A new mycobactin,mycobactin J,fromMycobacterium paratuberculosis

This paper reports the production, purification, and biological assay of a new mycobactin fromMycobacterium paratuberculosis. This new mycobactin, designated mycobactin J, is produced by a strain ofM. paratuberculosis adapted to growth on synthetic medium without exogenous mycobactin. Mycobactin J reduces the incubation period ofM. paratuberculosis in complex medium by 3 weeks when compared with medium containing mycobactin P, and a higher proportion of the organisms, in inocula from infected tissues or fecal specimens, produce colonies. Also, some strains ofM. paratuberculosis will grow on medium containing mycobactin J that will not grow on medium containing mycobactin P.     

Structure of mycobactin J

Mycobactin J-1, an iron chelate fromMycobacterium paratuberculosis, was characterized by mass spectrum and by¹H nuclear magnetic resonance (NMR) and¹³C NMR spectra of the parent molecule and of cobactin J-1. The core structure of mycobactin J-1 contained the phenyloxazoline ring system common to the mycobactins. The benzene ring was disubstituted. The two hydroxamate functions were furnished by 1 linear 6-N-hydroxylysine residue and 1 cyclic 6-N-hydroxylysine residue as in other members of this class of compounds. The acyl function at the mycobactic acid hydroxamate center wasn-cis-hexadec-2-enoyl. The hydroxyacid of the cobactin portion of mycobactin J-1 was 2,4-dimethyl-3-hydroxypentanoic acid. This latter residue differs from those of other known mycobactins by the presence of the isopropyl group.     

The Emerging Role of Copeptin

Direct measurement of the nonapeptide vasopressin has been limited by analyte instability ex vivo and in vivo rapid degradation, low serum concentrations requiring a sensitive assay and inherent secretory pulsatility. Copeptin is a 39 amino acid glycopeptide cleavage product of vasopressin synthesis with high stability, providing a marker of vasopressin secretion. Copeptin measurement has applications in diagnosis of diabetes insipidus and other diseases with altered vasopressin secretion. This review summarises our current understanding of serum copeptin measurement in diabetes insipidus and possible future applications of copeptin assays. As vasopressin is a stress hormone, there is emerging evidence on the use of copeptin for diagnosis and prognostication of disorders such as syndrome of inappropriate anti-diuretic hormone secretion, diabetes mellitus, critical illness, stroke, cardiovascular disease, respiratory disease, renal disease and thermal stress. Copeptin concentration measurement is likely to improve the diagnostic reliability of diabetes insipidus and, as a marker of stress, may have diagnostic or prognostic utility in specific clinical circumstances. Further studies are needed to determine if goal-directed therapy using plasma copeptin concentrations may improve patient outcomes.     

Serum leptin concentration in pigs selected for high or low daily food intake

Selection for high or low daily food intake (DFI) in Large White pigs resulted in higher serum leptin concentration, fat deposition and food intake in the high DFI line. The response in serum leptin concentration indicated that the higher fat deposition of the high DFI line was not due to insufficient leptin production, as in the Lepob/Lepob mouse. Serum leptin was more highly correlated with fat deposition than with food intake indicating that the response in serum leptin was primarily due to increased fat deposition rather than to higher energy intake per se. The low correlations between serum leptin measured at 30 kg and performance test traits indicate that serum leptin would not be efficient for selection of animals prior to performance test. However, the consistent positive correlations between serum leptin and a measure of fat deposition suggest that serum leptin could usefully be incorporated in selection criteria for genetic improvement of carcass lean content in pigs.     

Requirements engineering: the key to designing complex medical systems

A variety of business systems, clinical work systems, instrumentation systems, information systems, infrastructure systems, and management systems interact to make the modern healthcare facility work. The key to designing for such a system is systems engineering, a skill often little appreciated among clinical engineers. At the heart of systems engineering is requirements engineering and management (REAM), which is defined as "the process of discovering, documenting and managing systems requirements." The principal activities of REAM include eliciting, understanding, negotiating, describing, validating, and managing system requirements. When REAM is done improperly, the resulting system will be satisfactory only if chance intervenes. Well-done REAM is likely to bring the project in on time, under budget, and at full performance.     

Composition of in vitro denture plaque biofilms and susceptibility to antifungals

The aim of this study was to investigate the composition of microcosm denture plaque biofilms and the susceptibility of Candida spp. within these biofilms to antifungal agents. An in vitro model was employed to grow oral biofilms derived from denture associated stomatitis (DAS) patient samples to assess fungal growth in the presence and absence of antifungal agents. The compositions of genera present in vitro were found to be similar to those exhibited on the mucosa and denture fitting surfaces of DAS samples. Exposure to single agents, e.g., miconazole, fluconazole or chlorhexidine did not inhibit growth of Candida spp. when used in clinically relevant doses. Combinations of miconazole and chlorhexidine, pulsed into the system to mimic patient use, did reduce bacterial and candidal growth for several days. Hence, the use of dual-therapy appeared to be useful in reducing the number of viable organisms within denture plaque grown in vitro although resistance to these agents was also evident.     

Double-stranded secondary structures on mRNA induce type I interferon (IFN alpha/beta) production and maturation of mRNA-transfected monocyte-derived dendritic cells

BACKGROUND: The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. Exogenously delivered mRNA can induce DC activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs. METHODS: In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using different non-viral gene transfer procedures. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labeled RNA, intracellular delivery and transfection efficiency were assessed by confocal microscopy and flow cytometry. DC activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-alpha/beta). RESULTS: mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules predicted highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand-specific RNase, but not a single-strand-specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation. CONCLUSIONS: Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.     

The reaction mechanism of DNA glycosylase/AP lyases at abasic sites

DNA glycosylase and glycosylase/abasic (AP) lyases are the enzymes responsible for initiating the base excision repair pathway by recognizing the damaged target base and catalyzing the breakage of the base-sugar glycosyl bond. The subset of glycosylases that have an associated AP lyase activity also catalyze DNA strand breakage at the resulting or preexisting AP site via a beta-elimination reaction, proceeding from an enzyme-DNA imino intermediate. Two distinct mechanisms have been proposed for the formation of this intermediate. These mechanisms essentially differ in the nature of the first bond broken and the timing of the opening of the deoxyribose ring. The data presented here demonstrate that the combined rate of sugar ring opening and reduction of the sugar is significantly slower than the rate of formation of a T4-pyrimidine dimer glycosylase (T4-pdg)-DNA intermediate. Using a methyl-deoxyribofuranose AP-site analogue that is incapable of undergoing sugar ring opening, it was demonstrated that the T4-pdg reaction can initiate at the ring-closed form, albeit at a drastically reduced rate. T4-pdg preferentially cleaved the beta-anomer of the methyl-deoxyribofuranose AP site analogue. This is consistent with a mechanism in which the methoxy group is backside-displaced by the amino group from the alpha-face of the deoxyribofuranose ring. In addition, studies examining rates of sugar-aldehyde reduction and the sodium borohydride concentration dependence of the rate of formation of the covalent imine intermediate suggest that the reduction of the intermediate is rate-limiting in the reaction.