Genetic complementation between two mutant unc alleles (unc A401 and unc D409) affecting the Fl portion of the magnesium ion-stimulated adenosine triphosphatase of Escherichia coli K12.

A new mutant strain of Escherichia coli in which phosphorylation is uncoupled from electron transport was isolated. A genetic-complementation analysis, using partial diploid strains, showed that the new mutant allele, uncD409, is in a gene distinct from the other previously identified genes uncA, uncB and uncC. A strain carrying the uncd409 allele has no Mg2+ ion-stimulated adenosine triphosphatase activity and is therefore phenotypically similar to strains carrying the uncA401 mutant allele. Complementation between the uncA401 and the uncD409 alleles occurred, as indicated by growth of partial diploid strains on succinate and their growth yields on limiting concentrations of glucose. Complementation was confirmed by using membranes prepared from the above partial diploids. Such membranes were found to have Mg2+-stimulated adenosine triphosphatase activity, ATP-dependent transhydrogenase activity ADP-induced atebrin-fluorescence quenching and low but significant amounts of oxidative phosphorylation.     

Studies of the antigenicity and immunogenicity of bromelain-pretreated red blood cells

The effects of the proteolytic enzyme bromelain (Br) on the antigenicity and immunogenicity of sheep and mouse red blood cells (RBC) have been investigated. The results presented support the previous claim that there are antigens present on Br RBC that are not present in an exposed form on untreated RBC and that Br RBC have lost some of the antigens present on the surface of normal RBC. The susceptibility of Br RBC to osmotic lysis was very similar to that of normal RBC, implying that the modified RBC were not more fragile than normal RBC. Injection of mice with Br mouse-RBC did not increase the unusually high "background" number of cells producing IgM antibodies against Br mouse-RBC and mice did not mount delayed-type hypersensitivity reactions against Br mouse-RBC, either before or after sensitizing injections of Br mouse-RBC. However, mouse-RBC and Br mouse-RBC elicited similar antibody responses in rabbits and guinea pigs. Although mice appeared unresponsive to Br mouse-RBC injections, delayed-type hypersensitivity responses and antibody production in primary and secondary responses were of similar levels irrespective of whether sheep-RBC or Br sheep-RBC were used as immunogens. From these studies it appears that mice have B-cells producing antibodies against the "new" antigens on Br mouse-RBC, but there are no T-cells that respond to these antigens by way of "helper" activity in antibody production or by way of cell-mediated immune reactions.     

The migration of lymphocytes across specialized vascular endothelium. IV. Prednisolone acts at several points on the recirculation pathways of lymphocytes

Radioactively labelled thoracic duct lymphocytes from syngeneic rat donors were injected iv into recipients which had been given a continuous iv infusion of prednisolone at 1 mg/hr for 15–18 hr previously. The tissue distribution and recirculation into lymph of the labelled lymphocytes were compared quantitatively in the prednisolone-treated and control recipients by scintillation counting and autoradiography. The most prominent effect of prednisolone was to retard recirculating lymphocytes within the tissues to which they are normally distributed by the blood, namely the bone marrow, the spleen, and the lymph nodes. Although lymphocyte traffic was almost completely frozen by prednisolone, recirculating lymphocytes were not killed. A second effect of prednisolone was to impair the influx of lymphocytes from the blood into lymph nodes. Different groups of lymph nodes varied in the extent to which prednisolone inhibited the entry of lymphocytes, and previous antigenic stimulation completely exempted lymph nodes from this inhibition. Lymphocytes took a longer time to cross the walls of high endothelial venules in the lymph nodes of prednisolone-treated rats. A third effect of prednisolone was to increase the rate at which lymphocytes entered the bone marrow from the blood by crossing sinusoidal endothelium.     

Ig H chain variable and C region genes in common variable immunodeficiency. Characterization of two new deletion haplotypes.

Common variable immunodeficiency, a disorder characterized by diminished antibody production, manifests clinically as an increased susceptibility to bacterial infections. We have investigated the Ig H chain V and C region gene segments in 33 patients with common variable immunodeficiency, to identify the possible role these genes may have in the molecular basis of the defect. No major deletions were recognized for the VH gene segments of the VH2, VH5, and VH6 families, nor were there any differences in the RFLP patterns of mu- or alpha- switch regions or of C gamma genes. Two new deletion haplotypes were identified for the C region genes, the first encompassing C gamma 1 on a different haplotype from the C gamma 1 deletion described previously, and the second a novel deletion encompassing both C gamma 2 and C gamma 4. Based on these and previously described deletions in the IGHC region, we postulate that homologous regions are involved in the deletion process and that other new deletions likely exist in the population.     

Crystallization and preliminary X-ray investigation of a sarcoplasmic calcium-binding protein from amphioxus

Crystals of a sarcoplasmic Ca(2+)-binding protein from the protochordate amphioxus have been grown from solutions of ammonium sulfate. The crystals are orthorhombic, space group C222(1), with unit cell axes a = 59.6(1) A, b = 81.3(1) A and c = 82.4(1) A. There is one molecule in the asymmetric unit. The crystals diffract beyond 2.5 A and show less than 20% decline in diffraction intensities after a three day exposure to X-rays from a laboratory rotating anode source.     

Development of an enzyme immunoassay for the detection of Clostridium novyi type B alpha toxin

Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis. The toxin was toxoided and used to produce a pair of non-interfering monoclonal antibodies. Their specificity was confirmed by immunoblotting and bioassay. The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format. Use of the assay can eliminate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological activity are not required. Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoiding and vaccine formulation.