Quantitative measurement of intracellular RNA genomes of Rauscher murine leukemia virus by competition hybridization in DNA excess.

The technique of competition hybridization in DNA excess was used to determine the intracellular distribution of RNA genomes of Rauscher murine leukemia virus. An examination of subcellular RNA fractions revealed that 59% of intracellular viral RNA genomes were associated with the nuclear-enriched fraction, 41% with the cytoplasmic fraction, and 18% with the polysomal-enriched fraction. Also, an analysis of total cellular RNA disclosed that 20% of intracellular viral RNA genomes were polyadenylated and 80% were not polyadenylated.     

Double-antibody solid-phase enzyme immunoassay for the detection of staphylococcal enterotoxin A.

A simple double-antibody enzyme immunoassay that uses a microtechnique was developed for detecting staphylococcal enterotoxin A in food products. Sample preparation can be completed in less than 15 min. Assay sensitivity ranges from 0.4 ng (20-h test time) to 3.2 ng (1- to 3-h test time) of toxin per ml of prepared sample. Separation and detection of enterotoxin from spiked food products ranged between 72 and 98% of the amount added.     

Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.     

Cognate interactions between helper T cells and B cells. V. Reconstitution of T helper cell function using purified plasma membranes from activated Th1 and Th2 T helper cells and lymphokines.

Th physically interact with B cells and produce lymphokines that influence B cell growth and differentiation. The respective contribution of cell contact and lymphokines to induction of B cell growth and differentiation was addressed using purified plasma membranes (PM) from resting Th (PMrest) and anti-CD3-activated Th (PMCD3) together with lymphokines. Results show that PMCD3, but not PMrest, induce 10% of resting B cells to enter the G1 phase of the cell cycle, with few B cells entering G1b and S/G2. The inclusion of IL-4, but not IL-2, IL-5, or IFN-gamma, amplifies the B cell response to PMCD3 by increasing the total percentage of activatable B cells to greater than 40% and inducing B cell progression into G1b, S, and G2. Direct comparison between PMrest and PMCD3 purified from Th1 and Th2 indicate that both Th1 and Th2 induce similar levels of B cell proliferation in the presence of IL-4. Further, the lymphokine requirements for B cell proliferation induced by PMCD3 from Th1 and Th2 is indistinguishable. B cell differentiation to IgM, IgG1, and IgG2a synthesis by PMCD3 required IL-4 and IL-5. Using lymphokine conditions that supported B cell differentiation, PMCD3 purified from Th1 and Th2 induced similar levels of IgM, and IgG1. Given the functional data on PMCD3 from Th1 and Th2, the data indicate that there are no substantive differences between Th1- and Th2-derived PMCD3, and that the major differences in the ability of viable Th1 and Th2 to activate B cells is the lymphokines produced by the cells.     

The effect of AZT on in vitro lymphokine-activated killer (LAK) activity in human immunodeficiency virus type-1 (HIV-1) infected individuals

Human immunodeficiency virus type-1 (HIV-1)-infected individuals exhibit functional impairment in various forms of cell-mediated cytotoxicities (CMC) at all stages of disease. The purpose of this study was to determine (i) if peripheral blood mononuclear cells (PBMC) obtained from HIV-1-infected patients could be stimulated in vitro to yield lymphokine-activated killer (LAK) activity; (ii) if non-MHC-restricted gp120-specific CMC could be preserved; and (iii) what effect zidovudine (AZT) would have on LAK activity. Fourteen asymptomatic HIV-1 seropositive adults and five healthy seronegative adults (controls) were evaluated. PBMCs were isolated and incubated in media or supplemented with IL-2 for 4 or 72 hr. Lysis of the NK resistant target cell line, Daudi, was similar for the control and experimental group. The increase in activity after stimulation was elevated to a similar degree in both seronegative and seropositive groups (P less than 0.001). LAK activity was significantly decreased (P = 0.011) when AZT was added to LAK cultures. In addition, virus production may not have been completely inhibited by AZT in LAK cultures. Thus, PBMCs from asymptomatic HIV-1-infected patients could be stimulated to yield LAK activity. However, AZT can impair LAK generation. It is unclear if LAK activation results in virus production that cannot be inhibited by AZT in this system. Further definition in other patient populations is required prior to applying this information to clinical trials.     

Basic fibroblast growth factor upregulates steady-state levels of laminin B1 and B2 chain mRNA in cultured neuroepithelial cells

The growth of purified populations of murine neuroepithelial cells isolated from 10 day embryonic (E10) telencephalon and mesencephalon can be specifically enhanced by supplementing growth culture media with basic fibroblast growth factor (bFGF). One effect of bFGF on cultured neuroepithelial cells was to enhance the amount of laminin expressed at the protein level as detected by immunofluorescence. This was correlated with significant upregulation of steady-state levels of laminin B1 and B2 chain expression as analyzed at the mRNA level. When E12 neuroepithelial cells were split into precursor neuronal or glial subpopulations on the basis of differential expression of major histocompatibility class-1 antigens, only the glial progenitor fraction was found to be capable of detectable laminin synthesis. It is thus possible that a primary action of FGF is to increase the synthesis and release of extracellular matrix molecules from neural cells which act back in a paracrine manner to stimulate differentiation.     

Expression of Transferrin mRNA in the CNS of Normal and Jimpy Mice

Both the iron mobilization protein transferrin and iron itself are found predominantly in oligodendrocytes in the brain and consequently have been hypothesized to have a role in myelination. This study is designed to begin to understand the mechanism(s) that control the expression of transferrin at the gene level in the nervous system using a hypomyelinating murine mutant (jimpy mouse). With this animal model it is possible to determine if transferrin gene expression in the nervous system is dependent on the presence of a mature oligodendrocytic population. The results demonstrate that normally expression of the transferrin gene increases from postnatal day 5 to 22-25 and then levels off in the adult. In the jimpy mouse, the relative amount of transferrin gene expression is less than that of littermate controls at 5 days of age. Furthermore, transferrin gene expression does not increase with age beyond the level observed at postnatal day 5 in the jimpy mouse. It is concluded from this study that the majority of the transferrin mRNA in the mouse brain is expressed by and/or requires the presence of a mature oligodendrocytic population.     

Regulation of androgen receptor mRNA and protein in the rat testis by testosterone.

Adult rats were treated with ethane dimethane sulphonate (EDS), an agent that destroys Leydig cells. Within 5 days after EDS treatment, the levels of testosterone (T) in the circulation and in the testis were decreased to very low values, which makes it possible to manipulate the testicular T concentration through administration of exogenous T. Spermatogenesis was not markedly affected within 5 days after EDS treatment, also not in the absence of T administration. In testes of EDS-treated rats, the androgen receptor mRNA (ARmRNA) level remained unaltered for 5 days. In ventral prostate, however, this treatment caused a pronounced upregulation of the level of ARmRNA, which could be counteracted by implantation of silastic T implants immediately after EDS treatment. In EDS-treated rats carrying a T implant and in untreated rats, the same number of specific [3H]R1881 binding sites was observed using a total testis nuclear fraction (Scatchard analysis). In testes from EDS-treated rats without T implants, androgen receptors (AR) did not fractionate into the nuclear fraction; however, the total testicular AR content in these animals (measured by nuclear [3H]R1881 binding after receptor transformation through injection of a high dose of T, 2 h before killing the rats) remained unaltered. Immunoprecipitation and Western blotting using anti N-terminal antibodies seemed to indicate that the total testicular amount of AR protein in the EDS-treated rats was very low as compared to that in EDS-treated rats carrying T implants and in untreated rats. Even after receptor retransformation (by injection of a high dose of T) the receptors were not quantitatively detected by immunoprecipitation and Western blotting. This may point to a structural modification of the AR that occurs in the prolonged absence of androgens.