DNA-Launched Alphavirus Replicons Encoding a Fusion of Mycobacterial Antigens Acr and Ag85B Are Immunogenic and Protective in a Murine Model of TB Infection

There is an urgent need for effective prophylactic measures against Mycobacterium tuberculosis (Mtb) infection, particularly given the highly variable efficacy of Bacille Calmette-Guerin (BCG), the only licensed vaccine against tuberculosis (TB). Most studies indicate that cell-mediated immune responses involving both CD4+ and CD8+ T cells are necessary for effective immunity against Mtb. Genetic vaccination induces humoral and cellular immune responses, including CD4+ and CD8+ T-cell responses, against a variety of bacterial, viral, parasitic and tumor antigens, and this strategy may therefore hold promise for the development of more effective TB vaccines. Novel formulations and delivery strategies to improve the immunogenicity of DNA-based vaccines have recently been evaluated, and have shown varying degrees of success. In the present study, we evaluated DNA-launched Venezuelan equine encephalitis replicons (Vrep) encoding a novel fusion of the mycobacterial antigens α-crystallin (Acr) and antigen 85B (Ag85B), termed Vrep-Acr/Ag85B, for their immunogenicity and protective efficacy in a murine model of pulmonary TB. Vrep-Acr/Ag85B generated antigen-specific CD4+ and CD8+ T cell responses that persisted for at least 10 wk post-immunization. Interestingly, parenterally administered Vrep-Acr/Ag85B also induced T cell responses in the lung tissues, the primary site of infection, and inhibited bacterial growth in both the lungs and spleens following aerosol challenge with Mtb. DNA-launched Vrep may, therefore, represent an effective approach to the development of gene-based vaccines against TB, particularly as components of heterologous prime-boost strategies or as BCG boosters.     

Performance of Two Fruit Fly (Diptera: Tephritidae) Pupal Parasitoids (Coptera haywardi [Hymenoptera: Diapriidae] and Pachycrepoideus vindemiae [Hymenoptera: Pteromalidae]) under Different Environmental Soil Conditions

We evaluated the performance of Coptera haywardi (Ogloblin) (Diapriidae) and Pachycrepoideus vindemiae (Rondani) (Pteromalidae), both hymenopteran pupal parasitoids of Anastrepha spp. (Diptera: Tephritidae). Performance was studied by manipulating the following environmental conditions in the laboratory: (1) soil type, (2) soil moisture content, (3) soil compaction, and (4) depth at which pupae were buried in the soil. There were two experiments: in the first, exposure time of pupae was held constant and in the second, it varied. In the first experiment, C. haywardi was significantly more effective than P. vindemiae in parasitizing fly pupae. With exposure time held constant (36 h), only soil type and pupal burial depth were significantly related to parasitism rates. While P. vindemiae only parasitized pupae located on the soil surface, C. haywardi attacked pupae that were buried up to 5 cm deep, performing better in clayey than in loamy soil. In the second experiment, exposure time (24, 36, 48, and 72 h) had no significant effect on parasitism rates, but soil type did. P. vindemiae again only attacked pupae on the soil surface while C. haywardi was also able to parasitize pupae that were buried up to 5 cm deep. We conclude that C. haywardi represents a viable candidate to replace the environmentally unfriendly P. vindemiae in augmentative biological control programs against fruit flies.     

Stopped-flow studies on the mechanism of oxidation of N-methyl-4-phenyltetrahydropyridine by bovine liver monoamine oxidase B

The kinetic mechanism of monoamine oxidase B involves either a binary or a ternary complex, depending on the substrate. In this study, stopped-flow kinetic data provide direct evidence for ternary complexes not only of reduced enzyme, oxygen, and product but also of reduced enzyme, oxygen, and substrate, both for benzylamine and for the tertiary amine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the mechanism for a given substrate is not exclusive but, rather, is determined by competition between the alternate pathways as a result of different rate constants for the oxidation of the reduced enzyme, the reduced enzyme-product complex, and the reduced enzyme-substrate complex, as well as the different dissociation constants for the complexes. Comparison of the rate constants obtained from the stopped-flow studies with steady-state data indicates that the overall rate of reaction for the oxidation of MPTP by monoamine oxidase is dominated by the reductive step, but for benzylamine the steady-state rate is determined by a complex function of the rates of both the reductive and oxidative half-reactions.     

Production of Poly-β-Hydroxyalkanoic Acid by Pseudomonas cepacia

The possibility of using the nutritionally versatile bacterium Pseudomonas cepacia to produce poly-β-hydroxyalkanoic acid was evaluated. Chemostat culture showed that growth of P. cepacia became nitrogen limited when the molar carbon-to-nitrogen ratio of the medium fed into the fermentor was above 15. When grown under nitrogen limitation in batch culture with fructose as the sole source of carbon, P. cepacia accumulated poly-β-hydroxybutyric acid (PHB) in excess of 50% of the dry weight of its biomass. In batch culture, almost no PHB was produced until the onset of nitrogen limitation. After this point, PHB was produced at a linear rate of 0.12 g liter⁻¹ h⁻¹ (from a constant value of 1.6 g of cellular protein liter⁻¹). PHB produced by P. cepacia had a weight-average molecular weight of 5.37 × 10⁵ g mol⁻¹ and a polydispersivity index of 3.9. Poly(β-hydroxybutyric acid-β-hydroxyvaleric acid) copolymer was produced with a poly-β-hydroxybutyric acid-poly-β-hydroxyvaleric acid ratio of up to 30% by weight when propionic acid was added to the medium.     

Cystic fibrosis typing with DNA probes and screening for ΔF508 deletion in families from Southern France

Summary A sample of 235 individuals from 49 French cystic fibrosis (CF) families with at least one living affected child was typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene, and was screened for the ΔF508 mutation. Using a combination of six probes, 44 out of the 49 families were sufficiently informative to enable prenatal diagnosis or carrier determination. As in many other populations, linkage disequilibrium was found between the CF locus and the haplotype B (XV2c: allele 1; KM19: allele 2), which accounts for about 78% of CF chromosomes in our families. The ΔF508 deletion was present in 64.3% of CF chromosomes.