Escherichia coli

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 lysine residue (Lys487) play essential roles in inhibition of cell adhesion and invasion in vitro and in prevention of metastasis of cancer cells in vivo . We tried to identify the HGK motif binding protein on the surface of cancer cells . A 95-kDa surface biotin-labeled membrane protein was specifically detached from GST-D5H by P-5 (His479-Lys493) peptide but not by P-1 (Gly402-Lys420) peptide originating from the N-terminal region of D5H.

J Biol Chem, 2003 Dec 5, 278(49), 49505 - 11 Epub 2003 Sep 23.
Hydrolytically deficient MutS E694A is defective in the MutL-dependent activation of MutH and in the mismatch-dependent assembly of the MutS.MutL.heteroduplex complex; Baitinger C et al.; The roles of ATP binding and hydrolysis by MutS in mismatch repair are poorly understood . MutS E694A, in which Glu-694 of the Walker B motif is substituted with alanine, is defective in hydrolysis of bound ATP and has been reported to support MutL-dependent activation of the MutH d(GATC) endonuclease in a trans DNA activation assay (Junop, M . S., Obmolova, G., Rausch, K., Hsieh, P., and Yang, W . (2001) Mol . Cell 7, 1-12) . Because the MutH trans activation assay used in these previous studies was characterized by high background and low efficiency, we have re-evaluated the activities of MutS E694A . In contrast to native MutS, which can be isolated in a nucleotide-free form, purified MutS E694A contains 1.0 mol of bound ATP per dimer equivalent, and substoichiometric levels of bound ADP (0.08-0.58 mol/dimer), consistent with the suggestion that the ADP.MutS.ATP complex comprises a significant fraction of the protein in solution (Bjornson, K . P . and Modrich, P . (2003) J . Biol . Chem . 278, 18557-18562) . In the presence of Mg2+, endogenous ATP is hydrolyzed with a rate constant of 0.12 min-1 at 30 degrees C, and hydrolysis yields a protein that displays increased specificity for heteroduplex DNA . As observed with wild type MutS, ATP can promote release of MutS E694A from a mismatch . However, the mutant protein is defective in the methyl-directed, mismatch- and MutL-dependent cis activation of MutH endonuclease on a 6.4-kilobase pair heteroduplex, displaying only 1 to 2% of the activity of wild type MutS . The mutant protein also fails to support normal assembly of the MutS.MutL.DNA ternary complex . Although a putative ternary complex can be observed in the presence of MutS E694A, assembly of this structure displays little if any dependence on a mismatched base pair.

Clin Cancer Res, 2003 Sep 1, 9(10 Pt 2), 3886S - 96S
Development of new multivalent-bispecific agents for pretargeting tumor localization and therapy; Rossi EA et al.; PURPOSE: Two bispecific diabodies (BS1.5 and BS1.5H) and two bispecific trivalent proteins (BS6 and BS8) were produced and tested as potential agents for pretargeted delivery of radiolabeled bivalent haptens to tumors expressing carcinoembryonic antigen . EXPERIMENTAL DESIGN: Each of the four proteins was expressed in Escherichia coli and purified from the soluble fraction . BS1.5 and BS1.5H (a humanized version of BS1.5) were evaluated in the GW-39 human colonic tumor-nude mouse model using a di-HSG-1,4,7,10-tetra-azacyclododecane-N,N',N" N"'-tetraacetic acid peptide (IMP-241) radiolabeled with (111)In . The biodistribution and T/NT ratios were compared with those of hMN-14 x m679 (Fab' x Fab') prepared chemically . RESULTS: In animals, both BS1.5 and BS1.5H cleared more rapidly than hMN-14 x m679 and showed tumor to nontumor ratios far superior to those of hMN-14 x m679 . For example, with BS1.5 injected 8 h before (111)In-IMP-241, the tumor uptake of (111)In was 10.3 +/- 2.7 and 6.3 +/- 2.2% ID/g at 3 and 24 h, respectively, with the tumor to blood ratios being 167 +/- 35 at 3 h and 631 +/- 231 at 24 h . In comparison, the tumor to blood ratios of (111)In observed for hMN-14 x m679 given 24 h earlier were 8 +/- 2 at 3 h and 16 +/- 3 at 24 h . CONCLUSIONS: These results indicate that BS1.5 and BS1.5H are promising candidates for use in a variety of pretargeting applications, including tumor therapy with radionuclides and drugs . BS6 and BS8 may be even more attractive because of their potential to achieve higher levels of tumor uptake because of divalent carcinoembryonic antigen binding.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3323 - 5
DNA gyrase and topoisomerase IV mutations in clinical isolates of Ureaplasma spp . and Mycoplasma hominis resistant to fluoroquinolones; Bebear CM et al.; Twelve clinical isolates of Ureaplasma spp . and one isolate of Mycoplasma hominis were examined for resistance to fluoroquinolones . Previously described mutations at positions 83 and 95 in GyrA (Escherichia coli numbering) and positions 80 and 87 in ParC were found . Unusual alterations were described at positions ParC 123 and 134.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3233 - 9
Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers; Geller BL et al.; Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli . PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth . However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent response . A significant improvement was made by covalently joining the peptide (KFF)(3)KC to the end of PMOs . In strains with an intact outer membrane, (KFF)(3)KC-myc PMO inhibited luciferase expression by 63% . A second (KFF)(3)KC-PMO conjugate targeted to lacI mRNA induced beta-galactosidase in a dose-dependent response . The end of the PMO to which (KFF)(3)KC is attached affected the efficiency of target inhibition but in various ways depending on the PMO . Another peptide-lacI PMO conjugate was synthesized with the cationic peptide CRRRQRRKKR and was found not to induce beta-galactosidase . We conclude that the outer membrane of E . coli inhibits entry of PMOs and that (KFF)(3)KC-PMO conjugates are transported across both membranes and specifically inhibit expression of their genetic targets.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3080 - 4
Acanthamoeba polyphaga strain age and method of cyst production influence the observed efficacy of therapeutic agents and contact lens disinfectants; Hughes R et al.; The effects of age in culture and the type of medium used for induction of Acanthamoeba polyphaga (Ros) cysts on susceptibilities to polyhexamethylene biguanide (PHMB; 3 micro g/ml), chlorhexidine digluconate (30 micro g/ml), myristamidopropyl dimethylamine (20 micro g/ml), H(2)O(2) (3%), and two multipurpose contact lens solutions (MPS-1 and MPS-2, based on 1 micro g of PHMB per ml) were examined . Strain Ros-02 was cryopreserved on isolation in 1991, while strain Ros-91 had been in continuous axenic culture . Significant differences in susceptibilities to the disinfectants were found depending on the medium used for cyst preparation and the age of the test strain, with Ros-02 generally being more resistant . For example, the killing of Ros-91 cysts produced from an axenic culture of trophozoites in the presence of 50 mM MgCl(2) by MPS-2 was 4 logs, but the killing of Ros-02 by MPS-2 was only 2 logs (P < 0.05) and killing of both strains with cysts obtained from monoxenic cultures with Escherichia coli was only 1 log (P < 0.001) . Assays repeated with different batches of the various cyst types gave consistent results . A batch of Ros-91 cysts stored at 4 degrees C and tested over an 8-week period with MPS-1 showed progressively increasing susceptibility to disinfection, although there was no loss of viability during storage (P < 0.01) . These observations have important implications for the standardization and interpretation of Acanthamoeba disinfectant and therapeutic agent testing.

Vaccine, 2003 Oct 1, 21(27-30), 4328 - 34
Mucosal antibody response induced with a nasal virosome-based influenza vaccine; Durrer P et al.; A vaccination against influenza that elicits both a systemic antibody and a mucosal IgA response would improve on the protective efficacy of currently available vaccines . Previous studies have shown the safety and efficacy of virosomes as delivery systems in vaccination . This study was a controlled, randomised, double-blind, single centre, phase II trial assessing an intranasal virosome vaccine, adjuvanted with heat-labile toxin (HLT) from enterotoxigenic Escherichia coli, versus an intranasal without HLT and comparing it open to an intramuscular vaccine in a total of 88 healthy adults . The development of a new technique enabled for the first time the detection of neutralising IgA antibodies in very dilute nasal wash samples . It was demonstrated that intranasally administered inactivated influenza vaccine, adjuvanted with HLT, not only elicits a spectrum of humoral and cell-mediated responses in healthy adults, critical for the protection and recovery from influenza virus infection, but is also highly effective in eliciting IgA neutralising antibodies at the mucosa . Intranasal virosome-formulated, HLT-adjuvanted, influenza vaccine was effective and well tolerated in this study . Its potential to offer a high level of mucosal protection, not provided by conventional parenteral vaccination, could play a significant role in preventing morbidity and mortality associated with influenza.

Vaccine, 2003 Oct 1, 21(27-30), 4133 - 44
Splenectomised and spleen intact Aotus monkeys' immune response to Plasmodium vivax MSP-1 protein fragments and their high activity binding peptides; Sierra AY et al.; Two E . coli expressed recombinant polypeptides (rPvMSP-1(14) and rPvMSP-1(20)) contained in the 33kDa fragment, located within Plasmodium vivax merozoite surface protein (PvMSP-1) 42kDa C-terminal region, and a cocktail of high reticulocyte binding synthetic peptides located within these fragments, were evaluated for immunogenicity and protective immune responses in splenectomised and spleen intact Aotus nancymaae monkeys . Thirty splenectomised monkeys who had been previously immunised with either rPvMSP-1(14), rPvMSP-1(20), or a mixture of both recombinant fragments were intravenously challenged with the heterologous P . vivax VCG-1 strain (as determined by DNA sequencing); full protection was observed in five monkeys and low parasitaemia levels were obtained in eight more monkeys . Splenectomised control monkey group rapidly developed high parasitaemia levels, while no significant parasitaemia was obtained in the non-splenectomised control group . Although PvMSP-1 42 and 33kDa fragments were recognised by Western Blot and whole parasites by IFAT when tested with immune monkey sera, no correlation between protection and antibody titres by IFAT and ELISA was observed, suggesting that protection is not being solely mediated by a humoral immune response . This data showed that partial protection against a heterologous strain challenge was best achieved when immunising with a rPvMSP-1(14)-rPvMSP-1(20) mixture (2 were fully protected and 4 with low parasitaemia out of 12) suggesting for the first time, that these fragments could be good candidates for inclusion in a P . vivax multi-stage, multi-antigen vaccine.

Biochimie, 2003 Jul, 85(7), 647 - 50
Inhibition of alkaline phosphatase activity by okadaic acid, a protein phosphatase inhibitor; Mestrovic V et al.; This paper reports on a potential physiological target of okadaic acid (OA), the toxin metabolite responsible for shellfish poisoning and, consequently, human intoxication . OA is a potent promoter of tumor activity, most likely by inhibiting protein phosphatase 1 and 2A (Adv . Cancer . Res . 61 (1993) 143) . However, all of its cellular targets have not yet been characterized . The interaction of OA with alkaline phosphatase (ALP) has been investigated in view of its protein phosphatase inhibition activity . Kinetic analysis of ALP from Escherichia coli, human placental and calf intestinal ALP has shown that OA acts as a non-competitive inhibitor of ALP . The bacterial enzyme displays a higher affinity for OA (K(i) 360 nM) than the eukaryotic proteins (human placental ALP, K(i) 2.05 microM; calf intestinal ALP, K(i) 3.15 microM) . The inhibition by OA suggests a putative role of ALP in the phosphorylation status, through regulation of the phosphorylation/dephosphorylation equilibrium of proteins with phosphoseryl or phosphothreonyl residues.

Zhonghua Nei Ke Za Zhi, 2003 Aug, 42(8), 561 - 5
{Isolation of differentially expressed genes in human osteoblast-like osteosarcoma cell line induced with 17beta-estradiol}; Peng YQ et al.; OBJECTIVE: To obtain a serial differentially expressed cDNA fragments from human osteoblast-like osteosarcoma MG-63 cells induced with 17beta-estradiol and to find some estrogen-responsive genes . METHODS: Optimized cDNA representational difference analysis (RDA) was performed to isolate up-regulated expressed sequences between cDNA from MG-63 cell line treated with and without 17beta-estradiol . The sources of up-regulated expressed cDNA fragments were proved by Southern blot . The fragments were cloned into the pGEM-T easy vector and a cDNA library was prepared in E . coli JM109 cells . The cDNA library was plated on LB/Amp(+)/X-gal/IPTG plates and white colonies were picked up and individually grown in LB/Amp(+) medium in 96-well plates . After PCR, colonies were individually blotted onto a Hybond N membrane . Membranes were hybridized with alpha-(32)P-labeled subtracted or unsubtracted tester cDNA . Clones showing a strong hybridization signal with the forward-subtracted probes compared with the reverse-subtracted ones were selected for DNA sequencing, BLAST and Northern blot analysis after release of the pGEM-T easy insert with EcoR I . RESULTS: Five up-regulated expressed fragments were isolated in the fourth subtraction hybridization using cDNA from MG-63 cells induced with 17beta-estradiol as tester amplicon and cDNA from untreated MG-63 cells as driver amplicon by cDNA RDA . These fragments were proved to be really coming from tester amplicon and down-regulated expressed in the untreated cell by Southern blotting . We obtained more than 600 cDNA clones with positive insert from MG-63 cells line induced with 17beta-estradiol and about 120 differentially expressed clones through dot blotting . Twenty clones were sequenced and we got 15 gene sequences . One of them was proved to be differentially expressed through Northern blotting . CONCLUSIONS: cDNA RDA is one of the most effective methods which can isolate differentially expressed genes and we can screen differentially expressed genes rapidly through cDNA RDA combined with cDNA arrays . Some genes of human osteoblast-like osteosarcoma MG-63 cell line showed up-regulated expression after induction by 17beta-estradiol.

Bioprocess Biosyst Eng, 2002 Jun, 25(2), 121 - 8 Epub 2002 Apr 24.
The production of human papillomavirus type 16 L1 vaccine product from Escherichia coli inclusion bodies; Lai WB et al.; The major capsid protein L1 of the human papillomavirus type 16 (HPV16) has been previously expressed recombinantly in Escherichia coli cells as inclusion bodies (IBs) . The HPV16 L1 protein offers potential as a vaccine candidate against cervical cancer, but the reported E . coli process is limited in its ability to economically produce significant quantities of material . In this study, a scaleable laboratory process for the purification of recombinant His-tagged L1 protein and its processing to give an immunogenic product is developed . The performances of ion-exchange chromatography (IEX) and immobilised metal affinity chromatography (IMAC) for the purification of L1 protein in the presence of concentrated denaturant are compared . IEX was found to be superior to IMAC when taking into account the complexity of operation, cost of adsorbent, selectivity and purity of the final product . Following purification, reduction of denaturant concentration was performed by dilution to yield a product suitable for formulation . The simplicity and ease of scale-up of dilution makes it an attractive option for process scale production and superior to the existing approach employing dialysis . It was found that direct dilution of denaturant into suitable buffer can give rise to products which have neutralising conformational epitopes identified by strong antibody-binding properties, as assessed by ELISA with a conformational monoclonal antibody . Analysis of the results showed negative main effects of protein concentration and PEG addition on antibody-binding yields, but positive main effects of the addition of detergent and L-arginine to the buffer . The diluted product had antigenic properties as assessed by ELISA and may be formulated easily for use by diafiltration and the addition of adjuvant . This work demonstrates the feasibility of producing viral vaccines using E . coli and scaleable unit operations.

Bioprocess Biosyst Eng, 2003 Jan, 25(4), 255 - 62 Epub 2002 Nov 16.
Model-based optimization of viral capsid protein production in fed-batch culture of recombinant Escherichia coli; Levisauskas D et al.; An optimized fed-batch cultivation process for the production of the polyoma virus capsid protein VP1 in recombinant Escherichia coli BL21 bacteria is presented . The optimization procedure maximizing the amount of desired protein is based on a mathematical model . The model distinguishes an initial cell growth phase from a protein production phase initiated by inducer injection . A new approach to model the target protein formation rate was elaborated, where product formation is primarily dependent on the specific biomass growth rate . Lower growth rates led to higher specific protein concentrations . The model was identified from a series of fed-batch experiments designed for parameter identification purposes and possesses good prediction quality . Then the model was used to determine optimal open-loop control profiles by manipulating the substrate feed rates in both phases as well as the induction time . Feed-rate optimization has been solved using Pontryagin's maximum principle . The solution was validated experimentally . A significant improvement of the process performance index was achieved.

Bioprocess Biosyst Eng, 2003 Jan, 25(4), 205 - 12 Epub 2002 Sep 24.
Escherichia coli high-cell-density culture: carbon mass balances and release of outer membrane components; Han L et al.; Carbon mass balances were calculated in fed-batch cultures of E . coli W3110, using mineral medium with glucose as the limiting substrate . The carbon recovery, based on biomass, CO(2), and acetate was approximately 90% at the end of the culture (25 h, 27 g L(-1) dw) . The missing carbon remained as soluble organic compounds in the medium . Outer membrane (OM) constituents, such as lipopolysaccharides (LPS), phospholipids (PL), and carbohydrates (each at approximately 1 g L(-1)) contributed to 63% of the extracellular carbon . The amount of released LPS and PL equaled the total amount of OM bound to the cells in the culture . Small amounts of DNA and protein detected in the medium indicated that no cell lysis had occurred . Acetate, lactate, ethanol, formate, succinate and amino acids (Glu, Gln, Asp, Asn, Ala, Gly, Ser) were detected in the culture medium, but made up only a few percent of the extracellular carbon mass . The remaining 30% was not identified, but was assumed to constitute complex carbohydrates.

Plant Cell Rep, 2003 Dec, 22(5), 344 - 9 Epub 2003 Sep 20.
Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants; Putalun W et al.; We constructed a recombinant antibody fragment--single chain fragment-variable (scFv) antibody--derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene . The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb) . Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S . khasianum . The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities . These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.

J Biol Chem, 2003 Nov 28, 278(48), 47700 - 6 Epub 2003 Sep 22.
Presteady state kinetic analysis of riboflavin synthase; Illarionov B et al.; Riboflavin synthase catalyzes a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine . The kinetics of the enzyme from Escherichia coli were studied under single turnover conditions . Stopped flow as well as quenched flow experiments documented the transient formation of a pentacyclic reaction intermediate . No other transient species were sufficiently populated to allow detection . The data are best described by a sequence of one second order and one first order reaction.

J Biol Chem, 2003 Dec 19, 278(51), 51415 - 21 Epub 2003 Sep 22.
Mapping temperature-induced conformational changes in the Escherichia coli heat shock transcription factor sigma 32 by amide hydrogen exchange; Rist W et al.; Stress conditions such as heat shock alter the transcriptional profile in all organisms . In Escherichia coli the heat shock transcription factor, sigma 32, out-competes upon temperature up-shift the housekeeping sigma-factor, sigma 70, for binding to core RNA polymerase and initiates heat shock gene transcription . To investigate possible heat-induced conformational changes in sigma 32 we performed amide hydrogen (H/D) exchange experiments under optimal growth and heat shock conditions combined with mass spectrometry . We found a rapid exchange of around 220 of the 294 amide hydrogens at 37 degrees C, indicating that sigma 32 adopts a highly flexible structure . At 42 degrees C we observed a slow correlated exchange of 30 additional amide hydrogens and localized it to a helix-loop-helix motif within domain sigma 2 that is responsible for the recognition of the -10 region in heat shock promoters . The correlated exchange is shown to constitute a reversible unfolding with a half-life of about 30 min due to a temperature-dependent decrease in stabilization energy . We propose that this gradual decrease in stabilization energy of domain sigma 2 with increasing temperatures facilitates the unfolding of sigma 32 by the AAA+ protease FtsH thereby decreasing its half-life . Taken together our data show that the sigma 2 domain of sigma 32 can act as a thermosensor, which might be important for the heat shock regulation.

Biochemistry, 2003 Sep 30, 42(38), 11373 - 81
Recognition and removal of oxidized guanines in duplex DNA by the base excision repair enzymes hOGG1, yOGG1, and yOGG2; Leipold MD et al.; 8-Oxo-7,8-dihydroguanine (OG) is susceptible to further oxidation in vitro to form two secondary oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) . Previous work from this laboratory has shown that OG, Gh, and Sp are recognized and excised from duplex DNA substrates by the Escherichia coli DNA repair enzyme Fpg . In this report, we extend these studies to the functionally related eukaryotic OG glycosylases (OGG) from yeast and humans: yOGG1, yOGG2, and hOGG1 . The hOGG1 enzyme was active only toward the removal of 8-oxoguanine, exhibiting a 1000-fold faster rate of removal of 8-oxoguanine from OG.C-containing duplexes relative to their OG.A counterparts . Duplexes containing Gh or Sp opposite any of the four natural bases were not substrates for the hOGG1 enzyme . In contrast, both yOGG1 and yOGG2 enzymes removed Gh and Sp in a relatively efficient manner from an 18 bp duplex . No significant difference was observed in the rate of reaction of Gh- and Sp-containing duplexes with yOGG1 . However, yOGG2 removed Sp at a faster rate than Gh . Both yOGG enzymes exhibit a negligible dependence on the base opposite the lesion, suggesting that the activity of these enzymes may be promutagenic . Surprisingly, in the 18 bp sequence context, both yOGG enzymes did not exhibit OG removal activity . However, both removed OG in a 30 bp duplex with a different sequence surrounding the OG . The wide range of repair efficiencies observed by these enzymes with different substrates in vitro suggests that this could greatly affect the mutagenicity of these lesions in vivo . Indeed, the greater efficiency of the yOGG proteins for removal of the further oxidized products, Gh and Sp, over their 8-oxoguanine parent, suggests that these lesions may be the preferred substrates in vivo.

Biochemistry, 2003 Sep 30, 42(38), 11297 - 306
Escherichia coli cystathionine gamma-synthase does not obey ping-pong kinetics . Novel continuous assays for the elimination and substitution reactions; Aitken SM et al.; Cystathionine gamma-synthase (CGS) is a pyridoxal phosphate-dependent enzyme that catalyzes a gamma-replacement reaction, in which the succinyl group of an O-succinyl-L-homoserine (L-OSHS) is displaced by the thiol of L-cysteine to form L-cystathionine, in the first step of the bacterial transsulfuration pathway . The mechanism of Escherichia coli CGS (eCGS) is ordered with L-OSHS associating before L-Cys (k(catR)/K(mR)(L-OSHS) = 9.8 x 10(4) M(-1) s(-1), where the subscript R denotes the replacement reaction) . The mechanism becomes ping-pong (k(catR)/K(mR)(L-OSHS) = 4.9 x 10(4) M(-1) s(-1)) at L-Cys concentrations lower than K(m)(L-Cys) . The enzyme also catalyzes a competing gamma-elimination reaction, in which L-OSHS is hydrolyzed to succinate, NH(3), and alpha-ketobutyrate (k(catE)/K(mE)(L-OSHS) = 1350 +/- 90 M(-1) s(-1), where the subscript E denotes the elimination reaction) . The k(cat)/K(m)(L-OSHS) versus pH profile of eCGS is bell-shaped for both reactions . The pH optimum and the pK(a) values for the acidic and basic limbs are 7.4, 6.8 +/- 0.1, and 8.0 +/- 0.1, respectively, for the elimination reaction and 7.8, 7.4 +/- 0.1, and 8.3 +/- 0.1, respectively, for the replacement reaction . The internal aldimine of eCGS remains protonated at pH <10.5, and the alpha-amino group of L-OSHS has a pK(a) of 9.71 +/- 0.01; therefore, neither limb of the k(cat)/K(m)(L-OSHS) versus pH profiles can be assigned to aldimine, or to L-OSHS prototropy . Novel continuous assays for the elimination reaction, employing D-2-hydroxyisocaproate dehydrogenase, and for the substitution reaction, employing cystathionine beta-lyase and L-lactate dehydrogenase as coupling enzymes, are described.

Biochemistry, 2003 Sep 30, 42(38), 11289 - 96
Substrate specificity and kinetic isotope effect analysis of the Eschericia coli ketopantoate reductase; Zheng R et al.; Ketopantoate reductase (EC 1.1.1.169), an enzyme in the pantothenate biosynthetic pathway, catalyzes the NADPH-dependent reduction of alpha-ketopantoate to form D-(-)-pantoate . The enzyme exhibits high specificity for ketopantoate, with V and V/K for ketopantoate being 5- and 365-fold higher than those values for alpha-ketoisovalerate and 20- and 648-fold higher than those values for alpha-keto-beta-methyl-n-valerate, respectively . For pyridine nucleotides, V/K for beta-NADPH is 3-500-fold higher than that for other nucleotide substrates . The magnitude of the primary deuterium kinetic isotope effects on V and V/K varied substantially when different ketoacid and pyridine nucleotide substrates were used . The small primary deuterium kinetic isotope effects observed using NADPH and NHDPH suggest that the chemical step is not rate-limiting, while larger primary deuterium isotope effects were observed for poor ketoacid and pyridine nucleotide substrates, indicating that the chemical reaction has become partially or completely rate-limiting . The pH dependence of (D)V using ketopantoate was observed to vary from a value of 1.1 at low pH to a value of 2.5 at high pH, while the magnitude of (D)V/K(NADPH) and (D)V/K(KP) were pH-independent . The value of (D)V is large and pH-independent when alpha-keto-beta-methyl-n-valerate was used as the ketoacid substrate . Solvent kinetic isotope effects of 2.2 and 1.2 on V and V/K, respectively, were observed with alpha-keto-beta-methyl-n-valerate . Rapid reaction analysis of NADPH oxidation using ketopantoate showed no "burst" phase, suggesting that product-release steps are not rate-limiting and the cause of the small observed kinetic isotope effects with this substrate pair . Large primary deuterium isotope effects on V and V/K using 3-APADPH in steady-state experiments, equivalent to the isotope effect observed in single turnover studies, suggests that chemistry is rate-limiting for this poorer reductant . These results are discussed in terms of a kinetic and chemical mechanism for the enzyme.

Biochemistry, 2003 Sep 30, 42(38), 11234 - 42
Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase; Ortiz-Maldonado M et al.; p-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor FAD by NADPH in response to binding p-hydroxybenzoate to the enzyme and reaction of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate . Three different reactions, each with specific requirements, are achieved by moving the position of the isoalloxazine ring in the protein structure . In this paper, we examine the operation of protein conformational changes and the significance of charge-transfer absorption bands associated with the reduction of FAD by NADPH when the substrate analogue, 5-hydroxypicolinate, is bound to the enzyme . It was discovered that the enzyme with picolinate bound was reduced at a rate similar to that with p-hydroxybenzoate bound at high pH . However, there was a large effect of pH upon the rate of reduction in the presence of picolinate with a pK(a) of 7.4, identical to the pK(a) of picolinate bound to the enzyme . The intensity of charge-transfer bands observed between FAD and NADPH during the reduction process correlated with the rate of flavin reduction . We conclude that high rates of reduction of the enzyme require (a) the isoalloxazine of the flavin be held by the protein in a solvent-exposed position and (b) the movement of a loop of protein so that the pyridine ring of NADPH can move into position to form a complex with the isoalloxazine that is competent for hydride transfer and that is indicated by a strong charge-transfer interaction.

Biochemistry, 2003 Sep 30, 42(38), 11226 - 33
Evidence for structural symmetry and functional asymmetry in the lactose permease of Escherichia coli; Green AL et al.; Previous work on the lactose permease of Escherichia coli has shown that mutations along a face of predicted transmembrane segment 8 (TMS-8) play a critical role in conformational changes associated with lactose transport (Green, A . L., and Brooker, R . J . {2001} Biochemistry 40, 12220-12229) . Substitutions at positions 261, 265, 268, 272, and 276, which form a continuous stripe along TMS-8, were markedly defective for lactose transport velocity . In the current study, three single mutants (F261D, N272Y, N272L) and a double mutant (T265Y/M276Y) were chosen as parental strains for the isolation of mutants that restored transport function . A total of 68 independent mutants were isolated and sequenced . Forty-four were first-site revertants in which the original mutation was changed back to the wild-type residue or to a residue with a similar side-chain volume . The other 24 mutations were second-site suppressors in TMS-2 (Q60L, Q60P), loop 2/3 (L70H), TMS-7 (V229G/A), TMS-8 (F261L), and TMS-11 (F354V, C355G) . On the basis of their locations, the majority of the second-site suppressors can be interpreted as improving the putative TMS-2/TMS-7/TMS-11 interface to compensate for conformational defects imposed by mutations in TMS-8 that disrupt the putative TMS-1/TMS-5/TMS-8 interface . Overall, this paper suggests that the TMS-2/TMS-7/TMS-11 interface is more important from a functional point of view, even though there is compelling evidence for structural symmetry between the two halves of the permease.

Biochemistry, 2003 Sep 30, 42(38), 11183 - 93
Mutagenesis study on the zebra fish SOX9 high-mobility group: comparison of sequence and non-sequence specific HMG domains; Hsiao NW et al.; A unique class of proteins, containing high-mobility group (HMG) domain(s), recognizes unusual DNA structures and/or bends specific to AT-rich linear double-stranded DNA . The DNA binding feature of these proteins is exhibited in the HMG domain(s) . Although the sequence specific and non-sequence specific HMG domains exhibit very high degrees of sequence similarity, the reasons for the difference between their DNA recognition mechanisms are unclear . A series of zebra fish SOX9 HMG domain mutants was prepared in an effort to elucidate the importance of various residues on protein stability and DNA binding . This study is the first of a comprehensive mutagenesis study on a sequence specific HMG domain . Comparing how various residues influence sequence specific and non-sequence specific HMG domains helps us to rationalize their mode of action . Positively charged amino acids concentrated at the surface of sequence specific HMG domains recognize specific, linear AT-rich DNA segments . After the negative charges at the surface of the DNA are neutralized, the hydrophobic residues of the protein may intercalate DNA . Phenylalanine at position 12 plays a crucial role in the sequence specific HMG domain . The differences in pI values, the instability index, and DNA contact regions between sequence and non-sequence specific HMG domains are associated with their functional modes.

Biochemistry, 2003 Sep 30, 42(38), 11150 - 60
NMR studies of the interaction of a type II dihydrofolate reductase with pyridine nucleotides reveal unexpected phosphatase and reductase activity; Pitcher WH 3rd et al.; The interaction of type II R67 dihydrofolate reductase (DHFR) with its cofactor nicotinamide adenine dinucleotide phosphate (NADP(+)) has been studied using nuclear magnetic resonance (NMR) . Doubly labeled {U-(13)C,(15)N}DHFR was obtained from Escherichia coli grown on a medium containing {U-(13)C}-D-glucose and (15)NH(4)Cl, and the 16 disordered N-terminal amino acids were removed by treatment with chymotrypsin . Backbone and side chain NMR assignments were made using triple-resonance experiments . The degeneracy of the amide (1)H and (15)N shifts of the tetrameric DHFR was preserved upon addition of NADP(+), consistent with kinetic averaging among equivalent binding sites . Analysis of the more titration-sensitive DHFR amide resonances as a function of added NADP(+) gave a K(D) of 131 +/- 50 microM, consistent with previous determinations using other methodology . We have found that the (1)H spectrum of NADP(+) in the presence of the R67 DHFR changes as a function of time . Comparison with standard samples and mass spectrometric analysis indicates a slow conversion of NADP(+) to NAD(+), i.e., an apparent NADP(+) phosphatase activity . Studies of this activity in the presence of folate and a folate analogue support the conclusion that this activity results from an interaction with the DHFR rather than a contaminating phosphatase . (1)H NMR studies of a mixture of NADP(+) and NADPH in the presence of the enzyme reveal that a ternary complex forms in which the N-4A and N-4B nuclei of the NADPH are in the proximity of the N-4 and N-5 nuclei of NADP(+) . Studies using the NADP(+) analogue acetylpyridine adenosine dinucleotide phosphate (APADP(+)) demonstrated a low level of enzyme-catalyzed hydride transfer from NADPH . Analysis of DHFR backbone dynamics revealed little change upon binding of NADP(+) . These additional catalytic activities and dynamic behavior are in marked contrast to those of type I DHFR.

Int J Med Microbiol, 2003 Aug, 293(4), 287 - 98
Detection of a plasmid-encoded pathogenicity island in F18+ enterotoxigenic and verotoxigenic Escherichia coli from weaned pigs; Fekete PZ et al.; Most virulence genes of enterotoxigenic Escherichia coli (ETEC) are located on plasmids . The gene for heat-stable enterotoxin I (sta) is part of the transposon Tn1681, and the heat-stable enterotoxin II (stb) gene was described to be part of the transposon Tn4521 . In the studies presented here, we describe the linkage of the sta and stb genes on an approximately 10-kb fragment designated as toxin-specific locus (TSL) . The TSL has been isolated from the 120-kb virulence plasmid pTC of the porcine ETEC strain 2173 that produces F18 fimbriae . The nucleotide sequence of the TSL fragment was determined . Sequences in the flanking regions of the sta gene indicated the presence of Tn1681, but--unexpectedly--flanking sequences of the neighbouring stb gene were completely different from those of Tn4521 . The 10-kb TSL is part of a 40-kb fragment that contains the replication origin of pTC . This 40-kb fragment was mobilised into plasmid pACYC177 and the nucleotide sequence of the bordering fragments was determined . The 40-kb fragment was flanked by IS10 elements at both ends, indicating the existence of a new 40-kb pathogenicity island (PAI) in strain 2173 . Several F4(K88)+ ETEC and F18+ ETEC as well as F18+ E . coli strains producing enterotoxins and verotoxin-2 (ETEC/VTEC) from weaned pigs of different geographical origin were tested for the flanking regions by PCR to see if they belong to the "Tn4521-like" or the "pTC-like" stb type . It turned out that the Tn4521-like stb-type was characteristic of F4(K88)+ ETEC, while the pTC-like stb type was present in most F18+ ETEC and F18+ ETEC/VTEC, although polymorphism was observed both in the K88 and F18 groups . These results suggest the existence and worldwide spread of a new plasmid-encoded pathogenicity island in porcine post weaning ETEC and ETEC/VTEC strains producing F18 fimbriae.

J Food Prot, 2003 Sep, 66(9), 1708 - 11
Thermal inactivation of Escherichia coli in camel milk; Sela S et al.; Camels subsist and produce milk in desert pastures not utilized by other domesticated herbivores . Developing the camel milk industry can improve the economy of desert inhabitants . To comply with sanitary ordinances, camel milk is pasteurized by procedures specified for bovine milk . It is widely accepted that milk composition might affect bacterial thermal death time (TDT) . Camel and bovine milks markedly differ in their chemical composition, yet data regarding TDT values of bacteria in camel milk is missing . As a first step toward developing specific heat treatments appropriate for camel milk, TDT curves of Escherichia coli in artificially contaminated camel and cow milks have been compared . Heating the milks to temperatures ranging from 58 to 65 degrees C yields similar thermal death curves and derived D- and z-values . These findings suggest that, in this temperature range, E . coli might behave similarly in bovine and camel milk . Additional TDT studies of various pathogenic species in camel milk are required before establishing pasteurization conditions of camel milk.

Cell Death Differ, 2003 Oct, 10(10), 1156 - 64
Rapid, noninflammatory and PS-dependent phagocytic clearance of necrotic cells; Hirt UA et al.; In pathological situations, different modes of cell death are observed, and information on the role and uptake of nonapoptotic corpses is scarce . Here, we modeled two distinct forms of death in human Jurkat T cells treated with staurosporine: classical apoptosis under normal culture conditions and programmed death with necrotic morphology under ATP-depleting conditions (necPCD) . When offered to phagocytes, both types of cell corpses (but not heat-killed unscheduled necrotic cells) reduced the release of the proinflammatory cytokine TNF from the macrophages . The necPCD cells were efficiently engulfed by macrophages and microglia, and from mixtures of necPCD and apoptotic cells macrophages preferentially engulfed the necrotic cells . Using a newly developed assay, we demonstrated that phosphatidylserine is translocated to the surface of such necrotic cells . We demonstrate that this can occur independently of calcium signals, and that surface phosphatidylserine is essential for the uptake of necrotic cells by both human macrophages and murine microglia.

Shock, 2003 Oct, 20(4), 375 - 81
Metalloendopeptidase inhibition regulates phosphorylation of p38-mitogen-activated protein kinase and nitric oxide synthase in heart after endotoxemia; Gupta A et al.; We tested the hypothesis that metalloendopeptidase inhibition using phosphoramidon during induction of endotoxemia 24 h later would down-regulate the protein expression of myocardial inducible nitric oxide synthase (iNOS) and phosphorylation of p38-mitogen-activated protein kinase (p38-MAPK) . Male Sprague-Dawley rats (350-400 g) were randomly divided into sham-treated and LPS-treated groups (Escherichia . coli lipopolysaccharide {LPS} 2 mg/kg bolus + 2 mg/kg infusion for 30 min) . The animals in each group were further subdivided into vehicle- and phosphoramidon (1 mg/kg bolus)-treated subgroups . Blood and heart samples were collected at 2- and 24-h postendotoxemia/phosphoramidon treatment . LPS at 2 h after its administration produced a significant decrease in mean arterial pressure that was blocked by phosphoramidon treatment . LPS at 2 and 24 h produced a significant elevation in the concentration of left ventricular endothelin-1 (ET-1) both in heart and plasma as compared with control group . This LPS-induced left ventricular ET-1 elevation at 24 h was significantly reduced by phosphoramidon . No significant alterations were observed in the myocardial protein expression of preproET-1, iNOS, and eNOS at 2 h post LPS . In 24-h post treatment groups phosphoramidon upregulated the expression of myocardial preproET-1 protein both in control and endotoxemic rat groups . Also, LPS-induced upregulated protein expression of myocardial-inducible nitric oxide synthase and increased levels of nitric oxide byproducts at 24 h were blocked by phosphoramidon . Phosphoramidon inhibited LPS-induced down-regulated expression of myocardial endothelial nitric oxide synthase and upregulated p38-MAPK phosphorylation . These results indicated that inhibition of metalloendopeptidase during induction of endotoxemia could regulate the phosphorylation of myocardial p38-MAPK and iNOS protein expression at 24-h post endotoxemia . We concluded that inhibition of metalloendopeptidases during early endotoxemia not only decreased the biosynthesis of ET-1 in heart locally but also simultaneously down-regulated myocardial protein expression of iNOS and p38-MAPK phosphorylation in the later stage of endotoxemia.

Virus Genes, 2003 Oct, 27(2), 115 - 23
Characterization of bro-b gene of Spodoptera litura multicapsid nucleopolyhedrovirus; Gong Y et al.; Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) ORF125 (designated as the bro-b gene) is a member of the unique multigene family called the baculovirus repeated ORFs (bro) family . Computer-assisted analysis revealed that BRO-B contains a conserved Bro-N domain in its N-terminus and a single-stranded DNA (ssDNA) binding motif in the middle region . In vitro the bro-b gene transcription was present at 12 h post-infection (p.i.) and remained detectable up to 96 h p.i . Western blot analysis of BRO-B expression with an antiserum made against 6xHis tagged BRO-B expressed in Escherichia coli showed that it was present from 12 h through 96 h p.i . in vitro . Structural localization revealed that BRO-B could be found in the nucleocapsid components of both occlusion-derived virus (ODV) and budded virus (BV) . Immunofluorescence analysis indicated that BRO-B is located only in the nucleus of infected S . litura cells . Furthermore, Western blot analysis indicated that BRO-B was associated with nuclear structures . These results suggested that BRO-B might be a nuclear-associated protein.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1871 - 3 Epub 2003 Sep 19.
Purification and crystallization of Escherichia coli pseudouridine synthase RluD; Del Campo M et al.; RluD is the pseudouridine (Psi) synthase responsible for forming Psi1911, Psi1915 and Psi1917 in Escherichia coli 23S RNA . Out of the 11 Psi synthases in E . coli, only cells lacking RluD show a severe growth defect . In addition, RluD belongs to the RluA family of Psi synthases, one of the two remaining families without a representative crystal structure . In this paper, the crystallization of selenomethionine-substituted RluD by the hanging-drop method is reported . The crystals diffract to 1.9 A and belong to space group P4(3)2(1)2, with unit-cell parameters a = b = 75.14, c = 181.81 A . Synchrotron radiation was used on a single crystal to collect a complete multiwavelength anomalous dispersion (MAD) data set to 2.0 A resolution.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1863 - 5 Epub 2003 Sep 19.
Protein preparation, crystallization and preliminary X-ray crystallographic studies of a thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis; You D et al.; Native and His-tagged mutant (L160I) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermoanaerobacter tengcongensis were cloned, expressed in Escherichia coli and purified . Both proteins were crystallized with polyethylene glycol as the main precipitant at 293 K using the hanging-drop vapour-diffusion method . The crystal of native HGPRT belongs to space group C222(1), with unit-cell parameters a = 65.77, b = 137.73, c = 95.27 A, and diffracted to 2.2 A resolution on an in-house X-ray generator . The crystal of the His-tagged mutant (L160I) HGPRT belongs to the space group I222, with unit-cell parameters a = 52.21, b = 88.36, c = 93.03 A, and diffracted to 1.7 A resolution in-house.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1859 - 62 Epub 2003 Sep 19.
Preliminary structure analysis of the DH/PH domains of leukemia-associated RhoGEF; Kristelly R et al.; Leukemia-associated RhoGEF (LARG) is a multidomain protein that relays signals from Galpha(12/13)-coupled heptahelical receptors to GTPases that regulate the cytoskeleton . To understand the molecular basis of LARG-mediated signal transduction, structural analysis of its DH/PH domains has been initiated . The LARG DH/PH domains have been overexpressed in Escherichia coli as a TEV protease-cleavable fusion protein containing maltose-binding protein and a hexahistidine tag at the N- and C-termini, respectively . Crystals of the DH/PH domains were obtained (space group C2; unit-cell parameters a = 195.5, b = 46.0, c = 75.1 A, beta = 105.0 degrees ) and xenon and NaBr derivatives were generated which should allow the structure to be determined by MIRAS.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1856 - 8 Epub 2003 Sep 19.
Expression, crystallization and preliminary crystallographic analysis of the extracellular IgV-like domain of the human natural killer cell inhibitory receptor p75/AIRM1; Dimasi N et al.; p75/AIRM1 (Siglec-7) is a sialic acid-binding Ig-like lectin recently identified as an inhibitory receptor on natural killer cells . The expression, in vitro folding, circular-dichroism spectroscopy, crystallization and preliminary X-ray characterization of the Ig-V like domain of p75/AIRM1 are reported . X-ray data were collected from a single crystal at 100 K, with a maximum useful diffraction pattern extending to 1.45 A resolution on a synchrotron source . The crystal belongs to a primitive monoclinic space group, with unit-cell parameters a = 32.65, b = 49.72, c = 39.79 A, alpha = gamma = 90, beta = 113 degrees . The systematic absences indicate that the space group is P2(1) . Assuming one molecule per asymmetric unit, V(M) (the Matthews coefficient) was calculated to be 1.879 A(3) Da(-1) and the solvent content was estimated to be 32.01%.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1853 - 5 Epub 2003 Sep 19.
Crystallization and preliminary crystallographic analysis of RSB-66, a novel round spermatid-specific protein; Yang M et al.; Crystals of the RSB-66 protein have been grown at 291 K using NaCl as precipitant . In the refinement of the crystallization this protein, the crystallographic PCR method was used and was found to help in obtaining the best crystals more quickly and easily . The diffraction pattern of the crystal extends to 2.7 A resolution in-house . A full set of X-ray diffraction data were collected to 2.7 A from a single crystal . The crystals belong to space group P4212, with unit-cell parameters a = 90.4, b = 90.4, c = 122.2 A, alpha = beta = gamma = 90 degrees . The presence of two or three molecules per asymmetric unit gives a crystal volume per protein mass (V(M)) of 3.22 or 2.14 A(3) Da(-1), respectively.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1846 - 8 Epub 2003 Sep 19.
Crystallization and preliminary X-ray diffraction studies of Aes acetyl-esterase from Escherichia coli; Sorrentino N et al.; The acetyl-esterase Aes from Escherichia coli, which belongs to the HSL group of the esterase/lipase superfamily, has been crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 8000 as a precipitant and magnesium chloride as an additive . Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 110.0, b = 190.6, c = 218.6 A . A complete data set has been collected to 2.5 A resolution at the Elettra synchrotron source, Trieste using a single frozen crystal . Packing density considerations agree with 10-16 monomers in the asymmetric unit, with a corresponding solvent content of 61-38%.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1838 - 9 Epub 2003 Sep 19.
Crystallization and preliminary X-ray crystallographic study on a xyloglucan-specific exo-beta-glycosidase, oligoxyloglucan reducing-end specific cellobiohydrolase; Yaoi K et al.; A novel xyloglucan-specific exo-beta-glycosidase, oligoxyloglucan reducing-end specific cellobiohydrolase (OXG-RCBH), recognizes the reducing end of oligoxyloglucan and releases two glucosyl residue segments from the main chain . OXG-RCBH was crystallized by the hanging-drop vapour-diffusion method with polyethylene glycol 3000 and polyethylene glycol 400 as precipitants . The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 61.0, b = 146.9, c = 211.9 A . The crystals diffract to a resolution of 2.2 A and are suitable for X-ray structure analysis.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1822 - 3 Epub 2003 Sep 19.
Purification, crystallization and preliminary crystallographic analysis of phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus; Akerboom J et al.; The glycolytic enzyme phosphoglucose isomerase catalyses the reversible isomerization of glucose 6-phosphate to fructose 6-phosphate . The phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus, which shows no sequence similarity to any known bacterial or eukaryotic phosphoglucose isomerase, has been cloned and overexpressed in Escherichia coli, purified and subsequently crystallized by the hanging-drop method of vapour diffusion using 1.6 M sodium citrate as the precipitant at pH 6.5 . Multiple-wavelength anomalous dispersive X-ray data have been collected to a maximum resolution of 1.92 A on a single selenomethionine-incorporated crystal . This crystal belongs to space group C2, with approximate unit-cell parameters a = 84.7, b = 42.4, c = 57.3 A, beta = 120.6 degrees and a monomer in the asymmetric unit.

Acta Crystallogr D Biol Crystallogr, 2003 Oct, 59(Pt 10), 1762 - 6 Epub 2003 Sep 19.
Unusual space-group pseudosymmetry in crystals of human phosphopantothenoylcysteine decarboxylase; Manoj N et al.; Phosphopantothenoylcysteine (PPC) decarboxylase is an essential enzyme in the biosynthesis of coenzyme A and catalyzes the decarboxylation of PPC to phosphopantetheine . Human PPC decarboxylase has been expressed in Escherichia coli, purified and crystallized . The Laue class of the diffraction data appears to be 3m, suggesting space group R32 with two monomers per asymmetric unit . However, the crystals belong to the space group R3 and the asymmetric unit contains four monomers . The structure has been solved using molecular replacement and refined to a current R factor of 29% . The crystal packing can be considered as two interlaced lattices, each consistent with space group R32 and with the corresponding twofold axes parallel to each other but separated along the threefold axis . Thus, the true space group is R3 with four monomers per asymmetric unit.

Methods Mol Biol, 2003, 236, 345 - 62
T-DNA activation tagging; Memelink J; T-DNA activation tagging is a method to generate dominant mutations in plants or plant cells by random insertion of a T-DNA carrying constitutive enhancer elements, which can cause transcriptional activation of flanking plant genes . The method consists of generating a large number of transformed plants or plant cells using a specialized T-DNA construct, followed by selection for the desired phenotype . Subsequently, the activated plant gene is rescued from selected mutant transformants for further functional analysis . Since the exact procedure depends on the plant material and the selected phenotype, this chapter describes one specific example of T-DNA activation tagging of suspension-cultured cells, including, where possible, cross-references to more general applications of the technique.

Methods Mol Biol, 2003, 236, 21 - 36
Constructing gene-enriched plant genomic libraries using methylation filtration technology; Rabinowicz PD; Full genome sequencing in higher plants is a very difficult task, because their genomes are often very large and repetitive . For this reason, gene targeted partial genomic sequencing becomes a realistic option . The method reported here is a simple approach to generate gene-enriched plant genomic libraries called methylation filtration . This technique takes advantage of the fact that repetitive DNA is heavily methylated and genes are hypomethylated . Then, by simply using an Escherichia coli host strain harboring a wild-type modified cytosine restriction (McrBC) system, which cuts DNA containing methylcytosine, repetitive DNA is eliminated from these genomic libraries, while low copy DNA (i.e., genes) is recovered . To prevent cloning significant proportions of organelle DNA, a crude nuclear preparation must be performed prior to purifying genomic DNA . Adaptor-mediated cloning and DNA size fractionation are necessary for optimal results.

Methods Mol Biol, 2004, 237, 39 - 53
Expression and purification of soluble adenylyl cyclase from Escherichia coli; Beeler JA et al.; This chapter outlines methods to purify soluble adenylyl cyclase (AC7) expressed in an Escherichia coli (E . coli) heterologous expression system . Guidelines are provided for constructing the expression plasmids, optimizing expression, culturing, and purifying the proteins . Purification requires two chromatographic steps . A histidine tag (H6) is incorporated into the expression vector and utilized for affinity purification on a Ni-NTA column . Subsequently, an anion exchange column is employed to further purify the protein.

Methods Mol Biol, 2004, 237, 3 - 20
Purification of recombinant G protein alpha subunits from Escherichia coli; Greentree WK et al.; The purification of recombinant G protein a subunits expressed in Escherichia coli (E . coli) is a convenient and inexpensive method to obtain homogeneous preparations of protein for biochemical and biophysical analyses . Wild-type and mutant forms of G alpha are easily produced for analysis of their intrinsic biochemical properties, as well as for reconstitution with receptors, effectors, regulators, and G protein beta gamma subunits . Methods are described for the expression of Gi alpha and Gs alpha proteins in E . coli . Protocols are provided for the purification of untagged G protein a subunits using conventional chromatography and histidine (His)-tagged subunits using metal chelate chromatography . Modification of G alpha with myristate can be recapitulated in E . coli by expressing N-myristoyltransferase (NMT) with its G protein substrate . Protocols for the production and purification of myristoylated G alpha are presented.

Protein Sci, 2003 Oct, 12(10), 2379 - 82
Association and dissociation kinetics of colicin E3 and immunity protein 3: convergence of theory and experiment; Zhou HX; The rapid binding of cytotoxic colicin E3 by its cognate immunity protein Im3 is essential in safeguarding the producing cell . The X-ray structure of the E3/Im3 complex shows that the Im3 molecule interfaces with both the C-terminal ribonuclease (RNase) domain and the N-terminal translocation domain of E3 . The association and dissociation rates of the RNase domain and Im3 show drastically different sensitivities to ionic strength, as previously rationalized for electrostatically enhanced diffusion-limited protein-protein associations . Relative to binding to the RNase domain, binding to full-length E3 shows a comparable association rate but a significantly lower dissociation rate . This outcome is just what was anticipated by a theory for the binding of two linked domains to a protein . The E3/Im3 system thus provides a powerful paradigm for the interplay of theory and experiment.

Protein Sci, 2003 Oct, 12(10), 2303 - 11
The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites; Zhao Y et al.; The mRNA of Escherichia coli yedU gene is induced 31-fold upon heat shock . The 31-kD YedU protein, also calls Hsp31, is highly conserved in several human pathogens and has chaperone activity . We solved the crystal structure of YedU at 2.2 A resolution . YedU monomer has an alpha/beta/alpha sandwich domain and a small alpha/beta domain . YedU is a dimer in solution, and its crystal structure indicates that a significant amount of surface area is buried upon dimerization . There is an extended hydrophobic patch that crosses the dimer interface on the surface of the protein . This hydrophobic patch is likely the substrate-binding site responsible for the chaperone activity . The structure also reveals a potential protease-like catalytic triad composed of Cys184, His185, and Asp213, although no enzymatic activity could be identified . YedU coordinates a metal ion using His85, His122, and Glu90 . This 2-His-1-carboxylate motif is present in carboxypeptidase A (a zinc enzyme), and a number of dioxygenases and hydroxylases that utilize iron as a cofactor, suggesting another potential function for YedU.

Protein Sci, 2003 Oct, 12(10), 2230 - 8
Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes; Osborne MJ et al.; Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution . (1)H(N), (15)N, and (13)C(alpha) resonance assignments have been made for the ternary complex with folate and oxidized NADP(+) cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH . The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme) . Analysis of (1)H(N) and (15)N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme . Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide-ribose binding sites of the NADPH cofactor . The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E . coli DHFR are populated in solution.

Protein Sci, 2003 Oct, 12(10), 2206 - 14
On the structural and functional modularity of glycinamide ribonucleotide formyltransferases; Lee SG et al.; Glycinamide ribonucleotide formyltransferases (GARTs) are part of the de novo purine biosynthetic pathway, catalyzing the direct transfer of a formyl group from the tetrahydrofolate cofactor to the glycinamide ribonucleotide substrate . Despite the low amino acid-sequence identity between the GARTs from Escherichia coli and human, their tertiary structures are superimposable . As part of our functional studies of these enzymes, we have investigated the interchangeability of individual protein fragments or modules between the two enzymes and the functional properties of the resulting hybrids . The modular nature of GART facilitated the creation of combinatorial libraries of chimeras between the Escherichia coli and human enzymes, which were functionally selected through complementation of an auxotrophic Escherichia coli strain . From a pool of several dozen sequence distinct hybrids, six in vivo-functional fusion genes were selected, overexpressed, and purified to homogeneity . The kinetic analysis of these constructs and the comparison of their k(cat) and K(M) values to the parental enzymes suggest that the characteristic kinetic properties from the two parents are "modular encoded" and can be exchanged by domain swapping . The chimeras in general, however, are subject to temperature instability and misfolding; thus, they serve primarily as useful candidates for further rounds of optimization.

Protein Sci, 2003 Oct, 12(10), 2194 - 205
A de novo redesign of the WW domain; Kraemer-Pecore CM et al.; We have used a sequence prediction algorithm and a novel sampling method to design protein sequences for the WW domain, a small beta-sheet motif . The procedure, referred to as SPANS, designs sequences to be compatible with an ensemble of closely related polypeptide backbones, mimicking the inherent flexibility of proteins . Two designed sequences (termed SPANS-WW1 and SPANS-WW2), using only naturally occurring L-amino acids, were selected for study and the corresponding polypeptides were prepared in Escherichia coli . Circular dichroism data suggested that both purified polypeptides adopted secondary structure features related to those of the target without the aid of disulfide bridges or bound cofactors . The structure exhibited by SPANS-WW2 melted cooperatively by raising the temperature of the solution . Further analysis of this polypeptide by proton nuclear magnetic resonance spectroscopy demonstrated that at 5 degrees C, it folds into a structure closely resembling a natural WW domain . This achievement constitutes one of a small number of successful de novo protein designs through fully automated computational methods and highlights the feasibility of including backbone flexibility in the design strategy.

Nucleic Acids Res, 2003 Oct 1, 31(19), 5776 - 88
Selective inhibitory DNA aptamers of the human RNase H1; Pileur F et al.; Human RNase H1 binds double-stranded RNA via its N-terminal domain and RNA-DNA hybrid via its C-terminal RNase H domain, the latter being closely related to Escherichia coli RNase HI . Using SELEX, we have generated a set of DNA sequences that can bind efficiently (K(d) values ranging from 10 to 80 nM) to the human RNase H1 . None of them could fold into a simple perfect double-stranded DNA hairpin confirming that double-stranded DNA does not constitute a trivial ligand for the enzyme . Only two of the 37 DNA aptamers selected were inhibitors of human RNase H1 activity . The two inhibitory oligomers, V-2 and VI-2, were quite different in structure with V-2 folding into a large, imperfect but stable hairpin loop . The VI-2 structure consists of a central region unimolecular quadruplex formed by stacking of two guanine quartets flanked by the 5' and 3' tails that form a stem of six base pairs . Base pairing between the 5' and 3' tails appears crucial for conferring the inhibitory properties to the aptamer . Finally, the inhibitory aptamers were capable of completely abolishing the action of an antisense oligonucleotide in a rabbit reticulocyte lysate supplemented with human RNase H1, with IC50 ranging from 50 to 100 nM.

Plant Physiol, 2003 Oct, 133(2), 850 - 63 Epub 2003 Sep 18.
Functional dissections between GAMYB and Dof transcription factors suggest a role for protein-protein associations in the gibberellin-mediated expression of the RAmy1A gene in the rice aleurone; Washio K; In the germinated cereal aleurone layer, gibberellic acids (GA) induce expression of a number of genes encoding hydrolytic enzymes that participate in the mobilization of stored molecules . Previous analyses suggest that the key events controlling the GA-regulated gene expression in the aleurone are formation of active transcription machinery referred to as the GA responsive complex, followed by recruiting GAMYB . In general, bipartite promoter contexts composed of the GA-responsive element and the pyrimidine box are observed within the regulatory regions of cereal GA-responsive genes . Protein factors that recognize each promoter sequence were identified and distinct effects on the GA-mediated activation of gene expression have been also investigated; however, the connection and intercalation between two promoter motifs remain obscure . In this study, I have evaluated cooperative function of GAMYB and a pyrimidine box-binding protein OsDOF3 that influenced the promoter activity of the most predominant GA-responsive gene (RAmy1A) of rice (Oryza sativa) . Transient expression of OsDOF3 in the germinated aleurone prolonged GAMYB function on the reporter expression in the absence of GA . The synergistic effect required a set of DNA bindings of two proteins on the RAmy1A promoter region . The yeast two-hybrid assay showed the physical interaction of GAMYB and OsDOF3 in yeast cells, indicating that the association of GAMYB and OsDOF3 may be a functional unit in transcription regulation . The results showed the accessory function of OsDOF3 responsible for a dosage-dependent mediation of GA signaling that leads to high-level expression of physiological target genes.

Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11373 - 7 Epub 2003 Sep 18.
Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping; Lia G et al.; The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping . Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of approximately 40 nm of DNA that encompasses the promoters . Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU) . By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence . The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo . Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation . Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.

J Immunol, 2003 Oct 1, 171(7), 3645 - 54
Lipopolysaccharide-induced suppression of airway Th2 responses does not require IL-12 production by dendritic cells; Kuipers H et al.; The prevalence of atopic asthma, a Th2-dependent disease, is reaching epidemic proportions partly due to improved hygiene in industrialized countries . There is an inverse correlation between the level of environmental endotoxin exposure and the prevalence of atopic sensitization . As dendritic cells (DC) have been implicated in causing sensitization to inhaled Ag, we studied the effect of endotoxin on Th2 development induced by bone marrow DC in vitro and by intratracheal injection in vivo, with particular emphasis on the role played by the polarizing cytokine IL-12 . Bone marrow-derived DC stimulated with Escherichia coli O26:B6 LPS produced IL-12p70 for a limited period of time, after which production became refractory to further stimulation with CD40 ligand, a phenomenon previously called "exhaustion." The level of IL-12 production of DC did not correlate with Th1 development, as exhausted OVA-pulsed DC were still capable of shifting the cytokine pattern of responding OVA-specific Th cells toward Th1 in vitro and in vivo . When mice were first immunized by intratracheal injection of OVA-DC and subsequently challenged with OVA aerosol, prior in vitro stimulation of DC with LPS reduced the development of airway eosinophilia and Th2 cytokine production . Most surprisingly, the capacity of LPS to reduce Th2-dependent eosinophilic airway inflammation was IL-12-independent altogether, as IL-12p40 knockout DC had a similar reduced capacity to prime for Th2 responses . These results suggest that LPS reduces sensitization to inhaled Ag by reducing DC-driven Th2 development, but that IL-12 is not necessary for this effect.

J Immunol, 2003 Oct 1, 171(7), 3379 - 84
Electroporation enables plasmid vaccines to elicit CD8+ T cell responses in the absence of CD4+ T cells; Dayball K et al.; In vivo electroporation dramatically enhances plasmid vaccine efficacy . This enhancement can be attributed to increased plasmid delivery and, possibly, to some undefined adjuvant properties . Previous reports have demonstrated CD8(+) T cell priming by plasmid vaccines is strongly dependent upon CD4(+) T cell help . Indeed, the efficacy of a plasmid vaccine expressing Escherichia coli beta-galactosidase was severely attenuated in MHC class II-deficient (C2D) mice . To determine whether electroporation could compensate for the absence of CD4(+) T cell help, C2D mice were immunized by a single administration of plasmid in combination with electroporation using two conditions which differed only by the duration of the pulse (20 or 50 msec) . Both conditions elicited robust cellular and humoral responses in wild-type mice, as measured by IFN-gamma ELISPOT, anti-beta-galactosidase ELISA, and protection from virus challenge . In C2D mice, the cellular response produced by the vaccine combined with the 50-msec pulse, as measured by ELISPOT, was identical to the response in wild-type mice . The 20-msec pulse elicited a milder response that was approximately one-fifth that of the response elicited by the 50-msec pulse . By contrast, the 20-msec conditions provided comparable protection in both wild-type and C2D recipients whereas the protection elicited by the 50-msec conditions in C2D mice was weaker than in wild-type mice . Further investigation is required to understand the discordance between the ELISPOT results and outcome of virus challenge in the C2D mice . Nonetheless, using this technique to prime CD8(+) T cells using plasmid vaccines may prove extremely useful when immunizing hosts with limiting CD4(+) T cell function, such as AIDS patients.

Infect Immun, 2003 Oct, 71(10), 5772 - 84
Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1; Verma A et al.; Genome sequencing of C . trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases) . Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced . Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins . CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins . In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases . Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle . Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay . Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues . Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system . These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays . This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia . The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.

Infect Immun, 2003 Oct, 71(10), 5505 - 13
Chimeric Dr fimbriae with a herpes simplex virus type 1 epitope as a model for a recombinant vaccine; Zalewska B et al.; The potential of the major structural protein DraE of Escherichia coli Dr fimbriae has been used to display an 11-amino-acid peptide of glycoprotein D derived from herpes simplex virus (HSV) type 1 . The heterologous sequence mimicking an epitope from glycoprotein D was inserted in one copy into the draE gene in place of a predicted 11-amino-acid sequence in the N-terminal region of surface-exposed domain 2 within the conserved disulfide loop (from Cys21 to Cys53) . The inserted epitope was displayed on the surface of the chimeric DraE protein as evidenced by immunofluorescence and was recognized by monoclonal antibodies to the target HSV glycoprotein D antigen . Conversely, immunization of rabbits with purified chimeric Dr-HSV fimbriae resulted in a serum that specifically recognized the 11-amino-acid epitope of HSV glycoprotein D, indicating the utility of the strategy employed.

Cancer Res, 2003 Sep 1, 63(17), 5532 - 7
Generation of Escherichia coli nitroreductase mutants conferring improved cell sensitization to the prodrug CB1954; Grove JI et al.; Escherichia coli nitroreductase (NTR) activates the prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or tumors to the prodrug . This is one of several enzyme-prodrug combinations that are under development for cancer gene therapy, and the system has now entered clinical trials . Enhancing the catalytic efficiency of NTR for CB1954 could improve its therapeutic potential . From the crystal structure of an enzyme-ligand complex, we identified nine amino acid residues within the active site that could directly influence prodrug binding and catalysis . Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E . coli to CB1954 . Amino acid substitutions at six positions conferred markedly greater sensitivity to CB1954 than did the WT enzyme; the best mutants, at residue F124, resulted in approximately 5-fold improvement . Using an adenovirus vector, we introduced the F124K NTR mutant into human SK-OV-3 ovarian carcinoma cells and showed it to be approximately 5-fold more potent in sensitizing the cells to CB1954 at the clinically relevant prodrug concentration of 1 micro M than was the WT enzyme . Enhanced mutant NTRs such as F124K should improve the efficacy of the NTR/CB1954 combination in cancer gene therapy.

Res Microbiol, 2003 Sep, 154(7), 499 - 509
Replication functions of new broad host range plasmids isolated from polluted soils; Gstalder ME et al.; The nucleotide sequencing of replicons isolated from three new broad host range plasmids, pMOL98, pEMT8, and pEMT3, originating from polluted soils, showed a typical organization of iteron replicons replicating by the theta mode . In the pMOL98 replicon, the origin region and the rep gene were identified in complementation experiments . Sequence comparisons showed that the regions bearing these features are highly identical to regions in pIP02T and pSB102 and that the Rep proteins (but not the origin regions) of these three plasmids show some identity to the Rep proteins of the IncW group of plasmids . This suggests that pMOL98, pIPO2T, and pSB102 constitute a new Inc/Rep family, distantly related to the IncW group, but having an incompatibility phenotype different from the IncW phenotype . The pEMT8 replicon displayed an orf whose conceptually translated product is related to the Rep proteins of four plasmids, pSD20, pSW500, pMLb, and pALC1, not yet classified into any known incompatibility group . The vegetative origins of these plasmids were not similar, suggesting that the five plasmids could belong to a new family with similar Rep proteins but different incompatibility phenotypes . The pEMT3 replicon is clearly related to IncP replicons (sequence similarities and incompatibility phenotype), although sequence comparisons revealed some divergence with respect to the two well-documented subgroups IncPalpha and IncPbeta . This suggests that in these plasmids, despite the existence of a powerful system of centralized control over replication, maintenance, and transfer functions, plasticity and evolution of these functions are at work . Our analysis confirms the extreme genetic flexibility of plasmids and the absolute necessity of using multiple techniques (PCR, DNA sequencing, DNA chips, and databases) to analyze the role of broad host range plasmids in the capture, recombination and spread of genetic traits among bacteria.

Biophys Chem, 2003 Sep, 105(2-3), 263 - 8
Effect of redox potential of the heme on the peroxidase activity of cytochrome b562; Mazumdar S et al.; Measurements of peroxidase activities of two site-specific mutants and wild type cytochrome b562 suggest that the enzymatic activity correlates with the redox potential of the metal center . A lower value of the Fe(3+)/Fe(2+) redox potential seems to be important for promoting peroxidase activity of the hemeprotein possibly by stabilization of the high-valent redox intermediate involved in the catalytic function . The results provide an approach towards rational tuning of enzyme function when 'grafted' into a new protein environment.

Adv Drug Deliv Rev, 2003 Sep 26, 55(10), 1315 - 36
Design of PEGylated soluble tumor necrosis factor receptor type I (PEG sTNF-RI) for chronic inflammatory diseases; Edwards CK 3rd et al.; A recombinant C-terminal truncated form of the human soluble tumor necrosis factor receptor type I (sTNF-RI) was produced in E . coli . This soluble receptor contains the first 2.6 of the 4 domains of the intact sTNF-RI molecule . A monoPEGylated form of this molecule was produced using a 30 kD methoxyPEG aldehyde with approximately 85% selectivity for the N-terminal amino group . This molecule was shown to be less immunogenic in primates than the full length (4.0 domain) molecule or other versions of sTNF-RI which were either PEGylated at different sites or with different molecular weight PEGs . The 30 kD PEG also has a longer serum half-life to the molecule than lower molecular weight PEGs . This molecule markedly blunts the inflammatory response in a number of rheumatoid arthritis animal models . In addition, phase I/II and early phase II data in humans indicate that PEG sTNF-RI is non-immunogenic and that weekly dosing with this drug can reduce the number of tender and swollen joints in rheumatoid arthritis patients . PEG sTNF-RI has comparable American College of Rheumatology (ACR) efficacy scores as other anti-TNF molecules currently used to treat rheumatoid arthritic patients.

Exp Cell Res, 2003 Oct 1, 289(2), 211 - 21
Yeast two-hybrid screens imply involvement of Fanconi anemia proteins in transcription regulation, cell signaling, oxidative metabolism, and cellular transport; Reuter TY et al.; Mutations in one of at least eight different genes cause bone marrow failure, chromosome instability, and predisposition to cancer associated with the rare genetic syndrome Fanconi anemia (FA) . The cloning of seven genes has provided the tools to study the molecular pathway disrupted in Fanconi anemia patients . The structure of the genes and their gene products provided few clues to their functional role . We report here the use of 3 FA proteins, FANCA, FANCC, and FANCG, as "baits" in the hunt for interactors to obtain clues for FA protein functions . Using five different human cDNA libraries we screened 36.5x10(6) clones with the technique of the yeast two-hybrid system . We identified 69 proteins which have not previously been linked to the FA pathway as direct interactors of FANCA, FANCC, or FANCG . Most of these proteins are associated with four functional classes including transcription regulation (21 proteins), signaling (13 proteins), oxidative metabolism (10 proteins), and intracellular transport (11 proteins) . Interaction with 6 proteins, DAXX, Ran, IkappaBgamma, USP14, and the previously reported SNX5 and FAZF, was additionally confirmed by coimmunoprecipitation and/or colocalization studies . Taken together, our data strongly support the hypothesis that FA proteins are functionally involved in several complex cellular pathways including transcription regulation, cell signaling, oxidative metabolism, and cellular transport.

J Mol Biol, 2003 Oct 3, 332(5), 1083 - 94
The 2.2 A resolution structure of RpiB/AlsB from Escherichia coli illustrates a new approach to the ribose-5-phosphate isomerase reaction; Zhang RG et al.; Ribose-5-phosphate isomerases (EC 5.3.1.6) interconvert ribose 5-phosphate and ribulose 5-phosphate . This reaction permits the synthesis of ribose from other sugars, as well as the recycling of sugars from nucleotide breakdown . Two unrelated types of enzyme can catalyze the reaction . The most common, RpiA, is present in almost all organisms (including Escherichia coli), and is highly conserved . The second type, RpiB, is present in some bacterial and eukaryotic species and is well conserved . In E.coli, RpiB is sometimes referred to as AlsB, because it can take part in the metabolism of the rare sugar, allose, as well as the much more common ribose sugars . We report here the structure of RpiB/AlsB from E.coli, solved by multi-wavelength anomalous diffraction (MAD) phasing, and refined to 2.2A resolution . RpiB is the first structure to be solved from pfam02502 (the RpiB/LacAB family) . It exhibits a Rossmann-type alphabetaalpha-sandwich fold that is common to many nucleotide-binding proteins, as well as other proteins with different functions . This structure is quite distinct from that of the previously solved RpiA; although both are, to some extent, based on the Rossmann fold, their tertiary and quaternary structures are very different . The four molecules in the RpiB asymmetric unit represent a dimer of dimers . Active-site residues were identified at the interface between the subunits, such that each active site has contributions from both subunits . Kinetic studies indicate that RpiB is nearly as efficient as RpiA, despite its completely different catalytic machinery . The sequence and structural results further suggest that the two homologous components of LacAB (galactose-6-phosphate isomerase) will compose a bi-functional enzyme; the second activity is unknown.

J Mol Biol, 2003 Oct 3, 332(5), 1015 - 24
The X-ray structure of Escherichia coli RraA (MenG), A protein inhibitor of RNA processing; Monzingo AF et al.; The Escherichia coli protein regulator of RNase E activity A (RraA) has recently been shown to act as a trans-acting modulator of RNA turnover in bacteria; it binds to the essential endonuclease RNase E and inhibits RNA processing in vivo and in vitro . Here, we report the 2.0A X-ray structure of RraA . The structure reveals a ring-like trimer with a central cavity of approximately 12A in diameter . Based on earlier sequence analysis, RraA had been identified as a putative S-adenosylmethionine:2-demethylmenaquinone and was annotated as MenG . However, an analysis of the RraA structure shows that the protein lacks the structural motifs usually required for methylases . Comparison of the observed fold with that of other proteins (and domains) suggests that the RraA fold is an ancient platform that has been adapted for a wide range of functions . An analysis of the amino acid sequence shows that the E.coli RraA exhibits an ancient relationship to a family of aldolases.

Biochim Biophys Acta, 2003 Sep 23, 1651(1-2), 41 - 9
Molecular and functional analysis of the (6-4) photolyase from the hexactinellid Aphrocallistes vastus; Schroder HC et al.; The hexactinellid sponges (phylum Porifera) represent the phylogenetically oldest metazoans that evolved 570-750 million years ago . At this period exposure to ultraviolet (UV) light exceeded that of today and it may be assumed that this old taxon has developed a specific protection system against UV-caused DNA damage . A cDNA was isolated from the hexactinellid Aphrocallistes vastus which comprises high sequence similarity to genes encoding the protostomian and deuterostomian (6-4) photolyases . Subsequently functional studies were performed . It could be shown that the sponge gene, after transfection into mutated Escherichia coli, causes resistance of the bacteria against UV light . Recombinant sponge photolyase was prepared to demonstrate that this protein binds to DNA treated with UV light (causing the formation of thymine dimers) . Finally, it is shown that the photolyase gene is strongly expressed in the upper part of the animals and not in their middle part or their base . It is concluded that sponges not only have an excision DNA repair system, as has been described earlier by us, but also a photolyase-based photo-reactivating system.

Phytother Res, 2003 Sep, 17(8), 921 - 4
The in vitro effect of aqueous extract of Nigella sativa seeds on nitric oxide production; Mahmood MS et al.; The in vitro effect of aqueous extract of Nigella sativa seeds on nitric oxide (NO) production by murine macrophages was studied . Murine peritoneal macrophages were pre-incubated with the extract and then activated with Escherichia coli lipopolysaccharride . NO production was measured after 24 hours by spectrophotometry . The plant extract caused a dose-dependent decrease in NO production . Dialyzed preparation of the extract did not affect NO production . However, the boiled fraction of the extract resulted in a dose-dependent inhibition of NO apparently comparable to that of the whole extract . These results indicate that the aqueous extract of N . sativa seeds exhibits an inhibitory effect on nitric oxide production by murine macrophages and the active component(s) is/are non-protein in nature . In view of the fact that nitric oxide is a pro-inflammatory mediator, this study validates the traditional use of the Nigella sativa seeds for the treatment of rheumatism .

Bioprocess Biosyst Eng, 2004 Dec, 26(6), 401 - 11 Epub 2003 Sep 05.
Bioprocess control from a multivariate process trajectory; Cimander C et al.; A multivariate bioprocess control approach, capable of tracking a pre-set process trajectory correlated to the biomass or product concentration in the bioprocess is described . The trajectory was either a latent variable derived from multivariate statistical process monitoring (MSPC) based on partial least squares (PLS) modeling, or the absolute value of the process variable . In the control algorithm the substrate feed pump rate was calculated from on-line analyzer data . The only parameters needed were the substrate feed concentration and the substrate yield of the growth-limiting substrate . On-line near-infrared spectroscopy data were used to demonstrate the performance of the control algorithm on an Escherichia coli fed-batch cultivation for tryptophan production . The controller showed good ability to track a defined biomass trajectory during varying process dynamics . The robustness of the control was high, despite significant external disturbances on the cultivation and control parameters.

Arch Microbiol, 2003 Nov, 180(5), 339 - 46 Epub 2003 Sep 11.
Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents; Chatterji M et al.; DNA gyrase, a type II topoisomerase, is the sole supercoiling activity in the cell and is essential for cell survival . There are two proteinaceous inhibitors of DNA gyrase that are plasmid-borne and ensure maintenance of the plasmids in bacterial populations . However, the physiological role of GyrI, an inhibitor of DNA gyrase encoded by the Escherichia coli genome, has been elusive . Previously, we have shown that GyrI imparts resistance against microcin B17 and CcdB . Here, we find that GyrI provided partial/limited protection against the quinolone class of gyrase inhibitors but had no effect on inhibitors that interfere with the ATPase activity of the enzyme . Moreover, GyrI negated the effect of alkylating agents, such as mitomycin C and N-methyl- N-nitro- N-nitrosoguanidine, that act independently of DNA gyrase . Hence, in vivo, GyrI appears to be involved in reducing DNA damage from many sources . In contrast, GyrI is not effective against lesions induced by ultraviolet radiation . Furthermore, the expression of GyrI does not significantly alter the topology of DNA . Thus, although isolated as an inhibitor of DNA gyrase, GyrI seems to have a broader role in vivo than previously envisaged.

Arch Toxicol, 2004 Jan, 78(1), 7 - 15 Epub 2003 Sep 10.
Vanadyl sulfate can differentially damage DNA in human lymphocytes and HeLa cells; Wozniak K et al.; Using the comet assay, we showed that vanadyl sulfate induced DNA damage in human normal lymphocytes and in HeLa cells . Vanadyl at 0.5 and 1 mM produced DNA single- and double-strand breaks (SSBs and DSBs) in lymphocytes, whereas in HeLa cells we observed only SSBs . Post-treatment of vanadyl-damaged DNA from lymphocytes with formamidopyrimidine-DNA glycosylase (Fpg), an enzyme recognizing oxidized purines, gave rise to a significant increase in the extent of DNA damage . A similar effect was observed in HeLa cells, but, using endonuclease III, we also detected oxidized pyrimidines in DNA of these cells . There were no differences in the extent of DNA damage in the lymphocytes and HeLa cells in the pH >13 and pH 12.1 conditions of the comet assay, which indicates that strand breaks, and not alkali-labile sites, contributed to the measured DNA damage . Study of DNA repair, determined in the comet assay as an ability of cells to decrease of DNA damage, revealed that HeLa cells retained the ability to repair vanadyl-damaged DNA induced at a ten-fold higher concentration than that in lymphocytes . Incubation of the cells with nitrone spin traps DMPO, POBN and PBN decreased the extent of DNA damage, which might follow from the production of free radicals by vanadyl sulfate . The presence of vitamins A, C or E caused an increase of DNA damage in HeLa cells whereas in lymphocytes such an increase was observed only for vitamin C . Our data indicate that vanadyl sulfate can be genotoxic for normal and cancer cells . It seems to have a higher genotoxic potential for cancer cells than for normal lymphocytes . Vitamins A, C and E can increase this potential.

J Gen Virol, 2003 Oct, 84(Pt 10), 2669 - 78
Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells; Pijlman GP et al.; Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression . In particular, Autographa californica multicapsid nucleopolyhedovirus (AcMNPV) DIs are enriched in an origin of viral DNA replication (ori) not associated with the homologous regions (hrs) . This non-hr ori is located within the coding sequence of the non-essential p94 gene . We investigated the effect of a deletion of the AcMNPV non-hr ori on the heterologous protein expression levels following serial passage in Sf21 insect cells . Using homologous ET recombination in E . coli, deletions within the p94 gene were made in a bacterial artificial chromosome (BAC) containing the entire AcMNPV genome (bacmid) . All bacmids were equipped with an expression cassette containing the green fluorescent protein gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2) . For the parental (intact) bacmid only, a strong accumulation of DIs with reiterated non-hr oris was observed . This was not observed for the mutants, indicating that removal of the non-hr ori enhanced the genetic stability of the viral genome upon passaging . However, for all passaged viruses it was found that the entire BAC vector including the expression cassette was spontaneously deleted from the viral genome, leading to a rapid decrease in GFP and CSFV-E2 production . The rationale for the (intrinsic) genetic instability of the BAC vector in insect cells and the implications with respect to large-scale production of proteins with bacmid-derived baculoviruses are discussed.

Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11297 - 302 Epub 2003 Sep 17.
A noncognate aminoacyl-tRNA synthetase that may resolve a missing link in protein evolution; Skouloubris S et al.; Efforts to delineate the advent of many enzymes essential to protein translation are often limited by the fact that the modern genetic code evolved before divergence of the tree of life . Glutaminyl-tRNA synthetase (GlnRS) is one noteworthy exception to the universality of the translation apparatus . In eukaryotes and some bacteria, this enzyme is essential for the biosynthesis of Gln-tRNAGln, an obligate intermediate in translation . GlnRS is absent, however, in archaea, and most bacteria, organelles, and chloroplasts . Phylogenetic analyses predict that GlnRS arose from glutamyl-tRNA synthetase (GluRS), via gene duplication with subsequent evolution of specificity . A pertinent question to ask is whether, in the advent of GlnRS, a transient GluRS-like intermediate could have been retained in an extant organism . Here, we report the discovery of an essential GluRS-like enzyme (GluRS2), which coexists with another GluRS (GluRS1) in Helicobacter pylori . We show that GluRS2's primary role is to generate Glu-tRNAGln, not Glu-tRNAGlu . Thus, GluRS2 appears to be a transient GluRS-like ancestor of GlnRS and can be defined as a GluGlnRS.

Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11382 - 7 Epub 2003 Sep 17.
Competing ligands stabilize alternate conformations of the energy coupling motif of a TonB-dependent outer membrane transporter; Fanucci GE et al.; BtuB is a TonB-dependent outer-membrane transporter for vitamin B12 (or cyanocobalamin, CN-Cbl) in Escherichia coli . The binding of CN-Cbl is believed to promote an unfolding or undocking of the Ton box, the conserved N-terminal energy coupling motif at the periplasmic surface of the transporter . This structural change may facilitate the interaction of BtuB with the inner membrane protein TonB . In this work, the effect of the receptor binding fragment of colicin E3 (E3R) on the conformation of the Ton box was examined with site-directed spin labeling . Addition of E3R reverses the undocking of the Ton box that is promoted by CN-Cbl, consistent with a competitive binding between the substrate and the colicin fragment . EPR spectroscopy indicates that the Ton box is in a two-state equilibrium between docked and undocked conformations . In the absence of substrate, the docked conformation is the predominant state; however, the equilibrium can be shifted to the undocked state by the addition of detergents or site-specific proline substitutions . Even when the undocking is induced by detergents or by certain proline mutations, E3R binding shifts the equilibrium to the docked conformation . Thus, two competitive extracellular ligands, CN-Cbl and ER3, transduce opposite conformations of the N-terminal Ton box . Substrate binding stabilizes an undocked conformation, whereas E3R binding stabilizes a docked conformation of the Ton box.

Mol Biol Cell, 2004 Jan, 15(1), 46 - 57 Epub 2003 Sep 17.
Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal; Ems-McClung SC et al.; The small GTPase Ran is essential for spindle assembly . Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly . To date, the molecular mechanisms by which the Ran pathway functions remain unclear . Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly . We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP . The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding . Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.

Lab Invest, 2003 Sep, 83(9), 1373 - 82
Alteration of migration and maturation of dendritic cells and T-cell depletion in the course of experimental Trypanosoma cruzi infection; Chaussabel D et al.; Trypanosoma cruzi, the etiologic agent of Chagas disease, induces infection that affects most immunocompetent cells . However, its effect on dendritic cells (DC) is still unknown in vivo . In this report, we show, by immunohistochemical staining, that T . cruzi infection triggers a huge increase in the number of CD11c(+) DC in the spleen of infected mice at Days 14 and 21 post-inoculation (pi) . In mice reaching the chronic phase (starting on Day 35 pi), the number of splenic DC (sDC) returned progressively to normal (ending on Day 98 pi) . In the spleens of noninfected mice, most of the CD8alpha(+)CD11c(+) and CD8alpha(-)CD11c(+) DC were found in the red pulp and the marginal and T-cell zones . However, starting on Day 14 pi, a progressive decline of CD8alpha(+)CD11c(+) was observed . In addition, sDC expressed low levels of the costimulatory molecule B7.2 at Days 14 and 21 pi, suggesting that they remained immature in the course of the infection . As expected, in lipopolysaccharide-treated and noninfected mice, the expression of B7.2 molecules was sharply up-regulated on sDC that migrated toward the T-cell zone . In contrast, upon lipopolysaccharide stimulation, sDC from T . cruzi-infected mice did not migrate toward the T-cell zone nor did they undergo maturation . Finally, white pulp was severely depleted in both CD4(+) and CD8(+) T cells at the peak of infection . Taken together, these results indicate that profound alterations of migration and maturation of sDC and depletion/redistribution of T cells occur during the acute phase of T . cruzi infection and could be part of another strategy to escape immune surveillance and to persist in the host.

J Biol Chem, 2003 Nov 21, 278(47), 47340 - 9 Epub 2003 Sep 17.
Fis stabilizes the interaction between RNA polymerase and the ribosomal promoter rrnB P1, leading to transcriptional activation; Zhi H et al.; It has been shown that Fis activates transcription of the ribosomal promoter rrnB P1; however, the mechanism by which Fis activates rrnB P1 transcription is not fully understood . Paradoxically, although Fis activates transcription of rrnB P1 in vitro, transcription from the promoter containing Fis sites (as measured from rrnB P1-lacZ fusions) is not reduced in a fis null mutant strain . In this study, we further investigated the mechanism by which Fis activates transcription of the rrnB P1 promoter and the role of Fis in rRNA synthesis and cell growth in Escherichia coli . Like all other stringent promoters investigated so far, open complex of rrnB P1 has been shown to be intrinsically unstable, making open complex stability a potential regulatory step in transcription of this class of promoters . Our results show that Fis acts at this regulatory step by stabilizing the interaction between RNA polymerase and rrnB P1 in the absence of NTPs . Mutational analysis of the Fis protein demonstrates that there is a complete correlation between Fis-mediated transcriptional activation of rrnB P1 and Fis-mediated stabilization of preinitiation complexes of the promoter . Thus, our study indicates that Fis-mediated stabilization of RNA polymerase-rrnB P1 preinitiation complexes, presumably at the open complex step, contributes prominently to transcriptional activation . Furthermore, our in vivo results show that rRNA synthesis from the P1 promoters of several rRNA operons are reduced 2-fold in a fis null mutant compared with the wild type strain, indicating that Fis plays an important role in the establishment of robust rRNA synthesis when E . coli cells are emerging from a growth-arrested phase to a rapid growth phase . Thus, our results resolve an apparent paradox of the role of Fis in vitro and in vivo in the field.

Life Sci, 2003 Oct 10, 73(21), 2713 - 26
Enhancement of endotoxin-induced vascular hyporeactivity to phenylephrine in the thoracic aortas of Mg-deficient rats ex vivo; Miyamoto A et al.; Since endotoxin lethality is enhanced by Mg deficiency in animals, we determined whether endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is enhanced in Mg-deficient rats . Normal and Mg-deficient adult male Wistar rats were injected with Escherichia coli 011: B4 lipopolysaccharide (1 or 5 mg/kg, i.p.) . Six h later, rings prepared from their thoracic aortas showed severe hyporeactivity to PE . This was more pronounced in the Mg-deficient rats, and was reversed by in vitro treatment with a highly selective inducible nitric oxide (NO) synthase inhibitor, 1400 W, or a highly selective soluble guanylyl cyclase inhibitor, ODQ . However, reversal required high doses of both inhibitors in Mg-deficient rats . Endotoxemia for 6 h was associated with elevated serum interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha levels, and strong TNF receptor mRNA expression in the abdominal aortas, which were significantly greater in the Mg-deficient rats . Treatment of the thoracic aortas, isolated from control and Mg-deficient rats before endotoxic challenge, with IL-1beta or TNF-alpha for 6 h in vitro caused hyporeactivity to PE, but its severity did not differ significantly between the two groups . These results suggest that high serum IL-1beta and TNF-alpha levels, and increased TNF receptor production in the vascular tissue, contribute to vascular hyporeactivity to PE in endotoxemia, and to its enhancement in Mg-deficient rats, via NO/cGMP signaling.

Farmaco, 2003 Sep, 58(9), 625 - 9
Comparative QSAR of the sulfonamide function; Hansch C; The aromatic SO(2)NH(2) function has been characterized in biological reactions and in physical organic chemical reactions using the Hammett constant sigma, the Swain-Lupton parameter F and steric constants . The results allow comparable support for the biological processes from the much better understood mechanistic organic reactions . The approach is applied to the enzyme carbonic anhydrase and the rate of excretion of Na(+) by rats . From the study of QSAR on the ionization of the amide function, it is clearly apparent that it is the ionized form of SO(2)NH(2) that promotes the biological processes . It is clear from Fujita-Nishioka proposal that, in dealing with ortho substituents, one should routinely test sigma, F and a steric parameter to account for substituent effects.

Arch Biochem Biophys, 2003 Oct 1, 418(1), 80 - 92
Monoterpene double-bond reductases of the (-)-menthol biosynthetic pathway: isolation and characterization of cDNAs encoding (-)-isopiperitenone reductase and (+)-pulegone reductase of peppermint; Ringer KL et al.; Random sequencing of a peppermint essential oil gland secretory cell cDNA library revealed a large number of clones that specified redox-type enzymes . Full-length acquisitions of each type were screened by functional expression in Escherichia coli using a newly developed in situ assay . cDNA clones encoding the monoterpene double-bond reductases (-)-isopiperitenone reductase and (+)-pulegone reductase were isolated, representing two central steps in the biosynthesis of (-)-menthol, the principal component of peppermint essential oil, and the first reductase genes of terpenoid metabolism to be described . The (-)-isopiperitenone reductase cDNA has an open reading frame of 942 nucleotides that encodes a 314 residue protein with a calculated molecular weight of 34,409 . The recombinant reductase has an optimum pH of 5.5, and K(m) values of 1.0 and 2.2 microM for (-)-isopiperitenone and NADPH, respectively, with k(cat) of 1.3s(-1) for the formation of the product (+)-cis-isopulegone . The (+)-pulegone reductase cDNA has an open reading frame of 1026 nucleotides and encodes a 342 residue protein with a calculated molecular weight of 37,914 . This recombinant reductase catalyzes the reduction of the 4(8)-double bond of (+)-pulegone to produce both (-)-menthone and (+)-isomenthone in a 55:45 ratio, has an optimum pH of 5.0, and K(m) values of 2.3 and 6.9 microM for (+)-pulegone and NADPH, respectively, with k(cat) of 1.8s(-1) . Deduced sequence comparison revealed that these two highly substrate specific double-bond reductases show less than 12% identity . (-)-Isopiperitenone reductase is a member of the short-chain dehydrogenase/reductase superfamily and (+)-pulegone reductase is a member of the medium-chain dehydrogenase/reductase superfamily, implying very different evolutionary origins in spite of the similarity in substrates utilized and reactions catalyzed.

Arch Biochem Biophys, 2003 Oct 1, 418(1), 55 - 62
The role of transmembrane span 2 in the structure and function of subunit a of the ATP synthase from Escherichia coli; DeLeon-Rangel J et al.; The importance of the second transmembrane span of subunit a of the ATP synthase from Escherichia coli has been established by two approaches . First, biochemical analysis of five cysteine-substitution mutants, four of which were previously constructed for labeling experiments, revealed that only D119C, found within the second transmembrane span, was deleterious to ATP synthase function . This mutant had a greatly reduced growth yield, indicating inefficient ATP synthesis, but it retained a significant level of ATP-driven proton translocation and sensitivity to N,N(')-dicyclohexyl-carbodiimide, indicating more robust function in the direction of ATP hydrolysis . Second, the entire second transmembrane span was probed by alanine-insertion mutagenesis at six different positions, from residues 98 to 122 . Insertions at the central four positions from residues 107 to 117 resulted in the inability to grow on succinate minimal medium, although normal levels of membrane-bound ATPase activity and significant levels of subunit a were detected . Double mutants were constructed with a mutation that permits cross-linking to the b subunit . Cross-linked products in the mutant K74C/114iA were seen, indicating no major disruption of the a-b interface due to the insertion at 114 . Analysis of the K74C/110iA double mutant indicated that K74C is a partial suppressor of 110iA . In summary, the results support a model in which the amino-terminal, cytoplasmic end of the second transmembrane span has close contact with subunit b, while the carboxy-terminal, periplasmic end is important for proton translocation.

Arch Biochem Biophys, 2003 Oct 1, 418(1), 49 - 54
Kinetic and biochemical analysis of the mechanism of action of lysine 5,6-aminomutase; Tang KH et al.; Lysine 5,6-aminomutase (5,6-LAM) catalyzes the reversible and nearly isoenergetic transformations of D-lysine into 2,5-diaminohexanoate (2,5-DAH) and of L-beta-lysine into 3,5-diaminohexanoate (3,5-DAH) . The activity of 5,6-LAM depends on pyridoxal-5(')-phosphate (PLP) and adenosylcobalamin . The currently postulated multistep mechanism involves at least 12 steps, two of which involve hydrogen transfer . The deuterium kinetic isotope effects on k(cat) and k(cat)/K(m) have been found to be 10.4+/-0.3 and 8.3+/-1.9, respectively, in the reaction of DL-lysine-3,3,4,4,5,5,6,6-d(8) . The corresponding isotope effects for reaction of DL-lysine-4,4,5,5-d(4) are 8.5+/-0.7 and 7.1+/-1.2, respectively . Neither cob(II)alamin nor a free radical can be detected in the steady state by UV-Vis spectrophotometry or electron paramagnetic resonance (EPR) spectroscopy . Therefore, hydrogen abstraction from carbon-5 of the substrate side chain is rate limiting in the mechanism . DL-4-Oxalysine is an alternative substrate for 5,6-LAM . DL-4-Oxalysine reacts irreversibly because the product breaks down into ammonia, acetaldehyde, and DL-serine . The value of K(m) for the reaction of DL-4-oxalysine is lower than that for DL-lysine and that of k(cat) for DL-4-oxalysine is slightly lower than that for DL-lysine . As measured by values of k(cat)/K(m), 5,6-LAM uses DL-4-oxalysine essentially as efficiently as the best substrates, D-lysine and L-beta-lysine, and more efficiently than DL-lysine . DL-4-Oxalysine induces the same suicide inactivation by electron transfer as do the biological substrates . The putative substrate-related radical intermediate is not sufficiently stabilized by the nonbonding 4-oxa electrons to be detectable by EPR spectroscopy.

Arch Biochem Biophys, 2003 Oct 1, 418(1), 34 - 8
Characterization of the branching patterns of glycogen branching enzyme truncated on the N-terminus; Devillers CH et al.; Truncation of 112 amino acids at the N-terminus (Nd(1-112)) changes the chain transfer pattern of the Escherichia coli glycogen branching enzyme (GBE) {Arch . Biochem . Biophys . 397 (2002) 279} . We investigated further the role of the N-terminus by engineering other truncated GBEs and analyzing the branching pattern by high-performance anion-exchange chromatography . The wild type GBE transfers mainly chains with a degree of polymerization (d.p.) of 8-14, the Nd(1-112) enzyme transfers a greater proportion of chains with higher d.p . 15-20, whereas the 63- and 83-amino acid deleted enzymes had an intermediate pattern of transferred chains (d.p . 10-20) . These data showed that a progressive shortening of the N-terminus leads to a gradual increase in the length of the transferred chains, suggesting that the N-terminus provides a support for the glucan substrate during the processes of cleavage and transfer of the alpha-(1-4) glucan chains.

Biochem Biophys Res Commun, 2003 Oct 3, 309(4), 885 - 92
Metabolism of 20-epimer of 1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats; Kusudo T et al.; The 20-epi form of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)-20-epi-D(3)) is expected as drugs for leukemia, other cancers or psoriasis, because it shows several-hundred fold enhanced ability to induce cell differentiation and growth inhibition than 1alpha,25-dihydroxyvitamin D(3) while its calcemic activity is only slightly elevated . In this study, we compared the human and rat CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3) by using the Escherichia coli expression system . The HPLC and LC-MS analyses of the metabolites revealed that rat CYP24 converted 1alpha,25(OH)(2)-20-epi-D(3) to 25,26,27-trinor-1alpha(OH)-24(COOH)-20-epi-D(3) through 1alpha,24,25(OH)(3)-20-epi-D(3) and 1alpha,25(OH)(2)-24-oxo-20-epi-D(3) . The binding affinity of trinor-1alpha(OH)-24(COOH)-20-epi-D(3) for vitamin D receptor (VDR) was less than 1/4000 of that of 1alpha,25(OH)(2)-20-epi-D(3) . These results suggest that rat CYP24 can almost completely inactivate 1alpha,25(OH)(2)-20-epi-D(3) . On the other hand, human CYP24 mainly converted 1alpha,25(OH)(2)-20-epi-D(3) to its putative demethylated compound with a hydroxyl group, via 1alpha,24,25(OH)(3)-20-epi-D(3), 1alpha,25(OH)(2)-24-oxo-20-epi-D(3), and 1alpha,23,25(OH)(3)-24-oxo-20-epi-D(3) . All of these metabolites showed considerable affinity for vitamin D receptor . These results clearly demonstrate the species-based difference between human and rat on the CYP24-dependent metabolism of 1alpha,25(OH)(2)-20-epi-D(3).

Biochem Biophys Res Commun, 2003 Oct 3, 309(4), 749 - 54
Structural and evolutionary consequences of unpaired cysteines in trypsinogen; Kenesi E et al.; Vertebrate trypsins usually contain six disulfide bonds but human trypsin 1 (PRSS1) contains only five and human trypsin 2 (PRSS2) contains only four . To elucidate possible evolutionary pathways leading to the loss of disulfide bonds, we have constructed mutants lacking one or two cysteines of four disulfide bonds (C22-C157, C127-C232, C136-C201, and C191-C220) in rat anionic trypsinogen and followed their expression in the periplasm of Escherichia coli . When both cysteines of any of the above-mentioned disulfide bonds were replaced by alanines we found, as expected, proteolytically active enzymes . In the case of C127-C232 (missing from both human trypsins) and C191-C220 both single mutants gave active enzymes although their yield was significantly reduced . In contrast, only one of the single mutants of disulfide bonds C22-C157 and C136-C201 (missing from human trypsin 2) was expressed in E . coli . In the case of these disulfide bonds, we obtained no expression when the solvent accessible molecular surface of the free cysteine residue was the smaller one, indicating that a buried unpaired cysteine was more deleterious than one on the surface of the molecule.

Eur J Pharm Sci, 2003 Sep, 20(1), 43 - 51
Role of a non-natural beta-C-nucleotide unit in DNA as a template for DNA and RNA syntheses and as a substrate for nucleolytic digestion; Aketani S et al.; A non-natural beta-C-nucleoside bearing a 3,4-dibenzyloxyphenyl group as a nucleobase (X) was synthesized and incorporated into a 34-mer oligomer with the sequence 5'-dTTTTTAAAAAAXATATAGCAGCGACATGTCACCG-3' . This synthetic oligonucleotide was examined for template activity in the enzymatic syntheses of DNA by the Klenow fragments of Escherichia coli DNA polymerase I and the recombinant DNA polymerase I, and in the synthesis of RNA by the E . coli RNA polymerase core enzyme . As a result, the template-directed polymerization of both DNA and RNA was precisely terminated at the position of X . The X-containing oligonucleotide was also tested for digestion by an exonuclease, Exo III nuclease (Exo III), and an endonuclease, Mung Bean nuclease (MB) . The results indicate that the artificial nucleobase X acts as a terminator for digestion by Exo III, whereas the site X becomes susceptible to digestion by MB . These findings provide a useful tool for the size control of products in the synthesis and degradation of nucleic acids.

Neurobiol Dis, 2003 Oct, 14(1), 146 - 56
Identification of peptides that specifically bind Abeta1-40 amyloid in vitro and amyloid plaques in Alzheimer's disease brain using phage display; Kang CK et al.; The accumulation of the amyloid-beta (Abeta) peptides in amyloid plaques correlates with pathologic changes that occur in the brains of patients with Alzheimer's disease (AD) . The ability to directly target reagents to the amyloid form of the Abeta peptide may allow the delivery of neuroprotective agents to make amyloid plaques less toxic, the delivery of amyloid-destroying molecules to eliminate plaques, or the delivery of reagents to prevent amyloid plaque formation . In addition, such reagents may be useful as diagnostic tools to quantitate the extent of amyloid plaque formation in AD patients . As a step toward these goals, we have used phage peptide display technology to identify peptides that bind specifically to the amyloid form of the Abeta(1-40) peptide . Here we identify two 20-amino acid peptides with similar structural features that bind to the amyloid form of Abeta(1-40) but not to monomeric Abeta(1-40) . A recombinant form of one of these peptides was produced in Escherichia coli as a fusion protein with thioredoxin . After purification, this reagent bound Abeta(1-40) amyloid in vitro with a K(d) of 60 nM and specifically labeled amyloid plaques in AD brains . A chemically synthesized version of this peptide also bound Abeta(1-40) amyloid and specifically stained amyloid plaques in AD brain . These peptide sequences represent new potential carrier molecules to deliver medicines to amyloid plaques in AD patients and to image plaques in AD brains.

Neurobiol Dis, 2003 Oct, 14(1), 10 - 8
Evidence for peripheral clearance of cerebral Abeta protein following chronic, active Abeta immunization in PSAPP mice; Lemere CA et al.; Immunization with amyloid-beta (Abeta) peptide in mouse models of Alzheimer's disease has been reported to decrease cerebral Abeta levels and improve behavioral deficits . Several mechanisms have been proposed, including antibody-induced phagocytosis of Abeta by cerebral microglia and increased efflux of Abeta from the brain to the periphery . The latter mechanism was suggested in mice undergoing acute, passive transfer of an Abeta monoclonal antibody . Here, PSAPP transgenic mice were actively immunized by a single intraperitoneal injection of synthetic Abeta followed by chronic intranasal administration of Abeta with the mucosal adjuvant, Escherichia coli heat-labile enterotoxin, LT, twice weekly for 8 weeks . Serum from Abeta-immunized mice had an average of 240 microg/ml of anti-Abeta-specific antibodies; these antibodies had epitope(s) within Abeta1-15 and were of immunoglobulin (Ig) isotypes IgG2b, IgG2a, and IgG1 . Immunization led to a 75% decrease in plaque number (P < 0.0001) and a 58% decrease in Abetax-42 levels (P < 0.026) in brain, and gliosis and neuritic dystrophy were diminished . No pathological effects of the immunization were observed in kidney, spleen, or snout . Serum Abeta levels increased 28-fold in immunized mice (53.06 ng/ml) compared to controls (1.87 ng/ml) . Most of the Abeta in the serum of the immunized mice was bound to antibodies . We conclude that following active immunization, anti-Abeta antibodies sequester serum Abeta and may increase central nervous system to serum Abeta clearance.

Int J Parasitol, 2003 Sep 30, 33(11), 1207 - 17
Molecular and genetic characterisation of the host-protective oncosphere antigens of taeniid cestode parasites; Lightowlers MW et al.; Highly effective recombinant vaccines have been developed against Taenia ovis infection in sheep, Taenia saginata infection in cattle, Taenia solium infection in pigs, Echinococcus granulosus and Echinococcus multilocularis infections in a variety of intermediate host species . These vaccines have been based on the identification and expression in Escherichia coli of antigens derived from the oncosphere life cycle stage, contained within the parasites' eggs . Investigation of the molecular aspects of these proteins and the genes encoding them have revealed a number of common features, including the presence of a predicted secretory signal sequence, and one or two copies of a fibronectin type III domain, each encoded by separate exons within the associated gene . Evidence has been obtained to confirm glycosylation of some of these antigens . Ongoing investigations will shed light on the biological roles played by the proteins within the parasites and the mechanism by which they make the parasites vulnerable to vaccine-induced immune responses.

Curr Biol, 2003 Sep 16, 13(18), R714 - 6
Rho factor: transcription termination in four steps; Kaplan DL et al.; Rho factor is a hexameric ring-shaped helicase which terminates transcription in Escherichia coli . Two recent crystal structures of Rho in complex with nucleic acid reveal how this helicase ring loads onto mRNA and encircles it.

Cell, 2003 Sep 5, 114(5), 647 - 54
A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase; Spies M et al.; RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous recombination at double-stranded DNA breaks . Several of its activities are regulated by the DNA sequence chi (5'-GCTGGTGG-3'), which is recognized in cis by the translocating enzyme . When RecBCD enzyme encounters chi, the intensity and polarity of its nuclease activity are changed, and the enzyme gains the ability to load RecA protein onto the chi-containing, unwound single-stranded DNA . Here, we show that interaction with chi also affects translocation by RecBCD enzyme . By observing translocation of individual enzymes along single molecules of DNA, we could see RecBCD enzyme pause precisely at chi . Furthermore, and more unexpectedly, after pausing at chi, the enzyme continues translocating but at approximately one-half the initial rate . We propose that interaction with chi results in an enzyme in which one of the two motor subunits, likely the RecD motor, is uncoupled from the holoenzyme to produce the slower translocase.

Antioxid Redox Signal, 2003 Aug, 5(4), 367 - 74
Defining the domain boundaries of the human protein disulfide isomerases; Alanen HI et al.; The protein disulfide isomerase (PDI) family of folding catalysts are constructed from combinations of redoxactive and redox-inactive domains, all of which are probably based on the thioredoxin fold . To understand the function of each domain in the variety of catalytic reactions that each family member can perform (to differing extents), the domain boundaries of each family member must be known . By using a technique based on sequence alignments and the known structure of the a and b domains of human PDI, we generated a large number of domain constructs for all six redox-active human PDIs: PDI, PDIp, ERp72, ERp57, P5, and PDIr . The ability to generate significant amounts of soluble protein in E . coli from most of these domain constructs strongly indicates that the domain boundaries are correct . The implications for these domain boundaries on the tertiary structure of the human PDIs are discussed.

Syst Appl Microbiol, 1998 Mar, 21(2), 220 - 9
Acetobacter intermedius, sp . nov; Boesch C et al.; Strains of a new species in the genus Acetobacter, for which we propose the name A . intermedius sp . nov., were isolated and characterized in pure culture from different sources (Kombucha beverage, cider vinegar, spirit vinegar) and different countries (Switzerland, Slovenia) . The isolated strains grow in media with 3% acetic acid and 3% ethanol as does A . europaeus, do, however, not require acetic acid for growth . These characteristics phenotypically position A . intermedius between A . europaeus and A . xylinus, DNA-DNA hybridizations of A . intermedius-DNA with DNA of the type strains of Acetobacter europaeus, A . xylinus, A . aceti, A . hansenii, A . liquefaciens, A . methanolicus, A . pasteurianus, A . diazotrophicus, Gluconobacter oxydans and Escherichia coli HB 101 indicated less than 60% DNA similarity . The important features of the new species are described . Acetobacter intermedius strain TF2 (DSM11804) isolated from the liquid phase of a tea fungus beverage (Kombucha) is the type strain.

J Chromatogr A, 2003 Aug 15, 1009(1-2), 197 - 205
Two-dimensional nano-liquid chromatography-mass spectrometry system for applications in proteomics; Nagele E et al.; This work demonstrates the development of a method for the analysis of complex proteome samples by two-dimensional nano-liquid chromatography-mass spectrometry . This approach includes strong cation-exchange, sample enrichment, reversed-phase chromatography and nanospray ion trap mass spectroscopy with data dependent tandem mass spectrometry spectra acquisition, and subsequent database search . The new methodology was first evaluated using standard protein digest samples . Finally, data for the analysis of a total Escherichia coli proteome are provided.

J Chromatogr A, 2003 Aug 15, 1009(1-2), 179 - 88
Purification and partial characterization by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the recombinant transposase, TniA; Eklund P et al.; A recombinant transposase, TniA, a basic DNA binding protein, was chromatographically purified and characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) methods . Escherichia coli cells, overexpressing native TniA, were ultrasonically disrupted and the clarified supernatant was used as starting material for anion-exchange chromatography on SOURCE1 15Q 4.6/100 PE (Tricorn), at pH 7.5 . This initial step was proven to be a fast and simple way of removing acidic proteins like proteases . TniA was collected from the flow-through fraction and applied onto HiTrap heparin HP 5 ml in order to capture the basic TniA . This was followed by cation-exchange chromatography through Mono S 5/50 GL (Tricorn), at pH 6.5 which resulted in a purity of TniA of about 95% . The molecular mass of TniA was determined to 62 869 rel . mol . mass units with MALDI-TOF-MS and the identity of the protein was confirmed by peptide mass fingerprinting of trypsin-digested TniA . Partial amino acid sequencing was achieved after derivatization of tryptic peptides using Ettan CAF MALDI Sequencing Kit and post source decay . The fact that transposases are DNA-binding and that many of them possess basic isoelectric point values suggest that the outlined purification protocol may serve as a general method for the purification of recombinant nontagged transposases and other basic DNA-binding proteins.

Plant Mol Biol, 2003 Jul, 52(4), 817 - 30
Identification and molecular characterization of mitochondrial ferredoxins and ferredoxin reductase from Arabidopsis; Takubo K et al.; We have identified and characterized novel types of ferredoxin and ferredoxin reductase from Arabidopsis . Among a number of potential ferredoxin reductase genes in the Arabidopsis genome, AtMFDR was identified to encode a homologue of mitochondrial ferredoxin reductase, and AtMFDX1 and AtMFDX2 were predicted to code for proteins similar to mitochondrial ferredoxin . First, we isolated cDNAs for these proteins and expressed them in heterologous systems of insect cells and Escherichia coli, respectively . The recombinant AtMFDX1 and AtMFDR proteins exhibited spectral properties characteristic of ferredoxin and ferredoxin reductase, respectively, and a pair of recombinant AtMFDX1 and AtMFDR proteins was sufficient to transfer electrons from NAD(P)H to cytochrome c in vitro . Subcellular fractionation analyses suggested membrane association of AtMFDR protein, and protein-gel blot analyses and transient expression studies of green fluorescence protein fusions indicated mitochondrial localization of AtMFDX1 and AtMFDR . RNA-gel blot analyses revealed that the accumulation levels of AtMFDXs and AtMFDR gene transcripts were specifically high in flowers, while protein-gel blot analysis demonstrated substantial accumulation of AtMFDR protein in leaf, stem, and flower . Possible physiological roles of these mitochondrial electron transfer components are discussed in relation to redox dependent metabolic pathways in plants.

Plant Mol Biol, 2003 Jul, 52(4), 745 - 59
Fungus- and wound-induced accumulation of mRNA containing a class II chitinase of the pathogenesis-related protein 4 (PR-4) family of maize; Bravo JM et al.; Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack . PR proteins are grouped into independent families based on their sequences and properties . The PR-4 family comprises class I and class II chitinases . We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins . Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA . In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen . ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect . Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves . Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos . Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein . The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.

Plant Mol Biol, 2003 Jul, 52(4), 705 - 14
Characterization of UDP-glucose:protein transglucosylase genes from potato; Wald FA et al.; Many plant autocatalytic glycosyltransferases are implicated in plant polysaccharide biosynthesis . Cloning of cDNAs encoding potato (Solanum tuberosum L.) UDP-Glc:protein transglucosylase (UPTG, EC 2.4.1.112) and expression of the cDNA clone E11 in Escherichia coli have been previously reported . Here, we studied the functional expression of a second cDNA of the enzyme (E2 clone) . Northern blots analysis, with specific cDNA probes for the two UPTG isoforms, showed a differential expression pattern of mRNA levels in different potato tissues . Moreover, both UPTG recombinant enzymes showed different kinetic parameters . The recombinant protein encoded by E2 clone has an apparent Imax for UDP-Xyl and UDP-Gal, significantly higher than for UDP-Glc . The Km values for UDP-Glc were 0.45-0.71 microM and the values for UDP-Xyl and UDP-Gal were slightly higher than that of the UDP-Glc (1.2-2.71 microM) for both UPTG recombinant enzymes . The present study revealed further evidence for the proposed role of UPTG in the synthesis of cell wall polysaccharide . It was found a correlation between UPTG transcript levels and the growing state of the tissues in which there was an active synthesis of cell wall components . Southern blot analysis indicates that at least three genes encoding UPTG are present in potato genome . Phylogenetic analysis of both UPTG recombinant proteins showed that they are members of the RGP subfamilies from dicots.

RNA, 2003 Oct, 9(10), 1188 - 97
The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control; Zengel JM et al.; Ribosomal proteins L4 and L22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core . The tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides . Also, the extensions of L4 and L22 contact multiple domains of 23S rRNA, suggesting they might facilitate rRNA folding during ribosome assembly . To learn more about the roles of these extensions, we constructed derivatives of both proteins that lack most of their extended loops . Our analysis of ribosomes carrying L4 or L22 deletion proteins did not detect any significant difference in their sedimentation property or polysome distribution . Also, the role of L4 in autogenous control was not affected . We conclude that these extensions are not required for ribosome assembly or for L4-mediated autogenous control of the S10 operon.

RNA, 2003 Oct, 9(10), 1180 - 7
Adenosine 5'-O-(3-thio)triphosphate (ATPgammaS) is a substrate for the nucleotide hydrolysis and RNA unwinding activities of eukaryotic translation initiation factor eIF4A; Peck ML et al.; Whereas ATPgammaS is often considered a nonhydrolyzable substrate for ATPases, we present evidence that ATPgammaS is a good substrate for the RNA-stimulated nucleotide hydrolysis and RNA unwinding activities of eIF4A . In the presence of saturating single-stranded poly(U) RNA, eIF4A hydrolyzes ATPgammaS.Mg and ATP.Mg with similar steady-state parameters (KM(NTP.Mg) = 66 and 58 microM and kcat = 1.0 and 0.97 min(-1), respectively) . ATPgammaS.Mg also supports catalysis of RNA unwinding within 10-fold of the rate supported by ATP.Mg . The identical steady-state rate parameters, in comparison with the expected difference in the intrinsic rate of hydrolysis for ATP and ATPgammaS, suggest a nonchemical rate-limiting step for nucleotide hydrolysis . These results raise caution concerning the assumption that ATPgammaS is a nonhydrolyzable ATP analog and underscore the utility of thio-substituted NTPs as mechanistic probes.

RNA, 2003 Oct, 9(10), 1174 - 9
Protein synthesis by single ribosomes; Vanzi F et al.; The ribosome is universally responsible for synthesizing proteins by translating the genetic code transcribed in mRNA into an amino acid sequence . Ribosomes use cellular accessory proteins, soluble transfer RNAs, and metabolic energy to accomplish the initiation, elongation, and termination of peptide synthesis . In translocating processively along the mRNA template during the elongation cycle, ribosomes act as supramolecular motors . Here we demonstrate that ribosomes adsorbed on a surface, as for mechanical or spectroscopic studies, are capable of polypeptide synthesis and that tethered particle analysis of fluorescent beads connected to ribosomes via polyuridylic acid can be used to estimate the rate of polyphenylalanine synthesis by individual ribosomes . This work opens the way for application of biophysical techniques, originally developed for the classical motor proteins, to the understanding of protein biosynthesis.

J Bacteriol, 2003 Oct, 185(19), 5862 - 70
Dual overlapping promoters control napF (periplasmic nitrate reductase) operon expression in Escherichia coli K-12; Stewart V et al.; Escherichia coli elaborates a flexible respiratory metabolism, involving differential synthesis of isoenzymes for many oxidation and reduction reactions . Periplasmic nitrate reductase, encoded by the napFDAGHBC operon, functions with concentrations of nitrate that are too low to support respiration by membrane-bound nitrate reductase . The napF operon control region exhibits unusual organization of DNA binding sites for the transcription regulators Fnr and NarP, which activate transcription in response to anaerobiosis and nitrate, respectively . Previous studies have shown that the napF operon control region directs synthesis of two transcripts whose 5' ends differ by about 3 nucleotides . We constructed mutant control regions in which either of the two promoter -10 regions is inactivated . Results indicate that the downstream promoter (P1) was responsible for Fnr- and NarP-regulated napF operon expression, whereas transcription from the upstream promoter (P2) was activated only weakly by the Fnr protein and was inhibited by phospho-NarP and -NarL proteins . The physiological function of promoter P2 is unknown . These results establish the unconventional napF operon control region architecture, in which the major promoter P1 is activated by the Fnr protein bound to a site centered at -64.5 with respect to the transcription initiation site, working in conjunction with the phospho-NarP protein bound to a site centered at -44.5.

J Bacteriol, 2003 Oct, 185(19), 5838 - 46
Monomeric NarB is a dual-affinity nitrate reductase, and its activity is regulated differently from that of nitrate uptake in the unicellular diazotrophic cyanobacterium Synechococcus sp . strain RF-1; Wang TH et al.; Synechococcus sp . strain RF-1 is a unicellular freshwater cyanobacterium that fixes N(2) aerobically and exhibits a circadian rhythm for nitrogenase activity under a light-dark regimen . Synechococcus sp . strain RF-1 also utilizes nitrate, nitrite, or ammonium for growth . Under the diazotrophic growth, the nitrate uptake in Synechococcus sp . strain RF-1 was induced by nitrate or nitrite but repressed by ammonium . In contrast, a prominent nitrate reductase (NR) activity was detected in diazotrophically grown cells using the reduced methyl viologen assay . The NR activity was not inhibited by ammonium and only slightly enhanced by nitrate . The different expression patterns of nitrate uptake and NR in Synechococcus sp . strain RF-1 were reflected in general at the transcript level determined by reverse transcriptase PCR . Under both nitrate-induced and uninduced conditions, the in situ NR activity exhibited similar biphasic kinetics for nitrate . The recombinant NR encoded by the narB gene of Synechococcus sp . strain RF-1, expressed in E . coli, also showed the biphasic kinetics with similar pH and temperature profiles . By in-gel NR activity assay, the recombinant NarB was found to exist as a single form . Both the high- and low-affinity NR activities of the recombinant NarB showed the same thermostability . When modified at the N terminus by a polyhistidine tag, the recombinant NR activity was shifted from biphasic to hyperbolic kinetics and showed only a single K(m) for nitrate, indicating the functional importance of the NarB N-terminal structure in NR kinetics.

J Bacteriol, 2003 Oct, 185(19), 5815 - 21
Role of 2-phosphoglycolate phosphatase of Escherichia coli in metabolism of the 2-phosphoglycolate formed in DNA repair; Teresa Pellicer M et al.; The enzyme 2-phosphoglycolate phosphatase from Escherichia coli, encoded by the gph gene, was purified and characterized . The enzyme was highly specific for 2-phosphoglycolate and showed good catalytic efficiency (k(cat)/K(m)), which enabled the conversion of this substrate even at low intracellular concentrations . A comparison of the structural and functional features of this enzyme with those of 2-phosphoglycolate phosphatases of different origins showed a high similarity of the sequences, implying the use of the same catalytic mechanism . Western blot analysis revealed constitutive expression of the gph gene, regardless of the carbon source used, growth stage, or oxidative stress conditions . We showed that this housekeeping enzyme is involved in the dissimilation of the intracellular 2-phosphoglycolate formed in the DNA repair of 3'-phosphoglycolate ends . DNA strand breaks of this kind are caused by agents such as the radiomimetic compound bleomycin . The differential response between a 2-phosphoglycolate phosphatase-deficient mutant and its parental strain after treatment with bleomycin allowed us to connect the intracellular formation of 2-phosphoglycolate with the production of glycolate, which is subsequently incorporated into general metabolism . We thus provide evidence for a salvage function of 2-phosphoglycolate phosphatase in the metabolism of a two-carbon compound generated by the cellular DNA repair machinery.

J Bacteriol, 2003 Oct, 185(19), 5800 - 6
Sigma 32-dependent promoter activity in vivo: sequence determinants of the groE promoter; Wang Y et al.; The Escherichia coli transcription factor sigma 32 binds to core RNA polymerase to form the holoenzyme responsible for transcription initiation at heat shock promoters, utilized upon exposure of the cell to higher temperatures . We have developed two ways to assay sigma 32-dependent RNA synthesis in E . coli . The plasmid-borne reporter gene for both is lacZ (beta-galactosidase), driven by the groE promoter . In one application, the cells are exposed to a temperature of 42 degrees C in order to induce accumulation of endogenous sigma 32 . The other involves isopropylthiogalactopyranoside (IPTG)-induced synthesis of sigma 32 at 30 degrees C from a gene contained on a second plasmid . The latter employs DnaK(-) cells, which additionally contained a second mutation, inactivating the endogenous sigma 32 gene (Bukau and Walker, EMBO J . 9:4027-4036, 1990) . These assays were used to delineate the sequences CTTGA (-37 to -33) and GNCCCCATNT (-18 to -9) as important for sigma 32 promoter activity . At each of the specified base pairs, substitutions were found which reduced promoter activity by greater than 75% . Activity was also dependent upon the number of base pairs separating the two regions.

J Bacteriol, 2003 Oct, 185(19), 5765 - 71
Temperature-sensitive growth and decreased thermotolerance associated with relA mutations in Escherichia coli; Yang X et al.; The relA gene of Escherichia coli encodes guanosine 3',5'-bispyrophosphate (ppGpp) synthetase I, a ribosome-associated enzyme that is activated during amino acid starvation . The stringent response is thought to be mediated by ppGpp . Mutations in relA are known to result in pleiotropic phenotypes . We now report that three different relA mutant alleles, relA1, relA2, and relA251::kan, conferred temperature-sensitive phenotypes, as demonstrated by reduced plating efficiencies on nutrient agar (Difco) or on Davis minimal agar (Difco) at temperatures above 41 degrees C . The relA-mediated temperature sensitivity was osmoremedial and could be completely suppressed, for example, by the addition of NaCl to the medium at a concentration of 0.3 M . The temperature sensitivities of the relA mutants were associated with decreased thermotolerance; e.g., relA mutants lost viability at 42 degrees C, a temperature that is normally nonlethal . The spoT gene encodes a bifunctional enzyme possessing ppGpp synthetase and ppGpp pyrophosphohydrolase activities . The introduction of the spoT207::cat allele into a strain bearing the relA251::kan mutation completely abolished ppGpp synthesis . This ppGpp null mutant was even more temperature sensitive than the strain carrying the relA251::kan mutation alone . The relA-mediated thermosensitivity was suppressed by certain mutant alleles of rpoB (encoding the beta subunit of RNA polymerase) and spoT that have been previously reported to suppress other phenotypic characteristics conferred by relA mutations . Collectively, these results suggest that ppGpp may be required in some way for the expression of genes involved in thermotolerance.

J Bacteriol, 2003 Oct, 185(19), 5747 - 54
In vivo evidence for TonB dimerization; Sauter A et al.; TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and phi80 . To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) {TonB(1-239)} and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx . ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P(ctx)::lacZ) . Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P(ctx)::lacZ transcription . Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation . The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system . This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition . Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined . The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail . They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

J Bacteriol, 2003 Oct, 185(19), 5735 - 46
Genome-wide analyses revealing a signaling network of the RcsC-YojN-RcsB phosphorelay system in Escherichia coli; Hagiwara D et al.; In Escherichia coli, capsular colanic acid polysaccharide synthesis is regulated through the multistep RcsC-->YojN-->RcsB phosphorelay . By monitoring a hallmarked cps::lacZ reporter gene, we first searched for physiological stimuli that propagate the Rcs signaling system . The expression of cps::lacZ was activated when cells were grown at a low temperature (20 degrees C) in the presence of glucose as a carbon source and in the presence of a relatively high concentration of external zinc (1 mM ZnCl(2)) . In this Rcs signaling system, the rcsF gene product (a putative outer membrane-located lipoprotein) was also an essential signaling component . Based on the defined signaling pathway and physiological stimuli for the Rcs signaling system, we conducted genome-wide analyses with microarrays to clarify the Rcs transcriptome (i.e., Rcs regulon) . Thirty-two genes were identified as putative Rcs regulon members; these genes included 15 new genes in addition to 17 of the previously described cps genes . Using a set of 37 two-component system mutants, we performed alternative genome-wide analyses . The results showed that the propagation of the zinc-responsive Rcs signaling system was largely dependent on another two-component system, PhoQ/P . Considering the fact that the PhoQ/P signaling system responds to external magnesium, we obtained evidence which supports the view that there is a signaling network that connects the Rcs system with the PhoQ/P system, which coordinately regulates extracellular polysaccharide synthesis in response to the external concentrations of divalent cations.

J Bacteriol, 2003 Oct, 185(19), 5706 - 13
The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway; Schierle CF et al.; The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP) . Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway . When DsbAss is fused to MBP, MBP also is directed to the SRP pathway . We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized . However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA . These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway . Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.

J Bacteriol, 2003 Oct, 185(19), 5697 - 705
Secretion of LamB-LacZ by the signal recognition particle pathway of Escherichia coli; Bowers CW et al.; LamB-LacZ fusion proteins have classically been used in studies of the general secretion pathway of Escherichia coli . Here we describe how increasing signal sequence hydrophobicity routes LamB-LacZ Hyb42-1 to the signal recognition particle (SRP) pathway . Secretion of this hydrophobic fusion variant (H*LamB-LacZ) was reduced in the absence of fully functional Ffh and Ffs, and the translocator jamming caused by Hyb42-1 was prevented by efficient delivery of the fusion to the periplasm . Finally, we found that in the absence of the ribosome-associated chaperone, trigger factor (Tig), LamB-LacZ localized to the periplasm in a SecA-dependent, SRP-independent fashion . Collectively, our results provide compelling in vivo evidence that there is an SRP-dependent cotranslational targeting mechanism in E . coli and argue against a role for trigger factor in pathway discrimination.

J Bacteriol, 2003 Oct, 185(19), 5673 - 84
Experimental determination and system level analysis of essential genes in Escherichia coli MG1655; Gerdes SY et al.; Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells . We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media . We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions . Functional context analysis of these data allows individual functional assignments to be refined . Evolutionary context analysis demonstrates a significant tendency of essential E . coli genes to be preserved throughout the bacterial kingdom . Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.

J Biol Chem, 2003 Nov 28, 278(48), 47610 - 21 Epub 2003 Sep 16.
Identification of PSD-93 as a substrate for the Src family tyrosine kinase Fyn; Nada S et al.; In order to study the role of tyrosine kinase signaling in the post-synaptic density (PSD), tyrosine-phosphorylated proteins associated with the PSD-95/NMDA receptor complex were analyzed . The NMDA receptor complex from the mouse brain was successfully solubilized with deoxycholate and immunopurified with anti-PSD-95 or anti-phosphotyrosine antibody . Immunoblot analyses revealed that the predominantly tyrosine-phosphorylated proteins in the NMDA receptor complex are the NR2A/B subunits and a novel 120 kDa protein . Purification and microsequencing analysis showed that the 120 kDa protein is mouse PSD-93/Chapsyn-110 . Recombinant PSD-93 was phosphorylated by Fyn in vitro, and Tyr-384 was identified as a major phosphorylation site . Tyrosine phosphorylation of PSD-93 was greatly reduced in brain tissue from Fyn-deficient mice compared with wild-type mice . Furthermore, an N-terminal palmitoylation signal of PSD-93 was found to be essential for its anchoring to the membrane, where Fyn is also localized . In COS7 cells, exogenously expressed PSD-93 was phosphorylated, dependent on its membrane localization . In addition, tyrosine-phosphorylated PSD-93 was able to bind to Csk, a negative regulator of Src family kinases, in vitro as well as in a brain lysate . These results suggest that PSD-93 serves as a membrane-anchored substrate of Fyn and plays a role in the regulation of Fyn-mediated modification of NMDA receptor function.

J Biol Chem, 2003 Nov 28, 278(48), 48162 - 8 Epub 2003 Sep 16.
Structural insight into modest binding of a non-PXXP ligand to the signal transducing adaptor molecule-2 Src homology 3 domain; Kaneko T et al.; Although some exceptional motifs have been identified, it is well known that the PXXP motif is the motif of ligand proteins generally recognized by the Src homology 3 (SH3) domain . SH3-ligand interactions are usually weak, with ordinary KD approximately 10 microM . The structural basis for a tight and specific association (KD = 0.24 microm) between Gads SH3 and a novel motif, PX(V/I)(D/N)RXXKP, was revealed in a previous structural analysis of the complex formed between them . In this paper, we report the crystal structure of the signal transducing adaptor molecule-2 (STAM2) SH3 domain in complex with a peptide with a novel motif derived from a ligand protein, UBPY . The derived KD value for this complex is 27 microM . The notable difference in affinity for these parallel complexes may be explained because the STAM2 SH3 structure does not provide a specificity pocket for binding, whereas the Gads SH3 structure does . Instead, the structure of STAM2 SH3 is analogous to that of Grb2 SH3 which, in addition to normal PXXP ligands, has also been shown to moderately recognize the novel motif discussed herein . Thus, the extremely tight interaction observed between Gads SH3 and the novel motif is caused not by an innate ability of the novel motif but rather by an evolutionary change in the Gads SH3 domain . Instead, SH3 domains of STAM2 and Grb2 retain the moderate characteristics of recognizing their ligand proteins like other SH3 domains for appropriate transient interactions between signaling molecules.

J Biol Chem, 2003 Dec 12, 278(50), 50714 - 23 Epub 2003 Sep 16.
Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases; Min T et al.; Despite the importance of plant lignans and isoflavonoids in human health protection (e.g . for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g . in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun . In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids . Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates . Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively . Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain . IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure . The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

FEMS Microbiol Lett, 2003 Sep 12, 226(1), 31 - 7
Oxygen-dependent coproporphyrinogen-III oxidase from Escherichia coli: one-step purification and biochemical characterisation; Macieira S et al.; Coproporphyrinogen-III oxidase (CPO) catalyses the conversion of coproporphyrinogen-III to protoporphyrinogen-IX in the haem biosynthetic pathway, and its deficient activity is associated with human hereditary coproporphyria . The 47% sequence identity between the oxygen-dependent CPO from Escherichia coli and its human counterpart makes the bacterial enzyme a good model system for structural studies of this disease . Therefore, we overexpressed and purified to homogeneity the oxygen-dependent CPO from E . coli and its selenomethionine derivative fused with a His(6)-tag . Both preparations showed a specific activity of 37500 U mg(-1), had a subunit molecular mass of 35 kDa and behaved as a compact shaped dimer . First crystallisation trials produced plate-shaped diffracting crystals.

Bioorg Med Chem, 2003 Oct 1, 11(20), 4511 - 21
Design, synthesis and biological evaluation of 10-CF3CO-DDACTHF analogues and derivatives as inhibitors of GAR Tfase and the de novo purine biosynthetic pathway; Desharnais J et al.; The synthesis and evaluation of analogues and key derivatives of 10-CF3CO-DDACTHF as inhibitors of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide transformylase (AICAR Tfase) are reported . Polyglutamate analogues of 1 were evaluated as inhibitors of Escherichia coli and recombinant human (rh) GAR Tfase, and AICAR Tfase . Although the pentaglutamate 6 was found to be the most active inhibitor of the series tested against rhGAR Tfase (Ki=0.004 microM), little distinction between the mono-pentaglutamate derivatives was observed (Ki=0.02-0.004 microM), suggesting that the principal role of the required polyglutamation of 1 is intracellular retention . In contrast, 1 and its defined polyglutamates 3-6 were much less inactive when tested against rhAICAR Tfase (Ki=65-0.120 microM) and very selective (> or =100-fold) for rh versus E . coli GAR Tfase . Additional key analogues of 1 were examined (7 and 8) and found to be much less active (1000-fold) highlighting the exceptional characteristics of 1.

Bioorg Med Chem, 2003 Oct 1, 11(20), 4303 - 13
Design, synthesis, and anticancer activity of phosphonic acid diphosphate derivative of adenine-containing butenolide and its water-soluble derivatives of paclitaxel with high antitumor activity; Moosavi-Movahedi AA et al.; Synthesis of adenine derivative of triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 4 was accomplished by treatment of phosphonate 3 with 5-phosphoribosyl 1-pyrophosphate in the presence of 5-phosphoribosyl 1-pyrophosphate synthetase . It was found that triphosphonate 4 functions as an irreversible stoichiometric inactivator of the Escherichia coli ribonucleoside diphosphate reductase (RDPR) . Triphosphonate 4 exhibited potent inhibitory activity against murine leukemias (L1210 and P388), breast carcinoma (MCF7), and human T-lymphoblasts (Molt4/C8 and CEM/0) cell lines . Paclitaxel ester derivatives of adenine-containing triphosphono-gamma-(Z)-ethylidene-2,3-dimethoxybutenolide 8-10 were also synthesized . Like triphosphonate 4, compound 8 exhibited inhibitory property toward RDPR . It also induced microtubule assembly similar to paclitaxel (5) . The structure of the chlorodiester linker in 8 was found to account for this dual property . After treatment of MCF7 cells with compounds 4, 5, and 8, fluorescence microscope examination demonstrated the presence of nucleus shrinkage or segmentation . Bifunctional prodrug 8 exhibited higher lipophilicity than 4 and higher water-solubility than 5 . Pro-dual-drug 8 exhibited more pronounced anticancer activity relative to that of the triphosphonate 4 and paclitaxel (5) . In contrast, compound 9, resulting from the linkage of triphosphonate 4 and paclitaxel (5) through a diester unit, was only found to function as a highly water-soluble prodrug for paclitaxel (5) . It induced microtubule assembly in vitro, but did not show inhibitory property toward RDPR . On the other hand, compound 10, an aggregate of triphosphonate 4 and paclitaxel (5), neither functioned as an inhibitor of RDPR nor exhibited microtubule assembly stimulating activity in vitro.

Bioconjug Chem, 2003 Sep-Oct, 14(5), 974 - 8
Engineered protein a for the orientational control of immobilized proteins; Johnson CP et al.; This work describes the genetic engineering and characterization of a histidine-tagged fragment of protein A . The histidine tag results in the site-selective immobilization of the protein A receptor and the preservation of its high ligand affinity when immobilized on solid supports . The fragment was expressed at high yield in E . coli and purified to homogeneity . When selectively immobilized to histidine binding matrices, the protein A fragment exhibits high affinity for soluble IgG . We further demonstrate from adsorption isotherms that the receptor exhibits a homogeneous, high affinity population at densities where steric crowding between large ligands does not affect the apparent receptor affinity . This engineered receptor is appropriate for a range of applications including sensor design or those using immobilized Fc-tagged proteins.

Bioconjug Chem, 2003 Sep-Oct, 14(5), 909 - 18
A fluorescence resonance energy transfer sensor based on maltose binding protein; Medintz IL et al.; A fluorescence resonance energy-transfer (FRET) sensing system for maltose based on E . coli maltose binding protein (MBP) is demonstrated . The FRET donor portion of the sensing system consists of MBP modified with long wavelength-excitable cyanine dyes (Cy3 or Cy3.5) . The novel acceptor portion of the sensor consists of beta-cyclodextrin (beta-CD) modified with either the cyanine dye Cy5 or the dark quencher QSY9 . Binding of the modified beta-CD to dye-conjugated MBP results in assembly of the FRET complex . Added maltose displaces the beta-CD-dye adduct and disrupts the FRET complex, resulting in a direct change in fluorescence of the donor moiety . In the use of these FRET pairs, MBP dissociation values for maltose were estimated (0.14-2.90 microM) . Maltose limits of detection were in the 50-100 nm range.

J Biol Chem, 2003 Nov 21, 278(47), 46625 - 31 Epub 2003 Sep 15.
Oxygen-dependent coproporphyrinogen III oxidase (HemF) from Escherichia coli is stimulated by manganese; Breckau D et al.; During heme biosynthesis in Escherichia coli two structurally unrelated enzymes, one oxygen-dependent (HemF) and one oxygen-independent (HemN), are able to catalyze the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX . Oxygen-dependent coproporphyrinogen III oxidase was produced by overexpression of the E . coli hemF in E . coli and purified to apparent homogeneity . The dimeric enzyme showed a Km value of 2.6 microm for coproporphyrinogen III with a kcat value of 0.17 min-1 at its optimal pH of 6 . HemF does not utilize protoporphyrinogen IX or coproporphyrin III as substrates and is inhibited by protoporphyrin IX . Molecular oxygen is essential for the enzymatic reaction . Single turnover experiments with oxygen-loaded HemF under anaerobic conditions demonstrated electron acceptor function for oxygen during the oxidative decarboxylation reaction with the concomitant formation of H2O2 . Metal chelator treatment inactivated E . coli HemF . Only the addition of manganese fully restored coproporphyrinogen III oxidase activity . Evidence for the involvement of four highly conserved histidine residues (His-96, His-106, His-145, and His-175) in manganese coordination was obtained . One catalytically important tryptophan residue was localized in position 274 . None of the tested highly conserved cysteine (Cys-167), tyrosine (Tyr-135, Tyr-160, Tyr-170, Tyr-213, Tyr-240, and Tyr-276), and tryptophan residues (Trp-36, Trp-123, Trp-166, and Trp-298) were found important for HemF activity . Moreover, mutation of a potential nucleotide binding motif (GGGXXTP) did not affect HemF activity . Two alternative routes for HemF-mediated catalysis, one metal-dependent, the other metal-independent, are proposed.

J Biol Chem, 2003 Nov 28, 278(48), 47602 - 9 Epub 2003 Sep 15.
Iron-sulfur cluster N2 of the Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is located on subunit NuoB; Flemming D et al.; The proton-pumping NADH:ubiquinone oxidoreductase, also called respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane . One FMN and up to 9 iron-sulfur (Fe/S) clusters participate in the redox reaction . There is discussion that the EPR-detectable Fe/S cluster N2 is involved in proton pumping . However, the assignment of this cluster to a distinct subunit of the complex as well as the number of Fe/S clusters giving rise to the EPR signal are still under debate . Complex I from Escherichia coli consists of 13 polypeptides called NuoA to N . Either subunit NuoB or NuoI could harbor Fe/S cluster N2 . Whereas NuoB contains a unique motif for the binding of one Fe/S cluster, NuoI contains a typical ferredoxin motif for the binding of two Fe/S clusters . Individual mutation of all four conserved cysteine residues in NuoB resulted in a loss of complex I activity and of the EPR signal of N2 in the cytoplasmic membrane as well as in the isolated complex . Individual mutations of all eight conserved cysteine residues of NuoI revealed a variable phenotype . Whereas cluster N2 was lost in most NuoI mutants, it was still present in the cytoplasmic membranes of the mutants NuoI C63A and NuoI C102A . N2 was also detected in the complex isolated from the mutant NuoI C102A . From this we conclude that the Fe/S cluster N2 is located on subunit NuoB.

Genes Dev, 2003 Oct 1, 17(19), 2374 - 83 Epub 2003 Sep 15.
Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli; Masse E et al.; RyhB is a small antisense regulatory RNA that is repressed by the Fur repressor and negatively regulates at least six mRNAs encoding Fe-binding or Fe-storage proteins in Escherichia coli . When Fe is limiting, RyhB levels rise, and target mRNAs are rapidly degraded . RyhB is very stable when measured after treatment of cells with the transcription inhibitor rifampicin, but is unstable when overall mRNA transcription continues . We propose that RyhB turnover is coupled to and dependent on pairing with the target mRNAs . Degradation of both mRNA targets and RyhB is dependent on RNase E and is slowed in degradosome mutants . RyhB requires the RNA chaperone Hfq . In the absence of Hfq, RyhB is unstable, even when general transcription is inhibited; degradation is dependent upon RNase E . Hfq and RNase E bind similar sites on the RNA; pairing may allow loss of Hfq and access by RNase E . Two other Hfq-dependent small RNAs, DsrA and OxyS, are also stable when overall transcription is off, and unstable when it is not, suggesting that they, too, are degraded when their target mRNAs are available for pairing . Thus, this large class of regulatory RNAs share an unexpected intrinsic mechanism for shutting off their action.

Int J Exp Pathol, 2003 Jun, 84(3), 135 - 44
Induction of glomerular alkaline phosphatase after challenge with lipopolysaccharide; Kapojos JJ et al.; Alkaline phosphatase (AP) can be considered as a host defence molecule since this enzyme is able to detoxify bacterial endotoxin at physiological pH . The question emerged whether this anti-endotoxin principle is inducible in the glomerulus and if so, which glomerular cells might be involved in the expression of ectoAP after stimulation with pro-inflammatory agents . Therefore kidneys of rats treated with either lipopolysaccharide (LPS), E . coli bacteria or non-toxic monophosphoryl lipid A (MPLA) were examined for AP activity 6 or 24 h after challenge . In addition cultures of endothelial cells or mesangial cells were evaluated for AP activity after stimulation with either LPS, TNFalpha or IL-6, and mRNA for AP was studied in TNFalpha-stimulated and control mesangial cells . The results show significant up-regulation of glomerular AP in LPS- or E . coli-injected rats compared to rats injected with MPLA . Endothelial and mesangial cells in vitro showed significant up-regulation of AP activity following stimulation with LPS, TNFalpha or IL-6, whereas increased mRNA for AP was observed in mesangial cells after TNFalpha stimulation compared to non-stimulated control cells . Since it appeared that hydrolysis occurred when endotoxin was used as a substrate in the histochemical staining, we concluded that inducible glomerular ectoAP may reflect a local endotoxin detoxifying principle of the kidney.

Transpl Infect Dis, 2003 Jun, 5(2), 104 - 7
Brain abscess caused by Cladophialophora (Xylohypha) bantiana in a renal transplant patient; Silveira ER et al.; Infectious disease is the most significant cause of morbidity and mortality in allotransplantation because of heavy immunosuppression . Brain abscesses caused by melanized fungi have been found occasionally and are an example of this complication . In this paper, we describe a case in a 61-year-old black man, who received a cadaveric kidney transplantation in December 1993, followed by triple therapy with cyclosporine, azathioprine, and prednisone . The patient developed right hemiparesis at the beginning of April 1998 . A computed tomography scan showed a mass in the left parieto-temporal region of the brain . The patient underwent surgery and a brown-colored encapsulated brain abscess was resected . Histology of the tissue revealed a large number of pigmented fungal hyphae . Culture in a Sabouraud dextrose medium with cyclohexamide and chloramphenicol at 25 degrees C resulted in the growth of dark-green colonies . The fungus identified was Cladophialophora bantiana, based on characteristic microscopic features and on growth at 40 degrees C . The abscess recurred in spite of treatment with fluconazole . The patient was submitted to a second brain surgical procedure and was treated with amphotericin B in addition to fluconazole . Ten days later the patient's blood cultures became positive for Escherichia coli . After 3 days the patient died due to septic shock.

Biochemistry, 2003 Sep 23, 42(37), 11065 - 73
The role of tryptophan residues in an integral membrane protein: diacylglycerol kinase; Clark EH et al.; Tryptophan residues are thought to play special roles in integral membrane proteins, anchoring transmembrane alpha-helices into the lipid bilayer . We have studied the effect of mutating the five Trp residues in the diacylglycerol kinase (DGK) of Escherichia coli to Leu residues . The fluorescence emission maxima for DGK and a variety of Trp mutants in bilayers of dioleoylphosphatidylcholine {di(C18:1)PC} are all centered at ca . 327 nm, suggesting that all five Trp residues are located close to the glycerol backbone region of the bilayer . This is also consistent with fluorescence quenching experiments, measuring the separation between the Trp residues and the bromine atoms in a bilayer of dibromostearoylphosphatidylcholine . Mutation of Trp residues in DGK was found to have significant effects on activity for DGK reconstituted into bilayers of di(C18:1)PC containing 30 mol % 1,2-dihexanoylglycerol (DHG) . Of the mutants containing a single Trp residue, only that containing Trp-112 was found to give active protein . The presence of both Trp-25 and Trp-112 gave higher activity than Trp-112 alone . Trp-25 and Trp-112 are the most important Trp residues in DGK as far as activity is concerned . Effects of mutations on K(m) for DHG were generally greater than effects on v(max) . The activity of wild-type and mutant DHGs reconstituted into bilayers of phosphatidylcholines was sensitive to the chain length of the phospholipid, with highest activities for chain lengths of C18 or C20 and lower activities in phosphatidylcholines with shorter or longer chains . Compared to wild-type DGK, the Trp mutants were less affected by long-chain phosphatidylcholines but more affected by short-chain phospholipids . In mutants lacking Trp-25, low activities in short-chain phospholipids followed from a decrease in v(max) compared to wild type, combined with an increase in K(m) value for DHG, as observed in the wild type . It is suggested that Trp-25 plays a role in maintaining the alignment of ATP and DHG at the active site . Fluorescence emission spectra for the Trp mutants do not change significantly with changing fatty acyl chain length from C14 to C24, showing efficient hydrophobic matching between DGK and the surrounding lipid bilayer . It is suggested that hydrophobic matching is achieved by tilting of the transmembrane alpha-helix or rotation of residues at the ends of the helices about the Calpha-Cbeta bond linking the residue to the helix backbone . As well as any structural effects, the presence of Trp residues in DGK has a clear effect on thermal stability.

Biochemistry, 2003 Sep 23, 42(37), 11057 - 64
Structure-based mutagenesis of the malonyl-CoA:acyl carrier protein transacylase from Streptomyces coelicolor; Koppisch AT et al.; Malonyl-CoA:acyl carrier protein transacylase (MAT) provides acyl-ACP thioesters for the biosynthesis of aromatic polyketides, and thus is the primary gatekeeper of substrate specificity in type II PKS . A recent report described the X-ray crystal structure of the Streptomyces coelicolor MAT and suggested active site residues which may be important for substrate selectivity {Keatinge-Clay, A . T., et al . (2003) Structure 11, 147-154} . Mutants were made to test the proposed roles of these residues, and the enzymes were characterized kinetically with respect to native and non-native substrates . The activity of the MAT was observed to be greatly attenuated in many of the observed mutants; however, the K(m) for malonyl-CoA was only modestly affected . Our results suggest the MAT uses an active site that is rigorously ordered around the acyl-thioester moiety of the acyl-CoA to facilitate rapid and efficient transacylation to an ACP . Our results also suggest that the MAT does not discriminate against alpha-substituted acyl-CoA thioesters solely on the basis of substrate size.

Biochemistry, 2003 Sep 23, 42(37), 11048 - 56
Catalytic mechanism of dichloromethane dehalogenase from Methylophilus sp . strain DM11; Stourman NV et al.; The glutathione (GSH)-dependent dichloromethane dehalogenase from Methylophilus sp . strain DM11 catalyzes the dechlorination of CH(2)Cl(2) to formaldehyde via a highly reactive, genotoxic intermediate, S-(chloromethyl)glutathione (GS-CH(2)Cl) . The catalytic mechanism of the enzyme toward a series of dihalomethane and monohaloethane substrates suggests that the initial addition of GSH to the alkylhalides is fast and that the rate-limiting step in turnover is the release of either the peptide product or formaldehyde . With the exception of CH(2)ClF, which forms a relatively stable GS-CH(2)F intermediate, the turnover numbers for a series of dihalomethanes fall in a very narrow range (1-3 s(-1)) . The pre-steady-state kinetics of the DM11-catalyzed addition of GSH to CH(3)CH(2)Br exhibits a burst of S-(ethyl)-glutathione (k(b) = 96 +/- 56 s(-1)) followed by a steady state with k(cat) = 0.13 +/- 0.01 s(-1) . The turnover numbers for CH(3)CH(2)Cl, CH(3)CH(2)Br, and CH(3)CH(2)I are identical, indicating a common rate-limiting step . The turnover numbers of the enzyme with CH(3)CH(2)Br and CH(3)CH(2)I are dependent on viscosity and are very close to the measured off-rate of GSEt . The turnover number with CH(2)I(2) is also dependent on viscosity, suggesting that a diffusive step is rate-limiting with dihaloalkanes as well . The rate constants for solvolysis of CH(3)SCH(2)Cl, a model for GS-CH(2)Cl, range between 1 s(-1) (1:1 dioxane/water) and 64 s(-1) (1:10 dioxane/water) . Solvolysis of the S-(halomethyl)glutathione intermediates may also occur in the active site of the enzyme preventing the release of the genotoxic species . Together, the results indicate that dissociation of the GS-CH(2)X or GS-CH(2)OH intermediates from the enzyme may be a relatively rare event.

Biochemistry, 2003 Sep 23, 42(37), 10998 - 1003
Functional split and crosslinking of the membrane domain of the beta subunit of proton-translocating transhydrogenase from Escherichia coli; Althage M et al.; Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha subunit with the NAD(H)-binding domain I and a beta subunit with the NADP(H)-binding domain III . The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the alpha and beta subunits . The interface in domain II between the alpha and the beta subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the alpha subunit and loops connecting the nine transmembrane helices in the beta subunit . However, to investigate the organization of the nine transmembrane helices in the beta subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon . The resulting enzyme was composed of the wild-type alpha subunit and the two new peptides beta1 and beta2 . As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD(+) by NADPH, the cyclic reduction of 3-acetylpyridine-NAD(+) by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities . These high activities suggest that the alpha subunit was normally folded, followed by a concerted folding of beta1 + beta2 . Cross-linking of a betaS105C-betaS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity . This is the first time that cross-linking between helices in the same beta subunit has been demonstrated.

Biochemistry, 2003 Sep 23, 42(37), 10965 - 70
O6-alkylguanine-DNA alkyltransferase: low pKa and high reactivity of cysteine 145; Guengerich FP et al.; The active site cysteine of human O(6)-alkylguanine-DNA alkyltransferase (hAGT), Cys145, was shown to be highly reactive with model electrophiles unrelated to substrates, including 1-chloro-2,4-dinitrobenzene . The high reactivity suggested that the Cys145 thiolate anion might be stable at neutral pH . The pK(a) was estimated from plots of UV spectra (A(239)) and reactivity toward 4,4'-dithiopyridine vs pH . The estimated pK(a) for hAGT was 4-5, depending upon the method used, and near that of the extensively characterized papain Cys25 . Rates of reaction with 4,4'-dithiopyridine were similar for the thiolate forms of hAGT, papain, glutathione, and the bacterial hAGT homologue Ogt (the pK(a) of the latter was 5.4) . Bound Zn(2+) has previously been shown to be required for the catalytic activity of hAGT (Rasimas, J . J . et al . (2003) Biochemistry 42, 980-990) . Zn(2+) was shown to be required for the low pK(a) of hAGT . The high reactivity of hAGT Cys145 is postulated to be important in normal catalytic function, in cross-linking reactions involving bis-electrophiles, and in inhibition of the DNA repair function of hAGT by electrophiles.

Biochemistry, 2003 Sep 23, 42(37), 10931 - 7
Amino acid discrimination by a highly differentiated metal center of an aminoacyl-tRNA synthetase; Zhang CM et al.; Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves high amino acid specificity without the need for an editing reaction . Crystallographic and spectroscopic studies have previously demonstrated that a major determinant of the specificity is an active site zinc ion that recognizes the substrate cysteine through a strong zinc-thiolate interaction . The active site cleft of CysRS is composed of highly or strictly conserved amino acids, including four inner-sphere zinc ligands, five histidine imidazoles at the base of the cleft, and a tryptophan that flips down upon cysteine binding to complete formation of the binding pocket . Here we establish the significance of each of these major features of the active site cleft by mutational analysis . Substitutions generally lead to substantially deleterious effects on K(m) and k(cat) parameters with respect to each of the cysteine, ATP, and tRNA(Cys) substrates . These findings emphasize the importance of the highly differentiated nature of the active site and provide new insights into the origins of selectivity without editing . Most mutants are less attenuated in tRNA aminoacylation than in adenylate synthesis, suggesting that tRNA binding drives a conformational change to help assemble the active site.

Biochemistry, 2003 Sep 23, 42(37), 10904 - 14
Structure of avian AICAR transformylase with a multisubstrate adduct inhibitor beta-DADF identifies the folate binding site; Wolan DW et al.; The penultimate catalytic step of the purine de novo synthesis pathway is the conversion of aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formyl-AICAR that requires the cofactor N(10)-formyl-tetrahydrofolate as the formyl donor . This reaction is catalyzed by the AICAR transformylase domain of the bifunctional enzyme AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC) . Identification of the location of the AICAR transformylase active site was previously elucidated from the crystal structure of the avian ATIC with bound substrate AICAR; however, due to the absence of any bound folate, the folate binding region of the active site could not be identified . Here, we have determined the homodimeric crystal structure of avian ATIC in complex with the ATIC-specific multisubstrate adduct inhibitor beta-DADF to 2.5 A resolution . Beta-DADF encompasses both the AICAR and folate moieties into a single covalently linked entity, thereby allowing for the characterization of the folate binding pocket of the AICAR transformylase active site . Beta-DADF is intimately bound at the dimer interface of the transformylase domains with the majority of AICAR moiety interactions occurring within one subunit, whereas the primary interactions to the folate occur with the opposing subunit . The crystal structure suggests that a buried Lys(267) is transiently protonated during formyl transfer allowing for the stabilization of the oxyanion transition state and subsequent protonation of N10 on the tetrahydrofolate leaving group . Furthermore, the beta-DADF-bound structure provides a more optimal three-dimensional scaffold to improve the design of specific antineoplastic agents.

Cell Res, 2003 Aug, 13(4), 301 - 8
Cloning and molecular characterization of a novel lectin gene from Pinellia ternata; Yao JH et al.; The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR . Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species . PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species . Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family . Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence . The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E . coli BL21 . SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E . coli BL21 when induced by IPTG . Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v) . The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.

Biol Chem, 2003 Aug, 384(8), 1165 - 72
Assignment of disulphide bridges in Par j 2.0101, a major allergen of Parietaria judaica pollen; Amoresano A et al.; Par j 2.0101, a major allergen of the Parietaria judaica pollen, was expressed in E . coli, purified to homogeneity and fully characterised both at the structural and the functional level . The recombinant rPar j 2.0101 protein showed an allergenic activity in histamine release, skin prick tests and capacity to bind IgE, almost identical to that of the native allergens purified from aqueous pollen extract . The complete pattern of S-S bridges of rPar j 2.0101 was determined by enzymatic digestion with endoproteinase Lys-C followed by mass spectrometric analysis of the resulting peptide mixtures . The eight cysteines occurring in the allergenic protein were found to be paired into the following four disulphides: Cys35-Cys83, Cys45-Cys60, Cys61-Cys106 and Cys81-Cys121 . This structural information probes Par j 2.0101 to attain a 3-D fold consistent with that of the non-specific lipid transfer protein (ns-LTP) family and it represents an effective molecular basis to develop modified antigens by selective site-directed mutagenesis for immunotherapy.

J Huazhong Univ Sci Technolog Med Sci, 2003, 23(2), 206 - 8
Anti-endotoxic effects of syringic acid of Radix Isatidis; Liu Y et al.; The anti-endotoxic effect of syringic acid (SA) isolated from Radix Isatidis (Banlangen, BLG) was studied . SA was extracted and isolated from BLG and diluted into 1% solution . The content of SA-pretreated endotoxin (ET) was quantitatively determined using Limulus test . The ability of fever induction of ET pretreated with SA was measured using endotoxin-induced fever test in rabbits . The LPS-induced death in mice pretreated with and without SA was compared . Results showed that after pretreatment with SA, 83.16% of ET was destroyed, the ET-induced fever in rabbits relieved markedly and the LPS-induced death rate in mice dropped from 68% to 20% . It was concluded that SA isolated from BLG had anti-endotoxic effects.

Electrophoresis, 2003 Sep, 24(17), 3097 - 103
Separation of Escherichia coli 055:B5 lipopolysaccharide and detoxified lipopolysaccharide by high-performance capillary electrophoresis; Volpi N; A rapid, highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method (electrokinetic chromatography with sodium dodecyl sulfate) is described for the determination of the lipopolysaccharide (LPS) and detoxified LPS (D-LPS), produced by both alkaline treatment in anhydrous conditions and mild acid hydrolysis, from Escherichia coli 055:B5 bacteria . LPS and D-LPS are separated and readily determined within 25 min on an uncoated fused-silica capillary using normal polarity at 20 kV and detection at 200 nm . A linear relationship (correlation coefficient greater than about 0.97) was found for the LPS and the two D-LPS species over a wide range of concentrations, from approximately 120 to 360 ng, with a detection sensitivity less than about 100 ng . Furthermore, HPCE was able to separate several molecular species mainly due to the presence of populations with O-specific polysaccharides of distinct and increasing mean chain lengths . This approach could be of great importance for the quantitative determination of LPS and D-LPS during the purification and preparation processes, also considering the importance of D-LPS in the preparation of human vaccines, and for the qualitative evaluation of the heterogeneity of LPS and the O-polysaccharide components.

Cad Saude Publica, 2003 Jul-Aug, 19(4), 1205 - 8 Epub 2003 Sep 08.
{Enteropathogens associated with diarrheal disease in infants (< 5 years old) in a population sample in Greater Metropolitan Criciúma, Santa Catarina State, Brazil}; Schnack FJ et al.; Enteropathogens were investigated in 94 children with diarrhea and 45 age-matched controls, 0 to 5 years old, attending an outpatient unit in Criciuma, Santa Catarina State, Brazil . Cryptosporidium (85.1%) topped the list of parasite isolates, followed by Entamoeba histolytica (56.4%) and Giardia lamblia (4.3%) . Four samples contained enteropathogenic Escherichia coli (4.3%) . Samonella and Shiguella were not detected . Only one sample contained rotavirus (1.1%).

Plant Physiol, 2003 Oct, 133(2), 702 - 12 Epub 2003 Sep 04.
The subcellular localization of plant protein phosphatase 5 isoforms is determined by alternative splicing; de la Fuente van Bentem S et al.; Protein serine/threonine phosphatase 5 (PP5) plays an important role in signal transduction in animal cells, but in plants, knowledge about PP5 is scarce . Here, we describe the isolation of a full-length cDNA encoding tomato (Lycopersicon esculentum) PP5 (LePP5) and its expression in Escherichia coli . Biochemical characterization showed that recombinant LePP5 has a low intrinsic protein phosphatase activity . This activity was increased 6- to 10-fold by either removal of the N-terminal tetratricopeptide repeat domain or by addition of fatty acids, indicating that biochemical features specific for PP5 homologs from other species are conserved in tomato . The single-copy LePP5 gene was cloned and shown to encode two mRNA species that arise by alternative pre-mRNA splicing . Similarly, Arabidopsis was found to express two PP5 transcripts, suggesting that alternative splicing of PP5 pre-mRNA is not specific for tomato . Alternative splicing results in a larger transcript containing an additional exon encoding two putative transmembrane domains and, hence, in a larger PP5 isoform . Subcellular fractionation studies on tomato protein lysates indicated that the majority of the 55-kD LePP5 isoform is soluble, whereas the 62-kD isoform is an integral membrane protein . Production of yellow fluorescent protein-PP5 chimeras in plant cells indicated that the 55-kD isoform is localized in both the nucleus and the cytoplasm, whereas the 62-kD isoform is targeted to the endoplasmic reticulum, including the nuclear envelope . Our findings show that alternative splicing generates two LePP5 isoforms with a different subcellular localization.

Mol Cell Biol, 2003 Oct, 23(19), 6809 - 22
Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA; Moreau V et al.; Members of the Rho GTPase family play a central role in the orchestration of cytoskeletal rearrangements, which are of prime importance in endothelial cell physiology . To explore their role in this specialized cell type, we used the bacterial toxin cytotoxic necrotizing factor 1 (CNF1) as a Rho GTPase activator . Punctate filamentous actin structures appeared along the ventral plasma membrane of endothelial cells and were identified as the core of podosomes by the distinctive vinculin ring around the F-actin . Rho, Rac, and Cdc42 were all identified as targets of CNF1, but only a constitutively active mutant of Cdc42 could substitute for CNF1 in podosome induction . Accordingly, organization of F-actin in these structures was highly dependent on the main Cdc42 cytoskeletal effector N-Wiskott-Aldrich syndrome protein . Other components of the actin machinery such as Arp2/3 and for the first time WIP also colocalized at these sites . Like CNF1 treatment, sustained Cdc42 activity induced a time-dependent F-actin-vinculin reorganization, prevented cytokinesis, and downregulated Rho activity . Finally, podosomes were also detected on endothelial cells explanted from patients undergoing cardiac surgery . These data provide the first description of podosomes in endothelial cells . The identification of such specialized structures opens up a new field of investigation in terms of endothelium pathophysiology.

Mol Biol Cell, 2003 Sep, 14(9), 3857 - 67 Epub 2003 May 29.
Molecular evolution of the Rab-escort-protein/guanine-nucleotide-dissociation-inhibitor superfamily; Alory C et al.; Prenylation of Rab GTPases regulating vesicle traffic by Rab geranylgeranyltransferase (RabGGTase) requires a complex formed by the association of newly synthesized Rab proteins with Rab-escort-protein (REP), the choroideremia-gene-product that is mutated in disease, leading to loss of vision . After delivery to the membrane by the REP-Rab complex, subsequent recycling to the cytosol requires the REP-related guanine-nucleotide-dissociation-inhibitor (GDI) . Although REP and GDI share common Rab-binding properties, GDI cannot assist in Rab prenylation and REP cannot retrieve Rab proteins from the membranes . We have now isolated REP mutant proteins that are able to partially function as both REP and GDI . These results provide molecular insight into the functional and evolutionary organization of the REP/GDI superfamily.

J Cell Sci, 2003 Oct 15, 116(Pt 20), 4087 - 94
Structural basis of urothelial permeability barrier function as revealed by Cryo-EM studies of the 16 nm uroplakin particle; Min G et al.; The apical surface of terminally differentiated mammalian urothelial umbrella cells is covered by numerous plaques consisting of two-dimensional (2D) crystals of hexagonally packed 16 nm uroplakin particles, and functions as a remarkable permeability barrier . To determine the structural basis of this barrier function, we generated, by electron cryo microscopy, a projection map of the isolated mouse urothelial plaques at 7 A and a 3D structure at 10 A resolution . Our results indicate that each 16 nm particle has a central 6 nm lipid-filled 'hole' surrounded by 6 inverted U-shaped subunits, each consisting of an inner and an outer subdomain connected via a distal joint . The transmembrane portion of each subdomain can fit about 5 helices . This finding, coupled with our STEM and EM data, suggests that uroplakin pairs Ia/II and Ib/III are associated with the inner and outer subdomains, respectively . Since the inner subdomains interconnect to form a ring, which can potentially segregate the lipids of the central hole from those outside, the 2D crystalline uroplakin network may impose an organized state and a severely restricted freedom of movement on the lipid components, thus reducing membrane fluidity and contributing to the barrier function of urothelial plaques . Our finding that distinct uroplakin substructures are in contact with the cytoplasmic and exoplasmic leaflets of the plaque suggests that the two leaflets may have different lipid composition and contribute asymmetrically to the barrier function . We propose that the crystalline lattice structure of uroplakin, through its interactions with specialized lipids, plays a major role in the remarkable permeability barrier function of urothelial apical surface . Our results also have implications for the transmembrane signal transduction in urothelial cells as induced by the binding of uropathogenic E . coli to its uroplakin receptor.

J Biol Chem, 2003 Nov 28, 278(48), 47578 - 84 Epub 2003 Sep 12.
Tryptophan phosphorescence spectroscopy reveals that a domain in the NAD(H)-binding component (dI) of transhydrogenase from Rhodospirillum rubrum has an extremely rigid and conformationally homogeneous protein core; Broos J et al.; The characteristics of tryptophan phosphorescence from the NAD(H)-binding component (dI) component of Rhodospirillum rubrum transhydrogenase are described . This enzyme couples hydride transfer between NAD(H) and NADP(H) to proton translocation across a membrane and is only active as a dimer . Tryptophan phosphorescence spectroscopy is a sensitive technique for the detection of protein conformational changes and was used here to characterize dI under mechanistically relevant conditions . Our results indicate that the single tryptophan in dI, Trp-72, is embedded in a rigid, compact, and homogeneous protein matrix that efficiently suppresses collisional quenching processes and results in the longest triplet lifetime for Trp ever reported in a protein at ambient temperature (2.9 s) . The protein matrix surrounding Trp-72 is extraordinarily rigid up to 50 degrees C . In all previous studies on Trp-containing proteins, changes in structure were reflected in a different triplet lifetime . In dI, the lifetime of Trp-72 phosphorescence was barely affected by protein dimerization, cofactor binding, complexation with the NADP(H)-binding component (dIII), or by the introduction of two amino acid substitutions at the hydride-transfer site . It is suggested that the rigidity and structural invariance of the protein domain (dI.1) housing this Trp residue are important to the mechanism of transhydrogenase: movement of dI.1 affects the width of a cleft which, in turn, regulates the positioning of bound nucleotides ready for hydride transfer . The unique protein core in dI may be a paradigm for the design of compact and stable de novo proteins.

J Biol Chem, 2003 Dec 5, 278(49), 49261 - 70 Epub 2003 Sep 11.
The link module from ovulation- and inflammation-associated protein TSG-6 changes conformation on hyaluronan binding; Blundell CD et al.; The solution structure of the Link module from human TSG-6, a hyaladherin with important roles in inflammation and ovulation, has been determined in both its free and hyaluronan-bound conformations . This reveals a well defined hyaluronan-binding groove on one face of the Link module that is closed in the absence of ligand . The groove is lined with amino acids that have been implicated in mediating the interaction with hyaluronan, including two tyrosine residues that appear to form essential intermolecular hydrogen bonds and two basic residues capable of supporting ionic interactions . This is the first structure of a non-enzymic hyaladherin in its active state, and identifies a ligand-induced conformational change that is likely to be conserved across the Link module superfamily . NMR and isothermal titration calorimetry experiments with defined oligosaccharides have allowed us to infer the minimum length of hyaluronan that can be accommodated within the binding site and its polarity in the groove; these data have been used to generate a model of the complex formed between the Link module and a hyaluronan octasaccharide.

J Mol Biol, 2003 Sep 26, 332(4), 851 - 60
A comparison of directed evolution approaches using the beta-glucuronidase model system; Rowe LA et al.; Protein engineers can alter the properties of enzymes by directing their evolution in vitro . Many methods to generate molecular diversity and to identify improved clones have been developed, but experimental evolution remains as much an art as a science . We previously used DNA shuffling (sexual recombination) and a histochemical screen to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with improved beta-galactosidase (BGAL) activity . Here, we employ the same model evolutionary system to test the efficiencies of several other techniques: recursive random mutagenesis (asexual), combinatorial cassette mutagenesis (high-frequency recombination) and a versatile high-throughput microplate screen . GUS variants with altered specificity evolved in each trial, but different combinations of mutagenesis and screening techniques effected the fixation of different beneficial mutations . The new microplate screen identified a broader set of mutations than the previously employed X-gal colony screen . Recursive random mutagenesis produced essentially asexual populations, within which beneficial mutations drove each other into extinction (clonal interference); DNA shuffling and combinatorial cassette mutagenesis led instead to the accumulation of beneficial mutations within a single allele . These results explain why recombinational approaches generally increase the efficiency of laboratory evolution.

Blood Cells Mol Dis, 2003 Sep-Oct, 31(2), 183 - 6
Unsuccessful chimeraplast strategy for the correction of a mutation causing Gaucher disease; Diaz-Font A et al.; A number of gene therapy approaches have been developed for the treatment of genetic diseases, most of them based on the use of viral vectors . However, in general, they have not been successful and some complications, such as immune reactions induced by adenoviral vectors or random integration of retroviral vectors, have been reported frequently . To overcome these limitations, novel strategies have recently emerged . One of them is chimeraplasty, based on the correction of single-base mutations by mismatch repair mechanisms using chimeric RNA/DNA oligonucleotides, named chimeraplasts . Several papers have reported the use of this method to correct a number of pathological mutations underlying different diseases . In Gaucher disease, the most prevalent spingoliposis, mutation c.1448C->T (L444P), is one of the most common mutations in many populations . We have attempted to correct mutation c.1448C->T in fibroblasts from a Gaucher disease patient using a chimeraplast approach . Although we have shown that the chimeraplast reaches the fibroblast nucleus by colocalization with nuclear structures, no genomic correction was detected . To evaluate whether fibroblast and hepatocyte extracts are able to effect correction in vitro, we followed a cell-free extract assay using Escherichia coli cells . Our results show a very low efficiency (if any) of chimeraplast correction . A growing number of negative results for chimeraplast experiments have recently been reported . This promising technique has turned out to be inconsistent and impossible to replicate in most laboratories and many of the first successful results are now being questioned . Our negative data are consistent with this criticism.

Chem Res Toxicol, 2003 Sep, 16(9), 1118 - 23
Oxidative damage to a supercoiled DNA by water soluble peroxyl radicals characterized with DNA repair enzymes; Sanchez C et al.; Earlier work (Paul, T., et al . (2000) Biochemistry 39, 4129-4135) has demonstrated that the water soluble positively charged peroxyl radical, (H(2)N)(2)(+)CC(CH(3))(2)OO(*) ((+)AOO(*)), caused direct strand scission of the Escherichia coli plasmid supercoiled DNA, pBR 322, with ca . 50% scission occurring at a (+)AOO(*)/base pair (bp) ratio of 0.2 . There was no measurable direct scission with a negatively charged peroxyl ((-)BOO(*)) at (-)BOO(*)/bp = 24, nor with a neutral peroxyl (COO(*)) at COO(*)/bp = 5 . Base modification (BM) of the same DNA by the same peroxyls has now been investigated using four base excision repair (BER) glycosylases . At (+)AOO(*)/bp = 0.04, there is 10% direct strand scission, and the Fpg protein recognized an additional 25% BM, while endonuclease (Endo) IV recognized an additional 20% BM and the other two BER enzymes did not give statistically significant BMs . None of the BER enzymes showed BMs in the DNA treated with -BOO(*) . However, Fpg and Endo IV showed that at COO(*)/bp = 3.4 there was a BM comparable to that observed at (+)AOO(*)/bp = 0.04 . Thus, COO(*) radicals are only ca . 1.2% as reactive toward the DNA's bases as (+)AOO(*) . These results underline the importance of Coulombic forces in DNA reactions . It is also proposed that (+)AOO(*) has a higher intrinsic reactivity in H-atom abstractions and electron transfer processes than (-)BOO(*) or COO(*) radicals.

Nat Struct Biol, 2003 Oct, 10(10), 794 - 9 Epub 2003 Sep 14.
Crystal structure of the nickel-responsive transcription factor NikR; Schreiter ER et al.; NikR is a metal-responsive transcription factor that controls nickel uptake in Escherichia coli by regulating expression of a nickel-specific ATP-binding cassette (ABC) transporter . We have determined the first two structures of NikR: the full-length apo repressor at a resolution of 2.3 A and the nickel-bound C-terminal regulatory domain at a resolution of 1.4 A . NikR is the only known metal-responsive member of the ribbon-helix-helix family of transcription factors, and its structure has a quaternary arrangement consisting of two dimeric DNA-binding domains separated by a tetrameric regulatory domain that binds nickel . The position of the C-terminal regulatory domain enforces a large spacing between the contacts that each NikR DNA-binding domain can make with the nik operator . The regulatory domain of NikR contains four nickel-binding sites at the tetramer interface, each exhibiting a novel square-planar coordination by three histidines and one cysteine side chain.

Science, 2003 Sep 12, 301(5639), 1515 - 9
Observation of polymer conformation hysteresis in extensional flow; Schroeder CM et al.; Highly extensible Escherichia coli DNA molecules in planar extensional flow were visualized in dilute solution by fluorescence microscopy . For a narrow range of flow strengths, the molecules were found in either a coiled or highly extended conformation, depending on the deformation history of the polymer . This conformation hysteresis persists for many polymer relaxation times and is due to conformation-dependent hydrodynamic forces . Polymer conformational free-energy landscapes were calculated from computer simulations and show two free-energy minima for flow strengths near the coil-stretch transition . Hysteresis cycles may directly influence bulk-solution stresses and the development of stress-strain relations for dilute polymer flows.

Plant Physiol, 2003 Sep, 133(1), 368 - 78
Insect attack and wounding induce traumatic resin duct development and gene expression of (-)-pinene synthase in Sitka spruce; McKay SA et al.; Conifers possess inducible terpenoid defense systems . These systems are associated with the formation of traumatic resin ducts (TRD) and are underpinned by enhanced gene expression and activity of terpene synthases (TPS), enzymes responsible for oleoresin formation . We first determined that Sitka spruce (Picea sitchensis {Bong.} Carriere) had the capacity for TRD formation by mechanically wounding representative trees . We then proceeded to investigate whether the white pine weevil (Pissodes strobi Peck.), a stem-boring insect, can influence the expression of genes encoding monoterpene synthases (mono-tps) in Sitka spruce . We went on to compare this response with the effects of a simulated insect attack by drill wounding . A significant increase in mono-tps transcript level was observed in the leaders of lateral branches of weevil-attacked and mechanically wounded trees . In this study, weevils induced a more rapid enhancement of mono-tps gene expression . A full-length Sitka spruce mono-tps cDNA (PsTPS2) was isolated, expressed in Escherichia coli, and functionally identified as (-)-pinene synthase . The recombinant (-)-pinene synthase catalyzes the formation of (-)-alpha-pinene and (-)-beta-pinene, both of which are known constituents of stem oleoresin in Sitka spruce and increase in abundance after weevil attack . These data suggest that increased (-)-pinene synthase gene expression is an important element of the direct defense system deployed in Sitka spruce after insect attack.

J Virol, 2003 Oct, 77(19), 10706 - 13
Simian virus 40 T antigens and J domains: analysis of Hsp40 cochaperone functions in Escherichia coli; Genevaux P et al.; The N-terminal exon of DNA tumor virus T antigens represents a J domain that can direct interaction with the host-encoded Hsp70 chaperones . We have taken advantage of rapid Hsp40 cochaperone assays with Escherichia coli to assess simian virus 40 (SV40)-encoded J-domain loss of function . We found a strong correlation between loss of cochaperone function in E . coli and defective SV40 growth, suggesting that the major role of the J domain in DNA tumor viruses is to provide cochaperone function . We also report the expression of native SV40 virus T antigens in E . coli . Our results show that small t antigen, but not large T antigen (LT) or LT truncation TN125 or TN136, can functionally replace under limited growth conditions DnaJ (Hsp40) function in vivo . In addition, purified small t antigen can efficiently stimulate E . coli DnaK's (Hsp70) ATPase in vitro, thus behaving like a bona fide cochaperone . Furthermore, small t amino acids 83 to 174, which are adjacent to the viral J domain, can replace the E . coli DnaJ J-domain glycine-phenylalanine-rich domain, immediately adjacent to the J-domain sequences, even in the absence of significant amino acid similarity to their DnaJ counterpart . Taken together, our studies demonstrate that functionally related Hsp40 proteins from mammalian viral systems can be rapidly studied in bacteria and exploited to probe the universally conserved Hsp70 chaperone machine mechanism.

J Pharmacol Exp Ther, 2003 Nov, 307(2), 737 - 44 Epub 2003 Sep 11.
Calf intestinal alkaline phosphatase, a novel therapeutic drug for lipopolysaccharide (LPS)-mediated diseases, attenuates LPS toxicity in mice and piglets; Beumer C et al.; It has been demonstrated that human placental alkaline phosphatase (HPLAP) attenuates the lipopolysaccharide (LPS)-mediated inflammatory response, likely through dephosphorylation of the lipid A moiety of LPS . In this study, it is demonstrated that also alkaline phosphatase derived from calf intestine (CIAP) is able to detoxify LPS . In mice administered CIAP, 80% of the animals survived a lethal Escherichia coli infection . In piglets, previous to LPS detoxification, the pharmacokinetic behavior of CIAP was studied . CIAP clearance was shown to be dose-independent and showed a biphasic pattern with an initial t1/2 of 3 to 5 min and a second phase t1/2 of 2 to 3 h . Although CIAP is cleared much faster than HPLAP, it attenuates LPS-mediated effects on hematology and tumor necrosis factor-alpha responses at doses up to 10 microg/kg in piglets . LPS-induced hematological changes were antagonized, and the tumor necrosis factor-alpha response was reduced up to 98% . Daily i.v . bolus administration of 4000 units CIAP, the highest dose used in the LPS intervention studies, in piglets for 28 days was tolerated without any sign of toxicity . Therefore, CIAP potentially encompasses a novel therapeutic agent in the treatment of LPS-mediated diseases . Based on the data mentioned above, human clinical trials have been initiated.

J Biol Chem, 2003 Nov 14, 278(46), 45476 - 84 Epub 2003 Sep 10.
Reconstitution of R6K DNA replication in vitro using 22 purified proteins; Abhyankar MM et al.; We have reconstituted a multiprotein system consisting of 22 purified proteins that catalyzed the initiation of replication specifically at ori gamma of R6K, elongation of the forks, and their termination at specific replication terminators . The initiation was strictly dependent on the plasmid-encoded initiator protein pi and on the host-encoded initiator DnaA . The wild type pi was almost inert, whereas a mutant form containing 3 amino acid substitutions that tended to monomerize the protein was effective in initiating replication . The replication in vitro was primed by DnaG primase, whereas in a crude extract system that had not been fractionated, it was dependent on RNA polymerase . The DNA-bending protein IHF was needed for optimal replication and its substitution by HU, unlike in the oriC system, was less effective in promoting optimal replication . In contrast, wild type pi-mediated replication in vivo requires IHF . Using a template that contained ori gamma flanked by two asymmetrically placed Ter sites in the blocking orientation, replication proceeded in the Cairns type mode and generated the expected types of termination products . A majority of the molecules progressed counterclockwise from the ori, in the same direction that has been observed in vivo . Many features of replication in the reconstituted system appeared to mimic those of in vivo replication . The system developed here is an important milestone in continuing biochemical analysis of this interesting replicon.

J Biol Chem, 2003 Nov 21, 278(47), 47171 - 80 Epub 2003 Sep 11.
The three-dimensional structures of two beta-agarases; Allouch J et al.; Agars are important gelifying agents for biochemical use and the food industry . To cleave the beta-1,4-linkages between beta-d-galactose and alpha-l-3,6-anhydro-galactose residues in the red algal galactans known as agars, marine bacteria produce polysaccharide hydrolases called beta-agarases . Beta-agarases A and B from Zobellia galactanivorans Dsij have recently been biochemically characterized . Here we report the first crystal structure of these two beta-agarases . The two proteins were overproduced in Escherichia coli and crystallized, and the crystal structures were determined at 1.48 and 2.3 A for beta-agarases A and B, respectively . The structure of beta-agarase A was solved by the multiple anomalous diffraction method, whereas beta-agarase B was solved with molecular replacement using beta-agarase A as model . Their structures adopt a jelly roll fold with a deep active site channel harboring the catalytic machinery, namely the nucleophilic residues Glu-147 and Glu-184 and the acid/base residues Glu-152 and Glu-189 for beta-agarases A and B, respectively . The structures of the agarases were compared with those of two lichenases and of a kappa-carrageenase, which all belong to family 16 of the glycoside hydrolases in order to pinpoint the residues responsible for their widely differing substrate specificity . The relationship between structure and enzymatic activity of the two beta-agarases from Z . galactanivorans Dsij was studied by analysis of the degradation products starting with different oligosaccharides . The combination of the structural and biochemical results allowed the determination of the number of subsites present in the catalytic cleft of the beta-agarases.

J Biol Chem, 2003 Nov 28, 278(48), 47905 - 14 Epub 2003 Sep 11.
Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana; Poppenberger B et al.; Plant pathogenic fungi of the genus Fusarium cause agriculturally important diseases of small grain cereals and maize . Trichothecenes are a class of mycotoxins produced by different Fusarium species that inhibit eukaryotic protein biosynthesis and presumably interfere with the expression of genes induced during the defense response of the plants . One of its members, deoxynivalenol, most likely acts as a virulence factor during fungal pathogenesis and frequently accumulates in grain to levels posing a threat to human and animal health . We report the isolation and characterization of a gene from Arabidopsis thaliana encoding a UDP-glycosyltransferase that is able to detoxify deoxynivalenol . The enzyme, previously assigned the identifier UGT73C5, catalyzes the transfer of glucose from UDP-glucose to the hydroxyl group at carbon 3 of deoxynivalenol . Using a wheat germ extract-coupled transcription/translation system we have shown that this enzymatic reaction inactivates the mycotoxin . This deoxynivalenol-glucosyltransferase (DOGT1) was also found to detoxify the acetylated derivative 15-acetyl-deoxynivalenol, whereas no protective activity was observed against the structurally similar nivalenol . Expression of the glucosyltransferase is developmentally regulated and induced by deoxynivalenol as well as salicylic acid, ethylene, and jasmonic acid . Constitutive overexpression in Arabidopsis leads to enhanced tolerance against deoxynivalenol.

EMBO J, 2003 Sep 15, 22(18), 4846 - 55
VIVID is a flavoprotein and serves as a fungal blue light photoreceptor for photoadaptation; Schwerdtfeger C et al.; Blue light regulates many physiological and developmental processes in fungi . Most of the blue light responses in the ascomycete Neurospora crassa are dependent on the two blue light regulatory proteins White Collar (WC)-1 and -2 . WC-1 has recently been shown to be the first fungal blue light photoreceptor . In the present study, we characterize the Neurospora protein VIVID . VIVID shows a partial sequence similarity with plant blue light photoreceptors . In addition, we found that VIVID non-covalently binds a flavin chromophore . Upon illumination with blue light, VIVID undergoes a photocycle indicative of the formation of a flavin-cysteinyl adduct . VVD is localized in the cytoplasm and is only present after light induction . A loss-of-function vvd mutant was insensitive to increases in light intensities . Furthermore, mutational analysis of the photoactive cysteine indicated that the formation of a flavin-cysteinyl adduct is essential for VIVID functions in vivo . Our results show that VIVID is a second fungal blue light photoreceptor which enables Neurospora to perceive and respond to daily changes in light intensity.

EMBO J, 2003 Sep 15, 22(18), 4770 - 8
Programmed translational -1 frameshifting on hexanucleotide motifs and the wobble properties of tRNAs; Licznar P et al.; Programmed -1 ribosomal frameshifting, involving tRNA re-pairing from an AAG codon to an AAA codon, has been reported to occur at the sequences CGA AAG and CAA AAG . In this study, using the recoding region of insertion sequence IS3, we have investigated the influence on frameshifting in Escherichia coli of the first codon of this type of motif by changing it to all other NNA codons . Two classes of NNA codons were distinguished, depending on whether they favor or limit frameshifting . Their degree of shiftiness is correlated with wobble propensity, and base 34 modification, of their decoding tRNAs . A more flexible anticodon loop very likely makes the tRNAs with extended wobble more prone to liberate the third codon base, A, for re-pairing of tRNALys in the -1 frame.

EMBO J, 2003 Sep 15, 22(18), 4719 - 27
Transcription through the roadblocks: the role of RNA polymerase cooperation; Epshtein V et al.; During transcription, cellular RNA polymerases (RNAP) have to deal with numerous potential roadblocks imposed by various DNA binding proteins . Many such proteins partially or completely interrupt a single round of RNA chain elongation in vitro . Here we demonstrate that Escherichia coli RNAP can effectively read through the site-specific DNA-binding proteins in vitro and in vivo if more than one RNAP molecule is allowed to initiate from the same promoter . The anti-roadblock activity of the trailing RNAP does not require transcript cleavage activity but relies on forward translocation of roadblocked complexes . These results support a cooperation model of transcription whereby RNAP molecules behave as 'partners' helping one another to traverse intrinsic and extrinsic obstacles.

EMBO J, 2003 Sep 15, 22(18), 4625 - 33
Crystal structure of the heterodimeric complex of LXRalpha and RXRbeta ligand-binding domains in a fully agonistic conformation; Svensson S et al.; The nuclear receptor heterodimers of liver X receptor (LXR) and retinoid X receptor (RXR) are key transcriptional regulators of genes involved in lipid homeostasis and inflammation . We report the crystal structure of the ligand-binding domains (LBDs) of LXRalpha and RXRbeta complexed to the synthetic LXR agonist T-0901317 and the RXR agonist methoprene acid (Protein Data Base entry 1UHL) . Both LBDs are in agonist conformation with GRIP-1 peptides bound at the coactivator binding sites . T-0901317 occupies the center of the LXR ligand-binding pocket and its hydroxyl head group interacts with H421 and W443, residues identified by mutational analysis as critical for ligand-induced transcriptional activation by T-0901317 and various endogenous oxysterols . The topography of the pocket suggests a common anchoring of these oxysterols via their 22-, 24- or 27-hydroxyl group to H421 and W443 . Polyunsaturated fatty acids act as LXR antagonists and an E267A mutation was found to enhance their transcriptional inhibition . The present structure provides a powerful tool for the design of novel modulators that can be used to characterize further the physiological functions of the LXR-RXR heterodimer.

Eur J Pharmacol, 2003 Aug 29, 476(3), 257 - 8
The PPAR-gamma ligand 15-deoxy(delta12,14) prostaglandin J2 reduces the liver injury in endotoxic shock; Collin M et al.; We demonstrate here for the first time that the endogenous cyclopentenone prostaglandin 15-deoxy-Delta12,14-prostaglandin J2 (15d-prostaglandin J2) reduces the liver injury (rise in serum transaminases) caused by severe endotoxaemia (6 mg/kg Escherichia coli endotoxin i.v . for 6 h) in the anaesthetised rat . The protection of the liver afforded by this potent agonist of PPAR-gamma was not secondary to a haemodynamic effect . Thus, 15d-prostaglandin J2 and other PPAR-gamma agonists may be useful in the therapy of septic shock.

J Immunol Methods, 2003 Aug, 279(1-2), 219 - 32
Di-diabody: a novel tetravalent bispecific antibody molecule by design; Lu D et al.; The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods . In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules . These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens . Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH(3) domain of an IgG . The novel BsAb, which we termed "di-diabody", represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH(3) domains . A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography . Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors . The di-diabody retained good antigen-binding activity after incubation at 37 degrees C in mouse serum for 72 h, demonstrating good product stability . Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity . This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.

J Immunol Methods, 2003 Aug, 279(1-2), 33 - 40
A solid-phase method for evaluation of gold conjugate used in quantitative detection of antigen by immunogold-labeling electron microscopy; Kaur R et al.; Rapid and sensitive screening for confirming the reactivity of reagents, before proceeding for electron microscopy, is highly desirable . ELISA-based methods have been shown to be highly efficient and successful for rapid prescreening and optimization of immunological as well as sample-processing reagents for the sensitive detection and quantitation of antigen by electron microscopy . The drawback of these methods lies in their inability to provide any information regarding the gold conjugate used for the final observed and measured signal . In this work, we demonstrate a simple and rapid, solid-phase method in ELISA format that is also suitable for evaluation and optimization of the gold conjugate . We have demonstrated the utility of this technique by screening for Vitreoscilla hemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold-labeling transmission electron microscopy (TEM) of cell sections . The sensitivity of detection and quantitation of antigens by immuno-electron microscopy depends upon the assay procedure being optimized to obtain the best possible signal . Our study indicates that evaluation of gold conjugate by the solid-phase assay could help in the rapid optimization of this reagent for immunogold localization and quantification of antigens by TEM.

Plant J, 2003 Sep, 35(6), 693 - 703
The biosynthesis of the branched-chain sugar d-apiose in plants: functional cloning and characterization of a UDP-d-apiose/UDP-d-xylose synthase from Arabidopsis; Molhoj M et al.; d-Apiose is a plant-specific branched-chain monosaccharide found in rhamnogalacturonan II (RG-II), apiogalacturonan, and several apioglycosides . Within RG-II, d-apiose serves as the binding site for borate, which leads to the formation of cross-links within the wall . Biochemical studies in duckweed and parsley have established that uridine 5'-diphospho-d-apiose (UDP-d-apiose) is formed from UDP-d-glucuronate by decarboxylation and re-arrangement of the carbon skeleton, leading to ring contraction and branch formation . The enzyme catalyzing this reaction also forms UDP-d-xylose by decarboxylation of UDP-d-glucuronate, and has therefore been named UDP-d-apiose/UDP-d-xylose synthase . Using a bioinformatics approach, we identified a candidate gene (AXS1) for this enzyme in Arabidopsis and functionally expressed its cDNA in Escherichia coli . The recombinant enzyme catalyzed the conversion of UDP-d-glucuronate to a mixture of UDP-d-apiose and UDP-d-xylose with a turnover number of 0.3 min-1 . AXS1 required NAD+ for enzymatic activity, and was strongly inhibited by UDP-d-galacturonate . It was highly expressed in all plant organs consistent with a function in synthesizing an essential cell wall precursor . Database searches indicated the presence of closely related sequences in a variety of crop plants . The cloning of the AXS1 gene will help to investigate the biosynthesis of RG-II, and permit insights into the mechanism by which d-apiose and other branched monosaccharides are formed.

J Neuroendocrinol, 2003 Oct, 15(10), 940 - 5
Histamine and prostaglandin interaction in regulation of oxytocin and vasopressin secretion; Knigge U et al.; Prostaglandins and histamine in the hypothalamus are involved in the regulation of oxytocin and vasopressin secretion, and appear to be involved in the mediation of pituitary hormone responses to immunochallenges . Therefore, we investigated in conscious male rats: (i) whether blockade of H1 or H2 receptors affected the oxytocin and vasopressin responses to prostaglandins and (ii) whether blockade of prostaglandin synthesis affected the oxytocin and vasopressin responses to histamine or to Escherichia coli lipopolysaccharide (LPS), in order to determine any interaction between prostaglandins and histamine in the hypothalamus . Oxytocin secretion was dose-dependently stimulated by intracerebroventricular infusion of 1 or 5 microg of PGE1, PGE2 or PGF2alpha, with PGE2 being the most potent of the compounds used . Prior central infusion of the H1 receptor antagonist mepyramine or the H2 receptor antagonist cimetidine significantly inhibited the oxytocin response to all three prostaglandins by approximately 50% . Vasopressin secretion was increased by PGE1 but not by PGE2 or PGF2alpha . The stimulatory effect of PGE1 was almost annihilated by prior administration of mepyramine or cimetidine . Central infusion of histamine or immunochallenge with LPS administered intraperitoneally increased oxytocin and vasopressin secretion four- and two-fold, respectively . Pretreatment with systemic injection of the prostaglandin synthesis inhibitor indomethacin dose-dependently reduced the oxytocin response and prevented the vasopressin response to histamine or LPS . We conclude that histamine and PGE1, PGE2 or PGF2alpha interact in the regulation of oxytocin secretion, whereas histamine and only PGE1 interact in the regulation of vasopressin secretion . Furthermore, histamine as well as LPS may affect oxytocin and vasopressin neurones via activation of prostaglandins, probably in the hypothalamic supraoptic nucleus.

Acta Anaesthesiol Scand, 2003 Oct, 47(9), 1125 - 31
Regionally differentiated fibrinolytic responses during volume-resuscitated acute endotoxemia in pigs; Nyberg A et al.; BACKGROUND: Microcirculatory dysfunction and formation of microthrombi are common in sepsis as a result of a procoagulant state . A profibrinolytic change has however, been reported in early sepsis . This study investigates systemic and regional (pulmonary, preportal, hepatic, renal) fibrinolytic capacity as mirrored by fluxes of tissue-type plasminogen activator (t-PA) in response to acute endotoxemia and volume resuscitation . METHODS: Eight anaesthetized, ventilated pigs (24-29 kg) were instrumented for systemic and regional haemodynamic monitoring . Aortic, pulmonary arterial, portal, hepatic and renal venous blood samples were collected . Following baseline stabilisation, Escherichia coli endotoxin was infused for 120 min . During the last 30 min of infusion, volume resuscitation was initiated targeting baseline cardiac output, and animals were observed for 3 h . Total tPA was analyzed by ELISA calibrated for porcine tPA . Net organ tPA fluxes were calculated based on in/outflowing plasma concentrations and regional blood flows . RESULTS: Preportal release and hepatic extraction of tPA was observed at baseline . Pulmonary and renal net fluxes of tPA were not significantly different from zero . Endotoxemia increased plasma tPA levels in all investigated vascular beds . Preportal tPA release increased approximately 10-fold and hepatic extraction increased approximately 12-fold in non-resuscitated acute endotoxemia . No significant changes in pulmonary or renal tPA fluxes were observed . Volume resuscitation restored net fluxes to baseline values while plasma levels remained elevated approximately twofold compared with baseline . CONCLUSION: Acute endotoxemia induces a prompt, marked and regionally differentiated pro-fibrinolytic response that cannot be discerned based on systemic levels of circulating tPA and that was normalized during volume resuscitation.

J Anim Sci, 2003 Sep, 81(9), 2322 - 32
Effects of dexamethasone on lymphoid tissue in the gut and thymus of neonatal calves fed with colostrum or milk replacer; Norrman J et al.; An increased susceptibility to disease in neonatal calves may be attributable to high glucocorticoid levels that influence immune reactions . We tested whether dexamethasone (DEXA) administration influences the proliferation, apoptosis, and number of B- and T-lymphocytes in Peyer's patches (PP) and thymus in calves fed colostrum (C) or a milk-derived formula . All calves were subcutaneously administered bovine colostrum-derived immunoglobulin G and fed chicken-egg derived immunoglobulins that protected against rotavirus and pathogenic Escherichia coli . The DEXA (30 microg/kg of BW daily) was injected for 4 d into groups fed colostrum on the first 3 d (CD+) and those fed the formula that contained nutrients in amounts as in colostrum but no immunoglubulin G (FD+) . Groups CD- and FD were fed the same as the other two groups, but did not receive DEXA . Immunohistochemical methods were used to evaluate cell proliferation rates (by labeling of 5-bromo-2'-deoxyuridine), apoptosis rates (by terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling) . Numbers of T- and B-lymphocytes were determined with antibodies specific for CD3 and CD79 surface proteins . There were significant effects (P < 0.05) of DEXA treatment (decrease of cell proliferation rates in follicles of PP and thymus, increase of apoptotic rate in follicles of PP and thymus, decrease of B-lymphocyte numbers in follicles of PP, increase of B-lymphocyte numbers in domes of PP, increase of T-lymphocyte numbers in follicles of PP, and a decrease of intraepithelial T-lymphocyte numbers) . There were significant effects (P < 0.05) of C feeding (decrease of cell proliferation rates in follicles of PP and of B-lymphocyte numbers in interfollicular areas, domes, and follicular-associated epithelium of PP, and an increase of cell proliferation rate in the thymus) . A DEXA x feeding interaction (P < 0.001) was found for cell proliferation rate in the thymus . In conclusion, DEXA treatment decreased cell proliferation rates in follicles of PP and thymus and enhanced apoptotic rates in follicles of PP . Colostrum feeding decreased cell proliferation rates, likely of B-lymphocytes, in follicles of PP and numbers of B-lymphocytes in domes, follicular-associated epithelium, and interfollicular areas of PP and enhanced cell proliferation rates and selectively modified DEXA effects in the thymus.

Org Biomol Chem, 2003 Aug 21, 1(16), 2848 - 52
Selenenyl iodide: a new substrate for mammalian thioredoxin reductase; Mugesh G et al.; Areneselenenyl iodide stabilised by internal chelation has been synthesized and evaluated as a substrate of thioredoxin reductase (TrxR) . The reactivity of TrxR obtained from human placenta towards selenenyl iodide was found to be much higher than that of the E . coli enzyme, indicating the essential nature of a selenocysteine residue in the active site of the human enzyme . The addition of thioredoxin (Trx) significantly enhanced the TrxR-catalysed reduction of selenenyl iodide 1 . These studies on the reduction of a selenenyl iodide by the thioredoxin system suggest that stable selenenyl iodides could be new substrates for human TrxR . The Trx system could act as a cofactor for iodothyronine deiodinase by reducing the selenenyl iodide intermediate in the second-half of the deiodinase catalytic cycle to regenerate the active site . The TrxR-catalysed reduction of 1 was not inhibited by the anti-thyroid drug, PTU, suggesting that the involvement of the Trx system in the deiodinase cycle may be responsible for the insensitivity of certain deiodinases towards clinically useful thiourea drugs.

Biotechnol Bioeng, 2003 Nov 5, 84(3), 314 - 23
Tracking the acetate threshold using DO-transient control during medium and high cell density cultivation of recombinant Escherichia coli in complex media; Johnston WA et al.; DO-transient nutrient controllers use the dissolved oxygen signal to attempt acetate threshold tracking during fed-batch cultivation of recombinant E . coli . Here we apply DO-transient control to the production of Jembrana disease virus protein in complex Super Luria medium and compare performance against a high-limit pH-stat controller . For induction at medium cell density (harvest between 31 and 32.5 g dcw L) a total productivity of 0.27 g L h was achieved as compared to 0.24 g L h with the high-limit pH-stat . For induction at high cell density (harvest at 60 g dcw L), decreased productivity (0.12 g L h) was attributed to the effect of acetate accumulation on recombinant protein formation and a concomitant lowering of the critical growth rate . Our results suggest that complex media provides a difficult environment for the application of acetate threshold tracking DO-transient control because of difficulties in re-oxidizing acetate, and apparent localized production of acetate below the production threshold (as detected by the DO-transient controller as SPOUR(crit)) . Configuring the DO-transient controller to avoid aggressive threshold probing is suggested as a means to improve performance and reduce acetate accumulation in complex media .

Protein Eng, 2003 Aug, 16(8), 607 - 14
The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase; Gonzalez-Pacanowska D et al.; We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198 . Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity . To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199 . All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity . For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis . Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP) . Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS . Several Pro196 and Pro197 variants were temperature sensitive . The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.

J Biol Chem, 2003 Dec 5, 278(49), 49102 - 12 Epub 2003 Sep 10.
Biochemical diversity among the 1-amino-cyclopropane-1-carboxylate synthase isozymes encoded by the Arabidopsis gene family; Yamagami T et al.; 1-Amino-cyclopropane-1-carboxylate synthase (ACS, EC 4.4.1.14) is the key enzyme in the ethylene biosynthetic pathway in plants . The completion of the Arabidopsis genome sequence revealed the presence of twelve putative ACS genes, ACS1-12, dispersed among five chromosomes . ACS1-5 have been previously characterized . However, ACS1 is enzymatically inactive whereas ACS3 is a pseudogene . Complementation analysis with the Escherichia coli aminotransferase mutant DL39 shows that ACS10 and 12 encode aminotransferases . The remaining eight genes are authentic ACS genes and together with ACS1 constitute the Arabidopsis ACS gene family . All genes, except ACS3, are transcriptionally active and differentially expressed during Arabidopsis growth and development . IAA induces all ACS genes, except ACS7 and ACS9; CHX enhances the expression of all functional ACS genes . The ACS genes were expressed in E . coli, purified to homogeneity by affinity chromatography, and biochemically characterized . The quality of the recombinant proteins was verified by N-terminal amino acid sequence and MALDI-TOF mass spectrometry . The analysis shows that all ACS isozymes function as dimers and have an optimum pH, ranging between 7.3 and 8.2 . Their Km values for AdoMet range from 8.3 to 45 microm, whereas their kcat values vary from 0.19 to 4.82 s-1 per monomer . Their Ki values for AVG and sinefungin vary from 0.019 to 0.80 microm and 0.15 to 12 microm, respectively . The results indicate that the Arabidopsis ACS isozymes are biochemically distinct . It is proposed that biochemically diverse ACS isozymes function in unique cellular environments for the biosynthesis of C2H4, permitting the signaling molecule to exert its unique effects in a tissue- or cell-specific fashion.

Br J Pharmacol, 2003 Sep, 140(1), 115 - 22 Epub 2003 Jul 29.
Role of cannabinoid CB1 receptors and tumor necrosis factor-alpha in the gut and systemic anti-inflammatory activity of SR 141716 (rimonabant) in rodents; Croci T et al.; (1) We investigated the effect of the cannabinoid CB1 receptor antagonist, SR 141716, on indomethacin-induced small intestine inflammation and Escherichia coli lipopolysaccharide (LPS)-induced plasma TNF-alpha (TNF) release in comparison to the cannabinoid CB2 receptor antagonist, SR 144528, in rodents . (2) In rats, indomethacin induced significant ulcer formation in the small intestine; this was accompanied by an increase in tissue TNF levels and myeloperoxidase (MPO) activity . SR 141716 prevented the ulcers and the rise in TNF levels (ID50 3.3, 0.4 mg kg-1, respectively) and MPO activity . SR 144528 prevented intestinal ulcers only . (3) The effect of SR 141716 against indomethacin-induced ulcers and increase of plasma TNF levels after LPS was also studied in wild-type and CB1 receptor knockout mice . Indomethacin induced intestinal ulcers in mice, but not tissue TNF production and MPO activity . SR 141716 reduced the ulcers to a similar extent in wild-type and CB1 receptor knockout mice . In rats and wild-type mice, but not in CB1 receptor knockout mice, SR 141716 inhibited the LPS-induced increase in plasma TNF levels . (4) These findings provide evidence that the indomethacin model of intestinal lesions differs in rat and mouse and support the existence of several mechanisms for the antiulcer activity of SR141716, the most important involving the inhibition of TNF production . The potent anti-inflammatory activity of SR141716 in rodents indicated its potential therapeutic interest in chronic immune-inflammatory diseases.

Br J Pharmacol, 2003 Sep, 140(1), 91 - 6 Epub 2003 Aug 04.
Dexamethasone improves vascular hyporeactivity induced by LPS in vivo by modulating ATP-sensitive potassium channels activity; d'Emmanuele di Villa Bianca R et al.; (1) Septic shock represents an important risk factor for patients critically ill . This pathology has been largely demonstrated to be a result of a myriad of events . Glucocorticoids represent the main pharmacological therapy used in this pathology . (2) Previously we showed that ATP-sensitive potassium (KATP) channels are involved in delayed vascular hyporeactivity in rats (24 h after Escherichia coli lipopolysaccharide (LPS) injection) . In LPS-treated rats, we observed a significant hyporeactivity to phenylephrine (PE) that was reverted by glybenclamide (GLB), and a significant increase in cromakalim (CRK)-induced hypotension . (3) We evaluated the effect of dexamethasone (DEX 8 mg kg-1 i.p.) whether on hyporeactivity to PE or on hyperreactivity to CRK administration, in vivo, in a model of LPS (8 x 106 U kg-1 i.p.)-induced endotoxemia in urethane-anaesthetised rats . (4) DEX treatment significantly reduced, in a time-dependent manner, the increased hypotensive effect induced by CRK in LPS-treated rats . This effect was significantly (P<0.05) reverted by the glucocorticoid receptor antagonist RU38486 (6.6 mg kg-1 i.p.) . (5) GLB-induced hypertension (40 mg kg-1 i.p.), in LPS-treated rats, was significantly inhibited by DEX if administered at the same time of LPS . (6) Simultaneous administration of DEX and LPS to rats completely abolished the hyporeactivity to PE observed after 24 h from LPS injection . (7) In conclusion, our results suggest that the beneficial effect of DEX in endotoxemia could be ascribed, at least in part, to its ability to interfere with KATP channel activation induced by LPS . This interaction may explain the improvement of vascular reactivity to PE, mediated by DEX, in LPS-treated rats, highlighting a new pharmacological activity to the well-known anti-inflammatory properties of glucocorticoids.

Mol Biochem Parasitol, 2003 Sep, 131(1), 35 - 44
Trypanosoma cruzi poly-zinc finger protein: a novel DNA/RNA-binding CCHC-zinc finger protein; Espinosa JM et al.; A poly-zinc finger protein, designated PZFP1 was identified in Trypanosoma cruzi for the first time . The protein has 191 amino acids, contains seven motifs Cys(X)(2)Cys(X)(4)His(X)(4)Cys . A recombinant PZFP1 was generated in E . coli and the expected 21kDa polypeptide co-purified with two other inducible products of about 42 and 63kDa . Western blot analysis of cell extracts using an anti-PZFP1 antibody recognized a major band of 41kDa . Electrophoretic mobility shift analysis demonstrated that both, recombinant and native PZFP1, specifically interact with single-stranded DNA or RNA oligonucleotides carrying recognition sequences of other CCHC proteins . The protein was localized mainly in the cytoplasm and nucleus as observed by indirect immunofluorescence analysis . PZFP1 interacted specifically with a T . cruzi serine-arginine-rich protein (TcSR) in a yeast two-hybrid assay, suggesting a role in pre-mRNA processing.

Mol Biochem Parasitol, 2003 Sep, 131(1), 25 - 33
Properties of trypanothione synthetase from Trypanosoma brucei; Oza SL et al.; Trypanothione {N(1),N(8)-bis(glutathionyl)spermidine} plays a central role in defence against oxidant damage, ribonucleotide metabolism and in resistance to certain drugs in trypanosomatids . In Crithidia fasciculata, synthesis of trypanothione involves sequential conjugation of two molecules of glutathione (GSH) to spermidine by two enzymes: glutathionylspermidine synthetase (GspS; EC 6.3.1.8) and trypanothione synthetase (TryS; EC 6.3.1.9), whereas in Trypanosoma cruzi both steps are catalysed by an unusual TryS with broad substrate specificity . To determine which route operates in T . brucei, we have cloned and expressed a single copy gene with similarity to C . fasciculata and T . cruzi TRYS . The purified recombinant protein catalyses formation of trypanothione from either spermidine and GSH, or glutathionylspermidine and GSH . The enzyme displays high substrate inhibition with GSH as variable substrate (apparent K(m)=56 microM, K(i)(s)=37 microM, k(cat)=2.9s(-1)) . At a fixed subsaturating GSH concentration (100 microM), the enzyme obeys simple hyperbolic kinetics yielding apparent K(m) values for spermidine, glutathionylspermidine and MgATP of 38, 2.4, and 7.1 microM, respectively . Recombinant TryS can also catalyse conversion of spermine to glutathionylspermine and bis(glutathionyl)spermine, as recently reported for T . cruzi . The enzyme has amidase activity that can be inhibited by iodoacetamide . Studies using GSH and polyamine analogues identified GSH as the critical determinant for recognition by the amidase domain . Thus, the biosynthesis and degradation of trypanothione are similar in African and American trypanosomes, and different from the insect trypanosomatid, C . fasciculata.

Mol Phylogenet Evol, 2003 Oct, 29(1), 31 - 42
Phylogenetic analyses among octocorals (Cnidaria): mitochondrial and nuclear DNA sequences (lsu-rRNA, 16S and ssu-rRNA, 18S) support two convergent clades of branching gorgonians; Armando Sanchez J et al.; Gorgonian octocorals lack corroborated hypotheses of phylogeny . This study reconstructs genealogical relationships among some octocoral species based on published DNA sequences from the large ribosomal subunit of the mitochondrial RNA (lsu-rRNA, 16S: 524bp and 21 species) and the small subunit of the nuclear RNA (ssu-rRNA, 18S: 1815bp and 13 spp) using information from insertions-deletions (INDELS) and the predicted secondary structure of the lsu-rRNA (16S) . There were seven short (3-10bp) INDELS in the 18S with consistent phylogenetic information . The INDELS in the 16S corresponded to informative signature sequences homologous to the G13 helix found in Escherichia coli . We found two main groups of gorgonian octocorals using a maximum parsimony analysis of the two genes . One group corresponds to deep-water taxa including species from the suborders Calcaxonia and Scleraxonia characterized by an enlargement of the G13 helix . The second group has species from Alcyoniina, Holaxonia and again Scleraxonia characterized by insertions in the 18S . Gorgonian corals, branching colonies with a gorgonin-containing flexible multilayered axis (Holaxonia and Calcaxonia), do not form a monophyletic group . These corroborated results from maternally inherited (16S) and biparentally inherited (18S) genes support a hypothesis of independent evolution of branching in the two octocoral clades.

Mol Gen Mikrobiol Virusol, 2003, (3), 33 - 8
{Construction of vector plasmids for the Plectonema boryanum cyanobacterium and creation, on such basis, of the vector-host system}; Samoilenko VA et al.; The plasmid composition of the Plectonema boryanum Gom . Cyanobacterium, strain CALU 465, was analyzed . A small pSM1 plasmid, size 1.5, was chosen for genetic engineering . The physical map, constructed for the plasmid, was used to create the pSTS series, i.e . vector construction for the P . boryanum cyanobacterium . A method was selected and tested for the introduction of the vector into host cells . The needed orientation of pSM1 cyanobacterium plasmid within the vector as well as a possible role of its separate elements in inheriting the construction by host were defined.

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