Immunoglobulin VH usage analysis by fluorescent in situ hybridization and flow cytometry.

We have devised a flow cytometry-based fluorescent in situ hybridization assay that permits analysis of gene expression in a large number of single cells. In this technique, fixed and permeabilized cells are incubated with biotinylated single-stranded RNA probes and by means of a fluorescently labelled second-step reagent, the cells are analyzed by flow cytometry. This is a rapid and simple method that allows all of the steps in the procedure to be performed on cells in suspension. Using this approach, we demonstrate here that immunoglobulin heavy chain variable region (VH) gene expression can be analyzed among individual cells using particular VH family-specific probes. This technique has a high degree of accuracy (greater than 97%) in detecting the fraction of cells expressing a specific message in a population and is sensitive enough to detect immunoglobulin message in LPS activated B cells. The technique has been applied successfully to monitor gene expression in homogeneous and heterogeneous populations. It also allows concurrent analysis of cell surface proteins and gene expression through two-color flow cytometry. This method of monitoring gene expression in individual cells may have a number of applications in immunology and cell biology.

The effect of nicotine on cell proliferation and synthesis of secreted proteins in BALB/C 3T3 cells.

The alkaloid nicotine induces a dose-dependent inhibition of cell proliferation and morphological change when added to BALB/C 3T3 cells. Significant differences were observed between control and nicotine-treated cells with respect to the newly synthesized secreted proteins by using heparin-agarose column chromatography. Both the anticellular and protein synthesis modulating activities of nicotine were affected by the degree of confluence of cells, suggesting a complex mode of action of nicotine in mammalian cells.

The antigenic structure of dengue type 1 virus envelope and NS1 proteins expressed in Escherichia coli.

The antigenic structures of the envelope protein, E, and the non-structural protein, NS1, of dengue type 1 virus (DEN1) have been studied in the form of recombinant fusion proteins expressed in Escherichia coli. Deletion analysis was used to identify two distinct antigenic domains in E that reacted with subsets of antiviral monoclonal antibodies (MAbs). Domain I of E extends from amino acid residues (aa) 76 to 93 of E; domain II extends from aa 293 to 402 and contains an essential disulphide bridge. MAbs also reacted with several determinants clustered near the N terminus of the NS1 protein (aa 57 to 126). Recombinant fusion proteins containing E. coli trpE sequences and most of the sequences for either E or NS1 were immunogenic in mice. The antibodies elicited by the E fusion protein reacted with a portion of the protein containing domain II, whereas antibodies elicited by the NS1 fusion protein did not react with the antigenic determinants defined by our MAbs.

The effect of ammonium ion on the induction of (2'-5')-(A)n synthetase and (2'-5')-(A)n-dependent endoribonuclease activities by interferon in mouse BALB/C 3T3 cells.

Addition of ammonium chloride to interferon (IFN)-treated mouse L cells has been reported to reduce the antiviral state. Because (2'-5')-(A)n synthetase and (2'-5')-(A)n-dependent endoribonuclease (RNase L) activities have been implicated in the establishment of the antiviral state, we wish to ascertain if ammonium ions will also impair the IFN-elicited induction of these two enzymatic activities. To test this possibility, cell-free extracts were prepared from mouse BALB/C 3T3 cells and assayed for (2'-5')-(A)n synthetase and RNase L activities. Results of these studies show that when cells were incubated with IFN (100 or 500 U/ml) and ammonium chloride (20-40 mM) for 20 h, the induction of (2'-5')-(A)n synthetase by IFN is significantly suppressed. Based on binding to the specific radioactive (2'-5')-(A)n analog, (2'-5')p3A4,3'-[32P]pCp, on cross-linking to the periodate-oxidized (2'-5')p3A4,3'-[32P]pC, or on the (2'-5')-(A)n-enhanced degradation of [3H]-polyadenylated RNA, IFN (10-250 U/ml) was shown to cause a two- to sevenfold increase in RNase L activity. The induction of RNase L by IFN was blocked by simultaneous addition of ammonium chloride. However, 20 mM or 40 mM ammonium chloride did not affect the antiviral state in BALB/C 3T3 cells (based on a plaque reduction assay with 10-500 U/ml IFN). These results suggest that the establishment of the antiviral state is not necessarily coordinated with changes in the synthetase and the RNase L activities.

Ontogeny of ppp(A2'p)nA-binding protein in mouse brain.

Mouse brain tissue extracts at various stages of development show a drastic change in the specific activity of pp(A2'p)3A-[32P]pCp binding protein. Identification of the ppp(A2'p)3A-[32P]pCp binding protein was established by (i) binding to the specific ligand ppp(A2'p)3A-[32P]pCp, (ii) displacement of binding by nanomolar concentrations of pppA(pA)3, and (iii) affinity labeling techniques in which periodate oxidized ppp(A2'p)3A-[32P]pC was specifically cross-linked to a protein with a molecular weight of 85000. These data suggest that the ppp(A2'p)3A-[32P]pCp protein is closely associated with the process of cellular proliferation and differentiation.

Effect of temperature on ppp(A2'p)nA binding protein activities in rabbit reticulocyte lysates and other mammalian extracts.

Lysates of rabbit reticulocytes and other mammalian cells are known to contain an activity which binds with high specificity ppp(A2'p)3A,3'-[32P]pCp. The binding activity shows a marked dependence on preincubation of lysates at different temperatures (4 degrees C - 45 degrees C). For example, binding was increased 50% by preincubation of rabbit reticulocyte lysates at 37 degrees C for 60 minutes. An identical preincubation of mouse brain extracts results in a greater than 90% loss of binding activity. Fractionation of rabbit reticulocyte lysates into the postribosomal supernatant (PRS) and the ribosomal salt wash (RSW), followed by heparin-agarose column chromatography, showed that with the PRS fraction, most of the binding activity is eluted with 600 mM KCl. With the RSW fraction, more than 50% of the binding activity is eluted with 250 mM KCl. These data suggest that multiple ppp(A2'p)nA binding protein activities exist in mammalian cells.