Clostridia

Arch Microbiol, 1995 Apr, 163(4), 310 - 2
Purification and characterization of the NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum; Bayer M et al.; An NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum was purified 15-fold with a yield of 46% . It was homogeneous by gel electrophoresis after three chromatographic steps . The apparent molecular mass was estimated by column chromatography to be 240 kDa . SDS-gel electrophoresis revealed the presence of 33 kDa subunits . Substrates of the enzyme were ethyl and methyl 3-oxobutyrate, 3-oxobutyryl-N-acetylcysteamine thioester, and 3-oxobutyryl coenzyme A . The specific activities were 340 and 10U (mg protein)-1 for the reduction of 3-oxobutyryl coenzyme A and ethyl 3-oxobutyrate, respectively; the Michaelis constants were 300 microM and 300 mM, respectively . The identity of 12 N-terminal amino acid residues was determined . The enzyme was used in a preparative reduction of substrate, yielding ethyl (S)-3-hydroxybutyrate (> 99% enantiomeric excess).

Arch Microbiol, 1995 Apr, 163(4), 286 - 90
Purification and characterization of threonine dehydrogenase from Clostridium sticklandii; Wagner M et al.; Threonine dehydrogenase from Clostridium sticklandii has been purified 76-fold from cells grown in a defined medium to a homogeneous preparation of 234 units.mg-1 protein . Purification was obtained by chromatography on Q-Sepharose fast flow and Reactive green 19-Agarose . The native enzyme had a molecular mass of 67 kDa and consisted of two identical subunits (33 kDa each) . The optimum pH for catalytic activity was 9.0 . Only L-threo-threonine, DL-beta-hydroxynorvaline and acetoin were substrates; only NAD was used as the natural electron acceptor . The apparent Km values for L-threonine and NAD were 18 mM and 0.1 mM, respectively . Zn2+, Co2+ and Cu2+ ions (0.9 mM) inhibited enzyme activity . The N-terminal amino acid sequence revealed similarities to the class of non-metal short-chain alcohol dehydrogenases, whereas the threonine dehydrogenase from Escherichia coli belongs to the class of medium chain, zinc-containing alcohol dehydrogenases.

Appl Environ Microbiol, 1995 Apr, 61(4), 1257 - 65
Cyclodextrin formation by the thermostable alpha-amylase of Thermoanaerobacterium thermosulfurigenes EM1 and reclassification of the enzyme as a cyclodextrin glycosyltransferase; Wind RD et al.; Extensive characterization of the thermostable alpha-amylase of Clostridium thermosulfurogenes EM1, recently reclassified as Thermoanaerobacterium thermosulfurigenes, clearly demonstrated that the enzyme is a cyclodextrin glycosyltransferase (CGTase) . Product analysis after incubation of the enzyme with starch revealed formation of alpha-, beta-, and gamma-cyclodextrins, as well as linear sugars . The specific activity for cyclization of this CGTase was similar to those of other CGTases, whereas the specific activity for hydrolysis was relatively high in comparison with other CGTases . Alignment of the amino acid sequence of the T . thermosulfurigenes enzyme with sequences from known bacterial CGTases showed high homology . The four consensus regions of carbohydrate-converting enzymes, as well as a C-terminal raw-starch binding motif, could be identified in the sequence.

Eur J Biochem, 1995 Apr 1, 229(1), 77 - 82
Purification and characterization of an oxygen-sensitive reversible 4-hydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum; He Z et al.; A 4-hydroxybenzoate decarboxylase from the anaerobe Clostridium hydroxybenzoicum strain JW/Z-1T was purified and partially characterized . It had an apparent molecular mass of 350 kDa and consisted of six identical subunits of 57 kDa each . The temperature optimum for the decarboxylation was approximately 50 degrees C, the optimum pH 5.6-6.2 . The pI of the enzyme was 5.1 . The activation energy for decarboxylation of 4-hydroxybenzoate was 65 kJ.mol-1 (20-37 degrees C) . The enzyme also catalyzed decarboxylation of 3,4-dihydroxybenzoate . The apparent Km and kcat values obtained for 4-hydroxybenzoate were 0.40 mM and 3.3 x 10(3) min-1, and for 3,4-dihydroxybenzoate 1.2 mM and 1.1 x 10(3) min-1, respectively, at pH 6.0 and 25 degrees C . The enzyme activity was not influenced by the addition of biotin or avidin to either the crude cell extracts or the purified enzyme . The p-hydroxyl group of hydroxybenzoate appears to be essential for binding by the enzyme . The N-terminal amino acid sequence shows some similarity to the uroporphyrinogen decarboxylases from Synechococcus and Saccharomyces . The enzyme catalyzed the reverse reactions, that is, the carboxylation of phenol to 4-hydroxybenzoate and of catechol to 3,4-dihydroxybenzoate . The carboxylation did not require ATP.

Eur J Biochem, 1995 Apr 1, 229(1), 61 - 9
Structural studies on the zinc-endopeptidase light chain of tetanus neurotoxin; De Filippis V et al.; Tetanus neurotoxin (TeNT) blocks neuroexocytosis via a zinc-endopeptidase activity highly specific for vescicle-associated membrane protein(VAMP)/synaptobrevin . TeNT is the prototype of clostridial neurotoxins, a new family of metalloproteinases . They consist of three domains and the proteolytic activity is displayed by the 50-kDa light chain (L chain) . The L chain was isolated here in the native state from bacterial filtrates of Clostridium tetani and its structure was studied via circular dichroism (CD) and fluorescence spectroscopy . The secondary structure content (27% alpha-helix and 43% beta-sheet), estimated by far-ultraviolet CD measurements, was in reasonable agreement with that obtained by standard predictive methods (25% alpha-helix and 49% beta-sheet) . Moreover, the hypothetical zinc-binding motif, encompassing residues His-Glu-Leu-Ile-His, was correctly predicted to be in alpha-helical conformation, as also expected on the basis of the geometrical requirements for a correct coordination of the zinc ion . Both near-ultraviolet CD and fluorescence data strongly suggest that the single Trp43 residue is buried and constrained in a hydrophobic environment, likely distant from the zinc ion located in the active-site cleft . The contribution of the bound zinc ion to the overall conformation of TeNT L chain was investigated by different and complementary techniques, including spectroscopic (far- and near-ultraviolet CD, fluorescence, second derivative absorption spectroscopy) as well as proteolytic probes . The results indicate that the zinc ion plays little, if any, role in determining the structural properties of the L chain molecule . Similarly, the metal-free apo-enzyme and the holo-protein share common stability features evaluated in respect to different physico-chemical parameters (pH, temperature and urea concentration) . These results parallel those obtained on thermolysin, a zinc-dependent neutral endoprotease from Bacillus thermoproteolyticus, where both conformational and stability properties are unchanged upon zinc removal.

Eur J Biochem, 1995 Apr 1, 229(1), 308 - 15
Characterization of the glycan structure of a major glycopeptide from the surface layer glycoprotein of Clostridium thermosaccharolyticum E207-71; Altman E et al.; The squarely arranged surface layer (S-layer) glycoprotein of Clostridium thermosaccharolyticum E207-71 was isolated from bacterial cells which were grown under defined culture conditions . By sodium dodecyl sulfate polyacrylamide gel electrophoresis, the S-layer showed a series of distinct bands with apparent molecular masses in the range 83-210 kDa . Upon deglycosylation by trifluoromethanesulfonic acid, only the single band at 83 kDa remained unchanged . After pronase digestion of the intact S-layer glycoprotein, the degradation products were isolated by gel-permeation chromatography, cation-exchange chromatography and isoelectric focusing . Three main fractions and an amino sugar containing minor fraction were obtained . The main fractions, which showed identical carbohydrate compositions, were further purified by reverse-phase chromatography and characterized by monosaccharide analysis, Smith degradation, methylation analysis, and one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy . The combined chemical and spectroscopical evidence suggest the following glycan structure for the main fractions: {Sequence: See text}

Dis Colon Rectum, 1995 Apr, 38(4), 350 - 4
Severe Clostridium difficile colitis; Rubin MS et al.; PURPOSE: Reports of fatality related to Clostridium difficile colitis and a sharp increase in prevalence of this infection prompted a study of patients who develop a more aggressive form of this disease . METHODS: Over 38 months, 710 patients at our institution developed C . difficile colitis . Twenty-one (3 percent) of these patients either required intensive care unit admission or died as a result of their infection . A retrospective, case-controlled study was undertaken to compare these patients, who were considered to have severe C . difficile colitis, with the remaining patients with milder disease . RESULTS: Factors that predisposed to the development of severe C . difficile colitis included intercurrent malignancy, chronic obstructive pulmonary disease, immunosuppressive and antiperistaltic medications, renal failure, and administration of clindamycin (P < 0.05 for all) . Patients with severe C . difficile colitis were more likely to have abdominal pain, tenderness and distention, peritonitis, hemoconcentration (> 5 points), hypoalbuminemia (< 3 mg/dl), and elevated or suppressed white blood cell count (> 25,000; < 1,500; P < 0.05 for all) . These factors were used to create a scoring system that could distinguish between patients with severe C . difficile colitis and those with mild disease . Thirteen patients in the late stages of terminal illness with metastatic malignancy or age > 90 were considered poor or inappropriate surgical candidates . Only the remaining eight patients could have potentially recovered from operation with hope for long-term survival . Of these, seven were treated without colonic resection, and six of the seven survived, whereas one patient underwent colectomy and did not survive . CONCLUSIONS: Patients with severe C . difficile colitis can be readily identified . Often they have coexisting illness that precludes operation . In this series, only 1 of 21 patients with severe C . difficile might have benefited from an aggressive surgical approach.

Am J Respir Cell Mol Biol, 1995 Apr, 12(4), 369 - 78
Augmentation of permeability in the bronchial epithelium by the house dust mite allergen Der p1; Herbert CA et al.; Sequence analyses have revealed the existence of homology between certain aeroallergens and proteolytic enzymes . This homology can be expressed functionally, but its significance to airway pathophysiology is unknown . Studies with Madin-Darby canine kidney cells and canine tracheal epithelial cells grown on plastic substrata or matrix proteins suggest that Der p1, a major allergen from the house dust mite Dermatophagoides pteronyssinus and a cysteine proteinase, or the unfractionated growth medium extract (SGME) from which it was purified, are both capable of causing cell detachment . The ability of both agents to produce functional changes in the barrier function of the epithelium was further demonstrated using isolated bovine airway preparations . Over a 3-h duration, both Der p1 and SGME elicited significant increases in the permeability of isolated sheets of bronchial mucosa to serum albumin . Exposure of isolated bronchial segments to luminally applied solutions of Der p1 resulted in histologic evidence of epithelial injury . Neither Der p1 nor SGME was active in these experimental systems unless chemically reduced, suggesting that the effect was initiated by cysteine proteinase activity . Similar augmentation of mucosal permeability and tissue injury was produced by bovine trypsin and collagenase from Clostridium histolyticum . In both the isolated mucosal sheet model and in cultured cells, the actions of Der p1 or SGME were associated with relatively little cytolysis, suggesting a specific action of the reagents on cell attachment . These results demonstrate a new functional activity of Der p1 that may be germane to the processes of allergen presentation, inflammatory cell activation, and chronic tissue injury.

Avian Dis, 1995 Apr-Jun, 39(2), 375 - 81
The effects of dietary lactose and rye on cecal colonization of Clostridium perfringens in chicks; Takeda T et al.; Five experiments were conducted to determine the effects of dietary lactose and rye on cecal colonization of Clostridium perfringens in white leghorn chickens . Six days after oral inoculation of the organism, the numbers of C . perfringens organisms in the cecal contents were significantly lower in chickens on 2% and 10% lactose-supplemented feed than in chickens on unsupplemented feed . When C . perfringens was given in drinking water, 10% lactose supplementation was needed to significantly reduce the counts of C . perfringens 4, 6, and 8 days after feeding began . Effect of rye-ration on cecal colonization of C . perfringens was also examined . Counts of C . perfringens in cecal contents of chickens fed a diet containing 50% rye were significantly higher than control values 4, 6, and 8 days after feeding began . When chicks were fed a diet containing both 10% lactose and 50% rye, C . perfringens counts in cecal contents were lower than in chickens fed 50% rye only at 6 days after feeding began . Results led to the conclusion that dietary lactose is effective in reducing the cecal colonization of C . perfringens.

Eur J Cell Biol, 1995 Apr, 66(4), 335 - 41
Effects of Clostridium botulinum C2 toxin and cytochalasin D on in vitro invasiveness, motility and F-actin content of a murine T-lymphoma cell line; Verschueren H et al.; In order to investigate the role of microfilaments in the crawling movements of lymphoid cells, we have analyzed the effects of botulinum C2 toxin and of cytochalasin D (cytoD) on the actin cytoskeleton and on the motility of a BW5147 T-lymphoma-derived cell line . Actin was ADP-ribosylated by C2 toxin in the living cells, and this resulted in a time and dose-dependent disappearance of F-actin, as assessed by staining with labeled phalloidin . CytoD did not affect the amount of polymerized actin, but rather changed its distribution from a diffuse peripheral network to focal accumulations on one side of the cell . Both treatments affected the motility of the lymphoma cells in two assay systems . Fourier analysis was used to quantify shape changes performed by the cells . C2 toxin as well as CytoD caused the cessation of pseudopodal protrusion . Invasion of the lymphoma cells through a monolayer of fibroblast-like cells was also inhibited by the treatments, in a dose-dependent way . C2 toxin significantly inhibited invasion at concentrations at which only part of the actin pool had been ADP-ribosylated . We conclude that partial depolymerization, as well as disorganization, of the microfilament network impairs the active cellular deformations that are involved in the crawling movements of the lymphoma cells . From previous work, there is evidence to state that the monolayer invasion assay to some extent mimics tissue infiltration by hematopoietic cells . The present study is the first to analyze the role of actin polymerization in a model system that is relevant for the migration of lymphoid cells in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Tierarztl Prax, 1995 Apr, 23(2), 187 - 91
{Blood parameters and enzyme values of healthy and sick racing camels (Camelus dromedarius)}; Wernery U; Camel races have a long tradition in Arabia . Since the oil boom of the 1960s a tremendous revival of the old Bedouin tradition of camel racing has occurred in the United Arab Emirates . These camel races are comparable to horse races in Europe and the U.S.A . Since 1985 the most valuable racing camels of Dubai are routinely tested in the Central Veterinary Research Laboratory (CVRL) for their stamina and endurance . Blood and serum enzyme values, which have been statistically ascertained through testing of 10000 healthy racing camels, are now generally accepted as reference values . Besides these check-ups of healthy racing camels, hematological tests, enzyme and substrate estimations are performed on sick racing camels . These tests support the diagnosis, therapy and prognosis of sick camels . In this connection three diseases are discussed: B . cereus intoxication, Clostridium perfringens enterotoxemia and Trypanosomiasis.

J Lipid Res, 1995 Apr, 36(4), 901 - 10
Synthesis and characterization of novel analogs of conjugated bile acids containing reversed amide bonds; Coleman JP et al.; New analogs of amino acid-conjugated bile acids were synthesized in which the amide bond was reversed from its normal configuration . These structural isomers of the beta-alanyl conjugates of cholic acid and ursodeoxycholic acid were synthesized by reaction of succinic anhydride with the 24-nor-23-amine derivatives of cholic acid and ursodeoxycholic acid . The chemical and physical properties of these reverse amide conjugated bile acid analogs were compared with those of the normal glycine and beta-alanine conjugates . The reverse amide analogs comigrated with their isomeric beta-alanine conjugates during thin-layer chromatography using a variety of solvent systems . However, the isomeric pairs could be resolved by reversed-phase high performance liquid chromatography, with the reverse amides having greater retention times compared to the beta-alanine conjugates . Critical micelle concentrations, solubility of undissociated forms, and acid dissociation constants were similar for the isomeric pairs . Significant differences in melting points were observed, however, While the isomeric pairs showed no significant differences in sensitivity to base hydrolysis, the reverse amides were not hydrolyzed by the cholylglycine hydrolase from Clostridium perfringens, even after long incubation periods.

Protein Expr Purif, 1995 Apr, 6(2), 206 - 12
Purification and characterization of the oxygen-sensitive 4-hydroxybutanoate dehydrogenase from Clostridium kluyveri; Wolff RA et al.; Cell extracts of Clostridium kluyveri grown on ethanol plus succinate contained a NAD(H) dependent 4-hydroxybutanoate dehydrogenase (EC 1.1.1.61) at 66 U/mg . This enzyme was purified 42-fold under anaerobic conditions to homogeneity . Heat treatment, ion exchange chromatography on DEAE-cellulose, nondenaturing polyacrylamide gel electrophoresis, hydrophobic interaction chromatography on phenyl agarose, and gel filtration on Sephadex G-100 were used in the purification . The molecular mass of the enzyme was estimated to be 41.6 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 86 kDa by gel filtration which indicates the active form of the enzyme is dimeric . The protein contains two atoms of Cu and one atom of Fe per monomeric unit . The enzyme exhibits maximum activity at pH 6.1 for the reduction of succinic semialdehyde and at pH 9.4 for the oxidization of 4-hydroxybutanoate . The Km values for NADH and succinic semialdehyde were 150 +/- 20 microM and 560 +/- 80 microM, respectively . In the reverse direction, the Km values were 670 +/- 80 microM and 55 +/- 16 mM for NAD and 4-hydroxybutanoate, respectively . The enzyme is inactivated by oxygen . The inactivation occurs with a t1/2 = 4.5 min at pH 8.2 and 30 degrees C.

Mol Cell Probes, 1995 Apr, 9(2), 101 - 9
Synthesis and evaluation of a non-radioactive gene probe for the detection of C.perfringens alpha toxin; Schlapp T et al.; The synthesis and evaluation of a non-radioactive hybridization probe is described, specific detecting the Clostridium perfringens alphatoxin gene (plc) by colony blot hybridization assay . A vector free digoxigenin-dUTP-labelled probe was generated by polymerase chain reaction (PCR) targeting the cloned plc gene of C.perfringens strain ATCC 13124 . In a colony blot hybridization assay 296 strains of C.perfringens were tested for plc . None of the strains failed in hybridization . Presence of plc was even demonstrated in C.perfringens strains reported to lack lecithinase activity . Specificity of the probe was shown with various strains of other bacterial species . None different Clostridia sp . tested, e.g . C.bifermentans, C.tertium, C.novyi, C.chauvoei, C.sporogenes, C.difficile, C.putrifucum, C.sordellii, C.botulinum, C . septicum and C.histolyticum, hybridized with the plc specific probe . Strains expressing an enzymatically related phospholipase like Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus gave also negative results . Comparing the results of conventionally used egg yolk turbidity assay and those gained with DNA hybridization, the plc probe proved to be a much more sensitive and specific diagnostic tool for the detection of C.perfringens plc.

Toxicon, 1995 Apr, 33(4), 515 - 26
Botulinal neurotoxin C1 complex genes, clostridial neurotoxin homology and genetic transfer in Clostridium botulinum; Hauser D et al.; The botulinal neurotoxins (BoNT) associate with non-toxic proteins (ANTP) by non-covalent bonds to form large complexes . In C . botulinum C, the BoNT/C1 locus consists of six genes which are organized in three clusters . Cluster 1 encompasses the genes of BoNT/C1 and ANTP/139 which could be involved in the resistance of the BoNT/C1 to the acidic pH and protease degradation . The second cluster consists of three genes which encode hemagglutinin components . The last gene encodes a DNA binding protein (Orf22) which might regulate the BoNT/C1 complex gene expression . BoNT and tetanus toxin (TeTx) display similar structure and mechanism of action at the molecular level . Their identity at the amino acid level range from 34 to 96.8%, indicating that the clostridial neurotoxin genes probably derive from a common ancestor . The fact that Clostridium other than C . botulinum such as C . butyricum and C . baratii can produce a BoNT suggests that the BoNT genes can be transferred between Clostridium strains . The toxigenic C . butyricum strains seem to derive from originally non-toxic strains by neurotoxin gene transfer from C . botulinum E, probably including a mobile DNA element . In C . botulinum C and D the gene encoding the exoenzyme C3 has been localized in a transposon-like element of 21.5 kbp . Transposons could be involved in BoNT gene transfer in C . botulinum.

Toxicon, 1995 Apr, 33(4), 499 - 506
The enterotoxin of Clostridium perfringens type A binds to the presynaptic nerve endings in neuromuscular junctions of mouse phrenic nerve-diaphragm; Senda T et al.; The enterotoxin of Clostridium perfringens type A, a channel-pore forming protein toxin, inhibited neuromuscular transmission in isolated mouse phrenic nerve-diaphragm preparation at low concentrations of calcium . We investigated immunohistochemically the localization of the binding sites of the enterotoxin in the preparation under the conditions in which the enterotoxin reduced maximally the amplitudes of the twitch tension elicited by electrical stimulations to the phrenic nerve . Under the conditions, double immunohistochemical staining of the preparation with (1) rabbit anti-enterotoxin IgG-rhodamine-labeled goat anti-rabbit IgG and (2) mouse anti-synaptophysin (one of the synaptic vesicle-specific membrane proteins)-fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG showed that the enterotoxin binds specifically to most of the sites which were stained with anti-synaptophysin exactly in the same configurations having the shapes of the nerve endings in the endplates . The thin section electron micrographs of the enterotoxin-intoxicated preparation showed no alterations in the ultrastructure of the neuromuscular junction and the nerve endings filled with numerous synaptic vesicles . The present results, together with our previous electrophysiological findings, indicate that the enterotoxin binds specifically to the presynaptic nerve endings and inhibits neurotransmitter release at the neuromuscular junction.

Int J Food Microbiol, 1995 Apr, 25(2), 191 - 7
Glucono-delta-lactone and citric acid as acidulants for lowering the heat resistance of Clostridium sporogenes PA 3679 in HTST working conditions; Silla Santos MH et al.; The heat resistance of Clostridium sporogenes PA 3679 spores has been studied to establish the influence of acidification with glucono-delta-lactone (GDL) and citric acid on the thermal resistance parameters (DT and z) of this microorganism and to compare their effect with phosphate buffer and natural asparagus as reference substrates . A reduction in DT values was observed in asparagus puree as the acidification level increased with both acidulants although this effect was more evident at the lower treatment temperatures studied (121-127 degrees C) . Citric acid was more effective for reducing the heat resistance of spores than GDL at all of the temperatures . The reduction in pH diminished the value of the z parameter, although it was necessary to lower the pH to 4.5 to obtain a significant reduction.

Microbiology, 1995 Apr, 141 ( Pt 4), 989 - 96
A Clostridium acetobutylicum regulator gene (regA) affecting amylase production in Bacillus subtilis; Davison SP et al.; Plasmid pMET7C containing a 6.05 kb DNA insert from Clostridium acetobutylicum P262 made Escherichia coli F19 cells sensitive to metronidazole . The nucleotide sequence of the C . acetobutylicum DNA controlling metronidazole sensitivity in E . coli F19 revealed an ORF of 972 bp which encoded a protein of 324 amino acids with a calculated Mr of 35,000 . The amino acid sequence encoded by the ORF contained a helix-turn-helix DNA-binding domain and was homologous to the catabolite control protein, CcpA, from Bacillus subtilis and Bacillus megaterium, a tRNA repressor of E . coli encoded by the shl gene, and the GalR, Lacl and PurR repressors of E . coli . The C . acetobutylicum ORF, which was termed regA, complemented a B . subtilis ccpA mutant and an E . coli shl mutant, but was unable to complement E . coli galR, lacl or purR mutants . To determine whether the regA gene product was involved in the regulation of amylase gene expression in C . acetobutylicum, a starch-degrading enzyme gene (staA) from C . acetobutylicum NCIMB 8052 was cloned . The RegA protein inhibited the degradation of starch by the C . acetobutylicum staA gene product in E . coli.

Int J Syst Bacteriol, 1995 Apr, 45(2), 403 - 5
Phylogenetic placement of Dialister pneumosintes (formerly Bacteroides pneumosintes) within the Sporomusa subbranch of the Clostridium subphylum of the gram-positive bacteria; Willems A et al.; The nucleotide sequence of the 16S rRNA gene of the type strain of Dialister pneumosintes was determined . Phylogenetic analysis revealed that this species belongs to the Sporomusa branch of the Clostridium subphylum of the gram-positive bacteria and should therefore be excluded from the family Bacteroidaceae . Within this branch, which encompasses several other gram-negative taxa, such as Acidaminococcus, Pectinatus, Phascolarcobacterium, Quinella, Selenomonas, and Zymophilus, Dialister showed a specific, albeit distant, affinity with the genera Megasphaera and Veillonella.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2979 - 83
Membrane permeabilization by Listeria monocytogenes phosphatidylinositol-specific phospholipase C is independent of phospholipid hydrolysis and cooperative with listeriolysin O; Goldfine H et al.; We have examined potential cooperative interactions of Listeria monocytogenes phosphatidylinositol-specific phospholipase C (PI-PLC) and listeriolysin O (LLO), a pore-forming hemolysin, in a liposome lysis assay . Large unilamellar vesicles, approximately 0.1 micron in diameter, encapsulating the fluorescent probe calcein, were treated with PI-PLC or LLO at pH 6.0, and each was capable of causing dye release . With phosphatidylcholine/phosphatidylinositol/cholesterol liposomes at 0.1 microM lipid, minimal release of dye was observed on addition of 80 pM LLO or 7 nM PI-PLC . Addition of the two proteins together produced rapid dye release . Unexpectedly, essentially identical results were obtained with phosphatidylcholine/cholesterol liposomes . Thus, the effect of PI-PLC did not depend on lipid hydrolysis . Both proteins also released inulin (M(r) 5200) from liposomes . Membrane permeabilization was not accompanied by membrane fusion . Very little dye release from phosphatidylcholine/phosphatidylinositol/cholesterol liposomes was seen with PI-PLC from Bacillus thuringiensis, and addition of this enzyme to LLO produced no additional dye release; however PI-PLC from L . monocytogenes cooperated with perfringolysin O from Clostridium perfringens . PI-PLC from L . monocytogenes and LLO bind to phosphatidylcholine/cholesterol liposomes, and the rate of binding of each protein was not influenced by the presence of the other . These data support a postulated accessory role for PI-PLC with LLO in lysing the primary phagosome of a macrophage.

J Immunol Methods, 1995 Mar 27, 180(2), 181 - 91
Immunological detection of Clostridium botulinum toxin type A in therapeutic preparations; Ekong TA et al.; The potent neurotoxins produced by strains of Clostridium botulinum act by blocking the release of acetylcholine from peripheral nerve junctions . This specific action of the botulinum neurotoxins is now being exploited therapeutically to treat a variety of conditions involving involuntary muscle spasms . We aimed to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA) which may be used in parallel with the currently accepted mouse bioassay test for the determination of botulinum neurotoxin type A in therapeutic preparations . High titre polyclonal antitoxins and their biotin derivatives, highly labelled horseradish peroxidase (HRP)-antibody conjugates, and streptavidin-biotin-HRP complexes were prepared and used in a sandwich ELISA for the detection of pure neurotoxin and neurotoxin in therapeutic material . The ELISA utilized either a monoclonal or polyclonal antibody as capture agent and HRP-labelled IgG or F(ab')2 fragment as second antibody . The limit of detection was 4-8 pg of purified toxin/ml (gcv < 13%), equivalent to 1-2 mouse bioassay units/ml . The assay was used to evaluate therapeutic preparations and the results compared with the mouse bioassay . The lower limit of detection for a therapeutic preparation of BoTxA was 2-5 mouse bioassay units/ml . Although across different manufacturers and bulk products there was no correlation between immunologically detected neurotoxin and its biological activity in different therapeutic preparations (r = -0.44, p = 0.34, n = 8), the assay could be used to quantify neurotoxin in therapeutic preparations derived from the same bulk concentrate and manufacturer . The assay is relatively simple, and may be readily adapted to routine monitoring of BoTxA content in therapeutic preparations.

FEBS Lett, 1995 Mar 27, 362(1), 1 - 4
Differential codon usage: a safeguard against inappropriate expression of specialized genes?
Saier MH Jr.
Recent work has suggested that rare codons are sometimes used for the regulation of specialized gene expression in bacteria . Moreover, the cellular levels of certain tRNAs may fluctuate with growth conditions . Evidence implicating such mechanisms in the control of photosynthesis in Rhodobacter, solventogenesis in Clostridium, sporulation in Streptomyces, and fimbrial phase variation in E . coli is summarized . It is suggested that such mechanisms will prove applicable to the control of numerous additional specialized functions, and that the empirical tools for testing this possibility are currently available.

J Biol Chem, 1995 Mar 24, 270(12), 6984 - 90
Guanosine 5'-3-O-(thio)triphosphate stimulates tyrosine phosphorylation of p125FAK and paxillin in permeabilized Swiss 3T3 cells . Role of p21rho; Seckl MJ et al.; Addition of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) to streptolysin O-permeabilized Swiss 3T3 cells induced tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands . Specifically, GTP gamma S stimulated tyrosine phosphorylation of both focal adhesion kinase (p125FAK) and paxillin . GTP gamma S induced tyrosine phosphorylation was dose-dependent (EC50 of 2.5 microM) and reached maximum levels after 1.5 min for the M(r) 110,000-130,000 band and 2 min for the M(r) 70,000-80,000 paxillin band . Guanosine 5'-O-(2-thiodiphosphate) inhibited GTP gamma S-induced tyrosine phosphorylation with an IC50 of 100 microM . Protein kinase C did not mediate GTP gamma S-induced tyrosine phosphorylation . Varying the Ca2+ concentration from 0 to 6 microM did not increase tyrosine phosphorylation above basal levels and did not affect the ability of GTP gamma S to induce tyrosine phosphorylation . GTP gamma S was able to stimulate tyrosine phosphorylation in the presence of nanomolar concentrations of Mg2+ . Furthermore, 30 microM AlF4- only weakly induced tyrosine phosphorylation in permeabilized cells . Pretreatment with the Clostridium botulinum C3 exoenzyme which inactivates p21rho, markedly reduced the ability of GTP gamma S to stimulate tyrosine phosphorylation of M(r) 110,000-130,000 and 70,000-80,000 bands including p125FAK and paxillin in permeabilized Swiss 3T3 cells . Furthermore, a peptide of p21rho (p21rho17-44) inhibited GTP gamma S-induced tyrosine phosphorylation in a dose-dependent manner (IC50 1 microM) . This peptide also inhibited tyrosine phosphorylation of p125FAK and paxillin . In contrast, 20 microM p21ras17-44 peptide failed to inhibit GTP gamma S-induced tyrosine phosphorylation . Using permeabilized cells, our findings demonstrate that GTP gamma S stimulates tyrosine phosphorylation of p125FAK and paxillin and that a functional p21rho is implicated in this process.

J Biol Chem, 1995 Mar 17, 270(11), 6071 - 80
Phosphatidylserine decarboxylase 2 of Saccharomyces cerevisiáe . Cloning and mapping of the gene, heterologous expression, and creation of the null allele; Trotter PJ et al.; The yeast Saccharomyces cerevisiae expresses two phosphatidylserine decarboxylase (PSD) activities which are responsible for conversion of phosphatidylserine to phosphatidylethanolamine, and either enzyme alone is sufficient for normal cellular growth . However, strains containing a PSD1 null allele and a mutation leading to loss of PSD2 activity (psd1-delta 1::TRP1 psd2) are auxotrophic for ethanolamine . This nutritional requirement was utilized to isolate the gene encoding the PSD2 enzyme by complementation . The PSD2 gene encodes a protein of 1138 amino acids with a predicted molecular mass of 130 kDa . The deduced amino acid sequence shows significant identity (34%) to a PSD-like sequence from Clostridium pasteurianum and the yeast PSD1 (19%) at the carboxyl end of the protein . Of particular interest is the presence of a sequence, GGST, which may be involved in post-translational processing and prosthetic group formation similar to other PSD enzymes . The PSD2 amino acid sequence also shows significant homology to the C2 regions of protein kinase C and synaptotagmin . Physical mapping experiments demonstrate that the PSD2 is located on chromosome 7 . The PSD2 gene was heterologously expressed by infection of Sf-9 insect cells with recombinant baculovirus, resulting in a 10-fold increase in PSD activity . The null allele of PSD2 was introduced into yeast strains by one-step gene deletion/disruption with a HIS3 marker gene . Strains expressing wild type PSD1 and the psd2-delta 1::HIS3 allele show a small decrease in overall PSD activity, but no noticeable effect upon {3H}serine incorporation into aminophospholipids . Strains containing both the psd1-delta 1::TRP1 and psd2-delta 1::HIS3 null alleles, however, express no detectable PSD activity, are ethanolamine auxotrophs and show a severe deficit in the conversion of {3H}serine-labeled phosphatidylserine to phosphatidylethanolamine . These data indicate that the gene isolated is the structural gene for PSD2 and that the PSD1 and PSD2 enzymes account for all yeast PSD activity.

Biochim Biophys Acta, 1995 Mar 15, 1247(2), 231 - 8
Correlation of intron-exon organisation with the three-dimensional structure in glutamate dehydrogenase; Teller JK et al.; The positions of the intron-exon boundaries in the genes for glutamate dehydrogenase from Chlorella sorokiniana rat, and human have been located on the three-dimensional structure of the highly homologous enzyme from Clostridium symbiosum and analysed for their position in the protein structure . This analysis shows no correlation between the positions of these boundaries in the mammalian and Chlorella glutamate dehydrogenase genes and no correlation with units of function in the enzyme and suggests that the present day exons do not represent the protein modules of an ancestral glutamate dehydrogenase . There appears to be no clear preference for the residues at the splice junctions to be either buried or exposed to solvent . However, the frequency with which the introns appear in the loops linking elements of secondary structure, rather than in either the alpha-helical or beta-sheet segments, is higher than predicted on the basis of the proportion of residues in the loops . This is consistent with but not proof of a role for exon modification/exchange in protein evolution since changes at these positions are less likely to disturb the structure and hence maintain function.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2189 - 93
Glycine reductase selenoprotein A is not a glycoprotein: the positive periodic acid-Schiff reagent test is the result of peptide bond cleavage and carbonyl group generation; Kimura Y et al.; The complete amino acid sequence of Clostridium sticklandii selenoprotein A, a selenocysteine-containing protein component of the glycine reductase complex, has been established . Both the intact protein and peptide fragments produced by Staphylococcus aureus V8 protease or trypsin were purified by reversed-phase high-performance liquid chromatography and subjected to electrospray ionization mass spectrometric analysis and standard Edman degradation . Selenoprotein A consists of 157 amino acids with a chemical molecular weight of 17,011, in reasonable agreement with the observed molecular weight (17,022.7) determined from its ionization mass spectrum . The sequence of the amino-terminal region of the isolated native protein is Ser-Arg-Phe-Thr-Gly-Lys- Lys-Ile-Val-Ile-Ile-Gly-Asp-Arg-Asp- . An N-terminal methionine residue deduced from the gene sequence was not present . Although selenoprotein A reacted positively in a glycoprotein stain when using either the periodic acid-Schiff reagent procedure or a commercial glycan detection kit, no saccharide was detected by carbohydrate analyses after acid hydrolysis or methanolysis . Identity of the amino acid sequence determined by analysis with that deduced from the gene sequence is further evidence of the absence of bound carbohydrate.

Biochem Biophys Res Commun, 1995 Mar 8, 208(1), 36 - 41
Activation of the Sendai virus fusion protein by receptor binding; Dallocchio F et al.; 2,3 Dehydro-2-deoxy-N-acetyl-neuraminic acid (DNANA) competitively inhibits the neuraminidase activity of Hemagglutinin-neuraminidase (HN) from Sendai virus . The inhibition constant depends on the presence of the Fusion (F) protein, which is 30 microM in the presence of active F protein and 50 microM when the F protein is inactivated . These data correlate with previously reported evidence of interaction of the F protein with HN (Dallocchio, F., Tomasi, M., & Bellini, T . (1994) Biochem . Biophys . Res . Comm . 201, 988-993) . Desialyzation of erythrocytes, by Clostridium neuraminidase, lowers the hemolytic activity of SV to < 0.1% of that observed on untreated erythrocytes . However, addition of DNANA causes a concentration-dependent increase of hemolytic activity . Both HN and the F protein are required for the activation of hemolytic activity by DNANA . The affinity constant for DNANA, calculated from the activation of hemolytic activity on desialyzed erythrocytes, is 35 microM, very close to the Ki for neuraminidase activity . These data suggest that the binding of the F protein to HN, induced by the binding to HN of a substrate or a substrate analogue, causes a conformational change which activates the F protein.

Am J Physiol, 1995 Mar, 268(3 Pt 1), G487 - 95
Clostridium difficile toxin B activates calcium influx required for actin disassembly during cytotoxicity; Gilbert RJ et al.; The principal cellular response to Clostridium difficile toxin B, a protein toxin associated with antibiotic-associated colitis, is the disassembly of actin microfilaments . Although receptor-activated signal transduction mechanisms have been proposed to mediate these effects, the intracellular events that precede actin breakdown are unknown . In NIH-3T3 fibroblasts, toxin B induced an elevation of intracellular calcium possessing either a slow (minutes) or fast (seconds) rise time, followed by a sustained elevation of calcium concentration . Subcellular analysis of steady-state calcium distribution after toxin B demonstrated that the increase of calcium was homogeneous throughout the cytosol and did not vary based on the kinetics of the initial calcium rise . All calcium responses were blocked by substitution with calcium-free buffer or buffer containing lanthanum chloride, indicating that the rise in calcium was attributable to calcium influx from the extracellular space . Quantitatively similar responses were observed in primary cultured gastric smooth muscle and AR42J pancreatic tumor cells, suggesting that toxin-induced calcium signal transduction was conserved between cell types . The morphological response to toxin B consisted of sequential dissociation of the actin cytoskeleton from membrane attachments, retraction of actin stress fibers from the periphery to the perinuclear region, loss of fibre alignment, and cell rounding . The actin reorganization associated with toxin B was blocked by incubation of cells in calcium-free media or the clamping of intracellular calcium with cell-permeant calcium chelating agents . These results demonstrate that the calcium influx activated by C . difficile toxin B is a necessary condition for the breakdown of filamentous actin associated with cytotoxicity.

Clin Radiol, 1995 Mar, 50(3), 153 - 6
Clostridium difficile colitis: correlation of CT findings with severity of clinical disease; Boland GW et al.; Clinical records and abdominal CT scans from 64 patients with documented Clostridium difficile disease were reviewed to determine if any correlation existed between CT findings of colitis and severity of clinical disease . Clostridium difficile disease was documented with stool toxin titre levels and CT scans were performed within 3 days of stool sample . Clinical disease severity was estimated by tabulating the degree of fever, WBC count, frequency and duration of diarrhoea . Thirty-nine of 64 patients showed CT evidence of colitis of which 28/39 showed evidence of focal colitis and 11/39 had pancolitis . CT findings suggesting colitis included colonic wall thickening (39 patients), nodular mucosal thickening (11 patients), the 'accordion pattern' (3 patients), pericolonic oedema (27 patients) and ascites (10 patients) . Twenty-five of 64 patients showed no CT evidence of colitis . The clinical severity of disease did not statistically differ (P < 0.05) between patients with CT evidence of colitis and those without colitis . The only CT finding that correlated with clinical severity of disease was nodular mucosal thickening which was found with significantly (P < 0.05) more frequency in patients with a WBC count > 11,000 mm3 . CT changes with Cl . difficile disease correlate poorly with the clinical severity . This and negative findings do not exclude the disease.

J Clin Invest, 1995 Mar, 95(3), 1026 - 31
The low molecular mass GTP-binding protein Rho is affected by toxin A from Clostridium difficile; Just I et al.; Enterotoxin A is one of the major virulence factors of Clostridium difficile, and the causative agent of antibiotic-associated pseudomembranous colitis . In cell culture (NIH-3T3, rat basophilic leukemia cells) toxin A inhibits Clostridium botulinum ADP-ribosyltransferase C3 (C3)-catalyzed ADP-ribosylation of the low molecular mass GTP-binding Rho proteins . Rho participates in the regulation of the microfilament cytoskeleton . Decrease in ADP-ribosylation of Rho occurs in a time- and concentration-dependent manner and precedes the toxin A-induced destruction of the actin cytoskeleton . Action of toxin A is not due to proteolytical degradation of Rho or to an inherent ADP-ribosyltransferase activity of toxin A . Toxin A-induced decrease in ADP-ribosylation is observed also in cell lysates and with recombinant RhoA protein . A heat stable low molecular mass cytosolic factor is essential for the toxin effect on Rho . Thus, the enterotoxin (toxin A) resembles the effects of the C . difficile cytotoxin (toxin B) on Rho proteins (Just, I., G . Fritz, K . Aktories, M . Giry, M . R . Popoff, P . Boquet, S . Hegenbath, and C . Von Eichel-Streiber . 1994 . J . Biol . Chem . 269:10706-10712) . The data indicate that despite different in vivo effects, toxin A and toxin B act on the same cellular target protein Rho to elicit their toxic effects.

J Bacteriol, 1995 Mar, 177(6), 1641 - 4
Exoglucanase activities of the recombinant Clostridium thermocellum CelS, a major cellulosome component; Kruus K et al.; The recombinant CelS (rCelS), the most abundant catalytic subunit of the Clostridium thermocellum cellulosome, displayed typical exoglucanase characteristics, including (i) a preference for amorphous or crystalline cellulose over carboxymethyl cellulose, (ii) an inability to reduce the viscosity of a carboxymethyl cellulose solution, and (iii) the production of few bound reducing ends on the solid substrate . The hydrolysis products from crystalline cellulose were cellobiose and cellotriose at a ratio of 5:1 . The rCelS activity on amorphous cellulose was optimal at 70 degrees C and at pH 5 to 6 . Its thermostability was increased by Ca2+ . Sulfhydryl reagents had only a mild adverse effect on the rCelS activity . Cellotetraose was the smallest oligosaccharide substrate for rCelS, and the hydrolysis rate increased with the substrate chain length . Many of these properties were consistent with those of the cellulosome, indicating a key role for CelS.

J Bacteriol, 1995 Mar, 177(5), 1402 - 4
Accurate determination of the molecular weight of the major surface layer protein isolated from Clostridium thermosaccharolyticum by time-of-flight mass spectrometry; Allmaier G et al.; Matrix-assisted laser desorption with concomitant ionization, in combination with a linear time-of-flight mass spectrometer, was used to analyze underivatized and hard-to-solubilize surface layer proteins and glycoproteins by depositing them on top of a microcrystalline layer of the matrix alpha-cyano-4-hydroxycinnamic acid . Use of this special sample preparation technique allowed the first successful desorption-ionization of intact surface layer proteins and accurate determination of their molecular weights by mass spectrometry . The molecular mass of the monomeric subunit of the major surface layer protein isolated from Clostridium thermosaccharolyticum E207-71 was determined to be 75,621 +/- 81 Da . The obtainable mass accuracy of the technique is conservatively considered to be within +/- 0.2% . This result deviates from that given by sodium dodecyl sulfate-polyacrylamide gel electrophoresis by approximately 7.4 kDa because this method is strongly affected and biased by the three-dimensional structure of this type of surface protein . With the apparent advantages of unsurpassed mass accuracy, low dependence on the physicochemical properties of the surface layer proteins, and high sensitivity, it can be concluded that a linear time-of-flight instrument combined with UV matrix-assisted laser desorption with concomitant ionization is better suited for molecular weight determination than is gel electrophoresis.

J Bacteriol, 1995 Mar, 177(5), 1179 - 85
Site-directed mutagenesis of histidine residues in Clostridium perfringens alpha-toxin; Nagahama M et al.; Mutagenesis of H-68 or -148 in Clostridium perfringens alpha-toxin resulted in complete loss of hemolytic, phospholipase C, sphingomyelinase, and lethal activities of the toxin . These activities of the variant toxin at H-126 or -136 decreased by approximately 100-fold of the activities of the wild-type toxin . Mutation at H-46, -207, -212, or -241 showed no effect on the biological activities, indicating that these residues are not essential for these activities . The variant toxin at H-11 was not detected in culture supernatant and in cells of the transformant carrying the variant toxin gene . Wild-type toxin and the variant toxin at H-148 bound to erythrocytes in the presence of Ca2+; however, the variant toxins at H-68, -126, and -136 did not . Co2+ and Mn2+ ions stimulated binding of the variant toxin at H-68, -126, and -136 to membranes in the presence of Ca2+ and caused an increase in hemolytic activity . Wild-type toxin and the variant toxins at H-68, -126, and -136 contained two zinc atoms in the molecule . Wild-type toxin inactivated by EDTA contained two zinc atoms . These results suggest that wild-type toxin contains two tightly bound zinc atoms which are not coordinated to H-68, -126, and -136 . The variant toxin at H-148 possessed only one zinc atom . Wild-type toxin and the variant toxin at H-148 showed {65Zn}2+ binding, but the variant toxins at H-68, -126, and -136 did not . Furthermore, {65Zn}2+ binding to wild-type toxin was competitively inhibited by unlabeled Zn2+, Co2+, and Mn2+ . These results suggest that H-68, -126, and -136 residues bind an exchangeable and labile metal which is important for binding to membranes and that H-148 tightly binds one zinc atom which is essential for the active site of alpha-toxin.

Biosci Biotechnol Biochem, 1995 Mar, 59(3), 514 - 5
Purification and partial characterization of a spore cortex-lytic enzyme of Clostridium perfringens S40 spores; Miyata S et al.; A spore cortex-lytic enzyme was purified in an active form from the exudate of fully germinated spores of Clostridium perfringens S40 . The enzyme caused attenuation of absorbance in coatless spore suspensions and phase-darkening of the spores, but had minimal activity on isolated peptidoglycan fragments . The enzyme was identified as a 31 kDa protein which is probably an N-acetylmuramyl-L-alanine amidase . The amino-terminal 15 residues of the enzyme were: VLPEPVVPEYIVVHN.

Lett Appl Microbiol, 1995 Mar, 20(3), 152 - 6
Growth and toxin production by non-proteolytic and proteolytic Clostridium botulinum in cooked vegetables; Carlin F et al.; Growth and toxin production by proteolytic and non-proteolytic strains of Clostridium botulinum have been followed in 28 cooked pureed vegetables prepared under strict anaerobic conditions and incubated at 30 degrees C for up to 60 d . Toxin production was confirmed in 25 of the cooked vegetables inoculated with a suspension of spores of proteolytic strains of types A and B, and in 13 inoculated with a suspension of spores of non-proteolytic strains of types B, E and F . For both proteolytic and non-proteolytic strains, a trend was identified correlating growth and toxin production with the pH of the cooked pureed vegetables.

Orthop Nurs, 1995 Mar-Apr, 14(2), 38 - 41
Antibiotic-induced diarrhea; Vogel LC; Diarrhea is a common complication of antibiotic therapy and can range from mild soiling of a cast to severe and life-threatening pseudomembranous colitis . Although clindamycin is the most notorious, almost all antibiotics, particularly penicillins and cephalosporins, may also be responsible (Bartlett, 1992; Kelly, Pothoulakis, & LaMont, 1994) . Because of the frequent use of these antibiotics in orthopaedic patients, antibiotic-associated enteric disease is a common problem in this population . About 15% to 25% of cases of antibiotic-associated diarrhea are caused by Clostridium difficile (Bartlett, 1992; George, 1984; Kelly et al., 1994) . The majority of patients with antibiotic-associated diarrhea have no identifiable etiologic agent . Salmonella, enterotoxin-producing Clostridium perfringens (Borriello et al., 1984) and Candida albicans (Danna et al., 1991) have rarely been identified as causative agents . This article describes the role of C . difficile as an enteric pathogen and its spectrum of clinical disease, including diagnosis, treatment, and prevention of nosocomial transmission.

J AOAC Int, 1995 Mar-Apr, 78(2), 381 - 5
Production of monoclonal antibodies specific to Clostridium botulinum type B neurotoxin; Noah CW et al.; Four monoclonal antibodies were produced for use in a rapid method to detect Clostridium botulinum type B neurotoxin . Cells of mouse myeloma cell line SP2/0 were fused with splenocytes of immunized BALB/c mice . An immunoblot assay of semipurified commercial neurotoxins of C . botulinum types A, B, C, D, E, and F was used to show specificity . All the monoclonal antibodies reacted with type B neurotoxin but did not cross-react with the other types . The monoclonal antibodies, separately and combined, did not neutralize the toxin in mice, and all showed specificity to the whole neurotoxin molecule and the heavy-chain component by immunoblot . No evidence of specific binding to the hemagglutinin molecule was noted . When tested against concentrated cultured supernatants of C . botulinum types A, B, E, and F, the 4 monoclonal antibodies reacted only against type B strains . They will be incorporated into a rapid assay with other specific monoclonal antibodies to detect C . botulinum neurotoxins from pure cultures or suspect foods.

Eur J Gastroenterol Hepatol, 1995 Mar, 7(3), 275 - 7
Pseudomembranous colitis following clarithromycin therapy; Teare JP et al.; OBJECTIVE: To describe the association of clarithromycin, used to treat Helicobacter pylori infection and duodenal ulceration, with pseudomembranous colitis in two patients . SETTING: St Mary's Hospital, London, UK . PATIENTS: Two female patients aged 77 and 78 years, admitted with duodenal ulceration and H . pylori infection . INTERVENTION: Clarithromycin (500 mg three times daily) was administered concurrently with omeprazole (40 mg once daily) . OUTCOME MEASURES: After an initial improvement in symptoms both patients experienced persistent Clostridium difficile-associated diarrhoea . CONCLUSIONS: High-dose clarithromycin should be used with caution for the treatment of H . pylori infection associated with gastroduodenal ulceration . The drug may induce antibiotic-associated colitis which can lead to morbidity and mortality, particularly in the elderly.

Eur J Gastroenterol Hepatol, 1995 Mar, 7(3), 259 - 63
Clostridium difficile-associated diarrhoea in patients with human immunodeficiency virus infection: a case-control study; Tumbarello M et al.; OBJECTIVE: To evaluate the prevalence of, risk factors for, treatment and outcome of Clostridium difficile-associated diarrhoea (CDAD) in patients with human immunodeficiency virus (HIV) infection . DESIGN: A prospective case-control study, conducted between January 1992 and April 1994 . SETTING: Department of Infectious Diseases in a large university hospital with HIV in- and out-patient units . PATIENTS AND METHODS: The study included 124 patients grouped as follows: 31 HIV-infected patients with CDAD (group A); 31 HIV-seronegative patients with CDAD (group B) and 62 HIV-infected patients without CDAD (group C) . The patients in group B and C were selected randomly during the study period . RESULTS: The prevalence of CDAD in HIV-infected patients was 3.1% compared with 1.6% in HIV-seronegative patients (P = 0.02) . On univariate analysis, the predisposing factors in group A were antibiotic use in the 4 weeks prior to the onset of CDAD (P = 0.03 versus group C), prolonged hospitalization (over 20 days; P = 0.04), low levels of circulating CD4+ cells (P = 0.03) and use of antacids (P = 0.04) . The antibiotics significantly associated with CDAD were trimethoprim-sulfamethoxazole (P = 0.02 versus group C), third generation cephalosporins (P = 0.03) and clindamycin (P = 0.03) . On multivariate analysis of the risk factors, the use of antibiotics was the sole independent risk factor for CDAD (P = 0.03) . The clinical symptoms of CDAD were more severe in HIV-infected patients than in controls . Three patients in group A (9.7%) had one relapse and one patient (3.2%) experienced chronic diarrhoea . The outcome of CDAD was not influenced by the number of circulating polymorphonuclear cells and CD4+ cells . No difference in the survival curves of AIDS patients with or without CDAD, stratified according to age, sex and CD4+ cell count was observed . CONCLUSIONS: Our data suggest that CDAD is more common in HIV-infected patients, particularly those receiving antibiotic therapy, than in HIV-seronegative patients . Since C . difficile can cause severe and recurrent or chronic infections in HIV-infected patients, CDAD must be always considered in the differential diagnosis of diarrhoea in patients with AIDS and AIDS-related conditions.

Arch Dis Child, 1995 Mar, 72(3), 219 - 22
Investigation and management of Clostridium difficile colonisation in a paediatric oncology unit; Schuller I et al.; Little is known about Clostridium difficile infection in children with cancer but a presumed outbreak has previously been described . The carriage rate before admission to hospital and morbidity is reported to be high, especially in younger children . The prevalence of C difficile infection on a paediatric oncology ward was monitored from June 1991 to May 1992 . Twenty eight (13%) of 214 children were found to be infected . Though the temporal distribution suggested an outbreak, polyacrylamide gel electrophoresis identified several different types . Unlike previous reports, infection appeared to be possibly endogenous or possibly environmental in origin rather than due to cross infection; the morbidity was low and age was not a determinant for infection . The duration of hospital stay and the primary diagnosis were found to be determinants for infections, those with lymphoid malignancies being at the highest risk . The diagnostic category at greatest risk were those most intensively treated, with protracted neutropenia and prolonged antibiotic exposure . Early identification of cases and prompt institution of simple control measures will prevent cross infection . It is therefore important that diarrhoea is not accepted as a normal symptom of cancer chemotherapy and stool specimens are sent for full bacteriological and viral investigation.

J Appl Bacteriol, 1995 Mar, 78(3), 316 - 26
Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test; Le Jeune C et al.; On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes (hdcA) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus, oligonucleotides unique to the hdcA genes were synthesized and used in PCR . All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR . In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc aenos strains in PCR . The 150 base pair amplification product of the decarboxylating Leuc . aenos strain generated with primer set CL1/CL2 was sequenced . Alignment studies showed a high degree of relatedness among the hdcA gene products of Gram-positive bacteria . The amplification products of the hdcA genes from Lac . buchneri and Leuct . aenos were used to serve as a DNA probe in hybridization studies . All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes . In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdcA gene . In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.

Microbiology, 1995 Mar, 141 ( Pt 3), 605 - 10
Reversible expression of motility and flagella in Clostridium chauvoei and their relationship to virulence; Tamura Y et al.; Clostridium chauvoei strain Okinawa produced spontaneous non-motile variants at an unusually high rate (approx . 10(-4) per generation) under normal conditions without mutagen . Revertants of non-motile variants were detected at a rate of approximately 10(-3) . Biochemically, every variant corresponded well with the parental strain . By transmission electron microscopy, three of nine non-motile variants of strain Okinawa were found to be flagellate, while the other six were found to be aflagellate . These phenotypes were confirmed by Western blot analysis using monoclonal antibodies directed against the flagella of C . chauvoei . Moreover, the parental flagellate strain and non-motile flagellate variants were significantly more virulent in mice than non-motile, aflagellate variants . Our results demonstrated that phase variation in motility and flagellation occurs in C . chauvoei, and that the flagella are associated with the full expression of virulence.

Zh Mikrobiol Epidemiol Immunobiol, 1995 Mar-Apr, (2), 68 - 72
{The effect of Clostridium tetani cultures on immunization with tetanus anatoxin in an experiment}; Stovbun SF et al.; The paper presents the data on the study, carried out on 383 guinea pigs, of the action of C.tetani cultures on immune reactions of the animals after the injection of tetanus toxoid . The state of the humoral and cell-mediated immunity of the animals (in the hemagglutination inhibition test, the neutralization test, the enzyme-linked immunosorbent assay and the skin allergic test with tetanin), as well as their resistance to challenge with C.tetani . The inhibiting effect of C.tetani on primary immune response to tetanus toxoid and their stimulating effect on secondary immune response have been established . This fact may be used for enhancing the quality of antitetanus preparations, such as toxoid and serum, as well as for working out tactical approaches for the rapid prevention of tetanus.

Res Microbiol, 1995 Mar-Apr, 146(3), 251 - 62
The effects of tunicamycin on secretion, adhesion and activities of the cellulase complex of Clostridium cellulolyticum, ATCC 35319; Gehin A et al.; The effects of tunicamycin, an inhibitor of N-asparagine-linked glycosylation, on the secretion, adhesion and activities of the cellulase complex produced by Clostridium cellulolyticum have been studied . Tunicamycin at 0.1 micrograms/ml slightly inhibited growth on cellobiose . Endoglucanase, p-nitrophenylcellobiosidase and avicelase activities of the "Avicel"-adsorbed fraction from a culture grown with this drug were decreased 4.4-, 1.4- and 12.2-fold, respectively . During growth on cellulose, tunicamycin considerably inhibited growth and adhesion of cells on their substrate (only 28% of the cells were bound to cellulose) . SDS-PAGE mobilities of some proteins excreted during growth with the drug were different from those of proteins from control cultures; the native Avicel-adsorbed fraction (PH2O) consisted of three major components of molecular weights about 135, 90 and 68 kDa, whereas in the presence of tunicamycin (0.1 micrograms/ml), the Avicel-adsorbed fraction (PH2OT) contained only a major band of 105 kDa, and the proteins of 135 and 68 kDa appeared weakly . By using the "Dig Glycan Detection" kit, some proteins appeared to be glycosylated, such as the 135-, 95-, 47- and 40-kDa proteins . Moreover, the affinity for Avicel and the avicelase activity decreased dramatically for the Avicel-adsorbed fraction from a culture grown with the drug . The remaining avicelase activity of the PH2O fraction in the presence of specific P135 antiserum was 50% of the initial activity, whereas CMCase and pNPCbase were not affected . The glycosylated protein of 135 kDa played a prominent role in the adhesion and avicelase activity of C . cellulolyticum . Moreover, the endoglucanase activity in a culture broth from tunicamycin-grown cells was more thermolabile and protease-sensitive than that from control cultures.

Microbiologia, 1995 Mar, 11(1), 75 - 90
{Escherichia coli, other Enterobacteriaceae and additional indicators as markers of microbiologic quality of food: advantages and limitations}; Mossel DA et al.; The 93/43 European Union directive assigns to the food and catering industries the main responsibility for an integrated safety and quality assurance strategy in the food chain . Relying on hazard analysis, followed by design and adoption of control of all critical points and practices ("HACCP") . Hiatus-free compliance with such HACCP-based Codes of Good Practices is to be assessed by monitoring, recording results on process performance charts and gauging such data against experimentally established, attainable and maintainable references ranges ("standards") . Marker microorganisms are a major analytical tool for validating compliance in the sense of the EU directive . They should be expertly chosen amongst microbes usually present in food so that their, whose presence in quantities exceeding predetermined levels point to a lack of microbiological integrity of a food product . This may encompass (i) the potential presence of taxonomically, physiologically and ecologically related pathogens, markers are called index organisms; or else (ii) a lack of process integrity; in this case, markers are termed indicator organisms . The classical index organism was E . coli, introduced in the 1980's to monitor drinking water supplies . It is still used as an appropriate marker to assess the bacteriological safety of raw foods . In the 1920's the coli-aerogenes ("coliform") group was adopted as an indicator to validate the adequate processing, i.e . pasteurization of dairy products . Since the 1950's the entire Enterobacteriaceae taxon is preferred for the latter purpose because it is better defined in determinative sense and includes more organisms of significance . In some food and water supplies, processed for safety, more vigorous or more resistant organisms than the Gram-negative rods are reliable supplementary markers . These include Enterococcus spp., spores of the Clostridium genus, and bacteriophages of E . coli and Bacteroides fragilis mimicking the fate of enteric viruses under particular ecological conditions . Population surveys conducted by the authors provided ranges for epsilon-factors . Those factors were defined as the proportion between colony forming units (cfu) numbers of index organisms and the pathogenic agent to whose potential occurrence they are expected to point . Epsilon factor values obtained for thermotropic Enterobacteriaceae in relation to Salmonella spp . allow the calculation of the probability that the pathogen has been reliably eliminated by the processing of initially contaminated raw materials, when cfu's of the marker organisms remain below a reference range previously fixed.

Microbiologia, 1995 Mar, 11(1), 7 - 22
{Effectiveness of modified atmospheres against psychrotrophic pathogenic microorganisms in proteinaceous food}; Garcia de Fernando GD et al.; Modified atmosphere packaging (MAP) of proteinaceous raw foods (meat, poultry and fish) extends their shelf-lives . It is well established that modified atmospheres (MA) inhibit the psychotropic aerobic Gram-negative bacteria, the main spoilage microflora of proteinaceous raw foods stored under refrigeration . Several researchers have warned about the possible growth of food poisoning microorganisms on them . Considering the minimal growth temperatures of pathogens, this review only deals with Aeromonas hydrophila, Clostridium botulinum, Listeria monocytogenes and Yersinia enterocolitica . C . botulinum produces its toxin in many different atmospheres, but it is unable to grow at temperatures below 3.3 degrees C, and its production rate of the toxin at temperatures below 4.5 degrees C is very low, to the extent that fish can be spoiled before the toxin is detected . Therefore, the control of the storage temperature of MAP fish seems to be indispensable to assure the absence of botulinal toxin . With regard to the other pathogens, vacuum is the atmosphere that may support more readily its growth; the higher the CO2 concentration in the atmosphere, the lower the growth rate is . Some investigations have shown that the growth rates of the psychotropic pathogens in MAP are lower than those of the spoilage flora . It has been shown also that A . hydrophila and L . monocytogenes growth rates are lower under MA than under aerobic storage . In relation to Y . enterocolitica, more investigations should be carried out in order to clear up its behaviour, because the available data in the literature are still confusing and sometimes even contradictory . In conclusion, there are no evidences that support the concern about MAP of proteinaceous raw foods representing a greater hazard than its conventional storage under air.

J Cell Sci, 1995 Mar, 108 ( Pt 3), 1183 - 93
The state of actin assembly regulates actin and vinculin expression by a feedback loop; Bershadsky AD et al.; Actin filaments are major determinants of cell shape, motility and adhesion, which control important biological processes including embryonic development and wound healing . These processes are associated with changes in actin assembly, which is regulated by controlling the balance between polymerized and non-polymerized actin . To maintain a significant pool of non-polymerized actin, mechanism(s) linking actin synthesis to its state of polymerization were proposed . We have studied this relationship between actin synthesis and organization by modulating actin assembly using different drugs . Unassembled actin was increased in 3T3 cells using either the Clostridium botulinum C2 toxin, which ADP-ribosylates actin, or by latrunculin A, a Red Sea sponge product, which binds monomeric actin . The synthesis of actin was dramatically reduced in these cells owing to a concomitant decrease in actin RNA level . Similar results were obtained with HeLa cells grown in both monolayer and in suspension, suggesting that cell shape changes associated with drug treatment are not the primary cause for the effect on actin synthesis . In contrast, the scrape-loading of 3T3 cells with phalloidin, a stabilizer of polymerized actin that increased the level of assembled actin, resulted in elevated actin synthesis and RNA content . The expression of vinculin, a major component of adhesion plaques and cell-cell junctions, which is involved in actin-membrane associations, was altered in parallel with that of actin in cells treated with these drugs . The decrease in actin RNA resulted from destabilization of actin mRNA in cells where unassembled actin level was elevated . This is suggested by the unchanged transcription of actin in isolated nuclei from drug-treated cells, and by demonstrating that actin mRNA was degraded faster in cells after C2 toxin treatment than in control cells . This feedback control mechanism is mainly confined to the cytoplasm, as it remained active in enucleated cells . The results suggest the existence of an autoregulatory pathway for the expression of actin and other microfilament-associated proteins which is linked to the state of actin polymerization in the cell.

J Lipid Mediat Cell Signal, 1995 Mar, 11(2), 133 - 43
Role of platelet activating factor in the inflammatory and secretory effects of Clostridium difficile toxin A; Fonteles M et al.; Clostridium difficile is a major recognized cause of antibiotic-associated diarrhea, an effect mediated through its toxin A . Toxin A has been reported to disrupt epithelial tight junctions, attract neutrophils, and cause striking intestinal inflammation and secretion . Having demonstrated that phospholipase A2 inhibitors block the secretory effects of toxin A, we next wished to examine whether platelet activating factor (PAF) was involved in either the direct epithelial or secretory effects of toxin A . The effects of toxin A on net secretion in ligated rabbit ileal segments were significantly inhibited by the PAF antagonists 10(-4)-10(-5) M BN 52021, 10(-5) M WEB 2170, or 10(-5) M SR 27417 by 59-102% . SR 27417 also inhibited secretion induced by toxin A in loops adjacent to the drug (by 58%) . Furthermore, the striking inflammation and epithelial disruption seen at 6 h and ligated ileal segments with toxin A was largely prevented by simultaneous treatment with the PAF antagonist SR 27417 . In addition, we noted a significant synergistic effect of 10(-8) M PAF with 10 micrograms/ml toxin A in the ligated rabbit ileal segments . To examine direct effects of PAF antagonists on toxin A in T-84 epithelial cell monolayers, rhodamine-labeled phalloidin stained F-actin demonstrated significant disruption of F-actin by toxin A that was reduced by the PAF antagonist BN 52021 or WEB 2170 . However, the PAF antagonists (10(-4) M WEB, 10(-5) M BN or 10(-4) M SR) failed to alter the disruption of T-84 cell tissue resistance by C . difficile toxin A (0.03 micrograms/ml) . We conclude that PAF may be involved in the secretory effects of C . difficile toxin A, and that PAF antagonists deserve further study in C . difficile diarrhea.

J Clin Microbiol, 1995 Mar, 33(3), 755 - 8
Techniques for controlling variability in gram staining of obligate anaerobes; Johnson MJ et al.; Identification of anaerobes recovered from clinical samples is complicated by the fact that certain gram-positive anaerobes routinely stain gram negative; Peptostreptococcus asaccharolyticus, Eubacterium plautii, Clostridium ramosum, Clostridium symbiosum, and Clostridium clostridiiforme are among the nonconformists with regard to conventional Gram-staining procedures . Accurate Gram staining of American Type Culture Collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber . Under anaerobic conditions, gram-positive staining occurred in all test organisms with "quick" fixing techniques with both absolute methanol and formalin . The results support the hypothesis that, when anaerobic bacteria are exposed to oxygen, a breakdown of the physical integrity of the cell wall occurs, introducing Gram stain variability in gram-positive anaerobes.

Zentralbl Veterinarmed B, 1995 Mar, 42(1), 51 - 8
Diagnosis of Clostridium perfringens type C enteritis in pigs using a DNA amplification technique (PCR); Buogo C et al.; Clostridium perfringens type C, which produces alpha- and beta-toxin, causes severe haemorrhagic and necrotic enteritis in animals and humans . A polymerase-chain-reaction (PCR) assay was developed for the specific detection of the genes encoding alpha-, beta-, epsilon- and entertoxin of C . perfringens for rapid typing of C . perfringens strains, and especially for the identification of type C strains . Both the alpha- and beta-toxin genes were detected directly in porcine C . perfringens type C cultures and also in type B and type C collection strains to a sensitivity of 10(3) cells without purification of the DNA . The alpha-toxin gene was detected in all types of C . perfringens . The epsilon-toxin gene was found in type B and type D, and the enterotoxin gene in some type A strains . Nine other species of Clostridium and a variety of intestinal pathogenic bacteria showed no signal for these toxin genes in this PCR assay . The alpha- and beta-toxin genes PCR assay were used to identify C . perfringens strains isolated from intestinal contents of 36 necropsied piglets that had suddenly died or died after premonitory signs of diarrhoea . At necropsy, 20 piglets showed necrotizing enteritis (15 acute and 5 chronic cases) and were suspected to have suffered from a C . perfringens type C infection . All of them had C . perfringens which gave a positive PCR signal for alpha- and beta-toxin genes, and, hence, were identified as type C strains . From the 16 other piglets with lesions other than necrotizing enteritis, C . perfringens strains with the alpha-toxin gene, but no beta-toxin gene, were isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

FEBS Lett, 1995 Feb 27, 360(2), 121 - 4
Expression, purification and subunit-binding properties of cohesins 2 and 3 of the Clostridium thermocellum cellulosome; Yaron S et al.; The enzymatic subunits of the cellulosome of Clostridium thermocellum are integrated into the complex by a major non-catalytic polypeptide, called scaffoldin . Its numerous functional domains include a single cellulose-binding domain (CBD) and nine subunit-binding domains, or cohesin domains . Two of the cohesin domains, together with the adjacent CBD, have been cloned and expressed in Escherichia coli, and the recombinant constructs were purified by affinity chromatography on a cellulosic matrix . Both cohesin domains, which differ by about 30% in their primary structure, showed a similar binding profile to the cellulosomal subunits . Calcium ions enhanced dramatically this binding . Under the conditions of the assay, only one major catalytic subunit of the cellulosome failed to bind to either cohesin domain . The results indicate a lack of selectivity in the binding of cohesin domains to the catalytic subunits and also suggest that additional mechanisms may be involved in cellulosome assembly.

Biochemistry, 1995 Feb 21, 34(7), 2188 - 94
Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase . 2 . Site-directed mutagenesis of an essential arginine contained within an apparent P-loop; Yankie L et al.; Pyruvate phosphate dikinase catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (P(i)), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PP(i)), and phosphoenolpyruvate (PEP) . The Arg 561 residue of Clostridium symbiosum PPDK is contained within a Gly-rich stretch of sequence spanning positions 553-563 (viz., GAEGIGLCRTE) located in the 35 kDa C-terminal domain of the enzyme . The possible role of this stretch of sequence as a phosphate binding loop participating in catalysis of the PEP/pyruvate partial reaction (viz., E+PEP<-->E-P+pyruvate, where E-P represents enzyme phosphorylated at the catalytic histidine) was deduced from the similarity of this sequence to other known phosphate binding loops and by its location in the 35 kDa PEP/pyruvate binding domain of PPDK . To test the proposed role of Arg 561, and hence, the signature sequence, in catalysis of the E+PEP<-->E-P+pyruvate partial reaction, the C . symbiosum PPDK site-directed mutants Arg 561-->Leu 561 and Arg 561-->Lys 561 were constructed and expressed in Escherichia coli JM101 . Neither mutant catalyzed the full PPDK reaction, ATP+P(i)+pyruvate<-->AMP+PP(i)+PEP, but both catalyzed the E+ATP+P(i)<-->E-P+AMP+PP(i) partial reaction as efficiently as wild-type PPDK . Both mutants were shown to be unable to catalyze the PEP/pyruvate partial reaction . On the basis of these results it was proposed that Arg 561 and, possibly, the Gly-rich stretch of sequence spanning positions 553-563 are essential components of the active site of the PEP/pyruvate partial reaction.

Biochemistry, 1995 Feb 21, 34(7), 2181 - 7
Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase . 1 . Identification of an essential cysteine by chemical modification with {1-14C}bromopyruvate and site-directed mutagenesis; Xu Y et al.; Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of adenosine 5'-triphosphate (ATP), orthophosphate (Pi), and pyruvate with adenosine 5'-monophosphate (AMP), pyrophosphate (PPi), and phosphoenolpyruvate (PEP) . The reaction takes place according to the following steps: (1) E+ATP+P(i)<-->E-PP.AMP.P(i), (2) E-PP.AMP.P(i)<-->E-P+AMP+PP(i), and (3) E-P+pyruvate<-->E+PEP, where E represents free enzyme; E-PP, pyrophosphorylenzyme; and E-P, phosphorylenzyme . Steps 1 and 2 comprise the nucleotide partial reaction, and step 3 comprises the pyruvate partial reaction . The present studies were carried out to locate amino acid residues within the primary structure of Clostridium symbiosum PPDK participating in the catalysis of the pyruvate partial reaction . The enzyme was treated with the affinity label {1-14C}bromopyruvate, reduced with NaBH4, proteolyzed with trypsin, and chromatographed on an HPLC column . The radiolabeled tryptic peptide isolate was sequenced to reveal Cys 831 as the site of alkylation . Using PCR techniques Cys 831 was replaced by Ala, and the C831A PPDK mutant formed was then subjected to kinetic analysis . Rapid quench studies of single turnover reactions on the enzyme showed that the mutant is as efficient as wild-type PPDK in catalyzing the nucleotide partial reaction while it is unable to catalyze the pyruvate partial reaction . These results were interpreted as evidence for a role of Cys 831 in pyruvate/PEP binding and/or catalysis.

Biochim Biophys Acta, 1995 Feb 16, 1265(1), 73 - 8
Insulin action on cardiac glucose transport: studies on the role of protein kinase C; Russ M et al.; Isolated ventricular cardiomyocytes from adult rat have been used to elucidate a possible relationship between protein kinase C (PKC) and the stimulatory action of insulin on cardiac glucose transport . Cells were incubated in the presence of either insulin or phospholipase C from Clostridium perfringens (PLC-Cp) and intracellular sn-1,2-diacylglycerol (DAG) levels and initial rates of 3-O-methylglucose transport were determined . Insulin had no effect on the DAG mass level, whereas it was elevated by PLC-Cp to 200% of control . Under these conditions the hormone produced a 2.7-fold stimulation of glucose transport with no significant effect of PLC-Cp . Insulin was unable to produce a redistribution of PKC, whereas phorbol 12-myristate 13-acetate (PMA) increased membrane associated PKC twofold . The PKC inhibitors tamoxifen and staurosporine did not interfere with glucose transport stimulation by insulin . Furthermore, cells treated with PMA exhibited unaltered basal and maximally insulin stimulated rates of glucose transport . In contrast, at physiological concentrations of insulin the stimulatory action of the hormone was significantly reduced . We conclude from our data that PKC is not involved in insulin action on cardiac glucose transport . However, activation of this enzyme may lead to a modified insulin sensitivity of the cardiac cell.

Presse Med, 1995 Feb 11, 24(6), 317 - 22
{Digestive involvements in human immunodeficiency virus infection}; Verdon R et al.; Dysphagia or odynophagia occurs in an estimated 21% of patients with human immunodeficiency virus infection . A causal agent can be identified in 60-90% of the cases and generally can be successfully eradicated . Oesophageal candidosis, the predominant disorder, usually responds to nitrate derivatives and amphotericine B after a 10 to 15 day cure . Ulcerations of the oesophagus is the second major cause of dysphagia in these patients and result from cytomegalovirus and herpes simplex infections or unknown causes . Epstein-Barr virus infection has been suggested but is rarely demonstrated in clinical situations . Similar to other localizations in HIV-infected patients, Kaposi sarcoma and non-Hodgkin malignant lymphomas are the predominant tumours in the bowel . Infections are essentially revealed by sometimes very severe diarrhoea . Infective agents include Cryptosporidium parvum, microsporidiosae, cytomegalovirus, adenovirus, Isospora belli, Clostridium difficile, Salmonellae and non-tuberculous mycobacteria among others . When the search for an infective agent is negative, the diarrhoea is usually considered to be the expression of HIV infection itself . The clinical approach to HIV-related diarrhoea can be based on decision making management scheme according to the results of stool cultures or on complete exploration protocols . Whatever the diagnostic procedure, symptomatic treatment is of major importance because of the severe nutritional impact of HIV-related diarrhoea.

Gene, 1995 Feb 3, 153(1), 89 - 92
Sequence and arrangement of genes encoding sigma factors in Clostridium acetobutylicum ATCC 824; Wong J et al.; The nucleotide sequence of a 2.7-kb region of Clostridium acetobutylicum ATCC 824 DNA containing three open reading frames was determined . They encoded homologs of three proteins of Bacillus subtilis, and the gene arrangement in both organisms was identical . The first gene, orfA, was 801-bp long; the 31-kDa (266 aa) product it encoded exhibited homology with the putative sigma E-processing enzyme . The second gene, sigE, was 708-bp long encoding a 27-kDa (235 aa) product; the third gene, sigG, was 774-bp long encoding a 30-kDa (257 aa) product . These two proteins showed high homology with sigma E and sigma G, two sporulation-specific sigma factors.

J Med Microbiol, 1995 Feb, 42(2), 78 - 82
Clostridial infection in children; Brook I; A survey of the isolation of Clostridium spp . from 1543 specimens sent to anaerobic microbiology laboratories revealed 113 isolates from 107 specimens (7.0% of all specimens) from 96 children . The isolates comprised 43 (38%) unidentified Clostridium spp., 37 (33%) C . perfringens, 13 (12%) C . ramosum, five (4%) C . innocuum, six (5%) C . botulinum, three (3%) C . difficile, two (2%) C . butyricum, and one isolate each of C . bifermentans, C . clostridiiforme, C . limosum and C . paraputrificum . Most clostridial isolates were from abscesses (38), peritonitis (26), bacteraemia (10), and chronic otitis media (7) . Predisposing or underlying conditions were present in 31 (32%) cases . These were immunodeficiency (12), malignancy (9), diabetes (7), trauma (7), presence of a foreign body (6) and previous surgery (6) . The clostridia were the only bacterial isolates in 14 (15%) cases; 82 (85%) cases had mixed infection . The species most commonly isolated with clostridia were anaerobic cocci (57); Bacteroides spp . (B . fragilis group) (50), Escherichia coli (22), pigmented Prevotella or Porphyromonas spp . (18) and Fusobacterium spp . (10) . Most Bacteroides and Escherichia coli isolates with clostridia were from abdominal infections and skin and soft tissue infections adjacent to the rectal area; most pigmented Prevotella and Porphyromonas isolates were from oropharyngeal, pulmonary, and head and neck sites . Antimicrobial therapy was given to all patients, in conjunction with surgical drainage in 34 (35%) . Only two patients died . These data illustrate the importance of Clostridium spp . in paediatric infections.

J Bacteriol, 1995 Feb, 177(4), 1098 - 103
Tracking the evolution of the bacterial choline-binding domain: molecular characterization of the Clostridium acetobutylicum NCIB 8052 cspA gene; Sanchez-Beato AR et al.; The major secreted protein of Clostridium acetobutylicum NCIB 8052, a choline-containing strain, is CspA (clostridial secreted protein) . It appears to be a 115,000-M(r) glycoprotein that specifically recognizes the choline residues of the cell wall . Polyclonal antibodies raised against CspA detected the presence of the protein in the cell envelope and in the culture medium . The soluble CspA protein has been purified, and an oligonucleotide probe, prepared from the determined N-terminal sequence, has been used to clone the cspA gene which encodes a protein with 590 amino acids and an M(r) of 63,740 . According to the predicted amino acid sequence, CspA is synthesized with an N-terminal segment of 26 amino acids characteristic of prokaryotic signal peptides . Expression of the cspA gene in Escherichia coli led to the production of a major anti-CspA-labeled protein of 80,000 Da which was purified by affinity chromatography on DEAE-cellulose . A comparison of CspA with other proteins in the EMBL database revealed that the C-terminal half of CspA is homologous to the choline-binding domains of the major pneumococcal autolysin (LytA amidase), the pneumococcal antigen PspA, and other cell wall-lytic enzymes of pneumococcal phages . This region, which is constructed of four repeating motifs, also displays a high similarity with the glucan-binding domains of several streptococcal glycosyltransferases and the toxins of Clostridium difficile.

Diabetes, 1995 Feb, 44(2), 227 - 33
Different roles of class I and class II Clostridium histolyticum collagenase in rat pancreatic islet isolation; Wolters GH et al.; Crude Clostridium histolyticum collagenase was purified by gel filtration and fractionated by anion exchange chromatography into class I with high collagen digestion activity (CDA) and low FALGPA (2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine) hydrolysis activity (FHA), class II with low CDA and high FHA, and a fraction called class I/II with intermediate activities . The roles of these collagenase classes in rat pancreatic islet isolation were investigated . Dissociations were carried out with 360 mg of pancreatic tissue in 10 ml of buffer containing 10% (wt/vol) albumin to suppress endogenous proteolytic activity, 100 U of C . histolyticum neutral protease, and one or two purified collagenase(s) . For purified nonfractionated (PNF) collagenase, 2.6 mg of enzyme containing 2.4 U CDA and 38.0 U FHA was used, and for the separate classes, comparable amounts of activity were added . PNF collagenase dissociated the tissue completely in 32 min and yielded 5.0 +/- 0.4 microliters islet tissue/g pancreas . Class I collagenase alone dissociated pancreatic tissue extremely slowly and incompletely; only a few islets were released (0.7 +/- 0.2 microliters/g pancreas) . Class II collagenase alone dissociated the tissue adequately in 50 min, and a high islet yield of 5.7 +/- 0.6 microliters/g was obtained . With class I/II, a similar dissociation time (47 min) and islet yield (5.5 +/- 0.3 microliters/g) were obtained . Combining class I and class II collagenase resulted in a more rapid dissociation (32 min) and a higher islet yield (7.1 +/- 0.8 microliters/g) than that obtained with PNF collagenase (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

J Exp Med, 1995 Feb 1, 181(2), 577 - 84
Sustained signaling leading to T cell activation results from prolonged T cell receptor occupancy . Role of T cell actin cytoskeleton; Valitutti S et al.; Using antigen-specific T cell clones and peptide-pulsed antigen-presenting cells (APCs) we investigated the mechanisms that lead to sustained signaling, known to be required for activation of effector function . Four lines of evidence indicate that the T cell actin cytoskeleton plays a crucial role in T cell activation by antigen-pulsed APCs, but is not required when T cell receptor (TCR) is cross-linked by soluble antibodies . First, addition of antibodies to the major histocompatibility complex molecules recognized by the TCR aborts the ongoing intracellular calcium concentration ({Ca2+}i) increase in performed T-APC conjugates, indicating that the sustained signaling requires the continuous occupancy of TCR . Second, time-lapse image recording shows that T lymphocytes conjugated to peptide-pulsed APCs undergo a sustained {Ca2+}i increase, which is accompanied by the formation of a large and changing area of contact between the two opposing membranes . Third, drugs that disrupt the actin cytoskeleton, Cytochalasin D and and C2 Clostridium botulinum toxin induce a rapid block of {Ca2+}i rise, coincident with a block of the cyclic changes in T cell shape . Finally, the addition of Cytochalasin D or of anti-MHC antibodies to preformed conjugates inhibits interferon gamma production in an 1-antigen dose- and time-dependent fashion . These results identify T cell actin cytoskeleton as a major motor for sustaining signal transduction and possibly for driving TCR cross-linking and offer an explanation for how T cells equipped with low affinity TCR can be triggered by a small number of complexes on APCs.

Berl Munch Tierarztl Wochenschr, 1995 Feb, 108(2), 55 - 7
{Clostridium bifermentans infection in grass carp (Ctenopharyngodon idella)}; Hoffmann RW et al.; The outbreak of an infection with Clostridium bifermentans in grass carp (Ctenopharyngodon idella) from a polyculture fish pond is documented.

Mol Microbiol, 1995 Feb, 15(4), 639 - 47
The enterotoxin gene (cpe) of Clostridium perfringens can be chromosomal or plasmid-borne; Cornillot E et al.; The location of the cpe gene, encoding the enterotoxin responsible for food poisoning in humans, has been studied in a series of enterotoxigenic Clostridium perfringens strains by means of pulsed field gel electrophoresis of genomic DNA . The cpe gene was found at the same chromosomal locus in strains associated with food poisoning in humans and was shown to be linked to a repetitive sequence, the HindIII repeat, and an open reading frame, ORF3, that may be part of an insertion sequence . In contrast, when the strains originated from domesticated livestock cpe was located on a large episome where it was often close to a copy of the transposable element IS1151 . In these cases, the HindIII repeat was not linked to the cpe gene although this was generally preceded by ORF3.

J Clin Pharm Ther, 1995 Feb, 20(1), 5 - 11
Teicoplanin or vancomycin in the treatment of gram-positive infections?
Murphy S, Pinney RJ.
The glycopeptide antibiotics vancomycin and teicoplanin have similar mechanisms of action on bacterial cell wall synthesis . Their spectra of activity are limited to Gram-positive bacteria, with the degree of bactericidal activity depending on the species of micro-organism . Staphylococcus aureus, Staphylococcus epidermis, enterococci and Clostridium difficile are generally sensitive, including methicillin-resistant strains of S . aureus and S . epidermidis . Glycopeptide resistance has recently emerged in staphylococci and enterococci . Vancomycin has a shorter half-life than teicoplanin and requires multiple dosing to maintain adequate serum levels . It can only be given by prolonged intravenous infusion over 1 h . In contrast, the pharmacokinetics of teicoplanin allow for once-daily dosing, either by rapid intravenous infusion or by the intramuscular route . The latter offers reliable absorption for patients with limited venous access and is also of benefit for out-patient therapy . Teicoplanin is a safer drug than vancomycin . It is associated with a lower incidence of nephrotoxicity or ototoxicity . Compared to vancomycin, the availability of the intramuscular route and the absence of a requirement for routine serum monitoring, together with the reduced need to treat drug-related side-effects make teicoplanin more cost-effective . It is as effective as vancomycin for most indications, is safe, easy to administer and an important agent for treating Gram-positive infections . Its role in hospitals is likely to increase if the price of drug acquisition is kept low.

Trop Anim Health Prod, 1995 Feb, 27(1), 31 - 6
An outbreak of bacillary haemoglobinuria in sheep in India; Randhawa SS et al.; An outbreak of bacillary haemoglobinuria was recorded in 60 out of 110 sheep in Ludhiana, Punjab, India . The condition was clinically characterised by fever, haemoglobinuria, constipation, weakness of hind quarters followed by recumbency, respiratory distress and death in 16 sheep . Haematological studies revealed moderate to severe degrees of anaemia associated with leucocytosis . Plasma gamma-glutamyl transferase, alkaline phosphatase and creatinine phosphokinase activities were significantly higher in haemoglobinuric sheep . Babesiosis and copper poisoning were ruled out on stained blood film examination and from blood mineral profiles, respectively . Post-mortem examination of affected sheep revealed no gross changes . Pure cultures of Clostridium haemolyticum isolated from heart blood, liver, kidney and spleen of freshly killed sheep confirmed the disease . Parenteral administration of procaine penicillin was effective in the treatment of affected sheep.

J Antimicrob Chemother, 1995 Feb, 35(2), 305 - 15
Transfer of macrolide-lincosamide-streptogramin B (MLS) resistance in Clostridium difficile is linked to a gene homologous with toxin A and is mediated by a conjugative transposon, Tn5398; Mullany P et al.; An MLS resistance gene designated ermBZ, from a toxigenic Clostridium difficile strain (630) could be transferred between C . difficile strains, and to and from Bacillus subtilis . The intergeneric transfer occurred in the absence of any detectable plasmid DNA and the element responsible for gene transfer entered the recipient's chromosome, behaviour which is characteristic of a conjugative transposon . The element was designated Tn5398 and was found in six C . difficile strains . Tn5398 could be transferred to the non-toxigenic strain C . difficile CD37 which lacks the genes for toxins A and B . Transconjugants from both C . difficile and B . subtilis that had received the ermBZ gene also acquired a sequence of DNA that was homologous to the part of the toxin A gene that coded for the C-terminal repeat region.

Lab Anim Sci, 1995 Feb, 45(1), 47 - 53
In vivo and in vitro studies of Clostridium difficile-induced disease in hamsters fed an atherogenic, high-fat diet; Blankenship-Paris TL et al.; After previous observation of increased susceptibility to Clostridium difficile enterocolitis in hamsters fed an atherogenic, high-fat diet, a study was undertaken to examine experimental reproducibility of this disease . Hamsters were fed either the high-fat diet or a control diet, then orally challenged with a toxigenic strain of C . difficile . Hamsters fed the high-fat diet suffered 80% morbidity, which was statistically significant from the 11% morbidity of the control diet group (P < or = 0.05) . The disease presented acutely, the most common presentation being sudden death . The most common lesions in the affected hamsters were necrohemorrhagic cecitis and cecal mucosal hyperplasia . Hepatic lipidosis was consistent in all hamsters fed the high-fat diet . Toxigenic C . difficile could be recovered from the cecum of most affected hamsters, and toxins A and B were detected in these ceca . Hamsters that were fed the high-fat diet and orally inoculated with a nontoxigenic strain of C . difficile before experimental challenge with a toxigenic strain were initially protected against disease . The protection decreased with each exposure to the toxigenic strain . Results of in vitro colonization-resistance studies indicated that the cecal flora from hamsters fed the high-fat diet and control diets inhibited C . difficile growth, suggesting that increased disease susceptibility was not the result of altered cecal flora.

Ann Plast Surg, 1995 Feb, 34(2), 201 - 2
Tetanus caused by human bite of the finger; Agrawal K et al.; A case of generalized tetanus after human bite of the finger is reported . The patient recovered with institutional care . We propose that secondary invasion by Clostridium tetani is the cause for infection . It could be prevented by immediate tetanus prophylaxis, thorough debridement, and primary repair of the wound.

Antimicrob Agents Chemother, 1995 Feb, 39(2), 413 - 21
E-4695, a new C-7 azetidinyl fluoronaphthyridine with enhanced activity against gram-positive and anaerobic pathogens; Guinea J et al.; E-4695, (-)-7-{3-(R)-amino-2-(S)-methyl-1-azetidinyl}-1-cyclopropyl-1,4- dihydro-6-fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acid, is a new fluorinated naphthyridine with an azetidine moiety . The MICs of E-4695 at which 90% of the isolates were inhibited (MIC90s) were 0.06 to 0.5 microgram/ml for gram-positive cocci, including species of the genera Staphylococcus, Streptococcus, and Enterococcus, and the MIC90s against gram-negative pathogens such as members of the family Enterobacteriaceae (with the exception of Providencia spp . {MIC90, 8 micrograms/ml}) and Pseudomonas aeruginosa were 0.015 to 0.5 microgram/ml . E-4695 inhibited 90% of the Clostridium perfringens and Bacteroides fragilis isolates at 0.25 and 4 micrograms/ml, respectively . Against gram-positive cocci the potency of E-4695 was 2- to 8-fold higher than that of ciprofloxacin, 4- to 8-fold higher than that of ofloxacin, and 8- to 16-fold higher than that of fleroxacin . Against enteric bacteria and P . aeruginosa the potency of E-4695 was, in general, similar to that of ciprofloxacin and eightfold higher than those of ofloxacin and fleroxacin . E-4695 was four- and eightfold more potent than ciprofloxacin against C . perfringens and B . fragilis isolates, respectively . E-4695 and ciprofloxacin showed similar properties when the effects of pH or magnesium concentration were tested on them . E-4695 and ciprofloxacin had substantial reductions of activity only when pH decreased below 4.8 . E-4695 and ciprofloxacin activities were not markedly affected by the presence of 5 or 10 mM Mg2+ . The presence of serum and human urine at pH 7.2 decreased the activity of E-4695 between two- and fourfold . After an oral dose of 50 mg/kg of body weight, the maximum level in serum, the biological half-life, and the area under the concentration-time curve from 0 to 10 h for E-4695 were 13.2 microgram/ml, 3.3 h, and 45.6 microgram . h/ml, respectively . The area under the concentration-time curve from 0 to 4 h for ciprofloxacin was 2.3 microgram . h/ml at the same dose . Fifty-percent effective doses (ED50S) against Staphylococcus aureus HS-93 infections in mice were 4.5 mg/kg with E-4695 and 37.6 mg/kg with ciprofloxacin . Infection with Streptococcus pneumoniae 29206 was more effectively treated with E-4695 (ED50, 41,2 mg/kg) than with ciprofloxacin (ED50, 200 mg/kg) . The ED50 of E-4695 for infections with Streptococcus pneumoniae 1625 was 132.2 mg/kg; ciprofloxacin was ineffective at 400 mg/kg against this strain . E-4695 was also more potent than ciprofloxacin in treatment of infections caused by gram-negative organisms such as Escherichia coli HM-42 (ED50S, 1.0 and 3.9 mg/kg, respectively) . The ED50S of E-4695 and ciprofloxacin were 33.0 and 145.5 mg/kg against P . aeruginosa HS-116 and 9.6 and 18.9 mg/kg against P . aeruginosa B-120, respectively . The therapeutic efficacy of E-4695 may depend not only on its in vitro activity but also on its improved pharmacokinetic properties.

J Dermatol, 1995 Feb, 22(2), 98 - 106
A histological study of cutaneous thermal wounds using a Clostridium perfringens-derived wound healing substance with wound healing stimulation activity; Takahashi M et al.; We studied the effects of a Clostridium perfringens-derived wound-healing substance (WHS) on the healing of thermal burn wounds . Third-degree burn injuries were inflicted on the back skin of rats . We histologically evaluated the effects of WHS ointments and compared them with those of lysozyme chloride ointment . We observed the formation of dermal collagen fibers and the increase of capillaries in the WHS ointment treated groups . From the results of hematoxylin and eosin staining and silver staining, an increase in capillaries was observed one week after the application of WHS ointment . Three weeks after the application, when the epithelization was in the final stage, capillary formation ceased . In the WHS ointment-applied groups, electron microscopic observation showed that new collagen fibers were regularly formed in the dermis . On the other hand, in the lysozyme chloride ointment-applied groups, new collagen fibers were present, but were irregularly formed . The main wound healing stimulative action of the WHS ointment could be due to its acceleration of new capillary formation.

Microbiology, 1995 Feb, 141 ( Pt 2), 371 - 5
A defined growth medium for Clostridium difficile; Karasawa T et al.; Minimal requirements of amino acids and vitamins were determined in chemically defined medium for five strains of Clostridium difficile . Cysteine, isoleucine, leucine, proline, tryptophan and valine were essential amino acids for growth of C . difficile . Arginine, glycine, histidine, methionine and threonine enhanced growth . Biotin, pantothenate and pyridoxine were essential vitamins . A defined medium containing the minimal requirements of amino acids and vitamins produced a rapid and heavy growth which was comparable to that in modified brain heart infusion, a complex medium . Adenine was able to substitute for glycine and threonine, suggesting that the two amino acids may be utilized as precursors of purine nucleotides . The defined medium developed here will assist physiological and biochemical studies on C . difficile.

Appl Environ Microbiol, 1995 Feb, 61(2), 807 - 10
Nonradioactive colony hybridization assay for detection and enumeration of enterotoxigenic Clostridium perfringens in raw beef; Baez LA et al.; A DNA probe endolabeled with digoxigenin by PCR was developed to detect and enumerate enterotoxigenic Clostridium perfringens in raw beef . After 2 h of hybridization, membranes were developed by using an anti-digoxigenin-alkaline phosphatase conjugated antibody . The resulting chromogenic reaction allowed us to detect and enumerate < or = 10 CFU of C . perfringens per g.

Appl Environ Microbiol, 1995 Feb, 61(2), 583 - 91
Ruminal microbial digestion in free-living, in captive lichen-fed, and in starved reindeer (Rangifer tarandus tarandus) in winter; Aagnes TH et al.; In free-living (FL) reindeer eating a natural mixed winter diet dominated by lichens, captive (CF) reindeer fed pure lichens ad libitum, and CF reindeer subsequently starved for 1 day (CS1 reindeer) or 4 days (CS4 reindeer), the dominant rumen anaerobic bacteria were characterized, their population densities were estimated, and ruminal pH and volatile fatty acid concentrations were determined . In the FL reindeer, the total median viable anaerobic bacterial population ranged from 18 x 10(8) to 35 x 10(8) cells per ml of rumen fluid (n = 4), compared with 26 x 10(8) to 34 x 10(8) and 0.09 x 10(8) to 0.1 x 10(8) cells per ml of rumen fluid in CF reindeer (n = 2) and CS4 reindeer (n = 2), respectively . The median bacterial population adhering to the rumen solids ranged from 260 x 10(8) to 450 x 10(8), 21 x 10(8) to 38 x 10(8), and 0.5 x 10(8) cells per g (wet weight) of rumen solids in FL, CF, and CS4 reindeer, respectively . Although there were variations in the rumen bacterial composition among the FL reindeer (n = 4), strains of Bacteroides, Fibrobacter, Streptococcus, and Clostridium dominated in the rumen fluid . Streptococcus spp . and Clostridium spp . were the dominant bacteria in the CF reindeer (n = 2), while in the CS4 reindeer (n = 2) the dominant bacteria were Fusobacterium spp., members of the family Enterobacteriaceae, and Eubacterium spp . Transmission electron micrographs of lichen particles from the rumen of one FL reindeer, one CF reindeer, and one CS4 reindeer show bacteria resembling Bacteroides spp . adhering to the lichen particles, evidently digesting the lichen hyphae from the inside.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1995 Jan 27, 270(4), 1770 - 4
IgA protease from Neisseria gonorrhoeae inhibits exocytosis in bovine chromaffin cells like tetanus toxin; Binscheck T et al.; When tetanus toxin from Clostridium tetani or IgA protease from Neisseria gonorrhoeae is translocated artificially into the cytosol of chromaffin cells, both enzymes inhibit calcium-induced exocytosis, which can be measured by changes in membrane capacitance . The block of exocytosis caused by both proteases cannot be reversed by enforced stimulation with increased calcium concentration . This effect differs from the botulinum A neurotoxin-induced block of exocytosis that can be overcome by elevation of the intracellular calcium concentration . Tetanus toxin is about 50-fold more potent than IgA protease in cells stimulated by carbachol . In this case, the release of {3H}noradrenaline was determined . Trypsin and endoprotease Glu-C are hardly effective and only at concentrations that disturb the integrity of the cells . Like tetanus toxin, IgA protease also splits synaptobrevin II, though at a different site of the molecule . However, unlike tetanus toxin, it does not cleave cellubrevin . It is concluded that the membranes of chromaffin vesicles contain synaptobrevin II, which, as in neurons, appears to play a crucial part in exocytosis.

J Biol Chem, 1995 Jan 20, 270(3), 1092 - 7
Analysis of acyl coenzyme A binding to the transcription factor FadR and identification of amino acid residues in the carboxyl terminus required for ligand binding; Raman N et al.; The Escherichia coli FadR protein regulates the transcription of many unlinked genes and operons encoding proteins required for fatty acid synthesis and degradation . Previously, we demonstrated that the ability of purified FadR to bind DNA in vitro is inhibited by long chain acyl coenzyme A esters (DiRusso, D . D., Heimert, T . L., and Metzger, A . K . (1992) J . Biol . Chem . 267, 8685-8691) . In the present work, we show that FadR binds acyl-CoA directly . Ligand binding resulted in a shift in the apparent pI of FadR from 6.9 to 6.2 and in a marked decrease in intrinsic fluorescence . The Km for FadR binding of oleoyl coenzyme A was determined to be 12.1 nM using the fluorescence quenching assay . The binding site for acyl-CoA was identified by selection of non-inducible mutations in the FadR gene . One altered protein carrying the change Ser219 to Asn (S219N) was purified and shown to have a reduced affinity for oleoyl coenzyme A as evidenced by a Km of 257 nM . S219N retained the ability to bind DNA and to repress or activate transcription . Alanine substitution of amino acid residues 215 through 230 identified Gly216 and Trp223 as also required specifically for induction . This region of FadR shares amino acid identities and similarities with the coenzyme A-binding site of Clostridium thermoaceticum CO dehydrogenase/acetyl-coenzyme A synthase . Due to the alteration in binding affinity of the purified S219N protein, the non-inducible phenotype of several proteins carrying alanine substitutions and similarities to CO dehydrogenase/acetyl-coenzyme A synthase we propose this region of FadR forms part of the acyl-CoA-binding domain.

FEMS Microbiol Lett, 1995 Jan 15, 125(2-3), 185 - 91
Structure and transcription of genes within the beta-hbd-adh1 region of Clostridium acetobutylicum P262; Youngleson JS et al.; The 1.2-kb DNA fragment upstream of the linked beta-hbd (3-hydroxybutyryl-CoA dehydrogenase) and adh1 (NADPH-dependent alcohol dehydrogenase) genes from Clostridium acetobutylicum P262 was sequenced . The upstream region contained an open reading frame (ORFB) which was found to have 44% amino acid identity to the fixB gene products of Rhizobium and Azorhizobium . The beta-hbd and ORFB genes were expressed during the acidogenic and solventogenic phases . The beta-hbd gene was transcribed on a single mRNA species of 2.0 kb, whereas the ORFB gene was transcribed on two species of mRNA of 2.0 and 3.5 kb, respectively . The adh1 gene was induced or derepressed at the pH breakpoint before the onset of solventogenesis and was transcribed on a single species of mRNA of 2.4 kb.

J Am Vet Med Assoc, 1995 Jan 15, 206(2), 187 - 93
Small intestinal bacterial overgrowth in dogs with chronic intestinal disease; Rutgers HC et al.; Small intestinal bacterial overgrowth (SIBO) was diagnosed by quantitative bacterial culture of duodenal juice samples obtained endoscopically in 41 of 80 dogs that were admitted with chronic diarrhea, vomiting, or weight loss . Thirteen dogs had aerobic bacterial overgrowth, most frequently comprising Escherichia coli, staphylococci, and enterococci, and 28 dogs had mixed anaerobic overgrowth, most frequently including Clostridium and Bacteroides spp . Affected dogs comprised 23 breeds, including 10 German Shepherd Dogs and median age at diagnosis was 2 years (range, 6 months to 11 years) . High serum folate and low serum cobalamin concentrations had fair specificity (79 and 87%, respectively), but low sensitivity (51 and 24%, respectively) in detecting SIBO . Histologic examination of duodenal biopsy specimens did not reveal abnormalities (26/41 dogs), or revealed mild to moderate lymphocytic (12/41) or eosinophilic (2/41) infiltrates, or lymphosarcoma (1/41) . Oral antibiotic treatment was effective in 77% (23/30 dogs), but prolonged treatment (> 4 weeks) was required to control signs and prevent recurrence in 50% (15/30) . Corticosteroids were used alone in a dog with eosinophilic enteritis and in combination with antibiotics in 4 dogs with marked gastrointestinal lymphocytic/plasmacytic infiltrates . This study suggested that SIBO may be observed in dogs of many breeds, without an obvious primary cause, and that, although results of indirect tests may be suggestive of SIBO, bacterial culture of duodenal juice samples remains necessary for definitive diagnosis.

Biochemistry, 1995 Jan 10, 34(1), 334 - 40
Rho-ADP-ribosylating exoenzyme from Bacillus cereus . Purification, characterization, and identification of the NAD-binding site; Just I et al.; The ADP-ribosyltransferase produced by a pathogenic strain of Bacillus cereus was purified to near homogeneity . The transferase is a 28,000 Da molecular mass enzyme with a pI of 10.3 . The specific enzyme activity is 7.0 nmol of ADP-ribose min-1 mg-1 with a Km for NAD of 0.3 microM . Partial amino acid sequence analysis of the exoenzyme reveals no significant homology to Clostridium botulinum C3 nor to Clostridium limosum exoenzyme . The novel exoenzyme selectively modifies the small GTP-binding proteins of the Rho family presumably at the same acceptor amino acid (Asn-41) as determined for C3 . Besides cellular Rho, recombinant RhoA and -B are substrates for the exoenzyme . However, recombinant Rac1 and CDC42, although belonging to the Rho family, are not modified . B . cereus exoenzyme was photolabeled with {carbonyl-14C}NAD resulting in inhibition of ADP-ribosyltransferase and NAD-glycohydrolase activity . A glutamic acid residue was identified as part of the NAD-binding site which corresponds to Glu-174 of C3 . This glutamic acid is located in a domain which shows high homology with the C-terminal part of C3 exoenzyme, C . limosum exoenzyme, and Staphylococcus aureus EDIN and which probably represents the catalytic site of the transferases . The data indicate that B . cereus exoenzyme is a novel member of the family of C3-like ADP-ribosyltransferases which share the same substrate protein Rho and which have an identical highly conserved catalytic domain.

Biochemistry, 1995 Jan 10, 34(1), 326 - 33
ADP-ribosyltransferase type A from turkey erythrocytes modifies actin at Arg-95 and Arg-372; Just I et al.; Turkey erythrocyte ADP-ribosyltransferase A catalyzes the transfer of ADP-ribose from NAD to both monomeric and polymeric skeletal muscle alpha-actin with the incorporation of 2 mol of ADP-ribose per mol of actin . In contrast, Clostridium perfringens iota toxin ADP-ribosylates only G-actin, with modification at arginine-177 {Vandekerckhove, J., et al . (1987) FEBS Lett . 255, 48-42} . Transferase A-catalyzed modifications are sensitive to 0.5 M neutral hydroxylamine, consistent with the arginine side chain modification . Radiolabeled peptides ADP-ribosylated by transferase A were generated by tryptic digestion and purified by reversed phase high-performance liquid chromatography . Amino acid sequence and molecular mass analysis identified the ADP-ribosylation sites as Arg-95 and Arg-372 of actin; both residues are located within subdomain-1 of the actin 3D structure {Kabsch, W., et al . (1990) Nature 347, 37-44} . ADP-ribosylation did not affect cytochalasin D-stimulated G-actin ATPase, the binding of actin to DNase I or to gelsolin, or the ability of actin to polymerize . Following ADP-ribosylation, however, a prolonged delay in polymerization was observed, consistent with a decreased rate of nucleation.

Acta Cient Venez, 1995, 46(1), 41 - 50
{Histochemical characterization of glycoconjugates in the tongue of the toad Bufo marinus L . (Amphibian, anuran) with conventional techniques for light microscopy}; Gonzalez-Elorriaga MA et al.; Histochemical stains with and without enzymatic digestions such as alpha-amylase, neuraminidase and hyaluronidase, made possible to localize and differentiate various types of glycoconjugates (GCs) in the tongue of the toad Bufo marinus . In the dorsal mucosae the covering epithelium of the filiform papillae, of the central folds and in the marginal cells of the fungiform papillae there were present large amounts of neutral GCs with little or no galactose and/or N-acetylgalactosamine and scanty carboxylic acid GCs while the superficial strata of the taste organs showed a mixture of neutral an acid GCs with a predominance of sulfated and carboxylic acid GCs . The glandular secretory cells showed neutral GCs almost exclusively with a gradient of concentrations increasing from the base to the apex being galactose or N-acetyl-galactosamine one of the component sugars . The ventral epithelium showed two types of mucous cells, one with neutral GCs and the other with neutral and acidic GCs . The connective tissue contained many mast cells showing highly acid GCs both sulfated and carboxylic with some neutral GCs . The extracellular connective matrix showed scanty neutral and acid GCs . Glycogen was present in the cytoplasm of glandular epithelial cells and of the striated muscle fibers . Additionally, the obtained results suggest the presence of a type of GC with a carboxylic acid (sialic acid) resistant to neuraminidase of Clostridium perfringens used in this study.

Exp Clin Immunogenet, 1995, 12(4), 232 - 7
Fungal anti-A agglutinins with different affinities for subgroups A1 and A2 red cells; Ying R et al.; We found two anti-A hemagglutinating lectins in the extracts of the fruiting bodies of two fungi (93-34, 93-138) belonging to the Tricholomataceae family . The extracts reacted with papain-treated human group A red cells but not with O or B cells . Both fungus lectins gave negative results with saline suspensions of human and animal red cells . In agglutination tests against red cells of A subgroups, 93-34 gave similar results to Dolichos biflorus, such as a stronger reaction with A1 than with A2 cells, while 93-138 showed stronger agglutination of A2 than of A1 cells . Using both fungus hemagglutinating lectins with human standard anti-A serum it was possible to distinguish subgroups of A1 and A2 . The activity of both lectins was inhibited by A secretor saliva and A substance from human stomach linings . A-decomposing enzyme from Clostridium tertium A destroyed this inhibiting activity of the reaction of lectins with A cells . N-Acetyl-D-galactosamine was the only potent inhibitor of the hemagglutination reaction among the monosaccharides tested . No agglutination was observed with Cad(+) red cells . The results suggested that two lectins have affinity with terminal alpha-linked N-acetylgalactosaminyl residues of polysaccharide side chains.

Przegl Epidemiol, 1995, 49(4), 361 - 5
{Occurrence and clinical picture of Clostridium difficile infections after antibiotic therapy}; Lapinski TW et al.; 39 patients with colon bowel inflammatory by C . difficile infection after antibioticotherapy were treated . The causes of antibioticotherapy most frequently former infection of the upper respiratory tract . The C . difficile infection were most frequently before therapy: poliantibiotics, Cephalosporins II generations and Linkomycin . In diagnosis of C . difficile infection the Enzyme Immuno-Fluorescence Test were practical utility confirmed . The symphtomaticotherapy patients with slight course of ills were sufficiently . The therapy of Metronidazol or/and Colestyramine in patients with mild or highly course of ills were efficacious.

Med Dosw Mikrobiol, 1995, 47(3-4), 177 - 81
{Study of the thermoresistance of Clostridium difficile spores}; Meisel-Mikolajczyk F et al.; Thermoresistance of C . difficile spores was investigated . C . difficile strains were isolated from different sources . As control, toxigenic VPI 10463 and nontoxigenic NIH BRIGGS 8050 C . difficile strains were used . The inhibition of growth majority of C . difficile after heating at 85 degrees C was shown . No correlation between thermoresistance of C . difficile spores, toxigenicity and source of the strains was observed.

Biofactors, 1995-96, 5(2), 93 - 7
Identification of molybdopterins in molybdenum- and selenium-containing enzymes; Gladyshev VN et al.; Selenium is coordinated to a molybdenum atom in nicotinic acid hydroxylase (NAH) from Clostridium barkeri and formate dehydrogenase H (FDH) from Escherichia coli . Selenium is present in FDH in a selenocysteine residue whereas in NAH in occurs in an unidentified labile cofactor . In this paper we describe a simple procedure for isolation and identification of molybdopterins from Mo-containing enzymes . The molybdopterin, after release from the protein with guanidine-hydrochloride, is reduced with KBH4, alkylated with iodoacetamide and separated on a reverse-phase HPLC column . The carboxam-idomethylated pterin compound is further characterized by UV spectroscopy and mass-spectrometry . We found that FDH contains molybdopterin guanine dinucleotide whereas NAH contains molybdopterin cytosine dinucleotide.

Vet Res Commun, 1995, 19(6), 451 - 6
Detection of Clostridium chauvoei in formalin-fixed, paraffin-embedded tissues of sheep by the peroxidase-antiperoxidase (PAP) technique; Giraudo Conesa LC et al.; A peroxidase-antiperoxidase (PAP) technique was used to detect Clostridium chauvoei in tissue sections from sheep inoculated intramuscularly with a pure culture of this microorganism . Samples of various tissues were taken for bacteriology, histopathology and immunohistochemistry . A primary antiserum against C . chauvoei for use in the PAP technique was produced in rabbits . Formalin-fixed, paraffin-embedded sections of muscle samples were positively and specifically stained by the PAP technique . The results were consistent with those obtained by bacteriology, but the PAP test was simpler, quicker and less expensive than the bacteriological procedures . The use of the PAP technique would be appropriate for detecting clostridial infections without the constraints of conventional identification methods, especially where laboratory conditions for anaerobic procedures are not readily available.

Microbiol Immunol, 1995, 39(9), 677 - 86
Sequence analysis of flanking regions of the pfoA gene of Clostridium perfringens: beta-galactosidase gene (pbg) is located in the 3'-flanking region; Shimizu T et al.; The 3'-flanking region of the perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was analyzed by chromosome walking . A total of 5,363 bp of the downstream region of the pfoA gene was sequenced and four open reading frames were found . ORF54 and ORF80 were found to be homologous to genes coding for membrane-bound transporter proteins of other bacteria and the beta-galactosidase gene (bgaB) of Bacillus stearothermophilus, respectively . ORF80 was named the pbg gene . Clones which showed beta-galactosidase activities were selected from a lambda FIXII genomic library of C . perfringens by blue plaque screening using X-Gal as a substrate . Four clones whose plaques showed blue appearances were obtained . Two of the four clones hybridized with the pbg probe but the others did not, indicating that there are two distinct beta-galactosidase genes in C . perfringens . The pbg gene was subcloned into pBR322 and was successfully expressed in Escherichia coli, suggesting that the pbg gene codes for a beta-galactosidase of C . perfringens.

Microbiol Immunol, 1995, 39(10), 767 - 74
Immunological characterization of the neurotoxin produced by Clostridium botulinum type A associated with infant botulism in Japan; Kozaki S et al.; The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains . The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin . Each neurotoxin showed its own manner of binding to brain synaptosomes . These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain . In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino-terminal half (H-1 fragment) of the heavy chain . This may support the hypothesis that the H-1 fragment functions in the transport of the light chain in the target cell.

Ann Fr Anesth Reanim, 1995, 14(4), 362 - 5
{Severe forms of pseudomembranous colitis caused by Clostridium difficile}; Mohammedi I et al.; Clostridium difficile causes a broad spectrum of enteric diseases in humans, ranging from mild antibiotic-associated diarrhoea to more severe pseudomembranous colitis . The authors report four cases of life-threatening pseudomembranous colitis with haemodynamic changes . Infection due to Clostridium difficile should be kept in mind whenever a patient undergoing antibiotic therapy develops a symptomatology of an acute abdomen.

Ann Fr Anesth Reanim, 1995, 14(4), 359 - 61
{Clostridium perfringens septicemia associated with foodborne toxic infection and abortion}; Lantelme P et al.; A 32-year-old pregnant woman with poor life and hygiene conditions presented with premature labour, fever and diarrhoea . After admission she gave birth to a stillborn child . The examination revealed a septicaemia with massive haemolysis and renal failure . Six blood cultures obtained on admission yielded Clostridium perfringens . The outcome was favourable after an adapted antibiomicrobial therapy . This case illustrates the potential severity of Clostridium perfringens foodborne toxi-infection which can lead to abortion and septicaemia with massive haemolysis.

Microbiol Immunol, 1995, 39(7), 457 - 65
Characterization of nontoxic-nonhemagglutinin component of the two types of progenitor toxin (M and L) produced by Clostridium botulinum type D CB-16; Ohyama T et al.; A 9.8-kbp DNA fragment which contained a neurotoxin gene and its upstream region was cloned from Clostridium botulinum type D strain CB-16 . Nucleotide sequencing of the fragment revealed that genes encoding for hemagglutinin (HA) subcomponents and one for a nontoxic-nonhemagglutinin (NTNH) component were located upstream of the neurotoxin gene . This strain produced two toxins of different molecular size (approximately 300 kDa and 500 kDa) which were designated as progenitor toxins (M and L toxins) . The molecular size of the NTNH component of L toxin was approximately 130 kDa on SDS-PAGE and its N-terminal amino acid sequence was M-D-I-N-D-D-L-N-I-N-S-P-V-D-N-K-N-V-V-I which agreed with that deduced from the nucleotide sequence . In contrast, the M toxin had a 115-kDa NTNH component whose N-terminal sequence was S-T-I-P-F-P-F-G-G-Y-R-E-T-N-Y-I-E, corresponding to the sequence from Ser141 of the deduced sequence . A 15-kDa fragment, which was found to be associated with an M toxin preparation, possessed the same N-terminal amino acid sequence as that of the 130-kDa NTNH component . Furthermore, five major fragments generated by limited proteolysis with V8 protease were shown to have N-terminal amino acid sequences identical to those deduced from the nucleotide sequence of 130-kDa NTNH . These results indicate that the 130-kDa NTNH of the L toxin is cleaved at a unique site, between Thr and Ser, leading to the 115-kDa NTNH of the M toxin.

Annu Rev Microbiol, 1995, 49, 523 - 55
Biodegradation of nitroaromatic compounds; Spain JC; Nitroaromatic compounds are released into the biosphere almost exclusively from anthropogenic sources . Some compounds are produced by incomplete combustion of fossil fuels; others are used as synthetic intermediates, dyes, pesticides, and explosives . Recent research revealed a number of microbial systems capable of transforming or biodegrading nitroaromatic compounds . Anaerobic bacteria can reduce the nitro group via nitroso and hydroxylamino intermediates to the corresponding amines . Isolates of Desulfovibrio spp . can use nitroaromatic compounds as their source of nitrogen . They can also reduce 2,4,6-trinitrotoluene to 2,4,6-triaminotoluene . Several strains of Clostridium can catalyze a similar reduction and also seem to be able to degrade the molecule to small aliphatic acids . Anaerobic systems have been demonstrated to destroy munitions and pesticides in soil . Fungi can extensively degrade or mineralize a variety of nitroaromatic compounds . For example, Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene and 2,4,6-trinitrotoluene and shows promise as the basis for bioremediation strategies . The anaerobic bacteria and the fungi mentioned above mostly transform nitroaromatic compounds via fortuitous reactions . In contrast, a number of nitroaromatic compounds can serve as growth substrates for aerobic bacteria . Removal or productive metabolism of nitro groups can be accomplished by four different strategies . (a) Some bacteria can reduce the aromatic ring of dinitro and trinitro compounds by the addition of a hydride ion to form a hydride-Meisenheimer complex, which subsequently rearomatizes with the elimination of nitrite . (b) Monooxygenase enzymes can add a single oxygen atom and eliminate the nitro group from nitrophenols . (c) Dioxygenase enzymes can insert two hydroxyl groups into the aromatic ring and precipitate the spontaneous elimination of the nitro group from a variety of nitroaromatic compounds . (d) Reduction of the nitro group to the corresponding hydroxylamine is the initial reaction in the productive metabolism of nitrobenzene, 4-nitrotoluene, and 4-nitrobenzoate . The hydroxylamines undergo enzyme-catalyzed rearrangements to hydroxylated compounds that are substrates for ring-fission reactions . Potential applications of the above reactions include not only the biodegradation of environmental contaminants, but also biocatalysis and synthesis of valuable organic molecules.

Biochimie, 1995, 77(5), 326 - 32
Studies on the active-site structure of C3-like exoenzymes: involvement of glutamic acid in catalysis of ADP-ribosylation; Aktories K et al.; Various C3-like ADP-ribosyltransferases like Clostridium botulinum exoenzyme C3, C limosum transferase, B cereus transferase and a transferase from Staphylococcus aureus (EDIN) selectively modify the low-molecular mass GTP-binding proteins RhoA,B,C . UV-irradiation of C limosum transferase in the presence of {carbonyl-14C}NAD resulted in radiolabeling of Glu-174 . Concomitantly, ADP-ribosyltransferase and NAD glycohydrolase activities were inhibited . Site-directed mutagenesis of Glu-174 (E174D, E174Q) which resulted in more than 1000-fold reduction of enzyme activity, suggests that the glutamic acid residue is essentially involved in the catalytic action of C3-like transferases . These findings support the view that all bacterial ADP-ribosyltransferases share a similar active-site structure.

Microbiology, 1995 Jan, 141 ( Pt 1), 171 - 80
Characterization and expression of the hydrogenase-encoding gene from Clostridium acetobutylicum P262; Santangelo JD et al.; The hydrogenase enzyme of Clostridium acetobutylicum plays a pivotal role in controlling electron flow, and hence carbon flow, during the complex biphasic fermentation of carbohydrates to the neutral solvents acetone and butanol . We report here the cloning and molecular characterization of the hydrogenase-encoding gene (hydA) from C . acetobutylicum P262 . This gene was isolated by colony hybridization, using the Clostridium pasteurianum hydrogenase-1 gene as a probe . The DNA sequence encoding the hydA gene from C . acetobutylicum was determined, and revealed an ORF (1722 bp) encoding a 574 amino-acid protein . This C . acetobutylicum hydrogenase protein product has 82% similarity and 67% identity with the C . pasteurianum hydrogenase-1 protein . Northern blot analysis of RNA isolated from C . acetobutylicum indicates that the C . acetobutylicum hydrogenase protein product is translated from a monocistronic operon . RNA was isolated from the different morphological and physiological stages of a batch C . acetobutylicum fermentation, and further Northern blot analyses revealed no differences in the expression of the gene during acidogenesis as opposed to solventogenesis . Primer extension experiments confirmed these results and identified the 5' start of the mRNA transcript . These results correlated well with the physiological need for this organism to dispose of excess reducing equivalents.

Chirurg, 1995 Jan, 66(1), 66 - 8
{Gas gangrene after cholecystectomy}; Krause-Bergmann A et al.; We report on a case of an infection by clostridium perfringens (gas gangrene) following cholecystectomy and ERCP . Reviewing the literature, mode and risk of infection and principles of therapy are discussed.

Appl Environ Microbiol, 1995 Jan, 61(1), 389 - 92
PCR and gene probe identification of botulinum neurotoxin A-, B-, E-, F-, and G-producing Clostridium spp . and evaluation in food samples; Fach P et al.; A degenerate primer pair was selected to amplify specifically a 260-bp DNA fragment from Clostridium botulinum types A, B, E, F, and G, and five individual probes allowed identification of each toxinotype by hybridization of the PCR products . The 72 strains of different Clostridium species tested and 11 other bacterial species commonly found in food samples gave an amplification product . This assay was able to detect 1 C . botulinum type A or B and 10 C . botulinum type E strains per reaction . With 184 artificially contaminated food samples, after an 18-h enrichment step, the sensitivity was 10 bacteria per g of sample and the correlation with the mouse bioassay reached 95.6%.

Am J Clin Pathol, 1995 Jan, 103(1), 52 - 6
Laboratory detection of Clostridium difficile . A comparison of media and incubation systems; Mundy LS et al.; Parallel testing for culture recovery of Clostridium difficile was performed using three selective media in each of four anaerobic incubation environmental systems . Testing was completed on 67 stool samples from 60 hospitalized patients in whom C difficile-associated diarrhea was suspected . Three different media were evaluated: CCFA (modified cycloserine-cefoxitin-fructose agar), CCFA-PRAS (CCFA, prereduced-anaerobically-sterilized) and CMBA (modified cycloserine-mannitol-blood agar) . The incubation systems compared were an anaerobic chamber (Model 800 Anaerobic Environmental System, Anaerobe Systems, San Jose, CA), the anaerobic jar (BBL, Cockeysville, MD), the anaerobic Bio-Bag (BBL), and the anaerobic pouch (BBL) . C difficile was found in 16 samples collected from 15 patients . Comparing recovery on the various types of media in all four anaerobic atmospheres, C difficile was recovered on all (64) CCFA plates, 56 of 64 CCFA-PRAS plates, and 43 of 64 CMBA plates (P < .03 comparing CCFA and CMBA) . Of the 48 plates in each incubation system that were inoculated with specimens positive for C difficile, the organism was recovered from 43 plates in the anaerobe chamber, 41 incubated in an anaerobe jar, 40 in the Bio-Bag, and 39 incubated in the GasPak pouch, all providing similar recovery of C difficile (P = .08) . The CCFA-PRAS and CMBA media demonstrated less inhibition of normal stool flora compared to the CCFA . Overall CCFA that was anaerobically reduced at least 4 hours before use, and contained the original concentration of antimicrobial agents described by George and colleagues, was superior to CMBA for recovery of C . difficile.

J Bacteriol, 1995 Jan, 177(2), 439 - 48
Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome; Wilkinson SR et al.; A combined physical and genetic map of the single, circular, 6.7-Mbp chromosome of the NCIMB 8052 strain of Clostridium beijerinckii (formerly Clostridium acetobutylicum) has been constructed by using a combination of cloned DNA fragments as hybridization probes and a bank of strains harboring insertions of the conjugative transposon Tn1545 . The positions of 81 restriction endonuclease cleavage sites and 32 genes have been determined . Eight genes concerned with solventogenic fermentation are found at three different locations . The chromosome contains at least 13 rrn operons, 11 of which have been located on the map . Their transcriptional orientation diverges from the presumed location of the replication origin.

Infect Immun, 1995 Jan, 63(1), 340 - 4
The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin; Ballard J et al.; The gene for Clostridium septicum alpha-toxin was cloned and expressed in Escherichia coli from C . septicum BX96 . The toxin was determined to be 443 amino acids in length, with a 31-residue signal peptide that was removed from the toxin during secretion . No extended hydrophobic regions were observed in the mature toxin sequence . Expression of alpha-toxin in E . coli BL21 resulted in the production of ATpro, which was identical to native toxin from C . septicum with respect to activity and activation . The proteolytic activation site for alpha-toxin was determined to be on the carboxy-terminal side of arginine 398, which lies within the sequence KKRRGKR-398SVD . Previous work showing similarities in activation and mechanism between alpha-toxin and Aeromonas hydrophila aerolysin was extended to the primary structures of both toxins . The DNA-derived primary sequence of alpha-toxin exhibited 27% identity and 72% similarity over a 387-residue region with the primary structure of the A . hydrophila aerolysin toxin, a level of similarity heretofore unobserved between toxins produced by a gram-positive organism and a gram-negative organism.

J Clin Oncol, 1995 Jan, 13(1), 239 - 50
Ciprofloxacin versus trimethoprim/sulfamethoxazole for prophylaxis of bacterial infections in bone marrow transplant recipients: a randomized, controlled trial; Lew MA et al.; PURPOSE: To compare the efficacy and safety of ciprofloxacin (CIP) and trimethoprim/sulfamethoxazole (TMS) for the prevention of bacterial infections in patients who received bone marrow transplantation (BMT) for the treatment of solid and hematopoietic neoplasms . PATIENTS AND METHODS: Adult inpatients about to undergo BMT for lymphoma, leukemia, or solid tumors were enrolled onto a prospective, randomized, double-blinded, controlled trial that compared CIP (750 mg orally twice per day) with TMS (160 mg trimethoprim and 800 mg sulfamethoxazole orally twice per day) . Subjects were stratified before randomization according to tumor and BMT type . Prophylaxis was begun within 96 hours of initiation of the BMT preparative regimen and continued until the onset of fever, signs or symptoms of infection, serious adverse effects, or recovery of the absolute granulocyte count (AGC) to > or = to 400/microL . RESULTS: Seventy-five CIP recipients and 71 TMS recipients were assessable for efficacy . No difference was noted between the two groups in occurrence of fever during neutropenia, time to onset of first fever, or overall infection rates . Ten bacteremias occurred in CIP recipients versus six in TMS recipients (P = .43) . Ten episodes of Clostridium difficile enterocolitis occurred in TMS recipients versus no episodes in CIP recipients (P = .001) . Four infections caused by gram-negative bacilli, including one bacteremia, occurred in TMS recipients versus none in CIP recipients (P = .06) . No differences were noted in the incidence of rash or organ toxicity . TMS recipients had longer durations of granulocytopenia at AGC levels < or = to 500/microL and < or = to 100/microL than did CIP recipients (P = .08 for both comparisons) . Mean peak and trough serum levels of CIP decreased significantly between weeks 1 and 2 of prophylaxis . CONCLUSION: CIP and TMS were equally safe and effective in the prevention of bacterial infections in BMT patients when the overall infection rate was used as the principal end point . TMS prophylaxis was associated with a higher incidence of C difficile enterocolitis and infections caused by gram-negative bacilli, as well as a trend toward prolongation of granulocytopenia.

J Clin Oncol, 1995 Jan, 13(1), 165 - 76
Monotherapy for fever and neutropenia in cancer patients: a randomized comparison of ceftazidime versus imipenem; Freifeld AG et al.; PURPOSE: To compare the efficacy of ceftazidime and imipenem monotherapy for fever and neutropenia, and to determine whether fewer antimicrobial modifications (additions or changes) are required by the broader-spectrum agent, imipenem . PATIENTS AND METHODS: Adult and pediatric patients undergoing chemotherapy for solid tumors, leukemias, or lymphomas were randomized to receive open-label ceftazidime or imipenem on presentation with fever and neutropenia . Success with or without modifications of the initial antibiotic was defined as survival through neutropenia; failure was death due to infection . Comparisons were based on numbers of modifications made to each monotherapy during the course of neutropenia, in patients stratified as having unexplained fever or a documented infection . RESULTS: Among 204 ceftazidime and 195 imipenem recipients, the overall success rate with or without modification was more than 98%, regardless of initial antibiotic regimen . Modifications occurred in half of all episodes, primarily in patients with documented infections on either monotherapy . Antianaerobic agents were more frequently added to ceftazidime (P < .001), but addition of other antibiotics, including vancomycin and aminoglycosides, was similar between the two monotherapy groups . Imipenem therapy was associated with significantly greater toxicity, manifested by Clostridium difficile-associated diarrhea and by nausea and vomiting, which required discontinuation of imipenem in 10% of recipients . CONCLUSION: Ceftazidime and imipenem are both effective in the management of fever and chemotherapy-related neutropenia, provided that modifications are made in response to clinical and microbiologic data that emerge during the course of neutropenia . Imipenem, despite its broader antimicrobial spectrum, does not significantly decrease the overall need for antibiotic modifications and is more often complicated by gastrointestinal toxicity.

J Protein Chem, 1995 Jan, 14(1), 7 - 18
Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified from Clostridium botulinum; Singh BR et al.; Type E botulinum neurotoxin is produced by Clostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adverse pH, temperature, and proteolytic conditions . The neurotoxin binding protein has been purified as a 118-kDa protein . Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% alpha-helix, 50% beta-sheets, 28% random coils, and 3% beta-turns . This compared to 22% alpha-helix, 44% beta-sheets, 34% random coils, and no beta-turns of the type E botulinum neurotoxin . The complex of the two proteins revealed 25% alpha-helix, 45% beta-sheets, 27% random coils, and 3% beta-turns, suggesting a significant alteration at least in the alpha-helical folding of the two proteins upon their interaction . Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins . Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin . The interaction is favored at low pH as indicated by an initial binding rate of 8.4 min-1 at pH 5.7 compared to 4.0 min-1 at pH 7.5 as determined using a fiber optic-based biosensor . The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.

Equine Vet J, 1995 Jan, 27(1), 8 - 12
Seroanalysis of Tyzzer's disease in horses: implications that multiple strains can infect Equidae; Hook RR et al.; A monoclonal antibody based competitive inhibition assay was used to detect antibodies in horse sera to purified flagellar antigens from distinct Clostridium piliforme isolates . Sequential absorption of hyperimmune rat serum to C . piliforme isolate E (horse-origin isolate), a positive C . piliforme-immune horse serum, and other suspected immune horse sera with unrelated bacteria or C . piliforme isolates E or isolate R1 (rat-origin isolate) alone demonstrated the specificity of this assay for C . piliforme . This specificity was associated with the inhibition of monoclonal antibody binding to C . piliforme flagella, rather than to C . piliforme somatic antigens, by horse immunoglobulins partially purified from serum . Thirty seven of 162 horse sera possessed large amounts of antibody to the flagella of C . piliforme isolate E and 23 of the 162 had large amounts of antibody to the flagella of C . piliforme isolate R1; 9 of the sera possessed large amounts of antibody to both flagellar antigens . Absorption of these sera with isolate E or R1 demonstrated that antibody reactivity to the 2 C . piliforme isolates was isolate-specific and not due to antibody cross-reactive with both isolates . These results suggest that infection of horses with C . piliforme may be relatively common; and that they are susceptible to at least 2 distinct strains.

Biosci Biotechnol Biochem, 1995 Jan, 59(1), 47 - 50
Process of thermal denaturation of xylanase (XynB) from Clostridium stercorarium F-9; Fukumura M et al.; The thermal denaturation process of Clostridium stercorarium F-9 xylanase (XynB) was studied by monitoring remaining activity and recovered activity of the enzyme . At pH 5.5, aggregation occurred rapidly after the thermal denaturation initiated . The aggregated protein could be dissolved in 8 M urea solution, and the enzyme activity was recovered by diluting the urea . The extent of the recovered activity was gradually decreased with two phases as the reaction time of the thermal denaturation became longer . These results suggested the thermal denaturation process to be as follows: {formula: see text} where N is the native state of the enzyme; D1 is the denatured state of the enzyme that is formed rapidly after the reaction started and can be renatured by the urea treatment, and D2 and D3 are the denatured states of the enzyme that cannot be renatured even by the urea treatment . The rate constants were k1 > 9.2, k2 = 0.33, and k2 = 0.57, and k3 = 0.13 (in min-1 unit).

Biosci Biotechnol Biochem, 1995 Jan, 59(1), 40 - 6
Nucleotide sequence of the Clostridium stercorarium xynB gene encoding an extremely thermostable xylanase, and characterization of the translated product; Fukumura M et al.; The nucleotides of the xynB gene of Clostridium stercorarium F-9 were sequenced . The structural gene consists of an open reading frame of 1161 bp encoding a xylanase (XynB) in family F of 387 amino acids with a molecular weight of 44,377 . The molecular weight of the enzyme purified from a recombinant Escherichia coli was around 41,000, smaller than the predicted value, on SDS-polyacrylamide gel electrophoresis due to the lack of 32 amino acids at the N-terminus . Intact XynB with a molecular weight of around 43,000 was immunologically detected in the total cell proteins of a recombinant E . coli and C . stercorarium F-9 . The purified XynB was active toward xylan, carboxymethylcellulose, p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-beta-D-cellobioside . The pH optimum was 7.0 and it was quite stable over the pH range of 5 to 12 at 4 degrees C . This enzyme was optimally active at 80 degrees C and retained about 50% of the original activity even after incubation at 100 degrees C for 10 min.

Pediatr Radiol, 1995, 25(1), 24 - 6; discussion 27
Infant botulism: a rare cause of colonic ileus; Kothare SV et al.; We have recently seen two patients with infant botulism, one of whom had radiologic evidence of autonomic and neuromuscular dysfunction . Both infants had been fed small amounts of honey, which is often contaminated with Clostridium botulinum spores, during the Jewish New Year celebration.

PDA J Pharm Sci Technol, 1995 Jan-Feb, 49(1), 32 - 41
The effect of formulation of parenteral solutions on microbial growth--measurement of D- and z-values; Berger TJ et al.; In order to assess the sterilization conditions for parenteral solutions, the microbial inactivation kinetics of steam resistant spores of Clostridium sporogenes were evaluated . The D-value or death rate kinetics as well as z-values were obtained for several formulation categories of solutions employing a miniature steam retort . Amino acid and heparin solutions provided appreciable microbial resistance as demonstrated by resulting high D- and low z-values . The addition of electrolytes to carbohydrate solutions resulted in increased microbial resistance compared to carbohydrate solutions without electrolytes . A categorization of solutions and their potential impact on microbial thermal resistance will be presented.

Mol Microbiol, 1995 Jan, 15(2), 191 - 202
Virulence studies on chromosomal alpha-toxin and theta-toxin mutants constructed by allelic exchange provide genetic evidence for the essential role of alpha-toxin in Clostridium perfringens-mediated gas gangrene; Awad MM et al.; The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes . The precise role of each of these toxins in tissue invasion and necrosis has not been determined . To enable genetic approaches to be used to study C . perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C . perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant . The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene . Allelic exchange was used to isolate mutations in the chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as theta-toxin or perfringolysin O, and in the chromosomal plc gene, which encodes the alpha-toxin or phospholipase C . The resultant mutants failed to produce detectable theta-toxin or alpha-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes . The resultant strains were virulence tested in a mouse myonecrosis model . The results showed that the plc mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of alpha-toxin in gas gangrene or clostridial myonecrosis.

Jpn J Pharmacol, 1995 Jan, 67(1), 1 - 7
Possible involvement of a small G-protein sensitive to exoenzyme C3 of Clostridium botulinum in the regulation of myofilament Ca2+ sensitivity in beta-escin skinned smooth muscle of guinea pig ileum; Itagaki M et al.; The effects of exoenzyme C3 of Clostridium botulinum on Ca(2+)- and drug-induced tension developments were investigated in beta-escin skinned smooth muscle of guinea pig ileum to test the involvement of a small G-protein in the regulation of myofilament Ca2+ sensitivity . C3 is known to ADP-ribosylate the rho p21 family of small G-proteins . Treatment with C3 (0.35 microgram/ml, for 30 min) shifted the pCa-tension curve rightward along the Ca2+ concentration axis, indicating a decrease in Ca2+ sensitivity of the contractile elements . The inhibitory effect of C3 was not preserved after treatment with GDP beta S (1 mM), an antagonist of GTP for the binding to G-proteins . Stimulation of muscarinic receptors with carbachol (CCh, 100 microM) shifted the pCa-tension curve leftward, indicating Ca2+ sensitization of tension development . The Ca(2+)-sensitizing effect of CCh was not observed after C3 treatment . When GTP gamma S (10 microM), an activator of G-proteins, was applied at a plateau of tension development produced by a moderate concentration of Ca2+, further increase in tension was elicited and the effect of GTP gamma S was inhibited by C3 treatment . The results suggest the possible involvement of a rho p21-like small G-protein in the regulation of Ca2+ sensitivity of smooth muscle myofilaments.

J Med Microbiol, 1995 Jan, 42(1), 10 - 9
Immunoglobulin and non-immunoglobulin components of human milk inhibit Clostridium difficile toxin A-receptor binding; Rolfe RD et al.; Clostridium difficile is isolated from the intestinal tracts of > 50% of healthy infants . The mechanism by which intestinal colonisation of infants by toxigenic C . difficile is generally asymptomatic is unknown but may reflect the presence in human milk of neutralising activity against C . difficile toxin A . On this basis, the ability of human milk to inhibit the binding of toxin A to a purified hamster brush border membrane receptor was determined . Ten milk samples from healthy volunteers in various stages of lactation inhibited the binding of toxin A to the receptor by an average of 90% . Heating and dialysis did not significantly alter the inhibitory activity of any of the milk samples . Human milk protected adult hamsters against a lethal challenge with toxin A but had no effect on the cytotoxic activity of the toxin . SDS-PAGE and ligand blot analyses showed that there were at least four distinct factors in human milk that specifically bound toxin A . Thiophilic adsorption chromatography was used to separate immunoglobulin from non-immunoglobulin components of human milk . IgA was the only immunoglobulin detected in human milk and > 90% of this immunoglobulin was recovered after purification by thiophilic adsorption . Both the unbound non-immunoglobulin and bound immunoglobulin fractions of human milk inhibited the binding of toxin A to the purified receptor . These results suggest that human milk may be important in protecting infants against C . difficile-associated intestinal disease.

J Hosp Infect, 1995 Jan, 29(1), 69 - 73
A hospital outbreak of Clostridium perfringens food poisoning--implications for food hygiene review in hospitals; Regan CM et al.; An outbreak of Clostridium perfringens (C . perfringens) food poisoning affected 17 of 44 (38.6%) patients interviewed on two hospital wards . A case-control study showed a statistically significant association between the consumption of roast pork and illness (P < 0.01) . C . perfringens type A, untypable serotype, was isolated from samples of pre-cooked vacuum sealed pork supplied by a local meat producer . Faults were noted in the food production process at the factory . Cuts of meat were too large and equipment to ensure rapid cooling of cooked meat was not installed . Cost improvements taken by hospitals, such as the use of commercially cooked meat, may not be consistent with the highest standards of food safety . Amendments to the District Catering Policy were implemented to prevent further outbreaks.

J Dairy Sci, 1995 Jan, 78(1), 55 - 61
Iron removal from milk and other nutrient media with a chelating resin; Feng M et al.; A water-insoluble iron(III)-chelating resin was used to study iron removal from milk and other nutrient media . Seventy to 85% of the iron could be removed from wine and beer with the resin, which was a crosslinked copolymer of 1-(beta-acrylamidoethyl)-3-hydroxy-2-methyl-4(1H)- pyridinone and N,N-dimethylacrylamide . Iron removal from milk was dependent on the pH of milk and on the concentration of soluble chelators added . Under the same conditions as used for the removal of iron from wine and beer, only 11 to 19% of the iron could be removed from milk . However, in combination with water-soluble chelators, the resin removed 60 to 75% of the iron from the milk . Preliminary results showed that the growth of spores of Clostridium tyrobutyricum in the treated milk was reduced . Moreover, addition of the resin and sodium bicarbonate to the milk completely inhibited the growth of the spores.

J Cell Sci, 1995 Jan, 108 ( Pt 1), 369 - 77
Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells; Soole KL et al.; To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated . The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C . In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane . Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral . EGE' delivered to the basolateral cell surface was transcytosed to the apical surface . These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells . However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

Can J Microbiol, 1995 Jan, 41(1), 88 - 91
Diagnosis of Clostridium difficile associated diarrhea: comparison of three rapid methods employing different markers for detection; Riederer KM et al.; Latex agglutination and the enzyme immunoassays Cytoclone (EIA-C) and VIDAS (EIA-V) were compared with a cytotoxicity assay for the diagnosis of Clostridium difficile associated diarrhea . Among patients with discrepant results, the cytotoxicity assay and clinical assessment were used to evaluate the performance of the latex agglutination and EIA tests . Clostridium difficile associated diarrhea was documented in 30/149 samples (20.1%) from 130 patients . All test results matched in 113 instances . Latex agglutination, EIA-C, and EIA-V yielded false positive results in 10, 4, and 7 samples and false negative results in 8, 9, and 14 samples, respectively . Latex agglutination demonstrated 87.8% efficiency compared with 91.3% for EIA-C and 85.7% for EIA-V and 3 min hands-on time compared with 4.5 min for EIA-V and 10 min for EIA-C . On the basis of these findings and given the fact that all rapid tests have their shortcomings, we believe that latex agglutination is the most practical method.

Clin Infect Dis, 1995 Jan, 20(1), 160 - 2
Elevated serum antibody response to toxin A following splenic abscess due to Clostridium difficile; Stieglbauer KT et al.; Splenic abscess and segmental small-bowel infarction were documented in a patient from whose splenic culture Clostridium difficile was isolated . A week and a half after splenectomy and partial bowel resection, diarrhea developed and stool cultures yielded an isolate of C . difficile that was identical to the abscess isolate when subjected to restriction endonuclease analysis . The level of IgG antibody to toxin A was markedly higher in serum from this patient than in sera from patients with C . difficile diarrhea alone . This case illustrates a rare but serious extraintestinal manifestation of infection with C . difficile and suggests a correlation between serum levels of IgG antibody to toxin A and systemic exposure to C . difficile, a typically noninvasive enteric pathogen.

J Clin Microbiol, 1995 Jan, 33(1), 184 - 7
PCR amplification of rRNA intergenic spacer regions as a method for epidemiologic typing of Clostridium difficile; Cartwright CP et al.; From January to March 1993, a suspected outbreak of antibiotic-associated diarrhea occurred on a pediatric oncology ward of the Clinical Center Hospital at the National Institutes of Health . Isolates of Clostridium difficile obtained from six patients implicated in this outbreak were typed by both PCR amplification of rRNA intergenic spacer regions (PCR ribotyping) and restriction endonuclease analysis of genomic DNA . Comparable results were obtained with both methods; five of the six patients were infected with the same strain of C . difficile . Subsequent analysis of 102 C . difficile isolates obtained from symptomatic patients throughout the Clinical Center revealed the existence of 41 distinct and reproducible PCR ribotypes . These data suggest that PCR ribotyping provides a discriminatory, reproducible, and simple alternative to conventional molecular approaches for typing strains of C . difficile.

Acta Microbiol Pol, 1995, 44(1), 47 - 53
Toxigenicity of Clostridium difficile strains isolated in the surgical ward; Martirosian G et al.; We report the use of the polymerase chain reaction (PCR) for the detection of toxigenic Clostridium difficile strains . PCR was performed as described (McMillin et al., 1991) . The results obtained by PCR were compared with those of cytotoxicity assays and of the latex agglutination test . Twenty seven strains cultured from stool specimens of patients and five strains isolated from hospital environment in a surgical ward were investigated . Twenty five of 32 strains produced cytotoxin as shown on the McCoy cell-line (CA) . Positive reaction in the latex test was observed in 20 strains supernatants . In 24 strains PCR revealed toxin B gene fragments (1.0 kb) . In the 5 strains PCR did not detect toxin B fragments, and these strains were not cytotoxic on McCoy cells . We observed 2 strains toxin B gene fragment negative, which had a low cytotoxic activity on McCoy cells, and were toxin A gene fragment positive.

Microbiol Immunol, 1995, 39(4), 249 - 53
Lethal and dermonecrotic activities of Clostridium perfringens lota toxin: biological activities induced by cooperation of two nonlinked components; Sakurai J et al.; The effect of separate injections of two components of Clostridium perfringens iota toxin, designated Ia and Ib components, on the biological activities of the toxin was investigated . The intravenous injection of one component within 120 min after the injection of the other component killed mice . The activity of iota toxin was abolished by anti-Ia or anti-Ib antiserum . On the other hand, when Ib component was intravenously administered to mice given anti-Ia antiserum within 120 min after the intravenous injection of Ia component, the lethal activity was completely neutralized, but when Ia component was injected into mice that were given anti-Ib antiserum over 5 min after the injection of Ib component, the activity was not neutralized . The separate injections of Ia and Ib components in skin of guinea pigs indicated dermonecrosis at the injection site of Ib components, but not at the site of Ia components . Furthermore, when one component was intradermally injected in guinea pigs and then the other intraperitoneally, the dermonecrotic activity of the toxin was observed at the intradermal injection site of Ib component, but not at that of Ia component . From the data, it appears that the lethal and dermonecrotic activities of iota toxin are initiated by the binding of Ib component to specific sites on tissues.

Microbiol Immunol, 1995, 39(4), 231 - 5
Differentiation of Clostridium difficile, Clostridium bifermentans, Clostridium sordellii, and Clostridium perfringens from diarrheal stool by API ZYM and API LRA oxidase test; Fontana C et al.; A simple, rapid and reliable outline for identification of clostridia isolates from human infections was developed . It consists of a combination of API ZYM and API LRA Oxidase tests . The enzymatic activities were performed with strains sub-cultured onto carbohydrate-free medium (Columbia blood agar) . Fifty-five strains of Clostridium difficile, C . bifermentans, C . sordellii, and C . perfringens from clinical specimens and eight reference standard strains representing different species of the same genus were analyzed . The accuracy of the new method was evaluated by comparison with the results obtained by DNA/DNA analysis.

Acta Clin Belg, 1995, 50(2), 114 - 6
Treatment of Clostridium difficile colitis . Summary of a round table held in Brussels on March, 3rd, 1994; Delmee M et al.; Due to the growing incidence and the severity of infections due to vancomycin resistant enterococci, it is now recommended to treat mild to moderate cases of Clostridium difficile-associated diarrhoea with metronidazole while maintaining the use of oral vancomycin in serious or life-threatening colitis . Clostridium difficile is a common cause of diarrhoea after antibiotic therapy and induces a spectrum of diseases ranging from mild diarrhoea to pseudomembranous colitis (PMC) . It is considered to be the first cause of diarrhoea occurring among hospitalized patients (1).

DNA Res, 1995, 2(2), 61 - 9
Cloning and sequencing of a 36-kb region of the Bacillus subtilis genome between the gnt and iol operons; Yoshida K et al.; Within the framework of an international project for the sequencing of the entire Bacillus subtilis genome, a 36-kb chromosome segment, which covers the region between the gnt and iol operons, has been cloned and sequenced . This region (36447 bp) contains 33 complete open reading frames (ORFs; genes) including the four gnt genes and one partial gene . A homology search for the products of the 33 complete ORFs revealed significant homology to known proteins in 16 of them such as tetracycline resistance protein (Clostridium perfringens), asparagine synthetase (Arabidopsis thaliana), aldehyde dehydrogenase (Pseudomonas oleovorans), 2,5-dichloro-2,5-cyclohexadiene-1, 4-diol dehydrogenase (P . paucimobilis), heat shock protein HtpG (Escherichia coli), galactose-proton symporter (E . coli), auxin-induced protein (common tobacco), glucitol operon repressor (E . coli) and methylmalonate-semialdehyde dehydrogenase (P . aeruginosa) . Unlike the regions we sequenced so far, this region contained two short sequence multiplications: one was a tandem sequence duplication (409 and 410 bp), and the other a triplication consisting of two highly conserved 118-bp tandem sequences preceded by a less conserved similar sequence (129 bp) . The reasons for the presence of these sequence multiplications in the gnt to iol region were deduced.

Drugs Exp Clin Res, 1995, 21(2), 59 - 63
Prophylactic effect of ornidazole on experimental tetanus in mice; Erenmemisoglu A et al.; Tetanus is still responsible for many deaths, especially in the developing parts of the world . In this study, the prophylactic effect of ornidazole on experimental tetanus in mice was investigated . The initial minimum lethal dose (MLD) of Clostridium tetani spores was determined in mice and 100 MLD was applied to mice in experiments . Ornidazole was then administered at the dose of 20 mg, 40 mg and 80 mg/kg . In addition, penicillin and horse tetanus serum were also administered to other groups . According to the present results, ornidazole decreased the number of deaths in mice significantly in a dose and drug administration time dependent manner . These results suggest that, together with essential wound care and active immunization, ornidazole (or another nitroimidazole) may be a useful and supportive therapeutic agent in tetanus prophylaxis.

Cancer Invest, 1995, 13(5), 492 - 4
Clostridium septicum bacteremia in a patient with large granular lymphocyte leukemia; Litam PP et al.; This is the first case report of Clostridium septicum septicemia in a patient with large granular lymphocyte leukemia . C . septicum infection is highly associated with malignancy and causes a rapidly fatal enterocolitis among patients who are profoundly neutropenic . The need for early recognition and combination of early antibiotic therapy and necessary surgical intervention may help to alter the fulminating nature of C . septicum infection.

Biol Cell, 1995, 83(2-3), 141 - 7
Relationship between the acid phosphatases of the Kurloff body and the major 30-35 kDa glycoproteins of the Kurloff cell; Oulhaj N et al.; This study deals with the acid phosphatase (AcPase) of the Kurloff body (KB), a large (10 microns diameter) periodic acid-Schiff-positive lysosomal inclusion body present in Kurloff cells (KC) . KC AcPase were extracted, with similar yields, either with non-ionic detergent solution or after Dounce homogenization in low ionic strength buffer suggesting that they mainly correspond to hydrosoluble AcPase . After DEAE-cellulose chromatography of such crude Dounce-extracts, 97% of KC AcPase activity was recovered in the unbound glycoprotein fraction (peak I)1) . The main protein content consisted of, as testified by SDS-PAGE analysis, major KC glycoproteins of 30-35 kDa . Thus, KC AcPase, and particularly sialoAcPase, may be assumed to correspond to these N-glycosylproteins among which the presence of alpha (2,6) sialoglycoproteins was previously established . Following electrophoresis on a 4-15% gradient native polyacrylamide gel or isoelectric focusing, the two size populations (200 kDa and 500 kDa) and up to 20 isoforms of KC AcPase were respectively detected by zymography in peak I . After Clostridium-derived sialidase digestion of peak I, the highly active bands observed at pH 3.5-5.2 disappeared . These zymographic patterns were similar to those obtained with crude extracts . After Concanavalin A (ConA)-Sepharose chromatography of peak I, the single ConA-bound glucosamine-labelled peak, eluted at 200 methyl-alpha-D-mannopyranose, contained the AcPase activity while the ConA-unbound peak was devoid of any acid phosphatase activity . After SDS-PAGE analysis, the ConA-bound fraction appeared to correspond only to a single broad protein band in the 30-35 kDa zone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Clin Belg, 1995, 50(1), 36 - 9
Gamma globulin administration in relapsing Clostridium difficile-induced pseudomembranous colitis with a defective antibody response to toxin A; Warny M et al.; A 53-year-old woman suffered six episodes of Clostridium difficile-pseudomembranous colitis . The serological follow-up demonstrated the absence of a rise in IgG and IgA to toxin A . Human pooled gamma globulin was administered during the fifth relapse and raised IgG levels to toxin A . Normal stools reappeared a week later . The role of the antibodies to toxin A and gamma globulin in C . difficile colitis are discussed.

Mutat Res, 1995 Jan, 341(3), 217 - 24
Effect of Clostridium perfringens-derived wound healing substance as compared with epidermal growth factor on the growth and morphological transformation of BALB/3T3 A31-1-1 cells; Arima T et al.; Clostridium perfringens-derived wound-healing substance (WHS), having growth-stimulating activity, was examined to determine its effect on the growth and morphological transformation of BALB/3T3 A31-1-1 cells . WHS accelerated the cell growth at the exponential growth phase, shortening the doubling time by 8-18% . The maximum cell density of the treated cultures was slightly higher than that of the control culture, and the cell number decreased in the same way as the control cells did . On the other hand, the cells treated with epidermal growth factor (EGF) or insulin showed growth rates similar to that of the control cells during the exponential growth phase, and after the control cells attained the maximum cell number, the number of the treated cells continued to increase gradually for more than 4 days and then decreased . Under the experimental conditions of the two-stage transformation assay, application of WHS at the tumor-initiation or promotion stage did not accelerate the formation of transformed foci . Although treatment with EGF at the initiation stage induced no enhancement, marked enhancement of morphological transformation was observed in the treatment at the promotion stage . These results indicate that the mode of action between WHS and EGF or insulin is different on the growth-stimulating activity and morphological transformation of BALB/3T3 A31-1-1 cells.

J Mol Biol, 1994 Dec 16, 244(5), 648 - 50
Purification, crystallization and preliminary X-ray diffraction studies of alpha-toxin of Clostridium perfringens; Basak AK et al.; Alpha-toxin of Clostridium perfringens, cloned in Escherichia coli, has been purified and crystallized from ammonium sulphate using the hanging drop vapour diffusion method at 20 degrees C . The crystals diffract to a minimum Bragg spacing of 2.7 A, belong to the space group R32 (with a = b = 153.3 A, c = 95.4 A, alpha = beta = 90 degrees and gamma = 120 degrees) and contain a single polypeptide chain in the crystallographic unit.

FEMS Microbiol Lett, 1994 Dec 15, 124(3), 439 - 47
Gene sequence and analysis of protein domains of EGB, a novel family E endoglucanase from Fibrobacter succinogenes S85; Broussolle V et al.; The endoglucanase gene (endB) of Fibrobacter succinogenes S85 encodes a protein of 555 amino acids (EGB) with a M(r) of 62,500 . EGB shows homology with cellulases belonging to family E . Residues involved in the catalytic activity of CelD from Clostridium thermocellum are also found in EGB . Structure predictions suggest that EGB, like CelD, comprises a large alpha-helical catalytic domain plus a beta-strand domain of unknown function located in the N-terminal part of the protein . Construction of a phylogenetic tree of family E catalytic domains revealed that EGB is closest to a cellodextrinase from Butyrivibrio fibrisolvens.

FEMS Microbiol Lett, 1994 Dec 15, 124(3), 355 - 9
Cyclic AMP in ruminal and other anaerobic bacteria; Cotta MA et al.; An examination of cAMP levels in predominant species of ruminal bacteria and other anaerobic bacteria was conducted . Cellular cAMP concentrations of glucose-grown cultures of Butyrivibrio fibrisolvens 49, Prevotella ruminicola D31d, Selenomonas ruminantium HD4 and D, Megasphaera elsdenii B159, Streptococcus bovis JB1, Bacteroides thetaiotaomicron 5482, and Clostridium acetobutylicum ATCC 824 were determined at various times during growth by a competitive binding radioimmunoassay procedure . The results were compared to those for Escherichia coli NRRL B3704 . The levels of cAMP ranged from undetectable for B . thetaiotaomicron to approximately 15 pmol/mg cell protein for P . ruminicola D31d . Varying the growth substrate in a manner previously shown to elicit regulatory response did not alter the level of cAMP in these organisms . In general, cAMP concentrations present in these organisms were much lower than the 6-25 pmol/mg cell protein observed for E . coli . The levels of cAMP in P . ruminicola were consistently higher than levels in other anaerobes, particularly during the early exponential and stationary phases of growth . Based on these data it seems unlikely that cAMP is involved in regulation of substrate catabolism in the anaerobic bacteria examined except in P . ruminicola where it may have an unknown regulatory function.

FEMS Microbiol Lett, 1994 Dec 15, 124(3), 277 - 84
Transcriptional analysis of the Clostridium cellulovorans endoglucanase gene, engB; Attwood GT et al.; An endoglucanase gene, which was shown to be identical to the previously sequenced engB gene {Attwood et al . (1993) Abstr . Ann . Meet . Am . Soc . Microbiol.}, was isolated from a Clostridium cellulovorans genomic library . Because of the lack of transcriptional information concerning engB we examined its expression in C . cellulovorans and in the heterologous hosts Escherichia coli and C . acetobutylicum following transformation of engB . Northern analysis suggested that both E . coli and C . acetobutylicum produced several transcripts of various sizes . C . cellulovarans produced a single transcript of 1600 bp with the relative amount of engB mRNA from cellulose-grown cells being much greater than that from cellobiose-grown cells . Primer extensions showed that engB was transcribed from a single transcription initiation site in C . cellulovorans preceded by sequences similar to promoter sequences found in Gram-positive bacteria . Primer extensions from both E . coli and C . acetobutylicum strains containing the engB gene showed multiple transcription initiation sites, none of which corresponded to the site determined in C . cellulovorans . We conclude that transcriptional control of the engB gene is less stringent in heterologous backgrounds and postulate that expression of the engB gene in C . cellulovorans is increased in the presence of cellulose.

Biochem Biophys Res Commun, 1994 Dec 15, 205(2), 1291 - 8
Molecular construction of Clostridium botulinum type C progenitor toxin and its gene organization; Fujinaga Y et al.; The 16S progenitor toxin of Clostridium botulinum type C is made up by conjugation of a neurotoxin with nontoxic components designated as nontoxic-nonHA and hemagglutinin (HA) . The HA was found to be composed of subcomponents having 53, 33, 22-23, and 17 kDa molecular masses . Since we previously determined the whole nucleotide sequences of the genes for neurotoxin, nontoxic-nonHA, and HA-33, the cloning and nucleotide sequencing of the genes for the remaining HA subcomponents were performed . Two open reading frames coding for 16.7 kDa (HA-17) and 70.6 kDa proteins were identified . The N-terminal amino acid sequences of HA-53 and HA-22-23 indicated that the 70.6 kDa protein is split into 53 and the 22-23 kDa proteins after translation and that the 22-23 kDa protein consists of at least four proteins showing slightly different molecular weights.

Biochemistry, 1994 Dec 6, 33(48), 14486 - 95
Comparison of native and mutant proteins provides a sequence-specific assignment of the cysteinyl ligand proton NMR resonances in the 2{Fe4S4} ferredoxin from Clostridium pasteurianum; Scrofani SD et al.; A sequence-specific assignment is presented for the eight low-field paramagnetically shifted cysteinyl ligand proton NMR resonances in the 2{Fe4S4} ferredoxin from Clostridium pasteurianum . The assignment is based upon comparison of chemical shifts in 1D and 2D NMR spectra of native oxidized protein and those of three mutants . The mutant proteins G12A and G41A were designed to produce minor local structural changes (hence small chemical shift perturbations) in either cluster I (glycine 12 to alanine) or in cluster II (glycine 41 to alanine) . Observed chemical shift changes in spectra of the double mutant G12,41A support the interpretation . The comparison is aided by structural models derived from the crystal structure of the related ferredoxin from Peptococcus aerogenes . Each of the eight low-field resonances is assigned to a beta-proton from a different cysteinyl ligand, and so connectivities established from previous TOCSY and HMQC data allow assignment of all 24 cysteinyl ligand protons.

Proc Natl Acad Sci U S A, 1994 Dec 6, 91(25), 11859 - 63
Delineation and comparison of ganglioside-binding epitopes for the toxins of Vibrio cholerae, Escherichia coli, and Clostridium tetani: evidence for overlapping epitopes; Angstrom J et al.; Binding studies of various glycolipids, mainly belonging to the ganglio series, to the toxins isolated from Vibrio cholerae, Escherichia coli, and Clostridium tetani have been performed, using the microtiter well assay . By using the found binding preferences in conjunction with minimum-energy conformations obtained from molecular modeling of the various ligands, binding epitopes on the natural receptor glycolipids for the toxins have been defined . The binding preferences for the cholera toxin and the heat-labile E . coli toxin are very similar, with the ganglioside GM1 being the most efficient ligand . The tetanus toxin binds strongly to gangliosides of the G1b series, with GT1b as the most efficient ligand . It is found that the binding epitope on GM1 for the cholera and heat-labile toxins to a large extent overlaps with the epitope on GQ1b for the tetanus toxin.

Am J Gastroenterol, 1994 Dec, 89(12), 2246 - 8
Cytomegalovirus infection causing pseudomembranous colitis; Franco J et al.; A liver transplant patient with previous episodes of diarrhea and pseudomembranous colitis due to Clostridium difficile subsequently developed pseudomembranous colitis due to cytomegalovirus . The patient responded to ganciclovir . Cytomegalovirus infection should be considered in the differential of pseudomembranous colitis in immunocompromised patients, particularly when C . difficile toxin assays or cultures are negative.

J Med Microbiol, 1994 Dec, 41(6), 405 - 7
Outbreaks of food-poisoning associated with lecithinase-negative Clostridium perfringens; Brett MM; Clostridium perfringens type A is a common cause of food-poisoning . Production of lecithinase (alpha toxin) is frequently used to identify the organism . Details of 10 outbreaks of food-poisoning caused by lecithinase-negative C . perfringens are reported here.

J Bacteriol, 1994 Dec, 176(23), 7328 - 34
Mutation analysis of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A; Goldstein MA et al.; Cellulose-binding protein A (CbpA) has been previously shown to mediate the interaction between crystalline cellulose substrates and the cellulase enzyme complex of Clostridium cellulovorans . CbpA contains a family III cellulose-binding domain (CBD) which, when expressed independently, binds specifically to crystalline cellulose . A series of N- and C-terminal deletions and a series of small internal deletions of the CBD were created to determine whether the entire region previously described as a CBD is required for the cellulose-binding function . The N- and C-terminal deletions reduced binding affinity by 10- to 100-fold . Small internal deletions of the CBD resulted in substantial reduction of CBD function . Some, but not all, point mutations throughout the sequence had significant disruptive effects on the binding ability of the CBD . Thus, mutations in any region of the CBD had effects on the binding of the fragment to cellulose . The results indicate that the entire 163-amino-acid region of the CBD is required for maximal binding to crystalline cellulose.

Infect Immun, 1994 Dec, 62(12), 5647 - 51
Cell-associated collagenolytic activity by group B streptococci; Jackson RJ et al.; Group B streptococci (GBS) are important pathogens in neonatal sepsis, pneumonia, and meningitis . The ability of GBS to invade the collagen-rich amniotic membrane of the placenta has been shown in vitro . In the presence of GBS, the collagen fibrils of the amnion appear disordered, suggesting a role for GBS in premature rupture of membranes . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and gelatin zymograms were used in this study to characterize cell-associated collagenolytic activities of GBS . The synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which mimics the primary structure of collagen, was degraded by GBS USF704, a clinical isolate from the placenta of a septic newborn . Cells of GBS USF704 (9 x 10(7) CFU/ml) hydrolyzed 902 nmol of FALGPA over a 24-h period . As reported for zinc metalloenzymes such as collagenase, the hydrolysis of FALGPA by GBS was inhibited by addition of EDTA or 1,10-phenanthroline . Boiling of the cells resulted in loss of activity, while higher activity was observed with crude GBS cell lysates (hydrolysis of 970 nmol of FALGPA in 1.5 h) . Antiserum raised against collagenase from Clostridium histolyticum was found to cross-react with cell-associated proteins produced by GBS and to inhibit GBS FALGPA hydrolysis . Twenty-five additional GBS clinical isolates were screened and found to have various levels of FALGPA hydrolytic activity . These observations suggest a cell-associated collagenolytic activity by GBS which may be involved in premature rupture of membranes and neonatal disease.

Infect Immun, 1994 Dec, 62(12), 5550 - 8
Expression from the Clostridium perfringens cpe promoter in C . perfringens and Bacillus subtilis; Melville SB et al.; Clostridium perfringens is a source of food poisoning in humans and animals because of production of a potent enterotoxin (CPE) . To study the regulation of the cpe gene in C . perfringens, we cloned and sequenced the cpe promoter regions and N-terminal domains from three strains . The cpe promoter region from one strain contained a 45-bp insertion compared with previously published sequences . This insertion was also found in two (of five) other Cpe+ strains . cpe gene expression in C . perfringens was measured by using translational fusions of each promoter type to the Escherichia coli gusA gene, which codes for beta-glucuronidase . For either promoter type, cpe-gusA expression was undetectable throughout exponential growth but increased dramatically at the beginning of the stationary phase . To measure cpe expression in Bacillus subtilis, cpe-gusA fusions were integrated into the B . subtilis chromosome . Both types of promoter exhibited moderate expression during exponential growth; cpe expression increased threefold at the beginning of the stationary phase . Transcriptional start sites were determined by primer extension and in vitro transcription assays . For C . perfringens, both types of promoter gave the same 5' end, 197 bp upstream of the translation start (50 bp downstream of the 45-bp insertion) . In B . subtilis, however, the 5' end was internal to the 45-bp insertion, suggesting the use of a different promoter than that utilized by C . perfringens.

Infect Immun, 1994 Dec, 62(12), 5281 - 9
GTP-binding proteins are involved in the modulated activity of human neutrophils treated with the Panton-Valentine leukocidin from Staphylococcus aureus; Hensler T et al.; Significant amounts of leukotriene B4 (LTB4) are generated by human polymorphonuclear neutrophils (PMNs) after incubation with the Panton-Valentine leukocidin (Luk-PV) from Staphylococcus aureus V8 strains . We showed that GTP-binding proteins (G proteins) are involved in the Luk-PV-activated signal transduction of PMNs . ADP-ribosylation of heterotrimeric G proteins by cholera and pertussis toxins decreased the Luk-PV-induced LTB4-generation . In contrast, ADP-ribosylation of the low-molecular-weight G proteins rho and rac by Clostridium botulinum exoenzyme C3 increased the Luk-PV-induced LTB4 synthesis . The subsequent stimulation of Luk-PV-treated PMNs by either calcium ionophore A23187, sodium fluoride, or formylmethionyl-leucyl-phenylalanine was significantly inhibited . This decrease was paralleled by a loss of G-protein functions, including GTPase activity and GTP-binding capacity . An increase of G-protein functions was obtained with low amounts of Luk-PV . In addition to the modulated G-protein functions, ADP-ribosylation of 24-, 40-, and 45-kDa proteins by Luk-PV was detected . As shown in control experiments, the ADP-ribosylated 24-kDa proteins were not substrates for C . botulinum exoenzyme C3 . Introduction of ras p21 into digitonin-permeabilized PMNs was without effect on subsequent Luk-PV stimulation . In addition, the translocation of ras p21, ras GAP, and 5-lipoxygenase into the membrane of Luk-PV-treated PMNs, as well as the expression of chemotactic membrane receptors for LTB4 and formylmethionyl leucyl phenylalanine, was significantly diminished.

Lab Anim Sci, 1994 Dec, 44(6), 568 - 72
Tyzzer's infection: host specificity of Clostridium piliforme isolates; Franklin CL et al.; Tyzzer's disease, a well-recognized syndrome in numerous laboratory animal species, is caused by the obligate intracellular bacterium, Clostridium piliforme . Distinct isolates of C . piliforme from various laboratory animal species have been identified based on protein and antigenic heterogeneity . The goal of this study was to examine the host specificity of three well-characterized isolates of C . piliforme . Groups of mice, rats, and hamsters were experimentally infected with isolates obtained from a naturally infected mouse (M1), a naturally infected rat (R1), and a naturally infected hamster (H2) . To assess infection status, animals were monitored serologically for antibody to C . piliforme over a 12-week period . Evaluation of results indicated that the M1 isolate infected rats and mice but not hamsters, whereas the R1 and H2 isolates infected only the host species from which the isolates were originally obtained . These findings suggest that C . piliforme isolates can be categorized into two types: 1) cross-infective isolates, such as M1, which can infect more than one laboratory animal species, and 2) isolates, such as R1 and H2, which have a more limited host range within laboratory animal species . These results emphasize the need to consider the host specificity of C . piliforme isolates when investigating outbreaks of Tyzzer's disease.

Prostaglandins, 1994 Dec, 48(6), 367 - 75
The effect of Clostridium difficile toxin on colonocyte prostanoid activity; Stratton MD et al.; Antibiotic-associated colitis is caused by Clostridium difficile toxin . However, the pathophysiology of this entity is poorly understood . The aim of this study was to determine the effects of C . difficile toxin on colonocyte cyclooxygenase and phospholipase A2 (PLA2) activity . A transformed colonocyte cell line (Caco-2) was grown to confluency on 6 well plates . The cells were stimulated with graded concentrations of C . difficile toxin . In separate experiments, the cells were pretreated for one hour prior to stimulation with the cyclooxygenase inhibitor, indomethacin, or the glucocorticoid, dexamethasone . The culture media was collected one hour following C . difficile stimulation . Prostaglandin E2 (PGE2), 6-keto prostaglandin F1 alpha (6KPGF), thromboxane B2 (TxB2) and leukotriene B4 (LTB4) levels were determined in the media by an ELISA . Platelet activating factor (PAF) concentration was determined by a RIA . C . difficile toxin stimulated PGE2 and 6KPGF levels in a dose dependent fashion but failed to stimulate TxB2, LTB4 or PAF . Prostanoid production was inhibited by indomethacin dose dependently but was not inhibited by dexamethasone . The presence of indomethacin resulted in production of PAF . Our results show that the effects of C . difficile toxin on colonocytes are mediated by cyclooxygenase activity . The increase in PAF formation associated with indomethacin administration suggests that the prostanoids modulate PLA2 activity and inhibit PAF formation.

J Clin Microbiol, 1994 Dec, 32(12), 3002 - 7
Rapid identification of bacteria by PCR-single-strand conformation polymorphism; Widjojoatmodjo MN et al.; A new molecular biological approach for the identification of bacteria is described . This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP) . The PCR product was denatured and separated on a nondenaturing polyacrylamide gel . SSCP patterns were detected by silver staining the nucleic acids . The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria . Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria . We tested over 100 strains of bacteria representing 15 genera and 40 species . With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels . Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp . PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU . This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.

Eur J Biochem, 1994 Dec 1, 226(2), 577 - 85
Characterization of the coenzyme-B12-dependent glutamate mutase from Clostridium cochlearium produced in Escherichia coli; Zelder O et al.; The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium . The enzyme consists of two protein components, E, a dimer epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa) . The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli . The genes glmS and glmE are separated by glmL encoding a protein of unknown function . The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins . Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E . coli carrying the corresponding genes . In contrast to component E, component S was shown to bind coenzyme B12 . This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif . Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of coenzyme B12 . EPR spectra of recombinant glutamate mutase, now available in sufficiency large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate . The EPR signals (gx,y approximately 2.1, gz = 1.985) were of much better resolution than observed earlier with the clostridial enzyme . Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y = 2.25) . The spin concentration was 34-50% of the concentration of the enzyme (epsilon 2 sigma 2) coenzyme complex . The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration . Even (S)-{2,3,3,4,4-2H5}glutamate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7% . These data suggest an involvement of the Co(II)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition . The large difference in the spin concentrations observed with (S)-glutamate as compared to the predeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS)

J Appl Bacteriol, 1994 Dec, 77(6), 650 - 5
Typing of Clostridium perfringens by in vitro amplification of toxin genes; Daube G et al.; The strains of Clostridium perfringens are classified according to major toxins produced . Classically, this determination involves the seroneutralization of their lethal effect in mice . However, this method requires specific antisera and a large number of mice . In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR) . By combination of several pairs of primers, the toxinotype of a Cl . perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis . This mixture contained primers amplifying simultaneously a part of alpha-toxin, beta-toxin, epsilon-toxin and enterotoxin genes . In order to distinguish between toxinotype A and E, the l-toxin gene fragment must be amplified in a separate PCR reaction . Moreover, with the primers combination, in most cases, a PCR product corresponding to the alpha-toxin gene was obtained from direct enrichments of animal intestinal contents.

FEMS Microbiol Lett, 1994 Dec 1, 124(2), 151 - 5
Acetivibrio cellulolyticus and Bacteroides cellulosolvens are members of the greater clostridial assemblage; Lin C et al.; The nucleotide sequences of the 16S rRNA genes of Acetivibrio cellulolyticus, A . cellulosolvens, and Bacteroides cellulosolvens were determined and shown to be affiliated with the low G + C members of Gram-positive bacteria . The sequences for A . celluolyticus and A . cellulosolvens were revealed to be identical, supporting the proposal by W.D . Murray {Int . J . Syst . Bacteriol . (1986) 36, 314-316} that A . cellulosolvens be correctly classified as A . cellulolyticus . The closest relative to A . cellulolyticus is Clostridium aldrichii, related at 98.5% sequence similarity . B . cellulosolvens and A . cellulolyticus are related at 94.4% sequence similarity.

Int J Biol Macromol, 1994 Dec, 16(6), 335 - 42
Isolation of four major subunits from Clostridium thermocellum cellulosome and their synergism in the hydrolysis of crystalline cellulose; Bhat S et al.; The cellulosome of Clostridium thermocellum, purified by affinity chromatography, was dissociated under mild conditions and separated by SDS-PAGE . Two major p-nitrophenylcellobiosidases (PNPCases I and II) corresponding to the S5 (103 kDa) and S8 (78 kDa) subunits and one major carboxymethylcellulase (CMCase) coinciding with the S11 (60.5 kDa) subunit were isolated and characterized using carboxymethylcellulose (CMC), H3PO4-swollen cellulose and cello-oligosaccharides . Both PNPCases showed little effect on the viscosity of CMC and released twice as much total sugar as reducing sugar from H3PO4-swollen cellulose . The CMCase released ten times more total sugar than reducing sugar from H3PO4-swollen cellulose and reduced the viscosity of CMC rapidly . None of these enzymes was active on cellotriose . Both PNPCases released cellobiose from cellotetraose, and cellobiose and cellotriose from cellopentaose . In contrast, CMCase was active only on cellopentaose and released mainly glucose . Use of MeUmb(Glc)n revealed that both PNPCases cleaved preferentially either the second or fourth linkage from the non-reducing end while the CMCase was specific for the internal glycosidic bonds . Thus, the PNPCases and CMCase behaved as typical exo- and endoglucanases, respectively . When tested individually, all three enzymes degraded Avicel only to a small extent . A 1.5-2.0-fold increase in sugar release was observed when CMCase was combined with either PNPCase I, II or both . Combining S1 with either PNPCase II or CMCase resulted in fourfold synergism in the hydrolysis of Avicel . Synergism was sevenfold when all three enzymes were combined with S1.

Toxicon, 1994 Dec, 32(12), 1581 - 92
Clostridium sordellii cytotoxin induces phosphorylation of an 80,000 mol . wt protein in McCoy cultured cells; Schue V et al.; The cytotoxins from Clostridium difficile (toxin B) and Clostridium sordellii (toxin L) induce rounding of cultured cells . The cellular effects induced by these two cytotoxins are clearly distinct, suggesting that both toxins use a similar, but not identical mechanism for cell intoxication . We have employed the technique of two-dimensional PAGE for the separation of 32P-labelled cell lysates of McCoy cultured cells to investigate changes in the phosphorylation status of cellular proteins after treatment with toxin B and with toxin L . The two-dimensional electrophoresis patterns suggest the implication of an 80,000 mol . wt cellular protein (named pp80c) in the cytopathic action of the cytotoxin from C . sordellii . This protein shows immunoreactivity with non-muscle caldesmon . Toxin B, however, does not affect the phosphorylation of pp80c, but alters the phosphorylation of another cellular protein, pp77, indicating another mechanism for cell intoxication . In addition, our experiments suggest that the mechanism of action of okadaic acid, a phosphatase inhibitor which causes cell rounding similar to that induced by C . sordellii, and these two cytotoxins are different.

Protein Eng, 1994 Dec, 7(12), 1501 - 7
Efficient generation of a reshaped human mAb specific for the alpha toxin of Clostridium perfringens; Tempest PR et al.; We have used the technique of antibody reshaping to produce a humanized antibody specific for the alpha toxin of Clostridium perfringens . The starting antibody was from a mouse hybridoma from which variable (V) region nucleotide sequences were determined . The complementarity-determining regions (CDRs) from these V regions were then inserted into human heavy and light chain V region genes with human constant region gene fragments subsequently added . The insertion of CDRs alone into human frameworks did not produce a functional reshaped antibody and modifications to the V region framework were required . With minor framework modifications, the affinity of the original murine mAb was restored and even exceeded . Where affinity was increased, an altered binding profile to overlapping peptides was observed . Computer modelling of the reshaped heavy chain V regions suggested that amino acids adjacent to CDRs can either contribute to, or distort, CDR loop conformation and must be adjusted to achieve high binding affinity.

Diagn Microbiol Infect Dis, 1994 Dec, 20(4), 213 - 9
Evaluation of the Etest for antimicrobial spectrum and potency determinations of anaerobes associated with bacterial vaginosis and peritonitis; Croco JL et al.; One hundred ninety-seven anaerobic organisms (24 Gardnerella vaginalis, 16 Mobiluncus spp., 19 Peptostreptococcus spp., 20 Lactobacillus spp., 20 Prevotella bivia/disiens, 81 Bacteroides fragilis group, 12 Clostridium spp., and five Fusobacterium spp.) were processed by the Etest (AB Biodisk, Solna, Sweden) and a reference (Brucella blood agar) method against 10 antimicrobial agents . For the bacterial vaginosis-associated pathogens, the Etest was more reproducible and correlated acceptably with the reference agar test: within +/- 1 log2 dilution for 74.4% of Mobiluncus spp . to 96.0% for Peptostreptococcus spp . (all organisms, 83.4%) . The quantitative correlation +/- 2 log2 dilution steps between test results was 94.3% . Results with B . fragilis group strains demonstrated 97.3% correlation (+/- 2 log2 dilution) with a trend toward slightly lower Etest minimum inhibitory concentrations for ampicillin-sulbactam, cefotaxime, imipenem, and clindamycin . The absolute qualitative interpretive agreement between Etest and the reference agar dilution method results was 94.4%, with only a 0.4% false-susceptible error rate . The Etest appears to be a very practical, quantitatively accurate, alternative procedure for clinical microbiology laboratories routinely testing the susceptibilities of anaerobes and, by these presented data, organisms associated with female tract infections.

Int J Food Microbiol, 1994 Dec, 24(1-2), 227 - 38
Utilization of cheddar cheese containing nisin as an antimicrobial agent in other foods; Zottola EA et al.; Cheddar cheese made with nisin-producing lactococci contained between 400 and 1200 IU of nisin per gram of cheese . Cultures used were Lactococcus lactis ssp . cremoris JS102, a nisin-producing transconjugant developed in the laboratories of Dr . L.L . McKay and Lactococcus lactis ssp . lactis NCDO 1404 obtained from the National Collection of Food Bacteria, Reading, England . Pasteurized process cheese spreads with 53% and 60% moisture and 0, 301 and 387 IU nisin/g were manufactured and inoculated with 2000 spores of Clostridium sporogenes PA 3679 during manufacture . The heat process did not reduce nisin activity in the cheese spreads . The spreads were incubated at 22 degrees and 37 degrees C for 90 days . Spoilage was detected by the presence of gas and/or odor in the packages . The shelf-life of the nisin-containing cheese spreads was significantly greater than that of the control cheese spreads at the lower temperature at both moisture levels, whereas the keeping quality of the higher moisture cheeses at the higher temperature was not significantly different . Club cheese or cold pack cheese spreads with moisture levels of 44% and 60% and 0, 100 and 300 IU nisin/g were made . These cold processed cheese spreads were inoculated with 1000 cfu per g of Listeria monocytogenes V7, Staphylococcus aureus 196E and spores of C . sporogenes PA 3679 . Heat shocked spores of PA 3769 at the same number were added to separate lots of the cheese spread . The cold pack cheese spreads were incubated at 23 degrees and 37 degrees C for up to 8 weeks . Samples were taken weekly and analyzed for surviving organisms . Significant reductions in numbers of the non-sporeforming test microbes were noted at both temperatures, at both moisture levels and both levels of nisin . Heat shocking the spores was needed to show reduction in numbers during the storage of the cold pack cheese spreads . The data obtained in this study suggest that the use of nisin-containing cheese as an ingredient in pasteurized process cheese or cold pack cheese spreads could be an effective method of controlling the growth of undesirable microorganisms in these processed foods.

Int J Food Microbiol, 1994 Dec, 24(1-2), 211 - 25
Isolation and characterization of two bacteriocins produced by Enterococcus faecium strains inhibitory to Listeria monocytogenes; Vlaemynck G et al.; A total of 4000 bacterial strains were isolated from milk, cheese and from different samples taken on the farm (silage, faeces) and screened for their antimicrobial activity against Listeria monocytogenes . Only eight of the almost 4000 strains exhibited inhibitory activity in the cell-free supernatant fluid . Two different Enterococcus strains (RZS C5 and RZS C13) were chosen for further study . Plasmid profile analysis of both revealed that RZS C5 contained a 4 kb plasmid while RZS C13 contained a 23 kb plasmid . The inhibitory compounds were proteinaceous and active against all Listeria spp . tested while most of the lactic acid bacteria were insensitive . Weak inhibition was also detected against Clostridium perfringens and some Bacillus spp . Cross-inhibition tests showed different profiles . The bacteriocins were heat-resistant and were very stable during storage, especially at low pH values . Their molecular weight was estimated at +/- 3.0 kDA . Addition of 200 AU/ml of bacteriocin resulted in an initial rapid decrease of the number of viable L . monocytogenes cells followed by slower a logarithmic decrease.

Mol Cell Probes, 1994 Dec, 8(6), 463 - 7
Specific detection of Clostridium difficile toxin A gene sequences in clinical isolates; Tang YJ et al.; The polymerase chain reaction (PCR) was used to specifically detect toxin A gene sequences of Clostridium difficile in DNA isolated from human faeces . A set of oligonucleotide primers derived from the non-repetitive region of the toxin A gene was developed to amplify a 634-bp DNA fragment . All 28 cytotoxic strains of C . difficile, previously characterized by a toxin B-PCR assay, were positive for the presence of toxin A gene sequences . No amplification products were obtained from DNAs extracted from non-toxigenic strains, strains of C . sordellii, or C . bifermentans . In addition, amplification of DNA extracted from C . difficile 8864, a strain which does not produce toxin A, resulted in multiple bands which probed negative for toxin A gene sequences . DNAs extracted from nine stool specimens which were positive for toxin B by the cytotoxicity assay and by the toxin B-PCR assay were also positive in this assay . Toxin A gene sequences were detected in DNAs obtained from 4/11 stool specimens which were negative by the toxin B cytotoxicity assay . These four specimens were from patients who had a history of relapses due to C . difficile-associated colitis, and whose stools had previously been found to be positive by the toxin B-PCR test despite no detectable toxin B in the specimens . These data indicate a comparable degree of clinical sensitivity between these two toxin-gene PCR-based assays . This rapid, sensitive and specific assay may be useful not only in the diagnosis of C . difficile infections, but also in molecular studies of the toxin A gene in C . difficile strains.

J Vet Med Sci, 1994 Dec, 56(6), 1047 - 50
Analysis of multiple antigenic determinants of Clostridium perfringens enterotoxin as revealed by use of different synthetic peptides; Sugii S; To identify the antigenic determinants of Clostridium perfringens enterotoxin (CPE), overlapping peptides corresponding to the entire amino acid sequence of CPE were synthesized and their antigenicity and immunogenicity were analyzed . Of the 21 synthetic peptides (C-1 to C-21) tested, peptide C-7 (amino acid residues 91-110) showed the highest reactivity with rabbit antibodies against CPE . Peptides C-4 (46-65), C-9 (121-140), and C-11 (151-170) were reactive with the antibodies, whereas peptides C-2 (16-35), C-3 (31-50), and C-20 (286-305) were less reactive . Other peptides were much less reactive . On the other hand, all of the synthetic peptides reactive with the anti-CPE antibodies were found to elicit rabbit antibodies reactive with both their respective antigens and native CPE molecule . These findings demonstrate that CPE molecule contains at least 10 different antigenic determinants which are all immunogenic and supposed to be responsible for functionally and biologically active sites.

FEMS Microbiol Lett, 1994 Dec 1, 124(2), 229 - 37
The use of PCR to monitor the population abundance of six human intestinal bacterial species in an in vitro semicontinuous culture system; Wang RF et al.; Six PCR primer sets complementary to the 16S rDNAs (rRNA genes) were developed and shown to be specific for the following anaerobic bacteria: Clostridium clostridiiforme, C . perfringens, C . leptum, Bacteroides vulgatus, B . distasonis, and B . thetaiotaomicron, respectively . These primers were used for PCR to detect and monitor the bacteria in a semicontinuous culture system designed to mimic intestinal microflora in the human gastrointestinal tract . Except for C . perfringens, the five species of Bacteroides and Clostridia present in the in vitro culture system were detected by the PCR, and the titers varied from 10(-2) to 10(-6) dilutions . The role of azo dye reduction by these bacterial species in the system was examined and discussed.

Biochem Biophys Res Commun, 1994 Nov 30, 205(1), 893 - 8
Differential reactivity of two types of N-glycolyneuraminic acid dimers toward enzymatic and nonenzymatic hydrolysis of their interketosidic linkages; Kitazume S et al.; The kinetics of acid- and sialidase-catalyzed hydrolysis of the interketosidic linkages of two different disialic acids, Neu5Gc alpha 2-->5-OglycolylNeu5Gc and Neu5Gc alpha 2-->8Neu5Gc, were studied . The former sequence was recently identified in the polysialic acid chains of a sialic acid-rich glycoprotein isolated from the egg jelly coat of two different species of sea urchins, and the latter was previously found in the cortical alveolar-derived polysialoglycoprotein from rainbow trout eggs . At pH values < 3.8, the rate of hydrolysis of Neu5Gc alpha 2-->5-OglycolylNeu5Gc was greater than that of Neu5Gc alpha 2-->8Neu5Gc . Paradoxically, however, Neu5Gc alpha 2-->5-OglycolylNeu5Gc was more stable than Neu5Gc alpha 2-->8Neu5Gc at pH values > 3.8 . These findings indicate a greater contribution of intramolecular general acid catalysis to the lability of the alpha 2-->5-ketosidic linkage . Neu5Gc alpha 2-->5-OglycolylNeu5Gc was a poor substrate for Arthrobacter ureafaciens, Clostridium perfringens, and Vibrio cholerae sialidases, in contrast to Neu5Gc alpha 2-->8Neu5Gc . Neu5Gc alpha 2-->5-OglycolylNeu5Gc was essentially resistant to hydrolysis by A . ureafaciens sialidase.

J Mol Biol, 1994 Nov 25, 244(2), 236 - 7
Crystallization and preliminary X-ray analysis of the major cellulose-binding domain of the cellulosome from Clostridium thermocellum; Lamed R et al.; The cellulose-binding domain from the scaffoldin subunit of the cellulosome from Clostridium thermocellum strain YS has been expressed in Escherichia coli, purified to homogeneity, and crystallized . Crystals were grown by vapor diffusion using polyethylene glycol as precipitant . They belong to the monoclinic space group C2 with unit cell dimensions of a = 64.68 A, b = 50.36 A, c = 96.27 A; beta = 99.43 degrees, and density packing considerations suggest that the asymmetric unit contains two molecules . The crystals diffract beyond 2.0 A resolution using a laboratory rotating anode source.

J Biol Chem, 1994 Nov 25, 269(47), 29592 - 7
The demonstration of a glutamate dehydrogenase-NADP-L-glutamate charge-transfer complex and its location on the reaction pathway; Saha SK et al.; In previous transient state kinetic work from this laboratory, we proposed a new mechanism for the glutamate dehydrogenase-catalyzed oxidative deamination reaction involving an initial replacement of a proton from lysine 126 by a single bound water molecule, followed by closure of the active site cleft and expulsion of bulk water, providing a hydrophobic environment for the ensuing hydride transfer step . Here, we report the results of further transient state fluorescence, absorbance, and kinetic isotope effect studies, which demonstrate the occurrence of an unusual intermediate in the early steps of that reaction . This phenomenon is revealed by an initial fluorescence burst that occurs in the time period where the absorbance signal is still in its lag phase . Using an extension of the proton/product ratio approach we have described earlier, we show that this intermediate is a strongly fluorescent but weakly absorbing species whose absorption maximum is red-shifted beyond that of other known complexes of this enzyme . The transient state kinetic isotope effects of the fluorescence and absorbance signals are compatible only with a reaction scheme in which the formation of the fluorescent complex precedes the hydride transfer step . The optical properties of this enzyme-oxidized coenzyme-substrate intermediate strongly suggest that it is a charge-transfer complex, similar in nature to the complex responsible for the well known "Racker band" reported in 1952 for glyceraldehyde-3-phosphatase dehydrogenase (Racker, E., and Krimsky, I . (1952) Nature 169, 1043-1044) . The crystal structure studies of the enzyme-coenzyme and enzyme-L-glutamate complexes of the closely analogous Clostridium symbosium glutamate dehydrogenase, reported by the Sheffield group (Stillman, T . J., Baker, P . J., Britton, K . L., and Rice, D . W . (1993) J . Mol . Biol . 234, 1131-1139), provide a basis for a physical explanation of the phenomenon . We conclude that the charge transfer phenomenon is caused by the near apposition of the unprotonated alpha-amino group of the substrate in a form of the enzyme in which a conformational change has caused the complete closing of the active site cleft.

Biochim Biophys Acta, 1994 Nov 23, 1196(1), 1 - 13
The relationship between phospholipid content and Ca2+-ATPase activity in the sarcoplasmic reticulum; Pikula S et al.; The relationship between the phospholipid composition of sarcoplasmic reticulum and the activity of the Ca2+, Mg2+-stimulated ATPase was analyzed by digestion of membrane phospholipids with phospholipase C and A2 enzymes of diverse specificity and by detergent extraction . Phospholipase C of Clostridium perfringens and Clostridium welchii, that hydrolyze preferentially phosphatidylcholine (PC), inhibited the Ca2+-ATPase activity parallel with the depletion of phosphatidylcholine from the membrane . Phospholipase C of Bacillus cereus hydrolyzed in addition to PC, phosphatidylethanolamine (PE) and phosphatidylserine (PS), causing complete inhibition of Ca2+-stimulated ATPase activity . Digestion of sarcoplasmic reticulum with the phospholipase A2 of snake or bee venom produced similar effects . The phosphatidylinositol (PI)-specific phospholipases of B . cereus and Bacillus thuringiensis caused less than 10% inhibition of the Ca2+-ATPase, accompanied by the hydrolysis of more than 70% of the phosphatidylinositol content of the membrane, without significant change in PC, PE and PS content . The inhibition of ATPase activity by the C type phospholipases was nearly completely reversed by octaethyleneglycol dodecyl ether (C12E8) . These experiments suggest that the full phospholipid content of native sarcoplasmic reticulum (congruent to 100 mol phospholipid per mol Ca2+-ATPase), is required for ATPase activity and there is no indication that PE, PS, and PI play a specific role in ATP hydrolysis . Extraction of sarcoplasmic reticulum phospholipids by detergents such as deoxycholate, cholate and C12E8 also caused proportional inhibition of ATPase activity with the decrease in phospholipid content; the parallel extraction of PC, PE and PI left the phospholipid composition largely unchanged during delipidation . These observations do not support the requirement for a 'lipid annulus' of congruent to 30 phospholipid molecules/Ca2+-ATPase as proposed by Hesketh et al . ((1976) Biochemistry 15, 4145-4151) or the specific interaction of phosphatidylethanolamine with the ATPase molecule proposed by Bick et al . ((1991) Arch . Biochem . Biophys . 286, 346-352).

Proc Natl Acad Sci U S A, 1994 Nov 22, 91(24), 11437 - 41
A pathway for the biosynthesis of straight and branched, odd- and even-length, medium-chain fatty acids in plants; Kroumova AB et al.; Pathways and enzymes of fatty acid synthase-mediated, long-even-chain (generally C16-C20) fatty acid synthesis are well studied, and general metabolism involved in short-chain (C4-C7) fatty acid biosynthesis is also understood . In contrast, mechanisms of medium-chain (C8-C14) fatty acid synthesis are unclear . Recent work suggests involvement of chain-elongation-terminating thioesterases in medium-chain fatty acid formation in oilseeds and animals . We have shown that iso- and anteiso-branched and straight, odd- and even-length, short-chain fatty acids esterified in plant-trichome-gland-produced sucrose esters are synthesized by using carbon skeletons provided by modified branched-chain amino acid metabolism/catabolism . The principal enzymes involved are those catalyzing leucine biosynthesis in all organisms and those leading to short-chain alcohols in mutant yeasts and alkyl acids in Clostridium species (products often serving as mammalian pheromones) . Here we provide evidence that C10-C12 straight medium-chain and C10-C12 branched medium-chain acyl acids of tomato, C6-C8 straight-chain acyl acids of Petunia, and C6 and C8 branched acyl acids of Nicotiana glutinosa are formed by alpha-ketoacid elongation without participation of fatty acid synthase-mediated reactions or -independent thioesterases . This different metabolism suggests greater integration of amino acid and fatty acid metabolism than previously considered and provides other avenues to study and manipulate not only straight even-length but also odd- and even-length straight and branched medium-chain fatty acid biosynthesis.

Biochemistry, 1994 Nov 22, 33(46), 13642 - 50
Mutated forms of the {2Fe-2S} ferredoxin from Clostridium pasteurianum with noncysteinyl ligands to the iron-sulfur cluster; Meyer J et al.; The {2Fe-2S} ferredoxin from Clostridium pasteurianum is unique among ferredoxins, both by its sequence and by the distribution of its cysteine residues (in positions 11, 14, 24, 56, 60) . Thus, no homologous sequences are available to infer, by comparison, the identity of the ligands of the iron-sulfur cluster . Therefore, in order to obtain information on the latter point, a combination of site-directed mutagenesis and UV-vis, EPR, and resonance Raman spectroscopy has been implemented . All of the cysteine residues have individually been replaced by serine and two of them by alanine . Cysteine 14 could be replaced by either serine or alanine without any modification of the spectroscopic properties of the protein and was therefore dismissed as a ligand of the {2Fe-2S} cluster . The C56S, and C60S-mutated proteins were both found to display UV-vis, EPR, and resonance Raman spectra consistent with serine-coordinated {2Fe-2S} clusters . The C11S-mutated protein was considerably less stable than the wild type ferredoxin . This observation, together with the hypsochromic shifts of UV-visible absorption features upon cysteine 11-->serine mutation, suggested cysteine 11 to be a ligand of the {2Fe-2S} cluster . Cysteine 24 could be replaced by either serine or alanine without decreasing the stability of the protein and without dramatically changing its spectroscopic properties . Thus, either cysteine 24 is not a ligand of the {2Fe-2S} cluster or it is replaced by another ligand in the C24A mutated protein . A {2Fe-2S} cluster was also assembled in the C14A/C24A doubly mutated protein, i.e., in a polypeptide chain containing only three cysteine residues.2+ off

FEMS Microbiol Lett, 1994 Nov 15, 124(1), 61 - 7
Genes encoding homologues of three consecutive enzymes in the butyrate/butanol-producing pathway of Clostridium acetobutylicum are clustered on the Clostridium difficile chromosome; Mullany P et al.; Screening of a Clostridium difficile lambda EMBL3 gene library with antisera raised against C . difficile culture supernatant identified several clones expressing a 31-kDa protein . A 1.8-kb HindIII fragment subcloned from one of the clones was sufficient for expression of the 31-kDa polypeptide . Southern blot analysis showed a region homologous to this fragment to be present in all of 13 different C . difficile strains tested . Sequence analysis of the 1.8-kb fragment revealed three adjacent open reading frames . A database search showed that these three open reading frames appeared to encode homologues of three consecutive enzymes in the butanol/butyrate-producing pathway of Clostridium acetobutylicum (crotonase, beta-hydroxybutyryl coenzyme A dehydrogenase and thiolase).

Biochem J, 1994 Nov 15, 304 ( Pt 1), 139 - 45
Expression of vesicle-associated membrane protein 2 (VAMP-2)/synaptobrevin II and cellubrevin in rat skeletal muscle and in a muscle cell line; Volchuk A et al.; Molecular studies have identified a family of synaptic vesicle-associated membrane proteins (VAMPs, also known as synaptobrevins) which have been implicated in synaptic vesicle docking and/or fusion with plasma membrane proteins . Here we demonstrate the expression of two members of this family, VAMP-2/synaptobrevin II and cellubrevin, in skeletal muscle, a tissue with both constitutive and regulated membrane traffic . The 18 kDa VAMP-2 polypeptide was detected in purified membrane fractions from adult skeletal muscle and from L6 myotubes in culture, demonstrating that the presence of this protein in the isolated muscle membrane fractions is not the result of contamination by ancillary tissues such as peripheral nerve . Furthermore, skeletal muscle and the muscle cell line also expressed cellubrevin, a VAMP-2 homologue of 17 kDa; which is much less abundant in brain cells . Both VAMP-2 and cellubrevin were preferentially isolated in membrane fractions rich in plasma membranes, and were less concentrated in light microsomes and other internal membrane fractions of mature muscle or muscle cells in culture . Interestingly, both VAMP-2 and cellubrevin were much more abundant in the differentiated L6 myotubes than in their precursor myoblasts, suggesting that they are required for functions of differentiated muscle cells . The identity of both polypeptides was further confirmed by their susceptibility to proteolysis by Clostridium tetanus toxin . Expression of these products was further established by the presence of mRNA transcripts of VAMP-2 and cellubrevin, but not of VAMP-1, in both skeletal muscle and L6 myotubes . In contrast, other synaptic vesicle and docking/fusion components were undetectable, such as VAMP-1, SNAP25 and syntaxin 1A/1B, as were synaptophysin and synapsin Ia/Ib, proteins which are believed to be involved in sensing the signal for neuronal exocytosis . It is concluded that VAMP-2 and cellubrevin are expressed in skeletal muscle cells and may each participate in specific processes of intracellular membrane traffic.

FEBS Lett, 1994 Nov 14, 354(3), 315 - 9
Botulinum C3 exoenzyme blocks the tyrosine phosphorylation of p125FAK and paxillin induced by bombesin and endothelin; Rankin S et al.; In this study we examined the role of rho p21 in neuropeptide-stimulated tyrosine phosphorylation . Intact Swiss 3T3 cells were treated with the Clostridium botulinum C3 exoenzyme which specifically ADP ribosylates and inactivates rho p21 . C3 exoenzyme treatment of cells caused a marked decrease in both bombesin- and endothelin-stimulated tyrosine phosphorylation of multiple proteins, including p125 focal adhesion kinase (FAK) and paxillin . Our results suggest that rho p21 is a component of the signal transduction pathway linking seven transmembrane domain receptors with tyrosine phosphorylation and cytoskeletal events.

J Biol Chem, 1994 Nov 11, 269(45), 28076 - 83
Identification of a nitrogenase protein-protein interaction site defined by residues 59 through 67 within the Azotobacter vinelandii Fe protein; Peters JW et al.; During nitrogenase catalysis the Fe protein and the MoFe protein associate and dissociate in a MgATP-dependent process involving electron transfer from the Fe protein to the MoFe protein . A docking model, based primarily on the crystal structures of the separate components from Azotobacter vinelandii, was previously proposed in which the 2-fold symmetric surface of the homodimeric Fe protein interacts with the exposed surface of a MoFe protein pseudosymmetric alpha beta-unit interface . In this model, a loop, which is included within residues 59 through 67 of the Fe protein primary sequence, is likely to interact with the MoFe protein during component protein docking . In the present study, evidence supporting the component protein docking model was obtained by construction of an A . vinelandii strain that produces a hybrid Fe protein for which residues 59 through 67 have been replaced by the corresponding residues from the Fe protein of Clostridium pasteurianum . Biochemical analyses of the hybrid Fe protein revealed the following features when compared with the unaltered Fe protein . First, the hybrid Fe protein exhibited half the maximum specific activity of the normal Fe protein and was insensitive to inhibition by low levels of NaCl . Second, the hybrid Fe protein activity was hypersensitive to a molar excess of MoFe protein, which also resulted in the uncoupling of MgATP hydrolysis from substrate reduction . Third, stopped-flow spectrophotometry experiments showed that during catalysis the hybrid Fe protein dissociates from the MoFe protein at only half the normal rate of Fe protein-MoFe protein dissociation . Thus, the salient feature of the hybrid Fe protein is that it appears to form a relatively tighter complex with the MoFe protein . This property is in line with previous biochemical reconstitution experiments where it was shown that a heterologous mixture of Fe protein from C . pasteurianum and MoFe protein from A . vinelandii form a tight, inactive complex and supports the proposal that a region defined by residues 59 through 67 within the Fe protein is involved in component protein interaction.

J Mol Biol, 1994 Nov 4, 243(4), 683 - 95
Refined crystal structure of the 2{4Fe-4S} ferredoxin from Clostridium acidurici at 1.84 A resolution; Duee ED et al.; The crystal structure of the 2{4Fe-4S} ferredoxin from Clostridium acidurici has been determined at a resolution of 1.84 A and refined to an R-factor of 0.169 . Crystals belong to space group P4(3)2(1)2 with unit cell dimensions a = b = 34.44 A and c = 74.78 A . The structure was determined by molecular replacement using the previously published model of an homologous ferredoxin and refined by molecular dynamics techniques . The model contains the protein and 46 water molecules . Only two amino acid residues, Asp27 and Asp28, are poorly defined in the electron density maps . The molecule has an overall chain fold similar to that of other {4Fe-4S} bacterial ferredoxins of known structure . The two {4Fe-4S} clusters display similar bond distances and angles . In both of them the co-ordination of one iron atom (bound to Cys11 and Cys40) is slightly distorted as compared with that of the other iron atoms . A core of hydrophobic residues and a few water molecules contribute to the stability of the structure . The {4Fe-4S} clusters interact with the polypeptide chain through eight hydrogen bonds each, in addition to the covalent Fe-Scys bonds . The ferredoxin from Clostridium acidurici is the most typical clostridial ferredoxin crystallized so far and the biological implications of the newly determined structure are discussed.

Int J Radiat Biol, 1994 Nov, 66(5), 499 - 503
Influence of nucleic acid base composition on radiation-induced strand breakage in single stranded DNA: a time resolved study; Melvin T et al.; The following study investigates the pathways involved in the induction of single strand breaks (ssb) in various samples of single stranded (ss) DNA (calf thymus, Micrococcus lysodeikticus, Clostridium perfringens) with differing nucleic acid base composition . The time scale for the induction of ssb was determined from changes in the light scattering intensity following pulse irradiation of aqueous solutions containing these ssDNA samples at pH7.8 under either aerated or deaerated conditions . The induction of ssb under these conditions is predominantly by the hydroxyl radical and shows various kinetically distinct components . The immediate ssb (t < 0.02 s) account for approximately 40-60% of the total yield of ssb . The majority of these ssb are suggested to arise from the 'common' initial attack of the hydroxyl radicals at the sugar phosphate backbone for each of the three DNA samples . Furthermore, slower components for ssb formation (t > 0.02 s) were observed and are suggested to occur through base radical mediated H-atom abstraction from the sugar moiety . The half lives for formation of the majority of ssb, formed through this base radical-mediated H-atom abstraction(s), are in the range of 20-43 ms . The yields of these 'base-mediated' ssb vary markedly (under both aerobic and anaerobic conditions) and reflect the base composition of the DNA sample . It is suggested from these studies that the OH-induced base radicals of guanine/cytosine are more effective precursors for strand breakage than those from adenine/thymine in ssDNA.

Biochem J, 1994 Nov 1, 303 ( Pt 3), 743 - 8
Purification and molecular characterization of the NAD(+)-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica; Bruchhaus I et al.; A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica . Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor . Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM respectively in the presence of 0.2 mM NAD+ . The primary structure of EhADH2 as deduced from respective amoebic DNA sequences showed striking similarity to the trifunctional AdhE protein of Escherichia coli and the bifunctional AAD protein of Clostridium acetobutylicum . Alignment with a number of aldehyde dehydrogenases and alcohol dehydrogenases from various species suggested that the two catalytic functions of EhADH2 are located on separate parts of the molecule . By cross-linking experiments and electron-microscopic analysis, native EhADH2 was found to be organized in a homopolymeric fashion consisting of more than 20 associated promoters which form rods about 50-120 nm in length.

J Surg Oncol, 1994 Nov, 57(3), 191 - 5
Clostridium difficile toxin A therapy for HCT 116 human colon cancer in nude mice; Redlich PN et al.; Clostridium difficile toxin A was evaluated for an antitumor effect in vivo on HCT 116 human colon carcinoma cells growing subcutaneously in nude mice . A mean reduction in tumor volume of at least 65%, by measurement in three dimensions, was observed in mice who received two 9- to 13-day courses of daily intraperitoneal injections of toxin A as compared to mice receiving diluent alone . Reversible adverse effects of toxin A were noted in some animals, consisting primarily of liver toxicity and skin rash . HCT 116 cells in toxin A-treated mice grew as flattened tumors with ulcerated centers compared to rounded tumors without ulceration in controls . Histologic examination of tumors from representative mice revealed that two thirds of the tumor in a treated mouse was necrotic compared to only one third in a control, suggesting greater antitumor efficacy of toxin A than estimated by tumor measurements alone.

J Med Microbiol, 1994 Nov, 41(5), 319 - 23
Toxin production by Clostridium difficile in a defined medium with limited amino acids; Yamakawa K et al.; Basal defined medium (BDM) containing vitamins, minerals and seven amino acids--(/L) tryptophan 0.1 g, methionine 0.2 g, valine 0.3 g, isoleucine 0.3 g, proline 0.3 g, leucine 0.4 g and cysteine 0.5 g--which appeared to be essential for good growth of Clostridium difficile was prepared . Addition of glycine 0.2 g/L and threonine 0.4 g/L to BDM produced better growth of strain VPI 10463, and this defined medium was designated minimum amino acid-defined medium (MADM) . Production of toxins A and B by strain VPI 10463 in 6 x MADM containing (/L) tryptophan 0.6 g, methionine 1.2 g, valine 1.8 g, isoleucine 1.8 g, proline 1.8 g, leucine 2.4 g, cysteine 0.5 g, glycine 0.2 g and threonine 0.4 g, was much greater than in MADM . Toxin production by 20 C . difficile strains was examined in two defined media--6 x MADM and complete amino acid-defined medium (CADM) containing 18 amino acids--and one complex medium, modified brain heart infusion medium (m-BHI) . Simultaneous production of toxins A and B by all test strains was demonstrated in m-BHI and the two defined media . It was also shown that 6 x MADM was generally better than CADM and as effective as m-BHI for stimulating toxin production by 13 strains . This defined medium would be useful for studies on the physiology, metabolism and pathogenicity of C . difficile.

J Med Microbiol, 1994 Nov, 41(5), 316 - 8
Comparison of the ToxA test with cytotoxicity assay and culture for the detection of Clostridium difficile-associated diarrhoea disease; Schue V et al.; Stool samples (355 from 350 patients) were examined in a new commercial assay, the ToxA test, for the rapid diagnosis of Clostridium difficile-associated diarrhoea . The results were compared with direct assay of cytotoxin in McCoy cell tissue culture, and detection of toxigenic C . difficile by culture and cytotoxin testing of isolates . Discordant results were resolved by consultation of clinical records . Test sensitivities were 84.6% for the ToxA test, 78.5% for the direct cytotoxicity assay and 95.4% for culture . The specificity was 100% for all the tests . The ToxA test is a rapid and reliable alternative for the detection of toxigenic C . difficile where tissue culture facilities are unavailable.

J Clin Invest, 1994 Nov, 94(5), 1919 - 26
Clostridium difficile toxin A-induced microvascular dysfunction . Role of histamine; Kurose I et al.; Clostridium difficile toxin A (Tx-A) mediates secretion and inflammation in experimental enterocolitis . Intravital video microscopy was used to define the mechanisms that underlie the inflammatory reactions elicited by direct exposure of the microvasculature to Tx-A . Leukocyte adherence and emigration, leukocyte-platelet aggregation, and extravasation of FITC-albumin were monitored in rat mesenteric venules exposed to Tx-A . Significant increases in leukocyte adherence and emigration (LAE) and albumin leakage were noted within 15-30 min of Tx-A exposure . These responses were accompanied by mast cell degranulation and the formation of platelet-leukocyte aggregates . The Tx-A-induced increases in LAE and albumin leakage were significantly attenuated by pretreatment with either monoclonal antibodies (mAbs) directed against the leukocyte adhesion glycoproteins, CD11/CD18, intercellular adhesion molecule-1, and P-selectin (but not E-selectin) or with sialyl Lewis x, a counter-receptor for P-selectin . The mast cell stabilizer, lodoxamide, an H1- (but not an H2-) receptor antagonist, and diamine oxidase (histaminase) were also effective in reducing the LAE and albumin leakage elicited by Tx-A . The platelet-leukocyte aggregation response was blunted by an mAb against P-selectin, sialyl Lewis x, and the H1-receptor antagonist . These observations indicate that Tx-A induces a leukocyte-dependent leakage of albumin from postcapillary venules . Mast cell-derived histamine appears to mediate at least part of the leukocyte-endothelial cell adhesion and platelet-leukocyte aggregation by engaging H1-receptors on endothelial cells and platelets to increase the expression of P-selectin . The adhesion glycoproteins CD11/CD18 and intercellular adhesion molecule-1 also contribute to the inflammatory responses elicited by toxin A.

J Bacteriol, 1994 Nov, 176(22), 6952 - 6
Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes; Matano Y et al.; Cellulosome synthesis by Clostridium cellulovorans was investigated by growing the cells in media containing different carbon sources . Supernatant from cells grown with cellobiose contained no cellulosomes and only the free forms of cellulosomal major subunits CbpA, P100, and P70 and the minor subunits with enzymatic activity . Supernatant from cells grown on pebble-milled cellulose and Avicel contained cellulosomes capable of degrading crystalline cellulose . Supernatants from cells grown with cellobiose, pebble-milled cellulose, and Avicel contained about the same amount of carboxymethyl cellulase activity . Although the supernatant from the medium containing cellobiose did not initially contain active cellulosomes, the addition of crystalline cellulose to the cell-free supernatant fraction converted the free major forms to cellulosomes with the ability to degrade crystalline cellulose . The binding of P100 and P70 to crystalline cellulose was dependent on their attachment to the endoglucanase-binding domains of CbpA . These data strongly indicate that crystalline cellulose promotes cellulosome assembly.

J Bacteriol, 1994 Nov, 176(21), 6590 - 8
Cloning, DNA sequencing, and characterization of a nifD-homologous gene from the archaeon Methanosarcina barkeri 227 which resembles nifD1 from the eubacterium Clostridium pasteurianum; Chien YT et al.; L . Sibold, M . Henriquet, O . Possot, and J.-P . Aubert (Res . Microbiol . 142:5-12, 1991) cloned and sequenced two nifH-homologous open reading frames (ORFs) from Methanosarcina barkeri 227 . Phylogenetic analysis of the deduced amino acid sequences of the nifH ORFs from M . barkeri showed that nifH1 clusters with nifH genes from alternative nitrogenases, while nifH2 clusters with nifH1 from the gram-positive eubacterium Clostridium pasteurianum . The N-terminal sequence of the purified nitrogenase component 2 (the nifH gene product) from M . barkeri was identical with that predicted for nifH2, and dot blot analysis of RNA transcripts indicated that nifH2 (and nifDK2) was expressed in M . barkeri when grown diazotrophically in Mo-containing medium . To obtain nifD2 from M . barkeri, a 4.7-kbp BamHI fragment of M . barkeri DNA was cloned which contained at least five ORFs, including nifH2, ORF105, and ORF125 (previously described by Sibold et al.), as well as nifD2 and part of nifK2 . ORFnifD2 is 1,596 bp long and encodes 532 amino acid residues, while the nifK2 fragment is 135 bp long . The deduced amino acid sequences for nifD2 and the nifK2 fragment from M . barkeri cluster most closely with the corresponding nifDK1 gene products from C . pasteurianum . The predicted M . barkeri nifD2 product contains a 50-amino acid insert near the C terminus which has previously been found only in the clostridial nifD1 product . Previous biochemical and sequencing evidence indicates that the C . pasteurianum nitrogenase is the most divergent of known eubacterial Mo-nitrogenases, most likely representing a distinct nif gene family, which now also contains M . barkeri as a member . The similarity between the methanogen and clostridial nif sequences is especially intriguing in light of the recent findings of sequence similarities between gene products from archaea and from low-G+C gram-positive eubacteria for glutamate dehydrogenase, glutamine synthetase I, and heat shock protein 70 . It is not clear whether this similarity is due to horizontal gene transfer or to the resemblance of the M . barkeri and C . pasteurianum nitrogenase sequences to an ancestral nitrogenase.

J Bacteriol, 1994 Nov, 176(21), 6572 - 82
Sporulation and primary sigma factor homologous genes in Clostridium acetobutylicum; Sauer U et al.; Using a PCR-based approach, we have cloned various sigma factor homologous genes from Clostridium acetobutylicum DSM 792 . The nucleotide sequence of the dnaE-sigA operon has been determined and predicts two genes encoding 69- and 43-kDa proteins . The deduced DnaE amino acid sequence has approximately 30% amino acid identity with protein sequences of other primases . The putative sigA gene product shows high homology to primary sigma factors of various bacteria, most significantly to Bacillus subtilis and Staphylococcus aureus . Northern (RNA) blot analysis revealed that both genes from an operon, which is clearly expressed under conditions that allow for cell division . A promoter sequence with significant homology to the sigma H-dependent Bacillus promoters preceded the determined transcriptional start point, 182 bp upstream of the GUG start codon of dnaE . The homologous genes to Bacillus spp . sporulation sigma factors G, E, and K have been cloned and sequenced . Indirect evidence for the existence of sigma F was obtained by identification of a DNA sequence homologous to the respective Bacillus consensus promoter . Southern hybridization analysis indicated the presence of sigma D and sigma H homologous genes in C . acetobutylicum . A new gene group conserved within the eubacteria, but with yet unspecified functions, is described . The data presented here provide strong evidence that at least some of the complex regulation features of sporulation in B . subtilis are conserved in C . acetobutylicum and possibly Clostridium spp.

J Bacteriol, 1994 Nov, 176(21), 6433 - 8
Regulation of Clostridium acetobutylicum metabolism as revealed by mixed-substrate steady-state continuous cultures: role of NADH/NAD ratio and ATP pool; Girbal L et al.; Glycerol-glucose-fed (molar ratio of 2) chemostat cultures of Clostridium acetobutylicum were glucose limited but glycerol sufficient and had a high intracellular NADH/NAD ratio (I . Vasconcelos, L . Girbal, and P . Soucaille, J . Bacteriol . 176:1443-1450, 1994) . We report here that the glyceraldehyde-3-phosphate dehydrogenase, one of the key enzymes of the glycolytic pathway, is inhibited by high NADH/NAD ratios . Partial substitution of glucose by pyruvate while maintaining glycerol concentration at a constant level allowed a higher consumption of glycerol in steady-state continuous cultures . However, glycerol-sufficient cultures had a constant flux through the glyceraldehyde-3-phosphate dehydrogenase and a constant NADH/NAD ratio . A high substitution of glucose by pyruvate {P/(G+P) value of 0.67 g/g} provided a carbon-limited culture with butanol and butyrate as the major end products . In this alcohologenic culture, the induction of the NADH-dependent butyraldehyde and the ferredoxin-NAD(P) reductases and the higher expression of alcohol dehydrogenases were related to a high NADH/NAD ratio and a low intracellular ATP concentration . In three different steady-state cultures, the in vitro phosphotransbutyrylase and butyrate-kinase activities decreased with the intracellular ATP concentration, suggesting a transcriptional regulation of these two genes, which are arranged in an operon (K . A . Walter, R . V . Nair, R . V . Carry, G . N . Bennett, and E . T . Papoutsakis, Gene 134:107-111, 1993).

Clin Orthop, 1994 Nov, (308), 178 - 82
Clostridium septicum gas gangrene of the gluteus maximus and an ascending colon malignant tumor . A case report; Fernandez RJ et al.; Clostridial myonecrosis is a complication associated with contaminated traumatized wounds . Presented is the case of an elderly female with pain in her right hip, radiographic evidence of gas in the soft tissues, and no history of trauma . Evaluation of this patient revealed Clostridial myonecrosis . Culture results identified the organism as Clostridium septicum . Due to the association between Clostridium septicum and occult malignancies, colonoscopic evaluation was performed . A colonic lesion was discovered, biopsied, resected, and staged, using the Modified Duke Classification, as a well differentiated adenocarcinoma, C2 lesion . Myonecrosis and its associated malignancies carry high morbidity and mortality, but early diagnosis, appropriate treatment, and awareness of the association with occult malignancies may avert unnecessary mortality.

Infect Immun, 1994 Nov, 62(11), 5048 - 54
Preparation, characterization, and immunological properties in mice of Escherichia coli O157 O-specific polysaccharide-protein conjugate vaccines; Konadu E et al.; Escherichia coli O157 causes severe enteritis and the extraintestinal complication of hemolytic-uremic syndrome, with their highest incidence occurring in children . We postulated that serum immunoglobulin G (IgG) antibodies to the O-specific polysaccharide of lipopolysaccharide (LPS) may confer protective immunity to enteric pathogens by inducing bactericidal reactions against the ingested organisms in the jejunum (J . B . Robbins, C . Chu, and R . Schneerson, Clin . Infect . Dis . 15:346-361, 1992; S . C . Szu, R . Gupta, and J . B . Robbins, p . 381-394, in I . K . Wachsmuth, P . A . Blake, and O . Olsvik, ed., Vibrio cholerae, 1994) . Because polysaccharide-protein conjugates induce serum IgG antibodies in infants, we bound the O-specific polysaccharide of E . coli O157 to proteins . E . coli O157 LPS, treated with acetic acid or hydrazine, was derivatized with adipic acid dihydrazide and bound to proteins by carbodiimide-mediated condensation . Conjugates of these adipic hydrazide derivative were prepared with bovine serum albumin, formalin-treated exotoxin C of Clostridium welchii (Pig Bel toxoid), or Pseudomonas aeruginosa recombinant exoprotein A . The conjugates had low levels of endotoxin and elicited serum antibodies with bactericidal activity to the O157 LPS . The largest increase in LPS antibodies was of the IgG class . Clinical evaluation of E . coli O157-toxoid conjugates is planned.

Infect Immun, 1994 Nov, 62(11), 5032 - 9
Role of alpha-toxin in Clostridium perfringens infection determined by using recombinants of C . perfringens and Bacillus subtilis; Ninomiya M et al.; Clostridium perfringens type A strains which differed in alpha-toxin (phospholipase C {PLC}) productivity were inoculated intraperitoneally or intravenously into mice, and then their 50% mouse lethal doses (LD50) were determined . Strain NCTC 8237 produced ninefold higher PLC activity than strain 13 . The mean LD50 for the former was 1 log unit lower than that for the latter . Two isogenic strains were constructed from strain 13: strain 13(pJIR418 alpha) (pJIR418 alpha contains the plc gene), which produced ninefold higher PLC activity than strain 13; and strain 13 PLC-, which showed no PLC productivity at all because of transformation-mediated gene disruption . The mean LD50 for strain 13(pJIR418 alpha) was 1 log unit lower than those for strain 13 PLC- and strain 13 . These results indicate that PLC functions as a virulence-determining factor when it is produced in a sufficient amount . Such a difference in LD50 was also observed between Bacillus subtilis with and without the cloned plc gene . Inoculation of B . subtilis PLC+ intravenously into mice caused marked thrombocytopenia and leukocytosis . Mice inoculated with B . subtilis at 2 LD50 died because of circulatory collapse . Histological examination revealed that intravascular coagulation and vascular congestion occurred most prominently in the lungs . These results suggest that PLC plays a key role in the systemic intoxication of clostridial myonecrosis, probably by affecting the functions of platelets and phagocytes.

Gastroenterology, 1994 Nov, 107(5), 1398 - 407
Etiology and outcome of diarrhea after marrow transplantation: a prospective study; Cox GJ et al.; BACKGROUND/AIMS: Acute diarrhea after marrow transplant is usually ascribed to acute graft-vs.-host disease (GVHD) or infection, with a reported 40%-50% incidence of infection . The aim of this study was to determine the incidence of acute diarrhea after transplantation, its causes, and its outcome . METHODS: Two hundred ninety-six patients were followed up; patients with diarrhea were studied using standard evaluation of stool plus immunoelectron microscopy; assays for astrovirus, picobirnavirus, and Norwalk virus; and gene-probe methods for toxin-producing Escherichia coli . In 38 patients with diarrhea, intestinal biopsy specimens and duodenal fluid were also analyzed . RESULTS: One hundred fifty acute diarrheal episodes developed in 126 patients (an incidence of 43%) . Intestinal infection was found in 20 of 150 episodes: viruses (astrovirus, adenovirus, cytomegalovirus, and rotavirus) in 12 patients, nosocomially acquired bacteria (Clostridium difficile and Aeromonas) in 7 patients, and mixed infection in 1 patient . Acute GVHD was responsible for 72 of 150 episodes (48%) . Clinical signs and symptoms of infection and GVHD were similar . In 58 of 150 episodes (39%), no clear etiology could be found for self-limited diarrhea . CONCLUSIONS: Intestinal infection accounted for 13% and acute GVHD for 48% of diarrheal episodes . The most common infecting organisms were astrovirus, C . difficile, and adenovirus . Most cases of diarrhea after marrow transplant are not caused by infection.

Clin Investig, 1994 Nov, 72(11), 922 - 4
Intravenous teicoplanin does not prevent Clostridium difficile associated diarrhea; Wenisch C et al.; A 59-year-old man with the diagnosis of endocarditis of the mitral valve due to Streptococcus mitis was treated with penicillin G, gentamicin, and later with clindamycin as inpatient for 3 weeks . Thereafter outpatient therapy with parenteral teicoplanin 3 x per week was initiated . After 17 days of teicoplanin treatment he developed severe diarrhea, and stool samples were positive for Clostridium difficile toxin . In addition to the ongoing parenteral therapy with teicoplanin, oral teicoplanin was administered . On the third day of this regimen the diarrhea and other disabling symptoms subsided, and test results for C . difficile toxin became negative . Oral teicoplanin was continued for 10 days and cleared C . difficile effectively after treatment as assessed by consecutive stool cultures (until 60 days thereafter) . The parenteral administration of teicoplanin could not prevent the onset of C . difficile associated diarrhea in this patient, who previously had been treated with clindamycin . Thus, the administration of parenteral teicoplanin does not seem to be a treatment option for C . difficile associated diarrhea in patients in which oral therapy is not possible.

Diagn Microbiol Infect Dis, 1994 Nov, 20(3), 135 - 42
Major methodology-dependent discordant susceptibility results for Bacteroides fragilis group isolates but not other anaerobes; Aldridge KE et al.; Two standardized susceptibility test methods, a broth microdilution (BMD) and agar dilution (AD) method were performed on a total of 441 clinical isolates of anaerobes with ceftizoxime, cefotaxime, ceftriaxone, cefoxitin, piperacillin, and metronidazole . Against the 339 strains of the Bacteroides fragilis group BMD minimum inhibitory concentration (MIC) values were lower than those from AD testing for all the beta-lactams . Overall for the B . fragilis group and the beta-lactams, the mode MIC values were two- to 64-fold lower, and the MIC50 values two- to eightfold lower . Resistance rates were 11%-28% higher overall with AD results and were higher especially for non-B . fragilis species . For non-Bacteroides anaerobes no major discrepancies were noted for Prevotella species, Peptostreptococcus species, and Viellonella parvula . With Clostridium species and Eubacterium species, some differences were noted with ceftizoxime because of differences in cut-off points . These data illustrate the magnitude of differences in results produced by the two methods using essentially the same test medium for the B . fragilis group . Fortunately, such major discordant results were not widely noted with other groups of anaerobes.

Antimicrob Agents Chemother, 1994 Nov, 38(11), 2599 - 604
Activity of WY-49605 compared with those of amoxicillin, amoxicillin-clavulanate, imipenem, ciprofloxacin, cefaclor, cefpodoxime, cefuroxime, clindamycin, and metronidazole against 384 anaerobic bacteria; Spangler SK et al.; The National Committee for Clinical Laboratory Standards agar dilution method was used to compare the in vitro activity of WY-49605 (also called SUN/SY 5555 and ALP-201), a new broad-spectrum oral penem, to those of amoxicillin, amoxicillin-clavulanate, imipenem, ciprofloxacin, cefaclor, cefpodoxime, cefuroxime, clindamycin, and metronidazole against 384 clinically isolated anaerobes . These anaerobic organisms included 90 strains from the Bacteroides fragilis group, 87 Prevotella and Porphyromonas strains, non-B . fragilis group Bacteroides strains, 56 fusobacteria, 55 peptostreptococci, 49 gram-positive non-spore-forming rods, and 47 clostridia . Overall, WY-49605 had an MIC range of 0.015 to 8.0 micrograms/ml, an MIC at which 50% of the isolates are inhibited (MIC50) of 0.25 microgram/ml, and an MIC at which 90% of the isolates are inhibited (MIC90) of 2.0 micrograms/ml . Good activity against all anaerobe groups was observed, except for Clostridium difficile and lactobacilli (MIC50s of 4.0 and 2.0 micrograms/ml, respectively, and MIC90s of 8.0 and 2.0 micrograms/ml, respectively) . Imipenem had an MIC50 of 0.03 microgram/ml and an MIC90 of 0.25 microgram/ml . Ciprofloxacin was much less active (MIC50 of 2.0 micrograms/ml and MIC90 of 16.0 micrograms/ml) . By comparison, all oral beta-lactams were less active than WY-49605, with susceptibilities as follows: amoxicillin MIC50 of 8.0 micrograms/ml and MIC90 of > 256.0 micrograms/ml), amoxicillin-clavulanate MIC50 of 1.0 microgram/ml and MIC90 of 8.0 micrograms/ml, cefaclor MIC50 of 8.0 micrograms/ml and MIC90 of > 32.0 micrograms/ml, cefpodoxime MIC50 of 4.0 micrograms/ml and MIC90 of > 32.0 micrograms/ml, and cefuroxime MIC50 of 4.0 micrograms/ml and MIC90 of > 32.0 micrograms/ml . Clindamycin was active against all groups except some members of the B . fragilis group, Fusobacterium varium, and some clostridia ( overall MIC50 of 0.5 micrograms/ml and overall MIC90 of 8.0 micrograms/ml) . Metronidazole was active (MIC of less than or equal to 4.0 micrograms/ml) against all gram-negative anaerobic rods, but most gram-positive non-spore-forming rods, some peptostreptococci, and some clostridia were less susceptible . To date, WY-49605 is the most active oral beta-lactam against anaerobes: these results suggest clinical evaluation for clinical indications suitable for oral therapy.

Mol Biol Cell, 1994 Nov, 5(11), 1199 - 213
Effect of disruption of actin filaments by Clostridium botulinum C2 toxin on insulin secretion in HIT-T15 cells and pancreatic islets; Li G et al.; To examine their role in insulin secretion, actin filaments (AFs) were disrupted by Clostridium botulinum C2 toxin that ADP-ribosylates G-actin . Ribosylation also prevents polymerization of G-actin to F-actin and inhibits AF assembly by capping the fast-growing end of F-actin . Pretreatment of HIT-T15 cells with the toxin inhibited stimulated insulin secretion in a time- and dose-dependent manner . The toxin did not affect cellular insulin content or nonstimulated secretion . In static incubation, toxin treatment caused 45-50% inhibition of secretion induced by nutrients alone (10 mM glucose + 5 mM glutamine + 5 mM leucine) or combined with bombesin (phospholipase C-activator) and 20% reduction of that potentiated by forskolin (stimulator of adenylyl cyclase) . In perifusion, the stimulated secretion during the first phase was marginally diminished, whereas the second phase was inhibited by approximately 80% . Pretreatment of HIT cells with wartmannin, a myosin light chain kinase inhibitor, caused a similar pattern of inhibition of the biphasic insulin release as C2 toxin . Nutrient metabolism and bombesin-evoked rise in cytosolic free Ca2+ were not affected by C2 toxin, indicating that nutrient recognition and the coupling between receptor activation and second messenger generation was not changed . In the toxin-treated cells, the AF web beneath the plasma membrane and the diffuse cytoplasmic F-actin fibers disappeared, as shown both by staining with an antibody against G- and F-actin and by staining F-actin with fluorescent phallacidin . C2 toxin dose-dependently reduced cellular F-actin content . Stimulation of insulin secretion was not associated with changes in F-actin content and organization . Treatment of cells with cytochalasin E and B, which shorten AFs, inhibited the stimulated insulin release by 30-50% although differing in their effects on F-actin content . In contrast to HIT-T15 cells, insulin secretion was potentiated in isolated rat islets after disruption of microfilaments with C2 toxin, most notably during the first phase . This effect was, however, diminished, and the second phase became slightly inhibited when the islets were degranulated . These results indicate an important role for AFs in insulin secretion . In the poorly granulated HIT-T15 cells actin-myosin interactions may participate in the recruitment of secretory granules to the releasable pool . In native islet beta-cells the predominant function of AFs appears to be the limitation of the access of granules to the plasma membrane.

J Hosp Infect, 1994 Nov, 28(3), 231 - 4
Typing of Clostridium difficile by polymerase chain reaction with an arbitrary primer; Wilks M et al.; We assessed the use of the polymerase chain reactions (PCR) with an arbitrary primer (AP-PCR) to investigate a major hospital outbreak of diarrhoea due to Clostridium difficile . A single pattern consisting of three bands of 240, 580 and 1100 bp was obtained from all isolates studied . AP-PCR is a simple, rapid technique which should find increased application in the rapid investigation of suspected outbreaks of many different bacterial species, particularly new pathogens or those for which no accepted typing scheme exists.

Infect Control Hosp Epidemiol, 1994 Nov, 15(11), 697 - 702
Effectiveness of liquid soap vs . chlorhexidine gluconate for the removal of Clostridium difficile from bare hands and gloved hands; Bettin K et al.; OBJECTIVE: To compare liquid soap versus 4% chlorhexidine gluconate in 4% alcohol for the decontamination of bare or gloved hands inoculated with an epidemic strain of Clostridium difficile . DESIGN: C difficile (6.7 log10 colony-forming units {CFU}, 47% spores), was seeded onto bare or latex gloved hands of ten volunteers and allowed to dry . Half the volunteers initially washed with soap and half with chlorhexidine, followed by the other agent 1 week later . Cultures were done with Rodac plates at three sites on the hand: finger/thumbtips, the palmar surfaces of the fingers, and the palm . Statistical comparison was by paired Student's t test . RESULTS: On bare hands, soap and chlorhexidine did not differ in residual bacterial counts on the finger/thumbtips (log10 CFU, 2.0 and 2.1, P = NS) and fingers (log10 CFU, 2.4 and 2.5, P = NS) . Counts were too high on bare palms to quantitate . On gloved hands, soap was more effective than chlorhexidine on fingers (log10 CFU 1.3 and 1.7, P < .01) and palms (log10 CFU 1.5 and 2.0, P < .01), but not finger/thumbtips (log10 CFU 1.6 with each, P = NS) . Residual C difficile counts were lower on gloved hands than bare hands (P < 0.01 to < 0.0001) . CONCLUSIONS: The two agents did not differ significantly in residual counts of C difficile on bare hands, but on gloved hands residual counts were lower following soap wash than following chlorhexidine wash . These observations support the use of either soap or chlorhexidine as a handwash for removal of C difficile, but efficacy in the prevention of C difficile transmission must be determined by prospective clinical trials.

J Clin Microbiol, 1994 Nov, 32(11), 2867 - 8
Clostridium bifermentans bacteremia with metastatic osteomyelitis; Scanlan DR et al.; Osteomyelitis caused solely by an anaerobic organism is uncommon . We report a case of recurrent Clostridium bifermentans bacteremia resulting in metastatic osteomyelitis involving the sacrum, spine, and ribs . The emergence of resistance of this organism to imipenem and metronidazole is noteworthy because of the usual susceptibility of clostridial species to these antibiotics.

Zhonghua Yi Xue Za Zhi (Taipei), 1994 Nov, 54(5), 291 - 9
The elevation of plasma DNA in patients with systemic lupus erythematosus is attributable to increased DNA release and defective DNA binding of mononuclear cells; Tsai CY et al.; BACKGROUND . Although immunoprecipitable DNA has been found in a subgroup of patients with systemic lupus erythematosus (SLE) exhibiting systemic vasculitis and/or central nervous system involvement, the mechanism for elevated plasma DNA in these patients is poorly understood . METHODS . The plasma DNA concentrations and reactivity of serum and lymphocytes to six species of double-stranded DNA from calf thymus, human placenta, Escherichia coli, Micrococcus lysodeikticus, Clostridium perfringens and poly (dG.dC) . poly (dG.dC) were measured in twenty-seven patients with active SLE . To understand the mechanism of increased plasma DNA in SLE, the DNA binding and release of the mononuclear cells were examined . RESULTS . Compared with the controls, the incidence of the presence of plasma DNA was markedly increased in SLE (59.3% in SLE vs . 7.4% in controls) as detected by counterimmunoelectrophoresis . Except for DNA from Clostridium perfringens, the reactivity of lupus sera to various DNA samples was significantly higher than that of the controls . The reactivity of lymphocytes to 6 species of DNA (as defined by 3H-thymidine incorporation of the cells) was also higher in SLE patients . In DNA binding and releasing experiments, patients with SLE were found to have decreased 3H-DNA binding activity (0.169 +/- 0.018 micrograms/2 x 10(6) cells in SLE vs . 0.283 +/- 0.02 micrograms/2 x 10(6) cells in controls, p = 0.001) but to have increased spontaneous release of DNA (1,465 +/- 412 cpm in SLE vs . 630 +/- 179 cpm in controls, p = 0.0173) in mononuclear cells . CONCLUSIONS . The results suggest that some subsets of lymphocytes can be sensitized by different DNA samples in vivo to increase endogenous DNA release from mononuclear cells, which in addition to decreased DNA clearance as has been previously reported, may be responsible for the elevation of plasma DNA in patients with SLE.

Appl Microbiol Biotechnol, 1994 Nov, 42(2-3), 346 - 52
Cloning and expression of the Clostridium thermocellum celS gene in Escherichia coli; Wang WK et al.; Clostridium thermocellum ATCC 27405 produces an extremely complicated multi-component cellulase aggregate (cellulosome) highly active on crystalline cellulose . From the cellulosome, two subunits, CelS (or Ss; M(r) = 82,000) and CelL (or SL, CipA; M(r) = 250,000), have been identified as essential for crystalline cellulose degradation {Wu et al . (1988) Biochemistry 27:1703} . We have determined the DNA sequence of the celS gene from four cloned DNA fragments encompassing this gene {Wang et al . (1993) J Bacteriol 175:1293} . To express the entire celS gene in Escherichia coli, the celS structural gene was amplified by the polymerase chain reaction (PCR) employing the PCR primers corresponding to sequences flanking the desired gene . This PCR product (2.1 x 10(3) bases; 2.1 kb) was cloned into an E . coli expression vector pRSET B . Subsequent expression of the cloned gene resulted in a fusion protein (rCelS; M(r) = 86,000) as inclusion bodies . The rCelS protein was recognized specifically by an anti-CelS antiserum in a Western blot analysis . The inclusion bodies were purified and solubilized in 5 M urea . The refolded rCelS produced very little reducing sugar from carboxymethylcellulose . However, it showed a higher activity on the crystalline cellulose (Avicel) and an even higher activity on phosphoric-acid-swollen Avicel . These results indicate that the CelS is an exoglucanase.

Development, 1994 Nov, 120(11), 3313 - 23
Roles of heterotrimeric and monomeric G proteins in sperm-induced activation of mouse eggs; Moore GD et al.; Results of several lines of experimentation suggest that sperm-induced egg activation has several features in common with G protein-coupled receptor signal transduction mechanisms . We report that microinjection of GDP beta S into metaphase II-arrested mouse eggs blocks sperm-induced egg activation . Since GDP beta S inactivates both heterotrimeric and monomeric classes of G proteins, the involvement of members of each of these families in sperm-induced egg activation was evaluated . Neither pertussis toxin treatment of eggs nor microinjection of eggs with inhibitory antibodies toward G alpha q blocked sperm-induced egg activation . Nevertheless, microinjection of phosducin, a protein that binds tightly to free G protein beta gamma subunits, specifically inhibited second polar body emission, the fertilization evoked decrease of H1 kinase activity and pronucleus formation . Microinjection of phosducin, however, did not inhibit the fertilization-induced modifications of the zona pellucida and microinjection of beta gamma t did not result in egg activation in the absence of sperm . Inactivation of the monomeric Rho family of G proteins with C3 transferase from Clostridium botulinum inhibited emission of the second polar body and cleavage to the 2-cell stage, but did not affect the modifications of the zona pellucida or pronucleus formation . Microinjection of Rasval12, which is a constitutively active form of Ras, did not result in egg activation in the absence of sperm . Moreover, microinjection of either an anti-Ras neutralizing antibody (Y13-259) or a dominant negative form of Ras (RasT) did not affect events of sperm-induced egg activation . In contrast, microinjection of RasT inhibited embryo cleavage to the 2-cell stage . These results suggest that both heterotrimeric and monomeric G proteins are involved in various aspects of sperm-induced egg activation.

FEBS Lett, 1994 Oct 31, 354(1), 123 - 7
Characterisation of a complementary DNA encoding a novel plant enzyme with sucrolytic activity; Machray GC et al.; The cloning of a 1332 bp cDNA from a potato (Solanum tuberosum L.) cv . Cara leaf cDNA expression library, using an antibody raised against a purified tuber protein preparation with sucrolytic activity, is described . The corresponding gene in potato is of low copy number, is expressed in a variety of tissues, and encodes a protein which includes several domains with similarity to database sequences, including ferredoxin from Clostridium pasteurianum . Expression of the cDNA in E . coli yields a fusion protein with sucrolytic activity.

J Biol Chem, 1994 Oct 21, 269(42), 25951 - 4
Activation of rat liver phospholipase D by the small GTP-binding protein RhoA; Malcolm KC et al.; Stimulation of phospholipase D by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) in rat liver plasma membranes indicates the involvement of GTP-binding proteins . We used RhoGDI, an inhibitor of GDP dissociation from small GTP-binding proteins of the Rho family, to determine the involvement of these proteins . Incubation, and subsequent washing, of plasma membranes with RhoGDI dose-dependently diminished GTP gamma S-stimulated phospholipase D activity, as determined by accumulation of phosphatidylethanol in the presence of ethanol . Incubation with RhoGDI also caused a rapid and dose-dependent appearance of RhoA in the wash, which was associated with the inhibition of phospholipase D . RhoGDI also rapidly extracted Cdc42 from membranes, but Rac1 was not extracted . Full reconstitution of GTP gamma S-stimulated phospholipase D in RhoGDI-washed membranes was achieved with recombinant RhoA . There was partial reconstitution with Rac1 and no enhancement with Cdc42 or ADP-ribosylation factor . The response to RhoA was dose-dependent (EC50 = 0.5 microM) . ADP-ribosylation of RhoA by Clostridium botulinum C3 exoenzyme did not affect its ability to recover GTP gamma S-stimulated phospholipase D activity in RhoGDI-washed membranes . These findings support a role for GTP-binding proteins of the Rho family in the activation of membrane-associated phospholipase D and implicate RhoA as the major protein involved.

FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 213 - 8
Transformation of tetrachloroethylene to trichloroethylene by homoacetogenic bacteria; Terzenbach DP et al.; Eight homoacetogenic strains of the genera Acetobacterium, Clostridium and Sporomusa were tested for their ability to dechlorinate tetrachloroethylene (perchloroethene, PCE) . Of the organisms tested only Sporomusa ovata was able to reductively dechlorinate PCE with methanol as an electron donor . Resting cells of S . ovata reductively dechlorinated PCE at a rate of 9.8 nmol h-1 (mg protein)-1 to trichloroethylene (TCE) as the sole product . The dechlorination activity depended on concomitant acetogenesis from methanol and CO2 . Cell-free extracts of S . ovata, Clostridium formicoaceticum, Acetobacterium woodii, and the methanogenic bacterium Methanolobus tindarius transformed PCE to TCE with Ti(III) or carbon monoxide as electron donors . Corrinoids were shown in S . ovata to be involved in the dechlorination reaction of PCE to TCE as evident from the reversible inhibition with propyl iodide . Rates of dechlorination followed a pseudo-first-order kinetic.

Gene, 1994 Oct 11, 148(1), 167 - 8
Phage display of a human antibody against Clostridium tetani toxin; Esposito G et al.; 9F12, a human antibody capable of neutralising tetanus toxin in an animal model, has been expressed as a Fab fragment in a phagemid system . The nucleotide sequence for the variable domain of both chains has been determined and compared to germline sequences.

J Appl Bacteriol, 1994 Oct, 77(4), 382 - 7
Influence of culture conditions on growth and protective antigenicity of Clostridium chauvoei; Cortinas TI et al.; The effect of culture conditions on growth and immunogenicity of Clostridium chauvoei were examined . The pH control and partial feeding of the carbon source at high concentrations were beneficial for growth . The biomass yield was significatively improved, however the butanol concentration reached toxic levels hampering further growth . For each experimental condition the immunogenicity of cells was tested . No differences were found with cells obtained at different temperatures, but it decreased significatively with the partial supply of the carbon source and pH control.

Int J Syst Bacteriol, 1994 Oct, 44(4), 812 - 26
The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations; Collins MD et al.; The 16S rRNA gene sequences of 34 named and unnamed clostridial strains were determined by PCR direct sequencing and were compared with more than 80 previously determined clostridial sequences and the previously published sequences of representative species of other low- G + C-content gram-positive genera, thereby providing an almost complete picture of the genealogical interrelationships of the clostridia . The results of our phylogenetic analysis corroborate and extend previous findings in showing that the genus Clostridium is extremely heterogeneous, with many species phylogenetically intermixed with other spore-forming and non-spore-forming genera . The genus Clostridium is clearly in need of major revision, and the rRNA structures defined in this and previous studies may provide a sound basis for future taxonomic restructuring . The problems and different possibilities for restructuring are discussed in light of the phenotypic and phylogenetic data, and a possible hierarchical structure for the clostridia and their close relatives is presented . On the basis of phenotypic criteria and the results of phylogenetic analyses the following five new genera and 11 new combinations are proposed: Caloramator gen . nov., with Caloramator fervidus comb . nov.; Filifactor gen . nov., with Filifactor villosus comb . nov.; Moorella gen . nov., with Moorella thermoacetica comb . nov . and Moorella thermoautotrophica comb . nov.; Oxobacter gen . nov., with Oxobacter pfennigii comb . nov.; Oxalophagus gen . nov., with Oxalophagus oxalicus comb . nov.; Eubacterium barkeri comb . nov.; Paenibacillus durum comb . nov.; Thermoanaerobacter kivui comb . nov.; Thermoanaerobacter thermocopriae comb . nov.; and Thermoanerobacterium thermosaccharolyticum comb . nov.

Am J Med Sci, 1994 Oct, 308(4), 247 - 50
Case report: transverse colon volvulus in a patient with Clostridium difficile pseudomembranous colitis; Yaseen ZH et al.; Sixty-eight cases of transverse colon volvulus have been reported in the literature . The authors report the first case of transverse colon volvulus in association with Clostridium difficile pseudomembranous colitis, with a review of the available literature . It is possible that the acute inflammation of the colonic mucosa, which occurred from the pseudomembranous colitis in this patient, contributed to the development of volvulus . Further studies are needed to explore the role of mucosal inflammation in the occurrence of volvulus.

Surgery, 1994 Oct, 116(4), 768 - 74; discussion 774-5
Surgical patients with pseudomembranous colitis: factors affecting prognosis; Prendergast TM et al.; BACKGROUND . Although several studies have identified the factors that contribute to the development of antibiotic-associated colitis (AAC), little data are available in regard to those factors that may affect the prognosis of patients with the disease . Therefore we conducted a retrospective analysis of 201 surgical patients with AAC to identify risk factors predictive of increased morbidity or mortality . METHODS . We conducted a review of the charts of 201 surgical patients hospitalized between Jan . 1, 1991, and June 30, 1993, in whom AAC developed . AAC was defined as the presence of diarrhea associated with a positive latex agglutination or toxin assay for Clostridium difficile . An additional 52 procedure-matched charts of patients admitted to a surgical service during the same period were also reviewed and constituted the control group . We analyzed the contribution of 21 variables to prognosis in both groups . RESULTS . There was no difference between the two groups in the preoperative length of stay, the number of antibiotics per patient and the number of antibiotic-days, number of patients receiving preoperative bowel preparation, total parenteral nutrition, and overall mortality rate . Patients in the control group were at increased risk of death if they had a history of preexisting renal dysfunction, prolonged preoperative hospital stay, and a poor nutritional status . AAC developed 10.0 +/- 13.8 days after operation in the study group . All patients were receiving multiple antibiotics at the time of diagnosis (3.6 +/- 7.5 antibiotic), with a mean of 14.3 +/- 20.7 antibiotic-days . The overall mortality rate in the study group was 8% . In five patients (2%) toxic megacolon developed; four deaths occurred among these patients (80% mortality rate) . A 25% mortality rate was directly attributable to complications of AAC . Six variables were identified as predictive of increased mortality rate: steroids, laxatives, length of preoperative stay, postoperative interval before the onset of symptoms, use of total parenteral nutrition, and abdominal distention . CONCLUSIONS . AAC carries a significant mortality rate in surgical patients; therefore the diagnosis of AAC should be aggressively pursued and patients with the disease should be promptly treated . Patients receiving steroids, total parenteral nutrition, and multiple antibiotics in whom signs and symptoms of AAC develop late in their postoperative course, and patients with abdominal distention and marked leukocytosis, are at increased risk of dying of AAC and should be aggressively treated.

J Bacteriol, 1994 Oct, 176(19), 6146 - 7
Transmembrane pH of Clostridium acetobutylicum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased; Girbal L et al.; Evidence is reported here that alkalinization of Clostridium acetobutylicum cytoplasm involves hydrogenase activity . A decrease of in vivo hydrogenase activity is accompanied by intracellular accumulation of protons leading to a negative (interior acidic) pH gradient . However, the organism is able to maintain a constant proton motive force by interconverting chemical and electrical potentials.

J Bacteriol, 1994 Oct, 176(19), 6127 - 30
The reductive acetyl coenzyme A pathway: sequence and heterologous expression of active methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase from Clostridium thermoaceticum; Roberts DL et al.; The methyltransferase (MeTr) from Clostridium thermoaceticum transfers the N5-methyl group of (6S)-methyltetrahydrofolate to the cobalt center of a corrinoid/iron-sulfur protein in the acetyl coenzyme A pathway . MeTr was purified to homogeneity and shown to lack metals . The acsE gene encoding MeTr was sequenced and actively expressed in Escherichia coli at a level of 9% of cell protein . Regions in the sequence of MeTr and the E . coli cobalamin-dependent methionine synthase were found to share significant homology, suggesting that they may represent tetrahydrofolate-binding domains.

Infect Immun, 1994 Oct, 62(10), 4347 - 55
Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus; Karjalainen T et al.; Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component . The experiments described here show that C . difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock . The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion . To clone the adhesin gene, polyclonal antibodies against C . difficile heated at 60 degrees C were used to screen a genomic library of C . difficile constructed in lambda ZapII . Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose . In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C . difficile as a possible adhesin . The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C . difficile antibodies, by a surface extract of C . difficile, and by mucus isolated from axenic mice . Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice . Preliminary studies on the receptor moieties implicated in C . difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin . In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment.

Epidemiol Infect, 1994 Oct, 113(2), 335 - 43
The production of Clostridium botulinum type A, B and D toxin in rotting carcasses; Ortiz NE et al.; Carrion is a major source of botulinal toxin for animals . A type D strain of Clostridium botulinum differed from type A and B strains in producing (1) much higher concentrations of toxin in putrefying mouse carcasses at several different temperatures over a period of 35 days, (2) toxicity that sometimes persisted in mouse carcasses for at least a year, and (3) mouse carcasses with exceptionally high oral toxicity . Fish carcasses were much less favourable than mouse carcasses for type D toxigenesis . The study, together with earlier studies on types C and E, indicated that carrion contaminated with C . botulinum type C or D is likely to be particularly dangerous for animals that may ingest it . This accords with the observation that carrion-transmitted botulism in animals is usually caused by type C or D.

Eur J Biochem, 1994 Oct 1, 225(1), 263 - 70
Peptide substrate specificity and properties of the zinc-endopeptidase activity of botulinum type B neurotoxin; Shone CC et al.; Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle-associated membrane protein (VAMP) . A synthetic peptide of human/rat VAMP-2 {VAMP-2-(60-94)} is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane-associated protein (at the Gln76-Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin . Cleavage of the VAMP-2 peptide was demonstrated by both botulinum type B neurotoxin (Km = 3.3 x 10(-4) M) and by its purified light subunit (Km = 3.5 x 10(-4) M) . The endopeptidase displayed a pH optimum of 7.0-7.5 and was inhibited by greater than 0.2 M NaCl and greater than 0.05 M sodium phosphate . Neurotoxin which had been inactivated by dialysis against EDTA could be re-activated by incubation with various divalent cations, notably Zn2+ and Cu2+ . The substrate specificity of botulinum type B neurotoxin was studied using various analogues of VAMP-2 (60-94) . The neurotoxin cleaved selectively to the N-terminal side of phenylalanine and tyrosine; no activity was observed with either leucine, valine or alanine in the P'1 position . The properties of the P1 amino acid were less critical; the neurotoxin cleaving the C-terminus of glutamine, asparagine and alanine . A substrate analogue with valine in the P1 position corresponding to the sequence of rat VAMP-1 was not cleaved . The rate of cleavage of a substrate analogue representing the sequence of human VAMP-1, however, was more than twofold that of the VAMP-2 peptide . The properties and substrate specificity of botulinum type B neurotoxin suggest that the toxin represents a novel class of endopeptidase which requires a specific peptide substrate conformation for the expression of proteolytic activity.

Pathology, 1994 Oct, 26(4), 477 - 9
Use of gene amplification to detect Clostridium difficile in clinical specimens; Ratcliff RM et al.; The combined use of an enrichment broth and gene amplification following simple DNA extraction to detect toxigenic strains of Clostridium difficile in feces was investigated by examining feces from 329 cases of suspected C . difficile infection . DNA was extracted by heating the washed centrifuged deposit from the broth in a microwave oven . For comparison, specimens were tested concurrently using standard methods for culture and cytotoxin testing . Amplified fragments were identified by molecular weight estimation, restriction enzyme digestion patterns and Southern blot hybridization . The combination of an enrichment broth followed by gene amplification was shown to be a sensitive, specific and rapid method for detecting toxigenic strains of C . difficile in feces . Use of the method in diagnostic laboratories may require the development of improved detection and verification systems for the amplified gene fragment.

Mol Cell Probes, 1994 Oct, 8(5), 365 - 73
Detection of Clostridium botulinum type F using the polymerase chain reaction; Ferreira JL et al.; The polymerase chain reaction (PCR) was used to amplify a portion of the Clostridium botulinum type F toxin gene . An 1137-bp fragment was amplified from 11 different strains of type F C . botulinum with primers derived from the published sequence of type F strain no . 202 . This fragment was not amplified from the DNA of C . botulinum types A, B and E, or from other clostridial organisms examined . When used as a hybridization probe, the 1137-bp PCR-generated fragment generated from one of the type F strains (the proteolytic strain type F Langeland) hybridized to the PCR products from all other type F toxin-producing strains tested . Portions of fragments amplified from the type F Langeland strain were sequenced . The sequence of this strain was found to exhibit approximately 3% variation from the published sequence of the non-proteolytic type F strain no . 202 . Primers designed to pair with the regions of maximum sequence variation between strain 202 and the Langeland strain gave amplification products only with DNA from type F strains that exhibited the same proteolytic properties as the strain from which the primer sequences were derived . These findings underscore the need to consider variations in sequence when designing oligonucleotide probes and PCR primers in order to avoid false negative results.

Wei Sheng Wu Xue Bao, 1994 Oct, 34(5), 379 - 84
{Rumen bacteria degrading toxic mimosine and dihydroxypyridine compounds in China}; Tan P et al.; Four anaerobic strains were isolated from the rumen of the cattle which no specific toxic symptoms were seen when Leucaena was fed in Weizhou island Beihai city, Guangxi Province, China . All of these strains (BR-1, BR-2, BR-5 and BR-7) possess degradative activities to toxic mimosine, 3-hydroxy-4 (1H) -pyridone (3, 4-DHP) and 2, 3-dihydroxypyridine (2, 3DHP) from Leucaena, that were confirmed by analysis of HPLC . Pure and mixed cultures of these four strains in vitro degraded 44-59% of mimosine, 30-47% of 3, 4 DHP, and 58-60% of 2, 3 DHP respectively in 3 days . Strains of Both BR-1 and BR-2 were almost identical and were characterized as Lactobacillus, may be a new species . Strains of BR-5 and BR-7 were characterized as Streptococcus bovis and Clostridium sporogenes respectively . It has not yet been reported that these Gram positive facultatively and obligately anaerobic bacteria were able to degrade mimosine, 3, 4 DHP and 2, 3 DHP . These detoxic bacteria were existing in the rumen microflora of cattle in Weizhou island, protecting therefore their hosts animal from Leucaena toxicity.

J Antimicrob Chemother, 1994 Oct, 34(4), 579 - 84
In-vitro activity of clinafloxacin (CI-960) and PD 131628-2 against anaerobic bacteria; Wexler HM et al.; The antimicrobial activities of two new quinolones, CI-960 and PD 131628-2 were determined against 339 strains of anaerobic bacteria and compared to cefoxitin, imipenem and metronidazole . The NCCLS-approved Wadsworth agar dilution technique with Brucella-lysed blood agar was used throughout the study . Breakpoints of the new quinolones are 2 mg/L, and breakpoints for cefoxitin, imipenem and metronidazole are 32, 8 and 16 mg/L, respectively . CI-960 displayed excellent activity, inhibiting all strains tested at 1 mg/L . PD 131628-2 inhibited 94% of Bacteroides fragilis, 75% of other B . fragilis group isolates, 87% of Prevotella spp, 79% of the Fusobacterium mortiferum-varium group, 74% of non-sporing gram-positive bacilli, and 89-100% of Clostridium spp other than Clostridium difficile at 2 mg/L . None of the eight strains of C . difficile was inhibited at 2 mg/L although they were inhibited at 4 mg/L . PD 131628-2 inhibited all strains of other Bacteroides spp, Porphyromonas spp, and Fusobacterium nucleatum at < or = 1 mg/L.

Biochem Mol Biol Int, 1994 Oct, 34(4), 645 - 52
Purification and biological properties of a cell growth-stimulating factor from Clostridium perfringens FERM P-14028; Watanabe K et al.; The active component that stimulates fibroblast growth was purified from cultured Clostridium perfringens FERM P-14028, and a quantitative assay method for the biological activity was established . The active component was named cell growth-stimulating factor (CGSF), and the molecular weight of this factor was estimated to be 420 kDa on gel permeation chromatography . CGSF(50-100 ng/ml) stimulated the growth rate of BHK-21 (C-13) cells in the logarithmic growth phase, and shortened the doubling time by 16-18%, but had no effect on confluent cells . These actions were indicated only with medium containing fetal bovine serum . These results suggest that CGSF is a novel protein that regulates cell growth via a mechanism of action different from that of other growth factors.

Vet Immunol Immunopathol, 1994 Oct, 43(1-3), 117 - 26
Bacterial immunogens and protective immunity in swine; Wannemuehler MJ et al.; This review provides a limited discussion of antibody-mediated immune responses to bacterial pathogens which cause disease in swine . Serum antibody titers or responses have been used to correlate immunization or convalescence with protection from a given disease or infectious agent . Though much effort has been devoted to the elucidation of the host's antibody response to bacterial antigens, there are limited examples where an antibody response to a singular antigen has induced protection from disease . Antibody responses have been shown to be very effective in neutralizing bacterial exotoxins, e.g . Escherichia, Pasteurella, Actinobacillus, and inhibiting the ability of bacterial pathogens to colonize mucosal surfaces, e.g . Escherichia, Salmonella . The protective role of monospecific antibody responses to other bacterial components are less clear; however, antibody responses are generally polyclonal in nature and are an important component of the host immune response following the onset of disease . Anticapsular antibodies have been shown to enhance phagocytosis of numerous pathogens, e.g . Actinobacillus, Streptococcus, Pasteurella . Humoral immune responses directed against the lipopolysaccharide (LPS) of many Gram-negative organisms have been shown to enhance phagocytosis and the activation of complement, e.g . Salmonella . Anti-LPS antibodies have also aided in the identification of the serotypic diversity of Gram-negative organisms, e.g . Serpulina, Salmonella, Pasteurella . Antibody responses to the outer membrane proteins of Gram-negative organisms enhance phagocytosis, activation of complement, or inhibit bacterial adhesion to host cell . Adhesion of Gram-positive microorganisms, e.g . Staphylococcus, Streptococcus, Clostridium, to eukaryotic cells can be inhibited by antibody against the peptidoglycan and these peptidoglycan-specific antibodies may also facilitate opsonization of these organisms . Mycoplasma species have been shown to be metabolically inhibited by antibody directed against membrane proteins . In addition to the protective aspects of humoral immunity, the host's antibody response has been used as a diagnostic and epidemiological tool to identify or determine the prevalence of infectious agents.

Antimicrob Agents Chemother, 1994 Oct, 38(10), 2504 - 9
In vitro activity of DU-6859a against anaerobic bacteria; Wexler HM et al.; The activity of a new quinolone agent, DU-6859a, against 330 strains of anaerobic bacteria was determined by using the National Committee for Clinical Laboratory Standards-approved Wadsworth brucella laked blood agar method; the activity of DU-6859a was compared with those of amoxicillin-clavulanate (2:1), chloramphenicol, ciprofloxacin, clindamycin, fleroxacin, imipenem, lomefloxacin, metronidazole, sparfloxacin, and temafloxacin . DU-6859a and chloramphenicol inhibited all of the isolates at concentrations of 1 and 16 micrograms/ml, respectively; amoxicillin-clavulanate, imipenem, and metronidazole inhibited > or = 94% of the isolates at their respective breakpoints (8, 8, and 16 micrograms/ml) . MICs of DU-6859a at which 90% of the strains were susceptible were 1 to 5 twofold dilutions lower than those of the other quinolones for every group of organisms . MICs of DU-6859a at which 90% of the strains were susceptible (total numbers of strains tested are in parentheses) were < or = 0.25 micrograms/ml for Bacteroides fragilis (57), other B . fragilis group species (84), Bilophila wadsworthia (15), Clostridium species (27) (including C . difficile, C . perfringens, and C . ramosum), Fusobacterium nucleatum (16), Fusobacterium mortiferum-F . varium group species (10), Peptostreptococcus species (20), non-spore-forming gram-positive rods (20), and Prevotella species (25).

J Rheumatol, 1994 Oct, 21(10), 1932 - 7
The spectrum of myositis and rhabdomyolysis associated with bacterial infection; Falasca GF et al.; OBJECTIVE . (1) To describe the clinical and radiographic features of 6 patients with myositis or rhabdomyolysis associated with bacterial infection . (2) To analyze the role of computed tomography (CT) in myositis associated with bacterial infection . METHODS . Review of cases treated by the authors with literature review . RESULTS . Two patients had classical pyomyositis with Staphylococcus aureus as the etiologic agent . One patient had pyomyositis with Enterobacter cloacae (the first reported to our knowledge), 2 had myositis/fasciitis (one due to Clostridium perfringens and one due to S . aureus), and one had fatal toxic rhabdomyolysis in association with C . perfringens bacteremia without evidence of gas gangrene . No patient had a completely normal CT scan of affected muscles, but CT scans in 3 patients failed to show abscesses that were subsequently discovered at surgery, while in another patient CT scanning falsely suggested a large abscess that was not present at surgery . CONCLUSION . Infection associated muscle involvement represents a spectrum of clinical manifestations that include pyomyositis, myonecrosis, fasciitis/myositis, and toxic rhabdomyolysis . Diagnosis may be delayed by the often mild clinical presentation . CT scanning alone may be unreliable in distinguishing muscle abscess from swollen muscle unless combined with CT guided needle biopsy.

Pediatr Res, 1994 Oct, 36(4), 522 - 7
Saccharomyces boulardii enhances rat intestinal enzyme expression by endoluminal release of polyamines; Buts JP et al.; Saccharomyces boulardii is a yeast widely used in humans for the prevention and treatment of infectious enteritis and Clostridium difficile-associated enterocolopathies . After oral administration to human volunteers or growing rats, S . boulardii enhances markedly the expression of intestinal enzymes as well as the production of the polymeric immunoglobulin receptor by mechanisms that remain unknown . We have analyzed the role of the yeast polyamines as potential mediators in the intestinal trophic response . In weanling rats (d 20 to d 30), a daily dose of 100 mg of lyophilized S . boulardii produced significant (p < 0.025) increases in sucrase (157%) and maltase (47%) activities . This dose corresponded to a total oral load of 678 nmol of polyamines per day (spermidine; 376 +/- 32, spermine: 293 +/- 26, putrescine: 9.5 +/- 1.4 nmol/100 mg) . Spermine, given orally to growing rats at doses nearly equivalent (500 nmol) to the load of polyamines provided by the yeast (678 nmol), reproduced similar enzymatic changes, including a 2.5-fold induction of sucrase, and enhanced maltase activity (+24%) . Spermidine and spermine concentrations measured in the jejunal mucosa of treated rats were increased over matched controls by 21.4% (p < 0.005) and 21.9%, respectively (p < 0.002) . After being centrifuged and filtered to discard residual yeast cells, 2-mL samples of jejunal and ileal fluid collected from S . boulardii-treated rats by intestinal flushing contained higher levels of spermidine (48 and 60%) and spermine (150 and 316%) than did control rats . Our data indicate that lyophilized S . boulardii exerts trophic effects on the small intestine that are likely mediated by the endoluminal release of spermine and spermidine.

J Pediatr Gastroenterol Nutr, 1994 Oct, 19(3), 310 - 4
Intestinal microflora in colicky and noncolicky infants: bacterial cultures and gas-liquid chromatography; Lehtonen L et al.; To find out whether intestinal microflora in colicky infants is different from that in noncolicky controls, stool samples were collected from colicky infants during colic (n = 55) and at the age of 3 months (n = 46) and compared with samples from age-matched controls (n = 49 and n = 45, respectively) . The samples were cultured on several selective and unselective aerobic and anaerobic culture agars, and gas-liquid chromatography of bacterial cellular fatty acids was used to produce fatty-acid profiles of the stool samples . In quantitative bacterial cultures, no differences were found between the colicky and control groups in the amounts of each bacterium . The colicky infants were more frequently colonized with Clostridium difficile during the time of colic than were the age-matched controls . This difference disappeared by age 3 months . The fatty-acid profiles did not differ between the colicky and control groups as a whole at the time of colicky symptoms . At age 3 months, a difference in fatty-acid profiles was found between the colicky infants who had suffered from severe colic and the control infants . The fatty-acid profiles were also influenced by the age of the infant, the mode of delivery, antimicrobial drugs taken by the mother during delivery, and breast-feeding and type of feeding . In conclusion, no difference in intestinal microflora was found between the colicky infants at the time of colic and the controls . However, a difference in bacterial cellular fatty-acid profiles at the age of 3 months was found that correlated with severe infantile colic . This difference may contribute to the cause(s) of colic, or it may be secondary to the colic, which may influence the microbial environment of the intestine.

Biometals, 1994 Oct, 7(4), 272 - 8
Characteristic features of the heterologous functional synthesis in Escherichia coli of a 2{4Fe-4S} ferredoxin; Moulis JM et al.; Different strategies have been used to express synthetic genes all encoding Clostridium pasteurianum 2{4Fe-4S} ferredoxin (Fd) in Escherichia coli . The polypeptide can be produced as the C-terminal addition to a hybrid Cro::Protein A fusion protein lacking the metallic centers . The incorporation of the {4Fe-4S} clusters into the cleaved apoFd cannot be carried out in the same conditions as those affording holoFd from purified C . pasteurianum apoFd . In contrast, fully functional Fds can be produced from non-fused synthetic genes under the dependence of strong promoters . The yields of recombinant Fd, although sufficient to purify significant quantities of protein, are limited by the very short half-life of the 2{4Fe-4S} Fd in E . coli, irrespective of the expression system used . These features are characteristic of 2{4Fe-4S} Fds when compared with the far more stable recombinant rubredoxin, and probably other small iron-sulfur proteins which have already been produced in high yields . The reasons for the high turnover of 2{4Fe-4S} Fds are discussed.

Clin Nucl Med, 1994 Oct, 19(10), 860 - 2
Periprosthetic Clostridium difficile hip abscess imaged with In-111 WBCs; Achong DM et al.; During a prolonged hospital stay, left hip pain developed in a woman with sickle cell disease and bilateral hip prostheses . In-111 labeled WBC scintigraphy supplemented by Tc-99m SC bone marrow imaging demonstrated abnormal WBC accumulation surrounding the left greater trochanter . Results of surgical exploration showed an abscess involving the pseudocapsule, trochanteric bursa, and periprosthetic cement column . Cultures grew Clostridium difficile, an unusual pathogen in this site.

Clin Infect Dis, 1994 Oct, 19(4), 761 - 4
Clinical courses of seven survivors of Clostridium septicum infection and their immunologic responses to alpha toxin; Johnson S et al.; Clostridium septicum bacteremia typically portends a fulminant disease associated with high mortality . We describe the clinical courses of seven survivors of C . septicum infection and their antibody responses to the alpha toxin produced by C . septicum . Three patients had clinical syndromes ranging from uncomplicated bacteremia to early typhlitis, and three patients had syndromes ranging from abscess to myonecrosis and septic shock . In addition, an AIDS patient who developed septic shock and who had extensive gas in the retroperitoneal musculature did not undergo surgery but survived after receiving antimicrobial therapy and intensive supportive care . Both immunocompetent patients with myonecrosis had detectable IgG to alpha toxin by immunoblot analysis . IgG to alpha toxin was not detected in the four immunocompetent patients who had C . septicum bacteremia but who did not have myonecrosis or in the AIDS patient with myonecrosis . Therefore, humoral responses to alpha toxin during C . septicum infection may be related to the host's clinical syndrome and immune status.

Appl Microbiol Biotechnol, 1994 Oct, 42(1), 40 - 5
Purification and characterization of an (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum; Bayer M et al.; An NADP(+)-dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified . As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps . Its molecular mass was estimated to be 40-43 kDa . The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate . Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate . Except for the latter all substrates were converted enantioselectively to (S)-3-hydroxy acids in the presence of NADPH . 4-Chloro-3-oxobutyrate was reduced to the (R)-3-hydroxy acid . The specific activity of the enzyme was about 1400 mumol min-1 mg-1 protein for the reduction of 3-oxobutyrate at pH 5.0 . The Michaelis constant (Km) values for 3-oxobutyrate, 3-oxovalerate and 3-oxocaproate were determined to be 0.22, 1.6 and 3.0 mM, respectively . The Km values for dehydrogenation of (S)-3-hydroxybutyrate, (S)-3-hydroxyvalerate and (S)-3-hydroxycaproate were found to be 2.6, 1.1 and 5.2 mM, respectively . The identity of 43 of the first 45 N-terminal amino acid residues has been determined . So far such enzyme activities have been described in eucaryotes only.

Biosci Biotechnol Biochem, 1994 Oct, 58(10), 1866 - 9
Molecular cloning and nucleotide sequence of the beta-galactosidase gene from Enterobacter cloacae GAO; Nagano H et al.; The gene encoding a beta-galactosidase from Enterobacter cloacae GAO was cloned and expressed in Escherichia coli . The nucleotide sequence of the insert of a positive clone had an open reading frame of 3084 bp that encoded a polypeptide of 1028 amino acid residues with a calculated molecular mass of 116,677 daltons . The amino acid sequence of beta-galactosidase deduced from the nucleotide sequence, especially the sequence around the putative active site and of the fourteen regions, showed significant homology to beta-galactosidases of other microorganisms, E . coli, Klebsiella pneumoniae, Lactobacillus bulgaricus, and Clostridium acetobutylicum.

Lab Anim Sci, 1994 Oct, 44(5), 424 - 9
Studies on the diagnosis of Tyzzer's disease in laboratory rat colonies with antibodies against Bacillus piliformis (Clostridium piliforme); Hansen AK et al.; To develop a system to be used for confirming diagnosis of Tyzzer's disease in seropositive rat colonies, 38 Mol:SPRD, BB/Wor/Mol-BB, and Stroke-Prone rats suffering from megaloileitis were examined . All affected rats had been found by a systematic examination of 5-week-old male rats from barrier-protected colonies, sero-positive to Bacillus piliformis, the agent of Tyzzer's disease . The rats were evaluated by serologic testing, histologic examination of the ileum, liver, and heart (hematoxylin and eosin and Warthin-Starry staining), and immunofluorescence staining of tissue smears of the ileum and liver . The presence of B . piliformis was verified in 24 of the rats . All animals that had the agent in the liver or heart also had it in the ileum . The sensitivity of immunofluorescence staining for identification of the agent was higher than that for Warthin-Starry staining . Thirty-seven rats had histologic changes indicative of Tyzzer's disease in the liver, and 23 had histologic changes in the myocardium . All rats had a high titer of antibodies against B . piliformis . Having verified the presence of the agent, germ-free sentinels were placed in one of the colonies . These also became infected, as did germ-free sentinels caged with the infected rats outside the barrier unit . The number of sentinels infected increased with the age of the sentinels at introduction . To prove the absence of Tyzzer's disease in a seropositive colony, it is considered necessary to examine approximately 3,000 5-week-old rats for abdominal distension and demonstrate negative staining of the ileum for B . piliformis.

J Appl Bacteriol, 1994 Oct, 77(4), 448 - 55
An enzyme-linked immunosorbent assay (ELISA) for detection of Clostridium aldrichii in anaerobiodigesters; Brigmon RL et al.; Monoclonal antibodies (Mabs) against Clostridium aldrichii were prepared by in vivo and in vitro immunization with whole cells and produced after fusion as ascites in BALB/c mice . An enzyme-linked immunosorbent assay (ELISA) was used to test for specificity and sensitivity of the Mabs to detect Cl . aldrichii . The lower limit for Cl . aldrichii detection in pure and mixed culture with ELISA was 10(5) cells ml-1 . Twenty other species of bacteria, including 12 cellulolytic species, were tested for cross-reactivity with the ELISA, but none was detected . The ELISA was used for detection of Cl . aldrichii over a 16-month period in five mesophilic continuously-stirred tank reactors (CSTR) with wood, glucose, sludge or sorghum as substrates . The population of Cl . aldrichii in the poplar wood anaerobic digester effluent was 10(6)-10(7) cells ml-1 over that time . These numbers were confirmed by anaerobic microbiological methods . Results from the ELISA technique were obtained in 36 h vs 3 weeks for culture methods . It is concluded that the ELISA is a useful, time-saving method for identification, detection and quantification of Cl . aldrichii in axenic, mixed culture, and in complex undefined cultures such as those found in anaerobic digesters.

Biochemistry, 1994 Sep 20, 33(37), 11217 - 24
Anaerobic pathway for conversion of the methyl group of aromatic methyl ethers to acetic acid by Clostridium thermoaceticum; el Kasmi A et al.; Clostridium thermoaceticum and other anaerobic acetogenic bacteria can utilize the methyl group of aromatic methyl ethers as a carbon and energy source . It has been unclear what pathway is used to metabolize this methyl group . In the work reported here, the pathway was established by identifying and quantitating the substrates, stable intermediates, and products of O-demethylation of syringic acid . By measuring the dependence of the O-demethylation reaction on purified enzymes of the acetyl-CoA pathway, it was established that CO dehydrogenase, the corrinoid/iron-sulfur protein, and methyltransferase all were required for acetyl-CoA formation . By 13C-NMR spectroscopy it was shown that the O-demethylase from C . thermoaceticum converts the methyl group of syringate to methyltetrahydrofolate (CH3-H4folate) . When the reaction was conducted in the presence of CO, H2, or titanium(III), or in the absence of any electron donor, the rate of demethylation of syringic acid at pH 7.2 was approximately 15 nmol min-1 mg-1 . In the absence of CO, CH3-H4folate accumulated as a stable product . When CO was added, 13CH3-H4folate was converted to {2-13C}acetyl-CoA, {2-13C}acetyl phosphate, and {2-13C}acetate . Therefore, the acetogenic O-demethylase uses H4folate as acceptor of the methyl group of phenyl methyl ethers and catalyzes the formation of CH3-H4folate . The pathway of conversion of CH3-H4folate, CO, and CoA to acetyl-CoA has been studied previously . Methyltransferase catalyzes the reaction of CH3-H4folate with the corrinoid/iron-sulfur protein to form a methylcobalt species . The nickel/iron-sulfur enzyme CO dehydrogenase then catalyzes the final steps in the formation of acetyl-CoA.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Rec, 1994 Sep 17, 135(12), 279 - 80
An outbreak of enterotoxaemia caused by Clostridium perfringens type D in goats in Patagonia; Uzal FA et al.; Forty-four of a flock of 117 angora goats in the Rio Negro province of Argentina died within four days . Most of the animals died shortly after the onset of clinical signs, but in a few the clinical course lasted for several days . Post mortem the small and large intestines were filled with watery contents, blood and fibrin clots, and there were numerous ulcers on the mucosa . Small areas of malacia were observed histologically in the brain . Clostridium perfringens type D in pure culture was isolated from the kidneys and gut contents of the affected animals . Epsilon toxin was identified by the mouse seroneutralisation test in the supernatant solution from cultures of these isolates and in the intestinal contents of the affected animals . Heavy infestations with coccidia, nutritional and environmental stress, and an anthelmintic overdose were possible predisposing factors for the outbreak.

FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 7 - 12
Gram-positive cell wall structure of the A3 gamma type in heliobacteria; Pickett MW et al.; The amino acid composition and structure of the peptidoglycan from Heliobacillus mobilis was determined by one- and two-dimensional thin-layer chromatography of completely and partially hydrolysed cell wall preparations . The structure was found to be of the A3 gamma type, with L,L-diaminopimelate in position 3, D-alanine in position 4 and a glycine interpeptide bridge, as found in certain groups of Gram-positive bacteria including Clostridium perfringens and Nocardioides simplex . The presence of a Gram-positive type of cell wall in heliobacteria is consistent with their phylogenetic relationship to the 'low G + C' Gram-positive bacteria, as previously demonstrated by 16S rRNA sequencing.

FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 61 - 8
Comparison of ribotyping, pulsed-field gel electrophoresis and random amplified polymorphic DNA for typing Clostridium difficile strains; Chachaty E et al.; Clostridium difficile is a Gram-positive sporulating anaerobic bacillus which causes pseudomembranous colitis . Nosocomial acquisition of this bacteria has proved frequent, and epidemiological markers are needed to recognize and control common-source outbreaks . We therefore compared the results of pulsed-field gel electrophoresis (PFGE) after restriction with SmaI or NruI, random-amplified polymorphic DNA (RAPD) using 3 10-mer oligonucleotides, and ribotyping to differentiate between 30 unrelated strains of C . difficile belonging to 8 serotypes . The strains were separated into 26 different types by PFGE, 25 by RAPD, but into only 18 types by ribotyping . Median percentages of similarity between strains ranged from 27 in the PFGE assay to 90 in the ribotyping assay, but there was good agreement between the 3 methods for the clustering of strains . PFGE was more time-consuming than RAPD but its patterns were easier to analyze.

FEBS Lett, 1994 Sep 12, 351(3), 317 - 20
Differentiation of HL-60 cells is promoted by H-toxin of Clostridium septicum; Morinaga N et al.; H-toxin of Clostridium septicum potentiated dimethyl sulfoxide (DMSO)-induced differentiation of human promyelocytic leukemia HL-60 cells which were monitored by nuclear morphology and production of oxidative radicals . But, H-toxin did not induce differentiation of HL-60 cells in the absence of DMSO . These phenomena were not observed by staphylococcal leukocidin, a cytotoxin affecting to HL-60 cells . In HL-60 cells, ADP-ribosylation of 118, 93, 75 and 58 kDa membrane proteins was observed, but the ADP-ribosylation was not detected either in differentiated HL-60 cells by DMSO or in normal polymorphonuclear leukocytes of human . When the membranes of HL-60 cells were incubated with H-toxin, ADP-ribosylation of the membrane proteins was inhibited . Such suppressive effects on ADP-ribosylation were not observed by DMSO or staphylococcal leukocidin . These data suggest that inhibition of the ADP-ribosylation by H-toxin may play an important role in potentiation of DMSO-induced differentiation of HL-60 cells.

J Bacteriol, 1994 Sep, 176(18), 5843 - 6
Expression of plasmid-encoded aad in Clostridium acetobutylicum M5 restores vigorous butanol production; Nair RV et al.; Mutant M5 of Clostridium acetobutylicum ATCC 824, which produces neither butanol nor acetone and is deficient in butyraldehyde dehydrogenase (BYDH), acetoacetate decarboxylase, and acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase activities, was transformed with plasmid pCAAD, which carries the gene aad (R . V . Nair, G . N . Bennett, and E . T . Papoutsakis, J . Bacteriol, 176:871-885, 1994) . In batch fermentation studies, aad expression restored butanol formation (84 mM) in mutant M5 without any acetone formation or any significant increase in ethanol production . The corresponding protein (AAD) appeared as a ca . 96-kDa band in a denaturing protein gel . Expression of AAD in M5 resulted in restoration of BYDH activity and small increases in the activities of acetaldehyde dehydrogenase, butanol dehydrogenase, and ethanol dehydrogenase . These findings suggest that BYDH activity in C . acetobutylicum ATCC 824 resides largely in AAD, and that AAD's primary role is in the formation of butanol rather than of ethanol.

Surgery, 1994 Sep, 116(3), 491 - 6
Pseudomembranous colitis: a surgical disease?
Lipsett PA, Samantaray DK, Tam ML, Bartlett JG, Lillemoe KD.
BACKGROUND . Pseudomembranous colitis (PMC) is an increasingly common nosocomial infection caused by Clostridium difficile and its toxins . The disease is usually successfully treated with oral vancomycin . The toxic form of PMC, which requires surgical intervention, is uncommon and often carries a high mortality rate . The indications and recommended surgical procedure and the results of current surgical treatment remain unclear . METHODS . All charts of adults undergoing surgical intervention for PMC during the last 6 years were retrospectively reviewed . During the last 6 years an estimated 37,000 C . difficile toxin assays have been performed with 3300 positive results . RESULTS . Thirteen adults (0.39%) underwent surgical intervention for PMC . Surgical intervention was performed for systemic toxic effects in all patients, with physical signs of peritonitis in six patients and worsening computed tomographic scans with ongoing illness despite appropriate medical therapy in five . The overall mortality rate in the series was 38%; in those undergoing left hemicolectomy (n = 4) the mortality rate was 100% versus a mortality rate of 14% for those undergoing subtotal colectomy (n = 9) . CONCLUSIONS . Despite the effectiveness of oral vancomycin therapy, surgical therapy is occasionally although rarely indicated for the treatment of toxic PMC . Surgical intervention should be considered when the patient has signs of organ failure, a worsening CT scan, or signs of peritonitis . At laparotomy the external appearance of the colon is often deceptively normal and should not influence the surgical procedure of choice, subtotal colectomy . These severely ill patients can be treated with an acceptable morbidity and mortality rate.

J Infect Dis, 1994 Sep, 170(3), 615 - 21
Wide diversity of Clostridium difficile types at a tertiary referral hospital; Samore MH et al.; Nosocomial Clostridium difficile infection was investigated at a hospital with 15 cases of C . difficile diarrhea per 1000 discharges . From January 1991 to May 1991, patients admitted or transferred to five wards or units had weekly rectal swabs taken for culture; in addition, all cytotoxin-positive stools were cultured . Restriction enzyme analysis (REA) was used for molecular typing . Among 205 isolates from 39 patients with C . difficile diarrhea and 67 asymptomatically colonized, 55 distinct REA banding patterns were identified . Evidence for patient-to-patient transmission was limited, in that numerous strains were found even among clustered cases of diarrhea . Patients who acquired C . difficile in the community or other hospitals constituted 32% of culture-positive patients and contributed 44% of the REA types . Diversity of C . difficile strains was in part the result of patients acquiring C . difficile in the community or other hospitals . High incidences of nosocomial C . difficile diarrhea do not necessarily indicate clonal epidemics.

Gastroenterology, 1994 Sep, 107(3), 858 - 63
Etiology and management of toxic megacolon in patients with human immunodeficiency virus infection; Beaugerie L et al.; We report six cases of toxic megacolon in patients with human immunodeficiency virus (HIV) . One case, at an early stage of HIV infection, mimicked a severe attack of Crohn's disease, with a negative search for infectious agents . Subtotal colectomy was successfully performed with an uneventful postoperative course . The five other cases concerned patients with acquired immunodeficiency syndrome at a late stage of immunodeficiency . They were related to Clostridium difficile or cytomegalovirus (CMV) intestinal infection in two and three patients, respectively . One case of CMV colitis presented macroscopically and histologically as pseudomembranous colitis . Emergency subtotal colectomy, performed in the first four patients with acquired immunodeficiency syndrome was followed by a fatal postoperative outcome . The last patient treated conservatively by colonoscopic decompression, in association with anti-CMV therapy, had a favorable short-term outcome . From the experience of our series and data from the literature, we discuss the best diagnostic and therapeutic approach to toxic megacolon in patients with HIV.

J Gen Intern Med, 1994 Sep, 9(9), 528 - 33
Clostridium difficile infection: a common clinical problem for the general internist; Caputo GM et al.; Considering the current wide use of antimicrobial agents, the general internist is commonly faced with the patient at risk for diarrhea due to C . difficile . The diagnosis should be considered for any patient with diarrhea who has received any type of antibiotic therapy in the preceding 4-6 weeks . Symptoms may range from a minor bout of diarrhea to fulminant and fatal colitis . Diagnosis usually requires demonstration of the toxin in stool; culture of the organism and fiberoptic endoscopy may play an adjunctive role in selected clinical settings . The ultimate goal in the treatment for C . difficile infection is to repopulate the normal colonic flora in the most efficacious manner . Minimally symptomatic patients may respond to discontinuing the offending antimicrobial agent or using nonspecific binding agents . Oral vancomycin continues to be the "gold standard" for specific treatment, while metronidazole therapy is considered the first-line agent for individuals with milder infection . Oral bacitracin shows promise, though large studies are lacking . Patients with multiple relapses of C . difficile diarrhea can be treated with prolonged courses of vancomycin or a combination of vancomycin and rifampin . Intensive care unit patients who are NPO have few therapeutic options besides intravenous administration of metronidazole and oral administration of vancomycin via clamped nasogastric tube . Preventive efforts are directed at cautious use of antibiotics and the use of vinyl gloves when caring for patients with known infection.

Nippon Shokakibyo Gakkai Zasshi, 1994 Sep, 91(9), 1407 - 14
{A role of platelet activating factor in experimental hemorrhagic enteritis induced by Clostridium difficile toxin}; Boku S; Clostridium difficile is thought to be an important causative agent of antibiotics associated colitis . However its mechanisms are not fully understood . The present study was designed to elucidate the effect of PAF and free radicals on experimental hemorrhagic enteritis induced by Clostridium difficile toxin . PAF concentration in the portal blood and accumulated fluid, disturbance of the vascular endothelial cells in the ligated jejunal loops and chemiluminescence activity of WBC in the control group's rats increased from 4 hrs over 10 hrs after the administration of Clostridium difficile toxin . On the other hand, the amount of fluid accumulation, protein concentrations in the accumulated fluids, histological changes in the ligated jejunal loops of toxin administered rats and chemiluminescence activity of WBC were significantly suppressed by PAF antagonist (CV6209:TAKEDA) . These results suggest that PAF and free radicals may have some role in microcirculatory disturbance and hyperpermeability of the blood vessels in the intestine of hemorrhagic enteritis induced by Clostridium difficile toxin.

Gastroenterology, 1994 Sep, 107(3), 657 - 65
Neuronal involvement in the intestinal effects of Clostridium difficile toxin A and Vibrio cholerae enterotoxin in rat ileum; Castagliuolo I et al.; BACKGROUND/AIMS: Activation of intestinal mast cells and neurons is involved in intestinal inflammation and diarrhea . This study compared the effects of neuronal inhibitors and inhibition of intestinal sensory afferent nerves on the intestinal actions of Clostridium difficile toxin A, an inflammatory enterotoxin, and cholera toxin, a noninflammatory enterotoxin . METHODS: The effects of lidocaine, hexamethonium, atropine, and long-term pretreatment of capsaicin on fluid secretion, mannitol permeability, myeloperoxidase (MPO) activity, and release of rat mast cell protease II (RMCPII) were measured in toxin A- and cholera toxin-exposed loops in vivo . RESULTS: Lidocaine, hexamethonium, and capsaicin, but not atropine, inhibited toxin A-mediated secretion and MPO activity, but only capsaicin reduced mannitol permeability . Lidocaine, but not capsaicin, reduced secretion and permeability caused by cholera toxin . Toxin A caused release of RMCPII from rat ileum in vivo and in vitro; this was inhibited by lidocaine or capsaicin, whereas cholera toxin had no effect on release of RMCPII . CONCLUSIONS: Neuronal mechanisms are important in the in vivo effects of these two enterotoxins . Capsaicin-sensitive sensory afferent neurons and mast cells are involved in the intestinal mechanism of toxin A, but not cholera toxin.

Mol Cell Biochem, 1994 Sep, 138(1-2), 167 - 76
Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins; Aktories K; The actin cytoskeleton appears to be as the cellular target of various clostridial ADP-ribosyltransferases which have been described during recent years . Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium spiroforme toxin ADP-ribosylate actin monomers and inhibit actin polymerization . Clostridium botulinum exoenzyme C3 and Clostridium limosum exoenzyme ADP-ribosylate the low-molecular-mass GTP-binding proteins of the Rho family, which participate in the regulation of the actin cytoskeleton . ADP-ribosylation inactivates the regulatory Rho proteins and disturbs the organization of the actin cytoskeleton.

Mol Cell Biochem, 1994 Sep, 138(1-2), 135 - 40
Characterization of botulinum C3-catalyzed ADP-ribosylation of rho proteins and identification of mammalian C3-like ADP-ribosyltransferase; Maehama T et al.; The exoenzyme C3 produced by Clostridium botulinum catalyzes ADP-ribosylation of rho gene products which belong to a family of small molecular-weight GTP-binding proteins . The C3 enzyme-catalyzed ADP-ribosylation of rho proteins partially purified from bovine brain was markedly activated by certain types of detergents or phospholipids and by endogenous factors present in the brain cytosol . Rho A protein that had been expressed in E . coli and subsequential purified was readily ADP-ribosylated by the C3 enzyme even in the absence of the activating factors . These results suggest that partially purified rho proteins contain an inhibitor, probably rho GDI (GDP-dissociation inhibitor for rho p21), of C3-catalyzed ADP-ribosylation . The activity of an endogenous enzyme, having the same substrate as botulinum C3 enzyme, was also found in brain cytosol . The enzyme activity was partially purified and characterized . The enzyme appeared to have a molecular mass of approximately 20,000 on a gel filtration and displayed unique properties similar to those observed with the botulinum C3 enzyme . The alpha-subunits of alpha beta gamma-trimeric G proteins which served as the substrates of cholera or pertussis toxin were not ADP-ribosylated by the brain enzyme.

Arq Neuropsiquiatr, 1994 Sep, 52(3), 410 - 3
Botulinum toxin A for trismus in cephalic tetanus; Andrade LA et al.; Cephalic tetanus is a localized form of tetanus . As in generalized forms, trismus is a prominent feature of the disease, leading to considerable difficulty in feeding, swallowing of the saliva and mouth hygiene . These difficulties often precede respiratory problems and aspiration bronchopneumonia is a frequent life-threatening complication . Muscle relaxants other than curare drugs may show a limited benefit for relieving trismus . Tetanospasmin, the tetanic neurotoxin, and botulinum toxin share many similarities, having a closely related chemical structure, an origin from related microorganisms (Clostridium tetani and Clostridium botulinum, respectively), and presumably, the same mechanisms of action in the neuron . The difference between the two lies in their peculiar neurospecificity, acting in different neurons . Injection of minute doses of botulinum toxin in the muscles involved in focal dystonias or other localized spastic disorders have proved to be very effective in these conditions . We describe the use of botulinum toxin A in the successful treatment of trismus in a patient suffering from cephalic tetanus . We believe that this form of treatment may be of value in lowering the risk of pulmonary complications in tetanic patients.

J Diarrhoeal Dis Res, 1994 Sep, 12(3), 200 - 7
Clostridium perfringens type A enterotoxin induces tissue damage and fluid accumulation in rabbit ileum; Sherman S et al.; Rabbit ileal loops were treated with purified Clostridium perfringens enterotoxin (CPE) to compare the onset of toxin-induced tissue damage with the onset of fluid transport changes (i.e . diarrhoea) . Mild changes in fluid transport were detectable after 15 minutes of toxin treatment and then increased progressively with time . Histopathologic studies on toxin-treated ileal loops and measurement of toxin-treated loop fluid protein contents (an indirect marker for tissue damage) both indicated that detectable CPE-induced tissue damage also occurred within 15 minutes of toxin treatment and then progressively increased for at least the first 30 minutes of CPE treatment . Luminal fluid from CPE-treated loops contained elevated Ca2+ levels compared to control luminal fluid, but these elevated Ca2+ levels were not required to initiate in vivo CPE pathophysiologic effects . Since the onsets and severity of tissue damage and fluid transport changes coincided for at least the first 30 minutes of CPE treatment in our study, these results are consistent with CPE-induced tissue damage having a role in the initiation and extent of diarrhoea occurring during C . perfringens type A food poisoning.

Diagn Microbiol Infect Dis, 1994 Sep, 20(1), 1 - 5
Comparison of TechLab Clostridium difficile Tox-A enzyme immunoassay and Bartels Prima system toxin-A EIA; Forward KR et al.; We evaluated the Bartles Clostridium difficile toxin A test and the TechLab Tox-A test to detect C . difficile toxin A in stool . The results were compared with C . difficile cytotoxicity assays . Of the 463 specimens tested 82 (17.7%) tested positive by cytotoxicity assay . The sensitivity, specificity, and positive and negative predictive values of the TechLab EIA were 86.6%, 93.7%, 74.7%, and 97.0%, respectively . For the Bartels Prima EIA, sensitivity, specificity, and positive and negative predictive values were 95.1%, 95.5%, 82.1%, and 98.9%, respectively . The differences in sensitivity were statistically significant . Indeterminate results requiring repeat testing were more common with the TechLab EIA than with the Bartels Prima EIA . Of the two kits, the Bartels EIA is preferable, primarily because of its increased sensitivity.

Infect Dis Clin North Am, 1994 Sep, 8(3), 655 - 75
Dermatologic manifestations of infections in neutropenic patients; Bodey GP; Skin lesions may serve as the site of origin for disseminated infection in neutropenic patients or result as a secondary consequence of disseminated infection from another source . It is important to recognize the characteristic skin lesions associated with infections, such as those caused by Clostridium spp, Candida spp, and Pseudomonas aeruginosa, so that appropriate therapy is initiated promptly . Unfortunately, therapy for some of these infections, especially those caused by fungi, is unsatisfactory in persistly neutropenic patients . Antibacterial, antifungal, and antiviral prophylaxes have caused a significant reduction in some of these infections.

Toxicon, 1994 Sep, 32(9), 1137 - 46
Clostridium butyricum neurotoxin: partial amino acid sequence of major proteolytic fragments and intracellular activity in PC12 cells; Gimenez J et al.; The approximately 150 kDa single-chain neurotoxin produced by Clostridium butyricum, reported to be similar to C . botulinum neurotoxin serotype E (Gimenez and Sugiyama, Infect . Immun . 54, 926-929, 1988), was probed with trypsin and endoproteinase Lys-C . The two proteases cleaved the butyricum neurotoxin between residues Arg 421-Lys 422 and Lys 418-Gly 419, respectively . Cleavage at this region, highly susceptible to proteolysis, generated approximately 50 kDa light and approximately 100 kDa heavy chains, whose identities were established by amino acid sequence determination . In the approximately 150 kDa dichain neurotoxin these two chains remained linked by an interchain disulfide bond . The endoproteinase Lys-C also cleaved the heavy chain between residues Lys 594-Ile 595, yielding a approximately 73 kDa fragment . A total of 77 amino acid residues was identified by Edman degradation of the major fragments generated by proteolysis . By analogy with other botulinum neurotoxin serotypes, the light chain contains the intracellular inhibitory domain and the heavy chain the receptor-binding and channel-forming domains . Butyricum neurotoxin, whether in single-chain or dichain form, inhibited approximately 70% of the Ca2+ stimulated {3H}norepinephrine release from permeabilized PC12 cells . Reduction with 5 mM dithiothreitol enhanced the intracellular inhibitory activity of dichain neurotoxin about 30-fold and increased the extent of inhibition to about 80%, while the activity of single-chain neurotoxin was slightly affected . Increase in the intracellular inhibitory activity of butyricum neurotoxin following mild treatment with trypsin (i.e . conversion of the single-chain protein to the dichain form) was virtually complete within 6 min.

Toxicon, 1994 Sep, 32(9), 1095 - 104
Quaternary structure of botulinum and tetanus neurotoxins as probed by chemical cross-linking and native gel electrophoresis; Ledoux DN et al.; Botulinum and tetanus neurotoxins are water-soluble proteins (mol . wt 150,000) produced by Clostridium botulinum and Clostridium tetani, respectively . It is believed that these neurotoxins, once internalized via receptor-mediated endocytosis, form membrane channels in order to traverse the endosomal membrane and enter the cytoplasm of the nerve terminal . Investigation of the associative properties between neurotoxin molecules could yield an understanding of this channel formation . That is, an association between neurotoxin monomers could result in an oligomeric form of the neurotoxin necessary for assembly of a channel through the hydrophobic interior of the endosomal membrane, thereby allowing passage of the neurotoxin or its active fragment through the resulting pore . Based on the native gel electrophoresis and chemical cross-linking experiments, tetanus neurotoxin exists as a dimer and a trimer, type A botulinum neurotoxin exists as a dimer, trimer, and a larger species, type E botulinum neurotoxin exists as a monomer and dimer, and type B botulinum neurotoxin appears to exist as a dimer in aqueous solution . The results imply that quaternary structures of these neurotoxins may play an important role in their mode of action during neuronal poisoning.

Lett Appl Microbiol, 1994 Sep, 19(3), 153 - 7
Characterization of a psychrotrophic Clostridium causing spoilage in vacuum-packed cooked pork: description of Clostridium algidicarnis sp . nov; Lawson P et al.; A Clostridium species causing spoilage of vacuum-packed refrigerated pork was isolated and characterized . The unknown organism differed phenotypically from other clostridial species usually associated with spoilage . Phylogenetic analyses based on 16S rRNA gene sequencing demonstrated that the psychrotroph represents a distinct line of descent within the genus Clostridium . It is proposed that the organism be classified as a new species of the genus Clostridium, Clostridium algidicarnis.

Biochem Biophys Res Commun, 1994 Aug 30, 203(1), 22 - 8
Hyperphosphorylation of calnexin, a chaperone protein, induced by Clostridium difficile cytotoxin; Schue V et al.; Exposure of McCoy cultured cells to Clostridium difficile cytotoxin B or okadaic acid (OA), a potent phosphatase inhibitor, results in similar morphological changes . Using two-dimensional gel electrophoresis, we have detected a protein of approximately 77 kDa, with a pI of 4.5 (termed pp77) which is hyperphosphorylated in both cases . The level of phosphorylation of pp77 is increased by 293% and 35% after treatment with C . difficile cytotoxin B or OA, respectively . This protein was identified by microsequencing as calnexin, a protein which exhibits the characteristics of an endoplasmic reticulum (ER) chaperone.

Biochemistry, 1994 Aug 16, 33(32), 9769 - 77
Binding of carbon disulfide to the site of acetyl-CoA synthesis by the nickel-iron-sulfur protein, carbon monoxide dehydrogenase, from Clostridium thermoaceticum; Kumar M et al.; Carbon monoxide dehydrogenase (CODH) is a key enzyme in the pathway of carbon monoxide and carbon dioxide fixation by anaerobic bacteria . It performs the oxidation of CO to CO2, the reduction of CO2 to CO, and the synthesis of acetyl-CoA from a methylated corrinoid/iron-sulfur protein, CO, and CoA . These reactions occur at metal centers on CODH and involve metal-carbon bond formation and transformation . There are three iron-containing centers that play distinct roles in CODH: Centers A, B, and C . Center A is the site of synthesis of acetyl-CoA and catalyzes an exchange reaction between CO and acetyl-CoA . Center C is the site of CO oxidation and CO2 reduction . In the work described here, inhibition of CODH by carbon disulfide was studied . CS2 was found to serve as a probe of the interaction of CODH with CO at Center A . EPR spectroscopic and steady-state kinetic studies demonstrated that CS2 mimics the binding of CO to the nickel/iron-sulfur cluster at Center A; however, CS2 itself does not undergo oxidation-reduction and does not appear to bind to Center C as does CO . In the isotope exchange reaction between acetyl-CoA and CO, CS2 was found to be a competitive inhibitor with respect to CO (Ki = 0.47 mM) and a mixed inhibitor with respect to acetyl-CoA (Ki1 = 0.30 and Ki2 = 1.1 mM) . The reaction of dithionite-reduced CODH with CS2 resulted in an EPR spectrum with g values of 2,200, 2,087, and 2,017.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem J, 1994 Aug 15, 302 ( Pt 1), 163 - 70
Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2 degrees Adh) as a bifunctional alcohol dehydrogenase--acetyl-CoA reductive thioesterase; Burdette D et al.; The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described . The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa . The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes . This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production . The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa . The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation . The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation . The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose . The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.

FEBS Lett, 1994 Aug 15, 350(1), 41 - 5
The pH dependent spectral properties of Clostridium pasteurianum 2{Fe4S4} ferredoxin; Calzolai L et al.; The recently assigned 1H NMR hyperfine signals of Clostridium pasteurianum ferredoxin were investigated over the pH range 8-12 to monitor possible pH-dependent conformational changes of the protein . For very high pH values minor perturbations were detected in the chemical shifts of three signals assigned to beta-CH2 cysteine protons of cluster II, while cluster I was not affected at all . These chemical shift variations, which can be fitted to a single pKa = 10.9, are interpreted as an effect of deprotonation of the phenolic group of Tyr-2, located reasonably close to cluster II . This hypothesis has been supported by means of other techniques such as CD and absorption spectroscopy that, on turn, are able to reveal minor pH-dependent spectral variations at high pH . Finally a UV difference experiment has provided further evidence for deprotonation of the phenolic group of Tyr-2 . The possible influence of deprotonation of Tyr-2 on the redox properties of cluster II is discussed.

J Biol Chem, 1994 Aug 12, 269(32), 20425 - 30
Adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum . Overexpression in Escherichia coli, purification, and characterization of the recombinant enzyme; Holloway DE et al.; The genes encoding both components, MutE and MutS, of adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum have been over-expressed in Escherichia coli . This has allowed MutE to be obtained in homogeneous form, free of inhibiting cobamides and traces of MutS . MutE binds MutS cooperatively, with a Hill coefficient of 1.3 . The recombinant enzyme has an unchanged Km for L-glutamate, but a much higher specific activity than those previously reported for preparations from clostridia . The apparent Km for adenosylcobalamin was dependent upon the concentration of MutS and varied between 18 microM with equimolar concentrations of MutS and MutE and 5.8 microM with a 5-fold molar excess of MutS over MutE present in the assay . The dissociation constant for adenosylcobalamin was measured directly using equilibrium gel filtration . In the presence of equimolar amounts of MutE and MutS, the apparent Kd was 5.4 microM, but this decreased to 1.8 microM when MutS was present at a 5-fold molar excess . No binding of adenosylcobalamin to MutE was observed in the absence of MutS . This suggests that the (minimal) function for MutS, whose role in the reaction has been unclear until now, is to form part of the adenosylcobalamin-binding site . It seems likely that MutS is representative of a cobalamin-binding domain conserved across several cobalamin-dependent enzymes.

Clin Lab Sci, 1994 Sep-Oct, 7(5), 311 - 3
Evaluation of a commercial latex agglutination assay for screening for Clostridium difficile-associated disease; Stella PA; OBJECTIVE: To determine if the Clostridium difficile latex agglutination assay is an effective screening procedure for the diagnosis of C . difficile-associated disease (CDAD) . DESIGN: Convenience sample . SETTING: The Washington Hospital, a 364-bed, secondary, acute-care center in Washington, Pennsylvania . PARTICIPANTS: A total of 321 women and 195 men aged 21 days to 100 years . INTERVENTIONS: Diarrheal stool specimens from patients on antibiotic therapy were randomly tested for the presence of C . difficile-associated antigen by CULTURETTE brand latex agglutination assay (Beckton Dickinson Microbiology Systems, Cockeyesville, MD 21030) . Latex-positive samples were confirmed or negated by cytotoxin assay using the Toxi-titer microtiter plate system (Baxter Healthcare Corp., Bartels Diagnostic Division, Issaquah, WA 98027) . MAIN OUTCOME MEASURES: Comparative statistical analysis of the raw data to develop the sensitivity and specificity of the latex screening assay . RESULTS: Stool specimens of 403 patients (78.1%) were negative for C . difficile-associated antigen . Of the latex-positive specimens, 70 (69.3%) were also cytotoxin-positive . Of the indeterminate latex specimens, four (33.3%) were cytotoxin-positive . A total of 70 specimens (13.6%) were positive by both methods and 74 patients (14.3%) were diagnosed with CDAD . Based on this data, the latex agglutination screening assay had a sensitivity of 77.1% and a specificity of 93.4% . CONCLUSION: Latex agglutination for C . difficile-associated antigen is a moderately sensitive screening test for a presumptive diagnosis of CDAD . However, it is not the best screening test for CDAD compared with the new enzyme-linked immunosorbent assay (ELISA) techniques for toxins A and B.

FEMS Microbiol Lett, 1994 Aug 1, 121(1), 25 - 30
Multilocus enzyme typing of human and animal strains of Clostridium perfringens; Pons JL et al.; Multilocus enzyme electrophoresis was developed to evaluate the genetic diversity of 71 human strains and 17 animal strains of Clostridium perfringens . Crude protein extracts, obtained by sonication of washed bacteria, were analyzed by polyacrylamide-agarose gel electrophoresis to characterize electrophoretic mobility variants of seven enzymes (esterase, glutamate dehydrogenase, glutamic-oxaloacetic transaminase, nucleoside phosphorylase, phosphoglucose isomerase, phosphoglucomutase, threonine dehydrogenase) . Genetic diversity of the enzyme loci ranged from 0.340 to 0.813 . Sixty-nine electrophoretic types were described among the 88 strains tested and the index of discrimination was 0.994 . All strains were typable, and epidemiological relationships between isolates could be established . This method showed a fair correlation with esterase electrophoretic typing based on hydrolytic and electrophoretic polymorphism of esterases . This work demonstrates that multilocus enzyme polymorphism is a reliable and discriminant marker of genetic diversity of strains of C . perfringens.

Epidemiol Infect, 1994 Aug, 113(1), 1 - 12
Identification of outbreak-associated and other strains of Clostridium difficile by numerical analysis of SDS-PAGE protein patterns; Costas M et al.; Seventy-three cultures of Clostridium difficile isolated both during, and in the period immediately following, an outbreak of infection in a group of three hospitals, were characterized by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins . Each protein pattern was characterized by the presence of one or two dense bands which were highly reproducible . The protein patterns were used as the basis for a numerical analysis which divided the strains into five phenons (electrophoretic or EP types) . The majority, 60 of the 73 cultures, belonged to a single phenon which included strains from both patients and the environment . We conclude that high-resolution SDS-PAGE of proteins provides an effective method for typing C . difficile and therefore for tracing the possible spread of epidemic strains in hospitals and other institutions, thereby allowing a better understanding of the epidemiology of the organism.

Eur J Biochem, 1994 Aug 1, 223(3), 957 - 63
Isolation and characterization of new phosphatidylglycerol acetals of plasmalogens . A family of ether lipids in clostridia; Johnston NC et al.; A new phosphatidylglycerol acetal of cardiolipin plasmalogen has been isolated from Clostridium innocuum . The structure was derived from the results of quantitative group analyses, the identification of the products of acid hydrolysis, alkaline methanolysis, hydrolysis by a cardiolipin-specific phospholipase D and by one- and two-dimensional proton NMR . Two other minor ether phospholipids: the lyso form of the phosphatidylglycerol acetal of cardiolipin plasmalogen, and the phosphatidylglycerol acetal of plasmenylglycerol have been identified in C . innocuum lipid extracts.

Eur J Biochem, 1994 Aug 1, 223(3), 909 - 16
Differentiation-induced increase in Clostridium botulinum C3 exoenzyme-catalyzed ADP-ribosylation of the small GTP-binding protein Rho; Fritz G et al.; The specific {32P}ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study differentiation-dependent changes in the regulation of the low-molecular-mass GTP-binding protein Rho . Differentiation of F9 teratocarcinoma cells to neuronal-like cells by treatment with retinoic acid and dibutyryl-adenosine 3',5'-monophosphate {(Bt)2cAMP} increased the C3-catalyzed ADP-ribosylation of RhoA proteins in cytosolic and membrane fractions by about threefold and sixfold, respectively . Phenotypical differentiation of F9 cells was not required for increase in ADP-ribosylation . Increase in ADP-ribosylation after (Bt)2cAMP and retinoic acid treatments was blocked by cycloheximide, indicating the requirement of protein biosynthesis . As deduced from specific rho mRNA amounts and from Western analysis with a monoclonal RhoA antibody, the stimulation in the {32P}ADP-ribosylation of Rho was not caused by an increased de-novo synthesis of Rho proteins . GDP increased the ADP-ribosylation of membrane-associated Rho from non-differentiated, but not from differentiated F9 cells . GTP{S} decreased ADP-ribosylation of membranous Rho from differentiated and much less from non-differentiated F9 cells . Differentiation-dependent increase in ADP-ribosylation of cytosolic Rho was reversed by protein phosphatase type-1 . Treatment with SDS (0.01%) which releases Rho from complexation with guanine nucleotide dissociation inhibitor, increased ADP-ribosylation both in differentiated and non-differentiated cells, indicating no differentiation-specific change of such complexes . In total, our data indicate that the induction of the differentiation process in F9 cells is accompanied by changes in the regulation of cytosolic and membrane-associated Rho proteins.

Eur J Biochem, 1994 Aug 1, 223(3), 879 - 92
(S)-2-amino-1,3-propanediol-3-phosphate-carrying diradylglyceroglycolipids . Novel major membrane lipids of Clostridium innocuum; Fischer W et al.; Two novel aminophosphoglycolipids (I, II) were isolated from Clostridium innocuum which constitute 51% (I) and 15% (II) of total polar membrane lipids . The structures, established by quantitative and methylation analyses, fast-atom-bombardment mass spectrometry, and one- and two-dimensional NMR spectroscopy, are (I) S-2-amino-1,3-propanediol-3-phospho-6-alpha-D-galactopyranosyl(1-2)alpha -D- glucopyranosyl(1-3)diradylglycerol and (II) an acylated derivative of (I) that carries an additional fatty acid ester on O6 of the glucosyl moiety . The stereochemical configuration of the 2-amino-1,3-propanediol 3-phosphate residue was elucidated by conversion to N-acetylserine 3-phosphate, with subsequent release and identification of L-serine by HPLC . In addition to diacylglycerol species, both aminophosphoglycolipids contain 15-32% 1-O-(alk-1-enyl)-2-O-acyl-glycerol species in which C14, C16, and C18 vinyl ether are combined predominantly with unsaturated C16 and C18 fatty acid ester . Hydrogenation of the vinyl ether was required to desorb the alkyl, acyl-substituted species in fast-atom-bombardment mass spectrometry . Hydrogenation made it further possible to release the alkyl glycerols by acid hydrolysis and to locate the ether bond at O1 of the glycerol moiety . In contrast to the glycerophosphoglycolipids of other Gram-positive bacteria, the aminophosphoglycolipids are metabolically not related to the lipoteichoic acid of C . innocuum and serve, therefore, exclusively as major membrane components . Their large abundance among membrane lipids suggests bilayer-forming physicochemical properties.

Eur J Biochem, 1994 Aug 1, 223(3), 865 - 72
The specificity of clostripain from Clostridium histolyticum . Mapping the S' subsites via acyl transfer to amino acid amides and peptides; Ullmann D et al.; The S' subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala-Xaa dipeptides, proline derivatives and pentapeptides using N alpha-benzoyl-L-arginine ethyl ester as acyl donor . A pentapeptide library consisting of 29 pentapeptides with general structure Xaa-Ala-Ala-Ala-Gly, Ala-Xaa-Ala-Ala-Gly and Ala-Ala-Xaa-Ala-Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid-phase synthesis . The data analysis was performed by HPLC and evaluated by statistical algorithms . The nucleophile efficiency covers a range of more than three orders of magnitude . In the P'1 position, low specificity for amino acid amides and Xaa-(Ala)3-Gly peptides was found, however, in the P'2 position, positively charged amino acid residues are strongly preferred . The negatively charged side chains of aspartic acid and glutamic acid in the P'1 and P'2 positions, respectively, show only poor nucleophilic behaviour . In the case of these amino acids, the S'-P' interactions depend significantly on their position of these residues in the peptide chain of the nucleophile . The transfer of aspartic acid and glutamic acid from P'1-P'3 increases the nucleophile efficiency by approximately two orders of magnitude . The aromatic side chains are not well accepted, especially in the case of P'3Phe . Surprisingly, P'1Gly leads to effective P'-S' interactions . However, the opposite result was obtained for P'2Gly . The high efficiency for Gly-NH2 does not fit with the hydrophobicity structure/activity relationships . In most cases, peptide chain elongation does not improve the nucleophile efficiency . The effective interaction of D-Leu-NH2 with the S' subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to L-amino acids . The results with proline derivatives indicate remarkably different specificities of the S' binding site which can only be explained by conformational restraints . A positive cooperativity between P'1Pro and P'2Gly and a negative cooperativity between P'1Pro and P'2Phe was observed . The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme-nucleophile interactions.

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