Inactivation of Candida albicans by ultraviolet radiation

Summary Inactivation of Candida albicans by ultraviolet (uv) light is markedly dependent upon (a) the cell division stage and (b) the nutrition and growth temperatures of cells both before and after irradiation. Cells grown at 37°C after irradiation show lower survivals than those grown at 25°C. At either recovery temperature, cells which had been cultured before irradiation at 37°C are able to sustain less uv damage prior to inactivation than those cultured at 25°C. The radiosensitivities of budding and non-budding cells are the same when survivals are scored at 25°C; at low uv dosages, cells show slightly poorer recoveries on enriched medium than on minimal medium whereas at higher dosages, their recoveries on both kinds of media are equivalent. In contrast, at 37°C, uv treated non-budding cells are much more susceptible to inactivation than budding cells; non-budding cells also express much poorer recovery on enriched medium than on minimal medium at 37°C whereas budding cells survive equally well on either medium. Though non-budding cells grown for irradiation on minimal or enriched media exhibit the same radiosensitivites, budding cells grown for irradiation on enriched medium are more susceptible to inactivation at 37°C than those grown on minimal medium.The particularly poor recovery by irradiated non-budding cells at 37°C is correlated with their unique tendency to undergo a transitory filamentation when initiating growth at that temperature. Evidence is presented that neither the filamentous growth per se nor the temporary inhibition of cell division associated with filamentation causes the poor recovery. Furthermore, while irradiated non-budding cells at 37°C exhibit singular susceptibility to inhibition of recovery by metabolic antagonists which disturb protein synthesis, the course of their filamentous growth is not affected by such agents. It is concluded that recovery from irradiation and the instigation of cytokinesis by non-budding cells of C. albicans result from different metabolic processes which may be related through a common temperature sensitive step. C. albicans does not photoreactivate and observations on recovery by cells prevented from undergoing immediate postirradiation replication do not indicate the existence of a system for dark repair of DNA damage comparable to that occurring in bacteria. Difficulties attending a valid demonstration of DNA dark repair in yeasts are discussed.     

Relative susceptibilities of caffeine-sensitive and caffeine-resistant strains of Candida albicans to inactivation and mutation by ultraviolet radiation

Summary Stable variants having increased resistance to growth inhibition by caffeine were obtained from four different absolute, amino acid auxotrophs of Candida albicans. Differences in growth rates and expression of auxotrophy between the resistant (Caf^R) variants and their sensitive (Caf^S) progenitors suggest that caffeine resistance arises through suppressor mutations which affect the fidelity of messenger RNA translation.Both Caf^S and Caf^R strains of C. albicans are more susceptible to inactivation by ultraviolet radiation (uv) when grown at 37°C rather than 25°C following exposure. Post irradiation growth on caffeine potentiates ultraviolet inactivation of all Caf^S strains at both temperatures. Depending on its origin, a Caf^R strain (i) may show greater, lesser or the same intrinsic susceptibility to uv inactivation as its Caf^S parent at 25°C or at 37°C and (ii) may or may not be refractory to post-irradiation contact with caffeine. Caf^R variants independently isolated from a given auxotroph are alike in inactivational responses whereas those obtained from different auxotrophs are dissimilar. This implies that different suppressor mutations are unique in the way they affect expression of potentially lethal uv damage and that only one kind of suppressor is obtained by selection for caffeine resistance in a particular auxotroph.The histidine requiring Caf^R strain WB-2CR is much more resistant to uv inactivation that its Caf^S parent WB-2. Moreover, post-irradiation survival of WB-2CR is unaffected by caffeine. However, WB-2CR and WB-2 are equally susceptible to uv-induced reversion to prototrophy. In both strains, caffeine does not enhance uv-induced reversion at 25°C or 37°C and exhibits an antimutagenic activity at high uv dosage at 37°C.The findings reinforce previously reported indications that, in C. albicans, (i) caffeine-sensitive excision-repair of uv damaged DNA does not occur and (ii) caffeine potentiates uv cellular inactivation by disturbing post-irradiation synthesis of protein essential for recovery from non-genetic damage.     

Effects of ergosterol,palmitic acid and related simple lipids on the recovery of Candida albicans from ultraviolet irradiation

Summary Post-irradiation growth on sterols or long chain fatty acids promotes recovery of C. albicans from uv-induced lethal damage. The effect is observed only for cells which are not budding at the time of irradiation. Lipids have no effect on uv mutagenesis. A survey of a number of sporogenous and asporogenous yeasts indicates that the capacity for lipid-induced recovery from uv is a species specific characteristic of yeasts. The behaviors of cells damaged by uv and by amphotericin B, a membrane specific fungicidal antibiotic, suggest that lipids remediate an uv-induced derangement of the structure of the cell membrane critical for the initiation of cell division.     

Resistance of the neck plasma membrane between the mother and the bud of Saccharomyces cerevisiae and Candida albicans to amphotericin B-induced deformation

Abstract The polyene antibiotic, amphotericin B, at high concentrations (5–20 μg/ml) induced particle-free smooth areas in the plasma membranes of Saccharomyces cerevisiae and Candida albicans . These areas occured more or less over the entire plasma membrane of unbudded cells. In budded cells, however, the neck between the mother and bud did not undergo deformation. This suggests the strong interaction between the filamentous ring, which is firmly attached to the neck plasma membrane, and plasma membrane particles in the neck regions.     

Post-irradiation modification of radiation-induced heteroallelic reversion in diploid yeast: Effect of nutrient broth

The effect of post-irradiation growth in complete rich medium on the expression of the reversion to arginine-independence induced by gamma and alpha radiation in a heteroallelic diploid yeast strain (Saccharomyces cerevisiae BZ34) has been studied. During the post-irradiation treatment the reversion frequency increased, reached a peak at about 90 min and decreased thereafter reaching a constant value for treatment periods exceeding 6 h. As determined by the increase in number of budding cells, extensive DNA synthesis took place in cells incubated only in the nutrient medium and not in the omission medium. Hence the observed increase in the reversion frequency is explained on the basis that post-irradiation DNA synthesis is necessary for the expression of gene conversion. The decrease in the reversion frequency for continued treatment with yeast extract, peptone, dextrose (YEPD) is related to the fact that only one daughter of the post-irradiation first cell division is a revertant.The broth effect was not lost when the irradiated cells were first incubated for 90 min in arginine-less medium and then transferred to the broth. Similarly, the broth effect persisted even at doses high enough to induce considerable division delay. These results suggest that the radiation-induced pre-conversional lesions are not susceptible to repair by alternative pathways.     

Unequal division in Saccharomyces cerevisiae and its implications for the control of cell division

The budding yeast, Saccharomyces cerevisiae, was grown exponentially at different rates in the presence of growth rate-limiting concentrations of a protein synthesis inhibitor, cycloheximide. The volumes of the parent cell and the bud were determined as were the intervals of the cell cycle devoted to the unbudded and budded periods. We found that S. cerevisiae cells divide unequally. The daughter cell (the cell produced at division by the bud of the previous cycle) is smaller and has a longer subsequent cell cycle than the parent cell which produced it. During the budded period most of the volume increase occurs in the bud and very little in the parent cell, while during the unbudded period both the daughter and the parent cell increase significantly in volume. The length of the budded interval of the cell cycle varies little as a function of population doubling time; the unbudded interval of the parent cell varies moderately; and the unbudded interval for the daughter cell varies greatly (in the latter case an increase of 100 min in population doubling time results in an increase of 124 min in the daughter cell's unbudded interval). All of the increase in the unbudded period occurs in that interval of G1 that precedes the point of cell cycle arrest by the S. cerevisiae alpha-mating factor. These results are qualitatively consistent with and support the model for the coordination of growth and division (Johnston, G. C., J. R. Pringle, and L. H. Hartwell. 1977. Exp. Cell. Res. 105:79-98.) This model states that growth and not the events of the DNA division cycle are rate limiting for cellular proliferation and that the attainment of a critical cell size is a necessary prerequisite for the "start" event in the DNA-division cycle, the event that requires the cdc 28 gene product, is inhibited by mating factor and results in duplication of the spindle pole body.     

Yeast-phase cell cycle of the polymorphic fungus Wangiella dermatitidis.

The yeast-phase cell cycle of Wangiella dermatitidis was studied using flow microfluorimetry and the deoxyribonucleic acid (DNA) synthesis inhibitor hydroxyurea (HU). Exposure of exponential-phase yeastlike cells to 0.1 M HU for 3 to 6 h resulted in the arrest of the cells in DNA synthesis and produced a nearly homogeneous population of unbudded cells. Treatment of the yeast-phase cells with HU for 9 h or longer resulted in the accumulation of the cells predominantly as budded forms having either a single nucleus in the mother cell or a single nucleus arrested in the isthmus between the mother cell and the daughter bud. Exposure of unbudded stationary-phase cells to 0.1 M HU resulted in the accumulation of the cells in the same phenotypes. Analysis by flow microfluorimetry and cell counts of HU-inhibited mithramycin-stained cells indicated that the eventual progress of HU-inhibited cells from unbudded to the two budded forms was due to the limited continuation of the growth sequence of the cell cycle even in the absence of DNA synthesis, nuclear division, and in some cases nuclear migration. On the basis of these observations and the results of flow microfluorimetric analysis of exponential-phase cells, a map of the yeast-phase cell cycle was constructed. The cycle appears to consist of two independent sequences of events, a budding growth sequence and a DNA division sequence. The nuclear division cycle of yeast-phase cells growing exponentially with a 4.5-h generation time is composed of a G1 interval of 148 min, as S phase of 16 min, and a G2 plus M interval of 107 min.     

Kinetics of Micronucleus Expression In Synchronized Irradiated Chinese Hamster Ovary Cells

The kinetics of expression of radiation-induced micronuclei (MN) in synchronized Chinese hamster cells (CHO) was examined. the purpose of the study was to determine if the cell cycle distribution of a population significantly influences the levels of radiation induced MN, thereby obscuring the exact quantification of the radiation effect. Cells were synchronized by centrifugal elutriation, irradiated, and then different phases of the cell cycle were examined for: cell cycle progression, division probability, and temporal expression of MN. the results demonstrate that the time interval for maximal MN expression is long enough that the position of cells in the cell cycle and radiation induced division delays do not prevent the majority of cells from completing their first post-irradiation mitosis, therefore, expressing MN. By following the progression of synchronized cell populations by flow cytometry and also examining the time of division of individual cells for 24 hr after irradiation, we observed that the maximum number of cells from all phases of the cell cycle are in their first post-irradiation interphase at that time, thus explaining the MN results.     

Kinetics of micronucleus expression in synchronized irradiated Chinese hamster ovary cells

The kinetics of expression of radiation-induced micronuclei (MN) in synchronized Chinese hamster cells (CHO) was examined. The purpose of the study was to determine if the cell cycle distribution of a population significantly influences the levels of radiation induced MN, thereby obscuring the exact quantification of the radiation effect. Cells were synchronized by centrifugal elutriation, irradiated, and then different phases of the cell cycle were examined for: cell cycle progression, division probability, and temporal expression of MN. The results demonstrate that the time interval for maximal MN expression is long enough that the position of cells in the cell cycle and radiation induced division delays do not prevent the majority of cells from completing their first post-irradiation mitosis, therefore, expressing MN. By following the progression of synchronized cell populations by flow cytometry and also examining the time of division of individual cells for 24 hr after irradiation, we observed that the maximum number of cells from all phases of the cell cycle are in their first post-irradiation interphase at that time, thus explaining the MN results.     

Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle

Temperature-sensitive yeast mutants defective in gene CDC24 continued to grow (i.e., increase in cell mass and cell volume) at restrictive temperature (36 degrees C) but were unable to form buds. Staining with the fluorescent dye Calcofluor showed that the mutants were also unable to form normal bud scars (the discrete chitin rings formed in the cell wall at budding sites) at 36 degrees C; instead, large amounts of chitin were deposited randomly over the surfaces of the growing unbudded cells. Labeling of cell-wall mannan with fluorescein isothiocyanate-conjugated concanavalin A suggested that mannan incorporation was also delocalized in mutant cells grown at 36 degrees C. Although the mutants have well-defined execution points just before bud emergence, inactivation of the CDC24 gene product in budded cells led both to selective growth of mother cells rather than of buds and to delocalized chitin deposition, indicating that the CDC24 gene product functions in the normal localization of growth in budded as well as in unbudded cells. Growth of the mutant strains at temperatures less than 36 degrees C revealed allele-specific differences in behavior. Two strains produced buds of abnormal shape during growth at 33 degrees C. Moreover, these same strains displayed abnormal localization of budding sites when growth at 24 degrees C (the normal permissive temperature for the mutants); in each case, the abnormal pattern of budding sites segregated with the temperature sensitivity in crosses. Thus, the CDC24 gene product seems to be involved in selection of the budding site, formation of the chitin ring at that site, the subsequent localization of new cell wall growth to the budding site and the growing bud, and the balance between tip growth and uniform growth of the bud that leads to the normal cell shape.     

Cell cycle model for recombinant Pichia pastoris during glycerol fed-batch cultivation

A cell cycle model is proposed for methylotrophic yeast Pichia pastoris grown on glycerol during fed-batch cultivation. Morphological differentiation of cells, such as unbudded daughter cell, unbudded parent cell and budding cell, is depicted by the model. During the cyclic growth, cells in different cycling period are assumed to undergo sequential shifting dominantly. The input of the cell cycle model is the specific growth rate, which is calculated from the macrokinetic model proposed previously. The cell cycle related variables, such as the fraction of budding cells and the cell density are then simulated. Model validation is carried out with the experimental data of off-line assays.     

Daughter Cell Repair by Mammalian Cells in Culture after Potentially Lethal Radiation Damage

COLONY formation by irradiated surface-attached mammalian cells in culture requires that at least one of the progeny produced at the first post-irradiation division retains the capacity for unlimited division. Abortive colonies result when the irradiated parent cell has experienced sufficient damage to suppress eventual colony formation, but not to prevent the production of a small number of progeny. We now present data which indicate that, if given a suitable environment, progeny from cells “lethally” damaged by X-radiation (not capable of producing a macroscopic colony) can repair damage transferred (sectored) from the parent cell.     

Induced synchrony in Cryptococcus neoformans after release from G2-arrest

Cryptococcus neoformans was grown first to OD 4 under moderate aeration, then diluted 2.5 times with fresh medium, and grown under limited aeration for 5 h. Oxygen concentration decreased from 5-6 mg l(-1) to 1.5 mg l(-1) 1 h after the shift to limited aeration, and remained at a similar level thereafter. In all the eleven strains examined the shift caused unbudded G(2)-arrest in more than half of the cells. In three strains more than 80% of the cells were arrested in unbudded G(2), and, therefore they were selected for synchrony experiments. After being shifted to extensive aeration again, the cells resumed growth by synchronous budding, followed by synchronous nuclear division. This method has turned out to be a good tool to prepare synchronized culture in C. neoformans, especially when a large amount of synchronized cells is needed. This is worthy of attention, since synchronous cultures after release from G(2)-arrest have not been reported yet in any yeast species.     

Dying of Gamma-Irradiated Escherichia coli Studied by the Use of Prophage

Loss of the biological activity of deoxyribonucleic acid in gamma-irradiated Escherichia coli cells was studied. The study is based on two sets of experimental data: (i) post-irradiation heat inducibility of the cells whose chromosomes were "labeled" with the thermoinducible lambdacI857ind prophage, and (ii) post-irradiation capacity of nonlysogenic cells to promote growth of the unirradiated lambdacI857ind phage. The results show that, at the beginning of incubation after irradiation, the number of plaques formed upon heat induction of lysogenic cells was much higher than the viable cell count of the nonheated culture. This high resistance of the heat inducibility gradually decreased during post-irradiation incubation. Finally, after a period of 4 h, there was no difference in sensitivity between the heat inducibility and the colony-forming ability of gamma-irradiated cells. The capacity of gamma-irradiated bacteria to support growth of unirradiated lambdacI857ind is radioresistant; this resistance, in contrast to that of heat inducibility, is much less affected during post-irradiation incubation. A continuous decrease in radioresistance of heat inducibility without a corresponding decrease in radioresistance of the capacity suggests that functional failure of initially undamaged and/or repaired parts of the chromosome gradually develops after irradiation. From the fact that after 4 h all colony formers are capable of being induced by heat, whereas no chromosomal activity can be detected in nonviable cells, two conclusions may be drawn: (i) gamma-irradiated E. coli cells destined to die reach their biological end point within 4 h of post-irradiation incubation; (ii) in most cells, functional failure of the whole chromosome is the immediate cause of death.     

Supersensitivity to levorin and amphotericin B in several Saccharomyces cerevisiae mutants resistant to nystatin]

104 mutants resistant to nystatin were isolated after UV-treatment of two haploid marked strains of Saccharomyces cerevisiae. The analysis of resistance to three polyene antibiotics allowed to determine 8 phenotype classes of mutants including those resistant to nystatin but in various combinations showing hypersensitivity to levorin and (or) amphotericin B. The analysis of UV absorption spectra of sterolic extracts prepared from cells of different mutants showed that similar quality changes in sterol composition could be associated both with polyresistant an supersensitive phenotype. New type of mutants resistant to nystatin and supersensitive to levorin and (or) amphotericin B seems to be promising for studies on the mechanisms of action of polyene antibiotics, the bases of resistance to them and also in consideration of the possibility to increase the efficiency of antimycotic antibiotic therapy.     

Amphotericin B resistance is recessive in Chinese hamster hybrid cells

Previous studies from our laboratory have shown that Chinese hamster V79 cells mutated to high level resistance to amphotericin B have a lower cellular level of cholesterol, the target molecule for the polyene antibiotic. Two amphotericin B-resistant (AMB^R) mutants were each hybridized to their parental amphotericin B-sensitive (AMB^S) V79 cells. All the hybrids derived from AMB^R/AMB^S fusions were as sensitive to polyene antibiotics (amphotericin B, filipin, and pimaricin) as AMB^S/AMB^S hybrids. The AMB^R/AMB^S hybrids were found to contain cholesterol per phospholipids that is comparable to those in AMB^S or AMB^S/AMB^S. The analysis of hybrids formed between mutant and wild-type cells thus indicated that resistance to amphotericin B is a recessive marker, and that the cellular level of cholesterol is compensated in the AMB^S/AMB^R hybrids. Hybrids of AMB^R and AMB^R cells were all resistant, so that the three AMB^R mutants all fell into a single complementation group.     

DNA histograms of irradiated cells: The case of prophase-trapping

A time-dependent DNA histogram is calculated for an irradiated population of cells under the limiting assumption that the cells cannot pass through prophase due to the effects of the radiation. The population is assumed to increase exponentially prior to irradiation, but after irradiation to neither gain nor lose cells. Chromosome-number dispersion is taken into account in the calculation. The qualitative behavior of the calculated and experimental histograms are in reasonable agreement. The quantitative agreement between the two is relatively good at short post-irradiation times but is poor at long post-irradiation times (say, greater than half the doubling time). This suggests that recovery phenomena cannot be neglected at long post-irradiation times.     

The use of amphotericin B to detect inhibitors of cellular cholesterol biosynthesis

Pores formed in the membranes of animal cells by complexes of sterols and the polyene antibiotic amphotericin B can efficiently kill the cells. Thus, in the absence of exogenous sources of cholesterol, inhibitors of enzymes in the cholesterol biosynthetic pathway render cells resistant to amphotericin B. Preincubation of Chinese hamster ovary cells with compactin or 25-hydroxycholesterol, inhibitors of the synthesis of the key intermediate mevalonate, protected cells from amphotericin B killing and this protection was reversed by the addition of exogenous mevalonate. The ability of compactin to confer amphotericin B resistance on normal cells was abolished when cells were provided exogenous cholesterol by the receptor-mediated endocytosis of low density lipoprotein. Low density lipoprotein receptor-defective Chinese hamster ovary cells were not subject to this low density lipoprotein-dependent amphotericin B killing. Exogenous mevalonate did not prevent 4,4,10 beta-trimethyl-trans-decal-3 beta-ol, an inhibitor of mevalonate conversion to sterols, from protecting cells from amphotericin B. A simple two-step protocol in which cells are preincubated (15-24 h) with potential inhibitors and then treated (3-6 h) with amphotericin B was devised to provide a sensitive method for detecting direct (e.g., competitive) and regulatory inhibitors of cholesterol biosynthesis. This protocol may prove useful in detecting potential antihypercholesterolemia drugs and is currently being used to isolate mutants in receptor-mediated endocytosis.     

Phospholipid enrichment of Saccharomyces cerevisiae and its effect on polyene sensitivity

Sensitivity to polyene antibiotics, e.g., nystatin, amphotericin B, and filipin, was determined in phosphatidylcholine (PC) or phosphatidylethanolamine (PE) or phosphatidylserine (PS) enriched Saccharomyces cerevisiae cells, using glutamic acid, phenylalanine, glycine, and lysine transport as an index of polyene antibiotic action. As compared with normal cells, phospholipid-enriched cells acquired resistance towards different polyenes. However, the sensitivity of glutamic acid transport towards nystatin remained unaffected in PC-, PE-, or PS-enriched cells. In contrast to nystatin, the other two polyenes were more effective in checking the influx of amino acids. Results demonstrated that the specific enrichment of PC, PE, or PS could selectively protect S. cerevisiae cells from polyene antibiotic action.     

UV survival of human mycoplasmas: evidence of dark reactivation in Mycoplasma buccale.

The inactivation by ultraviolet (UV) light irradiation of mycoplasma cells of five human strains was monitored by investigating the colony-forming ability. The survival curves of five strains tested indicated that the cells of Mycoplasma buccale only are single and homogenously susceptible to UV light. The effect of the repair inhibitor, caffeine, on the colony-forming ability of UV-irradiated cells was investigated with M. buccale because of its homogenous susceptibility to UV light. The colony formation of irradiated cells was markedly depressed by post-irradiation treatment with caffeine at concentrations that had little or no effect on the colony formation of unirradiated cells. The colony-forming units (CFU) of UV-irradiated cells which were kept in broth without caffeine in the dark increased without a lag as the time in the dark increased. The colony-forming ability of the irradiated cells completely recovered after 3 hr in the dark. However, when irradiated cells were kept in the presence of caffeine, no increase in their CFU was observed. The mode of action of caffeine on UV-irradiated cells closely resembles that described for other organisms which possess dark reactivation systems for UV-induced damage in deoxyribonucleic acid (DNA). Thus, the results obtained provide evidence for the existence of a dark repair function in M. buccale.