FEBS Lett, 1970 Jan 26, 6(2), 73 - 76 L-serine deaminating enzymes in Escherichia coli crude extracts; Alfoldi L et al.; (a) The measured L-serine deaminating activity of a crude bacterial extract may originate from L-serine deaminase, from biosynthetic L-threonine deaminase, or from degradative L-serine deaminase . Nevertheless, the contribution of the individual enzymes can be determined.(b) About a half of the L-serine deaminating activity of wild type E . coli bacteria, grown in synthetic minimal medium, originates from L-serine deaminase and about half from biosynthetic L-threonine deaminase.(c) Ninety percent of L-serine deaminating activity of wild type E . coli bacteria, grown in yeast extract-tryptone medium, originates from L-serine deaminase, and the remainging ten percent from the degradative L-threonine deaminase.(d) Conditions have been established in which threonine deaminases are eliminated and the activity of L-serine deaminase alone could be measured, even in crude extracts. FEBS Lett, 1970 Jan 15, 6(1), 5 - 8 Polysome-ribosome distribution in isogenic RC(str) and RC(rel) strains of Escherichia coli; Matzura H; Study of the relative proportions of ribosomes and polysomes released by a standardized lysing procedure from isogenic RC(str) and RC(rel) strains of Escherichia coli shows that a 20-min period of amino acid starvation of RC(str) bacteria reduces the fraction of ribosomes recovered in polysomes to about 60% of its value characteristic of exponentially growing cells: A similar starvation treatment of the RC(rel) bacteria causes no appreciable reduction in the fraction of polysomal ribosomes. FEBS Lett, 1969 Aug, 4(4), 347 - 350 Nau F, Petrissant G, Dubert JM. This paper describes an attempt to find a difference between the patterns of methylation of E . coli tRNA by extracts of two mouse tissues . Two samples of tRNAs, methylated in two separate experiments with extracts of myeloma and of liver in presence of either 14C or 3H S-Adenosyl-L-Methionine, were pooled and fractionated together on a RPC column . The results show a difference in the specificities of the two extracts . Chromatography on DEAE Sephadex suggests that the tRNA Met is methylated by the enzymes on the myeloma, while enzymes from liver react very little, if at all, with that particular tRNA species . Studies have been undertaken in order to find out whether similar differences can also be demonstrated in homologous systems. FEBS Lett, 1971 Dec 15, 19(3), 247 - 250 Schmitt B, Kolb H, Cohen R. We have applied successfully the active enzyme analytical centrifugation method (A.E.C.) to the study of the well known pyruvate dehydrogenase complex (P.D.C.) in crude extracts from E . coli . It is not possible to study the complex in a spectrophotometer (through its dehydrogenase activity) due to NADH oxidase activities . The A.E.C . method, which overcomes this difficulty, can also be viewed as a refined spectrophotometric method. FEBS Lett, 1971 Mar 5, 13(3), 137 - 139 Marcot J, Azoulay E. Three double mutants were prepared from single mutant chl-r strains of Escherichia coli K12 lacking nitrate reductase activity . When extracts of these three double mutants were mixed, nitrate reductase activity was reconstituted. FEBS Lett, 1970 Dec 18, 11(5), 312 - 315 Formation of initiation complex with lambdamRNA in vitro; Becarevic A et al.; The product from the in vitro transcription of native lambdaDNA by purified E . coli RNA polymerase does not stimulate total amino acid incorporation although it can program ribosomes for the first peptide bond synthesis in the presence of the three initiation factor A, B and C. FEBS Lett, 1970 Sep 18, 10(1), 25 - 28 Mitochondrial peptide chain elongation factors from Neurospora crassa; Grandi M et al.; Two complementary peptide chain elongation factors (G and T) have been isolated from a mitochondrial 100,000 g supernatant . Both factors are specific for 70 S ribosomes and can be crossed with T and G factors from E . coli. FEBS Lett, 1968 Jul, 1(1), 35 - 37 Evidence for more than one pathway in the formation of purine deoxyribonucleotides; Foung A et al.; The biosynthesis of purine deoxyribonucleotides was studied in the context of general purine nucleotide metabolism in the chick with the aid of radioactive nucleic acid precursors . Our results showed that in chick liver and intestine, the nucleoside phosphate reductase system so firmly established in E . coli {1} and L . leichmanni {2} is not exclusively responsible for the biosynthesis of purine deoxyribonucleotides. Mol Ther, 2002 Apr, 5(4), 360 - 70 Correct integration mediated by integrase-LexA fusion proteins incorporated into HIV-1; Holmes-Son ML et al.; Fusion of wild-type or truncated integrase to a sequence-specific DNA-binding protein, such as the Escherichia coli LexA repressor, results in an integration bias toward the recognition site of the DNA-binding protein in vitro . Integrase-defective HIV-1 could become integration-competent by supplying the fusion protein in trans . To understand the mechanism of complementation, the virus-host DNA junctions of cells infected with the integrase-LexA containing virus were sequenced . The characteristic hallmarks of wild-type integration were present, a 5'-TG/CA-3' at the ends of the viral sequence and a 5-bp direct repeat in the immediately flanking cellular DNA . Experiments were also carried out to determine the mechanism by which the amino- or carboxy-terminal truncated integrase fused to LexA restored integration to the integrase-mutant viral clone . Complementation experiments using purified fusion proteins in vitro, or viruses encoding a C-terminal truncated integrase and containing various fusion proteins in trans, indicated that the truncated integrase-LexA proteins are inactive per se and they restore integration by forming mixed multimers with the virally encoded mutant integrase . Correct integration of retroviral DNA by the in trans method illustrates the feasibility of introducing integrase fusion proteins into retroviral vectors to achieve site-directed integration without interfering with the attributes of the integration reaction. Gen Comp Endocrinol, 2002 Mar, 126(1), 75 - 89 Production of recombinant goldfish prolactin and its applications in radioreceptor binding assay and radioimmunoassay; Wong AO et al.; Goldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli . Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein . This fusion protein was purified, refolded, and (125)I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin . Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors . To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the (125)I-labeled recombinant prolactin as a tracer was established . This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II) . This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish . In this case, the Ca(2+) ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells . In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells . These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca(2+) influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish. Gen Comp Endocrinol, 2002 Mar, 126(1), 52 - 8 Comparison of R128Q mutations in human, ovine, and chicken leptins; Raver N et al.; Human leptin and its R128Q mutant, as well as the R128Q mutants of ovine and chicken leptins, were prepared, expressed in Escherichia coli, refolded, and purified to homogeneity yielding electrophoretically pure, over 95% monomeric protein . R128Q mutations did not change the binding properties to BAF/3 cells stably transfected with the long form of human leptin receptor compared, respectively, to non-mutated human, ovine, and chicken leptins . In contrast, the biological activity tested in a proliferation assay in the same cells was drastically changed . Human leptin R128Q lost its activity and even became a weak antagonist, whereas the activities of ovine and chicken leptins were reduced 25- and 80-fold . If dimerization models were applicable leptin receptor activation, the present results would suggest that site 2 of the hormone was impaired . Two models, the human growth hormone:human growth hormone receptor (hGH:hGHR) (1:2) and the granulocyte-colony stimulating factor:granulocyte-colony stimulating factor receptor (GCSF:GCSFR) (2:2) complexes, were used for modeling . Superimposing the leptin structure on the hGH and GCSF models in the complex structures did not indicate any role for R128 in receptor binding . This made it impossible to correlate the results shown in the present work with the currently available models . Therefore, leptin may bind its receptors in a manner different than those proposed until now. Biochem Biophys Res Commun, 2002 Apr 12, 292(4), 1036 - 43 Discovery of a stable dimeric mutant of cyanovirin-N (CV-N) from a T7 phage-displayed CV-N mutant library; Han Z et al.; Mutant proteins with altered properties can be useful probes for investigating structure, ligand binding sites, mechanisms of action, and physicochemical attributes of the corresponding wild-type proteins of interest . In this report, we illuminate properties of mutants of the potent HIV-inactivating protein, cyanovirin-N (CV-N), selected by construction of a mutant library by error-prone polymerase chain reaction and affinity-based screening using T7 phage display technology . After three rounds of biopanning, two phage-displayed, one-point mutants of CV-N, Ser52Pro and Ala77Thr, were isolated . After the elucidation of biological activities of the mutants displayed on phage as well as the Escherichia coli-expressed, purified mutant proteins, we subsequently subjected the mutants to analyses by native PAGE and size-exclusion chromatography . We found that the Ser52Pro mutant not only was active against HIV but also existed exclusively as a dimer in solution . This was in marked contrast to the wild-type CV-N, which exists in solution predominantly as the monomer . The Ser52Pro mutant provides a novel model for further investigations of the folding mechanism as well as structure-activity requirements for CV-N's antiviral properties. Biochem Biophys Res Commun, 2002 Apr 12, 292(4), 922 - 30 Mechanism of specific recognition of the aidB promoter by sigma(S)-RNA polymerase; Lacour S et al.; Transcription of the Escherichia coli aidB gene is controlled by an Esigma(S)-dependent promoter (PaidB) and is poorly transcribed by the Esigma(70) form of RNA polymerase in the absence of additional factors . In this report, we investigate the interaction between PaidB and either the Esigma(70) or the Esigma(S) forms of RNA polymerase in vitro . We show that although Esigma(70) can bind the aidB promoter, its interaction with the promoter results in the formation of an open complex inefficient in transcription initiation and sensitive to heparin challenge . Deletion of the C residue at position -13 of PaidB (Delta-13C) slightly impaired transcription initiation by Esigma(S), consistent with the role of -13C as a specific feature of Esigma(S)-dependent promoters . However, Esigma(S) could still bind and initiate transcription from the Delta-13C mutant aidB promoter more efficiently than Esigma(70), suggesting that sequence elements other than the -13C play an important role in specific promoter recognition by Esigma(S) . (c)2002 Elsevier Science (USA). Biochem Biophys Res Commun, 2002 Apr 12, 292(4), 874 - 9 An infrared study of the thermal and pH stabilities of the GPI-alkaline phosphatase from bovine intestine; Bortolato M et al.; Alkaline phosphatase (EC 3.1.3.1) from bovine intestine mucosa (BIAP) is a homodimeric metalloenzyme, which hydrolyses nonspecifically phosphate monoesters at alkaline pH with release of inorganic phosphate and alcohol . BIAP is either soluble (sBIAP) or membrane-anchored by a glycosylphosphatidylinositol moiety (GPI-BIAP) . This anchor might have some contribution in the stabilization of the GPI-linked protein structure . Our purpose was to study the role of the anchor by using two parameters, the enzymatic activity and the protein conformation, which was analyzed by using FTIR spectroscopy . We determined that the two forms of BIAP show some similarities with the previously described structure of alkaline phosphatase isolated from Escherichia coli and human placenta . Meanwhile GPI-BIAP and sBIAP exhibit similar specific activities, the presence of the anchor increases the thermal and pH stabilities of the enzyme activity and conformation . (c)2002 Elsevier Science (USA). Vet Res, 2002 Mar-Apr, 33(2), 223 - 8 Escherichia coli heat-stable enterotoxin b (STb) in vivo internalization within rat intestinal epithelial cells; Labrie V et al.; Heat-stable enterotoxin b (STb) is a low molecular weight toxin known to bind sulfatide, its receptor . The fate of STb bound to rat intestinal epithelium cells was followed using an anti-toxin gold labeled assay and transmission electron microscopy . The data suggest that STb toxin and the fusion protein maltose binding protein (MBP)-STb were internalized whereas its mutant I41 E-M42R with reduced hydrophobicity did not show internalization . There was a significant difference in the mean of gold particles per field between rat intestine incubated with STb or the fusion protein MBP-STb and the negative control consisting of intestine incubated with PBS alone . No subcellular compartment seems to be particularly aimed by the toxin as gold particles were randomly distributed within the cell. Pol J Vet Sci, 2002, 5(1), 17 - 24 Hormonal profile and morphological changes in pig ovaries after intraovarian infusions of Escherichia coli endotoxin; Kucharski J et al.; The purpose of this study was to estimate morphological changes in the ovary and size of the production of steroid hormones during the luteal phase of the estrous cycle in pigs after intraovarian infusions of Escherichia coli endotoxin . Polish Large White gilts (n = 15) of similar age (7-8 months) and body weight (90-110 kg) with two controlled subsequent estrous cycles were used . The animals were randomly divided into two groups: control (n = 9, the 10th day of the estrous cycle,) and treated with Escherichia coli endotoxin (n = 6, the same day of the estrous cycle) . The gilts were infused with Escherichia coli endotoxin at a dose of 1 mg three times a day during six consecutive days, from the 14th to the 19th day of the estrous cycle . Plasma concentrations of progesterone (P4), androstenedione (A4), testosterone (T), estrone (E1) and estradiol-17 beta (E2) were determined by radioimmunoassay method . Infusions of Escherichia coli endotoxin resulted in a significant (p < 0.001) decrease in the production of P4, A4, T, E1 and E2 in the luteal phase as compared with the levels found in the control animals . Plasma level of P4, A4 and T was decreased by 84.6%, 86.0% and 73.0%, respectively . Plasma concentrations of E1 and E2 in some cases exceeded 5 pg/ml, nevertheless in the majority of the samples they were under sensitivity of the method . Escherichia coli endotoxin infusions resulted in a considerable decrease in the size of the ovaries, and morphological changes characteristic for acute and chronic inflammation were observed. Int J Vitam Nutr Res, 2002 Mar, 72(2), 85 - 9 Study on the interaction of ions of transient metals with ascorbic acid in the presence of different scavengers of active oxygen species in SOS chromotest; Marczewska J et al.; SOS chromotest was employed to study the interaction of ascorbic acid with free ions of transient metals in the presence of added catalase, superoxide dismutase or D-mannitol . Catalase diminished the genotoxic activity of the mixture of ascorbic acid with copper ions in E . coli strains PQ37 and PQ 300, but genotoxicity of this mixture was not suppressed by superoxide dismutase and D-mannitol . The results suggest that copper ions diminished the content of peroxide generated by ascorbic acid. Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5710 - 5 Epub 2002 Apr 09. NMDA receptor function is regulated by the inhibitory scaffolding protein, RACK1; Yaka R et al.; Phosphorylation regulates the function of ligand-gated ion channels such as the N-methyl d-aspartate (NMDA) receptor . Here we report a mechanism for modulation of the phosphorylation state and function of the NMDA receptor via an inhibitory scaffolding protein, RACK1 . We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein tyrosine kinase, Fyn . RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices . Peptides that disrupt the interactions between RACK1, NR2B, and Fyn induce phosphorylation and potentiate NMDA receptor-mediated currents . Therefore, RACK1 is a regulator of NMDA receptor function and may play a role in synaptic plasticity, addiction, learning, and memory. J Biol Chem, 2002 Jun 14, 277(24), 21604 - 9 Epub 2002 Apr 09. Stabilization of the biotinoyl domain of Escherichia coli acetyl-CoA carboxylase by interactions between the attached biotin and the protruding "thumb" structure; Solbiati J et al.; We previously reported (Chapman-Smith, A., Forbes, B . E., Wallace, J . C., and Cronan, J . E., Jr . (1997) J . Biol . Chem . 272, 26017-26022) that the biotinylated (holo) species of the biotin carboxyl carrier protein (BCCP) biotinoyl domain is much more resistant to chemical modification and proteolysis than the unbiotinylated (apo) form . We hypothesized that the increased stability was due to a conformational change engendered by interaction of the domain with biotin protein ligase, the enzyme that attaches the biotin moiety . We now report that a BCCP-87 species to which the biotin moiety was attached by chemical acylation rather than by biotin protein ligase showed the characteristically greater stability of the holo biotinoyl domain . This result demonstrates that our hypothesis was incorrect; the attached biotin is solely responsible for the increased stability . The bacterial and chloroplast multisubunit acetyl-CoA carboxylases are unusual in that the highly symmetrical and conserved structure of the biotinoyl domain of the BCCP subunit is disrupted by a structured loop called the "thumb" that protrudes from body of the domain . Prior structural work showed that the thumb interacts with uriedo ring of the attached biotin moiety . We have tested whether the thumb-biotin interactions are responsible for the greater holo form stability by examination of two BCCP-87 species that lack the thumb . These BCCP species were produced in both the apo and holo forms, and their sensitivities to trypsin digestion were compared . The holo forms of these proteins were found to be only marginally more stable than their apo forms and much more sensitive to trypsin digestion than the wild type holo-BCCP-87 . Therefore, removal of the thumb has an effect similar to lack of biotinylation, indicating that thumb-biotin interactions are responsible for most (but not all) of the increased stability of the holo biotinoyl domain . In the course of these experiments we demonstrated that treatment of Escherichia coli with the peptide deformylase inhibitor, actinonin, results in the expected (but previously unreported) accumulation of an N-formylated protein species. J Biol Chem, 2002 Jun 14, 277(24), 21246 - 53 Epub 2002 Apr 09. The gibberellin 20-oxidase of Gibberella fujikuroi is a multifunctional monooxygenase; Tudzynski B et al.; The genes for gibberellin (GA) biosynthesis are clustered in the fungus Gibberella fujikuroi . In addition to genes encoding a GA-specific geranylgeranyl diphosphate synthase and a bifunctional ent-copalyl diphosphate/ent-kaurene synthase, the cluster contains four cytochrome P450 monooxygenase genes (P450-1, -2, -3, -4) . Recently it was shown that P450-4 and P450-1 encode multifunctional enzymes catalyzing the three oxidation steps from ent-kaurene to ent-kaurenoic acid and the four oxidation steps from ent-kaurenoic acid to GA14, respectively . Here we describe the functional analysis of the P450-2 gene by gene disruption and by expressing the gene in a mutant that lacks the entire GA biosynthesis gene cluster . Mutants in which P450-2 is inactivated by the insertion of a large piece of DNA accumulated GA14 and lacked biosynthetically more advanced metabolites, indicating that the gene encodes a 20-oxidase . This was confirmed by incubating lines containing P450-2 in the absence of the other GA biosynthesis genes with isotopically labeled substrates . The P450-2 gene product oxidized the 3beta-hydroxylated intermediate, GA14, and its non-hydroxylated analogue GA12 to GA4 and GA9, respectively . Expression of P450-2 is repressed by high amounts of nitrogen in the culture medium but is not affected by the presence of biosynthetically advanced GAs, i.e . there is no evidence for feedback regulation . The fact that the GA 20-oxidase is a cytochrome P450 monooxygenase in G . fujikuroi and not a 2-oxoglutarate-dependent dioxygenase as in plants, together with the significant differences in regulation of gene expression, are further evidence for independent evolution of the GA biosynthetic pathways in plants and fungi. J Biol Chem, 2002 Jun 14, 277(24), 21561 - 6 Epub 2002 Apr 09. Association of Fyn and Lyn with the proline-rich domain of glycoprotein VI regulates intracellular signaling; Suzuki-Inoue K et al.; The glycoprotein VI (GPVI)-Fc receptor (FcR) gamma-chain complex, a key activatory receptor for collagen on platelet surface membranes, is constitutively associated with the Src family kinases Fyn and Lyn . Molecular cloning of GPVI has revealed the presence of a proline-rich domain in the sequence of GPVI cytoplasmic tail which has the consensus for interaction with the Src homology 3 (SH3) domains of Fyn and Lyn . A series of in vitro experiments demonstrated the ability of the SH3 domains of both Src kinases to bind the proline-rich domain of GPVI . Furthermore, depletion of the proline-rich domain in GPVI (Pro(-)-GPVI) prevented binding of Fyn and Lyn and markedly reduced phosphorylation of FcR gamma-chain in transiently transfected COS-7 cells, but did not affect the association of the gamma-chain with GPVI . Jurkat cells stably transfected with wild type GPVI show robust increases in tyrosine phosphorylation and intracellular Ca2+ in response to the snake venom convulxin that targets GPVI . Importantly, convulxin is not able to activate cells transfected with Pro(-)-GPVI, even though the association with the immunoreceptor tyrosine-based activation motif-containing chains is maintained . These findings demonstrate that the proline-rich domain of GPVI mediates the association with Fyn/Lyn via their SH3 domain and that this interaction initiates activation signals through GPVI. Mutat Res, 2002 Apr 26, 516(1-2), 81 - 9 The influence of the solvents DMSO and ethanol on the genotoxicity of alpha,beta-unsaturated aldehydes in the SOS chromotest; Eder E et al.; alpha,beta-Unsaturated aldehydes are ubiquitous environmental pollutants, important industrial chemicals, have mani-fold biological functions in plants and insects and are natural products in food . They are endogenously formed in animals and humans during lipid peroxidation and arachidonic acid oxidation and are genotoxic, mutagenic and carcinogenic . Crotonaldehyde and 2-hexenal in food may contribute to general carcinogenicity in humans . The high bacterial toxicity of these compounds leads to problems in genotoxicity testing in bacterial systems . Recently, we have shown that using ethanol as solvent instead of dimethylsulfoxide (DMSO) results in an increase in the induction factors and the SOS-inducing potency of alpha,beta-unsaturated ketones in the SOS chromotest . Here, we demonstrate that utilization of ethanol as solvent also improves the testing of alpha,beta-unsaturated aldehydes . Five aldehydes out of nine tested were clearly positive in the SOS chromotest according to the criteria of Quillardet, i.e . acrolein, crotonaldehyde, 2,4-hexadienal, 2-methylacrolein and 2-ethylacrolein, three further, 2-hexenal, 2-heptenal and 2-propylacrolein showed a dose dependent increase of the induction factors which was however lower than 1.5 times that of the background . Only 2-butylacrolein did not lead to an increase in the induction factors . With DMSO as solvent only the three aldehydes acrolein, crotonaldehyde and 2,4-hexadienal showed an increase in the induction factor, which was however lower than 1.5 that of the background . Utilization of ethanol allows to establish structure genotoxicity relationships for alpha,beta-unsaturated aldehydes in the SOS chromotest . Genotoxicity decreases with increasing degree of substitution . The decreasing genotoxicities can be explained (a) by increasing bacterial toxicity due to increasing lipophilicities of the higher substituted aldehydes and (b) by decreasing reactivity due to steric hindrance by the alkyl substituents. J Control Release, 2002 Apr 23, 80(1-3), 345 - 56 Enhanced expression of plasmid DNA-cationized gelatin complex by ultrasound in murine muscle; Aoyama T et al.; This study is an investigation to experimentally confirm that ultrasound (US) irradiation is effective in enhancing the gene expression of plasmid DNA . A cationized gelatin was prepared by introducing primary amino groups into gelatin . The plasmid of the LacZ gene was complexed with the cationized gelatin and injected into the femoral muscle of normal mice . Following US irradiation at the injected site of muscle, the gene expression of the treated muscle was evaluated to compare with that of the complex injection plus no US irradiation . US irradiation enabled the complex to significantly enhance the gene expression at the injected muscle, in contrast to naked plasmid DNA, although the extent depended on the experimental conditions, such as the injection dose of plasmid DNA, and the time period or timing of US irradiation . The gene-expressed area at the muscle was not changed by the time interval between the complex injection and US irradiation . Fluorescent microscopic observation revealed that the complex was homogeneously distributed in the muscle tissue around the injected site by US irradiation without any dissociation of plasmid DNA . The fluorescent image of plasmid DNA superposed on that of myosin heavy chain indicated intracellular localization of plasmid DNA . We demonstrated that US irradiation is a promising technique to enhance gene expression even in the normal muscle. J Control Release, 2002 Apr 23, 80(1-3), 333 - 43 Controlled release of plasmid DNA from cationized gelatin hydrogels based on hydrogel degradation; Fukunaka Y et al.; This paper shows achievement of the in vivo controlled release of a plasmid DNA from a biodegradable hydrogel and the consequent regulation of gene expression period . Cationization of gelatin was preformed through introduction of ethylenediamine and the gelatin prepared was crosslinked by various concentrations of glutaraldehyde to obtain cationized gelatin (CG) hydrogels as the carrier of plasmid DNA . In vivo release of plasmid DNA from the CG hydrogels was compared with the in vivo degradation of hydrogels . When CG hydrogels incorporating 125I-labeled plasmid DNA were implanted into the femoral muscle of mice, the plasmid DNA radioactivity remaining decreased with time and the retention period prolonged with a decrease in the water content of hydrogels used . The higher the water content of 125I-labeled CG hydrogels, the faster the hydrogel radioactivity remaining decreased with time . The time profile of plasmid DNA remaining in the hydrogels was in good accordance with that of hydrogel radioactivity, irrespective of the water content . Intramuscular implantation of plasmid DNA-incorporated CG hydrogels enhanced significantly expression of the plasmid DNA around the implanted site . The retention period of gene expression became longer as the hydrogel water content decreased . Fluorescent microscopic study revealed that the plasmid DNA-CG complex was detected around the hydrogel implanted even after 7-day implantation in marked contrast to the injection of plasmid DNA solution . It was concluded that in our hydrogel system, active plasmid DNA was released accompanied with the in vivo degradation of hydrogel, resulting in extended gene expression . The time profile of plasmid DNA release and the consequent gene expression was controllable by changing the water content of hydrogels. J Control Release, 2002 Apr 23, 80(1-3), 273 - 82 Biodegradable poly(ethylenimine) for plasmid DNA delivery; Ahn CH et al.; Poly(ethylenimine) (PEI) has been known as an efficient gene carrier with the highest cationic charge potential . High transfection efficiency of PEI, along with its cytotoxicity, strongly depends on the molecular weight . Synthesis of cationic copolymers derived from the low molecular weight of PEI and hydrophilic poly(ethylene glycol) (PEG), which are water soluble and degradable under physiological conditions, was investigated for plasmid delivery . Hydrophilic PEG is expected to reduce the toxicity of the copolymer, improve the poor solubility of the PEI and DNA complexes, and help to introduce degradable bonds by reaction with the primary amines in the PEI . Considering the dependence of transfection efficiency and cytotoxicity on the molecular weight of the PEI, high transfection efficiency is expected from an increased molecular weight of the copolymer and low cytotoxicity from the introduction of PEG and the degradation of the copolymer into low molecular weight PEIs . Reaction conditions were carefully controlled to produce water soluble copolymers . Results from a gel retardation assay and zetapotentiometer indicated that complete neutralization of the complexes was achieved at the charge ratios of copolymer/pSV-beta-gal plasmid from 0.8 to 1.0 with the mean particle size of the polyplexes ranging from 129.8+/-0.9 to 151.8+/-3.4 nm . In vitro transfection efficiency of the synthesized copolymer increased up to three times higher than that of starting low molecular weight PEI, while the cell viability was maintained over 80%. Vet Immunol Immunopathol, 2002 May, 86(1-2), 55 - 77 Development of a rapid in vitro protein refolding assay which discriminates between peptide-bound and peptide-free forms of recombinant porcine major histocompatibility class I complex (SLA-I); Oleksiewicz MB et al.; The extracellular domains of swine leukocyte antigen class I (SLA-I, major histocompatibility complex protein class I) were cloned and sequenced for two haplotypes (H4 and H7) which do not share any alleles based on serological typing, and which are the most important in Danish farmed pigs . The extracellular domain of SLA-I was connected to porcine beta2 microglobulin by glycine-rich linkers . The engineered single-chain proteins, consisting of fused SLA-I and beta2 microglobulin, were overexpressed as inclusion bodies in Escherichia coli . Also, variants were made of the single-chain proteins, by linking them through glycine-rich linkers to peptides representing T-cell epitopes from classical swine fever virus (CSFV) and foot-and-mouth disease virus (FMDV) . An in vitro refold assay was developed, using a monoclonal anti-SLA antibody (PT85A) to gauge refolding . The single best-defined, SLA-I restricted porcine CD8(+) T-cell epitope currently known is a 9-residue peptide from the polyprotein of CSFV (J . Gen . Virol . 76 (1995) 3039) . Based on results with the CSFV epitope and two porcine haplotypes (H4 and H7), the in vitro refold assay appeared able to discriminate between peptide-free and peptide-occupied forms of SLA-I . It remains to be seen whether the rapid and technically very simple in vitro refold assay described here will prove generally applicable for the screening of virus-derived peptides for SLA-I binding. Vet Immunol Immunopathol, 2002 May, 86(1-2), 1 - 10 Regulation of adhesion molecules on circulating neutrophils during coliform mastitis and their possible immunomodulation with drugs; Diez-Fraile A et al.; Fast neutrophil diapedesis has been demonstrated to be critical in coliform mastitis and is determining for the severity of infection . Leukocyte adhesion molecules play a pivotal role in neutrophil recruitment . Two families of cell surface proteins help to regulate the adherence of neutrophils to vascular endothelium: selectins and beta(2)-integrins . Both classes of leukocyte adhesion molecules are reviewed in the context of their dynamic expression around parturition and during acute coliform mastitis . Their potential modulation by commonly used drugs and the therapeutic implications during acute coliform mastitis are also discussed. Vet Immunol Immunopathol, 2002 Mar, 85(3-4), 171 - 8 F4 receptor-independent priming of the systemic immune system of pigs by low oral doses of F4 fimbriae; Van den Broeck W et al.; Oral administration of F4 fimbriae of Escherichia coli induces intestinal mucosal immune responses in F4 receptor-positive (F4R(+)) pigs, but not in F4R(-) pigs . We examined whether F4 fimbriae in F4R(-) animals behave like a food antigen and can induce oral tolerance . Therefore, F4R(+) and F4R(-) pigs were fed 2mg of F4 and challenged i.m . to evaluate the effect of oral F4 on the systemic immune system . As control antigen, two different oral doses (2 and 600 mg) of OVA were used . Thirty days after the i.m . OVA challenge, the OVA-specific serum IgG titre in 600 mg-fed pigs was lower than that in non-fed animals, indicating that tolerance was induced . Conversely, in the 2mg-fed pigs a rapid increase of OVA-specific IgG with higher titres than those in non-fed pigs was seen following challenge, indicating a priming of the systemic immune system . A similar priming was seen in both F4-fed F4R(-) and F4R(+) pigs . Upon challenge, non-fed pigs displayed a primary immune response with a slow increase of F4-specific serum IgG, whereas F4-fed F4R(-) and F4R(+) pigs showed secondary responses with a rapid increase of serum IgG . This was expected in F4R(+) pigs, as in these animals oral F4 induces F4-specific antibody-secreting cells in the spleen, suggesting a priming of the systemic immune system . However, also the F4-fed F4R(-) pigs displayed a secondary response, despite the failure to detect a response upon oral F4 administration . These findings suggest that the F4 antigen, at a dose of 2 mg, behaves like a common food antigen in F4R(-) pigs, namely it induces a systemic priming. Vet Immunol Immunopathol, 2002 Mar, 85(3-4), 137 - 45 Increase in milk metalloproteinase activity and vascular permeability in bovine endotoxin-induced and naturally occurring Escherichia coli mastitis; Raulo SM et al.; An endotoxin-induced mastitis model was used to study permeability changes associated with increased milk matrix metalloproteinase (MMP) activity in early inflammation . One quarter of two cows was inoculated with endotoxin (Escherichia coli 055:B5) . Blood, milk, and whey were collected before and repeatedly after inoculation for 48 h . The profile and amounts of gelatinolytic MMPs were determined by zymography; gelatinase A (72 kD MMP-2) and gelatinase B (92 kD MMP-9) were identified by Western immunoblotting . Bovine serum albumin (BSA) and trypsin inhibitor capacity (TIC) were used as markers of capillary permeability with parallel examination of neutrophil penetration from blood to milk . Five clinical E . coli mastitis milk samples and five milk samples from cows with healthy udders were analyzed to detect whether increased levels of gelatinolytic MMP-2 and MMP-9 have a role in naturally occurring mastitis with endotoxin involvement . Milk MMP levels increased 2h after the endotoxin challenge . Both MMP-2 and MMP-9 were involved in this early proteolytic event . These increased MMP levels are associated with increased capillary permeability, evidenced first by the penetration of small molecular weight proteins, such as BSA and TIC . Later, 6-12h post endotoxin inoculation, neutrophilic leucocytes also entered the site, as they require larger tissue damage in basal membrane and interstitial tissue structures than BSA and TIC to extravasate . In naturally occurring disease, increased MMP-2 and MMP-9 levels were detected in milk . Thus, gelatinases, especially MMP-2, are involved in the early degradation of the blood-milk barrier, which precedes the penetration of blood-derived cellular components into milk in endotoxin-induced mastitis . In the future, measuring MMP in milk/whey might be a useful tool for diagnosing early mastitis. FEBS Lett, 2002 Mar 27, 515(1-3), 189 - 93 Self-assembly of ATP synthase subunit c rings; Arechaga I et al.; Subunit c of the H(+) transporting ATP synthase is an essential part of its membrane domain that participates in transmembrane proton conduction . The annular architecture of the subunit c from different species has been previously reported . However, little is known about the type of interactions that affect the formation of c-rings in the ATPase complex . Here we report that subunit c over-expressed in Escherichia coli and purified in non-ionic detergent solutions self-assembles into annular structures in the absence of other subunits of the complex . The results suggest that the ability of subunit c to form rings is determined by its primary structure. FEBS Lett, 2002 Mar 27, 515(1-3), 20 - 4 Arabidopsis glutathione-dependent formaldehyde dehydrogenase is an S-nitrosoglutathione reductase; Sakamoto A et al.; S-Nitrosoglutathione (GSNO), an adduct of nitric oxide (NO) with glutathione, is known as a biological NO reservoir . Heterologous expression in Escherichia coli of a cDNA encoding a glutathione-dependent formaldehyde dehydrogenase of Arabidopsis thaliana showed that the recombinant protein reduces GSNO . The identity of the cDNA was further confirmed by functional complementation of the hypersensitivity to GSNO of a yeast mutant with impaired GSNO metabolism . This is the first demonstration of a plant GSNO reductase, suggesting that plants possess the enzymatic pathway that modulates the bioactivity and toxicity of NO. FEBS Lett, 2002 Mar 13, 514(2-3), 343 - 6 Functional analysis of the Arabidopsis thaliana GCPE protein involved in plastid isoprenoid biosynthesis; Querol J et al.; Plastid isoprenoids are synthesized via the 2-C-methyl-D-erythritol 4-phosphate pathway . A few years after its discovery, most of the Escherichia coli genes involved in the pathway have been identified, including gcpE . In this work, we have identified an Arabidopsis thaliana protein with homology to the product of this gene . The plant polypeptide, GCPE, contains two structural domains that are absent in the E . coli protein: an N-terminal extension and a central domain of 30 kDa . We demonstrate that the N-terminal region targets the Arabidopsis protein to chloroplasts in vivo, consistent with its role in plastid isoprenoid biosynthesis . Although the presence of the internal extra domain may have an effect on activity, the Arabidopsis mature GCPE was able to complement a gcpE-defective E . coli strain, indicating the plant protein is a true functional homologue of the bacterial gcpE gene product. FEBS Lett, 2002 Mar 13, 514(2-3), 323 - 8 Mechanism of allosteric modulation of Escherichia coli carbamoyl phosphate synthetase probed by site-directed mutagenesis of ornithine site residues; Rochera L et al.; The role of residues of the ornithine activator site is probed by mutagenesis in Escherichia coli carbamoyl phosphate synthetase (CPS) . Mutations E783A, E783L, E892A and E892L abolish ornithine binding, E783D and T1042V decrease 2-3 orders of magnitude and E892D decreased 10-fold apparent affinity for ornithine . None of the mutations inactivates CPS . E783 mutations hamper carbamate phosphorylation and increase K(+) and MgATP requirements, possibly by perturbing the K(+)-loop near the carbamate phosphorylation site . Mutation E892A activates the enzyme similarly to ornithine, possibly by altering the position of K891 at the opening of the tunnel that delivers the carbamate to its phosphorylation site . T1042V also influences modulation by IMP and UMP, supporting signal transmission from the nucleotide effector to the ornithine site mediated by a hydrogen bond network involving T1042 . Ornithine activation of CPS may be mediated by K(+)-loop and tunnel gating changes. FEBS Lett, 2002 Mar 13, 514(2-3), 290 - 4 Expression of Fab fragment of catalytic antibody 6D9 in an Escherichia coli in vitro coupled transcription/translation system; Jiang X et al.; The heavy chain (Hc) and light chain (Lc) genes of the Fab fragment of a catalytic antibody 6D9 were simultaneously expressed in an Escherichia coli in vitro transcription/translation system without a reducing agent . The intermolecular disulfide bond between the Hc and Lc was found formed, suggesting a correct formation of the Fab fragment in the in vitro system . In enzyme-linked immunosorbent assay, the Fab fragment synthesized in vitro exhibited an antigen-binding activity . Addition of reduced glutathione, oxidized glutathione, protein disulfide-isomerase and molecular chaperones, GroEL and GroES, increased the solubility and the antigen-binding activity of the Fab fragment greatly . The in vitro synthesized Fab was purified by means of a hexa-histidine tag attached to the C-terminus of the Hc . Catalytic assay of the purified Fab fragment showed that the His-tagged Fab fragment synthesized in vitro had a catalytic activity comparable to that produced in vivo. FEBS Lett, 2002 Mar 13, 514(2-3), 275 - 80 Recombinant glucocorticoid induced tumor necrosis factor receptor (rGITR) induces NOS in murine macrophage; Shin HH et al.; Glucocorticoid induced tumor necrosis factor receptor (GITR) is a new member of the tumor necrosis factor-nerve growth factor receptor superfamily of which the function has not been well studied . The extracellular domain of GITR was produced in Escherichia coli and purified as a single band of predicted M(r) of 18.0 kDa . GITR and GITR ligand were expressed constitutively on the surface of Raw 264.7 macrophage cell line and murine peritoneal macrophages . An extracellular domain of GITR can activate murine macrophages to express inducible nitric oxide synthase and to generate nitric oxide in a dose- and time-dependent manner. FEBS Lett, 2002 Mar 13, 514(2-3), 255 - 9 The putative effector-binding site of Leishmania mexicana pyruvate kinase studied by site-directed mutagenesis; Hannaert V et al.; The activity of pyruvate kinase of Leishmania mexicana is allosterically regulated by fructose 2,6-bisphosphate (F-2,6-P(2)), contrary to the pyruvate kinases from other eukaryotes that are usually stimulated by fructose 1,6-bisphosphate (F-1,6-P(2)) . Based on the comparison of the three-dimensional structure of Saccharomyces cerevisiae pyruvate kinase crystallized with F-1,6-P(2) present at the effector site (R-state) and the L . mexicana enzyme crystallized in the T-state, two residues (Lys453 and His480) were proposed to bind the 2-phospho group of the effector . This hypothesis was tested by site-directed mutagenesis . The allosteric activation by F-2,6-P(2) appeared to be entirely abrogated in the mutated enzymes confirming our predictions. FEBS Lett, 2002 Mar 13, 514(2-3), 225 - 8 SufC hydrolyzes ATP and interacts with SufB from Thermotoga maritima; Rangachari K et al.; Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response . The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle . Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence . By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli . We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro. BMC Physiol . 2002 Apr 09;2(1):5. Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin; Krebs RA et al.; BACKGROUND: Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic gamma-proteobacteria . One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204 . These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR . It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR . RESULTS: A pR purification procedure is described that utilizes Phenylsepharose and hydroxylapatite columns and yields 85% (w/w) purity . Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence . Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant . At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct . Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent . CONCLUSIONS: The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e . that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly). BMC Biochem . 2002 Mar 27;3(1):5. The Caenorhabditis elegans Y87G2A.14 Nudix hydrolase is a peroxisomal coenzyme A diphosphatase; AbdelRaheim SR et al.; BACKGROUND: The number of Nudix hydrolase family members varies widely among different organisms . In order to understand the reasons for the particular spectrum possessed by a given organism, the substrate specificity and function of different family members must be established . RESULTS: The Y87G2A.14 Nudix hydrolase gene product of Caenorhabditis elegans has been expressed as a thioredoxin fusion protein in Escherichia coli and shown to be a CoA diphosphatase with catalytic activity towards CoA and its derivatives . The products of CoA hydrolysis were 3',5'-ADP and 4'-phosphopantetheine with Km and kcat values of 220 microM and 13.8 s(-1) respectively . CoA esters yielded 3',5'-ADP and the corresponding acyl-phosphopantetheine . Activity was optimal at pH 9.5 with 5 mM Mg2+ and fluoride was inhibitory with a Ki of 3 microM . The Y87G2A.14 gene product has a potential C-terminal tripeptide PTS1 peroxisomal targeting signal - SKI . By fusing a Y87G2A.14 cDNA to the C-terminus of yeast-enhanced green fluorescent protein, the enzyme appeared to be targeted to peroxisomes by the SKI signal when transfected into yeast cells . Deletion of SKI abolished specific targeting . CONCLUSIONS: The presence of related sequences with potential PTS1 or PTS2 peroxisomal targeting signals in other organisms suggests a conserved peroxisomal function for the CoA diphosphatase members of this group of Nudix hydrolases. Cell Mol Biol Lett, 2002, 7(1), 172 - 4 Alternating electric fields stimulate ATP synthesis in Escherichia coli; Zrimec A et al.; External alternating electric fields of low intensity stimulated membrane bound ATP synthesis in starving Escherichia coli cells with electric field amplitudes of 2.5-50 V/cm and a frequency optimum at 100 Hz . The model of electrocon-formational coupling was used to analyze the frequency and amplitude responses of ATP synthesis . Two relaxation frequencies of the system were obtained at 44 Hz and 220 Hz, and an estimate of roughly 12 was obtained as the effective charge displacement for the catalytic cycle of ATP synthesis. Cell Mol Biol Lett, 2002, 7(1), 129 - 31 The small world in biophysical systems structural properties of glycolysis and the TCA cycle in Escherichia coli; Krajnc B et al.; It has been shown that the central metabolic network of Escherichia coli is of the small-world type . In this paper, we present that the metabolic network of glycolysis and TCA cycle as a part of the E . coli metabolism is also a small-world network . We found that the hubs of the studied network are consistent with those found in the complete metabolic network . The evolutionary meaning of this finding is discussed. Nat Prod Lett, 2002 Feb, 16(1), 25 - 7 An in vitro screening method for DNA cytosine-C5-methylase inhibitor; Tamura T et al.; A specific inhibitor of DNA cytosine C5-methylases would be useful for studying genomic imprinting, X-chromosome inactivation, carcinogenesis, and regulation of tissue-specific gene expression, for these physiological phenomena appears to be regulated through DNA methylation in promoter sequences . This paper reports a novel convenient in vitro assay method for screening DNA cytosine C5-methylase inhibitor . Our method uses a commercially available Hae III methylase (cytosine C5 methylase), its corresponding Hae III endonuclease, and lambda DNA as their substrate. Ultramicroscopy, 2001 Feb, 90(2-3), 207 - 13 Dual-color 4Pi-confocal microscopy with 3D-resolution in the 100 nm range; Kano H et al.; We report the development of simultaneous two-color channel recording in 4Pi-confocal microscopy . A marked increase of spatial resolution over confocal microscopy becomes manifested in 4Pi-confocal three-dimensional (3D) data stacks of dual-labeled objects . The fundamentally improved resolution is verified both with densely labeled fluorescence beads as well as with membrane labeled fixed Escherichia coli . The synergistic combination of dual-color 4Pi-confocal recording with image restoration results in dual-color imaging with a 3D resolution in the 100 nm range. Cell Res, 2002 Mar, 12(1), 69 - 78 Antibodies against the C-terminal peptide of rabbit oviductin inhibit mouse early embryo development to pass 2-cell stage; Yong P et al.; A full-length rabbit oviductin cDNA(1909bp) was cloned . It consists of a 5'-UTR of 52bp, an open reading frame (ORF) of 1374bp and a 3'-UTR of 483bp and has more than 80% homology with that of other mammal oviductins . N-terminal peptide (NTP) (384 residues) and C-terminal peptide (CTP) (73 residues) of deduced protein precursor has about 80% and 50% identity with that of other mammals respectively . Fusion proteins GST-NTP 368(1R-368N)and GST-CTP73 (369F-441A) were expressed and purified . NH2-terminal of CTP sequencing reveals that the purified protein is consistent with the deduced one . In order to study the function of NTP and CTP the mouse anti-NTP and rabbit anti-CTP antisera were prepared . Tissue-specific (skeleton muscle, oviduct, uterus, ovary, liver, heart and brain) analysis indicated that rabbit oviductin was only found in oviduct . The conditioned medium derived from the rabbit oviduct mucosa epithelial cells has a function of overcoming the early embryonic development block of Kunming mouse cultured in vitro . Anti-CTP antiserum could totally inhibit the early embryo development at 2-cell stage cultured in the conditioned culture medium, but anti-NTP antiserum couldn't . There was a positive relationship between the ratio of early embryos at development block and the dosage of anti-CTP antiserum added in the conditioned culture medium . These results suggest that oviductin has a function not only on fertilization, but also on the release of early embryonic development block, and the later function domain of rabbit oviductin may be situate in its C-terminal. Planta, 2002 Apr, 214(6), 920 - 30 Epub 2001 Dec 15. Daylength and spatial expression of a gibberellin 20-oxidase isolated from hybrid aspen (Populus tremula L . x P . tremuloides Michx.); Eriksson ME et al.; Physiologically active gibberellins (GAs) are key regulators of shoot growth in trees . To investigate this mechanism of GA-controlled growth in hybrid aspen, we cloned cDNAs encoding gibberellin 20-oxidase (GA 20-oxidase), a key, highly regulated enzyme in the biosynthesis of GAs . Clones were isolated from leaf and cambium cDNA libraries using probes generated by polymerase chain reaction, based on conserved domains of GA 20-oxidases . Upon expression in Escherichia coli, the GST-fusion protein was shown to oxidise GA12 as well as oxidising the 13-hydroxylated substrate GA53, successively to GA9 and GA20, respectively . The gene PttGA20ox1 was expressed in meristematic cells and growing tissues such as expanding internodes, leaves and roots . The expression was negatively regulated by both GA4 and overexpression of phytochrome A . RNA analysis also showed that the expression was down-regulated in late-expanding leaf tissue in response to short days (SDs) . Actively growing tissues such as early elongating internodes, petioles and leaf blades had the highest levels of C19-GAs . Upon transfer to SDs an accumulation of GA19 was observed in early elongating internodes and leaf blades . The levels of C19-GAs were also to some extent changed upon transfer to SDs . The levels of GA20 were down-regulated in internodes, and those of GA1 were significantly reduced in early expanding leaf blades . In roots the metabolites GA19 and GA8 decreased upon shifts to SDs, while GA20 accumulated slightly . The down-regulation of GA 20-oxidase activity in response to SDs was further indicated by studies of {14C}GA12 metabolism in shoots, demonstrating that the substrate for GA 20-oxidase, {14C}GA53, accumulates in SDs. Crit Care Med, 2002 Apr, 30(4), 904 - 7 Effects of posttreatment with propofol on mortality and cytokine responses to endotoxin-induced shock in rats; Taniguchi T et al.; OBJECTIVE: To clarify the effects of posttreatment with propofol administration on mortality rate and cytokine responses to endotoxin-induced shock in rats . DESIGN: Randomized prospective laboratory study . SETTING: University laboratory . SUBJECTS: Thirty-three male rats . INTERVENTIONS: Animals were randomly assigned to one of three groups (n = 11 per group): a) endotoxemic group, receiving intravenous Escherichia coli endotoxin (20 mg/kg over 2 mins); b) early posttreatment group, treated identically to the endotoxemic group with the additional administration of propofol (10 mg/kg bolus, followed by infusion at 10 mg x kg(-1) x hr(-1)) 1 hr after the injection of endotoxin; and c) late posttreatment group, treated identically to the endotoxemic group with the additional administration of propofol (10 mg/kg bolus, followed by infusion at 10 mg x kg(-1) x hr(-1)) 2 hrs after the injection of endotoxin . MEASUREMENTS AND MAIN RESULTS: Hemodynamics and arterial blood gases were recorded, and mortality rate and plasma cytokine concentrations were calculated for the 5-hr observation . The mortality rate 5 hrs after endotoxin injection was 73% for the endotoxic, 9% for the early posttreatment, and 36% for the late posttreatment groups . The mortality rate for the early posttreatment group was significantly lower than that for the other groups . The increases in plasma cytokine (tumor necrosis factor-alpha and interleukin-6 and -10) concentrations were less for the early posttreatment group than the other two groups . CONCLUSIONS: The early posttreatment of propofol after endotoxin injection drastically reduced the mortality rate of rats and attenuated their cytokine responses . Moreover, propofol attenuated the production of tumor necrosis factor-alpha . These findings suggest that propofol administration may be beneficial during sepsis. Crit Care Med, 2002 Apr, 30(4), 874 - 80 Importance of hypoxic vasoconstriction in maintaining oxygenation during acute lung injury; Brimioulle S et al.; OBJECTIVE: To investigate the role of hypoxic pulmonary vasoconstriction in the intrapulmonary blood flow redistribution and gas exchange protection during oleic acid acute lung injury . DESIGN: Prospective, controlled animal study . SETTING: Research laboratory of an academic institution . SUBJECTS: Three groups of five mongrel dogs . INTERVENTIONS: Induction of acute lung injury by 0.08 mL/kg oleic acid intravenously . Hypoxic pulmonary vasoconstriction inhibition by Escherichia coli endotoxin microdose (15 microg/kg) pretreatment or by metabolic alkalosis (pH 7.60) . MEASUREMENTS AND MAIN RESULTS: Pulmonary arterial and venous resistances were determined by flow-pressure curves and by capillary pressure estimation . Regional lung water and pulmonary blood flow were assessed by positron emission tomography . Oleic acid alone increased the arterial and venous resistances, redistributed blood flow away from edematous areas, and decreased the Pao2 from 507 +/- 16 to 373 +/- 60 torr . on Fio2 1.0 and positive end-expiratory pressure 5 cm H2O . Endotoxin pretreatment inhibited the increase in arterial resistance, suppressed the redistribution, and decreased the Pao2 to 105 +/- 22 torr . Alkalosis inhibited the increase in arterial and venous resistances, suppressed the redistribution, and decreased the Pao2 to 63 +/- 12 torr . Reversal of the alkalosis increased the arterial and venous resistances, restored the perfusion redistribution, and improved the Pao2 to 372 +/- 63 torr . Changes in blood gases conformed to predictions of a computer lung model in which hypoxic pulmonary vasoconstriction was suppressed by endotoxin and alkalosis . CONCLUSIONS: We conclude that in oleic acid-induced lung injury, a) pulmonary hypertension results from increases in both arterial and venous resistances; b) the increase in arterial resistance is the primary mechanism responsible for the perfusion redistribution and the gas exchange protection; and c) the increase in arterial resistance is most consistent with hypoxic pulmonary vasoconstriction. J Biol Chem, 2002 Jun 14, 277(24), 22018 - 24 Epub 2002 Apr 08. Structure of tagatose-1,6-bisphosphate aldolase . Insight into chiral discrimination, mechanism, and specificity of class II aldolases; Hall DR et al.; Tagatose-1,6-bisphosphate aldolase (TBPA) is a tetrameric class II aldolase that catalyzes the reversible condensation of dihydroxyacetone phosphate with glyceraldehyde 3-phosphate to produce tagatose 1,6-bisphosphate . The high resolution (1.45 A) crystal structure of the Escherichia coli enzyme, encoded by the agaY gene, complexed with phosphoglycolohydroxamate (PGH) has been determined . Two subunits comprise the asymmetric unit, and a crystallographic 2-fold axis generates the functional tetramer . A complex network of hydrogen bonds position side chains in the active site that is occupied by two cations . An unusual Na+ binding site is created using a pi interaction with Tyr183 in addition to five oxygen ligands . The catalytic Zn2+ is five-coordinate using three histidine nitrogens and two PGH oxygens . Comparisons of TBPA with the related fructose-1,6-bisphosphate aldolase (FBPA) identifies common features with implications for the mechanism . Because the major product of the condensation catalyzed by the enzymes differs in the chirality at a single position, models of FBPA and TBPA with their cognate bisphosphate products provide insight into chiral discrimination by these aldolases . The TBPA active site is more open on one side than FBPA, and this contributes to a less specific enzyme . The availability of more space and a wider range of aldehyde partners used by TBPA together with the highly specific nature of FBPA suggest that TBPA might be a preferred enzyme to modify for use in biotransformation chemistry. J Biol Chem, 2002 Jun 28, 277(26), 23223 - 9 Epub 2002 Apr 08. His-859 is an essential residue for the activity and pH dependence of Escherichia coli RTX toxin alpha-hemolysin; Cortajarena AL et al.; Escherichia coli alpha-hemolysin (HlyA) is a toxin protein that, in common with other members of the RTX family, contains a calcium-binding domain consisting of a number of Gly- and Asp-rich nonapeptides (17 in this case) repeated in tandem . Amino acid number 6 in these nonapeptides is almost invariably Asp, and occasionally Asn, but HlyA contains a His residue (number 859 in the chain) in position 6 of the last-but-one nonapeptide . HlyA mutants have been prepared, by site-directed mutagenesis, in which His-859 has been replaced by an Asn (H859N) or by Asp (H859D) . HlyA exists in aqueous media in an aggregate-monomer equilibrium, but only the monomer containing bound Ca(2+) (HlyA.Ca) appears to be competent to achieve target membrane insertion and subsequent lysis . In mutant H859N, equilibrium appears to be shifted toward the aggregate, therefore the protein does not exchange Ca(2+) with the aqueous environment, no HlyA.Ca monomers are detected, and the protein lacks any membrane lytic activity . Mutant H859D in turn is almost indistinguishable from the wild-type regarding its calcium binding and membrane lytic activity, however, it differs significantly in its pH dependence . Wild-type HlyA activity decreases sigmoidally with pH, following rather closely the protonation curve of a His residue (apparent pK(a) approximately 6.5) . With mutant H859D activity decreases almost linearly with pH and to a smaller extent . It can be concluded that His-859 plays a critical role in several aspects of HlyA activity, namely self-aggregation properties, calcium binding, hemolysis, and pH dependence. J Biol Chem, 2002 Jun 14, 277(24), 21598 - 603 Epub 2002 Apr 08. Conclusive evidence that the major T-cell antigens of the Mycobacterium tuberculosis complex ESAT-6 and CFP-10 form a tight, 1:1 complex and characterization of the structural properties of ESAT-6, CFP-10, and the ESAT-6*CFP-10 complex . Implications for pathogenesis and virulence; Renshaw PS et al.; The proteins ESAT-6 and CFP-10 have been shown to be secreted by Mycobacterium tuberculosis and Mycobacterium bovis cells, to be potent T-cell antigens, and to have a clear but as yet undefined role in tuberculosis pathogenesis . We have successfully overexpressed both ESAT-6 and CFP-10 in Escherichia coli and developed efficient purification schemes . Under in vivo-like conditions, a combination of fluorescence, circular dichroism, and nuclear magnetic resonance spectroscopy have shown that ESAT-6 contains up to 75% helical secondary structure, but little if any stable tertiary structure, and exists in a molten globule-like state . In contrast, CFP-10 was found to form an unstructured, random coil polypeptide . An exciting discovery was that ESAT-6 and CFP-10 form a tight, 1:1 complex, in which both proteins adopt a fully folded structure, with about two-thirds of the backbone in a regular helical conformation . This clearly suggests that ESAT-6 and CFP-10 are active as the complex and raises the interesting question of whether other ESAT-6/CFP-10 family proteins (22 paired genes in M . tuberculosis) also form tight, 1:1 complexes, and if so, is this limited to their genome partner, or is there scope for wider interactions within the protein family, which could provide greater functional flexibility? Circulation, 2002 Apr 9, 105(14), 1623 - 6 Accelerated reendothelialization with suppressed thrombogenic property and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide; Ohno N et al.; BACKGROUND: Vein graft disease limits the late results of coronary revascularization . C-type natriuretic peptide (CNP) inhibits the growth of vascular smooth muscle cells . Given the effects of CNP on cGMP cascade, we hypothesized that transfected CNP genes modulate endothelial repair and thrombogenicity in the vein graft . METHODS AND RESULTS: Autologous rabbit jugular vein grafts were incubated ex vivo in a solution of adenovirus vectors containing CNP gene (Ad.CNP) or Escherichia coli lac Z gene (Ad.LacZ) and then interposed in the carotid artery . Reendothelialization, mural thrombi formation, and intima/media ratio were evaluated on the 14th and 28th postoperative days . More reendothelialization was seen in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts both at 14 days (0.81+/-0.05 versus 0.30+/-0.14, P<0.01) and at 28 days (0.96+/-0.01 versus 0.45+/-0.08, P<0.001) . The mural thrombus area was smaller in Ad.CNP-infected grafts than in Ad.LacZ-infected grafts . Neointimal thickening was significantly suppressed in the Ad.CNP group . The in vitro wound assay with human coronary artery endothelial cells revealed significant potentiation of the wound repair process by CNP and atrial natriuretic peptide administration . CONCLUSIONS: Infected Ad.CNP accelerated reendothelialization and suppressed thrombosis and neointimal hyperplasia . The method may potentially prevent vein graft disease in patients undergoing coronary artery revascularization. Lett Appl Microbiol, 2002, 34(4), 233 - 7 The M . tuberculosis antigen 85 complex and mycolyltransferase activity; Kremer L et al.; AIMS: The antigen 85 complex (Ag85) from Mycobacterium tuberculosis consists of three abundantly secreted proteins (FbpA, FbpB and FbpC2) which play a key role in the pathogenesis of tuberculosis and also exhibit cell wall mycolyltransferase activity . A related protein with similarity to the Ag85 complex was recently annotated in the M . tuberculosis genome as FbpC1 . An investigation was carried out to determine whether FbpC1 may also possess mycolyltransferase activity, a characteristic feature of the Ag85 complex . METHODS AND RESULTS: Heterologous expression of FbpA, FbpC1 and FbpC2 was performed in Escherichia coli . Recombinant proteins were purified under non-denaturating conditions and used in an in vitro mycolyltransferase assay . CONCLUSIONS: In contrast to FbpA and FbpC2, recombinant FbpC1 did not possess in vitro mycolyltransferase activity and was not recognized by two monoclonal antibodies to the native Ag85 . SIGNIFICANCE AND IMPACT OF THE STUDY: Mycolyltransferase activity is restricted to FbpA, FbpbB and FbpC2 only; the actual function of FbpC1 remains to be established. Clin Exp Allergy, 2002 Mar, 32(3), 455 - 62 Cloning and molecular characterization of the Hevea brasiliensis allergen Hev b 11, a class I chitinase; O'Riordain G et al.; BACKGROUND: In the last 10 years type-I allergy against proteins from Hevea brasiliensis latex has become an acknowledged medical issue . Fruit-allergic patients represent one risk group for developing latex allergy . Class I chitinases have been identified from chestnut, avocado and banana as relevant allergens . The chitin binding (hevein) domain from these class I chitinases has been postulated to bear the important IgE binding epitopes . OBJECTIVE: To clone the cDNA of an allergenic latex class I chitinase, to express the recombinant protein and to determine its IgE cross-reactivity with hevein (Hev b 6.02) . METHODS: A full-length cDNA coding for a class I chitinase has been isolated from Hevea latex RNA by reverse transcription followed by PCR . The chitinase encoding sequence has been subcloned into the pMAL expression vector and expressed in E . coli as a fusion protein to maltose binding protein . The highly enriched recombinant protein fraction has been tested for its IgE binding capacity in immunoblots and ELISA . Furthermore, the pathogenesis-related function of the recombinant protein was tested in a fungal growth inhibition assay . RESULTS: The Hevea brasiliensis latex chitinase, designated Hev b 11, displays 70% identity to the endochitinase from avocado and its hevein-domain 58% to hevein (Hev b 6.02) . The recombinant Hev b 11-maltose binding protein is recognized by latex- and fruit-allergic patients with IgE binding in both, ELISA and immunoblots . Pre-incubation of sera with rHev b 11-maltose binding protein showed an overall 16% inhibition of subsequent binding to rHev b 6.02-maltose binding protein on solid phase . The growth of F . oxysporum was inhibited in a dose dependent manner by addition of rHev b 11-maltose binding protein to the culture . CONCLUSIONS: Hev b 11, a class I chitinase, is another allergen from Hevea latex with a chitin binding domain and displays a different IgE binding capacity compared with hevein. Biochemistry, 2002 Apr 16, 41(15), 4946 - 52 Importance of hydrophobic matching for spontaneous insertion of a single-spanning membrane protein; Ridder AN et al.; In this study, we have investigated the effect of hydrophobic mismatch between the thickness of the membrane and a transmembrane segment of a protein that directly inserts into the membrane bilayer . For this purpose we used mutants of the single-spanning Pf3 coat protein that can spontaneously insert into Escherichia coli membrane vesicles and large unilamellar vesicles (LUVs) . The thickness of the liposomal bilayer could be altered by using lipids with different acyl chain lengths or by incorporation of cholesterol . The insertion efficiency of the protein clearly depended on the bilayer thickness, with most efficient insertion under hydrophobic matching conditions . To discriminate between effects of length and hydrophobicity, mutants with different synthetic transmembrane segments were constructed . These mutants inserted into LUVs in a mismatch-dependent manner . However, in particular for longer and less hydrophobic mutants, most efficient insertion was generally observed in thinner bilayers than expected on the basis of hydrophobic matching. Biochemistry, 2002 Apr 16, 41(15), 4819 - 26 A comparative resonance Raman analysis of heme-binding PAS domains: heme iron coordination structures of the BjFixL, AxPDEA1, EcDos, and MtDos proteins; Tomita T et al.; The heme-PAS is a specialized domain with which a broad class of signal-transducing heme proteins detect physiological heme ligands . Such domains exhibit a wide range of ligand binding parameters, yet they are all expected to feature an alpha-beta heme binding fold and a predominantly hydrophobic heme distal pocket without a distal histidine . We have compared, for the first time, the resonance Raman spectra of several heme-PASs: the heme-binding domains of Bradyrhizobium japonicum FixL, Escherichia coli Dos, Acetobacter xylinum PDEA1, and Methanobacterium thermoautotrophicum Dos . In all cases, the nu(Fe)-(CO) and nu(C-O) values of the carbonmonoxy forms were consistent with coordination of the heme iron to histidine on the proximal side and binding of the CO without electrostatic interaction with the heme distal pocket . EcDos was unusual in having predominantly hexacoordinate heme iron in the deoxy and met forms . Despite an evident lack of CO interaction with the EcDos heme pocket, relatively low Fe-O(2) (562 cm(-1)) and N-O (1576 cm(-1)) stretching frequencies indicated that strong polar interactions with that heme distal pocket are possible for highly bent ligands such as O(2) or NO . None of the newly studied NO adducts exhibited evidence of the Fe-His rupture and pentacoordination previously noted for Sinorhizobium meliloti FixL . A low Fe-His stretching frequency, formerly interpreted as a strained Fe-His bond, and the slow association of O(2) with S . meliloti FixL failed to correlate with the newly studied proteins having low association rate or low equilibrium association constants for binding of O(2) . We conclude that although heme-PASs share some features, they represent distinct signal transduction mechanisms. Biochemistry, 2002 Apr 16, 41(15), 4789 - 97 Identification of the active site of gelatinase B as the structural element sufficient for converting a protein to a metalloprotease; Kaur K et al.; Gelatinase B is a member of the matrix metalloproteinase family that efficiently cleaves gelatin, elastin, and types V and X collagen . To understand the contribution of the active site of the enzyme (amino acid residues 373-456) in these activities, we studied catalytic properties of a fusion protein consisting of maltose binding protein and the active site region of gelatinase B . We found that addition of the active site of gelatinase B, which corresponds to 12% of the total protein molecule, to maltose binding protein is sufficient to endow the protein with the ability to cleave the peptide substrates Mca-PLGL(Dpa)AR-NH(2) and DNP-PLGLWA-(D)-R-NH(2) . The fusion protein hydrolyzed the Mca-PLGL(Dpa)AR-NH(2) peptide with the same efficiency as that of the stromelysin, k(cat)/K(m) approximately 1.07 x 10(6) M(-)(1) h(-)(1) . The fusion protein, however, was not able to degrade the large substrate, gelatin . Inhibition of the activity of the protein by EDTA suggested that its activity was metal dependent . ESR analyses indicated that the fusion protein bound one molecule of Zn(2+) . In addition, Z-Pro-Leu-Gly-hydroxamate and TIMP-1 inhibited the activity of the protein, suggesting that the structure of the active site of the fusion protein is similar to that of the other metalloproteinases . These data provide fundamental information about the structural elements required for transforming a protein to a metalloprotease. DNA Res, 2002 Feb 28, 9(1), 19 - 24 Conservation of translation initiation sites based on dinucleotide frequency and codon usage in Escherichia coli K-12 (W3110): non-random distribution of A/T-rich sequences immediately upstream of the translation initiation codon; Yamagishi K et al.; Dinucleotide frequencies are useful for characterizing consensus elements as a minimum unit of nucleotide sequence because the neighborhood relations of nucleotide sequences are reflected in dinucleotides . Using a consensus score based on dinucleotide frequencies and intra-species codon usage heterogeneity, denoted by the Z1 parameter, we report the relationship between nucleotide conservation at the translation initiation sites of genes in the Escherichia coli K-12 genome (W3110) and codon usage in its downstream genes . Significant positive correlations were obtained in three regions centered at -13, -4, and +7, which correspond to the Shine-Dalgarno element, the A + T element immediately upstream of the translation initiation site, and the downstream box, respectively. DNA Res, 2002 Feb 28, 9(1), 1 - 10 Distribution of repetitive sequences on the leading and lagging strands of the Escherichia coli genome: comparative study of Long Direct Repeat (LDR) sequences; Kawano M et al.; In the present study, we developed a method for detecting sequences whose similarity to a target sequence is statistically significant and we examined the distribution of these sequences in the E . coli K-12 genome . Target sequences examined are as follows: (i) short repeat: Crossover hot-spot instigator (Chi) sequence, replication termination (Ter) sequence, and DnaA binding sequence (DnaA box); (ii) potential stem-loop structure repeats: palindromic unit (PU), boxC sequences, and intergenic repeat unit (IRU); (iii) potential RNA coding repeats: rRNAs, PAIR, TRIP, and QUAD; and (iv) potential protein coding repeats: insertion elements (ISs) and Long Direct Repeats (LDRs) . We also examined the distribution of these sequences on leading and lagging strands . We obtained another four statistically significant LDR sequences with more than 187 bp matched to LDR-A near the LDR loci, suggesting that these regions might be used as high recombination hot spots for LDR . Adaptation of individual LDRs to E . coli genome is also discussed on the basis of codon usage. Cell Biochem Biophys, 2002, 36(1), 1 - 18 Temperature-induced formation of a non-native intermediate state of the all beta-sheet protein CD2; Yang JJ et al.; Domain 1 of the cell adhesion protein CD2 (CD2-1) has an all beta-structure typical of proteins belonging to the immunoglobin superfamily . It has a remarkable ability to fold as a native monomer or a metastable intertwined dimer . To understand the origin of structural rearrangements of CD2-1, we have studied equilibrium unfolding of the protein using various biophysical spectroscopic techniques . At temperatures above approx 68 degrees C, a partially folded state of CD2-1 (H state) with a distinct secondary structure, involving largely exposed aromatic and hydrophobic residues and a substantially perturbed tertiary structure, is observed . In contrast, an unfolded state (D state) of CD2-1 with random-coil-like secondary and tertiary structures is observed in 6 M GuHCl . This partially folded high-temperature state has increased negative molar ellipticity at 222 nm in far-ultraviolet CD spectra, implying formation of a non-native helical conformation . The existence of this non-native high-temperature intermediate is consistent with relatively high intrinsic helical propensities in the primary sequence of CD2-1 . This conformational flexibility may be important in the observed domain swapping of CD2-1. Anal Bioanal Chem, 2002 Jan, 372(1), 174 - 80 A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli; Salins LL et al.; A sensing system for nickel based on the nickel binding protein (NBP) from Escherichia coli is shown to be feasible . The versatility of NBP was demonstrated by its use in three different assay formats . When the NBP binds nickel, it undergoes a conformational change that can be used as the basis for an optical sensing system for nickel . The NBP gene was overexpressed in E . coli and the protein purified in a single step using perfusion anion-exchange chromatography . A unique cysteine residue at position 15 in the NBP was labeled with the fluorophore, N-{2-(1-maleimidyl)ethyl}-7-(diethylamino)coumarin-3-carboxamide (MDCC) . In a spectrofluorimetric assay, there was a maximum of 65% quenching of the fluorescence signal produced by NBP-MDCC in the presence of nickel . A response curve for nickel using NBP-MDCC revealed a detection limit of 8 x 10(-8) mol L(-1) . NBP-MDCC was also used to develop assays in microtiter plate and fiber optic bundle formats . Detection limits for nickel using these formats were also in the submicromolar range . Selectivity studies conducted with other divalent metals, including copper, cobalt, iron, cadmium, and manganese, showed that fluorescence quenching for cobalt was similar in magnitude but with a detection limit more than 10-fold higher than for nickel . The quenching responses were lower for the other metals, with detection limits at least 10 to 100 times higher than for nickel . These results suggest that fluorescently labeled NBP is potentially useful in the development of a sensing system for nickel. Hunan Yi Ke Da Xue Xue Bao, 1999, 24(1), 1 - 4 {Construction of three transgenic vectors carrying the latent membrane protein gene of Epstein-Barr virus}; Xiao Z et al.; In order to produce transgenic mice carrying the latent membrane protein(LMP) gene of Epstein-Barr virus(EBV) and to study the oncogenic role of LMP gene in vivo, three different transgenic vectors carrying the LMP gene were constructed . In pBR322-MT-LMP vector, the promoter of LMP gene is the regulation region of mouse metallothionein-I gene . In pMci3-LMP shuttle vector, the promoter of LMP gene is the replication origin(oriP) of EBV . In pMV-LMP-c-myc retroviral vector, the long terminal repeat (LTR) of mouse sarcomavirus serves as the promoter of LMP gene . The characterization and usefulness of these three transgenic vectors are also discussed. Intervirology, 2002, 45(1), 24 - 32 Evaluation of HBs, HBc, and frCP virus-like particles for expression of human papillomavirus 16 E7 oncoprotein epitopes; Pumpens P et al.; OBJECTIVES: In an attempt to develop virus-like particles (VLPs) as experimental vaccine against human papilloma virus (HPV)-induced tumours, the HPV16 E7 oncoprotein epitopes spanning amino acid (aa) residues 35-98 were expressed on three proteins capable of VLP formation: hepatitis B virus (HBV) surface (HBs) and core (HBc) antigens, and RNA phage fr coats (frCP) . METHODS: The profile of immunoglobulin isotypes induced in Balb/C mice after immunization with purified chimeric proteins was studied . RESULTS: The HBs*-E7(35-54) protein expressing E7 residues 35-54 between residues 139 and 142 of the HBs carrier formed HBs-like particles in Saccharomyces cerevisiae . The HBc Delta-E7(35-98), but not the frCP-E7(35-98), ensured VLP formation in Escherichia coli . In Balb/C mice, the HBs*-E7(35-54) VLPs predominantly induced an anti-E7 antibody, but not anti-HBs carrier response, whereas the HBc Delta-E7(35-98) VLPs induced a lower anti-E7 compared to anti-HBc carrier response . The frCP-E7(35-98) protein elicited equally high antibody responses to both E7 and frCP carrier . Analysis of the immunoglobulin G isotype profile of the antibodies induced by the E7-carrying chimeras showed that the HBs and frCP derivatives were capable of eliciting the Th1 and Th2 subsets of T helper cells, whereas the HBc-derived chimeras elicited only the Th2 subset . CONCLUSIONS: The HBs and HBc, but not frCP carriers support an efficient outcome for VLPs carrying the HPV16 E7 epitopes . All chimeric proteins may be regarded as potential vaccine candidates . J Immunol, 2002 Apr 15, 168(8), 4012 - 7 Two lipoproteins extracted from Escherichia coli K-12 LCD25 lipopolysaccharide are the major components responsible for Toll-like receptor 2-mediated signaling; Lee HK et al.; Toll-like receptor 2 (TLR2)-mediated cell activation induced by commercial preparations of LPS was recently shown to arise from impurities whose identities are not known . In this work, we determined the molecules responsible for TLR2-mediated cell activation in LPS derived from Escherichia coli K-12 strain LCD25 . When LCD25 LPS was phenol extracted, two proteins capable of TLR2-mediated cell activation were purified and identified as E . coli lipoproteins . We cloned, expressed, and purified these two lipoproteins, Lip19 and Lip12 . Lip19 or Lip12 activated TNF-alpha production from RAW264.7 and THP-1 cells in a TLR2-dependent manner . However, neither Lip19 nor Lip12 activated HUVECs, which lack endogenous TLR2 . Additionally, IkappaB kinase beta and c-Jun N-terminal kinase 1 activation in THP-1 cells induced by Lip19 or Lip12 was observed . TLR2 activation by Lip19 and Lip12 in HEK293 cells was blocked by inhibitory anti-TLR2 mAbs . The unlipidated mutants, Lip19-C19S and Lip12-C21S, in which the NH(2)-terminal cysteine was substituted by serine, lost their ability to activate TLR2-transfected HEK 293 cells . Taken together, these results demonstrate that two lipoproteins constitute the major contaminants responsible for TLR2-mediated cell activation in E . coli LCD25 LPS. J Biol Chem, 2002 Jun 21, 277(25), 22992 - 9 Epub 2002 Apr 05. Function assignment to conserved residues in mammalian alkaline phosphatases; Kozlenkov A et al.; We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP) . Mutations were introduced in wild-type PLAP, i.e . {E429}PLAP, and in some instances also in {G429}PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme . All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP . We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP . Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased k(cat) and increased K(m) when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP . All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP . By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxyl-terminally located Cys-467-Cys-474 bond plays a lesser structural role . The substitution of the free Cys-101 did not significantly affect the properties of the enzyme . A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit . We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429 . The mutations led to a 2-fold decrease in k(cat), a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors l-Phe and l-Leu . Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs. J Biol Chem, 2002 Jun 21, 277(25), 22806 - 13 Epub 2002 Apr 05. Scanning mutagenesis of a Janus-faced atracotoxin reveals a bipartite surface patch that is essential for neurotoxic function; Maggio F et al.; The Janus-faced atracotoxins (J-ACTXs) are a family of insect-specific excitatory neurotoxins isolated from the venom of Australian funnel web spiders . In addition to a strikingly asymmetric distribution of charged residues, from which their name is derived, these toxins contain an extremely rare vicinal disulfide bond . To shed light on the mechanism of action of these toxins and to enhance their utility as lead compounds for insecticide development, we developed a recombinant expression system for the prototypic family member, J-ACTX-Hv1c, and mapped the key functional residues using site-directed mutagenesis . An alanine scan using a panel of 24 mutants provided the first complete map of the bioactive surface of a spider toxin and revealed that the entire J-ACTX-Hv1c pharmacophore is restricted to seven residues that form a bipartite surface patch on one face of the toxin . However, the primary pharmacophore, or hot spot, is formed by just five residues (Arg(8), Pro(9), Tyr(31), and the Cys(13)-Cys(14) vicinal disulfide) . The Arg(8)-Tyr(31) diad in J-ACTX-Hv1c superimposes closely on the Lys-(Tyr/Phe) diad that is spatially conserved across a range of structurally dissimilar K(+) channel blockers, which leads us to speculate that the J-ACTXs might target an invertebrate K(+) channel. Structure (Camb), 2002 Apr, 10(4), 581 - 8 Structure of the lac operon galactoside acetyltransferase; Wang XG et al.; The galactoside acetyltransferase (thiogalactoside transacetylase) of Escherichia coli (GAT, LacA, EC 2.3.1.18) is a gene product of the classical lac operon . GAT may assist cellular detoxification by acetylating nonmetabolizable pyranosides, thereby preventing their reentry into the cell . The structure of GAT has been solved in binary complexes with acetyl-CoA or CoA and in ternary complexes with CoA and the nonphysiological acceptor substrates isopropyl beta-D-thiogalactoside (IPTG) or p-nitrophenyl beta-D-galactopyranoside (PNPbetaGal) . A hydrophobic cleft that binds the thioisopropyl and p-nitrophenyl aglycones of IPTG and PNPbetaGal may discriminate against substrates with hydrophilic substituents at this position, such as lactose, or inducers of the lac operon . An extended loop projecting from the left-handed parallel beta helix domain contributes His115, which is in position to facilitate attack of the C6-hydroxyl group of the substrate on the thioester. Structure (Camb), 2002 Apr, 10(4), 535 - 46 Structural basis for proofreading during replication of the Escherichia coli chromosome; Hamdan S et al.; The epsilon subunit of the Escherichia coli replicative DNA polymerase III is the proofreading 3'-5' exonuclease . Structures of its catalytic N-terminal domain (epsilon186) were determined at two pH values (5.8 and 8.5) at resolutions of 1.7-1.8 A, in complex with two Mn(II) ions and a nucleotide product of its reaction, thymidine 5'-monophosphate . The protein structure is built around a core five-stranded beta sheet that is a common feature of members of the DnaQ superfamily . The structures were identical, except for differences in the way TMP and water molecules are coordinated to the binuclear metal center in the active site . These data are used to develop a mechanism for epsilon and to produce a plausible model of the complex of epsilon186 with DNA. Curr Biol, 2002 Apr 2, 12(7), R250 - 2 Water transporters: how so fast yet so selective? Law RJ, Sansom MS. A high-resolution X-ray structure of an aquaporin has revealed water molecules bound within the transmembrane pore and provided new clues to the mechanisms of rapid water transport and high selectivity in this important class of membrane proteins. Hum Gene Ther, 2002 Apr 10, 13(6), 689 - 96 Gene transfer to the nonhuman primate retina with recombinant feline immunodeficiency virus vectors; Lotery AJ et al.; We hypothesize that recombinant feline immunodeficiency viral (rFIV) vectors may be useful for gene transfer to the nonhuman primate retina . We performed vitrectomies and subretinal injections in the right eyes of 11 cynomolgus monkeys . Vesicular stomatitis virus glycoprotein-pseudotyped rFIV that expressed the Escherichia coli beta-galactosidase gene was injected into eight eyes . Sham vehicle or lactose buffer injections were also performed in two of these eight study eyes . rFIV pseudotyped with an amphotropic envelope was used in two eyes, and in one animal injections of lactose buffer only were given . After surgery the animals were clinically evaluated by retinal photography and electroretinography . beta-Galactosidase expression was evaluated, at a final end point, in histological sections . We found photoreceptor and Muller cells to have the greatest transgene expression . Focal inflammatory responses localized to the injection site were seen histologically in all eyes . No difference in transduction efficiency was seen between injections near the macula and more peripheral injections . Visual function as assessed by electroretinography was not significantly affected by vector or vehicle injections . We conclude that rFIV vectors administered beneath the retina can transduce a variety of retinal cells in the nonhuman primate retina . rFIV vectors have therapeutic potential and could be exploited to develop gene therapy for the human eye. Mol Cell Biochem, 2002 Jan, 229(1-2), 107 - 11 Changes in the antioxidant content of mononuclear leukocytes from mice with endotoxin-induced oxidative stress; Victor VM et al.; Oxidative stress, associated with a high production of reactive oxygen species (ROS) by immune cells, is involved in the endotoxic shock caused by endotoxin . This oxidative stress is linked to the inability of the immune cells to maintain adequate levels of antioxidants with free radical-scavenging action . Glutathione (GSH) and ascorbic acid (AA) are intracellular and extracellular antioxidants (ROS scavengers) that improve the leukocyte functions . Therefore, in the present work we have determined the reduced GSH and AA content in axillary nodes, spleen, thymus and peritoneal mononuclear leukocytes from BALB/c mice subjected to lethal endotoxic shock produced by intraperitoneal injection of E . coli lipopolysaccharide (LPS, 100 mg/kg), at several times (0, 2, 4, 12 and 24 h) after LPS injection . Endotoxic shock decreased the levels of AA in the leukocytes from the three organs as well as the levels of GSH in axillary nodes and spleen cells while it increased the GSH levels in thymus and peritoneum . These results are in agreement with the oxidative stress and the altered function previously observed in those leukocytes, and they suggest that antioxidant administration may be useful for the treatment of endotoxic shock and other oxidative stress situations with altered immunological responses. Water Sci Technol, 2002, 45(4-5), 191 - 9 Detection of Escherichia coli in water by culture-based amperometric and luminometric methods; Nistor C et al.; The application of amperometric biosensor- and chemiluminiscence based methods for rapid detection of viable E . coli in water has been investigated . An amplification of the amperometric signal by a factor of 4 was obtained when the cellobiose dehydrogenase (CDH) biosensor was used instead of a plain graphite electrode for detection of b-galactosidase (b-GAL) activity at 22.5 degrees C . A linear correlation was demonstrated for detection time (DT) vs . initial concentrations (logarithmic units) of E . coli IT1 and E . coli in environmental samples, respectively, by use of the CDH biosensor or a chemiluminometric technique . The study has shown that an E . coli concentration > or = 10(4) cfu/100 mL in environmental samples was determined by the CDH biosensor within one working day . However, further reduction of the DT can be obtained, e.g . by increasing the signal amplification factor using other biosensors. J Chemother, 2001 Nov, 13 Spec No 1(1), 17 - 22 Laparoscopic surgery and surgical infection; Balague Ponz C et al.; Laparoscopic surgery (LS) has improved our knowledge of some aspects of surgical physiopathology . Other advantages include a lower incidence of postoperative infections, as evidenced by a lower inflammatory response which is related to a better preserved immune response to infection . But, the differential aspects of LS may influence the intraperitoneal environment and, in case of infection, must be evaluated in two different situations: during clean and potentially-contaminated surgery or in the presence of established infection . The most important differential factors of LS are the pneumoperitoneum and the use of CO2 . The influence of both these on the evolution of an intraperitoneal infecton has been of interest in recent years . Our department developed an experimental study with mice to evaluate the local and systemic inflammatory response to perioperative intra-abdominal contamination with a known inoculum of Escherichia coli . The animals were distributed in four groups: control, laparotomy, laparoscopy with CO2 pneumoperitoneum, and laparoscopy with wall traction . Peritoneal liquid and blood cultures such as peritoneal and systemic cytokine levels were analyzed . The results showed a better tolerance to perioperative contamination in LS groups while the CO2 pneumoperitoneum had no influence . But, in the presence of peritonitis, an elevated CO2 pneumoperitoneum can be dangerous and the operative time is an important factor to be considered . The literature is reviewed on the relationship between LS and surgical infection. Mol Biotechnol, 2002 Mar, 20(3), 257 - 60 A modified nick translation method used with FISH that produces reliable results with archival tissue sections; Watters AD et al.; Nick translation is used to label DNA and RNA to produce probes for in situ hybridization and Northern and Southern blotting . Fluorescence in situ hybridization (FISH) is a widely applied technique used to determine chromosomal and genetic anomalies in many biological samples . Initially the technique was applied to metaphase preparations, but the usefulness of detecting genetic anomalies in solid tumors in situ has resulted in the development of modified protocols . Formalin fixed paraffin processed tissue sections present novel challenges when applying FISH; the probes must be small (between 200 and 600 base pairs) and pretreatment is necessary before the probes can be applied to tissue sections, to promote probe access to target DNA . Here we report on a modification of a nick translation method to produce a probe that can reliably be used with FISH in paraffin processed tissue sections. Ann Anat, 2002 Mar, 184(2), 153 - 7 An improved method for beta-galactosidase activity detection on muscle tissue . A light and electron microscopic study; Gioglio L et al.; In the present study we describe a method for the histochemical demonstration of bacterial beta-D-galactosidase activity on skeletal muscle tissue processed for light and transmission electron microscopy . Hence allowing this enzyme to be accurately detected, bacterial beta-galactosidase expression was studied in transgenic mouse where the enzyme, with the nuclear localization signal (nlacZ), is under the transcriptional control of the striated muscle-specific promoter MLC3F . The chromogenic substrate, 5-bromo-3-indolyl-beta-D-galactopyranoside (Bluo-Gal), was used both to recognize labelled myofibers, and beta-gal positive organelles inside single myofibers . Moreover, because the preservation of enzyme is highly dependent on tissue fixation, we developed a suitable fixation solution allowing good preservation of both tissue and enzymatic activity . This was achieved by briefly fixing tissue (3 hours) in glutaraldehyde (2.5%) and paraformaldehyde (1%) in combination . This method should be taken into consideration when studying the gene therapy of muscle diseases because it is sensitive, inexpensive and not time consuming. Anal Bioanal Chem, 2002 Jan, 372(2), 273 - 5 Epub 2001 Dec 15. Potentiometric phosphate-sensing system utilizing phosphate-binding protein; Kubo I; A phosphate-detection system has been developed which uses phosphate-binding protein (PBP) from Escherichia coli . PBP was immobilized on a sheet of nitrocellulose membrane by cross-linking and the membrane potential of the immobilized PBP was measured . The response time of the system to phosphate was 5 min . The response was selective to phosphate among other anions . Under optimum conditions 0.1-1.5 mmol L(-1) phosphate can be determined with this system. Anal Bioanal Chem, 2002 Jan, 372(2), 261 - 7 Epub 2001 Dec 22. Cloning, functional expression and kinetic characterization of pesticide-selective Fab fragment variants derived by molecular evolution of variable antibody genes; Rau D et al.; Fab antibody fragments were constructed by subcloning single chain Fv variable regions from the phagemid vector pCANTAB 5E into the expression vector pASK99 . The vector was designed for bacterial secretion of Fab fragments and bears coding sequences for murine constant domains including the Strep-tag II at the carboxyl-terminal end of the constant heavy chain domain . The cloning procedure was carried out with the scFv antibodies IPR-7, IPR-53 and IPR-23 . The second and third clone originated from the molecular evolution of the s-triazine selective antibody IPR-7 . The Fab fragments were expressed under the transcriptional control of the tetA promoter system . Large-scale production benefits from the anhydrotetracycline-inducible system because of the lower costs for the inducer compared to IPTG and the tightly regulated expression of the recombinant antibody fragments . The Strep-tag purification technology facilitates the isolation of Fab fragments from the E . coli periplasm . The characteristics of functionally expressed Fab fragments were determined by employing a BIAcore 2000 system . The KD of the Fab variant IPR-23 (K(D)= 1.1 2 x 10(-9) M) optimized by molecular evolution was improved by a factor of 24 compared to the Fab IPR-7 (K(D) = 2.73 x 10(-8) M), which was derived from the template scFv antibody IPR-7 . The affinity alteration was also reflected in the 22-fold reduction of the IC50 values of the variants Fab IPR-7 (IC50 = 60.5 microg/L) and Fab IPR-23 (IC50=2.7 microg/L) in the corresponding atrazine ELISA. Appl Microbiol Biotechnol, 2002 Mar, 58(3), 386 - 92 Epub 2002 Jan 16. Growth rate-dependent changes in Escherichia coli membrane structure and protein leakage; Shokri A et al.; The phospholipid and fatty acid content of the Escherichia coli membrane were investigated during continuous cultivation . At low growth rates, there was an increase in cardiolipin produced at the expense of phosphatidylethanolamine . Phosphatidylglycerol had a maximum at a growth rate of 0.3 h(-1) . The amount of cyclic fatty acids was markedly increased at lower growth rates, while there was an evident minimum at 0.3 h(-1) . This was also the case for saturated fatty acids . At this point, the unsaturated fatty acids had a maximum depending mainly on changes in cis-vaccenic acid . The mechanical strength towards sonication and osmotic shock/enzymatic treatment showed that the cells were more rigid at low dilution rates . However, this was accompanied by a higher cell lysis, a reduced capacity for total and specific protein production and a lower yield of cells . The amount of lipid A in the medium (endotoxin) was constant and negligible at all growth rates . The leakage of periplasmic protein to the medium had an optimum at 0.3 h(-1), resulting in a transport of 20% of the total recombinant product . It is argued that this constitutes the point of highest membrane fluidity and thus an increase possibility for protein transport. Appl Microbiol Biotechnol, 2002 Mar, 58(3), 330 - 7 Epub 2002 Jan 08. Role of the general stress response during strong overexpression of a heterologous gene in Escherichia coli; Schweder T et al.; The strong overexpression of heterologous genes in Escherichia coli often leads to inhibition of cell growth, ribosome destruction, loss of culturability, and induction of stress responses, such as a heat shock-like response . Here we demonstrate that the general stress response, which is connected to the stress response regulator sigmas (sigma38, rpoS gene product), is suppressed during strong overproduction of a heterologous alpha-glucosidase . The mRNA levels of the rpoS and osmY stress genes drastically decrease after induction of the strong overexpression system . It is shown that an rpoS mutation causes a significant loss of cell viability after induction of the expression system . Furthermore, it is demonstrated that an E . coli c/pP mutant, which could be suggested to improve heterologous protein production, is not a good production host if a tac-promoter is used to control the expression of the recombinant gene . Data from this study suggest that the overexpression of the alpha-glucosidase was greatly decreased by sigma factor competition in the clpP mutant, due to the increased sigmas level in this mutant background. Appl Microbiol Biotechnol, 2002 Mar, 58(3), 286 - 90 Epub 2001 Dec 20. Importance of redox balance on the production of succinic acid by metabolically engineered Escherichia coli; Hong SH et al.; We had previously shown that succinic acid production in a pfl ldhA double mutant strain of Escherichia coli could be enhanced by amplifying the malic enzyme activity . However, recombinant E . coli NZN111 (F- Apfl::Cam ldhA::Kan) harboring pTrcML, a plasmid containing the E . coli malic enzyme gene, produced a considerable amount of malic acid along with the desired product, succinic acid . To have an insight into the intracellular metabolism, metabolic control analysis was carried out . From the results of a simulation, it was predicted that supplying additional reducing power could enhance succinic acid production . More reduced carbon substrate sorbitol was thus examined for the possibility of matching the potential during succinic acid production . When NZN111 (pTrcML) was cultured in LB medium containing 20 g sorbitol/l under a CO2 atmosphere, 10 g succinic acid/l was produced . The apparent yield of succinic acid was 1.1 g succinic acid/g sorbitol, which is 85% of the maximum theoretical yield . Therefore, it was found that redox balancing was important for the enhanced production of succinic acid in metabolically engineered E . coli. J Biol Chem, 2002 Jun 21, 277(25), 22289 - 96 Epub 2002 Apr 04. A direct pyrophosphatase-coupled assay provides new insights into the activation of the secreted adenylate cyclase from Bordetella pertussis by calmodulin; Lawrence AJ et al.; Continuous recording of the activity of recombinant adenylate cyclase (CyaA) of Bordetella pertussis (EC ) by conductimetric determination of enzyme-coupled pyrophosphate cleavage has enabled us to define a number of novel features of the activation of this enzyme by calmodulin and establish conditions under which valid activation data can be obtained . Activation either in the presence or absence of calcium is characterized by a concentration-dependent lag phase . The rate of formation and breakdown of the activated complex can be determined from an analysis of the lag phase kinetics and is in good agreement with thermodynamic data obtained by measuring the dependence of activation on calmodulin concentration, which show that calcium increases k(on) by about 30-fold . The rate of breakdown of the activated complex, formed either in the presence or absence of calcium, has been determined by dilution experiments and has been shown to be independent of the presence of calcium . The coupled assay is established as a rapid, convenient and safe method which should be readily applicable to the continuous assays of most other enzymes that catalyze reactions in which inorganic pyrophosphate is liberated. Curr Opin Microbiol, 2002 Apr, 5(2), 187 - 93 Regulation of gene expression in the PTS in Escherichia coli: the role and interactions of Mlc; Plumbridge J; Mlc represses several glucose-related genes, including the phosphotransferase system (PTS) genes ptsHI and ptsG . Induction of these genes by glucose occurs as a response to the flux of glucose through the PTS and involves the sequestration of Mlc to membranes containing dephosphorylated PtsG . In addition, ptsG levels are post-transcriptionally regulated by the glycolytic flux . PtsG is, thus, subject to multiple layers of control that, in turn, feed back on the regulation of the PTS by Mlc . The regulatory function of Mlc is compared with that of FruR and of the PTS-regulation-domain-containing protein, BglG. FEMS Microbiol Lett, 2002 Feb 19, 208(1), 53 - 9 FlaK of the archaeon Methanococcus maripaludis possesses preflagellin peptidase activity; Bardy SL et al.; Archaeal flagellins are initially synthesized as preflagellins with a short, positively charged leader peptide, which is cleaved prior to the incorporation of the mature flagellins into the filament . While preflagellin peptidase activity had previously been detected in methanogen membranes, the enzyme responsible for this activity had not been identified . We show here that FlaK of Methanococcus maripaludis has preflagellin peptidase activity . In an in vitro preflagellin peptidase assay, Escherichia coli membranes overexpressing Methanococcus voltae preflagellin FlaB2 (as substrate) were combined with E . coli membranes overexpressing M . maripaludis FlaK (as enzyme) . Cleavage of the preflagellin was demonstrated by immunoblotting using antibody to FlaB2 and detection of a faster migrating cross-reactive species . This activity required detergent in the assay, and was not detected in membranes previously heated to 95 degrees C . This is the first reported identification of the preflagellin peptidase, and aside from the flagellins, this is the first assignment of function to a gene involved in archaeal flagellation. FEMS Microbiol Lett, 2002 Feb 19, 208(1), 47 - 51 Pseudomurein endoisopeptidases PeiW and PeiP, two moderately related members of a novel family of proteases produced in Methanothermobacter strains; Luo Y et al.; Sequence comparison of pseudomurein endoisopeptidases PeiW encoded by the defective prophage PsiM100 of Methanothermobacter wolfeii, and PeiP encoded by phage PsiM2 of Methanothermobacter marburgensis, revealed that the two enzymes share only limited similarity . Their amino acid sequences comprise an N-terminal domain characterized by the presence of direct repeats and a C-terminal domain with a catalytic triad C-H-D as in thiol proteases and animal transglutaminases . Both PeiW and PeiP catalyze the in vitro lysis of M . marburgensis cells under reducing conditions and exhibit characteristics of metal-activated peptidases . Optimal temperature and pH were determined to be 63 degrees C and 6.4 for His-tagged PeiP and 71 degrees C and 6.4 for His-tagged PeiW, respectively . Database search results suggest that PeiW and PeiP are the first two experimentally identified members of a novel family of proteases in a superfamily of archaeal, bacterial, and eukaryotic protein homologs of animal transglutaminases. FEMS Microbiol Lett, 2002 Feb 19, 208(1), 29 - 34 Biological effects of DNA damage in the hyperthermophilic archaeon Sulfolobus acidocaldarius; Reilly MS et al.; To investigate the generality of efficient double-strand break repair and damage-induced mutagenesis in hyperthermophilic archaea, we systematically measured the effects of five DNA-damaging agents on Sulfolobus acidocaldarius and compared the results to those obtained for Escherichia coli under corresponding conditions . The observed lethality of gamma-radiation was very similar for S . acidocaldarius and E . coli, arguing against unusually efficient double-strand break repair in S . acidocaldarius . In addition, DNA-strand-breaking agents (gamma-radiation or bleomycin), as well as DNA-cross-linking agents (mechlorethamine, butadiene diepoxide or cisplatin) stimulated forward mutation, reverse mutation, and formation of recombinants via conjugation in Sulfolobus cells . Although two of the five DNA-damaging agents failed to revert the E . coli auxotrophs under these conditions, all five reverted S . acidocaldarius auxotrophs. Mutat Res, 2002 Apr 25, 501(1-2), 129 - 36 Asymmetry of frameshift mutagenesis during leading and lagging-strand replication in Escherichia coli; Gawel D et al.; Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands . In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication . The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication . Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other . Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains . Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication. Biotechnol Prog, 2002 Mar-Apr, 18(2), 380 - 6 Assessment of the performance of a fed-batch cultivation from the preculture quality using an electronic nose; Cimander C et al.; An electronic nose, a gas-phase multisensor system, was used to monitor precultivations of a recombinant tryptophan-producing Escherichia coli strain . The electronic nose signals showed a high correlation toward the main stages of the precultivations, namely, exponential growth, oxygen-limited growth, and glucose depletion . Principal component analysis (PCA) of the electronic nose signals was performed and shown to be useful for monitoring preculture progression . More importantly, PCA also allowed a qualitative assessment of the preculture performance during subsequent fed-batch cultivations . The electronic nose signals from the precultures showed, furthermore, a high correlation to the time of phosphate limitation and the tryptophan yield coefficient of the subsequent fed-batch cultivations, which allowed an accurate prediction of these process variables using partial least squares (PLS) . The results demonstrate on data from 12 cultivations how the electronic nose can be a useful tool for the assessment of inoculum quality, thereby providing means of reducing