J Biol Chem, 2003 Oct 31, 278(44), 43682 - 90 Epub 2003 Aug 01. Crystal structure of Escherichia coli PdxA, an enzyme involved in the pyridoxal phosphate biosynthesis pathway; Sivaraman J et al.; Pyridoxal 5'-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids . Two pathways for its de novo synthesis are known . The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes . The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C . 1.1.1.262), which converts 4-hydroxy-l-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate . This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP+ or NAD+ as redox cofactor . We report the crystal structure of E . coli PdxA and its complex with HTP and Zn2+ . The protein forms tightly bound dimers . Each monomer has an alpha/beta/alpha-fold and can be divided into two subdomains . The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers . A Zn2+ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers . In addition two conserved amino acids, Asp247 and Asp267, play a role in maintaining integrity of the active site . The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn2+ ion . PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase . These structural similarities and the comparison with a NADP-bound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP. J Biol Chem, 2003 Oct 10, 278(41), 40408 - 14 Epub 2003 Aug 01. Mechanism underlying the inner membrane retention of Escherichia coli lipoproteins caused by Lol avoidance signals; Hara T et al.; Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2 . The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e . the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane . To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes . Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release . In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue . Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function . Based on these results, the Lol avoidance mechanism is discussed. Am J Physiol Lung Cell Mol Physiol, 2004 Feb, 286(2), L259 - 67 Epub 2003 Aug 01. Increased iNOS activity is essential for pulmonary epithelial tight junction dysfunction in endotoxemic mice; Han X et al.; A murine endotoxemia model and cultured Calu-3 monolayers were used to test the hypothesis that excessive nitric oxide (NO) production secondary to induction of inducible NO synthase (iNOS) is a key factor leading to altered tight junction (TJ) protein expression and function in the pulmonary epithelium . C57Bl/6J mice were injected with either Escherichia coli 0111:B4 lipopolysaccharide (LPS; 2 mg/kg) or vehicle . Twelve hours later, leakage of FITC-dextran (M(r) 4 kDa; FD4) from blood into bronchoalveolar lavage fluid was significantly increased in endotoxemic but not control mice . This decrease in bronchoalveolar barrier function was associated with upregulation of iNOS protein expression and NF-kappaB activation in lung tissue . Expression of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin, as assessed by immunoblotting and/or immunofluorescence, decreased in lung after the injection of mice with LPS . Treatment of endotoxemic mice with an isoform-selective iNOS inhibitor {l-N(6)-(1-iminoethyl)lysine; l-NIL} ameliorated LPS-induced changes in TJ protein expression and preserved bronchoalveolar epithelial barrier function . Incubating Calu-3 bronchiolar epithelial monolayers with cytomix (a mixture of 1,000 U/ml IFN-gamma, 10 ng/ml TNF-alpha, and 1 ng/ml IL-1beta) increased permeability to FD4, but adding l-NIL prevented this effect . These results suggest that decreased expression and mistargeting of TJ proteins in lung after systemic inflammation may be NO dependent. Neuron, 2003 Jul 31, 39(3), 513 - 28 Narp and NP1 form heterocomplexes that function in developmental and activity-dependent synaptic plasticity; Xu D et al.; Narp is a neuronal immediate early gene that plays a role in excitatory synaptogenesis . Here, we report that native Narp in brain is part of a pentraxin complex that includes NP1 . These proteins are covalently linked by disulfide bonds into highly organized complexes, and their relative ratio in the complex is dynamically dependent upon the neuron's activity history and developmental stage . Complex formation is dependent on their distinct N-terminal coiled-coil domains, while their closely homologous C-terminal pentraxin domains mediate association with AMPA-type glutamate receptors . Narp is substantially more effective in assays of cell surface cluster formation, coclustering of AMPA receptors, and excitatory synaptogenesis, yet their combined expression results in supraadditive effects . These studies support a model in which Narp can regulate the latent synaptogenic activity of NP1 by forming mixed pentraxin assemblies . This mechanism appears to contribute to both activity-independent and activity-dependent excitatory synaptogenesis. J Biochem Mol Biol, 2003 Jul 31, 36(4), 379 - 86 Gene therapy for mice sarcoma with oncolytic herpes simplex virus-1 lacking the apoptosis-inhibiting gene, icp34.5; Lan P et al.; A mutant herpes simplex virus 1, mtHSV, was constructed by inserting the E . coli beta-galactosidase gene into the loci of icp34.5, the apoptosis-inhibiting gene of HSV . The mtHSV replicated in and lysed U251 (human glioma cells), EJ (human bladder cells), and S-180 (mice sarcoma cells), but not Wish (human amnion cells) cells . With its intact tk (thymidine kinase) gene, mtHSV exhibited susceptibility to acyclovir (ACV), which provided an approach to control viral replication . An in vivo test with mtHSV was conducted in immune-competent mice bearing sarcoma S-180 tumors, which were treated with a single intratumoral injection of mtHSV or PBS . Tumor dimensions then were measured at serial time points, and the tumor volumes were calculated . Sarcoma growth was significantly inhibited with prolonged time and reduced tumor volume . There was microscopic evidence of necrosis of tumors in treated mice, whereas no damage was found in other organs . Immunohistochemical staining revealed that virus replication was exclusively confined to the treated tumor cells . HSV-1 DNA was detected in tumors, but not in the other organs by a polymerase chain reaction analysis . From these experiments, we concluded that mtHSV should be a safe and promising oncolytic agent for cancer treatment. Liver Int, 2003 Aug, 23(4), 276 - 82 Glycine modulates cytokine secretion, inhibits hepatic damage and improves survival in a model of endotoxemia in mice; Bruck R et al.; BACKGROUND AND AIM: There is substantial experimental evidence that the amino acid glycine may have a role in protecting tissues against insults such as ischemia, hypoxia and reperfusion . Our aim was to investigate the ability of the amino acid glycine to prevent hepatic damage induced by injection of lipopolysaccharide and d-galactosamine (d-Gal), to modulate pro- and anti-inflammatory cytokine levels, and to improve survival . METHODS: Mice were challenged with an intraperitoneal injection of d-Gal (16 mg/mouse) and lipopolysaccharide (LPS, 1 microg/mouse) . The intervention group also received an intraperitoneal injection of glycine (150 mg/kg) in two doses: 24 h before and just after LPS challenge . Serum cytokine levels were measured 2 h after challenge, and liver enzymes and histology were determined 16 h after LPS . Separate groups of mice received the same treatment and the survival rate was determined 24 h and ten days after endotoxin administration . In in vitro experiments, cultured mononuclear cells were stimulated by LPS, and TNF-alpha and IL-10 secretion were measured, in the presence or absence of glycine . RESULTS: In the glycine-treated mice, the serum levels of liver enzymes and TNF-alpha, the histologic necroinflammation score and the mortality rate were significantly reduced compared to control mice (P<0.001) . Serum IL-10 levels in the glycine-treated mice were increased (P<0.01) . In vitro studies in cultured lymphocytes isolated from either normal or glycine pretreated mice, demonstrated a significant and dose-dependent inhibition of LPS-induced TNF-alpha secretion and increase in IL-10 response after treatment with glycine (P<0.01) . In conclusion, glycine reduces hepatic damage and improves survival rate in this mouse model of endotoxemia . The protective effect of glycine is associated with modulation of TNF-alpha and IL-10 secretion. Pathol Int, 2003 Aug, 53(8), 525 - 33 Cross-reactive antigenicity of nucleoproteins of lyssaviruses recognized by a monospecific antirabies virus nucleoprotein antiserum on paraffin sections of formalin-fixed tissues; Inoue S et al.; Diagnosis of rabies is routinely confirmed by detection of rabies virus antigens in acetone-fixed frozen brain tissues or imprint smears using an immunofluorescence method with commercial antirabies virus antibodies . Since recent molecular analyses disclosed wide heterogeneity in the genome sequences of rabies virus strains and related lyssaviruses, it is necessary to confirm the presence of common epitopes in these lyssaviruses . In this study we confirmed the presence of cross-reactive antigens of various lyssaviruses in paraffin sections of formalin-fixed tissue using a monospecific rabbit antiserum prepared by immunization with a recombinant nucleoprotein of rabies virus . By immunohistochemical application, the antigen was detected predominantly in the cytoplasm of neurons in the brains of mice infected with rabies virus, Duvenhage virus, Mokola virus and European bat lyssavirus-1, while no cross-reaction was observed in uninfected humans and animals including dogs, bats, and raccoons . In addition, we examined one autopsy case that was infected in a rabies-endemic nation and developed the clinical manifestation of rabies after returning to Japan in 1970, and found that the antigen was well preserved in paraffin sections of formalin-fixed tissues . Thus, this suggests that the lyssavirus-specific antigen is recognized by the monospecific antibody against rabies virus nucleoprotein, and that this cross-reactive antigen is detectable on formalin-fixed paraffin-embedded tissues by immunohistochemical analysis. J Org Chem, 2003 Aug 8, 68(16), 6222 - 8 Access to optically pure 4- and 5-substituted lactones: a case of chemical-biocatalytical cooperation; Wang S et al.; Optically pure or highly enantiomerically enriched 4- and 5-substituted lactones are rather difficult to obtain . Chemical or enzymatic syntheses alone are not particularly successful . A combination of chemical catalysis and biocatalysis, however, provides a convenient route to a variety of these useful chiral compounds . In this paper we describe the synthesis of several optically pure 4- and 5-substituted lactones obtained via whole cell-catalyzed Baeyer-Villiger oxidations of highly enantiomerically enriched 3-alkyl cyclic ketones . Such chiral ketones are readily accessed by recently developed copper-catalyzed asymmetric conjugate reductions of the corresponding enones . A very high proximal regioselectivity and complete chirality transfer was obtained by employing biological Baeyer-Villiger oxidations, using recombinant E . coli strains that overexpress cyclopentanone monooxygenase (CPMO) . A comparative study showed that CPMO gives superior results to those obtained with cyclohexanone monooxygenase (CHMO) catalyzed oxidations. Cell Death Differ, 2003 Dec, 10(12), 1310 - 9 Unique structural features of a BCL-2 family protein CED-9 and biophysical characterization of CED-9/EGL-1 interactions; Woo JS et al.; The interactions between B-cell lymphoma 2 (BCL-2) family members are known to be mediated through the binding of the BH3 domain of a proapoptotic member to the BH3-binding groove of an antiapoptotic member . We determined the crystal structure of antiapoptotic CED-9, which reveals a unique C-terminal helix altering the common BH3-binding region . A coexpression system to produce CED-9 in complex with proapoptotic EGL-1 enabled us to show that the binding of EGL-1 to CED-9 is extremely stable, raising the melting temperature (T(M)) of CED-9 by 25 degrees C, and that the binding surface of CED-9 extends beyond the BH3-binding region and reaches the BH4 domain . Consistently, the T(M) and a 1H-15N correlation NMR spectrum of CED-9 in complex with EGL-1 are drastically different from those of CED-9 in complex with the EGL-1 BH3 peptide . The data suggest that the recognition between other BCL-2 family members may also involve much wider protein surfaces than is previously thought . Published Online 1 August 2003 Pharmacogenetics, 2003 Aug, 13(8), 495 - 500 Deleterious mutations in the flavin-containing monooxygenase 3 (FMO3) gene causing trimethylaminuria; Zhang J et al.; The primary genetic form of trimethylaminuria (TMAU) is caused by inherited defects in the flavin-containing monooxygenase 3 (FMO3) gene . Defective FMO3 has a decreased ability to catalyze the N-oxygenation of the dietary-derived malodourous amine, trimethylamine . We report two novel deleterious mutations identified in two unrelated individuals affected by the disorder . Sequence analysis of the FMO3 coding exons amplified from genomic DNA revealed that the mutation from individual 1 was heterozygous for a G>A missense mutation in exon 2 of the FMO3 gene . The mutation changed a GAG encoding Glu at codon 32 to AAG encoding Lys . Wild-type and mutant E32K FMO3 were expressed in Escherichia coli as maltose binding-fusion proteins and assayed for their ability to catalyze oxygenation of various FMO3 substrates . The results showed that the E32K mutation abrogated the catalytic activity of the enzyme . Individual 2 was identified as heterozygous for the P153L mutation . In addition, individual 2 was also heterozygous for a novel single nucleotide deletion of A191 in exon 3 at codon 64 . The deletion resulted in a frame shift and caused premature termination of the FMO3 gene immediately after codon 65 . Family pedigree analysis revealed that the P153L and the deletion mutation were carried on different alleles for this individual . Therefore, both alleles of the FMO3 gene for individual 2 were affected by mutations abolishing the catalytic activity of the enzyme, explaining the severe TMAU condition . The two deleterious mutations reported herein were rare mutations with estimated allelic frequencies of less than 1%. Pharmacogenetics, 2003 Aug, 13(8), 461 - 72 Genetic findings and functional studies of human CYP3A5 single nucleotide polymorphisms in different ethnic groups; Lee SJ et al.; OBJECTIVES: Genetic polymorphisms of cytochromes P450 (CYPs) are a principal reason for inter-individual variations in the metabolism of therapeutic drugs and environmental chemicals in humans . The present study identifies 34 single nucleotide polymorphisms (SNPs) of CYP3A5 including 27 previously unidentified SNPs by direct sequencing of the exons, intron-exon junctions and 5'-upstream region of CYP3A5 from 92 racially diverse individuals (24 Caucasians, 24 Africans, 24 Asians, and 20 individuals of unknown racial origin) . RESULTS: Four new CYP3A5 SNPs produced coding changes: R28C, L82R, A337T, and F446S . CYP3A5 R28C occurred in African populations (allelic frequency of 4%) . CYP3A5 A337T occurred in Asians (2% allelic frequency), CYP3A5 L82R (occurred in the racially unidentified group) and CYP3A5 F446S (identified in Caucasians with a 2% allelic frequency) were on an allele containing the splice change g.6986A>G known as CYP3A5*3 . The newly identified allelic proteins were constructed by site-directed mutagenesis, expressed in Escherichia coli and purified . CYP3A5 L82R was expressed only as denatured CYP420, suggesting it may be unstable . CYP3A5*1 exhibited the highest maximal clearance for testosterone followed by CYP3A5 A337T > CYP3A5 R28C >> CYP3A5 F446S . CYP3A5*1 exhibited a higher V(max) for nifedipine oxidation than CYP3A5 A337T > CYP3A5 R28C >> CYP3A5 F446S . CYP3A5 A337T and CYP3A5 R28C exhibited a 42-64% lower V(max) for nifedipine oxidation than CYP3A5*1 . CYP3A5 F446S exhibited a > 95% decrease in the intrinsic clearance for both 6beta-hydroxytestosterone and nifedipine oxidation . CONCLUSION: This study identifies four new potentially defective coding alleles . CYP3A5 F446S is predicted to be more catalytically defective than the splice change alone. Science, 2003 Aug 1, 301(5633), 657 - 61 Screening for nitric oxide-dependent protein-protein interactions; Matsumoto A et al.; Because nitric oxide (NO) may be a ubiquitous regulator of cellular signaling, we have modified the yeast two-hybrid system to explore the possibility of NO-dependent protein-protein interactions.We screened for binding partners of procaspase-3, a protein implicated in apoptotic signaling pathways, and identified multiple NO-dependent interactions.Two such interactions, with acid sphingomyelinase and NO synthase, were shown to occur in mammalian cells dependent on endogenous NO.Nitrosylation may thus provide a broad-based mechanism for regulating interactions between proteins.If so, systematic proteomic analyses in which redox state and NO bioavailability are carefully controlled will reveal a large array of novel interactions. Science, 2003 Aug 1, 301(5633), 616 - 20 Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli; Huang Y et al.; The major facilitator superfamily represents the largest group of secondary membrane transporters in the cell . Here we report the 3.3 angstrom resolution structure of a member of this superfamily, GlpT, which transports glycerol-3-phosphate into the cytoplasm and inorganic phosphate into the periplasm . The amino- and carboxyl-terminal halves of the protein exhibit a pseudo two-fold symmetry . Closed off to the periplasm, a centrally located substrate-translocation pore contains two arginines at its closed end, which comprise the substrate-binding site . Upon substrate binding, the protein adopts a more compact conformation . We propose that GlpT operates by a single-binding site, alternating-access mechanism through a rocker-switch type of movement. Science, 2003 Aug 1, 301(5633), 610 - 5 Structure and mechanism of the lactose permease of Escherichia coli; Abramson J et al.; Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins . We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters . The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the permease . A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter . The structure with a bound lactose homolog, beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified . We propose a possible mechanism for lactose/proton symport (co-transport) consistent with both the structure and a large body of experimental data. Biochem Biophys Res Commun, 2003 Aug 1, 307(3), 678 - 83 Absence of superoxide dismutase activity in a soluble cellular isoform of prion protein produced by baculovirus expression system; Sakudo A et al.; A method for expression and purification of a soluble form of histidine (HIS)-tagged murine prion protein (bacMuPrP), which lacks the entire C-terminal cleavage and glycosyl phosphatidyl inositol (GPI) addition site, has been developed using a recombinant baculovirus expression system and purification with Ni-NTA agarose affinity chromatography . In mammalian sources, PrP(C) is attached to the cell membrane by a GPI anchor . However, in our system, bacMuPrP was secreted into the media, enabling its easy purification in abundance . Indirect immunofluorescence studies and immunoblot analysis localized not in cell membrane but in the perinuclear endoplasmic reticulum region in cells and is secreted into the media . Tunicamycin treatment revealed non-glycosylated proteins were secreted into the media, suggesting that glycosylation is not necessary for bacMuPrP secretion . Density-gradient sedimentation analysis demonstrated a sedimentation coefficient of secretory bacMuPrP as 2.3 S, indicating a monomeric form . Although affinity-purified PrP from mouse brain or recombinant prion protein (PrP) produced by Escherichia coli and refolded in the presence of copper has been reported to display superoxide dismutase (SOD) activity, bacMuPrP did not show SOD activity . These results suggest that bacMuPrP has a different biochemical and biophysical characterization from mammalian and bacterial-derived PrP . Furthermore, this simple expression system may provide an adequate source for structural, functional, and biochemical analyses of PrP. Res Vet Sci, 2003 Oct, 75(2), 133 - 40 Lipopolysaccharide and interferon gamma activate nuclear factor kappa B and induce cyclo-oxygenase-2 in equine vascular smooth muscle cells; Janicke H et al.; Equine endotoxaemia is an important cause of morbidity and mortality in horses caused by the interaction of bacterial lipopolysaccharide (LPS) with cells such as macrophages and vascular smooth muscle . In this study we isolated equine vascular smooth muscle from a variety of vessels and stimulated it with LPS and human interferon (hIFN)-gamma . Using reverse transcriptase polymerase chain reaction (rt-PCR) and Western blot analysis we show that cyclooxygenase-2 (COX-2) is readily expressed in equine vascular smooth muscle . Vascular smooth muscle cells produced prostaglandin E2 in response to LPS and hIFNgamma . Using similar approaches we saw very limited expression of inducible nitric oxide synthase (iNOS) in only one vascular smooth muscle preparation . LPS and IFNgamma caused translocation of the transcription factor nuclear factor kappa B (NfkappaB) to the nucleus in equine cells suggesting the limited iNOS production seen in our cells is not due to deficits in this signal transduction pathway . These data suggest that in equine vascular smooth muscle COX-2 and NfkappaB are likely to play important roles in the pathogenesis of equine endotoxaemia. DNA Repair (Amst), 2003 Aug 12, 2(8), 863 - 78 Initiation of repair of A/G mismatches is modulated by sequence context; Sanchez AM et al.; The efficiency of DNA glycosylases to initiate base excision repair (BER) has been demonstrated to be modulated by the precise sequence context in which the lesion or mismatch is located . In the case of DNA containing an A/G mismatch, in which the recognition and excision of adenine from the mismatch is mediated by the Escherichia coli MutY enzyme, not only does the local sequence context affect the strength of base stacking interactions, but it also modulates the syn/anti conformation around the glycosyl bond of the bases in the mispair . Utilizing prior NMR data to identify DNA sequence contexts that adopt either an anti/anti or a syn/anti configuration at an A/G mismatch, we tested the hypothesis that the initial equilibrium of the mismatched base orientations would modulate the overall efficiency of glycosyl bond scission . By systematically varying the sequence context around a central A/G mismatch within a 30-mer duplex DNA, significant kinetic differences were observed that were consistent with this hypothesis . Since the relative efficiency of the kinetics fell into only two groupings, a NMR study was conducted on a DNA sequence context of unknown syn/anti conformation . These data established that the relative syn/anti conformation did not correlate with the excision efficiency, as well as there being a lack of correlation between kinetics and thermal stability of these DNAs. Lancet, 2003 Jul 26, 362(9380), 286 - 91 Protection from natural infections with enterotoxigenic Escherichia coli: longitudinal study; Steinsland H et al.; BACKGROUND: Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhoea and diarrhoeal deaths in children living in developing countries and of travellers' diarrhoea . During the past 25 years, vaccine development efforts have been focused on induction of protective immunity against surface colonisation factors (CFs) and the heat-labile enterotoxin . Although vaccines that induce immunity to heat-labile toxin offer protection against diarrhoea from ETEC that produce this toxin, the benefit of including CF antigens remains uncertain . We aimed to estimate the protection that natural ETEC infections induce against new infections . METHODS: In Guinea-Bissau, we followed up 200 neonates until up to age 2 years, most of whom were breastfed throughout the study . We collected stool specimens from the children every week irrespective of whether they had diarrhoea . As a measure of protection, we used Cox regression models to estimate the change in infection rates after a primary ETEC infection . We thus estimated the protection attributable to CFs, toxins, and to any other factors that could be shared by ETEC with the same toxin-CF profile . FINDINGS: ETEC infections induced a 47% (95% CI 12 to 69) protection against new infections with ETEC that had the same toxin-CF profile; the corresponding estimate attributable to CFs was -1% (-40 to 27) . Infection with heat-labile toxin-positive ETEC conferred a 45% (-1 to 70) protection against symptomatic infections with ETEC positive for this toxin . INTERPRETATION: For breastfed children living in endemic areas, other antigens are substantially more important than CFs for induction of protective immunity against ETEC infection. FEMS Microbiol Lett, 2003 Jul 29, 224(2), 175 - 81 Helicobacter pylori strain ATCC700392 encodes a methyl-accepting chemotaxis receptor protein (MCP) for arginine and sodium bicarbonate; Cerda O et al.; Helicobacter pylori ATCC43504 responds chemotactically to aspartic acid and serine, but not to arginine, nor to sodium bicarbonate . In contrast, H . pylori ATCC700392 (strain 26695) shows chemotaxis to all four attractants . Open reading frame HP0099 from H . pylori 26695 is predicted to encode one of three methyl-accepting chemotaxis receptor proteins (MCPs) . When Escherichia coli is transformed with a plasmid carrying HP0099 from strain 26695, the recombinants acquire chemotaxis to arginine, bicarbonate, and urea . In H . pylori 43504, the HP0099 gene is interrupted with a mini-IS605 insertion, which accounts for its inability to recognize arginine and bicarbonate as attractants . Together, these results argue that the H . pylori HP0099 gene encodes an MCP for arginine and bicarbonate. Eur J Pharmacol, 2003 Jul 25, 473(2-3), 171 - 6 Therapeutic effects of ghrelin on endotoxic shock in rats; Chang L et al.; We investigated the effects of ghrelin in a rat endotoxic shock model, and also observed the direct role of endotoxin on ghrelin generation in gastric mucosa . About 55% (11/20) of rats treated with lipopolysaccharide (5 mg/kg i.v.) alone died within 24 h of endotoxin injection . However, administration of ghrelin either at the same time as lipopolysaccharide injection (early treatment) or 12 h after lipopolysaccharide injection (late treatment) significantly decreased the mortality rate and ameliorated the hypotension seen in rats with endotoxic shock . Early and late treatment with ghrelin increased markedly the plasma glucose concentration and decreased the plasma lactate concentration . Early treatment with ghrelin attenuated significantly the deficiency in myocardial ATP content, but late treatment with ghrelin had no effect on myocardial ATP content . The plasma ghrelin level was significantly increased in the rats with endotoxin shock, and it increased further after ghrelin administration . Exposure of rat gastric mucosa in vitro to lipopolysaccharide (1.0 to 100 microg/ml) triggered the release of ghrelin from mucosa tissue in a dose- and time-dependent manner, meaning that lipopolysaccharide stimulated directly gastric mucosa to synthesize and secrete ghrelin . The results suggest that ghrelin could have therapeutic value for endotoxic shock. J Struct Biol, 2003 Jul, 143(1), 45 - 55 The mechanical properties of simple epithelial keratins 8 and 18: discriminating between interfacial and bulk elasticities; Yamada S et al.; The abundance and cytoplasmic organization of keratin filaments enables them to contribute to the maintenance of structural integrity in epithelial tissues . Co-polymers of the type II keratin 8 and type I keratin 18 form the major intermediate filament network in simple epithelia . We investigated the mechanical properties of K8-K18 filament suspensions using rheological assays in conjunction with light and electron microscopy . Suspensions of K8-K18 filaments behave like a viscoelastic solid under standard assembly conditions . Bulk elasticity is weakly dependent on deformation frequency but is very sensitive to the concentration (G' approximately C1.5) and size of individual keratin polymers, in agreement with recent models of semiflexible-polymer physics . K8-K18 filaments can self-organize to form a bundled network that exhibits gel-like mechanical properties . In all cases the mechanical properties of the suspensions correlate with the structural features of individual polymers, as seen under light and electron microscopy . Importantly, these bulk viscoelastic properties of K8-K18 filaments are revealed only when interfacial elastic effects are minimized by the application of phospholipids at the air-liquid interface . Suspensions of K5-K14 and vimentin filaments also exhibit interfacial elasticity, which distorts the interpretation of the viscoelastic moduli as determined by standard rheometry . The potential for modulation of mechanical properties through self-organization may be a general property of keratin polymers and contribute to their organization and function in vivo. BJOG, 2003 Aug, 110(8), 735 - 43 The effect of systemic administration of lipopolysaccharide on cerebral haemodynamics and oxygenation in the 0.65 gestation ovine fetus in utero; Peebles DM et al.; OBJECTIVE: To investigate the effect of intravenous lipopolysaccharide on systemic and cerebral haemodynamics and oxygenation in the preterm ovine fetus . DESIGN: Prospective observational study . SETTING: Research centre for perinatal brain injury . SAMPLE: Nine fetal sheep at circa 93 days of gestation (0.65) . METHODS: Fetal sheep were chronically instrumented with arterial and venous catheters and a flow probe in the carotid artery . Near-infrared spectroscopy was used to measure changes in cerebral oxygenation and total haemoglobin concentration . Three days after surgery, each fetus was given 100 ng/kg Escherichia coli lipopolysaccharide . Observations were continued for 48 hours post-injection and compared with baseline control values . MAIN OUTCOME MEASURES: Fetal heart rate, mean arterial pressure, carotid blood flow . RESULTS: Three fetuses died after administration of the lipopolysaccharide . In the survivors fetal heart rate rose from 193 (SEM 7) to a mean maximal level of 226 (SEM 31 bpm) (P = 0.01) after 6.5 (SEM 1.0) hours . The mean arterial pressure decreased from 40.5 (SEM 4.2) to 29.4 (SEM 1.6) mmHg (P < 0.05) after 7.0 (SEM 2.0) hours, and carotid blood flow increased from 29.6 (SEM 1.6) to 45.8 (SEM 5.7) mL/min (P = 0.0002) at 12 (SEM 3) hours . All values returned to control levels by 48 hours . Histological assessment showed evidence of periventricular leucomalacia in three out of six brains studied . CONCLUSION: These data do not suggest that cerebral ischaemia is the main aetiological factor in endotoxin-related fetal brain injury . Fetal tachycardia and cerebral vasodilation may indicate endotoxaemia in fetuses exposed to prenatal infection. J Biol Chem, 2003 Oct 17, 278(42), 40996 - 1002 Epub 2003 Jul 30. Crystal structure of carbapenam synthetase (CarA); Miller MT et al.; Carbapenam synthetase (CarA) is an ATP/Mg2+-dependent enzyme that catalyzes formation of the beta-lactam ring in (5R)-carbapenem-3-carboxylic acid biosynthesis . CarA is homologous to beta-lactam synthetase (beta-LS), which is involved in clavulanic acid biosynthesis . The catalytic cycles of CarA and beta-LS mediate substrate adenylation followed by beta-lactamization via a tetrahedral intermediate or transition state . Another member of this family of ATP/Mg2+-dependent enzymes, asparagine synthetase (AS-B), catalyzes intermolecular, rather than intramolecular, amide bond formation in asparagine biosynthesis . The crystal structures of apo-CarA and CarA complexed with the substrate (2S,5S)-5-carboxymethylproline (CMPr), ATP analog alpha,beta-methyleneadenosine 5'-triphosphate (AMP-CPP), and a single Mg2+ ion have been determined . CarA forms a tetramer . Each monomer resembles beta-LS and AS-B in overall fold, but key differences are observed . The N-terminal domain lacks the glutaminase active site found in AS-B, and an extended loop region not observed in beta-LS or AS-B is present . Comparison of the C-terminal synthetase active site to that in beta-LS reveals that the ATP binding site is highly conserved . By contrast, variations in the substrate binding pocket reflect the different substrates of the two enzymes . The Mg2+ coordination is also different . Several key residues in the active site are conserved between CarA and beta-LS, supporting proposed roles in beta-lactam formation . These data provide further insight into the structures of this class of enzymes and suggest that CarA might be a versatile target for protein engineering experiments aimed at developing improved production methods and new carbapenem antibiotics. J Biotechnol, 2003 Aug 15, 103(3), 249 - 55 Molecular engineering of biotin-glutamic acid decarboxylase 65 fusion protein (Biotin-GAD65) for non-radioactive GAD65 antibody assay; Luo D et al.; We constructed biotinylated fusion proteins that linked to three detection tags to GAD65 at the N-terminus, and expressed them in an E . coli expression system . The Biotin14-GAD65 protein exhibited the strongest binding to both the GAD65 antibody and the streptavidin among the three constructs . We describe the optimal conditions using a Biotin14-GAD65-based immunoassay for the detection of GAD65 antibody. Comp Biochem Physiol A Mol Integr Physiol, 2003 Aug, 135(4), 613 - 24 Recombinant goldfish growth hormones (gfGH-I and -II) expressed in Escherichia coli have similar biological activities; Chan YH et al.; Complementary DNA regions coding for two different mature goldfish growth hormones (gfGH-I and gfGH-II) with four and five cysteine residues were cloned into the bacterial expression vector, pRSETA . The recombinant gfGH-I (five cysteines) and -II (four cysteines) were produced in Escherichia coli as the fusion proteins carrying N-terminal 6XHis tag, which facilitates purification by using metal chelating affinity chromatography under denaturing condition with urea . The recombinant hormones were further refolded by gradually removing the urea . Native gfGH was also purified from goldfish pituitary glands and served as a positive control in the present study . The native and recombinant hormones were tested in goldfish hepatic radioreceptor assay and in vitro Spi 2.1 promoter activation assay . Our results showed that the two recombinant gfGHs are biologically active, and they have similar biological activities despite their having different cysteine contents. Mol Microbiol, 2003 Aug, 49(4), 1109 - 18 Distant cis-active sequences and sialic acid control the expression of fimB in Escherichia coli K-12; El-Labany S et al.; The phase variation of type 1 fimbriation in Escherichia coli is controlled by the inversion of a 314 bp element of DNA, determined by FimB (switching in both directions) or FimE (switching from the ON-to-OFF orientation predominantly), and influenced by auxiliary factors IHF, Lrp and H-NS . The fimB gene is separated from the divergently transcribed yjhATS operon by a large (1.4 kbp) intergenic region of unknown function . Here, we show that fimB expression is regulated by multiple cis-active sequences that lie far upstream (>600 bp) of the transcription start sites for the recombinase gene . Two regions characterized further (regions 1 and 2) show sequence identity, and each coincides with a methylation-protected Dam (5'-GATC) site . Regions 1 and 2 apparently control fimB expression by an antirepression mechanism that involves additional sequences proximal to yjhA . Region 1 encompasses a 27 bp DNA sequence conserved upstream of genes known (nanAT ) or suspected (yjhBC) to be involved in sialic acid metabolism, and we show that FimB expression and recombination are suppressed by N-acetylneuraminic acid . We propose that E . coli recognizes the amino sugars as a harbinger of potential host defence activation, and suppresses the expression of type 1 fimbriae in response. Mol Microbiol, 2003 Aug, 49(4), 947 - 63 Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori; Contreras M et al.; The Helicobacter pylori genome contains a gene (hp1338 or nikR) that encodes a nickel-dependent regulator that is homologous to the Escherichia coli nickel-responsive regulator, NikR . The H . pylori nikR product acts as a pleiotropic metal-dependent regulator . We constructed a non-polar isogenic mutant deleted for the nikR gene . NikR was essential for the survival of H . pylori in the presence of high nickel and cobalt ion concentrations in vitro . We screened a DNA macroarray for genes that were differentially expressed in parental and nikR-deficient H . pylori strains grown in the presence of excess nickel . We found that H . pylori NikR mediates the expression of nickel-activated and -repressed genes . In the presence of excess nickel, NikR activated the transcription of ureA-ureB (hp72-73), nixA (hp1077 ), copA2 (hp1072), hpn (hp1427 ) and hpn-like (hp1432) genes and repressed the expression of genes encoding proteins involved in ferric iron uptake and storage {pfr (hp0653), fur (hp1027 ), frpB4 (hp1512), exbB/exbD (hp1339-1340), ceuE (hp1561)}, motility {cheV (hp616), flaA (hp0601), flaB (hp0115 )}, stress responses {hrcA-grpE-dnaK (hp111-110-109)} and encoding outer-membrane proteins {omp11(hp0472), omp31 (hp1469), omp32 (hp1501)} . Slot blot DNA/RNA hybridization experiments using RNA from three independent bacterial cultures confirmed the transcriptome data for 10 selected genes . The results of gel shift experiments using purified native NikR, beta-galactosidase assays with the region between nikR and the exbB/exbD divergent operon, and the study of exbB gene expression using a gentamicin/apramycin reporter gene in H . pylori indicated that NikR is an autorepressor that binds to this intergenic region and also controls the expression of the exbB/exbD/tonB operon, which provides energy for ferric iron uptake . Thus, as previously suggested for Fur in H . pylori, NikR appears to be a global regulator of the metabolism of some divalent cations within a highly complex regulated network. Biotechnol Lett, 2003 Jul, 25(13), 1111 - 6 Metal chelate affinity precipitation of RNA and purification of plasmid DNA; Balan S et al.; The affinity of metal chelates for amino acids, such as histidine, is widely used in purifying proteins, most notably through six-histidine 'tails' . We have found that metal affinity interactions can also be applied to separation of single-stranded nucleic acids through interactions involving exposed purines . Here we describe a metal affinity precipitation method to resolve RNA from linear and plasmid DNA . A copper-charged copolymer of N-isopropyl acrylamide (NIPAM) and vinyl imidazole (VI) is used to purify plasmid from an alkaline lysate of E . coli . The NIPAM units confer reversible solubility on the copolymer while the imidazole chelates metal ions in a manner accessible to interaction with soluble ligands . RNA was separated from the plasmid by precipitation along with the polymer in the presence of 800 mM NaCl . Bound RNA could be recovered by elution with imidazole and separated from copolymer by a second precipitation step . RNA binding showed a strong dependence on temperature and on the type of buffer used. Biotechnol Lett, 2003 Jul, 25(13), 1041 - 7 A single residual replacement improves the folding and stability of recombinant cassava hydroxynitrile lyase in E . coil; Yan G et al.; Substitution of Ser113 for Gly113 in the cap domain of hydroxynitrile lyase from Manihot esculenta (MeHNL) was performed by site-directed mutagenesis to improve its self-generated folding and stability under denaturation conditions . The yield of the recombinant mutant HNL1 (mut-HNL1), which had higher specific activity than the wild type HNL0 (wt-HNL0), was increased by 2 to 3-fold . Thermostability of MeHNL was also enhanced, probably due to an increase in content of the beta-strand secondary structure according to CD analysis . Our data in this report suggest that Ser113 significantly contributes to the in vivo folding and stability of MeHNL and demonstrates an economic advantage of mut-HNL1 over the wt-HNL0. Biotechnol Lett, 2003 Jul, 25(13), 1037 - 40 Fusion protein of the hyaluronan binding domain from human TSG-6 with luciferase for assay of hyaluronan; Chang TS et al.; The gene expression plasmid, pET-Lmluc, for the fusion protein of the hyaluronan binding domain from human TSG-6 {product of tumor necrosis factor (TNF)-stimulated gene-6} and luciferase from Renilla reniformis was constructed . The fused gene was expressed in Escherichia coli and the resulted insoluble Lm-luc fusion protein was purified and refolded to recover both the hyaluronan binding capability and the luciferase activity . Hyaluronan as low as 1 ng ml(-1) was detected by using the indirect enzymatic immunological assay with the refolded Lm-luc fusion protein. Biotechnol Lett, 2003 Jul, 25(13), 1025 - 9 Nucleofection as an efficient nonviral transfection method for human monocytic cells; Martinet W et al.; Despite some progress in the field of gene transfer into hard-to-transfect cells, so far an efficient nonviral method for monocytes has not been available . A comparison of plasmid DNA with capped and polyadenylated mRNA for enhanced green fluorescent protein gene delivery into the commonly used monocytic cell lines U937 and THP-1 suggested that limited DNA trafficking may be the underlying cause of poor transfection results . As Nucleofector technology delivers DNA (or mRNA) straight into the nucleus, we obtained nucleofection efficiencies of up to 80% without significant cell toxicity . Moreover, as the DNA quickly reaches the nucleus, nucleofected cells were ready for analysis after only 2-6 h . The technique is suitable not only for monocytes but also for other hard-to-transfect cells. Biotechnol Lett, 2003 Jul, 25(13), 1013 - 7 Functional expression of arginine kinase improves recovery from pH stress of Escherichia coli; Canonaco F et al.; Acid stress in Escherichia coli involves a complex resource- and energy-consuming response mechanism . By overexpression of arginine kinase from Limulus polyphemus in E . coli, we improved the recovery from a transient pH stress . While wild type E . coli resumed growth after a transient pH reduction to pH 3 for 1 h with a rate that was 25% lower than before the stress, the arginine kinase expressing strain continued to grow as rapidly as before . This effect is presumably caused by the physiological function of arginine kinase as a short term energy buffer in the form of phosphoarginine, but a pH-buffering effect cannot be excluded. Biotechnol Lett, 2003 Jun, 25(11), 909 - 15 Functional identification of rub52 gene involved in the biosynthesis of rubradirin; Maharjan J et al.; An open reading frame, rub52, has been identified as a gene encoding thymidine diphospho-glucose 2,3-dehydratase by sequence analysis of the rubradirin biosynthetic gene cluster of Streptomyces achromogenes var . nibradiris NRRL3061.The gene codes for a protein consisting of 458 amino acids with calculated molecular mass of 50862 Da . The gene was amplified and heterologously expressed in Escherichia coli as a soluble His-tagged fusion protein . C-2 deoxygenation functionality of thymidine diphospho-4-keto-6-deoxyglucose was assigned to the rub52 gene product from in vitro enzyme assay. Biotechnol Bioeng, 2003 Sep 20, 83(6), 646 - 52 Chitosan scaffolds for biomolecular assembly: coupling nucleic acid probes for detecting hybridization; Yi H et al.; Chitosan, a naturally occurring biopolymer, was used as a scaffold for the covalent binding of single-stranded DNA oligonucleotide probes in a fluorescence-based nucleic acid hybridization assay . Chitosan's pH dependent chemical and electrostatic properties enable its deposition on electrodes and metal surfaces, as well as on the bottom of microtiter plates . A combinatorial 96-well microtiter plate format was used to optimize chemistries and reaction conditions leading to hybridization experiments . We found the coupling of oligonucleotides using relatively common glutaraldehyde chemistry was quite robust . Our hybridization results for complementary ssDNA oligonucleotides (E . coli dnaK sequences) demonstrated linear fluorescence intensity with concentration of E . coli dnaK-specific oligonucleotide from 0.73 microM to 6.6 microM . Moreover, hybridization assays were specific as there was minimal fluorescence associated with noncomplementary groEL oligonucleotide . Finally, these results demonstrate the portability of a DNA hybridization assay based on covalent coupling to chitosan, which, in turn, can be deposited onto various surfaces . More arduous surface preparation techniques involving silanizing agents and hazardous washing reagents are eliminated using this technique . J Biomed Mater Res A, 2003 Aug 1, 66(2), 376 - 84 Heparin inhibits lipopolysaccharide (LPS) binding to leukocytes and LPS-induced cytokine production; Anastase-Ravion S et al.; The glycosaminoglycan heparin is known to exhibit anti-inflammatory properties unrelated to its anticoagulant activity . However, in a generalized inflammatory response with implanted or extracorporeal devices, the beneficial effect of heparin coating and/or systemic administration is still unclear as well as the precise mechanisms of action . In the present study, we have first studied the effect of heparin on lipopolysaccharide (LPS)-induced cytokine production by human blood monocytes . Our results indicated that the production of interleukin-1alpha, tumor necrosis factor-alpha, and interleukin-8 was significantly decreased when heparin was simultaneously incubated with Escherichia coli LPS . Because the modulation of heparin on monocyte activation could be mediated by its binding via CD14, the main LPS receptor on monocytes, we then studied the binding of LPS and heparin to leukocytes from human blood and to Chinese hamster ovary cells transfected with the human CD14 gene . The data by flow cytometry showed the binding of biotinylated heparin to leukocytes . Moreover, the experiments performed on leukocytes and on CD14-positive Chinese hamster ovary cells indicated that heparin inhibited LPS binding . From our results, we conclude that: 1 . heparin is an effective inhibitor of LPS-induced monocyte activation, and 2 . heparin inhibits the binding of LPS to cells via a CD14-independent pathway . This study suggests a potentially important therapeutic application for heparin or heparin analogs to prevent inflammation with biomaterials . Thromb Haemost, 2003 Aug, 90(2), 206 - 17 Biochemical importance of glycosylation of plasminogen activator inhibitor-1; Gils A et al.; The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy . PAI-1 has 3 potential sites for N-linked glycosylation . We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosylation pattern of the sites at N209 and N265, while that at N329 is not utilised . The IC(50)-values for inactivation of PAI-1 by 4 monoclonal antibodies differed strongly between glycosylated PAI-1 and non-glycosylated PAI-1 expressed in E . coli . For 3 antibodies, an overlap of the epitopes with the glycosylation sites could be excluded as explanation for the differential reactivity . The latency transition of non-glycosylated, but not of glycosylated PAI-1, was strongly accelerated by a non-ionic detergent . The different biochemical properties of glycosylated and non-glycosylated PAI-1 depended specifically on glycosylation of either one or the other of the utilised sites . The PAI-1-binding protein vitronectin reversed the changes associated with the lack of glycosylation at one of the sites . Our results stress the importance of the source of PAI-1 when studying the mechanisms of action of PAI-1-inactivating compounds of potential clinical importance. J Biol Chem, 2003 Oct 17, 278(42), 41443 - 51 Epub 2003 Jul 29. Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88; Dunne A et al.; The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily . The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components . Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves . Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP . To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88 . We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct . Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling . Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes . The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations . Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88 . Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways. Nucleic Acids Res, 2003 Aug 1, 31(15), 4573 - 81 Repair of clustered uracil DNA damages in Escherichia coli; D'souza DI et al.; Multiply damaged sites (MDS) are defined as greater than/equal to two lesions within 10-15 bp and are generated in DNA by ionizing radiation . In vitro repair of closely opposed base damages > or =2 bp apart results in a double strand break (DSB) . This work extends the in vitro studies by utilizing clusters of uracil DNA damage as model lesions to determine whether MDS are converted to DSBs in bacteria . Lesions were positioned within the firefly luciferase coding region, transformed into bacteria (wild-type, uracil DNA glycosylase-deficient, ung-, or exonuclease III and endonuclease IV-deficient, xth-nfo-) and luciferase activity measured following repair . DSB formation was expected to decrease activity . Two closely opposed uracils separated by < or =7 bp decreased luciferase activity in wild-type and xth-nfo-, but not ung- bacteria . Growth of bacteria to obtain plasmid-containing colonies demonstrated that the plasmid was destroyed following the mis-repair of two uracils positioned 7 bp apart . This study indicates a DSB is formed when uracil DNA glycosylase initiates repair of two closely opposed uracils < or =7 bp apart, even in the absence of the major apurinic endonucleases . This work supports the in vitro studies and demonstrates that DNA repair is not always advantageous to cells. J Mol Biol, 2003 Aug 8, 331(2), 407 - 16 Crystal structures of methionine adenosyltransferase complexed with substrates and products reveal the methionine-ATP recognition and give insights into the catalytic mechanism; Gonzalez B et al.; Methionine adenosyltransferases (MATs) are a family of enzymes in charge of synthesising S-adenosylmethionine (SAM), the most important methyl donor present in living organisms . These enzymes use methionine and ATP as reaction substrates, which react in a S(N)2 fashion where the sulphur atom from methionine attacks C5' from ATP while triphosphate chain is cleaved . A MAT liver specific isoenzyme has been detected, which exists in two distinct oligomeric forms, a dimer (MAT III) and a tetramer (MAT I) . Our previously reported crystal structure of MAT I complexed with an inhibitor led to the identification of the methionine-binding site . We present here the results obtained from the complex of MAT I with a competitive inhibitor of methionine, (2S,4S)-amino-4,5-epoxypentanoic acid (AEP), which presents the same features at the methionine binding site reported before . We have also analysed several complexes of this enzyme with methionine and ATP and analogues of them, in order to characterise the interaction that is produced between both substrates . The crystal structures of the complexes reveal how the substrates recognise each other at the active site of the enzyme, and suggest a putative binding site for the product SAM . The residues involved in the interactions of substrates and products with MAT have been identified, and the results agree with all the previous data concerning mutagenesis experiments and crystallographic work . Moreover, all the information provided from the analysis of the complexes has allowed us to postulate a catalytic mechanism for this family of enzymes . In particular, we propose a key role for Lys182 in the correct positioning of the substrates, and Asp135(*), in stabilising the sulphonium group formed in the product (SAM). J Mol Biol, 2003 Aug 8, 331(2), 385 - 93 Structure of Mycobacterium tuberculosis single-stranded DNA-binding protein . Variability in quaternary structure and its implications; Saikrishnan K et al.; Single-stranded DNA-binding protein (SSB) is an essential protein necessary for the functioning of the DNA replication, repair and recombination machineries . Here we report the structure of the DNA-binding domain of Mycobacterium tuberculosis SSB (MtuSSB) in four different crystals distributed in two forms . The structure of one of the forms was solved by a combination of isomorphous replacement and anomalous scattering . This structure was used to determine the structure of the other form by molecular replacement . The polypeptide chain in the structure exhibits the oligonucleotide binding fold . The globular core of the molecule in different subunits in the two forms and those in Escherichia coli SSB (EcoSSB) and human mitochondrial SSB (HMtSSB) have similar structure, although the three loops exhibit considerable structural variation . However, the tetrameric MtuSSB has an as yet unobserved quaternary association . This quaternary structure with a unique dimeric interface lends the oligomeric protein greater stability, which may be of significance to the functioning of the protein under conditions of stress . Also, as a result of the variation in the quaternary structure the path adopted by the DNA to wrap around MtuSSB is expected to be different from that of EcoSSB. J Mol Biol, 2003 Aug 8, 331(2), 331 - 44 Mechanism of transcriptional activation by FIS: role of core promoter structure and DNA topology; Auner H et al.; The Escherichia coli DNA architectural protein FIS activates transcription from stable RNA promoters on entry into exponential growth and also reduces the level of negative supercoiling . Here we show that such a reduction decreases the activity of the tyrT promoter but that activation by FIS rescues tyrT transcription at non-optimal superhelical densities . Additionally we show that three different "up" mutations in the tyrT core promoter either abolish or reduce the dependence of tyrT transcription on both high negative superhelicity and FIS in vivo and infer that the specific sequence organisation of the core promoter couples the control of transcription initiation by negative superhelicity and FIS . In vitro all the mutations potentiate FIS-independent untwisting of the -10 region while at the wild-type promoter FIS facilitates this step . We propose that this untwisting is a crucial limiting step in the initiation of tyrT RNA synthesis . The tyrT core promoter structure is thus optimised to combine high transcriptional activity with acute sensitivity to at least three major independent regulatory inputs: negative superhelicity, FIS and ppGpp. Mutat Res, 2003 Sep, 544(1), 65 - 85 Theoretical analysis of mutation hotspots and their DNA sequence context specificity; Rogozin IB et al.; Mutation frequencies vary significantly along nucleotide sequences such that mutations often concentrate at certain positions called hotspots . Mutation hotspots in DNA reflect intrinsic properties of the mutation process, such as sequence specificity, that manifests itself at the level of interaction between mutagens, DNA, and the action of the repair and replication machineries . The hotspots might also reflect structural and functional features of the respective DNA sequences . When mutations in a gene are identified using a particular experimental system, resulting hotspots could reflect the properties of the gene product and the mutant selection scheme . Analysis of the nucleotide sequence context of hotspots can provide information on the molecular mechanisms of mutagenesis . However, the determinants of mutation frequency and specificity are complex, and there are many analytical methods for their study . Here we review computational approaches for analyzing mutation spectra (distribution of mutations along the target genes) that include many mutable (detectable) positions . The following methods are reviewed: derivation of a consensus sequence, application of regression approaches to correlate nucleotide sequence features with mutation frequency, mutation hotspot prediction, analysis of oligonucleotide composition of regions containing mutations, pairwise comparison of mutation spectra, analysis of multiple spectra, and analysis of "context-free" characteristics . The advantages and pitfalls of these methods are discussed and illustrated by examples from the literature . The most reliable analyses were obtained when several methods were combined and information from theoretical analysis and experimental observations was considered simultaneously . Simple, robust approaches should be used with small samples of mutations, whereas combinations of simple and complex approaches may be required for large samples . We discuss several well-documented studies where analysis of mutation spectra has substantially contributed to the current understanding of molecular mechanisms of mutagenesis . The nucleotide sequence context of mutational hotspots is a fingerprint of interactions between DNA and DNA repair, replication, and modification enzymes, and the analysis of hotspot context provides evidence of such interactions. Cell, 2003 Jul 25, 114(2), 157 - 9 Loading Rho to terminate transcription; Richardson JP; In bacteria, one of the major transcriptional termination mechanisms requires a hexameric RNA/DNA helicase known as Rho . One question that has remained unanswered is how the helicase loads onto a nascent transcript so that it can initiate actions on the transcript to cause termination . Recent structures of Rho bound to nucleic acid by show how the individual RNA-binding domains of the 6 subunits are organized and that the ring is split open . The opening is wide enough to accommodate single-stranded RNA and suggests that this conformation is poised to load onto mRNA. Avian Dis, 2003 Apr-Jun, 47(2), 415 - 24 Effect of ochratoxin A on Escherichia coli-challenged broiler chicks; Kumar A et al.; A study was conducted to evaluate the effects of ochratoxin A (OA) on Escherichia coli-challenged broiler chickens . Day-old broiler chicks were separated into two groups of 92 chicks each, with one group fed a control mash diet, and the other fed a mash diet containing 2 ppm OA . On day 14, each group was further separated into two groups, with one group inoculated with E . coli O78 (1 x 10(7) colony-forming units/0.5 ml), whereas the other group was not inoculated with E . coli . After E . coli inoculation on day 14, four birds from each group were euthanatized at 1, 2, 3, 5, 7, 10, 14, and 21 days postinoculation . Escherichia coli infection caused dullness, depression, huddling, and diarrhea . Mortality was 14.3% in chicks infected with E . coli but fed no OA . Mortality increased to 35.7% in chicks fed OA and infected with E . coli . Decreased body weight and reduced feed intake were observed in chicks fed OA, and the effects were more pronounced in chicks fed OA and infected with E . coli . Increased serum levels of aspartate aminotransferase, alanine aminotransferase, uric acid, and creatinine and decreased levels of total proteins, albumin, globulins, calcium, and phosphorus were observed in OA-fed birds . Escherichia coli infection did not cause significant alteration in any of the serum biochemical parameters . The presence of OA in poultry rations increased mortality and the severity of an E . coli infection. Avian Dis, 2003 Apr-Jun, 47(2), 396 - 405 Enhancement of enteropathogenic Escherichia coli pathogenicity in young turkeys by concurrent turkey coronavirus infection; Pakpinyo S et al.; In a previous study, turkey coronavirus (TCV) and enteropathogenic Escherichia coli (EPEC) were shown to synergistically interact in young turkeys coinfected with these agents . In that study, inapparent or mild disease was observed in turkeys inoculated with only TCV or EPEC, whereas severe growth depression and high mortality were observed in dually inoculated turkeys . The purpose of the present study was to further evaluate the pathogenesis of combined TCV/EPEC infection in young turkeys and determine the role of these agents in the observed synergistic interaction . Experiments were conducted to determine 1) effect of EPEC dose, with and without concurrent TCV infection, and 2) effect of TCV exposure, before and after EPEC exposure, on development of clinical disease . Additionally, the effect of combined infection on TCV and EPEC shedding was determined . No clinical sign of disease and no attaching and effacing (AE) lesions characteristic of EPEC were observed in turkeys inoculated with only EPEC isolate R98/5, even when turkeys were inoculated with 10(10) colony forming units (CFU) EPEC (high dose exposure) . Only mild growth depression was observed in turkeys inoculated with only TCV; however, turkeys inoculated with both TCV and 10(4) CFU EPEC (low dose exposure) developed severe disease characterized by high mortality, marked growth depression, and AE lesions . Inoculation of turkeys with TCV 7 days prior to EPEC inoculation produced more severe disease (numerically greater mortality, significantly lower survival probability {P < 0.05}, increased frequency of AE lesions) than that observed in turkeys inoculated with EPEC prior to TCV or simultaneously inoculated with these agents . Coinfection of turkeys with TCV and EPEC resulted in significantly increased (P < 0.05) shedding of EPEC, but not TCV, in intestinal contents of turkeys . These findings indicate that TCV infection predisposes young turkeys to secondary EPEC infection and potentiates the expression of EPEC pathogenicity in young turkeys. Avian Dis, 2003 Apr-Jun, 47(2), 370 - 9 Prediction of chicken embryo lethality with the avian Escherichia coli traits complement resistance, colicin V production, and presence of the increased serum survival gene cluster (iss); Gibbs PS et al.; Differentiating between virulent and avirulent avian Escherichia coli isolates continues to be a problem for poultry diagnostic laboratories and the study of colibacillosis in poultry . The ability of a laboratory to conduct one simple test that correlates with virulence would simplify studies in these areas; however, previous studies have not enabled researchers to establish such a test . In this study, the occurrence of certain phenotypic and genotypic traits purported to contribute to avian E . coli virulence in 20 avian E . coli isolates was correlated with the results of embryo challenge studies . This analysis was undertaken in an effort to determine which trait(s) best identified each avian E . coli isolate as virulent or avirulent . Traits selected were complement resistance, production of colicin V (ColV), motility, type F1 pili expression, presence of the temperature-sensitive hemagglutinin gene (tsh), and presence of the increased serum survival genetic locus (iss) . ColV production, complement resistance, and presence of the iss genetic element were the three traits most highly correlated with high embryo lethality . A logistic regression model was used to predict the embryo lethality results on the basis of the most frequent isolate characteristics . Results indicate that ColV, complement resistance, and if are significant predictor variables for the percentage of embryo lethality resulting from challenge with a specific avian E . coli isolate . However, no single trait has the ability to predict virulent isolates 100% of the time . Such results suggest the possibility that the embryo lethality assay may prove to be the one test needed to determine if an avian E . coli isolate is virulent. Cell Mol Biol (Noisy-le-grand), 2003 May, 49(3), 389 - 97 Glycosylation of zona pellucida glycoprotein-3 is required for inducing acrosomal exocytosis in the bonnet monkey; Gahlay GK et al.; To investigate the role of polypeptide backbone vis-a-vis glycosylation of the putative primary sperm receptor, the bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 (bmZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was expressed as polyhistidine fusion protein either in Escherichia coli orusing baculovirus expression system . The recombinant bmZP3 (r-bmZP3) was purified using nickel-nitrilotriacetic acid resin and subsequently refolded in the presence of oxidized and reduced glutathione . SDS-PAGE and Western blot analysis revealed approximately 43 and approximately 52 kDa bands corresponding to E . coli and baculovirus expressed r-bmZP3, respectively . The r-bmZP3 purified from both E . coli and baculovirus binds to the principal segment of the acrosomal cap of the capacitated bonnet monkey spermatozoa as evaluated by indirect immunofluoresence assay . In a competitive inhibition assay, the binding of biotinylated baculovirus expressed r-bmZP3 to capacitated spermatozoa was inhibited not only by cold baculovirus expressed r-bmZP3 but also by E . coli expressed r-bmZP3 and vice-versa . Lectin binding studies revealed that the baculovirus r-bmZP3 has N-linked glycosylation with galactose and mannose residues . Capacitated spermatozoa, in the presence of baculovirus expressed r-bZP3, undergoes a significant increase (p < 0.01) in the acrosomal exocytosis as compared to control whereas E . coli expressed r-bmZP3 failed to have a significant effect . These results suggest that though the polypeptide backbone of ZP3 is sufficient for its binding to capacitated spermatozoa, yet glycosylation is required for induction of acrosomal exocytosis. J Microbiol Immunol Infect, 2003 Jun, 36(2), 149 - 52 Culture-to-culture physical interactions causes the alteration in red and infrared light stimulation of Escherichia coli growth rate; Trushin MV; Escherichia coli MC1061 cells were irradiated at 660 and 900 nm, incubated in M9 and LB media with the use of a specially constructed device, and assayed for growth rate . There was a reduction of growth rate stimulation when the irradiated culture was cultivated jointly with the non-irradiated one . In the same time, the irradiated culture extended the invigorate effect on the non-irradiated one . It is proposed that the effects observed were mediated by culture-to-culture physical interactions. DNA Res, 2003 Jun 30, 10(3), 129 - 36 High-throughput production of recombinant antigens for mouse KIAA proteins in Escherichia coli: computational allocation of possible antigenic regions, and construction of expression plasmids of glutathione-S-transferase-fused antigens by an in vitro recombination-assisted method; Hara Y et al.; Since the end of 2001, we have conducted a project to isolate and determine entire sequences of mouse cDNA clones which encode the polypeptides corresponding to human KIAA proteins . Towards the ultimate goal of this project to clarify the biological functions of KIAA genes, we have set production of antibodies against mouse KIAA gene products based on their sequence information as the next important stage . As the first step, we developed a high-throughput system utilizing shotgun clones generated during entire sequencing of mouse KIAA cDNAs . The system consists of the following three parts: (1) Shotgun clones encoding regions suitable for production of antigens were selected using a newly developed browser system; (2) the protein-coding sequences of the selected shotgun clones were transferred into an expression vector by in vitro recombination-assisted method in a 96-well format, and expressed as glutathione S-transferase fusion proteins in Escherichia coli; and (3) the solubility of the recombinant antigens were preliminarily assessed in a small-scale culture and then large-scale production and purification was performed using glutathione-affinity beads or retrieval from polyacrylamide gels depending on their solubility . Using these systems, we successfully produced and purified 400 antigens for production of mKIAA antibodies to date. C R Biol, 2003 May, 326(5), 501 - 8 Dynamic simulation of pollutant effects on the threonine pathway in Escherichia coli; Chassagnole C et al.; The enzymatic activities of threonine pathway in Escherichia coli are sensitive to pollutants such as cadmium, copper and mercury, which, even at low concentration, can substantially decrease or even block the pathway at several steps . Our aim was to investigate the complex effects on a metabolic pathway of such general enzyme inhibitors with several sites of action, using a previously developed computer simulation of the pathway . For this purpose, the inhibition parameters were experimentally determined and incorporated in the model . The calculation of the flux control coefficient distribution between the five steps of the threonine pathway showed that control remains shared between the three first steps under most inhibition conditions . Response coefficient analysis shows that the inhibition of aspartate semialdehyde dehydrogenase is quantitatively dominant in most circumstances. Zh Mikrobiol Epidemiol Immunobiol, 2003 Mar-Apr, (2), 7 - 12 {Isolation and identification of growth stimulators of Escherichia coli M-17}; Vakhitov TIa et al.; The stimulating activity of E . coli M-17 culture fluid (CF) is determined by the action of low-molecular exometabolites, readily soluble in water . The high-molecular fraction was removed from CF by ultrafiltration, the growth stimulators were adsorbed on anion exchange resin and eluated at pH 3.1 . In further purification HPLC and chromatography on TSK gel HW-40 were used . The identification of compounds was carried out by the methods of thin-layer chromatography, amino acid analysis and 1H-NMR spectroscopy . Glutamic acid (glutamine) and succinic acid proved to be the most active growth autostimulators . The data of biological testing made it possible to believe that CF also contained less active stimulators and/or synergic substances which had no their own activity, but stimulated growth, acting jointly with other compounds . In view of the definite specificity of action observed in initial CF, some differences in the spectrum of growth stimulators of other E . coli strains may be supposed. Kisaengchunghak Chapchi, 1986 Dec, 24(2), 213 - 215 {A survey on intestinal parasites of soldiers in Korea}; Hong ST; Total of 2,643 Korean soldiers were examined of their stool for parasitic infections by both cellophane thick smear and formalin-ether concentration techniques from August 1983 to December 1985 . Out of them, 73.6% were free from any parasite, 22.6% were ova positive and 4.0% cyst positive . The ova positive rates by species were Ascaris lumbricoides 2.0%, Trichuris trichiura 13.0%, hookworm and Trichostrongylus orientalis 0.08% respectively, Clonorchis sinensis 7.6%, Metagonimus yokogawai 1.1%, Paragonimus westermani 0.08%, Echinostoma hortense 0.04%, Fibricola seoulensis 0.9%, Taenia sp . 0.3%, Hymenolepis nana 0.2% and H . diminuta 0.04% . Most of them (87.3%) were positive by a species, 11.9% by two and 0.8% by 3 species . The cyst positive rates were Entamoeba histolytica 0.6%, E . coli 1.4%, Endolimax nana 1.7%, Iodoameba butschlii 0.04% and Giardia lambla 0.9% . Among the cyst positives, 89.5% were positive by a species, 7.6% by two and 0.5% by 3 species . The intestinal parasite infections among the Korean soldiers decreased distinctly compared with previous data. Proc Natl Acad Sci U S A, 2003 Aug 5, 100(16), 9548 - 53 Epub 2003 Jul 28. Differential regulation of microtubule dynamics by three- and four-repeat tau: implications for the onset of neurodegenerative disease; Panda D et al.; The microtubule (MT)-associated protein tau is important in neuronal development and in Alzheimer's and other neurodegenerative diseases . Genetic analyses have established a cause-and-effect relationship between tau dysfunction/misregulation and neuronal cell death and dementia in frontotemporal dementia and parkinsonism associated with chromosome 17; several mutations causing this dementia lead to increased ratios of four-repeat (4R) to three-repeat (3R) wild-type tau, and an attractive hypothesis is that the abnormally high ratio of 4R to 3R tau might lead to neuronal cell death by altering normal tau functions in adult neurons . Thus, we tested whether 3R and 4R tau might differentially modulate the dynamic instability of MTs in vitro using video microscopy . Although both isoforms promoted MT polymerization and decreased the tubulin critical subunit concentration to approximately similar extents, 4R tau stabilized MTs significantly more strongly that 3R tau . For example, 4R tau suppressed the shortening rate, whereas 3R tau had little or no detectable effect . Similarly, 3R tau had no effect on the length shortened during a shortening event, whereas 4R tau strongly reduced this parameter . Further, when MTs were diluted into buffer containing 4R tau, the MTs were stabilized and shortened slowly . In contrast, when diluted into 3R tau, the MTs were unstable and shortened rapidly . Thus, 4R tau stabilizes MTs differently and significantly more strongly than 3R tau . We suggest a "dosage effect" or haploinsufficiency model in which both tau alleles must be active and properly regulated to produce appropriate amounts of each tau isoform to maintain MT dynamics within a tolerable window of activity. J Virol, 2003 Aug, 77(16), 8801 - 11 The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex; Bosch BJ et al.; Coronavirus entry is mediated by the viral spike (S) glycoprotein . The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit . The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2 . HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it . Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins . We investigated the role of these regions in coronavirus membrane fusion . Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli . When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex . Both on their own and within the complex, the peptides were highly alpha helical . Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length . Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion . In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity . Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion . Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein. J Virol, 2003 Aug, 77(16), 8669 - 75 Identification of a region of the tobacco mosaic virus 126- and 183-kilodalton replication proteins which binds specifically to the viral 3'-terminal tRNA-like structure; Osman TA et al.; UV irradiation of a mixture of an isolated tobacco mosaic virus (TMV; tomato strain L {TMV-L}) RNA-dependent RNA polymerase complex and the TMV-L RNA 3'-terminal region (3'-TR) resulted in cross-linking of the TMV-L 126-kDa replication protein to the TMV-L 3'-TR . Using both Escherichia coli-expressed proteins corresponding to parts of the 126-kDa protein and mutants of the 3'-TR, the interacting sites were located to a 110-amino-acid region just downstream of the core methyltransferase domain in the protein and a region comprising the central core C and domain D2 in the 3'-TR . Mutation to alanine of a tyrosine residue at position 409 or a tyrosine residue at position 416 in the protein binding region abolished cross-linking to the 3'-TR, and corresponding mutations introduced into TMV-L RNA abolished its ability to replicate in tomato protoplasts, with no detectable production of either plus- or minus-strand RNA . The results are compatible with a model for initiation of TMV-L minus-strand RNA synthesis in which an internal region of the TMV-L 126-kDa protein first binds to the central core C and domain D2 region of the TMV-L 3'-TR and is then followed by binding of the 183-kDa protein to this complex and positioning of the catalytically active site of the polymerase domain close to the 3'-terminal CCCA initiation site. J Biol Chem, 2003 Oct 3, 278(40), 38514 - 21 Epub 2003 Jul 28. Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: Role of toxin acylation and identification of the main integrin interaction domain; El-Azami-El-Idrissi M et al.; Adenylate cyclase toxin (CyaA) is one of the major virulence factors produced by Bordetella pertussis, the whooping cough agent . CyaA belongs to the repeat in toxin protein family and requires a post-translational fatty acylation to form cation-selective channels in target cell membranes and to penetrate into cytosol . We have demonstrated recently that CyaA uses the alphaMbeta2 integrin (CD11b/CD18) as a specific cellular receptor . Here we show that the acylation of CyaA is required for a productive and tight interaction of the toxin with cells expressing CD11b . In addition, we demonstrate that the catalytic domain is not required for binding of CyaA to CD11b and that the main integrin interacting domain of CyaA is located in its glycine/aspartate-rich repeat region . These data decipher, for the first time, the interaction of CyaA with CD11b-positive cells and open new prospects for understanding the interaction of Bordetella pertussis with innate and adaptive immune systems. Biophys J, 2003 Aug, 85(2), 744 - 54 Metabolic switching in the sugar phosphotransferase system of Escherichia coli; Thattai M et al.; Bacteria grown in a mixture of multiple sugars will first metabolize a preferred sugar until it is nearly depleted, only then turning to other carbon sources in the medium . This sharp switching of metabolic preference is characteristic of systems that optimize fitness . Here we consider the mechanism by which switching can occur in the Escherichia coli phosphotransferase system (PTS), which regulates the uptake and metabolism of several sugars . Using a model combining the description of fast biochemical processes and slower genetic regulation, we derive metabolic phase diagrams for the uptake of two PTS sugars, indicating regions of distinct sugar preference as a function of external sugar concentrations . We then propose a classification of bacterial phenotypes based on the topology of the metabolic phase diagram, and enumerate the possible topologically distinct phenotypes that can be achieved through mutations of the PTS . This procedure reveals that there is only one nontrivial switching phenotype that is insensitive to large changes in biochemical parameters . This phenotype exhibits diauxic growth, a manifestation of the winner-take-all dynamics enforced by PTS architecture . Winner-take-all behavior is implemented by the induction of sugar-specific operons, combined with competition between sugars for limited phosphoryl flux . We propose that flux-limited competition could be a common mechanism for introducing repressive interactions in cellular networks, and we argue that switching behavior similar to that described here should occur generically in systems that implement such a mechanism. Biochemistry, 2003 Aug 5, 42(30), 9227 - 34 Quantitative identification of the protonation state of histidines in vitro and in vivo; Shimba N et al.; The C{bond}N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states . In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C{bond}N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states . This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments . We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1. Biochemistry, 2003 Aug 5, 42(30), 9153 - 9 Characterization of a lactose permease mutant that binds IIAGlc in the absence of ligand; Sondej M et al.; Enzyme IIA(Glc) of the Escherichia coli phosphoenolpyruvate:glucose phosphotransferase system plays a direct role in regulating inducible transport systems . Dephosphorylated IIA(Glc) binds directly to lactose permease in a reaction that requires binding of a galactosidic substrate . A double-Cys mutation (Ile129 --> Cys/Lys131 --> Cys) was introduced into helix IV of the permease near the IIA(Glc) binding site in cytoplasmic loop IV/V and in the vicinity of the galactoside binding site at the interface of helices IV, V, and VIII . The mutant no longer requires galactoside for IIA(Glc) binding as demonstrated by both a {(125)I}IIA(Glc) binding assay and a newly developed fluorescence anisotropy assay . Further characterization of the mutant shows that it binds substrate with high affinity, but is almost completely defective in all modes of translocation across the cytoplasmic membrane . The data are consistent with the interpretation that the double mutant is locked in an inward-facing conformation. Biochemistry, 2003 Aug 5, 42(30), 9121 - 6 Metal-1,4-dithio-2,3-dihydroxybutane chelates: novel inhibitors of the Rho transcription termination factor; Weber TP et al.; Rho is an enzyme that is essential for the growth and survival of Escherichia coli, and bicyclomycin (1) is its only known selective inhibitor . We show that metal (Cd(2+), Ni(2+), and Zn(2+)) complexes of 1,4-dithio-2,3-dihydroxybutanes (2) serve as effective and potent rho inhibitors with I(50) values that can exceed that of 1 . Maximal inhibition for ZnCl(2) and L-dithiothreitol (2a) corresponded to Zn(2):L-DTT stoichiometry . The I(50) value for the 2:1 Zn-L-DTT solution was 20 microM, which made it 3 times more potent than 1 (I(50) = 60 microM) . Kinetic studies showed that a Zn-L-DTT solution functioned as a noncompetitive inhibitor with respect to ATP in the rho poly(C)-dependent ATPase assay and as a competitive inhibitor with respect to ribo(C)(10) in the poly(dC).ribo(C)(10)-stimulated ATPase assay . These findings demonstrated that both 1 and a Zn-L-DTT solution disrupted rho-mediated ATP hydrolysis but that they inhibit using different mechanisms . Substitution of L-DTT with 1,2-ethanedithiol in ZnCl(2) solutions led to a comparable loss of rho poly(C)-dependent ATPase activity, indicating that other metal chelates can serve as efficient inhibitors . The site and pathway of rho inhibition by the putative metal-1,4-dithio-2,3-dihydroxybutane chelates are discussed in light of the current data. Biochemistry, 2003 Aug 5, 42(30), 9050 - 9 Thermodynamic linkage in the GrpE nucleotide exchange factor, a molecular thermosensor; Gelinas AD et al.; GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the bacterial homologue of Hsp70 . In the temperature range of the bacterial heat shock response, the long helices of GrpE undergo a helix-to-coil transition, and GrpE exhibits non-Arrhenius behavior with respect to its nucleotide exchange function . It is hypothesized that GrpE acts as a thermosensor and that unwinding of the long helices of E . coli GrpE reduces its activity as a nucleotide exchange factor . In turn, it was proposed that temperature-dependent down-regulation of the activity of GrpE may increase the time in which DnaK binds its substrates at higher temperatures . A combination of thermodynamic and hydrodynamic techniques, in concert with the luciferase refolding assay, were used to characterize a molecular mechanism in which the long helices of GrpE are thermodynamically linked with the beta-domains via an intramolecular contact between Phe86 and Arg183 . These "thermosensing" long helices were found to be necessary for full activity as a nucleotide exchange factor in the luciferase refolding assay . Point mutations in the beta-domains and in the long helices of GrpE destabilized the beta-domains . Engineered disulfide bonds in the long helices alternately stabilized the long helices and the four-helix bundle . This allowed the previously reported 75 degrees C thermal transition seen in the excess heat capacity function as monitored by differential scanning calorimetry to be further characterized . The observed thermal transition represents the unfolding of the four-helix bundle and the beta-domains . The thermal transitions for these two domains are superimposed but are not thermodynamically linked. Biochemistry, 2003 Aug 5, 42(30), 9028 - 40 Deletion of DnaK's lid strengthens binding to the nucleotide exchange factor, GrpE: a kinetic and thermodynamic analysis; Chesnokova LS et al.; In this study, we have used surface plasmon resonance (SPR) and isothermal microtitration calorimetry (ITC) to study the mechanism of complex formation between the Hsp70 molecular chaperone, DnaK, and its cochaperone, GrpE, which is a nucleotide exchange factor . Experiments were geared toward understanding the influence of DnaK's three domains, the ATPase (residues 1-388), substrate-binding (residues 393-507), and lid (residues 508-638) domains, on complex formation with GrpE . We show that the equilibrium dissociation constants for the interaction of GrpE with wtDnaK, lidless DnaK(2-517), the ATPase domain (2-388), and the substrate-binding fragment (393-507) are 64 (+/-16) nM, 4.0 (+/-1.5) nM, 35 (+/-10) nM, and 67 (+/-11) microM, respectively, and that the on-rate constant for the different reactions varies by over 4 orders of magnitude . SPR experiments revealed that GrpE-DnaK(393-507) complex formation is inhibited by added peptide and abolished when the 33-residue flexible "tail" of GrpE is deleted . Such results strongly suggest that the 33-residue flexible N-terminal tail of GrpE binds in the substrate-binding pocket of DnaK . This unique mode of binding between GrpE's tail and DnaK contributes to, but does not fully explain, the decrease in K(d) from 64 to 4 nM upon deletion of DnaK's lid . The possibility that deletion of DnaK's lid creates a more symmetrically shaped molecule, with enhanced affinity to GrpE, is also discussed . Our results reveal a complex set of molecular interactions between DnaK and its cochaperone GrpE . We discuss the impact of each domain on complex formation and dissociation. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 2003, 21(1), 12 - 5 {Cloning and expression of specific antigen genes of Ancylostoma caninum}; Guo XR et al.; OBJECTIVE: To search for the gene encoding specific antigen of Ancylostoma caninum that induces host's protective immunity . METHODS: A lambda ZAP II cDNA library of A . caninum was screened with sera from dogs immunized subcutaneously with hookworm larvae(L3) . After sequencing, insert gene (AcAg) from positive clones was ligated into PUC18 and PET28C . Recombinant pET28C plasmid was induced to expressed protein in the E . coli BL21 . The characteristic of recombinant protein is analyzed by SDS-PAGE and Western blotting assay . RESULTS: Five positive clones were obtained, and proved to be the same . The insert gene (AcAg) in pET28C vector expressed a recombinant protein of about 43 kDa . Using Western blotting assay, this protein was recognized by the sera from dog immunized with Ancylostoma caninum third-stage infected larvae and used for screening library . CONCLUSION: The AcAg, which exhibits 35% homologous to Caenorhabditis elegans gene unc-89, is a novel specific antigen of A . caninum . Its ability to elicit the protective immunity and the probability of the recombinant protein as a vaccine need to be further evaluated. Genetika, 2003 Jun, 39(6), 748 - 57 {Effect of insertional mutation in the cspA gene encoding the major cold-shock protein on radiation resistance of Escherichia coli}; Verbenko VN et al.; Plasmid pCspA::Km carrying a cloned mutant allele of the cspA gene for the major Escherichia coli cold-shock protein CspA with an insertion of the kanamycin resistance gene cassette from transposon Tn903 into the core region of the coding sequence causes a 2.3-fold increase in radioresistance of wild-type E . coli cells (cspA+) . The radioprotective effect of this plasmid is abolished or drastically reduced in mutants recA13 and rpoH15 defective in RecA protein and in induction of the heat-shock protein regulon, respectively . Plasmid pCspA::Km causes a 1.3-fold elevation in the resistance to gamma-irradiation of E . coli mutants with an intermediate level of radioresistance (Gamr445 and KS0160) but slightly diminishes resistance of a highly radiation-resistant Gamr445 mutant . In the chromosome of E . coli with normal DNA repair systems, the cspA::Km mutation in the homozygous state enhances resistance to the lethal effect of gamma-rays and UV light 2.9 and 1.4 times, respectively . These data suggest that the system of cold-shock proteins can modulate resistance of E . coli cells to the lethal effect of gamma-rays and UV light. Mol Carcinog, 2003 Jul, 37(3), 122 - 9 Ets protein Elf-1 bidirectionally suppresses transcriptional activities of the tumor suppressor Tsc2 gene and the repair-related Nth1 gene; Honda S et al.; Alterations in the rat tuberous sclerosis gene (Tsc2) cause renal cell carcinomas (RCCs) with complete penetrance . In this study, it was shown that the minimal core promoters of the rat Tsc2 and endonuclease III 1 (Nth1) genes, lying in a 5'-to-5' arrangement, were localized in a 0.11-kb region containing two Ets binding sites (EBSs) . This region worked as a bidirectional promoter in a single reporter plasmid . Mutational inactivation of each of the two EBSs significantly reduced promoter activity . Moreover, gel shift assays revealed the presence of specific EBSs-protein complexes . These results demonstrate that some members of the Ets family positively regulate the promoter activities of the Tsc2/Nth1 genes by binding to the EBSs . We identified Elf-1 as a binding factor for EBSs through super-shift assays, and detected approximately 35 kDa bands with an EBSs-containing DNA probe by Southwestern blot analysis . Forced expression of Elf-1 in cells, however, bidirectionally suppressed the activities of the Tsc2/Nth1 promoters . Elf-1 may be a negative regulator of Tsc2/Nth1 gene expression and may compete against positive regulators for binding to the EBSs . Our observations suggest that mechanisms that inactivate Tsc2 gene expression, such as promoter suppression, may exist . Appl Microbiol Biotechnol, 2003 Aug, 62(2-3), 239 - 43 Epub 2003 Mar 20. Effect of cysteine desulfhydrase gene disruption on L-cysteine overproduction in Escherichia coli; Awano N et al.; In Escherichia coli, the enzyme called cysteine desulfhydrase (CD), which is responsible for L-cysteine degradation, was investigated by native-PAGE and CD activity staining of crude cell extracts . Analyses with gene-disrupted mutants showed that CD activity resulted from two enzymes: tryptophanase (TNase) encoded by tnaA and cystathionine beta-lyase (CBL) encoded by metC . It was also found that TNase synthesis was induced by the presence of L-cysteine . The tnaA and metC mutants transformed with the plasmid containing the gene for feedback-insensitive serine acetyltransferase exhibited higher L-cysteine productivity than the wild-type strain carrying the same plasmid . These results indicated that TNase and CBL did act on L-cysteine degradation in E . coli cells. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jul, 35(7), 649 - 54 {Cloning, expression, and antibody production of mouse ISP2}; Huang ZP et al.; A cDNA encoding mouse implantation serine proteinase 2 ISP2 was amplified from total cDNAs of mouse uterus implantation sites on D4.5 of pregnancy by PCR, and sequenced GenBank accession No . A442918 . DNA sequencing indicated that the ISP2 cDNA had an unreported 204 bp sequence at 3' untranslated region besides the open reading frame encoding 279 amino acid residues, which was identical with literature . In order to obtain recombinant ISP2 rISP2 an expression plasmid pGEX-4T-2/ISP2 was constructed and transformed into E.coli BL21(DE3) strain . Expressed fusion protein GST-ISP2 was purified by SDS-PAGE and digested with thrombin, and the digestion mixture was subjected to SDS-PAGE again to recover rISP2 . Rabbits were immunized using rISP2 as immunogen, and the polyclonal anti-ISP2 antisera were obtained . Immunohistochemical analysis using this antisera showed specific and high expression of ISP2 in mouse endometrial gland epithelium in early pregnancy. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jul, 35(7), 624 - 8 {Biological function of fusion protein ATF-PAI2CD}; Wang X et al.; To express the fusion protein ATF-PAI2CD (urokinase-type plasminogen activator amino terminal fragment-plasminogen activator inhibitor type 2 with the region inter C and D helices deleted ) gene in E.coli and determine the biological characterization of fusion protein ATF-PAI2CD, the cDNA fragment encoding ATF-PAI2CD was cloned into the expression vector pLY-4 and transformed into E.coli JF1125 . After temperature induction, the expression amount of ATF-PAI2CD account for 15% of total bacterial protein . The result was confirmed by Western blot . ATF-PAI2CD protein was isolated and purified by washing and solubilization of inclusion body, renaturation and ion exchange chromatography . The final product displayed a single band with a corresponding molecular weight 62 kD in SDS-PAGE . The purity was over 90%, the protein yield was 50% and the specific activity was 12 000 IU/mg . The PAI activity was measured by chromogenic assay . The purified fusion protein inhibited urokinase-type plasminogen activator as measured by milk-agarose plate assay, and bound to human lung cancer cells via uPA receptor (uPAR), which was confirmed by radio competition experiments . The results indicate that the biological characteristics of ATF-PAI2CD were very similar to those of the wide type PAI-2 (or mutants PAI-2, PAI-2CD) and to pro-uPA in binding to uPAR-bearing cells. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Jul, 35(7), 601 - 5 Expression of recombinant human ICOS and in vitro characterization of its bioactivity on B lymphocytes; Deng ZB et al.; Inducible costimulator (ICOS) is a novel costimulatory molecule expressed in activated T cell and has critical regulation effect on special immune response . In this study, the cDNA encoding human ICOS was cloned from activated tonsil cells via RT-PCR, and was expressed in E . coli on pET28 expression vector . The recombinant ICOS protein expressed from E . coli showed a molecular weight of 14 kD on SDS-polyacrylamide gel electrophoresis and was further confirmed by Western blot . In presence of IL-10, the purified rhICOS significantly increased in vitro B cell growth stimulated by pokeweed mitogen (PWM), and enhanced the secretion of IgG from B cells. Toxicol Sci, 2003 Oct, 75(2), 448 - 57 Epub 2003 Jul 25. Developmental toxicity and genotoxicity studies of 1,1,1,3,3,3-hexachloropropane (HCC-230fa) in rats; Brock WJ et al.; The potential developmental toxicity and the in vitro and in vivo genotoxicity of HCC-230fa were assessed . In the developmental toxicity study, groups of 25 mated Crl:CD(R)(SD)BR rats were exposed (whole body) by inhalation to HCC-230fa over days 7-21 of gestation; the day of confirmed mating was designated as gestation day 1 (GD1) . Exposures were 6 h per day at concentrations of 0, 0.5, 2.5, or 25 ppm . Body weight, food consumption, and clinical observation data were collected during the study . On day 22 of gestation, the dams were euthanized and examined grossly . The fetuses were removed and subsequently weighed, sexed, and examined for external, visceral, head, and skeletal alterations . Evidence of maternal and developmental toxicity was observed at 25 ppm and was noted as significant, compound-related reductions in mean maternal body weight, weight change, and food consumption . Significant fetal effects also were observed at 25 ppm as compound-related reductions in mean fetal weight and increased fetal malformations (filamentous tail, situs inversus, absent vertebrae) and variations (rudimentary cervical ribs, delayed sternebral ossification) . There was no evidence of either maternal or developmental toxicity at 0.5 or 2.5 ppm . The genotoxicity of HCC-230fa was examined in a bacterial reversion assay and in erythrocyte micronucleus studies in two species by different routes of administration . No increases in the number of revertants were observed in the bacterial reversion assay . In one micronucleus study, HCC-230fa was administered by inhalation to rats as part of a 90-day study at doses indicated above . For the second study, ICR mice were given a single ip dose at 0, 166, 330, or 660 mg/kg . In both micronucleus studies, a significant increase in micronucleated erythrocytes was observed . The results of these studies suggest that HCC-230fa affects rapidly dividing cells and may have long-term consequences for occupational exposures. J Biol Chem, 2003 Oct 3, 278(40), 38505 - 13 Epub 2003 Jul 25. Juxtaposition of the two distal CX3C motifs via intrachain disulfide bonding is essential for the folding of Tim10; Allen S et al.; The TIM10 complex, composed of the homologous proteins Tim10 and Tim9, chaperones hydrophobic proteins inserted at the mitochondrial inner membrane . A salient feature of the TIM10 complex subunits is their conserved "twin CX3C" motif . Systematic mutational analysis of all cysteines of Tim10 showed that their underlying molecular defect is impaired folding (demonstrated by circular dichroism, aberrant homo-oligomer formation, and thiol trapping assays) . As a result of defective folding, clear functional consequences were manifested in (i) complex formation with Tim9, (ii) chaperone activity, and (iii) import into tim9ts mitochondria lacking both endogenous Tim9 and Tim10 . The organization of the four cysteines in intrachain disulfides was determined by trypsin digestion and mass spectrometry . The two distal CX3C motifs are juxtaposed in the folded structure and disulfide-bonded to each other rather than within each other, with an inner cysteine pair connecting Cys44 with Cys61 and an outer pair between Cys40 and Cys65 . These cysteine pairs are not equally important for folding and assembly; mutations of the inner Cys are severely affected and form wrong, non-native disulfides, in contrast to mutations of the outer Cys that can still maintain the native inner disulfide pair and display weaker functional defects . Taken together these data reveal this specific intramolecular disulfide bonding as the crucial mechanism for Tim10 folding and show that the inner cysteine pair has a more prominent role in this process. J Biol Chem, 2003 Oct 3, 278(40), 38796 - 802 Epub 2003 Jul 25. A novel mitochondrial carnitine-acylcarnitine translocase induced by partial hepatectomy and fasting; Sekoguchi E et al.; The carnitine-dependent transport of long-chain fatty acids is essential for fatty acid catabolism . In this system, the fatty acid moiety of acyl-CoA is transferred enzymatically to carnitine, and the resultant product, acylcarnitine, is imported into the mitochondrial matrix through a transporter named carnitine-acylcarnitine translocase (CACT) . Here we report a novel mammalian protein homologous to CACT . The protein, designated as CACL (CACT-like), is localized to the mitochondria and has palmitoylcarnitine transporting activity . The tissue distribution of CACL is similar to that of CACT; both are expressed at a higher level in tissues using fatty acids as fuels, except in the brain, where only CACL is expressed . In addition, CACL is induced by partial hepatectomy or fasting . Thus, CACL may play an important role cooperatively with its homologue CACT in a stress-induced change of lipid metabolism, and may be specialized for the metabolism of a distinct class of fatty acids involved in brain function. J Biol Chem, 2003 Oct 17, 278(42), 41069 - 76 Epub 2003 Jul 26. Reaction intermediate structures of 1-aminocyclopropane-1-carboxylate deaminase: insight into PLP-dependent cyclopropane ring-opening reaction; Ose T et al.; The pyridoxal 5'-phosphate-dependent enzymes have been evolved to catalyze diverse substrates and to cause the reaction to vary . 1-Aminocyclopropane-1-carboxylate deaminase catalyzes the cyclopropane ring-opening reaction followed by deamination specifically . Since it was discovered in 1978, the enzyme has been widely investigated from the mechanistic and physiological viewpoints because the substrate is a precursor of the plant hormone ethylene and the enzymatic reaction includes a cyclopropane ring-opening . We have previously reported the crystal structure of the native enzyme . Here we report the crystal structures of the two reaction intermediates created by the mutagenesis complexed with the substrate . The substrate was validated in the active site of two forms: 1) . covalent-bonded external aldimine with the coenzyme in the K51T form and 2) . the non-covalent interaction around the coenzyme in the Y295F form . The orientations of the substrate in both structures were quite different form each other . In concert with other site-specific mutation experiments, this experiment revealed the ingenious and unique strategies that are used to achieve the specific activity . The substrate incorporated into the active site is reactivated by a two-phenol charge relay system to lead to the formation of a Schiff base with the coenzyme . The catalytic Lys51 residue may play a novel role to abstract the methylene proton from the substrate in cooperation with other factors, the carboxylate group of the substrate and the electron-adjusting apparatuses of the coenzyme. Biotechnol Lett, 2003 Mar, 25(5), 435 - 41 Scalable method to determine mutations that occur during adaptive evolution of Escherichia coli; Raghunathan A et al.; Denaturing HPLC was used to determine mutations occurring during the adaptive evolution of Escherichia coli K-12 . The strains were evolved over 700 generations on glycerol as the sole carbon source from a sub-optimal to an optimal growth rate . The mutations detected by direct sequencing of amplicons of the glycerol-phosphate regulon repressor (glpR) gene were a synonymous substitution Val20Val in two separately evolved strains . Non-synonymous substitutions, Val119Gly and Gly179Trp, were also observed in each of the two strains . This procedure can be scaled to determine genome-scale sequence variations that have occurred during adaptive evolution. Biotechnol Lett, 2003 Mar, 25(5), 427 - 33 Structural and immunological characterization of recombinant ovomucoid expressed in Escherichia coli; Rupa P et al.; The expression of recombinant allergens is becoming new insights of an important diagnosis and the therapy of allergies as well as molecular approaches to immunological and structural studies of allergens . Ovomucoid is a major food allergens in the hen's egg white which causes immediate food-hypersensitivity reactions mainly in children . A gene coding for the cDNA representing an entire ovomucoid molecule has been cloned in Escherichia coli under the control of T5 promoter fused with six-Histidine tag at the amino terminal end . Upon induction, the E . coli cells, harbouring this construct, expressed the recombinant protein as a soluble fraction and the recombinant ovomucoid protein was purified to electrophoeretic homogeneity using Ni2+ nitrilotriacetic acid agarose affinity chromatography . Immunoblot analysis showed that human IgE and IgG binding activities of the recombinant ovomucoid was identical to that of native analogue . The antigenicity and allergenicity of recombinant ovomucoid were almost same as that of native form when tested with an ELISA using six individual patient's serum . CD spectra indicated that that the recombinant ovomucoid has more alpha-helix and less beta-structure than native form . These results show that the recombinant ovomucoid constructed in this study could be used for further studies on the immunological and structural studies of ovomucoid. Biotechnol Lett, 2003 Feb, 25(4), 353 - 8 Purification and characterization of alpha-galactosidase from sunflower seeds; Kim WD et al.; From 100 g sunflower seeds, 1.2 mg purified alpha-galactosidase was obtained with an overall yield of 51% . The alpha-galactosidase acted on both terminal alpha-galactosyl residues and side-chain alpha-galactosyl residues of the galactomanno-oligosaccharides and galactomannans . The cDNA coding for sunflower alpha-galactosidase was cloned and the deduced amino acid sequence revealed that the mature enzyme consisted of 363 amino acid residues with a molecular weight of 40,263 . Seven cysteine residues were found but no putative N-glycosylation sites were present in the sequence . The deduced amino acid sequences of mature enzyme and alpha-galactosidases from coffee, guar and Mortierella vinacea alpha-galactosidase II showed over 81%, 77%, and 47% homology, respectively . These enzymes are classified into the third group in which the enzyme has no insertion sequence and a broad specificity on galactomanno-oligosaccharides compared to the other groups. Eur Respir J, 2003 Jul, 22(1), 20 - 7 Therapeutic potential of a new phosphodiesterase inhibitor in acute lung injury; Rocco PR et al.; The effects of LASSBio596, a phosphodiesterase type-4 and -5 inhibitor, were tested in Escherichia coli lipopolysaccharide (LPS)-induced acute lung injury . Twenty-four BALB/c mice were randomly divided into four groups . In the control group, saline (0.05 mL) was injected intratracheally (i.t.) . The LPS group received LPS (10 microg i.t., 0.05 mL) . In the LASSBio596 groups, LASSBio596 (10 mg x kg(-1), 0.2 mL) was injected intraperitoneally 1 h before or 6 h after LPS administration . After 24 h, in vivo (lung resistive and viscoelastic pressures, and static and dynamic elastances) and in vitro (tissue resistance, elastance and hysteresivity) pulmonary mechanics, lung morphometry and collagenous fibre content were computed . Neutrophils and tumour necrosis factor (TNF)-alpha levels were evaluated in the bronchoalveolar lavage fluid . LASSBio596 prevented the changes in lung mechanics, and inhibited neutrophilic recruitment, TNF-alpha release, bronchoconstriction, alveolar collapse and the increment of collagen fibre content induced by LPS, independently of the moment of injection . In conclusion, LASSBio596 modulated the lung inflammatory process and had the potential to block fibroproliferation . Thus, agents that inhibit phosphodiesterase 4 and 5 simultaneously may be a useful adjunct therapy for acute lung injury. Aging Cell, 2003 Jun, 2(3), 159 - 64 Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages; Tasat DR et al.; Immunosenescence is an age-associated dysregulation of the immune function, which contributes to increased susceptibility to disease in the elderly . Alveolar macrophages (AM) are known phagocytes that generate reactive oxygen species (ROS) and nitric oxide (NO), essential mediators for host defence . We studied phagocytosis, ROS and NO production in AM obtained from young, adult and senescent rats (1-2, 9-12 and 18-24 months old, respectively) after exposure to lipopolysaccharide (LPS, 0.1-10 microg mL(-1)), 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 microg mL(-1)) or LPS + TPA in culture . Phagocytosis was significantly lower in control AM from adult rats than in AM from young animals . Nevertheless, AM from adult animals pretreated with LPS exhibited higher phagocytic capacity than AM from younger animals . ROS was identified by the NBT test at single cell level and quantified by automated image analysis . When TPA was added to all three populations, AM from adult and senescent animals responded more than AM from young animals . All LPS-stimulated AM produce more NO than controls . However, NO production increased three-, four- and two-fold in young, adult and senescent animals, respectively . Our results demonstrate that AM from young, adult and senescent animals display differential responsiveness to inflammatory mediators . Therefore, aging processes markedly affect AM metabolic functions and may further compromise the lung immune defence response, increasing adverse long-term health effects. Biotechnol Lett, 2003 Jan, 25(2), 101 - 4 Expression of gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, in Escherichia coli; Yang Q et al.; Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, was expressed in Escherichia coli using expression vector pET-32a(+) . The gene was expressed under T7 promotor with a fusion partner of Thx.Tag and a 6xHis.Tag at its 5' terminal . After induction by IPTG for 6 h, the recombinant enzyme was expressed in the cytoplasm . Expression at 25 degrees C gave twice the amount of recombinant gloshedobin in cytoplasm tha