Microbiol Immunol, 1985, 29(12), 1185 - 95 Solubilization and partial properties of receptor substance for bacteriophage alpha 2 induced from Clostridium botulinum type A 190L; Takumi K et al.; Bacteriophage alpha 2, one of the two inducible phages from Clostridium botulinum type A 190L, had a latent period of 55 min and an average burst size of 75 in C . botulinum type A Hall used as the host bacterium . The phage particles were adsorbed on the cell walls extracted with hot trichloroacetic acid (TCA-walls) . The receptor substance for the phage was solubilized from the TCA-walls with Achromopeptidase and fractionated by gel filtration on Sephadex G-150 . The fraction having the highest level of receptor activity for the phage contained large amounts of muramic acid and glucosamine . Both authentic muramic acid and glucosamine significantly inactivated the phage, whereas glucose, galactose, L-and D-alanine, diaminopimeric acid, or D-glutamic acid did not exhibit similar activity . There results strongly suggest that the receptor site for phage alpha 2 is closely associated with glycan moieties of the cell wall peptidoglycan. Scand J Infect Dis Suppl, 1985, 46, 47 - 56 Pathogenesis and diagnosis of clostridium difficile enterocolitis; Mollby R et al.; Antibiotic associated Clostridium difficile enterocolitis is an infectious disease with symptoms ranging from self-limiting diarrhoea to severe colitis with bloody stools and formation of pseudomembranes . The carrier rate of C . difficile in a general Swedish population was found to be low (2%; 11/594) . In patients with acute diarrhoea unrelated to antibiotics the bacterium or its toxin was found in 3% (12/398) . In patients with diarrhoea associated with antibiotics C . difficile or its toxin was demonstrated in 18% (873/4 793) during 1980-1982 . Local outbreaks reported recently from different hospital wards in Sweden suggest that nosocomial spread of C . difficile takes place among patients on antibiotic treatment . Immunochemical fingerprinting of the isolates from one outbreak showed that one specific strain of C . difficile had spread among the patients in one hospital ward . C . difficile produces at least 2 toxins: a cytotoxin currently used in the diagnosis of C . difficile enterocolitis, and an enterotoxin . A "sandwich" enzyme-linked immunosorbent assay (ELISA) was used for the detection of enterotoxin in stool specimens . The enterotoxin was demonstrated in 80% (57/71) of patients with cytotoxin in stools . In an additional 5 patients with colitis the immunoassay was positive while the cytotoxin assay remained negative . An immunoassay demonstrating circulating antibodies to C . difficile toxins seemed to be positive in about half of the patients with verified C . difficile infection . It was notable that most patients suffering from repeated episodes of colitis did not develop an antibody response until after final recovery. Toxicon, 1985, 23(2), 235 - 46 A sensitive and useful radioimmunoassay for neurotoxin and its haemagglutinin complex from Clostridium botulinum; Ashton AC et al.; A sensitive radioimmunoassay for the detection of botulinum toxin, produced by Clostridium botulinum, was developed . This employs homogeneous botulinum neurotoxin type A and its 125I-labelled derivative of high specific radioactivity, rather than its complex with haemagglutinin as used hitherto . The sensitivity of the assay is 1 ng of neurotoxin per ml, which is equivalent to 80 LD50 units (half-lethal doses) in mice . Neurotoxin and its complex with haemagglutinin were measurable with equal sensitivity when using antibodies against botulinum neurotoxin type A . Specificity of the assay was demonstrated by the lack of response to type B and E botulinum toxins and to heat-inactivated botulinum toxin or extracts of Clostridium sporogenes strain BL46, which contains many surface antigenic determinants common to Clostridium botulinum . Using appropriate conditions, neurotoxin added to fish extract could be quantified accurately, proportionality being observed between the amounts of standard toxin added . In addition, the amounts of toxin species produced by culturing Clostridium botulinum in canned fish was measurable; the values obtained were comparable to those observed by the mouse bioassay . Moreover, the fish samples gave a dose-response curve in the competition radioimmunoassay which was paralleled by the response of botulinum neurotoxin standards . This assay offers the most sensitive, reliable immunological method available for the quantitation of molecular forms of botulinum toxin . As the technique can be used with unpurified fish extracts, it should be widely applicable to different types of samples contaminated with botulinum toxin; furthermore, the clinical diagnosis of human botulism could be substantiated with this method. Ciba Found Symp, 1985, 111, 97 - 111 Chiral products from non-pyridine nucleotide-dependent reductases and methods for NAD(P)H regeneration; Simon H et al.; Enoate reductase (EC 1.3.1.31) from a Clostridium tyrobutyricum strain catalyses the stereospecific reduction of many different alpha, beta-unsaturated carboxylates, aldehydes and even some ketones . The enzyme accepts electrons from NADH and, 1.5 times faster, from reduced methyl viologen (1,1'-dimethyl-4,4'-bipyridinium) . Another new type of non-pyridine nucleotide-dependent reductase has an extremely broad substrate specificity for 2-oxo-carboxylates and 2-oxo-dicarboxylates . In crude extracts from Proteus mirabilis and Proteus vulgaris, specific activities of 2-12 mumol product formed per mg protein per min can be found when reduced methyl or benzyl viologen is used as electron donor . The products are (2R)-hydroxy acids . Enoate reductase and 2-oxo-carboxylate reductase are suitable for electro-enzymic reductions in which catalytic amounts of viologens are continuously reduced in an electrochemical cell . This procedure has three advantages: (1) regeneration of NAD(P)H by a second enzyme and substrate is not required, (2) the unstable pyridine nucleotides are not required in the reaction mixture, and (3) the rate of the reaction can be observed continuously by measuring an electric current . Several yeasts, as well as aerobic and anaerobic bacteria, catalyse the reduction of NAD(P)+ by reduced methyl viologen . Such cells can be used for electro-microbial reductions when only pyridine nucleotide-dependent reductases are present . Information about the enzymes which catalyse the reduction of NAD(P)+ at the expense of reduced methyl viologen is given. Zentralbl Allg Pathol, 1985, 130(1), 45 - 50 Pathogenesis of pseudomembranous colitis; Pesce CM et al.; This work is concerned with new morphologic data pointing to an immune component in the pathogenesis of pseudomembranous colitis . The focal distribution of the pseudomembranes suggests selective damage induced by Clostridium difficile toxins . The sites of attachment to the mucosa correspond anatomically to the intestinal structures specialized for immune information and response . Furthermore, viable IgA production supports the view that toxins are carried to lymphoid aggregates where plasma cell proliferation takes place . A sharp increase in the mast cell population of the colon is also reported . Mast cells, whose role in the pathogenesis of intestinal diseases is still obscure, are diffusely distributed, irrespective of the focal lesions of pseudomembranous colitis. J Clin Microbiol, 1985 Jan, 21(1), 122 - 6 Comparison of PRAS II, RapID ANA, and API 20A systems for identification of anaerobic bacteria; Karachewski NO et al.; This study evaluated the PRAS II, RapID ANA, and API 20A systems for the identification of anaerobic bacteria . A total of 80 isolates (68 fresh clinical isolates and 12 stock cultures) were examined and included 25 Bacteriodes spp., 7 Fusobacterium spp., 12 Clostridium spp., 2 Veillonella spp., 16 gram-positive cocci, and 18 gram-positive nonsporeforming bacilli . All isolates were initially identified by the procedures outlined in Holdeman et al . (ed.), Anaerobe Laboratory Manual, Virginia Polytechnic Institute and State University, Blacksburg, Va., 1977; identifications from the PRAS II, RapID ANA, and API 20A systems were compared with these initial identifications . If no supplemental tests were required, the RapID ANA and API 20A systems had incubation times of 4 and 24 h, respectively; the PRAS II system generally required 2 to 5 days of incubation, depending on the growth rate of the isolate . PRAS II identified 74% correct to species level, 14% correct to genus only, and 6% incorrect; 6% could not be identified . PRAS II data were reevaluated according to a revised data base that was provided after completion of the study; PRAS II (revised) identified 82% correct to species, 12% correct to genus only, and 6% incorrect . RapID ANA identified 62% correct to the species level, 28% correct to genus only, and 10% incorrect . API 20A identified 71% correct to the species level, 10% correct to genus only, and 3% incorrect; 16% could not identified . The API 20A is a more established system for identification of anaerobic bacteria; PRAS II and RapID ANA appear to be promising new methods for the identification of anaerobic bacteria. Scand J Infect Dis Suppl, 1985, 46, 57 - 63 Beta-lactamases in anaerobic bacteria; Nord CE et al.; The known mechanisms of beta-lactam resistance in anaerobic bacteria involve production of beta-lactamases, alteration of penicillin-binding proteins and blocked penetration of beta-lactams through the outer membranes . The most important factor in beta-lactam resistance is production of beta-lactamase . Beta-lactamases in various Bacteroides, Fusobacterium and Clostridium species have been described . Beta-lactam resistance in Bacteroides fragilis is most commonly mediated by beta-lactamase production mainly of cephalosporinase character . Recent studies have also shown that B . fragilis can produce a penicillinase which inactivates piperacillin and carbenicillin . Enzymes inactivating cefoxitin and imipenem have also been isolated from B . fragilis . The Bacteroides non-fragilis species produce beta-lactamases of mainly penicillinase character . Recently a penicillinase from Fusobacterium nucleatum has been characterized . Among the clostridia, Clostridium butyricum, C . clostridiiformis and C . ramosum have been shown to produce penicillinases. Drugs Exp Clin Res, 1985, 11(7), 431 - 4 The comparative activity of twelve 4-quinolone antimicrobials against gram-positive and gram-negative anaerobes; Robbins MJ et al.; The minimal inhibitory concentrations (MICs) of twelve 4-quinolone antimicrobials were determined for the Bacteroides fragilis group (50), Bacteroides melaninogenicus (20), Bacteroides bivius (10), Fusobacterium spp . (10), anaerobic Gram-positive cocci (50) and Clostridium spp . (20) . MICs were determined using an agar dilution technique in Mueller-Hinton agar supplemented with 10% lysed horse blood . The inoculum used was approximately 10(4) colony-forming units, contained in 10 microliter of Mueller-Hinton broth, which was applied to the agar plates using a multipoint inoculator . Following inoculation, plates were incubated at 37 degrees C for 48 h in an anaerobic atmosphere . The MIC of each antimicrobial for each isolate examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum . The minimum concentrations required to inhibit the growth of 50% (MIC50) and 90% (MIC90) of the organism examined were also determined . All of the more recently synthesised 4-quinolones showed increased activity against the anaerobic bacteria used in this study . Ciprofloxacin and ofloxacin were the most active compounds examined (Bacteroides fragilis group MIC90 ciprofloxacin 4 micrograms/ml; ofloxacin 4 microgram/ml; Bacteroides melaninogenicus MIC90 ciprofloxacin 2 micrograms/ml, ofloxacin 2 micrograms/ml; Bacteroides bivius MIC90 ciprofloxacin 16 micrograms/ml, ofloxacin 32 micrograms/ml; Fusobacterium spp . MIC90 ciprofloxacin 2 micrograms/ml, ofloxacin 4 micrograms/ml; Clostridium spp . MIC90 ciprofloxacin 1 microgram/ml, ofloxacin 1 microgram/ml and anaerobic Gram-positive cocci MIC90 ciprofloxacin 4 micrograms/ml, ofloxacin 4 micrograms/ml). Rev Argent Microbiol, 1985, 17(1), 55 - 8 {Specificity against group A, C, and G streptococci of the reverse CAMP test for identifying Clostridium perfringens}; Martini P et al.; We studied "reverse CAMP" test specificity for the identification of Clostridium perfringens, and the probable existence of cross-reactions with groups A, C and G streptococci . Ninety eight Clostridium strains were tested with ten S . agalactiae, ten S . pyogenes, two group C streptococci and one group G . All the strains of S . agalactiae yielded a positive "reverse CAMP" reaction when it was performed with C . perfringens strains . Cross-reactions were not detected when C . perfringens were tested with groups A, C or G streptococci . The "reverse CAMP" test proved to be specific for all the C . perfringens strains used. Microbios, 1985, 44(181S), 271 - 9 Isolation of Clostridium species from herbage; Princewill TJ et al.; A total of fourteen herbage samples were collected from paddocks on six farms from two States of Nigeria . These samples yielded twenty two clostridial isolates out of which eleven were identified as Clostridium perfringens and five as Clostridium bifermentans . The other species identified were less frequently isolated . No farm yielded all the species isolated . The farm with the highest distribution of the species of Clostridium was Government Farm, Butura, Plateau State, with a frequency distribution of 50% of the species isolated . In each of two other farms the distribution was 37.5%; and in each of the remaining farms it was less than 30% in frequency . The effect of ubiquitous clostridial infections on ruminants is discussed. Arch Orthop Trauma Surg, 1985, 104(4), 262 - 4 Wound infection after lower extremity amputation because of ischemia; Moller BN et al.; The importance of postoperative wound infection in major amputations was elucidated by recording the organisms isolated in preoperatively infected gangrene and in postoperatively infected wounds of patients undergoing lower-limb amputations for ischemia . Sixty-four amputations were performed on 61 patients . The frequency of coexisting diabetes mellitus was 34% . Postoperative infections occurred in nearly two-thirds of the 19 cases of infected gangrene, as compared with less than one-third of cases of noninfected gangrene . The presence of diabetes mellitus did not significantly influence the infection rate . Preoperatively as well as postoperatively, the most frequently isolated bacterium was Staphylococcus aureus . Clostridium perfringens was cultured in four cases . Postoperative wound infection following lower-limb amputation for ischemia is the main reason for reamputation, especially in patients with infected gangrene. Acta Med Scand, 1985, 218(3), 341 - 3 Clostridium perfringens septicemia and acute leukemia; Grutzmeier S; Three cases of fatal clostridial septicemia in patients with acute leukemia are described . Predisposing factors and treatment are discussed . Clostridium septicemia should always be suspected when a patient with neutropenia suddenly develops diffuse abdominal pain, fever, and tachycardia over 120/min . The importance of early treatment with penicillin or another adequate antibiotic is discussed. Microbiol Immunol, 1985, 29(6), 509 - 16 The suitability of Tórtora's medium for the production of enterotoxin in Clostridium perfringens strains; Tortora JC et al.; Examination of 200 samples from soil and the same number of samples from healthy human feces yielded 49 (24.5%) and 105 (52.5%) strains of heat-resistant Clostridium perfringens respectively . Fourteen (7.0%) strains isolated from soil and 37 (18.5%) from feces synthesized enterotoxin, as demonstrated by Tortora's method, at sufficient levels to permit its detection by mouse lethality, microslide double gel diffusion or counterimmunoelectrophoresis tests . By using the Duncan-Strong (DS) method, only four (2%) enterotoxigenic strains from soil and 14 (7.0%) from feces were obtained . The supernatant fluid from two enterotoxigenic-negative strains grown in DS medium gave a false-positive reaction when they were injected intravenously into mice . Tortora's medium was preferable because a larger number of isolated strains produced spores and enterotoxin to permit their recognition as enterotoxigenic strains. Microbios, 1985, 42(169-170), 155 - 62 Animal feeds as likely vehicles of clostridial infections in livestock; Princewill TJ et al.; A total of sixteen samples were collected from various food items on seven farms in three States of Nigeria . These samples yielded thirty one clostridial isolates which were identified as eleven species . The species most frequently isolated was Clostridium perfringens representing 35.5% of the isolates . The next highest was C . bifermentans with a frequency of 22.6% . The other nine species identified were each isolated at frequencies less than 10% . No farm yielded all the species isolated . The farms with the highest distribution of the species of Clostridium were Government Farm, Buruku (Kaduna State), Kano Farm Centre and Government Farm, Butura (Plateau State) with frequency distributions of 36.4% each of the species isolated . Except at the Dairy Cattle Multiplication Centre, Shika (Kaduna State), where the frequency distribution was 27.3%, at none of the other three farms was the distribution more than 20%. Comp Biochem Physiol A, 1985, 81(4), 713 - 39 Effectors of amino acid transport processes in animal cell membranes; Lerner J; Various effectors, which act upon ion gradients, protein synthesis, membrane components or cellular functional groups, have been employed to provide insights into the nature of amino acid-membrane transport processes in animal cells . Such effectors, for example, include ions, hormones, metabolites and various organic reagents and their judicious use has allowed the following list of conclusions . Sodium ion has been found to stimulate amino acid transport in a wide variety of cell systems, although depending on the tissue and/or substrate, this ion may have no effect on such transport, or even inhibit it . Amino acid transport can be stimulated in some cell systems by other ions such as K+, Li+, H+ or Cl- . Both H+ and K+ have been found to be inhibitory in other systems . Amino acid transport is dependent in many cell systems upon an inwardly directed Na+ gradient and is stimulated by a membrane potential (negative cell interior) . In some cell systems an inwardly directed Cl- and H+ gradient or an outwardly directed K+ gradient can energize transport . Structurally dissimilar effectors such as ouabain, Clostridium enterotoxin, aspirin and amiloride inhibit amino acid transport presumably through dissipation of the Na+ gradient . Inhibition by certain sugars or metabolic intermediates of the tricarboxylic acid cycle may compete with the substrate for the energy of the Na+ gradient or interact with the substrate at the carrier level either allosterically or at a common site . Stimulation of transport by other sugars or intermediates may result from their catabolism to furnish energy for transport . Insulin and glucagon stimulate transport of amino acids in a variety of cell systems by a mechanism which involves protein synthesis . Microtubules may be involved in the regulation of transport by insulin or glucagon . Some reports also suggest that insulin has a direct effect on membranes . In addition, a number of growth hormones and factors have stimulatory effects on amino acid transport which are also mediated by protein synthesis . Steroid hormones have been noted to enhance or diminish transport of amino acids depending on the nature of the hormone . These agents appear to function at the level of protein synthesis . While stimulation may involve increased carrier synthesis, inhibition probably involves synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system.(ABSTRACT TRUNCATED AT 400 WORDS) Toxicon, 1985, 23(3), 449 - 55 Effect of Clostridium perfringens alpha toxin on the isolated rat vas deferens; Sakurai J et al.; Alpha toxin produced by Clostridium perfringens potentiated norepinephrine-evoked contraction in the isolated rat vas deferens, but itself caused no contraction within 60 min . The potentiating activity was dependent on the dose of the toxin and was quantitatively related to the phospholipase C activity of the toxin preparation. Prog Clin Biol Res, 1985, 181, 65 - 8 Steam quality and effective sterilization; Sedlacek RS et al.; Faced with using steam from a commercial utility having boilers greater than 5 miles distant and being the last user on the system resulted in ineffective sterilization . A three phase testing program was established utilizing: Direct physical measurements - an Ellison model 915A portable steam calorimeter . Direct microbiology - Autoclaved feed pellets were aseptically placed in fluid thioglycolate medium and incubated at 37 degrees C . Indirect microbiology - Feces from "defined flora" mice fed the autoclaved pelleted feed were tested . Colorimetric measurements verified that the steam sometimes contained greater than 5% entrained water . During periods of wet steam it was impossible to maintain consistent sterility of the mouse pellets even using a cycle of 126 degrees C for 60 minutes . One spore-forming Gram positive rod, Clostridium perfringens type D was the predominant bacterium isolated . Lactating mice, or mice stressed experimentally came down with diarrhea within days of eating pellets treated with wet steam (calorimetric measurements) and a subsequent positive culture . These mice voided stools predominantly showing Clostridium perfringens type D. Microbiol Immunol, 1985, 29(4), 317 - 26 Heat resistance, spore germination, and enterotoxigenicity of Clostridium perfringens; Ando Y et al.; Heat resistance at 95 C, heat activation at 75 C, and germination response were determined for spores of 10 serotype strains of Clostridium perfringens type A, including five heat-resistant and five heat-sensitive strains . The D95-values ranged from 17.6 to 63.0 and from 1.3 to 2.8 for the heat-resistant and the heat-sensitive strains, respectively . The heat-activation values, the ratios between the heated and unheated viable counts of spore suspensions, ranged from 0.0035 to 0.65 and from 6.5 to 60.0 for the heat-sensitive and the heat-resistant strains, respectively . Spores of these strains were divided into two distinct germination types on the basis of their germination response; spores of the heat-resistant strains germinated in KC1 medium after heat activation (K-type), and spores of the heat-sensitive strains germinated in a mixture of L-alanine, inosine, and CaCl2 in the presence of CO2 without heat activation (A-type) . The strains were tested for enterotoxigenicity by a reversed passive latex-agglutination (RPLA) test . All the heat-resistant strains were RPLA-positive, whereas the heat-sensitive strains were all RPLA-negative . A total of 37 strains of the organism isolated from food-poisoning outbreaks were tested for spore germination and enterotoxin formation . All of the 20 heat-resistant strains showed K-type spore germination and, except for three strains, were RPLA-positive, whereas all of the 17 heat-sensitive strains showed A-type spore germination and, except for only one strain, were RPLA-negative. Ciba Found Symp, 1985, 112, 230 - 41 Clostridial toxins active locally in the gastrointestinal tract; Wilkins T et al.; Clostridium difficile and Clostridium spiroforme have only in recent years been recognized as intestinal pathogens . They both produce toxins that are also produced by other clostridia . C . difficile toxins A and B are produced by C . sordellii and a few strains of C . perfringens whereas C . spiroforme produces the same toxins as C . perfringens Type E (iota toxin) . Iota toxin activity may be the product of two proteins . Toxigenic strains of C . spiroforme and Type E produce two antigens which possess much more biological activity when administered together than when given alone . C . difficile was thought for some time to produce only a single toxin, but then the enterotoxic activity was shown to be due to a separate toxin (toxin A) . This toxin increases the oral toxicity of toxin B (the main cytotoxin) and may increase the permeability of the colon . Toxin A binds to a specific receptor in hamster brush border membranes and in the membranes of rabbit erythrocytes . This receptor appears to be a glycoprotein . The receptor can be extracted from the membrane with Triton and binds to immobilized toxin A . The receptor can be extracted and used to coat plastic plates as a first phase in an ELISA assay . Another assay has been developed in which the toxin A binds to the red cells and then the erythrocytes are agglutinated with antitoxin . An even more sensitive assay consists of using rabbit erythrocyte ghosts to bind the toxin and then precipitating the ghosts with antibody to toxin A attached to latex beads . Monoclonal antibodies to toxin A also have been developed and are used in these and other assays. Infect Immun, 1985 Jan, 47(1), 260 - 3 Tryptophan content of Clostridium perfringens epsilon toxin; Sakurai J et al.; The tryptophan content of Clostridium perfringens epsilon toxin was investigated . When the tryptophan content was determined by amino acid analysis after the hydrolysis of epsilon prototoxin with methanesulfonic acid containing 3-(2-aminoethyl)indole and by the spectrophotometric method with N-bromosuccinimide, the number of tryptophan residues was calculated at 1/mol of the protein . Cleavage of the prototoxin or the toxin with N-bromosuccinimide in the presence of urea gave two new fragments on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . There was only a tyrosine residue as the new N-terminal amino acid after the cleavage of the prototoxin or the toxin with N-bromosuccinimide . The data showed that epsilon prototoxin or epsilon toxin contained only one tryptophan residue. J Biochem (Tokyo), 1985 Jan, 97(1), 213 - 8 The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin; Agui T et al.; The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane . The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin . These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S . (1983) J . Biochem . 94, 521-527) . The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) {N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)}galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b . The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM . Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively . Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody. Biol Cell, 1985, 53(1), 23 - 32 Effect of the cytotoxin of Clostridium difficile on cultured hepatoma cells; Rihn B et al.; Clostridium difficile is the major etiologic agent of human pseudomembranous colitis . It produces two toxins: an enterotoxin and a cytotoxin . In cultured hepatoma cells, at very low doses, the cytotoxin inhibits the incorporation of precursors into biological macromolecules . Protein synthesis is more affected than RNA and DNA synthesis . The toxin also induces severe alterations of the cell morphology consisting in damages to the cytoskeleton and to the cell shape. Biochemistry, 1984 Dec 18, 23(26), 6345 - 9 Electron transfer between flavodoxin semiquinone and c-type cytochromes: correlations between electrostatically corrected rate constants, redox potentials, and surface topologies; Tollin G et al.; We have measured the ionic strength dependence of the rate constants for electron transfer from the semiquinone of Clostridium pasteurianum flavodoxin to 12 c-type cytochromes and several inorganic oxidants using stopped-flow methodology . The experimental data were fit quite well by an electrostatic model that represents the interaction domains as parallel disks with a point charge equal to the charge within this region of the protein . The analysis provides an evaluation of the electrostatic interaction energy and the rate constant at infinite ionic strength (k affinity) . The electrostatic charge on the oxidant within the interaction site can be obtained from the electrostatic energy, and for most of those reactants for which structures are available, the results are in good agreement with expectation . The k affinity values were found to correlate with redox potential differences, as expected from the theory of adiabatic (or nonadiabatic) outer-sphere electron-transfer reactions . Deviations from the theoretical curves are interpreted in terms of the influence of surface topology on reaction rate constants . In general, we find that electrostatic effects, steric influences, and redox potential all exert a much larger effect on reaction rate constants for the flavodoxin-cytochrome system than has been previously observed for free flavin-cytochrome interactions . The implications of this for determining biological specificity are discussed. J Biol Chem, 1984 Dec 10, 259(23), 14328 - 31 Mössbauer and electron nuclear double resonance study of oxidized bidirectional hydrogenase from Clostridium pasteurianum W5; Wang G et al.; The bidirectional hydrogenase from Clostridium pasteurianum W5 is an iron-sulfur protein containing approximately 12 Fe atoms and 12 labile sulfides . We have studied oxidized samples of the enzyme with Mossbauer and electron nuclear double resonance (ENDOR) spectroscopy to elucidate the nature of the center that gives rise to the EPR signal with principal g-values at 2.10, 2.04, and 2.01 . The g = 2.10 center exhibits two well-resolved 57Fe ENDOR resonances . One is isotropic with A1 = 9.5 MHz; the other is nearly isotropic with A2 = 17 MHz . These magnetic hyperfine coupling constants are substantially (approximately 50%) smaller than those observed for {2Fe-2S}, {3Fe-4S}, and {4Fe-4S} clusters . The Mossbauer and ENDOR data, taken together, suggest that the g = 2.10 center contains at least two but not more than four iron atoms . Comparison of our data with recent results reported for Escherichia coli sulfite reductase and the ferricyanide-treated {4Fe-4S} cluster from Azotobacter vinelandii ferredoxin I suggests that the g = 2.10 center may possibly be formed, by oxidation, from a structure with a {4Fe-4S} core . The Mossbauer spectra give evidence that at least 8 of the 12 Fe atoms of oxidized hydrogenase are organized in two ferredoxin-type {4Fe-4S} clusters, supporting conclusions derived previously from EPR studies of the reduced enzyme. Chemioterapia, 1984 Dec, 3(6), 365 - 7 In vitro activity of rifaximin and rifampicin against some anaerobic bacteria; Lamanna A et al.; Activity of rifampicin and of a new rifamycin, rifaximin, was tested in strains of anaerobic bacteria belonging to the Bacteroides genus (75 B . fragilis group and 17 Bacteroides non-fragilis group) and in Clostridium perfringens (15 strains) . It turned out that the bactericidal activity of both rifamycins could be overlapped and that it equalled 100% in the case of the non-fragilis Bacteroides and Clostridium species. J Antimicrob Chemother, 1984 Dec, 14 Suppl D, 85 - 95 Diagnosis and epidemiology of Clostridium difficile enterocolitis in Sweden; Aronsson B et al.; Experience of the diagnosis and epidemiology of Clostridium difficile in Sweden is reviewed . Samples from 5885 patients have been investigated at the National Bacteriological Laboratory in Stockholm from 1978-1983 . Patients originate from all parts of the country and their number continues to increase . Cl . difficile seem to be of growing importance, especially in nosocomial infections . Most patients with antibiotic-associated diarrhoea and colitis (AAD/AAC) and Cl . difficile in their stools were above 60 years of age (63%) and there was a significant preponderance of females over males in the age groups 21-50 and above 60 years of age . The antibiotics most commonly associated with Cl . difficile enterocolitis (CDE) were penicillins, cephalosporins and clindamycin/lincomycin . On the basis of consumption clindamycin/lincomycin and cephalosporins are associated 70 and 40 times, respectively, more often than penicillins in CDE . Diagnosis of CDE relies mainly on detection of the cytotoxin (toxin B) in stool specimens . It was present in 873/4793 (18.2%) patients whereas the bacterium was found in only 12% . An immunoassay for detection of the enterotoxin (toxin A) of Cl . difficile seems to be a useful alternative to the cytotoxin assay, but some stool specimens with a low toxin B titre were negative . Five specimens negative for toxin B were positive for toxin A and came from patients where additional information suggested CDE . A serological assay for demonstration of circulating antibodies to Cl . difficile toxins has also been evaluated and is positive in about half of the cases of CDE . Antibody response seems to be absent or delayed in patients with relapse of colitis after antibiotic treatment. Vet Med (Praha), 1984 Dec, 29(12), 747 - 52 {Botulism of aquatic birds at the Starý u Pohorelic pond (Breclav District)}; Hubalek Z et al.; In the years 1981 and 1982 when a mass mortality of wild and domestic water birds was observed on the Stary pond, eleven diseases or just died birds were examined by the neutralization test for botulotoxin and four sludge samples for the presence of Clostridium botulinum . C . Botulinum toxin of C type was detected in four wild ducks (Anas platyrhynchos) from September 1981 and July 1982 and in one gull (Larus ridibundus) and July 1982 . The highest titres (of mice i . p . LD50/g) of botulotoxin in ducks were 10(6.3) in the intestinal contents, 10(6.1) in the liver tissue and 10(5.1) in the stomach contents . In one duck we detected, besides the C type, a smaller botulotoxin amount of A type . Therefore we can recommend a complete serotypifying for the cases of botulism in water birds . Out of four sludge samples collected in September 1982 one sample was negative, whereas in three samples the C botulotoxin was demonstrated after propagation and one C . botulinum strain of C type was isolated. J Antimicrob Chemother, 1984 Dec, 14 Suppl D, 7 - 18 Mode of action and in-vitro activity of vancomycin; Watanakunakorn C; Vancomycin is a unique glycopeptide structurally unrelated to any currently available antibiotic . It also has a unique mode of action inhibiting the second stage of cell wall synthesis of susceptible bacteria . There is also evidence that vancomycin alters the permeability of the cell membrane and selectively inhibits ribonucleic acid synthesis . Induction of bacterial L-phase variants from susceptible organisms with vancomycin is extremely difficult, and such variants are unstable . Stable L-phase variants induced by other agents are susceptible to vancomycin . Vancomycin is active against a large number of species of Gram-positive bacteria, such as Staphylococcus aureus (including methicillin-resistant strains), Staph . epidermidis (including multiple-resistant strains), Streptococcus pneumoniae (including multiple-resistant strains), Str . pyogenes, Str . agalactiae, Str . bovis, Str . mutans, viridans streptococci, enterococci, Clostridium species, diphtheroids, Listeria monocytogenes, Actinomyces species and Lactobacillus species . There has been no increase in resistance to vancomycin during the past three decades . Enhancement of antimicrobial activity has been demonstrated with the combination of vancomycin and an aminoglycoside against Staph . aureus, Str . bovis, enterococci and viridans streptococci . The combination of vancomycin and rifampicin are antagonistic to most strains of Staph . aureus, though indifference and occasionally synergism have been shown, but is synergistic against strains of Staph . epidermidis . It shows indifference against enterococci . Vancomycin and fusidic acid are indifferent against Staph . aureus. Onderstepoort J Vet Res, 1984 Dec, 51(4), 249 - 52 Bacteriological findings regarding the hygienic safety of poultry litter intended as an ingredient of feeds for ruminants; Ogonowski K et al.; An investigation of poultry litter intended for use in farm feeds showed that 0,37%, 0,49%, 0,25% and 12,3% of the 813 samples tested were contaminated with Clostridium spp., haemolytic Escherichia coli, Staphylococcus aureus and 21 different species of Salmonella . The findings clearly underline the hygienically dangerous nature of crude poultry litter . The practical implications of the results are briefly discussed, particularly in view of current regulations. J Clin Microbiol, 1984 Dec, 20(6), 1209 - 12 Evaluation of fluorescent-antibody tests as a means of confirming infant botulism; Glasby C et al.; Fluorescent-antibody techniques were evaluated for confirming infant botulism . Seventy-seven stool specimens from suspected cases were examined . All 34 specimens containing viable Clostridium botulinum at time of study gave positive results (29 on direct smears and 34 on enrichments) . Two false-positive reactions were observed. J Clin Microbiol, 1984 Dec, 20(6), 1145 - 53 Rapid differentiation of enterotoxigenic Escherichia coli that produce heat-stable and heat-labile toxins by frequency-pulsed electron capture gas-liquid chromatography analysis of diarrheal stool specimens; Brooks JB et al.; Thirty-three stool specimens from infants in the village of Tamooh near Cairo, Egypt, were studied by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC) . In 13 of the diarrheal cases, the suspected causative agent isolated was Escherichia coli which produced heat-stable toxin (ST), and in 10 other cases E . coli that produced heat-labile toxin (LT) were isolated . Ten control stool samples, collected from infants from whom no pathogenic organisms were isolated, were analyzed at the same time . Comparisons also were made against healthy control stools from individuals in the United States who had been previously analyzed by FPEC-GLC (Brooks et al., J . Clin . Microbiol . 20:549-560, 1984) . The stools were suspended in water and centrifuged, and the supernatant was extracted with organic solvents and derivatized to form electron-capturing derivatives of carboxylic acids, hydroxy acids, alcohols, and amines . Results from the study showed distinct differences among the FPEC-GLC profiles of E . coli ST-positive stools, of E . coli LT-positive stools, and of the control stool samples . An unidentified compound appearing in the ether-soluble hydroxy acid fraction from E . coli ST-positive stools was tentatively identified by mass spectrometry as 6-methoxy-2-hydroxyhexanoic acid . 6-Methoxy-2-hydroxyhexanoic acid was found in all stools that contained E . coli ST but was not present either in stools from which E . coli LT was isolated or in control samples . 6-Methoxy-2-hydroxyhexanoic acid may prove to be an important marker for use in the identification of E . coli ST . In addition to 6-methoxy-2-hydroxyhexanoic acid, the carboxylic acid, alcohol, and amine FPEC-GLC profiles obtained from stools were very different between these two organisms . The data indicate that FPEC-GLC analysis of diarrheal stool specimens might be a rapid way to distinguish diarrhea caused by E . coli ST, E . coli LT, Clostridium difficile, and rotavirus. J Antimicrob Chemother, 1984 Dec, 14 Suppl D, 103 - 9 Treatment of pseudomembranous colitis and antibiotic-associated diarrhoea; Burdon DW; Pseudomembranous colitis is caused by release of toxins from Clostridium difficile when it colonizes the large intestine . This clostridium is susceptible to concentrations of vancomycin which are readily attained in the colon after oral administration . When vancomycin is given orally to infected patients in a dose of 125 mg every 6 h, a rapid clinical cure can be expected . Some patients may relapse after the vancomycin is stopped, but a further course of treatment will control symptoms. J Med Microbiol, 1984 Dec, 18(3), 385 - 91 Production and release of toxins A and B by Clostridium difficile; Ketley JM et al.; The production and release of toxins A and B by Clostridium difficile during in-vitro culture was investigated . Cell-associated toxin A was detected by immunoelectrophoresis of bacterial extracts released by ultrasonication and by fluorescent antibody labelling of whole cells . Extracellular toxin A was detected by immunoelectrophoresis and by enzyme-linked immunosorbent assay; extracellular toxin B was detected by cytotoxin assay . Both toxins A and B were produced and released during the decline phase of the bacterial growth cycle . The possible significance of these results in relation to the pathogenesis of pseudomembranous colitis is discussed. Infect Immun, 1984 Dec, 46(3), 715 - 9 Botulism in metronidazole- treated conventional adult mice challenged orogastrically with spores of Clostridium botulinum type A or B; Wang Y et al.; Conventional adult mice were pretreated with metronidazole to make their intestinal tracts receptive to colonization by Clostridium botulinum . These mice, in groups of 10, were fed 0 (controls), 10(2), 10(3), 10(4), or 10(5) C . botulinum type B spores and were placed for observation in filter-lid cages whose screen floors minimized the amounts of feces available for coprophagy . The opportunity to eat feces was made equal for all mouse groups by placing one mouse of every group in each of 10 cages . Mice given a spore inoculum began to develop botulism after incubation periods of slightly less than 2.75 days . Morbidity rates, which reached maxima within 5 days of challenge, were related to inocula levels . Mortality rates were also dose related . Mice given 10(5) spores and then type B antitoxin intraperitoneally, a treatment not affecting intraintestinal toxin production, remained healthy . Morbidity among control mice was seldom more than 10% and could be ascribed to toxin ingested with feces . A C . botulinum type A spore suspension gave similar results, although morbidity and mortality rates were generally lower than after challenge with a comparable number of type B spores . Mice challenged with 10(2) or 10(5) spores had similar toxin levels in their large intestines 48 h later . Morbidity rates correlated better with toxin levels in the small intestines. J Immunol, 1984 Dec, 133(6), 3250 - 4 Lysine residues, but not carbohydrates, are required for the regulatory function of H on the amplification C3 convertase of complement; Jouvin MH et al.; Lysine epsilon-amino groups of human factor H were selectively converted to guanidino groups by treatment with 0.1 M O-methylisourea at pH 10.4 . Guanidination resulted in a dose-dependent decrease in the capacity of the regulatory protein to accelerate decay dissociation of P-stabilized amplification C3 convertase sites, to serve as a co-factor for cleavage of cell-bound C3b by I, and to compete for binding of 125I-untreated H to C3b . Modification of approximately 75% lysine epsilon-amino groups suppressed 97% of H functional activity . Biochemical analysis of native H demonstrated a total carbohydrate content of 18.5% (w/w) and the presence in the molecule of 11 biantennary oligosaccharidic chains of the N-acetyl-lactosaminic type . Total desialation of H by using Clostridium perfringens neuraminidase, and total deglycosylation of desialated H by using beta-endo-N-acetylglucosaminidase resulted in a 1.5- to 2-fold increase in H activity on a weight basis . Deglycosylation did not alter the capacity of H to discriminate between activating and nonactivating surfaces of the alternative pathway . Thus, lysine residues are important determinants of the binding capacity of H for cell-bound C3b, whereas the carbohydrate portion of the molecule is not required for the regulatory function of the protein on the amplification C3 convertase. J Immunol, 1984 Dec, 133(6), 2898 - 903 Immune response to ferredoxin; nonresponder status is not due to suppression; Sikora LK et al.; Ferredoxin (Fd), a small protein from Clostridium pasteurianum, has been selected for immunologic studies because of its limited number (two) of antigenic determinants . Functionally (as determined by antibody binding), monodeterminant fragments of Fd can be generated enzymatically, leaving molecules only a few amino acids smaller than the native protein, with unaltered solid phase binding properties . These fragments were used to assess the immune response to each of the two determinants . Clear differences in immunologic properties can be assigned to sequences within Fd: the amino terminal tripeptide is responsible for inducing a proliferative response and limited antibody production, whereas the carboxy terminal dipeptide accounts for most of the antibody activity, yet little, if any, T-proliferative activity . Studies with the enzyme-generated fragments of Fd have unmasked a sequence proximal to the amino terminal that represents a second determinant for T cell proliferation but does not have any demonstrable antibody-inducing activity . This third determinant is shown to induce responsiveness to Fd in nonresponder animals after the removal of the amino terminal tripeptide . The results indicate that nonresponsiveness to this molecule in H-2d mice is not a direct effect of suppression. Gastroenterology, 1984 Dec, 87(6), 1344 - 50 Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity . Assessment of inflammation in rat and hamster models; Krawisz JE et al.; An assay was devised to quantitate acute intestinal inflammation based on the assessment of myeloperoxidase activity . Myeloperoxidase is an enzyme found in neutrophils and, in much smaller quantities, in monocytes and macrophages . Myeloperoxidase was solubilized with hexadecyltrimethylammonium bromide and myeloperoxidase activity was measured with a dianisidine-H2O2 assay . In neutrophil suspensions, myeloperoxidase activity was directly related to cell number down to as few as 500 cells . Myeloperoxidase activity was assayed in two animal models of inflammation: acetic acid-induced colitis in rats and Clostridium difficile enterotoxin-induced enteritis in hamsters . In both models, the activity of myeloperoxidase solubilized from the inflamed tissue was directly proportional to the number of neutrophils seen in histologic sections . Histologic evaluation of neutrophil accumulation was performed by counting the number of neutrophils in a histologic section 0.18 mm long and 5 micron thick . In both animal models, myeloperoxidase activity was linearly related to neutrophil number from 400 and 4000 cells/mm . Myeloperoxidase activity from chronically inflamed colon, in which both neutrophils and histiocytes were present, was directly related to neutrophil content . Histiocytes did not contribute significantly to myeloperoxidase activity . The determination of myeloperoxidase activity in the intestine is a simple biochemical assay that can be used to quantitate inflammation. Wien Med Wochenschr, 1984 Nov 30, 134(22), 509 - 13 {Tinidazole in the therapy of anaerobic pulmonary infections}; Vetter N et al.; The purpose of the study in 10 patients with anaerobic lung infections was to demonstrate the efficacy of therapy with Tinidazole . In 9 patients, the clinical evaluation as well as X-ray and bacteriological evidence showed positive results of therapy, while in one patient a mixed anaerobic infection with Streptococcus an., Bacteroides frag., and Clostridium sp . was still present after the end of therapy . Side effects were not observed with Tinidazole therapy. Biochem Biophys Res Commun, 1984 Nov 30, 125(1), 413 - 9 Different susceptibility of alkylacyl--versus diacyl--and alkenylacyl--phosphatidylcholine subclasses to stimulation of biosynthesis by phospholipase C; Terce F et al.; Krebs II ascites cells were incubated with {3H} or {14C} choline in the presence or in the absence of Clostridium welchii phospholipase C (PLC) . At enzyme concentrations where cell lysis remained limited, PLC specifically enhanced phosphatidylcholine (PC) biosynthesis, as shown by comparison with {14C} ethanolamine . Further analysis revealed that the stimulating effect of PLC remained limited to 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and 1-alkenyl-2-acyl-GPC, whereas the biosynthesis of 1-alkyl-2-acyl-GPC, the putative precursor of platelet activating factor (PAF-acether) remained unchanged . These differences reflect different localizations of the three PC subclasses in the plasma membrane and are discussed in relation to the regulation mechanism of PC biosynthesis. Biochem Biophys Res Commun, 1984 Nov 14, 124(3), 690 - 5 A new purification procedure for Clostridium difficile enterotoxin; Rihn B et al.; Clostridium difficile produces two toxins, an enterotoxin and a cytotoxin . The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography) . The purified enterotoxin is composed of two subunits (A1 = 41,500, A2 = 16,000) and its pI is 3.5. Antimicrob Agents Chemother, 1984 Nov, 26(5), 785 - 6 In vitro susceptibility of anaerobic bacteria to ciprofloxacin (Bay o 9867); Prabhala RH et al.; About 80% of 70 clinical isolates of Bacteroides fragilis were inhibited by 4 micrograms of ciprofloxacin (Bay o 9867) per ml . The 90% MIC of ciprofloxacin was 8 micrograms/ml for other Bacteroides species, 2 micrograms/ml for Peptococcus species, 8 micrograms/ml for Peptostreptococcus species, and 16 micrograms/ml for Clostridium and Eubacterium species. Antimicrob Agents Chemother, 1984 Nov, 26(5), 665 - 9 Ferredoxin-linked reduction of metronidazole in Clostridium pasteurianum; Lockerby DL et al.; Clostridium pasteurianum cell-free extracts enzymatically reduced metronidazole when coupled by hydrogenase via reduced ferredoxin . A 5 mM concentration of methyl viologen, flavin adenine dinucleotide, or flavin mononucleotide could completely replace ferredoxin (0.05 mM) in the in vitro reduction assay system, whereas 5 mM benzyl viologen was less effective . However, when these electron carriers were used at a concentration of 0.05 mM, there was a drastic loss in their abilities to couple the metronidazole reduction system compared with the comparable concentration of ferredoxin . It is not understood why these flavin coenzymes participate in this enzymatic reaction . NAD and NADP had no activity when substituted for ferredoxin in the enzyme system . Two reduced ferredoxin-linked pathways, "metronidazole reductase" and the inducible dissimilatory sulfite reductase system, when combined in a single in vitro competition experiment demonstrated a preferential flow of electrons to metronidazole away from sulfite . A proposed bactericidal mechanism for metronidazole against C . pasteurianum incorporating the above findings is discussed. Age Ageing, 1984 Nov, 13(6), 363 - 6 Clostridium difficile diarrhoea: a highly infectious organism; Bennett GC et al.; This paper describes an outbreak of Clostridium difficile diarrhoea in a ward for the elderly in a 550-bedded District General Hospital . The measures taken to contain it and clinical features, previously undescribed, are highlighted. Appl Environ Microbiol, 1984 Nov, 48(5), 951 - 5 Acute toxicity of aminoglycoside antibiotics as an aid in detecting botulism; Wang YC et al.; Gentamicin sulfate or neomycin sulfate injected intraperitoneally into 24- to 27-g mice at a dose of 6.2 mg per mouse elicited botulism-like responses in less than 30 min, but a dose of 3.1 mg per mouse had no observable effect . The normally nontoxic 3.1-mg aminoglycoside dose aggravated the illness induced by an earlier injection of Clostridium botulinum type A or B toxin; it was usually lethal in 2 to 20 min if the preexisting illness was moderate to severe and worsened the condition of mice for about 30 min if the preexisting botulism was mild . The aminoglycoside had no effect when given shortly after the botulinum toxin was injected intraperitoneally; the sensitized state followed a latent period . It rapidly produced botulism-like effects when given to mice which had responded to a mixture of botulinum toxin and another mouse toxic agent with an illness that did not include signs of botulism . An unexpected illness devoid of botulism-like effects was encountered during intestinal colonization of mice by C . botulinum . The appearance of botulism-like signs soon after 3.1 mg of gentamicin sulfate was injected supported other suggestions that this illness included botulism that was masked by the effects of a second cause. Infect Immun, 1984 Nov, 46(2), 324 - 31 Differential cytotoxic effects of toxins A and B isolated from Clostridium difficile; Rothman SW et al.; Toxin A and toxin B preparations of Clostridium difficile have been shown to affect metabolic functions of intact HeLa cells with different kinetics . The cytotoxins were purified from dialyzed filtrates of C . difficile strain VPI 10463 by hydrophobic interaction chromatography and ion-exchange chromatography and were concentrated by dialysis or by ultrafiltration . The toxins, which are immunologically unrelated, were analyzed by polyacrylamide gel electrophoresis and by immunochemistry with the Western blot technique . Toxin A was resolved into one major cytotoxic protein and a minor, rapidly migrating species that did not comigrate with toxin B . Toxin B was resolved into one major and three minor cytotoxic proteins . One protein comigrating with toxin A had no cytotoxic activity . The highly purified toxin A at 1.0 mg/ml caused loss of intracellular K+ and inhibition of protein synthesis in HeLa cells within 1 h . These effects correlated with morphological changes indicating cytotoxicity . At lower protein concentrations of toxin A (10- to 100-fold less), however, cytotoxic effects were seen at 120 min, whereas no changes in K+ levels or protein synthesis were yet evident . The toxin B preparation, 1,000-fold more toxic than toxin A, was diluted to equivalent cytotoxicity as measured in the overnight assay . Toxin B caused loss of K+ and inhibition of protein synthesis well after cytotoxic morphological changes were complete . In contrast, at higher protein concentrations (2- to 2,000-fold more), intracellular K+ was lost completely by 120 min . The effects on cell rounding and protein synthesis were incomplete at 120 min, but increased with the toxin B concentration. Equine Vet J, 1984 Nov, 16(6), 519 - 21 Outbreak of botulism in horses; Kelly AP et al.; An outbreak of nervous disease in Standardbred horses occurred near Bendigo, in south-eastern Australia, in October 1980 . Over a two week period 11 horses in four training stables were affected with gait abnormalities, depression and recumbency . Eight of the 11 died . The results of an investigation implicated Clostridium botulinum toxin as the cause . The toxin was food-borne as a contaminant of oaten chaff. Equine Vet J, 1984 Nov, 16(6), 515 - 8 Thirteen cases of botulism in horses fed big bale silage; Ricketts SW et al.; An outbreak of pharyngeal and limb paresis involving four horses and nine ponies in the south east of England is described . Nine of the animals died or were destroyed on humane grounds . The clinical features suggested a diagnosis of botulism and mouse innoculation tests confirmed the presence of type B toxin in the serum of one case . All animals were fed big bale silage . It is describe how, in plastic wrapped silage manufacture, conditions of fermentation may be inadequate to prevent the growth of Clostridium botulism . Examination of a sample of silage fed to the affected horses suggested that this was probably the source of the toxin. Arch Microbiol, 1984 Nov, 139(4), 361 - 5 Hydrogenase from Acetobacterium woodii; Ragsdale SW et al.; Hydrogenase from fructose-grown cells of Acetobacterium woodii has been purified 70-fold to a specific activity of 3,500 mumol hydrogen oxidized per min per mg of protein measured at 35 degrees C and pH 7.6 with methyl viologen as electron acceptor . At the same conditions with reduced methyl viologen as electron donor the enzyme catalyzes the evolvement of 440 mumol of H2 per min per mg of protein . The enzyme was found in the soluble portion of the cell, indicating that it is either not membrane-bound or is loosely associated with the membrane . The purified enzyme, which does not contain nickel, exhibits spectroscopic properties similar to the iron-sulfur hydrogenase of Clostridium pasteurianum . The enzyme is strongly inhibited by carbon monoxide, with 50% inhibition occurring at approximately 7 nM CO . Ferredoxin, flavodoxin, and carbon monoxide dehydrogenase are reduced in hydrogen-dependent reaction by the A . woodi hydrogenase. Appl Environ Microbiol, 1984 Nov, 48(5), 956 - 63 Plasmids in Clostridium botulinum and related Clostridium species; Strom MS et al.; Toxigenic Clostridium botulinum and nontoxigenic C . sporogenes, C . subterminale, and C . botulinum-like organisms from a variety of sources were screened for plasmids . Of the 68 toxigenic C . botulinum isolates, 56% carried one or more plasmids, ranging in mass from 2.1 to 81 megadaltons . Within individual groups (based on the type of neurotoxin produced), many strains showed identical plasmid banding patterns on agarose gels . Of the 15 nontoxigenic strains tested, 40% also carried one or more plasmids ranging from 1.7 to 25.0 megadaltons, with both unique and common banding patterns represented . A total of 67 plasmids from both toxigenic and nontoxigenic strains were detected . At this time, no phenotypic functions have been assessed for these plasmids, and they must therefore be considered cryptic . A variety of lysing and extraction techniques were necessary to detect plasmids in the different C . botulinum groups. Ann Microbiol (Paris), 1984 Nov-Dec, 135B(3), 269 - 82 Physical characterization of the Clostridium perfringens tetracycline-chloramphenicol resistance plasmid pIP401; Magot M; Four restriction endonucleases were used to construct a physical map of the tetracycline-chloramphenicol resistance plasmid pIP401, which had been isolated from Clostridium perfringens . Twenty-seven restriction sites were placed within the 52-kb map, including 1 site for AvaI, 2 sites for KpnI, 10 sites for EcoRI and 14 sites for PstI . The loss of chloramphenicol resistance in the derived pIP406 plasmid was associated with a deletion of a 6.2-kb DNA segment located in a 10.55 EcoRI-PstI fragment . Two 1.4-kb inverted repeats were also characterized in both pIP401 and pIP406. Zh Mikrobiol Epidemiol Immunobiol, 1984 Nov, (11), 65 - 8 {Economic coefficients during batch and multicyclic cultivation of Clostridium perfringens type A}; Mel'nikova VA et al.; The analysis of the accomplished processes of cultivation of C . perfringens, type A, has shown that at all cycles of the multicyclic process and during large-scale submerged cultivation in reactors the culture of this micro-organism remains in a physiologically active state . This is confirmed by high values of economic coefficients and by the consumption of keto acids. Poult Sci, 1984 Nov, 63(11), 2241 - 6 The iron milk most probable number method for enumeration of Clostridium perfringens in the diet and the intestine of the chick; Stutz MW et al.; Seven experiments were conducted to evaluate the iron milk most probable number method for enumeration of Clostridium perfringens in the diet and the intestine of broiler chicks . Levels of 50 ppm of neomycin and 20 ppm of polymixin improved the iron milk tube method when compared to the TSN (tryptone-sulfite-neomycin) agar plate method . Low numbers of the organism, approximately 5 per gram, were detected in the practical diet fed to the chicks . Vegetative cell numbers of C . perfringens increased from 1.7 log10 in the duodenum of chicks to greater than 9.2 log10 in the ceca . Spores of the organism were detected in the ileum and ceca . Results of two experiments demonstrated that C . perfringens became established in the ileum of chicks early in life, before initiation of feeding at 2 days of age. Biochemistry, 1984 Oct 23, 23(22), 5175 - 81 Purification and characterization of three forms of collagenase from Clostridium histolyticum; Sugasawara R et al.; Three collagenases from Clostridium histolyticum, designated C1, C2, and C3, with apparent molecular weights of 96 000, 92 000, and 76 000 were purified . Peptide maps of the enzymes prepared by digestion with Staphylococcus aureus V-8 protease were found to be similar . Cleavage of native C1 with alpha-chymotrypsin or V-8 protease yielded C2 and C3 . This suggested that proteolysis of the Mr 96 000 collagenase may have occurred in vivo, producing the other two lower molecular weight enzymes . Previously prepared antiserum directed against a form of the bacterial enzyme similar by molecular weight and charge to collagenase C3 and Fab' fragments generated from this antiserum inhibited the collagenolytic activity . C1, C2, and C3 were immunologically identical by Ouchterlony double diffusion, and C3 was able to compete with C1 for the antiserum binding site . The ability of each enzyme to bind to antiserum raised against the bacterial collagenase supported the hypothesis that these three proteins were closely related . Zinc analyses of C1 and C3 resulted in a value of 1.14 mol of zinc/mol of C1 and 0.82 mol of zinc/mol of C3 . C1 did not contain carbohydrate as measured by gas-liquid chromatography or periodic acid-Schiff staining. Biochim Biophys Acta, 1984 Oct 17, 777(1), 99 - 106 Osmotic stabilizers differentially inhibit permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin; McClane BA; Using a sensitive Vero (African green monkey kidney) cell model system, studies were performed to further investigate whether Clostridium perfringens enterotoxin acts via disruption of the colloid-osmotic equilibrium of sensitive cells . Enterotoxin was shown to cause a rapid loss of intracellular 86Rb+ (Mr approx . 100) with time- and dose-dependent kinetics . The enterotoxin-induced release of intracellular 86Rb+ preceded the loss of two larger labels, 51Cr label (Mr approx . 3500) and 3H-labeled nucleotides (Mr less than 1000) . The osmotic stabilizers, sucrose and poly(ethylene glycol), differentially inhibited enterotoxin-induced larger label loss versus 86Rb+ loss . Further, enterotoxin was shown to cause a rapid influx of 24Na+ that was not significantly inhibited by osmotic stabilizers . Additional studies demonstrated that lysosomotropic agents were not protective against characteristic enterotoxin-induced membrane permeability alterations or morphological damage . Taken collectively, these results are consistent with an action for enterotoxin which involves a disruption of the osmotic equilibrium. Biochim Biophys Acta, 1984 Oct 12, 805(2), 131 - 6 Polyphosphate-mediated protection from cellular intoxication with Clostridium difficile toxin B; Florin I et al.; The influence of polyphosphorylated compounds on intoxication of human lung fibroblasts with Clostridium difficile toxin B was studied . ATP, as well as other nucleoside di-, tri-, and tetraphosphates, inorganic polyphosphates and polyphosphorylated sugars, caused a dose-dependent (1-5 mM range) delay in the appearance of the cytopathogenic effect . With a longer phosphate chain, the delay was more pronounced, although the cytopathogenic effect always developed finally, reaching the level of the control within 20 h . Toxin preparations contained one fraction of molecules able to bind ATP, besides one non-binding fraction . The protective effect of ATP did not depend on its energy producing ability . Neither was the protective effect due to an inactivation of the toxin per se, or to an interference with binding of the toxin to the cells . ATP was protective even upon addition 10 min after the toxin binding step . In the presence of ATP, the toxin remained accessible to neutralization with antitoxin . In analogy with the P-site on diphtheria toxin, we postulate that C . difficile toxin B contains a polyphosphate-binding site . This site is separate from the receptor-binding site, but involved in the interaction of toxin B with the cell surface shortly after the binding step. Aust N Z J Med, 1984 Oct, 14(5), 606 - 10 Clostridium difficile colitis; Rocca JM et al.; We reviewed all rectal biopsies performed on patients with proven C . difficile infection between 1977 and 1982 (36 patients) . All patients were symptomatic and all had received antibiotic treatment recently, the commonest antibiotic treatment being ampicillin or amoxycillin . There was poor correlation between the histological appearances and the severity of symptoms . A range of histological appearances was observed: normal (8%), congestion and edema (8%), nonspecific colitis (3%), infective colitis (28%) and pseudomembranous colitis (53%) (PMC) . Most cases of PMC showed 'early' features, involving predominantly the surface epithelium, where attenuation and inflammation, intraepithelial microabscesses, and small eruptive lesions were seen . Recognition of these features, in the context of an acute infective-type colitis, may lead to early diagnosis of C . difficile colitis. FEBS Lett, 1984 Oct 1, 175(2), 208 - 12 The primary structure of the DNA-binding protein II from Clostridium pasteurianum; Kimura M et al.; The complete amino acid sequence of the Clostridium pasteurianum DNA-binding protein II (DNAb-II) has been determined . The molecule contains 91 amino acid residues and has an Mr of 10 133 . Sequence data were obtained from manual Edman degradation, using the DABITC/PITC double-coupling of the tryptic, peptic, chymotryptic and Staphylococcus protease peptides . A comparison of the amino acid sequence of the C . pasteurianum DNAb-II with those of the DNAb-II from Escherichia coli, Bacillus stearothermophilus, Thermoplasma acidophilum and Pseudomonas aeruginosa shows that the C . pasteurianum protein is more homologous to that of B . stearothermophilus (60%) than to that of E . coli (45%) . All DNAb-II proteins have identical sequences Gly-Phe-Gly-X-Phe at positions 46-50 and Arg-Asn-Pro-X-Thr at positions 61-65. J Wildl Dis, 1984 Oct, 20(4), 267 - 71 Internal temperature of decomposing duck carcasses in relation to botulism; Wobeser G et al.; Under spring conditions (mean daily maximum 22 C, mean daily minimum 9 C), the temperature within duck carcasses paralleled air temperature for 3 days; on days 4 and 5 the internal temperature rose above 30 C for approximately 30 hr and maximum temperatures of 40-47 C occurred . This coincided with the period of maximum blowfly maggot activity in the carcasses . Carcasses screened from blowflies did not experience this period of high internal temperature . Under autumn conditions (mean daily maximum 13 C, mean daily minimum 1 C), the internal temperature of carcasses paralleled air temperature for approximately 2 wk . Following a warm day (23.5 C), maggots appeared in the carcasses and the internal temperature rose markedly higher than air temperature . Maggots moved into the soil on cold nights and reinhabited the carcasses during the day . The microclimate within maggot-infested carcasses appeared very suitable for growth and toxin production by Clostridium botulinum and this phenomenon may help explain the occurrence of botulism outbreaks during cool weather. Antimicrob Agents Chemother, 1984 Oct, 26(4), 552 - 6 Prevention of clindamycin-induced mortality in hamsters by Saccharomyces boulardii; Toothaker RD et al.; Saccharomyces boulardii, a yeast used in a number of countries for general and antibiotic-associated gastrointestinal illnesses, was examined for possible application in the prevention of clindamycin-induced mortality in the hamster colitis model . Hamsters were given free access to an aqueous 5% suspension of lyophilized yeast for 3 days before and 10 days after administration of a single oral clindamycin dose of from 0.2 to 0.8 mg/kg . Mortality was recorded in groups of 7 to 20 animals every 24 h for 10 to 30 days . Mean cecal concentrations of S . boulardii were greater than 10(6) CFU/ml throughout the yeast administration period . Yeast treatment significantly decreased cumulative percent mortality by an average of 29% . Death onset was not affected by yeast treatment . Cecitis was present in 86% of moribund animals (N = 95) and was absent in all surviving animals examined (N = 27) . Toxigenic Clostridium difficile was isolated from 13 of 14 moribund hamsters examined . No adverse effects of the yeast treatment were observed in animals receiving S . boulardii without clindamycin . The results suggest that S . boulardii warrants further evaluation for the prevention of antibiotic-associated colitis. Anal Biochem, 1984 Oct, 142(1), 226 - 31 Chromatographic separation of extruded iron-sulfur cores from the apoproteins of Clostridium pasteurianum and spinach ferredoxins in aqueous Triton X-100/urea; Bonomi F et al.; Iron-sulfur core extrusions from spinach {( 2Fe-2S}) and Clostridium pasteurianum (2{4Fe-4S}) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively {FenSn(SPh)4}2- with n = 2 and n = 4, respectively . The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose . Essentially quantitative recovery of {FenSn (SPh)4}2- is achieved in the eluate . The apoprotein remaining on the column can be eluted with 0.5 M NaCl . Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent . Apoprotein recovery is comparable to that obtained by other chromatographic methods . At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted . The procedures developed in this work are potentially most applicable to selective removal of {2Fe-2S} and {4Fe-4S} centers from a multicenter enzyme without irreversible denaturation. Microbiologica, 1984 Oct, 7(4), 375 - 9 Cytotoxin and enterotoxin production by Clostridium difficile; Gianfrilli P et al.; 30 strains of Cl . difficile isolated from faeces of patients with pseudomembranous colitis (PMC), antibiotic associated diarrhoea (AAC) and other intestinal disorders and from faeces of asymptomatic carriers were studied for production of toxins . Tissue culture assay was used for the detection of cytotoxin (toxin B) and ileal loop test for enterotoxin (toxin A) . All Cl . difficile isolates from patients with PMC and AAC were found to produce cytotoxin, whereas enterotoxin was demonstrated only in approximately 70% of strains. J Lipid Res, 1984 Oct, 25(10), 1124 - 31 Mode of action of steroid desmolase and reductases synthesized by Clostridium "scindens" (formerly Clostridium strain 19); Winter J et al.; A recently isolated hitherto unknown Clostridium from human feces, designated Clostridium "scindens" (formerly strain 19), synthesizes at least two enzymes active on the side-chain of the steroid molecule and two enzymes active on the hydroxyl groups of the 7-position of bile acids . Steroid desmolase, responsible for side-chain cleavage of corticoids, and 20 alpha-hydroxysteroid dehydrogenase have not been detected in any other bacterial species of the resident colonic flora . Steroid desmolase is Eh-dependent (optimum ca . -130 mV), requires a hydroxy group at C-17, and preferably an alpha-ketol group in the side-chain; an alpha-hydroxy group at C-20 reduces and a beta-hydroxy group at C-20 prevents side-chain cleavage . With suitable substrates, the yield of C-19 steroids is proportional to the bacterial multiplication rate . 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSDH) is also Eh-dependent (optimum ca . -300 mV) and reduces the C-20 keto function to an alpha-hydroxy group, regardless of the presence or absence of a hydroxy group at C-17 . 7 alpha-Dehydroxylase metabolizes cholic and chenodeoxycholic acid, while 7 beta-hydroxysteroid dehydrogenase acts upon ursodeoxycholic acid . The latter two enzymes are not specific for C . scindens. J Lipid Res, 1984 Oct, 25(10), 1084 - 9 Formation of urso- and ursodeoxy-cholic acids from primary bile acids by a Clostridium limosum soil isolate; Sutherland JD et al.; A gram-positive, rod-shaped anaerobe (isolate F-14) was isolated from soil . This organism was identified by cellular morphology as well as by fermentative and biochemical data as Clostridium limosum . Isolate F-14 formed ursocholic acid (UC) and 7-ketodeoxycholic acid (7-KDC) from cholic acid (CA), and ursodeoxycholic acid (UDC) and 7-ketolithocholic acid (7-KLC) from chenodeoxycholic acid (CDC) in whole cell cultures, but did not transform deoxycholic acid (DC) . No hydrolysis or transformation occurred when either taurine- or glycine-conjugated bile acids were incubated with F-14 . The type stain of Clostridium limosum (American Type Culture Collection 25620) did not transform bile acids . The structures of ursocholic, ursodeoxycholic, 7-ketodeoxycholic, and 7-ketolithocholic acids were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent . The organism transformed cholic and chenodeoxycholic acids at concentrations of 20 mM and 1 mM, respectively; higher concentrations of bile acids inhibited growth . Optimal yields of ursocholic and ursodeoxycholic acids were obtained at 9-24 hr of incubation and depended upon the substrate used . Increasing yields of 7-ketodeoxycholic and 7-ketolithocholic acids, and decreasing yields of ursocholic and ursodeoxycholic acids were observed with longer periods of incubation . Culture pH changed with time and was characterized by a small initial drop (0.2-0.4 pH units) and a subsequent increase to a pH (8.1-8.2) that was above the starting pH (7.4).(ABSTRACT TRUNCATED AT 250 WORDS) J Am Vet Med Assoc, 1984 Oct 1, 185(7), 798 - 801 Catastrophic death losses in a dairy herd attributed to type D botulism; Abbitt B et al.; Clostridium botulinum type D intoxication was diagnosed as the cause of death of 42 of 67 lactating cows in a southeast Texas dairy herd over an 11-day period . By necessity, the diagnosis was based on clinicopathologic findings, as the toxin could not, by standard laboratory tests, be demonstrated in affected cattle . The predominant clinical findings were hindlimb weakness/ataxia rapidly progressing to persistent recumbency . Affected cattle were alert until just before death, which occurred without notable agonal movements or respirations after 6 to 72 hours' recumbency . Abnormal laboratory findings included neutrophilic leukocytosis (all affected cattle), proteinuria (most affected cattle), slight elevations of serum aspartate transaminase and low serum inorganic phosphorus (some affected cattle), and patchy areas of hyperemia/congestion of the mucosa in the small intestine (postmortem examination of 3 affected cattle) . This report confirms the findings of others with regard to the difficulty of demonstrating the causative toxin in C botulinum type D-intoxicated cattle and presents available information on the clinicopathologic features of this intoxication that may aid in the differentiation of this condition from other causes of down cows. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 Oct, 258(1), 62 - 8 {Formation of alpha-aminobutyric acid in Clostridium sordellii}; Huckenbeck W et al.; After a modified growth C . sordellii is able to ferment threonine as mono-substrate . It could be demonstrated for the first time that alpha-aminobutyric acid is definitely formed from threonine by clostridia . The pH-optimum of this reaction is greater than 8, the temperature optimum is greater than 28 degrees C . Further fermenting products are: glycine and presumably acetaldehyde. J Bacteriol, 1984 Oct, 160(1), 466 - 9 Effects of cultivation gas phase on hydrogenase of the acetogen Clostridium thermoaceticum; Kellum R et al.; The effect of cultivation gas phase on the expression and activity of hydrogenase in heterotrophic cultures of Clostridium thermoaceticum was examined . Of the five gas phases tested, hydrogenase was maximal from cells cultivated under CO . Correlations were observed between the level of hydrogenase and the evolution of H2 by growing cultures . Activity stains of polyacrylamide gels revealed a single hydrogenase band in CO2 cells and multiple hydrogenase bands in CO cells. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 Oct, 258(1), 51 - 61 {Degradation of glutamic acid and proline in Clostridium sordellii under cadaveric bacteriological inspection}; Huckenbeck W et al.; Glutamic acid (GLU) is decarboxylated to gamma-aminobutyric acid (GABA) by Clostridium sordellii isolated from a decaying human brain . The dependence of this reaction on temperature, pH and substrate concentration has been established . The pH-optimum is in the range of 5.0 to 5.2 . Near optimal pH the temperature optimum is greater than 28 degrees C . At higher pH-values (6 to 7) activity is relatively independent of temperature . The similarity to results in decaying human brains (10, 11, 12) shows a good correlation between postmortem bacterial flora and GLU-degradation . Furthermore it is shown that C . sordellii produces delta-aminovaleric acid (AVA) from proline (PRO). Avian Dis, 1984 Oct-Dec, 28(4), 1120 - 4 The occurrence of Clostridium perfringens in the intestine of chicks; Shane SM et al.; Commercial broiler chicks brooded either on wire or on used or new litter demonstrated a 75% (62/75) incidence of recovery of "perfringens-like" colonies from the intestine during a 5-week period . Eleven Clostridium spp . were identified from among these "perfringens-like" organisms, which were cultured on SPS selective agar medium . Clostridium perfringens was positively identified only infrequently (five isolates) from among the "perfringens-like" colonies . In contrast, "perfringens-like" colonies were not recovered from the intestinal contents of specific-pathogen-free chicks reared in an isolation unit . However, C . perfringens was isolated from the yolk sac of one embryonated egg and from the intestine of a single 7-day-old chick, indicating the possibility of vertical transmission of this potential pathogen. Zh Mikrobiol Epidemiol Immunobiol, 1984 Oct, (10), 20 - 4 {Effect of iron on the growth and toxin formation of Clostridium perfringens type A cultures}; Artemenko VD et al.; The influence of iron at different concentrations on the growth and toxin formation of the cultures of C . perfringens strain 28 BP6K, type A, in media having different composition and protein base has been studied . As revealed by the results of these studies, bivalent iron at concentrations of 1-30 mg%, while having no essential influence on the growth and development of the culture, not only produces no stimulating effect on toxin formation, but even inhibits it . The concentrations of iron, both producing the optimum effect on toxin formation and inhibiting it, are not identical for different kinds of culture media and depend on their nature and composition . In culture media with fodder yeast extract added, and especially in media based on fermentative fodder yeast hydrolysate, the cultures have shown the increased stability of toxin formation in the presence of higher concentrations of iron. Lab Anim Sci, 1984 Oct, 34(5), 443 - 52 Experimental and spontaneous clostridial enteropathies of laboratory and free living lagomorphs; Carman RJ et al.; The enteric diseases of hares, European and cottontail rabbits, which are caused by members of the genus Clostridium are reviewed . Disease caused by C . perfringens Types A and E, C . spiroforme, C . difficile, C . sordellii, C . tympany cuniculi and clostridial enterotoxins are included . Tyzzer's disease also is discussed. Poult Sci, 1984 Oct, 63(10), 2036 - 42 Effects of diet and antimicrobials on growth, feed efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks; Stutz MW et al.; Five experiments were conducted to evaluate the effects of diet and antimicrobials on weight gain, feed efficiency, ileal weight, and Clostridium perfringens in the ileum of broiler chicks . In the first experiment, glucose, sucrose, and fructose were added to a semipurified diet and the results were compared with those from a practical corn and soybean meal diet . All of the diets were fed with and without bacitracin at a level of 55 ppm . Fructose resulted in the greatest depression in weight gain, followed by sucrose . Bacitracin significantly improved weight gain and feed efficiency of chicks fed the fructose, sucrose, and practical diets . Highly significant inverse correlations were obtained between ileal weight and weight gain and the number of C . perfringens in the ileum and weight gain . In other experiments bacitracin, penicillin, chlortetracycline, oxytetracycline, erythromycin, tylosin, virginiamycin, lincomycin, bambermycins, and carbadox, all at a level of 55 ppm, improved weight gain and feed efficiency and significantly reduced the weight of the ileum and the number of C . perfringens in the ileum of chicks fed the practical diet . The antibacterial agents 3-nitro-4-hydroxy-phenylarsonic acid, arsanilic acid, furazolidone, and sulfathiazole had little to no effect on the 4 parameters evaluated . Virginiamycin and lincomycin at 16.5 and 4.4 ppm, respectively, were shown to be effective . In vitro activities of the antimicrobials against C . perfringens did not directly relate to in vivo activities and the effects on growth and feed efficiency . The results of these experiments support the concept of antimicrobials as growth permittants and provide further evidence for C . perfringens as a causative bacteria for growth depression. Pflugers Arch, 1984 Oct, 402(2), 171 - 5 Ca2+- and H+-dependent effects of crude bacterial phospholipase C on the hydroosmotic response of toad urinary bladder to serosal hypertonicity; Hardy MA; Phospholipase C (EC 3.1.4.3.) from Clostridium perfringens (crude extracts) was used to study the role of phospholipids in the osmotic permeability of the urinary bladder of the toad . When added to the serosal bath (430 mU/ml) it inhibited the effects of antidiurectic hormone (ADH) and exogenous cyclic AMP . Under the same conditions the increase in osmotic flow produced by serosal hypertonicity (SH) was slightly enhanced by the lipase . The hydroosmotic effect of SH was greatly potentiated by the lipase by decreasing 10-fold the Ca2+ concentration . The SH-induced flow was inhibited by the lipase if the Ca2+ or the H+ concentration was increased 10-fold, but not if the increase in positive charges was produced by a concentration of Mg2+ . Phospholipase C had no effect on the action of either ADH or SH if added to the mucosal bath . Serosal neuraminidase or phospholipase A2 could not mimic the effect of phospholipase C on SH . The effect of phospholipase C on the response to SH was not modified if fatty acid-free bovine serum albumin was added to the bath . Therefore, the release of products of lipolysis into the bath do not seem to be responsible for the effects of phospholipase C on SH-induced water flow . The results suggest that the effects of the enzyme on the composition and rearrangement of lipids at the basolateral membrane produce modifications of the water flow . Ca2+ and H+ may modify the enzyme-substrate interaction, suggesting that different phospholipids may be differentially involved in the control of water permeability of the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1984 Sep 25, 23(20), 4650 - 5 Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-{(methylamino)methyl}-2-selenouridine; Wittwer AJ et al.; Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids {( 75Se}tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32- . When E . coli {75Se}tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA . This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-{(methylamino)methyl}-2-selenouridine . Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties . The new selenonucleoside also accounted for 40-60% of the 75Se found in {75Se}tRNA from C . sticklandii or M . vannielii . Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-{(methylamino)methyl}-2-selenouridine . Pure seleno-tRNAGlu isolated from C . sticklandii contains one 5-{(methylamino)methyl}-2-selenouridine and one 4-thiouridine per tRNA molecule. J Biol Chem, 1984 Sep 25, 259(18), 11396 - 402 Unusual features in EPR and Mössbauer spectra of the 2{4Fe-4Se}+ ferredoxin from Clostridium pasteurianum; Moulis JM et al.; The electronic and magnetic properties of the selenium-substituted 2{4Fe-4Se}2+/+ ferredoxin (Fd) from Clostridium pasteurianum have been investigated by EPR and Mossbauer spectroscopy . The {4Fe-4Se}2+ clusters of oxidized Fd are diamagnetic and the Mossbauer spectra are nearly identical to those of oxidized 2{4Fe-4S}2+ Fd . The addition of 2e- per molecule of Se-substituted Fd causes the simultaneous appearance of three EPR signals: one (g1,2,3 = 2.103, 1.940, 1.888) is reminiscent of {4Fe-4S}+ EPR spectra and accounts for 0.7 to 0.8 spin/molecule . The two others consist of a broad signal with g = 4.5, 3.5, and approximately 2 (0.7 to 0.8 spin/molecule) and of a narrow peak at g = 5.172 which is observed up to 60 K . Peculiar features are also present in the Mossbauer spectra of 2{4Fe-4Se}+ Fd below 20 K: a subcomponent with lines near to +/- 4 mm/s and accounting for 20% of the total iron corresponds to two antiferromagnetically coupled sites in approximately a 3:1 ratio and displays fully developed paramagnetic hyperfine interactions at 4.2 K without any applied field . At 77 K, however, the reduced Se-substituted Fd yields a Mossbauer spectrum similar to that of 2{4Fe-4S}+ Fd . The new EPR and Mossbauer spectroscopic features of the 2{4Fe-4Se}+ Fd are attributed to S = 3/2 and S = 7/2 spin states which accompany the classical S = 1/2 state of {4Fe-4X}+ (X = S, Se) structures. Biochem Biophys Res Commun, 1984 Sep 17, 123(2), 463 - 7 Energy-dependent activation of spore-lytic enzyme precursor by germinated spores of Clostridium perfringens; Ando Y et al.; A precursor of the spore-lytic enzyme of Clostridium perfringens was extracted with alkali from dormant spores of the organism . The enzyme precursor was activated by incubating it with germinated spores which had been treated with alkali . The activation was greatly enhanced by the addition of 3-phosphoglycerate, suggesting that the conversion of precursor to active enzyme depends on endogenous energy-producing metabolism during germination. Biochemistry, 1984 Sep 11, 23(19), 4309 - 17 23Na NMR relaxation study of the effects of conformation and base composition on the interactions of counterions with double-helical DNA; Nordenskiold L et al.; NMR relaxation rates (T1(-1) and T2(-1)) have been determined for 23Na in aqueous salt solutions containing various types of helical double-stranded deoxyribonucleic acids . These measurements were performed on three synthetic polynucleotides having different overall conformations, poly-(dA-dT).poly(dA-dT) (alternating B-DNA), poly(dG-dC).poly(dG-dC) at low salt (B-DNA), and Br-poly(dG-dC).Br-poly(dG-dC) (left-handed Z-DNA), and on four types of natural DNA differing in base composition, Clostridium perfringens (26% GC), calf thymus (40% GC), Escherichia coli (50% GC), and Micrococcus lysodeikticus (72% GC) . For all types of DNA investigated, except poly(dA-dT).poly(dA-dT), the 23Na NMR spectra measured at 21 degrees C and an applied field of 4.7 T are non-Lorentzian . These non-Lorentzian spectra were analyzed on the basis of the two-state model and the standard theory of nonexponential quadrupolar relaxation processes in order to obtain estimates of the correlation times (tau c) characteristic of the sodium nuclei associated with the various nucleic acids . All of the correlation times estimated in this way are in the range of nanoseconds . The magnitudes of these correlation times show a significant dependence on the overall conformation of the nucleic acid (B vs . Z) but not on its base composition . To investigate the concentration dependence of tau c, sodium or magnesium salts were added to solutions of Br-poly(dG-dC).Br-poly(dG-dC) (Z-DNA).(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1984 Sep 10, 259(17), 10845 - 9 Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum; Clark JE et al.; Methylenetetrahydrofolate reductase in Clostridium formicoaceticum has been purified to a specific activity of 140 mumol min-1 mg-1 when assayed at 37 degrees C, pH 7.2, in the direction of oxidation of 5-methyltetrahydrofolate with benzyl viologen as electron acceptor . The purified enzyme is judged to be homogeneous by polyacrylamide disc-gel electrophoresis and gel filtration . The enzyme which is an octamer has a molecular weight of about 237,000 and consists of four each of two different subunits having the molecular weights 26,000 and 35,000 . The octameric enzyme contains per mol 15.2 +/- 0.3 iron, 2.3 +/- 0.2 zinc, 19.5 +/- 1.3 acid-labile sulfur, and 1.7 FAD . The UV-visible absorbance spectrum has a peak at 385 nm and a shoulder at 430 nm and is that of a flavoprotein containing iron-sulfur centers . The reductase, which is sensitive to oxygen, must be handled anaerobically and is stabilized by 2 mM dithionite . It catalyzes the reduction of methylene blue, menadione, benzyl viologen, rubredoxin, and FAD with 5-methyltetrahydrofolate and the oxidation of reduced ferredoxin and FADH2 with 5,10-methylenetetrahydrofolate . No activity was observed with pyridine nucleotides . It is suggested that the physiologically important reaction catalyzed by the enzyme is the reduced ferredoxin-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. FEBS Lett, 1984 Sep 3, 174(2), 284 - 8 A high-molecular-mass cell wall protein released from Clostridium tyrobutyricum by heat treatment; Hayes H et al.; Analysis of the cell wall of 4 strains of Clostridium tyrobutyricum reveals an unusually high protein content (35-40% dry weight) . Brief heat treatment of whole cells of these stains causes release of two proteins, flagellin and a cell wall component of high molecular mass (110-125 kDa in the different strains) . This component represents approx . 5% of the dry cell weight. J Antimicrob Chemother, 1984 Sep, 14 Suppl C, 7 - 17 The activity of enoxacin against clinical bacterial isolates in comparison with that of five other agents, and factors affecting that activity; Reeves DS et al.; The activity of enoxacin against 362 clinical bacterial isolates in comparison with norfloxacin, nalidixic acid, ampicillin, latamoxef (moxalactam) and gentamicin was tested by an agar dilution method . Typical MICs for enterobacteria lay between 0.12 and 1.0 mg/l . Enterobacter spp . and Serratia spp . tended to be more resistant . Enoxacin was also active against Pseudomonas aeruginosa (mean MIC 0.5 mg/l) and highly active against fastidious Gram-negative aerobes . Typical MICs for Staphylococcus aureus were 1-2 mg/l while streptococci were more resistant (16-32 mg/l) . Enoxacin had no useful activity against Bacteroides fragilis and Clostridium perfringens . Enoxacin was generally more active at pH 8 than pH 6, and in broth than in urine . It was bactericidal in its action . Daily serial passage from growth in broth containing enoxacin caused decreased sensitivity which was limited to four- to 16-fold greater than the original MIC . Enoxacin was about half as active as norfloxacin against enterobacteria, equally active against staphylococci, and some two to four times less active against streptococci. Ann Microbiol (Paris), 1984 Sep-Oct, 135B(2), 219 - 22 {Biosynthesis of toluene in Clostridium aerofoetidum strain WS}; Pons JL et al.; Formation of toluene in growing cultures of Clostridium aerofoetidum strain WS was enhanced when the medium was supplemented with phenylacetic acid or with L-phenylalanine and L-methionine together . Evidence for the role of L-phenylalanine was shown by the detection of {2H2}-methyl{2,3,4,5,6-2H5}benzene ("heptadeuterotoluene") in growing cultures with L-{2',3',4',5',6'-2H5}phenyl{2,3-2H3}alanine and L-methionine. J Antibiot (Tokyo), 1984 Sep, 37(9), 949 - 57 Empedopeptin (BMY-28117), a new depsipeptide antibiotic . I . Production, isolation and properties; Konishi M et al.; Empedopeptin is a new antibiotic produced by empedobacter haloabium nov . sp . (ATCC 31962) . It is a water-soluble depsipeptide antibiotic containing eight amino acid residues and a C14-fatty acid moiety in the molecule . Although structurally unrelated, empedopeptin and vancomycin have similar antimicrobial spectra against aerobic and anaerobic Gram-positive bacteria including antibiotic-resistant strains . Empedopeptin is highly active in vivo in mice against systemic infections of Staphylococcus aureus, Streptococcus pyogenes and Clostridium perfringens . Empedopeptin is not absorbed orally. Vet Microbiol, 1984 Sep, 9(5), 497 - 502 Infectious nature of Clostridium spiroforme-mediated rabbit enterotoxaemia; Carman RJ et al.; Newly weaned rabbits had diarrhoea only if they were infected with Clostridium spiroforme . In adult rabbits exposure to both clindamycin and C . spiroforme was necessary to induce disease . All diseased animals harboured C . spiroforme and its toxin . Adult rabbits given a course of clindamycin survived when held in a protected environment as did those challenged with C . spiroforme alone . At necropsy none of these apparently healthy animals showed signs of diarrhoea or caecitis . These findings suggest that, in the development of enterotoxaemia, weaning or clindamycin treatment and infection with C . spiroforme are separate events and that disease follows infection with this organism from the environment, as opposed to overgrowth by undetectable levels of C . spiroforme resident in the gut . Our data indicate that C . spiroforme is not a normal component of the rabbit gut flora and that the normal bowel ecology of the adult must be disrupted before C . spiroforme will colonize. Pediatr Infect Dis, 1984 Sep-Oct, 3(5), 429 - 32 Nosocomial Clostridium difficile reservoir in a neonatal intensive care unit; Zedd AJ et al.; A new bacteriophage/bacteriocin typing system was used to study Clostridium difficile colonization in a neonatal intensive care unit . C . difficile was isolated from 21 of 62 (34%) stools from 15 of 37 (41%) infants . Colonization was reduced during antimicrobial therapy and for about 1 week thereafter . One of five nurses and one of two parents studied were carriers . Eight isolates were cultured from environmental surfaces . Thirty of 31 C . difficile isolates were found to be a single type, Cld 6,9,10,13; bacteriocin 1320,1537,2304 . No C . difficile was found in 29 specimens obtained in the delivery room from mothers and infants, and there was no association of early colonization with vaginal delivery . The data provide strong evidence for nosocomial acquisition of C . difficile by infants in the neonatal intensive care unit . No obvious pathologic role for C . difficile could be identified among colonized infants . Among 22 C . difficile isolates from 7 adult inpatients with diarrhea and 13 healthy infants attending the center's well baby clinic, 4 were the same type as the strain found in the intensive care nursery . Only one of these patients had had direct contact with the neonatal intensive care unit, indicating that the nursery strain may also be found elsewhere in the community. J Clin Microbiol, 1984 Sep, 20(3), 549 - 60 Studies of stools from pseudomembranous colitis, rotaviral, and other diarrheal syndromes by frequency-pulsed electron capture gas-liquid chromatography; Brooks JB et al.; Thirty-five patients with various diarrheal syndromes and 22 controls were studied . All stool samples were carefully cultured for Clostridium difficile, using selective isolation media . Cytotoxin assays with proper antitoxin neutralization were done in MRC-5 cells . The stool samples were extracted four times, three times at pH 2 and once at pH 10, using CHCl3 or ether . Derivatizations of extracts were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol, and all derivatives were analyzed by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC) . A dedicated computer was used to assist in both qualitative and quantitative data analysis . Isocaproic acid (iC6) was always found in stool from which C . difficile was isolated and was absent in C . difficile-negative specimens . p-Cresol was found frequently in both persons with pseudomembranous colitis and controls . Tryptamine was found in stool containing C . bifermentans . The FPEC-GLC profiles of persons with acute diarrhea were very different from those of normal persons . Diarrhea associated with adenovirus and rotavirus, Klebsiella spp., and Escherichia spp . showed different FPEC-GLC patterns . Stools from well persons consistently contained full-scale peaks of pyruvic, acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids . In rotavirus stools isobutyric, isovaleric, and valeric acids were reduced in quantity from those found in control stools, whereas propionic and butyric acids were increased. J Clin Microbiol, 1984 Sep, 20(3), 539 - 48 Frequency-pulsed electron capture gas-liquid chromatographic analysis of metabolites produced by Clostridium difficile in broth enriched with amino acids; Brooks JB et al.; Clostridium difficile strain CDC A-567 was cultured in Trypticase (BBL Microbiology Systems)-yeast-salt broth supplemented with 0.2% L-leucine, L-norleucine, L-isoleucine, L-tyrosine, or L-tryptophan . Four extractions were done on the spent medium, three at pH 2 and one at pH 10, using CHCL3 or ether . Derivatizations were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol . All samples were analyzed with frequency-pulsed electron capture gas-liquid chromatography . A dedicated computer was used to assist in data analysis . C . difficile produced both short-chain and aromatic acids in Trypticase-yeast-salt broth; hydroxy acids were also detected . p-Cresol, indoleacetic acid, 4-methylthio-2-hydroxybutyric acid, and some unidentified alcohols were observed . The basic chloroform extraction contained cadaverine and putrescine . Leucine, norleucine, and isoleucine influenced the production of C5 and C6 acids and alcohols . L-Tyrosine underwent successive degradation to produce p-cresol and aromatic acids as final products . Tryptophan increased the production of indoleacetic, indolepropionic, and indolebutyric acids . Isocaproic acid was produced in relatively high concentrations regardless of medium substitution . The consistent production of iC6 under various substrate conditions indicates that the production of this compound might be consistent enough in vitro to form the basis of a rapid test for detection of C . difficile in stool specimens by frequency-pulsed electron capture gas-liquid chromatography. J Clin Microbiol, 1984 Sep, 20(3), 339 - 41 Latex agglutination test for detection of Clostridium difficile toxin in stool samples; Shahrabadi MS et al.; A total of 163 stool specimens were tested for detection of Clostridium difficile and its toxin by cytotoxicity assay with tissue culture, latex agglutination test, and isolation of the organism . From 33 specimens which were positive for toxin by cytotoxicity, 30 were positive by the latex agglutination test; the organism was isolated from 21 . The total number of samples which were positive with the latex agglutination test was 44 . The predictive value of a positive latex agglutination result relative to the cytotoxicity test was 68%, and the predictive value of a negative result was 97.5% . The specificity and sensitivity of the latex agglutination test relative to the cytotoxicity assay and the low cost and simple facilities required indicate that the latex agglutination test is a useful procedure for screening for C . difficile toxins, provided that positive latex results are confirmed by cytotoxicity assay. Tijdschr Diergeneeskd, 1984 Sep 1, 109(17), 669 - 71 {Liability, prostaglandins and Clostridium}; van Nie GJ; A case of malignant oedema following injection of fenprostalene in cattle is reported . The question is asked whether the vasoconstrictive action of prostaglandins may predispose to the establishment of anaerobes. Can J Surg, 1984 Sep, 27(5), 435 - 7 Clostridium difficile in Crohn's disease; Wright JM et al.; Clostridium difficile has been detected in the stools of some patients with relapse of Crohn's disease . The authors looked prospectively for present or previous exposure to C . difficile cytotoxin in 10 patients with mild to severe Crohn's disease . None of 25 stool samples from these 10 patients was positive for C . difficile cytotoxin . These negative stool ultrafiltrates had mild cytotoxin neutralizing activity, but this finding did not differ from that in 30 cytotoxin-negative stools from patients with other diarrheal diseases . Serum from these patients also showed no cytotoxin neutralizing activity . Review of the literature reveals that C . difficile can cause complications ranging from diarrhea to toxic megacolon in a small but variable proportion of patients with Crohn's disease . There is no evidence that C . difficile plays a part in the pathogenesis of the disease. Biochem J, 1984 Sep 1, 222(2), 535 - 40 A transient increase in diacylglycerols is associated with the action of vasopressin on hepatocytes; Hughes BP et al.; Vasopressin induced a transient increase of 50% in the total concentration of diacylglycerols (determined by g.l.c.) in isolated hepatocytes . The increase was maximal at 0.25 min, and the concentration of diacylglycerols in cells treated with vasopressin had returned to the basal value by 4 min . No change in the concentration of diacylglycerols was observed after the treatment of cells with glucagon . The dependency of this effect on the concentration of vasopressin was similar to that of the effect of the hormone on 45Ca2+ efflux measured at 0.1 mM extracellular Ca2+ . Vasopressin increased the proportion of arachidonic acid and stearic acid and decreased the proportion of oleic acid present in the diacylglycerols . In hepatocytes prelabelled with {14C}arachidonic acid, vasopressin increased the amount of {14C}diacylglycerol . The effects of vasopressin on the total concentration of diacylglycerols and {14C}diacylglycerol were mimicked by an exogenous phospholipid phosphodiesterase (phospholipase C) from Clostridium perfringens . The results are consistent with the conclusion that the transient increase in diacylglycerols induced by vasopressin is caused by the rapid hydrolysis of both the phosphoinositides and one or more other phospholipids. Zh Mikrobiol Epidemiol Immunobiol, 1984 Sep, (9), 97 - 9 {Comparative study of resistance to tetanus and indices of the passive hemagglutination reaction in experimental animals}; Stovbun SF et al.; The article presents the results of the study of resistance to tetanus in 450 guinea pigs immunized against tetanus in a single injection and having antitoxin titers in their blood, as determined in the passive hemagglutination test, from less than or equal to 0.01 IU/ml to 1.6 IU/ml and more . The degree of protection in the immunized animals was determined by their challenge with Clostridium tetani spores in DCL and LD50. J Anim Sci, 1984 Sep, 59(3), 813 - 22 Thiamin and niacin in the rumen; Brent BE et al.; Thiamin analogs, produced in the rumen by thiaminase I, in the presence of a cosubstrate appear to be responsible for the central nervous system disorder, polioencephalomalacia (PEM) . For PEM to occur, an analog must be produced that inhibits an essential thiamin-requiring reaction, and results from a cosubstrate present in the rumen . In high concentrate diets, thiaminase I is produced by rumen microbes . However, PEM can also be caused by thiaminase I of plant origin . Based on physical characteristics and cosubstrate specificity, the thiaminase I enzymes produced by Bacillus thiaminolyticus and Clostridium sporogenes appear to be different from the enzyme produced by the rumen . Because niacin and certain antihelmentics are thiaminase I cosubstrates, they should be used cautiously . Supplementary niacin increased microbial protein synthesis in vitro and in vivo, and was more effective with urea than soybean meal . Supplementary niacin (5 to 6 g X cow-1 X d-1) increased milk production in postpartum cows but not in those in mid-lactation, and in cows fed soybean meal but not in those fed urea . We believe the heating of soybean meal during commercial processing decreased the availability of niacin for rumen protozoa . Supplementary niacin for postpartum cows increased blood glucose, decreased blood ketones and reduced the incidence of ketosis . Niacin flow to the small intestine and its absorption from the small intestine increased with niacin supplementation . Supplemental niacin prevented the postpartum decrease in red blood cell niacin observed in control cows. Pediatr Infect Dis, 1984 Sep-Oct, 3(5), 433 - 6 Occurrence of Clostridium difficile toxin-associated gastroenteritis following antibiotic therapy for otitis media in young children; Hyams JS et al.; The pathogenesis of diarrhea following antibiotic therapy for otitis media in young children remains unknown . We performed a prospective study evaluating the incidence of diarrhea and Clostridium difficile toxin in 115 outpatients (ages 6 months to 6 years) with acute otitis media treated with ampicillin, amoxicillin or trimethoprim-sulfamethoxazole . In 21 patients younger than one year of age six of 11 developing diarrhea had toxin-positive stools compared with three of 10 without diarrhea (P = 0.39) . In 94 patients between 13 months and 6 years of age three of 12 with diarrhea had toxin-positive stools compared with five of 82 without diarrhea (P = 0.06) . Diarrhea was self-limited in all cases . Although the data suggest that C . difficile might have been associated with diarrhea in the older children, further studies will be required to confirm this finding. J Pharmacol Exp Ther, 1984 Sep, 230(3), 665 - 9 Molecular basis for the pharmacological actions of Clostridium botulinum type C2 toxin; Simpson LL; The light chain of type C2 toxin produced by Clostridium botulinum was isolated by high-performance liquid chromatography . The protein eluted as a single peak; as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, it had an apparent molecular weight of 51,000 daltons . The light chain was an enzyme that possessed ADP-ribosylating activity . In experiments with synthetic substrates (homo-poly-L-amino acids; alanine, arginine, asparagine, aspartic acid, histidine, leucine, lysine, methionine, phenylalanine, proline, serine and tryptophan), only poly-L-arginine was ADP-ribosylated by the enzyme . In experiments with endogenous substrates (50,000 X g pellet and 50,000 X g supernatant from homogenates of mouse brain, liver and lung), the enzyme ADP-ribosylated proteins or polypeptides in both the particulate and soluble fractions . ADP-ribosylation of the soluble substrate was antagonized by adenine (K1 approximately 2.1 X 10(-5) M) and by adenosine (K1 approximately 2.7 X 10(-4) M); the reaction was reversed by a large molar excess of nicotinamide (0.1 M) . ADP-ribosylation of soluble substrate was diminished when the substrate had been pretreated with 1,2-cyclohexane-dione (0.1 M), a site reactive reagent that modified selectively arginine residues . Neither the light chain nor the heavy chain of the binary toxin possessed adenylate cyclase activity . Tissue fractions did possess endogenous adenylate cyclase activity, but the toxin did not stimulate this activity . The data indicate that the binary toxin produced by Clostridium botulinum resembles other protein toxins. Anal Biochem, 1984 Sep, 141(2), 344 - 7 Simultaneous single-step purification of thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri; Sliwkowski MX et al.; Thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri were purified by ion-exchange chromatography to near homogeneity in a simultaneous, single-step procedure . The yield of both enzymes was greater than 80% . Thiolase was purified approximately 8-fold with sp act 115 units/mg, whereas 3-hydroxybutyryl-CoA dehydrogenase was purified 14-fold with sp act 292 units/mg . Isoelec