Functional analysis of a single chain chimeric alpha/beta-granulocyte-macrophage colony-stimulating factor receptor. Importance of a glutamate residue in the transmembrane region.

To analyze the function of each subunit of the receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF), GMR, we previously generated a single-chain chimeric receptor by fusion of the extracellular and transmembrane domain from the alpha-subunit (alpha-GMR) to the intracellular part of the beta-subunit (beta-GMR) introducing an additional glutamate residue at the fusion site (alpha/beta-GMR). We demonstrated the capacity of alpha/beta-GMR to bind GM-CSF with low affinity and to induce GM-CSF-dependent activation of tyrosine kinase activity and proliferation in transfected Ba/F3 cells. To further compare the functions of wild type and chimeric receptors, we now report that this alpha/beta-GMR is sufficient to mediate morphological changes, expression of alpha(4)- and beta(1)-integrin receptor subunits, and serine-phosphorylation of Akt kinase. To analyze the function of the glutamate residue at the fusion region of alpha/beta-GMR various point mutants changing this amino acid and its position were expressed in Ba/F3 cells. None of these mutants was capable of supporting GM-CSF-dependent proliferation; however, when beta-GMR was coexpressed, GM-CSF mediated short and long term proliferation. Interestingly, some mutants but not alpha/beta-GMR can induce proliferation in the presence of an anti-alpha-GMR antibody. These data demonstrate the significance of a glutamate residue in the transmembrane region of alpha/beta-GMR for ligand-induced receptor activation.     

Treatment of intestinal dysbacteriosis in children with diabetes mellitus]

The use of the probiotic Acylact in combination with Viferone containing human recombinant interferon-alpha 2 in the treatment of intestinal dysbacteriosis in children with insulin-dependent diabetes mellitus (IDDM) was shown to be highly efficient. Elimination of microecologic disorders in the intestine was accompanied in all the children by immune rearrangements. The quantity of secretory and serum immunoglobulins and complement increased and the white blood cell chemiluminescence proved to be higher. The correction of the immune status of the children with IDDM resulted in a certain decrease of glycemia. The data are indicative of the fact that such a treatment of intestinal dysbacteriosis in children with IDDM is promising.     

Expression of tracheal differentiated functions in serum-free hormone-supplemented medium

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.     

Spindle disturbances in mammalian cells. IV. The action of some glutathione-specific agents in V79 Chinese hamster cells, changes in levels of free sulfhydryls and ATP, c-mitosis and effects on DNA metabolism

The glutathione-specific agents diamide, diethyl maleate and 1-chloro-2,4-dinitrobenzene were found to induce a low frequency of c-mitosis (15%) at non-toxic concentrations concomitant with a 30-40% decrease of non-protein sulfhydryls. The frequency of c-mitosis did not increase further with increased concentrations until non-protein sulfhydryl levels were obtained suggesting depletion of reduced glutathione. The observed shape of the concentration-response curve for c-mitosis is particular to these 3 agents and caffeine among 22 different compounds being tested under comparable conditions. This suggests a similar mechanism of action and from what is known about caffeine this mechanism probably involves an impaired control of cytoplasmic free Ca2+. It is speculated that this impairment with the glutathione-specific agents is primarily due to depletion of a particular pool of reduced glutathione. Tertiary butylhydroperoxide which is a substrate for glutathione peroxidase(s) also causes c-mitosis when there is no significant decrease of non-protein sulfhydryls. The c-mitotic response was found to be biphasic with maintained control levels at an intermediate concentration. The humps in the concentration-response curve for c-mitosis appeared coincident with a mitogenic response (G1----S). Since the latter type of effect most probably is Ca2+ dependent and since the spindle is sensitive to Ca2+ it is tentatively suggested that the c-mitotic effect of tertiary butylhydroperoxide is due to an increase of cytoplasmic Ca2+. Measurements performed imply that an increase of glutathione disulfide (diamide) is more inhibitory to uptake and incorporation of thymidine than a decrease of reduced glutathione per se (diethyl maleate). This difference is probably due to secondary effects on pertinent protein sulfhydryls with diamide, one possible target being the ribonucleotide reductase. All compounds were found to cause an increase of ATP with some of the applied concentrations. The results with diethyl maleate suggest that an increase of ATP is favored by an attack on mitochondrial reduced glutathione. The possible analogy between this effect and an increase of ATP and Ap4A in bacteria during oxidative stress is considered.     

Characterization of herpesvirus sylvilagus glycoproteins released into the culture medium of infected cells: antisera to gp13 and gp32 neutralize viral infectivity in vitro and identify antigens on plasma membranes of infected cells.

Polypeptides released into the culture medium of herpesvirus sylvilagus-infected cells were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extracellular fluid from [35S]methionine- and [3H]glucosamine-labeled cell cultures. Virus-induced glycoproteins 31, 32, and 33 (molecular weights of 62,000, 59,000, and 54,000, respectively) were the most abundant species and appeared predominantly in the culture medium. This observation, together with the known cell-associated nature of herpesvirus sylvilagus, suggested that virus-induced glycoproteins 31, 32, and 33 were specifically released. Immunization of rabbits with virus-induced glycoproteins 13 (molecular weight of 130,000) and 32 resulted in the production of antibodies that neutralized viral infectivity in vitro. Both antiserum to gp13 and antiserum to gp32 immunoprecipitated gp13, gp26, gp33a, gp45, and virus-induced polypeptide 39 (molecular weights of 130,000, 77,000, 49,000, 27,000, and 36,000, respectively) from [35S]methionine-labeled cell extracts as well as virus-induced glycoproteins 31, 32, and 33 from the culture medium. In addition, membrane immunofluorescence assays indicate that an antigen(s) reactive with anti-gp13/32 serum was located on the plasma membrane of infected cells.     

A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA

A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.     

Linearity of the accumulation of various dosages of uranium in the major organs of mature male Japanese quail. Effect of various doses of estradiol-17 beta

1. Male Japanese quail were given 2.20 x 10(-4)-14.53 mg uranium/kg intravenously as uranyl ion (235U label). 2. The relationship between dosage and the 18-hr accumulation of U in leg bones, liver, kidneys and testes was linear. 3. Increases in U deposition with increased dosage were approximately 1:1, except for kidneys where 10-fold increases in dosage resulted in 25-fold increases in deposition. 4. Estradiol-17 beta increased U deposition in bones by 15-fold thereby providing some protection for the kidneys as now the ratio of dosage to accumulation was 1:1.