Normal Yeast Cells Research Articles

Blastogenic responses of lymphocytes from mice immunized by sublethal infection with yeast cells of Histoplasma capsulatum.

Blastogenic responses of spleen cells to histoplasmin and ribosomal antigens and to the mitogens concanavalin A. phytohemagglutinin, and lipopolysaccharide were studied in normal and immunized mice (10(5) live yeast cells of Histoplasma capsulatum given by the subcutaneous route). Cells (10(6) per well) were cultured with and without antigens and mitogens in microtiter plates with RPMI 1640-5% heat-inactivated normal mouse serum for 72 h at 37 degrees C. Cells were harvested after a 16- to 18-h pulse with 1 microCi of [3H]thymidine (6.7 Ci/mol), and thymidine incorporation was measured by scintillation counting. The initial blastogenic response to concanavalin A (54 X 10(3) cpm) was suppressed (P less than 0.001) from 4 to 14 days post-immunization and returned to control levels on day 21. The response to phytohemagglutinin was suppressed up to 21 days. Lipopolysaccharide responses, however, were affected to a lesser degree. Blastogenic responses to histoplasmin and H. capsulatum ribosomes were similar on day 0 in normal and immune lymphocytes, but by day 4 cells from immunized mice were more responsive (P less than 0.01). The maximum response to H. capsulatum antigens was detected on day 42 and was 9- to 16-fold higher than in controls. These results demonstrate in vitro responses of primed lymphocytes on exposure to H. capsulatum antigens and suppressed responses to mitogens during early stages of the immune response.

New views of the biochemistry of eucaryotic DNA replication revealed by aphidicolin, an unusual inhibitor of DNA polymerase α

Just two years ago one of the hottest topics in eucaryotic DNA synthesis was identification of the polymerase(s) responsible for chromosomal DNA replication. Today the controversy is nearly over; only the details remain to be filled in. DNA polymerase (Y is clearly the principal DNA polymerase required for DNA replication; major credit for this conclusion goes to the unusual antibiotic, aphidicolin. Aphidicolin is a tetracyclic diterpene tetraol, obtained from Cephalosporium aphidicola and certain other fungi. Its structure (shown below) is steroid-like, but it contains an unusual new ring system (Brundret et al., J. Chem. Sot. Chem. Comm. 1027-l 028, 1972). Aphidicolin inhibits the growth of eucaryotic cells (except those, like normal yeast cells, that are impermeable to it) and the growth of certain animal viruses (SV40, herpes virus and vaccinia virus) by inhibiting replicative DNA synthesis (Bucknali et al., Antimicrob. Agents Chemother. 4, 294-298, 1973; Krokan et al., Biochemistry 78, 4431-4443, 1979). The recent excitement about aphidicolin began with the demonstration that it is a specific, direct inhibitor of animal DNA polymerase 01 but is without effect on polymerases p or y (Ikegami et al., Nature 275, 458460, 1978). More recent studies show that it also inhibits the a-like DNA polymerases of plants (Sala et al., FEBS Lett. 7 7 7, 93-98, 1980) and of yeast (Plevani et al., Antimicrob. Agents Chemother. 78, 5057, 1980) and inhibits the DNA polymerases coded by vaccinia virus and herpes virus (Pedrali-Noy and Spadari, J. Virol. 36, 457-464, 1980). Although aphidicolin does inhibit DNA polymerase II of yeast (Plevani et al., lot. cit.), which, like procaryotic DNA

Saturated fatty acid-starved cells of Saccharomyces cerevisiae grown in the presence of cerulenin and oleic acid.

Cell growth of Saccharomyces cerevisiae ATCC 12341 inhibited by the antibiotic cerulenin, a specific inhibitor of fatty acid synthesis, was restored by oleic acid (18 : 1) to give saturated fatty acid-starved cells, which could not grow when again transferred into a fresh synthetic medium containing the antibiotic and oleic acid. The growth of the saturated fatty acid-starved cells was restored when they were transferred into a medium supplemented with myristic acid (14 : 0), pentadecanoic acid (15 : 0), and palmitic acid (16 : 0) in the presence of cerulenin and oleic acid. Cellular saturated fatty acid content in the growth-restored cells was also restored to about two-thirds of that of the normal yeast cells. The DNA, RNA, and cell wall synthetic capabilities of the saturated fatty acid-starved cells were almost normal, but the L-leucine uptake and cytochrome pattern were severely impaired. These impairments were reversed on supplying palmitic acid. The decrease of L-leucine uptake of the yeasts was also caused by the addition of cerulenin alone. However, since the decrease occurred later than the inhibition of fatty acid synthesis, it was considered to be a secondary effect. These results, obtained by using the saturated fatty acid-starved cells, indicate that the membranes of S. cerevisiae require certain amounts of saturated fatty acid and that the membrane functions (energy metabolism, transport, and so on) are impaired by starvation of saturated fatty acids.

Evidence for a functional change in the plasma membrane of yeasts associated with the mechanism of arsenate resistance

In previous reports experimental evidence has been presented indicating a possible relationship between the formation of arseno-phosphoinositides and the active transport of arsenate-phosphate in yeast cells. There is an increment in the amount of inositides in yeast cells adapted to grow in the presence of toxic concentrations of arsenate. These cells exhibit a highly reduced arsenate uptake but maintain their capacity to transport phosphate. Since, in normal (nonadapted) yeast cells, both arsenate and phosphate anions share the same transport system, a study was conducted to obtain further information about the plausible role played by the phosphoinositides in the active transport system of arsenate and their inhibition that allows the cells to grow in the presence of the toxic. Studies on [ 32P]orthophosphate and [ 74As]arsenate incorporation into phospholipids in normal and arsenate-adapted yeast show that: The 32P incorporation into phospholipids is two times larger in normal yeast as compared to arsenateadapted ones. The 32P labeling was maximum for phosphatidylinositol in normal yeasts while in the arsenate-adapted cells it was maximum for phosphatidylcholine. This incorporation was largely inhibited by arsenate in normal yeasts and minimal in the arsenate-adapted ones. Cell fractionation shows that the maximum incorporation of [ 32P]orthophosphate resides in the microsomal fraction, while the incorporation of [ 74As]arsenate resides mainly in the cell envelope fraction which incorporates 86% of the 74As label. Phosphate is capable of inhibiting the 74As-inositide complex formation and destroying the previously formed one. Yeast cells prelabeled with [2C- 3H]myoinositol showed a reduced turnover rate of phosphoinositides even when transporting nontoxic amounts of arsenate. The involvement of the inositides as a regulatory mechanism in the phosphate-arsenate active transport system in yeast cells is discussed.

Chapter 8 Preparation and Growth of Yeast Protoplasts

Publisher Summary This chapter discusses preparation and growth of yeast protoplasts. Many metabolic activities of yeast protoplasts prepared with either snail gut or microbial enzymes are comparable to those of intact cells when the protoplasts are incubated in appropriate medium. These protoplasts are able to synthesize protein and nucleic acid as well as cell wall materials. In solid medium containing gelatin or agar, protoplasts grow and eventually develop into normal yeast cells. Yeast protoplasts are useful for study of the regulation of macromolecular synthesis, especially of enzymes and cell wall materials, because protoplasts can be lysed readily by osmotic shock or detergents, unlike intact yeasts. Procedures for the preparation of yeast protoplasts can be classified in three major groups: (1) mechanical or autolytic methods, (2) specific inhibition of cell wall synthesis, and (3) enzymatic dissolution of the wall. The chapter describes methods adopted by several investigators for the preparation of yeast protoplasts and for the use of protoplasts to study growth and macromolecular synthesis. Factors affecting the growth and metabolic activities of protoplasts are described and growth and macromolecular synthesis is discussed in the chapter.

The effect of oxygen concentration on the growth and metabolism of Saccharomyces cerevisiae grown with excess of potassium or in potassium-deficient media.

1. Saccharomyces cerevisiae cells grown in limiting K(+) concentration have their growth inhibited by O(2) concentrations above 40%. With these conditions the cells grow very large and are unable to maintain ionic gradients when washed with water. 2. Cells grown in excess of K(+) showed the same pattern of change in cell size with change in O(2) concentration, but the magnitude of the changes was much less. Cells grown in excess of K(+) were not leaky. 3. Cell death, growth and development of ;leakiness' were not correlated in the cells grown in limiting K(+) concentration. 4. The activities of both alcohol dehydrogenase and cytochrome oxidase were higher in K(+)-deficient cells than in the cells grown with excess of K(+). The differences were much larger when the measurements were made on a cellular basis than when made on a protein basis. 5. In 100% O(2) 3mm-K(+) in the medium was sufficient to produce normal yeast cells.

Effect of saramycetin on the fine structure of Histoplasma capsulatum.

WhenHistoplasma capsulatum yeast cells were grown in the presence of partially inhibitory concentrations of saramycetin, electron microscopy revealed that the proportion of apparently damaged cells was a function of the concentration of the antibiotic. Evidence of early damage consisted of irregularity of outline, followed by progressive lysis of internal structures leading to residual dense granules, membranes, and the cell wall. Normal yeast cells of the strain studied were found to possess intranuclear bodies of a type apparently not previously described.

Modification of K + transport in yeast by the polyene antifungal antibiotic N-acetylcandidin

The action of the polyene antifungal, N-acetylcandidin (NAC), on K + retention and transport by Saccharomyces cerevisiae was examined, since K + loss is an early result of polyene binding by sensitive cells. Log-phase yeast in a K +-free medium at 30° retained its K + relatively effectively in the presence of glucose, but slow losses occurred in the absence of an energy source. This loss was sharply increased by 20 μg NAC/mg cells; the effect was temporarily prevented by glucose. NAC-treated yeast could still accumulate added K + (30°; 0.1 M glucose) but to a lesser extent than did the untreated cells. This accumulation is energy dependent, since at 0° or when glucose was omitted, both cell types took up K + only with the concentration gradient. Adding 100 μg NAC/mg cells further increased the rate of K + loss and prevented the accumulation of K +. The antibiotic does not appear to produce true holes in the cell membrane, since K + was retained at 0°, even by NAC-treated cells which had already lost a part of their K + at 30°. Sodium enhanced both K + retention and accumulation in normal yeast cells. With NAC-treated cells, Na + increased K + loss and reduced K + accumulation when added with the K +. NAC did not seem to increase the permeability of the yeast cell membrane, nor did it completely prevent the active uptake of K + except at high antibiotic levels which eliminated energy-yielding reactions. The binding of NAC apparently alters the membrane, thus reducing indirectly the efficiency of ion transport, probably both into and out of the cell, and changing the usual Na +-K + transport balance. This reduces the ratio of intracellular to extracellular K + which the treated cell can maintain. It is proposed that this alteration of the transport system is an early, general effect of polyenes and that polyenes other than NAC produce additional changes which are important to, but not essential for, antifungal action.

Enzyme Alteration by Ionizing Radiation

(2) revealed that the increase in catalase activity was accompanied by changes in some of the other properties of the catalase. Its sensitivity to destruction by heat and its sensitivity to inactivation by high substrate concentrations were increased considerably; the of treated cells resembles the extracted yeast catalase and crystalline catalases in solution. These properties of the changed in situ, without release of the from the cell, by ultraviolet light, freeze-drying, and protoplasmic poisons such as chloroform and toluene, are now considered to result from the direct or indirect effect of these agents on the structure of the enzyme. This phenomenon has been called enzyme alteration (2). In further investigations (3-6) evidence was found in support of the hypothesis that the catalase of normal yeast cells exists as partially unfolded molecules at some interface of the oil/water type within the cell. The action of altering agents was envisaged as bringing about a desorption of the from the interface with consequent changes in the kinetic and thermodynamic properties of the catalase, the then resembling extracted yeast catalase or crystalline catalase in bulk. A study of the effect of ultraviolet light in altering yeast catalase (7, 8) revealed that it is closely associated with ribonucleic acid (RNA) at the interface. The present investigation shows that the Euler effect may be induced by X- and 7y-ray irradiation and that it is also due to alteration. A calculation of the size of the target involved in the process supports the finding that intracellular yeast catalase is in intimate association with some substance of high molecular weight, perhaps RNA.

A comparative study of the preparation and properties of ribonucleic acids of yeast

Highly purified preparations of ribonucleic acid have been obtained from baker's yeast employing newly modified methods of isolation. Solubility studies show that these materials are inhomogeneous, apparently containing a range of molecular structures of varying weight, and a dependence of solubility on molecular size is reported. It is suggested that homogeneous samples have never been obtained from cells. In addition, it is proposed tentatively that normal yeast cells possess a variety of ribonucleic acids and that known isolation methods modify the native structures thereby adding to the complexity of isolated preparations. The foregoing conclusion is supported by solubility studies designed to compare qualitatively the compositions of two different preparations of ribonucleic acid by examining an experimental mixture of these samples. The presence of common components in otherwise dissimilar and inhomogeneous preparations is suggested. Number-average molecular weights are reported for the various preprations with a value in one case of 66,000 after prolonged dialysis at 0 °C. This average size exceeds any recorded for preparations of the ribonucleic acid of yeast.