Activation of human and murine B cells by anti-immunoglobulin antibody: dependence of the response on the preexisting level of B-cell activation

Previous reports of the response of B lymphocytes to soluble anti-immunoglobulin (anti-Ig) antibodies have yielded conflicting data. While most studies show activation of B cells, others have shown inhibitory effects. In the assay reported in this report, we were able to obtain widely diverse responses of human B-cell populations to anti-Ig antibody. An explanation of this variability was established by resort to an animal (murine) model. Mice maintained in a pathogen-free environment failed to respond or responded only weakly to anti-Ig antibody. Mice which had previously received heavy antigenic stimulation, but at the time of the experiment were not undergoing any known challenge, showed a marked positive response. Mice deliberately challenged with lipopolysaccharide (LPS) 24 hr prior to anti-Ig antibody exposure showed a high background mitogenesis in control cultures, which was inhibited by anti-Ig antibody. This preliminary study suggests that response to anti-Ig antibody differs in each phase of B-cell differentiation. In future studies it is hoped that this variability in response can be used to characterize different subsets of B-cell differentiation separated by physical or phenotypic parameters.     

Polyethylene glycol precipitation for recovery of pathogenic viruses, including hepatitis A virus and human rotavirus, from oyster, water, and sediment samples

Polyethylene glycol 6000 precipitation was found to be an effective concentration method that enhanced the chances for detecting human virus pathogens in environmental samples. Percent recoveries from eluates of fresh and estuarine waters with 8% polyethylene glycol 6000 averaged 86 for hepatitis A virus, 77 for human rotavirus Wa, 87 for simian rotavirus SA11, and 68 for poliovirus. Percent recoveries of 97, 40, 97 and 105, respectively, for the same viruses were obtained from oyster eluates by the same procedure. Percent recoveries of 97 for hepatitis A virus and 78 for human rotavirus Wa were obtained from sediment eluates containing 2 M NaNO3 with a final concentration of 15% polyethylene glycol 6000. The polyethylene glycol method was shown to be more effective than the organic flocculation method for recovery of hepatitis A virus and rotaviruses Wa and SA11, but not of poliovirus 1 in laboratory studies. In field trials, hepatitis A virus or rotavirus or both were recovered from 12 of 18 eluates by polyethylene glycol, compared with recovery from 9 of 18 eluates by organic flocculation from fresh and estuarine waters subject to pollution.     

Inhibition of 3(17)beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni by steroidal A ring fused pyrazoles

Several 2,3- and 3,4-steroidal fused pyrazoles have been investigated as potential inhibitors of NAD(P)H-dependent steroid oxidoreductases. These compounds are proven to be potent, specific inhibitors for 3(17) beta-hydroxysteroid dehydrogenase from Pseudomonas testosteroni with Ki values of 6-100 nM. In contrast, the activities of 3 alpha,20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans, steroid 5 alpha-reductase from rat prostate, and 3 alpha-hydroxysteroid dehydrogenase from rat liver were unaffected by micromolar concentrations of these compounds. Product and dead-end inhibition studies indicate an ordered association to the beta-dehydrogenase with the cofactor binding prior to substrate or inhibitor. From the results of double inhibition experiments, it is proposed that inhibition occurs through formation of an enzyme-NAD+-inhibitor ternate. On the basis of pH profiles of Vm/Km, Vm, and 1/Ki and of absorbance difference spectra, a hypothetical mechanism of inhibition by the steroidal pyrazoles, drawn by analogy from the inhibition of liver alcohol dehydrogenase by alkylpyrazoles [Theorell, H., & Yonetani, T. (1963) Biochem. Z. 338, 537-553; Andersson, P., Kvassman, J. K., Lindstr?m, A., Oldén, B., & Pettersson, G. (1981) Eur. J. Biochem. 113, 549-554], is reconsidered. The pH studies and enzyme modification experiments by diethyl pyrocarbonate suggest the involvement of histidine in binding of the inhibitor. A modified proposal for the structure of the enzyme-NAD+-steroidal pyrazole complex is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production of cytopathology in FRhK-4 cells by BS-C-1-passaged hepatitis A virus.

Cytopathic effects were produced in fetal rhesus monkey kidney (FRhK-4) cells 7 days postinfection by a serially BS-C-1-passaged strain of hepatitis A virus. Typical enterovirus cytopathology was produced by the HM-175 strain after 15 passages at 7-day intervals in BS-C-1 cells. No cytopathic effects were obtained after neutralization of virus with human anti-hepatitis A virus immunoglobulin G. Normal human serum had no effect on development of cytopathology. Maximum antigen and cDNA probe-based hybridization activity were associated with a CsCl gradient fraction having a density of 1.34 g/cm3. Large quantities of 27- to 30-nm virions typical of hepatitis A virus were associated with the same fraction. These data led to the conclusion that the observed cytopathology was caused by hepatitis A virus.     

Endogenous lectin from cultured soybean cells. Chemical characterization of the lectin of SB-1 cells

A lectin has been identified in the cell line, SB-1, originally derived from the roots of Glycine max. This lectin, which we shall refer to as SB-1 lectin, was isolated on the basis of its carbohydrate-binding activity (affinity chromatography on Sepharose column derivatized with N-caproyl-galactosamine) and its immunological cross-reactivity (immunoblotting with rabbit antibodies directed against seed soybean agglutinin (SBA]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of SB-1 lectin revealed a major polypeptide (Mr approximately equal to 30,000) which co-migrated with seed SBA. This form of the lectin was observed in fractions purified from culture medium of SB-1 cells or supernatant fraction of SB-1 cell suspension after enzymatic removal of cell wall. Extracts of SB-1 cells under some other conditions yielded a major band (Mr approximately equal to 60,000) as revealed by SDS-PAGE and immunoblotting with rabbit anti-seed SBA; prolonged incubation of these samples in the presence of SDS resulted in the appearance of the 30-kDa polypeptide. It appears that the 60-kDa band represented a highly stable, even under SDS-PAGE conditions, dimeric form of the 30-kDa subunit. The SB-1 lectin derived from the culture medium was compared with seed SBA by gel filtration and by peptide mapping after limited proteolysis; no difference between the lectins from the two sources was found. Extracts of soybean roots fractionated on N-caproyl-galactosamine-Sepharose affinity columns yielded, upon elution with galactose, polypeptides of Mr 30,000 and 60,000. These results suggest that soybean roots contain a lectin whose polypeptide composition corresponds to that of seed SBA and SB-1 lectin.     

A lethal myeloproliferative syndrome in mice transplanted with bone marrow cells infected with a retrovirus expressing granulocyte-macrophage colony stimulating factor.

Murine bone marrow cells infected with a novel recombinant retrovirus, MPZen(GM-CSF), were engrafted into lethally irradiated recipients. The transplanted animals developed extremely high circulating levels of GM-CSF (up to 3 x 10(5) units/ml), and greatly elevated peripheral nucleated cell counts (up to 110 x 10(6) per ml). Their haemopoietic tissues contained GM-CSF proviral DNA and produced substantial levels of GM-CSF. The mice died within 4 weeks of transplantation with extensive neutrophil and macrophage infiltration of the spleen, lung, liver and peritoneal cavity and significant infiltration of both heart and skeletal muscle by neutrophils, macrophages and eosinophils. The thymus and lymph nodes were deficient in lymphoid cells. No disease occurred when infected cells from haemopoietic tissues of the primary transplanted animals were injected into normal or sub-lethally irradiated mice. Dysregulated GM-CSF expression by haemopoietic cells thus produces a fatal albeit non-neoplastic myeloproliferative syndrome.     

Interchromosomal exchange of genetic information between gene arrangements on the third chromosome of Drosophila pseudoobscura

During the last 60 years, the inversion polymorphism on the third chromosome of Drosophila pseudoobscura has become a case study of the evolution of linked blocks of genes, isolated from each other by the suppression of recombination in heterozygotes for different inversions. Due to its location within inverted regions in most gene arrangements, the amylase (Amy) gene region can be used to elucidate the molecular pattern of evolution in these inversions. We studied this region in the Tree Line phylad of gene arrangements, with regard to both restriction site polymorphisms (RSP) and nucleotide sequences. The analysis of restriction maps, encompassing 26 kb, corroborates the cytogenetic phylogeny established on the basis of inversion breakpoints. However, we found that the 2.7 kb of nucleotide sequences of the AmyI gene are identical in both Estes Park and Hidalgo arrangements, despite the fact that these inversions arose independently from Tree Line. These contrasting results suggest that a homogenizing force, most likely gene conversion, is able to bring about localized exchanges between otherwise isolated gene arrangements.