Comparative bacterial clearances of muscle and skin/subcutaneous tissues with and without dead bone: a laboratory study

Seventeen New Zealand White rabbits underwent implantation of three different concentrations of bacteria and a sterile saline control solution with and without dead autologous bone in eight separate muscular and eight separate subcutaneous sites. Following a period of 1 week, each site was surgically explored and samples of tissue were taken for histology and quantitative culture. Results reveal that final bacterial concentrations in the subcutaneous sites were significantly lower than in the muscle sites (p less than or equal to 0.0001) for each concentration of bacteria, with and without dead bone. Dead bone resulted in very significantly greater bacterial concentrations in both subcutaneous and muscle sites. Clinically, these results indicate that a thorough bony wound debridement is more important than the type of tissue used to close the wound. Flap tissue should be selected with regard to the perfusion, contour, and appearance of the recipient site.     

Complete nucleotide sequence of a cryptic plasmid, pbaw301, from the ruminai anaerobe Ruminococcus flavefaciens R13e2

Abstract The complete nucleotide sequence of a cryptic plasmid designated pBAW301, from the Gram-positive ruminai bacterium Ruminococcus flavefaciens R13e2, has been determined. This plasmid is 1768 bp in size and has an overall G+C content of 43.5%. Computer analysis of the sequence data revealed an open reading frame, ORF1 (256 amino acids), which is similar to the Rep protein of the Bacillus borstelensis plasmid pHT926. ORF1 is preceded by Shine-Dalgarno and Escherichia coli —10 and —35 like sequences. Nine smaller open reading frames showed no significant homologies to known protein sequences. Analysis of replication intermediates and the nucleotide sequence indicate that the plasmid does not replicate by a rolling-circle mode of replication similar to other plasmids from Gram-positive bacteria. Moreover, sequences typical of theta replication origins were not found in the nucleotide sequence of pBAW301. These data suggest that this plasmid either replicates by an as yet undescribed mechanism, or represents a new class of theta replicating plasmids.     

A novel enzyme from bovine neurointermediate pituitary catalyzes dealkylation of alpha-hydroxyglycine derivatives, thereby functioning sequentially with peptidylglycine alpha-amidating monooxygenase in peptide amidation

We report here the isolation of a novel enzyme from bovine neurointermediate pituitary which catalyzes the conversion of alpha-hydroxybenzoylglycine to benzamide. This enzyme, termed HGAD (alpha-hydroxyglycine amidating dealkylase), is a soluble protein with an apparent molecular mass of 45 kDa and no apparent cofactor requirement. Addition of HGAD to purified neurointermediate pituitary PAM (peptidylglycine alpha-amidating monooxygenase, EC 1.14.17.3) increases the rate of formation of amide products by an order of magnitude. Sequential additions of PAM and HGAD gave results consistent with PAM first catalyzing the formation of an intermediate that is subsequently, in a separate reaction, converted by HGAD to the final amide product. Experiments with olefinic inactivators demonstrate that HGAD is not required for turnover-dependent inactivation of PAM and, correspondingly, that HGAD activity is not affected by inactivators of PAM. As expected, HGAD has no effect on the rate of PAM-catalyzed sulfoxidation, where a reaction analogous to that occurring during amidation of glycine-extended substrates is not possible. On the basis of these results, we propose that peptide C-terminal amidation in neurointermediate pituitary is a two-step process, with PAM first catalyzing the conversion of a glycine-extended peptide to the alpha-hydroxyglycine derivative, which is in turn converted to the final amide product by HGAD.     

Study of enteric pathogens among children in the tropics and effects of prolonged storage of stool samples

The study was performed to compare real-time PCR after nucleic acid extraction directly from stool samples as well as from samples stored and transported on Whatman papers or flocked swabs at ambient temperature in the tropics. In addition, the possible suitability for a clear determination of likely aetiological relevance of PCR-based pathogen detections based on cycle threshold (Ct) values was assessed. From 632 Tanzanian children <5 years of age with and without gastrointestinal symptoms, 466 samples were subjected to nucleic acid extraction and real-time PCR for gastrointestinal viral, bacterial and protozoan pathogens. Equal or even higher frequencies of pathogen detections from Whatman papers or flocked swabs were achieved compared with nucleic acid extraction directly from stool samples. Comparison of the Ct values showed no significant difference according to the nucleic acid extraction strategy. Also, the Ct values did not allow a decision whether a detected pathogen was associated with gastrointestinal symptoms.     

Generation of Mouse Small Intestinal Epithelial Cell Lines That Allow the Analysis of Specific Innate Immune Functions

Cell lines derived from the small intestine that reflect authentic properties of the originating intestinal epithelium are of high value for studies on mucosal immunology and host microbial homeostasis. A novel immortalization procedure was applied to generate continuously proliferating cell lines from murine E19 embryonic small intestinal tissue. The obtained cell lines form a tight and polarized epithelial cell layer, display characteristic tight junction, microvilli and surface protein expression and generate increasing transepithelial electrical resistance during in vitro culture. Significant up-regulation of Cxcl2 and Cxcl5 chemokine expression upon exposure to defined microbial innate immune stimuli and endogenous cytokines is observed. Cell lines were also generated from a transgenic interferon reporter (Mx2-Luciferase) mouse, allowing reporter technology-based quantification of the cellular response to type I and III interferon. Thus, the newly created cell lines mimic properties of the natural epithelium and can be used for diverse studies including testing of the absorption of drug candidates. The reproducibility of the method to create such cell lines from wild type and transgenic mice provides a new tool to study molecular and cellular processes of the epithelial barrier.