Myonecrosis by Clostridium septicum in a dog,diagnosed by a new multiplex-PCR

Clostridial myositis is an acute, generally fatal toxemia that is considered to be rare in pet animals. The present report describes an unusual canine clostridial myositis that was diagnosed by a new multiplex-PCR (mPCR) designed for simultaneous identification of Clostridium sordellii, Clostridium septicum, Clostridium perfringens type A, Clostridium chauvoei, and Clostridium novyi type A. A ten-month-old male Rottweiler dog, that had displayed lameness and swelling of the left limb for 12 h, was admitted to a veterinary hospital. The animal was weak, dyspneic and hyperthermic, and a clinical examination indicated the presence of gas and edema in the limb. Despite emergency treatment, the animal died in only a few minutes. Samples of muscular tissue from the necrotic area were aseptically collected and plated onto defibrinated sheep blood agar (5%) in anaerobic conditions. Colonies suggestive of Clostridium spp. were submitted to testing by multiplex-PCR. Impression smears of the tissues, visualized with Gram and also with panoptic stains, revealed long rod-shaped organisms, and specimens also tested positive using the fluorescent antibody technique (FAT). The FAT and mPCR tests enabled a diagnosis of C. septicum myonecrosis in the dog.     

Evidence for a peroxidatic oxidation of norepinephrine, a catecholamine, by lactoperoxidase

The electron spin resonance-spin stabilization technique has been applied to identify the o-semiquinone intermediate produced during the lactoperoxidase-catalyzed oxidation of the catecholamine norepinephrine. The results of a rapid scan and spectrophotometric investigation of the reaction clearly indicate a normal peroxidatic pathway of catecholamine degradation.     

Porewater mixing by microorganisms,monitored by a radiotracer method

Bioturbation is an important process for the transport and mixing of solutes and particles in sediments. Mixing of porewater caused by motile microorganisms has not previously been considered to be of significance in this context, although no conclusive evidence that it is negligible has been presented. We have developed a radiotracer method for the direct comparison of mixing of a soluble inert substance in microbially active and sterile sediments. We found clear evidence of porewater mixing caused by motile microorganisms. Estimated diffusion coefficients (expressed as “mixing”; coefficients) were found to be about 20% larger in microbially active sediments than in sterile ones.     

Daptomycin,a Bacterial Lipopeptide Synthesized by a Nonribosomal Machinery

Daptomycin (Cubicin®) is a branched cyclic lipopeptide antibiotic of nonribosomal origin and the prototype of the acidic lipopeptide family. It was approved in 2003 for the nontopical treatment of skin structure infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), and in 2006 for the treatment of bacteremia. Understanding the ribosome-independent biosynthesis of daptomycin assembly will provide opportunities for the generation of daptomycin derivatives with an altered pharmaceutical spectrum to address upcoming daptomycin-resistant pathogens. Herein, the structural properties of daptomycin, its biosynthesis, recent efforts for the generation of structural diversity, and its proposed mode of action are discussed.     

Ceriporic acid C, a hexadecenylitaconate produced by a lignin-degrading fungus, Ceriporiopsis subvermispora

A lignin-degrading basidiomycete, Ceriporiopsis subvermispora produces a series of alkyl- and alkenylitaconates (ceriporic acids). Previously, two alkylitaconic acids with tetradecyl and hexadecyl side chains were isolated and identified as 1-heptadecene-2,3-dicarboxylic acid (ceriporic acid A) and 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B). In the present study, one hexadecenylitaconate (ceriporic acid C) was isolated and its chemical structure was analyzed by glycolation and subsequent (1) trimethylsilation, or (2) acetalation with acetone and acetone-d6. Analyses of the isolated metabolite demonstrated that the hexadecenylitaconic acid was (Z)-1,10-nonadecadiene-2,3-dicarboxylic acid. The structure of the side chain in ceriporic acid C was the same as that of hexadecenylcitraconate, chaetomellic acid B. Thus, it was found that ceriporic acids share close structural similarity with alk(en)yl citraconate derivatives, chaetomellic acids and other lichen lactones, protolichesterinic, lichesterinic, and murolic acids.     

Studies of a tumor-associated antigen,COX-1, recognized by a monoclonal antibody

Summary Monoclonal antibodies against an ovarian tumor cell line, OC-3-VGH, were generated using modified hybridoma technology. Among the seven that were selected for their high specificity and affinity to ovarian cancer cells and low cross-reactivity to most normal human tissues, RP 215 was shown to react specifically with a tumor-associated antigen, COX-1, from certain ovarian/cervical cancer cell lines. By Western blot assay, COX-1 was shown to have a subunit molecular mass of about 60 kDa and exist as an aggregate in the native state. COX-1 could also be detected in the shed medium of certain cultured tumor cells. A solid-phase sandwich enzyme-immunoassay procedure was designed for quantitative determinations of COX-1 in the shed medium or in patients' sera using RP 215 for both well-coating and the signal detection. Highly purified COX-1 was obtained from the shed medium of cultured OC-3-VGH tumor cells mainly by hydroxyapatite and immunoaffinity chromatography with RP 215 as the affinity ligand. At neutral pH, purified COX-1 also exists as an aggregate and is relatively stable at temperatures below 50°C. Its immunoactivity was found to decrease with time in the presence of trypsin. However, the immunoactivity of COX-1 was not affected upon incubation with carbohydrate-digestive enzymes or concanavalin A and only partially inactivated in the presence of NaIO₄ or iodoacetamide. Treatments of COX-1 with dithiothreitol and guanidine thiocyanate resulted in a complete loss of activity. Furthermore, rabbit antisera raised against purified COX-1 exhibited similar immunospecificity to that of RP 215. The results of this study suggest that COX-1 is a glycoprotein consisting of a 60 kDa subunit, which is recognized by RP 215 through its peptide determinant. Preliminary retrospective clinical studies were performed to assess the utility of a COX-1 enzyme immunoassay kit for detection and monitoring of patients with ovarian and cervical cancers.     

Galactoside Accumulation by Escherichia coli, Driven by a pH Gradient

Acidification of the external medium results in thiomethylgalactoside accumulation in an energy-depleted adenosine triphosphatase-negative mutant of Escherichia coli.     

Glutamicin CBII,a bacteriocin-like substance produced by

Corynebacterium glutamicum CBII, in the stationary phase of growth, was found to produce spontaneously a substance resembling bacteriocins by its bactericidal properties. This substance designated glutamicin CBII was observed to exhibit bactericidal activity against coryneform bacteria (12 species tested) but not against unrelated gram-positive (3) and gram-negative (3) bacteria, while its action on bacteria with no quite known relatedness to the coryneform group (14) was found to be variable. Glutamicin CBII was partially purified by precipitation with ammonium sulphate (70% saturation), selective heat precipitation and gel chromatography on Sepadex G-50. The antibacterial substance diffused through cellophane membrane with an approximate cut-off of 10000 dalton and its sedimentation coefficient was determined to be 1.1. S by ultracentrifugation. Heating at 100°C for 30 min had no effect on its activity. Glutamicin CBII was proved to be resistant to chloroform, trypsin, chymotrypsin, pronase, and subtilisin. According to its staining behaviour and ¹H NMR spectra it probably represents a glycoprotein containing only a minor protein component.

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Luteolin, a flavonoid, inhibits AP-1 activation by basophils

Flavonoids including luteolin, apigenin, and fisetin are inhibitors of IL-4 synthesis and CD40 ligand expression by basophils. This study was done to search for compounds with greater inhibitory activity of IL-4 expression and to clarify the molecular mechanisms through which flavonoids inhibit their expression. Of the 37 flavonoids and related compounds examined, ayanin, luteolin, and apigenin were the strongest inhibitors of IL-4 production by purified basophils in response to anti-IgE antibody plus IL-3. Luteolin did not suppress Syk or Lyn phosphorylation in basophils, nor did suppress p54/46 SAPK/JNK, p38 MAPK, and p44/42 MAPK activation by a basophilic cell line, KU812 cells, stimulated with A23187 and PMA. However, luteolin did inhibit phosphorylation of c-Jun and DNA binding activity of AP-1 in nuclear lysates from stimulated KU812 cells. These results provide a fundamental structure of flavonoids for IL-4 inhibition and demonstrate a novel action of flavonoids that suppresses the activation of AP-1.     

天目山——和尚的遗产

天目高山的佛教延续至今,已近1700多年,东晋兴起,宋末明初达到顶峰,最盛时候,僧侣达到1000多人,佛教给天目山留下厚重的文化遗产的同时。 还给后人留下了别一份遗产。 参天古木与原始森林。然而,和尚是怎样保住这片山林的呢？ 带着这个疑问,我们走村串户,逐渐揭开了这个谜团……     

Transformation of a Methylotrophic Bacterium, Methylobacterium extorquens, with a Broad-Host-Range Plasmid by Electroporation

An efficient method for the transformation of the methylotrophic bacterium Methylobacterium extorquens NR-2 with a broad-host-range plasmid, pLA2917, by electroporation was examined. Transformants of M. extorquens NR-2 expressing resistance to kanamycin were obtained after electric pulse. These transformants were found to harbor a single plasmid which was electrophoretically identical and homologous to pLA2917 obtained from Escherichia coli. Several factors which determined the transformation efficiency were optimized, resulting in a transformation efficiency of up to 8 × 10³ transformants per μg of plasmid DNA by 10 pulses at a field strength of 10 kV/cm and a pulse duration of 300 μs.     

Poinsettia Leaf Curl, a New Disease Caused by a Geminivirus

A serious disorder of unknown aetiology was found on the poinsettia cv. Angelica, a recent introduction to Taiwan. The symptoms of downward leaf curling and vein enation were similar to those of leaf curl disease on tobacco. The causal agent of the disease was transmitted to healthy plants of'Angelica'poinsettia, common poinsettia and tobacco by whiteflies. Twinned particles measuring 16-18 × 30-32 nm were detected in a purified preparation obtained from diseased leaf tissues of'Angelica'poinsettia, indicating that the disease is caused by a geminivirus. An earlier introduction of common poinsettias also showed mild leaf curl symptoms under natural condition. When the causal agent of this disease was transmitted to healthy'Angelica'poinsettia plants by whiteflies or bud grafting, these plants also developed severe leaf curl symptoms, indicating that the disease is not a new introduction to Taiwan.     

Production of a New Thiopeptide Antibiotic,TP-1161, by a Marine Nocardiopsis Species

Twenty-seven marine sediment- and sponge-derived actinomycetes with a preference for or dependence on seawater for growth were classified at the genus level using molecular taxonomy. Their potential to produce bioactive secondary metabolites was analyzed by PCR screening for genes involved in polyketide and nonribosomal peptide antibiotic synthesis. Using microwell cultures, conditions for the production of antibacterial and antifungal compounds were identified for 15 of the 27 isolates subjected to this screening. Nine of the 15 active extracts were also active against multiresistant Gram-positive bacterial and/or fungal indicator organisms, including vancomycin-resistant Enterococcus faecium and multidrug-resistant Candida albicans. Activity-guided fractionation of fermentation extracts of isolate TFS65-07, showing strong antibacterial activity and classified as a Nocardiopsis species, allowed the identification and purification of the active compound. Structure elucidation revealed this compound to be a new thiopeptide antibiotic with a rare aminoacetone moiety. The in vitro antibacterial activity of this thiopeptide, designated TP-1161, against a panel of bacterial strains was determined.Natural products remain the most prolific source of new antimicrobials, and the chemical diversity of natural compounds is still unmatched by combinatorial chemistry approaches (9, 31). While the latter has been successfully applied for lead optimization, it basically failed to deliver genuinely new pharmacophores, especially in the field of antimicrobials (31), mainly due to limitations in the structural variety of compounds represented in combinatorial libraries.Most of the antibiotics in clinical use today have been developed from compounds isolated from bacteria and fungi, with members of the actinobacteria being the dominant source (34). Traditionally, most of these antimicrobials have been isolated from soil-derived actinomycetes of the genus Streptomyces. However, isolation strategies in recent years have been directed to unexploited environments like marine sources (40). Bioprospecting efforts focusing on the isolation and screening of actinobacteria from ocean habitats (25, 27) have added new biodiversity to the order Actinomycetales and revealed a range of novel natural products of pharmacological value. The existence of marine actinobacterial species physiologically and phylogenetically distinct from their terrestrial relatives is now widely accepted, and new taxonomic groups of marine actinomycetes have been described for at least six different families within the order Actinomycetales (12). Apart from being phylogenetically distinct from their terrestrial relatives, marine isolates have been shown to possess specific physiological adaptations (e.g., to high salinity/osmolarity and pressure) to their maritime surroundings and many were found to produce novel and chemically diverse secondary metabolites (10, 13, 35).Most streptomycetes and other filamentous actinomycetes possess numerous gene clusters for the biosynthesis of secondary metabolites (2, 32), and genome sequence studies have shown that large portions of their genomes are devoted to secondary metabolite biosynthesis. Twenty gene clusters coding for known or predicted secondary metabolites were identified in the 8.7-Mb genome of Streptomyces coelicolor A3(2) (2), and 6.4% of the 8.7-Mb genome of Streptomyces avermitilis is dedicated to gene clusters for secondary metabolite biosynthesis (32). The marine actinomycete Salinispora allocates nearly 10% of its 5.2-Mb genome to 17 diverse biosynthetic loci, including polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and several hybrid clusters (4, 43). Many medicinally important natural products, including antibacterials and antifungals, are synthesized by these multimodular assembly lines (14), and genome mining for secondary metabolite gene clusters has become a common tool to assess the genetic capability of bacteria to produce novel bioactive compounds. However, even for well-studied model antibiotic producers like S. coelicolor A3(2), discrepancies between the number of known metabolites on the one hand and the number of pathways identified from genomic data on the other hand are tremendous (2). These discrepancies can only be explained by the facts that most gene clusters for secondary metabolites are silenced under standard laboratory cultivation conditions and that an expression or upregulation of these pathways is only triggered in response to certain environmental signals. It has been shown that by cultivating bacteria under a range of conditions, it is possible to obtain products of many of these “orphan” biosynthetic pathways (4). Using the OSMAC (one strain-many compounds) approach, Bode et al. were able to isolate more than 100 compounds comprising 25 structural classes from only six microorganisms (4).In this study, marine sediment-derived actinomycete isolates were analyzed for the production of antimicrobial secondary metabolites by using microwell plate fermentations and a range of media and conditions. This approach led to the isolation of a new thiopeptide antibiotic, designated TP-1161, produced by a marine sediment-derived Nocardiopsis isolate. Here we report the isolation and structural and biological characterization of TP-1161.     

CbpA, a DnaJ homolog, is a DnaK co-chaperone, and its activity is modulated by CbpM

The DnaK chaperone system, consisting of DnaK, DnaJ, and GrpE, remodels and refolds proteins during both normal growth and stress conditions. CbpA, one of several DnaJ analogs in Escherichia coli, is known to function as a multicopy suppressor for dnaJ mutations and to bind nonspecifically to DNA and preferentially to curved DNA. We found that CbpA functions as a DnaJ-like co-chaperone in vitro. CbpA acted in an ATP-dependent reaction with DnaK and GrpE to remodel inactive dimers of plasmid P1 RepA into monomers active in P1 DNA binding. Additionally, CbpA participated with DnaK in an ATP-dependent reaction to prevent aggregation of denatured rhodanese. The cbpA gene is in an operon with an open reading frame, yccD, which encodes a protein that has some homology to DafA of Thermus thermophilus. DafA is a protein required for the assembly of ring-like particles that contain trimers each of T. thermophilus DnaK, DnaJ, and DafA. The E. coli YccD was isolated because of its potential functional relationship to CbpA. Purified YccD specifically inhibited both the co-chaperone activity and the DNA binding activity of CbpA, suggesting that YccD modulates the activity of CbpA. We named the product of the yccD gene CbpM for "CbpA modulator."