Biochem Biophys Res Commun, 1986 Nov 14, 140(3), 1015 - 9 Evidence for direct binding of Clostridium botulinum type E derivative toxin and its fragments to gangliosides and free fatty acids; Kamata Y et al.; Clostridium botulinum type E derivative toxin and its heavy chain bound to gangliosides GT1b, GD1a and GQ1b and saturated and unsaturated free fatty acids with chain lengths of 14-20 carbons . The L-H-1 fragment lacking the carboxyl-terminal portion of the heavy chain bound to free fatty acids but not to gangliosides . These observations led us to a new hypothesis on the mechanism of binding between botulinum toxin and gangliosides; the carboxyl-terminal portion (H-2 fragment) of the heavy chain binds to an oligosaccharide residue of gangliosides and then the amino-terminal portion (H-1 fragment) interacts with the hydrophobic portion of gangliosides consisting of fatty acids. Nucleic Acids Res, 1986 Nov 11, 14(21), 8605 - 13 Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum; Joliff G et al.; The nucleotide sequence of the celD gene, encoding the previously crystallized endoglucanase D of Clostridium thermocellum, is reported . The enzyme shares a conserved, reiterated domain with the COOH-terminal end of endoglucanases A and B from the same organism . The overexpression in Escherichia coli of celD subcloned in pUC8 appears to result from a translational fusion of the NH2-terminal end of the endoglucanase with the NH2-terminal end of beta-galactosidase. J Immunol Methods, 1986 Nov 6, 93(2), 225 - 30 Enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of Clostridium botulinum toxins type A and B; Michalik M et al.; Affinity chromatography has been used for a two-step purification of commercial horse botulinum antitoxic globulins type A and B . The first step performed using CH-Sepharose 4B conjugated to toxin type A (or B), permitted the removal of non-botulinal antibodies from antitoxic globulin type A (or B) . The anti-botulinal antibodies obtained from the first step were cross-absorbed in the second affinity chromatography using CH-Sepharose 4B conjugated to toxin B (for the purification of antibodies to type A) or to toxin A (for antibodies to type B) . The antibodies obtained were used to coat polystyrene wells in an ELISA for the detection of botulinum toxin type A and type B . The first purification step increases the sensitivity of such an ELISA whereas the second step improved the specificity of the test . Only slight cross-reactions were observed between the type A and type B detection systems . The sensitivity achieved with ELISA was 100 and 300 DLM (dosis lethalis minima) for type A and B respectively. J Biol Chem, 1986 Nov 5, 261(31), 14551 - 6 The hormone-sensitive hepatic Na+-pump . Evidence for regulation by diacylglycerol and tumor promoters; Lynch CJ et al.; Ouabain-sensitive 86Rb+ uptake by isolated rat hepatocytes was studied to elucidate how Ca2+-mobilizing hormones stimulate the Na+-pump . Stimulation of this uptake was observed with concentrations of vasopressin ({8-arginine}vasopressin, AVP), angiotensin II, and norepinephrine which elicited Ca2+ mobilization and phosphorylase activation . These results suggested that changes in cytosolic Ca2+, mediated by inositol trisphosphate, might trigger sodium pump stimulation by AVP . However, in hepatocytes incubated in Ca2+-free Krebs-Henseleit buffer, Na+-pump activity was not altered over 15 min by either 1.5 mM EGTA or 1.5 mM Ca2+ . Furthermore, incubation of cells in 5 mM EGTA for 15-30 min drastically impaired the ability of AVP to increase cytosolic Ca2+, but only modestly attenuated AVP-stimulated Na+-pump activity . Two tumor promoters, phorbol myristate acetate (PMA) and mezerein, stimulated Na+/K+-ATPase-mediated transport activity . Similarly, addition of synthetic diacylglycerols or of exogenous phospholipase C from Clostridium perfringens to increase endogenous diacylglycerol levels also resulted in a stimulation of the Na+-pump in the absence of changes in cytosolic or total cellular Ca2+ levels . Stimulation of the Na+-pump by the combination of maximal concentrations of PMA and AVP did not produce an additive response, and both agents displayed a transient time course, suggesting that the two agents share a common mechanism . Stimulation of the Na+-pump by AVP and PMA was not blocked by amiloride analogs which inhibit Na+/H+ exchange, but these compounds blocked the action of insulin . These data suggest that the elevated Na+/K+-ATPase-mediated transport activity observed in hepatocytes following exposure to Ca2+-mobilizing hormones is a consequence of stimulated diacylglycerol formation and may involve protein kinase C. Biochemistry, 1986 Nov 4, 25(22), 6789 - 99 A comparative carbon-13, nitrogen-15, and phosphorus-31 nuclear magnetic resonance study on the flavodoxins from Clostridium MP, Megasphaera elsdenii, and Azotobacter vinelandii; Vervoort J et al.; The flavodoxins from Megasphaera elsdenii, Clostridium MP, and Azotobacter vinelandii were studied by 13C, 15N, and 31P NMR techniques by using various selectivity enriched oxidized riboflavin 5'-phosphate (FMN) derivatives . It is shown that the pi electron distribution in protein-bound flavin differs from that of free flavin and depends also on the apoflavoprotein used . In the oxidized state Clostridium MP and M . elsdenii flavodoxins are very similar with respect to specific hydrogen bond interaction between FMN and the apoprotein and the electronic structure of flavin . A . vinelandii flavodoxin differs from these flavodoxins in both respects, but it also differs from Desulfovibrio vulgaris flavodoxin . The similarities between A . vinelandii and D . vulgaris flavodoxins are greater than the similarities with the other two flavodoxins . The differences in the pi electron distribution in the FMN of reduced flavodoxins from A . vinelandii and D . vulgaris are even greater, but the hydrogen bond patterns between the reduced flavins and the apoflavodoxins are very similar . In the reduced state all flavodoxins studied contain an ionized prosthetic group and the isoalloxazine ring is in a planar conformation . The results are compared with existing three-dimensional data and discussed with respect to the various possible mesomeric structures in protein-bound FMN . The results are also discussed in light of the proposed hypothesis that specific hydrogen bonding to the protein-bound flavin determines the specific biological activity of a particular flavoprotein. Ann Neurol, 1986 Nov, 20(5), 641 - 3 Botulism in a patient with jejunoileal bypass; Freedman M et al.; A 45-year-old woman was diagnosed as having the unclassified form of botulism . Her intestines may have been predisposed to colonization with Clostridium botulinum because of a jejunoileal bypass procedure that had been done several years earlier . One other similar case has been reported. J Bacteriol, 1986 Nov, 168(2), 688 - 93 Purification and characterization of a molybdenum-pterin-binding protein (Mop) in Clostridium pasteurianum W5; Hinton SM et al.; A large-scale fractionation scheme purified the major molybdenum(Mo)-binding protein (Mop) from crude extracts of Clostridium pasteurianum, with a 10 and 0.2% yield of Mo and protein, respectively . The apparent molecular weight of the purified molybdoprotein is 5,700, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The protein contains 0.7 mol of Mo per mol of protein with a molecular weight of 5,700 . Mop, as isolated, has a peak absorbency at 293 nm . Denaturation and oxidation of the molybdoprotein released multiple pterin like fluorescent compounds . Mop appears to contain a pterin derivative and Mo, but phosphate analysis indicated that the pterin at the very least is not phosphorylated; phosphorylation is required for functional molybdenum cofactor . All treatments used to release the putative Mo-pterin species from Mop failed to yield a molybdopterin that had detectable molybdenum cofactor activity. Arch Biochem Biophys, 1986 Nov 1, 250(2), 440 - 5 Conversion of purines to xanthine by Methanococcus vannielii; DeMoll E et al.; Based on the finding that Methanococcus vannielii can employ any of several purines as the sole nitrogen source, an investigation was undertaken to elucidate the pathways of purine metabolism in this organism . Cell-free extracts of M . vannielii converted guanine, uric acid, and hypoxanthine to xanthine and also formed guanine from guanine nucleotides or guanosine . The conversions of guanine and uric acid to xanthine appear to occur by pathways similar to those described in clostridia . The conversion of hypoxanthine to xanthine, however, is different than that described for Clostridium cylindrosporum and C . acidiurici, but is similar to that of C . purinolyticum, and apparently involves the direct oxidation of hypoxanthine to xanthine. Gastroenterology, 1986 Nov, 91(5), 1147 - 53 Clostridium difficile cytotoxin inhibits protein synthesis in fibroblasts and intestinal mucosa; Pothoulakis C et al.; The pathophysiology of Clostridium difficile colitis is thought to be mediated by release of toxin A, an enterotoxin, and toxin B, a cytotoxin . We compared the differential effects of toxin B on protein synthesis in IMR-90 fibroblasts and in hamster esophagus, stomach, gallbladder, small intestine, and cecum in organ culture . Toxin B in low concentrations stimulated (p less than 0.001) incorporation of {3H}leucine into fibroblast proteins, whereas at higher dosages it inhibited incorporation (p less than 0.001) . This biphasic effect was independent of cell rounding and was not caused by a change in uptake of precursor . Purified toxin B had no effect on protein synthesis in a cell-free rabbit reticulocyte translation system, indicating that inhibition of protein synthesis in intact fibroblast monolayers and intestinal explants is a consequence of toxin B effect on some other cellular target . Toxin B significantly inhibited protein synthesis in hamster cecal explants in a dose-dependent fashion . Again, this inhibition was not mediated by altered precursor uptake . Toxin B significantly inhibited in vitro protein synthesis in hamster terminal ileum, cecum, and sigmoid colon, but not in esophagus, gallbladder, stomach, or duodenum . These results suggest that toxin B-mediated inhibition of protein synthesis may be a generalized toxic effect in tissue culture cells and intestinal epithelium . Inhibition of protein synthesis in the distal intestinal epithelium may contribute to the pathophysiology of colitis caused by this organism. Pathol Biol (Paris), 1986 Nov, 34(9), 977 - 82 {Clostridium difficile and its cytotoxin in the stools of young hospitalized children . Influence of antibiotic treatment}; Collignon A et al.; Clostridium difficile has been searched in 153 stool samples from 138 children aged 0 to 12 months . We divided the population in two groups depending on the antibiotic treatment . We have found C difficile in 39 samples (25%) . The colonization rate increases with age ranging from 5% before 1 month, to 36% between 1 and 6 months and 54% between 6 and 13 months . An environmental sampling yielded once C difficile . Contamination may be related to the environment . 29% of the isolates produced a cytopathic toxin . Toxin titers in infants' stools range from 1/160 to 1/10240 . One only of these children had diarrhea . C difficile and its toxin does not seem to infer any signs of enteric illness with infants . The results obtained with the group of non treated infants are not significantly different from the ones of the other group: the colonization rates are 21% in the non treated group and 29% in the other group . The rate of strains yielding a cytophatic toxin is similar in the 2 groups . It seems reasonable to agree that antibiotics do not influence the settlement of C difficile in infants' intestine. Rev Infect Dis, 1986 Nov-Dec, 8 Suppl 5, S543 - 8 Mechanisms of beta-lactam resistance in anaerobic bacteria; Nord CE; The known mechanisms of beta-lactam resistance in anaerobic bacteria involve production of beta-lactamases, alteration of penicillin-binding proteins, and blocking of the penetration of beta-lactams through the outer membranes . The most important factor in beta-lactam resistance is the production of beta-lactamase . beta-Lactamases in various Bacteroides, Fusobacterium, and Clostridium species have been described . beta-Lactam resistance in Bacteroides fragilis is most commonly mediated by the production of beta-lactamase, primarily of the cephalosporinase type . Studies have also shown that B . fragilis can produce a penicillinase that inactivates piperacillin and carbenicillin . Enzymes that inactivate cefoxitin and imipenem have also been found in B . fragilis . The nonfragilis Bacteroides species produce beta-lactamases mainly of the penicillinase type . Recently a penicillinase from Fusobacterium nucleatum has been characterized . Among the clostridia, Clostridium butyricum, clostridium clostridiiforme and Clostridium ramosum have been shown to produce penicillinases. Antimicrob Agents Chemother, 1986 Nov, 30(5), 749 - 55 Inactivation of cefoxitin and moxalactam by Bacteroides bivius beta-lactamase; Malouin F et al.; Moxalactam and cefoxitin are known for their high stability against Bacteroides beta-lactamases . We investigated the beta-lactamase activity of crude extracts obtained from three strains of Bacteroides bivius and two strains of Bacteroides fragilis against cefoxitin and moxalactam . In a spectrophotometric antibiotic assay with a 24-h incubation period, B . bivius extracts decreased the initial concentration (10 micrograms/ml) of moxalactam and cefoxitin by 60%, whereas B . fragilis extracts had no effect . In a microbiological assay, when B . bivius or B . fragilis extracts were added to cephalothin (10 micrograms/ml) or cefamandole (4 micrograms/ml), we observed complete disappearance of the inhibitory zones against the indicator strain (Clostridium perfringens ATCC 13124) . Only the B . bivius extracts were able to decrease the inhibitory activity (from 10 to 100%) of cefoxitin and moxalactam (each at 10 micrograms/ml) . Prior addition of clavulanic acid to crude extracts prevented the losses of antibacterial activity . Furthermore, the inhibition of the beta-lactamase hydrolysis of nitrocefin by cefoxitin or moxalactam was prevented by a 12-h preincubation of the beta-lactam with the B . bivius extracts but not with the B . fragilis extracts . Finally, with the B . bivius strain producing the most beta-lactamase, we showed an effect of inoculum size on the MICs of cefoperazone, cefoxitin, and moxalactam with a broth dilution technique . Increasing the inoculum size with the B . fragilis strains had no effect on the MISs of cefoxitin and moxalactam . These results indicate a slow and clavulanate-sensitive beta-lactamase activity of B . bivius extracts against cefoxitin and moxalactam. Steroids, 1986 Nov-Dec, 48(5-6), 381 - 94 The 21-acetylation of corticosteroids by Clostridium sporogenes; Winter J et al.; A strain of Clostridium sporogenes, an anaerobic bacterium, isolated from sewage in New York City synthesizes two constitutive enzymes with action on steroid molecules: (i) an enzyme capable of selectively acetylating the 21-hydroxyl function of certain steroids and (ii) the corresponding esterase . Under our experimental conditions the enzymes have a strict structural requirement for 3-keto-4-ene and C-20-keto or 20 alpha-hydroxyl group and convert their respective substrates to a mixture of free and acetylated products. Ann Inst Pasteur Microbiol, 1986 Nov-Dec, 137B(3), 271 - 82 Major protein components in the cell envelope of Clostridium tyrobutyricum; Bergere JL et al.; The overall composition of the Clostridium tyrobutyricum cell envelope did not vary significantly during cell growth and was characterized by a high protein content (about 40% dry weight) . Teichoic and teichuronic acids were absent and the neutral sugar content low . Insoluble peptidoglycan represented only 10-12% of the cell envelope (dry weight basis); it contained glucosamine, muramic acid, alanine, diaminopimelic acid and glutamic acid (molecular ratio 1/1/2/1/1) . SDS-PAGE revealed the presence of about 50 proteins in this cell envelope; however, one high molecular weight protein was largely predominant . They were not covalently bound to the peptidoglycan and their relative amounts were practically constant through cell growth and with various extraction treatments . A brief heat treatment of whole cells in PBS caused selective release of the major cell envelope proteins together with flagellin; this method was used to characterize these proteins in 37 strains of C . tyrobutyricum and some other clostridia . The major envelope proteins had molecular weights ranging from 96 to 145 Kd and the flagellins from 32 to 72 Kd. J Infect, 1986 Nov, 13(3), 245 - 53 Pathogenicity of Clostridium species with other bacteria in mixed infections; Brook I et al.; The relationship of clostridial isolates with other bacteria in mixed infections was studied by means of a subcutaneous abscess model in mice . We used 26 isolates of seven clostridial species, two Bacteroides spp., eight Gram-positive facultative or anaerobic cocci and three enteric Gram-negative aerobic rods . Abscesses were induced by all seven Clostridium perfringens and three C . butyricum isolates and by some of the others . Selective antimicrobial therapy experiments showed that enteric Gram-negative rods were of equal or greater significance in the formation of abscesses than were clostridial strains in mixed infections . Enhancement or suppression of each component of the mixed infection was studied by comparing the number of each bacterium to its number when injected alone . Enhancement was observed mainly with C . perfringens in mixed infections . By contrast, other Clostridium spp . were less able to induce enhancement . Clostridium difficile and C . sporogenes often inhibited other bacterial species . This study demonstrated the synergistic and antagonistic relationship between clostridial species and other bacteria. Biochim Biophys Acta, 1986 Oct 31, 889(1), 65 - 71 Effects of Ca2+ and other cations on the action of Clostridium perfringens enterotoxin; Horiguchi Y et al.; We investigated the role of extracellular Ca2+ in the Clostridium perfringens enterotoxin-induced alteration of the permeability of the plasma membrane . Enterotoxin released 86Rb and 51Cr from the Vero cells preloaded with the isotope . In the presence of EGTA, however, it released 86Rb but not 51Cr . The binding of enterotoxin to the cells was not influenced by Ca2+ or Mg2+ . The effects of various cations on the enterotoxin-induced 51Cr release was also studied . The release depended on extracellular Ca2+ but not on Mg2+; it was inhibited by each of Zn2+, La3+ and Co2+ . Zn2+ and Co2+ also inhibited 51Cr release caused by the enterotoxin previously bound to the cell membrane . In contrast, antibody against enterotoxin did not neutralize the toxin once it was bound to the Vero cells . When the cells were treated with enterotoxin, 45Ca influx occurred and reached the plateau in a few minutes, as did 86Rb release. J Biol Chem, 1986 Oct 15, 261(29), 13536 - 41 An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5; Telser J et al.; Previous Mossbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic {4Fe-4S}2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel Mossbauer and ENDOR properties . In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms . In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe . Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry . The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center . The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron-carbonyl clusters . The observation of 57Fe ENDOR signals from two types of iron site ({A1c} approximately 30-34 MHz; {A2c} approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by {A1c} confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants. Biochemistry, 1986 Oct 7, 25(20), 6054 - 61 Amino acid sequence of {2Fe-2S} ferredoxin from Clostridium pasteurianum; Meyer J et al.; The complete amino acid sequence of the {2Fe-2S} ferredoxin from the saccharolytic anaerobe Clostridium pasteurianum has been determined by automated Edman degradation of the whole protein and of peptides obtained by tryptic and by staphylococcal protease digestion . The polypeptide chain consists of 102 amino acids, including 5 cysteine residues in positions 11, 14, 24, 56, and 60 . The sequence has been analyzed for hydrophilicity and for secondary structure predictions . In its native state the protein is a dimer, each subunit containing one {2Fe-2S} cluster, and it has a molecular weight of 23,174, including the four iron and inorganic sulfur atoms . The extinction coefficient of the native protein is 19,400 M-1 cm-1 at 463 nm . The positions of the cysteine residues, four of which are most probably the ligands of the {2Fe-2S} cluster, on the polypeptide chain of this protein are very different from those found in other {2Fe-2S} proteins, and in other ferredoxins in general . In addition, whole sequence comparisons of the {2Fe-2S} ferredoxin from C . pasteurianum with a number of other ferredoxins did not reveal any significant homologies . The likely occurrence of several phylogenetically unrelated ferredoxin families is discussed in the light of these observations. Biochemistry, 1986 Oct 7, 25(20), 6048 - 53 Cold-labile hemolysin produced by limited proteolysis of theta-toxin from Clostridium perfringens; Ohno-Iwashita Y et al.; A nicked toxin whose hemolytic activity is temperature dependent was obtained by limited proteolysis of theta-toxin (Mr 54,000) with subtilisin . The nicked toxin (C theta) is a complex of two fragments: the N-terminal fragment (Mr 15,000) with basic isoelectric point and the C-terminal fragment (Mr 39,000) with the single cysteinyl residue of the toxin whose reduced form is essential for the hemolytic activity . C theta hemolyzes erythrocytes only at temperatures above 25 degrees C, whereas the native toxin hemolyzes them even at 10 degrees C . At temperatures below 25 degrees C, C theta does not hemolyze them although it does bind to membrane cholesterol and although no distinct difference was observed between the secondary structure of C theta and that of native toxin . It was found that C theta binds to the cells only in a reversible manner at low temperature, while the native one binds irreversibly to the cells within 10 min, which explains the cold lability of C theta on hemolysis . The structural basis of the cold lability was discussed through comparison of C theta with another nicked derivative of theta-toxin that was also obtained. J Steroid Biochem, 1986 Oct, 25(4), 561 - 6 Influence of 1-double bond and 11 beta-hydroxy group on stereospecific microbial reductions of 4-en-3-oxo-steroids; Kaufmann G et al.; The yeast Rhodotorula glutinis and the anaerobic bacterium Clostridium paraputrificum were used for stereospecific reductions of 4-chloro-11 beta-hydroxy-17 alpha-methyl-testosterone and the corresponding 1-dehydro compound to prepare 5 alpha- and 5 beta-H derivatives, respectively . C . paraputrificum was able to 5 beta-reduce both substances, whereas the 5 alpha-reduction by R . glutinis was inhibited by the structure elements 1-en and 11 beta-OH so that the substrate with both structure elements was not 5 alpha-reduced . The microbial conversion of the four steroids with and without 1-en and 11 beta-OH was compared in semiquantitative experiments . A number of new substances are described, 11 beta-hydroxy and 11-oxo derivatives of 5 alpha- and 5 beta-dihydro-4-chloro-17 alpha-methyltestosterone including some 3-OH compounds and characterized by NMR, mass spectrometric and further data. Am J Med, 1986 Oct, 81(4), 596 - 600 Emergence of Clostridium tertium as a pathogen in neutropenic patients; Thaler M et al.; Although usually considered a non-pathogen, Clostridium tertium was isolated from 10 immunosuppressed patients including seven patients with bacteremia . This organism can grow aerobically and can be easily disregarded as a contaminant . It also has a somewhat unusual susceptibility pattern, with significant resistance to the penicillins, cephalosporins, and clindamycin, possibly explaining its emergence in immunocompromised patients already receiving multiple antibiotics. Am J Gastroenterol, 1986 Oct, 81(10), 940 - 3 Clostridium difficile culture-positive toxin-negative diarrhea; Lashner BA et al.; Antibiotic-associated colitis (AAC) is confirmed by the isolation of Clostridium difficile cytotoxin from stool in patients with diarrhea . Culture of the organism has not been required to confirm the diagnosis . A review of cases of C . difficile culture-positive patients was performed in an attempt to clarify the significance of culture-positive toxin-negative (CPTN) compared to culture-positive toxin-positive (CPTP) disease . During an 11-month period, 45 patients were identified who had stool cultures positive for C . difficile . Sixteen of the patients studied were CPTP and 29 were CPTN . There were no major differences between the two groups for underlying diseases, antibiotic exposure, or diagnostic testing . Of the CPTP patients, 10 were treated for AAC and all responded . Two untreated patients resolved spontaneously . Of the CPTN patients, none was given specific antibiotic therapy, symptoms spontaneously resolved in 17, and symptoms were unresolved in five (colectomy or expired before resolution) . A prospective analysis was performed of all C . difficile isolated from stool samples by the microbiology laboratory . Isolates were incubated in vitro and cytotoxin production was measured . Of isolates from CPTP patients 97% produced cytotoxin compared to 67% of isolates from CPTN patients (p less than 0.005) . The results suggest that C . difficile, despite the absence of cytotoxin, may be an etiological factor in certain diarrheal syndromes . Until a randomized therapeutic trial for CPTN patients is conclusive, a positive culture should be considered evidence for treatment of patients with persistent diarrhea. J Antimicrob Chemother, 1986 Oct, 18 Suppl C, 141 - 7 Some aspects of the occurrence of resistant bacteria in the normal animal flora; Wray C; The paper outlines some of the problems encountered in assessing the occurrence of antibiotic resistance in the normal flora of animals, using Escherichia coli as an example . Additional information is provided on the occurrence and mechanisms of resistance in Clostridium perfringens, Pasteurella haemolytica and Staphylococcus aureus, and some of the factors which may affect this resistance . The paper concludes with general considerations about the choice of organism, the sample size and the desirability of designing standard protocols. EMBO J, 1986 Oct, 5(10), 2495 - 502 Tetanus toxin: primary structure, expression in E . coli, and homology with botulinum toxins; Eisel U et al.; A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani . The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol . wt of 150,700 . In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol . wt light chain and the 98,300 mol . wt heavy chain, respectively . Cysteine (467) was involved in the disulfide linkage between the two subchains . The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C . tetani and C . botulinum are derived from a common ancestral gene . Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies . The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E . coli. Infect Immun, 1986 Oct, 54(1), 260 - 1 Lyophilized airborne Clostridium botulinum spores as inocula that intestinally colonize antimicrobially pretreated adult mice; Sugiyama H et al.; Adult mice, made susceptible to Clostridium botulinum by feedings of metronidazole, were immobilized with an anesthetic and held for 30 min in isolators in which a fine powder of lyophilized pathogen spores was made airborne . Exposed mice were surface decontaminated before being kept for 2 days in holding isolators . Mice were intestinally colonized by the pathogen . Colonization rates were related to spore numbers (10(4) to 10(7) type A or B) seeded into isolators. Am J Dis Child, 1986 Oct, 140(10), 1013 - 4 Infant botulism . Three cases in a small town; Istre GR et al.; Through Dec 31, 1985, there have been six cases of infant botulism reported in Colorado . Three of these infants have lived in the same town of 800 people in western Colorado . Two of these three infants developed infant botulism within a six-month period in late 1981 . The infants lived approximately 400 m apart; they had used the same crib at the time each developed botulism . A specimen from the crib yielded Clostridium botulinum, as did four soil samples from the town and house-dust samples from the home of a relative of the second infant . The third infant developed infant botulism in September 1984 . This infant had not shared the crib . In this case, all seven samples of soil from various locations in the town yielded C botulinum, as did a sample of house dust from the home of this infant . The occurrence of these three cases in such a small town seems unlikely to be only coincidental . Investigations and reports of other such clusters may provide insight into modes of transmission of infant botulism. Orthop Rev, 1986 Oct, 15(10), 658 - 63 Nontraumatic clostridial myonecrosis, a case report; Jamison JP et al.; Physicians are aware of the association between massively contaminated wounds and clostridial myonecrosis, or gas gangrene . A far less common but equally devastating presentation is that of nontraumatic or spontaneous gas gangrene . The most frequently encountered organism in this rare form of gangrene is Clostridium septicum, and there is a high correlation with hematologic or gastrointestinal malignancy . The mainstays of treatment are intravenous antibiotics, surgical debridement, and hyperbaric oxygen therapy . The prognosis is dependent upon early recognition and institution of treatment. J Appl Bacteriol, 1986 Oct, 61(4), 331 - 7 Purification and characterization of a streptomycete collagenase; Chakraborty R et al.; A soil streptomycete designated as Streptomyces sp . A8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-a rginine into two tripeptides . The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration . The purified enzyme had an apparent molecular weight of about 75,000 by SDS-polyacrylamide gel electrophoresis . Treatment with lithium chloride did not dissociate it into subunits . A strong inhibition was observed with chelating agents such as alpha-alpha-dipyridyl and 8-hydroxyquinoline . Ethylene diamine tetraacetate completely inhibited the enzyme activity . Among the cations tested only Ca2+ and Mg2+ enhanced the collagenase activity . Heavy metal ions like Pb2+, Ag+, Cu2+ and Zn2+ strongly inhibited the enzyme . The EDTA inhibition could be reversed with Ca2+ . Cysteine and reduced glutathione caused significant reduction in enzyme activity . Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase . Amino acid analysis revealed the absence of cysteine and tyrosine . Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus. J Neurochem, 1986 Oct, 47(4), 1098 - 105 Effect of phospholipases on chronic opiate action in neuroblastoma X glioma NG108-15 hybrid cells; Griffin MT et al.; Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone . The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases . Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine-treated (1 microM for 4 h) hybrid cells . In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment . This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations . The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D . Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin . Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains. Microb Pathog, 1986 Oct, 1(5), 417 - 23 Amino groups in Clostridium perfringens epsilon prototoxin and epsilon toxin; Sakurai J et al.; Modification with succinic anhydride (SA) of Clostridium perfringens epsilon prototoxin or toxin resulted in a loss of activation by trypsin or lethal activity, respectively . The prototoxin was more sensitive to succinylation than the toxin . On the other hand, the succinylated prototoxin was activated and cleaved by chymotrypsin, but not by trypsin . The lethal activity of the toxin was also lost after treatment with 2,3-dimethylmaleic anhydride (DMA) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) . When the DMA-treated toxin treated with SA or TNBS was incubated under acidic condition, it regained lethal activity . Thus modification of amino groups (lysine residues) prevented activation of the prototoxin by trypsin, and abolished lethal activity of the toxin. J Gen Microbiol, 1986 Oct, 132 ( Pt 10), 2893 - 8 A scanning electron microscope study of the effect of an enterotoxin from Clostridium perfringens 8-6 on mice of different ages; Lindsay JA et al.; Intestinal damage to mice caused by an enterotoxin from a coatless spore mutant of Clostridium perfringens type A (8-6) was examined by scanning electron microscopy . Two distinct types of damage were observed, both of which could be correlated with animal age . Damage appeared to occur in a specific sequence similar to that found in previous studies in rabbits . We conclude that the type of ileal tissue damage reflects the mode of toxin incorporation from the gut, which is a function of animal age. Anal Biochem, 1986 Oct, 158(1), 158 - 64 Analysis of sialidase and N-acetylneuraminate pyruvate-lyase substrate specificity by high-performance liquid chromatography; Shukla AK et al.; A rapid and sensitive assay by high-performance liquid chromatography for determination of the activity and substrate specificity of sialidase (EC 3.2.1.18) and N-acetylneuraminate lyase (EC 4.1.3.3) is described . Sialic acids were separated on a strong anion-exchange resin using 0.75 mM sodium sulfate as elution medium . This method allows the determination of a minimum amount of 200 pg (0.6 pmol) of sialic acid . Usually the enzyme mixtures were directly applied to the column without prior purification of substrates and products . The action of sialidase was studied either by the decrease of sialyllactose concentration or by the amount of sialic acid liberated . The relative hydrolysis rates of N-acetylneuraminyl-alpha(2-3)-lactose, N-glycolylneuraminyl-alpha(2-3)-lactose, N-acetylneuraminyl-alpha(2-6)-lactose, N-acetyl-9-O-acetylneuraminyl-alpha(2-3)-lactose, and N-acetyl-4-O-acetylneuraminyl-alpha(2-3)-lactose by Vibrio cholerae sialidase were 100, 88, 25, 12, and 0, respectively . The activity of N-acetylneuraminate lyase from Clostridium perfringens was determined by measuring the rate of disappearance of sialic acids or the formation of acylmannosamines, which is possible in the same chromatogram . Relative cleavage rates of N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, and N-acetyl-4-O-acetylneuraminic acid were found to be 100, 67, 24, 3, and 0, respectively . Comparison of the substrate specificities shows that substituents on the neuraminic acid molecule influence the reactions of both enzymes in a similar way. Appl Environ Microbiol, 1986 Oct, 52(4), 969 - 70 Evaluation of the diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin; Jackson SG et al.; The diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin was evaluated . Test results from 100 individuals associated with C . perfringens gastroenteritis outbreaks and 111 control individuals were included . The assay sensitivity was 93.7%, and the assay specificity was 98.7%. Am Fam Physician, 1986 Oct, 34(4), 130 - 6 Anaerobic infections in childhood; Brook I; Bacteroides melaninogenicus and Bacteroides oralis are predominant anaerobes in orofacial infections and aspiration pneumonia . Fusobacterium species are common pathogens in aspiration pneumonia, brain abscesses and orofacial infections . Clostridium perfringens can cause bacteremia and wound infections . Clostridium botulinum can produce a paralytic toxin that causes a paralytic syndrome in infants . Clostridium difficile can cause diarrhea or antibiotic-associated colitis. Surg Gynecol Obstet, 1986 Oct, 163(4), 310 - 4 The significance of clostridial isolates in intra-abdominal sepsis; Jaggers RL et al.; In order to evaluate the significance of clostridial species in intra-abdominal infections, the bacteriology records of three hospitals were reviewed during a period of five years . Included in this report were 41 patients from whom clostridial species were recovered from specimens of free peritoneal fluid, abscess cavities or bile . Seven patients died for a mortality rate of 17.1 per cent . Most patients had polymicrobial infections of which clostridial organisms were one of the several anaerobes isolated . Clostridium perfringens was the single most frequently noted species, identified in 23 of the patients, but it was not associated with a different mortality rate than was observed for the other clostridial species . Clostridial bacteremia was uncommon and demonstrated in only one patient . The mean age of the patients was 56.4 years; 56.2 for males and 56.8 for females . Neither age nor sex of the patient influenced the likelihood of survival . The source of the clostridial isolates--bile, abscess cavity or free peritoneal fluid--had no effect upon the outcome . Several underlying conditions were responsible for the intraperitoneal clostridial organisms identified in this series . Only mesenteric infarction proved significantly predictive of a fatal result . Antibiotic coverage specifically directed against clostridia did not influence survival. J Submicrosc Cytol, 1986 Oct, 18(4), 773 - 81 The surface charge of Toxoplasma gondii: a cytochemical and electrophoretic study; Cintra WM et al.; The surface charge of Toxoplasma gondii tachyzoites was evaluated by means of binding of colloidal iron hydroxide particles at pH 1.8, cationized ferritin particles at pH 7.2 and ruthenium red to the cell surface, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility (EPM) of the cells suspended in solutions of different ionic strength and pH . At pH 7.2, T . gondii has a negative surface charge with a mean EPM of--1.1272 +/- 0.0917 micron.s-1 X V-1 X cm . No significant difference was observed between the EPM of living cells at 25 degrees C and that of glutaraldehyde-fixed cells . At lower pH, there is a decrease in the negative surface charge, with an isoelectric point at pH 3.5 . At higher pH (greater than 10), there is an increase in the surface charge reaching an EPM of--1.5675 +/- 0.0848 micron.s-1 X V-1 X cm at pH 7.2 . These results indicate that the surface of T . gondii contains both negatively and positively charged dissociating groups . Binding of cationized ferritin particles and ruthenium red throughout the cell surface of glutaraldehyde-fixed cells was observed . However, when living parasites were incubated at 4 degrees C in the presence of cationized ferritin some cells showed a uniform distribution of the label, others showed a patch-distribution and still in others no label was seen, indicating a process of mobility and shedding of surface anionic sites . Colloidal iron hydroxyde particles did not bind to the surface of T . gondii . Incubation of the parasites in the presence of neuraminidase from Clostridium perfringens or Vibrio cholerae or in the presence of proteolytic enzymes (trypsin or protease) did not interfere with the surface charge.(ABSTRACT TRUNCATED AT 250 WORDS) J Appl Physiol, 1986 Oct, 61(4), 1418 - 30 Effect of proteolytic enzymes on transepithelial solute transport; Niewoehner DE et al.; The effects of proteases on air-space clearance (AC) of small ({14C}sucrose, 342 daltons) and large (125I-neutral dextran, 70,000 daltons) solutes were studied in isolated, fluid-filled hamster lungs that were perfused in a nonrecirculating system . When instilled into the air spaces, porcine pancreatic elastase (0.1-0.4 mg/ml) and bovine pancreatic trypsin (BPT) (0.5-2.0 mg/ml), but neither Clostridium histolyticum collagenase (5.0 mg/ml) nor phenylmethylsulfonyl fluoride-inactivated BPT caused large increases in the AC of both tracer molecules . BPT-induced solute clearance was further characterized functionally and morphologically . The functional characteristics of solute AC under steady-state conditions did not indicate that transepithelial transport was diffusion-limited . Inhibition by millimolar concentrations of Zn2+ and by lung cooling, along with electron microscopic studies employing horseradish peroxidase as a macromolecule tracer, were consistent with epithelial solute transport by a vesicular mechanism (transcytosis) . Solute transport from the interstitial compartment to the lung exterior was shown to occur via two pathways . By unknown mechanisms BPT caused small amounts of water to flow through an incompletely identified, extravascular pathway . In BPT-exposed lungs efflux of 125I-dextran 70 occurred almost exclusively through this pathway, whereas {14C}sucrose was transported to the lung exterior partly through this same pathway and partly through the vasculature . The large differences in the diffusion coefficients of the two tracers may have accounted for these observed patterns of solute efflux from the lung . The possible significance of our findings to the pathogenesis of experimental emphysema are discussed. Infect Immun, 1986 Oct, 54(1), 70 - 6 Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies; Lyerly DM et al.; Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B . One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A . This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin . The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide . Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components . The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C . difficile by immunoaffinity chromatography . The second antibody, the G-2 MAb, cross-reacted with toxins A and B . The cross-reaction was confirmed by immunoblot analysis . These results show that toxins A and B share an epitope and suggest that they have a common subunit . The G-2 MAb did not neutralize or precipitate either toxin . The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide. Biochem Pharmacol, 1986 Sep 15, 35(18), 3031 - 8 Effects of phospholipases C on the beta-receptor-adenylate cyclase system of chick erythrocyte membranes; Nakajima M et al.; The beta-adrenergic receptor located in chick erythrocyte membranes was characterized using (-)-{3H}-dihydroalprenolol ({3H}-DHA) with rapid filtration techniques . The affinity of beta-adrenergic antagonist, (-)-propranolol, was approximately 100-fold higher than that of (+)-propranolol . Catecholamines were bound with the receptor in the following order, (-)-isoproterenol greater than (-)-norepinephrine greater than (-)-epinephrine, suggested the binding site to be beta 1-classification . When the membrane preparation was treated with phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens or phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis, {3H}-DHA binding was decreased to the level of 66 or 86% of the control, respectively . The treatment with sphingomyelinase C (SMase) of Bacillus cereus, however, did not cause any appreciable reduction of {3H}-DHA binding . Throughout these experiments, the equilibrium dissociation constant (KD) of {3H}-DHA was not influenced by phospholipases C . The affinity of isoproterenol for beta-receptor was decreased in the absence of GTP, but not altered in the presence of GTP by PIase action . Treatment with PCase or SMase, however, did not affect the affinity of isoproterenol for beta-receptor . Treatment with PIase inhibited basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase activities . On the other hand, PCase treatment inhibited only isoproterenol-stimulated adenylate cyclase activity, but not basal and forskolin-stimulated activities . These results suggest that membrane phospholipids, especially phosphatidylcholine (PC) and phosphatidylinositol (PI), are directly related to the receptor binding and that PI interacts with adenylate cyclase activity. Biochim Biophys Acta, 1986 Sep 11, 860(3), 620 - 5 Difference in phospholipid dependence between two isozymes of brain (Na+ + K+)-ATPase; Matsuda T et al.; The effect of phospholipase C on two isozymes (alpha (+) and alpha forms) of rat brain (Na+ + K+)-ATPase and the temperature-dependence of their activities were investigated . Phospholipase C from Clostridium welchii inhibited the activities of the enzymes treated with and without pyrithiamin or N-ethylmaleimide, a preferential inhibitor of the alpha (+) form, but the extent of the inhibition was higher in the control enzyme than in the treated enzymes . The treatment of the (Na+ + K+)-ATPase with phospholipase C altered a ratio between high- and low-affinity components for ouabain inhibition . It also caused the similar change in a ratio between the alpha (+) and alpha forms of Na+-stimulated phosphorylation from {gamma-32P}ATP . These findings indicate that the alpha (+) form of rat brain (Na+ + K+)-ATPase is more sensitive to phospholipase C than the alpha form . Analysis of Arrhenius plots of the activities of the control and pyrithiamin-treated enzymes showed that there was a difference between the two enzymes in a break point . We suggest that two isozymes of rat brain (Na+ + K+)-ATPase differ in the interaction with phospholipids or in the lipid-environment. Biochemistry, 1986 Sep 9, 25(18), 5189 - 95 Purification of human collagenases with a hydroxamic acid affinity column; Moore WM et al.; Human collagenase has been isolated from skin fibroblasts and rheumatoid synovium by using an affinity matrix, prepared by coupling Pro-Leu-Gly-NHOH to agarose . Following the methodology described herein, the skin enzyme was isolated in two steps in 76% yield and the synovial enzyme was purified in three steps in 71% yield . Importantly, each enzyme hydrolyzed collagen into 3/4-1/4 cleavage fragments, indicating that a true collagenase had been isolated . The column was specific for the human enzyme since the collagenase from Clostridium histolyticum did not bind . The affinity ligand was designed according to the formalism proposed by Holmquist and Vallee {Holmquist, B., & Vallee, B . L . (1979) Proc . Natl . Acad . Sci . U.S.A . 76, 6216} that effective metalloenzyme inhibitors can be synthesized by coupling a suitable metal-coordinating group to a substrate analogue . In this case, the hydroxamic acid probably coordinates to the active-site metal and the Pro-Leu-Gly moiety is similar to the carboxyl side of the cleavage site of collagen, the enzyme's substrate . The IC50 for N-(benzyloxycarbonyl)-Pro-Leu-Gly-NHOH is 4 X 10(-5) M for both enzymes . The affinity chromatographic procedures described here should aid in future studies on vertebrate collagenases. Mikrobiologiia, 1986 Sep-Oct, 55(5), 804 - 7 {Biological activity of a polar lipid of Clostridium butyricum spores}; Gaenko GP et al.; A fraction of polar lipids was isolated from spores of the anaerobic bacterium Clostridium butyricum 35/11 exerting a noticeable radioprotective effect . The main biological activity of spore extracts was associated with this fraction . The fraction of polar lipids inhibited autolysis of the bacterial cell walls . The fraction was found to contain a phenolic glycolipid and a peptide component . The bacteriostatic and radiotherapeutic properties of the fraction are presumed to be due to its membranotropic activity. J Antibiot (Tokyo), 1986 Sep, 39(9), 1205 - 10 Lustromycin, a new antibiotic produced by Streptomyces sp; Tomoda H et al.; A new antibiotic, lustromycin, was isolated from the cultured broth of Streptomyces sp . SK-1071 . It exhibits selective antibacterial activity against anaerobic bacteria including Clostridium sp . The molecular formula C32H38O13 as determined by high resolution mass spectrometry, and elemental analysis and the NMR spectrum suggest structural resemblance of this antibiotic to luminamicin, an anti-anaerobic antibiotic reported previously. Pediatr Infect Dis, 1986 Sep-Oct, 5(5), 550 - 6 Anaerobic osteomyelitis in children; Brook I; Twenty-six pediatric patients with osteomyelitis caused by anaerobic bacteria are presented . The etiologic factors were chronic mastoiditis (7 patients), decubitus ulcers (5), chronic sinusitis (4), periodontal abscesses (3), bites (3), paronychia (2), trauma (1) and scalp infection after fetal monitoring (1) . Seventy-four organisms (2.8 isolates/specimen), including 63 anaerobes (2.4/specimen), and 11 facultative and aerobic bacteria (0.4/specimen) were recovered . The predominant organisms were anaerobic cocci (29 isolates), Bacteroides sp . (21), Fusobacterium sp . (8), Streptococcus sp . (5) and Clostridium sp . (4) . The organisms generally reflected the microbial flora of the mucous surface adjacent to the infected site . Ten beta-lactamase-producing organisms were recovered from 7 (27%) patients . These included all isolates of the Bacteroides fragilis (4) and of Staphylococcus aureus (3), 2 of the 12 Bacteroides melaninogenicus group and 1 of 3 Bacteroides oralis . The clinical, diagnostic and therapeutic aspects of anaerobic osteomyelitis in children are discussed. J Bacteriol, 1986 Sep, 167(3), 828 - 36 Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose; Bayer EA et al.; The ultrastructural distribution of the cellulosome (a cellulose-binding, multicellulase-containing protein complex) on the cell surface of Clostridium thermocellum YS was examined by cytochemical techniques and immunoelectron microscopy . When cells of the bacterium were grown on cellobiose, cellulosome complexes were compacted into quiescent exocellular protuberant structures . However, when the same cells were grown on cellulose, these polycellulosomal organelles underwent extensive structural transformation; after attachment to the insoluble substrate, the protuberances protracted rapidly to form fibrous "contact corridors." The contact zones mediated physically between the cellulosome (which was intimately attached to the cellulose matrix) and the bacterial cell surface (which was otherwise detached from its substrate) . In addition, cell-free cellulosome clusters coated the surface of the cellulose substrate . The cellulose-bound cellulosome clusters appear to be the site of active cellulolysis, the products of which are conveyed subsequently to the cell surface via the exocellular contact zones. Infect Immun, 1986 Sep, 53(3), 573 - 81 Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc; Krivan HC et al.; This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin . Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C . difficile . The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved . We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces . Only rabbit erythrocytes bound the toxin, and the cells agglutinated . A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C . difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C . difficile toxin A than hamsters . Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs . Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C . A similar binding phenomenon was observed with rabbit erythrocytes . Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose . Treatment of both membrane types with alpha-galactosidase destroyed the binding activity . The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells . Toxin A did not bind to human erythrocytes from blood group A, B, or O donors . However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3{Fuc alpha 1-2}Gal beta 1-4GlcNAc-R, bound the toxin . This indicated that toxin A was likely binding to Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes . All of the results indicate that hamster BBMs contain a carbohydrate-binding site for toxin A that has at least a Gal alpha 1-3Gal beta 1-4GlcNAc nonreducing terminal sequence. Obstet Gynecol, 1986 Sep, 68(3 Suppl), 26S - 28S Clostridial myonecrosis arising from an episiotomy; Soper DE; A case of clostridial episiotomy infection is reported and the literature reviewed . Early recognition of the severity of this disease along with aggressive operative therapy is necessary to improve outcome . Clostridium sordellii is now the most common Clostridia species to be isolated from patients with these serious infections. Can J Microbiol, 1986 Sep, 32(9), 743 - 50 Aerobic degradation of choline by Proteus mirabilis: enzymatic requirements and pathway; Sandhu SS et al.; Cleavage of choline to trimethylamine and acetaldehyde by extracts of Proteus mirabilis requires both particulate and soluble protein fractions, K+, and a bound divalent metal cation . The reaction shows a long lag period, abolished only by preincubation of the particulate fraction in the complete reaction system . The two-carbon fragment produced is acetaldehyde; choline cleavage appears to be tightly coupled to dismutation of the acetaldehyde to ethanol and acetate, as indicated by stimulation by NAD+, ADP, and Fe2+ and inhibition by reagents reacting with acetaldehyde . The system is thus similar to that previously described in anaerobes (Desulfovibrio, Clostridium) . Attempts to demonstrate a cobamide coenzyme requirement (as in the similar ethanolamine ammonia-lyase reaction) were unsuccessful; the reaction was carried out by fractions devoid of vitamin B12 activity (not supporting growth of Lactobacillus leichmannii) and insensitive to light. J Am Vet Med Assoc, 1986 Sep 1, 189(5), 557 - 9 Splenic hematoma and abscess as a cause of chronic weight loss in a horse; Spier S et al.; An 8-year-old gelding with a 3-month history of anorexia and weight loss was found to have a massive subcapsular splenic hematoma . At flank laparotomy, 36 L of fluid was removed from the hematoma . The horse's condition improved after drainage . Fifteen months later, the horse became depressed and febrile . A splenic abscess containing Bacteroides ruminicola and Clostridium sporogenes was found at necropsy. Appl Environ Microbiol, 1986 Sep, 52(3), 407 - 12 Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens; Goldner SB et al.; Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C . The enterotoxin was detected by serological and biological assays . Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage . The culture maintained enterotoxigenicity throughout cultivation in a continuous system . The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium . No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth. J Am Vet Med Assoc, 1986 Sep 1, 189(5), 564 - 5 Clostridium perfringens type C enterotoxemia in a newborn foal; Howard-Martin M et al.; A 1-day old, full-term foal with a history of colic died 2 hours after admission . Necropsy revealed an extremely flaccid, fluid-filled intestinal tract . Histopathologically, the superficial intestinal mucosa was completely necrotic, with minimal inflammatory response . Numerous large, gram-positive rods covered the villi . Clostridium perfringens was isolated on bacteriologic culturing of the intestinal tract contents and was identified as type C by toxin neutralization tests. J Clin Microbiol, 1986 Sep, 24(3), 384 - 7 Immunoblotting to demonstrate antigenic and immunogenic differences among nine standard strains of Clostridium difficile; Heard SR et al.; The epidemiology of Clostridium difficile-associated disease is being elucidated with the development of typing schemes for the organism . We recently described a new typing scheme based on the incorporation of {35S}methionine into bacterial proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . Nine standard strains were identified . We report here some observations on the antigenic differences among these nine strains when studied by immunoblotting . Type-specific rabbit antiserum was raised against each of the nine standard strains . Immunoblotting of the strains with these antisera demonstrated, in addition to the presence of shared, common proteins, a type-specific response with homologous antisera . When {35S}methionine-labeled C . difficile proteins were immunoblotted with homologous and heterologous antisera, both the immunoblots and the autoradiographs demonstrated the same strain-specific response . These strain-specific proteins, which have been so useful for epidemiological and typing purposes, were also immunogenic. Biochem Biophys Res Commun, 1986 Aug 29, 139(1), 64 - 70 ADP-ribosylation in cultured cells treated with Clostridium difficile toxin B; Florin I et al.; In cultured fibroblasts intoxicated with Clostridium difficile toxin B, a radioactive moiety was transferred from {14C-adenosine}NAD, but not from {14C-nicotinamide} NAD, into a cellular protein (MW 90,000) . No labeling was detected in toxin-treated cultures not yet showing any toxin-induced cytopathogenic effect, whereas maximal labeling was obtained in cultures with about half of the cells showing a cytopathogenic effect . The radioactivity was removed from the substrate by treatment with snake venom phosphodiesterase . The results suggest that ADP-ribosylation of a cellular protein occurs in toxin B-treated cells and that this reaction may be responsible for development of the cytopathogenic effect. N Z Med J, 1986 Aug 27, 99(808), 617 - 20 Hyperbaric oxygen therapy in the management of Clostridium perfringens infections; Gibson A et al.; Patients with Clostridium perfringens infections treated in Christchurch over a 14 year period to 1984 were reviewed retrospectively . Of the 46 documented cases, 21 died . Twenty-nine patients were treated with hyperbaric oxygen (HBO) therapy . Of these, nine died, whilst 12 of 17 other patients who did not receive HBO died, including five in whom the diagnosis was only made at postmortem . The clinical features and predisposing factors for clostridial infections are discussed . The importance of early vigorous treatment with a combination of surgical debridement, antibiotics and HBO to reduce the morbidity and mortality of this lethal infection is emphasised. N Z Med J, 1986 Aug 27, 99(808), 620 - 2 Clostridium difficile toxin in chronic idiopathic colitis; Lee DK et al.; Clostridium difficile toxin was isolated from the stools of three patients with chronic idiopathic colitis . Two patients were known to have chronic idiopathic colitis before Cl difficile toxin was isolated . The third patient was subsequently found to have ulcerative colitis after presentation with Cl difficile toxin in the stool . Two patients were on sulphasalazine at the time of diagnosis of Cl difficile infection and one had taken sulphasalazine two months previously . Only one patients had antibiotic exposure and that was at least three months before presentation . In each patient, treatment with vancomycin was accompanied by symptomatic improvement and disappearance of the toxin . The underlying colitis remained unaffected . In patients with inflammatory bowel disease in relapse, the presence of Cl difficile toxin should be sought as this may be a factor in the relapse . In any patient presenting with diarrhoea, the presence of Cl difficile toxin may obscure the presence of underlying inflammatory bowel disease. J Biol Chem, 1986 Aug 25, 261(24), 11409 - 15 In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene . Evidence for "extended" promoter elements in gram-positive organisms; Graves MC et al.; Analysis of Clostridium pasteurianum genomic DNA indicates that the ferredoxin (Fd) gene is present in a single copy . The cloned Fd gene previously described (Graves, M.C., Mullenbach, G . T., and Rabinowitz, J . C . (1985) Proc . Natl . Acad . Sci . U . S . A . 82, 1653-1657) was used to map in vivo and in vitro synthesized Fd transcripts . The in vivo mRNA was sized in two ways: by Northern hybridization analysis, and more directly from the known DNA sequence after the 5'- and 3'-termini were identified . The 5'-end was determined by primer extension-dideoxy sequencing and the 3'-end by S1 nuclease mapping . The monocistronic Fd mRNA contains about 255 nucleotides and, thus, is one of the shortest bacterial mRNAs yet described . We also examined the Fd transcripts produced by Escherichia coli transformed with the plasmid containing the Fd gene . E . coli RNA polymerase most likely recognizes the same promoter (P1) as the clostridial polymerase, and furthermore, efficiently uses an additional promoter (P2) that is poorly recognized by the normal host enzyme . For comparison, in vitro transcripts were generated by E . coli and Bacillus subtilis RNA polymerases . In vitro, only promoter P1 is used by either E . coli or B . subtilis RNA polymerase . The 3'-end of each of the four types of transcripts occurs essentially at the same location and maps to within a large dyad symmetry element . Comparison of the Fd promoter with other Gram-positive promoters reveals that some sequences outside of the traditional Pribnow and -35 regions are conserved . This analysis indicates that an "extended" promoter recognition site may be required in these organisms. J Biol Chem, 1986 Aug 25, 261(24), 11049 - 55 Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase; Vlahos CJ et al.; Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner . To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either {14C}pyruvate or {14C}glyoxylate was reduced with CNBH3- . After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination . In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine) . Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit . This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum . Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate. Orv Hetil, 1986 Aug 10, 127(32), 1923 - 8 {The role of intrauterine contraceptive devices in the development of inflammatory processes in the small pelvis}; Batar I; PIP: The incidence of pelvic inflammatory disease (PID) attributable to IUD use has been increasing, especially after the removal of the Dalkon shield from the market, but this relationship has not been settled conclusively . In recent decades PID included a variety of infections, but lately the definition of PID has meant acute ascending infections of the female genital tract . Its most common risk factors include promiscuity of IUD use, although this can be reduced to one fourth by regular checkups and proper hygiene . The frequency of PID is estimated at 2-5% of IUD users . Microorganisms contributing to PID include Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Escherichia coli, Proteus, Staphylococcus epidermis, Haemophilus influenzae, Bacteroides, Peptococcus, Peptostreptococcus, Clostridium, and Actinomyces israelii, The differentiation of actinomycosis (AC) and pseudoactinomycosis (PAC) is well advised . The potential of IUD use in increasing the risk of AIDS should not be discounted . The clinical picture of PID is varied, it can be mild requiring conservative drug therapy; with medium severity requiring removal of the IUD and drug therapy; severe necessitating removal, antibiotics and sulfonamide treatment and laparotomy; and very severe with potentially fatal generalized sepsis . In addition to antibiotics, e.g., penicillin, treatment can include the so called catastrophy combination of Mandokef- Metronidazol-Gentamycin . An analysis of the data of 8536 IUD fittings in Debrecen, Hungary showed 1.4% removals due to PID after 4 years, 694 patients (8.1%) had lower abdominal pain 73 of which (0.9%) had palpable resistance, and suppuration occurred in only 30 cases (0.4%) . Treatment included Semicillin or Tetran, or removal of the IUD, and even surgery if no improvement resulted . Prevention of PID include elimination of risk factors, the careful selection of IUD users, regular checkups, the use of copper (Cu) T device, and strict adherence to professional standards . S Afr Med J, 1986 Aug 2, 70(3), 137 - 42 Surgery in patients with heart transplants . Anaesthetic and operative considerations; Cooper DK et al.; As cardiac transplantation becomes more common, so an increasing number of patients with functioning heart transplants may require surgery for related or unrelated non-cardiac conditions . Fifteen patients who have undergone a total of 39 operations (excluding retransplantation) since heart transplantation were reviewed; 36% were for infective conditions and 23% each for gastro-intestinal and vascular lesions . There was one postoperative death in a patient undergoing leg amputation for overwhelming Clostridium welchii infection . There were no major non-fatal complications . The conditions for which operation may be necessary, the specific problems of anaesthesia and surgery in such patients, and the prophylactic measures which may be undertaken to ensure an uncomplicated clinical course are discussed . A clear understanding of the physiology and pharmacology of the denervated heart is essential if these patients are successfully to undergo major operations requiring general anaesthesia. J Antibiot (Tokyo), 1986 Aug, 39(8), 1054 - 8 SQ 30,957, a new antibiotic produced by Penicillium funiculosum . Taxonomy, fermentation, isolation, structure determination, synthesis and antibacterial activity; Singh PD et al.; A new antibiotic, SQ 30,957, 4-diazo-3-methoxy-2,5-cyclohexadien-1-one, has been isolated from fermentation broths of Penicillium funiculosum . The structure (1) was deduced from its spectroscopic properties and its degradation reaction . SQ 30,957 has excellent activity against anaerobic bacteria such as Clostridium and Bacteroides and has moderate activity against aerobic bacteria . The compound has an LD50 of less than 17 mg/kg in mice by intraperitoneal administration. Eur J Clin Microbiol, 1986 Aug, 5(4), 441 - 5 Hydrophobic and adherence properties of Clostridium difficile; Wood-Helie SJ et al.; Nine strains of Clostridium difficile isolated from symptomatic and asymptomatic patients and four other species of clostridia were tested for relative hydrophobicity by determining the degree of adherence to polystyrene . Under three different conditions of growth all strains of Clostridium difficile had high rates of adherence, whereas the other clostridial species showed no pronounced adherence . Isolates of Clostridium difficile were also tested for their ability to adhere to human embryonic intestinal cells and adult colon cells . All strains adhered to both cell lines, although the percentages of organisms adhering varied . Adherence was greatest at pH 5.5-6.0 but was not significantly altered at a pH of 7.0-7.8 (p = 0.15, p = 0.20); it decreased significantly upon washing with 1% Tween 80 but not with 0.1% Tween 80 . This capacity for adherence may play a part in the organism's colonization of the human intestinal tract. J Clin Pathol, 1986 Aug, 39(8), 861 - 2 Isolation of Clostridium difficile from human jejunum: identification of a reservoir for disease? Testore GP, Nardi F, Babudieri S, Giuliano M, Di Rosa R, Panichi G. The possibility that the small intestine may represent a reservoir for Clostridium difficile was studied, using segments of human jejunum collected at necropsy . Our results (three of 100 specimens positive for C difficile culture) support the hypothesis that C difficile can be found in human jejunum and that it adheres to the normal mucosa as a resident bacterium . These findings suggest that gastrointestinal disease caused by C difficile has an endogenous origin. J Clin Microbiol, 1986 Aug, 24(2), 181 - 5 Predicting the susceptibility of anaerobes to cefoperazone, cefotaxime, and cefoxitin with the thioglycolate broth disk procedure; Zabransky RJ et al.; A variety of clinical anaerobic isolates were tested against cefoperazone (216 strains), cefoxitin (120 strains), and cefotaxime (120 strains) by the thioglycolate anaerobic broth disk method, and the results were compared with the National Committee for Clinical Laboratory Standards reference agar dilution method . The broth disk and reference breakpoint concentrations were as follows: cefoperazone, 60 and 64 or 30 and 32 micrograms/ml; cefotaxime, 30 and 32 micrograms/ml; cefoxitin, 18 and 16 micrograms/ml, respectively . Discrepant results were retested to obtain a mode . There was 99% agreement between the broth disk and reference methods for cefotaxime, 98% for cefoperazone with 60- and 64-micrograms/ml breakpoints and 91% with 30- and 32-micrograms/ml breakpoints, and 75% for cefoxitin . All but one of the strains that produced false susceptibility results by broth disk were members of the Bacteroides fragilis group, 1 with cefoperazone using the 60-micrograms/ml concentration, 14 with cefoperazone at the 30-micrograms/ml concentration, and 27 with cefoxitin . One strain of Clostridium difficile produced false susceptibility results to cefoperazone at the 30-micrograms/ml concentration . The lack of agreement between the broth disk and reference methods with cefoxitin may be a reflection of the number of isolates at the 16-micrograms/ml level and that the broth disk breakpoint was slightly higher than this concentration . Increased incubation time did not improve the results significantly. Br J Exp Pathol, 1986 Aug, 67(4), 617 - 21 Individual variation in botulism; Smith GR; Mice were treated per os with one oral LD100 of toxic filtrate from a culture of Clostridium botulinum type C . The period between dosing and the first appearance of clinical signs varied greatly (2-31 h) from one animal to another . The duration of the pre-clinical and clinical phases together ranged from 5.5 to greater than 55 h . The duration of the clinical phase alone ranged from 1.25 to greater than 24 h, except for a minority of mice in which death occurred suddenly from apparent heart failure with no premonitory signs 4.75-31 h after dosing . Toxaemia was demonstrable in all mice that had just begun to show a clinical response 3.75-6.5 h after dosing, and in some that had not . Outside these time limits toxaemia was demonstrable only rarely, and beyond 12 h after dosing never . Therefore the many (approximately 50%) mice that began to show clinical signs more than 12 h after dosing had no demonstrable toxaemia throughout the entire clinical phase of the disease . The concentrations of toxin demonstrated in the blood ranged from less than 5 to greater than or equal to 20 (but less than 40) intravenous mouse-lethal doses/ml. Am J Clin Pathol, 1986 Aug, 86(2), 208 - 11 Detection of Clostridium difficile toxins A (enterotoxin) and B (cytotoxin) in clinical specimens . Evaluation of a latex agglutination test; Peterson LR et al.; A new latex test, Culturette Brand Rapid Latex Test for detection of Clostridium difficile toxin A, was tested on 408 stool samples . In 247 frozen tissue culture supernate specimens previously obtained from patients with C . difficile-associated diarrhea (CAD), the latex test (enterotoxin) was positive in 182 (74%) as compared with 194 (79%) for the repeat tissue culture (P greater than 0.1) cytotoxin (toxin B) test . Testing of 161 fresh stool samples found the latex test superior to tissue culture (P less than 0.05) in cases of CAD (90% positivity vs . 70%), with the two tests being equal in both non-CAD diarrheal and non-diarrheal control groups . In vitro evaluation of 61 C . difficile isolates found all (100%) to be producers of enterotoxin A, while only 53 (87%) produced toxin B . The latex test for C . difficile toxin detection is a rapid, simple test for use in the diagnosis in CAD. J Med Microbiol, 1986 Aug, 22(1), 33 - 8 Sporogenesis and toxin A production by Clostridium difficile; Ketley JM et al.; The kinetics of spore production by Clostridium difficile were not paralleled by release of C . difficile toxin A in vitro . Toxin A was not found to be associated with either purified whole spores or spore coats . Residual traces of toxin A detected in spore contents were almost certainly derived from contaminating vegetative cell debris . Thus, toxin A is unlikely to be a spore constituent or associated with sporogenesis. Clin Chem, 1986 Aug, 32(8), 1503 - 5 Effect of phospholipase C on high-molecular-mass alkaline phosphatase in serum; Sykes E et al.; Electrophoresis of some serum samples on polyacrylamide gel, followed by staining for alkaline phosphatase (EC 3.1.3.1), produces a band of activity at the gel origin . This high-Mr band consists of liver membrane fragments containing alkaline phosphatase and other enzymes . Alkaline phosphatase is closely associated with phosphatidylinositol in liver plasma membranes, and we have found that phospholipase C (EC 3.1.4.3) from Bacillus cereus, known to possess some phosphatidylinositol specificity, was able to release liver alkaline phosphatase from the high-Mr band . Two preparations of phospholipase C from Clostridium perfringens, however, which has no phosphatidylinositol specificity, had no effect on the alkaline phosphatase activity in the high-Mr band. J Infect Dis, 1986 Aug, 154(2), 207 - 11 Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy; Aureli P et al.; The first two confirmed cases of type E infant botulism occurred in two 16-week-old girls in Rome, Italy . The original diagnosis for the first patient was intestinal blockage due to an ileocecal invagination, which was treated surgically . Postoperatively, the patient became unresponsive and required ventilatory assistance . A diagnosis of infant botulism was then made . The second infant presented to the same hospital 7 1/2 months later with profound weakness, hypotonicity, mydriasis, and areflexia . This case was recognized as possible botulism at admission . Both cases were confirmed by detection and identification of type E botulinal toxin in stool specimens and in enrichment cultures of those specimens . The toxigenic organisms isolated were quite different from Clostridium botulinum type E . The apparent causative organism in each case resembles Clostridium butyricum but produces a neurotoxin that is indistinguishable from type E botulinal toxin by its effects on mice and by its neutralization with type E botulinal antitoxin. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1986 Aug, 262(2), 179 - 88 Molecular characterization of a protein, insoluble at low temperature, produced by Clostridium botulinum type G; Gimenez JA et al.; A preliminary study of a low-toxicity protein, called cryoprotein, produced by Clostridium botulinum type G, led to a better characterization of this substance and to discriminate its relationship with type G botulinum toxin . This sparingly soluble protein has been characterized as an aggregated form of a soluble precursor with an Mr of 170,000 . This phenomenon is temperature-dependent . The monomeric protein is usually contaminated with a lower Mr form (150,000) quite probably originated by a limited proteolytic process . The amino acid composition of this protein is relatively analogous to that of the botulinum toxins A and B, the only notable exception being the absence of cysteine . The N-terminal amino acid is alanine and the C-terminal sequence is Val-Ala-Leu-OH . The low toxicity which is usually demonstrable in samples of this protein disappears after a reductive treatment, strongly suggesting that it is not an intrinsic property . Taking into account that some of its physiochemical properties are similar to those of the known botulinal toxins, it is quite probable that this substance accompanies G toxin preparations currently obtained by routine methods, increasing its non-toxic antigenic mass . This fact could be critical to the sensitivity and specificity of G toxin immunological detection methods. Microb Pathog, 1986 Aug, 1(4), 373 - 85 Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B; Florin I et al.; The process of internalisation of Clostridium difficile toxin B into human lung fibroblasts was further studied, with the aim of elucidating the fate of endocytosed toxin . Development of the toxin-induced cytopathogenic effect was reversibly inhibited at 18 degrees C and in the presence of 200 mM KCl or 1-20 mM benzyl alcohol, i.e . at conditions when the fusion between endosomes and lysosomes is prevented . Fibroblasts treated with toxin at 37 degrees C but transferred to 18 degrees C within 10 min were also completely protected, whereas transfer to 18 degrees C later during the latency resulted in only partial protection . KCl was also protective upon addition after the toxin binding step . Inhibitors of lysosomal proteases, such as chymostatin, leupeptin and antipain, prevented the appearance of the cytopathogenic effect, when present during toxin exposure or added after the toxin binding step . Chinese hamster ovary cell mutants, defective in acidification of their endosomes, were resistant to toxin B, whereas wildtype cells were sensitive . The resistance was not overcome by applying a low extracellular pH . The results suggest that exposure to a low pH compartment is necessary but not sufficient for entry of active toxin B to the cytosol . In addition to a low pH, a fusion of toxin-containing endosomes with lysosomes and a further processing of the toxin by lysosomal proteases is required for cellular intoxication. Mol Gen Genet, 1986 Aug, 204(2), 317 - 21 Cloning of Clostridium acetobutylicum genes and their expression in Escherichia coli and Bacillus subtilis; Efstathiou I et al.; DNA from Clostridium acetobutylicum ABKn8 was partially digested with Sau3A and the fragments obtained were inserted into the unique BamHI site of the cloning vector pHV33 . The recombinant plasmids were used to transform Escherichia coli HB101 with selection for ampicillin resistance . A collection of ampicillin-resistant, tetracycline-sensitive clones representative of the Clostridium acetobutylicum genome was made . The clones were shown to carry recombinant plasmids each containing an insert of 2 to 16 kb in size . Several of them complemented the HB101 proA2 or leuB6 auxotrophic mutations . The cloned sequences were shown by Southern blot hybridization to be homologous to the corresponding ABKn8 DNA fragments. J Biochem (Tokyo), 1986 Aug, 100(2), 407 - 13 NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin; Ohishi I; C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two separate protein components, designated components I and II, which individually have little activity, but, when mixed and treated with trypsin, exert the potent activity . The present study provides the evidence that component I of the toxin catalyzes the hydrolysis of NAD into nicotinamide and ADP-ribose, whereas component II does not, indicating that component I of C2T has NAD-glycohydrolase activity, which ability is shared with cholera and diphtheria toxins . However, C2T affected neither glycerol production of fat cells nor protein synthesis in cell-free system . Component I of C2T in the presence of {alpha-32P}NAD radiolabeled a protein of Mr 46,000 in the supernatant fractions of mouse tissue homogenates; the protein was abundant in brain, lung and intestine, whereas there was little or none of the protein in muscle . These results indicate that component I can catalyze the covalent attachment of the ADP-ribose moiety of NAD to intracellular protein, which differs from those modified with cholera and diphtheria toxins . The present data, together with previous findings, suggest that the biological activity of C2T is elicited by ADP-ribosylation activity of component I, which is internalized into the cells after binding to the receptor site introduced with the binding of component II to the cell surface membrane. J Am Acad Dermatol, 1986 Aug, 15(2 Pt 1), 180 - 5 Effects of topical clindamycin on intestinal microflora in patients with acne; Siegle RJ et al.; Thirty-two patients with acne completed a randomized, double-blind study using topical 1% clindamycin phosphate or its vehicle applied twice daily for 8 weeks for a study of its effects on the intestinal microflora . Two clindamycin patients and one vehicle patient had Clostridium difficile in stools prior to therapy . Of the remaining twenty-nine patients, four of nineteen patients who used clindamycin and none of ten patients who used vehicle had C . difficile detected during treatment; the difference was not statistically significant . There was no diarrhea in the clindamycin group, even though clostridial cytotoxin was found transiently in two patients . Self-limited diarrhea occurred in one vehicle-treated patient, whose stool culture was negative but whose stool specimen showed a positive reaction for C . difficile cytotoxin . With the use of a bioassay, clindamycin was not detected in urine or stool of any patient . No significant changes in Bacteroides fragilis counts in stools were observed in either group. J Lipid Res, 1986 Aug, 27(8), 905 - 9 Reaffirmation of the validity of enzymatic cleavage of lithocholic acid from N-epsilon-lithocholyl-L-lysine and N-alpha-CBZ-N-epsilon-lithocholyl-L-lysine; Nair PP et al.; N-epsilon-lithocholyl-L-lysine or N-alpha-CBZ-N-epsilon-lithocholyl-L-lysine when incubated overnight at 37 degrees C with 3 K units of clostridial cholanoylaminoacid hydrolase (from Clostridium perfringens ATCC 19574) in the presence of disodium EDTA (0.1 M), beta-mercaptoethanol (0.1 M), and sodium acetate buffer, pH 5.6, released free lithocholic acid . The latter material was isolated by thin-layer chromatography and identified by combined gas-liquid chromatography-mass spectrometry in the full scan and selected-ion mode . In order to maintain its activity, the enzyme was always stored in 1.0-ml aliquots at temperatures below -20 degrees C and each aliquot when thawed was used immediately; any left over enzyme was never reused . Contrary to the observations of Yanagisawa et al . (J . Lipid Res . 1984 . 25: 1263-1271) the results of this study reaffirm the validity of the original observations on the enzymatic cleavage of lithocholic acid from tissue-bound form. J Hyg (Lond), 1986 Aug, 97(1), 71 - 80 Large outbreaks of Clostridium perfringens food poisoning associated with the consumption of boiled salmon; Hewitt JH et al.; Five large outbreaks of food poisoning are described in which clinical, epidemiological or laboratory data indicated Clostridium perfringens as the causative organism . The foodstuff common to all incidents was boiled salmon served cold as an hors d 'oeuvre . In all cases the fish had been subject to a long period of cooling or storage between boiling and consumption . It is thought that multiplication of the organism occurred during this time . Recommendations are made for the avoidance of further similar incidents. Nature, 1986 Jul 24-30, 322(6077), 390 - 2 Botulinum C2 toxin ADP-ribosylates actin; Aktories K et al.; ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions . While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase . Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity . This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs . In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects . Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations . We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin . Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form . ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro. Lancet, 1986 Jul 5, 2(8497), 11 - 3 Is Clostridium difficile endemic in chronic-care facilities? Bender BS, Bennett R, Laughon BE, Greenough WB 3rd, Gaydos C, Sears SD, Forman MS, Bartlett JG. An apparent outbreak of Clostridium difficile diarrhoea on the chronic hospital ward of a long-term care facility prompted an investigation lasting seven months . Approximately a third of patients had stools that were positive for C difficile by either toxin or culture . Attempts to eradicate the infection by simultaneously treating all toxin-positive patients with metronidazole, limiting antibiotic use, and implementing enteric isolation were unsuccessful . New cases were both nosocomially acquired and imported into the facility . Of the C difficile toxin-positive patients, 34% had diarrhoea and 19/49 (38%) died during the study period . C difficile is not routinely sought by most clinical microbiology laboratories and may therefore be endemic in many long-term care facilities for the elderly. Int J Pediatr Nephrol, 1986 Jul-Sep, 7(3), 173 - 6 Fever due to Clostridium difficile during hemodialytic treatment; Agrawal R et al.; A ten-year-old on hemodialysis had a prolonged unexplained fever secondary to Clostridium difficile antibiotic-associated colitis and posed a great diagnostic challenge. Lab Anim, 1986 Jul, 20(3), 266 - 70 Isolation of Clostridium difficile from various colonies of laboratory mice; Itoh K et al.; An attempt was made to isolate Clostridium difficile from a total of 565 mice from nine different conventional mouse colonies and six different specified-pathogen-free mouse colonies . C . difficile was isolated from all the conventional colonies but from none of the specified-pathogen-free colonies . Ampicillin injected intraperitoneally increased the isolation rate of C . difficile from mouse faeces to 63.6% compared with 19.4% from untreated mice. J Biochem (Tokyo), 1986 Jul, 100(1), 27 - 33 Binding of Clostridium botulinum neurotoxin to gangliosides; Ochanda JO et al.; The binding characteristics of Clostridium botulinum neurotoxins of types B, C1, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) or high (150 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) ionic strengths and at 0 or 37 degrees C . The three types of toxins bound exclusively to three kinds of gangliosides, GD1a, GD1b, and GT1b, in both the thin layer chromatography plate and the microtiter plate methods . Type C1 toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37 degrees C . At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4 X 10(8) M-1 . In contrast, the binding kinetics of type C1 toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system . The heavy chain facilitated the binding of the toxin to the gangliosides . These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins . The gangliosides may bind to type C1 toxin together with other potential receptor(s) on synaptosomal membranes. J Appl Bacteriol, 1986 Jul, 61(1), 81 - 6 Stoichiometry of glucose and starch splitting by strains of amylolytic bacteria from the rumen and anaerobic digester; Marounek M et al.; The stoichiometry of glucose and starch splitting by the amylolytic bacteria Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Eubacterium ruminantium and Clostridium sp . was followed . There were many differences in the ratios of metabolites and in growth yields, as well as in the cell composition, between the growth on glucose and starch . The bacteria employ different nutritional strategies with respect to both energy sources. Ann Gastroenterol Hepatol (Paris), 1986 Jul-Sep, 22(4), 235 - 40 {Pseudomembranous rectocolitis}; Debat J et al.; The following conclusions were drawn from a study of 15 cases of pseudo-membranous coloproctitis (PMCP): PMCP was seen in subjects of both sexes and all ages . The causative agent was found in all antibiotic classes . Clinical signs comprised constant diarrhea, fever, abdominal pain, toxic shock and, more rarely, pseudo-occlusive, pseudo-perforative surgical evidence . Diagnosis involved visualization of pseudo-membranes by endoscopy . Lesions were most frequent in the left colon and increased in severity towards the distal end . Three stages were distinguished by histological examination: superficial necrosis of the mucous membranes, interruption of glands, complete necrosis of the mucous membrane . Without preparation the abdomen did not provide specific information; nor did barium enema which revealed lesions that were frequently diffuse but more marked in the left colon . Conventional coprocultures did not provide diagnostic information . Only a more sophisticated technique will be capable of detecting the pathogen currently considered to be the cause of PMCP: Clostridium difficile . The course of the disorder is generally satisfactory under medical treatment (parenteral feeding, vancomycin) but may sometimes call for surgery. Ann Gastroenterol Hepatol (Paris), 1986 Jul-Sep, 22(4), 217 - 22 {Undesirable colorectal effects of drugs}; Cheymol G et al.; The most severe adverse reactions associated with medicinal treatment of the colon involve pseudomembranous colitis following antibiotic treatment, notably with clindamycin, lincomycin and betalactamine . The frequency of this adverse reaction is poorly defined: 1 per 100 to 1 per 5 000 treatments depending on the study . Lesions are explained by the cytotoxic effect of Clostridium difficile toxin . Necrotizing anorectitis has been seen in cases of abuse of suppositories containing propoxyphene . The mechanism involved is still unknown. Br J Pharmacol, 1986 Jul, 88(3), 531 - 9 Excitatory effect of Clostridium perfringens alpha toxin on the rat isolated aorta; Fujii Y et al.; Clostridium perfringens alpha toxin caused contraction of the isolated aorta of the rat in a dose-dependent manner . The contractile action caused by the toxin was inhibited or abolished by calcium antagonists such as nifedipine, verapamil and cinnarizine, or a Ca-free medium, but was not affected by phentolamine, chlorpheniramine, atropine, tetrodotoxin or a low Na medium . The toxin stimulated Ca uptake into the aorta in a dose-dependent manner . 8-N,N'-diethylaminooctyl-3,4,5-trimethoxybenzoate (TMB-8) blocked significantly both the toxin- and noradrenaline (NA)-induced contractions . Trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphtharene sulphonamide (W-7) did not affect the contractile activity of the toxin but blocked the NA-induced contraction . The toxin also stimulated the 32P phosphate labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in the preparation . These results indicate that the toxin-induced contraction, which is different from that induced by NA, is the result of a direct action of the toxin on the aorta and is due to an increased Ca2+ permeability across the smooth muscle membrane . It is suggested that the contractile response to the toxin is associated with activation of phospholipid metabolism and enhanced entry of Ca into the aorta. J Infect, 1986 Jul, 13(1), 5 - 9 Application of a technique for serogrouping Clostridium difficile in an outbreak of antibiotic-associated diarrhoea; Delmee M et al.; A severe outbreak of Clostridium difficile antibiotic-associated diarrhoea (AAD) in an orthopaedic surgical unit is reported . Thirty-seven cases and eight relapses were observed . The 45 related strains together with another 13 strains of C . difficile isolated during the same period in other wards of the same hospital were typed by detection of cytotoxin production, determination of sorbitol fermentation and serogrouping by agglutination with six rabbit antisera defining the serogroups A, B, C, D, F and G . All the strains from the outbreak belonged to serogroup C, were toxigenic and fermented sorbitol . In contrast, four different patterns were observed in seven isolates from cases of AAD in other wards . Finally, six strains isolated from four asymptomatic adults, one adult suffering from shigellosis and one child with salmonellosis demonstrated two patterns different from that of the epidemic isolates . The data strongly suggest nosocomial spread of this micro-organism. J Appl Bacteriol, 1986 Jul, 61(1), 39 - 49 The effect of citric acid on growth of proteolytic strains of Clostridium botulinum; Graham AF et al.; In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d . Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks . Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6) . The citric acid also reduced the concentration of free Ca2+ in the medium . The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+ . The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium . The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+ . Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2 . The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3. Ann Inst Pasteur Microbiol, 1986 Jul-Aug, 137B(1), 113 - 21 Ability of two Clostridium difficile strains from man and hare to produce cytotoxin in vitro and in gnotobiotic rodent intestines; Corthier G et al.; Cytotoxin production by human (VP1) and hare (FD) strains of Clostridium difficile were compared both in vitro in a broth culture and in vivo in intestinal contents of gnotobiotic rodents . Strain VP1 produced about 1,000 times more cytotoxin than the FD strain, both in vitro and in vivo, although the population levels of the two strains were not significantly different either in vitro or in vivo . Ninety percent of gnotobiotic rats and 100% of gnotobiotic mice established with the VP1 strain died within 3 days, whereas no mortality in rats or mice was observed with the FD strain . The cytotoxin titre increased during the 3 weeks following establishment of the FD strain in mice, decreasing thereafter . Mice previously established with the FD strain were protected from VP1 strain challenge . Cytotoxin production was greatly decreased in diassociated mice, although the population levels of the two strains did not differ to a great extent. Biochem J, 1986 Jul 1, 237(1), 235 - 42 Mechanism of hepatic glycogen synthase inactivation induced by Ca2+-mobilizing hormones . Studies using phospholipase C and phorbol myristate acetate; Blackmore PF et al.; Incubation of hepatocytes with the protein kinase C activator and tumour promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) produced a time- and concentration-dependent inactivation of glycogen synthase, but no change in phosphorylase . The same rate and extent of inactivation occurred in hepatocytes depleted of Ca2+ by treatment with the Ca2+ chelator EGTA . When hepatocytes were treated with the Ca2+-mobilizing hormone vasopressin (10 nM), the rate of glycogen synthase inactivation was similar to that observed with PMA (1 microM) . Depletion of intracellular Ca2+ stores with EGTA abolished the ability of vasopressin to mobilize Ca2+ and activate phosphorylase without abolishing its ability to inactivate glycogen synthase and increase 1,2-diacylglycerol (DAG), the endogenous activator of protein kinase C . Protein kinase C, either in membranes or after partial purification, was shown to be activated in vitro by PMA in the presence of very low concentrations of Ca2+ . Exogenous phospholipase C from Clostridium perfringens, at low concentrations, inactivated glycogen synthase and increased DAG without affecting cell Ca2+ or phosphorylase . It is proposed that the inactivation of glycogen synthase elicited by the Ca2+-mobilizing hormones is due, at least in part, to generation of DAG and activation of protein kinase C. Avian Dis, 1986 Jul-Sep, 30(3), 620 - 2 Subcutaneous clostridial infection in broilers; Hofacre CL et al.; A flock of 12,500 broilers 36 days of age experienced a sudden increase in mortality . Post-mortem lesions were emphysema, severe enteritis, and a serosanguineous fluid in the subcutaneous tissue of the breast and thighs; there was no evidence of a loss in the integrity of the skin . Clostridium perfringens and C . septicum were isolated from the affected subcutaneous tissue . Histopathological and serological examination indicated previous infection with infectious bursal disease virus . The subsequent immunosuppression and severe enteritis may have permitted the clostridia access to the circulatory system, with localization in the subcutaneous areas of the breast and thighs . Mortality returned to normal 48 hours after potassium penicillin G was administered via the drinking water. J Bacteriol, 1986 Jul, 167(1), 205 - 9 Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi-urici"); Whitehead TR et al.; The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8 . A C . acidiurici genomic library was prepared in E . coli from a partial Sau3A digest and screened with antibody against the synthetase . Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E . coli had the same subunit molecular weight as the C . acidiurici enzyme . The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter . Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C . acidiurici chromosome. Avian Dis, 1986 Jul-Sep, 30(3), 617 - 9 Necrotic enteritis in cage-reared commercial layer pullets; Broussard CT et al.; Necropsy of five 12-week-old pullets from a flock of 99,300 suffering from an increased mortality rate revealed enlarged, gas-filled intestines, the mucosal surfaces of which had the "dirty turkish towel" appearance typical of necrotic enteritis . Although the pullets had been raised entirely in cages, intestinal scrapings revealed the presence of Eimeria maxima . Histopathological findings were compatible with necrotic enteritis . Clostridium perfringens was isolated by anaerobic culture from the intestines . Mortality returned to normal after bacitracin and amprolium were added to the feed. Appl Environ Microbiol, 1986 Jul, 52(1), 214 - 6 Lethal effect of butylated hydroxyanisole as related to bacterial fatty acid composition; Post LS et al.; Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas fragi, Escherichia coli, and Salmonella "anatum" were challenged with butylated hydroxyanisole (BHA) . Susceptibility was measured as the concentration of BHA required to cause a 90% reduction in bacterial survivors . Staphylococcus aureus LP and P . fragi were two of the most resistant species examined; C . perfringens and P . fluorescens were the most susceptible . Gram stain reaction was found not to be a strict indicator of bacterial susceptibility to BHA . There was no obvious relationship between individual fatty acids and susceptibility . The ratio of saturated to unsaturated fatty acids in the total lipid fraction of only the gram-positive species was related to susceptibility . The ratios of saturated to unsaturated fatty acids of other fractions were not related to susceptibility. Appl Environ Microbiol, 1986 Jul, 52(1), 197 - 9 Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum; Roberts I et al.; A rapid plasmid isolation procedure for Clostridium perfringens and C . absonum is described . The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation . The method can be scaled up, without difficulty, for large-scale plasmid preparation. J Leukoc Biol, 1986 Jul, 40(1), 65 - 72 Ganglioside identification on human monocyte membrane with Clostridium perfringens delta-toxin; Cavaillon JM et al.; Clostridium perfringens delta-toxin was first described as a hemolysin with a restricted lytic spectrum . A selective cytotoxicity of the delta-toxin was then found on rabbit leukocytes: peritoneal and alveolar macrophages were uniformly killed, whereas thymocytes were essentially resistant . The toxin was shown to be specific for GM2 ganglioside or a GM2-like structure . In the present study we report the interaction of delta-toxin with human monocytes . A specific, saturable, and irreversible binding of 125I-delta-toxin was demonstrated . Binding was inhibited by preincubation of the radiolabeled toxin with GM2 and with high amount of GM1 ganglioside . As judged by dye exclusion, no cytotoxicity was observed on freshly isolated monocytes, but when added at the beginning of a culture of human adherent cells, the cytotoxic effect was detected after 48 hours of culture . Taken together, these data indicate the presence of monosialoganglioside(s) at the surface of human monocytes, and suggest a possible reorganisation of such structure into the cell membrane when monocytes mature in vitro toward macrophage-like cells. J Gen Microbiol, 1986 Jul, 132 ( Pt 7), 1981 - 8 Activation of Clostridium botulinum type E toxin purified by two different procedures; Yokosawa N et al.; Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods . The specific activity {2 X 10(4) mouse LD50 (mg p