Am J Vet Res, 1977 Jul, 38(7), 1069 - 73 Isolation and identification of anaerobes in the veterinary diagnostic laboratory; Berkhoff GA et al.; In a 1-year survey (1975 to 1976) of anaerobic bacteria recovered from diseased animals, anaerobes were as follows: Clostridium spp, 50%; gram-positive nonspore-forming anaerobic bacilli, 19%; gram-negative anaerobic bacilli, 19%; Actinomyces spp, 10%; and anaerobic cocci, 1% . Anaerobes were in approximately 61% of the specimens that were culturally positive for any bacteria . Approximately 25% of the specimens did not yield any bacteria (sterile specimens) . The method for isolating and identifying anaerobes was based upon the use of reducible solid mediums and was specially designed for veterinary diagnostic laboratories . Emphasis was placed on the selection, collection, and transportation of specimens. Hoppe Seylers Z Physiol Chem, 1977 Jul, 358(7), 789 - 95 Immobilized Clostridium perfringens neuraminidase . Substrate cleavage and enzyme release during incubation; Parker TL et al.; Pure Clostridium perfringens neuraminidase was immobilized on Sepharose 4 B, azido-Sepharose 4 B and controlled pore glass (CPG)- glycophase using different coupling procedures . The immobilized enzyme showed increased stability under various conditions relative to the soluble enzyme . The low release of active enzyme from the supports under incubation conditions was quantitated using a highly sensitive radioactive assay . The activity of the immobilized enzyme was dependent on the nature of the support and the substrate . Activity decreased with increasing substrate molecular weight, but the enzyme showed improved cleavage with GD1a micelles and human erythrocytes, substrates having ordered surface properties . Uses of immobilized neuraminidase in biochemistry and cell biology are considered and evaluated relative to the measured release of enzyme from the supports reported and to the molecular size and organization of possible substrates. Can J Microbiol, 1977 Jul, 23(7), 908 - 15 Enterotoxin formation by Clostridium perfringens type A studied by the use of fluorescent antibody; Niilo L; Fluorescein isothiocyanate-conjugated antibody to purified enterotoxin of Clostridium perfringens was used to study the intracellular formation of enterotoxin by this organism . Enterotoxin was detected at 4 h of growth at the end of the cell containing forespore . With the development of the spore, enterotoxin accumulation continued and involved the entire length of the cell until its lysis with the release of enterotoxin and mature spore . The spores did not contain demonstrable enterotoxin . Only a certain number of the sporulated cells of the enterotoxigenic strains studied produced this toxin . The amount of enterotoxin produced varied with sporulation percentage, and between strains and individual cells. Can J Microbiol, 1977 Jul, 23(7), 884 - 92 ICMSF methods studies . VIII . Comparative study for the enumeration of Clostridium perfringens in foods; Hauschild AH et al.; Four methods were compared in an international comparative study for the enumeration of Clostridium perfringens: the SFP (Shahidi-Ferguson perfringens) agar (A), TSC (tryptose-sulfite-cycloserin) agar (B), SC (sulfite-cycloserine) agar (C), and neomycin blood agar (D) methods . The confirmed C . perfringens counts were slightly lower for D than for A-C . The percentages of presumptive colonies confirmed as C . perfringens were essentially the same in each method . The relative numbers of nonspecific colonies were the lowest in C, followed by B, D, and A . The methods were also compared for simplicity and for aspects associated with the recognition and selection of presumptive colonies. Appl Environ Microbiol, 1977 Jul, 34(1), 30 - 3 Effect of storage time and temperature on the survival of Clostridium botulinum spores in acid media; Odlaug TE et al.; Clostridium-botulinum type A and type B spores were stored in tomato juice (pH 4.2) and citric acid-phosphate buffer (pH 4.2) at 4, 22, and 32 degrees C for 180 days . The spore count was determined at different intervals over the 180-day storage period . There was no significant decrease in the number of type A spores in either the tomato juice or citric acid-phosphate buffer stored for 180 days at 4, 22, and 32 degrees C . The number of type B spores did not decrease when storage was at 4 degrees C, but there was an approximately 30% decrease in the number of spores after 180 days of storage at 22 and 32 degrees C. Eur J Nucl Med, 1977 Jun 30, 2(2), 117 - 20 Enhancement of incorporation of 131iododeoxyuridine into tumors after application of Clostridium oncolyticum s . butyricum (M 55) . Animal experiments on positive scintigraphic localization of tumors; Volm M et al.; The application of spores of the bacterium Clostridium oncolyticum s . butyricum (M 55) results in an increased incorporation of 131Iododeoxyuridine in tumors (amelanotic melanoma Fortner III of the golden hamster and Brown-Pearce carcinoma of the rabbit) . This leads to an improved scintigraphic demonstration of the tumors. J Cell Sci, 1977 Jun, 25, 335 - 54 Extracellular matrix synthesis in blastula and gastrula stages of normal and hybrid frog embryos . III . Characterization of galactose- and glucosamine-labelled materials; Johnson KE; Normal Rana pipiens gastrulae show more incorporation of isotopically-labelled galactose and glucosamine into TCA-insoluble materials than blastulae . Interspecific hybrid embryos which undergo developmental arrest at the onset of gastrulation often synthesize reduced amounts of galactose- and glucosamine-labelled materials . These materials are high-molecular weight, but not collagen . After pronase digestion, labelled materials elute in the void volume of Sephadex G-50 . Labelled materials migrate slowly on cellulose acetate, bind to several kinds of anion exchangers and elute at low ionic strength, are precipitated by cetyl pyridinium chloride (CPC) when mixed with carrier compounds, and are not degraded by Clostridium neuraminidase or Streptomyces, leech, and bovine testicular hyaluronidases. Appl Environ Microbiol, 1977 Jun, 33(6), 1287 - 8 Production and counting of spores of Clostridium chauvoei; Bagadi HO; The concentration and viability of spores produced by four different strains of Clostridium chauvoei (C . feseri) grown in a modified medium for 18 days are described . The medium yielded enough viable spores for experimental work. Appl Environ Microbiol, 1977 Jun, 33(6), 1270 - 4 Hydrogen utilization by clostridia in sewage sludge; Ohwaki K et al.; A sporeformer morphologically different but physiologically similar to Clostridium aceticum Wieringa was isolated from sewage sludge . It used large amounts of H2 and CO2, converting them chiefly to acetic acid . Growth occurs anaerobically on yeast extract alone, but after the nutrients in yeast extract are used, growth continues at a reduced rate, supported by the conversion of the gases to acetate. J Bacteriol, 1977 Jun, 130(3), 1084 - 90 A four-iron, four-sulfide ferredoxin with high thermostability from Clostridium thermoaceticum; Yang SS et al.; A ferredoxin containing only one {Fe4S4} cluster was purified from Clostridium thermoaceticum . It has a molecular weight of about 7,300, a partial specific volume of 0.67, and an isoelectric point of 3.25 . Its absorption spectrum has two maxima at 390 nm (epsilon = 16.8 X 10(3)M-1cm-1) and at 280 nm (epsilon = 24.2 X 10(3)M-1cm-1) . The absorption at 390 nm is almost half that of other clostridial ferredoxins, which have two {Fe4S4} clusters, and is similar to other ferredoxins with only one {Fe4S4} cluster . The ferredoxin had high thermal stability and retained over 50% of its activity after treatment at 80 degrees C . It functions in the transfer of electrons from pyruvate to nicotinamide adenine dinucleotide phosphate (NADP), which indicates the presence of pyruvate:ferredoxin oxidoreductase and reduced ferredoxin-NADP reductase in C, thermoaceticum . NADPH is used in the synthesis of acetate from CO2 in this organism. Onderstepoort J Vet Res, 1977 Jun, 44(2), 53 - 4 The taxonomic position of Clostridium botulinum type c; Jansen BC et al.; Experimental evidence is produced to justify abandoning the practice of subdividing Costridium botulinum Type C into type Calpha and Cbeta. J Gen Microbiol, 1977 Jun, 100(2), 395 - 401 Clostridium sporogenes isolates and their relationship to C . botulinum based on deoxyribonucleic acid reassociation; Nakamura S et al.; Sixty-two isolates of Clostridium sporogenes from canned foods were examined for cultural properties, heat resistance and DNA-DNA homology to Clostridium botulinum type A190 . Sporulation was observed in most of 21 umbonate and rhizoidal colony-forming strains (colony-type I strains), but not in most of the 41 strains with convex and circular or crenate colonies with a mat to semi-glossy surface (colony-type II strains) . More than half of the latter strains showed much higher heat resistance than the rhizoidal colony-forming strains . The DNA isolated from colony-type II strains was 81% or more homologous to C . botulinum A190 DNA, forming duplexes which had thermostabilities similar to homologous duplexes of strain A190 DNA . Colony-type I strains differed from C . botulinum by 30 to 40% DNA homology and the DNA duplexes formed between these strains and strain A190 showed deltaT m(e) values of 7-0 degrees C when compared with the T m(e) of homologous DNA duplexes of strain A190. Can J Microbiol, 1977 Jun, 23(6), 829 - 32 Enumeration of Clostridium botulinum spores in meats by a pour-plate procedure; Hauschild AH et al.; Colonies of Clostridium botulinum could be easily distinguished from meat particles by supplementing Wynne agar with 0.4% egg yolk . The pour-plate method was suitable for enumeration of C . botulinum, provided the medium was covered with a layer of agar containing 0.01% dithiothreitol . Viable counts of heat-treated spores were consistently higher in Wynne agar supplemented with egg yolk (Wynne-EY agar) than in Wynne agar alone. Biokhimiia, 1977 Jun, 42(6), 1077 - 82 {The actions of low-molecular fragments of substrate and their analogs on the activity of isoenzymes of phospholipase C from Clostridium perfringens}; Litvinko NM et al.; The interaction of the lecithin molecule fragments and their analogues with phospholipase C Cl . perfringens was studied by gel-diffusion in agarose-lecithin gels . It was found intense inhibition of phospholipase C activity in the presence of cathionic compounds; this phenomenon shows the existence of anionic centre in the active site of enzyme . The esteric centre is probably hydrophobic nature and is not capable to bind the negatively charged groups . However, phosphoserine, phosphothreonine, gamma-aminobutyric, aspartic and glutamic acids can interact with an additional cathionic centre, whose location in phospholipase C differs from that in pancreatic phospholipase A2. Am J Vet Res, 1977 Jun, 38(6), 857 - 61 Reproduction and treatment of necrotic enteritis in broilers; Truscott RB et al.; A change in ration from one containing 50% fish meal to a standard chick starter containing 10(8) Clostridium perfringens/g of feed resulted in the production of necrotic enteritis in 2-week-old broiler chicks . Lincomycin added to the inoculated ration at a concentration of 20 g/907.2 kg significantly reduced mortality in the chicks . Inoculation of broth cultures of C perfringens directly into the duodenum, using a surgically inserted tetrafluoroethylene resin tube, indicated a relationship existed between the number of C perfringens inoculated and the gross lesions . The disease could be consistently produced by this technique. Appl Environ Microbiol, 1977 Jun, 33(6), 1295 - 7 Identification of intermediates formed during the degradation of hexachlorocyclohexanes by Clostridium sphenoides; Heritage AD et al.; Washed cell suspensions of Clostridium sphenoides degraded the alpha-isomer of 1,2,3,4,5,6-hexachlorocyclohexane via delta-3,4,5,6-tetrachloro-1-cyclohexene and the gamma-isomer via gamma-3,4,5,6-tetrachloro-1-cyclohexene . Both intermediates were further metabolized to unknown substances . The tetrachlorocyclohexene intermediates were identified by gas chromatography and mass spectrometry. Infect Immun, 1977 Jun, 16(3), 910 - 4 Correlation between oral toxicity and in vitro stability of Clostridium botulinum type A and B toxins of different molecular sizes; Sugii S et al.; The in vitro sensitivity to acid and pepsin differed markedly among Clostridium botulinum type A and B toxins of different molecular sizes . The larger the molecular size of the toxin, the higher the resistance to these agents . Tye B derivative toxin was rapidly inactivated, but the progenitor toxins resisted in vitro exposure to rat intestinal juice . The molecular dissociation of the progenitor toxins did not occur in rat intestinal juice of pH 7.0, but did occur in a buffer solution of the same pH . The oral toxicity may depend mostly on the stability of toxin molecules in the stomach and, to a less extent, in the intestine . The present results seem to justify the conclusion that C . botulinum type A and B progenitor toxins with molecular sizes larger than 16S are more potent oral toxins than 12S progenitor toxins. Biochim Biophys Acta, 1977 May 27, 492(1), 215 - 24 The proteolytic action of bacterial proteases from Clostridium histolyticum on bovine fibrinogen; Tranqui L et al.; Bacterial proteases from Clostridium histolyticum bring about the polymerization of fibrinogen into structured fibres, the period being 9 nm when the ionic strength (I) of the solution is between 0.1 and 0.2 . At I=0.3 no polymerization occurs, but the successive actions of bacterial proteases and thrombin induce polymerization, the fibres obtained showing a periodic structure: the major period is 23 nm with two minor striations . The striation pattern is different from that obtained with fibrin which shows a major period of 23 nm with three minor striations . The degree of degradation of fibrinogen monitored by disc gel electrophoresis shows that the Aalpha polypeptide chain (mol . wt.=68 000) is degraded into Aalpha1 (mol . wt.=32 000) and Aalpha2 (mol . wt.=29 000), whilst the mobility of Bbeta increases and gamma remains unchanged; the molecular weight is estimated between 272 000 and 269 000 . These results emphasize that the charge distribution differences which are compatible with the loss of different portions of the molecule, lead to variations in fibre structures. Lancet, 1977 May 14, 1(8020), 1046 - 8 Hospital outbreak of clostridium perfringens food-poisoning; Thomas M et al.; An outbreak of Clostridium perfringens (C.welchii) food-poisoning affected a third of the patients in a large hospital, and one frail patient died . C.perfringens type A, serotype 1, was isolated from 46 (61 per cent) of 76 patients examined and from food, and a new serotype (61) was isolated from 16 . The attack-rate among patients who ate a minced-ham dish was 78 per cent . The cooking and storage of this mince was faulty: cuts of meat were much too large, they were kept at room temperature too long before refrigeration, and after cooking they were put into mincers used also for raw meat . C.perfringens type A, serotype 1, was isolated from meat scraps in a mincer . Final reheating was inadequate to destroy even vegetative bacteria, and multiplication may have occurred during slow distribution to the wards . Outbreaks of C.perfringens fool-poisoning are common in hospitals, and some underlying problems are discussed . A plea is made for the Food Hygiene Regulations to apply to National Health Service premises and for simple but effective reforms in institutional catering management. JAMA, 1977 May 2, 237(18), 1946 - 51 Infant botulism . Epidemiological, clinical, and laboratory aspects; Arnon SS et al.; Clostridium botulinum organisms and toxin were identified in the feces of six infants, aged 5 to 20 weeks, who had illnesses clinically consistent with botulism . Five of the infants lived in California and became ill within a six-month period in 1976; one infant became ill in New Jersey in 1975 . Three cases were type A botulism, and three were type B . No source of ingested botulinal toxin could be found in any case . However, one infant with type B botulism had ingested a food containing C botulinum type B organisms, and no toxin was found in it . The clinical findings in these cases include constipation, weak sucking and crying ability, pooled oral secretions, cranial nerve deficits, generalized weakness, and, on occasion, sudden apnea . A characteristic electromyographic pattern termed "brief, small, abundant, motor-unit action potentials" (BSAP) was observed . The sources of C botulinum toxin for these six infants is thought to have been in vivo (gastrointestinal) production following ingestion of C botulinum organisms . Studies are underway to determine the full clinical spectrum, incidence, and potential public health importance of this infectious disease newly recognized in infants. Res Vet Sci, 1977 May, 22(3), 343 - 6 Observations on the possible invasiveness of Clostridium botulinum for waterfowl; Graham JM et al.; Twenty-four ducklings given multiple doses of Clostridium botulinum type C spores and toxin per os and 27 affected waterfowl from four natural outbreaks of the disease were examined bacteriologically . No evidence of invasion of the blood or liver was found in any bird and it is suggested that invasiveness and toxigenesis in internal organs are probably of little, if any, importance in the causation of botulism in waterfowl. J Assoc Off Anal Chem, 1977 May, 60(3), 563 - 9 An improved cooked meat medium for the detection of Clostridium botulinum; Quagliaro DA; A new medium composed of cooked meat and fluid thioglycolate broth was tested with 20 proteolytic and 11 saccharolytic strains of Clostridium botulinum . All of the proteolytic strains produced a black ring at the surface of the broth, presumably due to hydrogen sulfide production, while the saccharolytic strains produced white opaque rings . Since the black ring is formed rapidly and is easily seen, the new medium may be useful for the rapid identification of proteolysis in unknown isolates . Growth and toxin production were better in the new medium than in the usual cooked meat medium or in cooked meat medium plus 0.5% glucose . These results suggest that the new medium will be useful in facilitating identification of C . botulinum. J Assoc Off Anal Chem, 1977 May, 60(3), 541 - 5 Collaborative study of a method for the detection of Clostridium botulinum and its toxins in foods; Kautter DA et al.; The mouse toxicity and protection technique for the detection and identification of Clostridium botulinum and its toxins in foods was collaboratively studied by 11 laboratories . Each laboratory received 4 samples of cream of mushroom soup; 2 contained spores and toxin of C . botulinum type A, 1 contained spores and toxin of C . botulinum type E, and 1 contained spores of C . sporogenes . The media used were cooked meat medium (beef heart or chopped liver broth) and trypticase peptone glucose yeast extract broth with trypsin . The results indicate that this method has a high degree of repeatability and reproducibility . All 11 laboratories correctly identified the toxins and the nontoxic sample in the food and detected and identified the viable spores in the samples by means of the subsequent cultures . This method has been adopted as official first action. Ann Sclavo, 1977 May-Jun, 19(3), 494 - 501 {Two outbreaks of "Clostridium perfringens" food poisoning: epidemiological remarks (author's transl)}; Caroli G et al.; Two outbreaks of Cl . perfringens food poisoning which occurred in Florence during 1976 have been described . The first one involved three hundred primary school children; processed re-heated turkey meat was thought to have been the vehicle of infection in the school meal . The clinical symptoms consisted of mild diarrhoea in all cases and the duration of the illness was about 12 hours . The possible part played by food storage temperature, post-cooking periods and food trolleys in the spread of infection is discussed . The other outbreak interested three people who ate a dish with gravy in a restaurant; one of these suffered severe haemorrhagic enteritis and died after two weeks . Necroscopy was performed and the results of post-mortem examination as well as histological and bacteriological findings certified that the cause of death was severe enteritis (Necrotizing enteritis) in elderly debilitated patient (with diabetes, chronic bronchitis, arteriosclerosis and previously gastroresected). Zh Mikrobiol Epidemiol Immunobiol, 1977 May, (5), 71 - 4 {Characteristics of the immunologic response of animals to Clostridium perfringens anatoxin}; Petrov RV et al.; Experiments were conducted on guinea pigs, rabbits and mice (mongrel and inbred); immunogenic properties of Cl . perfringens toxoids of different purity were studied . Toxin neutralization and passive hemagglutination tests were used to determine the antitoxic immunity level . It appeared that in the immunization of guinea pigs and rabbits the degree of immunogenicity of the preparations increased with the elevation of their specific activity . Under the same conditions both the mongrel and the inbred mice displayed the maximum immune response in the immunization with the least purified preparations, and the minimum after the injection of a highly purified antigen. J Infect Dis, 1977 May, 135(5), 850 - 4 Anaerobic bacteria in biliary disease in elderly patients; Shimada K et al.; Gallbladder bile from 52 elderly subjects who had undergone biliary tract surgery was examined for the presence of bacteria . Twelve patients had sterile bile, 18 specimens of bile yielded anaerobes as well as aerobes, and 22 yielded aerobic bacteria only . Escherichia coli was the most commonly isolated organism (30 strains) . Bacteroides fragilis was the most frequently encountered anaerobic bacterium and was found in 15 patients . The Klebsiella-Enterobacter group was the second most commonly isolated group and B . fragilis was third . Clostridium perfringens was recovered in 10 specimens of bile . Anaerobic bacteria were recovered more frequently in patients with ductal obstruction . The relatively frequent isolation of anaerobes, especially of B . fragilis, in this study may be related to the anaerobic techniques used, to the age of the patients, and to the high incidence of pigment stones among the subjects. Can J Microbiol, 1977 May, 23(5), 601 - 6 Clostridium perfringens--specific lysin; Nakamura S et al.; A mitomycin C induced lysate of Clostridium perfringens strain KZ219 was lytic to 50 strains of C . perfringens of types A-E, and three strains of C . plagarum . The lysin was active against only 2 out of 87 strains of 51 other clostridial species . The optimum pH of the lytic agent was 5.5 . The activity was largely inactivated by proteolytic enzymes, and nearly completely inactivated by heating at 60 degrees C for 5 min. J Mol Evol, 1977 Apr 29, 9(2), 111 - 9 Phylogenetic studies of two rubredoxins from sulfate reducing bacteria; Vogel H et al.; The sequences of two rubredoxins isolated from the sulfate reducing bacteria: Desulfovibrio vulgaris and Desulfovibrio gigas have been elucidated . They have similar sequences but many more differences occur than would be expected from two bacteria of the same genus . Of the 52 sites, only 37 are occupied by identical residues . The primary structures are compared with those of the anaerobic bacteria rubredoxins of Clostridium pasteurianum, Micrococcus aerogenes, Pseudomonas oleovorans and Peptostreptococcus elsdenii: only 12 identities are found, mostly in the two clusters that contain two iron-bound cysteines each . A phylogenetic tree based on the primary structures is presented and possible relations with plant and bacterial ferredoxins are discussed . A secondary and tertiary structure stereochemically compatible with the sequence data, is proposed. J Biol Chem, 1977 Apr 25, 252(8), 2657 - 61 Evidence for separate enzymes of pyruvate decarboxylation and pyruvate synthesis in soluble extracts of Clostridium pasteurianum; Bush RS et al.; Additional evidence to that already presented (Sauer, F . D., Bush, R . S., and Stevenson, I . L . (1976) Biochim . Biophys . Acta 445, 518-520) suggests that pyruvate-ferredoxin oxidoreductase isolated from Clostridium pasteurianum consists of two separate enzymes: (a) pyruvate lyase, which catalyzes the CoA and electron acceptor-dependent decarboxylation of pyruvate, and (b) pyruvate synthase, which catalyzes the reduced ferredoxin-dependent carboxylation of acetyl-CoA to pyruvate . The enzymes separated on Sephadex G-200 and with acrylamide gel electrophoresis but complete separation of one enzyme free of the other was not achieved . Extensive purification procedures were not used because both enzymes are unstable . The results confirm published reports that pyruvate lyase contains thiamin and a chromophore which participates in electron transfer . Pyruvate synthase, however, did not appear to be a thiamin enzyme and there was no evidence to indicate participation of an enzyme chromophore in the pyruvate synthase reaction. J Biol Chem, 1977 Apr 10, 252(7), 2245 - 53 Direct assignment of the cysteinyl, the slowly exchangeable, and the aromatic ring 1H nuclear magnetic resonances in clostridial-type ferredoxins; Packer EL et al.; We have directly assigned the 1H NMR corresponding to the cysteinyl protons, the slowly exchangeable protons, and the aromatic ring protons in the 1H NMR spectrum of Clostridium acidi-urici ferredoxin by isotopic labeling and 13C NMR decoupling techniques . We also show that the resonance pattern in the 8- to 20-ppm (from 2,2-dimethyl-2-sialapentanesulfonic acid) region of the 1H NMR spectra of oxidized Clostridium acidi-urici, Clostridium pasteurianum, Clostridium perfringens, and Peptococcus aerogenes ferredoxins are very similar, and we assign the resonances in this region by analogy with the spectrum of C . acidi-urici ferredoxin . The 1H NMR spectra of the beta protons of the cysteinyl residues of these ferredoxins differ, however, from the 1H NMR spectra of equivalent beta protons of the methylene carbon atoms bonded via a sulfur atom to {4Fe-4S} clusters in synthetic inorganic analogues . In the spectra of the synthetic compounds, the beta protons appear as a single resonance shifted 10 ppm from its unbonded reference position . In the spectra of oxidized clostridial ferredoxins, the cysteinyl beta protons appear as a series of at least eight resolved resonances with shifts that range from 6 to 14 ppm, relative to the free amino acid resonance position . This difference in the spectra of the protein and the synthetic compounds probably results from the fact that the equivalent beta protons of the synthetic compounds are not constrained and are free to rotate and thus assume the same average orientation with respect to the {4Fe-4S} cluster . The shift pattern in the 9- to 14-ppm region is identical in three different clostridial ferredoxins . This suggests that the molecular environments of the corresponding cysteinyl residues are identical . Significant differences in the resonance positions occur, however, in the 14- to 18-ppm region, suggesting that the physical environments of these cysteinyl residues differ . This may reflect differences in the orientation of the corresponding cysteinyl residues relative to the {4Fe-4S} clusters or differences in charge density at the cysteinyl beta protons or both . The slowly exchangeable protons were identified by comparing the 1H NMR spectra of ferredoxins reconstituted in H2O and 2H2O . The remaining resonances in the 8- to 20-ppm region were assigned to each of the 2 tyrosyl residues in C . acidi-urici ferredoxin . This was done by comparing the 1H NMR spectra of C . acidi-urici {(3',5'-2H2)Tyr}ferredoxin and C . acidi-urici {PHE2}ferredoxin with that of C . acidi-urici native ferredoxin. Biochemistry, 1977 Apr 5, 16(7), 1467 - 73 Benzo{a}yrenedione/benzo{a}pyrenediol oxidation-reduction couples and the generation of reactive reduced molecular oxygen; Lorentzen RJ et al.; The ability of the isomeric quinone metabolites of benzo{a}pyrene, benzo{a}pyrene-6,12-dione, benzo{a}pyrene-1,6-dione, and benzo{a}pyrene-3,6-dione to undergo reversible, univalent oxidation-reduction cycles involving the corresponding benzo{a}pyrenediols and intermediate semiquinone radicals has been characterized . Under anaerobic conditions, all three benzo{a}pyrenediones are easily reduced to benzo{a}pyrenediols, even by mild biological agents such as NAD(P)H, cysteamine, and glutathione . The benzo{a}pyrenediols, in turn, are very rapidly autoxidized to the benzo{a}pyrenediones when exposed to air . Substantial amounts of hydrogen peroxide are produced during these autoxidations, and other reactive reduced oxygen species, such as the superoxide and hydroxyl radicals, are probably formed transiently as well . The benzo{a}pyrenediol-benzo{a}pyrenedione interconversions proceed by one-electron steps; the corresponsing semiquinone radicals can be monitored by electron spin resonance spectroscopy as inter mediates during these reactions carried out at high pH . Benzo{a}pyrenediones induce DNA strand scission when incubated with bacteriophage T7 DNA . This damage is modified by conditions which indicate that reduced oxygen species propagate the free-radical reactions responsible for the strand scission . Benzo{a}pyrenediones are electron-acceptor substrates for NADH dehydrogenase from Clostridium kluyveri . Catalytic amounds of these benzo{a}pyrene metabolites, together with this respiratory enzyme function as cyclic oxidation-reduction couples which link NADH and molecular oxygen in the continuous production of hydrogen peroxide . These data, together with preliminary results with cells in culture, indicate that benzo{a}pyrenediones are potentially harmful metabolites of benzo{a}pyrene, acting by processes which lead to their regeneration rather than depletion; nucleic acid and call damage is probably produced by the reactive reduced oxygen species resulting from such regenerative oxidation-reduction cycles. Ann Microbiol (Paris), 1977 Apr, 128A(3), 267 - 76 {Taxonomic characters of "Clostridium tyrobutyricum" (author's transl)}; Roux C et al.; The taxonomic characters of 77 strains of Clostridium tyrobutyricum have been studied and compared to those of known strains . This work shows the insufficiency of the Bergey's Manual (8th edition) nine characters distinguishing Clostridium groupe I to identify C . tyrobutyricum . To be more representative of the species, addition of one or two characters to this key of determination and some modifications to the complementary description of C . tyrobutyricum in this manual are proposed. Appl Environ Microbiol, 1977 Apr, 33(4), 955 - 62 Citrate, a specific substrate for the isolation of Clostridium sphenoides; Walther R et al.; With a medium containing citrate as the carbon and energy source, 10 clostridial strains were isolated from various mud samples . Characterization of these strains revealed that they all belonged to the same species, Clostridium sphenoides . Strains of this organism obtained from culture collections were also able to grow citrate, whereas 15 other clostridial species tested were not . Citrate was fermented by C . sphenoides to acetate, ethanol, carbon dioxide, and hydrogen . Experiments with stereospecifically 14C-labeled citrate indicated that citrate lyase was involved in citrate degradation. Can J Comp Med, 1977 Apr, 41(2), 188 - 94 Inactivation of Clostridium haemolyticum toxic fluids and their antigenicity; Lozano EA; One hundred fifty-one isolates of Clostridium haemolyticum were examined for consistent toxin production following repeated serial transfers in laboratory media . Most of these isolates produced only small amounts of toxic materials and serial transfers appeared to reduce toxigenic characteristics . Eleven of the isolates consistenly produced measurable amounts of toxic materials . One of these isolates was used for production of toxic fluids that were concentrated by lyophilization and reconstitution to a smaller volume or by precipitation with ammonium sulphate followed by dialysis against water and glycerol . Known amounts of these substances were inactivated with formalin, heat, beta-propiolactone, ultra-violet irradiation and glutathione . The resulting toxoids were inoculated into guinea pigs and most were judged to be nonimmunogenic because the animals were unable to resist dermal challenge . Toxic materials with added glycine were inactivated with formaldehyde as readily as those without the amino acid but the resulting toxoids were immunogenic while those prepared without the amino acid were not. Appl Environ Microbiol, 1977 Apr, 33(4), 963 - 6 Improved procedure for crystallization of Clostridium botulinum type A toxic complexes; Sugiyama H et al.; A modified protocol for the preparation of botulinum type A toxic crystals is decribed . The essential change was the substitution of chromatography on diethylaminoethyl-Sephadex for the step in which toxin was precipitated with ethanol . Colored materials and nucleic acids were removed by the chromatography so that crystal lots of greater uniformity could be produced . Significantly more of toxin in the starting culture was recovered as crystals . The lots of crystals obtained initially by either the original or the modified method contained an antigen that was eliminated when the lots were recrystallized. Infect Immun, 1977 Apr, 16(1), 107 - 9 Oral toxicities of Clostridium botulinum toxins in response to molecular size; Ohishi I et al.; Clostridium botulinum type A, B, and F toxins of different molecular sizes were fed to mice to compare the oral toxicities . The progenitor toxin, a complex of a toxic and nontoxic component, of any type was higher in oral toxicity to mice than the dissociated toxic component or the derivative toxin . The former may no doubt play a more important role in the pathogenesis of food-borne botulism . The higher oral toxicity possessed by the progenitor toxin, including the exceptionally high one found with type B-L toxin, can be explained solely by the protection afforded by the nontoxic component attached to the toxic component . The possibility of the highest oral toxicity of type B-L toxin to humans is discussed. Am J Dis Child, 1977 Apr, 131(4), 445 - 6 Otogenous tetanus: a sequelae of chronic ear infections; Fischer GW et al.; Eight patients had bacteriologically confirmed otogenous tetanus and all survived, suggesting that this may be a less severe form of the disease . Clostridium organisms most probably secondarily infect the purulent ear discharge after contamination by dirty cloth or fingers . Since most cases of tetanus are seen first by pediatricians or family physicians, they should be familiar with this potential source . Adequate tetanus immunization predisposed to chronic otorrhea. Avian Dis, 1977 Apr-Jun, 21(2), 256 - 63 The interaction of Clostridium perfringens and its toxins in the production of necrotic enteritis of chickens; Al-Sheikhly F et al.; The intraduodenal administration of large numbers of Clostridium perfringens cells harvested from broth cultures and resuspended in PBS or fresh sterile thioglycollate broth produced a very mild form of necrotic enteritis . Administering an appropriate number of cells in culture supernatant, however, produced typical field-type disease . Alpha toxin was shown to be the significant toxin recoverable from broth-culture supernatant fluids . Requirements to produce the disease are minor intestinal damage and sufficient numbers of toxigenic C . perfringens in the intestine. Avian Dis, 1977 Apr-Jun, 21(2), 241 - 55 The pathology of necrotic enteritis of chickens following infusion of crude toxins of Clostridium perfringens into the duodenum; Al-Sheikhly F et al.; Necrotic enteritis was produced in 4-week-old chickens with bacteria-free crude toxins of Clostridium perfringens . Typical gross lesions could be seen as early as 3 hr after intraduodenal infusion of toxin was begun . Early microscopic lesions were observed at 20 minutes, progressing with time to involve most of the mucosa . Death was related to the amount of toxin administered . Because systemic absorption of toxin is likely, acutely affected birds may die from toxemia. Avian Dis, 1977 Apr-Jun, 21(2), 230 - 40 The pathology of necrotic enteritis of chickens following infusion of broth cultures of Clostridium perfringens into the duodenum; Al-Sheikhly F et al.; Necrotic enteritis was consistently reproduced when enough active broth culture of Clostridium perfringens type A was infused intraduodenally . Typical lesions of necrotic enteritis were seen as early as 5 hr after infusion was begun . The histologic lesions observed at 1 hr were characterized by edema in the lamina propria and desquamation of epithelial cells . Large numbers of clostridia were seen among these sloughed cells . Coagulation necrosis of the tips of villi became evident at 3 hr and was marked at 5 hr . Many clumps of clostridia were obvious among the necrotic tissue . At 8 and 12 hr the necrotic lesions extended to involve most of the villus structures . Morphologically abnormal erythrocytes were evident in the visceral organs at 12 hr. Mikrobiologiia, 1977 Mar-Apr, 46(2), 346 - 51 {Variable forms of Clostridium felsineum}; Avrova NP; Variants of five types, differing in morphology of their colonies, resulted from spontaneous variability of Clostridium felsineum, the main agent involved in flax retting, when it was grown in a liquid medium and then on a solid medium . Morphological, cultural, and biochemical properties of the variants are described . The variants of the second and third types were less stable than those of the first type in the course of passages on growth media . The rate of spore formation was higher in the variants of the first type whereas the activity of pectolytic enzymes was greater in the variants of the second type. Hoppe Seylers Z Physiol Chem, 1977 Mar, 358(3), 339 - 52 {On cobyrinic acid biosynthesis . Novel methylated hydroporphyrins and their role in cobyrinis acid formation (author's transl)}; Deeg R et al.; Clostridium tetanomorphum and Propionibacterium shermanii were examined for intermediates in the synthetic pathway uroporphyrinogen III leads to cobyrinic acid . The isolation of two novel methylated hydroporphyrins, whose methyl groups are derived from S-adenosyl-L-methionine, is described . Spectroscopic (field desorption-mass, visible absorption) und electrophoretic studies as well as incorporation of labelled substrates indicate that they are analogues of a dihyrouroporphyrin and a tetrahydrouroporphyrin with adjacent reduced rings . Field desorption spectra of the {C2H3}- and {CH3}-tetrahydrouroporphyrin analogues show that the compound contains two methyl groups; it is concluded that the chlorine-like compound has one methyl group . Dehydrogenation experiments indicate that the methyl groups are located at one beta-carbon of the reduced rings . Incorporation experiments suggest that the tetrahydrouroporphyrin-like compound is an intermediate in cobyrinic acid biosynthesis . Studies on the utilization of a heptacarboxyporphyrinogen from C . tetanomorphum for cobyrinic acid formation are also described. J Infect Dis, 1977 Mar, 135 Suppl, S7 - 12 In vitro susceptibility of anaerobes: comparison of clindamycin and other antimicrobial agents; Sutter VL; The in vitro activity of clindamycin against anaerobic bacteria was compared with that of other antimicrobial agents . Clindamycin remained effective against most anaerobic bacteria found in infections, although resistance to the drug was found in certain groups, such as Peptococcus and Clostridium species. Can J Microbiol, 1977 Mar, 23(3), 346 - 53 Studies on some characteristics of hydrogen production by cell-free extracts of rumen anaerobic bacteria; Joyner AE et al.; Hydrogen production was studied in the following rumen anaerobes: Bacteroides clostridiiformis, Butyrivibrio fibrisolvens, Enbacterium limosum, Fusobacterium necrophorum, Megasphaera elsdenii, Ruminococcus albus, and Ruminococcus flavefaciens . Clostridium pasteurianum and Escherichia coli were included for comparative purposes . Hydrogen production from dithionite, dithionite-reduced methyl viologen, pyruvate, and formate was determined . All species tested produced hydrogen from dithionite-reduce methyl viologen, but only C . pasteurianum, B . clostridiiformis, E . limosum, and M . elsdenii produced hydrogen from dithionite . All species except E . coli produced hydrogen from pyruvate, but activity was low or absent in extracts of E . limosum, F . necrophorum, R . albus, and R . flavefaciens unless methyl viologen was added . Hydrogen was produced from formate only by E . coli, B . clostridiiformis, E . limosum, F . necrophorum, and R . flavefaciens . Extracts were subjected to ultracentrifugation in an effort to determine the solubility of hydrogenase . The hydrogenase of all species except E . coli appeared to be soluble, although variable amounts of hydrogenase activity were detected in the pellet . Treatment of extracts of the rumen microbial species with DEAE-cellulose resulted in loss ofhydrogen production from pyruvate . Activity was restored by the addition of methyl viologen . It is concluded that hydrogen production in these rumen microorganisms is similar to that in the saccharolytic clostridia. Infect Immun, 1977 Mar, 15(3), 999 - 1001 Effect of Clostridium perfrongens enterotoxin in mitochondrial respiration; McDonel JL et al.; Clostridium perfringens enterotoxin caused a 26 to 41% reduction in the rate of oxygen consumption by rat liver mitochondria when various tricarboxylic acid cycle intermediates were used as substrate . However, P/O ratios were unaltered. Infect Immun, 1977 Mar, 15(3), 765 - 71 Capsular polysaccharide of Clostridium perfringens Hobbs 9; Cherniak R et al.; Several closely related capsular polysaccharides were isolated from a strain of Clostridium perfringens Hobbs 9 type A by extraction of encapsulated cells with cold 0.85% NaCl . The soluble polymers were precipitated with alcohol and purified by (NH4)2SO4 fractionation, enzymatic digestion with papain and ribonuclease, and chromatography on diethylaminoethyl-Sephadex A25 . The polysaccharides were composed mainly of glucose, galactose, and galactosamine . The major fraction contained these constituents (representing 77% of the dry weight) in a molar ratio of 1:1.6:1.1 . All of the fractions contained phosphate and peptide material that was not removed during purification . The polysaccharides were closely related but not identical as indicated by double-diffusion-in-gel experiments . Immunoelectrophoresis in agarose demonstrated that the polysaccharides had identical mobilities and that no resolution into additional fractions occurred . The immunological activity of all the purified polysaccharides was destroyed by periodate oxidation but was unaffected by protease. Mikrobiologiia, 1977 Mar-Apr, 46(2), 311 - 7 {Ecologic features of cultures of the species Clostridium gürfelii}; Vozniakovskaia IuM et al.; The Clostridium cultures decomposing pectin were isolated from flax straw during flax retting . The activity was manifested only at high pH values, the optimum pH being 6.7--8.2 . Such ecological adaptation of the cultures is determined by the properties of the pectolytic enzyme complex which they synthesize . The complex does not contained polygalacturonase (the optimum pH 5.0) but has a high activity of pectate transeliminase (the optimum pH 8.0--8.4) . The cultures differ from Clostridium felsineum which possesses a high activity of polygalacturonase and therefore can decompose pectin in acid medium . By their morphological, cultural and physiological properties, the bacteria are classed as Clostridium gufelii (Prevot, 1966). Vet Rec, 1977 Feb 5, 100(6), 106 - 9 Avian botulism and the high prevalence of Clostridium botulinum in the Norflok Broads; Borland ED et al.; An account is given of a severe outbreak of type C botulism in waterfowl that occurred on the Norfolk Broads during the exceptionally warm summer of 1975 . Forty-five mud samples were collected from 22 well distributed aquatic sites representing a considerable proportion of the total number of Broads . All samples except one (ie, 97-8 per cent) were shown to contain Clostridium botulinum and 58 per cent contained more than one type of the organism . Types B, C and E were demonstrated in 62-2 per cent, 51-1 per cent and 60 per cent of samples respectively . Recent surveys, made by identical methods, of aquatic environments in the London area and the Camargue (France) showed prevalences of Cl botulinum of 72-5 per cent and 4-5 per cent respectively . It seems likely that the Norfolk Broads will continue to present a risk to waterfowl from botulism in future hot summers. Appl Environ Microbiol, 1977 Feb, 33(2), 289 - 97 Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum; Weimer PJ et al.; The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C . thermocellum/Methanobacterium thermoautotrophicum cocultures was studied . All cultures were grown under anaerobic conditions in batch culture at 60 degrees C . When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture . Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture . Monocultures produced primarily ethanol, acetic acid, H2 and CO2 . Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture . In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion . Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture . Fermentation of cellobiose was more rapid than that of cellulose . In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced. Biull Eksp Biol Med, 1977 Feb, 83(2), 198 - 200 {Immune response of mice of different inbred lines to Clostridium oedematiens anatoxin}; Petrov RV et al.; Mice belonging to a number of inbred strains were immunized intradermally with Cl . oedematiens alpha-toxoid . The immunization was repeated 30 days later . On the 20th and the 30th days after the first injection and on the 10th day after the second one the antibody level against the toxoid was determined in the blood of mice by the passive hemagglutination test . The maximum response to the primary immunization was observed in the mice of the C3H strain, and the minimum one--in mice of the DBA/2 strain; the difference was more than 30-fold . The rest of the strains used in the test (A,CBA, BALB/c, AKR, CC57BR) displayed an intermediate level of the immune response . The differences reduced after the repeated immunization . The immune response to this antigen in mice is supposed to be genetically controlled. Appl Environ Microbiol, 1977 Feb, 33(2), 400 - 5 Isolation and molecular size of Clostridium botulinum type C toxin; Syuto B et al.; A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin . The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein . The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain . The molecular weights of the subunits were approximately 98,000 and 53,000 . When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond). J Bacteriol, 1977 Feb, 129(2), 1148 - 50 Biochemical properties of Clostridium bifermentans spores; Hausenbauer JM et al.; As previously found for spores of Bacillus species, dormant spores of Clostridium bifermentans contained essentially no adenosine triphosphate, a high level of adenosine monophosphate, a high level of 3-phosphoglyceric acid, and much transfer ribonucleic acid lacking a 3'-terminal adenosine monophosphate residue . As in spores of Bacillus species, germination of C . bifermentans spores was accompanied by utilization of the 3-phosphoglyceric acid, a large increase in the adenosine triphosphate level, and the disappearance of defective transfer ribonucleic acid . In contrast to spores of Bacillus species, dormant spores of C . bifermentans contained little free amino acid. J Hyg (Lond), 1977 Feb, 78(1), 39 - 41 A comparison of the distribution of Clostridium botulinum in soil and in lake mud; Smith GR et al.; In 1975, 25 soil samples were collected from the London area . Of these, 20 were obtained 200-300 yards from 20 lakes that had been shown in 1974 to contain mud contaminated with one or more of types of B, C, D, and E of clostridium botulinum . By means of a technique comparable with that use for the examination of mud, the 20 soil samples were found negative . The remaining 5 soil samples, obtained from sites that were not in close proximity to lakes, were also negative except for one that contained type B. J Hyg (Lond), 1977 Feb, 78(1), 33 - 7 The low prevalence of Clostridium botulinum in the lakes, marshes and waterways of the Camargue; Smith GR et al.; Mud samples collected in June 1975 from the lakes, marshes and waterways of the Camargue were examined for Clostridium botulinum . The Grand Rhone and Petit Rhone were shown to contain types B and E, but of 44 samples taken from well distributed sites on the Ile de la Carmargue, only two (4-5%) were positive and these contained type E alone . The survey indicated a much lower prevalence of Cl . botulinum than any encountered in recent surveys of inland aquatic environments elsewhere. Proc Natl Acad Sci U S A, 1977 Feb, 74(2), 547 - 51 Identification of the iron-sulfur center in trimethylamine dehydrogenase; Hill CL et al.; Trimethylamine dehydrogenase {trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7} from a facultative methylotroph bacterium has a molecular weight of 147,000 and contains two types of prosthetic groups, one a covalently bound organic chromophore of uncertain structure and the other containing iron and labile sulfur (S*) . The structure of the Fe-S* center has been investigated by reactions of the enzyme with sodium mersalyl, o-xylyl-alpha,alpha'-dithiol, and p-methoxybenzenethiol in a 4:1 vol/vol hexamethylphosphoramide/water reaction medium, which destabilizes tertiary structure . Mersalyl treatment results in reduction of visible absorbance consistent with the presence of a 4-Fe center of the ferredoxin type . Reaction with thiols effects partial bleaching of the organic chromophore, as established by separate studies of a detached chromophore peptide, and results in removal (extrusion) of the core unit of the Fe-s* center in the form of the complexes {Fe4S*4(S2-o-xylyl)2}n2n- and {Fe4S*4(SC6H4OMe)4}2-, which were identified by absorption spectra . These results, in conjunction with control extrusion reactions of oxidized ferredoxins from spinach and Clostridium pasteurianum, establish that trimethylamine dehydrogenase contains one Fe4S*4 core unit most probably present as a ferredoxin-type, cysteinate-ligated cluster {Fe4S*4(S-Cys)4}. Appl Environ Microbiol, 1977 Feb, 33(2), 254 - 6 Amorphous ferrous sulfide as a reducing agent for culture of anaerobes; Brock TD et al.; Amorphous ferrous sulfide, prepared by reacting ferrous ammonium sulfate and sodium sulfide, is an excellent reducing agent for the culture of anaerobes . It reduces resazurin and reacts much more rapidly with O2 than does either soluble sulfide (HS)- or cysteine . One of the end products of the oxidation of ferrous sulfide with O2 is red and serves as an indicator for the oxygen contamination of a culture medium . Amorphous ferrous sulfide served as a suitable reducing agent for the growth of species of Methanobacterium or Clostridium . Its use is recommended for enrichment or culture of anaerobes (e.g . autotrophs, fermentative organisms) from sediments and other habitats were organic reducing agents are undesirable and where soluble sulfide might be toxic. Acta Pathol Microbiol Scand {B}, 1977 Feb, 85B(1), 89 - 94 Chemical modification and characterization of enterotoxin from clostridium perfringens type A; Granum PE et al.; Enterotoxin from Clostridium perfringens type A has been purified . The enterotoxin was shown to be heat-labile, but re-activation of heat treated enterotoxin did occur down to an activity of 15 per cent of the native enterotoxin . The molecular weight was shown to be 34,000 by ultracentrifugation, and the molecular weight did not change significantly after treatment with 0.1 M beta-mercaptoethanol and 6 M guanidine hydrochloride . It was concluded that the enterotoxin consists of one single polypeptide chain . The enterotoxin did not lose any activity after treatment with iodoacetic acid and iodoacetamide, but a complete loss of activity was observed after succinylation. J Bacteriol, 1977 Feb, 129(2), 843 - 9 Evidence for stable messenger ribonucleic acid during sporulation and enterotoxin synthesis by Clostridium perfringens type A; Labbe RG et al.; Stable messenger ribonucleic acid (mRNA) was shown to be involved in both enterotoxin synthesis and synthesis of other spore coat proteins in Clostridium perfringens . When used at a concentration that inhibited {14C}uracil incorporation, rifampin, a specific inhibitor of deoxyribonucleic acid-dependent RNA polymerase, prevented incorporation of a mixture of labeled amnoo acids by 3-h sporulating cells . At that time, enterotoxin protein was first detectable and cells were primarily at stage II or III of sporulation . When rifampin or streptolydigin was added to 5-h sporulating cells, which were primarily at stage IV or V and had significant toxin levels, incorporation of labeled amino acids continued through 30 min despite its presence . Rifampin also failed to prevent the specific synthesis of enterotoxin, a structural protein of the spore coat . The half-life of enterotoxin RNA was estimated to be at least 58 min . When cell extracts from 5-h sporulating cells that had been exposed to 3H-labeled amino acids for 10 min were subjected to electrophoresis on polyacrylamide gels and the gels were subsequently analyzed for radioactivity, two major peaks of radioactivity were obtained . The two peaks corresponded to enterotoxin and another spore coat protein(s) . Similar results were obtained when the cells had been preincubated for 60 min with rifampin before label addition, indicating the functioning of stable mRNA. Can J Microbiol, 1977 Feb, 23(2), 152 - 60 {Study of the NADH and NADPH-ferredoxin oxidoreductase activities in Clostridium acetobutylicum}; Petitdemange H et al.; The NADH and NADPH-ferredoxin oxidoreductase have been studied in Clostridium acetobutylicum . Acetyl-CoA is an obligatory activator of NADH-ferredoxin reductase activity and NADH a competitive inhibitor of ferredoxin-NAD+ reductase activity . These regulations are the same when C . acetoburylicum moves from 'butylic-type metabolism' to 'butyric-type metabolism'; this demonstrates that NADH-ferredoxin oxidoreductase cna, through its reversible action, meet the very different cell needs imposed by these two types of culture . The physiological function of the clostridial NADPH-ferredoxin oxidoreductase was anabolic as it has been with other clostridia. Biochim Biophys Acta, 1977 Jan 25, 490(1), 120 - 31 Comparative immunochemistry of bacterial, algal and plant ferredoxins; Tel-Or E et al.; 1 . Antibodies were produced in rabbits to the 4Fe-4S ferrodoxins from Bacillus stearothermophilus, the 2 {4Fe-4S} ferredoxin from Clostridium pasteurianum, and the 2Fe-2S ferredoxins from the blue-green algia Spirulina maxima, the green alga Scenedesmus obliquus, and the higher plant Beta vulgaris . The antibodies were tested for immunoprecipitation activity with seven bacterial, twelve blue-green algal, six eukaryotic algal and six higher plant ferredoxins . 2 . Antibodies to the bacterial ferredoxins reacted to a significant extent only with their homologous proteins . On the other hand, antibodies to the plant and algal ferredoxins showed cross-reaction with other ferredoxins . There was a correlation between the degrees of immunoprecipitation and the similarity in amino acid sequences . These results suggest that the method can be used as a marker in taxonomic studies . 3 . The interaction of the antibodies with the five native ferredoxins was compared with the reactions with their apoproteins . In each case the degree of interaction was different . This behaviour was interpreted as due to an influence of tertiary structure on the antibody-antigen interaction. Biochemistry, 1977 Jan 11, 16(1), 30 - 3 Influence of cationic triphenylmethane dyes upon DNA polymerization and product hydrolysis by Escherichia coli polymerase I; Wolfe AD; The cationic triphenylmethane dyes crystal violet, methyl green, and malachite green inhibited DNA synthesis catalyzed by Escherichia coli B polymerase I (polymerase I) . Lower concentrations of the dyes inhibited DNA replication as a direct function of the proportion of AT to GC in the DNA of Clostridium perfringens, Escherichia coli, and Micrococcus lysodeikticus . When the intercalant proflavin was employed, the GC-rich micrococcal DNA was most severely inhibited . In addition, both the triphenylmethanes and proflavin inhibited product hydrolysis catalyzed by polymerase I. J Biol Chem, 1977 Jan 10, 252(1), 20 - 31 Purification and properties of 3-keto-5-aminohexanoate cleavage enzyme from a lysine-fermenting Clostridium; Yorifuji T et al.; The lysine-fermenting Clostridium SB4 is shown to contain a new type of beta-keto acid-degrading enzyme that converts 3-keto-5-aminohexanoate and acetyl-CoA reversibly to L-3-aminobutyryl-CoA and acetoacetate . Following the development of a sensitive radiochemical assay the enzyme was purified 175-fold to about 90% homogeneity in 44% yield . The specific activity of the purified enzyme is 44 IU/mg of protein at 30 degrees . The equilibrium constant for the forward reaction was found to be 0.68 at 30 degrees and pH 7.0, corresponding to a deltaG0' of 0.23 kcal/mol . The enzyme is highly substrate-specific . Of several substrate analogs tested in the forward and back reactions only beta-alanyl-CoA and D-3-aminobutyryl-CoA are utilized about 130% and 1.7% as fast as L-3-aminobutyryl-CoA, respectively . The product formed from beta-alanyl-CoA and acetoacetate is a neutral beta-keto acid, presumably 3-keto-5-aminopentanoic acid; its borohydride reduction product was partially characterized as a hydroxy-amino acid by various chromatographic and ion exchange methods . The activity of the purified enzyme is increased about 5-fold by addition of 0.1 mM Co2+ and to a lesser extent by Mn2+ . Activity is inhibited by orthophosphate, thiol reagents, and EDTA; however, exposure of the enzyme to the latter compound prior to addition of Co2+ increases activity, presumably by removing competing divalent cations . Tracer experiments have shown that carbon atoms 1 and 2 of acetoacetate are derived from carbon atoms 1 and 2 of 3-keto-5-aminohexanoate whereas carbon atoms 3 and 4 are derived from acetyl-CoA . The amino acid moiety of 3-aminobutyryl-CoA is derived from carbon atoms 3 to 6 of 3-keto-5-aminohexanoate . Since no evidence for covalent enzyme-substrate intermediates could be obtained by the study of four possible group exchange reactions, a concerted reaction between 3-keto-5-aminohexanoate and acetyl-CoA is considered . The enzyme has a molecular weight of about 97,000 and probably contains four identical subunits . The relatively high specific activity of the enzyme in extracts of Clostridium SB4 indicates it functions in the main pathway of lysine degradation . This relatively stable enzyme provides a convenient and specific method for the quantitative estimation of nanomolar amounts of L- and D-3-aminobutyryl-CoA and beta-alanyl-CoA. Biochem J, 1977 Jan 1, 161(1), 37 - 47 Preparation and characterization of armadillo submandibular glycoproteins; Wu AM et al.; The nine-banded armadillo (Dasypus novemcinctus mexicanus Peters) was chosen for this study so that a comparison could be made of the salivary mucus glycoproteins of an ancient mammalian species with those derived from previously studied, more highly evolved species . Two mucus glycoproteins, armadillo submandibular glycoprotein A and armadillo submandibular glycoprotein B, were prepared from the armadillo submandibular gland by a modification of the method of Tettamanti & Pigman (1968) (Arch . Biochem . Biophys . 124, 41-50) . The composition of glycoprotein A is the simplest one among the known mucus glycoproteins . Six amino acids constitute 98.5 mol/100mol of the protein of glycoprotein A and 82 mol/100 mol of that of glycoprotein B . These are serine and threonine (which make up 40-50% of the molar amino acid composition), glutamic acid, glycine alanine and valine . Proline is absent from glycoprotein A and comprises only 2.3% of glycoprotein B . For both glycoproteins, the protein content, as determined by the method of Lowry, Rosebrough, Farr & Randall (1951) (J . Biol . Chem 193, 265-275), with bovine serum albumin as standard, was nearly 60% higher than when determined by the sum of the amino acids . The ratios of total mol of amino acid/total mol of carbohydrate are 1:0.63 for glycoprotein A and 1:0.68 for glycoprotein B, N-Acetylneuraminic acid and N-acetylgalactosamine, in a molar ratio of about 0.35:1.00, are the principal carbohydrates present in both glycoproteins . Neutral sugars seem to be absent from glycoprotein A, but galactose and fucose are present in glycoprotein B . The carbohydrate side chains in glycoprotein A are composed of about two-thirds monosaccharide and one-third disaccharide residues, whereas those of glycoprotein B are more complex . For both glycoproteins, essentially all of the N-acetylgalactosamine was attached O-glycosidically to the hydroxyamino acid residues of the protein core . The linkage of N-acetylneuraminic acid glycoprotein A was extremely sensitive to dilute acid and neuraminidase . Glycoprotein B has chemical properties similar to those of glycoprotein A . However, whereas glycoprotein A was susceptible to both Clostridium perfringens and Vibrio cholerae neuraminidases, only the latter enzyme had an effect on glycoprotein B at pH 4.75 . Both glycoproteins were homogeneous by cellulose acetate electrophoresis and ultracentrifugal analyses . The apparent mol.wts . of glycoprotein A and glycoprotein B were 7.8 X 10(4) and 3.1 X 10(4) respectively. Zentralbl Bakteriol {Orig A}, 1977, 237(2-3), 358 - 71 {Susceptibility to Thiamphenicol and Chloramphenicol of Anaerobic Bacteria (author's transl)}; Werner H et al.; The in vitro susceptibility to thiamphenicol and chloramphenicol of 272 anaerobes, most of which were recent clinical isolated, was determined by broth dilution tests, With chloramphenicol, 133 anaerobic gram-negative non-sporing rods (48 Bacteroides fragilis, 13 B . thetaiotaomicron, 14 B . oralis, 16 Sphaerophorus varius etc.) had MIC values of 0.03 through 16 microng/ml . Very similar results (MIC, 0.06-16 microng/ml) were obtained with thiamphenicol . In concentrations of 4 microng/ml or less chloramphenicol inhibited 90.2% and thiamphenicol 78.95% of the strains . Strains with only moderate sensitivity to both antibiotics (MIC, 8 or 16 microng/ml) belonged to B . fragilis or other Bacteroides species . Members of the Fusobacterium and Sphaerophorus group were susceptible to less than or equal to 2 microng chloramphenicol/ml and less than or equal to 4 microng thiamphenicol/ml respectively . With both antibiotics, 102 strains of gram-positive non-sporing anaerobes (P . acnes, Peptostreptococcus spp., Peptococcus spp.) were susceptible to less than or equal to 8 microng/ml . Of 37 Clostridium isolates, 35 (belonging to C . perfringens, C . septicum, C . cadaveris etc.) were inhibited by concentrations of 8 microng/ml or less of chloramphenicol and thiamphenicol . Only one strain each of C . perfringens and Clostridium sp . had an MIC of 16 microng thiamphenicol/ml . Accordingly, resistance to thiamphenicol or chloramphenicol was not observed . A standardized monodisk agardiffusion test was performed on 40 Bacteroidaceae, 18 Peptococcaceae and 20 C, (P.) acnes strains . Only a poor correlation was observed between MIC and zone size for thiamphenicol and chloramphenicol . Therefore, inhibition zone diameter measurement cannot be regarded as a reliable method to detect chloramphenicol or thiamphenicol resistance in anaerobes . Thiamphenicol, which is virtually as active against anaerobes as chloramphenicol but lacks serious toxicity, may well play an important role in the therapy of various anaerobic infections. Ann Ist Super Sanita, 1977, 13(4), 805 - 32 {Studies of the microbial content of raw materials used in pharmaceutical preparations}; Bonomi E et al.; The microbiological controls (determination of the total and mycetic bacterial count; isolation of enterococci, Clostridium, Pseudomonas aeruginosa, Staph . aureus, E . coli, Salmonella and other Enterobacteriaceae) executed on 1517 samples of 100 varous raw materials, have ascertained the presence of a microbial contamination varying to matters, with presence also of pathogenic or hygienically undesirable microrganisms . The more contaminated substances have resulted to be those organic either of vegetable or animal origin . Sensible differences have been noted between the various lots and according to the various suppliers . The conservation trials under various conditions of temperature (15-22-37 degrees C) and humidity (30 degrees - 60 degrees - 90 degrees of relative humidity) with opened or closed recipients, have demonstrated the possibility of remarkable increases of the number of the microrganisms in the raw materials with increase of temperature and humitidy, especially in the non hermatically closed recipients. Arzneimittelforschung, 1977, 27(12), 2263 - 5 Susceptibility to erythromycin of anaerobes of the genera Bacteroides, Fusobacterium, Sphaerophorus, Veillonella, Clostridium, Corynebacterium, Peptococcus, Peptostreptococcus; Werner H et al.; The minimal inhibitory concentrations (MIC) of erythromycin were determined by broth dilution tests for 313 anaerobic strains, most of which were clinical isolates . All the gram-positive anaerobes tested (84 Peptococcaceae, including 21 Peptostreptococcus anaerobius and 15 Peptococcus variabilis; 65 Corynebacterium acnes and 29 Clostridium strains, including 13 C . perfringens) were sensitive (MIC values 0.012 through 3.12 microgram erythromycin/ml); so were 111 cultures of gram-negative anaerobes (52 Bacteroides fragilis, 12 B . thetaiotaomicron, 7 B . vulgatus, 13 B . oralis, 4 B . melaninogenicus, 10 Sphaerophorus necrophorus, 2 Veillonella sp., 11 members of other species) . Erythromycin at concentrations of 6.25 through 200.0 microgram/ml was active against 24 strains (1 B . fragilis, 4 Fusobacterium fusiforme, 9 Sph . freundi, 10 Sph . varius) . The present results are compared to the limited number of reports existing with regard to the susceptibility of anaerobes to erythromycin. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg, 1977, 132(7), 659 - 65 Microbial society and its activity in the soil; Prasad M; In this study, with the help of the buried slide method, a qualitative observation of the microsociological association in the soil was assessed . Fungal mycelium cohabiting with bacterial forms, rods as well as cocci, individual colonies of Azotobacter-type cells, and certain algae of Hantzschia species were recorded in a central European typical garden soil . The use of CHOLODNY-STRUGGER combination method was helpful in ascertaining the decaying nature of a soil nematode and also fungal mycelium which, during the process of decomposition, served there as a substrate for bacterial multiplication . Colony of Clostridium-type rod-shaped bacteria existed between the soil particles, acting as living bridges . When examined under fluorescent microscope, the bacterial forms emitted shining fluorescence of green colour which in effect presented them as lighted objects, prominent against the dull red background of soil objects and other decaying substances. South Med J, 1977 Jan, 70(1), 5 - 7 Type A botulism from commercially canned beef stew; Blake PA et al.; Two of three persons who ate lunch together became ill with symptoms characteristic of botulism . One died before botulism was suspected and before specimens could be collected for laboratory testing, but a serum specimen from the other patient, who survived, yielded botulinal toxin, type A . The third person remained asymptomatic, but Clostridium botulinum type A was cultured from his stool . The three persons had shared two canned foods: home-canned green beans and commercially canned beef stew . The green beans were initially assumed to be the cause of the outbreak . However, the empty stew can was recovered from the garbage, and washings from the can yielded C botulinum, type A, and its toxin. Z Allg Mikrobiol, 1977, 17(7), 501 - 6 Dissimilatory nitrate reduction in Clostridium tertium; Hasan M et al.; Fermentation balance studies were carried out on Clostridium tertium grown with and without nitrate in the medium . Nitrate reduction increased the efficiency of energy produced from glucose by permitting the utilization of additional sites of substrate level phosphorylation . The effect was even more dramatic in C . tertium than in C . perfringens, with increased cell yields of about 30% being observed in the former compared with 20% in the latter . Unlike C . perfringens, C . tertium responded to the presence of nitrate in the medium with an increased growth rate . A slight increase in the Y ATP of these cultures was also observed, and quantitatively, this appeared to be consistent with the prediction of Stouthammer and Bettenhaussen that Y ATP will vary with the growth rate . Thus, C . tertium, like C . perfringens, was able to use nitrate as an electron acceptor in conjunction with its energy metabolism, suggesting that this may be widespread among the nitrate-reducing anaerobes. Exp Pathol (Jena), 1977, 13(6), 296 - 301 Effect of microwave radiation on cells treated with membrane-injuring substances; Szmigielski S et al.; WISH cells grown in vitro were pretreated with subcytotoxic concentrations of digitonin, cortisol and purified bacterial toxins -- staphylococcal beta-haemolysin or Clostridium perfringens alpha-toxin and irradiated with 3 GHz electromagnetic wave (microwaves) at the field power densities 5 or 40 mW/cm2 . At 40 mW/cm2 increase in temperature of the culture medium of about 2-3 degrees C was noted, while at 5 mW/cm2 no detectable increase in temperature was found . Control and pretreated WISH cells after irradiation in the microwave field were used for evaluation of their viability, incorporation of tritiated thymidine, glycine and uridine and level of intracellular cyclic AMP . Irradiation with microwaves resulted in lowering of thymidine and glycine incorporation along with changes in the intracellular amount of cAMP (decrease in cells exposed to 5 mW/cm2 and increase in those exposed to 40 mW/cm2) . Under both conditions viability of the cultures was normal . Pretreatment of cells with digitonin or purified bacterial toxins followed by irradiation with microwaves resulted in enhancement of the cytotoxic effect with lowering of cell viability, especially after exposition to power density of 40 mW/cm2 . Cortisol led to decrease in 3H-glycine and 3H-uridine incorporation into WISH cells, but did not influence the reaction of the cells to microwave radiation . In view of the results presented it may be concluded that substances injuring cell membranes sensitize cell cultures to electromagnetic radiation of the microwave range and may enhance the specific (non-thermal) effect of microwaves. J Biochem (Tokyo), 1977 Jan, 81(1), 115 - 26 Purification and some properties of phospholipase C (alpha-toxin) of Clostridium perfringens; Yamakawa Y et al.; 1 . Phospholipase C{EC 3.1.4.3} was purified from the culture filtrate of Clostridium perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100 . During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms . 2 . The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C . perfringens) antitoxin, indicating the homogeneity of the preparation . 3 . The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported . 4 . The molecular weight of the purified enzyme was estimated to be 43,000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31,000 as determined by gel filtration on Sephadex G-150 . From this and the above finding that a part of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C . perfringens phospholipase C can be accounted for . 5 . For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxycholate (SDC) and Ca2+ ions . In the presence of 6 mM Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin . The effects of various divalent cations on the enzymatic hydrolysis were also investigated . 6 . The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation. Can J Comp Med, 1977 Jan, 41(1), 112 - 6 {Necrotic enteritis in broiler chickens . III . Study of the factors favoring the multiplication of Clostridium perfringens and the experimental transmission of the disease}; Bernier G et al.; Necrotic enteritis was reproduced experimentally in two week old broiler chickens by intravenous injection and also by oral administration of a pure culture of Clostridium perfringens . In the first experiment, gross and microscopic intestinal lesions, typical of necrotic enteritis, were observed in all diseased birds and mortality was obtained only in the group of birds that were injected with 0.4 ml or more of the pure culture of the microorganism . In the second experiment, the highest mortality was noted in the group of birds that received orally, in addition to the Clostridium culture, a solution of sodium bicarbonate, to obtain an alkaline intestinal content and opium to decrease the intestinal peristaltism . The gross and microscopie intestinal lesions of the diseased and killed birds were more severe than those observed in the other groups and were similar to those encountered in field outbreaks of necrotic enteritis. Am J Public Health, 1977 Jan, 67(1), 44 - 9 A progressive approach to the problem of foodborne infections; Zaki MH et al.; Qualitative and quantitative bacteriological examinations of 100 samples of perishable foods from 39 retail stores were performed to determine the presence of bacterial contaminants and to explore the feasibility of establishing and utilizing microbiological standards in enforcement . Forty-six per cent of the samples had standard plate counts in excess of 100,000 per gram, 17 per cent showed coliform organisms in excess of 100 per gram, 20 per cent revealed the presence of Staphylococcus aureus and 2 per cent Clostridium perfringens . None of the shell fish samples grew Vibrio parahaemolyticus . The bacteriological findings are discussed in relation to pertinent variables and the use of microbiological standards for potentially hazardous foods is explored . All 450 retail food establishments in a selected area of Western Suffolk County (New York) were subjected to comprehensive study, using a scoring system developed by the Food and Drug Administration . Initial inspections revealed 32 per cent as having one or more major violations . Follow-up inspections were performed to insure compliance and most violations were corrected within four weeks . Six months later all establishments were reinspected . The scoring system was found to have limited value . Half the establishments with major violations on initial inspection had major violations six months later as compared to less than a quarter of those with no initial major violation. Chemotherapy, 1977, 23(4), 227 - 35 Concentrations of tinidazole in body fluids and tissues in gynaecological patients; Ripa T et al.; The concentrations of tinidazole in various tissues and body fluids were studied in gynaecological patients after a single 2g oral dose . Tinidazole was determined by the agar-diffusion technique using a strain of Clostridium bifermentans . Reliable estimates of concentrations down to 0.5 microng/ml could be obtained . Dichloromethane extraction of tinidazole added to various tissues in known amounts gave a recovery of 100 +/- 10% . Peak serum values of 32-52 microng/ml were reached 3-6h after the administration . The concentrations in peritoneal fluid, obtained at operation 8.5-15h after the intake, varied between 16 and 40 microng/ml . Specimens from the Fallopian tubes yielded 15-26 microng tinidazole/g tissue; similar levels were obtained specimens from myometrium, endometrium, portio, vaginal secretions, omental fat, and cutis . It is concluded that, with the given dose, tinidazole concentrations are achieved in fluids and tissues of the female genital tract that are far in excess of those that should be therapeutical in infections caused by microorganisms know to respond to nitroimidazole treatment. Acta Microbiol Pol, 1977, 26(3), 285 - 94 Purification of Clostridium toxoids; Buchowicz I et al.; A two-step fractionation procedure was applied for purification and concentration of the individual Clostridium toxoids . The toxoids were precipitated with hydrochloric acid in the presence of sodium sextametaphosphate, then antigenic fractions were separated from inactive contaminants by Sephadex G-75 filtration . Specific activity of the preparations thus obtained, as determined by Mancini radial immunodiffusion, was 150--565 binding units per mg of protein nitrogen for Clostridium perfringens toxoid, 204--352 binding units for Clostridium oedematiens toxoid and 26.6 -- 51.2 binding units for Clostridium septicum toxoid. Eur J Biochem, 1977 Jan, 72(2), 247 - 50 Synthesis of stereospecifically deuterated phenylalamines and determination of their configuration; Bartl K et al.; 1 . Starting from trans-cinnamic acid a chiral (-)3-phenyl-{2,3-2H}propionic acid has been synthesized using Clostridium kiuyveri cells as catalyst . 2 . The chiral dideuterated acid has been converted by chemical methods to a mixture of (2R) and (2S)-phenyl{2,3-2H}-alanine . 3 . By means of 1H nuclear magnetic resonance spectroscopy and the action of D and L-amino acid oxidase the configuration of the phenylalamine has been shown to be (2R, 3S) and (2S, 3S), respectively . The labelled phenylalanine is thus sterically and isotopically homogenous at position 3 but heterogenous at position 2. Biochem J, 1976 Dec 15, 160(3), 475 - 9 Validation of a simple radiochemical assay measuring hydrolysis of choline-labelled microsomal phosphatidylcholine by phospholipase C . pH-dependence; Cater BR et al.; Selective hydrolysis of phosphatidylcholine species, which are selectively radioactively labelled in vivo, does not appear to interfere with a radiochemical assay for hydrolysis of microsomal phosphatidylcholine by C-type phospholipases from Bacillus cereus or Clostridium perfringens . Both phospholipases substantially hydrolysed phosphatidylcholine over the pH range 4.0-10.0. J Biol Chem, 1976 Dec 10, 251(23), 7398 - 404 Gonadotropin receptors in plasma membranes of bovine corpus luteum . I . Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane-associated and solubilized receptors; Azhar S et al.; The ability of bovine corpus luteum plasma membranes to bind 125I-choriogonadotropin has been examined after prior treatment of the membranes with phospholipases A, C, and D . Treatment of the purified membranes with low concentrations of phospholipases A and C resulted in the inhibition of the binding of 125I-choriogonadotropin to its receptors, whereas phospholipase D had no effect . Receptor activity was decreased by low concentrations of phospholipase A from either bee venom, Vipera russelli or Crotalus terrificus terrificus . Similarly, low concentrations of phospholipase C from Clostridium perfringens and Clostridium welchii also inhibited the binding activity while comparatively higher concentrations of phospholipase C from Bacillus cereus were required to achieve comparable inhibition . The time required to produce 50% inhibition of in vitro binding by phospholipases A and C was found to be 6 and 23 min, respectively . Upon either removal or chelation of calcium ions by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) both enzymes were completely inhibited as evidenced by the complete retention of the membrane binding activity . The decrease in the specific binding of choriogonadotropin to membranes after phospholipase digestion resulted in a decrease in the number of binding sites and was not accompanied by a change in the affinity of the hormone-receptor complex . The rates of association and dissociation of the 125I-choriogonadotropin-receptor complex and the equilibrium dissociation constant (Kd) were nearly identical in untreated and phospholipase-treated membranes . Phospholipases did not have any effect on the preformed hormone-receptor complex or on solubilized receptor . Filtration through Sepharose 6B of solubilized 125I-choriogonadotropin-receptor complex from untreated membranes or membranes which had been pretreated with phospholipase C prior to carrying out hormone binding did not alter the profile (Kav 0.38) . Gel filtration of membranes treated with phospholipase A showed two peaks of bound radioactivity with distribution coefficients (Kav) of 0.08 and 0.35, respectively. Proc Natl Acad Sci U S A, 1976 Dec, 73(12), 4369 - 73 Interactions of heterologous nitrogenase components that generate catalytically inactive complexes; Emerich DW et al.; A unique method is described for inhibiting nitrogenase . When Clostridium pasteurianum nitrogenase is assayed in the presence of the Mo-Fe protein of Azotobacter vinelandii, all the characteristic activities of nitrogenase are inhibited . C . pasteurianum nitrogenase is unaffected by the Fe protein of A . vinelandii . The Fe protein, but not the Mo-Fe protein of C . pasteurianum, inhibits A . vinelandii nitrogenase . Both inhibitions described result from the formation of an inactive complex of A . vinelandii Mo-Fe protein and C . pasteurianum Fe protein . Complex formation requires active components, as oxygen-denatured proteins are ineffective . The results for titration of components of the complex against each other and kinetic data each indicate that the inactive complex consists of two molecules of C . pasteurianum Fe protein per molecule of A . vinelandii Mo-Fe protein . The results of kinetic experiments suggest that the Fe protein from each organism competes for the same site(s) on the A . vinelandii Mo-Fe protein . The Fe protein of C . pasteurianum will form an active or an inactive complex with the Mo-Fe proteins from six different organisms . Inhibition by nitrogenase components that form inactive complexes provides numeroius ways to study the mechanism of nitrogenase action. Strahlentherapie, 1976 Dec, 152(6), 537 - 41 {Tumor hyperthermia using high frequency for increase of oncolysis by clostridium butyricum (M 55)}; Dietzel F et al.; In solid experimental tumors hypoxic cells preferably are inactivated by means of a short-termed radio-frequency treatment with eddy-current fields . Hence follows an increase of the necro-biotic areas . The presence of anoxic-necrobiotic areas is indispensable for the termination of certain anaerobic spores such as Clostridium butyricum s . oncolyticum (M 55) . Local tumor hyperthermy is tested as a technique in altogether 861 mice bearing neck tumors, in order to enhance the germination of oncolytic Clostridia in tumors differing by their mode of formation and rate of growth . In all the three test systems used (Ehrlich solid carcinoma, Harding-Pasey-melanoma, fibrosarcoma induced by methylcholanthrene), the oncolysis being brought about by Clostridia can be intensified significantly by means of a short-termed warming of the tumor up to temperatures of 42 to 44 degress C using radio-frequency. Onderstepoort J Vet Res, 1976 Dec, 43(4), 165 - 73 The antibody response of cattle to Clostridium botulinum types C and D toxoids; Jansen BC et al.; The resistance of cattle with varying serum-antitoxin titres was determined by per os challenge . The results proved that a solid immunity can be produced against C . botulinum toxins C1 and D . The immune response of cattle to various quantities of C . botulinum C1 and D toxoids, aluminium-phosphate-adsorbed and in water-in-oil emulsion was investigated . The response to antigen in water-in-oil emulsion was far superior to the other when they were used for primary and secondary stimuli . When cattle had been given a solid basic immunity with 2 injections of antigen in water-in-oil emulsion, essentially the same booster effect was obtained with antigen in water-in-oil emulsion and in aqueous solution . Only some of the animals injected intramuscularly with antigens in water-in-oil emulsion developed local lesions . These lesions were not large and their histological picture indicated a noticeable decline in severity within 20 weeks . A case is thus made out for the use of C . botulinum C1 and D toxoids in water-in-oil emulsion for the primary and secondary stimuli and an aqueous solution of these antigens for any booster stimulus as an improved method of protecting cattle against botulism. Jpn J Med Sci Biol, 1976 Dec, 29(6), 335 - 49 Production and properties of theta-toxin of Clostridium perfringens with special reference to lethal activity; Soda S et al.; theta-Toxin of Clostridium perfringens produced in a synthetic medium showed high toxicity . The mouse was killed with 5-10 hemolytic units of the toxin . Neutralization experiments showed that lethal and hemolytic activities were due to the same toxic entity . The amount of theta-toxin expressed in lethal activity reached more than 30% that of alpha-toxin in the synthetic medium SM67 . Although the activities of alpha-and theta-toxins were not additive in terms of LD50, increase in the ratio of theta-toxin to alpha-toxin resulted in reduction of the survival time of the mouse injected with a lethal dosis of the mixture of the two toxins when compared with alpha-toxin alone . Unlike those produced in other media, the hemolytic and lethal activities of theta-toxin produced in the synthetic medium was not activated by reduction with thioglycollate, even after partial purification . In other respects, the toxin was not different significantly from those reported in the past . The toxic form was detected also in complex medium . It was suggested that theta-toxin may be produced as a nascent entity. Appl Environ Microbiol, 1976 Dec, 32(6), 803 - 7 "Phoenix phenomenon" in the growth of Clostridium perfringens; Shoemaker SP et al.; The "Phoenix phenomenon" was observed with Clostridium perfringens Hobbs' serological type 9 (HT9) in a cooked-meat medium at 81.7 degrees C by a decrease in plate count (phase I), followed by an increase in count to the intiial level (phase II) and a continued increase above the initial count (phase III) . The effects of sporulation, age of inoculum, assay medium, anaerobiosis, diluent, and growth inhibitor were studied . This phenomenon was reproduced in experiments with sporulation-negative mutants derived from HT9 inocula of various cell ages, and different assay media (sulfite-iron agar, tryptose-soytone-yeast extract agar, prereduced peptone-yeast extract agar, prereduced veal agar, and veal agar) . When strict anaerobic conditions were employed, it was necessary to increase the heating temperature to 52.3 degrees C to observe the phenomenon . The phenomenon was eliminated at 52.3 degrees C when a combination of strict anaerobic conditions, prereduced media, and prereduced veal diluent was employed . The addition of nalidixic acid at the minimum point of the growth curve (end of phase I) had no effect on the appearance of phase II; however, phase III was completely inhibited . This indicated that phases I and II were an injury-recovery process. Acta Pathol Microbiol Scand {B}, 1976 Dec, 84B(6), 414 - 20 An enterotoxin produced by Clostridium perfringens type D . Purification by affinity chromatography; Uemura T et al.; Clostridium perfringens type D 9867 produced enterotoxin immunologically identical to that produced by types A and C which are responsible for C . perfringens food poisoning . Enterotoxin from type C 5386 and type D 9867 was produced in Duncan og Strong sporulation medium (DS medium) . The supernatant fluid from DS-cultures was used for purification of the enterotoxin by affinity chromatography on a monospecific anti-enterotoxin-coupled CNBr activated-Sepharose 4B and activated CH-Sepharose 4B column . The enterotoxin purified by this one-step procedure proved to be of a purity comparable to that obtained by conventional methods, and possessed lethal activity in mice. Zentralbl Bakteriol {Orig B}, 1976 Dec, 163(5-6), 433 - 40 The ultrastructure of oncolytic spores found on pig skin; Althoff J et al.; A new type of clostridium spore, derived from melanotic hamster tumors treated with pig skin extracts, was examined in a lyophylized form by electron microscopy (EM) . Scanning EM indicated that two out of five preparations contained only spores . The spores exhibited oval forms (1.5 X 1.0 MU)with elongated, sometimes infolded, poles indicating an overlying membrane . These structures corresponded to the exosporium as seen by transmission EM . The size of the spores, including the cortex (350-550 A), inner membranes, and cytoplasm averaged 5100 X 5900 A . Cytochemical findings suggest that acid mucopolysaccharides and polypeptides containing N-acetyl-neuraminic acid are localized within the exterior and interior layers of the exosporium and at the surface of the outer coat, which could have a tumor inhibiting effect. Biochim Biophys Acta, 1976 Nov 19, 450(2), 142 - 53 3alpha-, 7alpha- and 12alpha-hydroxysteroid dehydrogenase activities from Clostridium perfringens; Macdonald IA et al.; 25 strains of Clostridium perfringens were screened for hydroxysteroid dehydrogenase activity; 19 contained NADP-dependent 3alpha-hydroxysteroid dehydrogenase and eight contained NAD-dependent 12alpha-hydroxysteroid dehydrogenase active against conjugated and unconjugated bile salts . All strains containing 12alpha-hydroxysteroid dehydrogenase also contained 3alpha-hydroxysteroid dehydrogenase although 12alpha-hydroxysteroid dehydrogenase was invariably in lesser quantity than the 3alpha-hydroxysteroid dehydrogenase . In addition, 7alpha-hydroxysteroid dehydrogenase activity was evident only when 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholanoate was substrate but notably absent when 3alpha, 7alpha-dihydroxy-5beta-cholanoate was substrate . The oxidation product 12alpha-hydroxy-3, 7-diketo-5beta-cholanoate is rapidly further degraded to an unknown compound devoid of either 3alpha- or 7alpha-OH groups . Group specificity of these enzymes was confirmed by thin-layer chromatography studies of the oxidation products . These enzyme systems appear to be constitutive rather than inducible . In contrast to C . perfringens . Clostridium paraputrificum (five strains tested) contained no measurable hydroxysteroid dehydrogenase activity . pH studies of the C . perfringens enzymes revealed a sharp pH optimum at pH 11.3 and 10.5 for the 3alpha-OH- and 12alpha-OH-oriented activities, respectively . Kinetic studies gave Km estimates of approx . 5 X 10(-5) and 8 X 10(-4) M with 3alpha, 7a-dihydroxy-5beta-cholanoate and 3alpha, 12alpha-dihydroxy-5beta-cholanoate as substrates for two respective enzymes . 3alpha-hydroxysteroid dehydrogenase was active against 3alpha-OH-containing steroids such as androsterone regardless of the sterochemistry of the 5H (Both A/B cis and A/B trans steroides were substrates) . There was no activity against 3beta-OH-containing steroids . The 3alpha- and 12alpha-hydroxysteroid dehydrogenase activities, although differing in cofactor requirements cannot be distinguished by their appearance in the growth curve, their mobility on disc gel electrophoresis, elution volume on passage through Sephadex G-200 or heat inactivation studies. Biochim Biophys Acta, 1976 Nov 8, 452(1), 42 - 58 Nitrogenase IX . Effect of the MgATP generator on the catalytic and EPR properties of the enzyme in vitro; Davis LC et al.; Nitrogenase(nitrogen:(acceptor) oxidoreduction, EC 1.7.99.2) of Clostridium pasteuranium is very sensitive to the ratio of MgADP/MgATP in dithionite oxidation assays . Variation of concentration of creatine kinase, an ATP-regenerating enzyme, can be used to control the ratio of ADP/ATP and thereby the dithionite oxidation activity of nitrogenase . The in vitro properties of nitrogenase support the suggestion of Haaker (Haaker, H., deKok, A . and Veeger, C . (1974) Biochim . Biophys . Acta 357, 344-357) that in vivo the nucleotide ratio and not the electron supply normally regulates nitrogenase activity . In EPR experiments it has been shown that the "steady state" varies as a function of the concentration of creatine kinase . The spectral differences are interpreted as being a function of the ratio of MgADP/MgATP obtained in the pseudo steady-state condition, which occurs as a result of variation in relative rates of ATP-utilizing and ATP-generating reactions, that is, the relative nitrogenase and creatine kinase activities . Implications of these finding for interpretation of previously reported kinetic and EPR studies are discussed. Biochim Biophys Acta, 1976 Nov 8, 452(1), 262 - 70 Coenzyme B-12-dependent reactions . Part IV . Observations on the purification of ethanolamine ammonia-lyase; Joblin KN et al.; Purification of ethanolamine ammonia-lyase (EC 4.3.1.7) from a Clostridium sp . grown at the University of Sussex, U.K . and the National Institutes of Health, U.S.A., has been compared and an improved isotopic assay for the enzyme has been developed . Successful purification of this enzyme from Sussex-grown cells requires modification of the published procedure (Kaplan and Stadtman (1968) J . Biol, Chem . 243, 1787-1793) principally a 70% decrease in volume during precipitation with 0.4 M NaCl . This modification also increases the yield from N.I.H.-grown cells . Purified enzyme, resolved of inactive cobalamins, has the same high specific activity from both sources and behaves in the same way on disc gel electrophoresis . Sussex enzyme, before resolution, has less than 20% of the specific activity of unresolved N.I.H . enzyme and contains over 50% more inactive cobalamin . The bound cobalamin from both preparations has been identified as a "base-on" Co11 psi-cobalamin. Gastroenterology, 1976 Nov, 71(5), 793 - 6 The importance of clostridia in experimental intestinal strangulation; Yale CE et al.; Hemorrhagic and ischemic strangulation of closed intestinal segments was sutdied in germ-free rats individually monocontaminated with one of eight separate species of clostridia . Ischemic strangulation was found to be as deadly as hemorrhagic strangulation in the presence of clostridia . Some clostridia were very toxic whereas others were relatively innocuous . The clostridia, ranked in the order of decreasing lethality, were: Clostridium perfringens type A, C . septicum, C . histolyticum, C . haemolyticum, C . bifermantans, C . sporogenes, C . tertium, and C . novyi type A . All species were readily established in the gastrointestinal tract of the nonoperated germ-free rat . The clostridia appear to be one of the most potentially lethal groups of organisms commonly found in the intestinal tract of human beings and animals. J Clin Pathol, 1976 Nov, 29(11), 1011 - 3 Characteristics of a strain of Clostridium carnis causing septicaemia in a young infant; Wort AJ et al.; Clostridium carnis is a species which is only rarely isolated from man or animals and is occasionally found in the soil . This paper is an account of a single isolate found in blood cultures obtained from an 8-week-old boy who was suffering from gastroenteritis. Injury, 1976 Nov, 8(2), 117 - 9 Chronic Clostridium septicum infection of a tibial fracture: a case report; Lucas HK et al.; An open transverse fracture of the mid-shaft of the tibia of a professional footballer became infected by Clostridium septicum and, after early compression plating, required surgical intervention on three further occasions and extensive antibiotic treatment before healing occurred . Clostridial infection is a recognized complication of open fractures contaminated with soil, and the necrotizing toxins produced by the C . septicum were probably responsible for the persistence of this infection . Infection occurred in less than 1 per cent of our series of 215 operations of compression plating of fresh fractures of the tibial shaft . Infection by clostridium species is a serious complication of open fractures . This patient did not show the spreading inflammation and necrosis, or the marked systemic upset, characteristic of acute clostridial infection, but persistent local infection necessitated prolonged surgical and antibiotic treatment. Appl Environ Microbiol, 1976 Nov, 32(5), 685 - 8 Inactivation of Clostridium botulinum toxin by ruminal microbes from cattle and sheep; Allison MJ et al.; Toxin from Clostridium botulinum type C was rapidly inactivated during incubation in vitro with ruminal contents from either a cow or a sheep . Fractions of ruminal contents from which cells had been removed by high-speed centrifugation did not inactivate toxin . Inactivation was associated with fractions containing bacteria, whereas fractions containing protozoa and relatively few bacteria were much less active . This activity may help explain the relatively greater tolerance by ruminants to oral doses of botulinum toxin than to toxin administered by other routes . The results are also pertinent to assays for botulinum toxin from gastrointestinal samples obtained postmortem. Ann Clin Lab Sci, 1976 Nov-Dec, 6(6), 537 - 9 A rapid glutamic decarboxylase test for identification of bacteria; Freier PA et al.; A simple rapid glutamic decarboxylase test is described . This test was found useful in the identification of Escherichia coli, Shigella sp., Providencia alcalifaciens, Clostridium perfringens and Bacteroides fragilis. Ann Microbiol (Paris), 1976 Nov-Dec, 127B(4), 509 - 24 {Purification and some properties of "Clostridium perfringens" delta toxin (author's transl)}; Tixier G et al.; Delta toxin, a hemolytic exocellular protein excreted by C . perfringens type C has been purified to homogeneity, assessed by polyacrylamide disc gel electrophoresis . Purification steps involved successively calcium phosphate gel formation in culture supernatant fluid, salting-out of unadsorbed material by ammonium sulfate to 50 % saturation, isoelectric focusing and gel filtration on Sephadex G75 . Purified toxin appears as a basic protein occuring in two forms with isoelectric points of 8.8 and 9.4 as disclosed by isoelectric focusing . Molecular weight estimated by SDS-polyacrylamide disc gel electrophoresis was found to be close to 42,000 for the two forms . The lytic activity of delta toxin is inhibited by Ca++ and EDTA . The toxin is activated by short-term treatment with low concentration of trypsin. J Med Microbiol, 1976 Nov, 9(4), 475 - 85 A serotyping system for Clostridium welchii (C . perfringens) type A, and studies on the type-specific antigens; Hughes JA et al.; A serotyping scheme for Clostridium welchii (C . perfringens) type A employing 57 antisera has been used to investigate the epidemiology of 153 food-poisoning outbreaks and 32 cases of gas gangrene and other clinical infections . Respectively 65% and 59% of the isolates were typable, and in 55% of the food-poisoning outbreaks the causative serotypes were established . Isolation and reporting methods that would render the typing scheme of even greater epidemiological value are described . The type-specific antigen was shown to reside in the capsule and to be lost from strains that had become rough . Development of roughness and its prevention are described . A great range of antisera and an internationally acceptable serotyping scheme is expected after integration of this set with those developed independently in America and Japan. Appl Environ Microbiol, 1976 Nov, 32(5), 711 - 5 Growth and toxin production by Clostridium botulinum in moldy tomato juice; Huhtanen CN et al.; Tomato juice inoculated with Cladosporium sp . or Penicillium sp . developed pH gradients with the upper portions near the mold mats having pH values near neutrality and the lower portions remaining more acid . Clostridium botulinum spores in these moldy tomato juices germinated, grew out, and produced toxin. Lancet, 1976 Oct 30, 2(7992), 934 - 6 Infant botulism . Identification of Clostridium botulinum and its toxins in faeces; Midura TF et al.; Clostridium botulinum and its toxin were identified in the faeces of four infants, aged 6 to 13 weeks, who had symptoms consistent with botulism . Two cases had type-A toxin and two cases had type-B toxin present in their faeces . No toxin was detectable in sera C . botulinum and toxin could be recovered from faeces more than 8 weeks after admission to hospital . All four cases occurred within a 6-month period . The source of the toxin in these infants may have been in-vivo production from ingested organisms. J Membr Biol, 1976 Oct 20, 29(1-2), 179 - 84 On the question of an electrokinetic requirement for phospholipase C action; Dawson RM et al.; The phospholipase C of clostridium welchii (alpha toxin) has an absolute requirement for trace quantities of Ca2+ . It attacks pure phosphatidylcholine particles (smectic mesophases) having a close-packed bilayer structure only when their surface zeta potential is made positive by the addition of certain divalent ions (e.g., Ca2+) to the aqueous phase or by the presence of low concentrations of long chain cations to the lipid . Alternatively, if the rotational freedom of individual phospholipid molecules is increased by the insertion of short n-alkanols (e.g., hexanol) into the bilayer or when a monolayer of the substrate at an air/water interface is expended, enzymic hydrolysis can occur without any requirement for a net postive charge on the surface. Biochemistry, 1976 Oct 19, 15(21), 4559 - 65 Histone redistribution and conformational effect on chromatin induced by formaldehyde; Polacow I et al.; Histone redistributions between endogenous DNA in calf thymus chromatin and exogenous DNA from Clostridium perfringens (69% A + T) or from Micrococcus luteus (30% A + T) induced by 0.6 M NaCl or by 2% formaldehyde were studied by thermal denaturation . The observed redistribution occurred on histone Hl when the exogenous DNA was (A + T)-richer than the DNA in chromatin, and when the mixture was exposed to 0.6 M NaCl or formaldehyde . When a (G + C)-richer DNA was added as the acceptor for histones, no substantial transfer of histones from chromatin DNA to exogenous DNA was found . Thus the activation energy of histone dissociation from chromatin DNA seems to be substantially lowered by 0.6 M NaCl or formaldehyde such that histones (mostly histone Hl) can be dissociated and bind the (A + T)-richer DNA and form a more stable complex . It is suggested that the formaldehyde effect on histones may be due to the loss of positive charges on lysine and arginin residues (probably more on lysine than on arginine) in histones after their rapid reaction with formaldehyde . Formaldehyde treatment of chromatin also distorts the DNA conformation, as revealed by circular dichroism (CD) studies . This structural effect occurs mainly on those base pairs bound by histones other than Hl, or within the chromatin subunit . Histone redistribution is treated as a thermodynamic phenomenon of histone binding to DNA . The validity of using formaldehyde to study chromatin structure is discussed. Biochemistry, 1976 Oct 19, 15(21), 4736 - 41 Collagenase enzymes from Clostridium: characterization of individual enzymes; Lwebuga-Mukasa JS et al.; Four collagenases have been purified to apparent homogeneity from extracts of Clostridium histolyticum and partially characterized . The four purified enzymes are devoid of hydrolytic activity against casein and the synthetic substrate, benzolyarginine naphthylamide, but all retain activity against native collagen . The enzymes are initially spearated by isoelectric focusing where three of the enzymes show distinct isoelectric points: collagenase I = 5.50, collagenase II = 5.65, and collagenases IIIa and IIIb = 5.90-6.00 . Collagenases IIIa and IIIb can be subsequently separated on diethylaminoethylcellulose . The four purified enzymes show single bands upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate . Calibration of the molecular weights on the basis of migration distance shows a marked dependence on gel porosity . At high acrylamide concentration, collagenases I, II, and IIIa appear to converge to a limiting molecular weight congruent to 81 000, while collagenase IIIb has a distinctly lower value congruent to 72 000 . The similarity between these molecular weight values and those derived from the sedimentation and diffusion coefficients of the native enzyme indicates that each collagenase is a single polypeptide chain . All of the collagenases have comparable catalytic activities against a series of natural and synthetic substrates and are immunologically cross-reactive . Although all four enzymes are evident upon initial electrofocusing of the crude extract, it is possible that the multiplicity of forms is, at least in part, a consequence of lysis following initial secretion from the cell. Lancet, 1976 Oct 2, 2(7988), 715 - 6 Necrotising enterocolitis of the newborn--is it gas-gangrene of the bowel? Pedersen PV, Hansen FH, Halveg AB, Christiansen ED. Necrotising enterocolitis (N.E.C.) of the newborn is thought to be caused by ischaemia of the bowel . This would favour the conversion of clostridial spores, which can occur very early in the intestinal tract of newborns, to toxin-producing, invading bacilli . The histology of resected gut specimens from 6 of 7 N.E.C . patients who had undergone operation was similar to that in cases of gas-gangrene of the bowel and that in experimentally provoked pneumatosis cystoides intestinalis . In one case Clostridium perfringens type A was cultured in great number by anaerobic technique . The clostridia in these cases may have played an important role in the development of N.E.C. Angiology, 1976 Oct, 27(10), 602 - 9 Gas-forming clostridial mycotic aneurysm of the abdominal aorta . A case report; Babenco GO et al.; Gas-forming mycotic aneurysms are extremely rare . A case is reported in which rupture of a gas-forming mycotic aneurysm of the distal abdominal aorta due to Clostridium paraputrificum occurred in an elderly male with a myeloproliferative disorder and a necrotic carcinoma of the colon. Can J Microbiol, 1976 Oct, 22(10), 1579 - 83 Stability of mRNA from the Clostridium sporogenes phage F1; Taylor DE et al.; Polyacrylamide gel electrophoresis was used to study the decay of individual species of mRNA in F1, a bacteriophage specific for the obligate anaerobie Clostridium sporogenes . Immediate early mRNA species had a half-life of 3.5 min, while delayed early and late mRNA had a half-life of between 6 and 8 min. Am J