Biochemistry, 1996 Jan 9, 35(1), 89 - 94 A difference in the sequence of steps in the reactions catalyzed by two closely homologous forms of glutamate dehydrogenase; Maniscalco SJ et al.; Glutamate dehydrogenase from beef liver (bl GDH) and the corresponding enzyme from Clostridium symbiosum (cs GDH) each catalyze the same sequence of chemical events in the oxidative deamination of L-glutamate . This catalysis involves interactions between at least six conserved functional groups, each of which appears to occupy the same geometric position with respect to the substrate molecule in both enzyme--coenzyme--L-glutamate reactive ternary complexes . In both cases steady-state V/K pH profiles indicate the requirement for the transfer to the solvent of a single proton from the same abnormal lysine for L-glutamate to bind and react; the pK of that lysine is the same for both enzymes . Here we report studies of the proton traffic between enzyme and solvent using direct pH-stat back-titration and indicator dye measurements on dead-end inhibitor ternary complexes, simultaneous transient-state time courses of proton and product, and transient-state kinetic isotope studies on both enzymes . We find that in the cs GDH catalyzed reaction the single proton is released only after the hydride transfer step whereas in the bl GDH reaction this proton release occurs prior to the hydride transfer step, despite the fact that the substrate molecule undergoes the same sequence of chemical events in both reactions . Interpreting these results in the context of the X-ray crystallographic structures of cs GDH and its NAD binary complex and of thermodynamic studies of bl GDH and its complexes, we conclude that the difference in the relative times of proton release in the two enzyme-catalyzed reactions must be ascribed to a difference in the sequence of active site cleft-opening and -closing events in the two identical reaction sequences . We suggest a possible biological significance to this unusual method of modulating a common reaction to suit differing metabolic roles. Biochemistry, 1996 Jan 9, 35(1), 41 - 6 Mechanism of the reaction catalyzed by acetoacetate decarboxylase . Importance of lysine 116 in determining the pKa of active-site lysine 115; Highbarger LA et al.; Acetoacetate decarboxylase from Clostridium acetobutylicum (AAD) catalyzes the decarboxylation of acetoacetate via a Schiff base intermediate {Hamilton, G . A., & Westheimer, F . H . (1959) J . Am . Chem . Soc . 81, 6332; Fridovich, I., & Westheimer F . H . (1962) J . Am . Chem . Soc . 84, 3208} . The pKa of the active-site lysine (Lys 115) is 6.0, 4.5 pKa units less than the pKa of lysine in solution {Kokesh, F . C., & Westheimer, F . H . (1971) J . Am . Chem . Soc . 93, 7270; Frey, P . A., Kokesh, F . C., & Westheimer, F . H . (1971) J . Am . Chem . Soc . 93, 7266; Schmidt, D . E., Jr., & Westheimer, F . H . (1971) Biochemistry 10, 1249} . Westheimer and co-workers hypothesized that the pKa of Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116 . We have investigated this proposal by studying site-directed mutants of Lys 115 and Lys 116 . Two substitutions for Lys 115 (K115C and K115Q) were both catalytically inactive at pH 5.95, the pH optimum of wild type AAD, demonstrating the importance of this residue in catalysis . Activity could be restored to K115C by aminoethylation with 2-bromoethyl-ammonium bromide (2-BEAB) . Substitutions for Lys 116 (K116C, K116N, and K116R) had reduced but significant activities at pH 5.95 . The effects of Lys 116 on the pKa of Lys 115 in the mutant AADs were evaluated following imine formation with 5-nitrosalicylaldehyde and reduction with NaBH4 . Whereas the pKa of Lys 115 in K116R is similar to that observed for wild type AAD, the pKaS of Lys 115 in K116C and K116N were elevated to > 9.2 . Alkylation of Cys 116 in K116C with 2-BEAB resulted in both significant activation and restoration of the pKa of Lys 115 to 5.9 . These data support Westheimer's hypothesis that the pKa of the Schiff base-forming Lys 115 is decreased by its spatial proximity to the epsilon-ammonium group of Lys 116. Biochemistry, 1996 Jan 9, 35(1), 282 - 9 Active site mutation of the C3-like ADP-ribosyltransferase from Clostridium limosum--analysis of glutamic acid 174; Bohmer J et al.; Clostridium limosum ADP-ribosyltransferase modifies low molecular mass GTP-binding proteins of the Rho subtype family . Here we cloned and sequenced the gene of the transferase and expressed it in Escherichia coli . The gene encodes a protein of 250 amino acids (M(r) = 27,840), with a putative signal peptide of 45 amino acids, that shows about 60-65% identity with C3 transferases from Clostridium botulinum . The mature C . limosum transferase was expressed as a maltose-binding fusion protein in E . coli and purified to apparent homogeneity . To study the functional role of Glu174 of C . limosum transferase, which was recently photoaffinity-labeled with {carbonyl-14C}NAD {Jung, M., et al . (1993) J . Biol . Chem . 268, 23215-23218}, two mutants E174D and E174Q were constructed by a polymerase chain reaction-based system . The E174D and E174Q mutants showed a dramatic decrease in kcat, but no major changes in Km,NAD . Furthermore, replacement of Glu174 by aspartic acid and glutamine largely reduced and completely blocked UV-induced incorporation of {carbonyl-14C}NAD into the transferase . The data indicate that Glu174 is an active site residue of C . limosum transferase. Biochemistry, 1996 Jan 9, 35(1), 212 - 23 Properties of the selenium- and molybdenum-containing nicotinic acid hydroxylase from Clostridium barkeri; Gladyshev VN et al.; NADP(+)-coupled nicotinic acid hydroxylase (NAH) has been purified to near-homogeneity from Clostridium barkeri by an improved purification scheme that allowed the isolation of milligram amounts of enzyme of higher specific activity then previously reported . NAH is most stable at alkaline pH in the presence of glycerol . The protein which consists of four dissimilar subunits occurs in forms of different molecular masses . There are 5-7 Fe, 1 FAD, and 1 Mo per 160 kDa protein promoter . Mo in the enzyme is bound to a dinucleotide form of molybdopterin and is coordinated with selenium . Mo(V), flavin radical, and two Fe2S2 clusters could be observed with EPR spectroscopy . The Se cofactor which is essential for nicotinic acid hydroxylase activity could be released from NAH as a reactive low molecular weight compound by a number of denaturing procedures . Parallel losses of Se and catalytic activity were observed during purification and storage of the enzyme . Addition of sodium selenide or selenophosphate did not restore the catalytic activity of the enzyme . Instead, NAH is reversibly inactivated by these compounds and also by sulfide . Cyanide, a common inhibitor of Mo-containing hydroxylases, does not affect NAH catalytic activity . The "as isolated" enzyme exhibits a Mo(V) EPR signal (2.067 signal) that was detected at early stages of purification . NAH exhibits a high substrate specificity toward electron donor substrates . The ability of a nicotinate analog to reduce NAH (disappearance of 2.067 signal) correlates with the rate of oxidation of the analog in the standard assay mixture . The properties of NAH differentiate the enzyme from known Mo-containing hydroxylases. Biodegradation, 1996-97, 7(6), 507 - 11 Isolation of an anaerobic bacterium which reductively dechlorinates tetrachloroethene and trichloroethene; Wild A et al.; Strain TEA, a strictly anaerobic, motile rod with one to four lateral flagella and a crystalline surface layer was isolated from a mixed culture that completely reduces chlorinated ethenes to ethene . The organism coupled reductive dehalogenation of tetrachloroethene or trichloroethene to cis-1,2-dichloroethene to growth, using molecular hydrogen as the electron donor . It was unable to grow fermentatively or in the presence of tri- or tetrachloroethene with glucose, pyruvate, lactate, acetate or formate . The 16S rDNA sequence of strain TEA was 99.7% identical to that of Dehalobacter restrictus . The two organisms thus are representatives of the same species or the same genus within the Bacillus/Clostridium subphylum of the gram-positive bacteria. Rocz Akad Med Bialymst, 1996, 41(2), 176 - 82 Clostridium difficile colitis--diagnosis and therapy; Lapinski TW et al.; Clostridium difficile is a Gram-positive bacillus which had been identified as the source of potent exotoxins: toxin A and toxin B . C . difficile infection usually follows antibiotic therapy and results from unrestrained growth of pathogenic strains of C . difficile in the colon . Typical clinical findings include: diarrhoea with blood and mucus, fever, abdominal pain, nausea, loss of body weight . In the past the diagnosis was based on positive result of stool culture but now several tests are available: latex-agglutination test, enzyme immunoassays and fluorescence-immunoenzymatic tests . Diagnostic methods enable quick and safe detection of C . difficile antigens or toxins and proper management . Poor susceptibility of C . difficile strains to common antibiotics hinders choosing the effective therapy. Microbiol Immunol, 1996, 40(12), 923 - 9 Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and its application to enzyme purification; Jung CM et al.; The colH gene encoding 116-kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli-Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH) . When plasmid pJCM310 containing the colH gene was introduced into B . subtilis DB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained . However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C . When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts . rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography . The yield of rColH from an 800-ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein . The purified rColH was capable of degrading native type-I collagen fibril from bovine achilles tendon, as was demonstrated by zymography . A comparison of the N-terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N-terminal amino acid residues . However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH . Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colH gene . Therefore, the recombinant B . subtilis culture is capable of serving as a useful source for enzyme purification. Crit Rev Microbiol, 1996, 22(4), 257 - 77 Clostridium perfringens type A enterotoxin (CPE): more than just explosive diarrhea; Lindsay JA; The bacterial pathogen Clostridium perfringens is the most prolific toxin-producing species within the clostridial group . The toxins are responsible for a wide variety of human and veterinary diseases, many of which are lethal . C . perfringens type A strains are also associated with one of the most common forms of food-borne illness (FBI) . The toxicosis results from the production and gastrointestinal absorption of a protein-enterotoxin known as CPE . The regulation, expression, and mechanism of action of CPE has been of considerable interest as the protein is unique . CPE expression is sporulation associated, although the mechanism of cpe-gene regulation is not fully elucidated . Cloning studies suggest the involvement of global regulators, but these have not been identified . Although very few type A strains are naturally enterotoxigenic, the cpe gene appears highly conserved . In FBI strains, cpe is chromosomally encoded; whereas in veterinary strains, cpe may be plasmid-encoded . Variation in cpe location suggests the involvement of transposable genetic element(s) . CPE-like proteins are produced by some C . perfringens types C and D; and silent remnants of the cpe gene can be found in C . perfringens type E strains associated with the iota toxin gene . CPE has received attention for its biomedical importance . The toxin has been implicated in sudden infant death syndrome (SIDS) because of its superantigenic nature . CPE can destroy a wide variety of cell types both in vitro and in vivo, suggesting that it could have potential in the construction of immunotoxins to neoplastic cells . It is obvious that CPE is an interesting protein that deserves continued attention. Vet Res Commun, 1996, 20(6), 481 - 92 Enterotoxaemia in goats; Uzal FA et al.; Enterotoxaemia of sheep and goats occurs worldwide, but the condition in goats is poorly understood . The disease in goats is mostly caused by Clostridium perfringens type D, although the role of the toxins of this microorganism in the pathogenesis of the disease is not fully understood . The disease occurs in three forms, peracute, acute and chronic, the cardinal clinical sign of the acute and chronic forms being diarrhoea . The main biochemical alterations are hyperglycaemia and glycosuria, while at necropsy the disease is often characterized by haemorrhagic colitis . The typical histological changes observed in the brain of sheep with enterotoxaemia are not considered to be a common feature of enterotoxaemia in goats . Although the pathogenesis of caprine enterotoxaemia has not yet been properly defined, it is usually accepted that the presence of C . perfringens type D in the small bowel, together with a sudden change to a diet rich in carbohydrates, is the main predisposing factor for the disease . Vaccination seems to be poorly effective in preventing caprine enterotoxaemia, which might be due to the fact that the enteric form of the disease is partially independent of circulating C . perfringens toxin . More studies are needed on caprine enterotoxaemia, especially of its pathogenesis and immunity, in order to develop more efficient control measures for this disease. Microbiol Immunol, 1996, 40(3), 189 - 93 Threonine-74 is a key site for the activity of Clostridium perfringens alpha-toxin; Nagahama M et al.; A mutant toxin (MT) that abolished almost 99% of the hemolytic activity of alpha-toxin was isolated by random polymerase chain reaction (PCR) mutagenesis of the gene for Clostridium perfringens alpha-toxin . In the mutant toxin, the amino acids at Tyr (Y)-62, Thr (T)-74 and Ile (I)-345 were substituted with His, Ile and Met, respectively . Replacement of T-74 with Ile by site-directed mutagenesis resulted in the loss of hemolytic, phospholipase C and sphingomyelinase activities by 1/250-fold of that of the wild-type . The replacement of Y-62 with Ile or I-345 with Met alone did not affect the activities of the toxin . T74I mutant bound to sheep erythrocyte membranes and specifically bound {65Zn}2+ in Tris-buffered saline, in the same manner as the wild-type, and contained 2 mol of zinc ions per mol of protein . These results suggest that the T-74 residue plays a key role in these biological activities of C . perfringens alpha-toxin. J Biochem (Tokyo), 1996 Jan, 119(1), 200 - 7 Activation of Clostridium botulinum C3 exoenzyme-catalyzed ADP-ribosylation of RhoA by K+ in a Mg2+ -dependent manner; Miyaoka T et al.; The effect of KCl on ADP-ribosylation of the recombinant RhoA protein catalyzed by the Clostridium botulinum C3 enzyme was studied . When the recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was incubated with C3 and {adenylate-32P}NAD, incorporation of radioactivity into the recombinant RhoA increased in the presence of KCl . The increase in ADP-ribose incorporation into RhoA due to KCl appeared in the presence of MgCl2 and was abolished by EDTA . C3 was stabilized by KCl, but the stabilization was also seen with BSA . The KCl-induced increase in the ADP-ribosylation was observed even in the presence of BSA during the modification reaction, thus the effect of KCl was not due to the stabilization of C3 . While the initial rate of the reaction was increased by KCl, maximum incorporation of ADP-ribose per GST-RhoA molecule did not increase in the presence of KCl . Kinetic analysis revealed that KCl increased Vmax but did not alter Km for either NAD or RhoA . The NAD glycohydrolase activity of C3 was also increased by KCl . These results indicate that KCl directly activates the C3 enzyme. Microbiol Immunol, 1996, 40(1), 5 - 11 Simple method for detection of Clostridium botulinum type A to F neurotoxin genes by ploymerase chain reaction; Takeshi K et al.; A polymerase chain reaction (PCR)-based method was established to detect each type of neurotoxin genes of Clostridium botulinum types A to F by employing the oligonucleotide primer sets corresponding to special regions of the light chains of the neurotoxins . In this procedure, the PCR products were easily confirmed by restriction enzyme digestion profiles, and as little as 2.5 pg of template DNAs from toxigenic strains could be detected . The specific PCR products were obtained from toxigenic C . botulinum types A to F, a type E toxin-producing C . butyricum strain, and a type F toxin-producing C . baratii strain, but no PCR product was detected in nontoxigenic strains of C . botulinum and other clostridial species . The neurotoxin genes were also detected in food products of a seasoned dry salmon and a fermented fish (Izushi) which had caused type E outbreaks of botulism . Therefore, it is concluded that this PCR-based detection method can be used for the rapid diagnosis of botulism. Microbiol Immunol, 1996, 40(2), 141 - 5 Characterization of a toxin-deficient Clostridium perfringens strain, KZ1340; Shimizu T et al.; Clostridium perfringens KZ1340, previously classified as Clostridium plagarum, is an isolate from Antarctic soil, and was identified as an alpha-, theta-, and kappa-toxin non-producing variant . On Southern hybridization, the variant was found to be defective in the pfoA (theta-toxin) gene, but the plc (alpha-toxin) and colA (kappa-toxin) genes were present on the same EcoRI fragment as in the standard strain, NCTC8237 . Northern analysis revealed that mature plc mRNA was transcribed in KZ1340 though less efficiently than in NCTC8237, while no mature colA mRNA was present in KZ1340 . After transformation of the pfoA and plc genes into the KZ1340 via shuttle vector, pJIR418, the pfoA gene was successfully expressed but the plc gene was not efficiently expressed, suggesting that in KZ1340 there is negative regulation of plc gene expression . Toxin-deficient C . perfringens KZ1340 might be a suitable host for expression analysis of the pfoA gene and other clostridial virulence genes, if expressed efficiently, because it produces a small amount of extracellular toxins. Adv Exp Med Biol, 1996, 389, 251 - 60 Tetanus and botulism neurotoxins: a novel group of zinc-endopeptidases; Tonello F et al.; Tetanus and botulinum neurotoxins are produced by bacteria of the genus Clostridium and cause the paralytic syndromes of tetanus and botulism with a persistent inhibition of neurotransmitter release at central and peripheral synapses, respectively . These neurotoxins consist of two disulfide-linked polypeptides: H (100 kDa) is responsible for neurospecific binding and cell penetration of L(50 kDa), a zinc-endopeptidase specific for three protein subunits of the neuroexocytosis apparatus . Tetanus neurotoxin and botulinum neurotoxins serotypes B, D, F, and G cleave at single sites, which differ for each neurotoxin . VAMP/synaptobrevin, a membrane protein of the synaptic vesicles . Botulinum A and E neurotoxins cleave SNAP-25, a protein of the presynaptic membrane, at two different carboxyl-terminal peptide bonds . Serotype C cleaves specifically syntaxin, another protein of the nerve plasmalemma . The target specificity of these metallo-proteinases relies on a double recognition of their substrates based on interactions with the cleavage site and with a non contiguous segment that contains a structural motif common to VAMP, SNAP-25 and syntaxin. Eur Respir J, 1996 Jan, 9(1), 93 - 9 Differential role of actin in lung endothelial and epithelial barrier properties in perfused rabbit lungs; Ermert L et al.; Lung fluid balance is critically dependent on capillary endothelial and alveolar epithelial barrier properties, and cytoskeletal components have been implicated in these barrier functions . In an earlier study, we perfused Clostridium botulinum C2 toxin, which effects selective loss of non-muscle F-actin, through isolated rabbit lungs: a severalfold increase in the capillary filtration coefficient (Kfc) was noted, together with attenuations and disruptions of endothelial cells upon electron microscopic examination . In this model we have investigated the influence of the C2 toxin on alveolar epithelial barrier properties . Epithelial permeability was assessed by continuous monitoring of the transepithelial passage of technetium-labelled diethylenetriamine penta-acetic acid (99mTc-DTPA), offered to the alveolar surface by aerosol technique . Intravascular administration of hydrogen peroxide, used as control agent, was shown to provoke a four- to fivefold increase in the clearance rate of 99mTc-DTPA under conditions of severe fluid leakage into the lung interstitial and alveolar space . Intravascular administration of C2 toxin caused a dose- and time-dependent increase in Kfc values (8-15 fold), but the Tc-DTPA clearance rate was entirely unaffected . Moreover, transbronchial application of C2 toxin again reproduced the manifold increase in Kfc data (about six fold), but the rate of transepithelial passage of the hydrophilic Tc-DTPA complex remained unchanged . We conclude that the barrier properties of the lung microvascular endothelial and epithelial layer are differentially regulated . It is suggested that the actin microfilament system plays a decisive role in the structural and functional integrity of the endothelial but not the epithelial barrier. Clin Infect Dis, 1996 Jan, 22(1), 168 - 70 Cryptosporidiosis: an unrecognized cause of diarrhea in elderly hospitalized patients; Neill MA et al.; Human infection with Cryptosporidium species has been increasingly noted in the past decade . We conducted a broad-based longitudinal review in a community setting and found that a Cryptosporidium species was detected in one-third of the specimens screened over a 5-year period . Thirty-six patients were identified, comprising three distinct clinical groups: persons with human immunodeficiency virus (HIV) infection (18 patients); young, otherwise healthy persons (5 patients); and, surprisingly, chronically ill elderly persons (13 patients) . In six (46%) of the 13 elderly patients, both Cryptosporidium and Clostridium difficile toxin was identified, suggesting that Cryptosporidium may be a copathogen in some instances of nosocomial diarrhea . Acquisition in an institutional setting was suspected for nine (69%) of the elderly and three (17%) of the HIV-infected patients . Elderly patients with chronic illnesses constitute a newly recognized category of persons at risk for cryptosporidial infection . In this group cryptosporidiosis may be far more common than previously recognized, may be acquired institutionally, and can mimic and occur with Clostridium difficile-associated diarrhea. Invest New Drugs, 1996, 13(4), 315 - 20 Phase I trial of high-dose infusional hydroxyurea, high-dose infusional 5-fluorouracil and recombinant interferon-alpha-2a in patients with advanced malignancies; Wadler S et al.; The ribonucleotide reductase inhibitor, hydroxyurea (HU), augments the cytotoxic effects of 5-fluorouracil (5FU) in vitro; both drugs are synergistic with interferon-alpha (IFN) in vitro . The aim of this phase I study was to determine the maximal duration of HU, 4.3 g/m2, administered as a parenteral infusion in combination with 5FU, 2.6 g/m2 administered over 24 hrs each week, + IFN, 9 MU, subcutaneously three times per week . There were 26 patients enrolled and evaluable . This included 14 patients with colorectal cancer of whom 13 had been previously treated, and 12 patients with other refractory malignancies (pancreas, cholangiocarcinoma, hepatocellular carcinoma, renal cell carcinoma, and others), of whom 10 were previously untreated . The dose-limiting toxicity of this regimen was myelosuppression . This prohibited dose escalation of HU above the starting dose (24 hrs) on a 6-weeks-on, 2-weeks-off therapy schedule . When filgrastim, 480 microg, was administered subcutaneously on days 3-6, the duration of HU could be extended to 48 hrs on a 2-weeks-on, 1-week-off therapy schedule . There were two instances of fatal infection, one in a patient with a rectovaginal fistula with neutropenic sepsis and the second in a patient with non-neutropenic Clostridium septicum sepsis . All therapy was administered in the ambulatory setting . There were three responders, all among previously untreated patients . High-dose parenteral hydroxyurea, 4.3 g/m2 administered over 24 hrs, can be safely combined with high-dose weekly 5FU, 2.6 g/m2 over 24 hrs + IFN, 9 MU subcutaneously three times per week, without filgrastim in the ambulatory setting . Parenteral hydroxyurea, 4.3 g/m2 over 24 hrs daily x 2 can also be combined with high-dose 5FU + IFN, but requires the addition of filgrastim to avoid severe myelosuppression. Zh Mikrobiol Epidemiol Immunobiol, 1996 Jan-Feb, (1), 23 - 6 {The use of the polymerase chain reaction for the identification of Clostridium botulinum type A strains}; Shliapnikova OV et al.; Polymerase chain reaction (PCR) for the identification of C.botulinum of type A was developed . As primers, oligonucleotides corresponding to sequences 913 -- 932 and 1852 -- 1871 of the gene of type A botulinic neurotoxin were used . The study revealed that under optimum conditions the positive result of the reaction was registered only when the DNA of C.botulinum strains of type A (11 strains) was used, but not that of C.botulinum strains of other types (11 strains of type B, 5 strains of type C, 2 strains of type D, 6 strains of type E and 1 strain of type G) . High sensitivity, specificity and rapidity of PCR open good prospects for its practical use. Acta Microbiol Pol, 1996, 45(1), 75 - 83 Toxin occurrence time in relation to sensorial changes in meat cans contaminated with Clostridium botulinum type B endospores; Palec W; The study concerning dissemination and expansion of spore-forming rods and the possibility of botulinum toxin occurrence was performed on meat cans model contaminated experimentally with Clostridium botulinum type B366 toxigenic strain endospores . Meat cans were contaminated with the spore suspension of 4 x 10(3) spores per can and they were stored at 30 degrees C . Methodically visible toxigenicity (biological assay performed on white mice) took place in 84.3% cases by the time deformation of contaminated cans took place . When the first can bulging occurred almost 50% of contaminated cans were sensorially estimated as useful for consumption but almost 40% of them already contained toxin . In comparison to contaminated cans estimated sensorially as useful for consumption (in each experimental group) the percentage was established where the presence of toxin was determined as "consumer's risk" . The mean value of those percentages was 76.4% for all experimental groups . In the studies on the vegetation dynamics and C . botulinum rods and toxin expansion in contaminated cans, we demonstrated the spore-forming rods dissemination, as well as limited range of toxin occurrence . Undertaken studies demonstrate that the process of toxigenicity often precede of accumulation of metabolic gases responsible for cans' deformation, as well as generation of visible sensorial changes which disqualified the contaminated cans for consumption. J Clin Gastroenterol, 1996 Jan, 22(1), 45 - 7 Fatal Clostridium difficile enteritis after total abdominal colectomy; Yee HF Jr et al.; A 71-year-old man who had undergone an ileorectal anastomosis some years earlier, developed fulminant fatal Clostridium difficile pseudomembranous enteritis and proctitis after a prostatectomy . This case and three reports of C . difficile involvement of the small bowel in adults emphasize that the small intestine can be affected . No case like ours, of enteritis after colectomy from C . difficile, has hitherto been reported. Reprod Nutr Dev, 1996, 36(3), 253 - 61 In vitro study of the age-dependent caecal fermentation pattern and methanogenesis in young rabbits; Piattoni F et al.; The caecal fermentation pattern, including methanogenesis, was studied in young rabbits using in vitro batch incubations . Six conventional litters of eight rabbits each were used . At the age of 22, 25, 28, 32, 36, 42 and 56 days, an animal was slaughtered from each litter and its caecal contents were used for in vitro batch incubations at 39 degrees C/24 h . The incubated samples were analysed for volatile fatty acids (VFA), methane, hydrogen, ammonia nitrogen (NH3-N) and lactic acid (LA) . The net total in vitro VFA production did not differ clearly with age, although a significant decrease was observed on day 36, reflecting the reduced zootechnical performances probably related to an infection with Clostridium spiroforme that occurred in the same period . The molar proportions of butyrate and propionate formed a change in the opposite direction with age, starting with a sudden shift from propionate to butyrate at day 25 . In vitro NH3-N production was suggestive of a progressive and significant decrease with age; in vitro LA production was always low . Methane production was almost absent from fermentation until 32 days of age, after which it suddenly shifted from 1.6 to 52.0 mumol/flask/day and increased further with age . A significant litter effect on methanogenesis was observed which suggested the existence of a genetic effect . The hydrogen production was quite low and decreased significantly from day 36 with increasing methanogenesis . The calculated hydrogen recoveries showed a gradual increase from day 32 and were positively correlated (r = 0.92) with methane production . In conclusion, it would seem that in young suckling rabbits, reductive acetogenesis is a major characteristic of caecal fermentation, to be replaced gradually and partially by methanogenesis with the increasing intake of solid feed. Therapie, 1996 Jan-Feb, 51(1), 81 - 6 {Antibiotic-associated pseudomembranous colitis: retrospective study of 48 cases diagnosed by colonoscopy}; Andrejak M et al.; Pseudomembranous colitis (PMC) is a rare but potentially severe complication of antibiotic treatment, which is characterized by the proliferation of the bacterium Clostridium difficile in the colon . In this retrospective study, 48 cases of endoscopically confirmed PMC were included . The following variables were analysed: characteristics of the patients, antibiotics, clinical, biological and endoscopic features of PMC and its treatment . The antibiotic treatment was often ambulatory (83 per cent) for a broncho-pulmonary infection (42 per cent) . In 90 per cent of the cases, the treatment included a -lactam, frequently amoxicillin with clavulanic acid, and in 25 per cent of the cases, a fluoroquinolone . The PMC generally occurred after more than 4 days of treatment and was associated with diarrhoea, abdominal pain, fever and rarely vomiting (23 per cent) . The complications were hypokalaemia (37 per cent), renal failure (27 per cent) and/or hypoproteinaemia (50 per cent) . Pseumembranes were found between the rectum and the left angle of the colon . All patients recovered after one week of oral treatment with metronidazole and/or vancomycin, often in association with Saccharomyces boulardii. Crit Rev Food Sci Nutr, 1996 Jan, 36(1-2), 87 - 121 Spoilage and shelf-life extension of fresh fish and shellfish; Ashie IN et al.; Fresh fish and shellfish are highly perishable products due to their biological composition . Under normal refrigerated storage conditions, the shelf life of these products is limited by enzymatic and microbiological spoilage . However, with increasing consumer demands for fresh products with extended shelf life and increasing energy costs associated with freezing and frozen storage, the fish-processing industry is actively seeking alternative methods of shelf life preservation and marketability of fresh, refrigerated fish and at the same time economizing on energy costs . Additional methods that could fulfill these objectives include chemical decontamination, low-dose irradiation, ultra-high pressure, and modified atmosphere packaging (MAP) . This review focuses on the biochemical and microbiological composition of fresh fish/shellfish, the spoilage patterns in these products, factors influencing spoilage, and the combination treatments that can be used in conjunction with refrigeration to extend the shelf life and keeping quality of fresh fish/shellfish . The safety concerns of minimally processed/MAP fish, specifically with respect to the growth of Clostridium botulinum type E, is also addressed. Thymus, 1996, 24(2), 61 - 88 Naturally-occurring anti-thymocyte autoantibody which identifies a restricted CD4+CD8+CD3-/lo/int thymocyte subpopulation exhibits extensive polyspecificity; Underwood JR et al.; In this study, naturally-occurring, monoclonal IgM kappa anti-thymocyte autoantibodies from the neonatal inbred Balb/c mouse-derived hybridoma NMT-1 (NMT-1 mAb), previously reported to identify a restricted CD4+CD8+CD3/lo/int thymocyte subpopulation, have been shown to exhibit extensive polyspecificity . Using immunofluorescence, immunoblotting and antibody titration and competition ELISAs, NMT-1 mAbs exhibited polyspecific binding to 12 apparently structurally unrelated self and non-self antigens . The autoreactive component of the polyspecificity profile of NMT-1 mAbs encompassed reactivity to developmentally-related 14.5 and 18.3 kDa Thy-1 glycoforms expressed on a CD4+CD8+CD3-/lo/int thymocyte subpopulation . The autoreactivity profile of NMT-1 mAbs also included recognition of the heavy and light chains of mouse IgG1 and mouse cytokeratins within thymic medullary epithelium and basal epithelial cells of stratified squamous epithelium of mouse tongue, oesophagus, stomach, skin and vagina . Examination of the polyspecificity profile of NMT-1 mAbs was also undertaken using a panel of 23 antigens including heterologous proteins, phospholipids, haptens and bacterial antigens by antibody titration and competition ELISAs . Antibody titration ELISAs demonstrated that NMT-1 mAbs bound nine antigens including bovine carbonic anhydrase, ovalbumin, cardiolipin, phosphatidylserine, the haptens, DNP and FITC and the bacterial antigens including Escherichia coli beta-galactosidase and the toxoids from Corynebacterium tetani and Clostridium diphtheria . Competition ELISAs, based on the inhibition of NMT-1 mAb binding to antigens adsorbed to ELISA plate surfaces by inhibitor antigens in solution, demonstrated that NMT-1 mAb interactions were not dependent on multivalent binding . In these assays, NMT-1 mAbs recognized unmodified (native) epitopes on the solution phase forms of the protein antigens, including E . coli beta-galactosidase and toxoids from Corynebacterium tetani and Clostridium diphtheria, providing further evidence for the hypothesis that the binding of multiple, apparently unrelated, antigens by NMT-1 mAbs occurs via unique polyspecific antigen combining sites. Pathology, 1996 Jan, 28(1), 70 - 3 Clostridium tertium bacteremia: 2 cases and review; Gosbell IB et al.; Clostridium tertium bacteremia is unusual, seen most often with gastrointestinal disease and/or neutropenia . Two cases are described . The first was a 19-yr-old female with acute leukemia, who developed gastrointestinal symptoms and C . tertium bacteremia while neutropenic . The second was a 57-yr-old female with quiescent ulcerative colitis, who presented with fever, rigors and epigastric pain . Four organisms including C . tertium were isolated from blood cultures . This patient responded to broad spectrum antimicrobial therapy, whereas the first patient required the addition of specific agents to recover . C . tertium is aerotolerant and thus can be misidentified as a Bacillus or Corynebacterium spp . Our isolates had a distinctive Gram stain morphology, were catalase negative and failed to sporulate aerobically--this aided in the recognition of this significant Gram-positive bacillus. Microbiol Immunol, 1996, 40(4), 255 - 63 Analysis of the phospholipase C gene of Clostridium perfringens KZ1340 isolated from Antarctic soil; Kameyama K et al.; Clostridium perfringens KZ1340 isolated from Antarctic soil was first classified as Clostridium plagarum and later as a lecithinase-negative variant of C . perfringens . Although the strain produced no detectable lecithinase (phospholipase C, PLC) activity in the culture supernatant, it was shown by Southern blot hybridization to possess a PLC-encoding gene (plc) . To determine the cause of the PLC deficiency, we cloned and sequenced the plc gene from KZ1340 . The deduced amino acid sequence consists of 398 amino acid residues, coinciding with those of the plc genes previously determined . Tyrosine was substituted for histidine at amino acid position 148, which is thought to bind a zinc ion essential for PLC activity . Northern blot analysis revealed that KZ1340 expressed the plc gene at an extremely low level . Furthermore, the plc gene cloned from C . perfringens strain 13 into a plasmid was expressed weakly in KZ1340, compared to that in strain 13 . This indicates that the former strain represses plc gene expression in trans . When a phylogenetic tree of plc genes was constructed, the KZ1340 plc gene formed a monophyletic branch along with those of various other C . perfringens strains, supporting the classification of the strain as a variant of C . perfringens. Lab Anim, 1996 Jan, 30(1), 42 - 5 Mutual viral and bacterial infections after housing rats of various breeders within an experimental unit; Boot R et al.; Fifteen athymic rat strains from 11 breeding colonies were housed within an experimental facility for an immunological study . Health status records supplied with 14 of the strains listed infections by Kilham's rat virus (KRV), Clostridium piliforme (Bacillus piliformis) and Pasteurella pneumotropica for 2, 2 and 1 colonies respectively . In sera taken previous to the study from euthymic rats of 10 strains, antibodies to KRV were detected in 3 strains, to Pneumonia virus of mice (PVM), Rat corona virus (RCV) and Sendai virus in one strain each and to P . pneumotropica in 2 strains . Only 2 of the KRV infections had been reported by the supplier . During the study rats of all 10 strains developed antibodies to 2-4 of viral antigens . Eight out of 10 rat strains seroconverted to 1-5 of the antigens C . piliforme (B . piliformis), Bordetella bronchiseptica, Haemophilus spp., P . pneumotropica and Streptobacillus moniliformis . Two rat strains housed in filtertop cages did not develop antibodies to bacterial antigens . The potential detrimental effects of intercurrent infections on the outcome of the comparative immunological study are discussed. Acta Clin Belg, 1996, 51(2), 70 - 9 In vitro activity of amoxycillin/clavulanate and ticarcillin/clavulanate compared with that of other antibiotics against anaerobic bacteria: comparison with the results of the 1987 survey; Pierard D et al.; The activity of amoxycillin/clavulanate (Augmentin) and ticarcillin/ clavulanate (Timentin) was tested against 351 strict anaerobic clinical isolates collected from September 1993 to April 1994 in eight Belgian university hospitals and compared with that of 8 other antibiotics using the NCCLS reference agar dilution procedure . Production of beta-lactamase was detected by the nitrocefin test in 48% of the isolates . At NCCLS-recommended breakpoints, more than 90% of isolates were susceptible to amoxycillin/clavulanate, ticarcillin/clavulanate, piperacillin/tazobactam, imipenem, chloramphenicol and metronidazole but only 77%, 72% and 48% to cefoxitin, clindamycin and penicillin, respectively . In comparison with the results of a similar survey conducted in 1987 no major changes in susceptibility were observed except for the susceptibility to clindamycin that declined from 83% to 72% overall, and from 83% to 66% in the B . fragilis group . Furthermore one isolate of Clostridium clostridioforme was found produce beta-lactamase and few B . fragilis group isolates showed reduced susceptibility to metronidazole. Pediatr Radiol, 1996, 26(4), 278 - 9 Diffuse pneumocephalus due to meningitis: CT findings; Goyal M et al.; Diffuse pneumocephalus due to infection by gas forming organisms is very unusual . We report computed tomography (CT) findings of such a case in an infant with Clostridium meningitis. Appl Biochem Biotechnol, 1996 Spring, 57-58, 213 - 21 Inactivation of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824; Green EM et al.; A nonreplicative plasmid containing an internal aad gene fragment has been integrated into the chromosome of Clostridium acetobutylicum ATCC 824 . Transformation was accomplished by electroporation with relatively high concentrations of methylated plasmid DNA . Southern hybridization experiments revealed that integration occurred by single crossover homologous recombination inactivating the aad gene . Integrants were relatively stable after 25 generations . Inactivation of the aad gene drastically reduced solvent production . This result suggests that aldehyde/alcohol dehydrogenase(AAD) plays a important role in butanol production. Ann Gastroenterol Hepatol (Paris), 1996 Jan-Feb, 32(1), 11 - 7 {The value of rectosigmoidoscopy and the bacteriologic culture of colon biopsies in the etiologic diagnosis of acute diarrhea of adults . A prospective study of 65 patients}; Bellaiche G et al.; The goal of this study was to evaluate the contribution of sigmoidoscopy with bioptic microbiology to the etiologic diagnosis of acute diarrhea in adults . Patients and methods . Sixty-five patients with acute diarrhea were included prospectively from February 1993 to November 1994 . Ages ranged from 17 to 83 years . In each patient, two stool samples were cultured and three examined for parasites . Clostridium difficile toxin was looked for in the 18 patients who had taken antimicrobials before onset of the diarrhea . Sigmoidoscopy with collection of biopsy specimens for bacteriologic cultures was performed routinely . Results . A pathogenic organism was identified in 35 patients (54%) . Eighteen patients (28%) had positive stool cultures . Clostridium difficile toxin was detected in six patients . Colonic biopsy cultures were positive in 26 patients (40%) . Endoscopic findings established the diagnosis of pseudomembranous colitis with negative tests for C . difficile toxin in two patients, diverticulitis in one, ischemic colitis in two, and cryptogenic colitis in seven . Conclusions . Sigmoidoscopy ensured the diagnosis in over 72% of cases of acute diarrhea . This investigation complements stool cultures and should be done routinely in adults with severe acute diarrhea. Prikl Biokhim Mikrobiol, 1996 Jan-Feb, 32(1), 78 - 93 {Biosensor models based on potentiometric and amperometric transducers for use in medicine, biotechnology, and environmental monitoring (review)}; Reshetilov AN; Various types of potentiometric and amperometric biosensors are characterized: microbial sensors with Gluconobacter oxydans cells with potentiometric (pH-sensitive field-effect transistor) and amperometric (Clark-type) electrodes for determining glucose; a potentiometric enzymatic electrode with butyrylcholinesterase, which is used in the biosensor designed to detect pesticides; immunosensors with pH-sensitive field-effect transistors which detect the herbicide 2, 4-D; a biosensor for human immunoglobulin G; biosensors with anaerobic bacteria Clostridium thermocellum; chemical and enzymatic sensors containing a photosensitive membrane for determining ammonium ions and urea; and amperometric microbial sensors prepared with Pseudomonas cells for determining naphthalene, biphenyl, and polychlorinated benzoates . Practical applications of the developed models of biosensors to medicine, biotechnology, and environmental monitoring are discussed. Microbiology, 1996 Jan, 142 ( Pt 1), 191 - 8 Molecular variation between the alpha-toxins from the type strain (NCTC 8237) and clinical isolates of Clostridium perfringens associated with disease in man and animals; Ginter A et al.; The alpha-toxin produced by the type strain of Clostridium perfringens (NCTC 8237) was shown to differ from the alpha-toxins produced by most strains of C . perfringens isolated from man and from calves with respect to reactivity with a neutralizing monoclonal antibody (DY2F5D11) . The difference in antibody binding correlated with three differences in the deduced amino acid sequence (Ala174 to Asp174; Thr177 to Ala177; Ser335 to Pro335) of the alpha-toxins . Using octapeptides synthesized on the basis of the amino acid sequences from these regions of variability, it was shown that the Ala174 to Asp174 change had the greatest effect on reducing the binding of monoclonal antibody DY2F5D11 to the alpha-toxin . These differences did not affect the enzymic or toxic properties of the protein . However, the phospholipase C activity of the alpha-toxin produced by strain NCTC 8237 was more susceptible to inactivation by chymotrypsin . The changes in amino acid sequence did not affect the ability of a C-terminal domain vaccine, derived from the alpha-toxin of strain NCTC 8237, to induce protection against the alpha-toxin from a bovine enteric strain of C . perfringens. Am J Med, 1996 Jan, 100(1), 32 - 40 Clinical and molecular epidemiology of sporadic and clustered cases of nosocomial Clostridium difficile diarrhea; Samore MH et al.; PURPOSE: A prospective clinical and molecular epidemiologic study was conducted to define the frequency of nosocomial Clostridium difficile patient-to-patient transmission in an urban tertiary referral hospital . PATIENTS and METHODS: Over a 6-month period, environmental cultures for C difficile were obtained from patients with new positive stool cytotoxin assay (index cases); stool samples were obtained from selected patient contacts (the roommate, occupants of adjacent rooms, and the patient occupying the index room after discharge of the index case); and hand cultures were obtained from personnel contacts . C difficile isolates were analyzed by pulse-field gel electrophoresis (PFGE) or, for isolates that were nontypeable by PFGE, by restriction enzyme analysis . RESULTS: During the study period, we identified 98 index cases of C difficile toxin-associated diarrhea, including focal outbreaks on two wards totaling 26 cases within a 2-month interval . Environmental contamination was detected at > or = 1 sites in 58% of rooms and often involved wide dispersed areas . Among 99 prospectively identified patient contacts, C difficile was cultured from the stool of 31 (31%), including 12 with diarrhea and 19 who were asymptomatic . C difficile was cultured from the hands of 10 (14%) of 73 personnel . Molecular analysis resolved 31 typing profiles among the index isolates; the most common profile (designated strain D1) was represented by 30 isolates . Among the isolates from patient contacts, 5 of 12 from symptomatic contacts matched the corresponding index isolate, and only 1 of 19 from asymptomatically colonized contacts matched . Transmission to personnel or patient contacts of the strain cultured from the corresponding index case was correlated strongly with the intensity of environmental contamination . Strain D1 was frequently represented among isolates associated with heavy environmental contamination, with personnel carriage, and with development of symptomatic illness among prospectively identified contacts . CONCLUSIONS: Intense environmental contamination and transmission to close personnel and patient contacts represented coordinated properties of an individual epidemic strain . For most epidemiologically linked contacts, positive cultures for C difficile did not result from transmission from the presumed index case. Int J Syst Bacteriol, 1996 Jan, 46(1), 341 - 3 Phylogenetic analysis of Fusobacterium prausnitzii based upon the 16S rRNA gene sequence and PCR confirmation; Wang RF et al.; In order to develop a PCR method to detect Fusobacterium prausnitzii in human feces and to clarify the phylogenetic position of this species, its 16S rRNA gene sequence was determined . The sequence described in this paper is different from the 16S rRNA gene sequence is specific for F . prausnitzii, and the results of this assay confirmed that F . prausnitzii is the most common species in human feces . However, a PCR assay based on the original GenBank sequence was negative when it was performed with two strains of F . prausnitzii obtained from the American Type Culture Collection . A phylogenetic tree based on the new 16S rRNA gene sequence was constructed . On this tree F . prausnitzii was not a member of the Fusobacterium group but was closer to some Eubacterium spp . and located between Clostridium "clusters III and IV" (M.D . Collins, P.A . Lawson, A . Willems, J.J . Cordoba, J . Fernandez-Garayzabal, P . Garcia, J . Cai, H . Hippe, and J.A.E . Farrow, Int . J . Syst . Bacteriol . 44:812-826, 1994). Int J Syst Bacteriol, 1996 Jan, 46(1), 195 - 9 Phylogenetic analysis of Butyrivibrio strains reveals three distinct groups of species within the Clostridium subphylum of the gram-positive bacteria; Willems A et al.; The phylogenetic positions of 40 Butyrivibrio strains were determined by performing a comparative sequence analysis of the 16S rRNA genes of these organisms . We found that all of the strains which we studied belong to cluster XIVa (M . D . Collins, P . A . Lawson, A . Willems, J . J . Cordoba, J . Fernandez=Garayzabal, P . Garcia, J . Cai, H . Hippe, and J . A . E . Farrow, Int . J . Syst . Bacteriol . 44:812-826, 1994) of the Clostridium subphylum of the gram-positive bacteria, which also includes several Clostridium, Coprococcus, Eubacterium, and Ruminococcus species . We also found that the Butyrivibrio strains which we examined were genotypically heterogeneous and exhibited 12 distinct rRNA sequence types . The 12 rRNA sequence types formed three distinct lineages in cluster XIVa, which were separate from each other and from all other species belonging to this cluster . One lineage consisted of strains which exhibited a single rRNA type and corresponded to the species Butyrivibrio crossotus . The second lineage consisted of 12 strains designated Butyrivibrio fibrisolvens which exhibited seven distinct rRNA sequence types . The type strain of B . fibrisolvens was a member of this lineage, but its position was peripheral . The third lineage comprised 26 B . fibrisolvens strains which exhibited four distinct rRNA sequence types . Tree topology and sequence divergence considerations indicated that the three lineages correspond to three separate genera and that the genus Butyrivibrio should be restricted to the group that contains the type strain of B . fibrisolvens. Infect Immun, 1996 Jan, 64(1), 358 - 62 Phospholipase C and perfringolysin O from Clostridium perfringens upregulate endothelial cell-leukocyte adherence molecule 1 and intercellular leukocyte adherence molecule 1 expression and induce interleukin-8 synthesis in cultured human umbilical vein endothelial cells; Bryant AE et al.; Clostridium perfringens phospholipase C (PLC) and perfringolysin O (PFO) differentially induced human umbilical vein endothelial cell expression and synthesis of endothelial cell-leukocyte adherence molecule-1 (ELAM-1), intracellular leukocyte adherence molecule-1 (ICAM-1), and interleukin-8 (IL-8) . PLC strongly induced expression of ELAM-1, ICAM-1, and IL-8, while PFO stimulated early ICAM-1 expression but did not promote ELAM-1 expression or IL-8 synthesis . PLC caused human umbilical vein endothelial cells to assume a fibroblastoid morphology, whereas PFO, in high concentrations or after prolonged low-dose toxin exposure, caused cell death . The toxin-induced expression of proadhesive and activational proteins and direct cytopathic effects may contribute to the leukostasis, vascular compromise, and capillary leak characteristics of C . perfringens gas gangrene. Infect Immun, 1996 Jan, 64(1), 230 - 7 Purification, characterization, and primary structure of Clostridium perfringens lambda-toxin, a thermolysin-like metalloprotease; Jin F et al.; The lambda-toxin of Clostridium perfringens type B NCIB10691 was purified by ammonium sulfate precipitation, followed by size exclusion, anion-exchange, and hydrophobic interaction chromatography . The purified toxin had an apparent molecular mass of 36 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The toxin possessed casein-hydrolyzing activity, which was inhibited specifically by metal chelators, indicating that the toxin is a metalloprotease . The gene encoding the lambda-toxin (lam), which was shown by Southern analysis to be located on a 70-kb plasmid, was cloned into Escherichia coli cells . Nucleotide and N-terminal amino acid sequencing revealed that the lam gene encodes a 553-amino-acid protein, which is processed into a mature form, the molecular mass of which was calculated to be 35,722 Da . The deduced amino acid sequence of the mature enzyme contains an HEXXH motif characteristic of zinc metalloproteases and is homologous to other known enzymes belonging to the thermolysin family . The purified toxin degraded various biologically important substances, such as collagen, fibronectin, fibrinogen, immunoglobulin A, and the complement C3 component . It caused an increase in vascular permeability and hemorrhagic edema on injection into the dorsal skin of mice . These results suggest that the toxin contributes to the pathogenesis of histolytic infection by lambda-toxin-producing C . perfringens. Arch Pathol Lab Med, 1996 Jan, 120(1), 49 - 52 The lack of value of repeated Clostridium difficile cytotoxicity assays; Renshaw AA et al.; OBJECTIVE--To determine the value of repeated Clostridium difficile cytotoxicity assays (CA) . DESIGN--All CAs performed during 1993 were retrospectively reviewed and correlated with clinical data . Assays were grouped into episodes, which were defined as one or more successive tests performed on a single patient within 7 days or less of each other . SETTING--A 751-bed tertiary care facility . PATIENTS--All patients with Clostridium difficile CAs submitted to the microbiology laboratory . RESULTS--There were 947 episodes with two or more CAs . In 15 of these episodes, a negative CA result was followed by a positive result, and in 25 cases, a positive result was followed by a negative one . We reviewed the clinical data for these cases . Of the 947 episodes with two or more CAs, the repeated assays provided new information that was used in patient care in fewer than nine cases . Repeated testing within 7 days of an initial CA accounted for 36% of all assays performed, but provided clinically useful information in only about 1% of cases . CONCLUSIONS--Clostridium difficile CAs should not be repeated within a 7-day period. J Bacteriol, 1996 Jan, 178(1), 143 - 8 Cloning, functional organization, transcript studies, and phylogenetic analysis of the complete nitrogenase structural genes (nifHDK2) and associated genes in the archaeon Methanosarcina barkeri 227; Chien YT et al.; Determination of the nucleotide sequence of the nitrogenase structural genes (nifHDK2) from Methanosarcina barkeri 227 was completed in this study by cloning and sequencing a 2.7-kb BamHI fragment containing the 3' end of nifK2 and 1,390 bp of the nifE2-homologous genes . Open reading frame nifK2 is 1,371 bp long including the stop codon TAA and encodes a polypeptide of 456 amino acids . Phylogenetic analysis of the deduced amino acid sequences of the nifK2 and nifE2 gene products from M . barkeri showed that both genes cluster most closely with the corresponding nif-1 gene products from Clostridium pasteurianum, consistent with our previous analyses of nifH2 and nifD2 . The nifE gene product is known to be homologous to that of nifD, and our analysis shows that the branching pattern for the nifE proteins resembles that for the nifD product (with the exception of vnfE from Azotobacter vinelandii), suggesting that a gene duplication occurred before the divergence of nitrogenases . Primer extension showed that nifH2 had a single transcription start site located 34 nucleotides upstream of the ATG translation start site for nifH2, and a sequence resembling the archaeal consensus promoter sequence {TTTA(A/T)ATA} was found 32 nucleotides upstream from that transcription start site . A tract of four T's, previously identified as a transcription termination site in archaea, was found immediately downstream of the nifK2 gene, and a potential promoter was located upstream of the nifE2 gene . Hybridization with nifH2 and nifDK2 probes with M . barkeri RNA revealed a 4.6-kb transcript from N2-grown cells, large enough to harbor nifHDK genes and their internal open reading frames, while no transcript was detected from NH4(+)-grown cells . These results support a model in which the nitrogenase structural genes in M . barkeri are cotranscribed in a single NH4(+)-repressed operon. J Med Microbiol, 1996 Jan, 44(1), 60 - 4 Characterisation of an enterotoxin-negative, cytotoxin-positive strain of Clostridium sordellii; Green GA et al.; In ileal loop assay, ELISA and anion-exchange column chromatography, Clostridium sordellii strain 6018 was shown to produce a cytotoxin, but no detectable enterotoxin . DNA sequence and polymerase chain reaction analyses indicated that the lack of enterotoxin activity is not due to a lack of gene transcription, but to lack of a major portion of the enterotoxin gene . This is the first characterisation of such a strain. Biochemistry, 1995 Dec 26, 34(51), 16781 - 8 Probing the role of electrostatic forces in the interaction of Clostridium pasteurianum ferredoxin with its redox partners; Moulis JM et al.; The ability of several low-potential redox proteins to mediate electron transfer between Clostridium pasteurianum pyruvate-ferredoxin oxidoreductase and hydrogenase has been evaluated in a coupled enzymatic assay . The active electron mediators, whatever their structure, must have a reduction potential compatible with the two enzymes, but for proteins of similar potentials, a marked specificity is displayed by 2{4Fe-4S} ferredoxins of the clostridial type . Such ferredoxins are small proteins exchanging electrons with many enzymes involved in the metabolism of anaerobic bacteria . The forces underlying the interactions of ferredoxin with hydrogenase and pyruvate-ferredoxin oxidoreductase have been examined with an emphasis on electrostatics: site-directed mutagenesis experiments have been used to individually convert all conserved glutamates and aspartates of C . pasteurianum ferredoxin into either neutral or positively charged amino acids . Also, up to four of these residues have been replaced simultaneously . The biological activities of the resulting variants depend very little on the number and the distribution of the anionic side chains on the surface of the ferredoxin . Only those molecular forms for which the immediate environment of the clusters is perturbed, independently of the charge distribution, display variations in their catalytic properties . It is concluded that electron transfer between C . pasteurianum 2{4Fe-4S} ferredoxin and its partners is far less dependent on electrostatic interactions than in many other well-documented electron transfer systems. Eur J Biochem, 1995 Dec 15, 234(3), 786 - 93 Isolation and structural characterization of N-acetyl- and N-glycolylneuraminic-acid-containing GalNAc-GD1a isomers, IV4GalNAcIV3Neu5AcII3Neu5GcGgOse4Cer and IV4GalNAcIV3Neu5GcII3Neu5AcGgOse4Cer, from bovine brain; Casellato R et al.; A ganglioside preparation containing two structurally related minor gangliosides (Gg 1 + 2) was isolated from bovine brain ganglioside mixture and characterized . Treatment of 50 g ganglioside mixture with Clostridium perfrigens sialidase, followed by chromatography on DEAE-Sepharose and silica gel columns, yielded 20 mg Gg 1 + 2 . By chemical analyses, 1H- and 13C-NMR spectroscopy, enzymic hydrolyses using human beta-hexosaminidase A and clostridial sialidase, and TLC overlay with the conjugated cholera toxin B subunit, the two novel gangliosides Gg 1 and Gg 2 were identified to be: Gg 1, GalNAc-GD1a(Neu5Ac/Neu5Gc), beta-GalNAc-(1-4)-{alpha-Neu5Ac-(2-3)}-beta- Gal-(1-3)-beta-GalNAc-(1-4)-{alpha-Neu5Gc-(2-3)}-beta-Gal-(1-4)-be ta- Glc-(1-1)-Cer; Gg 2, GalNAc-GD1a(Neu5Gc/Neu5Ac), beta-GalNAc-(1-4)-{alpha-Neu5Gc-(2-3)}- beta-Gal-(1-3)-beta-GalNAc-(1-4)-{alpha-Neu5Ac-(2-3)}-beta-Gal-(1- 4)-beta- Glc-(1-1)-Cer . These two gangliosides contain the identical pentasaccharide backbone except that the substitution of the two sialic acids, Neu5Ac and Neu5Gc, are in the reversed position of the external and the internal Gal residues . Our analyses showed that the content of Gg 1 and Gg 2 were approximately 0.12% and 0.08%, respectively, of the total brain ganglioside mixture. J Biol Chem, 1995 Dec 15, 270(50), 29843 - 7 Nuclear phospholipase D in Madin-Darby canine kidney cells . Guanosine 5'-O-(thiotriphosphate)-stimulated activation is mediated by RhoA and is downstream of protein kinase C; Balboa MA et al.; We have recently demonstrated the existence of an ATP-activated phospholipase D (PLD) in the nuclei of MDCK-D1 cells (Balboa, M . A., Balsinde, J., Dennis, E . A., and Insel, P . A . (1995) J . Biol . Chem . 270, 11738-11740) . We have now found that nuclear PLD is synergistically activated by guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and ATP in a time- and concentration-dependent manner, but these compounds do not alter the sensitivity of the enzyme to activation by Ca2+ . The synergistic stimulation of PLD activity could be blocked by addition of the protein kinase C inhibitors chelerythrine and calphostin C . Stimulation by GTP gamma S was abolished by guanosine 5'-O-(2-thiodiphosphate) . Incubation of isolated nuclei with Clostridium botulinum C3 exoenzyme inhibited the potentiating effect of GTP gamma S on ATP-dependent nuclear PLD activity . Moreover, use of the Rho GDP dissociation inhibitor to extract Rho family G proteins from cell nuclei also inhibits PLD activity . Western blot analyses of isolated nuclei revealed the presence of the small G protein RhoA, but not of RhoB or the ADP-ribosylation factor . GTP gamma S-stimulated ATP-dependent PLD activity could be reconstituted in Rho GDP dissociation inhibitor-washed nuclei by addition of recombinant prenylated RhoA, but not by addition of non-prenylated RhoA . Taken together, these results indicate that nuclear PLD activity is modulated via a RhoA-dependent activation that occurs downstream of protein kinase C . Nuclear PLD, which appears to be a previously unrecognized effector regulated by protein kinase C and G proteins, may be involved in the regulation of nuclear function or structure. Cent Afr J Med, 1995 Dec, 41(12), 391 - 7 A study of the anaerobic bacterial flora of the female genital tract in health and disease; Egwari L et al.; Semi-quantitative and qualitative bacterial assessment of the vaginal and cervical flora of a total of 202 women was carried out over a period of six months to determine the bacterial flora in three groups of women and changes caused by prior use of antibiotics . The number was made up of 32 healthy volunteers, 80 women with gynaecological problems and 90 women with gynaecological infections who had had antibiotic treatment prior to this study . Standard methods were used for the investigations . Five main genera of anaerobic bacteria were isolated from all patients . They included, the Bacteroides spp., Prevotella spp., Porphyromonas spp., Peptostreptococcus spp . and Clostridium spp . Five non-sporing gram negative anaerobic bacteria constituted the bulk of the flora including Prevotella bivia, P . disiens, P . melanogenica, P . asaccharolytica and B . fragilis . The predominant flora was P . bivia occurring in 61 pc of cervical swab specimens of the 80 women with proven gynaecological infections who had not used antibiotics and accounting for 27 pc of the total number of Gram-negative anaerobic bacteria isolated . Escherichia coli and Staphylococcus epidermidis were the most frequently encountered aerobic bacteria . The semi-quantitative counts of the different bacterial species in the patient group were significantly higher than in the control group of healthy individuals (p < 0,025) . Similarly, prior antibiotic administration significantly reduced the population and quantitative count of the anaerobic bacteria. Protein Eng, 1995 Dec, 8(12), 1287 - 94 Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability; Lebbink JH et al.; The glutamate dehydrogenase gene from the hyperthermophilic archaeon Pyrococcus furiosus has been functionally expressed in Escherichia coli under the control of the lambda PL promoter . The P . furiosus glutamate dehydrogenase amounted to 20% of the total E . coli cell protein, and the vast majority consisted of hexamers . Following activation by heat treatment, an enzyme could be purified from E . coli that was indistinguishable from the glutamate dehydrogenase purified from P . furiosus . Hybrid genes, that consisted of the coding regions for the homologous glutamate dehydrogenases from P . furiosus and the mesophilic bacterium Clostridium difficile, were constructed and successfully expressed in E . coli . One of the resulting hybrid proteins, containing the glutamate binding domain of the C . difficile enzyme and the cofactor binding domain of the P . furiosus enzyme, did not show a detectable activity . In contrast, the complementary hybrid containing the P . furiosus glutamate and the C . difficile cofactor binding domain was a catalytically active hexamer that showed a reduced substrate affinity but maintained efficient cofactor binding with the specificity found in the Clostridium symbiosum enzyme . Compared with the C . difficile glutamate dehydrogenase, the archaeal-bacterial hybrid is slightly more thermoactive, less thermostable but much more stable towards guanidinium chloride-induced inactivation and denaturation. Toxicon, 1995 Dec, 33(12), 1541 - 7 Botulinum versus tetanus neurotoxins: why is botulinum neurotoxin but not tetanus neurotoxin a food poison? Singh BR, Li B, Read D. Botulinum and tetanus neurotoxins, produced by Clostridium botulinum and Clostridium tetani, respectively, are the most poisonous poisons known to mankind . Although botulinum and tetanus neurotoxins share several characteristics, such as similar mol . wts, similar macrostructure, virtually identical mode of action, and a strong amino acid sequence homology, the two neurotoxins differ in one very significant way; only botulinum neurotoxin is a food poison . Factors responsible for the food poisoning potential of botulinum neurotoxins seem to be a group of complexing proteins that are also produced by C . botulinum, and are known to associate with the neurotoxin . Translation products of nucleotide sequences upstream to the neurotoxin genes of serotypes A, B, C, D, E and F botulinum neurotoxin reveal the location of genes for one of the complexing proteins that could be transcribed as polycistronic mRNA to include neurotoxin sequences . No such protein seems to be present in C . tetani, suggesting that the lack of complexing proteins might be responsible for tetanus not being a food poison. J Am Mosq Control Assoc, 1995 Dec, 11(4), 485 - 8 Evaluation of entomopathogenic bacteria against Aedes polynesiensis, the vector of lymphatic filariasis in French Polynesia; Mercer DR et al.; Thirteen strains among 3 species of entomopathogenic bacteria were tested against 3 medically important mosquito species in French Polynesia . Two strains of Bacillus thuringiensis were highly toxic to Aedes polynesiensis, Aedes aegypti, and Culex quinquefasciatus . Six of 7 strains of Bacillus sphaericus tested were highly toxic to Cx . quinquefasciatus but not to the Aedes spp . Clostridium bifermentans serovar . malaysia was more toxic to Ae . polynesiensis than to the other 2 species . Entomopathogenic bacteria merit field testing for larval mosquito control in French Polynesia. Int J Food Microbiol, 1995 Dec, 28(2), 145 - 55 What problems does the food industry have with the spore-forming pathogens Bacillus cereus and Clostridium perfringens? Andersson A, Ronner U, Granum PE. Spore-forming bacteria are special problems for the food industry . It is not always possible to apply enough heat during food processing to kill spores, thus we have to take advantage of knowledge of the spore-formers to control them . For the meat industry Clostridium perfringens might become a special problem, although this bacterium mainly causes food poisoning through food served in restaurants, hospitals or homes for elderly people (Cliver, 1987; Reynolds, 1987; Gondrosen et al., 1990) . The reason for the food poisoning is always the same: meat-containing dishes stored after cooking with insufficient cooling and reheating (Granum, 1990) . Even though it should be relatively easy to control this kind of food poisoning, C . perfringens is still one of the most common sources of foodborne diseases . Proper disinfection is necessary to control this type of food poisoning, as it is now clear that only kitchen strains of C . perfringens are able to produce the large amounts of enterotoxin necessary to cause food poisoning (Granum, 1990; Cornillot et al., 1995) . Bacillus cereus is more difficult to control, specifically in the dairy industry, where it is now causing the main problems . Insufficient heating of rice-containing dishes has been known to cause B . cereus food poisoning of the emetic kind for a long time (Kramer and Gilbert, 1989), but will not be dealt with in this paper . There are several reasons for the problems in the dairy industry . First of all it seems to be impossible to completely avoid the presence of B . cereus in all milk samples . Secondly the spores are very hydrophobic (Husmark, 1993), and will attach to the surfaces of the pipelines of the dairy industry, where they might multiply and resporulate . A third problem is that pasteurisation heating is insufficient to kill the spores, while competition from other vegetative bacteria is eliminated . It seems that several B . cereus strains have become psychrotrophic over the years, making possible growth at temperatures as low as 4-6 degrees C (Granum et al., 1993a) . None of the methods used to control hygiene in the dairy industry so far are able to control B . cereus . This is a continuously increasing problem for the industry but, with emerging knowledge, we should be able to control it . In this paper we will discuss the problems the food industry is facing with C . perfringens and B . cereus, and how these problems might be solved . We will also give our view on how research might ease these problems in the future. Int J Food Microbiol, 1995 Dec, 28(2), 129 - 44 A survey of bacterial toxins involved in food poisoning: a suggestion for bacterial food poisoning toxin nomenclature; Granum PE et al.; There is at present no accepted nomenclature for bacterial protein toxins, although there have been several attempts at dividing them into groups by their mode of action . In this paper we will not try to describe all known bacterial protein toxins, but concentrate on the toxins involved in food poisoning . Although most of these toxins are enterotoxins (protein exotoxins with the site of action on the mucosal cells of the intestinal tract) there are also other toxins involved in food poisoning, like the neurotoxins . In Table 1 the most important food pathogens in Europe are listed . For most, but not all, of these food pathogens, toxins are virulence factors . Generally, we divide food poisoning into infections and intoxications, where Salmonella spp . and Shigella spp . are typical examples of infections and Clostridium botulinum and Staphylococcus aureus for intoxications . We consider it better to make four different groups of food pathogenic bacteria, according to Table 2 . Today the first three groups are all defined as infections, although for both group 2 and 3 the bacterium itself does not harm the host directly . The bacterium in such locations is like an 'enterotoxin factory' . The bacteria belonging to group 3 do not even interact with the epithelial cells in the intestine, while the bacteria of group 2 must colonise the epithelial cells prior to enterotoxin production. Vet Immunol Immunopathol, 1995 Dec, 49(3), 209 - 27 Prospective characterization of the clinicopathologic and immunologic features of an immunodeficiency syndrome affecting juvenile llamas; Hutchison JM et al.; The clinicopathologic and immunologic features of 15 llamas affected with juvenile llama immunodeficiency syndrome (JLIDS) are described . Healthy adult (n = 10) and juvenile (n = 10) llamas served as controls . JLIDS llamas were characterized by wasting, and clinically apparent, repeated infections were frequently observed . The median age at which a health problem was first perceived was 11.6 months . All 15 affected llamas died or were killed, and JLIDS was confirmed at necropsy . The median duration of illness was 3.5 months . Lymphocyte blastogenesis assays showed suppressed responses (particularly to Staphylococcus sp . Protein A) in JLIDS llamas . No evidence of retroviral infection was detected . Mild, normocytic, normochromic, non-regenerative anemia, low serum albumin concentration and low to low-normal globulin concentrations were typically found on initial clinical evaluation . Lymph node biopsies showed areas of paracortical depletion . All llamas affected with JLIDS had low serum IgG concentrations, pre-vaccination titers against Clostridium perfringens C and D toxoids of < or = 1:100, and no titer increase following vaccination. Cardiovasc Surg, 1995 Dec, 3(6), 687 - 92 Double clostridial mycotic aneurysms of the aorta; Messa CA 3rd et al.; Mycotic aortic aneurysm continues to present challenging and difficult management issues with a significant morbidity and mortality . The offending organism in the etiology of this aneurysm can be variable and unusual . The first report of two mycotic aortic aneurysms caused by Clostridium septicum in the same patient is described here . Presentation and management as well as conditions commonly associated with Clostridium septicum infection and a review of all clostridial mycotic aortic aneurysms in the English literature are discussed. FEMS Immunol Med Microbiol, 1995 Dec, 12(3-4), 239 - 44 Flow cytometric assay for cytotoxic activity of crude Clostridium perfringens enterotoxin using non-adherent cell FM3A; Hu D et al.; The flow cytometric assay method was tested for the cytotoxic activity of Clostridium perfringens enterotoxin (CPE) in culture using mouse mammary carcinoma cell line FM3A stained with propidium iodide (PI) . From the results obtained, FM3A cells proved to be susceptible to CPE . A reproducible dose-response curve with FM3A was obtained between crude CPE at 13.9-109 ng/ml and between purified CPE at 40-400 ng/ml, respectively . These findings indicate that non-adherent FM3A is preferable to determine the cytotoxic activity of CPE because it can be used without detachment procedures with trypsinin compared with adherent African monkey kidney cell line (Vero cells) . Furthermore, the flow cytometry with non-adherent cell FM3A stained with PI only proved to be a useful method to determine the biological activity of CPE in culture isolates. Berl Munch Tierarztl Wochenschr, 1995 Dec, 108(12), 466 - 70 {Detection of Clostridium perfringens toxins on cell cultures}; Borrmann E et al.; An important prerequisite for the possible application of cell culture systems as an in vitro method for the potency test of C . perfringens vaccines is the detection of cytotoxic effects of non-neutralized toxins on cell cultures . For this purpose eight cell lines were investigated in terms of the sensitivity to toxins using microscopy and the MTT assay . The results suggest that the MDCK cell line is a sensitive and specific detection system for epsilon-toxin . The VERO cell line is a suitable indicator for the detection of beta-toxin . Another task was the selection of a sensitive method for the analysis of cytotoxic effects of the toxins used . The neutral red and the tetrazolium-(MTT)-assays were compared for three cell lines (MDCK, FBTR, MA 104) . The MTT assay was proved to be the optimal test system under our conditions. Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 507 - 13 Production of caproic acid by cocultures of ruminal cellulolytic bacteria and Clostridium kluyveri grown on cellulose and ethanol; Kenealy WR et al.; Ruminal cellulolytic bacteria (Fibrobacter succinogenes S85 or Ruminococcus flavefaciens FD-1) were combined with the non-ruminal bacterium Clostridium kluyveri and grown together on cellulose and ethanol . Succinate and acetate produced by the cellulolytic organisms were converted to butyrate and caproate only when the culture medium was supplemented with ethanol . Ethanol (244 mM) and butyrate (30 mM at pH 6.8) did not inhibit cellulose digestion or product formation by S85 or FD-1; however caproate (30 mM at pH 6.8) was moderately inhibitory to FD-1 . Succinate consumption and caproate production were sensitive to culture pH, with more caproic acid being produced when the culture was controlled at a pH near neutrality . In a representative experiment under conditions of controlled pH (at 6.8) 6.0 g cellulose l-1 and 4.4 g ethanol l-1 were converted to 2.6 g butyrate l-1 and 4.6 g caproate l-1 . The results suggest that bacteria that efficiently produce low levels of ethanol and acetate or succinate from cellulose should be useful in cocultures for the production of caproic acid, a potentially useful industrial chemical and bio-fuel precursor. Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 399 - 404 Product inhibition of the recombinant CelS, an exoglucanase component of the Clostridium thermocellum cellulosome; Kruus K et al.; CelS is the most abundant subunit and an exoglucanase component of the Clostridium thermocellum cellulosome, multicomponent cellulase complex . The product inhibition pattern of CelS was examined using purified recombinant CelS (rCelS) produced in Escherichia coli . The rCelS activity on cellopentaose was strongly inhibited by cellobiose . The rCelS activity was also inhibited by lactose . Glucose was only marginally inhibitory . Cellobiose appeared to inhibit the rCelS activity through a competitive mechanism . The inhibition was relieved when beta-glucosidase was added, presumably because of the conversion of cellobiose into glucose . These hydrolysis product inhibition patterns are consistent with those of the crude enzyme (cellulosome), suggesting that CelS is a rate-limiting factor in the activity of the cellulosome. J Clin Microbiol, 1995 Dec, 33(12), 3209 - 15 Identification and antimicrobial resistance patterns of clinical isolates of Clostridium clostridioforme, Clostridium innocuum, and Clostridium ramosum compared with those of clinical isolates of Clostridium perfringens; Alexander CJ et al.; Clostridium ramosum, C . innocuum, and C . clostridioforme are frequently isolated from clinical specimens including blood . Because of Gram stain variability, a lack of spores, and atypical colonial morphology, identification of these species is often difficult . Three anaerobe identification kits were evaluated for their abilities to identify these species . For comparison, 11 strains of C . perfringens were evaluated in parallel . By using profile numbers and codebooks, the correct genus and species were identified, as follows: with the RapID ANA II kit, 100% (20 of 20) of C . ramosum isolates, 24% (5 of 21) of C . innocuum isolates, and 50% (10 of 20) of C . clostridioforme isolates; with the AnIDent kit, 60% (12 of 20) of C . ramosum isolates, 28% (6 of 21) of C . innocuum isolates, and 90% (18 of 20) of C . clostridioforme isolates; with the ATB32A kit, 70% (14 of 20) of C . ramosum isolates, 0% (0 of 21) of C . innocuum isolates, and 40% (8 of 20) of C . clostridioforme isolates . Profile numbers that overlapped several species were obtained as follows: with the RapID ANA II kit, 0% of C . ramosum isolates, 76% of C . innocuum isolates, and 40% of C . clostridioforme isolates; with the AnIDent kit 40% of C . ramosum isolates, 62% of C . innocuum isolates, and 5% of C . clostridioforme isolates; with the ATB32A kit, 15% of C . ramosum isolates, 52% of C . innocuum isolates, and 25% of C . clostridioforme isolates . One strain of C . innocuum was misidentified by the AnIDent kit, and the remainder yielded profile numbers that were not listed in the codebooks . The MICs of 11 antimicrobial agents including penicillin G, metronidazole, clindamycin, cefoxitin, cefotetan, imipenem, meropenem, amoxicillin-clavulanate, ampicillin-sulbactam, piperacillin-tazobactam, and vancomycin were determined by the agar dilution method . All C . perfringens strains were susceptible to all antimicrobial agents tested . Various levels of resistance to cefoxitin, cefotetan, and penicillin G were noted with C . ramosum, C . clostridioforme, and C . innocuum . In addition, resistance to clindamycin was noted with C . ramosum (5%) and C . innocuum (10%) . Most strains of C . innocuum were only moderately susceptible to vancomycin (MIC at which 90% of strains are inhibited, 4 micrograms/ml). J Clin Microbiol, 1995 Dec, 33(12), 3169 - 73 Comparison of arbitrarily primed PCR with restriction endonuclease and immunoblot analyses for typing Clostridium difficile isolates; Tang YJ et al.; Arbitrarily primed PCR (AP-PCR) was used to genotype 26 clinical isolates of Clostridium difficile previously analyzed by immunoblotting (IB) and 20 isolates typed by restriction endonuclease analysis (REA) with HindIII . Two levels of differentiation were achieved with the AP-PCR approach by use of two different arbitrary primers . With the 19-mer arbitrary primer T-7 (first level of differentiation), a good correlation was found between IB and AP-PCR typing . Twenty isolates grouped into six IB types were separated into seven major AP-PCR types . These seven AP-PCR groups were further discriminated into 12 subtypes after genotyping with the arbitrary primer PG-05 (second level of differentiation) . The remaining six isolates, all of different IB types, showed a unique and distinct DNA banding pattern with both of the arbitrary primers, T-7 and PG-05 . Twenty isolates representing 20 REA types from 15 REA groups were resolved into 13 AP-PCR DNA profiles with the arbitrary primer T-7 . A good correlation was found at this level of differentiation between the major REA groups, Y and M, and AP-PCR typing . While AP-PCR with this primer failed to differentiate isolates in REA groups J, G, R, and B, AP-PCR with PG-05 resolved these four isolates into four distinct AP-PCR types . In addition, one of three M strains and one of four Y strains displayed a slightly different DNA banding pattern by AP-PCR (with PG-05) from that of the other strains in the group . We conclude that AP-PCR is a rapid and sensitive method which not only complements other typing schemes but also may be a substitute and prove to be especially suited for immediate epidemiological tracking of nosocomial infections due to C . difficile. Z Lebensm Unters Forsch, 1995 Dec, 201(6), 557 - 61 Comparative effects of gamma and microwave irradiation on the quality of black pepper; Emam OA et al.; Powdered black pepper from Egyptian markets, was irradiated with different recommended doses of gamma rays (5.0 and 10.0 kGy) and with microwaves for different periods (20, 40 and 75 s) to improve its hygienic quality . The most common bacterial isolates were of three genera Bacillus, Clostridium and Micrococcus (7.5 x 10(6)), whereas the predominant fungi (7.8 x 10(4)) were Aspergillus species, A . glaucus, A . flavus, A . niger and A . ochraceus . Doses of gamma irradiation used (5.0 and 10 kGy) were sufficient to decrease spore-forming bacteria (SFB) and to inhibit the fungal flora and coliforms which contaminated the black pepper powder . Microwave treatments for 40 s and 75 s were of the same effectiveness whereas treatment for 20 s was less so . GLC analysis proved the presence of 31 peaks, only 19 compounds were identified as monoterpene hydrocarbons (56.21%), the major one being beta-phellandrene and limonene . Sesquiterpenes were also present, mainly beta-caryollphyllene (3.69%) as well as oxygenated compounds such as terpenol, geraniol, Me-chavicol, eugenol and anisol . Gamma irradiation at 5 kGy and 10 kGy respectively decreased the numbers of identified compounds from 21 (86.58% concentration) in untreated pepper to 16 (59.22% concentration), 15 (54.06% concentration) . In comparison, microwave treatments, particularly for 40 s and 75 s, increased the concentration of the same compounds . The results obtained indicate that microwave treatment, under these conditions, is a safe and suitable technique for decontamination of black pepper which does not result in a great loss of flavour compounds, as compared with recommended doses of gamma irradiation. Tierarztl Prax, 1995 Dec, 23(6), 559 - 64 {Care of pregnancy and prevention of lamb diseases in goats}; Elze K et al.; The breeding of dairy goats has spread in Saxony for over 200 years . Recently the keeping of bigger flocks (30-300 animals) for milk and cheese production has become more common . Within the care of the pregnant she goats the feeding recommendation is a main point of the veterinary herd management . The special performances done by the pregnant animals are discussed . The daily need of energy intake is given with about 11 Megajoule Nettoenergy-lactation as well as the daily need of protein with 230 g . Additionally the minimal daily intake of minerals and vitamins is mentioned . Supervising she goats during lambing and avoiding temperatures lower than 18 degrees C in the stables is considered as necessary to prevent hypoglycemia of the newborn lambs . The enzootic process of Clostridium-perfringens-type-B-infection is discussed in connection with the intake of colostrum and the increasing density of pathogen microorganism during the lambing period. Immun Infekt, 1995 Dec, 23(6), 224 - 7 {Gas gangrene as a manifestation of endogenous Clostridium septicum infection}; Fille M et al.; Endogenous, nontraumatic clostridial myonecrosis has a frequent association with colon carcinoma, leukemia, diabetes mellitus, and drug-induced immunosuppression . We present two cases of Clostridium septicum myonecrosis . An 18-year-old girl developed severe abdominal pain on day 7 after hospitalization for cytostatic treatment of acute lymphoblastic leukemia . Blood cultures yielded Clostridium septicum and histopathological exam of muscle tissue showed extended myonecrosis . Eventually the patient recovered with antibiotics and surgical therapy . A 72-year-old diabetic woman was treated as an outpatient with an intramuscular injection of steroidal antiphlogistics for "acute lumbar disc disease" . The next morning persistence of hip pain and discoloration of the right thigh caused hospitalization under the suspected diagnosis "fracture of the neck of the femur" . Clostridium septicum was cultured from intraoperatively taken swabs . At autopsy, in addition to the gangrene, there was an adenocarcinoma of the cecum, which had not been diagnosed during life. J Biotechnol, 1995 Dec 1, 43(2), 111 - 24 A new balance equation of reducing equivalents for data consistency check and bioprocess calculation; Zeng AP; The reducing equivalent (RE) balance is a basic equation for data consistency check and calculations of bioprocesses . The macroscopic approach is often used because it does not require detailed knowledge of metabolic pathways . In this work, the conventional Minkevich-Eroshin balance equation is examined with data of anaerobic glycerol conversion by Klebsiella pneumoniae and Clostridium butyricum as examples . It is shown that the Minkevich-Eroshin equation is very insensitive to measurement errors in products of less dominance and/or with relatively low reductance degree . Relatively large deviations from experimental values are encountered when the Minkevich-Eroshin equation is used for the calculations of these products . To overcome some of these shortcomings an improved RE balance equation is proposed that is based on 'reductance equations' of substrate conversion into the individual carbon containing products (including biomass) . The proposed new equation significantly improves the performance of the RE balance equation for data consistency check and for the calculations of unknown variables with relatively low reductance degree . The rationale for this is that it considers merely the REs that really participate in the bioreactions . The use of the proposed method requires no detailed knowledge of metabolic pathways and is therefore of macroscopic nature . It can be reduced to the pathway balance equation if the pathways are known. J Formos Med Assoc, 1995 Dec, 94(12), 757 - 9 Gas composition in Clostridium septicum gas gangrene; Chi CH et al.; Clostridial gas gangrene (myonecrosis) is a rare but catastrophic condition that usually occurs in patients with underlying diseases . This paper reports a fatal case of spontaneous clostridial gas gangrene in a 60-year-old female diabetic patient . The composition of gas samples from the patient's damaged muscle was analyzed . The results showed 5.9% hydrogen, 3.4% carbon dioxide, 74.5% nitrogen and 16.1% oxygen . This gas composition supports the belief that such gas production occurs via glucose fermentation . This is the first time such an analysis has been performed in a clinical case of spontaneous clostridial gas gangrene. Eur J Biochem, 1995 Dec 1, 234(2), 603 - 15 The mechanism of substrate and coenzyme binding to clostridial glutamate dehydrogenase during reductive amination; Basso LA et al.; The binding of NADH and 2-oxoglutarate to glutamate dehydrogenase (GDH) from Clostridium symbiosum has been studied by fluorescence spectroscopy . The Kd values for the binding of these ligands have been measured by titration of either the nucleotide or protein fluorescence . During ternary complex formation, the substrate and coenzyme binding sites interact in a positive cooperative manner, but steady-state studies reveal a decrease in affinity of the catalytic complex indicative of negative cooperativity . It was possible to determine the kinetics of formation of the glutamate-dehydrogenase-NADH complex by stopped-flow fluorescence spectroscopy but formation of the glutamate-dehydrogenase-2-oxoglutarate complex was optically silent . Ternary complex formation was characterized by a large quench in protein fluorescence . The binding of NADH to the glutamate-dehydrogenase-2-oxoglutarate binary complex is characterised by a linear increase in the association rate constant, consistent with a one-step binding process . However, the binding of 2-oxoglutarate to the glutamate-dehydrogenase-NADH binary complex is characterised by a decrease in the rate for the observed transient . This suggests that 2-oxoglutarate binds to a different conformation of the enzyme to that stabilized by NADH, and that the transition between these different conformational forms is rate limiting for ternary complex formation . NADH and 2-oxoglutarate can therefore stabilize different conformational states of the enzyme . Collectively, these studies are suggestive of a kinetic model for ternary complex formation that involves the oscillation of the free, binary, and ternary glutamate dehydrogenase complexes between two different conformational states, termed E1 and E2 . The equilibrium constants for ternary complex formation via the predominant pathway have been determined . The cooperativity between the substrate and coenzyme binding sites can be accounted for by the displacement of the equilibria between the E1 and E2 states because of their difference in affinities for NADH and 2-oxoglutarate. Appl Environ Microbiol, 1995 Dec, 61(12), 4441 - 7 Genome analysis of Clostridium botulinum type A by pulsed-field gel electrophoresis; Lin WJ et al.; Genomic DNA from type A Clostridium botulinum was digested with restriction endonucleases that cut at rare sites, and the large fragments were separated by pulsed-field gel electrophoresis . Of 15 restriction enzymes tested, MluI, RsrII, SmaI, NruI, KspI, NaeI, and XhoI generated satisfactory digestion patterns of genomic DNA of various C . botulinum strains, enabling the use of the method for genomic fingerprinting . The genomes of four group I (type A) C . botulinum strains examined had similar restriction patterns . However, each strain had unique digestion patterns, reflecting genotypic differences . The genome size of C . botulinum strain 62A was estimated to be 4,039 +/- 40 kbp from the summation of restriction fragments from MluI, RsrII, and SmaI digestions . Genes encoding proteins involved in the toxinogenicity of C . botulinum, including neurotoxin, hemagglutinin A, and genes for a temperate phage, as well as various transposon Tn916 insertion sites in C . botulinum 62A, were mapped by pulsed-field gel electrophoresis . The genes encoding neurotoxin and hemagglutinin A-1, were located on the same fragment in several cases, indicating their probable physical linkage . The macrorestriction analysis established here should be useful for genetic and epidemiological studies of C . botulinum. Appl Environ Microbiol, 1995 Dec, 61(12), 4141 - 6 A direct PCR detection method for Clostridium tyrobutyricum spores in up to 100 milliliters of raw milk; Herman LM et al.; A direct detection method for Clostridium tyrobutyricum spores in up to 100 ml of raw milk is presented . The bacterial spores are concentrated by centrifugation after chemical extraction of the milk components . The vegetative cells are selectively lysed, and their DNA is digested and washed away . Afterwards, the DNA is liberated from the spores by microwave treatment . For the identification of the C . tyrobutyricum DNA, a two-step PCR method with two nested pairs of primers is used . The primers were derived from the 16S-23S rRNA spacer region of C . tyrobutyricum, and the specificity of each of them for C . tyrobutyricum is demonstrated . The detection limit can be estimated to be between 3 and 30 spores in 100 ml of raw milk. J Bacteriol, 1995 Dec, 177(24), 7164 - 70 Phylogenetic analysis of phospholipase C genes from Clostridium perfringens types A to E and Clostridium novyi; Tsutsui K et al.; The phylogenetic interrelationships between strains of 5 toxin types (A to E) of Clostridium perfringens were examined by analysis of differences in the nucleotide sequences of phospholipase C genes (plc genes) among 10 strains, including 3 strains for which the plc gene sequences have been previously reported . A plc gene was also cloned from a Clostridium novyi type A strain and sequenced to analyze the interspecies diversity of plc genes . Phylogenetic trees constructed by the neighbor-joining method revealed that the phylogeny of C . perfringens strains is not related to toxin typing, in agreement with the results of a comparative genome mapping study by Canard et al . (B . Canard, B . Saint-Joanis, and S . T . Cole, Mol . Microbiol . 6:1421-1429, 1992) . Various C . perfringens phospholipase C enzymes were purified from cultures of Escherichia coli cells into which the encoding plc genes had been cloned . All of the enzymes showed the same specific activity . On the other hand, the level of plc transcripts differed greatly (up to 40-fold) from one C . perfringens strain to another . No significant difference in the nucleotide sequence of the plc promoter region was observed for any of the plc genes . These results suggest that the variation in phospholipase C activity among different strains is not due to mutation in the plc coding region but to that in an extragenic region . The evolution of C . perfringens phospholipase C is discussed on the basis of similarities and differences between clostridial plc genes. Infect Immun, 1995 Dec, 63(12), 4619 - 27 Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters; Torres JF et al.; Clostridium difficile produces toxins that cause inflammation, necrosis, and fluid in the intestine and is the most important cause of nosocomial antibiotic-associated diarrhea and colitis . We evaluated C . difficile antigens as vaccines to protect against systemic and intestinal disease in a hamster model of clindamycin colitis . Formalin-inactivated culture filtrates from a highly toxigenic strain were administered by mucosal routes (intranasal, intragastric, and rectal) with cholera toxin as a mucosal adjuvant . A preparation of culture filtrate and killed whole cells was also tested rectally . The toxoid was also tested parenterally (subcutaneously and intraperitoneally) and by a combination of three intranasal immunizations followed by a combined intranasal-intraperitoneal boost . Serum antibodies against toxins A and B and whole-cell antigen were measured by enzyme-linked immunosorbent assay, neutralization of cytotoxic activity, and bacterial agglutination . The two rectal immunization regimens induced low antibody responses and protected only 20% of hamsters against death and 0% against diarrhea . The intragastric regimen induced high antibody responses but low protection, 40% against death and 0% against diarrhea . Hamsters immunized by the intranasal, intraperitoneal, and subcutaneous routes were 100% protected against death and partially protected (40, 40, and 20%, respectively) against diarrhea . Among the latter groups, intraperitoneally immunized animals had the highest serum anticytotoxic activity and the highest agglutinating antibody responses . Hamsters immunized intranasally and revaccinated intraperitoneally were 100% protected against both death and diarrhea . Protection against death and diarrhea correlated with antibody responses to all antigens tested . The results indicate that optimal protection against C . difficile disease can be achieved with combined parenteral and mucosal immunization. FEBS Lett, 1995 Nov 27, 376(1-2), 41 - 4 Molecular characterization of two forms of nontoxic-nonhemagglutinin components of Clostridium botulinum type A progenitor toxins; Fujita R et al.; The entire sequences of the type A nontoxic-nonhemagglutinin gene and an adjacent open reading frame designated as orf 22-a, which are located between the neurotoxin and the HA-35 genes were determined . SDS-PAGE and N-terminal amino acid sequence analyses of the purified type A progenitor toxins (12S, 16S and 19S) indicate that the nontoxic-nonhemagglutinins of 16S and 19S are single peptides of approximately 120k, but that of 12S has a cleavage at the site between Pro-144 and Phe-145 of this protein. Biochemistry, 1995 Nov 21, 34(46), 15075 - 83 Mechanistic studies of the methyltransferase from Clostridium thermoaceticum: origin of the pH dependence of the methyl group transfer from methyltetrahydrofolate to the corrinoid/iron-sulfur protein; Zhao S et al.; A methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes the transfer of the N5 methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (C/Fe-SP) . The methylcobamide product is the first in a series of enzyme-bound organometallic intermediates in the acetyl-CoA pathway of anaerobic CO2 fixation . The mechanisms of the forward and reverse reactions with CH3-H4folate and either the C/Fe-SP or vitamin B12 as substrates were studied by steady-state and pre-steady-state kinetics . This ability to effectively utilize free cobalamin as well as the C/Fe-SP in the transmethylation appears to explain why {14C}methylcobyric acid was found as a product of labeling C . thermoaceticum cells with 14CO2 {Ljungdahl, L . G., Irion, E., & Wood, H . G . (1965) Biochemistry 4, 2771-2780} . Stopped-flow experiments indicate that the Co(I)-C/Fe-SP performs a direct SN2 displacement of the methyl group of CH3-H4folate to form H4folate and methyl-Co(III) . The pre-steady-state rate constants in the forward and reverse reactions increased as the pH was lowered (pKa approximately 5.5) . Similar pH profiles were obtained by steady-state kinetics . The kcat/Km values for the C/Fe-SP and CH3-H4folate in the forward direction and for the methylated C/Fe-SP and H4folate in the reverse direction increased as the pH was lowered (pKa approximately 5.3) . A different pH profile was obtained with free cobalamin as the substrate; the kcat/Km for CH3-H4folate and cobalamin (forward reaction) increased (pKa approximately 7.0) and the kcat/Km for H4folate and methylcobalamin (reverse reaction) decreased (pKa approximately 5.3) as the pH was lowered.(ABSTRACT TRUNCATED AT 250 WORDS) Structure, 1995 Nov 15, 3(11), 1147 - 58 The structure of Pyrococcus furiosus glutamate dehydrogenase reveals a key role for ion-pair networks in maintaining enzyme stability at extreme temperatures; Yip KS et al.; BACKGROUND: The hyperthermophile Pyrococcus furiosus is one of the most thermostable organisms known, with an optimum growth temperature of 100 degrees C . The proteins from this organism display extreme thermostability . We have undertaken the structure determination of glutamate dehydrogenase from P . furiosus in order to gain further insights into the relationship between molecular structure and thermal stability . RESULTS: The structure of P . furiosus glutamate dehydrogenase, a homohexameric enzyme, has been determined at 2.2 A resolution and compared with the structure of glutamate dehydrogenase from the mesophile Clostridium symbiosum . CONCLUSIONS: Comparison of the structures of these two enzymes has revealed one major difference: the structure of the hyperthermophilic enzyme contains a striking series of ion-pair networks on the surface of the protein subunits and buried at both interdomain and intersubunit interfaces . We propose that the formation of such extended networks may represent a major stabilizing feature associated with the adaptation of enzymes to extreme temperatures. Eur J Biochem, 1995 Nov 15, 234(1), 192 - 9 Glycine reductase of Clostridium litorale . Cloning, sequencing, and molecular analysis of the grdAB operon that contains two in-frame TGA codons for selenium incorporation; Kreimer S et al.; A 2.8-kb HindIII fragment, containing three open reading frames, has been cloned and sequenced from Clostridium litorale . The first gene grdA encoded the selenocysteine-containing protein PA of the glycine reductase complex, a protein of 159 amino acids with a deduced molecular mass of 16.7 kDa . The second gene (grdB) encoded the 47-kDa subunit of the substrate-specific selenoprotein PB glycine that is composed of 437 amino acids . The third gene contained the 5'-region of the gene for thioredoxin reductase, trxB . All gene products shared high similarity with the corresponding proteins from Eubacterium acidaminophilum . In both genes grdA and grdB, the opal termination codon (TGA) was found inframe, indicating the presence of selenocysteine in both polypeptides . Northern-blot analysis showed that grdA and grdB are organized as one operon . Unlike Escherichia coli, no stable secondary structures of the corresponding mRNA were found immediately downstream of the UGA codons to direct an insertion of selenocysteine into the grdA and grdB transcripts of C . litorale . Instead, a secondary structure was identified in the 3'-untranslated region of grdB. Arch Biochem Biophys, 1995 Nov 10, 323(2), 215 - 22 Reductant-independent ATP hydrolysis catalyzed by homologous nitrogenase proteins from Azotobacter vinelandii and heterologous crosses with Clostridium pasteuranium; Larsen C et al.; Reductant-independent ATPase activity was initiated and studied for Azotobacter vinelandii and Clostridium pasteuranium nitrogenase proteins (Av1, Cp1 and Av2, Cp2, 1 designating the iron molybdenum protein and 2 the iron protein) and their heterologous crosses by two methods: (1) allowing dithionite to be depleted from a normal assay in the presence of substrate levels of MgATP and (2) using reduced but reductant-free nitrogenase proteins in the presence of substrate levels of MgATP . In both cases, at a 1:1 protein ratio, MgATP is converted initially to MgADP with a specific activity of 400-500 nmol MgATP hydrolyzed/min.mg Av1, but in slower steps the MgADP is converted to AMP and, after 12 h, AMP is ultimately converted to adenosine . This reactivity requires the presence of both proteins, increases with increasing Av2/Av1 ratio, and is not a result of unique redox states of either protein . For Av1-Av2, ATP hydrolysis in the absence of Mg2+ occurred at nearly the same rate as reductant-dependent MgATP hydrolysis . Reductant-independent ATPase activity also occurred for the Av1-Cp2 and Cp1-Av2 heterologous crosses and was 2-fold and 18-fold slower than the Av1-Av2 or Cp1-Cp2 combinations . In both cases further hydrolysis of MgADP to AMP and AMP to adenosine occurred . A unique nucleotide hydrolysis system is apparently operating in the complex formed between the two nitrogenase proteins in the absence of reductant . The relationship between the reductant-independent and reductant-dependent activities of nitrogenase catalysis is explored. Gene, 1995 Nov 7, 165(1), 147 - 8 Identification of the gene encoding a mechanosensitive channel MscL homologue in Clostridium perfringens; Matsushita O et al.; The mscL gene, which encodes the protein forming a large-conductance mechanosensitive channel (MscL) in Escherichia coli, has previously been cloned and sequenced by Sukharev et al . {Nature 368 (1994) 265-268} . We found a gene homologous to mscL in Clostridium perfringens which is located just downstream from the collagenase-encoding gene in the opposite direction. J Vasc Interv Radiol, 1995 Nov-Dec, 6(6), 933 - 7 Anaerobic culture yield in interventional radiologic drainage procedures; Malden ES et al.; PURPOSE: This study was designed to determine the yield of anaerobic cultures from percutaneous radiologic drainage procedures . PATIENTS AND METHODS: Anaerobic culture results in 317 patients from June 1992 to May 1994 were retrospectively examined . Anaerobic specimens were placed in specially designed anaerobic culture tubes and not blood culture media . Patients had undergone the following procedures: percutaneous nephrostomy (105 patients), biliary drainage (65 patients), and abdominal abscess drainage (147 patients) . Aerobic culture results were tabulated in those patients with positive anaerobic cultures . RESULTS: Overall, 10% of patients (n = 32) had positive anaerobic cultures (Bacteroides species, n = 25; Clostridium, n = 6; other organisms, n = 4) . Anaerobes were isolated in 13% (n = 19) of abdominal abscess drainages, 8% (n = 8) of nephrostomy drainages, and 8% (n = 5) of biliary drainages . Aerobic isolates were present in 78% (n = 25) of patients with anaerobic infection . CONCLUSION: The yield for anaerobic cultures varies for different types of percutaneous drainage procedures from 8% to 13% . When isolated, anaerobic bacteria are frequently mixed with aerobic bacteria . Anaerobic culture usage is recommended with abdominal abscess and biliary drainages . Anaerobic bacterial cultures are not recommended for percutaneous nephrostomy unless the patient has a urinary tract malignancy or has undergone urinary instrumentation. Drug Saf, 1995 Nov, 13(5), 317 - 28 A risk-benefit assessment of teicoplanin in the treatment of infections; de Lalla F et al.; Teicoplanin is a glycopeptide antibiotic whose activity is selectively oriented against Gram-positive aerobic and anaerobic bacteria, including Staphylococcus aureus, coagulase-negative staphylococci, Clostridium difficile, Peptostreptococcus spp . and Corynebacterium jeikeium; such activity is affected by neither methicillin resistance nor beta-lactamase production . Teicoplanin is not significantly absorbed from the gastrointestinal tract; consequently, it has to be administered intravenously (either by infusion or by rapid injection) or intramuscularly . Its long half-life allows regimens based upon once daily administration . The adverse effects most frequently associated with teicoplanin treatment are local and hypersensitivity reactions, such as itching and drug fever; anaphylactoid reactions (the 'red man syndrome') are seldom observed . Teicoplanin also has less potential than vancomycin to cause nephrotoxicity, especially when administered in combination with an aminoglycoside . Teicoplanin has been proven to be effective in the treatment of microbiologically documented Gram-positive infections, including 'difficult to treat infections' such as endocarditis and prosthetic infections . Furthermore, recent trials in patients with haematological malignancies or other cancers have clearly demonstrated that teicoplanin is at least as efficacious as vancomycin in the empirical initial antibiotic regimen for febrile neutropenic patients, and is associated with fewer adverse effects . Finally, owing to its good tolerability profile and the advantage of once daily administration by both intravenous and intramuscular routes, teicoplanin has proven to be very useful for the outpatient treatment of serious Gram-positive infections . In conclusion, teicoplanin is potentially an effective alternative to vancomycin both in immunocompetent and immunocompromised patients, with the advantage over vancomycin of single daily dose administration and lower toxicity . Further comparative studies with vancomycin are, however, required to better define the therapeutic role of teicoplanin for particular infections (i.e . infective endocarditis). Toxicon, 1995 Nov, 33(11), 1519 - 30 Inhibition by clostridial neurotoxins of calcium-independent {3H}noradrenaline outflow from freeze-thawed synaptosomes: comparison with synaptobrevin hydrolysis; Hausinger A et al.; Clostridial neurotoxins are known to inhibit regulated, i.e . calcium-dependent exocytosis . In the present study we have investigated their potential role in also inhibiting calcium-independent exocytosis . Synaptosomes from rat forebrain were preloaded with {3H}noradrenaline and permeab