Am J Ophthalmol, 2001 Jul, 132(1), 116 - 7 Post-traumatic endophthalmitis involving Clostridium tetani and Bacillus spp; Iyer MN et al.; PURPOSE: To report a case of post-traumatic infectious endophthalmitis caused by Clostridium tetani and Bacillus spp . METHODS: Case report . RESULTS: A 25-year-old man developed endophthalmitis after a traumatic corneoscleral laceration of his right eye by a concrete reinforcement bar . He underwent pars plana lensectomy and vitrectomy with aspiration of vitreous fluid and a conjunctival swab for cultures . Cultures from the conjunctival swab were negative for organisms . Cultures of the vitreous aspirate were positive for Bacillus species and C . tetani . He had received a tetanus toxoid booster at the emergency department . By the time the culture results became available, he had developed severe eye pain associated with marked orbital congestion, increased swelling and erythema of the lids, marked injection and chemosis of the conjunctiva, and subsequently underwent evisceration . The inflammation resolved after evisceration of the right eye, and he was discharged to home on doxycycline 100 mg orally two times daily for 10 days . CONCLUSION: We are unaware of previous reports of endophthalmitis involving C tetani and could find none in a computerized MEDLINE search . Patients with penetrating eye injury should be assessed for tetanus immunization status, and early intervention with tetanus toxoid booster and/or tetanus immune globulin should be considered if cultures are positive. Ned Tijdschr Geneeskd, 2001 Jun 16, 145(24), 1137 - 40 {Diarrhea due to Clostridium difficile toxin in hemato-oncological patients}; Kerst JM et al.; In two patients with multiple myeloma, men aged 72 and 54 years, diarrhoea developed upon chemotherapy with vincristin, doxorubicin and dexamethasone (VAD) . In the second patient, diarrhoea developed after subsequent peripheral stem cell mobilisation . Pseudomembranous colitis was seen in the first patient during endoscopy but an enzyme immunoassay of the faeces was false negative for Clostridium difficile enterotoxin . The bacterium was later cultured from stool samples and toxins were detected in a repeated immunoassay . Stool samples of the second patient were positive for C . difficile enterotoxin . For both patients an antibiotic treatment resulted in a rapid recovery . In haemato-oncological patients, diarrhoea is often caused by oncolytic therapy . However, consideration should also be given to C . difficile infection as an alternative cause which is easily treatable. Med Sci Monit, 2001 Jul-Aug, 7(4), 751 - 4 Pseudomembranous colitis after eradication of Helicobacter pylori infection with a triple therapy; Harsch IA et al.; BACKGROUND: H.pylori (H.p.) infection of the gastric mucosa is causally related to chronic gastritis, peptic ulcer disease, MALT-lymphoma and gastric cancer . There is also an evidence for a link between the H.p.-infection and non-ulcer dyspepsia and even extragastric diseases . The number of patients treated against H.p . infection is expanding . Although in the last years the PPI-based triple therapies have been considered to be effective and safe, in some patients, however, severe side-effects may occur . CASE REPORT: We report on a 86 year old female patient, who received a one-week triple eradication therapy (metronidazole 3x400 mg/die, clarithromycin 2x250 mg/die and omeprazole 2x20 mg/die) because of non-ulcer dyspepsia . A few days after the eradication treatment, she developed profuse watery and bloody diarrhea and abdominal pain with distention . In stool specimens Clostridium difficile toxin was detected . A colonoscopy showed typical features of antibiotic associated pseudomembranous colitis . Until now, only few reports concerning this complication have been published and the frequency of the complication in patients eradicated for H.p . is unknown . The potential risk factors to develop this condition have not been clarified . Since the complication may be potentially lethal, this severe side-effect of the usually well-tolerated triple-therapy has to be considered in patients suffering from profuse diarrhea and abdominal pain after eradication therapy. Eur J Biochem, 2001 Jul, 268(13), 3831 - 9 Characterization and mutagenesis of the recombinant N-acetylneuraminate lyase from Clostridium perfringens: insights into the reaction mechanism; Kruger D et al.; The N-acetylneuraminate lyase from Clostridium perfringens was expressed in Escherichia coli as a fusion protein with a His-tag and purified to homogeneity using metal chelate affinity and anion exchange chromatography . The purified enzyme has a pH optimum of 7.6 and a temperature optimum of 65-70 degrees C . In kinetic studies the lyase exhibits a Km of 3.2 mM for Neu5Ac and a Vmax of 27.5 U x mg(-1) . To clarify the functional role of some putative active site residues, site-directed mutagenesis was performed . Lysine 161 was identified as the residue forming the Schiff base intermediate with the substrate . Tyrosine 133 was shown to be also a catalytically important residue; it seems to function as an acceptor for the proton of the C4 hydroxyl group, as already suggested by other groups . Furthermore, it is involved in stabilizing the Schiff base intermediate . Mutations of aspartate 187 and glutamate 188 indicate that both residues are involved in substrate binding . In this respect the carboxy group of aspartate 187 seems to be particularly important . Based on the results of these studies, a model of the reaction mechanism is discussed. FEMS Microbiol Lett, 2001 May 1, 198(2), 171 - 6 Variant toxin B and a functional toxin A produced by Clostridium difficile C34; Mehlig M et al.; A particular property of Clostridium difficile strain C34 is an insertion of approximately 2 kb in the tcdA-C34 gene that does not hinder expression of a fully active TcdA-C34 molecule . Intoxication with TcdA-C34 induced an arborized appearance in eukaryotic cells (D-type cytopathic effect); intoxication with TcdB-C34 induced a spindle-like appearance of cells (S-type cytopathic effect) . Inactivation of GTPases with purified toxins revealed that Rho, Rac, Cdc42, and Rap are substrates of TcdA-C34 . The variant cytotoxin TcdB-C34 inactivated Rho, Rac, Cdc42, Rap, Ral, and R-Ras . Hence, this is the first 'S-type' cytotoxin which inactivates both Rho and R-Ras, and is coexpressed with a 'D-type' enterotoxin . Our results support the hypothesis that R-Ras is a key GTPase related to the S-type cytopathic effect and suggest that induction of a S-type cytopathic effect dominates induction of the D-type cytopathic effect. Microbiology, 2001 Jul, 147(Pt 7), 1909 - 22 Physical and genetic map of the Clostridium saccharobutylicum (formerly Clostridium acetobutylicum) NCP 262 chromosome; Keis S et al.; A physical and genetic map of the Clostridium saccharobutylicum NCP 262 chromosome was constructed . The order of macrorestriction fragments was determined by analysing fragments generated after single and double digestion with the restriction enzymes BssHII, I-CeuI, Sse8387I, RsrII and SfiI and separation by PFGE . The I-CeuI backbone of C . saccharobutylicum was constructed by indirect end-labelling with rrs- and 3' rrl-specific probes located on either side of the I-CeuI site in the rrn operon, and reciprocal separation of BssHII and I-CeuI digestion products by two-dimensional PFGE . The positions of BssHII fragments on the physical map were determined using a library of linking clones containing BssHII cleavage sites . The size of the circular genome was estimated to be 5.3 Mb with a mean resolution of approximately 140 kb . The chromosome of C . saccharobutylicum contains 12 rrn operons, located on 46% of the chromosome, which are transcribed divergently from the deduced origin of replication . The genetic map was constructed by determining the location of 28 genes involved in house-keeping, heat-shock response, sporulation, electron transfer and acid- and solvent-formation . Comparison of the C . saccharobutylicum genetic map with those of the spore-forming bacteria Bacillus subtilis, Clostridium acetobutylicum, Clostridium perfringens and Clostridium beijerinckii indicated C . saccharobutylicum to be most similar to the latter two Clostridium species, with the order of the genes within the gyrAB and recA loci being conserved. Transpl Infect Dis, 1999 Jun, 1(2), 138 - 43 Infective endocarditis in renal transplant recipients; Bishara J et al.; Because of the increasing number of renal transplantations performed and the rarity of reported cases of infective endocarditis in these patients, we studied the clinical characteristics of this infection in this population . We report on two cases from our experience and review reported cases of infective endocarditis in renal transplant recipients retrieved from the MEDLINE system . In addition, we reviewed a large series of infective endocarditis looking for patients with renal transplants . In addition to our 2 cases, 12 previously reported cases were found . The mean time from transplantation to diagnosis of infective endocarditis was 3.5 years (range 2 months to 15 years) . Causative organisms included fungi, Staphylococcus aureus (3 cases each), Corynebacterium sp . (2 cases), Streptococcus viridans, VRE, Brucella sp., Clostridium sp., Nocardia sp . and Erysipelothrix sp . (one case each) . Skin manifestations of endocarditis and/or splenomegaly were not reported in these patients . Septic emboli and mycotic aneurysms were relatively common . The overall mortality rate was 50% (7 of 14 patients died) . Infective endocarditis seems to be rare in renal transplant recipients . The few reported cases are characterized by unusual causative micro-organisms and atypical clinical presentation . Further studies are needed to delineate the magnitude and scope of this association. J Hosp Infect, 2001 Jun, 48(2), 93 - 7 Antibiotic prophylaxis and the risk of Clostridium difficile-associated diarrhoea; Harbarth S et al.; To test the hypothesis that extended antibiotic prophylaxis increases the risk of Clostridium difficile -associated diarrhoea (CDAD), we conducted a retrospective cohort study of 2641 patients under-going cardiovascular surgery . Main outcome measures were the duration of prophylaxis (< 48 h vs . > 48 h) and the occurrence of CDAD . CDAD occurred in 31 patients (1.2%), who were significantly older (70 +/- 9 y vs . 66 +/- 10 y; P = 0.03), received more therapeutic antibiotics (2.2 +/- 1.9 vs . 0.4 +/- 0.9; P = 0.001) and had a longer postoperative hospital stay (26 +/- 19 d vs . 9 +/- 8 d; P < 0.001) than non-cases . After adjusting for confounding, we did not observe an association between prolonged prophylaxis and CDAD {adjusted odds ratio (AOR), 0.8; CI, 0.4-1.8} . In contrast, three independent predictors were identified: increasing length of hospital stay (AOR per one-day-increment, 1.03; CI, 1.01-1.05), and treatment with third generation cephalosporins (AOR, 5.9; CI, 2.2-16.0) or beta-lactam-beta-lactamase inhibitor combinations (AOR, 4.6; CI, 1.7-12.3) . Our results did not confirm that extended prophylaxis after clean surgery increases the risk of CDAD, which remains an uncommon postoperative complication, associated even with short antibiotic exposure . J Hosp Infect, 2001 Jun, 48(2), 86 - 92 Molecular analysis of relapse vs re-infection in HIV-positive patients suffering from recurrent Clostridium difficile associated diarrhoea; Alonso R et al.; Recurrence is a major complication of Clostridium difficile associated diarrhoea, especially in human immunodeficiency virus (HIV) positive patients, and it is important to distinguish between relapse and re-infection in recurrent episodes . The aim of our study was to analyse C . difficile isolates obtained from HIV-positive patients with recurrent diarrhoea in order to distinguish between relapse and re-infection . This analysis was based on the study of DNA similarities among isolates obtained from different episodes within each patient . Relapses occurred in 64% of patients, 32% suffered re-infections and a combination of relapse plus re-infection was seen in 4% . DNA typing methods can be useful tools to characterize recurrent episodes of C . difficile associated disease . Int J Food Microbiol, 2001 Jun 15, 66(3), 149 - 61 Antibacterial effect of protamine in combination with EDTA and refrigeration; Hansen LT et al.; The antimicrobial effect of protamine (clupeine) on a range of gram-positive and gram-negative foodborne pathogens and spoilage bacteria, was evaluated using an agar dilution assay and a broth dilution assay with Alamar Blue as growth indicator . Protamine was tested alone at concentrations from 0 to 10,000 microg/ml, and in combination with EDTA (0.9 mM) . Assays were performed at 5 degrees C, 10 degrees C, 18 degrees C and 30 degrees C to test the effect of temperature . Minimum inhibitory concentration (MIC) values ranged from 10 microg/ml for Brochothrix thermosphacta to no inhibition at 10,000 microg/ml for bacteria such as Aeromonas hydrophila, proteolytic strains of Clostridium botulinum, Hafnia alvei and Morganella morganii . The minimum bactericidal concentrations (MBCs) were generally higher than MICs . In combination with EDTA, MICs of protamine decreased for gram-negative test strains, whereas EDTA alone inhibited gram-positive strains . The effect of assay incubation temperature was variable and not clear for most strains . Concentrations of 100-750 microg/ml protamine inhibited the five non-proteolytic C . botulinum strains, while none of the eight proteolytic strains was inhibited, indicating the possible role of proteolytic enzymes in protecting cells from protamine . Clearing zones, indicative of proteolytic activity, were observed in the opaque TSB-agarose around colonies of some but not all protamine-resistant bacteria, suggesting that this is not the only resistance mechanism . Addition of 5% (w/v) gelatin to study the effect of an increased protein concentration in the agar dilution assay showed that electrostatic interactions between protamine and the protein decreased the antimicrobial efficacy of the peptide. J Zoo Wildl Med, 2000 Dec, 31(4), 558 - 62 Tyzzer's disease in a red panda (Ailurus fulgens fulgens); Langan J et al.; A debilitated 9-yr-old female red panda (Ailurus fulgens fulgens) with a recent history of corticosteroid administration displayed anorexia, depression, and diarrhea for 2 days . Blood work revealed a moderate nonregenerative anemia, leukocytosis, hypokalemia, hyperbilirubinemia, and mildly elevated alanine aminotransferase and aspartate aminotransferase . Serology was negative for occult heartworm, Toxoplasma gondii, feline leukemia virus, feline infectious peritonitis, feline immunodeficiency virus, and canine distemper virus . Electron microscopy of the feces demonstrated corona-like virus particles . The panda died 3 days after initial presentation . Histologic findings included multifocal, acute, hepatic necrosis and diffuse, necrotizing colitis . Liver and colon lesions contained intracellular, curved, spore-forming, gram-negative, silver-positive rods morphologically consistent with Clostridium piliforme . This panda most likely contracted Tyzzer's disease subsequent to having a compromised immune system after corticosteroid administration and concurrent disease. Appl Environ Microbiol, 2001 Jul, 67(7), 3201 - 7 Characterization of neutralizing antibodies and identification of neutralizing epitope mimics on the Clostridium botulinum neurotoxin type A; Wu HC et al.; Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death . In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge . The neutralizing activities for BT57-1 and BT150-3 were 10(3) and 10(4) times the 50% lethal dose, respectively . Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A . Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes . These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150 . The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically . This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1 . Aspartic acid (D(5)) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K(5)) . Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A . These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies . In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine. Vet Microbiol, 2001 Sep 3, 82(1), 39 - 43 PCR detection and prevalence of alpha-, beta-, beta 2-, epsilon-, iota- and enterotoxin genes in Clostridium perfringens isolated from lambs with clostridial dysentery; Gkiourtzidis K et al.; Clostridium perfringens isolated from lambs with dysentery (n=117) were analysed by a DNA amplification technique, the polymerase chain reaction (PCR), in order to determine the prevalence of the alpha-, beta-, beta 2-, epsilon-, iota- and enterotoxin genes . The most prevalent toxin type of C . perfringens found was type B, containing the alpha-, beta-, and epsilon-toxin genes, representing 46% of the cases with clostridial dysentery . C . perfringens type C containing the alpha-, and beta-toxin genes was isolated in 20% and type D, which is characterized by the alpha- and epsilon-toxin genes, was isolated in 28% of all isolates . The recently discovered, not yet assigned beta 2-toxigenic type of C . perfringens was represented in 6% of all isolates . No C . perfringens type A containing the alpha-toxin alone and no type E, which harbours the ADP-ribosylating iota-toxin, were found in the diseased animals . None of the samples contained the enterotoxin gene . Only one type of C . perfringens was found in a given herd, revealing the epidemiological use of PCR toxin gene typing of C . perfringens . The animals originated from 79 different herds with sizes ranging from 30 to 250 animals, bred in the area of northern Greece. Vet Microbiol, 2001 Sep 3, 82(1), 1 - 9 Clostridium perfringens beta toxin and Clostridium septicum alpha toxin: their mechanisms and possible role in pathogenesis; Tweten RK; The Clostridium septicum alpha toxin and the Clostridium perfringens beta toxin are examples of pore-forming toxins that exhibit several different features . The cell types that are targeted by these toxins reflect the effect these toxins have on the host during infection with either organism . Alpha toxin elicits a rapid shock-like syndrome, whereas beta toxin appears to induce a variety of neurological effects . The effects of the purified toxins appear to mimic some of the features of the animal and human diseases caused by C . septicum and C . perfringens . This review, examines the current state of knowledge for the cytolytic mechanism, role in pathogenesis and structure of these two toxins. Eur J Biochem, 2001 Jun, 268(12), 3538 - 44 In vitro processing of the proproteins GrdE of protein B of glycine reductase and PrdA of D-proline reductase from Clostridium sticklandii: formation of a pyruvoyl group from a cysteine residue; Bednarski B et al.; GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and D-proline reductase, respectively, that are processed post-translationally to form a catalytic active pyruvoyl group . The cleavage occurred on the N-terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety . Both proproteins could be over-expressed in Escherichia coli and conditions were developed for in vitro processing . GrdE could be expressed as full-size protein, whereas PrdA had to be truncated N-terminally to achieve successful over-expression . Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mM NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS . Cleavage of GrdE was observed with a half-time of approximately 30 min . Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site-directed mutagenesis . The Cys242-->Ser and Cys242-->Thr mutant proteins were also cleaved under similar conditions with extended half-times . However, the Cys242-->Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group-dependent reductases. Microbios, 2001, 105(412), 163 - 74 The effects of subminimal inhibitory concentrations of beta-lactam antibiotics against Clostridium perfringens; Kondo F et al.; The effects of subminimal inhibitory concentrations (sub-MIC) of four beta-lactam antibiotics {penicillin-G (PCG), ampicillin (AMP), cephaloridine (CER), cephalothin (CET)} were tested against Clostridium perfringens type A PB6K, after determining the minimum inhibitory concentrations (MIC) of 29 different Clostridium strains . The majority of the strains were sensitive to all beta-lactam antibiotics . Morphological changes, such as filamentous development and lysis, occurred at concentrations considerably lower than the MIC of CER and CET in C . perfringens . Clear cooperation of AMP and CER with rabbit polymorphonuclear leucocytes (PMNL) against C . perfringens was observed . The filamentous bacteria produced as a result of exposure to sub-MIC of each antibiotic, were phagocytosed easily . The ratios between the drug concentrations (microg/ml) at which the morphological changes began to occur, the minimum antibiotic concentrations (MAC), and the MIC values (microg/ml), were calculated . A large ratio indicated a wide range of effective concentrations below the MIC value for the antibiotics. Pediatr Surg Int, 2001 May, 17(4), 265 - 8 Effect of probiotics on enterocyte bacterial translocation in vitro; Mattar AF et al.; Enteral probiotics such as Lactobacillus casei GG (LGG) have been used in the treatment of a variety of intestinal disorders in infants and children, including diarrhea, malabsorption, and Clostridium difficile colitis . We have previously demonstrated that the probiotic bacterium LGG has an inhibitory effect on bacterial translocation (BT) in a neonatal rabbit model . However, this in-vivo model is limited for investigating the cellular and molecular mechanisms responsible for probiotic inhibition of BT . The purpose of this study was to determine the efficacy of LGG in reducing the rate of Escherichia coli C25 (E . coli C25) translocation using an in-vitro enterocyte cell-culture model . Human colonic carcinoma (Caco-2) enterocytes were seeded in porous filters in the apical chamber of a two-chamber cell-culture system and grown for 14 days to confluence . The monolayers were incubated at 37 degrees C with LGG for 180 min . Non-adherent LGG was washed away prior to a 120-min incubation period with 10(5) CFU E . coli C25 . E . coli that had translocated across the enterocyte monolayer were quantified by growing basal-chamber media samples on gram-negative bacteria-specific MacConkey's agar . In order to determine monolayer integrity, transepithelial electrical resistance (TEER) was measured across Caco-2 cells treated with LGG and E . coli . Statistical analysis was by ANOVA with P < 0.05 considered significant . LGG inhibited E . coli translocation at all LGG concentrations tested . The TEER ratio was not significantly altered by addition of LGG or E . coli (0.9 +/- 0.03 vs 0.8 +/- 0.05) . These results demonstrate that the probiotic bacterium LGG inhibits BT of E . coli C25 in a dose-dependent manner in an in-vitro cell-culture model . This model should be valuable in investigating the cellular and molecular mechanisms involved in the inhibition of pathological enteral bacteria by probiotic agents. Gut, 2001 Jul, 49(1), 56 - 65 Involvement of nerves and calcium channels in the intestinal response to Clostridium difficile toxin A: an experimental study in rats in vivo; Sorensson J et al.; BACKGROUND: The involvement of nerves and calcium channels in the intestinal response to Clostridium difficile toxin A (luminal concentration 1 or 15 microg/ml) was studied in the small intestine of rats in vivo . METHODS: Inflammation was quantified by estimating myeloperoxidase (MPO) activity in the intestinal lumen, extravascular accumulation of Evan's blue (EB) in the intestine, and number of red blood cells (RBCs) in veins in histological sections . Intestinal damage was estimated using a histological grading system . In some experiments net fluid transport was recorded using a gravimetric technique . RESULTS: In acutely denervated intestines, toxin A caused marked destruction of the villi, increased luminal release of MPO activity, and augmentation of intestinal content of EB and venous RBCs . Denervating the intestine 3-4 weeks prior to the actual experiment prevented the development of villus damage and significantly decreased the number of RBCs in intestinal veins in experiments with a low toxin concentration, whereas no effect was demonstrated on luminal MPO activity . Using a high toxin concentration, chronic denervation decreased only the number of RBCs . Pretreatment with hexamethonium (low toxin concentration; acute denervation) attenuated the effect of toxin A on morphology, luminal MPO activity, and number of RBCs . Pretreatment with nifedipine (low toxin concentration; acute denervation) significantly decreased intestinal MPO activity and number of RBCs . Tissue accumulation of EB was not influenced by experimental manipulation . Net fluid transport was measured in experiments exposing the intestinal mucosa to a high toxin concentration . Fluid secretion caused by the toxin was significantly attenuated by intravenous hexamethonium whereas no effect was observed after administration of nifedipine or granisetron . CONCLUSIONS: At a low toxin concentration, intramural reflexes are involved in the inflammatory response whereas axon reflexes contribute to tissue damage . At a high toxin concentration no nervous involvement in the toxin A response was demonstrated except for fluid secretion evoked by the toxin. J Appl Microbiol, 2001 Jun, 90(6), 1006 - 14 Separation and characterization of the 1,3-propanediol and glycerol dehydrogenase activities from Clostridium butyricum E5 wild-type and mutant D; Malaoui H et al.; AIMS: Clostridium butyricum E5 wild-type and mutant E5-MD were cultivated in chemostat culture on glycerol in order to compare the properties of two key enzymes of glycerol catabolism, i.e . propanediol and glycerol dehydrogenase . METHODS AND RESULTS: These two enzymes, which belong to the dha regulon, were separated by gel filtration . Both dehydrogenase activities displayed similar properties, such as pH optimum values, specificity towards physiological substrates and dependence on Mn2+ . Both strains accumulate glycerol at high levels . CONCLUSION: The mutant D strain contained a propanediol dehydrogenase activity which had a low affinity for its physiological substrate, leading to the conclusion that this strain would seem more resistant to the toxic effect of 3-hydroxypropionaldehyde than the wild-type . SIGNIFICANCE AND IMPACT OF THE STUDY: These properties make Cl . butyricum mutant D strain the best candidate so far to be used as a biotechnological agent for the bioconversion of glycerol to 1,3-propanediol. Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 963 - 6 Clostridium felsineum and Clostridium acetobutylicum are two distinct species that are phylogenetically closely related; Tamburini E et al.; The gene sequences encoding the 16S rRNA of Clostridium felsineum DSM 794T and NCIMB 10690T were determined . Both sequences exhibited a relatively very low degree of similarity to the previously determined 16S rRNA gene sequence from C . felsineum DSM 794T . C . felsineum is a member of the major Clostridium cluster, cluster I, and is phylogenetically closely related to Clostridium acetobutylicum . DNA-DNA hybridization results clearly indicated that C . felsineum and C . acetobutylicum belong to distinct species. Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 901 - 4 Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences; Sasaki Y et al.; The partial sequences (1465 bp) of the 16S rDNA of Clostridium novyi types A, B and C and Clostridium haemolyticum were determined . C . novyi types A, B and C and C . haemolyticum clustered with Clostridium botulinum types C and D . Moreover, the 16S rDNA sequences of C . novyi type B strains and C . haemolyticum strains were completely identical; they differed by 1 bp (level of similarity > 99.9%) from that of C . novyi type C, they were 98.7% homologous to that of C . novyi type A (relative positions 28-1520 of the Escherichia coli 16S rDNA sequence) and they exhibited a higher similarity to the 16S rDNA sequence of C . botulinum types D and C than to that of C . novyi type A . These results suggest that C . novyi types B and C and C . haemolyticum may be one independent species generated from the same phylogenetic origin. Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1127 - 31 Transfer of thermobacteroides leptospartum and Clostridium thermolacticum as Clostridium stercorarium subsp . leptospartum subsp . thermolacticum subsp . nov., comb . nov . and C . stercorarium subsp . thermolacticum subsp . nov., comb . nov; Fardeau ML et al.; 16S rRNA sequencing and sequence analysis of the sole member of the genus Thermobacteroides, Thermobacteroides leptospartum, revealed that it was related to members of cluster III (according to the scheme of Collins et al . 1994) represented exclusively by cellulolytic Clostridium species . Phenotypic studies indicated that Thermobacteroides leptospartum was also able to grow on cellulose, providing further evidence of its affiliation to members of cluster III . Its closest phylogenetic relatives, Clostridium thermolacticum and Clostridium stercorarium, were almost equidistantly placed with a similarity value of 99% . DNA hybridization studies also indicated that Thermobacteroides leptospartum, C . thermolacticum and C . stercorarium were closely related to each other (values of over 95% homology) . Similarities based on the comparison of the 16S rRNA gene sequences and DNA homology are sufficiently high to regard all three strains as subspecies of a single species . It is therefore proposed that Thermobacteroides leptospartum and C . thermolacticum be transferred to cluster III as C . stercorarium subsp . leptospartum subsp . nov., comb . nov . and C . stercorarium subsp . thermolacticum subsp . nov., comb . nov., respectively, thus automatically creating C . stercorarium subsp . stercorarium subsp . nov., comb . nov . The transfer of the sole member of Thermobacteroides invalidates the taxonomic status of the genus. Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1119 - 25 Clostridium uliginosum sp . nov., a novel acid-tolerant, anaerobic bacterium with connecting filaments; Matthies C et al.; An anaerobic, acid-tolerant bacterium, CK55T, was isolated from an acidic forest bog . Cells of CK55T stained Gram-negative but did not have an outer membrane . Cells were spore-forming, motile rods with peritrichous flagella, formed chains or aggregates and were linked by connecting filaments that were composed of a core and outer sheath . Cellobiose, glucose, xylose, mannose, mannitol, sucrose and peptone supported growth . Arabinose, lactose, raffinose, H2/CO2, CO/CO2, vanillate, Casamino acids and various purines and pyrimidines did not support growth . Growth on carbohydrates yielded acetate, butyrate, lactate, formate and H2 as end-products . Growth was observed at pH 4.0-9.0, with an optimum at pH 6.5, and at 10-30 degrees C, with an optimum at 20-25 degrees C . At 20 degrees C, doubling times were 4 and 6 h at pH 6.5 and 4.0, respectively . Hydrogenase activity in cell-free extracts was 12 U (mg protein)-1 . CK55T did not: (i) contain detectable levels of CO, formate, lactate dehydrogenases or cytochromes; (ii) carry out dissimilatory reduction of nitrate or sulfate; or (iii) produce methane . Thus, CK55T was characterized as a non-acetogenic, fermentative chemo-organotroph . The G+C content of CK55T was 28.0 mol% . CK55T was phylogenetically most closely related to Clostridium botulinum (types B, E and F), Clostridium acetobutylicum and other saccharolytic clostridia; the 16S rRNA gene sequence similarity values to the nearest relatives of CK55T were approximately 97% . Based on morphological, physiological and phylogenetic properties of CK55T, it is proposed that CK55T be termed Clostridium uliginosum sp . nov . (= DSM 12992T = ATCC BAA-53T). J Inorg Biochem, 2001 Jun, 85(2-3), 117 - 22 Oxygen disruption of the 2{4Fe-4S} clusters in Clostridium pasteurianum ferredoxin shown by 1H-NMR; Harklau H et al.; The ferredoxin from Clostridium pasteurianum, which contains two {4Fe-4S} clusters, was investigated in its oxidized and reduced states by two-dimensional (2D) (1)H-(1)H nuclear Overhauser enhancement spectroscopy (NOESY) . Comparison of the data from the oxidized ferredoxin with those published previously revealed the same NOE connectivities . No previous (1)H-(1)H NOESY study of the fully reduced ferredoxin has previously been published . However, it was possible to compare our results with those of a 2D exchange spectroscopy investigation of half-reduced C . pasteurianum ferredoxin . The present results with reduced C . pasteurianum ferredoxin confirm many of the (1)H peaks and NOE interactions reported earlier, revise others, and locate resonances previously undetected . When the ferredoxin was slightly exposed to oxygen, several of the hyperfine shifted resonances were irreversibly influenced . A resonance at 34 ppm in the (1)H NMR spectra of both redox states is indicative of oxygen exposure . These results indicate the importance of keeping the ferredoxin strictly anaerobic during purification and solvent exchange. Cancer Res, 2001 Jun 15, 61(12), 4885 - 91 Inhibition of epidermal growth factor-induced RhoA translocation and invasion of human pancreatic cancer cells by 3-hydroxy-3-methylglutaryl-coenzyme a reductase inhibitors; Kusama T et al.; 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors prevent the conversion of HMG-CoA to mevalonate and thereby inhibit the synthesis of other products derived from this metabolite . This includes a number of small prenylated GTPases involved in cell growth, motility, and invasion . We studied the effect of HMG-CoA reductase inhibitors (fluvastatin and lovastatin) on in vitro invasion of human pancreatic cancer PANC-1 cells . Epidermal growth factor (EGF) induced a dose-dependent increase of PANC-1 cell invasion in a modified Boyden chamber assay . Stimulation of cancer cells with EGF induced translocation of RhoA from the cytosol to the membrane fraction and actin stress fiber assembly . Furthermore, Clostridium botulinum C3 transferase, a specific inhibitor of Rho, inhibited the ability of EGF to promote invasion, indicating that EGF-induced cancer cell invasion is regulated by Rho signaling . Treatment of PANC-1 cells with fluvastatin markedly attenuated EGF-induced translocation of RhoA from the cytosol to the membrane fraction and actin stress fiber assembly, whereas it did not inhibit the tyrosine phosphorylation of EGF receptor and c-erbB-2 . The induction of cancer cell invasion by EGF was inhibited by the addition of fluvastatin or lovastatin in a dose-dependent manner . The effects of fluvastatin or lovastatin on cell morphology and invasion were reversed by the addition of all-trans-geranylgeraniol but not by the addition of all-trans-farnesol . These results suggest that HMG-CoA reductase inhibitors affect RhoA activation by preventing geranylgeranylation, which results in inhibition of EGF-induced invasiveness of human pancreatic cancer cells. J Med Chem, 2001 Jun 21, 44(13), 2253 - 8 Protease inhibitors: synthesis of a series of bacterial collagenase inhibitors of the sulfonyl amino acyl hydroxamate type; Clare BW et al.; A series of sulfonyl amino acyl hydroxamates incorporating alkyl/arylsulfonyl-N-2-nitrobenzyl-L-alanine was prepared . Related compounds were obtained by reaction of N-2-nitrobenzyl-L-Ala with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the conversion of the COOH into the CONHOH moiety . The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase (ChC), a bacterial protease involved in the degradation of extracellular matrix . Many of the obtained hydroxamates proved to be effective bacterial collagenase inhibitors, the main contributor to activity being the substitution pattern at the sulfonamido moiety . The best ChC inhibitors were those containing pentafluorophenylsulfonyl and 3- and 4-protected-aminophenylsulfonyl P(1)(') groups among others, with affinities in the low nanomolar range . This study also proves that the 2-nitrobenzyl- moiety, similarly to the 4-nitrobenyl one previously investigated (Scozzafava, A.; Supuran, C . T . J . Med . Chem . 2000, 43, 1858-1865) is an efficient P(2)(') anchoring moiety for obtaining potent bacterial collagenase inhibitors. Chemotherapy, 2001 Jul-Aug, 47(4), 243 - 9 In vitro activity and post-antibiotic effect of quinupristin/dalfopristin (Synercid); Ling TK et al.; The in vitro activities of quinupristin/dalfopristin (Synercid), ampicillin, erythromycin, clarithromycin, vancomycin, teicoplanin, ciprofloxacin and tetracycline were examined and compared against 526 gram-positive bacteria . The minimal inhibitory concentrations (MICs) for quinupristin/dalfopristin against Staphylococcus aureus, including methicillin-resistant strains, were low (MIC(90) = 0.5 mg/l), and were comparable with those of vancomycin and teicoplanin . This compound was superior to the macrolides and highly active against Streptococcus pneumoniae (both penicillin-sensitive and penicillin-resistant strains), with MIC(90) = 2 mg/l . It was also active against other streptococci, with MIC(90) = 4 mg/l . However, this agent is less active against enterococci (MIC(90) = 32 mg/l) . Quinupristin/dalfopristin showed high activity against gram-positive anaerobes, including Clostridium spp., Peptococcus spp . and Peptostreptococcus spp., with MIC(90) < or = 2 mg/l . Quinupristin/dalfopristin was also investigated for its post-antibiotic effect (PAE) and bactericidal kinetics against nine strains of gram-positive organisms, including staphylococci, enterococci and pneumococci . Exponentially growing (log phase) cultures were exposed to quinupristin/dalfopristin at 2 x MIC . Growth kinetics was evaluated using viable counting . The drug was uniformly bactericidal against pneumococci and staphylococci within 2 and 8 h of exposure, respectively . The killing activity against enterococci was weak; there was little or no reduction in bacterial count over 24 h of incubation . PAEs ranging from 2.13 to 3.28 h, 0.92 to 3.02 h and 1.89 to 7.07 h were produced on the tested pneumococci, staphylococci and enterococci, respectively . This study showed that quinupristin/dalfopristin is a promising agent active against gram-positive bacteria . The prolonged PAEs also suggest that the drug could be used intermittently at more widely spaced dosing intervals against gram-positive organisms. J Food Prot, 2001 Jun, 64(6), 838 - 44 Inhibition of growth of nonproteolytic Clostridium botulinum type B in sous vide cooked meat products is achieved by using thermal processing but not nisin; Lindstrom M et al.; The safety of refrigerated processed foods of extended durability (REPFEDs) with respect to nonproteolytic Clostridium botulinum is under continuous evaluation . In the present study, mild (P7.0(85.0) values 0 to 2 min {P, pasteurization value; z-value 7.0 degrees C; reference temperature 85.0 degrees C}) and increased (P7.0(85.0) values 67 to 515 min) heat treatments were evaluated in relation to survival of nonproteolytic C . botulinum type B spores in sous vide processed ground beef and pork cubes . The use of two concentrations of nisin in inhibition of growth and toxin production by nonproteolytic C . botulinum in the same products was also evaluated . A total of 96 samples were heat processed and analyzed for C . botulinum by BoNT/B gene-specific polmerase chain reaction and for botulinum toxin by a mouse bioassay after storage of 14 to 28 days at 4 and 8 degrees C . Predictably, after mild processing all samples of both products showed botulinal growth, and one ground beef sample became toxic at 8 degrees C . The increased heat processing, equivalent to 67 min at 85 degrees C . resulted in growth but not toxin production of C . botulinum in one ground beef sample in 21 days at 8 degrees C: in the pork cube samples no growth was detected . The increased heating of both products resulted in higher sensory quality than the milder heat treatment . Nisin did not inhibit the growth of nonproteolytic C . botulinum in either product; growth was detected in both products at 4 and 8 degrees C, and ground beef became toxic with all nisin levels within 21 to 28 days at 8 degrees C . Aerobic and lactic acid bacterial counts were reduced by the addition of nisin at 4 degrees C . The study demonstrates that the mild processing temperatures commonly employed in sous vide technology do not eliminate nonproteolytic C . botulinum type B spores . The intensity of each heat treatment needs to be carefully evaluated individually for each product to ensure product safety in relation to nonproteolytic C . botulinum. Acta Vet Hung, 2000, 48(1), 59 - 67 Cholangiohepatitis in broiler chickens in Japan: histopathological, immunohistochemical and microbiological studies of spontaneous disease; Sasaki J et al.; Forty-five broiler carcasses from 6 different flocks were condemned due to liver lesions at processing meat inspection, and collected for pathological and bacterial examinations . All affected chickens showed liver enlargement with discolouration and an apparent acinar pattern . The enlarged gallbladder and the extrahepatic bile ducts contained yellow inspissated cream-coloured material . Histopathologically, extensive proliferation of bile ductules with fibrosis was observed in interlobular connective tissue, and it spread to form bridges with adjoining triads . Destruction and obstruction of portal bile ducts with multiple granulomas due to bacterial infection and outflow of the bile were frequently observed . Many Gram-positive bacilli were seen in the lesions, and they were identified as Clostridium perfringens by indirect immunofluorescence staining technique . Clostridium perfringens was isolated from affected livers . These findings are consistent with cholangiohepatitis . Therefore, it is suggested that C . perfringens might be important in the pathogenesis of cholangiohepatitis in broiler chickens. Am J Physiol Cell Physiol, 2001 Jul, 281(1), C89 - 98 Regulation of I(Cl,swell) in neuroblastoma cells by G protein signaling pathways; Estevez AY et al.; Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) activated the I(Cl,swell) anion channel in N1E115 neuroblastoma cells in a swelling-independent manner . GTPgammaS-induced current was unaffected by ATP removal and broadly selective tyrosine kinase inhibitors, demonstrating that phosphorylation events do not regulate G protein-dependent channel activation . Pertussis toxin had no effect on GTPgammaS-induced current . However, cholera toxin inhibited the current approximately 70% . Exposure of cells to 8-bromoadenosine 3',5'-cyclic monophosphate did not mimic the effect of cholera toxin, and its inhibitory action was not prevented by treatment of cells with an inhibitor of adenylyl cyclase . These results demonstrate that GTPgammaS does not act through Galpha(i/o) GTPases and that Galpha(s)/Gbetagamma G proteins inhibit the channel and/or channel regulatory mechanisms through cAMP-independent mechanisms . Swelling-induced activation of I(Cl,swell) was stimulated two- to threefold by GTPgammaS and inhibited by 10 mM guanosine 5'-O-(2-thiodiphosphate) . The Rho GTPase inhibitor Clostridium difficile toxin B inhibited both GTPgammaS- and swelling-induced activation of I(Cl,swell) . Taken together, these findings indicate that Rho GTPase signaling pathways regulate the I(Cl,swell) channel via phosphorylation-independent mechanisms. Mol Microbiol, 2001 Jun, 40(5), 1187 - 99 Molecular characterization of the surface layer proteins from Clostridium difficile; Calabi E et al.; Many bacteria express a surface-exposed proteinaceous layer, termed the S-layer, which forms a regular two-dimensional array visible by electron microscopy . Clostridium difficile is unusual in expressing two S-layer proteins (SLPs), which are of varying size in a number of strains . In an approach combining molecular biology with mass spectrometric sequencing strategies, we have identified the structural gene (slpA) for the S-layer from three strains of C . difficile . Both proteins are derived from a common precursor, and processing involves the removal of a signal peptide and a second cleavage to release the two mature SLPs . To our knowledge, this is the first example in which two SLPs have been shown to derive from a single gene product through post-translational processing, rather than from the expression of separate genes . The higher molecular weight (MW) SLP is highly conserved among the three strains, whereas the lower MW SLP shows considerable sequence diversity, reflecting the results from Western blotting . The high-MW SLP shows weak homology to N-acetyl muramoyl-L-alanine amidase from Bacillus subtilis, and both the native SLP from C . difficile and a recombinant protein expressed in Escherichia coli were found to display amidase activity by zymography . The high-MW SLPs showed evidence of glycosylation, whereas the lower MW proteins did not . A family of genes with sequence homology to the amidase domain of the high-MW SLP was identified in the C . difficile strain 630 genome, some of which are located in the same region of the genome as slpA and were shown by reverse transcription-polymerase chain reaction (RT-PCR) analysis to be transcribed. Biochemistry, 2001 Jun 19, 40(24), 7279 - 90 Structural basis for thermostability in aporubredoxins from Pyrococcus furiosus and Clostridium pasteurianum; Zartler ER et al.; The structures of apo- and holorubredoxins from Pyrococcus furiosus (PfRd) and Clostridium pasteurianum (CpRd) have been investigated and compared using residual dipolar couplings to probe the origin of thermostability . In the native, metal (Fe or Zn) containing form, both proteins can maintain native structure at very high temperatures (>70 degrees C) for extended periods of time . Significant changes in either structure or backbone dynamics between 25 and 70 degrees C are not apparent for either protein . A kinetic difference with respect to metal loss is observed as in previous studies, but the extreme stability of both proteins in the presence of metal makes thermodynamic differences difficult to monitor . In the absence of metal, however, a largely reversible thermal denaturation can be monitored, and a comparison of the two apoproteins can offer insights into the origin of stability . Below denaturation temperatures apo-PfRd is found to have a structure nearly identical to that of the native holo form, except immediately adjacent to the metal binding site . In contrast, apo-CpRd is found to have a structure distinctly different from that of its holo form at low temperatures . This structure is rapidly lost upon heating, unfolding at approximately 40 degrees C . A PfRd mutant with the hydrophobic core mutated to match that of CpRd shows no change in thermostability in the metal-free state . A metal-free chimera with residues 1-15 of CpRd and the remaining 38 residues of PfRd is severely destabilized and is unfolded at 25 degrees C . Hence, the hydrophobic core does not seem to be the key determinant of thermostability; instead, data point to the hydrogen bond network centered on the first 15 residues or the interaction of these 15 residues with other parts of the protein as a possible contributor to the thermostability. Can J Microbiol, 2001 May, 47(5), 448 - 56 Purification, cloning, and sequencing of an enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from Clostridium bifermentans DPH-1; Okeke BC et al.; An enzyme mediating the reductive dechlorination of tetrachloroethylene (PCE) from cell-free extracts of Clostridium bifermentans DPH-1 was purified, cloned, and sequenced . The enzyme catalyzed the reductive dechlorination of PCE to cis-1,2-dichloroethylene via trichloroethylene, at a Vmax and Km of 73 nmol/mg protein and 12 microM, respectively . Maximal activity was recorded at 35 degrees C and pH 7.5 . Enzymatic activity was independent of metal ions but was oxygen sensitive . A mixture of propyl iodide and titanium citrate caused a light-reversible inhibition of enzymatic activity suggesting the involvement of a corrinoid cofactor . The molecular mass of the native enzyme was estimated to be approximately 70 kDa . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) revealed molecular masses of approximately 35 kDa and 35.7 kDa, respectively . A broad spectrum of chlorinated aliphatic compounds (PCE, trichloroethylene, cis-1,2-dichloroethylene, trans-1,2-dichloroethylene, 1,1-dichloroethylene, 1,2-dichloropropane, and 1,1,2-trichloroethane) was degraded . With degenerate primers designed from the N-terminal sequence (27 amino acid residues), a partial sequence (81 bp) of the encoding gene was amplified by polymerase chain reaction (PCR) and sequenced . Southern analysis of C . bifermentans genomic DNA using the PCR product as a probe revealed restriction fragment bands . A 5.0 kb ClaI fragment, harboring the relevant gene (designated pceC) was cloned (pDEHAL5) and the complete nucleotide sequence of pceC was determined . The gene showed homology mainly with microbial membrane proteins and no homology with any known dehalogenase, suggesting a distinct PCE dehalogenase. Can J Microbiol, 2001 May, 47(5), 373 - 81 Separation of a phenol carboxylating organism from a two-member, strict anaerobic co-culture; Letowski J et al.; In a culture converting phenol to benzoic acid under anaerobic conditions and previously described as being constituted of only a Clostridium-like strain 6, another bacterium (strain 7) was observed . Each organism was enriched by centrifugation on a Percoll gradient . Strain 6 was purified by dilution and plating . Strain 7 did not grow on solid media, but a strain 7 culture, cleared of strain 6, was obtained by subculturing in the presence of ampicillin and by dilution . In fresh medium, phenol was transformed by the reconstituted co-culture but not by each strain alone . In a supernatant from a co-culture or from a strain 6 culture, strain 7 alone transformed phenol but not strain 6 . Maintenance of an active strain 7 in fresh medium instead of co-culture supernatant became possible when phenol was replaced by 4-hydroxybenzoate (4-OHB), which is decarboxylated to phenol before being transformed to benzoate . Even with 4-OHB, the use of co-culture (or strain 6 culture) supernatant resulted in faster transformation activity and growth rate . A phylogenetic analysis placed strain 7 in a cluster of uncultivated or nonisolated bacteria (92-96% homology) . Strain 7 is also related to Desulfotomaculum, Desulfitobacterium, Desulfosporosinus, Moorella, and Sporotomaculum genera (87-92% homology). West Indian Med J, 2001 Mar, 50(1), 50 - 4 Antibiotic susceptibility of Clostridium difficile isolates from adult patients at two Jamaican hospitals . Clinical and epidemiological implications; Heslop OD et al.; The susceptibility of 39 toxin producing Clostridium difficile isolates from stools of hospitalized patients was determined, by disc diffusion, to six antibiotics . All but one isolate (toxin A negative) produced toxin A and toxin B . A wide variation in susceptibility to clindamycin, tetracycline and chloramphenicol was noted . Erythromycin and cotrimoxazole showed a clear-cut discrimination in resistance and susceptibility, while all isolates were sensitive to vancomycin . Erythromycin sensitive isolates demonstrated a significant association with diarrhoea (60.9%, 14/23, p < 0.001) . These strains were predominantly found at the University Hospital of the West Indies (UHWI, 94.1%, 16/17) . Strains resistant to erythromycin and clindamycin together were commonly found at the National Chest Hospital (NCH, 68.2%, 15/22) . All erythromycin sensitive strains found at the NCH were from patients transferred to that hospital . These findings suggest that there is a common strain of C difficile (erythromycin resistant) at the NCH different from that found at the UHWI; the resistant pattern seen with isolates from the NCH was typical of toxigenic serogroup C strain and could be typed by the the disc diffusion method . Patients at the NCH who were colonized with either of the two strains of C difficile were likely to get diarrhoea, once there was suppression of the normal microflora by antibiotics and colonic overgrowth with C difficile. J Mol Biol, 2001 Jun 8, 309(3), 777 - 91 The B(12)-binding subunit of glutamate mutase from Clostridium tetanomorphum traps the nucleotide moiety of coenzyme B(12); Tollinger M et al.; Glutamate mutase from Clostridium tetanomorphum binds coenzyme B(12) in a base-off/His-on form, in which the nitrogenous ligand of the B(12)-nucleotide function is displaced from cobalt by a conserved histidine . The effect of binding the B(12)-nucleotide moiety to MutS, the B(12)-binding subunit of glutamate mutase, was investigated using NMR spectroscopic methods . Binding of the B(12)-nucleotide to MutS was determined to occur with K(d)=5.6(+/-0.7) mM and to be accompanied by a specific conformational change in the protein . The nucleotide binding cleft of the apo-protein, which is formed by a dynamic segment with propensity for partial alpha-helical conformation (the "nascent" alpha-helix), becomes completely structured upon binding of the B(12)-nucleotide, with formation of helix alpha1 . In contrast, the segment containing the conserved residues of the B(12)-binding Asp-x-His-x-x-Gly motif remains highly dynamic in the protein/B(12)-nucleotide complex . From relaxation studies, the time constant tau, which characterizes the time scale for the formation of helix alpha1, was estimated to be about 30 micros (15)N and was the same in both, apo-protein and nucleotide-bound protein . Thus, the binding of the B(12)-nucleotide moiety does not significantly alter the kinetics of helix formation, but only shifts the equilibrium towards the structured fold . These results indicate MutS to be structured in such a way, as to be able to trap the nucleotide segment of the base-off form of coenzyme B(12) and provide, accordingly, the first structural clues as to how the process of B(12)-binding occurs . Anticancer Res, 2001 Mar-Apr, 21(2A), 857 - 61 Increasing specificity of anti-tumor therapy: cytotoxic protein delivery by non-pathogenic clostridia under regulation of radio-induced promoters; Nuyts S et al.; BACKGROUND: Pathogenic clostridia, genetically engineered to express therapeutic genes, will specifically target hypoxic regions in tumors . This specificity can be further improved if expression of these genes is controlled by a radio-induced promoter, leading to spatial and temporal control of gene expression . MATERIALS AND METHODS: Following administration of Clostridium spores to tumor bearing rats, normal tissue and tumoral specimens were compared for colonization . Clostridium was genetically modified to express tumor necrosis factor a or cytosine deaminase . Expression of these proteins was assayed . Northern blot hybridizations were used to detect genes which are radio-induced . RESULTS: Clostridium gave a selective colonization of tumors . The recombinant clostridia expressed in vitro and in vivo TNF alpha and cytosine deaminase . Clostridial SOS-repair genes were induced at a dose of 2 Gy . CONCLUSIONS: Pathogenic Clostridium can be used for tumor specific delivery of therapeutic genes . The specificity can be improved via radio-induced promoters . Overall, this new gene delivery system can lead to an increase of the therapeutic ratio in cancer treatment. Biochem Biophys Res Commun, 2001 Jun 8, 284(2), 470 - 7 Epidermal growth factor stimulation of the ACK1/Dbl pathway in a Cdc42 and Grb2-dependent manner; Kato-Stankiewicz J et al.; The tyrosine kinase ACK1 phosphorylates and activates the guanine nucleotide exchange factor Dbl, which in turn directs the Rho family GTP-binding proteins . However, the regulatory mechanism of ACK1/Dbl signaling in response to extracellular stimuli remains poorly understood . Here we describe that epidermal growth factor stimulates the ACK1/Dbl pathway, leading to actin cytoskeletal rearrangements . The role of the two ACK1-binding proteins Cdc42 and Grb2 was assessed by overexpression of the Cdc42/Rac interactive binding domain and a dominant-negative Grb2 mutant, respectively . Specific inhibition of the interaction of ACK1 with Cdc42 or Grb2 by the use of these constructs diminished tyrosine phosphorylation of both ACK1 and Dbl in response to EGF . Therefore, the activation of ACK1 and subsequent downstream signaling require both Cdc42-dependent and Grb2-dependent processes within the cell . In addition, we show that EGF transiently induces formation of the focal complex and stress fibers when ACK1 was ectopically expressed . The induction of these structures was totally sensitive to the action of botulinum toxin C from Clostridium botulinum, suggesting a pivotal role of Rho . These results provide evidence that ACK1 acts as a mediator of EGF signals to Rho family GTP-binding proteins through phosphorylation and activation of GEFs such as Dbl . Am J Forensic Med Pathol, 2001 Jun, 22(2), 177 - 9 Acute necrotizing bacterial tonsillitis with Clostridium perfringens; Gerber JE; Bacterial infection with Clostridium perfringens in children less than 2 years of age is frequently associated with meningitis, necrotizing gastrointestinal infection, and postoperative infections . However, a review of the literature reveals no reports of these bacteria infecting the tonsils . A 9-month old black female was found unresponsive at the baby-sitter's and was rushed to the hospital . Shortly after admission to the emergency department death was pronounced . An autopsy performed on this otherwise healthy infant revealed shock and acute necrotizing bacterial tonsillitis . The initial report of this infant's death was questionable sudden infant death syndrome and questionable smothering . Postmortem cerebrospinal fluid, blood, and lung cultures grew pure colonies of C . perfringens . The necrotizing tonsil revealed no significant gross lesions . Microscopically, large numbers of gram-positive rods were easily recognized and were compatible with C . perfringens . Because the oropharynx is a common portal of entry for infectious agents, it is essential to sample tissues of Waldeyer's ring and especially the tonsils to find infectious diseases that may become systemic. J Med Microbiol, 2001 Jun, 50(6), 526 - 34 Flow cytometric analysis of Clostridium difficile adherence to human intestinal epithelial cells; Drudy D et al.; Clostridium difficile is the most common cause of diarrhoea in hospitalised patients . Bacterial adherence to gut epithelial cells is a likely prerequisite to infection and toxin production . A novel flow cytometric method was developed for detecting adherence of C . difficile to human colonic and small intestinal epithelial cells (EC) and human intestinal cell lines . Small intestinal and colonic EC were isolated from biopsy specimens with mucolytic and chelating agents . Adherence of fluorochrome-labelled C . difficile to EC was measured by flow cytometry and was calculated as increase in median fluorescent intensity (deltaMFI) . Cells with bacteria attached could be distinguished easily from cells alone or cells with unlabelled bacteria attached . Toxin-positive C . difficile adhered to colonic and small intestinal EC (deltaMFI mean 21.2 SD 16.7, n = 33 and 16.5 SD 20.7, n = 19 respectively) . The toxin-negative strain also adhered to both epithelial cell types (deltaMFI 26.1 SD 32.5, n = 17 and 18.3 SD 31.3, n = 16) . Adherence of toxin-positive C . difficile to the intestinal cell lines Caco-2 (deltaMFI 9.4 SD 4.4, n = 14) and HT29 (deltaMFI 8.1 SD 3.1, n = 12) was quantifiable, although at a significantly lower level than with primary colonic epithelial cells . Adherence of the toxin-negative strain was slightly lower, deltaMFI 6.5 SD 1.8, n = 9 with Caco-2 cells and deltaMFI 6.0 SD 2.0, n = 10 with HT29 cells . Adherence of C . difficile to epithelial cell lines was blocked with C . difficile antiserum, confirming specificity of adherence . In conclusion, flow cytometry is a useful approach to quantifying adherence of C . difficile to human colonic and small intestinal epithelial cells . Binding of toxin-negative as well as toxin-positive bacteria was detectable by this approach . Analysis of C . difficile adherence to target cells may have important implications for the understanding of the pathogenesis of C . difficile-related disease. Cancer Gene Ther, 2001 Apr, 8(4), 294 - 7 Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum; Theys J et al.; The presence of severe hypoxia and necrosis in solid tumors offers the potential to apply an anaerobic bacterial enzyme/prodrug approach in cancer treatment . In this context the apathogenic C . acetobutylicum was genetically engineered to express and secrete E . coli cytosine deaminase (CDase) . Considerable levels of functional cytosine deaminase were detected in lysates and supernatants of recombinant C acetobutylicum cultures . After administration of the recombinant Clostridium to rhabdomyosarcoma bearing rats used as a model, cytosine deaminase could be detected at the tumor site . Moreover, following administration of the vascular targeting agent combretastatin A-4 phosphate significantly increased levels of cytosine deaminase were detected at the tumor site as a consequence of enlarged tumor necrosis and subsequently improved growth of C . acetobutylicum . The results provide evidence for the potential application of Clostrisdium-based therapeutic protein transfer to tumors in anticancer therapy. Am J Clin Nutr, 2001 Jun, 73(6), 1152S - 1155S Probiotics: future directions; Vanderhoof JA; Clinical studies have shown that certain probiotics may be useful in treating a variety of diarrheal disorders, including rotavirus diarrhea, antibiotic-associated diarrhea, Clostridium difficile diarrhea, and traveler's diarrhea . New data suggest that probiotics might be useful in controlling inflammatory diseases, treating and preventing allergic diseases, preventing cancer, and stimulating the immune system, which may reduce the incidence of respiratory disease . Different modes of administering probiotics are currently being investigated, which may ultimately lead to the widespread use of probiotics in functional foods . It is important that such practices be directed by carefully controlled clinical studies published in peer-reviewed journals. News Physiol Sci, 1998 Apr, 13, 58 - 63 Nerves and Intestinal Mast Cells Modulate Responses to Enterotoxins; Pothoulakis C et al.; Experiments in intact animals exposed to enterotoxins demonstrate that neurons and immune cells of the lamina propria regulate toxin-induced diarrhea and tissue damage . Clostridium difficile toxins cause profound diarrhea and acute inflammation by activating a complex cascade initiated by toxin binding to enterocyte receptors. Microbiology, 2001 Jun, 147(Pt 6), 1461 - 71 Metabolic flux in cellulose batch and cellulose-fed continuous cultures of Clostridium cellulolyticum in response to acidic environment; Desvaux M et al.; Clostridium cellulolyticum, a nonruminal cellulolytic mesophilic bacterium, was grown in batch and continuous cultures on cellulose using a chemically defined medium . In batch culture with unregulated pH, less cellulose degradation and higher accumulation of soluble glucides were obtained compared to a culture with the pH controlled at 7.2 . The gain in cellulose degradation achieved with pH control was offset by catabolite production rather than soluble sugar accumulation . The pH-controlled condition improved biomass, ethanol and acetate production, whereas maximum lactate and extracellular pyruvate concentrations were lower than in the non-pH-controlled condition . In a cellulose-fed chemostat at constant dilution rate and pH values ranging from 7.4 to 6.2, maximum cell density was obtained at pH 7.0 . Environmental acidification chiefly influenced biomass formation, since at pH 6.4 the dry weight of cells was more than fourfold lower compared to that at pH 7.0, whereas the specific rate of cellulose assimilation decreased only from 11.74 to 10.13 milliequivalents of carbon (g cells)(-1) h(-1) . The molar growth yield and the energetic growth yield did not decline as pH was lowered, and an abrupt transition to washout was observed . Decreasing the pH induced a shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation . The acetate/ethanol ratio decreased as the pH declined, reaching close to 1 at pH 6.4 . Whatever the pH conditions, lactate dehydrogenase was always greatly in excess . As pH decreased, both the biosynthesis and the catabolic efficiency of the pyruvate-ferredoxin oxidoreductase declined, as indicated by the ratio of the specific enzyme activity to the specific metabolic rate, which fell from 9.8 to 1.8 . Thus a change of only 1 pH unit induced considerable metabolic change and ended by washout at around pH 6.2 . C . cellulolyticum appeared to be similar to rumen cellulolytic bacteria in its sensitivity to acidic conditions . Apparently, the cellulolytic anaerobes studied thus far do not thrive when the pH drops below 6.0, suggesting that they evolved in environments where acid tolerance was not required for successful competition with other microbes. Electrophoresis, 2001 May, 22(8), 1585 - 9 Comparison of protocols for pulsed-field gel electrophoresis of Clostridia; Stolle A et al.; Genotyping of bacterial strains via pulsed-field gel electrophoresis has to be considered an important tool for epidemiological investigations in food hygiene as well as in other areas . Yet, a major disadvantage of this method is its long duration . Therefore, rapid procedures for DNA isolation and restriction are being sought . One such protocol was modified and further shortened to two days . This short protocol was used for macrorestriction analysis of 34 strains of 25 different Clostridium species . Parallel analyses were performed using a conventional 5-day protocol in order to compare the long and the short method by running the DNA samples obtained via both protocols on the same gel . In the case of nine strains, none of the two methods yielded satisfactory results, whereas for three strains the long protocol proved to be preferable to the short one . Comparable results were obtained using both methods in the case of 22 strains belonging to 17 different Clostridium species. Med Sci Monit, 2001 May-Jun, 7(3), 382 - 6 Acute appendicitis: the role of enterotoxigenic strains of Bacteroides fragilis and Clostridium difficile; Martirosian G et al.; BACKGROUND: The aim of this study was to investigate whether there is a relationship between enterotoxin-producing B . fragilis strains and toxigenic C . difficile strains and the pathogenesis of acute appendicitis . MATERIAL AND METHODS: Post-appendectomy tissues from 34 patients with histopathologically confirmed phlegmonous or gangrenous appendicitis were studied . RESULTS: Among 86 anaerobes isolated, the B . fragilis group was most frequently isolated: 34 B . fragilis strains were cultured from 21 post-appendectomy tissues . Two enterotoxin-producing B . fragilis strains were found . Enterotoxin titers (1:10 and 1:160, respectively) were measured on HT29/C cells . The presence of the enterotoxin gene was confirmed by PCR in DNA extracted from both strains . Among 21 DNA samples isolated from those post-appendectomy tissues from which B . fragilis strains were cultured, the presence of the enterotoxin gene was confirmed in only one case (the corresponding B . fragilis strain enterotoxin titer was 1:160) . A unique toxigenic C . difficile strain was also cultured from the tissue of an adult patient with gangrenous non-perforated appendicitis . The presence of toxin A and toxin B genes was confirmed by PCR in DNA extracted from the C . difficile strain, but these genes were not found in the DNA extracted from the corresponding tissue . CONCLUSION: The presence of enterotoxigenic B . fragilis and toxigenic C . difficile strains was shown in post-appendectomy tissue from patients with phlegmonous and gangrenous appendicitis, and the B . fragilis enterotoxin gene was detected directly in the corresponding tissue . Further investigations (including immunologic aspects) require to confirm the role of these toxins in pathogenesis of acute appendicitis. Mar Pollut Bull, 2001 Jan, 42(1), 31 - 5 Clostridium perfringens as a potential indicator for the presence of sewage solids in marine sediments; Skanavis C et al.; Marine sediment cores collected from several depths of water and distances from a California sewage outfall were tested to see if sediments influenced by sewage solids were a reservoir of enteric pathogens, and if concentrations of indicator bacteria were related to the presence of sewage solids . Vertical distributions of microorganisms in marine sediments were determined; there was a decrease of indicator bacteria with increasing sediment depth . Aeromonas was randomly isolated, but none of the enteric bacterial pathogens or viruses were detected . While classic indicator bacteria were of little value in predicting the presence of pathogens, or relative amounts of sewage solids, Clostridium perfringens may be a suitable indicator . Clostridium perfringens concentrations were not related to the presence of pathogens in sediments. J Biol Chem, 2001 Jul 27, 276(30), 28226 - 32 Epub 2001 May 29. A novel endo-beta-galactosidase from Clostridium perfringens that liberates the disaccharide GlcNAcalpha 1-->Gal from glycans specifically expressed in the gastric gland mucous cell-type mucin; Ashida H et al.; We found that commercially available sialidases prepared from Clostridium perfringens ATCC10543 were contaminated with an endoglycosidase capable of releasing the disaccharide GlcNAcalpha1-->4Gal from glycans expressed in the gastric gland mucous cell-type mucin . We have isolated this enzyme in electrophoretically homogeneous form from the culture supernatant of this organism by ammonium sulfate precipitation followed by affinity chromatography using a Sephacryl S-200 HR column . The enzyme was specifically retained by and eluted from the column with methyl-alpha-Glc . By NMR spectroscopy, the structure of the disaccharide released from porcine gastric mucin by this enzyme was established to be GlcNAcalpha1-->4Gal . The specificity of this enzyme as an endo-beta-galactosidase was established by analyzing the liberation of GlcNAcalpha1-->4Gal from GlcNAcalpha1-->4Galbeta1-->4GlcNAcbeta1-->6(GlcNAcalpha1--> 4Galbeta1-->3)GalNAc-ol by mass spectrometry . Because this novel endo-beta-galactosidase specifically releases the GlcNAcalpha1-->4Gal moiety from porcine gastric mucin, we propose to call this enzyme a GlcNAcalpha1-->4Gal-releasing endo-beta-galactosidase (Endo-beta-Gal(GnGa)) . Endo-beta-Gal(GnGa) was found to remove the GlcNAcalpha1-->4Gal epitope expressed in gastric adenocarcinoma AGS cells transfected with alpha1,4-N-acetylglucosaminyltransferase cDNA . Endo-beta-Gal(GnGa) should become useful for studying the structure and function of glycoconjugates containing the terminal GlcNAcalpha1-->4Gal epitope. Dtsch Med Wochenschr, 2001 May 4, 126(18), 519 - 22 {Nosocomial epidemiology and transmission of Clostridium difficile infection}; Grunewald T et al.; BACKGROUND: Clostridium difficile is of growing importance as a hospital-acquired pathogen . Pseudomembraneous colitis is the main clinical disease . Transmission and epidemiological features are not yet fully understood . PATIENTS AND METHODS: Stool samples from 1164 individuals (571 women and 593 men) attending were examined for the presence of C . difficile . Follow-up examinations and molecular typing methods were used for the detection nosocomial transmissions . Additionally, hospital-borne environmental samples as well as staff samples were tested . RESULTS: Incidence of C . difficile infection was 8.4% . Nearly all patients (92.9%) had antibiotics given . Using molecular typing nosocomial transmission was evident . Though, environmental samples in general had a low positivity, toilet chairs were contaminated in 15.4% and may be a potential source of transmission . Staff was positive in only one case . CONCLUSIONS: Prevention of infections with C . difficile becomes to be a major threat for the clinical and hygienic management. Infect Control Hosp Epidemiol, 2001 Apr, 22(4), 219 - 21 A comparison of multifaceted versus Clostridium difficile-focused VRE surveillance strategies in a low-prevalence setting; Katz KC et al.; We compared our current screening strategy for vancomycin-resistant Enterococcus (VRE) with a focused strategy that screens all stool samples sent for Clostridium difficile toxin assay but limits rectal swab screening to wards with new VRE cases detected via C . difficile samples . The proposed strategy detects 72.7% of new VRE cases, with substantial cost savings. Nahrung, 2001 Apr, 45(2), 125 - 8 Differentiation of Clostridium perfringens and Clostridium botulinum from non-toxigenic clostridia, isolated from prepared and frozen foods by PCR-DAN based methods; Cordoba MG et al.; During the elaboration process of prepared and frozen foods, Clostridium sp . have been reported . From those microorganisms, C . perfringens and C . botulinum may pose a high risk for the consumers . To avoid these pathogenic organisms an HACCP program should be implemented, but in addition sensitive and moderately time consuming microbiological methods for monitoring C . perfringens and C . boulinum should be established . In this work, an RFLP analysis of the 16S rDNA will be developed to differentiate C . perfringens from other Clostridium sp . In addition, a PCR protocol, will be assayed to detect C . botulinum . Both two methods will be compared with biochemical characterization by API system . The restriction analysis of the 16S rDNA with Taq I and Rsa I showed at least the same sensitivity to differentiate C . perfringens from clostridial isolates as biochemical identification . However, the former method takes only 8-10 h of analysis as compared with 24-48 h required for biochemical characterization . With the specific PCR protocol to detect C . botulinum a band of 1.1 kbp was obtained derived from the specific amplification of BoNT genes, taking 6-8 h for analysis . Both two molecular DNA based methods should be considered as verification techniques of pathogenic clostridia in the HACCP program. Ugeskr Laeger . 2001 Jan 8;163(2):169. {Infantile botulism caused by honey}; Jung A et al.; We report a case of a 5-week-old infant admitted with respiratory arrest . He had been fed with honey for two weeks . Infant botulism was suspected and confirmed by the finding of Clostridium botulinum toxin in the serum and faeces, and in the honey . The infant needed 7.5 months of ventilatory support. FEMS Microbiol Lett, 2001 May 30, 199(2), 191 - 5 Molecular evidence that the capacity for endosporulation is universal among phototrophic heliobacteria; Kimble-Long LK et al.; Although enrichment cultures for anoxygenic phototrophic heliobacteria commonly contain sporulating cells, once strains of heliobacteria are obtained in pure culture, they all but cease to sporulate . In fact, some species of heliobacteria have never been observed to sporulate . Thus, despite their phylogenetic connection to endospore-forming bacteria, the question of sporulation capacity in heliobacteria remains open . We have investigated this problem using PCR and Southern hybridization as tools and show here that all recognized species of heliobacteria tested, as well as several unclassified strains, contain homologs to the ssp genes of Clostridium and Bacillus species, genes that encode key sporulation-specific proteins . It can therefore be concluded that as a group, heliobacteria are likely all to be endospore-forming bacteria in agreement with their phylogenetic placement within the 'low GC' Gram-positive bacteria. Mol Pathol, 2001 Jun, 54(3), 176 - 9 Regulation of connective tissue growth factor (ccn2; ctgf) gene expression in human mesangial cells: modulation by HMG CoA reductase inhibitors (statins); Goppelt-Struebe M et al.; AIM: Connective tissue growth factor (ccn; ctgf) gene expression is upregulated in fibrotic renal glomeruli . Therefore, the regulation and pharmacological modulation of ccn2 (ctgf) mRNA expression was investigated in a human renal mesangial cell line . METHODS: A human renal mesangial cell line was cultured in vitro under standard conditions . After stimulation, RNA was extracted and ccn2 (ctgf) mRNA expression assessed by northern blot analysis . RESULTS: The expression of ccn2 (ctgf) mRNA was transiently upregulated by fetal calf serum . Very rapid onset but short lasting ccn2 (ctgf) mRNA expression was observed after stimulation with lysophosphatidic acid, a bioactive lipid, which activates G protein coupled receptors . Induction of ccn2 (ctgf) mRNA expression by transforming growth factor beta (TGF-beta) was more prolonged and lasted for more than one day . The small GTPases of the Rho family were essential for basal as well as induced ccn2 (ctgf) expression: preincubation of the cells with toxin B from Clostridium difficile abrogated ccn2 (ctgf) mRNA expression . HMG CoA reductase inhibitors, which are therapeutically used as lipid lowering drugs, interfere with the isoprenylation and thus activation of Rho proteins . Simvastatin, an HMG CoA reductase inhibitor, inhibited ccn2 (ctgf) mRNA expression in a concentration dependent manner (IC(50): 1-2 microM) . CONCLUSION: Statins were identified as potent inhibitors of ccn2 (ctgf) mRNA expression in mesangial cells, and therefore might be of potential use to modulate the excessive ccn2 (ctgf) expression in mesangial cells related to glomerular fibrosis. J Infect Dis, 2001 Jun 15, 183(12), 1760 - 6 Epub 2001 May 11. Infection of hamsters with epidemiologically important strains of Clostridium difficile; Sambol SP et al.; Five different toxigenic strains of Clostridium difficile of known human epidemiologic importance were tested for virulence in hamsters . Three strains-types B1, J9, and K14-have caused hospital outbreaks . Type Y2 is associated with a high rate of asymptomatic colonization in patients . The fifth strain, type CF2, is a toxin A-negative, toxin B-positive strain implicated in multiple human cases of C . difficile-associated diarrhea . Groups of 10 hamsters per strain were given 1 dose of clindamycin, followed 5 days later with gastric inoculation of 100 cfu of C . difficile . Hamsters given types B1, J9, K14, or Y2 showed 90%-100% colonization (albeit at a slower rate with type Y2) and 100% mortality of colonized animals . Hamsters challenged with type CF2 showed 60% (P= .01) colonization and 30% mortality (P= .0003) . The hamster model demonstrated pathogenicity differences between a toxin variant strain and standard toxigenic strains but no significant differences among the standard strains. J Bacteriol, 2001 Jun, 183(12), 3742 - 51 Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity; Shimamoto S et al.; A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains . After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination . The present study was undertaken to characterize GSP . In the presence of 3-{(3-cholamidopropyl)dimethylammonio}-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores . The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa . The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55 degrees C for 40 min . GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme . Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl(2) indicated that the protease is a cysteine-dependent serine protease . Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5' end of sleC . The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases . Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions. Pathology, 2001 May, 33(2), 187 - 96 The effect of bacterial enterotoxins implicated in SIDS on the rabbit intestine; Kamaras J et al.; The aim of this project was to characterise the type of damage caused to the intestine of the infant rabbit by bacterial enterotoxins implicated in sudden infant death syndrome (SIDS) . Samples of the duodenum, jejunum, ileum, caecum and large intestine exposed to the toxins for up to 6 hours were examined by scanning (SEM) and transmission electron microscopy (TEM) . The damage was quantitatively assessed (% villi damaged) by SEM and qualitatively by SEM and TEM . Clostridium perfringens enterotoxin, staphylococcal enterotoxin B and Clostridium difficile toxin A + toxin B combined all caused severe damage to the villi in the small intestine (80-90% damage) . Clostridium difficile toxin B caused only slight damage (17% to the jejunum, 26% to the caecum) . Clostridium perfringens alpha-toxin caused moderate damage to the small intestine (duodenum 34%, caecum 35%), and Escherichia coli STa caused significant damage to the small (53-70%) and large intestine (51%) . The level of toxin damage increased with time, the small intestine being more susceptible generally to damage than the large intestine . Each toxin differed in its ability to damage the villi, microvilli, enterocytes and lamina propria. FEMS Microbiol Lett, 2001 May 15, 199(1), 27 - 31 Role of residues constituting the 2beta1 strand of domain II in the biological activity of anthrax protective antigen; Khanna H et al.; Anthrax toxin consists of three proteins, protective antigen, lethal factor and oedema factor . A proteolytically activated 63-kDa fragment of protective antigen binds lethal factor/oedema factor and translocates them into the cytosol . Domain II of protective antigen has been implicated in membrane insertion and channel formation . In the present study, alanine substitutions in 14 consecutive residues of the 2beta1 strand that are highly homologous to the putative membrane interacting segment of Clostridium perfringens iota-b toxin were generated and the effect on the biological activity of protective antigen studied . One of the mutants, Pro260Ala, showed considerably reduced toxicity in combination with lethal factor . The mutant also showed decreased membrane insertion and translocation of lethal factor into the cytosol . The data suggest that Pro260 is important for membrane insertion and translocation by protective antigen. Vet Microbiol, 2001 Jul 3, 81(1), 21 - 32 Bacterial intestinal flora associated with enterotoxaemia in Belgian Blue calves; Manteca C et al.; The enterotoxaemia syndrome in Belgian Blue calves is characterised by a high case fatality rate, sudden death, lesions of haemorrhagic enteritis of the small intestine and, quite often an absence of other clinical signs but its cause has not been yet identified . As a first step in this identification, the aerobic and anaerobic intestinal flora of a population of 78 calves, originating from farms located in southern Belgium and that died in circumstances defined as "calf enterotoxaemia" (study population) and of 64 calves that died in other circumstances (control population) were studied qualitatively and quantitatively . The colonies were identified after subcultures with appropriate API sugar sets . Anaerobically Clostridium perfringens was isolated in higher numbers (mean values of 10(7)-10(7.5) colony forming units (CFU) versus 10(4)-10(5) CFU per ml of intestinal content) and from more animals (79 versus 19%) in the study population than in the control population, although individual results from both populations could overlap . Other clostridial species, i.e . mainly urease-negative C . sordellii and C . bifermentans, were isolated in high numbers (>10(6) CFU per ml of intestinal content) from a few animals in the study population only . All but one of the 705 C . perfringens isolates from both populations belonged to the A toxin type and none of the urease-negative C . sordellii was toxigenic . Gram-negative anaerobes were not isolated in high numbers from any of the samples . Aerobically beta-haemolytic E . coli were significantly more frequent among the study population, but were isolated from only 25% of the animals . Salmonella Typhimurium was isolated from only two animals in the study population . Less than 1% of the E . coli isolated were verotoxigenic and one-third were necrotoxigenic . At this stage only non-enterotoxigenic type A C . perfringens are thus statistically associated with the enterotoxaemia syndrome in Belgian Blue calves and fulfil the first of the Koch's postulates. Vet Pathol, 2001 May, 38(3), 326 - 7 Tyzzer's disease in a neonatal rainbow lorikeet (Trichoglossus haematodus); Raymond JT et al.; A captive-born 8-day-old male rainbow lorikeet (Trichoglossus haematodus) was found dead . Histologically, there were necrotizing hepatitis, myocarditis, and ventriculitis . Silver stain revealed argyrophilic filamentous bacilli within hepatocytes, smooth myofibers of the gizzard, and cardiac myofibers surrounding foci of necrosis . Immunohistochemistry using anti-Clostridium piliforme RT and MSK strain antisera reacted positively against bacilli within hepatocytes, cardiac myofibers, smooth myofibers of the gizzard, and splenic and intestinal macrophages . Polymerase chain reaction (PCR) assay of paraffin-embedded liver, heart, gizzard, spleen, and small intestine amplified the 196-bp DNA fragment specific to 16S ribosomal RNA of C . piliforme . The results of histopathology, immunohistochemistry, and PCR are consistent with C . piliforme infection in this lorikeet. Environ Toxicol Chem, 2001 Mar, 20(3), 479 - 84 Structure-activity relationship study on the bioreduction of azo dyes by Clostridium paraputrificum; Moir D et al.; Seven commercially available, structurally related azo dyes have been bioreduced by the anaerobic bacterium Clostridium paraputrificum . The rates of reduction of these dyes were found to vary between 24 and 74 nmoles reduced/mg protein/h . Acid red 1 and desmethyl acid red 106 were found to be the most readily reduced, while chromotrope 2R and cibacron brilliant red 3B-A were reduced at the slowest rates . The differences in reduction rates can be rationalized on the basis of structural differences and are consistent with the possible intermediacy of low molecular-weight electron carriers as the mediators of reduction . The incorporation of electron-withdrawing groups into the dyes, even if remotely placed, was found to increase the rate of reduction of dyes under controlled conditions, supporting the inversely proportional relationship between the electron density of the azo bond and the ease of bioreduction. New Microbiol, 2001 Apr, 24(2), 125 - 36 Weakly beta-haemolytic human intestinal spirochaetes antagonize the haemolytic activity of Clostridium perfringens alpha-toxin producer; Calderaro A et al.; The production of haemolytic antagonism between weakly beta-haemolytic human intestinal spirochaetes (wbetaHIS) related to human intestinal spirochaetosis and Clostridium perfringens alpha-toxin producer was investigated . A reduction of the clostridial haemolytic activity and a distortion of the haemolytic halo of clostridial alpha-toxin surrounded by a small zone of poorly cooperative haemolysis was clearly observed on the level of the spirochaetal growth area when 40 out of 41 wbetaHIS were cultivated in sheep blood agar plates together with Clostridium perfringens alpha-toxin producer . This phenomenon of haemolytic antagonism was observed only when wbetaHIS grew 72-96 hours sooner than C . perfringens and after the inoculum of the latter at a distance of 0 to 10 mm from wbetaHIS the plates were anaerobically incubated for an additional 48 hours and the bacteria were used at concentrations ranging from 10(7) to 10(4) CFU/ml . These results were also observed between C . perfringens and weakly beta-haemolytic intestinal spirochaetes related to animal intestinal spirochaetosis including avian strains and Brachyspira (Serpulina) pilosicoli of porcine origin. Kaku Igaku, 2001 Mar, 38(2), 125 - 30 {18F-FDG injections produced by a solid phase 18F-fluorination (FDG MicroLab): effects of 18F-FDG and the components on endotoxin and sterility tests}; Kuge Y et al.; Effects of 18F-FDG and components of the injections on endotoxin tests (Limulus tests) and sterility tests (Blood culture system) were determined with 18F-FDG injections produced by a solid phase 18F-fluorination (FDG MicroLab, GE) . 18F-FDG injections with endotoxins shortened the time for gelling (turbidimetry), compared with that of the control (saline) . Blood culture systems inoculated with 18F-FDG injections and microorganisms showed positive results within 72 h of incubation for every species of microorganisms used in the present study (Bacillus subtilis, Candida albicans, Clostridium sporogenes, Micrococcus luteus) . These results were quite similar to those for the control samples inoculated with saline and the microorganisms . Consequently, 18F-FDG and the components of the injections produced by the present methods may not significantly affect the endotoxin tests and sterility tests. Biotechnol Bioeng, 2001 Jun 20, 73(6), 476 - 83 Enhanced selection of an anaerobic pentachlorophenol-degrading consortium; Tartakovsky B et al.; A rapid enrichment approach based on a pentachlorophenol (PCP) feeding strategy which linked the PCP loading rate to methane production was applied to an upflow anaerobic sludge bed reactor inoculated with anaerobic sludge . Due to this strategy, over a 140-day experimental period the PCP volumetric load increased from 2 to 65 mg L(R)(-1) day(-1) with a near zero effluent concentration of PCP . Dechlorination dynamics featured sequential appearance of 3,4,5-chlorophenol, 3,5-chloro- phenol, and 3-chlorophenol in the reactor effluent . Profiling of the reactor population by denaturing gradient gel electrophoresis (DGGE) revealed a correlation between the appearance of dechlorination intermediates and bands on the DGGE profile . Nucleotide sequencing of newly detected 16S rDNA fragments suggested the proliferation of Clostridium and Syntrophobacter/Syntrophomonas spp . in the reactor during PCP degradation . Published by John Wiley & Sons, Inc. Protein Sci, 2001 Mar, 10(3), 613 - 21 Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin; Min T et al.; Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large . It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions . The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved . To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state . Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated . First, expansion of the {Fe-S} cluster and concomitant contraction of the NH...S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge . Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons . Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus . How general this correlation is among redox proteins must be determined in future work . The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin. Protein Eng, 2001 Mar, 14(3), 167 - 72 Do domain interactions of glycosyl hydrolases from Clostridium thermocellum contribute to protein thermostability? Kataeva IA, Blum DL, Li XL, Ljungdahl LG. Cellulolytic and hemicellulolytic enzymes usually have a domain composition . The mutual influence of a cellulose-binding domain and a catalytic domain was investigated with cellobiohydrolase CelK and xylanase XynZ from Clostridium thermocellum . CelK is composed of an N-terminal family IV cellulose-binding domain (CBDIV(CelK)), a family 9 glycosyl hydrolase domain (Gh9(CelK)) and a dockerin domain (DD) . CelK without the DD, (CBDIV-Gh9)(CelK) and CBDIV(CelK) bound cellulose . The thermostability of (CBDIV-Gh9)(CelK) was significantly higher than that of CBDIV(CelK) and Gh9(CelK) . The temperature optima of (CBDIV-Gh9)(CelK) and Gh9(CelK) were 65 and 45 degrees C, respectively . XynZ consists of an N-terminal feruloyl esterase domain (FAE(XynZ)), a linker (L), a family VI CBD (CBDVI(XynZ)), a DD and a xylanase domain . FAE(XynZ) and (FAE-L-CBDVI)(XynZ), used in the present study did not bind cellulose, but both were highly thermostable . Replacement of CBDVI(XynZ) with CBDIV(CelK) resulted in chimeras with feruloyl esterase activity and the ability to bind cellulose . CBDIV(CelK)-FAE(XynZ) bound cellulose with parameters similar to that of (CBDIV-Gh9)(CelK) . (FAE-L)(XynZ)-CBDIV(CelK) and FAE(XynZ)-CBDIV(CelK) had lower relative affinities and binding capacities than those of (CBDIV-Gh9)(CelK) . The three chimeras were much less thermostable than FAE(XynZ) and (FAE-L-CBDVI)(XynZ) . The results indicate that domains of glycosyl hydrolases are not randomly combined and that domain interactions affect properties of these domain-structured enzymes. Biochim Biophys Acta, 2001 Jan 12, 1544(1-2), 10 - 7 Contribution of an aspartate residue, D114, in the active site of clostridial glutamate dehydrogenase to the enzyme's unusual pH dependence; Coughlan S et al.; Glutamate dehydrogenase from Clostridium symbiosum displays unusual kinetic behaviour at high pH when compared with other members of this enzyme family . Structural and sequence comparisons with GDHs from other organisms have indicated that the Asp residue at position 114 in the clostridial enzyme may account for these differences . By replacing this residue by Asn, a mutant protein has been created with altered functional properties at high pH . This mutant protein can be efficiently overexpressed in Escherichia coli, and several criteria, including mobility in non-denaturing electrophoresis, circular dichroism (CD) spectra and initial crystallisation studies, suggest a folding and an assembly comparable to those of the wild-type protein . The D114N mutant enzyme shows a higher optimum pH for activity than the wild-type enzyme, and both CD data and activity measurements show that the distinctive time-dependent reversible conformational inactivation seen at high pH in the wild-type enzyme is abolished in the mutant. Clin Infect Dis, 2001 Jun 1, 32(11), 1567 - 76 Epub 2001 May 04. Probiotic agents and infectious diseases: a modern perspective on a traditional therapy; Alvarez-Olmos MI et al.; There is an increasing scientific and commercial interest in the use of beneficial microorganisms, or "probiotics," for the prevention and treatment of disease . The microorganisms most frequently used as probiotic agents are lactic-acid bacteria such as Lactobacillus rhamnosus GG (LGG), which has been extensively studied in recent literature . Multiple mechanisms of action have been postulated, including lactose digestion, production of antimicrobial agents, competition for space or nutrients, and immunomodulation . We have reviewed recent studies of probiotics for the treatment and control of infectious diseases . Studies of pediatric diarrhea show substantial evidence of clinical benefits from probiotic therapy in patients with viral gastroenteritis, and data on LGG treatment for Clostridium difficile diarrhea appear promising . However, data to support use of probiotics for prevention of traveler's diarrhea are more limited . New research suggests potential applications in vaccine development and prevention of sexually transmitted diseases . Further studies are needed to take full advantage of this traditional medical approach and to apply it to the infectious diseases of the new millennium. J Med Microbiol, 2001 May, 50(5), 421 - 9 Novel Helicobacter species isolated from rhesus monkeys with chronic idiopathic colitis; Fox JG et al.; Chronic, idiopathic diffuse colitis is a well recognised clinical and pathological entity in captive rhesus monkeys . Six rhesus monkeys were diagnosed with clinically debilitating, chronic diarrhoea . Histologically, colonic tissues were characterised as chronic, moderate to severe colitis and typhlitis, with diffuse mononuclear inflammation of lamina propria, reactive lymphoid hyperplasia and multifocal micro-abscesses . Colonic tissues were cultured for Salmonella spp . and Shigella spp.; all results were negative . Samples were negative for Clostridium difficile A and B toxins, and special stains of colonic tissue for acid-fast bacteria were also negative . The six diarrhoeic monkeys tested gave negative results for serum IgG antibodies to herpes B virus, STLV, SRV and SIV . Colonic tissue from the six diarrhoeic and two clinically normal monkeys with histologically confirmed colitis from the same colony were also subjected to micro-aerobic culture . Micro-aerobic cultures from all eight monkeys incubated at 37 degrees C and 42 degrees C revealed pinpoint or spreading colonies on antibiotic-containing media . Bacteria were identified as gram-negative, oxidase positive and urease negative . Of the nine strains characterised biochemically, two separate biotypes (corresponding to different species by 16S rRNA analysis) were identified . One biotype (type 1), from non-diarrhoeic monkeys and the second biotype (type 2) from diarrhoeic animals with subclinical chronic colonic inflammation, differed by catalase activity, ability to reduce nitrate to nitrite and sensitivity to cephalothin . Complete 16S rRNA analysis of five of the nine strains characterised biochemically indicated that the organisms isolated were two novel Helicobacter spp . By electron microscopy, these novel helicobacters had spiral morphology with bipolar sheathed flagella . This is the first report describing the isolation of novel Helicobacter spp . from inflamed colons of rhesus monkeys . Studies are needed to determine whether these novel Helicobacter spp . play a causal role in the initiation and progression of chronic colitis in macaques . Further microbiological and histological analysis of this chronic idiopathic colitis syndrome in macaques may prove useful in understanding the aetiology and pathogenesis of inflammatory bowel disease in man. J Med Microbiol, 2001 May, 50(5), 407 - 14 Molecular typing and long-term comparison of clostridium difficile strains by pulsed-field gel electrophoresis and PCR-ribotyping; Spigaglia P et al.; Thirty-two related and 68 unrelated isolates of Clostridium difficile, isolated in different Italian hospitals since 1987, were analysed by PFGE and PCR-ribotyping to investigate their genetic relatedness . The isolates were classified into 28 groups by PFGE and 20 ribotypes by PCR-ribotyping . A single clone of C . difficile was recognised as the cause of three geographically and chronologically distant outbreaks . The correlation between PFGE and PCR-ribotyping results was good, with agreement for 77 (84%) of the 92 isolates typed by both methods . However, among sporadic isolates the discriminatory power of PFGE was more evident . Eight isolates that were untypable by PFGE could be analysed by PCR-ribotyping . The dendrograms generated showed that the genetic relatedness of the C . difficile isolates obtained by both techniques was comparable . The majority of the isolates in recent years appeared to be genetically unrelated to isolates from past infections . However, two clonal groups identified in all time periods had a common origin and this seems to indicate that they share some advantageous biological characteristics . The constant monitoring of C . difficile epidemiology will allow acquisition of further important data on this nosocomial pathogen. Eur J Epidemiol, 2000, 16(10), 913 - 8 An outbreak in Italy of botulism associated with a dessert made with mascarpone cream cheese; Aureli P et al.; In the late 1996, an outbreak of botulism affected eight young people (age of patients ranged from 6 to 23 years) in Italy . The onset of the illness was the same for all of these patients: gastrointestinal symptoms (nausea and vomiting) followed by neurologic symptoms . The most common neurologic symptoms were dysphagia, respiratory failure (100%), diplopia (87%), dysarthria, ptosis (75%) and mydriasis (50%) . All patients required mechanical ventilation . Botulinum toxin was detected from two of respectively five sera and six stool samples analysed, while spores of Clostridium botulinum type A were recovered from all patient' faeces . The epidemiological investigation led to suspect a commercial cream cheese ('mascarpone') as a source of botulinum toxin: indeed, it had been eaten by all the patients before onset of the symptoms, either alone or as the (uncooked) ingredient of a dessert, 'tiramisu' . Botulinum toxin type A was found in the 'tiramisu' leftover consumed by two patients and in some mascarpone cheese samples collected from the same retail stores where the other patients had previously bought their cheeses . A break in the cold-chain at the retail has likely caused germination of C . botulinum spores contaminating the products, with subsequent production of the toxin . One of the patients died, while the others recovered very slowly . Prompt international alerting and recall of the mascarpone cheese prevented the spread of the outbreak due to the wide range of distribution, demonstrating the importance of a rapid surveillance system . None of the people complaining of symptoms after the public alert resulted positive for botulinum spores and toxin. Biochem J, 2001 May 15, 356(Pt 1), 97 - 103 HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction; Gil C et al.; A recent report {Gil, Chaib-Oukadour, Pelliccioni and Aguilera (2000) FEBS Lett . 481, 177-182} describes activation of signal transduction pathways by tetanus toxin (TeTx), a Zn(2+)-dependent endopeptidase synthesized by the Clostridium tetani bacillus, which is responsible for tetanus disease . In the present work, specific activation of protein kinase C (PKC) isoforms and of intracellular signal-transduction pathways, which include nerve-growth-factor (NGF) receptor trkA, phospholipase C(PLC)gamma-1 and extracellular regulated kinases (ERKs) 1 and 2, by the recombinant C-terminal portion of the TeTx heavy chain (H(C)-TeTx) is reported . The activation of PKC isoforms was assessed through their translocation from the soluble (cytosolic) compartment to the membranous compartment, showing that clear translocation of PKC-alpha, -beta, -gamma and -delta isoforms exists, whereas PKC-epsilon showed a slight decrease in its soluble fraction immunoreactivity . The PKC-zeta isoform showed no consistent response . Using immunoprecipitation assays against phosphotyrosine residues, time- and dose-dependent increases in tyrosine phosphorylation were observed in the trkA receptor, PLCgamma-1 and ERK-1/2 . The effects shown by the H(C)-TeTx fragment on tyrosine phosphorylation were compared with the effects produced by NGF . The trkA and ERK-1/2 activation were corroborated using phospho-specific antibodies against trkA phosphorylated on Tyr(490), and antibodies against Thr/Tyr phosphorylated ERK-1/2 . Moreover, PLCgamma-1 phosphorylation was supported by its H(C)-TeTx-induced translocation to the membranous compartment, an event related to PLCgamma-1 activation . Since H(C)-TeTx is the domain responsible for membrane binding and lacks catalytic activity, the activations described here must be exclusively triggered by the interaction of TeTx with a membrane component. Virology, 2001 May 10, 283(2), 188 - 96 RhoA is activated during respiratory syncytial virus infection; Gower TL et al.; Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants and immunocompromised adults . We have recently shown the RSV F glycoprotein, which mediates viral fusion and entry, interacts with the cellular protein RhoA in two-hybrid and in vitro binding assays . Whether this interaction occurs in living cells remains an open question . However, because RhoA signaling is associated with many cellular functions relevant to RSV pathogenesis such as actin cytoskeleton organization, expression of proinflammatory cytokines, and smooth muscle contraction, we asked whether RhoA activation occurred during RSV infection of HEp-2 cells . We found that the amount of isoprenylated and membrane-bound RhoA in RSV-infected cultures was increased . Further evidence of RhoA activation was demonstrated by downstream signaling activity mediated by RhoA . There was an increase in p130(cas) phosphorylation during RSV infection, which was prevented by Y-27632, a specific inhibitor of Rho kinase, or lovastatin, an HMG-CoA reductase inhibitor that reduces the synthesis of groups needed for isoprenylation . In addition, RSV infection of HEp-2 cells resulted in an increase in the formation of actin stress fibers . Pretreatment of HEp-2 cells with Clostridium botulinum C3 exotoxin, an enzyme that specifically ADP-ribosylates and inactivates RhoA, prevented RSV-induced stress fiber formation . These observations indicate that RhoA and subsequent downstream signaling events are activated during RSV infection, which has implications for RSV pathogenesis . FEBS Lett, 2001 Apr 27, 495(3), 172 - 7 Tyrosine 331 and phenylalanine 334 in Clostridium perfringens alpha-toxin are essential for cytotoxic activity; Jepson M et al.; Differences in the biological properties of the Clostridium perfringens phospholipase C (alpha-toxin) and the C . bifermentans phospholipase C (Cbp) have been attributed to differences in their carboxy-terminal domains . Three residues in the carboxy-terminal domain of alpha-toxin, which have been proposed to play a role in membrane recognition (D269, Y331 and F334), are not conserved in Cbp (Y, L and I respectively) . We have characterised D269Y, Y331L and F334I variant forms of alpha-toxin . Variant D269Y had reduced phospholipase C activity towards aggregated egg yolk phospholipid but increased haemolytic and cytotoxic activity . Variants Y331L and F334I showed a reduction in phospholipase C, haemolytic and cytotoxic activities indicating that these substitutions contribute to the reduced haemolytic and cytotoxic activity of Cbp. Bioresour Technol, 2001 Jun, 78(2), 141 - 7 In vitro dehalogenation of tetrachloroethylene (PCE) by cell-free extracts of Clostridium bifermentans DPH-1; Chang YC et al.; Cell-free extracts of Clostridium bifermentans DPH-1 catalyzed tetrachloroethylene (PCE) dechlorination . PCE degradation was stimulated by addition of a variety of electron donors . Ethanol (0.61 mM) was the most effective electron donor for PCE dechlorination . Maximum activity was recorded at 30 degrees C and pH 7.5 . Addition of NADH as a cofactor stimulated enzymatic activity but the activity was not stimula