J Gen Microbiol, 1976 Jan, 92(1), 81 - 8 The effect of sublethal doses of rifampin on the sporulation of Clostridium botulinum; Hawirko RZ et al.; Sublethal doses of rifampin (0-005 mug/ml), added to vegetatively growing cultures of a sporogenic mutant of Clostridium botulinum at inoculation time or after 4 h, resulted in a decrease of growth and in blockage of spore formation . But when rifampin was added 6 to 24 h after inoculation, normal growth and sporulation occurred, indicating that the time of addition was critical and that rifampin was most effective on rapidly dividing, exponential-phase cells . Ultrastructural studies showed that when rifampin was added at the time of inoculation, endospore development was blocked at stage III . During subsequent incubation (greater than 10 h) the cells lost their rigidity, and lysis of the mother cell was followed by that of the forespore . When the cultures were treated with rifampin at 4 h, about 40% of the cells were blocked at stage III and about 60% reached stages IV and V . Some showed excessive elongation and contained developing spores at each pole . They appeared to be derived from two daughter cells unable to form a division septum because of a specific inhibitory effect of rifampin on division . It would seem, therefore, that two daughter cells which are genetically coded to form endospores will do so irrespective of the development of a division septum, and the spores are formed at the 'old' polar regions. J Gen Microbiol, 1976 Jan, 92(1), 67 - 75 The stability of toxigenicity in Clostridium botulinum types C and D; Oguma K; Several type C and D strains of Clostridium botulinum, which had been converted to the toxgenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum . Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage . However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion . When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage . Filtrates of the supernatants of culture fluids of strains transferred without anti-phase serum converted non-toxigenic strains to toxigenicity at varying rates. Acta Chir Scand, 1976, 142(6), 461 - 6 Pathophysiology in the acute afferent loop syndrome . A study in rats; Bengtsson S et al.; The afferent loop syndrome, i.e . occlusion of an afferent intestinal loop after a Billroth II partial gastrectomy, was induced in rats . After various time intervals the animals were killed and the haematocrit and serum osmolality were determined . The content of the occluded loop was analysed with respect to volume, bacterial flora and osmolality . In some cases a sample of the content was incubated at 37 degrees C and the osmolality determined at regular intervals . Groups of animals were studied in this way after 30 min or 1, 4, 8 or 12 h of occlusion . The haematocrit rose with time after the occlusion . The osmolality of the plasma and of the content of the occluded loop did not increase . The volume of fluid in the occluded loop increased continuously with time--from an average of 0.5 ml in the control cases to 6.1 ml after 12 h of occlusion . Experiments in vitro showed that the initial osmolality of the content of the loop was 300 mOsm . After incubation of the samples this increased by 43 to 146 percent . The bacterial content of the loop, including Clostridium perfringens, increased significantly . The results indicate that a marked breakdown of substances takes place in such an occluded intestinal loop, which increases the osmolality . As a result fluid is immediately attracted to the loop to keep the osmolality constant . A combination of this fluid increase due to osmosis and contractions of the intestinal wall leads to a pressure in the occluded loop which considerably exceeds the pressure in the common bile duct due to secretions from the pancreas and liver. Dev Biol Stand, 1976, 32, 151 - 5 {Failure of anti-symptomatic vaccination and the methods of vaccine activity control}; Joubert L et al.; Blackleg (Clostridium chauvoei) infection in Charollais cattle appears to have undergone some etiological and pathogenic changes which are reflected in the apparent failure of vaccination . Two methods have been used to determine the quality of the vaccines in order to meet the different requirements specified by pharmacopeias . The two methods differ in the vaccination schedules of the guinea pigs and the test strain used which may be either a virulent culture or a suspension of spore in calcium chloride . Both methods appear to be efficient in selecting vaccines which provide good protection . It follows, therefore, that the vaccination failures would appear to be the result of intensive selection of beef cattle with reduced immunological response . If this is the case, the remedy for these vaccination failures might lie in an adaptation of the vaccination schedule to the early developing breeds of cattle obtained by intensive selective breeding. Dev Biol Stand, 1976, 32, 143 - 50 Some problems concerning the assay of Clostridium chauvoei vaccine in laboratory animals; Knight PA et al.; The potency test prescribed for Cl . chauvoei vaccines in the British Veterinary Codex requires the complete survival of a group of immunized guinea pigs and death of all unvaccinated controls . Problems arising from the influence of extraneous factors such as animal strain, diet and seasonal variation on the outcome of this test are discussed together with alternative methods that might permit the use of a standard preparation. Dev Biol Stand, 1976, 32, 137 - 41 Some observations on the quality control testing of Clostridium chauvoei vaccines; Chandler HM; Agglutination tests are not suitable for the estimation of the protective antibody level in the sera of vaccinated animals and should not be used for the quality control testing of blackleg vaccines . Some highly virulent strains of Cl . chauvoei are only effectively protected against by vaccines containing cells with a heat labile protective antigen in addition to the heat stable antigen common to all cells of Cl . chauvoei . As these highly virulent strains may be encountered in the field it is important that such strains should be selected for the challenge of vaccinated animals in the quality control testing of blackleg vaccines . It would be useful if an international center could be responsible for the collection and distribution of such strains. Dev Biol Stand, 1976, 32, 123 - 30 Preparation and evaluation to Clostridium septicum toxoid; Kennedy K; Clostridium septicum was grown under controlled conditions to produce high levels of alpha toxin . The toxin was toxoided and concentrated by ultrafiltration . The toxoided alpha toxin retained immunizing properties . The concentrated toxoid, when formulated on Total Combining Power, in monovalent or multivalent clostridial products, provided protection to rabbits against direct challenge with Cl . septicum spores . The toxoided alpha toxin produced antitoxin levels which exceeded current minima. Dev Biol Stand, 1976, 32, 115 - 22 Potency testing of Clostridium septicum bacterins in sheep and laboratory animals; Cardella MA et al.; The immunogenic potency of bacterins containing Clostridium septicum was evaluated in mice, hamsters, guinea pigs, rabbits and sheep to establish potency requirements for these bacterins . Two subcutaneous injections of Cl . septicum bacterins were shown to provide resistance against intramuscular spore challenge . Protection in mice, hamsters and guinea pigs was generally at low levels, in the range of 1 to 5 LD50 . Efficacy estimates, at these levels of challenge, indicated that a degree of relationship existed between the response of rabbits and the response of sheep . This relationship was confirmed at higher levels of spore challenge, at greater than 5 to 500 LD50 . Undiluted bacterins were classed as good or poor based on percent survival responses in sheep . Bacterins providing 90 to 100% survival were classed as good; those providing 30 to 57% survival were classed as poor . In rabbits, this classification was less evident with the undiluted bacterins . Dilution of bacterins to 1:32 provided a discriminatory level with 7 of 8 bacterins examined and represented correlation of the sheep and rabbits responses . A similar relationship was demonstrated between protection in sheep against spore challenge and the antitoxin response in rabbits to undiluted bacterins . Antitoxin responses, when obtained, were generally higher in rabbits than those obtained in sheep . Further evaluation of a good and a poor bacterin in rabbits indicated that no direct relationship existed between antitoxin titer in rabbits and resistance to spore challenge, when compared at the same dilutions of bacterins . Evaluation of serum agglutination responses to types I and II somatic antigens indicated a poor relationship between agglutination response and resistance to spore challenge in both sheep and rabbits . Cl . septicum toxoids were shown to provide resistance to spore challenge in rabbits. Dev Biol Stand, 1976, 32, 103 - 12 Clostridium oedematiens: observations on potency assaying; Macheak ME; The United States has established a Standard Requirement for the potency testing of biological products containing Clostridium oedematiens belonging to two types: type B (Cl . novyi) and type D (Cl . haemolyticum) . Guinea pig testing has provided widely varying results depending on the origin of the animals . The tests reported are intended to determine the efficacity of the vaccines (Cl . novyi and Cl . haemolyticum) depending on the animal tested: guinea pigs, sheep and bovines; they further establish a parallelism between the antitoxin titer and the immunity of the animal. Klin Monatsbl Augenheilkd, 1976 Jan, 168(1), 134 - 7 {Gas gangrene panophthalmitis (author's transl)}; Kessler S et al.; Gas gangrene panophthalmitis is a rare condition of penetrating injury to the globe . The infecting organism is usually Clostridium perfringens . Characteristic symptoms are a brawny swelling of the lids, marked chemosis, coffee-coloured discharge, hypopyon, ring abscess of the cornea, formation of gas bubbles in the anterior chamber, rise of intraocular tension and early amaurosis . Treatment consists in the evisceration or enucleation of the globe, rarely in the exenteration of the orbit . Antibiotics along (Penicillin, Tetracyclines) are insufficient . Administration of antiserum is almost completely abandoned, it is probably more dangerous than helpful . The use of hyperbaric oxygen is not indicated in cases of gas gangrene panophthalmitis . Extraocular extension of the infection and its danger for the individual is prevented by well-times surgical procedure. Appl Environ Microbiol, 1976 Jan, 31(1), 83 - 90 Characterization of anaerobic heterotrophic bacteria isolated from freshwater lake sediments; Molongoski JJ et al.; Strict anaerobic culture techniques were used to quantitatively and qualitatively evaluate the anaerobic heterotrophic bacteria present at the sediment-water interface of hyperutrophic Wintergreen Lake (Augusta, Mich.) . Anaerobic plate counts remained constant from March through December, 1973, ranging from 2.4 X 10(6) to 5.7 X 10(6) organisms/g (dry weight) of sediment . The isolatable bacteria represented a small percentage of the total microbial community, which was shown by direct microscopic counts to be 2.0 X 10'' organisms/g (dry weight) of sediment during June and July . Bacteria of the genus Clostridium dominated the isolates obtained, accounting for 71.8% of the 960 isolates examined . A single species, Clostridium bifermentens, comprised 47.7% of the total . Additional bacterial groups and the percentage in which they were isolated included: Streptococcus sp . (10.8%), unidentified curved rods (9.5%y, gram-positive nonsporing rods (5.6%), and motile gram-negative rods (1.9%) . Temperature growth studies demonstrated the ability of all the isolates to grow at in situ sediment temperatures . Gas-liqid radiochromatography was used to determine the soluble metabolic end products from {U-14C}glucose and a U-14C-labeled amino acid mixture by representative sedimentary clostridial isolates and by natural sediment microbial communities . At in situ temperatures the natural sediment microflora produced soluble fermentative end products characteristic of those elaborated by the clostridial isolates tested . These results are considered strong presumptive evidence that clostridia are actively metabolizing in the sediments of Wintergreen Lake. Appl Environ Microbiol, 1976 Jan, 31(1), 25 - 8 Rapid determination of dipicolinic acid in the spores of Clostridium species by gas-liquid chromatography; Tabor MW et al.; A gas-liquid chromatographic procedure has been developed to quantitate dipicolinic acid in bacterial spores . The culture, washed from a plate, was hydrolyzed with acid containing the internal standard, pyridine-2,4-dicarboxylate, and then extracted into methyl isobutyl ketone . The internal standard and dipicolinic acid were then extracted into a small volume of trimethylphenylammonium hydroxide . Injection of the resultant quaternary ammonium salts into a gas chromatograph yielded, via thermal decomposition, the methyl ester derivatives of the dipicolinic acid and the internal standard . The amount of dipicolinic acid in the sample was determined from a standard curve . The method was sensitive to 100 ng of dipicolinic acid per sample and was 1,000 to 5,000 times more sensitive than the commonly used methods . Preparation of the sample required less than 1.5 h and less than 15 min of the analyst's time. Mikrobiologiia, 1976 Jan-Feb, 45(1), 28 - 32 {Hydrogenase activity of different strains of Clostridium butyricum}; Gogotov IN et al.; Extracts of the cells of Clostridium butyricum, strains MO-1, USA, TM-7B, WS-6K, and C . pasteurianum 038 absorb hydrogen in the presence of its different acceptors; hydrogen is absorbed most actively by the cell extracts of C . pasteurianum 038 . The cell extracts of all studied strains of Clostridium liberate hydrogen from reduced methylviologene, benzylviologene, and azocarmine . The rate of this process in the presence of reduced methyl-viologene was highest in the strains of C . butyricum, TM-7B, and MO-1 . Hydrogenases of all studied Clostridium strains are susceptible to the action of heat and oxygen. Ann Ist Super Sanita, 1976, 12(2-3), 142 - 69 {Microbiological methods of mineral water analysis}; Merino JR; The Spanish legislature has announced the required microbiological standards for bottled mineral waters . These requirements were the subject of "Decree 607/13th March 1975" and they are the following: -A total absence of parasites and pathogen microorganisms . -A total account of Fungus: absence in 100 ml . -A total account of coliforms: absence in 100 ml . -E . coli: entire absence in 100 ml . -A total account of Streptococcus, absence in 100 ml . -A total account of Clostridium sulphite reducing: absence in 100 ml . -A total absence of Pseudomonas aeruginosa in 100 ml . Several technical methods which have been adopted in Spain for the microbiological investigation of mineral waters have been described . For the first time the Spanish legislation has specified the total absence of Pseudomonas aeruginosa in mineral waters and techniques wich reveal the differentiation between Pseudomonas aeruginosa and other Pseudomonas by fluorescent pigments . The confirmation of diagnosis must be to carry out by the Director of National Reference Centre of Health. Vet Med Nauki, 1976, 13(1), 53 - 8 {Normal status of Bac . cereus and Cl . perfringens in the body of healthy slaughter animals}; Milev M; Studied were cattle, pigs, sheep, and young calves intended for slaughter . The experiments were carried out under productional conditions, strictly observing the routinely adopted practice of preslaughter handling . The blood of the animals was sampled prior to slaughter . Samples from the meat (musculature), spleen, kidneys, liver, mesenterial and body lymph nodes as well as feces were taken immediately after slaughter . It was established that Bacillus cereus and Clostridium perfringens were almost lacking in the musculature, mesenterial and body lymph nodes and in some of the parenchymal organs . These organisms were chiefly found in the intestinal tract . Eighty-three up to 100 per cent of the isolated cultures of Cl . perfringens and from 62.5 up to 100 per cent of the isolated cultures of Bac . cereus originated from the feces. Dev Biol Stand, 1976, 32, 193 - 201 Antitoxin responses to Clostridium botulinum vaccines types C and D in guinea pigs; Mathews AG; In guinea pigs, the type C and/or type D antitoxin responses to a single dose of a bivalent or monovalent Cl . botulinum vaccine increase markedly between the fourth and ninth week after injection and still increase markedly by the ninth week . For type C, a similar pattern has been found in cattle . Antigens of types C and D mutually interfere with the antitoxin responses in guinea pigs . Graded doses of vaccine arouse graded antitoxin responses in guinea pigs . Stability trials of vaccines have emphasized the unsatisfactory nature of an absolute-response type of assay, rather than revealing any loss of potency during storage . Attention is drawn to the need for a graded-response type of assay in which vaccines under test are compared with a reference vaccine. Dev Biol Stand, 1976, 32, 175 - 83 Serological classification and typing of Clostridium botulinum; Gimenez DF; Serological classification of Cl . botulinum, based on the antigenic structure of the toxins produced, is distinguished by the behaviour of the toxin-antitoxin mixtures in in vivo neutralization tests . Observations on the dissimilarity between strains within types, the behaviour of antitoxins in the cross-neutralization tests, the established concepts of the antigenic structure of the toxin types and the lack of a standard methodology for typing, have led to the definition of the terms efficiency, type, subtype, intratypic serological variant (ISV), and degree of serological homology . These definitions are primarily applied to typing and to the establishment of the taxonomic serological categories of type, subtype and ISV . In the case of antitoxin standardization, the accepted standard methods must be followed. Scand J Gastroenterol, 1976, 11(5), 437 - 46 Anaerobic and aerobic bacteriological studies in biliary tract disease; Nielsen ML et al.; Thirty-eight patients without biliary tract disease, 68 patients with gallbladder stones or acalculous chronic cholecystitis, 30 patients with common duct obstruction due to stones or strictures, and 28 patients with common duct obstruction due to tumors or chronic pancreatitis were investigated . Gallbladder bile or common duct bile was cultured anaerobically and aerobically . Anaerobic culture procedures were based on the use of a glove-box and prereduced, anaerobically sterilized media . Transportation of samples was based on evacuation of atmospheric air with oxygen-free carbon dioxide and transportation-time less than 30 minutes . Patients with biliary tract and patients with common duct obstruction not due to stones or strictures did not yield bacteria on culture . Twenty-two per cent of patients with disease of the gallbladder but normal common ducts and 87% of patients with common duct stones or strictures yielded bacteria on culture of the bile . Among 41 patients with infected bile, 39% yielded anaerobic bacteria, either in pure culture (12%) or in mixed culture with anaerobic bacteria (27%) . B . fragilis and other non-sporing anaerobic bacteria were most frequently isolated, whereas Clostridium species were found in three patients only. Infect Immun, 1976 Jan, 13(1), 172 - 9 Indigenous microorganisms prevent reduction in cecal size induced by Salmonella typhimurium in vaccinated gnotobiotic mice; Tannock GW et al.; Germfree CD-1 mice challenged by the oral route with Salmonella typhimurium had ceca that were abnormal in appearance and reduced in size compared to those of germfree controls . Similarly, germfree mice injected with heat-killed S . typhimurium or gnotobiotes associated with three indigenous microbes (Lactobacillus, Bacteroides, Clostridium), and subsequently challenged with S . typhimurium also had small ceca . By contrast, gnotobiotic mice that had been both injected with the heat-killed S . typhimurium and associated with the three indigenous microbes before challenge with S . typhimurium had ceca similar in size and appearance to germfree mice . Thus, indigenous microorganisms could interfere with the mechanism by which the pathogen induced the decrease in cecal size, but could do so only in mice injected with heat-killed bacteria . This phenomenon suggests synergism between the interference effected by the indigenous bacteria and the resistance mechanisms of the animal. Vet Med Nauki, 1976, 13(10), 91 - 5 {Demonstration and type determination of Clostridium perfringens isolated from meat semipreserves}; Popova V; Investigated were a total of 92 samples of pasteurized cans of different batches . Six strains of Clostridium perfringens were isolated from a sample of pasteurized ham . To demonstrate a toxin and present its type differentiation a strain was investigated with the use of dermonecrotic test after Williams along with the cross toxin neutralization after the internationally accepted method, and the neutralization of the lecithinase activity after Williams . The use of native toxins at the type differentiation of slightly toxigenic strains of Clostridium perfringens isolated from food products proved inappropriate . The production of dry toxin and the application of cross toxinneutralization proved to be most suitable in the typing of such strains . The type differentiation carried out as described above showed that the studied strain belonged to type A. Arch Exp Veterinarmed, 1976, 30(6), 903 - 12 {Experimental reproduction of necrotic enteritis in the chicken . 1 . Mono- and polyinfections with Clostridium perfringens and coccidia with reference to cage managemen}; Balauca N; Experimental mono-infection and poly-infection, using vegetative germs of a CL . perfringens Type A strain 1663 and its toxin as well as E . acervulina, necatrix or mitis oocysts, were applied to 100 SPF chicken aged seven days . While clostridial or coccidial mono-infection did not cause any loss and only unspecific pathologic-anatomic changes, polyinfection was accompanied by diarrhoea and slower weight increase, with 55.4 per cent of the chicken having been lost three to nine days from the outbreak of the infection . The pathologic-anatomic findings recorded from the chicken with poly-infections included haemorrhagic, ulcerative, and necrotic intestinal inflammations, primarily in the small intestin . The results of these experimental infections indicated a possible correlation between CL . perfringens and coccidia in the pathogenesis of necrotic enteritis. Dev Biol Stand, 1976, 32, 85 - 9 An international serotyping system for Clostridium perfringens (welchii) type A in the near future; Stringer MF et al.; The importance of Clostridium perfringens (welchii) type A as an agent of clinical infection in man is well known and, more recently, its role as a cause of human food poisoning has become well recognized . There has been a growing awareness of the need for a system of finer subdivision within the type A group for better understanding of the epidemiology of Cl . perfringens in infections and food poisoning . A set of antisera used for typing and developed in the Food Hygiene Laboratory over a period of some years has proved to be of considerable value in this respect; it is now being integrated with similar sets of antisera prepared independently in the USA and Japan to form a comprehensive and internationally workable serotyping scheme . Negotiations regarding commercial production of the main type antisera are now in progress . It is hoped that serotyping facilities will be available in the near future to workers and institutions concerned with Cl . perfringens type A. Dev Biol Stand, 1976, 32, 77 - 83 Solid-phase radioimmunoassays for quantitative antibody determination of bacterial exotoxins . Measurement of Clostridium perfringens type D epsilon antitoxin; Bernath S; Two reversed solid-phase radioimmunoassays have been developed for quantitative determination of antibodies against Clostridium perfringens type D epsilon toxin . 125I labelled prototoxin was used in the bromoacetyl cellulose-bound antibody method and in the antibody coated tube method . The procedures are based on the competition for 125I labelled prototoxin between the insoluble antibodies and the antibodies present in the unknown sample . The radioactivity bound to solid-phase is in inverse ratio to quantity of measured antibodies . The antibody values which can be detected are in the range of 0.004 IU/ml of investigated serum . The methods allow the rapid and inexpensive screening of large groups of vaccinated sheep, and are very suitable for measuring small amounts of Cl . perfringens D epsilon antibodies with a small experimental error. Dev Biol Stand, 1976, 32, 45 - 61 {Standardization of the control technics of Clostridium perfringens vaccine . Use of a reference vaccine}; Bousicaux A; Control of Cl perfringens toxins . It is possible to employ a standardized technique for the control of the toxins of Cl . perfringens . Titres of LD50 per ml for the mouse and L+/50 per ml may be expressed with a confidence limit of 1.5 . The assay of toxins by L+/50 (relating to their antigenic value) permits the establishment of standards by which it is possible to obtain a satisfactory vaccine with a high degree of certainty . 10 L+/50 per dose for Cl . perfringens A 20 L+/50 per dose for Cl . perfringens B 180 L+/50 per dose for Cl . perfringens C 50 L+/50 per dose for Cl . perfringens D Control of vaccine values . Technique adopted : Determination of antibodies in the rabbit following vaccination . The confidence limit of the antitoxin titre is similarly equal to 1.5 when P = 0.05 . It would not appear to be necessary to establish minimum levels of antibodies to confer protection; it seems nevertheless that the results are influenced by considerable variations attributable to the animals used for the most part and that a reference vaccine is required . Freeze-dried vaccine . There is a natural antigenic activity in the rabbit which retains a considerable part of its antigenic properties after maintenance for one week at +37 degrees C . In our view this would be suitable for a reference vaccine. Dev Biol Stand, 1976, 32, 31 - 4 Production and standardization of polyvalent Clostridium perfringens vaccine in Iran; Ardehali M et al.; In this presentation an account of the large-scale production and the standardization of polyvalent Clostridium perfringens vaccine in Iran is described . Over 20 million doses of this vaccine are produced and utilized in Iran every year . Certain modifications have been made to the culture media used in our laboratory for the production of this vaccine in order to bring down its cost . The prepared vaccine is highly immunogenic as determined by the laboratory examination on the quality of the vaccine according to British Veterinary Codex and the field reports. Dev Biol Stand, 1976, 32, 245 - 50 The use of a multicomponent standard for clostridial vaccines; Gray AK; A multicomponent clostridial vaccine in being developed with the objective of acting as a standard preparation for the control of vaccines containing one to eight components . It is intended initially for use in monitoring those components which can be assessed by antitoxin response assays in rabbits . The components involved, therefore, are the principle toxoids of Clostridium (perfringens) welchii types B, C and D, Cl . oedematiens type B, Cl . septicum and Cl . tetani . The proposed standard also contains the toxoid of Cl . oedematiens type D and an anaculture of Cl . chauvoei . It is hoped later to develop the use of the two latter components as standards in other test systems . The aim of establishing the standard was to provide a means of controlling inter-laboratory variation, especially that due to animals. Can J Comp Med, 1976 Jan, 40(1), 53 - 9 Necrotic enteritis in broiler chickens . III . Reproduction of the disease; Long JR et al.; Lesions typical of necrotic enteritis could be produced experimentally in from 11-26% of broiler chickens consuming feed containing approximately 10(7) Clostridium perfringens per gram . Highest mortality was produced using isolates from field cases of necrotic enteritis which were reisolated from experimental cases in the laboratory . Penicillin in the drinking water at 100,000 I.U./litre completely prevented mortality whereas chloramphenicol at 110 mg/litre delayed the onset and reduced the number of deaths compared to controls. Mech Ageing Dev, 1976, 5(6), 443 - 6 Change in the stability of tendon collagen during ageing in the reptile, Calotes versicolor . A brief note; Panigrahy GK et al.; Age related changes in the digestibility of tendon collagen of the garden lizard, Calotes versicolor, were traced using type I collagenase from Clostridium histolyticum . Collagen from older lizards showed less susceptibility for collagenase digestion than the younger lizards suggesting increased number of cross-linkages comparable with mammals. Adv Exp Med Biol, 1976, 74, 68 - 82 The iron-sulfur centers and the function of hydrogenase from Clostridium pasteurianum; Chen JS et al.; Hydrogenase from C . pasteurianum is an iron-sulfur protein containing at least two tetrameric iron-sulfur centers . Information on the structure of the remaining iron atoms must await future investigation . Although the EPR spectra of dithionite-reduced hydrogenase and eight-iron Fd showed some similarity, the CD spectra clearly indicated a difference . The tetrameric iron-sulfur centers of hydrogenase were shown to undergo redox changes when hydrogenase was oxidized or reduced . However, no evidence is now available to support a role for the tetrameric Fe-S centers, responsible for the EPR spectrum A, as the primary site for H2 binding and activation . Because we have found that the {Fe4S4(SR)4}-containing ferredoxins do not have hydrogenase activity, it is conceivable that the additional iron atoms and/or certain amino acid residues of hydrogenase also contribute to the unique catalytic properties of this enzyme . Chemical synthesis of Fe-S clusters with different peptide environments and with hydrogenase function would lead to the identification of these functional groups . X-ray diffraction studies on hydrogenase will certainly complement the other approaches . Knowledge of the structure of the active site of hydrogenase will certainly accelerate research into: (1) the synthesis of a stable catalyst to replace hydrogenase in systems designed to produce H2 by coupling this catalyst to a photoreducing system; and (2) the elucidation of the active sites of more complicated iron-sulfur enzymes such as nitrogenase. Int J Pept Protein Res, 1976, 8(2), 155 - 65 The effect of calcium chloride on the activity and inhibition of bacterial collagenase; Head DL et al.; The enzymatic behavior and inhibition patterns of collagenase of Clostridium histolyticum in the presence of 0.5 M and 3.4 mM CaCl2 have been examined viscosimetrically . The more concentrated salt was found to enhance the rate of digestion of calfskin collagen when either measured viscosimetrically or colorimetrically by trinitrobenzenesulfonate . However, the rate of digestion of calfskin gelatin is unaffected by 0.5 M CaCl2 as determined colorimetrically . Calcium chloride also proved to have a marked effect on the inhibitory behavior of a series of imidazole compounds . Histidine (10mM) is about three-fold more effective as an inhibitor in 0.5 M CaCl2 than in 3.4 mM CaCl2, whereas a reverse effect is true for histamine, Imidazolylpropionate (10mM) was only weakly inhibitory (16%) in 0.5 M CaCl2 and not at all in 3.4 mM CaCl2 . Inhibition by 10 mM imidazole was not detectable . These observations may be useful in the design of inhibitors for tissue collagenases which share a number of common characteristics with the bacterial enzyme. Physiol Bohemoslov, 1976, 25(2), 155 - 8 Pregnancy interrupting effects of some bacterial toxins; Sechser T et al.; PIP: Pregnancy interrupting effects of some bacterial toxins were studied in mice . Embryotoxic properties of Shigella dysenteriae and Clostridium perfringens toxins, Escherichia coli endotoxin, Vibrio cholerae, and Escherichia coli interotoxins were compared up to Day 18 of pregnancy following injections on Day 6, 8, or 13 of pregnancy . Escherichia coli endotoxin caused embryotoxic effects in all stages of pregnancy while Escherichia coli enterotoxin, Vibrio cholerae enterotoxin, and Shigella dysenteriae toxin were most effective mainly at earlier stages of pregnancy . Histological studies revealed indirect embryotoxicity at later stages by placental damage . The main site of damage was the fetal side for Shigella dysenteriae toxin and the maternal side for Escherichia coli endotoxin, enterotoxin, and cholera toxin . Clostridium perfringens toxin had no embryonic effect . Am J Med Sci, 1976 Jan-Feb, 271(1), 59 - 63 Case report . Clostridium perfringens septicemia with detection of phospholipase C activity in the serum; Moore A et al.; Clostridium perfringens septicemia with massive hemolysis is described in an elderly woman with refractory anemia . This case is highly unusual in that (1) the diagnosis was made during life by discovery of the organisms on a routine peripheral blood film, and that (2) phospholipase C activity was detected in the serum. J Bacteriol, 1976 Jan, 125(1), 211 - 9 Spin-labeling studies on the lipids of psychrophilic, psychrotrophic, and mesophilic clostridia; Finne G et al.; Spin-labeling studies were conducted to elucidate the viscosity and phase transition temperatures of lipids isolated from psychrophilic, psychrotrophic, and mesophilic clostridia . Electron spin resonance spectroscopy indicated that the lipids, for all the growth temperatures tested, were in a fluid state and from 13 to 24 C higher than the corresponding lipid transition temperatures . When the organisms were grown at different temperatures, a psychrotropic and two mesophilic clostridia were shown to be able to adjust their lipid-phase transition temperature to the growth temperature . A psychrophilic Clostridium strain, when grown at different temperatures, synthesized lipids that had the same phase transition temperature . It is suggested that this lack of growth temperature-inducible regulation of lipid-phase transition temperature may be a molecular determinant for the psychrophily of this organism . It is proposed that the growth temperature range of an organism is dependent upon the ability of the organism to regulate its lipid fluidity within a specific range. Vox Sang, 1976, 31(1), 64 - 6 Acquired B antigen disappearance by in vitro acetylation associated with A1 activity restoration; Gerbal A et al.; The chemical acetylation of RBC bearing the acquired B antigen led to the disappearance of the agglutinability by anti-B and restored the A1 specificity . The same results are obtained using RBC transformed in vitro by a Clostridium Tertium filtrate, where a deacetylase was reported. Acta Biol, 1976, 27(2-3), 107 - 17 The fermentative production of acetone-butanol by Clostridium acetobutylicum; Fouad M et al.; Fourteen different media were used in the fermentative production of acetone-butanol . The highest total yields were achieved in medium I . Potato starch and soluble starch were suitable as carbon sources . The best concentrations of potato starch and soluble starch were 500.0 and 10.0 g/l, respectively . Peptone was the most favourable nitrogen source . The best concentration of peptone was 4.0 g/l . Calcium carbonate in 3.6 g/l acted as buffering agent in the fermentation process . The best initial pH value of the fermentation medium was 6.0 . The optimum temperature was 32--33degreesC . The fermentation process required 120 h to obtain maximum yields of acetone-butanol. Appl Environ Microbiol, 1976 Jan, 31(1), 49 - 52 Inhibition of Clostridium perfringens by heated combinations of nitrite, sulfur, and ferrous or ferric ions; Asan T et al.; Heating mixtures of sodium nitrite, cysteine, and either ferrous sulfate or ferric chloride at 121 C for 20 min at pH 6.5 or 6.3 produced a potent inhibitor of Clostridium perfringens vegetative cells and spores when added to previously heat-sterilized fluid thioglycolate medium . When the mixtures containing FeSO4 at pH 5.2 or FeCl3 at pH 2.7 were heated, the inhibitory effect was not produced . These responses seem to eliminate the possibility that cysteine nitrosothiol is the agent responsible for the heated-nitrite inhibition known as the Perigo effect . The variable pH responses also cast doubt upon the role of the black Roussin salt as the agent of the Perigo effect. Experientia Suppl, 1976, 26, 237 - 48 Properties of enzymes from Clostridium thermoaceticum and Clostridium formicoaceticum; Ljungdahl LG et al.; Methylenetetrahydrofolate dehydrogenase from C . thermoaceticum and C . formicoaceticum have been purified to homogeneity and compared . The two enzymes are very similar physically, chemically, and kinetically, but he C . thermoaceticum enzyme has a higher thermostablility, which is an intrinsic property of the protein . Formate dehydrogenase enzymes from both bacteria require selenite and tungstate for formation and these enzymes also appear to have similar properties, although the C . thermoaceticum is stable at 70 degrees C for more than one hour . Acetate kinase from C . thermoaceticum appears to be under metabolic control . It can be concluded that enzymes from C . thermoaceticum, although they are more thermostable, are very similar to corresponding enzymes from mesophilic organisms. C R Acad Sci Hebd Seances Acad Sci D, 1975 Dec 22, 281(24), 2033 - 6 {The mode of action of bacteriocin N5 purified from Clostridium perfringens}; Ionesco H et al.; The purified bacteriocin N5 produced by Clostridium perfringens BP6K inhibited simultaneously the syntheses of DNA, RNA and protein in sensitive cells, without DNA degradation . Bacteriocin N5 inhibited the accumulation of leucin and caused the exit of the previously accumulated amino acid . The effects of bacteriocin N5 are very similar to those observed for colicins E1, K, A and I. Eur J Biochem, 1975 Dec 15, 60(2), 467 - 76 Nitrogenase from Azotobacter chroococcum . Purification and properties of the component proteins; Yates MG et al.; 1 . A large-scale purification of the nitrogenase components from Azotobacter chroococcum yielded two non-haem iron proteins, both of which were necessary for nitrogenase activity and each had a specific activity of approximately 2000 +/- 300 nmol of acetylene reduced/mg protein per min in the presence of sautrating amounts of the other . This procedure freed the Mo-Fe protein from a protein contaminant which had an electron paramagnetic resonance signal at g = 1.94 . 2 . Both proteins were purified to homogeneity as determined by disc gel electrophoresis and ultracentrifugal analysis . Both proteins were oxygen-sensitive but not cold-labile . Ultracentrifugal analysis indicated that both proteins dissociated to a slight degree at concentrations below 2 mg/ml . 3 . The larger of the two proteins had a molecular weight of 227 000 and contained 1.9 +/- 0.3 atoms of Mo, 23 +/- 2 atoms of Fe, 20 +/- 2 acid-labile sulphide and 47 tryptophan residues/mol . The protein consists of 4 subunits of mol . wt 60 000 (approx.) . The reduced protein showed electron paramagmetic resonance signals at g = 4.29, 3.65 and 2.013 but not in the area of g = 5 to 6 . Upon oxidation abosrbance increased throughout the visible region of the ultraviolet visible spectrum, with a maximum difference between oxidised and reduced protein occurring at 430 nm . 4 . The smaller protein had a molecular weight of 64 000 and contained 4 g-atoms of Fe and 4 acid-labile sulphide groups/mol but no tryptophan . It had two subunits of mol . wt 30 800 . The reduced protein showed electron paramagnetic resonance signhe protein retained almost full activity after oxidation with phenazine methosulphate . The ultraviolet visible spectrum of oxidised protein was clearly different from that of the oxygen-inactivated protein: it had a sharp peak at 269 nm and a broad absorbance between 340 and 470 nm with a maximum difference between oxidised and reduced forms at 430 nm . Oxygen-inactivated protein showed a sharp peak at 277.5 nm and broad peaks from 305 to 360, 400 to 425 and 435 to 475 nm . 5 . Amino acid analyses of both proteins showed that most common amino acids were present with a preponderance of acidic residues . Analyses of compositional relatedness showed that the nitrogenase proteins from A . chroococcum were most closely related to those from A . vinelandii and least so to those from Clostridium pasteurianum. Biochemistry, 1975 Dec 2, 14(24), 5257 - 60 Activation of acetyl- and butyrylcholinesterase by enzymatic removal of sialic acid from intact neuroblastoma and astroblastoma cells in culture; Stefanovic V et al.; Removal of sialic acid from intact mammalian nervous system cells in tissue culture is accompanied by an immediate increase in cellular cholinesterase activity . Treatment of hamster astroblast cells (clonal line NN) and mouse neuroblastoma cells (clonal lines S21, N18, and N115) for brief periods with a low level of Clostridium perfringens sialidase, 5 X 10(-3) units/ml, removed 1-15 mug of sialic acid per mg of cell protein and brought about a large increase in v0 and Vmax of cellular acetylcholinesterase (EC 3.1.1.7) . Butyrylcholinesterase (EC 3.1.1.8) activities also increased upon careful enzymatic removal of cellular sialic acid, and cells with characteristically low butyrylcholinesterase activity, e.g., adrenergic clonal line N115 neuroblasts displayed relatively high activity after treatment with sialidase . These findings open the possibility that adaptive regulation of cholinesterases in mammalian cells may be mediated rapidly through changes in their sialic acid content. Zentralbl Bakteriol {Orig A}, 1975 Dec, 233(4), 542 - 52 {Bacteriocins of Clostridium septicum (author's transl)}; Schallehn G; 17 strains of Clostridium septicum were examined for bacteriocin production . Two bacteriocin-producing strains were detected by the stab culture method and the spot test using the supernatant fluids of the cultures . The bacteriocin of Cl . septicum Ovinus was active against all other strains of Cl . septicum without inhibiting the donro strain (Table 2) . This bacteriocin and one indicator strain (Wex) were chosen for further study . The bacteriocin, which was spontaneously produced during the logarithmic growth phase of the bacteriocinogenic strain Ovinus (Fig . 1) was not inducible with high temperatures or Mitomycin C . It was resistent to chloroform, but sensitive to heat (Fig . 4, 5) and proteolytic enzymes (Table 4) . Its MG is about 20000 (Fig . 3). Appl Microbiol, 1975 Dec, 30(6), 964 - 9 Growth potential of Clostridium botulinum in fresh mushrooms packaged in semipermeable plastic film; Sugiyama H et al.; Fresh mushrooms (Agaricus bisporus) were inoculated in the stem, gill, or cap with Clostridium botulinum spores . They were placed with uninoculated mushrooms in paper board trays, which were then covered and sealed in a polyvinyl chloride stretch film to simulate prepackaged mushrooms available at retail stores . When incubated at 20 C, botulinum toxin could be detected as early as day 3, or 4, when the mushrooms still appear edible . Mushrooms inoculated in the stem with 1,000 type A spores frequently became botulinogenic; higher spore levels were needed if gills or caps were inoculation sites . Type B spores were less apt to produce toxic mushrooms . Respiration of the fresh mushrooms used up O2 more rapidly than could enter through the semipermeable wrapping film, so that the equilibrium O2 concentration became low enough for growth of C . botulinum . Inoculated mushrooms did not become botulinogenic when held at 4 C. J Hyg (Lond), 1975 Dec, 75(3), 371 - 9 Clostridium botulinum in the lakes and waterways of London; Smith GR et al.; Mud samples collected during 1974 from a large proportion of the lakes and waterways of London were examined for Clostridium botulinum . Of 69 such sites, 50 (72.5%) contained at least one type of the organism . Of the 50 positive sites, 31, 12, 1 and 10 contained, respectively, types B, C, D and E . Most of the demonstrations of type B required trypsinization of culture filtrates . An examination of 7 lakes in Edinburgh, made for the purpose of comparison, showed that 4 contained type B and one type C . An analysis of the results gave quantitative information on the value of (1) resampling apparently negative lakes, (2) the use of both heated and unheated culture inocula, and (3) trypsinization of culture filtrates. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4854 - 8 NH---S hydrogen bonds in Peptococcus aerogenes ferredoxin, Clostridium pasteurianum rubredoxin, and Chromatium high potential iron protein; Adman E et al.; Results from refinement of the crystal structures of P . aerogenes ferredoxin and C . pasteurianum rubredoxin determined by x-ray diffraction show that there are 15-18 NH---S bonds in the former and six in the latter with lengths in the range 3.1-3.9 A . Earlier tritium exchange experiments are consistent with the presence of these hydrogen bonds in the ferredoxin structure and show that more peptide hydrogen atoms are available for exchange in apoferredoxin than in intact ferredoxin . Four types of NH---S bonds are observed and two of these are geometrically similar to the two types of 3(10) NH---O bonds . The existence of more NH---S bonds in ferredoxin than in high potential iron protein suggests why the -2 form of the Fe4S4 cluster is preferred in ferredoxin over the -1 form found in high potential iron protein . From comparison of Cys-X-Y-Cys sequences in rubredoxin, ferredoxin, and high potential iron protein we suggest that two Cys-X-Y-Cys-Z sequences, where Z may have conformation angles similar to glycine, are required to make a one-iron cluster, no more than one Cys-X-Y-Cys-Z-Gly sequence is required to form a Fe2S2 ferredoxin, and a Cys-X-Y-Cys-Gly sequence where Y has a conformation such that the cysteines bond to different iron atoms is necessary to form the tetrameric cluster. Jpn J Exp Med, 1975 Dec, 45(6), 433 - 8 Clostridium perfringens exotoxins . III . Binding of theta-toxin to erythrocyte membrane; Hase J et al.; When Clostridium perfringens theta-toxin was incubated with sheep erythrocytes the toxin activity disappeared before lysis, the fact of which suggests fixation of the toxin to erythrocyte membranes . 2 . Theta-Toxin lost its activity by binding to cell membranes, and the membrane constituted inhibitor of theta-hemolysis was neither a protein, a carbohydrate nor a phosphatide, but was cholesterol . From these results this report proposes that the theta-toxin binding site of erythrocytes should be cholesterol. Proc Natl Acad Sci U S A, 1975 Dec, 72(12), 4795 - 9 On the iron-sulfur cluster in hydrogenase from Clostridium pasteurianum W5; Erbes DL et al.; Hydrogenase, purified to an average specific activity of 328 mumol of H2 evolved/(min X mg of protein) from Clostridium pasteurianum W5, was found to have 4-5 Fe and 4-5 labile sulfur atoms per molecule of 60,000 molecular weight, in contrast with earlier reports of 12 Fe per molecule . Displacement of the iron-sulfur cluster from hydrogenase by thiophenol in 80% hexamethyl phosphoramide:20% H2O yielded the Fe4S4 (thiophenyl)4 dianion according to absorption spectroscopy . Electron paramagnetic resonance spectroscopy at 12 K showed that the iron-sulfur cluster in the enzyme could be reduced by the H2 to a state (g-values of 2.098, 1.970, and 1.898) similar to that in reduced ferredoxin and could be oxidized by dichlorophenolindophenol or H+ to a state (g-values at 2.099, 2.041, and 2.001) similar to that in high potential iron-sulfur proteins . These oxidations and reductions appeared to occur within the turnover time of the enzyme . Deuterium failed to narrow the electron paramagnetic resonance signal in either state, but the competitive inhibitor carbon monoxide reversibly formed a compound with either state and substantially altered the electron paramagnetic resonance . 13CO produced a broadening of these signals, suggesting the formation of a direct CO complex with the iron-sulfur cluster . These data are consistent with a model of the active site of the enzyme in which a four-iron four-sulfur cluster is a component that can accept one or two electrons from and donate either one or two electrons to substrates, and in which the iron-sulfur cluster serves as the site of binding of gaseous ligands. J Bacteriol, 1975 Dec, 124(3), 1621 - 3 Competitive inhibition of transformation in group H Streptococcus strain Challis by heterologous deoxyribonucleic acid; Ceglowski P et al.; Glucosylated deoxyribonucleic acid (DNA) from phages T4 and T6 competes poorly with homologous DNA causing only a slight decrease of transformation in Group H Streptococcus strain Challis . Other types of heterologous DNAs (Micrococcus luteus, Clostridium perfringens, Escherichia coli, calf thymus and non-glucosylated phage T6 DNA), in contrast to glucosylated T4 and T6 DNAs, compete with transforming DNA to the normal, high extent . These results indicate that as in transformation of Bacillus subtilis, the presence of glucose attached to 5-hydroxymethylcytosine in phage T6 DNA considerably decreases the interaction of such DNA with competent cells of the Challis strain . It also indicates that the guanine plus cytosine content of DNA is not decisive in determining its interaction with competent cells. Biochim Biophys Acta, 1975 Dec 1, 413(2), 309 - 16 Changes in lipid metabolism and cell morphology following attack by phospholipase C (Clostridium perfringens) on red cells or lymphocytes; Allan D et al.; When intact human erythrocytes were treated with phospholipase C (Clostridium perfringens), up to 30% of the membrane phospholipids were broken down without significant cell lysis . Only phosphatidylcholine and sphingomyelin were attacked . Ceramide (derived from sphingomyelin) accumulated, but 1,2-diacylglycerol (derived from phosphatidylcholine) was largely converted into phosphatidate . Up to 12% of the cell phospholipid could be converted into phosphatidate in this way . Pig erythrocytes and lymphocytes showed a similar but smaller synthesis of phosphatidate after phospholipase C attack . Phospholipase C also caused a marked morphological change in erythrocytes, giving rise to spherical cells containing internal membrane vesicles . This change appeared to be due to ceramide and de and diacylglycerol accumulation rather than to increased phosphatidate content of the cells. Am J Med, 1975 Dec, 59(6), 851 - 6 Necrotizing pneumonia and empyema due to Clostridium perfringens . Report of a case and review of the literature; Bayer AS et al.; Clostridia are rare causes of pleuropulmonary infections in the absence of penetrating chest injuries; only 10 previous cases have been reported from civilian practice . An additional case of a rapidly progressive, necrotizing pneumonia and empyema is reported . Clostridial pneumonia is more likely to occur in patients with underlying pleuropulmonary disease . Unlike clostridial myonecrosis, it is rarely associated with toxemia; its mortality rate is comparable to that of nonclostridial pleuropulmonary infections . Appropriate antimicrobial therapy with surgical drainage of the empyema is the treatment of choice . Among the cases reviewed, an iatrogenic cause of infection involving an invasive procedure into the pleural cavity could be identified in seven of 11 cases . Aspiration of oropharyngeal contents was the likely route of infection in three other cases . In the remaining case, bacteremic seeding of the pleural cavity was the most probable mode of infection. Jpn J Exp Med, 1975 Dec, 45(6), 479 - 88 Lung damage caused by phospholipase C and the changes in phospholipids in the rat lung; Ito M et al.; The lipid and protein fractions of the endobronchial lavage fluid from the normal rats which contained the lung surfactant were analysed . Lecithin, the main main component of the lung surfactant, was exclusively combined with a dextran precipitable protein . This protein then behaved as beta-globulin on cellulose acetate electrophoresis or low density lipoprotein on agarose gel filtration . After administration of phospholipase C (from Clostridium Welchii), the protein content of the lavage fluid increased markedly . The amount of dextran precipitable protein also increased markedly and its properties remained the same on gel filtration after treatment with phospholipase C . The phospholipids in the lavage fluid were not affected, although the total phospholipids in the lung tissue, especially lecithin, did decrease during the 10 days after treatment . Histopathologically, an eosinophilic dense exudative fluid appeared in both the interstitium and the broncho-alveolar spaces . A large number of the alveolar lining cells disappeared and a few of them were desquamated into the alveolar spaces . Thus, the immediate destruction of the alveolar lining cells after the administration of phospholipase C resulted in interstitial pneumonia in 10 days . The significance of phospholipase in pulmonary inflammation is discussed. Infect Immun, 1975 Dec, 12(6), 1262 - 70 Molecular construction of Clostridium botulinum type A toxins; Sugii S et al.; Two Clostridium botulinum type A toxic fractions, named large (L) and medium (M) toxins, were eluted from Sephadex G-200 . Sucrose density gradient centrifugation resolved L toxin (2.5 X 10(8) to 3.0 X 10(8) mean lethal doses per mg of N) into two fractions, 19S and 16S . The same procedure performed at pH 8resolved it into three fractions; the heavier two were both nontoxic and hemagglutinin positive, and the lightest on (7S) was toxic . M toxin (12S) (4.5 X 10(8) to 5.0 X 10(8) mean lethal doses per mg of N) was homogeneous in electrophoresis and centrifugation at pH 6 . The latter procedure performed at pH 8 dmonstrated that it dissociated into uniform 7S components . The nontoxic component of M toxin was free from hemagglutinin . M toxin alone was demonstrated in culture by sucrose density gradient centrifugation at pH 6 . Dialysis of the culture supernatant resulted in partial formation of 16S toxin . Centrifugation of the crystalline toxin in 1 MNaCl demonstrated 16S toxin only . The toxic components of L, M, and crystalline toxins were antigenically identical . The nontoxic components of the crystalline and L toxins, consisting of two distinct antigens, were antigenically identical; that of M toxin was identical with one of these two antigens. J Bacteriol, 1975 Dec, 124(3), 1295 - 1301 Transport of molybdate by Clostridium pasteurianum; Elliott BB et al.; The transport of 99MoO42- into dinitrogen-fixing cells of Clostridium pasteurianum was investigated . Transport of molybdate in this organism is energy dependent; sucrose is required in the minimal media, and the system is inhibited by the glycolysis inhibitors, NaF, iodoacetic acid, and arsenate . The cells accumulate molybdate against a concentration gradient, and the uptake shows a marked dependence on temperature (optimum 37 C) and pH (optimum 6.0) . The rate of molybdate uptake with increasing molybdate concentrations shows saturation kinetics with an apparent Km and Vmax of 4.8 X 10(-5) M and 55 nmol/g of dry cells per min, respectively . Inhibition studies with the anions SO42-, S2O32-, WO42-, and VO32- show that SO42- and WO42- competitively inhibit MoO42- uptake (apparent Ki {SO42-} is 3.0 X 10(-5) M; apparent Ki {WO42-} is 2.4 X 10(-5), whereas S2O32- and VO32- have no inhibitory effect . Exchange experiments with MoO42- show that only a small percentage of the 99MoO42- taken up by the cells is exchangeable . Exchange experiments with WO42- and SO42- indicate that once inside the cells WO42- and SO42- cannot substitute for MoO42-. Biochim Biophys Acta, 1975 Nov 18, 412(1), 62 - 9 Studies on epsilon-prototoxin of Clostridium perfringens type D . Physicochemical and chemical properties of epsilon-prototoxin; Habeeb AF; epsilon-Prototoxin of clostridium perfringens type D consists of one polypeptide chain of 311 amino acids with the following composition: Asp52 Thr31 Ser25 Glu28 Pro12 Gly17 Ala14 Val28 Met5 Ile15 Leu18 Tyr17 Phe8 Lys31 His2 Arg5 Tyr2 . It has no free cysteine but contains one blocked cysteine . The N-terminal as well as the C-terminal residue is lysine . The ultracentrifuge pattern gave one single boundary having S020,w = 2.15 S and Do20,w = 5.56-10(-7) cm2/s . Calculation of the molecular weight from D020,w and S020,w gave a value of 34 250 . The molecular weight determined from sedimentation equilibrium using ultraviolet optics gave a value of 33 000 +/- 1000 . On the other hand molecular weights calculated from a calibrated Sephadex G-100 column in borate buffer was 25 000 and that in sodium phosphate, ionic strength 0.2, was 27 500 . This discrepancy between values obtained in the ultracentrifuge and by gel filtration is attributed to adsorption of epsilon-prototoxin by Sephadex. Experientia . 1975 Nov 15;31(11):1270. Creatinine metabolism by Clostridium welchii isolated from human faeces; ten Krooden E et al.; A Clostridium welchii has been isolated from human faeces which can deaminate creatinine to N-methyl hydantoin . Evidence suggests the reaction is inducible since the rate of conversion is increased by growth of the organism in creatinine-rich media. Appl Microbiol, 1975 Nov, 30(5), 811 - 20 Low-temperature irradiation of beef and methods of evaluation of radappertization process; Anellis A et al.; An inoculated, irradiated beef pack (1,240 cans) was conducted for the determination of microbiological safety for unrestricted human consumption . Each can contained a mixture of 10(6) spores of each of 10 strains of Clostridium botulinum (5 type A and 5 type B), or a total of 10(7) spores/can . The cans were irradiated to various doses (100 cans/dose) with 60Co gamma rays at -30 +/- 10 C, incubated at 30 +/- 2 C for 6 months, and examined for swelling, toxicity, and recoverable botulinal cells . The minimal experimental sterilizing dose based on nonswollen, nontoxic sterile cans were 2.2 less than experimental sterilizing dose based on nonswollen, nontoxic sterile cans was 2.2 less than experimental sterilizing dose less than or equal to 2.6 Mrad . Using recoverable cells as the most stringent criterion of spoilage, and assuming the conventional simple exponential (without an initial shoulder) rate of spore kill, the "12D" dose was 3.7 Mrad when estimated on the basis of mixture of 10 strains totaling 10(7) spores/can, and 4.3 Mrad if it is assumed that each can of beef contained 10(6) spores of a single most resistant strain and all of these spores were of identical resistances . However, an analysis of the data by extreme value statistics indicated with 90% confidence that the spore death rate was not a simple exponential but might be a shifted exponential (with an initial shoulder), Weibull, lognormal, or normal, with a "12D" equivalent of about 3.0 Mrad regardless of the initial spore density per can . There was an apparent antagonism between the irradiated type A and B strains in the cans . Some of the cans contained type B toxin but did not include type B viable cells . Other cans had a mixture of type A and B toxins, but a large number of these cans did not yield recoverable type B cells . However, type A viable cells could always be demonstrated in those cans containing type A toxin. J Biol Chem, 1975 Nov 10, 250(21), 8462 - 6 Effects of strong electrolyte upon the activity of Clostridium perfringens sialidase toward sialyllactose and sialoglycolipids; Barton NW et al.; Clostridium perfringens sialidase was purified by affinity chromatography . Kinetic properties of the enzyme were examined with sialyllactose and with mixed sialoglycolipids (gangliosides) as substrates . With the latter substrate in 0.01 M Tris-acete in the absence of strong electrolyte, the pH optimum for enzymatic activity was 6.8 . Addition of strong electrolyte (0.01 to 0.10 M Nac1) to the reaction medium caused an acidic shift and a broadening of the pH optimum, Enzymatic activity at pH 5.8 rose approximately 2.5-fold; a concomitant loss of activity at pH 6.8 was also observed . The alteration of enzymatic activity caused by strong electrolyte were dependent upon changes in Vmax . Km remained nearly invariant . Thus, a reversible transition of the enzyme from a relatively inactive to a highly active form occurred as a function of strong electrolyte concentration . Determination of the pK values of the active functional groups of C . perfringens sialidase revealed that the effects of strong electrolyte were exerted upon the pKa group of the enzyme . Strong electrolyte appeared to shield unfavorable electrostatic interactions between polyanionic sialoglycolipid micelles and the enzyme molecule, thus protecting the pKa group from inactivation . In comparision with the effects of strong electrolyte upon enzymatic activity toward the sialoglycolipid substrate, those observed with the monovalent substrate, sialyllacthose, were minor . Collectively, these findings indicate that ionic environment may effectively control the activity and relative substrate specificity of C . perfringens sialidase at a given pH . Furthermore, they explain the low pH optima and skewed pH profiles previously reported for enzymatic activity toward high molecular weight substrates. Cancer Res, 1975 Nov, 35(11 Pt 1), 2962 - 8 The reduction of N-hydroxy-4-acetylaminobiphenyl by the intestinal microflora of the rat; Wheeler LA et al.; The role of the intestinal flora in the conversion of N-hydroxy-4-acetyl-aminobiphenyl (N-OH-AABP) to 4-acetylaminobiphenyl has been examined . This reaction, which reverses the metabolic activation of the parent carcinogen, can be demonstrated in cultures of some bacteria indigenous to the intestinal microflora . These include cultures of Clostridium sp., Clostridium perfringens, Peptostreptococcus productus I, and Bacteroides fragilis ss . thetaiotaomicron and ss . vulgatus . In contrast, cultures of Lactobacillus plantarum and Escherichia coli show little or no capacity for this reaction . The reduction of N-OH-AABP is also carried out by homogenates of liver, kidney, and brain . On a weight basis, the cecal flora is considerably more active in reducing N-OH-AABP than are homogenates of tissues of the gastrointestinal tract . The cecal flora also has a greater activity for reducing N-OH-AABP than the stomach flora, an observation which may relate to the induction of tumors in the forestomach but not in the cecum of rats fed this compound . The products of the metabolism of N-OH-AABP have been compared in germ-free and conventional animals . Glucuronide conjugates of N-OH-AABP are found in the cecal contents and feces only of the germ-free rats, while 4-acetylaminobiphenyl is found in the feces only of conventional rats . These results suggest that the flora, by hydrolyzing glucuronides and reducing N-OH-AABP, may influence the level of metabolities of 4-acetylaminobiphenyl which are critical for carcinogenesis. J Cell Sci, 1975 Nov, 19(2), 341 - 55 The perturbation of the human erythrocyte membrane by phospholipase C; Limbrick AR et al.; A study has been made of freeze-fractured preparations of erythrocyte ghosts modified by phospholipase C (Clostridium welchii) . Such membranes show a decrease in surface area of up to about 47% and lipid droplets appear on their external surface but there is no loss of protein . Freeze-fracture of maximally hydrolysed membranes exposes only very small areas of A faces and these appear particle-free . Most of the membranes are simply cross-fractured . At lower levels of hydrolysis there is more extensive exposure of A fracture faces but the particle density is less than in control preparations . If such exposed faces were representative of the whole membrane then the particle density would have been expected to increase . It is suggested either that areas of membrane with increased particle density do not fracture or that the particles revealed by freeze-fracture involve phospholipid as well as protein and are not revealed in the absence of phospholipid. J Biochem (Tokyo), 1975 Nov, 78(5), 905 - 9 Action of bacterial collagenase on Ascaris cuticle collagen; Fujimoto D; The collagen from the cuticle of Ascaris lumbricoides was digested by Clostridium histolyticum collagenase {EC 3.4.24.3} in the presence and absence of CaCl2 . About 1.2 mumoles of amino groups per mg collagen was liberated when the digestion was performed in the presence of 5 mM CaCl2, whereas about 0.5 mumole of amino groups per mg collagen was liberated by digestion in the absence of CaCl2 . In contrast, CaCl2 influenced the extent of hydrolysis of rat tail tendon collagen only slightly . The results suggest that CaCl2 is necessary for the hydrolysis of certain regions in the molecule of Ascaris collagen and that such structures may not be present in mammalian collagens. Appl Microbiol, 1975 Nov, 30(5), 873 - 5 Repair of heat-injured Clostridium perfringens spores during outgrowth; Barach JT et al.; Clostridium perfringens strain NCTC 8798 spores were injured by ultrahigh temperature treatment and were unable to outgrow in the presence of antibiotics used in selective enumeration media . Injured spores underwent repair in a nonselective laboratory medium and in foods. Appl Microbiol, 1975 Nov, 30(5), 838 - 43 Inhibitor of Clostridium perfringens formed by heating sodium nitrite in a chemically defined medium; Moran DM et al.; An inhibitor of Clostridium perfringens formed when low levels of nitrite were autoclaved with a defined chemical medium . A systematic study of the medium revealed that only amino acids and mineral salts were involved in the production of this inhibitor, which was proven to be a toxic compound formed from cysteine, ferrous sulfate, and sodium nitrite . The inhibitor was compared to several known compounds . S-nitrosocysteine inhibited the test organism, but would not form in the test system in amounts large enough to explain the observed inhibition . Roussin red salt was unstable in the test system and therefore was not the inhibitor . Roussin black salt, which was also inhibitory, could form in sufficient amounts to explain the inhibition . A complex of cysteine, iron, and nitric oxide was detected in the autoclaved solution of cysteine, ferrous sulfate, and sodium nitrite; this cysteine complex did not appear to be inhibitory, however, at levels which could form in the autoclaved medium . The observed inhibition may have been due to the combined effects of sublethal concentrations of each compound. Appl Microbiol, 1975 Nov, 30(5), 781 - 5 Differential effects of oxygen and oxidation-reduction potential on the multiplication of three species of anaerobic intestinal bacteria; Walden WC et al.; The sensitivity of three strains of anaerobic intestinal bacteria, Clostridium perfringens, Bacteroides fragilis, and Peptococcus magnus, to the differential effects of oxygen and adverse oxidation-reduction potential was measured . The multiplication of the three organisms was inhibited in the presence of oxygen whether the medium was at a negative oxidation-reduction potential (Eh of -50 mV), poised by the intermittent addition of dithiothreitol, or at a positive oxidation-reduction potential (Eh of near +500 mV) . However, when these organisms were cultured in the presence of oxygen, no inhibition was observed, even when the oxidation-reduction potential was maintained at an average Eh of +325 mV by the addition of potassium ferricyanide . When the cultures were aerated, the growth patterns of the three organisms demonstrated different sensitivities to oxygen . P . magnus was found to be the most sensitive . After 2 h of aerobic incubation, no viable organisms could be detected . B . fragilis was intermediately sensitive to oxygen with no viable organisms detected after 5 h of aerobic incubation . C . perfringens was the least sensitive . Under conditions of aerobic incubation, viable organisms survived for 10 h . During the experiments with Clostridium, no spores were observed by spore staining. J Laryngol Otol, 1975 Nov, 89(11), 1147 - 50 Hyperbaric oxygen in anaerobic infection of the medistinum; Monies-Chass I et al.; In the last decade hyperbaric oxygen has been recognized as an important therapeutic tool in a variety of instances in which either destruction of anaerobic bacteria is urgent or an improvement in the oxygenation level is mandatory . We have used it successfully in a case of mediastinal anaerobic infection (gas gangrene) after medical and surgical measures had failed to eradicate the disease . The causative organism of gas gangrene, Clostridium perfringens (Welchii), is widely distributed . It may be cultured from the soil, house dust, human skin and faeces, etc . For this reason infection with Clostridium is practically inevitable whenever suitable conditions arise . As an anaerobic bacterium Clostridium Welchii multiplies readily in damaged tissues without contact with the air and devoid of a normal blood supply . This occurs especially in road accidents and war wounds in which broken bones and crushed muscles provide a suitable medium for the infection (Altmeier, 1965) . Sometimes this infection can occur too after abdominal or gynaecological operations (Hitchcock, 1965) . We present here a case of mediastinal gas gangrene which was caused by perforation of the oesophagus by a swallowed foreign body. Infect Immun, 1975 Nov, 12(5), 1214 - 8 Histopathological effect of Clostridium perfringens enterotoxin in the rabbit ileum; McDonel JL et al.; Highly purified enterotoxin from Clostridium perfringens was found to have histopathological activity in the rabbit ileum . Unlike the action of cholera, Escherichia coli, and Shigella enterotoxins, epithelium was denuded from the tips of ileal villi at concentrations of the enterotoxin necessary to induce fluid accumulation in the rabbit . Whether or not this observed histopathology is essential for the diarrheal syndrome associated with Clostridium perfringens food poisoning remains unclear. Can J Microbiol, 1975 Nov, 21(11), 1719 - 23 A comparative study on the heat stability of triosephosphate isomerase in psychrophilic, psychrotrophic, and mesophilic clostridia; Finne G et al.; The temperature stability of triosephosphate isomerase (EC 5.3.1.1) in the cell-free extracts of psychrophilic, psychrotrophic, and mesophilic Clostridium species has been investigated . Even though this enzyme was found to be heat labile in the psychrophilic isolates, no detectable loss in activity was evident when cell-free extracts were heated for 1/2 h at the maximum temperature of growth for the organisms . Two organisms, each with a maximum growth temperature of 23 degrees C, showed different heat stability of triosephosphate isomerase . However, a trend is evident that as the maximum growth temperature increases, the thermostability also increases . It is suggested that the heat liability of this enzyme is not a controlling factor in psychrophilism, but rather an adaptation to the cold habitat of these organisms. Lancet, 1975 Nov 1, 2(7940), 861 - 3 A continuing common-source outbreak of botulism in a family; Horwitz MA et al.; In December, 1974, three cases of botulism occurred in a family; two were fatal . The first patient died after a 10-day illness without botulism being suspected . 4 days later, after a 2-day illness, the second patient was diagnosed as having botulism after a cardiorespiratory arrest; she died 3 days later . In the third patient, the only symptom was dysphagia . Clostridium botulinum type B was found in stool specimens from all three patients . Home-canned (bottled) mushrooms, which were found to contain C . botulinum type B and its toxin, were believed to be responsible for the outbreak; mushrooms were found at necropsy in the gastrointestinal tracts of both patients who died . Heat treatment of the mushrooms during canning had been inadequate. Can J Microbiol, 1975 Nov, 21(11), 1711 - 8 Regulation and properties of an invertase from Clostridium pasteurianum; Laishley EJ; An intracellular invertase was induced in cultures of Clostridium pasteurianum utilizing sucrose as its carbon source for growth . This enzyme synthesis could be repressed by the addition of fructose of a sucrose-growing culture . In contrast, invertase activity was not affected by the addition of glucose to sucrose-growing cells and this enzyme could be induced in a glucose-metabolizing culture by the addition of sucrose . This enzyme was purified 10.5-fold over the induced lese, EC 3.2.1.26) by substrate-specificity studies . Invertase had a pH optimum of 6.5 and an apparent Km of 79.5 mM for sucrose, and required high concentration of potassium phosphate for maximum activity . Invertase was completely inactivated by a 2-min heat treatment at 60 degrees C . This enzyme was strongly inhibited by p-hydroxymercuribenzoate (pCMB) and weakly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid), while cysteine could substantially reverse pCMB) inhibition, suggesting that sulfhydryl group(s) were necessary for invertase activity. J Biol Chem, 1975 Oct 25, 250(20), 8049 - 54 The monovalent cation-induced association of formyltetrahydrofolate synthetase subunits . Kinetic and thermodynamic aspects; Harmony JA et al.; Formyltetrahydrofolate synthetase monomers are converted to catalytically active tetramers in the presence of monovalent cations . The stoichiometry of the reaction is 4M + 2C+ in equilibrium M4C2(2+) . A positive deltaS compensates for an unfavorable positive deltaH so that the overall reaction is exergonic . Both deltaH and deltaS become more positive as the temperature is increased . Association of subunits of the enzyme prepared from Clostridium cylindrosporum is second order with respect to monomer concentration, consistent with a rate-determining dimerization step . Activation parameters for this step at 20 degrees are: deltaG, 12.6 kcal mol-1; deltaH, 12.5 kcal mol-1; deltaS, -05 e.u . The rate-limiting step for the cation-dependent association of Clostridium acidi-urici monomers is believed to be a conformational alteration since first order kinetics is observed . The Eyring plot of the kinetic data obtained for the C . acidi-urici system has a sharp break at 15 degrees . Activation parameters for cation-induced association at 20 degrees are: deltaG, 21.5 kcal mol-1; deltaH, 14.0 kcal mol-1; deltaS, -26.6 e.u. Biochim Biophys Acta, 1975 Oct 22, 403(2), 521 - 9 Regulatory responses of arginine deiminase in whole cells of Clostridium sporogenes; Venugopal V et al.; Arginine deiminase (EC 3.5.3.6) has been shown to have regulatory properties . The activity was observed to be sigmoidal with respect to substrate concentrations . Addition of histidine to the system caused the abolition of sigmoidal responses . The regulatory properties of the enzyme as well as the desensitising action of histidine could also be demonstrated with whole cell suspensions . The pH of the system also seemed to influence modulations in the enzyme. Biochim Biophys Acta, 1975 Oct 21, 409(1), 75 - 85 Phospholipase C from Clostridium novyi type A . I; Taguchi R et al.; 1 . Phospholipase C (EC 3.1.4.3) from Clostridium novyi (oedematiens) type A was purified 2000-fold by (NH4)2SO4 precipitation, DEAE-Sephadex treatment in a batchwise system and Sephadex G-100 column chromatography . 2 . The purified preparation had a specific activity of 95 mumol per min per mg protein toward phosphatidylcholine . This preparation was free from protease, lipase and oxygen-labile delta-hemolysin . 3 . Phosphatidylcholine was hydrolyzed at the highest rate, while sphingomyelin and lysophosphatidylcholine were hydrolyzed at much lower rates . 4 . Sodium deoxycholate and divalent cations such as Mg2+ and Ca2+ were extremely effective in stimulating phosphatidylcholine-hydrolyzing activity of this enzyme . 5 . This enzyme hemolyzed horse red cells by hydrolyzing phosphatidylcholine, spingomyelin and phosphatidylethanolamine. J Biol Chem, 1975 Oct 10, 250(19), 7842 - 7 High and low reduction potential 4Fe-4S clusters in Azotobacter vinelandii (4Fe-4S) 2ferredoxin I . Influence of the polypeptide on the reduction potentials; Sweeney WV et al.; Azotobacter vinelandii (4Fe-4S)2 ferredoxin I (Fd I) is an electron transfer protein with Mr equals 14,500 and Eo equals -420 mv . It exhibits and EPR signal of g equals 2.01 in its isolated form . This resonance is almost identical with the signal that originates from a "super-oxidized" state of the 4Fe-4S cluster of potassium ferricyanide-treated Clostridium ferredoxin . A cluster that exhibits this EPR signal at g equals 2.01 is in the same formal oxidation state as the cluster in oxidized Chromatium High-Potential-Iron-Protein (HiPIP) . On photoreduction of Fd I with spinach chloroplast fragments, the resonance at g equals 2.01 vanishes and no EPR signal is observed . This EPR behavior is analogous to that of reduced HiPIP, which also fails to exhibit an EPR spectrum . These characteristics suggest that a cluster in A . vinelandii Fd I functions between the same pair of states on reduction as does the cluster in HiPIP, but with a midpoint reduction potential of -420 mv in contrast to the value of +350 mv characteristic of HiPIP . Quantitative EPR and stoichoimetry studies showed that only one 4Fe-4S cluster in this (4Fe-4S)2 ferredoxin is reduced . Oxidation of Fd I with potassium ferricyanide results in the uptake of 1 electron/mol as determined by quantitative EPR spectroscopy . This indicates that a cluster in Fd I shows no electron paramagnetic resonance in the isolated form of the protein accepts an electron on oxidation, as indicated by the EPR spectrum, and becomes paramagnetic . The EPR behavior of this oxidizable cluster indicates that it also functions between the same pair of oxidation states as does the Fe-S cluster in HiPIP . The midpoint reduction potential of this cluster is approximately +340 mv . A . vinelandii Fd I is the first example of an iron-sulfur protein which contains both a high potential cluster (approximately +340 mv) and a low potential cluster (-420 mv) . Both Fe-S clusters appear to function between the same pair of oxidation states as the single Fe-S cluster in Chromatium HiPIP, although the midpoint reduction potentials of the two clusters are approximately 760 mv different. Zentralbl Bakteriol {Orig A}, 1975 Oct, 233(2), 253 - 60 Studies on the cultivation of Clostridium oncolyticum M 55 . 5th communication: The influence of iron, zinc, and cobaltous ions on the growth and the kininase activity of clostridium oncolyticum M 55 ATCC 13.732; Brantner H et al.; The precipitation or iron sulfide by hydrogen sulfide excreting Clostridium oncolyticum M 55 led to further examinations in order to avoid this disturbing factor . The method should render the cultivation of Cl . oncolyticum M 55 under definite conditions and should also intensify the formation and the enrichment of the clostridial kininases . Cobaltous ions and zinc granules were found useful for cultivation, because no disturbances increased from both during sterilization and incubation . The determination of the growth cycles and the replication rates showed the usefulness of zinc for substitution of iron . Cobaltous ions did only allow a low replication rate . These results confirmed those showing the influence of chelate-forming agents on the kininase activity . Cobaltous ions were not able to exchange the central atom of the metallo-proteide . The influence of the peptone quality on the kininase activity in presence of zinc was also examined, since examinations with iron media have shown a direct connection between peptone quality and enzyme activity . The results with zinc-containing media were nearly 5-10% lower than those with iron . The results allowed also the conclusion that the kininase excreted by Cl . oncolyticum M 55 is an iron-containing metallo-proteide, whose central atom is exchangeable for zinc . The consequences of the loss of enzyme activity for the oncolytic therapy with Cl . oncolyticum M 55 will be shown in further examinations. J Biochem (Tokyo), 1975 Oct, 78(4), 673 - 8 Characterization of two glycoasparagines isolated from the urine of patients with aspartylglycosylaminuria (AGU); Sugahara K et al.; Two major glycoasparagines (2-acetamido-N-(4'-L-aspartyl)-2-deoxy-beta-D-glycosylamines) were isolated from the urine of patients with aspartylglycosylaminuria (AGU) . They were composed of equimolar amounts of sialic acid, galactose, glucosamine, and aspartic acid . They were isomeric with respect to the position of sialic acid attachment, since they produced the same glycoasparagine on incubation with the neuraminidase from Clostridium perfringens . The structure of the resulting sialic acid-free glycoasparagine was determined as beta-Gal-(1 leads to 4)-beta-GlcNAc-Asn based on the following findings . It produced galactose on incubation with beta-galactosidase, and N-acetyllactosamine and aspartic acid on incubation with 4-L-aspartylglycosylamine amindo hydrolase. Proc Natl Acad Sci U S A, 1975 Oct, 72(10), 4003 - 7 Determination of the iron-sulfur distances in rubredoxin by x-ray absorption spectroscopy; Shulman RG et al.; The high intensity x-ray flux from the synchrotron radiation at the Stanford Synchroton Radiation Project has been used to study the extended x-ray absorption fine structure (EXAFS) of the iron-sulfur protein Peptococcus aerogenes rubredoxin . Absorption measurements were made from 7080 eV, which is below the K-edge of iron, to about 650 eV above the edge and structure was obtained over the entire region . By means of a model iron-sulfur compound for evaluating the phase shifts, the variation of the absorption above the edge of lyophilized, oxidized rubredoxin was converted to iron-sulfur distances . The data were fitted with a least squares program to a model in which three distances R3 were kept equal and the fourth R1 was allowed to differ . The mean square error was constant over a region of this parameter space, becoming twice as large at R3 = 2.217, R1 = 2.389 and R3 = 2.268, R1 = 2.108 A . These values, which are the extreme differences allowed by the present data, are definitely closer to being equal than those found by the determination of the x-ray diffraction crystal structure of the similar protein from Clostridium pasteurianum . However, the average distance from our experiment is in excellent agreement with the average distance from the crystal structure determination . Preliminary EXAFS measurements were also made on the oxidized rubredoxin in solution at pH 7.0 . The spectra were unchanged, indicating that the average iron-sulfur distance change is less than 0.02 A . Upon reduction the average iron-sulfur bond length increased by about 0.05 A . Since the EXAFS measurements can give accurate determinations of distances in proteins both in crystals and solution, the technique should be widely applicable. J Biochem (Tokyo), 1975 Oct, 78(4), 763 - 72 Formation of beta-methylmalate and its conversion to citramalate in Rhodospirillum rubrum; Osumi T et al.; Using a cell-free extract of Rhodospirillum rubrum, studies were made of the condensation reaction between propionyl-CoA and glyoxylate . When {14C}propionate was incubated with the extract in the presence of glyoxylate, ATP, CoA, Mg2+, and Mn2+, radioactivity was incorporated into several compounds . Two of the main products were characterized as citramalate (CMA) and erythro-beta-methylmalate (erythro-MMA) on the basis of their behavior compared with authentic samples of CMA and erythro-MMA in the following three analyses: (i) paper chromatography using two solvent systems, (ii) radio-gas chromatography on their methyl esters, and (iii) chemical conversion to readily crystallizable derivatives, that is, citramalyl chloralide for CMA, and thymine for MMA . The CMA was thought to be of L(+)-form based on the results of optical resolution with brucine and also its susceptibility to L(+)-citramalate lyase of Clostridium tetanomorphum . When the reaction was carried out with lower concentrations of the enzyme, only MMA was accumulated . However, when the reaction was allowed to proceed further after addition of higher concentrations of the enzyme and of excess semicarbazide to prevent further condensation, the amount of accumulated MMA was decreased and CMA was formed instead . Furthermore, the time course of MMA and CMA formation exhibited a pattern typical of a precursor-product relationship . From these results, it was concluded that MMA was formed by alpha-condensation between propionyl-CoA and glyoxylate, and that CMA was derived from MMA, possibly from its CoA derivative. Am J Med Technol, 1975 Oct, 41(10), 377 - 86 Hematological findings in acute infections and septicemias; Talluto MR; This paper is a report on twelve cases of septicemia, the diagnosis made possible by medical technologists observing the degenerative morphological findings in the peripheral blood smears . This led to the finding of microorganisms, prior to the bacteriological confirmation, in an active emergency department . It also includes four case reports in which the organism was identified: a meningococcus, a pneumonococcus, an Escherichia coli, and a clostridium perfringens (welchii). Biochem J, 1975 Oct, 151(1), 75 - 83 Physicochemical characterization of the four-iron-four-sulphide ferredoxin from Bacillus stearothermophilus; Mullinger RN et al.; 1 . A stable ferredoxin was prepared from Bacillus stearothermophilus and purified by chromatography on DEAE-cellulose and by electrophoresis . 2 . The minimum molecular weight determined from the amino acid composition was about 7900 and this was in reasonable agreement with a value of 8500 determined by polyacrylamide-gel electrophoresis . The ferredoxin contained four iron atoms and four labile sulphide groups per molecule . 3 . The optical absorption, optical-rotatory-dispersion and circular-dichroism spectra are typical of ferredoxins containing 4Fe-4S clusters . 4 . Oxidation-reduction titrations, combined with electron-paramagnetic-resonance (e.p.r.) spectroscopy, showed that the protein has a mid-point potential, at pH8, of -280 +/- 10mV, and that only one electron-accepting paramagnetic species is present . 5 . The e.p.r . spectrum of the reduced ferredoxin is more readily saturated with microwave power at low temperatures than those of the eight-iron ferredoxins, indicating that there is another mechanism of electron-spin relaxation in the latter . 6 . Mossbauer spectra of both redox states were observed over a range of temperatures and in magnetic fields . At high temperatures (77 degrees K and above) both redox states appear as quadrupole-split doublets; in the reduced state two resolved doublets are seen, suggesting appreciable localization of the additional reducing electron . 7 . The average chemical shift indicates formal valences of two Fe3+ and two Fe2+ in the oxidized state and three Fe2+ and one Fe3+ in the reduced state . However, the spectra indicate that there are differing degrees of electron delocalization over the iron atoms . 8 . At low temperatures (4.2 degrees K) the oxidized form shows no hyperfine magnetic interaction, even in an applied magnetic field, evidence that the oxidized ferredoxin is in a non-magnetic state as a result of antiferromagnetic coupling between the iron atoms . 9 . At 4.2 degrees K the reduced form shows a broad asymmetric pattern resulting from magnetic hyperfine interaction . This contrasts with the reduced ferredoxin of Clostridium pasteurianum, which shows a doublet, suggesting that in the latter there may be interaction between the two 4Fe-4S centres . 10 . In large applied magnetic fields, positive and negative hyperfine fields are seen in the Mossbauer spectra of the reduced ferredoxin, evidence for antiferromagnetic coupling between the iron atoms in the 4Fe-4S centre . The high-field spectra of the reduced ferredoxin of B . stearothermophilus are similar to those of the reduced ferredoxin of C . pasteurianum. Ann Microbiol (Paris), 1975 Oct-Nov, 126(3), 343 - 56 {Purification and characterization of "Clostridium perfringens" BP6K-N5 strain bacteriocin N5 (author's transl)}; Wolff A et al.; The bacteriocin N5 produced by Clostridium perfringens, strain BP6K-N5, after UV irradiation induction, was purified by ammonium sulfate precipitation, followed by ion-exchange chromatography on DEAE cellulose and gel filtration on Sephadex G100 . By polyacrylamid-gel electrophoresis and immunodiffusion analysis the purified material was shown to be homogenous . The purified bacteriocin N5 is inactivated by proteolytic enzymes and by heat treatment at 50 degrees C for 15 minutes . It is a simple protein with a molecular weight of approximately 82.000 . The protein appears to be a single polypeptide chain, as no dissociation is obtained by sodium dodecyl sulfate and beta-mercaptoethanol disc-gel-electrophoresis. Appl Microbiol, 1975 Oct, 30(4), 530 - 5 Convenient non-chromatographic assays for the microbial deconjugation and 7alpha-OH bioconversion of taurocholate; MacDonald IA et al.; We described two convenient assay methods to estimate bile acid deconjugation and bile acid bioconversion at the 7alpha-OH position by individual microorganisms grown in media containing taurocholic acid . The methods are based on (i) a selective chemical assay for taurine conjugates previously described and (ii) the use of a cell-free preparation of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli to directly quantify 7alpha-OH groups . These non-chromatographic approaches have been applied to the study of three model strains of intestinal organisms, E . coli, Bacteroides fragilis, and Clostridium perfringens, grown in standard media in the presence of purified tritiated taurocholate . Assay results were confirmed by thin-layer chromatography solvent systems designed to separate conjugated from unconjugated bile acid and unmodified cholic acid nucleus from 7alpha-OH bioconversion product(s) (primarily 3alpha, 12alpha dihydroxy, 7-keto-cholanoic acid) . In addition, 7alpha-hydroxysteroid dehydrogenase activity was demonstrated in cell-free extracts of all three organisms . Of the three organisms, only C . perfringens was demonstrated to (i) deconjugate taurocholic acid, (ii) contain 3alpha-hydroxysteroid dehydrogenase activity, (iii) convert cholic acid into at least five labeled metabolites visible on thin-layer chromatography, and (iv) catalyze significant tritium exchange with water in the medium. Appl Microbiol, 1975 Oct, 30(4), 499 - 502 Solid-phase radioimmunoassays for quantitative antibody determination of Clostridium perfringens type D epsilon toxin; Bernath S; Two reversed solid-phase radioimmunoassays were developed for quantitative determination of antibodies against Clostridium perfringens type D epsilon toxin . 125I-labeled prototoxin was used in the bromoacetylcellulose-bound antibody method and in the antibody-coated tube method . The antibody values which can be detected by the assays are in the range of 0.004 IU/ml of investigated serum . The methods allow the screening investigation of large groups of vaccinated sheep in a rapid and inexpensive way, and are very suitable for measuring small amounts of C . perfringens D epsilon antibodies with a small experimental error. Arch Microbiol, 1975 Sep 30, 105(1), 67 - 71 Effect of cyclic AMP on catabolite repressed bacterial sporogenesis of an anaerobe; Emeruwa AC et al.; The sporulation of a high frequency sporogenic mutant of Clostridium botulinum was reduced to less than 30% in a medium containing 270 mM glucose . The repression was reversed from 30 to greater than 80% sporulation by the addition of 10(-5) or 10(-4) M cyclic 3',5'-adenosine monophosphate (cAMP) or monobutyrul cyclic AMP (B-cAMP) . No difference was observed in amount of growth with the addition of either the cAMP or B-cAMP . Glucose consumption was enhanced by the addition of either of the cyclic nucleotides and corresponding changes in pH were observed . The catabolite repression by glucose was reversed by ATP or ADP . Except for GTP, guanine nucleotides were not effective . The intracellular cyclic AMP levels were high in vegetative, sporulating and derepressed cells, but low in glucose-repressed cells . The findings suggest that the sporulation of the anaerobe was sensitive to catabolite repression which was specifically reversed by cyclic AMP. J Biol Chem, 1975 Sep 25, 250(18), 7435 - 42 Tetanus toxin . The effect of chemical modifications on toxicity, immunogenicity, and conformation; Robinson JP et al.; Tetanus toxin has been isolated from the extract of Clostridium tetani and analyzed for purity using various methods, e.g . sedimentation velocity, gel filtration, polyacrylamide gel electrophoresis, immunoelectrophoresis, and immunodiffusion . The homogeneous toxin, characterized by a minimum lethal dose of 10 pg (18- to 20-g mouse), was judged to be of high purity . The amino acid composition was determined and found to be in good agreement with reported values for both filtrate and extract toxin . The corrected sedimentation coefficient, so20,w, was found to be 7.5, and the molecular weight was estimated to be 150,000 . These values agree closely with those reported by others . The toxin was modified using the conventional formaldehyde reaction to produce toxoid and both reductive methylation and carbamylation, which are highly specific for lysyl residues . Under certain reaction conditions, carbamylation of the toxin completely eliminated toxicity . Whereas reductive methylation yielded a high degree of conversion of lysine to dimethyllysine and monomethyllysine, the toxicity, albeit greatly reduced, was never completely eliminated . The circular dichroic spectrum of each chemically modified toxin was obtained, resolved into Gaussian components, and compared with that of native toxin, which is estimated to contain about 20% alpha helix and 23% beta structure . The far ultraviolet circular dichroic spectra of toxin and toxoid were characterized by negative extrema at 208 nm and 217 nm attributable to ordered secondary structure, and toxin also exhibited a distinct shoulder at 223 nm . Carbamylated toxin and methylated toxin were characterized by negative extrema at 210 nm and 206 nm, respectively, and both exhibited shoulders at 216 to 217 nm and 223 nm . The toxin and derivatives exhibited multiple negative extrema above 250 nm which were assigned to the various aromatic residues . There were differences in the spectra of the toxin and derivatives over the entire wavelength region, thus suggesting changes in the local environment of various chromophores . In particular, the rotational strengths of many of the bands assigned to tryptophan, tyrosine, and phenylalanine were altered in the derivatives . Also, in the far ultraviolet region of the circular dichroic spectrum, the data were suggestive of some reduction in the amount of both alpha helix and beta structure in the derivatives . However, there was no evidence of extensive conformational changes, e.g . unfolding, in the modified toxins . Presently, it is not known if the small conformational differences between toxin and toxoid are important in the loss of toxicity with the retention of immunogenicity in the derivative . The modification data are consistent with the hypothesis that separate amino acid residues are involved in toxicity and immunogenicity. Biochim Biophys Acta, 1975 Sep 16, 406(1), 97 - 107 Relation between various phospholipase actions on human red cell membranes and the interfacial phospholipid pressure in monolayers; Demel RA et al.; The action of purified phospholipases on monomolecular films of various interfacial pressures is compared with the action on erythrocyte membranes . The phospholipases which cannot hyorolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Bacillus cereus, phospholipase A2 from pig pancreas and Crotalus adamanteus and phospholipase D from cabbage, can hydrolyse phospholipid monolayers at pressure below 31 dynes/cm only . The phospholipases which can hydrolyse phospholipids of the intact erythrocyte membrane, phospholipase C from Clostridium welchii phospholipase A2 from Naja naja and bee venom and sphingomyelinase from Staphylococcus aureus, can hydrolyse phospholipid monolayers at pressure above 31 dynes/cm . It is concluded that the lipid packing in the outer monolayer of the erythrocyte membrane is comparable with a lateral surface pressure between 31 and 34.8 dynes/cm. Biochim Biophys Acta, 1975 Sep 16, 406(1), 83 - 96 Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases; Zwaal RF et al.; 1 . The action of eight purified phospholipases on intact human erythrocytes has been investigated . Four enzymes, e.g . phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells . On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50% of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80% of the sphingomyelin . 2 . Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes . Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect . 3 . With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved . 4 . Due to its absolute requirement for Ca2+, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure . Subsequent addition of Ca2+ stimulates phospholipase A2 activity at the inside of the resealed cell, eventually leading to lysis . Before lysis occurs, however, 25% of the lecithin, half of the phosphatidylethanolamine and some 65% of the phosphatidylserine can be hydrolysed . This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes. Mikrobiologiia, 1975 Sep-Oct, 44(5), 893 - 8 {Correlation of the DNA nucleotide makeup with the physiological and cytological characteristics of spore-forming anaerobic bacteria}; Duda VI et al.; The nucleotide composition of DNA from 12 studied species of anaerobic bacteria belongs to AT type, with G+C varying from 28.4 to 36.8 mole% . In the anaerobic group of Clostridium bifermentans, a correlation has been established between the nucleotide composition of DNA, the type of appendages on spores, and some physiologo-biochemical characteristics . The nucleotide composition of DNA in the spores of four anaerobic species is shifted toward GC type as compared to DNA in the vegetative cells . Data on the content of GC pairs in DNA of the spores may sometimes be of a higher taxonomic value than the corresponding evidence on DNA of the vegetative cells. Appl Microbiol, 1975 Sep, 30(3), 420 - 3 Detection of Clostridium botulinum toxin by local paralysis elicited with intramuscular challenge; Sugiyama H et al.; Clostridium botulinum toxin can be identified by a characteristic, acute local paralysis that follows its injection into the gastrocnemius ("calf" muscle) of mice . The local botulism can be elicited with slightly less than one-tenth the toxin amount that is needed to kill mice by the intraperitoneal (i.p.) challenge route . The practical sensitivities of the intramuscular (i.m.) versus i.p . tests are about equal, however, because maximum sample volume injectable i.m . is 0.1 ml as compared to the 0.5-ml range that can be given i.p . i.m . injection of 10 or more mouse i.p . mean lethal doses causes paralysis in about 1 h, and an i.m . injection of about 0.5 i.p . mean lethal doses causes paralysis in 3 to 4 h . Toxin neutralization by homologous type of antitoxin only can be demonstrated with an incubated mixture of toxin and antitoxin . Although not as convenient as the i.p . method for routine use to detect botulinum toxin, the i.m . method has characteristics which could make it a useful supplement to the presently accepted i.p . procedure. Hoppe Seylers Z Physiol Chem, 1975 Sep, 356(9), 1353 - 8 {Vergleichende Untersuchungen mit (14c)5-Aminolävulinat und (14c)Uroporphyrinogen (author's transl)}; Dauner HO et al.; Cell-free extracts from Clostridium tetanomorphum, a microorganism which synthesizes corrins but no heme, are capable of converting both 5-aminolevulinate and uroporphyrinogen III into cobyrinic acid . Comparative examinations with (14C)5-aminolevulinate and (14C)uroporphyrinogen yielded corresponding results . Cell-free extracts from Clostridium tetanomorphum contain uroporphyrinogen III . To obtain good radiochemical yields it is therefore necessary to use substrates of high specific radioactivity . A method for the preparation of 14C-labelled uroporphyrin I-IV with high specific radioactivity is described. Am J Clin Pathol, 1975 Sep, 64(3), 385 - 8 Primary osteomyelitis due to an anaerobic microorganism; Isenberg HD et al.; Primary osteomyelitis in a teen-aged boy that mimicked Ewing's tumor radiologically showed small Gram-negative rods on the original smear . The organism isolated was an obligately anaerobic bacterium, finally identified as Clostridium sphenoides . This finding underlines the need for microbiologic analysis of orthopedic lesions. J Bacteriol, 1975 Sep, 123(3), 978 - 84 Role of molybdenum in dinitrogen fixation by Clostridium pasteurianum; Cardenas J et al.; The role of Mo in the activity and synthesis of the nitrogenase components of Clostridium pasteurianum has been studied by observing the competition of Mo with its structural analogue W . Clostridial cells when fixing N2 appeared strictly dependent upon the available Mo, showing maximal N2-fixing activity at molybdate concentrations in the media of 10 muM . Cells grown in media with 3 times 10(-6) muM Mo, although showing good growth, had only 15% as much N2-fixing activity . In the presence of W the synthesis of both nitrogenase components, molybdoferredoxin and azoferredoxin, was affected . Attempts to produce nitrogenase in W-grown cells by addition of high molybdenum to the media in the presence of inhibitors of protein synthesis showed that Mo incorporation into a possible inactive preformed apoenzyme did not occur . Unlike other molybdoenzyme-containing cells, in which W either is incorporated in place of Mo to yield inactive protein or initiates the production of apoprotein, C . pasteurianum forms neither a tungsten substituted molybdoferredoxin nor an apoprotein . It is concluded that in C . pasteurianum molybdenum is an essential requirement for both the biosynthesis and activity of its nitrogenase. J Bacteriol, 1975 Sep, 123(3), 1115 - 23 Regulation of breakdown and synthesis of L-glutamate decarboxylase in Clostridium perfringens; Cozzani I et al.; L-Glutamate decarboxylase (GAD) activity of Clostridium perfringens (ATCC 8009) cells grown in various culture conditions was investigated . Remarkable variations of GAD level occur during the growth cycle in thioglycollate broth . These changes are affected by the pH of the culture medium . Addition of alkali to the culture media results in decrease of cell GAD activity, whereas increase of enzyme level occurs only in cells growing in unbuffered media . The results indicate that the mechanism regulating the GAD levels is sensitive to the changes of pH (or buffering substances) rather than to the steady pH values . Neither repression by glucose nor induction by L-glutamate was observed . Moreover, high concentrations of the free amino acid substrate in the culture media considerably decrease cell GAD activity, owing to the buffering effect of the amino acid . The molecular mechanism supporting the variations of GAD activity during the growth cycle of the cells were investigated and tentatively related to the structural and functional properties of the pure enzyme . It is shown that the drop of GAD activity during the lag phase is due to protein breakdown . Evidence is presented suggesting a control of protein degradation by its quaternary structure . Data are also reported supporting de novo synthesis of GAD during the late logarithmic phase of cell growth . Finally, the possible role of GAD as part of the pH regulation system of C . perfringens cells is discussed in relation both to physiologic conditions of the bacterial cell and to the molecular mechanisms regulating the GAD activity in vivo. Zentralbl Bakteriol {Orig A}, 1975 Sep, 233(1), 75 - 9 Phenotypic chain formation in Clostridium welchii by suramin; Shaikh MR et al.; Cl . welchii NCTC 6785 was grown in 1% glucose containing medium having 1% Suramin w/v . The cells grew into long chains of thirty units . This effect was not found beyond 0.5% concentration . On transfer into Suramin free medium the chains reverted to normal morphology indicating the change in morphology to be phenotypic. Vopr Neirokhir, 1975 Sep-Oct, (5), 24 - 8 {Serologic diagnosis of brain tumors using Clostridium butyrium spores (experimental and clinical studies)}; Krechmer Kh; Spores of the Clostridium butyrium M 55 strain, introuduced intravenously, proliferate selectivity in tumours of individual with newgrowths forming bacilli and then multiply evoking the appearance of antibacillary antibodies . In persons with no tumours there emerge only antispore antibodies . Since the spores and bacilli of this strain have antigens of a different nature their antibodies are encountered separately . The finding of antibacillary antibodies following injection of spores thus serves as an indirect proof for the presence of a malignant tumour . In experiments on animals (rats) with neurogenic tumours induced with ethylnitosole urea and also in patients with brain tumours this phenomenon has been studied in detail. Onderstepoort J Vet Res, 1975 Sep, 42(3), 91 - 8 The partial purification of Clostridium perfringens beta toxin; Worthington RW et al.; An attempt was made to purify Clostridium perfringens Beta toxin . Crude toxin prepared by ammonium sulphate precipitation of culture supernatants was purified by chromatography on Sephadex G50, Sephadex G100 and DEAE cellulose . This material, although highly purified was not homogeneous on polyacrylamide gel electrophoresis . It had a toxicity of 800 000 mouse MLDs/mg N, a typical protein absorption spectrum in the UV region, an iso-electric point of 5, 6 and the main component had a molecular mass of 42 000 +/- 2 000 (estimated by electrophoresis in sodium dodecyl sulphate containing polyacrylamide gels). Chem Phys Lipids, 1975 Sep, 15(1), 15 - 26 The activation of phospholipase C from Clostridium Welchii by quinine: an absolute requirement for calcium ions; Klein R et al.; Quinine activates the hydrolysis of phosphatidyl choline suspensions by phospholipase C (E.C . 3.1.4.3) obtained from Clostridium welchii . Low levels of calcium are an absolute requirement for this activation: Mg2+, Ba2+, Sr2+, and Zn2+ are ineffective . The induction period, or lag phase for this enzyme is dependent upon both calcium concentration and substrate interfacial surface area . At low concentrations (less then 50 muM) calcium ions affect the induction period but not the maximal rate of hydrolysis, whereas guinine predominantly affects the rate of hydrolysis by alterations in the surface charge carried by the substrate. Infect Immun, 1975 Sep, 12(3), 536 - 43 Affinity chromatography purification of Clostridium perfringens enterotoxin; Scott VN et al.; Anti-enterotoxin immunoglobulins immobilized on CH-Sepharose or CNBr-Sepharose were used for affinity chromatography purification of Clostridium perfringens enterotoxin . Cell extracts containing enterotoxin or partially purified toxin preparations were applied to the column and nonspecifically-bound protein was eluted . NaOH was used to elute specifically bound toxin . The purity of enterotoxin purified by Sephadex G-100 chromatography followed by affinity chromatography appears similar to toxin highly purified by conventional means . The procedure can be used successfully for the rapid (less than 2 h) purification of small amounts of enterotoxin. Ann Microbiol (Paris), 1975 Sep, 126(2), 175 - 80 {Comparative study of neuraminidases from "Diplococcus pneumoniae" and "Clostridium perfringens"}; Houdret N et al.; Neuraminidases have been purified from the culture medium of two microorganisms, one aerobic, Diplococcus neumoniae, the other anaerobic, Clostridium perfringens . The enzymatic properties of the 2 neuraminidases have been studied (pH optimum; effect of cations;