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 a