Production of the 19-kDa antigen of Mycobacterium tuberculosis in Escherichia coli and its purification

The 19-kDa antigen (19Ag) of Mycobacterium tuberculosis (Mt) is a lipoprotein which is released from the organism during growth. In order to study the possible involvement of this antigen in the host protective response against Mt infection, it would be helpful to obtain high-level production of 19Ag from a recombinant organism. We have found that overexpression of the native 19Ag gene in Escherichia coli or yeast leads to products which are aggregated and insoluble. By site-directed mutagenesis of the 19Ag lipoprotein leader sequence, we have generated a mutant gene which directs the production of 19Ag into the periplasmic space of E. coli, from where it can be easily purified in high yield. 19Ag obtained from this mutant construct lacks the lipid-modified N-terminal Cys residue found in the native 19Ag, and is not glycosylated, but is otherwise indistinguishable from 19Ag isolated from Mt culture supernatant.     

Nuclear fuel considerations for the 21 century

There are many external influences that may control the path that nuclear power deployment follows. In the next 50 years several events may unfold. Fear of the consequences of the greenhouse effect may produce a carbon tax that would make nuclear power economically superior very quickly. This, in turn, would increase the rate at which uranium reserves diminish due to the increased rate of nuclear power deployment. However, breakthroughs in the extraction of uranium from the sea or deployment of fast breeder reactors would greatly extend the uranium reserves and, as well, utilize the thorium cycle.On the other hand, carbon sequestering technology breakthroughs could keep fossil fuels dominant for the remainder of the century. Nuclear power may only then continue, as today, in a lesser role or even diminish. Fusion power or new developments in solar power could completely displace nuclear power as we know it today.Even more difficult to predict is when the demand for mobile fuel for transportation will develop such that hydrogen and hydrogen rich fuel cells will be in common use. When this happens, nuclear power may be the energy source of choice to produce this fuel from water or methane. In a similar vein, the demand for potable and irrigation water may be another driver for the advent of increased deployment of nuclear power.With all these possibilities of events that could happen it appears impossible to predict with any certainty which path nuclear power deployment may take. However, it is necessary to define a strategy that is flexible enough to insure that when a technology is needed, it is ready to be deployed.For the next few decades there will be an evolutionary improvement in the performance of uranium oxide and mixed uranium oxide-plutonium oxide (MOX) LWR fuels. These improvements will be market driven to keep the cost of fuel and the resulting cost of nuclear power electricity as competitive as possible. The development of fuels for accelerator transmutation and for reactor transmutation with inert matrix fuels is in its infancy. A great deal of research has been initiated in a number of countries, which has been summarized in recent conferences. In Europe the work on these fuels is directed at the same problem as their utilization of MOX; namely to reduce the inventory of separated plutonium, minor actinides, and Long Lived Fission Products (LLFP). In the United States there is no reprocessing and thus no inventory of separated civilian plutonium. However, in the United States there is a resistance to a permanent spent fuel repository and thus accelerator transmutation presents a possible alternative. If nuclear power does have a long-term future, then the introduction of the fast reactor is inevitable. Included in the mission of the fast reactors would be the elimination of the inventory of separated plutonium while generating useful energy. The work that is ongoing now on the development of fuel concepts for assemblies that contain actinides and LLFP would be useful for fast reactor transmutation.There is still a great deal of work required to bring the fast breeder reactor option to maturity. Fortunately there is perhaps a fifty-year period to accomplish this work before fast breeders are necessary. With regard to fast reactor fuel development, future work should be considered in three stages. First, all the information obtained over the past forty years of fast reactor fuel development should be completely documented in a manner that future generations can readily retrieve and utilize the information. Fast reactor development came to such an abrupt halt world-wide that a great deal of information is in danger of being lost because most of the researchers and facilities are rapidly disappearing. Secondly, for all of the existing fast reactor fuels, and this includes, oxides, carbides, nitrides, and metallic fuels, the evolutionary work was far from being completed. Although mixed oxide fuels were probably the furthest advanced, there were many concepts for improved claddings and advanced fabrication methods that were never fully explored. Finally, with such an extended period before fast reactors are needed there is ample time for truly innovative fuels to be developed that are capable of performing over a wide range of conditions and coolants.     

Evaluation of humoral immune responses in cattle grazing endophyte-infected or endophyte-free fescue

Anecdotal reports suggest cattle with fescue toxicosis may not respond to vaccination and thus, experience increased incidence of Bovine Respiratory Disease Complex (BRDC) when shipped to feedlots. Fescue toxicosis causes hypoprolactemia in cattle. Hypoprolactemia decreases humoral immune responses in mice. Therefore, a study was conducted to compare the magnitude of primary and secondary humoral immune responses against specific antigens in cattle grazing endophyte-infected or endophyte-free fescue. Angus steers were blocked by weight and allocated into four groups. Two groups grazed endophyte-infected (EI) fescue and the other two groups grazed endophyte-free (EF) fescue. All steers were injected IM on d 0 and 21 with lysozyme without adjuvant and concanavalin. A (Con A) with sheep red blood cells (SRBC) in incomplete adjuvant of Freund. Steers were bled on days 0, 21 and 35 post-vaccination. Average daily gains (ADG), alkaline phosphatase (ALP) activity, cholesterol concentrations, rectal temperatures, and serum prolactin concentrations were measured to confirm fescue toxicosis in steers grazing EI fescue. Antibodies to Con A and SRBC were determined by ELISA and hemagglutination assay, respectively. The ADG were decreased for the EI group during the first month. Rectal temperature were elevated and serum prolactin concentrations were decreased in the EI group. Cholesterol and ALP concentrations also were decreased in the EI group. Primary and secondary immune responses against Con A tended to be increased and were increased against SRBC in the EI group. Antibodies against lysozyme were not induced in either group. In conclusion, cattle grazing EI fescue mounted similar humoral immune responses to vaccination, despite hypoprolactemia, as cattle grazing EF fescue. Increases in bovine respiratory disease in cattle maintained on EI fescue probably is not associated with lack of humoral immune response to vaccination protocols as a result of fescue toxicosis.     

Inhibition of sickle beta-chain (betaS)-dependent polymerization by nonhuman alpha-chains. A superinhibitory mouse-horse chimeric alpha-chain

Horse alpha-chain inhibits sickle beta-chain-dependent polymerization; however, its inhibitory potential is not as high as that of mouse alpha-chain. Horse alpha-(1-30) and alpha-(31-141) segments make, respectively, minor and major contributions to the inhibitory potential of horse alpha-chain. The sum of the inhibitory potential of the two segments does not account for the inhibitory potential of the full-length horse alpha-chain. Although the polymerization inhibitory potential of horse alpha-chain is lower than mouse alpha-chain, the inhibitory potential of horse alpha-(31-141) is comparable to that of mouse alpha-(31-141). When mouse alpha-(1-30) is stitched to horse alpha-(31-141), the product is a chimeric alpha-chain with an inhibitory potential greater than mouse alpha-chain. In contrast, the stitching of horse alpha-(1-30) with mouse alpha-(31-141) had no additional inhibitory potential. Molecular modeling studies of HbS containing the mouse-horse chimeric alpha-chain indicate altered side-chain interactions at the alpha1beta1 interface when compared with HbS. In addition, the AB/GH corner perturbations facilitate a different stereochemistry for the interaction of the epsilon-amino group of Lys-16(alpha) with the beta-carboxyl group of Asp-116(alpha), resulting in a decrease in the accessibility of the side chain of Lys-16(alpha) to the solvent. Based on molecular modeling, we speculate that these perturbations by themselves, or in synergy with the altered conformational aspects of the alpha1beta1 interactions, represent the molecular basis of the superinhibitory potential of the mouse-horse chimeric alpha-chains.