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 commu