Structure and Immunochemistry of the Cell Wall Mannans from Saccharomyces chevalieri, Saccharomyces italicus, Saccharomyces diastaticus, and Saccharomyces carlsbergensis

The mannans of Saccharomyces chevalieri, S. italicus, S. diastaticus, and S. carlsbergensis, were acetolyzed, and the fragments were separated by gel filtration. All gave similar acetolysis fingerprints, which were distinguished from S. cerevisiae by the presence of a pentasaccharide component in addition to the mono-, di-, tri-, and tetrasaccharides. All oligosaccharide fragments were composed of mannose in alpha-linkage. From methylation analysis and other structural studies, the disaccharide was shown to be alphaMan(1 --> 2)Man; the trisaccharide was shown to be a mixture of alphaMan(1 --> 2)alphaMan (1 --> 2)Man and alphaMan(1 --> 3)alphaMan(1 --> 2)Man; the tetrasaccharide was alphaMan(1 --> 3)alphaMan(1 --> 2)alphaMan(1 --> 2)Man; and the pentasaccharide was alphaMan(1 --> 3)alphaMan(1 --> 3)alphaMan(1 --> 2)alphaMan(1 --> 2)Man. The ratios of the different fragments varied slightly from strain to strain. Mannanase digestion of two of the mannans yielded polysaccharide residues that were unbranched (1 --> 6)-linked polymers, thus establishing the structural relationship between these mannans and that from S. cerevisiae. Antisera raised against the various yeasts cross-reacted with the mannans from each, and also with S. cerevisae mannan. The mannotetraose and mannopentaose acetolysis fragments gave complete inhibition of the precipitin reactions, which indicated that, in these systems as in the S. cerevisiae system, the terminal alpha(1 --> 3)-linked mannose unit was the principal immunochemical determinant on the cell surface.     

Effects of Mitogenic and Antigenic Stimulation on the Thymidine Kinases of Mouse Spleen Cells in vivo and in vitro

The DNA-synthesizing ability of mouse spleen cells in vitro and in vivo is paralleled by their levels of cytoplasmic thymidine kinase. However, a very high level of nuclear-associated enzyme activity developed in cultures of both non-stimulated and mitogen-transformed lymphocytes. This activity did not appear in the spleens of mice during the primary immune response to sheep erythrocytes (SRC) or following administration of Convanavalin A (Con A).
Feedback inhibition studies with TTP demonstrated that the nuclear enzymes were more sensitive than the cytoplasmic activities. The thermal stabilities of the nuclear enzymes were also found to be greater than the corresponding cytoplasmic ones. Furthermore, analysis of the rate of thermal inacti-vation indicated that both the cytoplasmic and nuclear enzymes present in transformed lymphocytes in vitro were far more heat-stable than those activities normally found in the mouse spleen, even after antigenic challenge with SRC in vivo or incubation in vitro in the absence of mitogenic stimulation.     

Effect of calcofluor white on chitin synthases from Saccharomyces cerevisiae.

The growths of Saccharomyces cerevisiae wild-type strain and another strain containing a disrupted structural gene for chitin synthase (chs1::URA3), defective in chitin synthase 1 (Chs1) but showing a new chitin synthase activity (Chs2), were affected by Calcofluor. To be effective, the interaction of Calcofluor with growing cells had to occur at around pH 6. Treatment of growing cells from these strains with the fluorochrome led to an increase in the total levels of Chs1 and Chs2 activities measured on permeabilized cells. During treatment, basal levels (activities expressed in the absence of exogenous proteolytic activation) of Chs1 and Chs2 increased nine- and fourfold, respectively, through a mechanism dependent on protein synthesis, since the effect was abolished by cycloheximide. During alpha-factor treatment, both Chs1 and Chs2 levels increased; however, as opposed to what occurred during the mitotic cell cycle, there was no further increase in Chs1 or Chs2 activities by Calcofluor treatment.     

Costimulatory Effect of Fas in Mouse T Lymphocytes

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.     

Costimulatory Effect of Fas in Mouse T Lymphocytes

To induce proper immune responses, T lymphocytes require two types of stimuli, antigen-specific and costimulatory signals. Among costimulatory molecules, CD28-engagement promotes the survival and proliferation of both naive and memory T cells. In addition, it is now believed that Fas may play a role in T cell activation in the human system. It is, however, controversial whether Fas can act as a costimulatory signal in the murine system. Thus, we investigated fundamental differences in the capacity to induce proliferation of T cells between Fas and CD28 in mice. Fas-mediated T cell proliferation was observed only with a full mitogenic dose of anti-CD3 antibodies, whereas CD28 engagement was able to enhance T cell proliferation in the presence of a suboptimal level of anti-CD3 antibody. Furthermore, Fas-engaged T cells showed faster response in the upregulation of CD25 and CD69 expression than CD28-engaged ones. Here, we report that Fas might play a role in mature T cell activation in the mouse system through a different mechanism from that in CD28 costimulation.     

Effect of caffeine on ozone-sensitivity in Saccharomyces cerevisiae

Summary The addition of 0.1% caffeine to the plating medium markedly reduced the ozone-survival of the wild-type and the rad1 and rad6 mutants of Saccharomyces cerevisiae, whereas no effect was observed in the rad52 mutant. Since, in S. cerevisiae, caffeine has been reported to interfere with the recombinational repair pathway under the control of the RAD52 gene, these results support previous observations suggesting that this pathway is involved in the repair of ozone-induced DNA damage.     

Actin from Saccharomyces cerevisiae.

Inhibition of DNase I activity has been used as an assay to purify actin from Saccharomyces cerevisiae (yeast actin). The final fraction, obtained after a 300-fold purification, is approximately 97% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Like rabbit skeletal muscle actin, yeast actin has a molecular weight of about 43,000, forms 7-nm-diameter filaments when polymerization is induced by KCl or Mg2+, and can be decorated with a proteolytic fragment of muscle myosin (heavy meromyosin). Although heavy meromyosin ATPase activity is stimulated by rabbit muscle and yeast actins to approximately the same Vmax (2 mmol of Pi per min per mumol of heavy meromyosin), half-maximal activation (Kapp) is obtained with 14 micro M muscle actin, but requires approximately 135 micro M yeast actin. This difference suggests a low affinity of yeast actin for muscle myosin. Yeast and muscle filamentous actin respond similarly to cytochalasin and phalloidin, although the drugs have no effect on S. cerevisiae cell growth.     

RNA-dependent ATPase from Saccharomyces cerevisiae

A new RNA-dependent ATPase has been isolated from yeast chromatin extracts and partially characterized. The protein has a sedimentation coefficient of about 7 S. The enzyme hydrolyzes specifically ATP (or dATP) to ADP (or dADP) and Pi in the presence of Mg2+ or Mn2+ ions and requires a single-stranded polynucleotide as cofactor. The order of efficiency of synthetic polymers is poly(rU) > poly(rI) greater than or equal to poly(dU) > poly(rA) greater than or equal to poly(rC). Among natural polymers, single-stranded DNA and poly(rA)-containing mRNA from yeast are also active but less so than poly(rU). The enzyme exhibits a pH optimum of 8 and is fully inhibited by 0.25 M NaCl. The Km for ATP is0.2 mM. The resemblance between this ATPase and DNA-dependent ATPases from other sources, as well as the termination factor rho, is discussed.     

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria ¹. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year ², and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.      

Effect of Brestan on Saccharomyces cerevisiae during continuous cultivation

An increase in Brestan concentration in nutrient media decreased the content of protein, phosphorus, total ribonucleic acid, activity of pyruvate carboxylase and isocitrate lyase in cells ofSaccharomyces cerevisiae parent strain and respiratory deficient (RD) mutant while the trehalose content increased. The respiration quotient value for the RD mutant was higher than for the parent strain. The RD mutant lacked cytochromeaa ₃; cytochromec andb contents were lower than those of the parent strain.     

Effect of different starvation conditions on the flocculation of Saccharomyces cerevisiae

AIMS: To study the effect of different starvation conditions on the flocculation of an ale brewing yeast of Saccharomyces cerevisiae NCYC 1195. METHODS AND RESULTS: Flocculation was assessed by a micro-flocculation technique (Soares and Mota 1997). Carbon-starved cells of a NewFlo phenotype strain did not lose flocculation during a 48 h period. Cells incubated only in the presence of fermentable carbon sources (glucose, galactose and maltose at 2%, w/v), showed a progressive flocculation loss. The incubation of cells in 4% (v/v) ethanol did not induce a flocculation loss. The simultaneous incubation of cells in the presence of 2% (w/v) glucose and 15 microg ml(-1) cycloheximide hindered flocculation loss. The presence of 0.1 mmol l(-1) PMSF or 10 mmol l-1 EDTA prevented partially or completely, respectively, the loss of flocculation in the presence of glucose. CONCLUSIONS: Fermentable sugars induced a flocculation loss, which seems to require de novo protein synthesis and the involvement of different proteases. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings reported here contribute to the elucidation of the role of nutrients on the physiological control of yeast flocculation.     

Effect of tunicamycin on thiamine transport in Saccharomyces cerevisiae

The activity of thiamine transport in Saccharomyces cerevisiae was decreased by the treatment with tunicamycin without affecting the growth of yeast cells. Although the total activity of a soluble thiamine-binding protein in yeast periplasm, which is known to be a glycoprotein, was decreased by tunicamycin treatment, the activity of thiamine uptake by yeast protoplasts was inhibited as much as by whole cells. Furthermore, tunicamycin decreased the activity of the membrane-bound thiamine-binding protein in a dose dependent way and in parallel with the thiamine transport activity. These findings suggested that the membrane-bound thiamine-binding protein is a glycoprotein which plays a functional role in thiamine transport in S. cerevisiae.