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 singl