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J Infect Dis, 2005 Feb 15, 191(4), 562 - 570 Epub 2005 Jan 14.
Serologic Correlates of Protection against Enterotoxigenic Escherichia coli Diarrhea; Rao MR et al.; Background . We conducted a nested case-control study in 397 rural Egyptian children <36 months of age to assess the correlation between serum levels of antibodies against toxin and colonization factors (CFs) and the risk of homologous enterotoxigenic Escherichia coli (ETEC) diarrhea.Methods . Active case detection was performed via semiweekly home visits, and blood was obtained at 3-month intervals . After each serosurvey, case subjects were selected from children experiencing a CF antigen (CFA)/I-, CFA/II-, CFA/IV-, or heat-labile enterotoxin (LT)-ETEC diarrheal episode during the subsequent 3 months . Up to 5 control subjects per case subject were selected from children who did not experience an ETEC diarrheal episode during the corresponding interval . Serum titers of immunoglobulin G antibodies against CFA/I, coli surface antigen (CS) 3, CS6, and LT were measured by enzyme-linked immunosorbant assay.Results . The distribution of serum titers of LT, CS3, and CS6 antibodies did not differ between the case and control subjects . For children <18 months of age, serum titers of CFA/I antibody were inversely related to the risk of CFA/I-ETEC diarrhea; reciprocal serum titers of CFA/I antibody >/=76 were associated with a 77% reduction in the odds of CFA/I-ETEC diarrhea.Conclusion . Induction of reciprocal serum titers of antibodies against CFA/I within or above the 76-186 range should be further evaluated as a predictor for assessment of the ability of candidate vaccines to protect against CFA/I-ETEC diarrhea.

Appl Microbiol Biotechnol . 2005 Jan 18; {Epub ahead of print}
Effect of substrate feed rate on recombinant protein secretion, degradation and inclusion body formation in Escherichia coli; Bostrom M et al.; The effect of changes in substrate feed rate during fedbatch cultivation was investigated with respect to soluble protein formation and transport of product to the periplasm in Escherichia coli . Production was transcribed from the P(malK) promoter; and the cytoplasmic part of the production was compared with production from the P(lacUV5) promoter . The fusion protein product, Zb-MalE, was at all times accumulated in the soluble protein fraction except during high-feed-rate production in the cytoplasm . This was due to a substantial degree of proteolysis in all production systems, as shown by the degradation pattern of the product . The product was also further subjected to inclusion body formation . Production in the periplasm resulted in accumulation of the full-length protein; and this production system led to a cellular physiology where the stringent response could be avoided . Furthermore, the secretion could be used to abort the diauxic growth phase resulting from use of the P(malK) promoter . At high feed rate, the accumulation of acetic acid, due to overflow metabolism, could furthermore be completely avoided.

FEBS J, 2005 Jan, 272(2), 593 - 602
Probing the mechanism of the bifunctional enzyme ketol-acid reductoisomerase by site-directed mutagenesis of the active site; Tyagi R et al.; Ketol-acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched-chain amino acids . It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH-dependent reduction of a 2-ketoacid . The 2-ketoacid formed by the alkyl migration is not released . Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant . The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2-ketoacids . The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities . Several mutations result in loss of the isomerase activity with retention of reductase activity . However, none of the 17 mutants examined have the isomerase activity only . We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization . Our mechanism explains why the two activities must occur in a single active site without release of a 2-ketoacid and provides a rationale for the requirement for NADPH by the isomerase.

FEBS J, 2005 Jan, 272(2), 454 - 63
Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails; Folichon M et al.; The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation . In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation . We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I . In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase . We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).

FEBS J, 2005 Jan, 272(2), 363 - 74
A single mutation in Escherichia coli ribonuclease II inactivates the enzyme without affecting RNA binding; Amblar M et al.; Exoribonuclease II (RNase II), encoded by the rnb gene, is a ubiquitous enzyme that is responsible for 90% of the hydrolytic activity in Escherichia coli crude extracts . The E . coli strain SK4803, carrying the mutant allele rnb296, has been widely used in the study of the role of RNase II . We determined the DNA sequence of rnb296 and cloned this mutant gene in an expression vector . Only a point mutation in the coding sequence of the gene was detected, which results in the single substitution of aspartate 209 for asparagine . The mutant and the wild-type RNase II enzymes were purified, and their 3' to 5' exoribonucleolytic activity, as well as their RNA binding capability, were characterized . We also studied the metal dependency of the exoribonuclease activity of RNase II . The results obtained demonstrated that aspartate 209 is absolutely essential for RNA hydrolysis, but is not required for substrate binding . This is the first evidence of an acidic residue that is essential for the activity of RNase II-like enzymes . The possible involvement of this residue in metal binding at the active site of the enzyme is discussed . These results are particularly relevant at this time given that no structural or mutational analysis has been performed for any protein of the RNR family of exoribonucleases.

FEBS J, 2005 Jan, 272(2), 313 - 26
Defining the Q-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy; Rothery RA et al.; We have used fluorescence quench titrations, EPR spectroscopy and steady-state kinetics to study the effects of site-directed mutants of FrdB, FrdC and FrdD on the proximal menaquinol (MQH(2)) binding site (Q(P)) of Escherichia coli fumarate reductase (FrdABCD) in cytoplasmic membrane preparations . Fluorescence quench (FQ) titrations with the fluorophore and MQH(2) analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indicate that the Q(P) site is defined by residues from FrdB, FrdC and FrdD . In FQ titrations, wild-type FrdABCD binds HOQNO with an apparent K(d) of 2.5 nm, and the following mutations significantly increase this value: FrdB-T205H (K(d) = 39 nm); FrdB-V207C (K(d) = 20 nm); FrdC-E29L (K(d) = 25 nm); FrdC-W86R (no detectable binding); and FrdD-H80K (K(d) = 20 nm) . In all titrations performed, data were fitted to a monophasic binding equation, indicating that no additional high-affinity HOQNO binding sites exist in FrdABCD . In all cases where HOQNO binding is detectable by FQ titration, it can also be observed by EPR spectroscopy . Steady-state kinetic studies of fumarate-dependent quinol oxidation indicate that there is a correlation between effects on HOQNO binding and effects on the observed K(m) and k(cat) values, except in the FrdC-E29L mutant, in which HOQNO binding is observed, but no enzyme turnover is detected . In this case, EPR studies indicate that the lack of activity arises because the enzyme can only remove one electron from reduced MQH(2), resulting in it being trapped in a form with a bound menasemiquinone radical anion . Overall, the data support a model for FrdABCD in which there is a single redox-active and dissociable Q-site.

Biochemistry, 2005 Jan 25, 44(3), 987 - 95
Amino Acid Substitution and Modification Resulting from Escherichia coli Expression of Recombinant Plasmodium falciparum Histidine-Rich Protein II; Schneider EL et al.; The histidine-rich protein II (HRP II) from Plasmodium falciparum is an unusual protein composed of 40% alanine, 36% histidine, and 11% aspartate residues . Expression of HRP II in Escherichia coli results in the isolation of a heterogeneous protein . Mass spectrometry reveals a reduction in mass by multiples of 9 Da from the expected molecular mass that can be attributed to the substitution of glutamine for some histidine residues in the sequence . The extent of the glutamine for histidine substitution can be reduced by slowing the expression rate . Mass spectral analysis of HRP II also revealed alpha-amino methylation of the N-terminal alanine residue of HRP II.

Biochemistry, 2005 Jan 25, 44(3), 959 - 70
Equilibrium and Kinetic Analysis of Nucleotide Binding to the DEAD-Box RNA Helicase DbpA; Talavera MA et al.; The Escherichia coli DEAD-box protein A (DbpA) is an RNA helicase that utilizes the energy from ATP binding and hydrolysis to facilitate structural rearrangements of rRNA . We have used the fluorescent nucleotide analogues, mantADP and mantATP, to measure the equilibrium binding affinity and kinetic mechanism of nucleotide binding to DbpA in the absence of RNA . Binding generates an enhancement in mant-nucleotide fluorescence and a corresponding reduction in intrinsic DbpA fluorescence, consistent with fluorescence resonance energy transfer (FRET) from DbpA tryptophan(s) to bound nucleotides . Fluorescent modification does not significantly interfere with the affinities and kinetics of nucleotide binding . Different energy transfer efficiencies between DbpA-mantATP and DbpA-mantADP complexes suggest that DbpA adopts nucleotide-dependent conformations . ADP binds (K(d) approximately 50 muM at 22 degrees C) 4-7 times more tightly than ATP (K(d) approximately 400 muM at 22 degrees C) . Both nucleotides bind with relatively temperature-independent association rate constants ( approximately 1-3 muM(-)(1) s(-)(1)) that are much lower than predicted for a diffusion-limited reaction . Differences in the binding affinities are dictated primarily by the dissociation rate constants . ADP binding occurs with a positive change in the heat capacity, presumably reflecting a nucleotide-induced conformational rearrangement of DbpA . At low temperatures (<22 degrees C), the binding free energies are dominated by favorable enthalpic and unfavorable entropic contributions . At physiological temperatures (>22 degrees C), ADP binding occurs with positive entropy changes . We favor a mechanism in which ADP binding increases the conformational flexibility and dynamics of DbpA.

Biochemistry, 2005 Jan 25, 44(3), 840 - 50
Calcium and magnesium binding to human centrin 3 and interaction with target peptides; Cox JA et al.; There are four isoforms of centrin in mammals, with variable sequence, tissue expression, and functional properties . We have recently characterized a number of structural, ion, and target binding properties of human centrin isoform HsCen2 . This paper reports a similar characterization of HsCen3, overexpressed in Escherichia coli and purified by phase-reversed chromatography . Equilibrium and dynamic binding studies revealed that HsCen3 has one mixed Ca(2+)/Mg(2+) binding site of high affinity (K(d) = 3 and 10 muM for Ca(2+) and Mg(2+), respectively) and two Ca(2+)-specific sites of low affinity (K(d) = 140 muM) . The metal-free protein is fragmented by an unidentified protease into a polypeptide segment of 11 kDa, which was purified by HPLC, and identified by mass spectrometry as the segment of residues 21-112 . Similarly, controlled trypsinolysis on Ca(2+)-bound HsCen3 yielded a mixture of segments of residues 1-124 and 1-125 . The Ca(2+)/Mg(2+) site could be assigned to this segment and thus resides in the N-terminal half of HsCen3 . Temperature denaturation experiments, circular dichroism, and utilization of fluorescence hydrophobic probes allowed us to propose that the metal-free protein has molten globule characteristics and that the dication-bound forms are compact with a polar surface for the Mg(2+) form and a hydrophobic exposed surface for the Ca(2+) form . Thus, HsCen3 could be classified as a Ca(2+) sensor protein . In addition, it is able to bind strongly to a model target peptide (melittin), as well as to peptides derived from the protein XPC and Kar1p, with a moderate Ca(2+) dependence.

Naunyn Schmiedebergs Arch Pharmacol . 2005 Jan 15; {Epub ahead of print}
Thaliporphine increases survival rate and attenuates multiple organ injury in LPS-induced endotoxaemia; Chiao CW et al.; This study addressed the question of whether thaliporphine, a phenolic aporphine alkaloid obtained from Chinese herbs and possessing antioxidant and alpha-1 adrenoceptor antagonistic activity, has protective effects in endotoxaemic rats and we attempted to elucidate the mechanisms contributing to such protective effects . Injection of rats with endotoxin (E . coli lipopolysaccharide, LPS) induced severe hypotension and tachycardia as well as vascular hyporeactivity to noradrenaline . Pretreatment of LPS-treated rats with thaliporphine attenuated the delayed hypotension significantly whilst only a higher dose (1 mg/kg) of thaliporphine decreased LPS-induced tachycardia . LPS significantly increased nitric oxide (NO.) and superoxide anion (O(2).(-)) levels, a response that was reduced by pretreatment with 1 mg/kg thaliporphine . Endotoxaemia for 240 min resulted in a bell-shaped time course for the change of serum tumour necrosis factor-alpha (TNF-alpha) level with a peak at 60 min . Pretreatment of LPS-treated rats with 1 mg/kg thaliporphine significantly reduced the serum TNF-alpha level at 60 min . In addition, LPS caused a biphasic change in blood glucose and thaliporphine attenuated the late-phase decrease in blood glucose . Endotoxaemia induced multiple organ injury in the liver, kidney and heart, as indicated by increases of aspartate aminotransferase (GOT), alanine aminotransferase (GPT), creatinine (CRE), lactate dehydrogenase (LDH) and creatine phosphate kinase muscle-brain (CKMB) levels in serum . These increases of biochemical markers and inflammatory cell infiltration into injured tissues were reduced significantly by treatment with thaliporphine . In addition, thaliporphine increased the survival rate of LPS-treated mice dose-dependently . In conclusion, our results suggest that thaliporphine could be a novel agent for attenuating endotoxin-induced circulatory failure and multiple organ injury and may increase the survival rate . These beneficial effects of thaliporphine may be attributed to the suppression of TNF-alpha, NO . and O(2).(-) production.

Inflamm Res, 2004 Dec 1, 53(12), 658 - 63
Evidence that arachidonic acid derived from neutrophils and prostaglandin E2 are associated with the induction of acute lung inflammation by lipopolysaccharide of Escherichia coli; Alba-Loureiro TC et al.; OBJECTIVE: The involvement of arachidonic acid (AA) and PGE2 during the E . coli lipopolysaccharide (LPS)-induced acute lung injury was investigated . MATERIAL: Adult male Wistar rats were used . For in vitro studies, rat neutrophils, bronchoalveolar lavage (BAL) fluid, and lug vascular endothelium were used, as described below . TREATMENT: Rats were given an intratracheal injection of LPS (750 microg) . METHODS: Total and differential cell counts in BAL fluid; enzyme-linked immunoassay (ELISA) analyses of TNF-alpha, IL-1beta, LTB4 and PGE2 in BAL, and immunohistochemical detection of ICAM-1 on lung vascular endothelium were performed six h after LPS challenge . Fatty acid composition of blood neutrophils and plasma was analyzed by HPLC . RESULTS: Rats instilled with LPS presented a sixty three-fold increase in the number of neutrophils in BAL (from 0.5 x 10(6) to 31.5 x 10(6) cells), accompanied by increased levels of TNF-alpha and IL-1beta (p < 0.001), and a three-fold increase in ICAM-1 expression on vascular endothelium . The content of AA in blood neutrophils was reduced by 50%, whereas the level of PGE2 in BAL was increased by 3.5 fold, without changes in the levels of LTB4 . CONCLUSIONS: These findings suggest that AA and PGE2 are associated with LPS challenge.

J Biol Chem . 2005 Jan 14; {Epub ahead of print}
Solution structure of enzyme IIA chitobiose from the N,N'-diacetylchitobiose branch of the Escherichia coli phosphotransferase system; Tang C et al.; The solution structure of trimeric E . coli enzyme IIAChb (34 kDa), a component of the N,N'-diacetylchitobiose/lactose branch of the phospho-transferase signal transduction system, has been determined by NMR spectroscopy . Backbone residual dipolar couplings were used to provide long-range orientational restraints, and long-range (|i - j| = 5 residues) nuclear Overhauser enhancement restraints were derived exclusively from samples in which at least one subunit was 15N/13C/2H/(Val-Leu-Ile)-methyl-protonated . Each subunit consists of a three-helix bundle . Hy-drophobic residues lining helix 3 of each subunit are largely responsible for the formation of a parallel coiled-coil trimer . The active site histi-dines (His89 from each subunit) are located in three symmetrically placed deep crevices located at the interface of two adjacent subunits (A and C, C and B, and B and A) . Partially shielded from bulk solvent, structural modeling suggests that phosphorylated His89 is stabilized by electrostatic interactions with the side-chains of His93 from the same subunit and Gln91 from the adjacent subunit . Comparison with the X-ray structure of L . lactis IIALac reveals some substantial structural differences, particularly in regard to helix 3 which exhibits a 40 degrees kink in IIALac versus a 7 degrees bend in IIAChb . This is associated with the presence of an unusually large (230 A3) buried hydrophobic cavity at the trimer interface in IIALac which is reduced to only 45 A3 in IIAChb.

Genome Res . 2005 Jan 14; {Epub ahead of print}
Making connections between novel transcription factors and their DNA motifs; Tan K et al.; The key components of a transcriptional regulatory network are the connections between trans-acting transcription factors and cis-acting DNA-binding sites . In spite of several decades of intense research, only a fraction of the estimated approximately 300 transcription factors in Escherichia coli have been linked to some of their binding sites in the genome . In this paper, we present a computational method to connect novel transcription factors and DNA motifs in E . coli . Our method uses three types of mutually independent information, two of which are gleaned by comparative analysis of multiple genomes and the third one derived from similarities of transcription-factor-DNA-binding-site interactions . The different types of information are combined to calculate the probability of a given transcription-factor-DNA-motif pair being a true pair . Tested on a study set of transcription factors and their DNA motifs, our method has a prediction accuracy of 59% for the top predictions and 85% for the top three predictions . When applied to 99 novel transcription factors and 70 novel DNA motifs, our method predicted 64 transcription-factor-DNA-motif pairs . Supporting evidence for some of the predicted pairs is presented . Functional annotations are made for 23 novel transcription factors based on the predicted transcription-factor-DNA-motif connections.

Plant Cell Physiol, 2004 Dec, 45(12), 1882 - 8
Identification and Subcellular Localization of Two Solanesyl Diphosphate Synthases from Arabidopsis thaliana; Jun L et al.; Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified . Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli . SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells . These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.

Plant Cell Physiol, 2004 Dec, 45(12), 1787 - 97
Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity; Li Y et al.; Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants . A modified Arabidopsis thaliana PCS sequence (AtPCS1) was active in Escherichia coli . When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20-100 times more biomass on 250 and 300 microM arsenate than wild type (WT); however, they were hypersensitive to Cd(II) . After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC . Whereas cadmium induced greater increases in traditional PCs (PC(2), PC(3), PC(4)), arsenic exposure resulted in the expression of many unknown thiol products . Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, gamma-glutamyl cysteine (gamma-EC), were also dramatically increased . Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants . The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.

J Dairy Sci, 2005 Feb, 88(2), 534 - 42
Effects of Adjuvants on Safety and Efficacy of an Escherichia coli J5 Bacterin; Hogan JS et al.; The effects of using a water-soluble adjuvant or an emulsified oil-based adjuvant on the safety, antibody titer, and clinical responses of an Escherichia coli J5 bacterin were tested in an experimental infection trial . Fifty-one cows were assigned to 17 blocks of 3 . Two cows within each block of 3 were vaccinated with a commercially prepared E . coli J5 bacterin containing either a water-soluble adjuvant or the same bacterin preparation emulsified in oil . One cow in each block was an unvaccinated control . Cows were immunized at drying off and 42 d later . The right or left front mammary quarter of each experimental cow was challenged by intramammary infusion of E . coli 727 between 14 and 35 DIM . Areas of inflammation at the primary injection site were greater 1, 2, and 3 d following primary vaccination for bacterin containing oil-in-water adjuvant compared with bacterin containing water-soluble adjuvant . Whey anti-E . coli J5 IgG titers were higher at calving for cows vaccinated with bacterin containing oil-in-water adjuvant than for cows either vaccinated with bacterin containing water-soluble adjuvant or unvaccinated controls . Serum x-E . coli J5 IgG titers were higher at calving for vaccinated cows than for unvaccinated controls . Peak bacterial counts in milk from challenged quarters were greater for unvaccinated controls than for cows vaccinated with bacterin containing water-in-oil adjuvant . Bacterial counts in milk from challenged quarters and clinical score both were greater in unvaccinated controls than cows vaccinated with bacterin containing water-in-oil adjuvant between 12 and 24 h postchallenge . Clinical responses were similar between unvaccinated controls and cows vaccinated with bacterin containing water-soluble adjuvant.

J Surg Res, 2005 Jan, 123(1), 49 - 54
Targeting of vaccinia virus using biotin-avidin viral coating and biotinylated antibodies; Purow B et al.; INTRODUCTION: To test a general method for altering the tropism of viral vectors, we conjugated targeting antibody to the surface of recombinant vaccinia virus with a biotin-avidin-biotin linker and assessed the resulting infectivity in target cells and controls . MATERIALS AND METHODS: We biotinylated a vaccinia viral vector and used avidin to crosslink the biotinylated viral surface to a biotinylated antibody specific for a molecule on the surface of a target cell . In an in vitro model system, we coated a recombinant vaccinia construct containing the E . coli beta-galactosidase gene with antibody to the murine class I MHC molecule D(b) . Target cells were B78H1 murine melanoma cells transduced with either the D(b) gene or, as a control, the K(b) gene . Infectivity was assessed by staining target cells with x-gal to demonstrate expression of virally delivered beta-galactosidase . This technique was also assessed in a second system with vaccinia/beta-gal targeted to the murine B7.2 molecule . The infectivity of the resulting construct was assessed for murine SA1 fibrosarcoma cells transfected with the B7.2 gene and for wild-type, B7.2-negative SA1 . Experiments were repeated in each system with similar results . RESULTS: This strategy demonstrated antibody-mediated viral targeting in both the B78H1 and the SA1 models . Importantly, addition of the targeting coat diminished the infectivity of the modified vaccinia for control cells but preserved infectivity for targeted cells . In the B78H1 system, D(b)-targeted vaccinia consistently had 2- to 3-fold greater infectivity for B78H1D(b) than B78H1K(b) . Increasing the number of avidin molecules used per virion in the synthesis of the viral coat led to greater selectivity but decreased overall infectivity . In the SA1 system, B7.2-targeted vaccinia demonstrated completely ablated infectivity for control SA1 cells, but maintained infectivity for target SA1/B7.2 cells . CONCLUSIONS: Recombinant viral vectors such as vaccinia may be coated with biotin/avidin and linked to biotinylated antibodies to preferentially target specific cell types in vitro . Such an approach may be useful in targeting recombinant lytic viruses to tumors for destruction and in immune up-regulation in vivo . Similarly, this approach may enhance nonlytic viruses for gene therapy applications.

J Biotechnol, 2005 Mar 2, 116(1), 11 - 20 Epub 2004 Nov 18.
Modulation of gene expression by promoter mutants of the lambdacI857/p(RM)/p(R) system; Jechlinger W et al.; Gene expression driven by the p(R) promoter of the lambdacI857/p(RM)/p(R) system results from inactivation of the temperature-sensitive CI857 repressor . The CI857 repressor, whose gene is transcribed by the divergently orientated p(RM) promoter, is destabilised at temperatures above 30 degrees C . In this study, the lambdacI857/p(RM)/p(R) system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C) . The mutated lambdap(R) expression modules, 32G and 32G/41C, tightly repressed the highly lethal phage PhiX174 lysis gene E at temperatures up to 37 and 39 degrees C, respectively . Expression of protein E and subsequent lysis of Escherichia coli was still induced by a temperature up-shift to 42 degrees C . The impact of the mutations on gene expression levels driven by the lambdap(R) and p(RM) promoters was evaluated at various temperatures using the lacZ reporter gene . Results indicate that the A-32G mutation confers a lambdap(R) promoter-down phenotype . The additional increase in the temperature stability of the 32G/41C expression system is due to the T-41C mutation leading to a higher p(RM) activity . The described lambdap(R) expression modules can be used to obtain a defined expression level at a given temperature and to tightly repress in particular highly lethal genes at different bacterial growth temperatures.

Biochem Pharmacol, 2005 Feb 1, 69(3), 385 - 394 Epub 2004 Dec 9.
Inhibition of interleukin-8 (CXCL8/IL-8) responses by repertaxin, a new inhibitor of the chemokine receptors CXCR1 and CXCR2; Casilli F et al.; Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis . Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes . Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8 . Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected . Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration . These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites.

Chem Res Toxicol, 2005 Jan, 18(1), 76 - 86
Effects of Peroxynitrite Dose and Dose Rate on DNA Damage and Mutation in the supF Shuttle Vector; Kim MY et al.; Peroxynitrite (ONOO(-)) induces oxidative and nitrosative DNA damage, and previous studies by our group have shown that it is strongly mutagenic in the supF shuttle vector pSP189 replicated in Escherichia coli MBL50 cells . In those experiments, however, the pSP189 plasmid was exposed under unphysiological conditions to large single bolus doses of ONOO(-), which limits extrapolation of the data to in vivo pathological states in which ONOO(-) may play a role . We have thus sought to define the effects of ONOO(-) dose and dose rate on the DNA damage and mutations induced in the supF gene by three different dosage mechanisms: (i) by infusion of ONOO(-) solution into suspensions of pSP189 at rates approximating those estimated to occur in inflamed tissues; (ii) by exposure to 3-morpholinosydnonimine (SIN-1), which generates ONOO(-) spontaneously during decomposition; and (iii) by bolus doses of ONOO(-) solution . In all cases, plasmid DNA was exposed in the presence of 25 mM bicarbonate, since the reaction of CO(2) with ONOO(-) (to form nitrosoperoxycarbonate) has a major impact on mutagenic potency of ONOO(-) in this system . Nucleobase and deoxyribose damage were evaluated by a plasmid nicking assay immediately after ONOO(-) and SIN-1 exposures . Mutation frequency (MF) and mutational spectra in the supF gene were determined after plasmid pSP189 replicated in host E . coli cells . Bolus ONOO(-) addition caused the highest amount of DNA damage, including base and deoxyribose lesions, while infusion caused the least . SIN-1 was found to induce almost exclusively deoxyribose oxidation, while bolus addition generated a high percentage of base damage . MF increased in a dose-dependent manner following all treatments, but infused ONOO(-) and SIN-1 exposures were less mutagenic than bolus ONOO(-) exposure . MFs induced by infusion and by SIN-1 incubated for 100 min at the highest level (4 mM) were 63 and 43% less, respectively, than that induced by bolus . All mutational hot spots were located at G:C sites except for A121 and A177 induced by SIN-1 exposure . Hot spots at C108 and C168 were common to all exposures; G113, G115, and G116 were common to bolus and infused ONOO(-) exposures; and G129 was common to infused ONOO(-) and SIN-1 exposures . Almost all mutations were single base pair substitutions under all exposure conditions . Whereas those induced by infused or bolus ONOO(-) and SIN-1 consisted predominantly of G:C to T:A transversions (66, 65, and 51%, respectively), G:C to C:G mutations were much less frequent following infusion and SIN-1 (8 and 19%, respectively) than those induced by bolus exposure (29%) . A:T to T:A mutations induced were detected only after ONOO(-) infusion and SIN-1 exposure (9 and 11%, respectively) . In conclusion, both dose and dose rate at which a genetic target is exposed to ONOO(-) substantially influence the damage and mutational response, indicating that these parameters will need to be taken into account in assessing the potential effects of ONOO(-) in vivo . Furthermore, the results indicate that the chemistry of SIN-1-induced DNA damage differs substantially from native ONOO(-), which suggests the need for caution in interpreting the biological relevance of SIN-1 as a surrogate for ONOO(-).

Chem Res Toxicol, 2005 Jan, 18(1), 51 - 60
Analysis of M(1)G-dR in DNA by Aldehyde Reactive Probe Labeling and Liquid Chromatography Tandem Mass Spectrometry; Jeong YC et al.; A novel method for the measurement of pyrimido{1,2-a}purin-10(3H)one (M(1)G) has been developed . Previous methods for analysis of M(1)G have been confounded by the fact that this lesion exists in equilibrium between a ring-closed form and a ring-opened aldehyde form . Poor detection sensitivity of the aldehydic form can result from loss of the adduct during analysis by its reaction with amines or proteins . We utilized the aldehyde reactive probe (ARP) to produce a stable ARP-M(1)G-deoxyribose (ARP-M(1)G-dR) conjugate to minimize adduct loss . This conjugate has increased the hydrophobicity that enhances separation of ARP-M(1)G-dR from unmodified DNA nucleosides by using solid phase extraction . In addition, measuring ARP-M(1)G-dR by selective reaction monitoring (SRM) of the {ARP-M(1)G-dR + H}(+) (635) --> {M(1)G + H}(+) (188) transition increases the detection sensitivity by nearly an order of magnitude relative to the measurement of M(1)G-dR by SRM . For accurate measurement, analytical standard (AS) DNA and internal standard (IS) DNA were used . High purity (15)N-labeled DNA was isolated from Escherichia coli that had been grown in minimum salt medium containing ((15)NH(4))(2)SO(4) . The (15)N-DNA and calf thymus DNA were treated with malondialdehyde to induce a high number of M(1)G adducts to prepare the IS and AS DNA, respectively . A consistent calibration curve was established from the analysis of 200 mug of blank DNA, 23 ng of IS DNA (400 fmol of (15)N(5)-M(1)G-dR), and AS DNA containing 0-810 fmol of M(1)G-dR . With the use of this novel IS DNA and selective labeling, this assay is a useful tool for monitoring oxidative stress-induced DNA damage from small amounts of DNA without the need of a specific antibody or laborious procedures . By this assay, two M(1)G adducts/10(8) guanines can readily be detected . Furthermore, this approach should be applicable to the analysis of other aldehydic DNA adducts as well as the measurement of an array of DNA lesions.

Appl Microbiol Biotechnol . 2005 Jan 14; {Epub ahead of print}
Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei; Leskinen S et al.; Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli . The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively . The catalytic domains belong to family 11 and family 10 of glycoside hydrolases . The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively . Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized . The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation . In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T . reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region . The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence . However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80 degrees C than the full-length enzyme . All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80 degrees C.

Ann Surg, 2005 Feb, 241(2), 349 - 55
Increased Susceptibility of Glutamine-Depleted Monocytes to Fever-Range Hyperthermia: The Role of 70-kDa Heat Shock Protein; Pollheimer J et al.; OBJECTIVE:: This study investigates the effect of fever-range hyperthermia on Gln-starving monocytes and the role of the 70-kDa heat shock protein Hsp70 . SUMMARY BACKGROUND DATA:: Fever is a protective acute-phase response to infection . However, in critically ill patients, the harmful effects of fever seem to be predominant . Critical illness is frequently associated with reduced plasma glutamine (Gln) levels, which contribute to the immune suppression in these patients due to impaired monocyte function . METHODS:: Isolated monocytes were suspended in Gln-depleted medium and exposed to 41 degrees C . Cell survival was determined by an MTT-based assay, and phagocytosis of Escherichia coli was measured by flow cytometry . Expression of Hsp70 was determined by Western blot . RESULTS:: Hyperthermia for 300 minutes strongly decreased the viability of Gln-depleted monocytes (85%), whereas it had only a moderate effect on Gln-supplied cells (45%, P < 0.05) . Shorter treatments (45 minutes) of Gln-starving monocytes had almost no effects on viability but decreased the phagocytosis activity by 30.8% . In addition, the expression of Hsp70 was inhibited almost completely . CONCLUSION:: These data show that Gln-starving monocytes have a reduced thermoresistance . This suggests that elevated body temperature damages monocytes in critically ill patients with reduced plasma Gln-levels possibly via an inhibition of the cytoprotective protein Hsp70.

J Virol, 2005 Feb, 79(3), 1871 - 87
Exposure of RNA Templates and Encapsidation of Spliced Viral RNA Are Influenced by the Arginine-Rich Domain of Human Hepatitis B Virus Core Antigen (HBcAg 165-173); Le Pogam S et al.; Previously, human hepatitis B virus (HBV) mutant 164, which has a truncation at the C terminus of the HBV core antigen (HBcAg), was speculated to secrete immature genomes . For this study, we further characterized mutant 164 by different approaches . In addition to the 3.5-kb pregenomic RNA (pgRNA), the mutant preferentially encapsidated the 2.2-kb or shorter species of spliced RNA, which can be reverse transcribed into double-stranded DNA before virion secretion . We observed that mutant 164 produced less 2.2-kb spliced RNA than the wild type . Furthermore, it appeared to produce at least two different populations of capsids: one encapsidated a nuclease-sensitive 3.5-kb pgRNA while the other encapsidated a nuclease-resistant 2.2-kb spliced RNA . In contrast, the wild-type core-associated RNA appeared to be resistant to nuclease . When arginines and serines were systematically restored at the truncated C terminus, the core-associated DNA and nuclease-resistant RNA gradually increased in both size and signal intensity . Full protection of encapsidated pgRNA from nuclease was observed for HBcAg 1-171 . A full-length positive-strand DNA phenotype requires positive charges at amino acids 172 and 173 . Phosphorylation at serine 170 is required for optimal RNA encapsidation and a full-length positive-strand DNA phenotype . RNAs encapsidated in Escherichia coli by capsids of HBcAg 154, 164, and 167, but not HBcAg 183, exhibited nuclease sensitivity; however, capsid instability after nuclease treatment was observed only for HBcAg 164 and 167 . A new hypothesis is proposed here to highlight the importance of a balanced charge density for capsid stability and intracapsid anchoring of RNA templates.

Genes Dev . 2005 Jan 13; {Epub ahead of print}
Implication of membrane localization of target mRNA in the action of a small RNA: mechanism of post-transcriptional regulation of glucose transporter in Escherichia coli; Kawamoto H et al.; Accumulation of phosphosugars such as glucose-6-phosphate causes a rapid degradation of ptsG mRNA encoding the major glucose transporter IICB(Glc) in an RNase E/degradosome-dependent manner . The destabilization of ptsG mRNA is caused by a small antisense RNA (SgrS) that is induced by phosphosugar stress . In this study, we analyzed a series of ptsG-crp translational fusions to identify the mRNA region required for the rapid degradation of ptsG mRNA . We found that the ptsG-crp mRNA is destabilized in response to phosphosugar stress when it contains the 5' portion of ptsG mRNA corresponding up to the first two transmembrane domains (TM1 and TM2) of IICB(Glc) . The destabilization of ptsG-crp mRNA was largely eliminated by frameshift mutations in the transmembrane region . The IICB(Glc)-CRP fusion proteins containing more than two transmembrane domains were localized at the membrane . The efficient destabilization of ptsG-crp mRNA was restored when TM1 and TM2 of IICB(Glc) were replaced by part of the LacY transmembrane region . We conclude that the membrane-targeting property of IICB(Glc) protein rather than the particular nucleotide or amino acid sequence is required for the efficient degradation of ptsG mRNA in response to metabolic stress . The stimulation of ptsG-crp mRNA degradation was completely eliminated when either the hfq or sgrS gene is inactivated . The efficient mRNA destabilization was observed in the absence of membrane localization when translation was reduced by introducing a mutation in the ribosome-binding site in the cytoplasmic ptsG-crp mRNA . Taken together, we conclude that mRNA localization to the inner membrane coupled with the membrane insertion of nascent peptide mediates the Hfq/SgrS-dependent ptsG mRNA destabilization presumably by reducing second rounds of translation.

Proc Natl Acad Sci U S A . 2005 Jan 13; {Epub ahead of print}
Altering the interaction between {sigma}70 and RNA polymerase generates complexes with distinct transcription-elongation properties; Berghofer-Hochheimer Y et al.; We compare the elongation behavior of native Escherichia coli RNA polymerase holoenzyme assembled in vivo, holoenzyme reconstituted from sigma(70) and RNA polymerase in vitro, and holoenzyme with a specific alteration in the interface between sigma(70) and RNA polymerase . Elongating RNA polymerase from each holoenzyme has distinguishable properties, some of which cannot be explained by differential retention or rebinding of sigma(70) during elongation, or by differential presence of elongation factors . We suggest that interactions between RNA polymerase and sigma(70) may influence the ensemble of conformational states adopted by RNA polymerase during initiation . These states, in turn, may affect the conformational states adopted by the elongating enzyme, thereby physically and functionally imprinting RNA polymerase.

J Biol Chem . 2005 Jan 12; {Epub ahead of print}
Crystal and solution structures of the helicase-binding domain of escherichia coli primase; Oakley AJ et al.; During bacterial DNA replication, the DnaG primase interacts with the hexameric DnaB helicase to synthesize RNA primers for extension by DNA polymerase . In Escherichia coli, this occurs by transient interaction of primase with the helicase . Here we demonstrate directly by surface plasmon resonance that the C-terminal domain of primase (DnaG-C) is responsible for interaction with DnaB6 . Determination of the 2.8-A crystal structure of DnaG-C revealed an asymmetric dimer . The monomers have an N-terminal helix bundle similar to the N-terminal domain of DnaB, followed by a long helix that connects to a C-terminal helix hairpin . The connecting helix is interrupted differently in the two monomers . Solution studies using NMR showed that an equilibrium exists between a monomeric species with an intact, extended but naked, connecting helix and a dimer in which this helix is interrupted in the same way as in one of the crystal conformers . The other conformer is not significantly populated in solution, and its presence in the crystal is due largely to crystal packing forces . It is proposed that the connecting helix contributes necessary structural flexibility in the primase-helicase complex at replication forks.>

J Appl Physiol . 2005 Jan 13; {Epub ahead of print}
PULMONARY AND EXTRA-PULMONARY ACUTE LUNG INJURY: INFLAMMATORY AND ULTRASTRUCTURAL ANALYSES; Menezes SL et al.; To test whether pulmonary and extrapulmonary acute lung injury (ALI) of identical mechanical compromise would express diverse morphological patterns and immunological pathways . For this purpose, a model of pulmonary (p) and extra-pulmonary (exp) ALI with similar functional changes was developed and pulmonary morphology (light and electron microscopy), cytokines levels and neutrophilic infiltration in the bronchoalveolar lavage fluid (BALF), elastic and collagen fibers content in the alveolar septa, and neutrophil apoptosis in the lung parenchyma were analyzed . BALB/c mice were divided into four groups . In control groups (Cp and Cexp), saline was intratracheally (i.t., 0.05 ml) instilled and intraperitoneally (i.p., 0.5 ml) injected, respectively . In ALIp and ALIexp groups, mice received E . coli lipopolysaccharide (10 microg, i.t . and 125 microg, i.p., respectively) . The changes in lung resistive and viscoelastic pressures, and static elastance, alveolar collapse and cells content in lung tissue were similar in ALIp and ALIexp groups . ALIp group presented a threefold increase in IL-8 and IL-10 levels in the BALF in relation to ALIexp, whereas IL-6 level showed a two-fold increase in ALIp . Neutrophils in the BALF were more frequent in ALIp than in ALIexp group . ALIp group showed more extensive injury of alveolar epithelium, intact capillary endothelium, and apoptotic neutrophils, while ALIexp group presented interstitial edema, intact type I and II cells and endothelial layer . In conclusion, given the same pulmonary mechanical dysfunction independently of the etiology of ALI, insult in pulmonary epithelium yielded more pronounced inflammatory responses, which induce ultrastructural morphological changes.

J Magn Reson, 2005 Feb, 172(2), 191 - 200
Backbone-only restraints for fast determination of the protein fold: The role of paramagnetism-based restraints . Cytochrome b(562) as an example; Banci L et al.; CH(alpha) residual dipolar couplings (Deltardc's) were measured for the oxidized cytochrome b(562) from Escherichia coli as a result of its partial self-orientation in high magnetic fields due to the anisotropy of the overall magnetic susceptibility tensor . Both the low spin iron (III) heme and the four-helix bundle fold contribute to the magnetic anisotropy tensor . CH(alpha) Deltardc's, which span a larger range than the analogous NH values (already available in the literature) sample large space variations at variance with NH Deltardc's, which are largely isooriented within alpha helices . The whole structure is now significantly refined with the chemical shift index and CH(alpha) Deltardc's . The latter are particularly useful also in defining the molecular magnetic anisotropy parameters . It is shown here that the backbone folding can be conveniently and accurately determined using backbone restraints only, which include NOEs, hydrogen bonds, residual dipolar couplings, pseudocontact shifts, and chemical shift index . All these restraints are easily and quickly determined from the backbone assignment . The calculated backbone structure is comparable to that obtained by using also side chain restraint . Furthermore, the structure obtained with backbone only restraints is, in its whole, very similar to that obtained with the complete set of restraints . The paramagnetism based restraints are shown to be absolutely relevant, especially for Deltardc's.

Biochem Biophys Res Commun, 2005 Feb 18, 327(3), 656 - 62
Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation; Cho O et al.; Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds . Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons . Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate . The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity . Complementation experiments verify that the salicylate oxygenase in S . yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away . Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.

Anal Biochem, 2005 Feb 1, 337(1), 35 - 47
Complex glycosaminoglycans: profiling substitution patterns by two-dimensional nuclear magnetic resonance spectroscopy; Guerrini M et al.; Biological and pharmacological interactions of heparin and structurally related glycosaminoglycans (GAGs) such as heparan sulfate (HS) involve complex sequences of variously sulfated uronic acid and aminosugar residues . Due to their structural microheterogeneity, these sequences are usually characterized in statistical terms, by high-performance liquid chroamtographic analysis of fragments obtained by enzymatic or chemical degradation . Nuclear magnetic resonance (NMR) spectroscopy is also currently used for structural characterization of GAGs . However, the use of monodimensional NMR analysis of complex GAGs is often limited by severe signal overlap that does not allow reliable quantitative measurements . Using magnetically equivalent signals, the higher resolution achieved by two-dimensional NMR methods could be also exploited for quantitative applications . In this work, heteronuclear single quantum coherence (HSQC) spectroscopy has been evaluated to determine variously substituted monosaccharide components of HS and HS mimics obtained by chemical modification of the Escherichia coli K5 polysaccharide (K5-PS) structurally related to the common biosynthetic precursor of heparin and HS . Heparin was used as a model for assessing the influence of (1)H-(13)C spin-spin couplings on "volumes" of the corresponding signals . For major signals, the HSQC approach permitted quantification of additional structural features both in heparins and in a typical HS . The method was applied to profile the substitution patterns of K5-PS derivatives involving different degrees of N,O-sulfation and N-acetylation, including O-sulfated heparosans bearing free amino groups.

Anal Chem, 2005 Jan 15, 77(2), 673 - 680
Analysis of Biological and Synthetic Ribonucleic Acids by Liquid Chromatography-Mass Spectrometry Using Monolithic Capillary Columns; Holzl G et al.; Ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) has been evaluated as a method for the fractionation and desalting of ribonucleic acids prior to their characterization by electrospray ionization mass spectrometry . Monolithic, poly(styrene-divinylbenzene)-based capillary columns allowed the rapid and highly efficient fractionation of both synthetic and biological ribonucleic acids . The common problem of gas-phase cation adduction that is particularly prevalent in the mass spectrometric analysis of ribonucleic acids was tackled through a combination of chromatographic purification and the addition of ethylenediaminetetraacetic acid to the sample at a concentration of 25 mmol/L shortly before on-line analysis . For RNA molecules ranging in size from 10 to 120 nucleotides, the mass accuracies were typically better than 0.02%, which allowed the characterization and identification of failure sequences and byproducts with high confidence . Following injection of a 500 nL sample onto a 60 x 0.2 mm column, the limit of detection for a 120-nucleotide ribosomal RNA transcript from Escherichia coli was in the 50-80 fmol range . The method was applied to the analysis of synthetic oligoribonucleotides, transfer RNAs, and ribosomal RNA . Finally, sequence information was derived for low picomole amounts of a 32-mer RNA upon chromatographic purification and tandem mass spectrometric investigation in an ion trap mass spectrometer . Complete series of fragment ions of the c- and y-types could be assigned in the tandem mass spectrum . In conclusion, IP-RP-HPLC using monolithic capillary columns represents a very useful tool for the structural investigation and quantitative determination of RNAs of synthetic and biological origin.

Proteomics . 2005 Jan 13; {Epub ahead of print}
Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: Sample preparation, data analysis, and application to the analysis of complex peptide mixtures; Mirgorodskaya E et al.; We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures . The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis . Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method . A procedure for data processing is described . The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein . For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis . The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit . Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.

Planta . 2005 Jan 13; {Epub ahead of print}
Molecular cloning, recombinant gene expression, and antifungal activity of cystatin from taro (Colocasia esculenta cv . Kaosiung no . 1); Yang AH et al.; A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5'-/3'-RACE extension . The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa . It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin . Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot . Recombinant GST-CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE . These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity . Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc . etc., at a concentration of 80 mug recombinant CeCPI/ ml . Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150-200 mug/ml . Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.

Proc Natl Acad Sci U S A . 2005 Jan 12; {Epub ahead of print}
Inhibited cell growth and protein functional changes from an editing-defective tRNA synthetase; Bacher JM et al.; The genetic code is established in aminoacylation reactions catalyzed by aminoacyl-tRNA synthetases . Many aminoacyl-tRNA synthetases require an additional domain for editing, to correct errors made by the catalytic domain . A nonfunctional editing domain results in an ambiguous genetic code, where a single codon is not translated as a specific amino acid but rather as a statistical distribution of amino acids . Here, wide-ranging consequences of genetic code ambiguity in Escherichia coli were investigated with an editing-defective isoleucyl-tRNA synthetase . Ambiguity retarded cell growth at most temperatures in rich and minimal media . These growth rate differences were seen regardless of the carbon source . Inclusion of an amino acid analogue that is misactivated (and not cleared) diminished growth rate by up to 100-fold relative to an isogenic strain with normal editing function . Experiments with target-specific antibiotics for ribosomes, DNA replication, and cell wall biosynthesis, in conjunction with measurements of mutation frequencies, were consistent with global changes in protein function caused by errors of translation and not editing-induced mutational errors . Thus, a single defective editing domain caused translationally generated global effects on protein functions that, in turn, provide powerful selective pressures for maintenance of editing by aminoacyl-tRNA synthetases.

Proc Natl Acad Sci U S A . 2005 Jan 12; {Epub ahead of print}
Profiling the humoral immune response to infection by using proteome microarrays: High-throughput vaccine and diagnostic antigen discovery; Davies DH et al.; Despite the increasing availability of genome sequences from many human pathogens, the production of complete proteomes remains at a bottleneck . To address this need, a high-throughput PCR recombination cloning and expression platform has been developed that allows hundreds of genes to be batch-processed by using ordinary laboratory procedures without robotics . The method relies on high-throughput amplification of each predicted ORF by using gene specific primers, followed by in vivo homologous recombination into a T7 expression vector . The proteins are expressed in an Escherichia coli-based cell-free in vitro transcription/translation system, and the crude reactions containing expressed proteins are printed directly onto nitrocellulose microarrays without purification . The protein microarrays are useful for determining the complete antigen-specific humoral immune-response profile from vaccinated or infected humans and animals . The system was verified by cloning, expressing, and printing a vaccinia virus proteome consisting of 185 individual viral proteins . The chips were used to determine Ab profiles in serum from vaccinia virus-immunized humans, primates, and mice . Human serum has high titers of anti-E . coli Abs that require blocking to unmask vaccinia-specific responses . Naive humans exhibit reactivity against a subset of 13 antigens that were not associated with vaccinia immunization . Naive mice and primates lacked this background reactivity . The specific profiles between the three species differed, although a common subset of antigens was reactive after vaccinia immunization . These results verify this platform as a rapid way to comprehensively scan humoral immunity from vaccinated or infected humans and animals.

J Biol Chem . 2005 Jan 14; {Epub ahead of print}
Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1; Trabold PA et al.; Initiation of DNA replication in eukaryotes requires the Origin Recognition Complex (ORC) and other proteins that interact with DNA at origins of replication . In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with a defect in initiation of DNA replication . Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC . A dosage suppressor screen identified the budding yeast mitochondrial co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug . Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation . In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin . Mge1p is the budding yeast homologue of the E . coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences . Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA.

J Biol Chem . 2005 Jan 12; {Epub ahead of print}
The retroviral hypermutation specificity of APOBEC3F and APOBEC3G is governed by the C-terminal DNA cytosine deaminase domain; Hache G et al.; The human proteins APOBEC3F and APOBEC3G restrict retroviral infection by deaminating cytosine residues in the first cDNA strand of a replicating virus . These proteins have two putative deaminase domains and it is unclear whether one or both catalyze deamination, unlike their homologs, AID and APOBEC1, which are well-characterized single domain deaminases . Here, we show that only the C-terminal cytosine deaminase domain of APOBEC3F and -3G governs retroviral hypermutation . A chimeric protein with the N-terminal cytosine deaminase domain from APOBEC3G and the C-terminal cytosine deaminse domain from APOBEC3F elicited a dinucleotide hypermutation preference nearly indistinguishable from that of APOBEC3F . This 5'-TC -> TT mutational specificity was confirmed in a heterologous E . coli-based mutation assay, where the 5'-CC -> CT dinucleotide hypermutation preference of APOBEC3G also mapped to the C-terminal deaminase domain . An N-terminal APOBEC3G deletion mutant displayed a preference indistinguishable from that of the full-length protein, and replacing the C-terminal deaminase domain of APOBEC3F with AID resulted in AID-like mutational signature . Together, these data indicate that only the C-terminal domain of APOBEC3F and -3G dictates the retroviral minus strand 5'-TC and 5'-CC dinucleotide hypermutation preferences, respectively, leaving the N-terminal domain to perform other aspects of retroviral restriction.

In Vivo, 2004 Nov-Dec, 18(6), 795 - 8
Synergistic effect of dehydroascorbic acid and mixtures with vitamin E and beta-carotene on mitomycin C efficiency under irradiation in vitro; Kammerer C et al.; Experiments in vitro have shown that dehydroascorbic acid (DHA) possesses antitumor properties under irradiation, which are gradually enhanced by combination with beta-carotene or vitamin E . On the other hand the cytostatic efficiency of mitomycin C (MMC) is increased from deltaD37 = -93 up to deltaD37 = -141 in the presence of DHA . It has also been shown that Escherichia coli bacteria are able, to some extent, to reduce DHA to ascorbate under the same experimental conditions . The results are of interest for the radiation therapy of cancer.

Sheng Wu Yi Xue Gong Cheng Xue Za Zhi, 2004 Dec, 21(6), 947 - 52
{Effects of entrapment of murine interleukin-2 gene with chitosan nanoparticles on expression of mIL-2 gene and on regulation of immune response in mice}; Li H et al.; The experiment was conducted to prepare chitosan nanoparticles (CNP), to entrap VRMIL-2 with CNP, the eukaryotic VR1020 expression plasmid containing murine IL-2 gene (mlL-2), and to investigate the expression in vivo and the regulatory effect of mIL-2 on immune-response and immuno-protection in mice inoculated muscularly with CNP entrapped VMIL-2 at 21 days old . The results showed that IgG, IgM and IgA contents increased to different degrees in the sera from the inoculated mice, which were remarkably higher than those of the controls inoculated VR1020 packed with CNP (P<0.05); so were the IL-2, IL-4 and IL-6 contents in the sera of the immunized mice . The number of white blood cells and lymphocytes significantly increased respectively in the vaccinated mice, compared with those of controls . These mice were orally challenged with virulent E . coli 35 days post-inoculation, and all the immune responses were significantly higher than those of the control except the number of neutrophils . The mice inoculated with VRMIL-2 survived healthily, while the mice of control group were ill with the evident lesions . Although there are no remarkable differences between the cellular and humoral immune indexes of mice inoculated with CNP-VRMIL-2 and nude VRMIL-2 (P>0.05), the dosage of CNP-VRMIL-2 is only one fifth of the VRMIL-2 . These indicated that entrapment of mIL-2 gene with chitosan nanoparticles could remarkably enhance the expression of mIL-2 in vivo, and significantly raise the levels of cellular and humoral immune, and increase the resistance of mice against E . coli infection . The results suggested that chitosan nanoparticles and IL-2 gene could be used as an effective immunoenhancer to increase the immunity of animals against infection.

New Microbiol, 2004 Apr, 27(2 Suppl 1), 5 - 9
Sulfated K5 Escherichia coli polysaccharide derivatives inhibit human immunodeficiency type-1 (HIV-1) infection: candidate microbicides to prevent sexual HIV transmission; Pacciarini F et al.; The ideal microbicide must fulfill a number of criteria including a broad and potent activity against transmission of HIV and other sexually transmitted agents in the absence of toxicity and inflammation . We have described that derivatives of K5 polysaccharide from Escherichia coli inhibit HIV entry in target cells . K5 derivatives have a structure that resembles that of heparin, but they are devoid of the anticoagulant activity typical of heparin . Moreover, in contrast to heparin, they inhibit a broad spectrum of HIV-1 laboratory-adapted and primary isolates that use either CCR5 or CXCR4 or both coreceptors in terms of their infection and replication in primary CD4+ lymphocytes and monocytes-derived macrophages (MDM) . Therefore, these compounds could be developed as candidate microbicides for preventing sexual HIV transmission, a predominant modality of HIV spreading in both the developed and underdeveloped world.

Proteins . 2005 Jan 11; {Epub ahead of print}
S-adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: Biochemical characterization and analysis of protein structure by comparative molecular modeling; Porcelli M et al.; S-Adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine . AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified . The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C . The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit . Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity . Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme . The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface . These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability . Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding . Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures . Proteins 2005 . (c) 2005 Wiley-Liss, Inc.

Radiat Environ Biophys, 2004 Dec, 43(4), 265 - 70 Epub 2004 Nov 13.
Various effects on transposition activity and survival of Escherichia coli cells due to different ELF-MF signals; Del Re B et al.; Previous assays with weak sinusoidal magnetic fields (SMF) have shown that bacteria that had been exposed to a 50 Hz magnetic field (0.1-1 mT) gave colonies with significantly lower transposition activity as compared to sham-exposed bacteria . These experiments have now been extended by using a pulsed-square wave magnetic field (PMF) and, unexpectedly, it was found that bacteria exposed to PMF showed a higher transposition activity compared to the controls . The increase of the transposition activity was positively correlated with the intensity of the magnetic fields (linear dose-effect relation) . This phenomenon was not affected by any bacterial cell proliferation, since no significant difference was observed in number and size of PMF-exposed and sham-exposed colonies . In addition, the cell viability of E . coli was significantly higher than that of the controls when exposed to SMF, and lower than that of the controls when exposed to PMF . Under our experimental conditions it was shown that exposure to PMF stimulates the transposition activity and reduces cell viability of bacteria, whereas exposure to SMF reduces the transposition mobility and enhances cell viability . These results suggest that the biological effects of magnetic fields may critically depend on the physical characteristics of the magnetic signal, in particular the wave shape.

Genome, 2004 Dec, 47(6), 1036 - 42
Molecular characterization of a gene encoding N-myristoyl transferase (NMT) from Triticum aestivum (bread wheat); Dumonceaux T et al.; Myristoyl-CoA:protein N-myristoyl transferase (NMT; EC 2.3.1.97) acylates the Gly residue abutting the N-terminal Met with a myristic acid following the removal of the Met residue in certain eukaryotic proteins, and in some cases myristoylation is essential to cell growth and survival . We report the cloning of a full-length cDNA encoding NMT from Triticum aestivum (TaNMT) . The cDNA included a predicted open reading frame of 1317 nucleotides, which encoded a predicted protein of 438 amino acids containing all of the residues that are important for NMT activity . The TaNMT amino acid and nucleotide sequences were compared with NMTs from 14 other species encompassing a wide array of taxonomic groups . Among the experimentally validated NMTs, TaNMT was most similar to that of Arabidopsis thaliana . Southern blot analysis of wheat genomic DNA showed that TaNMT is encoded by a single copy gene, with one copy per haploid genome . We expressed TaNMT in Escherichia coli cells and determined that the recombinant protein possessed NMT activity, catalyzing the N-myristoylation of peptides from known or putatively myristoylated proteins from plants and animals without a strong preference for the plant peptides . TaNMT is the second experimentally validated plant NMT sequence and the first from a monocotyledonous species.

Can J Microbiol, 2004 Oct, 50(10), 835 - 843
Purification and characterization of Thermobifida fusca xylanase 10B; Kim JH et al.; Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases . The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E . coli, and the protein was purified and characterized . Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus . It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 degrees C, and is most active between pH 5 and 8 . Xyl10B is produced by growth of T . fusca on xylan or Solka Floc but not on pure cellulose . Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T . fusca Xyl11A . Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl &alpha;-D-arabinofuranoside, p-nitrophenyl-&beta;-D-xylobioside, p-nitrophenyl-&beta;-D-xyloside, and p-nitrophenyl-&beta;-D-cellobioside . Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.

J Pediatr, 2005 Jan, 146(1), 54 - 61
Prevalence of diarrheagenic Escherichia coli in acute childhood enteritis: A prospective controlled study; Cohen MB et al.; Objective Since diarrheagenic E . coli are not identified by common clinical laboratory techniques, we hypothesized that these organisms might be an unrecognized cause of enteritis in children in the U.S . Study design 1327 children with acute gastroenteritis were identified prospectively by active surveillance in the Emergency Department (ED) and the inpatient units at Cincinnati Children's Hospital Medical Center . Stool samples were evaluated for diarrheagenic E . coli using a panel of DNA probes and adherence pattern to HEp-2 cells . Stool samples from a reference group of 555 well children were studied for comparison . Results Gene probe studies, but not HEp-2 cell adherence, demonstrated that enteroaggregative, diffusely adherent and enteropathogenic E . coli were associated with clinical illness . Each was isolated significantly more often from study subjects in the ED than controls . In children <1 year of age, enteroaggregative E . coli were isolated significantly more often from both inpatients (4.7%, Odds Ratio = 3.4, 95% confidence intervals 1.3-9.1, p <0.03) and ED patients (10.0%, Odds Ratio = 7.2, 95% confidence intervals 2.9-18.2, p <0.001) than from well children (1.4%) . Conclusions Diarrheagenic E . coli , especially enteroaggregative E . coli , may be an important, unrecognized cause of childhood diarrhea in the U.S.

J Vet Med Sci, 2004 Dec, 66(12), 1517 - 21
Serodiagnosis of Babesia gibsoni Infection in Dogs by an Improved Enzyme-Linked Immunosorbent Assay with Recombinant Truncated P50; Verdida RA et al.; The surface antigen P50 of Babesia gibsoni is an important candidate for the development of a diagnostic reagent for canine piroplasmosis . In order to establish an effective diagnostic method for practical use, the gene encoding truncated P50 (P50t) lacking a signal peptide and C-terminal hydrophobic regions were cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) . More than 90% portion of the GST-P50t was expressed as a soluble form, in contrast with GST-P50f (full-length), which was completely expressed as an insoluble form . This result indicates that removal of the hydrophobic signal peptide and C-terminus had dramatically improved its hydrophilicity . The purified GST-P50t was tested in an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to B . gibsoni in dogs . The ELISA with GST-P50t clearly differentiated between B . gibsoni-infected dog sera and uninfected dog sera . In addition, the ELISA detected no cross-reactivity with sera from dogs experimentally infected with the closely related parasites, B . canis canis, B . canis vogeli, and B . canis rossi . Field serum samples collected from dogs in Japan and China were examined for the diagnosis of B . gibsoni infection by using the ELISA . 14.5% (9/62), 5.8% (7/120), and 5.4% (2/37) of tested samples were positive for dogs from Okinawa, Yamaguchi, and Osaka prefectures, Japan, respectively . On the other hand, 4.8% (2/41) of tested samples were positive for dogs from Nanjing, China . These results suggest that the GST-P50t could be a reliable reagent for practical use in ELISA for the serodiagnosis of canine piroplasmosis caused by B . gibsoni.

J Mol Biol, 2005 Feb 4, 345(5), 1229 - 41 Epub 2004 Dec 07.
Functional Solubilization of Aggregation-prone HIV Envelope Proteins by Covalent Fusion with Chaperone Modules; Scholz C et al.; The envelope proteins of human immunodeficiency virus (HIV) and human T-cell lymphotrophic virus (HTLV) mediate cell attachment and membrane fusion . For HIV-1, the precursor protein gp160 is cleaved proteolytically into two fragments, the surface-associated receptor binding subunit gp120 and the membrane spanning subunit gp41, which is involved in membrane fusion during virus entry . Soluble and immunoreactive variants of gp41 are essential for the reliable diagnosis of HIV-1 infections . Hitherto, gp41 was solubilized by adding detergents, or in acidic or alkaline solvents . We find that covalent fusions with SlyD or FkpA, two homodimeric Escherichia coli chaperones with peptidyl-prolyl isomerase activity, solubilize retroviral envelope proteins without compromising their immunological reactivity . gp41 from HIV-1, gp36 from HIV-2 and gp21 from HTLV could be expressed in large amounts in the Escherichia coli cytosol when fused with one or two subunits of SlyD or FkpA . The fusion proteins could be easily isolated and refolded, and showed high solubility and immunoreactivity, thus providing sensitive and reliable tools for diagnostic applications . Covalent fusions with SlyD or FkpA might be valuable generic tools for the solubilization and activation of aggregation-prone proteins.

J Mol Biol, 2005 Feb, 345(5), 1119 - 1130 Epub 2004 Dec 16.
Comparative Structural Analysis of Oxidized and Reduced Thioredoxin from Drosophila melanogaster; Wahl MC et al.; Thioredoxins (Trx) participate in essential antioxidant and redox-regulatory processes via a pair of conserved cysteine residues . In dipteran insects like Drosophila and Anopheles, which lack a genuine glutathione reductase (GR), thioredoxins fuel the glutathione system with reducing equivalents . Thus, characterizing Trxs from these organisms contributes to our understanding of redox control in GR-free systems and provides information on novel targets for insect control . Cytosolic Trx of Drosophila melanogaster (DmTrx) is the first thioredoxin that was crystallized for X-ray diffraction analysis in the reduced and in the oxidized form . Comparison of the resulting structures shows rearrangements in the active-site regions . Formation of the C32-C35 disulfide bridge leads to a rotation of the side-chain of C32 away from C35 in the reduced form . This is similar to the situation in human Trx and Trx m from spinach chloroplasts but differs from Escherichia coli Trx, where it is C35 that moves upon change of the redox state . In all four crystal forms that were analysed, DmTrx molecules are engaged in a non-covalent dimer interaction . However, as demonstrated by gel-filtration analyses, DmTrx does not dimerize under quasi in vivo conditions and there is no redox control of a putative monomer/dimer equilibrium . The dimer dissociation constants K(d) were found to be 2.2mM for reduced DmTrx and above 10mM for oxidized DmTrx as well as for the protein in the presence of reduced glutathione . In human Trx, oxidative dimerization has been demonstrated in vitro . Therefore, this finding may indicate a difference in redox control of GR-free and GR-containing organisms.

J Mol Biol, 2005 Feb 4, 345(5), 1047 - 57 Epub 2004 Dec 07.
Structure at 1.3A Resolution of Rhodothermus marinus caa(3) Cytochrome c Domain; Srinivasan V et al.; The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised . The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom . The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively . The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold . This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes . This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase . The R.marinus caa(3) uses HiPIP as the redox partner . The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.

J Mol Biol, 2005 Feb 4, 345(5), 969 - 85 Epub 2004 Dec 08.
The Pre-tRNA Nucleotide Base and 2'-Hydroxyl at N(-1) Contribute to Fidelity in tRNA Processing by RNase P; Zahler NH et al.; Fidelity in tRNA processing by the RNase P RNA from Escherichia coli depends, in part, on interactions with the nucleobase and 2' hydroxyl group of N(-1), the nucleotide immediately upstream of the site of RNA strand cleavage . Here, we report a series of biochemical and structure-function studies designed to address how these interactions contribute to cleavage site selection . We find that simultaneous disruption of cleavage site nucleobase and 2' hydroxyl interactions results in parallel reactions leading to correct cleavage and mis-cleavage one nucleotide upstream (5') of the correct site . Changes in Mg(2+) concentration and pH can influence the fraction of product that is incorrectly processed, with pH effects attributable to differences in the rate-limiting steps for the correct and mis-cleavage reaction pathways . Additionally, we provide evidence that interactions with the 2' hydroxyl group adjacent to the reactive phosphate group also contribute to catalysis at the mis-cleavage site . Finally, disruption of the adjacent 2'-hydroxyl contact has a greater effect on catalysis when pairing between the ribozyme and N(-1) is also disrupted, and the effects of simultaneously disrupting these contacts on binding are also non-additive . One implication of these results is that mis-cleavage will result from any combination of active site modifications that decrease the rate of correct cleavage beyond a certain threshold . Indeed, we find that inhibition of correct cleavage and corresponding mis-cleavage also results from disruption of any combination of active site contacts including metal ion interactions and conserved pairing interactions with the 3' RCCA sequence . Such redundancy in interactions needed for maintaining fidelity may reflect the necessity for multiple substrate recognition in vivo . These studies provide a framework for interpreting effects of substrate modifications on RNase P cleavage fidelity and provide evidence for interactions with the nucleobase and 2' hydroxyl group adjacent to the reactive phosphate group in the transition state.

J Urol, 2005 Feb, 173(2), 630 - 4
Modulating bladder neuro-inflammation: rdp58, a novel anti-inflammatory Peptide, decreases inflammation and nerve growth factor production in experimental cystitis; Gonzalez RR et al.; PURPOSE:: In interstitial cystitis (IC) inflammation induces and perpetuates neurotrophic changes in the bladder, resulting in the symptoms of frequency, urgency and pain . RDP58 (NH2-arg-norleucine (nle)-nle-arg-nle-nle-nle-gly-tyr-CONH2) (Sangstat Corp., Fremont, California) is a novel synthetic peptide that inhibits early signal transduction pathways for the expression of inflammatory cytokines . In this study we evaluated the effects of intravesical RDP58 on an established model of cystitis . MATERIALS AND METHODS:: Mice were catheterized and equal volumes of Escherichia coli lipopolysaccharide (LPS) or saline were instilled into the bladder . After 45 minutes the bladders were drained and distilled water or RDP58 (1 mg/ml) was instilled for 30 minutes . At 24 hours later the bladders were excised and cultured for analysis of tumor necrosis factor-alpha (TNF-alpha), substance P (SP) and nerve growth factor (NGF) production, as quantified by enzyme-linked immunosorbent assay . RESULTS:: LPS caused severe inflammation in mouse bladders compared with controls . Exposure to LPS increased the levels of TNF-alpha, SP and NGF production compared with controls (each p <0.05) . In LPS exposed mice RDP58 significantly decreased inflammatory parameters by 82% 24 hours after treatment (p <0.05) . Within 4 hours RDP58 abolished TNF-alpha production and at 24 hours TNF-alpha remained undetectable . RDP58 also significantly decreased SP and NGF production in LPS exposed bladders by more than 40% and 85%, respectively (each p <0.05) . CONCLUSIONS:: Inflammatory models of cystitis result in increased levels of TNF-alpha, SP and NGF production in the bladder, paralleling the hypothesized neuro-inflammatory etiology of IC . RDP58 decreases inflammation and neurotrophic factors in vivo and it may potentially treat bladder disorders with an inflammatory component, such as IC.

Clin Diagn Lab Immunol, 2005 Jan, 12(1), 180 - 6
Antibody responses of pigs to defined erns fragments after infection with classical Swine Fever virus; Lin M et al.; Antibody responses of pigs to defined E(rns) fragments, after classical swine fever virus (CSFV) infection, were studied by using an enzyme-linked immunosorbent assay (ELISA) . Selection of various E(rns) fragments was based on an immunodominant E(rns) region encompassing three overlapping antigenic regions, amino acids 65 to 145 (E(rns)(aa) (65-145)) (AR1), 84 to 160 (E(rns)(aa) (84-160)) (AR2), and 109 to 220 (E(rns)(aa) (10)(9-220)) (AR3), identified earlier by our group (M . Lin, E . Trottier, J . Pasick, and M . Sabara, J . Biochem., in press) . Defined E(rns) fragments, including AR1, AR2, AR3, E(rns)(aa) (65-160) (AR12), E(rns)(aa) (84-220) (AR23), E(rns)(aa) (65-220) (AR123), E(rns)(aa) (109-145) (the consensus region defined by the three overlapping regions), and E(rns)(aa) (109-160) (a fragment 15 amino acids larger than the consensus region), were expressed in Escherichia coli, purified by nickel chelate affinity chromatography, and used to measure antibody responses in 20 sera serially collected from pigs experimentally infected with CSFV . Based on the optimum cutoffs determined by receiver operating characteristic analysis after testing 238 negative field sera from Canadian sources, all the E(rns) fragments were capable of distinguishing positive from negative antibody responses with sensitivities ranging between 75 and 90% and specificities ranging between 83.2 and 100% . Detection of antibody responses to refolded E(rns)(aa) (109-145) and E(rns)(aa) (109-160) by ELISA (this study) but not by Western blots (Lin et al., in press) indicated that the epitopes within the consensus region are conformational . When cutoff values were raised to give a specificity of 100%, four E(rns) fragments (AR2, AR23, E(rns)(aa) (109-145), and E(rns)(aa) (109-160)) offered much higher sensitivities (75 to 90%) than those obtained with other fragments (20 to 65%) . E(rns)(aa) (109-145) and E(rns)(aa) (109-160) were capable of detecting antibody responses in infected pigs as early as 7 days postinfection . Demonstration of antibody responses to either one of the four fragments can thus be an alternative to use of the full-length protein in ELISA for serological diagnosis of CSFV infection . An advantage of such a test would be its utilization for serological survey in a classical swine fever-free country (e.g., Canada) in biocontainment level 2 laboratories.

Clin Diagn Lab Immunol, 2005 Jan, 12(1), 157 - 64
Induction of Thymus-Derived {gamma}{delta} T Cells by Escherichia coli Enterotoxin B Subunit in Peritoneal Cavities of Mice; Shimizu T et al.; We examined the activation of intraperitoneal T cells in BALB/c mice by the Escherichia coli enterotoxin B subunit, which induced a specific Th2 type of T-cell response to intraperitoneally coadministered bovine immunoglobulin G . The numbers of both gammadelta and alphabeta T cells increased significantly after intraperitoneal administration of the B subunit in a time-dependent manner; these numbers were not affected by the B-subunit G33D mutant, which is defective in GM(1) ganglioside-binding ability . Early after administration a small number of gammadelta T cells produced either interleukin-4 (IL-4) or gamma interferon, while late after administration primarily IL-10-producing gammadelta T cells were detected . gammadelta T cells induced by the B subunit did not express a characteristic V gene over the time course of the study . The induction of gammadelta T cells did not occur in athymic nu/nu mice but could be induced upon transplantation of fetal AKR thymus-like alphabeta T cells . gammadelta T cells in athymic nu/nu mice with a fetal thymic graft predominantly expressed the donor Thy-1.1 antigen but not the host Thy-1.2 antigen . The induction of these T cells, however, could not be restored by coadministration of the B subunit with peritoneal cells from normal mice . These results suggest that the B subunit activates intraperitoneal gammadelta and alphabeta T cells in a manner dependent upon its ability to bind to GM(1) ganglioside . gammadelta T cells induced by the B subunit are Th2-type cells derived from the thymus . These gammadelta T cells may be functionally involved in specific Th2 responses to the B subunit, which possibly acts as an adjuvant through the influence of alphabeta T cells.

Clin Diagn Lab Immunol, 2005 Jan, 12(1), 60 - 7
Acute inflammatory response to endotoxin in mice and humans; Copeland S et al.; Endotoxin injection has been widely used to study the acute inflammatory response . In this study, we directly compared the inflammatory responses to endotoxin in mice and humans . Escherichia coli type O113 endotoxin was prepared under identical conditions, verified to be of equal biological potency, and used for both mice and humans . The dose of endotoxin needed to induce an interleukin-6 (IL-6) concentration in plasma of approximately 1,000 pg/ml 2 h after injection was 2 ng/kg of body weight in humans and 500 ng/kg in mice . Healthy adult volunteers were injected intravenously with endotoxin, and male C57BL/6 mice (n = 4 to 12) were injected intraperitoneally with endotoxin . Physiological, hematological, and cytokine responses were determined . Endotoxin induced a rapid physiological response in humans (fever, tachycardia, and slight hypotension) but not in mice . Both mice and humans exhibited lymphopenia with a nadir at 4 h and recovery by 24 h . The levels of tumor necrosis factor (TNF) and IL-6 in plasma peaked at 2 h and returned to baseline levels by 4 to 6 h . IL-1 receptor antagonist RA and TNF soluble receptor I were upregulated in both mice and humans but were upregulated more strongly in humans . Mice produced greater levels of CXC chemokines, and both mice and humans exhibited peak production at 2 h . These studies demonstrate that although differences exist and a higher endotoxin challenge is necessary in mice, there are several similarities in the inflammatory response to endotoxin in mice and humans.

Circulation . 2005 Jan 10; {Epub ahead of print}
Targeted Modification of Atrial Electrophysiology by Homogeneous Transmural Atrial Gene Transfer; Kikuchi K et al.; BACKGROUND: Safe and effective myocardial gene transfer remains elusive . Heterogeneous ventricular gene delivery has been achieved in small mammals but generally with methods not readily transferable to the clinic . Atrium-specific gene transfer has not yet been reported . We hypothesized that homogeneous atrial gene transfer could be achieved by direct application of adenoviral vectors to the epicardial surface, use of poloxamer gel to increase virus contact time, and mild trypsinization to increase virus penetration . METHODS AND RESULTS: We "painted" recombinant adenovirus encoding the reporter gene Escherichia coli beta-galactosidase directly onto porcine atria . Investigational variables included poloxamer use, trypsin concentration, and safety . Using the painting method, we modified the atrial phenotype with an adenovirus expressing HERG-G628S, a long-QT-syndrome mutant . Our results showed that application of virus with poloxamer alone resulted in diffuse epicardial gene transfer with negligible penetration into the myocardium . Dilute trypsin concentrations allowed complete transmural gene transfer . After trypsin exposure, echocardiographic left atrial diameter did not change . Left atrial function decreased on postoperative day 3 but returned to baseline by day 7 . Tissue tensile strength was affected only in the 1% trypsin group . HERG-G628S gene transfer prolonged atrial action potential duration and refractory period without affecting ventricular electrophysiology . CONCLUSIONS: We show complete transmural atrial gene transfer by this novel painting method . Adaptation of the method could allow application to other tissue targets . Use with functional proteins in the atria could cure or even prevent diseases such as atrial fibrillation or sinus node dysfunction.

J Biol Chem . 2005 Jan 10; {Epub ahead of print}
The non-consecutive disulfide bond of Echerichia coli phytase (AppA) renders it dependent on the protein disulfide isomerase, DsbC; Berkmen M et al.; The formation of protein disulfide bonds in the E . coli periplasm by the enzyme DsbA is an inaccurate process . Many eukaryotic proteins with non-consecutive disulfide bonds expressed in E . coli require an additional protein for proper folding, the disulfide bond isomerase DsbC . Here we report studies on a native E . coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one non-consecutive disulfide bonds . We show that AppA requires DsbC for its folding . However, the activity of an AppA mutant lacking its non-consecutive disulfide bond is DsbC-independent . An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the non-consecutive disulfide bond, but has the three consecutive disulfide bonds found in AppA . The consecutively disulfide-bonded Agp is not dependent on DsbC, but is rendered dependent by the engineering into it of AppA's conserved non-consecutive disulfide bond . Taken together, these results provide support for the proposal that proteins with non-consecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.

J Biol Chem . 2005 Jan 10; {Epub ahead of print}
Restoration of growth to acidic phospholipid-deficient cells by DnaA(L366K) is independent of its capacity for nucleotide binding and exchange and requires DnaA; Li Z et al.; In the absence of adequate levels of cellular acidic phospholipids, E . coli remain viable, but are arrested for growth . Expression of a DnaA protein that contains a single amino acid substitution in the membrane binding domain, DnaA(L366K), in concert with expression of wild-type DnaA protein, restores growth . DnaA protein has high affinity for ATP and ADP, and in vitro lipid bilayers that are fluid and contain acidic phospholipids reactivate inert ADP-DnaA by promoting an exchange of ATP for ADP . Here, nucleotide and lipid interactions and replication activity of purified DnaA(L366K) were examined to gain insight into the mechanism of how it restores growth to cells lacking acidic phospholipids . DnaA(L366K) behaved like wild-type DnaA with respect to nucleotide binding affinities and hydrolysis properties, specificity of acidic phospholipids for nucleotide release, and origin binding . Yet, DnaA(L366K) was feeble at initiating replication from oriC unless augmented with a limiting quantity of wild-type DnaA, reflecting the in vivo requirement that both wild-type and a mutant form of DnaA must be expressed and act together to restore growth to acidic phospholipid deficient cells.

J Exp Bot . 2005 Jan 10; {Epub ahead of print}
Evolution and expression analysis of starch synthase III and IV in rice; Dian W et al.; Plants contain at least five subfamilies of starch synthases, granule bound starch synthase (GBSS) and starch synthases I, II, III, and IV (SSI, SSII, SSIII, SSIV) . In this work, two members of SSIII and SSIV, respectively, were cloned and designated OsSSIII-1/-2 and OsSSIV-1/-2 in rice . Together with six other previously reported genes, the SS gene family in rice therefore is known to be duplicated and to comprise ten SS genes distributed among the five subfamilies . The starch synthase activity of each SS was confirmed by expression and enzyme activity assay in E . coli . Expression profile analysis with reverse transcription-PCR, western blotting and zymogram, indicates that OsSSIII-2 and OsSSIV-1 are mainly expressed in endosperm, while OsSSIII-1 and OsSSIV-2 are mainly expressed in the leaves . With a similar pattern of genes encoding other enzymes for starch synthesis, (such as GBSS, SSII, ADP-glucose pyrophosphorylases, and branching enzymes), it is suggested that two divergent groups of these genes should be classified in rice . Group I genes are preferentially expressed in the endosperm and function on storage starch synthesis . Group II genes are mainly expressed in leaves and some of them in the early developing endosperm, and function on transient starch synthesis in rice.

Protein Expr Purif, 2005 Feb, 39(2), 307 - 19
Functional expression of bitistatin, a disintegrin with potential use in molecular imaging of thromboembolic disease; Knight LC et al.; Bitistatin is a single-chain disintegrin which contains 83 amino acids and is internally crosslinked with seven disulfide bonds . This platelet aggregation inhibitor, which binds with high affinity to the alphaIIbbeta3 integrin, has potential use as the basis for a radiotracer to locate thrombi and emboli by scintigraphic imaging . A method amenable to large-scale, consistent production of bitistatin was sought . A synthetic gene coding for bitistatin was inserted into two different Escherichia coli expression vectors . One vector expressed recombinant bitistatin (rBitistatin) as a cleavable fusion protein and the other expressed rBitistatin as an isolated protein . In both cases, rBitistatin contained an additional amino acid (Gly) at the N-terminus compared with the native protein . The fusion protein was purified by affinity chromatography, then cleaved enzymatically to release rBitistatin, which was purified by reversed-phase high performance liquid chromatography (HPLC) to a single active form . The rBitistatin produced as an isolated protein was purified from cell lysate by HPLC in a reduced form, then refolded, and purified again by HPLC . Yields of active rBitistatin averaged 12mg/L for expression as an isolated protein, 10 times as high as when the fusion protein was employed . Structural assays confirmed the expected mass and sequence of the product . Functional assays (inhibition of platelet aggregation in vitro, equilibrium binding to platelets in vitro, and binding of labeled protein to experimental thrombi and emboli in vivo) confirmed that rBitistatin retained the functional characteristics of native bitistatin.

Protein Expr Purif, 2005 Feb, 39(2), 288 - 95
Expression, purification of human vasostatin120-180 in Escherichia coli, and its anti-angiogenic characterization; Sun QM et al.; According to codon preference of Escherichia coli, the optimized coding sequence of human vasostatin120-180aa (VAS) was obtained by chemical synthesis and molecular cloning methods . Using PCR and enzyme digestion, the full encoding sequence for VAS was cloned into the E . coli expression vector pALEX and expressed as a GST fusion protein in BL21 (DE3) strain . GST-VAS protein approximately accounted for 45% of the total bacterial proteins . Most of target protein existed in inclusion body . To improve the solubility of GST-VAS, the contribution of low temperature and molecular chaperone co-expression to the solubility of GST-VAS was tested . The results showed that co-expression with chaperons, TF and GroES/GroEL, and low expression temperature cooperatively improved the solubility of GST-VAS from 10 to 85%, and the yield of soluble GST-VAS was sixfold increased . When purified by GST affinity chromatography, 50mg GST-VAS was obtained with purity over 85% from 1L culture . Intact VAS was released by enterokinase digestion and further purified by Sephadex G50 gel filtration chromatography . About 7.2mg intact homogeneous VAS protein was finally produced from 1L bacterial culture . The identity of GST-VAS and VAS was validated by Western blotting analysis . Recombinant VAS protein displayed distinct inhibition of endothelial cell proliferation and anti-angiogenic activity by chick embryo chorioallantoic membrane assay.

Protein Expr Purif, 2005 Feb, 39(2), 269 - 82
Expression, purification, and physical characterization of Escherichia coli lipoyl(octanoyl)transferase; Nesbitt NM et al.; Lipoic acid is a sulfur-containing 8-carbon fatty acid that functions as a central cofactor in multienzyme complexes that are involved in the oxidative decarboxylation of glycine and several alpha-keto acids . In its functional form, it is bound covalently in an amide linkage to the epsilon-amino group of a conserved lysine residue of the "lipoyl bearing subunit," resulting in a long "swinging arm" that shuttles intermediates among the requisite active sites . In Escherichia coli and many other organisms, the lipoyl cofactor can be synthesized endogenously . The 8-carbon fatty-acyl chain is constructed via the type II fatty acid biosynthetic pathway as an appendage to the acyl carrier protein (ACP) . Lipoyl(octanoyl)transferase (LipB) transfers the octanoyl chain from ACP to the target lysine acceptor, generating the substrate for lipoyl synthase (LS), which subsequently catalyzes insertion of both sulfur atoms into the C-6 and C-8 positions of the octanoyl chain . In this study, we present a three-step isolation procedure that results in a 14-fold purification of LipB to >95% homogeneity in an overall yield of 25% . We also show that the protein catalyzes the transfer of the octanoyl group from octanoyl-ACP to apo-H protein, which is the lipoyl bearing subunit of the glycine cleavage system . The specific activity of the purified protein is 0.541Umg(-1), indicating a turnover number of approximately 0.2s(-1), and the apparent K(m) values for octanoyl-ACP and apo-H protein are 10.2+/-4.4 and 13.2+/-2.9muM, respectively.

Protein Expr Purif, 2005 Feb, 39(2), 254 - 60
Heterologous expression of hexaheme fragments of a multidomain cytochrome from Geobacter sulfurreducens representing a novel class of cytochromes c; Londer YY et al.; Multiheme cytochromes c are of great interest for researchers for a variety of reasons but difficult to obtain in quantities sufficient for the majority of studies . The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes . Three of them represent a new class of multiheme cytochromes characterized by a mixed type of heme coordination and multidomain organization . We cloned and expressed in Escherichia coli three hexaheme fragments corresponding to two-domain fragments of one such protein containing 12 heme binding motifs and believed to consist of four triheme domains . Despite high sequence similarity among the fragments, expression levels varied significantly . Expression was optimized either by host strain variation or by reducing the rate of apoprotein synthesis . All three fragments were purified by cation exchange followed by gel filtration and were shown to contain six covalently attached hemes as confirmed by mass spectrometry . Their visible spectra are typical of c-type cytochromes . One of the fragments was crystallized and its preliminary X-ray structure shows two separate domains.

Protein Expr Purif, 2005 Feb, 39(2), 209 - 218
Characterization of a recombinant granzyme B derivative as a "restriction" protease; Fynbo CH et al.; Blood coagulation factor X(a) (FX(a)) and Thrombin are well-known serine proteases often used for processing of recombinant fusion proteins, but because they are purified from bovine blood or other animal sources, there is a risk of pathogenic contaminants in the preparation of the proteases . We report here the characterization of a recombinant serine protease produced in Escherichia coli, which can be used as a specific and efficient alternative to FX(a) and Thrombin as processing protease . This recombinant protease is derived from human granzyme B (GrB) . The protease is found to be very stable in general, and it performs very well in the cleavage of several different fusion proteins tested and was even found superior to processing by FX(a) in two cases.

Protein Expr Purif, 2005 Feb, 39(2), 199 - 208
Comparative evaluation of two purification methods of anti-CD19-c-myc-His(6)-Cys scFv; Das D et al.; Different chromatographic methods have been used to purify bacterially expressed single chain antibodies in soluble or insoluble form . Here, we compared two methods for purification of anti-CD19-c-myc-His(6)-Cys scFv expressed in Escherichia coli as soluble protein . The protein-L-agarose purification method is a one step purification method that yielded significant amounts of pure protein compared to the two-step Ni-NTA-agarose plus Resource 15S purification method . However, the protein-L purification method exhibited an additional lower molecular weight protein contaminant . Based on results from in vitro gel digestion, mass spectrometry and database search results, we confirmed that the lower molecular weight protein contaminant, which could not be purified by Ni-NTA-agarose and 15S column method, is a degraded product of the full length scFv construct.

Protein Expr Purif, 2005 Feb, 39(2), 184 - 8
Cloning, expression, and refolding of a secretory protein ESAT-6 of Mycobacterium tuberculosis; Wang BL et al.; A DNA encoding the 6-kDa early secretory antigenic target (ESAT-6) of Mycobacterium tuberculosis was inserted into a bacterial expression vector of pQE30 resulting in a 6x His-esat-6 fusion gene construction . This plasmid was transformed into Escherichia coli strain M15 and effectively expressed . The expressed fusion protein was found almost entirely in the insoluble form (inclusion bodies) in cell lysate . The inclusion bodies were solubilized with 8M urea or 6M guanidine-hydrochloride at pH 7.4, and the recombinant protein was purified by Ni-NTA column . The purified fusion protein was refolded by dialysis with a gradient of decreasing concentration of urea or guanidine hydrochloride or by the size exclusion protein refolding system . The yield of refolded protein obtained from urea dialysis was 20 times higher than that from guanidine-hydrochloride . Sixty-six percent of recombinant ESAT-6 was successfully refolded as monomer protein by urea gradient dialysis, while 69% of recombinant ESAT-6 was successfully refolded as monomer protein by using Sephadex G-200 size exclusion column . These results indicate that urea is more suitable than guanidine-hydrochloride in extracting and refolding the protein . Between the urea gradient dialysis and the size exclusion protein refolding system, the yield of the monomer protein was almost the same, but the size exclusion protein refolding system needs less time and reagents.

Protein Expr Purif, 2005 Feb, 39(2), 137 - 143
High level expression and single-step purification of hexahistidine-tagged l-2-hydroxyisocaproate dehydrogenase making use of a versatile expression vector set; Chatterjee S et al.; Affinity tags as fusions to the N- or C-terminal part of proteins are valuable tools to facilitate the production and purification of proteins . In many cases, there may be the necessity to remove the tag after protein preparation to regain activity . Removal of the tag is accomplished by insertion of a unique amino acid sequence that is recognized and cleaved by a site specific protease . Here, we report the construction of an expression vector set that combines N- or C-terminal fusion to either a hexahistidine tag or Streptag with the possibility of tag removal by factor Xa or recombinant tobacco etch virus protease (rTEV), respectively . The vector set offers the option to produce different variants of the protein of interest by cloning the corresponding gene into four different Escherichia coli expression vectors . Either immobilized metal affinity chromatography or streptactin affinity chromatography can be used for the one-step purification . Furthermore, we show the successful application of the expression vector for C-terminal hexahistidine tagging . The expression and purification of His-tagged l-2-hydroxyisocaproate dehydrogenase yields fully active enzyme . The tag removal is here accomplished by a derivative of rTEV.

Protein Expr Purif, 2005 Feb, 39(2), 131 - 136
Expression, refolding, purification, and bioactivity of recombinant bifunctional protein, hIL-2/GM-CSF; Wang QR et al.; Interleukin-2 (IL-2) can stimulate T cell proliferation and differentiation when binding to its receptor on T cells . It produces a marked effect by enhancing the cytotoxicity of CD8(+) T cells and natural killer cells . Granulocyte-macrophage colony stimulating factor (GM-CSF) is associated with many cells proliferation, such as dendritic cells, macrophages . Here, we report the construction, expression and purification of a bifunctional protein, hIL-2/GM-CSF, which may facilitate interaction between T cells and the antigen presentation cells and improve the efficiency of antigen presentation . We found that the use of chemicals and temperature shift is a peculiar system for induction of the Escherichia coli transformed with an IPTG-regulated hIL-2/GM-CSF expression vector in this research . After renaturation, anion exchange chromatography, metal affinity chromatography, and strict endotoxin-free cation exchange chromatography, the fusion protein devoid of endotoxin showed high purity . Cell proliferation experiments proved that this bifunctional protein retains both hIL-2 and GM-CSF biological activities . These results will facilitate the numerous subsequent studies on this bifunctional molecule.

FEBS Lett, 2005 Jan 17, 579(2), 523 - 8
Involvement of ATP synthase residues alphaArg-376, betaArg-182, and betaLys-155 in Pi binding; Ahmad Z et al.; alphaArg-376, betaLys-155, and betaArg-182 are catalytically important ATP synthase residues that were proposed to be involved in substrate Pi binding and subsequent steps of ATP synthesis {Senior, A.E., Nadanaciva, S . and Weber, J . (2002) Biochim . Biophys . Acta 1553, 188-211} . Here, it was shown using purified Escherichia coli F(1)-ATPase that whereas Pi protected wild-type from reaction with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, mutations betaK155Q, betaR182Q, betaR182K, and alphaR376Q abolished protection . Therefore, in ATP synthesis initial binding of substrate Pi in open catalytic site betaE is supported by each of these three residues.

FEBS Lett, 2005 Jan 17, 579(2), 469 - 72
The biosynthetic routes for carbon monoxide and cyanide in the Ni-Fe active site of hydrogenases are different; Roseboom W et al.; The incorporation of carbon into the carbon monoxide and cyanide ligands of {NiFe}-hydrogenases has been investigated by using (13)C labelling in infrared studies of the Allochromatium vinosum enzyme and by (14)C labelling experiments with overproduced Hyp proteins from Escherichia coli . The results suggest that the biosynthetic routes of the carbon monoxide and cyanide ligands in {NiFe}-hydrogenases are different.

BMC Biotechnol . 2005 Jan 11;5(1):1 {Epub ahead of print}
Short description of an alternative and easier method for screening recombinant clones within the "AdEasy-System" by Duplex-PCR; Antolovic D et al.; BACKGROUND: Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery . They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression . The AdEasy system is a widely used system for generating recombinant adenoviral vectors, which are created with a minimum of enzymatic manipulations and by employing homologous recombination in E . coli . In this paper we describe an alternative simplified method for screening recombinant DNA within the AdEasy system . This Duplex-PCR-method is independent of the transgene or insert and can be used for the complete AdEasy system . It is characterized by a simple standard protocol and the results can be obtained within a few hours . The PCR is run with two different primer sets . The primers KanaFor and KanaRev hybridizise with the Kanamycin resistence gene and AdFor and AdRev detect the adenoviral backbone . In case of recombinant clones, two diagnostic fragments with a size of 384 bp and 768 bp are generated . RESULTS: The practicability of this method was verified with three different transgenes: Cytosin Deaminase (AdCD), p53 (Adp53) and Granulocyte Macrophage Colony Stimulating Factor (AdGM-CSF) . Recombinant clones are indicated by two diagnostic fragments and are then suitable for further processing . CONCLUSION: In summary, the presented protocol allows fast detection of recombinants with an easy technique by minimizing the amount of necessary steps for generating a recombinant adenovirus . This method is time sparing and cost-effective.

Genome Biol . 2005;6(1):R2 . Epub 2004 Dec 22.
Computational prediction of human metabolic pathways from the complete human genome; Romero P et al.; BACKGROUND: We present a computational pathway analysis of the human genome that assigns enzymes encoded therein to predicted metabolic pathways . Pathway assignments place genes in their larger biological context, and are a necessary first step toward quantitative modeling of metabolism . RESULTS: Our analysis assigns 2,709 human enzymes to 896 bioreactions; 622 of the enzymes are assigned roles in 135 predicted metabolic pathways . The predicted pathways closely match the known nutritional requirements of humans . This analysis identifies probable omissions in the human genome annotation in the form of 203 pathway holes (missing enzymes within the predicted pathways) . We have identified putative genes to fill 25 of these holes . The predicted human metabolic map is described by a Pathway/Genome Database called HumanCyc, which is available at We describe the generation of HumanCyc, and present an analysis of the human metabolic map . For example, we compare the predicted human metabolic pathway complement to the pathways of Escherichia coli and Arabidopsis thaliana and identify 35 pathways that are shared among all three organisms . CONCLUSIONS: Our analysis elucidates a significant portion of the human metabolic map, and also indicates probable unidentified genes in the genome . HumanCyc provides a genome-based view of human nutrition that associates the essential dietary requirements of humans with a set of metabolic pathways whose existence is supported by the human genome . The database places many human genes in a pathway context, thereby facilitating analysis of gene expression, proteomics, and metabolomics datasets through a publicly available online tool called the Omics Viewer.

Biochemistry, 2005 Jan 18, 44(2), 782 - 9
Role of the N-Terminal Domain of the Calcitonin Receptor-like Receptor in Ligand Binding; Chauhan M et al.; Calcitonin receptor-like receptor (CRLR) is a seven-transmembrane (7-TM) domain class B G protein-coupled receptor (GPCR) which requires coexpression of different receptor activity modifying proteins (RAMP) to become a functional calcitonin gene-related peptide (CGRP) receptor or an adrenomedullin (AM) receptor . The N-terminal (Nt) extracellular region of class B GPCRs in ligand binding has been reported for receptors such as glucagon and parathyroid hormone . We hypothesize that the Nt-domain of CRLR (Nt-CRLR) is an autonomously folded unit possessing a well-defined structure and is involved in ligand binding and specificity . To obtain structural and functional information on the Nt-CRLR, we cloned and expressed the Nt-CRLR as a fusion protein in Escherichia coli . Overexpressed protein formed an inclusion body, which was refolded and purified, resulting in a soluble monomeric protein . Far-UV CD and fluorescence spectra of Nt-CRLR showed characteristics of a folded protein . The ability of Nt-CRLR to bind CGRP and AM independent of RAMPs was determined by studying inhibition of (125)I-CGRP and (125)I-AM binding to pregnant rat uterine membrane in the presence of Nt-CRLR protein . We observe that Nt-CRLR inhibits (125)I-CGRP and (125)I-AM binding to rat uterus in a dose-dependent fashion (IC(50) = 0.25 and 0.29 muM, respectively) . Taken together, our data provide evidence that Nt-CRLR is structured and further that a significant part of the binding affinity comes from binding to the Nt-domain.

Biochemistry, 2005 Jan 18, 44(2), 766 - 74
Mechanism of Action of Escherichia coliPhosphoribosylaminoimidazolesuccinocarboxamide Synthetase; Nelson SW et al.; The conversion of ATP, l-aspartate, and 5-aminoimidazole-4-carboxyribonucleotide (CAIR) to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP, and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represents the eighth step of de novo purine nucleotide biosynthesis . SAICAR synthetase and other enzymes of purine biosynthesis are targets of natural products that impair cell growth . Prior to this study, no kinetic mechanism was known for any SAICAR synthetase . Here, a rapid equilibrium random ter-ter kinetic mechanism is established for the synthetase from Escherichia coli by initial velocity kinetics and patterns of linear inhibition by IMP, adenosine 5'-(beta,gamma-imido)triphosphate (AMP-PNP), and maleate . Substrates exhibit mutual binding antagonism, with the strongest antagonism between CAIR and either ATP or l-aspartate . CAIR binds to the free enzyme up to 200-fold more tightly than to the ternary enzyme-ATP-aspartate complex, but the latter complex may be the dominant form of SAICAR synthetase in vivo . IMP is a competitive inhibitor with respect to CAIR, suggesting the possibility of a hydrogen bond interaction between the 4-carboxyl and 5-amino groups of enzyme-bound CAIR . Of several aspartate analogues tested (hadacidin, l-malate, succinate, fumarate, and maleate), maleate was by far the best inhibitor, competitive with respect to l-aspartate . Inhibition by IMP and maleate is consistent with a chemical mechanism for SAICAR synthetase that parallels that of adenylosuccinate synthetase.

Biochemistry, 2005 Jan 18, 44(2), 537 - 545
Assembly of Human Frataxin Is a Mechanism for Detoxifying Redox-Active Iron; O'neill HA et al.; Mitochondrial function depends on a continuous supply of iron to the iron-sulfur cluster (ISC) and heme biosynthetic pathways as well as on the ability to prevent iron-catalyzed oxidative damage . The mitochondrial protein frataxin plays a key role in these processes by a novel mechanism that remains to be fully elucidated . Recombinant yeast and human frataxin are able to self-associate in large molecular assemblies that bind and store iron as a ferrihydrite mineral . Moreover, either single monomers or polymers of human frataxin have been shown to serve as donors of Fe(II) to ISC scaffold proteins, oxidatively inactivated {3Fe-4S}(+) aconitase, and ferrochelatase . These results suggest that frataxin can use different molecular forms to accomplish its functions . Here, stable monomeric and assembled forms of human frataxin purified from Escherichia coli have provided a tool for testing this hypothesis at the biochemical level . We show that human frataxin can enhance the availability of Fe(II) in monomeric or assembled form . However, the monomer is unable to prevent iron-catalyzed radical reactions and the formation of insoluble ferric iron oxides . In contrast, the assembled protein has ferroxidase activity and detoxifies redox-active iron by sequestering it in a protein-protected compartment.

J Huazhong Univ Sci Technolog Med Sci, 2004, 24(5), 414 - 6
Expression and purification of SARS coronavirus membrane protein; Dai W et al.; To construct a recombinant plasmid Pet23a-M, the gene encoding severe acute respiratory syndrome (SARS) coronavirus membrane protein was amplified by RT-PCR and cloned into the expression plasmid Pet23a . Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis revealed that the cloned DNA sequence was the same as that reported . The recombinants were transformed into Escherichia coli (E . Coli) BL21 (DE3) and induced by Isopropyl-beta-D-thiogalactopyranoside (IPTG) . The expression of 27 kD (1 kD=0.992 1 ku) protein was detected by SDS-PAGE and pured by metal chelated chromatography . Results of Western-blot showed that this expressed protein could react with antibodies in sera of SARS patients during convalescence . This provided the basis for the further study on SARS virus vaccine and diagnostic agents.

Cell Mol Biol (Noisy-le-grand), 2004 Sep, 50(6), 737 - 47
Cloning and identification of Rab cDNAs from Limulus polyphemus; Cao Z et al.; Small GTPases of the Rab family are essential for the control of membrane transport between intracellular compartments . Trafficking of the sodium-dependent facilitative insulin responsive glucose transporter (GLUT 4) has been shown to be associated with the intracellular redistribution of Rabs 4, 5 and 11 in adipose and muscle tissues . As a prelude to studies of the endosomal trafficking of the choline cotransporter (ChCoT), we describe herein our initial efforts to identify Rab proteins in Limulus polyphemus central nervous system (CNS) tissue . The studies were initiated after results from Microarray analysis of Limulus RNA hybridized to mouse gene chips suggested the presence of RNA transcripts for Rab 7 protein . Subsequently, more than 30 sequences for different Rab proteins were aligned and several consensus segments were selected for degenerate primer design to produce Rabs 2, 4, 7, 9 and 11 . The expected PCR fragment sizes were obtained using RT-PCR and subcloned into pCR II TOPO vector and transferred into E . coli Top 10 . The nucleotide sequences indicated that the recombinants encoded partial amino acid sequences for Rabs 1a, 1b, 1c, 2, 2a, 2b, 3a, 4, 5a, 7a, 7b, 11a, 11b, 14, 33b1 and 33b2 . Northern blot analyses showed that the molecular sizes of Limulus Rabs 3a, 4, 7, 11a and 11b ranged from approximately 1.94.6 Kb . These Rab proteins, particularly Rabs 4, 7 and 11, will be studied further to determine their possible roles in the trafficking of the Limulus ChCoT

World J Gastroenterol, 2005 Jan 28, 11(4), 614 - 5
Unrecognized pylephlebitis causing life-threatening septic shock: A case report; Wireko M et al.; A man who developed profound septic shock was treated for Escherichia coli sepsis of unknown origin . Following stabilisation, a diagnosis of pylephlebitis (infection and thrombosis in the portal vein) was made at computed tomography . A review of the condition, its primary causes, typical features, investigation and management was presented.

World J Gastroenterol, 2005 Jan 28, 11(4), 492 - 7
Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus; Yang HJ et al.; AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier . METHODS: One to 6 tandem copies of HBV preS1 (21-47) fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E.coli . ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens . The ability to capture HBV by antibodies elicited by chimeric particles was detected with immuno-capture PCR . RESULTS: Recombinant antigens CI, CII, CIII carrying 1-3 copies of HBV preS1 (21-47) individually could form virus-like particles (VLPs), similar to HBcAg in morphology . But recombinant antigens carrying 4-6 copies of HBV preS1 (21-47) were poorly expressed in E.coli . Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs . CI, CII, CIII could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs . Three chimeric VLP antigens (CI, CII and CIII) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47) . Mouse antibodies to CI, CII and CIII were able to capture HBV virions in immuno-capture PCR assay in vitro . CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1 . They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

Nat Struct Mol Biol . 2005 Jan 10; {Epub ahead of print}
The yeast DASH complex forms closed rings on microtubules; Miranda JL et al.; The Saccharomyces cerevisiae DASH complex is an essential microtubule-binding component of the kinetochore . We coexpressed all ten subunits of this assembly in Escherichia coli and purified a single complex, a ~210-kDa heterodecamer with an apparent stoichiometry of one copy of each subunit . The hydrodynamic properties of the recombinant assembly are indistinguishable from those of the native complex in yeast extracts . The structure of DASH alone and bound to microtubules was visualized by EM . The free heterodecamer is relatively globular . In the presence of microtubules, DASH oligomerizes to form rings and paired helices that encircle the microtubules . We discuss potential roles for such collar-like structures in maintaining microtubule attachment and spindle integrity during chromosome segregation.

Appl Environ Microbiol, 2005 Jan, 71(1), 451 - 9
Phenotypic Screening of Escherichia coli K-12 Tn5 Insertion Libraries, Using Whole-Genome Oligonucleotide Microarrays; Winterberg KM et al.; Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments . In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn5 insertions in Escherichia coli K-12 . Libraries of insertion mutants generated by a unique Tn5 mutagenesis system were grown competitively in defined media . Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants . These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population . By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium . It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the E . coli chromosome were identified . Tn5 insertions were detected in 15 genes for which no previous insertions have been reported . Other applications of this method are discussed.

Yi Chuan, 2004 Jul, 26(4), 460 - 4
{Preparation and specificity analysis of anti-Drosophila dxl6 antibody.}; Wang SZ et al.; In order to study the function of Dxl6 which is a novel member of SR protein family, its cDNA was cloned by RT-PCR, and the sequences of its RS domain and its middle part were subcloned into two fusion express vectors, pGEX-4T-1His(6)C and pET32a.After expressing in E.coli BL21, the truncated proteins of Dxl6 RS domain part and Dxl6 middle part in pGEX-4T-1His(6)C were purified and used to immunize rabbits.Purified antibodies against the RS domain and Dxl6 middle part were obtained by affinity chromatography with the expressed products of Dxl6 RS domain part and Dxl6 middle part in pET32a, respectively.The result shows the antibody against Dxl6 RS domain has a good specificity to Dxl6 in Drosophila larvae by Western blot analysis.

Yi Chuan, 2003 Nov, 25(6), 685 - 90
{Cloning and fusion expression of bovine enterokinase light chain gene in Escherichia coli.}; Huang H et al.; The objective of the study was to obtain the gene of bovine enterokinase light chain,which would be used in the cleavage and purification of fusion proteins.The fragment of bovine enterokinase light chain cDNA was obtained by RT-PCR from a sold bovine's duodenal mucosa, then cloned into the pUCmT cloning vector and sequenced.Compared with the sequence deposited in GenBank,the cloned gene sequence is correct.Then the interested gene fragment was inserted into the pET39b expression plasmid.The recombinant vector pET39b-EKL was transformed into E.coli BL21(DE3) and induced by IPTG.It was confirmed that the nucleotide sequence was correct on the conjunction site between the recombinant DNA 5'terminal multi-cloning site and recombinant fragment after the analysis of the nucleotide sequence.SDS-PAGE analysis indicated that target product was about 65 kDa which occupied 28% of the total protein.A pure fusion protein was obtained by nickel chelating chromatogram using His*Binding Resin.After desalting and changing buffer,the crude kinase was incubated at 21 degrees overnight and demonstrated a high autocatalytic cleavage activity.This investigation would be able to lay a foundation for enterokinase activity research and farther application of expression products on a large scale.

Arch Biochem Biophys, 2005 Feb 15, 434(2), 275 - 81
Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans; Lee BC et al.; Methionine (Met) residues in proteins are susceptible to oxidation . The resulting methionine sulfoxide can be reduced back to methionine by methionine sulfoxide-S-reductase (MsrA) . The MsrA gene, isolated from Caenorhabditis elegans, was cloned and expressed in Escherichia coli . The resultant enzyme was able to revert both free Met and Met in proteins in the presence of either NADPH or dithiothreitol (DTT) . However, approximately seven times higher enzyme activity was observed in the presence of DTT than of NADPH . The enzyme had an absolute specificity for the reduction of l-methionine-S-sulfoxide but no specificity for the R isomer . K(m) and k(cat) values for the enzyme were approximately 1.18mM and 3.64min(-1), respectively . Other kinetics properties of the enzyme were also evaluated.

Arch Biochem Biophys, 2005 Feb 15, 434(2), 221 - 31
Peroxynitrite-mediated oxidation of the C85S/C152E mutant of dihydrofolate reductase from Escherichia coli: functional and structural effects; Pucciarelli S et al.; Peroxynitrite is a potent reactive oxygen species that is believed to mediate deleterious protein modifications in a wide variety of neurodegenerative disorders . In this study, we have analysed the effects of oxidative damage induced by peroxynitrite on a cysteine-free mutant of dihydrofolate reductase (SE-DHFR), from a functional and a structural point of view . The peroxynitrite-mediated oxidation results in the inhibition, concentration-dependent, of the catalytic activity . This effect is strongly influenced by the HCO(3)(-)/CO(2) buffering system, that we observed to significantly affect the yield of protein oxidation by modulating the peroxynitrite-induced modification of aromatic residues . Because of this effect, in presence of bicarbonate system, we have observed a protection of enzymatic activity of SE-DHFR with regard to peroxynitrite . The thermodynamic stability of the oxidized protein has been studied in comparison with the non-oxidized protein by differential scanning calorimetry . The thermodynamic parameters obtained showed a decrease of stability of SE-DHFR upon oxidation, evaluated in terms of Gibbs free energy of about 1.25kcal/mol at 25 degrees C, with respect to the non-oxidized protein . Together, these data indicate that structural and functional alterations induced by peroxynitrite may play a direct role in compromising DHFR function in multiple pathological conditions.

Immunobiology, 2004, 209(8), 619 - 27
Effect of ovarian hormones on periodical changes in immune resistance associated with estrous cycle in the beagle bitch; Sugiura K et al.; In bitches, the onset of pyometra, an infection of the uterus, characteristically occurs in the first half of the diestrous stage in the estrous cycle, in which the blood concentration of progesterone peaks and that of estradiol-17beta is lowest . To investigate the immunological mechanisms governing stage-specific onset of pyometra, peripheral blood mononuclear cells (PBMNCs) were collected from beagle bitches during different stages of the estrous cycle and examined using various immunological assays . When we examined the proliferative response of PBMNCs to PYO-252, that is a clone of Escherichia coli isolated from the uterus of a dog afflicted with pyometra, the response of PBMNCs significantly decreased in the first half (day 10) of diestrus, but increased in proestrus/estrus . No significant differences were observed in the responses to concanavaline A between stages of the cycle . Throughout the estrous cycle, canine PBMNCs did not respond to lipopolysaccharide derived from E . coli . The response of PBMNCs collected in anestrus to PYO-252 was significantly enhanced upon the addition of estradiol-17beta to the culture . In contrast, these responses were significantly suppressed in the presence of progesterone . Progesterone progenitor or metabolite molecules, which have a low affinity for the progesterone receptor, did not affect proliferative responses . Expression of gamma interferon (IFNgamma) in response to PYO-252 was also significantly enhanced by estradiol-17beta, but suppressed by progesterone . This evidence suggests that in the first half of the diestrous stage, suppressed activity of cellular immunity results from increasing progesterone concentration and minimal estrogen release . This marked decrease of immune resistance allows the expansion of E . coli, which enter the uterine cavity through the loosened cervical canal during estrus, leading to pyometra onset.

Science, 2005 Jan 7, 307(5706), 113 - 7
Disulfide isomerization after membrane release of its SAR domain activates P1 lysozyme; Xu M et al.; The P1 lysozyme Lyz is secreted to the periplasm of Escherichia coli and accumulates in an inactive membrane-tethered form . Genetic and biochemical experiments show that, when released from the bilayer, Lyz is activated by an intramolecular thiol-disulfide isomerization, which requires a cysteine in its N-terminal SAR (signal-arrest-release) domain . Crystal structures confirm the alternative disulfide linkages in the two forms of Lyz and reveal dramatic conformational differences in the catalytic domain . Thus, the exported P1 endolysin is kept inactive by three levels of control-topological, conformational, and covalent-until its release from the membrane is triggered by the P1 holin.

Proc Natl Acad Sci U S A . 2005 Jan 6; {Epub ahead of print}
Solution NMR-derived global fold of a monomeric 82-kDa enzyme; Tugarinov V et al.; The size of proteins that can be studied by solution NMR spectroscopy has increased significantly because of recent developments in methodology . Important experiments include those that make use of approaches that increase the lifetimes of NMR signals or that define the orientation of internuclear bond vectors with respect to a common molecular frame . The advances in NMR techniques are strongly coupled to isotope labeling methods that increase sensitivity and reduce the complexity of NMR spectra . We show that these developments can be exploited in structural studies of high-molecular-weight, single-polypeptide proteins, and we present the solution global fold of the monomeric 723-residue (82-kDa) enzyme malate synthase G from Escherichia coli, which has been extensively characterized by NMR in the past several years.

J Biol Chem . 2005 Jan 6; {Epub ahead of print}
Characteristics of the polyamine transporter TPO1 and regulation of its activity and cellular localization by phosphorylation; Uemura T et al.; The subcellular localization of the polyamine transporter TPO1 of Saccharomyces cerevisiae was determined by sucrose gradient centrifugation and indirect immunofluorescence microscopy . When expressed from a multi-copy vector, TPO1 was mainly located on the plasma membrane, but with some localization on the vacuolar membrane . Polyamine transport by TPO1 was dependent on pH . Uptake of spermidine and spermine occurred at alkaline pH (8.0), whereas inhibition of spermidine uptake, but not spermine uptake, was observed at acidic pH (5.0) . This suggests that TPO1 catalyzes polyamine excretion at acidic pH, similar to the PotE transporter in Escherichia coli . Paraquat, a polyamine analogue, was excreted by TPO1 at a rate comparable to the excretion of spermidine (deduced from the inhibition of spermidine uptake) at pH 5.0 . However, excretion of preloaded radiolabeled spermidine and spermine was not observed in intact cells, suggesting that preloaded spermidine (or spermine) mainly exists as spermidine (or spermine)-ribosome complex in cells . The transport activity of TPO1 was enhanced through phosphorylation at Ser19 by protein kinase C and at Thr52 by casein kinase 1 . Sorting of TPO1 from the endoplasmic reticulum to the plasma membrane was enhanced through phosphorylation at Ser342 by cAMP-dependent protein kinases 1 and 2.

J Biol Chem . 2005 Jan 6; {Epub ahead of print}
Nonequivalence of the nucleotide binding domains of the ArsA ATPase; Jiang Y et al.; The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance . The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker . Each half contains a nucleotide binding domain . The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding . The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5'-{alpha-32P}triphosphate at 4 degrees C indicate that metalloid stimulation correlates with a >10-fold increase in affinity for nucleotide . To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker . This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA . The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid . Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain . The ATP analogue labeled with 32P at the position was to measure hydrolysis of trinucleotide at 37 degrees C . Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2 . These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent.

J Biol Chem . 2005 Jan 6; {Epub ahead of print}
alpha -Amylase is not required for breakdown of transitory starch in arabidopsis leaves; Yu TS et al.; The Arabidopsis thaliana genome encodes three alpha-amylase-like proteins (AtAMY1, AtAMY2 and AtAMY3) . Only AtAMY3 has a predicted N-terminal transit peptide for plastidial localization, as would be expected of an enzyme involved in breakdown of starch in chloroplasts . AtAMY3 is an unusually large alpha-amylase (93.5 kD) with the C-terminal half showing similarity to other known alpha-amylases . When expressed in E . coli, both the whole AtAMY3 protein and the C-terminal half alone show alpha-amylase activity . The N-terminal domain shares similarity with the N-terminal (non-catalytic) region of glucan, water dikinase (GWD1), a protein that phosphorylates amylopectin and is required for starch breakdown . We show that AtAMY3 is localized in chloroplasts . The starch-excess mutant of Arabidopsis sex4, previously shown to have reduced plastidial alpha-amylase activity, is deficient in AtAMY3 protein . Unexpectedly, T-DNA knockout mutants of AtAMY3 have the same diurnal pattern of transitory starch metabolism as the wild type . These results show that AtAMY3 is not required for transitory starch breakdown and that the starch-excess phenotype of the sex4 mutant is not caused simply by deficiency of AtAMY3 protein . Knockout mutants in the predicted non-plastidial alpha-amylases AtAMY1 and AtAMY2 were also isolated, and these displayed normal starch breakdown in the dark, as expected for extra-plastidial amylases . Furthermore, all three AtAMY double-knockout mutant combinations and the triple knockout degraded their leaf starch normally . We conclude that alpha-amylase is not necessary for transitory starch breakdown in Arabidopsis leaves.

Biochem J . 2005 Jan 7; {Epub ahead of print}
Molecular evidence that melatonin is enzymatically oxidized in a different manner than tryptophan . Investigation on both indoleamine-2,3-dioxygenase and myeloperoxidase; Ferry G et al.; Melatonin catabolism can be accounted for ~70 % by conjugation (sulfo- and glucurono-conjugations) and ~30 % by oxidation . It is commonly thought that the interferon-induced enzyme, indoleamine-2,3-dioxygenase (E.C . 1.13.11.11), which oxidizes tryptophan, is also responsible for the oxidation of serotonin and its derivative, melatonin . Using the recombinant enzyme expressed in E . coli, we show, in the present work, that indoleamine-2,3-dioxygenase indeed cleaves tryptophan, but using the same enzymatic conditions, it is incapable of cleaving the two other indoleamines . By contrast, myeloperoxidase (E.C . 1.11.1.7) is capable of cleaving the indole moiety of melatonin . Using peroxidase conditions of assay - with H 2O 2 as co-substrate - indoleamine-2,3-dioxygenase is nevertheless capable to cleave melatonin into its main metabolite, a kynurenine derivative . The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H 2O 2, to both myeloperoxidase, and indoleamine-2,3-dioxygenase with poorer potency, since both enzymes have K m values for melatonin in the muM range . Under those conditions, several indolic compounds can be cleaved off by both enzymes, such as tryptamine and serotonin . Furthermore, its metabolism leads to a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.

Shi Yan Sheng Wu Xue Bao, 2004 Oct, 37(5), 375 - 83
{hHO-1 structure prediction and its mutant construct, expression, purification and activity analysis}; Xia ZW et al.; Human Heme Oxygenase-1 (hHO-1) is the rate-limiting enzyme in the catabolism reaction of heme, which directly regulates the concentration of bilirubin in human body . The mutant structure was simulated by Swiss-pdbviewer procedure, which showed that the structure of active pocket was changed distinctly after Ala25 substituted for His25 in active domain, but the mutated enzyme still binded with heme . On the basis of the results, the expression vectors, pBHO-1 and pBHO-1(M), were constructed, induced by IPTG and expressed in E . coli DH5alpha strain . The expression products were purified with 30%-60% saturation (NH4)2SO4 and Q-Sepharose Fast Flow column chromatography . The concentration of hHO-1 in 30%-60% saturation (NH4)2SO4 components and in fractions through twice column chromatography was 3.6-fold and 30-fold higher than that in initial product, respectively . The activity of wild hHO-1 (whHO-1) and mutant hHO-1 (deltahHO-1) showed that the activity of deltahHO-1 was reduced 91.21% compared with that of whHO-1 . The study shows that His25 is of importance for the mechanism of hHO-1, and provides the possibility for effectively regulating the activity to exert biological function.

Zh Mikrobiol Epidemiol Immunobiol, 2004 Nov-Dec, (6), 76 - 80
{Use of recombinant HBcore antigen for development of the diagnostic test system}; Expression and immunological characterization of the carboxy-terminal region of the P1 adhesin protein of Mycoplasma pneumoniae; Department of Microbiology, All India Institute of Medical Sciences, New Delhi, India . rc123@hotmail.com

Mycoplasma pneumoniae is the causative agent of primary atypical pneumonia in humans . Adherence of M . pneumoniae to host cells requires several adhesin proteins, such as P1, P30, and P116 . A major limitation in developing a specific diagnostic test for M . pneumoniae is the inability to express adhesin proteins in heterologous expression systems due to unusual usage of the UGA stop codon, leading to premature termination of these proteins in Escherichia coli . In the present study, we successfully expressed the C-terminal (P1-C1) and N-terminal (P1-N1) regions of the P1 protein in E . coli . On screening these recombinant proteins with sera from M . pneumoniae-infected patients, only the P1-C1 protein was found to be immunogenic . This protein can be used as an antigen for immunodiagnosis of M . pneumoniae infection, as well as in adherence inhibition studies to understand the pathophysiology of the disease.

J Clin Microbiol, 2005 Jan, 43(1), 57 - 65
Assessing the serodiagnostic potential of 35 Mycobacterium tuberculosis proteins and identification of four novel serological antigens; Weldingh K et al.; Improved diagnostic reagents are needed for the detection of Mycobacterium tuberculosis infections, and the development of a serodiagnostic test would complement presently available diagnostic methods . The aim of the present study was to identify novel serological targets for use for the future serodiagnosis of tuberculosis (TB) . We cloned and expressed 35 M . tuberculosis proteins as recombinant proteins in Escherichia coli and analyzed their serodiagnostic potentials . By a two-step selection process, four superior seroantigens, TB9.7, TB15.3, TB16.3, and TB51, were identified, none of which has been described before . The four novel antigens were tested with panels of sera from smear-positive and smear-negative TB patients from areas both where TB is endemic and where TB is not endemic, with recognition frequencies ranging from 31 to 93% and with a specificity of at least 97% . The single most potent antigen was TB16.3, which had a sensitivity of 48 to 55% with samples from Danish resident TB patients and a sensitivity of 88 to 98% with samples from African TB patients . Importantly, the TB16.3 and the TB9.7 antigens were recognized by more than 85% of the samples from TB patients coinfected with human immunodeficiency virus, a patient group for which it is in general difficult to detect M . tuberculosis-specific antibodies.

J Immunol, 2005 Jan 15, 174(2), 1091 - 6
Phospholipids Inhibit Lipopolysaccharide (LPS)-Induced Cell Activation: A Role for LPS-Binding Protein; Mueller M et al.; The inhibition of LPS-induced cell activation by specific antagonists is a long-known phenomenon; however, the underlying mechanisms are still poorly understood . It is commonly accepted that the membrane-bound receptors mCD14 and TLR4 are involved in the activation of mononuclear cells by LPS and that activation may be enhanced by soluble LPS-binding protein (LBP) . Hexaacylated Escherichia coli lipid A has the highest cytokine-inducing capacity, whereas lipid A with four fatty acids (precursor IVa, synthetic compound 406) is endotoxically inactive, but expresses antagonistic activity against active LPS . Seeking to unravel basic molecular principles underlying antagonism, we investigated phospholipids with structural similarity to compound 406 with respect to their antagonistic activity . The tetraacylated diphosphatidylglycerol (cardiolipin, CL) exhibits high structural similarity to 406, and our experiments showed that CL strongly inhibited LPS-induced TNF-alpha release when added to the cells before stimulation or as a CL/LPS mixture . Also negatively charged and to a lesser degree zwitterionic diacyl phospholipids inhibited LPS-induced cytokine production . Using Abs against LBP, we could show that the activation of cells by LPS was dependent on the presence of cell-associated LBP, thus making LBP a possible target for the antagonistic action of phospholipids . In experiments investigating the LBP-mediated intercalation of LPS and phospholipids into phospholipid liposomes mimicking the macrophage membrane, we could show that preincubation of soluble LBP with phospholipids leads to a significant reduction of LPS intercalation . In summary, we show that LBP is a target for the inhibitory function of phospholipids.

FEBS J, 2005 Jan, 272(1), 217 - 27
Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1; Ball T et al.; Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species . We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells . Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain . This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens . Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a folded species with significant secondary structure . This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution . The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1 . Moreover, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients . Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1 . This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.

J Med Chem, 2005 Jan 13, 48(1), 256 - 261
Beta-glucuronidase-cleavable prodrugs of O6-benzylguanine and O6-benzyl-2'-deoxyguanosine; Wei G et al.; Glucuronic acid linked prodrugs of O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine were synthesized . The prodrugs were found to be quite stable at physiological pH and were more than 200-fold less active as inactivators of O(6)-alkylguanine-DNA alkyltransferase (alkyltransferase) than either O(6)-benzylguanine or O(6)-benzyl-2'-deoxyguanosine . Beta-glucuronidase from both Escherichia coli and bovine liver cleaved the prodrugs efficiently to release O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine, respectively . In combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), the prodrugs were not effective adjuvants for HT29 cell killing . However, as expected, incubation of these prodrugs with beta-glucuronidase in the culture medium led to much more efficient cell killing by BCNU as a result of the liberation of the more potent inactivators, O(6)-benzylguanine and O(6)-benzyl-2'-deoxyguanosine . These prodrugs may be useful for prodrug monotherapy of necrotic tumors that liberate beta-glucuronidase or for antibody-directed enzyme prodrug therapy with antibodies that can deliver beta-glucuronidase to target tumor cells.

Cell Stress Chaperones, 2004 Winter, 9(4), 332 - 43
Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon); Lo WY et al.; We cloned the complementary deoxyribonucleic acid (cDNA) of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon) . It was 2207 bp long and included a 1959-bp coding region, a 40-bp flanking region at the 5' end, and a 208-bp flanking region at the 3' end . The deduced, 652-amino acid sequence had a molecular mass of 71 481 Da and an estimated isoelectric point (pI) of 5.2 . Based on phylogenetic analysis, the gene is clustered with the hsc70 proteins of invertebrates and vertebrates . In native gel electrophoresis, recombinant P . monodon hsc70 expressed in an Escherichia coli system is tightly associated with carboxymethylated alpha-lactalbumin (CMLA), which indicates that hsc70 probably functions as a chaperone . In an in vitro adenosine triphosphatase assay, recombinant hsc70 hydrolyzed adenosine triphosphate to adenosine-5'-diphosphate and increased hydrolysis activity by binding to unfolded peptide, CMLA . In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp . The expression of hsc70 messenger ribonucleic acid (mRNA) in hemocytes increased 2- to 3-fold at the first hour after shrimp experienced heat shock and 0.5-hour recovery . Hsc70 mRNA decreased gradually to the background level . Cloning and characterizing the P . monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins with the stress or immune responses of shrimp.

Biochem Biophys Res Commun, 1976 Dec 20, 73(4), 903 - 10
Oligonucleotides containing modified bases . I . Inhibition of DNA and RNA polymerases by partially thiolated oligocytidylic acids; Ho YK et al.; A series of oligomers of cytidylic acid were prepared and partially (6-9% of the bases) thiolated in the 5 positions . The modified oligomers showed increasing inhibition with increasing chain length of both the DNA polymerase-alpha from regenerating rat liver and the DNA-dependent RNA polymerase of E . coli, but the minimum chain length for observable inhibitory activity was 5 nucleotide units for the DNA polymerase-alpha and 16 units for the RNA polymerase.

Microbiology, 2005 Jan, 151(Pt 1), 281 - 90
DNA-binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912; Rebets Y et al.; The gene lndI is involved in the pathway-specific positive regulation of biosynthesis of the antitumour polyketide landomycin E in Streptomyces globisporus 1912 . LndI was overexpressed in Escherichia coli as a protein C-terminally fused to the intein-chitin-binding-domain tag and purified in a one-step column procedure . Results of in vivo LndI titration, DNA gel mobility-shift assays and promoter-probing experiments indicate that LndI is an autoregulatory DNA-binding protein that binds to its own gene promoter and to the promoter of the structural gene lndE . Enhanced green fluorescent protein was used as a reporter to study the temporal and spatial pattern of lndI transcription . Expression of lndI started before cells entered mid-exponential phase and peak expression coincided with maximal accumulation of landomycin E and biomass . In solid-phase analysis, lndI expression was evident in substrate mycelia but was absent from aerial hyphae and spores.

Microbiology, 2005 Jan, 151(Pt 1), 69 - 74
Can genetically modified Escherichia coli with neutral buoyancy induced by gas vesicles be used as an alternative method to clinorotation for microgravity studies?
Benoit M, Klaus D.
Space flight has been shown to affect various bacterial growth parameters . It is proposed that weightlessness allows the cells to remain evenly distributed, consequently altering the chemical makeup of their surrounding fluid, and hence indirectly affecting their physiological behaviour . In support of this argument, ground-based studies using clinostats to partially simulate the quiescent environment attained in microgravity have generally been successful in producing bacterial growth characteristics that mimic responses reported under actual space conditions . A novel approach for evaluating the effects of reduced cell sedimentation is presented here through use of Escherichia coli cultures genetically modified to be neutrally buoyant . Since clinorotation would not (or would only minimally) affect cell distribution of this already near-colloidal cell system, it was hypothesized that the effects on final population density would be eliminated relative to a static control . Gas-vesicle-producing E . coli cultures were grown under clinostat and static conditions and the culture densities at 60 h were compared . As a control, E . coli that do not produce gas vesicles, but were otherwise identical to the experimental strain, were also grown under clinostat and static conditions . As hypothesized, no significant difference was observed in cell populations at 60 h between the clinorotated and static gas-vesicle-producing E . coli cultures, while the cells that did not produce gas vesicles showed a mean increase in population density of 10.5 % (P=0.001) . These results further suggest that the lack of cumulative cell sedimentation is the dominant effect of space flight on non-stirred, in vitro E . coli cultures.

Microbiology, 2005 Jan, 151(Pt 1), 15 - 23
Influence of the age and sex of human hosts on the distribution of Escherichia coli ECOR groups and virulence traits; Gordon DM et al.; Escherichia coli were isolated from the faeces of 266 individuals living in the Canberra region of Australia . The isolates were characterized for their ECOR group membership (A, B1, B2 or D) and for the presence of 29 virulence-associated traits . Overall, 19.5 % of the strains were members of group A, 12.4 % B1, 45.1 % B2 and 22.9 % D . The frequency with which strains belonging to the four ECOR groups were observed varied with the age and sex of the hosts from which they were isolated . In males, the probability of isolating A or D strains increased with host age, whilst the probability of detecting a group B2 strain declined . In females, the probability of recovering A or B2 strains increased with increasing host age and there was a concomitant decline in the likelihood of isolating B1 or D strains . Of the 29 virulence-associated traits examined, 24 were detected in more than one strain . The likelihood of detecting most traits varied with a strain's ECOR membership, with the exception of afa/draBC, astA, cvaC, eaeA, iss and iutA, for which there was no statistically significant evidence of an association with ECOR group . The frequency with which fimH, iha, eaeA, iroN, hlyD, iss, ompT and K1 were detected in a strain depended on the age or sex of the host from which the strain was isolated . In group B2 strains many of the virulence traits were non-randomly associated, with some co-occurring in a strain less often than expected by chance, whilst others were co-associated . In 17 cases, the extent to which two virulence traits were co-associated was found to depend on host sex and age . The results of this study suggest that the morphological, physiological and dietary differences that occur among human individuals of different sex or age may influence the distribution of E . coli genotypes.

J Biochem (Tokyo), 2004 Nov, 136(5), 711 - 722
A Comprehensive Study on the Immunological Reactivity of the Hsp90 Molecular Chaperone; Kawano T et al.; Periodontitis is a chronic infectious disease, Porphyromonas gingivalis being the most implicated pathogen . In the present study, we investigated the role of P . gingivalis HtpG (PgHtpG), a bacterial ortholog of mammalian Hsp90, in the growth of P . gingivalis and also assessed the immunological cross-reactivity of the members of the Hsp90 family . Antiserum against rat liver Hsp90 potently reacted with yeast Hsp90, called Hsc82, and also weakly with human Hsp90 (hHsp90) and human mitochondrial paralog Trap1, but did not react with PgHtpG, Escherichia coli HtpG, or human endoplasmic reticulum paralog Grp94 . Moreover, among 19 monoclonal antibodies raised against hHsp90, nine cross-reacted with yeast Hsc82, and one with human Grp94, but none bound to PgHtpG or E . coli HtpG . Among them, three mAbs that strongly reacted with yeast Hsc82 recognized Asn(291)-Ile(304), a conserved region of the family protein . The polyclonal antibody raised against a peptide, Met(315)-Glu(328), of human Grp94, which corresponded to the conserved region of hHsp90, cross-reacted with hHsp90, but not with other Hsp90-family members . Thus, although mammalian Hsp90 shares some immunological reactivity with yeast Hsc82, human Grp94, and human Trap1, it is considerably distinct from its bacterial ortholog, HtpG . Disruption of the P . gingivalis htpG gene neither affected bacterial survival nor altered the sensitivity of P . gingivalis to various forms of stress.

J Biochem (Tokyo), 2004 Nov, 136(5), 693 - 9
Kinetic Analysis of Precursor M1 RNA Molecules for Exploring Substrate Specificity of the N-Terminal Catalytic Half of RNase E; Kim KS et al.; To gain insight into the mechanism by which the sequence at the rne-dependent site of substrate RNA affects the substrate specificity of Escherichia coli RNase E, we performed kinetic analysis of the cleavage of precursor M1 RNA molecules containing various sequences at the rne-dependent site by the N-terminal catalytic half of RNase E (NTH-RNase E) . NTH-RNase E displayed higher K(m) and k(cat) values for more specific substrates . The retention of single strandedness at the rne-dependent site was essential for cleavage efficiency . Moreover, the loss of single-strandedness was accompanied by a decrease in both the K(m) and k(cat) values.

J Biochem (Tokyo), 2004 Nov, 136(5), 673 - 81
Identification of Oligopeptidase B in Higher Plants . Purification and Characterization of Oligopeptidase B from Quiescent Wheat Embryo, Triticum aestivum; Tsuji A et al.; Proteolytic enzymes in general, and cysteine proteases in particular, play key roles in seed germination and early seedling growth . However, the precise mechanism by which the serine proteases are regulated remains unclear . Trypsin-like activity was detected in wheat germ (quiescent embryo) and this activity increased in the germinating embryo . In this work, a trypsin-like serine protease expressed in wheat germ was purified to homogeneity by chromatography through DEAE-cellulose, phenyl-Sepharose, Ultrogel AcA-34 and Blue-Sepharose . The molecular mass of the enzyme was estimated to be 81 kDa by SDS-PAGE under reducing conditions . Amino acid analysis of the peptides generated following digestion of the enzyme with lysyl endopeptidase indicated that the enzyme is a plant homologue of Escherichia . coli oligopeptidase B . The subsite specificity of the enzymes differ, although both enzymes hydrolyze synthetic substrates and model peptides at the carboxyl side of basic amino acids . The wheat enzyme is more sensitive to leupeptin and antipain than the E . coli emzyme . These results provide the basis for characterizing plant oligopeptidase B and contribute to our understanding of its role in the early development of seedlings.

J Biochem (Tokyo), 2004 Nov, 136(5), 595 - 600
Oxygen Equilibrium and EPR Studies on {alpha}1{beta}1 Hemoglobin Dimer; Venkatesh B et al.; We undertook this project to clarify whether hemoglobin (Hb) dimers have a high affinity for oxygen and cooperativity . For this, we prepared stable Hb dimers by introducing the mutation Trp-->Glu at beta37 using our Escherichia coli expression system at the alpha1beta2 interface of Hb, and analyzed their molecular properties . The mutant hybrid Hbs with a single oxygen binding site were prepared by substituting Mg(II) protoporphyrin for ferrous heme in either the alpha or beta subunit, and the oxygen binding properties of the free dimers were investigated . Molecular weight determination of both the deoxy and CO forms showed all these molecules to be dimers in the absence of IHP at different protein concentrations . Oxygen equilibrium measurements showed high affinity and non-cooperative oxygen binding for all mutant Hb and hybrid Hb dimers . However, EPR results on the {alpha(N)(Fe-NO)beta(M)(Mg)} hybrid showed some alpha1beta1 interactions . These results provide some clues as to the properties of Hb dimers, which have not been studied extensively owing to practical difficulties in their preparation.

Protein Sci . 2005 Jan 4; {Epub ahead of print}
Regulation of protein activity with small-molecule-controlled inteins; Skretas G et al.; Inteins are the protein analogs of self-splicing RNA introns, as they post-translationally excise themselves from a variety of protein hosts . Intein insertion abolishes, in general, the activity of its host protein, which is subsequently restored upon intein excision . These protein elements therefore have the potential to be used as general molecular "switches" for the control of arbitrary target proteins . Based on rational design, an intein-based protein switch has been constructed whose splicing activity is conditionally triggered in vivo by the presence of thyroid hormone or synthetic analogs . This modified intein was used in Escherichia coli to demonstrate that a number of different proteins can be inactivated by intein insertion and then reactivated by the addition of thyroid hormone via ligand-induced splicing . This conditional activation was also found to occur in a dose-dependent manner . Rational protein engineering was then combined with genetic selection to evolve an additional intein whose activity is controlled by the presence of synthetic estrogen ligands . The ability to regulate protein function post-translationally through the use of ligand-controlled intein splicing will most likely find applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, as well as many additional areas of biotechnology.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
alpha -methylacyl-CoA racemase from mycobacterium tuberculosis-mutational and structural characterization of the active site and the fold; Savolainen K et al.; alpha-Methylacyl-CoA racemase (Amacr)catalyzes the racemization of alpha-methyl-branched CoA esters . Sequence comparisons have shown that this enzyme is a member of the family III CoA transferases . The mammalian Amacr is involved in bile acid synthesis and branched-chain fatty acid degradation . In human, mutated variants of Amacr have been shown to be associated with disease states . Amino acid sequence alignment of Amacrs and its homologues from various species revealed 26 conserved protic residues, assumed to be potential candidates as catalytic residues . Amacr from Mycobacterium tuberculosis (MCR) was taken as a representative of the racemases . In order to determine their importance for efficient catalysis, each of the 26 protic residues of MCR was mutated into an alanine, respectively, and the mutated variants were overexpressed in E . coli . It was found that four variants (R91A, H126A, D156A, and E241A) were properly folded, but had much decreased catalytic efficiency . Apparently, Arg91, His126, Asp156 and Glu241 are important catalytic residues of MCR . The importance of these residues for catalysis can be rationalized by the 1.8A resolution crystal structure of MCR, which shows that the catalytic site is at the interface between the large and small domain of two different subunits of the dimeric enzyme . This crystal structure is the first structure of a complete enzyme of the bile acid synthesis pathway . It shows that MCR has unique structural features, not seen in the structures of the sequence related formyl-CoA transferases, suggesting that the family III CoA transferases can be subdivided in at least two classes, being racemases and CoA transferases.

J Biol Chem . 2005 Jan 4; {Epub ahead of print}
In vitro molybdenum ligation to molybdopterin using purified components; Nichols JD et al.; We have previously shown that Escherichia coli MoeA and MogA are required in vivo for the final step of molybdenum cofactor biosynthesis, the addition of the molybdenum atom to the dithiolene of molybdopterin . MoeA was also shown to facilitate the addition of molybdenum in an assay using crude extracts from E . coli moeA- cells . The experiments detailed in this report an in vitro assay for MoeA-mediated molybdenum ligation to de novo synthesized molybdopterin using only purified components and monitoring the reconstitution of human apo-sulfite oxidase . In this assay, maximum activation was achieved by delaying the addition of apo-sulfite oxidase to allow for adequate molybdenum coordination to occur . Tungsten, which substitutes for molybdenum in hyperthermophilic organisms, could also be ligated to molybdopterin using this system, though not as efficiently Mo . Addition of thiol compounds to the assay inhibited activity . Addition of MogA also inhibited the reaction . However, in the presence of ATP and magnesium, addition of MogA to the assay increased the level of apo-sulfite oxidase reconstitution beyond that observed with MoeA alone . This effect was not observed in the absence of MoeA . The results presented here demonstrate that MoeA is responsible for mediating molybdenum ligation to molybdopterin, while MogA stimulates this activity in an ATP-dependent manner.

Plant Cell, 2005 Jan, 17(1), 233 - 40
Identification of a Vinyl Reductase Gene for Chlorophyll Synthesis in Arabidopsis thaliana and Implications for the Evolution of Prochlorococcus Species; Nagata N et al.; Chlorophyll metabolism has been extensively studied with various organisms, and almost all of the chlorophyll biosynthetic genes have been identified in higher plants . However, only the gene for 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR), which is indispensable for monovinyl chlorophyll synthesis, has not been identified yet . In this study, we isolated an Arabidopsis thaliana mutant that accumulated divinyl chlorophyll instead of monovinyl chlorophyll by ethyl methanesulfonate mutagenesis . Map-based cloning of this mutant resulted in the identification of a gene (AT5G18660) that shows sequence similarity with isoflavone reductase genes . The mutant phenotype was complemented by the transformation with the wild-type gene . A recombinant protein encoded by AT5G18660 was expressed in Escherichia coli and found to catalyze the conversion of divinyl chlorophyllide to monovinyl chlorophyllide, thereby demonstrating that the gene encodes a functional DVR . DVR is encoded by a single copy gene in the A . thaliana genome . With the identification of DVR, finally all genes required for chlorophyll biosynthesis have been identified in higher plants . Analysis of the complete genome of A . thaliana showed that it has 15 enzymes encoded by 27 genes for chlorophyll biosynthesis from glutamyl-tRNA(glu) to chlorophyll b . Furthermore, identification of the DVR gene helped understanding the evolution of Prochlorococcus marinus, a marine cyanobacterium that is dominant in the open ocean and is uncommon in using divinyl chlorophylls . A DVR homolog was not found in the genome of P . marinus but found in the Synechococcus sp WH8102 genome, which is consistent with the distribution of divinyl chlorophyll in marine cyanobacteria of the genera Prochlorococcus and Synechococcus.

Zhonghua Yi Xue Za Zhi, 2004 Nov 17, 84(22), 1904 - 8
{Preparation of human phage antibodies specific for SSA/Ro antigen and its sequence analysis.}; Li YJ et al.; OBJECTIVE: To prepare monoclonal antibodies (mAbs) specific for SSA/Ro antigen from the phage-displayed human single-chain Fv (scFv) antibody library constructed previously, and analyze its gene sequence . METHODS: Frozen strain of Escherichia coli TG1 containing pHEN2-scFv was defrosted and infected by helper phage to produce scFv phage antibody library . 10 random clones from the primary library were checked for the presence of inserts by PCR screening . Four clones containing exogenous gene with correct length were selected randomly to undergo gene sequencing . The diversity of the library was monitored by digesting the PCR products of 8 different phagemids encoding scFv genes . The phage antibody library was biopanned for 3 cycles, using purified SSA/Ro antigen . After biopanning, the binding characterization and the specificity of the enriched library to SSA/Ro antigen was assayed by ELISA . mAbs were obtained from this enriched library and ELISA was used to detect the binding characterization and the specificity of these mAbs, using different purified antigens . The variable region of the monoclonal antibody undergoes sequencing . RESULTS: A phage scFv library with the titer of 1.6 x 10(12) cfu/ml was established . Sample screening showed that eight of the ten clones contained scFv fragments . The insertion and recombination rate of exogenous gene was 80% . These 8 clones were also analyzed by restriction enzyme analysis to assess the diversity of the library . Each of the 8 clones showed a distinct restriction pattern . Sequencing results demonstrated that the genes of scFv were the human antibody genes, and the gene ligating and cloning were correct and successful . The library was subjected to 3 rounds of rescue and panning . A secondary phage antibody library against SSA/Ro was generated and the interest phages were obviously enriched . The 3rd panned library showed good reaction to the SSA/Ro antigen with higher OD value than unenriched library demonstrated by ELISA . The result of ELISA showed that the enriched library reacted specifically to SSA/Ro and had no cross-reactivity to the other autoantigen . 5 mAbs specific for SSA/Ro antigen could be selected from 52 clones, and the gene sequences of their VH and VL were coded by VH1, VH3, VH4, Vkappa1, Vkappa2, and Vkappa3 family genes with somatic mutations . CONCLUSION: The primary scFv phage antibody library fits the need for later operations, and this library was enriched successfully . Enriched scFv phage antibody library is specific for SSA/Ro antigen, from which 5 monoclonal anti-SSA/Ro phage antibodies can be obtained successfully . The sequences of these mAbs show somatic mutations comparing with germline . It may help understand the pathogenesis of some antibody-mediated diseases.

J Am Chem Soc, 2005 Jan 12, 127(1), 325 - 30
Bifunctional small molecules are biomimetic catalysts for silica synthesis at neutral pH; Roth KM et al.; Silicatein is an enzyme isolated from the biosilica produced by the marine demosponge, Tethya aurantia . Once isolated from the sponge, silicatein can be used in vitro to catalyze the hydrolysis and direct polycondensation of a wide variety of alkoxide, ionic, and organometallic precursors to the corresponding chalcogens at standard temperature and pressure and neutral pH . On the basis of these results, an array of small molecules that mimic the unique physiochemical environment found in the enzyme active site was investigated for catalytic activity in the formation of silica from silicon alkoxides at neutral pH . The most successful of these biomimetic catalysts (cysteamine) was used to encapsulate firefly luciferase, green and blue fluorescent proteins (GFP, BFP), and Escherichia coli cells expressing GFP in silica matrixes . The benign conditions required for the catalysis of synthesis of these silica composites does not impair the activities of the encapsulated enzyme, fluorescent proteins, or live cells as shown by fluorescence measurements . In conjunction with microcontact printing, this biomimetically catalyzed encapsulation method has been used to produce patterned functional arrays of silica nanoparticulate composite materials.

J Am Chem Soc, 2005 Jan 12, 127(1), 146 - 57
Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism; Magliery TJ et al.; Identification of protein binding partners is one of the key challenges of proteomics . We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides . Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments . Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (K(D) approximately 1 mM) . In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions . Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains . These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis . Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.

J Membr Biol, 2004 Sep 15, 201(2), 97 - 107
Importance of conserved acidic residues in mntH, the Nramp homolog of Escherichia coli; Haemig HA et al.; A bioinformatic approach was used for the identification of residues that are conserved within the Nramp family of metal transporters . Site-directed mutagenesis was then carried out to change six conserved acidic residues (i.e., Asp-34, Glu-102, Asp-109, Glu-112, Glu-154, and Asp-238) in the E . coli Nramp homolog mntH . Of these six, five of them, Asp-34, Glu-102, Asp-109, Glu-112, and Asp-238 appear to be important for function since conservative substitutions at these sites result in a substantial loss of transport function . In addition, all of the residues within the signature sequence of the Nramp family, DPGN, were also mutated in this study . Each residue was changed to several different side chains, and of ten site-directed mutations made in this motif, only P35G showed any measurable level of (54)Mn(2+) uptake with a V(max) value of approximately 10% of wild-type and a slightly elevated K(m) value . Overall, the data are consistent with a model where helix breakers in the conserved DPGN motif in TMS-1 provide a binding pocket in which Asp-34, Asn-37, Asp-109, Glu-112 (and possibly other residues) are involved in the coordination of Mn(2+) . Other residues such as Glu-102 and Asp238 may play a role in the release of Mn(2+) to the cytoplasm or may be involved in maintaining secondary structure.

Biofactors, 2004, 21(1-4), 79 - 82
Characterization of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin); Suzuki Y et al.; gamma-Terpinene is a monoterpene and a major component of essential oils made from citrus fruits and shows strong antioxidant activity in various assay systems . Plant gamma-terpinene synthase is a member of the monoterpene cyclase family, which produces a specific monoterpene through cyclization of geranyl diphosphate (GPP), but the monoterpene cyclases have not been fully characterized . It is necessary to prepare large amounts of gamma-terpinene synthase from Citrus unshiu (Satsuma mandarin) for the characterization, on this purpose we expressed the protein in Escherichia coli (E . coli) cells . As most monoterpene synthases have plastid-targeting signals, a gene lacking these signals was prepared and functionally expressed in E . coli cells harboring extra copies of the argU gene . The purified enzyme was incubated with GPP and the main product was confirmed to begamma-terpinene by GC/MS.

Genes Dev, 2005 Jan 1, 19(1), 127 - 37
Premature targeting of a cell division protein to midcell allows dissection of divisome assembly in Escherichia coli; Goehring NW et al.; Cell division in Escherichia coli requires the recruitment of at least 10 essential proteins to the bacterial midcell . Recruitment of these proteins follows a largely linear dependency pathway in which depletion of one cell division protein leads to the absence from the division site of "downstream" proteins in the pathway . Analysis of events that underlie this pathway is complicated by the fact that a protein's ability to recruit "downstream" proteins is dependent on its own recruitment by "upstream" proteins . Hence, one cannot separate the individual contributions of various upstream proteins to any specific recruitment step . Here we present a method--premature targeting--for bypassing the normal localization requirements of a cell division protein and apply it to FtsQ, a protein recruited midway through the pathway . We fused FtsQ to the FtsZ-binding protein ZapA such that FtsQ was targeted to FtsZ rings independently of proteins FtsA and FtsK, which are normally required for FtsQ localization . Analysis of the resulting ZapA-FtsQ fusion suggests that FtsQ associates with a large complex of cell division proteins and that premature targeting of FtsQ can restore localization of this complex under conditions in which neither FtsQ nor the associated proteins would normally be localized.

J Bacteriol, 2005 Jan, 187(2), 672 - 86
The Cpx Envelope Stress Response Affects Expression of the Type IV Bundle-Forming Pili of Enteropathogenic Escherichia coli; Nevesinjac AZ et al.; The Cpx envelope stress response mediates adaptation to potentially lethal envelope stresses in Escherichia coli . The two-component regulatory system consisting of the sensor kinase CpxA and the response regulator CpxR senses and mediates adaptation to envelope insults believed to result in protein misfolding in this compartment . Recently, a role was demonstrated for the Cpx response in the biogenesis of P pili, attachment organelles expressed by uropathogenic E . coli . CpxA senses misfolded P pilus assembly intermediates and initiates increased expression of both assembly and regulatory factors required for P pilus elaboration . In this report, we demonstrate that the Cpx response is also involved in the expression of the type IV bundle-forming pili of enteropathogenic E . coli (EPEC) . Bundle-forming pili were not elaborated from an exogenous promoter in E . coli laboratory strain MC4100 unless the Cpx pathway was constitutively activated . Further, an EPEC cpxR mutant synthesized diminished levels of bundle-forming pili and was significantly affected in adherence to epithelial cells . Since type IV bundle-forming pili are very different from chaperone-usher-type P pili in both form and biogenesis, our results suggest that the Cpx envelope stress response plays a general role in the expression of envelope-localized organelles with diverse structures and assembly pathways.

J Bacteriol, 2005 Jan, 187(2), 567 - 75
Introns in the Cytolethal Distending Toxin Gene of Actinobacillus actinomycetemcomitans; Tan KS et al.; In eukaryotic cells, genes are interrupted by intervening sequences called introns . Introns are transcribed as part of a precursor RNA that is subsequently removed by splicing, giving rise to mature mRNA . However, introns are rarely found in bacteria . Actinobacillus actinomycetemcomitans is a periodontal pathogen implicated in aggressive forms of periodontal disease . This organism has been shown to produce cytolethal distending toxin (CDT), which causes sensitive eukaryotic cells to become irreversibly blocked at the G(2)/M phase of the cell cycle . In this study, we report the presence of introns within the cdt gene of A . actinomycetemcomitans . By use of reverse transcription-PCR, cdt transcripts of 2.123, 1.572, and 0.882 kb (RTA1, RTA2, and RTA3, respectively) were detected . In contrast, a single 2.123-kb amplicon was obtained by PCR with the genomic DNA . Similar results were obtained when a plasmid carrying cdt was cloned into Escherichia coli . Sequence analysis of RTA1, RTA2, and RTA3 revealed that RTA1 had undergone splicing, giving rise to RTA2 and RTA3 . Two exon-intron boundaries, or splice sites, were identified at positions 863 to 868 and 1553 to 1558 of RTA1 . Site-directed and deletion mutation studies of the splice site sequence indicated that sequence conservation was important in order for accurate splicing to occur . The catalytic region of the cdt RNA was located within the cdtC gene . This 0.56-kb RNA behaved independently as a catalytically active RNA molecule (a ribozyme) in vitro, capable of splicing heterologous RNA in both cis and trans configurations.

J Bacteriol, 2005 Jan, 187(2), 507 - 11
The N5-Glutamine S-Adenosyl-L-Methionine-Dependent Methyltransferase PrmC/HemK in Chlamydia trachomatis Methylates Class 1 Release Factors; Pannekoek Y et al.; The gene prmC, encoding the putative S-adenosyl-l-methionine (AdoMet)-dependent methyltransferase (MTase) of release factors (RFs) of the obligate intracellular pathogen Chlamydia trachomatis, was functionally analyzed . Chlamydial PrmC expression suppresses the growth defect of a prmC knockout strain of Escherichia coli K-12, suggesting an interaction of chlamydial PrmC with E . coli RFs in vivo . In vivo methylation assays carried out with recombinant PrmC and RFs of chlamydial origin demonstrated that PrmC methylates RFs within the tryptic fragment containing the universally conserved sequence motif Gly-Gly-Gln . This is consistent with the enzymatic properties of PrmC of E . coli origin . We conclude that C . trachomatis PrmC functions as an N(5)-glutamine AdoMet-dependent MTase, involved in methylation of RFs.

J Bacteriol, 2005 Jan, 187(2), 458 - 72
The LEE1 Promoters from both Enteropathogenic and Enterohemorrhagic Escherichia coli Can Be Activated by PerC-Like Proteins from Either Organism; Porter ME et al.; The PerC protein of enteropathogenic Escherichia coli (EPEC), encoded by the pEAF plasmid, is an activator of the locus of enterocyte effacement (LEE) pathogenicity island via the LEE1 promoter . It has been assumed that the related LEE-containing pathogen enterohemorrhagic E . coli (EHEC) lacks PerC-dependent activation due to utilization of an alternative LEE1 promoter and lack of a perC gene . However, we show here that EPEC PerC can activate both the EPEC and EHEC LEE1 promoters and that the major transcriptional start site is similarly located in both organisms . Moreover, a PerC-like protein family identified from EHEC genome analyses, PerC1 (also termed PchABC), can also activate both promoters in a manner similar to that of EPEC PerC . The perC1 genes are carried by lambdoid prophages, which exist in multiple copies in different EHEC strains, and have a variable flanking region which may affect their expression . Although individual perC1 copies appear to be poorly expressed, the total perC1 expression level from a strain encoding multiple copies approaches that of perC in EPEC and may therefore contribute significantly to LEE1 activation . Alignment of the protein sequences of these PerC homologues allows core regions of the PerC protein to be identified, and we show by site-directed mutagenesis that these core regions are important for function . However, purified PerC protein shows no in vitro binding affinity for the LEE1 promoter, suggesting that other core E . coli proteins may be involved in its mechanism of activation . Our data indicate that the nucleoid-associated protein IHF is one such protein.

J Bacteriol, 2005 Jan, 187(2), 449 - 57
Error-Prone DNA Polymerase IV Is Regulated by the Heat Shock Chaperone GroE in Escherichia coli; Layton JC et al.; An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation . The groE operon consists of two genes, groES and groEL; point mutations in either gene conferred the same phenotype, reducing Lac(+) adaptive mutation 10- to 20-fold . groE mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV) . In recG(+) strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in recG mutant strains, a deficiency of GroE had some additional effect on adaptive mutation . Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA . We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly . Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.

J Bacteriol, 2005 Jan, 187(2), 443 - 8
Identification of Chlamydia trachomatis Genomic Sequences Recognized by Chlamydial Divalent Cation-Dependent Regulator A (DcrA); Rau A et al.; The Chlamydia trachomatis divalent cation-dependent regulator (DcrA), encoded by open reading frame CT296, is a distant relative of the ferric uptake regulator (Fur) family of iron-responsive regulators . Chlamydial DcrA specifically binds to a consensus Escherichia coli Fur box and is able to complement an E . coli Fur mutant . In this report, the E . coli Fur titration assay (FURTA) was used to locate chlamydial genomic sequences that are recognized by E . coli Fur . The predictive regulatory regions of 28 C . trachomatis open reading frames contained sequences functionally recognized by E . coli Fur; targets include components of the type III secretion pathway, elements involved in envelope and cell wall biogenesis, predicted transport proteins, oxidative defense enzymes, and components of metabolic pathways . Selected FURTA-positive sequences were subsequently examined for recognition by C . trachomatis DcrA using an electrophoretic mobility shift assay . The resultant data show that C . trachomatis DcrA binds to native chlamydial genomic sequences and, overall, substantiate a functional relationship between chlamydial DcrA and the Fur family of regulators.

J Bacteriol, 2005 Jan, 187(2), 434 - 42
Starvation for Different Nutrients in Escherichia coli Results in Differential Modulation of RpoS Levels and Stability; Mandel MJ et al.; Levels of RpoS increase upon glucose starvation in Escherichia coli, which leads to the transcription of genes whose products combat a variety of stresses . RpoS stability is a key level of control in this process, as SprE (RssB)-mediated degradation is inhibited under glucose starvation . Starvation for ammonia or phosphate also results in increased stress resistance and induction of RpoS-dependent genes . However, we demonstrate that RpoS levels following ammonia starvation are only slightly increased compared to growing cells and are 10-fold below the levels observed under glucose or phosphate limitation . This difference is largely due to regulated proteolysis of RpoS, as its stability in ammonia-starved cells is intermediate between that in logarithmic-phase cells and glucose-starved cells . Use of an rpoS construct that is devoid of the gene's native transcriptional and translational control regions reveals that stability differences are sufficient to explain the different levels of RpoS observed in logarithmic phase, ammonia starvation, and glucose starvation . Under phosphate starvation, however, rpoS translation is increased . The cellular response to nutrient limitation is much more complex than previously appreciated, as there is not simply one response that is activated by starvation for any essential nutrient . Our data support the hypothesis that SprE activity is the key level at which ammonia and glucose starvation signals are transmitted to RpoS, and they suggest that carbon source and/or energy limitation are necessary for full inactivation of the SprE pathway.

Mol Cell, 2005 Jan 7, 17(1), 153 - 9
Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells; Cotta-Ramusino C et al.; The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer . The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks . We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells . Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates . Hence, Exo1-mediated fork processing resembles the action of E . coli RecJ nuclease at damaged forks . Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing . We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.

Biochim Biophys Acta, 2005 Jan 5, 1686(3), 238 - 47
Mutations associated with a congenital form of ichthyosis (NCIE) inactivate the epidermal lipoxygenases 12R-LOX and eLOX3; Yu Z et al.; Non-bullous congenital ichthyosiform erythroderma (NCIE) is one of the main clinical forms of ichthyosis . Genetic studies indicated that 12R-lipoxygenase (12R-LOX) or epidermal lipoxygenase-3 (eLOX3) was mutated in six families affected by NCIE {F . Jobard, C . Lefevre, A . Karaduman, C . Blanchet-Bardon, S . Emre, J . Weissenbach, M . Ozguc, M . Lathrop, J.F . Prud'homme, J . Fischer, Lipoxygenase-3 (ALOXE3) and 12(R)-lipoxygenase (ALOX12B) are mutated in non-bullous congenital ichthyosiform erythroderma (NCIE) linked to chromosome 17p13.1, Hum . Mol . Genet . 11 (2002) 107-113.}, but the impact of these mutations on LOX function has not been defined . To explore this, we overexpressed the wild-type or mutated enzymes in E . coli and COS7 cells and then analyzed the essential catalytic properties . We showed recently that human eLOX3 is a hydroperoxide isomerase (hepoxilin synthase) that converts the product of 12R-LOX, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE) to a specific epoxyalcohol . Using incubations with {(14)C}-labeled substrates and HPLC analyses, we found that the naturally occurring mutations totally eliminate the lipoxygenase activity of 12R-LOX and the hydroperoxide isomerase activity of eLOX3 . We further demonstrate that the 12R-LOX/eLOX3-derived 8R-hydroxy-11R,12R-epoxide is converted by an epoxide hydrolase in COS7 cells and in human keratinocytes to a single isomer of 8,11,12-trihydroxyeicosa-5,9,14-trienoic acid . Taken together, the results support the hypothesis that 12R-LOX, eLOX3, and perhaps an epoxide hydrolase function together in the normal process of skin differentiation, and that the loss of function mutations are the basis of the LOX-dependent form of NCIE.

Peptides, 2005 Feb, 26(2), 359 - 68
Cloning and characterization of a molt-inhibiting hormone-like peptide from the prawn Marsupenaeus japonicus; Ohira T et al.; Recently, it was demonstrated by PCR amplification that an additional molt-inhibiting hormone (MIH)-like peptide was present in the kuruma prawn Marsupenaeus japonicus . In this study, a cDNA encoding this peptide designated Pej-MIH-B was cloned . The Pej-MIH-B gene was expressed strongly in the nerve cord, and weakly in the eyestalk . It was possible to isolate Pej-MIH-B from the sinus glands in the eyestalks . The recombinant Pej-MIH-B expressed in Escherichia coli showed low molt-inhibiting activity, but did not exhibit hyperglycemic activity . These results suggest that Pej-MIH-B does not function as MIH or CHH intrinsically, but may have some unknown functions.

Peptides, 2005 Feb, 26(2), 343 - 9
Heterologous expression, characterization and structural studies of a hydrophobic peptide from the HIV-1 p24 protein; Castilho PV et al.; Proteins from the inner core of HIV-1, such as the capsid protein (p24), are involved in crucial processes during the virus life cycle . The p24 protein plays an active structural role in the Gag protein and in its mature form . This work describes the production of a peptide derived from the p24 C-terminal, TLRAEQASQEVKNWMTETLLVQNA, using recombinant technology . This region (p24-3) is involved in interfaces during the p24 dimerization, which occurs during capsid assembly . The p24-3 sequence was obtained by a synthetic gene strategy and inserted into the pET 32a expression vector to produce soluble fusion protein in Escherichia coli BL21(DE3) . This strategy leads to an incorporation of three amino acid residues (AMA) in the N-terminal of the native sequence to form the recombinant p24-3 (rp24-3) . The rp24-3 was purified by reverse phase chromatography to homogeneity, as inferred by mass spectrometry and protein sequence analysis . Structural studies using circular dichroism and steady-state fluorescence showed that the rp24-3 is structured by helical and beta elements . As a function of its hydrophobic character it can self-associate forming oligomers . We present in this paper the first development of a suitable expression system for rp24-3, which provides high amounts of the peptide . This strategy will allow the development of new antiviral (HIV) agents.

Biochem Biophys Res Commun, 2005 Feb 4, 327(1), 4 - 7
Correlation of codon bias measures with mRNA levels: analysis of transcriptome data from Escherichia coli; Goetz RM et al.; Although codon usage is often represented by a 61-dimensional vector, the ability of determining the codon bias in a gene relies on a uni-dimensional vector which measures the total bias in usage of synonymous codons . Codon usage is receiving more and more focus because codon biases might be valuable tools to predict and optimize gene/protein expression . How good any of these measures is for correlating codon usage with gene and protein expression has yet to be investigated . In this study, we correlated gene transcript levels in Escherichia coli with codon usage, using a number of different codon bias measures . We found that there is a significant correlation between transcript levels and codon bias measures, suggesting that these measures can be used to assess or predict gene expression . The codon bias measure performing best in this context was the codon adaptation index.

Biochem Biophys Res Commun, 2005 Feb 11, 327(2), 604 - 8
Glutamate decarboxylase-derived IDDM autoantigens displayed on self-assembled protein nanoparticles; Choi H et al.; The recombinant ferritin heavy chain (FTN-H) formed self-assembled spherical nanoparticles with the size comparable to native one . We tried to express the GAD65 COOH-terminal fragments, i.e., 448-585 (GAD65(448-585)), 487-585 (GAD65(487-585)), and 512-585 (GAD65(512-585)) amino acid fragments, using FTN-H as N-terminus fusion expression partner in Escherichia coli . All of recombinant fusion proteins (FTN-H::GAD65(448-585), FTN-H::GAD65(487-585), and FTN-H::GAD65(512-585)) also formed spherical nanoparticles due probably to the self-assembly function of the fused ferritin heavy chain . The antigenic epitopes within GAD65(448-585), GAD65(487-585), and GAD65(512-585) against insulin-dependent diabetes mellitus (IDDM) marker (autoantibodies against GAD65) were localized at the surface of the spherical protein nanoparticles so that anti-GAD65 Ab could recognize them . Protein nanoparticles like FTN-H seem to provide distinct advantages over other inorganic nanoparticles (e.g., Au, Ag, CdSe, etc.) in that through the bacterial synthesis, the active capture probes can be located at the nanoparticle surface with constant orientation/conformation via covalent cross-linking without complex chemistry . Also it is possible for the protein nanoparticles to have uniform particle size, which is rarely achieved in the chemical synthesis of inorganic nanoparticles . Thus, the recombinant ferritin particles can be used as a three-dimensional (spherical) and nanometer-scale probe structure that is a key component in ultra-sensitive protein chip for detecting protein-small molecule interactions and protein-protein interactions.

Biochem Biophys Res Commun, 2005 Feb 11, 327(2), 407 - 14
Cloning and characterization of two endoxylanases from the cereal phytopathogen Fusarium graminearum and their inhibition profile against endoxylanase inhibitors from wheat; Belien T et al.; Two genes encoding family 11 endo-beta-1,4-xylanases (XylA, XylB) from Fusarium graminearum were cloned and expressed in Escherichia coli . The amount of active endoxylanase in the cytoplasmic soluble fraction was considerably improved by varying different expression parameters, including host strain and temperature during induction . Both recombinant endoxylanases showed a temperature optimum around 35 degrees C and neutral pH optima (around pH 7 and 8 for XylB and XylA, respectively) . For the first time this allowed one to test endoxylanases of a phytopathogenic organism for inhibition by proteinaceous endoxylanase inhibitors TAXI and XIP . Whereas XylA and XylB were inhibited by TAXI-I, no inhibition activity could be detected upon incubation with XIP-I . The insensitivity of both F . graminearum endoxylanases towards XIP is surprising, since the latter is typically active against endoxylanases produced by (aerobic) fungi . As F . graminearum is an important phytopathogen, these findings have implications for the role of endoxylanase inhibitors in plant defence.

Arch Biochem Biophys, 2005 Feb 1, 434(1), 195 - 200
Studies of human mitochondrial 2,4-dienoyl-CoA reductase; Yu W et al.; Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids . Sequence alignment indicates that there are five highly conserved acidic residues, one of which might act as a proton donor . We constructed five mutant expression plasmids of human mitochondrial 2,4-dienoyl-CoA reductase using site-directed mutagenesis . Mutant proteins were overexpressed in Escherichia coli and purified with a nickel metal affinity column . Studies of these mutant proteins were carried out, and the proton donor is likely to be E276 . Three substrate analogs were synthesized and characterized . Two analogs, 2-fluoro-2,4-octadienoyl-CoA and 5-methyl-2,4-hexadienoyl-CoA, were substrates of the enzyme . Another analog, 3-furan-2-yl-acrylyl-CoA, was not a substrate, but a competitive inhibitor of the enzyme . These studies increased our understanding of human mitochondrial 2,4-dienoyl-CoA reductase.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005 Jan, 21(1), 90 - 3
{Construction and identification of recombinant adenovirus vector harboring CTLA4Ig-IRES 2-Ikappa-Balpha gene in ECV-304 cells.}; Li N et al.; AIM: To construct adenovirus vector harboring CTLA4Ig-IRES 2-IkappaBalpha gene,and investigate its expression in ECV304 cells . METHODS: Recombinant adenovirus vector harboring CTLA4Ig-IRES 2-IkappaBalpha was constructed by homologous recombination in E.coli BJ5183 . Then recombinant vector was packaged and propagated in 293 cells . ECV304 cells were infected with the recombinant adenovirus, and Western Blot was used to detect IkappaBalpha and CTLA4Ig protein expression in infected ECV304 cells . The effect of expressed IkappaBalpha and CTLA4Ig on the expression of TNF-alpha of ECV304 cells stimulated with LPS was also investigated . RESULTS: The recombinant CTLA4Ig-IRES 2-IkappaBalpha adenovirus was generated by homologous recombination and identified by PCR methods . Ikappa-Balpha and CTLA4Ig were expressed in ECV304 cells infected with the recombinant adenovirus . The production of TNF-alpha induced by the LPS was inhibited in infected ECV304 cells . CONCLUSION: The recombinant CTLA4Ig-IRES 2-IkappaBalpha adenovirus may offer a method to down regulate the inflammatory cytokines in the inflammatory reaction which may lay the fundation for genetherapy of immunosuppression.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005 Jan, 21(1), 65 - 8
{Comparison of polyclonal anti-sera against extracellular domain of hepatoma associated antigen HAb18G/CD147 prepared by different immunization schemes.}; Zhang SH et al.; AIM: To compare characteristics of polyclonal anti-sera against extracellular domain of hepatoma-associated antigen HAb18G/CD147(HAb18GEF) generated by different immunization schemes . METHODS: BALB/c mice were immunized with GST-HAb18GEF fusion protein expressed in E.coli (routine immunization method), recombinant eukaryotic expression plasmid pcDNA3/HAb18G (intramuscular injection) and pcDNA3/HAb18G plasmid followed by human hepatoma cells booster (DNA-cell booster), respectively . The titers and Ig subclasses of polyclonal anti-sera against denatured and natural HAb18GEF were detected by indirect ELISA and cell ELISA, respectivly . The specific binding of polyclonal anti-sera prepared by different immunization schemes to denatured HAb18GEF was analyzed by Western blot . The specific binding of polyclonal anti-sera produced by DNA-cell booster immunization to natural HAb18G antigen on hepatoma cells was detected by immunofluorescence staining . RESULTS: GST-HAb18GEF immunization could induce polycolonal antibody IgG1 with higher titer mainly against denatured or linear epitopes on HAb18GEF . Antibody induced by pcDNA3/HAb18G intramuscular immunization was IgG2a with lower titer against natural epitopes on HAb18GEF . DNA-cell booster immunization could induce generation of polycolonal antibody IgG2a and IgG1 of moderate titers against the native epitopes on HAb18G . CONCLUSION: The polycolonal sera with different titers against different epitopes on HAb18GEF can be induced by different immunization schemes.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005 Jan, 21(1), 6 - 8
{Cloning of mouse FasL full-length cDNA and construction of its recombinant adenovirus vector.}; Yang JH et al.; AIM: To clone mouse FasL full-length cDNA and construct recombinant adenovirus expression vector . METHODS: Mouse FasL full-length cDNA was cloned from BALB/c mouse's splenic mononuclear cells stimulated with ConA(5 mg/L)for 48 h, and then the adenovirus shuttle vector mFasL-pAdTrack containing mouse FasL full-length cDNA under the control of CMV promoter was constructed . The shuttle vector was linearized with Pme I and cotransformed into E.coli BJ5183 with pAdEasy-1, the adenovirus background plasmid vector . The positive recombinants were subsequently identified by restriction enzyme digestion, and transfected into HEK293 cells for preparing recombinant FasL adenoviruses . RESULTS: The mouse FasL full-length cDNA has been cloned and recombinant mFasL adenovirus has been constructed successfully, as shown by PCR, restriction endonuclease digestion analysis and fluorescence detection . CONCLUSION: The mouse FasL recombinant adenovirus prepared in this study can be used to study the role of FasL in tumor and autoimmune diseases.

Microb Cell Fact . 2005 Jan 4;4(1):1 {Epub ahead of print}
Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli; Sorensen HP et al.; Pure, soluble and functional proteins are of high demand in modern biotechnology . Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice . Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing . Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available . In spite of all these qualities, expression of recombinant proteins with E . coli as the host often results in insoluble and/or nonfunctional proteins . Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags.

Biochemistry, 2005 Jan 11, 44(1), 431 - 9
Conversion of the Escherichia coli Cytochrome b(562) to an Archetype Cytochrome b: A Mutant with Bis-Histidine Ligation of Heme Iron; Hay S et al.; A mutant of the Escherichia coli cytochrome b(562) has been created in which the heme-ligating methionine (Met) at position 7 has been replaced with a histidine (His) (M7H) . This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His . While the H63N mutation has no measurable effect on the cytochrome, the M7H mutation converts the atypical His/Met heme ligation in cytochrome b(562) to the classic cytochrome b-type bis-His ligation . This mutation has little effect on the K(d) of heme binding but significantly reduces the chemical and thermal stability of the mutant cytochrome relative to the wild type (wt) . Both proteins have similar absorbance (Abs) and electron paramagnetic resonance (EPR) properties characteristic of 6-coordinate low-spin heme . The Abs spectra of the oxidized and reduced bis-His cytochrome are slightly blue-shifted relative to the wt, and the alpha Abs band of ferrous M7H mutant is unusually split . The M7H mutation decreases the midpoint potential of the bound heme by 260 mV at pH 7 and considerably alters the pH dependence of the E(m), which becomes dominated by a single pK(red) = 6.8.

Biochemistry, 2005 Jan 11, 44(1), 121 - 129
EPR and X-ray Crystallographic Characterization of the Product-Bound Form of the Mn(II)-Loaded Methionyl Aminopeptidase from Pyrococcus furiosus(,); Copik AJ et al.; Methionine aminopeptidases (MetAPs) are ubiquitous metallohydrolases that remove the N-terminal methionine from nascent polypeptide chains . Although various crystal structures of MetAP in the presence of inhibitors have been solved, the structural aspects of the product-bound step has received little attention . Both perpendicular- and parallel-mode electron paramagnetic resonance (EPR) spectra were recorded for the Mn(II)-loaded forms of the type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs in the presence of the reaction product l-methionine (l-Met) . In general, similar EPR features were observed for both {MnMn(EcMetAP-I)}-l-Met and {MnMn(PfMetAP-II)}-l-Met . The observed perpendicular-mode EPR spectra consisted of a six-line hyperfine pattern at g = 2.03 (A = 8.8 mT) with less intense signals with eleven-line splitting at g = 2.4 and 1.7 (A = 4.4 mT) . The former feature results from mononuclear, magnetically isolated Mn(II) ions and this signal are 3-fold more intense in the {MnMn(PfMetAP-II)}-l-Met EPR spectrum than in the {MnMn(EcMetAP-I)}-l-Met spectrum . Inspection of the EPR spectra of both {MnMn(EcMetAP-I)}-l-Met and {MnMn(PfMetAP-II)}-l-Met at 40 K in the parallel mode reveals that the {Mn(EcMetAP-I)}-l-Met spectrum exhibits a well-resolved hyperfine split pattern at g = 7.6 with a hyperfine splitting constant of A = 4.4 mT . These data suggest the presence of a magnetically coupled dinuclear Mn(II) center . On the other hand, a similar feature was not observed for the {MnMn(PfMetAP-II)}-l-Met complex . Therefore, the EPR data suggest that l-Met binds to {MnMn(EcMetAP-I)} differently than {MnMn(PfMetAP-II)} . To confirm these data, the X-ray crystal structure of {MnMn(PfMetAP-II)}-l-Met was solved to 2.3 A resolution . Both Mn1 and Mn2 reside in a distorted trigonal bipyramidal geometry, but the bridging water molecule, observed in the {CoCo(PfMetAP-II)} structure, is absent . Therefore, l-Met binding displaces this water molecule, but the carboxylate oxygen atom of l-Met does not bridge between the two Mn(II) ions . Instead, a single carboxylate oxygen atom of l-Met interacts with only Mn1, while the N-terminal amine nitrogen atom binds to M2 . This l-Met binding mode is different from that observed for l-Met binding {CoCo(EcMetAP-I)} . Therefore, the catalytic mechanisms of type-I MetAPs may differ somewhat from type-II enzymes when a dinuclear metalloactive site is present.

Biochemistry, 2005 Jan 11, 44(1), 62 - 71
Electrostatic Interaction of a K(+) Channel RCK Domain with Charged Membrane Surfaces; Ptak CP et al.; In a subset of K(+) channels, gating is regulated through the direct binding of ligands by large cytoplasmic RCK domains . To further investigate the role of the RCK domain, we have begun the biochemical characterization of a two-transmembrane segment, RCK domain-containing channel from Methanococcus jannaschii, MjK2, by testing its general functional behavior and identifying purification conditions . Standard detergent solubilization of recombinantly expressed MjK2 required the addition of a high NaCl concentration to the extraction buffer for MjK2 solubilization . The cytoplasmic RCK domain was identified as the region of MjK2 responsible for the dependence of solubilization on high salt concentrations since expression of an MjK2 construct lacking the transmembrane domain, MjK2cd, also required high salt concentrations for extraction from Escherichia coli lipids, a necessary step in the purification of both MjK2 and MjK2cd . MjK2 expression was able to weakly recover growth of K(+) uptake deficient LB2003 cells at 10 mM KCl, suggesting that the channel can conduct K(+) but has a low open probability . Purified MjK2 reconstituted in liposomes generated only limited Rb(+) uptake, blocked by BaCl(2) . MjK2cd exhibited direct binding to PC/PS lipid vesicles with a molar partition coefficient (K(1)) of approximately 10(3) M(-)(1), which decreased with both an increase in the salt concentration and a decrease in the anionic lipid ratio . Lipid blot assays revealed that MjK2cd binds most strongly to lipids of increasingly negative charge . These results support the idea that the binding of the MjK2 RCK domain to membranes takes place via an electrostatic interaction with anionic lipid surfaces.

Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao, 2004 Oct, 30(5), 503 - 10
Cloning and expression of an alternative oxidase gene from Lycopersicon esculentum; Song CF et al.; A full-length cDNA gene (LeAox1au) was isolated from a cDNA library made from ripening fruit of tomato "UC-82B" (Lycopersicon esculentum), after probing with alternative oxidase (AOX) gene fragments, obtained by degenerate primer PCR . Sequence analysis showed that LeAox1au was 1418 bp long and contained a 1077-bp open reading frame encoding a about 40 kD precursor protein which is processed to a mature protein of 32 kD . Southern blot analysis suggested LeAox1au is present as a single copy in the genome of tomato . RT-PCR analysis indicated LeAox1au was expressed in roots, stems, leaves and cotyledons of tomato plants . A recombinant construct containing the open reading frame sequence of the LeAox1au was transformed into Escherichia coli to express the alternative oxidase precursor protein.

Biochim Biophys Acta, 2005 Jan 11, 1681(2-3), 107 - 15 Epub 2004 Nov 24.
The ornithine cycle enzyme arginase from Agaricus bisporus and its role in urea accumulation in fruit bodies; Wagemaker MJ et al.; An extensive survey of higher fungi revealed that members of the family Agaricaceae, including Agaricus bisporus, accumulate substantial amounts of urea in their fruit bodies . An important role of the ornithine cycle enzymes in urea accumulation has been proposed . In this work, we present the cloning and sequencing of the arginase gene and its promoter region from A . bisporus . A PCR-probe based on fungal arginase was used to identify the A . bisporus arginase gene from a cDNA library . The arginase cDNA encodes a 311-aa protein which is most likely expressed in the cytosol . Expression of the cDNA in Escherichia coli was established as a His-tagged fusion protein . The arginase gene was used as a molecular marker to study expression and regulation during sporophore formation and postharvest development . The expression of the arginase gene was significantly up-regulated from developmental stage 3 onwards for all the tissues studied . A maximum of expression was reached at stage 6 for both stipe and cap tissue . In postharvest stages 5, 6 and 7 the level of expression observed was similar to normal growth stages 5, 6 and 7 . A good correlation was found between arginase expression and urea content of stipe, velum, gills, cap and peel tissue . For all tissues the urea content decreased over the first four stages of development . From stage 4 onwards urea accumulated again except for stipe tissue where no significant changes were observed . The same trend was also observed for postharvest development, but the observed increase of urea in postharvest tissues was much higher.

BMC Genomics . 2005 Jan 3;6(1):1 {Epub ahead of print}
Bioinformatic mapping of AlkB homology domains in viruses; Bratlie MS et al.; BACKGROUND: AlkB-like proteins are members of the 2-oxoglutarate- and Fe(II)-dependent oxygenase superfamily . In Escherichia coli the protein protects RNA and DNA against damage from methylating agents . 1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation and direct reversal of the methylated base back to its unmethylated form . Genes for AlkB homologues are widespread in nature, and Eukaryotes often have several genes coding for AlkB-like proteins . Similar domains have also been observed in certain plant viruses . The function of the viral domain is unknown, but it has been suggested that it may be involved in protecting the virus against the post-transcriptional gene silencing (PTGS) system found in plants . We wanted to do a phylogenomic mapping of viral AlkB-like domains as a basis for analysing functional aspects of these domains, because this could have some relevance for understanding possible alternative roles of AlkB homologues e.g . in Eukaryotes . RESULTS: Profile-based searches of protein sequence libraries showed that AlkB-like domains are found in at least 22 different single-stranded RNA positive-strand plant viruses, but mainly in a subgroup of the Flexiviridae family . Sequence analysis indicated that the AlkB domains probably are functionally conserved, and that they most likely have been integrated relatively recently into several viral genomes at geographically distinct locations . This pattern seems to be more consistent with increased environmental pressure, e.g . from methylating pesticides, than with interaction with the PTGS system . CONCLUSIONS: The AlkB domain found in viral genomes is most likely a conventional DNA/RNA repair domain that protects the viral RNA genome against methylating compounds from the environment.

Filaria J . 2004 Dec 31;3(1):10 {Epub ahead of print}
Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses; Noordin R et al.; BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori . In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases . In order to characterize the homologs of the BmR1 antigen in W . bancrofti (Wb-BmR1), O . volvulus (Ov-BmR1) and L . loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied . METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W . bancrofti, O . volvulus and L . loa . The clones obtained were sequenced and compared to the cDNA sequence of BmR1 . Ov-BmR1 and Ll-BmR1 were expressed in E . coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B . malayi, W . bancrofti, O . volvulus and L . loa infections or various other parasitic infections . BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera . RESULTS: Wb-BmR1 was found to be identical to BmR1 . Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1 . The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical . This included weak IgG4 reactivities demonstrated by L . loa- and O . volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1) . With respect to reactivity to other IgG subclasses, sera from O . volvulus- and L . loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1 . No reactivity was observed with IgG2 or with IgG3 . Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B . malayi or W . bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses . CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W . bancrofti, O . volvulus and L . loa and that these antigens are highly conserved . Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies . We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis . However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.

Biochemistry (Mosc), 2004 Dec, 69(12), 1324 - 35
Kinetic Model of Imidazologlycerol-Phosphate Synthetase from Escherichia coli; Demin OV et al.; Based on the available experimental data, we developed a kinetic model of the catalytic cycle of imidazologlycerol-phosphate synthetase from Escherichia coli accounting for the synthetase and glutaminase activities of the enzyme . The rate equations describing synthetase and glutaminase activities of imidazologlycerol-phosphate synthetase were derived from this catalytic cycle . Using the literature data, we evaluated all kinetic parameters of the rate equations characterizing individually synthetase and glutaminase activities as well as the contribution of each activity depending on concentration of the substrates, products, and effectors . As shown, in the presence of 5 -phosphoribosylformimino-5-aminoimidazolo-4-carboxamideribonucleotide (ProFAR) and imidazologlycerol phosphate (IGP) glutaminase activity dominates over synthetase activity at sufficiently low concentrations of 5 -phosphoribulosylformimino-5-aminoimidazolo-4-carboxamideribonucleotide (PRFAR) . Increased PRFAR concentrations resulted in decreased contribution of glutaminase activity and, consequently, increased the contribution of synthetase activity in the enzyme functioning.

Proc Natl Acad Sci U S A, 2005 Jan 11, 102(2), 285 - 90 Epub 2004 Dec 30.
The effects of upstream DNA on open complex formation by Escherichia coli RNA polymerase; Davis CA et al.; Binding of activators to upstream DNA sequences regulates transcription initiation by affecting the stability of the initial RNA polymerase (RNAP)-promoter complex and/or the rate of subsequent conformational changes required to form the open complex (RP(O)) . Here we observe that the presence of nonspecific upstream DNA profoundly affects an early step in formation of the transcription bubble . Kinetic studies with the lambdaP(R) promoter and Escherichia coli RNAP reveal that the presence of DNA upstream of base pair -47 greatly increases the rate of forming RP(O), without significantly affecting its rate of dissociation . We find that this increase is largely due to an acceleration of the rate-limiting step (isomerization) in RP(O) formation, a step that occurs after polymerase binds . Footprinting experiments reveal striking structural differences downstream of the transcription start site (+1) in the first kinetically significant intermediate when upstream DNA is present . On the template strand, the DNase I downstream boundary of this early intermediate is +20 when upstream DNA is present but is shortened by approximately two helical turns when upstream DNA beyond -47 is removed . KMnO(4) footprinting reveals an identical initiation bubble (-11 to +2), but unusual reactivity of template strand upstream cytosines (-12, -14, and -15) on the truncated promoter . Based on this work, we propose that early wrapping interactions between upstream DNA and the polymerase exterior strongly affect the events that control entry and subsequent unwinding of the DNA start site in the jaws of polymerase.

Proc Natl Acad Sci U S A, 2005 Jan 11, 102(2), 291 - 6 Epub 2004 Dec 30.
Sequence-independent upstream DNA-{alpha}CTD interactions strongly stimulate Escherichia coli RNA polymerase-lacUV5 promoter association; Ross W et al.; The C-terminal domains of the two alpha-subunits (alphaCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters . These interactions can increase transcription dramatically . Previously, effects of upstream DNA-alphaCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements . However, contributions of nonspecific upstream DNA-alphaCTD interactions to promoter activity have not been evaluated extensively . Here, we examine effects of removal of alphaCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements . Deletion of alphaCTD decreased the composite second-order association rate constant, k(a), of RNAP for the lacUV5 promoter by approximately 10-fold . Much of this effect was attributable to a decrease in the isomerization rate constant, k(2) . Removal of promoter DNA upstream of the -35 element also decreased both k(a) and k(2) approximately 10-fold . Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with lacUV5 promoter fragments . The alphaCTD and upstream DNA did not affect dissociation rates from the open complex . We suggest that sequence-independent upstream DNA interactions with alphaCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the alpha-binding sites . In addition to highlighting the functional importance of nonspecific protein-DNA interactions, these results suggest also that UP element-alphaCTD interactions play an even larger role in transcription initiation than appreciated previously.

Biosens Bioelectron, 2005 Feb 15, 20(8), 1656 - 61
Porous silicon-based biosensor for pathogen detection; Mathew FP et al.; A porous silicon-based biosensor for rapid detection of bacteria was fabricated . Silicon (0.01ohmcm, p-type) was anodized electrochemically in an electrochemical Teflon cell containing ethanoic hydrofluoric acid solution to produce sponge-like porous layer of silicon . Anodizing conditions of 5mA/cm(2) for 85min proved best for biosensor fabrication . A single-tube chemiluminescence-based assay, previously developed, was adapted to the biosensor for detection of Escherichia coli . Porous silicon chips were functionalized with a dioxetane-Polymyxin B (cell wall permeabilizer) mixture by diffusion and adsorption on to the porous surface . The reaction of beta-galactosidase enzyme from E . coli with the dioxetane substrate generated light at 530nm . Light emission for the porous silicon biosensor chip with E . coli was significantly greater than that of the control and planar silicon chip with E . coli (P < 0.01) . Sensitivity of the porous silicon biosensor was determined to be 10(1)-10(2) colony forming units (CFU) of E . coli . The porous silicon-based biosensor was fabricated and functionalized to successfully detect E . coli and has potential applications in food and environmental testing.

Biosens Bioelectron, 2005 Feb 15, 20(8), 1482 - 90
Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes; Matsubara Y et al.; A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described . Polymerase chain reaction (PCR) mixture of only 40nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system . The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system . Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates . The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established . Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA . The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip . DNA target could be detected as small as 0.4 copies . The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software . This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit . Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.

Vet Immunol Immunopathol, 2005 Jan 10, 103(1-2), 83 - 92
Production of a monoclonal antibody to canine thymus and activation-regulated chemokine (TARC) and detection of TARC in lesional skin from dogs with atopic dermatitis; Maeda S et al.; A monoclonal antibody to canine thymus and activation-regulated chemokine (TARC/CCL17) was developed to examine the association of TARC with the immunopathogenesis of canine atopic dermatitis (AD) . Recombinant canine TARC was prepared using an E . coli expression system . Results of transwell chemotaxis assay demonstrated that the recombinant canine TARC showed chemotactic activity for canine lymphoid cells expressing CC chemokine receptor 4 (CCR4) . Mice were then immunized with the recombinant canine TARC to obtain monoclonal antibodies . Among the monoclonal antibodies thereby obtained, one monoclonal antibody (CTA-1) was found to react with both recombinant and authentic canine TARC in ELISA and flowcytometric assays, respectively . Immunohistochemical analysis using the monoclonal antibody CTA-1 demonstrated that keratinocytes were major TARC producing cells in lesional skin of dogs with AD.

Toxicon, 2005 Feb, 45(2), 171 - 8
Role of TNF-alpha, IL-1beta and IL-6 in the local tissue damage induced by Bothrops asper snake venom: an experimental assessment in mice; Chaves F et al.; The role of the cytokines TNF-alpha, IL-1beta and IL-6 in the acute local pathological effects induced by Bothrops asper snake venom was assessed in mice . Intramuscular injection of this venom induced increments in IL-1beta and IL-6 in muscle, but no elevations of TNF-alpha were detected . Pentoxifylline (PTX), a methylxanthine derivative that inhibits the synthesis of TNF-alpha, and antibodies against these three cytokines were used to assess the role of these cytokines in venom-induced effects . As a control, PTX pretreatment was effective at abrogating lethality and serum TNF-alpha increments in mice subjected to endotoxemia induced by injection of Escherichia coli lipopolysaccharide, although it did not affect the increment in IL-1beta and IL-6 in such endotoxic model . PTX failed to reduce lethality, hemorrhage, myonecrosis, dermonecrosis and edema induced by B . asper venom . Moreover, pretreatment with anti-cytokine antibodies was also ineffective at reducing venom-induced myonecrosis and hemorrhage . It is concluded that TNF-alpha, IL-1beta and IL-6 do not have a significant role in the pathogenesis of the acute local pathological effects induced by B . asper venom in mice, although this does not exclude the possibility that these cytokines play a role in other aspects of venom-induced local pathology, as well as in the reparative and regenerative responses that take place after the onset of tissue damage.

Mol Reprod Dev . 2004 Dec 29;70(3):247-254 {Epub ahead of print}
Efficacy of antibodies against a chimeric synthetic peptide encompassing epitopes of bonnet monkey (Macaca radiata) zona pellucida-1 and zona pellucida-3 glycoproteins to inhibit in vitro human sperm-egg binding; Sivapurapu N et al.; Immunocontraception achieved by immunization with zona pellucida (ZP) glycoproteins is invariably associated with ovarian dysfunction . Use of ZP glycoprotein-based synthetic peptides as immunogens has been proposed to overcome adverse side effects on ovaries . In the present study, a chimeric peptide encompassing the epitopes of bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1; amino acid residues 251-273) and ZP glycoprotein-3 (bmZP3; amino acid residues 324-347), separated by a tri-glycine spacer, was synthesized and conjugated to diphtheria toxoid (DT) . Immunization of female BALB/cJ mice and bonnet monkeys with the chimeric peptide led to generation of antibodies that reacted with the chimeric peptide, individual bmZP1 & bmZP3 peptides, and also recombinant bmZP1 and bmZP3 proteins expressed by E . coli in an ELISA . Indirect immunofluorescence studies revealed that the immune serum also recognized human as well as bonnet monkey ZP . A significant inhibition of human sperm binding to ZP was observed with antibodies generated against the chimeric peptide in mice (P = 0.0001) as well as monkeys (P = 0.0002) in a hemizona assay (HZA) . The inhibition efficacy was significantly higher than that observed by using antibodies against the individual bmZP1 and bmZP3 peptides . Interestingly, no ovarian pathology was observed in female bonnet monkeys immunized with the chimeric peptide . These studies have demonstrated that the chimeric peptide encompassing peptides of multiple ZP glycoproteins may be a promising candidate antigen for designing immunocontraceptive vaccines . Mol . Reprod . Dev . 70: 247-254, 2005 . (c) 2005 Wiley-Liss, Inc.

J Cereb Blood Flow Metab, 2004 Dec, 24(12), 1359 - 68
Anti-monocyte chemoattractant protein-1 gene therapy protects against focal brain ischemia in hypertensive rats; Kumai Y et al.; Monocyte chemoattractant protein-1 (MCP-1) is expressed in the ischemic cortex after focal brain ischemia and appears to exacerbate ischemic damage . The authors examined the effect of gene transfer of dominant negative MCP-1, called 7ND, 90 minutes after induction of focal brain ischemia in hypertensive rats . Adenoviral vectors encoding mutant MCP-1 (Ad7ND; n = 11), or Escherichia coli beta-galactosidase (AdlacZ; n = 17) as control were injected into the lateral ventricle of male spontaneously hypertensive rats . Both AdlacZ (n = 12) and Ad7ND (n = 6) administration provided transgene expression as early as 6 hours after injection and the expression further increased on day 1, followed by a sustained detection on day 5 . Five days after ischemia, infarct volume (75 +/- 13 mm, n = 5, mean +/- SD) significantly reduced to 72% of control (104 +/- 22 mm3, n = 5, P < 0.05) by 7ND gene transfer . Numbers of leukocytes in the vessels (48.3 +/- 32.9/cm2) and macrophage/monocyte infiltration (475.2 +/- 125.5/mm2) of the infarct area in the Ad7ND group were significantly less than those measured in the AdlacZ group (143.8 +/- 72.1/cm2 and 671.8 +/- 125.5/mm2, P < 0.05, respectively) . In summary, the postischemic gene transfer of dominant negative MCP-1 attenuated the infarct volume and infiltration of inflammatory cells, suggesting potential usefulness of the anti-MCP-1 gene therapy.

J Biochem (Tokyo), 2004 Oct, 136(4), 541 - 7
Oxidative Stress Response in an Anaerobic Hyperthermophilic Archaeon: Presence of a Functional Peroxiredoxin in Pyrococcus horikoshii; Kawakami R et al.; Oxidative stress response in an anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3 was analyzed by two-dimensional gel electrophoresis . When P . horikoshii was grown on medium supplemented with air, a marked increase in the level of a 25-kDa protein was observed in comparison with cells grown under anaerobic conditions . The N-terminal amino acid sequence of the protein was determined to be VVIGEKFPEVEVKTTHGVIKLPDYF, which coincides with that of the putative alkyl hydroperoxide reductase that has been predicted in the genome database of P . horikoshii . The gene (PH1217) encoding the protein was cloned and expressed in Escherichia coli . The produced enzyme was a hyperthermostable peroxiredoxin whose activity was not lost after incubation at 90 degrees C for 20 min . The enzyme catalyzes the reduction of cumene hydroperoxide and hydrogen peroxide using dithiothreitol as an electron donor . Northern blot analysis revealed that the transcription of the gene increased by the addition of exogenous oxygen and by the addition of an oxidative stress-inducing reagent, and reached maximum within 30 min . These results suggest that the peroxiredoxin plays an important role in the peroxide-scavenging system in an anaerobic archaeon P . horikoshii.

J Biochem (Tokyo), 2004 Oct, 136(4), 447 - 55
Purification, Characterization, Cloning and Expression of Pyruvate Decarboxylase from Torulopsis glabrata IFO005; Wang Q et al.; In the production of pyruvate and optically active alpha-hydroxy ketones by Torulopsis glabrata, pyruvate decarboxylase (PDC, EC 4.1.1.1) plays an important role in pyruvate metabolism and in catalyzing the biotransformation of aromatic amino acid precursors to alpha-hydroxy ketones . In this paper, we have purified and characterized PDC from T . glabrata IFO005 and cloned the corresponding gene . A simple, rapid and efficient purification protocol was developed that provided PDC with high specific activity . Unlike other yeast or higher plant enzymes, known as homotetramers (alpha(4) or beta(4)) or heterotetramers (alpha(2)beta(2)), two active isoforms of PDC purified from T . glabrata IFO005 were homodimeric proteins with subunits of 58.7 kDa . We isolated the T . glabrata PDC gene encoding 563 amino acid residues and succeeded in overproducing the recombinant PDC protein in Escherichia coli, in which the product amounted to about 10-20% of the total protein of the cell extract . Recombinant PDC from E . coli was purified as a homotetramer . Targeted gene disruption of PDC confirmed that T . glabrata has only one gene of PDC . This PDC gene showed about 80% homology with the genes of other yeasts, and amino acid residues involved in the allosteric site for pyruvate in other yeast PDCs were conserved in T . glabrata PDC . Both native PDC and recombinant PDC were activated by pyruvate and exhibited sigmoidal kinetics similar to those of Saccharomyces cerevisiae and higher plants . They also exhibited the similar catalytic properties: low thermostability, similar pH stability and optimal pH, and complete inhibition by glyoxylate.

Glycobiology . 2004 Dec 29; {Epub ahead of print}
The wbbD gene of Escherichia coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide.*
Riley JG, Menggad M, Montoya-Peleaz P, Szarek WA, Marolda CL, Valvano MA, Schutzbach JS, Brockhausen I.
In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in E . coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit . The galactosyltransferase catalyzed the transfer of galactose (Gal) from UDP-Gal to the Nacetylglucosamine (GlcNAc) residue of a GlcNAc-pyrophosphate-lipid acceptor . A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity . The normal phenotype was restored by complementing the mutant with the cloned wbbD gene . To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate, covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO3-PO3-(CH2)11-O-Phenyl) . The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl2.Detergents in the assay did not increase glycosyl transfer . Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a a-linkage . Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses revealed a disaccharide with the structure Gal beta1-3GlcNAc . Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.

Obstet Gynecol, 2005 Jan, 105(1), 145 - 55
Nitric oxide and fetal organ blood flow during normoxia and hypoxemia in endotoxin-treated fetal sheep; Coumans AB et al.; OBJECTIVE: To investigate the role of nitric oxide in the process of circulatory decentralization during fetal hypoxemia . METHODS: Fifteen sheep with singleton pregnancies were chronically instrumented at 107 days of gestation (term is 147 days) . Three days later, 8 of the fetuses received nitro-l-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthesis . Fifteen minutes after L-NAME administration, all 15 fetuses received lipopolysaccharides (LPS) from a strain of Escherichia coli . The 7 fetuses that received LPS only were used as controls . Sixty minutes after LPS was administered, the maternal aorta was occluded for 2 minutes in all fetuses . Organ blood flow and physiological variables were measured at 75 minutes before the start of occlusion (ie, at the time of L-NAME administration to the experimental group), at 1 minute before the start of occlusion, and at 2, 4, and 30 minutes after the start of occlusion . RESULTS: Arterial pH was lower in the L-NAME group than in the control group at 1 minute before and 2 minutes after occlusion . Mean arterial pressure was higher in the L-NAME group than in the control group at 2 and 4 minutes after occlusion . Cardiac output fell in the L-NAME group and was lower than in the control group; the percentage of cardiac output to the cerebrum in the L-NAME group was 35% lower than that in the control group . Throughout the study, placental blood flow decreased by more than 80% in both groups and remained low . Blood flow to the fetal body decreased by 65% in the L-NAME group and was lower than in the control group . Blood flow to the carcass also decreased in the L-NAME group and was 36% of that in the control group . CONCLUSION: Inhibition of nitric oxide synthesis causes a general vasoconstriction in practically all organs and leads to a reduction in LPS-induced circulatory decentralization . The changes in blood flow distribution in endotoxin-treated fetal sheep seem to be mediated in part by nitric oxide.

J Biol Chem . 2004 Dec 28; {Epub ahead of print}
Exploring the role of a unique carboxyl residue in substrate binding to EmrE by mass spectrometry; Weinglass AB et al.; EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds . Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu14) from both EmrE monomers . Carbodiimide modification of EmrE has been studied using functional assays and the evidence suggests that Glu14 is the target of the reaction . Here we exploit electrospray ionization mass spectrometry (ESI-MS) to directly monitor the reaction with each monomer rather than following inactivation of the functional unit . A cyanogen bromide peptide containing Glu14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2-NPYIYLGGAILAEVIGTTLM-21 is ~80% modified in a time-dependent fashion, indicating that each Glu14 residue in the oligomer is accessible to DiPC . Furthermore, pre-incubation with tetraphenylphosphonium (TPP+) reduces the reaction of Glu14 with DiPC by up to 80% . Taken together with other biochemical data the findings support a 'time sharing' mechanism in which both Glu14 residues in a dimer are involved in TPP+ and H+ binding.

J Biol Chem . 2004 Dec 28; {Epub ahead of print}
Heterologous expression, purification and characterization of recombinant rat cysteine dioxygenase; Chai SC et al.; Cysteine dioxygenase (CDO, EC 1.13.11.20) catalyzes the oxidation of cysteine to cysteine sulfinic acid (CSA), which is the first major step in cysteine catabolism in mammalian tissues . Rat liver CDO was cloned and expressed in E . coli as a 26.8 kDa N-terminal fusion protein bearing a poly-histidine tag . Purification by immobilized-metal affinity chromatography yielded homogeneous protein, which was catalytically active even in the absence of the secondary protein-A, which has been reported to be essential for activity in partially purified native preparations . As compared to those existing purification protocols for native CDO, the milder conditions used in the isolation of the recombinant CDO allowed a more controlled study of the properties and activity of CDO, clarifying conflicting findings in the literature . Apo-protein was inactive in catalysis and was only activated by Fe . Metal analysis of purified recombinant protein indicated that only 10% of the protein contained Fe, and that the Fe was loosely bound to the protein . Kinetic studies showed that the recombinant enzyme displayed a Km value of 2.5 +/- 0.4 mM at pH 7.5 and 37 C . The enzyme was shown to be specific for L-cysteine oxidation, while homocysteine inhibited CDO activity.

Zhonghua Xue Ye Xue Za Zhi, 1997 Jun, 18(6), 311 - 4
{Cloning and expression of heavy- and light-chain variable region genes of monoclonal antibody specific for human fibrin}; Song Z et al.; OBJECTIVE: To obtain the minimum molecule having activity to bind human fibrin . METHODS: The immunoglobulin heavy- and light-chain variable region (VH and Vkappa) genes were isolated from 8E5 hybridoma cells, which secreted a monoclonal antibody against human fibrin, by RT-PCR . An expression vector pOPE51-8E5 was constructed for the recombinant VH-Vkappa expression . The transformed E . coli JM 109 cells were propagated and induced by IPTG . RESULTS AND CONCLUSION: Expression product was found in the periplasmic space and inclusion bodies by SDS-PAGE and immunobloting . It was a 30000 single chain fragment (scFv) with antigen-binding specificity . The yield of the scFv was 28.9% of the total bacterial proteins.

FEMS Microbiol Lett, 2005 Jan 15, 242(2), 361 - 6
Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability; Grudniak AM et al.; The frequency of spontaneous as well as induced reversions of auxotrophic mutations in Escherichia coli AB1157 and its DeltadnaK and DeltadnaKdnaJ derivatives was estimated . The obtained results demonstrate that both mutants tested are characterized by elevated frequency of spontaneous reversions compared to their AB1157 parent . In contrast, the frequency of reversions induced by UV and MMS, i.e . agents inducing the SOS response, is reduced in DeltadnaJ and DeltadnaKdnaJ mutants, pointing to the possible defect of these mutants in error prone repair . Due to the fact that UmuC protein is one of the main players executing the error prone repair, its stability in DeltadnaJ and DeltadnaKdnaJ mutants was also studied . Reduced UmuC stability was demonstrated only in the DeltadnaKdnaJ mutant.

FEMS Microbiol Lett, 2005 Jan 15, 242(2), 333 - 8
GcvA interacts with both the alpha and sigma subunits of RNA polymerase to activate the Escherichia coli gcvB gene and the gcvTHP operon; Stauffer LT et al.; The glycine cleavage enzyme system in Escherichia coli provides one-carbon units for cellular methylation reactions . The gcvB gene encodes two small RNAs that in turn regulate other genes . The GcvA protein is required for expression of both the gcvTHP (P(gcvT)) and gcvB (P(gcvB)) promoters . However, the architectures of the two promoters are different, with the P(gcvT) promoter representing a class III activator-dependent promoter and the P(gcvB) promoter representing a class II activator-dependent promoter . The RNA polymerase holoenzyme was examined for its role in transcription activation of the gcvTHP operon and the gcvB gene by the GcvA protein . The results suggest that GcvA interacts with the RNA polymerase alpha subunit for activation of the gcvTHP operon and interacts with the RNA polymerase sigma subunit for activation of the gcvB gene.

FEMS Microbiol Lett, 2005 Jan 1, 242(1), 95 - 9
Sepiapterin reductases from Chlorobium tepidum and Chlorobium limicola catalyze the synthesis of l-threo-tetrahydrobiopterin from 6-pyruvoyltetrahydropterin; Choi YK et al.; The ORF sequences of the gene encoding sepiapterin reductase were cloned from the genomic DNAs of Chlorobium tepidum and Chlorobium limicola, which are known to produce l-threo- and l-erythro-tetrahydrobiopterin (BH4)-N-acetylglucosamine, respectively . The deduced amino acid sequence of C . limicola consists of 241 residues, while C . tepidum SR has three residues more at the C-terminal . The overall protein sequence identity was 87.7% . Both recombinant proteins generated from Escherichia coli were identified to catalyze reduction of diketo compound 6-pyruvoyltetrahydropterin to l-threo-BH4 . This result suggests that C . limicola needs an additional enzyme for l-erythro-BH4 synthesis to yield its glycoside . The catalytic activity of Chlorobium SRs also supports the previously proposed mechanism of two consecutive reductions of C1' carbonyl group of 6-pyruvoyltetrahydropterin via isomerization reaction.

FEMS Microbiol Lett, 2005 Jan 1, 242(1), 87 - 94
Characterization of bdhA, encoding the enzyme d-3-hydroxybutyrate dehydrogenase, from Sinorhizobium sp . strain NGR234; Aneja P et al.; A genomic library of Sinorhizobium sp . strain NGR234 was introduced into Escherichia coli LS5218, a strain with a constitutively active pathway for acetoacetate degradation, and clones that confer the ability to utilize D-3-hydroxybutyrate as a sole carbon source were isolated . Subcloning experiments identified a 2.3 kb EcoRI fragment that retained complementing ability, and an ORF that appeared orthologous with known bdhA genes was located within this fragment . The deduced NGR234 BdhA amino acid sequence revealed 91% identity to the Sinorhizobium meliloti BdhA . Site-directed insertion mutagenesis was performed by introduction of a OmegaSmSp cassette at a unique EcoRV site within the bdhA coding region . A NGR234 bdhA mutant, NGRPA2, was generated by homogenotization, utilizing the sacB gene-based lethal selection procedure . This mutant was devoid of d-3-hydroxybutyrate dehydrogenase activity, and was unable to grow on D-3-hydroxybutyrate as sole carbon source . NGRPA2 exhibited symbiotic defects on Leucaena but not on Vigna, Macroptilium or Tephrosia host plants . Furthermore, the D-3-hydroxybutyrate utilization phenotype of NGRPA2 was suppressed by presence of plasmid-encoded multiple copies of the S . meliloti acsA2 gene . The glpK-bdhA-xdhA gene organization and the bdhA-xdhA operon arrangement observed in S . meliloti are also conserved in NGR234.

FEMS Microbiol Lett, 2005 Jan 1, 242(1), 13 - 8
PHB synthase from Streptomyces aureofaciens NRRL 2209; Ramachander TV et al.; An approximately 4.9 kb Sau3A I genomic DNA fragment from the Streptomyces aureofaciens NRRL 2209 aiding in the biosynthesis of PHB in recombinant Escherichia coli has been sequenced and analysed for phaC gene . The putative phaC(Sa) gene of 2 kb is 79.1% GC rich and encodes a 63.5 kDa protein . It expressed under its own promoter and significant PHA synthase activity was detected in the recombinant E . coli . This is the first putative PHA synthase gene reported from a Streptomyces sp . with serine as the active nucleophile in the conserved lipase box . The phaC(Sa) was found in close proximity to a regulatory gene, which apparently regulated the phaC expression.

Carbohydr Res, 2005 Jan 17, 340(1), 167 - 71
Structural analysis of the O-antigen polysaccharide from Escherichia coli O152; Olsson U et al.; The structure of the O-antigen polysaccharide (PS) from Escherichia coli O152 has been determined . Component analysis together with (1)H, (13)C and (31)P NMR spectroscopy were used to elucidate the structure . Inter-residue correlations were determined by (1)H,(31)P COSY, (1)H,(1)H NOESY and (1)H,(13)C heteronuclear multiple-bond correlation experiments . The PS is composed of pentasaccharide repeating units with the following structure: The structure is similar to that of the O-antigen polysaccharide from E . coli O173 . The cross-reactivity between E . coli O152 and E . coli O3 may be explained by structural similarities in the branching region of their O-antigen polysaccharides.

Curr Biol, 2004 Dec 29, 14(24), 2271 - 6
The small RNA IstR inhibits synthesis of an SOS-induced toxic peptide; Vogel J et al.; More than 60 small RNAs (sRNA) have been identified in E . coli . The functions of the majority of these sRNAs are still unclear . For the few sRNAs characterized, expression and functional studies indicate that they act under stress conditions . Here, we describe a novel E . coli chromosome locus that is part of the SOS response to DNA damage . This locus encodes two sRNAs, IstR-1 and IstR-2, and a toxic peptide, TisB, encoded by tisAB mRNA . Transcription of tisAB and istR-2 is SOS regulated, whereas IstR-1 is present throughout growth . IstR-1 inhibits toxicity by base-pairing to a short region in the tisAB mRNA . This antisense interaction entails RNase III-dependent cleavage, thereby inactivating the mRNA for translation . In the absence of the SOS response, IstR-1 is present in high excess over its target . However, SOS induction leads to depletion of the IstR-1 pool, concomitant with accumulation of tisAB mRNA . Under such conditions, TisB exerts its toxic effect, slowing down growth . We propose that the inhibitory sRNA prevents inadvertent TisB synthesis during normal growth and, possibly, also limits SOS-induced toxicity . Our study adds the SOS regulon to the growing list of global regulatory circuits controlled by sRNA genes.

J Virol Methods, 2005 Feb, 123(2), 131 - 40
Establishment of an alternative intracellular cytokine staining assay for HIV/AIDS clinical studies; Chen H et al.; To evaluate immune responses in large-scale clinical studies, it is crucial to use assays that are not only sensitive and specific, but also standardized and easily transferable . To this end, an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CD8+ T cells has been established and its performance characteristics determined . PBMCs were stimulated using recombinant vaccinia expressing HIV-Env, -Gag, -Pol and -Nef proteins, or a protein from Escherichia coli (Eco); CD8+ T cell responses were assessed by intracellular IFN-gamma staining and flow cytometric (FC) analysis . In 15 HIV seronegative and 11 HIV seropositive individuals, a minimal background IFN-gamma staining was found after Eco stimulation with a median {inter-quartile range (IQR)} of 0.005% (0.000%, 0.030%) irrespective of HIV infection status . Recent smallpox vaccination was associated with a small but significant increase in background staining {0.02% (0.02%, 0.04%) versus 0.0% (0.0%, 0.01%) (p=0.039)} in HIV seronegative individuals . This assay detected moderate (>0.10%) HIV-specific responses in 64% (7/11) of HIV seropositive individuals . The results suggest that this ICS assay format is sensitive and specific, and is amenable to standardization for screening for HIV-specific CD8+ T cells in clinical trials.

Biochim Biophys Acta, 2005 Jan 7, 1706(1-2), 110 - 6
Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F(1)F(o) ATP synthase; Ishmukhametov RR et al.; His-tagged cysteine-less F(1)F(o) ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography . During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold . The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%) . Incorporation of F(1)F(o) into soybean liposomes yielded well-coupled and highly active proteoliposomes . The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h . Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.

Biochim Biophys Acta, 2005 Jan 7, 1706(1-2), 81 - 7
Reconstitution of phycobilisome core-membrane linker, L(CM), by autocatalytic chromophore binding to ApcE; Zhao KH et al.; The core-membrane linker, L(CM), connects functionally the extramembraneous light-harvesting complex of cyanobacteria, the phycobilisome, to the chlorophyll-containing core-complexes in the photosynthetic membrane . Genes coding for the apoprotein, ApcE, from Nostoc sp . PCC 7120 and for a C-terminally truncated fragment ApcE(1-240) containing the chromophore binding cysteine-195 were overexpressed in Escherichia coli . Both bind covalently phycocyanobilin (PCB) in an autocatalytic reaction, in the presence of 4M urea necessary to solubilize the proteins . If judged from the intense, red-shifted absorption and fluorescence, both products have the features of the native core-membrane linker L(CM), demonstrating that the lyase function, the dimerization motif, and the capacity to extremely red-shift the chromophore are all contained in the N-terminal phycobilin domain of ApcE . The red-shift is, however, not the result of excitonic interactions: Although the chromoprotein dimerizes, the circular dichroism shows no indication of excitonic coupling . The lack of homologies with the autocatalytically chromophorylating phytochromes, as well as with the heterodimeric cysteine-alpha84 lyases, indicates that ApcE constitutes a third type of bilin:biliprotein lyase.

Int J Parasitol, 2005 Jan, 35(1), 39 - 48 Epub 2004 Nov 05.
Functional analysis and localisation of a delta-class glutathione S-transferase from Sarcoptes scabiei; Pettersson EU et al.; The mite Sarcoptes scabiei causes sarcoptic mange, or scabies, a disease that affects both animals and humans worldwide . Our interest in S . scabiei led us to further characterise a glutathione S-transferase . This multifunctional enzyme is a target for vaccine and drug development in several parasitic diseases . The S . scabiei glutathione S-transferase open reading frame reported here is 684 nucleotides long and yields a protein with a predicted molecular mass of 26kDa . Through phylogenetic analysis the enzyme was classified as a delta-class glutathione S-transferase, and our paper is the first to report that delta-class glutathione S-transferases occur in organisms other than insects . The recombinant S . scabiei glutathione S-transferase was expressed in Escherichia coli via three different constructs and purified for biochemical analysis . The S . scabiei glutathione S-transferase was active towards the substrate 1-chloro-2,4-dinitrobenzene, though the positioning of fusion partners influenced the kinetic activity of the enzyme . Polyclonal antibodies raised against S . scabiei glutathione S-transferase specifically localised the enzyme to the integument of the epidermis and cavities surrounding internal organs in adult parasites . However, some minor staining of parasite intestines was observed . No staining was seen in host tissues, nor could we detect any antibody response against S . scabiei glutathione S-transferase in sera from naturally S . scabiei infected dogs or pigs . Additionally, the polyclonal sera raised against recombinant S . scabiei glutathione S-transferase readily detected a protein from mites, corresponding to the predicted size of native glutathione S-transferase.

Microb Pathog, 2004 Dec, 37(6), 287 - 93 Epub 2004 Dec 09.
Role of S fimbriae in Escherichia coli K1 binding to brain microvascular endothelial cells in vitro and penetration into the central nervous system in vivo; Wang Y et al.; Bacterial binding to host cell surface is considered an important initial step in the pathogenesis of many infectious diseases including meningitis . Previous studies using a laboratory Escherichia coli (E . coli) strain HB101 possessing a recombinant plasmid carrying the cloned S fimbriae gene cluster have shown that S fimbriae are the major contributor to binding to bovine brain microvascular endothelial cells (BMEC) for HB101 . Our present study, however, revealed that S fimbriae did not play a major role for E . coli K1's binding to human BMEC in vitro and crossing of the blood-brain barrier in vivo . This was shown by our demonstration that E . coli K1 strain and its S fimbriae-operon deletion mutant exhibited similar rates of binding to human BMEC and similar rates of penetration into the central nervous system in the experimental hematogenous meningitis model . Studies are needed to identify major determinants of E . coli K1 contributing to BMEC binding and subsequent crossing of the blood-brain barrier in vivo.

Drug Metab Pharmacokinet, 2003, 18(3), 163 - 72
Production of Inhibitory Polyclonal Antibodies against Cytochrome P450s; Ng PS et al.; Nine different antibodies against P450 isoforms were prepared using purified cytochrome P450s (P450) expressed in E . coli . Purified isozymes were injected into rabbits to raise specific antibody . The resulting antibodies were characterized for their specificity and sensitivity through each particular P450 enzyme-mediated probe reaction.Anti-CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 antibodies proved to be strong immunoinhibitors with inhibitory effects specific to their corresponding antigen . Antiserum derived from the CYP2C19-immunized rabbits was reacted with CYP2C9 as well as CYP2C19 and immunoabsorbed with membrane-bound CYP2C9 expressed in E . coli . Antibody specific for CYP2C19 was obtained . Anti-CYP2C19 together with the anti-CYP2C8 and anti-CYP2C9 can be very useful for determining the contribution of a particular P450 in the metabolism of a drug . The developed inhibitory antibodies will serve as in vitro-specific tools for evaluating the quantitative contribution of individual P450 enzymes to drug metabolism.

Drug Metab Pharmacokinet, 2002, 17(3), 207 - 13
A Mutation in the Flavin-containing Monooxygenase 3 Gene and its Effects on Catalytic Activity for N-oxidation of Trimethylamine In Vitro; Kubota M et al.; To clarify the mutation of the flavin-containing monooxygenase (FMO) 3 gene causing fish-odor syndrome, we analyzed the FMO3 gene of a Thai subject who possibly suffered from fish-odor syndrome . A novel mutation, a single-base substitution from G to A at the position of 265 (G265A), was identified in exon 3 . The mutation caused an amino acid substitution from valine to isoleucine at residue 58 (V58I) . The mutated FMO3 protein with V58I exhibited the reduced trimethylamine N-oxidase activity when it was expressed in E . coli . The V(max)/K(m) value for the activity of the mutant-type FMO3 was about 5 times lower than that for the wild-type FMO3.

Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2637 - 9
Convenient and Sensitive Evaluation of a Superoxide Anion-Generating Reagent Methyl Viologen by Escherichia coli Harboring a soxS'::gfp Reporter Plasmid; Shibuya A et al.; We constructed a reporter system to detect a superoxide-generating methyl viologen using SoxRS of Escherichia coli and GFP of Aequorea victoria . E . coli carrying this plasmid exhibited strong fluorescence when grown in the presence of a superoxide-generating reagent methyl viologen . The fluorescence intensity observed in the stationary phase culture of the transformant increased in response to the methyl viologen concentration in a range of 0.01 muM to 10 muM.

Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2630 - 2
Mutational Analysis of the Length of the J3/4 Domain of Escherichia coli Ribonuclease P Ribozyme; Haga S et al.; We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A(62)G(63)G(64)A(65)) was replaced with GGA (denoted DeltaA), GA (DeltaAG), A (DeltaAGG), AAGGA (SigmaA), AAAGGA (SigmaAA), and AAAAGGA (SigmaAAA) . The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT>>SigmaAA>SigmaA>SigmaAAA>>DeltaAG>DeltaA, DeltaAGG), but did not affect the cleavage site selection of a substrate by the ribozyme . The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions . Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.

Biosci Biotechnol Biochem, 2004 Dec, 68(12), 2498 - 504
A Nonconserved Carboxy-Terminal Segment of GroEL Contributes to Reaction Temperature; Nakamura T et al.; The role of the C-terminal segment of the GroEL equatorial domain was analyzed . To understand the molecular basis for the different active temperatures of GroEL from three bacteria, we constructed a series of chimeric GroELs combining the C-terminal segment of the equatorial domain from one species with the remainder of GroEL from another . In each case, the foreign C-terminal segment substantially altered the active temperature range of the chimera . Substitution of L524 of Escherichia coli GroEL with the corresponding residue (isoleucine) from psychrophilic GroEL resulted in a GroE with approximately wild-type activity at 25 degrees C, but also at 10 degrees C, a temperature at which wild-type E . coli GroE is inactive . In a detailed look at the temperature dependence of the GroELs, normal E . coli GroEL and the L524I mutant became highly active above 14 degrees C and 12 degrees C respectively . Similar temperature dependences were observed in a surface plasmon resonance assay of GroES binding . These results suggested that the C-terminal segment of the GroEL equatorial domain has an important role in the temperature dependence of GroEL . Moreover, E . coli acquired the ability to grow at low temperature through the introduction of cold-adapted chimeric or L524I mutant groEL genes.

Am J Respir Crit Care Med . 2004 Dec 23; {Epub ahead of print}
The Critical Role of Hematopoietic Cells in Lipopolysaccharide Induced Airway Inflammation; Hollingsworth Ii JW et al.; Rapid and selective recruitment of neutrophils into the airspace in response to lipopolysaccharide (LPS) facilitates the clearance of bacterial pathogens . However, neutrophil infiltration can also participate in the development and progression of environmental airway disease . Previous data have revealed that toll-like receptor 4 (tlr4) is required for neutrophil recruitment to the lung following either inhaled or systemically administrated LPS from E . coli . While many cell types express tlr4, endothelial cell expression of tlr4 is specifically required to sequester neutrophils in the lung in response to systemic endotoxin . To identify the cell types requiring trl4 expression for neutrophil recruitment following inhaled LPS, we generated chimeric mice separately expressing tlr4 on either hematopoietic cells, or on structural lung cells . Neutrophil recruitment into the airspace was completely restored in tlr4-/- mice receiving wild type bone marrow . By contrast, wild type animals receiving tlr4-/- marrow had dramatically reduced neutrophil recruitment . Moreover, adoptive transfer of wild type alveolar macrophages also restored the ability of tlr4-/- recipient mice to recruit neutrophils to the lung . These data demonstrate the critical role of hematopoietic cells and alveolar macrophages in initiating LPS induced neutrophil recruitment from the vascular space to the airspace.

J Biol Chem . 2004 Dec 22; {Epub ahead of print}
Consequences of lysine 72 mutation on the phosphorylation and activation state of PKA; Iyer GH et al.; General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core and the equivalent Lys72 in cAMP-dependent kinase (PKA) has often been used to generate a 'dead' kinase . Here we have analyzed the consequences of this mutation on kinase structure and function . Mutation of Lys72 to histidine (K72H) generated an inactive enzyme which was unphosphorylated . Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP . Ser338 in K72H cannot be autophosphorylated nor can it be phosphorylated in an intermolecular process by active wild type C-subunit . The K72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIalpha subunits . Thus a 'dead' kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective . Using a potent inhibitor of C-subunit activity, H-89, E . coli expressed C-subunit was also obtained in its unphosphorylated state . This protein is able to mature into its active form in the presence of PDK-1, and is able to undergo secondary autophosphorylation on Ser338 . Unlike the H-89 treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197 . Using these two substrates and mammalian expressed PDK-1, we can elucidate a possible two step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or PDK-1- like enzyme, followed by second cis autophosphorylation step at Ser338.

Infect Immun, 2005 Jan, 73(1), 612 - 6
Shiga-toxigenic Escherichia coli-inoculated neonatal piglets develop kidney lesions that are comparable to those in humans with hemolytic-uremic syndrome; Pohlenz JF et al.; Kidney lesions similar to those in humans with hemolytic-uremic syndrome were observed histologically in 82 of 122 piglets inoculated intragastrically with Shiga-toxigenic Escherichia coli but not in 29 controls . The locations of lesions matched locations where Stx-2 binding and Gb3 (globotriasylceramide receptors for Stx) were identified immunohistochemically.

Infect Immun, 2005 Jan, 73(1), 453 - 8
Abundant secretion of bioactive interleukin-1beta by human macrophages induced by Actinobacillus actinomycetemcomitans leukotoxin; Kelk P et al.; Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes . Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1 . In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages . The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A . actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha (TNF-alpha) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively . Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1beta, while the effects on IL-6 and TNF-alpha production were limited . Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1beta than did LPS (100 ng/ml) . The secreted IL-1beta was mainly the bioactive 17-kDa protein . At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1beta . The presence of specific antibodies to IL-1beta or of a caspase 1 inhibitor blocked the secretion and production of the cytokine . Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model . The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1beta . We concluded that A . actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1beta by human macrophages, which is mediated by activation of caspase 1.

Infect Immun, 2005 Jan, 73(1), 287 - 97
The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites; Darko CA et al.; A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites . The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices . This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P . falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits . The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P . falciparum FVO erythrocytic-stage malaria parasites . The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control . Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E . coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen . None of the six monkeys that were vaccinated with the E . coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia . Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment . Soluble MSP1(42) (FVO) expressed in E . coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.

Infect Immun, 2005 Jan, 73(1), 268 - 76
Upregulation of intercellular adhesion molecule 1 and proinflammatory cytokines by the major surface proteins of Treponema maltophilum and Treponema lecithinolyticum, the phylogenetic group IV oral spirochetes associated with periodontitis and endodontic infections; Lee SH et al.; Treponema maltophilum and Treponema lecithinolyticum belong to the group IV oral spirochetes and are associated with endodontic infections, as well as periodontitis . Recently, the genes encoding the major surface proteins (Msps) of these bacteria (MspA and MspTL, respectively) were cloned and sequenced . The amino acid sequences of these proteins showed significant similarity . In this study we analyzed the functional role of these homologous proteins in human monocytic THP-1 cells and primary cultured periodontal ligament (PDL) cells using recombinant proteins . The complete genes encoding MspA and MspTL without the signal sequence were cloned into Escherichia coli by using the expression vector pQE-30 . Fusion proteins tagged with N-terminal hexahistidine (recombinant MspA {rMspA} and rMspTL) were obtained, and any possible contamination of the recombinant proteins with E . coli endotoxin was removed by using polymyxin B-agarose . Flow cytometry showed that rMspA and rMspTL upregulated the expression of intercellular adhesion molecule 1 (ICAM-1) in both THP-1 and PDL cells . Expression of proinflammatory cytokines, such as interleukin-6 (IL-6) and IL-8, was also induced significantly in both cell types by the Msps, as determined by reverse transcription-PCR and an enzyme-linked immunosorbent assay, whereas IL-1beta synthesis could be detected only in the THP-1 cells . The upregulation of ICAM-1, IL-6, and IL-8 was completely inhibited by pretreating the cells with an NF-kappaB activation inhibitor, l-1-tosylamido-2-phenylethyl chloromethyl ketone . This suggests involvement of NF-kappaB activation . The increased ICAM-1 and IL-8 expression in the THP-1 cells obtained with rMsps was not inhibited in the presence of the IL-1 receptor antagonist (IL-1ra), a natural inhibitor of IL-1 . Our results show that the Msps of the group IV oral spirochetes may play an important role in amplifying the local immune response by continuous inflammatory cell recruitment and retention at an infection site by stimulation of expression of ICAM-1 and proinflammatory cytokines.

Infect Immun, 2005 Jan, 73(1), 135 - 45
Molecular aspects of biogenesis of Escherichia coli Dr Fimbriae: characterization of DraB-DraE complexes; Piatek R et al.; The Dr hemagglutinin of uropathogenic Escherichia coli is a fimbrial homopolymer of DraE subunits encoded by the dra operon . The dra operon includes the draB and draC genes, whose products exhibit homology to chaperone-usher proteins involved in the biogenesis of surface-located polymeric structures . DraB is one of the periplasmic proteins belonging to the superfamily of PapD-like chaperones . It possesses two conserved cysteine residues characteristic of the FGL subfamily of Caf1M-like chaperones . In this study we obtained evidence that DraB cysteines form a disulfide bond in a mature chaperone and have the crucial function of forming the DraB-DraE binary complex . Expression experiments showed that the DraB protein is indispensable in the folding of the DraE subunit to a form capable of polymerization . Accumulation of DraB-DraE(n) oligomers, composed of head-to-tail subunits and the chaperone DraB, was observed in the periplasm of a recombinant E . coli strain which expressed DraB and DraE (but not DraC) . To investigate the donor strand exchange mechanism during the formation of DraE oligomers, we constructed a series of DraE N-terminal deletion mutants . Deletion of the first three N-terminal residues of a potential donor strand resulted in a DraE protein lacking an oligomerization function . In vitro data showed that the DraE disulfide bond was not needed to form a binary complex with the DraB chaperone but was essential in the polymerization process . Our data suggest that assembly of Dr fimbriae requires a chaperone-usher pathway and the donor strand exchange mechanism.

Infect Immun, 2005 Jan, 73(1), 103 - 13
Role of EspA and intimin in expression of proinflammatory cytokines from enterocytes and lymphocytes by rabbit enteropathogenic Escherichia coli-infected rabbits; Ramirez K et al.; Enteropathogenic Escherichia coli (EPEC) produces attaching and effacing (A/E) lesions and watery diarrhea, both of which are intimin and EspA dependent . In this work, we explored the mucosal immune response by detecting cytokine induction in rabbits with diarrhea caused by rabbit EPEC (REPEC) . Orally inoculated rabbits exhibited weight loss and mucosal inflammation, developed watery diarrhea, and died (day 7) . At day 6 postinoculation, animals were analyzed for the induction of proinflammatory cytokines in enterocytes . The role of lymphocyte-dependent immunity was determined through the expression of proinflammatory cytokines by lymphocytes from Peyer's patches (PP) and the spleen . EspA and intimin mutants were used to explore the role of A/E lesions in the expression of these cytokines . REPEC-infected rabbit enterocytes showed increased interleukin 1beta (IL-1beta), IL-6, IL-8, and tumor necrosis factor alpha (TNF-alpha) mRNA expression, but that of anti-inflammatory IL-10 was increased only slightly . In contrast, intimin mutant-infected rabbits were unable to produce this proinflammatory cytokine profile but did produce a remarkable increase in IL-10 expression . Bacteria lacking EspA increased the expression of IL-8 and TNF-alpha, but that of IL-10 was increased only slightly . PP lymphocytes also produced proinflammatory cytokines, which were dependent on EspA (except for TNF-alpha) and intimin, while IL-10 was induced by EspA and intimin mutants . In contrast, spleen lymphocytes (systemic compartment) were unable to produce IL-1beta and TNF-alpha . These data show the importance of the proinflammatory cytokines secreted by enterocytes and those expressed locally by PP lymphocytes, which can activate effector mechanisms at the epithelium . Furthermore, this cytokine profile, including IL-6 and IL-1beta, which may be involved in the diarrhea produced by EPEC, depends on intimin.

J Fluoresc, 2004 Sep, 14(5), 603 - 9
Live imaging of glucose homeostasis in nuclei of COS-7 cells; Fehr M et al.; Measuring subcellular glucose levels deep in tissues can provide new insights into compartmentalization and specialization of glucose metabolism among different cells . As shown previously, a FRET-based glucose-sensor consisting of two GFP-variants and the Escherichia coli periplasmic glucose/galactose binding protein was successfully expressed in the cytosol of COS7-cells and used to determine cytosolic glucose levels . Recording cytosolic fluorescence intensities in cells located in deeper layers of tissues is often difficult due to loss of signal intensity caused by effects of other cell layers on excitation and emission light . These interfering effects may be reduced by restricting fluorophores to occupy only a fraction of the assayed tissue volume . This can be accomplished by confining fluorophores to a sub-compartment of each cell in the tissue, such as the nucleus . The glucose-sensor was targeted to nuclei of COS7-cells . To determine, whether nuclear glucose levels can be used to track cytosolic changes, nuclear glucose concentrations were quantified as the cells were challenged with external glucose over a range of 0.5 to 10 mM and compared to cytosolic levels . Internal glucose concentrations in both compartments were similar, corresponding to approximately 50% of the external concentration . Taken together, these results indicate that nuclear glucose levels can be used to determine cytosolic levels indirectly, permitting more reliable quantification of fluorescence intensities and providing a tool for measurements not only in cell cultures but also in tissues.

EMBO J . 2004 Dec 16; {Epub ahead of print}
Escherichia coli dihydroxyacetone kinase controls gene expression by binding to transcription factor DhaR; Bachler C et al.; Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize either ATP (in animals, plants, bacteria) or the bacterial phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) as a source of high-energy phosphate . The PTS-dependent kinase of Escherichia coli consists of three subunits: DhaK contains the Dha binding site, DhaL contains ADP as cofactor for the double displacement of phosphate from DhaM to Dha, and DhaM provides a phospho-histidine relay between the PTS and DhaLColon, two colonsADP . DhaR is a transcription activator belonging to the AAA+ family of enhancer binding proteins . It stimulates transcription of the dhaKLM operon from a sigma70 promoter and autorepresses dhaR transcription . Genetic and biochemical studies indicate that the enzyme subunits DhaL and DhaK act antagonistically as coactivator and corepressor of the transcription activator by mutually exclusive binding to the sensing domain of DhaR . In the presence of Dha, DhaL is dephosphorylated and DhaLColon, two colonsADP displaces DhaK and stimulates DhaR activity . In the absence of Dha, DhaLColon, two colonsADP is converted by the PTS to DhaLColon, two colonsATP, which does not bind to DhaR.

EMBO J . 2004 Dec 23; {Epub ahead of print}
X-ray crystallography study on ribosome recycling: the mechanism of binding and action of RRF on the 50S ribosomal subunit; Wilson DN et al.; This study presents the crystal structure of domain I of the Escherichia coli ribosome recycling factor (RRF) bound to the Deinococcus radiodurans 50S subunit . The orientation of RRF is consistent with the position determined on a 70S-RRF complex by cryoelectron microscopy (cryo-EM) . Alignment, however, requires a rotation of 7 degrees and a shift of the cryo-EM RRF by a complete turn of an alpha-helix, redefining the contacts established with ribosomal components . At 3.3 A resolution, RRF is seen to interact exclusively with ribosomal elements associated with tRNA binding and/or translocation . Furthermore, these results now provide a high-resolution structural description of the conformational changes that were suspected to occur on the 70S-RRF complex, which has implications for the synergistic action of RRF with elongation factor G (EF-G) . Specifically, the tip of the universal bridge element H69 is shifted by 20 A toward h44 of the 30S subunit, suggesting that RRF primes the intersubunit bridge B2a for the action of EF-G . Collectively, our data enable a model to be proposed for the dual action of EF-G and RRF during ribosome recycling.

Nature, 2004 Dec 23, 432(7020), 1050 - 4
Accurate multiplex gene synthesis from programmable DNA microchips; Tian J et al.; Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis . Here we describe a microchip-based technology for multiplex gene synthesis . Pools of thousands of 'construction' oligonucleotides and tagged complementary 'selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold . A one-step polymerase assembly multiplexing reaction assembles these into multiple genes . This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias . This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.

Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 75 - 80 Epub 2004 Dec 22.
Sequential multiple functions of the conserved sequence in sequence-specific termination by T7 RNA polymerase; Sohn Y et al.; Escherichia coli rrnB terminator t1 contains an RNA hairpin-dependent (class I) and a sequence-specific (class II) termination signal . The latter consists of an 8-bp conserved sequence (CS), TATCTGTT, immediately followed by an 8-bp T rich sequence . In this study, elongation complexes of T7 RNA polymerase at various positions of the class II signal and several mutant signals were obtained by stepwise walking on immobilized DNA templates free of the class I signal . Multiple CS-associated conformational changes were observed, starting at the beginning of the signal and occurring sequentially . When the complexes reach the first base pair of the CS-DNA duplex, which is downstream of the RNA-DNA heteroduplex, their stability, as measured by time-course retention of radiolabeled transcripts, markedly decreases . Further elongation leads to an abrupt change in polymerase-RNA interaction . Cross-linking of the polymerase to a 4-thio-UMP incorporated into RNA 8 nucleotides upstream of the 3' end and just upstream of the heteroduplex is initially strong but diminishes when the polymerase reaches the fourth base pair of the CS . After a further 7-nt elongation, the exposed single-stranded region of nontemplate strand is contracted; RNA in the upstream half of the heteroduplex becomes dissociated, and the CS-DNA duplex is reformed . During the next 5-nt elongation before termination, the CS duplex is prevented from translocation, and the contracted transcription bubble expands only downstream . These findings suggest that the CS duplex plays essential roles by successively binding to polymerase both downstream and upstream of the heteroduplex.

J Biol Chem . 2004 Dec 22; {Epub ahead of print}
The trifunctional sulfate activating complex of mycobacterium tuberculosis; Sun M et al.; The sulfate activation pathway is essential for the assimilation of sulfate and, in many bacteria, is comprised of three reactions: the synthesis of APS (adenosine 5' phosphosulfate), the hydrolysis of GTP, and the 3' phosphorylation of APS to produce PAPS, whose sulfuryl group is reduced or transferred to other metabolites . The entire sulfate activation pathway is organized into a single complex in M . tuberculosis . Although present in many bacteria, these tripartite complexes have not been studied in detail . Initial rate characterization of the mycobacterial system reveals that it is poised for extremely efficient throughput - at saturating ATP, PAPS synthesis is 5800 times more efficient than APS synthesis . The APS kinase domain of the complex does not appear to form the covalent E*P intermediate observed in the closely related APS kinase from E . coli . The stoichiometry of GTP hydrolysis and APS synthesis is 1:1, and the APS synthesis reaction is driven 1.1 x 10(6)-fold further during GTP hydrolysis - the system harnesses the full chemical potential of the hydrolysis reaction to the synthesis of APS . A key energy-coupling step in the mechanism is a ligand-induced isomerization that enhances the affinity of GTP and commits APS synthesis and GTP hydrolysis to the completion of the catalytic cycle . Ligand induce increases in guanine nucleotide affinity observed in the mycobacterial system suggest that it too undergoes the energy coupling isomerization.

J Biol Chem . 2004 Dec 22; {Epub ahead of print}
Structural and biochemical characterization of a quinol binding site of escherichia coli nitrate reductase A (NarGHI); Bertero MG et al.; The crystal structure of Esherichia coli nitrate reductase A (NarGHI) in complex with pentachlorophenol (PCP) has been determined to 2.0A resolution . We have shown that PCP is a potent inhibitor of quinol:nitrate oxidoreductase activity, and that it also perturbs the EPR spectrum of one of the hemes located in the membrane anchoring subunit (NarI) . This new structural information, together with site-directed mutagenesis data, biochemical analyses and molecular modeling, provide the first molecular characterization of a quinol binding and oxidation site (Q-site) in NarGHI . A possible proton conduction pathway linked to electron transfer reactions has also been defined providing fundamental atomic details of ubiquinol oxidation by NarGHI at the bacterial membrane.

BMC Public Health . 2004 Dec 22;4(1):64.
Intestinal parasites prevalence and related factors in school children, a western city sample--Turkey; Okyay P et al.; BACKGROUND: Intestinal parasitic infections are amongst the most common infections worldwide . Epidemiological research carried out in different countries has shown that the social and economical situation of the individuals is an important cause in the prevalence of intestinal parasites . Previous studies in Turkey revealed a high prevalence of intestinal parasitic infection . The objectives of the current study were to determine the prevalence of intestinal parasitic infections in Aydin among 7-14 years old school children and to identify associated socio-demographic and environmental factors, behavioral habits and also related complaints . METHODS: Multistage sampling was used in the selection of the study sample . A questionnaire, cellulose adhesive and a stool specimen examination were done . RESULTS: A total of 456 stool specimens were collected . 145 students (31.8%) were infected with one or more intestinal parasites . 29 (6.4%) of the students were infected more than one parasite, 26 (5.7%) with two parasites and 3 (0.7%) with three parasites . The three most common were E . vermicularis, G . intestinalis and E . coli . Intestinal parasite prevalence was higher in rural area, in children with less than primary school educated mother, in children who use hands for washing anal area after defecation, and in children who use toilet paper sometimes or never . The relation between child health and mother education is well known . Children were traditionally taught to wash anal area by hand . Toiler paper usage was not common and might be due to low income or just a behavioral habit also . Most of the complaints of the study population were not significantly related with the intestinal parasitic infection . CONCLUSIONS: Intestinal parasitic infection is an important public health problem in Aydin, Turkey . Rural residence, mother education less than primary school, sometimes or never usage of toilet paper, and washing anal area by hands after defecation were the significant associations . Interventions including health education on personal hygiene to the students and to the parents, especially to mothers are required . The ratio of uneducated women should be declined with specific programs . A multisectoral approach is needed.

Poult Sci, 2004 Dec, 83(12), 1973 - 8
A plasmid DNA encoding chicken interleukin-6 and Escherichia coli K88 fimbrial protein FaeG stimulates the production of anti-K88 fimbrial antibodies in chickens; Cho SH et al.; Immunization using a plasmid to deliver an encoded protein for expression in situ as the antigen is a promising technology . A plasmid encoding the enterotoxigenic Escherichia coli K88 fimbrial protein FaeG when injected into chickens stimulates the production of antibodies against the fimbrial protein, similar to what has been observed in mice . The efficacy of a genetic adjuvant on fimbrial antibody production was tested by introducing the gene for chicken interleukin-6 in tandem with the faeG gene . Expression of both the fimbrial FaeG protein and chicken interleukin-6 protein was confirmed in COS-M6 cells . Slightly higher antiFaeG antibody titer in chickens was obtained compared with immunization with the plasmid encoding FaeG alone, especially at 10 (19%, P < 0.05) and 12 (27%, P < 0.05) wk, respectively, after the secondary immunization . Elevated antiFaeG antibody titer induced by chicken interleukin-6 and FaeG proteins expressed jointly persisted longer than when induced by FaeG protein alone . This is the first report of an avian cytokine enhancing an immune response, and confirms that coexpression of the antigen and adjuvant from a plasmid delivered by DNA immunization is an effective protocol.

Acta Crystallogr D Biol Crystallogr, 2004 Dec, 60(Pt 12 Pt 2), 2358 - 60
Crystallization and preliminary X-ray analysis of the Streptomyces olivaceoviridis NgcE binding protein of the ABC transporter for N-acetylglucosamine; Saito A et al.; The NgcE protein binds N-acetylglucosamine (GIcNAc) as well asN,N'-diacetylchitobiose and is a component of the ABC transporter Ngc for GIcNAc uptake in Streptomyces olivaceoviridis . After cloning the corresponding gene in an Escherichia coli host, the NgcE protein was overproduced in a soluble form within the cytoplasm and purified to homogeneity by four consecutive chromatographic processes . Crystals of NgcE that grew in the presence of 1 mM GlcNAc,20%(w/v) PEG MME 2000 and 100 mM Tris-HCI pH 8.5 had a plate-like shape and belonged to either space group P21212 (unit-cell parameters a = 59.9, b = 153.0, c = 41.7 A) or P212121 (a = 58.1, b = 96.3, c = 151.7 A) . The former crystals diffracted to 1.8 A resolution andthe latter to 2.2 A . Selenomethionine-containing crystals were generated under the same conditions and belonged to space group P212121 with unit-cell parameters a = 58.4, b = 96.6, c = 152.5 A, and diffracted to 2.0 A resolution.

Proteins . 2004 Dec 21; {Epub ahead of print}
A detailed unfolding pathway of a (beta/alpha)(8)-barrel protein as studied by molecular dynamics simulations; Akanuma S et al.; The (beta/alpha)(8)-barrel is the most common protein fold . Similar structural properties for folding intermediates of (beta/alpha)(8)-barrel proteins involved in tryptophan biosynthesis have been reported in a number of experimental studies; these intermediates have the last two beta-strands and three alpha-helices partially unfolded, with other regions of the polypeptide chain native-like in conformation . To investigate the detailed folding/unfolding pathways of these (beta/alpha)(8)-barrel proteins, temperature-induced unfolding simulations of N-(5'-phosphoribosyl)anthranilate isomerase from Escherichia coli were carried out using a special-purpose parallel computer system . Unfolding simulations at five different temperatures showed a sequential unfolding pathway comprised of several events . Early events in unfolding involved disruption of the last two strands and three helices, producing an intermediate ensemble similar to those detected in experimental studies . Then, denaturation of the first two betaalpha units and separation of the sixth strand from the fifth took place independently . The remaining central betaalphabetaalphabeta module persisted the longest during all simulations, suggesting an important role for this module as the incipient folding scaffold . Our simulations also predicted the presence of a nucleation site, onto which several hydrophobic residues condensed forming the foundation for the central betaalphabetaalphabeta module . Proteins 2005 . (c) 2004 Wiley-Liss, Inc.

Genet Mol Res, 2004 Sep 30, 3(3), 342 - 55
Pro domain peptide of HGCP-Iv cysteine proteinase inhibits nematode cysteine proteinases; Silva FB et al.; Cysteine proteinases (CPs) are synthesized as zymogens and converted to mature proteinase forms by proteolytic cleavage and release of their pro domain peptides . A cDNA encoding a papain-like CP, called hgcp-Iv, was isolated from a Heterodera glycines J2 cDNA library, expressed and utilized to assess the ability of its propeptide to inhibit proteinase in its active form . The hgcp-Iv cDNA sequence encodes a polypeptide of 374 amino acids with the same domain organization as other cathepsin L-like CPs, including a hydrophobic signal sequence and a pro domain region . HGCP-Iv, produced in Escherichia coli as a fusion protein with thioredoxin, degrades the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin and is inhibited by E-64, a substrate and inhibitor commonly used for functional characterization of CPs . Recombinant propeptides of HGCP-Iv, expressed in E . coli, presented high inhibitory activity in vitro towards its cognate enzyme and proteinase activity of Meloidogyne incognita females, suggesting its usefulness in inhibiting nematode CPs in biological systems . Cysteine proteinases from other species produced no noticeable activity.

J Biomol NMR, 2004 Nov, 30(3), 311 - 25
Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol; Torizawa T et al.; We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy . These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems . Endogenous amino acids normally present in E . coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein . This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids . The second problem encountered in conventional E . coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra . This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag . Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage . We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis.

J Biol Chem . 2004 Dec 21; {Epub ahead of print}
Isolation and characterization of griffithsin, a novel HIV-inactivating protein from the red alga griffithsia sp; Mori T et al.; Griffithsin (GRFT), a novel and potent anti-HIV protein, was isolated from an aqueous extract of the red alga Griffithsia sp . The 121 amino acid sequence of GRFT has been determined by a combination of enzymatic cleavage and Edman degradation . Biologically active GRFT was subsequently produced recombinantly by expression of a corresponding DNA sequence in Escherichia coli . Both native and recombinant GRFT displayed potent antiviral activity against laboratory strains and primary isolates of T- and M-tropic HIV-1 with EC50 values ranging from 0.043 to 0.63 nM . In addition, GRFT aborted cell-to-cell fusion and transmission of HIV-1 infection at similar concentrations . High concentrations (e.g . 783 nM) of GRFT were not lethal to any tested host cell types . GRFT blocked CD4-dependent gp120 binding to receptor-expressing cells and bound to viral coat glycoproteins (gp120, gp41, and gp160) in a glycosylation-dependent manner . GRFT preferentially inhibited gp120 binding of the HIV-neutralizing monoclonal antibody (mAb) 2G12, which recognizes a carbohydrate-dependent conformational motif, and the (mAb) 48d, which binds to CD4-induced epitope . In addition, GRFT moderately interfered with the binding of gp120 to both sCD4 and the mAb IgG1b12, which recognizes the CD4-binding site . Further data showed that the binding of GRFT to sgp120 was inhibited by the monosaccharides galactose, glucose, mannose and N-acetyl glucosamine, but not by xylose, fucose, N-acetyl galactosamine or sialic acid-containing glycoproteins . Taken together these data suggest that GRFT is a new type of lectin that binds to various viral glycoproteins in a monosaccharide-dependent manner . GRFT could be a potential candidate microbicide to prevent the sexual transmission of HIV and AIDS.

J Biol Chem . 2004 Dec 21; {Epub ahead of print}
Structural and biochemical evidence for an enzymatic quinone redox cycle in escherichia coli: Identification of a novel quinol monooxygenase; Adams MA et al.; Naturally synthesized quinones perform a variety of important cellular functions . Escherichia coli produce both ubiquinone and menaquinone, which are involved in electron transport . However, semiquinone intermediates produced during the one-electron reduction of these compounds, as well as through auto-oxidation of the hydroxyquinone product, generate reactive oxygen species that stress the cell . Here, we present the crystal structure of YgiN, a protein of hitherto unknown function . The three-dimensional fold of YgiN is similar to that of ActVA-Orf6 monooxygenase, which acts on hydroxyquinone substrates . YgiN shares a promoter with 'Modulator of drug activity B' (MdaB), a protein with activity similar to that of mammalian DT-diaphorase capable of reducing mendione . YgiN was able to re-oxidize menadiol, the product of the MdaB enzymatic reaction . We therefore refer to YgiN as Quinol Monooxygenase (QuMo) . MdaB is reported to be involved in the protection of cells from reactive oxygen species formed during single electron oxidation and reduction reactions . The enzymatic activities, together with the structural characterization of YgiN, lend evidence to the possible existence of a novel quinone redox cycle in E . coli.

Mol Microbiol, 2005 Jan, 55(1), 289 - 98
Sequential binding of SeqA protein to nascent DNA segments at replication forks in synchronized cultures of Escherichia coli; Yamazoe M et al.; Summary To demonstrate that sequestration A (SeqA) protein binds preferentially to hemimethylated GATC sequences at replication forks and forms clusters in Escherichia coli growing cells, we analysed, by the chromatin immunoprecipitation (ChIP) assay using anti-SeqA antibody, a synchronized culture of a temperature-sensitive dnaC mutant strain in which only one round of chromosomal DNA replication was synchronously initiated . After synchronized initiation of chromosome replication, the replication origin oriC was first detected by the ChIP assay, and other six chromosomal regions having multiple GATC sequences were sequentially detected according to bidirectional replication of the chromosome . In contrast, DNA regions lacking the GATC sequence were not detected by the ChIP assay . These results indicate that SeqA binds hemimethylated nascent DNA segments according to the proceeding of replication forks in the chromosome, and SeqA releases from the DNA segments when fully methylated . Immunofluorescence microscopy reveals that a single SeqA focus containing paired replication apparatuses appears at the middle of the cell immediately after initiation of chromosome replication and the focus is subsequently separated into two foci that migrate to 1/4 and 3/4 cellular positions, when replication forks proceed bidirectionally an approximately one-fourth distance from the replication origin towards the terminus . This supports the translocating replication apparatuses model.

Mol Microbiol, 2005 Jan, 55(1), 276 - 88
Disulphide trapping of an in vivo energy-dependent conformation of Escherichia coli TonB protein; Ghosh J et al.; Summary In Escherichia coli, the TonB system transduces the protonmotive force (pmf) of the cytoplasmic membrane to support a variety of transport events across the outer membrane . Cytoplasmic membrane proteins ExbB and ExbD appear to harvest pmf and transduce it to TonB . Experimental evidence suggests that TonB shuttles to the outer membrane, apparently to deliver conformationally stored potential energy to outer membrane transporters . In the most recent model, discharged TonB is then recycled to the cytoplasmic membrane to be re-energized by the energy coupling proteins, ExbB/D . It has been suggested that the carboxy-terminal 75 amino acids of active TonB could be represented by the rigid, strand-exchanged, dimeric crystal structure of the corresponding fragment . In contrast, recent genetic studies of alanine substitutions have suggested instead that in vivo the carboxy-terminus of intact TonB is dynamic and flexible . The biochemical studies presented here confirm and extend those results by demonstrating that individual cys substitution at aromatic residues in one monomeric subunit can form spontaneous dimers in vivo with the identical residue in the other monomeric subunit . Two energized TonBs appear to form a single cluster of 8-10 aromatic amino acids, including those found at opposite ends of the crystal structure . The aromatic cluster requires both the amino-terminal energy coupling domain of TonB, and ExbB/D (and cross-talk analogues TolQ/R) for in vivo formation . The large aromatic cluster is detected in cytoplasmic membrane-, but not outer membrane-associated TonB . Consistent with those observations, the aromatic cluster can form in the first half of the energy transduction cycle, before release of conformationally stored potential energy to ligand-loaded outer membrane transporters . The model that emerges is one in which, after input of pmf mediated through ExbB/D and the TonB transmembrane domain, the TonB carboxy-terminus can form a meta-stable high-energy conformation that is not represented by the crystal structure of the carboxy-terminus.

Mol Microbiol, 2005 Jan, 55(1), 250 - 60
Differential ability of sigma and sigma of Escherichia coli to utilize promoters containing half or full UP-element sites; Typas A et al.; Summary The sigma(s) subunit of RNA polymerase (RNAP) is the master regulator of the general stress response in Escherichia coli . Nevertheless, the selectivity of promoter recognition by the housekeeping sigma(70)-containing and sigma(5)-containing RNAP holoenzymes (Esigma(70) and Esigma(s) respectively) is not yet fully clarified, as they both recognize nearly identical -35 and -10 promoter consensus sequences . In this study, we show that in a subset of promoters, Esigma(s) favours the presence of a distal UP-element half-site, and at the same time is unable to take advantage of a proximal half-site or a full UP-element . This is reflected by the frequent occurrence of distal UP-element half-sites in natural sigma(s)-dependent promoters and the absence of proximal half-sites . Esigma(70), however, exhibits the opposite preference . The presence of the -35 element is a prerequisite for this differential behaviour . In the absence of the -35 element, half or full UP-element sites play no role in sigma selectivity, but the distal subsite leads to an equivalent, if not greater, transcriptional stimulation than the proximal one for both sigma factors . Finally, experiments using single amino acid substitutions of sigma(s) indicate that the foundation for this preference lies in an inability of sigma(s) to interact with the a subunit C-terminal domain.

Mol Microbiol, 2005 Jan, 55(1), 175 - 83
A cis-acting sequence involved in chromosome segregation in Escherichia coli; Fekete RA et al.; Summary Eukaryotic chromosomes contain a locus, the centromere, at which force is applied to separate replicated chromosomes . A centromere analogue is also found in some bacterial plasmids and chromosomes, although not yet identified in the well-studied Escherichia coli chromosome . We aimed to identify centromere-like sequences in E . coli with the premise that such sequences would be the first to migrate towards the cell poles, away from the cell centre where DNA replication is believed to occur . We have labelled different loci on the chromosome by integrating arrays of binding sites for LacI-EYFP and phage lambdacI-ECFP and supplying these fusion proteins in trans . Comparison of such pairs of loci suggests the presence of a centromere-like site close to the origin of replication . Polar migration of the site was dependent on migS, a locus recently implicated in chromosome migration, thus providing strong support for migS being the E . coli centromere.

Mol Microbiol, 2005 Jan, 55(1), 150 - 61
Heterologous expression of Aquifex aeolicus ribosome recycling factor in Escherichia coli is dominant lethal by forming a complex that lacks functional co-ordination for ribosome disassembly; Yamami T et al.; Summary Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome recycling factor (RRF) and elongation factor EF-G . Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E . coli RRF, the resulting complex is non-functional and dominant lethal in E . coli, even in the presence of homologous A . aeolicus EF-G . These findings suggest that the E . coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling . A chimeric EF-G (E . coli domains I-III, A . aeolicus domains IV-V) or an A . aeolicus EF-G with distinct mutations in the domain I-II interface could activate aaRRF . Furthermore, novel mutations that localize to one surface of the L-shape structure of aaRRF restored activity in E . coli . These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.

Mol Microbiol, 2005 Jan, 55(1), 137 - 49
Cell size and nucleoid organization of engineered Escherichia coli cells with a reduced genome; Hashimoto M et al.; Summary The minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell . Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome . We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome . Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells . The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope . Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes . Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change . Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.

Mol Microbiol, 2005 Jan, 55(1), 16 - 26
The TyrR regulon; Pittard J et al.; Summary The TyrR protein of Escherichia coli can act both as a repressor and as an activator of transcription . It can interact with each of the three aromatic amino acids, with ATP and, under certain circumstances, with the C-terminal region of the alpha-subunit of RNA polymerase . TyrR protein is a dimer in solution but in the presence of tyrosine and ATP it self-associates to form a hexamer . Whereas TyrR dimers can, in the absence of any aromatic amino acids, bind to certain recognition sequences referred to as 'strong TyrR boxes', hexamers can bind to extended sequences including lower-affinity sites called 'weak TyrR boxes', some of which overlap the promoter . There is no single mechanism for repression, which in some cases involves exclusion of RNA polymerase from the promoter and in others, interference with the ability of bound RNA polymerase to form open complexes or to exit the promoter . When bound to a site upstream of certain promoters, TyrR protein in the presence of phenylalanine, tyrosine or tryptophan can interact with the alpha-subunit of RNA polymerase to activate transcription . In one unusual case, activation of a non-productive promoter is used to repress transcription from a promoter on the opposite strand . Regulation of individual transcription units within the regulon reflects their physiological function and is determined by the position and nature of the recognition sites (TyrR boxes) associated with each of the promoters . The intracellular levels of the various forms of the TyrR protein are also postulated to be of critical importance in determining regulatory outcomes . TyrR protein remains a paradigm for a regulator that is able to interact with multiple cofactors and exert a range of regulatory effects by forming different oligomers on DNA and making contact with other proteins . A recent analysis identifying putative TyrR boxes in the E . coli genome raises the possibility that the TyrR regulon may extend beyond the well-characterized transcription units described in this review.

J Am Chem Soc, 2004 Dec 29, 126(51), 16930 - 6
Preparation and characterization of the native iron(II)-containing DNA repair AlkB protein directly from Escherichia coli; Mishina Y et al.; The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism . This protein belongs to a family of 2-ketoglutarate-Fe(II)-dependent dioxygenase proteins that utilize iron and 2-ketoglutarate to activate dioxygen for oxidation reactions . We report here the overexpression and isolation of the native Fe(II)-AlkB with a bound cofactor, 2-ketoglutarate, directly from E . coli . UV-vis measurements showed an absorption peak at 560 nm, which is characteristic of a bidentate 2-ketoglutarate bound to an iron(II) ion . Addition of excess amounts of single-stranded DNA to this isolated Fe(II)-AlkB protein caused a 9 nm shift of the 560 nm band to a higher energy, indicating a DNA-binding-induced geometry change of the active site . X-ray absorption spectra of the active site iron(II) in AlkB suggest a five-coordinate iron(II) center in the protein itself and a centrosymmetric six-coordinate iron(II) site upon addition of single-stranded DNA . This geometry change may play important roles in the DNA damage-searching and damage-repair functions of AlkB . These results provide direct evidence for DNA binding to AlkB which modulates the active site iron(II) geometry . The isolation of the native Fe(II)-AlkB also allows for further investigation of the iron(II) center and detailed mechanistic studies of the dioxygen-activation and damage-repair reactions performed by AlkB.

Arzneimittelforschung, 2004, 54(11), 711 - 4
Effects of 15-deoxy-delta12,14-prostaglandin J2 on the cyclooxygenase-2 expression in the murine lung in the presence of lipopolysaccharide; Inoue K et al.; A previous study has demonstrated that 15-deoxy-delta12,14-prostaglandin J2 (15d-PG J2) enhanced acute lung injury induced by lipopolysaccharide (LPS) in mice . The enhancement in acute lung injury by 15d-PG J2 was concomitant with the enhanced expression of interleukin-1beta and chemokines in the lung . However, other underlying mechanisms of this enhancement remain to be elucidated . Cyclooxygenase (COX)-2 has been reported to be involved in enhanced pulmonary permeability during acute lung injury . This study investigated the effects of 15d-PG J2 on COXs expressions in the lung in the presence or absence of LPS . ICR mice were divided into 4 experimental groups that intratracheally received vehicle, lipopolysaccharide (LPS: 125 microg/kg), 15d-PG J2 (1 mg/kg), or 15d-PG J2 + LPS . The expression of mRNA for both COX-1 and -2 in the lung was evaluated 4 h after the intratracheal administration . 15d-PG J2 enhanced the COX-2 mRNA expression in the presence of LPS . In contrast, 15d-PG J2 did not affect the COX-1 expression . These results suggest that the enhancing effects of 15d-PG J2 on LPS-induced acute lung injury might be explained, at least in part, by those on the lung expression of COX-2.

Genetika, 2004 Nov, 40(11), 1457 - 68
{Involvement of sigma S and sigma 70 subunits of RNA polymerase and the CRP protein in the regulation of microcin C51 operon expression}; Structure-function analysis of the yeast NAD+-dependent tRNA 2'-phosphotransferase Tpt1; Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USATpt1 is an essential 230-amino-acid enzyme that catalyzes the final step in yeast tRNA splicing: the transfer of the 2'-PO4 from the splice junction to NAD+ to form ADP-ribose 1''-2''cyclic phosphate and nicotinamide . To understand the structural requirements for Saccharomyces cerevisiae Tpt1 activity, we performed an alanine-scanning mutational analysis of 14 amino acids that are conserved in homologous proteins from fungi, metazoa, protozoa, bacteria, and archaea . We thereby identified four residues-Arg23, His24, Arg71, and Arg138-as essential for Tpt1 function in vivo . Structure-activity relationships at these positions were clarified by introducing conservative substitutions . The activity of the Escherichia coli ortholog KptA in complementing tpt1Delta was abolished by alanine substitutions at the equivalent side chains, Arg21, His22, Arg69, and Arg125 . Deletion analysis of Tpt1 shows that the C-terminal 20 amino acids, which are not conserved, are not essential for activity in vivo at 30 degrees C . These findings attest to the structural and functional conservation of Tpt1-like 2'-phosphotransferases and identify likely constituents of the active site.

J Immunol, 2005 Jan 1, 174(1), 340 - 4
The functional and structural properties of MD-2 required for lipopolysaccharide binding are absent in MD-1; Tsuneyoshi N et al.; MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins . MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4 . In vivo studies revealed that MD-1 and MD-2 have roles in responses to LPS . Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown . In this study, we compared the LPS-binding functions of MD-1 and MD-2 . LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2 . In contrast, binding was not observed for RP105/MD-1-transfected cells . When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS . In contrast, preparations of MD-1 did not contain LPS . When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LPS binding disappeared . Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions . An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site . We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids . A mutation at Phe(191) or Phe(121) strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring . The Phe residues are present in MD-2 and absent in MD-1 . Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.

J Immunol, 2005 Jan 1, 174(1), 195 - 204
Generation of murine CTL by a hepatitis B virus-specific peptide and evaluation of the adjuvant effect of heat shock protein glycoprotein 96 and its terminal fragments; Li H et al.; Previously, we reported that a 7-mer HLA-A11-restricted peptide (YVNTNMG) of hepatitis B virus (HBV) core Ag (HBcAg(88-94)) was associated with heat shock protein (HSP) gp96 in liver tissues of patients with HBV-induced hepatocellular carcinoma (HCC) . This peptide is highly homologous to a human HLA-A11-restricted 9-mer peptide (YVNVNMGLK) and to a mouse H-2-K(d)-restricted 9-mer peptide (SYVNTNMGL) . To further characterize its immunogenicity, BALB/c mice were vaccinated with the HBV 7-mer peptide . It was found that a specific CTL response was induced by the 7-mer peptide, although the response was approximately 50% of that induced by the mouse H-2-K(d)-restricted 9-mer peptide, as detected by ELISPOT, tetramer, and (51)Cr release assays . To evaluate the adjuvant effect of HSP gp96, mice were coimmunized with gp96 and the 9-mer peptide, and a significant adjuvant effect was observed with gp96 . To further determine whether the immune effect of gp96 was dependent on peptide binding, the N- and C-terminal fragments of gp96, which are believed to contain the putative peptide-binding domain, were cloned and expressed in Escherichia coli . CTL assays indicated that only the N-terminal fragment, but not the C-terminal fragment, was able to produce the adjuvant effect . These results clearly demonstrated the potential of using gp96 or its N-terminal fragment as a possible adjuvant to augment CTL response against HBV infection and HCC.

J Biol Chem . 2004 Dec 16; {Epub ahead of print}
Investigating the effects of mutations on protein aggregation in the cell; Calloni G et al.; The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology . Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilise the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures . We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behaviour of proteins are present . We have expressed, in E . coli cells, an E . coli protein domain, HypF-N . The results for a range of mutational variants indicate that while mutants with a conformational stability similar to that of the wild-type protein are soluble in the E . coli cytosol, variants with single point mutations predicted to destabilise the protein invariably aggregate after expression . We show, however, that aggregation of destabilised variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here this is achieved by an increase in the net charge of the protein . These results suggest that the principles being established to rationalise aggregation behaviour in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.

J Biol Chem . 2004 Dec 20; {Epub ahead of print}
HIV-1 Vif can directly inhibit APOBEC3G-mediated cytidine deamination by using a single amino acid interaction and without protein degradation; Santa-Marta M et al.; The human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3G (APOBEC3G), also known as CEM-15, is a host-cell factor involved in innate resistance to retroviral infection . HIV-1 viral infectivity factor (Vif) protein was shown to protect the virus from APOBEC3G-mediated viral cDNA hypermutation . The mechanism proposed for protection of the virus by HIV-1 Vif is mediated by APOBEC3G degradation through ubiquitination and the proteasomal pathway . Here we show that in Escherichia coli the APOBEC3G-induced cytidine deamination is inhibited by expression of Vif without depletion of deaminase . Moreover, inhibition of deaminase-mediated bacterial hypermutation is dependent on a single amino acid substitution D128K that renders APOBEC3G resistant to Vif inhibition . This single amino acid was elegantly proven by other authors to determine species specific sensitivity . Our results show that in bacteria this single amino acid substitution controls Vif-dependent blocking of APOBEC3G that is dependent on a strong protein interaction . The C-terminal region of Vif is the responsible for this strong protein-protein interaction . In conclusion, our experiments suggest a complement to the model of Vif-induced degradation of APOBEC3G by bringing to relevance that deaminase inhibition can also result from a direct interaction with Vif protein.

J Biol Chem . 2004 Dec 14; {Epub ahead of print}
Protein associations in DnaA-ATP hydrolysis mediated by the replicase clamp-Hda complex; Su'etsugu M et al.; In Escherichia coli, the activity of ATP-bound DnaA protein in initiating chromosomal replication is negatively controlled in a replication-coordinated manner . The RIDA (regulatory inactivation of DnaA) system promotes DnaA-ATP hydrolysis to produce the inactivated form DnaA-ADP in a manner depending on the Hda protein and the DNA-loaded form of the beta sliding clamp, a subunit of the replicase holoenzyme . A highly functional form of Hda was purified and shown to form a homodimer in solution, and two Hda dimers were found to associate with a single clamp molecule . Purified Hda mutant proteins were used in a staged in vitro RIDA system followed by pull-down assay to show that Hda-clamp binding is a prerequisite for DnaA-ATP hydrolysis and that binding is mediated by an Hda N-terminal motif . Arginine-168 in the AAA+ Box VII motif of Hda plays a role in stable homodimer formation and in DnaA-ATP hydrolysis but not in clamp-binding . Furthermore, the DnaA N-terminal domain is required for the functional interaction of DnaA with the Hda-clamp complex . Single cells contain about 50 Hda dimers, consistent with the results of in vitro experiments . These findings and the features of AAA+ proteins, including DnaA, suggest the following model: DnaA-ATP is hydrolyzed at a binding interface between the AAA+ domains of DnaA and Hda, the DnaA N-terminal domain supports this interaction, and the interaction of DnaA-ATP with the Hda-clamp complex occurs in a catalytic mode.

Mol Biochem Parasitol, 2005 Jan, 139(1), 33 - 9
Characterization of kinetics of DNA strand-exchange and ATP hydrolysis activities of recombinant PfRad51, a Plasmodium falciparum recombinase; Bhattacharyya MK et al.; Although homologous recombination-mediated DNA rearrangements are quite widespread in Plasmodium falciparum, the molecular mechanisms involved are essentially unknown . Recent identification of PfRad51 in P . falciparum has suggested that it may play central role during homologous recombination and DNA rearrangements . Full-length recombinant PfRad51 was over expressed in Escherichia coli and purified to near homogeneity . Using optimized enzymatic activity conditions recombinant PfRad51 protein was shown to catalyze DNA strand-exchange reaction, a central step during homologous recombination . Unlike bacterial RecA protein, PfRad51 promoted strand-exchange reaction does not require ATP hydrolysis . The PfRad51 protein also catalyzed ssDNA-dependent ATP hydrolysis and the k(cat) values were similar to those reported for human Rad51 . The demonstration of strand-exchange activity of PfRad51 protein, first such report in any protozoan parasite, suggests importance of similar recombination mechanism during DNA rearrangements associated with antigenic variation in P . falciparum.

Pediatr Allergy Immunol, 2004 Dec, 15(6), 513 - 6
Monocyte switch in neonates: high phagocytic capacity and low HLA-DR expression in VLBWI are inverted during gestational aging; Hallwirth U et al.; Pre-term neonates are at high risk to develop early-onset sepsis which possibly is caused by an immature immune system . Monocytes play a pivotal role as professional phagocytic and antigen-presenting cells in the innate immunity . In the present study, we investigated in monocytes from cord blood the expression of human leukocyte antigen (HLA)-DR as a marker for antigen-presenting capability, the expression of the high-affinity receptor for IgG (FcgammaRI/CD64), and the capacity to phagocytize non-opsonized Escherichia coli . We compared 70 infants in three groups according to their gestational age (group I: 20 very low birth weight infants (VLBWI), 24-31 weeks of gestation; group II: 25 pre-term infants, 32-36 weeks of gestation, and group III: 25 term neonates) . The expression of CD64 as well as the phagocytic capacity of monocytes from cord blood were highest in VLBWI (p < 0.05 and p < 0.01, respectively) . In contrast, HLA-DR expression was significantly (p < 0.05) diminished in VLBWI, which possibly leads to a reduced antigen-presenting capacity . We conclude that monocytes have different functional properties during gestational aging, which perhaps participate in the high incidence of infections in VLBWI.

Kidney Int, 2005 Jan, 67(1), 111 - 21
Renal tubular triglyercide accumulation following endotoxic, toxic, and ischemic injury; Zager RA et al.; Renal tubular triglyercide accumulation following endotoxic, toxic, and ischemic injury . Background . Cholesterol accumulates in renal cortical proximal tubules in response to diverse forms of injury or physiologic stress . However, the fate of triglycerides after acute renal insults is poorly defined . This study sought new insights into this issue . Methods . CD-1 mice were subjected to three diverse models of renal stress: (1) endotoxemia {Escherichia coli lipopolysaccharide (LPS), injection}; (2) ischemia/reperfusion (I/R); or (3) glycerol-induced rhabdomyolysis . Renal cortical, or isolated proximal tubule, triglyceride levels were measured approximately 18 hours later . To gain mechanistic insights, triglyceride levels were determined in (1) proximal tubules following exogenous phospholipase A(2) (PLA(2)) treatment; (2) cultured HK-2 cells after mitochondrial blockade (antimycin A) +/- serum; or (3) HK-2 cells following "septic" (post-LPS) serum, or exogenous fatty acid (oleate) addition . Results . Each form of in vivo injury evoked three-to fourfold triglyceride increases in renal cortex and/or proximal tubules . PLA(2) treatment of proximal tubules evoked acute, dose-dependent, triglyceride formation . HK-2 cell triglyceride levels rose with antimycin A . With serum present, antimycin A induced an exaggerated triglyceride loading state (vs . serum alone or antimycin A alone) . "Septic" serum stimulated HK-2 triglyceride formation (compared to control serum) . Oleate addition caused striking HK-2 cell triglyceride accumulation . Following oleate washout, HK-2 cells were sensitized to adenosine triphosphate (ATP) depletion or oxidant attack . Conclusion . Diverse forms of renal injury induce dramatic triglyceride loading in proximal tubules/renal cortex, suggesting that this is a component of a cell stress response . PLA(2) activity, increased triglyceride/triglyceride substrate (e.g., fatty acid) uptake, and possible systemic cytokine (e.g., from LPS) stimulation, may each contribute to this result . Finally, in addition to being a marker of prior cell injury, accumulation of triglyceride (or of its constituent fatty acids) may predispose tubules to superimposed ATP depletion or oxidant attack.

Biochemistry, 2004 Dec 28, 43(51), 16515 - 24
Analysis of non-template-directed nucleotide addition and template switching by DNA polymerase; Garcia PB et al.; DNA polymerases use an uninterrupted template strand to direct synthesis of DNA . However, some DNA polymerases can synthesize DNA across two discontinuous templates by binding and juxtaposing them, resulting in synthesis across the junction . Primer/template duplexes with 3' overhangs are especially efficient substrates, suggesting that DNA polymerases use the overhangs as regions of microhomology for template synapsis . The formation of these overhangs may be the result of non-template-directed nucleotide addition by DNA polymerases . To examine the relative magnitude and mechanism of template switching, we studied the in vitro enzyme kinetics of template switching and non-template-directed nucleotide addition by the 3'-5' exonuclease-deficient large fragment of Escherichia coli DNA polymerase I . Non-template-directed nucleotide addition and template switching were compared to that of standard primer extension . We found that non-template-directed nucleotide addition and template switching showed similar rates and were approximately 100-fold slower than normal template-directed DNA synthesis . Furthermore, non-template-directed nucleotide addition showed a 10-fold preference for adding dAMP to the ends of DNA over that of the other three nucleotides . For template switching, kinetic analysis revealed that the two template substrates acted as a random bireactant system with mixed-type inhibition of substrate binding by one substrate over the other . These data are the first to establish the binding kinetics of two discontinuous DNA substrates to a single DNA polymerase . Our results suggest that although the activities are relatively weak, non-template-directed nucleotide addition and template switching allow DNA polymerases to overcome breaks in the template strand in an error-prone manner.

Biochemistry, 2004 Dec 28, 43(51), 16505 - 14
Biophysical characterization of human XRCC1 and its binding to damaged and undamaged DNA; Mani RS et al.; The human DNA repair protein, hXRCC1, which is required for DNA single-strand break repair and genetic stability was produced as a histidine-tagged polypeptide in Escherichia coli, purified by affinity chromatography, and subjected to sedimentation and spectroscopic analyses . This study represents the first biophysical examination of full-length XRCC1 . Sedimentation equilibrium measurements indicated that hXRCC1 exists as a monomer at lower protein concentrations but forms a dimer at higher protein concentrations with a K(d) of 5.7 x 10(-)(7) M . The size and shape of hXRCC1 in solution were determined by analytical ultracentrifugation studies . The protein exhibited an intrinsic sedimentation coefficient, s(0)(20,w), of 3.56 S and a Stokes radius, R(s), of 44.5 A, which together with the M(r) of 68000 suggested that hXRCC1 is a moderately asymmetric protein with an axial ratio of 7.2 . Binding of model ligands, representing single-strand breaks with either a nick or a single nucleotide gap, quenched protein fluorescence, and binding affinities and stoichiometries were determined by carrying out fluorescence titrations as a function of ligand concentration . XRCC1 bound both nicked and 1 nucleotide-gapped DNA substrates tightly in a stoichiometric manner (1:1) with K(d) values of 65 and 34 nM, respectively . However, hXRCC1 exhibited lower affinities for a duplex with a 5 nucleotide gap, the intact duplex with no break, and a single-stranded oligonucleotide with K(d) values of 215, 230, and 260 nM, respectively . Our results suggest that hXRCC1 exhibits preferential binding to DNA with single-strand breaks with a gap size of <5 nucleotides.

Biochemistry, 2004 Dec 28, 43(51), 16432 - 41
Further investigation on the turnover of Escherichia coli biotin synthase with dethiobiotin and 9-mercaptodethiobiotin as substrates; Tse Sum Bui B et al.; Biotin synthase, a member of the "radical-SAM" family, produces biotin by inserting a sulfur atom between C-6 and C-9 of dethiobiotin . Each of the two saturated carbon atoms is activated through homolytic cleavage of a C-H bond by a deoxyadenosyl radical, issued from the monoelectronic reduction of S-adenosylmethionine (SAM or AdoMet) . An important unexplained observation is that the enzyme produces only 1 mol of biotin per enzyme monomer . Some possible reasons for this absence of multiple turnovers are considered here, in connection with the postulated mechanisms . There is a general agreement among several groups that the active form of biotin synthase contains one (4Fe-4S)(2+,1+) center, which mediates the electron transfer to AdoMet, and one (2Fe-2S)(2+) center, which is considered the sulfur source {Ugulava, N . B., Sacanell, C . J., and Jarrett, J . T . (2001) Biochemistry 40, 8352-8358; Tse Sum Bui, B., Benda, R., Schunemann, V., Florentin, D., Trautwein, A . X., and Marquet, A . (2003) Biochemistry 42, 8791-8798; Jameson, G . N . L., Cosper, M . M., Hernandez, H . L., Johnson, M . K., and Huynh, B . H . (2004) Biochemistry 43, 2022-2031} . An alternative hypothesis considers that biotin synthase has a pyridoxal phosphate (PLP)-dependent cysteine desulfurase activity, producing a persulfide which could be the sulfur donor . The absence of turnover was explained by the inhibition due to deoxyadenosine, an end product of the reaction {Ollagnier-de Choudens, S., Mulliez, E., and Fontecave, M . (2002) FEBS Lett . 535, 465-468} . In this work, we show that our purified enzyme has no cysteine desulfurase activity and the required sulfide has to be added as Na(2)S . It cannot be replaced by cysteine, and consistently, PLP has no effect . We observed that deoxyadenosine does not inhibit the reaction either . On the other hand, if the (2Fe-2S)(2+) center is the sulfur source, its depletion after reaction could explain the absence of turnover . We found that after addition of fresh cofactors, including Fe(2+) and S(2)(-), either to the assay when one turn is completed or after purification of the reacted enzyme by different techniques, only a small amount of biotin (0.3-0.4 equiv/monomer) is further produced . This proves that an active enzyme cannot be fully reconstituted after one turn . When 9-mercaptodethiobiotin, which already contains the sulfur atom of biotin, is used as the substrate, the same turnover of one is observed, with similar reaction rates . We postulate that the same intermediate involving the (2Fe-2S) cluster is formed from both substrates, with a rate-determining step following the formation of this intermediate.

Biochemistry, 2004 Dec 28, 43(51), 16243 - 53
Identification of membrane-anchoring domains of RLIP76 using deletion mutant analyses; Yadav S et al.; RLIP76 (RALBP1) is a multifunctional transporter involved in signaling and transmembrane movement of solute allocrites, which include glutathione conjugates and several natural product antineoplastic agents {Awasthi, S., et al . (2000) Biochemistry 39, 9327-9334; (2001) Biochemistry 40, 4159-4168} . Our previous studies suggested that the membrane-anchoring domain resides in the N-terminus of RLIP76, despite the lack of identifiable membrane-spanning domains . Amino acid sequence analysis indicated that this region of RLIP76 contains sequences that are similar to those of vector peptides . We, therefore, have studied the effect of a series of deletion mutant proteins on hydrophobicity and transport activity . RLIP76 or one of its derived deletion mutants was expressed in Escherichia coli, and bacteria were lysed and extracted in buffer without or with the nonionic detergent polidocanol . The ratio of RLIP76 in the detergent/aqueous extracts was found to be 2.5 for the wild-type protein, but decreased to 0.7 in the mutant in which amino acids 154-219 were deleted . Deletion of only one segment of this region (amino acids 171-185) alone resulted in a significant decrease in this ratio to 1.0 . For the mutants with deletions within the region from amino acid 154 to 219, loss of hydrophobicity correlated with less incorporation of mutants into artificial liposomes, and decreased transport activity toward doxorubicin and dinitrophenyl-S-glutathione . In contrast, deletion of one of the two ATP-binding sites (at amino acids 65-80 or 415-448) or both sites did not affect hydrophobicity but reduced or abrogated transport activity . NSCLC (H358) stably transfected with del171-185 and del154-219 showed that loss of these regions results in a decrease in the extent of membrane association of RLIP76 . Confocal laser immunohistochemistry colocalized amino acids 171-185 with her2/neu on the cell surface . Depletion of wild-type RLIP76 using si-RNA directed to this region in cells transfected with del171-185 resulted in the loss of cell surface expression . These finding demonstrate that amino acids 171-185 constitute a cell surface epitope which is necessary for optimal transport of anthracycline and glutathione conjugates by RLIP76, and that this peptide could be a novel target for antineoplastic therapy.

Biochemistry, 2004 Dec 28, 43(51), 16142 - 52
Crystal structures of Escherichia coli RecA in complex with MgADP and MnAMP-PNP; Xing X et al.; RecA catalyzes the DNA pairing and strand-exchange steps of homologous recombination, an important mechanism for repair of double-stranded DNA breaks . The binding of RecA to DNA is modulated by adenosine nucleotides . ATP increases the affinity of RecA for DNA, while ADP decreases the affinity . Previously, the crystal structures of E . coli RecA and its complex with ADP have been determined to resolutions of 2.3 and 3.0 A, respectively, but the model for the RecA-ADP complex did not include magnesium ion or side chains . Here, we have determined the crystal structures of RecA in complex with MgADP and MnAMP-PNP, a nonhydrolyzable analogue of ATP, at resolutions of 1.9 and 2.1 A, respectively . Both crystals grow in the same conditions and have RecA in a right-handed helical form with a pitch of approximately 82 A . The crystal structures show the detailed interactions of RecA with the nucleotide cofactors, including the metal ion and the gamma phosphate of AMP-PNP . There are very few conformational differences between the structures of RecA bound to ADP and AMP-PNP, which differ from uncomplexed RecA only in a slight opening of the P-loop residues 66-73 upon nucleotide binding . To interpret the functional significance of the structure of the MnAMP-PNP complex, a coprotease assay was used to compare the ability of different nucleotides to promote the active, extended conformation of RecA . Whereas ATPgammaS and ADP-AlF(4) facilitate a robust coprotease activity, ADP and AMP-PNP do not activate RecA at all . We conclude that the crystal structure of the RecA-MnAMP-PNP complex represents a preisomerization state of the RecA protein that exists after ATP has bound but before the conformational transition to the active state.

Biochemistry, 2004 Dec 28, 43(51), 16092 - 105
Substrate discrimination by formamidopyrimidine-DNA glycosylase: distinguishing interactions within the active site; Perlow-Poehnelt RA et al.; Reactive oxygen species are byproducts of normal aerobic respiration and ionizing radiation, and they readily react with DNA to form a number of base lesions, including the mutagenic 8-oxo-7,8-dihydroguanine (8-oxoG), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), 4,6-diamino-5-formamidopyrimidine (FapyA), and 8-oxo-7,8-dihydroadenine (8-oxoA) . Such oxidative lesions are removed by the base excision repair pathway, which is initiated by DNA glycosylases such as the formamidopyrimidine-DNA glycosylase (Fpg) in Escherichia coli . The 8-oxoG, FapyG, and FapyA lesions are bound and excised by Fpg, while structurally similar 8-oxoA is excised by Fpg very poorly . We carried out molecular modeling and molecular dynamics simulations to interpret substrate discrimination within the active site of E . coli Fpg . Lys-217 and Met-73 were identified as residues playing important roles in the recognition of the oxidized imidazole ring in the substrate bases, and the Watson-Crick edge of the damaged base plays a role in optimally positioning the base within the active site . The recognition and excision of FapyA likely result from the opened imidazole ring, while 8-oxoA's lack of flexibility and closed imidazole ring may contribute to Fpg's inability to excise this base . Different interactions between each base and the enzyme specificity pocket account for differential treatment of the various lesions by this enzyme, and thus elucidate the structure-function relationship involved in an initial step of base excision repair.

Biochemistry, 2004 Dec 28, 43(51), 16046 - 55
Conformational changes in the active site loops of dihydrofolate reductase during the catalytic cycle; Venkitakrishnan RP et al.; Escherichia coli dihydrofolate reductase (DHFR) has several flexible loops surrounding the active site that play a functional role in substrate and cofactor binding and in catalysis . We have used heteronuclear NMR methods to probe the loop conformations in solution in complexes of DHFR formed during the catalytic cycle . To facilitate the NMR analysis, the enzyme was labeled selectively with {(15)N}alanine . The 13 alanine resonances provide a fingerprint of the protein structure and report on the active site loop conformations and binding of substrate, product, and cofactor . Spectra were recorded for binary and ternary complexes of wild-type DHFR bound to the substrate dihydrofolate (DHF), the product tetrahydrofolate (THF), the pseudosubstrate folate, reduced and oxidized NADPH cofactor, and the inactive cofactor analogue 5,6-dihydroNADPH . The data show that DHFR exists in solution in two dominant conformational states, with the active site loops adopting conformations that closely approximate the occluded or closed conformations identified in earlier X-ray crystallographic analyses . A minor population of a third conformer of unknown structure was observed for the apoenzyme and for the disordered binary complex with 5,6-dihydroNADPH . The reactive Michaelis complex, with both DHF and NADPH bound to the enzyme, could not be studied directly but was modeled by the ternary folate:NADP(+) and dihydrofolate:NADP(+) complexes . From the NMR data, we are able to characterize the active site loop conformation and the occupancy of the substrate and cofactor binding sites in all intermediates formed in the extended catalytic cycle . In the dominant kinetic pathway under steady-state conditions, only the holoenzyme (the binary NADPH complex) and the Michaelis complex adopt the closed loop conformation, and all product complexes are occluded . The catalytic cycle thus involves obligatory conformational transitions between the closed and occluded states . Parallel studies on the catalytically impaired G121V mutant DHFR show that formation of the closed state, in which the nicotinamide ring of the cofactor is inserted into the active site, is energetically disfavored . The G121V mutation, at a position distant from the active site, interferes with coupled loop movements and appears to impair catalysis by destabilizing the closed Michaelis complex and introducing an extra step into the kinetic pathway.

Vet Res Commun, 2004 Nov, 28(8), 711 - 8
Some endotoxin-induced clinical and biochemical changes in plasma of camels (Camelus dromedarius); Al-Dughaym AM; Intravenous administration of endotoxin, prepared from E . coli serotype O55:B5, at a dose of 0.1 microg/kg body weight to calves and adult camels induced fever and increased haematocrit, triiodothyronine and cortisol values . The endotoxin-treated animals showed significantly decreased (p < 0.05) total protein, urea, glucose and creatinine . A significant increase was seen in the activity of aspartate aminotransaminase and creatine kinase . These results demonstrate high sensitivity of camels to endotoxin.

Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D334 - 7
EcoCyc: a comprehensive database resource for Escherichia coli; Keseler IM et al.; The EcoCyc database is a comprehensive source of information on the biology of the prototypical model organism Escherichia coli K12 . The mission for EcoCyc is to contain both computable descriptions of, and detailed comments describing, all genes, proteins, pathways and molecular interactions in E.coli . Through ongoing manual curation, extensive information such as summary comments, regulatory information, literature citations and evidence types has been extracted from 8862 publications and added to Version 8.5 of the EcoCyc database . The EcoCyc database can be accessed through a World Wide Web interface, while the downloadable Pathway Tools software and data files enable computational exploration of the data and provide enhanced querying capabilities that web interfaces cannot support . For example, EcoCyc contains carefully curated information that can be used as training sets for bioinformatics prediction of entities such as promoters, operons, genetic networks, transcription factor binding sites, metabolic pathways, functionally related genes, protein complexes and protein-ligand interactions.

Protein Sci, 2005 Jan, 14(1), 148 - 58
Cloning and expression of multiple integral membrane proteins from Mycobacterium tuberculosis in Escherichia coli; Korepanova A et al.; Seventy integral membrane proteins from the Mycobacterium tuberculosis genome have been cloned and expressed in Escherichia coli . A combination of T7 promoter-based vectors with hexa-His affinity tags and BL21 E . coli strains with additional tRNA genes to supplement sparsely used E . coli codons have been most successful . The expressed proteins have a wide range of molecular weights and number of transmembrane helices . Expression of these proteins has been observed in the membrane and insoluble fraction of E . coli cell lysates and, in some cases, in the soluble fraction . The highest expression levels in the membrane fraction were restricted to a narrow range of molecular weights and relatively few transmembrane helices . In contrast, overexpression in insoluble aggregates was distributed over a broad range of molecular weights and number of transmembrane helices.

Biochem Biophys Res Commun, 2005 Jan 28, 326(4), 766 - 76
Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site; Shioi S et al.; PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation . Here we report a 2.0-A-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli . The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold . The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB . Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue . The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed.

FEMS Immunol Med Microbiol, 2005 Jan 1, 43(1), 67 - 72
Pathogenic enteric Escherichia coli in children with and without diarrhea in Maputo, Mozambique; Rappelli P et al.; A study was conducted on the circulation of potentially diarrheagenic Escherichia coli in two groups of children, both under the age of seven . The first group (548 children) suffered from mild diarrhea and attended the Xipamanine Health Center of Maputo, in Mozambique . The second group (380 children) included randomly chosen, asymptomatic, children from the same population . A total of 503 E . coli strains were isolated from the two groups of children (n=375 and 128, respectively) . All E . coli strains were genotypically and phenotypically screened . The presence of virulence-associated genes was assessed by a set of multiplex PCR specific for st and lt genes of enterotoxic Escherichia coli (ETEC), eae and bfpA genes of enteropathogenic E . coli (EPEC), stx(1) and stx(2) of enterohemorrhagic E . coli (EHEC), ial of enteroinvasive E . coli (EIEC) and the species-specific gene uidA . Adhesion and citotoxicity of isolated E . coli were evaluated in vitro on different cell cultures . A total of 37 isolates harbored virulence-associated genes: 18 were classified as ETEC, (15 from symptomatic, and three from asymptomatic children), 16 as EPEC (respectively, 13 and 3) and three EIEC in the symptomatic group . No stx(1) or stx(2) genes, associated with enterohemorrhagic E . coli were found . On the basis of the adhesion pattern on HeLa cells, 167 E . coli were classified as diffusely adhering, (125 in patients and 42 in controls) and 67 as enteroaggregative, (50 and 17, respectively) . To the best of our knowledge, this is the first report in the literature on the circulation of potentially diarrheagenic E . coli in Mozambique.

J Biotechnol, 2005 Jan 26, 115(2), 113 - 28
Advanced genetic strategies for recombinant protein expression in Escherichia coli; Sorensen HP et al.; Preparations enriched by a specific protein are rarely easily obtained from natural host cells . Hence, recombinant protein production is frequently the sole applicable procedure . The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis . Escherichia coli facilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large number of compatible tools available for biotechnology . Especially the variety of available plasmids, recombinant fusion partners and mutant strains have advanced the possibilities with E . coli . Although often simple for soluble proteins, major obstacles are encountered in the expression of many heterologous proteins and proteins lacking relevant interaction partners in the E . coli cytoplasm . Here we review the current most important strategies for recombinant expression in E . coli . Issues addressed include expression systems in general, selection of host strain, mRNA stability, codon bias, inclusion body formation and prevention, fusion protein technology and site-specific proteolysis, compartment directed secretion and finally co-overexpression technology . The macromolecular background for a variety of obstacles and genetic state-of-the-art solutions are presented.

J Biotechnol, 2005 Jan 12, 115(1), 23 - 34
An efficient expression system for the production of functionally active human LKB1; Martinez-Torrecuadrada JL et al.; Human LKB1, also known as STK11, is a tumour-suppression protein that mediates important functions in cellular proliferation and polarization . It might constitute an important target in cancer therapy . In order to produce large amounts of recombinant protein for biochemical and functional studies, a full-length cDNA clone was subcloned and expressed in Escherichia coli and insect cells . Although fusion proteins corresponding to LKB1 with 6xHis, GST and MBP tags could be overexpressed in E . coli, only MBP-LKB1 was recovered in a soluble, but heavily degraded form . Further studies demonstrated that this protein was not functional . Subsequent expression in insect cells of LKB1 with 6xHis and GST tags yielded insoluble products also . However, when chaperones Hsp70 and its cofactors Hsp40 and Hsdj were co-expressed with GST-LKB1, a clear increase in the solubility of the final protein was obtained . Moreover, this soluble, purified recombinant GST-LKB1 demonstrated to be a phosphoprotein, with at least residue Ser325 phosphorylated . The purified protein was functionally active as being able to demonstrate autophosphorylation in the absence of any associated kinase.

J Biochem Mol Biol, 2004 Nov 30, 37(6), 709 - 14
A point mutation at the C-terminal half of the repressor of temperate mycobacteriophage L1 affects its binding to the operator DNA; Ganguly T et al.; The wild-type repressor CI of temperate mycobacteriophage L1 and the temperature-sensitive (ts) repressor CIts391 of a mutant L1 phage, L1cIts391, have been separately overexpressed in E . coli . Both these repressors were observed to specifically bind with the same cognate operator DNA . The operator-binding activity of CIts391 was shown to differ significantly than that of the CI at 32 to 42 degrees C . While 40-95% operator-binding activity was shown to be retained at 35 to 42 degrees C in CI, more than 75% operator-binding activity was lost in CIts391 at 35 to 38 degrees C, although the latter showed only 10% less binding compared to that of the former at 32 degrees C . The CIts391 showed almost no binding at 42 degrees C . An in vivo study showed that the CI repressor inhibited the growth of a clear plaque former mutant of the L1 phage more strongly than that of the CIts391 repressor at both 32 and 42 degrees C . The half-life of the CIts391-operator complex was found to be about 8 times less than that of the CI-operator complex at 32 degrees C . Interestingly, the repressor-operator complexes preformed at 0 degrees C have shown varying degrees of resistance to dissociation at the temperatures which inhibit the formation of these complexes are inhibited . The CI repressor, but not that of CIts391, regains most of the DNA-binding activity on cooling to 32 degrees C after preincubation at 42 to 52 degrees C . All these data suggest that the 131(st) proline residue at the C-terminal half of CI, which changed to leucine in the CIts391, plays a crucial role in binding the L1 repressor to the cognate operator DNA, although the helix-turn-helix DNA-binding motif of the L1 repressor is located at its N-terminal end.

Microb Cell Fact . 2004 Dec 17;3(1):16 {Epub ahead of print}
Directed evolution of single-chain Fv for cytoplasmic expression using the beta-galactosidase complementation assay results in proteins highly susceptible to protease degradation and aggregation; Philibert P et al.; BACKGROUND: Antibody fragments are molecules widely used for diagnosis and therapy . A large amount of protein is frequently required for such applications . New approaches using folding reporter enzymes have recently been proposed to increase soluble expression of foreign proteins in Escherichia coli . To date, these methods have only been used to screen for proteins with better folding properties but have never been used to select from a large library of mutants . In this paper we apply one of these methods to select mutations that increase the soluble expression of two antibody fragments in the cytoplasm of E . coli . RESULTS: We used the beta-galactosidase alpha-complementation system to monitor and evolve two antibody fragments for high expression levels in E . coli cytoplasm . After four rounds of mutagenesis and selection from large library repertoires (>107 clones), clones exhibiting high levels of beta-galactosidase activity were isolated . These clones expressed a higher amount of soluble fusion protein than the wild type in the cytoplasm, particularly in a strain deficient in the cytoplasmic Lon protease . The increase in the soluble expression level of the unfused scFv was, however, much less pronounced, and the unfused proteins proved to be more aggregation prone than the wild type . In addition, the soluble expression levels were not correlated with the beta-galactosidase activity present in the cells . CONCLUSION: This is the first report of a selection for soluble protein expression using a fusion reporter method . Contrary to anticipated results, high enzymatic activity did not correlate with the soluble protein expression level . This was presumably due to free alpha-peptide released from the protein fusion by the host proteases . This means that the alpha-complementation assay does not sense the fusion expression level, as hypothesized, but rather the amount of free released alpha-peptide . Thus, the system does not select, in our case, for higher soluble protein expression level but rather for higher protease susceptibility of the fusion protein.

Eur J Biochem, 2004 Dec, 271(23-24), 4921 - 31
Aptamers to Escherichia coli core RNA polymerase that sense its interaction with rifampicin, sigma-subunit and GreB; Kulbachinskiy A et al.; Bacterial RNA polymerase (RNAP) is the central enzyme of gene expression that is responsible for the synthesis of all types of cellular RNAs . The process of transcription is accompanied by complex structural rearrangements of RNAP . Despite the recent progress in structural studies of RNAP, detailed mechanisms of conformational changes of RNAP that occur at different stages of transcription remain unknown . The goal of this work was to obtain novel ligands to RNAP which would target different epitopes of the enzyme and serve as specific probes to study the mechanism of transcription and conformational flexibility of RNAP . Using in vitro selection methods, we obtained 13 classes of ssDNA aptamers against Escherichia coli core RNAP . The minimal nucleic acid scaffold (an oligonucleotide construct imitating DNA and RNA in elongation complex), rifampicin and the sigma(70)-subunit inhibited binding of the aptamers to RNAP core but did not affect the dissociation rate of preformed RNAP-aptamer complexes . We argue that these ligands sterically block access of the aptamers to their binding sites within the main RNAP channel . In contrast, transcript cleavage factor GreB increased the rate of dissociation of preformed RNAP-aptamer complexes . This suggested that GreB that binds RNAP outside the main channel actively disrupts RNAP-aptamer complexes by inducing conformational changes in the channel . We propose that the aptamers obtained in this work will be useful for studying the interactions of RNAP with various ligands and regulatory factors and for investigating the conformational flexibility of the enzyme.

Eur J Biochem, 2004 Dec, 271(23-24), 4865 - 71
Classification of ATP-dependent proteases Lon and comparison of the active sites of their proteolytic domains; Rotanova TV et al.; ATP-dependent Lon proteases belong to the superfamily of AAA(+) proteins . Until recently, the identity of the residues involved in their proteolytic active sites was not elucidated . However, the putative catalytic Ser-Lys dyad was recently suggested through sequence comparison of more than 100 Lon proteases from various sources . The presence of the catalytic dyad was experimentally confirmed by site-directed mutagenesis of the Escherichia coli Lon protease and by determination of the crystal structure of its proteolytic domain . Furthermore, this extensive sequence analysis allowed the definition of two subfamilies of Lon proteases, LonA and LonB, based on the consensus sequences in the active sites of their proteolytic domains . These differences strictly associate with the specific characteristics of their AAA(+) modules, as well as with the presence or absence of an N-terminal domain.

Eur J Biochem, 2004 Dec, 271(23-24), 4845 - 54
Cloning, over-expression, purification and characterization of Plasmodium falciparum enolase; Pal-Bhowmick I et al.; We have cloned, over-expressed and purified enolase from Plasmodium falciparum strain NF54 in Escherichia coli in active form, as an N-terminal His(6)-tagged protein . The sequence of the cloned enolase from the NF54 strain is identical to that of strain 3D7 used in full genome sequencing . The recombinant enolase (r-Pfen) could be obtained in large quantities ( approximately 50 mg per litre of culture) in a highly purified form (> 95%) . The purified protein gave a single band at approximately 50 kDa on SDS/PAGE . MALDI-TOF analysis gave a mean +/- SD mass of 51396 +/- 16 Da, which is in good agreement with the mass calculated from the sequence . The molecular mass of r-Pfen determined in gel-filtration experiments was approximately 100 kDa, indicating that P . falciparum enolase is a homodimer . Kinetic measurements using 2-phosphoglycerate as substrate gave a specific activity of approximately 30 U.mg(-1) and K(m2PGA) = 0.041 +/- 0.004 mm . The Michaelis constant for the reverse reaction (K(mPEP)) is 0.25 +/- 0.03 mm . pH-dependent activity measurements gave a maximum at pH 7.4-7.6 irrespective of the direction of catalysis . The activity of this enzyme is inhibited by Na(+), whereas K(+) has a slight activating effect . The cofactor Mg(2+) has an apparent activation constant of 0.18 +/-0.02 mm . However, at higher concentrations, it has an inhibitory effect . Polyclonal antibody raised against pure recombinant P . falciparum enolase in rabbit showed high specificity towards recombinant protein and is also able to recognize enolase from the murine malarial parasite, Plasmodium yoelii, which shares 90% identity with the P . falciparum protein.

Eur J Biochem, 2004 Dec, 271(23-24), 4834 - 4844
Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus; Cacciapuoti G et al.; The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography . The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies . The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction . 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit . Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges . The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 m after 22 h incubation . This value decreases to 2.0 m in the presence of 30 mm dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability . The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, fluorescence measurements and SDS/PAGE . The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.

Eur J Biochem, 2004 Dec, 271(23-24), 4769 - 78
Escherichia coli cyclopropane fatty acid synthase; Courtois F et al.; Escherichia coli fatty acid cyclopropane synthase (CFAS) was overproduced and purified as a His(6)-tagged protein . This recombinant enzyme is as active as the native enzyme with a K(m) of 90 microm for S-AdoMet and a specific activity of 5 x 10(-2) micromol.min(-1).mg(-1) . The enzyme is devoid of organic or metal cofactors and is unable to catalyze the wash-out of the methyl protons of S-AdoMet to the solvent, data that do not support the ylide mechanism . Inactivation of the enzyme by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), a pseudo first-order process with a rate constant of 1.2 m(-1).s(-1), is not protected by substrates . Graphical analysis of the inactivation by DTNB revealed that only one cysteine is responsible for the inactivation of the enzyme . The three strictly conserved Cys residues among cyclopropane synthases, C139, C176 and C354 of the E . coli enzyme, were mutated to serine . The relative catalytic efficiency of the mutants were 16% for C139S, 150% for C176S and 63% for C354S . The three mutants were inactivated by DTNB at a rate comparable to the rate of inactivation of the His(6)-tagged wild-type enzyme, indicating that the Cys responsible for the loss of activity is not one of the conserved residues . Therefore, none of the conserved Cys residues is essential for catalysis and cannot be involved in covalent catalysis or general base catalysis . The inactivation is probably the result of steric hindrance, a phenomenon irrelevant to catalysis . It is very likely that E . coli CFAS operates via a carbocation mechanism, but the base and nucleophile remain to be identified.

Eur J Biochem, 2004 Dec, 271(23-24), 4685 - 95
The structure and biological characteristics of the Spirochaeta aurantia outer membrane glycolipid LGL; Vinogradov E et al.; In an attempt to isolate lipopolysaccharide from Spirochaeta aurantia, Darveau-Hancock extraction of the cell mass was performed . While no lipopolysaccharide was found, two carbohydrate-containing compounds were detected . They were resolved by size-exclusion chromatography into high molecular mass (LGL(A)) and low molecular mass (LGL(B)) fractions . Here we present the results of the analysis of the glycolipid LGL(B) . Deacylation of LGL(B) with hydrazine and separation of the products by using anion-exchange chromatography gave two major products . Their structure was determined by using chemical methods, NMR and mass spectrometry . All monosaccharides had the d-configuration, and aspartic acid had the l-configuration . Intact LGL(B) contained two fatty groups at O-2 and O-3 of the glycerol residue . Nonhydroxylated C14 to C18 fatty acids were identified, which were predominantly unsaturated or branched . LGL(B) was able to gel Limulus amebocyte lysate, albeit at a lower level than that observed for Escherichia coli O113 lipopolysaccharide . However, even large amounts of LGL(B) were unable to stimulate any Toll-like receptor (TLR) examined, including TLR4 and TLR2, previously shown to be sensitive to lipopolysaccharide and glycolipids from diverse bacterial origins, including other spirochetes.

Clin Exp Immunol, 2005 Jan, 139(1), 65 - 73
Endogenous glucocorticoids modulate neutrophil function in a murine model of haemolytic uraemic syndrome; Gomez SA et al.; Summary Haemolytic uraemic syndrome (HUS) is caused by Shiga-toxin-producing Escherichia coli (STEC) . Although, Shiga toxin type 2 (Stx2) is responsible for the renal pathogenesis observed in patients, the inflammatory response, including cytokines and polymorphonuclear neutrophils (PMN), plays a key role in the development of HUS . Previously, we demonstrated that Stx2 injection generates an anti-inflammatory reaction characterized by endogenous glucocorticoid (GC) secretion, which attenuates HUS severity in mice . Here, we analysed the effects of Stx2 on the pathogenic function of PMN and the potential role of endogenous GC to limit PMN activation during HUS development in a murine model . For this purpose we assessed the functional activity of isolated PMN after in vivo treatment with Stx2 alone or in simultaneous treatment with Ru486 (GC receptor antagonist) . We found that Stx2 increased the generation of reactive oxygen intermediates (ROI) under phobol-myristate-acetate (PMA) stimulation and that the simultaneous treatment with Ru486 strengthened this effect . Conversely, both treatments significantly inhibited in vitro phagocytosis . Furthermore, Stx2 augmented in vitro PMN adhesion to fibrinogen (FGN) and bovine serum albumin (BSA) but not to collagen type I (CTI) . Stx2 + Ru486 caused enhanced adhesion to BSA and CTI compared to Stx2 . Whereas Stx2 significantly increased migration towards N-formyl-methionyl-leucyl-phenylalanine (fMLP), Stx2 + Ru486 treatment enhanced and accelerated this process . The percentage of apoptotic PMN from Stx2-treated mice was higher compared with controls, but equal to Stx2 + Ru486 treated mice . We conclude that Stx2 activates PMN and that the absence of endogenous GC enhances this activation suggesting that endogenous GC can, at least partially, counteract PMN inflammatory functions.

Radiat Res, 2005 Jan, 163(1), 79 - 84
8-OxoA Inhibits the Incision of an AP Site by the DNA Glycosylases Fpg, Nth and the AP Endonuclease HAP1; Lomax ME et al.; Lomax, M . E., Salje, H., Cunniffe, S . and O'Neill, P . 8-OxoA Inhibits the Incision of an AP Site by the DNA Glycosylases Fpg, Nth and the AP Endonuclease HAP1 . Radiat . Res . 163, 79-84 (2005).Ionizing radiation induces clustered DNA damage sites, whereby two or more individual DNA lesions are formed within one or two helical turns of DNA by a single radiation track . A subset of DNA clustered damage sites exist in which the lesions are located in tandem on the same DNA strand . Recent studies have established that two closely opposed lesions impair the repair machinery of the cell, but few studies have investigated the processing of tandem lesions . In this study, synthetic double-stranded oligonucleotides were synthesized to contain 8-oxoA and an AP site in tandem, separated by up to four bases in either a 5' or 3' orientation . The influence 8-oxoA has on the incision of the AP site by the E . coli glycosylases Fpg and Nth protein and the human AP endonuclease HAP1 was assessed . 8-OxoA has little or no effect on the efficiency of incision of the AP site by Nth protein; however, the efficiency of incision of the AP site by Fpg protein is reduced in the presence of 8-oxoA even up to a four-base separation in both the 5' and 3' orientations . 8-OxoA influences the efficiency of HAP1 incision of the AP site only when it is 3' to the AP site and separated by up to two bases . This study demonstrates that the initial stages of base excision repair can be impaired by the presence of a second base lesion in proximity to an AP site on the same DNA strand . This impairment could have biological consequences, such as mutation induction, if the AP site is present at replication.

J Med Entomol, 2004 Nov, 41(6), 1082 - 9
Persistence of Escherichia coli in immature house fly and stable fly (Diptera: Muscidae) in relation to larval growth and survival; Rochon K et al.; The persistence of Escherichia coli in artificially fed larvae was examined for up to 48 h after ingestion by house flies, Musca domestica L., and stable flies, Stomoxys calcitrans (L.) . The rate of change in the E . coli load was similar for both species for up to 5 h after ingestion . Up to 48 h after ingestion, abundance of E . coli declined in immature house flies but remained constant in immature stable flies . When different E . coli concentrations were fed to larvae, the abundance of E . coli increased in stable fly larvae regardless of the initial concentration . The E . coli load in house fly larvae increased when larvae were fed a low concentration of bacteria, but it declined when larvae were fed a high concentration of bacteria . Survival of house fly and stable fly larvae averaged 62 and 25%, respectively, when reared on pure E . coli cultures . These observations suggest that house fly larvae digest E . coli and use it as a food source but stable fly larvae do not.

Planta, 2004 Oct, 219(6), 955 - 66 Epub 2004 Jun 19.
Glucosylation of the saffron apocarotenoid crocetin by a glucosyltransferase isolated from Crocus sativus stigmas; Moraga AR et al.; Saffron, the dry stigma of Crocus sativus L., is considered to be the world's most expensive spice . Three major apocarotenoids--crocin, crocetin and picrocrocin--are responsible for the colour and bitter taste of saffron . The final step in the biosynthesis of the 20-carbon esterified carotenoid crocin is the transformation of the insoluble crocetin into a soluble and stable storage form by glucosylation . These glucosylation reactions are catalysed by glucosyltransferases (GTases) that play a crucial role in natural-product biosynthesis . Using degenerate primers designed to match the plant secondary product GTase (PSPG) box we cloned two cDNAs, UGTCs2 and UGTCs3, from C . sativus stigmas that encode putative polypeptides of 460 and 475 amino acids, respectively . These genes were expressed differentially in saffron tissues . UGTCs2 was mainly expressed in fully developed stigmas, whereas UGTCs3 was mainly expressed in stamens . The UGTCs2 transcript was not detected in the stigma tissue of a Crocus species that does not synthesize crocin, while UGTCs3 and other structural genes for carotenoid biosynthesis were expressed in the stigma of all tested Crocus species . To identify the biochemical function of UGTCs2, the isolated cDNA was expressed in Escherichia coli cells . The recombinant protein UGTCs2 had glucosylation activity against crocetin, crocetin beta-D-glucosyl ester and crocetin beta-D-gentibiosyl ester . These results might suggest that the isolated clone UGTCs2 codes for a saffron crocetin GTase.

Biotechnol Lett, 2004 Nov, 26(21), 1659 - 63
Construction of a pTOC-T vector using GST-ParE toxin for direct cloning and selection of PCR products; Kim HG et al.; Using linker insertion mutations, we determined the most stable region of the parE gene which encodes a toxic protein (ParE) that inhibits growth of Escherichia coli . The toxicity of ParE was sustained until a 144 bp linker was inserted into this region . We have developed a 3' T-overhang vector based on these characteristics of the GST-ParE toxin, and named pTOC-T . Because pTOC-T uses a post-segregational killing system, all transformants grown up on the plates can be considered as recombinants containing foreign DNA . pTOC-T not require X-Gal, IPTG or other substrates for selection . This T-vector using a positive selection system can be applied to various E . coli strains such as XL1-Blue, BL21, DH5alpha, JM109, and JM110.

Biotechnol Lett, 2004 Oct, 26(20), 1593 - 4
Improved long-term cryostorage of Escherichia coli competent cells using trehalose; Ahn T et al.; Trehalose added to storage buffers for chemical-induced transformations at 200 mM increased the survival of Escherichia coli competent cells 1.3 approximately 4.8-fold over 180 d cryostorage compared with dimethyl sulfoxide or glycerol.

Biotechnol Lett, 2004 Oct, 26(20), 1543 - 8
A novel expression vector, designated as pHisJM, for producing recombinant His-fusion proteins; Masuda J et al.; Compared to glutathione S -transferase (GST), tagging with hexahistidine residues (His) has several merits: low levels of toxicity and immunogenicity, a smaller size and no electric charge . We have constructed a novel expression vector, designated as pHisJM (EMBL/GenBank/DDJB accession no . AB116367), for producing recombinant His-fusion proteins . This vector was constructed by replacing GST and multiple cloning site (MCS) cassettes in pGEX-5X-3 with those of hexahistidine and MCS derived from pRSET C vector . Human annexin IV (Anx IV) was used as target protein . His-Anx IV fusion protein was expressed using pHisJM and gave a 40 kDa band when immuno-stained with anti-His mAb or anti-Anx IV mAb as predicted . To compare expression efficiency, a Anx IV cDNA inserted-pHisJM or pGEX-5X-3 was transformed into Escherichia coli DH5alpha, JM109, BL21 and BL21(DE3) . Using pHisJM, Anx IV protein was highly expressed in all cell strains . In addition to the merits of using His-tag, pHisJM has several advantages: 1) it has high expression efficiency; 2) it can be used in any Escherichia coli strain; and 3) it can be used in a single strain of Escherichia coli in all steps from plasmid construction to the expression of the target gene.

Plant Mol Biol, 2004 May, 55(2), 297 - 309
ACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA; Leung KC et al.; In plants, fatty acids synthesized in the chloroplasts are exported as acyl-CoA esters to the endoplasmic reticulum (ER) . Cytosolic 10-kDa acyl-CoA-binding proteins (ACBPs), prevalent in eukaryotes, are involved in the storage and intracellular transport of acyl-CoAs . We have previously characterized Arabidopsis thaliana cDNAs encoding membrane-associated ACBPs with ankyrin repeats, designated ACBP1 and ACBP2, which show conservation to cytosolic ACBPs at the acyl-CoA-binding domain . Analysis of the Arabidopsis genome has revealed the presence of three more genes encoding putative proteins with acyl-CoA-binding domains, designated ACBP3, ACBP4 and ACBP5 . Homologues of ACBP1 to ACBP5 have not been reported in any other organism . We show by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis that ACBP3 , ACBP4 and ACBP5 are expressed in all plant organs, like ACBP1 and ACBP2 . ACBP4 and ACBP5 that share 81.4 identity and which contain kelch motifs were further investigated . To demonstrate their function in binding acyl-CoA, we have expressed them as (His)6-tagged recombinant proteins in Escherichia coli for in vitro binding assays . Both (His)6-ACBP4 and (His)6-ACBP5 bind {14C}oleoyl-CoA with high affinity, {14C}palmitoyl-CoA with lower affinity and did not bind {14C}arachidonyl-CoA . Eight mutant forms of each protein with single amino acid substitutions within the acyl-CoA-binding domain were produced and analyzed . On binding assays, all mutants were impaired in oleoyl-CoA binding . Hence, these novel ACBPs with kelch motifs have functional acyl-CoA-binding domains that bind oleoyl-CoA . Their predicted cytosol localization suggests that they could maintain an oleoyl-CoA pool in the cytosol or transport oleoyl-CoA from the plastids to the ER in plant lipid metabolism.

Plant Mol Biol, 2004 May, 55(2), 281 - 95
A novel alkaline alpha-galactosidase gene is involved in rice leaf senescence; Lee RH et al.; We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves . Here we characterized the structure and function of an SAG- Osh69 encoding alkaline alpha-galactosidase that belongs to a novel family of glycosyl hydrolases . Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8 . The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H2O2 and wounding) . The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal alpha-galactosidase activity at pH 8.0 . The enzyme showed good hydrolytic activities towards alpha-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol . Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves . These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence.






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