Biomedical subjects

J Ohara

Publications and source records attributed to J Ohara.

61 records · Page 4Linked to original sources

PubMed1982

Microinjection of macromolecules into normal murine lymphocytes by means of cell fusion. II. Enhancement and suppression of mitogenic responses by microinjection of monoclonal anti-cyclic AMP into B lymphocytes.

Reproducible methods are now available for introducing protein molecules such as antibodies into normal murine lymphocytes by fusion with protein molecule-containing erythrocyte ghosts. Monoclonal antibodies against cyclic AMP were raised by hybridoma technique and packed into erythrocyte ghosts. Then, monoclonal anti-cyclic AMP containing ghosts were fused with splenic B lymphocytes by polyethylene glycol-mediated fusion at various intervals after LPS stimulation. This method made it possible for us to quantitatively microinject antibodies into B lymphocytes. Microinjection of anti-cyclic AMP antibody molecules into lymphocytes at a very early stage of LPS stimulation resulted in a marked enhancement of DNA synthetic responses as well as increased numbers of plaque-forming cells. Intracellular cyclic AMP levels were found to be markedly decreased after microinjection of monoclonal anti-cyclic AMP, suggesting that lowering the intracellular cyclic-AMP level in the B lymphocytes at an early stage of stimulation might have induced the enhanced proliferative as well as differentiative responses to LPS. Similar enhancing effects on cell proliferation were obtained when antibodies were injected 18 hr after stimulation. Microinjection of anti-cyclic AMP at 12 hr after culture, however, inhibited the DNA synthetic responses, and induction of plaque-forming cells was suppressed when anti-cyclic AMP was injected 6 hr after LPS stimulation. The present data suggest the biphasic regulatory roles of cyclic AMP at the early stage of B lymphocyte activation. This approach may be useful in identifying regulatory molecules in B lymphocyte induced by mitogenic or antigenic stimulation.

Animals↗

PubMed1981

Functional nuclei of LPS-nonresponder C3H/HeJ mice after transfer into LPS-responder C3H/HeN cells by cell fusion.

Splenic lymphocytes of C3H/HeJ mice are defective in responding to the mitogenic activity of bacterial lipopolysaccharide (LPS), which has been attributed to a mutation at a single gene locus on chromosome 4 (ref. 5). It was suggested that C3H/HeJ mice lack a cell-surface receptor necessary for LPS recognition. However, specific binding of 125I-labelled lipid-A of LPS to B cells was found to be equivalent in LPS-responder and LPS-nonresponder C3H/HeJ, C57BL/10 ScCR and C57BL/10 ScN mice. This suggests that the genetic defect in C3H/HeJ mice is related to triggering mechanisms rather than to LPS binding. We recently established a method for separating mouse splenic lymphocytes into karyoplasts (minicells) and cytoplasts (enucleated cells) with high purity. This method makes possible the exchange of nuclei from one type of lymphocyte to another. In the present study, karyoplasts were purified from LPS-nonresponder C3H/HeJ mice and transferred to mitomycin C-treated or X ray-irradiated splenic lymphocytes taken from LPS-responder C3H/HeN mice by fusion with polyethyleneglycol. These reconstituted hybrid cells could respond to LPS, suggesting that nuclei from C3H/HeJ mice could be activated by LPS if proper signals were provided from cell-surface receptors. Moreover, responsiveness to concanavalin A (Con A) of mitomycin C-treated or X ray-irradiated lymphocytes was also recovered by the microinjection of karyoplasts from untreated lymphocytes.

Animals↗

PubMed1981

Purification of cytoplasts (enucleated cells) and karyoplasts (nuclei) from mouse splenic lymphocytes with cytochalasin-B.

Murine spleen and lymphoma cells were almost completely enucleated in suspension by centrifugation in discontinuous Ficoll gradients in the presence of cytochalasin-B. Viability of cytoplasts (cells lacking nuclei) and karyoplasts (nuclei) was over 90%. Pure fractions of cytoplasts or karyoplasts were then obtained by fractionation of the cytochalasin-B treated cells with unit gravity sedimentation in bovine serum albumin. Protein synthesis was seen in cytoplasts when they were stimulated with mitogens, such as lipopolysaccharide (LPS). This method can consistently yield pure fractions of morphologically and functionally intact karyoplasts or cytoplasts from normal lymphocytes with very low contamination of nucleated cells. Pure karyoplasts or cytoplasts obtained in this way could be extremely useful for subcellular analysis of lymphocyte activation.

Animals↗

PubMed1980

Pulmonary nodules: detection in 1 and 2 cm full lung linear tomography.

Pulmonary nodule detection was evaluated in full lung linear tomography at 1 and 2 cm intervals. Three radiologists independently reviewed 1 and 2 tomograms on 26 patients with 39 pulmonary nodules. Decisions made in each case included: (1) no nodule; (2) definite nodule(s); and (3) suspect nodule(s). The presence of nodules was determined by surgery, radiographic follow-up, or observer consensus. A significantly greater number of nodules was detected by all reviewers on the 1 cm tomograms. Of the 39 nodules, 72%-97% were detected as definite and 82%-100% were identified as definite or suspect. Factors relating to nodule detectability and observer performance are discussed. It is recommended that full lung linear tomography be performed at 1 cm intervals.

Evaluation Studies as Topic↗

PubMed1978

In vitro immune response of human peripheral lymphocytes. III. Effect of anti-mu or anti-delta antibody on PWM-induced increase of cyclic nucleotides in human B lymphocytes.

Stimulation of human peripheral blood lymphocytes (PBL) with pokeweed mitogen (PWM) induced consistent increases of intracellular levels of cyclic AMP and cyclic GMP within 15 min. Increases of cyclic AMP were observed in both B and T lymphocyte populations, but increase of cyclic GMP was observed only in the B lymphocyte population. The addition of anti-mu antibody to B cells abolished PWM-induced increase of cyclic GMP without any effect on cyclic AMP response. Anti-delta antibody did not show any inhibitory or stimulatory effect on PWM-induced increase of cyclic GMP or cyclic AMP. Pretreatment of B cells with anti-mu antibody at 37 degrees C for 1 hr inhibited PWM-induced increase of cyclic GMP, whereas pretreatment with anti-mu antibody at 4 degrees C did not show any inhibitory effect on PWM-induced increase of cyclic GMP. The effect of anti-mu-pretreatment was reversible and pretreated cells were recovered from the inhibitory effect of anti-mu antibody after 36 hr culture.

Antibodies↗

PubMedDate not supplied

Production of a monoclonal antibody to and molecular characterization of B-cell stimulatory factor-1.

B-cell stimulatory factor-1 (BSF-1), formerly designated B-cell growth factor, is a T-cell-derived factor required for entry into the S phase of the cell cycle by B cells stimulated with low concentrations of anti-IgM antibodies. BSF-1 acts directly on resting B cells to prepare them to synthesize DNA more promptly on subsequent exposure to competent stimuli and to strikingly enhance their expression of class II molecules of the major histocompatibility complex. Previous studies have shown that murine BSF-1 can be separated physically from interleukin-2 (IL-2) and that the molecule has an apparent relative molecular mass (Mr) of approximately 15,000 and pI values of 6.4-6.7 and 7.4. Here, we report the production of a monoclonal antibody to BSF-1, its use in characterizing BSF-1, and functional studies demonstrating that this molecule is distinct from IL-1, IL-2 and IL-3.

Animals↗

PubMedDate not supplied

Receptors for B-cell stimulatory factor-1 expressed on cells of haematopoietic lineage.

B-cell stimulatory factor-1 (BSF-1) is a T-cell product of relative molecular mass 20,000 (Mr, 20K) initially described as a cofactor required for DNA synthesis by resting mouse B cells stimulated with low concentrations of anti-IgM antibodies. It acts on resting B cells to enhance the expression of class II major histocompatibility complex (MHC) molecules, to prepare these cells to respond more promptly to subsequent stimuli, such as anti-IgM antibodies, and causes the secretion of IgG1 and IgE by B cells stimulated with lipopolysaccharide (LPS). BSF-1 has been shown to stimulate T cell lines, resting T cells and some mast cell lines. Recently, the designation interleukin-4 (IL-4) has been suggested for BSF-1. We report here the existence of high-affinity cell-surface receptors specific for BSF-1 on both B and T lymphocytes, and on cells of several other haematopoietic lineages, including mast cell, macrophage and undifferentiated haematopoietic cell lines. Resting B and T lymphocytes express receptors, which increase in number upon activation of B cells with LPS or anti-IgM, and of T cells with concanavalin A. Cross-linking of 125I-labelled-BSF-1 to its receptors creates a complex of Mr approximately 80,000.

Animals↗