Ginecol Obstet Mex, 1992 Jun, 60, 162 - 70 {Gynecologic and obstetric infections caused by aerobic bacteria}; Figueroa-Damian R et al.; The anaerobic bacteria (AB) are between the most numerous microorganisms (mo) that constitute the flora of the female genital tract, so they can participate in the etiology of obstetric and gynecologic infections (OGI) . The objective of this study was to investigate the frequency of AB isolations and the clinical characteristics of the anaerobic infections (AI) in patients of the National Institute of Perinatology, from January 1st, 1988 to May 31, 1991 . AB were isolated from 117 patients who developed 163 infections; 167 anaerobic and 83 aerobic bacteria were recovered from these infections . The 99.2% were obstetric patients . The 85.5% of the isolations of AB were done from patients with endometritis, and 8.5% from postsurgical wound abscesses . Most of the AI were polymicrobial with a mean of 2.1 mo by infection . Peptostreptococcus, Clostridium and Bacteroids were the AB most frequent recovered . The majority of the patients had resolution of the infection within the first 5 days of antimicrobial treatment . There was no mortality in this group . We concluded that the AB have an important role in the etiology of OGI, then it is necessary that the treatment of these infections include antibiotics that cover AB. Poult Sci, 1992 Jun, 71(6), 959 - 69 Barley inclusion and avoparcin supplementation in broiler diets . 1 . Effect on small intestinal bacterial flora and performance; Hofshagen M et al.; Diets based on barley or corn without avoparcin supplementation were associated with high counts of Clostridium perfringens in the contents of the small intestine of the birds at the age of 2 to 4 wk . The weight gain of birds younger than 2 wk and the body weight of 4-wk-old birds were significantly lower, and the feed conversion ratio at slaughter was significantly higher, in birds on barley diets than in birds on corn diets . The frequency of birds with sticky droppings on Day 21 was significantly higher for barley diets . The number of C . perfringens, and the feed conversion ratio at slaughter were significantly lower but the number of coliform bacteria, weight gain during the 3rd wk, and body weight of 4-wk-old chickens were significantly higher when the diets were supplemented with 7.5 mg avoparcin/kg feed . The effect of avoparcin on the feed conversion ratio was statistically significant only on a barley diet. J Clin Invest, 1992 Jun, 89(6), 1866 - 74 Production of the Pseudomonas aeruginosa neuraminidase is increased under hyperosmolar conditions and is regulated by genes involved in alginate expression; Cacalano G et al.; The pathogenesis of Pseudomonas aeruginosa infection in cystic fibrosis (CF) is a complex process attributed to specific characteristics of both the host and the infecting organism . In this study, the properties of the PAO1 neuraminidase were examined to determine its potential role in facilitating Pseudomonas colonization of the respiratory epithelium . The PAO1 neuraminidase was 1000-fold more active than the Clostridium perfringens enzyme in releasing sialic acid from respiratory epithelial cells . This effect correlated with increased adherence of PAO1 to epithelial cells after exposure to PAO1 neuraminidase and was consistent with in vitro studies demonstrating Pseudomonas adherence to asialoganglioside receptors . The regulation of the neuraminidase gene nanA was examined in Pseudomonas and as cloned and expressed in Escherichia coli . In hyperosmolar conditions neuraminidase expression was increased by 50% (P less than 0.0004), an effect which was OmpR dependent in E . coli . In Pseudomonas the osmotic regulation of neuraminidase production was dependent upon algR1 and algR2, genes involved in the transcriptional activation of algD, which is responsible for the mucoid phenotype of Pseudomonas and pathognomonic for chronic infection in CF . Under the hyperosmolar conditions postulated to exist in the CF lung, nanA is likely to be expressed to facilitate the initial adherence of Pseudomonas to the respiratory tract. Eur J Biochem, 1992 Jun 1, 206(2), 547 - 52 (R)-lactyl-CoA dehydratase from Clostridium propionicum . Stereochemistry of the dehydration of (R)-2-hydroxybutyryl-CoA to crotonyl-CoA; Hofmeister AE et al.; 1 . A new two-step method for purifying component E II of lactyl-CoA dehydratase was developed . The source of the enzyme was Clostridium propionicum grown on either D,L-alanine or L-threonine . No difference in these preparations was observed whether during purification or by SDS/PAGE of the pure enzymes . Both preparations exhibited similar activities towards (R)-lactyl-CoA as well as towards (R)-2-hydroxybutyryl-CoA, the latter being the superior substrate . 2 . Three species of (2R)-2-hydroxybutyrate labelled with 3H at C3 were prepared containing 96%, 37% and 63% of the 3H in the 3S-position . By incubation of these species with acetyl-CoA, propionate CoA-transferase and lactyl-CoA dehydratase 104%, 32% and 70% of the 3H, respectively, was release as 3HOH . The data indicate that stereospecific abstraction of the 3Si hydrogen of (2R)-2-hydroxybutyryl-CoA during the dehydration . 3 . The identity of the product of the dehydration as crotonyl-CoA was established by the combined action of the enzymes crotonase and (S)-3-hydroxyacyl-CoA dehydrogenase . The results indicate that the elimination of water from (R)-2-hydroxybutyryl-CoA occurs in a syn mode . 4 . All enzyme activities necessary for the conversion of L-threonine via (R)-2-hydroxybutyryl-CoA to butyrate were detected in cell-free extracts of C . propionicum . 5 . A new mechanism for the dehydration of lactyl-CoA is proposed. Eur J Biochem, 1992 Jun 1, 206(2), 537 - 46 Rac1, a low-molecular-mass GTP-binding-protein with high intrinsic GTPase activity and distinct biochemical properties; Menard L et al.; Rac1, a member of the family of low-molecular-mass GTP-binding proteins, functions in phagocytic leukocytes as a component necessary for activation of the respiratory burst . To characterize the biochemical properties of rac1, the protein was expressed as a fusion protein in Escherichia coli and purified to greater than 99% homogeneity by affinity chromatography . Rac1 protein bound maximally bound and hydrolyzed GTP under low free-Mg2+ concentrations . Under those conditions, (45 nm free Mg2+), purified rac1 exhibited a steady-state GTPase activity of 18 nmol.min-1.mg protein-1 (turnover number approximately 0.39 min-1 at 37 degrees C), which is 40-fold higher than H-ras . The high intrinsic GTPase activity of rac1 under low free Mg2+ was mainly due to an increased kcat, the rate constant for hydrolysis of bound GTP, which was 0.29 min-1 for rac1 vs 0.007 min-1 for H-ras (at 20 degrees C) . Rac1 also released bound GDP faster than H-ras (koff.GDP = 1.02 min-1 for rac1 vs 0.33 min-1 for H-ras at 20 degrees C) . In contrast, rac1 released bound guanosine 5'-{gamma-thio}triphosphate (GTP{S}) at a slower rate than H-ras (koff.GTP{S} approximately 0.04 min-1 for rac1 vs 0.31 min-1 for H-ras at 20 degrees C) . Rac1 was a very good substrate for in vitro geranylgeranylation (C20) but not for farnesylation (C15), whereas the converse is true for H-ras . Surprisingly, rac1 was a very poor substrate for in vitro ADP-ribosylation by the C3 component of Clostridium botulinum toxin compared to rhoA . As a further characterization of rac1, a mutant was made in which the Thr115 was replaced by asparagine . This protein (referred to as {Thr115----Asn}rac1) contains the consensus amino acids of all four GTP-binding domains of H-ras . The koff.GDP of {Thr115----Asn}rac1 was reduced to that of H-ras, but {Thr115----Asn}rac1 exhibited essentially identical kcat (0.13 min-1 at 20 degrees C) and koff-GTP{S} (0.03 min-1 at 20 degrees C) values as the wild-type protein . Thus, the region(s) in rac1 which control the dissociation of GTP{S} (and presumably GTP) do not entirely coincide with those controlling GDP dissociation . Biochemical analysis of {Thr115----Asn}rac1 also suggests that the region responsible for the increased kcat of rac1 is not within the consensus amino acids of the four guanine-nucleotide-binding domains. J Trop Pediatr, 1992 Jun, 38(3), 129 - 31 Pathogens in neonatalomphalitis; Airede AI; During a 3-year study period, 33 neonates with omphalitis (with proven cultures) were encountered; aerobic and anaerobic cultures were obtained . An incidence of 2/1000 live births was recorded, with a high prevalence rate of 15.6/1000 admissions noted . Aerobes were isolated from 23 (70 per cent) specimens, whilst anaerobes were recovered alone in five (15 per cent) cases . There were 40 (1.2 per specimen) and 31 (0.9 per specimen) aerobic and anaerobic isolates, respectively . There was a significant detection of anaerobic pathogens such as the B . fragilis group (14), Gram-positive cocci (4) and Clostridium perfringens (3) . Beta lactamase production was seen in 25 isolates, recovered from 25 newborns. Clin Otolaryngol, 1992 Jun, 17(3), 223 - 4 Management of facial synkinesis with Clostridium botulinum toxin injection; Mountain RE et al.; Associated movements after facial paralysis (synkinesis), due to unphysiological co-innervation of the facial muscles, often complicates the rehabilitation of patients following facial palsy . Clostridium botulinum toxin is a neurotoxin that interferes with the release of acetylcholine from motor nerve end plates, causing skeletal muscular paralysis . This paper concentrates on its clinical use in treating synkinesis affecting orbicularis oculi function and documents the results of treatment in 4 patients . Control of synkinesis, achieved in all 4 patients, was effective within a few days and lasted for 4-6 months . 2 patients developed transient diplopia and ptosis shortly after injection . However, no lasting complications or systemic side-effects were noted . All patients reported a significant improvement in their symptoms and reinjection at 7 months was carried out successfully. J Pediatr Surg, 1992 Jun, 27(6), 744 - 6 Perforated pseudomembranous colitis in the breast-fed infant; Harmon T et al.; Pseudomembranous colitis (PMC) is uncommon in the infant and complications requiring surgical intervention are rare . All prior cases have involved the direct administration of antibiotics to the child . A 2-month-old girl required bowel resection for perforation of a thickened and inflamed left colon . Findings were consistent with PMC and the stool was Clostridium difficile toxin positive . The patient was treated with vancomycin and did well . The patient's mother later admitted to self-administration of ciprofloxacin while breast-feeding her infant . Oral doses of this drug are concentrated in breast milk at levels higher than serum . Antibiotics derived from breast milk can cause complicated PMC in the infant . A directed history can establish an early diagnosis and help distinguish PMC from other more common infantile enterocolitides. J Diarrhoeal Dis Res, 1992 Jun, 10(2), 87 - 92 Colonisation with digoxin-reducing strains of Eubacterium lentum and Clostridium difficile infection in nursing home patients; Bennett RG et al.; Stool specimens obtained from 77 residents of a nursing home were analysed to determine the relationship between colonisation with digoxin-reducing strains of Eubacterium lentum and infection with Clostridium difficile . Patients were categorised according to previous antibiotic treatment, prescription of enteral feedings, and pattern of bowel habits . Colonisation with digoxin-reducing E . lentum was less common in subjects infected with C . difficile, in those treated with antibiotics previously, and in those prescribed enteral feedings . Normal bowel habits were more common in those without C . difficile . The lowest incidence of diarrhoea was seen in patients without C . difficile who were colonised with digoxin-reducing species . This study establishes an inverse relationship between the presence of C . difficile and E . lentum that reduce digoxin which is related to previous treatment with antibiotics and prescription of enteral feedings . Bacterial markers may prove to be a useful tool for predicting clinical disturbances in bowel function. Int J Food Microbiol, 1992 Jun, 16(2), 117 - 21 Detection of Clostridium botulinum in natural sweetening; Nakano H et al.; Various sugar products were examined for contamination with C . botulinum spores . Type A, B and C spores were detected in three of 56 samples of sugar for apiculture, which may attest the significance of bee-feed as a source of contamination of honey . The heavy contamination of honey with C . botulinum spores sometimes encountered, however, can not be explained unless some other factors, e.g., that allowing germination and multiplication of the spores somewhere during honey production, are found . Type A spores were detected in some samples of raw sugar and molasses and also in two of 41 samples of brown sugar lump, but not in refined sugar or other various samples taken at a sugar factory or in sugar cane left on the field in Okinawa . The fact that some natural sweetenings are contaminated with C . botulinum spores, even in low concentrations, may be food-hygienically important. Antimicrob Agents Chemother, 1992 Jun, 36(6), 1332 - 5 Effects of antibiotics and other drugs on toxin production in Clostridium difficile in vitro and in vivo; Barc MC et al.; In an attempt to understand more completely why patients treated with phenothiazines (chlorpromazine and cyamemazine), methotrexate, and certain antibiotics such as clindamycin have an increased risk of developing pseudomembranous colitis, the production of toxins A and B by Clostridium difficile in the presence of these drugs was measured in vitro as well as in vivo by using axenic mice . None of the drugs tested increased the production of toxins either in vitro or in vivo. J Clin Microbiol, 1992 Jun, 30(6), 1544 - 50 Monoclonal antibodies specific for Clostridium difficile toxin B and their use in immunoassays; Muller F et al.; Five mouse monoclonal antibodies (MAbs) against Clostridium difficile toxin B have been raised and characterized . Three of them were immunoglobulin M (IgM) antibodies (6B10, 6G3, and 10B9), and the other two were of the IgG1 isotype (9E5 and 17G2), recognizing specifically two distinct epitopes on the toxin B molecule . No MAb was able to neutralize cytotoxic activity significantly . The two IgG1 MAbs were purified and applied to various immunodiagnostic assays . MAbs coupled to latex beads were used for specific removal of toxin B from cytotoxic samples and for agglutination assay . An indirect sandwich enzyme-linked immunosorbent assay with MAb 9E5 or 17G2 as the capture antibody was established for identification of toxin B with a lower detection limit of 5 ng/ml. Infect Immun, 1992 Jun, 60(6), 2488 - 92 Localization of two epitopes recognized by monoclonal antibody PCG-4 on Clostridium difficile toxin A; Frey SM et al.; The toxin A gene of Clostridium difficile contains a 2.5-kb region encoding a series of contiguous repeating units located at the COOH terminus of the molecule . We previously showed that the monoclonal antibody (MAb) PCG-4, which neutralizes the enterotoxic activity of toxin A, binds to epitopes located within these repeating units . In the present study, we subcloned a series of fragments from this portion of the gene . The recombinant peptides expressed from the gene fragments were examined for reactivity with MAb PCG-4 to identify the epitopes involved in binding . Our results showed that MAb PCG-4 recognizes epitopes in amino acid residues 2097 through 2141 and amino acid residues 2355 through 2398. Biochem Biophys Res Commun, 1992 May 29, 185(1), 341 - 9 Design and functional expression in Escherichia coli of a synthetic gene encoding Clostridium pasteurianum 2{4Fe-4S} ferredoxin; Davasse V et al.; A gene encoding the exact sequence of Clostridium pasteurianum 2{4Fe-4S} ferredoxin and containing 11 unique restriction endonuclease cleavage sites has been synthesized and cloned in Escherichia coli . The synthetic gene is efficiently expressed in E . coli and its product has been purified and characterized . The N-terminal sequence is identical to that of the protein isolated from C . pasteurianum and the recombinant ferredoxin contains the exact amount of {4Fe-4S} clusters (2 per monomer) expected for homogeneous holoferredoxin . It displays reduction potential and kinetic parameters as electron donor to C . pasteurianum hydrogenase I identical to those determined for the native ferredoxin . All of these properties demonstrate that the 2{4Fe-4S} ferredoxin expressed in E . coli is identical to the parent clostridial protein. J Biol Chem, 1992 May 25, 267(15), 10274 - 80 Purification and characterization of an ADP-ribosyltransferase produced by Clostridium limosum; Just I et al.; We purified a novel ADP-ribosyltransferase produced by a Clostridium limosum strain isolated from a lung abscess and compared the exoenzyme with Clostridium botulinum ADP-ribosyltransferase C3 . The C . limosum exoenzyme has a molecular weight of about 25,000 and a pI of 10.3 . The specific activity of the ADP-ribosyltransferase is 3.1 nmol/mg/min with a Km for NAD of 0.3 microM . Partial amino acid sequence analysis of the tryptic peptides revealed about 70% homology with C3 . The novel exoenzyme modifies selectively the small GTP-binding proteins of the rho family in human platelet membranes presumably at the same amino acid (asparagine 41) as known for C3 . Recombinant rhoA and rhoB serve as substrates for C3 and the C . limosum exoenzyme . Whereas recombinant rac1 protein is only marginally ADP-ribosylated by C3 or by the C . limosum exoenzyme in the absence of detergent, in the presence of 0.01% sodium dodecyl sulfate rac1 is modified by C3 but not by the C . limosum exoenzyme . Recombinant CDC42Hs protein is a poor substrate for C . limosum exoenzyme and is even less modified by C3 . The C . limosum exoenzyme is auto-ADP-ribosylated in the presence of 0.01% sodium dodecyl sulfate by forming an ADP-ribose protein bond highly stable toward hydroxylamine . The data indicate that ADP-ribosylation of small GTP-binding proteins of the rho family is not unique to C . botulinum C3 ADP-ribosyltransferase but is also catalyzed by a C3-related exoenzyme from C . limosum. Eur J Biochem, 1992 May 15, 206(1), 79 - 85 Purification and characterization of protein PC, a component of glycine reductase from Eubacterium acidaminophilum; Schrader T et al.; Protein PC of the glycine reductase from Eubacterium acidaminophilum was purified to homogeneity by chromatography on phenyl-Sepharose and Sepharose S . The apparent molecular mass of the native protein, which showed an associating/dissociating behaviour, was about 420 kDa . Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of protein PC revealed two protein bands corresponding to 48 and 57 kDa, indicating an alpha 4 beta 4 composition . The smaller subunit was identified as an acetyl-group-transferring protein, the 57-kDa protein was hydrophobic . N-terminal amino acid sequences were determined for both subunits . Antibodies raised against the 48-kDa subunit showed cross-reactions with extracts of E . acidaminophilum grown on different substrates and with extracts from other glycine-utilizing anaerobic bacteria such as Clostridium purinolyticum, C . sticklandii, and C . sporogenes . The respective protein from the former two organisms corresponded in molecular mass . When protein PA was chemically carboxymethylated by iodo{2-14C}acetate and incubated with protein PC, acetyl phosphate was a reaction product, thus establishing it as the product of the glycine reductase reaction by using homogeneous preparations of these two proteins from E . acidaminophilum. Eur J Biochem, 1992 May 15, 206(1), 151 - 9 The glutamate dehydrogenase gene of Clostridium symbiosum . Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli; Teller JK et al.; The gene encoding the NAD(+)-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques . The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein . The recombinant plasmid complemented the nutritional lesion of an E . coli glutamate auxotroph . There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%) . The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M(r) 49,295 which corresponds to a single subunit of the hexameric enzyme . The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme . The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host . The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-dependent enzymes . The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site-directed mutagenesis. Eur J Pharmacol, 1992 May 12, 226(1), 87 - 91 ADP-ribosylation of rho proteins is inhibited by melittin, mast cell degranulating peptide and compound 48/80; Koch G et al.; The amphiphilic agents melittin, mast cell degranulating peptide and compound 48/80 inhibit the ADP-ribosylation of the small GTP-binding proteins rho by Clostridium botulinum exoenzyme C3 . Half-maximal and maximal inhibition (greater than 90%) of ADP-ribosylation occurred at about 8 and 25 micrograms/ml for compound 48/80, at 10 and 45 microM for mast cell degranulating peptide and at 15 and 50 microM for melittin, respectively . In addition, these compounds increase the steady state GTP hydrolysis and the association and dissociation rate of GTP-binding of rho proteins through an increase of GDP/GTP exchange . The data suggest that the amphiphilic agents tested interact with small GTP-binding proteins of the rho protein family. J Biol Chem, 1992 May 5, 267(13), 8719 - 22 Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction; Hirata K et al.; In the rabbit mesenteric arterial smooth muscle skinned by saponin, Ca2+ induced contraction in a concentration-dependent manner . Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable GTP analogue, lowered the Ca2+ concentrations required for this contraction and increased the Ca2+ sensitivity of the skinned smooth muscle contraction . GTP gamma S alone did not induce the contraction in the absence of Ca2+ . This GTP gamma S-enhanced Ca2+ sensitivity was completely abolished by an exoenzyme of Staphylococcus aureus, named EDIN, and an exoenzyme of Clostridium botulinum, named C3, both of which are known to ADP-ribosylate the rho p21 family that belongs to the ras p21-like small GTP-binding protein superfamily . The GTP gamma S-bound form of rhoA p21 overcame the inhibitory action of EDIN . smg p21B, another small GTP-binding protein, was inactive . EDIN ADP-ribosylated a protein, which was most likely to be rho p21, in the skinned smooth muscle . The GTP gamma S-bound form of rhoA p21, but not the GDP-bound form, substituted for GTP gamma S and enhanced the Ca2+ sensitivity of the skinned smooth muscle contraction . smg p21B was inactive . These results indicate that rhoA p21 is involved in the GTP gamma S-enhanced Ca2+ sensitivity of the smooth muscle contraction. J Gen Microbiol, 1992 May, 138 ( Pt 5), 861 - 9 Autolysis of Clostridium acetobutylicum ATCC 824; Croux C et al.; The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined . Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer . The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth . Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations . The autolysin of C . acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase . The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence . The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol . A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed. Mol Gen Genet, 1992 May, 233(1-2), 260 - 8 Comparative sequence analysis of the Clostridium difficile toxins A and B; von Eichel-Streiber C et al.; The six clones pTB112, pTB324, pTBs12, pCd122, pCd14 and pCd13 cover the tox locus of Clostridium difficile VPI 10463 . This region of 19 kb of chromosomal DNA contains four open reading frames including the complete toxB and toxA genes . The two toxins show 63% amino acid (aa) homology, a relatedness that had been predicted by the cross-reactivity of some monoclonal antibodies (mAb) but that is in contrast to the toxin specificity of polyclonal antisera . A special feature of ToxA and ToxB is their repetitive C-termini . We define herein 19 individual CROPs (combined repetitive oligopeptides of 20-50 aa length) in the ToxB C-terminus, which are separable into five homologous groups . Comparison of the aa sequences of the N-terminal two-thirds of ToxA and ToxB revealed three marked structures, a cluster of 172 hydrophobic, highly conserved aa in the centre of both toxins, a sequence of 120 residues with an accumulation of highly conserved arginine, cysteine, histidine, methionine, and tryptophan residues, and a stretch of 248 less conserved aa . The probable function of these domains is discussed . Structural and functional homologies of ToxA and ToxB indicate that both genes have a common ancestor and may have evolved by gene duplication, with subsequent recombination and mutation, as has been reported for streptococcal glucosyltransferases (Gtf). J Med Microbiol, 1992 May, 36(5), 307 - 11 Proteolytic activity of Clostridium difficile; Seddon SV et al.; Ten isolates of Clostridium difficile expressing different degrees of toxigenicity and virulence in an animal model were assayed for the production of proteolytic enzymes by various methods . All strains demonstrated some activity in one or more of the assay systems . There was no direct correlation between toxigenic status and enzyme production . However, those strains known to be highly virulent in a hamster model were the most proteolytic . The most commonly detected enzyme was cell associated, and its substrate specificity suggested it was a trypsin-like enzyme . Initial purification of the enzyme from strain VPI 10463 gave a 10% yield with a 14-fold increase in purity . Inhibition studies on this preparation indicated that the enzyme was a thiol protease . The enzyme has pH and temperature optima of 7.5 and 37 degrees C, respectively . These characteristics suggest that the enzyme is more related to clostripain, the thiol clostridio-peptidase of C . histolyticum, than to trypsin . Whilst the role of this enzyme remains unclear, it is possible that it may be a contributory factor in the virulence of the organism as described for other clostridial infections. Mol Biol Evol, 1992 May, 9(3), 495 - 506 Nucleotide sequence of nifD from Frankia alni strain ArI3: phylogenetic inferences; Normand P et al.; The complete nucleotide sequence of the nifD gene encoding the alpha subunit of component I of nitrogenase from Frankia alni strain ArI3 was determined . The coding region is 1,458 bp in length and encodes a polypeptide of 486 residues with a predicted molecular weight of 53,500 . Phylogenetic inferences with 12 complete published nifD sequences were drawn using a variety of approaches . Frankia nifD clusters with proteobacteria rather than with Clostridium pasteurianum, the other Gram-positive bacterium studied . Extant eubacterial nif genes seem to have at least three distinct evolutionary origins as a result of ancient gene duplications . Within the Gram-positive bacterial phylum, functional nif genes descend from different duplicates. J Clin Microbiol, 1992 May, 30(5), 1085 - 8 Multicenter evaluation of a new enzyme immunoassay for detection of Clostridium difficile enterotoxin A; De Girolami PC et al.; The Premier Clostridium difficile toxin A enzyme immunoassay (PTA EIA) (Meridian Diagnostics, Inc., Cincinnati, Ohio) for rapid diagnosis of antibiotic-associated colitis (AAC) was evaluated in a multicenter study . Stool samples from 421 patients suspected of having AAC were tested for toxin A by the PTA EIA and for toxin B by three tissue culture assays (TCA) employing WI-38 cells (New England Deaconess Hospital) in conventional tubes or foreskin fibroblasts (Children's Hospital) or Vero cells (Beth Israel Hospital) in microwells . The tubes and plates were examined at 24 and 48 h for cytotoxicity . Clinical criteria, repeat testing at another site, and culture of frozen stool samples for C . difficile were used to evaluate discrepant results . Of 504 samples, 66 were positive and 409 were negative by both tests . Eight samples had indeterminate PTA EIA results and were excluded from this analysis . Of 21 discrepancies, 9 were PTA EIA positive and TCA negative and 12 were PTA EIA negative TCA positive . Following resolution of the discrepancies, 11 of 12 PTA EIA-negative-TCA-positive and 5 of 9 PTA EIA-positive-TCA-negative samples were considered true positive for AAC . The sensitivity and specificity were, respectively, 86.6 and 99.0% for the PTA EIA and 93.9 and 99.8% for TCA . The predictive values of positive and negative tests were, respectively, 94.7 and 97.4% for the PTA EIA and 98.7 and 98.8% for TCA . We conclude that the PTA EIA is a rapid, simple EIA technique whose accuracy in detecting enterotoxin A approaches that of reference TCA methods for detection of cytotoxin B. Arch Pathol Lab Med, 1992 May, 116(5), 517 - 20 Evaluation of the latex agglutination test for detection of Clostridium difficile; Kelly WF et al.; We compared two Clostridium difficile latex agglutination tests, Meritec from Meridian Diagnostic (Cincinnati, Ohio) and CDT from Becton-Dickinson (Cockeysville, Md), on 289 specimens submitted for tissue culture cytotoxicity using MRC-5 cells . When compared with CDT, the Meritec latex agglutination test had a sensitivity of 90% (26/29), a specificity of 97% (251/260), and a correlation of 96% . Meritec was compared with tissue culture cytotoxicity on 357 specimens . Meritec had a sensitivity of 77% (30/39), a specificity of 93% (298/318), and a correlation of 92% . Clinical review of 10 Meritec +/- tissue culture cytotoxicity minus patients revealed one likely, two probable, and seven doubtful cases of C difficile disease . In contrast, review of 10 Meritec +/- tissue culture cytotoxicity plus patients showed seven likely and three probable cases of C difficile disease . The Meritec is comparable with the CDT latex agglutination test, but is not nearly as sensitive as either tissue culture assay or culture for detection of C difficile disease . A positive latex agglutination test should be confirmed by a tissue culture cytotoxicity assay. J Bacteriol, 1992 May, 174(10), 3290 - 9 Molecular characterization of the dnaK gene region of Clostridium acetobutylicum, including grpE, dnaJ, and a new heat shock gene; Narberhaus F et al.; The dnaK gene region of Clostridium acetobutylicum was cloned in Escherichia coli by using the pBluescript SK+ and pUC18 vectors . By using the E . coli dnaK gene as a probe and by in vivo chromosome walking, three positive clones harboring the recombinant plasmids pKG1, pKG2, and pKG3 containing 1.2-kbp HindIII, 3.55-kbp EcoRV, and 1.2-kbp PstI fragments of the chromosome of C . acetobutylicum, respectively, were isolated . The cloned fragments partially overlapped, and together they spanned 4,083 bp of the clostridial genome that were completely sequenced . On one strand, four open reading frames of which the last was obviously truncated were identified . The last three genes showed high homology to the grpE, dnaK, and dnaJ heat shock genes of E . coli, respectively . They were preceded by an open reading frame (orfA) without any homology to sequences available in the EMBL or GenBank data bases . Typical translational start sites could be found in front of all four genes . Northern (RNA) blot analysis revealed transcripts of this region with a maximum length of 5.0 kb . Thus, these genes are probably organized in an operon . A transcription terminator could be found between the dnaK and dnaJ genes . By primer extension analysis, a major heat-inducible transcription start site was identified 49 bases upstream of orfA . This site was preceded by a region (5'-TTGACA{17 bp}TATTTT) that exhibited high homology to the consensus promoter sequences of gram-positive bacteria as well as sigma 70-dependent E . coli . Between this promoter and the initiation codon of orfA, a hairpin-loop structure with a possible regulatory role in the expression of these genes was found . Additional heat-inducible transcription start sites were located 69 bases upstream of orfA and 87 bases upstream of grpE; the corresponding promoter regions showed less similarity to other known promoter sequences . Maximum mRNA levels of this heat shock operon were found about 15 min after a heat shock from 30 to 42 degrees C . Our results indicate that orfA codes for an unknown heat shock protein. J Bacteriol, 1992 May, 174(9), 2874 - 80 Binding of DNA to alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species prevents formation of cytosine dimers, cytosine-thymine dimers, and bipyrimidine photoadducts after UV irradiation; Fairhead H et al.; Small, acid-soluble proteins (SASP) of the alpha/beta-type from spores of Bacillus and Clostridium species bind to DNA; this binding prevents formation of cyclobutane-type thymine dimers upon UV irradiation, but promotes formation of the spore photoproduct, an adduct between adjacent thymine residues . alpha/beta-Type SASP also bound to poly(dG).poly(dC) and poly(dA-dG).poly(dC-dT) . While UV irradiation of poly(dG).poly(dC) produced cyclobutane-type cytosine dimers as well as fluorescent bipyrimidine adducts, the yields of both types of photoproduct were greatly reduced upon irradiation of alpha/beta-type SASP-poly(dG).poly(dC) complexes . UV irradiation of poly(dA-dG).poly(dC-dT) produced a significant amount of a cyclobutane dimer between cytosine and thymine, as well as a 6-4 bipyrimidine adduct . Again, binding of alpha/beta-type SASP to poly(dA-dG).poly(dC-dT) greatly reduced formation of these two photoproducts, although formation of the cytosine-thymine analog of the spore photoproduct was not observed . These data provide further evidence for the dramatic change in DNA structure and photoreactivity which takes place on binding of alpha/beta-type SASP and suggest that binding of these proteins to DNA in vivo prevents formation of most deleterious photoproducts upon UV irradiation. Toxicon, 1992 May-Jun, 30(5-6), 653 - 68 Isolation of immunogenic and lethal peptides of alpha-toxin from Clostridium novyi type B; Schranner I et al.; The lethal alpha-toxin was isolated from the culture filtrate of Clostridium novyi type B using ammonium sulfate precipitation and ion exchange chromatography . The alpha-toxin has a mol . wt of 190,000 and does not contain any disulfide cross-linkages . It consists of a single polypeptide chain . The peptide fragments resulting from the cyanogen-bromide cleavage were isolated using reversed phase and gel filtration HPLC . The immunogenic actions of these peptides and peptide mixtures were studied in Balb/c mice . Three polyclonal antisera recognizing the uncleaved native toxin could be found using an ELISA test (Br3, Bro2, Bro5) . One peptide mixture (Tx5), which was proved lethal in shell-less quail eggs (in vitro), was rechromatographed with gel filtration HPLC that resulted in one peptide with mol . wt 3000 (Txleth), which again proved lethal in the shell-less quail egg lethality test . The immunogenic peptides differ from the lethal one, therefore we assumed different locations on the polypeptide chain . The separation of the immunogenic, non-toxic fragment from the lethal one may allow the production of a highly specific non-toxic vaccine . By using synthetically produced immunogenic peptides, time-consuming purification methods and working with the whole toxin will become unnecessary. Plasmid, 1992 May, 27(3), 207 - 19 Construction of a sequenced Clostridium perfringens-Escherichia coli shuttle plasmid; Sloan J et al.; A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled . The vector, pJIR418, contains the replication regions from the C . perfringens replicon pIP404 and the E . coli vector pUC18 . The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E . coli . Both chloramphenicol and erythromycin resistance can be selected in C . perfringens and E . coli since pJIR418 carries the C . perfringens catP and ermBP genes . Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms . The versatility of pJIR418 and its applicability for the cloning of toxin genes from C . perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C. Indian J Med Res, 1992 May, 95, 148 - 51 Bacteriology of ophthalmic infections with special reference to anaerobes; Aggarwal R et al.; A total of 138 eye specimens were processed for aerobic and anaerobic cultures . Clinical data were obtained from 50 patients with unilateral ophthalmic infection . Cultures from the uninfected eye of 38 of these 50 patients were also processed for comparison . In addition, 50 cultures were obtained from one or both eyes of 30 healthy controls who had no eye infection . Anaerobes and aerobes were isolated from infected eyes of 6 (12%) and 37 (74%) patients respectively . No growth was observed in infected eye of 8 (16%) patients . A mixture of aerobes and anaerobes were recovered only in 2 cases . Of the total 47 aerobic isolates from infected eye specimens, Staphylococcus aureus (11), coagulase negative staphylococci (12) and Streptococcus pneumoniae (9) were predominant isolates . Six anaerobes isolates included Gram positive nonsporing anaerobic bacilli (4 including Propionibacterium acne) as predominant isolates . Clostridium perfringens was isolated from a case of post operative endoophthalmitis . From the uninfected eye of same patients though the number and types of aerobic bacteria were similar, none grew any anaerobes . Aerobic and anaerobic bacteria were isolated in 70 and 6 per cent of eye swabs respectively from the healthy controls. Pharmacol Res, 1992 May-Jun, 25(4), 373 - 81 In vitro activity of sulphimidazole alone and in association with trimethoprim against enteric pathogens; Castelli M et al.; Sulphimidazole is a new sulphonamide belonging to the class of intestinal sulphonamides and characterized by the fact that it is active even in vitro . It has the heterocyclic ring of 5-nitroimidazoles on amidic nitrogen . Its antibacterial activity is similar to that of the classical sulphonamides but differs in that it also combats certain anaerobic bacteria such as Clostridium botulinum . This effect is completely absent in the case of sulphadiazine and sulphamethoxazole . Also, since p-amino-benzene-sulphonamide is present in the molecule, the drug acts in synergism with trimethoprim against certain aerobic or facultative strains of enteric pathogens. Infect Immun, 1992 May, 60(5), 2110 - 4 Mapping of functional regions of Clostridium perfringens type A enterotoxin; Hanna PC et al.; Studies were conducted to allow construction of an initial map of the structure-versus-function relationship of the Clostridium perfringens type A enterotoxin (CPE) . Removal of the N-terminal 25 amino acids of CPE increased the primary cytotoxic effect of CPE but did not affect binding . CPE sequences required for at least four epitopes were also identified. Biosci Biotechnol Biochem, 1992 May, 56(5), 788 - 93 Cloning and nucleotide sequence of a frxC-ORF469 gene cluster of Synechocystis PCC6803: conservation with liverwort chloroplast frxC-ORF465 and nif operon; Ogura Y et al.; A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase . Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known . Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469-ORF248) was isolated from the cyanobacterium Synechocystis PCC6803 . In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively . Synechocystis frxC showed 31% amino acid sequence identity with nifH1 from Clostridium pasteurianum . Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C . pasteurianum nifK product, which is the beta subunit of a molybdenum-iron protein of a nitrogenase complex . Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution. J Bacteriol, 1992 May, 174(10), 3282 - 9 Cloning, sequencing, and molecular analysis of the groESL operon of Clostridium acetobutylicum; Narberhaus F et al.; The groESL operon of Clostridium acetobutylicum was cloned in Escherichia coli by using a gene probe of E . coli groESL . Sequencing of a positively reacting 2.2-kbp HindIII fragment contained in the recombinant plasmid pFN1 and a 2.5-kbp XbaI fragment present in pFN4 revealed that both fragments partially overlapped and together spanned 3,493 bp of the clostridial chromosome . Two complete open reading frames (288 and 1632 bp) were found and identified as the groES- and groEL-homologous genes of C . acetobutylicum, respectively . The 3' end of a third gene (orfZ), which was divergently transcribed, showed no significant homology to other sequences available in the EMBL and GenBank data bases . The length of the groESL-specific mRNA (2.2 kb), a transcription terminator downstream of groEL, and a transcription start site upstream of groES, identified by primer extension analysis, indicated that groES and groEL of C . acetobutylicum are organized in a bicistronic operon . From the transcription start site, the promoter structure 5'-TTGCTA (17 bp) TATTAT that shows high homology to the consensus promoter sequence of gram-positive bacteria as well as E . coli was deduced . Transcription of the groESL operon was strongly heat inducible, and maximum levels of mRNA were detected 15 min after heat shock from 30 to 42 degrees C . An 11-bp inverted repeat, located between promoter and translation start sites of groES and partially identical with similar structures in front of several heat shock genes of other bacteria, may play an important role in the regulation of heat shock gene expression in this organism. Eur J Clin Microbiol Infect Dis, 1992 May, 11(5), 458 - 62 In vitro activity of the new glycopeptide decaplanin; Neu HC et al.; The activity of decaplanin, a new glycopeptide, was compared to that of vancomycin, teicoplanin and daptomycin . Decaplanin was two- to four-fold less active than vancomycin, telcoplanin and daptomycin against Staphylococcus aureus and Staphylococcus epidermidis, with an MIC90 of 2 micrograms/ml for methicillin-susceptible and 4 micrograms/ml for methicillin-resistant isolates . Decaplanin had activity similar to that of vancomycin against Streptococcus pyogenes, Streptococcus agalactiae, group C and G streptococci, with an MIC90 of 0.12 micrograms/ml . It was less active than the other agents against the viridans group streptococci (MIC90 4 micrograms/ml) . The activity of decaplanin against enterococci (MIC90 4 micrograms/ml) was similar to that of vancomycin . Clostridium spp . were inhibited by 0.5 micrograms/ml, peptostreptococci and peptococci by 0.25 microgram/ml . Decaplanin was active from pH 5.5 to 7.5 . Inoculum size had a minimal effect on MICs, and increased concentrations of Ca2+ and Mg2+ and 50% serum did not alter MICs or MBCs. J Pediatr, 1992 May, 120(5), 747 - 9 Electrodiagnosis reliability in the diagnosis of infant botulism; Graf WD et al.; Infant botulism is confirmed by isolation of Clostridium botulinum from stool culture or by toxin assay . Although electrodiagnosis has been described as a diagnostic tool in infant botulism, our 11-year review of toxin-confirmed cases suggests that electrodiagnosis is not a reliable tool . In the case report presented, results of electrodiagnosis were negative but enema effluent contained adequate concentrations of organism and toxin to confirm the diagnosis. J Mol Biol, 1992 Apr 20, 224(4), 1181 - 4 Effect of additives on the crystallization of glutamate dehydrogenase from Clostridium symbiosum . Evidence for a ligand-induced conformational change; Stillman TJ et al.; A new crystal form of the hexameric NAD(+)-linked glutamate dehydrogenase (GDH) from Clostridium symbiosum has been grown using the hanging drop method of vapour diffusion . The crystals are obtained either by using high concentrations of the amino acid substrate of the enzyme, glutamate, as the precipitant or by co-crystallization from ammonium sulphate in the presence of either p-chloromercuribenzene sulphonate or potassium tetracyanoplatinate . The crystals diffract well and X-ray photographs have established that they are in the space group R32 . Considerations of the values of Vm indicate that the asymmetric unit of the R32 crystals contains a single subunit . Packing considerations based on the structure of the native enzyme determined from a different crystal form suggest that the molecule must undergo a significant conformational change in order to be accommodated in the new cell . Such a conformational rearrangement may represent an important step in the catalytic cycle. Eur J Biochem, 1992 Apr 15, 205(2), 799 - 808 Novel oligosaccharide constituents of the cellulase complex of Bacteroides cellulosolvens; Gerwig GJ et al.; The multiple cellulase-containing protein complex, isolated from the cellulolytic bacterium Bacteroides cellulosolvens, contains oligosaccharides which are O-linked mainly to a 230-kDa subunit . The oligosaccharide chains were liberated by alkaline-borohydride treatment and fractionated as oligosaccharide alditols via gel-permeation chromatography and HPLC . The fractions were investigated by one- and two-dimensional (correlation, homonuclear Hartmann-Hahn, rotating-frame nuclear Overhauser enhancement) 500-MHz 1H-NMR spectroscopy in combination with monosaccharide and methylation analyses and with fast-atom-bombardment mass spectrometry . The following carbohydrate structures could be established: {formula: see text} The results indicate an interesting similarity between the oligosaccharide moieties of the cellulase complex of B . cellulosolvens and of Clostridium thermocellum {Gerwig, G . J., Kamerling, J . P., Vliegenthart, J . F . G., Morag (Morgenstern), E., Lamed, R . & Bayer, E . A . (1991) Eur . J . Biochem . 196, 115-122}, having 3, 5 and 6 as common elements . The furanose form of a terminal alpha-D-galactose residue demonstrated an inhibitory effect on the interaction of Griffonia simplicifolia I isolectin B4 with the cellulosome-like entity of B . cellulosolvens. Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3483 - 7 Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A; Shoseyov O et al.; The cbpA gene for the Clostridium cellulovorans cellulose binding protein (CbpA), which is part of the multisubunit cellulase complex, has been cloned and sequenced . When cbpA was expressed in Escherichia coli, proteins capable of binding to crystalline cellulose and of interacting with anti-CbpA were observed . The cbpA gene consists of 5544 base pairs and encodes a protein containing 1848 amino acids with a molecular mass of 189,036 Da . The open reading frame is preceded by a Gram-positive-type ribosome binding site . A signal peptide sequence of 28 amino acids is present at its N terminus . The encoded protein is highly hydrophobic with extremely high levels of threonine and valine residues . There are two types of putative cellulose binding domains of approximately 100 amino acids that are slightly hydrophilic and eight conserved, highly hydrophobic beta-sheet regions of approximately 140 amino acids . These latter hydrophobic regions may be the CbpA domains that interact with the different enzymatic subunits of the cellulase complex. Eur J Biochem, 1992 Apr 15, 205(2), 767 - 73 Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri; Michel C et al.; The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively . Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa) . Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin . This preparation could be reactivated to 95-100% by addition of adenosylcobalamin . The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity . 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin . Upon incubation of the mutase with {5'-3H}adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity . During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin . EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme . Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose . The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation. Eur J Biochem, 1992 Apr 15, 205(2), 759 - 65 Glutamate mutase from Clostridium cochlearium . Purification, cobamide content and stereospecific inhibitors; Leutbecher U et al.; Both components, E and S, of the adenosylcobalamin-(coenzyme B12)-dependent glutamate mutase from Clostridium cochlearium were purified . Component S (16 kDa) must be added to component E to obtain activity, although the latter contains substoichiometric amounts of component S besides the major 50-kDa subunit . The enzyme proved to be very similar to that of C . tetanomorphum as described by Barker et al . {Barker, H . A., Rooze, V., Suzuki, F . & Iodice, A . A . (1964) J . Biol . Chem . 239, 3260-3266} but component E of C . cochlearium was more stable and led to the first pure preparation . The pink component E showed a cobamide-like absorbance spectrum with a characteristic maximum at 470 nm indicating the presence of a cob(II)amide, probably Co alpha-{alpha-(aden-9-yl)}-cob(II)amide . A typical cob(II)amide signal at g = 2.23 with hyperfine and superhyperfine splitting was observed by EPR spectroscopy . A cobamide content of about 0.43 mol/mol 50-kDa subunit was determined by cyanolysis . Substitution of the migrating hydrogen at C-4 of glutamate by fluorine yielded the potent competitive inhibitor (2S,4S)-4-fluoroglutamate (Ki = 70 microM) . (2R,3RS)-3-Fluoroglutamate (Ki = 600 microM) was also inhibitory . The competitive inhibition by 2-methyleneglutarate (Ki = 400 microM) and (S)-3-methylitaconate (Ki = 100 microM) but not by (RS)-2-methylglutarate suggested the transient formation of an sp2 center during catalysis . However, the presence of an N-terminal pyruvoyl residue was excluded and no evidence for the participation of another electrophilic center in the reaction was obtained. Burns, 1992 Apr, 18(2), 167 - 9 Massive haemorrhage due to rectosigmoid ulcers in a patient with extensive burns; Law EJ et al.; A 36-year-old white-skinned male was admitted with 45.5 per cent burns, mostly of full skin thickness . Severe rectal bleeding from rectal ulcerations developed on postburn day 12 . Various conservative attempts at management failed, and after multiple transfusions, abdominoperineal resection was carried out with eventual complete recovery . Complications during his acute phase included Pseud . aeruginosa sepsis and Clostridium difficile diarrhoea . Extensive skin grafts were required . The cause of the rectal ulcerations is unclear. Mayo Clin Proc, 1992 Apr, 67(4), 369 - 72 An unusual case of myxedema megacolon with features of ischemic and pseudomembranous colitis; Patel R et al.; Myxedema megacolon is rare; usually, it manifests with abdominal distention, flatulence, and constipation . Herein we describe a 72-year-old man who had intermittent diarrhea, bloating, and abdominal pain for more than a year . Cultures of stool specimens for Clostridium difficile enterotoxin were variably positive and negative . Colonoscopic biopsy specimens were thought to be consistent with chronic ischemia . Thyroid function tests showed severe hypothyroidism; the patient's symptoms resolved with thyroid hormone replacement . We hypothesize that gross dilatation of the colon, attributed to myxedema, was followed by intestinal ischemia and complicated by recurrent episodes of pseudomembranous colitis . A review of the relevant literature is provided . This unusual manifestation of myxedema should be considered in the differential diagnosis when a patient has diarrhea, bloating, and abdominal pain. Biochim Biophys Acta, 1992 Apr 6, 1130(3), 259 - 66 The use of chimeric gene constructs to express a bacterial endoglucanase in mammalian cells; Hall J et al.; The synthesis and secretion of a truncated Clostridium thermocellum endoglucanase (EGE') encoded by the celE' gene was investigated in Chinese hamster ovary (CHO) cells . Fusion genes consisting of the human growth hormone (hGH) gene and celE', transcribed from the SV40 early enhancer/promoter, were constructed and stably transfected into CHO cells . A gene consisting of celE' inserted into the first exon of the hGH gene resulted in the synthesis of truncated proteins (less than or equal to 22 kDa) lacking endoglucanase activity . Cloning celE' into the second exon of the hGH gene, resulted in the synthesis and secretion of a 50 kDa protein with endoglucanase activity . A 50 kDa protein was also synthesised by cells transfected with celE' cloned into the fifth exon of the hGH gene . However, despite a 5-fold increase in enzyme activity compared to the exon 2 transfected cell line less than 40% of the protein was secreted . Constructs devoid of introns, in which celE' was fused to the SV40 early promoter and to the rabbit beta-globin polyadenylation sequence resulted in a 2-18-fold increase in endoglucanase activity compared to the constructs containing introns . In addition more than 75% of the synthesised protein was secreted . Analyses of EGE' encoded mRNA from the transfected cell lines suggests that the presence of introns results in the aberrant splicing of message by the use of cryptic splice sites in the celE' gene . These results demonstrate that introns are not required for the efficient expression of a bacterial endoglucanase in mammalian cells, rather introns appear to reduce expression of the encoded protein. Actas Urol Esp, 1992 Apr, 16(4), 354 - 6 {Bacteremia caused by Clostridium perfringens as a complication of ureterosigmoidostomy}; Batista-Miranda JE et al.; A 63 year old male underwent cystectomy and ureterosigmoidostomy after diffuse carcinoma "in situ" of the bladder was discovered, and thereafter, various episodes of pyelonephritis and metabolic imbalance, in one of them, a left pneumo-ureter and a positive blood culture for Clostridium Perfringens and enterococci was detected . Empiric therapy with Aztreonam was started, and changed after to high-dose intravenous amoxicillin . Two months later the ureterosigmoidostomy was converted to an ileal conduit . The patient has remained asymptomatic on subsequent controls. Avian Dis, 1992 Apr-Jun, 36(2), 469 - 73 High mortality of domestic turkeys associated with Ascaridia dissimilis; Norton RA et al.; Third- and fourth-stage Ascaridia dissimilis larvae were isolated from commercial white turkey intestinal scrapings from two farms that were experiencing high mortality . Lesions consisted of a necrotic-like enteritis that was most severe in the jejunum . Subsequent bacteriological isolation yielded heavy growth of Escherichia coli and Clostridium perfringens . The rate of mortality declined rapidly when the turkeys were administered 18 ppm fenbendazole for 7 days. Toxicon, 1992 Apr, 30(4), 419 - 26 Sensitivity in culture of epithelial cells from rhesus monkey kidney and human colon carcinoma to toxins A and B from Clostridium difficile; Torres J et al.; The effect of toxins A and B from Clostridium difficile on human colon carcinoma cells (HT-29, epithelial), rhesus monkey kidney cells (MA-104, epithelial) and green monkey kidney cells (VERO, fibroblast) was studied . Both toxins caused rounding of HT-29 cells and rounding with projections remaining attached to the substrate in MA-104 and VERO cells; however, the sensitivity to each toxin varies considerably . Toxin A was detected in ng by VERO, pg by HT-29 and fractions of pg by MA-104 cells; for toxin B, pg were detected by VERO, ng by MA-104 and micrograms by HT-29 cells . HT-29 cells were grown with galactose to allow their differentiation to enterocytes, and their sensitivity to the toxins during the process was studied . At early stages, the sensitivity to both toxins was similar, and as the differentiation proceeded, the response to both toxins decreased continuously, and after 16 days no evident morphological effect was observed, even with micrograms amounts of either toxin . In contrast to all cell lines reported to date, HT-29 and MA-104 epithelial cells are exquisitely sensitive to toxin A and less responsive to toxin B . The rounding of HT-29 by these toxins depends on the degree of differentiation of the cell. FEMS Microbiol Lett, 1992 Apr 1, 71(1), 5 - 9 Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers; McMillin DE et al.; Clostridium difficile is the causative agent for pseudomembranous colitis in humans . Toxic strains of C . difficile produce two toxins, toxin A and toxin B . A reliable and definitive method of typing the toxic strains of C . difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities . A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study . The C . difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains . These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined . The use of this typing scheme in clinical applications is discussed. Vet Microbiol, 1992 Apr, 31(1), 89 - 99 Development of double sandwich ELISA for Clostridium perfringens beta and epsilon toxins; el Idrissi AH et al.; Specific, double-sandwich ELISAs for beta and epsilon toxins were developed by coating wells of microplates with specific sheep antitoxin IgG and using specific rabbit antitoxin IgG as detecting antibodies . The assay for beta toxin detected a minimum level of 8 ng/ml of purified toxin . The assay for epsilon toxin was capable of detecting 2 ng/ml of purified toxin . When applied to detect the toxin in intestinal contents using 50% fetal bovine serum as diluent the lowest amounts detected were about at least 30 ng/ml for beta toxin and 4 ng/ml for epsilon toxin . Clear differences in ELISA readings of both assays have been found between culture filtrates from toxin and non-toxin producing strains . These results suggested that both assays described in this study could detect their respective toxin in buffers, culture supernatants or in intestinal contents. Nippon Yakurigaku Zasshi, 1992 Apr, 99(4), 191 - 203 {ras oncogene-related small molecular weight GTP-binding protein, rho gene product and botulinum C3 ADP-ribosyltransferase}; Morii N et al.; The ras oncogene products (ras p21s) are 21-KDa proteins with activities of GTP binding and hydrolysis . A number of proteins homologous to ras p21 have been discovered and collectively named small molecular weight GTP-binding proteins . These proteins undergo post-translational modification with isoprenoid residues attached to cysteine in their carboxyl terminal . With this modification, they attach to cellular membranes . The biochemical activities of these proteins, i.e., GTP hydrolysis and binding, are regulated by various regulatory factors such as GDP-GTP exchange proteins and GTPase-activating proteins, but little is known about the cellular functions and physiological pathways through which they regulate these functions . Botulinum C3 ADP-ribosyltransferase, a 23-KDa exoenzyme secreted from certain strains of types C and D Clostridium botulinum, specifically ADP-ribosylates the rho family of these GTP-binding proteins . This ADP-ribosylation occurs at a specific asparagine residue in their putative effector domain, and presumably interferes with their interaction with a putative effector molecule downstream in signal transduction . C3 exoenzyme, when incubated with or microinjected into cultured cells, ADP-ribosylates a rho gene product in the cells, and causes profound cell rounding with loss of adhesion plaques and collapse of stress fiber . Microinjection of an activated mutant of rho A protein, on the contrary, induced extensive adhesion and actin assembly in cultured cells . These results suggest that the rho family of proteins are involved in morphogenesis and motility of cells via assembly and disassembly of cytoskeletal systems, and botulinum ADP-ribosyltransferase is a useful tool for clarifying the molecular mechanism of these processes. Mol Microbiol, 1992 Apr, 6(7), 873 - 84 Cloning, sequencing and distribution of the Salmonella typhimurium LT2 sialidase gene, nanH, provides evidence for interspecies gene transfer; Hoyer LL et al.; The Salmonella typhimurium LT2 sialidase (neuraminidase, EC 3.2.1.18) structural gene, nanH, has been cloned and sialidase overproduced from multicopy plasmids in Escherichia coli . Sialidase expression was regulated positively by cAMP . In contrast, certain Tn1000 insertions located upstream of nanH coding sequences reduced sialidase activity . A nanH chromosomal insertion mutation constructed by marker exchange demonstrated a single sialidase gene copy in S . typhimurium LT2 . The complete nucleotide sequence of nanH, encoding a 41,300 dalton polypeptide, was determined and the derived primary structure was similar to sialidases from Clostridium perfringens, Clostridium sordellii, Bacteroides fragilis, and Trypanosoma cruzi . Comparative sequence analysis, including codon usage and secondary structure predictions, indicated that the S . typhimurium and clostridial sialidases are homologous, strongly suggestive of an interspecies gene transfer event . At least two primary sequence motifs of the bacterial enzymes were detected in influenza A virus sialidases . The predicted secondary structure of the bacterial enzymes was strikingly similar to viral sialidase . From the population distribution of nanH detected within a collection of salmonellae, it was apparent that S . typhimurium obtained its nanH copy most recently from Salmonella arizonae . S . typhimurium LT2 is thus a genetic mosaic that differs from other strains of even the same serotype by nanH plus potentially additional characters linked to nanH . These results have relevance to the evolution and function of sialidases in pathogenic microbes, and to the origin of the sialic acids. Appl Environ Microbiol, 1992 Apr, 58(4), 1411 - 4 Sporulation and enterotoxin production by Clostridium perfringens type A at 37 and 43 degrees C; Garcia-Alvarado JS et al.; Enterotoxin-positive strains of Clostridium perfringens were grown in Duncan-Strong sporulation medium in the presence of 0.4% (7.9 mM) raffinose at 37 and 43 degrees C . Enterotoxin- and heat-resistant spores were produced at similar concentrations but sooner at 43 degrees C than at 37 degrees C . There was a direct relationship between spore heat resistance and sporulation temperature (32, 37, and 43 degrees C). Appl Environ Microbiol, 1992 Apr, 58(4), 1195 - 1200 Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914; Macfarlane GT et al.; Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture . In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1 . At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa . High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa . Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process . Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4 . The optimal pH of protease activity was 7.0 . Although a wide variety of proteins were hydrolyzed by C . bifermentans proteases, none of the enzymes were collagenolytic . Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed . With the exception of Ca2+, divalent metal ions inhibited proteolysis . Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type. Appl Environ Microbiol, 1992 Apr, 58(4), 1075 - 81 Purification and characterization of an extracellular muramidase of Clostridium acetobutylicum ATCC 824 that acts on non-N-acetylated peptidoglycan; Croux C et al.; An extracellular enzyme showing lytic activity on non-N-acetylated peptidoglycan has been isolated from Clostridium acetobutylicum ATCC 824 . The lytic enzyme was purified to homogeneity by anion-exchange chromatography and gel filtration, with a recovery of 24% . The enzyme was monomeric and had an estimated molecular weight of 41,000 and an isoelectric point of 3.8 . It has been characterized as a muramidase whose 23-amino-acid N terminus displayed 39% homology with the N,O-diacetyl muramidase of the fungus Chalaropsis sp . The muramidase hydrolyzed purified cell walls at an optimum pH of 3, with a maximum velocity of 9.1 mumol of reducing sugars released min-1 mg of muramidase-1 and a concentration of cell walls giving a half-maximum rate of 0.01 mg ml-1 . Its activity was inhibited by glucosamine, N-acetylglucosamine, Hg2+, Fe3+, and Ag+ but not by choline . The muramidase-peptidoglycan complex rapidly dissociated before total hydrolysis of the chain and randomly reassociated on another peptidoglycan chain . The affinity of the muramidase was affected by the protein content and the acetylation of the cell wall. Am J Physiol, 1992 Apr, 262(4 Pt 2), F572 - 7 Involvement of C3 exotoxin-sensitive G proteins (rho/rac) in PTH signal transduction in OK cells; Reshkin SJ et al.; Parathyroid hormone (PTH) in opossum kidney (OK) cells leads to inhibition of Na-Pi cotransport, to the generation of inositol trisphosphate (IP3) and adenosine 3',5'-cyclic monophosphate (cAMP) and to a phosphorylation of proteins present in an enriched apical membrane fraction (27, 28; for review see Ref . 23) . In the present report we have identified two of these phosphoproteins with molecular weights of approximately 22,000 and approximately 24,000, respectively, as guanosine 5'-triphosphate (GTP)-binding proteins, ADP-ribosylated by the Clostridium botulinum exotoxin C3 and recognized by an anti-rho polyclonal antibody but not by pan-ras monoclonal antibody; as suggested by Western-blot analysis the content of the proteins recognized by the anti-rho antibody did not alter in the membrane fraction as a function of treatment with PTH . Transient permeabilization of OK cells using streptolysin O and including the C3 exotoxin attenuated PTH-dependent inhibition of Na-Pi cotransport at hormone concentrations higher than 10(-10) M; residual PTH-dependent inhibition is equal to that observed after pharmacological activation of protein kinase A and protein kinase C, respectively . C3 exotoxin did not alter PTH-dependent generation of cAMP but modified production of IP3; it was increased at 10(-11) M and reduced at 10(-8) M PTH, respectively . It is suggested that protein kinase A may be involved in the phosphorylation of C3 exotoxin-sensitive G proteins (rho/rac) . These proteins could be involved in PTH signal transduction. Am J Phys Med Rehabil, 1992 Apr, 71(2), 102 - 7 Diarrhea in hospitalized patients; Yablon SA et al.; Clostridium difficle has been associated with diarrhea in hospitalized patients receiving antibiotic therapy and may be nosocomially acquired . Rehabilitation hospital inpatients may require frequent antibiotic intervention and are thus at risk, although few reports of epidemics at such centers have been published . This study describes the impact of C . difficle-related disease among rehabilitation hospital inpatients . A retrospective review was conducted of all inpatients evaluated for diarrhea in two freestanding rehabilitation hospitals over a 13-month period . Clostridium difficle was determined to be the etiologic agent of diarrhea in 36% of the 33 patients, and no other etiologies were identified . Four patients were transferred to acute care because of the severity of symptoms . A total of 120 altered or canceled therapy sessions were observed to occur during the rehabilitative hospital course among studied patients, of which 90% (108) occurred during periods when patients were documented to have been symptomatic for diarrhea . Diarrhea and C . difficile-related disease thus appear to exert an important and adverse impact on the hospital course of these patients, both in terms of medical complications and therapy attendance . Physicians should therefore possess a heightened index of suspicion for C . difficile infection when evaluating patients with diarrhea in this setting . Diagnostic evaluation of rehabilitation hospital inpatients with diarrhea should include C . difficile toxin assay . If the results of the toxin assay are positive, appropriate therapy, including initiation of oral vancomycin or metronidazole and avoidance of antimotility drugs, should be instituted promptly to minimize risk of potential sequelae. Ann Emerg Med, 1992 Apr, 21(4), 434 - 6 Clostridial myonecrosis resulting from subcutaneous epinephrine suspension injection; Hallagan LF et al.; A 32-year-old asthmatic man developed a life-threatening Clostridium perfringens infection after subcutaneous epinephrine suspension injection . Extensive surgical debridement, including forequarter amputation, hyperbaric oxygen therapy, and appropriate antibiotics were used as life-saving measures . The patient made a fair functional recovery . We could find no previously reported cases of gas gangrene following subcutaneous injection in otherwise healthy individuals . The pathogenesis and treatment of this infection are discussed. Am J Gastroenterol, 1992 Apr, 87(4), 526 - 9 Emphysematous gastritis secondary to gastric infarction; Binmoeller KF et al.; We report a 72-yr-old female hospitalized for an upper gastrointestinal hemorrhage who developed emphysematous gastritis and gas in the portal vein . Endoscopy of the stomach revealed severe circumferential erythema, erosions, exudates, and friability of irregularly thickened proximal gastric folds . The patient became septic on the third day of hospitalization and deteriorated rapidly . Computerized tomographic scan of the abdomen revealed extensive collections of gas within the gastric wall and in the intrahepatic portal veins . Autopsy revealed severe atherosclerotic disease and a stenosis with thrombus at the origin of the celiac artery . Clostridium welchii was isolated in blood cultures prior to the patient's death . Postmortem review of endoscopic biopsies of the stomach revealed changes of incipient gastric infarction. Surg Gynecol Obstet, 1992 Apr, 174(4), 291 - 6 Clostridial septicemia in an urban hospital; Myers G et al.; The records of 56 patients at an urban hospital who had positive blood cultures for clostridia were reviewed . Each patient was classified as immunologically normal or immunosuppressed . Data were collected on clinical history, type of clostridial bacteremia, physical and laboratory determinants of infection, therapeutic intervention, clinical course and outcome . Of the 56 patients, 22 were determined to be immunosuppressed . Among all 56 patients, 28 had a malignancy, usually gastrointestinal or hematologic in origin . Fever, leukocytosis and abdominal pain were common in both groups . Clostridial bacteremia almost always heralded clostridial septicemia . A gastrointestinal source of infection, particularly carcinoma of the colon or rectum or enterocolitis, was evident or presumed in 43 of the 56 patients . Clostridium perfringens was the most frequently isolated microorganism, but C . septicum was associated with more complications and a higher mortality rate . Septic complications and mortality were higher among the patients with immunosuppression. J Bacteriol, 1992 Apr, 174(7), 2236 - 40 Analysis of a novel linkage unit of O-linked carbohydrates from the crystalline surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70; Messner P et al.; The surface layer glycoprotein of Clostridium thermohydrosulfuricum S102-70 was shown to contain a new type of glycan chain . Different from all known eubacterial glycoproteins, the saccharide moiety consists only of six sugar residues without any repeat sequences . Proteolytic digestion of purified S-layer glycoprotein resulted in isolation of several glycopeptide fractions . These are composed of the same hexasaccharide portion but are linked to oligopeptides of different length . One of them contains only a single amino acid . As concluded from chemical analyses and proton and carbon nuclear magnetic resonance spectroscopy of this preparation, the hexasaccharide moiety is linked via a novel O-glycosidic linkage . This is a beta-D-glucose residue linked to the phenolic hydroxyl group of tyrosine in intact S-layer glycoprotein. J Bacteriol, 1992 Apr, 174(7), 2199 - 207 Physical and genetic mapping of the genomes of five Mycoplasma hominis strains by pulsed-field gel electrophoresis; Ladefoged SA et al.; We present the complete maps of five Mycoplasma hominis genomes, including a detailed restriction map and the locations of a number of genetic loci . The restriction fragments were resolved by field inversion gel electrophoresis or by the contour-clamped homogeneous-electric-field system of pulsed-field gel electrophoresis . All the ApaI, SmaI, BamHI, XhoI, and SalI restriction sites (total of 21 to 33 sites in each strain) were placed on the physical map, yielding an average resolution of 26 kb . The maps were constructed using three different approaches: (i) size determination of DNA fragments partially or completely cleaved with one or two restriction enzymes, (ii) hybridization analysis with purified restriction fragments and specific probes, and (iii) use of linking clones . A genetic map was constructed by hybridization with gene-specific probes for rpoA, rpoC, rrn, tuf, gyrB, hup, ftsY, the unc operon, the genes for two M . hominis-specific antigenic membrane proteins, and one gene encoding a protein with some homology to Escherichia coli alanyl-tRNA synthetase . The positions of mapped loci were partially conserved in the five strains except in one strain in which a 300-kb fragment was inverted . The numbers and order of mapped restriction sites were only partly conserved, and this conservation was restricted to certain regions . The gene order was compared with the gene order established for other bacteria and was found to be identical to that of the phylogenetically related Clostridium perfringens . The genome size of the M . hominis strains varied from 704 to 825 kb. J Appl Bacteriol, 1992 Apr, 72(4), 309 - 14 Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak; Mahony DE et al.; Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens . Ninety-two colonies of Cl . perfringens (3-5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis . They were also tested for the in vitro production of bacteriocin and enterotoxin . Sixteen of the 21 stool specimens were tested directly for enterotoxin . This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers . The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro, contained no plasmids, and was of a common bacteriocin type and serotype. Eur J Cell Biol, 1992 Apr, 57(2), 292 - 7 Clostridium difficile toxin A induces multinucleation in the human leukemic T cell line JURKAT; Fiorentini C et al.; Clostridium difficile toxin A is a cytotoxic enterotoxin known to be active on all mammalian cell lines tested up to now . It induces a disruption of the cytoskeleton, particularly the microfilament system, leading to inhibition of cell proliferation . Here, we describe some effects of toxin A on the leukemic T cell line JURKAT . Cells exposed to the toxin did not divide, as cell numbers remained constant for 3 days in the presence of 0.5 to 1.0 micrograms/ml of the toxin . However, these cells were found to become multinucleated, a phenomenon which was time- and dose-dependent . After treatment for 72 h with 0.5 micrograms/ml toxin A, 95% of the cells were multinucleated and had a considerably increased cell diameter . These effects in JURKAT cells were partially reversible upon removal of the toxin within 12 h after the beginning of toxin exposure, but irreversible after 24 h of toxin treatment . These results suggest a continuing nuclear division in the absence of cytoplasmic division, i.e., an effect of toxin A on contractile ring formation . The JURKAT cell is the first cell type reported to respond to toxin A with multinucleation. Br J Ophthalmol, 1992 Apr, 76(4), 252 - 3 Metastatic endophthalmitis caused by Clostridium perfringens; Nangia V et al.; A case of metastatic endophthalmitis due to Clostridium perfringens originating from the biliary tract is reported . The grave visual prognosis and the importance of early detection and treatment of the primary source of infection are emphasised. Int J Syst Bacteriol, 1992 Apr, 42(2), 312 - 4 Phylogenetic analysis of the pathogenic anaerobe Clostridium perfringens using the 16S rRNA nucleotide sequence; Canard B et al.; Clostridium perfringens, the first pathogenic clostridium examined, was placed in the nonmycoplasma subgroup of the low-dG+dC-content gram-positive cluster on the basis of the results of a phylogenetic analysis in which we used 16S rRNA comparisons . The closest relative that has been identified to date is Clostridium pasteurianum. Rev Latinoam Microbiol, 1992 Apr-Jun, 34(2), 87 - 9 {Diagnosis of blackleg by direct immunofluorescence in bovine bone marrow}; de Guzman AM et al.; On smears of marrow-bone death bovines, presumptively diagnostic of black-leg, we have carried out the direct immunofluorescence test (IF) . We used two labelled immunosera with fluorescein isothiocyanate . The immunosera wer prepared with the reference strain 5078 of Clostridium chauvoei as following: a) a cellular extract obtained with veronal buffer 0.045 M pH = 8.6, and b) a flagellar suspension obtained by agitation with glass beads, centrifuged at 3,500 x g, and centrifuged again at 16,000 x g during 20 min at 4 degrees C . Both antigenic preparations were injected into rabbits, five doses (0.2, 0.4, 0.8, 1.6 and 3.2 ml) each by i.v . route . Control positive strain of C . chauvoei were used, and control negative strains of C . septicum and C . perfringens were used too . Of all 56 examined samples, 26 (47.5%) gave positive IF test . These results had a 100% of correlation by culture and biochemical identification. J Am Vet Med Assoc, 1992 Apr 1, 200(7), 991 - 4 Enteric Clostridium perfringens infection associated with parvoviral enteritis in dogs: 74 cases (1987-1990); Turk J et al.; Parvovirus infection was confirmed by fluorescent antibody staining of or viral isolation from specimens of small intestine in 181 (17%) of 1,110 dogs necropsied between July 1, 1987 and Dec 31, 1990 . Clostridium perfringens was isolated from 74 (69%) of 108 dogs with parvovirus infection from which specimens of jejunum also had been obtained for culture of anaerobic bacteria . Gram-positive bacilli in association with focal to diffuse necrosis of the superficial portions of the villi were observed in histologic sections of specimens of small intestine from 56 (98%) of 57 dogs from which parvovirus and C perfringens had been identified . These findings indicate that C perfringens frequently proliferates in dogs with parvovirus infection. J Bacteriol, 1992 Apr, 174(7), 2312 - 22 Interaction between DNA and alpha/beta-type small, acid-soluble spore proteins: a new class of DNA-binding protein; Setlow B et al.; DNA in spores of Bacillus and Clostridium species is associated with small, acid-soluble proteins (SASP) of the alpha/beta type; the presence of these proteins is a major factor in causing spore resistance to UV light, alpha/beta-type SASP did not bind to single-stranded DNA, single- or double-stranded RNA, or DNA-RNA hybrids in vitro . However, these proteins bound a variety of double-stranded DNAs and conferred protection against DNase cleavage . The binding of alpha/beta-type SASP to DNA saturated at a protein/DNA ratio (wt/wt) of 4:1 to 5:1, which is approximately 1 SASP per 4 bp . alpha/beta-type SASP-DNA interaction did not require divalent cations, was independent of pH between 6 and 8, and, for some SASP-DNA pairs, was relatively insensitive to salt up to 0.3 M . The relative affinity of alpha/beta-type SASP for different DNAs was poly(dG).poly(dC) greater than poly(dG-dC).poly(dG-dC) greater than plasmid pUC19 greater than poly(dA-dT).poly(dA-dT), with poly(dA).poly(dT) giving no detectable binding . This order in alpha/beta-type SASP-DNA affinities parallels the facility with which the DNAs adopt an A-like conformation, the conformation in alpha/beta-type SASP-DNA complexes . An oligo(dG).oligo(dC) of 12 bp was bound by alpha/beta-type SASP . While a 26-bp oligo(dG).oligo(dC) bound more tightly than the 12-mer, there was no significant increase in affinity for alpha/beta-type SASP with further increase in size of oligo(dG).oligo(dC) . In contrast, binding of alpha/beta-type SASP to oligo(dA-dT).oligo(dA-dT) was minimal up to at least a 70-mer, and binding to poly(dA-dT).poly(dA-dT) was very cooperative . In addition to blocking DNase digestion, binding of alpha/beta-type SASP to DNA blocked (i) cleavage of the DNA backbone by hydroxyl radicals and orthophenanthroline-Cu2+, (ii) DNA cleavage by restriction enzymes, in particular those with specificity for GC-rich sequences; and (iii) in vitro transcription of some but not all genes . However, methylation of dG residues by dimethyl sulfate was not affected by alpha/beta-type SASP binding. Biochem Biophys Res Commun, 1992 Mar 31, 183(3), 931 - 6 ADP-ribosylation of small GTP-binding proteins by Bacillus cereus; Just I et al.; A human pathogenic strain of Bacillus cereus produces an exoenzyme which selectively ADP-ribosylates 20-25 kDa GTP-binding proteins in platelet membranes . Pre-ADP-ribosylation of rho proteins of human platelet membranes with Clostridium botulinum exoenzyme C3 or Clostridium limosum exoenzyme inhibits subsequent ADP-ribosylation by the exoenzyme from B . cereus indicating similar substrate specificity of the transferases . The ADP-ribosyltransferase from B . cereus reveals no immunological cross-reactivity with C . botulinum C3 and C . limosum exoenzyme. Biochem Biophys Res Commun, 1992 Mar 31, 183(3), 1273 - 9 The complete nucleotide sequence of the gene coding for the nontoxic-nonhemagglutinin component of Clostridium botulinum type C progenitor toxin; Tsuzuki K et al.; The structural gene for a nontoxic-nonhemagglutinin component of Clostridium botulinum type C progenitor toxin was found to exist on a 7.8 kb DNA fragment obtained from a type C phage DNA . The gene existed between the neurotoxin and hemagglutinin genes, and consisted of an 3588 bp open reading frame (1196 amino acid residues) . It was speculated that this gene and the neurotoxin gene were transcribed by the same mRNA (polycistronic transcription) in C . botulinum organisms. Biochim Biophys Acta, 1992 Mar 24, 1130(2), 203 - 8 Cloning of HSP70 (dnaK) gene from Clostridium perfringens using a general polymerase chain reaction based approach; Galley KA et al.; A general method to clone the HSP70 gene from any species employing polymerase chain reaction and degenerate oligonucleotide primers for conserved regions of this protein family is described . Using this method, a clone containing the entire coding sequence for the HSP70 gene from Clostridium perfringens, has been isolated and sequenced . The HSP70s from C . perfringens as well as other Gram-positive groups of bacteria contain a large deletion of 25 amino acids, near the N-terminal end, which is not seen in HSP70 from other sources. Presse Med, 1992 Mar 21, 21(11), 509 - 14 {Prevention of acute otitis media . Amoxicillin versus glycoproteins from Klebsiella pneumoniae . Study in children under 5 years of age}; Cohen R et al.; Several studies in the English language literature have shown that continuous antibiotic prophylaxis is more effective than a placebo in preventing recurrent otitis media . In this prospective, randomized trial the effectiveness of continuous amoxicillin therapy was compared with that of glycoproteins from Klebsiella pneumoniae (GKP) . The two treatments were administered during 3 months to children aged 1 to 5 years, who had at least 3 episodes of otitis media within the 3 months preceding their admission to the trial . The trial lasted from February 1989 to July 1990 . It involved 60 children (mean age: 22.6 months) who had been seen as out-patients and had recovered from their latest episode of otitis . Thirty-three children received amoxicillin 25 mg/kg/day divided into two doses, during 3 months . Twenty-seven children were given GKP 2 tablets per day during 8 days in the 1st month and 1 tablets per day during 8 days in the following 2 months . During the study period, 14 cases of otitis media were observed in children under amoxicillin, as against 22 cases in children under GKP . When the type of day-nursery was taken into account as adjustment factor, the results showed a significantly lower percentage of failures in the amoxicillin group (P less than 0.03) . Both treatments were well tolerated (no child was excluded from the trial for intolerance) . No Clostridium difficile toxin developed in the amoxicillin group. J Burn Care Rehabil, 1992 Mar-Apr, 13(2 Pt 2), 276 - 80 Changing flora in burn and trauma units: historical perspective--experience in the United States; Smith DJ Jr et al.; Changes in patient management, increased survival rates, and widespread use of antimicrobials have altered the flora that are found to colonize the wounds of patients with burns and trauma-related injuries . Enterococci have emerged as a prominent cause of wound infection and are capable of producing significant morbidity . Staphylococcus aureus, although it remains a common colonizer, has developed resistance to several antimicrobial agents . Recent reports suggest that the incidence of Pseudomonas infections is decreasing, whereas multiple antimicrobial resistance has emerged in a number of other gram-negative organisms that were not heretofore considered major pathogens . Candida organisms appear to be the prominent pathogens among opportunistic yeasts and fungi, and Bacteroides and Clostridium species appear to be the most common causes of infection by anaerobes . Patterns of viral infection in patients with burns and trauma-related injuries have not been investigated in detail . Overall, it appears that rates of infection and associated mortality have decreased in some patient populations; progress in this regard can be attributed to improvements in antimicrobial therapy, would management, and nutrition. J Biol Chem, 1992 Mar 5, 267(7), 4472 - 8 Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD; Chauvaux S et al.; 12 carboxyl residues of the Clostridium thermocellum endoglucanase CelD were mutated to alanine . The specific activity of five of the mutated proteins was 1% or less that of wild type . The Ca2+ binding isotherms of these five were similar to those of wild type CelD, consistent with the fact that none of the mutated residues is observed to be directly involved in Ca2+ binding in the three-dimensional structure of the protein (Juy, M., Anit, A . G., Alzuri, P . M., Poljak, R . J., Claeyssens, M., Beguin, P., and Aubert, J.-P., manuscript in preparation) and suggesting that the mutations did not result in gross alterations of the tertiary structure . Analysis of the physico-chemical and enzymatic properties of the five purified mutated proteins and consideration of their position in the three-dimensional structure suggest that carboxyl groups identified may play roles as a general acid catalyst and a source of negative charge in stabilizing a carbonium ion intermediate . Among mutated residues, Glu-555 appears as the most likely candidate for participating in the catalytic mechanism of endoglucanase CelD. Kansenshogaku Zasshi, 1992 Mar, 66(3), 302 - 5 {Studies on Clostridium absonum isolates from clinical specimens}; Masaki T et al.; Two strains of Clostridium absonum isolated from clinical specimens and a type strain were tested for heat resistance of spores, biochemical properties and neutralization of their lecithinase by anti-C . perfringens alpha toxin antiserum . In addition, the usefulness of RapID ANA (AMUCO) and ANIDENT (API) kits for the identification of C . absonum was examined . The results were summarized as follows: 1 . C . absonum isolates from clinical specimens were resistant against heating at 70 degrees C for 10 min and a type strain against heating at 85 degrees C for 10 min, less resistant than those of C . perfringens BP6K . 2 . C . absonum differentiated from C . perfringens in fermentation of trehalose, melibiose and raffinose, and hydrosis of esculin and starch . 3 . Lecithinase of C . absonum was not completely neutralized by anti-C . perfringens alpha toxin antiserum . 4 . Both RapID ANA and ANIDENT kits were useful for differentiation of C . absonum from C . perfringens but not for the identification of C . absonum. Biochem Int, 1992 Mar, 26(4), 577 - 85 1H NMR studies on the oxidized ferredoxin from Clostridium pasteurianum; Ganadu ML et al.; The 8Fe-8S ferredoxin from Clostridium pasteurianum was investigated by 1D and 2D 1H NMR . Spectra of a well-structured, full native preparation of the oxidized protein in 1 M NaCl at pH 8.0 are presented . Assignments of non-isotropically shifted resonances in the diamagnetic region of the spectrum, namely those of the unique aromatic residues F30 and Y2, are presented for the first time. Eur J Clin Microbiol Infect Dis, 1992 Mar, 11(3), 263 - 5 Susceptibility of anaerobic bacteria to tosufloxacin; Nord CE et al.; The in vitro activity of tosufloxacin against anaerobic cocci, Propionibacterium acnes, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, Bacteroides spp . and fusobacteria was determined by the agar dilution method . This activity was compared with that of ciprofloxacin, piperacillin, cefoxitin, imipenem, clindamycin, metronidazole and chloramphenicol . Tosufloxacin, imipenem, clindamycin, metronidazole and chloramphenicol were the most active agents tested . Tosufloxacin has an antibacterial activity that warrants investigation in clinical trials. Eur J Clin Microbiol Infect Dis, 1992 Mar, 11(3), 246 - 9 Evaluation of a new commercial Clostridium difficile toxin A enzyme immunoassay using diarrhoeal stools; Delmee M et al.; A new, commercially available enzyme immunoassay for the detection of toxin A in stool specimens, the Premier Clostridium difficile Toxin A test (Meridian Diagnostics), was evaluated using 228 diarrhoeal stool specimens . Using a cytotoxin assay on HeLa cells as the reference method, this new test resulted in a sensitivity of 88% and a specificity of 95% . Using the presence or absence of a toxigenic strain in the stools as the reference method, the sensitivity was similar to that of the cytotoxin assay (71.7+ versus 70.5%) and the overall correlation was even better (89.4% versus 82%) . The Premier Clostridium difficile Toxin A assay is rapid and easy to perform and is an excellent alternative to the usual toxin B assay. Biol Chem Hoppe Seyler, 1992 Mar, 373(3), 123 - 32 The tungsten-containing aldehyde oxidoreductase from Clostridium thermoaceticum and its complex with a viologen-accepting NADPH oxidoreductase; Strobl G et al.; Purification of aldehyde oxidoreductase from C . thermoaceticum, the first detected enzyme able to reduce reversibly non-activated carboxylic acids to the corresponding aldehydes (White, H., Strobl, G., Feicht, R . & Simon, H . (1989) Eur . J . Biochem . 184, 89-96), results in the generation of multiple forms of the enzyme . The specific activities for the viologen-mediated dehydrogenation of butyraldehyde for the two main forms of the purification procedure are 530 and 450 U/mg . Two forms of the enzyme composed of alpha,beta- and alpha,beta,gamma-subunits, can be differentiated . The latter binds to red-Sepharose and can be eluted very specifically with NADPH . In contrast to the alpha,beta-types the trimeric forms also catalyse the reversible reduction of oxidised viologen with NADPH (VAPOR activity) . The dimer alpha,beta can oligomerize and the alpha,beta,gamma-trimer can easily form various oligomers or split off the gamma-subunit . The apparent molecular masses of the subunits alpha,beta and gamma are 64, 14 and 43 kDa . The alpha,beta-form reveals an apparent molecular mass of 86 kDa containing about 29 iron, 25 acid-labile sulphur, 0.8 tungsten and forms about 1 mol pterine-6-carboxylic acid by permanganate oxidation . The corresponding values of the trimer showing a mass of 300 kDa, are about 82 Fe, 54 S, 3.4 W and 2.5 pterine-6-carboxylic acid . In addition, 1.7 mol of FAD could be found which seems to be a component of the gamma-subunit . The aldehyde oxidoreductase from C . thermoaceticum and that from C . formicoaceticum (White, H., Feicht, R., Huber, C., Lottspeich, F . & Simon, H . (1991) Biol . Chem . Hoppe-Seyler 372, 999-1005) show qualitative similarities as far as the Fe, S, W and pterin content and the broad substrate specificity are concerned . However, there are also surprisingly marked differences with respect to composition and amino-acid sequence. J Appl Bacteriol, 1992 Mar, 72(3), 244 - 51 Pseudomonas fluorescens subsp . cellulosa: an alternative model for bacterial cellulase; Hazlewood GP et al.; Pseudomonas fluorescens subsp . cellulosa, a Gram-negative soil bacterium, can utilize crystalline cellulose or xylan as main sources of carbon and energy . Synthesis of endoglucanases and xylanases is induced by Avicel, filter paper, carboxymethylcellulose or xylan and is repressed by cellobiose, glucose or xylose . These enzymes are secreted into the culture supernatant fluid and do not form aggregates or associate with the cell surface . Cells of Ps . fluorescens subsp . cellulosa do not adhere to cellulose . In cultures containing Avicel or filter paper, a significant proportion of the secreted cellulase and xylanase activities becomes tightly bound to the insoluble cellulose . Western blotting has revealed that endoglucanase B, xylanase A and a cellodextrinase encoded by genes previously isolated from Ps . fluorescens subsp . cellulosa and expressed in Escherichia coli, are synthesized by the pseudomonad under a variety of conditions . These enzymes appear to be post-translationally modified, probably through glycosylation . Overall, it appears that the cellulase/hemicellulase system of Ps . fluorescens subsp . cellulosa differs from the model established for celluloytic anaerobes such as Clostridium thermocellum. J Med Microbiol, 1992 Mar, 36(3), 190 - 7 A non-haemagglutinating form of Clostridium difficile toxin A; Kamiya S et al.; Analysis of crude culture filtrate of Clostridium difficile by Mono Q-anion exchange fast protein liquid chromatography (FPLC) demonstrated that toxin A had distinct peaks of activity for cytotoxicity and haemagglutination, as also did highly purified toxin A obtained by thyroglobulin affinity chromatography (TG) followed by two sequential anion-exchange chromatographic steps with Q-Sepharose FF and Mono Q . From TG unbound fractions a highly cytotoxic but weakly haemagglutinating variant (toxin A') of toxin A was obtained by Q-Sepharose FF and Mono Q chromatography . Analysis of toxins A and A' from cultures of C . difficile in a chemically defined medium, and of toxin A dialysed against brain heart infusion broth, indicated that A' was not merely toxin A coupled to a component of the growth medium . Polyacrylamide gel electrophoresis under non-denaturing conditions showed that toxins A and A' had the same Mr . Immunoblotting with mouse monospecific A antitoxin showed that five bands larger than the major 240-Kda band were more strongly developed in toxin A than in A' in denaturing but non-reducing conditions, and in reducing conditions eight bands (38-175 Kda) were seen in toxin A but not A' . Immunoblotting with a monoclonal antibody (PCG-4) showed that, in both reducing and non-reducing conditions, two bands of 160 and 155 Kda were more prominent in toxins A and A' respectively, and four bands (195, 180, 175 and 125 Kda) were detected only in toxin A'. J Bacteriol, 1992 Mar, 174(6), 1848 - 53 Replacement of the aliphatic chains of Clostridium acetobutylicum by exogenous fatty acids: regulation of phospholipid and glycolipid composition; Johnston NC et al.; The membrane lipid aliphatic chains of Clostridium acetobutylicum ATCC 4259 have been extensively modified by growth in biotin-free medium containing vitamin-free casein hydrolysate supplemented with either elaidic acid, oleic acid, or mixtures of palmitic and oleic acids . Growth with elaidic acid resulted in polar lipids containing 88.6% 18:1 acyl chains and 94.5% 18:1 ether-linked chains . Growth with oleic acid resulted in comparable levels of enrichment of the lipids with 18:1 chains and C19 chains containing cyclopropane rings . When cells were grown with mixtures of palmitic and oleic acids, the ether-linked chains of the plasmalogens were greater than or equal to 64% 18:1 plus C19 chains containing cyclopropane rings at all ratios of oleic to palmitic acid in the medium . The acyl chains reflected the palmitic acid content of the medium more closely . Marked changes were observed in both phospholipid and glycosyldiglyceride compositions as the lipid acyl and ether-linked chains became more enriched with unsaturated and cyclopropane chains . The ratio of the glycerol acetal of plasmenylethanolamine to phosphatidylethanolamine increased, the ratio of cardiolipin to phosphatidylglycerol decreased, and the ratio of diglycosyldiglyceride to monoglycosyldiglyceride increased . However, the monoglycosyldiglyceride/diglycosyldiglyceride ratio was lower for cells grown on 100% oleic acid than for cells grown on 60 or 80% oleic acid . In the membranes of cells grown on 100% oleic acid, the ratio of glycolipids to phospholipids was lower than that found in cells grown on 60% oleic acid . These results indicate that C . acetobutylicum regulates its polar lipid composition in a complex manner involving phospholipids and glycosyldiglycerides . These changes can affect the equilibria between those lipids that form bilayers and those lipids that tend to form nonlamellar phases when enriched with unsaturated aliphatic chains . Phosphoglycolipids of unknown structure were also observed in cells grown either with biotin or with fatty acids . The content of the most abundant phosphoglycolipid also varied with the degree of unsaturation of the cellular lipids. Biochem J, 1992 Mar 1, 282 ( Pt 2), 511 - 6 Elucidation of the topological parameters of N-acetylneuraminic acid and some analogues involved in their interaction with the N-acetylneuraminate lyase from Clostridium perfringens; Zbiral E et al.; A series of neuraminic acid derivatives modified in the side chain or at C-3, C-4 or C-5 were tested as substrates of inhibitors of N-acetylneuraminate lyase (EC 4.1.3.3) from Clostridium perfringens . The results, together with Km and Ki values reported previously, indicate that the region most important for the binding of sialic acids is an equatorial zone reaching from C-8 via the ring oxygen atom to C-4 of the sugar molecule, whereas the substituents at C-9 and C-5 may be varied to a higher extent without significantly disturbing enzyme action . It is shown that stereo-electronic factors are responsible for the immediate heterolytic fragmentation of the cyclic sialic acid into pyruvic acid and 2-acetamidomannose or a related C-6 sugar. Infect Immun, 1992 Mar, 60(3), 784 - 90 Purification and characterization of the lethal toxin (alpha-toxin) of Clostridium septicum; Ballard J et al.; Clostridium septicum lethal (alpha-toxin) was purified and found to be a basic protein (pI 8.4) of approximately 48 kDa that is both lethal and hemolytic . The alpha-toxin had a hemolytic activity of approximately 2 x 10(7) hemolytic units per mg and a 50% lethal dose of approximately 10 micrograms/kg of body weight for mice . The alpha-toxin formed concentration-dependent, sodium dodecyl sulfate-resistant aggregates of approximately 230 kDa . Mice immunized with alpha-toxin showed a significant increase in survival time over mock-immunized mice when challenged with C . septicum . Rabbit polyclonal antibody was generated against the purified toxin and was used to confirm that toxin with the same molecular weight was present in seven different C . septicum isolates . No proteins in the supernatants from cultures of Clostridium perfringens, Clostridium histolyticum, Clostridium chauvoei, or Clostridium difficile were found to react with the C . septicum alpha-toxin-specific antibody. Infect Immun, 1992 Mar, 60(3), 1237 - 40 High-affinity binding of Clostridium perfringens epsilon-toxin to rat brain; Nagahama M et al.; 125I-epsilon-toxin showed high affinity to rat brain homogenates and synaptosomal membrane fractions, having single binding phases with dissociation constants (Kds) of 2.5 and 3.3 nM, respectively . Treatment of synaptosomal membrane fractions with pronase and neuraminidase lowered the binding of the labeled toxin, whereas treatment with trypsin and phospholipase C did not . Heating of the fractions resulted in a decrease in the binding of the toxin . These data suggest that interaction of epsilon-toxin with cell membranes in the brain is facilitated by a sialoglycoprotein . On the other hand, treatment of the membrane fractions with lipase resulted in complete loss of binding, suggesting that the interaction may require an appropriate lipid environment . These data suggest the presence of specific binding sites in brain tissue for epsilon-toxin. Eur J Biochem, 1992 Mar 1, 204(2), 831 - 9 1H-NMR studies on partially and fully reduced 2(4Fe-4S) ferredoxin from Clostridium pasteurianum; Bertini I et al.; The ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated through 1H-NMR spectroscopy in the reduced and partially oxidized states . The 1H-NMR spectrum of fully reduced ferredoxin, obtained by addition of stoichiometric amounts of dithionite, has been characterized . One- and two-dimensional NMR saturation transfer experiments on partially reduced samples have allowed the isotropically shifted signals of the reduced form to be correlated to those of the oxidized form, for which the complete assignment of the beta-CH2 cysteinyl residues is available . In addition, observation of the 1H-NMR signals of the intermediate species with characteristic chemical shift values for each cluster allowed us to assign all the Cys beta-CH2 signals to cluster I or cluster II and to calculate the difference in redox potential between them . Starting from these results, reanalysis of the 1H-NMR features of the two clusters in the oxidized form showed that they are strikingly similar, supporting the idea of a high degree of internal symmetry between them, in agreement with crystallographic results on an homologous ferredoxin . On the other hand, the 1H-NMR properties of the two clusters in the reduced form deviate considerably from each other, suggesting that reduction of the clusters brings about different structural changes and loss of internal symmetry . A theoretical approach is reported to account for the isotropic shifts and the temperature dependence of the NMR signals of the reduced protein. Eur J Biochem, 1992 Mar 1, 204(2), 657 - 67 The complete amino acid sequence of the Clostridium botulinum type-E neurotoxin, derived by nucleotide-sequence analysis of the encoding gene; Whelan SM et al.; The entire structural gene of the Clostridium botulinum NCTC 11219 type-E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR) . Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene . Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date . The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%) . The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity . In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively . Comparisons with partial amino acid sequences of the light chain of BoNT/E from C . botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case . Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity . The most divergent region corresponds to the extreme COOH-terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites. Infection, 1992 Mar-Apr, 20(2), 58 - 60 Low levels of coagulation inhibitors in patients with Clostridium difficile infection; Aronsson B et al.; To investigate levels of coagulation inhibitors in sera from patients with Clostridium difficile-associated diarrhoea and colitis, commercially available antigen assays were used for immunochemical determination of antithrombin III, protein C and free protein S . Sera from patients with Clostridium difficile-associated diarrhoea and colitis showed significantly lowered levels of all measured inhibitors as compared to controls (Student's t test) . Protein C (mean +/- SD): 0.70 +/- 0.30 vs . 1.28 +/- 0.23, t = 6.61, p less than 0.001; antithrombin: 0.70 +/- 0.21 vs . 0.90 +/- 0.17, t = 3.12, p less than 0.01; free protein S: 0.27 +/- 0.06 vs . 0.37 +/- 0.08, t = 3.7, p less than 0.001 . Infection with C . difficile may lead to loss of coagulation inhibitors and constitutes a risk for thromboembolic complications. Toxicon, 1992 Mar, 30(3), 323 - 30 Effect of p-chloromercuribenzoate on Clostridium perfringens beta toxin; Sakurai J et al.; p-Chloromercuribenzoate (PCMB) was shown to bind