The release of sex-specific substances responsible for sexual agglutination from haploid cells of Saccharomyces cerevisiae

Cell surface substances responsible for sexual cell agglutination were successfully released in a large quantity from heterothallic haploid cells of Saccharomyces cerevisiae by a newly established autoclaving method. The conditions for this releasing phenomenon were examined. The sexual agglutination substances were solubilized most efficiently when the cells, suspended in a 30 mM Tris-HCl, pH 7.0, 5 mM EDTA solution, were autoclaved at a pressure of 1 kg/cm² at 120 °C for 3 min. The substances were specifically adsorbed by the cell surface of the opposite mating type, resulting in the masking of agglutinability of the cells of the opposite mating type. The substances were not released from the surface of cells which lacked sexual cell agglutination. The evidence suggesting the formation of a molecular complex between a- and α-agglutination substances in vitro is also presented. The above procedure is applicable to the solubilization of surfage agglutination substances from various strains of S. cerevisiae.     

Absence of spontaneous regeneration of endogenous pancreatic β-cells after chemical-induced diabetes and no effect of GABA on α-to-β cell transdifferentiation in rhesus monkeys

β-cell deficiency is common feature of type 1 and late-stage of type 2 diabetes mellitus. Thus, β?cell replacement therapy has been the focus of regenerative medicine past several decades. Particularly, evidences suggest that β?cell regeneration via transdifferentiation from sources including α-cells is promising. However, data using higher mammals besides rodents are scarce. Here, we examined whether endogenous pancreatic β-cells could regenerate spontaneously or under normoglycemia following porcine islet transplantation for varied periods up to 1197 days after streptozotocin-induced diabetes, and remaining α-cells transdifferentiate into β-cells by GABA treatment in vivo and in vitro. The results showed that endogenous β-cells rarely regenerate in both conditions as evidenced by stagnant serum C-peptide levels and β-cell number in the pancreas, and the remaining α-cells did not transdifferentiate into β-cells by GABA treatment. Collectively, we concluded that monkey β-cells had relatively low regenerative potential compared with rodent counterpart and GABA treatment could not induce α-to-β-cell transdifferentitation.     

Genetics of a-agglutunin function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p). Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and cells after pheromone induction. Expression of the Aga2p adhesion subunit in a cells allowed a-agglutinability, indicating that a cells express the a-agglutinin anchorage subunit, although no role for Aga1p in cells has been identified. Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant. Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La 199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function. However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids. The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation. The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes. AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere.     

Cell Surface Carbohydrate Differences in Wild and Mutant Strains of Crithidia fasciculata1

Wild type Crithidia fasciculata and three drug-resistant mutant strains that have shown “flagellar adherence” were studied as to their ability to agglutinate with lectins specific for receptor molecules containing N-acetyl glucosamine, N-acetyl galactosamine, galactose, mannose-like residues, fucose, and sialic acid. Escherichia coli with mannose-sensitive fimbriae was also used as an agglutination probe. The presence of D-GalNAc, D-Gal, and mannose-like residues was detected in the wild strain. Generally, in the mutants, residues of these sugar units were present in greater concentrations when compared to the wild type strain. β-Galactosidase treatment showed that β-D-Galp units are exposed on the cell membrane. All types of cell agglutination including flagellum-flagellum (F-F), flagellum-soma (F-S), and soma-soma (S-S) were observed when lectins were used; however, with E. coli only the F-F type of cell agglutination was observed with the wild type strain and the TFR^R1 mutant. All types of agglutination were observed with the other two mutants.     

Mating-type differentiation in ascosporogenous yeasts on the basis of mating-type-specific substances responsible for sexual cell-cell recognition

Summary Sexual agglutination occurred only between cells of opposite mating types of the same species in all the Sacharomyces, Hansenula, Saccharomycodes, and Pichia yeasts tested. We succeeded in solubilizing the sex-specific glycoprotein, cell wall agglutination substance responsible for sexual agglutinability by briefly autoclaving these yeasts. The agglutination substances of all the above yeasts were univalent and sensitive to the enzyme pronase. The formation of complementary complexes was observed only between agglutination substances of opposite mating types of the same species. In general, the agglutination substance of one mating type was more resistant to heat treatment at 100°C in 3% acetic acid and more sensitive to 5% 2-mercaptoethanol treatment than the agglutination substance of the other mating type in these yeasts. On the basis of these results together with the pheromone response and production, we expect that almost all ascosporogenous yeasts can be classified into the two mating types corresponding to a and mating types in Saccharomyces cerevisiae, respectively.     

Mating reaction in Saccharomyces cerevisiae

Summary Conspicuous cell agglutination occurred when cells of the a and types were mixed together and cultured, while it did not when the strains not to mate each other were mixed. In the former case the ability of cells to agglutinate developed gradually with time after the mixing. The agglutination was inhibited by cycloheximide but not by chloramphenicol. Relation between yeast sexual hormones and the mating-specific cell agglutination is discussed.     

Agglutination of Trypanosoma cruzi by Concanavalin A*

Epimastigotes of Trypanosoma cruzi obtained in culture agglutinate readily with low concentrations of concanavalin A (Con A). Agglutination was linear with time up to 10 min providing that the initial cell density was greater than 1 × 10⁸ cells/ml. Under these conditions, the percentage agglutination was dependent on the Con A concentration. Agglutination was inhibited by α-methyl D-mannoside, α-D-mannose, and α-D-glucose. Pretreatment of cells with trypsin had no effect on the epimastigote agglutinations. Blood forms (trypomastigotes) of T. cruzi did not agglutinate even in the presence of 100 times more Con A. Results suggest differences in membrane structure between blood forms and cultured epimastigotes of T. cruzi. These membrane differences might be related to the different pathogenic properties of both cell forms of T. cruzi.     

Strain Dependence of the Cell-expanding Effect of β-1,3-Glucanase in Yeast

The effect of β-1, 3-glucanase on the cell expansion was studied with diploid strains of Saccharomyces ellipsoideus and S. cerevisiae, and their mutants differing in the response to auxin. The following results were obtained. Cell expansion was induced by β-1, 3-glucanase only in auxin-responsive or potentially auxin-responsive strains. β-1, 3-Glucanase induced cell expansion more rapidly than auxin. The cell wall of the auxin-responsive strain was more susceptible to digestion by β-l, 3-glucanase than that of the auxin-unresponsive one. The sensitivity of yeast cells to auxin action is discussed in relation to the nature of cell wall.     

DTA-mediated targeted ablation revealed differential interdependence of endocrine cell lineages in early development of zebrafish pancreas

Cell lineage analysis is critical in understanding the relationship between progenitors and differentiated cells as well as the mechanism underlying the process of differentiation. In order to study the zebrafish endocrine pancreas cell lineage, transgenic expression of diphtheria toxin gene A chain (DTA) under two cell type-specific promoters derived from the insulin (ins) and somatostatin2 (sst2) genes was used to ablate the two types of endocrine cells: insulin-producing β-cells and somatostatin-producing δ-cells, respectively. We found that ablation of β-cells resulted in a reduction of not only β-cells but also glucagon-producing α-cells; in contrast, δ-cells were largely unaffected. Ablation of δ-cells led to reduction of all three types of endocrine cells: α-, β-, and δ. Interestingly, α-cells were more profoundly affected in both β- and δ-cell ablations and were frequently reduced together with β- and δ-cells. By taking advantage of Tg(ins:gfp) and Tg(sst2:gfp) lines, we also monitored the changes of different types of endocrine cells in vivo after ablation and found that both β- and δ-cell populations significantly recovered by 3 dpf after their ablation and it seemed that δ-cells had a better capability of recovery than β-cells. Thus, our current observations indicated differential interdependence of these three cell lineages. The development of zebrafish α-cells, but not δ-cells, is dependent on β-cells, while the development of both α- and β-cells is dependent on δ-cells. In contrast, the development of δ-cells was independent of β-cells.     

Isolation,and biochemical and biological characterization of an a-mating-type-specific glycoprotein responsible for sexual agglutination from the cytoplasm of a-cells,in the yeast Saccharomyces cerevisiae

An a-mating-type-specific substance responsible for sexual agglutination was purified to 397-times in specific activity (units/mg protein) from the cytoplasm of a-mating type cells. The purified substance gave a single band stained with PAS reagent but not with both Coomassie brilliant blue and silver staining reagent by polyacrylamide gel electrophoresis in the presence of 8 M urea. However, incorporation of [³⁵S]methionine and Lowry reaction clearly indicate that the substance is a glycoprotein. The substance specifically masked sexual agglutinability of cells of the opposite mating type , indicating univalent action. The substance is a glycoprotein with a carbohydrate content of 90%, a pI of 4.5, and a molecular weight of 130,000. The substance was inactivated by 2-mercaptoethanol and proteolytic enzymes but not by glycolytic enzymes. The substance formed a complementary complex having no biological activity when mixed with -agglutination substance from the wall or cytoplasm of -cells in vitro.Non-common abbreviations PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - PBS 10^-2 M phosphate buffer solution, pH 5.5 - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate     

Extracellular Enzymes and Nutritional Physiology of Ampelomyces quisqualis Ces., Hyperparasite of Powdery Mildew, in vitro

In vitro, the powdery’ mildew hyperparasite Ampelomyces quisqualis produced constitutively two groups of extracellular enzymes; β-glucosidase, β-N-acetylglucosaminidase and acid phosphatase possessing unusual high molecular weights (M, about 340 000), and ribonuclease, β-l→3-glucanase and α-l→4-glucanase representing smaller molecules (M, about 15 000–55 000). As can be concluded from adsorption to Concanavalin A-Sepharose 4 B, these enzymes are of glycoprotein nature Furthermore, traces of phospholipase could be detected in all of the isolates tested, whereas only phosphorylated carbohydrate components and nucleotides including cAMP as nutrients. The results of these studies suggest that in the biotrophic phase of parasitization A. qwsqualis interferes with energy metabolism, protein synthesis, cell wall synthesis and, possibly, regulation of the host, whereas proteins and membranes remain nearly unaffected. Thus, depletion of the energy reserves of the powdery mildew may be mainly responsible for the degeneration of host ultrastructure which represents the beginning of the necrotrophic phase of parasitization.     

Dnmt1 activity is dispensable in δ-cells but is essential for α-cell homeostasis

In addition to β-cells, pancreatic islets contain α- and δ-cells, which respectively produce glucagon and somatostatin. The reprogramming of these two endocrine cell types into insulin producers, as observed after a massive β-cell ablation in mice, may help restoring a functional β-cell mass in type 1 diabetes. Yet, the spontaneous α-to-β and δ-to-β conversion processes are relatively inefficient in adult animals and the underlying epigenetic mechanisms remain unclear. Several studies indicate that the conserved chromatin modifiers DNA methyltransferase 1 (Dnmt1) and Enhancer of zeste homolog 2 (Ezh2) are important for pancreas development and restrict islet cell plasticity. Here, to investigate the role of these two enzymes in α- and δ-cell development and fate maintenance, we genetically inactivated them in each of these two cell types. We found that loss of Dnmt1 does not enhance the conversion of α- or δ-cells toward a β-like fate. In addition, while Dnmt1 was dispensable for the development of these two cell types, we noticed a gradual loss of α-, but not δ-cells in adult mice. Finally, we found that Ezh2 inactivation does not enhance α-cell plasticity, and, contrary to what is observed in β-cells, does not impair α-cell proliferation. Our results indicate that both Dnmt1 and Ezh2 play distinct roles in the different islet cell types.     

Genetics of a-agglutunin function in Saccharomyces cerevisiae

The Saccharomyces cerevisiae cell adhesion protein a-agglutinin is composed of an anchorage subunit (Aga1p) and an adhesion subunit (Aga2p). Although functional a-agglutinin is expressed only by a cells, previous results indicated that AGA1 RNA is expressed in both a and α cells after pheromone induction. Expression of the Aga2p adhesion subunit in a cells allowed a-agglutinability, indicating that a cells express the a-agglutinin anchorage subunit, although no role for Aga1p in α cells has been identified. Most of the a-specific agglutination-defective mutants isolated previously were defective in AGA1; a single mutant (La199) was a candidate for an aga2 mutant. Expression of AGA2 under PGK control allowed secretion of active Aga2p from control strains but did not complement the La199 agglutination defect or allow secretion of Aga2p from La 199, suggesting that the La199 mutation might identify a new gene required for a-agglutinin function. However, the La199 agglutination defect showed tight linkage to aga2::URA3 and did not complement aga2::URA3 in a/a diploids. The aga2 gene cloned from La199 was nonfunctional and contained an ochre mutation. The inability of pPGK-AGA2 to express functional Aga2p in La199 was shown to result from an additional mutation(s) that reduces expression of plasmid-borne genes. AGA2 was mapped to the left arm of chromosome VII approximately 28 cM from the centromere.     

1,3-β-Glucanase from Vigna aconitifolia and its possible use in enzyme bioreactor fabrication

Endo-1,3(4)-β-glucanase (EC 3.2.1.6) from Vigna aconitifolia sprouts was purified to 14.5 fold by gel filtration and ion-exchange chromatography. The enzyme was found to be a glycoprotein, its activity was Ca²⁺ dependent and specific for β-1,3 linkages in different polysaccharides. The K_m value of the enzyme was estimated to be 3.0 mg ml⁻¹ for β-d-glucan as substrate. Circular dichroism studies revealed 8% α-helix, 48% β-pleated and 44% random coil in its secondary structure. Purified β-glucanase was then successfully co-immobilized with glucose oxidase in agarose-chitosan beads, showing better immobilization yield, operational range and stability as compared with the crude β-glucanase beads. The immobilized β-glucanase was successfully used for mini-bioreactor fabrication.     

Expression of K-Cl cotransporters in the α-cells of rat endocrine pancreas

The expression of K⁺-Cl⁻ cotransporters (KCC) was examined in pancreatic islet cells. mRNA for KCC1, KCC3a, KCC3b and KCC4 were identified by RT-PCR in islets isolated from rat pancreas. In immunocytochemical studies, an antibody specific for KCC1 and KCC4 revealed the expression of KCC protein in α-cells, but not pancreatic β-cells nor δ-cells. A second antibody which does not discriminate among KCC isoforms identified KCC expression in both α-cell and β-cells. Exposure of isolated α-cells to hypotonic solutions caused cell swelling was followed by a regulatory volume decrease (RVD). The RVD was blocked by 10 μM [dihydroindenyl-oxy] alkanoic acid (DIOA; a KCC inhibitor). DIOA was without effect on the RVD in β-cells. NEM (0.2 mM), a KCC activator, caused a significant decrease of α-cell volume, which was completely inhibited by DIOA. By contrast, NEM had no effects on β-cell volume. In conclusion, KCCs are expressed in pancreatic α-cells and β-cells. However, they make a significant contribution to volume homeostasis only in α-cells.     

Sex-specific binding and inactivation of agglutination factor in Chlamydomonas eugametos

Gametes of opposite mating type (mt ⁺ and mt ^-) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using ³⁵S-labeled flagella and the isolated mt ^-agglutination factor. It is shown that not only isolated flagella, but also the mt ^-agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt ^-agglutination factor in determining the sexual agglutinability of mt ^-gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt ^-agglutination factor can be completely inactivated.Abbreviations Mt ^+/- mating type plus or minus - PAS periodic-acid Schiff-reagent - Hepes 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - HMC buffer Hepes buffer (10 mM. pH 7.2, containing 1 mM MgCl₂ and 1 mM CaCl₂)     

Expression cassette and plasmid construction for Yeast Surface Display in Saccharomyces cerevisiae

Objectives

Develop a Cell Surface Display system in Saccharomyces cerevisiae, based on the construction of an expression cassette for pYES2 plasmid.

Results

The construction of an expression cassette containing the α-factor signal peptide and the C-terminal portion of the α-agglutinin protein was made and its sequence inserted into a plasmid named pYES2/gDαAgglutinin. The construction allows surface display of bovine herpesvirus type 5 (BoHV-5) glycoprotein D (gD) on S. cerevisiae BY4741 strain. Recombinant protein expression was confirmed by dot blot, and indirect immunofluorescence using monoclonal anti-histidine antibodies and polyclonal antibodies from mice experimentally vaccinated with a recombinant gD.

Conclusions

These results demonstrate that the approach and plasmid used represent not only an effective system for immobilizing proteins on the yeast cell surface, as well as a platform for immunobiologicals development.

     

The role of autophagy in pancreatic β-cell and diabetes

Pancreatic β-cells play a key role in glucose homeostasis in mammals. Although large-scale protein synthesis and degradation occur in pancreatic β-cells, the mechanism underlying dynamic protein turnover in β-cells remains largely unknown. We found low-level constitutive autophagy in β-cells of C57BL/6 mice fed a standard diet; however, autophagy was markedly upregulated in mice fed a high-fat diet. β-cells of diabetic db/db mice contained large numbers of autophagosomes, compared with non-diabetic db/misty controls. The functional importance of autophagy was analyzed using β-cell-specific Atg7 knockout mice. Autophagy-deficient mice showed degeneration of β-cells and impaired glucose tolerance with reduced insulin secretion. While a high-fat diet stimulated β-cell autophagy in control mice, it induced a profound deterioration of glucose intolerance in β-cell autophagy-deficient mutants, partly because of the lack of a compensatory increase in β-cell mass. These results suggest that the degradation of unnecessary cellular components by autophagy is essential for maintenance of the architecture and function of β-cells. Autophagy also serves as a crucial element of stress responses to protect β-cells under insulin resistant states. Impairment of autophagic machinery could thus predispose individuals to type 2 diabetes.     

Effect of α-Tocopherol, β-Carotene, Monogalactosyldiglyceride and Phosphatidylcholine on Light-Induced Degradation of Chlorophyll a in Acetone

The effect of α-tocopherol, β-carotene, monogalactosyldi-glyceride and phosphatidylcholine on red light induced degradation of chlorophyll a was studied in acetone at 4°C. Monogalaclosyldi-glyceride was ineffective up to a molar ratio of monogalactosyldi glyceride to chlorophyll of 1:10. α-Tocopherol, β-carotene and phosphatidylcholine inhibited chlorophyll degradation. Maximal protection by α tocopherol and β-carotene was similar (76%) but on a molar basis a tocopherol was less effective. Protection by phosphatidylcholine was less than by a tocopherol and α-carotene but the lipid was effective at a lower ratio of chlorophyll to protectant. Inhibition by phosphatidylcholine was independent of the degree of unsaturation of the fatty acids. Effects of β-carotene and α-tocopherol were additive at suboptimal concentrations, but addition did not increase the maximal protection of 76% by these substances alone. Phosphatidylcholine increased the effectiveness of α-tocopherol and β-carotene independent of their concentrations. It is suggested that interactions between lipids participate in the mechanism protecting chlorophyll a against photooxidation in the chloroplast membrane.     

Agglutinating Effects of Concanavalin A on Isolated Protoplasts of Daucus carota

The agglutinating effects of Concanavalin A (Con A) on protoplasts isolated from cell suspensions of Daucus carota were studied. Con A was shown to agglutinate the plant protoplasts in a manner similar to the way some animal cells are agglutinated. The agglutination process is dependent on the Concanavalin A concentration, protoplast density, treatment time, the temperature, and the membrane condition, α-D-Methylgluco-pyranosid completely inhibited Con A induced agglutination. The results are discussed in relation to membrane structure and morphology.