Annu Rev Pharmacol Toxicol, 2004, 44, 167 - 93 Identification of the major steps in botulinum toxin action; Simpson LL; Botulinum toxin is a uniquely potent substance synthesized by the organisms Clostridium botulinum, Clostridium baratii, and Clostridium butyricum . This toxin, which acts preferentially on peripheral cholinergic nerve endings to block acetylcholine release, is both an agent that causes disease (i.e., botulism) as well as an agent that can be used to treat disease (e.g., dystonia) . The ability of botulinum toxin to produce its effects is largely dependent on its ability to penetrate cellular and intracellular membranes . Thus, toxin that is ingested or inhaled can bind to epithelial cells and be transported to the general circulation . Toxin that reaches peripheral nerve endings binds to the cell surface then penetrates the plasma membrane by receptor-mediated endocytosis and the endosome membrane by pH-induced translocation . Internalized toxin acts in the cytosol as a metalloendoprotease to cleave polypeptides that are essential for exocytosis . This review seeks to identify and characterize all major steps in toxin action, from initial absorption to eventual paralysis of cholinergic transmission. Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 241 - 5 Hespellia stercorisuis gen . nov., sp . nov . and Hespellia porcina sp . nov., isolated from swine manure storage pits; Whitehead TR et al.; Four Gram-positive-staining, strictly anaerobic, non-spore-forming, rod-shaped organisms were isolated from a pig manure storage pit . Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to two related but distinct groups . Sequence analysis showed that the two groups of isolates were highly related to each other (approx . 97% 16S rRNA gene sequence similarity), forming a distinct cluster within the Clostridium coccoides suprageneric rDNA grouping . Biochemical and physiological studies confirmed the division of the isolates into two related, albeit distinct, groups . Based on both phenotypic and phylogenetic evidence, it is proposed that the unidentified rod-shaped isolates from pig manure should be classified in a novel genus, Hespellia gen . nov., as Hespellia stercorisuis sp . nov . and Hespellia porcina sp . nov . The type species of the novel genus is H . stercorisuis (type strain, PC18T=NRRL B-23456T=CCUG 46279T=ATCC BAA-677T) and the type strain of H . porcina is PC80T (=NRRL B-23458T=ATCC BAA-674T). Biomaterials, 2004 May, 25(10), 1841 - 9 Engineering a bifunctional starch-cellulose cross-bridge protein; Levy I et al.; Biodegradable starch- and cellulose-based polymers have a range of properties which make them suitable for use in a wide array of biomedical applications ranging from bone replacement to engineering of tissue scaffolds and drug delivery systems . A novel polysaccharide cross-bridging protein was designed which was comprised of a cellulose-binding domain from Clostridium cellulovorans (CBD(clos)) and a starch-binding domain from Aspergillus niger B1 (SBD(Asp)) . The two genes were fused in-frame via a synthetic elastin gene to construct a Cellulose/Starch Cross bridging Protein (CSCP) . Recombinant CSCP was expressed in Escherichia coli, and successfully refolded from inclusion bodies . CSCP demonstrated cross-bridging ability in different model systems composed of insoluble or soluble starch and cellulose . The aspect that different carbohydrate-binding module maintain their binding capacity over a wide range of conditions, without the need for chemical reactions, makes them attractive domains for designing new classes of chimeric polysaccharide-binding domains which demonstrate potential for use in a wide range of biomaterials. J Am Assoc Gynecol Laparosc, 2003 Nov, 10(4), 481 - 3 Outpatient total laparoscopic hysterectomy; Thiel J et al.; STUDY OBJECTIVE: To assess the safety of and patient satisfaction with total laparoscopic hysterectomy as an outpatient procedure . DESIGN: Retrospective case study (Canadian Task Force classification III) . SETTING: Tertiary care hospital . PATIENTS: Sixty-six consecutive women . INTERVENTION: Outpatient total laparoscopic hysterectomy . MEASUREMENTS AND MAIN RESULTS: Of 66 procedures completed, 6 patients (7.5%) required overnight hospitalization, with 4 of them discharged the next day . Seven (11%) minor postoperative complications occurred . One patient required hospitalization to receive intravenous antibiotics and one for drainage of a cuff hematoma . There were three (4.5%) minor intraoperative complications . One woman developed Clostridium difficile diarrhea as well as deep vein thrombosis and pulmonary embolus . Sixty-three women (95%) were satisfied with the procedure and would recommend it to others . CONCLUSION: Outpatient total laparoscopic hysterectomy is well tolerated, safe, and cost effective. Internist (Berl), 2004 Jan, 45(1), 85 - 9 {Generalized pain syndrome, fever and somnolence in an 81-year-old patient}; Fuchs KF et al.; We present a case of an 81-year-old diabetic man with anaerobic sepsis due to acalculous cholecystitis . The patient was admitted to our hospital with a seven-day history of severe abdominal pain accompanied with fever and somnolence . Blood cultures taken during the initial procedure developed Clostridium perfringens . The patient was immediately treated with parenteral penicillin . The ultrasonography pointed out the case: the gall bladder was found to be distended and slightly thickened . This result was interpreted as an acute non-emphysematous cholecystitis . The material obtained by needle aspiration and therapeutical emptying of the gall bladder revealed large gram positive rods, that also proved to be Clostridium perfringens . The patients course afterwards was uneventful . Antibiotics were continued and he was discharged after 13 days in a stable condition. J Cell Biol, 2004 Jan 19, 164(2), 279 - 90 Atypical protein kinase C (PKCzeta/lambda) is a convergent downstream target of the insulin-stimulated phosphatidylinositol 3-kinase and TC10 signaling pathways; Kanzaki M et al.; Insulin stimulation of adipocytes resulted in the recruitment of atypical PKC (PKCzeta/lambda) to plasma membrane lipid raft microdomains . This redistribution of PKCzeta/lambda was prevented by Clostridium difficile toxin B and by cholesterol depletion, but was unaffected by inhibition of phosphatidylinositol (PI) 3-kinase activity . Expression of the constitutively active GTP-bound form of TC10 (TC10Q/75L), but not the inactive GDP-bound mutant (TC10/T31N), targeted PKCzeta/lambda to the plasma membrane through an indirect association with the Par6-Par3 protein complex . In parallel, insulin stimulation as well as TC10/Q75L resulted in the activation loop phosphorylation of PKCzeta . Although PI 3-kinase activation also resulted in PKCzeta/lambda phosphorylation, it was not recruited to the plasma membrane . Furthermore, insulin-induced GSK-3beta phosphorylation was mediated by both PI 3-kinase-PKB and the TC10-Par6-atypical PKC signaling pathways . Together, these data demonstrate that PKCzeta/lambda can serve as a convergent downstream target for both the PI 3-kinase and TC10 signaling pathways, but only the TC10 pathway induces a spatially restricted targeting to the plasma membrane. Biochim Biophys Acta, 2004 Jan 5, 1676(1), 71 - 82 GltX from Clostridium saccharobutylicum NCP262: glutamate synthase or oxidoreductase? Stutz HE, Reid SJ. A full-length gene encoding a homologue of the small subunit of the glutamate synthase (GOGAT) enzyme was isolated from the anaerobic bacterium, Clostridium saccharobutylicum NCP262, which has been used extensively for the commercial production of solvents . Using a screening system designed to isolate genes involved in electron transport, plasmid pMET13C1 was isolated . Analysis of this plasmid identified a gene (1245 bp) with a predicted approximately 46-kDa product, which was associated with reductive activation of the pro-drug metronidazole . The deduced 414-amino-acid sequence was not typical of electron transport proteins, but rather shared striking homology to the small (beta) subunit of the GOGAT enzyme and other beta subunit-like polypeptides, and was thus designated gltX . Although all the functional domains typical of GOGAT beta subunits were conserved in this GltX protein, certain sequence features were not conserved . Furthermore, it was independently transcribed, did not lie adjacent to a GOGAT large subunit (alpha) domain, and its expression was not regulated by nitrogen conditions . These results provide additional support for current theories on the evolutionary relationships of GOGAT beta subunit domains in bacteria, and suggest that GltX belongs to a more general family of oxidoreductases, which is used in a context other than glutamate biosynthesis to transfer electrons to a currently unknown protein domain. Trends Cell Biol, 1995 Dec, 5(12), 441 - 3 Monoglucosylation of low-molecular-mass GTP-binding Rho proteins by clostridial cytotoxins; Aktories K et al.; Rho proteins, which are involved in receptor-mediated regulation of the actin cytoskeleton, are substrates for ADP-ribosylation by Clostridium botulinum C3 toxins . Recently, it was shown that Rho and other members of the Rho subfamily of low-molecular-mass GTP-binding proteins are glucosylated by C . difficile toxins A and B . Glucosylation occurs at threonine-37, which is a crucial amino acid residue for the regulatory functions of the small GTP-binding proteins . These toxins should prove useful as tools for studying the functions of Rho proteins. J Med Microbiol, 2004 Feb, 53(Pt 2), 167 - 72 Clostridium difficile colonization in healthy adults: transient colonization and correlation with enterococcal colonization; Ozaki E et al.; The aim of the present study was to investigate the colonization status of Clostridium difficile in healthy individuals . In total, 139 healthy adults from two study groups were examined at intervals of 3 months . Among the 18 positive subjects, the number of subjects from whom C . difficile was isolated once, twice, three times or four times was 10 (55.6%), three (16.7%), two (11.1%) and three (16.7%), respectively . In the student group, different subjects were colonized by different PCR ribotype/PFGE types . However, the same PCR ribotype/PFGE types of C . difficile were isolated from different subjects in the employee group, indicating that cross-transmission may have occurred in this group . Continuous colonization by the same PCR ribotype/PFGE type was only observed in three subjects . C . difficile-positive subjects were significantly more densely colonized by enterococci (P<0.05) than C . difficile-negative subjects: subjects that were found to be C . difficile-positive three or four times appeared to have higher concentrations of enterococci . The present results demonstrate that, although colonization by a C . difficile strain is transient in many cases, there are healthy individuals that are colonized persistently by C . difficile . They also suggest that dense colonization of the intestine by enterococci may be associated with C . difficile colonization. Scand J Infect Dis, 2003, 35(11-12), 883 - 6 Clostridium perfringens pleuropulmonary infection and septic shock: case report and population-based laboratory surveillance study; Jackson S et al.; We report a rare case of necrotizing pneumonia, empyema, and septic shock caused by Clostridium perfringens . Population-based laboratory surveillance was subsequently conducted that identified an annual incidence of invasive C . perfringens disease of 0.83 per 100,000 population with a striking age-related increased risk for acquisition. Am J Respir Med, 2003, 2(5), 395 - 411 Prevention and treatment of gastrointestinal complications in patients on mechanical ventilation; Mutlu GM et al.; There exists a complex, dynamic interaction between mechanical ventilation and the splanchnic vasculature that contributes to a myriad of gastrointestinal tract complications that arise during critical illness . Positive pressure-induced splanchnic hypoperfusion appears to play a pivotal role in the pathogenesis of these complications, the most prevalent of which are stress-related mucosal damage, gastrointestinal hypomotility and diarrhea . Furthermore, characteristics of the splanchnic vasculature make the gastrointestinal tract vulnerable to adverse effects related to positive pressure ventilation . While most of these complications seen in mechanically ventilated patients are reflections of altered gastrointestinal physiology, some may be attributed to medical interventions instituted to treat critical illness.Since maintenance of normal hemodynamics cannot always be achieved, pharmacologic prophylactic therapy has become a mainstay in the prevention of gastrointestinal complications in the intensive care unit . Improved understanding of the systemic effects of mechanical ventilation and greater application of lung-protective ventilatory strategies may potentially minimize positive pressure-induced reductions in splanchnic perfusion, systemic cytokine release and, consequently, reduce the incidence of gastrointestinal complications associated with mechanical ventilation . Herein, we discuss the pathophysiology of gastrointestinal complications associated with mechanical ventilation, summarize the most prevalent complications and focus on preventive strategies and available treatment options for these complications.The most common causes of gastrointestinal hemorrhage in mechanically ventilated patients are bleeding from stress-related mucosal damage and erosive esophagitis . In general, histamine H(2) receptor antagonists and proton pump inhibitors prevent stress-related mucosal disease by raising the gastric fluid pH . Proton pump inhibitors tend to provide more consistent pH control than histamine H(2) receptor antagonists . There is no consensus on the drug of choice for stress ulcer prophylaxis with several meta-analyses providing conflicting results on the superiority of any medication . Prevention of erosive esophagitis include careful use of nasogastric tubes and institution of strategies that improve gastric emptying . Many mechanically ventilated patients have gastrointestinal hypomotility and diarrhea . Treatment options for gastrointestinal motility are limited, thus, preventive measures such as correction of electrolyte abnormalities and avoidance of medications that impair gastrointestinal motility are crucial . Treatment of diarrhea depends on the underlying cause . When associated with Clostridium difficile infection antibacterial therapy should be discontinued, if possible, and treatment with oral metronidazole should be initiated.More studies are warranted to better understand the systemic effects of mechanical ventilation on the gastrointestinal tract and to investigate the impact of lung protective ventilatory strategies on gastrointestinal complications. Am J Vet Res, 2004 Jan, 65(1), 26 - 30 Effects of injection of botulinum toxin type B into the external anal sphincter on anal pressure of horses; Adam-Castrillo D et al.; OBJECTIVE: To determine effects on anal pressure of horses after local injection of the external anal sphincter with Clostridium botulinum toxin type B . ANIMALS: 11 healthy adult horses . PROCEDURE: Peak and resting anal sphincter pressures were measured with a custom-made rectal probe that was connected to a pressure transducer . Pressures were measured before treatment and after injection with botulinum toxin type B (BTB) or saline (0.9% NaCl) solution . Dose titration with 500, 1,000, 1,500, and 2,500 U of BTB was completed . Physical changes, behavior, and anal pressure were recorded for each horse . RESULTS: Injection of 1,000 U of BTB caused a significant reduction in peak anal pressure from days 2 to 84, compared with pressure in control horses . Maximal effect of the toxin was observed within the first 15 days after injection, followed by a slow return to baseline during the 168-day period . Injection of 2,500 U of BTB in the anal sphincter in 1 horse resulted in lethargy, generalized weakness, and dysphagia for 14 days . Adverse clinical effects were not observed in horses after injections with 500, 1,000, or 1,500 U of BTB . CONCLUSIONS AND CLINICAL RELEVANCE: The effect of focal intramuscular injection of BTB in horses is similar to that reported for other species . However, horses appear to be more sensitive to BTB, compared with other species, and clinical signs of botulism may develop at doses exceeding 1,500 U . Injections of BTB in the external anal sphincter of mares may be useful to reduce incisional dehiscence after repair of perineal lacerations. J Food Prot, 2004 Jan, 67(1), 199 - 202 Antibacterial efficiency of Finnish spice essential oils against pathogenic and spoilage bacteria; Nevas M et al.; The antibacterial properties of 13 essential oils, derived from spices grown in Finland, were examined with an agar diffusion method against 12 bacterial strains . The organisms tested included both spoilage and pathogenic bacteria . The gram-positive bacteria appeared to be more sensitive than the gram-negative organisms, Clostridium botulinum and Clostridium perfringens being the most sensitive . Oregano, savory, and thyme showed the broadest antibacterial activity by distinctly inhibiting the growth of all the organisms tested . By gas chromatography-mass spectrometry analysis, differences were noted in the composition of oregano and thyme oils in comparison to previous reports. J Food Prot, 2004 Jan, 67(1), 83 - 9 Growth of heat-treated enterotoxin-positive Clostridium perfringens and the implications for safe cooling rates; Andersen KG et al.; Clostridium perfringens 790-94 and 44071.C05 carrying a chromosomal and a plasmid cpe gene, respectively, were used to determine differences in heat resistance and growth characteristics between the genotypes . Heat inactivation experiments were conducted using an immersed coil apparatus . Spore germination, outgrowth, and lag phase, together named GOL time, as well as generation times were determined during constant temperatures in fluid thioglycollate (FTG) medium as well as in vacuum-packed, heat-treated minced turkey . GOL time and growth were also monitored during cooling scenarios from 65 to 10 degrees C for 3, 4, 5, 6, and 7 h in vacuum-packed, heat-treated minced turkey . Spores of strain 790-94 were approximately 10-fold more heat resistant at 85 degrees C than those of strain 44071.C05, and strain 790-94 also had a higher temperature growth range in FTG . The higher growth range for a chromosomal enterotoxin-producing CPE+ strain was confirmed using two other strains carrying a chromosomal (NCTC8239) and plasmid (945P) cpe gene . Moreover, strain 790-94 had shorter GOL times at 50 degrees C in turkey and approximately half the generation time compared with strain 44071.C05 at temperatures > or = 45 degrees C in both FTG and turkey . Strain 790-94 increased with 0.3, 1.0, 1.7, and 2.0 logs, respectively, during cooling from 65 to 10 degrees C in 4, 5, 6, and 7 h, which was significantly higher than for strain 44071.C05 . A maximum acceptable cooling time of 5 h between 65 and 10 degrees C is suggested. J Clin Microbiol, 2004 Jan, 42(1), 484 - 6 Osteosynthesis-associated bone infection caused by a nonproteolytic, nontoxigenic Clostridium botulinum-like strain; Carlier JP et al.; A nonproteolytic, nontoxigenic Clostridium botulinum strain identified by conventional and molecular techniques as type B-, E-, or F-like (BEF-like) was isolated from a human postsurgical wound . All previous reports of such strains have been from environmental sources . Since toxin production is the main taxonomic denominator for C . botulinum, a new name is needed for nonproteolytic, nontoxigenic BEF-like clinical isolates. J Biol Chem, 2004 Apr 2, 279(14), 14315 - 22 Epub 2004 Jan 10. The identification and structure of the membrane-spanning domain of the Clostridium septicum alpha toxin; Melton JA et al.; Alpha toxin (AT) is a pore-forming toxin produced by Clostridium septicum that belongs to the unique aerolysin-like family of pore-forming toxins . The location and structure of the transmembrane domains of these toxins have remained elusive . Using deletion mutagenesis, cysteine-scanning mutagenesis and multiple spectrofluorimetric methods a membrane-spanning amphipathic beta-hairpin of AT has been identified . Spectrofluorimetric analysis of cysteine-substituted residues modified with an environmentally sensitive fluorescent probe via the cysteine sulfydryl showed that the side chains of residues 203-232 alternated between the aqueous milieu and the membrane core when the AT oligomer was inserted into membranes, consistent with the formation of an amphipathic transmembrane beta-hairpin . AT derivatives that contained deletions that removed up to 90% of the beta-hairpin did not form a pore but were similar to native toxin in all other aspects of the mechanism . Furthermore, a mutant of AT that contained an engineered disulfide, predicted to restrict the movement of the beta-hairpin, functioned similarly to native toxin except that it did not form a pore unless the disulfide bond was reduced . Together these studies revealed the location and structure of the membrane-spanning domain of AT. Pain, 2004 Jan, 107(1-2), 125 - 33 Subcutaneous administration of botulinum toxin A reduces formalin-induced pain; Cui M et al.; Botulinum toxin type A (BoNT-A) produced by the bacterium Clostridium botulinum is a potent inhibitor of acetylcholine release in the neuromuscular junction and has been used to treat many disorders related to excessive muscle contraction . However, BoNT-A has recently been used in pain therapy to treat myofascial pain, low back pain and various types of headaches, including migraine . The purpose of this study is to investigate the antinociceptive effect of BoNT-A and its underlying mechanism in the rat formalin inflammatory pain model . BoNT-A (3.5, 7, 15 and 30 U/kg) or vehicle was administered to the plantar surface of the right hindpaw of male Sprague-Dawley rats . BoNT-A dose-dependently (P<0.05) inhibited formalin-induced nociceptive behavior during phase 2 but not during phase 1 when administered 5 h to 12 days before formalin challenge . The onset of the antinociceptive effect started at 5 h after pre-treatment and this effect lasted for at least 12 days . BoNT-A (7 U/kg) also reduced edema . Consistent with the lack of effect in the formalin phase 1, BoNT-A, at 15 U/kg, had no effect on acute thermal nociception; no local muscle weakness was observed at this dose . Pre-treatment of rats with BoNT-A (3.5, 7 or 15 U/kg) all significantly reduced formalin-evoked glutamate (Glu) release . These results demonstrate that local peripheral injection of BoNT-A significantly reduces formalin-induced nociceptive behaviors with the absence of obvious muscle weakness . Such an antinociceptive effect of BoNT-A is associated with the inhibition of formalin-induced release of Glu (and/or neuropeptides) from primary afferent terminals. Vet Q, 2003 Dec, 25(4), 155 - 9 Pathogenesis and pathology of blackleg in ruminants: the role of toxins and neuraminidase . A short review; Useh NM et al.; There is no consensus on the pathogenesis of blackleg infection that occurs in ruminants, but toxins and neuraminidase produced by Clostridium chauvoei are believed to play a significant role in the pathogenesis of the disease . This paper provides an update on the role of toxins and neuraminidase in the pathogenesis and pathology of the disease . The use of neuraminidase inhibitors to manage clinical blackleg infections is therefore an alternative therapeutic protocol that should be thoroughly investigated . It is suggested that in vivo clinical trials should be carried out to determine the mechanism of action and clinical efficacy of neuraminidase inhibitors. Surg Today, 2004, 34(1), 80 - 1 Primary gas gangrene of the pancreas: report of a case; Ikegami T et al.; Spontaneous gas gangrene of the pancreas, caused by an infection of Clostridium perfringens, is an extremely rare but severe form of acute pancreatitis . A 67-year-old man complaining of severe epigastric pain with diffuse guarding underwent an emergency laparotomy . During surgery, hemorrhagic pancreatic necrosis was observed with a large amount of peripancreatic gas . Cultures demonstrated C . perfringens . The identification of hemolysis and the accumulation of peripancreatic gas on computed tomography, which were both caused by an infection of C . perfringens, led us to make a diagnosis of clostridial infection of the pancreas. Biotechnol Bioeng, 2003 Dec 30, 84(7), 842 - 54 Transcriptional analysis of product-concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains; Tummala SB et al.; Antisense RNA (asRNA) downregulation alters protein expression without changing the regulation of gene expression . Downregulation of primary metabolic enzymes possibly combined with overexpression of other metabolic enzymes may result in profound changes in product formation, and this may alter the large-scale transcriptional program of the cells . DNA-array based large-scale transcriptional analysis has the potential to elucidate factors that control cellular fluxes even in the absence of proteome data . These themes are explored in the study of large-scale transcriptional analysis programs and the in vivo primary-metabolism fluxes of several related recombinant C . acetobutylicum strains: C . acetobutylicum ATCC 824(pSOS95del) (plasmid control; produces high levels of butanol snd acetone), 824(pCTFB1AS) (expresses antisense RNA against CoA transferase (ctfb1-asRNA); produces very low levels of butanol and acetone), and 824(pAADB1) (expresses ctfb1-asRNA and the alcohol-aldehyde dahydrogenase gene (aad); produce high alcohol and low acetone levels) . DNA-array based transcriptional analysis revealed that the large changes in product concentrations (snd notably butanol concentration) due to ctfb1-asRNA expression alone and in combination with aad overexpression resulted in dramatic changes of the cellular transcriptome . Cluster analysis and gene expression patterns of established and putative operons involved in stress response, motility, sporulation, and fatty-acid biosynthesis indicate that these simple genetic changes dramatically alter the cellular programs of C . acetobutylicum . Comparison of gene expression and flux analysis data may point to possible flux-controling steps and suggest unknown regulatory mechanisms . J Immunol, 2004 Jan 15, 172(2), 1118 - 24 Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire; Rhee KJ et al.; Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance . In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT . We studied the mechanism of bacteria-induced GALT development . Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits) . We then identified specific members of the intestinal flora that promote GALT development . The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells . Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis . B . fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response . To identify bacterial pathways required for GALT development, we introduced B . fragilis along with stress-response mutants of B . subtilis into germfree-appendix rabbits . We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development . We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses. J Hosp Infect, 2004 Jan, 56(1), 10 - 5 How do we assess hospital cleaning? A proposal for microbiological standards for surface hygiene in hospitals; Dancer SJ; Increasing numbers of hospital-acquired infections have generated much attention over the last decade . The public has linked the so-called 'superbugs' with their experience of dirty hospitals, but the precise role of cleaning in the control of these organisms in unknown . Hence the importance of a clean environment is likely to remain speculative unless it becomes an evidence-based science . This proposal is a call for bacteriological standards with which to assess clinical surface hygiene in hospitals, based on those used by the food industry . The first standard concerns any finding of a specific 'indicator' organism, the presence of which suggests a requirement for increased cleaning . Indicators would include Staphylococcus aureus, including methicillin-resistant S . aureus, Clostridium difficile, vancomycin-resistant enterococci and various Gram-negative bacilli . The second standard concerns a quantitative aerobic colony count of <5 cfu/cm(2) on frequent hand touch surfaces in hospitals . The principle relates to modern risk management systems such as HACCP, and reflects the fact that pathogens of concern are widespread . Further work is required to evaluate and refine these standards and define the infection risk from the hospital environment. Med Dosw Mikrobiol, 2003, 55(3), 253 - 8 {Assessment of susceptibility to metronidazole and vancomycin of Clostridium difficile strains isolated between 1998-2002}; Pituch H et al.; The drugs of choice used to treat C . diffcile associated diarrhoea (CDAD) are metronidazole and vancomycin . C . difficile strains isolated in most laboratories are susceptible to metronidazole and vancomycin . Communication about emergence of antimicrobial resistance among C . difficile strains in some countries to metronidazole and intermediate resistance to vancomycin are alarming . This study was performed to determine the susceptibility to metronidazole and vancomycin of 140 C . difficile strains isolated from patients with CDAD hospitalised in academic hospital between 1999-2002 . Resistance to metronidazole and vancomycin was not observed. Microbiology, 2004 Jan, 150(Pt 1), 127 - 34 The Clostridium perfringens TetA(P) efflux protein contains a functional variant of the Motif A region found in major facilitator superfamily transport proteins; Bannam TL et al.; The Clostridium perfringens tetracycline resistance protein, TetA(P), is an inner-membrane protein that mediates the active efflux of tetracycline from the bacterial cell . This protein comprises 420 aa and is predicted to have 12 transmembrane domains (TMDs) . Comparison of the TetA(P) amino acid sequence to that of several members of the major facilitator superfamily (MFS) identified a variant copy of the conserved Motif A . This region consists of the sequence E59xPxxxxxDxxxRK72 and is located within the putative loop joining TMDs 2 and 3 in the predicted structural model of the TetA(P) protein . To study the functional importance of the conserved residues, site-directed mutagenesis was used to construct 17 point mutations that were then analysed for their effect on tetracycline resistance and their ability to produce an immunoreactive TetA(P) protein . Changes to the conserved Phe-58 residue were tolerated, whereas three independent substitutions of Pro-61 abolished tetracycline resistance . Examination of the basic residues showed that Arg-71 is required for function, whereas tetracycline resistance was retained when Lys-72 was substituted with arginine . These results confirm that the region encoding this motif is important for tetracycline resistance and represents a distant version of the Motif A region found in other efflux proteins and members of the MFS family . In addition, it was shown that Glu-117 of the TetA(P) protein, which is predicted to be located in TMD4, is important for resistance although a derivative with an aspartate residue at this position is also functional. J Biol Chem, 2004 Mar 26, 279(13), 12181 - 9 Epub 2003 Dec 30. Clostridium perfringens alpha-toxin activates the sphingomyelin metabolism system in sheep erythrocytes; Ochi S et al.; Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism . Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine . We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes . Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P) . N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells . Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate . These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis . S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells . Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM . In addition, incubation of C . botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P . These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P. Ir J Med Sci, 2003 Jul-Sep, 172(3), 143 - 4 Botulism as a cause of respiratory failure in injecting drug users; FitzGerald S et al.; BACKGROUND: Wound botulism occurs as a consequence of inoculation of Clostridium botulinum spores into a wound . AIM: To describe such a case of wound botulism . RESULTS: A 23-year-old drug-injecting user presented with bulbar symptoms and progressive signs over a three-day period . The diagnosis of botulism was suspected and was treated with large doses of penicillin and botulinum antitoxin . The diagnosis was confirmed by the presence of serum botulinum toxin A . CONCLUSION: Physicians should be aware of the association of botulism in injecting drug users, particularly in Ireland. Microbiol Immunol, 2003, 47(12), 1005 - 16 Bacterial artificial chromosome library of Finegoldia magna ATCC 29328 for genetic mapping and comparative genomics; Goto T et al.; We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328 DNA to facilitate further genome analysis of F . magna . The BAC library contained 385 clones with an average insert size of 55 kb, representing a 10.1-fold genomic coverage . Repeated DNA hybridization using primer sets designed on the basis of BAC-end sequences yielded nine contigs covering 95% of the chromosome and two contigs covering 98% of the plasmid . The contigs were localized on the physical map of F . magna ATCC 29328 DNA . A total of 121 BAC-end sequences revealed 103 unique genes, which had not been previously reported for F . magna . The homolog ORF of albumin-binding protein (urPAB), one of the known virulence factors from F . magna, was sequenced and localized on the physical map . Homology analysis of 121 BAC-end sequences revealed that F . magna is most closely related to clostridia, particularly Clostridium tetani . This close relationship is consistent with the recent classification of peptostreptococci based on 16S rRNA sequence analysis . The BAC library constructed here will be useful for the whole genome sequencing project and other postgenomic applications. Microbiol Immunol, 2003, 47(12), 951 - 8 Effects of high amylose maize starch and Clostridium butyricum on metabolism in colonic microbiota and formation of azoxymethane-induced aberrant crypt foci in the rat colon; Nakanishi S et al.; High amylose maize starch (HAS) is not digested in the small intestine and most of it reaches the large intestine . In the large intestine, HAS is fermented by intestinal bacteria, resulting in production of short-chain fatty acids (SCFA), particularly butyrate . Clostridium butyricum can utilize HAS and produce butyrate and acetate . It has been proposed that butyrate inhibits carcinogenesis in the colon . In this study, we examined the inhibitory effects of HAS and C . butyricum strain MIYAIRI588 (CBM588) on azoxymethane-induced aberrant crypt foci (ACF) formation in rats . In the group of rats administered only CBM588 spores, the concentration of butyrate in the cecum increased, but there was no decrease in the number of ACF . In the group of rats fed an HAS diet, a decrease in the number of ACF was observed, and in the group of rats administered HAS and CBM588, the number of ACF decreased significantly . In these two groups, the concentrations of acetate and propionate in intestinal contents significantly increased, but the concentration of butyrate did not change . It was found that the beta-glucuronidase activity level of colonic contents decreased significantly in the two groups of rats fed HAS . This study showed that HAS and CBM588 changed the metabolism of colonic microbiota and decreased the level of beta-glucuronidase activity, phenomena that may play a role in the inhibition of ACF formation in the rat colon. J Mol Model (Online), 2004 Feb, 10(1), 69 - 75 Epub 2003 Dec 23. Improvement of comparative modeling by the application of conserved motifs amongst distantly related proteins as additional restraints; Chakrabarti S et al.; Protein comparative modeling has useful applications in large-scale structural initiatives and in rational design of drug targets in medicinal chemistry . The reliability of a homology model is dependent on the sequence identity between the query and the structural homologue used as a template for modeling . Here, we present a method for the utilization and conservation of important structural features of template structures by providing additional spatial restraints in comparative modeling programs like MODELLER . We show that root mean square deviation at C(alpha) positions between the model and the corresponding experimental structure and the quality of the models can be significantly improved for distantly related systems by utilizing additional spatial restraints of the template structures . We demonstrate the influence of such approaches to homology modeling during distant relationships in understanding functional properties of protein such as ligand binding using cytochrome P450 as an example.FIGURE Example of a modeling triad . Structures of flavodoxin from Clostridium beijerinckii (PDB code, 5nul) and flavodoxin from Chondrus crispus (PDB code, 2fcr) that are equidistant in sequence identity (Seq . ID.) and structural similarity (RMSD) to the third flavodoxin member (from Desulfovibrio vulgaris, PDB code, 1akr) J Infect Chemother, 2003 Dec, 9(4), 351 - 4 Pseudomembranous colitis caused by toxin A-negative/toxin B-positive variant strain of Clostridium difficile; Toyokawa M et al.; We report the first case of pseudomembranous colitis (PMC) due to a toxin A-negative, toxin B-positive strain (toxin A variant strain) of Clostridium difficile in Japan . The toxin A variant strain of C . difficile is capable of causing PMC and is undetectable in clinical laboratories that use only toxin A immunoassays for C . difficile testing . If C . difficile-associated diarrhea is clinically suspected and toxin A is not detected, then the possibility of a toxin A variant strain should be considered, and further diagnostic testing, such as polymerase chain reaction (PCR) analysis for the detection of C . difficile toxin genes should be performed. Biochemistry, 2003 Dec 30, 42(51), 15284 - 91 Cellular uptake of Clostridium botulinum C2 toxin: membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain; Haug G et al.; The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins . C2 toxin is composed of two separated nonlinked proteins . The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells . The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol . After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol . For this step, the cellular heat shock protein Hsp90 is essential . We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I . Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component . The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa . Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain . Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin . In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited . The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes . Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells . The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited. Tohoku J Exp Med, 2003 Dec, 201(4), 239 - 49 The role of rho and rho-dependent kinase in serotonin-induced contraction observed in bovine middle cerebral artery; Nishikawa Y et al.; The current study was designed to characterize the role of Rho and Rho-dependent kinase (Rho-kinase) in isometric contractile responses induced by serotonin (5-HT) and a solution containing 40 mM K(+) (high K(+)) in ring preparations of the middle cerebral artery of bovine . Application of W-7, a Ca(2+)-calmodulin inhibitor, reversibly and equally attenuated the amplitudes of contractions produced by both 5-HT and high K(+) . Similar effects were observed with ML-7, an inhibitor of myosin light chain kinase . Surprisingly, the protein kinase C inhibitors, calphostin C and Ro-31-8220, had no effect on the 5-HT-induced contraction . Incubation of preparations with Clostridium difficile toxin A and B or with Clostridium botulinum C3 exoenzyme for 48 hours attenuated the 5-HT-induced response but not the high K(+)-induced response . Application of the Rho-kinase inhibitor, Y-27632, resulted in marked inhibition of the 5-HT-induced response but had negligible effect on the high K(+)-induced response . These results suggest that the activation of Rho and Rho-kinase may be involved in the generation of the contraction produced by 5-HT in the bovine middle cerebral artery, while protein kinase C plays, if any, an insignificant role on the contraction. J Biol Chem, 2004 Mar 12, 279(11), 9867 - 74 Epub 2003 Dec 19. Cohesin-dockerin interactions within and between Clostridium josui and Clostridium thermocellum: binding selectivity between cognate dockerin and cohesin domains and species specificity; Jindou S et al.; The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component . We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C . thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C . josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C . thermocellum and C . josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria . Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C . josui, although this was not detected by qualitative analysis . Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C . thermocellum and C . josui, i.e . the dockerin polypeptides of C . thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C . josui CipA . SPR analysis confirmed this exceptional binding . We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains. Neurosci Res, 2004 Jan, 48(1), 33 - 43 The small GTP-binding protein RhoA regulates serotonin-induced Na+-current response in the neurons of Aplysia; Kawasaki S et al.; Application of serotonin (5-HT) induces a slow inward current response in identified neurons of Aplysia ganglia under voltage clamp . The 5-HT-induced current response was depressed in Na+-free media, but augmented in Ca2+-free media, and unaffected by a change in external K+ . The 5-HT-induced response was markedly blocked by intracellular injection of guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) . After the injection of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), the responses to 5-HT gradually and significantly increased at the initial period, reached its plateau, and finally decreased . Intracellular injection of Clostridium difficile toxin B, a blocker of small G-protein Rho family members such as Rho (RhoA, RhoB and RhoC), Rac and Cdc42, markedly depressed the 5-HT-induced response . Intracellular injection of Clostridium botulinum C3 exoenzyme, a specific blocker of RhoA, RhoB, RhoC, exhibited a similar depressing effect observed with toxin B . In contrast, intracellular injection of recombinant L63RhoA, a constitutively active form of RhoA, significantly augmented the 5-HT-induced response without affecting the resting membrane . These results suggested that the 5-HT-induced Na+-current response might be facilitated by the activation of Aplysia Rho which is closely homologous to RhoA, RhoB or RhoC in mammalian neuron. Hum Exp Toxicol, 2003 Nov, 22(11), 593 - 605 The development of tolerance to Clostridium perfringens type D epsilon-toxin in MDCK and G-402 cells; Beal DR et al.; The epithelial Madin Darby canine kidney (MDCK) cell line, Caucasian renal leiomyoblastoma (G-402) cells, human small airways epithelial (HSAE) cells, human bronchial epithelial (HBE) cells and human renal proximal tubule (HRPT) epithelial cells were examined for sensitivity to Clostridium perfringens biotype D epsilon-toxin . MDCK and G-402 cells were confirmed as being the only established cell lines that are sensitive to the toxin . HSAE, HBE and HRPT epithelial cells were only found to be sensitive to the toxin at concentrations of > 1 mg/ mL . Cultures of MDCK and G-402 cells, with increased resistance (tolerance) to the cytotoxic effects of epsilon-toxin, were developed by exposing these cultures to progressively higher concentrations of toxin . The greatest relative increase in tolerance to epsilon-toxin was developed in MDCK cells, in which the LC50 in control cultures was 2 microg/mL as determined by the MTS/PMS assay system; after selection for tolerance, this was raised to 100 microg/mL . This represents a 50-fold increase in tolerance as measured by this index . Using G-402 cells, it was possible to increase the LC50 by twofold from 290 to 590 microg/mL . Subsequent 2-D electrophoresis of membrane preparations from tolerant and control MDCK cells revealed that the expression of a discrete group of proteins found in control cells with a range of molecular weights from 32-36 kDa, all with acidic isoelectric points (IEPs), were either not expressed in epsilon-toxin tolerant cells or had undergone a shift in IEP to a more alkaline pH in tolerant cells . This suggests that epsilon-toxin lethality in MDCK cells may be mediated by membrane-located proteins . Their absence or alteration in toxin-resistant cells would, at least partly, explain the failure of most cell lines to demonstrate sensitivity to this toxin, despite being derived from tissues that are damaged by epsilon-toxin . This approach may have utility in the study of other toxin-cell interactions and could be used in the development of novel medical countermeasures by identifying cellular targets which mediate toxin lethality. J Pediatr (Rio J), 1999 Nov-Dec, 75(6), 463 - 6 {Pseudomembranous colitis}; Ferreira CT et al.; OBJECTIVE: To alert about the pseudomembranous colitis in children, a consequence of the use of antibiotics . METHODS: This report is the result of the clinical follow-up of a patient with chronic diarrhea after the use of several antibiotics . The bibliography was obtained through Medline system . RESULTS: Case report of a girl two years and seven months old, previously healthy, with a clinical picture of chronic diarrhea with dysenteric characteristics after the use of antibiotics, associated with important weight lost and hypoproteinemia . The diagnosis of pseudomembranous was established clinically and was confirmed by colonoscopy and the presence of Clostridium difficile toxin A in the stools . CONCLUSIONS: It is necessary to consider the C . difficile infection in any child with diarrhea associated to the use of antibiotics . The antibiotics in pediatric patients should always be prescribed with caution and precise indications. Avian Pathol, 2004 Feb, 33(1), 83 - 92 Maternal vaccination against subclinical necrotic enteritis in broilers; Lovland A et al.; The inclusion of antibacterial feed additives has until now been the major strategy for controlling Clostridium perfringens-associated necrotic enteritis in broilers . In the present study, the effect of maternal immunization against the disease was examined . Broiler breeder hens were injected intramuscularly with candidate vaccines based on C . perfringens type A and type C toxoids adjuvanted with aluminium hydroxide . Vaccination resulted in a strong serum immunoglobulin G response to C . perfringens alpha-toxin in parent hens, and specific antibodies were transferred to their progeny . Subclinical necrotic enteritis in broilers was induced under field conditions or in a disease model, and the occurrence of specific enteric and hepatic lesions was evaluated in randomly selected birds . In three experiments, estimates of odds ratio for developing such lesions were 0.23, 0.33 and 0.56 in maternally toxoid C-immunized broilers compared with non-immunized controls . In toxoid A-immunized birds, odds ratios were estimated at 0.41, 0.61 and 0.63 . From these results, immunoprophylaxis seems to be an interesting alternative for the control of necrotic enteritis in broilers. Avian Pathol, 2004 Feb, 33(1), 19 - 24 Fatal necrotic enteritis associated with Clostridium perfringens in wild crows (Corvus macrorhynchos); Asaoka Y et al.; Sporadic outbreaks of fatal enteritis occurred among free-living wild crows ('large billed' or 'wok' crow; Corvus macrorhynchos) in an open-air park in Japan in 2002 . Eight crows were found dead during February, followed by two more in September, and five of the eight were examined histopathologically . At necropsy, all cases showed a markedly dilated small intestine, especially the jejunum and ileum, with large amounts of gas, and dark red to greenish-brown soft content . The necrotic intestinal wall was markedly thickened with multifocal haemorrhages . All cases had multifocal white foci in the liver, and four cases showed marked splenomegaly . Histologically, there was severe necrotic enteritis characterized by extensive mucosal necrosis and multifocal haemorrhages, as well as inflammatory cell infiltrations . A prominent pseudo-membrane formation was noted in the affected intestine . Severe adhesive peritonitis was also observed in three cases . Gram-positive bacilli were present in large numbers in the lumen, and in and around necrotic lesions in the affected intestine . The bacilli were positive for Clostridium perfringens enterotoxin type A by immunohistochemistry, and were also positive for C . perfringens type A using the immunofluorescence method . C . perfringens was isolated by anaerobic culture from the intestinal contents . The present enteritis was thought to be induced by proliferated C . perfringens in the intestine, and to be the cause of death. Avian Pathol, 2004 Feb, 33(1), 3 - 7 Susceptibility of Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials; Martel A et al.; Clostridium perfringens strains isolated in 2002 from the intestines of broiler chickens from 31 different farms located in Belgium were tested for susceptibility to 12 antibiotics used for therapy, growth promotion or prevention of coccidiosis . All strains were uniformly sensitive to the ionophore antibiotics monensin, lasalocid, salinomycin, maduramycin and narasin . All were sensitive to avilamycin, tylosin and amoxicillin, while flavomycin (bambermycin) showed low or no activity . Chlortetracycline and oxytetracycline were active at very low concentrations, but low-level acquired resistance was detected in 66% of the strains investigated . Fifty percent of these strains carried the tetP(B) resistance gene, while the tet(Q) gene was detected in only one strain . One strain with high-level resistance against tetracyclines carried the tet(M) gene . Sixty-three percent of the strains showed low-level resistance to lincomycin . The lnu(A) and lnu(B) genes were each only found in one strain . Compared with a similar investigation carried out in 1980, an increase was seen in resistance percentages with lincomycin (63% against 49%) and a slight decrease with tetracycline (66% against 74%). FEMS Microbiol Lett, 2003 Dec 12, 229(2), 265 - 70 The insecticidal toxin makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen; Waterfield NR et al.; The Photorhabdus luminescens W14 toxin encoding gene makes caterpillars floppy (mcf) was discovered due to its ability to kill caterpillars when expressed in Escherichia coli . Here we describe a homologue of mcf (renamed as mcf1), termed mcf2, discovered in the same genome . The mcf2 gene predicts another large toxin whose central domain, like Mcf1, also shows limited homology to Clostridium cytotoxin B . However, the N-terminus of Mcf2 shows significant similarity to the type-III secreted effector HrmA from the plant pathogen Pseudomonas syringae and no similarity to the N-terminus of Mcf1 . HrmA is a plant avirulence gene whose transient expression in tobacco cells results in cell death . Here we show that E . coli expressing Mcf2 can, like E . coli expressing Mcf1, kill insects . Further, expression of the c-Myc tagged N-terminus of Mcf2, the region showing similarity to HrmA, results in nuclear localisation of the fusion protein and subsequent destruction of transfected mammalian cells . The Mcf1 and Mcf2 toxins therefore belong to a family of high molecular mass toxins, differing at their N-termini, which encode different effector domains. J Bacteriol, 2004 Jan, 186(1), 253 - 7 Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824; Perret S et al.; Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents . This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes . To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C . acetobutylicum . A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum . Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional. J Bacteriol, 2004 Jan, 186(1), 136 - 45 Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture; Devillard E et al.; The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized . Subtractive enrichment procedures were used to isolate mutants of R . albus 8 that are defective in adhesion to cellulose . Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked . Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose . The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R . albus 8 . One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A) . Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B(2) (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively . Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex . Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus . Such X modules are also present in several other R . albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria. J Toxicol Clin Toxicol, 2003, 41(6), 841 - 7 Polar poisons: did Botulism doom the Franklin expedition? Horowitz BZ. In 1845 the Franklin expedition left London with 2 ships and 134 men on board in an attempt to find the route through the Northwest Passage . The ships were built with state-of-the-art technology for their day, but provisioned with supplies from the lowest bidder . After taking on fresh provisions in the Whalefish Islands, off the coast of Greenland, the entire crew was never heard from again . Graves found on remote Beechey Island indicate that three able-bodied seamen died during the first winter . A note written on a ship's log, later found in a cairn, indicate that the expedition's leader, Sir John Franklin, died during the second winter entrapped on the ice, by which time 24 men had also perished . The remaining crew failed in their attempt to walk out of the Arctic by an overland route . In 1981 Owen Beattie, from the University of Alberta, exhumed the remains of the sailors from the three graves on Beechey Island . Elevated lead levels were found in all three sailors . While lead poisoning has been a leading theory of the cause of the crew's deaths, blamed on the crudely tinned provisions the ships carried with them from England, chronic lead exposure may only have weakened the crew, not necessarily killed them . One of three exhumed sailors also had in his intestine the spores of an unspecified Clostridium species . The theory put forth by this article is that Botulism, type E, which is endemic in the Arctic, may have been responsible for their deaths. J Infect Dis, 2003 Dec 15, 188(12), 1922 - 7 Epub 2003 Dec 09. Prevention of fatal Clostridium difficile-associated disease during continuous administration of clindamycin in hamsters; Merrigan MM et al.; Clostridium difficile-associated disease (CDAD) due to toxigenic strains is prevented in hamsters by colonization by nontoxigenic C . difficile after administration of clindamycin (Cm) . To prevent CDAD during treatment with antibiotics, we gave a Cm-resistant nontoxigenic C . difficile strain, M13 (minimal inhibitory concentration {MIC}, >256 microg/mL), and a Cm-susceptible strain, M3 (MIC, 0.5 microg/mL), to hamsters receiving Cm daily for days 1-5 . Either M13 or M3 was given orogastrically (1 x 10(6) spores/day) to each hamster in 3 groups of 5 each, on either day 3, days 3-5, or days 3-7 . M13 colonized at a higher rate and faster than did M3 (P<.001) . When hamsters were challenged by toxigenic strain B1 on days 5, 7, or 9, M13 prevented CDAD in 100% of the hamsters . M3 protected no hamsters challenged by B1 on day 5, 20% on day 7, and 100% on day 9 . M13 contains the erm(B) resistance gene but not the mobilizable element Tn5398 . The benefits of use of Cm-resistant nontoxigenic C . difficile to prevent CDAD must be balanced against the risk that resistance might be transferred to other enteric bacteria. Int J Food Microbiol, 2004 Jan 1, 90(1), 83 - 91 The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process; Cocolin L et al.; A molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp . in cheese samples suspected of late blowing . Strains of Clostridium spp . and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation . DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight . Moreover, volatile fatty acids were determined for comparison purposes . The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp . bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates . Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage . The sensitivity of the method is estimated to be 10(4) CFU/g. J Food Prot, 2003 Dec, 66(12), 2332 - 5 Microbiology of charcoal-broiled European river lampreys (Lampetra fluviatilis) stored at 3 and 22 degrees C; Merivirta LO et al.; The microbiological quality of 30 production lots of charcoal-broiled river lampreys was studied at three lamprey processing plants (plants A, B, and C) . Samples were taken directly after charcoal broiling and stored at 22 and 3 degrees C . Lampreys were examined on the day of manufacture, and those kept at 22 degrees C were examined every second day for 6 days . Samples kept at 3 degrees C were examined every fourth day for up to 24 days . On the production day, the mean aerobic plate counts (APCs) for broiled lampreys from plants A, B, and C were 2.29 log CFU/g, 1.88 log CFU/g, and undetectable (1.67 log CFU/g), respectively . At 22 degrees C, the mean APCs for samples from plants A, B, and C increased markedly within 4 days, and after 6 days the counts for samples from these plants were 8.56, 5.04, and 6.23 log CFU/g, respectively . Chilling and storage at 3 degrees C remarkably improved the shelf life of the product . The levels of bacteria in charcoal-broiled river lampreys from plant A were higher than those in lampreys from plants B and C . No significant increases in APCs were observed during storage at 3 degrees C for 24 days; mean APCs did not exceed 2.80 log CFU/g for samples from any plant . Staphylococcus aureus was found in two samples . No lactic acid bacteria, thermotolerant coliforms, enterococci, Clostridium perfringens, or Listeria monocytogenes was detected . Microbiological data from this study will be used for the development of a hazard analysis for the determination of critical control points. Anal Chem, 2003 Dec 15, 75(24), 6886 - 93 Bacillus spore identification via proteolytic peptide mapping with a miniaturized MALDI TOF mass spectrometer; English RD et al.; An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer . A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder . To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases . Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications. Curr Microbiol, 2003 Nov, 47(5), 425 - 30 Metabolic changes in Clostridium absonum ATCC 27555 accompanying induction of epimerization of a primary bile acid; Warchol M et al.; Some parameters of fermentation have been determined for Clostridium absonum in a chemostat by using a chemically defined medium with glucose as the sole source of carbon and energy . Steady-state continuous cultures were achieved at two dilution rates (D) . Trends of the carbon flow were determined by comparison of ratios between the specific rates of formation of the three products of metabolism (lactate, acetate, butyrate) . Chenodeoxycholate induced the 7alpha- and 7beta-hydroxysteroid dehydrogenases of C . absonum . In the presence of this inducer, the growth yield and the carbon recovery decreased, the carbon flow distribution was altered favoring acetate production, and a deficit in the reoxydation of nucleotidic cofactors was observed . In the presence of chenodeoxycholate, C . absonum would favor the production of energy at the expense of the reoxidation of nucleotidic cofactors so as to ensure its growth, and the epimerization of chenodeoxycholate to ursodeoxycholate. Biochem Pharmacol, 2004 Jan 1, 67(1), 129 - 34 Adenosine A3 receptor-mediated regulation of p38 and extracellular-regulated kinase ERK1/2 via phosphatidylinositol-3'-kinase; Hammarberg C et al.; The adenosine A(3) receptor generally couples to the G(i) class of heterotrimeric G proteins, thereby decreasing cAMP levels and also mediating signaling via release of betagamma subunits . Here we describe the central role of phosphatidylinositol-3'-kinase (PI3K) for adenosine A(3) receptor-induced intracellular signaling to the stress-activated protein kinase p38 and the extracellular signal-regulated protein kinases ERK1/2 . We used Chinese hamster ovary cells expressing the human adenosine A(3) receptor, phospho-specific antibodies and different pharmacological tools to dissect the signaling pathways involving PI3K . The adenosine receptor agonist 5'N-ethylcarboxamidoadenosine induced a time- and dose-dependent increase in p38 and ERK1/2 phosphorylation, two signaling pathways that appeared also to be activated in the immortalized microglia cell line N13, which expressed endogenous adenosine A(3) receptors . The 5'N-ethylcarboxamidoadenosine-induced effects on p38 and ERK1/2 in CHO cells were blocked by pertussis toxin pretreatment and were sensitive to pharmacological inhibition of PI3K . In addition, inhibition of Rac/Cdc42, small GTPases of the Rho family, by clostridium toxin B, diminished p38 phosphorylation but did not affect ERK1/2 . Furthermore, we identified the serine 727 site of signal transducer and activator of transcription STAT3 as a probable downstream target of ERK1/2, and thereby provide evidence that adenosine A(3) receptor mediated ERK1/2 activation has functional consequences. J Vet Diagn Invest, 2003 Nov, 15(6), 523 - 6 Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows; Moeller RB Jr et al.; Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay {ELISA} for type C botulinum toxin) . Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined . Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period . Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism . From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method . These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis . The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals. J Vet Med Sci, 2003 Nov, 65(11), 1249 - 51 Necrotic hepatitis due to Clostridium perfringens infection in newly hatched broiler chicks; Sasaki J et al.; Multiple necrotic hepatitis lesions of 5 newly hatched broiler chicks in three flocks derived from two hatcheries were examined pathologically . The livers were brittle, and multiple yellowish or green foci were scattered on the surface and cut surface . The main histological finding was well demarcated multi-focal necrosis in the liver . Many Gram-positive large bacilli that reacted positively with polyclonal anti-Clostridium perfringens serum were observed in necrotic areas. J Vet Med Sci, 2003 Nov, 65(11), 1245 - 7 Clostridium perfringens type A myonecrosis in a horse in Korea; Choi YK et al.; Acute hemorrhagic myonecrosis accompanied by severe inter- and intrafascicular edema and hemorrhage of the right gluteal area was diagnosed in a 13-year-old male thoroughbred horse . Once the muscular and fascicular changes were subsided, the horse then developed acute respiratory problem . Histologically, the lung had diffuse severe hemorrhage with mild neutrophilic infiltration . The cause of death was acute respiratory failure that is believed to occur secondary to toxaemic event . Alpha and beta2 toxin secreting Clostiridum perfringens type A was isolated from the muscle and lung . The diagnosis was based on the light microscopic examination, bacterial toxinotyping and toxin genotyping from the muscular and pulmonary lesion . Also, susceptibility of the isolates to antimicrobial agents was determined. Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15422 - 7 Epub 2003 Dec 08. Trans-editing of mischarged tRNAs; Ahel I et al.; Aminoacyl-tRNA synthetases (aaRSs) are multidomain proteins that specifically attach amino acids to their cognate tRNAs . Their most conserved, and presumably evolutionarily oldest, domains are the catalytic cores, which activate amino acids and transfer them to the 3' ends of tRNAs . Additional domains appended to or inserted in the body of aaRSs increase efficiency and specificity of the aminoacylation process, either by providing additional tRNA contacts, or by hydrolyzing noncognate amino acid products (cis-editing) . Here, we report specific tRNA-dependent trans-editing by aaRS-like proteins that reciprocate the editing domains of aaRSs, but not the remainder of the corresponding enzyme . A freestanding homologue of the prolyl-tRNA synthetase-editing domain, the PrdX protein from Clostridium sticklandii, efficiently and specifically hydrolyzes Ala-tRNAPro . Similarly, autonomous alanyl-tRNA synthetase-editing domain homologues (AlaX proteins) from Methanosarcina barkeri and Sulfolobus solfataricus hydrolyze Ser-tRNAAla and Gly-tRNAAla substrates . The discovery of autonomous editing proteins efficient in hydrolyzing misacylated products provides a direct link between ancestral aaRSs consisting solely of the catalytic core and extant enzymes to which functionally independent modules are appended. Microbiology, 2003 Dec, 149(Pt 12), 3361 - 70 Structural analysis by X-ray crystallography and calorimetry of a haemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum; Inoue K et al.; Botulism food poisoning is caused primarily by ingestion of the Clostridium botulinum neurotoxin (BoNT) . The 1300 amino acid BoNT forms a progenitor toxin (PTX) that, when associated with a number of other proteins, increases its oral toxicity by protecting it from the low pH of the stomach and from intestinal proteases . One of these associated proteins, HA1, has also been suggested to be involved with internalization of the toxin into the bloodstream by binding to oligosaccharides lining the intestine . Here is reported the crystal structure of HA1 from type C Clostridium botulinum at a resolution of 1.7 Angstrom . The protein consists of two beta-trefoil domains and bears structural similarities to the lectin B-chain from the deadly plant toxin ricin . Based on structural comparison to the ricin B-chain lactose-binding sites, residues of type A HA1 were selected and mutated . The D263A and N285A mutants lost the ability to bind carbohydrates containing galactose moieties, implicating these residues in carbohydrate binding. J Biol Chem, 2004 Feb 20, 279(8), 7208 - 12 Epub 2003 Dec 01. A Rho exchange factor mediates fMet-Leu-Phe-induced NF-kappaB activation in human peripheral blood monocytes; Chen LY et al.; We reported previously that fMLP stimulates NF-kappaB activation, and this function of fMLP requires small GTPase RhoA in human peripheral blood monocytes (Huang, S., Chen, L.-Y., Zuraw, B . L., Ye, R . D., and Pan, Z . K . (2001) J . Biol . Chem . 276, 40977-40981) . Here we present evidence that RhoA associates specifically with the guanine nucleotide exchange factor Lbc in human peripheral blood monocytes stimulated with fMLP and that Lbc specifically catalyzes the guanine nucleotide exchange activity of RhoA in human peripheral blood monocytes . Cotransfection of the monocytic THP1 cells with lbc with a kappaB promoter reporter plasmid results in a marked increase in NF-kappaB-mediated reporter gene expression . Finally, Lbc-enhanced NF-kappaB activation is inhibited by a RhoA inhibitor, C3 transferase from Clostridium botulinum . A dominant-negative form of RhoA (T19N) also inhibited Lbc-enhanced reporter gene expression in a kappaB-dependent manner . These results indicate that guanine nucleotide exchange factor Lbc is a novel signal transducer for RhoA-mediated NF-kappaB activation in human peripheral blood monocytes stimulated with bacterial products. Appl Environ Microbiol, 2003 Dec, 69(12), 6969 - 78 Cloning, expression, and cell surface localization of Paenibacillus sp . strain W-61 xylanase 5, a multidomain xylanase; Ito Y et al.; We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa . Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp . strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5 . xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence . Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains . Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101 . Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa . Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer . Thus, Paenibacillus sp . strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains . Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5. FEMS Microbiol Lett, 2003 Dec 5, 229(1), 103 - 10 Gene transfer into Clostridium difficile CD630 and characterisation of its methylase genes; Herbert M et al.; Ignorance of pathogenesis in Clostridium difficile may be attributable to a lack of effective genetic tools . We have now shown that oriT-based shuttle vectors may be conjugated from Escherichia coli donors to the C . difficile strain CD630, at frequencies of around 10(-6) transconjugants per donor cell . Transfer is unaffected by either sequences present on the vector or its methylation status . Whilst the genome of this strain carries five methylase genes, there is no in silico or experimental evidence for cognate restriction enzymes . It would seem that the identified methylases do not participate in restriction-modification, and must, therefore, fulfil another role . A similar situation most likely applies to other clostridia. Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 15083 - 8 Epub 2003 Dec 01. Overcoming the hypoxic barrier to radiation therapy with anaerobic bacteria; Bettegowda C et al.; The low level of oxygenation within tumors is a major cause of radiation treatment failures . We theorized that anaerobic bacteria that can selectively destroy the hypoxic regions of tumors would enhance the effects of radiation . To test this hypothesis, we used spores of Clostridium novyi-NT to treat transplanted tumors in mice . The bacteria were found to markedly improve the efficacy of radiotherapy in several of the mouse models tested . Enhancement was noted with external beam radiation derived from a Cs-137 source, systemic radioimmunotherapy with an I-131-conjugated monoclonal antibody, and a previously undescribed form of experimental brachytherapy using plaques loaded with I-125 seeds . C . novyi-NT spores added little toxicity to the radiotherapeutic regimens, and the combination resulted in long-term remissions in a significant fraction of animals. MMWR Morb Mortal Wkly Rep, 2003 Dec 5, 52(48), 1176 - 9 Clostridial endophthalmitis after cornea transplantation--Florida, 2003; Centers for Disease Control and Prevention (CDC); Endophthalmitis is a severe condition caused by inflammation of the ocular cavity that often is associated with infection of the internal structures of the eye . The source of infection can include bacteria disseminated through the bloodstream and contamination of the cornea at the time of ocular surgery or trauma . Complications include rapid, reversible vision loss that can progress quickly to panophthalmitis, requiring surgical removal of the eye . Clostridium perfringens, an anaerobic gram-positive bacillus found in soil and bowel flora, is an infrequent cause of endophthalmitis . Although the majority of cases are caused by penetrating injury with soil-contaminated foreign bodies, C . perfringens endophthalmitis has been reported in patients after cataract surgery . This report describes two cases of C . perfringens endophthalmitis that occurred within 24 hours after transplant of contaminated corneas . These cases demonstrate the potential for transmission of Clostridium infection from donor to recipient . Clinicians should be aware of potential infection risks associated with transplantation of corneal tissues and report any infections to the appropriate eye bank. Eur J Biochem, 2003 Dec, 270(24), 4962 - 72 A 49 kDa microtubule cross-linking protein from Artemia franciscana is a coenzyme A-transferase; Oulton MM et al.; Embryos and larvae of the brine shrimp, Artemia franciscana, were shown previously to possess a protein, now termed p49, which cross-links microtubules in vitro . Molecular characteristics of p49 were described, but the protein's identity and its role in the cell were not determined . Degenerate oligonucleotide primers designed on the basis of peptide sequence obtained by Edman degradation during this study were used to generate p49 cDNAs by RT-PCR and these were cloned and sequenced . Comparison with archived sequences revealed that the deduced amino acid sequence of p49 resembled the Drosophila gene product CG7920, as well as related proteins encoded in the genomes of Anopheles and Caenorhabditis . Similar proteins exist in several bacteria but no evident homologues were found in vertebrates and plants, and only very distant homologues resided in yeast . When evolutionary relationships were compared, p49 and the homologues from Drosophila, Anopheles and Caenorhabditis formed a distinct subcluster within phylogenetic trees . Additionally, the predicted secondary structures of p49, 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum and glutaconate CoA-transferase from Acidaminococcus fermentans were similar and the enzymes may possess related catalytic mechanisms . The purified Artemia protein exhibited 4-hydroxybutyrate CoA-transferase activity, thereby establishing p49 as the first crustacean CoA-transferase to be characterized . Probing of Western blots with an antibody against p49 revealed a cross-reactive protein in Drosophila that associated with microtubules, but to a lesser extent than did p49 from Artemia. Eur Arch Otorhinolaryngol, 2004 Sep, 261(8), 456 - 8 Epub 2003 Dec 03. Thyroid abscess--an acute emergency; Kale SU et al.; An abscess in a long-standing goitre is usually life threatening . A patient with a large goitre was lost to follow-up for about 10 years and presented with a large thyroid abscess causing acute respiratory obstruction . The abscess caused erosion of the tracheal wall and perforation of the esophagus . Clostridium septicum was the causative organism, which is almost always associated with cancer of the colon . This led to the detection of colonic malignancy in this patient on further investigations . We describe the complications of a thyroid abscess and discuss the pathophysiology and treatment. Dtsch Med Wochenschr, 2003 Nov 28, 128(48), 2531 - 3 {Fungemia after oral treatment with Saccharomyces boulardii in a patient with multiple comorbidities}; Lestin F et al.; HISTORY AND CLINICAL FINDINGS: A 48-year-old diabetic with multiple co-morbidities presented with generalized micro- and macroangiopathy including peripheral artery disease stage IV with necroses in several digits of both feet . He was admitted to the department of surgery for the insertion of femoropopliteal bypasses . INVESTIGATIONS: Infectious parameters were elevated (CRP 66.1 mg/l, sedimentation rate 90/96), accompanied by anemia (Hb 7.1 mmol/l), leukocytosis (14.8 Gpt/l) and thrombocytosis (514 Gpt/l) . Body temperature was normal (36.8 degrees C) . With insulin treatment the patient became nearly normoglycemic (HbA1c 6.8 %) . TREATMENT AND FOLLOW UP: After receiving different broad-spectrum antibiotics over seven weeks the patient developed Clostridium difficile toxin-positive diarrhea that resolved after administration of oral metronidazole and Saccharomyces boulardii (Perenterol ((R))) . Three days after bypass insertion, both legs had to be amputated due to infection and beginning sepsis . The condition of the patient improved . However, eight days after bypass-insertion the patient developed a toxic megacolon and sepsis . Blood cultures yielded the growth of Saccharomyces cerevisae . Despite of intensive care treatment the patient died five days later from to multi-organ failure . CONCLUSION: S . boulardii (synonym: S . cerevisiae) is considered an non-pathogenic probiotic yeast, and live yeast cells are used for supportive therapy of diarrhea . The present case and a review of the literature demonstrate that fungemia and sepsis are rare complications of the administration of S . boulardii in immunocompromised patients . For this reason the therapeutic usage of probiotics should be carefully considered regarding its risk-benefit potential. J Pediatr (Rio J), 2000 Sep-Oct, 76(5), 391 - 4 {Neonatal tetanus}; Guardiola A et al.; OBJECTIVE: Neonatal tetanus is an infection with high mortality, constituting a problem in underdeveloped countries, where there is faulty prenatal attendance . In spite of this and of the decreasing incidence of the disease in the state of Rio Grande do Sul, we report a case of neonatal tetanus identified in 1997 . METHOD: We interviewed the infantacute;s mother and verified her vaccination history . We conducted a clinical examination of the newborn, anaerobic culture of umbilical stump and review of medical records . We also reviewed the literature and verified the epidemic profile of neonatal tetanus in Rio Grande do Sul.RESULTS: Healthy mother, with complete outline of tetanus vaccination 4 years ago, prenatal uncomplicated and hospital delivery . Family of good socioeconomic level, differing from the other cases notified in the State, in that most did not have prenatal care nor hospital delivery . The patient is a boy . In the 24th day of life he presented fever, cyanosis, lower and upper limbs muscle hypertonia and opisthotonos episodes . An anaerobic culture of the umbilical stump grew Clostridium tetani . He was treated with penicillin G, gentamicin, sedation and tetanus immunoglobulin . He left hospital in good conditions.CONCLUSIONS: Faulty prenatal care in underdeveloped countries is the largest risk factor for high incidence of this disease in these areas . However, in pregnant women with good socioeconomic level and prenatal care, as in the case described here, this diagnosis should not be excluded if there are suggestive signs of it. J Med Assoc Thai, 2003 Oct, 86(10), 970 - 5 Detection of Clostridium difficile toxin A and B genes from stool samples of Thai diarrheal patients by polymerase chain reaction technique; Wongwanich S et al.; The prevalence of Clostridium difficile isolated from stools of Thai adult patients with suspected antibiotic-associated diarrhea (AAD) was 18.64 per cent . The recovery rate of toxin genes (tcdA and tcdB) by polymerase chain reaction (PCR) from stool samples yielded almost the same compared to the recovery rate of the toxin detection by enzyme immunoassay (EIA), which were 44.9 per cent and 46.7 per cent, respectively . Correlation of toxin gene detection by PCR and toxin detection by EIA was 90.6 per cent . All but one stool sample, the tcdA gene was detected together with the tcdB gene . Both genes were always detected together from tox gene-positive strains . Although, there were some discrepancy results for certain samples, the direct PCR-based-detection of C . difficile tox genes in stool samples seems to be the appropriate method for the diagnosis of C . difficile diarrhea . The PCR assay should be a recommended technique to be used routinely in laboratories . Further optimization of the technique to increase the sensitivity of the PCR assays is still needed . However, a quantitative isolation of the organism from stools of suspected antibiotic-associated diarrhea (AAD) or antibiotic-associated colitis (AAC) patients may give some evidence for clinicians in hospitals who cannot perform PCR-based or EIA-based techniques, since 48.6 per cent of the isolates were demonstrated as toxigenic strains. Equine Vet J, 2003 Nov, 35(7), 638 - 41 Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth; Weese JS et al.; REASONS FOR PERFORMING STUDY: Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses . Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use . OBJECTIVES: To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C . perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro . METHODS: Clostridium difficile toxins, C . perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests . Serial dilutions of smectite were tested for the ability to inhibit growth of C . perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C . perfringens in culture broth . RESULTS: Clostridium difficile toxins A and B, and C . perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16 . Partial binding of C . difficile toxins occurred at dilutions up to 1:256 while partial binding of C . perfringens enterotoxin occurred up to a dilution of 1:128 . Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32 . No inhibition of growth of C . difficile or C . perfringens was present at any dilution, and there was no effect on the action of metronidazole . CONCLUSIONS: Di-tri-octahedral smectite possesses the ability to bind C . difficile toxins A and B, C . perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole . POTENTIAL RELEVANCE: In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colitis and endotoxaemia in horses. Vet Ophthalmol, 2003 Dec, 6(4), 279 - 84 Normal ocular features, conjunctival microflora and intraocular pressure in the Canadian beaver (Castor canadensis); Cullen CL; OBJECTIVE: The aim of the study was to assess the ocular features, normal conjunctival bacterial and fungal flora, and intraocular pressure (IOP) in the Canadian beaver (Castor canadensis) . SAMPLE POPULATION: Sixteen, apparently healthy beavers with no evidence of ocular disease, and live-trapped in regions throughout Prince Edward Island . PROCEDURES: The beavers were sedated with intramuscular ketamine (12-15 mg/kg) . Two culture specimens were obtained from the ventral conjunctival sac of both eyes of 10/16 beavers for aerobic and anaerobic bacterial and fungal identifications . The anterior ocular structures of all beavers were evaluated using a transilluminator and slit lamp biomicroscope . Palpebral fissure length (11/16 beavers), and horizontal and vertical corneal diameters (10/16 beavers) were measured . IOPs were measured in both eyes of 11/16 beavers using applanation tonometry . Both eyes of 3/16 beavers and one eye of 1/16 beavers were dilated using topical tropicamide prior to sedation to effect timely maximal dilation . Culture specimens and IOPs were not evaluated in these four animals . Indirect ophthalmoscopy was performed on 7/8 eyes of these four beavers . RESULTS: Conjunctival specimens from all eyes cultured positively for one or more isolates of aerobic bacteria . The most common isolate was Micrococcus spp . (five beavers; 9/20 eyes) . Other isolates included a Gram-positive coccobacilli-like organism (four beavers; 7/20 eyes), Aeromonas hydrophila (three beavers; 4/20 eyes), Staphylococcus spp . (three beavers; 4/20 eyes), Gram positive bacilli (one beaver; 2/20 eyes), Enterobacter spp . (two beavers; 2/20 eyes), Streptococcus spp . (two beavers; 2/20 eyes), aerobic diphtheroids (one beaver; 1/20 eyes), and Pseudomonas spp . (one beaver; 1/20 eyes) . Clostridium sordellii (one beaver; 1/20 eyes) and Peptostreptococcus spp . (one beaver; 1/20 eyes) were the sole anaerobic bacteria isolated . All conjunctival specimens were negative for growth of fungi . Ophthalmic examinations revealed the normal beaver eye and ocular adnexa included dorsal and ventral puncta, a vestigial third eyelid, and a circular pupil . Average palpebral fissure length was 9.36 mm (SD = 1.00) for both eyes . Mean horizontal and vertical corneal diameters of both eyes were 9.05 mm (SD = 0.64) and 8.45 mm (SD = 0.69), respectively . Mean IOP for the right and left eyes were 17.11 mmHg (SD = 6.39) and 18.79 mmHg (SD = 5.63), respectively . Indirect ophthalmoscopic examinations revealed normal anangiotic retinas . CONCLUSIONS: Gram-positive aerobes were most commonly cultured from the conjunctival sac of normal beavers, with Micrococcus spp . predominating . The overall mean IOP in ketamine-sedated beavers was 17.95 mmHg . The beaver, an amphibious rodent, has an anangiotic retina. Environ Microbiol, 2003 Nov, 5(11), 1212 - 20 Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche; Nelson KE et al.; At present, there is little information on the phylogenetic diversity of microbial species that inhabit the gastrointestinal tracts of wildlife . To increase understanding in this area, we initiated a characterization of the bacterial diversity in the digestive tracts of three wild African ruminant species namely eland (Taurotragus oryx), Thompson's gazelle (Gazella rufifrons) and Grant's gazelle (Gazella granti), together with a domesticated ruminant species, zebu cattle (Bos indicus), and a non-ruminant species, zebra (Equus quagga) . Bacterial diversity was analysed by PCR amplification, sequencing and phylogenetic analysis of 16S ribosomal DNA (rDNA) sequences . A total of 252 full-length 16S rDNA sequences averaging 1,500 base pairs (bp) in length, and an additional 27 partial sequences were obtained and subject to phylogenetic analysis . Using a 98% criterion for similarity, all except for one of the sequences were derived from distinct phylotypes . At least 24 distinct operational taxonomic units (OTU's) could be identified, with the majority of these sequences representing hitherto uncharacterized species and genera . The sequences were generally affiliated with four major bacterial phyla, the majority being members of the Firmicutes (low G+C Gram-positives) related to the genera Clostridium and Ruminococcus . By contrast, with earlier studies using 16S rDNA sequences to assess biodiversity in Bos taurus dairy cattle, Gram-negative bacteria in the Bacteroidales (Prevotella-Bacteroides group) were poorly represented . The lack of redundancy in the 16S rDNA dataset from the five African ungulate species, and the presence of novel sequences not previously described from the gastrointestinal tract of any animal species, highlights the level of diversity that exists in these ecosystems and raises the question as to the functional role of these species in the gastrointestinal tract. Cell Microbiol, 2003 Dec, 5(12), 957 - 71 Activation of NF-kappaB and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascades; Grassl GA et al.; Yersinia enterocolitica triggers activation of the nuclear factor (NF)-kappaB and production of the proinflammatory chemokine interleukin (IL)-8 in intestinal epithelial cells . This activation is due to adhesion of the bacteria via their outer membrane protein invasin to the host cells . Using Clostridium difficile toxins that specifically inactivate small GTPases, and transfection of inhibitory proteins of the Rho-GTPases, we demonstrate that Rac1, but not Cdc42 or Rho, is required for activation of NF-kappaB by invasin . Invasin activated the mitogen activated protein kinases (MAPK) p38 and c-Jun N-terminal protein kinase (JNK) but not extracellular signal regulated kinase (ERK) . The functional relevance of these pathways for invasin-mediated IL-8 expression was assessed by protein kinase inhibitors and dominant-negative kinase mutants . While NF-kappaB and JNK contribute to IL-8 transcription, p38 MAPK also acts through stabilization of IL-8 mRNA, as confirmed by quantitative RT-PCR and electrophoretic mobility shift assays . Transfection experiments with I-kappaB kinase (IKK)1 and IKK2 mutants indicate that the release of NF-kappaB from its cytoplasmic inhibitor I-kappaB and its translocation into the nucleus is mediated by these kinases . Our data identify Rac1 as a key intermediate in invasin-triggered IL-8 synthesis and demonstrate that maximum IL-8 induction involves several MAP kinase cascades. Nucleic Acids Res, 2003 Dec 1, 31(23), 6748 - 57 Regulation of lysine biosynthesis and transport genes in bacteria: yet another RNA riboswitch? Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS. Comparative analysis of genes, operons and regulatory elements was applied to the lysine biosynthetic pathway in available bacterial genomes . We report identification of a lysine-specific RNA element, named the LYS element, in the regulatory regions of bacterial genes involved in biosynthesis and transport of lysine . Similarly to the previously described RNA regulatory elements for three vitamins (riboflavin, thiamin and cobalamin), purine and methionine regulons, this regulatory RNA structure is highly conserved on the sequence and structural levels . The LYS element includes regions of lysine-constitutive mutations previously identified in Escherichia coli and Bacillus subtilis . A possible mechanism of the lysine-specific riboswitch is similar to the previously defined mechanisms for the other metabolite-specific riboswitches and involves either transcriptional or translational attenuation in various groups of bacteria . Identification of LYS elements in Gram-negative gamma-proteobacteria, Gram-positive bacteria from the Bacillus/Clostridium group, and Thermotogales resulted in description of the previously uncharacterized lysine regulon in these bacterial species . Positional analysis of LYS elements led to identification of a number of new candidate lysine transporters, namely LysW, YvsH and LysXY . Finally, the most likely candidates for genes of lysine biosynthesis missing in Gram- positive bacteria were identified using the genome context analysis. J Food Prot, 2003 Nov, 66(11), 2171 - 5 Jerusalem artichokes stimulate growth of broiler chickens and protect them against endotoxins and potential cecal pathogens; Kleessen B et al.; Control of intestinal pathogens during the earliest phases of broiler production may be the best strategy for the reduction of human pathogens on processed broiler carcasses . The recent ban on antibiotics in poultry feed has served to focus much attention on alternative methods of controlling the gastrointestinal microflora . A field trial was conducted to evaluate the effect of the fructan-rich Jerusalem artichoke, or topinambur (administered as 0.5% topinambur syrup in drinking water), on cultural numbers of selected cecal bacteria (total aerobes, Enterobacteriaceae, Bdellovibrio spp., and Clostridium perfringens) and levels of bacterial endotoxins as well as on body weights and relative weights of organs (the pancreas and the bursa of Fabricius) of chickens in the first 35 days of life (with weekly investigations being conducted) . One-day-old broiler chickens (Ross 308) were randomly assigned to experimental (with topinambur) and control (without topinambur) groups . They were allowed free access to a standard broiler diet without growth-promoting antibiotics . Topinambur treatment resulted in a significant increase (P < 0.01) in cecal counts of B . bacteriovorus, which parasitizes susceptible gram-negative pathogens . Topinambur led to significantly smaller numbers of total aerobes, Enterobacteriaceae, and C . perfringens as well as to reduced levels of endotoxins in the blood compared with those for control birds . Increased body weights resulting from topinambur consumption were observed on day 35 of the trial period (P < 0.05) . The relative weights of the pancreas and the bursa of Fabricius, however, were higher (P < 0.05) for topinambur-treated broilers than for control birds at the ages of 14, 21, 28, and 35 days . These results indicate that a small amount of topinambur in broilers' drinking water has a beneficial effect on growth performance, reduces bacterial endotoxin levels, and suppresses potential pathogens in broilers' ceca. Ann Transplant, 2003, 8(2), 34 - 42 Peracetic acid-ethanol treatment of allogeneic avital bone tissue transplants--a reliable sterilization method; Pruss A et al.; OBJECTIVES: Based on the European Standard EN 1040, the validation guidelines of the German Federal Institute for Drugs and Medical Devices and CPMP guidelines we tested the antimicrobial effectiveness of a peracetic acid-ethanol sterilization procedure (PES) in allogenic avital bone transplants . STUDY DESIGN: Delipidated human bone spongiosa cubes (15 x 15 x 15 mm) served as tissue . Three enveloped viruses (human immunodeficiency virus type 2, pseudorabies virus, bovine virus diarrhoea virus) and three non-enveloped viruses (hepatitis A virus, poliovirus, porcine parvovirus) were used . The reduction of virus infectivity was measured as TCID50/ml in neutralized supernatants and bone homogenates . Staphylococcus aureus . Enterococcus faecium, Pseudomonas aeruginosa . Bacillus subtilis . Clostridium sporogenes, Mycobacterium terrae . Candida albicans, Aspergillus niger as well as spores of Bacillus subtilis were tested additionally . PES led to a reduction of virus titres by more than 4 log10 . Only HAV showed a reduction below 4 log10 (2.87) with residual infectivity . After including a delipidating step for HAV-infected cells, a reduction of over 7 log10 HAV titre was found . For viable bacteria, fungi and spores a titre reduction below the detection level (5 log10) was achieved after an incubation time of 2 hours . CONCLUSIONS: The peracetic acid-ethanol procedure proved to be a reliable method for the sterilization of human bone transplants (layer thickness < or = 15 mm) . However, additional safety measures (anamnestic informations, infectious serology, HIV-/HBV-/HCV-PCR in case of multiorgan donors) should be taken. Extremophiles . 2003 Nov 19; {Epub ahead of print} Pressure effects on Clostridium strains isolated from a cold deep-sea environment; Lauro FM et al.; Three Clostridium strains were isolated from deep-sea sediments collected at a depth of 6.3-7.3 km in the Japan Trench . Physiological characterization and 16S rDNA analysis revealed that the three isolates were all closely related to Clostridium bifermentans . The spores of all three isolates were resistant to inactivation at high pressure and low temperature . However, despite the fact that the vegetative cells were halotolerant and eurythermal they did not appear to be adapted for growth or viability under the conditions prevailing in the deep-sea sediments from which they were obtained . The results suggest that the isolates had survived as spores in the deep-sea sediments and that the marine benthos could be a source of clostridia originating in other environments. Biologicals, 2003 Dec, 31(4), 287 - 94 Validation of the 'Marburg bone bank system' for thermodisinfection of allogenic femoral head transplants using selected bacteria, fungi, and spores; Pruss A et al.; The Marburg Bone Bank System 'Lobator sd-2' is widely used to process human femoral heads removed during aseptic surgery by thermal disinfection . The inactivating capacity of the thermodisinfection system was validated in compliance with current standards using a newly developed femoral head model . The following micro-organisms, bacteria and fungi, taken from the American Type Culture Collection were investigated: Staphylococcus aureus, Staphyloccus epidermidis, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis including spores, Clostridium sporogenes, Mycobacterium terrae, Candida albicans and Aspergillus niger spores . Highly enriched suspensions of these micro-organisms were applied to the centre of the femoral heads . The reduction in the number of micro-organisms was determined by counting the colony-forming units (cfu) before and after processing the spiked test device in the 'Lobator sd-2' system.Vegetative bacteria, fungi and fungal spores were completely inactivated (reduction factor >/=6 log(10)) . The numbers of B . subtilis and C . sporogenes spores, both known to be heat-resistant, were reduced by one to two orders of magnitude . These bacteria serve as a model for spore forming pathogens which are not relevant in femoral heads from living donors . By processing human femoral heads from living donors by thermal disinfection using the Marburg Bone Banking system, a high level of safety is achieved regarding clinically relevant pathogens . To further increase the safety of the thermally treated femoral heads, we recommend that the medical history and present state of the donor, as well as the necessary serological tests should be taken into account. Arch Microbiol, 2004 Jan, 181(1), 45 - 51 Epub 2003 Nov 18. Identification and characterization of the cytoplasmic tungstate/molybdate-binding protein (Mop) from Eubacterium acidaminophilum; Makdessi K et al.; The mop gene, encoding the molybdate-binding protein from Eubacterium acidaminophilum, was cloned using Clostridium pasteurianum mopI as a probe for heterologous hybridization . mop encodes a 69-amino-acid protein ( M(r) 7,328) with high sequence similarities to members of the molbindin protein family, which have been implicated in molybdenum storage and homeostasis . Northern blot analysis showed three mRNA transcripts (1.0, 1.6, and 2.6 kb) for mop . This result was obtained independent of the availability of tungstate in the growth medium . mop was overexpressed in Escherichia coli as a C-terminal Strep-tag fusion protein . On the basis of gel filtration, the native protein was a homohexamer of 48 kDa . The specificity of oxyanion binding was examined by protein mobility shift assay . Molybdate, tungstate, and chromate strongly changed the mobility of the protein in a native polyacrylamide gel, indicating the binding of these oxyanions to Mop . Other oxyanions, such as sulfate and phosphate, had no effect on Mop mobility . Mutational analysis revealed that the positive charge of the Arg-6, located in the conserved SARN region of Mop, was not directly involved in oxyanion binding. Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13809 - 14 Epub 2003 Nov 17. Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex; Carvalho AL et al.; The utilization of organized supramolecular assemblies to exploit the synergistic interactions afforded by close proximity, both for enzymatic synthesis and for the degradation of recalcitrant substrates, is an emerging theme in cellular biology . Anaerobic bacteria harness a multiprotein complex, termed the "cellulosome," for efficient degradation of the plant cell wall . This megadalton catalytic machine organizes an enzymatic consortium on a multifaceted molecular scaffold whose "cohesin" domains interact with corresponding "dockerin" domains of the enzymes . Here we report the structure of the cohesin-dockerin complex from Clostridium thermocellum at 2.2-A resolution . The data show that the beta-sheet cohesin domain interacts predominantly with one of the helices of the dockerin . Whereas the structure of the cohesin remains essentially unchanged, the loop-helix-helix-loop-helix motif of the dockerin undergoes conformational change and ordering compared with its solution structure, although the classical 12-residue EF-hand coordination to two calcium ions is maintained . Significantly, internal sequence duplication within the dockerin is manifested in near-perfect internal twofold symmetry, suggesting that both "halves" of the dockerin may interact with cohesins in a similar manner, thus providing a higher level of structure to the cellulosome and possibly explaining the presence of "polycellulosomes." The structure provides an explanation for the lack of cross-species recognition between cohesin-dockerin pairs and thus provides a blueprint for the rational design, construction, and exploitation of these catalytic assemblies. Int J Food Microbiol, 2003 Dec 31, 89(2-3), 251 - 63 Combined effects of ionizing-irradiation and different environments on Clostridium botulinum type E spores; Lim YH et al.; We examined the combined effects of gamma-radiation (24 degrees C) on spores of Clostridium botulinum-type Eklund strain suspended in different gas-saturated Na-phosphate buffer in absence or presence of protectors or sensitizers . Response surface methodology (RSM) was also used to ascertain the effects of radiation on the recovery of spores using a medium containing various levels of NaCl or Na-thioglycollate . The former (< 0.5%) decreased viable spore counts, but the latter (0.15%) did not . Irradiation inactivation of Eklund spores was most effective in air-saturated buffers compared to N2O and N2 gas . The Na2-EDTA (0.01 M) was the most efficient radioprotector of spores due to its reactivity toward hydroxy radicals, followed by t-butanol (0.1 M) in NO2 or N(2)-saturated buffers, respectively . Catalase (10.0 mg ml(-1)) and DL-cysteine (0.1 mM) sensitized the spores during irradiated N2O or N(2)-saturated buffers, and NaCl (0.01 M) only sensitized spores in N2 environment . Spores frozen at -75 degrees C for 30 days and thawed prior to use were more sensitive to radiation damage compared to freshly prepared spores . Glycerol (15%), in Na-phosphate buffer (pH 7.0, 0.06 M), protected Eklund spores and increased the number of spores from 10(6) to 10(11) colony forming unit (CFU) ml(-1), and enhanced their radiosensitivities . Seven strains of C . botulinum type E were screened for plasmids and strain BL764 had two plasmids (15.8 and 46.8 mDa), BL4028 also had two (4.4 and 13.2 mDa), BL4850 contained only one (4.9 mDa), whereas EQA, BL211, Eklund, and Beluga had none . Gamma-Radiation (10 kGy, absorbed dose) cured the 15.8-mDa plasmid in strain BL764, but its absence yielded no changes in toxigenicity. Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 1067 - 71 SDF-1alpha-induced intracellular calcium transient involves Rho GTPase signalling and is required for migration of hematopoietic progenitor cells; Henschler R et al.; Signalling through the chemokine stromal derived factor (SDF)-1alpha and its receptor CXCR4 has been recognized as a key event in the migratory respons