Arch Biochem Biophys, 2003 Oct 15, 418(2), 179 - 85 A hyperthermostable novel protein-disulfide oxidoreductase is reduced by thioredoxin reductase from hyperthermophilic archaeon Pyrococcus horikoshii; Kashima Y et al.; A redox protein gene (PH0178) with high sequence homology to a glutaredoxin from Pyrococcus furiosus and a thioredoxin reductase homologue gene (PH1426) were found in the genome sequence of Pyrococcus horikoshii . These two genes were cloned and the corresponding expressed proteins were characterized . The redox protein from PH0178 had strong thioredoxin-like activity, but no glutaredoxin activity . The protein from PH1426 had some reductase activity against thioredoxin from Escherichia coli as well as the redox protein (PH0178) . The protein from PH1426 was a typical, homodimeric flavoprotein . These results indicate that the redox protein (PH0178) is not a glutaredoxin but, rather, a new protein-disulfide oxidoreductase that is involved in a thioredoxin-like system with thioredoxin reductase (PH1426) in P . horikoshii . The redox protein and thioredoxin reductase retained their full activities for over 1h at 100 degrees C . The redox potential of the redox protein was similar to that of thioredoxin from E . coli and lower than that of glutathione . Site-directed mutagenesis studies revealed that the active site of the redox protein corresponds to a CPYC sequence, located in the middle of the sequence. Arch Biochem Biophys, 2003 Oct 15, 418(2), 125 - 32 Abnormal growth of polyamine-deficient Escherichia coli mutant is partially caused by oxidative stress-induced damage; Jung IL et al.; Polyamines participate in numerous cellular processes and are required for normal cell growth in Escherichia coli . In this study, we constructed a new polyamine-deficient E . coli mutant and investigated the physiological function of polyamines during normal aerobic growth conditions . We showed that the requirement for sulfur-containing, branched chain, and aromatic amino acids, which was exhibited in the sodA sodB double mutant faced with severe oxidative stress, was also true of the polyamine-deficient mutant during normal aerobic cell growth . Sorbitol, sucrose, mannose, 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron), an antioxidant that functions as an oxygen radical scavenger including z.rad;O(2)(-), and thiamine partially relieved the cell growth defect caused by polyamine depletion in a dose-dependent manner . As was the case for the cells treated with paraquat, the mutant had an elongated shape compared with the polyamine-proficient wild type . Decreased aeration also relieved the cell growth defect of the polyamine-deficient mutant . Finally, we confirmed that chloromethyl-2('),7(')-dichlorofluorescin diacetate (DCFH-DA), which is oxidized in a fluorescent product in the presence of various oxidants, also fluoresce in the polyamine-deficient cells . These results showed that abnormal growth of the polyamine-deficient E . coli mutant results partially from oxidative stress-induced damage and the mutant thus exhibits the requirement for antioxidant or specific nutritional amino acid during normal aerobic growth. Chem Biol, 2003 Sep, 10(9), 827 - 35 How do DNA repair proteins locate potential base lesions? a chemical crosslinking method to investigate O6-alkylguanine-DNA alkyltransferases; Duguid EM et al.; O(6)-alkylguanine-DNA alkyltransferases directly reverse the alkylation on the O(6) position of guanine in DNA . This group of proteins has been proposed to repair the damaged base in an extrahelical manner; however, the detailed mechanism is not understood . Here we applied a chemical disulfide crosslinking method to probe the damage-searching mechanism of two O(6)-alkylguanine-DNA alkyltransferases, the Escherichia coli C-Ada and the human AGT . Crosslinking reactions with different efficiency occur between the reactive Cys residues of both proteins and a modified cytosine bearing a thiol tether in various DNA probes . Our results indicate that it is not necessary for these proteins to actively flip out every base to find damage . Instead they can locate potential lesions by simply capturing a lesioned base that is transiently extrahelical or sensing the unstable nature of a damaged base pair. Planta, 2003 Jul, 217(3), 449 - 56 Epub 2003 Mar 06. Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-phosphoglyceric acid; Burger BT et al.; Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose . It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important . Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive . Two hypotheses can explain the 3PGA activation differences . Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity . The absence of purified preparations has heretofore negated answering this question . To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits . Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis . We also placed the potato tuber UpReg1 mutation into the maize AGPase . This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase . Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme . These had minimal effect on maize endosperm activity . In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase. Biosci Biotechnol Biochem, 2003 Sep, 67(9), 2030 - 3 Fosmidomycin resistance in adenylate cyclase deficient (cya) mutants of Escherichia coli; Sakamoto Y et al.; Adenylate cyclase deficient (cya) mutants of E . coli K-12 were found to be resistant to fosmidomycin, a specific inhibitor of the non-mevalonate pathway, just like to fosfomycin . E . coli glpT mutants were resistant to fosfomycin and also to fosmidomycin . This fact shows that fosmidomycin was transported inside via the glycerol-3-phosphate transporter, GlpT . DNA micro-array analysis showed that the transcription of glpT and other genes concerning glycerol utilization were highly dependent on the presence of cAMP. Biosci Biotechnol Biochem, 2003 Sep, 67(9), 1996 - 8 Generation of reactive oxygen species from Hinokitiol under near-UV irradiation; Shibata H et al.; Near-UV irradiation caused the decomposition of hinokitiol in an aqueous solution . During the photochemical reaction, the distinct electron spin resonance signal characteristic of the adduct of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with the hydroxyl radical was accompanied by small signals corresponding to the adduct of DMPO with the superoxide anion radical . More than 95% of Escherichia coli cells were killed by the incubation with hinokitiol under near-UV irradiation by BLB fluorescent lamps . These results indicated the generation of reactive oxygen species during photochemical reaction of hinokitiol under near-UV irradiation. Biosci Biotechnol Biochem, 2003 Sep, 67(9), 1857 - 63 Suppression of ethanol and lipopolysaccharide-induced liver injury by extracts of Hydrangeae Dulcis Folium in rats; Hashizume E et al.; In female SD rats that were injected with 4 g/kg BW ethanol p.o . followed by a 5 mg/kg BW lipopolysaccharide (LPS) i.v . injection, serum glutamic pyruvic transaminases (GPT) activity increased to about eight times that of normal rats . In this model, rats that had been fed a diet containing 1% Hydrangeae Dulcis Folium (HDF) extracts for fifteen days showed significantly lower serum GPT activity (380.0+/-58.2 IU/l) than the control group (3527.0+/-774.1 IU/l) . HDF's efficacy was far superior to milk thistle in this model (2950.0+/-915.9 IU/l) . When mouse macrophages were treated with HDF extracts at 50 microg/ml, TNF-alpha production induced by LPS was suppressed to about 10% of the control . Rat serum TNF-alpha levels induced by LPS was decreased to 58.7% of the control by administering 1000 mg/kg BW HDF extract p.o . These results indicate that HDF prevents alcohol-induced liver injury through the inhibition of TNF-alpha production. J Nutr, 2003 Oct, 133(10), 3076 - 9 Transgenic chickens expressing beta-galactosidase hydrolyze lactose in the intestine; Mozdziak PE et al.; Chickens do not possess the necessary enzymes to efficiently hydrolyze lactose into glucose and galactose . The bacterial enzyme beta-galactosidase can convert lactose into glucose and galactose . Transgenic chickens that carry the E . coli lacZ gene and express beta-galactosidase could potentially utilize lactose as an energy source . The objective of this study was to determine the ability of the transgenic chicken small intestinal mucosa to hydrolyze lactose into glucose and galactose . Lactase activity was examined in the intestinal muscosa from wild-type chickens and two lines of chickens that carry the lacZ gene and express beta-galactosidase . Lactase activity was significantly higher in both transgenic lines compared with wild-type birds (P < 0.05) . The presence of the beta-galactosidase enzyme was revealed by X-gal staining in the intestine of transgenic chickens, whereas it was not present in the wild-type chickens . Overall, it appears that inserting the lacZ gene, which encodes beta-galactosidase, has resulted in a chicken that can utilize lactose as an energy source . This study demonstrates that transgenic technology can be used to modify nutrient utilization in domestic poultry. J Biol Chem, 2003 Dec 5, 278(49), 48921 - 7 Epub 2003 Sep 30. Generation of a recombinant, membrane-targeted form of the complement regulator CD59: activity in vitro and in vivo; Fraser DA et al.; Inappropriate activation of complement contributes to pathology in diverse inflammatory diseases . Soluble recombinant forms of the natural cell membrane regulators of complement are effective in animal models and some human diseases . However, their use is limited for reasons related to cost, short half lives, and propensity to cause unwanted systemic effects . Some of these limitations may be overcome by use of bacterial expression systems, specific targeting moieties, and judicious choice of regulator . Here we describe the application of these strategies to the generation of a membrane-targeted form of CD59 . A recombinant soluble form of rat CD59, comprising the first 71 residues of the mature protein and missing the membrane-anchoring signal, was expressed in bacteria, purified, and refolded in a fully active form . The protein was coupled through its carboxyl terminus to a short, synthetic address tag that confers membrane binding activity . Attachment of the membrane address tag markedly increased complement-inhibitory activity assessed in vitro in hemolysis assays . Intra-articular administration of the tagged agent markedly suppressed disease in a model of rheumatoid arthritis in Lewis rats . This novel type of agent, termed sCD59-APT542, offers for the first time the prospect of efficient and specific inhibition of membrane attack complex activity in vivo. Brain Res Dev Brain Res, 2003 Oct 10, 145(1), 39 - 48 Patterns of cerebral inflammatory response in a rabbit model of intrauterine infection-mediated brain lesion; Debillon T et al.; Although the fetal inflammatory response syndrome seems crucial to the association between intrauterine infection and white matter disease in human preterm infants, the underlying mechanisms remain unclear . Using our previously described rabbit model of cerebral cell death in the white matter and hippocampus induced by intrauterine Escherichia coli infection, we investigated inflammatory and astroglial responses in placenta and brain tissues, in correlation with cell death distribution . Brains and placentas were studied 12, 24, or 48 h following intrauterine inoculation of E . coli or saline (groups G12, G24, and G48) . Diffuse monocyte-macrophage infiltrates positive for inducible nitric oxide synthase (i-NOS) were significantly more marked in G24 and G48 placentas than in controls . In the G48 fetuses with both diffuse cell death and focal periventricular white matter cysts mimicking cystic periventricular leukomalacia, a strong rabbit macrophage and inducible nitric oxide synthase immunostaining was observed at the border of these cystic lesions . In contrast, in the fetuses with only diffuse and significant cell death, no inflammatory or astroglial responses were detected in the white matter or hippocampus . Cell death was accompanied by i-NOS immunostaining in the hippocampus but not the white matter . Hippocampal cells positive for i-NOS usually displayed a neuronal phenotype . In this model, focal white matter cysts are accompanied by a robust inflammatory response, and diffuse cell death, which may mimic the white matter and hippocampal damage seen in very and extremely pre-term infants, occur in the absence of a detectable brain inflammatory response. J Soc Gynecol Investig, 2003 Oct, 10(7), 423 - 7 Interleukin-6 is neither necessary nor sufficient for preterm labor in a murine infection model; Yoshimura K et al.; OBJECTIVE: The aim of this study was to investigate the role of interleukin 6 (IL-6) in a murine model of bacterially induced preterm delivery . METHODS: On day 14.5 of a 19-20-day gestation, female mice underwent one of two interventions . In experiment 1, pregnant right uterine horns were injected at laparotomy with 0.5-20 microg of recombinant human IL-6 (rhIL-6) . In experiment 2, IL-6-deficient (IL-6(-/-)) and wild-type control (IL-6(+/+)) mice underwent intrauterine inoculation with 10(5) to 10(8) heat-killed Escherichia coli organisms . Preterm delivery and maternal survival rates were recorded . RESULTS: In experiment 1, doses as high as 20 microg of IL-6 per mouse resulted in up-regulation of acute phase reactants but did not cause preterm delivery or other adverse maternal or fetal effects . In experiment 2, in bacterially inoculated mice, the absence of maternal and fetal IL-6 had no effect on preterm delivery rates . CONCLUSION: IL-6 was neither sufficient nor necessary for preterm delivery in these murine models. Eur J Pharmacol, 2003 Sep 12, 477(2), 183 - 93 Propofol ameliorates liver dysfunction and inhibits aortic superoxide level in conscious rats with endotoxic shock; Tsao CM et al.; Propofol, widely used as a sedative agent, is known to exert antioxidant and anti-inflammatory effects in vitro . We studied the effects of propofol on hemodynamics and the function of several organs in conscious rats with endotoxemia . Intravenous injection of rats with endotoxin (lipopolysaccharide) caused hypotension, vascular hyporeactivity and tachycardia as well as significant lung, liver and kidney damage . Hepatocellular damage caused by lipopolysaccharide for 6 h was significantly attenuated in the lipopolysaccharide+propofol group . Aortic superoxide anion (O(2)(radical)(-)) production, but not plasma nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) level, was also suppressed by propofol in lipopolysaccharide-injected rats . Light microscopy showed that propofol attenuated the marked infiltration of neutrophils in liver tissues from lipopolysaccharide-injected rats . Moreover, the survival rate of the lipopolysaccharide+propofol group at 16 h was significantly increased when compared with that of the lipopolysaccharide group (53% vs . 12%) . These results suggest that inhibition of aortic O(2)(radical)(-) production and amelioration of liver dysfunction contribute to the beneficial effect of propofol in conscious rats with endotoxic shock. Vet Microbiol, 2003 Oct 17, 96(2), 203 - 15 Typing of the eae and espB genes of attaching and effacing Escherichia coli isolates from ruminants; Orden JA et al.; The types of the eae and espB genes of 178 attaching and effacing Escherichia coli (AEEC) strains isolated from diarrhoeic and healthy ruminants were investigated by PCR . Six types of the eae gene: beta (beta), gamma1 (gamma-1), gamma2 (gamma-2), epsilon (epsilon), zeta (zeta) and iota (iota), and three types of the espB gene: alpha, beta and gamma were identified in the strains studied . Moreover, three strains were negative to all the types of the eae gene tested . The types beta and gamma2 in healthy cattle, beta, gamma2 and epsilon in healthy sheep and goats, and beta in diarrhoeic calves, lambs and goat kids were the most frequent types of the eae gene among the strains studied . Although the eaebeta gene was the most prevalent among AEEC from healthy and diarrhoeic ruminants, the percentages of AEEC strains with this type found in this study in diarrhoeic animals (66.7-100%) were higher than those found in healthy animals (33.3-40.6%) . Thus, these data suggest that AEEC strains with the eaebeta gene are associated with neonatal diarrhoea in ruminants . The eaegamma1, eaezeta and eaeiota genes were found in low percentages in the strains studied (4.5, 2.8 and 7.3%, respectively) . All the types of the eae gene, except the type iota, showed a close correlation with the types of the espB gene: the eaebeta and eae epsilon genes with the espBbeta gene, the eaegamma2 and eaezeta genes with the espBalpha gene and the eaegamma1 gene with the espBgamma gene. Emerg Infect Dis, 2003 Sep, 9(9), 1170 - 3 Enteroaggregative Escherichia coli serotype O126:H27, Israel; Shazberg G et al.; Enteroaggregative Escherichia coli (EAEC) is a newly diarrheagenic agent wherein several predominant serotypes are reported . We studied the association between those serotypes, as clonal indicators, and the trait of enteroaggregative adherence to host cells, tested by polymerase chain reaction . We also evaluated the clinical manifestations of infection in 17 hospitalized children by our most common EAEC serotype, O126:H27. BMC Struct Biol . 2003 Sep 30;3(1):7. Crystal structure of Escherichia coli protein ybgI, a toroidal structure with a dinuclear metal site; Ladner JE et al.; BACKGROUND: The protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function . RESULTS: The structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers . Each polypeptide chain binds two metal ions on the inside of the toroid . CONCLUSION: The toroidal structure is comparable to that of some proteins that are involved in DNA metabolism . The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme. J Agric Food Chem, 2003 Oct 8, 51(21), 6367 - 70 2-Dodecylcyclobutanone does not induce mutations in the Escherichia coli tryptophan reverse mutation assay; Sommers CH; Like thermal processing, ionizing radiation can break molecular bonds and induce the formation of chemicals not found in the unprocessed product . Irradiation of foods containing palmitic acid can lead to the formation of 2-dodecylcyclobutanone (2-DCB) . In this study, the Escherichia coli tryptophan reverse mutation assay was used to evaluate the capacity of 2-DCB to induce mutations . E . coli tester strains WP2 (pkM101) and WP2 uvrA (pKM101), with and without exogenous metabolic activation, were exposed to 0, 0.05, 0.1, 0.5, and 1 mg/well 2-DCB using the Miniscreen version of the assay . 2-DCB did not induce mutations in the E . coli tryptophan reverse mutation assay . These results are in agreement with negative results obtained in short-term and long-term genetic toxicology tests of irradiated food products. Langenbecks Arch Chir Suppl Kongressbd, 1998, 115(Suppl I), 709 - 11 {Effect of humanised anti-L- selectin antibody HuDreg 200 on leukocyte kinetics in pulmonary microcirculation in endotoxinemia}; Borges J et al.; In the pulmonary circulation, endotoxemia induces leukocyte/endothelium interaction in pulmonary arterioles and venules . Thus, leukocytes may contribute to the pathogenesis of acute lung injury . In order to investigate, whether this interaction can be inhibited by early blockade of the adhesion cascade, we studied the effect of the humanized anti-L-selectin antibody HuDreg 200 on leukocyte kinetics in pulmonary arterioles and venules following i.v.-infusion of endotoxin . In endotoxemia HuDreg 200 reduces sticking of leukocytes in both pulmonary arterioles and venules . Thus, we were able to demonstrate that early blockade of the adhesion cascade effectively prevents leukocyte accumulation in pulmonary microvessels in endotoxemia . Therefore, HuDreg 200 may exhibit protective effects on the manifestation of leukocyte-mediated acute lung injury. Langenbecks Arch Chir Suppl Kongressbd, 1998, 115(Suppl I), 397 - 8 {Endotoxin inhibits apoptosis of neutrophilic granulocytes via tyrosine phosphorylation}; Ertel W et al.; The activation of tyrosine phosphorylation plays a pivotal role for endotoxin induced inhibition of PMN-apoptosis . Modulation of tyrosine phosphorylation with specific inhibitors attenuates endotoxin induced prolongation of PMN-lifespan. Langenbecks Arch Chir Suppl Kongressbd, 1998, 115(Suppl I), 181 - 4 {Endotoxin tolerance and hemorrhagic shock in rats}; Ackermann MN et al.; The aim of the presents study was to investigate the protective capacity of endotoxin tolerance in a hemorrhagic shock model in rats . A pretreatment with low dose endotoxin induces a state of tolerance, which is characterized by decreased TNF alpha production in vivo and in vitro upon subsequent high dose endotoxin challenge . This endotoxin tolerance improves survival after hemorrhagic shock from 22.8% in untreated controls to 68.8 in tolerant rats . The protection was accompanied with the appearance of n TNF alpha inhibitory activity in the serum of endotoxin tolerant animals, which might be responsible for the improved survival after hemorrhagic shock. Langenbecks Arch Chir Suppl Kongressbd, 1998, 115(Suppl I), 177 - 80 {Transfusion of plasma of endotoxin tolerant rats improves survival of hemorrhagic shock in the rat model}; Reuter M et al.; To evaluate the protective effect of endotoxin tolerant plasma after hemorrhagic shock, plasma of endotoxin tolerant animals was used for resuscitation of normal rats . The transfusion of plasma of endotoxin tolerant rats improved the survival rate from 20 to 60 per cent . In the sera of endotoxin tolerant animals a TNF inhibitory activity was detected, which could be transferred by the plasma . This effect may be responsible for the protection to hemorrhagic shock. Langenbecks Arch Chir Suppl Kongressbd, 1998, 115(Suppl I), 39 - 41 {Severe trauma leads to damaged signal transduction with inhibited secretion of cytokines}; Oberholzer A et al.; The reduced secretion if IL-1 beta after severe injury seems to be due to disturbances in signal transduction pathways . Protein-tyrosine kinases are necessary for endotoxin-induced secretion of IL-1 beta, while protein kinase C antagonizes this effect. Electrophoresis, 2003 Sep, 24(18), 3219 - 23 Critical factors for reproducible capillary electrophoresis of Escherichia coli cells; Dai D et al.; Sharp peaks of Escherichia coli JM 109 (up to 1 300 000 theoretical plates) were recorded with either extremely diluted (< 5 mM) or extremely concentrated (ca . 150 mM) Tris-borate (TB) running buffers . However, under the conditions of yielding sharp peaks, migration time of E . coli was irreproducible . Critial factors influencing reproducibility were found to include bacterial growth phase, storage condition, cell pretreatment before injection, and concentration of running buffer . Buffer concentrations in the range of 20-100 mM TB were essential for reproducibility . E . coli JM 109 was shown to be sensitive to ultrasonification . Bacterial growth and storage conditions could be monitored by CE, with results comparable to those obtained with optical methods. Proteins, 2003 Nov 1, 53(2), 273 - 81 Insight into the stability of the hydrophobic binding proteins of Escherichia coli: assessing the proteins for use as biosensors; Salopek-Sondi B et al.; Spectroscopic methods were used to monitor the unfolding of the leucine specific (LS) and the leucine-isoleucine-valine (LIV) binding proteins . Our studies indicate that ligand-free protein undergoes a simple two-state unfolding, whereas the protein-ligand complex undergoes a three-state unfolding model . Ligand binding causes significant stabilization of both proteins . There is correlation between ligand hydrophobicity and protein stabilization: the most hydrophobic ligand, isoleucine, causes the most significant stabilization of LIV protein . A disulfide bond present in N-domain of both proteins makes a large contribution to the protein stability of these periplasmic binding receptors . Biopolymers, 2003, 71(4), 516 - 31 Biosynthesis and biophysical analysis of domains of a yeast G protein-coupled receptor; Arevalo E et al.; The alpha-factor receptor(Ste2p) is required for the sexual conjugation of the yeast Saccharomyces cerevisiae . Ste2p belongs to the G protein-coupled receptor (GPCR) family sharing a common heptahelical transmembrane structure . Biological synthesis of regions of Ste2p fused to a leader protein in Escherichia coli yielded milligram quantities of polypeptides that corresponded to one or two transmembrane domains . Fusion proteins were characterized by polyacrylamide gel electrophoresis, high performance liquid chromatography, and mass spectrometry . CD studies on the fusion proteins in trifluoroethanol:water mixtures indicated the existence of alpha-helical structures in the single- and the double-transmembrane domains . NMR experiments were performed in CDCl(3):CD(3)OH:H(2)O (4:4:1) on the (15)N-labeled single-transmembrane peptide showing a clear dispersion of the nitrogen-amide proton correlation cross peaks indicative of a high-purity, uniformly labeled molecule . The results indicate that single- and double-transmembrane domains of a GPCR can be produced by biosynthetic methods in quantities and purity sufficient for biophysical studies . Mol Biol Cell, 2003 Oct, 14(10), 4316 - 28 Epub 2003 Aug 07. Conserved function of pex11p and the novel pex25p and pex27p in peroxisome biogenesis; Rottensteiner H et al.; We describe the isolation and characterization of a homologous pair of proteins, Pex25p (YPL112c) and Pex27p (YOR193w), whose C-termini are similar to the entire Pex11p . All three proteins localize to the peroxisomal membrane and are likely to form homo-oligomers . Deletion of any of the three genes resulted in enlarged peroxisomes as revealed by fluorescence and electron microscopy . The partial growth defect on fatty acids of a pex25delta mutant was not exacerbated by the additional deletion of PEX27; however, when PEX11 was deleted on top of that, growth was abolished on all fatty acids . Moreover, a severe peroxisomal protein import defect was observed in the pex11deltapex25deltapex27delta triple mutant strain . This import defect was also observed when cells were grown on ethanol-containing medium, where peroxisomes are not required, suggesting that the function of the proteins in peroxisome biogenesis exceeds their role in proliferation . When Pex25p was overexpressed in the triple mutant strain, growth on oleic acid was completely restored and a massive proliferation of laminar membranes and peroxisomes was observed . Our data demonstrate that Pex11p, Pex25p, and Pex27p build a family of proteins whose members are required for peroxisome biogenesis and play a role in the regulation of peroxisome size and number. J Biol Chem, 2003 Dec 5, 278(49), 49555 - 62 Epub 2003 Sep 29. Molecular mechanism of maternal rescue in the clk-1 mutants of Caenorhabditis elegans; Burgess J et al.; The clk-1 mutants of Caenorhabditis elegans display an average slowing down of physiological rates, including those of development, various behaviors, and aging . clk-1 encodes a hydroxylase involved in the biosynthesis of the redox-active lipid ubiquinone (co-enzyme Q), and in clk-1 mutants, ubiquinone is replaced by its biosynthetic precursor demethoxyubiquinone . Surprisingly, homozygous clk-1 mutants display a wild-type phenotype when issued from a heterozygous mother . Here, we show that this maternal effect is the result of the persistence of small amounts of maternally derived CLK-1 protein and that maternal CLK-1 is sufficient for the synthesis of considerable amounts of ubiquinone during development . However, gradual depletion of CLK-1 and ubiquinone, and expression of the mutant phenotype, can be produced experimentally by developmental arrest . We also show that the very long lifespan observed in daf-2 clk-1 double mutants is not abolished by the maternal effect . This suggests that, like developmental arrest, the increased lifespan conferred by daf-2 allows for depletion of maternal CLK-1, resulting in the expression of the synergism between clk-1 and daf-2 . Thus, increased adult longevity can be uncoupled from the early mutant phenotypes, indicating that it is possible to obtain an increased adult lifespan from the late inactivation of processes required for normal development and reproduction. J Cell Biol, 2003 Sep 29, 162(7), 1245 - 54 Functional interaction of chloroplast SRP/FtsY with the ALB3 translocase in thylakoids: substrate not required; Moore M et al.; Integration of thylakoid proteins by the chloroplast signal recognition particle (cpSRP) posttranslational transport pathway requires the cpSRP, an SRP receptor homologue (cpFtsY), and the membrane protein ALB3 . Similarly, Escherichia coli uses an SRP and FtsY to cotranslationally target membrane proteins to the SecYEG translocase, which contains an ALB3 homologue, YidC . In neither system are the interactions between soluble and membrane components well understood . We show that complexes containing cpSRP, cpFtsY, and ALB3 can be precipitated using affinity tags on cpSRP or cpFtsY . Stabilization of this complex with GMP-PNP specifically blocks subsequent integration of substrate (light harvesting chl a/b-binding protein {LHCP}), indicating that the complex occupies functional ALB3 translocation sites . Surprisingly, neither substrate nor cpSRP43, a component of cpSRP, was necessary to form a complex with ALB3 . Complexes also contained cpSecY, but its removal did not inhibit ALB3 function . Furthermore, antibody bound to ALB3 prevented ALB3 association with cpSRP and cpFtsY and inhibited LHCP integration suggesting that a complex containing cpSRP, cpFtsY, and ALB3 must form for proper LHCP integration. Fungal Genet Biol, 2003 Nov, 40(2), 166 - 75 The Snf1 kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the filamentous fungal counterpart Cre1; Cziferszky A et al.; In Saccharomyces cerevisiae, the SNF1 gene product phosphorylates the carbon catabolite repressor protein Mig1 under conditions when glucose is limiting, thereby relieving the fungus from catabolite repression . We have investigated whether the corresponding counterpart of filamentous fungi-the Cre1 protein-is also phosphorylated by Snf1 . To this end, snf1, an ortholog of SNF1, was isolated from the ascomycete Hypocrea jecorina . The gene encodes a protein with high similarity to Snf1 kinases from other eukaryotes in its N-terminal catalytic domain, but little similarity in the C-terminal half of the protein, albeit some short aa-areas were detected, however, which are conserved in filamentous fungi and in yeast . Expression of snf1 is independent of the carbon source . An overexpressed catalytic domain of H . jecorina Snf1 readily phosphorylated yeast Mig1, but not a Mig1 mutant form, in which all four identified Snf1 phosphorylation sites (Phi XRXXSXXX Phi) had been mutated . The enzyme did neither phosphorylate H . jecorina Cre1 nor histone H3, another substrate of Snf1 kinase in yeast . H . jecorina Snf1 also phosphorylated peptides comprising the strict Snf1 consensus, but notably did not phosphorylate peptides containing the regulatory serine residue in Cre1 (=Ser(241) in H . jecorina Cre1 and Ser(266) in Sclerotinia sclerotiorum CRE1) . The use of cell-free extracts of H . jecorina as protein source for Snf1 showed phosphorylation of an unknown 36 kDa protein, which was present only in extracts from glucose-grown mycelia . We conclude that the Snf1 kinase from H . jecorina is not involved in the phosphorylation of Cre1. Fungal Genet Biol, 2003 Nov, 40(2), 159 - 65 The photolyase gene from the plant pathogen Fusarium oxysporum f . sp . lycopersici is induced by visible light and alpha-tomatine from tomato plant; Alejandre-Duran E et al.; Survival of irradiated spores from Fusarium oxysporum with ultraviolet radiation (UV) was increased following exposition to visible light, indicating that this phytopathogenic fungus has a mechanism of photoreactivation able to counteract the lethal effects of UV . A genomic sequence containing the complete photolyase gene (phr1) from F . oxysporum was isolated by heterologous hybridisation with the Neurospora crassa photolyase gene . The F . oxysporum phr1 cDNA was isolated and expressed in a photolyase deficient Escherichia coli strain . The complementation of the photoreactivation deficiency of this E . coli mutant by phr1 cDNA demonstrated that the photolyase gene from F . oxysporum encodes a functional protein . The F . oxysporum PHR1 protein has a domain characteristic of photolyases from fungi (Trichoderma harziaium, N . crassa, Magnaporthe grisea, Saccharomyces cerevisiae) to bacteria (E . coli), and clusters in the photolyases phylogenetic tree with fungal photolyases . The F . oxysporum phr1 gene was inducible by visible light . The phr1 expression was also detected in presence of alpha-tomatine, a glycoalkaloid from tomato damaging cell membranes, suggesting that phr1 is induced by this cellular stress. Fungal Genet Biol, 2003 Nov, 40(2), 83 - 92 Co-transformation with autonomous replicating and integrative plasmids in Penicillium chrysogenum is highly efficient and leads in some cases to rescue of the intact integrative plasmid; Banuelos O et al.; The efficiency of co-transformation in Penicillium chrysogenum Wisconsin 54-1255 pyrG(-) and the fate of the transforming DNA were studied using an integrative (pEF43) and an autonomous replicating plasmid (pAM9L) . The results showed a co-transformation frequency of nearly 70% of all transformants tested . The total efficiency of transformation was shown to be dependent on the plasmid marker used as transformant selection (i.e., markers in the integrative or autonomous replicating vector) . Analysis of the plasmids re-isolated from several co-transformants showed that different populations of plasmids co-exist in the fungal host . Interestingly, in all co-transformants studied, the integrative plasmid was found to be replicating autonomously without integrating into the host genome . In some cases, co-integrates were formed by recombination between autonomous replicating (pAM9L) and integrative (pEF43) plasmids . However, unexpectedly in some cases, the non-reorganised pEF43 integrative plasmid used in the co-transformation assays was rescued from some co-transformants. J Microsc, 2003 Oct, 212(Pt 1), 71 - 80 Cryoimmobilization and three-dimensional visualization of C . elegans ultrastructure; Muller-Reichert T et al.; Caenorhabditis elegans is one of the most important genetic systems used in current biological research . Increasingly, these genetics-based research projects are including ultrastructural analyses in their attempts to understand the molecular basis for cell function . Here, we present and review state-of-the-art methods for both ultrastructural analysis and immunogold localization in C . elegans . For the initial cryofixation, high-pressure freezing is the method of choice, and in this article we describe two different strategies to prepare these nematode worms for rapid freezing . The first method takes advantage of transparent, porous cellulose capillary tubes to contain the worms, and the second packs the worms in E . coli and/or yeast paste prior to freezing . The latter method facilitates embedding of C . elegans in a thin layer of resin so individual worms can be staged, selected and precisely orientated for serial sectioning followed by immunolabelling or electron tomography. An Med Interna, 2003 Aug, 20(8), 396 - 8 {Free radicals and cytotoxicity of ethanol over human leucocytes in peripheral blood}; Colome Pavon JA et al.; INTRODUCTION: The ingestion of alcohol produces oxydative stress generating free radicals of oxygen and ethanol . These free radicals have a molecular reactive ability and, therefore, they play an important role in the production of the injury which appears in the liver and in other organs and tissues . We have done an "in vitro" study where we analyse the oxydative status at rest and the respiratory explosion produced by ethanol at final concentrations of 50 and 25 mM and by the phagocytosis of a previously opsonized concentrate of bacteria (E.coli) in human leucocytes taken from peripheral blood of six healthy persons . METHOD: We have used 1.2.3 . dihydrorhodamine (non-fluorescent) as the oxydative marker because it is transformed in rhodamine (fluorescent), which is quantitatively studied by Flow Cytometry . RESULTS: The peak of oxydative stress is reached with the bacteria in the phagocytes (monocytes 50% and granulocytes 90%) and it has a significant difference with the control group . By adding ethanol at 50 mM we have seen an statistic significant difference in oxydative stress in the cells of all three types (lymphocytes 9.19%, monocytes 32% and granulocytes 36%) . With a concentration of 25 mM of ethanol the oxydative stress is increased but without a significant difference (lymphocytes 2.39%, monocytes 9.22% and granulocytes 4.46%) . We have also seen toxic cellular effect which reaches the 40.75% of cells with ethanol at 50 mM, the 10.7% with ethanol at 25 mM and the 5.65% with bacteria . CONCLUSION: The oxydative stress caused by the production of oxygen and ethanol free radicals in the leucocytes, and the proved cytotoxic effect of ethanol may play an important role over the qualitative and the quantitative leucocyte disorder on different organs and tissues of the alcoholic patient. In Vitro Cell Dev Biol Anim, 2003 Jul-Aug, 39(7), 263 - 5 Recombinant perchloric acid-soluble protein suppresses the immunoglobulin production of human-human hybridoma HB4C5 cells; Kanouchi H et al.; Because perchloric acid-soluble protein (PSP) has been conserved evolutionally in various species from Escherichia coli to humans, it may reflect an involvement in basic cellular regulation . However, the precise function of PSP is currently unknown . In this study, we examined the direct effect of PSP on the production of immunoglobulin (Ig) using human B, HB4C5, NAT-30, and U266 cells because it has been reported that subcutaneous administration of PSP affects rodent immune systems . Suppression of Ig productivity and decrement of the cell viability was recognized only in HB4C5 cells by the addition of PSP into the medium . On the other hand, PSP had no effect on Ig productivity and cell viability in NAT-30 and U266 cells . In addition, PSP was clearly incorporated by HB4C5 but not by the other cells . These results suggest that the Ig production suppressed by PSP, which has been previously reported to inhibit protein synthesis, contributed to the incorporation of PSP into the HB4C5 cells. Biochemistry, 2003 Oct 7, 42(39), 11494 - 503 Novel mammalian group XII secreted phospholipase A2 lacking enzymatic activity; Rouault M et al.; An increasing number of mammalian secreted phospholipases A(2) (sPLA(2)s) has been identified over the past few years . Here, we report the identification and recombinant expression of a novel sPLA(2)-like protein in mouse and human species that has been called group XIIB (GXIIB) . The mature protein has a molecular mass of 19.7 kDa and structural features similar to those of the previously identified GXII sPLA(2), now called GXIIA . Strikingly, the GXIIB sPLA(2) has a mutation in the active site, replacing the canonical histidine by a leucine, suggesting that this sPLA(2) is catalytically inactive . Recombinant expression of human (hGXIIB) and mouse (mGXIIB) sPLA(2)s in Escherichia coli indicates that GXIIB sPLA(2)s display no measurable lipolytic activity on various types of phospholipid substrates . Furthermore, these sPLA(2)-like proteins display relatively weak affinity to phospholipid vesicles . Binding experiments indicate that these proteins are also unable to bind to the well-known M-type sPLA(2) receptor . The RNA tissue distribution of GXIIB sPLA(2)s is distinct from that of other sPLA(2)s including the homologous GXIIA . Strong expression was observed in liver, small intestine, and kidney in both human and mouse species . Interestingly, the expression of the novel sPLA(2) is dramatically decreased in human tumors from the same tissues . The absence of enzymatic activity suggests that the GXIIB sPLA(2)-like proteins probably exert their biological roles by acting as ligands for as yet unidentified receptors. Biochemistry, 2003 Oct 7, 42(39), 11476 - 83 Determination of solid-state NMR structures of proteins by means of three-dimensional 15N-13C-13C dipolar correlation spectroscopy and chemical shift analysis; Castellani F et al.; In this paper, a three-dimensional (3D) NMR-based approach for the determination of the fold of moderately sized proteins by solid-state magic-angle spinning (MAS) NMR is presented and applied to the alpha-spectrin SH3 domain . This methodology includes the measurement of multiple (13)C-(13)C distance restraints on biosynthetically site-directed (13)C-enriched samples, obtained by growing bacteria on {2-(13)C}glycerol and {1,3-(13)C}glycerol . 3D (15)N-(13)C-(13)C dipolar correlation experiments were applied to resolve overlap of signals, in particular in the region where backbone carbon-carbon correlations of the C(alpha)-C(alpha), CO-CO, C(alpha)-CO, and CO-C(alpha) type appear . Additional restraints for confining the structure were obtained from phi and psi backbone torsion angles of 29 residues derived from C(alpha), C(beta), CO, NH, and H(alpha) chemical shifts . Using both distance and angular restraints, a refined structure was calculated with a backbone root-mean-square deviation of 0.7 A with respect to the average structure. Genetika, 2003 Aug, 39(8), 1033 - 8 {Heat shock inhibits the induced expression of the SOS genes and SoxRS regulons in Escherichia coli}; Vasil'eva SV et al.; Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR) . The modification by heat shock of the expression of sfiA and soxS genes induced by oxidative agents H2O2, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time . Quantitative parameters of gene expression were examined in E . coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ . The expression of these genes induced by cell treatment with H2O2, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock . Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion O2-, it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H2O2 is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O2-. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Oct, 35(10), 960 - 3 {Expression of a human ribonuclease inhibitor variant in Escherichia coli and silkworm insect cell (Bombyx mori)}; Huang GH et al.; Ribonuclease inhibitor (RI) is an acidic 50 kD protein with a high content of leucine and cysteine residues . RI inhibits RNases of the pancreatic type . A variant of RI was cloned from human fetal liver cDNA library by polymerase chain reaction (PCR) . Compared with the reported RI, only two variations Arg(359)Ala and Leu(365)Pro were found in RIv amino acid sequence . Recombinant RIv has been expressed both in Escherichia coli and silkworm insect cells (Bombyx mori) . The recombinant RIv exhibited inhibition activity on ribonucleolytic activity of RNase A in vitro system. Acta Biochim Pol, 2003, 50(3), 807 - 13 Kinetics of increased generation of (.)NO in endotoxaemic rats as measured by EPR; Plonka PM et al.; Ferrous-diethyldithiocarbamate (Fe(DETC)(2)) chelate is a lipophilic spin trap developed for (.)NO detection by electron paramagnetic resonance (EPR) spectroscopy . Using this spin trap we investigated the kinetics of (.)NO production in endotoxaemia in rats induced by lipopolysaccharide (Escherichia coli, 10 mg/kg) . The NO-Fe(DETC)(2) complex was found to give a characteristic EPR signal, and the amplitude of the 3rd (high-field) component of its hyperfine splitting was used to monitor the level of (.)NO . We found that in blood, kidney, liver, heart and lung (.)NO production starts to increase as early as 2 h after LPS injection, reaches the maximum 6 h after LPS injection and then returns to basal level within further 12-18 h . Interestingly, in the eye bulb the maximum of (.)NO production was detected 12 h after LPS, and the signal was still pronounced 24 h after LPS . In brief, the highly lipophilic exogenous spin trap, Fe(DETC)(2) is well suited for assessment of (.)NO production in endotoxaemia . We demonstrated that the kinetics of increased production of (.)NO in endotoxaemic organs, with the notable exception of the eye, do not follow the known pattern of NOS-2 induction under those conditions . Accordingly, only in early endotoxaemia a high level of (.)NO is detected, while in late endotoxaemia (.)NO detectability is diminished most probably due to concomitant oxidant stress. J Biol Chem, 2003 Dec 12, 278(50), 50664 - 70 Epub 2003 Sep 26. Crystal structure of ClpX molecular chaperone from Helicobacter pylori; Kim DY et al.; ClpX, a heat shock protein 100 chaperone, which acts as the regulatory subunit of the ATP-dependent ClpXP protease, is responsible for intracellular protein remodeling and degradation . To provide a structural basis for a better understanding of the function of the Clp ATPase family, the crystal structures of Helicobacter pylori ClpX, lacking an N-terminal Cys cluster region complexed with ADP, was determined . The overall structure of ClpX is similar to that of heat shock locus U (HslU), consisting of two subdomains, with ADP bound at the subdomain interface . The crystal structure of ClpX reveals that a conserved tripeptide (LGF) is located on the tip of ClpP binding loop extending from the N-terminal subdomain . A hexameric model of ClpX suggests that six tripeptides make hydrophobic contacts with the hydrophobic clefts of the ClpP heptmer asymmetrically . In addition, the nucleotide binding environment provides the structural explanation for the hexameric assembly and the modulation of ATPase activity. J Biol Chem, 2003 Dec 12, 278(50), 49882 - 9 Epub 2003 Sep 26. Sulfated derivatives of Escherichia coli K5 polysaccharides as modulators of fibroblast growth factor signaling; Borgenstrom M et al.; Heparan sulfate (HS) proteoglycans are intimately involved in the regulation of fibroblast growth factor (FGF) signaling . HS and the related glycosaminoglycan heparin interact with FGFs and FGF receptors (FGFRs), and it is believed that both interactions are required for productive FGF signaling . Attempts to inhibit FGF activity have been made with modified heparin preparations, various heparin-like polysaccharide analogues and other polyanionic molecules, which may all act by interfering with the physiological HS-FGF-FGFR interactions on the cell surface . Here, we have studied the potential of sulfated derivatives of a bacterial polysaccharide (capsular polysaccharide from Escherichia coli K5 (K5PS)) in the modulation of FGF-heparin/HS interactions and FGF signaling . We demonstrate that O-sulfated and N,O-sulfated species of K5PS, with high degrees of sulfation, displaced FGF-1, FGF-2, and FGF-8b from heparin . However, only O-sulfated K5PS efficiently inhibited the FGF-induced proliferation of S115 mammary carcinoma cells and 3T3 fibroblasts, whereas N,O-sulfated K5PS had little or no inhibitory effect . Studies with CHO677 cells lacking endogenous HS, as well as with chlorate-treated S115 cells expressing undersulfated HS, indicated that whereas exogenously administered heparin and N,O-sulfated K5PS restored the cellular response toward FGF stimulation, O-sulfated K5PS was largely devoid of such stimulatory activity . Our data suggest that highly O-sulfated species of K5PS may be efficient inhibitors of FGF signaling. J Biol Chem, 2003 Dec 12, 278(50), 50182 - 7 Epub 2003 Sep 26. Conserved pore residues in the AAA protease FtsH are important for proteolysis and its coupling to ATP hydrolysis; Yamada-Inagawa T et al.; Like other AAA proteins, Escherichia coli FtsH, a membrane-bound AAA protease, contains highly conserved aromatic and glycine residues (Phe228 and Gly230) that are predicted to lie in the central pore region of the hexamer . The functions of Phe228 and Gly230 were probed by site-directed mutagenesis . The results of both in vivo and in vitro assays indicate that these conserved pore residues are important for FtsH function and that bulkier, uncharged/apolar residues are essential at position 228 . None of the point mutants, F228A, F228E, F228K, or G230A, was able to degrade sigma32, a physiological substrate . The F228A mutant was able to degrade casein, an unfolded substrate, although the other three mutants were not . Mutation of these two pore residues also affected the ATPase activity of FtsH . The F228K and G230A mutations markedly reduced ATPase activity, whereas the F228A mutation caused a more modest decrease in this activity . The F228E mutant was actually more active ATPase . The substrates, sigma32 and casein, stimulated the ATPase activity of wild type FtsH . The ATPase activity of the mutants was no longer stimulated by casein, whereas that of the three Phe228 mutants, but not the G230A mutant, remained sigma32-stimulatable . These results suggest that Phe228 and Gly230 in the predicted pore region of the FtsH hexamer have important roles in proteolysis and its coupling to ATP hydrolysis. Am J Physiol Lung Cell Mol Physiol, 2004 Oct, 287(4), L649 - 55 Epub 2003 Sep 26. Smooth muscle F-actin disassembly and RhoA/Rho-kinase signaling during endotoxin-induced alterations in pulmonary arterial compliance; Boer C et al.; Endotoxemia is associated with changed pulmonary vascular function with respect to vasoreactivity, endothelial permeability, and activation of inducible nitric oxide synthase II (NOSII) . However, whether altered passive arterial wall mechanics contribute to this endotoxin-induced pulmonary vascular dysfunction is still unknown . Therefore, we investigated whether endotoxin affects the passive arterial mechanics and compliance of isolated rat pulmonary arteries . Pulmonary arteries of pentobarbital-anesthetized Wistar rats (n = 55) were isolated and exposed to Escherichia coli endotoxin (50 microg/ml) for 20 h . Endotoxin increased pulmonary artery diameter and compliance (transmural pressure = 13 mmHg) in an endothelium-, Ca2+-, or NOSII-induced NO release-independent manner . Interestingly, the endotoxin-induced alterations in the passive arterial mechanics were accompanied by disassembly of the smooth muscle cell (SMC) F-actin cytoskeleton . Disassembly of F-actin by incubation of control arteries with the cytoskeleton-disrupting agent cytochalasin B or the Rho-kinase inhibitor Y-27632 induced a similar increase in passive arterial diameter and compliance . In contrast, RhoA activation by lysophosphatidic acid prevented the endotoxin-induced alterations in the pulmonary SMC F-actin cytoskeleton and passive mechanics . In conclusion, these findings indicate that disassembly of the SMC F-actin cytoskeleton and RhoA/Rho-kinase signaling act as mediators of endotoxin-induced changes in the pulmonary arterial mechanics . They imply the involvement of F-actin rearrangement and RhoA/Rho-kinase signaling in endotoxemia-induced vascular lung injury. Biotechnol Lett, 2003 Sep, 25(17), 1463 - 7 Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization; Zhang J et al.; The lipA gene encoding a thermostable esterase was cloned from Thermoanaerobacter tengcongensis and over-expressed in Escherichia coli . The recombinant esterase, with a molecular mass of approx . 43 kDa determined by SDS-PAGE, was purified to homogeneity through Sephadex G-100 gel filtration . The purified enzyme actively hydrolyzed tributyrin but not olive oil . Maximum activity was observed on p-nitrophenyl (NP)-propionate (C3) and p-NP-butyrate (C4), with little activity towards p-NP-palmitate (C16) . The esterase was optimally active at 70 degrees C (over 15 min) and at pH 9 . It is highly thermostable, with a residual activity greater than 80% after incubation at 50 degrees C for more than 10 h . The activity was not inhibited by 5 mM EDTA and PMSF, indicating the esterase is not a metalloenzyme and may contain a specific structure around the catalytic serine residue . In addition, it was stable for 1 h at 37 degrees C in 1% CHAPS and Triton X-100 but not stable in 1% Tween 20 or SDS. Cell Mol Life Sci, 2003 Aug, 60(8), 1529 - 46 Heterologous expression of G-protein-coupled receptors: comparison of expression systems fron the standpoint of large-scale production and purification; Sarramegna V et al.; G-protein-coupled receptors (GPCRs) are of prime importance for cell signal transduction mechanisms and are the target of many current and potential drugs . However, structural data on these membrane proteins is still scarce because of their low natural abundance and the low efficiency of most of the expression systems currently available . This review presents the most important expression systems currently employed for heterologous expression of GPCRs; Escherichia coli, yeast, insect cells and mammalian cells . After briefly recalling the specificity, advantages and limitations of each system, particular emphasis is put on the quantitative comparison of these expression systems in terms of overall expression yield, and on the influence of various factors (primary sequence, origin, cell type, N- and C-terminal tags) on the results. Biotechniques, 2003 Sep, 35(3), 520 - 2, 524-6 Simultaneous cloning of open reading frames into several different expression vectors; Stanyon CA et al.; Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone . High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed . Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities . Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production . Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system . To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system . We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction . The resulting expression plasmids can be separated using prototrophic markers specific to each vector . Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies. Mol Genet Genomics, 2003 Nov, 270(3), 234 - 42 Epub 2003 Sep 26. Genomic characterization of Rim2/Hipa elements reveals a CACTA-like transposon superfamily with unique features in the rice genome; Wang GD et al.; The availability of huge amounts of rice genome sequence now permits large-scale analysis of the structure and molecular characteristics of the previously identified transposase-encoding Rim2 (also called Hipa) element, which is transcriptionally activated by infection with the fungal pathogen Magnaporthe grisea and by treatment with the corresponding fungal elicitor . Based on genomic cloning and data mining from 230 Mb of rice genome sequence, 347 Rim2 elements, with an average size of 5.8 kb, were identified . This indicates that an estimated total of 600-700 Rim2 elements are present in the whole genome . Rim2 insertions occur non-randomly on the chromosomes, as visualized by fluorescence in situ hybridization . The elements harbor 16-bp terminal inverted repeats with the core sequence CACTG, 16-bp sub-terminal repeats, internal variable regions, 3-bp target sequence duplications in the flanking regions, and genes coding for Rim2 proteins (the putative transposase) and hydroxyproline-rich glycoproteins . High levels of insertion into genic regions are observed for members of this family, and the transposition history of the family can be deduced from the high level of shared sequences and analysis of repeat target sites of the elements . Phylogenetic analysis indicates that the putative RIM2 proteins fall into a subgroup distinct from the TNP2-like subgroup of transposases . Southern hybridization with genomic DNA from monocotyledonous and dicotyledonous plants demonstrates that the RIM2-coding sequence is unique to the Oryza genome . Our results demonstrate that the Rim2 elements from rice belong to a distinct superfamily of CACTA-like elements with evolutionary diversity. J Biomol NMR, 2003 Dec, 27(4), 377 - 82 Stereospecific assignments of the isopropyl methyl groups of the membrane protein OmpX in DHPC micelles; Hilty C et al.; In NMR studies of large molecular structures, the number of conformational constraints based on NOE measurements is typically limited due to the need for partial deuteration . As a consequence, when using selective protonation of peripheral methyl groups on a perdeuterated background, stereospecific assignments of the diastereotopic methyl groups of Val and Leu can have a particularly large impact on the quality of the NMR structure determination . For example, 3D 15N- and 13C-resolved {1H,1H}-NOESY spectra of the E . Coli membrane protein OmpX in mixed micelles with DHPC, which have an overall molecular weight of about 60 kDa, showed that about 50% of all obtainable NOEs involve the diastereotopic methyl groups of Val and Leu . In this paper, we used biosynthetically-directed fractional 13C labeling of OmpX and {13C,1H}-HSQC spectroscopy to obtain stereospecific methyl assignments of Val and Leu in OmpX/DHPC . For practical purposes it is of interest that this data could be obtained without use of a deuterated background, and that combinations of NMR experiments have been found for obtaining the desired information either at a 1H frequency of 500 MHz, or with significantly reduced measuring time on a high-frequency instrument. Plant Physiol, 2003 Oct, 133(2), 773 - 82 Epub 2003 Sep 25. Functional characterization and expression of a cytosolic iron-superoxide dismutase from cowpea root nodules; Moran JF et al.; An iron-superoxide dismutase (FeSOD) with an unusual subcellular localization, VuFeSOD, has been purified from cowpea (Vigna unguiculata) nodules and leaves . The enzyme has two identical subunits of 27 kD that are not covalently bound . Comparison of its N-terminal sequence (NVAGINLL) with the cDNA-derived amino acid sequence showed that VuFeSOD is synthesized as a precursor with seven additional amino acids . The mature protein was overexpressed in Escherichia coli, and the recombinant enzyme was used to generate a polyclonal monospecific antibody . Phylogenetic and immunological data demonstrate that there are at least two types of FeSODs in plants . An enzyme homologous to VuFeSOD is present in soybean (Glycine max) and common bean (Phaseolus vulgaris) nodules but not in alfalfa (Medicago sativa) and pea (Pisum sativum) nodules . The latter two species also contain FeSODs in the leaves and nodules, but the enzymes are presumably localized to the chloroplasts and plastids . In contrast, immunoblots of the soluble nodule fraction and immunoelectron microscopy of cryo-processed nodule sections demonstrate that VuFeSOD is localized to the cytosol . Immunoblot analysis showed that the content of VuFeSOD protein increases in senescent nodules with active leghemoglobin degradation, suggesting a direct or indirect (free radical-mediated) role of the released Fe in enzyme induction . Therefore, contrary to the widely held view, FeSODs in plants are not restricted to the chloroplasts and may become an important defensive mechanism against the oxidative stress associated with senescence. J Biol Chem, 2003 Dec 5, 278(49), 49323 - 31 Epub 2003 Sep 25. Critical role of Lys212 and Tyr140 in porcine NADP-dependent isocitrate dehydrogenase; Kim TK et al.; Lys212 and Tyr140 are close to the enzyme-bound isocitrate in the recently determined crystal structure of porcine NADP-specific isocitrate dehydrogenase (Ceccarelli, C., Grodsky, N . B., Ariyaratne, N., Colman, R . F., and Bahnson, B . J . (2002) J . Biol . Chem . 277, 43454-43462) . We have constructed mutant enzymes in which Lys212 is replaced by Gln, Tyr, and Arg, and Tyr140 is replaced by Phe, Thr, Glu, and Lys . Wild type and mutant enzymes were each expressed in Escherichia coli and purified to homogeneity . At pH 7.4, the specific activity is decreased in K212Q, K212Y, and K212R, respectively, to 0.01-9% of wild type . The most striking change is in the pH-V(max) curves . Wild type depends on the deprotonated form of a group of pKaes 5.7, whereas this pKaes is increased to 7.4 in neutral K212Q and to 8.3 in K212Y . In contrast, the positive K212R has a pKaes of 5.9 . These results indicate that (by electrostatic repulsion) a positively charged residue at position 212 lowers the pK of the nearby ionizable group in the enzyme-substrate complex . Lys212 may also stabilize the carbanion formed initially on substrate decarboxylation . The Tyr140 mutants have specific activities at pH 7.4 that are reduced to 0.2-0.5% of those of wild type, whereas their Km values for isocitrate and NADP are not increased . Most notable are the altered pH-V(max) profiles . V(max) is constant from pH 5.3 to 8 for Y140F and Y140T and increases as pH is decreased for Y140E and Y140K . These results suggest that in wild type enzyme, Tyr140 is the general acid that protonates the substrate after decarboxylation and that the carboxyl and ammonium forms of Y140E and Y140K provide partial substitutes . Relative to wild type, the Y140T enzyme is specifically activated 106-fold by exogenous addition of acetic acid and 88-fold by added phenol; and the K212Q enzyme is activated 4-fold by added ethylamine . These chemical rescue experiments support the conclusion that Tyr140 and Lys212 are required for the catalytic activity of porcine NADP-dependent isocitrate dehydrogenase. J Biol Chem, 2003 Dec 5, 278(49), 48779 - 85 Epub 2003 Sep 25. SeqA protein stimulates the relaxing and decatenating activities of topoisomerase IV; Kang S et al.; The SeqA protein, which prevents overinitiation of chromosome replication, has been suggested to also participate in the segregation of chromosomes in Escherichia coli . Using a bacterial two-hybrid system, we found that SeqA interacts with the ParC subunit of topoisomerase IV (topo IV), a type II topoisomerase involved in decatenation of daughter chromosomes and relief of topological constraints generated by replication and transcription . We demonstrated that purified SeqA protein stimulates the activities of topo IV, both in relaxing supercoiled plasmid DNA and converting catenanes to monomers . The same moderate levels of SeqA protein did not affect the activities of DNA gyrase or topoisomerase I . At higher levels of SeqA, topo IV favored the formation of catenanes, caused by intermolecular strand exchange among plasmid DNA aggregates formed by SeqA . Excess SeqA inhibited the activity of all topoisomerases . We also found that stimulation of topo IV was dependent upon the affinity of SeqA for DNA . Our results suggest that this stimulation is mediated by the specific interaction of topo IV with SeqA . Some of the known phenotypes of mutant cells lacking SeqA, such as deficient chromosome segregation and increased negative superhelicity, support that the SeqA protein is required for topo IV-mediated relaxation and decatenation of chromosomes and plasmids, during and after their replication. Blood, 2004 Jan 15, 103(2), 607 - 12 Epub 2003 Sep 25. VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13; Kokame K et al.; ADAMTS-13 was recently identified as a new hemostatic factor, von Willebrand factor (VWF)-cleaving protease . Either congenital or acquired defects of the enzymatic activity lead to thrombotic thrombocytopenic purpura (TTP) . ADAMTS-13 specifically cleaves a peptidyl bond between Y1605 and M1606 in the A2 domain of VWF . Here, we determined the minimal region recognized as a specific substrate by ADAMTS-13 . A series of partial deletions in the A2 domain flanked with N- and C-terminal tags were expressed in Escherichia coli and affinity-purified . These purified proteins were incubated with human plasma, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by Western blot . Judging from mobility shifts, all constructs except one were cleaved at the expected site . Data suggested that a minimal region as a functional substrate consisted of 73 amino acid residues from D1596 to R1668 of VWF, designated VWF73, and that further deletion of the E1660-R1668 region led to the loss of cleavage by ADAMTS-13 . VWF73 was not cleaved by plasma from patients with congenital or acquired TTP, but cleaved by plasma from patients with hemolytic uremic syndrome, suggesting that VWF73 is a specific substrate forADAMTS-13 . Thus, VWF73 will be a useful seed to develop a new rapid assay to determine ADAMTS-13 activity. J Biotechnol, 2003 Oct 9, 105(1-2), 11 - 31 Gene expression patterns for metabolic pathway in pgi knockout Escherichia coli with and without phb genes based on RT-PCR; Kabir MM et al.; Metabolic regulations were investigated from the viewpoint of gene expressions for Escherichia coli JM109 and pgi knockout E . coli with and without phb genes using RT-PCR . One of the main features of pgi knockout E . coli is the overproduction of NADPH produced in pentose phosphate (PP) pathway . NADPH overproduction in PP pathway in pgi mutant causes some reducing power imbalance that ultimately affects the cell growth . It was shown that this reducing power imbalance can be recovered to some extent by introducing NADPH absorbing pathway such as PHB synthetic pathway into pgi mutant E . coli . To get insight into the regulation mechanism of pgi mutant E . coli at the transcriptional level, 87 E . coli genes involved in central metabolic pathways and key regulatory mechanisms were investigated by semi-quantitative RT-PCR analysis . The analysis showed that pentose phosphate pathway genes and part of the glycolysis pathway genes were affected significantly by expression of phb genes in pgi mutant E . coli DF11/pAeKG1 as well as in pgi mutant E . coli DF11 as compared with those in E . coli JM109 . In contrast, most of the TCA cycle genes except icdA were downregulated in both pgi mutants E . coli . The upregulation of icdA gene may be due to the positive regulation of fruR . Moreover, it was found that ack gene as well as aceA and aceB genes involved in the glyoxylate shunt were upregulated in pgi mutants while ppc gene was downregulated, indicating that pgi inactivation changes the anaplerotic pathway from ppc pathway to glyoxylate shunt . Enzyme activities of glk, zwf, tpiA, fbaA, ldhA, gltA, aceA, mdh and maeB were also measured and compared with the corresponding gene expressions . Most of them are well correlated except for aceA gene indicating that glyoxylate pathway is regulated on the protein level, not on the gene level. Mol Biochem Parasitol, 2003 Oct, 131(2), 129 - 36 Molecular characterization of a gene encoding a 29-kDa cytoplasmic protein of Babesia gibsoni and evaluation of its diagnostic potentiality; Fukumoto S et al.; A cDNA expression library prepared from Babesia gibsoni merozoite mRNA was screened with B . gibsoni-infected dog serum . cDNA encoding 29-kDa protein was cloned and designated as the P29 gene . The complete nucleotide sequence of the P29 gene was 792 bp . Computer analysis suggested that the sequence of the P29 gene contained an open reading frame of 597 bp with a coding capacity of approximately 23.4 kDa and a single intron of 250 bp . The P29 protein had homology to Toxoplasma gondii cytoskeletal protein IMC1 . Southern blot analysis indicated that the P29 gene was present as a single copy in the B . gibsoni genome . The native P29 protein of B . gibsoni with a molecular mass of 29 kDa was identified by Western blotting with anti-recombinant P29 mouse serum . Confocal laser microscopic analysis showed that the P29 protein was located on the cytoplasma of B . gibsoni merozoites . The recombinant P29 protein expressed in E . coli was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) . The ELISA was able to differentiate between B . gibsoni-infected dog serum and B . canis subspecies-infected dog serum or normal dog serum . Furthermore, the antibody response against the P29 protein was maintained during the chronic stage of infection in an experimentally infected dog, indicating that the recombinant P29 protein might be a useful diagnostic reagent for the detection of antibodies to B . gibsoni in dogs. Allergy, 2003 Oct, 58(10), 1059 - 63 Allergenicity of recombinant Bla g 7, German cockroach tropomyosin; Jeong KY et al.; BACKGROUND: Cockroach infestation may sensitize and elicit allergic responses to genetically predisposed individuals . Invertebrate tropomyosins are a frequent cause of allergy and highly cross-reactive in nature . In this study, we aimed to produce recombinant German cockroach tropomyosin and investigate its allergenicity . METHODS: German cockroach tropomyosin (Bla g 7) was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) . The cloned cDNA was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-nitrilotriacetic (NTA) acid resin . The allergenicity of the recombinant tropomyosin was examined by enzyme-linked immunosorbent assay (ELISA) . RESULTS: The cloned Bla g 7 shared up to 91% amino acid sequence identity with other cockroach tropomyosins . ELISA showed a recombinant Bla g 7 sensitization rate of 16.2% to German cockroach allergic sera . Recombinant tropomyosin was able to inhibit 32.4% of the specific IgE binding to cockroach extract . CONCLUSIONS: Tropomyosin represents a minor allergen in cockroach extracts . It is hoped that recombinant tropomyosin will be useful for further studies and clinical applications. Allergy, 2003 Oct, 58(10), 993 - 1002 Molecular and immunological characterization of Pen ch 18, the vacuolar serine protease major allergen of Penicillium chrysogenum; Shen HD et al.; BACKGROUND: We have suggested previously that the 32 and 34 kDa major allergens of Penicillium chrysogenum (also known as P . notatum) are the vacuolar (Pen ch 18) and the alkaline (Pen ch 13) serine proteases, respectively, of P . chrysogenum . The purpose of this study is to characterize the 32 kDa allergen of P . chrysogenum and its immunoglobulin E (IgE)cross-reactivity with Pen ch 13 allergen . METHODS: The full-length cDNA of Pen ch 18 was isolated by reverse transcriptase-polymerase chain reaction and the 5'-rapid amplification cDNA end reaction . Recombinant Pen ch 18 was expressed as his-tagged proteins in Escherichia coli . Its reactivity with IgE and monoclonal antibodies against fungal serine protease allergens was analyzed by immunoblotting . The IgE cross-reactivity between Pen ch 18 and Pen ch 13 was analyzed by immunoblot inhibition . Overlapping recombinant fragments and synthetic peptides were used to map the B cell epitopes on Pen ch 18 . RESULTS: In this study, we isolated a 1857 bp cDNA fragment containing an open reading frame of 494 amino acids that encodes the preproenzyme of Pen ch 18 . Similar to other vacuolar serine proteases, this precursor appears to undergo N- and possibly C-terminal cleavage upon maturation . The his-tagged recombinant Pen ch 18 containing the putative sequence of the mature protein reacted with IgE antibodies in serum samples from asthmatic patients . In addition, IgE-binding to the 32 kDa major allergen of P . chrysogenum was inhibited when a positive serum sample was absorbed with recombinant Pen ch 18 before immunoblotting . Both inhibition and almost no inhibition of IgE-binding to the 32 kDa major allergen of Pen ch 18 were detected when eight positive serum samples were preabsorbed individually with purified Pen ch 13 before immunoblotting . The major IgE binding region was located in a fragment (PN1) encompassing the N-terminal 102 amino acid residues of the recombinant Pen ch 18 . A dominant linear IgE epitope was further mapped within residues 73-95 (peptide PN1-e) of the N-terminally processed allergen . Monoclonal antibody FUM20 that reacts with Pen ch 18 but not with Pen ch 13 binds a synthetic peptide with sequence encompassing the N-terminal 23 residues of the recombinant Pen ch 18 . Monoclonal antibody PCM39 that reacts with both Pen ch 13 and Pen ch 18 recognizes a peptide containing residues 132-154 of the allergen . CONCLUSIONS: Our results confirm that the Pen ch 18 allergen is a vacuolar serine protease of P . chrysogenum that matures through N- and possibly C-terminal processing . The finding that there are cross-reactive and allergen-specific IgE epitopes for Pen ch 18 and Pen ch 13 suggests that both major allergens should be included in clinically diagnostic P . chrysogenum extracts. Allergy, 2003 Oct, 58(10), 986 - 92 IgE-binding epitopes of the American cockroach Per a 3 allergen; Wu CH et al.; BACKGROUND: The Per a 3 is a species-specific allergen of the American cockroach (Periplaneta americana) related to insect hemolymph proteins and includes four known isoallergens . This study aimed to identify Per a 3 linear IgE-binding epitopes . METHODS: Per a 3 recombinant fragments were generated from the recombinant Per a 3.01 allergen (685 amino acid residues) by using existing restriction sites or by using polymerase chain reaction products, and expressed in Escherichia coli . Antigenicities were assessed by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and binding inhibition with human IgE . RESULTS: Human IgE recognized recombinant fragments 340-425, 466-579, 502-595, and 595-636 as revealed by immunoblotting and ELISA . On the other hand, the N-terminal fragment 1-399, recombinants 410-443, 472-551, 502-579, 606-636, and the C-terminal fragment 636-685 were unable to bind human IgE . Amino acid sequences 400-409, 466-471, 580-595, and 595-605 were shown to be required for IgE binding to the Per a 3.01 allergen, suggesting that the C-terminus contains most of the IgE-binding sites . Four peptides corresponding to these IgE-binding amino acid sequences were synthesized . These peptides reacted with most sera (62.5-87.5%) tested as revealed by ELISA, demonstrating a heterogeneous IgE-binding response . Moreover, preincubation of IgE-positive recombinant proteins and synthetic peptides with atopic IgE resulted in marked inhibition of the IgE binding to Per a 3.01 allergen . Amino acid sequences 400TVLRDPVFYQ409, 466NNVDQI471, 580VDKGHNYCGYPENLLI595, and 595IPKGKKGGQAY605 of the major recombinant American cockroach Per a 3.01 allergen were involved in IgE binding . CONCLUSION: These findings will advance our understanding of the antigenic structures responsible for allergenicity to the American cockroach, thereby providing strategies for the development of immunotherapies. Biochem J, 2004 Jan 1, 377(Pt 1), 95 - 105 The nucleotide-binding domain of the Zn2+-transporting P-type ATPase from Escherichia coli carries a glycine motif that may be involved in binding of ATP; Okkeri J et al.; In P-type ATPases, the nucleotide-binding (N) domain is located in the middle of the sequence which folds into the phosphorylation (P) domain . The N domain of ZntA, a Zn2+-translocating P-type ATPase from Escherichia coli, is approx . 13% identical with the N domain of sarcoplasmic reticulum Ca2+-ATPase . None of the Ca2+-ATPase residues involved in binding of ATP are found in ZntA . However, the sequence G503SGIEAQV in the N domain of ZntA resembles the motif GxGxxG, which forms part of the ATP-binding site in protein kinases . This motif is also found in Wilson disease protein where several disease mutations cluster in it . In the present work, we have made a set of disease mutation analogues, including the mutants G503S (Gly503-->Ser), G505R and A508F of ZntA . At low {ATP}, these mutant ATPases are poorly phosphorylated . The phosphorylation defect of the mutants G503S and G505R can, however, be partially (G503S) or fully (G505R) compensated for by using a higher {ATP}, suggesting that these mutations lower the affinity for ATP . In all three mutant ATPases, phosphorylation by P(i) has become less sensitive to the presence of ATP, also consistent with the proposal that the Gly503 motif plays a role in ATP binding . In order to test this hypothesis, we have modelled the N domain of ZntA using the sarcoplasmic reticulum Ca2+-ATPase structure as a template . In the model, the Gly503 motif, as well as the residues Glu470 and His475, are located in the proximity of the ATP-binding site . In conclusion, the mutagenesis data and the molecular model are consistent with the idea that the two loops carrying the residues Glu470, His475, Gly503 and Gly505 play a role in ATP binding and activation. Nucleic Acids Res Suppl, 2003, (3), 317 - 8 Cloning and expression of bglF gene from alkaliphilic Nocardiopsis sp . strain F96; Masuda S et al.; The gene encoding a novel beta-1,3-glucanase was cloned from alkaliphilic Nocardiopsis sp . F96 and sequenced . The gene contained an open reading frame of 936 bp . The deduced amino acid sequence of the beta-1,3-glucanase exhibited highest homology to those of family 16 glucanases, suggesting that the enzyme belonged to family 16 . The beta-1,3-glucanase gene was functionally expressed in Escherichia coli. Nucleic Acids Res Suppl, 2003, (3), 259 - 60 Leucyl/phenylalanyl (L/F)-tRNA-protein transferase-mediated N-terminal specific labelling of a protein in vitro; Kuno A et al.; N-terminal specific radioisotope (RI) labelling of a protein posessing lysine as the N-terminal residue (Lys-XBD) was achieved under mild enzymatic reaction condition using L/F-tRNA-protein transferase . To expose a single lysine moiety only at the N-teminal site, we overexpressed pelB signal peptide-Lys-XBD fusion protein using the bacterial expression host BL21(DE3) . The pelB signal peptide was sponteneously cleaved in E . coli to obtain the desired Lys-XBD. Nucleic Acids Res Suppl, 2003, (3), 245 - 6 Decoding property of C5 uridine modification at the wobble position of tRNA anticodon; Kurata S et al.; Post-transcriptional modification at the first (wobble) position of the tRNA anticodon participates in precise decoding of the genetic code . We recently identified a novel taurine-containing modified uridine (tau m5U; 5-taurinomethyluridine) at the wobble position of mammalian mitochondrial tRNAs and found lack of this modification in mutant mitochondrial tRNAs from human pathogenic cells of the mitochondrial encephalomyopathies, investigate molecular pathogenesis of the diseases, decoding activity of wobble uridines with or without C5 modification was measured using E . coli cell-free translation system . It has been revealed that C5 modification has a functional role for stabilizing U:G wobble base pair. Nucleic Acids Res Suppl, 2003, (3), 73 - 4 Detection of acceptor sites for antisense oligonucleotides on native folded RNA by fluorescence-labeled oligonucleotide; Mahara A et al.; Pyrene-labeled 2'-O-methyloligoribonucleotide (OMUpy) and 5'-Ru(II) complex labeled oligodeoxyribonucleotie (Ru-probe) were prepared as the fluorescence probes to detect acceptor sites of antisense molecules on native folded RNA under the physiological condition . OMUpy showed the remarkable increase of the fluorescence intensity (334-fold at 375 nm) only when hybridized with complementary oligoRNA . When OMUpy was applied to E . coli 16S-rRNA, the fluorescence intensities were increased in a sequence specific manner . The rotational correlation time of Ru-probe complementary to 16S-rRNA were largely dependent on the sequence of Ru-probe . These results suggest that the accessibility of the antisense molecules for RNA can be evaluated by those fluorescent probes. Biocell, 2003 Aug, 27(2), 197 - 203 Defense reactions of Dermatobia hominis (Diptera: Cuterebridae) larval hemocytes; Faraldo AC et al.; The defense reactions against biological (Histoplasma capsulatum and Escherichia coli) and non-biological materials (China ink and nylon thread) were tested in vivo in third instar larvae of Dermatobia hominis . The cellular defense performed by larval hemocytes was observed under electron microscopy . China ink particles were phagocytosed by granular cells 5 h after injection . E . coli cells were internalized by granular cells as early as 5 min after injection and totally cleared 180 min post-injection, when many hemocytes appeared disintegrated and others in process of recovering . H . capsulatum yeasts provoked, 24 h after being injected, the beginning of nodule formation . Nylon thread was encapsulated 24 h after the introduction into the hemocoel . Our results suggest that granular cells were the phagocytic cells and also the responsible for the triggering of nodule and capsule formation . In the presence of yeasts cells and nylon thread, they released their granules that chemotactically attracted the plasmatocytes that on their turn, flattened to surround and isolate the foreign material. Mar Biotechnol (NY), 2004 Jan-Feb, 6(1), 8 - 16 Epub 2003 Sep 29. Molecular cloning and expression of a pearl oyster (Pinctada fucata) homologue of mammalian putative tumor suppressor QM; Zhang Y et al.; The QM gene was originally identified as a putative tumor suppressor gene from a Wilms' tumor cell line by subtractive hybridization assay . Later studies showed that the QM protein is multifunctional, involved in cell growth and differentiation, energy metabolism, respiration, and cytoskeletal function . In this report a full-length complementary DNA encoding a QM counterpart in pearl oyster (Pinctada fucata) was isolated . Phylogenetic analysis shows that oyster QM is more closely related to its insect homologues than to the mammalian homologues . Analysis of the tissue expression pattern of the oyster QM gene showed that oyster QM messenger RNA is expressed in all tissues tested, with highest levels in the digestive gland and mantle . Furthermore, we expressed the QM protein in Escherichia coli; Western blotting showed that the antibody of human QM is immunoreactive to the expressed oyster QM protein . Incubation of the oyster QM with Zn2+ resulted in the reduction of intrinsic emission fluorescence and a red-shift in the lambda(max) emission, indicating the occurrence of Zn(2+)-induced conformational changes . This evidence presents a possible mechanism for the critical function of zinc ion in the interaction of QM with Jun. Exp Mol Med, 2003 Aug 31, 35(4), 310 - 6 Detection of antibodies against glucose 6-phosphate isomerase in synovial fluid of rheumatoid arthritis using surface plasmon resonance (BIAcore); Kim JY et al.; We have used a surface plasmon resonance biosensor (SPR, BIACORE 2000) to detect antibodies against glucose 6-phosphate isomerase (GPI) in synovial fluids of rheumatoid arthritis (RA) and osteoarthritis (OA) . Recombinant human GPI proteins fused with or without NusA were expressed in E . coli, purified to homogeneity and immobilized in flow cells of CM5 sensor chips . The flow cells immobilized with NusA protein or bovine serum albumin were used to monitor non-specific binding . Synovial fluid samples from RA patients showed a significantly higher level of binding to recombinant GPI proteins than samples from OA patients . Proteins which bound to the recombinant GPI proteins were confirmed to be immunoglobulin through the administration of anti-human immunoglobulin . NusA fusion protein was excellent for this assay because of a low background binding activity in the SPR analysis and its advantage of increased solubility in recombinant protein production . These results suggested a useful utilization of recombinant NusA-GPI fusion protein for the detection of autoantibodies against GPI in RA patients. Biophys J, 2003 Oct, 85(4), 2147 - 57 Robust biased Brownian dynamics for rate constant calculation; Zou G et al.; A reaction probability is required to calculate the rate constant of a diffusion-dominated reaction . Due to the complicated geometry and potentially high dimension of the reaction probability problem, it is usually solved by a Brownian dynamics simulation, also known as a random walk or path integral method, instead of solving the equivalent partial differential equation by a discretization method . Building on earlier work, this article completes the development of a robust importance sampling algorithm for Brownian dynamics-i.e., biased Brownian dynamics with weight control-to overcome the high energy and entropy barriers in biomolecular association reactions . The biased Brownian dynamics steers sampling by a bias force, and the weight control algorithm controls sampling by a target weight . This algorithm is optimal if the bias force and the target weight are constructed from the solution of the reaction probability problem . In reality, an approximate reaction probability has to be used to construct the bias force and the target weight . Thus, the performance of the algorithm depends on the quality of the approximation . Given here is a method to calculate a good approximation, which is based on the selection of a reaction coordinate and the variational formulation of the reaction probability problem . The numerically approximated reaction probability is shown by computer experiments to give a factor-of-two speedup over the use of a purely heuristic approximation . Also, the fully developed method is compared to unbiased Brownian dynamics . The tests for human superoxide dismutase, Escherichia coli superoxide dismutase, and antisweetener antibody NC6.8, show speedups of 17, 35, and 39, respectively . The test for reactions between two model proteins with orientations shows speedups of 2578 for one set of configurations and 3341 for another set of configurations. Biophys J, 2003 Oct, 85(4), 2087 - 99 Gating of MscL studied by steered molecular dynamics; Gullingsrud J et al.; Steered molecular dynamics simulations of the mechanosensitive channel of large conductance, MscL, were used to investigate how forces arising from membrane tension induce gating of the channel . A homology model of the closed form of MscL from Escherichia coli was subjected to external forces of 35-70 pN applied to residues near the membrane-water interface . The magnitude and location of these forces corresponded to those determined from the lateral pressure profile computed from a lipid bilayer simulation . A fully expanded state was obtained on the 10-ns timescale that revealed the mechanism for transducing membrane forces into channel opening . The expanded state agrees well with proposed models of MscL gating, in that it entails an irislike expansion of the pore accompanied by tilting of the transmembrane helices . The channel was most easily opened when force was applied predominantly on the cytoplasmic side of MscL . Comparison of simulations in which gating progressed to varying degrees identified residues that pose steric hindrance to channel opening. Trends Cell Biol, 2003 Oct, 13(10), 510 - 6 The Alb3/Oxa1/YidC protein family: membrane-localized chaperones facilitating membrane protein insertion? Kuhn A, Stuart R, Henry R, Dalbey RE. The recently identified Alb3/Oxa1/YidC family constitutes a novel class of proteins that function in promoting membrane insertion in chloroplasts, mitochondria and bacteria . These proteins mediate membrane insertion of a diverse group of membrane proteins that range from phage coat proteins in bacteria and respiratory-chain protein subunits in mitochondria to the light-harvesting chlorophyll-binding proteins in chloroplasts . Here, we discuss the Alb3/Oxa1/YidC protein family and their possible function as membrane chaperones, helping newly synthesized proteins to fold into the membrane bilayer. Mol Microbiol, 2003 Oct, 50(1), 349 - 62 Titration of the Escherichia coli DnaA protein to excess datA sites causes destabilization of replication forks, delayed replication initiation and delayed cell division; Morigen et al.; In Escherichia coli, the level of the initiator protein DnaA is limiting for initiation of replication at oriC . A high-affinity binding site for DnaA, datA, plays an important role . Here, the effect of extra datA sites was studied . A moderate increase in datA dosage ( approximately fourfold) delayed initiation of replication and cell division, but increased the rate of replication fork movement about twofold . At a further increase in the datA gene dosage, the SOS response was induced, and incomplete rounds of chromosome replication were detected . Overexpression of DnaA protein suppressed the SOS response and restored normal replication timing and rate of fork movement . In the presence of extra datA sites, cells showed a dependency on PriA and RecA proteins, indicating instability of the replication fork . The results suggest that wild-type replication fork progression normally includes controlled pausing, and that this is a prerequisite for normal replication fork function. Mol Microbiol, 2003 Oct, 50(1), 291 - 301 Eclipse period during replication of plasmid R1: contributions from structural events and from the copy-number control system; Olsson JA et al.; The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson-Stahl density-shift experiment . A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work . The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30 degrees C to 42 degrees C . The mutant plasmid had a similar eclipse at temperatures up to 38 degrees C . At 42 degrees C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350-400 plasmid replications per cell and cell generation . During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42 degrees C) . Hence, the copy-number control system contributed significantly to the eclipse . The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors. Mol Microbiol, 2003 Oct, 50(1), 193 - 204 Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III; Viguera E et al.; Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases . Here we report the properties of an E . coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (psi), as a result of the complete inactivation of the holD gene . We show that, in this mutant, chronic induction of the SOS response in a RecFOR-dependent way leads to lethality at high temperature . The SOS-induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA . Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild-type cell . In contrast, the SOS-induced Pol V DNA polymerase does not participate to the lethality of the holD mutant . We conclude that: (i) Psi is essential for efficient replication of the E . coli chromosome; (ii) SOS-induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks. Parasite Immunol, 2003 Jun, 25(6), 307 - 12 Serum IgG3 to the Plasmodium falciparum merozoite surface protein 2 is strongly associated with a reduced prospective risk of malaria; Metzger WG et al.; The merozoite surface protein 2 (MSP2) of Plasmodium falciparum is recognized by human antibodies elicited during natural infections, and may be a target of protective immunity . In this prospective study, serum IgG antibodies to MSP2 were determined in a cohort of 329 Gambian children immediately before the annual malaria transmission season, and the incidence of clinical malaria in the following 5 months was monitored . Three recombinant MSP2 antigens were used, representing each of the two major allelic serogroups and a conserved region . The prevalence of serum IgG to each antigen correlated positively with age and with the presence of parasitaemia at the time of sampling . These antibodies were associated with a reduced subsequent incidence of clinical malaria during the follow-up . This trend was seen for both IgG1 and IgG3, although the statistical significance was greater for IgG3, the most common subclass against MSP2 . After adjusting for potentially confounding effects of age and pre-season parasitaemia, IgG3 reactivities against each of the major serogroups of MSP2 remained significantly associated with a lower prospective risk of clinical malaria . Individuals who had IgG3 reactivity to both of the MSP2 serogroup antigens had an even more significantly reduced risk . Importantly, this effect remained significant after adjusting for a simultaneous strong protective association of antibodies to another antigen (MSP1 block 2) which itself remained highly significant. Parasite Immunol, 2003 Jun, 25(6), 297 - 305 Heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53; Romaris F et al.; This study investigates the heterogeneity and immunogenicity of the Trichinella TSL-1 antigen gp53 . Western blotting analysis of several Trichinella isolates with the gp53-specific monoclonal antibodies (mAbs) US5 and US9, produced in Btkxid mice, revealed that gp53 from the species T . britovi, T . murrelli and genotype T8 had higher MW (60 kDa) than gp53 from T . spiralis, T . nelsoni and genotype T6 (53 kDa) and from T . nativa (55 kDa) . mAb US5 reacted only with gp53 from T . spiralis . Experiments including immunoassays of gp53 binding by sera from T . spiralis-infected mice, in the presence of different potential inhibitors (recombinant gp53, US5, T . britovi-crude larval extract (CLE), and CLE N- and O-glycans), indicate (i) that gp53 from T . spiralis bears specific epitopes that induce antibody formation during infection; (ii) that the protein epitopes of gp53 are much more important (76 or 68% of total antibody reactivity in BALB/c and Swiss CD-1 mice, respectively) than the corresponding glycan epitopes including tyvelose (11 or 32% of total reactivity) for the induction of anti-gp53 circulating antibodies; and (iii) that the species-specific epitopes present on gp53 are differentially recognized in different mouse strains . Whereas in BALB/c mice US5- and non-US5-recognized species-specific epitopes on gp53 bind about 84% of circulating antibodies on day 80 post-infection, this percentage was only 38% in Swiss CD-1 mice . These data on the antigenicity of gp53 contrast with data for Trichinella CLE antigens, in that most circulating antibodies reactive with CLE antigens recognized tyvelose-containing epitopes (57% and 58% of circulating antibodies in BALB/c and Swiss CD-1 mice, respectively) . Together these results demonstrate that gp53 is recognized during infection but is antigenically different from other Trichinella TSL-1 antigens. J Enzyme Inhib Med Chem, 2003 Jun, 18(3), 273 - 8 Synthesis and enzymatic transformations of 5-halo-6-methoxy-5,6-dihydro derivatives of 5-{1-methoxy-2-halo (or 2,2-dihalo)ethyl}-2'-deoxyuridines as potential herpes simplex virus inhibitors; Kumar R; The 5-halo-6-methoxy-5,6-dihydro derivatives of 5-{1-methoxy-2-halo(or 2,2-dihalo)ethyl}-2'-deoxyuridines (3-12) were synthesized and investigated as potential anti-herpes agents . These 5,6-dihydro derivatives were designed to act as potential prodrugs to 5-{1-methoxy-2-halo(or 2,2-dihalo)ethyl}-2'-deoxyuridines (2a-e), with enhanced metabolic stability, and ready conversion to the parent molecules . These 5,6-disubstituted-5,6-dihydro analogs are stable to E . coli thymidine phosphorylase, and undergo regeneration of the 5,6-olefinic bond to provide parent moieties (2a-e), upon incubation with glutathione at 37 degrees C . The compounds (3-12) themselves were found to be non-inhibitory against herpes simplex virus type-1 (HSV-1), likely due in part to their inability to undergo conversion to parent compounds in cell culture medium. Lipids, 2003 Jul, 38(7), 761 - 8 Enterotoxigenic Escherichia coli strains bind bovine milk gangliosides in a ceramide-dependent process; Martin MJ et al.; Diarrhea caused by enterotoxigenic Escherichia coli (ETEC) is the main infectious disease of newborn calves . The first step of infection involves bacterial attachment to the intestinal mucosa . This adhesion is mediated by fimbriae that recognize some glycoconjugates on the host cell surface, in particular, several gangliosides . Because milk also contains gangliosides, these have been suggested to serve as ligands for bacterial fimbriae and thus prevent the bacterial attachment to mucosa . The most relevant ETEC strains in calves, including those with K99 and F41 fimbriae, were assayed to determine whether they are able to bind gangliosides isolated from several stages of bovine lactation . Both GM3 and GD3, the main gangliosides of milk, were recognized by ETEC strains, although the different fimbriae showed diverse levels of affinity . Unexpectedly, the adhesion to colostral gangliosides was considerably weaker than that to gangliosides from the other stages of lactation . Because the carbohydrate moiety did not change and because differences in the percentages of unsaturated FA and sphingosine between colostrum and other stages were observed, we conclude that the differences in adhesion could be due to a different composition of the ganglioside ceramide. Presse Med, 2003 Sep 6, 32(28), 1319 - 22 {Tolosa-Hunt syndrome revealing Burkitt lymphoma in an HIV-seropositive patient}; Ghosn J et al.; INTRODUCTION: Tolosa-Hunt's syndrome is characterised by a painful, uni or bilateral, recurrent ophthalmoplegia, involving one or several ocular motor nerves . It is secondary to non-specific granulomatous infiltration of the cavernous sinus . It regresses rapidly with systemic corticosteroid therapy . Lymphomas involving the cavernous sinus may mimic a Tolosa-Hunt syndrome and hence delay diagnosis . OBSERVATION: A 39 year-old HIV-seropositive male consulted for a painful ophthalmoplegia revealing a generalised Burkitt lymphoma evoking Tolosa-Hunts' syndrome . The outcome was unfavourable . CONCLUSION: When confronted with a clinical picture evoking Tolosa-Hunt's syndrome, thorough examination is required to eliminate the localisation of a lymphoma or meningioma. J Biol Chem, 2003 Dec 12, 278(50), 50435 - 41 Epub 2003 Sep 23. Allosteric and catalytic functions of the PPi-binding motif in the ATP sulfurylase-GTPase system; Pilloff DE et al.; ATP sulfurylase, from Escherichia coli K-12, catalyzes and couples the Gibbs potentials of two reactions, GTP hydrolysis and activated sulfate (APS, adenosine 5'-phosphosulfate) synthesis . Coupling these potentials requires that the catalytic cycle include reaction stage-dependent conformational changes that gate the activities of the two active sites . These interactions were probed in a mutagenesis study of a highly conserved pyrophosphate-binding motif (SXGXDS), which is located at the APS-forming active site . The motif appears to be unique to the N-type PPi synthetase family, and mutations in it are linked, in other systems, to citrullinemia, an often fatal orphan disease . The conserved sites in the motif were evaluated individually for their ability to activate GTP hydrolysis (which reports interactions among the activator (AMP or Mg2+.PPi), the enzyme, and GTP), to affect the energetic coupling of the two reactions, and to alter the kinetic constants of the adenylyl transfer reaction in the absence of guanine nucleotide . What emerges from this first mutagenic exploration of the PPi motif in any adenylyltransferase is that the residues of the motif participate differently, and in sometimes profoundly important ways, in the different functions of the enzyme. J Biol Chem, 2003 Dec 5, 278(49), 48965 - 72 Epub 2003 Sep 22. Defining the regions of Escherichia coli YidC that contribute to activity; Jiang F et al.; The YidC/Oxa1/Alb3 family of proteins catalyzes membrane protein insertion in bacteria, mitochondria, and chloroplasts . In this study, we investigated which regions of the bacterial YidC protein are important for its function in membrane protein biogenesis . In Escherichia coli, YidC spans the membrane six times, with a large 319-residue periplasmic domain following the first transmembrane domain . We found that this large periplasmic domain is not required for YidC function and that the residues in the exposed hydrophilic loops or C-terminal tail are not critical for YidC activity . Rather, the five C-terminal transmembrane segments that contain the three consensus sequences in the YidC/Oxa1/Alb3 family are important for its function . However, by systematically replacing all the residues in transmembrane segment (TM) 2, TM3, and TM6 with serine and by swapping TM4 and TM5 with unrelated transmembrane segments, we show that the precise sequence of these transmembrane regions is not essential for in vivo YidC activity . Single serine mutations in TM2, TM3, and TM6 impaired the membrane insertion of the Sec-independent procoat-leader peptidase protein . We propose that the five C-terminal transmembrane segments of YidC function as a platform for the translocating substrate protein to support its insertion into the membrane. J Biol Chem, 2003 Dec 12, 278(50), 50572 - 7 Epub 2003 Sep 23. Structuring of the 3' splice site by U2AF65; Kent OA et al.; Recognition of the 3' splice site in mammalian introns is accomplished by association of the splicing factor U2AF with the precursor mRNA (pre-mRNA) in a multiprotein splicing commitment complex . It is well established that this interaction involves binding of the large U2AF65 subunit to sequences upstream of the 3' splice site, but the orientation of the four domains of this protein with respect to the RNA and hence their role in structuring the commitment complex remain unclear and the basis of contradictory models . We have examined the interaction of U2AF65 with an RNA representing the 3' splice site using a series of U2AF deletion mutants modified at the N terminus with the directed hydroxyl radical probe iron-EDTA . These studies, combined with an analysis of extant high resolution x-ray structures of protein.RNA complexes, suggest a model whereby U2AF65 bends the pre-mRNA to juxtapose reactive functionalities of the pre-mRNA substrate and organize these structures for subsequent spliceosome assembly. J Biol Chem, 2003 Dec 12, 278(50), 49860 - 7 Epub 2003 Sep 23. Human RNase H1 uses one tryptophan and two lysines to position the enzyme at the 3'-DNA/5'-RNA terminus of the heteroduplex substrate; Lima WF et al.; In a previous study, we showed that the RNA-binding domain of human RNase H1 is responsible for the positional preference for cleavage exhibited by the enzyme (Wu, H., Lima, W . F., and Crooke, S . T . (2001) J . Biol . Chem . 276, 23547-23553) . Here, we identify the substituents on the heteroduplex substrate and the amino acid residues within the RNA-binding domain of human RNase H1 involved in positioning of the enzyme . The human RNase H1 cleavage patterns observed for heteroduplexes with various 3'-DNA/5'-RNA and 5'-DNA/3'-RNA termini indicate that the 5'-most cleavage site on the oligoribonucleotide is positioned 7 bp from the first 3'-DNA/5'-RNA base pair . The presence or absence of phosphate or hydroxyl groups at either the 3'-DNA or 5'-RNA terminus had no effect on the human RNase H1 cleavage pattern . Substitution of the 3'-deoxynucleotide with a ribonucleotide, 2'-methoxyethyl nucleotide, or mismatched deoxyribonucleotide resulted in the ablation of the 5'-most cleavage site on the oligoribonucleotide . Mutants in which Trp43 and Lys59-Lys60 of the RNA-binding domain were substituted with alanine showed a loss of the positional preference for cleavage . Comparison of the kcat, Km, and Kd for the alanine-substituted mutants with those for human RNase H1 suggests that Lys59 and Lys60 are involved in binding to the heteroduplex and that Trp43 is responsible for properly positioning the enzyme on the substrate for catalysis . These data suggest that Trp43, Lys59, and Lys60 constitute an extended nucleic binding surface for the RNA-binding domain of human RNase H1, with the entire interaction taking place at the 3'-DNA/5'-RNA pole of the heteroduplex . These results offer further insights into the interaction between human RNase H1 and the heteroduplex substrate as well as approaches to enhance the design of effective antisense oligonucleotides. J Biol Chem, 2003 Dec 5, 278(49), 49316 - 22 Epub 2003 Sep 23. The periplasmic molecular chaperone protein SurA binds a peptide motif that is characteristic of integral outer membrane proteins; Bitto E et al.; The Escherichia coli SurA protein is a periplasmic molecular chaperone that facilitates correct folding of outer membrane porins . The peptide binding specificity of SurA has been characterized using phage display of heptameric peptides of random sequence . The consensus binding pattern of aromatic-polar-aromatic-nonpolar-proline amino acids emerges for both SurA and a SurA "core domain," which remains after deletion of a peripheral peptidyl-proline isomerase domain . Isothermal titration calorimetry with a high affinity heptameric peptide of sequence WEYIPNV yields peptide affinities in the range of 1-14 microm for both SurA and its core domain . Although the peptide consensus aromatic-polar-aromatic-nonpolar-proline occurs infrequently in E . coli proteins, the less restrictive tripeptide motif aromatic-random-aromatic appears with greater-than-random frequency in outer membrane proteins and is prevalent in the "aromatic bands" of the porin beta barrel structures . Thus, SurA recognizes a peptide motif that is characteristic of integral outer membrane proteins. J Biol Chem, 2003 Dec 5, 278(49), 49478 - 86 Epub 2003 Sep 23. Crystal structure of Escherichia coli thiol peroxidase in the oxidized state: insights into intramolecular disulfide formation and substrate binding in atypical 2-Cys peroxiredoxins; Choi J et al.; Thioredoxin-dependent thiol peroxidase (Tpx) from Escherichia coli represents a group of antioxidant enzymes that are widely distributed in pathogenic bacterial species and which belong to the peroxiredoxin (Prx) family . Bacterial Tpxs are unique in that the location of the resolving cysteine (CR) is different from those of other Prxs . E . coli Tpx (EcTpx) shows substrate specificity toward alkyl hydroperoxides over H2O2 and is the most potent reductant of alkyl hydroperoxides surpassing AhpC and BCP, the other E . coli Prx members . Here, we present the crystal structure of EcTpx in the oxidized state determined at 2.2-A resolution . The structure revealed that Tpxs are the second type of atypical 2-Cys Prxs with an intramolecular disulfide bond formed between the peroxidatic (CP, Cys61) and resolving (Cys95) cysteine residues . The extraordinarily long N-terminal chain of EcTpx folds into a beta-hairpin making the overall structure very compact . Modeling suggests that, in atypical 2-Cys Prxs, the CR-loop as well as the CP-loop may alternately assume the fully folded or locally unfolded conformation depending on redox states, as does the CP-loop in typical 2-Cys Prxs . EcTpx exists as a dimer stabilized by hydrogen bonds . Its substrate binding site extends to the dimer interface . A modeled structure of the reduced EcTpx in complex with 15-hydroperoxyeicosatetraenoic acid suggests that the size and shape of the binding site are particularly suited for long fatty acid hydroperoxides consistent with its greater reactivity. J Biol Chem, 2003 Dec 12, 278(50), 49699 - 706 Epub 2003 Sep 23. A functional interaction between chromogranin B and the inositol 1,4,5-trisphosphate receptor/Ca2+ channel; Thrower EC et al.; Chromogranins A and B (CGA and CGB) are high capacity, low affinity calcium (Ca2+) storage proteins found in many cell types most often associated with secretory granules of secretory cells but also with the endoplasmic reticulum (ER) lumen of these cells . Both CGA and CGB associate with inositol 1,4,5-trisphosphate receptor (InsP3R) in a pH-dependent manner . At an intraluminal pH of 5.5, as found in secretory vesicles, both CGA and CGB bind to the InsP3R . When the intraluminal pH is 7.5, as found in the ER, CGA totally dissociates from InsP3R, whereas CGB only partially dissociates . To investigate the functional consequences of the interaction between the InsP3R and CGB monomers or CGA/CGB heteromers, purified mouse InsP3R type I were fused to planar lipid bilayers and activated by 2 microM InsP3 . In the presence of luminal CGB monomers or CGA/CGB heteromers the InsP3R/Ca2+ channel open probability and mean open time increased significantly . The channel activity remained elevated when the pH was changed to 7.5, a reflection of CGB binding to the InsP3R even at pH 7.5 . These results suggest that CGB may play an important modulatory role in the control of Ca2+ release from the ER . Furthermore, the difference in the ability of CGA and CGB to regulate the InsP3R/Ca2+ channel and the variability of CGA/CGB ratios could influence the pattern of InsP3-mediated Ca2+ release. J Biol Chem, 2003 Dec 5, 278(49), 49301 - 7 Epub 2003 Sep 23. Effect of His-Gly-Lys motif derived from domain 5 of high molecular weight kininogen on suppression of cancer metastasis both in vitro and in vivo; Kawasaki M et al.; We have demonstrated previously that kinin-free high molecular weight kininogen, its domain 5 (D5H, Gly402-Lys502), and peptides derived from D5H inhibited vitronectin-mediated migration and invasion of cancer cells in vitro (Kamiyama, F., Maeda, T., Yamane, T., Li, Y . H., Ogikubo, O., Otsuka, T., and Ohkubo, I . (2001) Biochem . Biophys . Res . Commun . 288, 975-980) . In this study, we found that the amino acid sequence His-Gly-Lys (HGK) in D5H is the core motif for inhibition of adhesion and invasion of MDA-MB-231 cells in vitro . P-5m (484GHGKHKNK491, Gly484-Lys491), an octapeptide including the HGK motif derived from D5H, and HGK, a tripeptide, inhibited both cell adhesion and invasion in vitro . However, an octapeptide designated P-5m (K487R), in which Lys487 was changed to Arg, did not inhibit either cell adhesion or invasion, and peptides HGR and HGG also had no inhibitory effect . Recombinant GST-D5H expressed in Escherichia coli had a stronger inhibitory effect on cell adhesion and invasion in vitro than did GST-D5H (K487R) in which Lys487 was changed to Arg . Furthermore, P-5m (Gly484-Lys491) peptide clearly suppressed lung metastasis in mice experimentally induced by using B16-F10 cells, but P-5m (G487R) had no effect . These data strongly indicate that both the HGK motif and ly