Acta Microbiol Pol, 1991, 40(1-2), 85 - 92 Comparative studies of serotype-specific Clostridium difficile strains; Meisel-Mikolajczyk F et al.; The following properties of serotype-specific Clostridium difficile strains were studied: toxigenicity, encapsulation, susceptibility to certain antibiotics, biochemical properties, enzymatic activity . No correlation between toxin titer and frequency of capsule production as well as serogroup affiliation and sensitivity to antibiotics was observed . The strain representative of serogroup C attracts attention because of its distinct properties. Microbiol Immunol, 1991, 35(2), 99 - 109 Isolation, and morphological and chemical properties of an autolysis-deficient mutant of Clostridium botulinum type A; Kinouchi T et al.; An autolysis-deficient mutant was isolated from Clostridium botulinum type A 190L by treatment with ethyl methanesulfonate . The cell wall prepared from the mutant autolyzed at much slower rate than that from the parent strain, accompanying with much less liberation of both amino terminals and reducing groups . Electron microscopic observation revealed that the mutant strain was converted to short rod or curved spherical form with thickened cell walls when the growth temperature was shifted from 37 to 45 C . The mutant had a significantly larger amount of non-peptidoglycan-carbohydrate complexes than did the parent strain and became markedly resistant to the autolysin partially purified from the parent, compared with the parent strain . Furthermore, the mutant was fairly tolerant to killing by penicillin . These results suggest that the autolysis deficiency of the mutant was due not only to the deficient production of autolysin but also to the excess accumulation of carbohydrate in the cell wall. Infect Immun, 1991 Jan, 59(1), 73 - 8 Toxin A of Clostridium difficile binds to the human carbohydrate antigens I, X, and Y; Tucker KD et al.; Clostridium difficile causes pseudomembranous colitis in humans . The enterotoxin (i.e., toxin A) from this organism is believed to be responsible for the initial intestinal pathology associated with this disease . Previous work shows that this toxin binds to carbohydrates that contain Gal alpha 1-3Gal beta 1-4GlcNAc . However, this carbohydrate is not present on normal human cells . Therefore, this study was undertaken to identify potential receptors for toxin A that do exist on human intestinal epithelium . Using enzyme-linked immunosorbent assay, affinity chromatography, and altered migration in an electric field, we assayed the binding of toxin A to purified carbohydrates and glycoproteins . We found that toxin A bound to the carbohydrate antigens designated I, X, and Y . Each of these carbohydrates exist on the intestinal epithelium of humans. C R Seances Soc Biol Fil, 1991, 185(1-2), 78 - 83 {Anacollagenase by the action of amitriptyline on Clostridium histolyticum collagenase}; Warter J et al.; Intraperitoneal injection of a mixture of collagenase (300 U) and amitriptyline (Laroxyl*, 3 mg) induce no lesions in contrast with the severe effects of collagenase alone . Also, a complete resistance to intraperitoneal collagenase injection is observed when preceded by 3 intramuscular injections of the same mixture (associated with Freund's incomplete adjuvant) . This is due to the development of collagenase antibodies, as demonstrated by nephelometry and immunodiffusion . These facts show that amitriptyline neutralizes the enzymatic properties of collagenase, without alterring its antigenicity . We propose to call this new substance anacollagenase . Such a phenomenon has never been observed with a drug . However we got identical results with other tricyclic depressants (clomipramine, imipramine, doxepine, iprindole) . The mechanism of the transformation of collagenase into anacollagenase is not yet explained. J Cancer Res Clin Oncol, 1991, 117 Suppl 4, S198 - 202 Cisplatin/etoposide/ifosfamide stepwise dose escalation with concomitant granulocyte/macrophage-colony-stimulating factor for patients with far-advanced testicular carcinoma; Harstrick A et al.; In order to develop a more dose-intensive induction regimen for the treatment of far-advanced testicular tumours, the German Cooperative Group for Testicular Tumours started a dose-escalation trial of cisplatin, etoposide and ifosfamide . At the first dose level 18 patients with advanced testicular cancer (Indiana University classification) received cisplatin 25 mg/m2, etoposide 120-150 mg/m2 and ifosfamide 1.2 g/m2 for 5 days every 3 weeks . Of these, 13 patients (72%) became tumour-free, 2 achieved a stable, marker-negative partial remission, 2 had progressive disease and 1 patient died of Clostridium sepsis . The main toxicity was myelosuppression with a white blood cell nadir of 900/microliters and a thrombocyte nadir of 47,000/microliters . Granulocytopenic fever occurred in 43% of all cycles . At the second dose level 15 patients received cisplatin 30 mg/m2, etoposide 150 mg/m2 and ifosfamide 1.6 g/m2 five times every 3 weeks together with s.c . recombinant granulocyte/macrophage-colony-stimulating factor (GM-CSF) 10 micrograms/kg on days 6-15 . Acute toxicity was severe with a white blood cell nadir of 300/microliters and thrombocyte nadir of 11,000/microliters . The duration of the thrombocytopenia increased with cycle number; 63% of all cycles were associated with granulocytopenic fever and in 83% platelet transfusions were required . One patient died from acute renal failure and Aspergillus sepsis; 3 patients experienced adverse reactions to GM-CSF, requiring omission of this drugs in 2; 33% had grade 3 or 4 mucositis . At this dose level 8 patients (53%) became tumour-free, 4 patients (26%) had marker normalization with irresectable residual disease and 2 patients were treatment failures . Though acute toxicity was severe at this dose level, there was no unexpected or unmanageable organ toxicity and thus patients are now entered at dose level 3, which consists of cisplatin 30 mg/m2, etoposide 200 mg/m2 and ifosfamide 1.6 g/m2 for 5 days and GM-CSF 10 micrograms kg-1 day-1 on days 6-15 s.c. J Biol Chem, 1990 Dec 5, 265(34), 20807 - 12 Guanine nucleotide-dependent ADP-ribosylation of soluble rho catalyzed by Clostridium botulinum C3 ADP-ribosyltransferase . Isolation and characterization of a newly recognized form of rhoA; Williamson KC et al.; Two C3 ADP-ribosyltransferase substrates with different characteristics were isolated from bovine brain cytosol . Amino acid sequences of tryptic peptides from the two substrates were identical to rhoA and rhoB; hence, the purified proteins are referred to as rhoA* and rhoB*, respectively . Soluble rhoA* exhibits properties different from those previously reported for rho proteins . In contrast to other C3 substrates, rhoA* behaved as a 77-80-kDa protein on gel filtration, although on sodium dodecyl sulfate-polyacrylamide gel electrophoresis the ADP-ribosylated moiety had a mobility consistent with a 21.5-kDa protein . Furthermore, C3-catalyzed ADP-ribosylation of rhoA* was dependent on guanine nucleotides in the presence of 1 mM Mg2+ or 1 mM EDTA (0.19 microM free Mg2+) . Half-maximal stimulation by GTP, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), guanylyl-imidodiphosphate (Gpp(NH)p), and GDP was observed at 16, 20, 220, and 380 nM, respectively; guanosine 5'-O-(2-thiodiphosphate), GMP, and adenine nucleotides were ineffective . In the presence of GTP gamma S, the rate and extent of ADP-ribosylation was enhanced by dimyristoylphosphatidylcholine and/or cholate . This increase in ADP-ribosylation was specific for rhoA*; it was not observed with rhoB* and has not been reported for other C3 substrates . These distinct properties suggest that rhoA* is a newly recognized type of C3 substrate, differing from the rhoA-like proteins previously reported . rhoB*, on the other hand, has properties similar to those reported for membrane-associated rhoB and its ADP-ribosylation was independent of guanine nucleotides in the presence of 1 mM Mg2+ and not affected by dimyristoylphosphatidylcholine and/or cholate. Changgeng Yi Xue Za Zhi, 1990 Dec, 13(4), 296 - 303 {High incidence of tetanus in the middle-aged and elderly}; Chen FC et al.; Tetanus is a disease which has a high mortality rate, caused by clostridium tetani . It presents with trismus, difficulty in swallowing, and more ominously difficulty in breathing, or opisthotonus . Included in this retrospective study are 23 cases of tetanus treated in Chang Gung Memorial Hospital between Jan . 1986 and Dec . 1989 . Age ranged from 36 to 87, with the highest incidence in the middle-aged and elderly (91% older than 40 years of age) . In 20 cases a wound was found as the source of infection, most of which were minor injuries . Of the nine patients who received no medical attention, three received herb drugs . Of the eleven patients who did seek medical attention at the time of injury, four received tetanus toxoid but none of them received human tetanus immunoglobulin . Tetanus is a totally preventable disease . Prevention includes three basic principles: local wound management, active immunization, and passive immunization . When treating injured patients, it is important to recall that many middle-aged and older adults are not adequately immunized against tetanus . Such patients should receive human tetanus immunoglobulin as well as tetanus toxoid. J Clin Microbiol, 1990 Dec, 28(12), 2804 - 5 Quality control criteria for testing the susceptibility of anaerobic bacteria to meropenem; Zabransky RJ et al.; Reference values for quality control of in vitro susceptibility tests with meropenem against anaerobic bacteria were determined in a multilaboratory study by the approved National Committee for Clinical Laboratory Standards agar dilution method for the four quality control strains . The study protocol also included the evaluation of microdilution testing, medium additives, and multiple lots of media . The recommended MIC control ranges for three of the control organisms are as follows: Bacteroides fragilis ATCC 25285, 0.06 to 0.125 micrograms/ml; Bacteroides thetaiotaomicron ATCC 29741, 0.125 to 0.5 micrograms/ml; and Eubacterium lentum ATCC 43055, 0.125 to 0.5 micrograms/ml . The modal MIC for Clostridium perfringens ATCC 13124 was at or below the lowest concentration of meropenem tested, and no values are recommended. J Perinatol, 1990 Dec, 10(4), 351 - 6 Bacteremia due to anaerobic bacteria in newborns; Brook I; Awareness of the role of anaerobic bacteria in neonatal bacteremia has increased in recent years . The incidence of recovery of anaerobes in neonatal bacteremia varies, according to different studies, between 1.8% and 12.5% . Of the 178 cases reported in the literature, 73 were due to Bacteroides species (69 were the Bacteroides fragilis group), 57 Clostridium species (mostly Clostridium perfringens), 35 Peptostreptococci, 5 Propionibacterium acnes, 3 Veillonella species, 3 Fusobacterium species, and 2 Eubacterium species . Predisposing factors were perinatal maternal complications (especially premature rupture of membranes and chorioamnionitis), prematurity, and necrotizing enterocolitis . Organisms similar to those recovered in blood were concomitantly isolated in lung aspirates and cerebrospinal and peritoneal fluids . The overall mortality noted is 26% and is highest with B fragilis group (34%) . Inappropriate antimicrobial therapy was often a contributory factor for such mortality . Correction of underlying pathology, surgical drainage, and the use of proper antimicrobials are critical to successful resolution of the infection . Penicillin G is the drug of choice for anaerobic infection other than beta-lactamase-producing Bacteroides . Antimicrobials useful for therapy of beta-lactamase-producing Bacteroides include clindamycin, metronidazole, chloramphenicol, imipenem-cilastatin, and the combination of a penicillin plus a beta-lactamase inhibitor. J Med Microbiol, 1990 Dec, 33(4), 207 - 15 12th C . L . Oakley lecture . Pathogenesis of Clostridium difficile infection of the gut; Borriello SP; On the basis of the above findings it is possible to propose a sequence of events following exposure to C . difficile . Exposure of neonates to C . difficile leads to transient colonisation which is almost invariably asymptomatic; the reasons why colonisation is asymptomatic are not known . Exposure of antibiotic-treated adults to C . difficile does not invariably lead to colonisation; however, in those instances where colonisation occurs, it may be transient and asymptomatic or transient and symptomatic . The transient nature of the colonisation could be because the infecting strain is poorly virulent, or because the degree of compromise of the intestinal flora is insufficient to permit establishment and full expression of virulence . It is likely that it is easier to fully compromise the intestinal flora of the elderly so that they more readily become fully susceptible to colonisation by C . difficile . In a fully susceptible host and with a highly virulent strain, the following sequence of events could occur . The organism may associate with the intestinal mucosa possible via fimbriae, and form a microcolony of capsulate cells protected by a glycocalyx . The toxins, or other factors, produced may facilitate the interaction with mucosa and toxin A will result in increased vascular and mucosal permeability resulting in intra-luminal accumulation of serum-albumin-rich fluid . Although C . difficile does not appear to be capable of using serum albumin nutritionally, it may utilise other serum proteins, and the serum proteins in general may compete with host proteases and help prevent degradation of the toxins produced.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1990 Dec, 172(12), 6907 - 18 Cloning, sequencing, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum; Gerischer U et al.; Acetoacetate decarboxylase (ADC) (EC4.1.1.4) of Clostridium acetobutylicum DSM 792 was purified to homogeneity, and its first 25 N-terminal amino acids were determined . Oligonucleotide probes deduced from this sequence were used to detect positive clones in partial gene banks derived from Sau3A and HaeIII digests with following ligation into the vector pUC9 . In Escherichia coli, the 2.1-kbp HaeIII clones expressed high levels of ADC activity . The expression was independent of the orientation of the insert with respect to the lac promoter of the vector and also of the addition of isopropyl-beta-D-thiogalactopyranoside, thus indicating that sequences located on the clostridial DNA controlled transcription and translation . From the E . coli clone with the recombinant plasmid pUG93 containing the 2.1-kbp HaeIII fragment, the ADC protein was purified and compared with the native enzyme . Both were indistinguishable with respect to the molecular mass of subunits and native protein as well as to activity stain . The 2.9-kbp Sau3A fragment could be shown to contain the amino terminus of the acetoacetate decarboxylase (adc) gene but did not express enzyme activity . It partially overlapped with the HaeIII fragment, spanning together 4,053 bp of the clostridial genome that were completely sequenced . Four open reading frames (ORFs) could be detected, one of which was unambiguously assigned to the acetoacetate decarboxylase (adc) gene . Amino acid sequences of the N terminus and the catalytic center as deduced from the nucleotide sequence were identical to sequences obtained from direct analysis of the protein . Typical procaryotic transcriptional and translational start and stop signals could be found in the DNA sequence . Together with these regulatory sequences, the adc gene formed a single operon . The carboxyl terminus of the enzyme proved to be rather hydrophobic . In vitro transcription-translation assays resulted in formation of ADC and ORF3 gene product; the other two ORFs were not expressed . Whereas no homology of the adc gene and ORF2 could be detected with sequences available in the EMBL or GenBank data bases, the obviously truncated ORF1 showed significant similarity to alpha-amylase of Bacillus subtilis . The restriction pattern and N-terminal amino acid sequence (as deduced from the nucleotide sequence) of ORF3 proved to be identical to those of the large subunit of acetoacetyl coenzyme A:acetate/butyrate:coenzyme A transferase. Arch Dis Child, 1990 Dec, 65(12), 1338 - 9 Microbiological studies of the enterocolitis of Hirschsprung's disease; Wilson-Storey D et al.; The results of a prospective study of 20 cases of newly diagnosed Hirschsprung's disease (nine of whom developed enterocolitis) and 10 normal controls showed no variations in the bacterial flora (including Clostridium difficile) in the stools of the groups studied . Viral studies showed that rotavirus was present in the stools of seven of the nine cases of enterocolitis during the episode . We suggest that Hirschsprung's enterocolitis may have a complex infective aetiology and that rotavirus plays a part. Int J Food Microbiol, 1990 Dec, 11(3-4), 271 - 7 Assay in mice for low levels of Clostridium botulinum toxin; Takahashi M et al.; When botulinum toxin at a low level such as 0.1 to 1.0 mouse intraperitoneal LD50 was injected subcutaneously into a mouse at the inguinocrual region, abdominal ptosis with local palsy developed . If this symptom is taken as a marker, 1.0 mouse intraperitoneal LD50 can be detected within 6 h and 0.1 LD50 within 24 h . The severity of symptoms and the time-to-death in days after injection of toxin were converted into scores to quantify the toxic activity . Over a wide range of dose, between 0.075 and 38.4 mouse intraperitoneal LD50, a linear relationship was obtained between the log dose and the score . By use of this method, low levels of toxin such as 0.1 mouse intraperitoneal LD50 can be titrated accurately and easily. Jpn J Med Sci Biol, 1990 Dec, 43(6), 219 - 31 Comparative studies on Clostridium botulinum type A strains associated with infant botulism in Japan and in California, USA; Tabita K et al.; Twenty strains of Clostridium botulinum type A associated with infant botulism cases, six in Japan and 14 in California, USA, were compared in their characters . All six Japanese strains produced medium-sized progenitor toxin (M toxin; Mr 300 k) but no hemagglutinin and showed lower 50% infective doses (ID50) in the infant mouse test; whereas most American strains produced large-sized progenitor toxin (L + LL toxins; Mr 500 k and 900 k) and hemagglutinin in addition to M toxin and showed higher ID50 in infant mice . No marked difference in the biochemical properties was found between the two groups except for two American strains. Int J Food Microbiol, 1990 Dec, 11(3-4), 231 - 41 Distinct characters of Clostridium botulinum type A strains and their toxin associated with infant botulism in Japan; Sakaguchi G et al.; Four strains of Clostridium botulinum type A having been associated with infant botulism in Japan, and another strain isolated from honey not associated with infant botulism, were found to be hemagglutinin (HA) negative . These strains do not produce L (Mr 500 kDa) nor LL toxin (Mr 900 kDa) but M toxin (Mr 300 kDa) only . No marked difference was found between the HA-positive and HA-negative strains in other biochemical properties, but the HA-negative strains tended to colonize more easily in the intestines of infant mice than did HA-positive strains . The toxin of HA-positive strains and that of HA-negative strains differed in the antigenicity of part of the toxic component and that of the nontoxic component, and in the molecular size of the toxic component. Br J Clin Pract, 1990 Dec, 44(12), 709 - 10 Clostridium septicum gas gangrene following intramuscular infection from an influenza vaccine booster; Thomas MG; We have reported a case of C septicum gas gangrene occurring in the forearm of a patient who had an Influvac influenza booster 48 hours before admission . Despite prompt antibiotic treatment and surgical debridement, the patient became hypotensive and could not be resuscitated. Jpn J Med Sci Biol, 1990 Dec, 43(6), 233 - 7 Attempts to quantity Clostridium botulinum type A toxin and antitoxin in serum of two cases of infant botulism in Japan; Takahashi M et al.; Serum samples taken from two infant botulism cases during hospitalization were titrated for botulinum toxin by both the intraperitoneal (ip) injection method and the score method in mice . By the ip method, in which death is the only parameter, such low levels of toxin as lower than 4 ip LD50/ml may not be titrated even though the surviving mice show abdominal palsy . By the score method based on the degree of abdominal palsy, such low levels of toxin as 1.1 and 0.8 ip LD50/ml were detected in specimens of one of the patient's serum . No antitoxin was demonstrated in either case of infant botulism by applying the score method . It is not known whether spontaneous recovery from infant botulism is due to the antitoxin production. FEMS Microbiol Rev, 1990 Dec, 7(3-4), 383 - 9 Metabolism and energy generation in homoacetogenic clostridia; Hugenholtz J et al.; Clostridium thermoautotrophicum and C . thermoaceticum contain an anaerobic electron transport chain . It involves hydrogen and carbon monoxide as electron donors and, presumably, methylenetetrahydrofolate as physiological electron acceptor . Cytochrome b554, cytochrome b559, menaquinone, a flavoprotein, ferredoxin and rubredoxin are parts of the electron transport chain . The electron transport results in the generation of a proton motive force which drives the synthesis of ATP or the uptake of amino acids. Appl Environ Microbiol, 1990 Dec, 56(12), 3760 - 5 Cloning of a lactate dehydrogenase gene from Clostridium acetobutylicum B643 and expression in Escherichia coli; Contag PR et al.; A lactate dehydrogenase (LDH) gene of Clostridium acetobutylicum B643 was cloned on two recombinant plasmids, pPC37 and pPC58, that were selected by complementation of Escherichia coli PRC436 (acd), a fermentation-defective mutant that does not grow anaerobically on glucose . E . coli PRC436(pPC37) and PRC436(pPC58) grew anaerobically and fermented glucose to mostly lactate . When pPC37 and pPC58 were transformed into E . coli FMJ39 (ldh pfl), an LDH-deficient strain, the resulting strains grew anaerobically on glucose and produced lactate . Crude extracts of E . coli FMJ39(pPC37) and FMJ39(pP58) contained high LDH activity only when assayed for pyruvate reduction to lactate, and the LDH activity was activated 15- to 30-fold by the addition of fructose 1,6-diphosphate (FDP) . E . coli FMJ39 had no detectable LDH activity, and E . coli LDH from a wild-type strain was not activated by FDP . Maxicell analysis showed that both plasmids pPC37 and pPC58 expressed a protein with an apparent Mr of 38,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Restriction endonuclease mapping of pPC37 and pPC58 and DNA hybridization studies indicated that a 2.1-kb region of these two clones of C . acetobutylicum DNA encodes the FDP-activated LDH. Appl Environ Microbiol, 1990 Dec, 56(12), 3634 - 42 Purification and characterization of acidolysin, an acidic metalloprotease produced by Clostridium acetobutylicum ATCC 824; Croux C et al.; Acidolysin an extracellular protease produced by Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography with a recovery of 91% . The enzyme was a monomeric protein with a molecular weight of 44,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an acidic isoelectric point of 3.3 . Acidolysin was very sensitive to metal-chelating agents and phosphoramidon and was unaffected by sulfhydryl reagents . It was shown to be a calcium- and zinc-containing protease . It exhibited optimal activity against Azocoll at pH 5 and 45 degrees C . It was stable at low pH and heat labile above 50 degrees C . It exhibited specificity toward peptide bonds formed by the amino group of hydrophobic amino acids (isoleucine, leucine, and phenylalanine) and its NH2-terminal amino acid sequence showed a high degree of similarity with that of Bacillus subtilis neutral metalloprotease A . Acidolysin is the first phosphoramidon-sensitive, acidic zinc metalloprotease reported. Eur J Cell Biol, 1990 Dec, 53(2), 357 - 63 Microfilament-disrupting Clostridium difficile toxin B causes multinucleation of transformed cells but does not block capping of membrane Ig; Shoshan MC et al.; The effects of Clostridium difficile toxin B on some actin-dependent cellular functions were studied . A three-day incubation of intoxicated B-lymphocytes and transformed 3T3 fibroblasts resulted in dose-dependent multinucleation . Using vimentin-negative Daudi cells we showed that this effect of toxin B does not involve vimentin . As DNA and protein syntheses are not impaired in the cells used, the results suggest that toxin B has an effect on the actin-containing contractile ring during mitosis, in a manner similar to that of the microfilament-disrupting agent cytochalasin B . Toxin B is the first bacterial toxin shown to have this effect . It was also found that the capping of surface IgM on B-lymphocytes was not inhibited by toxin B, whereas cytochalasin B did inhibit capping . These results suggest that capping is dependent on a specific membrane-associated actin structure, which is not affected by toxin B. Gene, 1990 Nov 30, 96(1), 107 - 13 Clostridium difficile toxin A carries a C-terminal repetitive structure homologous to the carbohydrate binding region of streptococcal glycosyltransferases; von Eichel-Streiber C et al.; A detailed analysis of the 8130-bp open reading frame (ORF) of gene toxA and of an upstream ORF designated utxA, indicates the presence of a transcription terminator stem-loop for toxA, promoter sequences, and Shine-Dalgarno boxes for toxA and utxA . No transcription terminator between toxA and utxA is suggested by the sequence . ToxA contains two domains, one-third (C-terminal) with a repetitive structure and the residual two-thirds with no repetitions . The 2499-bp sequence encoding the repetitive structure is composed of nine groups of different short repetitive oligodeoxyribonucleotides (SRONs) . A combination of these SRONs codes for five groups of combined repetitive oligopeptides (CROPs) . Seven 50-amino acid (aa) CROPs and 23 CROPs of 21 aa in length are noticed . The CROPs are generally highly conserved, but four exhibit variability and possibly represent 'hot spots' of the repetitive structure . The reactivity of the C-terminal repeat with monoclonal antibody 1337C8 indicates that this part contains the carbohydrate-binding domain of ToxA . In this region homology exists between the ToxA repeats and the glucosyltransferases of Streptococci . We propose that binding of ToxA to cells occurs via the C-terminal repeat domain, with the N-terminal domain being responsible for toxic function. Eur J Biochem, 1990 Nov 26, 194(1), 237 - 41 ADP-ribosylation of actin isoforms by Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin; Mauss S et al.; The substrate specificities of the actin-ADP-ribosylating toxins, Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin were studied by using five different preparations of actin isoforms: alpha-skeletal muscle actin, alpha-cardiac muscle actin, gizzard gamma-smooth muscle actin, spleen beta- and gamma-cytoplasmic actin, and aortic smooth muscle actin containing alpha- and gamma-smooth muscle actin isoforms . C . perfringens iota toxin ADP-ribosylated all actin isoforms tested, whereas C . botulinum C2 toxin did not modify alpha-skeletal muscle actin or alpha-cardiac muscle actin . Spleen beta/gamma-cytoplasmic actin and gizzard gamma-smooth muscle actin were substrates of C . botulinum C2 toxin . In the aortic smooth muscle actin preparation, gamma-smooth muscle actin but not alpha-smooth muscle actin was ADP-ribosylated by C . botulinum C2 toxin . The data indicate that, in contrast to C . perfringens iota toxin, C . botulinum C2 toxin ADP-ribosylates only beta/gamma-cytoplasmic and gamma-smooth muscle actin and suggest that the N-terminal region of actin isoforms define the substrate specificity for ADP-ribosylation by C . botulinum C2 toxin. Eur J Biochem, 1990 Nov 26, 194(1), 185 - 98 Tertiary structure of two-electron reduced Megasphaera elsdenii flavodoxin and some implications, as determined by two-dimensional 1H-NMR and restrained molecular dynamics; van Mierlo CP et al.; The tertiary structure of the non-crystallizable two-electron-reduced Megasphaera elsdenii flavodoxin (15 kDa, 137 amino acid residues) has been determined using nuclear Overhauser enhancement restraints extracted from two-dimensional 1H-NMR spectra . A tertiary structure satisfying the experimental restraints very well (maximum NOE violation of 66 pm) was obtained with use of restrained molecular dynamics, using 509 distance restraints (including one non-NOE) on a starting structure modeled from the crystal structure of one-electron-reduced Clostridium MP flavodoxin . The protein consists of a central parallel beta-sheet surrounded on both sides by two alpha-helices . The flavin is positioned at the periphery of the molecule . The tertiary structure of the protein is highly defined with the exception of the flavin . The latter is expected to result from performing the restrained molecular dynamics simulation without water molecules and without proper charges on the flavin . The flavin, including the phosphate, the ribityl side chain and the isoalloxazine ring, is solvent accessible under the experimental conditions used and evidenced by a two-dimensional amide exchange experiment . This accessibility is expected to be important in the redox potential regulation of the semiquinone/hydroquinone couple of the protein . The amide exchange against deuterons and several typical line shapes in the two-dimensional NMR spectra are consistent with the structure generated . The structure is discussed in detail. FEBS Lett, 1990 Nov 26, 275(1-2), 168 - 72 Morphological alterations of Xenopus oocytes induced by valine-14 p21rho depend on isoprenylation and are inhibited by Clostridium botulinum C3 ADP-ribosyltransferase; Mohr C et al.; Microinjection of the constitutively active recombinant Val-14 p21rho A into Xenopus oocytes induced dramatic morphological changes with redistribution of pigments from the animal pole resulting in spotted oocytes . The effects induced by Val-14 p21rho A were regulated by progesterone in a dose-dependent manner whereas prior ADP-ribosylation of the rho protein blocked its activity . About 30 min after microinjection, p21 rho was associated with the plasma membrane . The membrane association of p21rho and its biological activity were inhibited by lovastatin, an inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A reductase . The findings suggest that membrane attachment and biological activity of p21rho depend on isoprenylation of the GTP-binding protein. Dtsch Med Wochenschr, 1990 Nov 16, 115(46), 1750 - 3 {Acute segmental hemorrhagic penicillin-associated colitis}; Mrowka C et al.; Four days after beginning a perioperative antibiotic prophylaxis with amoxicillin and clavulanic acid a 33-year-old patient developed an acute haemorrhagic diarrhoea with cramp-like lower abdominal pain . Coloscopy revealed diffuse mucosal oedema with map-like ulcerations, increased susceptibility to trauma and submucous haemorrhages extending from the middle of the ascending to the middle of the descending colon . Granulocytic inflammation with cryptal atrophy was seen histologically . Stool cultures, including tests for Clostridium difficile toxin, were normal . All symptoms disappeared within three days of discontinuing the medication . Coloscopy after one week revealed marked improvement, after three months nothing abnormal . Acute segmental haemorrhagic penicillin-associated colitis is rare and must be distinguished from antibiotic-associated pseudomembranous colitis. J Biol Chem, 1990 Nov 15, 265(32), 19429 - 33 A hybrid Azotobacter vinelandii-Clostridium pasteurianum nitrogenase iron protein that has in vivo and in vitro catalytic activity; Jacobson MR et al.; Site-directed mutagenesis and gene replacement procedures were used to construct a mutant strain of Azotobacter vinelandii which expresses a hybrid nitrogenase Fe protein . This hybrid Fe protein has its carboxyl-terminal 18 residues replaced with the 5 analogous residues from the Clostridium pasteurianum Fe protein sequence . The hybrid Fe protein is 13 amino acids smaller than the wild-type A . vinelandii Fe protein and has a net loss of 4 negatively charged residues, resulting in a change in size and charge . The strain which produces the hybrid Fe protein remained capable of diazotrophic growth, albeit at a reduced rate . Also, the purified hybrid Fe protein exhibited a maximum activity about one-half that of native Fe protein . These results demonstrate that the tight, inactive complex which is formed when A . vinelandii MoFe protein and C . pasteurianum Fe protein are mixed in heterologous reconstitution experiments cannot be accounted for only by differences in the A . vinelandii and C . pasteurianum Fe protein primary sequences located at their respective carboxyl termini. Biochemistry, 1990 Nov 13, 29(45), 10413 - 8 Site-directed alteration of the active-site residues of histidine decarboxylase from Clostridium perfringens; van Poelje PD et al.; To clarify the mechanism of biogenesis and catalysis by the pyruvoyl-dependent histidine decarboxylase (HisDCase) from Clostridium perfringens, 12 mutant genes encoding amino acid substitutions at the active site of this enzyme were constructed and expressed in Escherichia coli . The resulting mutant proteins were purified to homogeneity, characterized, and subjected to kinetic analysis . The results (a) exclude all polar amino acid residues in the active site except Glu-214 as donor of the proton that replaces the carboxyl group of histidine during decarboxylation and, since E214I and E214H are nearly inactive, indicate that Glu-214 is the essential proton donor; (b) demonstrate the importance to substrate binding of hydrophobic interactions between Phe-98, Ile-74, and the imidazole ring of histidine, and of hydrogen bonding between Asp-78 and N2 of the substrate; and (c) demonstrate a significant unidentified role for Glu-81 in the maintenance of the active-site structure . The proposed roles of these amino acid residues are consistent with those assigned on the basis of crystallographic evidence to the corresponding residues at the active site of the related HisDCase from Lactobacillus 30a {Gallagher, T., Snell, E . E., & Hackert, M . L . (1989) J . Biol . Chem . 264, 12737-12743} . Of the residues altered, only Ser-97 was essential for the autocatalytic serinolysis reaction by which this HisDCase, (alpha beta)6, is derived from its inactive, pyruvate-free precursor, proHisDCase, pi 6.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1990 Nov 13, 29(45), 10364 - 75 Structure and oxidation-reduction behavior of 1-deaza-FMN flavodoxins: modulation of redox potentials in flavodoxins; Ludwig ML et al.; Flavodoxins from Clostridium beijerinckii and from Megasphaera elsdenii with 1-carba-1-deaza-FMN substituted for FMN have been used to study flavin-protein interactions in flavodoxins . The oxidized 1-deaza analogue of FMN binds to apoflavodoxins from M . elsdenii and C . beijerinckii (a.k.a . Clostridium MP) with association constants (Ka) of 1.0 x 10(7) M-1 and 3.1 x 10(6) M-1, values about 10(2) less than the corresponding Ka values for FMN . X-ray structure analysis of oxidized 1-deaza-FMN flavodoxin from C . beijerinckii at 2.5-A resolution shows that the analogue binds with the flavin atoms in the same locations as their equivalents in FMN but that the protein moves in the vicinity of Gly 89 to accommodate the 1-CH group, undergoing displacements which increase the distance between position 1 of the flavin ring and the main-chain atoms of Gly 89 and move the peptide hydrogen of Gly 89 by about 0.6 A . The X-ray analysis implies that protonation of normal flavin at N(1), as would occur in formation of the neutral fully reduced species, would result in a similar structural perturbation . The oxidation-reduction potentials of 1-deaza-FMN flavodoxin from M . elsdenii have been determined in the pH range 4.5-9.2 . The oxidized/semiquinone equilibrium (E'0 = -160 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit; the semiquinone/reduced equilibrium (E'0 = -400 mV at pH 7.0) displays a pH dependence of -60 mV per pH unit at low pH and is pH independent at high pH, with a redox-linked pK of 7.4 . Spectral changes of fully reduced 1-deaza-FMN flavodoxin with pH suggest that this latter pK corresponds to protonation of the flavin ring system (the pK of free reduced 1-deaza-FMN is 5.6 {Spencer, R., Fisher, J., & Walsh, C . (1977) Biochemistry 16, 3586-3593} . The pK of reduced 1-deaza-FMN flavodoxin provides an estimate of the electrostatic interaction between the protein and the bound prosthetic group; the free energy of binding neutral reduced 1-deaza-FMN is more negative than that for binding the anionic reduced 1-deaza-FMN by 2.4 kcal.(ABSTRACT TRUNCATED AT 250 WORDS) Lancet, 1990 Nov 10, 336(8724), 1165 - 7 Impact of active immunisation against enteritis necroticans in Papua New Guinea; Lawrence GW et al.; Enteritis necroticans, known locally as pigbel, has been a major cause of illness and death among children in the highlands of Papua New Guinea . After a successful trial of active immunisation against the beta toxin of the causative organism, Clostridium perfringens type C, immunisation of children was begun in 1980 . The effects of the immunisation programme on pigbel admissions in 3 of the 5 major highland hospitals were assessed . In each of the centres studied the proportion of admissions due to enteritis necroticans dropped significantly after immunisation was introduced (p less than 0.001) and hospital admissions for pigbel in 1984-86, when immunisation was well established, were less than one fifth of previous figures. Biochim Biophys Acta, 1990 Nov 5, 1020(2), 115 - 45 The structure and mechanism of iron-hydrogenases; Adams MW; Hydrogenases devoid of nickel and containing only Fe-S clusters have been found so far only in some strictly anaerobic bacteria . Four Fe-hydrogenases have been characterized: from Megasphaera elsdenii, Desulfovibrio vulgaris (strain Hildenborough), and two from Clostridium pasteurianum . All contain two or more {4Fe-4S}1+,2+ or F clusters and a unique type of Fe-S center termed the H cluster . The H cluster appears to be remarkably similar in all the hydrogenases, and is proposed as the site of H2 oxidation and H2 production . The F clusters serve to transfer electrons between the H cluster and the external electron carrier . In all of the hydrogenases the H cluster is comprised of at least three Fe atoms, and possibly six . In the oxidized state it contains two types of magnetically distinct Fe atoms, has an S = 1/2 spin state, and exhibits a novel rhombic EPR signal . The reduced cluster is diamagnetic (S = 0) . The oxidized H cluster appears to undergo a conformation change upon reduction with H2 with an increase in Fe-Fe distances of about 0.5 A . Studies using resonance Raman, magnetic circular dichroism and electron spin echo spectroscopies suggest that the H cluster has significant non-sulfur coordination . The H cluster has two binding sites for CO, at least one of which can also bind O2 . Binding to one site changes the EPR properties of the cluster and gives a photosensitive adduct, but does not affect catalytic activity . Binding to the other site, which only becomes exposed during the catalytic cycle, leads to loss of catalytic activity . Mechanisms of H2 activation and electron transfer are proposed to explain the effects of CO binding and the ability of one of the hydrogenases to preferentially catalyze H2 oxidation. Scand J Gastroenterol, 1990 Nov, 25(11), 1151 - 60 Phospholipase activation and arachidonic acid release in intestinal epithelial cells from patients with Crohn's disease; Gustafson C et al.; A method for studying the mobilization of free arachidonic acid (AA) in viable isolated human intestinal epithelial cells has been developed and applied to the study of patients with Crohn's disease . Cells were isolated from morphologically unaffected parts of the distal ileum and incubated with 14C-AA; most of the incorporated 14C-AA was then found in phospholipids (mainly phosphatidylcholine) and in a pool of neutral lipids (mainly triacylglycerols) . Cells from patients with Crohn's disease incorporated more 14C-AA into their neutral lipids than did cells from control patients . When the labeled cells were stimulated with phospholipase C from Clostridium perfringens or with the calcium ionophore A23187, they released significant amounts of AA, mainly from phosphatidylcholine . There was no difference between cells from Crohn patients and controls in the 14C-AA amounts released, but unstimulated and phospholipase C-stimulated cells from prednisolone-treated Crohn patients released less AA than cells from control patients . The A23187-stimulated AA release was completely inhibited by the phospholipase A2 inhibitor 4-bromophenacyl bromide, whereas the phospholipase C-stimulated release was not . These findings suggest that AA release in human small-intestinal epithelial cells may be caused by calcium-mediated phospholipase A2 activation or by products of microbial phospholipase C activity and that prednisolone reduces the mobilization of free AA in intestinal epithelial cells . They also illustrate the potential use of isolated epithelial cells for revealing mechanisms underlying AA release in the intestinal mucosa in different disease states. J Infect, 1990 Nov, 21(3), 287 - 92 Prevalence of Clostridium difficile on a mixed-function ward for the elderly; Corrado OJ et al.; We studied the prevalence of Clostridium difficile over a period of 2 months on a mixed-function geriatric ward . Seven (14%) of the 49 patients were long-stay but the remainder were in hospital for acute illness or required a short period of active rehabilitation . Although 69% patients had recently received antibiotics, from only two (4%) was C . difficile isolated from their faeces . Our results show that C . difficile is not endemic in patients on all geriatric wards as has been previously suggested. Br Vet J, 1990 Nov-Dec, 146(6), 551 - 8 Obligately anaerobic bacterial species isolated from foot-rot lesions in goats; Piriz Duran S et al.; Lesions showing clinical signs of foot-rot from 120 goats were cultured on six selective media during October 1987 to November 1988 . A total of 582 strictly anaerobic microorganisms belonging to 50 different species was isolated and identified . The anaerobes most frequently isolated belonged to the following genera: Bacteroides (80%), Peptostreptococcus (63.6%), Megasphaera (40%), Fusobacterium (29.2%), Clostridium (22.5%), Propionibacterium (12.5%), Eubacterium (11.7%) and Leptotrichia (10.8%) . Percentages for Acidaminococcus, Peptococcus, Tissierella, Wolinella and Veillonella were below 10% . The following species were identified in 10% or more of cases: Peptostreptococcus anaerobius (61.7%), Bacteroides buccae (51.7%), Bacteroides nodosus (42.5%), Megasphaera elsdenii (40%), Bacteroides ruminicola subsp . brevis (22.5%), Fusobacterium necrophorum (19.2%), Leptotrichia buccalis (11.7%) and Clostridium perfringens (10%) . Lower percentages were obtained for the remaining species . The efficiency and selectivity of the six culture media used for isolation are discussed. Appl Environ Microbiol, 1990 Nov, 56(11), 3491 - 8 Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli; Petersen DJ et al.; In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone . We report here the purification of the enzyme from C . acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli . A bacteriophage lambda EMBL3 library of C . acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein . Phage DNA from positive plaques was analyzed by Southern hybridization . Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment . A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E . coli harboring the clostridial gene . Although the expression of the gene is tightly regulated in C . acetobutylicum, it was well expressed in E . coli, although from a promoter sequence of clostridial origin. Appl Environ Microbiol, 1990 Nov, 56(11), 3485 - 90 Regulation of protease production in Clostridium sporogenes; Allison C et al.; The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures . Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures . Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1 . In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis . This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP . Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process . It is concluded that protease production by C . sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. Arch Surg, 1990 Nov, 125(11), 1445 - 51 Aerobic and anaerobic bacteriology of wounds and cutaneous abscesses; Brook I et al.; The aerobic and anaerobic microbiologic characteristics of 584 wounds and 676 skin or soft-tissue abscesses were studied and correlated with the infection site . In wounds, aerobic or facultative bacteria only were present in 223 specimens (38%), anaerobes only in 177 specimens (30%), and mixed flora in 184 specimens (32%) . In total there were 1470 isolates, 558 aerobic and 912 anaerobic, an average of 2.5 isolates per wound (1.6 anaerobic and 0.9 aerobic isolates) . In abscesses, aerobic or facultative bacteria were recovered in 177 specimens (26%), anaerobes only in 243 specimens (36%), and mixed flora in 256 specimens (38%) . In total there were 1702 isolates, 602 aerobic and 1100 anaerobic, an average of 2.5 isolates per abscess . The highest rates of anaerobes in wounds were in the inguinal, buttocks, and trunk areas and in abscesses in the perirectal, external genitalia, neck, and inguinal areas . The predominant aerobic organisms were Staphylococcus aureus (363 isolates), group A streptococci (98 isolates), and Escherichia coli (97 isolates) . The predominant anaerobic organisms were Bacteroides species (986 isolates), Peptostreptococcus species (559 isolates), Clostridium species (153 isolates), and Fusobacterium species (109 isolates) . The predominance of certain isolates in certain anatomical sites was correlated with their distribution in the normal flora adjacent to the infected site . These data highlight the polymicrobial nature of wounds and cutaneous abscesses. Am Rev Respir Dis, 1990 Nov, 142(5), 1225 - 7 Colobronchial fistula: a rare complication of Crohn's colitis; Domej W et al.; A 29-yr-old white woman presented with chronic pneumonia in the left lower lobe and with left pleural effusion . She was known to have inflammatory bowel disease, but she was asymptomatic under maintenance treatment with 5-ASA . She received numerous antibiotic regimens according to susceptibility testing of microorganisms cultured from sputum and bronchial lavage and on an empiric basis was also given antituberculosis treatment, but there was no clinical improvement or change in the chest radiographic findings . Sputum was repeatedly examined and yielded, among other organisms, Clostridium inocuum, Enterobacter, Klebsiella pneumoniae, Proteus mirabilis, and Staphylococcus aureus . On one microscopic examination of sputum, the presence of feculent material was suspected . A subsequent gastrografin enema revealed a cologastric and colobronchial fistula between the splenic flexture of the colon and the greater curve of the stomach and the bronchial system . Segmental resection of the colon and resection of the lower lobe of the left lung were performed . Histologic findings of the resected colon were consistent with Crohn's disease . After a long period of postoperative recovery, the patient returned to good general health and well-being . To our knowledge, a colobronchial fistula caused by Crohn's colitis has not been previously reported. Am J Surg, 1990 Nov, 160(5), 535 - 9 Role of surgery in antibiotic-induced pseudomembranous enterocolitis; Morris JB et al.; With the increased use of prophylactic and broad-spectrum antibiotics, pseudomembranous colitis has emerged as a significant clinical problem . Management with specific anti-Clostridium difficile therapy (vancomycin or metronidazole) has reduced mortality to less than 2% . Nevertheless, the disease may progress to a fulminant toxic colitis or colonic perforation . Additionally, another subset of patients will present with a dramatic clinical picture, suggesting acute peritonitis, eventuating in unnecessary laparotomy . This report reviews both the medical and surgical literature during the past 15 years of patients treated for pseudomembranous colitis . Analysis of this clinical data has provided us with the opportunity to both define the role of surgery in this disorder and illustrate the necessity for a combined medical and surgical cooperative approach in the early management of this iatrogenic disease. Am J Public Health, 1990 Nov, 80(11), 1372 - 3 Garlic-in-oil associated botulism: episode leads to product modification; Morse DL et al.; In February 1989, three cases of botulism occurred in persons who consumed garlic bread made from a garlic-in-oil product . Testing of leftover garlic-in-oil showed it to have a pH of 5.7 and to contain high concentrations of Clostridium botulinum organisms and toxin . This was the second episode of botulism associated with a low acid garlic-in-oil product which needs constant refrigeration . In response, the Food and Drug Administration has taken steps to prevent a recurrence by requiring that microbial inhibitors or acidifying agents be added to such products. Mol Cell Biol, 1990 Nov, 10(11), 5679 - 87 Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme; Barlowe CK et al.; In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase . This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis . Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF . In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement . In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement . Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo . These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase . A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C . K . Barlowe and D . R . Appling, Biochemistry 29:7089-7094, 1990) . We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase. Trans R Soc Trop Med Hyg, 1990 Nov-Dec, 84(6), 875 - 9 Bacteriological studies of the venom and mouth cavities of wild Malayan pit vipers (Calloselasma rhodostoma) in southern Thailand; Theakston RD et al.; Venom and oropharyngeal swabs from freshly captured Malayan pit vipers (Calloselasma rhodostoma) in southern Thailand and captive specimens in England were cultured aerobically and anaerobically to identify the bacterial flora which might contaminate wounds inflicted by bites of this species . The snakes' mouths contained a wider range of organisms than their venoms, especially gut-related Gram-negative rods such as Enterobacter and Pseudomonas species and some staphylococci and clostridia . There were fewer positive cultures from captive snakes . C . rhodostoma venom inhibited the growth of group A streptococci and, to a lesser extent, that of Staphylococcus aureus and Clostridium perfringens but not that of 2 Gram-negative organisms . Secondary bacterial infection is an important complication of snake bite, especially of necrotic wounds . A combination of gentamicin with benzyl penicillin would have prevented infection with, or treated, most of the bacteria isolated from snake venoms and mouths in Thailand. J Biochem (Tokyo), 1990 Nov, 108(5), 811 - 5 Secondary structure of sphingomyelinase from Bacillus cereus; Tomita M et al.; Of the total of 306 amino acids in the sequence of sphingomyelinase (SMPLC) from Bacillus cereus, almost half (150) are expected to be involved in the formation of loop or turn structure, while 65 and 73 residues may participate in the formation of alpha-helix and beta-structure, respectively . The helix content of SMPLC was calculated to be 0-5%, based on the CD spectra . The addition of divalent metal ions such as Mg2+ or both Ca2+ and Mg2+ had no effect on the CD spectra of SMPLC, although the addition of these metal ions caused the breakdown of membranous SM and specific adsorption of SMPLC onto erythrocyte membranes . A hydropathy study showed that SMPLC has hydrophobic regions at the N-terminal domain which must be responsible for the binding of the enzyme to the membranes . The partial homologies between the amino acid sequences of SMPLC and Clostridium perfringens alpha-toxin (phospholipase C) are discussed. Biol Chem Hoppe Seyler, 1990 Nov, 371(11), 1077 - 82 Properties of 5-hydroxyvalerate CoA-transferase from Clostridium aminovalericum; Eikmanns U et al.; 5-Hydroxyvalerate CoA-transferase from Clostridium aminovalericum, strain T2-7, was purified approximately 100-fold to homogeneity . The molecular mass of the native enzyme was determined by three different methods to be 178 +/- 11 kDa; that of the subunit was 47 kDa, indicating a homotetrameric structure . The following CoA esters acted as substrates (decreasing specificity, V/Km): 5-hydroxyvaleryl-CoA greater than propionyl-CoA greater than acetyl-CoA greater than (Z)-5-hydroxy-2-pentenoyl-CoA greater than butyryl-CoA greater than valeryl-CoA . 4-Pentenoate and 3-pentenoate were also activated by acetyl-CoA to the corresponding CoA esters, whereas crotonate, (E)-5-hydroxy-2-pentenoate, (E)-2-pentenoate and 2,4-pentadienoate were not attacked . 5-Hydroxyvalerate CoA-transferase showed ping-pong kinetics and was inactivated by sodium boranate only in the presence of a CoA substrate . This indicated the formation of a thiolester between a specific carboxyl group of the enzyme and CoASH during the course of the reaction . The CoA-transferase was inhibited by ATP and CTP, slightly by ADP, GTP and UTP, but not by AMP . The inhibition by ATP was competitive towards CoA esters and noncompetitive towards acetate. FEMS Microbiol Lett, 1990 Nov, 60(3), 323 - 7 Plasmid copy number and stability determination in Clostridium perfringens transformants; Allen SP et al.; The copy number of the streptococcal plasmid pAM beta 1 (26.5 kb), and its deletion derivatives, pVA1 (11 kb) and pVA677 (7.6 kb) contained in Clostridium perfringens 3624A transformants was determined by incorporation of {methyl-3H}thymidine (4 muCi/ml) into chromosomal and plasmid DNA and sizing of the C . perfringens genome using transverse alternating field electrophoresis . Plasmids pAM beta 1, pVA1, and pVA677 were found to be present at 1.0, 97, and 216 copies/cell, respectively . 10.2, 54, and 96% of the initial pAM beta 1-, pVA1- and pVA677-containing transformants, respectively, remained resistant to erythromycin over 220 generations of growth . The results indicate a size-dependent relationship between plasmid stability and plasmid copy number in C . perfringens. FEMS Microbiol Lett, 1990 Nov, 60(3), 243 - 7 Binding of Clostridium botulinum type B toxin to rat brain synaptosome; Park MK et al.; Purified toxin and its subunits from Clostridium botulinum type B were labeled with 125iodine and binding of them to rat brain synaptosomes was studied . Labeled toxin and heavy chain were shown to bind to synaptosomes and there was no significant difference in the molar quantity of bound toxin and heavy chain at several concentrations of synaptosomes, whereas labeled light chain did not bind to synaptosomes . The binding of labeled heavy chain to synaptosomes was inhibited by unlabeled toxin and heavy chain to a similar degree as that of labeled toxin . The binding of labeled toxin and heavy chain to synaptosomes were inhibited by a monoclonal antibody which is specific for the heavy chain. Food Addit Contam, 1990 Nov-Dec, 7(6), 717 - 68 Nitrates, nitrites and N-nitrosocompounds: a review of the occurrence in food and diet and the toxicological implications; Walker R; Data on occurrence of nitrate, nitrite and N-nitrosocompounds in food and drinking water, and on total dietary intakes are reviewed . Metabolic, toxicological and epidemiological studies are surveyed and the implications with respect to safety evaluation are addressed . It is concluded that, on the basis of recent long-term animal studies and of clinical experience in man, the current Acceptable Daily Intake (ADI) allocated to nitrate by the Joint FAO/WHO Expert Committee on Food Additives of 0-5 mg/kg body weight/day (expressed as sodium nitrate) might be increased to 0-25 mg/kg body weight/day . Based on similar criteria, the ADI for nitrite would be 0-0.1 mg/kg body weight/day (expressed as sodium nitrite) . In view of the known carcinogenicity of N-nitrosocompounds, exposure to these compounds in food should be minimized by appropriate technological means, such as lowering the nitrite concentration in preserved foods to the minimum required to ensure microbiological safety and use of inhibitors of nitrosation like alpha-tocopherol or ascorbic acid . Further work is needed to define the minimal levels of nitrite in foods needed to inhibit outgrowth of Clostridium botulinum and toxin production. Vojnosanit Pregl, 1990 Nov-Dec, 47(6), 419 - 21 {Detection of Clostridium botulinum toxin in the blood of patients and in meat products}; Radosavljevic MK; Epidemiological data from the whole country reveal that insufficiently salted and dried hams are the main cause of the botulism in our country . In suspected hams as well as in a small number of patients' sera Cl Botulinum toxin type B was the most frequently found one up to the present time . Cl botulinum toxin was examined in our laboratory for several years and it was confirmed by the biologic test in 50% samples of the incriminated home made ham and in 35% patients' sera samples . Toxins type A and 1 were found in sera and in ham samples only toxin type B was found . It is suggested that consumers should be better informed concerning the risk of insufficiently processed delicatessen intake. J Biochem (Tokyo), 1990 Nov, 108(5), 879 - 85 Low-molecular-weight GTP-binding proteins serving as ADP-ribosylation substrate for ADP-ribosyltransferase from Clostridium botulinum and their relation to phosphoinositides metabolism in thymocytes; Wang P et al.; ADP-ribosyltransferase from Clostridium botulinum type C strain was found to induce an increase of inositol phosphates (IPs) formation in murine thymocytes membranes . Incubation of electropermeabilized murine thymocytes with the enzyme also caused an increase of IPs formation in the cells . This increase of IPs formation in the enzyme-treated membranes and electropermeabilized cells was dependent on the amount of both NAD and the enzyme, suggesting that the stimulation of phosphoinositide-specific phospholipase C (PLC) was related to ADP-ribosylation of membrane proteins by the enzyme . On the other hand, in calf and murine thymocytes two proteins with the same molecular weight of 21,000 were found to be ADP-ribosylated by the botulinum ADP-ribosyltransferase . A minor ADP-ribosylation substrate was shown by two-dimensional polyacrylamide gel electrophoresis to be G21k, a low-molecular-weight GTP-binding protein (G protein) suggested previously by us to be involved in PLC regulation {Wang, P . et al . (1987) J . Biochem . 102, 1275-1287; (1988) 103, 137-142; and (1989) 105, 461-466}, and the other major ADP-ribosylation substrate was identified as a rho A protein . Under the experimental conditions of the IPs formation study, ADP-ribosylation of both G21k and rho A proteins by botulinum ADP-ribosyltransferase in membranes and permeabilized cells was observed . These results suggest that botulinum ADP-ribosyltransferase-induced PLC stimulation in thymocytes is closely correlated with ADP-ribosylation of the low-molecular-weight G proteins. J Antimicrob Chemother, 1990 Nov, 26(5), 627 - 33 In-vitro activity of vancomycin, teicoplanin, daptomycin, ramoplanin, MDL 62873 and other agents against staphylococci, enterococci and Clostridium difficile; Bartoloni A et al.; The minimum inhibitory concentrations (MICs) of different antibiotics were determined by a broth microdilution method for staphylococci, enterococci and Clostridium difficile . The antimicrobial agents tested were vancomycin, teicoplanin, daptomycin, ramoplanin, MDL 62873, rifampicin and piperacillin, the latter limited to enterococci . In terms of MIC90S, daptomycin (0.89 mg/l) . MDL 62873 (0.99 mg/l), and teicoplanin (1.50 mg/l) were found to be highly active against methicillin-resistant Staphylococcus aureus (MRSA) . Daptomycin (MIC90 0.48 mg/l), MDL 62873 (0.95 mg/l) and ramoplanin (1.45 mg/l) were the most active drugs against methicillin-resistant S . epidermidis (MRSE) . Teicoplanin (MIC90 0.45 mg/l) was the most active agent against enterococci, followed by MDL 62873 (0.65 mg/l) and daptomycin (1.60 mg/l) . MDL 62873 gave the lowest MIC90 (0.17 mg/l) for C . difficile . Teicoplanin (MIC90 0.42 mg/l), daptomycin (0.87 mg/l) and ramoplanin (0.98 mg/l) were also very active . Our results indicate that teicoplanin, daptomycin, ramoplanin and MDL 62873, a teicoplanin derivative, are potentially effective alternative antibiotics for treatment of infections caused by staphylococci, enterococci and C . difficile. Gene, 1990 Oct 30, 95(1), 31 - 8 Sequences and homology analysis of two genes encoding beta-glucosidases from Bacillus polymyxa; Gonzalez-Candelas L et al.; The nucleotide sequences of the bglA and bglB genes encoding beta-glucosidases from Bacillus polymyxa have been determined . Both genes contain coding regions of 1344 bp, corresponding to polypeptides with Mrs of 51,643 and 51,547, respectively . Patterns of codon usage indicate that both genes are expressed at a low frequency . Previous data suggested that the proteins encoded by bglA and bglB were intra- and extracellular enzymes, respectively; however, neither of the two deduced amino acid sequences has N termini with the typical features of a leader peptide . The proteins encoded by bglA and bglB show remarkable homology to each other and to other beta-glucosidases (Bgl) and beta-galactosidases (beta Gal) . On the basis of the observed homologies, we can define two groups of microbial Bgl: one of them, type I, including most bacterial Bgl, and type II, including enzymes from different yeast species and one from Clostridium thermocellum . Likewise, at least two groups of beta Gal can be distinguished: type I, including enzymes homologous to type-I Bgl, and type II, showing no homology to any of the previous groups. Biochem J, 1990 Oct 15, 271(2), 351 - 5 A pH-dependent activation-inactivation equilibrium in glutamate dehydrogenase of Clostridium symbiosum; Syed SE et al.; 1 . On transferring Clostridium symbiosum glutamate dehydrogenase from pH 7 to assay mixtures at pH 8.8, reaction time courses showed a marked deceleration that was not attributable to the approach to equilibrium of the catalysed reaction . The rate became approximately constant after declining to 4-5% of the initial value . Enzyme, stored at pH 8.8 and assayed in the same mixture, gave an accelerating time course with the same final linear rate . The enzyme appears to be reversibly converted from a high-activity form at low pH to a low-activity form at high pH . 2 . Re-activation at 31 degrees C upon dilution from pH 8.8 to pH 7 was followed by periodic assay of the diluted enzyme solution . At low ionic strength (5 mM-Tris/HCl), no re-activation occurred, but various salts promoted re-activation to a limiting rate, with full re-activation in 40 min . 3 . Re-activation was very temperature-dependent and extremely slow at 4 degrees C, suggesting a large activation energy . 4 . 2-Oxoglutarate, glutarate or succinate (10 mM) accelerated re-activation; L-glutamate and L-aspartate were much less effective . 5 . The monocarboxylic amino acids alanine and norvaline appear to stabilize the inactive enzyme: 60 mM-alanine does not promote re-activation, and, as substrates at pH 8.8 for enzyme stored at pH 7, alanine and norvaline give progress curves showing rapid complete inactivation . 6 . Mono- and di-nucleotides (AMP, ADP, ATP, NAD+, NADH, NADP+, CoA, acetyl-CoA) at low concentrations (10(-4)-10(-3) M) enhance re-activation at pH 7 and also retard inactivation at pH 8.8 . 7 . The re-activation rate is independent of enzyme concentration: ultracentrifuge experiments show no changes in molecular mass with or without substrates . 8 . The activation-inactivation appears to be due to a slow pH-dependent conformational change that is sensitively responsive to the reactants and their analogues. J Appl Bacteriol, 1990 Oct, 69(4), 481 - 92 The combined effect of incubation temperature, pH and sorbic acid on the probability of growth of non-proteolytic, type B Clostridium botulinum; Lund BM et al.; It has been reported that non-proteolytic strains of Clostridium botulinum will grow at 3.3 degrees C, and they are therefore of concern in relation to certain chilled foods . The effects of combinations of inhibitory factors may be used to reduce the risk of growth of these bacteria in foods . The combined effect of pH values between 4.8 and 7.0, temperatures between 6 degrees and 30 degrees C, and sorbic acid concentrations up to 2270 mg/l on the probability of growth from a single spore of non-proteolytic, type B strains in a culture medium has been determined . A mathematical model has been developed that enables the effect of varying combinations of these factors on the probability of growth of non-proteolytic, type B Cl . botulinum to be predicted. Antimicrob Agents Chemother, 1990 Oct, 34(10), 2007 - 8 In vitro activity of LY 264826 compared with that of vancomycin against 100 clinical isolates each of methicillin-resistant Staphylococcus aureus and Clostridium difficile; Fasola EL et al.; The in vitro activity of LY 264826, a new glycopeptide antibiotic, was compared with that of vancomycin against 100 strains each of methicillin-resistant Staphylococcus aureus and Clostridium difficile . LY 264826 was more active, by weight, than vancomycin against the isolates tested . The human serum protein binding of LY 264826 was 15.3% (range, 9.8 to 21.8%). Biologicals, 1990 Oct, 18(4), 263 - 70 In vitro tests for the measurement of clostridial toxins, toxoids and antisera . II . Titration of Clostridium perfringens toxins and antitoxins in cell culture; Knight PA et al.; The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated . Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical . However, the cytopathic effects of the same preparations are caused by other entities . Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not . Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity . It follows that all three activities can be valid indicators for toxin neutralization tests . Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test . This test has, in turn, been shown to reflect the results of the mouse lethal test accurately . Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test . It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines. Appl Environ Microbiol, 1990 Oct, 56(10), 3040 - 6 Subcellulosome preparation with high cellulase activity from Clostridium thermocellum; Kobayashi T et al.; We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum . This "subcellulosome" preparation from C . thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin) . The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase . We could not further fractionate this preparation without denaturing it . The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase . The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside . Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only. FEMS Microbiol Lett, 1990 Oct, 60(1-2), 59 - 62 Effect of prior treatment with Clostridium perfringens epsilon toxin inactivated by various agents on lethal, pressor and contractile activities of the toxin; Nagahama M et al.; Lethal and pressor activities, and the contractile responses of rat isolated ileum to Clostridium perfringens epsilon toxin, were significantly prevented by the prior administration of epsilon toxin inactivated by 1-ethyl-3-(3-diethyl-aminopropyl) carbodiimide in the presence of glycine methyl ester (EDC), 2,4,6-trinitrobenzene sulfonic acid (TNBS), succinic anhydride (SA) and ethoxyformic anhydride (EFA) . However, the prior administration of the toxin inactivated by N-acetylimidazole (NAI), tetranitromethane (TNM) and N-bromosuccinimide (NBS) resulted in no inhibition of these biological activities . These data suggest that the toxin interacts with specific site(s) on target organs or tissues . The relationship between amino acid residues and the actions of the toxin is described. Lab Anim, 1990 Oct, 24(4), 353 - 7 Gnotobiotic, athymic mice; a possible system for the study of the role of bacteria in human amoebiasis; Owen DG; Two groups of 12 + 14 gnotobiotic, athymic mice were intracaecally injected with Entamoeba histolytica strain HK9 and NIH:200, respectively . Two groups of 16 and 15 mice were given amoebae together with a pure strain of Escherichia coli and a further two groups of 16 and 27 were given amoebae with a pure strain of Clostridium perfringens . Batches of 3-7 mice from each group were killed at intervals of 1-4 weeks . All the mice given NIH:200 alone were found to be infected with trophozoites . Of those given HK9 alone, 20% of the first and 57% of the second group to be examined were infected . Groups of mice given either strain of amoeba monocontaminated with E . coli were all found to be infected at post-mortem examination with no apparent clinical signs and little histological change . The group given HK9 and C . perfringens, although all were infected, failed to produce clinical signs or histological lesions, though some died expectedly . In the group given NIH:200 with C . perfringens the amoebae showed a change of activity and there was evidence of both caecal and liver lesions after 120 days . The usefulness of the system in studying the effect of individual species of bacteria on invasive amoebae is discussed. Dtsch Tierarztl Wochenschr, 1990 Oct, 97(10), 398 - 400 {Rapid diagnosis of botulism in cattle using heated microcomplement fixation reactions}; Weiss HE et al.; Clostridium botulinum toxin, type C, could be demonstrated by means of temperature induced microcomplement fixation in blood serum and in aspirated rumen fluid of cattle suffering from botulism . The results were already available after seven hours . Botulinum toxin likewise could be identified from hair of a suspicious carcass . The investigations confirm the high sensitivity of this method. West J Med, 1990 Oct, 153(4), 390 - 3 Botulism among Alaska Natives . The role of changing food preparation and consumption practices; Shaffer N et al.; Alaska Natives have one of the highest rates of food-borne botulism worldwide . All outbreaks have been associated with the consumption of native foods, but in recent years outbreaks have occurred in previously unaffected areas and have involved new food items . Five botulism outbreaks occurred between 1975 and 1985 in an area of southwestern Alaska without previous confirmed outbreaks and among one ethnic group, the Yupik Eskimo . Of the 5 outbreaks, 3 were associated with fermented beaver tail, a nontraditional native food recently introduced into the region . Preparation techniques vary widely within villages and among ethnic groups . Traditional fermentation techniques have changed over the past 50 years; current preparation methods used by some families and ethnic groups may be more favorable for Clostridium botulinum growth . Prevention efforts should be targeted at high-risk subgroups of Alaska Natives who appear to have modified traditional practices and increased their risk of food-borne botulism. Proc Natl Acad Sci U S A, 1990 Oct, 87(20), 7844 - 8 Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons; Mochida S et al.; Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters) . No evidence for expression of injected exogenous mRNA, however, has been reported in terminally differentiated neurons . If achieved, it would allow the study of long-lasting changes of properties of nerve cells in their functional context . To obtain evidence of such expression, we chose two proteins that produce a detectable effect even at very low intracellular concentrations . Tetanus toxin and botulinum neurotoxin fulfill this criterion, being the most potent neurotoxins known . Both toxins block neurotransmitter release at nanomolar intracellular concentrations . These di-chain proteins, consisting of a light chain and a heavy chain, have recently been sequenced . Their active sites are located (or partly located) on the light chain . mRNAs encoding the light chain of either toxin were transcribed in vitro from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica . Depression of neurotransmitter release appeared in less than 1 hr, demonstrating successful expression of foreign mRNA injected into a neuron in situ. J Clin Microbiol, 1990 Oct, 28(10), 2377 - 8 Clostridium septicum as a cause of pericarditis and mycotic aneurysm; Brahan RB et al.; Clostridium septicum is a bacterial species associated with gas gangrene in both humans and animals . Although not usually a pathogen in humans, it has been implicated in some cases of abscesses and bacteremia . We now report the first case of pericarditis with mycotic aneurysm due to C . septicum. J Clin Microbiol, 1990 Oct, 28(10), 2210 - 4 Characterization of flagella of Clostridium difficile and their role in serogrouping reactions; Delmee M et al.; Slide agglutination with rabbit antisera allows the differentiation of 10 serogroups of Clostridium difficile, namely, A, B, C, D, F, G, H, I, K, and X . Each serogroup displays a specific protein profile in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, except for A, which displays 12 different protein profiles (A1 to A12) . In the present work, electron microscopy revealed the presence of uniformly distributed flagella in the reference strains of serogroups G and K and in all strains representative of the 12 subgroups within serogroup purified by differential centrifugation . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these preparations revealed one distinct band with an apparent molecular mass of approximately 39 kilodaltons . Antiserum was prepared by immunizing a rabbit with the serogroup A flagellin, which had been eluted from the gel . In immunoblotting, this antiserum cross-reacted with the flagellin of the other strains . When the cells were deflagellated by a short sonication, the cross-reactions observed by slide agglutination with A, G, and K antisera were suppressed . Similarly, shearing of flagella allowed specific slide agglutination of the 12 subgroups of serogroup A. Infect Immun, 1990 Oct, 58(10), 3173 - 7 Cloning and complete nucleotide sequence of the gene for the main component of hemagglutinin produced by Clostridium botulinum type C; Tsuzuki K et al.; In Clostridium botulinum types C and D, phage conversion to toxin and hemagglutinin (HA) production has been reported . DNA was extracted from a converting type C Stockholm phage, c-st, and a fragment (7.8 kilobase pairs) coding for the parts of both toxin and HA was cloned . The gene for HA was recloned, and the complete nucleotide sequence was determined . The molecular mass of this gene product was 33 kilodaltons, and it showed HA activity . The HA preparation partially purified from a type C Stockholm culture demonstrated two major bands (33 and 53 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without reducing agent . The amino acid sequence of the N terminus of the 33-kilodalton component of the native HA preparation, which was determined by a direct protein microsequencing procedure, was identical to that deduced from the nucleotide sequence of cloned HA gene . These data indicate that the cloned gene product (33 kilodaltons) is an important component of HA. J Gen Microbiol, 1990 Oct, 136 ( Pt 10), 2077 - 87 Interactions of iron-thiol-nitrosyl compounds with the phosphoroclastic system of Clostridium sporogenes; Payne MJ et al.; Certain reagents, such as ascorbate or iron salts and thiols, enhance the bacteriostatic action of nitrite on food-spoilage bacteria . This may be due to the formation of nitric oxide and iron-thiol-nitrosyl {( Fe-S-NO}) complexes . The minimum concentrations of these reagents required to inhibit growth of Clostridium sporogenes were investigated . A mixture of nitrite (0.72 mM) with iron (1.44 mM) and cysteine (2.16 mM) was found to be extremely inhibitory when autoclaved and diluted into the culture medium . This mixture caused rapid cessation of growth and loss of cell viability at a final concentration corresponding to 40 microM-nitrite . If added to the initial culture medium, it prevented growth at 5 microM-nitrite . The mixture was more inhibitory, on the basis of the nitrite concentration used, than the 'Perigo factor', obtained by autoclaving nitrite in growth medium . {Fe-S-NO} compounds of known chemical structure were tested to determine if they were responsible for this effect . Total inhibition of cell growth was observed with the tetranuclear clusters {Fe4S3(NO)7} (Roussin's black salt), {Fe4S4(NO)4} or {Fe4Se3(NO)7}, added at concentrations equivalent to 10 microM-nitrite, or with {Fe2(SMe)2(NO)4} (methyl ester of Roussin's red salt), equivalent to 200 microM-nitrite . The rate of hydrogen production in growing cell cultures was inhibited by {Fe4S3(NO)7} at levels equivalent to 2.5 microM-nitrite . EPR spectra of the inhibited cells showed features with g-values of 2.03, characteristic of mononuclear iron-nitrosyl species, and, under non-reducing conditions, an unusual signal at g = 1.65 . There was no correlation between growth inhibition and the g = 2.03 signal, though there was a better correlation between inhibition and the g = 1.65 signal . The direct effects of the compounds were tested on the iron-sulphur proteins of the phosphoroclastic system, namely ferredoxin, pyruvate-ferredoxin oxidoreductase and hydrogenase . EPR spectra and enzyme assays showed that these proteins were not destroyed by {Fe4S3(NO)7}, {Fe4S4(NO)4}, {Fe2(SMe)2(NO)4}, {Fe(SPh)2(NO)2}, or M2 (an autoclaved mixture of 66 mM-cysteine, 3.6 mM-FeSO4 and 0.72 mM-NaNO2) at concentrations higher than those that caused total inhibition of cell growth . Inhibition of cells by {Fe-S-NO} compounds is unlikely to be due to interaction with the preformed enzymes . The possible formation of iron-nitrosyl complexes in vivo, and their inhibitory actions, are discussed. J Gen Microbiol, 1990 Oct, 136 ( Pt 10), 2067 - 76 Electron paramagnetic resonance spectroscopic investigation of the inhibition of the phosphoroclastic system of Clostridium sporogenes by nitrite; Payne MJ et al.; The proposal that nitrite exerts its inhibitory effect on anaerobic bacteria by direct interaction with the iron-sulphur proteins of the phosphoroclastic system was investigated . The effects of nitrate, nitrite with or without ascorbate, and nitric oxide on the growth of Clostridium sporogenes in liquid cultures at pH 7.4, on the rates of hydrogen production, and on the activities of the enzymes pyruvate-ferredoxin oxidoreductase and hydrogenase, and of ferredoxin were investigated . In agreement with previous studies, nitrate was the least effective inhibitor of cell growth, and nitric oxide the most effective . Nitrite reductase activity was very low in C . sporogenes, indicating that the presence of external reducing agents would be necessary for the reduction of nitrite to nitric oxide . Inhibition by nitrite was enhanced by ascorbate; 0.5 mM-nitrite with 10 mM-ascorbate stopped growth completely . In partially-purified preparations 4.1 mM-NaNO2 and equimolar ascorbate caused complete inactivation of hydrogenase activity but only partial (up to 78%) inactivation of pyruvate-ferredoxin oxidoreductase . This agreed with the loss of hydrogen production observed with nitrite in vivo . Inhibition occurred within 5 min, and was irreversible in each case . Electron paramagnetic resonance (EPR) spectroscopy showed that paramagnetic {Fe(NO)2(SR)2} species were formed during growth in the presence of nitrite, and were associated with cells . However, the intensity of these EPR signals did not correlate with the inhibition of cell growth . The {4Fe-4S} clusters in ferredoxin were shown by EPR spectroscopy to be resistant to treatment with 3.6 mM-NaNO2 and 3.6 mM-ascorbate . It is concluded that the effects of nitrite on pre-formed iron-sulphur proteins are not convincing as a basis for the lethal effects on bacterial cells. Hepatology, 1990 Oct, 12(4 Pt 1), 638 - 43 Growth of group A rotaviruses in a human liver cell line; Schwarz KB et al.; Recent observations in children with rotavirus gastroenteritis and in infant mice given rotavirus vaccine by oral administration suggest that this well-known gastrointestinal pathogen may infect the liver . To examine this possibility, the susceptibility of Hep G2 cells to infection with a variety of rotavirus strains was tested . These cells were used because they are considered to be well differentiated and exhibit many liver-specific functions . The Hep G2 cells supported the growth of the simian strain rhesus rotavirus (MMU 18006), a strain currently being used in vaccine trails, but did not support the growth of any human strain (D, DS1, Price or ST3) . The rhesus rotavirus infection was cytopathic and resulted in release of lactate dehydrogenase . Rhesus rotavirus growth in Hep G2 cells displayed trypsin-enhanced infectivity and was inhibited by pretreatment of cells with Arthrobacter ureafaciens neuraminidase but not with neuraminidase from Clostridium perfringens . Hep G2 cells were also permissive for another simian strain (SA11), a bovine strain (UK) and single gene substitution reassortants containing VP7 (the major outer capsid neutralization protein) from a human rotavirus strain and the remaining 10 genes from either rhesus rotavirus or UK . In general, UK and its reassortants produced lower levels of antigen than did rhesus rotavirus and its reassortants . Hep G2 cells and other hepatic cell lines may prove to be useful tools to explore the hepatotropic potential of wild-type rotaviruses and candidate vaccine strains. Int J Food Microbiol, 1990 Oct, 11(2), 167 - 78 Sodium hypophosphite inhibition of the growth of selected gram-positive foodborne pathogenic bacteria; Rhodehamel EJ et al.; Sodium hypophosphite (SHP) was evaluated for inhibition of growth of selected Gram-positive foodborne pathogenic bacteria in Trypticase Soy Broth . In addition, the effects of pH and sodium chloride (NaCl) alone and in combination with (SHP) were also examined . All inhibition studies were performed with optimal or nearly optimal growth conditions for each bacterium . Growth was monitored by determining culture optical density at 600 nm, and a time to significant growth determined for each test media . Ratios of time to significant growth for each control over that in test variables were used to evaluate the effect of SHP and other variables on growth . SHP was effective in inhibiting growth of Clostridium perfringens and Clostridium botulinum strains 62A 52A and Lamanna B, but generally ineffective against Staphylococcus aureus and Bacillus cereus . Results from this investigation show that SHP has potential as a food ingredient for the inhibition of certain Gram-positive foodborne pathogens. J Bacteriol, 1990 Oct, 172(10), 5901 - 7 Expression of an aromatic-dependent decarboxylase which provides growth-essential CO2 equivalents for the acetogenic (Wood) pathway of Clostridium thermoaceticum; Hsu TD et al.; The acetogen Clostridium thermoaceticum generates growth-essential CO2 equivalents from carboxylated aromatic compounds (e.g., 4-hydroxybenzoate), and these CO2 equivalents are likely integrated into the acetogenic pathway (T . Hsu, S . L . Daniel, M . F . Lux, and H . L . Drake, J . Bacteriol . 172:212-217, 1990) . By using 4-hydroxybenzoate as a model substrate, an assay was developed to study the expression and activity of the decarboxylase involved in the activation of aromatic carboxyl groups . The aromatic-dependent decarboxylase was induced by carboxylated aromatic compounds in the early stages of growth and was not repressed by glucose or other acetogenic substrates; nonutilizable carboxylated aromatic compounds did not induce the decarboxylase . The decarboxylase activity displayed saturation kinetics at both whole-cell and cell extract levels, was sensitive to oxidation, and was not affected by exogenous energy sources . However, at the whole-cell level, metabolic inhibitors decreased the decarboxylase activity . Supplemental biotin or avidin did not significantly affect decarboxylation . The aromatic-dependent decarboxylase was specific for benzoates with a hydroxyl group in the para position of the aromatic ring; the meta position could be occupied by various substituent groups (-H, -OH, -OCH3, -Cl, or -F) . The carboxyl carbon from {carboxyl-14C} vanillate went primarily to 14CO2 in short-term decarboxylase assays . During growth, the aromatic carboxyl group went primarily to CO2 under CO2-enriched conditions . However, under CO2-limited conditions, the aromatic carboxyl carbon went nearly totally to acetate, with equal distribution between the carboxyl and methyl carbons, thus demonstrating that acetate could be totally synthesized from aromatic carboxyl groups . In contrast, when cocultivated (i.e., supplemented) with CO under CO2-limited conditions, the aromatic carboxyl group went primarily to the methyl carbon of acetate. Jpn J Med Sci Biol, 1990 Oct, 43(5), 183 - 95 Incidence of Clostridium botulinum in honey of various origins; Nakano H et al.; By the dilution-centrifugation method, 270 honey samples, both domestic and imported, were examined and Clostridium botulinum was detected in 23 samples (8.5%); type A in 11 samples, type B in two, type C in 10, and type F in one . Of 58 domestic honey samples, six (10%) were positive; three gave type A and the other two type C . Among imported honey samples, Chinese honey gave 12% positives (types A, B, and C) and Argentina honey 20% positives (types A and F) . The incidence was higher with samples taken from drums (18%) and from apiaries (23%) than marketing honey (5%) . It was estimated that most positive samples contained spores in one per gram or lower concentrations . One sample contained 4 type A spores per gram and another 36-60 type F spores per gram . No distinct biochemical properties were found with the honey isolates. Microbiologica, 1990 Oct, 13(4), 323 - 8 Incidence of cytotoxin producing isolates of Clostridium difficile in faeces of neonates and children in Nigeria; Emeruwa AC et al.; One hundred and fifty six (156) confirmed isolates of Clostridium difficile from faeces of neonates and children in parts of Anambra State, Nigeria were screened and assayed for cytotoxin production by the tissue culture technique and the frequency of occurrence estimated . Twenty three out of 156 isolates were found to be cytotoxin positive isolates representing a frequency of 14.8% . There was no difference between the frequency of occurrence of cytotoxin positive isolates in neonates and children from rural and urban areas . Infants in the age group of one dy to 1 yr showed 16.7% frequency of occurrence of cytotoxin positive isolates, with toxin titers between 5 to 1280, 10% for children of 1-2 yrs age group, with titers between 5 to 40 and 8.3% for children of 2 to 3 yrs age group with titers of 5 and 10 . No cytotoxin positive isolate was detected from children of the 3 to 5 yrs age group . Children fed by formula foods alone showed a 50% frequency of occurrence of cytotoxin positive isolates, children fed by breast milk plus formula supplementation, 19.23% and breast milk alone 17.5% . There was no significant difference in the frequency of occurrence of cytotoxin positive isolates in faeces from diarrhoeal and non diarrhoeal cases . It appears therefore, that age and mode of feeding are important factors that influence intestinal colonization of cytotoxin producing isolates of C . difficile in neonates and children. Aktuelle Traumatol, 1990 Oct, 20(5), 254 - 6 {Clostridium perfringens infection following intramedullary nailing of an open femur shaft fracture}; Raunest J et al.; In a 47-year old patient, clostridial bacteraemia and local gas gangrene developed following osteosynthesis of a penetrating femoral fracture by a medullary nail . The operation had been performed in a post-injury interval of 14 days . The first clinical symptoms did not appear until 6 days after surgery . Operative treatment by large local incisions, debridement and drainage with the medullary nail left in situ accompanied by administration of penicillin G resulted in recovery . In the case presented here, local haematoma and debris due to the osteosynthesis procedure had probably induced a proliferation of clostridia leading to gas gangrene . The delay in development of signs and symptoms and the benign appearance of the wound resulted in delay in early diagnosis and appropriate treatment of this potentially life-threatening entity . Ultrasound proved to be a valuable tool in the evaluation of local changes . Even in performed in an appropriate postinjury interval, medullary nailing bears a potential risk of inducing a clostridial infection, so that the indication must be very strict. Am J Clin Pathol, 1990 Oct, 94(4), 410 - 6 Clostridium difficile invasion and toxin circulation in fatal pediatric pseudomembranous colitis; Qualman SJ et al.; The direct involvement of Clostridium difficile in the lesional tissue of pseudomembranous colitis has not been demonstrated; the organism's effects have been assumed to be strictly toxin mediated . Because C . difficile cytotoxin may be found incidentally in the intestinal lumina of asymptomatic infants, the role of the organism in a variety of pediatric intestinal diseases is uncertain . The authors studied seven cases of fatal pediatric pseudomembranous colitis in which the presence of C . difficile was uniformly demonstrable in lesional tissues with the use of both an intestinal spore stain and a specific immunostain . The patients had either underlying Hirschsprung's disease or hematologic malignancy; the striking pathologic features peculiar to these patients were altered mucosal mucin and immunologic barriers in the former group and neutropenia in the latter . Two patients had demonstrable circulating cytotoxin in serum or ascitic fluid, and C . difficile was identified invading colonic mucosa or submucosa . Such phenomena did not occur in control pediatric patients with multiple other intestinal lesions . Altered host factors may be responsible for the intestinal invasion of C . difficile and its systemic toxin circulation in cases of fatal pediatric pseudomembranous colitis. Clin Exp Immunol, 1990 Oct, 82(1), 33 - 7 Selective recognition of DNA antigenic determinants by murine monoclonal anti-DNA antibodies; Wu DP et al.; To assess the immune recognition of DNA in systemic lupus erythematosus, the antigenic specificity of monoclonal anti-DNA antibodies from autoimmune MRL-lpr/lpr mice was investigated Determinant specificity was assessed by ELISA in terms of binding to a panel of ssDNA antigens including calf thymus, human placenta, Escherichia coli, Clostridium perfringens, Micrococcus lysodeikticus, salmon testes, chicken blood and murine DNA . Among the monoclonal antibodies, a variety of binding patterns was observed, although for all antibodies tested murine DNA was among the most reactive antigens . Binding to other DNAs varied markedly, with some antibodies showing only low reactivity to certain antigens in the test panel . Similar results were obtained with sera of individual MRL-lpr/lpr mice . These results suggest that anti-DNA antibodies bind specific antigenic determinants variably expressed by DNAs of various species . Furthermore, the preferential binding to mouse DNA by some MRL-lpr/lpr antibodies may suggest a role of self-DNA in the in vivo selection of anti-DNA antibodies for expression. J Biol Chem, 1990 Sep 25, 265(27), 16614 - 6 Separation of toxic activity and ADP-ribosylation activity of botulinum neurotoxin D; Moriishi K et al.; Neurotoxin from Clostridium botulinum type D strain South African (neurotoxin D) has shown ADP-ribosylation activity as well as toxic activity (Matsuoka, I., Sakuma, H., Syuto, B., Moriishi, K., Kubo, S., and Kurihara, K . (1989) J . Biol . Chem . 264, 706-712) . Separation of these activities from each other was attempted by means of gel filtration, hydroxylapatite column chromatography, or immunoaffinity chromatography . Approximately 90% of toxic activity was recovered in each chromatography . Although ADP-ribosylation activity was incompletely separated from neurotoxin D by gel filtration, it was separated by hydroxylapatite column chromatography . In immunoaffinity chromatography with a column of Sepharose 4B coupling antibodies against botulinum ADP-ribosyltransferase, no ADP-ribosylation activity was detected by autoradiography in the unabsorbed toxic fraction . These results indicate that neurotoxin D does not have ADP-ribosylation activity. Eur J Biochem, 1990 Sep 24, 192(3), 723 - 7 De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin; Just I et al.; The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied . In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of {32P}ADP-ribosylated actin and catalyzed the formation of {32P}NAD . The pH optima for both reactions were 5.5-6.0 . Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased . Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions . ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin . However, C . botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins. Eur J Biochem, 1990 Sep 11, 192(2), 411 - 7 Purification and properties of a NADH-dependent 5,10-methylenetetrahydrofolate reductase from Peptostreptococcus productus; Wohlfarth G et al.; The methylenetetrahydrofolate reductase from the carbon-monoxide-utilizing homoacetogen Peptostreptococcus productus (strain Marburg) has been purified to apparent homogeneity . The purified enzyme catalyzed the oxidation of NADH with methylenetetrahydrofolate as the electron acceptor at a specific activity of 380 mumols.min-1 mg protein-1 (37 degrees C; pH 5.5) . The apparent Km for NADH was near 10 microM . The apparent molecular mass of the enzyme was determined by gel filtration to be approximately 250.0 kDa . The enzyme consists of eight identical subunits with a molecular mass of 32 kDa . It contains 4 FAD/mol octamer which were reduced by the enzyme with NADH as the electron donor; iron could not be detected . Oxygen had no effect on the enzyme . Ultracentrifugation of cell extracts revealed that about 40% of the enzyme activity was recovered in the particulate fraction, suggesting that the enzyme is associated with the membrane . The enzyme also catalyzed the methylenetetrahydrofolate reduction with methylene blue as an artificial electron donor . The oxidation of methyltetrahydrofolate was mediated with methylene blue as the electron acceptor; neither NAD+ nor viologen dyes could replace methylene blue in this reaction . NADP(H) or FAD(H2) were not used to substrates for the reaction in either direction . The activity of the purified enzyme, which was proposed to be involved in sodium translocation across the cytoplasmic membrane, was not affected by the absence or presence of added sodium . The properties of the enzyme differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase of the homoacetogen Clostridium formicoaceticum and of the NADP(+)-dependent reductase of eucaryotes investigated so far. Eur J Biochem, 1990 Sep 11, 192(2), 283 - 9 Fructose 1-phosphate and the regulation of glucokinase activity in isolated hepatocytes; Davies DR et al.; Fructose 1-phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1-phosphate and fructose . The concentration of fructose 1-phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate . Addition of fructose (0.05-1 mM) caused a concentration-dependent and transient increase in the fructose 1-phosphate content . Glucagon (1 microM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1-phosphate in cells incubated with fructose, whereas the addition of 0.1 microM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect . All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1-phosphate concentration . Sorbitol had a similar effect to fructose in causing the formation of fructose 1-phosphate . D-Glyceraldehyde was much less potent in this respect than the ketose and its effect disappeared earlier . The effect of D-glyceraldehyde was reinforced by an increase in the glucose concentration and decreased by glucagon . Both fructose and D-glyceraldehyde stimulated the phosphorylation of glucose as estimated by the release of 3H2O from {2-3H}glucose, but the triose was less potent in this respect than fructose and its effect disappeared earlier . Glucagon and ethanol antagonised the effect of low concentrations of fructose or D-glyceraldehyde on the detritiation of glucose . These results support the proposal that fructose 1-phosphate mediates the effects of fructose, D-glyceraldehyde and sorbitol by relieving the inhibition exerted on glucokinase by a regulatory protein. J Infect Dis, 1990 Sep, 162(3), 678 - 84 Risk factors for Clostridium difficile carriage and C . difficile-associated diarrhea in a cohort of hospitalized patients; McFarland LV et al.; A prospective cohort study of 399 consecutive patients in a single ward over an 11-month period was conducted to identify risk factors for nosocomial C . difficile colonization and diarrhea . The incidence of asymptomatic carriage was 13.0/100 patient admissions and the incidence of C . difficile-associated diarrhea was 7.8/100 patient admissions . Increased age and more severe underlying illness were associated with increased risk of C . difficile carriage and diarrhea . Multivariate models adjusting for age and severity of underlying disease associated two risk factors with asymptomatic C . difficile carriage: stool softeners (relative risk {RR} = 2.04) and antacids (RR = 1.80) . Five risk factors were associated with C . difficile-associated diarrhea: cephalosporin use (RR = 2.07), penicillin use (RR = 3.41), enemas (RR = 3.26), gastrointestinal stimulants (RR = 3.06), and stool softeners (RR = 1.74) . C . difficile was a common nosocomial infection on this ward, resulting in asymptomatic carriage more often than diarrhea and accounting for one-fifth of all cases of nosocomial diarrhea. Rev Esp Enferm Dig, 1990 Sep, 78(3), 131 - 4 {Fournier's disease: a report of 9 cases}; Garcia Reinoso C et al.; Nine cases of Fournier's gangrene diagnosed between 1982 and 1989 are reported . All were males with a mean age of 76 (47-82 years) . Seven had a history of alcoholism and one had non insulin-dependent diabetes . Six patients also had an anal fistula which may have been the starting point of the infection . The causal agents were two anaerobes (Clostridium perfringens and Bacteroides fragilis) two gram-negatives (Morganella morgagni and Pseudomonas aeruginosa) and one, an unidentified gram-positive . In three patients a mixed intestinal flora was isolated and in another no germs were found . All were treated with broad-spectrum antibiotics and surgery . Seven patients survived and two died. J Biochem (Tokyo), 1990 Sep, 108(3), 475 - 82 Two distinct ferredoxins from Rhodobacter capsulatus: complete amino acid sequences and molecular evolution; Saeki K et al.; Two distinct ferredoxins were purified from Rhodobacter capsulatus SB1003 . Their complete amino acid sequences were determined by a combination of protease digestion, BrCN cleavage and Edman degradation . Ferredoxins I and II were composed of 64 and 111 amino acids, respectively, with molecular weights of 6,728 and 12,549 excluding iron and sulfur atoms . Both contained two Cys clusters in their amino acid sequences . The first cluster of ferredoxin I and the second cluster of ferredoxin II had a sequence, CxxCxxCxxxCP, in common with the ferredoxins found in Clostridia . The second cluster of ferredoxin I had a sequence, CxxCxxxxxxxxCxxxCM, with extra amino acids between the second and third Cys, which has been reported for other photosynthetic bacterial ferredoxins and putative ferredoxins (nif-gene products) from nitrogen-fixing bacteria, and with a unique occurrence of Met . The first cluster of ferredoxin II had a CxxCxxxxCxxxCP sequence, with two additional amino acids between the second and third Cys, a characteristics feature of Azotobacter-{3Fe-4S} {4Fe-4S}-ferredoxin . Ferredoxin II was also similar to Azotobacter-type ferredoxins with an extended carboxyl (C-) terminal sequence compared to the common Clostridium-type . The evolutionary relationship of the two together with a putative one recently found to be encoded in nifENXQ region in this bacterium {Moreno-Vivian et al . (1989) J . Bacteriol . 171, 2591-2598} is discussed. Antibiot Khimioter, 1990 Sep, 35(9), 17 - 9 {Changes in sensitivity of clinical strains of bacteria to dioxidine from 1984 to 1988}; Bol'shakov LV; Dioxidine sensitivity of 7291 strains of aerobic bacteria and 163 strains of anaerobic bacteria was assayed with the disk diffusion method . The sensitivity of the aerobes was studied in the time course from 1984 to 1988 . It was shown that during the 5-year period, the sensitivity of gram-positive bacteria to dioxidine gradually decreased . At the same time no increase in resistance of gram-negative organisms to dioxidine was observed . A high dioxidine sensitivity of obligate anaerobes, i.e . Clostridium spp., Bacteroides spp., Fusobacterium spp., anaerobic cocci and others was demonstrated. Appl Environ Microbiol, 1990 Sep, 56(9), 2941 - 3 Characterization of thermostable cyclodextrinase from Clostridium thermohydrosulfuricum 39E; Saha BC et al.; Clostridium thermohydrosulfuricum 39E produced a cell-bound cyclodextrin (CD)-degrading enzyme (cyclodextrinase) . It was partially purified 205-fold (specific activity, 14.5 U/mg of protein) by solubilizing with Triton X-100, ammonium sulfate treatment, and DEAE-Sepharose CL-6B column chromatography . The enzyme activity was found to be stable at pH 5.5 and 60 degrees C and optimally active at pH 6.0 and 65 degrees C . The enzyme preparation hydrolyzed CDs, with alpha-CD greater than beta-CD greater than gamma-CD, and displayed a putative multiple attack pattern . The enzyme activity was inhibited by p-chloromercuribenzoate but not by N-bromosuccinimide. Appl Environ Microbiol, 1990 Sep, 56(9), 2600 - 5 Hydrophobicity of Bacillus and Clostridium spores; Wiencek KM et al.; The hydrophobicities of spores and vegetative cells of several species of the genera Bacillus and Clostridium were measured by using the bacterial adherence to hexadecane assay and hydrophobic interaction chromatography . Although spore hydrophobicity varied among species and strains, the spores of each organism were more hydrophobic than the vegetative cells . The relative hydrophobicities determined by the two methods generally agreed . Sporulation media and conditions appeared to have little effect on spore hydrophobicity . However, exposure of spore suspensions to heat treatment caused a considerable increase in spore hydrophobicity . The hydrophobic nature of Bacillus and Clostridium spores suggests that hydrophobic interactions may play a role in the adhesion of these spores to surfaces. Appl Environ Microbiol, 1990 Sep, 56(9), 2591 - 9 Coenzyme A-acylating aldehyde dehydrogenase from Clostridium beijerinckii NRRL B592; Yan RT et al.; Acetaldehyde and butyraldehyde are substrates for alcohol dehydrogenase in the production of ethanol and 1-butanol by solvent-producing clostridia . A coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH), which also converts acyl-CoA to aldehyde and CoA, has been purified under anaerobic conditions from Clostridium beijerinckii NRRL B592 . The ALDH showed a native molecular weight (Mr) of 100,000 and a subunit Mr of 55,000, suggesting that ALDH is dimeric . Purified ALDH contained no alcohol dehydrogenase activity . Activities measured with acetaldehyde and butyraldehyde as alternative substrates were copurified, indicating that the same ALDH can catalyze the formation of both aldehydes for ethanol and butanol production . Based on the Km and Vmax values for acetyl-CoA and butyryl-CoA, ALDH was more effective for the production of butyraldehyde than for acetaldehyde . ALDH could use either NAD(H) or NADP(H) as the coenzyme, but the Km for NAD(H) was much lower than that for NADP(H) . Kinetic data suggest a ping-pong mechanism for the reaction . ALDH was more stable in Tris buffer than in phosphate buffer . The apparent optimum pH was between 6.5 and 7 for the forward reaction (the physiological direction; aldehyde forming), and it was 9.5 or higher for the reverse reaction (acyl-CoA forming) . The ratio of NAD(H)/NADP(H)-linked activities increased with decreasing pH . ALDH was O2 sensitive, but it could be protected a