Construction and characterization of multicopy plasmids containing the entire F transfer region

The restriction endonucleases HindIII and/or BamI have been used to clone the entire F transfer region into pBR322 to create a series of transfer-proficient multicopy plasmids. Despite the insertion of 40.7- to 55.9-kilobase F fragments, the plasmid copy numbers remained high, at 25–40 copies per cell. One of the chimeric plasmids contained the F replication and incompatibility region, and its high copy number confirmed that replication of the cointegrate was governed by pBR322. Despite the 30- to 40-fold increase in tra gene copy number compared to Flac, the transfer frequencies, number of pili per cell, and syntheses of the individual traT and traI proteins were increased only by about 5-fold. The level of tra mRNA in cells carrying the multicopy transfer-proficient plasmids was also increased only by about 5-fold, suggesting that its relative synthesis or stability was reduced in this situation. Nonetheless, the increased production of tra DNA, mRNA, and protein makes cells carrying the multicopy conjugative plasmids excellent sources of these products.     

Biotransformation of cycloalkenones by fungi Baeyer-Villiger oxidation of bicycloheptenone by dematiaceous fungi

Summary The biotransformation by ring expansion of a bicyclo [3.2.0]-alkenone has been demonstrated in 29 species of dematiaceous fungi. One of these biocatalysts,Curvularia lunata NRRL 2380 has been shown to produce synthetically-important chiral lactones in high yields.     

Local erythropoietin and endothelial progenitor cells improve regional cardiac function in acute myocardial infarction

Background

Expanded endothelial progenitor cells (eEPC) improve global left ventricular function in experimental myocardial infarction (MI). Erythropoietin beta (EPO) applied together with eEPC may improve regional myocardial function even further by anti-apoptotic and cardioprotective effects. Aim of this study was to evaluate intramyocardial application of eEPCs and EPO as compared to eEPCs or EPO alone in experimental MI.

Methods and Results

In vitro experiments revealed that EPO dosed-dependently decreased eEPC and leukocyte apoptosis. Moreover, in the presence of EPO mRNA expression in eEPC of proangiogenic and proinflammatory mediators measured by TaqMan PCR was enhanced. Experimental MI was induced by ligation and reperfusion of the left anterior descending coronary artery of nude rats (n = 8-9). After myocardial transplantation of eEPC and EPO CD68+ leukocyte count and vessel density were enhanced in the border zone of the infarct area. Moreover, apoptosis of transplanted CD31 + TUNEL + eEPC was decreased as compared to transplantation of eEPCs alone. Regional wall motion of the left ventricle was measured using Magnetic Resonance Imaging. After injection of eEPC in the presence of EPO regional wall motion significantly improved as compared to injection of eEPCs or EPO alone.

Conclusion

Intramyocardial transplantation of eEPC in the presence of EPO during experimental MI improves regional wall motion. This was associated with an increased local inflammation, vasculogenesis and survival of the transplanted cells. Local application of EPO in addition to cell therapy may prove beneficial in myocardial remodeling.

     

Further characterization of the F fertility inhibition systems of "unusual" Fin+ plasmids.

Flac mutants insensitive to transfer inhibition by R factors. JR66a and R485 were isolated and characterized. Representative mutations were cis dominant and are therefore presumed to be at the sites of action, fisU and fisV, respectively, of the FinU and FinV transfer inhibition systems encoded by JR66a and R485. The mutants were used to confirm that the FinU and FinV fertility inhibition systems are different from each other and from the FinOP, FinQ, and FinW systems of R100, R62, and R455, respectively. Together with traO and fisQ mutants of Flac, the new mutants were also used to investigate the nature of the F fertility inhibition systems encoded by a further group of "unusual" Fin+ plasmids. Of these, two incompatibility group X plasmids were found to carry finO+ genes, and of five incompatibility group I plasmids, three encoded FinQ systems, one the FinU system, and one a new system (FinR). Transfer of a variety of derepressed F-like plasmids was inhibited by the FinQ, FinU, and FinV systems, but a quantitatively very different levels; this emphasizes the differences as well as the similarities between the conjugation systems of F-like plasmids.     

Microbial metabolism of alkylbenzene sulphonates the oxidation of key aromatic compounds by a Bacillus

A Bacillus species originally elected for growth at the expense of alkylbenzene sulphonate detergents was found to metabolise a wide range of aromatic compounds. p-Hydroxybenzoate (PHB) was initially hydroxylated to protocatechuate (PCA) i.e. 3,4-dihydroxybenzoate, which was oxidatively cleaved to succinate and acetyl-CoA by a classical ortho cleavage pathway initiated by a substrate-specific 3, 4-oxygenase: no evidence of an alternative meta cleavage pathway was detected. Several key enzymes of this ortho cleavage pathway were induced by growth of the Bacillus on either PHB or PCA. Both PHB and PCA were able to act as sole source of carbon for energy and overall growth of the microorganism.In strict contrast, the higher homologue p-hydroxyphenylacetate (PHPA), after initial hydroxylation to 3, 4-dihydroxyphenylacetate (DHPA), was oxidatively cleaved to 4-carboxymethyl-2-hydroxymuconic semialdehyde (CMHMS) by a meta cleavage catalysed by a substrate-specific 2, 3-oxygenase: no evidence of an alternative ortho cleavage was detected. Several lines of evidence suggested that CMHMS was not further metabolised by the Bacillus and accumulated in the growth medium. Both PHPA and DHPA were unable to act as sole source of carbon for energy and overall growth.The implication of the occurrence in a single bacterium of two separate oxidative pathways catalysing the cleavage of different aromatic nuclei have been discussed.     

Immunodetection of occult eosinophils in lung tissue biopsies may help predict survival in acute lung injury

Background

Acute lung injury (ALI) is a serious respiratory disorder for which therapy is primarily supportive once infection is excluded. Surgical lung biopsy may rule out other diagnoses, but has not been generally useful for therapy decisions or prognosis in this setting. Importantly, tissue and peripheral blood eosinophilia, the hallmarks of steroid-responsive acute eosinophilic pneumonia, are not commonly linked with ALI. We hypothesized that occult eosinophilic pneumonia may explain better outcomes for some patients with ALI.

Methods

Immunohistochemistry using a novel monoclonal antibody recognizing eosinophil peroxidase (EPX-mAb) was used to assess intrapulmonary eosinophil accumulation/degranulation. Lung biopsies from ALI patients (n = 20) were identified following review of a pathology database; 45% of which (i.e., 9/20) displayed classical diffuse alveolar damage (ALI-DAD). Controls were obtained from uninvolved tissue in patients undergoing lobectomy for lung cancer (n = 10). Serial biopsy sections were stained with hematoxylin and eosin (H&E) and subjected to EPX-mAb immunohistochemistry.

Results

EPX-mAb immunohistochemistry provided a >40-fold increased sensitivity to detect eosinophils in the lung relative to H&E stained sections. This increased sensitivity led to the identification of higher numbers of eosinophils in ALI patients compared with controls; differences using H&E staining alone were not significant. Clinical assessments showed that lung infiltrating eosinophil numbers were higher in ALI patients that survived hospitalization compared with non-survivors. A similar conclusion was reached quantifying eosinophil degranulation in each biopsy.

Conclusion

The enhanced sensitivity of EPX-mAb immunohistochemistry uniquely identified eosinophil accumulation/degranulation in patients with ALI relative to controls. More importantly, this method was a prognostic indicator of patient survival. These observations suggest that EPX-mAb immunohistochemistry may represent a diagnostic biomarker identifying a subset of ALI patients with improved clinical outcomes.     

Age and medication are significant risk factors for xerostomia in an English population, attending general dental practice

Objective: To study the prevalence of xerostomia in an English population, attending general dental practice and relate it to age. medication and gender. Design Study: Cross‐sectional. Setting: Five General Dental Practices in Merseyside, North of England. Subjects: 1,103 adult patients attending for routine dental care. Intervention : Questionnaire administered by dentists. Main Outcome Measures: Age, gender, systemic medication, reported oral dryness. Results: 1,103 patients (654 females) were recruited, of whom 427 (39%) were aged 60 years or older, 26% of patients reported taking medication. The overall prevalence of xerostomia was 12.7% (males‐10.3%, females 14.4%). Age. medication and female gender were found to be significant risk factors for xerostomia, using logistic regression analysis. Conclusions: The prevalence of xerostomia (12.7%) in an English population was lower than reported in previous North American and Swedish studies. Medication was a significant risk factor for xerostomia and a better predictor of risk status, than either age or gender.     

MADAM - An open source meta-analysis toolbox for R and Bioconductor

Background  

Meta-analysis is a major theme in biomedical research. In the present paper we introduce a package for R and Bioconductor that provides useful tools for performing this type of work. One idea behind the development of MADAM was that many meta-analysis methods, which are available in R, are not able to use the capacities of parallel computing yet. In this first version, we implemented one meta-analysis method in such a parallel manner. Additionally, we provide tools for combining the results from a set of methods in an ensemble approach. Functionality for visualization of results is also provided.