Insulin receptor substrate-4 enhances insulin-like growth factor-I-induced cell proliferation.

The insulin receptor substrates (IRSs)-1-4 play important roles in signal transduction emanating from the insulin and insulin-like growth factor (IGF)-I receptors. IRS-4 is the most recently characterized member, which has been found primarily in human cells and tissues. It interacts with SH2-containing proteins such as phosphatidylinositol 3'-kinase (PI3K), Grb2, Crk-II, and CrkL. In this study, we transfected IRS-4 in mouse NIH-3T3 cells that overexpress IGF-I receptors. Clones expressing IRS-4 showed enhanced cellular proliferation when cells were cultured in 1% fetal bovine serum without added IGF-I. Addition of IGF-I enhanced cellular proliferation in cells overexpressing the IGF-I receptor alone but had an even greater proliferative effect in cells overexpressing both the IGF-I receptors and IRS-4. When etoposide and methylmethane sulfonate (MMS), both DNA damaging agents, were added to the cells, they uniformly induced cell cycle arrest. Fluorescence-activated cell sorter analysis demonstrated that the arrest of the cell cycle occurred at the G(1) checkpoint, and furthermore no significant degree of apoptosis was demonstrated with the use of either agent. In cells, overexpressing IGF-I receptors alone, IGF-I addition enhanced cellular proliferation, even in the presence of etoposide and MMS. In cells overexpressing IGF-I receptors and IRS-4, the effect of IGF-I in overcoming the cell cycle arrest was even more pronounced. These results suggest that IRS-4 is implicated in the IGF-I receptor mitogenic signaling pathway.     

Expression of a lipocalin in prokaryote and eukaryote cells: quantification and structural characterization of recombinant bovine beta-lactoglobulin.

In this paper we quantify and characterize the expression of recombinant beta-lactoglobulin (rBLG) in prokaryote and eukaryote cells. In Escherichia coli we used the pET26 vector, which permits the secretion of rBLG in periplasm. We studied the expression of rBLG in COS-7 cells and in vivo in mouse tibialis muscle. The expression of rBLG was measured by two immunoassays specific, respectively, for BLG in its native and denatured conformation. We observed that rBLG was essentially expressed in a denatured form in E. coli even in the periplasm, whereas rBLG in eukaryote cells was found in its native conformation.     

Immunosuppressive activity of T cell clones generated from human T cells stimulated with autologous TPHA cells

T cell clones were generated from human T cells stimulated with autologous phytohemagglutinin (PHA)-activated T (TPHA) cells. Characterization of three T cell clones originated from donor SF and one from donor JM showed that they proliferated when stimulated with autologous TPHA cells, non-T cells, and peripheral blood mononuclear cells, but did not proliferate when stimulated with allogeneic TPHA cells, non-T cells, and mononuclear cells, with autologous and allogeneic resting T cells, and with PHA. These results in conjunction with the blocking of the proliferation by anti-histocompatibility leukocyte antigen class II monoclonal antibodies indicate that these class II antigens are involved in the proliferation of T cell clones stimulated with autologous lymphoid cells. The four T cell clones are cytotoxic neither to autologous lymphoid cells nor to a panel of cultured human cell lines. The four T cell clones display immunosuppressive activity, since they inhibit the proliferation of autologous and allogeneic cells stimulated with antigens and mitogens and the secretion of immunoglobulin by B cells stimulated with pokeweed mitogen in presence of T cells. Furthermore, the four T cell clones display differential inhibitory activity on the proliferation of cultured human cell lines. The immunosuppressive activity is species-specific, since the T cell clones do not inhibit the proliferation of murine cells. The suppression is mediated by a factor(s) with an apparent m.w. of 13,000 to 16,000. The suppressor activity is labile at alkaline pH and is lost following incubation with pronase (100 U/ml) for 30 min at 37 degrees C.     

A new internal perfusion method for transport studies in squid giant axons

A new internal perfusion method has been developed which allows control of the internal solute composition in squid axons. The superiority of this technique compared to the old perfusion methods is shown by the experiments performed which have reproduced, both qualitatively and quantitatively, the Na+ and Ca2+ fluxes observed in intact and dialyzed axons. Compared with the internal dialysis, the perfusion method has the advantage that the permeability barrier give by the porous capillary has been eliminated. This allows the introduction into the axon of solutes with very high molecular weight, at the same time that a fast and reliable internal control can be achieved.     

Enhanced activation of a T cell line specific for acetylcholine receptor (AChR) by using anti-AChR monoclonal antibodies plus receptors

Immune complex-mediated regulation of the immune response has been studied by using T cell lines and monoclonal antibodies (MAb), both specific for the acetylcholine receptor (AChR). Rat T lymphocytes bearing the W3/25 phenotype and specific for AChR from Torpedo californica have been propagated in vitro for nearly 1 yr. These T cells proliferate in response to optimal concentrations of AChR presented by irradiated syngeneic thymus cells. At suboptimal concentrations of antigen there is little activation of the T cell line. We report here that the addition of small amounts of anti-AChR MAb produces dramatic stimulation of the T cell lines at suboptimal doses of AChR. Enhanced activation depends on the isotype and not the fine specificity of the MAb that are used. The observed phenomenon is antigen specific, and in fact, the immune complexes may actually suppress the proliferative response of irrelevant T cells to some extent. The MAb plus antigen are rapidly bound to the surface of the antigen-presenting cell, which we have shown is the dendritic cell.     

Effects of seed-irradiation on meiosis in M1 plants of Lactuca and related plants

Meiosis was studied in control and seed-irradiated materials of Lactuca sativa, cultivars Tom, Cos and Webbs, L. serriola from the wild, and Cichorium intybus. In meiosis of the M₁ plants from irradiated seed, observations of univalent and multivalent formation, bridge formation at Anaphase I and II, micronucleus formation and persistence, and pollen fertility, were carried out, and compared with controls. Quantitative estimations of bridge formation were made in L. sativa Tom and Cos and in L. serriola, at anaphase I only. It was found that the frequency of anaphase I bridges and reduction of pollen fertility in the M₁ and also M₂ generation, were different for each variety or species; these could be sequenced radiation-resistant to sensitive in the order Tom, Cos and L. serriola.     

Low degree of ubiquitination of histone 2A in the dipteran Chironomus tentans

A polyclonal antibody to ubiquitin has been prepared and shown to react with both ubiquitin and ubiquitinated histone 2A (uH2A). Applying this antibody in Western blotting experiments, we have observed that the salivary glands of Chironomus tentans contain an unusually low amount of uH2A (1% of histone 2A), while the amount of free ubiquitin is as abundant as in other animal cells, e.g. HeLa cells. The same low content of uH2A was also found in diploid epidermal cells of Chironomus origin suggesting that the low amount is not a characteristic of the polytene state of chromatin in salivary gland cells but rather a property of C. tentans as a species. The significance of the low degree of ubiquitination is discussed in relation to the information available on the organization of Chironomus chromatin into unusually large chromomeric entities.