Med Princ Pract, 2002 Jan-Mar, 11(1), 23 - 8 Hospital-acquired Clostridium difficile infection amongst ICU and burn patients in Kuwait; Rotimi VO et al.; OBJECTIVES: To prospectively study the prevalence of nosocomially acquired Clostridium difficile, a major cause of diarrhoea in hospitalized patients, in the intensive care units (ICUs) and burn unit (BUs) of three teaching hospitals in Kuwait . METHODS: During a 1-year prospective study, stool/rectal swabs were obtained from 344 patients admitted into the ICUs of Mubarak Hospital (ICU-1), the Kuwait Cancer Control Centre (ICU-2), and the BU of Ibn Sina Hospital . The presence of C . difficile and/or its toxin was detected by serially culturing the specimens on differential, selective and enriched media and the use of TOX-A/B test, on admission and at subsequent 1-weekly interval until discharge . RESULTS: Out of the 344 patients admitted into these units, over a study period of 1 year, only 263 (77%) were evaluable . All of them had negative stool culture/toxin on admission . Overall, 25 (9.5%) of these 263 patients acquired C . difficile during their hospitalization . Thirteen (7%) of 187 patients acquired C . difficile in ICU-1, 9 (36%) of 25 on ICU-2 and 3 (5.9%) of 51 patients in BU . Eight (32%) developed diarrhoea attributable only to C . difficile and/or toxin, and the remaining 17 (68%) were asymptomatic: none had pseudomembranous colitis . The diarrhoea in these patients was associated with antibiotic use, the main trigger-antibiotics being the third-generation cephalosporins . Acquisition occurred within 4-53 days of admission, with the majority occurring in the first 15 days . CONCLUSION: Overall, the prevalence of hospital-acquired C . difficile infection/colonization was less than 10% . The use of third-generation cephalosporins was high and was related to the development of diarrhoea . Once acquired, diarrhoea developed in about one third of C . difficile-positive cases, an indication that C . difficile infection/colonization endemic in the hospital ICUs studied is usually transmitted among the hospitalized patients. Biotechnol Bioeng, 2002 Jun 5, 78(5), 567 - 75 Arylsulfotransferase from Clostridium innocuum-A new enzyme catalyst for sulfation of phenol-containing compounds; Mozhaev VV et al.; Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554) . The enzyme has a molecular weight of 320 kDa and is composed of four subunits . Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS) . The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates . Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol% . The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost . High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides . Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions . Microsc Res Tech, 2002 Jun 15, 57(6), 432 - 40 Actin machinery of phagocytic cells: universal target for bacterial attack; Belyi IF; Uptake of microorganisms by eukaryotic cells depends on proper functioning of the actin machinery . It creates a driving force for the cell membrane deformations necessary for ingestion and killing of microbes by phagocytes . Therefore, specific alterations in the activity of the actin apparatus could be favorable for pathogenic bacteria, representing an efficient mechanism in their virulence . Such alterations are supposed to be achieved in two principle ways . One is accomplished via binding of bacterial ligands to certain surface receptors, which initiate subsequent actin cytoskeleton rearrangements . Another is to introduce cytoskeleton-targeted products directly into eukaryotic cells and in this way modulate the activity of the actin apparatus . Indeed, Legionella and some other intracellular parasites possess ligands able to stimulate certain receptors on the surface of phagocytes and possess devices suitable for translocation of effector molecules into eukaryotic cytoplasm . The results of such events could be increased uptake of these microbes and their subsequent transportation to permit multiplication in their intracellular niche . On the contrary, representatives of Clostridium and a number of other extracellular pathogens create products which penetrate eukaryotic cells and disorganize the actin cytoskeleton network, thus making uptake of these pathogens by phagocytes impossible . Med Res Rev, 2002 Jul, 22(4), 329 - 72 Bacterial protease inhibitors; Supuran CT et al.; Serine-, cysteine-, and metalloproteases are widely spread in many pathogenic bacteria, where they play critical functions related to colonization and evasion of host immune defenses, acquisition of nutrients for growth and proliferation, facilitation of dissemination, or tissue damage during infection . Since all the antibiotics used clinically at the moment share a common mechanism of action, acting as inhibitors of the bacterial cell wall biosynthesis or affecting protein synthesis on ribosomes, resistance to these pharmacological agents represents a serious medical problem, which might be resolved by using new generation of antibiotics, possessing a different mechanism of action . Bacterial protease inhibitors constitute an interesting such possibility, due to the fact that many specific as well as ubiquitous proteases have recently been characterized in some detail in both gram-positive as well as gram-negative pathogens . Few potent, specific inhibitors for such bacterial proteases have been reported at this moment except for some signal peptidase, clostripain, Clostridium histolyticum collagenase, botulinum neurotoxin, and tetanus neurotoxin inhibitors . No inhibitors of the critically important and ubiquitous AAA proteases, degP or sortase have been reported, although such compounds would presumably constitute a new class of highly effective antibiotics . This review presents the state of the art in the design of such enzyme inhibitors with potential therapeutic applications, as well as recent advances in the use of some of these proteases in therapy . Appl Microbiol Biotechnol, 2002 Jul, 59(2-3), 182 - 9 Epub 2002 May 03. Dietary-fiber-degrading enzymes from a human intestinal Clostridium and their application to oligosaccharide production from nonstarchy polysaccharides using immobilized cells; Nakajima N et al.; The secretion of nonstarchy polysaccharide-degrading enzymes from an anaerobic human intestinal bacterium, Clostridium butyricum- beijerinckii (isolated from human feces), was investigated . Growth of the bacterium was found when laminarin, konjac glucomannan, and pectic acid were added separately to the culture media as sole carbon source . The corresponding degrading enzymes for these dietary fibers, laminarinase (endo-1,3- beta-glucanase), endo-1,4-beta-mannanase, endo- and exo-pectate lyases, and pectin methylesterase, were then purified and characterized . These extracelluar enzymes, which were secreted by the bacterium in the human large intestine, were considered to contribute to digestion of the ingested dietary fibers to their oligosaccharides, following by short-chain fatty acid fermentation by the bacterium . We have developed cell immobilization techniques of the bacterium on cellulose-foam carriers that are effective for continuous production of the oligosaccharides from the dietary fibers in a fed-batch reactor system . From 9 g of pectic acid, a total of 3.96 g of 4,5-unsaturated digalacturonic acid was produced over 40 h in four 500-ml batchcultures . In the same manner, the corresponding oligosaccharides were obtained from konjac glucomannan and laminarin with average conversion rates of around 30-40%. Commun Dis Public Health, 2001 Dec, 4(4), 259 - 67 General outbreaks of infectious intestinal disease linked with red meat, England and Wales, 1992-1999; Smerdon WJ et al.; Between 1992 and 1999, 1,426 foodborne general outbreaks of infectious intestinal disease (IID) were reported to the Public Health Laboratory Service (PHLS) Communicable Disease Surveillance Centre (CDSC) . Sixteen percent were linked with the consumption of red meat . Over 5,000 people were affected, with 186 hospital admissions and nine deaths . Beef (34%) and pig meat (32%) were the most frequently implicated meat types, with lamb implicated in 11% of outbreaks . The organisms most frequently reported were Clostridium perfringens (43.4%) and salmonellas (34.3%) . During the summer, outbreaks were mainly of Salmonella spp . and attributed to the consumption of pig meat . In December, outbreaks of C . perfringens linked with beef predominated . Most outbreaks occurred as a result of food cooked on commercial catering premises (46%) . The highlight of this surveillance period is a fall in the number of outbreaks linked with foods containing red meat . This corresponds with a steady decline in red meat consumption over the last two decades, as well as a transient though marked decline in the purchase and consumption of red meat in the UK during the BSE crisis in the early to mid 1990s . As cited in the Pennington Report, further reducing the morbidity and mortality from red meat outbreaks means targeting meat production at various points along the food chain from abattoir and butchering, to cooking and holding of cooked food, especially on commercial catering premises. Diagn Microbiol Infect Dis, 2002 Jul, 43(3), 189 - 92 High prevalence of toxin A-negative toxin B-positive Clostridium difficile in hospitalized patients with gastrointestinal disease; Samra Z et al.; The incidence of Clostridium difficile toxin A-negative toxin B-positive in hospitalized patients with severe gastrointestinal disease was evaluated . Of 530 stool specimens tested in parallel by two immunoassay tests, Tox A and Tox A/B (TechLab, Inc., Blacksburg, VA), 422 produced negative results on both tests . One hundred eight specimens (20.4%) tested positive by Tox A/B assay, and only 47 of them were also positive by Tox A . The 61 specimens with discrepant results were confirmed to be positive for toxin B by tissue culture cytotoxicity assay . Furthermore, 3 of the 422 specimens that were negative by enzyme immunoassay tested positive by cytotoxicity assay . The sensitivity and specificity of the Tox A/B test were 95.3% and 100%, respectively, with negative and positive predictive values of 99.3% and 100%, respectively, and a correlation rate of 99.4% . The high prevalence (56.5%) of specimens from symptomatic patients having detectable toxin B, but undetectable toxin A emphasizes the importance of using diagnostic tests that include toxin B . Furthermore, our data support the potential role of toxin B in the pathogenesis of toxigenic Clostridium difficile. J Biol Chem, 2002 Sep 20, 277(38), 34846 - 52 Epub 2002 Jul 08. Inhibition of release of neurotransmitters from rat dorsal root ganglia by a novel conjugate of a Clostridium botulinum toxin A endopeptidase fragment and Erythrina cristagalli lectin; Duggan MJ et al.; Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types . Here we report that a catalytically active derivative (termed LH(N)/A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate . This conjugate exhibits an in vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed using in vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters . Chemical conjugates prepared between E . cristagalli lectin and either natively sourced LH(N)/A or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated . Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LH(N)/A conjugate variant . The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed with Clostridium botulinum neurotoxin . Finally, in vivo electrophysiology shows that these in vitro actions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated . These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved. Cell Microbiol, 2002 Jul, 4(7), 425 - 34 Clostridium difficile toxin B activates dual caspase-dependent and caspase-independent apoptosis in intoxicated cells; Qa'Dan M et al.; Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells . In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis . The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdB-treated HeLa cells . Caspase-3 activation was subsequently confirmed in TcdB-intoxicated HeLa cells . Interestingly, caspase inhibitor delayed TcdB-induced cell death, but did not alter the time-course of cytopathic effects . A similar effect was also observed in MCF-7 cells, which are deficient in caspase-3 activity . The time-course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1-556) . Unlike TcdB treated cells, intoxication with TcdB1-556 or expression of TcdB1-556 in a transfected cell line, did not stimulate caspase-3 activation yet cells exhibited cytopathic effects and cell death . Although TcdB1-556 treated cells did not demonstrate caspase-3 activation these cells were apoptotic as determined by differential annexin-V/propidium iodide staining and nucleosomal DNA fragmentation . These data indicate TcdB triggers caspase-independent apoptosis as a result of substrate inactivation and may evoke caspase-dependent apoptosis due to a second, yet undefined, activity of TcdB . This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells. J Ky Med Assoc, 2002 Jun, 100(6), 234 - 7 Fatal pseudomembranous colitis in a continent urinary neobladder; Mathai MG et al.; Antibiotic-associated colitis is a significant clinical problem, especially in patients hospitalized for longer than three days . Clostridium difficile is now established as the most common nosocomial enteric pathogen causing antibiotic-associated colitis . The condition rarely occurs beyond the boundaries of the large bowel, but can represent significant diagnostic and therapeutic problems if it involves bowel that is used in the creation of a diversionary reservoir such as an ileo-cecal neobladder . We present what we believe to be the first reported case of fatal pseudomembranous colitis occurring in an ileo-cecal neobladder. Microbiology, 2002 Jul, 148(Pt 7), 2245 - 53 Proteins released during high toxin production in Clostridium difficile; Mukherjee K et al.; The mechanism by which toxins A and B are released by Clostridium difficile is unknown and information about the other extracellular proteins of this bacterium is limited . The authors identified exported proteins from C . difficile strain VPI 10463 during conditions promoting high toxin production . Toxins A and B were released in a 1:1 ratio and the proportion of toxin in the extracellular fraction reached 50% during the stationary phase as compared to a proportion of <1% for typical cytoplasmic proteins, showing that toxin export was not due to bacterial lysis . A 47 kDa protein, released with similar kinetics to the toxins, was processed and showed weak similarity to the channel-forming protein TolC . Another protein released during high toxin production was unprocessed and showed similarity to XkdK encoded by the prophage PBSX in Bacillus subtilis, a protein supposedly exported via phage-specific holins . The two most abundant extracellular C . difficile proteins, found during both high and low toxin production, were processed and identified as shed S-layer proteins . As shown by N-terminal sequencing and PCR-based methods, there was a considerable sequence variation of the S-layer gene slpA in different serogroup reference strains . To conclude, C . difficile uses the classical Sec-dependent and probably also holin-like pathways to secrete a comparatively small repertoire of proteins. Wound Repair Regen, 2002 May-Jun, 10(3), 169 - 76 Recombinant human platelet-derived growth factor and transforming growth factor-beta mediated contraction of human dermal fibroblast populated lattices is inhibited by Rho/GTPase inhibitor but does not require phosphatidylinositol-3' kinase; Han YP et al.; Matrix reorganization and tissue contraction are essential for wound healing . However, the intracellular signals that mediate these processes are largely unknown . We investigated cytokine-induced signaling and its potential role in contraction of adult human dermal fibroblast populated collagen lattices . The results document that recombinant human platelet-derived growth factor-BB and transforming growth factor-1 individually stimulate contraction of fibroblast populated collagen lattices, while a combination of the two cytokines leads to increased contraction . Although recombinant human platelet-derived growth factor-BB promoted collagen contraction, it failed to stimulate phosphatidylinositol-3' kinase in the human dermal fibroblasts . An inhibitor for phosphatidylinositol 3' kinase, wortmannin, had no effect on the cytokine-mediated collagen contraction . In addition, this failed activation of phosphatidylinositol 3' kinase is consistent with absence of tyrosine phosphorylation of the platelet-derived growth factor receptor when the cells are in a collagen matrix . In contrast, tyrosine phosphorylation of the platelet-derived growth factor receptor was readily detected in the dermal fibroblasts in monolayers . We also probed the potential role of Rho/GTPase in the cytokine-mediated contraction of fibroblast populated collagen lattices . Toxin B from Clostridium difficile at picomolar concentrations blocked both recombinant human platelet-derived growth factor and transforming growth factor-5 induced contraction . Further, this inhibition was correlated with the inhibition of cell spreading in collagen, which suggests the formation of actin fibers inside the cells is essential for cytokine-mediated contraction of fibroblast populated collagen lattices . Taken together, these results imply that Rho/GTPase signaling but not phosphoinositol-3' kinase is involved in the cytokine-mediated contraction of fibroblasts populated collagen lattice . These findings suggest a potential mechanism for these signaling components during human wound contraction. BMC Biochem . 2002 Jun 25;3(1):19. Molecular cloning and tissue distribution of mammalian L-threonine 3-dehydrogenases; Edgar AJ; BACKGROUND: In mammals, L-threonine is an indispensable amino acid . The conversion of L-threonine to glycine occurs through a two-step biochemical pathway involving the enzymes L-threonine 3-dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase . The L-threonine 3-dehydrogenase enzyme has been purified and characterised, but the L-threonine 3-dehydrogenase gene has not previously been identified in mammals . RESULTS: Transcripts for L-threonine 3-dehydrogenase from both the mouse and pig are reported . The ORFs of both L-threonine dehydrogenase cDNAs encode proteins of 373 residues (41.5 kDa) and they share 80% identity . The mouse gene is located on chromosome 14, band C . The amino-terminal regions of these proteins have characteristics of a mitochondrial targeting sequence and are related to the UDP-galactose 4-epimerases, with both enzyme families having an amino-terminal NAD+ binding domain . That these cDNAs encode threonine dehydrogenases was shown, previously, by tiling 13 tryptic peptide sequences, obtained from purified L-threonine dehydrogenase isolated from porcine liver mitochondria, on to the pig ORF . These eukaryotic L-threonine dehydrogenases also have significant similarity with the prokaryote L-threonine dehydrogenase amino-terminus peptide sequence of the bacterium, Clostridium sticklandii . In murine tissues, the expression of both L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A ligase mRNAs were highest in the liver and were also present in brain, heart, kidney, liver, lung, skeletal muscle, spleen and testis . CONCLUSIONS: The first cloning of transcripts for L-threonine dehydrogenase from eukaryotic organisms are reported . However, they do not have any significant sequence homology to the well-characterised Escherichia coli L-threonine dehydrogenase. Arch Esp Urol, 2002 May, 55(4), 446 - 8 {Fulminant sepsis caused by Clostridium perfringens of urologic origin}; Elejalde Guerra JI et al.; OBJECTIVE: To present a case of fulminant sepsis caused by Clostridium perfringens of urological origin . METHODS: An 81-year-old diabetic female (the only factor of immunodepression) presented complicated renal colic two days later with fulminant and fatal sepsis caused by Clostridium perfringens with signs of disseminated intravascular coagulation . RESULTS: The patient died one hour after the presentation of disseminated intravascular coagulation despite attempts to resuscitate the patient in the emergency department . Due to the fulminant course of the condition, it was not possible to demonstrate the presence of massive intravascular hemolysis characteristic of these conditions . Blood cultures obtained immediately after the patient died were positive for Clostridium perfringens . CONCLUSIONS: Sepsis is a possible complication of infection from Clostridium perfringens . It is more frequent in immune-depressed patients and carries a high mortality despite medical and surgical treatment . Although it is not the most frequent, the genitourinary tract is a known portal of entry that should not be forgotten as in the case described herein. Circulation, 2002 Jul 2, 106(1), 57 - 62 Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase; Takemoto M et al.; BACKGROUND: Hypoxia-induced pulmonary hypertension is a major cause of morbidity and mortality . Hypoxia induces pulmonary vasoconstriction, in part, by decreasing endothelial nitric oxide synthase (eNOS) expression . The mechanism by which hypoxia decreases eNOS expression is not known but may involve Rho-kinase-induced actin cytoskeletal changes in vascular endothelial cells . METHODS AND RESULTS: To determine whether hypoxia regulates eNOS expression through Rho-kinase, we exposed human saphenous and pulmonary artery endothelial cells to hypoxia (3% O2) with and without a Rho-kinase inhibitor, hydroxyfasudil (0.1 to 100 micromol/L), for various durations (0 to 48 hours) . Hypoxia increased Rho-kinase expression and activity by 50% and 74%, decreased eNOS mRNA and protein expression by 66+/-3% and 57+/-5%, and inhibited eNOS activity by 48+/-9% . All of these effects of hypoxia on eNOS were reversed by cotreatment with hydroxyfasudil . Furthermore, inhibition of Rho by Clostridium botulinum C3 transferase or Rho-kinase by overexpression of dominant-negative Rho-kinase reversed hypoxia-induced decrease in eNOS expression . Indeed, disruption of the actin cytoskeleton, the downstream target of Rho-kinase, by cytochalasin D also upregulated eNOS expression . Hypoxia reduced eNOS mRNA half-life from 22+/-2 to 13+/-2 hours, which was reversed by cotreatment with hydroxyfasudil . However, neither hypoxia nor hydroxyfasudil had any effects on eNOS gene transcription . CONCLUSIONS: These results indicate that hypoxia-induced decrease in eNOS expression is mediated by Rho-kinase and suggest that Rho-kinase inhibitors may have therapeutic benefits in patients with hypoxia-induced pulmonary hypertension. Clin Diagn Lab Immunol, 2002 Jul, 9(4), 872 - 6 Comparison of a multiplex flow cytometric assay with enzyme-linked immunosorbent assay for auantitation of antibodies to tetanus, diphtheria, and Haemophilus influenzae Type b; Pickering JW et al.; We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system . A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib . The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes . By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (> or = 0.1 IU/ml) . For Dip, 81.5% of the samples had protective antibody levels (> or = 0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex . Protective levels (> or = 1.0 microg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex . The correlations (R(2)) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively . There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration . Both methods detected strong postvaccination responses . The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time. Biochemistry, 2002 Jul 9, 41(27), 8767 - 76 Identification of a novel pyridoxal 5'-phosphate binding site in adenosylcobalamin-dependent lysine 5,6-aminomutase from Porphyromonas gingivalis; Tang KH et al.; Lysine 5,6-aminomutase (5,6-LAM) catalyzes the interconversion of D-lysine with 2,5-diaminohexanoate and of L-beta-lysine with 3,5-diaminohexanoate . The coenzymes for 5,6-LAM are adenosylcobalamin (AdoCbl) and pyridoxal 5'-phosphate (PLP) . In the proposed chemical mechanism, AdoCbl initiates the formation of substrate radicals, and PLP facilitates the radical rearrangement by forming an external aldimine linkage with the epsilon-amino group of a substrate, either D-lysine or L-beta-lysine . In the resting enzyme, an internal aldimine between PLP and an essential lysine in the active site facilitates productive PLP binding and catalysis . We present here biochemical, biophysical, and site-directed mutagenesis experiments, which document the existence of an essential lysine residue in the active site of 5,6-LAM from Porphyromonas gingivalis . Reduction of 5,6-LAM with NaBH(4) rapidly inactivates the enzyme and shifts the electronic absorption band from 420 to 325 nm . This is characteristic of the reduction of an aldimine linkage between the carbonyl group of PLP and the epsilon-amino group of a lysine residue . The reduced peptide was identified by Q-TOF/MS and further confirmed by Q-TOF/MS/MS sequencing . We show that lysine 144 in the small subunit of 5,6-LAM is the essential lysine residue . Lysine 144(beta) is separated by only 11 amino acids from histidine 133(beta), which forms a part of the "base-off"-AdoCbl binding motif . The sequence of the novel PLP-binding motif is conserved in 5,6-LAM from Clostridium sticklandii and P . gingivalis, and it is distinct from all known PLP-binding motifs . Mutation of lysine 144(beta) to glutamine led to K144Q(beta)-5,6-LAM, which displayed no enzymatic activity and no absorption band corresponding to an internal PLP-aldamine . In summary, we introduce a novel PLP-binding motif, the first to be discovered in an AdoCbl-dependent enzyme. J Clin Microbiol, 2002 Jul, 40(7), 2452 - 8 Clostridium difficile genotyping based on slpA variable region in S-layer gene sequence: an alternative to serotyping; Karjalainen T et al.; Recent investigations of Clostridium difficile cell wall components have revealed the presence of an S-layer encoded by the slpA gene . The aim of this study was to determine whether slpA genotyping can be used as an alternative to serotyping . The variable regions of slpA were amplified by PCR from serogroup reference strains and various clinical isolates chosen randomly . Amplified products were analyzed after restriction enzyme digestion and DNA sequencing . The sequences of the variable region of the SlpA protein were found to be strictly identical within a given serogroup but divergent between serogroups . These preliminary results suggest that PCR-restriction fragment length polymorphism, in conjunction with DNA sequencing of the slpA variable region, could constitute an alternative typing method for determining C . difficile serotypes. Appl Environ Microbiol, 2002 Jul, 68(7), 3496 - 501 Influence of the transposition of the thermostabilizing domain of Clostridium thermocellum xylanase (XynX) on xylan binding and thermostabilization; Shin ES et al.; A xylanase gene, xynX, of Clostridium thermocellum had one thermostabilizing domain (TSD) between the signal peptide sequence and the catalytic domain (CD) . The TSD of a truncated xylanase gene, xynX'(TSD-CD), was transpositioned from the N terminus to the C terminus of the CD by overlapping PCRs, and a modified product, xynX'(CD-TSD), was constructed . XynX'(TSD-CD) had a higher optimum temperature (70 degrees C versus 65 degrees C) and was more thermostable (residual activity of 68% versus 46% after a 20-min preincubation at 70 degrees C) than the one without the TSD, XynX'(CD) . However, the domain-transpositioned enzyme, XynX'(CD-TSD), showed a lower optimum temperature (30 degrees C) and thermostability (20%) than XynX'(CD) . Both XynX'(TSD-CD) and XynX'(CD-TSD) showed significantly higher binding capacity toward xylan than XynX'(CD), and the domain transposition did not cause any change in the binding ability . XynX'(TSD-CD) and XynX'(CD-TSD) also showed considerable binding to lichenan but not to carboxymethyl cellulose and laminarin . XynX'(TSD-CD) and XynX'(CD-TSD) had higher activities for insoluble xylan than XynX'(CD), while XynX'(CD) was more active against soluble xylan than XynX'(TSD-CD) and XynX'(CD-TSD) . These results indicate that the TSD of XynX has dual functions, xylan binding and thermostabilization, and the domain should also be classified as a xylan-binding domain (XBD) . The binding capacity of the XBD was not affected by domain transpositioning within the gene. Br J Nutr, 2002 May, 87 Suppl 2, S153 - 7 Nutritional advantages of probiotics and prebiotics; Marteau P et al.; The potential 'nutritional advantages' of probiotics and prebiotics consist of preventive, and sometimes curative, effects against certain diseases . The evidence supporting such advantages, which requires randomised controlled trials and consistency of results from study to study, is rapidly increasing . This article summarizes the effects against diseases of intestinal origin . There is a high level of evidence for positive effects of some prebiotics to alleviate constipation and treat hepatic encephalopathy . Interesting aspects, but with a lower level of evidence at the present time, include prevention of colon cancer, intestinal infection, and recurrence of inflammatory bowel disease . There is a high level of evidence for positive effects of some probiotics in the alleviation of lactose intolerance, antibiotic-associated intestinal disorders and gastroenteritis . Evidence is rapidly growing regarding the prevention of recurrence of inflammatory bowel diseases . Positive trials have suggested preventive effects against intestinal colonization with specific gut pathogens including Clostridium difficile and Helicobacter pylori. J Mol Biol, 2002 May 17, 318(5), 1417 - 32 The 3.0 A resolution crystal structure of glycosomal pyruvate phosphate dikinase from Trypanosoma brucei; Cosenza LW et al.; The crystal structure of the glycosomal enzyme pyruvate phosphate dikinase from the African protozoan parasite Trypanosoma brucei has been solved to 3.0 A resolution by molecular replacement . The search model was the 2.3 A resolution structure of the Clostridium symbiosum enzyme . Due to different relative orientations of the domains and sub-domains in the two structures, molecular replacement could be achieved only by positioning these elements (four bodies altogether) sequentially in the asymmetric unit of the P2(1)2(1)2 crystal, which contains one pyruvate phosphate dikinase (PPDK) subunit . The refined model, comprising 898 residues and 188 solvent molecules per subunit, has a crystallographic residual index Rf = 0.245 (cross-validation residual index Rfree = 0.291) and displays satisfactory stereochemistry . Eight regions, comprising a total of 69 amino acid residues at the surface of the molecule, are disordered in this crystal form . The PPDK subunits are arranged around the crystallographic 2-fold axis as a dimer, analogous to that observed in the C . symbiosum enzyme . Comparison of the two structures was carried out by superposition of the models . Although the fold of each domain or sub-domain is similar, the relative orientations of these constitutive elements are different in the two structures . The trypanosome enzyme is more "bent" than the bacterial enzyme, with bending increasing from the center of the molecule (close to the molecular 2-fold axis) towards the periphery where the N-terminal domain is located . As a consequence of this increased bending and of the differences in relative positions of subdomains, the nucleotide-binding cleft in the amino-terminal domain is wider in T . brucei PPDK: the N-terminal fragment of the amino-terminal domain is distant from the catalytic, phospho-transfer competent histidine 482 (ca 10 A away) . Our observations suggest that the requirements of domain motion during enzyme catalysis might include widening of the nucleotide-binding cleft to allow access and departure of the AMP or ATP ligand. J Bacteriol, 2002 Jul, 184(14), 3886 - 97 Patterns of sequence conservation in the S-Layer proteins and related sequences in Clostridium difficile; Calabi E et al.; Clostridium difficile is the etiological agent of antibiotic-associated diarrhea . Among the factors that may play a role in infection are S-layer proteins (SLPs) . Previous work has shown these to consist mainly of two components, resulting from the cleavage of a precursor encoded by the slpA gene . The high-molecular-weight (MW) subunit is related both to amidases from B . subtilis and to at least another 28 gene products in C . difficile strain 630 . To gain insight into the functions of the SLPs and related proteins, we have further investigated the pattern of variability both at the slpA locus and at six nearby paralogs . Sequencing of the slpA gene from an S-layer group II strain and a variant S-layer group strain confirms a high degree of divergence in the low-MW SLP, which may result from diversifying selection . A highly conserved motif, however, is found at the C terminus in all low-MW subunits and may be essential for SlpA precursor cleavage . In strain 167, a variant cleavage product is present, suggesting a secondary processing site . Southern blotting analysis shows slpA-like open reading frames (ORFs) 2 to 7 to be conserved in all nine strains tested, with one exception: ORF2, which encodes a 66-kDa polypeptide coextracted at low pH with the main SLPs in strain 630, may be partially deleted in strain 167 . Polymorphism within the slpA-ORF7 cluster may be more pronounced in the region proximal to the slpA gene . Unexpectedly, a high-MW subunit probe cross hybridizes to sequences outside the slpA locus, which appear to vary in number in different strains. Rev Lat Am Enfermagem, 2001 Nov-Dec, 9(6), 69 - 75 {Tetanus in the geriatric population: is it a collective health problem?}; Pagliuca LM et al.; Tetanus is an infectious non-contagious disease caused by the bacillus Clostridium tetani, which penetrates in the organism through wounds . Psychomotor dysfunction facilitates accidents among elderly people and vaccinal coverage is low in this population, thus contributing to high lethality . This study aimed at reflecting on the situation faced by elderly people in relation to tetanus in the perspective of Collective Health . It is a Case Study conducted with two elderly males who had accidental tetanus and were hospitalized in a hospital in the municipality of Fortaleza . Data collection took place from March to April, 1998 . The analysis showed the absence of vaccinal coverage as well as of the implementation of emergency prophylaxis . The two patients' conditions developed to death, which confirmed the high mortality due to tetanus in this age group . The critical reflection pointed out the urgency of a collective health approach. Int J Food Microbiol, 2002 Jul 25, 77(1-2), 55 - 9 A semi-quantitative seafood safety risk assessment; Sumner J et al.; As part of a semi-quantitative risk assessment of 10 seafood hazard/product combinations, a risk assessment tool was used to generate a Risk Ranking . The tool is in a spreadsheet software format and provides a risk estimate, which is scaled between 0 and 100, where 0 represents no risk and 100 represents all meals containing a lethal dose of the hazard . A full description of the tool is contained in Ross and Sumner (this issue) . Based on their ranking, seafoods in Australia fell into three risk categories . Hazard/product pairs with ranking < 32 included mercury poisoning (Relative Risk = 24), Clostridium botulinum in canned fish (RR = 25), or in vacuum-packed cold-smoked fish (RR = 28), parasites in sushi/sashimi (RR = 31), viruses in shellfish from uncontaminated waters, (RR = 31), enteric bacteria in imported cooked shrimp (RR = 31) and algal biotoxins from controlled waters (RR = 31) . It is noted that there have been no documented cases of food-borne illness from any of the above hazard/product pairings in Australia . Those with rankings 32-48 included Vibrio parahaemolyticus in cooked prawns (RR = 37), V . cholerae in cooked prawns (RR = 37), Listeria monocytogenes in cold-smoked seafoods (RR = 39), scombrotoxicosis (RR = 40), V . vulnificus in oysters (RR = 41), ciguatera in the general Australian population (RR = 45), L . monocytogenes in susceptible (RR = 45) and extremely susceptible populations (RR = 47) and enteric bacteria in imported cooked shrimp eaten by vulnerable consumers (RR = 48) . Almost all the hazard/product pairs in this category have caused the outbreaks of food poisoning in Australasia . Those hazard/product pairs with rankings >48 included ciguatera from recreational fishing in susceptible areas (RR = 60), viruses in shellfish from contaminated waters (RR = 67) and algal biotoxins from uncontrolled waters in an algal event (RR = 72) . There have been significant (>100 cases) food poisoning incidents involving viruses and biotoxins in shellfish, while ciguatera poisoning is prevalent among coastal communities in Australia's warmer waters. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2000, 32(4), 322 - 326 Cloning and Expression of Tetanus Toxin Fragment C in E.coli; He HJ et al.; The fragment C of tetanus toxin was amplified from Clostridium tetani DNA by PCR . This fragment was cloned into expression vector pET-28a(+),under the control of the T7 promoter . Expression of this plasmid in E.coli resulted in the production of a protein consisting of 6xHis of the vector fused to the N-terminal 451 amino acids of tetanus toxin . After induction with 1 mmol/L IPTG, TTC was expressed in E.coli BL21(DE3) . The protein product accounted for 8.2% of the bacteria total protein in soluble form, SDS-PAGE and Western blot analysis of TTC recombinant protein confirmed this result . The expression products were also purified by Ni(2+)-IDA-Sephrose 6B column . Immunization of mice with rTTC resulted in the production of antibodies that were able to protect mice against a challenge with tetanus toxin furthermore, rTTC in vivo appeared to be able to undergo retrograde axonal transport. Am J Med Sci, 2002 Jun, 323(6), 326 - 40 Microbiological, biological, and chemical weapons of warfare and terrorism; Greenfield RA et al.; Microbiological, biological, and chemical toxins have been employed in warfare and in terrorist attacks . In this era, it is imperative that health care providers are familiar with illnesses caused by these agents . Botulinum toxin produces a descending flaccid paralysis . Staphylococcal enterotoxin B produces a syndrome of fever, nausea, and diarrhea and may produce a pulmonary syndrome if aerosolized . Clostridium perfringens epsilon-toxin could possibly be aerosolized to produce acute pulmonary edema . Ricin intoxication can manifest as gastrointestinal hemorrhage after ingestion, severe muscle necrosis after intramuscular injection, and acute pulmonary disease after inhalation . Nerve agents inhibit acetylcholinesterase and thus produce symptoms of increased cholinergic activity . Ammonia, chlorine, vinyl chloride, phosgene, sulfur dioxide, and nitrogen dioxide, tear gas, and zinc chloride primarily injure the upper respiratory tract and the lungs . Sulfur mustard (and nitrogen mustard) are vesicant and alkylating agents . Cyanide poisoning ranges from sudden-onset headache and drowsiness to severe hypoxemia, cardiovascular collapse, and death . Health care providers should be familiar with the medical consequences of toxin exposure, and understand the pathophysiology and management of resulting illness. Aust Vet J, 2002 May, 80(5), 280 - 1 Malignant oedema associated with blood-sampling in sheep; Morris WE et al.; Malignant oedema is a fatal disease of several animal species, produced by one or more members of the Clostridium genus . We report here a case of malignant oedema in a 1-year-old Friesian sheep after a blood sample was collected from the jugular vein . Clostridium septicum and Clostridium sordellii were isolated from the lesions and also demonstrated by a fluorescent antibody test . This report stresses the need for maintaining a clean environment for animals and for strict hygienic measures during procedures that generate wounds, together with immunity acquired by proper vaccination, for prevention of malignant oedema. J Ind Microbiol Biotechnol, 2002 Feb, 28(2), 118 - 26 Molecular characterization and utilization of the CAK1 filamentous viruslike particle derived from Clostridium beijerinckii; Li Y et al.; An examination of the replication origin and stability determinant associated with the CAK1 filamentous viruslike particle recovered from Clostridium beijerinckii NCIMB 6444 was carried out . Seven deletion derivatives, pCKE, pCEP1, pDT5, pCKP, pDTH102, pYL102E and pYL102, were constructed and transformed into C . beijerinckii NCIMB 8052 . The successful transformation of pCKE, pDT5, pCKP, pDTH102, pYL102E and pYL102 into C . beijerinckii 8052, together with the corresponding recovery of single-stranded DNA from Escherichia coli indicated that the double- and single-stranded replication origins are located on a 0.4-kb CAK1 DNA fragment . Sequence analysis of the putative 0.4-kb replication origin region of CAK1 reveals a nick site containing 22 base pairs that has homology with plasmids pC194 and pUB110 and suggests the presence of a 2.0-kb DNA region involved in stability . The putative Rep protein of CAK1 contains three conserved motifs and three essential residues of the catalytic site in agreement with Rep proteins associated with the pC194 family . The utility of the developed CAK1-derived phagemid designated pYL102E was evaluated by using it to examine heterologous expression of: (1) the manA gene derived from Thermoanaerobacterium polysaccharolyticum in E . coli and C . beijerinckii NCIMB 8052 and (2) the sol operon derived from Clostridium acetobutylicum DSM 792 in C . beijerinckii SA-2. Microb Pathog, 2002 May, 32(5), 219 - 25 Inhibition of in vitro cell adherence of Clostridium difficile by Saccharomyces boulardii; Tasteyre A et al.; The influence on the adherence of Clostridium difficile to Vero cells of the yeast Saccharomyces boulardii, the yeast fractions (cytoplasm and cell wall) and the culture supernatant was investigated in vitro . C . difficile adherence was significantly inhibited when bacteria were pre-incubated with the whole yeast and the cell wall fraction; this adherence inhibition was dose-dependent . The cell wall fraction also acts upon the target cultured cells inasmuch as the level of adherence was significantly decreased when Vero cells were preincubated with it . The same experiments carried out in the presence of an inhibitor of serine proteases resulted in no inhibition of bacterial adherence . These results suggest that the yeast could inhibit adherence of C . difficile to cells thanks to its proteolytic activity but also through steric hindrance . Arch Microbiol, 2002 Jul, 178(1), 8 - 12 Epub 2002 Apr 16. Isolation of an anaerobic intestinal bacterium capable of cleaving the C-ring of the isoflavonoid daidzein; Hur HG et al.; Colonic bacteria were screened for bacteria involved in the conversion of phytoestrogens . A gram-positive anaerobic bacterium, strain HGH 136, capable of conversion of the isoflavonoid daidzein, was isolated and identified as a Clostridium sp . The bacterium cleaved the C-ring of daidzein to produce O-demethylangolensin ( O-Dma) . This compound was identified by comparison of the HPLC retention time and UV spectrum of the metabolite with chemically synthesized O-Dma . The identity of the metabolite was confirmed by liquid chromatography-mass spectrometry and NMR using synthetic O-Dma as a standard . The bacterium incubated with synthetic dihydrodaidzein also produced O-Dma . After 3 days of incubation, 28% of added daidzein and 12% of added dihydrodaidzein were converted to O-Dma . This is the first study in which an anaerobic bacterium involved in the ring cleavage of daidzein to produce O-Dma has been identified. Naunyn Schmiedebergs Arch Pharmacol, 2002 Jun, 365(6), 468 - 76 Epub 2002 Apr 09. Signalling components involved in the coupling of alpha 1-adrenoceptors to phospholipase D in neonatal rat cardiac myocytes; Gosau N et al.; Activation of phospholipase D (PLD) is assumed to be one major pathway by which alpha(1)-adrenoceptors (alpha(1)ARs) induce hypertrophic responses in cardiac myocytes . Heterotrimeric G proteins, protein kinase C (PKC) isoforms, protein tyrosine kinases, monomeric GTPases of the ADP-ribosylation factor (ARF) and Rho families, and as important cofactor phosphatidylinositol 4,5-bisphosphate (PIP(2)) seem to participate in the G protein-coupled receptor dependent regulation of PLD . We therefore studied the role of these components in the coupling of alpha(1)ARs to PLD in neonatal rat cardiac myocytes (NRCM) . Stimulation of alpha(1)ARs, most likely of the alpha(1A) subtype, by noradrenaline increased PLD activity three- to fourfold concomitant with the stimulation of phospholipase C (PLC) . In contrast, the partial agonist phenylephrine stimulated PLC, but failed to increase PLD activity . The PLC and PLD responses were pertussis toxin insensitive and treatment of the cells with the G(q)-activating toxin of Pasteurella multocida stimulated both phospholipases about fourfold . Over-expression of the G(q)-and G(i)-type-specific regulator of G protein signalling RGS4 blunted alpha(1)AR-induced PLC and PLD stimulation . Ro 31-8220, known to inhibit Ca(2+)-dependent and -independent PKC isoforms, strongly inhibited PLD activity, whereas Go 6976, known to inhibit preferentially Ca(2+)-dependent PKC isozymes, was without effect . The ARF signalling inhibitor brefeldin A, protein tyrosine kinase inhibitors and the Rho-inactivating toxin B of Clostridium difficile blunted alpha(1)AR-induced PLD stimulation and largely reduced the cellular PIP(2) content . In membranes of toxin B-treated NCRM, PLD activity was similarly reduced, but was fully restored by addition of exogenous PIP(2) . We conclude, that alpha(1A)ARs stimulate PLD activity via a G(q/11)-PLCbeta-novel PKC isoform-dependent pathway in NRCM . ARF and Rho GTPases as well as protein tyrosine kinases contribute to PLD stimulation in NRCM, most likely by regulating the supply of PIP(2). Med Sci Monit, 2002 Jun, 8(6), RA119 - 27 Hydrolysis of cortex peptidoglycan during bacterial spore germination; Makino S et al.; Despite the most extreme dormancy and resistance properties among living systems, bacterial endospores retain an alert sensory mechanism to respond to the germinants and initiate germination . Although the molecular mechanism of the germination process is not completely described, current progress in the studies on the enzymes involved in the process gave us a somewhat clearer picture of the process of spore peptidoglycan (cortex) hydrolysis, a major biochemical event in germination . Germination-specific cortex-lytic enzymes require muramic acid d-lactam in their substrates . At least two types of enzymes are involved in the germination process: a spore cortex-lytic enzyme (SCLE) and a cortical fragment-lytic enzyme (CFLE) . Except for their peptidoglycan-binding regions, the primary structures of SCLE and CFLE vary according species . Both enzymes differ in their hydrolytic bond-specificities and recognition of the substrates morphology . SCLE appears to initiate germination by uncrosslinking the intract cortex, and the CFLE further degrades the polysaccharide moiety of the SCLE-modified cortex . In vivo CFLE activity is likely regulated by its requirement for partially un-crosslinked cortex, while SCLE requires activation process . Clostridium perfringens SCLE is activated by a germination-specific serine protease during germination, but the activation mechanism of SCLE in Bacillus species is unknown . Cortex-lytic enzymes are expressed at the early stage of sporulation but the compartment of expression depends on proteins . However, all enzymes are located outside the cortex layer in dormant spores, suggesting that the hydrolysis process initiates at the exterior side of the cortex . The assembly of the germination apparatus is also discussed. FEBS Lett, 2002 Apr 24, 517(1-3), 261 - 6 Endothelial Rho signaling is required for monocyte transendothelial migration; Strey A et al.; Bacterial toxins affecting Rho activity in microvascular endothelial cells were employed to elucidate whether endothelial Rho participates in regulating the migration of monocytes across monolayers of cultured endothelial cells . Inactivation of Rho by the Clostridium C3 exoenzyme resulted in an increased adhesion of peripheral blood monocytes to the endothelium and a decreased rate of transendothelial monocyte migration . Cytotoxic necrotizing factor 1-mediated activation of endothelial Rho also reduced the rate of monocyte transmigration, but did not affect monocyte-endothelium adhesion . Thus, efficient leukocyte extravasation requires Rho signaling not only within the migrating leukocytes but also within the endothelial lining of the vessel wall. J Bacteriol, 2002 Jul, 184(13), 3586 - 97 Northern, morphological, and fermentation analysis of spo0A inactivation and overexpression in Clostridium acetobutylicum ATCC 824; Harris LM et al.; The Clostridium acetobutylicum ATCC 824 spo0A gene was cloned, and two recombinant strains were generated, an spo0A inactivation strain (SKO1) and an spo0A overexpression strain {824(pMPSOA)} . SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration--a technique never used before with solventogenic clostridia . SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain . After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate . SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores . No spo0A transcripts were detected in SKO1, while transcription of two solvent formation operons (aad-ctfA-ctfB and adc; both containing 0A boxes in their promoter regions) was limited . Strain 824(pMSPOA) produced higher butanol concentrations than the control strain {824(pIMP1)} and dramatically elevated spo0A transcript levels and displayed a bimodal pattern of spo0A transcription similar to that of B . subtilis . Microscopic studies indicated that sporulation was both enhanced and accelerated due to spo0A overexpression compared to that of both the 824(pIMP1) and parental strains . Consistent with that, expression of the key solvent formation genes (aad-ctfA-ctfB and adc) and three sporulation-specific genes (spoIIGA, sigE, and sigG) was observed earlier in strain 824(pMSPOA) than in the plasmid control . These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production . Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production. Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 921 - 5 Clostridium lactatifermen tans sp . nov., a lactate-fermenting anaerobe isolated from the caeca of a chicken; van der Wielen PW et al.; An obligately anaerobic, lactate-fermenting bacterium (strain G17T) was isolated from the caeca of a 31-day-old chicken . Grown at neutral pH, cells were rod-shaped with tapered ends and showed no motility and no spore formation . Electron microscopy showed that the cell walls had a gram-positive structure . The DNA G+C content was 44.6 mol % . Based on 16S rDNA sequence analysis, strain G17T was considered to belong to the low-G+C-content gram-positive bacteria of cluster XIV subgroup b and most closely related to Clostridium propionicum (93.5%) and Clostridium neopropionicum (93.5%) . The optimum temperature for growth was 41 degrees C and the optimum pH was pH 6.4-7.3 . The optimum temperature of 41 degrees C suggests that strain G17T might have become adapted to the body temperature of chickens . Strain G17T was able to grow on a variety of organic compounds . Most of these compounds were converted to acetate, propionate and traces of butyrate and isovalerate . In media with mixtures of substrates, lactate was degraded by strain G17T before the other substrates . This indicates that strain G17T might be important in the fermentation of lactate in the caeca of chickens . Based on its physiological and phylogenetic characteristics, it is proposed that strain G17T should be assigned to the genus Clostridium as a novel species, Clostridium lactatifermentans sp . nov. Int J Syst Evol Microbiol, 2002 May, 52(Pt 3), 801 - 7 Reclassification of Clostridium hydroxybenzoicum as Sedimentibacter hydroxybenzoicus gen . nov., comb . nov., and description of Sedimentibacter saalensis sp . nov.; Breitenstein A et al.; Strain ZF2T, isolated from freshwater sediment, is a motile, rod-shaped, gram-positive, endospore-forming, amino acid- and pyruvate-utilizing, anaerobic bacterium . It requires yeast extract for growth . Carbohydrates are not utilized . The optimal temperature and pH for growth are 37 degrees C and 6.8-7.3, respectively . The G+C content of the DNA is 34.0 mol % . A 16S rDNA sequence analysis of strain ZF2T revealed that the highest similarity (94.4%) was shared with Clostridium hydroxybenzoicum JW/Z-1T . Strain ZF2T, however, was not able to carboxylate phenol or to decarboxylate 4-hydroxybenzoate, which are characteristic properties of strain JW/Z-1T . The degree of 16S rDNA relatedness, together with the physiological and chemotaxonomic properties, suggest that strain ZF2T represents a novel species that is clearly distinct from Clostridium hydroxybenzoicum JW/Z-1T . In a phylogenetic dendrogram, both strains form a separate cluster that is peripherally associated with the Peptostreptococcus group (cluster XIII) of the clostridia and the lineage of Helcococcus kunzii . Strains ZF2T and JW/Z-1T show a somewhat deeper branching from the cluster XII clostridia Clostridium purinolyticum and Clostridium acidiurici . The latter strains possessed the closest 16S rDNA similarity (between 88.4 and 90.7%), but were clearly separated by phenotypic markers . Therefore, a new genus, Sedimentibacter gen . nov., is described, comprising Sedimentibacter hydroxybenzoicus gen . nov., comb . nov., as the type species of the genus, with JW/Z-1T (= ATCC 51151T = DSM 7310T) as the type strain, and the novel species Sedimentibacter saalensis sp . nov., with strain ZF2T (= DSM 13558T = ATCC BAA-283T) as the type strain. FEMS Microbiol Lett, 2002 May 21, 211(1), 65 - 70 Energetics and kinetics of lactate fermentation to acetate and propionate via methylmalonyl-CoA or acrylyl-CoA; Seeliger S et al.; Fermentation balances and growth yields were determined with various bacteria fermenting lactate to acetate plus propionate either via methylmalonyl-CoA or via acrylyl-CoA . All strains fermented lactate to acetate plus propionate at approximately a 1:2 ratio . Growth yields of Propionibacterium freudenreichii were more than twice as high as those of Clostridium homopropionicum or Veillonella parvula . Hydrogen was formed as a side product to a significant extent only by V . parvula and Pelobacter propionicus; the latter formed hydrogen preferentially when using ethanol as substrate . Acrylyl-CoA reductase of C . homopropionicum and Clostridium neopropionicum was found nearly exclusively in the cytoplasm thus confirming that this reduction step is unlikely to be involved in energy conservation . C . homopropionicum exhibited higher K(S) and higher micro(max) values, as well as higher specific substrate turnover rates than P . freudenreichii . The results allow us to conclude that C . homopropionicum using the acrylyl-CoA pathway with low growth yield obtains its specific competitive advantage compared to P . freudenreichii not through higher substrate affinity or metabolic shift toward enhanced acetate-plus-hydrogen formation but through faster specific substrate turnover. J Mol Biol, 2002 May 31, 319(2), 275 - 81 Crystal structure of the C . perfringens alpha-toxin with the active site closed by a flexible loop region; Eaton JT et al.; Clostridium perfringens biotype A strains are the causative agents of gas-gangrene in man and are also implicated as etiological agents in sudden death syndrome in young domestic livestock . The main virulence factor produced by these strains is a zinc-dependent, phosphatidylcholine-preferring phospholipase C (alpha-toxin) . The crystal structure of alpha-toxin, at pH 7.5, with the active site open and therefore accessible to substrate has previously been reported, as has calcium-binding to the C-terminal domain of the enzyme at pH 4.7 . Here we focus on conformation changes in the N-terminal domain of alpha-toxin in crystals grown at acidic pH . These changes result in both the obscuring of the toxin active site and the loss of one of three zinc ions from it . Additionally, this "closed" form contains a small alpha helix, not present in the open structure, which hydrogen bonds to both the N and C-terminal domains . In conjunction with the previously reported findings that alpha-toxin can exist in active and inactive forms and that Thr74Ile and Phe69Cys substitutions markedly reduced the haemolytic activity of the enzyme, our work suggests that these loop conformations play a critical role in the activity of the toxin . Annu Rev Biochem, 2002, 71, 1 - 16 Epub 2001 Nov 09. Discoveries of vitamin B12 and selenium enzymes; Stadtman TC; My undergraduate education at Cornell University was followed by graduate studies on methane fermentations under the guidance of H.A . Barker at the University of California, Berkeley . My Ph.D . degree was granted in June 1949 . Two anaerobic microorganisms isolated from the mud flats of San Francisco Bay served as sources of biochemical research material for later studies at the National Institutes of Health in Bethesda . These organisms, Methanococcus vannielii and Clostridium sticklandii, proved to be especially rich sources of selenium-dependent enzymes and seleno-tRNAs . New B12 coenzyme-dependent enzymes that catalyzed intermediate steps in the anaerobic conversion of lysine to fatty acids and ammonia were isolated from C . sticklandii and characterized . My research efforts since 1970 have dealt primarily with various aspects of selenium biochemistry . We have shown that selenium is an essential constituent of several enzymes in prokaryotes . Se is present in these either as a selenocysteine residue in the protein or alternatively, in a few molybdoenzymes, as a component of a bound cofactor . Recent studies with a human adenocarcinoma cell line led to the unexpected discovery that selenocysteine occurs in mammalian thioredoxin reductase . The selenium located in a redox center of this enzyme is essential for catalytic activity. Ann Intern Med, 2002 Jun 4, 136(11), 834 - 44 The commonality of risk factors for nosocomial colonization and infection with antimicrobial-resistant Staphylococcus aureus, enterococcus, gram-negative bacilli, Clostridium difficile, and Candida; Safdar N et al.; Recent years have witnessed a rapidly growing crisis in antimicrobial resistance, especially among microorganisms that cause nosocomial infection . To better understand common risk factors among multiresistant organisms, this review explores risk factors for nosocomial infection with methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococcus, Clostridium difficile, extended-spectrum beta-lactamase-producing gram-negative bacilli, and Candida . This review comprises data from 74 published studies; 53 (71%) were retrospective studies and addressed few risk factors or did not quantify risk . The analysis shows impressive commonality of risk factors across these diverse multiresistant organisms: advanced age; underlying diseases and severity of illness; inter-institutional transfer of the patient, especially from a nursing home; prolonged hospitalization; gastrointestinal surgery or transplantation; exposure to invasive devices of all types, especially central venous catheters; and exposure to antimicrobial drugs, especially cephalosporins . More restricted use of antibiotics, especially cephalosporins, and strategies to prevent medical device-related infection and cross-infection in the hospital would yield benefit with all types of resistant organisms . Preemptive isolation of all patients with risk factors for infection by resistant organisms would very likely reduce secondary spread within the hospital . Conversely, programs that focus on only one organism or one antimicrobial drug are unlikely to succeed . Prospective studies of sufficient size that address all potential risk factors, especially individual anti-infective agents, and that use matched controls who are shown by surveillance cultures to be free of colonization by resistant organisms would enhance understanding of the epidemiology of antimicrobial resistance in institutions and guide efforts to develop more effective strategies for prevention. Water Res, 2002 Apr, 36(7), 1767 - 75 Characterization of microbial community in granular sludge treating brewery wastewater; Liu WT et al.; The diversity and distribution of microbes within brewery-degrading anaerobic sludge granules were studied using various molecular techniques . Molecular cloning of small-subunit rRNA gene sequences indicated that all archaeal clones were affiliated with Methanosaeta concillii (>99% sequence similarity), and the bacterial clones were mostly affiliated with a not-yet-cultured Clostridium cluster (48 out of 99 clones) in the low G + C gram-positive group, Xanthomonas spp . in the gamma-subclass of Proteobacteria (30 clones), and Desulfovibrio spp . (16 clones) in the delta-subclass of Proteobacteria . Slot-blot hybridization indicated that archaeal cells from the Methanomicrobiales (58.4% of total rRNA), Methanobacterials (3.3%) and Methanococcales (1.0%) accounted for 62.4% of the total community rRNA . The rest of the microbial populations were the clostridial cluster (27.3% of total rRNA) and Desulfovibrio spp . (9.4%) . Fluorescence in-situ hybridization with domain and group-specific oligonucleotide probes further revealed a multi-layer granular architecture . On the surface layer, the hydrolytic clostridial species and hydrogenotrophic Methanobacteriales were the predominant . In the middle layer, mostly H2-producing acetogens from the delta-Proteobacteria (i.e., Desulfovibrio spp.), hydrogenotrophic Methanobacteriales and aceticlastic Methanosaeta were observed to presumably form a syntrophic association . Finally, the center core consisted of microcolonies of Methanosaeta cells. Clin Rev Allergy Immunol, 2002 Jun, 22(3), 255 - 73 Probiotics in clinical conditions; Marteau PR; Probiotics are nonpathogenic microorganisms which, when ingested, exert a positive influence on the health or physiology of the host . Their mechanisms of action and effects are now studied using the same pharmacological approach as for drugs . This article summarizes and comments on evidence for the positive effects of probiotics in various clinical situations . Substantial evidence can be achieved when randomized controlled trials or meta-analyses show positive results . The clinical situations studied include prevention or treatment of antibiotic-associated disorders, gastroenteritis, and diarrhea, lactose intolerance, intestinal infections and colonization by pathogenic bacteria (including Helicobacter pylori and Clostridium difficile), traveler's diarrhea, irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), colonic cancer, urogenital infections and tumors, allergy (especially atopic eczema), vaccination, and cholesterol lowering . Current probiotics have an excellent safety record--another topic discussed in this article. Eur Radiol, 2002 Jun, 12(6), 1432 - 7 Epub 2001 Dec 01. Portal-venous gas unrelated to mesenteric ischemia; Wiesner W et al.; The aim of this study was to report on 8 patients with all different non-ischemic etiologies for portal-venous gas and to discuss this rare entity and its potentially misleading CT findings in context with a review of the literature . The CT examinations of eight patients who presented with intrahepatic portal-venous gas, unrelated to bowel ischemia or infarction, were reviewed and compared with their medical records with special emphasis on the pathogenesis and clinical impact of portal-venous gas caused by non-ischemic conditions . The etiologies for portal-venous gas included: abdominal trauma ( n=1); large gastric cancer ( n=1); prior gastroscopic biopsy ( n=1); prior hemicolectomy ( n=1); graft-vs-host reaction ( n=1); large paracolic abscess ( n=1); mesenteric recurrence of ovarian cancer superinfected with clostridium septicum ( n=1); and sepsis with Pseudomonas aeruginosa ( n=1) . The clinical outcome of all patients was determined by their underlying disease and not negatively influenced by the presence of portal-venous gas . Although the presence of portal-venous gas usually raises the suspicion of bowel ischemia and/or intestinal necrosis, this CT finding may be related to a variety of non-ischemic etiologies and pathogeneses as well . The knowledge about these conditions may help to avoid misinterpretation of CT findings, inappropriate clinical uncertainty and unnecessary surgery in certain cases. J Vasc Surg, 2002 Jun, 35(6), 1287 - 8 Anaerobic cellulitis as the result of Clostridium perfringens: a rare cause of vascular access graft infection; Claeys LG et al.; Infection of prosthetic vascular access grafts is the second most common complication of vascular access and represents a challenge encountered by the vascular surgeon . Anaerobic graft infections are rare . We report on a case of a prosthetic vascular access graft infection with Clostridium perfringens . To our knowledge, only one other case with an infected arteriovenous shunt caused by C perfringens has been reported . The patient, a 67-year-old woman with end-stage renal failure as the result of polycystic renal disease, was seen with an infected pseudoaneurysm at the arterial puncture site of the loop graft on the left arm . There was associated purulence at the time of operation . Surgical management consisted of complete graft removal because of the presence of small tunnel abscesses . C perfringens was found in the resected pseudoaneurysm and graft material . Infected pseudoaneurysms most likely are attributable to repetitive punctures in one small area and to a break in sterile technique . A compromised vascular supply, not infrequent in patients for hemodialysis, may lower the oxidation reduction potential, which allows anaerobic bacteria, such as C perfringens, to cause infection. Pediatrics, 2002 Jun, 109(6), 1173 - 7 Comparative study of cefuroxime axetil versus amoxicillin in children with early Lyme disease; Eppes SC et al.; Cefuroxime axetil has been shown to have efficacy comparable to doxycycline in adults with early Lyme disease (LD) . Because of toxicity, doxycycline is usually avoided in children . For children who are unable to tolerate amoxicillin, there is currently no proven alternative oral therapy for LD . This randomized, unblinded study compared 2 dosage regimens of cefuroxime axetil (20 mg/kg/d and 30 mg/kg/d) with amoxicillin (50 mg/kg/d), each given for 20 days . Children were enrolled if they were 6 months to 12 years of age, had erythema migrans, and met other eligibility requirements . Serologic testing occurred at entry and after 6 months . Follow-up evaluations for safety, tolerability, and efficacy occurred at 10 and 20 days, 6 months, and 1 year . Forty-three children were randomized (13 in the amoxicillin group, 15 in each cefuroxime axetil group); 39 completed 12 months of follow-up . At the completion of treatment, there was total resolution of erythema migrans in 67% of the amoxicillin group, 92% of the low-dose cefuroxime group, and 87% of the high-dose cefuroxime group, and resolution of constitutional symptoms occurred in 100%, 69%, and 87%, respectively . All patients had a good outcome, with no long-term problems associated with LD . One patient, who was well at the first 2 follow-up visits, was treated with doxycycline because of new constitutional symptoms . Mild diarrhea occurred in a small number of participants in each group (1 patient was diagnosed and treated for Clostridium difficile-associated diarrhea, which occurred after completing the full course of study medication) . No hypersensitivity reactions occurred . The number of patients in this trial was not sufficient to demonstrate a statistically significant difference between the 3 groups; however, both amoxicillin and cefuroxime axetil seem to be safe, efficacious treatments for children with early LD. J Vet Intern Med, 2002 May-Jun, 16(3), 222 - 8 Role of the enteric nervous system in the pathophysiology of secretory diarrhea; Jones SL et al.; Details of the physiology and pathophysiology of epithelial secretion in the gastrointestinal tract are becoming clear, leading to new models of the mechanisms underlying diarrhea . The enteric nervous system is a critical component of the mechanism regulating fluid secretion in the normal gut and a key element in the pathophysiology of diarrhea . Neural reflex pathways increase epithelial fluid secretion in response to several enteric pathogens of veterinary importance such as Salmonella spp., Cryptosporidium parvum, rotavirus, and Clostridium difficile . Moreover, the enteric nervous system has an important role in epithelial secretion triggered by products of activated leukocytes during inflammation . New approaches targeting the enteric nervous system show promise for the treatment of secretory diarrhea. J Biol Chem, 2002 Jul 19, 277(29), 25867 - 9 Epub 2002 May 30. Caveolin-associated filamentous actin (Cav-actin) defines a novel F-actin structure in adipocytes; Kanzaki M et al.; Dynamic actin remodeling has been implicated in the translocation of the insulin-responsive glucose transporter 4 (GLUT4) to the plasma membrane in adipocytes . Here we show that fully differentiated 3T3L1 adipocytes have unique cortical filamentous actin structure, designated Cav-actin (caveolae-associated F-actin) . During 3T3L1 adipocyte differentiation, rhodamine-phalloidin staining demonstrated the formation of a cortical actin cytoskeleton that is composed of small dot-like F-actin spikes lining the inside of the plasma membrane . Double labeling with a caveolin antibody indicated that these F-actin spikes emanate from organized rosette-like clusters of caveolae/lipid raft microdomains . In contrast, there was no obvious relationship between F-actin and caveolin localization and/or organization in 3T3L1 preadipocytes (fibroblasts) . Treatments of differentiated adipocytes with latrunculin B, Clostridium difficile toxin B or a dominant-interfering TC10 mutant (TC10/T31N) disrupted the Cav-actin structure without significantly affecting the organization of clustered caveolae . Similarly, disruption of the clustered caveolae with methyl-beta-cyclodextrin also dispersed the Cav-actin structure . These data demonstrate that this novel Cav-actin structure is organized through clustered caveolae but that the formation of caveolae-rosettes are not dependent upon F-actin. J Clin Microbiol, 2002 Jun, 40(6), 2288 - 90 Development and evaluation of a PCR method for detection of the Clostridium difficile toxin B gene in stool specimens; Guilbault C et al.; A PCR assay detecting Clostridium difficile toxin B gene in stool specimens was compared to the cytotoxicity assay as the reference standard for the diagnosis of C . difficile antibiotic-associated diarrhea (CDAD) . Overall, 118 stool samples were tested . All of the specimens that were negative by the cytotoxicity assay (59 out of 118) were also negative by the PCR method (specificity of 100%) . Of the 59 cytotoxin-positive samples, 54 were PCR positive (sensitivity of 91.5%) . This PCR method is promising for rapid diagnosis of CDAD. J Clin Microbiol, 2002 Jun, 40(6), 2260 - 2 Botulism due to Clostridium baratii type F toxin; Harvey SM et al.; Botulism results from consumption of preformed toxin or in vivo toxin elaboration in wounds or intestine . Of U.S . food-borne botulism cases since 1950, the majority were due to toxin A, but a significant number of suspect cases were never confirmed by culture or toxin detection . We report here a possible case of food-borne botulism attributed to toxin F production by a Clostridium baratii organism isolated from food consumed by the patient . The isolation of a toxin-producing Clostridium species other than Clostridium botulinum from food and stool requires deviation from the usual laboratory protocols, which may account for the lack of complete laboratory confirmation of clinically diagnosed cases. J Clin Microbiol, 2002 Jun, 40(6), 2079 - 83 Prevalence and genetic characterization of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France; Barbut F et al.; Toxin A variant strains (toxin A-negative, toxin B-positive strains) of Clostridium difficile have been reported to be responsible for diarrhea or pseudomembranous colitis in humans . These strains lack parts of the repeating sequences of the toxin A gene (tcdA) and are toxin A negative by commercial enzyme immunoassays (EIA) . Here, we report the prevalence of the toxin A variant strains in 334 patients with C . difficile-associated diarrhea in France . The repeating segment of the tcdA gene (1,200 bp) was amplified by PCR using the primers NK9 and NK11 (H . Kato et al., J . Clin . Microbiol . 36:2178-2182, 1998) . In the case of amplified fragments of unexpected size, the entire tcdA gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J . Clin . Microbiol . 36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field gel electrophoresis and PCR ribotyping . By PCR with primers NK9 and NK11, C . difficile variant strains were detected in 2.7% of patients . Several variant types were found . A deletion of approximately 1,700 bp was observed in six strains from five patients . These strains belonged to serotype F and were characterized by the same pulsotype and the same PCR ribotype . They were toxin A negative by EIA and exhibited an atypical cytopathic effect on MRC-5 cells . Two other tcdA variant types that exhibited a positive result for toxin A by EIA were identified: one from serotype H with a longer amplified fragment (insertion of 200 bp) and one with a deletion of 600 bp . Diagnosis of C . difficile-associated diseases would have been missed in five patients (1.5%) by laboratories that screen the stools only for the presence of toxin A . This result underlines the need for testing stool by the cytotoxicity assay in patients with a high suspicion of C . difficile-associated diarrhea but a negative immunoassay for toxin A. Nucleic Acids Res, 2002 Jun 1, 30(11), 2453 - 9 A novel type of conserved DNA-binding domain in the transcriptional regulators of the AlgR/AgrA/LytR family; Nikolskaya AN et al.; Sequence analysis of bacterial genomes revealed a novel DNA-binding domain . This domain is found in several response regulators of the two-component signal transduction system, such as Pseudomonas aeruginosa AlgR, involved in the regulation of alginate biosynthesis and in the pathogenesis of cystic fibrosis; Clostridium perfringens VirR, a regulator of virulence factors, and in several regulators of bacteriocin biosynthesis, previously unified in the AgrA/ComE family . Most of the transcriptional regulators that contain this DNA-binding domain are involved in biosynthesis of extracellular polysaccharides, fimbriation, expression of exoproteins, including toxins, and quorum sensing . We refer to it as the LytTR ('litter') domain, after Bacillus subtilis LytT and Staphylococcus aureus LytR response regulators, involved in regulation of cell autolysis . In addition to response regulators, the LytTR domain is found in combination with MHYT, PAS and other sensor domains. J Am Chem Soc, 2002 Jun 5, 124(22), 6277 - 84 Stopped-Flow Kinetics of Methyl Group Transfer between the Corrinoid-Iron-Sulfur Protein and Acetyl-Coenzyme A Synthase from Clostridium thermoaceticum; Tan XS et al.; Kinetics of methyl group transfer between the Ni-Fe-S-containing acetyl-CoA synthase (ACS) and the corrinoid protein (CoFeSP) from Clostridium thermoaceticum were investigated using the stopped-flow method at 390 nm . Rates of the reaction CH(3)-Co(3+)FeSP + ACS(red) <==> Co(1+)FeSP + CH(3)-ACS(ox) in both forward and reverse directions were determined using various protein and reductant concentrations . Ti(3+)citrate, dithionite, and CO were used to reductively activate ACS (forming ACS(red)) . The simplest mechanism that adequately fit the data involved formation of a {CH(3)-Co(3+)FeSP}:{ACS(red)} complex, methyl group transfer (forming {Co(1+)FeSP}:{CH(3)-ACS(ox)}), product dissociation (forming Co(1+)FeSP + CH(3)-ACS(ox)), and CO binding yielding a nonproductive enzyme state (ACS(red) + CO <==> ACS(red)-CO) . Best-fit rate constants were obtained . CO inhibited methyl group transfer by binding ACS(red) in accordance with K(D) = 180 +/- 90 microM . Fits were unimproved when >1 CO was assumed to bind . Ti(3+)citrate and dithionite inhibited the reverse methyl group transfer reaction, probably by reducing the D-site of CH(3)-ACS(ox) . This redox site is oxidized by 2e(-) when the methyl cation is transferred from CH(3)-Co(3+)FeSP to ACS(red), and is reduced during the reverse reaction . Best-fit K(D) values for pre- and post-methyl-transfer complexes were 0.12 +/- 0.06 and 0.3 +/- 0.2 microM, respectively . Intracomplex methyl group transfer was reversible with K(eq) = 2.3 +/- 0.9 (k(f)/k(r) = 6.9 s(-1)/3.0 s(-1)) . The nucleophilicity of the {Ni(2+)D(red)} unit appears comparable to that of Co(1+) cobalamins . Reduction of the D-site may cause the Ni(2+) of the A-cluster to behave like the Ni of an organometallic Ni(0) complex. Clin Infect Dis, 2002 Jun 15, 34(12), 1585 - 92 Epub 2002 May 23. Conditions associated with leukocytosis in a tertiary care hospital, with particular attention to the role of infection caused by clostridium difficile; Wanahita A et al.; Few modern studies have enumerated the conditions associated with leukocytosis . Our clinical experience has implicated Clostridium difficile infection in a substantial proportion of patients with leukocytosis . In a prospective, observational study of 400 inpatients with WBC counts of >/=15,000 cells/mm(3), we documented >/=1 infection in 207 patients (53%) . Of these 207 patients, 97 (47%) had pneumonia, 60 (29%) had urinary tract infection, 34 (16%) had soft-tissue infection, and 34 (16%) had C . difficile infection . C . difficile infection was present in 25% of patients with WBC counts of >30,000 cells/mm(3) who did not have hematological malignancy . Other causes of leukocytosis in the 400 patients included physiological stress, in 152 patients (38%); medications or drugs, in 42 (11%); hematological disease, in 22 (6%); and necrosis or inflammation, in 22 (6%) . C . difficile infection is a prominent cause of leukocytosis and this diagnosis should be considered for patients with WBC counts of >/=15,000 cells/mm(3), even in the absence of diarrheal symptoms. Biochim Biophys Acta, 2002 May 10, 1571(1), 18 - 26 A novel heptasialosyl c-series ganglioside in embryonic chicken brain: its structure and stage-specific expression; Saito M et al.; A ganglioside of unknown structure (ganglioside X) was purified from chicken brain at embryonic day 12 (E12) and characterized for its structure . Ganglioside X was reactive with a monoclonal antibody A2B5 and migrated below GH1c on thin-layer chromatography (TLC) . Extensive treatment of ganglioside X with Clostridium perfringens sialidase produced a single ganglioside product . This ganglioside was identified as GM1 based upon its chromatographic mobility and reactivity to cholera toxin B subunit and anti-GM1 antibody . Partial hydrolysis of ganglioside X by sialidase generated several degradation products including GH1c, GP1c, and GQ1c . Electrospray ionization (ESI)-mass spectrometry (MS) of the permethylated derivative of ganglioside X produced a triple-charged parent ion peak at m/z 1355, which corresponded with the gangliotetraose oligosaccharide structure having seven sialic acids and ceramide with the molecular mass of 566 (as non-methylated form) . Collision-induced dissociation (CID)-MS(2) showed fragment ions including those at m/z 1066 and 1931; these two ions matched the structures of (NeuAc)(3)-Gal-Glc-Cer and (NeuAc)(4)-Gal-GalNAc, respectively . These structures were confirmed by CID-MS(3) of the corresponding peaks . Based upon these findings, the structure of ganglioside X was identified as NeuAc-NeuAc-NeuAc-NeuAc-Galbeta1-3GalNAcbeta1-4(NeuAc-NeuAc-NeuAcalpha2-3)Galbeta1-4Glcbeta1-1'Cer . This ganglioside was designated as GS1c . A developmental study demonstrated that GS1c was expressed in chicken brain during a period from E6 to E13 and thereafter decreased rapidly in its concentration . The present study suggests that GS1c may play a specific role in early development of chicken brain. Scand J Infect Dis, 2002, 34(3), 209 - 11 Clostridium novyi type A infection: a sporadic fatal case; McGuigan C et al.; Infection with type A Clostridium novyi is rare . We report the case of a previously healthy 31-y-old woman with no known risk factors who died suddenly with a necrotizing soft tissue infection . We compare this case with a simultaneous outbreak of this infection amongst Scottish IDUs (injecting drug users). J Food Prot, 2002 May, 65(5), 806 - 13 Antimicrobial activity of foodborne Paenibacillus and Bacillus spp . against Clostridium botulinum; Girardin H et al.; The saprophytic Paenibacillus and Bacillus spp . found in cooked chilled foods may have an effect on the growth of Clostridium botulinum, a major microbiological hazard, especially for pasteurized vacuum-packaged products . Culture supernatants of 200 strains of Paenibacillus and Bacillus strains isolated from commercial cooked chilled foods containing vegetables were screened for activity against C . botulinum type A, proteolytic type B, and type E strains in a well diffusion assay . Nineteen strains were positive against C . botulinum . Among those, seven Paenibacillus polymyxa strains showed the highest antibotulinal activity and the largest antimicrobial spectrum against C . botulinum strains . The antibotulinal activity was evaluated throughout the growth of a representative strain of the positive P . polymyxa strains . The antimicrobial activity was detected in the culture supernatant from late-log/early stationary phase of the bacteria, which occurred after 7 to 10 days of incubation at 10 degrees C and after 2 to 3 days at 20 degrees C in nutrient broth and in vegetable purees under aerobic or anaerobic conditions . In co-cultures with the positive strain of P . polymyxa in nutrient broth and vegetable purees, a C . botulinum type E strain was inhibited whenever P . polymyxa reached stationary phase and produced its antimicrobial activity before C . botulinum began its exponential growth phase . The antimicrobial activity of P . polymyxa against C . botulinum was attributed to the production of antimicrobial peptides resistant to high temperature and acidity . Other gram-positive and -negative bacteria (Escherichia coli, Streptococcus mutans, Leuconostoc mesenteroides, and Bacillus subtilis) were also sensitive to these antimicrobial peptides. J Biol Chem, 2002 Aug 23, 277(34), 30950 - 7 Epub 2002 May 23. NAD binding induces conformational changes in Rho ADP-ribosylating clostridium botulinum C3 exoenzyme; Menetrey J et al.; We have solved the crystal structures of Clostridium botulinum C3 exoenzyme free and complexed to NAD in the same crystal form, at 2.7 and 1.95 A, respectively . The asymmetric unit contains four molecules, which, in the free form, share the same conformation . Upon NAD binding, C3 underwent various conformational changes, whose amplitudes were differentially limited in the four molecules of the crystal unit . A major rearrangement concerns the loop that contains the functionally important ARTT motif (ADP-ribosyltransferase toxin turn-turn) . The ARTT loop undergoes an ample swinging motion to adopt a conformation that covers the nicotinamide moiety of NAD . In particular, Gln-212, which belongs to the ARTT motif, flips over from a solvent-exposed environment to a buried conformation in the NAD binding pocket . Mutational experiments showed that Gln-212 is neither involved in NAD binding nor in the NAD-glycohydrolase activity of C3, whereas it plays a critical role in the ADP-ribosyl transfer to the substrate Rho . We observed additional NAD-induced movements, including a crab-claw motion of a subdomain that closes the NAD binding pocket . The data emphasized a remarkable NAD-induced plasticity of the C3 binding pocket and suggest that the NAD-induced ARTT loop conformation may be favored by the C3-NAD complex to bind to the substrate Rho . Our structural observations, together with a number of mutational experiments suggest that the mechanisms of Rho ADP-ribosylation by C3-NAD may be more complex than initially anticipated. Plast Surg Nurs, 2000 Summer, 20(2), 60 - 5; quiz 66-7 BOTOX: a review; Mendez-Eastman SK; The history of medicine has many amazing stories of odd anecdotes for the treatment of a variety of ailments . Who would have thought that common mold would revolutionize the treatment of infection, or slimy leaches could assist in the resolution of venous congestion? When retching from the effects of food poisoning, or even worse, developing severe paralysis acquired from food contaminated with the anaerobic bacterium Clostridium Botulinum (Botulism), who could have guessed that toxins produced by this bacteria could be used as an effective cosmetic treatment for frown lines and wrinkles? Such is the story of medicine: always inventive and surprising, and sometimes downright odd. Microb Ecol, 2001 Dec, 42(4), 495 - 505 Interpreting 16S rDNA T-RFLP Data: Application of Self-Organizing Maps and Principal Component Analysis to Describe Community Dynamics and Convergence; Dollhopf SL et al.; Interpreting the large amount of data generated by rapid profiling techniques, such as T-RFLP, DGGE, and DNA arrays, is a difficult problem facing microbial ecologists . This study compares the ability of two very different ordination methods, principal component analysis (PCA) and self-organizing map neural networks (SOMs), to analyze 16S-DNA terminal restriction-fragment length polymorphism (T-RFLP) profiles from microbial communities in glucose-fed methanogenic bioreactors during startup and changes in operational parameters . Our goal was not only to identify which samples were similar, but also to decipher community dynamics and describe specific phylotypes, i.e., phylogenetically similar organisms, that behaved similarly in different reactors . Fifteen samples were taken over 56 volume changes from each of two bioreactors inoculated from river sediment (S2) and anaerobic digester sludge (M3) and from a well-established control reactor (R1) . PCA of bacterial T-RFLP profiles indicated that both the S2 and M3 communities changed rapidly during the first nine volume changes, and then became relatively stable . PCA also showed that an HRT of 8 or 6 days had no effect on either reactor communtity, while an HRT of 2 days changed community structure significantly in both reactors . The SOM clustered the terminal restriction fragments according to when each fragment was most abundant in a reactor community, resulting in four clearly discernible groups . Thirteen fragments behaved similarly in both reactors, eight of which composed a significant proportion of the microbial community as judged by the relative abundance of the fragment in the T-RFLP profiles . Six Bacteria terminal restriction fragments shared between the two communities matched cloned 16S rDNA sequences from the reactors related to Spirochaeta, Aminobacterium, Thermotoga, and Clostridium species . Convergence also occurred within the acetoclastic methanogen community, resulting in a predominance of Methanosarcina siciliae-related organisms . The results demonstrate that both PCA and SOM analysis are useful in the analysis of T-RFLP data; however, the SOM was better at resolving patterns in more complex and variable data than PCA ordination. Microb Ecol, 2002 Mar, 43(2), 271 - 9 Epub 2002 Jan 24. Sporulation of Clostridium cellulolyticum while grown in cellulose-batch and cellulose-fed continuous cultures on a mineral-salt based medium; Desvaux M et al.; Clostridium cellulolyticum sporulation was investigated during growth on cellulose fibers in a mineral-salt based medium which corresponds to conditions linked to its natural ecological niche . At steady state of the continuous cultures under limitation and with an excess of cellulose and/or ammonium, bacterial cells mainly sporulated at low dilution rates (D), at least 10% sporulation being observed at the lowest D tested . Increasing the cellulose concentration in the feed-medium reservoir increased the percentage of spores in the bioreactor . It appeared that the remaining undigested cellulose could serve as an exogenous carbon source supply at a continuous but limited rate throughout the sporulation process . In addition to the proportion of carbon and nitrogen, the influence of the environmental pH on spore formation was studied . In cellulose-fed continuous cultures at a constant D and a pH decreasing from 7.2 to 6.4, the percentage of spores increased to 14% at the lowest pH tested . When C . cellulolyticum was grown in batch culture, the level of sporulation was dramatically higher in unregulated-pH fermentation compared to pH-controlled growth conditions at pH 7.2 since in the former it reached 45% within 5 days of cultivation . It then appeared that a low specific growth rate and a low environmental pH in the presence of an insoluble carbon substrate were the major factors inducing sporulation in C . cellulolyticum . Furthermore, since the spores adhere to the carbon substrate (the cellulose) the bacteria gain advantages when the environment allows germination thanks to the recovery of suitable growth conditions . By allowing the maintenance and the integrity of the bacteria in the microbiota, spore formation could then explain the successful survival of C . cellulolyticum in cellulosic anaerobic habitats where low environmental pH conditions are often found. FEMS Microbiol Lett, 2002 Apr 23, 210(1), 93 - 8 amyP, a reporter gene to study strain degeneration in Clostridium acetobutylicum ATCC 824; Sabathe F et al.; Clostridium acetobutylicum produces an extracellular alpha-amylase when grown on glucose as the sole carbon source . This enzyme was previously characterized from a biochemical point of view but its encoding gene was never identified . The 2283-bp amyP gene encodes a 83013-Da mature protein with an N-terminal domain that exhibits strong identity to the family 13 glycosyl hydrolases such as the Bacillus alpha-amylases . Transcriptional analysis revealed that amyP is transcribed in solventogenic but not in acidogenic chemostat cultures . These results are in agreement with the extracellular alpha-amylase activities indicating that the expression of amyP is regulated at the transcriptional level . amyP is located on the pSOL1 megaplasmid that carries all the genes involved in the final steps of solvent formation . Degeneration of C . acetobutylicum has been associated to the loss of pSOL1 . We demonstrate here that amyP can be used as a reporter system to quantitatively follow this phenomenon. FEMS Microbiol Lett, 2002 Apr 23, 210(1), 7 - 17 A genome sequence survey of the mollicute corn stunt spiroplasma Spiroplasma kunkelii; Bai X et al.; The mollicute corn stunt spiroplasma (Spiroplasma kunkelii) is a leafhopper-transmitted pathogen of maize . Sequencing of the approximately 1.6-Mb genome of S . kunkelii was initiated to aid understanding the genetic basis of spiroplasma interactions with their plant and leafhopper hosts . In total, 144712 nucleotides of non-redundant, high-quality S . kunkelii genome sequence were obtained . Sequence tags were searched against the Mycoplasmataceae and Bacillus/Clostridium databases . Results showed that, in addition to spiroplasma phage SpV1 DNA insertions, spiroplasma genomes harbor more purine and amino acid biosynthesis, transcription regulation, cell envelope and DNA transport/binding genes than Mycoplasmataceae genomes . This investigation demonstrates that survey sequencing is an efficient procedure for gene discovery and genome characterization . The results of the S . kunkelii sequencing project are available at the Spiroplasma WebPage at http://www.oardc.ohio-state.edu/spiroplasma/genome.htm. Antimicrob Agents Chemother, 2002 Jun, 46(6), 1647 - 50 Reassessment of Clostridium difficile susceptibility to metronidazole and vancomycin; Pelaez T et al.; Clostridium difficile is the most frequently identified enteric pathogen in patients with nosocomially acquired, antibiotic-associated diarrhea . The drugs most commonly used to treat diseases associated with C . difficile are metronidazole and vancomycin . Most clinical laboratories assume that all C . difficile isolates are susceptible to metronidazole and vancomycin . We report on the antimicrobial susceptibilities of 415 C . difficile isolates to metronidazole and vancomycin over an 8-year period (1993 to 2000) . The overall rate of resistance to metronidazole at the critical breakpoint (16 microg/ml) was 6.3% . Although full resistance to vancomycin was not observed, the overall rate of intermediate resistance was 3.1% . One isolate had a combination of resistance to metronidazole and intermediate resistance to vancomycin . Rates of resistance to metronidazole and vancomycin were higher among isolates from human immunodeficiency virus-infected patients . Molecular typing methods proved the absence of clonality among the isolates with decreased susceptibilities to the antimicrobials tested. Appl Biochem Biotechnol, 2002 Spring, 98-100, 553 - 61 Production of acetone butanol ethanol from degermed corn using Clostridium beijerinckii BA101; Campos EJ et al.; In this article we report on acetone butanol ethanol (ABE) fermentation characteristics of degermed corn when using Clostridium beijerinckii BA101 . Recent economic studies suggested that recovery of germ from corn and hence corn oil would help to make the ABE fermentation process more economical . C . beijerinckii BA101 ferments corn mash efficiently to produce ABE under appropriate nutritional and environmental conditions . Corn mash contains germ/corn oil that is, possibly, ancillary to the production of butanol during the ABE fermentation process . Since the presence of corn oil is not a critical factor in solvent fermentation, it can be removed and this will allow for byproduct credit . Batch fermentation of degermed corn resulted in 8.93 g/L of total ABE production as compared with 24.80 g/L of total ABE when supplemented with P2 medium nutrients . During the course of the germ separation process, corn steeping is required prior to grinding and removing the germ . It is likely that some nutrients from the corn are leached out during the steeping process . This may reduce the rate of fermentation and impact the final concentration of butanol/ABE that can be achieved . Fermentation of degermed corn with corn steep liquor resulted in the production of 19.28 g/L of ABE. New Microbiol, 2002 Apr, 25(2), 139 - 47 Brachyspira (Serpulina) pilosicoli of human origin interfere with the growth of Clostridium perfringens alpha-toxin producer; Calderaro A et al.; Brachyspira (Serpulina) pilosicoli of human origin interfere with the growth of Clostridium perfringens alpha-toxin producer reducing the clostridial growth area and colonies number when bacteria were cultivated together in sheep blood agar plates . The growth inhibition of C . perfringens was only observed when B . (S.) pilosicoli grew 72-96 hours sooner than C . perfringens and after the inoculum of this latter the plates were anaerobically incubated for additional 48 hours . The phenomenon was observed at concentrations of B . (S.) pilosicoli ranging from 10(7) to 10(4) CFU/ml and at concentrations of C . perfringens ranging from 10(7) to 10(1) CFU/ml when the bacteria were 0-10 mm away from each other . When B . (S.) pilosicoli and C . perfringens were inoculated at the same time and when B . (S.) pilosicoli grew 24-48 hours sooner than C . perfringens, the clostridial growth inhibition was not appreciated and only a cooperative haemolysis was observed between the bacteria. J Neurol, 2001 Dec, 248(12), 1073 - 8 Efficacy and safety of a standardised 500 unit dose of Dysport (clostridium botulinum toxin type A haemaglutinin complex) in a heterogeneous cervical dystonia population: results of a prospective, multicentre, randomised, double-blind, placebo-controlled, parallel group study; Wissel J et al.; Results from a dose-ranging study in a selected group of de novo patients with rotational cervical dystonia (CD) suggest that 500 units of Dysport (Clostridium botulinum toxin type A haemaglutinin complex) is the optimal starting dose . The present study aimed to confirm the efficacy and safety profile of this dose in a population of CD patients more representative of those seen in a typical dystonia clinic . A total of 68 patients with moderate to severe CD (Tsui score > or = 9) were randomly assigned to receive placebo or Dysport 500 units . Treatment was administered according to the clinical pattern of head deviation, using a standardised injection protocol . A total of 21 patients (11 Dysport, 10 placebo) had not previously received botulinum toxin type A (BtxA) injections, and 47 patients (24 Dysport, 23 placebo) had received BtxA more than 12 weeks previously . Assessments were performed at baseline and weeks 4, 8 and 16 . Patients defined as non-responders at week 4 were re-treated in an open phase with 500 units of Dysport at week 6, and were followed up at week 10 . Significant between-group differences in Tsui scores were present at weeks 4 (p=0.001) and 8 (p=0.002) . Similarly, there were significant between-group differences (p < 0.001) in patient and investigator assessments of response in favour of Dysport at weeks 4 and 8 . Also, more Dysport (49%) than placebo (33%) patients were pain-free at week 4 (p=0.02) . Overall, 30/35 (86 %) Dysport patients and 14/33 (42%) placebo patients were classified as responders at week 4 . Adverse events were reported by 15/35 Dysport patients and 9/33 placebo patients . Open phase treatment produced improvements in Tsui (p < 0.001) and pain scores (p=0.011), and 23/24 patients were classified as responders . Although individual dose titration and muscle selection is desirable, this study demonstrated that a dose of 500 units of Dysport injected into clinically identified neck muscles without electromyographic guidance is safe and effective in the treatment of patients with the major clinical types of cervical dystonia. Clin Transplant, 2002 Jun, 16(3), 212 - 6 Diarrhoea following renal transplantation; Altiparmak MR et al.; In this study, we retrospectively evaluated all attacks of diarrhoea in our renal transplant recipients that came to our medical attention between 1985 and 2000 . Also, the clinical features of patients with diarrhoea were compared with the features of recipients without diarrhoea . We diagnosed 41 attacks of diarrhoea in 39 (12.6%) of 308 renal transplant recipients during this time period . An aetiology was detected in 33 (80.5%) of all diarrhoeal episodes and in seven (17.1%) of those the specific agent was diagnosed with the help of stool microscopy . The most frequent causes of diarrhoeal attacks were infectious agents (41.5%) and drugs (34%) . Six (14.6%) episodes of diarrhoea were chronic and six were nosocomial . About two-thirds of diarrhoea developed within the late post-transplant period (>6 months) . When recipients with diarrhoea were compared with those without diarrhoea, it was seen that diarrhoeal patients had significantly higher creatinine and significantly lower albumin levels when compared with the latter group (p < 0.05) . Also, the frequency of antibiotic usage was significantly higher in diarrhoeal patients than in the control group (p < 0.05) . Four (10.2%) patients with diarrhoea died despite institution of the appropriate therapy . Two of these deaths were primarily related to diarrhoea and the aetiological agent was Clostridium difficile in both these cases . During the 15-yr study period, 3.6% of all deaths and 5.1% of infection-related deaths in transplant recipients were secondary to diarrhoea . As a result, we observed that infections and drugs were the most frequent causes for diarrhoea in our series of renal transplant recipients . Also, diarrhoea was an important cause of mortality in this patient population. Biochemistry, 2002 May 21, 41(20), 6253 - 62 The first strain of Clostridium perfringens isolated from an avian source has an alpha-toxin with divergent structural and kinetic properties; Justin N et al.; Clostridium perfringens alpha-toxin is a 370-residue, zinc-dependent, phospholipase C that is the key virulence determinant in gas gangrene . It is also implicated in the pathogenesis of sudden death syndrome in young animals and necrotic enteritis in chickens . Previously characterized alpha-toxins from different strains of C . perfringens are almost identical in sequence and biochemical properties . We describe the cloning, nucleotide sequencing, expression, characterization, and crystal structure of alpha-toxin from an avian strain, SWan C . perfringens (SWCP), which has a large degree of sequence variation and altered substrate specificity compared to these strains . The structure of alpha-toxin from strain CER89L43 has been previously reported in open (active site accessible to substrate) and closed (active site obscured by loop movements) conformations . The SWCP structure is in an open-form conformation, with three zinc ions in the active site . This is the first example of an open form of alpha-toxin crystallizing without the addition of divalent cations to the crystallization buffer, indicating that the protein can retain three zinc ions bound in the active site . The topology of the calcium binding site formed by residues 269, 271, 336, and 337, which is essential for membrane binding, is significantly altered in comparison with both the open and closed alpha-toxin structures . We are able to relate these structural changes to the different substrate specificity and membrane binding properties of this divergent alpha-toxin . This will provide essential information when developing an effective vaccine that will protect against C . perfringens infection in a wide range of domestic livestock. J Immunol Methods, 2002 May 1, 263(1-2), 35 - 41 Rapid and sensitive detection of biological warfare agents using time-resolved fluorescence assays; Peruski AH et al.; We have achieved sensitive, rapid and reproducible detection of three biological threat agents in a variety of biological and environmental matrices using the DELFIA time-resolved fluorometry (TRF) assay system (Perkin-Elmer Life Sciences, Akron, OH) . Existing ELISA assays for the detection of Francisella tularensis, Clostridium botulinum A/B neurotoxin (BotNT A/B), and Staphylococcus aureus enterotoxin B (SEB) were converted to TRF assays . They use 100 microl of positive control or unknown per test well and require just over 2 h to run . Fluorescent signal read time is a fraction of a second per well . The assay format consists of a capture ELISA utilizing a biotinylated capture antibody, prebound to a streptavidin-coated 96-well plate and a lanthanide (Europium, Eu3+)-labeled detector antibody . The bound Eu-labeled detector antibody produces a fluorescent signal upon the addition of an enhancement solution . The signal results from the dissociation of the Europium from the antibody, creating a micelle, thus amplifying the signal nearly one million-fold . Sensitivities achieved by these assays were between 4 and 20 pg/ml in buffer . Additionally, we have tested this system in different matrices such as serum, urine, dirt, and sewage . Concentration curves generated from standard solutions produced a wide linear range making serial dilutions of unknown samples unnecessary . DELFIA TRF assays are significantly better in terms of sensitivity, linear range, and run time than standard capture ELISAs and should facilitate early detection of potential biological warfare agents in clinical and environmental samples. FEMS Microbiol Lett, 2002 Mar 19, 209(1), 113 - 8 Identification of a novel locus that regulates expression of toxin genes in Clostridium perfringens; Ohtani K et al.; A novel gene that regulates the alpha-toxin (plc), kappa-toxin (colA), and theta;-toxin (pfoA) genes was identified using toxin-negative mutant strains of Clostridium perfringens . The cloned 3.2-kb fragment contained the virX gene encoding a 51-amino acid polypeptide of unknown function that seemed to be responsible for the activation of toxin genes . The virX knock out mutant of wild-type strain 13 showed a reduced expression of the plc, colA, and pfoA genes, which was complemented by the transformation of the intact virX gene . Deletion and site-directed mutagenesis studies suggested that the virX gene acts as a regulatory RNA rather than as a peptide regulator . The virX locus found in this study might play a part in the signal transduction to regulate toxin production in C . perfringens. FEMS Microbiol Lett, 2002 Mar 19, 209(1), 93 - 8 The adaptation and resistance of Clostridium aminophilum F to the butyrivibriocin-like substance of Butyrivibrio fibrisolvens JL5 and monensin; Rychlik JL et al.; When the amino acid-fermenting bacterium Clostridium aminophilum F was inoculated into media containing 1 microM monensin or a bacteriocin-like inhibitory substance (BLIS) from Butyrivibrio fibrisolvens JL5, the cultures lagged and growth was not observed for more than 12 h . The monensin- and BLIS-treated cultures eventually grew rapidly and did not lag a second time . Because cross-resistance could not be demonstrated, it appeared that the adaptation was specific . Non-adapted cells that were incubated with monensin lost their ability to produce ammonia from amino acids, and ATP, intracellular potassium, and electrical potential (DeltaPsi) were lower than untreated cells . Monensin-adapted cells regained their ability to produce ammonia, and intracellular potassium and DeltaPsi increased, but ATP was still 40% lower than untreated cells . When non-adapted cells were treated with the BLIS, ammonia production did not decline . Non-adapted cells were agglutinated by lysozyme, but in each case, adapted cells were not agglutinated . Adapted cells had more cellular polysaccharide and bound less of either inhibitor . Based on these results, it appears that the adapted cells had altered cell wall characteristics that prevented the binding of either monensin or the B . fibrisolvens JL5 BLIS. Biomacromolecules, 2002 May-Jun, 3(3), 462 - 5 Heavy metal removal by novel CBD-EC20 sorbents immobilized on cellulose; Xu Z et al.; Heavy metals are major contributors to pollution of the biosphere, and their efficient removal from contaminated water is required . Biosorption is an emerging technology that has been shown to be effective in removing very low levels of heavy metal from wastewater . Although peptides such as metallothioneins or phytotchelatins are known to immobilize heavy metals, peptide-based biosorbents have not been extensively investigated . In this paper, we describe the construction and expression of bifunctional fusion proteins consisting of synthetic phytochelatin (EC20) linked to a Clostridium-derived cellulose-binding domain (CBD(clos)), enabling purification and immobilization of the fusions onto different cellulose materials in essentially a single step . The immobilized sorbents were shown to be highly effective in removing cadmium at parts per million levels . Repeated removal of cadmium was demonstrated in an immobilized column . The ability to genetically engineer biosorbents with precisely defined properties could provide an attractive strategy for developing high-affinity bioadsorbents suitable