Curr Treat Options Neurol, 2003 Jan, 5(1), 23 - 31 Botulism; Davis LE; Botulinum toxin is the most potent toxin known to humans and as little as 100 ng can be lethal . The toxin blocks peripheral cholinergic neurotransmission at the neuromuscular junction and cholinergic autonomic nervous system by introducing an endopeptadase enzyme into the presynaptic side of the synapse . The endopeptadase cleaves acetylcholine vesicle docking proteins that are required for the synapse to release acetylcholine into the synaptic cleft . Botulism occurs from consumption or inhalation of preformed botulinum toxin or growth of Clostridium botulinum bacteria in the infant gastrointestinal tract or within a wound . Growth of C . botulinum in the immature gut or wound will release botulinum toxin that reaches the circulation . All forms of botulism cause progressive weakness, bulbar signs (blurred vision, diplopia, mydriasis, dysphagia, and dysarthria), and respiratory failure with normal sensation and mentation . Treatment is aimed at 1) maintaining respiration via intubation and mechanical ventilation, 2) stopping progression of weakness by administration of botulinum antitoxin (equine trivalent botulinum antitoxin for adults and botulism immune-globulin intravenous-human for infant botulism), and 3) preventing complications from weeks of paralysis with good supportive care . The source of the botulinum toxin should be identified to prevent additional cases . Patients can recover normal muscle strength within weeks to months, but usually complain of fatigue for years. Am J Clin Pathol, 2003 Jan, 119(1), 45 - 9 Clinical usefulness of components of the Triage immunoassay, enzyme immunoassay for toxins A and B, and cytotoxin B tissue culture assay for the diagnosis of Clostridium difficile diarrhea; Massey V et al.; We studied 557 nonduplicate fresh stool specimens from adult patients clinically suspected of having Clostridium difficile-associated diarrhea . All samples were tested in parallel with an in-house cytotoxin B tissue culture assay (CTA), the C DIFFICILE TOX A/B II test (TA/B; TechLab, Blacksburg, VA), and the Triage Micro C DIFFICILE Panel (Biosite Diagnostics, San Diego, CA) . The Triage device detects toxin A (TA) and glutamate dehydrogenase (GDH) simultaneously . Of the specimens, 350 were negative and 95 were positive for all markers . Another 112 specimens yielded discrepant results . The CTA found 143 positive specimens . Results of the components of the Triage and TA/B were compared separately with those of CTA . GDH was the most sensitive but least specific marker, whereas TA and TA/B were less sensitive but highly specific . Because of these attributes and a quick turnaround time, GDH would be the best screening test for C difficile-associated diarrhea . CTA detected the highest number of cases of C difficile-associated diarrhea and would be most useful as a confirmatory test for GDH-positive and TA-negative specimens. Clin Microbiol Infect, 2002 Dec, 8(12), 814 - 22 In vitro anti-anaerobic activity of the cephalosporin derivative RWJ 54428, compared to seven other compounds; Hoellman DB et al.; Agar dilution MIC was used to test the activity of RWJ 54428, a new cephalosporin derivative, compared to imipenem, meropenem, ceftriaxone, piperacillin, piperacillin-tazobactam, clindamycin and metronidazole against 363 anaerobes isolated from clinical specimens . RWJ 54428 had low MICs against most beta-lactamase-negative Gram-negative rods, and all Gram-positive strains except Clostridium difficile . Imipenem and meropenem had the lowest MICs (MIC50s of 0.125 mg/L and MIC90s of 1.0 mg/L) . Piperacillin-tazobactam, clindamycin and metronidazole were active against most strains, and ceftriaxone was active mainly against beta-lactamase-negative organisms. J Clin Microbiol, 2003 Jan, 41(1), 509 - 11 Clostridium difficile brain empyema after prolonged intestinal carriage; Gravisse J et al.; Clostridium difficile, the most common cause of antibiotic-associated diarrhea, is occasionally isolated from extraintestinal sites and is usually found as part of a polymicrobial flora . We report a case of brain empyema that occurred after the recurrent intestinal carriage of a nontoxigenic strain of C . difficile . Brain abscess cultures contained both toxigenic and nontoxigenic isolates . Pulsed-field gel electrophoresis showed that nontoxigenic isolates from the intestine and from the brain were identical. Bioorg Med Chem, 2003 Feb 6, 11(3), 421 - 6 A quantitative structure-activity relationship study on some matrix metalloproteinase and collagenase inhibitors; Kumar D et al.; A quantitative structure-activity relationship (QSAR) study is made on some hydroxamic acid-based inhibitors of matrix metalloproteinases (MMPs) and a bacterial collagenase, namely Clostridium histolyticum collagenase (ChC), that also belongs to an MMP family, M-31, using Kier's valence molecular connectivity index (1)chi(v) of the substituents and electrotopological state (E-state) indices of some atoms . The results indicate that out of the four MMPs (MMP-1, MMP-2, MMP-8, and MMP-9) studied, MMP-2 and MMP-9 can be structurally quite similar, but widely differing from MMP-1 and MMP-8 and ChC . For MMP-2 and MMP-9, the inhibition activity of compounds is shown to depend on both (1)chi(v )and E-state indices, while for MMP-1 and MMP-8 it is shown to depend only on E-state indices and for ChC only on (1)chi(v) . However, in all the cases, an aromatic group like C(6)F(5) or 3-CF(3)-C(6)H(4) attached to SO(2) moiety in the compounds is indicated to be equally beneficial, due to probably the involvement of fluorine atom(s) in charge-charge interactions with the Zn(2+) ion of the enzymes or in the formation of the hydrogen bonds with some sites of the receptors. J Bacteriol, 2003 Jan, 185(2), 504 - 12 Characterization of a cellulase containing a family 30 carbohydrate-binding module (CBM) derived from Clostridium thermocellum CelJ: importance of the CBM to cellulose hydrolysis; Arai T et al.; Clostridium thermocellum CelJ is a modular enzyme containing a family 30 carbohydrate-binding module (CBM) and a family 9 catalytic module at its N-terminal moiety . To investigate the functions of the CBM and the catalytic module, truncated derivatives of CelJ were constructed and characterized . Isothermal titration calorimetric studies showed that the association constants (K(a)) of the CBM polypeptide (CBM30) for the binding of cellopentaose and cellohexaose were 1.2 x 10(4) and 6.4 x 10(4) M(-1), respectively, and that the binding of CBM30 to these ligands is enthalpically driven . Qualitative analyses showed that CBM30 had strong affinity for cellulose and beta-1,3-1,4-mixed glucan such as barley beta-glucan and lichenan . Analyses of the hydrolytic action of the enzyme comprising the CBM and the catalytic module showed that the enzyme is a processive endoglucanse with strong activity towards carboxymethylcellulose, barley beta-glucan and lichenan . By contrast, the catalytic module polypeptide devoid of the CBM showed negligible activity toward these substrates . These observations suggest that the CBM is extremely important not only because it mediates the binding of the enzyme to the substrates but also because it participates in the catalytic function of the enzyme or contributes to maintaining the correct tertiary structure of the family 9 catalytic module for expressing enzyme activity. J Bacteriol, 2003 Jan, 185(2), 391 - 8 CelI, a noncellulosomal family 9 enzyme from Clostridium thermocellum, is a processive endoglucanase that degrades crystalline cellulose; Gilad R et al.; The family 9 cellulase gene celI of Clostridium thermocellum, was previously cloned, expressed, and characterized (G . P . Hazlewood, K . Davidson, J . I . Laurie, N . S . Huskisson, and H . J . Gilbert, J . Gen . Microbiol . 139:307-316, 1993) . We have recloned and sequenced the entire celI gene and found that the published sequence contained a 53-bp deletion that generated a frameshift mutation, resulting in a truncated and modified C-terminal segment of the protein . The enzymatic properties of the wild-type protein were characterized and found to conform to those of other family 9 glycoside hydrolases with a so-called theme B architecture, where the catalytic module is fused to a family 3c carbohydrate-binding module (CBM3c); CelI also contains a C-terminal CBM3b . The intact recombinant CelI exhibited high levels of activity on all cellulosic substrates tested, with pH and temperature optima of 5.5 and 70 degrees C, respectively, using carboxymethylcellulose as a substrate . Native CelI was capable of solubilizing filter paper, and the distribution of reducing sugar between the soluble and insoluble fractions suggests that the enzyme acts as a processive cellulase . A truncated form of the enzyme, lacking the C terminal CBM3b, failed to bind to crystalline cellulose and displayed reduced activity toward insoluble substrates . A truncated form of the enzyme, in which both the cellulose-binding CBM3b and the fused CBM3c were removed, failed to exhibit significant levels of activity on any of the substrates examined . This study underscores the general nature of this type of enzymatic theme, whereby the fused CBM3c plays a critical accessory role for the family 9 catalytic domain and changes its character to facilitate processive cleavage of recalcitrant cellulose substrates. Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2141 - 6 Growth requirements and fermentation products of Fusobacterium prausnitzii, and a proposal to reclassify it as Faecalibacterium prausnitzii gen . nov., comb . nov; Duncan SH et al.; Two newly isolated strains of obligately anaerobic bacteria from human faeces are shown here to be related to Fusobacterium prausnitzii, which is regarded as one of the most abundant colonizers of the human colon . These strains, along with Fusobacterium prausnitzii ATCC 27768(T) and 27766, are non-motile and produce butyrate, formate and lactate, but not hydrogen as fermentation products . A new finding is that all four strains produce D-lactate, but not L-lactate . The strains have a requirement for acetate in the growth medium and this may account for the previously reported requirement for rumen fluid . The DNA G+C content of the four strains is 47-57 mol% . Together with phylogenetic analysis based on 16S rRNA sequencing, this establishes that Fusobacterium prausnitzii strains are only distantly related to Fusobacterium sensu stricto and are more closely related to members of Clostridium cluster IV (the Clostridium leptum group) . It is proposed that a new genus, Faecalibacterium gen . nov . be created; this genus should include Faecalibacterium prausnitzii gen . nov., comb . nov . ATCC 27768(T) (= NCIMB 13872(T)) (formerly Fusobacterium prausnitzii) as the type species together with ATCC 27766 and the newly isolated strains A2-165 and L2-6. Biochem Biophys Res Commun, 2003 Jan 17, 300(3), 706 - 11 The complete receptor-binding domain of Clostridium difficile toxin A is required for endocytosis; Frisch C et al.; Clostridium difficile toxin A, the chief pathogenicity factor of the antibiotic-associated pseudomembranous colitis, is an intracellular acting cytotoxin that reaches its targets, the Rho GTPases, after receptor-mediated endocytosis . The C-terminal part, constructed of repetitive peptide elements, is thought to bind to a lot of carbohydrate containing receptor molecules to induce clustering and endocytosis . To study which part of the receptor-binding domain is in charge of addressing toxin A into the target cells, we studied the functional, i.e., endocytosis-inducing, binding of toxin A . By a competition assay between various receptor-binding fragments of toxin A and the holotoxin A we found that the complete receptor-binding domain, encompassing the entire repetitive elements, but not parts of it, is necessary for binding-induced endocytosis . The receptor binding domain itself shows weaker competition with holotoxin A than the fragment consisting of receptor-binding domain plus intermediary part of the toxin . All toxin A fragments that compete with holotoxin A are capable of inducing their own endocytosis . Thus, the entire receptor-binding domain, covering the C-terminal third of the toxin A molecule, is responsible for cell uptake of toxin A and the intermediary part contributes to the correct folding and assembly of the repetitive domains. Pediatr Infect Dis J, 2002 Dec, 21(12), 1173 - 4 Recurrent crepitant cellulitis caused by Clostridium perfringens; Bryant P et al.; A previously healthy 13-year-old boy developed extensive subcutaneous emphysema of the lower limb after a penetrating injury to the knee . Clostridium perfringens was isolated from the wound . Despite surgical debridement and appropriate antibiotics, the emphysema recurred, and prolonged antibiotic treatment was required . This case highlights the distinction between gas gangrene and the lesser known entity of clostridial crepitant cellulitis. Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 73 - 5 {Study on the effect of a biostimulant on the growth and toxigenic function of Clostridium tetani strain Copenhagen-471}; Garib FIu et al.; Bakstim, a new biostimulating preparation obtained from the organs of the immune system of animals, was developed . The impact of Bakstim on the growth and toxigenic function of C . tetani production strain Copenhagen-471 was evaluated . The addition of the preparation to Gluzman commercial medium for obtaining tetanus toxoid led to an increase in the yield of bacterial biomass from 1.9 to 4-fold and an increase in the toxoid production from 2 to 2.8-fold . The optimum concentration of this biostimulant ensuring the maximum yield of tetanus toxin from the production culture was determined (1,000 mg/l) . Bakstim will supposedly be used as additive to nutrient media for the production of tetanus toxoid. Int J Food Microbiol, 2003 Jan 26, 82(1), 81 - 6 Evaluation of different methods for the detection of Clostridium perfringens phosphatases; Eisgruber H et al.; In order to detect phosphatase activity a total of 137 isolates from 12 Clostridium (C.) species were examined via fluorescence on SCA-agar with methylumbelliferyl phosphate (SCA-MUP), via APIZYM and RAPID ID 32 A as well as using a phosphatase reagent containing 1-naphthyl phosphate . Fluorescence on SCA-MUP showed the presence of acid or alkaline phosphatase in almost all isolates examined . Likewise, acid or alkaline phosphatase could be detected via APIZYM and RAPID ID 32 A in most strains and species . Opposed to this, the majority of the Clostridium bifermentans isolates showed a positive reaction exclusively on SCA-MUP . On the other hand, the phosphatase reagent for the detection of acid phosphatase lead to unambiguously positive results only when examining Clostridium perfringens isolates . Therefore, the SCA-MUP-agar, in contrast to the phosphatase reagent, was proven to be unsuitable for the identification of C . perfringens via detection of acid phosphatase . Using the phosphatase reagent the activity of this enzyme was detected in 95.1% of the C . perfringens isolates included in the study . In addition, the phosphatase reagent showed identical reactions after a 24 h incubation at 37 degrees C and when used on cultures incubated for 6 h at 44 degrees C in the case of 98.5% of the C . perfringens isolates. Ann Pharmacother, 2003 Jan, 37(1), 127 - 31 Management of botulism; Robinson RF et al.; OBJECTIVE: To provide a concise review of the presentation and treatment of botulism . DATA SOURCES: Searches of MEDLINE (1966-November 2001), tertiary references, and public and government Internet sites were conducted . STUDY SELECTION: All articles and additional references from those articles were thoroughly evaluated . DATA SYNTHESIS: Clostridium botulinum toxin blocks acetylcholine release in a dose-dependent fashion, resulting in acute symmetric diplopia, dysarthria, dysphonia, dysphagia, and possible neurologic sequelae despite the route of exposure (i.e., food-borne, wound, intestinal, inhalation) . Disease secondary to genetically engineered C . botulinum may differ from that of inadvertent exposure . Present treatment is primarily supportive care, respiratory support, rapid decontamination, and antitoxin administration (i.e., trivalent, pentavalent, heptavalent antitoxin) . Early initiation of antitoxin limits the extent of paralysis, but does not reverse it . CONCLUSIONS: Supportive care and the use of antitoxin have been effective in the treatment of botulism from food-borne, intestinal, and wound exposure . However, the effectiveness of antitoxin in the treatment of inhaled C . botulinum has not been proven. Folia Microbiol (Praha), 2002, 47(5), 559 - 64 Identification and characterization of Clostridium paraputrificum, a chitinolytic bacterium of human digestive tract; Simunek J et al.; A strictly anaerobic, mesophilic and chitinolytic bacterial strain was isolated from human feces . Based on morphological and physiological properties and 16S rRNA sequence analysis the strain was identified as Clostridium paraputrificum . The strain utilized chitin and N-acetyl-D-glucosamine, grew on glucose and hydrolyzed starch . Cultivation of the strain with colloidal chitin as the growth substrate resulted in the production of gas (hydrogen and carbon dioxide) and formation of acetate and lactate (21.6 and 18.9 mmol/L, respectively) and only small quantities of propionate and butyrate (1.7 and 2.6 mmol/L, respectively) . In the course of a 10-d cultivation with chitin, the endochitinase activity was detected after 1 d and gradually increased, reaching maximum after 3 d (251 nkat/L N-acetyl-D-glucosamine) . The beta-N-acetylglucosaminidase activity appeared just at the beginning of the cultivation, increased to day 2 and then remained nearly constant . More than 90% of chitin added was degraded within 2 d of cultivation . On the zymogram of the extracellular chitinolytic complex were visible at least 6 isoenzymes with molar mass 43.5-65.0 kDa . The temperature optimum of endochitinase and beta-N-acetylglucosaminidase activities was 50 degrees C; the optimum activity of both enzymes was found at pH 4-6. J Agric Food Chem, 2003 Jan 1, 51(1), 76 - 81 Bioactivity of Backhousia citriodora: antibacterial and antifungal activity; Wilkinson JM et al.; Backhousia citriodora products are used as bushfoods and flavorings and by the aromatherapy industry . The antimicrobial activity of 4 samples of B . citriodora oil, leaf paste, commercial tea (0.2 and 0.02 g/mL), and hydrosol (aqueous distillate) were tested against 13 bacteria and 8 fungi . Little or no activity was found to be associated with the leaf tea and hydrosol, respectively . Leaf paste displayed antimicrobial activity against 7 bacteria including Clostridium perfringens, Pseudomonas aeruginosa, and a hospital isolate of methicillin resistant Staphylococcus aureus (MRSA) . The 4 essential oils were found to be effective antibacterial and antifungal agents; however, variation was apparent between oils that did not correlate with citral content . The antimicrobial activity of B . citriodoraessential oils was found to be greater than that of citral alone and often superior to Melaleuca alternifolia essential oil . B . citriodora has significant antimicrobial activity that has potential as an antiseptic or surface disinfectant or for inclusion in foods as a natural antimicrobial agent. J Gen Appl Microbiol, 1998 Oct, 44(5), 327 - 335 Glucoamylase from Thermoanaerobacterium thermosaccharolyticum: Sequence studies and analysis of the macromolecular architecture of the enzyme; Ducki A et al.; A chromosomal DNA fragment with a length of 2,025 bp, carrying the structural gene coding for glucoamylase in Thermoanaerobacterium thermosaccharolyticum, was cloned and sequenced . It coded for 695 amino acids, representing a polypeptide with a predicted molecular mass of 77.5 kDa . The deduced amino acid sequence exhibited high homologies with the glucoamylase sequence of another bacterial glucoamylase (Clostridium sp . G0005) and with fungal glucoamylases . The catalytic domain (amino acids 271 to 695) of the T . thermosaccharolyticum enzyme shared a high degree of similarity (five conserved regions) with the catalytic domain of Aspergillus awamori glucoamylase . By comparing the secondary structure of the sequence of the catalytic domain of the T . thermosaccharolyticum enzyme with that of glucoamylase from A . awamori, and on the basis of X-ray crystallographic data available for the A . awamori enzyme, it turned out that, most probably, both enzymes have a catalytic domain organized into an "(alpha/alpha)(6)-barrel" and an overall size and shape that is very similar . These findings confirm and extend our working model for the macromolecular architecture of the T . thermosaccharolyticum glucoamylase obtained, in earlier experiments, by electron microscopy of negatively stained isolated enzyme molecules . Antibodies for an enzyme-specific peptide located near the active site were successfully applied for inhibition studies of enzyme activity and for electron microscopic epitope mapping . A study comparing the site of attachment of this kind of antibody to the T . thermosaccharolyticum glucoamylase molecule with the expected attachment site as deduced from the A . awamori enzyme structure confirmed the close similarity of both glucoamylases regarding the macromolecular architecture of that part of the enzyme carrying the catalytic center, though helices H9, H10, and H11 in peripheral parts of the A . awamori enzyme are missing in the T . thermosaccharolyticum enzyme. J Gen Appl Microbiol, 1999 Aug, 45(4), 163 - 168 Partial characterization of a 36-kDa protein from Clostridium botulinum type E that inhibits trypsin and chymotrypsin; Prabakaran S et al.; A 36-kDa trypsin inhibitor was purified from Clostridium botulinum type E culture supernatant by multiple molecular sieve and ion exchange chromatographic steps . The sequence of the amino-terminal 13 amino acid residues of this single-chain protein is Asn.Gln.Glu.Val.Phe.Asn.Met.Pro.Lys.Phe.Ser.Thr.Ala- . This novel protein that also inhibits chymotrypsin is produced by an organism that does not appear to produce any protease. J Gen Appl Microbiol, 1997 Oct, 43(5), 249 - 255 Isolation and characterization of anaerobic indole- and skatole-degrading bacteria from composting animal wastes; Kohda C et al.; Four species of indole-degrading Clostridium and 3 species of skatole-degrading Clostridium were isolated from piggery or chicken manure composting processes . Since type strains of respective isolates did not degrade these compounds, the degradability of the compounds was a novel characteristic . All isolates were mesophilic . The maximum growth allowance concentrations of these isolates were 300 to 800 mg/l in indole and 100 to 300 mg/l in skatole . All isolates showed better growth and utilization of indolic compounds in nutrient-rich medium than in minimal medium . Skatole-degrading isolates degraded some substituted indoles tested, 3-indoleacetic acid, indole and oxindole, but did not degrade 1-methylindole, 2-methylindole, isatin or anthranilic acid . On the other hand, indole-degrading isolates degraded only oxindole . The growth of Clostridium malenominatum A-3 was inhibited by a low concentration (0.005%) of indole or skatole, even when 200-fold excess glucose was present in the medium . When 0.03% indole or skatole was added to the medium, C . malenominatum A-3 showed a lag phase for about 10 and 70 h, respectively . When 0.01% of these compounds was added to the medium, the uptake of glucose was inhibited . C . malenominatum A-3 degraded these compounds under nutrient-rich and minimal conditions. Antimicrob Agents Chemother, 2003 Jan, 47(1), 337 - 41 In vitro activities of daptomycin, vancomycin, quinupristin- dalfopristin, linezolid, and five other antimicrobials against 307 gram-positive anaerobic and 31 Corynebacterium clinical isolates; Goldstein EJ et al.; The activities of daptomycin, a cyclic lipopeptide, and eight other agents were determined against 338 strains of gram-positive anaerobic bacteria and corynebacteria by the NCCLS reference agar dilution method with supplemented brucella agar for the anaerobes and Mueller-Hinton agar for the corynebacteria . The daptomycin MICs determined on Ca(2+)-supplemented (50 mg/liter) brucella agar plates were one- to fourfold lower than those determined in unsupplemented media . Daptomycin was highly active (MICs, <or=2 microg/ml) against many strains including 36 of 37 peptostreptococci, 37 of 48 isolates of the Eubacterium group, and all strains of Propionibacterium spp., Clostridium perfringens, Clostridium difficile, and other Clostridium spp . It was fourfold or greater more active than vancomycin against Clostridium innocuum and 16 of 34 strains of vancomycin-resistant lactobacilli . Three strains of C . difficile for which quinupristin-dalfopristin and linezolid MICs were >8 microg/ml were inhibited by <1 microg of daptomycin per ml . Daptomycin MICs were >or=4 microg/ml for most strains of Clostridium clostridioforme, Clostridium paraputrificum, Clostridium tertium, and Clostridium ramosum; the isolates were generally more resistant to other antimicrobials . Daptomycin was two- to fourfold less active against Actinomyces spp . than vancomycin, quinupristin-dalfopristin, or linezolid . Twenty-nine of 31 strains of Corynebacterium spp., including Corynebacterium jeikeium, Corynebacterium amycolatum, and Corynebacterium pseudodiphtheriticum, were inhibited by <or=0.25 microg of daptomycin per ml . For two strains of "Corynebacterium aquaticum," 8 microg of daptomycin per ml was required for inhibition . Daptomycin demonstrated very good activities against a broad range of gram-positive organisms including vancomycin-resistant C . innocuum and lactobacillus strains and quinupristin-dalfopristin- and linezolid-resistant C . difficile strains. J Mol Biol, 2003 Jan 17, 325(3), 471 - 83 Crystal structure and site-directed mutagenesis of enzymatic components from Clostridium perfringens iota-toxin; Tsuge H et al.; Iota-toxin from Clostridium perfringens type E is an ADP-ribosylating toxin (ADPRT) that ADP-ribosylates actin, which is lethal and dermonecrotic in mammals . It is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib) . Ia ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton . Here, we report on studies of the structure-function relationship by the crystal structures of Ia complexed with NADH and NADPH (at 1.8 A and 2.1 A resolution, respectively) and mutagenesis that map the active residues . The catalytic C-domain structure was similar to that of Bacillus cereus vegetative insecticidal protein (VIP2), which is an insect-targeted toxin, except for the EXE loop region . However, a significant structural difference could be seen in the N-domain, which interacts with Ib, suggesting an evolutionary difference between mammalian-targeted and insect-targeted ADPRT . The high resolution structure analysis revealed specific NAD conformation (a ring-like conformation of nicotinamide mononucleotide (NMN)) supported by Arg295, Arg296, Asn335, Arg352 and Glu380 . Additionally, the mutagenesis study showed that the residues Tyr251, Arg295, Glu301, Ser338, Phe349, Arg352 and Glu380, including a newly identified one, are essential for NAD(+)-glycohydrolase (NADase) activity . At least one residue, Glu378, is an essential residue for ADP-ribosyltransferase (ARTase), but not for NADase . Consequently, the structural feature and these mutagenesis findings suggest that the catalytic mechanism of Ia proceeds via an Sn1-type reaction . J Biol Chem, 2003 Mar 7, 278(10), 7956 - 63 Epub 2002 Dec 19. R-Ras glucosylation and transient RhoA activation determine the cytopathic effect produced by toxin B variants from toxin A-negative strains of Clostridium difficile; Chaves-Olarte E et al.; Clostridium difficile induces antibiotic-associated diarrhea through the production of toxin A and toxin B; the former toxin has been assumed to be responsible for the symptoms of the disease . Several toxin A-negative strains from C . difficile have recently been isolated from clinical cases and have been reported to produce toxin B variants eliciting an atypical cytopathic effect . Ultrastructural analysis indicated these toxins induce a rounding cytopathic effect and filopodia-like structures . Toxin B variants glucosylated R-Ras, and transfection with a constitutively active mutant of this GTPase protected cells against their cytopathic effect . Treatment of cells with toxin B variants induced detachment from the extracellular matrix and blockade of the epidermal growth factor-mediated phosphorylation of extracellular-regulated protein kinases, demonstrating a deleterious effect on the R-Ras-controlled avidity of integrins . Treatment with toxin B variants also induced a transient activation of RhoA probably because of inactivation of Rac1 . Altogether, these data indicate that the cytopathic effect induced by toxin B variants is because of cell rounding and detachment mediated by R-Ras glucosylation, and the induction of filopodia-like structures is mediated by RhoA activation . Implications for the pathophysiology of C . difficile-induced diarrhea are discussed. J Hosp Infect, 2003 Jan, 53(1), 1 - 5 General outbreaks of infectious intestinal disease (IID) in hospitals, England and Wales, 1992-2000; Meakins SM et al.; Between 1992 and 2000, 26.6% (1,396/5,257) of all general outbreaks of infectious intestinal disease (IID) reported to the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC) occurred in hospitals . Over 29,000 patients and staff were affected and the mortality risk was higher than for outbreaks in other settings {relative risk 2.00 (95% CI: 1.52-2.63) P<0.001} . Person-to-person spread was the predominant mode of transmission . The mortality risk was highest in foodborne disease outbreaks {relative risk 3.22 (95% CI: 1.41-7.36); P=0.003} . Most outbreaks occurred between November and April . The pathogens most frequently reported were Norwalk-like virus (NLV) (54%) and Clostridium difficile (12.6%) . These findings emphasize the public health importance of outbreaks of IID in hospitals, especially during the winter when pressures on hospitals are at their height. J Cell Physiol, 2003 Feb, 194(2), 127 - 38 Galphaq signaling is required for Rho-dependent transcriptional activation of the cyclooxygenase-2 promoter in fibroblasts; Slice LW et al.; Previously, we demonstrated that the gastrin releasing peptide (GRP) induces cyclooxygenase-2 (COX-2) expression through a Rho-dependent, protein kinase C (PKC)-independent signaling pathway in fibroblasts (Slice et al., 1999, J Biol Chem 274:27562-27566) . However, the specific role of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) that are coupled to the GRP receptor in Rho-dependent COX-2 expression has not been elucidated . In this report, we utilize embryonic fibroblasts from transgenic mice containing double gene knock-outs (DKO) for Galpha(q/11) and Galpha(12/13) to demonstrate that COX-2 promoter activation by GRP requires Galpha(q) . Furthermore, we show that GRP-dependent COX-2 gene expression, as assessed by a COX-2 reporter luciferase assay, was induced in cells lacking Galpha(12/13) but was blocked in cells that did not express Galpha(q/11) . GRP-dependent COX-2 promoter induction in Galpha(q/11) deficient cells was rescued by expression of wild type Galpha(q) but blocked by inhibition of calcium signaling in calcium-free media or in cells treated with 2-aminoethoxydiphenylborate (2-APB) . Co-stimulation of transfected Galpha(q/11) deficient cells with GRP and thapsigargin (TG) induced the COX-2 promoter . Activation of endogenous Rho by expression of Onco-lbc or expression of Rho A Q63L resulted in COX-2 promoter activation in Galpha(q/11) deficient cells . Inhibition of Rho by Clostridium botulinum C3 toxin blocked COX-2 promoter induction . Expression of Galpha(q) Q209L in the well-characterized fibroblast cell line, NIH3T3, induced the COX-2 promoter which was blocked by expression of C3 toxin . These results demonstrate that calcium signaling mediated by Galpha(q) and Rho play critical roles in GRP-dependent COX-2 expression in fibroblasts . J Biol Chem, 2003 Feb 21, 278(8), 5956 - 62 Epub 2002 Dec 18. Down-regulation of Rac-1 GTPase by Estrogen; Laufs U et al.; Rac1 GTPase is essential for the activation of the NAD(P)H oxidase complex and, thereby, regulates the release of reactive oxygen species (ROS) in the vessel wall . 17 beta-estradiol (E2) inhibits vascular ROS production . To elucidate the underlying molecular mechanisms we investigated the potential regulation of Rac1 by E2 in vascular smooth muscle cells . Treatment of vascular smooth muscle cells with angiotensin II as well as overexpression of the constitutively active mutant RacL61 increased ROS release as assessed by dichlorofluorescein fluorescence, whereas inhibition of Rac1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative RacN17 inhibited ROS production . Treatment with E2 (100 nm) completely prevented angiotensin II-induced NAD(P)H oxidase activity and ROS production . E2 time and concentration dependently decreased angiotensin II-induced and basal Rac1 mRNA and protein expression as well as Rac1 activity . Down-regulation of Rac1 expression by E2 was mediated by inhibition of gene transcription (nuclear run-on assays), but E2 had no effect on Rac1 mRNA stability . Regulation of Rac1 was mediated by estrogen receptors since co-incubation with ICI 182.780 prevented down-regulation of Rac1 . To test these observations in vivo, ovariectomized spontaneously hypertensive rats were treated with E2 or vehicle . Real-time PCR and Western blotting showed reduction of aortic Rac1 mRNA and protein by 32 and 58%, respectively . Furthermore, down-regulation of Rac1 by E2 was observed in human mononuclear cells of women with elevated E2 levels after controlled ovarian hyperstimulation . Rac1 GTPase gene-transcription and activity is regulated by 17 beta-estradiol, which may be an important molecular mechanism contributing to the cardiovascular effects of estrogens. Nippon Yakurigaku Zasshi, 2002 Nov, 120(1), 116P - 118P {Isolation and determination of an antidote for botulinum neurotoxin from black tea extract}; Sawamura S et al.; The botulinum neurotoxin produced by Clostridium botulinum exhibits the strongest neurotoxicity, and causes botulism in mammals . We have found an inactivator for clostridial neurotoxins in black tea extract (thearubigin fraction) as a natural foodstuff . In this study, we have isolated and identified the inactivators . The activity against the neuromuscular blocking action of botulinus neurotoxin type A was examined in mouse phrenic nerve diaphram preparation . The purification procedure of the inactivators was as follows . Tea was extracted with aqueous acetone, and then filtrated and lyophilized . It was also extracted with n-hexane, chloroform, ethyl acetate, n-butylalchol and water, so the activity of the antidote was recognized to be in the n-butylalchol layer (named the thearubigin fraction) . A two-step reversed phase HPLC was developed for the thearubigin fraction . Three flavonoids were found to have the major activity . The structural elucidation of the compounds by means of NMR spectrascopy revealed, kaempfenol-3-O-{glc-(6-1)-rha-(3-1)-glc};keampfetrin, kaempferol-3-O-{glc-(6-1)-rha};nicotiflorin and quercetin glycoside. Am J Physiol Cell Physiol, 2003 Apr, 284(4), C1073 - 82 Epub 2002 Dec 18. Protective role of HSP72 against Clostridium difficile toxin A-induced intestinal epithelial cell dysfunction; Liu TS et al.; We determined whether the cytoprotective heat shock protein HSP72 protects against the injurious effects of Clostridium difficile toxin A (TxA) on intestinal epithelial cells . Colonic epithelial Caco-2/bbe (C2) cells were stably transfected with HSP72 antisense (C2AS) or vector only (C2VC), resulting in low and high HSP72 expression, respectively . Measurements of epithelial barrier integrity, mitochondrial function, and apoptosis activation were assessed after TxA exposure . HSP72 and RhoA interactions were evaluated with immunoprecipitations . In C2AS cells, TxA was associated with a greater decrease in transepithelial resistance (TER), an increase in {(3)H}mannitol flux, and increased dissociation of perijunctional actin . Although HSP72 binds RhoA, it failed to prevent RhoA glucosylation . TxA caused a more rapid decrease in ATP, release of cytochrome c, and activation of caspase-9 in C2AS cells . To determine whether ATP depletion decreases TER, we treated cells with antimycin A, which caused a decline in TER . We conclude that HSP72 may protect intestinal epithelial cells from TxA-mediated damage through several mechanisms, including actin stabilization, mitochondrial protection, and inhibition of apoptosis activation, but not by prevention of RhoA glucosylation. Can J Microbiol, 2002 Oct, 48(10), 911 - 21 Lotus corniculatus condensed tannins decrease in vivo populations of proteolytic bacteria and affect nitrogen metabolism in the rumen of sheep; Min BR et al.; Condensed tannins in forage legumes improve the nutrition of sheep by reducing ruminal degradation of plant protein and increasing crude protein flow to the intestine . However, the effects of condensed tannins in forage legumes on rumen bacterial populations in vivo are poorly understood . The aim of this study was to investigate the specific effects of condensed tannins from Lotus corniculatus on four proteolytic rumen bacteria in sheep during and after transition from a ryegrass (Lolium perenne)-white clover (Trifolium repens) diet (i.e., low condensed tannins) to a Lotus corniculatus diet (i.e., higher condensed tannins) . The bacterial populations were quantified using a competitive polymerase chain reaction . Lotus corniculatus was fed with or without ruminal infusions of polyethylene glycol (PEG), which binds to and inactivates condensed tannins, enabling the effect of condensed tannins on bacterial populations to be examined . When sheep fed on ryegrass-white clover, populations of Clostridium proteoclasticum B316T, Butyrivibrio fibrisolvens C211a, Eubacterium sp . C12b, and Streptococcus bovis B315 were 1.5 x 10(8), 1.1 x 10(6), 4.6 x 10(8), and 7.1 x 10(6) mL(-1), respectively . When the diet was changed to Lotus corniculatus, the average populations (after 8-120 h) of C . proteoclasticum, B . fibrisolvens, Eubacterium sp., and S . bovis decreased (P < 0.001) to 2.4 x 10(7), 1.1 x 10(5), 1.1 x 10(8), and 2.5 x 10(5) mL(-1), respectively . When PEG was infused into the rumen of sheep fed Lotus corniculatus, the populations of C . proteoclasticum, B . fibrisolvens, Eubacterium sp., and S . bovis were higher (P < 0.01-0.001) than in sheep fed Lotus corniculatus without the PEG infusion, with average populations (after 8-120 h) of 4.9 x 10(7), 3.8 x 10(5), 1.9 x 10(8), and 1.0 x 10(6), respectively . Sheep fed the Lotus corniculatus diet had lower rumen proteinase activity, ammonia, and soluble nitrogen (P < 0.05-0.001) than sheep that were fed Lotus corniculatus plus PEG . The Lotus corniculatus diet reduced rumen nitrogen digestibility (P < 0.05) and ammonia pool size and increased the flow of undegraded feed nitrogen to the abomasum . The nitrogen intake, rumen non-ammonia nitrogen pool size, rumen microbial non-ammonia nitrogen pool size, and abomasal microbial non-ammonia nitrogen fluxes were similar both in sheep fed only Lotus corniculatus and in sheep fed Lotus corniculatus plus PEG, but nonmicrobial non-ammonia nitrogen flux to the abomasum was higher (P < 0.01) for the sheep fed only Lotus corniculatus . Although condensed tannins in Lotus corniculatus reduced the populations of some proteolytic bacteria, total ruminal microbial protein and microbial protein outflow to the abomasum were unchanged, suggesting a species-specific effect of condensed tannins on bacteria in the rumen. Lett Appl Microbiol, 2003, 36(1), 41 - 5 Variability in spore germination response by strains of proteolytic Clostridium botulinum types A, B and F; Alberto F et al.; AIMS: The objective of the study was to evaluate the variability of germination response of 10 strains of proteolytic Clostridium botulinum . METHODS AND RESULTS: An automated turbidometric method was used to follow the fall in optical density . Spores of proteolytic Cl . botulinum germinated in response to l-alanine alone, with rate and extent of germination increased by addition of l-lactate or bicarbonate ions . Other hydrophobic amino acids also triggered germination of spores of proteolytic Cl . botulinum but not AGFK and inosine, germinants for Bacillus subtilis or B . cereus . CONCLUSIONS: Unlike spores of nonproteolytic Cl . botulinum, all proteolytic Cl . botulinum germinate in hydrophobic l-amino acids without l-lactate . However, a great variability of response to germinant is evidenced between the species . SIGNIFICANCE AND IMPACT OF THE STUDY: The selection of a model strain to study germination of Cl . botulinum spores should consider the variability in sensitivity to germinants shown in this work . In particular, the sequenced strain ATCC 3502 may not be the most appropriate model for germination studies. J Physiol, 2002 Dec 15, 545(Pt 3), 879 - 86 Distinct effect of actin cytoskeleton disassembly on exo- and endocytic events in a membrane patch of rat melanotrophs; Chowdhury HH et al.; We used the cell-attached mode of patch-clamp technique to measure discrete attofarad steps in membrane capacitance (C(m)), reporting area changes in the plasma membrane due to unitary exocytic and endocytic events . To investigate the role of the actin cytoskeleton in elementary exocytic and endocytic events, neuroendocrine rat melanotrophs were treated with Clostridium spiroforme toxin (CST), which specifically depolymerises F-actin . The average amplitude of exocytic events was not significantly different in control and in CST-treated cells . However, the amplitude of endocytic events was significantly smaller in CST-treated cells as compared to controls . The frequency of exocytic events increased by 2-fold in CST-treated cells relative to controls . In control cells the average frequency of exocytic events (upsilon;(exo)) was lower than the frequency of endocytic events (upsilon;(endo)) with a ratio upsilon;(exo)/upsilon;(endo) < 1 . In the toxin treated cells, the predominant process was exocytosis with a ratio (upsilon;(exo)/upsilon;(endo) > 1) . To study the coupling between the two processes, the slopes of regression lines relating upsilon;(exo) and upsilon;(endo) in a given patch of membrane were studied . The slopes of regression lines were similar, whereas the line intercepts with the y-axis were significantly different . The increased frequency of unitary exocytic events in CST-treated cells is consistent with the view, that the actin cytoskeleton acts as a barrier for exocytosis . While the disassembly of the actin cytoskeleton diminishes the size of unitary endocytic events, suggesting an important role of the actin cytoskeleton in determining the size of endocytic vesicles, the coupling between exocytosis and endocytosis in a given patch of membrane was independent of the state of the actin cytoskeleton. Life Sci, 2003 Jan 3, 72(7), 843 - 50 Inhibition of collagenase and metalloproteinases by aloins and aloe gel; Barrantes E et al.; The effects of Aloe barbadensis gel and aloe gel constituents on the activity of microbial and human metalloproteinases have been investigated . Clostridium histolyticum collagenase (ChC) results dose-dependently inhibited by aloe gel and the activity-guided fractionation led to an active fraction enriched in phenolics and aloins . Aloins have been shown to be able to bind and to inhibit ChC reversibly and non-competitively . Aloe gel and aloins are also effective inhibitors of stimulated granulocyte matrix metalloproteinases (MMPs) . The remarkable structural resemblances between aloins and the pharmacophore structure of inhibitory tetracyclines, suggest that the inhibitory effects of aloins are via an interaction between the carbonyl group at C(9) and an adjacent hydroxyl group of anthrone (C(1) or C(8)) at the secondary binding site of enzyme, destabilizing the structure of granulocyte MMPs. J Biol Chem, 2003 Feb 14, 278(7), 4882 - 91 Epub 2002 Dec 10. Cooperation of Gq, Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid; Yuan J et al.; To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Galpha(q/11) (Galpha(q/11) KO cells) or Galpha(12/13) (Galpha(12/13) KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control . In RK and Galpha(12/13) KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Galpha(12/13) KO cells in autophosphorylation activity) achieved at 3 microm . In contrast, LPA did not induce any significant increase in PKD activity in Galpha(q/11) KO cells . However, LPA induced a significantly increased PKD activity when Galpha(q/11) KO cells were transfected with Galpha(q) . LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B . Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Galpha(12/13) KO cells . Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue . Our results indicate that G(q) is necessary but not sufficient to mediate LPA-induced PKD activation . In addition to G(q), LPA requires additional G-protein pathways to elicit a maximal response with G(i) playing a critical role in Galpha(12/13) KO cells . We conclude that LPA induces PKD activation through G(q), G(i), and G(12) and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways. Microbiol Immunol, 2002, 46(10), 647 - 55 Role of the C-domain in the biological activities of Clostridium perfringens alpha-toxin; Nagahama M et al.; Clostridium perfringens alpha-toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC) . In this study we examined the role of the C-domain (251-370 residues; CP251- 370) in biological activities of the toxin . The N-domain (1-250 residues; CP1- 250) of the alpha-toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them . A hybrid protein (BC-CP251-370) consisting of BcPLC and CP251- 370 bound to the red cells and lysed them . Incubation of CP1-250 with CP251-370 completely complemented hemolytic and PLC activities . CP251-370 also conferred hemolytic activity on BcPLC . CP251-340 (251-340 residues) significantly stimulated PLC activity of CP1-250), but did not confer hemolytic activity on CP1-250 . Kinetic analysis suggested that CP251-370 increased affinity toward the substrate of CP1-250 . The results suggested that CP251-370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1-250 . Acrylodan-labeled CP251-370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylene diamine (NBD)-labeled CP251-370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251-370 to a hydrophobic environment . These observations suggest that interaction of CP251-370 of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1-250. Mar Pollut Bull, 2002 Oct, 44(10), 1063 - 70 The effect of the new Massachusetts Bay sewage outfall on the concentrations of metals and bacterial spores in nearby bottom and suspended sediments; Bothner MH et al.; Since the new outfall for Boston's treated sewage effluent began operation on September 6, 2000, no change has been observed in concentrations of silver or Clostridium perfringens spores (an ecologically benign tracer of sewage), in bottom sediments at a site 2.5 km west of the outfall . In suspended sediment samples collected with a time-series sediment trap located 1.3 km south of the outfall, silver and C . perfringens spores increased by 38% and 103%, respectively, in post-outfall samples while chromium, copper, and zinc showed no change . All metal concentrations in sediments are <50% of warning levels established by the Massachusetts Water Resources Authority . An 11-year data set of bottom sediment characteristics collected three times per year prior to outfall startup provides perspective for the interpretation of post-outfall data . A greater than twofold increase in concentrations of sewage tracers (silver and C . perfringens) was observed in muddy sediments following the exceptional storm of December 11-16, 1992 that presumably moved contaminated inshore sediment offshore. Clin Infect Dis, 2002 Dec 15, 35(12), 1457 - 62 Epub 2002 Dec 02. Clostridium difficile-associated diarrhea: epidemiological data from Western Australia associated with a modified antibiotic policy; Thomas C et al.; The incidence of Clostridium difficile-associated diarrhea (CDAD) has increased dramatically in hospitals worldwide during the past 2 decades . In Western Australia, this increase was most obvious during the 1980s, when there was also an increase in the use of third-generation cephalosporin antibiotics . A study of the epidemiology of CDAD and the use of third-generation cephalosporins during 1993-2000 was undertaken . From 1993 through 1998, the incidence of CDAD remained relatively stable (2-3 cases per 1000 discharges annually) . Then, a significant decrease in the incidence occurred, from 2.09 cases per 1000 discharges (95% confidence interval {CI}, 1.71-2.47) in 1998 to 0.87 cases per 1000 discharges (95% CI, 0.63-1.11) in 1999 (P<.0001); this decrease persisted into 2000 . A decrease in third-generation cephalosporin use occurred during the period of the study because of changes in the prescribing policy . These findings suggest that a reduction in the use of third-generation cephalosporins can reduce the occurrence of CDAD. Pediatr Surg Int, 2002 Oct, 18(7), 586 - 90 Epub 2002 Sep 21. Probiotics up-regulate MUC-2 mucin gene expression in a Caco-2 cell-culture model; Mattar AF et al.; Enteral probiotics such as Lactobacillus casei GG (LGG) have been used in the treatment of a variety of intestinal disorders in infants and children, including diarrhea, malabsorption, and Clostridium difficile colitis . Previous studies have identified the gene locus for mucin (MUC-2) and its expression in Caco-2 cells . Others have demonstrated that mucin, located on the surface of the intestinal epithelium, inhibits bacterial translocation (BT) . We previously demonstrated that both mucin and the probiotic bacterium LGG have an inhibitory effect on BT in both an in-vitro Caco-2 cell model and a neonatal rabbit model . We hypothesized that the decline in BT by LGG is mediated by up-regulation of epithelial MUC-2 . Human enterocyte Caco-2 cells were grown to confluence and incubated at 37 degrees C with either medium (control group) or 10(4) or 10(8) LGG for 180 min . Non-adherent LGG was washed away . Caco-2 cells were then lysed, purified, and quantified for MUC-2 protein and mRNA . The addition of LGG to the enterocyte monolayer surface resulted in significantly ( P < 0.05) increased MUC-2 expression compared to the untreated monolayers . Protein densities for MUC-2 significantly ( P < 0.05) increased with LGG . Density (expressed as ratio to control group) was 8.6 +/- 1.3 in the low-dose group (10(4) LGG) and 15.6 +/- 2.3 in the high-dose group (10(8) LGG) . LGG may thus bind to specific receptor sites on the enterocyte and stimulate the up-regulation of MUC-2, resulting in increased inhibition of BT. Gynecol Oncol, 2002 Dec, 87(3), 252 - 9 Lysophosphatidic acid induces focal adhesion assembly through Rho/Rho-associated kinase pathway in human ovarian cancer cells; Sawada K et al.; OBJECTIVE: The level of lysophosphatidic acid (LPA) is elevated in patients with ovarian cancer, and LPA has been reported to have a pivotal role in cancer dissemination . In the current study, the effect of LPA on the motility of ovarian cancer cells was investigated . METHODS: We analyzed the effects of LPA on the migration activity, the focal adhesion formation, and the tyrosine phosphorylation of focal adhesion proteins in human ovarian cancer cell lines Caov-3 and OVCAR-3 . Inhibitors of the small GTPase Rho, one of its downstream effectors (Rho-associated kinase (ROCK)), myosin light chain kinase (MLCK), and myosin light chain (MLC) phosphatase were used to examine the mechanism of LPA-induced cellular effects . RESULTS: LPA enhanced the migration of ovarian cancer cells and facilitated their invasion . Rho and ROCK played essential roles in the migratory process, as evidenced by the inhibition of migration and focal adhesion formation of cancer cells by Clostridium botulinum C3 exoenzyme (C3), an inhibitor of Rho, or Y-27632, an inhibitor of ROCK . LPA also evoked the formation of focal adhesions and tyrosine phosphorylation of focal adhesion kinase and paxillin, all of which were inhibited by C3 or Y-27632 . CONCLUSION: These results suggest that LPA induced the migration of ovarian cancer cells, at least in part, through accelerated formation of focal adhesions mediated by Rho/ROCK-induced actomyosin contractility . This study may provide the basis for new therapies to control the metastasis of ovarian cancer. Appl Microbiol Biotechnol, 2002 Dec, 60(4), 420 - 7 Epub 2002 Oct 18. A novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose; Li H et al.; A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli . The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da . Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain . Nag3A was purified from recombinant E . coli and characterized . The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide {4-MU-(GlcNAc)}, but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine . The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively . SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin. J Clin Endocrinol Metab, 2002 Dec, 87(12), 5808 - 16 Small guanosine triphospatase RhoA and Rho-associated kinase as regulators of trophoblast migration; Shiokawa S et al.; The small guanosine triphosphatase Rho controls cell adhesion and motility through reorganization of the actin cyto-skeleton and regulation of actomyosin contractility . Among the putative target molecules of Rho, a Rho-associated coiled coil-forming protein kinase (ROCK) is thought to participate in Rho-mediated cell adhesion and motility . In the present study, we explored the expression and function of RhoA and ROCK in human trophoblast cells . The colocalization of RhoA, cytokeratin 8/18, and cytokeratin 7 in some cells located in the decidual stromal region indicated that extravillous trophoblast cells expressed RhoA . In double staining for RhoA and ROCK in human chorionic villi, RhoA staining was strongly positive in the cytoplasm of cytotrophoblasts, whereas ROCK stained in the cytoplasm of cytotrophoblasts and syncytiotrophoblasts . Both RhoA and ROCK were stained in cytoplasma of cultured human cytotrophoblast . Cultured human trophoblast cells contained actin stress fibers that were lost after treatment with C3, an exoenzyme produced by Clostridium botulinum . Y-27632, a selective ROCK inhibitor, suppressed RhoA-induced formation of actin stress fibers and formation of focal contact in trophoblast cells . The trophoblast reacquired actin stress fibers and focal contact after withdrawal of Y-27632 . Cultured human cytotrophoblast cells from 7-9 wk of gestation migrated into a fibronectin-coated membrane . Both C3 exoenzyme and Y-27632 inhibited cytotrophoblast migration in a dose-dependent manner . In conclusion, cyto-trophoblasts express RhoA and ROCK in their cytoplasm, and RhoA-ROCK is involved in their assembly of actin stress fibers . Suppression of RhoA-ROCK reduces trophoblast migration . These findings suggest that RhoA-ROCK signaling is a key regulator of trophoblast cell migration. Postgrad Med, 2002 Nov, 112(5), 53 - 4, 57-8, 65 passim Recurrent Clostridium difficile colitis . Tackling a tenacious nosocomial infection; Joyce AM et al.; C difficile infection recurs in about 20% of previously treated hospitalized patients . The elderly and patients with underlying colonic disease who have recently used antibiotics are at high risk . Signs and symptoms include diarrhea, abdominal pain, and leukocytosis . Diagnosis is dependent on a high degree of clinical suspicion and ELISA testing of a stool sample for toxins . Recurrence is thought to be due to the persistence of C difficile spores . Treatment can be difficult . Oral vancomycin or metronidazole for 10 to 14 days may be helpful as first-line therapy . Tapering the dose over 1 month helps destroy the spores while enabling the normal colonic flora to regrow . Probiotics, such as lactobacillus GG and S boulardii, are being developed to help restore normal colonic flora . Immunization may help prevent C difficile infection in the first place. Am J Infect Control, 2002 Dec, 30(8), 449 - 57 Combined application of simulated reuse and quantitative carrier tests to assess high-level disinfection: experiments with an accelerated hydrogen peroxide-based formulation; Sattar SA et al.; BACKGROUND: Heat-sensitive medical devices require chemical disinfection between patients, and certain formulations for this purpose can be reused for several days . Because dilution, evaporation, and breakdown or neutralization of active ingredients can occur during reuse, it is vital to ensure that the solution retains its broad-spectrum germicidal activity even at the end of the recommended reuse period . OBJECTIVE: The purpose of this study was to combine the US Environmental Protection Agency's and the Food and Drug Administration's recommended simulated reuse method with recently developed quantitative carrier tests (QCT) to assess the broad-spectrum germicidal activity of a 7% solution of accelerated hydrogen peroxide (pH 2.9) stressed for 14 days.Materials And Methods: On alternate days baths with 3 lots of the test formulation were stressed by the addition of bacteria (Salmonella choleraesuis, Staphylococcus aureus, and Pseudomonas aeruginosa ) on glass beads and spores (Bacillus subtilis and Clostridium sporogenes ) on metallic penicylinders . In addition, one set of respiratory therapy equipment was subjected to 3 daily cycles of disinfection in each bath . The pH and H(2)O(2) levels in the test samples were measured, and they were also subjected to QCTs for their sporicidal, bactericidal, virucidal, fungicidal, and mycobactericidal activities . RESULTS: After 14 days of reuse, the pH of the test solutions remained essentially unchanged . Although the level of H(2)O(2) dropped from a high of 7.66% to as low as 6.40%, all lots showed the required level of broad-spectrum germicidal activity after 14 days of stress . CONCLUSIONS: The stress test and QCT were successfully combined in demonstrating the broad-spectrum germicidal activity of a high-level disinfectant subjected to 14 days of simulated reuse. J Biol Inorg Chem, 2003 Jan, 8(1-2), 12 - 8 Epub 2002 Jul 13. Redox properties of rubredoxin variants as a function of solvent composition and temperature: investigation of monopolar and dipolar interactions; Zheng H et al.; The role of solvent composition and temperature on equilibrium electron transfer in seven rubredoxin variants { Clostridium pasteurianum ( Cp), V8D, V8R, V8A, V44A Cp, Pyrococcus furiosus ( Pf), and A44V Pf} were investigated to examine the role of both monopolar and dipolar interactions . The reduction potentials of all variants decreased as the polarity of the solvent decreased . The enthalpy and entropy associated with electron transfer were determined from temperature-controlled voltammetric studies . The entropic contribution {delta( Tdelta S degrees )} to the change in the reduction potential was larger for charged variants (V8D and V8R), while the enthalpic contribution {delta(-delta H degrees )} was larger for the other mutants . The large entropy change observed for monopolar variants is likely due to solvent reorganization that occurs between oxidation states . Entropic-enthalpic compensation phenomena, an observation that most proteins have an entropic term {delta( Tdelta S degrees )} and enthalpic term {delta(-delta H degrees )} with opposite signs, was observed . A correlation of the size of the amino acid side chain with delta E degrees ', delta(-delta H degrees ), and delta( Tdelta S degrees ) is also discussed. Vet Microbiol, 2003 Feb 2, 91(2-3), 239 - 48 PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues; Uzal FA et al.; The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants . Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively . The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C . chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii . Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR . In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C . chauvoei or C . septicum (four animals each) were also tested by the PCR using the three sets of primers . Purified DNA template of all C . chauvoei strains produced PCR amplicons of the expected size for all three primer pairs . However, when biomass from pure cultures of C . chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results . No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates . Therefore, the PCR primer sets appear to be very specific for identifying C . chauvoei in both cultures and tissues. Int J Food Microbiol, 2003 Mar 15, 81(2), 137 - 45 Isolation of nisin-producing Lactococcus lactis WNC 20 strain from nham, a traditional Thai fermented sausage; Noonpakdee W et al.; A total of 14,020 lactic acid bacteria (LAB) were isolated from nham and screened for bacteriocin production . One Lactococcus lactis strain WNC 20 produced a bacteriocin that not only inhibited closely related LAB, but also some food-borne pathogens including Listeria monocytogenes, Clostridium perfringens, Bacillus cereus and Staphylococcus aureus . Biochemical studies revealed that the bacteriocin was heat-stable even at autoclaving temperature (121 degrees C for 15 min) and was active over a wide pH range (2-10) . The bacteriocin was inactivated by alpha-chymotrypsin and proteinase K but not other proteases . The antimicrobial spectrum and some characteristics of this bacteriocin were nearly identical to that of nisin . The gene encoding this bacteriocin was amplified by polymerase chain reaction (PCR) with nisin gene-specific primer . Sequencing of this gene showed identical sequences to nisin Z as indicated by the substitution of asparagine residue instead of histidine at position 27 . The ability of the bacteriocin produced by Lc . lactis WNC 20 may be useful in improving the food safety of the fermented product. Biotechnol Appl Biochem, 2002 Dec, 36(Pt 3), 155 - 61 Development of an operational synaptobrevin-based fluorescent substrate for tetanus neurotoxin quantification; Perpetuo EA et al.; Tetanus neurotoxin (TTx), produced by Clostridium tetani, is a two-chain polypeptide with a heavy molecular chain (HC; 100 kDa) and a light molecular chain (LC; 50 kDa) linked by a disulphide bridge . The low-molecular-mass chain is classified as a zinc metalloprotease (EC 3.4.24.68) with specific hydrolysis on synaptobrevin . With the known enzymic activity for the LC of TTx, we developed a quantification method using a quenched fluorescence peptide substrate based on the synaptobrevin sequence (fragments 73-82), suitable for direct determination of the whole TTx (HC+LC) even in crude production batches, without the necessity of purification and reduction steps to isolate the LC of TTx . The rate of substrate hydrolysis was 200 nmol/min and it was totally inhibited by EDTA, anti-recombinant fragment C antibody, and the cleavage was in a single bond (Gln-Phe) with purified and crude TTx . Besides, ELISA applied to the anti-TTx serum produced at our Institute showed cross-reaction with every fraction of the crude TTx extract . Another aspect is that TTx activity depends on the storage time, reaching a maximum on day 10 . The results obtained suggest that the use of the new fluorescent substrate, Abz-synaptobrevin(73-82)-EDDnp, enables easy and quick determination of TTx . It is a good alternative to some of the existing methods such as flocculation assay, and it can replace, under some conditions, the biological assays (minimal mortal dose). J Indian Med Assoc, 2002 Oct, 100(10), 607 - 8, 610-2, 614 Botulinum toxin: a dreaded toxin for use in human being; Ghosh B et al.; Botulinum toxin is a dreaded biological toxin elaborated by Clostridium botulinum . The action of this toxin is to cause paralysis of both voluntary and involuntary muscles . The unique property of paralysing capability of muscles has been used for the benefit of human beings . Dr Allan Scot, an ophthalmologist, first used the toxin in a patient with squint in 1981 and since then the botulinum toxin is being used in various disorders characterised by muscle overactivity such as spasticity in both children and adult, dystonic conditions such as blepharospasm, cervical dystonia, spasmodic dysphonia, writer's cramp, etc, hemifacial spasm and headache . Its main action is at the terminal nerve endings of myoneural junction and it prevents release of acetylcholine from vesicles thus causing chemical denervation . Its action persists for 3 to 4 months on an average . Its side effects such as drooping, diplopia, dysphagia, depending on the sites of injection, are few and usually transient . Generalised anaphylaxis is almost unknown . Now botulinum toxin is being used in non-neurological conditions where muscles are under spasmodic state such as achalasia cardia, anal fissure, spasm of urethral sphincter, etc . Because of wider safety range and fewer complications, botulinum toxin has been an important therapeutic armamentarium in different branches of medicine and surgery. Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 696 - 703 Clostridium difficile in long-term-care facilities for the elderly; Simor AE et al.; Antimicrobial agents are among the most frequently prescribed medications in long-term-care facilities (LTCFs) . Therefore, it is not surprising that Clostridium difficile colonization and C . difficile-associated diarrhea (CDAD) occur commonly in elderly LTCF residents . C . difficile has been identified as the most common cause of non-epidemic acute diarrheal illness in nursing homes, and outbreaks of CDAD in LTCFs have also been recognized . This position paper reviews the epidemiology and clinical features of CDAD in elderly residents of LTCFs and, using available evidence, provides recommendations for the management of C . difficile in this setting. Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 660 - 4 The role of Clostridium difficile and viruses as causes of nosocomial diarrhea in children; Langley JM et al.; OBJECTIVE: We report surveillance of nosocomial diarrhea in children at our institution during the past decade and note different epidemiology of diarrhea due to viruses and Clostridium difficile . DESIGN: A prospective cohort study . SETTING: A university-affiliated pediatric hospital with 180 beds serving an urban area and providing referral care for the Maritime Provinces of Canada . PARTICIPANTS: Children younger than 18 years . METHODS: Surveillance was conducted from 1991 to 1999 using personal contact with personnel and review of microbiology and medical records . Nosocomial diarrhea was defined as loose stools occurring more than 48 hours after admission, with at least two loose stools in 12 hours and no likely non-infectious cause . RESULTS: Nosocomial diarrhea was the third most common nosocomial infection (217 of 1,466; 15%), after bloodstream and respiratory infections, with from 0.5 to 1 episode per 1,000 patient-days . Of 217 nosocomial diarrhea episodes, 122 (56%) had identified pathogens: C . difficile (39 of 122; 32%), rotavirus (38 of 122; 31%), adenovirus (36 of 122; 30%), and other viral (9 of 122; 7%) . The median age was 1.3 years (range, 11 days to 17.9 years), 0.80 year for children with viral diarrhea, 3.9 years for children with C . difficile, and 1.5 years for children with diarrhea without a causative organism identified (P< .0001) . Most children with nosocomial diarrhea were incontinent (diapered) at the time of their first episode (138 of 185; 75%), but preexisting incontinence was more common in those with viral diarrhea (93%) compared with those with no organism identified (71%) or those with C . difficile-associated diarrhea (CDAD) (49%) (P <.0001) . CONCLUSIONS: C . difficile is the single most common cause of nosocomial diarrhea in our tertiary-care center, although all viral pathogens account for 69% of cases . Diapered status appears to be a risk factor for CDAD in children, and CDAD occurs more often in older children than viral nosocomial diarrhea . Further characterization of risk factors for, and morbidity associated with, nosocomial CDAD in children is warranted. Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 653 - 9 Underlying disease severity as a major risk factor for nosocomial Clostridium difficile diarrhea; Kyne L et al.; OBJECTIVE: To determine the diagnostic accuracy of an index of underlying disease severity (Horn's index) in identifying patients with a high probability of having nosocomial Clostridium difficile diarrhea as a complication of antimicrobial therapy . DESIGN: A prospective cohort study of 252 adult patients admitted to the hospital and receiving antibiotics . Risk facctors for C . difficile diarrhea were first determined retrospectively in a different cohort of 300 hospitalized patients (primary cohort) and then prospectively in this cohort of 252 hospitalized patients receiving antibiotics (secondary cohort) . At the time of hospital admission, disease was rated by clinicians as mild (1), moderate (2), severe (3), or extremely severe (4) using a modified Horn's index . Multivariable logistic regression analysis was used to determine the odds ratio (OR) for C . difficile diarrhea associated with increasing levels of disease severity . SETTING: An urban teaching hospital affiliated with a medical school in Boston, Massachusetts . RESULTS: The incidence of nosocomial C . difficile diarrhea was 8.7% in the primary cohort and 11% in the secondary cohort In the prospective cohort study (secondary cohort), the OR for C . difficile diarrhea associated with extremely severe disease was 17.6 (95% confidence interval, 5.8 to 53.5) . The sensitivity, specificity, and positive and negative predictive values of a Horn's index score of 3 or more (severe to extremely severe disease) as a predictor of nosocomial C . difficile diarrhea were 79%, 73%, 27%, and 96%, respectively . CONCLUSIONS: These findings provide a means of early stratification of hospitalized patients receiving antibiotics according to their risk for nosocomial C . difficile diarrhea . Patients with severe to extremely severe disease at the time of admission may benefit from careful monitoring of antibiotic prescribing and early attention to infection control issues . In the future, these "high-risk" patients may benefit from prophylaxis studies of novel agents being developed to prevent C . difficile diarrhea. Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 648 - 52 Predominance of a single restriction endonuclease analysis group with intrahospital subgroup diversity among Clostridium difficile isolates at two Chicago hospitals; Mekonen ET et al.; OBJECTIVE: To determine the epidemiology and relatedness of Clostridium difficile isolates in two geographically separated hospitals in a large metropolitan area, each with unique patients and personneL DESIGN: Observational descriptive molecular epidemiology of clinical C . difficile isolates . SETTING: Two tertiary-care hospitals in Chicago . METHODS: Consecutive C . difficile isolates from the clinical laboratory of a Veterans Affairs hospital during a 13-month period were typed by restriction endonuclease analysis (REA) . During an overlapping 3-month period, stool specimens that tested positive for C . difficile toxin from patients at a nearby county hospital were cultured and the recovered isolates typed by the same method . RESULTS: Nineteen (68%) of 28 nosocomial isolates at the smaller, Veterans Affairs hospital belonged to REA group K . Within this group of closely related strains, 9 distinct REA types were recognized . Twenty-one (72%) of 29 nosocomial isolates at the larger, county hospital also belonged to group K . However, the predominant REA types within group K differed markedly at each institution . CONCLUSIONS: These findings demonstrate a high degree of similarity among nosocomial C . difficile strains from different hospitals in the same city and suggest the possibility of an extended outbreak of a prototype group K strain with subsequent genetic drift at the two different institutions. Infect Control Hosp Epidemiol, 2002 Nov, 23(11), 641 - 7 Difference in the incidence of Clostridium difficile among patients infected with human immunodeficiency virus admitted to a public hospital and a private hospital; Pulvirenti JJ et al.; OBJECTIVE: To compare the occurrence of Clostridium difficile among inpatients infected with human immunodeficiency virus (HIV) in two different hospitals . DESIGN: Prospective, observational study . SETTING: Specialized HIV inpatient units . PATlENTS: HIV-infected inpatients at Cook County Hospital (CCH) and Rush Presbyterian St . Luke's Medical Center (RPSLMC) . INTERVENTIONS: A clinical and epidemiologic assessment of patient risk factors for C . difficile was performed . C . difficile isolates found on stool, rectal, and environmental cultures were typed by pulsed-field gel electrophoresis . RESULTS: Twenty-seven percent of patients admitted to CCH versus 4% of patients admitted to RPSLMC had positive cultures for C . difficile (P = .001) . At CCH, 14.7% of environmental cultures were positive versus 2.9% at RPSLMC (P = .002) . Risk factors for C . difficile acquisition included hospitalization at CCH, more severe HIV, use of acyclovir and H2-blockers, and longer hospital stay . Patients admitted to CCH were taking more antibiotics, had longer hospital stays, and more frequently had a history of C . difficile infection . During the study, two strains (CD1A and CD4) extensively contaminated the CCH environment . However, only CD1A caused an outbreak . CONCLUSIONS: The C . difficile acquisition rate at CCH was sevenfold higher than that at RPSLMC, and CCH had a more contaminated environment . Differences in patient acquisition rates likely reflect a greater prevalence of traditional C . difficile risk factors and a concurrent outbreak at CCH . Although two strains heavily contaminated the environment at CCH, only one caused an outbreak, suggesting that factors other than the environment are important in initiating C . difficile outbreaks. Appl Environ Microbiol, 2002 Dec, 68(12), 6425 - 8 Quantitative determination of bile salt hydrolase activity in bacteria isolated from the small intestine of chickens; Knarreborg A et al.; A quantitative assay based on high-performance liquid chromatography analysis of bile salts and bacterial protein determination was established for investigating bile salt hydrolase (BSH) activity in bacteria isolated from the small intestine of chickens . Bacteria were isolated using various media and were subsequently grouped according to cell morphology, fermentation profile, and 16S ribosomal DNA sequence . Representative isolates from each bacterial group were assayed for BSH activity . The isolates differed in BSH activity with respect to the state of growth and preculturing with and without taurochenodeoxycholate . The highest levels of BSH activity were found with Enterococcus faecium and Clostridium perfringens. Appl Environ Microbiol, 2002 Dec, 68(12), 6399 - 402 Xylanase and acetyl xylan esterase activities of XynA, a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation; Kosugi A et al.; The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-beta-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain . The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates . rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan . Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction . These results indicate that XynA is a key enzymatic subunit for xylan degradation in C . cellulovorans. Appl Environ Microbiol, 2002 Dec, 68(12), 6256 - 62 Growth of iron(III)-reducing bacteria on clay minerals as the sole electron acceptor and comparison of growth yields on a variety of oxidized iron forms; Kostka JE et al.; Smectite clay minerals are abundant in soils and sediments worldwide and are typically rich in Fe . While recent investigations have shown that the structural Fe(III) bound in clay minerals is reduced by microorganisms, previous studies have not tested growth with clay minerals as the sole electron acceptor . Here we have demonstrated that a pure culture of Shewanella oneidensis strain MR-1 as well as enrichment cultures of Fe(III)-reducing bacteria from rice paddy soil and subsurface sediments are capable of conserving energy for growth with the structural Fe(III) bound in smectite clay as the sole electron acceptor . Pure cultures of S . oneidensis were used for more detailed growth rate and yield experiments on various solid- and soluble-phase electron acceptors {smectite, Fe(III) oxyhydroxide FeOOH, Fe(III) citrate, and oxygen} in the same minimal medium . Growth was assessed as direct cell counts or as an increase in cell carbon (measured as particulate organic carbon) . Cell counts showed that similar growth of S . oneidensis (10(8) cells ml(-1)) occurred with smectitic Fe(III) and on other Fe forms {amorphous Fe(III) oxyhydroxide, and Fe citrate} or oxygen as the electron acceptor . In contrast, cell yields of S . oneidensis measured as the increase in cell carbon were similar on all Fe forms tested while yields on oxygen were five times higher, in agreement with thermodynamic predictions . Over a range of particle loadings (0.5 to 4 g liter(-1)), the increase in cell number was highly correlated to the amount of structural Fe in smectite reduced . From phylogenetic analysis of the complete 16S rRNA gene sequences, a predominance of clones retrieved from the clay mineral-reducing enrichment cultures were most closely related to the low-G+C gram-positive members of the Bacteria (Clostridium and Desulfitobacterium) and the delta-Proteobacteria (members of the Geobacteraceae) . Results indicate that growth with smectitic Fe(III) is similar in magnitude to that with Fe(III) oxide minerals and is dependent upon the mineral surface area available . Iron(III) bound in clay minerals should be considered an important electron acceptor supporting the growth of bacteria in soils or sedimentary environments. Appl Environ Microbiol, 2002 Dec, 68(12), 5918 - 24 Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages; Knarreborg A et al.; The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed(-1) and salinomycin at 40 mg kg of feed(-1)) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture . The bacterial origin of fragments in DGGE profiles was identified by sequencing . Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation . An increased incidence of streptococci, enterobacteria, and Clostridium perfringens with age of the chickens was demonstrated . Lactobacilli and C . perfringens were the bacterial groups most strongly affected by the dietary treatments . Moreover, different strains (clonal variants of the alpha-toxin gene) of C . perfringens type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics. Appl Environ Microbiol, 2002 Dec, 68(12), 5891 - 903 Macedocin, a food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198; Georgalaki MD et al.; Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin . Macedocin has a molecular mass of 2,794.76 +/- 0.42 Da, as determined by electrospray mass spectrometry . Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen Streptococcus pyogenes . Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including Clostridium tyrobutyricum . It displays a bactericidal effect towards the most sensitive indicator strain, Lactobacillus sakei subsp . sakei LMG 13558(T), while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production . Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121 degrees C with 1 atm of overpressure . Inhibition of macedocin by proteolytic enzymes is variable. Appl Environ Microbiol, 2002 Dec, 68(12), 5870 - 6 Detection by PCR-enzyme-linked immunosorbent assay of Clostridium botulinum in fish and environmental samples from a coastal area in northern France; Fach P et al.; The prevalence of Clostridium botulinum types A, B, E, and F was determined in 214 fresh fish and environmental samples collected in Northern France . A newly developed PCR-enzyme-linked immunosorbent assay (ELISA) used in this survey detected more than 80% of samples inoculated with fewer than 10 C . botulinum spores per 25 g and 100% of samples inoculated with more than 30 C . botulinum spores per 25 g . The percent agreement between PCR-ELISA and mouse bioassay was 88.9%, and PCR-ELISA detected more positive samples than the mouse bioassay did . The prevalence of C . botulinum in seawater fish and sediment was 16.6 and 4%, respectively, corresponding to 3.5 to 7 and 1 to 2 C . botulinum most-probable-number counts, respectively, and is in the low range of C . botulinum contamination reported elsewhere . The toxin type identification of the 31 naturally contaminated samples was 71% type B, 22.5% type A, and 9.6% type E . Type F was not detected . The high prevalence of C . botulinum type B in fish samples is relatively unusual compared with the high prevalence of C . botulinum type E reported in many worldwide and northern European surveys . However, fish processing and fish preparation in France have not been identified as a significant hazard for human type B botulism. Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 625 - 30 Bacterial spores in silage and raw milk; te Giffel MC et al.; Spore-forming bacteria can survive food-processing treatments . In the dairy industry, Bacillus and Clostridium species determine the shelf-life of a variety of heat-treated milk products, mainly if the level of post-process contamination is low . In order to minimize problems caused by bacterial spores in foods and food production processes a chain management approach, from raw materials, ingredients and environmental sources to final product storage conditions, is most effective . Silage is considered to be a significant source of contamination of raw milk with spores . PCR-RAPD fingerprinting and heat resistance studies of populations of aerobic spore-formers isolated from grass and maize silage and from raw milk confirmed this assumption . Prevention of outgrowth of aerobic spores in silage will contribute to reduction of the total spore load of raw milk . Therefore, it is important that the silage fermentation process is controlled . Application of cultures of lactic acid bacteria or chemical additives can aid silage fermentation and improve aerobic stability. J Med Microbiol, 2002 Nov, 51(11), 1001 - 8 An investigation into the microflora of heroin; McLauchlin J et al.; In 2000, an unusual increase of morbidity and mortality among illegal injecting drug users in the UK and Ireland was reported and Clostridium novyi was identified as the likely source of the serious infection, although infections due to C . botulinum and Bacillus cereus were also reported . Because heroin was a possibile source of infection, this study investigated the microflora of heroin samples seized in England during 2000 and 2002 . Two methods were developed for the examination of the microflora of heroin . The first consisted of suspension of the drug in maximum recovery diluent (MRD) which was inoculated directly into Clostridium Botulinum Isolation Cooked Meat Broth (CBI) . The second method rendered the heroin soluble in citric acid, concentrated particulate material (and bacterial cells) by filtration and removed heroin residues by washing with citric acid and phosphate-buffered saline before placing the filter in CBI broth . Duplicate CBI broths from both methods were incubated without heating and after heating at 60 degrees C for 30 min . Subcultures were made after incubation for 7 and 14 days on to eight different solid media . The methods were evaluated with heroin samples spiked with either C . botulinum or C . novyi spore suspensions; recovery of 10 spores in the original sample was demonstrated . Fifty-eight heroin samples were tested by citric acid solubilisation and 34 by the MRD suspension technique . Fifteen different gram-positive species of four genera were recognised . No fungi were isolated . Aerobic endospore-forming bacteria (Bacillus spp . and Paenibacillus macerans) were the predominant microflora isolated and at least one species was isolated from each sample . B . cereus was the most common species and was isolated from 95% of all samples, with B . licheniformis isolated from 40% . Between one and five samples yielded cultures of B . coagulans, B . laterosporus, B . pumilus, B . subtilis and P . macerans . Staphylococcus spp . were isolated from 23 (40%) samples; S . warneri and S . epidermidis were the most common and were cultured from 13 (22%) and 6 (10%) samples respectively . One or two samples yielded cultures of S . aureus, S . capitis and S . haemolyticus . The remainder of the flora detected comprised two samples contaminated with C . perfringens and two samples with either C . sordellii or C . tertium . Multiple bacterial species were isolated from 43 (74%) samples, a single species from the remaining 15 . In 13 samples B . cereus alone was isolated, in one B . subtilis alone and in one sample B . pumilus alone . C . botulinum and C . novyi were not isolated from any of the heroin samples . Recommendations for the optimal examination of the microflora of heroin are given. J Med Microbiol, 2002 Nov, 51(11), 990 - 1000 Amplified fragment length polymorphism (AFLP) analysis of Clostridium novyi, C . perfringens and Bacillus cereus isolated from injecting drug users during 2000; McLauchlin J et al.; As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C . perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis . Among the 43 C . novyi isolates, 23 different AFLP profiles were detected . The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user . Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988) . The 40 C . perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin . Sixteen AFLP types of C . perfringens were distinguished and there was little evidence for commonality among the isolates . The AFLP types of C . perfringens from heroin differed and were unique . Six isolates of B . cereus were from four drug users and two samples of heroin . Four different AFLP patterns were distinguished . Three AFLP types were isolated from four drug users . B . cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user . The AFLP type of the isolate from a second and unrelated heroin sample was unique . The AFLP results showed no or very limited evidence for commonality between the different isolates of B . cereus and C . perfringens . In marked contrast, the C . novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C . novyi type, or both, which either had an adaptive advantage in spore germination, survival or growth following the drug preparation and the injection procedure, or produced a more severe clinical presentation. J Med Microbiol, 2002 Nov, 51(11), 985 - 9 Isolation and identification of Clostridium spp . from infections associated with the injection of drugs: experiences of a microbiological investigation team; Brazier JS et al.; Pathogenic species of the genus Clostridium may contaminate the materials used in the injection of drugs and under the right conditions may cause serious or life-threatening disease . C . novyi type A was implicated in an outbreak of severe infection with high mortality in injecting drug users who injected heroin extravascularly . The isolation of such highly oxygen-sensitive clostridia from clinical material may require adherence to enhanced methods and, once isolated, commercially available anaerobe identification kits alone may not give an accurate identification . Additional phenotypic tests that are useful in recognising the main pathogenic species are described . Differentiation of C . novyi type A from C . botulinum type C in reference laboratories was based on 16S rDNA sequence data and specific neutralisation of cytopathic effects in tissue culture. J Med Microbiol, 2002 Nov, 51(11), 978 - 84 An outbreak of serious illness and death among injecting drug users in England during 2000; Jones JA et al.; An outbreak of serious illness and death occurred in injecting drug users during 2000 in Scotland, Ireland and England . National and international collaboration was necessary for the investigation and management of this outbreak . In England and Wales active case-finding was initiated, coupled with standardised data collection and microbiological investigation of cases . Twenty-six definite or probable cases were identified in England between 1 April and 31 Aug . 2000; 17 of these occurred in the North . The overall case fatality was 50% (13/26) . The principal apparent risk factor was a history of intramuscular or subcutaneous injection of heroin and the limited duration of the outbreak suggested that the problem might have been related to a particular supply of heroin . Clostridium novyi was isolated from two English cases . Taken in conjunction with contemporaneous microbiological and epidemiological results from Scottish and Irish cases, the probable aetiology for this outbreak was infection with C . novyi associated with both a particular supply of heroin and the method of preparation and injection used . A 'toolkit' was distributed in Sept . 2000 to all Consultants for Communicable Disease Control in England and Wales to assist them with the ongoing surveillance, investigation and management of this condition . Lessons learned have been used to produce guidance for the investigation and management of outbreaks of unexplained serious illness of possible infective aetiology. J Med Microbiol, 2002 Nov, 51(11), 971 - 7 Lethal outbreak of infection with Clostridium novyi type A and other spore-forming organisms in Scottish injecting drug users; McGuigan CC et al.; This report describes the investigation and management of an unprecedented outbreak of severe illness among injecting drug users (IDUs) in Scotland during April to August 2000 . IDUs with severe soft tissue inflammation were prospectively sought among acute hospitals and a mortuary in Scotland . Cases were categorised as definite or probable: probable cases had severe injection site inflammation or multi-system failure; definite cases had both . Information about clinical course, mortality, post-mortem findings and laboratory data was gathered by standardised case-note review and interview . Sixty cases were identified--23 definite and 37 probable . Most had familial or social links with each other and 50 were from Glasgow . Median age was 30 years; 31 were female . The majority, especially definite cases, injected heroin/citric acid extravascularly . Of definite cases, 20 died (87% case-fatality rate; 13 after intensive care), 15 had necrotising fasciitis, 22 had injection site oedema and 13 had pleural effusion . Median white cell count was 60 x 10(9)/L . Of 37 probable cases, three died (8% case-fatality rate) . Overall, the most frequently isolated pathogen was Clostridium novyi type A (13 cases: 8 in definite cases) . The findings are consistent with an infection resulting from injection into soft tissue of acidified heroin contaminated with spore-forming bacteria . Toxin production led to a severe local reaction and, in many, multi-system failure. J Infect Dis, 2002 Dec 15, 186(12), 1781 - 9 Epub 2002 Nov 22. Colonization for the prevention of Clostridium difficile disease in hamsters; Sambol SP et al.; Studies suggest that asymptomatic colonization with Clostridium difficile (CD) decreases the risk of CD-associated disease (CDAD) in humans . A hamster model was used to test the efficacy of colonization with 3 nontoxigenic CD strains for preventing CDAD after exposure to toxigenic CD . Groups of 10 hamsters were given 10(6) nontoxigenic CD spores 2 days after receiving a single dose of clindamycin . Five days later, the hamsters were given 100 spores of 1 of 3 toxigenic CD strains previously shown to cause mortality within 48 h . Each nontoxigenic strain prevented disease in 87%-97% of hamsters that were challenged with toxigenic strains . Failure to prevent CDAD was associated with failure of colonization with nontoxigenic CD . Colonization with nontoxigenic CD strains is highly effective in preventing CDAD in hamsters challenged with toxigenic CD strains, which suggests that use of a probiotic strategy for CDAD prevention in humans receiving antibiotics might be beneficial. J Biol Chem, 2003 Feb 7, 278(6), 4112 - 20 Epub 2002 Nov 22. Recruitment of murine neutrophils in vivo through endogenous sialidase activity; Cross AS et al.; Upon activation with various noncytokine stimuli, polymorphonuclear leukocytes (PMNs) mobilize intracellular sialidase to the plasma membrane, where the sialidase releases sialic acid from the cell surface . This desialylation enhances PMN adherence, spreading, deformability, and motility, functions critical to diapedesis . We now have examined the role of sialidase activity in PMN adhesion to and migration across the endothelium in vivo . A polyclonal antibody prepared against Clostridium perfringens neuraminidase 1) detected surface expression of sialidase on human PMNs stimulated with IL-8 in vitro and on murine PMNs stimulated in vivo, but not on that of unstimulated cells, 2) recognized proteins in human PMN lysates and granule preparations that were not detected by preimmune antibody, 3) inhibited bacterial neuraminidase and human PMN sialidase activities in vitro, and 4) inhibited both pulmonary leukostasis in mice systemically infused with cobra venom factor and intrapulmonary transendothelial migration of PMNs into the bronchoalveolar compartment of mice intranasally challenged with interleukin-8 . We conclude that the chemokine interleukin-8, like other PMN agonists, induces the translocation of sialidase to the PMN surface and that surface expression of this sialidase is a prerequisite to PMN recruitment in vivo . The ability of antibodies raised against a prokaryotic neuraminidase to recognize eukaryotic sialidase extends the concept of the neuraminidase superfamily to mammalian enzymes . Inhibition of mobilized endogenous sialidase may provide a novel strategy for limiting the inflammatory response. J Bacteriol, 2002 Dec, 184(24), 6859 - 65 Characterization of two noncellulosomal subunits, ArfA and BgaA, from Clostridium cellulovorans that cooperate with the cellulosome in plant cell wall degradation; Kosugi A et al.; Plant cell wall degradation by Clostridium cellulovorans requires the cooperative activity of its cellulases and hemicellulases . To characterize the alpha-L-arabinosidases that are involved in hemicellulose degradation, we screened the C . cellulovorans genomic library for clones with alpha-L-arabinofuranosidase or alpha-L-arabinopyranosidase activity, and two clones utilizing different substrates were isolated . The genes from the two clones, arfA and bgaA, encoded proteins of 493 and 659 amino acids with molecular weights of 55,731 and 76,414, respectively, and were located on neighboring loci . The amino acid sequences for ArfA and BgaA were related to alpha-L-arabinofuranosidase and beta-galactosidase, respectively, which are classified as family 51 and family 42 glycosyl hydrolases, respectively . Recombinant ArfA (rArfA) had high activity for p-nitrophenyl alpha-L-arabinofuranoside, arabinoxylan, and arabinan but not for p-nitrophenyl alpha-L-arabinopyranoside . On the other hand, recombinant BgaA (rBgaA) hydrolyzed not only p-nitrophenyl alpha-L-arabinopyranoside but also p-nitrophenyl beta-D-galactopyranoside . However, when the affinities of rBgaA for p-nitrophenyl alpha-L-arabinopyranoside and p-nitrophenyl beta-D-galactopyranoside were compared, the K(m) values were 1.51 and 6.06 mM, respectively, suggesting that BgaA possessed higher affinity for alpha-L-arabinopyranose residues than for beta-D-galactopyranoside residues and possessed a novel enzymatic property for a family 42 beta-galactosidase . Activity staining analyses revealed that ArfA and BgaA were located exclusively in the noncellulosomal fraction . When rArfA and rBgaA were incubated with beta-1,4-xylanase A (XynA), a cellulosomal enzyme from C . cellulovorans, on plant cell wall polymers, the plant cell wall-degrading activity was synergistically increased compared with that observed with XynA alone . These results indicate that, to obtain effective plant cell wall degradation, there is synergy between noncellulosomal and cellulosomal subunits. FEMS Microbiol Lett, 2002 Nov 19, 217(1), 15 - 22 Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum; Sabathe F et al.; A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824 . Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp . Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes . As C . acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution . Biochemical evidence for the expression of a cellulosomal protein complex was investigated . The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C . cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present . In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C . acetobutylicum. Inorg Chem, 2002 Dec 2, 41(24), 6358 - 71 Mössbauer study of reduced rubredoxin as purified and in whole cells . Structural correlation analysis of spin Hamiltonian parameters; Vrajmasu VV et al.; The {Fe(II)(Cys)(4)}(2-) site of rubredoxin from Clostridium pasteurianum (Rd(red)) has been studied by Mossbauer spectroscopy in both purified protein and whole cells of Escherichia coli overproducing it . Excellent fits were obtained to an S = 2 spin Hamiltonian for D = 5.7(3) cm(-1), E/D = 0.25(2), delta = 0.70(3) mm/s, DeltaE(Q) = -3.25(2) mm/s, eta = 0.75(5), A(x) = -20.1(7) MHz, A(y) = -11.3(2) MHz, and A(z) = -33.4(14) MHz . These parameters were analyzed with crystal-field theory for the (5)D manifold of iron(II), revealing a d(z(2)) orbital ground state that is admixed by approximately 0.21 d(x(2) - y(2)) . The spin-Hamiltonian parameters are consistent within the (5)D theory, apart from the zero-field splitting parameter, D . This problem was solved by extending the crystal-field treatment with spin-orbit coupling to spin-triplet d-d excited states of the iron . Theoretical estimates are given for the spin-triplet (D(T)) and spin-quintet contributions (D(Q)) to D based on excitation energies derived from time-dependent density functional theory, TD-DFT . The computational results were interpreted in terms of crystal-field theory, yielding the Racah parameters B = 682 cm(-1) and C = 2583 cm(-1) . The theoretical analysis gives the relative magnitudes D(Q):D(T):D(ss) = 51%: 42%:7% (D(ss) originates from spin-spin interaction) . The DFT analysis corroborates the pivotal role of the torsion angles (omega(i)) of the C-S(i) bonds in shaping the electronic structure of the iron(II) site . Rd(red) in overexpressing whole cells accounts for 60% of the Mossbauer absorption . The Rd(red) spectra from whole cells are virtually identical to those of the purified protein . By using the theoretical omega dependence of the spin Hamiltonian parameters, the torsions for Rd(red) in whole cells and purified protein samples are estimated to be the same within 2 degrees . These findings establish Mossbauer spectroscopy as a structural tool for investigating iron sites in whole cells. Naunyn Schmiedebergs Arch Pharmacol, 2002 Dec, 366(6), 501 - 12 Epub 2002 Sep 24. The uptake machinery of clostridial actin ADP-ribosylating toxins--a cell delivery system for fusion proteins and polypeptide drugs; Barth H et al.; Several bacterial protein toxins, including Clostridium botulinum C2 toxin, Clostridum perfringens iota toxin, Clostridium difficile ADP-ribosyltransferase, and the Bacillus-produced vegetative insecticidal proteins, target the cytoskeleton by ADP-ribosylation of actin . All these toxins are binary in structure and consist of an enzyme component, possessing ADP-ribosyltransferase activity and a separated binding and translocation component, which is involved in the delivery of the enzyme component into the cell . The toxins are not only important virulence factors but also cell biological tools to study the function of the actin cytoskeleton . Moreover, the binary toxins turned out to be effective transporter systems for the delivery of specific fusion toxins (e.g., Rho-ADP-ribosylating C3 exoenzyme) into cells . The present review describes the biological functions of the toxins, focuses on recent studies on the uptake and delivery mechanism and discusses the usage as a drug delivery system. Ann Med Interne (Paris), 2002 Sep, 153(5), 291 - 9 {Clostridium difficile infection in a Department of Internal Medicine . A consecutive series of 45 patients}; Bligny D et al.; OBJECTIVES AND METHODS: A retrospective study of 45 patients with Clostridium difficile infection over a 4-year period in a department of Internal Medicine . RESULTS: Mean age was 79 years; sex-ratio (F/M)=1.5; 38% of the patients had neurological or severe psychiatric disorders; 20% had a neoplastic disease . Ninety-three percent of cases had received one or more antibiotics before onset of diarrhea, prescribed mainly for a pulmonary infection . Amoxicillin clavulanic acid and cephalosporins were the most frequently used treatments, respectively in 48% and 40% of cases . For 25 patients (56%) Clostridium difficile-associated diarrhea was considered as a nosocomial infection, and as community-acquired diarrhea in 20 cases (44%) . Treatment included isolation of the patient as soon as bacteriological diagnosis was known and specific therapy was instituted by metronidazole or vancomycin for a mean of 18 days . The addition of Saccharomyces boulardii was used in of cases . The clinical course was rapidly favorable for 80% of patients . Five patients died with complications of severe colitis in 2 cases . Mean hospital stay was 49 days (annual mean of the department=10 days) . CONCLUSION: Clostridium difficile diarrhea concerns above all elderly patients with one or more underlying pathologies . Amoxicillin clavulanic acid and third-generation cephalosporins are the most frequently prescribed antibiotics in these cases and have the highest correlation with this infectious complication . This medical problem requires greater knowledge as it causes significant morbidity and increases the risk of prolonged hospital stays. J Am Chem Soc, 2002 Nov 27, 124(47), 14039 - 48 Rotation of the exo-methylene group of (R)-3-methylitaconate catalyzed by coenzyme B(12)-dependent 2-methyleneglutarate mutase from Eubacterium barkeri; Pierik AJ et al.; 2-Methyleneglutarate mutase from the anaerobe Eubacterium (Clostridium) barkeri is an adenosylcobalamin (coenzyme B(12))-dependent enzyme that catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate . Two possibilities for the mechanism of the carbon skeleton rearrangement of the substrate-derived radical to the product-related radical are considered . In both mechanisms an acrylate group migrates from C-3 of 2-methyleneglutarate to C-4 . In the "addition-elimination" mechanism this 1,2-shift occurs via an intermediate, a 1-methylenecyclopropane-1,2-dicarboxylate radical, in which the migrating acrylate is simultaneously attached to both C-3 and C-4 . In the "fragmentation-recombination" mechanism the migrating group, a 2-acrylyl radical, becomes detached from C-3 before it starts bonding to C-4 . In an attempt to distinguish between these two possibilities we have investigated the action of 2-methyleneglutarate mutase on the stereospecifically deuterated substrates (Z)-3-methyl{2'-(2)H(1)}itaconate and (Z)-3-{2'-(2)H(1),methyl-(2)H(3)}methylitaconate . The enzyme catalyzes the equilibration of both compounds with their corresponding E-isomers and with a 1:1 mixture of the corresponding (E)- and (Z)-2-methylene{2'-(2)H(1)}glutarates, as shown by monitoring of the reactions with (1)H and (2)H NMR . In the initial phase of the enzyme-catalyzed equilibration a significant excess (8-11%) of (E)-3-methyl{2'-(2)H(1)}itaconate over its equilibrium value was observed ("E-overshoot") . The E-overshoot was only 3-4% with (Z)-3-{2'-(2)H(1),methyl-(2)H(3)}methylitaconate because the presence of the deuterated methyl group raises the energy barrier from 3-methylitaconate to the corresponding radical . The overshoot is explained by postulating that the migrating acrylate group has to overcome an additional energy barrier from the state leading back to the substrate-derived radical to the state leading forward to the product-related radical . It is concluded that the fragmentation-recombination mechanism can provide an explanation for the results in terms of an additional energy barrier, despite the higher calculated activation energy for this pathway. J La State Med Soc, 2002 Sep-Oct, 154(5), 251 - 5 Clostridial enteritis necroticans versus secondary clostridial infection superimposed upon ischemic bowel disease; Zhao W et al.; Clostridial enteritis necroticans, or pig-bel, as seen in Papua New Guinea, is a necrotizing, segmental gangrene of the small intestine occurring in members of a malnourished population, who become ill after consuming large quantities of pork contaminated with Type C . Clostridium perfringens . We report a case of possible Clostridial enteritis necroticans with concomitant ischemic intestinal disease secondary to superior mesenteric arterial thrombosis occurring in a 53-year-old woman with a long history of diabetes mellitus, hypertension, and peripheral vascular disease . The differential diagnosis and the pathogenesis of C . perfringens enteritis necroticans are discussed. Clin Infect Dis, 2002 Dec 1, 35(11), 1441 - 3 Epub 2002 Nov 13. Toxic shock syndrome due to Clostridium sordellii: a dramatic postpartum and posta