb, 42(1), 99 - 104
{Antigen analysis of envelope gene products of avian leukosis virus subgroup J}; Qin A et al.; Envelope glycoprotein of avian leukosis virus Subgroup J (ALV-J) determines the host range of virus infection and cross-neutralization patterns . The truncated envelope genes of avian lecukosis virus subgroup J (ALV-J) were amplified by PCR and cloned them into pGEX-5X-3 vector for expressing envGST-fusion protein . Western blot analysis results showed that the products of truncated env gene expressed in Escherichia coli could reacted with G2, JE9 and I45 monoclonal antibodies (Mabs) specific to envelope protein of ALV-J . Using different Mabs to map the epitopes in the expressed truncated gp85 GST fusion protein, the results showed that Mab G2 and JE9 antibodies recognizing epitope in gp85 was localized between amino acid 65-155 . Mab I45 reacted with the epitope at the location of amino acid 156-233 . It indicated that the specificity of subgroup J virus is determined by the gp85 peptide since GST-gp85 protein expressed in Escherichia coli is not glycosylated.

Wei Sheng Wu Xue Bao, 2002 Feb, 42(1), 40 - 4
{The study on cloning and expression of Bt cry1Ab13 gene}; Tan J et al.; B . thuringiensis strain C005 with high insecticidal activity to several kinds of pests, screened from China, was identified that it contained cry1Ab gene by PCR-RFLP . Southern blotting showed that a 8.5 kb positive band of plasmid DNA digested with PstI contained cry1Ab gene . The gene was cloned from Bt C005 and the results of sequence analysis showed that cry1Ab13 gene contained a 3468 bp open reading frame, encoding a 130.6 kD protein composing 1155 amino acids . The IE point of Cry1Ab13 protein was pH 4.845 . The cry1Ab gene has been registered in GenBank (Accession number is AF254640) and named as cry1Ab13 as a novel gene by International Nomenclature Committee of Bt delta-endotoxin genes . SDS-PAGE analysis indicated that 130 kD protein of Cry1Ab13 was expressed in a Bt acrystalliferous mutant cryB- and bioassay results proved that the transformant BiotI81 containing cry1Ab13 gene had high toxicity to Plutella xylostella.

Biotechnol Bioeng, 2003 Mar 30, 81(7), 783 - 9
Simultaneous synthesis of enantiomerically pure (S)-amino acids and (R)-amines using coupled transaminase reactions; Cho BK et al.; For the simultaneous synthesis of enatiomerically pure (S)-amino acids and (R)-amines from corresponding alpha-keto acids and racemic amines, an alpha/omega-transaminase coupled reaction system was designed using favorable reaction equilibrium shift led by omega-transaminase reaction . Cloned tyrB, aspC and avtA, and omegataA were co-expressed in E . coli BL21(DE3) using pET23b(+) and pET24ma, respectively . The coupled reaction produced the (S)-amino acids with 73-90% (> 99% ee(S)) of conversion yield and resolved the racemic amines with 83-99% ee(R) for 5 to 10 hours . In designing the coupled reactions in the cell, alanine and pyruvate were efficiently used in the cell as an amine donor for the alanine transaminase and an amino acceptor for the omega-transaminase, respectively, resulting in an alanine-pyruvate shuttling system . The common problem of the low equilibrium constant of the alpha-transaminase can be efficiently overcome by the coupling with the omega-transaminase . However, overcoming the product inhibition of omega-transaminase by the ketone by-product and increasing the decarboxylation rate of the oxaloacetate produced during the transaminase reaction become barriers to further improving the overall reaction rate and the yield of the coupled reactions .

Proteins, 2003 Feb 15, 50(3), 474 - 85
Structural studies on MtRecA-nucleotide complexes: insights into DNA and nucleotide binding and the structural signature of NTP recognition; Datta S et al.; RecA protein plays a crucial role in homologous recombination and repair of DNA . Central to all activities of RecA is its binding to Mg(+2)-ATP . The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single-stranded DNA . The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors . Here we report crystal structures of a MtRecA-ADP complex, complexes with ATPgammaS in the presence and absence of magnesium as well as a complex with dATP and Mg+2 . Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP-AlF4 confirms an expansion of the P-loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP . The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding . The nucleotide binding site itself, however, remains relatively unchanged . The analysis also reveals that ATPgammaS rather than ADP-AlF4 is structurally a better mimic of ATP . From among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA . The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level . Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site . Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition .

Arch Virol, 2003 Feb, 148(2), 345 - 55
Involvement of the terminus of grouper betanodavirus capsid protein in virus-like particle assembly; Lu MW et al.; Dragon grouper, Epinephelus lanceolatus, nervous necrosis virus (DGNNV) consists of 180 copies of capsid protein that encapsulates a bipartite genome of single-stranded (+)RNAs . Expressing the open reading frame (ORF) of RNA2 in Escherichia coli forms virus-like particles (VLPs) that resembles native virus . Deleting N- and C-termini revealed different impacts on VLP formation . Deletion of 35 or 52 residues at the N-terminus completely ruined the VLP assembly, presumably due to removal of positively charged residues for binding RNAs . When deletions were restricted to 4, 16, or 25 N-terminal residues, the assembly of VLPs remained . The ability of VLP formation diminished when 4 to 11 C-terminal residues were deleted . The termini that can be deleted without seriously destructing the VLPs are 25 and 3 residues at N- and C-termini, respectively.

Proc Natl Acad Sci U S A, 2003 Feb 18, 100(4), 1547 - 51 Epub 2003 Jan 29.
Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis; Cane DE et al.; The PCR has been used to amplify a 2,181-bp ORF from Streptomyces coelicolor A3(2), designated SC9B1.20 (= SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase . The full-length recombinant protein was expressed at high levels in Escherichia coli and shown to catalyze the Mg(2+)-dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4S, 7R)-germacra-1 (10)E, 5E-diene-11-ol . The enzymatic cyclization had a k(cat) of 6.2 +/- 0.5 x 10(-3) s(-1) and a K(m) for farnesyl diphosphate of 62 +/- 8 nM . Expression of the N-terminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower k(cat) of 3.2 +/- 0.4 x 10(-3) s(-1) and a twofold greater k(m) of 115 +/- 14 nM . By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity . The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of Streptomyces species.

J Biol Chem, 2003 Apr 4, 278(14), 12013 - 21 Epub 2003 Jan 28.
Regulation of the interleukin-1-induced signaling pathways by a novel member of the protein phosphatase 2C family (PP2Cepsilon); Li MG et al.; Although TAK1 signaling plays essential roles in eliciting cellular responses to interleukin-1 (IL-1), a proinflammatory cytokine, how the IL-1-TAK1 signaling pathway is positively and negatively regulated remains poorly understood . In this study, we investigated the possible role of a novel protein phosphatase 2C (PP2C) family member, PP2Cepsilon, in the regulation of the IL-1-TAK1 signaling pathway . PP2Cepsilon was composed of 303 amino acids, and the overall similarity of amino acid sequence between PP2Cepsilon and PP2Calpha was found to be 26% . Ectopic expression of PP2Cepsilon inhibited the IL-1- and TAK1-induced activation of mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase or MKK3-p38 signaling pathway . PP2Cepsilon dephosphorylated TAK1 in vitro . Co-immunoprecipitation experiments indicated that PP2Cepsilon associates stably with TAK1 and attenuates the binding of TAK1 to MKK4 or MKK6 . Ectopic expression of a phosphatase-negative mutant of PP2Cepsilon, PP2Cepsilon(D/A), which acted as a dominant negative form, enhanced both the association between TAK1 and MKK4 or MKK6 and the TAK1-induced activation of an AP-1 reporter gene . The association between PP2Cepsilon and TAK1 was transiently suppressed by IL-1 treatment of the cells . Taken together, these results suggest that, in the absence of IL-1-induced signal, PP2Cepsilon contributes to keeping the TAK1 signaling pathway in an inactive state by associating with and dephosphorylating TAK1.

J Biol Chem, 2003 Apr 11, 278(15), 13227 - 34 Epub 2003 Jan 29.
Molecular dissection of the contribution of negatively and positively charged residues in S2, S3, and S4 to the final membrane topology of the voltage sensor in the K+ channel, KAT1; Sato Y et al.; Voltage-dependent ion channels control changes in ion permeability in response to membrane potential changes . The voltage sensor in channel proteins consists of the highly positively charged segment, S4, and the negatively charged segments, S2 and S3 . The process involved in the integration of the protein into the membrane remains to be elucidated . In this study, we used in vitro translation and translocation experiments to evaluate interactions between residues in the voltage sensor of a hyperpolarization-activated potassium channel, KAT1, and their effect on the final topology in the endoplasmic reticulum (ER) membrane . A D95V mutation in S2 showed less S3-S4 integration into the membrane, whereas a D105V mutation allowed S4 to be released into the ER lumen . These results indicate that Asp(95) assists in the membrane insertion of S3-S4 and that Asp(105) helps in preventing S4 from being releasing into the ER lumen . The charge reversal mutation, R171D, in S4 rescued the D105R mutation and prevented S4 release into the ER lumen . A series of constructs containing different C-terminal truncations of S4 showed that Arg(174) was required for correct integration of S3 and S4 into the membrane . Interactions between Asp(105) and Arg(171) and between negative residues in S2 or S3 and Arg(174) may be formed transiently during membrane integration . These data clarify the role of charged residues in S2, S3, and S4 and identify posttranslational electrostatic interactions between charged residues that are required to achieve the correct voltage sensor topology in the ER membrane.

Faraday Discuss, 2003, 122, 131 - 44; discussion 171-90
Time-resolved and static-ensemble structural chemistry of hydroxymethylbilane synthase; Helliwell JR et al.; The enzyme hydroxymethylbilane synthase (HMBS, EC 4.3.1.8), 313 amino acid residues and MW 34 kDa, also known as porphobilinogen deaminase (PBGD), catalyses the stepwise polymerization of four molecules of porphobilinogen (PBG) to the linear tetrapyrrole 1-hydroxymethylbilane . Several crystallographic structures of HMBS have been previously determined, most recently including by time-resolved Laue protein crystallography of the Lys59Gln mutant form with reaction initiation undertaken by use of a flow cell carrying the substrate PBG . In this paper we review these structures and add new molecular graphics representations and analyses . Moreover we present a new structure refined at 1.66 A resolution using diffraction data recorded at cryo-temperature (100 K) in an attempt at trapping the polypeptide loop (residues 47 to 58) in the vicinity of the enzyme active site, missing in all previous structure determinations . This loop still has not appeared in the electron density maps, in spite of the advantage of cryo-temperature, but nevertheless the 1.66 A cryo-structure extends the ensemble of known HMBS structures . The cryomodel of protein, cofactor and 320 bound water molecules has been refined to a final R-factor and R-free of 0.198 and 0.247 respectively; the PDB deposition codes, coordinates and structure factors are 1GTK and R1GTKSF respectively . Finally a protein comparison study is presented of the Mycobacterium tuberculosis (MTb) HMBS, with the E . coli HMBS . This has been done as preparation for future structural studies on the MTb HMBS from this important disease bearing organism . The overall amino acid sequence identity is 41% . Most interestingly there is a two-residue reduction in length of the loop referred to above (Asp 50 and Gly 58 being missing in the MTb form) . This gives the hope that this loop will be less flexible and thus might become visible to crystallographic analysis.

Wei Sheng Wu Xue Bao, 1999 Aug, 39(4), 305 - 14
{Molecular cloning and sequencing of outer capsid protein gene of rice dwarf virus and its expression in Escherichia coli}; Lu R et al.; The S2 full-length cDNA of rice dwarf phytoreovirus which enocodes the viral outer capsid protein was cloned and its complete nucleotide sequence was determined . The results showed that S2 is 3512 bp long with a large open reading frame which encodes a protein of 1116 amino acids . It shares 94.6% and 95.4% identity with RDV of Japanese H isolate in terms of nucleotide and amino acid sequences, respectively, and it also shows some homology with VP2 of rotavirus at the level of amino acid sequence . The search of deduced RDV S2 amino acid sequence in Blast network found that there were 4 leucine-rich motifs in P2 protein, and ten amino acids within the hydrophibic region at amino-terminus could form an alpha-helix . Predicted secondary structure of S2 cDNA indicated that a hairpin and a stem loop are present in the 5'-end within 50 bp, and a stem loop in the 3'-end within 50 bp . RDV S2 partial and full-length sequences were then cloned into expression vector pET-11d & pTrcHisC . SDS-PAGE and Western blot proved that amino- and carborn-termini of P2 were successfully expressed in E . coli.

Wei Sheng Wu Xue Bao, 1999 Dec, 39(6), 521 - 6
{The isolation and characterization of type 1 pili from pathogenic Escherichia coli of chicken origin}; Gao S et al.; The pili from pathogenic Escherichia coli isolates 566, 1794 and TK3 of chicken and turkey origin were purified . After mechanic detachment from the bacterial cells, the pili were concentrated by precipitation with ammonium sulfate, dialyzed, and solubilized in buffer containing deoxycholate . The fraction containing the pilus was purified further by ultracentrifugation in a sucrose gradient . After ultracentrifugation, the pili at the density of 1.10 to 1.15 g.cm-3 (between 10%-20% of sucrose gradients) were collected, and the purified pili from strain 566, 1794 and TK3 had an apparent molecular weight of 17,500, 17,000 and 17,000 respectively, which retained their ability to bind the erythrocyte in a mannose-inhibitable fashion . Hyperimmunesera raised in BALB/C mice against the purified pili from strain 1794 reacted positively with type 1 pili from both isolates 566 and TK3 by immuno blot . These results revealed that the three strains either Chinese or north american isolates expressed type 1 pili which had molecular weights from 17,000 to 17,500, and they have common antigenic epitopes.

Wei Sheng Wu Xue Bao, 1999 Dec, 39(6), 489 - 94
{Cloning of DNA fragment related to salt tolerance in Sinorhizobium meliloti 042B}; Chen X et al.; Total DNA partially digested by EcoR I was prepared for S . meliloti 042B, in which 15-25 kb DNA fragments were collected . Vector pLAFR I was purified and digested by EcoR I, and then the various DNA fragments of 042B were ligated with pLAFR I by T4DNA ligase . Gene library of S . meliloti 042B was constructed with pLAFR I using E . coli S17-1 as recipient . The number of bacterial recombinants obtained was about 8,000 and 95% of them contained foreign DNA fragments . Using NTG, 042B was mutated on FY plates and 12 sensitive strains were screened at 0.5 mol/L NaCl from 2,000 colonies . One of them was named GZ17 and selected as a recipient strain . By biparental mating the foreign DNA fragments were introduced from gene library of strain 042B into recipient strain GZ17 which is sensitive to 0.5 mol/L NaCl . Then the transconjugants were grown on FY plates containing tetracycline (20 micrograms/mL) and 0.5 mol/L NaCl . A 7 kb inserted DNA fragment related to salt tolerance was obtained . In subcloning experiment, a 4 kb DNA fragment related to salt tolerance was obtained.

Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 226 - 33
{The outer membrane protein (OMP) patterns of Escherichia coli isolates of predominant O serogroups originated from chichens in different regions in China}; Gao S et al.; The purpose of this study is to determine the outer membrane protein (OMP) patterns of avian Escherichia coli isolates with predominant serogroups originated from 18 provinces, autonomous regions and municipal cities in China . Total of 204 isolates belonging to O18, O78, O2, O88, O11 and O26 serogroups respectively, were tested . The outer membrane proteins of these isolates were isolated with the improved N-lauroylsarcosine method and analyzed by SDS-PAGE . 4 different OMP patterns were identified with these isolates of predominant serogroups . 3 different OMP patterns with 56 isolates of O18, 4 with 54 isolates of O78, 2 with 28 isolates O2, 1 with 26 isolates of O88, 3 with 22 isolates of O11 and 1 with 18 isolates of O26 respectively, were found . Isolates with OMP pattern 1 were discovered in 6 different serogroups, and OMP-3 pattern was also shared by O18, O78 and O11 serogroups isolates . These results indicated that the OMP patterns of avian pathogenic Escherichia coli isolates of O18, O78, O2, O11 serogroups which were isolated from different regions in China were heterogeneous, and all of O88 and O26 serogroups isolates just belonged to OMP pattern 1 . Moreover, the OMP pattern 1 was presented in isolates of six different predominant O serogroups.

Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 215 - 9
{Molecular cloning of rat OB gene and its expression in Escherichia coli}; Zhang T et al.; In order to provide rat OB gene product for studying the relationship between obesity and noninfectious diseases, rat OB cDNA was amplified by RT-PCR technique . 460 bp fragment of OB cDNA was subcloned into EcoRI/BamHI site of plasmid pUC 19 . Sequence analysis of OB cDNA revealed that the translation reading frame was identical with that reported in the literature . Thereby plasmid pBV220-rOB was constructed and the specific expression of OB gene in E . coli identified by SDS-PAGE electrophoresis was obtained.

Wei Sheng Wu Xue Bao, 1999 Jun, 39(3), 209 - 14
{Expression detection and location analysis of BstNI isoschizomer restriction-modification system gene}; Liu J et al.; Some genetic markers of E . coli HB101 and JM110 were identified, two bacterial strains were used as recipients respectively to detect the expression of a restriction endonuclease(R) gene and a methylase(M) gene of BstNI isoschizomer restriction-modification system . DNA fragment containing the R-M genes was deleted unilaterally with exoIII and 23 deletion subclones were obtained . According to the Enzyme activity of each subclone, R and M gene were located respectively at the regions of 0.2-->1.4 kb and 1.5-->3.3 kb from cloning site PstI . Analysis showed that the R . M system belongs to type II, two genes are controlled by the different promoters; the recognition sequence of this system is the same as that of DNA-cytosine methyltransferase(Dcm), the latter's methylation function can resist the R enzyme . It was interesting that the recombinant plasmid with an R+ M- genotype appeared to be lethal to dcm+ hosts yet . This indicated that the M gene closely linking to R gene is of critical importance for the existence of the R-M system in process of evolution.

Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 178 - 80
{Preparation, purification and identification of sialic acid from Escherichia coli C-8}; Qian S et al.; Colominic acid produced from Escherichia coli C-8 was purified by ammonia sulfate precipitation, dialysis and concentration . After hydrolysis of colominic acid at a pH of 2.5, at 70 degrees C for 4 h, sialic acid was obtained, then purified by chromatography on Dowex1-x8 . Analysis of thin-layer chromatography and absorption spectrum of sialic acid in the orcinol/Fe3+/HCl and the periodic acid/thiobarbituric acid confirmed that its purity was identical with that of standard sample.

Wei Sheng Wu Xue Bao, 1999 Apr, 39(2), 100 - 7
{Study of HSP70 gene upstream regulation element on expression efficiency of GST gene in M . smegmatis}; Cheng J et al.; Four different expression vectors were constructed by cloning foreign gene which encode Schistosoma japonicum 26 kD antigen (Sj26GST) into Escherichia coli-Mycobacteria shuttle plasmid pBCG-2000 and investigated their expression efficiency in mycobacteria smegmatis . The plasmid which contains promoter of human mycobacterial tuberculosis heat shock protein 70 was firstly digested with Nco I and modified with two different ways to lead to two kinds of SD sequences, and ligated with Sj26GST encoding gene . Then, the DNA fragment contained HSP70 promoter and Sj26GST gene was obtained and cloned into E . coil-mycobacteria shuttle plasmid pBCG-2000, and finally four recombinant mycobacterial expression vectors that differenciated in SD sequence, orientation and copy number were selected . The expressed native recombinant Sj26GST(rSj26GST) was soluble and could be observed on SDS-PAGE about at the molecular weight of 26 kD obviously . Analysis with protein density scanning indicated: the expression efficiency that containing double-copy promoter-foreign gene vector was the highest and the expressed protein which was about 1.6 times than others was 28% of total protein of Mycobacteria smegmatis . The cloned direction and SD sequence had no significant effect on expression efficiency.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 400 - 3 Epub 2003 Jan 23.
De novo purification scheme and crystallization conditions yield high-resolution structures of chitinase A and its complex with the inhibitor allosamidin; Papanikolau Y et al.; The purification scheme of chitinase A (ChiA) from S . marcescens has been extensively revised . The pure enzyme crystallizes readily under new crystallization conditions . The ChiA crystal structure has been refined to 1.55 A resolution and the crystal structure of ChiA co-crystallized with the inhibitor allosamidin has been refined to 1.9 A resolution . Allosamidin is located in the deep active-site tunnel of ChiA and interacts with three important residues: Glu315, the proton donor of the catalysis, Asp313, which adopts two conformations in the native structure but is oriented towards Glu315 in the inhibitor complex, and Tyr390, which lies opposite Glu315 in the active-site tunnel.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 385 - 8 Epub 2003 Jan 23.
Improved three-dimensional growth of manganese superoxide dismutase crystals on the International Space Station; Vahedi-Faridi A et al.; Manganese superoxide dismutase was crystallized in microgravity with 35 PCAM experiments (Protein Crystallization Apparatus for Microgravity) on the ISS (International Space Station) from 5 December 2001 to 19 April 2002 . Crystals were very large in size and could easily be seen by eye . Crystals with 0.45 x 0.45 mm cross-sections and of up to 3 mm in length were obtained in several drops: an 80-fold increase in crystal Volume compared with the largest earth-grown crystal . A smaller crystal (0.15 x 0.30 mm in cross-section and 1.6 mm in length) was soaked in cryoprotectant and placed in a cryoloop . Diffraction data were collected at 100 K at the BioCARS bending-magnet beamline . The space group was C222(1), with unit-cell parameters a = 100.64, b = 107.78, c = 179.82 A . Diffraction spots to 1.26 A resolution were observed . Unfortunately, the high-resolution diffraction degraded owing to radiation damage and the resolution limit for the complete data set was 1.35 A . It is anticipated that increasing the crystal Volume and diffraction limit through microgravity crystal growth will enable several types of technically challenging structure determinations.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 381 - 4 Epub 2003 Jan 23.
Crystallization of hamster dihydroorotase: involvement of a disulfide-linked tetrameric form; Maher MJ et al.; Dihydroorotase (DHOase) catalyses the formation of L-dihydroorotate (DHO) in the de novo pyrimidine biosynthetic pathway . The type I DHOase domain from hamster forms part of the trifunctional enzyme CAD . The hamster DHOase domain has been cloned and expressed in Escherichia coli . Solutions of the homodimeric protein convert to a homotetrameric species when incubated at ambient temperature . Formation of the tetrameric species is mediated via disulfide linkages between single free cysteine residues on the surface of each monomer . This process is also observed under conditions used for crystallization of the hamster DHOase domain; crystals composed exclusively of the tetrameric species grow from solutions containing as little as 10% tetramer . The crystallization of pure tetrameric DHOase results in two crystal forms: form I, with space group C222(1) and unit-cell parameters a = 127.1, b = 603.5, c = 144.7 A, and form II, with space group P2(1) and unit-cell parameters a = 260.5, b = 148.2, c = 308.0 A, beta = 102.2 degrees . Data have been recorded to 4.3 and 4.0 A resolution, respectively.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 372 - 4 Epub 2003 Jan 23.
Crystallization and preliminary X-ray analysis of TBP-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1; Yamamoto T et al.; The 26 kDa TBP (TATA-binding protein) interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-TIP26) is a possible transcriptional regulatory protein in Thermococcales . Here, the crystallization of both the native and selenomethionine-substituted proteins and data collection are described . The native crystals belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 73.83, c = 86.41 A, and diffract to 2.2 A using synchrotron radiation . MAD data was collected and a Bijvoet difference Patterson map showed strong peaks sufficient to determine the positions of the Se atoms.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 369 - 71 Epub 2003 Jan 23.
Purification, crystallization and preliminary X-ray analysis of a mu-like calpain; Pal GP et al.; The X-ray structure of m-calpain in the absence of Ca(2+) has been described, but it has not been possible to obtain sufficient mu-calpain for structure determination . Comparison of the two structures is of interest in attempting to understand their different Ca(2+) requirements . Here, the crystallization in the absence of Ca(2+) of an inactive mutant hybrid calpain (MW approximately 100 kDa), which contains 85% of the rat mu-calpain sequence and is well expressed in Escherichia coli, is described . The properties of this calpain in its active form and particularly its Ca(2+) requirement are close to those expected for wild-type mu-calpain . Clusters of plate-shaped crystals were obtained by vapour diffusion with polyethylene glycol (M(r) approximately 6000) as precipitating agent in the presence of detergent . The crystals diffract to a resolution of 2.7 A at a synchrotron source . The space group is P2(1), with unit-cell parameters a = 72.7, b = 184.6, c = 86.3 A, beta = 100.7 degrees . There are two molecules in the asymmetric unit, corresponding to a solvent content of 57.1%.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 350 - 2 Epub 2003 Jan 23.
Preliminary X-ray characterization of the ribonuclease P (C5 protein) from Escherichia coli: expression, crystallization and cryoconditions; Choe HW et al.; The gene for Escherichia coli ribonuclease P (RNase P) protein (also known as C5 protein) and its mutant C5-C113A have been expressed as GST fusion proteins in E . coli at a high level . After cleavage of the fusion protein, highly purified functional C5 protein is obtained that can be crystallized with 2.5-2.6 M (NH(4))(2)HPO(4)/(NH(4))H(2)PO(4) pH 7.0 at room temperature . These crystals are suitable for X-ray analysis, belong to the space group P3(1)21 or P3(2)21 (unit-cell parameters a = b = 66.67, c = 142.09 A) and diffract to 2.9 A at 100 K using sorbitol and glycerol as cryoprotectants . For three molecules in the asymmetric unit a V(M) of 2.17 A(3) Da(-1) was calculated.

Acta Crystallogr D Biol Crystallogr, 2003 Feb, 59(Pt 2), 214 - 24 Epub 2003 Jan 23.
Ping-pong cross-validation in real space: a method for increasing the phasing power of a partial model without risk of model bias; Hunt JF et al.; Experimental phases could only be obtained to 4.4 A resolution for crystals of the SecA translocation ATPase . Density modification of these phases exploiting the 65% solvent content of the crystal produced a map from which an approximate backbone model could be built for 80% of the structure . Combining the phases inferred from this partial model with the MIR phases and repeating the density modification produced an improved map from which a more complete backbone model could be built . However, this procedure converged before yielding a map, that allowed unambiguous sequence assignment for the majority of the protein molecule . In order to avoid the likely model bias associated with a speculative attempt at sequence assignment, a real-space cross-validation procedure was employed to facilitate completion of the crystal structure based on partial model phasing . The protein was partitioned into two disjoint sets of residues . Models in which the side chains were built for residues in one of the two sets were used for phase combination and density modification in order to produce improved electron density for interpretation of residues in the other set that had not been included in the model . Residues in the two sets were therefore omitted from the model in alternation except at sites where the side chain could be identified definitively based on phasing with the other set . This ping-pong cross-validation procedure allowed partial model phasing to be used to complete the crystal structure of SecA without being impeded by model bias . These results show that the structure of a large protein molecule can be solved with exclusively low-resolution experimental phase information based on intensive use of partial model phasing and density modification . Real-space cross-validation can be applied to reduce the risk of model bias associated with partial model phasing, streamlining this approach and expanding its range of applicability.

RNA, 2003 Jan, 9(1), 33 - 43
Identification of the Hfq-binding site on DsrA RNA: Hfq binds without altering DsrA secondary structure; Brescia CC et al.; DsrA RNA regulates the translation of two global regulatory proteins in Escherichia coli . DsrA activates the translation of RpoS while repressing the translation of H-NS . The RNA-binding protein Hfq is necessary for DsrA to function in vivo . Although Hfq binds to DsrA in vitro, the role of Hfq in DsrA-mediated regulation is not known . One hypothesis was that Hfq acts as an RNA chaperone by unfolding DsrA, thereby facilitating interactions with target RNAs . To test this hypothesis, we have examined the structure of DsrA bound to Hfq in vitro . Comparison of free DsrA to DsrA bound to Hfq by RNase footprinting, circular dichroism, and thermal melt profiles shows that Hfq does not alter DsrA secondary structures, but might affect its tertiary conformation . We identify the site on DsrA where Hfq binds, which is a structural element in the middle of DsrA . In addition, we show that although long poly(U) RNAs compete with DsrA for binding to Hfq, a short poly(U) stretch present in DsrA is not necessary for Hfq binding . Finally, unlike other RNAs, DsrA binding to Hfq is not competed with by poly(A) RNA . In fact, DsrA:poly(A):Hfq may form a stable ternary complex, raising the possibility that Hfq has multiple RNA-binding sites.

RNA, 2003 Feb, 9(2), 231 - 8
Analysis of recombinant yeast decapping enzyme; Steiger M et al.; A critical step in the turnover of yeast mRNAs is decapping . Two yeast proteins, Dcp1p and Dcp2p, are absolutely required for decapping, although their precise roles in the decapping reaction have not been established . To determine the function of both Dcp1p and Dcp2p in decapping, we purified recombinant versions of these proteins from Escherichia coli and examined their properties . These experiments demonstrate that copurification of Dcp1p and Dcp2p yields active decapping enzyme under a variety of conditions . Moreover, Dcp2p alone can have decapping activity under some biochemical conditions . This suggests that Dcp2p can be a catalytic subunit of the decapping complex, and Dcp1p may function to enhance Dcp2p activity, or as an additional active subunit . In addition, recombinant Dcp1p/Dcp2p prefers long mRNA substrates and is sensitive to inhibition by sequestration of the 5' end but not the 3' end of the substrate . This suggests that Dcp1p/Dcp2p contains an additional RNA-binding site spatially distinct from the active site . Finally, using two RNA-binding proteins that enhance decapping in vivo (Edc1p and Edc2p), we can reconstitute the activation of decapping with recombinant proteins . This indicates that the Edc1 and Edc2 proteins act directly on the decapping enzyme.

J Biol Chem, 2003 Apr 25, 278(17), 15142 - 52 Epub 2003 Jan 28.
Deamidations in recombinant human phenylalanine hydroxylase . Identification of labile asparagine residues and functional characterization of Asn --> Asp mutant forms; Carvalho RN et al.; Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 degrees C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues . The multiple deamidations alter the functional properties of the enzyme including its affinity for l-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis . Asn(32) in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly(33) (Solstad, T., Carvalho, R . N., Andersen, O . A., Waidelich, D., and Flatmark, T . (2003) Eur . J . Biochem., in press) . Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 degrees C) expressed enzyme has shown Asn(376) and Asn(133) to be labile residues . Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn(32) and Asn(376) are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme . The Asn(32) --> Asp, Asn(376) --> Asp, and the double mutant forms expressed for 2 h at 28 degrees C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH . Moreover, deamidation of Asn(32) in the wt-hPAH (24 h expression at 28 degrees C) and the Asn(32) --> Asp mutation both increase the initial rate of phosphorylation of Ser(16) by cAMP-dependent protein kinase (p < 0.005) . By contrast, the substitution of Gly(33) with Ala or Val, both preventing the deamidation of Asn(32), resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression . The other Asn --> Asp substitutions (in the catalytic domain) revealed that Asn(207) and Asn(223) have an important stabilizing structural function . Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e . Asn(167) --> Ile and Asn(207) --> Asp, and their phenotypes were characterized.

J Biol Chem, 2003 Apr 18, 278(16), 13728 - 39 Epub 2003 Jan 28.
Analysis of human flap endonuclease 1 mutants reveals a mechanism to prevent triplet repeat expansion; Liu Y et al.; Flap endonuclease 1 (FEN1), involved in the joining of Okazaki fragments, has been proposed to restrain DNA repeat sequence expansion, a process associated with aging and disease . Here we analyze properties of human FEN1 having mutations at two conserved glycines (G66S and G242D) causing defects in nuclease activity . Introduction of these mutants into yeast led to sequence expansions . Reconstituting triplet repeat expansion in vitro, we previously found that DNA ligase I promotes expansion, but FEN1 prevents the ligation that forms expanded products . Here we show that among the intermediates that could generate sequence expansion, a bubble is necessary for ligation to produce the expansion product . Severe exonuclease defects in the mutant FEN1 suggested that the inability to degrade bubbles exonucleolytically leads to expansion . However, even wild type FEN1 exonuclease cannot compete with DNA ligase I to degrade a bubble structure before it can be ligated . Instead, we propose that FEN1 suppresses sequence expansion by degrading flaps that equilibrate with bubbles, thereby reducing bubble concentration . In this way FEN1 employs endonuclease rather than exonuclease to prevent expansions . A model is presented describing the roles of DNA structure, DNA ligase I, and FEN1 in sequence expansion.

EMBO J, 2003 Feb 3, 22(3), 746 - 56
The alternating ATPase domains of MutS control DNA mismatch repair; Lamers MH et al.; DNA mismatch repair is an essential safeguard of genomic integrity by removing base mispairings that may arise from DNA polymerase errors or from homologous recombination between DNA strands . In Escherichia coli, the MutS enzyme recognizes mismatches and initiates repair . MutS has an intrinsic ATPase activity crucial for its function, but which is poorly understood . We show here that within the MutS homodimer, the two chemically identical ATPase sites have different affinities for ADP, and the two sites alternate in ATP hydrolysis . A single residue, Arg697, located at the interface of the two ATPase domains, controls the asymmetry . When mutated, the asymmetry is lost and mismatch repair in vivo is impaired . We propose that asymmetry of the ATPase domains is an essential feature of mismatch repair that controls the timing of the different steps in the repair cascade.

EMBO J, 2003 Feb 3, 22(3), 735 - 45
The RdgC protein of Escherichia coli binds DNA and counters a toxic effect of RecFOR in strains lacking the replication restart protein PriA; Moore T et al.; PriA protein provides a means to load the DnaB replicative helicase at DNA replication fork and D loop structures, and is therefore a key factor in the rescue of stalled or broken forks and subsequent replication restart . We show that the nucleoid-associated RdgC protein binds non-specifically to single-stranded (ss) DNA and double-stranded DNA . It is also essential for growth of a strain lacking PriA, indicating that it might affect replication fork progression or fork rescue . dnaC suppressors of priA overcome this inviability, especially when RecF, RecO or RecR is inactivated, indicating that RdgC avoids or counters a toxic effect of these proteins . Mutations modifying ssDNA-binding (SSB) protein also negate this toxic effect, suggesting that the toxicity reflects inappropriate loading of RecA on SSB-coated ssDNA, leading to excessive or untimely RecA activity . We suggest that binding of RdgC to DNA limits RecA loading, avoiding problems at replication forks that would otherwise require PriA to promote replication restart . Mutations in RNA polymerase also reduce the toxic effect of RecFOR, providing a further link between DNA replication, transcription and repair.

EMBO J, 2003 Feb 3, 22(3), 724 - 34
A model for dsDNA translocation revealed by a structural motif common to RecG and Mfd proteins; Mahdi AA et al.; RecG protein differs from other helicases analysed to atomic resolution in that it mediates strand separation via translocation on double-stranded (ds) rather than single-stranded (ss) DNA . We describe a highly conserved helical hairpin motif in RecG and show it to be important for helicase activity . It places two arginines (R609 and R630) in opposing positions within the component helices where they are stabilized by a network of hydrogen bonds involving a glutamate from helicase motif VI . We suggest that disruption of this feature, triggered by ATP hydrolysis, moves an adjacent loop structure in the dsDNA-binding channel and that a swinging arm motion of this loop drives translocation . Substitutions that reverse the charge at R609 or R630 reduce DNA unwinding and ATPase activities, and increase dsDNA binding, but do not affect branched DNA binding . Sequences forming the helical hairpin and loop structures are highly conserved in Mfd protein, a transcription-coupled DNA repair factor that also translocates on dsDNA . The possibility of type I restriction enzymes and chromatin-remodelling factors using similar structures to drive translocation on dsDNA is discussed.

Drug Deliv, 2003 Jan-Mar, 10(1), 35 - 40
A kinetic model for predicting the release rate of sparingly-water-soluble drugs from a hydrogel-coated polymeric matrix; Fan YL; Medical devices used for on-target drug delivery are often coated with a hydrogel coating for friction-reduction purpose . Thus, the delivery of a sparingly-water-soluble drug by such a device must diffuse through a nonerodable hydrogel layer . An empirical rate equation has been derived for such a kinetic model and predicts that the rate of drug release from such a device is directly proportional to the loading of the drug in the polymeric matrix . The validity of this kinetic model was examined by measuring the rate of release of 2,4,4'-trichloro-2'-hydroxydiphenyl ether from different hydrogel-coated (ethylene-vinyl acetate) copolymer stents containing a wide range of the drug . The experimentally determined release rates are in reasonably good agreement with those calculated from the empirical rate equation . Bioefficacy test results based on zone-of-inhibition test against Escherichia coli are also in good agreement with the release rate and drug-loading data predicted according to the empirical rate equation.

Vet Microbiol, 2003 Apr 29, 92(4), 363 - 77
An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains; Angelos JA et al.; Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA . The DNA flanking mbxA was cloned and sequenced to determine if M . bovis contained a classical RTX operon . Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively . Thus, hemolytic M . bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD . In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5') . At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified . Thus, as with the cya RTX operon of Bordetella pertussis, M . bovis appears to have a secretion accessory protein linked to RTX genes . Analysis of genomic DNA isolated from 5 nonhemolytic M . bovis strains by PCR and Southern blotting revealed the absence of mbxCABD . These strains did, however, amplify with primers specific for the 5' region flanking mbxC . M . bovis harbors a classical RTX operon that is absent in nonhemolytic strains.

Mol Biochem Parasitol, 2003 Jan, 126(1), 15 - 23
Cloning, heterologous expression in Escherichia coli and characterization of a protein disulfide isomerase from Fasciola hepatica; Salazar-Calderon M et al.; A Fasciola hepatica cDNA clone of 1752 bp was isolated from an adult worm cDNA expression library by immunological screening using a rabbit serum against the excretory-secretory antigens . The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 489 codons which encoded a 55 kDa polypeptide, showing a high degree of homology to protein disulfide isomerases . This putative antioxidant protein cDNA was expressed in Escherichia coli as a GST fusion protein . The cleaved recombinant protein was shown to be biologically active in vitro by mediating the oxidative refolding of reduced RNase . Immunoblotting studies using a specific antiserum raised against the recombinant protein showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite . The extracellular location of this protein was also supported by the specific immune responses found against this protein in F . hepatica experimentally infected rabbits.

Clin Biochem, 2003 Feb, 36(1), 41 - 9
Antigenicity of a recombinant NS3 protein representative of ATPase/helicase domain from hepatitis C virus; Penton N et al.; It has been shown that the Hepatitis C virus nonstructural NS3 protein possesses at least two enzymatic domains: a serine-protease domain and an adenosine triphosphatase (ATPase)/helicase domain . In this report, a truncated fragment of NS3 (26 kDa), representing main epitopes from the (ATPase)/helicase domain, has been expressed in Escherichia coli . The recombinant protein was purified by Ion Metal Affinity Chromatography (IMAC) with more than 90% purity . The recognition of B-cell linear epitopes in the NS3 protein was evaluated by immunoblot . The recombinant NS3 protein was reduced and carboxymethylated, and the recognition of either conformational and/or linear B-cell determinants was evaluated by ELISA . The inclusion of the recombinant NS3 protein in a third-generation diagnostic system UltraMicroELISA (UMELISA) allowed an increase in the sensitivity, due to the detection of a new variety of false-negative sera in blood donor test samples.

J Endocrinol, 2003 Feb, 176(2), R1 - 7
The heparin-binding 10 kDa fragment of connective tissue growth factor (CTGF) containing module 4 alone stimulates cell adhesion; Ball DK et al.; Connective tissue growth factor (CTGF) is a 349-residue mosaic protein that contains four structural modules implicated in protein-protein interactions . To address the functionality of residues 247-349 (containing module 4 alone), this region of CTGF was produced as a maltose binding protein (MBP) fusion protein in E . coli . After removal of MBP, recombinant CTGF commenced at Glu(247), was of M(r) 10 000, was immunoreactive with anti-CTGF{247-260}, bound strongly to heparin, and promoted dose-dependent adhesion of fibroblasts, myofibroblasts, endothelial cells, and epithelial cells . An 8 kDa presumptive C-terminally truncated form of CTGF commencing at Glu(247) also promoted cell adhesion . CTGF-mediated cell adhesion was abolished by heparin or EDTA . These data demonstrate the presence of heparin-binding and cell-adhesion motifs within the C-terminal 103 residues of CTGF and show that CTGF-mediated cell adhesion is heparin-and divalent cation-dependent . Thus, CTGF isoforms comprising essentially module 4 are intrinsically functional in the absence of the other constituent modules of CTGF.

Protein Pept Lett, 2002 Dec, 9(6), 553 - 6
Purification, crystallization and preliminary X-ray studies of human augmenter of liver regeneration; Ji CN et al.; Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized . The crystals belong to space group C222, with unit-cell parameters a=51.7 A, b=78.8 A, c=63.7 A . Diffraction data were collected to 2.80 A with a completeness of 99.9% (99.9% for the last shell), a R(sym) value of 0.092(0.236) and an I/sigma(I) value of 6.2(2.7).

Biol Chem, 2002 Dec, 383(12), 1865 - 73
Evaluation of similarities in the cis/trans isomerase function of trigger factor and DnaK; Schiene-Fischer C et al.; Two functionally redundant enzymes, trigger factor and the hsp70 chaperone DnaK, have been found to assist de novo protein folding in E coli . Trigger factor is a peripheral peptidyl prolyl cis/trans isomerase (PPIase) of the large subunit of the ribosome . In contrast, DnaK displays two catalytic features: the secondary amide peptide bond cis/trans isomerase (APIase) function supplemented by the ATPase site . APIases accelerate the cis/trans isomerization of nonprolyl peptide bonds . Both enzymes have affinity for an unfolded polypeptide chain . The diminished low temperature cell viability in the presence of trigger factor variants with impaired PPlase activity indicates that the enhancement of folding rates plays a crucial role in protein folding in vivo . For the DnaK-mediated increase in the folding yield in vitro, the minimal model for APlase catalysis involves the catalyzed partitioning of a rapidly formed folding intermediate as could be inferred from the DnaK/DnaJ/GrpE/ATP-assisted refolding of GdmCl-denatured luciferase . Using three different peptide bond cis/trans isomerization assays in vitro, we could show that there is no overlapping substrate specificity of trigger factor and DnaK . We propose that only if trigger factor recruits supplementing molecules is it capable of exhibiting functional complementarity with DnaK in protein folding.

Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 415 - 20
{Cloning and expression of two garlic virus coat protein genes}; Ma Y et al.; The coat protein(CP) genes of garlic mosaic virus(GMVc) and garlic latent virus(GLVc) isolated from garlic(Allium) plants in Tianjin, China, were amplified from an established cDNA library by PCR method and subsequently expressed in E . coli . using the pET-30a expression system . The determined sequences of GMVc and GLVc CP genes show that the complete GMVc CP gene has 867 nucleotides encoding 289 amino acids . It has 88.5% and 97.2% homology, at the levels of nucleotide and amino acid, respectively, to a reported GMV, indicating that it belongs to Potyvirus . The complete GLVc CP gene has 885 nucleotides coding for 294 amino acids . It has 73.6% and 90.9% homologous percents, in nucleotide and amino acid, respectively, compared to a previously reported GLV, suggesting that it is a member of Carlavirus . The expressed products presented in inclusion body and were analyzed by SDS-PAGE . The molecular weights of GMVc and GLVc CPs appear in 32 kD and 34 kD size, respectively, which are consistent with the deduced sizes of these two CPs . These data will be virtually significant to the further investigation of viruses infecting parlic plant, the control of garlic virus diseases and the production of virus-freed garlic plants.

Wei Sheng Wu Xue Bao, 2001 Aug, 41(4), 402 - 7
{Cloning and sequencing of the gene encoding esterase estA from Ralstonia eutropha CH34}; Song S et al.; An esterase-positive clone was isolated by screening a genomic library of Ralstonia eutropha CH34 constructed in Escherichia coli S17-1 with top agar containing alpha-naphtyl acetate and Fast Blue RR . A gne encoding esterase activity, estA was subcloned from this clone . Nucleotide sequencing of estA showed that it was a 825 bp open reading frame, encoding an esterase EstA, composed of 275 amino acids with a predicted molecular mass of 30,785 D . Homology analysis revealed that EstA exhibited significant amino acid similarity with the enzymes involved in the meta-cleavage pathway in the metaboleism of aromatic acid compounds.

Wei Sheng Wu Xue Bao, 2001 Dec, 41(6), 693 - 8
{Cloning and sequencing of 16S rRNA gene of Phytoplasma CWB1 strain associated with cactus witches' broom}; Cai H et al.; A 1.5 kb DNA fragment was amplified in DNA samples extracted from Opuntia salmiana porm showed witches'-broom symptom . The result indicates the existence of phytoplasma associated with this disease and this phytoplasma was designated as CWB1 . The amplified fragment was ligated to pGEM-T easy vector and then transformed into JM109 strain of E . coli . Cloned DNA fragments were verified by PCR, restriction endonuclease (EcoRI) digestion and sequence analysis . The result revealed that the 16S rRNA gene of CWB1 consists of 1489 bp and shared 99.7% homology with Faba bean phyllody which belongs to phytoplasma 16S rII-C subgroup . So we can classify this strain into phytoplasma 16S rII-C subgroup.

Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 625 - 9
{Construction of mucosal vaccine derived from HBV surface antigen epitope A and the assay of its immunogenicity}; Liu X et al.; The fusion gene of HBV(adr) surface antigen epitope and B subunit of Cholera toxin was constructed and expressed successfully in E . coli at high yield . After denaturation-renaturation process, SDS-PAGE analysis showed that most of the renatured product reassociated in a pentameric form which was the same as natural CTB . Western blot analysis indicated that the immunogenicity of HBsAg antigen epitope was conserved . Moreover, ELISA analysis of the sera of orally, intranasally and intraperitoneally immunized mice showed that the circulating IgG antibodies to HBsAg were developed . The results may be helpful for constructing the novel mucosal vaccine with high efficacy.

Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 559 - 66
{Homologous recombination in Streptomyces lincolnensis B48}; Tang Y et al.; To study frequency and mechanism of homologous recombination in Streptomyces, an E . coli plasmid which cannot replicate in Streptomyces was transformed into Streptomyces lincolnensis B48 . After homologous recombination between delta lincomycin biosynthetic genes inactivated by thiostrepton resistant gene (tsr) carried on pYYE04al and homologous sequences on the chromosome, S . lincolnensis YY1 and S . lincolnensis, YY2 were obtained on SMA with low thiostrepton concentration . Hybridization of chromosomal DNA samples of S . lincolnensis YY1, S . lincolnensis YY2, standard S . lincolnensis and S . lincolnensis YYc digested with SmaI with the probe of tsr gene gave signal corresponding to a fragment of 1.5 kb in the former two; Nevertheless, hybridization of chromosomal DNA digested with Hind III and Sma I using the probe of delta lacZ' gene resulted in positive fragment of 4.4 kb only in S . lincolnensis YY2 . Southern hybridizations indicate that S . lincolnensis YY1 is the result of homologous exchange while S . lincolnensis YY2 comes from bomologous recombination . To prove the existence of E . coli replicon and ampicillin resistant gene on the chromosome of S . lincolnensis YY2, its DNA digested with SphI was ligated and then transformed into E . coli JM83 competent cell . Two transformants named pSLE1 grew on the plate containing ampicillin . It's confirmed that pSLE1 is a part of pYYE04a1 from its digestion with different enzymes.

Wei Sheng Wu Xue Bao, 2001 Oct, 41(5), 523 - 9
{The different functions of glnB and glnZ from Azospirillum brasilense YU62 in the control of nitrogen fixation}; Chen S et al.; The glnB and glnZ genes of A . brasilense have 70% homology at nucleotide sequence . glnB is located in a 3.7 kb Eco RI+ PstI fragment and glnZ is located in a 3.7 kb SalI fragment . Both glnB and glnZ genes were mutagenized by Kmr cassette insertions and glnB- and glnZ- mutants were obtained . glnB- mutant did not have any nitrogenase activity, while glnZ- mutant still has nitrogenase activity . The coding regions of glnB and glnZ were cloned into pVK100 vectors and recombinant plasmids pVK-II and pVK-Z were obtained, respectively . The recombinant plasmids pVK-II and pVK-Z were introduced into glnB- and glnZ- to produce C-glnB and C-glnZ, respectively . C-glnB can restore nitrogenase activity and C-glnZ does not have effect on nitrogenase activity . When pVK-II and pVK-Z were introduced into A . brasilense Yu62 and draT-, respectively, the Yu62-II (containing pVK-II) and draT-II (containing pVK-II) have higher nitrogenase activity than that of wild type Yu62 . In contrast, Yu62-Z (containing pVK-Z) and draT-Z (containing pVK-Z) has no effect on nitrogenase activity . The nifA(-)-II (containg pVK-II) and nifA(-)-Z (containing pVK-Z) still have no nitrogenase activity.

J Infect Dis, 2003 Feb 1, 187(3), 418 - 23 Epub 2003 Jan 24.
Inheritance of susceptibility to induced Escherichia coli bladder and kidney infections in female C3H/HeJ mice; Hopkins WJ et al.; In the present study, the inheritance of resistance and susceptibility to bladder and kidney infections in BALB/c, C3H/HeJ, F(1), and backcross mice was investigated, and the number of genes contributing to the phenotypes was estimated . Infections were induced in female mice by intravesical inoculation with Escherichia coli, and the number of bacteria in bladder and kidneys was quantified at 10 days . The (BALB/c x C3H/HeJ) F(1) mice had bladder and kidney infection intensities equivalent to those observed in the resistant BALB/c parents . Twelve percent of the (F(1) x C3H/HeJ) backcross mice had severe bladder infections, similar to the susceptible C3H/HeJ parents . Kidney infections ranging in intensity between those observed in BALB/c and C3H/HeJ parents were present in one-half of the backcross mice . Statistical analyses indicated that >/=1 gene is responsible for the increased susceptibility of C3H/HeJ mice and that the trait appears to be recessive.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 952 - 7 Epub 2003 Jan 27.
Determination of the energetics governing the regulatory step in growth hormone-induced receptor homodimerization; Bernat B et al.; Signaling in the human growth hormone (hGH)-human GH receptor system is initiated by a controlled sequential two-step hormone-induced dimerization of two hGH receptors via their extracellular domains (ECDs) . Little is currently known about the energetics governing the important regulatory step in receptor signaling (step 2) because of previously existing experimental barriers in characterizing the binding of the second receptor (ECD2) . A further complication is that ECD2 binds through contacts from two spatially distinct sites: through its N-terminal domain to hGH, and to ECD1 through its C-terminal domain, which forms a pseudo-2-fold symmetrical interaction between the stems of the two receptors . We report here a detailed evaluation of the energetics of step 2 binding using a modified surface plasmon resonance method that is able to measure accurately the kinetics of the trimolecular binding process and separate the effects of the two binding sites . The binding kinetics of 23 single and 126 ECD1-ECD2 pair-wise alanine mutations was measured . Although both of the ECD2 binding interfaces were found to be important, the ECD1-ECD2 stem-stem contact is the stronger of the two . It was determined that most residues in the binding interfaces act in additive fashion, and that the six residues common in both ECDs contribute very differently to homodimerization depending on which ECD they reside in . This interface is characterized by a binding "hot-spot" consisting of a core of three residues in ECD1 and two in ECD2 . There is no similar hot-spot in the N-terminal domain of ECD2 binding to Site2 of hGH . This study suggests ways to engineer ECD molecules that will bind specifically to either Site1 or Site2 of hGH, providing novel reagents for biophysical and biological studies.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 856 - 61 Epub 2003 Jan 24.
Structural evidence for substrate strain in antibody catalysis; Yin J et al.; The crystal structure of the Michaelis complex between the Fab fragment of ferrochelatase antibody 7G12 and its substrate mesoporphyrin has been solved to 2.6-A resolution . The antibody-bound mesoporphyrin clearly adopts a nonplanar conformation and reveals that the antibody catalyzes the porphyrin metallation reaction by straining/distorting the bound substrate toward the transition-state configuration . The crystal structures of the Fab fragment of the germ-line precursor antibody to 7G12 and its complex with the hapten N-methylmesoporphyrin have also been solved . A comparison of these structures with the corresponding structures of the affinity-matured antibody 7G12 reveals the molecular mechanism by which the immune system evolves binding energy to catalyze this reaction.

J Biol Chem, 2003 Mar 28, 278(13), 11237 - 45 Epub 2003 Jan 27.
C termini of the Escherichia coli mechanosensitive ion channel (MscS) move apart upon the channel opening; Koprowski P et al.; Heptameric YggB is a mechanosensitive ion channel (MscS) from the inner membrane of Escherichia coli . We demonstrate, using the patch clamp technique, that cross-linking of the YggB C termini led to irreversible inhibition of the channel activities . Application of Ni(2+) to the YggB-His(6) channels with the hexahistidine tags added to the ends of their C termini also resulted in a marked but reversible decrease of activities . Western blot revealed that YggB-His(6) oligomers are more stable in the presence of Ni(2+), providing evidence that Ni(2+) is coordinated between C termini from different subunits of the channel . Intersubunit coordination of Ni(2+) affecting channel activities occurred in the channel closed conformation and not in the open state . This may suggest that the C termini move apart upon channel opening and are involved in the channel activation . We propose that the as yet undefined C-terminal region may form a cytoplasmic gate of the channel . The results are discussed and interpreted based on the recently released quaternary structure of the channel.

J Biol Chem, 2003 Apr 11, 278(15), 13235 - 43 Epub 2003 Jan 27.
Functional roles of loops 3 and 4 in the cyclic nucleotide binding domain of cyclic AMP receptor protein from Escherichia coli; Chen R et al.; Cyclic AMP is a ubiquitous secondary message that regulates a large variety of functions . The protein structural motif that binds cAMP is highly conserved with the exception of loops 3 and 4, whose structure and length are variable . The cAMP receptor protein of Escherichia coli, CRP, was employed as a model system to elucidate the functional roles of these loops . Based on the sequence differences between CRP and cyclic nucleotide gated channel, three mutants of CRP were constructed: deletion (residues 54-56 in loop 3 were deleted), insertion (loop 4 was lengthened by 5 residues between Glu-78 and Gly-79) and double mutants . The effects of these mutations on the structure and function of CRP were monitored . Results show that the deletion and insertion mutations do not significantly change the secondary structure of CRP, although the tertiary and quaternary structures are perturbed . The functional data indicate that loop 3 modulates the binding affinities of cAMP and DNA . Although the lengthened loop 4 may have some fine-tuning functions, the specific function of the original loop 4 of CRP remains uncertain . The function consequences of mutation in loop 3 of CRP are similar to that of site A and site B in the regulatory subunits of cyclic AMP-dependent protein kinases . Thus, the roles played by loop 3 in CRP may represent a more common mechanism employed by cyclic nucleotide binding domain in modulating ligand binding affinity and intramolecular communication.

Life Sci, 2003 Feb 21, 72(14), 1627 - 33
Marathon running alters the DNA base excision repair in human skeletal muscle; Radak Z et al.; Reactive oxygen and nitrogen species generated either as products of aerobic metabolism or as a consequence of environmental mutagens, oxidatively modify DNA . Formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (endo III) or their functional mammalian homologues repair 7,8-dihydro-8-oxoguanine (8-oxoG) and damaged pyrimidines, respectively, to curb the deleterious effects of oxidative DNA alterations . A single bout of physical exercise can induce oxidative DNA damage . However, its effect on the activity of repair enzymes is not known . Here we report that the activity of a functional homolog of Fpg, human 8-oxoG DNA glycosylase (hOGG1), is increased significantly, as measured by the excision of 32P labeled damaged oligonucleotide, in human skeletal muscle after a marathon race . The AP site repair enzyme did not change significantly . Despite the large individual differences among the six subjects measured, data suggest that a single-bout of aerobic exercise increases the activity of hOGG1 which is responsible for the excision of 8-oxoG . The up-regulation of DNA repair enzymes might be an important part of the regular exercise induced adaptation process.

Biochem J, 2003 May 1, 371(Pt 3), 965 - 72
Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase; Kern R et al.; Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase . We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress . Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation . They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines . Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine . Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions . It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor . These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function.

Biochemistry, 2003 Feb 4, 42(4), 1160 - 9
The initiating steps of a type II fatty acid synthase in Plasmodium falciparum are catalyzed by pfACP, pfMCAT, and pfKASIII; Prigge ST et al.; Malaria, a disease caused by protozoan parasites of the genus Plasmodium, is one of the most dangerous infectious diseases, claiming millions of lives and infecting hundreds of millions of people annually . The pressing need for new antimalarials has been answered by the discovery of new drug targets from the malaria genome project . One of the early findings was the discovery of two genes encoding Type II fatty acid biosynthesis proteins: ACP (acyl carrier protein) and KASIII (beta-ketoacyl-ACP synthase III) . The initiating steps of a Type II system require a third protein: malonyl-coenzyme A:ACP transacylase (MCAT) . Here we report the identification of a single gene from P . falciparum encoding pfMCAT and the functional characterization of this enzyme . Pure recombinant pfMCAT catalyzes malonyl transfer from malonyl-coenzyme A (malonyl-CoA) to pfACP . In contrast, pfACP(trans), a construct of pfACP containing an amino-terminal apicoplast transit peptide, was not a substrate for pfMCAT . The product of the pfMCAT reaction, malonyl-pfACP, is a substrate for pfKASIII, which catalyzes the decarboxylative condensation of malonyl-pfACP and various acyl-CoAs . Consistent with a role in de novo fatty acid biosynthesis, pfKASIII exhibited typical KAS (beta-ketoacyl ACP synthase) activity using acetyl-CoA as substrate (k(cat) 230 min(-1), K(M) 17.9 +/- 3.4 microM) . The pfKASIII can also catalyze the condensation of malonyl-pfACP and butyryl-CoA (k(cat) 200 min(-1), K(M) 35.7 +/- 4.4 microM) with similar efficiency, whereas isobutyryl-CoA is a poor substrate and displayed 13-fold less activity than that observed for acetyl-CoA . The pfKASIII has little preference for malonyl-pfACP (k(cat)/K(M) 64.9 min(-1)microM(-1)) over E . coli malonyl-ACP (k(cat)/K(M) 44.8 min(-1)microM(-1)) . The pfKASIII also catalyzes the acyl-CoA:ACP transacylase (ACAT) reaction typically exhibited by KASIII enzymes, but does so almost 700-fold slower than the KAS reaction . Thiolactomycin did not inhbit pfKASIII (IC(50) > 330 microM), but three structurally similar substituted 1,2-dithiole-3-one compounds did inhibit pfKASIII with IC(50) values between 0.53 microM and 10.4 microM . These compounds also inhibited the growth of P . falciparum in culture.

Biochemistry, 2003 Feb 4, 42(4), 1109 - 17
MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli; Kambampati R et al.; Thionucleosides are uniquely present in tRNA . In many organisms, tRNA specific for Lys, Glu, and Gln contain hypermodified 2-thiouridine (s(2)U) derivatives at wobble position 34 . The s(2) group of s(2)U34 stabilizes anticodon structure, confers ribosome binding ability to tRNA and improves reading frame maintenance . Earlier studies have mapped and later identified the mnmA gene (formerly asuE or trmU) as required for the s(2)U modification in Escherichia coli . We have prepared a nonpolar deletion of the mnmA gene and show that it is not required for viability in E . coli . We also cloned mnmA from E . coli, and overproduced and purified the protein . Using a gel mobility shift assay, we show that MnmA binds to unmodified E . coli tRNA(Lys) with affinity in the low micromolar range . MnmA does not bind observably to the nonsubstrate E . coli tRNA(Phe) . Corroborating this, tRNA(Glu) protected MnmA from tryptic digestion . ATP also protected MnmA from trypsinolysis, suggesting the presence of an ATP binding site that is consistent with analysis of the amino acid sequence . We have reconstituted the in vitro biosynthesis of s(2)U using unmodified E . coli tRNA(Glu) as a substrate . The activity requires MnmA, Mg-ATP, l-cysteine, and the cysteine desulfurase IscS . HPLC analysis of thiolated tRNA digests using {(35)S}cysteine confirms that the product of the in vitro reaction is s(2)U . As in the case of 4-thiouridine synthesis, purified IscS-persulfide is able to provide sulfur for in vitro s(2)U synthesis in the absence of cysteine . Small RNAs that represent the anticodon stem loops for tRNA(Glu) and tRNA(Lys) are substrates of comparable activity to the full length tRNAs, indicating that the major determinants for substrate recognition are contained within this region.

Biochemistry, 2003 Feb 4, 42(4), 1095 - 100
Role of glutamate-126 and arginine-144 in the lactose permease of Escherichia coli; Johnson JL et al.; Several previous studies have suggested that glutamate-126 and arginine-144 in the lactose permease of Escherichia coli form an ion pair that is essential for sugar binding . To further investigate the role of these residues, E126Q, R144Q, and R144S mutants were made . The R144Q and R144S strains, which had negligible levels of transport, were used as parental strains to isolate suppressor mutations that partially restored sugar transport . The R144Q parent only yielded first-site revertants, but the R144S strain produced three types of second-site replacements: E126Q, V229A, and L330R . In downhill transport assays, the E126Q strain was able to transport lactose at low levels, with an apparent K(m) 3-fold higher than the wild-type strain but a severely depressed apparent V(max) . A triple mutant, E126Q/R144S/V229A, showed a relatively robust V(max) value for downhill transport and could actively accumulate lactose against a concentration gradient . Taken together, these results indicate that Glu-126 and Arg-144 are not essential for sugar binding . An alternative explanation for their role in maintaining secondary structure is discussed.

Biochemistry, 2003 Feb 4, 42(4), 1078 - 85
Structural/functional characterization of the alpha 2-plasmin inhibitor C-terminal peptide; Frank PS et al.; The alpha(2)-plasmin inhibitor (A2PI) is a main physiological regulator of the trypsin-like serine proteinase plasmin . It is composed of an N-terminal 15 amino acid fibrin cross-linking polypeptide, a 382-residue serpin domain, and a flexible C-terminal segment . The latter, peptide Asn(398)-Lys(452), and its Lys452Ala mutant were expressed as recombinant proteins in Escherichia coli (r-A2PIC and r-A2PICmut, respectively) . CD and NMR analyses indicate that r-A2PIC is flexible, loosely folded, and with low content of regular secondary structure . Functional characterization via intrinsic fluorescence ligand titrations shows that r-A2PIC interacts with the isolated plasminogen kringle 1 (r-K1) (K(a) approximately 69.9 mM(-)(1)), K4 (K(a) approximately 45.7 mM(-)(1)), K5 (K(a) approximately 4.3 mM(-)(1)), and r-K2 (K(a) approximately 3.2 mM(-)(1)), all of which are known to exhibit lysine-binding capability . The affinities of these kringles for r-A2PIC are consistently larger than those reported for the ligand N(alpha)-acetyllysine, a mimic of a C-terminal Lys residue . The r-A2PICmut, with a C-terminal Ala residue, also interacts with r-K1 and K4, although with approximately 5-fold lesser affinities relative to r-A2PIC, demonstrating that while Lys(452) plays a major role in the binding, internal residues in r-A2PIC tether the kringles . (1)H NMR spectroscopy shows that key aromatic residues within the K4 lysine-binding site (LBS), namely, Trp(25), Trp(62), Phe(64), Trp(72), and Tyr(74), selectively respond to the addition of r-A2PIC and r-A2PICmut, indicating that these interactions proceed via the kringles' canonical LBS . We conclude that r-A2PIC docks to kringles primarily through lysine side chains and that Lys(452) most definitely enhances the binding . This suggests that multiple Lys residues within A2PI could contribute, perhaps in a zipper-like fashion, to its binding to the in-tandem, multikringle array that configures the plasmin heavy chain.

Biochemistry, 2003 Feb 4, 42(4), 1024 - 30
Binding of Zn-chlorin to a synthetic four-helix bundle peptide through histidine ligation; Razeghifard MR et al.; We have used a two histidine-containing synthetic peptide (Sharp et al . (1998) Proc . Natl . Acad . Sci . U.S.A . 95, 10465-10470) as a scaffold to bind Zn(II) chlorin e6 (ZnCe6) through histidine ligation . Protocols for the preparation and purification of the peptide using an Escherichia coli expression system are presented . Size-exclusion chromatography and circular dichroism measurements indicate that the peptide self-assembles into a four-helix bundle protein . Two variants of the peptide lacking either one or both of the histidine residues were used to demonstrate the stoichiometry of ZnCe6 binding . Comparison of the titration profiles determined by UV-vis spectroscopy for the purified one- and two-histidine peptides suggests that the two-histidine peptide can bind two ZnCe6 . The binding stoichiometry of ZnCe6 was verified by gel chromatography and native gel electrophoresis using the peptide variant lacking histidine residues as the control . Like many other chlorophyll analogue molecules, ZnCe6 can be photooxidized . The light-induced electron transfer between the ZnCe6-peptide complex and the added phenyl-p-benzoquinone was measured using time-resolved EPR spectroscopy and shown to be faster and have a higher yield than the electron transfer between unbound ZnCe6 and quinone . The implications of constructing a ZnCe6-peptide complex in terms of artificial photosynthesis are discussed.

Biochemistry, 2003 Feb 4, 42(4), 864 - 9
Tautomeric rearrangement of a dihydroflavin bound to monomeric sarcosine oxidase or N-methyltryptophan oxidase; Khanna P et al.; Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous bacterial flavoenzymes that contain covalently bound flavin {8alpha-(S-cysteinyl)FAD} . Reaction of MSOX or MTOX with a small excess of sodium borohydride results in immediate flavin reduction to a species that exhibits spectral properties (lambda(max) = 405 nm with a second broad peak at 332 nm) similar to those of 3,4-dihydroflavin . The borohydride-reduced enzymes retain full catalytic activity . Substrate reduction converts the 405 nm species to an air-sensitive tetrahydroflavin that reacts with oxygen to yield unmodified oxidized enzyme . Unexpectedly, the putative 3,4-dihydroflavin bound to MSOX or MTOX is unstable in the absence of substrate . An isosbestic conversion of the 405 nm species to yield unmodified, oxidized flavin is observed when the reaction is conducted under aerobic conditions (k(obs) = 4.9 x 10(-2) min(-1)) . Under anaerobic conditions, an oxygen-sensitive species resembling 1,5-dihydroflavin is formed in an isosbestic reaction that occurs at a rate similar to that of the aerobic reaction (k(obs) = 5.3 x 10(-2) min(-1)) . Possible reaction of the 3,4-dihydroflavin with a second molecule of borohydride to yield an air-sensitive tetrahydroflavin is unlikely since prior scavenging of residual borohydride with excess formaldehyde had no effect on the aerobic conversion to unmodified oxidized flavin . The observed instability is attributed to a tautomeric rearrangement of the 3,4-dihydroflavin to generate 1,5-dihydroflavin, a species that is also air-sensitive . Evidence in favor of an active site facilitated tautomerization reaction is provided by the fact that the stability of the 405 nm species formed with MSOX is enhanced 200-fold upon denaturation with urea or heat . The observed tautomeric rearrangement of 3,4-dihydroflavin may provide insight regarding a related flavin tautomerization reaction that has been proposed as a key step in the biosynthesis of covalent flavin linkages.

Wei Sheng Wu Xue Bao, 1998 Feb, 38(1), 26 - 31
{Research on the regulation of glucoamylase gene(glaA) expression in A . niger . II . Analysis of the function of 5'-regulatory region of A . niger T21 and 3.795 glaA gene}; Qiao D et al.; Two plasmid vectors pXH2 and pGH1 were constructed through the fusion of E . coli hph gene, the report gene and the 5' upstream regions of A . niger T21 and 3.795 respectively, as well as the terminator of A . nidulans trpC gene . The plasmid vectors were than used to transform A . niger T21 to functionally identify those different basic groups between the two 5' upstream regions responsible for high-level expression of the glaA gene . Southern analysis of two transformants XH2C and GH1C revealed that pXH2 and pGH1 were integrated respectively into the chromosome at same site with two copies in tandem array . The level resistant to HmB(3000 micrograms/ml) of XH2C was twice as high as that (1500 micrograms/ml) of GH1C, indicating that the changes of basic groups through mutation result in twice increase of functional level of region responsible for transcription and regulation of A . niger T21 glaA gene compared with that of 3.795.

Wei Sheng Wu Xue Bao, 1998 Apr, 38(2), 103 - 7
{Studies on the selection of strains producing colominic acid and culture conditions}; Guo L et al.; One strain E . coli C-8, the highest yield of colominic acid, was selected from 40 E . coli strains in the medium in which glucose and ammonium sulfate were the only carbon and nitrogen resources . An optimum medium for the growth and colominic acid production of E . coli C-8 was studied . The optimum carbon resources for colominic acid production was sorbitol selected from 16 kinds of carbons, and its optimum concentration was 2.5% . The optimum inorganic and organic nitrogen resources for colominic acid production were ammonium sulfate (0.5%) and tryptone (1.5%), respectively . The optimum concentration of K2 HPO4 was 90 mmol/L . The optimum culture temperature was 37 degrees C, but no colominic acid was produced below 20 degrees C . The optimum pH range was 7.5-8.2 . The strain growth in the optimum medium kept logarithmic phase in 40 h (maxim A = 22) . The colominic acid secreted into medium was much lower before 20 h, but high biosynthetic rate of colominic acid was detected after 40 h, the colominic acid reached the highest level (1,200 micrograms/ml) at 65 h.

Wei Sheng Wu Xue Bao, 1998 Jun, 38(3), 193 - 6
{Expression, purification and characterization of recombinant human cyclophilin A}; Li F et al.; Plasmid-derived expression of the human CyPA in E . coli would make it possible to obtain ample protein quantities and to avoid difficult task of obtaining human tissues for protein purification . The cDNA encoding human CyPA from MT4 lymph cell line has been cloned and an expression vector (pET11/CyPA) has been constructed under control of the T7 promoter for efficient expression in E . coli . The recombinant CyPA is produced at 41% of total soluble cell protein, and showed the peptidyl-prodyl cis-trans isomerase activity.

Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 65 - 9
{Construction of plasmid gene bank of V . cholerae O139 and detection of O-antigen genes}; Qu D et al.; Because O-antigen biosynthesis genes are a tandem gene cluster . Gnomic fragments of 4-20 kilobases (kb) were obtained by digesting genomic DNA of V . cholerae O139 with restriction endonuclease EcoRI, then plasmid gene bank was constructed . Recombinant colony, E . coli DH5 alpha (pMG320), expressing O-antigen of V . cholerae O139 was detected from the bank by immunological agglutinative reaction . The futher analysis showed O-antigen expressed by recombinant colony had both immunogenicity and reactogenicity, and the size of O-antigen biosynthesis genes was about 4.6 kb.

Wei Sheng Wu Xue Bao, 2001 Feb, 41(1), 9 - 15
{Subcloning and sequencing of DNA fragment related to salt tolerance in Sinorhizobium meliloti 042B}; Ge S et al.; A 4 kb ClaI DNA fragment related to salt tolerance from S . meliloti 042B was digested by HindIII down 2.4 kb fragment, and a 1.6 kb ClaII-HindIII fragment was retained on plasmid pML122 . Then, the 2.4 kb DNA fragment was ligated with plasmid pBBR1-MCS2, and the recombinant plasmid was transformed to E . coli DH5 alpha, and transformant GS2 was obtained . Three-parental mating experiments were carried out with transformant GS2 as donor, salt sensitive strains GZ17 as recipient and pRK2013 as helper plasmid, then the transconjugant GG2 was selected on FY plates containing kanamycin and 0.4 mol/L NaCl . The remaining DNA fragment was self ligated with pML122 and then transformed into E . coli S17-1 and transformat GS0 was obtained . Two-parental mating experiment was carried out with transformant GS0 as donor and salt sensitive strain GZ17 as recipient, but no transconjugant was obtained on the FY plates . Then, the 2.4 kb HindIII DNA fragment was ligated into sequencing vector pGEM-7Zf(+) for sequencing . The result of sequencing and analysis showed that the 2.4 kb DNA fragment contained three ORFs . According to the result of sequencing, further subcloning was conducted and 1.9 kb HindIII-Sac II DNA fragment related to salt tolerance was obtained.

Wei Sheng Wu Xue Bao, 2000 Dec, 40(6), 605 - 9
{The construction and application of Streptomyces-E . coli shuttle plasmid pSGLgpp}; Zhang H et al.; The high-copy-number plasmid pSGL1(7.4 kb) was isolated from Streptomyces geobisporous . Its minimal replicon had been determined and sequenced . With the total DNA of S . geobisporus as template, the DNA fragment involving C1027 apoprotein signal peptide encoding sequence (gpp) was amplified by PCR . After this fragment was inserted into the plasmid pSGLN, a derivative plasmid of pSGL1, one new Streptomyces-E . coli shuttle plasmid was obtained namely as pSGLgpp . Using the new vector, we carried out the expression of hsIL-1RI in streptomyces.

Shi Yan Sheng Wu Xue Bao, 2000 Sep, 33(3), 229 - 35
{Cloning, expression of a segment of hTERT gene and detection of telomerase and hTERT by anti-hTERT polyclonal antibody}; Saleh SA et al.; Telomerase is an important biomarker in cancer cells . It is active in germline cells, most of cancer tissues and cell lines, but not in most somatic tissues . Telomerase is composed of two components, and while hTER is present in normal and tumor cells, expression of hTERT appears to be highly regulated and correlates with telomerase activity . In order to detect the telomerase enzyme and hTERT protein, anti-hTERT polyclonal antibodies were produced in this study . A segment of hTERT cDNA was amplified by RT-PCR and cloned into the multi-cloning site of the GST gene fusion vector pGEX-5X-3 . After the recombinant plasmid was expressed in E . coli BL21, the fusion protein was purified for immunization . Extracts from several cultured cells were analyzed by Western blot, and the results indicated that telomerase enzyme and hTERT protein could be specifically detected by this anti-hTERT antibod' . Thus, a simple and effective method was primarily established for the immunodetection of telomerase enzyme and hTERT protein.

Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 248 - 51
{Cloning and expression of D-arabitol dehydrogenase gene from Acetobacter suboxydans in Escherichia coli}; He X et al.; The partial genomic library of Acetobacter suboxydans was constructed using Yeast-E . coli shuttle plasmid YEp352 as vector . Two positive transformants, designated as DH5 alpha(pAD91) and DH5 alpha(pAD98), were obtained by screening the growth of transformants on the agar plate in which D-arabitol was used as the sole carbon source . The results of Southern blot and restriction endonuclease analysis showed that the two recombinants are identical . The insert is about 2.3 kb . Arabitol dehydrogenase activity assay indicated that the transformants could produce D-xylulose-forming D-arabitol dehydrogenase . Hence, the gene encoding D-arabitol dehydrogenase exists in the cloned DNA fragment.

Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 186 - 90
{Purification and gene cloning of a novel fibrinolytic protease from Streptomyces sp . C3662}; Gong Y et al.; A novel fibrinolytic protease from Streptomyces sp . C3662 was purified by (NH4)2SO4 fractionation, DEAE-Sepharose and CM-Sepharose chromatography . The molecular weight of the protease was indicated to be 30 kD by SDS-PAGE . Using a E . coli/Streptomyces shuttle plasmid pIJ699 as the vector, shot-gun cloning was performed to clone the gene of the protease . One clone with fibrinolytic activity harboring a plasmid that contains a DNA fragment of 6 kb was obtained from 3000 clones . Sequence analysis reveals that the open reading frame of the gene of the protease is 903 bp in size, encoding a putative protein of 300 amino acids with 30 kD . The overall GC% and the third codon GC% of the ORF were 68.33% and 95.6%, respectively . Comparison of homologue showed that the purative protein is highly homologous with other proteases of Streptomyces sp.

Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 181 - 5
{Cloning of promelittin cDNA and its expression in Escherichia coli}; Wang G et al.; The cDNA encoding promelittin was obtained from the total RNA of bee poison gland by RT-PCR and was cloned to pT7Blu-T vector . The expression vector of promelittin fused with partial sequence of beta-galactosidase was constructed by ligating the fragment inserted to pUC118 . Moreover, it was expressed in the strain DH5 alpha of Escherichia coli . The result of DNA sequence analysis demonstrated that the obtained cDNA sequence was same with the published one and the reading frame of fusion gene was correct.

Wei Sheng Wu Xue Bao, 2001 Apr, 41(2), 173 - 80
{Analysis and expression of Hyphantria cunea nuclear polyhedrosis virus sod gene}; Cao G et al.; The sequencing results indicated that Hyphantria cunea nuclear polyhedrosis virus (HcNPV) sod gene open reading frame of 456 nt encoding protein of 151 amino acid, was identified to that of Bombyx mori nuclear polyhedrosis virus (BmNPV), and exhibited 97.2% homology at nucletde level to that of Autographa californica nuclear polyhedrosis virus (AcNPV), three amino acid residues difference in amino acid level with AcNPV sod . The essential amino acid residues for the construction and active could be detected in HcNPV sod . Activity of the SOD is 147.09 U per milliliter E . coli.

Wei Sheng Wu Xue Bao, 2000 Jun, 40(3), 284 - 9
{Study on production of poly-beta-hydroxybutyrate by recombinant strain VG1(pTU14)}; Yu H et al.; Cloning and expression of Vitreoscilla hemoglobin gene (vgb) and lambda phage lytic genes (S-RRz) in production of Poly-beta-hydroxybutyrate(PHB) was studied in different Escherichia coli hosts such as E . coliJM105, E . coliJM109 and VG1 . In the recombinant strains, VG1(pTU14) was a superior one which simultaneously contained three exogenes including vgb, S-RRz and PHB biosynthesis genes(phbCAB) . The experimental results showed that after 82 hours fed-batch cult ure in LBG medium in shaking flask, cell concentration of VG1(pTU14) could reach 25.9 g/L, which was the highest PHB production ever reported, and till 52 h PHB content could be higher than 95% . Additionally, inducible cell lysis was also attained successfully in recombinant VG1(pTU14) accompanying with its high cell density culture . Therefore, VG1(pTU14) is a novel potential strain with promising prospect in industrial production of PHB.

Wei Sheng Wu Xue Bao, 2000 Jun, 40(3), 270 - 6
{Selection and recombinant expression of an immunodominant B cell epitope of HIV gp41}; Du Y et al.; A novel method was designed for disease-specific B cell epitope mapping and epitope expression in E . coli . A phage library displaying random dodecamers was biopanned first with human total IgG antibodies against HIV-1, and then non-specific phages were subtracted by HIV(-) polyclonal antibodies . After three rounds of screening, the positive phages were tested in an ELISA for their reactivity with HIV(+)-IgG and HIV(-)-IgG antibodies . Phages that showed positive reactivity with HIV(+)-IgG, but negative to HIV(-)-IgG, were selected and their displayed peptides were determined by DNA sequencing . All the 13 positive clones sequenced displayed five kinds of peptides (SPKCLGKLLCAF, THQCLGKLQCGV, SCSAKFTCTTQI, KSDCSARFMCSV, DCLKQWACEWSR) that have homology to the HIV-1 gp41(602GCSGKLICTTNV613), demonstrating there is a dominant epitope in the region.

Wei Sheng Wu Xue Bao, 2000 Aug, 40(4), 384 - 8
{Cloning and expression of metallothionein gene of Bombyx mori}; He X et al.; Yeast MTI gene from vector pCMI-1 was used as a probe . It appeared strong hybridization signals when the total DNA of Bombyx mori Huishu eggs hybridizes with the probe . The 1-6 kb DNA fragments were isolated from the EcoR I digested total DNA of Bombyx mori Huishu eggs and ligated with M13- vector digested by restriction EcoR I . The ligation mixtures were used to transform E . coli DH5 alpha . blue/White colonies selection was used to identify colonies with insert . Approximately 4000 white colonies were selected, so the part genomic library of Bombyx mori was constructed . Three positive colonies were gained from the genomic library by southern blotting analysis, designated T1 (pZHC-1), T5 (pZHC-5), T7 (pZHC-7) . Digesting the recombinant plasmid pZHC-5 with 12 restriction enzymes, the results suggested that the inserted fragment was about 1.2 kb and there was only a Hind III site . The experiment of resistant to CuSO4 proved that the DH5 alpha cells contain recombinant plasmids were more resistance than the recepient DH5 alpha cells . According to these results, the inserted fragment possibly contains the gene encoding Metallothionein of Bombyx mori . The sequence analysis of the inserted fragment and its high-expressions in E . coli are in progress.

Wei Sheng Wu Xue Bao, 2000 Aug, 40(4), 359 - 64
{Overproduction and partial characterization of the Ssh7a and Ssh7b proteins from Sulfolobus shibatae}; Chen X et al.; The Sulfolobus shibatae Ssh7a and Ssh7b proteins have been separately overproduced in Escherichia coli and purified using a simple procedure which includes a heat treatment step . The recombinant and native Ssh7 proteins are similar in the ability to bind both negatively supercoiled and relaxed DNAs . In addition, the recombinant Ssh7a resembles the native Ssh7 protein in constraining negative DNA supercoils . Our data suggest that the two isoforms of Ssh7 interact with DNA in a similar fashion, and the methylation state of Ssh7 interact with DNA in a similar fashion, and the methylation state of Ssh7 does not affect DNA binding and supercoil constraining by the protein.

Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 155 - 60
{Construction of a novel Bm NPV Bac to Bac system}; Deng X et al.; A Bi-Shuttle vector Bm-Bacmid was constructed by co-infecting Bm N cells with wild type genomic DNA from BmNPV and Ac-Bacmid DNA . It could not only replicate in E . coli cells as a large plasmid and but also remain infectious when induced into Bm N or Sf9 cells . Recombinant virus rBmHBe was obtained after transposition of a donor plasmid carrying Hepatitis Be antigen gene (HBeAg) into att Tn7, and was demonstrated by Southern blotting . SDS-PAGE analysis showed that HBeAg gene were highly expressed in Bm N cells . By ELISA testing, the highest antigenecity titer of HBeAg protein in cell cultural medium was up to a dilution of 1:32,000 . Although HBeAg protein also presented in the Bm N cells the titer was only 1:2000 . The HBcAg protein was much fewer than HBeAg (< 1:160) whatever in culture medium and in cells . The results showed that Bm N cells was able to recognize the signal peptide sequence and cut it correctly for HBeAg protein's excreting production.

Wei Sheng Wu Xue Bao, 2000 Apr, 40(2), 121 - 5
{Expression, purification and characterization of fruA, a transcription factor in Myxococcus xanthus}; Mao X et al.; FruA is a transcription factor essential for the development of Myxocoxccus xanthus . Gene encoding fruA with a poly-histidine tag was expressed in E . coli and simply purified by chromatography on nickel column . Data from gel retardation assay suggest that FruA regulates transcription of target genes in collaboration with other factors.

Wei Sheng Wu Xue Bao, 2000 Feb, 40(1), 50 - 6
{Expression of Vitreoscilla hemoglobin gene in Streptomyces avermitilis}; Wen Y et al.; Two expression vectors, pWY101 and pWY102, were constructed by cloning Vitreoscilla hemoglobin gene(vhb) with its native oxygen-regulated promoter into E . coli-Streptomyces shuttle vector pIJ653 . They were introduced into Streptomyces avermitilis, but Western blotting experiment failed to detect vhb gene expression . pHZ1252 is another shuttle vector for expressing VHb, in which vhb structural gene is controlled by a strong, thiostrepton-inducible Streptomyces promoter PtipA . pHZ1252 was transformed into S . avermitilis and expressed VHb which had biological activity after thiostrepton induction . pHZ1252 was structurally unstable in S . avermitilis, occurring deletion recombination . However, the rest part of pHZ1252 was stable in S . avermitilis and still contained vhb gene and PtipA . Plasmid pHZ1252 isolated from S . avermitilis was unable to transform E . coli, showing the loss of part of E . coli plasmid from pHZ1252.

Proteomics, 2003 Jan, 3(1), 36 - 44
A novel experimental design for comparative two-dimensional gel analysis: two-dimensional difference gel electrophoresis incorporating a pooled internal standard; Alban A et al.; The comparison of two-dimensional (2-D) gel images from different samples is an established method used to study differences in protein expression . Conventional methods rely on comparing images from at least 2 different gels . Due to the high variation between gels, detection and quantification of protein differences can be problematic . Two-dimensional difference gel electrophoresis (Ettan trade mark DIGE) is an emerging technique for comparative proteomics, which improves the reproducibility and reliability of differential protein expression analysis between samples . In the application of DIGE different samples are labelled with mass and charge matched spectrally resolvable fluorescent dyes and are then separated on the same 2-D gel . Using an Escherichia coli lysate "spiked" with varying amounts of four different known proteins, we have tested a novel experimental design that exploits the sample multiplexing capabilities of DIGE, by including a standard sample in each gel . The standard sample comprises equal amounts of each sample to be compared and was found to improve the accuracy of protein quantification between samples from different gels allowing accurate detection of small differences in protein levels between samples.

Biopolymers, 2003 Feb, 68(2), 139 - 49
Stepwise induced fit in the pico- to nanosecond time scale governs the complexation of the even-skipped transcriptional repressor homeodomain to DNA; Flader W et al.; Induced fit effects in the complex of a DNA decamer with two even-skipped transcriptional repressor homeodomain molecules were investigated by means of molecular dynamics simulations . Dynamics of these effects are found to be in the time scale from pico- to nanoseconds . First steps are made by the fast-moving DNA backbone phosphates, which upon binding change their B(I)/B(II) substate distribution . Further rearrangements in the DNA double helix induced upon complexation, like bending of the helix axis, changes of the minor groove width, and of different helical parameters, are slower and occur within a few nanoseconds . The flexibility of the DNA, especially of its backbone, seems thereby to play an important role for specific DNA ligand recognition .

J Pediatr Gastroenterol Nutr, 2003 Feb, 36(2), 253 - 60
Enteropathogenic Escherichia coli: stimulating neutrophil migration across a cultured intestinal epithelium without altering transepithelial conductance; Michail SK et al.; INTRODUCTION: Migration of neutrophils across the intestinal epithelium is the hallmark of inflammatory conditions of the bowel . In cultured intestinal epithelial monolayer models, neutrophils can be induced to migrate along a chemotactic gradient such as n-formyl-methionyl-leucyl-phenylalanine (fMLP) . Physical passage of the neutrophils across the epithelium could disrupt the tight-junctions, possibly leading to a large increase in the transepithelial conductance (G(t)) . The goal of this study is to determine whether transepithelial migration of neutrophils induced by enteropathogenic (EPEC) causes changes in G(t) comparable with those seen with fMLP . METHODS: The apical side of T84 monolayers were rapidly infected with EPEC E2348/69 or exposed to 1 microM fMLP . A third group of monolayers exposed to neither EPEC nor fMLP served as control . Indium-labeled neutrophils were added to the serosal side of monolayers grown on a cell culture insert membrane (12 microm pores) . G(t) was measured at fixed intervals up to 4 hours . After a 150-minute incubation, radioactivity of the neutrophils that migrated to the apical side was assayed and the number of migrating neutrophils was calculated . RESULTS: At 150 minutes, EPEC induced similar neutrophil chemotactic capability compared to fMLP (231 +/- 34.10(3) and 193 +/- 48.10(3), respectively, n = 13, P > 0.05) . However, EPEC-induced neutrophil migration was not associated with significant increase in G(t), 1.13 +/- 0.16 fold of baseline G(t), in distinction with fMLP groups, 13.3 +/- 0.48 fold, n = 7 (P< 0.05) . G(t) changes with EPEC were seen after 4 hours of infection, but were not different in the presence or absence of neutrophil migration (1.37 +/- 0.12 fold and 1.42 +/- 0.17 fold of baseline G(t), respectively) . CONCLUSIONS: The results indicate that EPEC-induced neutrophil migration can occur without significant disruption of barrier function . In addition, the chemo-attractant recruiting neutrophils during EPEC infection is unlikely to be fMLP; and, the G(t) increase seen with fMLP-driven recruitment may indicate a discretionary compromise of barrier function during neutrophil migration.

Biophys J, 2003 Feb, 84(2 Pt 1), 1131 - 45
Fluorescence resonance energy transfer over approximately 130 basepairs in hyperstable lac repressor-DNA loops; Edelman LM et al.; Lac repressor (LacI) binds two operator DNA sites, looping the intervening DNA . DNA molecules containing two lac operators bracketing a sequence-directed bend were previously shown to form hyperstable LacI-looped complexes . Biochemical studies suggested that orienting the operators outward relative to the bend direction (in construct 9C14) stabilizes a positively supercoiled closed form, with a V-shaped LacI, but that the most stable loop construct (11C12) is a more open form . Here, fluorescence resonance energy transfer (FRET) is measured on DNA loops, between fluorescein and TAMRA attached near the two operators, approximately 130 basepairs apart . For 9C14, efficient LacI-induced energy transfer ( approximately 74% based on donor quenching) confirms that the designed DNA shape can force the looped complex into a closed form . From enhanced acceptor emission, correcting for observed donor-dependent quenching of acceptor fluorescence, approximately 52% transfer was observed . Time-resolved FRET suggests that this complex exists in both closed- and open form populations . Less efficient transfer, approximately 10%, was detected for DNA-LacI sandwiches and 11C12-LacI, consistent with an open form loop . This demonstration of long-range FRET in large DNA loops confirms that appropriate DNA design can control loop geometry . LacI flexibility may allow it to maintain looping with other proteins bound or under different intracellular conditions.

Ann N Y Acad Sci, 2002 Dec, 981, 111 - 34
Genome organization and reorganization in evolution: formatting for computation and function; Shapiro JA; This volume deals with the role of epigenetics in life and evolution . The most dynamic forms of functional genome formatting involve DNA interacting with cellular complexes that do not alter sequence information . Such important epigenetic phenomena are the main subjects of other articles in this volume . This article focuses on the long-lived form of genome formatting that lies within the DNA sequence itself . I argue for a computational view of genome function as the long-term information storage organelle of each cell . Structural formatting consists of organizing various signals and coding sequences into computationally ready systems facilitating genome expression and genome transmission . The basic features of genome organization can be understood by examining the E . coli lac operon as a paradigmatic genomic system . Multiple systems are connected through distributed signals and repetitive DNA to form higher-order genome system architectures . Molecular discoveries about mechanisms of DNA restructuring show that cells possess the natural genetic engineering functions necessary for evolutionary change by rearranging genomic components and reorganizing system architectures . The concepts of cellular computation and decision-making, genome system architecture, and natural genetic engineering combine to provide a new way of framing evolutionary theories and understanding genome sequence information.

Fish Shellfish Immunol, 2003 Jan, 14(1), 1 - 23
The rainbow trout classical MHC class I molecule Onmy-UBA*501 is expressed in similar cell types as mammalian classical MHC class I molecules; Dijkstra JM et al.; Onmy-UBA is a polymorphic classical major histocompatibility (MHC) class I locus in rainbow trout (Oncorhynchus mykiss) . A common allomorph is Onmy-UBA*501, which has been detected in several wildtype strains, in the clonal homozygous rainbow trout C25 and, in the current study, in the rainbow trout gonad cell line RTG-2 . The extracellular domain of this allomorph was expressed in E . coli and a murine monoclonal antibody designated H9 was generated against the recombinant protein . In Western blot analysis Mab H9 specifically recognised an n-glycosylated protein of 45 kDa in leucocytes and erythrocytes of C25 fish and in RTG-2 cells . The level of Onmy-UBA*501 expression in erythrocytes was very low . Immunocytochemistry of isolated cells indicated expression in lymphocytes, macrophages, neutrophils, erythrocytes, RTG-2 cells and Onmy-UBA *501 transfected CHO cells, but not in untransfected CHO cells . Immunohistochemistry using frozen sections of C25 fish indicated that Onmy-UBA*501 expression is strong in the lymphoid organs (thymus, head kidney and spleen) and in the epithelia and endothelia of several organs . No significant expression was observed in muscle fibres, hepatocytes or neurons . These observations demonstrate that in jawed fish, the lowest phylogenetic group possessing an MHC system, the classical MHC class I molecules are expressed in similar cell types as in higher vertebrates.

Vaccine, 2003 Feb 14, 21(9-10), 836 - 42
The adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo; Pajak B et al.; The aim of this study was to test the capacity of the novel adjuvant OM-174, a lipid A analog, to induce the migration and the maturation of murine dendritic cells (DC) in vivo, a step which is considered as the initiation of the adaptive immune response . BALB/c mice were injected intravenously or subcutaneously with OM-174 . The spleen and popliteal lymph nodes were harvested, and analyzed for DC localization and phenotype . The data presented here clearly show that, OM-174 induces the migration of DC from the periphery to the T cell areas of lymphoid organs, and their maturation into cells expressing high levels of MHC class II and co-stimulatory molecules, with a potency close to that of Escherichia coli lipopolysaccharide (LPS).

Mutat Res, 2003 Feb 5, 535(1), 55 - 72
Mutagenicity test system based on a reporter gene assay for short-term detection of mutagens (MutaGen assay); Schmid C et al.; The construction of a bacterial mutation assay system detecting reversions of base substitutions and frameshifts in tetracycline (tet) and ampicillin resistance genes located on low copy plasmids is described . Frameshift mutations were introduced into repetitive GC-sequences and G-repeats known to be mutagenic hot-spots . Base pair substitutions were inserted in or around the active site of the ampicillinase gene thus generating reversibility of the ampicilline sensitivity . The plasmids carry genes to enable sensitive, fast and specific detection of mutagens in bacteria . MucAB was cloned into the test plasmid to enhance error-prone DNA-repair . The conventional reversion principle has been combined with the luminometric measurement of an inducible reporter gene . The revertants are detected after induction of the beta-galactosidase-producing lacZ-gene either controlled by its natural lac-promotor or by the more stringently repressed (anhydrotetracyclin inducible) tetA promotor . The tester strains containing the tetA/lacZ reporter gene construct can grow in full medium over the complete assay . This test procedure enables screening for mutations within one working day . Incubation for 16 h reveals high sensitivity .

J Mol Biol, 2003 Feb 7, 326(1), 217 - 23
Solution structure of the R3H domain from human Smubp-2; Liepinsh E et al.; The R3H domain is a conserved sequence motif, identified in over 100 proteins, that is thought to be involved in polynucleotide-binding, including DNA, RNA and single-stranded DNA . In this work the 3D structure of the R3H domain from human Smubp-2 was determined by NMR spectroscopy . It is the first 3D structure determination of an R3H domain . The fold presents a small motif, consisting of a three-stranded antiparallel beta-sheet and two alpha-helices, which is related to the structures of the YhhP protein and the C-terminal domain of the translational initiation factor IF3 . The similarities are non-trivial, as the amino acid identities are below 10% . Three conserved basic residues cluster on the same face of the R3H domain and could play a role in nucleic acid recognition . An extended hydrophobic area at a different site of the molecular surface could act as a protein-binding site . A strong correlation between conservation of hydrophobic amino acids and side-chain solvent protection indicates that the structure of the Smubp-2 R3H domain is representative of R3H domains in general.

J Mol Biol, 2003 Feb 7, 326(1), 203 - 16
Aspartate transcarbamylase from the hyperthermophilic archaeon Pyrococcus abyssi: thermostability and 1.8A resolution crystal structure of the catalytic subunit complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate; Van Boxstael S et al.; The Pyrococcus abyssi aspartate transcarbamylase (ATCase) shows a high degree of structural conservation with respect to the well-studied mesophilic Escherichia coli ATCase, including the association of catalytic and regulatory subunits . The adaptation of its catalytic function to high temperature was investigated, using enzyme purified from recombinant E.coli cells . At 90 degrees C, the activity of the trimeric catalytic subunit was shown to be intrinsically thermostable . Significant extrinsic stabilization by phosphate, a product of the reaction, was observed when the temperature was raised to 98 degrees C . Comparison with the holoenzyme showed that association with regulatory subunits further increases thermostability . To provide further insight into the mechanisms of its adaptation to high temperature, the crystal structure of the catalytic subunit liganded with the analogue N-phosphonacetyl-L-aspartate (PALA) was solved to 1.8A resolution and compared to that of the PALA-liganded catalytic subunit from E.coli . Interactions with PALA are strictly conserved . This, together with the similar activation energies calculated for the two proteins, suggests that the reaction mechanism of the P.abyssi catalytic subunit is similar to that of the E.coli subunit . Several structural elements potentially contributing to thermostability were identified: (i) a marked decrease in the number of thermolabile residues; (ii) an increased number of charged residues and a concomitant increase of salt links at the interface between the monomers, as well as the formation of an ion-pair network at the protein surface; (iii) the shortening of three loops and the shortening of the N and C termini . Other known thermostabilizing devices such as increased packing density or reduction of cavity volumes do not appear to contribute to the high thermostability of the P.abyssi enzyme.

Zhonghua Gan Zang Bing Za Zhi, 2003 Jan, 11(1), 5 - 7
{Cloning of genes transactivated by hepatitis B virus X protein}; Liu Y et al.; OBJECTIVE: To construct a subtractive cDNA library of genes transactivated by hepatitis B virus X protein (HBX) using suppression subtractive hybridization (SSH) technique and to clone genes associated with HBX transactivating function . METHODS: The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-X and pcDNA3.1(-) empty vector respectively, then cDNA was synthesized . After restriction enzyme RsaI digestion, a number of small size cDNA was obtained . Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor . After tester cDNA was hybridized with driver cDNA twice and underwent nested polymerase chain reaction (PCR) twice the production was subcloned into T/A plasmid vectors to set up the subtractive cDNA library . Amplification of the library was carried out with E . coli strain JM109, some cDNA was sequenced and analyzed in GenBank with Blast . RESULTS: The subtractive cDNA library of genes transactivated by HBX was constructed . The amplified library contained 85 positive clones, and colony PCR showed that these clones contained 200-1000 bp inserts . 65 clones were analyzed by sequencing and bioinformatics, which suggested nineteen known genes and fifteen genes with unknown function . CONCLUSION: A subtractive cDNA library of genes transactivated by HBX using SSH technique has been constructed successfully, which may bring some new clues for studying the biological functions of HBX and the pathogenesis of hepatoma.

Biochem Soc Trans, 2003 Feb, 31(Pt 1), 1 - 10
Biochemical Society Special Lecture . Nitrate- and nitrite-responsive sensors NarX and NarQ of proteobacteria; Stewart V; Nitrate and nitrite are efficient respiratory oxidants for anaerobic growth . In Escherichia coli, the homologous nitrate reductase (Nar) two-component regulatory systems NarX-NarL and NarQ-NarP collaborate to control anaerobic respiratory gene expression in response to nitrate and nitrite . Several other species classified in the gamma and beta subdivisions of the proteobacteria contain only a single Nar two-component regulatory pair . This raises questions concerning the physiology of anaerobic respiration as well as the evolution, function and cross-regulation of two-component regulatory systems . Here, I focus on the sensor histidine kinases NarX and NarQ, and present a comparison of the deduced NarX and NarQ primary sequences from a broad sampling of proteobacteria . This comparison defines shared features, including a large central region of unknown function that appears to be unique to this family of sensor kinases . I then consider the phylogenetic distribution of narX and narQ genes in relation to anaerobic respiratory enzyme repertoire and physiological function . One noteworthy observation is that narXL genes are specifically associated with the structural genes for membrane-bound nitrate reductase, narGHJI, whereas organization and linkage of the narQ and narP genes is quite variable . I conclude with some speculative thoughts on the evolutionary and functional divergence of the NarX-NarL and NarQ-NarP regulatory systems . Overall, this analysis aims to provide a basis for future hypothesis and experimentation in this area.

Biotechniques, 2003 Jan, 34(1), 124 - 30
Microarray of recombinant antibodies using a streptavidin sensor surface self-assembled onto a gold layer; Pavlickova P et al.; We have developed a sensitive method for the detection of recombinant antibody-antigen interactions in a microarray format . The biochip sensor platform used in this study is based on an oriented streptavidin monolayer that provides a biological interface with well-defined surface architecture that dramatically reduces nonspecific binding interactions . All the antibody or antigen probes were biotinylated and coupled onto streptavidin-coated biochip surfaces (1 microL total volume) . The detection limits for the immobilized probes on the microarray surface were 0.5 microgram/mL (200 fmol/spot) for the peptide antigen and 0.1 microgram/mL (3 fmol/spot) for the recombinant antibodies . Optimal concentrations for the detection of the Cy5-labeled protein target were in the range of 20 micrograms/mL . Protein microchips were used to measure antibody-antigen kinetics, to find optimal temperature conditions, and to establish the shelf life of recombinant antibodies immobilized on the streptavidin surface . For recombinant antibody fragments with a kDa of 10-100 nM, we have established an easy and direct immunoassay . In addition, we developed an indirect method for antibody detection with no need for expensive and time-consuming antibody purifications and modifications . Such a method was shown to be useful for large-scale screening of recombinant antibody fragments directly after their functional expression in bacteria . Our data demonstrate that recombinant antibody fragments are suitable components in the construction of antibody chips.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 193 - 6
{The relation between translation speed and protein secondary structure}; Li XQ et al.; Based on the statistical analysis of 119 human and 92 E . coli proteins it was found that for both human and E . coli, the mRNA sequences consisting of tri-codon and tetra-codon with high translation speed preferably code for alpha helices more than for coils . For beta strand, the preference/avoidance oscillates with the translation speed . Moreover, the non-homogeneous usages of tri-codon and tetra-codon with different translation speeds in a given secondary structure have also been found . These results cannot be simply explained by the effect of stochastic fluctuation.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 149 - 53
{Mutation of arginines near the active site Cys124 of human dual-specificity phosphatase and its effect on the enzymatic activity}; Wang YH et al.; To study the effect of three positively charged arginine residues near the active site Cys(124) of the human dual-specific phosphatase on the catalytic function, six VHR mutants R(125)L, R(130)L, R(130)K, R(130)L/S(131)A, R(158)K and R(158)L were obtained using QuikChange site-directed mutagenesis method . The recombinant plasmids containing mutant genes were transformed into the Escherichia coli strain BL21(DE3), and the expressed proteins were found to be water soluble after the induction of IPTG . The proteins with purity greater than 90% were obtained using Ni(2+) chelating affinity chromatography . The measurement of the steady-state kinetic parameters and arsenate inhibition constants K(i) of the enzyme and their mutants showed that the k(cat)/K(m) values of Arg(130) and Arg(158) mutants decreased, and K(i) values increased obviously compared with those of the wild enzyme . These results indicated that Arg(130) and Arg(158) were necessary for the enzymatic activity, and were probably related to the binding with the negatively charged phosphate group of the substrate . In addition, the slight difference for the k(cat) values between the R(130)L and R(130)L/S(131)A mutants suggested that Arg(130) mutation disrupted the hydrogen bond between Ser(131) and Cys(124) . Furthermore, the arsenate binding affinity for R(125)L, R(130)L and R(158)L mutants was decreased, suggesting that positive charges in the side chains of these three arginine residues may be helpful for the binding of the enzyme to the substrate.

Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Feb, 35(2), 133 - 7
{Effect of N-terminal deletion on biological activity of vascular endothelial cell growth inhibitor}; Zhang M et al.; Vascular endothelial cell growth inhibitor (VEGI) is a novel cytokine which belongs to the TNF superfamily . It can inhibit the proliferation of endothelial cells and neovascularization . However, little is known about the structure-function relationship of VEGI . In order to study the effect of the N-terminus of VEGI on biological properties, the sequence alignment among VEGI and TNF superfamily members based on structure knowledge was done, and then two truncated forms of VEGI were constructed, in which 43 and 51 amino acids from N-terminus were deleted and named VEGI(131) and VEGI(123) . Recombinant proteins were generated from E . coli . The expression rates were 25.2% (VEGI(131)) and 27.8% (VEGI(123)) of total bacterial proteins . After purification the purity reached 92.5% (VEGI(131)) and 91.6% (VEGI(123)) . VEGI(131) showed significant inhibitory effect on growth of human umbilical vein endothelial cells (HUVEC), IC(50) of VEGI(131) being 35 mg/L . Under the same conditions, IC(50) of VEGI(151) (the wild type of VEGI) was 27 mg/L, but VEGI(123) showed no inhibitory effect . On chick choriallantic membrane (CAM) assay, VEGI(151) markedly reduced the number of main vessels, and VEGI131 decreased capillary number, while the effect of VEGI(123) was almost the same as control . These results suggest that the first 43 amino acids from N-terminus of VEGI have no significant effect on biological activity, but the amino acids 44-51 at N-terminus are required for full biological activity.

AIDS, 2003 Jan 24, 17(2), 177 - 81
Broad spectrum inhibition of HIV-1 infection by sulfated K5 Escherichia coli polysaccharide derivatives; Vicenzi E et al.; OBJECTIVE: HIV-1 entry into CD4 cells represents a main target for developing novel antiretroviral agents and microbicides . DESIGN: Sulfated derivatives of the K5 polysaccharide have a backbone structure resembling the heparin precursor, but are devoid of the anticoagulant activity . The derivatives were chemically sulfated in the N position after N-deacetylation, in the O position, or in both sites . METHODS: HIV replication in human T cell blasts, monocyte-derived macrophages and cell lines was studied in the presence of sulfated K5 derivatives . RESULTS: O-sulfated {K5-OS(H)} and N,O-sulfated {K5-N,OS(H)} K5 derivatives with high degree of sulfation inhibited the replication of an HIV strain using CXCR4 as entry co-receptor (X4 virus) in both cell lines and T-cell blasts . K5 derivatives also strongly inhibited the multiplication of CCR5-dependent HIV (R5 virus) in cell lines, T-cell blasts and primary monocyte-derived macrophages . Their 50% inhibitory concentration was between 0.07 and 0.46 microM, without evidence of cytotoxicity even at the maximal concentration tested (9 microM) . In addition, both K5-N,OS(H) and K5-OS(H) potently inhibited the replication of several primary HIV-1 isolates in T-cell blasts, with K5-N,OS(H) being more active than K5-OS(H) on dual tropic R5X4 strains . K5 derivatives inhibited the early steps of virion attachment and/or entry . CONCLUSIONS: Because K5 derivatives are unlikely to penetrate into cells they may represent potential topical microbicides for the prevention of sexual HIV-1 transmission.

J Trauma, 2003 Jan, 54(1), 121 - 30; discussion 130-2
Hypertonic saline resuscitation attenuates neutrophil lung sequestration and transmigration by diminishing leukocyte-endothelial interactions in a two-hit model of hemorrhagic shock and infection; Pascual JL et al.; BACKGROUND: Hypertonic saline (HTS) attenuates polymorphonuclear neutrophil (PMN)-mediated tissue injury after hemorrhagic shock . We hypothesized that HTS resuscitation reduces early in vivo endothelial cell (EC)-PMN interactions and late lung PMN sequestration in a two-hit model of hemorrhagic shock followed by mimicked infection . METHODS: Thirty-two mice were hemorrhaged (40 mm Hg) for 60 minutes and then given intratracheal lipopolysaccharide (10 microg) 1 hour after resuscitation with shed blood and either HTS (4 mL/kg 7.5% NaCl) or Ringer's lactate (RL) (twice shed blood volume) . Eleven controls were not manipulated . Cremaster intravital microscopy quantified 5-hour EC-PMN adherence, myeloperoxidase assay assessed lung PMN content (2 1/2 and 24 hours), and lung histology determined 24-hour PMN transmigration . RESULTS: Compared with RL, HTS animals displayed 55% less 5-hour EC-PMN adherence (p = 0.01), 61% lower 24-hour lung myeloperoxidase ( p= 0.007), and 57% lower mean 24-hour lung histologic score ( p= 0.027) . CONCLUSION: Compared with RL, HTS resuscitation attenuates early EC-PMN adhesion and late lung PMN accumulation in hemorrhagic shock followed by inflammation . HTS resuscitation may attenuate PMN-mediated organ damage.

J Trauma, 2003 Jan, 54(1), 104 - 12; discussion 112-3
Modulation of endotoxin-induced endothelial activity by microtubule depolymerization; Cuschieri J et al.; BACKGROUND: Endotoxin not only activates the Toll-mediated signaling pathway within endothelial cells that leads to neutrophil migration but also causes the polymerization of microtubules . The potential role of this polymerization event, however, is unknown . METHODS: Human umbilical vein endothelial cells stimulated with endotoxin were pretreated with or without the microtubule depolymerizing agent colchicine . Toll-mediated signaling events and protein production were in turn investigated by Western blot, gel shift, and enzyme-linked immunosorbent assay . Finally, neutrophil adhesion was assayed fluorometrically under the various conditions . RESULTS: Endotoxin led to activation of the various Toll-mediated pathways, production of intercellular adhesion molecule-1 and interleukin-8, and subsequent neutrophil adhesion . Pretreatment with colchicine led to selective inhibition of anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1; selective enhancement of p38; and no effect on nuclear factor-kappaB . This selective modulation of intracellular signaling resulted in attenuated intercellular adhesion molecule-1, interleukin-8 and prostaglandin E2 production, but enhanced cyclooxygenase-2 expression . As a result, microtubule disruption led to a significant reduction in neutrophil adhesion . CONCLUSION: Microtubule formation is essential to optimal endotoxin-induced intracellular signaling through anti-dual phosphorylated extracellular signal-regulated kinase-1/2, anti-dual phosphorylated c-jun N-terminal kinase, and adaptor protein-1 . Failure of these signaling events is associated with a marked reduction in the formation of a proadhesive phenotype that may prove to be beneficial in modulating neutrophil recruitment during sepsis.

Blood Coagul Fibrinolysis, 2003 Jan, 14(1), 3 - 9
Development of an experimental model of pre-thrombosis in rats based on Wessler's principle using a calibrated venous stasis; Pottier P et al.; We have developed a model of a pre-thrombotic state in rats based on venous stasis induced by partial ligature of the inferior vena cava . The degree of stenosis was calibrated by using variations in upstream venous pressure . Different degrees of stasis were tested in order to obtain a pre-thrombotic state . Increasing doses of thromboplastin were infused . The thrombogenic potential of this model was evaluated by measuring thrombus weight and by the increase in levels of thrombin-antithrombin complexes . A pre-thrombotic state was induced by 2 h of exposure to a 40% stasis obtained by increasing by 40% the upstream venous pressure (mean thrombus weight, 0.2 +/- 0.6 mg) . In these conditions of stasis, low doses of thromboplastin induced venous thrombosis (mean weight, 23 +/- 20 mg; P < 0.05) . The increase in thrombus size was correlated to the rise in thrombin-antithrombin levels (r = 0.53, P < 0.001) . In conclusion, we have developed the first animal model in which venous stasis can be calibrated by varying the degree of stenosis of the inferior vena cava . This model could be used to study the kinetics of biological markers of hypercoagulability, to study the pathogeny of thrombosis or to evaluate the therapeutic efficacy of new drugs in pre-clinical trials .

Science, 2003 Feb 14, 299(5609), 1064 - 7 Epub 2003 Jan 23.
DNA damage-induced replication fork regression and processing in Escherichia coli; Courcelle J et al.; DNA lesions that block replication are a primary cause of rearrangements, mutations, and lethality in all cells . After ultraviolet (UV)-induced DNA damage in Escherichia coli, replication recovery requires RecA and several other recF pathway proteins . To characterize the mechanism by which lesion-blocked replication forks recover, we used two-dimensional agarose gel electrophoresis to show that replication-blocking DNA lesions induce a transient reversal of the replication fork in vivo . The reversed replication fork intermediate is stabilized by RecA and RecF and is degraded by the RecQ-RecJ helicase-nuclease when these proteins are absent . We propose that fork regression allows repair enzymes to gain access to the replication-blocking lesion, allowing processive replication to resume once the blocking lesion is removed.

Dev Comp Immunol, 2003 Feb, 27(2), 137 - 46
Interleukin-1beta does not contribute to genetic strain-based differences in iNOS expression and activity in chicken macrophages; Dil N et al.; The expression of IL-1beta and inducible nitric oxide synthase (iNOS) from iNOS hypo (GB2, B(6)B(6)) and hyper (K-strain, B(15)B(15)) responder chickens was examined . Compared to GB2, macrophages from K-strain expressed higher iNOS mRNA as quantitated by reverse transcriptase polymerase (RT-PCR) chain reaction after stimulation with 1 microgram/ml of Escherichia coli (E . coli) lipopolysaccharide (LPS) . On the contrary, IL-1beta mRNA expression was comparable between K and GB2 macrophages at 3h post-LPS stimulation but persisted up to 9h only in GB2 macrophages . The LPS-inducible interleukin-1 (IL-1) surface receptor expression, measured by flow cytometry, was higher in GB2 than on K-strain macrophages . Blocking of IL-1 receptor by the anti-IL-1 receptor antibody reduced the LPS-mediated iNOS expression by 50% as quantified by competitive RT-PCR . Furthermore, iNOS activity (nitrite) was also reduced to 50% . However, this magnitude of inhibition was similar in both K and GB2 macrophages . While these observations suggest that IL-1beta is involved in mediating LPS-induced iNOS expression and activity, the differential response of GB1 and K-strain macrophages in terms of LPS-induced iNOS expression and activity is unlikely to be modulated by IL-1beta.

J Biochem Mol Biol, 2003 Jan 31, 36(1), 12 - 9
Imidazole ring-opened DNA purines and their biological significance; Tudek B; Fragmentation of purine imidazole ring and production of formamidopyrimidines in deoxynucleosides (Fapy lesions) occurs upon DNA oxidation as well as upon spontaneous or alkali-triggered rearrangement of certain alkylated bases . Many chemotherapeutic agents such as cyclophosphamide or thiotepa produce such lesions in DNA . Unsubstituted FapyA and FapyG, formed upon DNA oxidation cause moderate inhibition of DNA synthesis, which is DNA polymerase and sequence dependent . Fapy-7MeG, a methylated counterpart of FapyG-, a efficiently inhibits DNA replication in vitro and in E.coli, however its mutagenic potency is low . This is probably due to preferential incorporation of cytosine opposite Fapy-7MeG and preferential extension of Fapy-7MeG:C pair . In contrast, FapyA and Fapy-7MeA possess miscoding potential . Both lesions in SOS induced E.coli preferentially mispair with cytosine giving rise to A-->G transitions . Fapy lesions substituted with longer chain alkyl groups also show simult aneous lethal and mutagenic properties . Fapy lesions are actively eliminated from DNA by repair glycosylases specific for oxidized purines and pyrimidines both in bacteria and eukaryotic cells . Bacterial enzymes include E.coli formamidopyrimidine-DNA-glycosylase (Fpg protein), endonuclease III (Nth protein) and endonuclease VIII (Nei protein).

J Endotoxin Res, 2002, 8(6), 419 - 26
Direct hemoperfusion with polymyxin B-immobilized fiber improves shock and hypoxemia during endotoxemia in anesthetized sheep; Yamamoto H et al.; This study evaluates the effect of direct hemoperfusion (DHP) using polymyxin B-immobilized fibers (PMX-F) as an extracorporeal blood filter on systemic hypotension and lung injury during endotoxemia Sheep were anesthetized, intubated, mechanically ventilated with 50% oxygen and connected to the DHP system between the right femoral artery and left jugular vein . Group 1 (n = 6)sheep were infused with 10 microg/kg Escherichia coli endotoxin over a 30 min period . At the same time, sheep underwent DHP with PMX-F (Toraymyxin: PMX-20R) for 2 h at a flow rate of 60 ml/h . Group 2 (n = 6) sheep were infused with the same dose of endotoxin and treated with a sham column, in the same manner as those in group 1 . DHP with PMX-F significantly improved and restored systemic pressure and arterial oxygen tension in group 1 sheep, although these values never returned to the baseline levels of group 2 sheep . Pulmonary hypertension and leukocytopenia were observed after endotoxin infusion in both groups, but there were no significant differences between these values . DHP with PMX-F significantly decreased the elevation of plasma nitric oxide products . The treatment with PMX-F improves shock and deteriorated oxygenation during endotoxemia, probably through the suppression of nitric oxide production.

Eur J Biochem, 2003 Feb, 270(3), 518 - 32
The transthyretin-related protein family; Eneqvist T et al.; A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization . Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates . A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved . Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved . RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C . elegans TRP genes encodes a functional protein . We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C . elegans but detected no apparent phenotype . The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR . The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.

Insect Mol Biol, 2003 Feb, 12(1), 61 - 6
Characterization of JNK-like protein derived from a mosquito cell line, C6/36; Mizutani T et al.; When Western blot analysis of heat-killed bacteria- and lipopolysaccharide (LPS)-treated Aedes albopictus mosquito cell line C6/36 was performed using antiphospholyrated c-Jun amino-terminal kinase (JNK) antibodies, approximately 46 kDa protein was clearly detected with a peak around 30 min . After the C6/36 cells were incubated at 45 degrees C in order to induce apoptosis, the 46 kDa protein continued to be detected for at least 3 h . The internalization of fluorescein-labelled bacteria was inhibited by a JNK-specific inhibitor SP600125, suggesting that phagocytosis involves the JNK signalling pathway in mosquito cells . Based on these results, we found one candidate for the nucleotide sequence of JNK (Ae-JNK) from the C6/36 cells . This study is the first report regarding the mitogen-activated protein kinase (MAPK) of mosquito.

J Nat Prod, 2003 Jan, 66(1), 57 - 61
Novel chromone derivatives from the fungus Aspergillus versicolor isolated from the marine sponge Xestospongia exigua; Lin W et al.; From the marine sponge Xestospongia exigua collected in Indonesia the fungus Aspergillus versicolor was isolated . Following cultivation in a seawater-based medium seven new angular tricyclic chromone derivatives (1-7) were obtained from the mycelia and culture filtrate . Compounds 2-7 contain an additional dihydropyran ring system which is replaced by a pyridine ring in 1 . The structures of the new natural products were established on the basis of extensive one- and two-dimensional NMR spectroscopic studies (1H, 13C, COSY, HMQC, HMBC, NOE difference spectra) as well as on mass spectral analysis.

J Biol Chem, 2003 Apr 4, 278(14), 11903 - 8 Epub 2003 Jan 22.
Catalytic mechanism of Escherichia coli isopentenyl diphosphate isomerase involves Cys-67, Glu-116, and Tyr-104 as suggested by crystal structures of complexes with transition state analogues and irreversible inhibitors; Wouters J et al.; Isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids . The reaction involves protonation and deprotonation of the isoprenoid unit and proceeds through a carbocationic transition state . Analysis of the crystal structures (2 A) of complexes of Escherichia coli IPP.DMAPPs isomerase with a transition state analogue (N,N-dimethyl-2-amino-1-ethyl diphosphate) and a covalently attached irreversible inhibitor (3,4-epoxy-3-methyl-1-butyl diphosphate) indicates that Glu-116, Tyr-104, and Cys-67 are involved in the antarafacial addition/elimination of protons during isomerization . This work provides a new perspective about the mechanism of the reaction.

J Biol Chem, 2003 Mar 28, 278(13), 11570 - 8 Epub 2003 Jan 22.
Organic hydroperoxide resistance gene encodes a thiol-dependent peroxidase; Cussiol JR et al.; ohr (organic hydroperoxide resistance gene) is present in several species of bacteria, and its deletion renders cells specifically sensitive to organic peroxides . The goal of this work was to determine the biochemical function of Ohr from Xylella fastidiosa . All of the Ohr homologues possess two cysteine residues, one of them located in a VCP motif, which is also present in all of the proteins from the peroxiredoxin family . Therefore, we have investigated whether Ohr possesses thiol-dependent peroxidase activity . The ohr gene from X . fastidiosa was expressed in Escherichia coli, and the recombinant Ohr decomposed hydroperoxides in a dithiothreitol-dependent manner . Ohr was about twenty times more efficient to remove organic hydroperoxides than to remove H(2)O(2) . This result is consistent with the organic hydroperoxide sensitivity of Delta ohr strains . The dependence of Ohr on thiol compounds was ascertained by glutamine synthetase protection assays . Approximately two thiol equivalents were consumed per peroxide removed indicating that Ohr catalyzes the following reaction: 2RSH + ROOH --> RSSR + ROH + H(2)O . Pretreatment of Ohr with N-ethyl maleimide and substitution of cysteine residues by serines inhibited this peroxidase activity indicating that both of the Ohr cysteines are important to the decomposition of peroxides . C125S still had a residual enzymatic activity indicating that Cys-61 is directly involved in peroxide removal . Monothiol compounds do not support the peroxidase activity of Ohr as well as thioredoxin from Saccharomyces cerevisiae and from Spirulina . Interestingly, dithiothreitol and dyhydrolipoic acid, which possess two sulfhydryl groups, do support the peroxidase activity of Ohr . Taken together our results unequivocally demonstrated that Ohr is a thiol-dependent peroxidase.

Infect Immun, 2003 Feb, 71(2), 845 - 9
Induction by sphingomyelinase of shiga toxin receptor and shiga toxin 2 sensitivity in human microvascular endothelial cells; Obrig TG et al.; Shiga toxin-producing enterohemorrhagic Escherichia coli is the major cause of acute renal failure in young children . The interaction of Shiga toxins 1 and 2 (Stx1 and Stx2) with endothelial cells is an important step in the renal coagulation and thrombosis observed in hemolytic uremic syndrome . Previous studies have shown that bacterial lipopolysaccharide and host cytokines slowly sensitize endothelial cells to Shiga toxins . In the present study, bacterial neutral sphingomyelinase (SMase) rapidly (1 h) sensitized human dermal microvascular endothelial cells (HDMEC) to the cytotoxic action of Stx2 . Exposure of endothelial cells to neutral SMase (0.067 U/ml) caused a rapid increase of intracellular ceramide that persisted for hours . Closely following the change in ceramide level was an increase in the expression of globotriaosylceramide (Gb3), the receptor for Stx2 . A rapid increase was also observed in the mRNA for ceramide:glucosyltransferase (CGT), the first of three glycosyltransferase enzymes of the Gb3 biosynthetic pathway . The product of CGT (glucosylceramide) was also increased . In contrast, mRNA for the third enzyme of the pathway, Gb3 synthase, was constitutively produced and was not influenced by SMase treatment of HDMEC . These results describe a rapid response mechanism by which extracellular neutral SMase derived from either bacteria or eukaryotic cells may signal endothelial cells to become sensitive to Shiga toxins.

Genome Biol . 2003;4(1):203 . Epub 2003 Jan 03.
The sigma70 family of sigma factors; Paget MS et al.; Members of the sigma70 family of sigma factors are components of the RNA polymerase holoenzyme that direct bacterial or plastid core RNA polymerase to specific promoter elements that are situated 10 and 35 base-pairs upstream of transcription-initiation points . Members of the sigma70 family also function as contact points for some activator proteins, such as PhoB and lambda(cl), and play a role in the initiation process itself . The primary sigma factor, which is essential for general transcription in exponentially growing cells, is reversibly associated with RNA polymerase and can be replaced by alternative sigma factors that co-ordinately express genes involved in diverse functions, such as stress responses, morphological development and iron uptake . On the basis of gene structure and function, members of the sigma70 family can broadly be divided into four main groups . Sequence alignments of the sigma70 family members reveal that they have four conserved regions, although the highest conservation is found in regions 2 and 4, which are involved in binding to RNA polymerase, recognizing promoters and separating DNA strands (so-called 'DNA melting') . The division of the linear sequence of sigma70 factors into four regions is largely supported by recent structural data indicating that primary sigma factors have three stable domains that incorporate regions 2, 3 and 4 . Furthermore, structures of the RNA polymerase holoenzyme have revealed that these domains of sigma70 are spread out across one face of RNA polymerase . These structural data are starting to illuminate the mechanistic role of sigma factors in transcription initiation.

Ann Biomed Eng, 2002 Nov-Dec, 30(10), 1313 - 22
A three-dimensional numerical fluid dynamic model of antigen-antibody surface adsorption on piezoelectric immunosensors; Barak-Shinar D et al.; A piezoelectric crystal is a unit that changes its frequency in parallel with a change in its mass . This characteristic is exploited in designing flow cell-based immunosensors for detecting the concentration of antibodies in liquid samples . In the present study, computational fluid dynamic techniques are used to optimize the antigen-antibody binding process on an electrode surface placed on the base of a conical flow cell . The geometry optimization of the flow cell was determined to minimize the test time . This time is needed for the electrode to be saturated by the antibody, a process that requires the maximization of the adsorption rate and be accomplished by increasing the shear rate in the vicinity of the electrode . To validate the numerical model and to determine its parameters, experiments were carried out using an identical flow cell . In the experiments, the system did not reach saturation within an acceptable time frame, therefore, the model parameters were determined based on the unsaturated state . The experimental results confirmed the applicability of numerical simulations in predicting the effect of changing the inlet section area of the flow cells, proving the computational model to be very valuable in designing immunosensors based on flow cells.

J Tongji Med Univ, 2001, 21(3), 219 - 21
Anti-viral activity of hairpin ribozyme directed against HBV core region in vitro; Lin J et al.; To study the preparation and cleavage of hairpin ribozyme (HpRz) directed against the transcript of HBV core region in vitro, HRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz . 32P-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme . 32P-labeled pKC transcript containing HBV core region as targets-RNA was transcribed by using T7 RNA polymerase and purified by PAGE . Cold HpRz transcript was incubated with 32P-labeled target-RNAs under different conditions and radioautographed after denaturing polyacrylamide gel electrophoresis . The results showed that HpRz had the ability of cleavage at 37 degrees C and 12 mmol/L MgCl2 and the design of ribozyme was correct . It is concluded that HpRz prepared in vitro possesses specific catalytic activity, indicating that it is possible for HpRz to intracellularly inhibit the replication of HBV . It may be developed into a nucleic acid drug in the treatment of hepatitis B in the future.

J Tongji Med Univ, 2001, 21(4), 273 - 6
Study on the preparation of recombinant human HSP70 and its presenting-antigen function; Zhang G et al.; The preparation of recombinant human HSP70 and its presenting-antigen function were investigated . Cultured in glucose-free M9ZB medium and induced with IPTG and lactose at a final concentration of 0.02 mmol/L and 5 mmol/L respectively, the engineered bacteria carrying expression vector of human HSP70 gene expressed rHSP70 at an efficiency fo 60% . After the purification with DEAE ion-exchange chromatography, HSP70 with a purity of higher than 90% was obtained . The purified product could bind tumor-antigen peptide in vitro, and the binding was identified by native PAGE containing 5% glycerol . HSP70-peptide complex could activate lymphocytes to produce specific cytotoxicity to tumor cells, suggesting that the recombinant human HSP70 could be used as an antigen-presenting reagent in tumor therapy.

Hua Xi Kou Qiang Yi Xue Za Zhi, 1999 Nov, 17(4), 297 - 9
{Plasmid DNA expression in the muscle of rat tongue}; Gao W et al.; OBJECTIVE: To develop rat tongue model for plasmid DNA expression in vivo . METHODS: The expression vectors which contained either human-muscle specific promoter or SV40 promoter directing the transcription of reporter gene (E . coli LacZ gene) were used . The straited muscle of the tongue was used as a model system, and expression plasmid DNA was injected directly into Wistar rat tongue . PCR technique, X-gal histochemical staining and Southern blot hybridization were used to analyze reporter gene expression in vivo . RESULTS: The expression of reporter gene was correlated with the amount of injected DNA and the time of incubation in tongue muscle . beta-galactosidase activity can be detected 24 hours after injection of X-gal staining and even be detected after two months, but the maximal expression level was observed one week later . Coinjection of two expression plasmids resulted in co-expression in the same striated myofibers . E . coli LacZ DNA sequence was found in rat tongue injected with the plasmid DNA by PCR, which demonstrated capacity of the rat tongue myofibers uptaking foreign DNA . The Southern blot analysis showed the injected plasmid DNA existed outside the genome of rat muscle cells . No integration was detected . CONCLUSION: Direct plasmid DNA injection into tongue muscle was a simple and efficient approach to transfer foreign gene into striated muscle cells, and tongue was a good model for analyzing exogenous gene expression.

Nat Med, 2003 Feb, 9(2), 231 - 5 Epub 2003 Jan 21.
Measuring the frequency of mouse and human cytotoxic T cells by the Lysispot assay: independent regulation of cytokine secretion and short-term killing; Snyder JE et al.; Antigen-specific T cells demonstrate several potent effector functions during immune responses . Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but it has been difficult to measure the frequency of cytolytic cells . We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli beta-galactosidase in mouse or human target cells, and an Elispot to detect release of beta-galactosidase from killed target cells . This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-gamma secretion are regulated independently . The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.

Protein Eng, 2002 Nov, 15(11), 931 - 41
An engineered IN-1 F(ab) fragment with improved affinity for the Nogo-A axonal growth inhibitor permits immunochemical detection and shows enhanced neutralizing activity; Fiedler M et al.; The myelin axonal growth inhibitor NI-220/250 (Nogo-A) has attracted considerable attention in elucidating the mechanisms that account for the lack of plasticity in the adult central nervous system . The cognate monoclonal antibody IN-1, which was obtained prior to the molecular characterization of its Nogo-A antigen, has played a crucial role in this respect . However, this murine IgM/kappa antibody does not only provide an inappropriate format for in vivo studies, its low antigen affinity has also hampered the thorough structure-function analysis of its neutralizing effect toward the Nogo-A inhibitor on a molecular basis . We describe here the affinity maturation of a bacterially produced functional IN-1 F(ab) fragment via protein engineering . A soluble fragment of Nogo-A derived from the central exon 3 of its gene, which was prepared by secretion into the periplasm of Escherichia coli, served as a target in these experiments . After repeated cycles of site-directed random mutagenesis and screening, the mutant II.1.8 of the IN-1 F(ab) fragment was obtained, carrying five side chain substitutions within CDR-L3 . Its dissociation constant for the complex with the recombinant Nogo-A fragment was determined in surface plasmon resonance measurements as approximately 1 microM . The affinity of the unmutated IN-1 F(ab) fragment was 8-fold lower . The engineered F(ab) fragment appeared to be well suited for the specific detection of Nogo-A in immunochemical assays and for the histochemical staining of myelin-rich tissue sections . Most importantly, its concentration-dependent neutralizing effect on the Nogo-A inhibitory activity was significantly enhanced in cell culture . This study confirms Nogo-A to be the antigen of the IN-1 antibody and it demonstrates increased potential of the engineered F(ab) fragment as a reagent for promoting axonal regeneration in vivo.

Protein Eng, 2002 Nov, 15(11), 855 - 9
19F NMR study of the leucine-specific binding protein of Escherichia coli: mutagenesis and assignment of the 5-fluorotryptophan-labeled residues; Salopek-Sondi B et al.; The Escherichia coli L-leucine receptor is an aqueous protein and the first component in the distinct transport pathway for hydrophobic amino acids . L-leucine binding induces a conformational change, which enables the receptor to dock to the membrane components . To investigate the ligand-induced conformational change and binding properties of this protein, we used (19)F NMR to probe the four tryptophan residues located in the two lobes of the protein . The four tryptophan residues were labeled with 5-fluorotryptophan and assigned by site-directed mutagenesis . The (19)F NMR spectra of the partially ligand free proteins show broadened peaks which sharpen when L-leucine is bound, showing that the labeled wild-type protein and mutants are functional . The titration of L-phenylalanine into the 5-fluorotryptophan labeled wild-type protein shows the presence of closed and open conformers . Urea-induced denaturation studies support the NMR results that the wild-type protein binds L-phenylalanine in a different manner to L-leucine . Our studies showed that the tryptophan to phenylalanine mutations on structural units linked to the binding pocket produce subtle changes in the environment of Trp18 located directly in the binding cleft.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1072 - 7 Epub 2003 Jan 21.
Parallel changes in gene expression after 20,000 generations of evolution in Escherichiacoli; Cooper TF et al.; Twelve populations of Escherichia coli, derived from a common ancestor, evolved in a glucose-limited medium for 20,000 generations . Here we use DNA expression arrays to examine whether gene-expression profiles in two populations evolved in parallel, which would indicate adaptation, and to gain insight into the mechanisms underlying their adaptation . We compared the expression profile of the ancestor to that of clones sampled from both populations after 20,000 generations . The expression of 59 genes had changed significantly in both populations . Remarkably, all 59 were changed in the same direction relative to the ancestor . Many of these genes were members of the cAMP-cAMP receptor protein (CRP) and guanosine tetraphosphate (ppGpp) regulons . Sequencing of several genes controlling the effectors of these regulons found a nonsynonymous mutation in spoT in one population . Moving this mutation into the ancestral background showed that it increased fitness and produced many of the expression changes manifest after 20,000 generations . The same mutation had no effect on fitness when introduced into the other evolved population, indicating that a mutation of similar effect was present already . Our study demonstrates the utility of expression arrays for addressing evolutionary issues including the quantitative measurement of parallel evolution in independent lineages and the identification of beneficial mutations.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 845 - 9 Epub 2003 Jan 21.
Crystal structure of the MurG:UDP-GlcNAc complex reveals common structural principles of a superfamily of glycosyltransferases; Hu Y et al.; MurG is an essential glycosyltransferase that forms the glycosidic linkage between N-acetyl muramyl pentapeptide and N-acetyl glucosamine in the biosynthesis of the bacterial cell wall . This enzyme is a member of a major superfamily of NDP-glycosyltransferases for which no x-ray structures containing intact substrates have been reported . Here we present the 2.5-A crystal structure of Escherichia coli MurG in complex with its donor substrate, UDP-GlcNAc . Combined with genomic analysis of other superfamily members and site-specific mutagenesis of E . coli MurG, this structure sheds light on the molecular basis for both donor and acceptor selectivity for the superfamily . This structural analysis suggests that it will be possible to evolve new glycosyltransferases from prototypical superfamily members by varying two key loops while maintaining the overall architecture of the family and preserving key residues.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 1134 - 9 Epub 2003 Jan 21.
Stabilization of perfect and imperfect tandem repeats by single-strand DNA exonucleases; Feschenko VV et al.; Rearrangements between tandemly repeated DNA sequences are a common source of genetic instability . Such rearrangements underlie several human genetic diseases . In many organisms, the mismatch-repair (MMR) system functions to stabilize repeats when the repeat unit is short or when sequence imperfections are present between the repeats . We show here that the action of single-stranded DNA (ssDNA) exonucleases plays an additional, important role in stabilizing tandem repeats, independent of their role in MMR . For perfect repeats of approximately 100 bp in Escherichia coli that are not susceptible to MMR, exonuclease (Exo)-I, ExoX, and RecJ exonuclease redundantly inhibit deletion . Our data suggest that >90% of potential deletion events are avoided by the combined action of these three exonucleases . Imperfect tandem repeats, less prone to rearrangements, are stabilized by both the MMR-pathway and ssDNA-specific exonucleases . For 100-bp repeats containing four mispairs, ExoI alone aborts most deletion events, even in the presence of a functional MMR system . By genetic analysis, we show that the inhibitory effect of ssDNA exonucleases on deletion formation is independent of the MutS and UvrD proteins . Exonuclease degradation of DNA displaced during the deletion process may abort slipped misalignment . Exonuclease action is therefore a significant force in genetic stabilization of many forms of repetitive DNA.

J Cell Sci, 2003 Feb 15, 116(Pt 4), 725 - 42
Constitutive activation of Rho proteins by CNF-1 influences tight junction structure and epithelial barrier function; Hopkins AM et al.; The apical-most epithelial intercellular junction, referred to as the tight junction (TJ), regulates paracellular solute flux in diverse physiological and pathological states . TJ affiliations with the apical filamentous actin (F-actin) cytoskeleton are crucial in regulating TJ function . F-actin organization is influenced by the Rho GTPase family, which also controls TJ function . To explore the role of Rho GTPases in regulating TJ structure and function, we utilized Escherichia coli cytotoxic necrotizing factor-1 (CNF-1) as a tool to activate constitutively Rho, Rac and Cdc42 signaling in T84 polarized intestinal epithelial monolayers . The biological effects of the toxin were polarized to the basolateral membrane, and included profound reductions in TJ gate function, accompanied by displacement of the TJ proteins occludin and zonula occludens-1 (ZO-1), and reorganization of junction adhesion molecule-1 (JAM-1) away from the TJ membrane . Immunogold electron microscopy revealed occludin and caveolin-1 internalization in endosomal/caveolar-like structures in CNF-treated cells . Immunofluorescence/confocal microscopy suggested that a pool of internalized occludin went to caveolae, early endosomes and recycling endosomes, but not to late endosomes . This provides a novel mechanism potentially allowing occludin to evade a degradative pathway, perhaps allowing efficient recycling back to the TJ membrane . In contrast to the TJ, the characteristic ring structure of proteins in adherens junctions (AJs) was largely preserved despite CNF-1 treatment . CNF-1 also induced displacement of a TJ-associated pool of phosphorylated myosin light chain (p-MLC), which is normally also linked to the F-actin contractile machinery in epithelial cells . The apical perjunctional F-actin ring itself was maintained even after toxin exposure, but there was a striking effacement of microvillous F-actin and its binding protein, villin, from the same plane . However, basal F-actin stress fibers became prominent and cabled following basolateral CNF-1 treatment, and the focal adhesion protein paxillin was tyrosine phosphorylated . This indicates differences in Rho GTPase-mediated control of distinct F-actin pools in polarized cells . Functionally, CNF-1 profoundly impaired TJ/AJ assembly in calcium switch assays . Re-localization of occludin but not E-cadherin along the lateral membrane during junctional reassembly was severely impaired by the toxin . A balance between activity and quiescence of Rho GTPases appears crucial for both the generation and maintenance of optimal epithelial barrier function . Overactivation of Rho, Rac and Cdc42 with CNF-1 seems to mirror key barrier-function disruptions previously reported for inactivation of RhoA.

J Immunol, 2003 Feb 1, 170(3), 1566 - 78
Induction of apoptosis by HIV-1-infected monocytic cells; Sperber K et al.; We have previously described a soluble 6000-Da peptide produced by an HIV-1-infected human macrophage cell line, clone 43(HIV), which induces apoptosis in T and B cells . We have identified this factor as the novel cDNA clone FL14676485 that encodes for the human hypothetical protein, FLJ21908 . The FL14676485 cDNA clone was isolated from a 43(HIV) lambda ZAP Escherichia coli expression library and screened with a panel of rabbit and mouse anti-apoptotic Abs . We transfected the FL14676485 clone into Bosc cells and non-HIV-1-infected 43 cells . Western blot analysis of lysates from the FL14676485-transfected 43 cells and Bosc cells using anti-proapoptotic factor Abs revealed a protein with a molecular mass of 66 kDa corresponding to the size of the full-length gene product of the FL14676485 clone, while Western blot of the supernatant demonstrated a doublet of 46-kDa and 6000-Da peptide that corresponds to our previously described proapoptotic factor . Primary HIV-1(BaL)-infected monocytes also produce the FLJ21908 protein . Supernatants from these transfected cells induced apoptosis in PBMC, CD4(+), and CD8(+) T and B cells similar to the activity of our previously described proapoptotic factor . PCR analysis of 43 cells and 43(HIV) cells revealed a base pair fragment of 420 bp corresponding to the FL14676485 gene product in 43(HIV) cells, but not in 43 cells . The FLJ21908 protein induces apoptosis through activation of caspase-9 and caspase-3 . We have further demonstrated that the FLJ21908 protein has apoptotic activity in the SH-SY5Y neuronal cell line and can be detected in brain and lymph tissue from HIV-1-infected patients who have AIDS dementia . The FLJ21908 protein may contribute to the apoptosis and dementia observed in AIDS patients.

J Immunol, 2003 Feb 1, 170(3), 1392 - 8
The shift of Th1 to Th2 immunodominance associated with the chronicity of Mycobacterium bovis bacille Calmette-Guérin infection does not affect the memory response; Jiao X et al.; In the present study we investigated the shaping and evolution of the immunodominance of the T cell response during a chronic mycobacterial infection . Using a recombinant bacille Calmette-Guerin expressing a reporter Ag, the Escherichia coli MalE protein, we analyzed the peptide specificity and the cytokine profile of the T cell response to the reporter Ag by ELISPOT . During the early steps of infection, the T cell response was focused on two dominant MalE epitopes and was characterized by a pure IFN-gamma response . Then, in the course of infection the initial IFN-gamma response to these two epitopes shifted to a mixed IFN-gamma/IL-4 response . At the same time, the peptide specificity of the T cell response was broadened to two additional MalE epitopes characterized by a unique IL-4 response resulting in the establishment of a dominant IL-4 response to the MalE protein at 16 wk postinfection . However, this phenomenon did not impair the outcome of a predominant IFN-gamma response upon subsequent MalE recall in vivo performed in the presence of CFA, a Th1-driving adjuvant . These results indicate that the Th2 nature of the immune response established during a chronic infection, which most likely reflects regulatory mechanisms to allow the return to T cell homeostasis, does not shape the Th1/Th2 nature of the memory response.

J Immunol, 2003 Feb 1, 170(3), 1374 - 82
Natural substrates and inhibitors of mannan-binding lectin-associated serine protease-1 and -2: a study on recombinant catalytic fragments; Ambrus G et al.; Mannan-binding lectin-associated serine protease (SP) (MASP)-1 and MASP-2 are modular SP and form complexes with mannan-binding lectin, the recognition molecule of the lectin pathway of the complement system . To characterize the enzymatic properties of these proteases we expressed their catalytic region, the C-terminal three domains, in Escherichia coli . Both enzymes autoactivated and cleaved synthetic oligopeptide substrates . In a competing oligopeptide substrate library assay, MASP-1 showed extreme Arg selectivity, whereas MASP-2 exhibited a less restricted, trypsin-like specificity . The enzymatic assays with complement components showed that cleavage of intact C3 by MASP-1 and MASP-2 was detectable, but was only approximately 0.1% of the previously reported efficiency of C3bBb, the alternative pathway C3-convertase . Both enzymes cleaved C3i 10- to 20-fold faster, but still at only approximately 1% of the efficiency of MASP-2 cleavage of C2 . We believe that C3 is not the natural substrate of either enzyme . MASP-2 cleaved C2 and C4 at high rates . To determine the role of the individual domains in the catalytic region of MASP-2, the second complement control protein module together with the SP module and the SP module were also expressed and characterized . We demonstrated that the SP domain alone can autoactivate and cleave C2 as efficiently as the entire catalytic region, while the second complement control protein module is necessary for efficient C4 cleavage . This behavior strongly resembles C1s . Each MASP-1 and MASP-2 fragment reacted with C1-inhibitor, which completely blocked the enzymatic action of the enzymes . Nevertheless, relative rates of reaction with alpha-2-macroglobulin and C1-inhibitor suggest that alpha-2-macroglobulin may be a significant physiological inhibitor of MASP-1.

J Biol Chem, 2003 Mar 28, 278(13), 10891 - 9 Epub 2003 Jan 21.
Purification, characterization, molecular cloning, and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J . Smith (Polypodiaceae); Tateno H et al.; A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose . The lectin, designated P . aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds . From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues . The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins . PALa contained peptide sequences exactly matching those found in the isolated lectin . PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography . Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase . The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry . Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins . The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.

Bioinformatics, 2003 Jan 22, 19(2), 228 - 33
A generalized global alignment algorithm; Huang X et al.; MOTIVATION: Homologous sequences are sometimes similar over some regions but different over other regions . Homologous sequences have a much lower global similarity if the different regions are much longer than the similar regions . RESULTS: We present a generalized global alignment algorithm for comparing sequences with intermittent similarities, an ordered list of similar regions separated by different regions . A generalized global alignment model is defined to handle sequences with intermittent similarities . A dynamic programming algorithm is designed to compute an optimal general alignment in time proportional to the product of sequence lengths and in space proportional to the sum of sequence lengths . The algorithm is implemented as a computer program named GAP3 (Global Alignment Program Version 3) . The generalized global alignment model is validated by experimental results produced with GAP3 on both DNA and protein sequences . The GAP3 program extends the ability of standard global alignment programs to recognize homologous sequences of lower similarity . AVAILABILITY: The GAP3 program is freely available for academic use at http://bioinformatics.iastate.edu/aat/align/align.html.

Int Immunopharmacol, 2003 Jan, 3(1), 97 - 106
Regulation of macrophage function by the antioxidant N-acetylcysteine in mouse-oxidative stress by endotoxin; Victor VM et al.; Changes in several functions of peritoneal macrophages from mice with oxidative stress caused by intraperitoneal injection of endotoxin (Escherichia coli lipopolysaccharide, LPS) (100 mg/kg), and associated with a high production of reactive oxygen species (ROS), have been observed in our previous studies . Antioxidants such as N-acetylcysteine (NAC) are free radical scavengers that improve and modulate the immune response, especially in oxidative stress situations . Therefore, in the present work, we have studied the effects of the administration of NAC (150 mg/kg i.p.) on different functions of peritoneal macrophages from Swiss mice suffering that oxidative stress, caused by LPS (100 mg/kg) . NAC was injected 30 min after LPS injection, and the peritoneal macrophages were obtained at 2, 4, 12, and 24 h after endotoxin injection . The following functions, key stages of the phagocytic process, were studied: adherence to substrate, chemotaxis, ingestion of particles, and production of ROS (reactive oxygen species), as well as tumor necrosis factor (TNFalpha) release . The decrease in chemotaxis and the increase in adherence, ingestion, superoxide anion production, and TNFalpha release shown by macrophages from animals with oxidative stress were counteracted by NAC injection . These data suggest that NAC administration may be useful for the treatment of oxidative stress-linked endotoxic shock, modulating the function of macrophages, specifically in decreasing the production of ROS and of inflammatory cytokines such as TNFalpha .

Bioorg Med Chem, 2003 Feb 20, 11(4), 621 - 8
Searching for allosteric effects via QSAR . Part II; Garg R et al.; Allosteric interactions have in the past been established by means of X-ray crystallography or careful study of a single molecule at a variety of concentrations . Here we report a method for using QSAR to establish a change in reaction mechanism by establishing an inversion point . That is, as polarizability of a member of a congeneric set of compounds is increased (as measured by CMR), activity at first decreases until, at the inversion, activity turns around and increases . Out of 23 examples, 14 have inversion points of 10+/-1 . This includes a wide variety of receptors such as thrombin, 5-HT, dopamine, and tyrosine kinase acting with a variety of ligands.

Reprod Biomed Online, 2001, 2(1), 33 - 39
Immunocontraceptive potential of recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C expressed in Escherichia coli and its corresponding synthetic peptide; Kaul R et al.; Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for development of immunocontraceptive vaccines . In this study, the efficacy to block fertility by immunization with recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-C (r-bmZPC) expressed in Escherichia coli and its synthetic peptide (P(4): KGDCGTPSHSRRQPHVVSQWSRSA, aa residues 324-347) conjugated to diphtheria toxoid (DT) has been evaluated in a homologous system . Female bonnet monkeys, immunized with P(4)-DT conjugate showed better immunocontraceptive potential as compared to an r-bmZPC-DT immunized group . In spite of high anti-P(4) antibody titres, animals continued to have ovulatory cycles and showed no disturbance in cyclicity (except summer amenorrhoea) . No ovarian pathology was observed in the P(4) immunized group . These results suggest that immunization with the P(4) may lead to block in fertility without obvious ovarian dysfunction . However, further inputs are required to identify additional ZP based B-cell epitopes to enhance the contraceptive efficacy.

Cancer Biother Radiopharm, 2002 Dec, 17(6), 665 - 71
Fusion of the CH1 domain of IgG1 to epidermal growth factor (EGF) prolongs its retention in the blood but does not increase tumor uptake; Wang J et al.; An expression vector (pJW4) for a human epidermal growth factor (hEGF)-CH1 fusion protein was constructed by fusing the gene for hEGF with the gene for CH1 of murine IgG1 with/without a peptide linker sequence {(GGGGS)3} and inserting the recombinant gene into vector pGEX2T . Expression vector pGEX2T was transfected into E . coli (BL-21) and hEGF-CH1 expressed by induction of the lac Iq promotor with 50 microM isopropyl beta-D-thiogalactopyranoside (IPTG) . hEGF- CH1 fused to glutathione S-transferase (GST) was isolated and purified by affinity chromatography . GST was cleaved using thrombin . SDS-PAGE demonstrated a protein with the expected M(r) (18 kDa) positive for hEGF by Western blot . hEGF-linker-CH1 exhibited preserved binding to A431 (2-3 x 10(6) EGFR/cell) and MDA-MB-468 breast cancer cells (1-2 x 10(6) EGFR/cell) . hEGF-CH1 without the linker exhibited poor receptor binding . hEGF-linker-CH1 also exhibited strong binding to soluble EGFR equivalent to that of hEGF . The tumor and normal tissue distribution of hEGF-linker-CH1 labeled with 123I was compared with 123 I-hEGF at 24 h after i.v . injection to mice implanted with s.c . MDA-MB-468 xenografts . Fusion of hEGF with CH1 increased its retention in the blood 14-fold but did not significantly increase tumor uptake . Tumor/blood ratios were higher for hEGF than for hEGF-linker-CH1 . We conclude that hEGF is more attractive than hEGF-linker-CH1 for imaging EGFR-positive tumors.

Radiat Res, 2003 Feb, 159(2), 251 - 61
Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks; Pastwa E et al.; Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ) . To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents . We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA . End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased . In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends . The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs . Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs . Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs . In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E . coli endonuclease IV permitted ligation.

Hua Xi Yi Ke Da Xue Xue Bao, 2001 Sep, 32(3), 356 - 8
{The effect of ketamine on the expression of HSP70 induced by endotoxin in liver of rat}; Zhang L et al.; OBJECTIVE: To observe the effect of ketamine on the expression of heat shock proteins (HSP70) in liver of rat with septic shock . METHODS: Sixty-four rats were randomly allocated to control group, endotoxin group and ketamine group . The model of septic shock was used to examine the expression of HSP70 in rat liver and the TNF-alpha level of blood at 2 hours after endotoxin treatment and dying time for endotoxin group rats . The survival rates for the three groups were compared . RESULTS: There was a significant increase in the expression of HSP70 and the level of TNF-alpha in the endotoxin group as compared with those of control group (P < 0.05) . A significant increase in the expression of HSP70, a decrease in the level of TNF-alpha and a significantly higher survival rate were seen in ketamine group when compared with those of endotoxin group, respectively . CONCLUSION: Ketamine could increase the expression of HSP70 in rat liver, restrain the release of TNF-alpha caused by endotoxin, and raise the survival rates of rats with septic shock.

Physiol Behav, 2003 Jan, 78(1), 149 - 55
Expression of biologically active rat apolipoprotein AIV in Escherichia coli; Liu M et al.; Rat apolipoprotein AIV (apo AIV) is a 43-kDa intestinal apolipoprotein that is important in lipid metabolism and the suppression of food intake . In this study, a full-length rat apo AIV was expressed in Escherichia coli and purified in a bioactive form . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis revealed that the isolated recombinant protein has a molecular mass of approximately 43 kDa, similar to that of natural rat apo AIV . Immunoblot analysis and N-terminal amino acid sequencing confirmed the identity of the recombinant apo AIV protein as natural rat apo AIV . The recombinant protein was functional in lipoprotein binding assays . Biological activity was assessed behaviorally in that the recombinant protein suppressed food intake of fasted rats comparably to natural rat apo AIV . Neither native nor recombinant apo AIV elicited a conditioned taste aversion (CTA) at doses that suppress feeding . These results indicate that the recombinant apo AIV is structurally and functionally indistinguishable from rat natural apo AIV, making this overexpression and purification scheme a powerful tool for future structure and function studies.

Biochim Biophys Acta, 2003 Jan 31, 1645(1), 113 - 5
Crystallization and preliminary X-ray crystallographic analysis of plant ferritin from Glycine max; Masuda T et al.; The iron storage protein ferritin from soybean (Glycine max) was expressed in E . coli and crystallized using the hanging drop vapor diffusion method with sodium tartrate as the precipitant . The crystals belong to the tetragonal I4(1)22 space group, with unit cell parameters a=b=324.0, c=182.7 A . The diffraction data were collected up to a resolution of 3.0 A with a multi-wire area detector.

Biochim Biophys Acta, 2003 Jan 31, 1645(1), 40 - 8
Structure and function correlations between the rat liver threonine deaminase and aminotransferases; Scarselli M et al.; The rat liver threonine deaminase is a cytoplasmic enzyme that catalyses the pyridoxal-phosphate-dependent dehydrative deamination of L-threonine and L-serine to ammonia and alpha-ketobutyrate and pyruvate, respectively, in vivo . During deamination, a molecule of the cofactor is converted to pyridoxamine phosphate . Recently, the ability of this enzyme to accomplish an inverse half-reaction, restoring pyridoxal-phosphate and L-alanine or L-aminobutyrate, respectively, from pyruvate or 2-oxobutyrate, was reported . In order to investigate the molecular mechanisms of this transaminating activity, a molecular model of rat liver threonine deaminase was constructed on the basis of sequence homology with the biosynthetic threonine deaminase of Escherichia coli, the crystal structure of which is known . The model has structural features shared by aminotransferases, suggesting that tertiary structural elements may be responsible for the transaminating activity observed for rat liver threonine deaminase.

Biochim Biophys Acta, 2003 Jan 31, 1645(1), 15 - 21
Expression and characterization of the FXYD ion transport regulators for NMR structural studies in lipid micelles and lipid bilayers; Crowell KJ et al.; The proteins PLM (phospholemman), CHIF (channel inducing factor), and Mat8 (mammary tumor protein 8 kDa) are members of the FXYD family of ion transport regulatory membrane proteins . Here we describe their cloning and expression in Escherichia coli, and their purification for NMR structural studies in lipid micelles and lipid bilayers . The molecular masses of the purified recombinant FXYD proteins, determined from SDS-PAGE and from MALDI TOF mass spectrometry, reflect monomeric species . The solution NMR and CD spectra in SDS micelles show that they adopt helical conformations . The solid-state NMR spectra in lipid bilayers give the first view of their transmembrane architecture.

Mol Cell, 2003 Jan, 11(1), 103 - 12
The critical role of the universally conserved A2602 of 23S ribosomal RNA in the release of the nascent peptide during translation termination; Polacek N et al.; The ribosomal peptidyl transferase center is responsible for two fundamental reactions, peptide bond formation and nascent peptide release, during the elongation and termination phases of protein synthesis, respectively . We used in vitro genetics to investigate the functional importance of conserved 23S rRNA nucleotides located in the peptidyl transferase active site for transpeptidation and peptidyl-tRNA hydrolysis . While mutations at A2451, U2585, and C2063 (E . coli numbering) did not significantly affect either of the reactions, substitution of A2602 with C or its deletion abolished the ribosome ability to promote peptide release but had little effect on transpeptidation . This indicates that the mechanism of peptide release is distinct from that of peptide bond formation, with A2602 playing a critical role in peptide release during translation termination.

Plant J, 2003 Jan, 33(2), 353 - 60
Molecular properties of the putative nitrogen sensor PII from Arabidopsis thaliana; Smith CS et al.; Although the signal sensing protein PII is well known to play a central role in bacterial nitrogen metabolism, the structure and function of PII in plants remains only partially understood . Comparative modeling was undertaken based on the high degree of amino acid identity between Escherichia coli and Arabidopsis PII . The mature Arabidopsis PII predicted structure superimposes very well onto the E . coli PII structure (Calpha root mean square deviation < 0.4 A) . The model of the highly conserved T-loop suggests a molecular mechanism by which the plant PII may regulate putative post-translational modification in response to metabolite binding . Consistent with the presence of key conserved residues necessary for trimer formation, gel filtration showed the oligomeric structure of Arabidopsis thaliana PII to be a homotrimer . We have demonstrated that Arabidopsis PII binds to the small molecules, ATP, ADP, 2KG, and with lesser affinity to OAA, using isothermal titration calorimetry . We have determined the metabolite dissociation constants and compared these with known physiological concentrations of these metabolites in the plant to identify the Arabidopsis PII effector molecules and their possible roles . We predict that the plant PII is likely continually bound by ATP, and its ligand-bound state only varying with respect to the degree of 2KG binding . Based on our in vitro binding studies, the function of plant PII as a 2KG sensor is suggested.

Mol Microbiol, 2003 Feb, 47(3), 839 - 48
The transmembrane domains of the sensor kinase KdpD of Escherichia coli are not essential for sensing K+ limitation; Heermann R et al.; The sensor kinase/response regulator system KdpD/KdpE of Escherichia coli regulates the expression of the kdpFABC operon, which encodes the high affinity K+ transport system KdpFABC . The membrane-bound sensor kinase KdpD consists of four transmembrane domains, a large cytoplasmic N-terminal domain and a cytoplasmic C-terminal transmitter domain . To elucidate the role of the four transmembrane domains, various deletions were introduced in kdpD and the activities of the resulting truncated derivatives of KdpD were determined . A KdpD protein lacking all four transmembrane domains was able to sense low K+ concentrations, whereas at higher K+ concentrations kdpFABC expression was constitutive . These and further results with various truncated KdpD proteins lacking distinct parts of the transmembrane domains or derivatives in which a linker peptide or two transmembrane domains of PutP, the Na+/proline transporter of Escherichia coli, replaced the missing part indicated that the transmembrane domains are not essential for sensing of K+ limitation, but may be important for the correct positioning of the large N- and C-terminal cytoplasmic domains to each other.

Mol Microbiol, 2003 Feb, 47(3), 619 - 32
Lack of SeqA focus formation, specific DNA binding and proper protein multimerization in the Escherichia coli sequestration mutant seqA2; Fossum S et al.; In Escherichia coli wild-type cells newly formed origins cannot be reinitiated . The prevention of reinitiation is termed sequestration and is dependent on the hemimethylated state of newly replicated DNA . Several mutants discovered in a screen for the inability to sequester hemimethylated origins have been mapped to the seqA gene . Here, one of these mutants, seqA2, harbouring a single amino acid change in the C-terminal end of the SeqA protein, was found to also be unable to form foci in vivo . The SeqA foci seen in the wild-type cells are believed to arise from multimerization of SeqA on hemimethylated DNA at the replication fork, presumably representing organization of newly formed DNA by SeqA . The result suggests that the process of origin sequestration is closely tied to the process of focus maintenance at the replication fork . In vitro, purified SeqA2 protein was found incapable of forming highly ordered multimers that bind hemimethylated oriC . The mutant protein was also incapable of restraining negative supercoils . Both in vivo and in vitro results support the idea that origin sequestration is an integral part of organization of newly formed DNA performed by SeqA.

Mol Microbiol, 2003 Feb, 47(3), 595 - 606
EspH, a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli; Tu X et al.; Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E . coli (EHEC) are closely related pathogens . During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions . These include transient filopodium formation which is Cdc42-dependent . Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42 . We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH . We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies . Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation . Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation . We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals . Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH . EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure . Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.

Biochem J, 2003 Apr 15, 371(Pt 2), 301 - 10
Physical interaction of tumour suppressor p53/p73 with CCAAT-binding transcription factor 2 (CTF2) and differential regulation of human high-mobility group 1 (HMG1) gene expression; Uramoto H et al.; The CCAAT-binding transcription factor (CTF)/nuclear factor I (NF-I) group of cellular DNA-binding proteins recognizes the sequence GCCAAT and is implicated in eukaryotic transcription, as well as DNA replication . Molecular analysis of human CTF/NF-I cDNA clones revealed multiple mRNA species that contain alternative coding regions, apparently as a result of differential splicing . Expression and functional analysis established that individual gene products can bind to GCCAAT recognition sites and serve as both promoter-selective transcriptional activators and initiation factors for DNA replication . The interaction between CTF2 and p53/p73 was shown to modulate their ability to regulate transcription of their respective target genes . In the present paper, we report that p53 down-regulates the activity of the high mobility group 1 (HMG1) gene promoter, whereas p73alpha up-regulates the activity of this promoter . Furthermore, CTF2 transactivates p53-induced p21 promoter activity, but inhibits p73alpha-induced p21 promoter activity . Using deletion mutants, we found that the DNA-binding domains of both p53 and p73alpha are required for physical interaction with CTF2 via the regions between amino acid residues 161 and 223, and 228 and 312 respectively . CTF2 enhances the DNA-binding activity of p53 and inhibits the DNA-binding activity of p73alpha . These results provide novel information on the functional interplay between CTF2 and p53/p73 as important determinants of their function in cell proliferation, apoptosis, DNA repair and cisplatin resistance.

Biochemistry, 2003 Jan 28, 42(3), 801 - 10
Escherichia coli MutY and Fpg utilize a processive mechanism for target location; Francis AW et al.; MutY and formamidopyrimidine-DNA-glycosylase (Fpg) are base-excision repair (BER) enzymes involved in the 8-oxoguanine repair pathway in Escherichia coli . An impressive feature of these enzymes is the ability to locate 8-oxoguanine lesions among a large excess of undamaged DNA . To provide insight into the mechanism of target location, the ability of these enzyme to utilize a one-dimensional processive search (DNA sliding) or distributive (random diffusion-mediated) mechanism was investigated . Each enzyme was incubated with double-stranded concatemeric polynucleotides containing a site-specific target site at 25-nucleotide (nt) intervals . The products of each reaction were analyzed after further treatment and denaturation . A rapid accumulation of predominantly 25-nt fragments would indicate the utilization of a processive mechanism, whereas oligomeric multiples of 25-nt fragments would form if a distributive mechanism were used . Both Fpg and MutY were found to function processively on concatemers containing 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG).C and G.A mispairs, respectively . An increase in sodium chloride concentration results in the modulation from a processive to distributive mechanism for both enzymes . Interestingly, processive behavior was not observed in the reaction of MutY with concatemers containing OG.A mispairs . A truncated form of MutY (Stop 225) containing only the N-terminal domain was found to behave in a manner consistent with a processive mechanism with both OG.A- and G.A-containing substrates . This suggests that the C-terminal domain of MutY plays an important role in the mechanism by which the enzyme detects OG.A base pairs in DNA.

Huan Jing Ke Xue, 2002 Sep, 23(5), 57 - 61
{Disinfection and degradation of 2,4-DCP with UV-radiation and on-line ozone in drinking water treatmeant}; Ma X et al.; A type reactor with on-line O3 was used to do further research of the disinfection of total bacteria, E . coli and degradation of 2,4-DCP . The result was obtained in the following conditions . Only UV-radiation, O3 applied by other machine and by the reactor itself, and other conditions were changed to study the disinfection and degradation . The result showed the satisfied effect of disinfection and degradation would be achieved by using UV/O3 applied outside and when the flow rate was about 400 L.h-1, on-line O3 could be produced and make high efforts to enhance disinfection and degradation . The method of UV/O3 was a promising technology in the treatment of drinking water.

Fa Yi Xue Za Zhi, 2001 Aug, 17(3), 148 - 51
{Study on the construction of standard D1S549 allelic ladder via molecular cloning and its genetic polymorphism in Chinese three populations}; Zhang L et al.; OBJECTIVE: To resolve the problem of the accuracy and standardization of STR-PCR typing in forensic practice, we have designed a new method to produce standard D1S549 allelic ladder . METHODS: Eight different PCR amplified D1S549 allelic fragments were isolated from the gel, eluted into the distilled water and re-amplified by PCR . The purified allelic fragments were then blunt-end subcloned individually into the pUC plasmid vectors and transfected into competent E . coli DH5 alpha cells . RESULTS: The sequencing results confirmed that the size and the construe of the inserts were correct . The recombinant plasmids DNA with 8 inserts were then used as templates for re-amplification to generate D1S549 standard ladder, with which the genetic polymorphisms of D1S549 locus in Chinese Han population in chengdu, Hui population in Gansu and Wei population in Xinjiang were studied . CONCLUSION: The results showed that the standard ladder made via this method is excellent, and D1S549 locus is robust for genetic research and forensic application.

J Gen Virol, 2003 Jan, 84(Pt 1), 215 - 25
Rabbit endogenous retrovirus-H encodes a functional protease; Voisset C et al.; Recent studies have revealed that 'human retrovirus-5' sequences found in human samples belong to a rabbit endogenous retrovirus family named RERV-H . A part of the gag-pro region of the RERV-H genome was amplified by PCR from DNA in human samples and several forms of RERV-H protease were expressed in bacteria . The RERV-H protease was able to cleave itself from a precursor protein and was also able to cleave the RERV-H Gag polyprotein precursor in vitro whereas a form of the protease with a mutation engineered into the active site was inactive . Potential N- and C-terminal autocleavage sites were characterized . The RERV-H protease was sensitive to pepstatin A, showing it to be an aspartic protease . Moreover, it was strongly inhibited by PYVPheStaAMT, a pseudopeptide inhibitor specific for Mason-Pfizer monkey virus and avian myeloblastosis-associated virus . A structural model of the RERV-H protease was constructed that, together with the activity data, confirms that this is a retroviral aspartic protease.

Proc Natl Acad Sci U S A, 2003 Feb 4, 100(3), 886 - 91 Epub 2003 Jan 17.
A photochemical approach to the lipid accessibility of engineered cysteinyl residues; Li J et al.; Ordinary electrophilic reagents react too slowly in a nonpolar environment to be useful for the determination of the accessibility to lipid of continuous stretches of residues mutated to cysteine . By contrast, photoactivated 5-iodonaphthyl-1-azide (INA) reacted readily with 2-mercaptoethanol and dodecanethiol in nonpolar solvents and in liposomes . Continuous stretches of residues in the amphipathic N-terminal helix and first transmembrane helix of the bacterial potassium channel KcsA were replaced with cysteine, and the mutants were expressed in Escherichia coli and isolated in inner membranes . These membranes were dissolved in detergent and reconstituted into asolectin liposomes incorporating INA . The extent of light-induced reaction of INA with each cysteine was assayed by subsequent reaction with the gel-shifting, SH-specific methoxy-polyethylene glycol-2-pyridine disulfide . The pattern of apparent second-order rate constants for the photoreactions of eight substituted cysteines in the N-terminal helix conformed to other measures of lipid exposure . The pattern of the rate constants for the photoreactions of 15 cysteines in the first transmembrane helix had peaks every third residue, which partly conformed to other measures of lipid exposure.

J Biol Chem, 2003 Apr 18, 278(16), 14101 - 11 Epub 2003 Jan 17.
Recognition of the intrinsically flexible addiction antidote MazE by a dromedary single domain antibody fragment . Structure, thermodynamics of binding, stability, and influence on interactions with DNA; Lah J et al.; The Escherichia coli mazEF operon defines a chromosomal addiction module that programs cell death under various stress conditions . It encodes the toxic and long-lived MazF and the labile antidote MazE . The denaturation of MazE is a two-state reversible dimer-monomer transition . At lower concentrations the denatured state is significantly populated . This leads to a new aspect of the regulation of MazE concentration, which may decide about the life and death of the cell . Interactions of MazE with a dromedary antibody domain, cAbMaz1 (previously used as a crystallization aid), as well as with promoter DNA were studied using microcalorimetric and spectroscopic techniques . Unique features of cAbMaz1 enable a specific enthalpy-driven recognition of MazE and, thus, a significant stabilization of its dimeric native conformation . The MazE dimer and the MazE dimer-cAbMaz1 complex show very similar binding characteristics with promoter DNA, i.e . three binding sites with apparent affinities in micromolar range and highly exothermic binding accompanied by large negative entropy contributions . A working model for the MazE-DNA assembly is proposed on the basis of the structural and binding data . Both binding and stability studies lead to a picture of MazE solution structure that is significantly more unfolded than the structure observed in a crystal of the MazE-cAbMaz1 complex.

J Biol Chem, 2003 Mar 21, 278(12), 10491 - 9 Epub 2003 Jan 16.
tRNA modification by S-adenosylmethionine:tRNA ribosyltransferase-isomerase . Assay development and characterization of the recombinant enzyme; Van Lanen SG et al.; The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase catalyzes the penultimate step in the biosynthesis of the hypermodified tRNA nucleoside queuosine (Q), an unprecedented ribosyl transfer from the cofactor S-adenosylmethionine (AdoMet) to a modified-tRNA precursor to generate epoxyqueuosine (oQ) . The complexity of the reaction makes it an especially interesting mechanistic problem, and as a foundation for detailed kinetic and mechanistic studies we have carried out the basic characterization of the enzyme . Importantly, to allow for the direct measurement of oQ formation, we have developed protocols for the preparation of homogeneous substrates; specifically, an overexpression system was constructed for tRNA(Tyr) in an E . coli queA deletion mutant to allow for the isolation of large quantities of substrate tRNA, and {U-ribosyl-(14)C}AdoMet was synthesized . The enzyme shows optimal activity at pH 8.7 in buffers containing various oxyanions, including acetate, carbonate, EDTA, and phosphate . Unexpectedly, the enzyme was inhibited by Mg(2+) and Mn(2+) in millimolar concentrations . The steady-state kinetic parameters were determined to be K(m)(AdoMet) = 101.4 microm, K(m)(tRNA) = 1.5 microm, and k(cat) = 2.5 min(-1) . A short minihelix RNA was synthesized and modified with the precursor 7-aminomethyl-7-deazaguanine, and this served as an efficient substrate for the enzyme (K(m)(RNA) = 37.7 microm and k(cat) = 14.7 min(-1)), demonstrating that the anticodon stem-loop is sufficient for recognition and catalysis by QueA.

J Bacteriol, 2003 Feb, 185(3), 1112 - 5
Genetic and biochemical studies of phosphatase activity of PhoR; Carmany DO et al.; In Escherichia coli, PhoR is the histidine kinase of the phosphate regulon . It has been postulated that PhoR may function as a phospho-PhoB phosphatase . Experiments with four precise phoR deletion mutants supported this hypothesis and suggested that this activity resides within the histidine phosphorylation domain . This biochemical activity was confirmed by using a separately expressed histidine phosphorylation domain.

J Bacteriol, 2003 Feb, 185(3), 1097 - 100
Plasmid DNA supercoiling and gyrase activity in Escherichia coli wild-type and rpoS stationary-phase cells; Reyes-Dominguez Y et al.; Stationary-phase cells displayed a distribution of relaxed plasmids and had the ability to recover plasmid supercoiling as soon as nutrients became available . Preexisting gyrase molecules in these cells were responsible for this recovery . Stationary-phase rpoS cells showed a bimodal distribution of plasmids and failed to supercoil plasmids after the addition of nutrients, suggesting that rpoS plays a role in the regulation of plasmid topology during the stationary phase.

J Bacteriol, 2003 Feb, 185(3), 1045 - 58
The VirB4 family of proposed traffic nucleoside triphosphatases: common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption; Rabel C et al.; Proteins of the VirB4 family are encoded by conjugative plasmids and by type IV secretion systems, which specify macromolecule export machineries related to conjugation systems . The central feature of VirB4 proteins is a nucleotide binding site . In this study, we asked whether members of the VirB4 protein family have similarities in their primary structures and whether these proteins hydrolyze nucleotides . A multiple-sequence alignment of 19 members of the VirB4 protein family revealed striking overall similarities . We defined four common motifs and one conserved domain . One member of this protein family, TrbE of plasmid RP4, was genetically characterized by site-directed mutagenesis . Most mutations in trbE resulted in complete loss of its activities, which eliminated pilus production, propagation of plasmid-specific phages, and DNA transfer ability in Escherichia coli . Biochemical studies of a soluble derivative of RP4 TrbE and of the full-length homologous protein R388 TrwK revealed that the purified forms of these members of the VirB4 protein family do not hydrolyze ATP or GTP and behave as monomers in solution.

J Bacteriol, 2003 Feb, 185(3), 966 - 72
Rates and consequences of recombination between rRNA operons; Hashimoto JG et al.; A mutant strain of Escherichia coli was created by inserting a cassette encoding sucrose sensitivity and neomycin resistance (sacB-neo) into the small-subunit rRNA-encoding gene rrs in the rrnB operon . During growth in a complex medium, the cassette was lost from the population, and a complete rrs gene was restored at a rate of 5 x 10(-9) per cell division . Repair of this lesion required flanking regions of DNA that were similar to the six remaining intact rRNA operons and reestablished the full complement of seven rRNA operons . The relative fitness of strains with restored rrnB operons was 1 to 2% higher than that of the mutant strain . The rrnB operon normally contains a spacer region between the 16S and 23S rRNA-encoding genes that is similar in length and tRNA gene content to the spacer in rrnC, -E, and -G . In 2 of the 14 strains in which rrnB was restored, the spacer region had the same length as the spacer region in rrnA, -D, and -H . The requirement for flanking regions of nearly identical DNA and the replication of the spacer region from other rRNA operons during the repair of rrnB suggest that the restoration was accomplished via gene conversion . The rate of gene conversion was 10-fold less than the fixation of point mutations in the same region of the chromosome but was apparently sufficient to homogenize the sequences of rRNA genes in E . coli . These findings are discussed in the context of a conceptual model describing the presence of sequence heterogeneity in coevolving rRNA genes.

J Bacteriol, 2003 Feb, 185(3), 948 - 56
A SecE mutation that modulates SecY-SecE translocase assembly, identified as a specific suppressor of SecY defects; Mori H et al.; The SecY39(Cs) (cold-sensitive) alteration of Arg357 results in a defect of translocation initiation . As a means to dissect the Sec translocation machinery, we isolated mutations that act as suppressors of the secY39 defect . A specific secE mutation, designated secE105, was thus isolated . This mutation proved to be identical with the prlG2 mutation and to suppress a number of cold-sensitive secY mutations . However, other prlG mutations did not effectively suppress the secY defects . Evidence indicates that the Ser105-to-Pro alteration in the C-terminal transmembrane segment of SecE weakens SecY-SecE association . In vitro analyses showed that the SecE(S105P) alteration preferentially stimulates the initial phase of translocation . It is suggested that the S105P alteration affects the SecYEG channel such that it is more prone to open and to accept the translocation initiation domain of a preprotein molecule.

J Bacteriol, 2003 Feb, 185(3), 843 - 53
The Escherichia coli BarA-UvrY two-component system is needed for efficient switching between glycolytic and gluconeogenic carbon sources; Pernestig AK et al.; The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive . Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E . coli drastically affect survival in long-term competition cultures . Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type . Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system . Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment.

J Bacteriol, 2003 Feb, 185(3), 735 - 49
ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro; Lackner LL et al.; Proper placement of the division apparatus in Escherichia coli requires pole-to-pole oscillation of the MinC division inhibitor . MinC dynamics involves a membrane association-dissociation cycle that is driven by the activities of the MinD ATPase and the MinE topological specificity factor, which themselves undergo coupled oscillatory localization cycles . To understand the biochemical mechanisms underlying Min protein dynamics, we studied the interactions of purified Min proteins with phospholipid vesicles and the role of ATP in these interactions . We show that (i) the ATP-bound form of MinD (MinD.ATP) readily associates with phospholipid vesicles in the presence of Mg(2+), whereas the ADP-bound form (MinD.ADP) does not; (ii) MinD.ATP binds membrane in a self-enhancing fashion; (iii) both MinC and MinE can be recruited to MinD.ATP-decorated vesicles; (iv) MinE stimulates dissociation of MinD.ATP from the membrane in a process requiring hydrolysis of the nucleotide; and (v) MinE stimulates dissociation of MinC from MinD.ATP-membrane complexes, even when ATP hydrolysis is blocked . The results support and extend recent work by Z . Hu et al . (Z . Hu, E . P . Gogol, and J . Lutkenhaus, Proc . Natl . Acad . Sci . USA 99:6761-6766, 2002) and support models of protein oscillation wherein MinE induces Min protein dynamics by stimulating the conversion of the membrane-bound form of MinD (MinD.ATP) to the cytoplasmic form (MinD.ADP) . The results also indicate that MinE-stimulated dissociation of MinC from the MinC-MinD.ATP-membrane complex can, and may, occur prior to hydrolysis of the nucleotide.

Emerg Infect Dis, 2003 Jan, 9(1), 127 - 31
Single multiplex polymerase chain reaction to detect diverse loci associated with diarrheagenic Escherichia coli; Lopez-Saucedo C et al.; We developed and tested a single multiplex polymerase chain reaction (PCR) that detects enterotoxigenic, enteropathogenic, enteroinvasive, and Shiga toxin-producing Escherichia coli . This PCR is specific, sensitive, and rapid in detecting target isolates in stool and food . Because of its simplicity, economy, and efficiency, this protocol warrants further evaluation in large, prospective studies of polymicrobial substances.

Int J Cancer, 2003 Mar 10, 104(1), 104 - 12
Immune enhancement of nitroreductase-induced cytotoxicity: studies using a bicistronic adenovirus vector; Green NK et al.; The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in preclinical studies and is currently being assessed in phase I clinical trials . It is well established that there is an immune component to the bystander effect observed with other systems such as thymidine kinase and cytosine deaminase; however, such an effect has not previously been described using NR . We have preliminary data suggesting an immune bystander effect with NR to further examine these effects and their potential enhancement by cytokines, an adenoviral vector containing CMV-NR, an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF) was constructed . The NR-GM-CSF virus was validated in 2 experimental models and demonstrated increased therapeutic efficacy in the MC26 murine colorectal tumour model . These data illustrate that the combination of suicide gene therapy using NR and CB1954 with immune stimulation via GM-CSF gives an improved response compared to either modality alone and suggests that the immune component of this response may be beneficial in combating unresectable, metastatic disease and preventing tumour recurrence .

Chemistry, 2003 Jan 20, 9(2), 372 - 7
Superbeads: immobilization in "sweet" chemistry; Nahalka J et al.; Enzymatic oligosaccharide synthesis using recombinant glycosyltransferases is able to overcome the difficulties associated with chemical methods . Nonetheless, sugar nucleotide regeneration cycles are necessary for the glycosylation . The multistep enzyme reaction can be efficiently carried out on superbeads that are prepared by immobilizing multienzyme mixtures on bead support through fused binding domains.

J Biol Chem, 2003 Mar 21, 278(12), 10067 - 72 Epub 2003 Jan 15.
Probing the H-protein-induced conformational change and the function of the N-terminal region of Escherichia coli T-protein of the glycine cleavage system by limited proteolysis; Okamura-Ikeda K et al.; T-protein, a component of the glycine cleavage system, catalyzes a tetrahydrofolate-dependent reaction . Previously, we reported a conformational change of Escherichia coli T-protein upon interacting with E . coli H-protein (EH), showing an important role for the N-terminal region of the T-protein in the interaction . To further investigate the T-protein catalysis, the wild type (ET) and mutants were subjected to limited proteolysis . ET was favorably cleaved at Lys(81), Lys(154), Lys(288), and Lys(360) by lysylendopeptidase and the cleavages at Lys(81) and Lys(288) were strongly prevented by EH . Although ET was highly resistant to trypsinolysis, the mutant with an N-terminal 7-residue deletion (ETDelta7) was quite susceptible and instantly cleaved at Arg(16) accompanied by the rapid degradation of the resulting C-terminal fragment, indicating that the cleavage at Arg(16) is the trigger for the C-terminal fragmentation . EH showed no protection from the N-terminal cleavage, although substantial protection from the C-terminal fragmentation was observed . The replacement of Leu(6) of ET with alanine resulted in a similar sensitivity to trypsin as ETDelta7 . These results suggest that the N-terminal region of ET functions as a molecular "hasp" to hold ET in the compact form required for the proper association with EH . Leu(6) seems to play a central role in the hasp function . Interestingly, Lys(360) of ET was susceptible to proteolysis even after the stabilization of the entire molecule of ET by EH, indicating its location at the surface of the ET-EH complex . Together with the buried position of Lys(81) in the complex and previous results on folate binding sites, these results suggest the formation of a folate-binding cavity via the interaction of ET with EH . The polyglutamyl tail of the folate substrate may be inserted into the bosom of the cavity leaving the pteridine ring near the entrance of the cavity in the context of the catalytic reaction.

Vaccine, 2003 Jan 17, 21(5-6), 562 - 5
B cell responses in gastric antrum and duodenum following oral inactivated Helicobacter pylori whole cell (HWC) vaccine and LT(R192G) in H pylori seronegative individuals; Losonsky GA et al.; To investigate whether B cell-specific responses could be elicited in the gastric mucosa of Helicobacter pylori (HP) naive subjects, five volunteers ingested three doses of a HP killed whole cell (HWC) vaccine with 25 microg of recombinant heat-labile toxin (LT(R192G)) . Two of three subjects had detectable LT(R192G) and HWC IgA antibody secreting cell (ASC) gastric responses . LT(R192G) and HWC responses in duodenal were 5-14-fold higher than those detected in antral biopsies (P<0.01 and P=0.05, respectively) . These results provide the first evidence that specific gastric B cell responses can be induced in HP-non-infected individuals following oral immunization.

Vaccine, 2003 Jan 17, 21(5-6), 341 - 6
Oral immunization of adult volunteers with microencapsulated enterotoxigenic Escherichia coli (ETEC) CS6 antigen; Katz DE et al.; As a step in the development of an oral vaccine against ETEC, we evaluated the safety and immunogenicity of CS6, a polymeric protein commonly found on the surface of ETEC . Formulations included 1 and 5mg doses of CS6, either encapsulated in biodegradable polymer poly(D, L)-lactide-co-glycolide (PLG), or as free protein, administered orally in a solution of either normal saline or a rice-based buffer . Three doses of CS6 were given at 2-week intervals . Blood was collected immediately before and 7 days after each dose . All formulations were well tolerated . Four of five volunteers who received 1mg CS6 in PLG microspheres with buffer had significant IgA ASC responses (median=30 ASC per 10(6) PBMC) and significant serum IgG responses (median=3.5-fold increase) . Oral administration of these prototype ETEC vaccine formulations are safe and can elicit immune responses . The ASC, serum IgA, and serum IgG responses to CS6 are similar in magnitude to the responses after challenge with wild-type ETEC {Coster et al., unpublished data} . Further studies are underway to determine whether these immune responses are sufficient for protection.

Biochim Biophys Acta, 2003 Jan 27, 1625(2), 214 - 20
Cloning, expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis; Takaya A et al.; Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP . GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds . In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis . The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes . This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity . The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively . The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5 . The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced . On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.

Biochim Biophys Acta, 2003 Jan 27, 1625(2), 173 - 82
Identification and characterization of a novel cancer/testis antigen gene CAGE-1; Park S et al.; Serological analysis of cDNA expression library (SEREX) was employed to identify cancer-associated genes . By screening cDNA expression libraries with sera of patients with lung cancers, we identified a total of 49 genes that specifically reacted with the sera of patients with lung cancers . Among these, we characterized a novel gene with expression pattern similar to that of cancer/testis antigens . Its open reading frame is 1920 bp in size and encodes for putative protein composed of 639 amino acids . Southern blot analysis reveals that this gene exists as single copy . In vitro transcription/translation and Western blot analysis confirm that this gene encodes a protein of 73 kDa in size . The comparison of cDNA and genomic sequences reveals that it is composed of 11 exons and 10 introns . This gene displays testis-specific expression among normal tissues, and wide expression among various cancer tissues and cancer cell lines . A study using GFP fusion construct reveals mainly nuclear localization of CAGE-1 protein . The expression of this clone is relatively higher in cancer tissues compared with their surrounding non-cancerous tissues . This suggests that overexpression of CAGE-1 may be associated with the progression of tumor . Because of its association with cancer, this gene was named cancer-associated gene-1 (CAGE-1) . Given the fact that several cancer/testis antigens reportedly induce cytolytic T lymphocyte (CTL) reactions, it is reasonable that this gene will be a valuable target for cancer immunotherapy . The exact functional role of CAGE-1 in tumorigenesis remains to be seen.

DNA Repair (Amst), 2003 Feb 3, 2(2), 159 - 73
Interactions among the Escherichia coli mutT, mutM, and mutY damage prevention pathways; Fowler RG et al.; We have investigated in detail the interactions between the Escherichia coli mutT, mutM, and mutY error-prevention systems . Jointly, these systems protect the cell against the effects of the oxidative stress product, 8-oxoguanine (8-oxoG), a base analog with ambiguous base-pairing properties, pairing with either A or C during DNA synthesis . mutT mutator strains display a specific increase in A.T-->C.G transversions, while mutM and mutY mutator strains show specific G.C-->T.A increases . To study in more detail the in vivo processing of the various mutational intermediates leading to A.T-->C.G and G.C-->T.A transversions, we analyzed defined A.T-->C.G and G.C-->T.A events in strains containing all possible combinations of these mutator alleles . We report three major findings . First, we do not find evidence that the mutT allele significantly increases G.C-->T.A transversions in either mut(+), mutM, mutY or mutMmutY backgrounds . We interpret this result to indicate that incorporation of 8-oxodGTP opposite template C may not be frequent relative to incorporation opposite template A . Second, we show that mutT-induced A.T-->C.G transversions are significantly reduced in strains carrying mutY and mutMmutY deficiencies suggesting that 8-oxoG, when present in DNA, preferentially mispairs with dATP . Third, the mutY and mutMmutY deficiencies also decrease A.T-->C.G transversions in the mutT(+) background, suggesting that, even in the presence of functional MutT protein, A.T-->C.G transversions may still result from 8-oxodGTP misincorporation.

Vaccine, 2003 Jan 30, 21(7-8), 812 - 5
Corn as a production system for human and animal vaccines; Streatfield SJ et al.; The synthesis of selected antigens in plants and their oral delivery has great potential for reducing the costs of vaccine production and administration . The application of this technology requires antigen concentrations in final plant material to be uniform to ensure consistent dosing . In addition, antigen levels should be such as to allow the volume of each dose, containing a set amount of antigen, to be practical for oral delivery . Here, we demonstrate that the Lt-B protein of enterotoxigenic E . coli is evenly distributed in defatted corn germ prepared from transgenic grain . Furthermore, the choice of sub-cellular location for Lt-B affects accumulation of the protein in excess of four orders of magnitude.

Vaccine, 2003 Jan 30, 21(7-8), 761 - 7
Functional mapping of protective epitopes within the rotavirus VP6 protein in mice belonging to different haplotypes; Choi AH et al.; We recently used "functional mapping" to locate protective epitopes in the carboxyl terminus (aa 197-397) of the VP6 protein (designated CD) of the EDIM strain of murine rotavirus {J . Virol . 74 (2000) 11574} . For this, H-2(d) BALB/c mice were given two intranasal (i.n.) immunizations (separated by 2 weeks) with VP6 or CD genetically-fused to maltose-binding protein, or with overlapping synthetic CD peptides, along with LT(R192G), a genetically-attenuated E . coli heat-labile toxin . The protective efficacies, i.e., percentage reductions in rotavirus shedding relative to control mice during 7 days following oral challenge with EDIM, were determined 4 weeks after the second immunization . Five of the 11 overlapping CD peptides stimulated significant protection (57-85%, P<0.05) . Furthermore, chimeric VP6, the CD fragment, and a 14-amino-acid VP6 peptide within CD (RLSFQLMRPPNMTP), identified as a H-2(d)-restricted CD4 T cell epitope, were highly protective (93-98%, P<0.05) . In this study, we continued to utilize functional mapping to show that the 14-mer peptide elicited significant protection (97.0%, P<0.05) in another H-2(d) mouse strain (DBA/2) but partial protection in H-2(b) 129 (39.2%) and C57Bl/6 (53.6%) as well as H-2(k) C3H (44.6%) mice . The first 13 amino acids of this 14-mer were necessary to induce maximal protection in H-2(d) mice . In addition, the H-2(b) 129 mice were immunized intranasally (i.n.) with 10 of the synthetic CD peptides and 5 were found to induce significant protection (90-97%, P<0.05) . We also performed functional mapping to identify MHC class I epitopes in rotavirus proteins . A class I-binding epitope for H-2(b) C57Bl/6 mice had previously been mapped by ex vivo CTL assays within the VP6 protein and two additional class I epitopes were identified by computer-based prediction . When examined for their protective efficacies by functional mapping, two of the three were found to be partially but not significantly protective (44 and 46%, P>0.05) . To better determine the usefulness of our in vivo methods to identify MHC class I-binding epitopes, four epitopes from the outer capsid VP7 rotavirus protein determined in ex vivo assays were evaluated for their protective efficacies and two were found to be partially protective . Together, these studies show that functional mapping is useful in locating epitopes that are relevant to the development of subunit rotavirus vaccines.

Vet Parasitol, 2003 Feb 13, 111(2-3), 247 - 60
Antibodies to Anaplasma marginale major surface proteins 1a and 1b inhibit infectivity for cultured tick cells; Blouin EF et al.; Major surface protein 1 (MSP1) of the cattle pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae) is a complex of two proteins, MSP1a and MSP1b . Previous studies demonstrated that MSP1a and MSP1b are adhesins for bovine erythrocytes, while only MSP1a proved to be an adhesin for tick cells . In this study, a tick cell culture system for propagation of A . marginale was used to develop an infection inhibition assay for testing the ability of antisera to block infection of A . marginale for cultured tick cells . A . marginale derived from cell culture was incubated with various antisera prior to inoculation onto cell monolayers . The monolayers were harvested 7 days post-inoculation and A . marginale in the cultures was quantified using an antigen detection ELISA . Antisera tested in the infection inhibition assay were derived from persistently infected cattle, from cattle immunized with A . marginale purified from bovine erythrocytes, and from rabbits and cattle that were immunized with the recombinant MSP1a, MSP1b and MSP1 complex . Antibodies from cattle persistently infected with A . marginale, cattle immunized with A . marginale from bovine erythrocytes or cattle immunized with the recombinant MSP1 complex did not inhibit the infectivity of A . marginale for tick cells . Antiserum from rabbits immunized with MSP1a and MSP1b (individually or combined) reduced infection of both the Virginia and Oklahoma isolates of A . marginale for tick cells by 25-70% . Likewise, antisera from cattle immunized with recombinant MSP1a or MSP1b inhibited infection of tick cells by 26-37% . These results further confirm the role of MSP1 complex proteins in infection of tick cells . Lack of inhibition of infection by antisera from naturally infected cattle or cattle immunized with whole organisms suggests that the bovine immune response is not directed toward blocking infection of A . marginale for tick cells and may contribute to the continued infectivity of the pathogen for ticks.

Mol Immunol, 2003 Jan, 39(12), 719 - 27
Expression and characterization of recombinant canine IL-13 receptor alpha2 protein and its biological activity in vitro; Tang L et al.; Our previous study showed that recombinant canine IL-13 (rcaIL-13) stimulated production of allergen-specific IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs . This has also been demonstrated using human IL-13 (huIL-13) and PBMC isolated from human allergy patients . The stimulatory activity of rcaIL-13 was specifically inhibited by a fusion protein of the extracellular domain of canine IL-13Ralpha2 and the Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc) . In this communication, we report the construction and expression of a non-fused recombinant extracellular domain of canine IL-13Ralpha2 (rcaIL-13Ralpha2) in an E . coli expression system . The E . coli expressed rcaIL-13Ralpha2 was isolated in inclusion bodies, then solubilized in buffer containing denaturants and reducing agents . After refolding and purification, the biological activity of rcaIL-13Ralpha2 was found in the monomer fraction resulting from gel filtration and ion exchange chromatographies . Biological activity of purified rcaIL-13Ralpha2 was demonstrated by the specific inhibition of rcaIL-13 activity in a TF-1 cell proliferation assay . Additionally, rcaIL-13Ralpha2 was found to be active in neutralizing rcaIL-13 induced upregulation of IgE mRNA levels in PBMCs of "high IgE" dogs, which have been bred to exhibit a predisposition for high IgE production and are used as a model for allergic asthma . The data confirm our previous report that the regulatory effects of IL-13 on IgE production in canine PBMCs are similar to those reported in humans . Thus, allergic dogs, such as the "high IgE" producing dogs, may be excellent models for research on IgE-mediated diseases in humans.

DNA Repair (Amst), 2002 Oct 1, 1(10), 847 - 54
Differential intracellular localization of the human and mouse endonuclease III homologs and analysis of the sorting signals; Ikeda S et al.; The mammalian endonuclease III homolog NTH1 is a DNA glycosylase/AP lyase that recognizes oxidized pyrimidine bases . Here, we compared the intracellular localization of human and mouse NTH1 and analyzed their sorting signals by examining expression of enhanced green fluorescent protein (EGFP)-tagged NTH1 protein . Full-length hNTH1 was sorted exclusively into nuclei . Deletion analysis showed that two basic amino acid clusters, which constitute the nuclear localization signal (NLS), are essential for nuclear sorting . Moreover, disruption of the NLS by deletion or substitution of arginine residue(s) altered the localization of the protein to mitochondria . In contrast, most mNTH1 molecules were sorted into mitochondria, with a relatively small amount localized in nuclei . Deletion analysis indicated that the mitochondrial targeting sequence of mNTH1 is contained within the N-terminal 38 amino acids . Alignment of the N-terminal sequence of human and mouse NTH1 showed that mNTH1 lacks a basic amino acid cluster corresponding to one of the NLS sequences found in hNTH1 . Nuclear localization of mNTH1 was increased when this NLS sequence was added to mNTH1 through the addition of appropriate amino acids . The fact that transcription of the hNTH1 gene is initiated at multiple sites indicated that three isoforms of hNTH1 protein are translated using different initiation codons . However, no difference in intracellular localization was observed among three isoforms of hNTH1 with different N-terminal sequences.

DNA Repair (Amst), 2002 Oct 1, 1(10), 779 - 93
Analysis of mouse Rad54 expression and its implications for homologous recombination; Essers J et al.; Homologous recombination is one of the major pathways for repair of DNA double-strand breaks (DSBs) . Important proteins in this pathway are Rad51 and Rad54 . Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) that mediates pairing with and strand invasion of homologous duplex DNA with the assist of Rad54 . We estimated that the nucleus of a mouse embryonic stem (ES) cells contains on average 4.7x10(5) Rad51 and 2.4x10(5) Rad54 molecules . Furthermore, we showed that the amount of Rad54 was subject to cell cycle regulation . We discuss our results with respect to two models that describe how Rad54 stimulates Rad51-mediated DNA strand invasion . The models differ in whether Rad54 functions locally or globally . In the first model, Rad54 acts in cis relative to the site of strand invasion . Rad54 coats the Rad51 nucleoprotein filament in stoichiometric amounts and binds to the target duplex DNA at the site that is homologous to the ssDNA in the Rad51 nucleoprotein filament . Subsequently, it promotes duplex DNA unwinding . In the second model, Rad54 acts in trans relative to the site of strand invasion . Rad54 binds duplex DNA distant from the site that will be unwound . Translocation of Rad54 along the duplex DNA increases superhelical stress thereby promoting duplex DNA unwinding.

DNA Repair (Amst), 2002 Nov 3, 1(11), 955 - 66
Survival and SOS induction in cisplatin-treated Escherichia coli deficient in Pol II, RecBCD and RecFOR functions; Bhattacharya R et al.; Cisplatin is a potent anticancer agent forming intrastrand-crosslinks in DNA . The efficacy of cisplatin in chemotherapy can be limited by the development of tumor resistances such as elevated DNA repair or damage tolerance . In Escherichia coli, cisplatin treatment causes induction of the SOS regulon resulting in elevated levels of DNA Pol II, DNA Pol IV, DNA Pol V, the cell division inhibitor SfiA (SulA), homologous recombination (HR) and DNA repair . In this work, the roles of Pol II and HR in facilitating resistance of E . coli to cisplatin are studied . SOS induction levels were measured by beta-galactosidase assays in cisplatin-treated and untreated E . coli PQ30 that has the lacZ gene fused to the sfiA promoter . Comparative studies were carried out with derivatives of PQ30 constructed by P1 transduction that have transposon insertions in the polB gene, the recB gene blocking the RecBCD pathway of HR and genes of the RecFOR pathway of HR . Resistance of E . coli strains to cisplatin as determined by plating experiments decreased in the following order: parent PQ30 strain, polB > recO, recR, recF > recB . Both the RecBCD and RecFOR pathways of HR are important for survival when E . coli is exposed to cisplatin, because treatment of double mutants deficient in both pathways reduced colony forming ability to 37% in 6-9min in comparison to 39-120min for single mutants . Pol II and RecF appear to function in two distinct pathways to initiate replication blocked due to damage caused by cisplatin because function of Pol II was required for survival in mutants deficient in the RecFOR pathway after 2h of cisplatin treatment . In contrast, Pol II was not required for survival in recB mutants . SOS induction was delayed in RecFOR deficient mutants but occurred at high levels in the recB mutant soon after cisplatin treatment in a RecFOR-dependent way . An SfiA independent, DNA damage dependent pathway is apparently responsible for the filamentous cells observed after cisplatin or MMC treatments of these SfiA defective strains.

DNA Repair (Amst), 2002 Nov 3, 1(11), 881 - 93
Mutational specificity of mice defective in the MTH1 and/or the MSH2 genes; Egashira A et al.; Oxidative damage of nucleotides within DNA or precursor pools caused by oxygen radicals is thought to play an important role in spontaneous mutagenesis, as well as carcinogenesis and aging . In particular, 8-oxodGTP and 2-OHdATP are potent mutagenic substrate for DNA synthesis . Mammalian MTH1 catalyzes hydrolysis of these mutagenic substrates, suggesting that it functions to prevent mutagenesis caused by these oxidized nucleotides . We have established MTH1(-/-) mice lacking the 8-oxodGTPase activity, which were shown to be susceptible to lung, liver and stomach cancers . To examine in vivo mutation events due to the MTH1-deficiency, a reporter gene, rpsL of Escherichia coli, was introduced into MTH1(-/-) mice . Interestingly, the net frequency of rpsL(-) forward mutants showed no apparent increase in MTH1(-/-) mice as compared to MTH1(+/+) mice . However, we found differences between these two genotypes in the class- and site-distributions of the rpsL(-) mutations recovered from the mice . Unlike MutT-deficient E . coli showing 1000-fold higher frequency of A:T-->C:G transversion than the wild type cells, an increase in frequency of A:T-->C:G transversion was not evident in MTH1 nullizygous mice . Nevertheless, the frequency of single-base frameshifts at mononucleotide runs was 5.7-fold higher in spleens of MTH1(-/-) mice than in those of wild type mice . Since the elevated incidence of single-base frameshifts at mononucleotide runs is a hallmark of the defect in MSH2-dependent mismatch repair system, this weak site-specific mutator effect of MTH1(-/-) mice could be attributed to a partial sequestration of the mismatch repair function that may act to correct mispairs with the oxidized nucleotides . Consistent with this hypothesis, a significant increase in the frequency of G:C-->T:A transversions was observed with MTH1(-/-) MSH2(-/-) mice over MSH2(-/-) mice alone . These results suggest a possible involvement of multiple anti-mutagenic pathways, including the MTH1 protein and other repair system(s), in mutagenesis caused by the oxidized nucleotides.

DNA Repair (Amst), 2002 Dec 5, 1(12), 1051 - 6
Cytotoxicity and mutagenesis induced by singlet oxygen in wild type and DNA repair deficient Escherichia coli strains; Cavalcante AK et al.; Singlet oxygen ((1)O(2)) is a product of several biological processes and can be generated in photodynamic therapy, through a photosensitization type II mechanism . (1)O(2) is able to interact with lipids, proteins and DNA, leading to cell killing and mutagenesis, and can be directly involved with degenerative processes such as cancer and aging . In this work, we analyzed the cytotoxicity and mutagenesis induced after direct treatment of wild type and the DNA repair fpg and/or mutY deficient Escherichia coli strains with disodium 3,3'-(1,4-naphthylidene) diproprionate endoperoxide (NDPO(2)), which releases (1)O(2) by thermodissociation . The treatment induced cell killing and mutagenesis in all strains, but the mutY strain showed to be more sensitive . These results indicate that even (1)O(2) generated outside bacterial cells may lead to DNA damage that could be repaired by pathways that employ MutY protein . As (1)O(2) is highly reactive, its interaction with cell membranes may generate secondary products that could react with DNA, leading to mutagenic lesions .

BMC Mol Biol . 2003 Jan 16;4(1):1 . Print 2003 Jan 16.
Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells; Zhang Y et al.; BACKGROUND: The phage protein pairs, RecE/RecT from Rac or Redalpha/Redbeta from lambda, initiate efficient double strand break repair (DSBR) in Escherichia coli that has proven very useful for DNA engineering . These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined . RESULTS: Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR) at high efficiencies . The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redalpha) but only requires a phage annealing protein (RecT or Redbeta) . Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity . By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site . Notably, ssOR shows strand bias . Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements . This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork . We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein . CONCLUSION: Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies . This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redalpha/Redbeta.

Biol Chem, 2002 Nov, 383(11), 1809 - 12
Individual recombinant thyroglobulin type-1 domains are substrates for lysosomal cysteine proteinases; Pungercic G et al.; Thyroglobulin contains 11 repeats of a motif called thyroglobulin type-1 domain that show sequence similarity to some proteins exhibiting inhibitory activity against cysteine proteinases . Here we report that thyroglobulin decreases the activity of cathepsins B, H, L, and papain . To examine the possible involvement of particular type-1 domains in that decrease of activity, some individual thyroglobulin type-1 domains were expressed in E . coli . These recombinant domains proved to be substrates for cathepsins B, H, L, and papain instead of inhibitors . The cleavage points with cathepsins B and L on the second and the fourth domains were determined . The possible reasons for degradation are discussed.

Biol Chem, 2002 Nov, 383(11), 1779 - 89
Molecular and immunological characterization of profilin from mugwort pollen; Wopfner N et al.; In late summer in Europe, pollen of mugwort is one of the major sources of atopic allergens . No information about the complete molecular structure of any mugwort allergen has been published so far . Here we report the isolation and characterization of mugwort pollen cDNA clones coding for two isoforms of the panallergen profilin . Thirty-six percent of the mugwort-allergic patients tested displayed IgE antibodies against natural and recombinant profilin, and no significant differences were observed in the IgE-binding properties of the isoforms . One profilin isoform was purified to homogeneity and detailed structural analysis indicated that the protein exists in solution as dimers and tetramers stabilized by sulfydryl and/or ionic interactions . Profilin monomers were detectable only after exposure of multimers to harsh denaturing conditions . Dimers and tetramers did not significantly differ in their ability to bind serum IgE from mugwort pollen-allergic patients . However, oligomeric forms might have a higher allergenic potential than monomers because larger molecules would have additional epitopes for IgE-mediated histamine release . Profilin isolated from mugwort pollen also formed multimers . Thus, oligomerization is not an artifact resulting from the recombinant production of the allergen . Inhibition experiments showed extensive IgE cross-reactivity of recombinant mugwort profilin and profilin from various pollen and food extracts.

Biol Chem, 2002 Nov, 383(11), 1769 - 78
Pre-loading of chlorophyll synthase with tetraprenyl diphosphate is an obligatory step in chlorophyll biosynthesis; Schmid HC et al.; The reaction of recombinant chlorophyll synthase from Avena sativa, expressed in Escherichia coli, was investigated . To verify the identity of the recombinant and native enzymes, reaction rates were determined for both enzyme preparations with several chlorophyllide analogs . The rates of esterification of these modified substrates ranged from 0 to 100% of the rate with the natural substrate, and were nearly identical for both enzyme preparations . The Lineweaver-Burk plot for variation of both chlorophyllide a and phytyl diphosphate concentration showed parallel lines, indicative of a 'ping-pong' mechanism . Pre-incubation with phytyl diphosphate exhibited an initial rapid reaction phase, which did not occur after pre-incubation with chlorophyllide . We conclude that the tetraprenyl diphosphate must bind to the enzyme as the first substrate and esterification occurs when this pre-loaded enzyme meets the second substrate, chlorophyllide . Approximately 10-17% of the recombinant enzyme were pre-loaded with phytyl diphosphate under the experimental conditions . The rapid reaction phase is also observed for the chlorophyll synthase reaction in etiolated barley leaves in addition to the well-known slow phase . This indicates that pre-loading of the enzyme with tetraprenyl diphosphate is also the basis for the reaction in vivo.

Biol Chem, 2002 Nov, 383(11), 1743 - 50
Specificity determinants in the interaction of apolipoprotein(a) kringles with tetranectin and LDL; Caterer NR et al.; Lipoprotein(a) is composed of low density lipoprotein and apolipoprotein(a) . Apolipoprotein(a) has evolved from plasminogen and contains 10 different plasminogen kringle 4 homologous domains {KIV(1-110)} . Previous studies indicated that lipoprotein(a) non-covalently binds the N-terminal region of lipoprotein B100 and the plasminogen kringle 4 binding plasma protein tetranectin . In this study recombinant KIV(2), KIV(7) and KIV(10) derived from apolipoprotein(a) were produced in E . coli and the binding to tetranectin and low density lipoprotein was examined . Only KIV(10) bound to tetranectin and binding was similar to that of plasminogen kringle 4 to tetranectin . Only KIV(7) bound to LDL . In order to identify the residues responsible for the difference in specificity between KIV(7) and KIV(10), a number of surface-exposed residues located around the lysine binding clefts were exchanged . Ligand binding analysis of these derivatives showed that Y62, and to a minor extent W32 and E56, of KIV(7) are important for LDL binding to KIV(7), whereas R32 and D56 of KIV(10) are required for tetranectin binding of KIV(10).

Cell Mol Life Sci, 2002 Nov, 59(11), 1983 - 92
New endo-beta-1,4-glucanases from the parabasalian symbionts, Pseudotrichonympha grassii and Holomastigotoides mirabile of Coptotermes termites; Watanabe H et al.; Abstract . An endo-beta-1,4-glucanase (EG) was purified from the hindgut of an Australian mound-building termite, Coptotermes lacteus . The hindgut extract had a peak separate from those for extracts obtained from the salivary glands and the midgut based on sephacryl S-200 gel chromatography, and also demonstrated an origin different from the endogenous EGs of the termite itself . The recovery was further purified by SDS-PAGE, and its N-terminal amino acid sequence analyzed . This showed high homology to EGs from glycoside hydrolase family (GHF) 7 . PCR-based cloning methods were applied to the hindgut contents of C . lacteus and individual protozoan symbionts from C formosanus . cDNAs encoding putative EGs homologous to GHF7 members were then identified . The functionality of one of the putative proteins was confirmed by its expression in Escherichia coli.

DNA Repair (Amst), 2002 May 30, 1(5), 411 - 418
Mutagenic target for hydroxyl radicals generated in Escherichia coli mutant deficient in Mn- and Fe- superoxide dismutases and Fur, a repressor for iron-uptake systems; Nunoshiba T et al.; We previously reported that mutations in Mn- and Fe-superoxide dismutases and Fur, a repressor for iron uptake systems, simulated generation of hydroxyl radicals, and caused hypermutability in Escherichia coli . The predominant type of spontaneous mutation was GC --> TA, followed by AT --> CG, suggesting the involvement of 7,8-dihydro-8-oxoguanine (8-oxoG) and 1,2-dihydro-2-oxoadenine (2-oxoA) in DNA as well as 7,8-dihydro-8-oxodeoxyguanosine triphosphate (8-oxodGTP) and 1,2-dihydro-2-oxodeoxyadenosine triphosphate (2-oxodATP) in the nucleotide pool . To determine the targets contributing to oxidative mutagenesis, DNA or nucleotides, we characterized spontaneous mutations and compared the distribution to those in mutMY and mutT strains, in which GC --> TA and AT --> CG were predominantly induced, respectively . The hotspots and sequence contexts where AT --> CG occurred frequently in sodAB fur strain were almost identical to those in mutT strain,whereas, those where GC --> TA occurred frequently in sodAB fur strain were quite different from those in mutMY strain . These observations suggested that AT --> CG is due to 8-oxodGTP, while GC --> TA is produced by some other lesion(s) . The 2-oxodATP is also a major oxidative lesion in nucleotides, and strongly induces GC --> TA . The expression of cDNA for MTH1, which can hydrolyze 2-oxodATP as well as 8-oxodGTP, partially but significantly, suppressed the GC --> TA mutator phenotype of the sodAB fur strain, whereas, it did not for the mutMY strain . Additionally, the sequence contextby 2-oxodATP in E . coli was similar to that in sodAB fur strain . These results suggested that the targets contributing to oxidative mutagenesis in sodAB fur strain are nucleotides such as dGTP and dATP, rather than DNA.

Insect Biochem Mol Biol, 2002 Nov, 32(11), 1585 - 96
Changes in cysteine protease activity and localization during midgut metamorphosis in the crucifer root maggot (Delia radicum); Hegedus D et al.; We show that differential localization and/or activation of two cysteine protease activities occur at the onset of dipteran midgut metamorphosis . A 26 kDa cysteine protease activity was associated specifically with midgut tissues of late third instar larvae . Starvation of mid third instar larvae simulated the onset of prepupation and resulted in loss of the 26 kDa protease activity . A cDNA clone encoding a cysteine protease, termed DrCP1, was isolated and shown to be highly similar to those from Sarcophaga peregrina and Drosophila melanogaster (DmCP1) . DrCP1 mRNA was present in all developmental stages including eggs, larvae, pupae and adults, but was highly induced at the onset of the larval-pupal transition and thereafter . The DrCP1 protein is localized to the exterior of the midgut tissues during the onset of the prepupal transition period, possibly in response to ecdysone . Analysis of transcription factor binding sites associated with the DmCP1 promoter indicated that elements exist that allow for both ecdysone-mediated as well as tissue-specific regulation . Based upon these and other studies we propose: (1) that the expression, activity and localization of the DrCP1-like cysteine proteases are highly regulated throughout development; and, (2) that cysteine protease activities are involved in aspects of tissue reconstruction at the onset of and during metamorphosis.

Anticancer Res, 2002 Nov-Dec, 22(6A), 3293 - 301
Effect of poly-herbal formula on NO production by LPS-stimulated mouse macrophage-like cells; Miyamoto M et al.; Pretreatment of mice with lyophilized hot water extracts of five poly-herbal formula protected them from lethal infection by E . coli . ESR spectroscopy shows that these extracts produced radicals under alkaline condition, and scavenged radicals such as superoxide anion, hydroxyl radical and nitric oxide (NO) radical . There was a positive relationship between their radical intensity and radical scavenging activity . Among the extracts, HD-02 efficiently inhibited the production of NO and citrulline, and the expression of inducible NO synthase (iNOS) mRNA by LPS-stimulated mouse macrophage-like cells Raw 264.7 . DLH-3073 not only inhibited the LPS-stimulated NO production at lower concentration, but also induced NO production at higher concentrations, suggesting the presence of two different antagonizing components in the DLH-3073 extract . These data suggest that poly-herbal extracts may alleviate radical-mediated diseases.

Plant Physiol, 2003 Jan, 131(1), 317 - 25
Divergent light-, ascorbate-, and oxidative stress-dependent regulation of expression of the peroxiredoxin gene family in Arabidopsis; Horling F et al.; Peroxiredoxins (prxs) are peroxidases with broad substrate specificity . The seven prx genes expressed in Arabidopsis shoots were analyzed for their expressional response to changing photon fluence rates, oxidative stress, and ascorbate application . The results reveal a highly variable and gene-specific response to reducing and oxidizing conditions . The steady-state transcript amounts of the chloroplast-targeted prxs, namely the two-cysteine (2-Cys) prxs, prx Q and prx II E, decreased upon application of ascorbate . prx Q also responded to peroxides and diamide treatment . prx II B was induced by tertiary butylhydroperoxide, but rather unaffected by ascorbate . The strongest responses were observed for prx II C, which was induced with all treatments . The two Arabidopsis 2-Cys Prxs and four Prx II proteins were expressed heterologously in Escherichia coli . In an in vitro test system, they all showed peroxidase activity, but could be distinguished by their ability to accept dithiothreitol and thioredoxin as electron donor in the regeneration reaction . The midpoint redox potentials (E(m)') of Prx II B, Prx II C, and Prx II E were around -290 mV and, thus, less negative than E(m)' of Prx II F, 2-Cys Prx A, and 2-Cys Prx B (-307 to -322 mV) . The data characterize expression and function of the mitochondrial Prx II F and the chloroplast Prx II E for the first time, to our knowledge . Antibodies directed against 2-Cys Prx and Prx II C showed a slight up-regulation of Prx II protein in strong light and of 2-Cys Prx upon transfer both to high and low light . The results are discussed in context with the subcellular localization of the Prx gene products.

J Biol Chem, 2003 Mar 21, 278(12), 10081 - 6 Epub 2003 Jan 15.
Regulation of the nitric oxide reduction operon (norRVW) in Escherichia coli . Role of NorR and sigma54 in the nitric oxide stress response; Gardner AM et al.; Nitric oxide (NO) induces NO-detoxifying enzymes in Escherichia coli suggesting sensitive mechanisms for coordinate control of NO defense genes in response to NO stress . Exposure of E . coli to sub-micromolar NO levels under anaerobic conditions rapidly induced transcription of the NO reductase (NOR) structural genes, norV and norW, as monitored by lac gene fusions . Disruption of rpoN (sigma(54)) impaired the NO-mediated induction of norV and norW transcription and NOR expression, whereas disruption of the upstream regulatory gene, norR, completely ablated NOR induction . NOR inducibility was restored to NorR null mutants by expressing NorR in trans . Furthermore, an internal deletion of the N-terminal domain of NorR activated NOR expression independent of NO exposure . Neither NorR nor sigma(54) was essential for NO-mediated induction of the NO dioxygenase (flavohemoglobin) encoded by hmp . However, elevated NOR activity inhibited NO dioxygenase induction, and, in the presence of dioxygen, NO dioxygenase inhibited norV induction by NO . The results demonstrate the role of NorR as a sigma(54)-dependent regulator of norVW expression . A role for the NorR N-terminal domain as a transducer or sensor for NO is suggested.

J Biol Chem, 2003 Mar 21, 278(12), 10799 - 806 Epub 2003 Jan 15.
The first crystal structure of archaeal aldolase . Unique tetrameric structure of 2-deoxy-d-ribose-5-phosphate aldolase from the hyperthermophilic archaea Aeropyrum pernix; Sakuraba H et al.; A gene encoding a 2-deoxy-d-ribose-5-phosphate aldolase (DERA) homolog was identified in the hyperthermophilic Archaea Aeropyrum pernix . The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized . The enzyme is an extremely thermostable DERA; its activity was not lost after incubation at 100 degrees C for 10 min . The enzyme has a molecular mass of approximately 93 kDa and consists of four subunits with an identical molecular mass of 24 kDa . This is the first report of the presence of tetrameric DERA . The three-dimensional structure of the enzyme was determined by x-ray analysis . The subunit folds into an alpha/beta-barrel . The asymmetric unit consists of two homologous subunits, and a crystallographic 2-fold axis generates the functional tetramer . The main chain coordinate of the monomer of the A . pernix enzyme is quite similar to that of the E . coli enzyme . There was no significant difference in hydrophobic interactions and the number of ion pairs between the monomeric structures of the two enzymes . However, a significant difference in the quaternary structure was observed . The area of the subunit-subunit interface in the dimer of the A . pernix enzyme is much larger compared with the E . coli enzyme . In addition, the A . pernix enzyme is 10 amino acids longer than the E . coli enzyme in the N-terminal region and has an additional N-terminal helix . The N-terminal helix produces a unique dimer-dimer interface . This promotes the formation of a functional tetramer of the A . pernix enzyme and strengthens the hydrophobic intersubunit interactions . These structural features are considered to be responsible for the extremely high stability of the A . pernix enzyme . This is the first description of the structure of hyperthermophilic DERA and of aldolase from the Archaea domain.

J Biol Chem, 2003 Mar 21, 278(12), 10484 - 90 Epub 2003 Jan 14.
The fourth epidermal growth factor-like domain of thrombomodulin interacts with the basic exosite of protein C; Yang L et al.; Thrombomodulin (TM) functions as a cofactor to enhance the rate of protein C activation by thrombin approximately 1000-fold . The molecular mechanism by which TM improves the catalytic efficiency of thrombin toward protein C is not known . Molecular modeling of the protein C activation based on the crystal structure of thrombin in complex with the epidermal growth factor-like domains 4, 5, and 6 of TM (TM456) predicts that the binding of TM56 to exosite 1 of thrombin positions TM4 so that a negatively charged region on this domain juxtaposes a positively charged region of protein C . It has been hypothesized that electrostatic interactions between these oppositely charged residues of TM4 and protein C facilitate a proper docking of the substrate into the catalytic pocket of thrombin . To test this hypothesis, we have constructed several mutants of TM456 and protein C in which charges of the putative interacting residues on both TM4 (Asp/Glu) and protein C (Lys/Arg) have been reversed . Results of TM-dependent protein C activation studies by such a compensatory mutagenesis approach support the molecular model that TM4 interacts with the basic exosite of protein C.

J Biol Chem, 2003 Mar 21, 278(12), 10744 - 51 Epub 2003 Jan 14.
Analysis of the interaction between the global regulator Mlc and EIIBGlc of the glucose-specific phosphotransferase system in Escherichia coli; Seitz S et al.; Mlc is a global regulator acting as a transcriptional repressor for several genes and operons of Escherichia coli encoding sugar-metabolizing enzymes and uptake systems . The repressing activity of Mlc is inactivated by binding to the dephosphorylated form of EIICB(Glc) (PtsG), which is formed during the transport of glucose . Here, we demonstrate that EIIB(Glc), the cytoplasmic domain of PtsG, alone is sufficient to inactivate Mlc but only when EIIB(Glc) is attached to the membrane by a protein anchor, which can be unrelated to PtsG . Several EIIB(Glc) mutants, which were altered in and around the phosphorylation site (Cys-421) of EIIB(Glc), were tested for their ability to bind Mlc and to affect transcriptional repression by Mlc . The exchange of Cys-421 with serine or aspartate still allowed binding to Mlc, and in addition, derepression became constitutive, i.e . independent of phosphoenolpyruvate-dependent phosphotransferase system (PTS) phosphorylation . Mutations were made in the surface-exposed residues in the vicinity of Cys-421 and identified Arg-424 as essential for binding to Mlc . Binding of Mlc to the EIIB(Glc) constructs in membrane preparations paralleled their ability to derepress Mlc-dependent transcription in vivo . These observations demonstrate that it is not the charge change at Cys-421, produced by PTS phosphorylation, that allows Mlc binding but rather the structural change in the environment surrounding Cys-421 that the phosphorylation provokes . Native Mlc exists as a tetramer . Deleting 18 amino acids from the C-terminal removes a putative amphipathic helix and results in dimeric Mlc that is no longer able to repress.

Am J Physiol Cell Physiol, 2003 Jun, 284(6), C1355 - 61 Epub 2003 Jan 15.
Glutamine delays spontaneous apoptosis in neutrophils; Pithon-Curi TC et al.; Nuclear, mitochondrial, and plasma membrane events associated with apoptosis were investigated in rat neutrophils cultivated for 3, 24, and 48 h in the absence or presence of glutamine (0.5, 1.0, and 2.0 mM) . Condensation of chromatin was reduced after 24 or 48 h of culture in the presence of glutamine compared with its absence as assessed by Hoechst 33342 staining . The level of Escherichia coli phagocytosis in the presence of glutamine was markedly increased compared with the level achieved by cells cultured in the absence of glutamine . Annexin V binding to externalized phosphatidylserine was reduced in the presence of glutamine . Sensitive fluorochrome rhodamine 123, as determined by fluorescence-activated cell sorting and confocal microscopy, was used to monitor loss of the mitochondrial transmembrane potential . In the absence of glutamine, neutrophils exhibited a marked reduction in the uptake of rhodamine 123 . In the presence of 1.0 or 2.0 mM glutamine, the uptake of rhodamine was 20 or 38% higher, respectively . Similar effect was found in human neutrophils by measuring DNA fragmentation and mitochondrial transmembrane potential . Therefore, glutamine protects from events associated with triggering and executing apoptosis in both rat and human neutrophils.

Biochem J, 2003 Apr 15, 371(Pt 2), 573 - 9
A pheromone-binding protein from the cockroach Leucophaea maderae: cloning, expression and pheromone binding; Riviere S et al.; Odorant-binding proteins (OBPs) are thought to transport volatile compounds from air to their receptors through the sensillary lymph . In this protein family, the subgroup of pheromone-binding proteins (PBPs) is specifically tuned to the perception of the sexual pheromone . To date, the description of OBPs has been restricted to Endopterygota and Paraneoptera . Their expression in Orthopteroid has been hypothesized, but no evidence of OBP has been produced in this assemblage to date . In the present study, we describe the first OBP from a Dictyopteran insect that belongs to the cockroach Leucophaea maderae . The PBP of L . maderae (PBPLma) shares all the hallmarks of the OBP family and is expressed specifically in the female adult antennae, the sex that perceives the sexual pheromone . The affinity of the recombinant PBPLma produced in the Escherichia coli periplasm for the pheromonal compounds has been tested by displacement of a fluorophore, 8-anilino-1-naphtalenesulphonic acid (ANS) . Our results suggest that two chemically close compounds of the pheromonal blend (3-hydroxy-butan-2-one and butane-2,3-diol) are capable of displacing ANS, whereas two other pheromone components (E-2-octenoic acid and senecioic acid) and other alkyl volatile compounds are not capable of displacing ANS, indicating a certain filtering of binding, which can be correlated with the putative function.

APMIS, 2002 Sep, 110(9), 665 - 72
Clonal clustering and colonization factors among thermolabile and porcine thermostable enterotoxin-producing Escherichia coli; Valvatne H et al.; A considerable proportion of enterotoxigenic Escherichia coli (ETEC) do not possess identifiable colonization factors (CFs) . Genetic fingerprint analyses based on repetitive sequence-based polymerase chain reaction (rep-PCR) showed that 9 of 10 such CF-negative isolates which produced the thermolabile and the porcine thermostabile enterotoxin could be divided into three clusters . Following transformation with a plasmid harbouring the gene encoding CfaR, a positive regulator for several ETEC adhesins, three of the six strains in the first cluster expressed coli surface antigen 20 (CS20) . No CFs were identified on the two transformed strains in the second cluster while the transformants of the two strains in the last cluster expressed CS12, the N-terminal amino acid sequence of which was deciphered . The study illustrates the potential of using genetic fingerprinting to group ETEC into clusters of strains with genes encoding different CFs and confirms the ability of CfaR to induce the expression of several different CFs.

Bioorg Khim, 2002 Nov-Dec, 28(6), 497 - 501
{The effect of pyrophosphate analogues on the inorganic pyrophosphatase from Escherichia coli}; Zakirova NF et al.; The effect of inorganic pyrophosphate analogues on the enzymic activity of inorganic pyrophosphatase from E . coli was studied . Hypophosphoric and diphosphonic acids were shown to inhibit inorganic pyrophosphatase, whereas pyrophosphorous acid exerts almost no effect on the hydrolysis of inorganic pyrophosphate . The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol . 28, no . 6; see also http://www.maik.ru.

Mol Pharmacol, 2003 Feb, 63(2), 283 - 8
Opioids bind to the amino acids 84 to 118 of UDP-glucuronosyltransferase UGT2B7; Coffman BL et al.; The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform that catalyzes the conjugation of many endogenous and exogenous compounds, among them opioids, resulting in the formation of D-glucuronides . The binding site of the aglycone is located in the N-terminal half of the protein . Using NMR analysis, we demonstrate that the opioid binding site in UGT2B7 is within the 84 to 118 N-terminal amino acids . Three maltose binding protein-UGT2B7 fusion proteins, 2B7F3 and 2B7F4 incorporating the amino acids 24 to 118 and 24 to 96 of UGT2B7, respectively, and 2B7F5 incorporating amino acids 84 to 118 of UGT2B7 were expressed in Escherichia coli and purified by affinity chromatography . NMR analysis showed that morphine was bound to the fusion protein 2B7F3 with a K(D) value similar to the K(D) values obtained for the previously produced fusion proteins, which included amino acids 24 to 180 . Morphine did not bind to 2B7F4, but it did bind to 2B7F5 . Both NMR 1-D spectra and NOESY experiments indicated that the 2B7F5 protein was mediating magnetization transfer within the morphine . These results allowed us to predict and model a binding site within the amino acids 96 to 101 of UGT2B7 . A mutant fusion protein 2B7F3 with the substitution D99A was produced, and the NMR spectroscopy analysis of the protein supported the model . A marked reduction of morphine binding was observed when the charged aspartate was substituted with alanine.

Nucleic Acids Res, 2003 Jan 15, 31(2), E6 - 6
Improving baculovirus recombination; Zhao Y et al.; Recombinant baculoviruses have established themselves as a favoured technology for the high-level expression of recombinant proteins . The construction of recombinant viruses, however, is a time consuming step that restricts consideration of the technology for high throughput developments . Here we use a targeted gene knockout technology to inactivate an essential viral gene that lies adjacent to the locus used for recombination . Viral DNA prepared from the knockout fails to initiate an infection unless rescued by recombination with a baculovirus transfer vector . Modified viral DNA allows 100% recombinant virus formation, obviates the need for further virus purification and offers an efficient means of mass parallel recombinant formation.

J Biol Chem, 2003 Mar 14, 278(11), 8869 - 72 Epub 2003 Jan 13.
Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection; Girardin SE et al.; Nod2 activates the NF-kappaB pathway following intracellular stimulation by bacterial products . Recently, mutations in Nod2 have been shown to be associated with Crohn's disease, suggesting a role for bacteria-host interactions in the etiology of this disorder . We show here that Nod2 is a general sensor of peptidoglycan through the recognition of muramyl dipeptide (MDP), the minimal bioactive peptidoglycan motif common to all bacteria . Moreover, the 3020insC frameshift mutation, the most frequent Nod2 variant associated with Crohn's disease patients, fully abrogates Nod2-dependent detection of peptidoglycan and MDP . Together, these results impact on the understanding of Crohn's disease development . Additionally, the characterization of Nod2 as the first pathogen-recognition molecule that detects MDP will help to unravel the well known biological activities of this immunomodulatory compound.

J Biol Chem, 2003 Apr 11, 278(15), 12913 - 9 Epub 2003 Jan 14.
Domain motions and quaternary packing of phosphofructokinase-2 from Escherichia coli studied by small angle x-ray scattering and homology modeling; Cabrera R et al.; The binding of MgATP and fructose-6-phosphate to phosphofructokinase-2 from Escherichia coli induces conformational changes that result in significant differences in the x-ray-scattering profiles compared with the unligated form of the enzyme . When fructose- 6-phosphate binds to the active site of the enzyme, the pair distribution function exhibits lower values at higher distances, indicating a more compact structure . Upon binding of MgATP to the allosteric site of the enzyme, the intensity at lower angles increases as a consequence of tetramer formation, but differences along higher angles also suggest changes at the tertiary structure level . We have used homology modeling to build the native dimeric form of phosphofructokinase-2 and fitted the experimental scattering curves by using rigid body movements of the domains in the model, similar to those observed in known homologous structures . The best fit with the experimental data of the unbound protein was achieved with open conformations of the domains in the model, whereas domain closure improves the agreement with the scattering of the enzyme-fructose-6-phosphate complex . Using the same approach, we utilized the scattering curve of the phosphofructokinase-2-MgATP complex to model the arrangement and conformation of dimers in the tetramer . We observed that, along with tetramerization, binding of MgATP to the allosteric site induces domain closure . Additionally, we used the scattering data to restore the low resolution structure of phosphofructokinase-2 (free and bound forms) by an ab initio procedure . Based on these findings, a proposal is made to account for the inhibitory effect of MgATP on the enzymatic activity.

FEBS Lett, 2003 Jan 16, 534(1-3), 156 - 60
DmsD is required for the biogenesis of DMSO reductase in Escherichia coli but not for the interaction of the DmsA signal peptide with the Tat apparatus; Ray N et al.; The DmsD protein is essential for the biogenesis of DMSO reductase in Escherichia coli, and binds the signal peptide of the DmsA subunit, a Tat substrate . This suggests a role as a guidance factor to target pre-DmsA to the translocase . Here, we have analysed the export of fusion proteins in which the DmsA and TorA signal peptides are fused to green fluorescent protein . Both chimeras are efficiently exported to the periplasm in wild-type E . coli cells and we show that their export efficiencies are essentially identical in a mutant lacking DmsD . An authentic Tat substrate, TMAO reductase, is also efficiently exported in the dmsD mutant . The data indicate that DmsD carries out a critical role in DMSO reductase biogenesis/assembly but is not required for the functioning of the DmsA signal peptide.

FEBS Lett, 2003 Jan 16, 534(1-3), 139 - 42
The processing of human mitochondrial leucyl-tRNA synthetase in the insect cells; Yao YN et al.; A His-tagged full-length cDNA of human mitochondrial leucyl-tRNA synthetase was expressed in a baculovirus system . The N-terminal sequence of the enzyme isolated from the mitochondria of insect cells was found to be IYSATGKWTKEYTL, indicating that the mitochondrial targeting signal peptide was cleaved between Ser39 and Ile40 after the enzyme precursor was translocated into mitochondria . The enzyme purified from mitochondria catalyzed the leucylation of Escherichia coli tRNA(1)(Leu)(CAG) and Aquifex aeolicus tRNA(Leu)(GAG) with higher catalytic activity in the leucylation of E . coli tRNA(Leu) than that previously expressed in E . coli without the N-terminal 21 residues.

J Mol Biol, 2003 Jan 31, 325(5), 1093 - 105
pK values of histidine residues in ribonuclease Sa: effect of salt and net charge; Huyghues-Despointes BM et al.; The primary goal of this study was to gain a better understanding of the effect of environment and ionic strength on the pK values of histidine residues in proteins . The salt-dependence of pK values for two histidine residues in ribonuclease Sa (RNase Sa) (pI=3.5) and a variant in which five acidic amino acids have been changed to lysine (5K) (pI=10.2) was measured and compared to pK values of model histidine-containing peptides . The pK of His53 is elevated by two pH units (pK=8.61) in RNase Sa and by nearly one pH unit (pK=7.39) in 5K at low salt relative to the pK of histidine in the model peptides (pK=6.6) . The pK for His53 remains elevated in 1.5M NaCl (pK=7.89) . The elevated pK for His53 is a result of screenable electrostatic interactions, particularly with Glu74, and a non-screenable hydrogen bond interaction with water . The pK of His85 in RNase Sa and 5K is slightly below the model pK at low salt and merges with this value at 1.5M NaCl . The pK of His85 reflects mainly effects of long-range Coulombic interactions that are screenable by salt . The tautomeric states of the neutral histidine residues are changed by charge reversal . The histidine pK values in RNase Sa are always higher than the pK values in the 5K variant . These results emphasize that the net charge of the protein influences the pK values of the histidine residues . Structure-based pK calculations capture the salt-dependence relatively well but are unable to predict absolute histidine pK values.

J Mol Biol, 2003 Jan 31, 325(5), 937 - 48
Coupling of protein assembly and DNA binding: biotin repressor dimerization precedes biotin operator binding; Streaker ED et al.; The kinetics of coupling of protein dimerization and DNA binding have been investigated in the biotin repressor system . Two repressor monomers bind to the 40 base-pair biotin operator sequence . In previous analyses of equilibrium-binding data the weak dimerization of the repressor has justified using a model in which two protein monomers bind cooperatively to the operator site . Here, rapid kinetic methods have been used to directly determine the binding mechanism . Results of rapid-mixing DNaseI footprinting measurements of association of the repressor with operator indicate that the binding process involves at least two steps . Results of measurements of the unimolecular dissociation of the complex reveal a half-life of approximately 400 seconds . Analysis of the data using a combination of simulation and global non-linear least-squares analysis provides support for a binding model in which a preformed repressor dimer associates with the biotin operator . This kinetic model is consistent with the previously proposed model for regulation of the functional switch in the repressor from enzyme to site-specific DNA-binding protein.

J Mol Biol, 2003 Jan 31, 325(5), 913 - 35
A Dimer of Escherichia coli UvrD is the active form of the helicase in vitro; Maluf NK et al.; The Escherichia coli UvrD protein is a 3' to 5' SF1 DNA helicase involved in methyl-directed mismatch repair and nucleotide excision repair of DNA . We have characterized in vitro UvrD-catalyzed unwinding of a series of 18 bp duplex DNA substrates with 3' single-stranded DNA (ssDNA) tails ranging in length from two to 40 nt . Single turnover DNA-unwinding experiments were performed using chemical quenched flow methods, as a function of both {UvrD} and {DNA} under conditions such that UvrD-DNA binding is stoichiometric . Although a single UvrD monomer binds tightly to the single-stranded/double-stranded DNA (dsDNA) junction if the 3' ssDNA tail is at least four nt, no unwinding was observed for DNA substrates with tail-lengths </=8 nt, even at high {UvrD}/{DNA} ratios . Unwinding is observed for DNA substrates with 3' ssDNA tail lengths >/=12 nt, and the unwinding amplitude displays a sigmoidal dependence on {UvrD(tot)}/{DNA(tot)} . Quantitative analysis of these data indicates that a single UvrD monomer bound at the ssDNA/dsDNA junction of any DNA substrate, independent of 3' ssDNA tail length, is not competent to fully unwind even a short 18 bp duplex DNA, and that two UvrD monomers must bind the DNA substrate in order to form a complex that is able to unwind short DNA substrates in vitro . Other proteins, including a mutant UvrD with no ATPase activity as well as a monomer of the structurally homologous E.coli Rep helicase, cannot substitute for the second UvrD monomer, suggesting a specific interaction between two UvrD monomers and that both must be able to hydrolyze ATP . Initiation of DNA unwinding in vitro appears to require a dimeric UvrD complex in which one subunit is bound to the ssDNA/dsDNA junction, while the second subunit is bound to the 3' ssDNA tail.

J Mol Biol, 2003 Jan 31, 325(5), 889 - 912
Self-association equilibria of Escherichia coli UvrD helicase studied by analytical ultracentrifugation; Maluf NK et al.; The Escherichia coli UvrD protein (helicase II) is an SF1 superfamily helicase required for methyl-directed mismatch repair and nucleotide excision repair of DNA . We have characterized quantitatively the self-assembly equilibria of the UvrD protein as a function of {NaCl}, {glycerol}, and temperature (5-35 degrees C; pH 8.3) using analytical sedimentation velocity and equilibrium techniques, and find that UvrD self-associates into dimeric and tetrameric species over a range of solution conditions (t</=25 degrees C) . Increasing {NaCl} from 20mM to 200 mM decreases the dimerization equilibrium constant (L(20)) from 2.33(+/-0.30) microM(-1) to 0.297(+/-0.006) microM(-1) (pH 8.3, 20% (v/v) glycerol, 25 degrees C) . The overall tetramerization equilibrium constant (L(40)) is 5.11(+/-0.80) microM(-3) at 20mM NaCl, but decreases so that it is not measurable at 200 mM NaCl . At 500 mM NaCl, only UvrD monomers are detectable . Increasing {glycerol} over the range from 20% to 40% (v/v) decreases both L(20) and L(40) . We find no evidence for hexamer formation, although a species consistent in size with an octamer is detected at 35 degrees C . Inclusion of either ADP or ATPgammaS does not affect either L(20) or L(40) significantly, and does not induce the formation of additional assembly states . We also investigated the stoichiometry of UvrD binding to a 3'-(dT)(20)-18 bp DNA substrate by sedimentation equilibrium . At saturating concentrations of UvrD, three UvrD monomers can bind to the DNA substrate, although only two UvrD monomers are required to form a processive helicase complex . When the total DNA substrate concentration is about twofold greater than the total UvrD concentration, the vast majority of the DNA is bound by a single UvrD monomer.

J Mol Biol, 2003 Jan 31, 325(5), 857 - 72
Purification, characterization, and gene expression of all sigma factors of RNA polymerase in a cyanobacterium; Imamura S et al.; The expression of RNA polymerase (RNAP) sigma factor genes and proteins was characterized as a first step toward understanding their functions in a unicellular cyanobacterium Synechocystis sp . PCC 6803, which can perform photosynthesis . All nine sigma factors (group 1, SigA; group 2, SigB to SigE; and group 3, SigF to SigI) and each RNAP core subunit (RpoA, RpoB, RpoC1 and RpoC2) were overproduced and purified from Escherichia coli cells, then polyclonal antibodies were prepared . Western blot and primer extension analyses revealed that the intracellular levels of group 1 and 2 sigma factors ranged from 0.9 fmol to 9.3 fmol per microgram of the total protein under conditions of steady-state growth, and that growth phase-dependent or constitutive transcripts were observed . Interestingly, no group 3 sigma factor proteins were detected under normal physiological conditions whereas their transcripts were robust, implying a possible regulation of translational attenuation and/or protein instability . Phylogenetic analysis also revealed that group 3 sigma factor homologues of cyanobacteria are conserved with evolutionary or functionary divergence among them . In vitro and in vivo results indicated significant evidence of high-light responsive SigD expression and its promoter recognition of the photosynthesis gene, psbA . On the other hand, autoregulated sigB transcription, a dramatically increased SigB expression upon the exposure of cells to heat-shock, and specific promoter recognition by SigB with redundancy of other sigma factors on the heat-shock hspA promoter were observed . These findings clearly indicated that SigB is a heat-shock responsive sigma factor . The unique promoter architecture and expression of the relevant sigma factor gene are also discussed herein.

J Mol Biol, 2003 Jan 31, 325(5), 843 - 56
The solution structure of the loop E region of the 5S rRNA from spinach chloroplasts; Vallurupalli P et al.; A structure has been obtained for the loop E region of the 5S rRNA from Spinacia oleracia chloroplast ribosomes using residual dipolar coupling data as well as NOE, J coupling and chemical shift information . Even though the loop E sequence of this chloroplast 5S rRNA differs from that of Escherichia coli loop E at approximately 40% of its positions, its conformation is remarkably similar to that of E.coli loop E . Consistent with this conclusion, ribosomal protein L25 from E.coli, which binds to the loop E region of both intact E.coli 5S rRNA and to oligonucleotides containing that sequence, also binds to the chloroplast-derived oligonucleotide discussed here.

Biochim Biophys Acta, 2003 Jan 20, 1619(2), 193 - 200
In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions; Gangopadhyay JP et al.; Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond . We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag) . The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence . When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine . Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products . Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site . We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161 . For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy . The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.

Biochim Biophys Acta, 2003 Jan 20, 1619(2), 139 - 43
A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins; Hefti MH et al.; The NifL PAS domain from Azotobacter vinelandii is a flavoprotein with FAD as the prosthetic group . Here we describe a novel immobilization procedure for the large-scale preparation of apo NifL PAS domain and its efficient reconstitution with either 2,4a-13C-FAD or 2,4a-13C-FMN . In this procedure, the His-tagged holoprotein is bound to an immobilized metal affinity column and the flavin is released by washing the column with buffer containing 2 M KBr and 2 M urea . The apoprotein is reconstituted on-column with the (artificial) flavin cofactor, and then eluted with buffer containing 250 mM imidazole . Alternatively, the immobilized apoprotein can be released from the column matrix before reconstitution.The His-tag based immobilization method of preparing reconstituted (or apo) NifL PAS domain protein has the advantage that it combines a protein affinity chromatography technique with limited protein loss, resulting in a high protein yield with extremely efficient flavin reconstitution . This on-column reconstitution method can also be used in cases where the apoprotein is unstable . Therefore, it may develop as a universal method for replacement of flavin or other cofactors.

Fish Shellfish Immunol, 2003 Feb, 14(2), 115 - 32
Production of rabbit antisera against recombinant MHC class II beta chain and identification of immunoreactive cells in Atlantic salmon (Salmo salar); Koppang EO et al.; In the present work, rabbit antisera recognising the Atlantic salmon (Salmo salar) MHC class II beta chain polypeptide were produced and used in immunoblotting, immunohistochemistry and immunogold electron microscopy . A construct encoding the beta1 and beta2 domains fused to the E . coli protein thioredoxin was used to express the recombinant MHC class II beta chain . Immunoblotting revealed a band of approximately 30kDa in total protein samples from head kidney, spleen, gills, thymus and blood leukocytes, while being absent in muscle . The distribution of MHC class II positive cells was immunohistochemically demonstrated in Atlantic salmon epithelial and haemopoietic tissues . Ultrastructural demonstration of immunoreactive organelles in mid-kidney cells was performed by immunogold electron microscopy . The results indicate expression in lymphocytes, macrophages, epithelial cells and endritic-like cells . This is the first study to address morphological MHC class II expression in a fish species.

Trends Microbiol, 2003 Jan, 11(1), 21 - 9
The structure and function of drug pumps: an update; McKeegan KS et al.; Our understanding of the exact mechanisms used by the transmembrane protein pumps that confer cellular resistance to cytotoxic drugs has improved enormously with the recent determination of the structures of three Escherichia coli transporters, two belonging to the ATP-binding cassette (ABC) superfamily and one to the resistance-nodulation-cell division (RND) family . Although these studies do not provide an insight into how drug pumps can recognize several structurally unrelated drugs, important advances have been also made in this area . Information on the molecular basis of multidrug recognition has been provided by determining the structure of transcriptional regulators that can bind, often structurally unrelated, cytotoxic drugs and control the expression of drug pumps.

Trends Microbiol, 2003 Jan, 11(1), 6 - 8
In search of the minimal Escherichia coli genome; Smalley DJ et al.; Recent plans announced for the systematic cataloging of the minimal Escherichia coli gene set, the phenotypes of all mutations, the expression levels of every transcript and gene product, and the interactions of all genetic loci or their gene products point the way towards a new frontier in the biology of model organisms . Powerful tools for this endeavor are emerging, and efforts to organize the E . coli community are under way . The anticipated benefit is a functional model of the bacterial cell.

Cell, 2003 Jan 10, 112(1), 113 - 22
Crystal structure of carnitine acetyltransferase and implications for the catalytic mechanism and fatty acid transport; Jogl G et al.; Carnitine acyltransferases have crucial roles in the transport of fatty acids for beta-oxidation . Dysregulation of these enzymes can lead to serious diseases in humans, and they are targets for therapeutic development against diabetes . We report the crystal structures of murine carnitine acetyltransferase (CRAT), alone and in complex with its substrate carnitine or CoA . The structure contains two domains . Surprisingly, these two domains share the same backbone fold, which is also similar to that of chloramphenicol acetyltransferase and dihydrolipoyl transacetylase . The active site is located at the interface between the two domains . Carnitine and CoA are bound in deep channels in the enzyme, on opposite sides of the catalytic His343 residue . The structural information provides a molecular basis for understanding the catalysis by carnitine acyltransferases and for designing their inhibitors . Specifically, our structural information suggests that the substrate carnitine may assist the catalysis by stabilizing the oxyanion in the reaction intermediate.

Cell, 2003 Jan 10, 112(1), 99 - 111
Structures of the alpha L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation; Shimaoka M et al.; The structure of the I domain of integrin alpha L beta 2 bound to the Ig superfamily ligand ICAM-1 reveals the open ligand binding conformation and the first example of an integrin-IgSF interface . The I domain Mg2+ directly coordinates Glu-34 of ICAM-1, and a dramatic swing of I domain residue Glu-241 enables a critical salt bridge . Liganded and unliganded structures for both high- and intermediate-affinity mutant I domains reveal that ligand binding can induce conformational change in the alpha L I domain and that allosteric signals can convert the closed conformation to intermediate or open conformations without ligand binding . Pulling down on the C-terminal alpha 7 helix with introduced disulfide bonds ratchets the beta 6-alpha 7 loop into three different positions in the closed, intermediate, and open conformations, with a progressive increase in affinity.

J Am Chem Soc, 2003 Jan 22, 125(3), 654 - 61
Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2'F-ANA constructs containing acyclic nucleotide inserts; Mangos MM et al.; The ability of modified antisense oligonucleotides (AONs) containing acyclic interresidue units to support RNase H-promoted cleavage of complementary RNA is described . Manipulation of the backbone and sugar geometries in these conformationally labile monomers shows great benefits in the enzymatic recognition of the nucleic acid hybrids, while highlighting the importance of local strand conformation on the hydrolytic efficiency of the enzyme more conclusively . Our results demonstrate that the duplexes support remarkably high levels of enzymatic degradation when treated with human RNase HII, making them efficient mimics of the native substrates . Furthermore, interesting linker-dependent modulation of enzymatic activity is observed during in vitro assays, suggesting a potential role for this AON class in an RNase H-dependent pathway of controlling RNA expression . Additionally, the butyl-modified 2'F-ANA AONs described in this work constitute the first examples of a nucleic acid species capable of eliciting high RNase H activity while possessing a highly flexible molecular architecture at predetermined sites along the AON.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Dec, 12(4), 350 - 2
{Soluble expression and identification of expressed single chain antibody fragment 3D3 against dengue virus type 3}; Si B et al.; Hybridoma secreting 3D3 monoclonal antibody against dengue virus type 3 was used . The isolated mRNA was reverse-transcribed into cDNA for amplifying the heavy and light chain variable domains genes . The amplified light and heavy chain variable domains' genes were connected by flexible linker to from a single chain fragment (ScFv) gene of 750 bp, which was cloned into phage pCANTAB5E vector using the recombination phage antibody system . pCANTAB5E vector transformed E coli HB2151 . Soluble single chain antibody fragment was expressed both in the bacterial supernatant and cell periplasm under the induction of IPTG . The expressed single chain antibody fragment has a molecular weight of about 28 kD detected by SDS-PAGE and can react with dengue virus type 3 by immunofluorescence test.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Sep, 12(3), 245 - 8
{Rapid construction of recombinant baculovirus carrying hepatitis B virus surface antigen gene by using a novel baculovirus shuttle vector(Bacmid)}; Miao J et al.; In order to express the HBV surface antigen (HBV) efficiently by using the Bac-to-Bac system and to produce HBsAg for diagnosis and vaccine development of HBV, a recombinant donor plasmid pFB-BS carrying HBsAg gene was constructed . The gene was inserted into shuttle plasmid (Bacmid) in E . coli DH10Bac with the help of Tn7 transposition system . The recombinant Bacmid was examined by PCR and transfected to Sf9 cells . HBsAg was expressed under the control of polyhedrin promoter of baculovirus in insect cells and was identified by ELISA . The quantity of the expression reached to 2 micrograms/10(6) cells and most production was within cells . Our data suggested that the Bac-to-Bac system can be used successfully and rapidly in the expression of viral gene.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Sep, 12(3), 233 - 6
{Expression of human ScFv against human immunodeficiency virus type 1 gp120}; He H et al.; In order to generate human antibody against HIV-1, using synthetic peptide from HIV-1 gp120 v3 loop as antigen, we screened the recombinant phage antibodies by immunoaffinity and selected positive clones carrying anti-gp120 ScFv gene . The phage DNA was extracted and transformed to E . coli HB2151 that allowed to express soluble ScFv proteins by inducing of IPTG . The results of SDS-PAGE and Western blot showed that the molecular weight of the expressed product is about 28 KD and exhibited anti-c-myc activity . ELISA and Dot blot analysis displayed that the soluble ScFv specifically bound to gp120 and had a higher specificity and affinity . Competitive ELISA further demonstrated that the product was specific to gp120.

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi, 1998 Sep, 12(3), 229 - 32
{Gene cloning, expression and immunological identification of a single chain antibody fragment 3D3 against dengue type 3 virus}; Si B et al.; Hybridoma 3D3 which secreted monoclonal antibody against Dengue type 3 virus was used to extract mRNA and then was reverse-transcripted into cDNA for amplifying heavy and light chain variable domain genes . The amplified light and heavy chain variable domain genes were connected by flexible linker to form a single chain antibody fragment (ScFv) gene of 750 bp, which was cloned into phage pCANTAB5 vector using the recombinant phage antibody system . The ScFv gene was expressed as fusion protein with phage g3p coat protein and displayed on the phage surface . The phagemid was used to transform competent E . coli TG1 cells and then infected with M13K07 helper phage to rescue the phagemid and antibody ScFv gene . 12 of the 20 randomized clones were shown to react with dengue type 3 virus by immunofluorescence.

Exp Mol Med, 2002 Nov 30, 34(5), 385 - 90
Effects of Val34Leu and Val35Leu polymorphism on the enzyme activity of the coagulation factor XIII-A; Lee IH et al.; Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis . In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (gammaFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet . Using these gammaFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated gammaFXIII-A were characterized . gammaFXIII-A (V34L) and gammaFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type gammagFXIII-A and V35L variant . However, the activated gammaFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay . These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2-plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.

J Med Virol, 2003 Mar, 69(3), 451 - 8
Induction of cellular immunity to varicella-zoster virus glycoproteins tested with pernasal coadministration of Escherichia coli enterotoxin in mice; Tsuji T et al.; A mutant of Escherichia coli enterotoxin promotes the induction of cellular immunity to a live varicella vaccine (the Oka strain) as a mucosal adjuvant in mice . An investigation was carried out to determine which of the purified glycoproteins of the virus among three induced cellular immunity with a single nasal administration . Spleen cells from mice immunized nasally with the vaccine and toxin produced interleukin-2 (IL-2) at the same level on restimulation in vitro with glycoprotein H: glycoprotein L (gH:gL), gB, and gE:gI, but not IL-4 . The spleen cells from mice immunized with gH:gL, gB, or gE:gI and toxin produced IL-2 on restimulation with gH:gL, gB, or gE:gI, respectively, and the vaccine, but not IL-4 . Immunization with gH:gL and the toxin showed increased thymidine uptake and production of IL-2 and interferon-gamma (IFN-gamma) of the spleen cells, but not IL-4, depending on the dose of gH:gL used for immunization and restimulation in vitro . Purified gE:gI and gB have been reported to be the strongest stimulators of cellular immunity to varicella upon subcutaneous injection and are useful as a subunit vaccine . All the glycoproteins tested are excellent stimulators of cellular immunity to the virus and itself on nasal co-immunization with the toxin .

J Biol Chem, 2003 Mar 21, 278(12), 10291 - 6 Epub 2003 Jan 13.
A new member of plant CS-lyases . A cystine lyase from Arabidopsis thaliana; Jones PR et al.; Cystine lyases catalyze the breakdown of l-cystine to thiocysteine, pyruvate, and ammonia . Until now there are no reports of the identification of a plant cystine lyase at a molecular level, and it is not clear what biological role this class of enzymes have in plants . A cystine lyase was isolated from Brassica oleracea (L.), and partial amino acid sequencing allowed the corresponding full-length cDNA (BOCL3) to be cloned . The deduced amino acid sequence of BOCL3 showed highest homology to the deduced amino acid sequences of several Arabidopsis thaliana genes annotated as tyrosine aminotransferase-like, including a coronatine, jasmonic acid, and salt stress-inducible gene, CORI3 (78.8% identity), and the unidentified rooty/superroot1 gene (44.8% identity) . A full-length expressed sequence tag clone of CORI3 was obtained and recombinant CORI3 was synthesized in Escherichia coli . Isolated recombinant CORI3 catalyzed a cystine lyase reaction, but no aminotransferase reactions . The present study identifies, for the first time, a cystine lyase from plants at a molecular level and redefines the functional assignment of the only functionally identified member of a group of A . thaliana genes annotated as tyrosine aminotransferase-like.

J Biol Chem, 2003 Apr 4, 278(14), 12319 - 24 Epub 2003 Jan 13.
Close proximity of a cytoplasmic loop of subunit a with c subunits of the ATP synthase from Escherichia coli; Zhang D et al.; Interactions between subunit a and the c subunits of the Escherichia coli ATP synthase are thought to control proton translocation through the F(o) sector . In this study cysteine substitution mutagenesis was used to define the cytoplasmic ends of the first three transmembrane spans of subunit a, as judged by accessibility to 3-N-maleimidyl-propionyl biocytin . The cytoplasmic end of the fourth transmembrane span could not be defined in this way because of the limited extent of labeling of all residues between 186 and 206 . In contrast, most of the preceding residues in that region, closer to transmembrane span 3, were labeled readily . The proximity of this region to other subunits in F(o) was tested by reacting mono-cysteine mutants with a photoactivated cross-linker . Residues 165, 169, 173, 174, 177, 178, and 182-184 could all be cross-linked to subunit c, but no sites were cross-linked to b subunits . Attempts using double mutants of subunit a to generate simultaneous cross-links to two different c subunits were unsuccessful . These results indicate that the cytoplasmic loop between transmembrane spans 3 and 4 of subunit a is in close proximity to at least one c subunit . It is likely that the more highly conserved, carboxyl-terminal region of this loop has limited surface accessibility due to protein-protein interactions . A model is presented for the interaction of subunit a with subunit c, and its implications for the mechanism of proton translocation are discussed.

Biochemistry, 2003 Jan 21, 42(2), 430 - 9
The limits of promiscuity: isoform-specific dimerization of filamins; Himmel M et al.; Filamins are a family of actin cross-linking proteins that are primarily localized in the cortical cytoplasm of all mammalian cells . Until now, three major isoforms (filamins a, b, and c) have been identified, that were shown to be differentially expressed and/or localized in different tissues . An amino-terminal double CH-domain actin binding domain, and a dimerization region in the carboxy-terminal portion of the protein are the molecular basis for its actin cross-linking activity . Chemical cross-linking of bacterially expressed recombinant proteins was used to demonstrate that in all three filamin isoforms the most carboxy-terminally situated immunoglobulinlike domain is required and sufficient for dimerization . The efficiency of the dimerization was increased upon inclusion of the preceding hinge 2 region, indicating a function for this region in the regulation of dimerization . By mixing recombinant proteins derived from different filamin isoforms, we found that heterodimer formation is possible between filamins b and c but not between filamin a and the other two filamins . This selectivity of dimerization might provide a further molecular explanation for the differential intracellular sorting of filamin isoforms and their distinct properties.

Biochemistry, 2003 Jan 21, 42(2), 410 - 20
Osmosensor ProP of Escherichia coli responds to the concentration, chemistry, and molecular size of osmolytes in the proteoliposome lumen; Culham DE et al.; Transporter ProP of Escherichia coli mediates the cellular accumulation of organic zwitterions in response to increased extracellular osmolality . We compared and characterized the osmoregulation of ProP activity in cells and proteoliposomes to define the osmotic shift-induced cellular change(s) to which ProP responds . ProP-(His)(6) activity in cells and proteoliposomes was correlated with medium osmolality, not osmotic shift, turgor pressure, or membrane strain . Both K(M) and V(max) for proline uptake via ProP-(His)(6) increased with increasing medium osmolality, as would be expected if osmolality controls the proportions of transporter with inactive and active conformations . The osmolality yielding half-maximal ProP-(His)(6) activity was higher in proteoliposomes than in cells . The osmolality response of ProP is also attenuated in bacteria lacking soluble protein ProQ . Indeed, the catalytic constant (k(cat)) for ProP-(His)(6) in proteoliposomes approximated that of ProP in intact bacteria lacking ProQ . Thus, the proteoliposome system may replicate a primary osmosensory response that can be further amplified by ProQ . ProP-(His)(6) is designated as an osmosensor because its activity is dependent on the osmolality, but not the composition, of the assay medium to which the cell surface is exposed . In contrast, ProP-(His)(6) activity was dependent on both the osmolality and the composition of the lumen in osmolyte-loaded proteoliposomes . For proteoliposomes containing inorganic salts, glucose, or poly(ethylene glycol) 503, transporter activity correlated with total lumenal cation concentration . In contrast, for proteoliposomes loaded with larger poly(ethylene glycol)s, the osmolality, the lumenal cation concentration, and the lumenal ionic strength at half-maximal transporter activity decreased systematically with poly(ethylene glycol) radius of gyration (range 0.8-1.8 nm) . These data suggest that ProP-(His)(6) responds to osmotically induced changes in both cytoplasmic K(+) levels and the concentration of cytoplasmic macromolecules.

Biochemistry, 2003 Jan 21, 42(2), 331 - 7
Helix packing in subunit a of the Escherichia coli ATP synthase as determined by chemical labeling and proteolysis of the cysteine-substituted protein; Zhang D et al.; Subunit a of the Escherichia coli ATP synthase is thought to control access of protons to the ring of c subunits during proton-driven ATP synthesis . In this study, the surface exposure of subunit a in the periplasm has been examined using 3-N-maleimidyl-propionyl biocytin labeling in cells permeabilized by polymyxin B nonapeptide, and the helix packing at the periplasmic surface has been probed by metal-chelate mediated proteolysis . Eighteen residues between 119 and 146 were changed individually to cysteine and tested for accessibility . Positions labeled included D124 and D146, indicating a periplasmic loop of at least 23 amino acids . Residues near the ends of the transmembrane spans were tested with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper for chemical proteolysis . Only residues W241C and D44C, and to a lesser extent I43C, led to proteolytic fragments after oxidation . The fragments were sized by comparison with molecular weight standards generated by Factor Xa sites engineered into subunit a . Fragments were detected by immunoblotting using an engineered HA epitope at the carboxyl-terminal end of subunit a . The results indicated that both transmembrane span 5 (W241) and transmembrane span 1 (D44) are close to transmembrane span 2.

Nat Biotechnol, 2003 Feb, 21(2), 191 - 4 Epub 2003 Jan 13.
Kindling fluorescent proteins for precise in vivo photolabeling; Chudakov DM et al.; Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins . Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell . Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker . A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins . However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration . In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP . The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling") . This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins . We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.

Proc Natl Acad Sci U S A, 2003 Jan 21, 100(2), 455 - 60 Epub 2003 Jan 10.
Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis; Yang JK et al.; One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form . We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli . A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations . Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase . We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH . The structure reveals that Asp-38 determines the cofactor specificity . The catalytic site includes the triad Ser-140Tyr-153Lys-157 . Additionally, it has an unusual feature, Glu-142 . Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153 . This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M . tuberculosis . Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.

J Biol Chem, 2003 Mar 14, 278(11), 9448 - 57 Epub 2003 Jan 10.
Protein interactions within the N-end rule ubiquitin ligation pathway; Siepmann TJ et al.; Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway . The concentration dependence for E1-catalyzed HsUbc2b/E2(14kb) transthiolation is hyperbolic and yields K(m) values of 102 +/- 13 nm and 123 +/- 19 nm for high affinity binding to rabbit and human E1/Uba1 orthologs . Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K(i) = 104 +/- 15 nm) and HsUbc2bC88S-ubiquitin oxyester (K(i) = 169 +/- 17 nm), respectively, indicates that the ubiquitin moiety contributes little to E1 binding . Under conditions of rate-limiting E3alpha-catalyzed conjugation to human alpha-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K(i) of 54 +/- 18 nm and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K(i) = 66 +/- 29 nm) . In contrast, the ligase product analog HsUbc2bC88A exhibits a K(i) of 440 +/- 55 nm with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3alpha to discriminate between substrate and product E2 . A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein . Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric (125)I-ubiquitin thiolester formation . Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways . The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.

J Biol Chem, 2003 Mar 21, 278(12), 10195 - 200 Epub 2003 Jan 10.
The isolated N-terminal domain of the glucagon-like peptide-1 (GLP-1) receptor binds exendin peptides with much higher affinity than GLP-1; Lopez de Maturana R et al.; Two fragments of the receptor for glucagon-like peptide-1 (GLP-1), each containing the N-terminal domain, were expressed and characterized in either bacterial or mammalian cells . The first fragment, rNT-TM1, included the N-terminal domain and first transmembrane helix and was stably expressed in the membrane of human embryonic kidney 293 cells . The second, 6H-rNT, consisted of only the N-terminal domain of the receptor fused with a polyhistidine tag at its N terminus . The latter fragment was expressed in Escherichia coli in the form of inclusion bodies from which the protein was subsequently purified and refolded in vitro . Although both receptor fragments displayed negligible (125)I-labeled GLP-1(7-36)amide-specific binding, they both displayed high affinity for the radiolabeled peptide antagonist (125)I-exendin-4(9-39) . Competition binding studies demonstrated that the N-terminal domain of the GLP-1 receptor maintains high affinity for the agonist exendin-4 as well as the antagonists exendin-4(3-39) and exendin-4(9-39) whereas, in contrast, GLP-1 affinity was greatly reduced . This study shows that although the exendin antagonists are not dependent upon the extracellular loops and transmembrane helices for maintaining their normal high affinity binding, the endogenous agonist GLP-1 requires regions outside of the N-terminal domain . Hence, distinct structural features in exendin-4, between residues 9 and 39, provide additional affinity for the N-terminal domain of the receptor . These data are consistent with a model for the binding of peptide ligands to the GLP-1 receptor in which the central and C-terminal regions of the peptides bind to the N terminus of the receptor, whereas the N-terminal residues of peptide agonists interact with the extracellular loops and transmembrane helices.

Annu Rev Nutr, 2003, 23, 1 - 16 Epub 2003 Jan 08.
Rough and rocky road to the retinoid revolution; Chytil F; Some of us who were born in the middle of Europe between World Wars I and II had to face quite a few unusual challenges that we all met in different ways . I was born and raised in Prague, Czechoslovakia, a country at the time of my birth that was governed by a Western style of democracy, which was later destroyed by the occupation by Nazi Germany and subsequently by the takeover by the equally cruel Communists . Life required special means of adaptation to the changing living conditions and a great deal of luck to survive . After graduating from the School of Technology, I started working in the Department of Medicine at Charles University in Prague as a clinical chemist in endocrinology . This work was followed with training in basic biochemistry and the study of metabolic changes in stress . This rather diversified research, due to my changing of workplaces, led to the findings that diet can change enzymatic activity of liver tryptophan oxygenase . For a short time I worked on the metabolism of cyclic AMP in Escherichia coli, and at the age of 41, I made a risky move and succeeded in escaping with my family from the "paradise of communism." The reasons for this decision will become clear . After settling in the United States, I worked on the mechanism of activation of liver tryptophan oxygenase by cyclic AMP and eventually moved to the Vanderbilt University School of Medicine . There I initially worked on the mechanism of action of steroid hormones and finally on the molecular mechanism of action of retinoids, retinol, and retinoic acid . Also in cooperation with neonatologists, I initiated studies on prematurely born human neonates which led to successful supplementation of these patients with vitamin A . The work from my laboratory and my coworkers eventually became recognized.

Am J Respir Crit Care Med, 2003 Jan 15, 167(2), 205 - 10
Early changes in alveolar fluid clearance by nitric oxide after endotoxin instillation in rats; Tsubochi H et al.; Alveolar fluid clearance may be inhibited and/or stimulated under pathologic conditions . We examined the early change of alveolar fluid clearance after endotoxin instillation in adult rats . We employed electron paramagnetic resonance nitric oxide (NO) trapping technique with iron complex with N,N-diethyldithiocarbamate as an NO trapping agent . We found that lung NO signals reached the highest magnitude by 6 hours after endotoxin instillation . NO production was accompanied by increases in lung cyclic guanosine monophosphate levels . Alveolar fluid clearance decreased significantly 6 hours after the administration of the endotoxin and increased further at 24 hours . These changes were shown to be related to the function of amiloride-sensitive sodium ion channels . Treatment with gadolinium chloride and aminoguanidine significantly decreased lung NO and cyclic guanosine monophosphate levels and completely ameliorated the decrease in alveolar fluid clearance . In addition, the increase in alveolar fluid clearance at 24 hours returned to normal levels after treatment with gadolinium chloride and aminoguanidine . We found immunoreactive inducible nitric oxide synthase to be abundantly expressed in the cytoplasm of alveolar macrophages . Our results suggest that alveolar endotoxin inhibits alveolar fluid clearance at 6 hours by NO . NO is produced via inducible NO synthase in endotoxin-stimulated alveolar macrophages and was also shown to increase alveolar fluid clearance at 24 hours.

J Biotechnol, 2003 Feb 27, 101(1), 1 - 9
Insertion of common mutations into the human beta-globin locus using GET Recombination and an EcoRI endonuclease counterselection cassette; Jamsai D et al.; A large number of mutations have been described in the human beta-globin locus causing thalassemia or various hemoglobinopathies . However, only a very limited number of these mutations have been studied in animal model systems in the context of the human beta-globin locus . We report here the use of the GET Recombination system with an EcoRI/Kan(R) counterselection cassette to facilitate the introduction of the HbE (codon 26, GAG-->AAG mutation and the codon 41-42 (-TTCT) deletion, two mutations found in high frequency in South-East Asia, into the human beta-globin locus . The counterselection cassette was first inserted into the target sequence in the beta-globin gene, and then a PCR fragment carrying the required modification was used to replace it . Efficient counterselection depends upon the tight regulation of the highly toxic EcoRI endonuclease gene by expression of lacI(q) . Induction by IPTG during counterselection efficiently eliminates non-recombinant bacterial clones . The technique can be performed on any known gene sequence using current BAC technology, allowing identification and comparative functional analysis of key regulatory elements, and the development of accurate animal models for human genetic disorders.

Toxicol Lett, 2003 Feb 3, 137(3), 143 - 50
Roles of human liver cytochrome P450 3A4 and 1A2 enzymes in the oxidation of myristicin; Yun CH et al.; The aim of this work was to identify the form(s) of human liver cytochrome P450 (CYP) involved in the hepatic transformation of myristicin to its major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene . When microsomes prepared from different human liver samples were compared, the activity of 5-allyl-1-methoxy-2,3-dihydroxybenzene formation was well correlated (r(2)=0.87) with nifedipine oxidation (a marker of CYP3A4) . With a microsomal sample having high CYP3A4 activity, microsomal oxidation of myristicin to the major metabolite (5-allyl-1-methoxy-2,3-dihydroxybenzene) was markedly inhibited by gestodene and ketoconazole, selective inhibitors of CYP3A enzymes, but not by any of several other P450 inhibitors . Antibodies raised against CYPs 3A4 and 1A2 could also inhibit the oxidation of myristicin, but antibodies recognizing other CYPs had no effect . The oxidation of myristicin to 5-allyl-1-methoxy-2,3-dihydroxybenzene was catalyzed by purified bacterial recombinant CYPs 3A4 and 1A2 . These results provide evidence that CYP3A4 (and possibly other CYP3A enzymes) and CYP1A2 play roles in the formation of the major metabolite, 5-allyl-1-methoxy-2,3-dihydroxybenzene.

Biochem J, 2003 Apr 15, 371(Pt 2), 451 - 62
Cystic fibrosis transmembrane conductance regulator: the NBF1+R (nucleotide-binding fold 1 and regulatory domain) segment acting alone catalyses a Co2+/Mn2+/Mg2+-ATPase activity markedly inhibited by both Cd2+ and the transition-state analogue orthovanadate; Annereau JP et al.; Cystic fibrosis (CF) is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator), a regulated anion channel and member of the ATP-binding-cassette transporter (ABC transporter) superfamily . Of CFTR's five domains, the first nucleotide-binding fold (NBF1) has been of greatest interest both because it is the major 'hotspot' for mutations that cause CF, and because it is connected to a unique regulatory domain (R) . However, attempts have failed to obtain a catalytically active NBF1+R protein in the absence of a fusion partner . Here, we report that such a protein can be obtained following its overexpression in bacteria . The pure NBF1+R protein exhibits significant ATPase activity {catalytic-centre activity (turnover number) 6.7 min(-1)} and an apparent affinity for ATP ( K (m), 8.7 microM) higher than reported previously for CFTR or segments thereof . As predicted, the ATPase activity is inhibited by mutations in the Walker A motif . It is also inhibited by vanadate, a transition-state analogue . Surprisingly, however, the best divalent metal activator is Co(2+), followed by Mn(2+) and Mg(2+) . In contrast, Ca(2+) is ineffective and Cd(2+) is a potent inhibitor . These novel studies, while demonstrating clearly that CFTR's NBF1+R segment can act independently as an active, vanadate-sensitive ATPase, also identify its unique cation activators and a new inhibitor, thus providing insight into the nature of its active site.

Biomacromolecules, 2003 Jan-Feb, 4(1), 189 - 92
Hyaluronic acid N-deacetylase assay in whole skin; Longas MO et al.; Hyaluronic acid (HA) N-deacetylase(s) was quantified in whole skin, using a novel method that involved reaction of skin with exogenous HA as substrate . Acetyl (CH(3)CO-) moieties generated were converted chemically to MeOAc and quantified using gas chromatography/mass spectrometry . HA (1.7 mg) and skin (1.0 g) yielded 3.32 and 769.00 microg of MeOAc from the 69.0- and 76.5-year-old-patient samples, respectively . Without added HA, 194.00 microg of product was obtained from the 76.5-year-old-patient samples . With chondroitin as substrate, the yields were 2.89 and 818.04 microg of MeOAc from the 69.0- and 76.5-year-old-patient samples, respectively . The K5 (capsular, Escherichia coli polysaccharide) substrate yielded no detectable product, except for 170.02 microg from the 76.5-year-old-patient samples . This highly sensitive method was used to demonstrate that human-skin-HA N-deacetylase(s) was first detectable at 69 years of age, highly active at 76.5 years of age, and specific for N-acetyl moieties of d-GlcNAc and d-GalNAc where C(1) is beta-linked as in HA and CH.

J Protein Chem, 2002 Oct, 21(7), 465 - 71
Preparation of biologically active recombinant human progastrin(1-80); McQueen K et al.; The bacterial expression of human progastrin(6-80) has been reported previously {Baldwin, G.S . et al . (2001) J . Biol . Chem . 276: 7791-7796} . The aims of the present study were to prepare full-length recombinant human progastrin(1-80) and to compare its biological activity with that of progastrin(6-80) in vitro, to determine whether or not the N-terminal five amino acids contributed to activity . A fusion protein of glutathione-S-transferase and human progastrin(1-80) was expressed in Escherichia coli, collected on glutathione-agarose beads, and cleaved with enterokinase . Progastrin(1-80) was purified by reversed-phase and anion exchange HPLC and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry . No differences were detected in the extent of stimulation by progastrin(1-80) and progastrin(6-80) in proliferation and migration assays with the mouse gastric cell line IMGE-5 . We conclude that residues 1-5 of progastrin(1-80) are not essential for biological activity.

J Am Vet Med Assoc, 2003 Jan 1, 222(1), 63 - 6
Evaluation of frequent milkout for treatment of cows with experimentally induced Escherichia coil mastitis; Leininger DJ et al.; OBJECTIVE: To evaluate the effect of frequent milkout (FMO) on the outcome of experimentally induced Escherichia coli mastitis in cows . DESIGN: Randomized complete block study . ANIMALS: 16 Holstein dairy cows . PROCEDURE: Cows were randomly assigned to 1 of 4 groups and were either not infected and not treated (NI-NT), experimentally infected with E coli and not treated (EC-NT), not infected and FMO (NI-FMO), or experimentally infected with E coli and FMO (EC-FMO) . The infected quarter in cows in FMO groups was milked out every 4 hours from 16 to 36 hours and every 6 hours from 36 to 84 hours after challenge, with the aid of oxytocin administration . Somatic cell counts (SCC); times to bacterial, clinical, and systemic cures; and serum concentrations of a-lactalbumin were determined . RESULTS: Use of FMO did not appear to affect SCC . For EC-NT and EC-FMO groups, mean bacterial cure times were 203 and 159 hours, clinical cure times were 276 and 360 hours, and systemic cure times were 144 and 159 hours, respectively; these times were not significantly different . Concentrations of a-lactalbumin were significantly increased in the EC-NT group at 12 hours and in the NI-FMO group at 36 and 60 hours after challenge, compared with values of cows in other treatment groups . CONCLUSIONS AND CLINICAL RELEVANCE: Compared with results in control cows, FMO does not appear to be an efficacious treatment for experimentally induced moderate to severe E coli mastitis.

Genomics, 2002 Dec, 80(6), 691 - 8
Isothermal strand-displacement amplification applications for high-throughput genomics; Detter JC et al.; Amplification of source DNA is a nearly universal requirement for molecular biology applications . The primary methods currently available to researchers are limited to in vivo amplification in Escherichia coli hosts and the polymerase chain reaction . Rolling-circle DNA replication is a well-known method for synthesis of phage genomes and recently has been applied as rolling circle amplification (RCA) of specific target sequences as well as circular vectors used in cloning . Here, we demonstrate that RCA using random hexamer primers with 29 DNA polymerase can be used for strand-displacement amplification of different vector constructs containing a variety of insert sizes to produce consistently uniform template for end-sequencing reactions . We show this procedure to be especially effective in a high-throughput plasmid production sequencing process . In addition, we demonstrate that whole bacterial genomes can be effectively amplified from cells or small amounts of purified genomic DNA without apparent bias for use in downstream applications, including whole genome shotgun sequencing.

Vopr Virusol, 2002 Sep-Oct, 47(5), 31 - 6
{Recombinant antibodies to tick-borne encephalitis virus}; Nikolenko GN et al.; A gene encoding the dimeric form of single-chain antibody fragments (scFv) was designed on the basis of the artificial gene coding monomeric scFv to tick-borne encephalitis glycoprotein E . The sequence encoding histidine oligomer was added to 3'-ends of the genes encoding the monomeric and dimeric forms of scFv antibodies . Escherichia coli strains were constructed for the production of monomeric and dimeric antibodies . These antibodies were purified using Ni-NTA resin . The specificity of the purified monomeric and dimeric antibodies in the binding reaction with tick-borne encephalitis virus was shown by ELISA and Western-blot analysis . The identity of glycoprotein E epitope bound by monomeric and dimeric scFv and parental monoclonal antibodies E6B was confirmed by competitive immunoassay.

J Biomol NMR, 2002 Dec, 24(4), 291 - 300
TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants; Vogeli B et al.; The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H(alpha)-C(alpha)/H(N)-N dipole/dipole and H(alpha)-C(alpha)/N dipole/CSA cross-correlated relaxation rates . In addition, the new experiment provides values of the (3,4)J(H alpha HN) coupling constants measured in an E.COSY manner . The conformational restraints for the psi and phi angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants . Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle . The application of the {(15)N,(13)C}-DQ/ZQ-{(15)N,(1)H}-TROSY-E.COSY experiment to the 16 kDa apo-form of the E . coli Heme Chaperon protein CcmE is described . Overall good agreement is achieved between psi and phi angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.

Proc Natl Acad Sci U S A, 2003 Jan 21, 100(2), 691 - 6 Epub 2003 Jan 09.
Robustness and the cycle of phosphorylation and dephosphorylation in a two-component regulatory system; Batchelor E et al.; The EnvZ/OmpR system in Escherichia coli, which regulates the expression of the porins OmpF and OmpC, is one of the simplest and best-characterized examples of two-component signaling . Like many other histidine kinases, EnvZ is bifunctional; it phosphorylates and dephosphorylates the response regulator OmpR . We have analyzed a mathematical model of the EnvZ-mediated cycle of OmpR phosphorylation and dephosphorylation . The model predicts that when EnvZ is much less abundant than OmpR, as is the case in E . coli, the steady-state level of phosphorylated OmpR (OmpR-P) is insensitive to variations in the concentration of EnvZ . The model also predicts that the level of OmpR-P is insensitive to variations in the concentration of OmpR when the OmpR concentration is sufficiently high . To test these predictions, we have perturbed the porin regulatory circuit in E . coli by varying the expression levels of EnvZ and OmpR . We have constructed two-color fluorescent reporter strains in which ompF and ompC transcription can be easily measured in the same culture . Using these strains we have shown that, consistent with the predictions of our model, the transcription of ompC and ompF is indeed robust or insensitive to a wide range of expression levels of both EnvZ and OmpR.

J Biol Chem, 2003 Mar 28, 278(13), 11289 - 302 Epub 2003 Jan 09.
Presence of 18-A long hydrogen bond track in the active site of Escherichia coli DNA polymerase I (Klenow fragment) . Its requirement in the stabilization of enzyme-template-primer complex; Singh K et al.; The analysis of the active site region in the crystal structures of template-primer-bound KlenTaq (Klenow fragment equivalent of Thermus aquaticus polymerase I) shows the presence of an approximately 18-A long H-bonding track contributed by the Klenow fragment equivalent of Asn(845), Gln(849), Arg(668), His(881), and Gln(677) . Its location is nearly diagonal to the helical axis of the template-primer . Four base pairs in the double stranded region proximal to 3' OH end of the primer terminus appear to interact with individual amino acid components of the track through either the bases or sugar moieties . To understand the functional significance of this H-bonding network in the catalytic function of Klenow fragment (KF), we generated N845A, N845Q, Q849A, Q849N, R668A, H881A, H881V, Q677A, and Q677N mutant species by site-directed mutagenesis . All of the mutant enzymes showed low catalytic activity . The kinetic analysis of mutant enzymes indicated that K(m)(.dNTP) was not significantly altered, but K(D)(.DNA) was significantly increased . Thus the mutant enzymes of the H-bonding track residues had decreased affinity for template-primer, although the extent of decrease was variable . Most interestingly, even the reduced binding of TP by the mutant enzymes occurs in the nonproductive mode . These results demonstrate that an H-bonding track is necessary for the binding of template-primer in the catalytically competent orientation in the pol I family of enzymes . The examination of the interactive environment of individual residues of this track further clarifies the mode of cooperation in various functional domains of pol I.

J Biol Chem, 2003 Mar 21, 278(12), 10162 - 73 Epub 2003 Jan 09.
Identification of a lactoferrin-derived peptide possessing binding activity to hepatitis C virus E2 envelope protein; Nozaki A et al.; Bovine and human lactoferrins (LF) prevent hepatitis C virus (HCV) infection in cultured human hepatocytes; the preventive mechanism is thought to be the direct interaction between LF and HCV . To clarify this hypothesis, we have characterized the binding activity of LF to HCV E2 envelope protein and have endeavored to determine which region(s) of LF are important for this binding activity . Several regions of human LF have been expressed and purified as thioredoxin-fused proteins in Escherichia coli . Far-Western blot analysis using these LF fragments and the E2 protein, expressed in Chinese hamster ovary cells, revealed that the 93 carboxyl amino acids of LF specifically bound to the E2 protein . The 93 carboxyl amino acids of LFs derived from bovine and horse cells also possessed similar binding activity to the E2 protein . In addition, the amino acid sequences of these carboxyl regions appeared to show partial homology to CD81, a candidate receptor for HCV, and the binding activity of these carboxyl regions was also comparable with that of CD81 . Further deletion analysis identified 33 amino acid residues as the minimum binding site in the carboxyl region of LF, and the binding specificity of these 33 amino acids was also confirmed by using 33 maltose-binding protein-fused amino acids . Furthermore, we demonstrated that the 33 maltose-binding protein-fused amino acids prevented HCV infection in cultured human hepatocytes . In addition, the site-directed mutagenesis to an Ala residue in both terminal residues of the 33 amino acids revealed that Cys at amino acid 628 was determined to be critical for binding to the E2 protein . These results led us to consider the development of an effective anti-HCV peptide . This is the first identification of a natural protein-derived peptide that specifically binds to HCV E2 protein and prevents HCV infection.

Proc Natl Acad Sci U S A, 2003 Jan 21, 100(2), 438 - 42 Epub 2003 Jan 08.
Expression and assembly of a fully active antibody in algae; Mayfield SP et al.; Although combinatorial antibody libraries have solved the problem of access to large immunological repertoires, efficient production of these complex molecules remains a problem . Here we demonstrate the efficient expression of a unique large single-chain (lsc) antibody in the chloroplast of the unicellular, green alga, Chlamydomonas reinhardtii . We achieved high levels of protein accumulation by synthesizing the lsc gene in chloroplast codon bias and by driving expression of the chimeric gene using either of two C . reinhardtii chloroplast promoters and 5' and 3' RNA elements . This lsc antibody, directed against glycoprotein D of the herpes simplex virus, is produced in a soluble form by the alga and assembles into higher order complexes in vivo . Aside from dimerization by disulfide bond formation, the antibody undergoes no detectable posttranslational modification . We further demonstrate that accumulation of the antibody can be modulated by the specific growth regime used to culture the alga, and by the choice of 5' and 3' elements used to drive expression of the antibody gene . These results demonstrate the utility of alga as an expression platform for recombinant proteins, and describe a new type of single chain antibody containing the entire heavy chain protein, including the Fc domain.

Free Radic Biol Med, 2003 Jan 15, 34(2), 277 - 82
No increase in lifespan in Caenorhabditis elegans upon treatment with the superoxide dismutase mimetic EUK-8; Keaney M et al.; The superoxide dismutase mimetic EUK-8 has been reported to extend lifespan in the nematode Caenorhabditis elegans . However, in five trials administering EUK-8 in liquid culture with E . coli, and two trials using defined liquid medium, we observed no increase in C . elegans lifespan . Instead we saw a dose-dependent reduction of lifespan and fertility . We conclude that extension of C . elegans lifespan by EUK-8 may only occur under very particular culture conditions.

Nucleic Acids Res, 2003 Jan 1, 31(1), 13 - 6
DNA Data Bank of Japan (DDBJ) in XML; Miyazaki S et al.; The DNA Data Bank of Japan (DDBJ, has collected and released more entries and bases than last year . This is mainly due to large-scale submissions from Japanese sequencing teams on mouse, rice, chimpanzee, nematoda and other organisms . The contributions of DDBJ over the past year are 17.3% (entries) and 10.3% (bases) of the combined outputs of the International Nucleotide Sequence Databases (INSD) . Our complete genome sequence database, Genome Information Broker (GIB), has been improved by incorporating XML . It is now possible to perform a more sophisticated database search against the new GIB than the ordinary BLAST or FASTA search.

J Biol Chem, 2003 Mar 28, 278(13), 10885 - 90 Epub 2003 Jan 07.
Mcp1 encodes the molybdenum cofactor carrier protein in Chlamydomonas reinhardtii and participates in protection, binding, and storage functions of the cofactor; Ataya FS et al.; The molybdenum cofactor (Moco) is essential for the activity of all molybdoenzymes except nitrogenase . The cDNA for the Moco carrier protein (MocoCP) of Chlamydomonas reinhardtii has been cloned by reverse transcription PCR approaches with primers designed from microsequenced peptides of this protein . The C . reinhardtii MocoCP has been expressed in Escherichia coli . The recombinant protein has been purified to electrophoretic homogeneity and is found assembled into a homotetramer when Moco is not present under native conditions . Recombinant MocoCP has the same biochemical characteristics as MocoCP from C . reinhardtii, as it bound Moco from milk xanthine oxidase with high affinity, prevented Moco inactivation by oxygen, and transferred Moco efficiently to aponitrate reductase from the Neurospora crassa nit1 mutant . The genomic DNA sequence corresponding to the Chlamydomonas MocoCP gene, CrMcp1, also was isolated . This gene contained three introns in the coding region . The deduced amino acid sequence of CrMcp1 did not show a significant identity to functionally known proteins in the GenBank data base, although a significant conservation was found with bacterial proteins of unknown function . The results suggest that proteins having a Moco binding function probably exist in other organisms.

J Biol Chem, 2003 Mar 14, 278(11), 8979 - 87 Epub 2003 Jan 07.
How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system . Spectroscopic evidence for highly efficient refolding of a single-chain Fv fragment; Umetsu M et al.; The gradual removal of the denaturing reagent guanidine HCl (GdnHCl) using stepwise dialysis with the introduction of an oxidizing reagent and l-arginine resulted in the highly efficient refolding of various denatured single-chain Fv fragments (scFvs) from inclusion bodies expressed in Escherichia coli . In this study, the influence of the additives on the intermediates in scFv refolding was carefully analyzed on the basis of the stepwise dialysis, and it was revealed that the additive effect critically changes the pathway of scFv refolding . Circular dichroism and tryptophan fluorescence emission spectroscopies demonstrated that distinct secondary and tertiary structures were formed upon dialysis from 2 m GdnHCl to 1 m GdnHCl, and 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt binding analysis indicated that the addition of l-arginine to the stepwise dialysis system effectively stabilized the exposed hydrophobic area on the scFv . Quantification of the free thiol groups in the scFv by means of Ellman's assay revealed that there was a particular stage in which most of the free thiol groups were oxidized and that adding an oxidizing reagent (the oxidized form of glutathione, GSSG) at that stage was important for complete refolding of the scFv . The particular stage depended on the nature of the refolding solution, especially on whether l-arginine was present . Spontaneous folding at the 1 m GdnHCl stage resulted in a structure in which a free thiol group accessed to the proper one for correct disulfide linkage; however, the addition of l-arginine resulted in the formation of a partially folded intermediate without disulfide linkages . Mass spectrometry experiments on alkylated scFv were carried out at each stage to determine the effects of l-arginine . The spectroscopic studies revealed two different pathways for scFv refolding in the stepwise dialysis system, pathways that depended on whether l-arginine was present . Controlled coupling of the effects of GSSG and l-arginine led to the complete refolding of scFv in the stepwise dialysis.

J Biol Chem, 2003 Mar 14, 278(11), 9005 - 12 Epub 2003 Jan 08.
Substrate specificity of human endonuclease III (hNTH1) . Effect of human APE1 on hNTH1 activity; Marenstein DR et al.; Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/apurinic/apyrimidinic (AP) lyase, human NTH1 (hNTH1), the homolog of Escherichia coli endonuclease III (Nth) . In contrast to Nth, the DNA N-glycosylase activity of hNTH1 is 7-fold greater than its AP lyase activity when the DNA substrate contains a thymine glycol (Tg) opposite adenine (Tg:A) (Marenstein, D . R., Ocampo, M . T . A., Chan, M . K., Altamirano, A., Basu, A . K., Boorstein, R . J., Cunningham, R . P., and Teebor, G . W . (2001) J . Biol . Chem . 276, 21242-21249) . When Tg is opposite guanine (Tg:G), the two activities are of the same specific activity as the AP lyase activity of hNTH1 against Tg:A (Ocampo, M . T . A., Chaung, W., Marenstein, D . R., Chan, M . K., Altamirano, A., Basu, A . K., Boorstein, R . J., Cunningham, R . P., and Teebor, G . W . (2002) Mol . Cell . Biol . 22, 6111-6121) . We demonstrate here that hNTH1 was inhibited by the product of its DNA N-glycosylase activity directed against Tg:G, the AP:G site . In contrast, hNTH1 was not as inhibited by the AP:A site arising from release of Tg from Tg:A . Addition of human APE1 (AP endonuclease-1) increased dissociation of hNTH1 from the DNA N-glycosylase-generated AP:A site, resulting in abrogation of AP lyase activity and an increase in turnover of the DNA N-glycosylase activity of hNTH1 . Addition of APE1 did not abrogate hNTH1 AP lyase activity against Tg:G . The stimulatory protein YB-1 (Marenstein et al.), added to APE1, resulted in an additive increase in both activities of hNTH1 regardless of base pairing . Tg:A is formed by oxidative attack on thymine opposite adenine . Tg:G is formed by oxidative attack on 5-methylcytosine opposite guanine (Zuo, S., Boorstein, R . J., and Teebor, G . W . (1995) Nucleic Acids Res . 23, 3239-3243) . It is possible that the in vitro substrate selectivity of mammalian NTH1 and the concomitant selective stimulation of activity by APE1 are indicative of selective repair of oxidative damage in different regions of the genome.

J Biol Chem, 2003 Mar 21, 278(12), 10033 - 40 Epub 2003 Jan 08.
Mechanism of loading the Escherichia coli DNA polymerase III beta sliding clamp on DNA . Bona fide primer/templates preferentially trigger the gamma complex to hydrolyze ATP and load the clamp; Ason B et al.; The Escherichia coli DNA polymerase III gamma complex clamp loader assembles the ring-shaped beta sliding clamp onto DNA . The core polymerase is tethered to the template by beta, enabling processive replication of the genome . Here we investigate the DNA substrate specificity of the clamp-loading reaction by measuring the pre-steady-state kinetics of DNA binding and ATP hydrolysis using elongation-proficient and deficient primer/template DNA . The ATP-bound clamp loader binds both elongation-proficient and deficient DNA substrates either in the presence or absence of beta . However, elongation-proficient DNA preferentially triggers gamma complex to release beta onto DNA with concomitant hydrolysis of ATP . Binding to elongation-proficient DNA converts the gamma complex from a high affinity ATP-bound state to an ADP-bound state having a 10(5)-fold lower affinity for DNA . Steady-state binding assays are misleading, suggesting that gamma complex binds much more avidly to non-extendable primer/template DNA because recycling to the high affinity binding state is rate-limiting . Pre-steady-state rotational anisotropy data reveal a dynamic association-dissociation of gamma complex with extendable primer/templates leading to the diametrically opposite conclusion . The strongly favored dynamic recognition of extendable DNA does not require the presence of beta . Thus, the gamma complex uses ATP binding and hydrolysis as a mechanism for modulating its interaction with DNA in which the ATP-bound form binds with high affinity to DNA but elongation-proficient DNA substrates preferentially trigger hydrolysis of ATP and conversion to a low affinity state.

J Biol Chem, 2003 Mar 14, 278(11), 9733 - 9 Epub 2003 Jan 08.
Amino acid substitutions at position 43 of NaeI endonuclease . Evidence for changes in NaeI structure; Carrick KL et al.; NaeI endonuclease contains a 10-amino acid region with sequence similarity to the active site KXDG motif of DNA ligase except for leucine (Leu-43) in NaeI ((43)LXDG(46)) . Changing Leu-43 to lysine abolishes the NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities . Here we report the results of substituting Leu-43 with alanine, arginine, asparagine, glutamate, and histidine . Quantitating specific activities and DNA binding values for the mutant proteins determined the range of amino acids at position 43 that alter NaeI mechanism . Substituting alanine, asparagine, glutamate, and histidine for Leu-43 maintained endonuclease activity, but at a lower level . On the other hand, substituting positively charged arginine, like lysine at position 43, converted NaeI to a topoisomerase with no observable double-strand cleavage activity . The specific activities of NaeI-43K and NaeI-43R and their relative sensitivities to salt, the topoisomerase-inhibiting drug N-{4-(9-acridinylamino)-3-methoxyphenyl}methane-sulfonamide (amsacrine) and single-stranded DNA showed that the two activities are similar . The effect of placing a positive charge at position 43 on NaeI structure was determined by measuring (for NaeI and NaeI-43K) relative susceptibilities to proteolysis, UV, circular dichroism spectra, and temperature melting transitions . The results provide evidence that a positive charge at position 43 induces dramatic changes in NaeI structure that affect both the Endo and Topo domains of NaeI . The identification of four putative DNA ligase motifs in NaeI leads us to speculate that structural changes that superimpose these motifs on the ligase structure may account for the changes in activity.

J Liposome Res, 2002 Nov, 12(4), 311 - 34
The transfection of Jurkat T-leukemic cells by use of pH sensitive immunoliposomes; Turner C et al.; A gene transfer vector has been developed utilising anionic liposomes as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3+ T lymphocytes (Jurkat cells) . The plasmid DNA that contained the Escherichia coli beta-galactosidase reporter gene was condensed using poly-l-lysine of molecular mass 20,700 (PLK99) to form a polyplex which was interacted with several anionic liposome formulations to form lipopolyplexes . The liposome formulations where based on dioleoylphosphatidylethanolamine (DOPE) in combination with cholesterol and dioleoylphosphatidylcholine (DOPC) and oleic acid, or dimyristoylphosphatidylethanolamine (DMPE) . For targeting to the Jurkat cells distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2,000 and coupled to anti-CD3 antibody was incorporated . The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, agarose gel electrophoresis and electron microscopy and the permeability of the lipopolyplexes to liposome-encapsulated glucose was determined . The polyplexes consisted of a mixed population of rod-like structures (53-160 nm long and 23-31 nm diameter) and spheres (18-30 nm diameter) . The lipopolyplexes retained a permeability barrier although were more permeable to glucose than their component liposomes . The poly-l-lysine condensing agent was still susceptible to pronase digestion suggesting that the polyplex was associated with the outer surface of the liposome . The lipopolyplexes with lipid composition DOPE/cholesterol/OA/DSPE-PEG2000 anti-CD3+ PLK99-plasmid DNA had significant gene transfer activity, as monitored by beta-galactosidase expression, that depended on the charge ratio of the component polyplex and the lipid/DNA weight ratio . The anti-CD3 antibody, the liposomal lipid and pH sensitivity were essential for transfection activity.

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