J Immunol, 2000 Jun 15, 164(12), 6543 - 9 Cyclic AMP blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of Rho/Rho kinase signaling; Essler M et al.; During Gram-negative sepsis bacterial LPS induces endothelial cell contraction, actin reorganization, and loss of endothelial integrity by an unknown signal mechanism . In this study, we provide evidence that LPS-stimulation of endothelial cells (HUVEC) decreases myosin light chain (MLC) phosphatase, resulting in an increase in MLC phosphorylation followed by cell contraction . All of these LPS effects could be blocked by the Rho-GTPase inhibitor C3 transferase from Clostridium botulinum or the Rho kinase inhibitor Y-27632 . These data suggest that LPS induces MLC phosphorylation via Rho/Rho kinase-mediated inhibition of MLC phosphatase in HUVEC . Furthermore, we observed that cAMP-elevating drugs, known to exert a vasoprotective function, mimicked the effects of C3 transferase and Y-27632, i.e., inhibited LPS-induced MLC phosphatase inactivation and MLC phosphorylation . cAMP elevation did not inhibit myosin phosphorylation induced by constitutively active V14Rho or the MLC phosphatase inhibitor calyculin and did not induce phosphorylation of RhoA in HUVEC, indicating inhibition of an upstream regulator of Rho/Rho kinase . Taken together, Rho/Rho kinase appears to be a central target for inflammatory mediators causing endothelial cell contraction such as bacterial toxins, but also for vasoprotective molecules elevating intracellular cAMP. Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 1259 - 64 Clostridium peptidivorans sp . nov., a peptide-fermenting bacterium from an olive mill wastewater treatment digester; Mechichi T et al.; A new peptide-degrading, strictly anaerobic bacterium, designated strain TMC4T, was isolated from an olive mill wastewater treatment digester . Cells of strain TMC4T were motile, rod-shaped (5-10 x 0.6-1.2 microm), stained Gram-positive and formed terminal to subterminal spores that distended the cells . Optimal growth occurred at 37 degrees C and pH 7 in an anaerobic basal medium containing 0.5% Casamino acids . Arginine, lysine, cysteine, methionine, histidine, serine, isoleucine, yeast extract, peptone, Biotrypcase, gelatin and crotonate also supported growth, but not carbohydrates, organic acids or alcohols . The end-products of degradation were: acetate and butyrate from lysine and crotonate; acetate, butyrate, H2 and CO2 from Biotrypcase, gelatin and peptone; acetate, alanine, H2 and CO2 from cysteine; acetate, H2 and CO2 from serine, cysteine and yeast extract; acetate and formate from histidine; propionate from methionine; methyl 2-butyrate, H2 and CO2 from isoleucine; acetate and ethanol from arginine; and acetate, propionate, butyrate, methyl 2-butyrate, H2 and CO2 from Casamino acids . The DNA G+C content of strain TMC4T was 31 mol% . Phylogeny based on 16S rRNA sequence analysis showed that strain TMC4T was a member of the low-G+C-content Gram-positive genus Clostridium, with the closest relative being Clostridium pascui (sequence similarity of 96 %) . Due to considerable differences in genomic and phenotypic properties between strain TMC4T and those of its nearest relative, strain TMC4T is proposed as a new species of the genus Clostridium, Clostridium peptidivorans sp . nov . Strain TMC4T has been deposited in the DSMZ as strain DSM 12505T. Int J Syst Evol Microbiol, 2000 May, 50 Pt 3, 971 - 8 Assignment of Eubacterium sp . VPI 12708 and related strains with high bile acid 7alpha-dehydroxylating activity to Clostridium scindens and proposal of Clostridium hylemonae sp . nov., isolated from human faeces; Kitahara M et al.; Unknown Eubacterium-like organisms VPI 12708 and five strains (Y-1113, I-10, M-18, TH-82 and 36S) had high bile acid 7alpha-dehydroxylating activity; the unknown Clostridium-like organisms TN-271T and TN-272 also had the same activity . Analysis of their 16S rDNA sequences demonstrated that all strains belong to cluster XIVa of the genus Clostridium (Collins et al., 1994) . Strain VPI 12708 and five other strains (Y-1113, I-10, M-18, TH-82 and 36S) formed a single cluster and strains TN-271T and TN-272 formed another single cluster . Clostridium scindens JCM 6567T was the most closely related species for two clusters in the phylogenetic tree . Values for DNA-DNA similarities among C . scindens JCM 6567T, strain VPI 12708 and the other five strains were greater than 70%, showing that these micro-organisms were a single species . Therefore, we identified strain VPI 12708 and the five other strains as C . scindens . In addition, DNA-DNA similarities among C . scindens JCM 6567T, strain TN-271T and strain N-272 revealed that strains TN-271T and TN-272 were distinct from C . scindens JCM 6567T . On the basis of phylogenetic analysis and DNA-DNA similarity data, it was concluded that strains TN-271T and TN-272 are members of a new species of the genus Clostridium, for which the name Clostridium hylemonae is proposed . The type strain is strain TN-271T (= JCM 10539T). Am J Infect Control, 2000 Jun, 28(3), 262 - 6 HIV and diarrhea in the era of HAART: 1998 New York State hospitalizations; Anastasi JK et al.; BACKGROUND: This study reflects an attempt to identify the causes of diarrheal illness in hospitalized HIV patients in light of therapeutic advancements in HIV management . METHODS: The study identifies the various etiologies associated with diarrhea among HIV patients hospitalized in New York State in 1998 . Data for this study were extracted from the New York State Department of Health Statewide Planning and Research Cooperative System . Pathogens recognized to cause diarrhea in persons with HIV and general codes identifying diarrhea were examined by using the principal and all secondary diagnoses based on the International Classification of Diseases 9th Revision Clinical Modification codes . RESULTS: Based on the Statewide Planning and Research Cooperative System data set, more than 15,000 patients with HIV were hospitalized in 1998 . Among the HIV patients hospitalized, 2.8% were admitted with a diarrheal diagnosis . The following diagnoses occurred the most frequently among HIV patients hospitalized with a diarrheal illness: Clostridium difficile (51.3%), other protozoal diseases (18.1%), and other organisms, not elsewhere specified (11.7%) . CONCLUSIONS: In the era of highly active antiretroviral therapy, diarrhea is still an occurring symptom in HIV patients . Despite the relatively small percentage of hospitalizations attributed to diarrhea, clinicians must remember that even "mild" to "moderate" diarrhea can have a debilitating impact among persons with the symptom. Biochem J, 2000 Jun 15, 348 Pt 3, 539 - 49 A novel lysine 2,3-aminomutase encoded by the yodO gene of bacillus subtilis: characterization and the observation of organic radical intermediates; Chen D et al.; The yodO gene product of Bacillus subtilis has been cloned and overexpressed in Escherichia coli and purified . The nucleotide sequence encodes a protein of 471 amino acids with a calculated molecular mass of 54071 Da . The translated amino acid sequence is more than 60% identical to that of the lysine 2,3-aminomutase from Clostridium subterminale SB4 . Analytical HPLC gel-permeation chromatography leads to an estimate of an over all molecular mass of 224000+/-21000 Da, which corresponds to a tetrameric protein . The purified protein contains iron, sulphide and pyridoxal 5'-phosphate (PLP) and displays an optical absorption band extending to 700 nm, suggesting the presence of an iron-sulphide cluster . After reductive incubation with L-cysteine anaerobically, the protein catalyses the transformation of L-lysine into beta-lysine in the presence of S-adenosylmethionine (AdoMet) and sodium dithionite . The K(m) value for L-lysine is estimated to be 8.0+/-2.2 mM . The iron-sulphur centre is stable in air,allowing aerobic purification . EPR spectroscopy at 10 K of the purified enzyme revealed an EPR signal similar to that of the {4Fe-4S}(3+) cluster observed in the clostridial lysine 2, 3-aminomutase . Incubation with cysteine under anaerobic conditions converts the iron-sulphur centre into the EPR-silent {4Fe-4S}(2+) . Unlike the clostridial enzyme, the fully reduced {4Fe-4S}(+) could not be characterized by further reduction with dithionite in the presence of AdoMet, although both dithionite and AdoMet were required to activate the enzyme . Upon addition of L-lysine, dithionite and AdoMet to the reduced enzyme and freezing the solution to 77 K, the EPR spectrum revealed the presence of an organic free-radical signal (g=2.0023), which displayed multiple hyperfine transitions very similar to the spectrum of the beta-lysine-related radical in the mechanism of the clostridial lysine 2,3-aminomutase . Experiments with isotopically substituted L-lysine and lysine analogues verified the association of spin density with the carbon skeleton of lysine . The data indicate that the protein encoded by the yodO gene of B . subtilis is a novel lysine 2,3-aminomutase . The E . coli homologue of clostridial lysine 2,3-aminomutase was also expressed in E . coli and purified . This protein contained ironand sulphide but not PLP, it did not display lysine 2,3-aminomutase activity, and addition of PLP did not induce 2,3-aminomutase activity. Microb Pathog, 2000 Jun, 28(6), 363 - 72 Characterization of surface layer proteins from different Clostridium difficile clinical isolates; Cerquetti M et al.; In a previous study we suggested that two surface proteins of a Clostridium difficile strain were involved in the formation of a regularly assembled surface layer (S-layer) external to the cell wall . In the present paper six C . difficile strains isolated from cases and healthy carriers were studied . By using freeze-etching and negative staining techniques two superimposed structurally different lattices were detected on the cell surface of the different C . difficile strains . In each strain, the outer S-layer lattice was arranged in a square symmetry and the inner S-layer lattice in hexagonal symmetry . The S-layer proteins from the different strains were isolated and characterized . Each strain showed two distinct S-layer glycoproteins ranging in molecular mass 36-56 kDa . Antigenic cross-reactivity among the S-layer proteins of higher molecular masses extracted from each strain was demonstrated whereas no antigenic relationship was observed among the different S-layer proteins of lower molecular masses . N-terminal sequence analysis showed the presence of common structural motifs conserved among the high S-layer proteins as well as among the low S-layer proteins . These data indicate that the presence of S-layer on C . difficile strains is common and that its glycoprotein subunits show a certain degree of heterogeneity . J Protein Chem, 1999 Nov, 18(8), 885 - 92 Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules; Sagane Y et al.; Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide . Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc) . We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc . Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively . In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules . The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues . The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence . In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G. Prim, Care Update Ob Gyns . 1998 Jul 1, 5(4), 146 - 147 A prospective, randomized, clinical study to compare the clinical safety, effectiveness, and cost of oral ofloxacin/clindamycin vs intravenous clindamycin/gentamicin for the treatment of postpartum endomyometritis; Pietrantoni M et al.; Objective: The primary objective of this prospective, randomized, clinical study was to compare the safety, clinical and microbiologic efficacy, and cost of oral ofloxacin in combination with clindamycin vs intravenous (IV) clindamycin/gentamicin in the early empiric treatment for hospitalized patients with mild to moderate postpartum endomyometritis . The secondary objective is to reduce total hospital and patient treatment cost . Postpartum endomyometritis is a major cause of infectious morbidity in the obstetric patient . It is the most common complication associated with cesarean delivery . Careful timing and amniotomy, limited vaginal examinations, and prophylactic antibiotics for cesarean section delivery may help to reduce the incidence and severity of endomyometritis . Endomyometritis is caused by bacteria that compose the normal cervicovaginal flora . These are anaerobic gram-positive cocci (Peptostreptococcus and Peptococcus), aerobic streptococci (Group B Streptococci and enterococci), Enterobacteriaceae, Bacteroides (B . fragilis, B . bivius, and B . disiens), and clostridium species.Ofloxacin is a synthetic broad-spectrum antibacterial agent for intravenous and oral administration . Following oral administration, the bioavailability in tablet form is 98% with maximum serum concentrations in 1 to 2 hours . Steady state concentrations are achieved after 4 doses . Ofloxacin usually is bactericidal in action . A synthetic broad-spectrum antibacterial agent for intravenous and oral administration . Ofloxacin inhibits DNA topoisomerase (ATP-hydrolyzing), commonly referred to as DNA-gyrase . DNA-gyrase causes double-stranded DNA breakage; it inhibits duplication, transcription, and repair of bacterial DNA.Methods: This is a preliminary study that has enrolled 19 evaluable patients towards the overall enrollment of 60 patients for statistical significance . Patients clinically diagnosed as having postpartum endomyometritis who meet the inclusion/exclusion criteria were entered into the trial . Patients were examined for the presence of fever (102.2 degrees F), pelvic pain, and foul lochia . A medical history, physical examination, and laboratory analysis were obtained prior to the first dose of antibiotic treatment . A signed consent was obtained prior to the study enrollment and randomization . Appropriate endometrial, blood, and urine culture specimens were obtained prior to the initiation of antibiotic therapy.Patients in Group 1 were treated with oral therapy using ofloxacin 400 mg q12h plus clindamycin 900 mg q8h until 24 hrs of afebrility . In Group 2, patients were treated with clindamycin 900 mg IV q8h plus gentamicin IV 5mg/kg/d q 8h until afebrility . Antibiotic therapy was continued for at least 48 hours unless significant clinical deterioration occurred necessitating the withdrawal of the patient from the study.Results:Conclusions: We found in our preliminary study that oral ofloxacin in combination with oral clindamycin was equally as efficacious, well tolerated, and safe as the combination of intravenous therapy with clindamycin and gentamicin for the treatment of postpartum endomyometritis. Vet Res Commun, 2000 May, 24(4), 229 - 38 Efficacy of chlorhexidine against some strains of cultured and clinically isolated microorganisms; Odore R et al.; The efficacy of chlorhexidine digluconate was determined against some strains of collected and clinically isolated bacteria and fungi . The efficacy was evaluated either by calculating a minimum inhibitory concentration (MIC) or by efficacy trials according to the guidelines of the European Committee for Standardization . The MIC values of chlorhexidine for Staphylococcus aureus, Microsporum gypseum, Microsporum canis and Trichophyton mentagrophytes were 0.625 microg/ml, 12.5 microg/ml, 50 microg/ml and 6.25 microg/ml, respectively . The in vitro efficacy of chlorhexidine was higher against ATCC strains of S . aureus and P . aeruginosa (0.5 mg/ml for 5 min and 0.5 mg/ml for 10 min, respectively) than against clinical isolates (0.5 mg/ml for 15 min and 1 mg/ml for 10 min, respectively) . The antiseptic activity of aqueous solutions of chlorhexidine against spores of Bacillus subtilis, Bacillis sfericus and Clostridium perfringens required longer contact times than against the vegetative forms . Nevertheless, 5 mg/ml of chlorhexidine in water-ethanol 20:80 v/v was totally effective against the vegetative forms or spores of these microorganisms. J Clin Microbiol, 2000 Jun, 38(6), 2386 - 8 Epidemiology of recurrences or reinfections of Clostridium difficile-associated diarrhea; Barbut F et al.; Approximately 15 to 35% of patients with a first episode of Clostridium difficile-associated diarrhea relapse within 2 months . Between 1994 and 1997, strains from 93 hospitalized patients with C . difficile recurrences were fingerprinted by using both serotyping and PCR-ribotyping . The results showed that 48.4% of clinical recurrences were, in fact, reinfections with a different strain of C . difficile . Rates of clinical recurrences could therefore be reduced by implementing strict isolation precautions. Gastroenterology, 2000 Jun, 118(6), 1094 - 105 Antibiotic therapy attenuates colitis in interleukin 10 gene-deficient mice; Madsen KL et al.; BACKGROUND & AIMS: Interleukin (IL)-10 gene-deficient mice, raised under germfree conditions, do not develop colitis, implying a role for bacteria . This study mapped the appearance of luminal colonic bacteria and, using antibiotic treatment, determined their association with colitis in IL-10 gene-deficient mice . METHODS: Mice were treated with ciprofloxacin or with neomycin and metronidazole . The intestine was harvested for histological scoring and bacterial assessment . RESULTS: At 2 weeks of age, before the development of colitis, IL-10 gene-deficient mice demonstrated an earlier appearance of Streptococcus and Clostridium sp., and had a greater proportion (P < 0.01) of bacteria adherent to the colonic mucosa . This pattern of increased adherent bacteria persisted for the 12 weeks of study . Treatment of mice before the onset of colonic inflammation, with either antibiotic regime, reduced mucosal adherent bacteria and prevented colitis (P < 0.01) . In contrast, treatment of established colitis with neomycin and metronidazole did not reduce adherent bacterial levels, yet was more efficacious (P < 0.05) in treating established colitis than ciprofloxacin, which did reduce adherent colonic bacteria . CONCLUSIONS: In the IL-10 gene-deficient mouse model, the appearance and number of mucosal adherent colonic bacteria are altered before the onset of colitis . Antibiotics both prevent and treat the colitis through correction of this primary bacterial alteration. Microbiol Immunol, 2000, 44(4), 223 - 7 Phylogenic and phenotypic characterization of some Eubacterium-like isolates from human feces: description of Solobacterium moorei Gen . Nov., Sp . Nov; Kageyama A et al.; Three isolated strains from human feces were characterized by biochemical tests and 16S rDNA analysis . Phylogenetic analysis revealed that these isolated strains were members of the Clostridium subphylum of gram-positive bacteria . The phenotypic characters resembled those of the genus Eubacterium, but these strains were shown to be phylogenetically distant from the type species of the genus, Eubacterium limosum . The strains showed a specific phylogenetic association with Holdemania filiformis and Erysipelothrix rhusiopathiae . Based on a 16S rDNA sequence divergence of greater than 12% with H . filiformis and E . rhusiopathiae, a new genus, Solobacterium, is proposed for three strains, with one species, Solobacterium moorei . The type strain of Solobacterium moorei is JCM 10645T. J Gastroenterol, 2000, 35(5), 341 - 6 Preventive efficacy of butyrate enemas and oral administration of Clostridium butyricum M588 in dextran sodium sulfate-induced colitis in rats; Okamoto T et al.; Butyrate enemas have been reported to be effective in ulcerative colitis . However, long-term use is difficult because of the troublesome procedure and the unpleasant smell . We therefore investigated the effects of the oral administration of Clostridium butyricum M588 (CBM588), an enterobacterium producing butyrate, in dextran sodium sulfate (DSS)-induced colitis in rats . First, we confirmed the effects of pre-treatment with a butyrate enema on DSS colitis . We then studied the efficacy of oral administration of CBM588 which was started 1 week prior to the induction of DSS colitis . In the CBM588 group, the ulcer index and myeloperoxidase (MPO) activity in the distal colon were significantly lower than in the control group . Proliferating cell nuclear antigen (PCNA) immuno-positive cells were increased around the ulcer in the CBM588 group . In regard to the contents of the cecum and colon, the proportions of Lactobacillus and Eubacterium were increased in the cecum in the CBM588 group . Further, there were significant increases of n-butyrate, propionate, and acetate concentrations in the cecum in the CBM588 group . These results indicated that the oral administration of CBM588 alleviated DSS-induced colitis, and may be useful instead of butyrate enema. Biochim Biophys Acta, 2000 May 31, 1485(2-3), 153 - 62 Activation of astroglial phospholipase D activity by phorbol ester involves ARF and Rho proteins; Kotter K et al.; Primary cultures of rat cortical astrocytes express phospholipase D (PLD) isoforms 1 and 2 as determined by RT-PCR and Western blot . Basal PLD activity was strongly (10-fold) increased by 4beta-phorbol-12beta,13alpha-dibutyrate (PDB) (EC(50): 56 nM), an effect which was inhibited by Ro 31-8220 (0.1-1 microM), an inhibitor of protein kinase C (PKC), and by brefeldin A (10-100 microg/ml), an inhibitor of ADP-ribosylating factor (ARF) activation . Pretreatment of the cultures with Clostridium difficile toxin B-10463 (0.1-1 ng/ml), which inactivates small G proteins of the Rho family, led to a breakdown of the astroglial cytoskeleton; concomitantly, PLD activation by PDB was reduced by up to 50% . In contrast, inactivation of proteins of the Ras family by Clostridium sordellii lethal toxin 1522 did not affect PLD activation . In parallel experiments, serum-induced PLD activation was sensitive to brefeldin A, but not to Ro 31-8220 and not to clostridial toxins . We conclude that, in astrocytes, the PLD isoform which is activated by phorbol ester requires PKC, ARF and Rho proteins for full activity and probably represents PLD1. Appl Environ Microbiol, 2000 Jun, 66(6), 2461 - 70 Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium; Desvaux M et al.; A reinvestigation of cellulose degradation by Clostridium cellulolyticum in a bioreactor with pH control of the batch culture and using a defined medium was performed . Depending on cellulose concentration, the carbon flow distribution was affected, showing the high flexibility of the metabolism . With less than 6.7 g of cellulose liter(-1), acetate, ethanol, H(2), and CO(2) were the main end products of the fermentation and cellulose degradation reached more than 85% in 5 days . The electron flow from the glycolysis was balanced by the production of H(2) and ethanol, the latter increasing with increasing initial cellulose concentration . From 6.7 to 29.1 g of cellulose liter(-1), the percentage of cellulose degradation declined; most of the cellulase activity remained on the cellulose fibers, the maximum cell density leveled off, and the carbon flow was reoriented from ethanol to acetate . In addition to that of previously indicated end products, lactate production rose, and, surprisingly enough, pyruvate overflow occurred . Concomitantly the molar growth yield and the energetic yield of the biomass decreased . Growth arrest may be linked to sufficiently high carbon flow, leading to the accumulation of an intracellular inhibitory compound(s), as observed on cellobiose (E . Guedon, M . Desvaux, S . Payot, and H . Petitdemange, Microbiology 145:1831-1838, 1999) . These results indicated that bacterial metabolism exhibited on cellobiose was distorted compared to that exhibited on a substrate more closely related to the natural ecosystem of C . cellulolyticum . To overcome growth arrest and to improve degradation at high cellulose concentrations (29.1 g liter(-1)), a reinoculation mode was evaluated . This procedure resulted in an increase in the maximum dry weight of cells (2,175 mg liter(-1)), cellulose solubilization (95%), and end product concentrations compared to a classical batch fermentation with a final dry weight of cells of 580 mg liter(-1) and 45% cellulose degradation within 18 days. J Inorg Biochem, 2000 Apr, 79(1-4), 83 - 91 Electron transfer properties of iron-sulfur proteins; Kummerle R et al.; The details of most electron transfer reactions involving iron-sulfur proteins have remained undisclosed because of the lack of experimental methods suitable to measure precisely the relevant rates . Nuclear magnetic resonance (NMR) provides a powerful means to overcome these problems, at least with selected proteins . A combination of NMR studies and site-directed mutagenesis experiments has been instrumental in defining both the site of interaction and the main trends of the intracomplex electron transfer in the case of rubredoxin electron self-exchange . Analysis of the NMR data obtained for mixtures of different redox levels of several 2{4Fe-4S} ferredoxins provided both first-order, for intramolecular, and second-order, for intermolecular, rate constants . Their dependence as a function of structural changes gave insight into the mechanism of electron transfer in this type of protein . Contrary to some expectations, the high-spin {4Fe-4Se}+ clusters assembled in isopotential ferredoxins do not change the intramolecular electron transfer rate as compared to low-spin {4Fe-4S}+ homologs . In combination with activity measurements, the kinetic data have been used to model the electron transfer competent complexes between Clostridium pasteurianum ferredoxin and the main enzymes acting as redox partners in vivo. Am J Med Sci, 2000 May, 319(5), 338 - 9 Convulsions induced by metronidazole treatment for Clostridium difficile-associated disease in chronic renal failure; Beloosesky Y et al.; Clostridium difficile-related diarrhea and colitis are common health problems, especially in elderly, frail hospitalized patients . The drug of choice is metronidazole, which can be associated, in long or high doses, with neurotoxic side effects . We report convulsions induced by short-term metronidazole therapy used in conventional doses for Clostridium difficile colitis in an elderly patient with chronic renal failure. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 107 - 18 Clostridium gasigenes sp . nov., a psychrophile causing spoilage of vacuum-packed meat; Broda DM et al.; Two psychrophilic Clostridium strains, DB1AT and R26, were isolated from incidences of 'blown-pack' spoilage of vacuum-packed chilled lamb . Vacuum packs of meat inoculated with these strains developed gas bubbles and pack distension within 14 d storage at 2 degrees C . The two main gases responsible for pack distension were carbon dioxide and hydrogen . 1-Butanol, butyric and acetic acid and butyl esters were the major volatile compounds produced by the strains in the artificially inoculated packs . The unknown strains were Gram-positive motile rods producing elliptical subterminal spores during the late-stationary growth phase . At pH 7.0, they grew from -1.5 to 26 degrees C, and their optimum growth temperature was 20-22 degrees C . At 20 degrees C, the pH range for growth was 5.4-8.9 and the optimum pH for growth was 6.2-8.6 . In peptone/yeast extract broth, the organisms grew little or not at all in the absence of fermentable carbohydrates . Both strains hydrolysed gelatin, aesculin and starch . The fermentation products formed in peptone yeast extract glucose starch broth were ethanol, acetate, butyrate, lactate, butanol, carbon dioxide and hydrogen . The G+C contents of the DNA of strains DB1AT and R26 were 29.4 and 28.3 mol%, respectively . Phylogenetic analyses indicated that the strains belong to cluster I of the genus Clostridium (sensu Collins et al . 1994) . The new strains differed from the phylogenetically related clostridia in cellular fatty acid composition, soluble protein profiles and phenotypic properties . On the basis of rDNA analysis and phenotypic and phylogenetic characterization, the strains were assigned to a new species for which the name Clostridium gasigenes is proposed . Strain DB1AT (= DSM 12272T) is designated as the type strain. Int J Syst Evol Microbiol, 2000 Jan, 50 Pt 1, 101 - 6 Reclassification of Clostridium quercicolum as Dendrosporobacter quercicolus gen . nov., comb . nov; Strompl C et al.; Morphological features, genomic DNA base composition and 16S rDNA sequence similarities, as well as a distinct phospholipid pattern, whole-cell fatty acid distribution and the occurrence of the lipoquinone 'lipid F', indicate that Clostridium quercicolum belongs to the Sporomusa-Pectinatus-Selenomonas phyletic group and possesses only a remote relationship to members of the genus Clostridium sensu stricto . On the basis of these results, the new genus and combination Dendrosporobacter quercicolus gen . nov., comb . nov . are proposed. J Antimicrob Chemother, 2000 Apr, 45 Suppl 1, 55 - 65 Review of the in vitro activity of gemifloxacin against gram-positive and gram-negative anaerobic pathogens; Goldstein EJ; Published reports on the in vitro activity of gemifloxacin mesylate (SB 265805), a new fluoronaphthyridone, against anaerobic pathogens are reviewed here . The studies used a variety of media, inocula and antimicrobial agents . Using a proposed breakpoint of 0.5 mg/L, these studies showed that gemifloxacin had generally higher potency against Gram-positive anaerobes (Clostridium perfringens, all Peptostreptococcus spp.) and fusobacteria (Fusobacterium nucleatum, Fusobacterium necrophorum) and moderate but variable potency against Gram-negative anaerobes . Bacteroides stercoris, Bacteroides tectum and many Bacteroides fragilis isolates were inhibited by concentrations of < or =0.5 mg/L, while the other species of the B . fragilis group required higher concentrations for inhibition . Species variability was evident: Porphyromonas asaccharolytica, Porphyromonas canoris, Porphyromonas gingivalis, Porphyromonas macaccae, Prevotella heparinolytica and Prevotella intermedia were susceptible to 0.5 mg/L of gemifloxacin while most other Porphyromonas and Prevotella spp . were not . These data suggest that gemifloxacin may have a clinical role in the treatment of certain dental, head and neck and pleuropulmonary infections in which Gram-positive anaerobes, fusobacteria and some Prevotella and Porphyromonas spp . may predominate. Oper Dent, 1999 Sep-Oct, 24(5), 286 - 91 Antibacterial activity of glass-ionomer restorative cements exposed to cavity-producing microorganisms; Herrera M et al.; The antibacterial activity of the glass-ionomer restorative cements Ketac-Fil, Ketac-Silver, Fuji II LC, and Vitremer was studied in vitro, in conjunction with a total of 32 strains of five bacterial genera that may be associated with dental caries: Streptococcus spp, Lactobacillus spp, Actinomyces spp, Porphyromonas spp, and Clostridium spp . Agar plate diffusion was the method used for the bacterial cultures, which included a chlorhexidine control . All four glass-ionomer cements were found to inhibit bacterial growth, though with noteworthy differences in their spheres of action . Vitremer was the cement determined to have the greatest antibacterial effects, whereas Ketac-Silver presented the least inhibitory action. Xenobiotica, 2000 Apr, 30(4), 359 - 69 Reduction of stilbene oxide and styrene oxide to the corresponding alkenes by intestinal bacteria; Kitamura S et al.; 1 . This study provides the first evidence that stilbene oxide and styrene oxide are reductively metabolized to the corresponding alkenes by intestinal bacteria in animals . 2 . When trans- or cis-stilbene oxide were incubated with the caecal contents of rat under anaerobic conditions, both trans- and cis-stilbene were isolated from the incubation mixture . Styrene oxide was also reduced to styrene by rat caecal contents . 3 . Caecal contents of mouse, hamster and guinea pig also exhibited alkene oxide reductase activities toward cis- and trans-stilbene oxides, and styrene oxide . In contrast, liver microsomes or cytosol exhibited no epoxide reductase activities toward these substrates . 4 . Seven pure strains of intestinal bacteria exhibited alkene oxide reductase activities of varying degrees under anaerobic conditions, with the highest activity being observed in Clostridium sporogenes . 5 . Cell-free extracts of either the intestinal bacteria in rat caecal contents or C . sporogenes exhibited reductase activity when supplemented with both NAD(P)H and FMN under anaerobic conditions . Reductase activity was also observed on addition of the photochemically reduced form of FMN instead of both NAD(P)H and FMN. J Formos Med Assoc, 2000 Mar, 99(3), 199 - 205 Toxic megacolon secondary to infective colitis in children; Tsai TC et al.; BACKGROUND AND PURPOSE: Toxic megacolon is a fulminating and potentially lethal complication of severe colitis . Toxic megacolon secondary to infective colitis in children is rare . We analyzed the clinical course, pathology, treatment, and outcome of toxic megacolon secondary to infective colitis in children . METHODS: The medical records of all 20 children treated for infective colitis complicated with toxic megacolon during a 12-month (October 1997-October 1998) period were retrospectively reviewed . RESULTS: There were 10 boys and 10 girls, with a mean (+/- standard deviation, SD) age of 26.2 +/- 12.9 months (range, 6-57 mo) . With an initial presentation of nonspecific gastroenteritis syndrome lasting several days, the disease progressed rapidly . In the acute stage, most patients developed toxic signs such as mental change, ranging from irritability to stupor (20, 100%), fever (19, 95%), tachycardia (20, 100%), abdominal distension (20, 100%), and abnormal stool pattern (19, 95%) . Initial investigations revealed anemia (11, 55%), leukocytosis (11, 55%), and elevated levels of C-reactive protein ranging from 25.0 mg/L to 483.0 mg/L with a mean +/- SD of 185.7 +/- 129.1 mg/L (normal range, < 8 mg/L) (20, 100%) . Salmonella enteritidis (12 patients, 60%) and Clostridium difficile (1, 5%) were isolated from stool samples in some cases . Plain abdominal x-rays revealed severe colonic dilatation . Prolonged hospitalization (mean, 33.6 d) and intensive therapy including a combination of broad-spectrum antibiotics, physical decompression, and total parenteral nutrition were necessary . Three patients (15%) underwent surgical management; the pathologic findings in these patients demonstrated severe transmural inflammation . We believe that bacterial and/or endotoxin translocation played an important role in gut failure . Three patients (15%) in the study died . CONCLUSION: Toxic megacolon in infective colitis is a fulminating illness that has a high mortality rate . The disease course can be divided into three stages: the acute toxic stage, the gut failure stage, and the convalescence or deterioration stage . Early diagnosis and aggressive management are important. Biochemistry, 2000 May 2, 39(17), 5013 - 21 The X6 "thermostabilizing" domains of xylanases are carbohydrate-binding modules: structure and biochemistry of the Clostridium thermocellum X6b domain; Charnock SJ et al.; Many polysaccharide-degrading enzymes display a modular structure in which a catalytic module is attached to one or more noncatalytic modules . Several xylanases contain a module of previously unknown function (termed "X6" modules) that had been implicated in thermostability . We have investigated the properties of two such "thermostabilizing" modules, X6a and X6b from the Clostridium thermocellumxylanase Xyn10B . These modules, expressed either as discrete entities or as their natural fusions with the catalytic module, were assayed, and their capacity to bind various carbohydrates and potentiate hydrolytic activity was determined . The data showed that X6b, but not X6a, increased the activity of the enzyme against insoluble xylan and bound specifically to xylooligosaccharides and various xylans . In contrast, X6a exhibited no affinity for soluble or insoluble forms of xylan . Isothermal titration calorimetry revealed that the ligand-binding site of X6b accommodates approximately four xylose residues . The protein exhibited K(d) values in the low micromolar range for xylotetraose, xylopentaose, and xylohexaose; 24 microM for xylotriose; and 50 microM for xylobiose . Negative DeltaH and DeltaS values indicate that the interaction of X6b with xylooligosaccharides and xylan is driven by enthalpic forces . The three-dimensional structure of X6b has been solved by X-ray crystallography to a resolution of 2.1 A . The protein is a beta-sandwich that presents a tryptophan and two tyrosine residues on the walls of a shallow cleft that is likely to be the xylan-binding site . In view of the structural and carbohydrate-binding properties of X6b, it is proposed that this and related modules be re-assigned as family 22 carbohydrate-binding modules. Lab Anim, 2000 Apr, 34(2), 162 - 70 A new technique to determine hydrogen excreted by gnotobiotic rats; Hartmann L et al.; A new system, that allowed the monitoring of hydrogen (H2) excretion by gnotobiotic rats without affecting their defined microbial status, was developed . The system consists of an isolator containing a chamber for an experimental animal, and a life-support system (LSS), with a sampling port outside the isolator connected to it . H2 accumulation in the system was measured by analysing a defined volume of gas after removal . H2 concentrations were determined with an electrochemical cell or by gas chromatography . To validate this technique, H2 excretion by germ-free (GF) and mono-associated rats fed a chemically defined diet was measured after oral application of lactulose . Mono-associated rats had been obtained by colonizing GF rats with a H2-producing Clostridium perfringens type A strain isolated from human faeces of a healthy volunteer . Application of 50 mg lactulose to the mono-associated rats resulted in a significant increase in H2 excretion . The net H2 excretion was 7.82+/-1.28 ml H2 in 12 h corresponding to a net maximal rate of 1.1+/-0.3 ml H2/h . In contrast, in experiments with GF rats, less than 0.13 ml H2 were detectable within 12 h . The technique presented is a useful tool for studying bacterial H2 metabolism in vivo under gnotobiotic conditions. Infect Immun, 2000 Jun, 68(6), 3475 - 84 Clostridium perfringens iota toxin: binding studies and characterization of cell surface receptor by fluorescence-activated cytometry; Stiles BG et al.; The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry . The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37 degrees C for 10 min . The binding of Ib was inhibited by antisera against C . perfringens type E or Clostridium spiroforme culture supernatants, but not C . perfringens types C or D . Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface . The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia) . Ia attached to cell-bound Ib within 10 min at 37 degrees C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min . Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane. Biol Blood Marrow Transplant, 2000, 6(2A), 204 - 10 Autologous blood and marrow transplantation in patients 60 years and older; Leger CS et al.; Although many hematologic malignancies are more common in older patients, autologous blood and marrow transplantation (ABMT) has traditionally been restricted to patients younger than 60 years because of concerns that older patients would be either unable to provide a graft or unable to tolerate the therapy . From June 1995 to May 1998, 30 patients > or = 60 years underwent ABMT at our institution for low-grade lymphoma (4 patients), relapsed intermediate-grade lymphoma (17 patients), or multiple myeloma (9 patients) . The median patient age was 62.5 years (range 60-73) . Pretransplantation conditioning regimens were CBV (cyclophosphamide, BCNU {carmustine}, etoposide) or BEAM (carmustine, etoposide, cytarabine, melphalan) for intermediate-grade lymphoma patients and melphalan 140 mg/m2 + etoposide 60 mg/kg + total body irradiation 500 cGy for the others . The rescue product was bone marrow (BM; 4 patients), peripheral blood stem cells (PBSC; 23 patients), or BM+PBSC (3 patients) . The median number of CD34+ cells/kg infused was 3.60 x 10(6) (range 0.53-31.0), by the International Society for Hematotherapy and Graft Engineering method . The treatment-related mortality at day 100 and at 6 months was 10% and 16.7%, respectively . The median days to neutrophil > 0.5 x 10(9)/L was 11 (range 9-25) and platelets > 20 x 10(9)/L was 16 (range 6-70) . Three patients died of infection (days 26, 27, and 38), and 1 died of an intracranial hemorrhage related to persistent thrombocytopenia (day 130) . Bearman regimen-related toxicity was moderate, with most toxicities < or = grade 2 . Seven patients developed significant gut toxicity: 4 patients with Clostridium difficile colitis and 3 patients with neutropenic enterocolitis . Depressive symptoms and signs were noted in 4 patients . Three male patients developed decreased gonadal function after transplantation . These transplantations accounted for 997 patient days, of which 266 days (27%) were in the outpatient BMT program--a smaller percentage than in patients < 60 years (56%, P = .002) . Twenty patients are alive 153 to > or = 1224 days after transplantation . ABMT in patients > or = 60 years of age is feasible . Further studies addressing supportive care particular to older patients and comparisons of ABMT with traditional approaches to multiple myeloma and relapsed non-Hodgkin's lymphoma in older patients are needed . Further work to identify elderly patients most likely to benefit from this approach is also required. Biotechnol Appl Biochem, 2000 Jun, 31 ( Pt 3), 197 - 203 Expression, purification and applications of staphylococcal protein A fused to cellulose-binding domain; Shpigel E et al.; Because staphylococcal Protein A (ProtA) binds specifically to IgG, it has been used for many immunological manipulations, most notably antibody purification and diagnostics . Immobilization is required for most of these applications . Here we describe a genetic-engineering approach to immobilizing ProtA on cellulose, by fusing it to cellulose-binding domain (CBD) derived from the cellulose-binding Protein A of Clostridium cellulovorans . The bifunctional fusion protein was expressed in Escherichia coli, recovered on a cellulose column and purified by elution at alkaline pH . ProtA-CBD was used to purify IgG from rabbit serum and its ability to bind IgG from different sources was determined . The bifunctional chimaeric protein can bind up to 23.4 mg/ml human IgG at a ratio of 1 mol of ProtA-CBD/2 mol of human IgG, and can purify up to 11.6 mg/ml rabbit IgG from a serum . The ability to bind functionally active CBD-affinity reagents to cellulosic microtitre plates was demonstrated . Our results indicate that a combination of CBD-affinity reagents and cellulosic microtitre plates is an attractive diagnostics matrix for the following reasons: (i) cellulose exhibits very low non-specific binding; and (ii) CBD-fusion proteins bind directly to cellulose at high density . A unique signal-amplification method was developed based on the ability of ProtA-CBD to link stained cellulose particles to primary antibody in a Western blot. J Cardiovasc Pharmacol, 2000 May, 35(5), 814 - 21 Agonist-dependent difference in the mechanisms involved in Ca2+ sensitization of smooth muscle of porcine coronary artery; Sato A et al.; This study was undertaken to explore possible signal-transduction mechanisms involved in the Ca2+-sensitizing effects of carbachol and endothelin-1 (ET-1) by using beta-escin-skinned smooth muscle of porcine coronary artery . Pretreatment with C3 exoenzyme of Clostridium botulinum, which selectively inactivates rho p21 by adenosine diphosphate (ADP) ribosylation, resulted in a significant inhibition of ET-1-induced Ca2+ sensitization, but had no effect on carbachol-induced Ca2+ sensitization . Whereas the protein kinase C (PKC) inhibitors calphostin C and staurosporine did not affect the Ca2+-sensitizing effect of carbachol, the tyrosine kinase inhibitors genistein and tyrphostin 25 greatly but incompletely suppressed it . In contrast, the Ca2+-sensitizing effect of ET-1 was significantly inhibited by either calphostin C or genistein . Although the inhibitory effect of calphostin C on ET-1-induced Ca2+ sensitization was less than that of genistein, the effects of calphostin C and genistein were additive . The genistein-sensitive component of ET-1-induced Ca2+ sensitization appeared to include the C3-sensitive one . However, a substantial enhancement by ET-1 of the Ca2+-induced contraction was observed even in the presence of the two inhibitors . In beta-escin-skinned smooth muscle of rabbit mesenteric artery, ET-1-induced Ca2+ sensitization was marginally affected by C3 pretreatment, calphostin C, and genistein . We conclude that, although PKC activation and rho p21 protein-dependent and -independent tyrosine phosphorylation each plays an important role in an increase in myofilament Ca2+ sensitivity, the contributions of these signaling pathways to Ca2+ sensitization are different depending on receptor agonists and tissues used . Furthermore, these data suggest the existence of an as yet undefined signal-transduction mechanism involved in Ca2+ sensitization caused by receptor agonists. Aust Vet J, 1999 Jun, 77(6), 388 - 91 Comparative immunogenicity of two bivalent botulinum vaccines; Brown AT et al.; OBJECTIVE: To compare the ability of a new single-dose botulinum vaccine containing a non-mineral oil adjuvant with a single dose of a conventional botulinum vaccine product to produce antibody to Clostridium botulinum types C and D in cattle in Northern Australia . DESIGN AND PROCEDURE: One hundred and fifty Brahman steer weaners were randomly divided into two groups receiving either a single dose of CSL Bivalent Botulinum vaccine or Websters Singvac . Blood samples were collected at 0, 8 and 24 weeks and tested by antibody ELISA . The final samples were also tested by the toxin neutralisation test, to test titres of neutralising antibody . RESULTS: Six months after inoculation, cattle vaccinated with Websters Singvac had ELISA antibody response twice that of CSL conventional product . However, this difference was only evident for neutralising antibody to type C botulinum toxin . Both products produced similar titres of type D neutralising antibody after a single dose . CONCLUSION: Websters' Singvac produces a greater neutralising antibody response to type C botulism upon single inoculation than a conventional vaccine . The product produces an equivalent neutralising antibody response to type D. J Biotechnol, 2000 Apr 28, 79(2), 117 - 26 Isolation of mesophilic solvent-producing clostridia from Colombian sources: physiological characterization, solvent production and polysaccharide hydrolysis; Montoya D et al.; One hundred and seventy-eight new butanol-acetone producing bacteria related to saccharolytic clostridia were isolated from agricultural sources in Colombia and their fermentation potential was evaluated . Thirteen isolates produced more total solvents from glucose than Clostridium acetobutylicum ATCC 824 . The isolates with the highest single solvent production were IBUN 125C and IBUN 18A with 0.46 mol butanol and 0.96 mol ethanol formed from 1 mol glucose, yielding 25 . 2 and 29.1 g l(-1) total solvents, respectively, which is close to the maximum values described to date . Most of the new isolates produced exoenzymes for the hydrolysis of starch, carboxymethyl cellulose, xylan, polygalacturonic acid, inulin and chitosan . Together with the high efficiency of solvent production, these hydrolytic isolates may be useful for the direct fermentation of biomass . According to their physiological profile, the most solvent-productive isolates could be classified as strains of C . acetobutylicum, Clostridium beijerinckii, and Clostridium NCP262. Sci Total Environ, 2000 Apr 24, 250(1-3), 73 - 81 Phosphine generation by mixed- and monoseptic-cultures of anaerobic bacteria; Jenkins RO et al.; A microbial basis for bioreductive generation of phosphine is proposed, which could account at least in part for the presence of this toxic gas in natural anaerobic environments and in sewage and landfill gases . Phosphine generation under anaerobic growth conditions was dependent upon both the culture inoculum source (animal faeces) and enrichment culture conditions . Phosphine was detected in headspace gases from mixed cultures under conditions promoting fermentative growth of mixed acid and butyric acid bacteria, either in the presence or absence of methane generation . Monoseptic cultures of certain mixed acid fermentors (Escherichia coli, Salmonella gallinarum, and Salmonella arizonae) and solvent fermentors (Clostridium sporogenes, Clostridium acetobutyricum and Clostridium cochliarium) also generated phosphine . Such fermentative bacteria participate in the multi-stage process of methanogenesis in nature . Generation of phosphine by these bacteria, rather than by methanoarchaea themselves, could explain the apparent correlation between methanogenesis and the formation of phosphine in nature. J Bacteriol, 2000 Jun, 182(11), 3247 - 53 Analysis of genes encoding an alternative nitrogenase in the archaeon Methanosarcina barkeri 227; Chien YT et al.; Methanosarcina barkeri 227 possesses two clusters of genes potentially encoding nitrogenases . We have previously demonstrated that one cluster, called nif2, is expressed under molybdenum (Mo)-sufficient conditions, and the deduced amino acid sequences for nitrogenase structural genes in that cluster most closely resemble those for the Mo nitrogenase of the gram-positive eubacterium Clostridium pasteurianum . The previously cloned nifH1 from M . barkeri shows phylogenetic relationships with genes encoding components of eubacterial Mo-independent eubacterial alternative nitrogenases and other methanogen nitrogenases . In this study, we cloned and sequenced nifD1 and part of nifK1 from M . barkeri 227 . The deduced amino acid sequence encoded by nifD1 from M . barkeri showed great similarity with vnfD gene products from vanadium (V) nitrogenases, with an 80% identity at the amino acid level with the vnfD gene product from Anabaena variabilis . Moreover, there was a small open reading frame located between nifD1 and nifK1 with clear homology to vnfG, a hallmark of eubacterial alternative nitrogenases . Stimulation of diazotrophic growth of M . barkeri 227 by V in the absence of Mo was demonstrated . The unusual complement of nif genes in M . barkeri 227, with one cluster resembling that from a gram-positive eubacterium and the other resembling a eubacterial V nitrogenase gene cluster, suggests horizontal genetic transfer of those genes. Blood, 2000 May 15, 95(10), 3044 - 51 Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells; Hippenstiel S et al.; Bacterial endotoxin (lipopolysaccharide, or LPS) has potent proinflammatory properties by acting on many cell types, including endothelial cells . Secretion of the CXC-chemokine interleukin-8 (IL-8) by LPS-activated endothelial cells contributes substantially to the inflammatory response . Using human umbilical vein endothelial cells (HUVECs), we analyzed the role of small GTP-binding Rho proteins and p38 mitogen-activated protein kinase (MAPK) for LPS-dependent IL-8 expression in endothelial cells . Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced tyrosine phosphorylation, nuclear factor (NF)-kappaB-dependent gene expression, IL-8 messenger RNA, and IL-8 protein accumulation but showed no effect on LPS-dependent p38 MAPK activation . Inhibition of p38 MAPK by SB 202190 also blocked LPS-induced NF-kappaB activation and IL-8 synthesis . Furthermore, selective activation of the p38 MAPK pathway by transient expression of a constitutively active form of MAPK kinase (MKK)6, the upstream activator of p38, was as effective as LPS with respect to IL-8 expression in HUVECs . In summary, our data suggest that LPS-induced NF-kappaB activation and IL-8 synthesis in HUVECs are regulated by both a Rho-dependent signaling pathway and the MKK6/p38 kinase cascade. J Inorg Biochem, 2000 Feb, 78(3), 251 - 4 A scanning tunnelling microscopy study of Clostridium pasteurianum rubredoxin; Mukhopadhyay R et al.; Scanning tunnelling microscopy (STM), which can provide 'direct' and 'non-averaged' information on molecular structure in three dimensions, has been used to achieve sub-molecular resolution in a 'single molecule' of rubredoxin, an important iron-sulphur protein, at the gold (111)/water interface . The metal-ligand site {Fe(III)-Cys4} appears distinct because of an enhancement of the tunnelling current over this region compared to the surrounding protein structure. FEMS Microbiol Lett, 2000 May 15, 186(2), 307 - 12 Production of actin-specific ADP-ribosyltransferase (binary toxin) by strains of Clostridium difficile; Stubbs S et al.; In addition to the two large clostridial cytotoxins (TcdA and TcdB) certain strains of Clostridium difficile produce an actin-specific ADP-ribosyltransferase, or binary toxin . PCR reactions were developed to detect genes encoding the enzymatic (cdtA) and binding (cdtB) components of the binary toxin and 170 representative strains were tested to assess the prevalence of the toxin . Positive PCR results (n=59) were confirmed by immunoblotting and ADP-ribosyltransferase assay . PCR ribotype and toxinotype (restriction fragment length polymorphism analysis of genes for TcdA and TcdB) correlated with possession of binary toxin genes . All strains with cdtA and cdtB belonged to toxin-variable toxinotypes and five toxin-producing groups of strains have been described according to the presence or absence of TcdA, TcdB and binary toxin . Result indicate that ca . 6.4% of toxigenic isolates of C . difficile referred to the Anaerobe Reference Unit from UK hospitals have cdtA and cdtB genes. J Neurochem, 2000 May, 74(5), 2010 - 20 A role for the small molecular weight GTPases, Rho and Cdc42, in muscarinic receptor signaling to focal adhesion kinase; Linseman DA et al.; An enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) is elicited during neuronal growth cone remodeling and requires the maintenance of agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate (PIP2) . Rho family GTPases are putative regulators of both PIP2 synthesis and growth cone remodeling, including neurite outgrowth elicited by muscarinic cholinergic receptor (mAChR) stimulation . In this study, we investigated the interrelationships among Rho family GTPases, PIP2 synthesis, and mAChR signaling to FAK in SH-SY5Y neuroblastoma cells . Preincubation with Clostridium difficile toxin B (Tox B), an inhibitor of Rho, Rac, and Cdc42, attenuated mAChR-stimulated FAK and paxillin tyrosine phosphorylation and lysophosphatidic acid (LPA)-induced FAK phosphorylation to a similar extent (75% decreases at 200 pg/ml Tox B) but did not affect mitogen-activated protein kinase activation elicited by either phorbol ester or an mAChR agonist . In contrast, preincubation with selective inhibitors of either Rho (C3 exoenzyme) or Rho kinase (HA-1 077) resulted in 80-90% reductions in LPA-induced FAK phosphorylation but only 40-50% decreases in mAChR-stimulated phosphorylation . Moreover, mAChR-mediated FAK phosphorylation was significantly attenuated in cells scrape-loaded with dominant-negative N17Cdc42 but not N17Rac1 . Tox B had little or no effect on agonist-sensitive pools of PIP2 but inhibited mAChR-driven actin cytoskeletal remodeling . The results suggest that the Rho family GTPases, Rho and Cdc42, link mAChR stimulation to increases in FAK phosphorylation independently of effects on PIP2 synthesis. Can J Gastroenterol, 2000 Apr, 14(4), 353 - 8 Synchronous occurrence of collagenous colitis and pseudomembranous colitis; Vesoulis Z et al.; Synchronous collagenous and pseudomembranous colitis has not been previously reported . A 73-year-old woman presented with chronic watery diarrhea and abdominal cramping of six weeks' duration . Biopsies of the colon revealed findings of collagenous colitis involving the endoscopically normal right colon, and superimposed collagenous and pseudomembranous colitis involving the rectosigmoid colon . Endoscopically, the left colon revealed discrete ulcerative plaques, and Clostridium difficile toxin A assay was positive . The patient partially responded to a three-week regimen of metronidazole, and symptoms resolved completely with subsequent steroid therapy . At follow-up endoscopy four months later, colon biopsies demonstrated persistence of subepithelial collagen but no pseudomembranes . The patient remained asymptomatic during this interval . Collagenous colitis has been reported in association with other inflammatory bowel diseases, including lymphocytic colitis, sprue and idiopathic inflammatory bowel disease . This unique association of collagenous colitis with an endotoxigenic inflammatory bowel disease is presented with a review of related disease features. J Antimicrob Chemother, 2000 May, 45(5), 633 - 8 Synergic activity, for anaerobes, of trovafloxacin with clindamycin or metronidazole: chequerboard and time-kill methods; Ednie LM et al.; Chequerboard titrations were used to test the activity of trovafloxacin, alone and in combination with clindamycin or metronidazole, against 156 Gram-positive or Gram-negative anaerobes, including 47 Bacteroides fragilis group, 36 Prevotella spp., 26 fusobacteria, 21 peptostreptococci and 26 clostridia . MIC50/MIC90 values (mg/L) of each drug alone against all 156 strains were: trovafloxacin, 0.5/1; clindamycin, 0.25/2; metronidazole, 1/2 . Synergy (FIC indices </= 0.5) was seen in two strains with trovafloxacin plus clindamycin, and seven with trovafloxacin plus metronidazole . All other combinations were additive (FIC indices >0 . 5-2.0); no antagonism (FIC indices >4.0) was seen . In addition, synergy was tested by time-kill methodology for each of the above combinations against 12 Gram-positive or Gram-negative strains . Results indicated that synergy (defined as a >/= 2 log(10) decrease in cfu/mL at 48 h compared with the more active drug alone) was found between trovafloxacin at or below the MIC and both clindamycin and metronidazole at or below the MIC in one strain each of Bacteroides fragilis, Bacteroides thetaiotaomicron, Prevotella intermedia, Fusobacterium varium, Peptostreptococcus asaccharolyticus and Clostridium bifermentans . Synergy between trovafloxacin (</=MIC) and metronidazole alone was seen in one strain each of Bacteroides distasonis, Prevotella bivia, Fusobacterium mortiferum, P . asaccharolyticus and C . bifermentans . In many cases of synergy, including those at the trovafloxacin MIC, regrowth after 48 h, which was commonly seen with trovafloxacin alone, was inhibited, and 99.9% killing was observed with the combination after 48 h, but not with trovafloxacin alone. Eur J Clin Microbiol Infect Dis, 2000 Mar, 19(3), 228 - 32 Comparative activity of moxifloxacin in vitro against obligately anaerobic bacteria; Ackermann G et al.; The antimicrobial activity of moxifloxacin and seven other antibiotics (four of them quinolones) against 292 strains of obligately anaerobic bacteria was assessed employing a broth microdilution technique performed in Wilkens-Chalgren broth . MIC50/MIC90 values (mg/l) for moxifloxacin were as follows: Bacteroides fragilis (n = 62) 0.25/2, Bacteroides ovatus (n = 70) 1/4, Bacteroides vulgatus (n = 29) 0.25/1, Bacteroides thetaiotaomicron (n = 17) 2/2, Bacteroides caccae (n = 11) 1/2, Prevotella spp . (n = 11) 0.25/2, Fusobacterium spp . (n = 17) 1/4, Bilophila wadsworthia (n = 29) 0.5/1, and Clostridium spp . (n = 29) 0.125/0.5, respectively . MIC50 values (mg/l) for Bacteroides distasonis (n = 8) and Peptostreptococcus spp . (n = 9) were 0.25 . The results indicated that moxifloxacin was almost as active as trovafloxacin, as active as gatifloxacin, and more active than levofloxacin and ciprofloxacin against the anaerobes tested. J Med Chem, 2000 May 4, 43(9), 1858 - 65 Protease inhibitors: synthesis of potent bacterial collagenase and matrix metalloproteinase inhibitors incorporating N-4-nitrobenzylsulfonylglycine hydroxamate moieties; Scozzafava A et al.; A series of compounds was prepared by reaction of alkyl/arylsulfonyl halides with N-4-nitrobenzylglycine, followed by conversion of the COOH to the CONHOH group, with hydroxylamine in the presence of carbodiimides . Other structurally related compounds were obtained by reaction of N-4-nitrobenzylglycine with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety . Another subseries of derivatives was prepared from sulfanilyl- or metanilyl-4-nitrobenzylglycine by reaction with arylsulfonyl isocyanates, followed by conversion of the COOH to the hydroxamate moiety . The new compounds were assayed as inhibitors of four matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-8, and MMP-9, and of the Clostridium histolyticum collagenase (ChC) . Some of the prepared hydroxamate derivatives proved to be very effective collagenase/gelatinase inhibitors, depending on the substitution pattern at the sulfonamido moiety . Substitutions leading to best inhibitors of MMP-1, a short pocket enzyme, were those involving pentafluorophenylsulfonyl or 3-trifluoromethylphenylsulfonyl moieties at P(1') (K(I)'s of 3-5 nM) . For MMP-2, MMP-8, and MMP-9 (deep-pocket enzymes), best inhibitors were especially those containing long perfluoroalkylsulfonyl and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl, 3- and 4-carboxyphenylsulfonyl, arylsulfonylureido, or arylsulfonylureidosulfanilyl/metanilyl moieties, at P(1') . Bulkier groups in this position, such as 1- and 2-naphthyl, substituted-naphthyl, or quinolin-8-yl moieties among others, led to less effective MMP/ChC inhibitors . Best ChC inhibitors were again those containing pentafluorophenylsulfonyl or 3- and 4-protected-aminophenylsulfonyl P(1') anchoring groups, suggesting that this protease is also a short-pocket wider-neck one (more similar to MMP-1) . This study also proves that the 4-nitrobenzyl moiety is an efficient P(2') anchoring moiety and that sulfonylureido, ureido, or carboxythioureido substitutions at P(1') are also tolerated for obtaining potent sulfonylated amino acid hydroxamate-like MMP/ChC inhibitors. J Appl Microbiol, 2000 May, 88(5), 877 - 86 Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7; Zhu WM et al.; A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces . The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus . An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7 . It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography . Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE . The bacteriocin was produced constitutively during exponential growth . It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis . The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified. J Appl Microbiol, 2000 May, 88(5), 817 - 25 Biochemical properties of Streptococcus macedonicus strains isolated from Greek Kasseri cheese; Georgalaki MD et al.; A total of 32 Streptococcus macedonicus strains, isolated from Greek Kasseri cheese, were screened for biochemical properties of technological importance in milk fermentation processing, such as acid production, proteolytic and lipolytic activity, citrate metabolism, exopolysaccharide production, antimicrobial activity and biogenic amines production . All strains were found to be moderate acidifiers in milk . Only four strains could hydrolyse milk casein, while 11 strains showed lipolytic activity against tributyrin . Using amino acid derivatives of 4-nitroaniline as substrates, the highest peptidase activities were determined against phenylalanine- and glycine-proline-4-nitroanilide . Using fatty acid derivatives of 4-nitrophenol, it was shown that all strains exhibited esterase activities up to caprylate, with highest values against butyrate and caproate . Only one showed activity up to palmitate; this was also the most active strain against tributyrin . Five of the 32 strains could metabolize citrate but none of them produced exopolysaccharides . Nine strains displayed antimicrobial activity towards Clostridium tyrobutyricum, while no antimicrobial activity was detected against Listeria innocua and Propionibacterium freudenreichii subsp . shermanii . Finally, none was able to decarboxylize ornithine, histidine or lysine, and only four strains produced tyramine from tyrosine. Dis Colon Rectum, 2000 Apr, 43(4), 551 - 4 Pseudomembranous enteritis after proctocolectomy: report of a case; Vesoulis Z et al.; Intestinal pseudomembrane formation, sometimes a manifestation of antibiotic-associated diarrheal illnesses, is typically limited to the colon but rarely may affect the small bowel . A 56-year-old female taking antibiotics, who had undergone proctocolectomy for idiopathic inflammatory bowel disease, presented with septic shock and hypotension . A partial small-bowel resection revealed extensive mucosal pseudomembranes, which were cultured positive for Clostridium difficile . Intestinal drainage contents from an ileostomy were enzyme immunoassay positive for C . difficile toxin A . Gross and histopathologic features of the small-bowel resection specimen were similar to those characteristic of pseudomembranous colitis . The patient was treated successfully with metronidazole . These findings suggest a reservoir for C . difficile also exists in the small intestine and that conditions for enhanced mucosal susceptibility to C . difficile overgrowth may occur in the small-bowel environment of antibiotic-treated patients after colectomy . Pseudomembranous enteritis should be a consideration in those patients who present with purulent ostomy drainage, abdominal pain, fever, leukocytosis, or symptoms of septic shock. J Biochem (Tokyo), 2000 May, 127(5), 909 - 14 Rho-mediated phosphorylation of focal adhesion kinase and myosin light chain in human endothelial cells stimulated with sphingosine 1-phosphate, a bioactive lysophospholipid released from activated platelets; Miura Y et al.; Since sphingosine 1-phosphate (Sph-1-P) is stored in abundant amounts in blood platelets and released extracellularly upon stimulation, it is important to clarify the effects of this bioactive lysophospholipid on vascular endothelial cells from the viewpoint of platelet-endothelial cell interactions . In this study, we investigated the effects of Sph-1-P on the cytoskeletal remodeling of human umbilical vein endothelial cells (HUVECs) . Of a focal adhesion kinase (FAK) family of non-receptor protein-tyrosine kinases, HUVECs were found to express FAK, but scarcely proline-rich tyrosine kinase 2 . Sph-1-P induced FAK tyrosine phosphorylation, myosin light chain phosphorylation, and the formation of stress fibers in HUVECs . The specific Rho inactivator C3 transferase from Clostridium botulinum abolished all of these cytoskeletal responses induced by Sph-1-P, while pertussis toxin only partly inhibited FAK tyrosine phosphorylation, and hardly affected myosin light chain phosphorylation and stress fiber formation . In contrast, Sph-1-P-induced intracellular Ca(2)(+) mobilization was suppressed by pertussis toxin, but not at all by C3 exoenzyme . Our results suggest that Sph-1-P, a bioactive lipid released from activated platelets, induces endothelial cell cytoskeletal reorganization, mainly through Rho-mediated signaling pathways. J Biol Chem, 2000 May 5, 275(18), 13228 - 34 Involvement of a conserved tryptophan residue in the UDP-glucose binding of large clostridial cytotoxin glycosyltransferases; Busch C et al.; Large clostridial cytotoxins catalyze the glucosylation of Rho/Ras GTPases using UDP-glucose as a cosubstrate . By site-directed mutagenesis of Clostridium sordellii lethal toxin and Clostridium difficile toxin B fragments, we identified tryptophan 102, which is located in a conserved region within the catalytic domain of all clostridial cytotoxins, to be crucial for UDP-glucose binding . Exchange of Trp-102 with alanine decreased the glucosyltransferase activity by about 1,000-fold and blocked cytotoxic activity after microinjection . Replacement of Trp-102 by tyrosine caused a 100-fold reduction in enzyme activity, indicating a partial compensation of the tryptophan function by tyrosine . Decrease in glucosyltransferase and glycohydrolase activity was caused predominantly by an increase in the K(m) for UDP-glucose of these mutants . The data indicate that the conserved tryptophan residue is implicated in the binding of the cosubstrate UDP-glucose by large clostridial cytotoxins . Data bank searches revealed different groups of proteins sharing the recently identified DXD motif (Busch, C., Hofmann, F., Selzer, J., Munro, J., Jeckel, D., and Aktories, K . (1998) J . Biol . Chem . 273, 19566-19572) and a conserved region defined by a tryptophan residue equivalent to Trp-102 of C . sordellii lethal toxin . From our findings, we propose a novel family of glycosyltransferases which includes both prokaryotic and eukaryotic proteins. Appl Environ Microbiol, 2000 May, 66(5), 2199 - 207 Localization of symbiotic clostridia in the mixed segment of the termite Nasutitermes takasagoensis (Shiraki); Tokuda G et al.; Phylogeny and the distribution of symbiotic bacteria in the mixed segment of the wood-eating termite Nasutitermes takasagoensis (Shiraki) were studied . Bacterial 16S rRNA genes (rDNA) were amplified from the mixed segment of the gut by PCR, and two kinds of sequences were identified . The phylogenetic tree was constructed by neighbor-joining and maximum parsimony methods to identify symbionts harbored in the mixed segment . They are classified as low-G+C-content gram-positive bacteria and are most closely related to the genus Clostridium . The distribution of these bacteria throughout the whole gut was examined by PCR using specific primers, which suggested that they are confined to the mixed segment despite the presence of bacteria throughout the gut . In situ hybridization indicated that the symbiotic bacteria were localized to the ectoperitrophic space between the midgut wall and the peritrophic membrane in the mixed segment . Electron microscopy revealed the close association between these bacteria and the mesenteric epithelium, suggesting that they have some interactions with the gut tissue of termites. Intensive Care Med, 2000, 26 Suppl 1, S14 - 21 Epidemiology of antibiotic resistance; Livermore DM; Three biological processes contribute to the accumulation of bacterial drug resistance: new selection, gene epidemics and strain epidemics . New resistance emerges by (i) the advantaging of entire species, (ii) by mutation, and (iii) by the escape of resistance genes to mobile DNA . Organisms to have 'benefited' from modern patterns of cephalosporins and quinolone use include enterococci, Clostridium difficile, coagulase-negative staphylococci and Enterobacter spp . Mutational resistance notoriously occurs with certain antibiotic/organism combinations and allows rapid multifocal accumulation of resistance . At worst, therapy can fail when resistant mutants are selected in individual patients . Escape of new genes to mobile DNA is rare but, having occurred, permits massive 'gene epidemics', as the same genes and plasmids spread into diverse pathogens . Strain epidemics notoriously occur in individual units, reflecting break-downs of hygiene . Some strains achieve a much wider distribution: thus, much of the MRSA problem in the UK depends on the dissemination of two epidemic strains, EMRSA15 and 16; penicillin resistant pneumococci of serotypes 6 and 23 have disseminated internationally from Spain and a serotype K25 strain of Klebsiella pneumoniae with SHV-4 beta-lactamase has spread widely in France . It remains unknown why some strains and genes achieve wide spread whereas others, equally resistant, fail to do so . There is no simple cure for resistance but the best opportunities for control lie in lesser and better use of antibiotics backed by swifter and more accurate microbiology; in developing new antibiotics; and in protecting old ones from resistance determinants . All this must be supported by good local knowledge of the epidemiology of infections and resistance and of the likelihood of particular antibiotics to select resistance. Eur J Med Chem, 2000 Mar, 35(3), 299 - 307 Protease inhibitors - part 5 . Alkyl/arylsulfonyl- and arylsulfonylureido-/arylureido- glycine hydroxamate inhibitors of Clostridium histolyticum collagenase; Scozzafava A et al.; Reaction of alkyl/arylsulfonyl halides with glycine afforded a series of derivatives which were first N-benzylated by treatment with benzyl chloride, and then converted to the corresponding hydroxamic acids with hydroxylamine in the presence of carbodiimide derivatives . Other derivatives were obtained by reaction of N-benzyl-glycine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by conversion of their COOH group into the CONHOH moiety, as mentioned above . The 90 new compounds reported here were assayed as inhibitors of the Clostridium histolyticum collagenase (EC 3.4.24.3), a zinc enzyme which degrades triple helical regions of native collagen . The prepared hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates . In the series of synthesized hydroxamates, substitution patterns leading to the best inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-carboxyphenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl or 1- and 2-naphthyl among others . Thus, it seems that similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, Clostridium histolyticum collagenase inhibitors should incorporate hydrophobic moieties at the P(1') and P(2') sites, whereas the alpha-carbon substituent may be a small and compact moiety (such as H, for the Gly derivatives reported here) . Such compounds might lead to the design of collagenase inhibitor-based drugs useful as anti-cancer, anti-arthritis or anti-bacterial agents for the treatment of corneal keratitis. Eur J Biochem, 2000 May, 267(9), 2525 - 32 M1 muscarinic acetylcholine receptors activate zif268 gene expression via small G-protein Rho-dependent and lambda-independent pathways in PC12D cells; Hirabayashi T et al.; We have previously shown that stimulation of M1 muscarinic acetylcholine receptors (mAChRs) in neuronal PC12D cells rapidly induces the immediate-early gene zif 268 {Ebihara, T . & Saffen, D . (1997) J . Neurochem . 68, 1001-1010} . Here we show that stimulation of M1 mAChRs in these cells activates four distal serum response elements (SREs) in the zif 268 promoter, and that this activation is strongly inhibited by Clostridium botulinum C3 exoenzyme (C3), which specifically inactivates the small G-protein Rho . Even with high doses of C3, however, a portion of the activation remains intact, indicating that stimulation of M1 mAChRs activates zif 268 SREs via Rho-dependent and Rho-independent pathways . Moreover, the Rho-independent activation of zif 268 SREs is inhibited by the dominant-negative form of the small G-protein Ras, suggesting that Rho-independent activation of zif 268 SREs is mediated by Ras . To determine if muscarinic agonists activate RhoA, we also measured the translocation of RhoA from the cytosolic fraction to the particulate fraction . Translocation of RhoA to the particulate fraction was observed within 15 min following stimulation of M1 mAChRs, indicating that RhoA is activated with sufficient rapidity to participate in the induction of zif 268 mRNA . Together, these results suggest that RhoA is activated following stimulation of M1 mAChRs and functions in SRE-dependent induction of the zif 268 gene within a Ras-independent pathway. Microbiology, 2000 Apr, 146 ( Pt 4), 957 - 66 A Clostridium difficile gene encoding flagellin; Tasteyre A et al.; Six strains of Clostridium difficile examined by electron microscopy were found to carry flagella . The flagella of these strains were extracted and the N-terminal sequences of the flagellin proteins were determined . Four of the strains carried the N-terminal sequence MRVNTNVSAL exhibiting up to 90% identity to numerous flagellins . Using degenerate primers based on the N-terminal sequence and the conserved C-terminal sequence of several flagellins, the gene encoding the flagellum subunit (fliC) was isolated and sequenced from two virulent strains . The two gene sequences exhibited 91% inter-strain identity . The gene consists of 870 nt encoding a protein of 290 amino acids with an estimated molecular mass of 31 kDa, while the extracted flagellin has an apparent molecular mass of 39 kDa on SDS-PAGE . The FliC protein displays a high degree of identity in the N- and C-terminal amino acids whereas the central region is variable . A second ORF is present downstream of fliC displaying homology to glycosyltransferases . The fliC gene was expressed in fusion with glutathione S-transferase, purified and a polyclonal monospecific antiserum was obtained . Flagella of C . difficile do not play a role in adherence, since the antiserum raised against the purified protein did not inhibit adherence to cultured cells . PCR-RFLP analysis of amplified flagellin gene products and Southern analysis revealed inter-strain heterogeneity; this could be useful for epidemiological and phylogenetic studies of this organism. Eur J Gastroenterol Hepatol, 2000 Apr, 12(4), 451 - 4 Short-acting general anaesthesia facilitates therapeutic ERCP in frail elderly patients with benign extra-hepatic biliary disease; Cocking JB et al.; AIMS AND OBJECTIVES: To ascertain whether therapeutic endoscopic retrograde cholangiopancreatography (ERCP) for benign biliary disease in frail elderly patients with comorbid conditions can be safely undertaken in a district general hospital, and whether the procedure is facilitated by the use of short-acting general anaesthesia . SETTING: District general hospital in South East England . DESIGN OF STUDY: Clinical study of 25 consecutive patients with benign biliary disease . METHODS: Describes the process of bile duct clearance by therapeutic ERCP under short-acting general anaesthesia in 25 patients with co-morbidity aged > or = 80 years and gives details of the general anaesthesia and monitoring . RESULTS: Twenty-two patients had their bile ducts successfully cleared locally and one patient was stented for a benign biliary stricture . The ampullae of two other patients were lying within diverticula, which hindered cannulation and only pancreatograms were obtained; one of the patients had a successful bile duct clearance at a tertiary centre, the other refused further intervention . Complications (melaena, bronchopneumonia and a Clostridium difficile infection) occurred in two patients (8%) . There was no morbidity associated with the anaesthesia, and no mortality occurred within 30 days of the procedure . CONCLUSIONS: Bile duct clearance by therapeutic ERCP can be safely carried out in frail elderly patients in a district general hospital and the process is facilitated by the use of short-acting general anaesthesia . The importance of optimizing the patient's condition before ERCP, and not overfilling the pancreatic duct, is highlighted. Dig Surg, 2000, 17(2), 160 - 3 Acute abdomen and Clostridium difficile colitis: still a lethal combination; Klipfel AA et al.; BACKGROUND: With the steadily prevalent appropriate and inappropriate use of antimicrobial agents, Clostridium difficile colitis has continued to be noticed as a common problem in hospitalized patients . The aim of this communication is to highlight a subset of C . difficile colitis patients who presented with an acute abdomen . METHODS: This is a retrospective study of 10 patients who underwent laparotomy for an 'acute abdomen' with an intraoperative or postoperative diagnosis of C . difficile colitis . RESULTS: All patients received antibiotics (mean 9.5 days) for other illnesses . The mean APACHE II score was 18.8 (range 8-25) and the mortality rate was 80% . Two patients had colostomies created . One patient underwent a subtotal colectomy, and another underwent a Hartmann procedure; the rest had a nontherapeutic procedure . CONCLUSION: We conclude that C . difficile colitis presenting as an 'acute abdomen' still represents a lethal entity . Patients who present with an 'acute abdomen', with a history of recent or current antibiotic intake, and without findings which mandate an exploration should have C . difficile colitis urgently excluded . Timely diagnosis of C . difficile colitis through bedside sigmoidoscopy or a CT scan could spare the critically ill patient an unneccessary and risky operation . Furthermore, if laparotomy is subsequently needed then having a preoperative diagnosis of C . difficile colitis will allow appropriate surgical therapy to be implemented . N Engl J Med, 2000 Apr 27, 342(17), 1250 - 3 Enteritis necroticans (pigbel) in a diabetic child; Petrillo TM et al.; BACKGROUND AND METHODS: Enteritis necroticans (pigbel), an often fatal illness characterized by hemorrhagic, inflammatory, or ischemic necrosis of the jejunum, occurs in developing countries but is rare in developed countries, where its occurrence is confined to adults with chronic illnesses . The causative organism of enteritis necroticans is Clostridium perfringens type C, an anaerobic gram-positive bacillus . In December 1998, enteritis necroticans developed in a 12-year-old boy with poorly controlled diabetes mellitus after he consumed pig intestines (chitterlings) . He presented with hematemesis, abdominal distention, and severe diabetic ketoacidosis with hypotension . At laparotomy, extensive jejunal necrosis required bowel resection, jejunostomy, and ileostomy . Samples were obtained for histopathological examination . Polymerase-chain-reaction (PCR) assay was performed on paraffin-embedded bowel tissue with primers specific for the cpa and cpb genes, which code for the alpha and beta toxins produced by C . perfringens . RESULTS: Histologic examination of resected bowel tissue showed extensive mucosal necrosis, the formation of pseudomembrane, pneumatosis, and areas of epithelial regeneration that alternated with necrotic segments--findings consistent with a diagnosis of enteritis necroticans . Gram's staining showed large gram-positive bacilli whose features were consistent with those of clostridium species . Through PCR amplification, we detected products of the cpa and cpb genes, which indicated the presence of C . perfringens type C . Assay of ileal tissue obtained during surgery to restore the continuity of the patient's bowel was negative for C . perfringens . CONCLUSIONS: The preparation or consumption of chitterlings by diabetic patients and other chronically ill persons can result in potentially life-threatening infectious complications. Int J Food Microbiol, 2000 Mar 25, 54(3), 213 - 7 Occurrence of clostridia in glass bottled foods; Fujisawa T et al.; Forty-one marketed samples of imported and domestic glass bottled foods were tested for clostridia contamination . It was detected in nine (22%) samples . Clostridia were isolated from fish sauce (Nam pla, Nuoc-mam), dressing, mustard, hot and sour soup mix (Tom Yum), mysids boiled down in soy, and salmon flakes . The origin of all clostridia positive samples was Asia . Clostridium botulinum and C . perfringens were not detected . The frequency of occurrence was higher by enrichment broth culture detection methods than by agar plate or pouch methods . These findings suggest that the number of bacteria in most of these clostridia positive food samples is very low, and the use of enrichment methods for detection of clostridia is essential. Int J Food Microbiol, 2000 Mar 25, 54(3), 147 - 54 Influence of milk centrifugation, brining and ripening conditions in preventing gas formation by Clostridium spp . in Gouda cheese; Su YC et al.; This study examined milk centrifugation, increased salt concentration, and low ripening temperature as potential strategies to prevent late blowing caused by gas-forming Clostridium spp . in Gouda cheese . The survival of clostridia spores in cheese brine and their ability to enter Gouda cheese during brining was also evaluated . Centrifugation (3000 x g for 30 s) of contaminated milk resulted in > 60% spore reduction, with increased spore reduction at greater centrifugal forces . Low levels of C . tyrobutyricum and C . sporogenes spores survived in saturated (23%, w/v) brine with 2% (v/v) added whey at 15 degrees C for 63 days, while C . beijerinckii and C . butyricum spores were not detectable on days 4 and 35, respectively . Spores of C . tyrobutyricum in brine infiltrated Gouda cheese during 2 h of brining at 13 degrees C resulted in production of small gas holes during ripening . In Gouda cheese slurry stored at 13 degrees C, three C . tyrobutyricum strains plus one of three C . sporogenes strains germinated in the slurry with no added salt . Of three C . tyrobutyricum strains stored at 13 degrees C in slurries with higher water-phase salt concentrations of 2.4 and 3.6%, two strains and one strain germinated, respectively . No germination of spores was detected in any cheese slurry stored at 5 or 8 degrees C . Milk centrifugation, increased percent water-phase salt, absence of spores in brine, and decreased ripening temperature are all potentially important measures against gas production by Clostridium spp . in Gouda cheese. Toxicon, 2000 Nov, 38(11), 1529 - 34 Antibacterial properties of KwaZulu natal snake venoms; Blaylock RS; The objective was to ascertain whether local snake venoms have antibacterial properties . The venoms of the common night adder (Causus rhombeatus), gaboon adder (Bitis gabonica), puff adder (Bitis arietans), black mamba (Dendroaspis polylepis), eastern green mamba (Dendroaspis augusticeps), forest cobra (Naja melanoleuca), snouted cobra (Naja annulifera) and Mozambique spitting cobra (Naja mossambica) were collected and, by gel diffusion, tested against the bacteria Staphylococcus aureus, Escherichia coli, Pseudomonas aeriginosa, Bacteriodes fragilis, Bacteroides intermedius, Clostridium sordellii and Clostridium perfringens . All snake venoms showed antibacterial activity, with the adders showing most activity against the aerobes while the cobras showed lesser, but equal activity against the aerobes and anaerobes . Black mamba venom only showed activity against C . perfringens . In conclusion, local snake venoms have antibacterial properties which are dependent on the venom and bacterial type; and in the Naja spp., for anaerobic bacteria, diminish in winter . There is liable to be more than one toxin component responsible. Arch Biochem Biophys, 2000 May 1, 377(1), 85 - 94 Activation of m3 muscarinic receptors induces rapid tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin in rat pancreatic acini; Rosado JA et al.; Tyrosine phosphorylation plays a key role in transmembrane and cytoplasmic signal transduction mechanisms stimulated by oncogenes, integrins, growth factors, neuropeptides, and bioactive lipids . Moreover, recent studies show that stimulation of odd-numbered muscarinic receptors increases the tyrosine phosphorylation of several proteins in different cellular types . The present study was aimed at examining whether activation of m3 muscarinic receptors in rat pancreatic acini evokes tyrosine phosphorylation of p125(FAK), and its substrates, p130(cas) and paxillin . Results show that stimulation of pancreatic acini with carbachol resulted in a rapid and transient increase in tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin . Tyrosine phosphorylation of these proteins occurred in a time- and concentration-dependent manner . Simultaneous blockage of both PKC activation and increases in {Ca(2+)}(i) partially decreased p125(FAK), p130(cas), and paxillin tyrosine phosphorylation stimulated by carbachol . Pretreatment of pancreatic acini with Clostridium botulinum C3 transferase, which specifically inactivates p21(rho), partially inhibited carbachol-induced p125(FAK), p130(cas), and paxillin tyrosine phosphorylation . In contrast, this treatment had no effect on amylase release stimulated by carbachol . Cytochalasin D, which disrupts actin microfilaments network, completely inhibited carbachol stimulated tyrosine phosphorylation of these proteins without having significant effects in carbachol-stimulated amylase secretion . These results dissociate tyrosine phosphorylation of p125(FAK), p130(cas), and paxillin from amylase secretion after m3 muscarinic receptors occupation in rat pancreatic acini . Taken together, these data suggest that (a) activation of m3 muscarinic receptors in rat pancreatic acini increases tyrosine phosphorylation of p125(FAK) and its substrates, p130(cas) and paxillin by diacylglycerol-activated PKC- and calcium- dependent, and independent pathways, (b) these responses require activation of p21(rho) and an intact actin cytoskeleton, and (c) p125(FAK), p130(cas), and paxillin are unlikely related to secretion in rat pancreatic acinar cells . Hum Exp Toxicol, 2000 Feb, 19(2), 108 - 16 An assessment of the in vitro toxicology of Clostridium perfringens type D epsilon-toxin in human and animal cells; Shortt SJ et al.; The epithelial Madin Darby canine kidney (MDCK) cell line and 17 human cell lines were examined for sensitivity to Clostridium perfringens type D epsilon-toxin . MDCK cells were confirmed as being sensitive to the toxin . In addition, the Caucasian renal leiomyoblastoma (G-402) human cell line was identified as being epsilon-toxin sensitive . Using the MTS/PMS assay system the concentration of toxin reducing cell culture viability by 50% (LC50) was found to be 2 microg/ml in MDCK cells . The LC50 for G-402 cells was 280 microg/ml . Epsilon-Toxin was found to be rapid acting in MDCK cells exposed to a maximum lethal dose of the toxin (40% loss of viability after a 0.5 h exposure), but slower acting in G-402 cells (40% loss of viability after 1.7 h exposure) . Photomicrography of toxin exposed cultures indicated necrotic cell death on exposure to epsilon-toxin . Investigations using an antibody probe indicated that epsilon-toxin could bind to many cell surface proteins in both MDCK, G-402 and a toxin insensitive human cell line (CAKI-2) . It has previously been found that the toxin may bind to the cell surface via glycosylated moieties . However, exposing MDCK and G-402 cells to epsilon-toxin in the presence of sialic acid and several different sugars did not reduce the lethal effects of the toxin. J Clin Invest, 2000 Apr, 105(8), 1147 - 56 p38 MAP kinase activation by Clostridium difficile toxin A mediates monocyte necrosis, IL-8 production, and enteritis; Warny M et al.; Clostridium difficile toxin A causes acute neutrophil infiltration and intestinal mucosal injury . In cultured cells, toxin A inactivates Rho proteins by monoglucosylation . In monocytes, toxin A induces IL-8 production and necrosis by unknown mechanisms . We investigated the role of mitogen-activated protein (MAP) kinases in these events . In THP-1 monocytic cells, toxin A activated the 3 main MAP kinase cascades within 1 to 2 minutes . Activation of p38 was sustained, whereas stimulation of extracellular signal-regulated kinases and c-Jun NH(2)-terminal kinase was transient . Rho glucosylation became evident after 15 minutes . IL-8 gene expression was reduced by 70% by the MEK inhibitor PD98059 and abrogated by the p38 inhibitor SB203580 or by overexpression of dominant-negative mutants of the p38-activating kinases MKK3 and MKK6 . SB203580 also blocked monocyte necrosis and IL-1beta release caused by toxin A but not by other toxins . Finally, in mouse ileum, SB203580 prevented toxin A-induced neutrophil recruitment by 92% and villous destruction by 90% . Thus, in monocytes exposed to toxin A, MAP kinase activation appears to precede Rho glucosylation and is required for IL-8 transcription and cell necrosis . p38 MAP kinase also mediates intestinal inflammation and mucosal damage induced by toxin A. Crit Rev Food Sci Nutr, 2000 Mar, 40(2), 159 - 72 Modeling microbial survival during exposure to a lethal agent with varying intensity; Peleg M et al.; Traditionally, the efficacy of preservation and disinfection processes has been assessed on the basis of the assumption that microbial mortality follows a first-order kinetic . However, as departures from this assumed kinetics are quite common, various other models, based on higher-order kinetics or population balance, have also been proposed . The database for either type of models is a set of survival curves of the targeted organism or spores determined under constant conditions, that is, constant temperature, chemical agent concentration, etc . Hence, to calculate the outcome of an actual industrial process, where conditions are changing, as in heating and cooling during a thermal treatment or when the agent dissipates as in chlorination or hydrogen peroxide application, one has to integrate the momentary effects of the lethal agent . This involves mathematical models based on assumed mortality kinetics, and simulated or measured history, for example, temperature-time or concentration-time relationships at the "coldest" point . It is shown that the survival curve under conditions where the agent intensity increases, decreases, or oscillates can be constructed without assuming any mortality kinetics and without the use of the traditional D and Z values, which require linear approximation, and without thermal death times, which require extrapolation . The actual survival curves can be compiled from the isothermal survival curves provided that growth and damage repair do not occur over the pertinent time scale and that the mortality rate is a function of only the momentary agent intensity and of the organism's or spore's survival fraction (but not of the rate at which this fraction has been reached) . The calculation is greatly facilitated if both the "isothermal" survival curves and the time-dependent agent intensity can be expressed algebraically . The differential equation derived from these considerations can be solved numerically to produce the required survival curve under the changing conditions . The concept is demonstrated with simulated survival curves during heating at different rates, heating and cooling cycles, oscillating temperature, and exposure to a dissipating chemical agent . The simulated thermal processes are based on published data of Clostridium botulinum spores, whose semilogarithmic survival curves have upward concavity and on a hypothetical "Listeria-like" organism whose semilogarithmic curves have downward concavity. Biochem J, 2000 May 1, 347 Pt 3, 865 - 73 Epoxyalkyl glycosides of D-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10; Ntarima P et al.; A series of omega-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families10 and 11 . Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant toelectrophilic attack of active-site carboxyl residues, glycosidehydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited . Theapparent inactivation and association constants (k(i), 1/K(i)) are one order of magnitude higher for thexylobiose and xylotriose derivatives . The effects of the aglycone chainlength can clearly be described . Xylobiose and n-alkyl beta-D-xylopyranosides are competitive ligands and provide protectionagainst inactivation . MS measurements showed 1:1 stoichiometries inmost labelling experiments . Electrospray ionization MS/MS analysisrevealed the nucleophile Glu(86) as the modified residue inthe T . lanuginosus xylanase when 2,3-epoxypropyl beta-D-xylopyranoside was used, whereas the acid/base catalyst Glu(178) was modified by the 3,4-epoxybutyl derivative . The active-site residues Glu(86) and Glu(177) in T . reesei Xyn II are similarly modified, confirming earlier X-raycrystallographic data {Havukainen, Torronen, Laitinen and Rouvinen (1996)Biochemistry 35, 9617-9624} . The inability of the omega-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10enzymes is discussed in terms of different ligand-subsiteinteractions. Infect Immun, 2000 May, 68(5), 2587 - 93 Inhibition of vesicular secretion in both neuronal and nonneuronal cells by a retargeted endopeptidase derivative of Clostridium botulinum neurotoxin type A; Chaddock JA et al.; Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex . A derivative of the type A neurotoxin from Clostridium botulinum (termed LH(N)/A) that retains catalytic activity can be prepared by proteolysis . The LH(N)/A, however, lacks the putative native binding domain (H(C)) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release . Here we report the chemical conjugation of LH(N)/A to an alternative cell-binding ligand, wheat germ agglutinin (WGA) . When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LH(N)/A conjugate potently inhibits secretory responses in those cells . Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate . These data confirm that the function of the H(C) domain of C . botulinum neurotoxin type A is limited to binding to cell surface moieties . The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin . These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated. Infect Immun, 2000 May, 68(5), 2470 - 4 pH-induced conformational changes in Clostridium difficile toxin B; Qa'Dan M et al.; Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells . In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B . Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation . Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment . Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents . Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease . As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B . The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence . Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments . Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein. J Biol Inorg Chem, 2000 Feb, 5(1), 75 - 84 Mutation of the surface valine residues 8 and 44 in the rubredoxin from Clostridium pasteurianum: solvent access versus structural changes as determinants of reversible potential; Xiao Z et al.; The Pr(i) sidechains of two adjacent valine residues, V8 and V44, define the surface of the rubredoxin from Clostridium pasteurianum and control access to its Fe(S-Cys)4 active site . To assess the effect of systematic change of the steric bulk of the alkyl sidechains, eight single and three double mutant proteins have been isolated which vary G (H), A (Me), V (Pr(i)), L (Bu(i)) and I (Bu(s)) at those positions . X-ray crystal structures of the Fe(III) forms of the V44A and V44I proteins are reported . Positive shifts in reversible potential of up to 116 mV are observed and attributed to increased polarity around the Fe(S-Cys)4 site induced by (1) changes in protein backbone conformation driven by variation of the steric demands of the sidechain substituents and (2) changes in solvent access to the side-chains of ligands C9 and C42 . Data for the V44A mutant show that a minor change in the steric requirements of a surface residue can introduce a NH...Sgamma hydrogen bond at the active site and lead to a shift in potential of + 50 mV. Diagn Microbiol Infect Dis, 2000 Apr, 36(4), 211 - 3 Comparison of the TOX A/B test to a cell culture cytotoxicity assay for the detection of Clostridium difficile in stools; Aldeen WE et al.; The TOX A/B Test (Techlab, Blacksburg, VA, USA) was compared to cell culture cytotoxicity assay on 1109 consecutive diarrheal stool samples collected from patients with the presumptive diagnosis of Clostridium difficile disease . The TOX A/B Test is an enzyme immunoassay in a microtiter format that detects both toxins A and B . The procedure used for this study takes approximately 1.5 h to perform . Cell culture cytotoxicity was performed by using a fibroblast cell line in a microtiter format read at 4 h, 24 h, and 48 h.One hundred ninety-four of the 1109 samples were positive by the "gold standard" cytotoxicity assay, whereas 189 were positive by EIA . There was a 98.5% agreement between the two assays . When compared to the cytotoxicity assay, the EIA had an initial sensitivity of 94.3% and a specificity of 99.3% . However, after resolution of six discrepants using another ELISA for toxin A detection the sensitivity, specificity, positive and negative predictive values for the TOX A/B test are as follows: 94.5%; 100%; 100%; 98.8% . The corresponding values for the cytotoxicity assay are: 97%; 100%; 100%; and 99.3% . This test seems to have excellent sensitivity and specificity as compared to an in-house cell culture cytotoxicity assay . It is sensitive enough to use as a stand-alone test for the detection of C . difficile toxin in laboratories that do not have cell culture cytotoxicity testing capability. J Biol Chem, 2000 Jun 23, 275(25), 18745 - 50 The small G-protein Rac mediates depolarization-induced superoxide formation in human endothelial cells; Sohn HY et al.; Superoxide anions impair nitric oxide-mediated responses and are involved in the development of hypertensive vascular hypertrophy . The regulation of their production in the vascular system is, however, poorly understood . We investigated whether changes in membrane potential that occur in hypertensive vessels modulate endothelial superoxide production . In cultured human umbilical vein endothelial cells, changes in membrane potential were induced by high potassium buffer, the non-selective potassium channel blocker tetrabutylammonium chloride (1 mm), and the non-selective cation ionophore gramicidin (1 micrometer) . Superoxide formation was significantly elevated to a similar degree by all three treatments (by approximately 60%, n = 23, p < 0.01), whereas hyperpolarization by the K(ATP) channel activator Hoe234 (1 micrometer) significantly decreased superoxide formation . Depolarization also induced an increased tyrosine phosphorylation of several not yet identified proteins (90-110 kDa) and resulted in a significant increase in membrane association of the small G-protein Rac . Accordingly, the Rac inhibitor Clostridium difficile toxin B blocked the effects of depolarization on superoxide formation . The tyrosine kinase inhibitor genistein (30 micrometer, n = 15) abolished depolarization-induced superoxide formation and also prevented depolarization-induced Rac translocation associated with it . It is concluded that depolarization is an important stimulus of endothelial superoxide production, which involves a tyrosine phosphorylation-dependent translocation of the small G-protein Rac. Biosci Rep, 1999 Oct, 19(5), 373 - 83 The terminal enzymes of sialic acid metabolism: acylneuraminate pyruvate-lyases; Schauer R et al.; The acylneuraminate pyruvate-lyase gene from Clostridium perfringens was sequenced and found to be most similar to the lyase gene from Haemophilus influenzae . Both the recombinant clostridial enzyme and the native enzyme from pig kidney were purified in larger amounts and characterized . The properties of the porcine lyase are similar to the microbial ones . However, the much higher degree of similarity in comparison to the microbial enzymes that was found between porcine lyase peptides and two putative mammalian lyase sequences show that the latter form an own group apart from the microbial lyases . Actual models of the acylneuraminate pyruvate-lyase reaction are discussed. Otolaryngol Pol, 1999, 53(6), 687 - 91 {Frey syndrome: diagnosis and treatment}; Budzinski R; Observed lately increased of the Lucie Frey's syndrom occurance is related to advanced frequency of the parotid gland surgery, especially because of neoplasmic lesions of the gland . Symptoms which follow partial or total parotidectomy are very bothersome for patients and unfortunately they strengthening with a lapse of a time . For applied pharmacotherapy outcomes are in fact dubious . In this light the recently applicated clostridium botulinum toxin of type A deserves of special attention as patient therapeutic agent for Frey's syndrome because of very promising results which can last for several months and even years . At first discussing the anatomopatholophysiology as well as diagnostic tests . The authors present their own experience as well as results obtained in application of the botulin toxin for therapy of the Lucie Frey syndrome. J Paediatr Child Health, 2000 Apr, 36(2), 193 - 5 Infantile botulism: clinical and laboratory observations of a rare neuroparalytic disease; Urdaneta-Carruyo E et al.; A 3-month-old male infant was admitted to the University Hospital of Los Andes with a history of constipation, weak crying, poor feeding, flaccidity and later bilateral ptosis and hyporeflexia . The admission diagnosis was septicaemia until an electrophysiological study reported postetanic facilitation with 50 Hz/seg stimulations four days later . The Clostridium botulinum toxin type B was isolated from the infant's stool samples and the organism grew in anaerobic cultures . The patient recovered completely and was discharged 2 months later . Although infant botulism is an uncommon disease in our environment, this diagnosis must be suspected in all afebrile infants with constipation, affected cranial nerves and generalized hypotonia . The principal differential diagnoses are Landry-Guillain-Barre syndrome, poliomyelitis, myasthenia gravis and infant muscular atrophy. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 873 - 81 Clostridium akagii sp . nov . and Clostridium acidisoli sp . nov.: acid-tolerant, N2-fixing clostridia isolated from acidic forest soil and litter; Kuhner CH et al.; Two anaerobic acid-tolerant bacteria, CK58T and CK74T, were isolated from acidic beech litter and acidic peat-bog soil, respectively . Both bacteria were spore-forming, motile rods with peritrichous flagella . The capacity to sporulate decreased with prolonged cultivation . Cells of CK58T formed chains or aggregates and were linked by a connecting filament that consisted of a core and a surrounding sheath . Cellobiose, glucose, xylose, arabinose, maltose, mannose and salicin supported growth of CK58T . These substrates, as well as mannitol, lactose, sucrose, glycerol, melezitose, raffinose and rhamnose, supported growth of CK74T . Sorbitol, trehalose, H2/CO2, CO/CO2, vanillate, Casamino acids, peptone, and various purines and pyrimidines did not support the growth of either organism . Growth of CK58T and CK74T on glucose yielded butyrate, lactate, acetate, formate, H2 and CO2 as end products . Growth of CK58T and CK74T was observed at pH 3.7-7.1 and 3.6-6.9, respectively . CK58T and CK74T grew in nitrogen-free medium at pH 3.7 under an N2 atmosphere and reduced acetylene at rates approximating 1 nmol min-1 (mg protein)-1 . CK58T and CK74T did not contain carbon monoxide dehydrogenase or cytochromes, produce methane, or dissimilate nitrate or sulfate . Thus, CK58T and CK74T were characterized as nonacetogenic, N2-fixing, fermentative chemo-organotrophs . The G + C contents of CK58T and CK74T were 31.4 and 30.7 mol%, respectively . CK58T and CK74T were phylogenetically most closely related to Clostridium pasteurianum . The 16S rRNA gene sequence similarity values of CK58T and CK74T to C . pasteurianum and each other did not exce