Cystathionine beta-lyase is important for virulence of Salmonella enterica serovar Typhimurium

The biosynthesis of methionine in bacteria requires the mobilization of sulfur from Cys by the formation and degradation of cystathionine. Cystathionine beta-lyase, encoded by metC in bacteria and STR3 in Schizosaccharomyces pombe, catalyzes the breakdown of cystathionine to homocysteine, the penultimate step in methionine biosynthesis. This enzyme has been suggested to be the target for pyridinamine antimicrobial agents. We have demonstrated, by using purified enzymes from bacteria and yeast, that cystathionine beta-lyase is not the likely target of these agents. Nonetheless, an insertional inactivation of metC in Salmonella enterica serovar Typhimurium resulted in the attenuation of virulence in a mouse model of systemic infection. This result confirms a previous chemical validation of the Met biosynthetic pathway as a target for the development of antibacterial agents and demonstrates that cystathionine beta-lyase is important for bacterial virulence.     

Stable micro-dystrophin gene transfer using an integrating adeno-retroviral hybrid vector ameliorates the dystrophic pathology in mdx mouse muscle

The ability to transfer the dystrophin gene stably to the skeletal muscle of DMD patients is a major confounding issue in establishing an effective gene therapy for this disease. To overcome this problem, we have examined the ability of muscle fibres from mdx mice to act as in situ factories of retroviral vector production. Tibialis anterior (TA) muscles from 4-week-old mdx mice were injected with an adenoviral vector expressing LacZ within a retroviral expression cassette (AdLZIN). Retroviral vector production was induced by the inclusion of two additional adenoviral vectors expressing retroviral gag-pol (AdGagPol) and 10A1 env genes (Ad10A1). Upon introduction of infected muscles into cell culture, colonies of beta-galactosidase-expressing myotubes formed only in cultures where the muscle was injected with AdLZIN, AdGagPol and Ad10A1, but not from muscle injected with AdLZIN only. Muscles from mdx/nude mice producing retroviral vector displayed a 4.6-fold increase in beta-galactosidase-positive myofibres after 1 month, compared with contralateral muscle in the same animal injected with AdLZIN and AdGagPol only. By constructing a hybrid adeno-retroviral vector expressing a truncated micro-dystrophin construct (AdmicroDyIN), we were able to partially correct the mdx dystrophic phenotype. AdmicroDyIN-mediated expression of micro-dystrophin in mdx TA muscle restored the formation of the dystrophin-associated glycoprotein complex and significantly reduced the level of muscle degeneration over uninjected controls. By stimulating in situ production of retroviral vector expressing micro-dystrophin, we achieved 92%+/-6% transduction of myofibres in the TA muscle by 4 weeks. Strikingly, by 3 months post injection, micro-dystrophin was still expressed to high levels in nearly all the myofibres of the TA muscle. By comparison, there was a pronounced drop in the levels of micro-dystrophin expressed by muscles injected with AdmicroDyIN only. Finally, using a novel PCR approach, we detected reverse-transcribed, integrated proviral sequences in TA muscle genomic DNA by 4 weeks post injection, the levels of which were found to increase after 3 months.     

Interaction of toxic venoms with the complement system

Thirty-nine venoms from various vertebrate and invertebrate species were tested for their ability to consume haemolytic complement (C) activity upon incubation in fresh guinea-pig serum. Nineteen had `anti-complementary' activity, and these were provisionally sorted into the following groups: Pattern I—exemplified by the Naja haje (Egyptian cobra) and six other Elapidae species (all cobras), which induced selective consumption of C3—C9, and led to formation of a stable C3—C9-consuming intermediate; Pattern II—exemplified by the Agkistrodon rhodostoma (Malayan pit viper), Bitis arietans (puff adder), Bothrops jararaca (South American pit viper), Bothrops atrox (Fer de Lance) and three other species, which induced marked consumption of C4 and C2, as well as C3—C9, but did not form a stable C3—C9-consuming intermediate; and individual animals, e.g. the Lachesis muta (bushmaster), which induced other patterns (III—VI) of complement component consumption. Active fractions of representative venoms were partially purified by ion exchange and gel filtration chromatography and their interactions with the complement system characterized further. It is anticipated that these enzymes, with a capacity to activate the complement system in unique ways, will prove to be of further experimental usefulness.     

Evaluation of human knee meniscus biopsies with near-infrared, reflectance confocal microscopy. A pilot study

Knee cartilage biopsy is used to confirm the pathology in both clinical and experimental conditions and often guides diagnosis and therapeutic strategies. Current histopathological techniques are time consuming, induce tissue artefacts and often prevent further evaluation, once the tissue has been fixed. Hence, there is a potential need for a fast and nondestructive imaging technique for unfixed tissue. Near-infrared, reflectance confocal microscopy (CM) allows real-time, virtual sectioning of unstained, bulk tissue samples. This pilot study evaluates the use of CM in the assessment of meniscus histopathology in a series of 26 freshly-excised human meniscus samples compared to standard light microscopy of stained sections. CM images of the meniscus show cell and matrix detail, depicting morphologic features of collagen and elastic fibres, vessels and nerve endings. In addition, crystal deposits of gout and pseudogout are also demonstrable. Thus, CM is a novel imaging technique that could enable the pathologist to make a rapid microscopic evaluation of cartilage in a fresh and unfixed fashion.     

Motives for the adoption of protective health behaviours for men and women: an evaluation of the psychosocial-appraisal health model

The present study has been designed to: (1) evaluate the adequacy of a psychosocial-appraisal health model posited to explain the decision-making processes associated with the intention as well as the (non)practice of protective behaviours; and (2) test for sex differences in the hypothesized structural relations (both direct and indirect) between the model's exogenous (psychosocial) and endogenous (cognitive appraisal) factors in representing the way health decisions are made. Results obtained from path analysis (N=1269) provided overall support for the 'fit' of the hypothesized health model. The implications of the findings with regard to differences in male and female health status are discussed.     

Striking a balance: inter-kingdom cell-to-cell signaling,friendship or war?

There is a growing body of evidence suggesting that bacterial signaling molecules are involved in cross-kingdom communication with eukaryotic cells. Bacterial signaling molecules can enter eukaryotic cells and appear to modulate immune responses to bacterial pathogens. Recently, an enticing study demonstrated that mammalian airway epithelial cells have the ability to inactivate these bacterial signaling molecules, suggesting that this activity might have a role in the host innate response against bacterial infections.     

Interleukin-4 and interleukin-4 soluble receptor alpha levels in bronchoalveolar lavage from children with asthma.

BACKGROUND: In asthma there is increased expression of the Th2-type cytokine interleukin-4 (IL-4). IL-4 is important in immunoglobulin isotype switching to immunoglobulin E and adhesion of eosinophils to endothelium. OBJECTIVE: We hypothesized that levels of IL-4 in bronchoalveolar lavage (BAL) fluid would be increased in stable, atopic asthmatic children compared with controls and that levels of its physiologic inhibitor IL-4 soluble receptor alpha (IL-4sR alpha) would be correspondingly decreased. METHODS: One hundred sixteen children attending a children's hospital for elective surgery were recruited. A nonbronchoscopic BAL was performed, and IL-4 and IL-4sR alpha were measured in the BAL supernatants. RESULTS: There was no significant difference in IL-4 concentrations between atopic asthmatic children, atopic normal controls, and nonatopic normal controls [0.13 pg/mL (0.13 to 0.87) vs 0.13 pg/mL (0.13 to 0.41) vs 0.13 pg/mL (0.13 to 0.5), P = 0.65]. IL-4sR alpha levels were significantly increased in asthmatic patients compared with atopic controls [6.4 pg/mL (5.0 to 25.5) vs 5.0 pg/mL (5.0 to 9.9), P = 0.018], but not when compared with the nonatopic controls [5.2 pg/mL (5.0 to 10.6), P = 0.19]. CONCLUSIONS: Contrary to expectation, IL-4sR alpha levels are increased in BAL from stable asthmatic children compared with nonatopic controls, and we speculate that IL-4sR alpha is released by inflammatory cells in the airways to limit the proinflammatory effects of IL-4.