Clostridia

Biochemistry, 2001 Jan 16, 40(2), 596 - 602
Identification of lysine 346 as a functionally important residue for pyridoxal 5'-phosphate binding and catalysis in lysine 2, 3-aminomutase from Bacillus subtilis; Chen D et al.; Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of L-lysine and L-beta-lysine . The enzyme contains pyridoxal 5'-phosphate (PLP) and a {4Fe-4S} center and requires S-adenosylmethionine (SAM) for activity . The hydrogen transfer is mediated by the 5'-deoxyadenosyl radical generated in a reaction of the iron-sulfur cluster with SAM . PLP facilitates the radical rearrangement by forming a lysine-PLP aldimine, in which the imine group participates in the isomerization mechanism . We here report the identification of lysine 346 as important for PLP binding and catalysis . Reduction of LAM with NaBH(4) rapidly inactivated the enzyme with concomitant UV/visible spectrum changes characteristic of reduction of an aldimine formed between PLP and lysine . Following reduction with NaBH(4) and proteolysis with trypsin, a single phosphopyridoxyl peptide of 36 amino acid residues was identified by reverse-phase liquid chromatography/mass spectrometry (LC/MS) . The purified phosphopyridoxyl peptide exhibited an absorption band at 325 nm, and its identity was further confirmed by tandem mass spectrometry (MS/MS) sequencing . The bound PLP is linked to lysine 346 in a PGGGGK (PLP) structure . The sequence of this binding motif is conserved in LAMs from Bacillus and Clostridium and other homologous proteins but is distinct from the PLP-binding motifs found in other PLP enzymes . The function of lysine 346 was further studied by site-directed mutagenesis . The purified K346Q mutant was inactive, and its content of PLP was only approximately 15% of that of the wild-type enzyme . The data indicate that the formation of the aldimine linkage between lysine 346 and PLP is important for LAM catalysis . Sequences similar to the PLP-binding motifs in other enzymes were also present in LAM . However, lysine residues within these motifs neither are the PLP-binding sites in LAM nor are directly involved in LAM catalysis . This study represents the first comprehensive investigation of PLP binding in a SAM-dependent iron-sulfur enzyme.

Arch Biochem Biophys, 2000 Dec 1, 384(1), 24 - 30
Identification of residues in the carboxy-terminal domain of Clostridium perfringens alpha-toxin (phospholipase C) which are required for its biological activities; Walker N et al.; A panel of random mutants within the DNA encoding the carboxy-terminal domain of Clostridium perfringens alpha-toxin was constructed . Three mutants were identified which encoded alpha-toxin variants (Lys330Glu, Asp305Gly, and Asp293Ser) with reduced hemolytic activity . These variants also had diminished phospholipase C activity toward aggregated egg yolk phospholipid and reduced cytotoxic and myotoxic activities . Asp305Gly showed a significantly increased enzymatic activity toward the monodisperse substrate rhoNPPC, whereas Asp293Ser displayed a reduced activity toward this phospholipid analogue . In addition, Asp293Ser showed an increased dependence on calcium for enzymatic activity toward aggregated phospholipid and appeared calcium-depleted in PAGE band-shift assays . In contrast, neither Lys330Glu nor Asp305Gly showed altered dependence on calcium for enzymatic activity toward aggregated phospholipid . Asp305 is located in the interface between the amino- and carboxy-terminal domains, whereas Asp293 and Lys330 are surface exposed residues which may play a role in the recognition of membrane phospholipids.

J Clin Endocrinol Metab, 2000 Dec, 85(12), 4742 - 9
Function of the small guanosine triphosphate-binding protein RhoA in the process of implantation; Shiokawa S et al.; The Rho family of small GTPases occupies a key position in the control of cell motility and morphology in response to extracellular stimuli . Rho proteins trigger the formation of contractile stress fibers, resulting in regulation of cell motility . We explored the expression and function of RhoA in human endometrium and decidua . RhoA immunoreactivity had a predominantly glandular epithelial distribution in the proliferative phase and midsecretory phase . In decidua, the expression of RhoA was more pronounced in the stromal cells as well as in the glandular epithelium . RhoA protein levels in proliferative phase and midsecretory phase endometrium as well as decidua were evaluated by immunoblotting; a single band of RhoA protein with a molecular mass of 21 kDa was detected in all cell lysates . Cultured human decidual cells were found to have few actin stress fibers . Decidual cells lost their actin stress fibers by the treatment with C3, an exoenzyme produced by Clostridium botulinum, whereas new actin stress fibers appeared in human decidual cells stimulated with lysophosphatidic acid (LPA) . Mouse blastocysts became attached to cultured human decidual cells after embryos hatched from the zona pellucida . The majority of hatched blastocysts attached to human decidual cells within 24 h . Blastocysts attached to decidual cells exhibited extensive outgrowth after 48 h in culture . Treatment of decidual cells with C3 exoenzyme or LPA did not affect the rates of hatching and attachment of blastocysts, but outgrowth of embryos on decidual cells was inhibited by C3 exoenzyme treatment in a dose-dependent manner . Contrariwise, addition of LPA to decidual cells dose dependently increased the outgrowth of embryos on decidual cells . These findings suggest that RhoA in decidual cells is important for embryonic development and differentiation after attachment.

Appl Environ Microbiol, 2001 Jan, 67(1), 206 - 16
Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish; Kimura B et al.; A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described . With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C . botulinum type E toxin gene . The sensitivity and specificity of the assay were verified by using 6 strains of C . botulinum type E and 18 genera of 42 non-C . botulinum type E strains, including strains of C . botulinum types A, B, C, D, F, and G . In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C . botulinum DNA could be monitored (the quantifiable range was 10(2) to 10(8) CFU/ml or g) much earlier than toxin could be detected by mouse assay . The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate . Overall, an efficient recovery of C . botulinum cells was obtained from all of the samples tested . These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.

J Biol Chem, 2001 Mar 23, 276(12), 9537 - 42 Epub 2000 Dec 21.
A novel C3-like ADP-ribosyltransferase from Staphylococcus aureus modifying RhoE and Rnd3; Wilde C et al.; Clostridium botulinum C3 is the prototype of the family of the C3-like transferases that ADP-ribosylate exclusively RhoA, -B and -C . The ADP-ribose at Asn-41 results in functional inactivation of Rho reflected by disaggregation of the actin cytoskeleton . We report on a new C3-like transferase produced by a pathogenic Staphylococcus aureus strain . The transferase designated C3(Stau) was cloned from the genomic DNA . At the amino acid level, C3(Stau) revealed an identity of 35% to C3 from C . botulinum and Clostridium limosum exoenzyme, respectively, and of 78% to EDIN from S . aureus . In addition to RhoA, which is the target of the other C3-like transferases, C3(Stau) modified RhoE and Rnd3 . RhoE was ADP-ribosylated at Asn-44, which is equivalent to Asn-41 of RhoA . RhoE and Rnd3 are members of the Rho subfamily, which are deficient in intrinsic GTPase activity and possess a RhoA antagonistic cell function . The protein substrate specificity found with recombinant Rho proteins was corroborated by expression of RhoE in Xenopus laevis oocytes showing that RhoE was also modified in vivo by C3(Stau) but not by C3 from C . botulinum . The poor cell accessibility of C3(Stau) was overcome by generation of a chimeric toxin recruiting the cell entry machinery of C . botulinum C2 toxin . The chimeric C3(Stau) caused the same morphological and cytoskeletal changes as the chimeric C . botulinum C3 . C3(Stau) is a new member of the family of the C3-like transferases but is also the prototype of a subfamily of RhoE/Rnd modifying transferases.

J Biol Chem, 2001 Mar 23, 276(12), 8761 - 70 Epub 2000 Dec 19.
Substrate recognition by the collagen-binding domain of Clostridium histolyticum class I collagenase; Matsushita O et al.; Clostridium histolyticum type I collagenase (ColG) has a segmental structure, S1+S2+S3a+S3b . S3a and S3b bound to insoluble collagen, but S2 did not, thus indicating that S3 forms a collagen-binding domain (CBD) . Because S3a+S3b showed the most efficient binding to substrate, cooperative binding by both domains was suggested for the enzyme . Monomeric (S3b) and tandem (S3a+S3b) CBDs bound to atelocollagen, which contains only the collagenous region . However, they did not bind to telopeptides immobilized on Sepharose beads . These results suggested that the binding site(s) for the CBD is(are) present in the collagenous region . The CBD bound to immobilized collagenous peptides, (Pro-Hyp-Gly)(n) and (Pro-Pro-Gly)(n), only when n is large enough to allow the peptides to have a triple-helical conformation . They did not bind to various peptides with similar amino acid sequences or to gelatin, which lacks a triple-helical conformation . The CBD did not bind to immobilized Glc-Gal disaccharide, which is attached to the side chains of hydroxylysine residues in the collagenous region . These observations suggested that the CBD specifically recognizes the triple-helical conformation made by three polypeptide chains in the collagenous region.

Shock, 2000 Dec, 14(6), 629 - 34
Clostridium difficile toxins A and B can alter epithelial permeability and promote bacterial paracellular migration through HT-29 enterocytes; Feltis BA et al.; Clostridium difficile toxins A and B are the widely recognized etiologic agents of antibiotic-associated diseases ranging from diarrhea to pseudomembranous colitis . We hypothesized that C . difficile toxins may alter intestinal epithelial permeability and facilitate bacterial penetration of the intestinal epithelial barrier . Experiments were designed to clarify the effects of C . difficile toxins A and B on the flux of inert particles across HT-29 enterocyte monolayers, and to correlate these results with bacteria-enterocyte interactions . In all experiments, mature, confluent HT-29 cultures were preincubated 16 h with toxin A or B (1-100 ng/mL) . To study alterations in epithelial permeability, toxin-treated enterocytes were incubated with 5 pM solutions of 10- and 40-kD inert dextran particles . Toxin A, but not toxin B, was associated with increased dextran flux through enterocyte monolayers . To study bacteria-enterocyte interactions, toxin-treated enterocytes were incubated with 10(8) Salmonella typhimurium, Proteus mirabilis, or Escherichia coli . Although numbers of internalized bacteria were generally unaffected, both toxins were associated with increased bacterial adherence, as well as increased bacterial transmigration through enterocyte monolayers . Bacterial transmigration was significantly greater using toxin A- compared to toxin B-treated enterocytes, consistent with the observation that dextran flux was significantly greater using toxin A- compared to toxin B-treated enterocytes . Thus intestinal colonization with toxigenic C . difficile may facilitate bacterial penetration of the intestinal epithelium by a mechanism involving increased permeability of the intestinal epithelial barrier.

Theriogenology, 2000 Oct 15, 54(7), 1019 - 32
Relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in postpartum cows with dystocia or retained placenta; Dohmen MJ et al.; A study was conducted to investigate the relationship between intra-uterine bacterial contamination, endotoxin levels and the development of endometritis in cows that experienced a dystocia or retained their placenta . Fifteen healthy cows, 31 cows with retained placenta (RP) and 13 cows that had dystocia were clinically examined 1 or 2 days after parturition when a uterine swab for bacteriological examination was taken . In addition, plasma and uterine lochia samples were collected to determine lipopolysaccharide (LPS) and the plasma IgG anti-LPS concentrations . Subsequently, 15 RP and 6 dystocia cows were initially left untreated and another uterine swab was collected at 2 and 4 wk postpartum . Immediately after calving, RP cows had significantly higher LPS levels in uterine lochia (average of 2.24 x 10(4) Endotoxin Units (EU)/mL) as compared to dystocia and healthy postpartum cows (average of 0.10 and 0.26 EU/mL, respectively) . However, plasma LPS levels were below the detection limit (<0.036 EU/mL platelet-rich plasma) in all groups of cows . IgG anti-LPS levels in plasma were not significantly different between the 3 groups immediately postpartum (average of 26, 16 and 44 Median Units (MU)/mL) for healthy, dystocia and RP cows, respectively), but they were significantly lower when compared to plasma IgG anti-LPS levels of healthy cows at more than 2 months postpartum (mean 83 MU/mL) . High LPS levels in lochia at 1 or 2 days postpartum were significantly related to abnormal cervical discharge, the presence of Escherichia coli, black pigmented gram-negative anaerobes and Clostridium spp . shortly after calving, and Arcanobacterium pyogenes and gram-negative anaerobes in the uterus at 14 days postpartum . These results suggest that the presence of E . coli and LPS (endotoxins) in lochia early postpartum favor the development of uterine infections by A . pyogenes and gram-negative anaerobes later postpartum . LPS were not observed in plasma, suggesting that either they are not absorbed into the blood, or they are efficiently detoxified by IgG anti-LPS or other detoxification mechanisms.

Acta Obstet Gynecol Scand, 2000 Dec, 79(12), 1134 - 5
Postpartum Clostridium sordellii infection associated with fatal toxic shock syndrome; Rorbye C et al.; Clostridium bacteria are anaerobic Gram positive spore-form-ing bacilli, known to cause distinct clinical syndromes such as botulism, tetanus, pseudomembranous colitis and myonecrosis . The natural habitats of Clostridium species are soil, water and the gastrointestinal tract of animals and humans . In 5-10% of all women, Clostridium species are also found to be normal inhabitants in the microbial flora of the female genital tract . In case of a non-sexually transmitted genital tract infection, Clostridium species are isolated in 4-20%, and clostridium welchii seems to be the most common isolate . Clostridium sordellii is rarely encountered in clinical specimens (1% of Clostridium species), but it has been described as a human pathogen with fatal potential . Two toxins, a lethal and a hemorrhagic (that antigenically and pathophysiologically appear similar to Clostridium difficile toxins B and A, respectively) are responsible for this potential . Reviewing the obstetric literature, only six cases of postpartum endometritis caused by C . sordellii, are described - all being fatal . In addition, one lethal case of spontaneous endometritis resulting from C . sordellii is reported . The clinical aspects of these cases include: - sudden onset with influenza-like symptoms in previously healthy women - progressive refractory hypotension - local and spreading tissue edema - absence of fever Laboratory findings include: - marked leukocytosis - elevated hematocrit . This paper reports the seventh fatal postpartum C . sorlellii associated toxic shock syndrome - the first recognized in Scandinavia.

J Vet Med Sci, 2000 Nov, 62(11), 1133 - 8
Endocytosis of Clostridium botulinum type B neurotoxin into rat brain synaptosomes; Kohda T et al.; Clostridium botulinum type B neurotoxin cleaves VAMP (vesicle-associated membrane protein)/synaptobrevin into two fragments, which results in inhibition of neurotransmitter release . The induced fragment did not react to the antibody raised against the synthetic peptide of the amino-terminal 20 residues of VAMP-2, suggesting that the toxin treatment has caused antigenical alteration in the amino-terminal region of VAMP-2 . In rat brain synaptosomes, type B neurotoxin was reduced presumably with sulfhydryls in the membrane and detected in the synaptic vesicle fraction which involved the degradation of VAMP-2 and the inhibition of neurotransmitter release . The light chain in a free form was present in the cytosol fraction . These findings suggest a possibility that type B neurotoxin endocytoses into synaptic vesicles by the recycling pathway and the light chain is penetrable through synaptic vesicle membrane . However, the amount of type B neurotoxin entrapped into synaptic vesicles appears to be extremely small, which may be attributed to a lower sensitivity of the toxin to brain synaptosomes than to peripheral nerve endings.

Br Poult Sci, 2000 Sep, 41(4), 459 - 64
Intestinal digesta viscosity decreases during coccidial infection in broilers; Waldenstedt L et al.; 1 . The effect of intestinal digesta viscosity on bird performance in chickens with coccidiosis was compared to those without coccidiosis . 2 . Six hundred chicks were divided into five groups: one control group was fed a basal maize/soyabean-based diet and the other groups were fed the basal diet supplemented with 2, 4, 6 or 8 g carboxymethyl cellulose (CMC) per kg of feed . At 14 d of age half the birds were individually inoculated with sporulated oocysts of Eimeria acervulina and Eimeria praecox . 3 . Intestinal digesta viscosity increased with increasing inclusion of CMC . This effect was considerably less pronounced in inoculated than in non-inoculated birds . 4 . There was a significant negative effect on live weight gain and feed conversion ratio (FCR) with increasing CMC inclusion in non-inoculated birds, but in inoculated birds there was no clear relation between CMC inclusion and performance . Neither intestinal lesion scores, nor numbers of Clostridium pefringens in the caeca, were significantly affected by CMC inclusion . 5 . Across all diets inoculation impaired growth rate by 9% and FCR by 8%, but did not affect the amount of C . perfringens in the caeca.

Microbiol Immunol, 2000, 44(10), 805 - 13
Metal content and biochemical analyses of a recombinant collagenase PrtV from Vibrio parahaemolyticus; Yu MS et al.; PrtV is an extracellular metalloprotease of Vibrio parahaemolyticus and regarded as a collagenase . Inductively coupled plasma-optical emission spectrometry analysis indicated that the recombinant PrtV contains 1 mol of zinc per mol of the native enzyme . On the basis of a kinetic study using 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA, the specific substrate for bacterial collagenase) as a substrate, it was suggested that metal ions may play a significant role in the binding and catalytic steps of the substrate . PrtV hydrolyzed type I, II, III, and IV collagens; however, it did not hydrolyze type V . In addition, the hydrolysis of native proteins and synthetic substrates revealed that PrtV possesses higher activity toward collagen and collagen-like sequences . The result of the thermal stability study indicated that PrtV was thermostable up to 40 C; at 50 C, stability gradually decreased . In addition, PrtV showed higher storage stability at -20 and 4 C, respectively, than at 25 C . Compared with collagenases from Clostridium histolyticum and Vibrio alginolyticus, PrtV was immunologically different and had no significant effect on the growth of CHO, HeLa, and Vero cells . Taken together, the results of the studies described in this paper advance our knowledge concerning the metal content and biochemical properties of PrtV.

J Nat Toxins, 2000 Nov, 9(4), 357 - 62
Immunological properties of Hn-33 purified from type A Clostridium botulinum; Sharma SK et al.; Type A botulinum neurotoxin is produced along with 6-7 neurotoxin associated proteins to form a complex in addition to the neurotoxin . Immunological reactivity of type A botulinum complex and purified hemagglutinin (Fu et al., 1998) to polyclonal antibody raised against the complex have been investigated using ELISA . In competitive ELISA, 50% inhibition of the binding of IgG antibody against complex A is observed at 78 ng/ml of complex and hemagglutinin-33, suggesting that the hemagglutinin-33 accounts for most of the immunogenic response of the complex . Considering the fact that the complex consists of a group of proteins with a cumulative molecular weight of 900 kDa, hemagglutinin-33 may be one of the major immunoreactive proteins present in the type A neurotoxin complex.

Res Vet Sci, 2000 Dec, 69(3), 289 - 94
Rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the 16S-23S rDNA spacer region; Sasaki Y et al.; In cattle, sheep, and other ruminants, clostridial myonecrosis (gas gangrene) is mostly caused by Clostridium chauvoei, C septicum, C novyi and C sordellii . A polymerase chain reaction (PCR) system using common primers designed from multiple alignment of the 16S rRNA and 23S rRNA genes of Clostridium species was developed to identify pathogenic clostridia . The PCR was performed with total DNA from 26 strains which included seven different Clostridia species . These bacteria were differentiated at species level by the different PCR product patterns . To characterise the 16S-23S rDNA spacer regions of these clostridia further, most PCR products of these bacteria were sequenced . The smallest PCR products of each bacterium represented the fundamental 16S-23S rDNA spacer region; larger PCR products of each bacterium were caused by insertion sequences, i.e . tRNA gene sequences . The authors' observations indicate that the PCR patterns of the 16S-23S rDNA spacer regions have the potential to be used as an identification marker of pathogenic clostridia in gas gangrene .

Infect Immun, 2001 Jan, 69(1), 599 - 601
Cytosolic delivery and characterization of the TcdB glucosylating domain by using a heterologous protein fusion; Spyres LM et al.; TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis . In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB(1-556) coding region were constructed, expressed, and purified from Escherichia coli . LFnTcdB(1-556) was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts . LFnTcdB(1-556) plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.

Arterioscler Thromb Vasc Biol, 2000 Dec, 20(12), E127 - 33
Role of RhoA and Rho kinase in lysophosphatidic acid-induced endothelial barrier dysfunction; van Nieuw Amerongen GP et al.; In the present study, the roles of the small GTPase RhoA and its target Rho kinase in endothelial permeability were investigated in vitro . We have shown previously that, in addition to a rise in the intracellular Ca(2+) concentration ({Ca(2+)}(i)), RhoA is involved in the prolonged thrombin-induced barrier dysfunction . To study the role of RhoA and Rho kinase more specifically, endothelial cells were stimulated with lysophosphatidic acid (LPA), a commonly used RhoA activator . LPA induced a 2- to 3-fold increase in the passage of horseradish peroxidase (HRP) across endothelial monolayers that lasted for several hours, whereas thrombin induced a 5- to 10-fold increase . Comparable to the thrombin-induced barrier dysfunction, the LPA-induced barrier dysfunction was accompanied by a reorganization of the F-actin cytoskeleton and the formation of focal attachment sites . LPA induced only a transient increase in myosin light-chain (MLC) phosphorylation, which returned to basal level within 10 minutes . In endothelial cells, {Ca(2+)}(i) was not elevated by LPA . Chelation of Ca(2+)(i) ions by 1, 2-bis(2-aminophenoxy)ethane-N:,N:,N:',N:'-tetraacetic acid did not prevent the LPA-induced passage of HRP . Apparently, a low degree of MLC kinase activation occurred, because the MLC kinase inhibitor KT5926 reduced the levels of both basal and LPA-stimulated HRP passage . Inhibition of RhoA by the C3 transferase from Clostridium botulinum inhibited the LPA-induced cytoskeletal changes and prevented the LPA-induced HRP passage . Inhibition of Rho kinase by Y-27632 completely prevented the LPA-induced increase in HRP passage without affecting basal permeability . These data indicate that LPA-induced endothelial hyperpermeability occurs without a change in {Ca(2+)}(i) and requires activation of RhoA and Rho kinase.

Gastrointest Endosc, 2000 Dec, 52(6), 725 - 9
Early attachment of anaerobic bacteria may play an important role in biliary stent blockage; Leung JW et al.; BACKGROUND: In vitro studies have demonstrated that ciprofloxacin suppresses Escherichia coli attachment on stents, and ciprofloxacin has been shown to prolong stent patency in cats . However, clinical studies with antibiotic prophylaxis have produced conflicting results . The aim of this study was to isolate and identify the bacteria that attach early on unblocked stents removed from patients and to study their enzyme activities . METHODS: Eighteen unblocked biliary stents were removed from 17 patients (benign obstruction in 14 and malignant obstruction in 4) . All patients received antibiotic prophylaxis (mean of 6 days) . Stents were in place for a mean of 33 days . The inside of stents was scraped and sludge was cultured aerobically and anaerobically . Identification of isolated bacteria and measurement of beta-glucuronidase and phospholipase C activities were performed by using standard techniques . Gastric and duodenal juice from 18 patients with no biliary diseases was used as control samples . RESULTS: All stents were patent and only 6 had visible sludge . There were 19 anaerobes isolated from 16 stents (Clostridium perfringens 13, Clostridium bifermentans 4 and Bacteroides fragilis 2) . Phospholipase C was detected in all Clostridium species . beta-Glucuronidase was produced only by 12 of 13 C perfringens isolates . Sixteen aerobes including Enterococcus species and Bacillus species were isolated but none produced beta-glucuronidase or phospholipase C . There were no aerobic gram-negative bacteria isolated from stents . Clostridium species and B fragilis were not recovered from the control samples . CONCLUSIONS: In patients who had received antibiotic prophylaxis against gram-negative bacterial infection, anaerobic bacteria may play a role in initiating stent blockage.

J Bacteriol, 2001 Jan, 183(1), 119 - 30
Carbon flux distribution and kinetics of cellulose fermentation in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium; Desvaux M et al.; The metabolic characteristics of Clostridium cellulolyticum, a mesophilic cellulolytic nonruminal bacterium, were investigated and characterized kinetically for the fermentation of cellulose by using chemostat culture analysis . Since with C . cellulolyticum (i) the ATP/ADP ratio is lower than 1, (ii) the production of lactate at low specific growth rate (mu) is low, and (iii) there is a decrease of the NADH/NAD(+) ratio and q(NADH produced)/ q(NADH used) ratio as the dilution rate (D) increases in carbon-limited conditions, the chemostats used were cellulose-limited continuously fed cultures . Under all conditions, ethanol and acetate were the main end products of catabolism . There was no shift from an acetate-ethanol fermentation to a lactate-ethanol fermentation as previously observed on cellobiose as mu increased (E . Guedon, S . Payot, M . Desvaux, and H . Petitdemange, J . Bacteriol . 181:3262-3269, 1999) . The acetate/ethanol ratio was always higher than 1 but decreased with D . On cellulose, glucose 6-phosphate and glucose 1-phosphate are important branch points since the longer the soluble beta-glucan uptake is, the more glucose 1-phosphate will be generated . The proportion of carbon flowing toward phosphoglucomutase remained constant (around 59.0%), while the carbon surplus was dissipated through exopolysaccharide and glycogen synthesis . The percentage of carbon metabolized via pyruvate-ferredoxin oxidoreductase decreased with D . Acetyl coenzyme A was mainly directed toward the acetate formation pathway, which represented a minimum of 27.1% of the carbon substrate . Yet the proportion of carbon directed through biosynthesis (i.e., biomass, extracellular proteins, and free amino acids) and ethanol increased with D, reaching 27.3 and 16.8%, respectively, at 0.083 h(-1) . Lactate and extracellular pyruvate remained low, representing up to 1.5 and 0.2%, respectively, of the original carbon uptake . The true growth yield obtained on cellulose was higher, {50.5 g of cells (mol of hexose eq)(-1)} than on cellobiose, a soluble cellodextrin {36.2 g of cells (mol of hexose eq)(-1)} . The rate of cellulose utilization depended on the solid retention time and was first order, with a rate constant of 0.05 h(-1) . Compared to cellobiose, substrate hydrolysis by cellulosome when bacteria are grown on cellulose fibers introduces an extra means for regulation of the entering carbon flow . This led to a lower mu, and so metabolism was not as distorted as previously observed with a soluble substrate . From these results, C . cellulolyticum appeared well adapted and even restricted to a cellulolytic lifestyle.

J Mol Biol, 2001 Jan 5, 305(1), 95 - 107
Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis; Han S et al.; Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton . C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling . The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation . Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins . Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3 . Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family . Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site . Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket . This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties . Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition . Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton . This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework .

J Org Chem, 2000 Dec 15, 65(25), 8518 - 26
Chemoenzymatic synthesis of sialyl oligosaccharides with sialidases employing transglycosylation methodology; Schmidt D et al.; A series of sialyloligosaccharides was synthesized using the transglycolytic activity of the sialidases from Vibrio cholerae, Clostridium perfringens, Salmonella typhimurium, and Newcastle disease virus . According to their hydrolytic activities the sialidases from V . cholerae and C . perfringens catalyze preferentially the formation of sialyl alpha(2-6)-linkages whereas the sialidases from S.typhimurium and Newcastle disease virus show a distinct preference for alpha(2-3) directed sialylations . Using combined chemical and enzymatic methodologies structures such as T-(Thomsen-Friedenreich) antigen {beta-D-Gal-(1-3)-alpha-D-GalNAc-OThr}, Tn-(Thomsen nouveau) antigen (alpha-D-GalNAc-OThr) and beta-D-Gal-(1-4)-alpha-D-2-deoxy-Gal-OMe were sialylated in alpha(2-3)- and alpha(2-6)-positions regioselectively or in high regioisomeric excess and purified by simple isolation procedures . Depending on the enzyme source and acceptor structure yields for transsialylation varied between 10 and 30%.

Biochemistry, 2000 Dec 19, 39(50), 15322 - 32
Conformational energetics of a reverse turn in the Clostridium beijerinckii flavodoxin is directly coupled to the modulation of its oxidation-reduction potentials; Kasim M et al.; A surface loop in the flavodoxin from Clostridium beijerinckii comprised of residues -Met(56)-Gly-Asp-Glu(59)- forms a four-residue reverse turn which undergoes a conversion from a mix of cis/trans peptide configurations that approximate a type II configuration in the oxidized state to a type II' turn upon reduction of the bound flavin mononucleotide (FMN) cofactor . This change results in the formation of a new hydrogen bond between the N(5)H of the reduced cofactor and the carbonyl group of Gly57 of the central peptide bond of the turn, an interaction that is thought to contribute to the modulation of the oxidation-reduction potentials of the cofactor {Ludwig, M . L., Pattridge, K . A., Metzger, A . L., Dixon, M . M., Eren, M., Feng, Y., and Swenson, R . P . (1997) Biochemistry 36, 1259-1280} . In this study, the direct linkage of the conformational energetics of this turn to the stabilization of the FMN semiquinone was established by systematically replacing the second and third residues of the turn (Gly57 and Asp58) with the -Gly-Gly-, -Gly-Ala-, -Ala-Gly-, and -Ala-Ala- dipeptidyl sequences . On the basis of published position specific preferences for residues with side chains (mimicked by Ala) and glycine, a strong correlation was observed between E(ox/sq) and the calculated free-energy differences between the type II and type II' conformations of each of these sequence combinations . The -Ala-Gly- sequence, which favors the type II turn configuration primarily adopted in the oxidized state, displays a E(ox/sq) value that is about 150 mV more negative than that for the wild-type-like -Gly-Ala- sequence, which prefers the type II' conformation observed in the reduced states . The -Gly-Gly- and -Ala-Ala- mutants exhibit intermediate E(ox/sq) values consistent with their ambivalent turn preferences . The potential changes are primarily the result of alterations in the stability of the semiquinone state . These results provide more conclusive evidence for the crucial role of this conformational change in the modulation of the redox potentials of this flavodoxin . Furthermore, this study establishes a direct association between the conformational energetics of the protein, induced in this case by the sequence specificity of a beta-turn, and the differential thermodynamic stabilization of specific redox states of the cofactor, demonstrating another means by which flavoproteins can modulate the redox potentials of the bound cofactor.

Int J Med Microbiol, 2000 Oct, 290(4-5), 357 - 61
Opening of the active site of Clostridium perfringens alpha-toxin may be triggered by membrane binding; Titball RW et al.; On the basis of amino acid sequence homologies with other phospholipases C, the alpha-toxin of Clostridium perfringens was predicted to be a two-domain protein . Using truncated forms of alpha-toxin the phospholipase C active site was shown to be located in the amino-terminal domain . Crystallographic studies have confirmed this organisation and have also revealed that the carboxy-terminal domain is structurally similar to the phospholipid-binding domains in eukaryotic proteins . This information has been used to devise a model predicting how alpha-toxin interacts with membranes via calcium-mediated recognition of phospholipid head groups and the interaction of hydrophobic amino acids with the phospholipid tail group . The binding of alpha-toxin to membranes appears to result in the opening of the active site allowing hydrolysis of membrane phospholipids.

Int J Med Microbiol, 2000 Oct, 290(4-5), 351 - 6
Cholesterol-binding cytolytic protein toxins; Alouf JE; Cholesterol-binding cytolysins (CBCs) are a large family of 50- to 60-kDa single-chain proteins produced by 23 taxonomically different species of Gram-positive bacteria from the genera Streptococcus, Bacillus, Clostridium, Listeria and Arcanobacterium . Apart pneumolysin, which is an intracytoplasmic toxin, all the other toxins are secreted in the extracellular medium . Among the species producing CBCs, only L . monocytogenes and L . ivanovii are intracellular pathogens which grow and release their toxins in the phagocytic cells of the host . CBCs are lethal to animals and highly lytic toward eukaryotic cells, including erythrocytes . Their lytic and lethal properties are suppressed by sulfhydryl-group-blocking agents and reversibly restored by thiols or other reducing agents . These properties are irreversibly abrogated by very low concentrations of cholesterol and other 3beta-hydroxysterols . Membrane cholesterol is thought to be the toxin-binding site at the surface of eukaryotic cells . Toxins molecules bind as monomers to the membrane surface with subsequent oligomerization into arc-and ring-shaped structures surrounding large pores generated by this process . Thirteen structural genes of the toxins (all chromosomal) have been cloned and sequenced to date . The deduced primary structure of the proteins shows obvious sequence homology particularly in the C-terminal part and a characteristic common consensus sequence containing a unique Cys residue (ECTGLAWEWWR) near the C-terminus of the molecules (except pyolysin and intermedilysin) . However, another Cys residue outside this undecapeptide and closer to the C-terminus occurs in ivanolysin . Genetic replacement of the Cys residue in the consensus undecapeptide by certain amino acids demonstrated that this residue was not essential for toxin function . Other residues in the undecapeptide have been mutagenized, particularly the Trp residues . One of these Trp appeared critical for lytic activity . The recent elucidation of the 3-D structure of perfringolysin O provided interesting information on the structure-activity relationship . The molecule was divided into four domains . Three domains are arranged in a row, giving an elongated shape . Domain 3 is covalently connected to the N-terminal domain 1 and packed laterally against domain 2 . Membrane interaction of the monomer appears to be mediated by domain 4, while, oligomerization involves several sites scattered throughout the sequence . The Trp-rich region around the conserved Cys residue within domain 4 is assumed to conformationally adapt to cholesterol, and domain 3 is envisaged to move across the "hinge" by which it is connected to domain 1.

ASAIO J, 2000 Nov-Dec, 46(6), 783 - 5
Extracorporeal adsorption as a new approach to treatment of botulism; Sato Y et al.; Botulism is a paralytic disease caused by a toxin produced by the bacterium Clostridium botulinum . Outbreaks of the illness take place with a mortality rate of 10%, and the potential terrorist use of the toxin has become a serious concern . The current treatment includes administration of antitoxin, which can cause serious allergic reactions . Recently, we have successfully treated a 64 year old woman with the illness with IMMUSORBA TR350 (Asahi Medical, Tokyo, Japan), an extracorporeal adsorptive column containing polyvinylalcohol-tryptophan as an adsorptive agent, which has been widely used in Japan to treat myasthenia gravis and Guillain-Barre syndrome . Initially, the patient developed ocular muscle weakness and a variant of the Guillain-Barre syndrome was suspected . After extracorporeal treatment, her neurologic symptoms remarkably improved . After a series of treatments, botulinum toxin type B was isolated in the food she had eaten, establishing the diagnosis . An in vitro study revealed that the adsorptive column removed botulinum toxin to a significant extent . Our recent findings suggest that treatment with the adsorptive column TR350 can be a feasible option for botulism, which is a rare but potentially lethal disease.

Int Microbiol, 2000 Jun, 3(2), 113 - 6
Changes in protein synthesis and acid tolerance in Clostridium perfringens type A in response to acid shock; Villarreal L et al.; The induction of acid-shock proteins and the degree of acid resistance conferred on Clostridium perfringens by acid shock, and the kinetics of this resistance were determined . A sublethal acid shock at pH 4.5 for 20 min increased the acid tolerance of cells at least fifteenfold . The acquired tolerance was maintained for 3 h after acid treatment . The response of the microorganism to acid shock was also examined by analysis of pulse-labeled proteins . Five acid-shock proteins (molecular weights 120, 84, 58, 45 and 17 kDa) were identified by polyacrylamide gel electrophoresis.

Int Microbiol, 1999 Sep, 2(3), 185 - 94
Bacterial toxins modifying the actin cytoskeleton; Richard JF et al.; Numerous bacterial toxins recognize the actin cytoskeleton as a target . The clostridial binary toxins (Iota and C2 families) ADP-ribosylate the actin monomers causing the dissociation of the actin filaments . The large clostridial toxins from Clostridium difficile, Clostridium sordellii and Clostridium novyi inactivate, by glucosylation, proteins from the Rho family that regulate actin polymerization . In contrast, the cytotoxic necrotic factor from Escherichia coli activates Rho by deamidation and increases the formation of actin filaments . The enterotoxin of Bacteroides fragilis is a protease specific for E-cadherin and it promotes the reorganization of the actin cytoskeleton . The bacterial toxins that modify the actin cytoskeleton induce various cell disfunctions including changes in cell barrier permeability and disruption of intercellular junctions.

J Electron Microsc (Tokyo), 2000, 49(3), 423 - 7
Atomic force microscopy of intact and digested collagen molecules; Yamamoto S et al.; The present study was performed to analyse the structure of non-digested and digested collagen type I molecules by atomic force microscopy (AFM) . Collagen type I molecules from the bovine skin were diluted with 0.05 N acetic acid, spread on a mica plate, air-dried and observed by non-contact mode AFM in air . Collagen molecules digested with Clostridium histolyticum collagenase were also examined by AFM . Intact collagen type I molecules were observed as twisted threads ranging mainly between 280 and 310 nm in length . The surface of the molecules was uneven and both ends usually slightly bulged like a globule . Depressions on the molecules were found throughout the length, and were most prominent approximately 70 nm from one end of the molecules . The collagenase-treated collagen molecules were degraded into fragments with various lengths, which corresponded to the data from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis . The end of these fragments often appeared like a tuft, suggesting that the triple-helix unraveled at these regions.

Schweiz Med Wochenschr, 2000 Nov 4, 130(44), 1681 - 4
{Recurrent Clostridium difficile enterocolitis}; Bergamin B et al.; Pseudomembranous enterocolitis generally occurs after antibiotic treatment . The standard treatment is oral metronidazol or vancomycin . Nevertheless, relapses of Clostridium difficile enterocolitis are observed in 10-25% of cases . Factors associated with recurrences include endogenous reinfection by spore formation, selective IgG1 or IgA deficiency or infection with mutated strains of Clostridium difficile . Recurrent Clostridium difficile enterocolitis may be treated with repeat oral vancomycin combined with Sacchoromyces boulardii, with intravenous immunoglobulin for severe colitis.

Hosp Med, 2000 Oct, 61(10), 718 - 21
Treatment of paediatric cerebral palsy with Dysport; Ubhi T; Dysport (Clostridium botulinum type A toxin-haemagglutin complex) has had its licence extended to include treatment of children aged 2 years and over with dynamic equinus foot deformity, caused by spasticity associated with cerebral palsy . Dysport reduces muscle tone, thus improving function, relieving pain, and facilitating physiotherapy, application and tolerability of splints.

Anal Chem, 2000 Nov 15, 72(22), 5529 - 34
Multidimensional information on the chemical composition of single bacterial cells by confocal Raman microspectroscopy; Schuster KC et al.; In many biotechnological processes, living microorganisms are used as biocatalysts . Biochemical engineering science is becoming more aware that individual cells of an organism in a process can be fairly inhomogeneous regarding their properties and physiological status . Raman microspectroscopy is a novel approach to characterize such differentiated populations . Cells of the anaerobic bacterium Clostridium beijerinckii were dried on transparent support surfaces . The laser beam of a confocal Raman microscope was focused on individual cells viewed through the objective . Single bacterial cells in size approximately 1 microm and sample mass approximately 1 pg could be analyzed within a few minutes, when placed on a calcium fluoride support and using excitation at 632.8 nm . Spectral features could be attributed to all major cell components . Cells from a morphologically differentiated culture sample showed different compositions, indicating the presence of subpopulations . As a reference, the storage polymer granulose was detected . The multidimensional information in Raman spectra gives a global view on all major components of the cell at once, complementing other more specific information-rich methods for single-cell analysis . The method can be used, for example, to study heterogeneities in a microbial population.

J Gastroenterol Hepatol, 2000 Oct, 15 Suppl, G38 - 45
Novel targets for the pharmacotherapy of diarrhoea: a view for the millennium; Farthing MJ; Acute diarrhoea continues to carry a high morbidity and mortality worldwide . Intestinal infection is the major cause of acute diarrhoea although the prevalence of individual pathogens varies according to geographic location . In many countries in the industrialized world, reports of intestinal infections continue to increase; these are largely related to waterborne and foodborne outbreaks . Acute diarrhoea may be due to increased intestinal secretion, commonly as a result of infection with enterotoxin-producing organisms (enterotoxigenic Escherichia coli, Vibrio cholerae) or to decreased intestinal absorption from infection with organisms that damage the intestinal epithelium (enteropathogenic E . coli, Shigella sp., Salmonella sp.) . Although oral rehydration therapy has reduced the mortality associated with acute diarrhoea, the diarrhoea attack rate remains unchanged and stool volume often increases during the rehydration process . The search for agents that will directly inhibit intestinal secretory mechanisms and thereby reduce stool volume has been going on for more than 20 years . Research during the past decade has highlighted the importance of neurohumoral mechanisms in the pathogenesis of diarrhoea, notably the role of 5-hydroxytryptamine, substance P, vasoactive intestinal polypeptide and neural reflexes within the enteric nervous system . Cholera toxin, E . coli enterotoxins and Clostridium difficile toxin A are known to invoke these mechanisms in diarrhoea pathogenesis . This new dimension of intestinal pathophysiology has already exposed possible novel targets for anti-secretory therapy, namely, 5-HT receptor antagonists, substance P antagonists and the possibility for potentiating the proabsorptive effects of endogenous enkephalins by use of enkephalinase inhibitors . There now seems to be a real possibility that anti-secretory therapy will become more widely available in the future.

Appl Environ Microbiol, 2000 Dec, 66(12), 5480 - 3
Cloning, nucleotide sequence, and expression of the gene encoding the bacteriocin boticin B from Clostridium botulinum strain 213B; Dineen SS et al.; Boticin B is a heat-stable bacteriocin produced by Clostridium botulinum strain 213B that has inhibitory activity against various strains of C . botulinum and related clostridia . The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18 . 8-kb plasmid, cloned, and sequenced . DNA sequencing revealed the boticin B structural gene, btcB, to be an open reading frame encoding 50 amino acids . A C . botulinum strain 62A transconjugant containing the HindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C . botulinum 213B . To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C . botulinum.

Appl Environ Microbiol, 2000 Dec, 66(12), 5334 - 9
Sequencing bands of ribosomal intergenic spacer analysis fingerprints for characterization and microscale distribution of soil bacterium populations responding to mercury spiking; Ranjard L et al.; Two major emerging bands (a 350-bp band and a 650-bp band) within the RISA (ribosomal intergenic spacer analysis) profile of a soil bacterial community spiked with Hg(II) were selected for further identification of the populations involved in the response of the community to the added metal . The bands were cut out from polyacrylamide gels, cloned, characterized by restriction analysis, and sequenced for phylogenetic affiliation of dominant clones . The sequences were the intergenic spacer between the rrs and rrl genes and the first 130 nucleotides of the rrl gene . Comparison of sequences derived from the 350-bp band to The GenBank database permitted us to identify the bacteria as being mostly close relatives to low G+C firmicutes (Clostridium-like genera), while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to beta-proteobacteria (Ralstonia-like genera) . Oligonucleotide probes specific for the identified dominant bacteria were designed and hybridized with the RISA profiles derived from the control and spiked communities . These studies confirmed the contribution of these populations to the community response to the metal . Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different soil microenvironments) also permitted to characterize the distribution and the dynamics of these populations at a microscale level following mercury spiking.

Appl Environ Microbiol, 2000 Dec, 66(12), 5253 - 8
Application of a propionyl coenzyme A synthetase for poly(3-hydroxypropionate-co-3-hydroxybutyrate) accumulation in recombinant Escherichia coli; Valentin HE et al.; The genetic operon for propionic acid degradation in Salmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A . R . Horswill and J . C . Escalante-Semerena, Microbiology 145:1381-1388, 1999) . In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme . When propionate was utilized as the substrate for PrpE, a K(m) of 50 microM and a specific activity of 120 micromol . min(-1) . mg(-1) were found at the saturating substrate concentration . PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate . When prpE was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha in recombinant E . coli, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium . To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (an acetyl-CoA synthetase gene from R . eutropha {H . Priefert and A . Steinbuchel, J . Bacteriol . 174:6590-6599, 1992}), or with orfZ (an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from Clostridium propionicum {H . E . Valentin, S . Reiser, and K . J . Gruys, Biotechnol . Bioeng . 67:291-299, 2000}) . Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity.

Am J Gastroenterol, 2000 Nov, 95(11), 3137 - 41
Leukocytosis as a harbinger and surrogate marker of Clostridium difficile infection in hospitalized patients with diarrhea; Bulusu M et al.; OBJECTIVES: Clostridium difficile is the etiological agent of antibiotic-associated diarrhea and pseudomembranous colitis and is a leading cause of nosocomial diarrhea . The objective of the study was to examine if leukocytosis could be a harbinger and surrogate marker of C . difficile infection in hospitalized patients . METHODS: We retrospectively examined the medical records of 70 hospitalized patients who presented with diarrhea of variable severity and who underwent stool examination for enteric pathogens, including C . difficile . We specifically recorded the white blood cell count and the pattern and severity of leukocytosis in two groups of patients--those who were C . difficile-positive and those who were negative . RESULTS: Leukocytosis was common in C . difficile-positive patients, compared to in C . difficile-negative patients (mean 15,800/mm3 vs 7700/mm3, p < 0.01) . Review of the 35 C . difficile-positive patients revealed three patterns: Pattern A) sudden WBC increase coinciding with the onset of symptoms suggestive of C . difficile; Pattern B) unexplained leukocytosis preceding the appearance of C . difficile-related diarrhea and serving as a harbinger of the infection; and Pattern C) worsening of pre-existing leukocytosis as a surrogate marker of C . difficile infection . Treatment with metronidazole led to amelioration of symptoms and normalization of the leukocyte count in all cases . CONCLUSIONS: Infection with C . difficile should be considered in the differential diagnosis of sudden onset of leukocytosis in hospitalized patients previously or concurrently treated with antibiotics . Doing so may obviate the need for expensive and time-consuming tests for other etiologies.

Med Clin (Barc), 2000 Oct 21, 115(13), 499 - 500
{The risk factors for Clostridium difficile infection in elderly patients . A case-control study}; Selva O'Callaghan A et al.; BACKGROUND: To study the main risk factors associated with Clostridium difficile infection in a geriatric unit . PATIENTS AND METHOD: Retrospective case-control study . RESULTS: In a multivariate analysis, tube feeding (OR = 6.73; IC 95%, 1.01-45.35) and length of antibiotic therapy (OR = 1.15; IC, 95% 1.01-1.28) were independent variables which associated with C . difficile infection . CONCLUSIONS: Antibiotic treatment, tube feeding and fragility are associated with C . difficile infection.

Curr Treat Options Gastroenterol, 1999 Apr, 2(2), 119 - 126
Infectious Enteritis; Gregg CR et al.; Initial management of acute infectious enteritis should focus on fluid and electrolyte repletion and symptomatic care . A decision to prescribe empiric antibiotic therapy should rest on clinical or epidemiologic features of the illness that suggest a treatable bacterial origin or a high-risk host . This decision should be reinforced by the detection of leukocytes or blood in the stool . If empiric therapy is indicated, a quinolone is generally the best initial choice . A stool culture yielding an enteropathogen should generally be specifically treated . A possible exception is uncomplicated Salmonella gastroenteritis in an otherwise healthy host . Nosocomial diarrhea is caused by Clostridium difficile in a minority of cases . Because diagnostic studies for this pathogen are sufficiently sensitive and specific, empiric antibiotic treatment for C . difficile is seldom indicated . Diarrhea in AIDS patients is best worked up and managed in a stepwise fashion, beginning with simple measures . Endoscopy or surgery are seldom indicated in the evaluation and management of infectious enteritis.

Mol Pharmacol, 2000 Dec, 58(6), 1389 - 97
Inhibition of protein isoprenylation impairs rho-regulated early cellular response to genotoxic stress; Gnad R et al.; Activation of c-Jun N-terminal kinases (JNKs) and nuclear factor-kappaB (NF-kappaB) are early cellular responses to genotoxic stress involved in the regulation of gene expression . Pretreatment of cells with the hydroxymethyl glutaryl-CoA reductase inhibitor lovastatin blocked stimulation of JNK1 activity by UV irradiation and by treatment with the alkylating compound methyl methanesulfonate but did not affect activation of extracellular signal-regulated kinase 2 by UV light . Lovastatin also attenuated UV-induced degradation of the NF-kappaB inhibitor IkappaBalpha . The effects of lovastatin on UV-triggered stimulation of JNK1 as well as on IkappaBalpha degradation were reverted by cotreatment with geranylgeranylpyrophosphate but not with farnesylpyrophosphate . Both a geranylgeranyltransferase type I inhibitor and a farnesyltransferase inhibitor blocked JNK1 stimulation by UV irradiation without impairing signaling to NF-kappaB . This indicates that different types of isoprenylated proteins impair UV-induced signaling to JNK1 and NF-kappaB, respectively . Since lovastatin caused a rapid decrease in the level of membrane-bound Rho GTPases, we hypothesize that Rho signaling is inhibited by lovastatin . In line with this hypothesis, Rho-inactivating toxin B from Clostridium difficile abolished both JNK1 activation and IkappaBalpha degradation evoked by UV irradiation . In summary, lovastatin-mediated inhibition of protein isoprenylation abrogates cellular stress responses involving JNK- and NF-kappaB-regulated pathways, which seems to be caused by inactivation of Rho GTPases.

J Bacteriol, 2000 Dec, 182(24), 6975 - 82
Mutational analysis of conserved residues in the putative DNA-binding domain of the response regulator Spo0A of Bacillus subtilis; Hatt JK et al.; The Spo0A protein of Bacillus subtilis is a DNA-binding protein that is required for the expression of genes involved in the initiation of sporulation . Spo0A binds directly to and both activates and represses transcription from the promoters of several genes required during the onset of endospore formation . The C-terminal 113 residues are known to contain the DNA-binding activity of Spo0A . Previous studies identified a region of the C-terminal half of Spo0A that is highly conserved among species of endospore-forming Bacillus and Clostridium and which encodes a putative helix-turn-helix DNA-binding domain . To test the functional significance of this region and determine if this motif is involved in DNA binding, we changed three conserved residues, S210, E213, and R214, to Gly and/or Ala by site-directed mutagenesis . We then isolated and analyzed the five substitution-containing Spo0A proteins for DNA binding and sporulation-specific gene activation . The S210A Spo0A mutant exhibited no change from wild-type binding, although it was defective in spoIIA and spoIIE promoter activation . In contrast, both the E213G and E213A Spo0A variants showed decreased binding and completely abolished transcriptional activation of spoIIA and spoIIE, while the R214G and R214A variants completely abolished both DNA binding and transcriptional activation . These data suggest that these conserved residues are important for transcriptional activation and that the E213 residue is involved in DNA binding.

Inflamm Res, 2000 Oct, 49(10), 535 - 40
Effects of the non-peptide B2 receptor antagonist FR173657 in models of visceral and cutaneous inflammation; Griesbacher T et al.; OBJECTIVE: The non-peptide B2 receptor antagonist (E)-3-(6-acetamido-3-pyridyl)-N-{N-{2,4-dichloro-3-{(2-methyl-8-quinolin yl)oxymethyl}phenyl}-N-methylaminocarbonylmethyl}acrylamide (FR173657) was compared to the peptide antagonist icatibant in models of visceral and cutaneous inflammation . METHODS: Pancreatitis was induced by caerulein in anaesthetized Sprague-Dawley rats . Acute cystitis was induced by intravesical instillation of xylene or i.p . cyclophosphamide injection . Cutaneous inflammation was induced in anaesthetized guinea-pigs by s.c . injection of collagenase from Clostridium histolyticum . RESULTS: FR173657 inhibited oedema formation and tissue enzyme retention during pancreatitis at 500 nmol/kg and above after peroral administration, and from 30 nmol/kg after s.c . injection; icatibant was effective at 3 nmol/kg s . c . Protein extravasation in both cystitis models was abolished by s.c . FR173657 at 300 nmol/kg . Collagenase-induced oedema was attenuated equieffectively by FR173657 and icatibant at doses of 10 micomol/kg and 300 nmol/kg s.c., respectively . CONCLUSIONS: FR173657 inhibits kinin-mediated effects in visceral and cutaneous inflammation at doses that are about 10 times higher than those of icatibant . However, FR173657 is also active following oral administration.

Biochim Biophys Acta, 2000 Nov 30, 1543(1), 36 - 46
Mechanistic interpretation of the dilution effect for Azotobacter vinelandii and Clostridium pasteurianum nitrogenase catalysis; Johnson JL et al.; Nitrogenase activity for Clostridium pasteurianum (Cp) at a Cp2:Cp1 ratio of 1.0 and Azotobacter vinelandii (Av) at Av2:Av1 protein ratios (R) of 1, 4 and 10 is determined as a function of increasing MoFe protein concentration from 0.01 to 5 microM . The rates of ethylene and hydrogen evolution for these ratios and concentrations were measured to determine the effect of extreme dilution on nitrogenase activity . The experimental results show three distinct types of kinetic behavior: (1) a finite intercept along the concentration axis (approximately 0.05 microM MoFe); (2) a non-linear increase in the rate of product formation with increasing protein concentration (approximately 0.2 microM MoFe) and (3) a limiting linear rate of product formation at high protein concentrations (>0.4 microM MoFe) . The data are fitted using the following rate equation derived from a mechanism for which two Fe proteins interact cooperatively with a single half of the MoFe protein . (see equation) The equation predicts that the cubic dependence in MoFe protein gives rise to the non-linear rate of product formation (the dilution effect) at very low MoFe protein concentrations . The equation also predicts that the rate will vary linearly at high MoFe protein concentrations with increasing MoFe protein concentration . That these limiting predictions are in accord with the experimental results suggests that either two Fe proteins interact cooperatively with a single half of the MoFe protein, or that the rate constants in the Thorneley and Lowe model are more dependent upon the redox state of MoFe protein than previously suspected {R.N . Thornley and D . J . Lowe, Biochem . J . 224 (1984) 887-894} . Previous Klebsiella pneumoniae and Azotobacter chroococcum dilution results were reanalyzed using the above equation . Results from all of these nitrogenases are consistent and suggest that cooperativity is a fundamental kinetic aspect of nitrogenase catalysis.

Biochim Biophys Acta, 2000 Nov 30, 1543(1), 24 - 35
Analysis of steady state Fe and MoFe protein interactions during nitrogenase catalysis; Johnson JL et al.; Steady state kinetic measurements are reported for nitrogenase from Azotobacter vinelandii (Av) and Clostridium pasteurianum (Cp) under a variety of conditions, using dithionite as reductant . The specific activities of Av1 and Cp1 are determined as functions of Av2:Av1 and Cp2:Cp1, respectively, at component protein ratios from 0.4 to 50, and conform to a simple hyperbolic rate law for the interaction of Av2 with Av1 and Cp2 with Cp1 . The specific activities of Av2 and Cp2 are also measured as a function of increasing Av1 and Cp1 concentrations, producing 'MoFe inhibition' at large MoFe:Fe ratios . When the rate of product formation under MoFe inhibited conditions is re-plotted as increasing Av2:Av1 or Cp2:Cp1 ratios, sigmoidal kinetic behavior is observed, suggesting that the rate constants in the Thorneley and Lowe (T&L) model are more dependent upon the oxidation level of MoFe protein than previously suspected {R.N.F . Thorneley, D.J . Lowe, Biochem . J . 224 (1984) 887-894}, at least when applied to Av and Cp . Calculation of Hill coefficients gave values of 1.7-1.9, suggesting a highly cooperative Fe-MoFe protein interaction in both Av and Cp nitrogenase catalysis . The T&L model lacks analytical solutions {R.N.F . Thorneley, D.J . Lowe, Biochem . J . 215 (1983) 393-404}, so the ease of its application to experimental data is limited . To facilitate the study of steady state kinetic data for H(2) evolution, analytical equations are derived from a different mechanism for nitrogenase activity, similar to that of Bergersen and Turner {Biochem . J . 131 (1973) 61-75} . This alternative cooperative model assumes that two Fe proteins interact with one MoFe protein active site . The derived rate laws for this mechanism were fitted to the observed sigmoidal behavior for low Fe:MoFe ratios (<0.4), as well as to the commonly observed hyperbolic behavior for high values of Fe:MoFe for both Av and Cp.

J Biol Chem, 2001 Feb 23, 276(8), 5622 - 8 Epub 2000 Nov 21.
Stimulation of M3 muscarinic receptors induces phosphorylation of the Cdc42 effector activated Cdc42Hs-associated kinase-1 via a Fyn tyrosine kinase signaling pathway; Linseman DA et al.; The tyrosine kinase, activated Cdc42Hs-associated kinase-1 (ACK-1), is a specific effector of the Rho family GTPase Cdc42 . GTP-bound Cdc42 has been shown to facilitate neurite outgrowth elicited by activation of muscarinic cholinergic receptors (mAChRs) . Because tyrosine kinase activity is a requirement for neuritogenesis in several cell systems, we investigated whether endogenous mAChRs (principally of the M3 subtype) expressed in human SH-SY5Y neuroblastoma cells would signal to ACK-1 . Incubation of cells with the cholinergic agonist oxotremorine-M (Oxo-M) induced an approximately 6-fold increase in the tyrosine phosphorylation of ACK-1 which was inhibited by atropine . ACK-1 phosphorylation was blocked by Clostridium difficile toxin B, an inhibitor of Rho family GTPases . In contrast, disruption of the actin cytoskeleton with cytochalasin D stimulated ACK-1 phosphorylation, and moreover, addition of Oxo-M to cells preincubated with this agent elicited a further increase in phosphorylation, indicating that an intact cytoskeleton is not required for mAChR signaling to ACK-1 . Although stimulation of M3 mAChRs induces both an increase in intracellular Ca2+ and activation of protein kinase C (PKC), neither of these second messenger pathways was required for receptor-stimulated ACK-1 phosphorylation . Instead, inhibition of PKC resulted in a 2-fold increase in Oxo-M-stimulated ACK-1 phosphorylation, whereas acute activation of PKC with phorbol ester decreased ACK-1 phosphorylation . The agonist-induced tyrosine phosphorylation of ACK-1 was blocked by inhibitors of Src family kinases, and ACK-1 was coprecipitated with Fyn (but not Src) in an agonist-dependent manner . Finally, scrape loading cells with glutathione S-transferase fusion proteins of either the Fyn-SH2 or Fyn-SH3 domain significantly attenuated mAChR-stimulated ACK-1 tyrosine phosphorylation . The data are the first to show phosphorylation of ACK-1 after stimulation of a receptor coupled to neurite outgrowth and indicate that a Rho family GTPase (i.e . Cdc42) and Fyn are essential upstream elements of this signaling pathway.

Vet Q, 2000 Oct, 22(4), 212 - 6
The impact of the quality of silage on animal health and food safety: a review; Driehuis F et al.; This paper reviews the microbiological aspects of forage preserved by ensilage . The main principles of preservation by ensilage are a rapid achievement of a low pH by lactic acid fermentation and the maintenance of anaerobic conditions . The silage microflora consists of beneficial micro-organisms, i.e . the lactic acid bacteria responsible for the silage fermentation process, and a number of harmful micro-organisms that are involved in anaerobic or aerobic spoilage processes . Micro-organisms that can cause anaerobic spoilage are enterobacteria and clostridia . Clostridium tyrobutyricum is of particular importance because of its ability to use lactic acid as a substrate . Silage-derived spores of C . tyrobutyricum can cause problems in cheese making . Aerobic spoilage of silage is associated with penetration of oxygen into the silage during storage or feeding . Lactate-oxidizing yeasts are generally responsible for the initiation of aerobic spoilage . The secondary aerobic spoilage flora consists of moulds, bacilli, listeria, and enterobacteria . Mycotoxin-producing moulds, Bacillus cereus, and Listeria monocytogenes in aerobically deteriorated silage form a serious risk to the quality and safety of milk and to animal health.

Biochimie, 2000 Sep-Oct, 82(9-10), 955 - 66
Development of vaccines for prevention of botulism; Byrne MP et al.; Botulism is a potentially lethal disease caused by one of seven homologous neurotoxic proteins usually produced by the bacterium, Clostridium botulinum . This neuromuscular disorder occurs through an exquisite series of molecular events, ultimately ending with the arrest of acetylcholine release and hence, flaccid paralysis . The development of vaccines that protect against botulism dates back to the 1940s . Currently, a pentavalent vaccine that protects against BoNT serotypes A-E and a separate monovalent vaccine that protects against BoNT serotype F are available as Investigational New Drugs . However, due to the numerous shortcomings associated with the toxoid vaccines, several groups have efforts towards developing next-generation vaccines . Identifying a synthetic peptide that harbors a neutralizing epitope is one approach to a BoNT vaccine, while another employs the use of a Venezuelan equine encephalitis virus replicon vector to produce protective antigens in vivo against BoNT . The strategy used in our laboratory is to design synthetic genes encoding non-toxic, carboxy-terminal fragments of the C . botulinum neurotoxins (rBoNT(H(C))) . The gene products are expressed in the yeast, Pichia pastoris, and purified to greater than 98% with yields typically ranging from 200-500 mg per kg of wet cells . Protective immunity to the purified products against high-level challenges of neurotoxin is elicited in mice and in non-human primates . A pre-Investigational New Drug meeting was held with the Food and Drug Administration, and the next milestone for the vaccine candidates will be clinical trials.

Arch Microbiol, 2000 Oct, 174(4), 239 - 47
Propionispora vibrioides, nov . gen., nov . sp., a new gram-negative, spore-forming anaerobe that ferments sugar alcohols; Biebl H et al.; Anaerobic enrichment cultures, with erythritol as substrate, resulted in the isolation of a strain with properties not yet found in an existing genus in this combination . The strain, FKBS1, was strictly anaerobic, stained gram-negative and formed spores . Cells were small motile vibrios with flagella inserted at the concave side of the cell . Spores were located terminally and caused only slight swelling of the cells if compared to related spore-forming genera . FKBS1 fermented fructose, mannitol, sorbitol, xylitol and erythritol to propionic acid, acetic acid, CO2 and small amounts of H2 to balance the difference in the oxidation-reduction value between substrate and cell mass . The 16S rDNA sequence revealed relationship to the Sporomusa-Pectinatus-Selenomonas group . However, the phylogenetic distance to any of its members was too great to allow it to be placed in one of the existing genera . Morphologically the strain resembled Sporomusa, which, however, performs an acetogenic type of fermentation . The propionic-acid-forming genera of the group are either not spore-formers or, in the case of Dendrosporobacter quercicolus (syn . Clostridium quercicolum), morphologically different . It is therefore proposed to classify strain FKBS1 as a new genus and species, Propionispora vibrioides.

Bone Marrow Transplant, 2000 Oct, 26(8), 871 - 6
Clostridium difficile infection in allogeneic stem cell transplant recipients is associated with severe graft-versus-host disease and non-relapse mortality; Chakrabarti S et al.; We retrospectively evaluated 75 allogeneic stem cell transplant recipients to ascertain the incidence, risk factors and outcome of infection with Clostridium difficile . Ten patients (13%) had Clostridium difficile infection at a median of 38 days (range day -6 to day +72) following the transplant . There was no difference in the duration or severity of diarrhoea in patients with Clostridium difficile infection compared to the uninfected patients and no relationship to the prior antibiotic or chemotherapy usage, age, gender, underlying disease, donor type, CMV serostatus, total body irradiation or time to engraftment . The incidence of viral infections was increased in patients infected with Clostridium difficile (7/10 vs 15/65, P = 0.005, odds ratio 7.7), but the strongest association was with GVHD >grade 2 (5/10 vs 6/65 uninfected patients, P = 0.004, odds ratio 9.8) . Patients infected with Clostridium difficile also suffered a higher non-relapse mortality with 7/10 patients succumbing to either GVHD or infections, compared to 19/65 patients in the uninfected group (P = 0.02, odds ratio 5.6) . Thus Clostridium difficile infections in our study had a strong association with GVHD and increased non-relapse mortality . It is possible that Clostridium difficile toxin might predispose to increased severity of GVHD leading to an adverse outcome.

Cell Signal, 2000 Oct, 12(9-10), 645 - 8
Lysophosphatidic acid-induced platelet shape change proceeds via Rho/Rho kinase-mediated myosin light-chain and moesin phosphorylation; Retzer M et al.; Platelet activation plays an important role in arterial thrombotic disorders . Here we show that the serum-borne phospholipid lysophosphatidic acid (LPA) activates the GTPase Rho and its target Rho-kinase to induce myosin light-chain (MLC) and moesin phosphorylation, leading to platelet shape change . MLC phosphorylation, moesin phosphorylation, and shape change were blocked by preincubating platelets with C3 transferase from Clostridium botulinum and Y-27632-specific inhibitors of Rho and Rho kinase, respectively . LPA did not increase the cytosolic Ca(2+) concentration during shape change . Our results suggest that LPA via Rho-Rho kinase induces MLC and moesin phosphorylation leading to shape change in the absence of an increase in the cytosolic Ca(2+) concentration . Rho/Rho kinase inhibition could be a therapeutic strategy to prevent pathologic platelet activation during arterial thrombotic disorders.

J Mol Biol, 2000 Nov 24, 304(2), 201 - 17
Solution structure of the module X2 1 of unknown function of the cellulosomal scaffolding protein CipC of Clostridium cellulolyticum; Mosbah A et al.; Multidimensional, homo- and heteronuclear magnetic resonance spectroscopy combined with dynamical annealing has been used to determine the structure of a 94 residue module (X2 1) of the scaffolding protein CipC from the anaerobic bacterium Clostridium cellulolyticum . An experimental data set comprising 1647 nuclear Overhauser effect-derived restraints, 105 hydrogen bond restraints and 66 phi torsion angle restraints was used to calculate 20 converging final solutions . The calculated structures have an average rmsd about the mean structure of 0.55(+/-0.11) A for backbone atoms and 1.40(+/-0.11) A for all heavy atoms when fitted over the secondary structural elements . The X2 1 module has an immunoglobulin-like fold with two beta-sheets packed against each other . One sheet contains three strands, the second contains four strands . An additional strand is intercalated between the beta-sandwich, as well as two turns of a 3(.10) helix . X2 1 has a surprising conformational stability and may act as a conformational linker and solubility enhancer within the scaffolding protein . The fold of X2 1 is very similar to that of telokin, titin Ig domain, hemolin D2 domain, twitchin immunoglobulin domain and the first four domains of the IgSF portion of transmembrane cell adhesion molecule . As a consequence, the X2 1 module is the first prokaryotic member assigned to the I set of the immunoglobulin superfamily even though no sequence similarity with any member of this superfamily could be detected .

J Mol Biol, 2000 Nov 24, 304(2), 189 - 200
Crystal structure of a cohesin module from Clostridium cellulolyticum: implications for dockerin recognition; Spinelli S et al.; In the assembly of the Clostridium cellulolyticum cellulosome, the multiple cohesin modules of the scaffolding protein CipC serve as receptors for cellulolytic enzymes which bear a dockerin module . The X-ray structure of a type I C . cellulolyticum cohesin module (Cc-cohesin) has been solved using molecular replacement, and refined at 2.0 A resolution . Despite a rather low sequence identity of 32 %, this module has a fold close to those of the two Clostridium thermocellum cohesin (Ct-cohesin) modules whose 3D structures have been determined previously . Cc-cohesin forms a dimer in the crystal, as do the two Ct-cohesins . We show here that the dimer exists in solution and that addition of dockerin-containing proteins dissociates the dimer . This suggests that the dimerization interface and the cohesin/dockerin interface may overlap . The nature of the overall surface and of the dimer interface of Cc-cohesin differ notably from those of the Ct-cohesin modules, being much less polar, and this may explain the species specificity observed in the cohesin/dockerin interaction of C . cellulolyticum and C . thermocellum . We have produced a topology model of a C . cellulolyticum dockerin and of a Cc-cohesin/dockerin complex using homology modeling and available biochemical data . Our model suggests that a special residue pair, already identified in dockerin sequences, is located at the center of the cohesin surface putatively interacting with the dockerin .

J Food Prot, 2000 Nov, 63(11), 1511 - 6
Nonproteolytic Clostridium botulinum toxigenesis in cooked turkey stored under modified atmospheres; Lawlor KA et al.; The ability of nonproteolytic Clostridium botulinum type B spores to grow and produce toxin in cooked, uncured turkey packaged under modified atmospheres was investigated at refrigeration and mild to moderate abuse temperatures . Cook-in-bag turkey breast was carved into small chunks, surface-inoculated with a mixture of nonproteolytic C . botulinum type B spores, packaged in O2-impermeable bags under two modified atmospheres (100% N2 and 30% CO2:70% N2), and stored at 4, 10, and 15 degrees C . Samples were analyzed for botulinal toxin and indigenous microorganisms, as well as subjected to sensory evaluation, on days 0, 7, 14, 28, 42, and 60 . Given sufficient incubation time, nonproteolytic C . botulinum type B grew and produced toxin in all temperature and modified atmosphere treatment combinations . At moderate temperature abuse (15 degrees C), toxin was detected by day 7, independent of packaging atmosphere . At mild temperature abuse (10 degrees C), toxin was detected by day 14, also independent of packaging atmosphere . At refrigeration temperature (4 degrees C), toxin was detected by day 14 in product packaged under 100% N2 and by day 28 in product packaged under 30% CO2:70% N2 . Reduced storage temperature significantly delayed toxin production and extended the period of sensory acceptability of cooked turkey, but even strict refrigeration did not prevent growth and toxigenesis by nonproteolytic C . botulinum . At all three storage temperatures, toxin detection preceded or coincided with development of sensory characteristics of spoilage, demonstrating the potential for consumption of toxic product when spoilage-signaling sensory cues are absent.

J Food Prot, 2000 Nov, 63(11), 1503 - 10
Bacterial spore inhibition and inactivation in foods by pressure, chemical preservatives, and mild heat; Shearer AE et al.; Sucrose laurates, sucrose palmitate, sucrose stearates, and monolaurin (Lauricidin) were evaluated for inhibitory effects against spores of Bacillus sp., Clostridium sporogenes PA3679, and Alicyclobacillus sp . in a model agar system . The combined treatment of sucrose laurate, high hydrostatic pressure, and mild heat was evaluated on spores of Bacillus and Alicyclobacillus in foods . The minimum inhibitory concentrations of the sucrose esters were higher than that of Lauricidin for all spores tested in the model agar system, but Lauricidin was not the most readily suspended in the test media . The sucrose laurates and sucrose palmitate were more effective and more readily suspended than the sucrose stearates . A combined treatment of sucrose laurate (<1.0%), 392 megaPascals (MPa) at 45 degrees C for 10 to 15 min provided 3- to 5.5-log10 CFU/ml reductions from initial populations of 10(6) CFU/ml for Bacillus subtilis 168 in milk, Bacillus cereus 14579 in beef, Bacillus coagulans 7050 in tomato juice (pH 4.5), Alicyclobacillus sp . N1089 in tomato juice (pH 4.5), and Alicyclobacillus sp . N1098 in apple juice . The most notable change in the appearance of the products was temporary foaming during mixing of the sucrose laurate in the foods . The effect of sucrose laurate appeared to be inhibitory rather than lethal to the spores . The inhibitory effects observed on Bacillus and Alicyclobacillus spores by the combined treatment of pressure, mild heat, and sucrose laurate appear promising for food applications where alternatives to high heat processing are desired.

J Mol Microbiol Biotechnol, 2000 Oct, 2(4), 531 - 41
Differential regulation of two thiolase genes from Clostridium acetobutylicum DSM 792; Winzer K et al.; Thiolase of Clostridium acetobutylicum is an important enzyme involved in both, acid and solvent fermentation . Two thiolase genes (thlA and thIB) have been cloned and sequenced from Clostridium acetobutylicum DSM 792, showing high homology to each other and to thiolases of PHA-synthesizing bacteria . The thlA gene is identical to the gene already cloned and sequenced from strain ATCC 824 (Stim-Herndon et al., 1995, Gene 154: 81-85) . Using primer extension and S1 nuclease analysis a transcriptional start site was identified 102 bp upstream of the thlA start codon . This site was preceded by a region that exhibits high similarity to the sigma70 consensus promoter sequences of Gram-positive and -negative bacteria . Regulation of thlA and thlB was studied at the transcriptional level to elucidate the specific function of each gene . Non-radioactive primer extension analysis using fluorescein-labelled oligonucleotides and Northern blot analysis revealed high levels of thlA transcripts in acid- and solvent-producing cells . During an induced shift of a continuous culture from acid to solvent formation, the transcript level transiently decreased to a minimum, 3 to 7 h after induction . The thlA transcript length is about 1.4 kb, indicating a monocistronic organisation, whereas genetic organization and reverse transcription (RT)-PCR analysis indicated that thlB forms an operon with two other adjacent genes, thlR and thlC . Transcription and regulation of the thlB operon was studied using RTPCR and showed a very low expression in acid- and solvent-producing cells . Heterologously expressed clostridial ThlB showed high thiolase activity in Escherichia coli . The N-terminal part of ThlR possesses a potential helix-turn-helix motif and shows significant homology to regulatory proteins belonging to the TetR/AcrR family of transcriptional regulators . ThlR possibly acts as a transcriptional repressor of thlB operon expression . The data provide strong evidence that ThlA is involved in the metabolism of both acid and solvent formation, whereas the physiological function of ThlB has yet to be elucidated.

J Bacteriol, 2000 Dec, 182(23), 6577 - 83
The large resolvase TndX is required and sufficient for integration and excision of derivatives of the novel conjugative transposon Tn5397; Wang H et al.; Tn5397 is a novel conjugative transposon, originally isolated from Clostridium difficile . This element can transfer between C . difficile strains and to and from Bacillus subtilis . It encodes a conjugation system that is very similar to that of Tn916 . However, insertion and excision of Tn5397 appears to be dependent on the product of the element encoded gene tndX, a member of the large resolvase family of site-specific recombinases . To test the role of tndX, the gene was cloned and the protein was expressed in Escherichia coli . The ability of TndX to catalyze the insertion and excision of derivatives (minitransposons) of Tn5397 representing the putative circular and integrated forms, respectively, was investigated . TndX was required for both insertion and excision . Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-terminal nonconserved region of TndX are essential for activity . Analysis of the target site choices showed that the cloned Tn5397 targets from C . difficile and B . subtilis were still hot spots for the minitransposon insertion in E . coli.

Mol Microbiol, 2000 Nov, 38(3), 588 - 601
Transposition of Tn4451 and Tn4453 involves a circular intermediate that forms a promoter for the large resolvase, TnpX; Lyras D et al.; Tn4451 is the paradigm element of a family of mobilizable chloramphenicol resistance transposons from Clostridium perfringens and Clostridium difficile . The unique feature of these 6.3 kb elements is that their excision to form a circular molecule is mediated by TnpX, a member of the large resolvase family of site-specific recombinases . By optimizing the transposition assay system in Escherichia coli, we showed that Tn4453a from C . difficile transposed at a higher frequency than the C . perfringens element, Tn4451, and that transposition of both Tn4451 and Tn4453a was significantly enhanced by the provision of a multicopy tnpX gene in trans . The complete nucleotide sequence of Tn4453a was determined, but its comparison with Tn4451 did not reveal why it transposed at a higher frequency . Using experiments involving a chromosomal derivative of Tn4453a, we have confirmed that the circular form is the transposition intermediate . As the tnpX gene is located very close to one end of these elements, primer extension analysis was used to determine the transcription start point . The results showed that the formation of the circular intermediate creates a strong tnpX promoter, which consists of a -10 box originally located at the left end of the transposon and a -35 box originally located at the right end . The data provide strong evidence that transcription of tnpX is likely to occur from the non-replicating circular intermediate, which would facilitate the subsequent insertion of the transient circular molecule . It is postulated that, when the transposon is in an integrated state, transcription of tnpX would depend on the presence of an appropriately spaced -35 sequence in the DNA flanking the insertion site or the presence of an alternative upstream promoter.

Mol Microbiol, 2000 Oct, 38(2), 254 - 61
A new cytotoxin from Bacillus cereus that may cause necrotic enteritis; Lund T et al.; A cytotoxin (CytK) has been isolated from a Bacillus cereus strain that caused a severe food poisoning outbreak killing three people . A protein of 34 kDa was highly cytotoxic, and the addition of other secreted proteins gave no synergistic effect . CytK was also necrotic and haemolytic . No known B . cereus enterotoxins were produced by this strain . A DNA sequence from 1.8 kb upstream to 0.2 kb downstream of the toxin gene was sequenced . The deduced amino acid sequence of the toxin showed similarity to Staphylococcus aureus leucocidins, gamma-haemolysin and alpha-haemolysin, Clostridium perfringens beta-toxin and B . cereus haemolysin II, all belonging to a family of beta-barrel channel-forming toxins . There was no sequence similarity between CytK and enterotoxins of B . cereus . The upstream sequence contained a partial sequence of a putative histidine kinase gene . A recognition site for PlcR, which regulates the transcription of enterotoxins HBL and Nhe of B . cereus, was found in the promoter region of the toxin . This new cytotoxin may be responsible for a disease that is similar to, although not as severe as, the necrotic enteritis caused by the beta-toxin of C . perfringens type C.

J Pediatr, 2000 Nov, 137(5), 694 - 700
Knowledge of Centers for Disease Control and Prevention guidelines for the use of vancomycin at a large tertiary care children's hospital; Lin PL et al.; OBJECTIVE: In 1994, the Centers for Disease Control and Prevention (CDC) published guidelines to encourage prudent use of vancomycin . We sought to determine whether physicians could demonstrate knowledge consistent with the guidelines . DESIGN: Survey consisting of 18 clinical vignettes based on the CDC guidelines . PARTICIPANTS: All residents, fellows, and attending physicians involved in pediatric inpatient services . SETTING: Tertiary care children's hospital providing service to an inner-city population and community referral base.Main outcome measures: Comparison of survey scores and individual responses among respondents . RESULTS: Survey scores did not vary with level of training or whether the respondent was a pediatrician or non-pediatrician . Average scores of attending physicians, fellows, and residents were 74.1% (SD = 13.1), 77.2% (SD = 11.5), and 73.4% (SD = 10.5), respectively, and did not differ significantly . Questions incorrectly answered by more than 30% of respondents concerned the use of vancomycin as: (1) first-line treatment of Clostridium difficile colitis, (2) a topical solution for wound infection, (3) initial, empiric treatment of patients with fever and neutropenia, (4) peri-operative prophylaxis, (5) a preferred agent over beta-lactam antimicrobial agents . CONCLUSION: Deficits in knowledge regarding appropriate vancomycin use can be localized to certain clinical settings . This observation lends optimism to the notion that targeted educational intervention may improve the appropriate use of vancomycin.

J Biol Chem, 2001 Feb 23, 276(8), 5779 - 87 Epub 2000 Nov 01.
Enoate reductases of Clostridia . Cloning, sequencing, and expression; Rohdich F et al.; The enr genes specifying enoate reductases of Clostridium tyrobutyricum and Clostridium thermoaceticum were cloned and sequenced . Sequence comparison shows that enoate reductases are similar to a family of flavoproteins comprising 2,4-dienoyl-coenzyme A reductase from Escherichia coli and old yellow enzyme from yeast . The C . thermoaceticum enr gene product was expressed in recombinant Escherichia coli cells growing under anaerobic conditions . The recombinant enzyme was purified and characterized.

J Biol Chem, 2001 Feb 2, 276(5), 3231 - 7 Epub 2000 Nov 01.
Rho GTPases as modulators of the estrogen receptor transcriptional response; Su LF et al.; The estrogen receptor alpha (ER) is a ligand-dependent transcription factor that plays a critical role in the development and progression of breast cancer, in part, by regulating target genes involved in cellular proliferation . To identify novel components that affect the ER transcriptional response, we performed a genetic screen in yeast and identified RDI1, a Rho guanine nucleotide dissociation inhibitor (Rho GDI), as a positive regulator of ER transactivation . Overexpression of the human homologue of RDI1, Rho GDIalpha, increases ERalpha, ERbeta, androgen receptor, and glucocorticoid receptor transcriptional activation in mammalian cells but not activation by the unrelated transcription factors serum response factor and Sp1 . In contrast, expression of constitutively active forms of RhoA, Rac1, and Cdc42 decrease ER transcriptional activity, suggesting that Rho GDI increases ER transactivation by antagonizing Rho function . Inhibition of RhoA by expression of either the Clostridium botulinum C3 transferase or a dominant negative RhoA resulted in enhanced ER transcriptional activation, thus phenocopying the effect of Rho GDI expression on ER transactivation . Together, these findings establish the Rho GTPases as important modulators of ER transcriptional activation . Since Rho GTPases regulate actin polymerization, our findings suggest a link between the major regulators of cellular architecture and steroid receptor transcriptional response.

Eur J Gastroenterol Hepatol, 2000 Oct, 12(10), 1069 - 71
Beneficial microbes: health or hazard?
McFarland LV.
Normal microbial flora support the health of the host by diverse mechanisms . When antibiotics, stress, disease or medications disrupt normal microflora, the ability to ward off infection by pathogens is compromised . The use of beneficial microbes (also known as biotherapeutic agents, probiotics, synbiotics) has been shown to be an effective therapeutic agent for some diseases . Various types of diarrhoea (antibiotic-associated diarrhoea, Clostridium difficile disease, traveller's diarrhoea) are most responsive to these beneficial microbes . Serious risks associated with these microbes are largely theoretical at this point, but the risks need to be studied as the use of these beneficial microbes increases in popularity . Beneficial microbes are living organisms used as therapeutic agents to restore the health of the host in times when normal microflora have been disturbed . The efficacy to prevent or treat diarrhoea has been documented in multiple large, placebo-controlled, blinded clinical trials with only a few of these beneficial microbes . Risks of these beneficial microbes are limited, but potential risks have not been extensively studied in large numbers of patients.

Appl Environ Microbiol, 2000 Nov, 66(11), 4992 - 7
Genetic analysis of type E botulinum toxin-producing Clostridium butyricum strains; Wang X et al.; Type E botulinum toxin (BoNT/E)-producing Clostridium butyricum strains isolated from botulism cases or soil specimens in Italy and China were analyzed by using nucleotide sequencing of the bont/E gene, random amplified polymorphic DNA (RAPD) assay, pulsed-field gel electrophoresis (PFGE), and Southern blot hybridization for the bont/E gene . Nucleotide sequences of the bont/E genes of 11 Chinese isolates and of the Italian strain BL 6340 were determined . The nucleotide sequences of the bont/E genes of 11 C . butyricum isolates from China were identical . The deduced amino acid sequence of BoNT/E from the Chinese isolates showed 95.0 and 96.9% identity with those of BoNT/E from C . butyricum BL 6340 and Clostridium botulinum type E, respectively . The BoNT/E-producing C . butyricum strains were divided into the following three clusters based on the results of RAPD assay, PFGE profiles of genomic DNA digested with SmaI or XhoI, and Southern blot hybridization: strains associated with infant botulism in Italy, strains associated with food-borne botulism in China, and isolates from soil specimens of the Weishan lake area in China . A DNA probe for the bont/E gene hybridized with the nondigested chromosomal DNA of all toxigenic strains tested, indicating chromosomal localization of the bont/E gene in C . butyricum . The present results suggest that BoNT/E-producing C . butyricum is clonally distributed over a vast area.

Anal Chem, 2000 Oct 15, 72(20), 4957 - 64
Rapid antibiotic susceptibility testing via electrochemical measurement of ferricyanide reduction by Escherichia coli and Clostridium sporogenes; Ertl P et al.; Electrochemical measurement of respiratory chain activity allows rapid and reliable screening for antibiotic susceptibility in microorganisms . Chronoamperometry and chronocoulometry of suspensions of aerobically cultivated E . coli combined with the non-native oxidant potassium hexacyanoferrate(III) (ferricyanide) yield signals for reoxidation of the reduction product ferrocyanide that are much smaller if the E . coli has been incubated briefly with an effective antibiotic compound . Chronocoulometric results, obtained following 20-min incubation with antibiotic and 2-min measurement in assay buffer containing 50 mM ferricyanide and 10 mM succinate, at +0.50 V vs Ag/AgCl at a Pt working electrode, were compared with traditional disk diffusion susceptibility testing, which requires overnight incubation on agar plates; the results show significantly lower accumulation of ferrocyanide in all cases in which growth inhibition was observed in the disk diffusion assay . A range of antibiotic compounds (13) were examined that possess different mechanisms of action . Quantitative determination of IC50 values for penicillin G and chloramphenicol yielded values that were 100-fold higher than those obtained by standard turbidity methods after 10-h incubation; this is likely a result of the very brief (10 min) exposure time to the antibiotics . Addition of 5 microM 2,6-dichlorophenolindophenol, a hydrophobic electron-transfer mediator, to the assay mixture allowed susceptibility testing of a Gram-positive obligate anaerobe, Clostridium sporogenes . This rapid new assay will facilitate clinical susceptibility testing, allowing appropriate treatment virtually as soon as a clinical isolate can be obtained.

J Antimicrob Chemother, 2000 Sep, 46 Suppl 1, 41 - 8; discussion 63-5
Effect on the human normal microflora of oral antibiotics for treatment of urinary tract infections; Edlund C et al.; Oral administration of antibiotics for treatment of urinary tract infections (UTIs) can cause ecological disturbances in the normal intestinal microflora . Poorly absorbed drugs can reach the colon in active form, suppress susceptible microorganisms and disturb the ecological balance . Suppression of the normal microflora may lead to reduced colonization resistance with subsequent overgrowth of pre-existing, naturally resistant microorganisms, such as yeasts and Clostridium difficile . New colonization by resistant potential pathogens may also occur and may spread within the body or to other patients and cause severe infections . It is therefore important to learn more about the ecological effects of antibacterial agents on the human microflora . The impact on intestinal microorganisms of oral antibiotics used for the treatment of UTIs is reviewed here . Ampicillin, amoxycillin and co-amoxiclav suppress both the aerobic and anaerobic intestinal microflora with overgrowth of ampicillin-resistant Enterobacteriaceae . Pivmecillinam also affects the intestinal microflora, suppressing Escherichia coli, but does not have a major effect on the anaerobic microflora . Several orally administered cephalosporins, such as cefixime, cefpodoxime, cefprozil and ceftibuten, reduce the number of Enterobacteriaceae and increase the number of enterococci . Colonization with C . difficile has also been observed . Fluoroquinolones eliminate or strongly suppress intestinal Enterobacteriaceae, but affect enterococci and anaerobic bacteria only slightly . When antimicrobial agents are prescribed for the treatment of UTIs, not only the antimicrobial spectrum of the agent but also the potential ecological disturbances, including the risk of emergence of resistant strains, should be considered.

J Biotechnol, 2000 Oct 13, 83(3), 253 - 67
On a new artificial mediator accepting NADP(H) oxidoreductase from Clostridium thermoaceticum; Gunther H et al.; The purification and partial characterisation of an NADP(H) dependent artificial mediator accepting pyridine nucleotide oxidoreductase (AMAPOR) from the anaerobic Clostridium thermoaceticum is described . Depending on the redox potential of the artificial mediators the AMAPOR is able to regenerate NADP+ or NADPH rendering the enzyme useful for preparative work applying NADP(H) dependent oxidoreductases . At 37 degrees C crude extracts of C . thermoaceticum have an AMAPOR activity of 5-7 U mg(-1) . This is 28 degrees under the optimal growth temperature of this microrganism . Out of apparently more than 10 AMAPOR active proteins in the crude cell extracts visible after electrophoresis and activity staining on the gel, two of these proteins were isolated . They seem to be two different oligomers . According to gel electrophoresis they show apparent molecular masses of about 200 and 400 kDa . These two forms showed after SDS gel electrophoresis two monomers with apparent molecular masses of 42 and 56 kDa which we call alpha and beta . The two oligomers may have the compositions alpha2beta2 and alpha4beta4 . They contain Fe/S cluster and FAD . Various amounts of the FAD were lost during the purification procedure . This loss is partially reversible after addition of FAD . The AMAPOR reacts with rather different artificial mediators such as viologens, quinones e.g . 1,4-benzoquinone or anthraquinone-2,6-disulphonate, 2,6-dichloro-indophenol and clostridial rubredoxin . Two different ferredoxins from C . thermoaceticum, oxygen or lipoamide are no substrates indicating the here described AMAPOR is not a diaphorase in the usual sense.

Proc Natl Acad Sci U S A, 2000 Nov 7, 97(23), 12530 - 5
Active acetyl-CoA synthase from Clostridium thermoaceticum obtained by cloning and heterologous expression of acsAB in Escherichia coli; Loke HK et al.; Acetyl-CoA synthase from Clostridium thermoaceticum (ACS(Ct)) is an alpha(2)beta(2) tetramer containing two novel Ni-X-Fe(4)S(4) active sites (the A and C clusters) and a standard Fe(4)S(4) cluster (the B cluster) . The acsA and acsB genes encoding the enzyme were cloned into Escherichia coli strain JM109 and overexpressed at 37(o)C under anaerobic conditions with Ni supplementation . The isolated recombinant His-tagged protein (AcsAB) exhibited characteristics essentially indistinguishable from those of ACS(Ct), from which Ni had been removed from the A cluster . AcsAB migrated through nondenaturing electrophoretic gels as a single band and contained a 1:1 molar ratio of subunits and 1.0-1.6 Ni/alphabeta and 14-22 Fe/alphabeta . AcsAB exhibited 100-250 units/mg CO oxidation activity but no CO/acetyl-CoA exchange activity . Electronic absorption spectra of thionin-oxidized and CO-reduced AcsAB were similar to those of ACS(Ct), with features typical of redox-active Fe(4)S(4) clusters . Partially oxidized and CO-reduced AcsAB exhibited EPR signals with g values and low spin intensities indistinguishable from those of the B(red) state of the B cluster and the C(red1) and C(red2) states of the C cluster of ACS(Ct) . Upon overnight exposure to NiCl(2), the resulting recombinant enzyme (ACS(Ec)) developed 0 . 06-0.25 units/mg exchange activity . The highest of these values is typical of fully active ACS(Ct) . When reduced with CO, ACS(Ec) exhibited an EPR signal indistinguishable from the NiFeC signal of Ni-replete ACS(Ct) . Variability of activities and signal intensities were observed among different preparations . Issues involving the assembly of these metal centers in E . coli are discussed.

Clin Infect Dis, 2000 Oct, 31(4), 1012 - 7 Epub 2000 Oct 25.
The search for a better treatment for recurrent Clostridium difficile disease: use of high-dose vancomycin combined with Saccharomyces boulardii; Surawicz CM et al.; Recurrent Clostridium difficile disease (CDD) is a difficult clinical problem because antibiotic therapy often does not prevent further recurrences . In a previous study, the biotherapeutic agent Saccharomyces boulardii was used in combination with standard antibiotics and was found to be effective in reducing subsequent recurrences of CDD . In an effort to further refine a standard regimen, we tested patients receiving a regimen of a standard antibiotic for 10 days and then added either S . boulardii (1 g/day for 28 days) or placebo . A significant decrease in recurrences was observed only in patients treated with high-dose vancomycin (2 g/day) and S . boulardii (16.7%), compared with those who received high-dose vancomycin and placebo (50%; P=.05) . No serious adverse reactions were observed in these patients . Comparison of data from this trial with data from previous studies indicates that recurrent CDD may respond to a short course of high-dose vancomycin or to longer courses of low-dose vancomycin when either is combined with S . boulardii.

Clin Infect Dis, 2000 Oct, 31(4), 995 - 1000 Epub 2000 Oct 25.
Environmental control to reduce transmission of Clostridium difficile; Mayfield JL et al.; Restrictive antibiotic policies and infection control measures have been shown to reduce the incidence of Clostridium difficile-associated diarrhea (CDAD) among hospitalized patients . To date, the role of environmental disinfectants in reducing nosocomial CDAD rates has not been well studied . In a before-and-after intervention study, patients in 3 units were evaluated to determine if unbuffered 1:10 hypochlorite solution is effective as an environmental disinfectant in reducing the incidence of CDAD . Among 4252 patients, the incidence rate of CDAD for bone marrow transplant patients decreased significantly, from 8.6 to 3.3 cases per 1000 patient-days (hazard ratio, 0.37; 95% confidence interval, 0.19-0.74), after the environmental disinfectant was switched from quaternary ammonium to 1:10 hypochlorite solution in the rooms of patients with CDAD . Reverting later to quaternary ammonium solution increased the CDAD rate to 8.1 cases per 1000 patient-days . No reduction in CDAD rates was seen among neurosurgical intensive care unit and general medicine patients, for whom baseline rates were 3.0 and 1.3 cases per 1000 patient-days, respectively . Unbuffered 1:10 hypochlorite solution is effective in decreasing patients' risk of developing CDAD in areas where CDAD is highly endemic . Presumed mechanisms include reducing the environmental burden and the potential for C . difficile transmission among susceptible patients.

Arch Latinoam Nutr, 2000 Jun, 50(2), 142 - 7
{Nutritional and microbiological profile of soybean sausages available in Costa Rica}; Monge R et al.; The nutritional and microbiological quality of 80 soybean sausage samples (50% frankfurter and 50% sausage mortadela) was studied . On average, the protein content was 17.5 g/100 g in sausage mortadela and 20 g/100 g in frankfurter . The mean total fat content was 5.5 g/100 g for both products . However when products of different manufacture industries were compared, a highly significant difference (p = 0.0000) in the fatty acids speciation between both groups and between samples of the same product were found . Bigger differences were found in the content of palmitic acid (C16:0), oleic acid (C18:1) and linoleic acid (C18:2) . Cholesterol was not detected in samples analyzed . On average the atherogenicity index was 0.55 for sausage mortadela and 0.59 for frankfurter . A consumption of 25 grams of of soybean protein from these sausages can bring an intake of saturated fatty acids between 20-90% of the daily recommendation . Likewise, they can supply between 12-70% of the recommended daily polyunsaturated fatty acids . These variations are owing to the big difference in fatty acids speciation in each sausage brand . Around 20% of soybean sausages studied showed total coliform levels above 10(4)/g, being more frequent in sausage mortadela . Also 60% of this product and 10% of frankfurters showed psychrotroph levels of 10(6)/g . Clostridium perfringens, in levels above 10(2)/g was evidenciated in 5% of samples, Escherichia coli was not isolated from them . The findings of this study suggest the urgent need for implementing a quality control system for soybean sausages, before national health authorities consider to support nutritional campainings that promote their consumption.

J Microbiol Methods, 2000 Nov, 42(3), 281 - 90
Method to sensitize bacterial spores to subsequent killing by dry heat or ultraviolet irradiation; Rutherford GC et al.; Hydrogen peroxide and ultraviolet irradiation are known to interact synergistically for killing of bacterial spores . Synergy could be demonstrated with spores of Bacillus megaterium ATCC19213 adsorbed to filter paper strips or glass coverslips treated first with the peroxide and then dried for as long as 48 h prior to UV irradiation . This delayed action was considered to be due to absorption of the peroxide by the spores in an active but not readily vaporized form, which could become sporicidal also if the spores were heated to 50 degrees C . B . megaterium spores mixed with 0.1% (32.6 mM) H(2)O(2) solution appeared to absorb as much as 15 micromol/mg dry weight or about 0.5 mg/mg, but only a third to half of the peroxide could be recovered by water washing . A part of the unrecovered peroxide was degraded in reactions resulting in measurable production of oxygen . Degradation was not reduced by heating the spores to 65 degrees C or by azide and so appeared to be non-enzymatic . Spores of the anaerobe Clostridium sporogenes were also sensitized to ultraviolet killing by H(2)O(2) treatment followed by drying . They appear to absorb less peroxide, only about 2 micromol/mg, but had lower capacities to degrade H(2)O(2) so that nearly all of the peroxide could be recovered by washing with water . The findings presented should be helpful in the design of new methods for synergistic killing of spores by H(2)O(2) and UV irradiation or dry heat, especially involving, for example, packaging materials.

J Thorac Cardiovasc Surg, 2000 Nov, 120(5), 909 - 15
Perioperative complications after living donor lobectomy; Battafarano RJ et al.; OBJECTIVE: Clinical lung transplantation has been limited by availability of suitable cadaveric donor lungs . Living donor lobectomy provides right and left lower lobes from a pair of living donors for each recipient . We reviewed our experience with living donor lobectomy from July 1994 to February 2000 . METHODS: Sixty-two donor lobectomies were performed . The hospital and outpatient records of these 62 donors were retrospectively analyzed to examine the incidence of perioperative complications . RESULTS: Twenty-four (38.7%) of 62 donors had no perioperative complications and had a median length of hospital stay of 5.0 days . Thirty-eight (61.3%) of 62 donors had postoperative complications . Twelve major complications occurred in 10 patients and included pleural effusions necessitating drainage (n = 4), bronchial stump fistulas (n = 3), bilobectomy (n = 1), hemorrhage necessitating red cell transfusion (n = 1), phrenic nerve injury (n = 1), atrial flutter ultimately necessitating electrophysiologic ablation (n = 1), and bronchial stricture necessitating dilatation (n = 1) . These 38 donors had 55 minor complications including persistent air leaks (n = 9), pericarditis (n = 9), pneumonia (n = 8), arrhythmia (n = 7), transient hypotension necessitating fluid resuscitation (n = 4), atelectasis (n = 3), ileus (n = 3), subcutaneous emphysema (n = 3), urinary tract infections (n = 2), loculated pleural effusions (n = 2), transfusion (n = 2), Clostridium difficile colitis (n = 1), puncture of a saline breast implant (n = 1), and severe contact dermatitis secondary to adhesive tape (n = 1) . There were no postoperative deaths and only 1 donor required surgical re-exploration . CONCLUSIONS: Living donor lobectomy can be performed with low mortality and remains an important alternative for potential recipients unable to wait for cadaveric lung allografts . However, morbidity is high and must be considered when potential living donors are being counseled.

Kidney Int, 2000 Nov, 58(5), 1996 - 2006
Evidence for Rho protein regulation of renal tubular epithelial cell function; Anderson RJ et al.; BACKGROUND: Rho proteins are small guanine 5'-triphosphate (GTP)-binding proteins felt to be important regulators of several aspects of cell function, including the organization of the actin cytoskeleton . The effects of Rho proteins on the regulation of renal tubular epithelial cell function are not known . METHODS: Selected bacterial toxins that inhibit Rho protein function were used to examine the effect of Rho in cultured renal tubular epithelial cells . RESULTS: Clostridium difficile toxin A significantly and dose dependently inhibited LLC-PK(1) cell (3)H-thymidine uptake and healing of small wounds made in confluent monolayers, and it induced apoptosis . A second Clostridium difficile toxin (toxin B) that acted via a different receptor also impaired LLC-PK(1) thymidine uptake and wound healing, and it induced apoptosis . A third bacterial toxin, C3 toxin from Clostridium botulinum, also impaired LLC-PK(1) thymidine uptake and stimulated apoptosis in LLC-PK(1) cells . Since Rho inhibition disrupted organization of the actin cytoskeleton, we examined the effects of another agent that disrupted the actin cytoskeleton (cytochalasin D) and found significant dose-dependent effects that impaired LLC-PK1 thymidine uptake and wound healing and that induced apoptosis . The effects of toxin A and cytochalasin D to induce apoptosis were not associated with significant changes in expression of Bcl-2, BAD, or BAK proteins and were significantly attenuated by a pancaspase inhibitor . CONCLUSIONS: Our results suggest that Rho proteins are important endogenous regulators of several aspects of renal tubular epithelial cell function, including proliferation, migration, and apoptosis . Further studies are needed to clarify the cellular mechanisms of Rho regulation of renal epithelial cell function.

Arch Microbiol, 2000 Sep, 174(3), 189 - 99
Fermentation of 4-aminobutyrate by Clostridium aminobutyricum: cloning of two genes involved in the formation and dehydration of 4-hydroxybutyryl-CoA; Gerhardt A et al.; Clostridium aminobutyricum ferments 4-aminobutyrate via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA and crotonyl-CoA to acetate and butyrate . The genes coding for the enzymes that catalyse the interconversion of these intermediates are arranged in the order abfD (4-hydroxybutyryl-CoA dehydratase), abfT (4-hydroxybutyrate CoA-transferase), and abfH (NAD-dependent 4-hydroxybutyrate dehydrogenase) . The genes abfD and abfT were cloned, sequenced and expressed as active enzymes in Escherichia coli . Hence the insertion of the {4Fe-4S}clusters and FAD into the dehydratase required no additional specific protein from C . aminobutyricum . The amino acid sequences of the dehydratase and the CoA-transferase revealed close relationships to proteins deduced from the genomes of Clostridium difficile, Porphyromonas gingivalis and Archaeoglobus fulgidus . In addition the N-terminal part of the dehydratase is related to those of a family of FAD-containing mono-oxygenases from bacteria . The putative assignment in the databank of Cat2 (OrfZ) from Clostridium kluyveri as 4-hydroxybutyrate CoA-transferase, which is thought to be involved in the reductive pathway from succinate to butyrate, was confirmed by sequence comparison with AbfT (57% identity) . Furthermore, an acetyl-CoA:4-hydroxybutyrate CoA-transferase activity could be detected in cell-free extracts of C . kluyveri . In contrast to glutaconate CoA-transferase from Acidaminococcus fermentans, mutation studies suggested that the glutamate residue of the motive EXG, which is conserved in many homologues of AbfT, does not form a CoA-ester during catalysis.

J Food Prot, 2000 Oct, 63(10), 1347 - 52
Differentiation between types and strains of Clostridium botulinum by riboprinting; Skinner GE et al.; The ability of automated ribotyping to differentiate between major types and individual strains of Clostridium botulinum was tested using the Qualicon Riboprinter Microbial Characterization System . Pure spores of C . botulinum type A, proteolytic type B, nonproteolytic type B, and type E strains were inoculated onto modified anaerobic egg yolk agar and incubated 24 h at 35 degrees C . Plates were rinsed with buffer (2 mM Tris + 20 mM EDTA) to remove vegetative cells that were heated for 10 min at 80 degrees C, treated with a lysing agent, and ribotyped in the Qualicon Riboprinter utilizing the enzyme EcoRI . Riboprint patterns were obtained for 30 strains of the four major types of C . botulinum most commonly involved in human foodborne botulism . Proteolytic strains yielded the best and most consistent results . Fifteen ribogroups were identified among the 31 strains tested . Interestingly, in two cases, a single ribogroup contained patterns from isolates belonging to evolutionarily distinct Clostridium lineages . This degree of differentiation between strains of C . botulinum may be useful in hazard analysis and identification, hazard analysis and critical control point monitoring and validation, environmental monitoring, and in inoculation studies.

J Clin Pathol, 2000 Sep, 53(9), 709 - 12
The clinical spectrum of Clostridium sordellii bacteraemia: two case reports and a review of the literature; Abdulla A et al.; Clostridium sordellii is rarely associated with disease in humans . Since its first report in 1922 only a few cases of bacteraemia have been reported . This report describes two cases of C sordellii bacteraemia; the oldest and youngest patients reported to date . The first, is a previously well 81 year old woman presented with perianal infection, which was later complicated by thrombosis of the aorta, and the second is a 12 year old boy with epilepsy who presented with an ear infection . These cases are also highlighted to demonstrate the wide spectrum of presentation of sordellii bacteraemia.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 15 - 20
DNA sequence of the insertional hot spot of Tn916 in the Clostridium difficile genome and discovery of a Tn916-like element in an environmental isolate integrated in the same hot spot; Wang H et al.; Tn916 is a broad host range tetracycline resistance conjugative transposon . In most bacteria, this element enters the bacterial genome at multiple sites . However, in Clostridium difficile, the element has a strong hot spot when introduced by filter mating from Bacillus subtilis . In this work, the DNA sequence of the preferred insertion site (att916) was obtained . An environmental isolate of C . difficile was also discovered which contained an element indistinguishable from Tn916, Tn916CD . Tn916CD was found integrated at att916.

Comp Immunol Microbiol Infect Dis, 2000 Oct, 23(4), 277 - 84
Detection and characterization of Clostridium species in soil of Zambia; Hang'ombe BM et al.; In the retrospective study of soil-borne diseases of cattle in Zambia, malignant edema and blackquarter were widespread . One hundred and sixty-five cases with malignant edema and 103 cases with blackquarter were reported between 1985 and 1997 . It was found that specific soil-conditions associate the emergence of the soil-borne diseases . Soil samples from five areas in Zambia were examined for the presence of genus Clostridium . Direct immunofluorescent assay (IFA) examination showed that C . septicum, C . novyi and C . chauvoei were detected in the soil of specific areas in Zambia, respectively . Causal organisms such as C . perfringens were isolated from the soil samples . The information of area-specific distribution of Clositridium species may give an efficient program in protecting cattle and man.

Infect Immun, 2000 Nov, 68(11), 6378 - 83
Characterization of the catalytic domain of Clostridium novyi alpha-toxin; Busch C et al.; Clostridium novyi alpha-toxin belongs to the family of large clostridial cytotoxins which act on cells through the modification of small GTP-binding proteins . We present here an analysis of the catalytic domain of alpha-toxin . A NH(2)-terminal 551-amino-acid fragment, alpha 551, was found to contain the full enzyme activity of the holotoxin, whereas a slightly shortened fragment encompassing 509 amino acids showed no detectable enzyme activity . Further characterization of the enzymatically active fragment alpha 551 revealed a substrate specificity for both UDP-N-acetylglucosamine and UDP-glucose . A Michaelis-Menten constant of 17 microM was determined for the substrate UDP-N-acetylglucosamine, while that for UDP-glucose was about 20 times higher, indicating a weaker affinity of the toxin for the latter substrate . Mutation of the aspartic acids of a conserved motif DXD within alpha 551 reduced enzyme activity >700-fold and inhibited cytotoxicity after microinjection in cells . Inhibition of enzyme activity of the DXD mutant could be partially overcome by increased concentrations of manganese ions, suggesting the involvement of these aspartic acids in Mn(2+) binding . By construction of chimeras of enzymatically active fragments of C . sordellii lethal toxin and C . novyi alpha-toxin, we located the region involved in nucleotide-sugar specificity to amino acids 133 through 517.

Klin Lab Diagn, 2000 Aug, (8), 46 - 50
{Detection of toxin-producing pathogenic bacterial strains by polymerase chain reaction}; Vertiev IuV et al.; Polymerase chain reaction (PCR) was used for detection of pathogenic Clostridium botulinum, Clostridium perfringens, Clostridium difficile, and Escherichia coli . With this aim in view, primers to botulinic toxins types A, B, C1, D, E, F, and G, perfringens enterotoxin, difficile toxin, and types 1 and 2 Shigella-like toxins were chosen and synthesized . Optimal amplification conditions were selected for each pair of primers, with DNA and the respective agent as the reaction mixture matrices . PCR was highly specific and sensitive in all cases . Its sensitivity was 10-100 cells/sample . Among the tested C . botulinum, C . perfringens, C . difficile, and E . coli strains, specific amplification products of expected size were observed only in the strains containing the respective toxin genes . These findings recommend the use of these methods in clinical microbiology . Strains containing type 2 Shigella-like toxin gene were detected among E . coli strains isolated from patients with the hemolytic uremic syndrome, which for the first time indicates that the problem with E . coli epidemic strain O157 is valid for Russia . As a result of our studies, test systems for detection of types A, B, C, D, E, F, and G C . botulinum strains, C . perfringens and C . difficile, and E . coli O157 strains are now available.

Arch Surg, 2000 Oct, 135(10), 1206 - 11
Clostridium difficile toxins influence hepatocyte protein synthesis through the interleukin 1 receptor; Mazuski JE et al.; HYPOTHESIS: Clostridium difficile toxins require interleukin 1 (IL-1) production or a functioning IL-1 receptor to elicit acute-phase protein production by murine hepatocytes . DESIGN: Experimental study . SETTING: Research laboratory at the DVA Medical Center, St Louis, Mo . CELLS STUDIED: Hepatocytes prepared from normal mice, from knockout mice deficient in IL-1 production due to loss of IL-1 converting enzyme, or from knockout mice deficient in the IL-1 p80 receptor . INTERVENTIONS: Cells were treated with lipopolysaccharide, a crude C difficile toxin extract, or purified C difficile toxins A or B for 24 hours in vitro, then radiolabeled with (35)S methionine . Newly synthesized acute-phase proteins were identified by electrophoresis and autoradiography . MAIN OUTCOME MEASURES: Synthesis of a 23-kd acute-phase protein in response to the various stimuli . RESULTS: Lipopolysaccharide, C difficile culture extract, and purified toxins A and B stimulated the synthesis of the 23-kd acute-phase protein by hepatocytes from normal mice and by hepatocytes from knockout mice deficient in the IL-1 converting enzyme . However, hepatocytes from knockout mice deficient in the IL-1 p80 receptor failed to produce this acute-phase protein when treated with the C difficile toxins, although they responded fully to lipopolysaccharide . CONCLUSIONS: Stimulation of acute-phase protein synthesis by C difficile toxins does not require IL-1 production, but does require a functioning IL-1 p80 receptor . This suggests that some of the actions of these toxins are mediated by this receptor.

J S Afr Vet Assoc, 2000 Jun, 71(2), 111 - 4
Evaluation of two PCR-based procedures for typing Clostridium perfringens; Dungu B et al.; Two polymerase chain reaction (PCR)-based procedures for typing Clostridium, perfringens, which affects most domestic animals, were compared and evaluated for efficiency as substitute to the guinea-pig intradermal test routinely used in our laboratory, namely a multiplex PCR and a protocol based on the individual amplification of gene sequences specific for each toxin . Reference isolates of C . perfringens types A, B, C and D as well as cultures from clinical specimens were tested . The sensitivity and specificity of the PCR was confirmed on reference isolates . There was similarity in results on 43 of the 46 samples typed by all 3 methods . Clear results were obtained by PCR on 5 clinical samples that showed either equivocal or weak skin reactions in guinea-pigs . The multiplex PCR protocol, in combination with the evaluation of bacterial growth, is a better alternative to in vivo toxin typing, since C . perfringens can only be incriminated as cause of a disease when it is present in large numbers in the intestine.

Am J Infect Control, 2000 Oct, 28(5), 370 - 5
Evaluation of a novel, rapid-acting, sterilizing solution at room temperature; Hobson DW et al.; Test data are presented for a novel chemical germicide formulation capable of sterilizing reusable medical devices in 30 minutes at 20 degrees C in an open tray . The tests conducted with this rapid-acting sterilizing solution (RSS) included sporicidal, mycobactericidal, and virucidal studies performed in accordance with Association of Official Analytical Chemist International or Environmental Protection Agency published guidelines, by using RSS stressed for as long as 7 days . Sporicidal assays were performed at 20 degrees C with a 30-minute exposure time by using both Clostridium sporogenes and Bacillus subtilis spores dried on porcelain penicylinders or suture loops (n = 60 carriers per treatment) . For comparison, identical carriers were exposed to a commercial glutaraldehyde-based sterilizing solution stressed to a maximum-use time of 14 days and exposed per manufacturer's requirements (10 hours at 25 degrees C) . The RSS sterilized 100% of the carriers of both spore types . The glutaraldehyde solution demonstrated 100% sterilization of C sporogenes -treated carriers but had difficulty sterilizing B subtilis spore-laden carriers (ie, no sterilization of suture loops and only 57% sterilization of porcelain penicylinders) . Similarly, Mycobacteria bovis and selected fungal and viral agents were exposed to stressed solution for 5 minutes or less at 20 degrees C . In each case, the resulting log decrease in viable microorganisms significantly supported a claim for rapid high-level disinfection . Based on these data, RSS demonstrates high-level disinfection in 5 minutes and sterilization in 30 minutes at 20 degrees C.

Biochem Biophys Res Commun, 2000 Oct 14, 277(1), 43 - 6
Activation of MMP-2 by Clostridium difficile toxin B in bovine smooth muscle cells; Koike T et al.; Matrix metalloproteinase-2 (MMP-2) plays critical roles in cell migration through the breakdown of the extracellular matrix . Cell movements require dynamic actin reorganization, which is controlled by Rho family GTPases . In order to examine the relation between MMP-2 regulation and actin reorganization, we used several inhibitors of Rho family GTPases . Treatment of smooth muscle cells with Clostridium difficile toxin B known to inactivate Rho family GTPases activated MMP-2 . However, neither C3 transferase, a Rho inhibitor, nor Y-27632, a specific inhibitor of Rho-kinase, induced MMP-2 activation . Treatment with C3 transferase and Y-27632 caused morphological changes into the round and stellate shape, respectively, by inhibition of actin stress fiber formation . In addition, toxin B treatment induced expression and processing of MT1-MMP, a major activator of MMP-2 . Taken together, we suggest the involvement of Rho family GTPases, although inhibition of neither Rho nor Rho-kinase is sufficient, in the activation of MMP-2 through expression and activation of MT1-MMP .

Digestion, 2000, 62(2-3), 208 - 12
Multimicrobial sepsis including Clostridium perfringens after chemoembolization of a single liver metastasis from common bile duct cancer; Eckel F et al.; A 65-year-old woman underwent resection of a distal common bile duct carcinoma (Whipple's procedure) . Twelve months later a single hepatic metastasis was detected and a chemoembolization was performed . Immediately after chemoembolization the patient developed a multimicrobial sepsis including Clostridium perfringens . CT scans depicted pathognomonic signs of gas-containing abscess in the necrotic liver metastasis . She was subsequently treated with broad-spectrum antibiotics, abscess drainage and hyperbaric oxygen therapy . We conclude that antibiotic prophylaxis is recommendable for chemoembolization of liver metastasis in patients with risk factors like intestinal biliary reflux (bilioenteric anastomosis or papillotomy and biliary stenting) and bile duct cancer, otherwise severe sepsis including clostridium bacteremia may occur .

J Vet Diagn Invest, 2000 Sep, 12(5), 453 - 5
Fatal Clostridium botulinum toxicosis in eleven Holstein cattle fed round bale barley haylage; Kelch WJ et al.; Twenty-two lactating Holstein cattle in Tennessee had clinical signs of intoxication with preformed Clostridium botulinum toxin . These signs included weakness, paralysis of the tongue and chest muscles, abdominal breathing, and, in 11 of the 22 cows, death . Differential diagnoses included hypocalcemia, hypomagnesemia, carbohydrate overload, and several toxicoses including mycotoxin, lead, nitrate, organophosphate, atropine or atropine-like alkaloid, and botulism . A diagnosis of botulism by the ingestion of preformed C . botulinum type B toxin was made by eliminating these other diseases, by finding C . botulinum type B spores in 3 bales of round bale barley haylage fed to these cattle, and by isolating preformed type B toxin from 1 of the 3 bales . Confirmation of the toxin type was made by demonstrating mouse lethality by intraperitoneal injection of specimen extracts with neutralization by C . botulinum type B antitoxin . The haylage, harvested green and encased in black plastic bags to facilitate fermentation, was presumably contaminated by the botulinum toxin when fermentation failed to produce enough acid to lower the pH to 4.5, the pH below which C . botulinum growth is inhibited . Farmers and ranchers who use round hay balers to produce haylage should be alert to this potential problem.

J Med Chem, 2000 Oct 5, 43(20), 3677 - 87
Carbonic anhydrase and matrix metalloproteinase inhibitors: sulfonylated amino acid hydroxamates with MMP inhibitory properties act as efficient inhibitors of CA isozymes I, II, and IV, and N-hydroxysulfonamides inhibit both these zinc enzymes; Scozzafava A et al.; The 14 different carbonic anhydrase (CA, EC 4.2.1.1) isozymes as well as the 23 different matrix metalloproteinases (MMPs) isolated up to now in higher vertebrates play important physiological functions in these organisms . Unsubstituted sulfonamides act as high-affinity inhibitors for the first type of these enzymes, whereas hydroxamates strongly inhibit the latter ones . Since the active site geometry around the zinc ion in these two types of metalloenzymes is rather similar, we tested whether sulfonylated amino acid hydroxamates of the type RSO(2)NX-AA-CONHOH (X = H, benzyl, substituted benzyl; AA = amino acid moiety, such as Gly, Ala, Val, Leu) with well-known inhibitory properties against MMPs and Clostridium histolyticum collagenase (ChC, another zinc enzyme related to the MMPs) might also act as CA inhibitors . We also investigated whether N-hydroxysulfonamides of the type RSO(2)NHOH (which are effective CA inhibitors) inhibit MMPs and ChC . Here we report several potent sulfonylated amino acid hydroxamate CA inhibitors (with inhibition constants in the range of 5-40 nM, against the human isozymes hCA I and hCA II, and 10-50 nM, against the bovine isozyme bCA IV), as well as preliminary SAR for this new class of non-sulfonamide CA inhibitors . Some N-hydroxysulfonamides also showed inhibitory properties (in the micromolar range) against MMP-1, MMP-2, MMP-8, MMP-9, and ChC . Thus, the SO(2)NHOH group is a new zinc-binding function for the design of MMP inhibitors . Both CA as well as MMPs are involved, among others, in carcinogenesis and tumor invasion processes . On the basis of these findings, we suggest that the mechanism of antitumor action with some hydroxamate inhibitors might also involve inhibition of some CA isozymes (such as CA IX, CA XII, and CA XIV) present only in tumor cell membranes, in addition to collagenases/gelatinases of the MMP type . Our data also suggest that it should be possible to develop dual enzyme inhibitors that would strongly inhibit both these metalloenzymes, CAs and MMPs, based on the nature of the R, AA, and X moieties in the above formula . Compact X (such as H) and AA (such as Gly) moieties favor CA over MMP inhibition, whereas bulkier X (benzyl, substituted benzyl, etc.) and AA (such as Val, Leu) moieties and substituted-aryl R groups are advantageous for obtaining potent MMP and ChC inhibitors, which show lower affinity for CA.

J Antimicrob Chemother, 2000 Oct, 46(4), 551 - 6
In vitro activity of new generation fluoroquinolones against genotypically distinct and indistinguishable Clostridium difficile isolates; Wilcox MH et al.; We compared the activities of ciprofloxacin and levofloxacin with those of the newer fluoroquinolones grepafloxacin, moxifloxacin, sparfloxacin and trovafloxacin against Clostridium difficile isolates . As there is good evidence of marked clonal spread of C . difficile, we studied both genotypically distinct (n = 26) and indistinguishable (n = 28) isolates as determined by random amplified polymorphic DNA and ribosomal spacer PCR fingerprinting . The indistinguishable strains examined represent the main UK epidemic C . difficile clone . For 17 of 54 strains (31%) we were unable to read MICs following inocula preparation using Mueller-Hinton broth . Using Schaedler's broth for inocula preparation 93% of strains had readable MICs, although geometric mean MICs were uniformly higher (2.5- to 5.4-fold) compared with results using Mueller-Hinton broth . Moxifloxacin and trovafloxacin, followed by grepafloxacin, were the most active fluoroquinolones tested and were 3- to 4-fold more active than older agents such as ciprofloxacin by both MIC methods . Unexpectedly, clonal C . difficile strains had markedly reduced susceptibility compared with the distinct strains to each of the fluoroquinolones tested . Clonal strains were more than seven-fold or 12- to 29-fold less susceptible (according to geometric mean MICs) than distinct strains to both moxifloxacin and trovafloxacin, depending on the MIC method used . It remains to be seen whether the enhanced activity of new fluoroquinolones such as moxifloxacin in comparison with other fluoroquinolones against C . difficile implies that these agents are unlikely to be associated with C . difficile infection . However, clinical use of new generation fluoroquinolones in elderly hospitalized patients where C . difficile is endemic requires further study, particularly given the reduced antibiotic susceptibility to all fluoroquinolones of the readily transmissible UK C . difficile clone.

Poult Sci, 2000 Sep, 79(9), 1311 - 9
Effect of zinc bacitracin and salinomycin on intestinal microflora and performance of broilers; Engberg RM et al.; A feeding experiment was carried out over 42 d with four groups of broiler chickens fed experimental diets formulated to provide no supplementation, 20 mg zinc bacitracin, 60 mg salinomycin, or both feed additives in combination . During the fifth week of the experiment, four chickens from each pen were killed, and the contents of gizzard, duodenum, jejunum, ileum, ceca, and rectum were separately collected and pooled . In all intestinal segments, the pH and the concentration of lactic acid were measured, and the numbers of anaerobic bacteria, coliforms, lactic acid bacteria, lactobacilli, enterococci, and Clostridium perfringens were counted . In homogenates of pancreas obtained from four animals, the activities of amylase, lipase, trypsin, and chymotrypsin were measured . A significant growth-promoting effect was observed in the group receiving zinc bacitracin in combination with salinomycin . Zinc bacitracin significantly reduced the number of coliform bacteria in the ileum and increased the activities of amylase and lipase in pancreas homogenates . Supplementation with salinomycin and zinc bacitracin, alone or in combination, resulted in significantly lower counts of C . perfringens as well as Lactobacillus salivarius, which was a dominant lactic acid bacterium found in broiler intestinal contents . High numbers of these lactobacilli may play a role in broiler growth depression related to competition in nutrient uptake or impaired fat absorption due to bile acid deconjugation.

Growth Factors, 2000, 18(2), 147 - 55
A conjugate composed of nerve growth factor coupled to a non-toxic derivative of Clostridium botulinum neurotoxin type A can inhibit neurotransmitter release in vitro; Chaddock JA et al.; Nerve growth factor (NGF) receptor binding, internalisation and transportation of NGF has been identified as a potential route of delivery for other molecules . A derivative of Clostridium botulinum neurotoxin type A (LHN) that retains catalytic activity but has significantly reduced cell-binding capability has been prepared and chemically coupled to NGF . Intact clostridial neurotoxins potently inhibit neurotransmitter release at the neuromuscular junction by proteolysis of specific components of the vesicle docking/fusion complex . Here we report that the NGF-LHN/A conjugate, when applied to PC12 cells, significantly inhibited neurotransmitter release and cleaved the type A toxin substrate . This work represents the successful use of NGF as a targeting moiety for the delivery of a neurotoxin fragment.

Clin Infect Dis, 2000 Sep, 31(3), 717 - 22 Epub 2000 Oct 04.
The role of physical proximity in nosocomial diarrhea; Chang VT et al.; To examine physical proximity as a risk factor for the nosocomial acquisition of Clostridium difficile-associated diarrhea (CDAD) and of antibiotic-associated diarrhea (AAD), we assessed a retrospective cohort of 2859 patients admitted to a community hospital from 1 March 1987 through 31 August 1987 . Of these patients, 68 had nosocomial CDAD and 54 had nosocomial AAD . In multivariate analysis, physical proximity to a patient with CDAD (relative risk {RR}, 1.86; 95% confidence interval {CI}, 1.06-3.28), exposure to clindamycin (RR, 4.22; 95% CI, 2.11-8.45), and the number of antibiotics taken (RR, 1.49; 95% CI, 1.23-1.81) were significant . For patients with nosocomial AAD, exposure to a roommate with AAD (RR, 3.94; 95% CI, 1 . 27-12.24), a stay in an intensive care unit or cardiac care unit (RR, 1.93; 95% CI, 1.05-3.53), and the number of antibiotics taken (RR, 2.01; 95% CI, 1.67-2.40) were significant risk factors . Physical proximity may be an independent risk factor for acquisition of nosocomial CDAD and AAD.

Int J Food Microbiol, 2000 Sep 25, 60(2-3), 205 - 18
New developments in chromogenic and fluorogenic culture media; Manafi M; This review describes some recent developments in chromogenic and fluorogenic culture media in microbiological diagnostic . The detection of beta-D-glucuronidase (GUD) activity for enumeration of Escherichia coli is well known . E . coli O157:H7 strains are usually GUD-negative and do not ferment sorbitol . These characteristics are used in selective media for these organisms and new chromogenic media are available . Some of the new chromogenic media make the Salmonella diagnostic easier and faster . The use of chromogenic and fluorogenic substrates for detection of beta-D-glucosidase (beta-GLU) activity to differentiate enterococci has received considerable attention and new media are described . Rapid detection of Clostridium perfringens, Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus are other application of enzyme detection methods in food and water microbiology.

J Nutr, 2000 Oct, 130(10), 2599 - 606
Fermentation by gut microbiota cultured in a simulator of the human intestinal microbial ecosystem is improved by supplementing a soygerm powder; De Boever P et al.; An in vitro model, designated the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), was used to study the effect of a soygerm powder rich in beta-glycosidic phytoestrogenic isoflavones on the fermentation pattern of the colon microbiota and to determine to what extent the latter metabolize the conjugated phytoestrogens . Initially, an inoculum prepared from human feces was introduced into the reactor vessels and stabilized over 3 wk using a culture medium . This stabilization period was followed by a 2-wk control period during which the microbiota were monitored . The microbiota were then subjected to a 2-wk treatment period by adding 2.5 g/d soygerm powder to the culture medium . The addition resulted into an overall increase of bacterial marker populations (Enterobacteriaceae:, coliforms, Lactobacillus: sp., Staphylococcus: sp . and Clostridium: sp.), with a significant increase of the Lactobacillus: sp . population . The short-chain fatty acid (SCFA) concentration increased approximately 30% during the supplementation period; this was due mainly to a significant increase of acetic and propionic acids . Gas analysis revealed that the methane concentration increased significantly . Ammonium and sulfide concentrations were not influenced by soygerm supplementation . Use of an electronic nose apparatus indicated that odor concentrations decreased significantly during the treatment period . The beta-glycosidic bonds of the phytoestrogenic isoflavones were cleaved under the conditions prevailing in the large intestine . The increased bacterial fermentation after addition of the soygerm powder was paralleled by substantial metabolism of the free isoflavones (genistein, daidzein and glycitein), resulting in recovery of only 12-17% of the supplemented isoflavones.

Br J Pharmacol, 2000 Oct, 131(3), 553 - 61
Inhibition of small G proteins of the rho family by statins or clostridium difficile toxin B enhances cytokine-mediated induction of NO synthase II; Hausding M et al.; In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG-CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools . Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho) . In contrast, TcdB is a glucosyltransferase and inactivates Rho-proteins directly . Human A549/8- and DLD-1 cells as well as murine 3T3 fibroblasts were preincubated for 18 h with statins (1 - 100 microM) or TcdB (0.01-10 ng ml(-1)) . Then NOS II expression was induced by cytokines . NOS II mRNA was measured after 4 - 8 h by RNase protection assay, and NO production were measured by the Griess assay after 24 h . Statins and TcdB markedly increased cytokine-induced NOS II mRNA expression and NO production . Statin-mediated enhancement of NOS II mRNA expression was reversed almost completely by cotreatment with mevalonate or geranylgeranylpyrophosphate . It was only slightly reduced by farnesylpyrophosphate . Therefore, small G proteins of the Rho family are likely to be involved in NOS II induction . In A549/8 cells stably transfected with a luciferase reporter gene under the control of a 16 kb fragment of the human NOS II promoter (pNOS2(16)Luc), statins produced only a small increase in cytokine-induced NOS II promoter activity . In contrast, statins had a considerable superinducing effect in DLD-1 cells stably transfected with pNOS2(16)Luc . In conclusion, our studies provide evidence that statins and TcdB potentiate cytokine-induced NOS II expression via inhibition of small G proteins of the Rho family . This in turn results in an enhanced NOS II promoter activity and/or a prolonged NOS II mRNA stability.

Circ Res, 2000 Sep 29, 87(7), 616 - 22
Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells; Hernandez-Perera O et al.; Endothelial dysfunction is characterized by an impaired vasodilatory response to endothelial agonists as well as by alterations in adhesion and coagulation processes . 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) have been shown to be useful in the reversal of endothelial dysfunction, an effect that may be independent of the reduction in cholesterol levels . Both the L-arginine-nitric oxide-cGMP and endothelin pathways are involved in the regulation of vascular tone . Here, we show that the basal transcription rate of the preproendothelin-1 gene was decreased by simvastatin (10 micromol/L) in bovine aortic endothelial cells . Transfection studies with the preproendothelin-1 gene promoter showed that mevalonate (100 micromol/L) was able to prevent the inhibitory effect mediated by simvastatin . Protein geranylgeranylation, but not farnesylation, proved to be crucial for a correct expression of the preproendothelin-1 gene . The C3 exotoxin from Clostridium botulinum that selectively inactivates Rho GTPases, the processing of which involves geranylgeranylation, reproduced the inhibitory effect of simvastatin on the expression of preproendothelin-1 . Overexpression of dominant-negative mutants of RhoA and RhoB led to a significant reduction in the preproendothelin-1 promoter activity, whereas the expression of wild-type and constitutively active forms of these proteins resulted in an increase, in support that Rho proteins are required for the basal expression of the preproendothelin-1 gene . Finally, we show that the Rho-dependent activation of the preproendothelin-1 gene transcription was inhibited by simvastatin . Thus, the control of vascular tone and proliferative response mediated by endothelin-1 is regulated at multiple levels, among which the Rho proteins play an essential role.

Cereb Cortex, 2000 Oct, 10(10), 927 - 38
Regulation of dendritic spine morphology by the rho family of small GTPases: antagonistic roles of Rac and Rho; Tashiro A et al.; Dendritic spines mediate most excitatory transmission in the mammalian CNS and have been traditionally considered stable structures . Following the suggestion that spines may 'twitch', it has been recently shown that spines are capable of rapid morphological rearrangements . Because of the role of the small GTPases from the Rho family in controlling neuronal morphogenesis, we investigated the effects of several members of this biochemical signaling pathway in the maintenance of the morphology of extant dendritic spines by combining biolistic transfection of pyramidal neurons in cultured cortical and hippocampal slices with two-photon microscopy . We find a variety of effects on the density and morphology of dendritic spines by expressing either constitutively active or dominant negative forms of several small GTPases of the Rho family, by blocking the entire pathway with Clostridium difficile toxin B or by blocking Rho with C3 transferase . We propose a model where Rac promotes spine formation, while Rho prevents it . We conclude that the small GTPases provide antagonistic control mechanisms of spine maintenance in pyramidal neurons.

Can J Microbiol, 2000 Sep, 46(9), 856 - 9
Purification and characterization of a 4-hydroxybenzoate decarboxylase from an anaerobic coculture; Li T et al.; The oxygen-sensitive 4-hydroxybenzoate decarboxylase (4OHB-DC) activity from a phenol-carboxylating coculture, consisting of Clostridium-like strain 6 and an unidentified strain 7, was studied . Assays done with cell extracts showed that the optimal pH was 5.0-6.5 and the Km was 5.4 mM . The activity decreased by 50% in the presence of 5 mM EDTA, and it was restored and even enhanced by the addition of Mg++, Mn++, Zn++, or Ca++ . After purification, the molecular mass of the enzyme was estimated as 420 kDa by gel chromatography, and as 119 kDa by SDS-PAGE, suggesting a homotetrameric structure . Its pI was 5.6 . The N-terminal amino acid sequence showed 95% and 76% homology with the pyruvate-flavodoxin oxidoreductase (nifJ gene product) from Enterobacter agglomerans and Klebsiella pneumoniae, respectively . The purified enzyme also slowly catalyzed the reverse reaction, that is the phenol carboxylation . These characteristics suggest that this enzyme is different from other known decarboxylases . This includes the 4OHB-DC from Clostridium hydroxybenzoicum, which is the only one that had been purified before.

J Bacteriol, 2000 Oct, 182(20), 5906 - 10
A large gene cluster for the Clostridium cellulovorans cellulosome; Tamaru Y et al.; A large gene cluster for the Clostridium cellulovorans cellulosome has been cloned and sequenced upstream and downstream of the cbpA and exgS genes (C.-C . Liu and R . H . Doi, Gene 211:39-47, 1998) . Gene walking revealed that the engL gene cluster (Y . Tamaru and R . H . Doi, J . Bacteriol . 182:244-247, 2000) was located downstream of the cbpA-exgS genes . Further DNA sequencing revealed that this cluster contains the genes for the scaffolding protein CbpA, the exoglucanase ExgS, several endoglucanases of family 9, the mannanase ManA, and the hydrophobic protein HbpA containing a surface layer homology domain and a hydrophobic (or cohesin) domain . The sequence of the clustered genes is cbpA-exgS-engH-engK-hbpA-engL-man A-engM-engN and is about 22 kb in length . The engN gene did not have a complete catalytic domain, indicating that engN is a truncated gene . This large gene cluster is flanked at the 5' end by a putative noncellulosomal operon consisting of nifV-orf1-sigX-regA and at the 3' end by noncellulosomal genes with homology to transposase (trp) and malate permease (mle) . Since gene clusters for the cellulosome are also found in C . cellulolyticum and C . josui, they seem to be typical of mesophilic clostridia, indicating that the large gene clusters may arise from a common ancestor with some evolutionary modifications.

Infect Control Hosp Epidemiol, 2000 Sep, 21(9), 592 - 6
Surveillance for nosocomial and central line-related infections among pediatric hematology-oncology patients; Simon A et al.; OBJECTIVE: To determine the incidence of all nosocomial infections (NIs) in pediatric hematology-oncology patients, as well as central venous access device (CVAD)-associated infections acquired during home care . DESIGN: Prospective surveillance study . SETTING: The Pediatric Hematology and Oncology Department at the University Hospital Bonn . PATIENTS: All patients admitted from January through October 1998 (surveillance period) . METHODS: Standardized surveillance system based on the Centers for Disease Control and Prevention's National Nosocomial Infections Surveillance System . RESULTS: A total of 143 patients were hospitalized for 3,701 days (776 admissions) during the surveillance period . Of the 40 NIs detected, 26 were CVAD-related, with 21 bloodstream infections (BSIs) and 5 local infections . Four were Clostridium difficile-associated diarrheal illnesses, 3 were pneumonias, and 7 were other infections . The incidence of NIs was 10.8 per 1,000 patient-days (5.2 NIs/100 admissions) . The overall CVAD-related BSI rate was 7.4 per 1,000 utilization days, without a significant difference between implanted infusion ports and tunneled catheters . In addition, 7 CVAD-related infections occurred during home care . All 8 BSIs associated with tunneled catheters and 13 (76%) of the 17 BSIs associated with ports were acquired nosocomially . For inpatients and outpatients combined, the exit sites of tunneled catheters were more likely to become locally infected than were the needle entry sites of ports (relative risk, 8.0; P=.007) . In 30 (75%) of the 40 NIs, the affected patients had severe neutropenia (<500/mm3) at the time of infection . CONCLUSIONS: Most NIs in the pediatric hematology-oncology patients were associated with CVAD devices . Although many infections in this high-risk population may not be preventable through infection control measures, the careful evaluation of specific infection rates permits the identification of risk factors that may be targeted by infection control programs . Prospective surveillance for NIs on pediatric oncology units is an indispensable tool for this internal quality control.

J Inorg Biochem, 2000 Jul 1, 80(3-4), 205 - 11
The presence of a SO molecule in {NiFe} hydrogenase from Desulfovibrio vulgaris Miyazaki as detected by mass spectrometry; Higuchi Y et al.; The active site of {NiFe} hydrogenase is a binuclear metal complex composed of Fe and Ni atoms and is called the Ni-Fe site, where the Fe atom is known to be coordinated to three diatomic ligands . Two mass spectrometric techniques, pyrolysis-MS (pyrolysis-mass spectrometry) and TOF-SIMS (time-of-flight secondary ion mass spectrometry), were applied to several proteins, including native and denatured forms of {NiFe} hydrogenase from Desulfovibrio vulgaris Miyazaki F, {Fe4S4}2-ferredoxin from Clostridium pasteurianum, {Fe,S2}-ferredoxin from Spirulna platensis, and porcine pepsin . Pyrolysis-MS revealed that only native hydrogenase liberated SO/SO2 (ions of m/z 48 and 64 at an equilibrium ratio of SO and SO2) at relatively low temperatures before the covalent bonds in the polypeptide moiety started to decompose . TOF-SIMS indicated that native Miyazaki hydrogenase released SO/SO2 (m/z 47.97 and 63.96) as secondary ions when irradiated with a high-energy Ga+ beam . Denatured hydrogenase, clostridial ferredoxin, and pepsin did not release SO as a secondary ion . The FT-IR spectrum of the enzyme suggested the presence of CO and CN . These lines of evidence suggest that the three diatomic ligands coordinated to the Fe atom at the Ni-Fe site in Miyazaki hydrogenase are SO, CO, and CN . The role of the SO ligand in helping to cleave H2 molecules at the active site and stabilizing the Fe atom in the diamagnetic Fe(II) state in the redox cycle of this enzyme is discussed.

Vet Microbiol, 2000 Oct 20, 76(4), 359 - 72
Cloning and expression of a gene encoding the flagellin of Clostridium chauvoei; Kojima A et al.; Clostridium chauvoei is a causative agent of blackleg and the major protective antigen of the organism is the flagellar protein . Using an Escherichia coli expression library of the C . chauvoei Okinawa strain, we isolated the fliC gene encoding the flagellin protein . DNA sequence analysis revealed an open reading frame of 413 amino acid residues with a calculated molecular mass of 43819Da . Comparison of the sequence with those of flagellins from other bacteria showed considerable homology in the N-terminal and C-terminal domains . The glutathione-S-transferase (GST)-flagellin fusion protein and the purified FliC protein after removing the GST part with thrombin reacted with both polyclonal antisera and the non-protective monoclonal antibody (Mab), Mo-114 . However, the protective Mab, Mo-41, which may recognize its conformational epitope, failed to react with both the GST-flagellin fusion protein and the purified FliC . Furthermore, the GST-flagellin fusion protein and the purified FliC induced very little protective immunity in mice . These results suggested that a conformation-dependent epitope play an important role in the development of immunity against blackleg.

J Microbiol Methods, 2000 Sep, 42(1), 29 - 38
Single-cell analysis of bacteria by Raman microscopy: spectral information on the chemical composition of cells and on the heterogeneity in a culture; Schuster KC et al.; In the acetone-butanol (ABE) fermentation process, the utilised organisms from the group of the solventogenic Clostridia go through a complex cell-cycle . The role of different cell types in product formation is not understood in detail yet . We aim to use Raman spectroscopy to characterise the population distribution in Clostridium cultures . Cell suspensions were dried on calcium fluoride carriers . Raman spectra of single cells were obtained using a confocal Raman microscope (Dilor, Lille, France) . The laser beam was focused on individual cells through the microscope objective . Spectra with good signal-to-noise ratio were obtained . Cells of different morphology, but also apparently similar cells, showed different spectra . Several cell components could be detected and varied in quantity . Compared to other methods for single-cell analysis, the new method is much more time-consuming to analyse one individual cell . However, a large amount of chemical information is obtained from each single cell in a non-destructive, non-invasive way . Raman microscopy appears to be a suitable method for studying population distributions in bacterial cultures.

Neurosci Lett, 2000 Oct 6, 292(2), 95 - 8
Extrinsic surgical denervation inhibits Clostridium difficile toxin A-induced enteritis in rats; Mantyh CR et al.; Clostridium difficile enteritis is caused by toxin A (TA) which stimulates substance P release and subsequent receptor activation . This receptor stimulation results in secretion, inflammation, and structural damage . However, it is unclear as to which subset of neurons is required to initiate substance P release following toxin stimulation . Five centimeter ileal segments were surgically denervated . After 10 days, three ileal loops were constructed in each rat: the denervated loop was injected intraluminally with 5 microg of TA and two intact loops were injected with TA or vehicle, respectively . Ileal secretion, myeloperoxidase activity, and histology were then assessed . Denervated ileal loops injected with TA had a 75% reduction in ileal secretion (P < 0.001), 92% reduction in myeloperoxidase activity (P < 0.01) and 96% reduction in histologic damage (P < 0.001) compared to innervated loops . There were no significant differences between the denervated loops injected with TA and those injected with vehicle . Extrinsic surgical denervation results in protection of ileal loops from TA enteritis . Furthermore, these results exclude the participation of intrinsic enteric nerves in TA-induced ileal damage . Finally, this suggests that extrinsic primary sensory neurons mediate the effects of intraluminal TA in the ileum.

Biochemistry, 2000 Sep 12, 39(36), 11129 - 36
Specificity and affinity of substrate binding by a family 17 carbohydrate-binding module from Clostridium cellulovorans cellulase 5A; Boraston AB et al.; The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulovorans cellulase 5A is a beta-1,4-glucan binding module with a preference for soluble chains . CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (+/-0.2) x 10(5) and 1.6 (+/-0.2) x 10(5) M(-1), respectively . The capacity values for PASA and Avicel were 11.9 and 0.4 micromol/g of cellulose, respectively . CBM17 did not bind to crystalline cellulose . CBM17 bound tightly to soluble barley beta-glucan and the derivatized celluloses HEC, EHEC, and CMC . The association constants for binding to barley beta-glucan, HEC, and EHEC were approximately 2.0 x 10(5) M(-1) . Significant binding affinities were found for cello-oligosaccharides greater than three glucose units in length . The affinities for cellotriose, cellotetraose, cellopentaose, and cellohexaose were 1.2 (+/-0.3) x 10(3), 4.3 (+/-0.4) x 10(3), 3.8 (+/-0.1) x 10(4), and 1.5 (+/-0.0) x 10(5) M(-1), respectively . Fluorescence quenching studies and N-bromosuccinimide modification indicate the participation of tryptophan residues in ligand binding . The possible architecture of the ligand-binding site is discussed in terms of its binding specificity, affinity, and the participation of tryptophan residues.

Structure Fold Des, 2000 Aug 15, 8(8), 817 - 30
Crystal structure of a methyltetrahydrofolate- and corrinoid-dependent methyltransferase; Doukov T et al.; BACKGROUND: Methyltetrahydrofolate, corrinoid iron-sulfur protein methyltransferase (MeTr), catalyzes a key step in the Wood-Ljungdahl pathway of carbon dioxide fixation . It transfers the N5-methyl group from methyltetrahydrofolate (CH3-H4folate) to a cob(I)amide center in another protein, the corrinoid iron-sulfur protein . MeTr is a member of a family of proteins that includes methionine synthase and methanogenic enzymes that activate the methyl group of methyltetra-hydromethano(or -sarcino)pterin . We report the first structure of a protein in this family . RESULTS: We determined the crystal structure of MeTr from Clostridium thermoaceticum at 2.2 A resolution using multiwavelength anomalous diffraction methods . The overall architecture presents a new functional class of the versatile triose phosphate isomerase (TIM) barrel fold . The MeTr tertiary structure is surprisingly similar to the crystal structures of dihydropteroate synthetases despite sharing less than 20% sequence identity . This homology permitted the methyl-H4folate binding site to be modeled . The model suggests extensive conservation of the pterin ring binding residues in the polar active sites of the methyltransferases and dihydropteroate synthetases . The most significant structural difference between these enzymes is in a loop structure above the active site . It is quite open in MeTr, where it can be modeled as the cobalamin binding site . CONCLUSIONS: The MeTr structure consists of a TIM barrel that embeds methyl-H4folate and cobamide . All related methyltransferases are predicted to fold into a similar TIM barrel pattern and have a similar pterin and cobamide binding site . The observed structure is consistent with either a 'front' (N5) or 'back' (C8a) side protonation of CH3-H4folate, a key step that enhances the electrophilic character of the methyl group, activating it for nucleophilic attack by Co(I).

J Neurosci, 2000 Sep 15, 20(18), 6743 - 51
Regulation of somatodendritic GABAA receptor channels in rat hippocampal neurons: evidence for a role of the small GTPase Rac1; Meyer DK et al.; The role of the cytoskeleton in the activity of GABA(A) receptors was investigated in cultured hippocampal neurons . Receptor currents were measured with the whole-cell patch-clamp technique during repetitive stimulation with 1 microm muscimol . After destruction of the microtubular system with nocodazol, muscimol-induced currents showed a rundown by 78% . A similar rundown was observed when actin fibers were destroyed with latrunculin B or C2 toxin of Clostridium botulinum . Because the small GTPases of the Rho family RhoA, Rac1, and Cdc42 are known to control the organization of actin fibers, we investigated their possible involvement . Inactivation of the GTPases with clostridial toxins, as well as intracellular application of recombinant Rho GTPases, indicated that active Rac1 was necessary for full GABA(A) receptor activity . Immunocytochemical labeling of the receptors showed that the disappearance of receptor clusters in the somatic membrane as induced by muscimol stimulation was enhanced by Rac1 inactivation . It is suggested that Rac1 participates in the regulation of GABA(A) receptor clustering and/or recycling.

J Biol Chem, 2000 Dec 29, 275(52), 41156 - 65
Identification of domain-domain docking sites within Clostridium symbiosum pyruvate phosphate dikinase by amino acid replacement; Wei M et al.; Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosum apoPPDK, were replaced by site-directed mutagenesis . The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency . PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>10(4)) of the ATP pyrophosphorylation of His(455) at the N-terminal domain . In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover . Arg(219) and Glu(271) were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His(455) into position for catalysis . Substitution of central domain residues Glu(434)/Glu(437) and Thr(453), the respective docking partners of Arg(219) and Glu(271), resulted in mutants impaired in catalysis at the ATP active site . The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr(453)-Glu(271) binding interaction . With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme . It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH and Met(452) backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution.

Bull Mem Acad R Med Belg, 1999, 154(6 Pt 2), 319 - 25
{Study of the role of Clostridium perfringens in bovine enterotoxemia}; Manteca C et al.; Bovine enterotoxaemia is an acute to peracute syndrome occurring mainly in calves and characterized by the sudden or very rapid death of the calf, with colics, convulsions and nervous disorders as clinical signs, if any . The most pronounced lesion is a necrohaemorrhagic enteritis of the jejunum, the ileum, and sometimes the colon . Suckling beef calves are the most frequently affected ones . In 67% of the 78 field cases investigated, some kind of stress was observed 24 to 36 hours prior to the death: change in diet or pasture, vaccination.. . The most frequently isolated bacteria, and the one isolated in highest numbers, was non-sporulated non-enterotoxigenic toxinotype A Clostridium perfringens . Reproduction of the lesions was successful in a ligated intestinal loop assay in one calf with a few of these strains, more especially with one of them, which was shown later to produce another recently described toxin, the beta 2 toxin . A role for this beta 2 toxin in bovine enterotoxaemia is thus speculated for future research.

J Pak Med Assoc, 2000 Aug, 50(8), 246 - 9
Diagnosis of Clostridium difficile antibiotic associated diarrhoea culture versus toxin assay; Sultana Q et al.; OBJECTIVE: To compare the results of Clostridium Difficile (CD) on culture with detection of C . difficile toxin by Enzyme Immunoassay (EIA) in the stool specimens of hospitalized patients with antibiotic associated diarrhoea (AAD) . PATIENTS AND METHODS: The study included 80 adult patients with AAD and 20 adult patients with non-AAD . Stool specimens of all these subjects were inoculated on cycloserine cefoxitin fructose agar and incubated anaerobically to isolate C . difficile . At the same time, all the stool specimens were tested for C . difficile toxin by EIA technique using cytoclone A and B kit manufactured by Cambridge Biotech Corporation, Worcester, Massachusette . RESULTS: Out of 80 adult patients with AAD, thirty were females and fifty males . C . difficile was isolated on culture from stool specimen of 16 patients, while twenty-three stool specimens were positive for C . difficile toxin . From 20 control subjects, C . difficile was isolated from stool specimen of only one subject . No stool specimen from the controls was positive for toxin . CONCLUSION: Diagnosis of CDAAD by culture is difficult and time consuming because of strict anaerobic nature of organism . Moreover, mere isolation of C . difficile on culture is not sufficient to establish the pathogenic role of these isolates . C . difficile toxin detection by EIA technique is a highly sensitive and specific method for diagnosis of CDAAD . Using this method, results are available in three hours time . Therefore, EIA is recommended for rapid diagnosis of CDAAD.

Infect Immun, 2000 Oct, 68(10), 5881 - 8
Toxins, butyric acid, and other short-chain fatty acids are coordinately expressed and down-regulated by cysteine in Clostridium difficile; Karlsson S et al.; It was recently found that a mixture of nine amino acids down-regulate Clostridium difficile toxin production when added to peptone yeast extract (PY) cultures of strain VPI 10463 (S . Karlsson, L . G . Burman, and T . Akerlund, Microbiology 145:1683-1693, 1999) . In the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (n = 6) and clinical isolates (n = 28) of C . difficile (>99% suppression by cysteine in the highest toxin-producing strain) . Also, cysteine derivatives such as acetylcysteine, glutathione, and cystine effectively down-regulated toxin expression . An impact of both cysteine and cystine but not of thioglycolate on toxin yield indicated that toxin expression was not regulated by the oxidation-reduction potential . Several metabolic pathways, including butyric acid and butanol production, were coinduced with the toxins in PY and down-regulated by cysteine . The enzyme 3-hydroxybutyryl coenzyme A dehydrogenase, a key enzyme in solventogenesis in Clostridium acetobutylicum, was among the most up-regulated proteins during high toxin production . The addition of butyric acid to various growth media induced toxin production, whereas the addition of butanol had the opposite effect . The results indicate a coupling between specific metabolic processes and toxin expression in C . difficile and that certain amino acids can alter these pathways coordinately . We speculate that down-regulation of toxin production by the administration of such amino acids to the colon may become a novel approach to prophylaxis and therapy for C . difficile-associated diarrhea.

Infect Immun, 2000 Oct, 68(10), 5546 - 51
Clostridium perfringens beta-toxin forms potential-dependent, cation-selective channels in lipid bilayers; Shatursky O et al.; Recombinant beta-toxin from Clostridium perfringens type C was found to increase the conductance of bilayer lipid membranes (BLMs) by inducing channel activity . The channels exhibited a distribution of conductances within the range of 10 to 380 pS, with the majority of the channels falling into two categories of conductance at 110 and 60 pS . The radii of beta-toxin pores found for the conductance states of 110 and 60 pS were 12.7 and 11.1 A, respectively . The single channels and the steady-state currents induced by beta-toxin across the BLMs exhibited ideal monovalent cation selectivity . Addition of divalent cations (Zn(2+), Cd(2+), or Mg(2+)) at a concentration of 2 mM increased the rate of beta-toxin insertion into BLMs and the single-channel conductance, while application of 5 mM Zn(2+) to a beta-toxin-induced steady-state current decreased the inward current by approximately 45% . The mutation of arginine 212 of beta-toxin to aspartate, previously shown to increase the 50% lethal dose of beta-toxin for mice nearly 13-fold, significantly reduced the ability of beta-toxin to form channels . These data support the hypothesis that the lethal action of beta-toxin is based on the formation of cation-selective pores in susceptible cells.

Infect Immun, 2000 Oct, 68(10), 5480 - 7
Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease; Sambol SP et al.; A toxin variant strain of Clostridium difficile was isolated from two patients with C . difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis . This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C . difficile toxin A . Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay . Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340 . Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of the gene, and a point mutation in the 5' end of the gene, resulting in a premature stop codon at tcdA position 139 . Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463 . Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C . difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans . REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2 . A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD . PCR amplification of the 3' end of tcdA demonstrated identical 1 . 8-kb deletions in all seven type CF2 isolates . REA type CF2 is a toxin variant strain of C . difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2719 - 27
Cloning and nucleotide sequence of the DNA gyrase (gyrA) gene from Mycoplasma hominis and characterization of quinolone-resistant mutants selected in vitro with trovafloxacin; Bebear CM et al.; We report the cloning and characterization of the gyrA gene of the Mycoplasma hominis DNA gyrase, which was previously shown to be associated with quinolone resistance in this organism . The 2,733-bp gyrA gene encodes a protein of 911 amino acids with a calculated molecular mass of 102.5 kDa . As expected, M . hominis GyrA exhibits higher homology with the GyrA subunits of the gram-positive bacteria Clostridium acetobutylicum, Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus than with its Escherichia coli counterpart . Knowing the entire sequence of the gyrA gene of M . hominis could be very useful for confirming the role of the GyrA subunit in fluoroquinolone resistance . Twenty-nine mutants of M . hominis were selected stepwise for resistance to trovafloxacin, a new potent fluoroquinolone, and their gyrA, gyrB, parC, and parE quinolone resistance-determining regions were characterized . Three rounds of selection yielded 3 first-step, 12 second-step, and 14 third-step mutants . The first-step mutants harbored a single substitution, Glu460-->Lys (E . coli coordinates), in ParE . GyrA changes, Ser83-->Leu, Glu87-->Lys, and Ala119-->Glu or Val, were found only in the second round of selection . At the third step, additional substitutions, at ParC Ser80, Ser81, and Glu84 and ParE Leu440, associated with high-level resistance to fluoroquinolones, appeared . Thus, high-level resistance to trovafloxacin required three steps and was associated with alterations in both fluoroquinolone targets . According to these genetic data, in M . hominis, as in Staphylococcus aureus and Streptococcus pneumoniae, topoisomerase IV seems to be the primary target of trovafloxacin.

Biochemistry, 2000 Sep 19, 39(37), 11434 - 40
Formation of a tight 1:1 complex of Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein: evidence for long-range interactions between the Fe protein binding sites during catalytic hydrogen evolution; Clarke TA et al.; It has been well documented that the combination of the MoFe protein of Azotobacter vinelandii nitrogenase (Av1) with the Fe protein (Cp2) from Clostridium pasteurianum nitrogenase produces an inactive, stable complex . However, we report that this heterologous nitrogenase has a low level of activity for H(2) evolution, with a specific activity of 12 nmol min(-)(1) mg(-)(1) of Av1 . This activity does not arise from contaminating hydrogenase since it required the presence of both Cp2 and Av1 and showed saturation kinetics when increasing amounts of Cp2 were added to the assay . Incubation of the two proteins at a 4:1 Cp2:Av1 ratio in the absence of MgATP followed by analytical gel filtration showed, surprisingly, that the stoichiometry of the isolated complex was Av1.Cp2 instead of Av1.(Cp2)(2) as determined previously . The presence of MgATP in the elution buffer did not change the elution profile of the complex . The hydrodynamic radius of the isolated complex determined by dynamic light scattering was 5.93 +/- 0.14 nm, intermediate between Av1 and a stable 2:1 nitrogenase complex, consistent with a 1:1 assignment for the Av1.Cp2 complex . When assayed with Av2, the isolated Av1.Cp2 complex showed full half-site reactivity with a specific activity of 750 nmol of C(2)H(2) reduced min(-)(1) mg(-)(1) of Av1 . The EPR spectrum of the isolated complex showed the Cp2 to be oxidized and the Av1 to retain the S = (3)/(2) signal characteristic of FeMoco . In the presence of MgATP, under turnover conditions at a 2:1 ratio of Cp2:Av1, the {4Fe-4S} center of Cp2 was protected from the chelator 2,2'-bipyridyl . This is consistent with the formation of a tight 2:1 complex of Av1.(Cp2)(2) which is more stable than the homologous Cp nitrogenase . Assuming that the Lowe-Thorneley model for nitrogenase applies and that a rate-limiting dissociation of the complex is required for H(2) evolution, then with a rate of 0.032 s(-)(1) the 1:1 complex is too stable to be involved in catalysis . The differences in the stability of the 2:1 and 1:1 complexes indicate cooperativity between the Fe protein binding sites of Av1, which structural data show to be separated by 105 A . On the basis of these observations, we propose a model for nitrogenase catalysis in which the stable 1:1 complex formed between oxidized Fe protein and the one-electron-reduced MoFe protein plays an essential role . In this scheme, the two Fe protein binding sites of the MoFe protein alternately bind and release Fe protein in a shuttle mechanism associated with long-range conformational changes in the MoFe protein.

Biochemistry, 2000 Sep 19, 39(37), 11238 - 46
Crystal structures of the cellulase Cel48F in complex with inhibitors and substrates give insights into its processive action; Parsiegla G et al.; Cellulase Cel48F from Clostridium cellulolyticum was described as a processive endo-cellulase . The active site is composed of a 25 A long tunnel which is followed by an open cleft . During the processive action, the cellulose substrate has to slide through the tunnel to continuously supply the leaving group site with sugar residues after the catalytic cleavage . To study this processive action in the tunnel, the native catalytic module of Cel48F and the inactive mutant E55Q, have been cocrystallized with cellobiitol, two thio-oligosaccharide inhibitors (PIPS-IG3 and IG4) and the cello-oligosaccharides cellobiose, -tetraose and -hexaose . Seven sub-sites in the tunnel section of the active center could be identified and three of the four previously reported sub-sites in the open cleft section were reconfirmed . The sub-sites observed for the thio-oligosaccharide inhibitors and oligosaccharides, respectively, were located at two different positions in the tunnel corresponding to a shift in the chain direction of about a half sugar subunit . These two positions have different patterns of stacking interactions with aromatic residues present in the tunnel . Multiple patterns are not observed in nonprocessive endo-cellulases, where only one sugar position is favored by aromatic stacking . It is therefore proposed that the aromatic residues serve as lubricating agents to reduce the sliding barrier in the processive action.

Med Klin (Munich), 2000 Aug 15, 95(8), 435 - 41
{Necrotizing enterocolitis: a historical and current review}; Kreft B et al.; Enteritis necroticans, locally called "Darmbrand", is a severe and life threatening infectious disease which was epidemic in Northern Germany after World War II . Darmbrand had a limited appearance, occurring only for a few years . In Lubeck many cases were diagnosed in 1946/1948 and the book "Darmbrand, Enteritis necroticans" was published in 1949 by clinicians and pathologists . Enteritis necroticans is also known as a tropical cause of bloody diarrhea and is caused by Clostridium perfringens Type C (type beta-toxin) . The disease is related to pig feasts in Papua New Guinea . Although necrotizing enterocolitis is now a rather rare disease we must be aware of the appearance of this fulminant entity . This paper represents a review on the historic and current aspects of enteritis necroticans and discusses the epidemiology, pathogenesis and treatment of this disease.

J Food Prot, 2000 Sep, 63(9), 1197 - 203
Bacterial thermal death kinetics based on probability distributions: the heat destruction of Clostridium botulinum and Salmonella Bedford; Kilsby DC et al.; Despite the long history and excellent record of inactivation models used in thermal processing, there are relatively few approaches that attempt to describe the kinetics commonly observed . There are even fewer examples of models that allow the user to deal with the environmental conditions that influence these kinetics . We describe an approach that assumes a distribution of inactivation times within a population of bacterial cells . The concept allows for alternative interpretations of death kinetics and provides excellent descriptions of data generated with two important foodborne pathogens, Clostridium botulinum and Salmonella Bedford . The Salmonella Bedford data set used is unusual and perhaps unique in that it provides information where more than 50% of the population survival has been measured . These measurements are often overlooked or missed in experimental work but are essential when using a vitalistic approach, enabling calculation of a 50% lethal dose for destruction of bacteria . Use of the normal or Prentice distribution provided better fits to the data than other models commonly used to describe thermal death . There was no obvious bias in the fits even though significant tailing was evident . In addition, the procedure described allows data from all the conditions to be fitted rather than individual independent series . This enables a single equation to be derived that can be judged against the whole domain of the data . Approaches that provide accurate and unbiased descriptions of thermal death are likely to become increasingly important to ensure the safety of more marginal heat processes.

J Zoo Wildl Med, 2000 Jun, 31(2), 265 - 6
Diarrhea associated with enterotoxigenic Clostridium perfringens in a red-footed tortoise (Geochelone carbonaria); Weese JS et al.; Enterotoxigenic Clostridium perfringens was associated with diarrhea in a 4-yr-old female captive-bred red-footed tortoise (Geochelone carbonaria) . Diagnosis was based on bacterial culture, detection of C . perfringens enterotoxin in feces, and exclusion of commonly recognized pathogens . After treatment with metronidazole, normal feces were passed and C . perfringens enterotoxin was no longer detected in the feces . Although the role of C . perfringens cannot be determined definitively from this case, this pathogen should be considered in cases of diarrhea in tortoises and, perhaps, other reptiles.

Microbiol Immunol, 2000, 44(7), 585 - 9
Role of tryptophan-1 in hemolytic and phospholipase C activities of Clostridium perfringens alpha-toxin; Nagahama M et al.; Replacement of the Trp-1 in Clostridium perfringens alpha-toxin with tyrosine caused no effect on hemolytic and phospholipase C (PLC) activities or on binding to the zinc ion, but that of the residue with alanine, glycine and histidine led to drastic decreases in these activities and a significant reduction in binding to the zinc ion . The hemolytic and PLC activities of W1H and W1A were significantly increased by the preincubation of these variant toxins with zinc ions, but the preincubation of W1G with the metal ion caused little effect on these activities . Gly-Ile-alpha-toxin, which contained an additional Gly-Ile linked to the N-terminal amino acid of alpha-toxin, did not show hemolytic activity, but showed about 6% PLC activity of the wild-type toxin . A mutant toxin, which contained an additional Gly-Ile linked to the N-terminus of a protein lacking 4 N-terminal residues of alpha-toxin, showed about 1 and 6% hemolytic and PLC activities of the wild-type toxin, respectively . Incubation of the mutant toxin with zinc ions caused a significant increase in PLC activity . These observations suggested that Trp-1 is not essential for toxin activity, but plays a role in binding to zinc ions.

Curr Top Microbiol Immunol, 2000, 250, 127 - 39
Treatment of Clostridium difficile-associated diarrhea and colitis; Gerding DN; Treatment of C . difficile diarrhea with metronidazole or vancomycin is highly effective at relieving symptoms . The high rate of diarrhea recurrence is concerning, but fortunately most patients respond to a second course of treatment . The problem of vancomycin resistance in hospital organisms has markedly reduced usage of this agent as a first-line treatment for C . difficile diarrhea, leaving metronidazole as the mainstay of treatment in the United States where teicoplanin and fusidic acid are not marketed . It is likely that any new antimicrobial agent used to treat C . difficile will be similarly plagued by a high rate of recurrence, presumably incurred as a result of disruption of normal bowel flora . There is a need for improved treatment and prevention of this increasingly frequent and debilitating nosocomial infection . Treatments that utilize passive antibodies, immunization, nontoxigenic C . difficile, or other forms of biotherapy may hold the key to improved treatment and prevention of C . difficile disease in the future . In the meantime, it behooves all practitioners to use antimicrobials judiciously in order to prevent as many cases of C . difficile diarrhea as possible.

Curr Gastroenterol Rep, 2000 Aug, 2(4), 310 - 4
Update on Clostridium difficile infection; Alcantara CS et al.; Clostridium difficile is a major cause of antibiotic-associated diarrhea in hospital and community settings, spreading endemic and epidemic disease in developed and developing areas throughout the world . Its toxins A and B cause epithelial disruption, inflammation, and secretion . Diagnosis of infection with C . difficile is based on appropriate clinical presentation and demonstration of the presence of either toxin A or B, or both . Established treatment is still predominantly metronidazole and vancomycin . The association of antibiotic therapy with recurrent disease and antimicrobial resistance, especially vancomycin-resistant enterococci, highlights the need for new approaches to managing C . difficile infection.

J Antimicrob Chemother, 2000 Sep, 46(3), 465 - 9
In vitro activity of an evernimicin derivative, SCH27899, against anaerobic bacteria and Propionibacterium acnes; Tanaka K et al.; The in vitro activity of SCH27899, a novel oligosaccharide antimicrobial agent, was compared with those of representatives of six classes of antimicrobial agents (piperacillin, clarithromycin, clindamycin, vancomycin, sitafloxacin and metronidazole) against clinical isolates of anaerobic bacteria and Propionibacterium acnes . Against Peptostreptococcus: spp . and Clostridium difficile, SCH27899 was the most potent (MIC(90) < 0.125 mg/L) of the agents examined . Besides these Gram-positive anaerobes, SCH27899 showed a moderate level of activity against Prevotella bivia, Prevotella intermedia and Porphyromonas: spp . (MIC(90)< or = 4 mg/L).

Toxicon, 2001 Feb-Mar, 39(2-3), 335 - 40
Absence of intestinal secretion on supernatants from macrophages stimulated with Clostridium difficile toxin B on rabbit ileum; Rocha MF et al.; Several studies have documented the involvement of both Clostridium difficile, toxins, A and B in the pathogenesis of antibiotic-associated diarrhea . Recently, we demonstrated that IL-1 beta is the intestinal secretory factor released by macrophages stimulated with toxin A . The aim of this study was to evaluate the importance of macrophages stimulated with toxin B on rabbit ileal ion transport . The changes in ion transport were analyzed by studying the short-circuit current of the rabbit ileal mucosa mounted in Ussing chambers . The supernatants of macrophages treated with toxin B (3.6 x 10(-7) M) had no effect on the ion transport (change in short-circuit current =28.0+/-9.2 vs . control=26.8+/-3.6 microA cm(-2)) . Supernatants of macrophages stimulated with toxin A (3.2 x 10(-7) M), our positive control, induced a significant change in ileal ion transport (delta I(sc)=55.2+/-5.7 mA cm(-2)) . It was also observed that, like toxin A, toxin B stimulated macrophages to produce TNF-alpha (555.0+/-37.9 pg/ml vs . control=182.0+/-39.8 pg/ml; p<0.05) . Nevertheless, in contrast to toxin A, toxin B did not stimulate IL-1 beta synthesis (28.0+/-7.5 pg/ml vs . control=40 . 0+/-14.4 pg/ml; p>0.05) . We conclude that the supernatants of macrophages stimulated with toxin B are not able to stimulate ion transport and that both toxins stimulate the genesis of TNF-alpha, but only toxin A induces the synthesis of IL-1 beta, which, we have earlier reported, causes an electrogenic intestinal response in rabbit ileum.

Toxicon, 2001 Feb-Mar, 39(2-3), 325 - 33
Modification of surface histidine residues abolishes the cytotoxic activity of Clostridium difficile toxin A; Roberts AK et al.; Clostridium difficile toxin A displays both cytotoxic and enterotoxic activities . It has recently been demonstrated that toxin A exerts its cytotoxic effect by the glucosylation of the small GTP-binding proteins of the Rho family . Diethyl pyrocarbonate, at pH 7.0, was used to chemically modify exposed histidine residues on toxin A . Modification of toxin A with diethyl pyrocarbonate abolished both its cytotoxic activity and the ability of the toxin to bind Zn-Sepharose gel . Treatment of toxin A with {(14)C}-diethyl pyrocarbonate revealed concentration dependent labelling of histidine residues on the toxin molecules . The effects of diethyl pyrocarbonate could be reversed by hydroxylamine treatment . These data suggest the modified histidine residues on toxin A are critical to its cytotoxic activity . Histidine modification had no effect on the glucosyl transferase enzyme activity of toxin A . However, modification abolished the 'cold' binding of toxin to bovine thyroglobulin in an ELISA and reduced ligand binding activity in a rabbit erythrocyte haemagglutination assay . The data suggest that the histidine residues may be crucial to the receptor-binding activity of toxin A . Exposed histidines on toxin A are available for zinc chelation, and these have been exploited in the development of a novel purification protocol for toxin A using zinc-chelating chromatography.

Mol Microbiol, 2000 Sep, 37(5), 1172 - 85
Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia; Ravagnani A et al.; The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized . The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues) . A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target . Insertional inactivation of spo0A in C . beijerinckii blocked the formation of solvents (as well as spores and granulose) . Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C . beijerinckii . These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1 . 4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c) . In vitro gel retardation experiments using C . acetobutylicum adc and C . beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C . beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A . The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence . In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C . beijerinckii validated this hypothesis . Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.

Mol Microbiol, 2000 Aug, 37(4), 821 - 7
The N-terminal prepeptide is required for the production of spore cortex-lytic enzyme from its inactive precursor during germination of Clostridium perfringens S40 spores; Okamura S et al.; A spore cortex-lytic enzyme of Clostridium perfringens S40 is synthesized during sporulation as a precursor consisting of four domains . After cleavage of an N-terminal preregion and a C-terminal proregion, inactive proenzyme (termed C35) is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination . The present results demonstrated that the cleaved N-terminal prepeptide remained associated with C35 . After the isolated complex was denatured and dissociated in 6 M urea solution, removal of urea regenerated a prepeptide-C35 complex which produces active enzyme when incubated with GSP . However, isolated C35 alone could not be activated by GSP . The prepeptide-C35 complex was more heat stable than active enzyme . Thus, non-covalent attachment of the prepeptide to C35 is required to assist correct folding of C35 and to stabilize its conformation, suggesting that the prepeptide functions as an intramolecular chaperone . Recombinant proteins, which have prepeptide covalently bonded to C35, were processed by GSP as well as the in vivo prepeptide-C35 complex, and the full length of the N-terminal presequence was needed to fulfil its role . Although the C-terminal prosequence is present as an independent domain which is not involved in the activation process of the enzyme, it appears that the N-terminal prosequence contributes to the regulation of enzyme activity as an inhibitor of the enzyme.

Lett Appl Microbiol, 2000 Sep, 31(3), 255 - 8
Influence of sodium chloride on the beta-glucuronidase activity of Clostridium perfringens and Escherichia coli; Fujisawa T et al.; While the beta-glucuronidase activity of intact cells of Clostridium perfringens was higher in 0.95% sodium chloride (NaCl) than that in 0, 0.1 or 0.5%, that of Escherichia coli was higher in 0.1% NaCl than that in 0, 0.5 or 0.95% NaCl in 0.1 mol l-1 KH2PO4 . However, the enzyme activity of both species of intact cells was higher in buffer containing 16 mEq sodium, 134 mEq potassium and 16 mEq chloride per litre than in that containing 146 mEq sodium, 13 mEq potassium and 146 mEq chloride . These findings suggest that bacterial cells are affected by the presence of NaCl and that the effect of NaCl on the activity of bacterial beta-glucuronidase may differ by location in the large intestine.

Cell Calcium, 2000 Aug, 28(2), 73 - 82
Hormone-stimulated calcium release is inhibited by cytoskeleton-disrupting toxins in AR4-2J cells; Bozem M et al.; We have studied the role of the actin cytoskeleton in bombesin-induced inositol 1,4,5-trisphosphate (IP(3))-production and Ca(2+)release in the pancreatic acinar tumour cell line AR4-2J . Intracellular and extracellular free Ca(2+)concentrations were measured in cell suspensions, using Fura-2 . Disruption of the actin cytoskeleton by pretreatment of the cells with latrunculin B (10 microM), cytochalasin D (10 microM) or toxin B from Clostridium difficile (20 ng/ml) for 5-29 h led to inhibition of both, bombesin-stimulated IP(3)-production and Ca(2+)release . The toxins had no effect on binding of bombesin to its receptor, on Ca(2+)uptake into intracellular stores and on resting Ca(2+)levels . Ca(2+)mobilization from intracellular stores, induced by thapsigargin (100 nM) or IP(3)(1 microM) was not impaired by latrunculin B . In latrunculin B-pretreated cells inhibition of both, bombesin-stimulated IP(3)- production and Ca(2+)release was partly suspended in the presence of aluminum fluoride, an activator of G-proteins . Aluminum fluoride had no effect on basal IP(3)and Ca(2+)levels of control and toxin-pretreated cells . We conclude that disruption of the actin cytoskeleton impairs coupling of the bombesin receptor to its G-protein, resulting in inhibition of phospholipase C-activity with subsequent decreases in IP(3)-production and Ca(2+)release.

J Clin Microbiol, 2000 Sep, 38(9), 3179 - 86
Phenotypic and genotypic diversity of the flagellin gene (fliC) among Clostridium difficile isolates from different serogroups; Tasteyre A et al.; Phenotypic and genotypic diversity of the flagellin gene (fliC) of Clostridium difficile was studied in 47 isolates from various origins belonging to the serogroups A, B, C, D, F, G, H, I, K, X, and S3 . Electron microscopy revealed 17 nonflagellated strains and 30 flagellated strains . PCR and reverse transcription-PCR demonstrated that the flagellin gene was present in all strains and that the fliC gene was expressed in both flagellated and nonflagellated strains . Southern blotting showed the presence of only one copy of the gene and three different hybridization patterns . DNA sequence analysis of fliC from the strains belonging to serogroups C, D, and X, representative of each profile, disclosed great variability in the central domain, whereas the N- and C-terminal domains were conserved . The variability of the flagellin gene fliC was further studied in the isolates by PCR-restriction fragment length polymorphism (RFLP) analysis . Nine different RFLP groups were identified (I to IX), among which three (I, VII, and VIII) corresponded to numerous serogroups whereas the six others (II, III, IV, V, VI, and IX) belonged to a single serogroup . Flagellin gene RFLP analysis could constitute an additional typing method employable in conjunction with other typing methods currently available.

J Antimicrob Chemother, 2000 Aug, 46 Suppl A, 41 - 48
Effect on the human normal microflora of oral antibiotics for treatment of urinary tract infections; Edlund C et al.; Oral administration of antibiotics for treatment of urinary tract infections (UTIs) can cause ecological disturbances in the normal intestinal microflora . Poorly absorbed drugs can reach the colon in active form, suppress susceptible microorganisms and disturb the ecological balance . Suppression of the normal microflora may lead to reduced colonization resistance with subsequent overgrowth of pre-existing, naturally resistant microorganisms, such as yeasts and Clostridium difficile . New colonization by resistant potential pathogens may also occur and may spread within the body or to other patients and cause severe infections . It is therefore important to learn more about the ecological effects of antibacterial agents on the human microflora . The impact on intestinal microorganisms of oral antibiotics used for the treatment of UTIs is reviewed here . Ampicillin, amoxycillin and co-amoxiclav suppress both the aerobic and anaerobic intestinal microflora with overgrowth of ampicillin-resistant Enterobacteriaceae . Pivmecillinam also affects the intestinal microflora, suppressing Escherichia coli, but does not have a major effect on the anaerobic microflora . Several orally administered cephalosporins, such as cefixime, cefpodoxime, cefprozil and ceftibuten, reduce the number of Enterobacteriaceae and increase the number of enterococci . Colonization with C . difficile has also been observed . Fluoroquinolones eliminate or strongly suppress intestinal Enterobacteriaceae, but affect enterococci and anaerobic bacteria only slightly . When antimicrobial agents are prescribed for the treatment of UTIs, not only the antimicrobial spectrum of the agent but also the potential ecological disturbances, including the risk of emergence of resistant strains, should be considered.

Appl Microbiol Biotechnol, 2000 Aug, 54(2), 201 - 5
Inhibition of Clostridium butyricum by 1,3-propanediol and diols during glycerol fermentation; Colin T et al.; 1,3-Propanediol inhibition during glycerol fermentation to 1,3-propanediol by Clostridium butyricum CNCM 1211 has been studied . The initial concentration of the 1,3-propanediol affected the growth of the bacterium more than the glycerol fermentation . mu(max) was inversely proportional to the initial concentration of 1,3-propanediol (0-65 g l(-1)) . For glycerol at 20 g l(-1), the growth and fermentation were completely stopped at an initial 1,3-propanediol concentration of 65 g l(-1) . However, for an initial 1,3-propanediol concentration of 50 g l(-1) and glycerol at 70 g l(-1), the final concentration (initial and produced) of 1,3-propanediol reached 83.7 g l(-1)(1.1 M), with complete consumption of the glycerol . Therefore, during the fermentation, the strain tolerated a 1,3-propanediol concentration higher than the initial inhibitory concentration (65 g l(-1)) . The addition of 1,2-propanediol or 2,3-butanediol (50 g l(-1)) in the presence of glycerol (50-100 g l(-1)), showed that 2-diols reduced the mu(max) in a similar way to 1,3-propanediol . The measurement of the osmotic pressure of glycerol solutions, diols and diol/glycerol mixtures did not indicate any differences between these compounds . The hypothesis of diol inhibition was discussed . Taking into account the strain tolerance of highly concentrated 1,3-propanediol during fermentation, the fermentation processes for optimising production were considered.

Appl Microbiol Biotechnol, 2000 Aug, 54(2), 162 - 7
Utilisation of saccharides in extruded domestic organic waste by Clostridium acetobutylicum ATCC 824 for production of acetone, butanol and ethanol; Lopez-Contreras AM et al.; Domestic organic waste (DOW) collected in The Netherlands was analysed and used as substrate for acetone, butanol and ethanol (ABE) production . Two different samples of DOW, referred to as fresh DOW and dried DOW, were treated by extrusion in order to expand the polymer fibres present and to obtain a homogeneous mixture . The extruded material was analysed with respect to solvent and hot water extractives, uronic acids, lignin, sugars and ash . The total sugar content in the polymeric fractions of the materials varied from 27.7% to 39.3% (w/w), in which glucose represented the 18.4 and 25.1% of the materials, for fresh and dried DOW, respectively . The extruded fresh DOW was used as substrate for the ABE fermentation by the solventogenic strain Clostridium acetobutylicum ATCC 824 . This strain was grown on a suspension of 10% (w/v) DOW in demineralised water without further nutrient supplement . This strain produced 4 g ABE/100 g extruded DOW . When C . acetobutylicum ATCC 824 was grown on a suspension of 10% (w/v) DOW hydrolysed by a combination of commercial cellulases and beta-glucosidases, the yield of solvents increased to 7.5 g ABE/100 g extruded DOW . The utilisation of sugar polymers in both hydrolysed and non-hydrolysed DOW was determined, showing that only a small proportion of the polymers had been consumed by the bacteria . These results indicate that growth and ABE production on DOW is mainly supported by soluble saccharides in the medium.

J Biol Inorg Chem, 2000 Aug, 5(4), 475 - 87
Mössbauer, EPR, and MCD studies of the C9S and C42S variants of Clostridium pasteurianum rubredoxin and MDC studies of the wild-type protein; Yoo SJ et al.; Rubredoxins contain a mononuclear iron tetrahedrally coordinated by four cysteinyl sulfurs . We have studied the wild-type protein from Clostridium pasteurianum and two mutated forms, C9S and C42S, in the oxidized and reduced states, with Mossbauer, integer-spin EPR, and magnetic circular dichroism (MCD) spectroscopies . The Mossbauer spectra of the ferric C42S and C9S mutant forms yielded zero-field splittings, D = 1.2 cm(-1), that are about 40% smaller than the D-value of the wild-type protein . The 57Fe hyperfine coupling constants were found to be ca . 8% larger than those of the wild-type proteins . The present study also revealed that the ferric wild-type protein has delta=0.24+/-0.01 mm/s at 4.2 K rather than delta = 0.32 mm/s as reported in the literature . The Mossbauer spectra of both dithionite-reduced mutant proteins revealed the presence of two ferrous forms, A and B . These forms have isomer shifts delta = 0.79 mm/s at 4.2 K, consistent with tetrahedral Fe2+(Cys)3(O-R) coordination . The zero-field splittings of the two forms differ substantially; we found D = -7+/-1 cm(-1), E/D = 0.09 for form A and D = +6.2+/-1.3 cm(-1), E/D = 0.15 for form B . Form A exhibits a well-defined integer-spin EPR signal; from studies at X- and Q-band we obtained g(z) = 2.08+/-0.01, which is the first measured g-value for any ferrous rubredoxin . It is known from X-ray crystallographic studies that ferric C42S rubredoxin is coordinated by a serine oxygen . We achieved 75% reduction of C42S rubredoxin by irradiating an oxidized sample at 77 K with synchrotron X-rays; the radiolytic reduction produced exclusively form A, suggesting that this form represents a serine-bound Fe2+ site . Studies in different buffers in the pH 6-9 range showed that the A:B ratios, but not the spectral parameters of A and B, are buffer dependent, but no systematic variation of the ratio of the two forms with pH was observed . The presence of glycerol (30-50 % v/v) was found to favor the B form . Previous absorption and circular dichroism studies of reduced wild-type rubredoxin have suggested d-d bands at 7400, 6000, and 3700 cm(-1) . Our low-temperature MCD measurements place the two high-energy transitions at ca . 5900 and 6300 cm(-1); a third d-d transition, if present, must occur with energy lower than 3300 cm(-1) . The mutant proteins have d-d transitions at slightly lower energy, namely 5730, 6100 cm(-1) in form A and 5350, 6380 cm(-1) in form B.

Bone Marrow Transplant, 2000 Aug, 26(3), 299 - 303
Infectious gastro-enteritis: an uncommon cause of diarrhoea in adult allogeneic and autologous stem cell transplant recipients; van Kraaij MG et al.; The incidence and aetiology of acute diarrhoea in 60 adult allogeneic or autologous stem cell transplant (SCT) recipients was determined in a prospective study . Stool specimens were obtained prior to SCT and on days +20, +40, +60 and +100 post transplant . Microbiological evaluation was performed for pathogenic bacteria, fungi, parasites and viruses . Forty-seven patients were evaluable of whom 31 had a total of 48 acute diarrhoeal episodes . Diarrhoea occurred in 79% of allogeneic and 47% of autologous SCT recipients (P < 0.05) . Intestinal infections were found in three of 48 (6%) diarrhoeal episodes . Clostridium difficile with positive toxin was cultured twice and one stool specimen was positive for cryptosporidium . Intestinal pathogens were identified in 13 out of 172 stool specimens from asymptomatic patients and included: rotavirus (4), adenovirus (3), C . difficile, toxin positive (2), and others (4) . Graft-versus-host disease was confirmed by biopsy in two of 36 episodes of diarrhoea in allogeneic patients, and in three patients a relationship between reactivation of cytomegalovirus and diarrhoea was suspected . In 40 of 48 (83%) episodes of diarrhoea no clear aetiology could be found.

J Mol Biol, 2000 Sep 1, 301(5), 1091 - 5
Structural consequences of mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin; Vetter IR et al.; Mono-glucosylation of Ha-Ras by Clostridium sordellii lethal toxin at effector region threonine 35 has diverse effects on the Ras GTPase cycle, the dominant one of which is the inhibition of Ras-Raf coupling, leading to complete blockade of Ras downstream signaling . To understand the structural basis of the functional consequences of glucosylation, the X-ray crystal structure of glucosylated Ras-GDP was compared with that of non-modified Ras . Glucosylated Ras exhibits a different crystal packing but the overall three-dimensional structure is not altered . The glucose group does not affect the conformation of the effector loop . Due to steric constraints, the glucose moiety prevents the formation of the GTP conformation of the effector loop, which is a prerequisite for binding to the Raf-kinase . The X-ray crystal data also revealed the alpha-anomeric configuration of the bound glucose, indicating that the glucose transfer proceeds under retention of the C-1 configuration of the d-alpha-glucose . Therefore, glucosylation preserves the inactive conformation of the effector loop independently of the nucleotide occupancy, leading to a complete inhibition of downstream signaling of Ras .

Appl Environ Microbiol, 2000 Sep, 66(9), 3711 - 21
Modeling reduction of uranium U(VI) under variable sulfate concentrations by sulfate-reducing bacteria; Spear JR et al.; The kinetics for the reduction of sulfate alone and for concurrent uranium {U(VI)} and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 +/- 3 degrees C were studied . The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp . determined via 16S ribosomal DNA analysis . The pure culture was Desulfovibrio desulfuricans (ATCC 7757) . A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM . A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml . For the mixed culture, average values for the maximum specific reaction rate, V(max), ranged from 2.4 +/- 0.2 micromol of sulfate/mg (dry weight) of SRB . h(-1)) at 0.25 mM sulfate to 5.0 +/- 1.1 micromol of sulfate/mg (dry weight) of SRB . h(-1) at 10 mM sulfate (average cell concentration, 0.52 mg {dry weight}/ml) . For the pure culture, V(max) was 1.6 +/- 0.2 micromol of sulfate/mg (dry weight) of SRB . h(-1) at 1 mM sulfate (0.29 mg {dry weight} of cells/ml) . When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 +/- 0.003 mg (dry weight) of cells/ml . min(-1) for the mixed culture and 0.137 +/- 0.016 mg (dry weight) of cells/ml . min(-1) (U(0) = 1 mM) for the D . desulfuricans culture . Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone . This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.

J Med Microbiol, 2000 Sep, 49(9), 827 - 30
Aerobic and anaerobic microbiology in intra-abdominal infections associated with diverticulitis; Brook I et al.; The aerobic and anaerobic microbiology of intra-abdominal infections associated with diverticulitis was studied in 110 specimens from the peritoneal cavity after intestinal perforation and in 22 specimens from abdominal abscesses . Anaerobic bacteria only were isolated from 17 (15%) of the peritoneal specimens, aerobic bacteria only from 12 (11%) and mixed aerobic and anaerobic flora from 81 (74%) . A total of 339 bacterial isolates was detected in peritoneal cultures (3.1 per specimen), comprising 155 aerobes (1.4 per specimen) and 184 anaerobes (1.7 per specimen) . Anaerobic bacteria only were isolated in 4 (18%) abscesses, aerobes alone in one (5%) and mixed aerobic and anaerobic flora in 17 (77%) . A total of 72 bacterial isolates (3.3 per specimen) was detected in abdominal abscesses - 35 aerobes (1.6 per specimen) and 37 aerobes (1.7 per specimen) . The predominant aerobic and facultative bacteria in abdominal infections were Escherichia coli and Streptococcus spp . The most frequently isolated anaerobes were Bacteroides spp . (B . fragilis group), Peptostreptococcus, Clostridium and Fusobacterium spp.

Jpn J Cancer Res, 2000 Aug, 91(8), 811 - 6
Y-27632, an inhibitor of rho-associated protein kinase, suppresses tumor cell invasion via regulation of focal adhesion and focal adhesion kinase; Imamura F et al.; Migration of rat ascites hepatoma (MM1) cells, invasion and phagokinetic movement were induced by the combination of lysophosphatidic acid (LPA) and fibronectin (FN) . Induction of migratory activity was tightly correlated with morphological change of MM1 cells from spherical or polygonal-shaped cells to fusiform-shaped ones with pseudopodia . MM1 cells were mobile in a fusiform shape, whereas those of a spherical or polygonal shape were not . A small GTPase Rho and one of its downstream effectors ROCK (Rho-associated coiled-coil forming protein kinase), play essential roles in these processes, as evidenced by suppression of migration and morphological change of MM1 cells by Clostridium botulinum C3 exoenzyme, an inhibitor of Rho, or by Y-27632, an inhibitor of ROCK . Y-27632 also suppressed the formation of fusiform-shaped pseudopodia-carrying MM1 cells that was induced by stimulation with the combination of LPA and FN . LPA and FN also evoked the formation of focal adhesions and actin bundles, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin . The inhibitory effect of Y-27632 on LPA-induced migration and morphological change of MM1 cells was considered to be mediated, at least in part, by impaired formation of focal adhesions and actin bundles . Y-27632 suppressed LPA-induced tyrosine phosphorylation of FAK and paxillin, suggesting that ROCK regulates these molecules and Y-27632 inhibits cellular migration and morphological change, at least in part, through this regulation.

Inflamm Bowel Dis, 2000 Aug, 6(3), 188 - 90
Toxic pseudomembranous colitis in a patient with ulcerative colitis; Garcia-Osogobio S et al.; Toxic colitis is a severe disease that may be caused by several inflammatory and/or infectious diseases . Ulcerative colitis is one of the most frequent causes of toxic colitis in the United States . Toxic megacolon complicating Clostridium difficile colitis is a rare occurrence with significant morbidity and mortality . CASE REPORT: A 52-year-old male presented with rectal bleeding and tenesmus . He had been treated for amebiasis with metronidazole, and had improved . Two weeks later, symptoms recurred, and he was referred to our hospital . A sigmoidoscopy and biopsies demonstrated mucosal ulcerative colitis . He underwent treatment with systemic prednisone, mesalamine, and hydrocortisone enemas with adequate response . He was asymptomatic for 2 months, but later presented with a tender abdomen and rectal bleeding . Plain abdominal and thorax films showed colonic distention and free intraperitoneal air . Emergency laparotomy was performed, and an inflamed and distended colon, with free inflammatory liquid in the peritoneum, was found . A total abdominal colectomy with temporary ileostomy and Hartmann's pouch was performed . The histopathology analysis demonstrated a Clostridium difficile pseudomembranous colitis . CONCLUSION: The presence of toxic megacolon due to Clostridium difficile in patients with ulcerative colitis is a rare complication that may be suspected in patients with initial relapse who are on antibiotics.

Biochemistry, 2000 Aug 29, 39(34), 10587 - 98
Investigations of the oxidative disassembly of Fe-S clusters in Clostridium pasteurianum 8Fe ferredoxin using pulsed-protein-film voltammetry; Camba R et al.; Rapid responses of biological {4Fe-4S} clusters to conditions of oxidative stress have been studied by protein-film voltammetry by using precise pulses of electrode potential to trigger reactions . Investigations with Clostridium pasteurianum 8Fe ferredoxin exploit the fact that {3Fe-4S} clusters display a characteristic pattern of voltammetric signals, so that their appearance and disappearance after an oxidative pulse can be tracked unambiguously under electrochemical control . Adsorbed to monolayer coverage at a graphite electrode, the protein initially shows a strong signal (B') at -0.36 V vs standard hydrogen electrode due to two {4Fe-4S}(2+/+) clusters at similar potentials . Short square pulses (0.1-5 s) to potentials in the range 0.5-0.9 V cause extensive loss of B', and new signals appear (A'and C') that arise from {3Fe-4S} species (+/0 and 0/2- couples) . The A' and B' intensities quantify transformations which are induced by the pulse and which occur subsequently when more reducing conditions are restored . Optimal {3Fe-4S} formation (in excess over {4Fe-4S}) is achieved with a 3-s pulse to 0.7 V, following which there is rapid partial recovery to yield a 1:1 3Fe:4Fe ratio, consistent with 7Fe protein . Thus, a 6Fe protein is formed, but one of the clusters is rapidly repaired . The {3Fe-4S}:{4Fe-4S} ratio follows a bell-shaped curve spanning the same potential range that defines complete loss of signals, while double-pulse experiments show that {3Fe-4S}(+) resists further oxidative damage . Oxidative disassembly involves successive one-electron oxidations of {4Fe-4S} (i.e., 2+ --> 3+ --> 4+), with {3Fe-4S}(+) being a relatively stable byproduct, that is, not an intermediate . Disassembly of {3Fe-4S} in the 7Fe protein continues after reducing conditions are restored, with lifetimes depending on oxidation level; thus 1+ (most stable) > 0 > 2- . In the presence of Fe(2+), the 0 level is stabilized by conversion back to {4Fe-4S}(2+/+) . By pulsing in the presence of Zn(2+), the {3Fe-4S} clusters that are formed are trapped rapidly as their Zn adducts.

Biochemistry, 2000 Aug 29, 39(34), 10373 - 84
Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis; Argyrou A et al.; Dihydroorotate dehydrogenase from Clostridium oroticum was purified to apparent homogeneity and found to be a heterotetramer consisting of two alpha (32 kDa) and two beta (28 kDa) polypeptides . This subunit composition, coupled with known cofactor requirements and the ability to transfer electrons from L-dihydroorotate to NAD(+), defines the C . oroticum enzyme as a family 1B dihydroorotate dehydrogenase . The results of steady-state kinetic analyses and isotope exchange studies suggest that this enzyme utilizes a ping-pong steady-state kinetic mechanism . The pH-k(cat) profile is bell-shaped with a pK(a) of 6.4 +/- 0.1 for the ascending limb and 8 . 9 +/- 0.1 for the descending limb; the pH-k(cat)/K(m) profile is similar but somewhat more complex . The pK(a) values of 6.4 and 8.9 are likely to represent the ionizations of cysteine and lysine residues in the active site which act as a general base and an electrostatic catalyst, respectively . At saturating levels of NAD(+), the isotope effects on (D)V and (D)(V/K(DHO)), obtained upon deuteration at both the C(5)-proR and C(5)-proS positions of L-dihydroorotate, increase from a value of unity at pH >9.0 to sizable values at low pH due to a high commitment to catalysis at high pH . At pH = 6.5, the magnitude of the double isotope effects (D)V and (D)(V/K(DHO)), obtained upon additional deuteration at C(6), is consistent with a mechanism in which C(5)-proS proton transfer and C(6)-hydride transfer occur in a single, partially rate-limiting step.

J Biol Chem, 2000 Nov 10, 275(45), 35040 - 50
Equilibrium and kinetic binding interactions between DNA and a group of novel, nonspecific DNA-binding proteins from spores of Bacillus and Clostridium species; Hayes CS et al.; Binding of alpha/beta-type small acid-soluble spore proteins (SASP) is the major determinant of DNA resistance to damage caused by UV radiation, heat, and oxidizing agents in spores of Bacillus and Clostridium species . Analysis of several alpha/beta-type SASP showed that these proteins have essentially no secondary structure in the absence of DNA, but become significantly alpha-helical upon binding to double-stranded DNAs or oligonucleotides . Folding of alpha/beta-type SASP induced by a variety of DNAs and oligonucleotides was measured by CD spectroscopy, and this allowed determination of a DNA binding site size of 4 base pairs as well as equilibrium binding parameters of the alpha/beta-type SASP-DNA interaction . Analysis of the equilibrium binding data further allowed determination of both intrinsic binding constants (K) and cooperativity factors (omega), as the alpha/beta-type SASP-DNA interaction was significantly cooperative, with the degree of cooperativity depending on both the bound DNA and the salt concentration . Kinetic analysis of the interaction of one alpha/beta-type SASP, SspC(Tyr), with DNA indicated that each binding event involves the dimerization of SspC(Tyr) monomers at a DNA binding site . The implications of these findings for the structure of the alpha/beta-type SASP.DNA complex and the physiology of alpha/beta-type SASP degradation during spore germination are discussed.

Nucleic Acids Res, 2000 Sep 1, 28(17), 3379 - 85
Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy; Deng H et al.; Four genomic DNAs of differing GC content (Micrococcus luteus, 72% GC; Escherichia coli, 50% GC; calf thymus, 42% GC; Clostridium perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines spermidine ({H(3)N(CH(2))(3)NH(2)(CH(2))(4)NH(3)}(3+)) and spermine ({(CH(2))(4)(NH(2)(CH(2))(3)NH(3))(2)}(4+)) . In solutions containing 60 mM DNA phosphate (approximately 20 mg DNA/ml) and either 1, 5 or 60 mM polyamine, only Raman bands associated with the phosphates exhibit large spectral changes, demonstrating that B-DNA phosphates are the primary targets of interaction . Phosphate perturbations, which are independent of base composition, are consistent with a model of non-specific cation binding in which delocalized polyamines diffuse along DNA while confined by the strong electrostatic potential gradient perpendicular to the helix axis . This finding provides experimental support for models in which polyamine-induced DNA condensation is driven by non-specific electrostatic binding . The Raman spectra also demonstrate that major groove sites (guanine N7 and thymine C5H(3)) are less affected than phosphates by polyamine-DNA interactions . Modest dependence of polyamine binding on genome base composition suggests that sequence context plays only a secondary role in recognition . Importantly, the results demonstrate that polyamine binding has a negligible effect on the native B-form secondary structure . The capability of spermidine or spermine to bind and condense genomic B-DNA without disrupting the native structure must be taken into account when considering DNA organization within bacterial nucleoids or cell nuclei.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2254 - 8
In vitro and in vivo activities of nitazoxanide against Clostridium difficile; McVay CS et al.; We have used the hamster model of antibiotic-induced Clostridium difficile intestinal disease to evaluate nitazoxanide (NTZ), a nitrothiazole benzamide antimicrobial agent . The following in vitro and in vivo activities of NTZ in the adult hamster were examined and compared to those of metronidazole and vancomycin: (i) MICs and minimum bactericidal concentrations (MBCs) against C . difficile, (ii) toxicity, (iii) ability to prevent C . difficile-associated ileocecitis, and (iv) propensity to induce C . difficile-associated ileocecitis . The MICs and MBCs of NTZ against 15 toxigenic strains of C . difficile were comparable to those of vancomycin or metronidazole . C . difficile-associated ileocecitis was induced with oral clindamycin and toxigenic C . difficile in a group of 60 hamsters . Subgroups of 10 hamsters were given six daily intragastric treatments of NTZ (15, 7.5, and 3.0 mg/100 g of body weight {gbw}), metronidazole (15 mg/100 gbw), vancomycin (5 mg/100 gbw), or saline (1 ml/100 gbw) . Animals receiving saline died 3 days post-C . difficile challenge . During the treatment period, NTZ (>/=7.5 mg/100 gbw), like metronidazole and vancomycin, prevented outward manifestations of clindamycin-induced C . difficile intestinal disease . Six of ten hamsters on a scheduled dose of 3.0 mg of NTZ/100 gbw survived for the complete treatment period . Of these surviving animals, all but three died of C . difficile disease by between 3 and 12 days following discontinuation of antibiotic therapy . Another group of hamsters received six similar daily doses of the three antibiotics, followed by an inoculation with toxigenic C . difficile . All of the NTZ-treated animals survived the 15-day postinfection period . Upon necropsy, all hamsters appeared normal: there were no gross signs of toxicity or C . difficile intestinal disease, nor was C . difficile detected in the cultures of the ceca of these animals . By contrast, vancomycin and metronidazole treatment induced fatal C . difficile intestinal disease in 20 and 70% of recipients, respectively.

Appl Microbiol Biotechnol, 2000 Jul, 54(1), 118 - 20
The effect of heat-shocking on batch fermentation by Clostridium beijerinckii NRRL B592; Gapes JR et al.; In spite of the large-scale industrial use of the acetone-butanol fermentation process earlier this century (until 1983 in South Africa), very little has been published on the inoculum preparation techniques required for successful fermentation using these bacteria . In particular, heat-shocking has often been referred to as "useful" but no quantitative data are available . Data presented in this paper demonstrate and quantify the effect of heat-shocking on batch fermentation yields using one organism capable of this fermentation.

J Infect Dis, 2000 Sep, 182(3), 808 - 15 Epub 2000 Aug 17.
Clostridial gas gangrene . II . Phospholipase C-induced activation of platelet gpIIbIIIa mediates vascular occlusion and myonecrosis in Clostridium perfringens gas gangrene; Bryant AE et al.; Clostridium perfringens gas gangrene is a fulminant infection, and radical amputation remains the single best treatment . It has been hypothesized that rapid tissue destruction is related to tissue hypoxia secondary to toxin-induced vascular obstruction, and previous studies demonstrated that phospholipase C (PLC) caused a rapid and irreversible decrease in skeletal muscle blood flow that paralleled the formation of intravascular aggregates of activated platelets, fibrin, and leukocytes . In this study, flow cytometry demonstrated that PLC stimulated platelet/neutrophil aggregation in a gpIIbIIIa-dependent fashion . Pretreatment of animals with heparin or depletion of leukocytes reduced blood-flow deficits, and aggregate formation caused by PLC . It is concluded that fulminant tissue destruction in gas gangrene results from profound attenuation of blood flow caused by PLC-induced, gpIIbIIIa-mediated formation of heterotypic platelet/polymorphonuclear leukocyte aggregates . Therapeutic strategies that target gpIIbIIIa may prevent vascular occlusion, maintain tissue viability, and provide an alternative to radical amputation for patients with this infection.

J Infect Dis, 2000 Sep, 182(3), 799 - 807 Epub 2000 Aug 17.
Clostridial gas gangrene . I . Cellular and molecular mechanisms of microvascular dysfunction induced by exotoxins of Clostridium perfringens; Bryant AE et al.; Mechanisms responsible for the rapid tissue destruction in gas gangrene are not well understood . To examine the early effects of Clostridium perfringens exotoxins on tissue perfusion, a rat model of muscle blood flow was developed . Intramuscular injection of a clostridial toxin preparation containing both phospholipase C (PLC) and theta-toxin caused a rapid (1-2 min) and irreversible decrease in blood flow that paralleled formation of activated platelet aggregates in venules and arterioles . Later (20-40 min), aggregates contained fibrin and leukocytes, and neutrophils accumulated along vascular walls . Flow cytometry confirmed that these clostridial toxins or recombinant PLC induced formation of P-selectin-positive platelet aggregates . Neutralization of PLC activity in the clostridial toxin preparation completely abrogated human platelet responses and reduced perfusion deficits . It is concluded that tissue destruction in gas gangrene is related to profound attenuation of blood flow initiated by activation of platelet responses by PLC.

J Food Prot, 2000 Aug, 63(8), 1071 - 9
Modeling the germination kinetics of clostridium botulinum 56A spores as affected by temperature, pH, and sodium chloride; Chea FP et al.; The germination kinetics of proteolytic Clostridium botulinum 56A spores were modeled as a function of temperature (15, 22, 30 degrees C), pH (5.5, 6.0, 6.5), and sodium chloride (0.5, 2.0, 4.0%) . Germination in brain heart infusion (BHI) broth was followed with phase-contrast microscopy . Data collected were used to develop the mathematical models . The germination kinetics expressed as cumulated fraction of germinated spores over time at each environmental condition were best described by an exponential distribution . Quadratic polynomial models were developed by regression analysis to describe the exponential parameter (time to 63% germination) (r2 = 0.982) and the germination extent (r2 = 0.867) as a function of temperature, pH, and sodium chloride . Validation experiments in BHI broth (pH: 5.75, 6.25; NaCl: 1.0, 3.0%; temperature: 18, 26 degrees C) confirmed that the model's predictions were within an acceptable range compared to the experimental results and were fail-safe in most cases.

Biosens Bioelectron, 2000 Jan, 14(10-11), 815 - 28
The development of immunoassays to four biological threat agents in a bidiffractive grating biosensor; O'Brien T et al.; A critical need exists for a field deployable biosensor to detect environmental infectious agents in collected air samples rapidly, with sensitivity and specificity approaching that of standard laboratory procedures . The ideal sensor would analyze unknown samples in minutes, have programmable operation for unattended sample analysis, and be capable of multiple agent analysis for a number of agents . The goal of this project was to further the development of the bidiffractive grating biosensor (BDG) created through collaboration between Battelle Memorial Institute (BMI), Hoffman LaRoche (HLR), and the Naval Medical Research Command (NMRC) . This manuscript details the development, optimization, and evaluation of this device as a potential field deployable biosensor . Well-characterized immunochemical reagents developed by the Biological Defense Research Department (BDRD) at NMRI were employed to develop assays in the BDG . These results were compared to those obtained with antigen capture enzyme linked immunosorbent assays (ELISAs) . Four separate antigens were evaluated: Staphylococcus aureus enterotoxin B (SEB), ricin (RIC), Francisella tularensis (FT), and Clostridium botulinum toxin (BOT).

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1027 - 9
Crystallization and preliminary X-ray analysis of the Clostridium thermocellum cellulosome xylanase Z feruloyl esterase domain; Blum DL et al.; Feruloyl esterases cleave ferulic acid from arabinoxylan and pectin . Feruloyl groups are believed to crosslink the polysaccharide chain within the polymer and to link hemicellulose to lignin, which may play a role in controlling the growth of plants . The Clostridium thermocellum cellulosome xylanase Z feruloyl esterase was expressed in Escherichia coli, purified and crystallized . The crystals diffract to 2.4 A resolution and belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.14, b = 63.77, c = 79.57 A . Assuming one molecule per asymmetric unit, the Matthews coefficient is calculated to be 1.87 A(3) Da(-1), which corresponds to a solvent content of 34%.

Acta Crystallogr D Biol Crystallogr, 2000 Aug, 56 ( Pt 8), 1024 - 6
Crystallization and preliminary X-ray analysis of Clostridium botulinum neurotoxin type B; Swaminathan S et al.; Single crystals of Clostridium botulinum neurotoxin type B have been obtained by the vapor-diffusion method . These crystals belong to space group P2(1), with unit-cell parameters a = 76.08, b = 123.11, c = 95.86 A, beta = 113.03 degrees and diffract to at least 1.8 A resolution . Native data have been collected from flash-frozen crystals at the National Synchrotron facility of Brookhaven National Laboratory . These crystals often tend to be non-isomorphic.

Thromb Res, 2000 Aug 1, 99(3), 259 - 65
Sphingosine 1-phosphate stimulates G(i)- and Rho-mediated vascular endothelial cell spreading and migration; Okamoto H et al.; Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid released from activated platelets, which may be involved in angiogenesis . We, hence, investigated Sph-1-P effects on human umbilical vein endothelial cells (HUVECs) from a viewpoint of angiogenesis . Sph-1-P facilitated HUVEC spreading on the basement membrane component Matrigel, at concentrations ranging from 10 to 250 nM . This stimulatory response induced by Sph-1-P was blocked by pertussis toxin and C3 transferase (from Clostridium botulinum), which inactivate G(i)-type heterotrimeric G protein and Rho, respectively . Furthermore, Sph-1-P, in the modified Boyden's chamber assay, stimulated HUVEC migration in a concentration-dependent manner, up to 250 nM . Checkerboard analysis revealed that Sph-1-P markedly induces directional migration (chemotaxis), but a random motility (chemokinesis) was also enhanced . The stimulatory effect of Sph-1-P on HUVEC migration was much stronger than that of other bioactive lipids, and again inhibited by pertussis toxin and by C3 transferase . Our present results that Sph-1-P induces endothelial spreading and migration through G(i)-coupled cell surface receptor(s) and Rho are consistent with a recent report on the role of this platelet-derived sphingolipid as a novel regulator of angiogenesis.

Microbiol Immunol, 2000, 44(6), 525 - 8
Genetic analysis of the ycgJ-metB-cysK-ygaG operon negatively regulated by the VirR/VirS system in Clostridium perfringens; Ohtani K et al.; The 5'-flanking region of the metB-cysK-ygaG operon, whose expression is negatively regulated by the VirR/VirS system in C . perfringens, was analyzed . The region contained the ycgJ, mscL, and colA genes encoding a hypothetical protein, a large conductance mechanosensitive channel protein, and kappa-toxin (collagenase), respectively . Northern analysis revealed that the ycgJ gene was transcribed as a 4.9-kb operon together with the metB-cysK-ygaG genes and that this operon was negatively regulated by the VirR/VirS system . It is indicated that the pfoA (theta-toxin or perfringolysin O), colA, and ycgJ-metB-cysK-ygaG genes that belong to the VirR/VirS regulon are situated very close together in a 26.5-kb region of the chromosome, but do not form a pathogenic island.

J Wildl Dis, 2000 Jul, 36(3), 489 - 93
Efficacy of a type C botulism vaccine in green-winged teal; Rocke TE et al.; We tested the efficacy of a single dose of Botumink toxoid for protecting wild green-winged teal (Anas crecca) during botulism epizootics caused by Clostridium botulinum type C . We challenged control and immunized ducks with four different doses of type C botulinum toxin to determine the LD50 for this species and to evaluate vaccine protection . Fewer immunized ducks were affected with botulism than control ducks, indicating that a single dose of Botumink toxoid could increase the survival of ducks during epizootics . However, the frequency of immunized ducks with signs of botulism increased with the challenge dose of botulinum toxin . Even at doses of botulinum toxin approximately 2 to 4 green-winged teal LD50, about 50% of the immunized ducks were affected . We believe an improved vaccine or a better delivery system is required to justify immunization of wild birds for experimental survival studies.

J Bacteriol, 2000 Sep, 182(17), 4773 - 82
Native corrinoids from Clostridium cochlearium are adeninylcobamides: spectroscopic analysis and identification of pseudovitamin B(12) and factor A; Hoffmann B et al.; The corrinoids from the obligate anaerobe Clostridium cochlearium were extracted as a mixture of Co(beta)-cyano derivatives . From 50 g of frozen cells, approximately 2 mg (1.5 micromol) of B(12) derivatives was obtained as a crystalline sample . Analysis of the corrinoid sample of C . cochlearium by a combination of high-pressure liquid chromatography and UV-Vis absorbance spectroscopy revealed the presence of three cyano corrinoids in a ratio of about 3:1:1 . The spectroscopic data acquired for the sample indicated the main components to be pseudovitamin B(12) (Co(beta)-cyano-7"-adeninylcobamide) (60%) and factor A (Co(beta)-cyano-7"-{2-methyl}adeninylcobamide) (20%) . Authentic pseudovitamin B(12) was prepared by guided biosynthesis from cobinamide and adenine . Both pseudovitamin B(12) and its homologue, factor A, were subjected to complete spectroscopic analysis by UV-Vis, circular dichroism, mass spectrometry, and by one- and two-dimensional (1)H, (13)C-, and (15)N nuclear magnetic resonance (NMR) spectroscopy . The third component was indicated by the mass spectra to be an isomer of factor A and is likely (according to NMR) to be 7"-{N(6)-methyl}-adeninylcobamide, a previously unknown corrinoid . C . cochlearium thus biosynthesizes as its native "complete" B(12) cofactors the 7"-adeninylcobamides and two homologous corrinoids, in which the nucleotide base is a methylated adenine.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1595 - 9
Catenibacterium mitsuokai gen . nov., sp . nov., a gram-positive anaerobic bacterium isolated from human faeces; Kageyama A et al.; Six strains of Eubacterium-like strains from human faeces were characterized by biochemical tests and analysis of cell wall peptidoglycan type and 16S rRNA . They were members of the Clostridium subphylum and have a specific phylogenetic association with Lactobacillus catenaformis and Lactobacillus vitulinus . These organisms resembled L . vitulinus in possessing the same A1gamma type of murein, but they showed different fermentation end-products . On the basis of a 16S rDNA sequence divergence of greater than 8% from L . vitulinus as well as phenotypic characteristics, a new genus, Catenibacterium, with one species (Catenibacterium mitsuokai), is proposed for six strains . The type strain of C . mitsuokai is JCM 10609T.

Int J Syst Evol Microbiol, 2000 Jul, 50 Pt 4, 1591 - 4
Anaerovorax odorimutans gen . nov., sp . nov., a putrescine-fermenting, strictly anaerobic bacterium; Matthies C et al.; The strictly anaerobic, gram-positive, non-spore-forming bacterium strain NorPut1T ferments putrescine to acetate, butyrate, molecular hydrogen and ammonia . It also utilizes 4-aminobutyrate and 4-hydroxybutyrate as growth substrates . Comparative 16S rDNA sequence analysis confirmed a phylogenetic affiliation of this strain to the phylum of gram-positive bacteria with low DNA G+C content . Together with its closest relative, 'Clostridium aminobutyricum' (DSM 2634), and several Eubacterium species, strain NorPut1T represents a well-defined monophyletic group . Moderate overall 16S rRNA sequence similarities (< 91%) were found for the NorPut1T/'Clostridium aminobutyricum' pair and several Eubacterium species . The type species, Eubacterium limosum, is not a member of the group and, together with Eubacterium barkeri and Pseudoramibacter alactolyticus, represents a distant phylogentic cluster . Therefore, a new genus, Anaerovorax, is proposed as harbouring strain NorPut1T (= DSM 5092T), which is described as a new species, i.e . Anaerovorax odorimutans.

J Enzyme Inhib, 2000, 15(2), 111 - 28
Protease inhibitors: Synthesis of L-alanine hydroxamate sulfonylated derivatives as inhibitors of clostridium histolyticum collagenase; Supuran CT et al.; L-alanine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with L-alanine, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group with hydroxylamine in the presence of carbodiimides . Other derivatives were obtained by reaction of N-benzyl-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by a similar conversion of the COOH to the CONHOH moiety . The obtained compounds were assayed as inhibitors of Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen . The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates . In the series of synthesized derivatives, substitution patterns leading to the most potent ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl-, or 1- and 2-naphthylsulfonyl among others . Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') sites, in order to achieve tight binding to the enzyme.

Metab Eng, 1999 Jul, 1(3), 206 - 13
Metabolic flux analysis elucidates the importance of the acid-formation pathways in regulating solvent production by Clostridium acetobutylicum; Desai RP et al.; Metabolic flux analysis was used to investigate the roles of the acid formation pathways in Clostridium acetobutylicum . The acid formation pathways were revealed to serve different roles in wildtype fermentations than previously expected . Specifically, enzymes known to catalyze butyrate formation were found to uptake butyrate without concomitant production of acetone . This role was further corroborated by flux analysis of a recombinant strain overexpressing the butyrate formation enzymes . Analysis of wildtype fermentation data also revealed an important role for the acetate formation enzymes, namely the cycling of carbon between acetate and acetylCoA during the stationary phase . Next, metabolic flux analysis was used to compare the patterns of activity in two butyrate kinase deficient strains of C . acetobutylicum . The strain developed by gene inactivation, PJC4BK, exhibited a shift in acid formation fluxes toward acetate while the strain developed by antisense RNA strategies, 824(pRD4), did not exhibit such a shift . However, both strains exhibited altered solvent formation patterns . PJC4BK exhibited a strong transient enhancement of solvent formation fluxes . In contrast, 824(pRD4) exhibited relatively lower levels of solvent formation fluxes, although fluxes were sustained over a longer period of time.

Biol Chem, 2000 May-Jun, 381(5-6), 421 - 6
Rho GTPases as targets of bacterial protein toxins; Aktories K et al.; Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton . The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42 . Large clostridial cytotoxins (e . g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling . The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins . Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B . bronchiseptica . These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases . Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.

Int Ophthalmol, 1998, 22(6), 369 - 75
Retinal detachment after posterior segment intraocular foreign body injuries; El-Asrar AM et al.; PURPOSE: To identify the risk factors for retinal detachment after posterior segment intraocular foreign body (IOFB) injuries and to study the association between the development of retinal detachment and visual outcome . METHODS: Ninety-six consecutive patients with posterior segment IOFB injuries were retrospectively reviewed . Vitrectomy techniques were used in primary and secondary treatment . Two eyes were eviscerated after primary repair because of Clostridium perfringens endophthalmitis . Factors analyzed included (1) entrance wound location, (2) presence of uveal prolapse, (3) presence of vitreous prolapse, (4) presence of traumatized iris, (5) presence of endophthalmitis, (6) location of IOFB, (7) size of IOFB, (8) use of scleral buckling and/or an encircling band, (9) use of gas tamponade, (10) use of lensectomy . Data were analyzed using univariate and multivariate logistic regression analysis . RESULTS: Retinal detachment was present in 6 eyes at presentation and occurred in another 19 eyes after vitrectomy . After a mean follow-up of 8.6 months, 63 (65.6%) eyes achieved visual acuities of 20/200 or better, and total retinal detachment complicated by inoperable proliferative vitreoretinopathy was present in 9 (9.4%) eyes . Multivariate analysis identified retinal detachment as a factor significantly associated with a poor visual outcome (odds ratio = 4.54, 95% confidence interval {CI} = 1.05-19.6) . Foreign body size of more than 4 mm (odds ratio = 5.8, 95% CI = 1.66-2.03) and presence of endophthalmitis (odds ratio = 11.7, 95% CI = 2.57-52.9) were identified as the only predictive factors for the development of retinal detachment after vitrectomy . Use of prophylactic scleral buckling and/or an encircling band reduced the risk of developing postoperative retinal detachment . CONCLUSIONS: Retinal detachment after posterior segment IOFB injuries is associated with a poor visual outcome . Large IOFB and presence of endophthalmitis are the strongest predictive factors for the development of retinal detachment.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 115 - 24
Comparative fermentation studies of industrial strains belonging to four species of solvent-producing clostridia; Shaheen R et al.; Industrial and culture collection strains of solvent-producing clostridia, classified as Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium saccharobutylicum, and Clostridium saccharoperbutylacetonicum were utilised in a comparative study of fermentation performance in a laboratory fermentation medium, a molasses fermentation medium, and a maize fermentation medium under standardised culture conditions . At least one representative strain was selected from each of the sub-groups within the four species . Preliminary evaluations were first undertaken for the three different fermentation media to determine the most appropriate media formulations, carbohydrate concentrations, and culture conditions for comparison of the solvent-producing ability of these strains . Standardised fermentation media and culture conditions were then selected for each of the comparative fermentation studies . These included TYA medium containing 4% glucose, a supplemented molasses medium containing 6% fermentable sugars, and a supplemented maize mash medium containing 8% maize . Additional comparative fermentation studies on industrial strains belonging to two species of solvent-producing clostridia were carried out in molasses containing higher concentrations of fermentable sugars, and the sugar concentrations supporting maximum levels of solvent production were determined . Although all the strains tested grew in the maize fermentation medium and degraded starch, only a few strains produced consistently high solvent levels . Optimum starch utilisation and solvent production was obtained at a maize concentration of 80 g/l . Pretreatment of the maize by milling or saccharification decreased the buffering capacity of the medium and resulted in decreased solvent production . Decreasing the time used to gelatinise the starch had little effect . Solvent yields and concentrations obtained in this study were compared with various published data in the scientific and patent literature and appeared to closely simulate the results obtained in the industrial fermentation process . The fermentation performances of individual strains could provide useful comparative data for the selection and development of strains for use on various commercial fermentation substrates.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 107 - 13
A new insertion sequence, ISCb1, from Clostridium beijernickii NCIMB 8052; Liyanage H et al.; The NCIMB 8052 strain of Clostridium beijerinckii contains nine copies of a novel insertion sequence, ISCb1, belonging to the IS4 family . The 1764 bp element has 18 bp inverted repeats at its extremities, and generates 11 bp target repeats upon insertion . It contains a 1365 bp ORF whose predicted product (455 amino acids) resembles bacterial transposases . The highly conserved DD(35)E motif is present, as are signatures characteristic of the N3 and C1 domains of bacterial transposases . Codon usage of the ORF is somewhat different from that of other C . beijerinckii genes, suggesting that ISCb1 may have been acquired from another organism by horizontal gene transfer in the evolutionary past . One ISCb1 copy lies close to the site of insertion of Tn 1545 in a mutant strain, C10, which shows a reduced tendency to degenerate (i.e . loss of the potential to form solvents) compared with the wild type . In the C10 strain, the characteristic pattern of DNA fragments detected by an IS-specific probe was altered, but this was due to the Tn1545 insertion itself, rather than an ISCb1-mediated genome re-arrangement . There is currently no evidence that the element is involved in strain degeneration, since 12 independently isolated spontaneous mutants that had lost the ability to form solvents had the same ISCb1 profile as that of the wild type strain . The element is apparently restricted to a series of closely related solvent-forming clostridia.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 101 - 5
Continuous two-stage ABE-fermentation using Clostridium beijerinckii NRRL B592 operating with a growth rate in the first stage vessel close to its maximal value; Mutschlechner O et al.; A two-stage continuous cultivation experiment with Clostridium beijerinckii NRRL B592 is described . The experiment was designed to mimic the two phases of batch culture growth of the organism in a two-stage continuous process . Thus in the first stage turbidostat the organism was grown acidogenically as rapidly as possible, and transferred to the second stage at the 'acid break point' . The second stage was designed to mimic the solventogenesis of the batch culture when it enters late exponential/early stationary phase . The volume of the second stage vessel was calculated to provide the necessary residence time for complete sugar utilization . It was hoped that the experimental set-up chosen would show whether data obtained from batch fermentation could be transferred directly to continuous culture . The culture maintained its ability to produce acetone, 1-butanol and ethanol at a dilution rate of 0.12 h(-1) for the first stage and 2.2 x 10(-2) h(-1) for the second stage and achieved an average overall solvent concentration of 15 g/l and an overall solvent productivity of 0.27 g/l/h for a period of steady-state operation of more than 1600 hours . The productivity of solventogenesis in the first stage was dependent on the value of the growth rate of the culture which was in turn determined in part by the organism employed but also by the medium composition.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 87 - 93
Butanol tolerance of Clostridium beijerinckii NCIMB 8052 associated with down-regulation of gldA by antisense RNA; Liyanage H et al.; Strain BR54 of Clostridium beijerinckii was derived from the wild type strain, NCIMB 8052, by mutagenesis with Tn1545 and selection for butanol tolerance . It harbours a single copy of Tn 1545 in a 435 bp intergenic region separating two convergently transcribed genes, accC and gldA . The former encodes biotin carboxylase (E.C.6.3.4.14), a subunit of acetyl-CoA carboxylase and the latter encodes glycerol dehydrogenase (E.C.1.1.1.6) . Since Tn1545 generates outwardly directed transcripts from its right end, we considered the possibility that the transposon inserted in strain BR54 might affect the expression of the adjacent gldA gene . RT-PCR experiments revealed that the mutant, but not the wild type, contains antisense RNA corresponding to the gldA gene . Correlated with this, the level of glycerol dehydrogenase activity in the mutant was only 25% of that in the wild type when bacteria were grown with either glucose or glycerol as the fermentable substrate . We conclude that transcripts emerging from the right end of the conjugative transposon, Tn1545, can reduce the expression of the adjacent gldA gene by the generation of antisense RNA and that this is associated with a butanol-tolerant phenotype.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 71 - 80
Analysis of a catabolic operon for sucrose transport and metabolism in Clostridium acetobutylicum ATCC 824; Tangney M et al.; The utilization of sucrose by Clostridium acetobutylicum ATCC 824 was investigated . Sucrose was found to be transported via a phosphoenol-pyruvate (PEP)-dependent phosphotransferase system (PTS) and a metabolic pathway identical to that previously identified in C . beijerinckii, was established . The genes encoding the proteins of this pathway were identified from the C . acetobutylicum genome sequence, in the order scrAKB encoding Enzyme II of the sucrose PTS, fructokinase and sucrose 6-phosphate hydrolase respectively . While the pathway for sucrose metabolism is conserved between C . acetobutylicum and C . beijerinckii, the operons show considerable differences in organization and regulatory elements . The C . acetobutylicum scr operon contains the elements of an antiterminator-mediated regulation mechanism, typical of the BgIG family of regulators . The scrT gene, located upstream of scrA encodes an antiterminator that is preceded by a transcription terminator, which is overlapped by a classical ribonucleic antiterminator (RAT) sequence . We also propose the existence of a new variant RAT-like sequence which overlaps a terminator between scrT and the downstream structural genes.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 59 - 69
Development of a transformation and gene reporter system for group II, non-proteolytic Clostridium botulinum type B strains; Davis TO et al.; Non-proteolytic, Group II strains of Clostridium botulinum are of particular concern to the food industry because of their ability to survive and grow in REPFEDs (refrigerated processed foods of extended durability) . Their analysis would benefit from the availability of a gene transfer system . In the present study we have been able, for the first time, to demonstrate transformation in a representative Group II strain, ATCC 25765 . Initial attempts to transform ATCC 25765 with existing clostridial cloning vectors (pMTL540E and pMTL500E) were, however, prevented by a restriction barrier . Through a combination of classical and molecular approaches we were able to show that strain ATCC 25765 possesses a restriction endonuclease (Cbol) and a methylase activity (M . Cbol) which have the same specificity as Mspl and M.Mspl, respectively . Cbol cleaves the palindrome 5'-CCGG-3' to generate a 3'-GC sticky end, whilst M.Cbol specifically methylates the external C residue . An E . coli host was generated which expressed a Bacillus subtilis methylase enzyme (M.BsuF1) with equivalent specificity to M.Cbol . Plasmids (pMTL540E and pMTL500E) prepared in this strain were subsequently shown to be capable of transforming ATCC 25765 . The highest frequencies (0.8 X 10(4) transformants per microg of DNA) were obtained when cells were cultivated in media supplemented with 1% (w/v) glycine, and when the electroporation was undertaken at 10 kV/cm, 25 microF and at 400 ohms . Having developed an effective transformation procedure, we went on to construct reporter cassettes based on the Thermanaerobacterium sulfurigenes lacZ and the Vibrio fischeri luxAB genes . Using the former, and promoter regions isolated from the botulinum toxin genes, we have obtained preliminary evidence that reporter genes may be used to evaluate the physiological factors that affect toxin production in the food environment.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 53 - 7
Construction of a reporter gene vector for Clostridium beijerinckii using a Clostridium endoglucanase gene; Quixley KW et al.; A beta-1,4-endoglucanase gene (eglA) cloned from C . acetobutylicum P262 was selected for use in the development of a reporter system for C . beijerinckii NCIMB 8052 . The reporter plasmid, pER1, was constructed by ligating the promoterless eglA gene into the B subtilis/Clostridium shuttle vector, pFNK1, which can replicate and is stably maintained in C . beijerinckii . The expression of the endoglucanase enzyme from its own promoter was not significantly induced in cells grown in glucose, sucrose or galactose, while growth of cells in cellobiose or fructose resulted in lower levels of activity . The enzyme was efficiently secreted into the culture medium and did not remain associated with the cell in any way . A transcriptional fusion between the glutamine synthetase (glnA) promoter region and the promoterless eglA gene resulted in high levels of endoglucanase expression, which reflected an 11-fold increase in expression levels over the eglA promoter.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 45 - 52
KdpE of Clostridium acetobutylicum is a highly specific response regulator controlling only the expression of the kdp operon; Behrens M et al.; KdpE from Clostridium acetobutylicum was enriched in form of its Strep-tag-derivative to allow easy immunodetection . It could be artificially phosphorylated by acetyl phosphate or carbamyl phosphate . Only phosphorylated clostridial KdpE was able to bind to a region upstream of the clostridial kdp structural genes . The minimal sequence requirements for binding were determined and found to share significant similarity with the Escherichia coli KdpE binding motif . However, the clostridial protein proved to be much more specific and did not bind in unphosphorylated form or to other similar sequences either from C . acetobutylicum or E . coli . In contrast, the enterobacterial protein recognized the clostridial binding motif . An HPt domain has been detected in KdpD from C . acetobutylicum, the cognate sensor kinase of KdpE . The data reported indicate that in E . coli, KdpE might represent a regulatory checkpoint for different phosphorelay signalling pathways, whereas in C . acetobutylicum KdpD might serve this function.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 39 - 44
Acetone, butanol and ethanol production from domestic organic waste by solventogenic clostridia; Claassen PA et al.; Domestic organic waste (DOW) was washed and dried to 85 % dryness by VAM (The Netherlands) . This material contained 25.1 g glucose, 8.4 g xylose and 5.8 g other monosaccharides/100 g dry matter . Using Mansonite steam explosion and enzymatic hydrolysis, a hydrolysate containing 15.4 g glucose, 2.2 g xylose and 0.8 g other monosaccharides per l was made . Clostridium acetobutylicum DSM 1731 produced 1.5 and C . beijerinckii B-592 0.9 g/l ABE and Clostridium LMD 84.48 1.9 g/l IBE, respectively, from this hydrolysate without further supplementation . Incubation with 2 fold concentrated hydrolysate completely impaired ABE production . After removal of unspecific inhibiting components, the yield of ABE production by Clostridium acetobutylicum DSM 1731 increased about 3 fold as compared to the nontreated hydrolysate . From 4 fold concentrated, partially purified, hydrolysate containing 34.2 g glucose/l, ABE production was 9.3 g/l after 120 h as compared to 3.2 g ABE/I from non-concentrated hydrolysate which contained 12.0 g glucose/l after elution over the same column . The concentration of butyric acid in the fermented hydrolysates was 2.2 and 0.4 g/l, respectively . This reasonably low amount of butyric acid showed that the fermentation had proceeded quite well.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 33 - 8
Identification and characterization of a second butyrate kinase from Clostridium acetobutylicum ATCC 824; Huang KX et al.; A gene encoding a new butyrate kinase isozyme (BKII) was identified from the C . acetobutylicum ATCC 824 DNA database . The enzyme was expressed in Escherichia coli, purified, and characterized . The purified enzyme exhibited a subunit molecular mass of 43 kDa by SDS-PAGE, and a native molecular mass of 80 kDa by gel filtration suggesting it functions as a dimer . In the butyryl phosphate-forming direction the optimal pH of BKII was 8.5 . The enzyme had a Km of 0.62 M and a turn over rate of 2.2 x 10(5)/sec (Vmax of 165 units/mg) . The presence of a mRNA encoding the BKII was demonstrated using a reverse transcription PCR reaction . The expression of the BKII in Clostridium acetobutylicum ATCC 824 was further examined by Western blot analysis using a polyclonal antibody prepared against recombinant BKII.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 21 - 6
Bacteriophage infections in the industrial acetone butanol (AB) fermentation process; Jones DT et al.; The reported incidence and effects of bacteriophage infections occurring in the industrial acetone butanol (AB) fermentation processes operated in the USA, Japan, and Puerto Rico during the earlier part of the twentieth century is reviewed . The growth characteristics and solvent-producing ability of a lysogenic strain of Clostridium madisonii isolated from a phage infection in Puerto Rico was determined in molasses fermentation medium . The host strain harbours a large lysogenic phage belonging to the Siphoviridae and the growth rate of the lysogenic strain was found to be slower than the non-lysogenic parent strain and exhibited reduced solvent production . The history of phage infections that occurred in the South African AB process is documented along with the various remedial actions that were taken to restore production . A more detailed account of the last phage infection that occurred in 1980 involving a small pseudo-lysogenic phage belonging to the Podoviridae is given . This phage infected Clostridium beijerinckii P260 and a number of closely related industrial strains . Factory-scale fermentations contaminated by this phage were compared with equivalent laboratory-scale control fermentations . The effect of the phage infection in the full-scale and laboratory-scale fermentations were monitored . Results obtained in laboratory-based studies included an assessment of the effect of the multiplicity of infection and the timing of phage infection . The general effects and symptoms of phage infections in the industrial AB fermentation are reviewed including gross changes in the fermentation and changes in cell morphology . Common techniques used for the diagnosis of phage infections and approaches for controlling phage contamination in the AB fermentation are discussed . Prevention strategies included good factory hygiene, sterilisation, decontamination and disinfection, and the use of resistant strains immunised against specific phages.

J Mol Microbiol Biotechnol, 2000 Jul, 2(3), 265 - 9
Identification of two genes encoding putative new members of the ECF subfamily of eubacterial RNA polymerase sigma factors in Clostridium acetobutylicum; Behrens S et al.; Two genes from Clostridium acetobutylicum DSM 792 were identified which are predicted to encode new members of the ECF subfamily of eubacterial RNA polymerase sigma factors . The sigX gene has the potential to encode a 184-amino acid protein with a molecular mass of 21,870 Da and with the highest overall similarity to Fecl of Escherichia coli (27 % identical residues) . The second gene, which is predicted to encode an alternative sigma factor of the ECF subfamily, is the previously described orf2 gene (Gerischer and Durre, 1990) located in the adc gene region of C . acetobutylicum . The deduced protein of orf2 has significant similarity to SigX of C . acetobutylicum (22 % identical residues) and shares structural features with other alternative sigma factors . Therefore, it is proposed to rename orf2 as sigY . Analysis of the phylogenetic relationship revealed that SigX from C . acetobutylicum, together with sigmaE from Streptomyces coelicolor and SigX from Bacillus subtilis, form a gram-positive cluster within the ECF subfamily and that SigY from C . acetobutylicum together with UviA from Clostridium perfringens, form a separate cluster located between the gram-positive cluster and the sporulation sigma factor sigmaH from B . subtilis.

Toxicon, 2001 Jan, 39(1), 27 - 41
Tetanus and botulinum neurotoxins: turning bad guys into good by research; Rossetto O et al.; The neuroparalytic syndromes of tetanus and botulism are caused by neurotoxins produced by bacteria of the genus Clostridium . They are 150 kDa proteins consisting of three-domains, endowed with different functions: neurospecific binding, membrane translocation and specific proteolysis of three key components of the neuroexocytosis apparatus . After binding to the presynaptic membrane of motoneurons, tetanus neurotoxin (TeNT) is internalized and transported retroaxonally to the spinal cord, where it blocks neurotransmitter release from spinal inhibitory interneurons . In contrast, the seven botulinum neurotoxins (BoNT) act at the periphery and inhibit acetylcholine release from peripheral cholinergic nerve terminals . TeNT and BoNT-B, -D, -F and -G cleave specifically at single but different peptide bonds, VAMP/synaptobrevin, a membrane protein of small synaptic vesicles . BoNT types -A, -C and -E cleave SNAP-25 at different sites within the COOH-terminus, whereas BoNT-C also cleaves syntaxin . BoNTs are increasingly used in medicine for the treatment of human diseases characterized by hyperfunction of cholinergic terminals.

Kyobu Geka, 2000 Jul, 53(8 Suppl), 715 - 7
{Hyperbaric oxygen as an adjunctive treatment for descending necrotizing mediastinitis: report of a case}; Kamiyoshihara M et al.; We report a case of 59-year-old man of descending necrotizing mediastinitis (DNM) secondary to peritonsillar abscess . A 59-year-old man with diabetes mellitus was admitted to a local hospital because of cervical swelling related to a peritonsillar abscess . Despite administration of antibiotics, swelling of the neck, dysphagia and dyspnea deteriorated . Therefore he was urgently undergone a tracheotomy and transferred to our hospital by an ambulance . The surgery consisted with neck and anterior mediastinal drainage through neck and cervical collar incision . Culture of drainage fluid showed clostridium difficile . On postoperative day 5, we started hyperbaric oxygen therapy (HBOT) . After lavage and HBOT, the patient improved by degrees, and discharged on postoperative day 82 . DNM is a rare but serious complication of otopharyngeal and deep neck infection that spreads down to the mediastinum through the cervical-facial planes . Its mortality rate remains high even with aggressive surgical drainage and appropriate antibiotics . Our patient was successfully treated with urgent surgical drainage, antibiotics and HBOT . HBOT might be of great value as an adjunctive management to control this fatal infection.

J Am Vet Med Assoc, 2000 Aug 1, 217(3), 365 - 8, 340
An outbreak of type C botulism in 12 horses and a mule; Schoenbaum MA et al.; A USDA Early Response Team investigated deaths of several horses and a mule in northern Arizona at the request of local animal health officials . Thirteen animals (12 horses and 1 mule) housed at 5 facilities in a 7.4 square mile area died between August 1998 and January 1999 . Clinical signs consisted of muscular weakness that rapidly progressed to lateral recumbency . Ten animals had paresis of the tongue, throat, or lips . Affected animals appeared alert and were interested in eating and drinking, even while recumbent . All 13 animals were euthanatized . Clostridium botulinum type C was isolated from feces or intestinal contents from 3 affected horses . Preformed toxin was detected in samples of soil and bird droppings collected from a nearby horse burial site . It was hypothesized that the outbreak was a result of birds, presumably ravens, feeding at the burial site and at horse facilities in the area that transferred toxin to the affected animals.

Rev Latinoam Microbiol, 1999 Oct-Dec, 41(4), 295 - 301
{Cell cultures as a system for distinguishing between strains ofClostridium chauvoei and Clostridium septicum isolated in northeastern Mexico}; Wong Gonzalez A et al.; Clostridium chauvoei and C . septicum have similar characteristics as far as results from biochemical methods and gas chromatography (GC) are concerned . A total of 267 samples collected from sick or dead animals in the fields from Northeast Mexico, were bacteriologically analysed and differentiated by the GC technique . From these strains, 16 belong to the group of C . chauvoei/C . septicum . Studies on the effect of toxin on cell cultures of the lines EBL, 3T3, BHK21-BSR/PK5/88, CHO-K1 and MDCK were performed . The objective was to obtain further data for identification, as the results from GC do not allow exact differentiation between C . chauvoei and C . septicum species . The results were obtained in tests with BHK21-BSR/PK5/88 cells as this had proved to be the most sensitive cell line, closely followed by 3T3 and CHO-K1 cells . MDCK cells were of little sensitivity . Results of the cytotoxin test of the 16 strains were reproducible and suggested a differentiation between C . chauvoei and C . septicum other than indicated by GC . The cytotoxin test is a highly specific system that provides also an additional method to distinguish between C . chauvoei and C . septicum strains.

Nat Struct Biol, 2000 Aug, 7(8), 693 - 9
Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B; Swaminathan S et al.; Clostridium botulinum neurotoxins are among the most potent toxins to humans . The crystal structures of intact C . botulinum neurotoxin type B (BoNT/B) and its complex with sialyllactose, determined at 1 . 8 and 2.6 A resolution, respectively, provide insight into its catalytic and binding sites . The position of the belt region in BoNT/B is different from that in BoNT/A; this observation presents interesting possibilities for designing specific inhibitors that could be used to block the activity of this neurotoxin . The structures of BoNT/B and its complex with sialyllactose provide a detailed description of the active site and a model for interactions between the toxin and its cell surface receptor . The latter may provide valuable information for recombinant vaccine development.

J Theor Biol, 2000 Aug 21, 205(4), 581 - 6
Number of triplets in 16S rRNA gene related with pathogenicity of Bacillus spp . and Clostridium spp; Abella CA et al.; The relation between the number of some trinucleotides in the sequence of 16S rRNA gene and pathogenicity of bacterial species from the genera of Bacillus and Clostridium was revealed . The species of genus Bacillus, which are pathogenic for humans, mammals and insects, have an increased number of AAA and TAT triplets in 16S rRNA gene . Theoretically, these species, B . anthracis and B . cereus for example, may be detected in the specimen by the higher ratio of AAA plus TAT triplets to the number of GGG triplet . Species of genus Clostridium, which are pathogenic for humans and mammals, have a maximum ratio of AAA and TAT triplet numbers . This ratio was higher than 2.6 for pathogenic species and lower than 2.2 for saprophytic ones . These theoretical data may open a new way for detecting pathogenic bacteria through the determination of triplet numbers in the sequences of 16S rRNA or rRNA . However, the mechanism of evolutionary relation between the number of AAA and TAT triplets in the sequence of 16S rRNA gene and the pathogenicity of bacterial species is not known .

Mol Microbiol, 2000 Jun, 36(6), 1447 - 59
A chimeric ribozyme in clostridium difficile combines features of group I introns and insertion elements; Braun V et al.; CdlSt1, a DNA insertion of 1975 bp, was identified within tcdA-C34, the enterotoxin gene of the Clostridium difficile isolate C34 . Located in the catalytic domain A1-C34, Cd/St1 combines features of two genetic elements . Within the first 434 nt structures characteristic for group I introns were found; encoding the two transposase-like proteins tlpA and tlpB nucleotides 435-1975 represent the remainder of a IS605-like insertion element . We show that the entire CdlSt1 is accurately spliced from tcdA-C34 primary transcripts and that purified TcdA-C34 toxin is of regular size and catalytic activity . A search for CdlSt1-related sequences demonstrates that the element is widespread in toxinogenic and non-toxinogenic C . difficile strains, indicating the mobility of CdlSt1 . In strain C34, we characterize 10 CdlSt1 variants; all are highly homologous to CdlSt1 (> 93% identity), integrated in bacterial open reading frames (ORFs), show the typical composite structure of CdlSt1 and are precisely spliced from their primary transcripts . CdlSt1-like chimeric ribozymes appear to combine the invasiveness of an insertion element with the splicing ability of a group I intron, rendering transposition harmless for the interrupted gene.

Eur J Biochem, 2000 Aug, 267(16), 5237 - 46
Control of cellular phosphatidylinositol 4,5-bisphosphate levels by adhesion signals and rho GTPases in NIH 3T3 fibroblasts involvement of both phosphatidylinositol-4-phosphate 5-kinase and phospholipase C; Weernink PA et al.; The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts . Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by approximately 50% . This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases . Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals . The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4, 5)P2-producing enzyme, PtdIns4P 5-kinase . Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells . In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30-50% . The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant-negative N17 Rac1 . Taken together, these data suggest that adhesion-controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5-kinase and phospholipase C.

Eur J Biochem, 2000 Aug, 267(16), 5191 - 7
Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene; Alape-Giron A et al.; Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene . The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied . The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region . The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin . The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin . The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin . This work therefore identifies residues critical for the toxic activities of C . perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.

Eur J Biochem, 2000 Aug, 267(16), 4988 - 97
Cloning, expression and characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca; Irwin DC et al.; The gene for a 104-kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans . The DNA sequence revealed a type II cellulose-binding domain at the N-terminus, followed by a FNIII-like domain and ending with a glycosyl hydrolase Family 48 catalytic domain . The enzyme and catalytic domain alone were each expressed in and purified from S . lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper . However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T . fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 micromol cellobiose.min-1.mL-1, more than 15-fold higher than any single enzyme alone . Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 degrees C . Using SWISSMODEL, the amino-acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF . Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed.

Vaccine, 2000 Sep 15, 19(2-3), 288 - 97
Cloning, expression and evaluation of a recombinant sub-unit vaccine against Clostridium botulinum type F toxin; Holley JL et al.; A synthetic gene encoding the Hc (binding) domain of Clostridium botulinum neurotoxin F (FHc) was expressed in Escherichia coli fused to maltose binding protein (MBP) . The purified MBP-FHc and FHc isolated after removal of MBP were evaluated in mice for their ability to protect against toxin challenge . Balb/c mice developed a protective immune response following administration of either protein via the intraperitoneal or intramuscular routes . A comparison of antibody titres and protection following single and multiple vaccinations and the effects of dosage are shown . The long term protection afforded by the vaccines was also investigated . Ten months following vaccination mice were still protected when challenged with 10(4) MLD(50) doses of botulinum toxin F.

Mol Biol Cell, 2000 Aug, 11(8), 2565 - 75
Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering; Kodama A et al.; Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met . We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity . We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells . SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains . Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering . C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering . Vav2 is a GDP/GTP exchange protein for Rho . Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation . Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering . These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering . In addition, Vav2 may be involved in the SHP-2-Rho pathway.

Int J Antimicrob Agents, 2000 Aug, 15(4), 305 - 9
Antibacterial activity of four dentin bonding systems; Herrera M et al.; The antibacterial action of bonding systems Gluma 2000, Syntac, Prisma Universal Bond 3, Scotchbond Multipurpose and Prime-Bond was tested against 32 strains of the caries-producing bacteria Streptococcus spp., Lactobacillus spp., Actinomyces spp., Porphyromonas spp . and Clostridium spp . An agar plate diffusion method was used with chlorhexidine as the positive control . Assays were performed in triplicate for each component (primer and adhesive) of the bonding systems . All the adhesives were found to inhibit bacterial growth but with differences in their spectra of action . The sum action of the Scotchbond Multipurpose components were most inhibitory and Prime-Bond was found to be the least effective system.

Biochemistry, 2000 Aug 8, 39(31), 9561 - 70
Lysine 2,3-aminomutase and trans-4,5-dehydrolysine: characterization of an allylic analogue of a substrate-based radical in the catalytic mechanism; Wu W et al.; An analogue of lysine, trans-4,5-dehydro-L-lysine (trans-4, 5-dehydrolysine), is a potent inhibitor of lysine 2,3-aminomutase from Clostridium subterminale SB4 that competes with L-lysine for binding to the active site . Inclusion of trans-4,5-dehydrolysine with activated enzyme and the coenzymes pyridoxal-5'-phosphate and S-adenosylmethionine, followed by freezing at 77 K, produces an intense signal in the electron paramagnetic resonance (EPR) spectrum at g 2.0, which is characteristic of an organic radical . A series of deuterated and (15)N-labeled samples of trans-4,5-dehydrolysine were synthesized and used to generate the EPR signal . Substitution of deuterium for hydrogen at C2, C3, C4, C5, and C6 of trans-4, 5-dehydrolysine led to significant simplifications and narrowing of the EPR signal, showing that the unpaired electron was located on the carbon skeleton of 4,5-trans-4,5-dehydrolysine . The hyperfine splitting pattern is simplified by use of 4,5-dehydro{3, 3-(2)H(2)}lysine or 4,5-dehydro{4,5-(2)H(2)}lysine, and it is dramatically simplified with 4,5-dehydro-{3,3,4,5,6,6-(2)H(6)}lysine . Spectral simulations show that the EPR signal arises from the allylic radical resulting from the abstraction of a hydrogen atom from C3 of trans-4,5-dehydrolysine . This radical is an allylic analogue of the substrate-related radical in the rearrangement mechanism postulated for this enzyme . The rate constant for formation of the 4,5-dehydrolysyl radical (2 min(-)(1)) matches that for the decrease in the concentration of {4Fe-4S}(+), showing that the two processes are coupled . The cleavage of S-adenosylmethionine to 5'-deoxyadenosine and methionine takes place with a rate constant of approximately 5 min(-)(1) . These kinetic correlations support the hypothesis that radical formation results from a reversible reaction between {4Fe-4S}(+) and S-adenosylmethionine at the active site to form {4Fe-4S}(2+), the 5'-deoxyadenosyl radical, and methionine as intermediates.

Appl Environ Microbiol, 2000 Aug, 66(8), 3234 - 40
Comparative experiments to examine the effects of heating on vegetative cells and spores of Clostridium perfringens isolates carrying plasmid genes versus chromosomal enterotoxin genes; Sarker MR et al.; Clostridium perfringens enterotoxin (CPE) is an important virulence factor for both C . perfringens type A food poisoning and several non-food-borne human gastrointestinal diseases . Recent studies have indicated that C . perfringens isolates associated with food poisoning carry a chromosomal cpe gene, while non-food-borne human gastrointestinal disease isolates carry a plasmid cpe gene . However, no explanation has been provided for the strong associations between certain cpe genotypes and particular CPE-associated diseases . Since C . perfringens food poisoning usually involves cooked meat products, we hypothesized that chromosomal cpe isolates are so strongly associated with food poisoning because (i) they are more heat resistant than plasmid cpe isolates, (ii) heating induces loss of the cpe plasmid, or (iii) heating induces migration of the plasmid cpe gene to the chromosome . When we tested these hypotheses, vegetative cells of chromosomal cpe isolates were found to exhibit, on average approximately twofold-higher decimal reduction values (D values) at 55 degrees C than vegetative cells of plasmid cpe isolates exhibited . Furthermore, the spores of chromosomal cpe isolates had, on average, approximately 60-fold-higher D values at 100 degrees C than the spores of plasmid cpe isolates had . Southern hybridization and CPE Western blot analyses demonstrated that all survivors of heating retained their cpe gene in its original plasmid or chromosomal location and could still express CPE . These results suggest that chromosomal cpe isolates are strongly associated with food poisoning, at least in part, because their cells and spores possess a high degree of heat resistance, which should enhance their survival in incompletely cooked or inadequately warmed foods.

Appl Microbiol Biotechnol, 2000 Jun, 53(6), 661 - 7
A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and alpha-galactosidase; Clarke JH et al.; Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and alpha-galactosidase . Mannanase A (ManA) from Pseudomonas fluorescens subsp . cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima . Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached kappa number when used with the xylanase . Sequential incubations with XylA and P . fluorescens ManA produced the largest final kappa number reduction in comparison to control pretreated pulp . The release of galactose from softwood pulp by alpha-galactosidase A (AgaA) from P . fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA . gave the most effective kappa number reduction using a single incubation . Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability.

Biotechnol Bioeng, 2000 Sep 20, 69(6), 591 - 6
Expression, immobilization, and enzymatic characterization of cellulose-binding domain-organophosphorus hydrolase fusion enzymes; Richins RD et al.; Bifunctional fusion proteins consisting of organophosphate hydrolase (OPH) moieties linked to a Clostridium-derived cellulose-binding domain (CBD) were shown to be highly effective in degrading organophosphate nerve agents, enabling purification and immobilization onto different cellulose materials in essentially a single step . Enzyme kinetics studies were performed for the CBD-OPH fusions using paraoxon as the substrate . The kinetics values of the unbound fusion enzymes were similar to OPH with a modest increase in K(m) . Immobilization of the enzymes onto microcrystalline cellulose resulted in a further increase in the K(m) values of approximately twofold . The pH profile of the cellulose-immobilized enzymes was also only minimally affected . The CBD-OPH fusion proteins could be immobilized onto a variety of cellulose matrixes, and retained up to 85% of their original activity for 30 days . The durability of the bound fusions increased with the amount of Avicel used, suggesting that protein/cellulose interactions may have a dramatic stabilizing effect . Repeated hydrolysis of paraoxon was achieved in an immobilized enzyme reactor with 100% degradation efficiency over 45 days . These fusion proteins should prove to be invaluable tools for the development of low cost, OPH-based cellulose materials for the simultaneous adsorption and degradation of stored or spilled organophosphate wastes .

MMWR Morb Mortal Wkly Rep, 2000 Jun 23, 49(24), 543 - 5
Update: Clostridium novyi and unexplained illness among injecting-drug users--Scotland, Ireland, and England, April-June 2000; Genetic characterization of toxin A-negative et al.; TechLab, Inc., Blacksburg, Virginia 24060-6364, USA . jsmoncrief@techlabinc.com

Toxin-specific enzyme immunoassays, cytotoxicity assays, and PCR were used to analyze 48 toxin A-negative, toxin B-positive Clostridium difficile isolates from various geographical sites around the world . All the isolates were negative by the TOX-A TEST and positive by the TOX A/B TEST . A deletion of approximately 1.7 kb was found at the 3' end of the toxA gene for all the isolates, similar to the deletion in toxinotype VIII strains (e.g., C . difficile serotype F 1470) . Additional PCR analysis indicated that the toxin B encoded by these isolates contains sequence variations downstream of the active site compared to the sequence of reference strain VPI 10463 . This variation may extend the glucosylation spectrum to Ras proteins, as observed previously for closely related lethal toxin from Clostridium sordellii and toxin B from toxin A-negative, toxin B-positive strain F 1470 . Toxin A-negative, toxin B-positive isolates have recently been associated with disease in humans, and they may be more common than was previously supposed.

J Food Prot, 2000 Jul, 63(7), 965 - 9
A survey of water activity and pH values in fresh pasta packed under modified atmosphere manufactured in Argentina and Uruguay; Schebor C et al.; The water activity (a(w)) and pH values of commercially available filled fresh pasta and gnocchi packed under modified atmosphere and manufactured in Argentina and Uruguay were examined . The retail survey included 58 samples (several brands) of filled pasta and 11 samples of gnocchi . Fillings consisted of different combinations of cheese (various types), beef, ricotta, ham, chicken, and spinach . The survey revealed that the a(w) values of the 58 samples of filled pasta ranged from 0.916 to 0.973, and their pH values ranged from 5.2 to 7.0 . The a(w) of gnocchi was consistently higher and ranged from 0.936 to 0.983, with pH values from 4.8 to 6.4 . Some samples of filled pasta and most gnocchi samples were found to have a(w) and pH values that would support growth of spores of Clostridium botulinum, if present, under conditions of temperature abuse (i.e., 30 degrees C).

FEMS Microbiol Lett, 2000 Aug 1, 189(1), 109 - 13
Clostridium perfringens epsilon toxin causes excessive release of glutamate in the mouse hippocampus; Miyamoto O et al.; The mechanism of neurotoxicity of Clostridium perfringens epsilon toxin to the mouse brain was investigated . Intravenous injection in mice with the toxin caused seizure and excited hippocampal neurons . Microdialysis revealed that epsilon toxin induced excessive glutamate release in the hippocampus . Both the seizure and glutamate release were attenuated by prior injection with riluzole, an inhibitor of pre-synaptic glutamate release, suggesting that this toxin enhances glutamate efflux, leading to seizure and hippocampal neuronal damage.

Eur J Pharm Sci, 2000 Jul, 11(1), 69 - 79
Protease inhibitors . Part 12 . Synthesis of potent matrix metalloproteinase and bacterial collagenase inhibitors incorporating sulfonylated N-4-nitrobenzyl-beta-alanine hydroxamate moieties; Scozzafava A et al.; N-4-Nitrobenzyl-beta-alanine was reacted with alkyl/arylsulfonyl halides, followed by conversion of the COOH to the CONHOH group . Structurally related compounds were obtained by reaction of N-4-nitrobenzyl-beta-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by similar conversion of the COOH into the CONHOH moiety . Another subseries of derivatives was prepared from sulfanilyl- or metanilyl-4-nitrobenzyl-beta-alanine by reaction with arylsulfonyl isocyanates, followed by the introduction of the hydroxamate moiety . The new compounds were assayed as inhibitors of four matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-8 and MMP-9, and of the Clostridium histolyticum collagenase (ChC) . Some of the prepared hydroxamate derivatives proved to be very effective collagenase/gelatinase inhibitors, depending on the substitution pattern at the sulfonamido moiety . Substitutions leading to the best inhibitors of MMP-1, a short-pocket enzyme, were those involving pentafluorophenylsulfonyl or 3-trifluoromethyl-phenylsulfonyl at P(1') (K(I) of 3-5 nM) . For MMP-2, MMP-8 and MMP-9 (deep-pocket enzymes), the best inhibitors were those containing perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, arylsulfonylureido- or arylsulfonylureido-sulfanilyl-/metanilyl moieties at P(1') . Bulkier groups in this position, such as 1- and 2-naphthyl-, substituted-naphthyl or quinoline-8-yl- moieties, among others, led to less effective MMP/ChC inhibitors . The best ChC inhibitors were again those containing pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl P(1') groups . This study demonstrates that the 4-nitrobenzyl moiety, investigated here for the first time, is an efficient P(2') anchoring moiety, whereas the beta-alanyl scaffold can successfully replace the alpha-amino acyl one, for obtaining potent MMP/ChC inhibitors.

FEBS Lett, 2000 Jul 7, 476(3), 258 - 61
Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein; Fujita K et al.; Claudins (claudin-1 to -18) with four transmembrane domains and two extracellular loops constitute tight junction strands . The peptide toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to claudin-3 and -4, but not to claudin-1 or -2 . We constructed claudin-1/claudin-3 chimeric molecules and found that the second extracellular loop of claudin-3 conferred CPE sensitivity on L fibroblasts . Furthermore, overlay analyses revealed that the second extracellular loop of claudin-3 specifically bound to CPE at the K(a) value of 1.0x10(8) M(-1) . We concluded that the second extracellular loop is the site through which claudin-3 interacts with CPE on the cell surface.

Przegl Lek, 2000, 57(3), 181 - 4
{Gas gangrene or inflammation of the neck--diagnostic difficulties}; Kedzierski B et al.; The authors describe a patient with an extensive inflammation of the neck soft tissues as a complication of the peritonsillar abscess . Follow-up computed tomography revealed gasi-form follicles in the inflammed neck soft tissues, suggesting gas gangrene . We report disseminate ways of the inflammation process on the financial tonsil, reasons of the gangrene also the infections of soft tissues caused by anaerobic bacteries--Clostridium . CT--examination in inflammatory tumors of the neck is valuable, permits to exclude expansion process, but it cannot give unequivocal answer to differentiate gas gangrene and phlegmon.

Acta Otolaryngol Suppl, 2000, 543, 61 - 2
Palatal myoclonus and clicking tinnitus in a 12-year-old girl--case report; Jero J et al.; Palatal myoclonus is a rare neurological disorder of the soft palate and other oropharyngeal muscles, which causes clicking tinnitus . The latter is audible both to the patient and to an observer . The aetiology may be a brain stem lesion, and it is only rarely that a cause cannot be identified . The condition has been described in adults, but seldom in children . We present here a case of palatal myoclonus with distressing tinnitus in a 12-year-old girl and its successful treatment with electromyography (EMG)-guided injections of Clostridium botulinum toxin.

J Biol Inorg Chem, 2000 Jun, 5(3), 381 - 92
Paramagnetic 1H NMR spectroscopy of the reduced, unbound photosystem I subunit PsaC: sequence-specific assignment of contact-shifted resonances and identification of mixed- and equal-valence Fe-Fe pairs in {4Fe-4S} centers FA- and FB-; Antonkine ML et al.; The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the {4Fe-4S} clusters FA and FB . The g-tensor orientation of FA- and FB- is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each {4Fe-4S}+ cluster . The preferential position of the mixed-valence and equal-valence pairs, in turn . can be inferred from the study of the temperature dependence of contact-shifted resonances by 1H NMR spectroscopy . For this, a sequence-specific assignment of these signals is required . The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp . PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances . The non-solvent-exchangeable, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA- and FB- by their downfield chemical shifts, by their temperature dependencies, and by their short T1 relaxation times . The usual fast method of assigning the 1H NMR spectra of reduced {4Fe-4S} proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC . Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2{4Fe-4S} cluster ferredoxin at 0.94 A resolution as a model . The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other {4Fe-4S}-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA- and FB- have the same preferential positions relative to the protein . The analysis reveals that the magnetic properties of the two {4Fe-4S} clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.

Enferm Infecc Microbiol Clin, 2000 Mar, 18(3), 109 - 12
{Evaluation of four rapid methods for the investigation of the toxigenic capacity of Clostridium difficile strains isolated in a selective medium}; Garcia A et al.; BACKGROUND: Use of selective Clostridium difficile culture as a diagnostic method for C . difficile associated disease requires to prove the toxigenic ability of the isolates . Toxin B detection by cell culture assay after growing the microorganism in enriched broth is the standard method, but it delays the final diagnosis for 3-5 days . This study compares retrospectively four rapid techniques for detecting these toxigenic C . difficile strains . METHODS: 106 clinical isolates of C . difficile (72 toxigenic and 34 non-toxigenic), these and 16 clinical strains of other species of Clostridium were investigated . The four methods were performed directly from colonies growing on solid agar . They were: a) cytotoxin detection in cell culture; b) two PCR amplifications of toxin A and toxin B, respectively, and c) toxin A detection by an immunoenzymatic method (VIDAS CDA2) . All these procedures were completed within a normal working day . RESULTS: Only the 72 toxigenic C . difficile strains gave positive results by cell culture and PCR techniques (sensitivity and specificity: 100%) . A total of 14 out of 49 toxigenic C . difficile strains showed negative results by the VIDAS assay in the first run, but all them were positive in repeated tests . CONCLUSIONS: Although all methods performed well, the cytotoxicity assay done directly on colonies growing in CCFA is a simple and rapid technique, and appears to be well-suited for use in laboratories with access to cell cultures.

DNA Seq, 2000, 11(1-2), 109 - 18
Sequence analysis of the atp operon of Clostridium acetobutylicum DSM 792 encoding the F0F1 ATP synthase; Externbrink T et al.; The atp gene region of Clostridium acetobutylicum DSM 792 has been fully sequenced . It contains the F0F1 ATPase genes in the order atpIBEFHAGDC, whose products share high sequence homology to the respective proteins of a variety of other bacteria . It is the first such sequence available for a mesophilic Clostridium . Significant differences to other reported atp operons are a distal transcription start point 219 bp upstream of the translation start point and a second transcription initiation site (without corresponding promoter sequence) upstream of atpE, indicating posttranscriptional processing for massive expression of this gene product.

Dtsch Med Wochenschr, 2000 Jun 16, 125(24), 755 - 60
{Ultrasound diagnosis of penicillin-induced segmental hemorrhagic colitis}; Dietrich CF et al.; INTRODUCTION: Penicillin-induced segmental haemorrhagic colitis (SHC) is a characteristic and striking but rarely diagnosed clinical entity . Bloody diarrhea and abdominal cramps start a few days after the intake of oral penicillin derivatives . We report the ultrasonographic and clinical findings in nine patients with SHC and compare the results with the findings in ten patients with antibiotic-related pseudomembranous colitis (PMC) . METHODS: Nine consecutive patients with SHC (age: 32 +/- 10 years; five males, four females) with PMC-negative proctoscopic findings, stool cultures and negative clostridium difficile toxin and ten patients with PMC (age: 50 +/- 18 years; six males, four females) with positive proctoscopy and Clostridium difficile toxin were clinically evaluated and examined by high resolution ultrasonography . The sonographic findings of the colonic and small bowel walls as well as the clinical course of the diseases were documented . RESULTS: In all nine patients with SHC the wall of the ascending colon was asymmetrically thickened with loss of layer structure . Neither the small bowel nor the cecum were involved in patients with SHC . In all cases a distinct border between involved and uninvolved colon wall was found . During follow-up all patients recovered soon after stopping antibiotic treatment and symptomatic care . In seven of ten patients with PMC pancolitis and in three of ten with left-sided colitis were found at ultrasonography . In all patients with PMC the bowel wall was symmetrically thickened with the layers remaining distinct . DISCUSSION: The knowledge of the clinical characteristics and sonographic findings of penicillin-induced segmental haemorrhagic colitis may reduce the need for invasive endoscopic and radiological investigations in diagnosis and follow-up . The age of patients, clinical course and sonographic findings may be helpful in differentiating patients with SHC and PMC.

Dtsch Med Wochenschr, 2000 Jun 16, 125(24), 750 - 4
{Ultrasound diagnosis of pseudomembranous colitis}; Ludolph T et al.; BACKGROUND AND OBJECTIVE: The diagnosis of pseudomembranous colitis (PMC) is based on the history of exposure to antibiotics, characteristic endoscopic findings and on demonstrating the presence of Clostridium difficile toxins in the faeces . This report presents typical sonographic features of PMC . PATIENTS AND METHODS: The sonograms of 13 patients with PMC (7 males, 6 females, median age 70 years, range 55-84 years), were retrospectively analyzed . Patients' histories, clinical findings, the results of colonoscopy including histological findings and microbiological tests were related to the sonographic features of inflammation of the colon . RESULTS: At sonography the wall of the colon was thickened (6-17 mm) in each of the patients, different types of changed wall-architecture were seen . In 85% sonographic signs of colitis were restricted to the left colon, whereas in 15% a pancolitis was diagnosed . Ascites was detected in 38%, the colon contents were visible in 85%, in 77% the lumen was narrowed . Colonoscopy confirmed the diagnosis in six patients, in two patients endoscopy was performed only to control the effect of therapy and in five patients endoscopy was thought to be unnecessary . CONCLUSION: Sonography can supplement history and clinical findings if PMC is suspected . Potentially life-saving therapy can be initiated even if colonoscopy has not been performed and results of microbiological assessment are not yet available . However, the diagnosis of PMC remains to be based on the gold standard of colonoscopy and histological findings.

Australas Radiol, 1999 May, 43(2), 256 - 9
Clostridium septicum septicaemia with myonecrosis; Salanitri GC et al.; A case of fatal spontaneous gas gangrene due to Clostridium septicum septicaemia associated with an occult rectal malignancy is presented . This condition has a rapid progression and a high mortality even with prompt treatment . It is important that the radiologist considers this diagnosis in an appropriate clinical setting to allow rapid instigation of appropriate therapy.

Infect Immun, 2000 Aug, 68(8), 4566 - 73
The C terminus of component C2II of Clostridium botulinum C2 toxin is essential for receptor binding; Blocker D et al.; The binary Clostridium botulinum C2 toxin consists of two separate proteins, the binding component C2II (80.5 kDa) and the actin-ADP-ribosylating enzyme component C2I (49.4 kDa) . For its cytotoxic action, C2II binds to a cell membrane receptor and induces cell entry of C2I via receptor-mediated endocytosis . Here we studied the structure-function relationship of C2II by constructing truncated C2II proteins and producing polyclonal antisera against selective regions of C2II . An antibody raised against the C terminus (amino acids 592 to 721) of C2II inhibited binding of C2II to cells . The antibody prevented pore formation by C2II oligomers in artificial membranes but did not influence the properties of existing channels . To further define the region responsible for receptor binding, we constructed proteins with deletions in C2II; specifically, they lacked amino acid residues 592 to 721 and the 7 C-terminal amino acid residues . The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells . Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding.

Curr Biol, 2000 Jul 13, 10(14), 839 - 48
Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation; West MA et al.; BACKGROUND: Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules . Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells . It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation . We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells . RESULTS: We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment . Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling . Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes . Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells . Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis . CONCLUSIONS: Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling . Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis . Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.

Arch Biochem Biophys, 2000 Jul 15, 379(2), 237 - 44
Secondary structure and calcium-induced folding of the Clostridium thermocellum dockerin domain determined by NMR spectroscopy; Lytle BL et al.; Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein . Docking to the cohesin domains is mediated by a highly conserved domain, dockerin (DS), borne by each enzymatic subunit . DS consists of two 22-amino-acid duplicated sequences, each bearing homology to the EF-hand calcium-binding loop . To compare the DS structure with that of the EF-hand helix-loop-helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy . The effect of Ca(2+)-binding on the DS structure was first investigated by using 2D (15)N-(1)H HSQC NMR spectroscopy . Changes in the spectra during Ca(2+) titration revealed that Ca(2+) induces folding of DS into its tertiary structure . This Ca(2+)-induced protein folding distinguishes DS from typical EF-hand-containing proteins . Sequential backbone assignments were determined for 63 of 69 residues . Analysis of the NOE connectivities and H(alpha) chemical shifts revealed that each half of the dockerin contains just one alpha-helix, comparable to the F-helix of the EF-hand motif . Thus, the structure of the DS Ca(2+)-binding subdomain deviates from that of the canonical EF-hand motif .

Antimicrob Agents Chemother, 2000 Aug, 44(8), 2222 - 4
In vitro activities of MK-826 (L-749,345) against 363 strains of anaerobic bacteria; Wexler HM et al.; The activity of MK-826 was compared to the activities of cefoxitin, ceftriaxone, imipenem, and meropenem against 363 gram-negative and gram-positive anaerobes by using NCCLS procedures . At least 98% of the strains were susceptible to the carbapenems . All strains of Clostridium perfringens, Fusobacterium nucleatum, Peptostreptococcus, and Sutterella wadsworthensis were susceptible to all agents tested.

J Hosp Infect, 2000 Jul, 45(3), 235 - 7
The rise of Clostridium difficile: the effect of length of stay, patient age and antibiotic use; Shek FW et al.; Hospitals in the UK have recently seen a marked increase in C . difficile for reasons which are unclear . Reduced standards of hygiene, increasingly elderly patients, greater cephalosporin use and longer hospital stay have been suggested . We retrospectively studied all cases of C . difficile diarrhoea at Princess Margaret Hospital, Swindon, over two years . Cephalosporins, patient age and LOS appeared unrelated to the rise in C . difficile; penicillins and macrolides were related . Our policy of using amoxycillin and clarithromycin for community-acquired pneumonia coincided with this study and may explain the observed rise in C . difficile.

Dermatology, 2000, 200(4), 287 - 91
Revival of the use of botulinum toxin: application in dermatology; Boni R et al.; Botulinum toxins (BTXs) comprise a family of neurotoxins designated as types A-G, which are produced by the anerobic bacterium Clostridium botulinum . BTX-A blocks the cholinergic transmission resulting in flaccid paralysis and autonomous nerve dysfunction . It has become a powerful therapeutic tool in a variety of conditions over the last decades . Primarily used in the treatment of strabismus, blepharospasm and hemifacial spasms, BTX has only recently been recognized in dermatology . The use of BTX in dermatology includes the treatment of focal hyperhidrosis, hyperfunctional facial lines as well as paralysis of the anal sphincter in the therapy of anal fissures . The mechanism of action is described and the current literature is reviewed .

Gastroenterology, 2000 Jul, 119(1), 139 - 50
Clostridium difficile toxin A causes early damage to mitochondria in cultured cells; He D et al.; BACKGROUND & AIMS: The mechanism by which Clostridium difficile toxin A causes actin depolymerization and cell rounding involves toxin internalization and subsequent monoglucosylation of the Rho family of proteins . This study explored toxin internalization and effects on mitochondrial function before cell rounding . METHODS: Chinese hamster ovary (CHO) cells were exposed to toxin A, and mitochondrial localization was assayed by confocal microscopy . Mitochondrial function was measured by adenosine triphosphate (ATP) concentration, mitochondrial permeability, and leakage of cytochrome c . RESULTS: Confocal microscopy showed toxin A colocalization with the mitochondrial protein GRP 75 at 5 minutes after toxin exposure . Between 5 and 15 minutes, toxin A caused an 80% diminution in cellular ATP levels; cell rounding and Rho glucosylation commenced between 15 and 30 minutes . Toxin A also resulted in reduction of mitochondrial membrane potential and a 2-3-fold increase in reactive oxygen radicals . Preincubation of CHO cells with the antioxidants butylated hydroxyanisole or butylated hydroxytoluene blocked the toxin A-induced increase in oxygen radicals and diminished cell rounding . Western blot analysis of toxin A-exposed isolated mitochondria showed a direct effect of toxin A on leakage of cytochrome c . CONCLUSIONS: The results show that extensive mitochondrial damage occurs within 15 minutes in CHO cells exposed to toxin A . Diminished ATP concentrations and increased oxygen radicals are likely to contribute to cytotoxicity from this bacterial toxin.

J Vet Med A Physiol Pathol Clin Med, 2000 May, 47(4), 251 - 5
Histamine in lambs with abomasal bloat, haemorrhage and ulcers; Vatn S et al.; The median concentration of histamine in abomasal fluid of lambs with abomasal haemorrhage and/or ulcers (group 2) was significantly (P < 0.05) higher than the concentrations in lambs presenting abomasal bloat (group 1) and in the healthy and the diseased controls . In group 2, there was also a strong correlation (R2 = 0.81) between the histamine concentrations in abomasal tissue and abomasal fluid, although the median value of histamine in the abomasal tissue was not statistically higher in this group than in the others . The urine of lambs in group 2 also had numerically higher median concentration of histamine than the other groups . Five out of eight tested strains of Lactobacillus spp . and one out of two strains of Clostridium sordellii, isolated from abomasal contents of lambs with abomasal disease, were strong producers of histamine . Bacterial production is one possible source for the increased histamine concentrations in lambs suffering from abomasal haemorrhage and/or ulcers.

Rev Argent Microbiol, 2000 Apr-Jun, 32(2), 63 - 70
{Survey of pH and water activity in acidified bottled vegetables and meats (home processed) in relation to the potential growth of Clostridium botulinum}; Mazzobre MF et al.; The water activity (aw) and pH of acidified (vinegar) bottled vegetables and meat with vegetables--mostly home-canned--was examined in relation to the potential growth of Clostridium botulinum . Most products (vegetables or meat with vegetables) had water activity above the "per se" inhibitory limit (aw < 0.95) for growth of C . botulinum . Regarding pH, 96% of canned vegetables had a pH lower than 4.6, but 81% of the canned meat with vegetables had a pH above 4.6 . This was attributed to the well known buffer effect of food proteins, which makes it difficult to lower food pH during acidification with vinegar . It is concluded that most bottled meat with vegetables constitute a potential hazard since these foods are marketed at room temperature, and botulism toxin may be produced if spores are present.

J Antimicrob Chemother, 2000 Jul, 46(1), 141 - 3
Local antibiotic guidelines for adult community-acquired pneumonia (CAP): a survey of UK hospital practice in 1999; Woodhead M et al.; We investigated the guidelines in British hospitals for the management of adults admitted with community-acquired pneumonia (CAP) . A questionnaire was sent to one consultant respiratory physician in each of the 263 hospitals in the British Thoracic Society (BTS) Directory of Training Posts and Services . Two hundred and thirteen (81%) responses were received: 178 (84%) had written CAP guidelines, of which 123 (69%) printed copies were received . For non-severe CAP a single antibiotic (74% of guidelines-most frequently amoxycillin or ampicillin) was the usual recommendation with the combination of a beta-lactam and a macrolide the second most frequent (24%) . The latter combination was recommended for severe CAP in 81% of guidelines . Clostridium difficile-associated diarrhoea had influenced guideline recommendations, or was commented on as a concern, in 18% of responses . Written guidelines for antibiotic therapy in adults with CAP exist in most British hospitals and follow broadly the 1993 BTS guidelines, although combination therapy is used not infrequently for non-severe CAP.

J Antimicrob Chemother, 2000 Jul, 46(1), 115 - 9
In vitro activity of telithromycin (HMR 3647) and seven other antimicrobial agents against anaerobic bacteria; Ackermann G et al.; We assessed the in vitro activity of telithromycin (HMR 3647) and seven other antimicrobials against 292 strains of obligately anaerobic bacteria . MICs were determined with the microdilution technique and Wilkins-Chalgren broth according to DIN 58940-83 . MIC50/MIC90s (mg/L) for telithromycin were 4/4 for Bacteroides fragilis, Bacteroides ovatus and Bacteroides thetaiotaomicron, 2/4 for Fusobacterium spp . and Bilophila wadsworthia, 2/2 for Bacteroides caccae, 1/4 for Bacteroides vulgatus, 0.25/4 for Prevotella spp., > or =0.03/0.5 for Clostridium spp . and 0.125/4 for Peptostreptococcus spp.

J Med Microbiol, 2000 Jul, 49(7), 635 - 42
Studies of the effect of Clostridium butyricum on Helicobacter pylori in several test models including gnotobiotic mice; Takahashi M et al.; The interaction between Clostridium butyricum and Helicobacter pylori was examined in vitro and in vivo . The culture supernate of C . butyricum MIYAIRI 588 inhibited the growth of H . pylori even when its pH was adjusted to 7.4 . The bactericidal effect of butyric acid on H . pylori was stronger than that of lactic, acetic or hydrochloric acids . Flow cytometric analysis showed that pre-incubation of gastric epithelial (MKN45) cells with H . pylori and C . butyricum inhibited the adhesion of H . pylori to the cells . Persistent infection with H . pylori in the gastric mucosa of germ-free mice was observed for 5 weeks . Cure of persistent infection with H . pylori in the gnotobiotic mice was demonstrated following infection with C . butyricum . The probiotic agent, C . butyricum MIYAIRI 588 may have some beneficial effects on H . pylori infection.

Lakartidningen, 2000 May 24, 97(21), 2606 - 10
{Drug-induced enterocolitis . Important differential diagnosis in the investigation of diarrhea and intestinal hemorrhage}; Tysk C; This article is a review of the side-effects of drugs affecting the small and large intestines . Pseudomembranous colitis is caused by antibiotics facilitating an overgrowth of Clostridium difficile . A hemorrhagic colitis, generally self-limiting, can be caused by penicillin, amoxycillin and ampicillin . Toxicity of NSAID may induce intestinal ulcers, diaphragm-like strictures, perforation, colitis and relapse of inflammatory bowel disease . Drug-induced lymphocytic colitis has been reported due to ticlopidine, Cyclo 3 Fort, and occasionally by ranitidine, carbamazepine, vinburnine, tardyferon, and flutamide . Sulphasalazine and 5-ASA can cause relapse of ulcerative colitis . Neutropenic enterocolitis is a severe complication to cytotoxic therapy for cancer . Ischemic colitis can be caused by drugs inducing mesenteric vasoconstriction.

Clin Infect Dis, 2000 Jun, 30(6), 954 - 5
Rapidly progressive necrotizing fasciitis and gangrene due to Clostridium difficile: case report; Bhargava A et al.; A case of rapidly progressive necrotizing fascitis and gas gangrene due to Clostridium difficile that responded very well to surgical intervention is described.

Clin Infect Dis, 2000 Jun, 30(6), 952 - 4
Persistence of an endemic (toxigenic) isolate of Clostridium difficile in the environment of a general medicine ward; Cohen SH et al.; The epidemiology of Clostridium difficile-associated diarrhea (CDAD) in an endemic setting was investigated by use of DNA typing methods to determine the strain identity of C . difficile isolates . Two predominant toxigenic clones were found in the environment and accounted for 29.8% (type 1) and 15.5% (type 2) of CDAD cases, respectively . In endemic settings, the environment and cross-transmission may play a role in acquisition of CDAD.

Scand J Infect Dis, 2000, 32(3), 320 - 2
CNS infection with clostridium septicum; Dirks C et al.; We present an unusual case of Clostridium septicum brain infection in a 72-yr-old woman who had no underlying malignant disease . The infection spread from a localized sit to the CNS causing gas formation . The patient died rapidly.

Microbiology, 2000 Jul, 146 ( Pt 7), 1593 - 603
Phylogeny and functional conservation of sigma(E) in endospore-forming bacteria; Arcuri EF et al.; Conservation of the sporulation processes between BACILLUS: spp . and CLOSTRIDIUM: spp . was investigated through evolutionary and complementation analyses of sigma(E) . Alignment of partial predicted sigma(E) amino acid sequences from three BACILLUS: spp., Paenibacillus polymyxa and five CLOSTRIDIUM: spp . revealed that amino acid residues previously reported to be involved in promoter utilization (M124, E119 and N120) and strand opening (C117) are conserved among all these species . Phylogenetic analyses of various sigma factor sequences from endospore-forming bacteria revealed that homologues of sigma(E), sigma(K) and sigma(G) clustered together regardless of genus, suggesting a common origin of sporulation sigma factors . The functional equivalence between CLOSTRIDIUM: acetobutylicum sigma(E) and BACILLUS: subtilis sigma(E) was investigated by complementing a non-polar B . subtilis sigma(E) null mutant with the spoIIG operon from either B . subtilis (spoIIG(Bs)) or C . acetobutylicum (spoIIG(Ca)) . Single-copy integration of spoIIG(Bs) into the amyE locus of the sigma(E) null mutant completely restored the wild-type sporulation phenotype, while spoIIG(Ca) only partially restored sporulation . Maximal expression of spoIIG(Ca)-lacZ occurred approximately 12 h later than maximal expression of spoIIG(Bs)-lacZ . Differences in temporal expression patterns for spoIIG(Ca) and spoIIG(Bs) in the B . subtilis background may at least partially explain the observed sporulation complementation phenotypes . This study suggests a common phylogenetic ancestor for sigma(E) in BACILLUS: spp . and CLOSTRIDIUM: spp., although regulation of sigma(E) expression may differ in these two genera.

Microbiology, 2000 Jul, 146 ( Pt 7), 1555 - 63
A novel beta-glucoside-specific PTS locus from Streptococcus mutans that is not inhibited by glucose; Cote CK et al.; A regulon from Streptococcus mutans that plays a role in the utilization of beta-glucosides has been isolated, sequenced and subjected to sequence analysis . This regulon encodes a beta-glucoside-specific Enzyme II (EII) component (bglP) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and a phospho-beta-glucosidase (bglA) which is responsible for the breakdown of the phospho-beta-glucosides within the cell . Both the bglP and bglA gene products have significant similarity with proteins that have similar functions from Clostridium longisporum, Listeria monocytogenes, Erwinia chrysanthemi, Escherichia coli, Klebsellia oxytoca and Bacillus subtilis . The potential functions of the BglP and BglA proteins are supported by phenotypic data from both S . mutans and E . coli . A chromosomal deletion in S . mutans spanning the bglP and bglA genes resulted in a strain that was unable to hydrolyse the beta-glucoside aesculin in the presence of glucose . When glucose was removed from the medium, the deletion strain regained the ability to break down aesculin . These data suggest that S . mutans possesses an alternative mechanism from the one described in this report for breaking down beta-glucosides . This second mechanism was repressed by glucose while the regulon described here was not . Complementation studies in E . coli CC118 also suggest a potential role for this regulon in the utilization of other beta-glucosides . When a plasmid containing the 8 kb beta-glucoside-specific regulon was transformed into E . coli CC118, the transformed strain was able to break down the beta-glucoside arbutin.

J Clin Microbiol, 2000 Jul, 38(7), 2706 - 14
Characterization of a toxin A-negative, toxin B-positive strain of Clostridium difficile responsible for a nosocomial outbreak of Clostridium difficile-associated diarrhea; Alfa MJ et al.; Clostridium difficile-associated diarrhea (CAD) is a very common nosocomial infection that contributes significantly to patient morbidity and mortality as well as to the cost of hospitalization . Previously, strains of toxin A-negative, toxin B-positive C . difficile were not thought to be associated with clinically significant disease . This study reports the characterization of a toxin A-negative, toxin B-positive strain of C . difficile that was responsible for a recently described nosocomial outbreak of CAD . Analysis of the seven patient isolates from the outbreak by pulsed-field gel electrophoresis indicated that this outbreak was due to transmission of a single strain of C . difficile . Our characterization of this strain (HSC98) has demonstrated that the toxin A gene lacks 1.8 kb from the carboxy repetitive oligopeptide (CROP) region but apparently has no other major deletions from other regions of the toxin A or toxin B gene . The remaining 1.3-kb fragment of the toxin A CROP region from strain HSC98 showed 98% sequence homology with strain 1470, previously reported by M . Weidmann in 1997 (GenBank accession number Y12616), suggesting that HSC98 is toxinotype VIII . The HSC98 strain infecting patients involved in this outbreak produced the full spectrum of clinical illness usually associated with C . difficile-associated disease . This pathogenic spectrum was manifest despite the inability of this strain to alter tight junctions as determined by using in vitro tissue culture testing, which suggested that no functional toxin A was produced by this strain.

J Clin Microbiol, 2000 Jul, 38(7), 2484 - 7
Comparison of PCR-ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for typing Clostridium difficile; Bidet P et al.; Clostridium difficile is now recognized as the major agent responsible for nosocomial diarrhea in adults . Among the genotyping methods available, arbitrarily primed PCR (AP-PCR), PCR-ribotyping, and pulsed-field gel electrophoresis (PFGE) have been widely used for investigating outbreaks of C . difficile infections . However, the comparative typing ability, reproducibility, discriminatory power, and efficiency of these methods have not been fully investigated . We compared the results of three methods-AP-PCR with three different primers (AP3, AP4, and AP5), PCR-ribotyping, and PFGE (with SmaI endonuclease)-to differentiate 99 strains of C . difficile that had been previously serogrouped . Typing abilities were 100% for PCR-ribotyping and AP-PCR with AP3 and 90% for PFGE, due to early DNA degradation in strains from serogroup G . Reproducibilities were 100% for PCR-ribotyping and PFGE but only 88% for AP-PCR with AP3, 67% for AP-PCR with AP4, and 33% for AP-PCR with AP5 . Discriminatory power for unrelated strains was >0.95 for all the methods but was lower for PCR-ribotyping among serogroups D and C . PCR-based methods were easier and quicker to perform, but their fingerprints were more difficult to interpret than those of PFGE . We conclude that PCR-ribotyping offers the best combination of advantages as an initial typing tool for C . difficile.

J Biol Chem, 2000 Sep 15, 275(37), 28494 - 9
The role of pyruvate ferredoxin oxidoreductase in pyruvate synthesis during autotrophic growth by the Wood-Ljungdahl pathway; Furdui C et al.; Pyruvate:ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO(2) . The catalytic proficiency of this enzyme for the reverse reaction, pyruvate synthase, is poorly understood . Conversion of acetyl-CoA to pyruvate links the Wood-Ljungdahl pathway of autotrophic CO(2) fixation to the reductive tricarboxylic acid cycle, which in these autotrophic anaerobes is the stage for biosynthesis of all cellular macromolecules . The results described here demonstrate that the Clostridium thermoaceticum PFOR is a highly efficient pyruvate synthase . The Michaelis-Menten parameters for pyruvate synthesis by PFOR are: V(max) = 1.6 unit/mg (k(cat) = 3.2 s(-1)), K(m)(Acetyl-CoA) = 9 micrometer, and K(m)(CO(2)) = 2 mm . The intracellular concentrations of acetyl-CoA, CoASH, and pyruvate have been measured . The predicted rate of pyruvate synthesis at physiological concentrations of substrates clearly is sufficient to support the role of PFOR as a pyruvate synthase in vivo . Measurements of its k(cat)/K(m) values demonstrate that ferredoxin is a highly efficient electron carrier in both the oxidative and reductive reactions . On the other hand, rubredoxin is a poor substitute in the oxidative direction and is inept in donating electrons for pyruvate synthesis.

Appl Environ Microbiol, 2000 Jul, 66(7), 2791 - 6
Production of volatile derivatives of metal(loid)s by microflora involved in anaerobic digestion of sewage sludge; Michalke K et al.; Gases released from anaerobic wastewater treatment facilities contain considerable amounts of volatile methyl and hydride derivatives of metals and metalloids, such as arsine (AsH(3)), monomethylarsine, dimethylarsine, trimethylarsine, trimethylbismuth (TMBi), elemental mercury (Hg(0)), trimethylstibine, dimethyltellurium, and tetramethyltin . Most of these compounds could be shown to be produced by pure cultures of microorganisms which are representatives of the anaerobic sewage sludge microflora, i.e., methanogenic archaea (Methanobacterium formicicum, Methanosarcina barkeri, Methanobacterium thermoautotrophicum), sulfate-reducing bacteria (Desulfovibrio vulgaris, D . gigas), and a peptolytic bacterium (Clostridium collagenovorans) . Additionally, dimethylselenium and dimethyldiselenium could be detected in the headspace of most of the pure cultures . This is the first report of the production of TMBi, stibine, monomethylstibine, and dimethylstibine by a pure culture of M . formicicum.

J Clin Invest, 2000 Jul, 106(1), 15 - 24
Neuroprotection mediated by changes in the endothelial actin cytoskeleton; Laufs U et al.; Cerebral blood flow is regulated by endothelium-derived nitric oxide (NO), and endothelial NO synthase-deficient (eNOS-deficient; eNOS(-/-)) mice develop larger cerebral infarctions following middle cerebral artery (MCA) occlusion . We report that disruption of Rho-mediated endothelial actin cytoskeleton leads to the upregulation of eNOS expression and reduces the severity of cerebral ischemia following MCA occlusion . Mice treated with the Rho inhibitor Clostridium botulinum C3 transferase (10 microgram/d) or the actin cytoskeleton disrupter cytochalasin D (1 mg/kg) showed a two- to fourfold increase in vascular eNOS expression and activity . This increase in eNOS expression was not due to increases in eNOS gene transcription, but to prolongation of eNOS mRNA half-life from 10 +/- 3 hours to 24 +/- 4 hours . Indeed, endothelial cells overexpressing a dominant-negative Rho mutant (N19RhoA) exhibited decreased actin stress fiber formation and increased eNOS expression . Inhibition of vascular Rho guanosine-5'-triphosphate binding activity by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin increased cerebral blood flow to ischemic regions of the brain, and mice treated with simvastatin, C3 transferase, or cytochalasin D showed smaller cerebral infarctions following MCA occlusion . No neuroprotection was observed with these agents in eNOS(-/-) mice . These findings suggest that therapies which target the endothelial actin cytoskeleton may have beneficial effects in ischemic stroke.

J Clin Gastroenterol, 2000 Jun, 30(4), 432 - 5
Ileal perforation caused by cytomegalovirus infection in a critically ill adult; Chamberlain RS et al.; Cytomegalovirus (CMV) infection of the gastrointestinal (GI) tract is common and is most often seen in patients with acquired immunodeficiency syndrome (AIDS), inflammatory bowel disease, or those receiving immunosuppressive therapy . CMV infection of the small bowel accounts for only 4.3% of all CMV infections of the GI tract . Isolated cases of small bowel perforation due to CMV have been reported in AIDS patients, and all but one patient has died . This article reports the first case of an ileal perforation due to transfusion-associated CMV infection in a critically-injured non-AIDS patient . Immediate surgical resection and antiviral therapy led to complete recovery . The development of abdominal pain, fever, watery diarrhea, and GI bleeding in a critically ill patient should prompt the clinician to consider the diagnosis of CMV enteritis . If standard stool pathogens and Clostridium difficile toxin studies are nondiagnostic, endoscopic evaluation and CMV serology should be obtained . If CMV infection is confirmed, ganciclovir therapy should be initiated without delay . If bowel perforation occurs . prompt surgical resection is indicated . A heightened level of suspicion for CMV infection in multiply injured trauma victims and other critically ill patients, with earlier recognition of potential small bowel involvement, can hopefully decrease the incidence of bowel perforation, which is usually a fatal event.

Boll Soc Ital Biol Sper, 1998 Jul-Aug, 74(7-8), 67 - 74
Modulation between aerobic and anaerobic metabolism in the mutant cell line CdtR-Q; Lofrumento DD et al.; It has recently been shown that the cell line Don Q obtained by mutagenesis of wild type Chinese hamster lung fibroblasts (Don wt), presents a point mutation in the gene coding for UDP-glucose pyrophosphorylase . The persistent low level of UDP-glucose makes Don Q clone resistant to Clostridium difficile toxin B . Starting from the observation that Don Q cells exhibit many large hydrophobic cytoplasmic inclusions, that we have found to be made of neutral lipids, the aerobic metabolism of the two cell lines has been examined . The specific activity of cytochrome oxidase in Don Q cells is more than 5 times lower than that found in Don wt . Also, the activity of Complexes II + III, expressed by the activity of succinate-cytochrome c oxido-reductase, has been found to be lower in Don Q compared to wt cells . On the other hand, NADH-cytochrome c oxido-reductase activity, insensitive to rotenone, is more than doubled in Don Q . In these cells the activity of lactate dehydrogenase is very high, being able to oxidise more than 3,000 nmoles of NADH/min/mg of protein . The results obtained indicate that Don Q cells, in addition to a decreased ability to synthesise UDP-glucose, have an impairment in the respiratory chain . Such an impairment could be correlated to the increased capacity to generate a higher amount of reducing equivalents through the glycolytic activity, which can then be utilised for the synthesis of fatty acids stored in lipid droplets.

Biochem Biophys Res Commun, 2000 Jul 5, 273(2), 499 - 504
Cloning of the Microcystis aeruginosa M228 lectin (MAL) gene; Jimbo M et al.; We have cloned and characterized the gene encoding Microcystis aeruginosa (strain M228) lectin (MAL) . The gene contains 1551 nucleotides and an open reading frame for a protein of 517 amino acids with a predicted molecular weight of 55,159 Da . The carboxy-terminal region of MAL has three tandemly repeated homologous domains composed of 61 amino acids . These regions show similarity to the corresponding regions of the alpha-amylase of Clostridium beijerinckii (23% identity) . The mal gene lies adjacent to an ORF that display homology to cytochrome P-450 and polyketide synthase . Southern hybridization showed that the genomic DNA of the strain M228 contained, in addition to MAL gene (mal), at least two other mal like gene .

FEMS Microbiol Lett, 2000 Jul 1, 188(1), 29 - 33
Use of a lux reporter system for monitoring rapid changes in alpha-toxin gene expression in Clostridium perfringens during growth; Phillips-Jones MK; To determine whether the luxA-luxB reporter system is suitable as a sensitive reporter for rapid real-time measurements of alpha-toxin gene (cpa) expression in Clostridium perfringens, and to widen the range of alpha-toxin-producing C . perfringens strains examined with respect to cpa expression during growth, the reporter plasmid pPS14 (possessing the alpha-toxin promoter region plus 0.7 kb of upstream region linked to the luxA-luxB genes), was used in batch growth experiments of C . perfringens P90.2.2, an alpha-toxin-producing strain with no known association with disease . Levels of in vivo bioluminescence obtained during growth were broadly in agreement with previous mRNA and reporter studies of cpa expression (Bullifent et al., FEMS Microbiol . Lett . 131 (1995) 99-105), confirming the suitability of lux as an accurate reporter system in this organism, but the sensitive nature of the lux reporter permitted the in vivo detection of a very rapid reduction in expression during late-exponential phase that was not attributable to loss in cell viability or limiting bioluminescence assay substrates . There was also a small peak in cpa expression in early- to mid-exponential phase cells, that was not detected in previous studies with other reporters . This may be indicative of the exquisite sensitivity of the lux reporter, or this may be a difference in cpa expression that occurs specifically in this C . perfringens strain . Whichever is the case, these results confirm the complexity of alpha-toxin gene expression in different strains of this pathogenic bacterium.

AACN Clin Issues, 1999 Nov, 10(4), 492 - 9
Toxic megacolon: diagnosis and treatment challenges; Levine CD; In adults, toxic megacolon is a relatively uncommon but potentially lethal complication of inflammatory bowel disease (IBD), infectious colitis, or ischemic colitis caused by cancer chemotherapeutic agents . Patients have distension of the colon and signs of toxicity such as elevated temperature, hypotension, decreased level of consciousness and electrolyte imbalances . Factors thought to increase the risk include premature discontinuation of IBD medications; procedures that increase colon trauma, such as barium enema and colonoscopy; medications that decrease gastrointestinal motility; and electrolyte imbalances, especially hypokalemia . Differential diagnosis is made based on the patient's history and results of stool cultures and assay for Clostridium difficile toxin . Medical management in the intensive care unit includes careful monitoring, fluid volume and electrolyte replacement, bowel rest and decompression, antibiotic therapy, and cessation of medications that slow gastric motility . Surgical management may be necessary if there are signs of deterioration, perforation, hemorrhage, or sepsis.

Proc Natl Acad Sci U S A, 2000 Jun 20, 97(13), 7208 - 13
Selenium-dependent metabolism of purines: A selenium-dependent purine hydroxylase and xanthine dehydrogenase were purified from Clostridium purinolyticum and characterized; Self WT et al.; During purification of the selenium-dependent xanthine dehydrogenase (XDH) from Clostridium purinolyticum, another hydroxylase was uncovered that also contained selenium and exhibited similar spectral properties . This enzyme was purified to homogeneity . It uses purine, 2OH-purine, and hypoxanthine as substrates, and based on its substrate specificity, this selenoenzyme is termed purine hydroxylase (PH) . The product of hydroxylation of purine by PH is xanthine . A concomitant release of selenium from the enzyme and loss of catalytic activity on treatment with cyanide indicates that selenium is essential for PH activity . Selenium-dependent XDH, also purified from C . purinolyticum, was found to be insensitive to oxygen during purification and to use both potassium ferricyanide and 2,6-dichloroindophenol as electron acceptors . Selenium is required for the xanthine-dependent reduction of 2, 6-dichloroindophenol by XDH . Kinetic analyses of both enzymes revealed that xanthine is the preferred substrate for XDH and purine and hypoxanthine are preferred by PH . This characterization of these selenium-requiring hydroxylases involved in the interconversion of purines describes an extension of the pathway for purine fermentation in the purinolytic clostridia.

Arch Biochem Biophys, 2000 Jun 15, 378(2), 246 - 58
fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation; Sternfeld L et al.; In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane . Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells . U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis inverted question mark2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release . The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release . In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM) . This effect was inhibited by either Ro 318220 or AACOCF(3) . Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP . fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation . However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity . The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35% . The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase . This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD .

J Biol Chem, 2000 Jun 23, 275(25), 19368 - 74
Molecular cloning of endo-beta -galactosidase C and its application in removing alpha -galactosyl xenoantigen from blood vessels in the pig kidney; Ogawa H et al.; Galalpha1-3Gal is the major xenoantigenic epitope responsible for hyperacute rejection upon pig to human xenotransplantation . Endo-beta-galactosidase C from Clostridium perfringens destroys the antigenic epitope by cleaving the beta-galactosidic linkage in the Galalpha1-3Galbeta1-4GlcNAc structure . Based on partial peptide sequences of the enzyme, we molecularly cloned the enzyme gene, which encodes a protein with a predicted molecular mass of about 93 kDa . The deduced protein sequence of the enzyme has limited homology in the C-terminal half with endo-beta-galactosidase from Flavobacterium keratolyticus and beta-1,3-glucanases . The enzyme expressed in Escherichia coli removed the alpha-galactosyl epitope nearly completely from pig erythrocytes and from pig aortic endothelial cells . The enzyme-treated endothelial cells in culture were greatly reduced in cell surface antigens, which were recognized by IgM, IgG, or IgA in human sera, and became much less susceptible to complement-mediated cytotoxicity caused by human sera . When the pig kidney was perfused with the enzyme, the vascular endothelial cells became virtually devoid of the alpha-galactosyl epitope, with concomitant decrease in binding to IgM in human plasma . These results demonstrated that the recombinant endo-beta-galactosidase C is a valuable aid in xenotransplantation.

Biochemistry, 2000 Jun 27, 39(25), 7455 - 60
Reversible carbon monoxide binding and inhibition at the active site of the Fe-only hydrogenase; Bennett B et al.; Carbon monoxide binding and inhibition have been investigated by electron paramagnetic resonance (EPR) spectroscopy in solution and in crystals of structurally described states of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum . Simulation of the EPR spectrum of the as-isolated state indicates that the main component of the EPR spectrum consists of the oxidized state of the "H cluster" and components due to reduced accessory FeS clusters . Addition of carbon monoxide to CpI in the presence of dithionite results in the inhibition of hydrogen evolution activity, and a characteristic axial EPR signal {g(eff(1)), g(eff(2)), and g(eff(3)) = 2.0725, 2.0061, and 2.0061, respectively} was observed . Hydrogen evolution activity was restored by successive sparging with hydrogen and argon and resulted in samples that exhibited the native oxidized EPR signature that could be converted to the reduced form upon addition of sodium dithionite and hydrogen . To examine the relationship between the spectroscopically defined states of CpI and those observed structurally by X-ray crystallography, we have examined the CpI crystals using EPR spectroscopy . EPR spectra of the crystals in the CO-bound state exhibit the previously described axial signal associated with CO binding . The results indicate that the addition of carbon monoxide to CpI results in a single reversible carbon monoxide-bound species characterized by loss of enzyme activity and the distinctive axial EPR signal.

Infect Immun, 2000 Jul, 68(7), 3848 - 53
Clostridium perfringens iota-toxin requires activation of both binding and enzymatic components for cytopathic activity; Gibert M et al.; Iota-toxin is produced by Clostridium perfringens type E strains and consists of two independent components, the enzymatic and binding components, referred to as Ia and Ib, respectively . A recombinant C . perfringens strain, strain 667/pMRP147, produced processed Ia and partially processed Ib, while a recombinant C . perfringens type A strain, strain TS133/pMRP147, in which the VirR-VirS two-component system is inactivated, produced only precursor forms of Ia and Ib . This suggests that iota-toxin is processed by a VirR-VirS-responsive protease, although not completely in the recombinant type A strain . The precursor forms of Ia and Ib were purified from cultures of the latter strain, and their proteolytic activation was examined . Treatment with proteases cleaved off small peptides (9 to 13 amino acid residues) and a 20-kDa peptide from the N termini of the Ia and Ib precursors, respectively, leading to their active forms . They were activated efficiently by alpha-chymotrypsin, pepsin, proteinase K, subtilisin, and thermolysin but only weakly by trypsin, as demonstrated by the cell-rounding assay . lambda-Protease from the C . perfringens type E strain, which was found to be a zinc-dependent protease related to thermolysin, activated iota-toxin as efficiently as did alpha-chymotrypsin . These results suggest that lambda-protease is most responsible for the activation of iota-toxin in type E strains.

Int J Food Microbiol, 2000 May 25, 56(1), 21 - 8
Amplified fragment length polymorphism (AFLP) analysis of Clostridium perfringens for epidemiological typing; McLauchlin J et al.; Thirty-five Clostridium perfringens isolates from patients and foods implicated in seven outbreaks of suspected Cl . perfringens food poisoning together with five unrelated incidents were analysed by serotyping and amplified fragment length polymorphism (AFLP) . Despite minor band differences, AFLP was found to be highly reproducible and 16 different profiles (each unique to the 12 incidents) were recognised . The results from both serotyping and AFLP analysis identified exactly the same groups of related cultures . It is concluded that AFLP can provide a rapid, sensitive and reproducible method for the typing of Cl . perfringens for outbreak investigation.

EMBO J, 2000 Jun 15, 19(12), 2900 - 10
Ras mediates the cAMP-dependent activation of extracellular signal-regulated kinases (ERKs) in melanocytes; Busca R et al.; In melanocytes and melanoma cells, cAMP activates extracellular signal-regulated kinases (ERKs) and MEK-1 by an unknown mechanism . We demonstrate that B-Raf is activated by cAMP in melanocytes . A dominant-negative mutant of B-Raf, but not of Raf-1, blocked the cAMP-induced activation of ERK, indicating that B-Raf is the MEK-1 upstream regulator mediating this cAMP effect . Studies using Clostridium sordelii lethal toxin and Clostridium difficile toxin B have suggested that Rap-1 or Ras might transduce cAMP action . We show that Ras, but not Rap-1, is activated cell-specifically and mediates the cAMP-dependent activation of ERKs, while Rap-1 is not involved in this process in melanocytes . Our results suggest a novel, cell-specific mechanism involving Ras small GTPase and B-Raf kinase as mediators of ERK activation by cAMP . Also, in melanocytes, Ras or ERK activation by cAMP is not mediated through protein kinase A activation . Neither the Ras exchange factor, Son of sevenless (SOS), nor the cAMP-responsive Rap-1 exchange factor, Epac, participate in the cAMP-dependent activation of Ras . These findings suggest the existence of a melanocyte-specific Ras exchange factor directly regulated by cAMP.

Appl Microbiol Biotechnol, 2000 May, 53(5), 545 - 52
Exploitation of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid); Liu SJ et al.; Active butyrate kinase (Buk) and phosphotransbutyrylase (Ptb) were purified in three steps: ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose and affinity chromatography on Matrex Red A from recombinant Escherichia coli K2006 (pJC7) . They were then successfully exploited for in vitro synthesis of 3-hydroxybutyryl-CoA (3HBCoA), 4-hydroxybutyryl-CoA (4HBCoA), 4-hydroxyvaleryl-CoA (4HVCoA) and poly(hydroxyalkanoic acid) (PHA) . In addition, the ability of the PHA synthase of Chromatium vinosum, PhaEC(Cv), to use these CoA thioesters was evaluated . Combination of Buk and Ptb with PhaEC(Cv) established a new system for in vitro synthesis of poly(3-hydroxybutyric acid) {poly(3HB)} . In this system, 3-hydroxybutyric acid was converted to 3HBCoA by Buk and Ptb at the expense of ATP . Formation of 3HBCoA was further driven by the polymerization of 3HBCoA molecules to poly(3HB) by PHA synthase, and the released CoA was recycled by Ptb . This system therefore also ensured the regeneration of CoA . With ATP as the energy supply, which was hydrolyzed to ADP and phosphate, 2.6 mg poly(3HB) was obtained from a 1-ml reaction mixture containing 7.6 mg 3-hydroxybutyrate at the beginning . Studies showed that Ptb and PHA synthase were the rate-limiting steps in this system, and initial CoA concentrations ranging from 1 to 7 mM did not inhibit poly(3HB) synthesis . Synthesis of various polyesters of 3HB and 4HB with this system was also tested, and copolyesters containing 4HB of 1-46 mol % were obtained.

Prev Vet Med, 2000 Jul 3, 46(1), 61 - 74
Foaling-management practices associated with the occurrence of enterocolitis attributed to Clostridium perfringens infection in the equine neonate; East LM et al.; Enterocolitis associated with Clostridium perfringens (C . perfringens) infection in neonatal foals is often severe and has been associated with a high case-mortality risk . We designed a premises-based survey to evaluate the associations of regional foaling practices, premises environmental management, periparturient foal and brood-mare management, and periparturient brood-mare ration with the occurrence of neonatal enterocolitis attributed to C . perfringens infection . Potential risk factors individually associated with enterocolitis were breed type, housing type at foaling and in the first three days of life, ground/floor surface type at foaling and in the first three days of life, brood-mare ration before and after foaling, and the presence of livestock other than horses on the premises in the past . From the multivariable-logistic regression models, six variables were significantly associated with an increased risk of the outcome of interest (p<0.05): foals of the stock horse type, housing in a stall or drylot in the first three days of life, other livestock present on the premises in the past, foal born on dirt, sand or gravel surface, and low amounts of grass hay and grain fed post-partum . Low grain amounts fed pre-partum represented a decreased risk of the outcome of interest.

J Vet Med Sci, 2000 May, 62(5), 473 - 8
The evaluation of the potential of botulinum C3 enzyme as an exogenous differentiation inducing factor to neurons; Watanabe Y et al.; Botulinum C3 enzyme produced by Clostridium botulinum type C and D strains modifies Rho proteins . In a previous study, we observed that the LDH isozyme pattern of neurons treated with C3 enzyme was different from that induced with endogenous growth factor of neurons such as NGF {21} . This type of change is considered to have an advantage in the medical use of C3 enzyme for neural disorder . To determine the functional similarity of C3-treated neurons to control and NGF-treated neurons, we examined the responses of C3-treated neurons to various drugs, including some neurotransmitters, by measuring the rise of intracellular Ca ions into the neurons . The time course of the rise of intracellular Ca ions induced by high concentration of potassium in the C3-treated neurons was similar to that in the NGF-treated neurons . The C3-treated neurons responded to glutamic acid, aspartic acid, kainic acid, gamma-aminobutylic acid, muscarine and ACh with similar time courses and magnitudes as the control neurons . These results suggest that the C3 enzyme induces the functional differentiation of neurons, and that C3 enzyme has the potential for the medical use as an exogenous differentiation-inducing factor of neurons.

Zh Mikrobiol Epidemiol Immunobiol, 1999 Sep-Oct, (5), 40 - 7
{Supertoxic complexes of botulinum toxins}; Vertiev IuV; The super-toxic super-complexes (SSC) of botulinic neurotoxins, types A and B, have been isolated . The preparation of type A SSC (SSC/A) consist of the proteolyzed form of type A botulinic neurotoxin (BoNT/A), 50 and 90 kD, nontoxic nonhemagglutinating protein of 140 kD (NTNH140) in the nonproteolyzed form, hemagglutinin of 17 kD (Ha17), hemagglutinin of 34 kD (Ha34) and the proteolyzed form of hemagglutinin of 70 kD (Ha70) (20 and 50 kD) . The preparations of type B SSC (SSC/B) consist of the nonproteolyzed form of type B botulinic neurotoxin (BoNT/B) of 150 kD, the proteolyzed form of BoNT/B of 150 kD (45 and 105 kD), the nonproteolyzed form of NTNH140, Ha17, Ha34 and two nonidentified proteins (32 and 40 kD) . As shown in this study, toxic complexes both in native toxins and in the preparations of SSC do not dissociate for several weeks at pH 8.0 and for 18 hours in 3% SDS, as well as after treatment with RNAase or 1 M NaCl . Some part of SSC/A (neurotoxin and Ha70) has been found to dissociate in 3% SDS after 1-hour incubation at 22 degrees C after the addition of 2-ME . The preparations of SSC contain nucleic acids (A260 nm/A280 nm = 2.0), supposedly ensuring the stability of the complexes . In contrast to the L-forms of Clostridium botulinum toxins, the preparations of SSC/A and SSC/B have been found to possess increased toxicity . The specific toxicity of SSC/A has proved to be 1-2 x 10(9) DLM per 1 OD280 nm and that of SSC/B, from 5 x 10(8) to 1 x 10(9) DLM per OD280 nm . One minimal lethal oral dose of these SSC preparations for mice was less than 10 DLM, introduces intraperitoneally.

Am J Physiol Heart Circ Physiol, 2000 Jun, 278(6), H1762 - 8
Inhibition of Rho protein stimulates iNOS expression in rat vascular smooth muscle cells; Muniyappa R et al.; Inducible nitric oxide synthase (iNOS) in vascular smooth muscle cells (VSMCs) is upregulated in arterial injury and plays a role in regulating VSMC proliferation and restenosis . Inflammatory cytokines {e.g., interleukin-1beta (IL-1beta)} released during vascular injury induce iNOS . Small GTP-binding proteins of the Ras superfamily play a major role in IL-1beta-dependent signaling pathways . In this study, we examined the role of Rho GTPases in regulating iNOS expression in VSMCs . Treatment of VSMCs with mevastatin, which inhibits isoprenylation of Rho and other small GTP-binding proteins, produced significantly higher amounts of IL-1beta-evoked NO and iNOS protein compared with control . Similarly, bacterial toxins {Toxin B from Clostridium difficile and C3 ADP-ribosyl transferase (C3) toxin from Clostridium botulinium} that specifically inactivate Rho proteins increased NOS products (NO and citrulline) and iNOS expression . Toxin B increased the activity of iNOS promoter-reporter construct in VSMCs . Both toxins enhanced IL-1beta-stimulated iNOS expression and NO production . These data demonstrate for the first time that inhibition of Rho induces iNOS and suggest a role for Rho protein in IL-1beta-stimulated NO production in VSMCs.

J Bacteriol, 2000 Jul, 182(13), 3775 - 83
Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX; Wang H et al.; Tn5397 is a conjugative transposon that was originally isolated from Clostridium difficile . Previous analysis had shown that the central region of Tn5397 was closely related to the conjugative transposon Tn916 . However, in this work we obtained the DNA sequence of the ends of Tn5397 and showed that they are completely different to those of Tn916 . Tn5397 did not contain the int and xis genes, which are required for the excision and integration of Tn916 . Instead, the right end of Tn5397 contained a gene, tndX, that appears to encode a member of the large resolvase family of site-specific recombinases . TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn4451 from Clostridium perfringens and Tn4453 from C . difficile . Like the latter elements, inserted copies of Tn5397 were flanked by a direct repeat of a GA dinucleotide . The Tn5397 target sites were also shown to contain a central GA dinucleotide . Excision of the element in C . difficile completely regenerated the original target sequence . A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C . difficile . A Tn5397 mutant in which part of tndX was deleted was constructed in B . subtilis . This mutant was nonconjugative and did not produce the circular form of Tn5397, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn5397 and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility . Finally, we showed that introduction of Tn916 into a strain containing Tn5397 induced the loss of the latter element in 95.6% of recipients.

Ugeskr Laeger, 2000 May 29, 162(22), 3200 - 1
{Diagnosis of Clostridium difficile infection in pseudomembranous colitis}; Pedersen F et al.; C . difficile is known as the main cause of pseudomembranous colitis, however, some individuals may be asymptomatically colonized . In this paper two patients with diarrhoea had three respectively five negative stool cultures . Endoscopically, one patient had severe colitis consistent with both pseudomembranous colitis and inflammatory bowel disease . In the other case the endoscopic findings were typical for pseudomembranous colitis . In both cases there were positive cultures for C . difficile from biopsies from colon-plaques . We find that culture of biopsy of a colon-plaque may contribute to the detection of C . difficile infection in patients with negative stool cultures.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 731 - 41
Development of a modified three-stage methane production process using food wastes; Kim SW et al.; A modified three-stage system was developed for the rapid production of methane from food wastes . The primary stage was a semianaerobic hydrolysis/acidogenic system, in which approx 4100 mg/L of volatile fatty acids (VFAs) was produced at a hydraulic retention time (HRT) of 2 d . The operation temperature and pH were 30 degrees C and 5.0-5.5, respectively . The non-degraded materials were removed through a hole at the bottom of the reactor . The secondary stage was an anaerobic acidogenic system equipped with an upflow anaerobic sludge blanket (UASB) type of fermentor . VFA was accumulated up to 6100 mg/L by the addition of Clostridium butyricum to the reactor at an HRT of 2 d . The optimum temperature and pH range were 35 degrees C and 5.0-5.5, respectively . The tertiary methanogenic stage produced CH4 and CO2 from the VFA in the UASB reactor . Methane content was 72% of the total gas volume, and the yield was 0.45-0.50 m3/kg of volatile solids at an HRT of 12 d . The operation temperature and pH were 41 degrees C and 7.6-7.9, respectively . The three-stage process exhibited an unusually high total chemical oxygen demand reduction rate up to 95% . Total nitrogen decreased to 96% and < 10 mg/L of total phosphorus remained in the final effluent.

Appl Biochem Biotechnol, 2000 Spring, 84-86, 225 - 35
Butanol production using Clostridium beijerinckii BA101 hyper-butanol producing mutant strain and recovery by pervaporation; Qureshi N et al.; Clostridium beijerinckii BA101 (mutant strain) and C . beijerinckii 8052 (wild type) were compared for substrate and butanol inhibition . The wild-type strain is more strongly inhibited by added butanol than is the mutant strain . Acetone and butanol were removed from a fed-batch reactor inoculated with C . beijerinckii BA101 by pervaporation using a silicone membrane . In the batch reactor, C . beijerinckii BA101 produced 25.3 g/L of total solvents, whereas in the fermentation-recovery experiment it produced 165.1 g/L of total solvents . Solvent productivity increased from 0.35 (batch reactor) to 0.98 g/L.h (fed-batch reactor) . The fed-batch reactor was fed with 500 g/L of glucose-based P2 medium . Acetone selectivities ranged from 2 to 10 whereas butanol selectivities ranged from 7 to 19 . Total flux varied from 26 to 31 g/m2.h.

J Clin Pharm Ther, 2000 Apr, 25(2), 101 - 9
Clostridium difficile-associated diarrhoea in hospitalised patients; Al-Eidan FA et al.; OBJECTIVE: The aim of the present study was to evaluate the incidence, risk factors and cost implications of Clostridium difficile-associated diarrhoea (CDAD) in hospitalized adult patients . METHODS: Eighty-seven hospitalized adult patients, positively identified as having CDAD, were reviewed retrospectively to determine the risk factors and cost implications of CDAD . RESULTS: The clinical manifestations, in addition to diarrhoea, included elevated temperature (= 37.8 degrees C; 42.5%), abdominal pain (63 . 2%) and leucocytosis (=12 x 109 cells/l; 52.9%) . Eight patients underwent endoscopy, and pseudomembranous colitis was confirmed in all of these patients . Nine patients died during their hospital stay . Cefotaxime and cefuroxime were the agents most commonly associated with CDAD . There was a significant difference (P < 0.001) between the sex distribution of CDAD patients and adult hospital patients (69% of CDAD patients were female vs . 52% of general adult hospital population) . Significantly (P < 0.001) more patients with CDAD were admitted from the nursing home (NH) setting . The mean age of patients with CDAD admitted from NHs (n = 19) was older than those cases admitted from the community (n = 68) by 14 years (P < 0.001) . The length of hospital stay was significantly (P < 0.001) longer for patients with CDAD (16.9 vs . 3.89 days) . No differences (P = 0.306) were found in the response times for CDAD patients treated with either oral metronidazole (n = 39) or oral vancomycin (n = 48) . The mean response time was, however, significantly longer in the CDAD patients admitted from NHs (4.2 days) compared with those admitted from the community (2.5 days), although the former patients were older and had significantly more comorbidity (P < 0.001) . The mean cost per one treated-case of CDAD (bed, laboratory requests and treatment therapy) was calculated as pound2860 . CONCLUSION: Patients admitted from NHs are at increased risk of development of CDAD; receiving cefotaxime or cefuroxime axetil (oral form), being elderly and being female are risk factors for the development of CDAD . Treatment of CDAD with oral metronidazole or oral vancomycin gives rise to similar response times and efficacy.

Eur J Biochem, 2000 Jun, 267(12), 3874 - 84
The involvement of coenzyme A esters in the dehydration of (R)-phenyllactate to (E)-cinnamate by Clostridium sporogenes; Dickert S et al.; Phenyllactate dehydratase from Clostridium sporogenes grown anaerobically on L-phenylalanine catalyses the reversible syn-dehydration of (R)-phenyllactate to (E)-cinnamate . Purification yielded a heterotrimeric enzyme complex (130 +/- 15 kDa) composed of FldA (46 kDa), FldB (43 kDa) and FldC (40 kDa) . By re-chromatography on Q-Sepharose, the major part of FldA could be separated and identified as oxygen insensitive cinnamoyl-CoA:phenyllactate CoA-transferase, whereas the transferase depleted trimeric complex retained oxygen sensitive phenyllactate dehydratase activity and contained about one {4Fe-4S} cluster . The dehydratase activity required 10 microM FAD, 0.4 mM ATP, 2.5 mM MgCl2, 0.1 mM NADH, 5 microM cinnamoyl-CoA and small amounts of cell-free extract (10 microg protein per mL) similar to that known for 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans . The N-terminus of the homogenous FldA (39 amino acids) is homologous to that of CaiB (39% sequence identity) involved in carnitine metabolism in Escherichia coli . Both enzymes are members of an emerging group of CoA-transferases which exhibit high substrate specificity but apparently do not form enzyme CoA-ester intermediates . It is concluded that dehydration of (R)-phenyllactate to (E)-cinnamate proceeds in two steps, a CoA-transfer from cinnamoyl-CoA to phenyllactate, catalysed by FldA, followed by the dehydration of phenyllactyl-CoA, catalysed by FldB and FldC, whereby the noncovalently bound prosthetic group cinnamoyl-CoA is regenerated . This demonstrates the necessity of a 2-hydroxyacyl-CoA intermediate in the dehydration of 2-hydroxyacids . The transient CoA-ester formation during the dehydration of phenyllactate resembles that during citrate cleavage catalysed by bacterial citrate lyase, which contain a derivative of acetyl-CoA covalently bound to an acyl-carrier-protein (ACP).

J Med Microbiol, 2000 Jun, 49(6), 557 - 63
Prolonged perturbations of tumour necrosis factor-alpha and interferon-gamma in mice inoculated with Clostridium piliforme; Van Andel RA et al.; Clostridium piliforme is an obligate intracellular bacterium that causes enterohepatic disease in many animal species . C . piliforme infections are commonly subclinical in laboratory rats and mice, and little is known about host regulation of disease or of the effects of C . piliforme infections on investigations that use subclinically infected animals . To assess host regulation of subclinical C . piliforme infections and the effects of those infections on laboratory mice, the expression of the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) was evaluated at 0, 1, 3, 7, 14 and 28 days after inoculation with C . piliforme . Subclinical infection was induced in weanling C . piliforme-susceptible DBA/2 or -resistant C57BL/6 mice with either a toxic or a non-toxic C . piliforme strain . Hepatic lesions and bacteria were demonstrated histologically in both mouse strains for 14 days after inoculation with the toxigenic bacterial strain, but were never demonstrated histologically following inoculation with the non-toxigenic strain . Hepatic TNF-alpha and IFN-gamma mRNA and serum protein levels were similarly elevated in both mouse strains 1 day after inoculation with both C . piliforme strains, as evaluated by reverse transcription PCR and enzyme-linked immunosorbent assays, respectively . Elevation of IFN-gamma persisted for 14 days after inoculation; TNF-alpha remained elevated at 28 days after inoculation.

Am J Kidney Dis, 2000 Jun, 35(6), 1083 - 8
Hospital-acquired infections among chronic hemodialysis patients; D'Agata EM et al.; The epidemiological characteristics of nosocomial infections among patients requiring chronic hemodialysis, a high-risk and rapidly growing population, have not been fully elucidated . During a 30-month cohort study, rates of bloodstream infections (BSIs), urinary tract infections (UTIs), pneumonia, and diarrhea caused by Clostridium difficile and the distribution of pathogens among hospitalized chronic hemodialysis patients were compared with hospitalized patients not requiring chronic hemodialysis . To identify risk factors for developing a nosocomial infection among chronic hemodialysis patients, a matched case-control study was performed . A total of 1,557 nosocomial infections were detected during 1,317 of 68,361 admissions (2%) . Of these, 47 nosocomial infections occurred in chronic hemodialysis patients during 31 of 578 admissions (5%) . Nosocomial infections were significantly more frequent among the chronic hemodialysis group (9.1/1,000 patient-days) compared with the non-chronic hemodialysis group (3 . 8/1,000 patient-days; relative risk {RR}, 2.4; 95% confidence interval {CI}, 1.8 to 3.2; P < 0.001) . UTIs were the most common nosocomial infections among chronic hemodialysis patients, accounting for 47% of all infections in this population . UTIs were significantly more common among chronic hemodialysis patients (4.2/1, 000 patient-days) compared with non-chronic hemodialysis patients (0.7/1,000 patient-days; RR, 6.2; 95% CI, 3.8 to 9.5; P < 0.001) . Among chronic hemodialysis patients, Candida spp and enterococci were the most common pathogens in contrast to coagulase-negative staphylococci and Staphylococcus aureus among patients not requiring hemodialysis . Using conditional logistic regression, a greater index of comorbidity was significantly associated with nosocomial infections among the chronic hemodialysis population (odds ratio, 3 . 6; 95% CI, 1.2 to 10.7; P = 0.02) . Chronic hemodialysis patients are at a substantially greater risk for developing a nosocomial infection compared with other hospitalized patients.

Clin Exp Dermatol, 2000 May, 25(3), 173 - 5
Botulinum A exotoxin in cosmetic dermatology; Markey AC; Botulinum A exotoxin, a neurotoxin produced by the bacterium Clostridium botulinum, is now being used by cosmetically oriented specialists for treatment of a large variety of movement associated wrinkles on the face and neck . This form of temporary chemical denervation compliments the cosmetic practitioner's armamentarium alongside resurfacing and tissue augmentation . Additionally, the use of Botulinum toxin to block sympathetic innervation of eccrine sweat glands is proving a valuable treatment of hyperhidrosis of the axillae, palms and soles.

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