Analysis of replication region of the cryptic plasmid pAG20 from Acetobacter aceti 3620

The DNA sequence of small cryptic plasmid pAG20 in Acetobacter aceti was determined at 3064 bp with 51.6% GC pairs. The plasmid encoded a 186 amino acid protein which is important for plasmid replication in Gram-negative bacteria except Escherichia coli. Two 21 bp large direct repeat sequence 1 and two 13 bp direct repeat sequence 2 were determined in the regulation region upstream from gene encoded Rep protein. Vector pAG24 with kanamycin gene and two deletion derivatives pAG25 and pAG26 without rep gene from plasmid pAG20 were constructed. Plasmid pAG24 was replicated in a broad host range like E. coli, Acetobacter pasteurianus, A. aceti, Comanomonas spp., Serratia marcescens, and Shigella spp.     

Stimulation of TNF-alpha release by fungal cell wall polysaccharides

Carboxymethylated derivatives were prepared from the (1-->3)-beta-D-glucan isolated from the cell wall of baker's yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-alpha by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-alpha release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1-->3)-beta-D-glucan, are potent macrophage stimulators and activators of TNF-alpha release, which implies their potential application in antitumor therapy.     

Changes of phenolic metabolism and oxidative status in nitrogen-deficient Matricaria chamomilla plants

The effects of nitrogen deficiency on selected physiological attributes, phenylalanine ammonia-lyase (PAL, EC. 4.3.1.5) activity, phenolic contents, peroxidase (EC. 1.11.1.7) and catalase (EC. 1.11.1.6) activities, lipid peroxidation status and H₂O₂ accumulation were studied in N-deficient Matricaria chamomilla (L.) over 12 days. N deficiency enhanced root growth and inhibited shoot growth. Chlorophyll composition and F _v/F _m were not affected by N stress, but nitrogen and soluble proteins decreased in both the rosettes and the roots. PAL activity, expressed per mg protein, was enhanced in N-deficient rosettes and tended to decrease by the end of the experiment, while in the roots PAL activity was maintained. Total phenolic contents increased in both rosettes and roots. Peroxidase and catalase activities in N-deficient rosettes tended to decrease by the end of the experiment, while in the roots they increased on the 12th day of deficiency. Furthermore, lipid peroxidation status increased in N-deficient roots on the 12th day, indicating that antioxidative protection was insufficient to scavenge reactive oxygen species being generated. Surprisingly, H₂O₂ content was even lower in N-deficient roots by the end of the experiment, while in the leaves increased. This observation in correlation to lipid peroxidation and H₂O₂ degradation is discussed. The importance of PAL activity and phenolic metabolites in combination with antioxidative enzymes for plant protection against oxidative stress and the significance of PAL activity for the mobilization of N availability in N-deficient tissue are also discussed in view of existing information.     

Muscle Histidine-Containing Dipeptides Are Elevated by Glucose Intolerance in Both Rodents and Men

Objective

Muscle carnosine and its methylated form anserine are histidine-containing dipeptides. Both dipeptides have the ability to quench reactive carbonyl species and previous studies have shown that endogenous tissue levels are decreased in chronic diseases, such as diabetes.

Design and Methods

Rodent study: Skeletal muscles of rats and mice were collected from 4 different diet-intervention studies, aiming to induce various degrees of glucose intolerance: 45% high-fat feeding (male rats), 60% high-fat feeding (male rats), cafeteria feeding (male rats), 70% high-fat feeding (female mice). Body weight, glucose-tolerance and muscle histidine-containing dipeptides were assessed. Human study: Muscle biopsies were taken from m. vastus lateralis in 35 males (9 lean, 8 obese, 9 prediabetic and 9 newly diagnosed type 2 diabetic patients) and muscle carnosine and gene expression of muscle fiber type markers were measured.

Results

Diet interventions in rodents (cafeteria and 70% high-fat feeding) induced increases in body weight, glucose intolerance and levels of histidine-containing dipeptides in muscle. In humans, obese, prediabetic and diabetic men had increased muscle carnosine content compared to the lean (+21% (p>0.1), +30% (p<0.05) and +39% (p<0.05), respectively). The gene expression of fast-oxidative type 2A myosin heavy chain was increased in the prediabetic (1.8-fold, p<0.05) and tended to increase in the diabetic men (1.6-fold, p = 0.07), compared to healthy lean subjects.

Conclusion

Muscle histidine-containing dipeptides increases with progressive glucose intolerance, in male individuals (cross-sectional). In addition, high-fat diet-induced glucose intolerance was associated with increased muscle histidine-containing dipeptides in female mice (interventional). Increased muscle carnosine content might reflect fiber type composition and/or act as a compensatory mechanism aimed at preventing cell damage in states of impaired glucose tolerance.     

Expression profiling reveals differences in metabolic gene expression between exercise-induced cardiac effects and maladaptive cardiac hypertrophy

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise-induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise-induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in beta-oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise-induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise-induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.     

Recombinant production of <Emphasis Type="Italic">Streptococcus equisimilis</Emphasis> streptokinase by <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Background  

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. It is naturally secreted by several strains of beta-haemolytic streptococci. The low yields obtained in SK production, lack of developed gene transfer methodology and the pathogenesis of its natural host have been the principal reasons to search for a recombinant source for this important therapeutic protein. We report here the expression and secretion of SK by the Gram-positive bacterium Streptomyces lividans. The structural gene encoding SK was fused to the Streptomyces venezuelae CBS762.70 subtilisin inhibitor (vsi) signal sequence or to the Streptomyces lividans xylanase C (xlnC) signal sequence. The native Vsi protein is translocated via the Sec pathway while the native XlnC protein uses the twin-arginine translocation (Tat) pathway.     

Identification, distribution and molecular evolution of the pacifastin gene family in Metazoa

Background  

Members of the pacifastin family are serine peptidase inhibitors, most of which are produced as multi domain precursor proteins. Structural and biochemical characteristics of insect pacifastin-like peptides have been studied intensively, but only one inhibitor has been functionally characterised. Recent sequencing projects of metazoan genomes have created an unprecedented opportunity to explore the distribution, evolution and functional diversification of pacifastin genes in the animal kingdom.     

Deliberate Attenuation of Chikungunya Virus by Adaptation to Heparan Sulfate-Dependent Infectivity: A Model for Rational Arboviral Vaccine Design

Mosquito-borne chikungunya virus (CHIKV) is a positive-sense, single-stranded RNA virus from the genus Alphavirus, family Togaviridae, which causes fever, rash and severe persistent polyarthralgia in humans. Since there are currently no FDA licensed vaccines or antiviral therapies for CHIKV, the development of vaccine candidates is of critical importance. Historically, live-attenuated vaccines (LAVs) for protection against arthropod-borne viruses have been created by blind cell culture passage leading to attenuation of disease, while maintaining immunogenicity. Attenuation may occur via multiple mechanisms. However, all examined arbovirus LAVs have in common the acquisition of positively charged amino acid substitutions in cell-surface attachment proteins that render virus infection partially dependent upon heparan sulfate (HS), a ubiquitously expressed sulfated polysaccharide, and appear to attenuate by retarding dissemination of virus particles in vivo. We previously reported that, like other wild-type Old World alphaviruses, CHIKV strain, La Réunion, (CHIKV-LR), does not depend upon HS for infectivity. To deliberately identify CHIKV attachment protein mutations that could be combined with other attenuating processes in a LAV candidate, we passaged CHIKV-LR on evolutionarily divergent cell-types. A panel of single amino acid substitutions was identified in the E2 glycoprotein of passaged virus populations that were predicted to increase electrostatic potential. Each of these substitutions was made in the CHIKV-LR cDNA clone and comparisons of the mutant viruses revealed surface exposure of the mutated residue on the spike and sensitivity to competition with the HS analog, heparin, to be primary correlates of attenuation in vivo. Furthermore, we have identified a mutation at E2 position 79 as a promising candidate for inclusion in a CHIKV LAV.