Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Spatial,road geometric and biotic factors associated with Barn Owl mortality along an interstate highway

Highway programmes typically focus on reducing vehicle collisions with large mammals because of economic or safety reasons, while overlooking the millions of birds that die annually from traffic. We studied wildlife–vehicle collisions along an interstate highway in southern Idaho, USA, with among the highest reported rates of American Barn Owl Tyto furcata road mortality. Carcass data from systematic and ad hoc surveys conducted in 2004–2006 and 2013–2015 were used to explore the extent to which spatial, road geometric and biotic factors explained Barn Owl–vehicle collisions. Barn Owls outnumbered all other identified vertebrate species of roadkill and represented > 25% of individuals and 73.6% of road‐killed birds. At a 1‐km highway segment scale, the number of dead Barn Owls decreased with increasing numbers of human structures, cumulative length of secondary roads near the highway and width of the highway median. The number of dead Barn Owls increased with higher commercial average annual daily traffic (CAADT), small mammal abundance index and grass rather than shrubs in the roadside verge. The small mammal abundance index was also greater in roadsides with grass vs. mixed shrubs, suggesting that Barn Owls may be attracted to grassy portions of the highway with more abundant small mammals for hunting prey. When assessed at a 3‐km highway segment scale, the number of dead Barn Owls again increased, with higher CAADT as well as with greater numbers of dairy farms. At a 5‐km scale, the number of dead Barn Owls increased with a greater percentage of cropland near the highway. Although human conversion of the environment from natural shrub‐steppe to irrigated agriculture in this region of Idaho has probably enhanced habitat for Barns Owls, it simultaneously has increased risk for owl–vehicle collisions where an interstate highway traverses the altered landscape. We review some approaches for highway mitigation and suggest that reducing wildlife–vehicle collisions involving Barn Owls may contribute to the persistence of this species.     

The transition in hemoglobin proton-binding characteristics within the basal actinopterygian fishes

Carbon dioxide (CO₂) transport in the blood of fishes is aided by the proton-binding properties of hemoglobin (Hb) through either a high-intrinsic buffer value and small oxylabile proton binding (Haldane effect), or a low buffer value and large Haldane effect. Primitive species, such as elasmobranchs and sarcopterygians have been shown to rely on the former, while derived species, such as teleosts rely on the latter. Both strategies are effective in the transport of CO₂ in the blood. However, there is a paucity of information on the nature of the transition between these two strategies that appears to occur within the intermediate group of fishes, the basal actinopterygians. The objective of the present study was to simultaneously assess the intrinsic Hb buffer values and Haldane effects of species within the basal actinopterygian lineage to characterize the transition in Hb-proton-binding strategy seen among the fishes. Expressed in order of most basal to most derived, the species investigated included American paddlefish (Polyodon spathula), white sturgeon (Acipenser transmontanus), spotted gar (Lepisosteus oculatus), alligator gar (Atractosteus spatula), bowfin (Amia calva), and mooneye (Hiodon tergisus). Hemolysates from these species were prepared and Hb titrations (under oxygenated and deoxygenated conditions) were performed in both the presence and absence of saturating levels of organic phosphates (GTP). The findings suggest that the nature of the Hb-proton-binding transition may have been punctuated rather than gradual, with the Hb buffer value decreasing and the Haldane effect increasing significantly in bowfin from fairly steady ancestral levels in the four more basal species. This change is coupled with the initial appearance of the choroid rete, as well as an increase in the magnitude and onset pH of the Root effect in bowfin, suggesting that the change in Hb-proton-binding strategy may be associated with the evolution of enhanced O₂ delivery to the eye and an in vivo operational Root effect.     

Immobilized enzyme reaction stability: Attrition of the support material

One of the main reasons for immobilizing an enzyme is to enable its reuse, or continuous use, in a reactor. Consequently immobilized enzyme stability is an important factor in enzyme reactor design. The performance of the reactor will decrease if during operation the support material disintegrates into smaller particles that pass out of the reactor system. When β-galactosidase is immobilized by covalent attachment to AE-cellulose, the smaller particles have a higher activity. After subjection of the immobilized enxyme to a shear stress the average particle size decreases and the total enzymic activity increases. A loss of small particles from the reactor, although constituting a small weight percent loss of support, will result in a disproportionately large loss in activity. The relevance of these observations to reactor performance is discussed.     

Validation of an automated determination of pulmonary resistance by electrical subtraction.

An analog computer to determine dynamic pulmonary compliance (C) and pulmonary resistance (R) on a breath-by-breath basis was tested in guinea pigs and dogs. C was determined by dividing volume by transpulmonary pressure at instants of zero flow. R was determined by the method of electrical subtraction at predetermined flows. In both species the computer outputs and the results of direct analysis were in close agreement. In guinea pigs, the device reliably followed the rapid three- to fourfold changes in C and R resulting from histamine infusion. In unanesthetized dogs, the dispersion and mean values of C and R were similar by the two methods.     

Cloned murine T lymphocytes synthesize a molecule with the biological characteristics of nitric oxide

Recently, activated neutrophils and macrophages have been shown to synthesize nitric oxide (NO) exclusively from L-arginine. We searched for the presence of this pathway in murine T cell clones. Using a platelet aggregation bioassay sensitive to NO, we demonstrate that IL2-stimulated CTLL and HT2 cells inhibit platelet aggregation, whereas unstimulated lymphocytes do not. This action can be inhibited by the specific NO synthase competitor NG-mono-methyl arginine, and only the L form of arginine or its analogue L-homoarginine are capable of providing substrate for NO synthesis.     

Cryptosporidium merozoite isolation and purification using differential centrifugation techniques.

Simple modifications to a recently published merozoite purification procedure (Bjorneby et al., J. Immunol. 145:298, 1990) increased yields 3- to 5-fold. Calves were infected with 2.5 x 10(8) Cryptosporidium parvum oocysts and sacrificed 65 h post-infection. The ilium and caecum were removed. The tissue was sieved through a large strainer (2 mm2) to produce a homogeneous suspension. Red blood cells were removed by differential centrifugation (600 g); merozoites remained in the supernatant. The merozoites were pelleted (2,100 g) and washed in modified Hank's balanced salt solution deficient in Mg+2 and Ca+2. Percoll purification (density 1.070 g/ml and centrifugation speed of 22,000 g for 30 min) yielded 8 x 10(8) merozoites. Nineteen monoclonal antibodies (MAb) detected by either an enzyme-linked immunosorbent assay or an immunofluorescence assay, have been generated against the merozoite stage. Gels of proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver-stained showed that sporozoites and merozoites have many common lower molecular weight proteins. Western blots of sporozoite and merozoite antigens reacted with anti-sporozoite MAb showed several cross-reacting antigens shared by these life-cycle stages.