Clostridia

East Afr Med J, 2004 Aug, 81(8), 427 - 9
Lower limb amputation in Jos, Nigeria; Kidmas AT et al.; OBJECTIVE: To determine the pattern of lower limb amputation (LLA) and highlight preventable causes in our hospital . DESIGN: A retrospective study . SETTING: University Teaching Hospital, Jos, Nigeria . SUBJECTS: Eighty seven patients who had LLA over a five and a half year period . There were 62 males and 25 females aged between three and 83 years (mean+/-SD = 44.5+/-21.1) . MAIN OUTCOME MEASURES: Indications, morbidity and mortality . RESULTS: A total of 94 LLA were performed in the 87 patients managed . Trauma, diabetic foot sepsis and malignant conditions of the limb were the main indications for LLA in 26(29.9%), 23(26.4%) and 20(23%) patients respectively . Others were peripheral vascular gangrene (PVG) in eight (9.2%) patients; chronic osteomyelitis, three (3.5%); chronic leg ulcers, three (3.5%); Ainhum, three (3.5%) and snake bite in one patient (1.1%) . Above knee amputation (AKA) was the most common procedure performed (48.9%) followed by below knee amputation (BKA) in 37.2% . There were 11 deaths (12.6%), out of which eight were due to sepsis with multiple organ failure following diabetic foot sepsis, two cases of clostridial myonecrosis complicating compound fractures and a case of metastatic osteosarcoma in a child . CONCLUSION: Majority of the cases of LLA could have been avoided with early presentation and appropriate management . A case is made for community health education on the need for early presentation to hospital for limb lesions.

Cell Signal, 2005 Apr, 17(4), 447 - 59
Two different pathways link G-protein-coupled receptors with tyrosine kinases for the modulation of growth and survival in human hematopoietic progenitor cells; Vichalkovski A et al.; The G-protein-coupled receptor agonists CXCL12 (SDF-1, a chemokine) and thrombin showed opposite effects on growth and survival of multipotent and erythroid human hematopoietic progenitor cells . CXCL12 promoted growth in multipotent cells by activating the RhoA-Rho kinase pathway . Its effect was largely blocked by Y-27632, a specific inhibitor of Rho kinase, and by clostridial toxin B, a specific inhibitor of Rho family proteins . Rho activation required a G(i)-mediated stimulation of tyrosine kinases, which was blocked by PP2 and tyrphostin AG 490, inhibitors of Src and Jak type kinases, respectively . By contrast, in erythroid cells, inhibitors of Src family and c-Abl tyrosine kinases (tyrphostin AG 82, PP2, imatinib) enhanced protein kinase C (PKC)-dependent cell growth and antagonized thrombin-promoted apoptosis by specifically stimulating PKCbeta activity . The PKC activating phorbol ester PMA (a growth factor in erythroid cells) induced the activation of Lyn and c-Abl tyrosine kinases, thus establishing a feedback inhibition of PKCbeta . Hence, developmental stage-specific crosstalk between PKC subtypes and tyrosine kinases appear to determine whether growth and survival of hematopoietic cells are promoted or inhibited by G-protein-coupled receptor agonists.

Appl Environ Microbiol, 2004 Dec, 70(12), 7329 - 41
Molecular characterization of a dechlorinating community resulting from in situ biostimulation in a trichloroethene-contaminated deep, fractured basalt aquifer and comparison to a derivative laboratory culture; Macbeth TW et al.; Sodium lactate additions to a trichloroethene (TCE) residual source area in deep, fractured basalt at a U.S . Department of Energy site have resulted in the enrichment of the indigenous microbial community, the complete dechlorination of nearly all aqueous-phase TCE to ethene, and the continued depletion of the residual source since 1999 . The bacterial and archaeal consortia in groundwater obtained from the residual source were assessed by using PCR-amplified 16S rRNA genes . A clone library of bacterial amplicons was predominated by those from members of the class Clostridia (57 of 93 clones), of which a phylotype most similar to that of the homoacetogen Acetobacterium sp . strain HAAP-1 was most abundant (32 of 93 clones) . The remaining Bacteria consisted of phylotypes affiliated with Sphingobacteria, Bacteroides, Spirochaetes, Mollicutes, and Proteobacteria and candidate divisions OP11 and OP3 . The two proteobacterial phylotypes were most similar to those of the known dechlorinators Trichlorobacter thiogenes and Sulfurospirillum multivorans . Although not represented by the bacterial clones generated with broad-specificity bacterial primers, a Dehalococcoides-like phylotype was identified with genus-specific primers . Only four distinct phylotypes were detected in the groundwater archaeal library, including predominantly a clone affiliated with the strictly acetoclastic methanogen Methanosaeta concilii (24 of 43 clones) . A mixed culture that completely dechlorinates TCE to ethene was enriched from this groundwater, and both communities were characterized by terminal restriction fragment length polymorphism (T-RFLP) . According to T-RFLP, the laboratory enrichment community was less diverse overall than the groundwater community, with 22 unique phylotypes as opposed to 43 and a higher percentage of Clostridia, including the Acetobacterium population . Bioreactor archaeal structure was very similar to that of the groundwater community, suggesting that methane is generated primarily via the acetoclastic pathway, using acetate generated by lactate fermentation and acetogenesis in both systems.

BMC Evol Biol . 2004 Nov 26;4(1):50.
Evolutionary relationships of Fusobacterium nucleatum based on phylogenetic analysis and comparative genomics; Mira A et al.; BACKGROUND: The phylogenetic position and evolutionary relationships of Fusobacteria remain uncertain . Especially intriguing is their relatedness to low G+C Gram positive bacteria (Firmicutes) by ribosomal molecular phylogenies, but their possession of a typical gram negative outer membrane . Taking advantage of the recent completion of the Fusobacterium nucleatum genome sequence we have examined the evolutionary relationships of Fusobacterium genes by phylogenetic analysis and comparative genomics tools . RESULTS: The data indicate that Fusobacterium has a core genome of a very different nature to other bacterial lineages, and branches out at the base of Firmicutes . However, depending on the method used, 35-56% of Fusobacterium genes appear to have a xenologous origin from bacteroidetes, proteobacteria, spirochaetes and the Firmicutes themselves . A high number of hypothetical ORFs with unusual codon usage and short lengths were found and hypothesized to be remnants of transferred genes that were discarded . Some proteins and operons are also hypothesized to be of mixed ancestry . A large portion of the Gram-negative cell wall-related genes seems to have been transferred from proteobacteria . CONCLUSIONS: Many instances of similarity to other inhabitants of the dental plaque that have been sequenced were found . This suggests that the close physical contact found in this environment might facilitate horizontal gene transfer, supporting the idea of niche-specific gene pools . We hypothesize that at a point in time, probably associated to the rise of mammals, a strong selective pressure might have existed for a cell with a Clostridia-like metabolic apparatus but with the adhesive and immune camouflage features of Proteobacteria.

Infect Immun, 2004 Dec, 72(12), 7063 - 72
Polymorphisms in the Chlamydia trachomatis cytotoxin locus associated with ocular and genital isolates; Carlson JH et al.; Chlamydia trachomatis is a strict human pathogen producing infections that cause medically important chronic inflammatory diseases, such as blinding trachoma and tubal factor infertility . Isolates exist as serotypes that fall into distinct biologic and pathological groups corresponding to differences in infection tissue tropism and invasion properties . Paradoxically, genome sequencing of several diverse strains has revealed a remarkable level of genomic synteny, suggesting that minor genetic differences determine the pathogen host- and tissue-specific infection characteristics . To better understand the genetic basis of chlamydial pathobiologic diversity, we performed comparative DNA-DNA microarray genomic hybridizations with all 15 C . trachomatis serovariants . We found there are few major genetic differences among the 15 serovars . An exception was the cytotoxin locus located in the plasticity zone, a region that exhibited significant polymorphisms among serovars . We therefore sequenced this region from all 15 serovars . The cytotoxin gene was interrupted by extensive mutations and deletions among the different serovars; however, three basic open reading frame motifs were discovered that correlated with noninvasive oculotropic, urogenitotropic, and invasive serovars . Of interest, only noninvasive genitotropic serovars possessed an intact N-terminal portion of the putative toxin gene . This region contains the UDP-glucose binding domain and the glycosyltransferase domain required for enzymatic activity of the clostridial toxin homologs, suggesting a role in urogenital infection or pathogenesis.

FEMS Microbiol Rev, 2004 Nov, 28(5), 543 - 52
Lipopolysaccharides of anaerobic beer spoilage bacteria of the genus Pectinatus--lipopolysaccharides of a Gram-positive genus; Helander IM et al.; Bacteria of the genus Pectinatus emerged during the seventies as contaminants and spoilage organisms in packaged beer . This genus comprises two species, Pectinatus cerevisiiphilus and Pectinatus frisingensis; both are strict anaerobes . On the basis of genomic properties the genus is placed among low GC Gram-positive bacteria (phylum Firmicutes, class Clostridia, order Clostridiales, family Acidaminococcaceae) . Despite this assignment, Pectinatus bacteria possess an outer membrane and lipopolysaccharide (LPS) typical of Gram-negative bacteria . The present review compiles the structural and compositional studies performed on Pectinatus LPS . These lipopolysaccharides exhibit extensive heterogeneity, i.e . several macromolecularly and structurally distinct LPS molecules are produced by each strain . Whereas heterogeneity is a common property in lipopolysaccharides, Pectinatus LPS have been shown to contain exceptional carbohydrate structures, consisting of a fairly conserved core region that carries a large non-repetitive saccharide that probably replaces the O-specific chain . Such structures represent a novel architectural principle of the LPS molecule.

Appl Environ Microbiol, 2004 Nov, 70(11), 6580 - 6
Novel endophytic nitrogen-fixing clostridia from the grass Miscanthus sinensis as revealed by terminal restriction fragment length polymorphism analysis; Miyamoto T et al.; Anaerobic nitrogen-fixing consortia consisting of N2-fixing clostridia and diverse nondiazotrophic bacteria were previously isolated from various gramineous plants (K . Minamisawa, K . Nishioka, T . Miyaki, B . Ye, T . Miyamoto, M . You, A . Saito, M . Saito, W . Barraquio, N . Teaumroong, T . Sein, and T . Tadashi, Appl . Environ . Microbiol . 70:3096-3102, 2004) . For this work, clostridial populations and their phylogenetic structures in a stand of the grass Miscanthus sinensis in Japan were assessed by a 16S rRNA gene-targeted terminal restriction fragment length polymorphism (TRFLP) analysis combined with most-probable-number (MPN) counts . PCR primers and restriction enzymes were optimized for analyses of the plant clostridia . Clostridia were detected in strongly surface-sterilized leaves, stems, and roots of the plants at approximately 10(4) to 10(5) cells/g of fresh weight; they made up a large proportion of N2-fixing bacterial populations, as determined by MPN counts associated with an acetylene reduction assay . Phylogenetic grouping by MPN-TRFLP analysis revealed that the clostridial populations belonged to group II of cluster XIVa and groups IV and V of cluster I; this result was supported by a culture-independent TRFLP analysis using direct DNA extraction from plants . When phylogenetic populations from M . sinensis and the soil around the plants were compared, group II clostridia were found to exist exclusively in M . sinensis.

Gene Ther, 2005 Jan, 12(2), 108 - 19
Adenoviral clostridial light chain gene-based synaptic inhibition through neuronal synaptobrevin elimination; Teng Q et al.; Clostridial neurotoxins have assumed increasing importance in clinical application . The toxin's light chain component (LC) inhibits synaptic transmission by digesting vesicle-docking proteins without directly altering neuronal health . To study the properties of LC gene expression in the nervous system, an adenoviral vector containing the LC of tetanus toxin (AdLC) was constructed . LC expressed in differentiated neuronal PC12 cells was shown to induce time- and concentration-dependent digestion of mouse brain synaptobrevin in vitro as compared to control transgene products . LC gene expression in the rat lumbar spinal cord disrupted hindlimb sensorimotor function in comparison to control vectors as measured by the Basso-Beattie-Bresnahan (BBB) scale (P<0.001) and rotarod assay (P<0.003) . Evoked electromyography (EMG) showed increased stimulus threshold and decreased response current amplitude in LC gene-transferred rats . At the peak of functional impairment, neither neuronal TUNEL staining nor reduced motor neuron density could be detected . Spontaneous functional recovery was observed to parallel the cessation of LC gene expression . These results suggest that light chain gene delivery within the nervous system may provide a nondestructive means for focused neural inhibition to treat a variety of disorders related to excessive synaptic activity, and prove useful for the study of neural circuitry.Gene Therapy (2005) 12, 108-119 . doi:10.1038/sj.gt.3302400 Published online 21 October 2004.

Khirurgiia (Mosk), 2004, (9), 41 - 3
{Anaerobic non-clostridial infection in acute appendicitis}; Lokhvitskii SV et al.; Etiological factors promoting anaerobic non-clostridial infection (ANI) after appendectomy are analyzed for the last 10 years (1993-2002) . Acute appendicitis was the indication for appendectomies in 2846 patients . In 55 (1.9%) patients ANI was detected 2-7 days after surgery . Later than 24 hours since beginning of the disease 40 (72.7%) patients were hospitalized . Diagnosis of ANI was based on typical clinical symptoms confirmed with bacteriological and morphological examinations . Treatment of ANI was complex: early radical surgery, massive antibiotic therapy, hyperbaric oxygenation, intensive care . 10-14 days after the last necrectomy (wound area from 139 to 1812 cm(2)) repair dermal-plastic surgeries were performed in 48 (87.3%) patients with good functional and cosmetic results . Two (3.6%) patients with ANI died . Of six patients with general forms of ANI admitted from other hospitals 3 patients died . Sepsis was the main cause of death . Late surgeries in general forms of ANI lead to worse results.

Appl Microbiol Biotechnol, 2004 Oct, 65(5), 520 - 9 Epub 2004 Jul 31.
Improvement of fermentative hydrogen production: various approaches; Nath K et al.; Fermentation of biomass or carbohydrate-based substrates presents a promising route of biological hydrogen production compared with photosynthetic or chemical routes . Pure substrates, including glucose, starch and cellulose, as well as different organic waste materials can be used for hydrogen fermentation . Among a large number of microbial species, strict anaerobes and facultative anaerobic chemoheterotrophs, such as clostridia and enteric bacteria, are efficient producers of hydrogen . Despite having a higher evolution rate of hydrogen, the yield of hydrogen {mol H2 (mol substrate(-1))} from fermentative processes is lower than that achieved using other methods; thus, the process is not economically viable in its present form . The pathways and experimental evidence cited in the literature reveal that a maximum of four mol of hydrogen can be obtained from substrates such as glucose . Modifications of the fermentation process, by redirection of metabolic pathways, gas sparging and maintaining a low partial pressure of hydrogen to make the reaction thermodynamically favorable, efficient product removal, optimum bioreactor design and integrating fermentative process with that of photosynthesis, are some of the ways that have been attempted to improve hydrogen productivity . This review briefly describes recent advances in these approaches towards improvement of hydrogen yield by fermentation.

J Dairy Sci, 2004 Oct, 87(10), 3407 - 16
The influence of treatment with dual purpose bacterial inoculants or soluble carbohydrates on the fermentation and aerobic stability of bermudagrass; Adesogan AT et al.; This study determined the effectiveness of an inoculant (BB), molasses, or a mixture of either BB and molasses (BBM) or BB and fibrolytic enzymes (BBE) for improving the fermentation and aerobic stability of bermudagrass . A 6-wk regrowth of Tifton 85 bermudagrass was conserved in quadruplicate mini silos alone or after treatment application . The inoculant contained a mixture of P . pentosaceus 12455, 1 x10(5) cfu/g of fresh forage, L . buchneri 40788, 4 x10(5) cfu/g of fresh forage, and beta-glucanase, alpha-amylase, and xylanase; BBE contained similar bacteria and enzymes as BB, but greater enzyme activities . Chemical composition was quantified after 2, 4, 7, 30, and 60 d of ensiling . Microbial composition and aerobic stability were measured after 60 d of ensiling, at which point the pH of additive-treated silages was consistently lower and DM recovery was higher than in untreated silages . The BB, BBM, and molasses-treated silages had less ammonia N than untreated silages, and BB, BBM, and BBE-treated silages had less residual water-soluble carbohydrates than untreated silages . All silages had high acetic acid (47.5 g/kg DM) and low lactic acid (1.7 g/kg DM) concentrations . However, untreated and BBE-treated silages had more butyric acid and ammonia N, suggesting that a clostridial fermentation had occurred . These butyric forages were more aerobically stable (27 d) but less desirable for feeding than those ensiled with BB or molasses, which were stable for 6.9 d . In conclusion, BB and molasses treatments improved the digestibility and fermentation of bermudagrass and produced higher quality silages that were stable for 6.9 d . Mixing BB with molasses or the inoculant tested was not more beneficial than BB or molasses alone.

Rev Physiol Biochem Pharmacol, 2004, 152, 165 - 82 Epub 2004.
Uptake of binary actin ADP-ribosylating toxins; Barth H; The focus of this article is on the cellular uptake mechanism of the family of binary actin ADP-ribosylating toxins from clostridia . These toxins are special-type AB toxins, because they are composed of two nonlinked proteins, which have to assemble on the surface of eukaryotic cells to act cytotoxically . The enzymatically active component (A), ADP-ribosylates G-actin in the cytosol of target cells . This leads to a complete depolymerization of the actin filaments and, thereby, to rounding up of cultured cells . The second component of these toxins, the binding/translocation component (B), mediates the transport of the enzyme component into the cytosol.

Zh Obshch Biol, 2004 May-Jun, 65(3), 195 - 210
{Highest level of division in classification of organisms . 3 . Monodermata and Didermata}; Shatalkin AI; The deepening our knowledge and embrassing the larger array of the investigated organisms leads to replacement of typological classifications with phylogenetic ones . This process seems to be the main stream of modern systematics . But typological classifications have not lost the value, remaining the important tool of the description of phylogeny . It is especially obvious today when molecular reconstructions are using so widely . However resulted phylogenetic classifications are difficult for understandable interpretation . Therefore phylogeneticist is interested in elaboration of typological classifications that can help to explain the results . As an example the phylogenetic classifications of organisms proposed recently by Cavalier-Smith (1998, 2002) and Gupta (1998, 2000) are considered . The modified system of Gupta is the most adequate description of organism phylogeny . Basal clostridia and togobacteria have to the greatest degree kept features of a common ancestor of organisms . From this common ancestor evolution spread by two phyletic lines . One of them included Gram-negative bacteria . The main groups of them have branched of from a common ancestor in the following order: (Deinococci, Chloroflexi) --> (Cyanobacteria) --> (Chlamydia, CFB, Fibrobacteria, Chlorobia) --> (Aquificae) --> --> (Epsilonproteobacteria, Deltaproteobacteria) --> (Alfaproteobacteria) --> (Betaproteobacteria) --> --> (Gammaproteobacteria) . In other phyletic line the main groups were separated in the following order: (Thermotogae) --> (Clostridia, Fusobacteria) --> (Bacillae) --> (Actinobacteria) . Exact position of archaebacteria and eukaryotes related to this line remains unclear . Typological division of organisms into Didermata and Monodermata (Gupta, 1998) corresponds to these two branches of a cladogram . The cell of the diderm organisms is covered with two membranes, plasmatic and outer . The cell of the monoderm organisms has only one plasmatic membrane . Development of the cellular organization at the earliest stages of evolution of a life went through use of non-lamellar (non-bilayer) lipids which could give a cell with one membrane (not two membranes as in the scenario of Cavalier-Smith (2001)) . Membranes appeared at the earliest stages of the evolution of life . Therefore their distinction is quite logical to take as a principle the first typological division of organisms . At the same time the typological classifications considered beyond the framework of phylogenetics, have no independent value . Typological classifications do not give monothetic division into groups . Always there are exceptions . So, among Monodermata there are Gram-negative forms (Acidaminococcaceae, Syntrophomonadaceae, some Thermoanaerobacteriaceae), which are didermic.

Mol Microbiol, 2004 Aug, 53(4), 1195 - 207
DNA binding properties of TnpX indicate that different synapses are formed in the excision and integration of the Tn4451 family; Adams V et al.; Site-specific recombination is an important mechanism for genetic exchange . Insertional recombination mediated by the recently delineated large resolvase or serine recombinase proteins is unique within the resolvase family as integration was thought to be a reaction catalysed only by members of the integrase or tyrosine recombinase family of site-specific recombinases . The large resolvase TnpX is a serine recombinase that is responsible for the movement of the Tn4451/3 family of chloramphenicol resistance elements, which are found within two genera of the medically important clostridia . Deletion analysis of TnpX showed that the last 110 amino acids (aa) of TnpX, which comprise a cysteine rich region, were not essential for its biological function and that a region required for DNA binding was located between aa 493-597 . Purified TnpX was shown to bind to the ends of the element and to the joint of the circular intermediate with high affinity but, most unusually, to bind to its target sites with a considerably lower affinity . Therefore, it was concluded that the resolvase-like excision and insertion reactions mediated by TnpX were distinct processes even though the same serine recombinase mechanism was involved . TnpX is the first large serine recombinase in which differential binding to its transposon and target sites has been demonstrated.

Am J Obstet Gynecol, 2004 Jul, 191(1), 266 - 70
Conservative management of clostridial endometritis; Lichtenberg ES et al.; OBJECTIVE: Clostridial infection during pregnancy may rapidly progress to life-threatening sepsis . This danger could prompt clinicians to consider hysterectomy as a treatment option as soon as clostridial infection becomes highly suspect or is confirmed, irrespective of its clinical severity . We present evidence that conservative management with the use of intravenous antibiotics is a reasonable initial treatment choice in women undergoing induced abortion who show no sign of sepsis . STUDY DESIGN: We describe the conservative treatment and epidemiologic investigation of unsuspected, culture proven clostridial infections in 5 women undergoing dilation and evacuation abortion during a 22-month period and review the existing literature . RESULTS: Prompt administration of broad-spectrum parenteral antibiotics successfully treated 5 healthy, stable women with culture proven uterine or blood stream clostridial infections . CONCLUSION: Intravenous broad-spectrum antibiotics with close surveillance is a reasonable initial treatment choice after atraumatic uterine evacuation for women with known or suspected clostridial infection that manifests no sign of sepsis.

Appl Microbiol Biotechnol . 2004 Jul 31; {Epub ahead of print}
Improvement of fermentative hydrogen production: various approaches; Nath K et al.; Fermentation of biomass or carbohydrate-based substrates presents a promising route of biological hydrogen production compared with photosynthetic or chemical routes . Pure substrates, including glucose, starch and cellulose, as well as different organic waste materials can be used for hydrogen fermentation . Among a large number of microbial species, strict anaerobes and facultative anaerobic chemoheterotrophs, such as clostridia and enteric bacteria, are efficient producers of hydrogen . Despite having a higher evolution rate of hydrogen, the yield of hydrogen {mol H(2) (mol substrate(-1))} from fermentative processes is lower than that achieved using other methods; thus, the process is not economically viable in its present form . The pathways and experimental evidence cited in the literature reveal that a maximum of four mol of hydrogen can be obtained from substrates such as glucose . Modifications of the fermentation process, by redirection of metabolic pathways, gas sparging and maintaining a low partial pressure of hydrogen to make the reaction thermodynamically favorable, efficient product removal, optimum bioreactor design and integrating fermentative process with that of photosynthesis, are some of the ways that have been attempted to improve hydrogen productivity . This review briefly describes recent advances in these approaches towards improvement of hydrogen yield by fermentation.

J Surg Orthop Adv, 2004 Summer, 13(2), 57 - 68
Gas gangrene and necrotizing fasciitis in the upper extremity; Perry BN et al.; Necrotizing soft tissue infections encompass a wide variety of clinical syndromes resulting from introduction of various pathogens into injured or devitalized tissue . The extent of microbial involvement in such tissue may range from simple contamination to overt and progressive local tissue necrosis, which, if untreated, may lead to septicemia and death . Early differentiation among these infections is not always possible, as there are overlapping classification criteria . These infections exist along a continuum of clinical severity with different etiological agents and associated medical conditions . The often subtle clues heralding the presence of a necrotizing soft tissue infection must be sought so that expeditious surgical debridement and broad-spectrum antibiotic management are initiated . Although experience enables the clinician to make a specific diagnosis based on early findings, aggressive and proper treatment of suspected infections remains the priority . The purpose of the article is to provide an overview of necrotizing soft tissue infections in the upper extremity, focusing on gas gangrene, or clostridial myonecrosis, and necrotizing fasciitis, to facilitate early diagnosis and optimal management of these lethal diseases.

J Mol Microbiol Biotechnol, 2004, 7(1-2), 78 - 87
Shaping and moving a spiroplasma; Trachtenberg S; The Mollicutes (Spiroplasma, Mycoplasma and Acholeplasma) are the most minimal cells known to exist, being the smallest and simplest free-living and self-replicating forms of life . Phylogenetically, the Mollicutes are related to gram-positive bacteria and have evolved, by regressive evolution and genome reduction, from Clostridia . The smallest genome in this group (Mycoplasma genitalium - 5.77 x 10(5) bp) is only twice that of a large virus (e.g., Entomopox viruses) . The largest Mollicute genome (Spiroplasma LB12 - 2.2 x 10(6) bp) is only about half that of, e.g., Escherichia coli . Structurally, the Mollicutes lack cell walls and flagella, but have internal cytoskeletons and are motile and chemotactic . Only a cholesterol-containing unit membrane envelops the cells . No analogs to the bacterial chemotactic and motility (che, mot, fla) genes, genes for a two-component signal transduction system, genes associated with gliding, or genomic homologs for the eukaryotic cytoskeleton and motor proteins were found in the Mollicutes . The Spiroplasmas are unique amongst the Mollicutes in having a well-defined basic helical cell geometry . In this respect, the Spiroplasma cell can, essentially, be viewed as a helical dynamic membranal tube (diameter approximately 0.2 microm; equivalent to that of one eukaryotic flagellar axoneme or to a bacterial flagellar bundle) . A flat cytoskeletal ribbon of parallel fibrils is attached to the inside of the cellular tube . Both tube and cytoskeleton are mutually coiled into a dynamic helix driven by differential length changes of the fibrils, which function as linear motors . The cytoskeletal ribbon follows the shortest (inner) helical line on the inner surface of the cellular tube . Being helical allows for further analytical reduction and consequent structural quantification of Spiroplasma . Of particular importance is the ability to correlate light and electron microscopy data and to calculate the fibril lengths (and corresponding molecular dimensions) in the helical and nonhelical dynamic states . The structural unit of the contractile cytoskeleton is a approximately 50-Angstrom-wide filament comprised of pairs of the 59-kD fib gene product . The monomers are arranged in pairs with opposite polarities allowing for a approximately 100-Angstrom-long axial repeat . The functional unit of the contractile cytoskeletal ribbon is a fibril comprised of an aligned pair of filaments . Neighboring repeats form a tetrameric ring with a lateral repeat of approximately 100 A . The axial length of the rings may shorten by approximately 40%, driving the changes in the fibril lengths and, consequently, helical dynamics . Local length changes result in helical symmetry breaking and nonreciprocating cell movements allowing for net directional displacement . Flexing allows for changes in swimming direction .

Mol Cell Biol, 2004 Jun, 24(11), 4664 - 76
Rap2B-dependent stimulation of phospholipase C-epsilon by epidermal growth factor receptor mediated by c-Src phosphorylation of RasGRP3; Stope MB et al.; Receptor tyrosine kinase regulation of phospholipase C-epsilon (PLC-epsilon), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors . PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-gamma1 and PLC-epsilon, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B . EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-epsilon, and Rap2B-dependent translocation of PLC-epsilon to the plasma membrane . GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-gamma1 but not of PLC-epsilon . Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor . EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation . Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-gamma1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-epsilon.

Biotechnol Bioeng, 2004 Jun 5, 86(5), 587 - 94
Fermentation of biomass-generated producer gas to ethanol; Datar RP et al.; The development of low-cost, sustainable, and renewable energy sources has been a major focus since the 1970s . Fuel-grade ethanol is one energy source that has great potential for being generated from biomass . The demonstration of the fermentation of biomass-generated producer gas to ethanol is the major focus of this article in addition to assessing the effects of producer gas on the fermentation process . In this work, producer gas (primarily CO, CO(2), CH(4), H(2), and N(2)) was generated from switchgrass via gasification . The fluidized-bed gasifier generated gas with a composition of 56.8% N(2), 14.7% CO, 16.5% CO(2), 4.4% H(2), and 4.2% CH(4) . The producer gas was utilized in a 4-L bioreactor to generate ethanol and other products via fermentation using a novel clostridial bacterium . The effects of biomass-generated producer gas on cell concentration, hydrogen uptake, and acid/alcohol production are shown in comparison with "clean" bottled gases of similar compositions for CO, CO(2), and H(2) . The successful implementation of generating producer gas from biomass and then fermenting the producer gas to ethanol was demonstrated . Several key findings following the introduction of producer gas included: (1) the cells stopped growing but were still viable, (2) ethanol was primarily produced once the cells stopped growing (ethanol is nongrowth associated), (3) H(2) utilization stopped, and (4) cells began growing again if "clean" bottled gases were introduced following exposure to the producer gas .

Appl Environ Microbiol, 2004 May, 70(5), 3096 - 102
Anaerobic nitrogen-fixing consortia consisting of clostridia isolated from gramineous plants; Minamisawa K et al.; We report here the existence of anaerobic nitrogen-fixing consortia (ANFICOs) consisting of N(2)-fixing clostridia and diverse nondiazotrophic bacteria in nonleguminous plants; we found these ANFICOs while attempting to overcome a problem with culturing nitrogen-fixing microbes from various gramineous plants . A major feature of ANFICOs is that N(2) fixation by the anaerobic clostridia is supported by the elimination of oxygen by the accompanying bacteria in the culture . In a few ANFICOs, nondiazotrophic bacteria specifically induced nitrogen fixation of the clostridia in culture . ANFICOs are widespread in wild rice species and pioneer plants, which are able to grow in unfavorable locations . These results indicate that clostridia are naturally occurring endophytes in gramineous plants and that clostridial N(2) fixation arises in association with nondiazotrophic endophytes.

Traffic, 2004 Jun, 5(6), 418 - 25
Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells; Carabeo RA et al.; Chlamydiae are gram-negative obligate intracellular pathogens to which access to an intracellular environment is paramount to their survival and replication . To this end, chlamydiae have evolved extremely efficient means of invading nonphagocytic cells . To elucidate the host cell machinery utilized by Chlamydia trachomatis in invasion, we examined the roles of the Rho GTPase family members in the internalization of chlamydial elementary bodies . Upon binding of elementary bodies on the cell surface, actin is rapidly recruited to the sites of internalization . Members of the Rho GTPase family are frequently involved in localized recruitment of actin . Clostridial Toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells . Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA . Rac but not Cdc42 was found to be activated by chlamydial attachment . The effect of dominant negative Rac expression on chlamydial uptake is manifested through the inhibition of actin recruitment to the sites of chlamydial entry . Studies utilizing Green Fluorescent Protein fusion constructs of Rac, Cdc42 and RhoA, showed Rac to be the sole member of the Rho GTPase family recruited to the site of chlamydial entry.

Syst Appl Microbiol, 2004 Feb, 27(1), 18 - 26
Comparative analysis of the four rRNA operons in Finegoldia magna ATCC29328; Todo K et al.; There are four rRNA operons rrnA, rrnB, rrnC and rrnD on the genome of Finegoldia magna (formerly Peptostreptococcus magnus) ATCC29328, which, in contrast to those of Clostridia, are dispersed around the chromosome . Using a BAC library we determined the nucleotide sequences and structures of all four operons, including their flanking regions, and performed comparative analyses . We identified putative boxA sequences in the operons, which should be required for rRNA transcription antitermination, as well as their respective tandem promoters, AT-rich UP elements in the upstream region and Rho-independent terminators in the downstream region . The mosaic features of the operons were revealed . Multiple tRNAs were identified in the downstream region of two operons, 18 in rrnC and 11 in rrnD . They were presumed to form transcription units together with rRNAs . rrnA and rrnB had repeat units with Rho-independent terminators instead of tRNAs in the downstream region . rrnB and rrnC were the most similar in rrn upstream promoter region . Focusing on the sequence variations of rRNA genes, rrnB alone was heterogeneous . In light of previous reports, we also assessed the correlation between intercistronic rRNA sequence differences and distances between the operons, but no positive correlation was seen in this strain.

Nat Rev Microbiol, 2003 Dec, 1(3), 237 - 42
Clostridia in cancer therapy; Minton NP; During the past decade, the search for an effective system for the selective delivery of high therapeutic doses of anti-cancer agents to tumours has explored a variety of ingenious and increasingly complex biological systems . These systems are most often based on gene therapy and use viral vectors as the delivery vehicle . Invariably, such systems have been found wanting with respect to a lack of tumour specificity, poor levels of transgene expression and inefficient distribution of the vector throughout the tumour mass . By contrast, the ability of intravenously injected clostridial spores to infiltrate, then selectively germinate in, the hypoxic regions of solid tumours seems to be a totally natural phenomenon, which requires no fundamental alterations and is exquisitely specific.

J Bacteriol, 2004 Apr, 186(7), 2099 - 106
Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon; Louis P et al.; The final steps in butyrate synthesis by anaerobic bacteria can occur via butyrate kinase and phosphotransbutyrylase or via butyryl-coenzyme A (CoA):acetate CoA-transferase . Degenerate PCR and enzymatic assays were used to assess the presence of butyrate kinase among 38 anaerobic butyrate-producing bacterial isolates from human feces that represent three different clostridial clusters (IV, XIVa, and XVI) . Only four strains were found to possess detectable butyrate kinase activity . These were also the only strains to give PCR products (verifiable by sequencing) with degenerate primer pairs designed within the butyrate kinase gene or between the linked butyrate kinase/phosphotransbutyrylase genes . Further analysis of the butyrate kinase/phosphotransbutyrylase genes of one isolate, L2-50, revealed similar organization to that described previously from different groups of clostridia, along with differences in flanking sequences and phylogenetic relationships . Butyryl-CoA:acetate CoA-transferase activity was detected in all 38 strains examined, suggesting that it, rather than butyrate kinase, provides the dominant route for butyrate formation in the human colonic ecosystem that contains a constantly high concentration of acetate.

Ann Chir Plast Esthet, 2004 Feb, 49(1), 32 - 5; discussion 36
A severe complication of muscle transfer: clostridial myonecrosis; Lorea P et al.; We present a case of gas gangrene as a severe complication of muscle transfer for opponensplasty . After debridement, the extensive palmar wound was covered by a free gracilis muscle transfer . This complication of muscle transfer has to our knowledge never been reported previously.

Pediatr Blood Cancer, 2004 Apr, 42(4), 338 - 42
Epidemiologic profile of symptomatic gastroenteritis in pediatric oncology patients receiving chemotherapy; El-Mahallawy HA et al.; BACKGROUND: Patients with cancer who receive intensive chemotherapeutic regimens are subject to profound immunosuppression and are susceptible to an extended array of pathogens . PROCEDURE: The infectious causes of symptomatic gastroenteritis as evidenced by diarrhea +/- fever, vomiting, and abdominal colic in children following chemotherapy were prospectively monitored at National Cancer Institute, Cairo University . RESULTS: A total of 104 diarrhea episodes were recorded in our institution during a 10-month period, of which an infectious cause was detected in 74 (71.1%) . Bacterial and fungal pathogens were isolated in culture from 41 (39.4%) and 24 (23.1%), respectively, while Clostridia difficile (C . difficile) and Cryptosporidium parvum (C . parvum) were detected in 15 (14.4%) and 10 (9.6%) of 104 diarrhea episodes following chemotherapy, respectively . Mixed infections were found in 24 of the patients; whereas, no cause was demonstrable in 30 . Hospital acquired and mixed infections were the worst as regards morbidity (P = 0.004 and 0.02) and mortality (P = 0.007 and <0.001) of the infectious episode regardless the cause, respectively . On multivariate analysis, C . difficile was associated with the highest mortality rate (OR = 0.04, 95% CI = 0.01-0.19), followed by fungal pathogens (OR = 0.20, 95% CI = 0.05-0.74) and bacterial infections (OR = 0.20, 95% CI = 0.05-0.79) . CONCLUSIONS: Infectious gastroenteritis is an important cause of morbidity and mortality in hospitalized pediatric cancer patients receiving intensified protocols of chemotherapy .

J Am Vet Med Assoc, 2004 Jan 1, 224(1), 96 - 9, 54
Hypomagnesemia among cows in a confinement-housed dairy herd; Donovan GA et al.; Between January and March 2002, 55 cows in a 1,200-cow commercial dairy herd in south Florida died . Most of the cows that were found dead did not have any clinical signs of disease prior to death . Because of a history of a feed change, a bloom of blue-green algae in cow cooling ponds, and initial necropsy findings of moderate enteritis, the preliminary differential diagnosis included clostridial enteritis, blue-green algae toxicosis, and mycotoxicosis . Rumen acidosis, hypomagnesemia, and heavy metal toxicosis were included as secondary considerations . On the basis of physical examination and gross necropsy findings, results of clinicopathol ogic testing, and results of feed and water analyses, a diagnosis of hypomagnesemia was made . Control procedures that were implemented included changing the forage source and increasing the magnesium concentration in the diet.

Curr Microbiol, 2003 Sep, 47(3), 203 - 7
Characterization and transcription of the genes encoding enzymes involved in butyrate production in Butyrivibrio fibrisolvens; Asanuma N et al.; Genes encoding enzymes that catalyze butyryl-CoA formation from acetyl-CoA in a type II strain of Butyrivibrio fibrisolvens were analyzed . The genes encoding thiolase, beta-hydroxybutyryl-CoA dehydrogenase, butyryl-CoA dehydrogenase, and electron transfer flavoproteins were clustered, but the crotonase gene was not present in this region . The deduced amino acid sequences of these enzymes were similar to those of clostridia . The clustered genes were shown to be cotranscribed . The rate of butyrate production increased with an increase in acetate concentration in the medium up to 5 mM, suggesting that the butyryl-CoA/acetate CoA transferase reaction limits butyrate production . Transcription of the clustered genes was not affected by acetate concentration, suggesting that acetate does not affect the synthesis of enzymes involved in butyryl-CoA formation . These results confirm that acetate stimulates butyrate production by acting as a CoA acceptor in the butyryl-CoA/acetate CoA transferase reaction.

Water Sci Technol, 2003, 48(4), 185 - 8
Isolation and characterization of solventogenic, cellulase-free xylanolytic Clostridia from cow rumen; Sankar M et al.; Hemicellulolytic obligate anaerobes capable of converting a range of agricultural substrates and monomeric sugars of hemicellulose to solvents and acids were isolated from cow rumen fluid . The strains were identified as 5 strains of C . beijerinckii (3 different genotypes), 3 strains of C . acetobutylicum (2 different genotypes), 1 strain of C . butyricum and 3 strains of C . bifermentans (2 different genotypes) . All of them produced cellulase-free xylanase activity . C . acetobutylicum LU1 produced solvent, a high of 5.1 gl(-1) and C.bifermentans LU1 produced xylanase activity a high of 4.2 IU ml(-1).

South Med J, 2003 Sep, 96(9), 900 - 5
Necrotizing soft tissue infections: a guide to early diagnosis and initial therapy; Majeski JA et al.; Necrotizing skin and soft tissue infections are caused by many different bacteria, are frequently polymicrobial, and may have a deceptively innocent early clinical presentation . Clostridial and nonclostridial necrotizing infections are frequently similar in their early presentation . The initial presentation of these infections can be insidious, which results in delay in diagnosis and the start of therapy . The clinician must use sound medical principles of clinical history and meticulous examination in each patient, combined with constant suspicion, to establish a timely diagnosis . This group of infectious diseases is associated with frequent morbidity and significant mortality rates, which increase with any delay in the diagnosis and the initiation of medical and surgical therapy . Also associated with these necrotizing infections is an excessive index of litigation . This review is intended as a guide for the clinician in making an early diagnosis of any necrotizing skin and soft tissue infection and initiating effective medical and surgical therapy.

Cancer, 2003 Oct 1, 98(7), 1483 - 90
Isolated limb perfusion with tumor necrosis factor-alpha and melphalan for patients with unresectable soft tissue sarcoma of the extremities; Noorda EM et al.; BACKGROUND: Since 1992, isolated limb perfusion (ILP) with tumor necrosis factor-alpha (TNFalpha) and melphalan has been used for the treatment of patients with unresectable soft tissue sarcomas of the extremities . The authors retrospectively studied the results of limb salvage surgery using TNFalpha-ILP at their institution . METHODS: From 1992 to 2001, 49 patients (mean age, 51 years; range, 14-85 years) underwent ILP for unresectable soft tissue sarcomas of the extremities . All patients received melphalan and TNFalpha (four patients also received interferon-gamma) . The median follow-up was 26 months (range, from 2 days to 103 months) . RESULTS: In 1 patient (2%) who died 2 days after undergoing ILP, response and acute limb toxicity could not be assessed . One patient (2%) attained a clinical complete response (2%), 23 patients (47%) attained a clinical partial response, 17 patients (35%) demonstrated no change, and 7 patients (14%) had tumor progression . Thirty-one patients (63%) underwent tumor resection . Histologic material also was available from eight amputations and three punctures/biopsies . Pathologic response was complete in 4 patients (8%), partial in 14 patients (29%), and no change was observed in 24 patients (49%) . Final response, based on both clinical and pathologic assessment in which pathology was decisive, was complete in 4 patients (8%) and partial in 27 patients (55%), resulting in a final overall response rate of 63% . Local control with preservation of the limb was attained in 28 patients (57%) . Four of 32 patients (13%) who had been rendered tumor free by ILP with or without undergoing resection and radiation therapy, developed a local recurrence . The 5-year disease specific survival rate was 48% for the 49 patients . Acute limb toxicity after ILP was a mild Grade 1-2 reaction in 35 patients (71%) patients, a Grade 3 reaction in 12 patients (25%), and a Grade 4 reaction in 1 patient (2%) . Three major ILP-related complications were encountered, including arterial thrombosis in two patients and a fulminant Clostridial infection leading to death in one patient . There were no severe cardiovascular reactions after ILP . CONCLUSIONS: In patients with unresectable soft tissue sarcomas of the limbs who underwent ILP with TNFalpha and melphalan followed by resection of the tumor remnant when possible, a 63% overall tumor response rate and a 57% local control rate with limb preservation was achieved .

Zh Mikrobiol Epidemiol Immunobiol, 2003 Jul-Aug, (4), 71 - 4
{Modifying action of oxytocin on the biological properties of the causative agents of anaerobic non-clostridial infection}; Abramzon OM et al.; In a number of in vitro experiments the effect of oxytocin on the antilysozyme and anticomplemental activity of Propiobacterium propionicum, Bacteroides fragilis, Prevotella melaninogenica and Peptostreprtococcus anaerobius, isolated from patients with acute pyoinflammatory pleuropulmonary diseases, was studied . Antibiotic resistance dynamics of the infective agents under study to lincomycin, clindamycin, thienam, vancomycin was also detected . The inhibiting activity of oxytocin on the persistence properties of B . fragilis, P . melanogenica and P . anaerobius was noted . Under the influence of the preparations used changes in the sensitivity of the strains to a number of antibiotics of the lincosamide, carbapenem and glycopeptide groups were found to occur . The data thus obtained were indicative of the possible mechanisms of action of oxytocin in the treatment of acute pyoinflammatory pleuropulmonary diseases of anaerobic nonclostridial etiology.

Microbiology, 2003 Sep, 149(Pt 9), 2529 - 37
Analysis of genes of tetrahydrofolate-dependent metabolism from cultivated spirochaetes and the gut community of the termite Zootermopsis angusticollis; Salmassi TM et al.; The hindguts of wood-feeding termites are the sites of intense, CO2-reductive acetogenesis . This activity profoundly influences host nutrition and methane emissions . Homoacetogens previously isolated from diverse termites comprised novel taxa belonging to two distinct bacterial phyla, Firmicutes and Spirochates . Little else is known about either the diversity or abundance of homoacetogenic species present in any given termite or the genetic details underlying CO2-reductive acetogenesis by Spirochaetes . A key enzyme of CO2-reductive acetogenesis is formyltetrahydrofolate synthetase (FTHFS) . A previously designed primer set was used to amplify FTHFS genes from three isolated termite-gut spirochaetes . Sequencing DNA flanking the FTHFS gene of Treponema strain ZAS-2 revealed genes encoding two acetogenesis-related enzymes, methenyltetrahydrofolate cyclohydrolase and methylenetetrahydrofolate dehydrogenase . Although termite-gut spirochaetes are only distantly related to clostridia at the ribosomal level, their tetrahydrofolate-dependent enzymes appear to be closely related . In contrast, homologous proteins identified in the non-homoacetogenic oral spirochaete Treponema denticola were only distantly related to those from clostridia and the termite-gut treponemes . Having demonstrated their utility with spirochaete pure cultures, the FTHFS primers were used to construct a 91-clone library from the termite-gut community DNA . From this, 19 DNA and eight amino acid FTHFS types were identified . Over 75 % of the retrieved clones formed a novel, coherent cluster with the FTHFS homologues obtained from the termite-gut treponemes . Thus, FTHFS gene diversity in the gut of the termite Zootermopsis angusticollis appears to be dominated by spirochaetes . The homoacetogenic capacity of termite-gut spirochaetes may have been acquired via lateral gene transfer from clostridia.

Biochim Biophys Acta, 2003 Aug 18, 1641(2-3), 121 - 35
Revisiting the role of SNAREs in exocytosis and membrane fusion; Szule JA et al.; For over a decade SNARE hypotheses have been proposed to explain the mechanism of membrane fusion, yet the field still lacks sufficient evidence to conclusively identify the minimal components of native fusion . Consequently, debate concerning the postulated role(s) of SNAREs in membrane fusion continues . The focus of this review is to revisit original literature with a current perspective . Our analysis begins with the earliest studies of clostridial toxins, leading to various cellular and molecular approaches that have been used to test for the roles of SNAREs in exocytosis . We place much emphasis on distinguishing between specific effects on membrane fusion and effects on other critical steps in exocytosis . Although many systems can be used to study exocytosis, few permit selective access to specific steps in the pathway, such as membrane fusion . Thus, while SNARE proteins are essential to the physiology of exocytosis, assay limitations often prevent definitive conclusions concerning the molecular mechanism of membrane fusion . In all, the SNAREs are more likely to function upstream as modulators or priming factors of fusion.

Methods, 2003 Jul, 30(3), 191 - 7
SNAREs and epithelial cells; Weimbs T et al.; SNARE proteins control the membrane fusion events of membrane trafficking pathways . Work in epithelial cells has shown that polarized trafficking to the apical and basolateral plasma membrane domains requires different sets of SNAREs, suggesting a mechanism that contributes to the overall specificity of polarized trafficking and, perhaps, the formation and maintenance of polarity itself . This article describes methods that have been designed and adapted specifically for the investigation of SNAREs in epithelial cells . The knowledge of the subcellular localization of a SNARE of interest is essential to understand its function . Unfortunately, the endogenous expression levels of SNAREs are often low which makes detection challenging . We provide guidelines for determination of the localization of SNAREs by immunofluorescence microscopy including methods for signal amplification, antigen retrieval, and suppression of antibody cross-reactivity . To define which trafficking pathway a SNARE of interest is involved in, one needs to specifically inhibit its function . We provide guidelines for SNARE inhibition by overexpression of the SNARE of interest . An alternative is to introduce inhibitors of SNARE function, such as antibodies or clostridial toxins, into cells . Two methods are presented to make this possible . The first allows the monitoring of effects on trafficking pathways by biochemical assays, and is based on plasma membrane permeabilization using the bacterial toxin streptolysin-O . The second is suitable for single-cell observations and is based on microinjection.

Arch Microbiol, 2003 Jun, 179(6), 444 - 56 Epub 2003 May 13.
The anabolic pyruvate oxidoreductase from Methanococcus maripaludis; Lin WC et al.; In autotrophic methanogens, pyruvate oxidoreductase (POR) plays a key role in the assimilation of CO(2) and the biosynthesis of organic carbon . This enzyme has been purified to homogeneity, and the genes from Methanococcus maripaludis were sequenced . The purified POR contained five polypeptides with molecular masses of 47, 33, 25, 21.5 and 13 kDa . The N-terminal sequences of four of the polypeptides had high similarity to the subunits commonly associated with this enzyme from other archaea . However, the 21.5-kDa polypeptide had not been previously observed in PORs . Nucleotide sequencing of the gene cluster encoding the POR revealed six open reading frames ( porABCDEF) . The genes porABCD corresponded to the subunits previously identified in PORs . On the basis of the N-terminal amino acid sequence, porE encoded the 21.5-kDa polypeptide and contained a high cysteinyl residue content and a motif indicative of a {Fe-S} cluster . porF also had a high sequence similarity to porE, a high cysteinyl residue content, and two {Fe-S} cluster motifs . Homologs to porE were also present in the genomic sequences of the autotrophic methanogens Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus . Based upon these results, it is proposed that PorE and PorF are components of a specialized system required to transfer low-potential electrons for pyruvate biosynthesis . Some biochemical properties of the purified methanococcal POR were also determined . This unstable enzyme was very sensitive to O(2 )and demonstrated high activity with pyruvate, oxaloacetate, and alpha-ketobutyrate . Methyl viologen, rubredoxin, FMN, and FAD were readily reduced . Activity was also observed with spinach and clostridial ferredoxins and cytochrome c . Coenzyme F(420) was not an electron acceptor for the purified enzyme.

Diabetes Metab, 2002 Dec, 28(6 Pt 2), 3S78 - 84; discussion 3S108-12
IL-1beta-induced nitric oxide release from insulin-secreting beta-cells: further evidence for the involvement of GTP-binding proteins; Tannous M et al.; Recently, we have demonstrated regulatory roles for G-proteins (e.g., H-Ras) in IL-1beta induced NO release from HIT-T15 cells . Herein, we report a similar regulatory mechanism for IL-1beta induced NO release from RIN5F and INS-1 cells . Our data indicate that functional inactivation of Ras, either by Clostridial toxins or by specific inhibitors of Ras function, results in a significant inhibition in IL-1beta induced NO release, suggesting that activation of specific G-proteins is essential for IL-1beta induced NO release . In the present study, we report possible loci where IL-1beta treatment might result in functional activation of these G-proteins . For example, IL-1beta treatment resulted in significant reduction in (high-and low-affinity) GTPase activities in lysates derived from normal rat islets; such a scenario might lead to retention of candidate G-proteins in GTP-bound, active conformation . Further, IL-1beta treatment increased the G-protein carboxyl methyl transferase activity as well as carboxyl methylation of endogenous beta-cell proteins; such a modification has been shown to increase the membrane association and interaction of these G-proteins with their respective effector proteins . Also, we report immunologic localization of H-Ras regulatory proteins including its nucleotide exchange factor (GRF-1) and its effector protein (eg., Raf-1) in isolated beta-cells . Together, our data indicate localization, and regulation by IL-1beta, of specific enzymes that are critical to activation of G-proteins . Based on these preliminary findings, we propose a model for the involvement of G-proteins in IL-1beta induced NO release and subsequent demise of the pancreatic beta-cell.

Dev Biol (Basel), 2002, 111, 101 - 8
Alternatives in testing of bacterial toxins and antitoxins; Sesardic D; The potency of several novel toxin-derived biological therapeutic products now in routine medical use is determined exclusively by in vivo methods . In addition, large numbers of animals continue to be used for the potency and safety testing of therapeutic antitoxins . There is thus an increasing need to develop acceptable alternative assays for toxicity testing which would reduce the need for a mouse lethality assay in quality control . Scientific advances in the understanding of the mode of action of clostridial neurotoxins have now provided the basis for improving conventional testing procedures for both therapeutic toxins and antitoxins.

Arch Microbiol, 2003 May, 179(5), 321 - 8 Epub 2003 Mar 26.
Fermentation of glycolate by a pure culture of a strictly anaerobic gram-positive bacterium belonging to the family Lachnospiraceae; Janssen PH et al.; The component bacteria of a three-membered mixed culture able to ferment glycolate to acetate, propionate and CO(2) were isolated in pure culture . All three strains were strict anaerobes that, on the basis of comparative 16S rRNA gene sequence analysis, belonged to the order Clostridiales in the phylum Firmicutes (low G+C gram-positive bacteria) . Two of the strains were not involved in glycolate metabolism . The third, the glycolate-fermenting strain 19gly4 (DSM 11261), was related to members of the family Lachnospiraceae . The cells of strain 19gly4 were oval- to lemon-shaped, 0.85 microm long and 0.65 microm in diameter, occurring singly, in pairs, or in chains of up to 30 cells . Strain 19gly4 fermented glycolate or fumarate to acetate, succinate, and CO(2) . Hydrogen was not formed, and strain 19gly4 was able to grow on glycolate in pure culture without any syntrophic hydrogen transfer and without the use of an external electron acceptor . There was no evidence for homoacetogenic metabolism . This bacterium therefore differs in metabolism from previously reported glycolate-utilising anaerobes.

Am J Respir Cell Mol Biol, 2003 Jul, 29(1), 39 - 47 Epub 2003 Jan 10.
The RhoA/Rho kinase pathway regulates nuclear localization of serum response factor; Liu HW et al.; RhoA and its downstream target Rho kinase regulate serum response factor (SRF)-dependent skeletal and smooth muscle gene expression . We previously reported that long-term serum deprivation reduces transcription of smooth muscle contractile apparatus encoding genes, by redistributing SRF out of the nucleus . Because serum components stimulate RhoA activity, these observations suggest the hypothesis that the RhoA/Rho kinase pathway regulates SRF-dependent smooth muscle gene transcription in part by controlling SRF subcellular localization . Our present results support this hypothesis: cotransfection of cultured airway myocytes with a plasmid expressing constitutively active RhoAV14 selectively enhanced transcription from the SM22 and smooth muscle myosin heavy chain promoters and from a purely SRF-dependent promoter, but had no effect on transcription from the MSV-LTR promoter or from an AP2-dependent promoter . Conversely, inhibition of the RhoA/Rho kinase pathway by cotransfection with a plasmid expressing dominant negative RhoAN19, by cotransfection with a plasmid expressing Clostridial C3 toxin, or by incubation with the Rho kinase inhibitor, Y-27632, all selectively reduced SRF-dependent smooth muscle promoter activity . Furthermore, treatment with Y-27632 selectively reduced binding of SRF from nuclear extracts to its consensus DNA target, selectively reduced nuclear SRF protein content, and partially redistributed SRF from nucleus to cytoplasm, as revealed by quantitative immunocytochemistry . Treatment of cultured airway myocytes with latrunculin B, which reduces actin polymerization, also caused partial redistribution of SRF into the cytoplasm . Together, these results demonstrate for the first time that the RhoA/Rho kinase pathway controls smooth muscle gene transcription in differentiated smooth muscle cells, in part by regulating the subcellular localization of SRF . It is conceivable that the RhoA/Rho kinase pathway influences SRF localization through its effect on actin polymerization dynamics.

Burns, 2002 Dec, 28(8), 791 - 4
Enzymatic debridement of burn wound with collagenase in children with partial-thickness burns; Ozcan C et al.; Seventy-eight pediatric burn patients treated by enzymatic debridement with collagenase clostridiopeptidase A (CCA), were compared to 41 patients those burn wounds were excised surgically . Patients whose burn wounds were initially assessed as partial-thickness at admission were enrolled in the study . Total removal of eschar was achieved in 49 of 78 (62.8%) patients by CCA only (group D) . In 29 patients (37.2%), therapy with CCA was ceased because of the development of burn wound infection or a manifest need for grafting of the wound, therefore, these patients underwent tangential wound excision (group DS) . The records of 41 patients, treated by early tangential excision, having similar burn wounds by extent and depth with groups D and DS were used as controls (group S).There was no significant difference between the time to achieve a clean wound bed in groups D, DS and S (mean 7.8, 8, and 7 days, respectively, P>0.05) . In group D, none of the patients required blood transfusion, except one . Patients in group DS were found to have fewer excisions (mean 1.1) when compared to those in group S (mean 1.5, P<0.05) . The shortest hospital stay was found in group D (12.5 days, P<0.01) . In conclusion, the use of CCA, provided a short hospital stay, reduced the overall need for surgery and blood transfusions in patients with partial-thickness burns . Thus, CCA should be considered as an initial treatment of choice for removal of eschar in children, having a partial-thickness burn wound without infection.

Blood, 2002 Dec 15, 100(13), 4462 - 9
Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin; Vidal C et al.; Platelet activation by thrombin or thrombin receptor-activating peptide (TRAP) results in extensive actin reorganization that leads to filopodia emission and lamellae spreading concomitantly with activation of the Rho family small G proteins, Cdc42 and Rac1 . Evidence has been provided that direct binding of Cdc42-guanosine triphosphate (GTP) and Rac1-GTP to the N-terminal regulatory domain of the p21-activated kinase (PAK) stimulates PAK activation and actin reorganization . In the present study, we have investigated the relationship between shape change and PAK activation . We show that thrombin, TRAP, or monoclonal antibody (MoAb) anti-Fc(gamma)RIIA IV.3 induces an activation of Cdc42 and Rac1 . The GpVI ligand, convulxin (CVX), that forces platelets to lamellae spreading efficiently activates Rac1 . Thrombin, TRAP, MoAb IV.3, and CVX stimulate autophosphorylation and kinase activity of PAK . Inhibition of Cdc42 and Rac1 with clostridial toxin B inhibits PAK activation and lamellae spreading . The cortical-actin binding protein, p80/85 cortactin, is constitutively associated with PAK in resting platelets and dissociates from PAK after thrombin stimulation . Inhibition of PAK autophosphorylation by toxin B prevents the dissociation of cortactin . These results suggest that Cdc42/Rac1-dependent activation of PAK may trigger early platelet shape change, at least in part through the regulation of cortactin binding to PAK.

Ann N Y Acad Sci, 2002 Oct, 971, 210 - 21
Molecular analysis of SNAP-25 function in exocytosis; Graham ME et al.; It is generally accepted that the SNARE proteins form the core of the machinery for intracellular membrane fusion and that formation of a SNARE complex is crucially important . Our aim is to dissect the molecular roles of the SNARE proteins and their regulators in physiological membrane fusion during exocytosis . We have developed approaches that allow us to manipulate protein expression in model secretory cells, PC12 and adrenal chromaffin cells, and to combine this with assay of exocytosis at high-time resolution using carbon-fiber amperometry . This technique allows us to assess the extent of exocytosis and to follow the kinetics of single secretory granule release events with millisecond time resolution . We established that manipulation of proteins involved in the exocytotic machinery can lead to detectable and interpretable changes in exocytosis kinetics that have revealed novel roles in late stages of exocytosis . Using this approach we have begun to analyze the function of SNAP-25B using a mutant resistant to the Clostridial neurotoxin BoNT/E . This SNAP-25 mutant can reconstitute exocytosis in BoNT/E-treated cells . With this construct it is possible to analyze the consequences of any introduced mutation in the absence of functional endogenous protein . We review here its use in the analysis of palmitoylated cysteines of SNAP-25 and the conserved residues of the 0 layer of the SNARE complex . The data suggest an important role of the cysteines, but not the 0 layer glutamines, in triggered exocytosis.

Avian Pathol, 2002 Aug, 31(4), 317 - 53
Anticoccidial vaccines for broiler chickens: pathways to success; Williams RB; The use of live vaccines, either attenuated or non-attenuated, for the control of coccidiosis due to Eimeria infections in broiler breeder or layer chickens is well established . Use in broilers, however, has been slow to gain acceptance . This has been partly for economic reasons, but also because of perceived adverse effects on early chick growth, particularly with non-attenuated vaccines, and concerns about timely onset of protective immunity in such short-lived birds . This review describes advances in understanding of epidemiological factors and recent improvements of administration methods that have helped to allay these fears and to make the use of anticoccidial vaccines in broilers technically achievable . Topics discussed include: (1) types of commercially available vaccine, (2) vaccines in development, (3) vaccination methods and equipment, (4) basis of vaccine efficacy and immunogenic variation of parasites, (5) key factors in the survival, sporulation and dissemination of vaccinal oocysts, (6) descriptions and significance of patterns of litter oocyst accumulation and occurrence of intestinal lesions in vaccinated flocks, (7) rotation of anticoccidial vaccination and chemotherapy to restore drug sensitivity to resistant wild-type coccidia, (8) combinations of anticoccidial vaccination and chemotherapy, (9) interactions between coccidiosis and clostridiosis in broilers and compatibilities of potential control methods, (10) published performance data for live anticoccidial vaccines in broilers, (11) possible further developments of live vaccines.

Cell Microbiol, 2002 Sep, 4(9), 557 - 69
Activation of Rac, Cdc42 and other downstream signalling molecules by Bartonella bacilliformis during entry into human endothelial cells; Verma A et al.; Bartonella bacilliformis is an intracellular bacterial pathogen of human endothelial cells . In vitro incubation of B . bacilliformis with human endothelial cells leads to the formation of filamentous actin extensions (filopodia) within 30 min, followed by formation of membrane rufflings or lamellipodia within 1 h of incubation . By immunofluorescence, F-actin phalloidin staining and anti-Rac antibodies were shown to co-localize in the membrane rufflings, indicating the recruitment of activated Rac at lamellipodia . Preincubation of endothelial cells with the Clostridial toxin, TcdB-10463, which inactivates the Rho-family GTPases, Rho, Rac and Cdc42, inhibited the entry of B . bacilliformis by 50-90% . Preincubation of endothelial cells with the Clostridial toxin, TcsL-1522, which specifically inactivates Rac and, to a lesser extent, Cdc42, but not Rho, inhibited entry by 30-40% . A 3.4-5.0-fold increase in activated (GTP-bound) -intracellular Rac and Cdc42 was observed in affinity precipitation assays . Increased kinase activity of p21-activated kinase (PAK), a specific downstream effector of activated Rac/Cdc42 was also observed during the time course of infection . Activation of SAPK/JNK-1 and 2, and p38 MAPKs in signalling pathways, was also detected during infection with Bartonella, as was increased binding activity of AP-1 transcription factor.

J Hand Surg {Am}, 2002 Sep, 27(5), 788 - 98
Collagen as a clinical target: nonoperative treatment of Dupuytren's disease; Badalamente MA et al.; The cellular events leading to abnormal synthesis of collagen are important to our understanding of pathologic processes leading to impaired joint function . The contracture of Dupuytren's disease is a notable example . In a series of controlled phase-2 clinical trials, excessive collagen deposition in Dupuytren's disease has been targeted by a unique nonoperative method using enzyme (Clostridial collagenase) injection therapy to lyse and rupture finger cords causing metacarpophalangeal and/or proximal interphalangeal joint contractures . Forty-nine patients were treated in a random, placebo-controlled trial of one dose of collagenase versus placebo at one center . Subsequently 80 patients were treated in a random, placebo-controlled, dose-response study of collagenase at 2 test centers . The results of these studies indicate that nonoperative collagenase injection therapy for Dupuytren's disease is both a safe and effective method of treating this disorder in the majority of patients as an alternative to surgical fasciectomy . Phase-3 efficacy trials are now being planned to further develop and test this method under Food and Drug Administration regulatory guidelines . The findings of our study may lead to simpler and less invasive nonoperative treatments of joint limitation in which collagen plays a major pathologic role.

Oral Dis, 2002 Jul, 8(4), 224 - 6
Clostridial deep neck infection developed after extraction of a tooth: a case report and review of the literature in Japan; Nakamura S et al.; A rare case of severe deep neck infection caused by clostridia after extraction of the left lower canine is presented . The patient was a 63-year-old Japanese woman who had a history of diabetes . The pertinent literature in Japan is reviewed and discussed.

Pathology, 2002 Aug, 34(4), 344 - 7
Effect of long-term mobile communication microwave exposure on vascular permeability in mouse brain; Finnie JW et al.; AIMS: To study the effect of long-term exposure to global system for mobile communication (GSM) radiofrequency fields on vascular permeability in murine brains . METHODS: Using a purpose-designed exposure system at 900 MHz, mice were given a 60-minute far-field, whole body exposure on each of 5 days per week for 104 weeks at specific absorption rates (SAR) of 0.25, 1.0,2.0 and 4.0 W/kg . Control mice were sham-exposed or permitted free movement in a cage to evaluate any stress-related effects . Albumin immunohistochemistry was used to detect increased vascular permeability and the efficacy of the vascular tracer was confirmed with a positive control group exposed to a clostridial toxin known to increase vascular permeability in the brain . RESULTS: In all exposed and control groups, albumin extravasation was minimal, often leptomeningeal, and was deemed insignificant as a maximum of three capillaries or venules in a given brain showed leakage from the very many blood vessels present in the three coronal brain sections . CONCLUSIONS: These results suggest that prolonged exposure to mobile telephone-type radiation produces negligible disruption to blood-brain barrier integrity at the light microscope level using endogenous albumin as a vascular tracer.

J Reprod Med, 2002 Jul, 47(7), 588 - 90
Uterine gas vs . vessel calcification . A case report; Cogan SL et al.; BACKGROUND: Pelvic ultrasound plays a significant role in the diagnosis of uterine abnormalities; however, occasionally the radiologic findings may be misleading . CASE: A case of suspected uterine vessel calcification was detected on ultrasound in a patient hospitalized with pelvic inflammatory disease that was originally interpreted as uterine gas . The lack of change over serial ultrasound scans in conjunction with the patient's benign clinical course led to reassessment of the initial impression; the findings were then attributed to uterine vessel calcification . CONCLUSION: Uterine vessel calcification in a polysubstance abuser may be a manifestation of the known atherosclerotic cardiovascular complications of cocaine . Although ultrasound findings may be misleading, clinical judgment is essential to rule out clostridial endomyometritis.

Mikrobiologiia, 2002 May-Jun, 71(3), 330 - 5
{Catalase and superoxide dismutase in the cells of strictly anaerobic microorganisms}; Briukhanov AL et al.; Strictly anaerobic microorganisms relating to various physiological groups were screened for catalase and superoxide dismutase (SOD) activity . All of the investigated anaerobes possessed the SOD activity, necessary for protection against toxic products of oxygen reduction . High specific activities of SOD were found in Acetobacterium woodii and Acetobacterium wieringae . Most of the investigated clostridia and acetogens were catalase-negative . A significant activity of catalase was found in Thermohydrogenium kirishiense, in representatives of the genus Desulfotomaculum, and in several methanogens . Methanobrevibacter arboriphilus had an exceptionally high catalase activity after growth in medium supplemented with hemin . Hemin also produced a strong positive effect on the catalase activity in many other anaerobic microorganisms . In methanogens, the activities of the enzymes of antioxidant defense varied in wide ranges depending on the stage of growth and the energy source.

Am J Hematol, 2002 Jun, 70(2), 145 - 8
Spontaneous gas gangrene in malignant lymphoma: an underreported complication?
Garcia-Suarez J, de Miguel D, Krsnik I, Barr-Ali M, Hernanz N, Burgaleta C.
We report a case of spontaneous gas gangrene (SGG), the most rapidly progressive form of clostridial infection, in a patient with non-Hodgkin's lymphoma (NHL) . We review the literature and examine the association between these two entities . A 43-year-old man with NHL developed fatal C . perfringens-associated SGG and massive hemolysis during induction chemotherapy . Although patients with NHL usually have several risk factors of SGG, such as bowel involvement or neutropenia, only two cases have been described previously in detail . Common features of all reports are a delayed diagnosis and a fatal outcome . Awareness of this condition should result in prompt antibiotic therapy at the onset of typical presenting symptoms in any lymphoma patient, especially if risk factors are present .

Asian Cardiovasc Thorac Ann, 2002 Jun, 10(2), 178 - 80
Gas gangrene in patient with atherosclerosis obliterans; Niimi M et al.; Clostridia are the main cause of nontraumatic spontaneous gas gangrene . Poor blood flow due to arterial occlusion exacerbates the anaerobic condition . Fulminant gas gangrene in a 54-year-old man with atherosclerosis obliterans was treated by revascularization of the iliac artery using endarterectomy, and his gangrenous lower leg was amputated . However, he died from renal failure.

Endocrinology, 2002 Jun, 143(6), 2119 - 38
Global gene profiling in human endometrium during the window of implantation; Kao LC et al.; Implantation in humans is a complex process that is temporally and spatially restricted . Over the past decade, using a one-by-one approach, several genes and gene products that may participate in this process have been identified in secretory phase endometrium . Herein, we have investigated global gene expression during the window of implantation (peak E2 and progesterone levels) in well characterized human endometrial biopsies timed to the LH surge, compared with the late proliferative phase (peak E2 level) of the menstrual cycle . Tissues were processed for poly(A(+)) RNA and hybridization of chemically fragmented, biotinylated cRNAs on high density oligonucleotide microarrays, screening for 12,686 genes and expressed sequence tags . After data normalization, mean values were obtained for gene readouts and fold ratios were derived comparing genes up- and down-regulated in the window of implantation vs . the late proliferative phase . Nonparametric testing revealed 156 significantly (P < 0.05) up-regulated genes and 377 significantly down-regulated genes in the implantation window . Up-regulated genes included those for cholesterol trafficking and transport {apolipoprotein (Apo)E being the most induced gene, 100-fold}, prostaglandin (PG) biosynthesis (PLA2) and action (PGE2 receptor), proteoglycan synthesis (glucuronyltransferase), secretory proteins {glycodelin, mammaglobin, Dickkopf-1 (Dkk-1, a Wnt inhibitor)}, IGF binding protein (IGFBP), and TGF-beta superfamilies, signal transduction, extracellular matrix components (osteopontin, laminin), neurotransmitter synthesis (monoamine oxidase) and receptors (gamma aminobutyric acid A receptor pi subunit), numerous immune modulators, detoxification genes (metallothioneins), and genes involved in water and ion transport {e.g . Clostridia Perfringens Enterotoxin (CPE) 1 receptor (CPE1-R) and K(+) ion channel}, among others . Down-regulated genes included intestinal trefoil factor (ITF) {the most repressed gene (50-fold)}, matrilysin, members of the G protein-coupled receptor signaling pathway, frizzled-related protein (FrpHE, a Wnt antagonist), transcription factors, TGF-beta signaling pathway members, immune modulators (major histocompatibility complex class II subunits), and other cellular functions . Validation of select genes was conducted by Northern analysis and RT-PCR using RNA from endometrial biopsies obtained in the proliferative phase and the implantation window and by RT-PCR using RNA from cultured endometrial epithelial and stromal cells . These approaches confirmed up-regulation of genes corresponding to IGFBP-1, glycodelin, CPE1-R, Dkk-1, mammaglobin, and ApoD and down-regulation for PR membrane component 1, FrpHE, matrilysin, and ITF, as with the microarray data . Cultured endometrial epithelial cells were found to express mRNAs for glycodelin, CPE-1R, Dkk-1, the gamma aminobutyric acid A receptor pi subunit, mammaglobin, matrilysin, ITF and PR membrane component 1 . The expression of IGFBP-1, CPE1-R, Dkk-1, and ApoD mRNAs increased upon decidualization of stromal cells in vitro with progesterone after E2 priming, whereas FrpHE decreased, consistent with the microarray results . Overall, the data demonstrate numerous genes and gene families not heretofore recognized in human endometrium or associated with the implantation process . Reassuringly, several gene products, known to be differentially expressed in the implantation window or in secretory endometrium, were verified, and the striking regulation of select secretory proteins, water and ion channels, signaling molecules, and immune modulators underscores the important roles of these systems in endometrial development and endometrial-embryonic interactions . In addition, the current study validates using high density oligonucleotide microarray technology to investigate global changes in gene expression in human endometrium.

Am Surg, 2002 May, 68(5), 463 - 5
Nontraumatic clostridial myonecrosis: an indication for colonoscopy?
Jacob ZC, Dedekian M, Seoudi H.
Clostridial myonecrosis (CM) is a rare infection that usually arises after traumatic injury . Spontaneous or nontraumatic CM is even more rare and has been described in association with colorectal malignancy . These infections carry a high mortality rate and require urgent surgical intervention and intensive antibiotic therapy . This report describes a patient who presented with nontraumatic CM of the lower extremity in association with adenocarcinoma of the cecum.

Dtsch Tierarztl Wochenschr, 2002 Apr, 109(4), 172 - 7
{Progress in the replacement of animal experiments in the quality control of clostridial vaccines}; Cussler K et al.; Animal experiments still play a central role in the quality control of vaccines . Generally, performance of these experiments is provided by law and laid down in the European Pharmacopoeia . Classical vaccines, the efficacy of which is calculated in International Units, require a very high number of experimental animals for quality control testing . The testing mainly consists of infection and intoxication experiments causing extreme suffering of the animals involved . This classical product group includes clostridial vaccines which are used to a great extent in veterinary medicine in particular . Within the last years, considerable efforts have been made to reduce the number of animal experiments in this field, lower the number of animals, and decrease the suffering of the animals during testing . Several research projects for the development and validation of alternative methods have been initiated . Furthermore, the 3R Concept (Replacement, Reduction and Refinement) is increasingly taken into consideration when developing or revising legal provisions . This led to various improvements regarding animal welfare in the quality control of clostridial vaccines.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 429 - 32
Ilyobacter insuetus sp . nov., a fermentative bacterium specialized in the degradation of hydroaromatic compounds; Brune A et al.; The mesophilic, anaerobic bacterium strain VenChi2T was isolated with quinic acid (1,3,4,5-tetrahydroxy-cyclohexane-1-carboxylic acid) as the sole source of carbon and energy . Of more than 30 substrates tested, only quinic acid and shikimic acid (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) were utilized, yielding acetate, propionate, butyrate, H2 and CO2 as fermentation products . Sugars, alcohols, (di-)carboxylic acids, amino acids and aromatic compounds were not fermented and no external electron acceptors were used . Strain VenChi2T is a gram-negative, strictly anaerobic, coccoid rod; it does not employ the classical hydroaromatic pathway of aerobic bacteria for the degradation of hydroaromatic compounds (no aromatic intermediates involved) . Comparative 16S and 23S rDNA sequence analyses placed strain VenChi2T in the fusobacteria phylum, with the closest relatives among species of the genera Ilyobacter and Propionigenium . The results indicate that, disregarding the taxonomically misplaced Ilyobacter delafieldii, which is a member of the clostridia, the validly described Ilyobacter and Propionigenium species are phylogenetically intermixed . Based on its phenotypic properties, strain VenChi2T (= DSM 6831T = ATCC BAA-291T) is assigned to the genus Ilyobacter as the type strain of a novel species, Ilyobacter insuetus sp . nov.

J Am Chem Soc, 2002 Mar 27, 124(12), 3083 - 92
Structural characterization of metallopeptides designed as scaffolds for the stabilization of nickel(II)-Fe(4)S(4) bridged assemblies by X-ray absorption spectroscopy; Musgrave KB et al.; In earlier work, de novo designed peptides with a helix-loop-helix motif and 63 residues have been synthesized as potential scaffolds for stabilization of the {Ni(II)-X-Fe(4)S(4)} bridged assembly that is the spectroscopically deduced structure of the A-Cluster in clostridial carbon monoxide dehydrogenase . The 63mers contain a consensus tricysteinyl ferredoxin domain in the loop for binding an Fe(4)S(4) cluster and Cys and His residues proximate to the loop for binding Ni(II), with one Cys residue designed as the bridge X . The metallopeptides HC(4)H(2)-{Fe(4)S(4)}-Ni and HC(5)H-{Fe(4)S(4)}-M, containing three His and one Cys residue for Ni(II) coordination and two His and two Cys residues for binding M = Ni(II) and Co(II), have been examined by Fe-, Ni-, and Co-K edge spectroscopy and EXAFS . All peptides bind an {Fe(4)S(4)}(2+) cubane-type cluster . Interpretation of the Ni and Co data is complicated by the presence of a minority population of six-coordinate species with low Z ligands, designated for simplicity as {M(OH(2))(6)}(2+) . Best fits of the data were obtained with ca . 20% {M(OH(2))(6)}(2+) and ca . 80% M(II) with mixed N/S coordination . The collective XAS results for HC(4)H(2)-{Fe(4)S(4)}-Ni and HC(5)H-{Fe(4)S(4)}-M demonstrate the presence of an Fe(4)S(4) cluster and support the existence of the distorted square-planar coordination units {Ni(II)(S.Cys)(N.His)(3)} and {Ni(II)(S.Cys)(2)(N.His)(2)} in the HC(4)H(2) and HC(5)H metallopeptides, respectively . In the HC(5)H metallopeptide, tetrahedral {Co(II)(S.Cys)(2)(N.His)(2)} is present . We conclude that the designed scaffolded binding sites, including Ni-(mu(2)-S.Cys)-Fe bridges, have been achieved . This is the first XAS study of a de novo designed metallopeptide intended to stabilize a bridged biological assembly, and one of a few XAS analyses of metal derivatives of designed peptides . The scaffolding concept should be extendable to other bridged metal assemblies.

J Biol Chem, 2002 May 10, 277(19), 16805 - 13 Epub 2002 Mar 04.
Stimulation of phospholipase C-epsilon by the M3 muscarinic acetylcholine receptor mediated by cyclic AMP and the GTPase Rap2B; Evellin S et al.; Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins . We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B . Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms . M(3) mAChR-mediated PLC stimulation and {Ca(2+)}(i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor . On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation . Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses . The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A) . Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado . Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and {Ca(2+)}(i) increase . In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s) . In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.

J Neurochem, 2002 Feb, 80(4), 706 - 14
Multiple mechanisms of transmitter release evoked by "pathologically" elevated extracellular {K+}: involvement of transporter reversal and mitochondrial calcium; Raiteri L et al.; The release of {3H}GABA evoked by depolarization with various concentrations of KCl was studied using superfused rat cerebrocortex synaptosomes . Elevating {K+} produced release of {3H}GABA over basal which was increasingly less dependent on external Ca2+ but more sensitive to the GABA transporter blocker SKF 100330 A . Accordingly, the sensitivity to clostridial toxins of the depolarization-evoked amino acid release was inversely correlated to the concentration of KCl used . However, at 50 mM K+, one-third of the stimulated release remained which was external Ca2+-independent but insensitive to SKF 100330 A . This release was prevented by BAPTA, thapsigargin or dantrolene; it also was inhibited by blocking in mitochondria the ATP production with oligomycin, the H+-dependent Ca2+ uniporter with RU 360, the Na+/Ca2+ exchanger with CGP 37157 or by lowering extraterminal {Na+} . In fluorescence experiments with fura-2/AM, 50 mM K+ (in Ca2+ free medium) caused elevation of cytosolic {Ca2+} that was sensitive to thapsigargin or CGP 37157; these compounds produced partially additive effects . When exocytosis was monitored with the fluorescent dye acridine orange, the fluorescence elicited by 50 mM K+ was sensitive to thapsigargin or CGP 37157, which produced additive effects, and to low-Na+ media . To conclude, extracellular K+ concentrations occurring in the CNS in certain pathological conditions provoke GABA release by mechanisms different from classical exocytosis . These include carrier-mediated release and internal Ca2+-dependent exocytosis; in the latter, mitochondrial Ca2+ seems to play a primary role.

G Chir, 2001 Oct, 22(10), 345 - 7
{Compartment syndrome or gas gangrene? A case report}; Assenza M et al.; A case of forearm compartment syndrome due to a minor trauma is reported . The severe clinical conditions due to a shock state lead to an initial misdiagnosis of clostridial myonecrosis . The patient, 68 y-old woman, was admitted to the intensive care unit of Authors' hospital for a traumatic injury of the right forearm . A diagnosis of gas gangrene due to clostridial myonecrosis was done and forearm amputation suggested . In spite of this indication a decompression fasciotomy of the forearm compartments was performed as well as a hyperbaric oxygene therapy . Since culture of necrotic tissue samples did not demonstrate any bacterial growth, the Authors decided to avoid amputation and perform a radial arteriovenous fistula to improve venous return and reduce distal edema and continuous bleeding, with a complete recovery within 40 days . Good evaluation of patient with suspected compartment syndrome means correct and not delayed treatment, avoiding invalidating outcome.

Mil Med, 2002 Jan, 167(1), 85 - 6
Necrotizing clostridial pneumonia: a case report and review of the literature; Cannon JW; Clostridial species infecting the pleuropulmonary structures characteristically cause a rapidly progressing cavitary pneumonia . Various risk factors contribute to these infections, and the clinical course can range from a mild to a highly virulent illness, even leading to death . If recognized early, however, the severity of the illness can be curbed with appropriate antibiotic therapy . The case of necrotizing clostridial pneumonia presented here documents remote pulmonary emboli as a newly established risk factor for these infections.

J Dairy Sci, 2001 Nov, 84(11), 2494 - 502
Effect of ensiling alfalfa at low and high dry matter on production of milk used to make Grana cheese; Colombari G et al.; The effect of alfalfa ensiled in bunker silos at high moisture {HM, 34% dry matter (DM)} and low moisture (LM, 56% DM) content on milk production and Grana Padano cheese quality was studied . Forty Italian Friesian lactating cows were allotted to two groups and fed, in a crossover design experiment, two corn silage-based diets containing 27% of the total DM as HM or LM . Each of the two periods included 10 d of adaptation and 3 experimental weeks . Forage was cut in the mid-vegetative stage with, on average, 34% neutral detergent fiber and 19% crude protein (DM basis) . The two alfalfa silages showed a different fermentation pattern with 4.04 and 1.25% of lactic acid, 1.95 and 0.42% of acetic acid, 9.1 and 4.8% of total N ammonia-N for HM and LM, respectively . No butyric acid was found . Clostridial spores and yeast showed no growth in both silages except in the first 2 wk of the experiment where slight aerobic deterioration occurred . The HM treatment resulted in slightly lower DM intake (19.3 vs . 19.9 kg/d) and milk protein content (3.33 vs . 3.38%), higher milk fat content (3.56 vs . 3.37%), and 4% fat-corrected milk (25.7 vs . 24.4 kg/d) . Totally, 38 cheeses obtained from over 19 tons of milk with an average yield efficiency of 6.8%, were produced . The milk renneting and microbiological properties and the cheese quality were not significantly different between treatments . However, both treatments had on average 40% of low quality (butyric fermentation) cheeses observed mainly in the first 2 wk of the experiment, when the number of clostridial spores found in alfalfa silages was significantly higher than in the subsequent weeks . The data obtained suggest that the microbial quality of milk depends more on careful management and monitoring all of the steps in milk production, from silage harvest through to cheese making, than on the moisture level of alfalfa silage, provided that the latter is in a range of 35 to 55% DM.

Water Res, 2002 Jan, 36(1), 189 - 95
Fluorescence properties of some farm wastes: implications for water quality monitoring; Baker A; Some farm wastes have been analysed for their fluorescence properties using fluorescence excitation-matrix (EEM) spectroscopy . Farm wastes investigated were silage liquor, pig and cattle slurry, and sheep barn waste . All farm wastes exhibited high intensities of fluorescence that can be attributed to the protein tryptophan . Silage liquor was characterised by a very high fluorescence intensity and an initial tryptophan: fulvic-like fluorescence intensity ratio of >20 . Cattle and pig slurries exhibited a lower tryptophan : fulvic-like fluorescence intensity ratio (approximately 2-5) and lower tryptophan fluorescence intensity, and tyrosine fluorescence was also observed . Sheep barn wastes had the lowest tryptophan: fulvic-like fluorescence intensity ratios (approximately 0.5-4.0) . Farm waste samples were reanalysed under controlled temperature conditions over a period of 50 days after sampling, to investigate the stability of their fluorescence properties . For silage liquor . tryptophan: fulvic-like fluorescence intensity ratios were observed to decrease with time, and were associated with a decrease in tryptophan fluorescence intensity, suggestive of clostridia breakdown of protein . For slurry samples . tryptophan: fulvic-like fluorescence intensity exhibited a more variable time-evolution, and tryptophan fluorescence intensity increased through time; the more complex fluorescence signal is due to the relatively heterogeneous nature of the slurry . Sheep barn waste samples exhibited more stable tryptophan: fulvic-like fluorescence intensity ratio and tryptophan intensities, suggesting these samples were more stable due to their greater age and decomposition . The ratios of tryptophan: fulvic-like fluorescence intensity observed from the farm wastes investigated are significantly higher than those observed in the majority of river waters, suggesting that farm waste pollution events could leave a signature in river waters due to their distinctively high protein fluorescence intensity.

J R Army Med Corps, 2001 Oct, 147(3), 309 - 10
Retroperitoneal gas gangrene complicating elective inguinal hernia repair; Privitera A et al.; Gas gangrene is a well recognised complication of contaminated military wounds . A case of Clostridial myonecrosis following elective inguinal hernia repair is described . The pathology, clinical features and management of this life-threatening condition are reviewed.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 1977 - 81
Lachnobacterium bovis gen . nov., sp . nov., a novel bacterium isolated from the rumen and faeces of cattle; Whitford MF et al.; Phenotypic and phylogenetic analysis was performed on four strains of a previously undescribed Gram-positive, rod-shaped, anaerobic bacterium isolated from the rumen and faeces of cattle . This bacterium fermented glucose primarily to lactic acid along with minor amounts of acetic and butyric acids . The four strains produced a temperature-sensitive bacteriocin-like inhibitory substance . Comparative 16S rRNA gene sequence analysis indicated that the bacterium was a member of the clostridial XIVa cluster of the low-G+C content Gram-positive bacteria . Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be assigned to a new genus, Lachnobacterium, as Lachnobacterium bovis gen . nov., sp . nov . The type strain is YZ 87T (= ATCC BAA-151T = DSM 14045T = LRC 5382T) . Its G+C content is 33.9 mol %.

J Periodontol, 2001 Nov, 72(11), 1588 - 93
Tetracycline modulates collagen membrane degradation in vitro; Moses O et al.; BACKGROUND: Structural integrity of implanted bioabsorbable barrier membranes should be preserved for a sufficient time to ensure expected results . Collagen membranes are degraded by metalloproteinases (MMP) . Their degradation rate can be altered either by enhancing structural integrity or by delaying the degradation process using MMP inhibitors . Tetracyclines (TTC) present inhibitory effects on matrix MMP . Immersing membranes in TTC solution before implantation can delay their degradation . The purpose of the present study was to evaluate the effect of collagen membranes immersed in varying TTC concentration solutions on the rate of their degradation in vitro . METHODS: Collagen bioabsorbable membranes were prepared as 5 mm diameter membrane discs . Membranes were then incubated at 4 degrees C for 24 hours, in either phosphate buffered saline (PBS, Ca2+ and Mg2+ free) or with TTC-HCl dissolved in PBS concentrations of 5 mg/ml, 50 mg/ml or 100 mg/ml . After rinsing, membranes were incubated with either bacterial collagenase or cultures of human bone lineage cells . Membrane degradation was studied on days 2, 4, 7, and 14 . Two- and 3-way analysis of variance was used to analyze results . RESULTS: Samples supplemented with bacterial collagenase exhibited a statistically significant interaction between changes of free protein in the medium, antibiotic concentration used for the immersion, presence of collagenase in the medium, and incubation time (P<0.0001) . Membranes incubated with bone cells exhibited similar degradation trends . CONCLUSIONS: Collagen membranes immersed in 50 mg/ml TTC solution exhibited the longest degradation time, both in the clostridial collagenase and the human bone cell lineage assays . Immersion in a 50 mg/ml TTC solution before implantation will delay their degradation.

Appl Microbiol Biotechnol, 2001 Oct, 57(3), 263 - 73
Unusual enzymes involved in five pathways of glutamate fermentation; Buckel W; Anaerobic bacteria from the orders Clostridiales and Fusobacteriales are able to ferment glutamate by at least five different pathways, most of which contain enzymes with radicals in their catalytic pathways . The first two pathways proceed to ammonia, acetate and pyruvate via the coenzyme B12-dependent glutamate mutase, which catalyses the re-arrangement of the linear carbon skeleton to that of the branched-chain amino acid (2S,3S)-3-methylaspartate . Pyruvate then disproportionates either to CO2 and butyrate or to CO2, acetate and propionate . In the third pathway, glutamate again is converted to ammonia, CO2, acetate and butyrate . The key intermediate is (R)-2-hydroxyglutaryl-CoA, which is dehydrated to glutaconyl-CoA, followed by decarboxylation to crotonyl-CoA . The unusual dehydratase, containing an iron-sulfur cluster, is activated by an ATP-dependent one-electron reduction . The remaining two pathways require more then one organism for the complete catabolism of glutamate to short chain fatty acids . Decarboxylation of glutamate leads to 4-aminobutyrate, which is fermented by a second organism via the fourth pathway to acetate and butyrate, again mediated by an unusual dehydratase which catalyses the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA . The fifth pathway is the only one without decarboxylation, since the gamma-carboxylate of glutamate is reduced to the amino group of delta-aminovalerate, which then is fermented to acetate, propionate and valerate . The pathway involves the oxidative dehydration of 5-hydroxyvaleryl-CoA to 2,4-pentadienoyl-CoA followed by reduction to 3-pentenoyl-CoA and isomerisation to 2-pentenoyl-CoA.

Minerva Anestesiol, 2001 Oct, 67(10), 745 - 9
HBO and gas gangrene . A case report; Ferrau S et al.; A 58-year old man, in a mediocre health condition, was admitted into Landspitallin Fossvogur, the University of Reykjavik City Hospital, Iceland, because of fever, chills, local pain and swelling due to the presence of a big old wound in his left heel . The first clinical appearance showed a gas gangrene of the area . The patient immediately underwent a surgical debridement and a fasciotomy, and antibiotic intravenous therapy and hyperbaric oxygen therapy (HBOT) were started . During the treatment the patient suffered for a multi-organ failure syndrome (MOFS), was admitted in ICU and survived . A total of 52 HBO sessions were performed and one month since the admission into the hospital the patient received a skin transplant . He achieved a complete restitutio ad integrum after 78 days of hospitalization . Gas gangrene is a fulminating infection caused by the genus of Clostridia . If not treated, it leads to the death of the host . The actual treatment for gas gangrene includes surgery, antibiotics, general resuscitative measures and HBOT.

Proc Natl Acad Sci U S A, 2001 Nov 20, 98(24), 13984 - 9 Epub 2001 Nov 13.
Chlamydia trachomatis cytotoxicity associated with complete and partial cytotoxin genes; Belland RJ et al.; Chlamydia trachomatis is an obligate intracellular human bacterial pathogen that infects epithelial cells of the eye and genital tract . Infection can result in trachoma, the leading cause of preventable blindness worldwide, and sexually transmitted diseases . A common feature of infection is a chronic damaging inflammatory response for which the molecular pathogenesis is not understood . It has been proposed that chlamydiae have a cytotoxic activity that contributes to this pathology, but a toxin has not been identified . The C . trachomatis genome contains genes that encode proteins with significant homology to large clostridial cytotoxins . Here we show that C . trachomatis makes a replication-independent cytotoxic activity that produces morphological and cytoskeletal changes in epithelial cells that are indistinguishable from those mediated by clostridial toxin B . A mouse chlamydial strain that encodes a full-length cytotoxin caused pronounced cytotoxicity, as did a human strain that has a shorter ORF with homology to only the enzymatically active site of clostridial toxin B . Cytotoxin gene transcripts were detected in chlamydiae-infected cells, and a protein with the expected molecular mass was present in lysates of infected epithelial cells . The protein was present transiently in infected cells during the period of cytotoxicity . Together, these data provide compelling evidence for a chlamydial cytotoxin for epithelial cells and imply that the cytotoxin is present in the elementary body and delivered to host cells very early during infection . We hypothesize that the cytotoxin is a virulence factor that contributes to the pathogenesis of C . trachomatis diseases.

J Biol Chem, 2001 Dec 28, 276(52), 49331 - 6 Epub 2001 Oct 17.
Insulin stimulates actin comet tails on intracellular GLUT4-containing compartments in differentiated 3T3L1 adipocytes; Kanzaki M et al.; Incubation of isolated GLUT4-containing vesicles with Xenopus oocyte extracts resulted in a guanosine 5'-{gamma-thio}triphosphate (GTP gamma S) and sodium orthovanadate stimulation of actin comet tails . The in vitro actin-based GLUT4 vesicle motility was inhibited by both latrunculin B and a dominant-interfering N-WASP mutant, N-WASP/Delta VCA . Preparations of gently sheared (broken) 3T3L1 adipocytes also displayed GTP gamma S and sodium orthovanadate stimulation of actin comet tails on GLUT4 intracellular compartments . Furthermore, insulin pretreatment of intact adipocytes prior to gently shearing also resulted in a marked increase in actin polymerization and actin comet tailing on GLUT4 vesicles . In addition, the insulin stimulation of actin comet tails was completely inhibited by Clostridum difficile toxin B, demonstrating a specific role for a Rho family member small GTP-binding protein . Expression of N-WASP/Delta VCA in intact cells had little effect on adipocyte cortical actin but partially inhibited insulin-stimulated GLUT4 translocation . Taken together, these data demonstrate that insulin can induce GLUT4 vesicle actin comet tails that are necessary for the efficient translocation of GLUT4 from intracellular storage sites to the plasma membrane.

J Biol Chem, 2001 Nov 30, 276(48), 44435 - 43 Epub 2001 Sep 28.
GTPases of the Rho subfamily are required for Brucella abortus internalization in nonprofessional phagocytes: direct activation of Cdc42; Guzman-Verri C et al.; Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals . Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease . Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry . Cell uptake of B . abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors . Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization . Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B . abortus by HeLa cells . In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B . abortus . Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B . abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake . Cdc42 was activated upon cell contact by virulent B . abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42 . The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion.

Mol Microbiol, 2001 Aug, 41(4), 827 - 48
A bacterial linear motor: cellular and molecular organization of the contractile cytoskeleton of the helical bacterium Spiroplasma melliferum BC3; Trachtenberg S et al.; The Mollicutes (Mycoplasma, Acholeplasma, and Spiroplasma) are the smallest, simplest and most primitive free-living and self-replicating known cells . These bacteria have evolved from Clostridia by regressive evolution and genome reduction to the range of 5.8 x 10(5)-2.2 x 10(6) basepairs (bp) . Structurally, the Mollicutes completely lack cell walls and are enveloped by only a cholesterol containing cell membrane . The Mollicutes contain what can be defined as a bacterial cytoskeleton . The Spiroplasmas are unique in having a well-defined, dynamic, helical cell geometry and a flat, monolayered, membrane-bound cytoskeleton, which follows, intracellularly, the shortest helical line on the cellular coil . By applying cryo-electron-microscopy to whole cells, isolated cytoskeletons and cytoskeletal fibrils and subunits, as well as by selective extraction of cellular components, we determined, at a resolution of approximately 25 A, the cellular and molecular organization of the cytoskeleton . The cytoskeleton is assembled from a 59 kDa protein . The 59 kDa protein, has an equivalent sphere diameter of approximately 50 A . Given the approximately 100 A axial and lateral spacings in the cytoskeletal ribbons and the near-circular shape of the subunit, we suggest that the subunit is a tetramer of 59 kDa monomers; the tetramers assemble further into flat fibrils, seven of which form a flat, monolayered, well-ordered ribbon . The cytoskeleton may function as a linear motor by differential and coordinated length-changes of the fibrils driven by conformational changes of the tetrameric subunits, the shape of which changes from near circular to elliptical . The cytoskeleton controls both the dynamic helical shape and the consequent motility of the cell . A stable cluster of proteins co-purifies with the cytoskeleton . These apparent membrane and membrane-associated proteins may function as anchor proteins.

Appl Environ Microbiol, 2001 Sep, 67(9), 4017 - 23
Obligate sulfide-dependent degradation of methoxylated aromatic compounds and formation of methanethiol and dimethyl sulfide by a freshwater sediment isolate, Parasporobacterium paucivorans gen . nov., sp . nov; Lomans BP et al.; Methanethiol (MT) and dimethyl sulfide (DMS) have been shown to be the dominant volatile organic sulfur compounds in freshwater sediments . Previous research demonstrated that in these habitats MT and DMS are derived mainly from the methylation of sulfide . In order to identify the microorganisms that are responsible for this type of MT and DMS formation, several sulfide-rich freshwater sediments were amended with two potential methyl group-donating compounds, syringate and 3,4,5-trimethoxybenzoate (0.5 mM) . The addition of these methoxylated aromatic compounds resulted in excess accumulation of MT and DMS in all sediment slurries even though methanogenic consumption of MT and DMS occurred . From one of the sediment slurries tested, a novel anaerobic bacterium was isolated with syringate as the sole carbon source . The strain, designated Parasporobacterium paucivorans, produced MT and DMS from the methoxy groups of syringate . The hydroxylated aromatic residue (gallate) was converted to acetate and butyrate . Like Sporobacterium olearium, another methoxylated aromatic compound-degrading bacterium, the isolate is a member of the XIVa cluster of the low-GC-content Clostridiales group . However, the new isolate differs from all other known methoxylated aromatic compound-degrading bacteria because it was able to degrade syringate in significant amounts only in the presence of sulfide.

Scand J Infect Dis, 2001, 33(8), 629 - 30
Non-clostridial gas gangrene caused by Klebsiella pneumoniae: a case report; Li CM et al.; A 45-y-old man was hospitalized due to pain and swelling of the right leg for 3 d . Bullae developed with gas formation involving multiple compartments of the entire limb 46 h later . Klebsiella pneumoniae was recovered from blood and surgical specimens . The patient died on Day 8 despite amputation and antibiotic therapy.

Pathology, 2001 Aug, 33(3), 338 - 40
Effect of global system for mobile communication (gsm)-like radiofrequency fields on vascular permeability in mouse brain; Finnie JW et al.; The effect of global system for mobile communication (GSM) radiofrequency fields on vascular permeability in the brain was studied using a purpose-designed exposure system at 898.4 MHz . Mice (n= 30) were given a single far field, whole body exposure for 60 minutes at a specific absorption rate of 4 W/kg . Control mice were also sham-exposed (n = 10) or permitted free movement in a cage (n = 10) to exclude any stress-related effects . Vascular permeability changes were detected using albumin immunohistochemistry and the efficacy of this vascular tracer was confirmed with a positive control group exposed to a clostridial toxin known to increase vascular permeability in the brain . No significant difference in albumin extravasation was detected between any of the groups at the light microscope level using the albumin marker.

Gene Ther, 2001 Aug, 8(15), 1197 - 201
Radio-responsive recA promoter significantly increases TNFalpha production in recombinant clostridia after 2 Gy irradiation; Nuyts S et al.; One of the major problems with gene therapy today is the lack of tumour specificity . The use of anaerobic apathogenic clostridia as a gene transfer system can target anoxic areas within the tumour . These bacteria can be genetically modified to express therapeutic proteins such as TNFalpha locally in the tumour . As shown in our results, ionising irradiation can be used in clostridia to activate genes encoding cytotoxic agents under control of a radiation-inducible promoter . A 44% significant increase (P < 0.05) in TNFalpha secretion was seen 3.5 h after a single dose of 2 Gy . A second dose of 2 Gy was also capable of repeating gene activation and gave a significant increase of TNFalpha production of 42% (P < 0.05) . These results provide evidence that spatial and temporal control of gene expression can be achieved using a radio-inducible promoter . Repetitive gene activation was feasible with a second dose of 2 Gy, indicating that fractionated radiotherapy could lead to repeated gene induction resulting in prolonged and enhanced protein expression . Gene targeting by ionising radiation could thus provide a new means of increasing the therapeutic ratio in cancer treatment.

Mol Cell Biochem, 2001 May, 221(1-2), 117 - 26
Purification and characterization of beta-methylaspartase from Fusobacterium varium; Bearne SL et al.; Beta-methylaspartase (EC 4.3.1.2) was purified 20-fold in 35% yield from Fusobacterium varium, an obligate anaerobe . The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAE-Sepharose . The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg2+ (Km = 0.27+/-0.01 mM) and K+ (Km = 3.3+/-0.8 mM) for maximum activity . Beta-methylaspartase-catalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)-3-methylaspartate (major product) and (2S,3R)-3-methylaspartate (minor product) . Optimal activity for the deamination of (2S,3S)-3-methylaspartate (Km = 0.51+/-0.04 mM) was observed at pH 9.7 . The N-terminal protein sequence (30 residues) of the F . varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.

Klin Khir, 2000 Sep, (9), 31 - 4
{Non-clostridial epi-fascia phlegmon}; Liul'ko IV et al.; Complex method of surgical treatment of epifascial phlegmon is elaborated . The flaps tailoring and dividing in accordance to anatomic site and its blood supply permits to do radical cleansing and sanitation of focus with mixture of hydrogen peroxide and spirituous solution of iodine preserving the viable skin maximally . In contrast to conventional "stripe" incisions, application of the proposed method permits to preserve the wound from significant lympho--and plasma losses, causing the wound exhaustion, secures the possibility of visual control of the necrotic process course . The wound coverage with cutaneo-subcutaneous flap of the full value assists reducing of the pain intensity . The flap fixation prevents its shortening, that's why good cosmetic effect is noted after the wound healing concludes . Complex program of the necrotic fascitis treatment proposed had permitted to reduce the hospitalization duration of patients twice and to lower the mortality rate more than in 3 times.

J Biol Inorg Chem, 2001 Jun, 6(5-6), 638 - 49
Redox thermodynamics of mutant forms of the rubredoxin from Clostridiumpasteurianum: identification of a stable Fe(III)(S-Cys)3(OH) centre in the C6S mutant; Xiao Z et al.; Redox thermodynamic data provide a detailed insight into control of the reduction potential E degrees' of the {Fe(S-Cys)4} site in rubredoxin . Mutant forms were studied in which specific structural changes were made in both the primary and secondary coordination spheres . Those changes have been probed by resonance Raman spectroscopy . The decrease of approximately 200 mV in E degrees' observed for the {Fe(S-Cys)3(O-Ser)}-/2- couples in the surface ligand mutants C9S and C42S is essentially enthalpic in origin and associated with the substitution of ligand thiolate by ligand olate . However, the pH dependence of the potentials below characteristic pKa(red) approximately equals 7 is an entropic contribution, plausibly associated with increased conformational flexibility induced by a longer Fe(II)-O(H)-Ser bond in the reduced form . The presence of a second surface Ser ligand in the new double mutant protein C9S/C42S affects the enthalpic term primarily for pH>pKa(red) > or = 9.3, but for pH<pKa(red) the entropic term again becomes significant . The available data for the internal ligand mutant C39S appear to follow those for the surface ligand mutants . A longer Fe(III)-O-Ser link in the oxidized form is expected from structural considerations and a smaller decrease of approximately 100 mV in E degrees' is observed for this particular {Fe(S-Cys)3(O-Ser)}/2- couple . The reduced form is particularly susceptible to hydrolysis with consequent irreversible electrochemistry, an apparent consequence of an even longer Fe(II)-O(H)-Ser bond . The second internal ligand mutant C6S exhibits starkly different behaviour . The cause was traced to the presence of a Fe(III)(S-Cys)3(OH)- centre in the oxidized form . Resonance Raman spectroscopy on 54Fe-, H2 18O- and D2O-enriched samples supports the presence of an iron hydroxyl group that donates a hydrogen bond to the OH group of the free S6 side chain and/or a cluster of bound water molecules . The thermodynamic parameters can then be rationalized in terms of the following processes: pH>pKa approximately 9: {Fe(III)(S-Cys)3(OH)}- + e- --> {Fe(II)(S-Cys)3(OH)}2- . pH<pKa: {Fe(III)(S-Cys)3(OH)}- +H+ e- --> {Fe(II)(S-Cys)3(OH2)}-.

Unfallchirurg, 2001 Feb, 104(2), 102 - 14
{Hyperbaric oxygen therapy in trauma surgery}; Mutschler W et al.; Hyperbaric oxygenation is achieved when a patient breathes 100 percent oxygen in an environment of elevated atmospheric pressure . Physiologically, this produces a directly proportional increase in the plasma volume fraction of transported oxygen which is readily available for cellular metabolism . A number of beneficial biochemical, cellular and physiologic effects result which account for the use of hyperbaric oxygen as an adjunctive therapy in the treatment of clostridial myonecrosis, crush injuries, compromised flaps, osteoradionecrosis and chronic problem wounds . Indications, modes of treatment, contraindications, side effects, costs and experimental and clinical results are presented . Overall, these data demonstrate that hyperbaric oxygen is no longer "a therapy in search of diseases" . However, more randomized controlled clinical trials are necessary to demonstrate its efficacy.

Vaccine, 2001 Jul 20, 19(30), 4318 - 27
The application of biodegradable collagen minipellets as vaccine delivery vehicles in mice and sheep; Lofthouse S et al.; Collagen minipellets are injectable delivery vehicles that release antigen and adjuvant over several days in a first-order release profile . In vaccination experiments in mice, secondary antibody responses induced by minipellets formulated with avidin and IL-1beta as adjuvant were equivalent to those induced by a conventional immunization with avidin in alum . When no adjuvant was used, anti-avidin responses induced by minipellets were 10-20-fold higher than those induced by injection of avidin in saline . In sheep, conventional vaccination with avidin in alum induced antibody responses initially exceeding that induced by minipellets formulated with avidin and IL-1beta, while following a secondary vaccination, the minipellet antibody response was equal to or greater than the alum-adjuvanted control groups . Increasing levels of IL-1beta adjuvant resulted in enhanced persistence of the antibody response . When clostridial vaccine antigens were incorporated into the minipellets, total antibody responses induced in sheep were equivalent to those induced by vaccination with the clostridial antigens in alum . Neutralizing antibody titres exceeded those induced by conventional vaccination . No adverse site reactions were observed at the implant site, with immunohistological study showing that the cellular infiltrate was dominated by a transient influx of neutrophils . This is a typical response to delivery of bioactive IL-1beta . The minipellets were completely degraded within 35 days of implantation.

J Anim Sci, 2001 Jun, 79(6), 1409 - 15
Influence of clostridial vaccines and injection sites on performance, feeding behavior, and lesion size scores of beef steers; Chirase NK et al.; Several clostridial vaccines are currently being used in the beef cattle industry . Of greatest concern is altering the location and route of administration of these vaccines to reduce injection-site lesions while maintaining seroconversion . Two experiments were conducted to determine the effect of clostridial vaccines and injection sites on the performance, feeding behavior, and lesion size scores of beef steers . In Exp . 1, 80 crossbred beef steers (BW 237 +/- 3.2 kg) were allotted randomly into five groups and given 14 d to adapt to the feed and individual feed intake-monitoring devices (Pinpointer devices) before starting the study . Each group was assigned randomly to one of the following vaccination treatments: 1) control (sterile saline water), 2) Alpha-7 Ear (A7E), 3) Alpha-7 Prescapula (A7P), 4) Vision-7 Prescapula (V7P), and 5) Ultrabac-7 Prescapula (U7P) . All vaccines were injected s.c . in the ear or prescapular region, and injection sites were palpated on d 0 and 28 (Exp . 1) and on d 63 and 91 (Exp . 2) . The protocol for Exp . 2 was exactly the same as for Exp . 1 except treatments included control, A7P, Alpha-CD Ear (ACDE), Alpha-CD Prescapula (ACDP), Fortress-7 Prescapula (F7P), and V7P . Also, control and steers receiving F7P and V7P were revaccinated on d 63 and palpated on d 91 . Results of Exp . 1 indicated that the A7E and U7P steers had a feed intake lower (P < 0.01) than all other treatment groups . The ADG of the A7P and A7E steers were not different (P > 0.05) from those of the control steers . The gain:feed ratio of the A7E steers was 41% higher (P < 0.01) than that of the V7P steers (Exp . 1) . The results of Exp . 2 indicated that the control, ACDP, and V7P steers had greater (P < 0.01) ADG than all other treatment groups, but the gain:feed ratios were not different (P > 0.05) among all treatment groups . Lesion sizes differed by vaccine and injection site in both experiments . These data suggest that vaccinating beef steers s.c . in the ear produced gain:feed ratios and lesion size scores that were similar to prescapular vaccinations . However, more research is required to determine the immune response of vaccinating cattle in the ear.

Biotechnol Bioeng, 2001 Aug 20, 74(4), 280 - 7
Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose; Lay JJ; Sixteen batch experiments were performed to evaluate the stability, kinetics, and metabolic paths of heat-shocked digester (HSD) sludge that transforms microcrystalline cellulose into hydrogen . Highly reproducible kinetic and metabolic data confirmed that HSD sludge could stably convert microcrystalline cellulose to hydrogen and volatile fatty acids (VFA) and induce metabolic shift to produce alcohols . We concluded that clostridia predominated the hydrogen-producing bacteria in the HSD sludge . Throughout this study the hydrogen percentage in the headspace of the digesters was greater than 50% and no methanogenesis was observed . The results emphasize that hydrogen significantly inhibited the hydrogen-producing activity of sludge when initial microcrystalline cellulose concentrations exceeded 25.0 g/L . A further 25 batch experiments performed with full factorial design incorporating multivariate analysis suggested that the ability of the sludge to convert cellulose into hydrogen was influenced mainly by the ratio of initial cellulose concentration (So) to initial sludge density (Xo), but not by interaction between the variables . The hydrogen-producing activity depended highly on interaction of So x (So/Xo) . Through response surface analysis it was found that a maximum hydrogen yield of 3.2 mmol/g cellulose occurred at So = 40 g/L and So/Xo = 8 g cellulose/g VSS . A high specific rate of 18 mmol/(g VSS-d) occurred at So = 28 g/L and So/Xo = 9 g cellulose/g VSS . These experimental results suggest that high hydrogen generation from cellulose was accompanied by low So/Xo.

Folia Microbiol (Praha), 2000, 45(5), 465 - 8
Chitinolytic enzymes produced by ovine rumen bacteria; Kopecny J et al.; Two strains of clostridia, isolated from the rumen fluid of sheep as potential antagonists toward anaerobic fungi showed a complete array of chitinolytic enzymes . Enzyme tests in cultures demonstrated endochitinase, exochitinase, N-acetyl-glucosaminidase, chitosanase and chitin deacetylase activities mainly in the extracellular fractions . In all samples, the highest was the activity of exochitinase (600-1100 nmol mL-1 h-1); the activity of endochitinase (280-500 nmol mL-1 h-1) was also significant . Chitinases were stimulated in the presence of reducing compounds and no dependence on cations was observed . In both strains different isoforms of chitinases of molar mass 36-96 kDa were detected . The chitinases from our isolates lyzed cell walls of anaerobic fungi in vitro and inhibited the activity of fungal beta-1,4-endoglucanases . Of the two bacteria examined, one was more effective in both antifungal effects.

J Protein Chem, 2000 Nov, 19(8), 699 - 707
Recombinant derivatives of clostridial neurotoxins as delivery vehicles for proteins and small organic molecules; Zdanovskaia MV et al.; Clostridial neurotoxins are the most powerful toxins known . Nevertheless, derivatives of these toxins may find broad applications both in science and medicine because of their unique abilities to recognize neurons and deliver small and large molecules into them . In this paper we describe the construction of two types of such derivatives . Proteins belonging to the first class were designed to allow direct conjugation with one or few molecules of interest . Proteins belonging to the second class contain biotin residue and therefore could be easily connected to streptavidin loaded with multiple molecules of interest . Only C-terminal regions of neurotoxin heavy chains were incorporated in the structure of recombinant proteins . Nevertheless, recombinant proteins were found to be able to recognize specific neuronal receptors and target model molecules to rat synaptosomes and human neuroblastoma cells.

Chem Biol Interact, 2001 Jan 30, 130-132(1-3), 351 - 8
Crystal structure of sorbitol dehydrogenase; Johansson K et al.; Sorbitol dehydrogenase (SDH) is a distant relative to the alcohol dehydrogenases (ADHs) with sequence identities around 20% . SDH is a tetramer with one zinc ion per subunit . We have crystallized rat SDH and determined the structure by molecular replacement using a tetrameric bacterial ADH as search object . The conformation of the bound coenzyme is extended and similar to NADH bound to mammalian ADH but the interactions with the NMN-part have several differences with those of ADH . The active site zinc coordination in SDH is significantly different than in mammalian ADH but similar to the one found in the bacterial tetrameric NADP(H)-dependent ADH of Clostridiim beijerinckii . The substrate cleft is significantly more polar than for mammalian ADH and a number of residues are ideally located to position the sorbitol molecule in the active site . The SDH molecule can be considered to be a dimer of dimers, with subunits A-B and C-D, where the dimer interactions are similar to those in mammalian ADH . The tetramers are composed of two of these dimers, which interact with their surfaces opposite the active site clefts, which are accessible on the opposite side . In contrast to the dimer interactions, the tetramer-forming interactions are small with only few hydrogen bonds between side-chains.

Ann Chir Gynaecol, 2000, 89 Suppl 214, 7 - 36
Hyperbaric oxygen therapy in acute necrotizing infections . With a special reference to the effects on tissue gas tensions; Korhonen K; Clostridial gas gangrene and perineal necrotizing fasciitis or Fournier's gangrene are rare but serious infections with an acute onset, rapid progression, systemic toxemia and a high mortality rate . The aim of this study was to investigate the efficacy of surgery, antibiotic treatment, surgical intensive care and in particular the role of hyperbaric oxygen (HBO) in the management of these infections . An experimental rat model was used to investigate the possibilities for measuring tissue oxygen and carbon dioxide tensions during hyperbaric oxygen treatment . In addition to this preliminary experimental study, Silastic tube tonometer and capillary sampling techniques were tested to measure the effect of hyperbaric oxygen treatment on subcutaneous oxygen and carbon dioxide tensions in patients with necrotizing fasciitis and healthy controls . Between January 1971 and April 1997, 53 patients with Clostridial gas gangrene were treated in the Department of Surgery, University of Turku . The patients underwent surgical debridement, broad spectrum antibiotic therapy and a series of hyperbaric oxygen treatments at 2.5 atmospheres absolute pressure (ATA) . Twelve patients died (22.6%) . Hyperbaric oxygen therapy in gas gangrene seems to be life-, limb- and tissue saving . Early diagnosis remains essential . Patient survival can be improved if the disease is recognized early and appropriate therapy instituted promptly . Between February 1971 and September 1996, 33 patients with perineal necrotizing fasciitis were treated in the Department of Surgery, University of Turku . The management included surgical debridement of the necrotic tissue with incisions and drainage of the involved areas, antibiotic therapy, hyperbaric oxygen treatment at 2.5 ATA pressure and surgical intensive care . Three patients died giving a mortality rate of 9.1% . The survivors received hyperbaric oxygen therapy for 2-12 times . Our results indicate that hyperbaric oxygenation is an important therapeutic adjunct in the treatment of Fournier's gangrene . Electrical equipment should not be used unsheltered in a hyperbaric chamber due to the increased risk of fire . The subcutaneous tissue gas tensions of rats were therefore measured using a subcutaneously implanted Silastic tube tonometer and a capillary sampling technique . The method was succesfully adapted to hyperbaric conditions . The subcutaneous oxygen tension levels increased five fold and the carbon dioxide tension levels two fold compared to intial levels . The PO2 and PCO2 of subcutaneous tissue and arterial blood were measured directly in six patients with necrotizing fasciitis and three healthy volunteers in normobaric conditions and during hyperbaric oxygen exposure at 2.5 ATA pressure . The measurements were carried out in healthy tissue and at the same time in the vicinity of the infected area of the patients . During HBO at 2.5 ATA subcutaneous oxygen tensions increased several fold from baseline values and carbon dioxide tensions also increased, but to a lesser degree in both healthy and infected tissues . When examining the subcutaneous PO2 levels measured from patients with necrotizing fasciitis, the PO2 was regularly higher in the vicinity of the infected area than in healthy tissue . In general, HBO treatment resulted in a marked increase in tissue oxygenation in both healthy tissue and in the vicinity of infected tissue . The hyperoxygenated tissue zone surrounding the infected area may be of significance in preventing the extension of invading microorganisms.

ALTEX, 2001, 18(1), 34 - 6
{Development of in vitro methods for the potency testing of clostridial vaccines}; Borrmann E et al.; Cell culture assays, using the MDCK cell line was confirmed as being sensitive to the C . perfringens epsilon toxin and VERO cell line to the C . novyi type B alpha toxin . Cell culture assays using these cells were developed and the test conditions were standardised . The antitoxin titres of rabbit antisera were calculated and compared with those of the manufacturers . The correlation coefficients between in vitro and in vivo method were calculated and were significant . The cell culture assay offers a valid in vitro alternative to the animal experiments for the titration of sera generated in the course of potency tests of clostridial vaccines.

Eur J Biochem, 2001 Mar, 268(5), 1173 - 80
Construction, separation and properties of hybrid hexamers of glutamate dehydrogenase in which five of the six subunits are contributed by the catalytically inert D165S; Hayden BM et al.; In vitro subunit hybridization was used to explore the basis of putative allosteric behaviour in clostridial glutamate dehydrogenase . C320S and D165S mutant enzymes were chosen to construct the hybrid proteins . The C320S mutant protein is fully active and shows normal allosteric properties but lacks the reactive cysteine . D165S is capable of binding both glutamate and NAD(+) but is catalytically inactive . The mutant proteins were denatured separately in 4 M urea, mixed in a 5 : 1 (D165S/C320S) ratio and diluted into a refolding mixture composed of 2 mM NAD(+), 1 M fluoride and artificial chaperones (4 mM polyoxyethylene 10 lauryl ether and 1.6 mM beta-cyclodextrin) . Under these conditions approximately 50% refolding was achieved for both mutant proteins separately . The renatured mixture was concentrated and separated from denatured proteins and the components of the refolding mixture by ultrafiltration and ion-exchange chromatography . Ellman's reagent, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), which binds close to the NAD(+) binding site, thus abolishing coenzyme binding in the wild-type enzyme, also reacts with D165S but has no effect on C320S . Modification by DTNB was coupled with dye-ligand affinity chromatography on a Procion Red HE-3B column in order to separate the hybrid mixture into fractions of defined composition . An optimized procedure based on salt gradient elution was developed . DTNB-modified 5 : 1 hybrids, with only one subunit capable of binding coenzyme, showed classical Michaelis-Menten kinetics when the NAD(+) concentration was varied, whereas removal of the thionitrobenzoate moieties that blocked the other five coenzyme binding sites in the hexamer reinstated nonlinear behaviour, suggesting that 'nonlinear' behaviour of the native enzyme and the hybrid with six coenzyme binding sites depends on binding to multiple sites . When assayed at high pH with increasing glutamate concentration, the sample with only one active subunit showed reduced sigmoidicity in the dependence of reaction rate on glutamate concentration (h = 3.0) compared with native C320S with six active subunits (h = 5.2) suggesting that the interaction between the subunits was reduced but not abolished completely . Catalytically silent subunits can thus still contribute to cooperativity.

Vet Clin North Am Equine Pract, 2000 Dec, 16(3), 471 - 85
Clostridial enterocolitis; Jones RL; Equine clostridial enterocolitis is being recognized with increasing frequency . It has been identified in foals with diarrhea, antibiotic-associated enterocolitis, or nosocomial enterocolitis . The sporadic occurrence of clostridial enterocolitis, the variety of types of clostridia involved, and the difficulty of experimentally reproducing the disease suggest that it is a poorly defined multifactorial syndrome . The risk factors associated with susceptibility to colonization and progressive infection are largely based on anecdotal observations and extrapolation from human studies . Quantitative studies are needed to decipher the complex interactions between host and indigenous microflora that provide for and maintain a healthy colonization resistance environment . It seems that such studies might be more beneficial in furthering our understanding of the pathogenesis of clostridial enterocolitis than attempting to implicate another agent or toxin as the sole cause of the disease in equids . Treatment protocols that interrupt the pathogenesis of the disease need to be devised and critically evaluated to complement the present protocols emphasizing supportive care . Perhaps it is time to consider clostridial enterocolitis as yet another consequence of the use of antimicrobials analogous to the selective pressures that result in the emergence of multiple drug-resistant pathogens.

Ann Chir Gynaecol Suppl, 2000, (214), 7 - 36
Hyperbaric oxygen therapy in acute necrotizing infections with a special reference to the effects on tissue gas tensions; Korhonen K; Clostridial gas gangrene and perineal necrotizing fasciitis or Fournier's gangrene are rare but serious infections with an acute onset, rapid progression, systemic toxemia and a high mortality rate . The aim of this study was to investigate the efficacy of surgery, antibiotic treatment, surgical intensive care and in particular the role of hyperbaric oxygen (HBO) in the management of these infections . An experimental rat model was used to investigate the possibilities for measuring tissue oxygen and carbon dioxide tensions during hyperbaric oxygen treatment . In addition to this preliminary experimental study, Silastic tube tonometer and capillary sampling techniques were tested to measure the effect of hyperbaric oxygen treatment on subcutaneous oxygen and carbon dioxide tensions in patients with necrotizing fasciitis and healthy controls . Between January 1971 and April 1997, 53 patients with Clostridial gas gangrene were treated in the Department of Surgery, University of Turku . The patients underwent surgical debridement, broad spectrum antibiotic therapy and a series of hyperbaric oxygen treatments at 2.5 atmospheres absolute pressure (ATA) . Twelve patients died (22.6%) . Hyperbaric oxygen therapy in gas gangrene seems to be life-, limb- and tissue saving . Early diagnosis remains essential . Patient survival can be improved if the disease is recognized early and appropriate therapy instituted promptly . Between February 1971 and September 1996, 33 patients with perineal necrotizing fasciitis were treated in the Department of Surgery, University of Turku . The management included surgical debridement of the necrotic tissue with incisions and drainage of the involved areas, antibiotic therapy, hyperbaric oxygen treatment at 2.5 ATA pressure and surgical intensive care . Three patients died giving a mortality rate of 9.1% . The survivors received hyperbaric oxygen therapy for 2-12 times . Our results indicate that hyperbaric oxygenation is an important therapeutic adjunct in the treatment of Fournier's gangrene . Electrical equipment should not be used unsheltered in a hyperbaric chamber due to the increased risk of fire . The subcutaneous tissue gas tensions of rats were therefore measured using a subcutaneously implanted Silastic tube tonometer and a capillary sampling technique . The method was successfully adapted to hyperbaric conditions . The subcutaneous oxygen tension levels increased five fold and the carbon dioxide tension levels two fold compared to initial levels . The PO2 and PCO2 of subcutaneous tissue and arterial blood were measured directly in six patients with necrotizing fasciitis and three healthy volunteers in normobaric conditions and during hyperbaric oxygen exposure at 2.5 ATA pressure . The measurements were carried out in healthy tissue and at the same time in the vicinity of the infected area of the patients . During HBO at 2.5 ATA subcutaneous oxygen tensions increased several fold from baseline values and carbon dioxide tensions also increased, but to a lesser degree in both healthy and infected tissues . When examining the subcutaneous PO2 levels measured from patients with necrotizing fasciitis, the PO2 was regularly higher in the vicinity of the infected area than in healthy tissue . In general, HBO treatment resulted in a marked increase in tissue oxygenation in both healthy tissue and in the vicinity of infected tissue . The hyper-oxygenated tissue zone surrounding the infected area may be of significance in preventing the extension of invading microorganisms.

J Agric Food Chem, 2000 Dec, 48(12), 6011 - 5
Depletion of moxidectin tissue residues in sheep; Lifschitz A et al.; The pattern of tissue depletion of moxidectin (MXD) subcutaneously administered to sheep was characterized in this study . MXD concentration profiles were determined in muscle, fat, and liver and at the site of injection following administration of a formulation combining MXD (0.5% w/v) with a standard 6 in 1 clostridial vaccine . Thirty-five (35) parasite-free Lincoln sheep were treated with the MXD injectable formulation at a dose rate of 0.2 mg of MXD/kg of live weight, administered subcutaneously on the inner surface of the thigh . Treated animals were sacrificed in randomly selected groups of six sheep weekly from day 21 until day 49 post-treatment . Three nontreated animals were sacrificed to obtain blank tissue samples to validate the analytical methodology . MXD concentration profiles were determined by a validated HPLC analytical method using fluorescence detection . MXD has an adequate pattern of absorption, based on the low residual concentrations found in the injection site area at all sampling intervals . Muscle samples showed the lowest MXD concentrations throughout the study period . The highest MXD concentrations at all sampling times were measured in the adipose tissue, indicating that fat is a target tissue for MXD . MXD concentrations in all of the tissues analyzed were below the accepted maximum limit of residue at 21 days post-treatment.

Surg Laparosc Endosc Percutan Tech, 2000 Dec, 10(6), 412 - 4
Small intestine perforation because of capacitive coupling as a cause of abdominal wall gas gangrene and clostridial sepsis after laparoscopic cholecystectomy; Zadrozny D et al.; The authors present a case report regarding abdominal wall gas gangrene and clostridial sepsis after laparoscopic cholecystectomy . Capacitive coupling was considered to be the most probable cause of small intestine perforation and further complications . Despite intensive treatment, the 69-year-old patient died.

Postgrad Med J, 2001 Jan, 77(903), 47 - 9
Gas gangrene after colonoscopy; Jamieson NF et al.; A case of spontaneous clostridial myonecrosis developing shortly after diagnostic colonoscopy is described . The prime underlying factor proved to be an unsuspected colonic cancer, developing in a patient with pre-existing ulcerative colitis and sclerosing cholangitis.

Curr Opin Struct Biol, 2000 Oct, 10(5), 528 - 35
Microbial toxins and the glycosylation of rho family GTPases; Busch C et al.; Large clostridial cytotoxins act on cells by glycosylating low molecular mass GTPases using nucleotide-sugars as the sugar donor . These toxins are important virulence factors in human and animal diseases, but are also valuable cell biology tools . Recent findings shed some light on their mode of action and provide new insights into the structure/activity relationship of these bacterial toxins.

Am J Vet Res, 2000 Oct, 61(10), 1169 - 72
Lesions and effects of location for administration of clostridial bacterin-toxoid vaccines on growth performance and eating and drinking behaviors in newly arrived calves at a feedlot; Buhman MJ et al.; OBJECTIVES: To determine the effect of location for administration of clostridial vaccines on behavior, growth performance, and health of calves at a feedlot, the relative risk of calves developing an injection-site reaction or being misdiagnosed as having bovine respiratory disease complex (BRDC), and the percentage of subcutaneous injection-site reactions that were detectable on carcasses after the hides were removed . ANIMAL: 170 newly arrived calves at a feedlot . PROCEDURE: Eating and drinking behaviors of calves during the initial 57 days after arrival were observed at a commercial feedlot, using an electronic monitoring system . Calves were assigned randomly to receive a clostridial vaccine (base of ear or neck) . Data on reactions at the injection site were collected . RESULTS: Mean daily gain (MDG) for the initial 57 days did not differ significantly between treatments . Risk of being misdiagnosed as having BRDC was not associated with location for administration of vaccine . Calves vaccinated in the base of the ear were at higher risk of having an injection-site reaction at day 57 or at slaughter . Eighty-nine percent (95% confidence interval, 52 to 100%) of injection-site reactions in the neck could not be located on the carcasses after hides were removed . Calves vaccinated in the neck drank significantly fewer times per day during the first 57 days than calves vaccinated in the base of the ear . CONCLUSIONS AND CLINICAL RELEVANCE: Location for administration of a clostridial vaccine did not significantly affect health, growth performance, or eating behavior . Most subcutaneous injection-site reactions were not detectable after the hide was removed.

Microbes Infect, 2000 Aug, 2(10), 1277 - 84
Structure and function of clostridial phospholipases C; Jepson M et al.; A range of clostridial species produce phospholipases C . The zinc metallo phospholipases C have related sequences but different properties . All of these enzymes may be arranged, like alpha-toxin as two-domain proteins . Differences in enzymatic, haemolytic and toxic properties may be explained by differences in amino acids at key positions.

J Anat, 2000 Aug, 197 ( Pt 2), 189 - 98
Differential expression of proteoglycan epitopes by ovine intervertebral disc cells; Melrose J et al.; The alginate bead culture system has been utilised by several groups to examine the in vitro proteoglycan (PG) metabolism of chondrocytes and intervertebral disc cells, but the nature of the PGs produced has not been examined in detail . This is largely due to the difficulty of separating the anionically charged sodium alginate support matrix from PGs which are similarly charged . In the present study ovine annulus fibrosus, transitional zone and nucleus pulposus cells were dissociated enzymatically from their respective matrices by sequential digestion with pronase/clostridial collagenase and DNAase and then cultured in alginate beads for 10 d . The beads were solubilised and subjected to DEAE Sepharose CL6B anion exchange chromatography to separate the sodium alginate bead support matrix material quantitatively from the disc cell PGs . The alginate free bead PGs were then subjected to composite agarose polyacrylamide gel electrophoresis to resolve PG populations and the PGs were transferred to nitrocellulose membranes by semidry electroblotting . The PGs were identified by probing the blots with a panel of antibodies to defined PG core protein and glycosaminoglycan side chain epitopes . Alginate beads of disc cells were also embedded in paraffin wax and 4 microm sections cut to immunolocalise decorin, biglycan, versican, and the 7-D-4 PG epitope within the beads . Decorin and biglycan had similar distributions in the beads, being localised on the cell surface whereas versican and the 7-D-4 PG epitope were immunolocalised interterritoriarly . This study is the first to demonstrate that ovine disc cells synthesise versican in alginate bead culture . Furthermore the immunoblotting studies also showed that a proportion of the 7-D-4 PG epitope was colocalised with versican.

J Bacteriol, 2000 Sep, 182(17), 4915 - 25
A scaffoldin of the Bacteroides cellulosolvens cellulosome that contains 11 type II cohesins; Ding SY et al.; A cellulosomal scaffoldin gene, termed cipBc, was identified and sequenced from the mesophilic cellulolytic anaerobe Bacteroides cellulosolvens . The gene encodes a 2,292-residue polypeptide (excluding the signal sequence) with a calculated molecular weight of 242,437 . CipBc contains an N-terminal signal peptide, 11 type II cohesin domains, an internal family III cellulose-binding domain (CBD), and a C-terminal dockerin domain . Its CBD belongs to family IIIb, like that of CipV from Acetivibrio cellulolyticus but unlike the family IIIa CBDs of other clostridial scaffoldins . In contrast to all other scaffoldins thus far described, CipBc lacks a hydrophilic domain or domain X of unknown function . The singularity of CipBc, however, lies in its numerous type II cohesin domains, all of which are very similar in sequence . One of the latter cohesin domains was expressed, and the expressed protein interacted selectively with cellulosomal enzymes, one of which was identified as a family 48 glycosyl hydrolase on the basis of partial sequence alignment . By definition, the dockerins, carried by the cellulosomal enzymes of this species, would be considered to be type II . This is the first example of authentic type II cohesins that are confirmed components of a cellulosomal scaffoldin subunit rather than a cell surface anchoring component . The results attest to the emerging diversity of cellulosomes and their component sequences in nature.

J Mol Microbiol Biotechnol, 2000 Jan, 2(1), 9 - 14
Clostridial iron-sulphur proteins; Meyer J; Iron-sulfur proteins are ubiquitous catalysts of a wide range of biological reactions, and are particularly abundant in clostridia which lack the ability to synthesize hemes . The development of research on these metalloproteins has therefore been strongly associated with biochemical investigations of clostridial metabolism . Major breakthroughs in the field, from the first isolation of an iron-sulfur protein in 1962, to the recent determination of an Fe-hydrogenase structure, have been made with clostridia . These data, as well as others obtained through studies on clostridia, are transferable to many other bioenergetic machineries, due to the strong phylogenetic conservation of some important components . For instance, clear homologies exist between constituents of the anaerobic electron transfer chains in clostridia and aerobic respiratory chains . The contribution of iron-sulfur proteins to the biotechnological and medical significance of clostridia is also discussed . Structural and functional genomics are expected to bring forth a wealth of novel data on clostridia and iron-sulfur proteins.

Appl Environ Microbiol, 2000 Aug, 66(8), 3166 - 73
Simple and efficient method for heterologous expression of clostridial proteins; Zdanovsky AG et al.; Many clostridial proteins are poorly produced in Escherichia coli . It has been suggested that this phenomena is due to the fact that several types of codons common in clostridial coding sequences are rarely used in E . coli and the quantities of the corresponding tRNAs in E . coli are not sufficient to ensure efficient translation of the corresponding clostridial sequences . To address this issue, we amplified three E . coli genes, ileX, argU, and leuW, in E . coli; these genes encode tRNAs that are rarely used in E . coli (the tRNAs for the ATA, AGA, and CTA codons, respectively) . Our data demonstrate that amplification of ileX dramatically increased the level of production of most of the clostridial proteins tested, while amplification of argU had a moderate effect and amplification of leuW had no effect . Thus, amplification of certain tRNA genes for rare codons in E . coli improves the expression of clostridial genes in E . coli, while amplification of other tRNAs for rare codons might not be needed for improved expression . We also show that amplification of a particular tRNA gene might have different effects on the level of protein production depending on the prevalence and relative positions of the corresponding codons in the coding sequence . Finally, we describe a novel approach for improving expression of recombinant clostridial proteins that are usually expressed at a very low level in E . coli.

J Hand Surg {Am}, 2000 Jul, 25(4), 629 - 36
Enzyme injection as nonsurgical treatment of Dupuytren's disease; Badalamente MA et al.; Surgical fasciectomy is the currently accepted treatment of Dupuytren's disease . The goal of this study was to test the clinical safety and efficacy of clostridial collagenase injection as a nonsurgical treatment of Dupuytren's disease in a phase II open-label trial . Thirty-five Dupuytren's disease patients entered the study (32 men and 3 women) . The mean age was 65 years . The first 6 patients were treated following a dose escalation protocol and received 300, 600, 1,200, 2,400, 4,800, and 9,600 U collagenase injected into the cord that was causing contracture of the metacarpophalangeal (MCP) joint . There were no beneficial clinical effects of these injections . The remaining 29 patients had collagenase injections at a dose level of 10,000 U, causing contractures of 34 MCP joints, 9 proximal interphalangeal (PIP) joints, and 1 thumb . Twenty-eight of the 34 MCP joint contractures corrected to normal extension (0 degrees ) and 2 of the 34 MCP joint contractures corrected to 5 degrees of normal extension, with full range of motion, within 1 to 14 days of injection . In the patients with PIP joint contractures, 4 of the 9 joints corrected to normal (0 degrees ) . One PIP joint corrected to within 10 degrees of normal and 2 corrected to within 15 degrees of normal . There were 2 failures; these patients will require surgery . The mean follow-up period was 20.0 +/- 5.6 months for the MCP joints and 14.1 +/- 6.6 months for the PIP joints . Clostridial collagenase injection of Dupuytren's cords causing MCP and PIP joint contractures appears to have merit as nonsurgical treatment of this disorder . Pending further placebo, double-blind studies, collagenase injection to treat Dupuytren's disease may be a safe and effective alternative to surgical fasciectomy.

Microbiology, 2000 Jun, 146 ( Pt 6), 1391 - 7
Three multidomain esterases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17 that carry divergent dockerin sequences; Aurilia V et al.; Three enzymes carrying esterase domains have been identified in the rumen cellulolytic anaerobe Ruminococcus flavefaciens 17 . The newly characterized CesA gene product (768 amino acids) includes an N-terminal acetylesterase domain and an unidentified C-terminal domain, while the previously characterized XynB enzyme (781 amino acids) includes an internal acetylesterase domain in addition to its N-terminal xylanase catalytic domain . A third gene, xynE, is predicted to encode a multidomain enzyme of 792 amino acids including a family 11 xylanase domain and a C-terminal esterase domain . The esterase domains from CesA and XynB share significant sequence identity (44%) and belong to carbohydrate esterase family 3; both domains are shown here to be capable of deacetylating acetylated xylans, but no evidence was found for ferulic acid esterase activity . The esterase domain of XynE, however, shares 42% amino acid identity with a family 1 phenolic acid esterase domain identified from Clostridum thermocellum XynZ . XynB, XynE and CesA all contain dockerin-like regions in addition to their catalytic domains, suggesting that these enzymes form part of a cellulosome-like multienzyme complex . The dockerin sequences of CesA and XynE differ significantly from those previously described in R . flavefaciens polysaccharidases, including XynB, suggesting that they might represent distinct dockerin specificities.

J Gastroenterol, 2000, 35(5), 382 - 90
Non-traumatic gas gangrene in the abdomen: report of six autopsy cases; Sasaki T et al.; Six autopsy cases of non-traumatic gas gangrene in the abdomen are reported . Five of the six were caused by clostridia, as identified by culture or histology . There were associated underlying diseases, such as alcoholism, liver cirrhosis, diabetes mellitus, and malignant disease . Three of the six patients had gas gangrene in the liver . Bacterial proliferation and gas accumulation were found in the sinusoids of the liver, and congestion and edema with extensive gas embolism were found in the lungs . Pulmonary gas embolism was considered to be the direct cause of death in these three patients . The other three patients had intestinal clostridial gas gangrene, with alcoholism as an underlying condition . None of the six patients was clinically diagnosed as having gas gangrene . We suggest that gas gangrene should be considered in any patient with abdominal infection . A review of 19 autopsy cases of gas gangrene in the abdomen reported in the Japanese literature is also presented.

Klin Khir, 1999, (12), 28 - 30
{The clinical characteristics, diagnosis and treatment of soft-tissue suppurative-inflammatory diseases in patients with narcotic dependence}; Arseniuk VV et al.; Comparative analysis of clinical course and the diagnostic methods of purulent-inflammatory disease of soft tissues was performed in 20 patients with drug abuse (the 1st group) and in 20 patients, who are keeping the normal way of living (2nd group) . In 16 (80%) of patients of the 1st group the anaerobic non-clostridial infection (ANI) was diagnosed . The factors causing the ANI occurrence, were determined, the programme of treatment was introduced . In the 1st group 3 patients died . The duration of the stationary course in the 1st group was 18 days and in the 2nd one--3.8 days.

J Vet Intern Med, 2000 Mar-Apr, 14(2), 190 - 6
Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia; Wilkerson MJ et al.; Classes of antibody bound to erythrocytes were determined using direct immunofluorescence (DIF) flow cytometry in 3 horses and 12 dogs with immune-mediated hemolytic anemia (IMHA) . Background levels of antibody binding were determined in samples from 12 horses and 12 dogs that were free of clinical disease . The range of nonspecific binding of a fluorescein isothiocyanate (FITC)-conjugated goat anti-equine immunoglobulin G (IgG) was 19.9-36.7%, but was eliminated by the use of the F(ab')2 fragment of FITC-conjugated goat anti-equine IgG . Background binding by other class-specific antibodies to equine and canine erythrocytes was negligible . The DIF results were compared to the direct antiglobulin (Coombs') test in 5 horses and 20 dogs with anemia . The former assay was more sensitive in dogs with IMHA than was the Coombs' test (100% versus 58%) . In contrast, the Coombs' test had better specificity than the DIF assay (100% versus 87.5%, respectively) . Using clinical parameters or response to therapy as the comparison, the positive and negative predictive values for the DIF test were 92% and 100% compared to the values of the Coombs' test of 100% and 62% . The DIF assay detected low levels of cells bound with antibody (<30%) in 5 dogs that were Coombs' test-negative . For both species, performance of the DIF test was independent of the prozone effect . Five dogs with IMHA had IgG and IgM on erythrocytes, 5 had IgG, and 2 had IgM . Three horses had surface-bound IgG, including a horse with suspected penicillin-induced IMHA, a foal with neonatal isoerythrolysis, and a foal with clostridial septicemia . The DIF method was valuable in monitoring the response to therapy in the foal with neonatal isoerythrolysis.

Can Vet J, 2000 Apr, 41(4), 306 - 11
The effect of tilmicosin administered to ewes prior to lambing on incidence of clinical mastitis and subsequent lamb performance; Croft A et al.; The effect of tilmicosin on the incidence of clinical mastitis and subsequent lamb performance was studied in 9 sheep flocks in Ontario . Ewes were treated randomly with either tilmicosin or placebo approximately one month prior to lambing . Outcome was assessed by comparing rates of clinical mastitis, palpable udder abnormalities, and preweaning (50-day) lamb weights between the 2 treatment groups, while controlling for other important variables . Lambs raised by multiparous ewes treated with tilmicosin were significantly heavier than lambs from placebo-treated multiparous ewes at 50 days . Lambs from tilmicosin-treated ewes were on average 0.52 kg heavier than lambs in the placebo group . There was no difference between treatment groups in the weight of lambs from first parity ewes . Tilmicosin treatment resulted in a 43% decrease in palpable udder abnormalities . Incidence of clinical mastitis did not differ between experimental groups . The administration of tilmicosin prelambing, at the time of routine clostridial disease vaccination, may be a beneficial and convenient way to reduce mastitis infection and improve the preweaning gain of lambs.

J Appl Microbiol, 2000 Jan, 88(1), 107 - 16
Use of restriction fragment length polymorphism analysis to differentiate strains of psychrophilic and psychrotropic clostridia associated with blown pack' spoilage of vacuum-packed meats; Broda DM et al.; Reference and meat strains of psychrophilic and psychrotrophic clostridia were differentiated using restriction fragment length polymorphism (RFLP) analysis of genomic DNA (DNA-RFLP) and the polymerase chain reaction-amplified 16S rDNA gene (PCR-RFLP) . Groupings obtained with PCR-RFLP were confirmed with 16S rDNA gene sequencing . DNA-RFLP resolved 19 of the 22 meat strains into 11 groups . Three meat strains were untypable using this method . All reference strains representing different genotypic species could be distinguished by the restriction patterns of 16S rDNA genes . With PCR-RFLP, the 22 meat strains produced eight distinct genotypes . 16S rDNA gene sequencing confirmed that each genotype was represented by a distinct sequence . PCR-RFLP restriction patterns of 15 meat strains matched those of one of two of the seven reference strains used . Seven meat strains whose RFLP restriction patterns of 16S rDNA genes differed from those of any reference strains probably represent four previously undescribed species . Although RFLP analysis of the amplified 16S rDNA gene allowed differentiation of psychrophilic and psychrotrophic clostridia at the genotypic species level and below, comparison of PCR-RFLP patterns and 16S rDNA sequences of unknown clostridial isolates with patterns and sequences of reference strains may not effect ready identification of these micro-organisms . The results of this study will be useful in diagnosis of the cause of premature spoilage of chilled vacuum-packed meats and in tracing spoilage-causing clostridia to their source(s) in the abattoir.

Infect Immun, 2000 Apr, 68(4), 2148 - 55
A large toxin from pathogenic Escherichia coli strains that inhibits lymphocyte activation; Klapproth JM et al.; The mechanisms by which bacteria resist cell-mediated immune responses to cause chronic infections are largely unknown . We report the identification of a large gene present in enteropathogenic strains of Escherichia coli (EPEC) that encodes a toxin that specifically inhibits lymphocyte proliferation and interleukin-2 (IL-2), IL-4, and gamma interferon production in response to a variety of stimuli . Lymphostatin, the product of this gene, is predicted to be 366 kDa and shares significant homology with the catalytic domains of the large clostridial cytotoxins . A mutant EPEC strain that has a disruption in this gene lacks the ability to inhibit lymphokine production and lymphocyte proliferation . Enterohemorrhagic E . coli strains of serotype O157:H7 possess a similar gene located on a large plasmid . Loss of the plasmid is associated with loss of the ability to inhibit IL-2 expression while transfer of the plasmid to a nonpathogenic strain of E . coli is associated with gain of this activity . Among 89 strains of E . coli and related bacteria tested, lifA sequences were detected exclusively in strains capable of attaching and effacing activity . Lymphostatin represents a new class of large bacterial toxins that blocks lymphocyte activation.

Scand J Infect Dis, 2000, 32(1), 27 - 30
Clostridial bacteremia in the community hospital; Haddy RI et al.; Anaerobic infections are not commonly studied in the community hospital . The aim of this study was to determine demographic factors, the portals of entry and underlying disorders for clostridial bacteremia and to determine whether appropriate (recommended) treatment is effective . Medical records were reviewed for 42 patients with clostridial bacteremia at 1 Florida, USA, hospital and 4 Dayton, Ohio, USA, hospitals . Fourteen (33.3%) of the patients had clostridial micro-organisms that were isolated in cultures with polymicrobial isolates . Only about half of the patients had fever at the onset of their bacteremia and only slightly more than half had elevated leukocyte counts . The most common portals of entry for the micro-organisms were gastrointestinal (42.9%), unknown (35.7%) and skin (16.7%) . The most common underlying disorders were advanced malignancy (31.0%), diabetes mellitus (14.3%), none determined (12.0%) and acute cholecystitis (9.5%) . The mortality rate was 23.8% . Timely appropriate treatment was started in only about half of the instances . Appropriate (recommended) treatment did not significantly affect survival (p = 0.469) . Clostridial infections and bacteremia exist in the community hospital most commonly in severely ill patients . The fact that clostridia are commonly cultured in blood cultures positive for other bacterial pathogens and that appropriate treatment for clostridia did not affect patient survival, call into question the significance and pathogenicity of clostridial organisms . On the other hand, if clostridial bacteremia was not considered in half these patients with bacteremia, it is possible that more indolent clostridial infections are being overlooked.

J Antimicrob Chemother, 2000 Mar, 45(3), 357 - 61
In vitro activity of R-95867, the active metabolite of a new oral carbapenem, CS-834, against anaerobic bacteria; Kato N et al.; The in vitro activity of R-95867, the active metabolite of a new oral carbapenem, CS-834, was compared with those of DU-6859a, cefditoren, ampicillin/sulbactam and clindamycin against a variety of anaerobic bacteria . R-95867 inhibited 90% of anaerobic strains at </=2 mg/L . In general, R-95867 was 2- to 4-fold less active than DU-6859a but more active than other agents tested against strains of peptostreptococci, clostridia, the Bacteroides fragilis group, Porphyromonas spp . and fusobacteria . R-95867 was stable to hydrolysis by beta-lactamase type 2e derived from B . fragilis, Prevotella bivia and Prevotella intermedia, but unstable to hydrolysis by carbapenemase from B . fragilis.

J Neurosci, 2000 Mar 1, 20(5), 1869 - 82
Expression of Kv1 potassium channels in mouse hippocampal primary cultures: development and activity-dependent regulation; Grosse G et al.; Excitability and discharge behavior of neurons depends on the highly variable expression pattern of voltage-dependent potassium (Kv) channels throughout the nervous system . To learn more about distribution, development, and activity-dependent regulation of Kv channel subunit expression in the rodent hippocampus, we studied the protein expression of members of the Kv1 subfamily in mouse hippocampus in situ and in primary cultures . In adult hippocampus, Kv1 (1-6) channel alpha-subunits were present, whereas at postnatal day 2, none of these proteins could be detected in CA1-CA3 and dentate gyrus . Kv1.1 was the first channel to be observed at postnatal day 6 . The delayed postnatal expression and most of the subcellular distribution observed in hippocampal sections were mimicked by cultured hippocampal neurons in which Kv channels appeared only after 10 days in vitro . This developmental upregulation was paralleled by a dramatic increase in total K(+) current, as well as an elevated GABA release in the presence of 4-aminopyridine . Thus, the developmental profile, subcellular localization, and functionality of Kv1 channels in primary culture of hippocampus closely resembles the in situ situation . Impairing secretion by clostridial neurotoxins or blocking activity by tetrodotoxin inhibited the expression of Kv1.1, Kv1.2, and Kv1.4, whereas the other Kv1 channels still appeared . This activity-dependent depression was only observed before the initial appearance of the respective channels and lost after they had been expressed . Our data show that hippocampal neurons in culture are a convenient model to study the developmental expression and regulation of Kv1 channels . The ontogenetic regulation and the activity-dependent expression of Kv1.1, Kv1.2, and Kv1.4 indicate that neuronal activity plays a crucial role for the development of the mature Kv channel pattern in hippocampal neurons.

Appl Environ Microbiol, 2000 Feb, 66(2), 754 - 62
Spatial changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores; Ludemann H et al.; Molecular ecology techniques were applied to assess changes in the bacterial community structure along a vertical oxygen gradient in flooded paddy soil cores . Microsensor measurements showed that oxygen was depleted from 140 microM at the floodwater/soil interface to nondetectable amounts at a depth of approximately 2.0 mm and below . Bacterial 16S rRNA gene (rDNA)-based community fingerprint patterns were obtained from 200-microm-thick soil slices of both the oxic and anoxic zones by using the T-RFLP (terminal restriction fragment length polymorphism) technique . The fingerprints revealed a tremendous shift in the community patterns in correlation to the oxygen depletion measured with depth . 16S rDNA clone sequences recovered from the oxic or anoxic zone directly corresponded to those terminal restriction fragments which were highly characteristic of the respective zone . Comparative sequence analysis of these clones identified members of the alpha and beta subclasses of Proteobacteria as the abundant populations in the oxic zone . In contrast, members of clostridial cluster I were determined to be the predominant bacterial group in the oxygen-depleted soil . The extraction of total RNA followed by reverse transcription-PCR of the bacterial 16S rRNA and T-RFLP analysis resulted for both oxic and anoxic zones of flooded soil cores in community fingerprint patterns similar to those obtained by the rDNA-based analysis . This finding suggests that the microbial groups detected on the rDNA level are the metabolically active populations within the oxic and anoxic soil slices examined.

Eur J Biochem, 1999 Dec, 266(3), 1073 - 80
Impact of amino acids 22-27 of Rho-subfamily GTPases on glucosylation by the large clostridial cytotoxins TcsL-1522, TcdB-1470 and TcdB-8864; Muller S et al.; Here we report data describing some principles of the interaction between small GTP-binding proteins and large Clostridial cytotoxins (LCTs) . Our investigation was based on the differential glucosylation of Rac1 versus RhoA by LCTs TcsL-1522, TcdB-1470 and TcdB-8864 . Chimeric RhoA/Rac1 proteins and GTPases mutated at defined regions or single amino acids were used as substrates . Starting with chimeric Rac/Rho proteins we demonstrated that proteins containing the N-terminal 73 amino acids of Rac1 (but not those of RhoA) were efficiently glucosylated . Within this stretch, three regions differ significantly in Rac1 and RhoA . Regions containing amino acids 41-45 and 50-54 had no effect on toxin induced glucosylation, whereas amino acids 22-27 had a drastic impact on the potential of all three toxins to covalently modify the GTPases . Point mutations K25T of RhoA (numbering according to Rac1) and K27A of Cdc42 significantly increased glucosylation by the cytotoxins; introduction of lysines at the equivalent positions of Rac1 hindered modification . Our experiments demonstrate the influence of this charged residue on GTPase-LCT interactions . Amino acids 22-27 are part of the transition between the alpha1-helix to the switch I region of small GTP-binding proteins; both are known structures for specificity determination of the interactions with physiologic partners . Comparing these structures with data from our investigation we suggest that TcsL-1522, TcdB-1470 and TcdB-8864 mimic aspects of the physiologic interactions of small GTP-binding proteins.

Appl Environ Microbiol, 1999 Nov, 65(11), 5117 - 23
Acetogenic and sulfate-reducing bacteria inhabiting the rhizoplane and deep cortex cells of the sea grass Halodule wrightii; Kusel K et al.; Recent declines in sea grass distribution underscore the importance of understanding microbial community structure-function relationships in sea grass rhizospheres that might affect the viability of these plants . Phospholipid fatty acid analyses showed that sulfate-reducing bacteria and clostridia were enriched in sediments colonized by the sea grasses Halodule wrightii and Thalassia testudinum compared to an adjacent unvegetated sediment . Most-probable-number analyses found that in contrast to butyrate-producing clostridia, acetogens and acetate-utilizing sulfate reducers were enriched by an order of magnitude in rhizosphere sediments . Although sea grass roots are oxygenated in the daytime, colorimetric root incubation studies demonstrated that acetogenic O-demethylation and sulfidogenic iron precipitation activities were tightly associated with washed, sediment-free H . wrightii roots . This suggests that the associated anaerobes are able to tolerate exposure to oxygen . To localize and quantify the anaerobic microbial colonization, root thin sections were hybridized with newly developed (33)P-labeled probes that targeted (i) low-G+C-content gram-positive bacteria, (ii) cluster I species of clostridia, (iii) species of Acetobacterium, and (iv) species of Desulfovibrio . Microautoradiography revealed intercellular colonization of the roots by Acetobacterium and Desulfovibrio species . Acetogenic bacteria occurred mostly in the rhizoplane and outermost cortex cell layers, and high numbers of sulfate reducers were detected on all epidermal cells and inward, colonizing some 60% of the deepest cortex cells . Approximately 30% of epidermal cells were colonized by bacteria that hybridized with an archaeal probe, strongly suggesting the presence of methanogens . Obligate anaerobes within the roots might contribute to the vitality of sea grasses and other aquatic plants and to the biogeochemistry of the surrounding sediment.

Appl Environ Microbiol, 1999 Nov, 65(11), 4926 - 34
Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites; Ohkuma M et al.; Nitrogen fixation by the microorganisms in the gut of termites is one of the crucial aspects of symbiosis, since termites usually thrive on a nitrogen-poor diet . The phylogenetic diversity of the nitrogen-fixing organisms within the symbiotic community in the guts of various termite species was investigated without culturing the resident microorganisms . A portion of the dinitrogenase reductase gene (nifH) was directly amplified from DNA extracted from the mixed population in the termite gut . Analysis of deduced amino acid sequences of the products of the clonally isolated nifH genes revealed the presence of diverse nifH sequences in most of the individual termite species, and their constituents were considerably different among termite species . A majority of the nifH sequences from six lower termites, which showed significant levels of nitrogen fixation activity, could be assigned to either the anaerobic nif group (consisting of clostridia and sulfur reducers) or the alternative nif methanogen group among the nifH phylogenetic groups . In the case of three higher termites, which showed only low levels of nitrogen fixation activity, a large number of the sequences were assigned to the most divergent nif group, probably functioning in some process other than nitrogen fixation and being derived from methanogenic archaea . The nifH groups detected were similar within each termite family but different among the termite families, suggesting an evolutionary trend reflecting the diazotrophic habitats in the symbiotic community . Within these phylogenetic groups, the sequences from the termites formed lineages distinct from those previously recognized in studies using classical microbiological techniques, and several sequence clusters unique to termites were found . The results indicate the presence of diverse potentially nitrogen-fixing microbial assemblages in the guts of termites, and the majority of them are as yet uncharacterized.

Glycobiology, 1999 Oct, 9(10), 985 - 93
Structural characterization of gangliosides isolated from mullet milt using electrospray ionization-tandem mass spectrometry; Zhu J et al.; Electrospray ionization (ESI) coupled with tandem mass spectrometry has been used in conjunction with microwave-mediated saponification, periodate oxidation, and clostridial sialidase hydrolysis to enable detailed structural characterization of gangliosides and their derivatives present in mullet milt . The gangliosides extracted from mullet milt were determined to be GM3, GM3 lactone, GM3 methyl ester, and 9-O-acetyl GM3 . For the major ganglioside GM3 and all GM3 derivatives, the ceramide composition was revealed to be C18:1/C16:0 . GM3 with a C18:0/C16:0 ceramide was also found as a minor ganglioside . Both the ganglioside intramolecular ester and the ganglioside methyl ester (lacking carboxylic acid groups) showed dominant chloride attachment peaks (M + Cl)- in negative ion ESI-MS in addition to low intensity peaks corresponding to (M-H)- . GM3 and O-acetyl GM3 bearing carboxylic acid functions showed only (M-H)- . In positive ion ESI, GM3 and O-acetyl GM3 revealed (M + 2Na-H)+ peaks in addition to (M + Na)+, indicating free exchange of the carboxylic acid proton with a sodium cation, while the ganglioside intramolecular ester and ganglioside methyl ester with no acidic protons yielded only (M + Na)+ . The strategy of employing ESI-MS to detect products of established wet chemical reactions represents a general approach for elucidation of ganglioside structural details.

Br Poult Sci, 1999 Jul, 40(3), 340 - 7
Effect of dietary high- and low-methylated citrus pectin on the activity of the ileal microflora and morphology of the small intestinal wall of broiler chicks; Langhout DJ et al.; 1 . A study was conducted with broiler chicks to evaluate the effects of dietary high-methylated citrus pectin (HMC) or low-methylated citrus pectin (LMC) on the performance, nutrient digestibility, morphology of the small intestinal wall and ileal microbial activity . 2 . Both pectin products were tested at a dietary content of 30 g/kg using a diet based on maize and soya flour . 3 . Inclusion of HMC in the diet depressed weight gain and food utilization significantly . With a dietary addition of LMC there were only small decreases in weight gain and food utilisation . 4 . Digestibility of dry matter, organic matter, crude fat, starch and amino acids, nitrogen retention and metabolisable energy value were reduced significantly when HMC was added to the diet . The addition of LMC to the diet reduced fat and ash digestibility and metabolisable energy value significantly . 5 . Inclusion of LMC in the diet increased ileal viscosity marginally, whilst HMC had such an effect that the supernatant could not be extracted . Microbial activity in the ileum, particularly that of Enterococci, Bacteroidaceae, Clostridia and E . coli, was increased significantly with dietary addition of HMC . Inclusion of LMC in the diet did not greatly affect microbial activity as only the number of Clostridia was increased . 6 . The addition of HMC to the diet markedly affected the morphology of the intestinal wall and significantly increased the number of goblet cells per 100 villus cells and the sucrase isomaltase activity was increased significantly . However, the morphology of the intestinal wall was hardly affected by LMC, whereas the number of goblet cells per 100 villi cells was significantly increased . 7 . Results of the present study indicate that the inclusion of water-soluble pectins in diets of chicks changes ileal microbial activity and the morphology of the small intestinal wall . The magnitude of these changes depends on the degree of methylation of the pectins.

J Biol Chem, 1999 Sep 10, 274(37), 26518 - 22
SNARE proteins regulate H(+)-ATPase redistribution to the apical membrane in rat renal inner medullary collecting duct cells; Banerjee A et al.; The interaction of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins provides the necessary steps for vesicle docking fusion . In inner medullary collecting duct (IMCD) cells, acid secretion is regulated in part by exocytotic insertion and endocytotic retrieval of an H(+)-ATPase to and from the apical membrane . We previously suggested a role for SNARE proteins in exocytotic insertion of proton pumps in IMCD cells . The purpose of the present study was to determine whether SNARE proteins are associated with the 31-kDa subunit of H(+)-ATPase in IMCD cells during exocytosis and to determine the effects of clostridial toxins on SNARE-mediated trafficking of H(+)-ATPase . Cell acidification induced a marked increment of H(+)-ATPase in the apical membrane . However, pretreating cells with clostridial toxins blocked the cellular translocation of the 31-kDa subunit . Immunoprecipitation of IMCD cell homogenate, using antibodies against either the 31-kDa subunit of H(+)-ATPase or vesicle-associated membrane protein-2, co-immunoprecipitated N-ethylmaleimide-sensitive factor, alpha-soluble NSF attachment protein (alpha-SNAP), synaptosome-associated protein-23, syntaxin, and vesicle-associated membrane protein-2 . Pretreatment with clostridial toxin resulted in reduced co-immunoprecipitation of H(+)-ATPase and syntaxin . These experiments document, for the first time, a putative docking fusion complex in IMCD cells and a physical association of the H(+)-ATPase with the complex . The sensitivity to the action of clostridial toxin indicates the docking-fusion complex is a part of the exocytotic mechanism of the proton pump.

Microbiology, 1999 Jul, 145 ( Pt 7), 1797 - 807
16S rDNA sequencing of Ruminococcus albus and Ruminococcus flavefaciens: design of a signature probe and its application in adult sheep; Krause DO et al.; The ruminococci are an important group of fibrolytic bacteria inhabiting the rumen . Seventeen strains of presumptively identified Ruminococcus were evaluated by a combination of nearly complete and partial 16S rDNA sequence that identified all strains as either Ruminococcus albus or Ruminococcus flavefaciens . All sequences fell into cluster IV of the clostridia, while other species of ruminococci (e.g . Ruminococcus obeum, Ruminococcus gnavus, Ruminococcus lactaris) fall into cluster XIVa of the clostridia . Ruminococcus cluster IV sequences were used to design a 16S rRNA oligonucleotide probe to assess the relative abundance of target populations in a stable ruminal environment . A stable population (animals fed eight times per day) was established in sheep so that statistically robust comparisons could be made in the absence of variation due to diurnal rumen fluctuations . The steady state populations were sampled six times over a 24 d period and direct microscopic counts (DC), total culturable counts (TCC), and total cellulolytic counts (CEL) were determined . DC and culturable data (TCC and CEL) were compared with relative abundance estimates of Ruminococcus IV and Fibrobacter succinogenes . A combination of the Ruminococcus and F . succinogenes probes accounted for 4.0% of the bacterial population and cellulolytic bacteria (measured by most-probable numbers) were 5.2% of the total culturable count . These data suggest that a major portion of the Ruminococcus and Fibrobacter diversity has been cultured and is represented by available sequences . Steady state populations were measured over several days in three sheep and an estimate of variation in DC, TCC, CEL and 16S-based data were obtained . These variance estimates could be used to determine the theoretical sample sizes required to obtain statistically significant differences under different experimental conditions.

J Burn Care Rehabil, 1999 Jul-Aug, 20(4), 282 - 91
Clostridial collagenase releases bioactive fragments from extracellular matrix molecules; Radice M et al.; The current study was designed to investigate the biological role of small extracellular matrix fragments in wound healing . Human burn eschar tissue was digested with bacterial collagenase, and small aminoacidic fragments were inoculated in both human dermal fibroblast cultures and polyvinyl alcohol sponges implanted subcutaneously in the rat . Proliferation assays on cell cultures and biochemical and histologic analyses of the animal model were then performed . Results showed that fibroblasts treated with low concentrations of eschar fragments duplicated significantly faster than controls . Biochemical and histologic data from sponge implants showed that the inflammatory response was augmented by eschar-derived fragments at postoperative day 2, whereas protein and hydroxyproline synthesis were decreased at day 14 . In conclusion, these data substantiate that the application of bacterial collagenase to debride necrotic tissue may have an indirect healing effect resulting from the local release of bioactive matrix-derived fragments.

Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8733 - 8
Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin; Jabaudon D et al.; Maintaining glutamate at low extracellular concentrations in the central nervous system is necessary to protect neurons from excitotoxic injury and to ensure a high signal-to-noise ratio for glutamatergic synaptic transmission . We have used DL-threo-beta-benzyloxyaspartate (TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration . By using the N-methyl-D-aspartate receptors of patched CA3 hippocampal neurons as "glutamate sensors," we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration . This increase was Ca(2+)-independent and was observed in the presence of tetrodotoxin . Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited . Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process . TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter . This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange . These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment . As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.

Ann Chir Gynaecol, 1999, 88(2), 139 - 42
Management of clostridial gas gangrene and the role of hyperbaric oxygen; Korhonen K et al.; BACKGROUND AND AIMS: Clostridial gas gangrene is one of the most dreaded infections in surgery . The aim of this study was to investigate the efficacy of surgery, antibiotic treatment, surgical intensive care and especially the role of hyperbaric oxygen in the management of clostridial gas gangrene . MATERIAL AND METHODS: 53 patients, 42 of them submitted from other hospitals in Finland . After the diagnosis had been made the patients underwent surgical debridement, broad spectrum antibiotic therapy and a series of hyperbaric oxygen (HBO) treatments at 2.5 ATA pressure . The necrotic tissue was excised and incisions were made in the affected areas . Amputations were performed when necessary . RESULTS: Twelve patients died (22.6%) . Hyperbaric oxygen therapy decreased the systemic toxicity and prevented further extension of the infection thereby improving the overall outcome of the patients . CONCLUSION: Hyperbaric oxygen therapy of gas gangrene seems to be life-, limb- and tissue saving . Early diagnosis remains essential . Patient survival can be improved if the disease is recognized early and appropriate therapy applied promptly . Surgical and antibiotic therapy as well as HBO treatment combined with surgical intensive care must be started as soon as possible.

Biochem J, 1999 Jun 1, 340 ( Pt 2), 555 - 60
Chemical rescue of the catalytically disabled clostridial glutamate dehydrogenase mutant D165S by fluoride ion; Hayden BM et al.; The catalytically disabled Asp165-->Ser mutant of clostridial glutamate dehydrogenase shows 100000-fold less activity than the wild-type (WT) enzyme in a standard glutamate oxidation assay and 1000-fold less activity in the reductive-amination reaction . The large reduction in the rate has been attributed to removal of the negative charge and the postulated proton-donor capacity of the aspartate carboxyl group . However, fluoride ion (1 M NaF) causes a 1000-fold activation of the mutant enzyme while simultaneously inhibiting WT activity by 20-fold in the forward reaction . For the reverse reaction, F- (1 M) activates the mutant 4-fold and inhibits WT activity to approx . 64% . The net result when 1 M F- is present is a decrease in the WT:mutant activity ratio from 100000 to 5 for the forward reaction . None of the other halides tested, nor NO3(-), CHCOO- or HCOO-, give comparable activation . Re-activation took 15-30 s under assay conditions, suggesting the possibility of conformational change; CD spectroscopy, however, provided no evidence of a substantial change and kinetics of modification using 5,5'-dithiobis(2-nitrobenzoic acid) suggested only subtle structural rearrangement . This phenomenon is discussed in the light of available information about the structure of the mutant enzyme . It is suggested that the F- ion provides a fixed negative charge at the position of the missing aspartate carboxyl group . Therefore, this appears to be an example of 'chemical rescue'.

Mol Cell Biochem, 1999 Mar, 193(1-2), 37 - 42
Clostridial toxins: molecular probes of Rho-dependent signaling and apoptosis; Bobak DA; The Rho family small GTPases are members of the Ras superfamily of small GTPases . Rho proteins were first determined to act as key regulators of many types of actin cytoskeletal-dependent cellular functions . Recent work by several investigators indicates that Rho GTPases are also critical modulators of several important intracellular and nuclear signal transduction pathways . Certain clostridial toxins and exoenzymes covalently modify, and thereby inactivate, specific types of Rho family GTPases . As such, these microbial enzymes have proven invaluable in helping to identify structural and functional attributes of Rho GTPases.

Klin Khir, 1999, (1), 5 - 6
{Comparative evaluation of the course of peritonitis caused by anaerobic non-Clostridia and aerobic bacteria}; Zaitsev VT et al.; Comparative estimation of clinical course and postoperative complications was done in 134 patients, operated on for diffuse and generalized peritonitis, caused by anaerobic nonclostridial microorganisms in 74 (55.2%) of them and aerobic--in 60 (44.8%) . More severe course and enhanced frequency of the peritonitis complications occurrence, caused by anaerobic microorganisms, was noted . The studying of characteristic peculiarities of peritonitis permits to perform purposeful antibacterial therapy even when bacteriological identification of initiating microorganism is impossible.

Lipids, 1999 Mar, 34(3), 269 - 73
Transformation of bile acids and sterols by clostridia (fusiform bacteria) in Wistar rats; Uchida K et al.; The effects on bile acid and sterol transformation of clostridia (fusiform bacteria), the dominant intestinal bacteria in rodents (ca . 10(10) counts per g wet feces) were examined in Wistar rats . After inoculation of clostridia into germ-free rats and into rats previously inoculated solely with Escherichia coli, most of the endogenous bile acids were deconjugated, and cholic acid and chenodeoxycholic acid were 7alpha-dehydroxylated to deoxycholic acid and lithocholic acid, respectively . Tauro-beta-muricholic acid, another major bile acid in rats, was deconjugated, but only part of it (ca . 30%) was transformed into hyodeoxycholic acid . Cholesterol and sitosterol were also reduced to coprostanol and sitostanol, respectively . Escherichia coli transformed neither bile acids nor sterols . These data suggest that clostridia play an important role in the formation of secondary bile acids and coprostanol in rats.

Protein Sci, 1999 Apr, 8(4), 866 - 72
Specificity of coenzyme analogues and fragments in promoting or impeding the refolding of clostridial glutamate dehydrogenase; Aghajanian S et al.; NAD+ facilitates high-yield reactivation of clostridial glutamate dehydrogenase (GDH) after unfolding in urea . The specificity of this effect has been explored by using analogues and fragments of NAD+ . The adenine portion, unlike the nicotinamide portion, is important for reactivation . Alteration in the nicotinamide portion, in acetylpyridine adenine dinucleotide, has little effect, whereas loss of the 6-NH2 substitution on the adenine ring, in 6-deamino NAD, diminishes the effectiveness of the nucleotide in promoting refolding . Also ADP-ribose, lacking nicotinamide, promotes reactivation whereas NMN-phosphoribose, lacking the adenine, does not . Of the smaller fragments, those containing an adenosine moiety, and especially those with one or more phosphate groups, impede the refolding ability of NAD+, and are able to bind to the folding intermediate though unable to facilitate refolding . These results are interpreted in terms of the known 3D structure for clostridial glutamate dehydrogenase . It is assumed that the refolding intermediate has a more or less fully formed NAD+-binding domain but a partially disordered substrate-binding domain and linking region . Binding of NAD+ or ADP-ribose appears to impose new structural constraints that result in completion of the correct folding of the second domain, allowing association of enzyme molecules to form the native hexamer.

Can Vet J, 1999 Apr, 40(4), 245 - 51
The effect of vaccines and antimicrobials on the formation of injection site lesions in subprimals of experimentally injected beef calves; Van Donkersgoed J et al.; Two hundred and thirty-nine beef calves were used to determine the occurrence of injection site lesions at slaughter (16 to 17 mo of age) following the use of 3 different 8-way clostridial bacterins, a 4-way viral respiratory vaccine, various long-acting oxytetracycline preparations, florfenicol, ceftiofur, and trimethoprim-sulfa when injected in the top hip (top butt), thigh (round), or neck (blade) of calves at 2 to 3 or 5 to 7 mo of age . The occurrence of lesions varied by product, route of administration, and location of injection . The number of steaks affected with lesions, the trim weight of lesions, the histological class of lesions, and the economic losses from trim are described.

Spectrochim Acta A Mol Biomol Spectrosc, 1998 Dec, 54A(14), 2393 - 402
Applications of electron paramagnetic resonance spectroscopy to study interactions of iron proteins in cells with nitric oxide; Cammack R et al.; Nitric oxide and species derived from it have a wide range of biological functions . Some applications of electron paramagnetic resonance (EPR) spectroscopy are reviewed, for observing nitrosyl species in biological systems . Nitrite has long been used as a food preservative owing to its bacteriostatic effect on spoilage bacteria . Nitrosyl complexes such as sodium nitroprusside, which are added experimentally as NO-generators, themselves produce paramagnetic nitrosyl species, which may be seen by EPR . We have used this to observe the effects of nitroprusside on clostridial cells . After growth in the presence of sublethal concentrations of nitroprusside, the cells show they have been converted into other, presumably less toxic, nitrosyl complexes such as (RS)2Fe(NO)2 . Nitric oxide is cytotoxic, partly due to its effects on mitochondria . This is exploited in the destruction of cancer cells by the immune system . The targets include iron-sulfur proteins . It appears that species derived from nitric oxide such as peroxynitrite may be responsible . Addition of peroxynitrite to mitochondria led to depletion of the EPR-detectable iron-sulfur clusters . Paramagnetic complexes are formed in vivo from hemoglobin, in conditions such as experimental endotoxic shock . This has been used to follow the course of production of NO by macrophages . We have examined the effects of suppression of NO synthase using biopterin antagonists . Another method is to use an injected NO-trapping agent, Fe-diethyldithiocarbamate (Fe-DETC) to detect accumulated NO by EPR . In this way we have observed the effects of depletion of serum arginine by arginase . In brains from victims of Parkinson's disease, a nitrosyl species, identified as nitrosyl hemoglobin, has been observed in substantia nigra . This is an indication for the involvement of nitric oxide or a derived species in the damage to this organ.

Curr Opin Struct Biol, 1998 Dec, 8(6), 778 - 84
Unraveling the structures and modes of action of bacterial toxins; Lacy DB et al.; The mechanism by which a soluble protein converts into a protein that spans a membrane remains a central question in understanding the molecular mechanism of toxicity of bacterial protein toxins . Using crystallographic structures of soluble toxins as templates, the past year has seen a number of experiments that are designed to probe the membrane state using other structural methods . In addition, crystallographic information concerning the clostridial neurotoxins has emerged, suggesting a novel mechanism of pore formation and new relationships between toxin binding domains.

Hand Clin, 1998 Nov, 14(4), 635 - 45, ix
Necrotizing fasciitis and gangrene of the upper extremity; Gonzalez MH; Necrotizing fasciitis is a severe, fulminant infection most commonly encountered in patients with diabetes mellitus, alcohol abuse, and intravenous drug abuse . The infection can spread-unrecognized along fascial planes beneath seemingly normal skin . The relatively benign appearance of the extremity is misleading and often results in delay in diagnosis and increased morbidity or death . Immediate aggressive surgical debridement through extensile incisions in combination with antibiotic therapy is necessary for control of these limb- and life-threatening, soft-tissue infections . Gas gangrene, or clostridial myonecrosis, is encountered commonly in those extremity wounds that involve devitalized or necrotic soft tissues . Clostridial microorganisms are anaerobes that produce local and systemic toxins . Delay in treatment can lead to hemolysis, renal failure, and death . Treatment consists of immediate wound debridement, intravenous antibiotics, and hyperbaric oxygen therapy . Diabetic gangrene typically occurs in those diabetic patients with severe peripheral vascular or renal disease . The infections are usually polymicrobial . Treatment involves broad-spectrum antibiotics and multiple surgical debridements or amputation.

Int J Antimicrob Agents, 1998 Aug, 10(3), 229 - 36
Survey of anaerobic susceptibility patterns: a French multicentre study; Mory F et al.; In 1996, the in vitro antibiotic susceptibility of 463 anaerobes was measured in five hospitals using the reference agar dilution method . None of the 209 B . fragilis group strains showed resistance to imipenem or ticarcillin-clavulanic acid . High resistance rates (29%) were observed for cefotetan and clindamycin . beta-Lactamase production was detected respectively in 64% of the Prevotella and 7% of the Fusobacterium strains . Because the same standardized methods were used for many years, the authors were able to evaluate the evolution of antibiotic resistance . Clindamycin resistance had increased within the B . fragilis group (from 14% in 1992 to 29% in 1996) and also among strains of clostridia (32%), P . acnes (18%) and Peptostreptococcus (28%) . In the B . fragilis group multidrug resistance was unlikely to occur.

Clin Cancer Res, 1997 Mar, 3(3), 409 - 17
Phase I evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies; Atkins MB et al.; A Phase I dose escalation trial of i.v . administered recombinant human interleukin 12 (rhIL-12) was performed to determine its toxicity, maximum tolerated dose (MTD), pharmacokinetics, and biological and potential antineoplastic effects . Cohorts of four to six patients with advanced cancer, Karnofsky performance >/=70%, and normal organ function received escalating doses (3-1000 ng/kg/day) of rhIL-12 (Genetics Institute, Inc.) by bolus i.v . injection once as an inpatient and then, after a 2-week rest period, once daily for five days every 3 weeks as an outpatient . Therapy was withheld for grade 3 toxicity (grade 4 hyperbilirubinemia or neutropenia), and dose escalation was halted if three of six patients experienced a dose-limiting toxicity (DLT) . After establishment of the MTD, eight more patients were enrolled to further assess the safety, pharmacokinetics, and immunobiology of this dose . Forty patients were enrolled, including 20 with renal cancer, 12 with melanoma, and 5 with colon cancer; 25 patients had received prior systemic therapy . Common toxicities included fever/chills, fatigue, nausea, vomiting, and headache . Fever was first observed at the 3 ng/kg dose level, typically occurred 8-12 h after rhIL-12 administration, and was incompletely suppressed with nonsteroidal anti-inflammatory drugs . Routine laboratory changes included anemia, neutropenia, lymphopenia, hyperglycemia, thrombocytopenia, and hypoalbuminemia . DLTs included oral stomatitis and liver function test abnormalities, predominantly elevated transaminases, which occurred in three of four patients at the 1000 ng/kg dose level . The 500 ng/kg dose level was determined to be the MTD . This dose, administered by this schedule, was associated with asymptomatic hepatic function test abnormalities in three patients and an onstudy death due to Clostridia perfringens septicemia but was otherwise well tolerated by the 14 patients treated in the dose escalation and safety phases . The T1/2 elimination of rhIL-12 was calculated to be 5.3-9.6 h . Biological effects included dose-dependent increases in circulating IFN-gamma, which exhibited attenuation with subsequent cycles . Serum neopterin rose in a reproducible fashion regardless of dose or cycle . Tumor necrosis factor alpha was not detected by ELISA . One of 40 patients developed a low titer antibody to rhIL-12 . Lymphopenia was observed at all dose levels, with recovery occurring within several days of completing treatment without rebound lymphocytosis . There was one partial response (renal cell cancer) and one transient complete response (melanoma), both in previously untreated patients . Four additional patients received all proposed treatment without disease progression . rhIL-12 administered according to this schedule is biologically and clinically active at doses tolerable by most patients in an outpatient setting . Nonetheless, additional Phase I studies examining different schedules and the mechanisms of the specific DLTs are indicated before proceeding to Phase II testing.

Mol Pharmacol, 1998 Nov, 54(5), 789 - 801
1-Methyl-4-phenylpyridinium induces autocrine excitotoxicity, protease activation, and neuronal apoptosis; Leist M et al.; The pathogenesis of several neurodegenerative diseases may involve indirect excitotoxic mechanisms, where glutamate receptor overstimulation is a secondary consequence of initial functional defects of neurons (e.g., impairment of mitochondrial energy generation) . The neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and other mitochondrial inhibitors (e.g., rotenone or 3-nitropropionic acid) elicited apoptosis in cerebellar granule cell cultures via stimulation of autocrine excitotoxicity . Cell death, increase in intracellular Ca2+ concentration, release of cytochrome c, and all biochemical and morphological signs of apoptosis were prevented by blockade of the N-methyl-D-aspartate receptor with noncompetitive, glycine-site or glutamate-site inhibitors . In addition, MPP+-induced apoptosis was reduced by high Mg2+ concentrations in the medium or by inhibiting exocytosis with clostridial neurotoxins . Two classes of cysteine proteases were involved in the execution of cell death: caspases and calpains . Inhibitors of either class of proteases prevented cell death, cleavage of intracellular proteins (i.e., fodrin), and the appearance of typical features of apoptosis such as phosphatidylserine translocation or DNA fragmentation . However, protease inhibitors did not interfere with the initial intracellular Ca2+ concentration increase . We suggest that MPP+ as well as other mitochondrial inhibitors trigger indirect excitotoxic processes, which lead to Ca2+ overload, protease activation, and subsequent neuronal apoptosis.

Pediatrics . 1998 Nov;102(5):E53.
Hyperbaric oxygen therapy in the pediatric patient: the experience of the Israel Naval Medical Institute; Waisman D et al.; The pediatric patient is to be found in hyperbaric facilities throughout the world, receiving hyperbaric oxygen (HBO) therapy for both life-threatening and chronic diseases . OBJECTIVE: To review the experience accumulated at the Israel Naval Medical Institute in the treatment of pediatric patients . DESIGN: A retrospective analysis and review of all records of patients younger than age 18 years . RESULTS: Between 1980 and 1997, 139 pediatric patients age 2 months to 18 years (mean, 7.7 years) received HBO treatment at the Israel Naval Medical Institute . Of the children, 111 (79%) suffered from acute carbon monoxide (CO) poisoning; 13 (9.2%) were treated after crush injury, traumatic ischemia, or compartment syndrome; 4 (2.8%) had clostridial myonecrosis; 1 (0.7%) had necrotizing fasciitis; 5 (3.6%) had refractory osteomyelitis; 2 (1.4%) had suffered massive air embolism; 2 (1.4%) had purpura fulminans; and 1 (0.7%) suffered from decompression sickness . Outcome, judged by neurologic sequelae, mortality, and extent of soft tissue loss and limb amputation, was favorable in 129 patients (93%) . Two patients (1.4%) died, 1 as a result of CO poisoning and the other, gas gangrene; 2 of the patients in the CO group (1.4%) remained with neurologic sequelae, and 6 patients in the acute traumatic ischemia group (4.3%) underwent limb amputation . CONCLUSIONS: We had a favorable experience with 129 of a total 139 pediatric patients treated at our facility for the indications listed . A basic knowledge of HBO therapy is needed to refer the pediatric patient for treatment when indicated . The needs of the pediatric patient, especially the critically ill, require specific skills and equipment inside the hyperbaric chamber . Close collaboration between the pediatrician and the hyperbaric medicine physician is essential to ensure adequate care for infants and children.

Crit Care Nurs Q, 1998 Aug, 21(2), 24 - 35
Orthopaedic infections; Redfield D et al.; Patients with orthopaedic infections may require admission to the intensive care unit (ICU) . Necrotizing fasciitis and clostridial myonecrosis (gas gangrene) are serious soft tissue infections that may cause life-threatening complications . Patients suffering from infectious arthritis, osteomyelitis, or prosthetic joint infections may be seen in the ICU as a result of a previous injury, surgery, or delayed infectious processes . This article introduces the ICU nurse to the pathophysiology, clinical presentation, and management of a variety of orthopaedic infections.

Biochemistry, 1998 Sep 15, 37(37), 12829 - 37
Delta T 14/Delta D 15 Azotobacter vinelandii ferredoxin I: creation of a new CysXXCysXXCys motif that ligates a {4Fe-4S} cluster; Kemper MA et al.; In clostridial-type ferredoxins, each of the two {4Fe-4S}2+/+ clusters receives three of its four ligands from a CysXXCysXXCys motif . Azotobacter vinelandii ferredoxin I (AvFdI) is a seven-iron ferredoxin that contains one {4Fe-4S}2+/+ cluster and one {3Fe-4S}+/0 cluster . During the evolution of the 7Fe azotobacter-type ferredoxins from the 8Fe clostridial-type ferredoxins, one of the two motifs present changed to a CysXXCysXXXXCys motif, resulting in the inability to form a 4Fe cluster and the appearance of a 3Fe cluster in that position . In a previous study, we were unsuccessful in using structure as a guide in designing a 4Fe cluster in the 3Fe cluster position of AvFdI . In this study, we have reversed part of the evolutionary process by deleting two residues between the second and third cysteines . UV/Vis, CD, and EPR spectroscopies and direct electrochemical studies of the purified protein reveal that this DeltaT14/DeltaD15 FdI variant is an 8Fe protein containing two {4Fe-4S}2+/+ clusters with reduction potentials of -466 and -612 mV versus SHE . Whole-cell EPR shows that the protein is present as an 8Fe protein in vivo . These data strongly suggest that it is the sequence motif rather than the exact sequence or the structure that is critical for the assembly of a 4Fe cluster in that region of the protein . The new oxygen-sensitive 4Fe cluster was converted in partial yield to a 3Fe cluster . In known ferredoxins and enzymes that contain reversibly interconvertible {4Fe-4S}2+/+ and {3Fe-4S}+/0 clusters, the 3Fe form always has a reduction potential ca . 200 mV more positive than the 4Fe cluster in the same position . In contrast, for DeltaT14/DeltaD15 FdI, the 3Fe and 4Fe clusters in the same location have extremely similar reduction potentials.

Arch Microbiol, 1998 Oct, 170(4), 297 - 303
Membrane-associated redox activities in Thermotoga neapolitana; Kaslin SA et al.; Elemental sulfur reduction by the hyperthermophilic bacterium Thermotoga neapolitana provides an alternative to hydrogen evolution during fermentation . Electrons are transferred from reduced cofactors (ferredoxin and NADH) to sulfur by a series of unknown steps . One enzyme that may be involved is an NADH:methyl viologen oxidoreductase (NMOR), an activity that in other fermenting organisms is associated with NADH:ferredoxin oxidoreductase . We found that 83% of NMOR activity was contained in the pellet fraction of cell extracts subjected to ultracentrifugation . This pellet fraction, presumably containing cell membranes, was required for electron transfer to NAD+ from ferredoxin-dependent pyruvate oxidation . However, the NMOR activity in this fraction used neither Thermotoga nor clostridial ferredoxins as substrates . NMOR activity was also detected in aerobically prepared vesicles . By comparison with ATPase activities, NMOR was found primarily on the cytoplasmic face of these vesicles . During these studies, an extracytoplasmic hydrogenase activity was discovered . In contrast to the soluble hydrogenase, this hydrogenase activity was completely inhibited when intact cells were treated with cupric chloride and was present on the extracytoplasmic face of vescides . In contrast to a soluble hydrogenase reported in Thermotoga maritima, this activity was air-stable and was inhibited by low concentrations of nitrite.

Appl Environ Microbiol, 1998 Sep, 64(9), 3444 - 50
New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of Nitrogenase (nifH) genes; Zehr JP et al.; Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated . Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques . Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the alpha and gamma subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans . nifH sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans . Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction . The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.

J Biol Chem, 1998 Aug 14, 273(33), 21015 - 24
Cloning and analysis of the genes for a novel electron-transferring flavoprotein from Megasphaera elsdenii . Expression and characterization of the recombinant protein; O'Neill H et al.; The genes that encode the two different subunits of the novel electron-transferring flavoprotein (ETF) from Megasphaera elsdenii were identified by screening a partial genomic DNA library with a probe that was generated by amplification of genomic sequences using the polymerase chain reaction . The cloned genes are arranged in tandem with the coding sequence for the beta-subunit in the position 5' to the alpha-subunit coding sequence . Amino acid sequence analysis of the two subunits revealed that there are two possible dinucleotide-binding sites on the alpha-subunit and one on the beta-subunit . Comparison of M . elsdenii ETF amino acid sequence to other ETFs and ETF-like proteins indicates that while homology occurs with the mitochondrial ETF and bacterial ETFs, the greatest similarity is with the putative ETFs from clostridia and with fixAB gene products from nitrogen-fixing bacteria . The recombinant ETF was isolated from extracts of Escherichia coli . It is a heterodimer with subunits identical in size to the native protein . The isolated enzyme contains approximately 1 mol of FAD, but like the native protein it binds additional flavin to give a total of about 2 mol of FAD/dimer . It serves as an electron donor to butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.

J Comp Neurol, 1998 Jul 20, 397(1), 89 - 104
Disruption of the retinal basal lamina during early embryonic development leads to a retraction of vitreal end feet, an increased number of ganglion cells, and aberrant axonal outgrowth; Halfter W; Bacterial collagenase was injected into the vitreous of the eye of chick and quail embryos . Immunocytochemical and ultrastructural studies revealed that the collagenase dissolved the retinal basal lamina of the injected eye . The basal lamina disruption was first detectable 1 hour after enzyme injection and was complete within 3 hours . With further development, the retinal basal lamina was not reestablished; newly developing neuroepithelium in the peripheral retina, however, generated an intact basal lamina . Western blot analysis showed that Clostridial collagenase degraded various collagens but spared noncollagenous proteins . Basal lamina disruption of embryonic day 3 to 6 retinae led to the retraction of the end feet of the neuroepithelial cells, caused an increase in the number of Islet-1+ cells (most likely ganglion cells), an increase in the thickness of the optic fiber layer, and aberrant growth of optic axons on their way toward the optic disc . None of these changes were observed when retinal basal laminae were disrupted at later stages of development . The present data demonstrate that the retinal basal lamina, by anchoring the neuroepithelial cells to the pial surface of the retina, has an important function in the development of the normal cytoarchitecture of this structure . It is proposed that the altered extracellular environment in the vitreal part of the retina, resulting in the retraction of the neuroepithelial end feet, is responsible for the increased number of Islet-1+ cells and the aberrant axonal navigation.

Mol Biol Cell, 1998 Jun, 9(6), 1437 - 48
A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells; Galli T et al.; The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25 . Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis . In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs . In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections . Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin . We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

J Protein Chem, 1998 May, 17(4), 311 - 8
Accuracy of secondary structure and solvent accessibility predictions for a clostridial neurotoxin C-fragment; Lebeda FJ et al.; Earlier studies used Rost and Sander's artificial neural network {(1993a), J . Mol . Biol . 232, 584-599} to predict the secondary structures {Lebeda and Olson (1994), Proteins 20, 293-300} and residue solvent accessibilities {Lebeda and Olson (1997), J . Protein Chem . 16, 607-618} of the clostridial neurotoxins . Because the X-ray crystal structure of the 50-kDa C-terminal half of the heavy chain of tetanus toxin was recently determined, this report evaluates the accuracy of these network-derived predictions . For this predominantly beta-strand-containing fragment, predictions, on a per-residue basis, for both secondary structure and solvent accessibility were about 70% accurate . A more flexible and realistic analysis based on overlapping segments yielded accuracies of over 80% for the three-state secondary structure and for the two-state accessibility predictions . Because the accuracies of these predictions are comparable to those made by Rost and Sander using a dataset of 126 nonhomologous globular proteins, our predictions provide a quantitative foundation for gauging the results when building by homology the structures of related proteins.

J Biol Chem, 1998 Mar 6, 273(10), 5514 - 9
Discovery of a novel ferredoxin from Azotobacter vinelandii containing two {4Fe-4S} clusters with widely differing and very negative reduction potentials; Gao-Sheridan HS et al.; Ferredoxins that contain 2{4Fe-4S}2+/+ clusters can be divided into two classes . The "clostridial-type" ferredoxins have two Cys-Xaa-Xaa-Cys-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Cys-Pro motifs . The "chromatium-type" ferredoxins have one motif of that type and one more unusual Cys-Xaa-Xaa-Cys-Xaa7-9-Cys-Xaa-Xaa-Xaa-Cys-Pro motif . Here we report the purification of a novel ferredoxin (FdIII) from Azotobacter vinelandii which brings to 12 the number of small {Fe-S} proteins that have now been reported from this organism . NH2-terminal sequencing of the first 56 amino acid residues shows that FdIII is a chromatium-type ferredoxin with 77% identity and 88% similarity to Chromatium vinosum ferredoxin . Studies of the purified protein by matrix-assisted laser desorption ionization-time of flight mass spectroscopy, iron analysis, absorption, circular dichroism, and electron paramagnetic resonance spectroscopies show that FdIII contains 2{4Fe-4S}2+/+ clusters in a 9,220-Da polypeptide . All 2{4Fe-4S}2+/+ ferredoxins that have been studied to date, including C . vinosum ferredoxin, are reported to have extremely similar or identical reduction potentials for the two clusters . In contrast, electrochemical characterization of FdIII clearly establishes that the two {4Fe-4S}2+/+ clusters have very different and highly negative reduction potentials of -486 mV and -644 mV versus the standard hydrogen electrode.

Biochemistry, 1998 Feb 24, 37(8), 2578 - 85
S-Adenosylmethionine-dependent reduction of lysine 2,3-aminomutase and observation of the catalytically functional iron-sulfur centers by electron paramagnetic resonance; Lieder KW et al.; Lysine 2,3-aminomutase catalyzes the interconversion of l-alpha-lysine and l-beta-lysine . The enzyme contains an iron-sulfur cluster with unusual properties, and it requires pyridoxal-5'-phosphate (PLP) and S-adenosylmethionine (AdoMet) for activity . The reaction proceeds by a substrate radical rearrangement mechanism, in which the external aldimine formed between PLP and lysine is initially converted into a lysyl-radical intermediate by hydrogen abstraction from C3 . The present research concerns the mechanism by which a hydrogen-abstracting species is generated at the active site of lysine 2,3-aminomutase . Earlier tritium tracer experiments have implicated the 5'-deoxyadenosyl moiety of AdoMet in this process . AdoMet is here shown to interact with the iron-sulfur cluster at the active site of Clostridial lysine 2,3-aminomutase . Reduction of the iron-sulfur cluster from its EPR-silent form {4Fe-4S}2+ to the fully reduced form {4Fe-4S}1+ requires the presence of either AdoMet or S-adenosylhomocysteine (SAH) and a strong reducing agent such as dithionite or deazariboflavin and light . The reduced forms are provisionally designated E-{4Fe-4S}1+/AdoMet and E-{4Fe-4S}1+/SAH, and they display similar low-temperature EPR spectra centered at gav = 1.91 . The reduced form E-{4Fe-4S}1+/AdoMet is fully active in the absence of any added reducing agent, whereas the form E-{4Fe-4S}1+/SAH is not active . It is postulated that the active form E-{4Fe-4S}1+/AdoMet is in equilibrium with a low concentration of a radical-initiating form that contains the 5'-deoxyadenosyl radical . Initiation of the radical rearrangement mechanism is postulated to take place by action of the 5'-deoxyadenosyl radical in abstracting a hydrogen atom from carbon-3 of lysine, which is bound as its external aldiminine with PLP . This process accounts for the results of tritium tracer experiments, it explains the radical rearrangement mechanism, and it rationalizes the roles of AdoMet and the {4Fe-4S} cluster in the reaction.

Eur J Biochem, 1998 Feb 1, 251(3), 946 - 54
Identification and characterization of the natural electron donor ferredoxin and of FAD as a possible prosthetic group of benzoyl-CoA reductase (dearomatizing), a key enzyme of anaerobic aromatic metabolism; Boll M et al.; Under anoxic conditions most aromatic compounds are metabolized via benzoyl-CoA which becomes reduced by benzoyl-CoA reductase (dearomatizing); this enzyme was recently described in the bacterium Thauera aromatica {Boll, M . & Fuchs, G . (1995) Eur . J . Biochem . 234, 921-933} . It catalyzes the reaction benzoyl-CoA + 2 e- + 2 H+ + 2 MgATP + 2 H2O --> cyclohexa-1,5-diene-1-carboxyl-CoA + 2 MgADP + 2 Pi . The iron-sulfur protein has a native molecular mass of 160-170 kDa and consists of four different subunits . In addition a flavin may be present . The nature of the potential prosthetic group and the natural electron donor were determined . Purified benzoyl-CoA reductase preparations contained 0.25-0.3 mol FAD/mol enzyme . Cells grown anaerobically with aromatic substrates contained a ferredoxin which represented the main, if not the only ferredoxin present . It was purified from 200 g cells with a yield of 60 mg and its N-terminal amino acid sequence was determined . The native molecular mass was 9659 +/- 2 Da as determined by electrospray mass spectrometry . The protein contained 7.6 +/- 0.6 mol iron and 7.6 +/- 1 mol acid-labile sulfur/mol . The ultraviolet-visible spectrum of the protein was typical for ferredoxins with maxima at 280 nm and 390 nm (in the oxidized state) . The estimated molar absorption coefficients were 63500 M(-1) cm(-1) at 280 nm and 40500 M(-1) cm(-1) at 390 nm . The difference spectrum between the oxidized and the reduced form had a maximum at 415 nm with delta epsilon415 = 8200 M(-1) cm(-1) . 1 mol ferredoxin became reduced/mol dithionite added, suggesting the presence of two {4Fe-4S} clusters . The average midpoint potential of the iron-sulfur clusters was -450 mV . The ferredoxin gene was cloned and sequenced . It was located in a gene cluster coding for enzymes involved in anaerobic aromatic metabolism . The amino acid sequence of the T . aromatica ferredoxin showed high similarities to several other ferredoxins containing 2{4Fe-4S} clusters, e.g . from Clostridia and phototrophic bacteria . The reduced ferredoxin served as electron donor for benzoyl-CoA reduction at a three times higher rate compared with the rate obtained with the artificial electron donor reduced methyl viologen . The turnover number with the natural electron donor of 5 s(-1) can explain the bacterial growth rate with benzoate as substrate . Half-maximal enzyme activity was obtained with 6 microM reduced ferredoxin, at an estimated cellular concentration of 70 microM ferredoxin . Both the low apparent Km value and the turnover number are consistent with the proposed role of ferredoxin in aromatic-ring reduction.

J Anim Sci, 1998 Jan, 76(1), 323 - 7
Application of research findings and summary of research needs: Bud Britton Memorial Symposium on Metabolic Disorders of Feedlot Cattle; Galyean ML et al.; Updated research findings with acidosis, feedlot bloat, liver abscesses, and sudden death syndromes were presented at the Bud Britton Memorial Symposium on Metabolic Disorders of Feedlot Cattle . Possible industry applications include the need to establish guidelines for use of clostridial vaccines in feedlot cattle, further assessment of the relationship between acidosis and polioencephalomalacia, examination of the effects of various ionophores on the incidence of metabolic disorders, and evaluation of the effects of feed bunk management and limit- and restricted-feeding programs on the incidence of metabolic disorders . A multidisciplinary approach among researchers, consulting nutritionists and veterinarians, and feedlot managers will be required for effective progress in research and in the application of research findings . Areas suggested for further research include 1) assessment of feed consumption patterns and social behavior of cattle in large-pen, feedlot settings; 2) evaluation of the relationship between feed intake management systems (feed bunk management programs, limit- and programmed-feeding) and the incidence of metabolic disorders, including delineation of the role of variability in feed intake in the etiology of such disorders; 3) efforts to improve antemortem and postmortem diagnosis, and to establish standardized regional or national epidemiological databases for various metabolic disorders; 4) ascertaining the accuracy of diagnosis of metabolic disorders and determining the relationship of previous health history of animals to the incidence of metabolic disorders; 5) further defining ruminal and intestinal microbiology as it relates to metabolic disorders and deeper evaluation of metabolic changes that occur with such disorders; 6) continued appraisal of the effects of grain processing and specific feed ingredients and nutrients on metabolic disorders, and development of new feed additives to control or prevent these disorders; and 7) application of biotechnology to develop grain varieties with altered nutrient degradation profiles that decrease the propensity for disastrous acid loads in the rumen, feed-grade enzymes and probiotics that modify nutrient digestion or microbial profiles in the rumen and intestine, and specific strains of ruminal bacteria and protozoa that alter ruminal and metabolic conditions that may precipitate metabolic disorders.

J Laparoendosc Adv Surg Tech A, 1997 Aug, 7(4), 245 - 7
Clostridial gas gangrene of the abdominal wall after laparoscopic cholecystectomy; Samel S et al.; Laparoscopic cholecystectomy is associated with a considerable rate of infectious complications of up to 2.8% . Such infections are usually of minor clinical importance . However, we observed a case of life-threatening Clostridial gas gangrene centering around the right lateral port site and developing across all of the right-sided abdominal wall, causing septic shock and severe multi-organ failure . Considering the overall infection rate and the possibility of even severe morbidity, we advocate perioperative antibiotics in laparoscopic cholecystectomy.

Invert Neurosci, 1995, 1(1), 3 - 13
Presynaptic proteins involved in exocytosis in Drosophila melanogaster: a genetic analysis; Littleton JT et al.; Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal . This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane . Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human . Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking . This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion . The hypothesis that these mechanisms function at the synapse relies largely upon in vitro evidence . Recently, genetic approaches in mice, C . elegans and the fruitfly, Drosophila melanogaster, have been used to dissect the in vivo function of numerous proteins involved in synaptic transmission . This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release in Drosophila . In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, making Drosophila an ideal model system to further unravel the intricacies of synaptic transmission.

Trends Neurosci, 1997 Oct, 20(10), 471 - 7
Organotypic slice cultures: a technique has come of age; Gahwiler BH et al.; Slices of CNS tissue prepared from young rodents can be maintained in culture for many weeks to months . The basic requirements are simple: a stable substratum, culture medium, sufficient oxygenation and incubation at a temperature of about 36 degrees C . Under these conditions, nerve cells continue to differentiate and to develop a tissue organization that closely resembles that observed in situ . Several alternative culturing methods have been developed recently . Slices maintained in stationary culture with the interface method are ideally suited for questions requiring a three-dimensional structure, whereas slices cultured in roller-tubes remain the method of choice for experiments that require optimal optical conditions . In this report, three typical experiments are discussed that illustrate the potential of the slice-culture technique . The first example indicates that, due to their high neuronal connectivity, slice cultures provide a very useful tool for studying the properties of synaptic transmission between monosynaptically coupled cell pairs . The other two studies show how long-term application of substances to slice cultures can be used to examine the consequences of epileptic discharges in vitro, as well as the effects of slowly acting clostridial neurotoxins on synaptic transmission.

Clin Transplant, 1997 Jun, 11(3), 206 - 8
Clostridial infection of a liver transplant treated with retransplantation; Knechtle SJ et al.; Clostridial infection of a liver transplant is reported and was treated successfully with immediate retransplantation and antibiotics . This strategy may salvage patients who otherwise have a fulminant course and certain death . This case supports the general principle that infection localized to the liver can be successfully treated with retransplantation when appropriate.

Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3402 - 7
Patterns of synaptic activity in neural networks recorded by light emission from synaptolucins; Miesenbock G et al.; The emission of light, coupled to exocytosis, can in principle be utilized to monitor the activity of a large number of individual synapses simultaneously . To illustrate this concept, fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP-2/synaptobrevin (which we term "synaptolucins") were expressed in cultured hippocampal neurons with the help of viral vectors . Synaptolucins were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with their cognate luciferin, which was added to the extracellular medium . Photon emissions required a depolarizing stimulus, occurred from regions with high synaptic density as ascertained by vital staining of recycling synaptic vesicles, and were sensitive to Ca2+ depletion and clostridial neurotoxins . The method can currently detect exocytosis of the readily releasable pool of synaptic vesicles at a hippocampal synapse, corresponding to about two dozen quanta, but has the potential for greater sensitivity.

Eur J Neurosci, 1997 Mar, 9(3), 472 - 9
Increased presynaptic protein kinase C activity and glutamate release in rats with a prenatally induced hippocampal lesion; Di Luca M et al.; We have previously shown that protein kinase C (PKC) activity is up-regulated in nerve terminals of animals that have been subjected to targeted cellular ablation of cortical and hippocampal neurons by treatment with methylazoxymethanol (MAM), which results in impaired long-term potentiation (LTP) and cognitive deficit . In this study we investigated the consequences of increased membrane-bound PKC in the regulation of release of glutamate, the major excitatory transmitter involved in LTP . We show that nerve terminals of MAM-treated rats show higher PKC activity, as monitored by the in situ phosphorylation of B-50/GAP-43, in both basal and phorbol ester-stimulated conditions . In these animals, hippocampal nerve endings release a greater amount of glutamate than those of controls, both in basal conditions and when synaptosomes are stimulated with KCl or 3,4-diaminopyridine . The potentiation observed in MAM-treated rats was counteracted by the PKC blocker H-7 and the clostridial tetanus toxin . On the contrary, GABA release was not significantly up-regulated, either in basal or in depolarization-evoked conditions . Therefore our data show that the increase in synaptosomal PKC activity is paralleled by increased glutamate but not GABA release in this animal model . Whether this reflects specific up-regulation of membrane PKC activity in glutamatergic terminals or an alteration in the regulation of glutamate release remains to be determined.

J Intern Med, 1997 Mar, 241(3), 253 - 5
Acute papillary muscle rupture in a patient with clostridial sepsis; Schulz R et al.; We describe the clinical course of a 58-year-old man who died from acute papillary muscle rupture as a complication of clostridial sepsis . There was no evidence for myocardial infarction, infective endocarditis, prior chest trauma or other known causes of papillary muscle rupture . Histological specimens taken at autopsy demonstrated clostridial infection of the heart . To our knowledge the occurrence of papillary muscle rupture in the setting of clostridial sepsis has not yet been reported in the literature.

Biochem J, 1997 Mar 1, 322 ( Pt 2), 507 - 10
Metal substitution of tetanus neurotoxin; Tonello F et al.; Tetanus neurotoxin was depleted of its catalytic Zn2+ ion, and the apotoxin was reconstituted with different transition metal ions . The Mn2+- and Co2+-tetanus neurotoxins are highly active in the proteolysis of vesicle-associated membrane protein/synaptobrevin, the natural substrate of this toxin, whereas Cu2+ and Fe2+ minimally supported proteolytic activity . The visible absorbance spectrum of Co2+-tetanus neurotoxin shows a maximum at 538 nm with a molar absorption coefficient of 82 M(-1) x cm(-1) . These results indicate that the Zn2+ environment at the active site of tetanus neurotoxin is different from those of known Zn2+-endopeptidases and provide a structural basis for the definition of tetanus neurotoxin, and the related clostridial neurotoxins, as an independent family of metalloproteases.

J Small Anim Pract, 1997 Feb, 38(2), 70 - 3
Management of a femoral fracture complicated by clostridial myositis; Thomson MJ et al.; A clinical case of clostridial myositis secondary to a comminuted femoral fracture is described . This case is unusual because, despite the severe degree of obvious muscle necrosis and gas production, the dog had minimal signs of systemic toxicity . Union of the fracture was achieved but six months postoperatively muscular contracture had resulted in permanent stifle extension.

Proteins, 1997 Feb, 27(2), 319 - 21
Purification, crystallization, and preliminary X-ray studies of 10-formyltetrahydrofolate synthetase from Clostridia acidici-urici; D'Ari L et al.; The monofunctional enzyme 10-formyltetrahydrofolate synthetase (THFS), which is responsible for the recruitment of single carbon units from the formate pool into a variety of folate-dependent biosynthetic pathways, has been subcloned, purified, and crystallized . The crystals belong to space group P2(1), with unit cell dimensions a = 102.4 A, b = 116.5 A, c = 115.8 A, and beta = 103.5 . The crystal unit cell and diffraction is consistent with an asymmetric unit consisting of the enzyme tetramer, and a specific volume of the unit cell of 2.7 A3/ Da . The crystals diffract to at least 2.3 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.

Adv Exp Med Biol, 1997, 419, 53 - 60
Identification of the catalytic site of clostridial ADP-ribosyltransferases; Aktories K; The catalytic sites of clostridial ADP-ribosyltransferases were studied by photoaffinity-labelling with {carbonyl-14C}NAD+ . In C3-like transferases, which are known to modify low molecular mass GTP-binding Rho proteins, Glu-174 was identified to be essential for catalysis . In C . perfringens iota toxin, Glu-380 and Glu-378 may have pivotal roles in the active site of this actin-ADP-ribosylating toxin.

EMBO J, 1996 Dec 16, 15(24), 6951 - 9
Mutational analysis of VAMP domains implicated in Ca2+-induced insulin exocytosis; Regazzi R et al.; Vesicle-associated membrane protein-2 (VAMP-2) and cellubrevin are associated with the membrane of insulin-containing secretory granules and of gamma-aminobutyric acid (GABA)-containing synaptic-like vesicles of pancreatic beta-cells . We found that a point mutation in VAMP-2 preventing targeting to synaptic vesicles also impairs the localization on insulin-containing secretory granules, suggesting a similar requirement for vesicular targeting . Tetanus toxin (TeTx) treatment of permeabilized HIT-T15 cells leads to the proteolytic cleavage of VAMP-2 and cellubrevin and causes the inhibition of Ca2+-triggered insulin exocytosis . Transient transfection of HIT-T15 cells with VAMP-1, VAMP-2 or cellubrevin made resistant to the proteolytic action of TeTx by amino acid replacements in the cleavage site restored Ca2+-stimulated secretion . Wild-type VAMP-2, wild-type cellubrevin or a mutant of VAMP-2 resistant to TeTx but not targeted to secretory granules were unable to rescue Ca2+-evoked insulin release . The transmembrane domain and the N-terminal region of VAMP-2 were not essential for the recovery of stimulated exocytosis, but deletions preventing the binding to SNAP-25 and/or to syntaxin I rendered the protein inactive in the reconstitution assay . Mutations of putative phosphorylation sites or of negatively charged amino acids in the SNARE motif recognized by clostridial toxins had no effect on the ability of VAMP-2 to mediate Ca2+-triggered secretion . We conclude that: (i) both VAMP-2 and cellubrevin can participate in the exocytosis of insulin; (ii) the interaction of VAMP-2 with syntaxin and SNAP-25 is required for docking and/or fusion of secretory granules with the plasma membrane; and (iii) the phosphorylation of VAMP-2 is not essential for Ca2+-stimulated insulin exocytosis.

Experientia, 1996 Dec 15, 52(12), 1026 - 32
Bacterial protein toxins and cell vesicle trafficking; Montecucco C et al.; A group of bacterial protein toxins interfere with vesicular trafficking inside cells . Clostridial neurotoxins affect mainly the highly regulated fusion of neurotransmitter- and hormone-containing vesicles with the plasma membrane . They cleave the three SNARE proteins: VAMP, SNAP-25 and syntaxin, and this selective proteolysis results in a blockade of exocytosis . The Helicobacter pylori cytotoxin is implicated in the pathogenesis of gastroduodenal ulcers . It causes a progressive and extensive vacuolation of cells followed by necrosis, after a cytotoxin-induced alteration of membrane trafficking by late endosomes . Vacuoles originate from this compartment in a rab7-dependent process and swell because they are acidic and accumulate membrane-permeant amines.

Arch Microbiol, 1996 Dec, 166(6), 418 - 20
The acetate kinase of Clostridum acetobutylicum strain P262; Diez-Gonzalez F et al.; Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent . Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity . The acetate kinase had a pH optimum of 8.0, a Km for acetate of 160 mM, and a kcat of 16, 800 min-1 . The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

Infect Dis Clin North Am, 1996 Dec, 10(4), 835 - 55
Necrotizing soft-tissue infections; Chapnick EK et al.; Necrotizing soft-tissue infections may be rapidly fatal because of toxin-induced circulatory collapse . Because of the often nonspecific clinical presentation, prompt diagnosis may be difficult but is imperative as prompt treatment can be lifesaving . This article discusses necrotizing fasciitis and clostridial myonecrosis, and highlights pathogenesis, clinical presentation, diagnosis, and treatment.

Appl Environ Microbiol, 1996 Nov, 62(11), 4049 - 59
Phylogenetic analysis of the bacterial communities in marine sediments; Gray JP et al.; For the phylogenetic analysis of microbial communities present in environmental samples microbial DNA can be extracted from the sample, 16S rDNA can be amplified with suitable primers and the PCR, and clonal libraries can be constructed . We report a protocol that can be used for efficient cell lysis and recovery of DNA from marine sediments . Key steps in this procedure include the use of a bead mill homogenizer for matrix disruption and uniform cell lysis and then purification of the released DNA by agarose gel electrophoresis . For sediments collected from two sites in Puget Sound, over 96% of the cells present were lysed . Our method yields high-molecular-weight DNA that is suitable for molecular studies, including amplification of 16S rRNA genes . The DNA yield was 47 micrograms per g (dry weight) for sediments collected from creosote-contaminated Eagle Harbor, Wash . Primers were selected for the PCR amplification of (eu)bacterial 16S rDNA that contained linkers with unique 8-base restriction sites for directional cloning . Examination of 22 16S rDNA clones showed that the surficial sediments in Eagle Harbor contained a phylogenetically diverse population of organisms from the Bacteria domain (G . J . Olsen, C . R . Woese, and R . Overbeek, J . Bacteriol . 176:1-6, 1994) with members of six major lineages represented: alpha, delta, and gamma Proteobacteria; the gram-positive high G+C content subdivision; clostridia and related organisms; and planctomyces and related organisms . None of the clones were identical to any representatives in the Ribosomal Database Project small subunit RNA database . The analysis of clonal representives in the first report using molecular techniques to determine the phylogenetic composition of the (eu)bacterial community present in coastal marine sediments.

Anal Biochem, 1996 Oct 1, 241(1), 120 - 7
A sensitive and rapid fluorescence-based assay for determination of tetanus toxin peptidase activity; Soleilhac JM et al.; The light chain of tetanus toxin (TeNT-L chain), endowed with a zinc metalloendopeptidase activity, cleaves specifically the vesicle-associated membrane protein (VAMP), also called synaptobrevin, at a single peptide bond (Gln76-Phe77), resulting in the blockade of neuroexocytosis . The 50-mer synaptobrevin peptide S 39-88, synthesized by solid-phase peptide synthesis, was determined to be the minimum substrate of TeNT still notably hydrolyzed by TeNT-L chain . In this peptide, Tyr88 was substituted by the highly fluorescent amino acid (L) pyrenylalanine (Pya) which was synthesized in good yields by an enantioselective method . The fluorescent substrate {Pya88} S 39-88 was cleaved four times more rapidly by TeNT-L chain than S 39-88 (kcat/Km = 9635 and 2455 M-1.min-1, respectively) . One of the two metabolites formed by the action of TeNT L chain, {Pya88} S 77-88, was easily separated from the substrate in one step using Sep-Pak Vac C18 cartridges and its concentration quantified by fluorescence . This novel enzymatic assay, which could be easily extended to other clostridial neurotoxins, is a major improvement in term of sensitivity and time saving, compared to currently used methods (SDS-PAGE, HPLC) . It lends itself readily to automation for large-scale screening of selective and potent inhibitors of these neurotoxins which remain to be developed.

Minerva Chir, 1996 Sep, 51(9), 751 - 4
{Enzymatic cleaning with collagenase in the local treatment of skin lesions complicated by scab and necrotic debris}; Pietrantoni P et al.; The authors present a clinical survey conducted to value the antiseptic and reparative properties of collagenasis (clostridiopeptidiasi A), in the topical treatment of skin lesions . The study presented 90 patients with burns, pressure sores, abrasions and trophic ulcers . The results obtained were highly positive in terms of the healing or cleansing of the wound, bearing further witness to the reliability of the product in question.

Arh Hig Rada Toksikol, 1996 Sep, 47(3), 289 - 93
{Hemipelvectomy due to blast injuries: possibilities of occupational rehabilitation}; Cesarec M et al.; A case of traumatic hemipelvectomy due to explosion of an antitank shell in a 21-year-old Croatian Army soldier is described . Explosion was due to inadvertent handling of the shell in the army barracks . Literature data on persons who survived traumatic hemipelvectomy are extremely scarce; the injury is characterized by a very high death rate (60-100%) . After prompt surgical treatment the patient developed a postoperative anaerobic clostridial infection . Owing to intensive clinical and physical therapy the patient was enabled for moving around in a special wheel-chair and walking with the help of crutches and a prosthesis . He was also enrolled in a training course in computer use . Continual physical therapy and psychotherapy were essential because of a high degree of disablement . Making the patient self-sufficient, fit for work and finding him an appropriate job is considered to be the responsibility not only of the professional medical team but also of joint efforts on the part of competent state ministries, Ministry of Defence, Ministry of Labour and Social Welfare, State Pension and Insurance Fund and Ministry of Education, Culture and Sports.

Voen Med Zh, 1996 Sep, 317(9), 34 - 7, 80
{The microflora of gunshot and explosive mine wounds in victims delayed for a long time at the prehospital stage}; Badikov VD et al.; The clinico-microbiological research of gunshot and mine-explosive injuries in wounded servicemen of Afghanistan army, arriving on stage of the qualified surgical care for 2-5 days after wound is lead . The quantitative and qualitative structure of wound microflora at modern combat trauma on a prehospital stage is determined . It was established, that main kinds of wound infectious complications in injuries are suppuration of wounds and clostridial infection . The advantages and the lacks of benzyl penicillin for prophylaxis of wound infection are investigated.

Aust Vet J, 1996 Sep, 74(3), 225 - 7
Field trials of a formulation containing moxidectin and 6 in 1 vaccines for sheep; Schwartzkoff CL et al.; OBJECTIVE: To assess the efficacy and safety of a formulation containing moxidectin and 6 in 1 vaccine in sheep under field conditions . DESIGN: Efficacy and safety study . ANIMALS: Two hundred and five crossbred Merino lambs and two hundred and eight Merino ewes were used in the studies . PROCEDURE: A formulation was made for the simultaneous treatment of sheep with moxidectin and immunisation against clostridial diseases and caseous lymphadenitis . The efficacy against nematodes, vaccine response and safety were assessed . RESULTS: Effective control of nematodes and responses to antigens were achieved following subcutaneous administration . The formulation was safe to administer; occasional minor tissue reactions were evident, but no other adverse effects of treatment were observed in either pregnant ewes or lambs, using either the recommended dose, or an overdose of the formulation . CONCLUSION: Administration of a formulation containing moxidectin, five clostridial antigens and caseous lymphadenitis antigen proved safe and efficacious under field conditions.

Biochemistry, 1996 Aug 27, 35(34), 11081 - 4
Lysine 2,3-aminomutase: rapid mix-freeze-quench electron paramagnetic resonance studies establishing the kinetic competence of a substrate-based radical intermediate; Chang CH et al.; Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-lysine and L-beta-lysine . The enzyme contains iron-sulfur clusters and is activated by pyridoxal 5'-phosphate and S-adenosylmethionine, all of which participate in catalysis . Current spectroscopic evidence implicates two substrate-based organic radicals as intermediates in the mechanism . One of these species, the radical N3-(5'-phosphopyridoxylidene)-beta-lysin-2-yl (3), appears in the steady state of the reaction of lysine and has been definitively characterized by EPR and ESEEM spectroscopy . The 2-deuterio form of this radical, 3-2-d, which is generated in the reaction of L-{2-2H}lysine, can be distinguished by line shape analysis from 3 . The rate at which the signal for 3-2-d is transformed into that for 3 has been measured by rapid mix-freeze quench kinetic analysis . The rate constant for this process is 24 +/- 8 s-1 at 21 degrees C . This is the rate constant for the turnover of radical 3 and is indistinguishable from the turnover number of lysine 2,3-aminomutase . Therefore, radical 3 is kinetically competent as an intermediate in the reaction of lysine 2,3-aminomutase.

J Am Vet Med Assoc, 1996 Jul 1, 209(1), 130 - 6
Aortic and iliac artery thrombosis in calves: nine cases (1974-1993); Morley PS et al.; OBJECTIVE--To identify common clinical and diagnostic features of calves with aortic or iliac artery thrombosis that might aid in antemortem diagnosis of this condition . DESIGN--Retrospective case series . ANIMALS--9 calves < or = 6 months old in which aortic or iliac artery thrombosis was confirmed at necropsy . RESULTS--All calves had an acute onset of paresis or flaccid paralysis of 1 or both hind limbs . Affected limbs were hypothermic and had diminished spinal reflexes and diminished pulse pressures . Diagnosis was definitively established in 2 calves by use of angiography . All 9 calves died or were euthanatized . CLINICAL IMPLICATIONS--This condition is rare and could be mistaken for more common diseases of young cattle, such as traumatic injury of the axial or appendicular skeleton, vertebral osteomyelitis, nutritional muscular dystrophy associated with vitamin E or selenium deficiency, injury to the sciatic or femoral nerves, or clostridial myositis.

Tidsskr Nor Laegeforen, 1996 May 30, 116(14), 1675 - 8
{Hyperbaric oxygen therapy . Now an established treatment in Norway}; Brattebo G et al.; Hyperbaric oxygen therapy can be a life-saving form of treatment for certain acute medical conditions, e.g . cerebral gas embolism, clostridial infections, and smoke/carbon monoxide inhalation . It has long been used for treating decompression illness in divers . As from 1994 our hospital has been delegated the national responsibility for hyperbaric medicine in Norway . This paper describes the physiological basis, indications, and contraindications for hyperbaric oxygen treatment, and summarises hyperbaric oxygen treatment in Bergen over the last two years.

J Biol Chem, 1996 May 3, 271(18), 10611 - 5
Characterization of an alternatively spliced GM2 activator protein, GM2A protein . An activator protein which stimulates the enzymatic hydrolysis of N-acetylneuraminic acid, but not N-acetylgalactosamine, from GM2; Wu YY et al.; GM2 activator protein is a protein cofactor which stimulates the enzymatic hydrolysis of both GalNAc and NeuAc from GM2 . We have previously isolated two cDNA clones, GM2 activator cDNA and GM2A cDNA, for human GM2 activator protein (Nagarajan, S., Chen, H.-C., Li, S.-C., Li, Y.-T., and Lockyer, J . M . (1992) Biochem . J . 282, 807-813) . GM2A mRNA is an RNA alternative splicing product that contains exons 1, 2, 3, and intron 3 of the genomic DNA sequence of GM2 activator protein (Klima, H., Tanaka, A., Schnabel, D., Nakano, T., Schroder, M., Suzuki, K., and Sandhoff, K . (1991) FEBS Lett . 289, 260-264) . GM2A cDNA encodes a protein (GM2A protein) containing 1-109 of the 160 amino acids of human GM2 activator protein, plus a tripeptide (VST) encoded by intron 3 at the COOH terminus . Thus, GM2A protein can be regarded as a form (truncated version) of GM2 activator protein . We have expressed GM2A cDNA in Escherichia coli using pT7-7 as the vector . The recombinant GM2A protein was purified to an electrophoretically homogeneous form and was found to stimulate the hydrolysis of NeuAc from GM2 by clostridial sialidase, but not the hydrolysis of GalNAc from GM2 by beta-hexosaminidase A . Like GM2 activator protein, GM2A protein also specifically recognized the terminal GM2 epitope in GalNAc-GD1a and stimulated the hydrolysis of only the external NeuAc from this ganglioside by clostridial sialidase . These results enabled us to discern the enzymatic hydrolyses of GalNAc and NeuAc from the GM2 epitope and established that the NeuAc recognition domain of GM2 activator protein is located within amino acids 1-109 . The presence of GM2A mRNA in human tissues and the selective stimulation of NeuAc hydrolysis by GM2A protein indicate that this activator protein may be involved in the catabolism of GM2 through the asialo-GM2 pathway.

J Hand Surg {Am}, 1996 May, 21(3), 490 - 5
Collagenase in the treatment of Dupuytren's disease: an in vitro study; Starkweather KD et al.; The effects of clostridial collagenase on the tensile strength of Dupuytren's cords was studied in vitro to assess its potential efficacy as an agent for clinical enzymatic fasciotomy . Collagenase was injected into Dupuytren's cords from patients undergoing fascioctomy . Following a pilot experiment, in which a 3,600-unit dose of collagenase induced a 93% decrease in tensile modulus as compared with control cords, groups of five cords each were injected with 150, 300, and 600 units . These cords and a control group of five cords were tested by loading to failure in tension . The ultimate stress and strain to failure were recorded by a video capture technique . All specimens were stained for histologic examination with hematoxylin and eosin for collagen typing with sirrius red . Comparison of the ultimate stress values obtained with published values of extensor forces obtainable by the individual fingers of 40 normal hands indicated that a 300-unit dose of collagenase was sufficient for cord rupture within the average maximum force limits of the extensors of the index, long, ring, and small fingers (p < .02) . All samples were in the residual disease stage histologically and contained type I collagen by sirrius red staining . These results indicate that collagenase may be effective in enzymatic fasciotomy of residual-stage Dupuytren's disease.

Eur J Cell Biol, 1996 Apr, 69(4), 351 - 9
Expression of synaptosomal-associated protein SNAP-25 in endocrine anterior pituitary cells; Aguado F et al.; A growing body of evidence indicates that the fundamental molecular mechanism of exocytosis in the secretory pathway may be structurally similar in all eukaryotic cells . The synaptosomal-associated protein of 25 kDa (SNAP-25) is a plasma membrane protein involved in regulated exocytosis in neurons . In order to compare exocytotic components in neurons and endocrine cells, we have analyzed the expression of SNAP-25 in the rat anterior pituitary . Western blotting analysis documented the presence of SNAP-25 in anterior pituitary homogenates and cultured anterior pituitary cells . In addition to SNAP-25, other neuronal proteins involved in exocytosis (syntaxin, VAMP/synaptobrevin and Rab3A) were also detected in the anterior pituitary . The specific expression of SNAP-25 mRNA in anterior pituitary cells was also corroborated by Northern analysis . SNAP-25 immunoreactivity was located at the plasma membrane of endocrine anterior pituitary cells . Characteristically, patches of fine punctate deposits exhibited intense SNAP-25 immunoreactivity . Double-labeling immunocytochemistry revealed that SNAP-25 was mainly associated with gonadotroph cell populations . Furthermore, we demonstrate that in the anterior pituitary, SNAP-25 is selectively cleaved by clostridial neurotoxins . In conclusion, our results establish the presence of SNAP-25 in secretory anterior pituitary cells and suggest a potential role of this protein in the secretion of adenohypophysial hormone.

J Chemother, 1996 Apr, 8(2), 91 - 5
Anaerobic bacteria in postsurgical infections: isolation rate and antimicrobial susceptibility; Di Rosa R et al.; Three hundred clinical samples, obtained from post-surgical infections, were evaluated . 37% of samples were positive for anaerobes, 99 samples (33%) for aerobes and anaerobes in mixed culture, and 13 (4%) only for anaerobes . One hundred forty-nine anaerobic strains were isolated: Bacteroides and Clostridia occurred most frequently (34% and 23% respectively) . Antimicrobial susceptibility of the isolates was tested by means of a commercial broth microdilution method . In addition, the standardized agar dilution method was performed to evaluate the susceptibility to 8 antibiotics of the 51 Bacteroides strains.

Eur J Biochem, 1996 Apr 1, 237(1), 35 - 44
Catalytic properties, molecular composition and sequence alignments of pyruvate: ferredoxin oxidoreductase from the methanogenic archaeon Methanosarcina barkeri (strain Fusaro); Bock AK et al.; Methanosarcina barkeri (strain Fusaro) was grown on pyruvate as methanogenic substrate {Bock, A . K., Prieger-Kraft, A . & Schonheit, P . (1994) Arch . Microbiol . 161, 33-46} . The first enzyme of pyruvate catabolism, pyruvate oxidoreductase, which catalyzes oxidation of pyruvate to acetyl-CoA was purified about 90-fold to apparent electrophoretic homogeneity . The purified enzyme catalyzed the CoA-dependent oxidation of pyruvate with ferredoxin as an electron acceptor which defines the enzyme as a pyruvate: ferredoxin oxidoreductase . The deazaflavin, coenzyme F420, which has been proposed to be the physiological electron acceptor of pyruvate oxidoreductase in methanogens, was not reduced by the purified enzyme . In addition to ferredoxin and viologen dyes, flavin nucleotides served as electron acceptors . Pyruvate: ferredoxin oxidoreductase also catalyzed the oxidation of 2-oxobutyrate but not the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, 3-hydroxypyruvate and oxaloacetate . The apparent Km values of pyruvate:ferredoxin oxidoreductase were 70 microM for pyruvate, 6 microM for CoA and 30 microM for clostridial ferredoxin . The apparent Vmax with ferredoxin was about 30 U/mg (at 37 degrees C) with a pH optimum of approximately 7 . The temperature optimum was approximately 60 degrees C and the Arrhenius activation energy was 40 kJ/mol (between 30 degrees C and 60 degrees C) . The enzyme was extremely oxygen sensitive, losing 90% of its activity upon exposure to air for 1 h at 0 degrees C . Sodium nitrite inhibited the enzyme with a Ki of about 10 mM . The native enzyme had an apparent molecular mass of approximately 130 kDa and was composed of four different subunits with apparent molecular masses of 48, 30, 25, and 15 kDa which indicates that the enzyme has an alpha beta gamma delta structure . The enzyme contained 1 mol/mol thiamine diphosphate, and about 12 mol/mol each of non-heme iron and acid-labile sulfur . FAD, FMN and lipoic acid were not found . The N-terminal amino acid sequences of the four subunits were determined . The sequence of the alpha-subunit was similar to the N-terminal amino acid sequence of the alpha-subunit of the heterotetrameric pyruvate:ferredoxin oxidoreductases of the hyperthermophiles Archaeoglobus fulgidus, Pyrococcus furiosus and Thermotoga maritima and of the mesophile Helicobacter pylori, and to the N-terminal amino acid sequence of the homodimeric pyruvate:ferredoxin oxidoreductase from proteobacteria and from cyanobacteria . No sequence similarities were found, however, between the alpha-subunit of the M . barkeri enzyme and the heterodimeric pyruvate:ferredoxin oxidoreductase of the archaeon Halobacterium halobium.

J Trauma, 1996 Mar, 40(3), 476 - 80
Clostridial meningitis as a result of craniocerebral arrow injury; Neal G et al.; We report a case of a 6-year-old girl who presented with an arrow lodged in the temporal lobe and carotid sinus . Her postoperative recovery was complicated by a rare penicillin-resistant clostridial meningitis . A review of the literature reveals no other cases of this nature in the pediatric population.

J Chemother, 1996 Feb, 8(1), 3 - 16
Beta-lactam resistance in anaerobic bacteria: a review; Hedberg M et al.; The majority of the human microflora consists of anaerobic bacteria . Normally these bacteria have low pathogenicity, but under certain conditions, such as destruction of tissues and poor circulation or impaired host defense, they may cause serious infections . Bacteroides species are the most frequently isolated microorganisms from suppurative anaerobic infections and they have the broadest spectrum of resistance to the commonly used antimicrobial agents . Resistance to antimicrobial agents is an increasing problem, especially to beta-lactam compounds . Multiresistant clinical isolates, resistant to beta-lactam antibiotics as well as other antimicrobial agents used in the treatment and prophylaxis of anaerobic infections are now occurring . Resistance to beta-lactam antibiotics is usually mediated by beta-lactamase production . A few isolates of Bacteroides fragilis are producing metallo-beta-lactamases which are capable of hydrolyzing beta-lactamase stable compounds such as cefoxitin and imipenem . The enzyme activity in metallo-beta-lactamases is not affected by the clinically used beta-lactamase inhibitors clavulanic acid, sulbactam and tazobactam . Other resistance mechanisms are alterations in the penicillin-binding proteins (PBPs) or a decreased permeability through the outer membrane . Beta-lactam resistance and beta-lactamase production have also been detected in some species of clostridia, fusobacteria, Prevotella, Porphyromonas and in some other anaerobic bacteria.

J Antibiot (Tokyo), 1996 Feb, 49(2), 145 - 9
Antibiotics A21459 A and B, new inhibitors of bacterial protein synthesis . I . Taxonomy, isolation and characterization; Selva E et al.; Novel cyclic peptide antibiotics A21459 A and B are produced by a member of the genus Actinoplanes sp . These antibiotics inhibit bacterial protein synthesis and have selective antimicrobial activity against clostridia, mycoplasma and some Gram-negative bacteria.

Adv Dermatol, 1996, 11, 117 - 51; discussion 152
Subcutaneous emphysema; Samlaska CP et al.; A medical emergency, the detection of subcutaneous emphysema requires thorough evaluation to exclude the multitude of disease processes that may demonstrate this clinical finding . Gas gangrene must be considered in the differential diagnosis of all forms of subcutaneous emphysema and infections with some species, such as C . novyi, may not produce gas at all . Isolation of C . septicum from the blood is almost always associated with colon cancer or hematologic malignancies . Nonclostridial gas gangrene in diabetic patients is indistinguishable clinically from clostridial gas gangrene . A unique and true dermatologic emergency is the detection of nontraumatic subcutaneous emphysema of the thigh with or without associated erythema, tenderness, or bullous lesions . This finding is associated with perforated viscus in a retroperitoneal location . Infections with gas-producing organisms continue to be a source of significant morbidity in modern times.

Khirurgiia (Mosk), 1996, (1), 73 - 4
{Diagnosis and treatment of local forms of surgical anaerobic non-clostridial infection}; Leshchenko IG et al.; 145 patients with such kind of surgical infection have been taken to clarify the cause of it . Aerobic agents were the cause in 16.6% and anaerobic nonclostridial agents--in 33.1% of all cases . The mixed flora was the cause in 44.1% . The most important place among anaerobic nonclostridial infections belongs to bacteroids (38.3%), peptostreptococcus (27.3%) and the peptococcus (17.2%) . One must use wide-spectrum antibiotic for a treatment of a such kind of infection tiel the usults of microbiological test are known and only then one can use group-specific antibiotics.

Vestn Ross Akad Med Nauk, 1996, (2), 41 - 3
{Study of pathogenetic mechanisms of intoxication in patients with anaerobic non-clostridial infection}; Istratov VG et al.; Pyoinflammatory diseases continue to be one of the most topical problem of modern medicine . Anaerobic non-clostridial infection is essential in the etiological pattern of pyoinflammatory diseases . Its specific feature is early developed severe intoxication . The authors have developed laboratory criteria for evaluating the degree of intoxication in patients with anaerobic non-clostridial infection, which are based on chromatographic determination of the concentrations of specific metabolic products, such as volatile fatty acids and toxic metabolites of the phenol group, in the patients' peripheral blood, which allows the detected disorders to be promptly corrected . The metabolic products of non spore-forming anaerobes, such as volatile fatty acids, are important pathogenetic factors since they inhibit the dynamic activity of platelets and the phagocytic activity of leukocytes in blood.

Vestn Ross Akad Med Nauk, 1996, (2), 37 - 41
{Several aspects of non-clostridial anaerobic infection in cancer patients}; Davydov MI et al.; The specific features of the development and natural history of nonclostridial anaerobic infection (NACI) were studied in 125 cancer patients with various site tumors . It was shown that after surgery for malignant abdominal and genital tumors, NCAI might develop in the late postoperative period despite antibacterial antianaerobic prevention and even long-term antianaerobic therapy . NCAI treated with antianaerobic therapy was found to run long with aggravations and remissions in patients undergone surgery and drug therapy for genital cancer . Radiation therapy is a risk factor for NCAI in patients with malignant genital neoplasms . The risk factors for NCAI in skin cancer patients are tumor ulceration, second infection, and resolution . The most severe problem is likely anaerobic bacteremia in cancer patients . Anaerobic bacteremia appears to be more common than we thought and this issue requires further in-depth study.

Vestn Ross Akad Med Nauk, 1996, (2), 34 - 7
{Progress and perspectives in the study of non-clostridial anaerobic infection of female genitalia}; Tsvelev IuV et al.; The paper summarizes the results from 15-year studies of non-clostridial anaerobic infection in obstetrics and gynecology . The authors show that strict non-spore-bearing anaerobic microbes are prevalent in the etiological pattern of pyoinflammatory diseases of the female genitals, present the basic pathogenetic features of the development and clinical signs of anaerobic infections . The present-day principles of diagnosis and treatment of these diseases are given . Prospects are shown for further studies of non-clostridial anaerobic infection in obstetric and gynecological care.

Vestn Ross Akad Med Nauk, 1996, (2), 30 - 3
{Anaerobic infection in abdominal surgery}; Bogomolova NS et al.; The etiological pattern of non-clostridial anaerobic infection, its natural history and drug therapy in patients with its various types were studied and analyzed at the Surgery Research Center, Russian Academy of Medical Sciences . Peritonitis and cholangitis were examined in detail . Multimodality treatment of patients with acute peritonitis caused by a combination of aerobic and anaerobic pathogens by using hyperbaric oxygenation and therapy with directly acting antibiotics was found to enhance its efficiency, decrease therapy duration, prevent inflammation progression, and reduce mortality rates from 25.64 to 8.8% . The application to the techniques of preliminary decompression and the developed regimens of antibacterial therapy and hyperbaric oxygenation in the multimodality treatment for suppurative cholangitis noticeably yielded better therapeutical results, reduced mortality rates from 5.7 to 0% and these patients' hospital stay.

Vestn Ross Akad Med Nauk, 1996, (2), 3 - 8
{Infections caused by non spore-forming anaerobic bacteria}; Vorob'ev AA et al.; Pyo-inflammatory diseases continue to be one of the topical problems of modern medicine . Their etiological agents are opportunistic microorganisms among which non spore-forming anaerobes are prevalent in the normal microscopic flora . They cause a pathological process when the body's immunity is decreased . The authors consider the biological features of this group of bacteria, epidemiological and pathogenetic aspects, the problems of laboratory and clinical diagnosis of non clostridial anaerobic infection and outline immediate steps to be taken to solve the problems.

Berl Munch Tierarztl Wochenschr, 1996 Jan, 109(1), 10 - 3
Equine intestinal clostridiosis in a group of polo ponies in Dubai, U.A.E; Wernery U et al.; An acute outbreak of a C . perfringens Type C enterotoxemia in a polo club killed 8 polo ponies in less than 24 hours . Hay with a massive fungal contamination (Aspergillus and Fusarium sp.) is suspected to have triggered the enterotoxemia . Our cases show numerous similarities with the previously reported cases of EIC . However, acute toxic tubulonephrosis in the necropsied horses and longstanding elevation of AST and gamma GT-levels in survivors is unique in our cases.

J Anim Sci, 1995 Nov, 73(11), 3235 - 40
Injection-site lesions in carcasses of cattle receiving injections at branding and at weaning; George MH et al.; Crossbred steer and heifer calves (n = 84) were given injections at branding and at weaning (using a completely randomized block design); 225 to 376 d later, pairs of two subprimal cuts from each animal were evaluated for incidence and severity of injection-site lesions . The four products compared were 1) a 2-mL clostridial, 2) a 5-mL clostridial, 3) vitamin AD3, and 4) a long-acting oxytetracycline antibiotic (OTC) . Branding-age calves (mean 48.3 d of age) received intramuscular injections of two of the four products, in the semimembranosus (inside round) muscles (one in the left muscle and one in the right); at weaning age (mean 199.3 d of age), calves received injections of the remaining two products in the gluteus medius (top sirloin butt) muscles (one in the left and the other in the right) . Injections at branding of 2 mL of clostridial, 5 mL of clostridial, vitamin AD3, and OTC caused injection-site lesions in 72.5, 92.7, 5.3, and 51.2%, respectively, of inside rounds from slaughter cattle . Incidence of injection-site lesions was lowest (P < .05) among inside rounds and top sirloin butts from animals given vitamin AD3 (as calves, and at both branding and weaning times) and was highest (P < .05) in inside rounds from cattle given, as calves, injections of 5 mL of clostridial at branding or of OTC at weaning . Less trimming was required to remove the lesions resulting from injections of all four products when they were given at weaning time . Before completion of the present study, it was thought that injection-site lesions were from damage that subsisted only briefly following an inoculation; these results make it abundantly clear that intramuscular administration of clostridials and certain antibiotics will cause damage so severe that it will be evident in beef muscle 7.5 to 12 mo later.

Biochem J, 1995 Nov 1, 311 ( Pt 3), 905 - 10
Urea-induced inactivation and denaturation of clostridial glutamate dehydrogenase: the absence of stable dimeric or trimeric intermediates; Aghajanian SA et al.; Urea-induced effects in clostridial glutamate dehydrogenase (GDH, EC 1.4.1.2) were studied by spectrophotometry, circular dichroism, FPLC, affinity chromatography and PAGE . Denaturation of enzyme occurred over a narrow range of urea concentrations (2.5-3.5 M), accompanied by inactivation of enzyme with a similar rate constant . The contribution of instantaneous inhibition by urea was also ascertained . FPLC studies of urea-treated GDH gave no evidence for dissociated oligomeric fragments of the hexamer in the presence of subdenaturing concentrations of urea . Likewise a mixture of fully 5,5'-dithiobis-(2-nitrobenzoic acid)-modified GDH hexamers and unmodified enzyme in 2 M urea failed to give rise to hybrid molecules . Exposure of unmodified GDH to high concentrations of urea led to the dissociation of hexamers to denatured monomers followed by association to form non-specific high-M(r) aggregates . This conclusion was confirmed by native gradient PAGE experiments . Various specific ligands stabilized the enzyme against urea-induced inactivation, succinate and 2-oxoglutarate being particularly effective . This protection of the native state was enhanced in ternary complexes, and the complex most resistant to urea-induced inactivation was the productive ternary complex GDH-NADH-2-oxoglutarate . Native gradient PAGE experiments indicate that these protecting ligands preserve the native hexameric structure of GDH.

J Biol Chem, 1995 Oct 13, 270(41), 24246 - 51
Specific recognition of N-acetylneuraminic acid in the GM2 epitope by human GM2 activator protein; Li SC et al.; GM2 Activator is a low molecular weight protein cofactor that stimulates the enzymatic conversion of GM2 into GM3 by human beta-hexosaminidase A and also the conversion of GM2 into GA2 by clostridial sialidase (Wu, Y.-Y., Lockyer, J.M., Sugiyama, E., Pavlova, N.V., Li, Y.-T., and Li, S.-C . (1994) J . Biol . Chem . 269, 16276-16283) . Among the five known activator proteins for the enzymatic hydrolysis of glycosphingolipids, only GM2 activator is effective in stimulating the hydrolysis of GM2 . However, the mechanism of action of GM2 activator is still not well understood . Using a unique disialosylganglioside, GalNAc-GD1a, as the substrate, we were able to show that in the presence of GM2 activator, GalNAc-GD1a was specifically converted into GalNAc-GM1a by clostridial sialidase, while in the presence of saposin B, a nonspecific activator protein, GalNAc-GD1a was converted into both GalNAc-GM1a and GalNAc-GM1b . Individual products generated from GalNAc-GD1a by clostridial sialidase were identified by thin layer chromatography, negative secondary ion mass spectrometry, and immunostaining with a monoclonal IgM that recognizes the GM2 epitope . Our results clearly show that GM2 activator recognizes the GM2 epitope in GalNAc-GD1a . Thus, GM2 activator may interact with the trisaccharide structure of the GM2 epitope and render the GalNAc and NeuAc residues accessible to beta-hexosaminidase A and sialidase, respectively.

Biochemistry, 1995 Sep 12, 34(36), 11417 - 22
Positive cooperativity with Hill coefficients of up to 6 in the glutamate concentration dependence of steady-state reaction rates measured with clostridial glutamate dehydrogenase and the mutant A163G at high pH; Wang XG et al.; Glutamate dehydrogenases from many sources display nonclassical kinetic behavior suggestive of allosteric interaction among the six subunits of the hexamer . A three-dimensional structure now potentially offers a framework for explaining the basis of such behavior in clostridial glutamate dehydrogenase, and this paper offers evidence of extreme, all-or-none cooperativity in the binding of glutamate by this enzyme . A site-directed mutant of clostridial glutamate dehydrogenase in which Ala163 in the glutamate binding site is replaced by glycine displays a markedly sigmoid dependence of reaction rate on glutamate concentration (S0.5 = 200 mM), with a Hill coefficient of 3.4 when assayed at pH 10.5 with 1 mM NAD+ . Under the same conditions the wild-type enzyme gave no measurable rate with glutamate concentrations in the range normally used for kinetics (0-100 mM) but gave a steep rise in reaction rate from 600 to 1200 mM glutamate . At pH 9.0, where the wild-type enzyme has previously been shown to be "inactive" in a standard assay, a study extending to much higher glutamate concentrations again revealed a sigmoid dependence, with a Hill coefficient of 5.4 and an S0.5 at 150 mM glutamate . With the mutant A163G the apparent cooperativity was less, with a Hill coefficient of 2.3, and the affinity for glutamate was higher, with S0.5 of 7 mM . Both proteins gave normal hyperbolic dependence on glutamate concentration at pH 7 and pH 8 . At pH 9 and with saturating glutamate, both enzymes showed a hyperbolic dependence of the rate on NAD+ concentration . The NAD+ concentration, however, affected the observed degree of cooperativity with varied glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemotherapy, 1995 Sep-Oct, 41(5), 337 - 44
In vitro activity of trimethoprim in association with sulfimidazole against aerobic gram-negative and gram-positive microorganisms and Clostridia; Castelli M et al.; The in vitro activity of a chemotherapeutic agent, sulfimidazole (SIZ), obtained by combining two molecules belonging to groups of extremely different antibacterial drugs, p-aminobenzene sulfonamide and a derivative with a 5-nitroimidazole ring, was studied . In association with trimethoprim, SIZ induces an intense synergistic antibacterial effect on gram-negative and gram-positive aerobic microorganisms and Clostridia . The results show that, in SIZ, the activity of each starting molecule remains unchanged providing that its structure-action relationship is kept intact.

Biochemistry, 1995 Aug 22, 34(33), 10532 - 7
Observation of a second substrate radical intermediate in the reaction of lysine 2,3-aminomutase: a radical centered on the beta-carbon of the alternative substrate, 4-thia-L-lysine; Wu W et al.; Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of lysine and beta-lysine by a mechanism in which four organic radicals are postulated as intermediates . One of the intermediates has been identified as the alpha-radical of beta-lysine in imine linkage to pyridoxal phosphate (PLP) {Ballinger, M . D., Frey, P . A., & Reed, G . H . (1992) Biochemistry 31, 10782-10788} . We report here the observation of another of the four putative radical intermediates in the reaction of the alternative substrate, 4-thia-L-lysine (S-2-aminoethyl-L-cysteine) . 4-Thialysine is a substrate for lysine 2,3-aminomutase . The Km of 4-thialysine is similar to that for lysine, and the Vm is approximately 3% of that for lysine . Upon mixing 4-thialysine with the activated enzyme in the presence of the required cofactor S-adenosylmethionine, followed by freeze-quenching with liquid N2 in the steady state, a strong EPR signal centered at g = 2.003 is observed . This signal exhibits strong hyperfine splitting due to the presence of 13C at carbon-3 of 4-thialysine, and the EPR pattern is narrowed upon the substitution of deuterium at carbon-3 . The hyperfine interactions show that the unpaired electron is centered on carbon-3 of 4-thialysine . The hyperfine pattern in the EPR spectrum is also simplified by the use of 4-thia{5,6-2H4}lysine as the substrate, showing either that the spin is partially delocalized through the sulfur intervening between carbons-3 and -5 or that the conformation is such that protons at carbon-6 are close to carbon-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Aug 8, 34(31), 10086 - 93
Pulsed electron paramagnetic resonance studies of the lysine 2,3-aminomutase substrate radical: evidence for participation of pyridoxal 5'-phosphate in a radical rearrangement; Ballinger MD et al.; The role of pyridoxal 5'-phosphate (PLP) in the radical-mediated amino group migration catalyzed by lysine 2,3-aminomutase from Clostridia SB4 has been investigated by electron spin echo envelope modulation (ESEEM) spectroscopy . This pulsed electron paramagnetic resonance (EPR) method was used to estimate the distance between the unpaired electron in the alpha-radical of beta-lysine, a steady-state intermediate in the reaction, and deuterium at the C4' position of the cofactor, PLP . {4'-2H}PLP was synthesized and exchanged into the enzyme . The steady-state radical was generated in the labeled samples and in samples with unlabeled PLP by addition of L-lysine.H2SO4 to activated enzyme . ESEEM spectra of the samples prepared with {4'-2H}PLP exhibited distinctive low-frequency modulations that were not present in spectra of matched samples with unlabeled PLP . Fourier transformation of the modulations yielded a prominent doublet signal centered about the Larmor frequency of deuterium . The magnitude of the doublet splitting of the 2H ESEEM signal exhibited angle selection across the CW EPR powder pattern . The observed angle selection, as well as simulation of the time domain spectra, indicated that the doublet splitting was due to the combined effects of the 2H hyperfine and nuclear quadrupole interactions . The influences of the quadrupole interaction and of isotropic and dipolar hyperfine interactions were explored by simulations of the ESEEM spectra . The analysis indicates a distance of < 3.5 A between the 2H at C4' of PLP and the radical center at C alpha lysine . The data are most compatible with an aldimine linkage between PLP and the beta-nitrogen of beta-lysine.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1995 Jul 14, 270(28), 16826 - 32
Inhibition of neurotransmitter release by synthetic proline-rich peptides shows that the N-terminal domain of vesicle-associated membrane protein/synaptobrevin is critical for neuro-exocytosis; Cornille F et al.; Tetanus toxin and clostridial neurotoxins type B, D, F, and G inhibit intracellular Ca(2+)-dependent neurotransmitter release via the specific proteolytic cleavage of vesicle-associated membrane protein (VAMP)/synaptobrevin, a highly conserved 19-kDa integral protein of the small synaptic vesicle membrane . This results in the release of the larger part of the cytosolic domain of this synaptic protein into the cytoplasm . Microinjection of synthetic peptides corresponding to this fragment into identified presynaptic neurons of Aplysia californica led to a potent, long lasting, and dose-dependent inhibition (approximately 50% at 10 MicroM) of acetylcholine release, probably by hindering endogenous VAMP/synaptobrevin from interacting with synaptic proteins involved in exocytosis . Structure activity studies showed that this effect is confined to the N-terminal domain of VAMP/synaptobrevin isoform II and is related to the presence of a proline-rich motif (PGGPXGX3PP or PAAPXGX3PP) . At higher concentrations, the inhibitory effect was lower and only transient, suggesting that the N-terminal proline-rich domain of VAMP/synaptobrevin plays opposing roles in neurotransmitter release very likely by interacting with different synaptic proteins . This probably occurs by disruption of the recently reported in vitro VAMP-synaptophysin interaction that involves the N-terminal domain of VAMP II and was proposed to hinder synatophysin-related formation of a fusion pore . The observed recovery of neurotransmitter release following injection of high concentration of N-terminal fragments of VAMP II brings a strong in vivo support to this hypothesis . The minimum active peptide GPGGPQGGMQPPREQS could be used for rationally designing potent synthetic blockers of neurotransmission.

Blutalkohol, 1995 Jul, 32(4), 193 - 207
{Methanol from ethanol? On cleavage of the ethanol carbon bond and methanol formation within the scope of microbial metabolic processes}; Stohlmacher P; While making investigations into microbiological literature, the following question was looked into: Are microorganisms able to generate methanol from the basic carbon frame of ethanol? Due to carbon monoxide dehydrogenase complexes some methanogens and acetate oxidisers are able to cleave the carbon bond of acetyl-CoA . Coenzyme bonded methyl groups (methyl-H4MPT, methyl-CoM, methyl-THF) are generated both by means of reduction (methano-genesis) and by means of oxidation (CO2 evolution) of the C2-unit . Usually these are looked at as precursors of methanol . Methanotrophs and certain methylotrophs are able to oxidise methane to methanol by means of oxigenase complexes . The C1-unit supplies coenzyme bonded CO; carbon monoxide can be reduced to methanol very effectively by several Clostridia . Both in vivo analysis of intestinal gases of alcoholised individuals and incubation experiments with certain intestinal groups with marked alcohol, might contribute in judging the real importance of the intestinal micro flora with regard to the problems of ethanol congeners.

Cell Transplant, 1995 May-Jun, 4(3), 281 - 9
Collagenase lot selection and purification for adipose tissue digestion; Williams SK et al.; Crude Clostridial collagenase (CCC) remains the most widely used enzyme for the digestion of tissues prior to cell isolation and culture . CCC contains numerous components in addition to specific collagenases and proteases . A chronic problem associated with CCC is significant lot variability which occurs with respect to the ability of different lots of CCC to digest tissue . We have evaluated numerous commercially available samples of CCC for their ability to digest human liposuction-derived SC fat . Digestion capacity was evaluated as the ability to release endothelial cells from fat as well as the ability of isolated cells to adhere to tissue culture plastic . A significant variation in digestion efficacy between lots of collagenase was observed . We subsequently purified CCC using a partial purification method with dialysis and centrifugation as well as a complete purification, using liquid chromatography, to remove all nonspecific proteases . While partially purified collagenase retained digestion capacity, pure collagenase exhibited reduced digestion capacity . Maximum digestion was achieved with pure collagenase when trypsin was added . The use of completely purified collagenase with trypsin is advantageous where all components in the enzyme digestion mixture must be known.

Protein Eng, 1995 Feb, 8(2), 147 - 52
Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue; Wang XG et al.; Two residues, K89 and S380, thought to interact with the gamma-carboxyl group of the substrate L-glutamate, have been altered by site-directed mutagenesis of clostridial glutamate dehydrogenase (GDH) . The single mutants K89L and S380V and the combined double mutant K89L/S380V were constructed . All three mutants were satisfactorily overproduced in soluble form . However, only the K89L mutant was retained by the dye column normally used in purifying the wild-type enzyme . All three mutant enzymes were purified to homogeneity and tested for substrate specificity with 24 amino acids . The single mutant S380V showed no detectable activity . The alternative single mutant K89L showed an activity towards L-glutamate that was decreased nearly 2000-fold compared with wild-type enzyme, whereas the activities towards the monocarboxylic substrates alpha-aminobutyrate and norvaline were increased 2- to 3-fold . A similar level of activity was obtained with methionine (0.005 U/mg) and norleucine (0.012 U/mg), neither of which give any activity with the wild-type enzyme under the same conditions . The double mutant showed decreased activity with all substrates compared with the wild-type GDH . In view of its novel activities, the K89L mutant was investigated in greater detail . A strictly linear relationship between reaction velocity and substrate concentration was observed up to 80 mM L-methionine and 200 mM L-norleucine, implying very high Km values . Values of kcat/Km for L-methionine and L-norleucine were 6.7 x 10(-2) and 0.15 s-1 M-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Khirurgiia (Sofiia), 1995, 48(3), 49 - 52
{Clostridial surgical infection}; Fichev G et al.; An ever increasing attention of clinicists is being focused on nontraumatic clostridal surgical infections . Personal clinical case material consisting of 390 patients with microbiologically proved clostridial infection are subjected to clinical and microbiological investigations aimed at establishing the nature of the infective process . In 377 patients (group one) signs of mixed aerobic-anaerobic polyinfection are clinically documented, fully confirmed by the comprehensive microbiological examinations performed . In 120 patients of this group 162 Clostridida species are isolated, but none of them, including the severest cases, are suspected for gas gangrene presence . In 13 patients of group two presenting clinical picture of a typical gas gangrene, the latter is both demonstrative and impressive . Here the microbiological examinations are absolutely analogical to those in group one which warrants the assumption of the endogenous character of the infective process, leading in turn to inferenceshaving important practical implications and recommendation to modify the antiepidemiological measures with a special reference to the endogenous character of the infective process.

Wien Klin Wochenschr, 1995, 107(23), 739 - 41
{Hyperbaric surgery and oxygen therapy in clostridial myonecrosis}; Smolle-Juttner FM et al.; 116 patients with verified clostridial myonecrosis were treated by means of a regimen of combined cyclic hyperbaric oxygenation at 3 ata and repeated necrectomy . With proximal localization of gas gangrene in the elbow or knee (n = 55) 34.5% of patients survived, whilst with peripheral localization at the limbs (n = 44) the survival rate was 88.6% amputation being avoided in 21 of these cases . Abdominal localization (n = 17) was associated with the lowest survival rate (23.5%) . In spite of severe clostridial toxicity in 80.1% of patients at the time of admission, the overall mortality was 50.8%.

Arch Microbiol, 1995 Jan, 163(1), 21 - 8
Pyruvate: ferredoxin oxidoreductase from the sulfate-reducing Archaeoglobus fulgidus: molecular composition, catalytic properties, and sequence alignments; Kunow J et al.; Archaeoglobus fulgidus is a hyperthermophilic sulfate-reducing archaeon . In this communication we describe the purification and properties of pyruvate: ferredoxin oxidoreductase from this organism . The catabolic enzyme was purified 250-fold to apparent homogeneity with a yield of 16% . The native enzyme had an apparent molecular mass of 120 kDa and was composed of four different subunits of apparent molecular masses of 45, 33, 25, and 13 kDa, indicating an alpha beta gamma delta structure . Per mol, the enzyme contained 0.8 mol thiamine pyrophosphate, 9 mol non-heme iron, and 8 mol acid-labile sulfur . FAD, FMN, lipoic acid, and copper were not found . The purified enzyme showed an apparent Km for coenzyme A of 0.02 mM, for pyruvate of 0.3 mM, and for clostridial ferredoxin of 0.01 mM, an apparent Vmax of 64 U/mg (at 65 degrees C) with a pH optimum near 7.5 and an Arrhenius activation energy of 75 kJ/mol (between 30 and 70 degrees C) . The temperature optimum was above 90 degrees C . At 90 degrees C, the enzyme lost 50% activity within 60 min in the presence of 2 M KCl . The enzyme did not catalyze the oxidation of 2-oxoglutarate, indolepyruvate, phenylpyruvate, glyoxylate, and hydroxypyruvate . The N-terminal amino acid sequences of the four subunits were determined . The sequence of the alpha-subunit had similarities to the N-terminal amino acid sequence of the alpha-subunit of the heterotetrameric pyruvate: ferredoxin oxidoreductase from Pyrococcus furiosus and from Thermotoga maritima, and unexpectedly, to the N-terminal amino acid sequence of the homodimeric pyruvate:ferredoxin oxidoreductase from proteobacteria and from cyanobacteria . No sequence similarities were found, however, between the alpha-subunits of the enzyme from A . fulgidus and the heterodimeric pyruvate:ferredoxin oxidoreductase from Halobacterium halobium.

Biochim Biophys Acta, 1994 Dec 14, 1209(2), 222 - 6
Initial formation of a non-covalent enzyme-reagent complex during the inactivation of clostridial glutamate dehydrogenase by Ellman's reagent: determination of the enzyme's dissociation constant for the binary complex with NAD+ from protection studies; Basso LA et al.; The time-course of reaction between Ellman's reagent (DTNB) and clostridial glutamate dehydrogenase has been investigated over a wide range of reagent concentrations (50-5000 microM) and showed pseudo-first-order kinetics throughout . The reaction was followed both by monitoring loss of enzyme activity and by detection of released thionitrobenzoate through its absorbance at 412 nm, and, when both methods were used for the same DTNB concentration, the pseudo-first-order rate constants were identical within experimental error, suggesting that the two methods detect the same process . The dependence of the rate constants on DTNB concentration clearly shows saturation, with a limiting value of 1.62 x 10(-3) s-1 and a dissociation constant of 1.0 mM governing the formation of the implied non-covalent enzyme-DTNB complex . This information has allowed a detailed analysis of the protection of the enzyme by NAD+, yielding a value of 334 microM for the dissociation constant for the enzyme-coenzyme binary complex . In view of the convenience of protection studies as a means of determining dissociation constants, this study emphasizes the importance of establishing whether a chemical modification reaction follows simple first-order kinetics with respect to the chemical reagent.

J Neurochem, 1994 Dec, 63(6), 2342 - 5
Bafilomycin A1 inhibits the action of tetanus toxin in spinal cord neurons in cell culture; Williamson LC et al.; Tetanus toxin (TeNT) is one of the clostridial neurotoxins that act intracellularly to block neurotransmitter release . However, neither the route of entry nor the mechanism by which these toxins gain access to the neuronal cytoplasm has been established definitively . In murine spinal cord cell cultures, release of the neurotransmitter glycine is particularly sensitive to blockade by TeNT . To test whether TeNT enters neurons through acidic endosomes or is routed through the Golgi apparatus, toxin action on potassium-evoked glycine release was assayed in cultures pretreated with bafilomycin A1 (baf A1) or brefeldin A (BFA) . baf A1, which inhibits the vacuolar-type H(+)-ATPase responsible for endosome acidification, diminishes the staining of acidic compartments and interferes with the action of TeNT in a dose-dependent manner . TeNT blockade of evoked glycine release is inhibited by 50 and 90% in cultures pretreated with 50 and 100 nM baf A1, respectively, compared with cultures treated with the inhibitor alone . The effects of baf A1 are fully reversible . In contrast, BFA, which disrupts Golgi function, has no effect on TeNT action . These findings provide evidence that TeNT enters the neuronal cytoplasm through baf A1-sensitive acidic compartments and that TeNT is not trafficked through the Golgi apparatus before its translocation into the neuronal cytosol.

Proteins, 1994 Dec, 20(4), 293 - 300
Secondary structural predictions for the clostridial neurotoxins; Lebeda FJ et al.; The primary structures of a family of ten clostridial neurotoxins have recently been deduced yet little information is presently available concerning their secondary or tertiary structures . Because the overall similarity percentage of multiply aligned sequences is high, the secondary structures of these metalloendopeptidases are also expected to be conserved . The neural net program, PHD (Rost and Sander, Proc . Natl . Acad . Sci . USA 90:7558-7562, 1993), predicted that the secondary structures of the neurotoxins were indeed conserved in both single and multiple sequence modes of analysis . Predictions for the amounts of helical, extended, and loop states from the single sequence analyses were consistent with previously published data from circular dichroism studies on some of these neurotoxins . In the single analysis mode, only the aligned regions were predicted to show conservation of the three-state structure . In contrast, the multiple sequence analysis predicted that a conserved state (variable loops) also exists in non-aligned regions . Alignments with the primary structure of the prototypic metalloendopeptidase thermolysin showed that about 25% of the residues within this enzyme are similar to those in the neurotoxins . A comparison of thermolysin's known secondary structure with the predictions from this study showed that about 80% of thermolysin's residues could be structurally aligned with those in the neurotoxins . These predictions provide the necessary framework to build a homologous low-resolution tertiary structure of the neurotoxin active site that will be essential in the development of synthetic inhibitors.

EMBO J, 1994 Nov 1, 13(21), 5051 - 61
Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly; Hayashi T et al.; Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal-associated protein of 25 kDa (SNAP-25) or syntaxin . Here we show that in binary reactions synaptobrevin binds weakly to both SNAP-25 and syntaxin, and SNAP-25 binds to syntaxin . In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate-resistant complex forms . Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha-helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled-coil structures . Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin-syntaxin-SNAP-25 complexes . Our data suggest that synaptobrevin, syntaxin and SNAP-25 associate into a unique stable complex that functions in synaptic vesicle exocytosis.

Br J Clin Pract, 1994 Nov-Dec, 48(6), 337 - 8
An unusual presentation of gas gangrene complicated by penicillin allergy; Woo PL et al.; A case is reported of non-clostridial gas gangrene that presented in a similar way to deep venous thrombosis and then developed into septic shock . Management was complicated by penicillin allergy.

FEBS Lett, 1994 Oct 24, 353(3), 319 - 23
Fusion complex formation protects synaptobrevin against proteolysis by tetanus toxin light chain; Pellegrini LL et al.; The clostridial neurotoxin, tetanus toxin, is a Zn(2+)-dependent protease which inhibits neurotransmitter exocytosis by selective cleavage of the synaptic vesicle protein, synaptobrevin . Synaptobrevin is thought to serve as a receptor for two neuronal plasma membrane proteins, syntaxin and SNAP-25, which in the presence of non-hydrolyzable ATP analogs form a 20 S fusion complex with the soluble fusion proteins NSF and alpha-SNAP . Here we show that synaptobrevin, when in this 20 S complex, or its 7 S precursor, is protected against proteolysis by the enzymatically active tetanus toxin light chain . Our data define distinct pools of synaptobrevin, which provide markers of different steps of vesicle/plasma membrane interaction.

J Biol Chem, 1994 Sep 30, 269(39), 24454 - 8
The role of transglutaminase in the mechanism of action of tetanus toxin; Coffield JA et al.; Tetanus toxin is a zinc-dependent metalloendoprotease that cleaves synaptobrevin, a polypeptide found in the membranes of synaptic vesicles . This action is thought to account for toxin-induced blockade of transmitter release . However, Facchiano and Luini (Fachiano, F., and Luini, A . (1992) J . Biol Chem . 267, 13267-13271) have proposed that tetanus toxin can stimulate transglutaminase, and Facchiano et al . (Facchiano, F., Benfenati, F., Valtorta, F., and Luini, A . (1993) J . Biol Chem . 268, 4588-4591) have further proposed that the stimulated enzyme produces cross-linking of synapsin . These actions might also account for toxin-induced blockade of exocytosis . Therefore, a series of experiments were performed to evaluate the possibility that tetanus toxin exerts its effects via transglutaminase . The results indicated that clostridial neurotoxins were poor substrates for the cross-linking effects of transglutaminase, and transglutaminase was a poor substrate for the proteolytic actions of tetanus toxin . In addition, at concentrations relevant to blockade of exocytosis, clostridial neurotoxins did not act on intact cells to stimulate transglutaminase, nor did they act on the isolated enzyme to stimulate cross-linking of putrescine and dimethylcasein . When used as competitive inhibitors of endogenous transglutaminase substrates, glycine methyl ester and monodansylcadaverine did not block toxin action . Furthermore, concentrations of calcium that were too low to support transglutaminase activity did not prevent toxin action . The data suggest that stimulation of transglutaminase is not the principal mechanism by which tetanus toxin blocks exocytosis in nerve cells.

J Biol Chem, 1994 Sep 9, 269(36), 22547 - 56
The actin cytoskeleton is important for the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages; Tabas I et al.; Stimulation of intracellular cholesterol esterification, which is catalyzed by the enzyme acyl-coenzyme A: cholesterol O-acyltransferase (ACAT), by atherogenic lipoproteins in macrophages is a key step in the ability of these cells to store lipoprotein-cholesterol and in the eventual development of atheroma foam cells . Herein, we provide evidence that the actin cytoskeleton plays an important role in the stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages . When the actin cytoskeleton of cultured mouse peritoneal macrophages was disrupted by treatment with cytochalasin D or Clostridial C2 toxin, the ability of beta very low density lipoprotein (beta-VLDL) to stimulate cholesterol esterification was decreased 3-6-fold, even under conditions in which beta-VLDL protein degradation, cholesteryl ester hydrolysis, or net cholesterol delivery to the cells was matched . Esterification of cellular phospholipids and triglycerides was not affected by this treatment . Cytochalasin D treatment of macrophages also inhibited the ability of acetyl-low density lipoprotein, another foam cell-forming lipoprotein, to stimulate cholesterol esterification, but stimulation of cholesterol esterification by 25-hydroxycholesterol was not inhibited by cytochalasin D . Cytochalasin D was found to inhibit neither the exit of beta-VLDL-derived cholesterol from lysosomes nor the ability of beta-VLDL to down-regulate endogenous cholesterol synthesis . From these data we conclude that an intact actin cytoskeleton is necessary for efficient stimulation of cholesterol esterification by atherogenic lipoproteins in macrophages . Although the exact function of actin in the cholesterol esterification pathway remains to be determined, our data rule out a general role for actin in intracellular cholesterol trafficking or maintenance of ACAT enzyme activity . Rather, we speculate that actin filaments play a role in specific cellular entry processes of atherogenic lipoproteins and/or in establishing transport or contact between the plasma membrane cholesterol substrate pool and the ACAT enzyme in macrophages.

Ann Plast Surg, 1994 Sep, 33(3), 313 - 6
Clostridial infection following severe facial trauma; Denny AD et al.; Severe posttraumatic wound infections are uncommon occurrences in the craniofacial region . Although most reported cases have occurred following an odontogenic infection or minor soft-tissue trauma, the potential for the development of this type of infection exists following complex facial fractures . We report a case with clostridial infection in the temporalis fascia complicating a complex facial fracture . The implications regarding antibiotic usage and delay of surgical treatment are discussed.

Curr Microbiol, 1994 Sep, 29(3), 125 - 31
Inactivation of anaerobic bacteria by various photosensitized porphyrins or by hemin; Nitzan Y et al.; The photodynamic effects of deuteroporphyrin (DP), hematoporphyrin derivative (HPD), hematoporphyrin (HP), or protoporphyrin (PP) on a variety of anaerobic microorganisms were examined in this study . The majority of the species, among the 350 strains tested, were inhibited by concentrations of < or = 2.5 micrograms/ml of light-activated DP . Species found to be resistant to this treatment included Bilophila wadsworthia, Fusobacterium mortiferum, Fusobacterium varium, and Bacteroides gracilis . These species were inhibited by concentrations of > 60 micrograms/ml of DP . The porphyrin-producing species, Porphyromonas and Prevotella spp, were all inhibited by < or = 2.5 micrograms/ml DP and light . Comparing the photodynamic activity of the porphyrins used on Porphyromonas strains resulted in the following pattern: DP > HPD > HP > PP . Porphyromonas spp., Gram-positive cocci, and many Gram-positive rods (excluding clostridia) were inactivated by hemin (a metal-containing porphyrin) at 10-20 micrograms/ml . Hemin inhibitory action was not affected by light . Binding and insertion of DP into bacteria (both inactivated and non-inactivated strains by DP and light) were monitored by the characteristic fluorescence band of bound DP at 622 nm . Porphyromonas spp . bound DP tightly, whereas only low binding was seen with B . wadsworthia and other DP-resistant species . High binding of DP to B . wadsworthia can be achieved by pretreatment of the bacteria with imipenem or cefoxitin, beta-lactam agents known to interfere with the integrity of the cell wall . If cell wall integrity is disturbed (e.g., by these agents), inactivation of B . wadsworthia by DP can occur.

Protein Eng, 1994 Aug, 7(8), 1013 - 6
Identification of the reactive cysteine in clostridial glutamate dehydrogenase by site-directed mutagenesis and proof that this residue is not strictly essential; Wang XG et al.; Cys320 of clostridial glutamate dehydrogenase, a residue close to the coenzyme binding site, has been replaced by serine . The mutant enzyme was successfully overproduced and purified by using the normal protocol for the wild-type enzyme and also behaved indistinguishably from wild-type enzyme on native and SDS-PAGE . The specific activity was significantly enhanced in assays at both pH 7 (+90%) and pH 8 (+38%) . Detailed initial-rate kinetics revealed that at pH 7 this increase was mainly attributable to a higher maximum rate, since the Km values for both substrates were marginally increased . In the mutant enzyme the inactivating reaction with DTNB that characterizes the wild-type enzyme is completely eliminated . This proves that inactivation of the wild-type enzyme is due to modification of Cys320, that nevertheless Cys320 is not strictly essential for catalytic activity and that the remaining cysteine residue at position 144 is inaccessible to DTNB . Provision of an engineered subunit with a correct native structure but with its DTNB titre decreased from 1 to 0 mol/mol now offers a valuable tool for counting subunits in hybrid oligomers.

J Biol Chem, 1994 Jul 22, 269(29), 18821 - 6
Purification and characterization of sialidase L, a NeuAc alpha 2-->3Gal-specific sialidase; Chou MY et al.; Sialidase L releases 2,7-anhydro-NeuAc from sialoglycoconjugates (Li, Y.-T., Nakagawa, H., Ross, S . A., Hansson, G., and Li, S.-C . (1990) J . Biol . Chem . 265, 21629-21633) . This enzyme has been purified more than 10,000-fold from Macrobdella leech . The final preparation gives a single protein band on SDS-polyacrylamide gel electrophoresis with the molecular mass of 84 kDa . The pI is determined to be 6.0 using isoelectric focusing . With 4-methylumbelliferyl-alpha-NeuAc as substrate, the pH optimum is between pH 5.5-7.0 . Unlike regular sialidases, sialidase L is not inhibited by 2-deoxy-2,3-dehydro-NeuAc . Two of the seven tryptic peptides derived from sialidase L contain the consensus repeat S-X-D-X-G-X-T-W that has been found in the regular sialidases . Among various sialoglycoconjugates tested, sialidase L cleaves only the NeuAc alpha 2-->3Gal linkage . NeuAc alpha 2-->6Gal, NeuAc alpha 2-->6GalNAc, NeuAc alpha 2-->6GlcNAc, NeuAc alpha 2-->8-NeuAc, and NeuAc alpha 2-->9NeuAc linkages are not hydrolyzed . At pH 7.0, sialidase L and Clostridial sialidase release 46 and 92% of sialic acid, respectively, from bovine fetuin, indicating that sialidase L selectively cleaves NeuAc alpha 2-->3Gal linkages in fetuin . Sialidase L is the first sialidase found to exhibit a strict specificity toward the hydrolysis of the NeuAc alpha 2-->3Gal linkage, and it should become useful for the selective cleavage of NeuAc alpha 2-->3Gal linkages in sialoglycoconjugates without destroying other sialosyl linkages.

Biochem J, 1994 Jul 1, 301 ( Pt 1), 13 - 6
The catalytic role of aspartate in the active site of glutamate dehydrogenase; Dean JL et al.; A putative catalytic aspartyl residue, Asp-165, in the active site of clostridial glutamate dehydrogenase has been replaced with serine by site-directed mutagenesis . The mutant enzyme is efficiently overexpressed in Escherichia coli as a soluble protein and can be successfully purified by the dye-ligand chromatographic procedure normally employed for the wild-type enzyme . By several criteria, including circular dichroism spectrum, sulphydryl reactivity with Ellman's reagent, crystallization and mobility in non-denaturing electrophoresis, the enzyme appears to be correctly folded . NAD+ protects the D165S mutant against modification by Ellman's reagent, suggesting unimpaired binding of coenzyme . In standard assays the specific activity is decreased 10(3)-fold in the reductive amination reaction and 10(5)-fold for oxidative deamination . Kinetic studies show that apparent Km values for NADH and 2-oxoglutarate are almost unchanged . The large reduction in the reaction rate coincides with a weakening of the affinity for ammonium ion (Km > 300 mM, compared with 60 mM for the wild-type) . The data are entirely consistent with the direct involvement of D165 in catalysis rather than in the binding of coenzyme or 2-oxoglutarate.

J Biol Chem, 1994 Jun 10, 269(23), 16276 - 83
Expression and specificity of human GM2 activator protein; Wu YY et al.; The cDNA encoding GM2 activator was expressed in the Escherichia coli/pT7-7 system . The yield of the GM2 activator with greater than 99% purity was about 3 mg per liter culture . The recombinant GM2 activator was found to be as active as that isolated from human kidney . The availability of the recombinant GM2 activator enabled us to critically examine the specificity of this activator protein . Our results show that the specificity of GM2 activator is not as strict as that reported previously . Although GM2 activator stimulates most efficiently the degradation of GM2 carried out by beta-N-acetylhexosaminidase A (Hex A), this activator also stimulates the following reactions: (a) conversion of GM2 to GA2 by clostridial sialidase; (b) hydrolysis of GalNAc from dipalmitoylphosphatidylethanolamine-II3NeuAcGgOse3 by Hex A; and (c) liberation of Gal from GM1 by beta-galactosidase at a high activator concentration . Thus, this activator does not differentiate between GM2 and dipalmitoylphosphatidylethanolamine-II3NeuAcGgOse3 or between Hex A and clostridial sialidase . The micellar forms of GD2 and GalNAc-GD1a were found to be more readily hydrolyzed by Hex A than GM2 in the absence of GM2 activator . Our results also show that saposin B can enhance the stimulatory activity of GM2 activator, but it cannot promote the stimulatory activity of sodium taurodeoxycholate . Taken together, our results suggest that the mechanism of action of GM2 activator is different from saposin B, and the action of GM2 activator is more than to solubilize lipid substrates . The effectiveness of GM2 activator in stimulating the hydrolysis of GM2 may be due to its ability to recognize the specific trisaccharide structure of the GM2 epitope, GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal-, and to modify the GalNAc-NeuAc interaction in this structure.

Arch Biochem Biophys, 1994 Jun, 311(2), 457 - 9
The fate of the carboxyl oxygens during D-proline reduction by clostridial proline reductase; Arkowitz RA et al.; D-Proline is converted to 5-amino valeric acid by D-proline reductase . This conversion involves the reductive cleavage of the alpha-carbon-nitrogen bond . We have examined the fate of the carboxyl oxygen atoms during conversion of D-proline to delta-NH2-valeric acid . 18O atoms from the carboxyl group of D-proline are not lost during conversion to product . In contrast, in the conversion of glycine to acetyl phosphate by glycine reductase a carboxyl oxygen atom is lost to solvent . An intermediate acyl-enzyme is found during the reduction of glycine . We conclude that the reduction of proline proceeds without the formation of an acyl enzyme intermediate.

Asia Oceania J Obstet Gynaecol, 1994 Jun, 20(2), 191 - 4
Two cases of gas gangrene complicating uterine rupture and fetal death at term; Habeebullah S et al.; Two patients with obstructed labor and uterine rupture complicated by clostridial septicemia are reported . The diagnosis was apparent clinically . The importance of early diagnosis and prompt treatment are emphasized . The place of hysterectomy, which should be individualized is discussed . All efforts should be directed towards prophylaxis in high risk cases.

Am J Surg, 1994 May, 167(5), 485 - 9
A multicenter review of the treatment of major truncal necrotizing infections with and without hyperbaric oxygen therapy; Brown DR et al.; To examine the effectiveness of hyperbaric oxygen (HBO) therapy in the treatment of major truncal necrotizing infections, a retrospective (1980 to 1992) analysis of the medical records of 30 patients treated with HBO therapy and 24 patients treated without HBO therapy was undertaken . The two groups were similar; however, there was a selection bias towards more frequent clostridial infections in a younger population in the HBO group . There was no difference in length of hospital stay, intensive care unit (ICU) stay, or duration of antibiotic therapy between groups . The mortality rates were 9/30 (30%) in the HBO group versus 10/24 (42%) (nonsignificant difference) in the non-HBO group . The total number of operations was greater in the HBO-treated group . This study failed to show that the use of HBO in the treatment of major truncal necrotizing infections statistically reduced mortality or the number of debridements . We believe, however, that the apparent selection bias and the trend towards increased survival in the HBO-treated group (12%) justifies the continued use of and research with HBO therapy.

Hawaii Med J, 1994 Apr, 53(4), 112 - 5, 119
Baromedicine today--rational uses of hyperbaric oxygen therapy; Tabrah FL et al.; Compressed air, and more recently hyperbaric oxygen, have been used and misused in medical treatment for more than 300 years . Advances in physiology have led to rational protocols for hyperbaric oxygen use . Hyperbaric oxygen will enhance wound healing by fibroblast and capillary proliferation, suppress infection, reduce edema, reverse CNS damage from carbon monoxide and cyanide poisoning, and reduce clostridial alpha toxins . Monoplace and multiplace chambers are used for treatment during which EKG and oxygen tissue monitoring, as well as hemodynamic and respiratory support, can be continued, iatrogenic air embolism and diving decompression sickness demand immediate treatment . Investigative uses of adjunct therapy for several other clinical problems include treatment of MS, acute spinal cord injuries, and acute MI . Specific indications agreed on by the Undersea and Hyperbaric Medicine Society are recognized by most third-party payers including Medicare, Champus, and HMSA . Hyperbaric medicine remains a fertile area for basic physiologic investigation and outcomes research.

Pharmacol Ther, 1994 Apr-May, 62(1-2), 29 - 39
Tetanus toxin as a neurobiological tool to study mechanisms of neuronal cell death in the mammalian brain; Bagetta G et al.; Tetanus toxin is a potent clostridial neurotoxin responsible for causing spastic paralysis in humans, often accompanied by seizures and death . The tetanic syndrome is believed to originate from a disinhibitory action of the toxin in the CNS . To produce its effects, tetanus toxin undergoes retrograde, intra-axonal transport to the CNS, where it blocks preferentially the release of gamma-aminobutyric acid and glycine, two inhibitory neurotransmitters . These effects stem from the cleavage of synaptobrevin, a constitutive small-vesicle protein, by tetanus toxin, whose zinc-dependent metalloprotease characteristics recently have been recognized . Blockade of inhibitory transmission produces a predominance of excitatory amino acid neurotransmission, which is responsible for the neurodegenerative effect caused by tetanus toxin after intrahippocampal injection in rats . In fact, hippocampal damage can effectively be prevented by reduction of glutamate-mediated excitatory transmission, thus suggesting that unopposed excitation may be the underlying mechanism for neuronal cell death.

Ophthalmology, 1994 Mar, 101(3), 461 - 72
Focal scleral permeability enhanced by collagenase digestion . Experimental model of enzymatic sclerostomy; Dan JA et al.; PURPOSE: The purpose of this study is to demonstrate that restricted application of clostridial collagenase to the animal sclerolimbal area produces increased tissue permeability in vitro and results in a lasting significant decrease in the intraocular pressure (IOP) in vivo . METHODS: A special miniature device was developed to apply the carefully characterized enzyme to a small (1-mm diameter) and defined sclerolimbal area isolated from the surrounding tissue . The device was applied to the rabbit limbus for 4 hours and then removed . The enzyme was inactivated and washed out with an inhibitory solution . The IOP was measured by pneumotonometry, and the treated tissue was investigated by light and electron microscopy . Application and removal of the drug-delivery microapplicator was performed under topical anesthesia . RESULTS: Enzymatic degradation propagated without lateral spreading and reached at least two thirds of tissue width . Results of histologic examination of the treated area showed a thin tissue, characterized by degraded and disorganized collagen fibers, which were sharply separated from the intact fibers in the untreated area . This was sufficient to cause a tenfold increase in the basic transscleral flow in vitro, and a significant, relatively long-lasting (> 30 days) lowering of the mean IOP in 15 albino rabbits . In the first 10-day period after treatment, the IOP was reduced by 40%, and during the rest of the follow-up the IOP remained lowered by more than 20% (P > 0.0001) without any adjuvant treatment . CONCLUSION: Despite the small size of the collagenase-affected tissue, the evacuation rate of the aqueous humor was sufficient to cause a significant and relatively long-lasting lowering of IOP in rabbits . This mild treatment, performed under topical anesthesia, might present the basis for the development of an ambulatory treatment of human glaucoma.

Steroids, 1994 Mar, 59(3), 212 - 6
Isolation and HPLC of N-epsilon-lithocholyl lysine as its fluorescamine and dimethylaminoazobenzene isothiocyanate derivatives; Nair PP et al.; N-epsilon-lithocholyl lysine (NELL) is a component of tissue-bound lithocholic acid (TBL) . The isolation of NELL from native protein sources was simulated by hydrolysis of lithocholyl-bovine serum albumin (BSA) (synthesized by coupling lithocholyl-N-hydroxysuccinimide to fatty acid-free BSA) by digestion with a mixture of 6N HCl-propionic acid at 70 C for 3 h under partial vacuum . NELL was isolated on a reversed phase Sep-Pak C18 column and converted to either a fluorophor with fluorescamine or to a chromophor with dimethylaminoazobenzene isothiocyanate for subsequent HPLC using appropriate fluorescence or UV/visible absorption detectors . The procedure described here is quantitative, highly sensitive, and not dependent upon the use of Clostridial cholanoylamino acid hydrolase, the activity of which is sometimes blocked by steric hindrance on the substrate . Using this procedure we have demonstrated the presence of TBL in native histones.

Kansenshogaku Zasshi, 1994 Feb, 68(2), 263 - 7
{A case of NIDDM with non-clostridial gas-producing infection in the lower limb--the effects of hyperbaric oxygen therapy}; Owada R et al.; A 36-year-old male was admitted because of swelling and pain in the lower left limb . He had been diagnosed as having non-insulin dependent diabetes mellitus 4 years previously and had been treated unsuccessfully with insulin . Since gas formation was radiographically demonstrated in the soft tissue of the left foot, we treated him with antibiotics and hyperbaric oxygen under the diagnosis of gas gangrene . Despite this therapy, the gangrenous lesions progressed . Amputation of the left foot was necessary . Peptstreptococcus was isolated from the gangrenous area and identified as such on the seven hospital day . Nine cases of diabetic patients with non-clostridial gas gangrene who were treated with hyperbaric oxygen therapy have been reported in Japan . The results indicated that hyperbaric oxygen therapy is ineffective in the treatment of non-clostridial gas gangrene in diabetic patients.

FEMS Microbiol Lett, 1994 Feb 1, 116(1), 1 - 5
Determination of 16S ribosomal RNA sequences of all members of the genus Peptostreptococcus and their phylogenetic position; Li N et al.; A comparative analysis of 16S ribosomal RNA sequences of all Peptostreptococcus species revealed that most members of the genus Peptostreptococcus should be divided into many different genera . The relationship between clostridia and peptostreptococci was analysed to find the phylogenetic position of peptostreptococci.

Acta Med Port, 1994 Jan, 7(1), 39 - 41
{Typhlitis}; Monteiro JA et al.; Typhlitis, ileocecal syndrome and neutropenic enterocolitis are different terms, of what seems to be a localized infection in the cecal mucosa, caused by Clostridia species . Initially described in neutropenic patients, with leukaemias or after antineoplastic chemotherapy, typhlitis can also occur in HIV patients, as in the present case report . Their early diagnosis is not easy, becoming particularly difficult in immunodepressed patients, whom can be affected by multiple problems causing similar symptoms . Treatment, as well, can be problematic, and aggressive medical treatment should be pursued . If some patients may need surgery, others might be submitted to unnecessary surgical procedures . In the long range, medical control of this situation can be very difficult.

J Physiol Paris, 1994, 88(4), 235 - 41
Inhibition of neurotransmitter release by clostridial neurotoxins correlates with specific proteolysis of synaptosomal proteins; Blasi J et al.; Rat brain synaptosomes were used to study the effect of several clostridial neurotoxins on the neurotransmitter release . In this system the blockade of transmitter release correlated with the proteolytic activity of the toxins . Blockade of glutamate release was linked to selective proteolysis of one of the following synaptic proteins: synaptobrevin (BoNT/D, BoNT/F); SNAP-25 (BoNT/A, BoNT/E), or HPC-1/syntaxin (BoNT/C1) . All the toxins used had an inhibitory effect on synaptosomes with the exception of BoNT/F . BoNT/F cleaved synaptobrevin in permeabilized synaptosomes but failed to produce the same effect on intact synaptosomes.

Surg Today, 1994, 24(12), 1097 - 100
Clostridial gas gangrene associated with congenital generalized lipodystrophy: report of a case; Nakae H et al.; Congenital generalized lipodystrophy (CGL) is a rare disease, the main symptoms of which are a reduction of systemic subcutaneous fat, characteristic facial features, hypertrichosis, and insulin-resistant diabetes . We report herein the unusual case of a 25-year-old man with CGL in whom gas gangrene developed, an association which has never before been encountered.

Cell Mol Neurobiol, 1993 Dec, 13(6), 649 - 64
Protein kinase C and clostridial neurotoxins affect discrete and related steps in the secretory pathway; Bittner MA et al.; 1 . The effects on catecholamine secretion of activation of protein kinase C and clostridial neurotoxins were examined in digitonin-permeabilized bovine adrenal chromaffin cells . 2 . The enhancement by phorbol esters increased only the initial rate of secretion; later rates were unaffected . This enhancement was present over a wide range of Ca2+ concentrations and was elicited at 18 as well as at 27 degrees C . 3 . Tetanus toxin inhibited both ATP-dependent and ATP-independent secretion, indicating that the tetanus toxin target is important during the final steps in the pathway . 4 . Prior activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl phorbol acetate rendered the primed state more sensitive to inhibition by tetanus toxin . The data indicate that a phosphorylated protein kinase C substrate is either identical to or closely associated with the tetanus toxin target protein at the final steps in the pathway . 5 . The interaction between the effect of protein kinase activation and that of tetanus toxin suggests that protein kinase C activation does not stimulate a separate pathway of secretion but, rather, modulates the activity of the ongoing pathway . 6 . The enhancement of secretion by protein kinase C is caused, at least in part, by a qualitative change in the characteristics of the primed state . This is indicated by the increased sensitivity of primed secretion to inhibition by tetanus toxin and a threefold increase in sensitivity of primed secretion to Ca2+ . 7 . Because activation of protein kinase C does not increase the later rates of secretion that are limited by ATP-dependent priming reactions, it is unlikely that enhancement of the maximal rate of secretion by TPA is due to an increased amount of the primed state . Instead, protein kinase C activation may increase the efficacy with which Ca2+ stimulates secretion at all Ca2+ concentrations.

Nature, 1993 Nov 25, 366(6453), 347 - 51
Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin; Hata Y et al.; Three synaptic proteins, syntaxin, SNAP-25 and synaptobrevin, were recently identified as targets of clostridial neurotoxins that irreversibly inhibit synaptic vesicle fusion . Experiments searching for membrane receptors for N-ethylmaleimide-sensitive fusion protein (NSF), which has an important role in membrane fusion, revealed an ATP-dependent interaction of the same three synaptic proteins with NSF and its soluble attachment proteins . Thus, two independent approaches identify syntaxin, synaptobrevin and SNAP-25 as components of the synaptic vesicle fusion machinery, but their mode of action is unclear . We have now discovered a brain protein of relative molecular mass 67,000 (67K) which binds stably to syntaxin . Amino-acid sequencing and complementary DNA cloning revealed that the 67K protein is encoded by the mammalian homologue of the Caenorhabditis elegans gene unc-18 . In C . elegans, unc-18 belongs to a group of genes defined by mutations with a paralytic phenotype and accumulations of acetylcholine, suggesting a defect in neurotransmitter release . The binding of the mammalian homologue of unc-18 (Munc-18) to syntaxin requires the N terminus of syntaxin whereas that of SNAP-25 involves a more C-terminal sequence . Our data suggest that Munc-18 is a previously unidentified essential component of the synaptic vesicle fusion protein complex.

Biochim Biophys Acta, 1993 Oct 4, 1144(3), 435 - 8
Molecular cloning and sequencing of the ferredoxin I fdxN gene of the photosynthetic bacterium Rhodospirillum rubrum; von Sternberg R et al.; Using an oligonucleotide probe derived from the amino acid sequence of Rhodospirillum rubrum ferredoxin I, the gene (fdxN) was identified, cloned and sequenced . The FdxN coding region is 183 nucleotides which codes for a 61 amino acid (7267 Da) protein . Phylogenetic comparisons between the R . rubrum FdI and other 8Fe-8S nif-coupled ferredoxins showed only moderate degrees of similarity between the amino acid sequences . R . rubrum FdI synthesis was stimulated by nif derepressing conditions, but was not completely repressed by nif repression . Previous reports of an extracellular clostridial-type ferredoxin in R . rubrum could not be confirmed.

J Bacteriol, 1993 Sep, 175(18), 5754 - 61
Purine metabolism in Methanococcus vannielii; DeMoll E et al.; Methanococcus vannielii is capable of degrading purines to the extent that each of these purines may serve as the sole nitrogen source for growth . Results presented here demonstrate that purine degradation by M . vannielii is accomplished by a route similar to that described for clostridia . Various characteristics of the purine-degrading pathway of M . vannielii are described . Additionally, it is shown that M . vannielii does not extensively degrade exogenously supplied guanine if that compound is present at levels near or lower than those required to supply the cellular guanine requirement . Under those conditions, M . vannielii incorporates the intact guanine molecule into its guanine nucleotide pool . The benefits of a purine-degrading pathway to methanogens are discussed.

Ann Emerg Med, 1993 Sep, 22(9), 1477 - 80
Spontaneous clostridial myonecrosis with abdominal involvement in a nonimmunocompromised patient; Rich RS et al.; Spontaneous clostridial myonecrosis is a rare but aggressive disease usually associated with underlying immunosuppression or malignancy . We present a fatal case of clostridial myonecrosis arising in a patient with intact immune defenses.

Vet Clin North Am Equine Pract, 1993 Aug, 9(2), 411 - 21
Clostridia-associated enterocolitis in adult horses and foals; Traub-Dargatz JL et al.; A review of the literature describing clostridia-associated enterocolitis is presented . The bacteria, their toxins, and possible factors that interact to contribute to the pathogenesis of enterocolitis are described . Clinical signs associated with clostridia-associated enterocolitis, methods of diagnosis, treatments, and preventive strategies are discussed.

Biochem Mol Biol Int, 1993 Jun, 30(2), 283 - 91
Inhibition of glutamate dehydrogenase by covalent coenzyme-substrate adducts: a re-examination; Syed SE et al.; Covalent adducts of NAD+ with pyruvate and 2-oxoglutarate have been reported to inhibit differentially the activities of bovine glutamate dehydrogenase (GDH) towards these two oxoacid substrates, implying separate active sites . Thorough reinvestigation fails to confirm this finding, with the pyruvate adduct uniformly the more potent inhibitor of both substrate activities under several assay conditions . This suggests that bovine GDH provides amino acid dehydrogenation sites of one structural type only . Clostridial GDH, with a strong preference for oxoglutarate over pyruvate as substrate, is also more strongly inhibited by the pyruvate adduct in the oxoglutarate assay . These findings challenge the generality of the view that carbonyl substrates used in forming such adducts confer specificity for the corresponding substrate binding pocket in enzyme active sites.

Biochim Biophys Acta, 1993 May 13, 1163(2), 124 - 30
Purification and properties of a bacterial-type ferredoxin from the nitrogen-fixing cyanobacterium Anabaena variabilis ATCC29413; Yakunin AF et al.; Three soluble ferredoxins were purified to homogeneity from nitrogen-fixing cultures of Anabaena variabilis (ATCC 29413) and characterized . The purified proteins have different absorption spectra, molecular mass, iron content, amino-acid composition and resistance to O2 inactivation . Two were plant-type ferredoxins FdI and FdxH, corresponding to the previously reported ferredoxins II and I (Bohme, H . and Schrautemeier, B . (1987) Biochim . Biophys . Acta 891, 1-7) . The third ferredoxin (ferredoxin III) (previously not described in cyanobacteria) was a bacterial-type ferredoxin . Ferredoxin III has a molecular mass of about 6 kDa and contains 3-4 atoms Fe/mol . Native (oxidized) ferredoxin III shows an EPR-signal at g = 2.014 that disappears after reduction by dithionite, characteristic of ferredoxins containing three-iron clusters . Ferredoxin III, like ferredoxin FdxH, is inactivated by oxygen . Ferredoxin III supports higher rates of C2H2 reduction by Rhodobacter capsulatus nitrogenase than FdI and higher rates of H2 evolution by clostridial hydrogenase than FdI and FdxH . Combined nitrogen suppresses the synthesis of both nitrogenase and ferredoxin III . These data suggest a possible role of ferredoxin III (bacterial-type) in nitrogen fixation by A . variabilis.

J Biol Chem, 1993 Mar 5, 268(7), 4588 - 91
Covalent modification of synapsin I by a tetanus toxin-activated transglutaminase; Facchiano F et al.; The synapsins are neuronal phosphoproteins that bind to small synaptic vesicles and to actin filaments and are believed to play a regulatory role in neurotransmitter release . Here we show that synapsin I is covalently modified with remarkable affinity and selectivity by the enzyme transglutaminase . Transglutaminase catalyzes the formation of covalent bonds between protein glutamine residues and primary amines and has been found recently to be potently activated by tetanus toxin, a dichain clostridial protein that selectively blocks neurotransmitter secretion . We also report the presence of two species of immunoreactive transglutaminases in nerve endings, one cytosolic and one located on synaptic vesicles; they are potently activated by tetanus toxin and, when activated, covalently modify synaptic vesicle-bound synapsin I . These results suggest a role for transglutaminase in the control of neurotransmitter secretion and provide evidence for synapsin I being a molecular target of tetanus toxin.

Pathologe, 1993 Mar, 14(2), 96 - 100
{"Iatrogenic" fatal gas gangrene infection}; Risse M et al.; The problems associated with lethal gas gangrene infections that arise in connection with medical treatment are described and discussed, based on our own observations and on cases reported in the literature . Special emphasis is placed on questions relating to causality and responsibility . The difficulties encountered in the evaluation of such "iatrogenic" gas gangrene infections are due to the fact that clostridia occur ubiquitously, i.e . that they also reside in the human organism, for instance on the skin . For this reason a positive bacteriological diagnosis of gas gangrene does not necessarily mean that a clinically relevant infection and/or disease state is present at the same time . The implications for morphological practice arising from this situation are discussed, especially with regard to the significance of the intravital, agonal or postmortem bacteriological diagnosis of gas gangrene . Questions concerning the obligation to report such cases when encountered during forensic autopsies are also addressed.

Mil Med, 1993 Feb, 158(2), 80 - 3
The role of hyperbaric oxygenation in the treatment of clostridial myonecrosis; Rudge FW; Hyperbaric oxygenation as an adjunct in the treatment of clostridial myonecrosis (gas gangrene) has been used extensively in facilities with hyperbaric chambers . The United States Air Force has had extensive experience in the treatment of clostridial myonecrosis, treating 77 patients since 1965 . This study reviews the experience in the treatment of this disease process in military multiplace hyperbaric chambers and provides an in-depth analysis of factors affecting patient survival . A comprehensive review of the literature on the treatment of clostridial myonecrosis with hyperbaric oxygenation is presented in this paper.

Lik Sprava, 1993 Feb-Mar, (2-3), 131 - 4
{The modern approach to the treatment of periodontitis}; Ivashkevich LG; For the treatment of patients with chronic and exacerbated periodontitis trichopol and lincomycin were used as well-known effective antibacterial drugs in anaerobic non-clostridial infection . Zinc-eugenol paste was added . The results were favourable.

J Urol, 1993 Jan, 149(1), 56 - 8
Collagenase versus placebo in the treatment of Peyronie's disease: a double-blind study; Gelbard MK et al.; We investigated 49 men with Peyronie's disease in a prospectively randomized placebo controlled double-blind study, comparing the effects on plaque size and penile deformity of intralesional purified clostridial collagenase and saline placebo . For the group as a whole, treatment out-performed placebo (p < 0.007) . When patients were analyzed with respect to disease severity, those with lesser deformity responded more favorably to treatment . The absolute angular change in patients responding to treatment was small . No significant side effects were noted within a 3-month followup.

Zentralbl Veterinarmed B, 1992 Dec, 39(10), 746 - 54
{Clostridia in carcasses and fresh meat--a literature review}; Eisgruber H et al.; Clostridia are of large clinical importance as well as in the field of food hygiene, where they are responsible for spoilage but they also have a certain significance as food poisoning organisms . Information on the ecology of Clostridia in samples of deep muscle tissue of slaughtered animals is insufficient . This article is intended to increase the knowledge on the occurrence of different Clostridia species in slaughtered animals . The main emphasis is put on the significance of clostridia in meat hygiene . The theoretical basis of the so called original content of microorganisms (intrinsic bacteria), the factors and pathways of Clostridia spreading in muscles and organs are demonstrated.

Biochemistry, 1992 Nov 10, 31(44), 10774 - 81
Characterization of iron-sulfur clusters in lysine 2,3-aminomutase by electron paramagnetic resonance spectroscopy; Petrovich RM et al.; Lysine 2,3-aminomutase from Clostridia catalyzes the interconversion of L-alpha-lysine with L-beta-lysine . The purified enzyme contains iron-sulfur ({Fe-S}) clusters, pyridoxal phosphate, and Co(II) {Petrovich, R . M., Ruzicka, F . J., Reed, G . H., & Frey, P . A . (1991) J . Biol . Chem . 266, 7656-7660} . Enzymatic activity depends upon the presence and integrity of these cofactors . In addition, the enzyme is activated by S-adenosylmethionine, which participates in the transfer of a substrate hydrogen atom between carbon-3 of lysine and carbon-2 of beta-lysine {Moss, M., & Frey, P . A . (1987) J . Biol . Chem . 262, 14859-14862} . This paper describes the electron paramagnetic resonance (EPR) properties of the {Fe-S} clusters . Purified samples of the enzyme also contain low and variable levels of a stable radical . The radical spectrum is centered at g = 2.006 and is subject to inhomogeneous broadening at 10 K, with a p1/2 value of 550 +/- 100 microW . The low-temperature EPR spectrum of the {Fe-S} cluster is centered at g = 2.007 and undergoes power saturation at 10 K in a homogeneous manner, with a p1/2 of 15 +/- 2 mW . The signals are consistent with the formulation {4Fe-4S} and are adequately simulated by a rhombic spectrum, in which gxx = 2.027, gyy = 2.007, and gzz = 1.99 . Treatment of the enzyme with reducing agents converts the cluster into an EPR-silent form . Oxidation of the purified enzyme by air or ferricyanide converts the {Fe-S} complex into a species with an EPR spectrum that is consistent with the formulation {3Fe-4S}.(ABSTRACT TRUNCATED AT 250 WORDS)

S Afr Med J, 1992 Nov, 82(5), 314 - 7
Antibiotic susceptibility of anaerobic bacteria isolated in Johannesburg; van der Westhuyzen I et al.; In vitro susceptibilities of 198 anaerobic bacteria to seven antibiotics were evaluated by the agar dilution method . In addition to testing amoxycillin/clavulanic acid in a 2:1 ratio against the bacteria, the combination was also tested against 63 isolates using fixed concentrations of clavulanic acid and serial dilutions of amoxycillin . Penicillin and cefoxitin were not effective against beta-lactamase-producing Bacteroides isolates and only 50% of isolates were susceptible to the 2:1 amoxycillin/clavulanic acid combination . However, when varying concentrations of amoxycillin were used together with constant concentrations of clavulanic acid (4 micrograms/ml) only 9 of 55 amoxycillin-resistant Bacteroides were resistant to the combination . Two clostridia were found to produce beta-lactamases and as expected were resistant to penicillin . Of the non-beta-lactamase-producing clostridia 11% were resistant to penicillin and 5% resistant to cefoxitin . Imipenem was effective against the majority of anaerobes tested and only 5 Bacteroides isolates were resistant . All anaerobic strains were susceptible to chloramphenicol and only 6% of strains resistant to clindamycin . Eighty-five per cent and 51% of Bacteroides strains had minimum inhibitory concentrations within two dilutions of the breakpoints of chloramphenicol (16 micrograms/ml) and clindamycin (4 micrograms/ml) respectively . Three strains of Peptostreptococcus spp . were resistant to metronidazole.

Zentralbl Bakteriol, 1992 Oct, 277(3), 340 - 4
Sialic acid as receptor of Bacteroides fragilis lectin-like adhesin; Domingues RM et al.; It was observed that sialic acid and macromolecules rich in this sugar were able to inhibit the hemagglutination activity (HA) of Bacteroides fragilis strains in low concentrations . Reversion of the HA and also of the adsorption to beads of Sepharose coupled to bovine submaxillary mucin, by this sugar residue corroborated the recognition capacity of the bacterial lectin-like adhesin . However, when erythrocytes were treated with clostridial neuraminidase, an increase in the HA of some strains was observed . Protease treatment of erythrocytes abolished the HA, indicating that cell receptors of B . fragilis are probably a glycoprotein moiety.

Int J Food Microbiol, 1992 Oct, 17(2), 135 - 43
Principles involved in the detection and enumeration of clostridia in foods; Mead GC; The clostridia are a group of anaerobic bacteria that vary considerably in their biochemical and physiological properties . Not surprisingly, attempts to develop a single isolation medium for all species that occur in foods have not been entirely successful, and the problem is compounded by the need to recover both vegetative cells and spores, some of the latter being unable to germinate without heat activation . Most available isolation media, except some of those used in the dairy industry, include sulphite and an appropriate iron salt, so that blackening due to sulphite reduction can serve as a differential test for clostridia . The limitations of this test in solid agar media are discussed and some advantages described in relation to its use in liquid media for Most Probable Number determinations . A medium favoured for the purpose is the Differential Reinforced Clostridial Medium of Gibbs and Freame (1965) . An unresolved issue is whether or not special precautions are needed to exclude oxygen during food sample preparation and dilution, preparation of media, and in conditions used for anaerobic incubation . Although such stringency may be required for maximum recovery of sub-lethally damaged cells or spores, practical constraints in food control laboratories necessitate use of relatively simple procedures for detecting clostridia routinely.

Food Addit Contam, 1992 Sep-Oct, 9(5), 597 - 605
Formaldehyde and hexamethylenetetramine as food additives: chemical interactions and toxicology; Restani P et al.; Formaldehyde (FA) and hexamethylenetetramine (HMT) are used in cheese production to control gas-forming clostridia; FA also occurs naturally in some foods at levels of 1-20 mg/kg . The toxicology of FA and HMT are briefly discussed together with their reaction in foods . The most abundant end-product of FA in cheese is spinacine derived from the N-terminal histidine residue in gamma 2-casein . Acute and short term toxicological studies on spinacine enable a No Observed Effect Level of 300 mg/kg body weight/day to be determined, leading to an Acceptable Daily Intake (ADI) for man of 3 mg/kg body weight/day . From these data a Tolerance Level (TL) of 1800 mg spinacine/kg cheese can be derived, leading to a Safety Margin (SM) of 12.9 . It is concluded that there is no appreciable health risk from consumption of cheese made using formaldehyde (Grana Padano) or hexamethylenetetramine (Provolone).

Anesthesiology, 1992 May, 76(5), 822 - 43
Up-and-down regulation of skeletal muscle acetylcholine receptors . Effects on neuromuscular blockers; Martyn JA et al.; Multiple factors alter the interaction of muscle relaxants with the NMJ . This review has focused on the aberrant responses caused principally by alterations in AChRs (table 1) . Many pathologic states increase (up-regulate) AChR number . These include upper and lower motor neuron lesions, muscle trauma, burns, and immobilization . Pre- or postjunctional inhibition of neurotransmission by drugs or toxins also up-regulate AChRs . These include alpha- and beta-BT, NDMR, anticonvulsants, and clostridial toxins . We speculate that other bacterial toxins also up-regulate AChR . With proliferation of AChRs, agonist drug dose-response curves are shifted to the left . The exaggerated release of potassium when depolarization occurs with the use of agonists such as SCh and decamethonium can be attributed to the increased number of AChR . Thus, SCh should be avoided in patients who are in the susceptible phase (see section V) . In the presence of increased AChR, the requirement for NDMR is markedly increased . Thus, the response to NDMR may be used as an indirect estimator of increased sensitivity to SCh (table 1) . The most extensively studied pathologic state in which there is a decrease in AChRs is myasthenia gravis; there is immunologically mediated destruction and/or functional blockade of AChRs . The pathophysiologic and pharmacologic changes in LEMS are quite distinct from those of myasthenia gravis . Decreased AChRs in myasthenia gravis result in resistance to agonists and increased sensitivity to competitive antagonists . In conditioning exercise, the perturbed muscles show sensitivity to NDMR that may be due to decreased AChRs . Chronic elevations of ACh observed with organophosphorus poisoning or chronic use of reversible cholinesterase inhibitors results in down-regulation of AChRs . In this condition, SCh should be avoided because its metabolic breakdown would be impaired; the requirement for NDMR may be decreased . All of the varied responses to SCh and NDMR, which are associated with concomitant changes in AChRs, are analogous to drug-receptor interactions observed in other biologic systems.

Cent Afr J Med, 1992 Apr, 38(4), 166 - 8
Metastatic clostridial myonecrosis associated with intra-uterine clostridial infection: a report of three cases; Oliver MJ; Three cases of metastatic Clostridial myonecrosis are reported, two following spontaneous and one following criminal abortion . This particular combination has not been reported before . The two survivors were managed by hysterectomy and disarticulation of the leg through the hip . The patient who died did so before surgery could be undertaken . The literature on gas gangrene infections of the uterus and metastatic Clostridial myonecrosis is reviewed and guidelines for management are discussed.

Anal Biochem, 1992 Mar, 201(2), 336 - 42
Specific identification of collagens and their fragments by clostridial and anti-collagenase antibody; Han S et al.; A method specific for identification of collagens irrespective of type, species, or tissue origin, and of their derived fragments of molecular weight more than 10,000, is described . The method is based on the low-temperature affinity between clostridial collagenase and almost all types of collagens as well as on the affinity between collagenase and its antibodies . Various collagens or fragments derived from them by treatment with CNBr were separated by SDS-PAGE and immobilized onto a nitrocellulose membrane by a slot-blot technique or electrotransfer . Following binding of clostridial collagenase to a collagen or its fragments at 0 degrees C, the collagen-collagenase complex was fixed with glutaraldehyde . The complex was then allowed to bind anti-collagenase antibody at room temperature . The new complex was subsequently treated with 125I-labeled donkey anti-rabbit IgG and visualized as an autoradiogram . Under the conditions of low temperature used, the collagenase binds to collagens without causing their digestion . This procedure is specific for detection of soluble collagens as well as of insoluble collagens converted to fragments by treatment with CNBr . The method is uniquely suited for detection of fragments of tissue collagens . Also, it may serve as a prototype for methods for detection of other specific polymeric substances.

What Is Water Purification?, What Is Listeria Monocytogenes?, What Is Bioremediation?, What Is MIC?, What Is Anthrax?, i, Bacteria, n, Bacteriology, c, Microbiology, n, Bacterium, r, Microorganisms, a, Biological reactors, o, Yeasts, i, Pseudomonas aeruginosa, o, Antibiotics, a, Bacillus subtilis, i, B. anthracis, a, Clostridia, a, Clostridia, s, Escherichia coli, s, Streptococcal, r, Escherichia coli, o, Antibiotics, o, Biological filters, e, Escherichia coli, i, Antibiotics, c, Phage, s, Meningococcus, e, Escherichia coli, c, Antibiotics, c, Streptococci, r, Escherichia coli

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005