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.     

Life stress and lymphocyte alterations among patients with rheumatoid arthritis.

Studied the relation between life stress and immune parameters for 33 30–70 yr old female rheumatoid arthritis (RA) patients interviewed during 3 routine monthly clinic checkups. Life stress from major and minor events, coping efficacy, and self-reported psychological distress were assessed, and immunofluorescence of T-cells and B-cells was performed on the blood drawn during each visit. Small stressful events were positively related to the proportion of circulating B-cells, psychological distress was inversely related to proportion of circulating T-cells, and major life events were associated with lower T-helper/T-suppressor cell ratios. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

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.     

Scattering by spherically stratified microwave lens antennas

A comprehensive treatment of scattering by sources in the region of a spherically stratified object composed of discrete shells is presented and compared with measured results. The work encompasses the developments necessary for a thorough understanding of the focusing mechanism of a spherical lens. The method used is a modal technique that employs separation of variables in order to determine an eigenfunction representation of the incident and scattered fields. This formulation provides the Green's function required for the calculation of the scattering from a multiple layered sphere excited by an infinitesimal current element. The algorithm allows both near- and far-field predictions. The scattering by more complicated source field distributions is modeled by applying superposition with the appropriate coordinate transformations. From a practical standpoint, the present analysis allows the limitations of spherical lenses to be determined while providing the information required to optimize the lens design. A system was built based on the new design data, and its measured radiation characteristics are compared to the theoretical predictions. Excellent agreement is observed in most cases     

Sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to the human immunodeficiency virus type 1 Vpu protein

CD4 is an integral membrane glycoprotein which functions as the human immunodeficiency virus receptor for infection of human host cells. We have recently demonstrated that Vpu, a human immunodeficiency virus type 1-encoded integral membrane phosphoprotein, induces rapid degradation of CD4 in the endoplasmic reticulum. Using an in vitro model system, we demonstrated that Vpu targets specific sequences in the cytoplasmic domain of CD4 to promote its degradation. In this report, we have further delineated regions within CD4 which are required for susceptibility to Vpu. Transfer of the CD4 cytoplasmic region into a heterologous protein, CD8, rendered the chimeric protein sensitive to Vpu-dependent degradation. In contrast, substitution of the CD8 transmembrane domain with the analogous region from CD4 did not confer sensitivity to Vpu. Finally, mutant forms of the CD4 protein containing the extracellular region alone or the extracellular and transmembrane regions linked to a heterologous cytoplasmic domain were not targeted by Vpu. Thus, sequences present in the cytoplasmic domain of CD4 are necessary and sufficient to confer sensitivity to Vpu.