Role of Reactive Oxygen Species and Glutathione in Inorganic Mercury-Induced Injury in Human Glioma Cells

The present study was undertaken to examine the role of reactive oxygen species (ROS) and glutathione (GSH) in glia cells using human glioma cell line A172 cells. HgCl₂ caused the loss of cell viability in a dose-dependent manner. HgCl₂-induced loss of cell viability was not affected by H₂O₂ scavengers catalase and pyruvate, a superoxide scavenger superoxide dismutase, a peroxynitrite scavenger uric acid, and an inhibitor of nitric oxide N^G-nitro-arginine Methyl ester. HgCl₂ did not cause changes in DCF fluorescence, an H₂O₂-sensitive fluorescent dye. The loss of cell viability was significantly prevented by the hydroxyl radical scavengers dimethylthiourea and thiourea, but it was not affected by antioxidants DPPD and Trlox. HgCl₂-induced loss of cell viability was accompanied by a significant reduction in GSH content. The GSH depletion was almost completely prevented by thiols dithiothreitol and GSH, whereas the loss of viability was partially prevented by these agents. Incubation of cells with 0.2 mM buthionine sulfoximine for 24 hr, a selective inhibitor of -glutamylcysteine synthetase, resulted in 56% reduction in GSH content without any change in cell viability. HgCl² resulted in 34% reduction in GSH content, which was accompanied by 59% loss of cell viability. These results suggest that HgCl₂-induced cell death is not associated with generation of H₂O₂ and ROS-induced lipid peroxidation. In addition, these data suggest that the depletion of endogenous GSH itself may not play a critical role in the HgCl₂-induced cytotoxicity in human glioma cells.     

X-Ray and Ultraviolet Sensitivity of Synchronized Chinese Hamster Cells at Various Stages of the Cell Cycle

Populations of Chinese hamster cells, synchronized by selecting for cells at or close to division, were exposed to 250 kvp x-rays and to ultraviolet light at different stages of the cell cycle and colony-forming ability examined thereafter. These cells were found to be most resistant to x-rays during the latter part of the DNA synthetic period (S) and to be about equally sensitive before (G₁) and after (G₂) this period. Multitarget type curves of the same slope (D_o ~ 200 rad) only approximately fitted the survival data at different stages in the cycle. The changes in response were primarily due to variations in the shoulders (or extrapolation numbers) of the curves however. The response to ultraviolet light differed from that to x-rays. Resistance was greatest in G₂ and changes in both shoulder and slope of the survival curves occurred throughout the cell cycle. The x-ray and ultraviolet responses for component stages of the cell cycle were respectively compounded into expected survival data for a log phase asynchronous population of hamster cells and found to agree well with direct experiment.     

Response of potassium retentivity and survival of yeast to farultraviolet,near-ultraviolet and visible,and x-radiation

Potassium retentivity and survival of yeast were studied after exposure to various kinds and conditions of irradiation. The radiations used were: 2537 A ultraviolet, 3500 to 4900 A long-ultraviolet and short visible, and 250 kvp¹ x-rays. Both potassium retentivity and survival are decreased by these radiations. The dose-response of survival is about 16 times as sensitive as is potassium retentivity after 2537 A irradiation. Potassium retentivity is about twice as sensitive as survival after irradiation of 3500 to 4900 A. Survival after x-irradiation under aerobic conditions is five times as sensitive as potassium retentivity. Survival of cells irradiated with x-rays under anaerobic conditions was about half as sensitive as under aerobic conditions. The response of potassium retentivity to x-radiation at 25°C. under anaerobic conditions is only slightly affected below 160 kr, at which dose the slope abruptly increases to that obtained under aerobic conditions; lowering the temperature to 0°C. moves this point to about 300 kr. These differential effects are indicative of interaction of radiations with the yeast cell at sites that independently control survival and the retention of potassium.     

THE MECHANISMS OF X-RAY EFFECTS ON CELLS

Irradiation of Tradescantia microspores does not increase subsequent sensitivity to x-rays as measured by the frequency of induced chromosomal aberrations curing the nuclear cycle. The slight decrease in sensitivity is to be expected because acentric fragments are less sensitive than the centric chromosomes. The physiological effects of x-rays appear to be of minor importance in causing injury or death of individual cells, and most of the deleterious effects can be attributed to "direct hits" which produce chromosomal alterations. In the reaction of tissues to x-rays the physiological effects may play a more important part.     

Multiplication and Fermentation of Saccharomyces cerevisiae under Carbon Dioxide Pressure in Wine

Conditions for rapid fermentation of sugar in wine under pressure were sought for use in continuous production of naturally fermented sparkling wine. Wine yeast growth and fermentation were measured under CO(2) pressure. The medium was white wine with added glucose. Pressure was very inhibitory to growth, especially at low pH or high alcohol concentration. Use of various strains of wine yeast, cultures of various ages, or cells adapted to wine did not give more rapid growth. Addition of nutrients increased growth, but under no conditions was growth rapid enough to bring about sufficiently rapid fermentation rates. Conditions for rapid fermentation were sought by use of high levels of cells as inocula. Fermentation rates in wine also were inhibited by pressure, and were dependent on pH and alcohol levels. Addition of nutrients did not increase the fermentation rate, but rapid fermentation rates were obtained, under pressure, by inoculation with high levels of cells adapted several weeks to the base wine. Thus, continuous sparkling-wine production might be practical with proper amounts of adapted cells used as inocula, or perhaps with reuse of the fermentation culture.     

Effect of starving and dowex 50 treatment on growth of normal and x-irradiated yeast

1. Effects of starvation or treatment with a cation exchange resin, dowex 50, parallel in some respects those seen earlier on the respiration and fermentation of bakers' yeast receiving 90,000 r of 250 kv. x-rays. Starvation increased the radiosensitivity of cell division processes whether measured by colony formation or by turbidimetric determination of growth in a liquid medium. The dowex 50 enhanced the radiation effect by the latter measure but appeared to increase colony formation of irradiated yeast. 2. The effects on growth differ from those on respiration and fermentation in that the exchange resin treatment did not inhibit colony formation further, and neither starvation nor resin appreciably altered the growth of non-irradiated yeast. 3. Two effects of radiation are seen in these experiments: (a) a permanent inhibition of growth, and (b) a temporary inhibition of the remaining cells resulting in delay of growth. 4. The irradiated cell is more dependent on certain aspects of its environment in terms of growth responses as well as in terms of metabolism (i.e. respiration and fermentation). Whether or not potassium plays a role in the growth response as it does in the metabolic response cannot be ascertained from the present data.     

CONTINUOUS ALCOHOL/MALOLACTIC FERMENTATION OF GRAPE MUST IN A BIOREACTOR SYSTEM USING IMMOBILIZED CELLS

Three cultures immobilized by entrapping within alginate gel beads and packed in near-horizontal acrylic columns (15.0° angle) were used for alcohol/malolactic fermentation of grape must. Immobilized cells of Saccharomyces cerevisiae spp. chablis were placed in the 1st column, S. cerevisiae cells (an alcohol-sucrose-tolerant yeast) in the 2nd and the Lactobacillus delbrueckii cells in the 3rd column. Grape must with different levels of sugar(s), were each fed to the bioreactor columns at dilution rate of 0.74 h⁻¹ and recycled at 37.0C. The percent fermentation efficiency and yield using the 1st and 2nd columns for grape must containing 33.3% sugar(s) were 92.9 and 91.5%, respectively, and the wine had 15.5% alcohol after 23 cycles (∼ 50 h fermentation). The viability of the immobilized yeast cells in the alginate gel-bead was 84%± 4.0. Immobilized Lactobacillus delbrueckii cells were then added to the 3rd column (in series 37.0C) and the three cultures resulted in alcohol/malolactic fermentation of the grape must, evidenced by the high level of alcohol formed and simultaneous transformation of malic to lactic acid. Sensory evaluation of the wine scored high (7.8 ± 2.0 based on a value of 10.0) and indicated the potential of using multiple immobilized cells of two specific yeast cultures and a malolactic Lactobacillus for wine production.     

Freezing stress and membrane injury of Norway spruce (Picea abies) tissues

Effects of sub-zero temperatures (−5 to −35°C) on the tissues of needles, buds and shoots of Norway spruce [ Picea abies (L.) Karst.] were studied. The freezing caused increased efflux of cellular electrolytes. Freezing injury of the primordial shoots and 1-year-old shoots was the result of the spontaneous freezing of a deep supercooled cellular water. The crystallization injures the cellular membranes leading to the loss of semipermeability and to the drastic efflux of K⁺. In the needles there was no deep supercooling of water and two patterns of changes in the membranes, depending upon the range of the applied temperatures, could be distinguished. At 0 to – 25°C, which do not kill the cells, we observed a disturbance in the membrane semipermeability as monitored by electrolytes efflux within a few hours after thawing of the needles. At lower temperatures (−35°C) we observed irreversible loss of the membrane semipermeability, and death of the tissue. Those changes occurred 10 h after thawing and were probably caused by the released lytic enzymes and some toxic compounds, which acted on the cellular membranes.     

Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

OPTIMIZATION OF FERMENTATION CONDITIONS AND PURIFICATION OF CORDYCEPIN FROM Cordyceps militaris

The fermentation medium and conditions for the production of cordycepin were optimized in static culture using single-factor experiments, Placket–Burman design, a central composite design, and response surface methodology. Among seven variables including temperature, pH, and the concentrations of glucose, tryptone, yeast extract, KH₂PO₄, and MgSO₄ · 7H₂O, temperature and the concentrations of yeast extract and tryptone were found to be the important factors that significantly affected cordycepin production. The optimized medium consisted of yeast extract 9.00 g/L and tryptone 17.10 g/L, while the optimized culture conditions consisted of seed age 3 days, with an inoculum size of 10% and incubation temperature of 27.1°C. A maximum cordycepin yield of 7.35 g/L was achieved in a 5-L fermenter under the optimized conditions. Next, cordycepin was partially purified and determined. The resulting product showed 90.54% high-performance liquid chromatography (HPLC)–ultraviolet (UV) purity. Therefore, cordycepin was applied to a cell viability assay on SH-SY5Y cells and RM-1 cells. Cordycepin can inhibit the proliferation of RM-1 cells with IC₅₀ of 133 µmol/L, but it has no inhibitory effect on SH-SY5Y cells.

Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.     

Role of electrolytes and starvation in altering apparent radiosensitivity of baker's yeast

1. Respiration and fermentation of yeast receiving 90,000 r of 250 kv. x-rays were studied under a variety of conditions. This dose will nearly completely inhibit growth or colony formation. 2. The apparent effects of irradiation are quite dependent on the K⁺ and H⁺ of the suspending medium. At pH 4.5 stimulatory effects were observed in KH₂PO₂ buffer and inhibition in potassium-free (T-S-T) buffer. At pH 6.5 the situation was reversed and the effects were very small (about 10 per cent). Addition of K⁺ to irradiated yeast in T-S-T buffer at pH 4.5 can completely reverse the inhibition seen. 3. Starving increases the apparent radiosensitivity of respiration and fermentation, probably by depletion of metabolite and/or electrolyte reserves. 4. Treatment with a cation exchange resin (dowex 50) results in marked inhibition of these processes in irradiated yeast, either fresh or starved. This was most effective if given after irradiation. Almost complete inhibition of anaerobic CO₂ production occurs with starvation, irradiation, and dowex treatment combined. 5. The effects of starvation and cation exchange resin treatment can be reversed, though not completely, by adding K⁺ to the medium.     

Dielectric Behavior of Yeast Cells Treated with HgCl2 and Cetyl Trimethyl Ammonium Bromide

The change in dielectric properties caused by the destruction of the transport barrier of yeast cells has been investigated. Dielectric measurements were made over the frequency range of 1 kc to 2 Mc by using “leaky” yeast cells prepared by treatments with HgCl₂ or CTAB (cetyl trimethyl ammonium bromide). The Hg-treated cells were observed to give smaller dielectric constants and lower critical frequencies as compared with that of the intact cells, while the CTAB-treated cells gave no clear-cut dielectric dispersion. These observations are interpreted on the basis of Maxwell-Wagner's theory as indicating the changes in the intracellular conductivity, the membrane capacitance and the membrane conductance.     

Mercuric chloride inhibits the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

Glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1). Mercuric chloride (HgCl₂) is a highly toxic compound that inhibits glutamate uptake in astrocytes, resulting in excessive extracellular glutamate accumulation, leading to excitotoxicity and neuronal cell death. The mechanisms associated with the inhibitory effects of HgCl₂ on glutamate uptake are unknown. This study examines the effects of HgCl₂ on the transport of ³H-d-aspartate, a nonmetabolizable glutamate analog, using Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2), as a model system. Additionally, studies were undertaken to determine the effects of HgCl₂ on mRNA and protein levels of these transporters. The results indicate that (1) HgCl₂ leads to significant (p<0.001) inhibition of glutamate uptake via both transporters, but is a more potent inhibitor of glutamate transport via GLAST and (2) the effect of HgCl₂ on inhibition of glutamate uptake in transfected CHO cells is not associated with changes in transporter protein levels despite a significant decrease in mRNA expression; thus, (3) HgCl₂ inhibition is most likely related to its direct binding to the functional thiol groups of the transporters and interference with their uptake function.     

Mercury translocation in and evaporation from soil. III. quantification of evaporation of mercury from podzolized soil profiles treated with Hg Cl

Mercury evaporation from undisturbed iron‐humus podzol lysimeters was measured over 3 months after treatment with HgCl₂ spiked with radioactive ²⁰³Hg. The relative evaporation rate from HgCl₂ treated soils followed the sum of two exponential functions. Because evaporation asymptotically approaches zero with time, the integral of the fit curve represents the evaporative loss in percent of atmospheric deposition. For the soil investigated, about 5% of atmospheric Hg deposition was reemitted into the atmosphere. It is hypothesized that mercury evaporation can decrease the leaching of mercury in and from soil significantly; this effect is probably increasing with decreasing rain acidity or soil acidity. Mercury deposited as soluble salt remains susceptible to reemission to air for 300 d after incorporation into the soil matrix. Indications are found that Hg evaporation from soils in geological background areas predominantly derives from recent atmospheric Hg deposition and not from geological sources.     

Studies supporting the use of mechanical mixing in large scale beer fermentations

Brewing fermentations have traditionally been undertaken without the use of mechanical agitation, with mixing being provided only by the fluid motion induced by the CO₂ evolved during the batch process. This approach has largely been maintained because of the belief in industry that rotating agitators would damage the yeast. Recent studies have questioned this view. At the bench scale, brewer’s yeast is very robust and withstands intense mechanical agitation under aerobic conditions without observable damage as measured by flow cytometry and other parameters. Much less intense mechanical agitation also decreases batch fermentation time for anaerobic beer production by about 25% compared to mixing by CO₂ evolution alone with a small change in the concentration of the different flavour compounds. These changes probably arise for two reasons. Firstly, the agitation increases the relative velocity and the area of contact between the cells and the wort, thereby enhancing the rate of mass transfer to and from the cells. Secondly, the agitation eliminates spatial variations in both yeast concentration and temperature, thus ensuring that the cells are maintained close to the optimum temperature profile during the whole of the fermentation time. These bench scale studies have recently been supported by results at the commercial scale from mixing by an impeller or by a rotary jet head, giving more consistent production without changes in final flavour. It is suggested that this reluctance of the brewing industry to use (adequate) mechanical agitation is another example where the myth of shear damage has had a detrimental effect on the optimal operation of commercial bioprocessing.     

HgCl2-induced alteration of actin filaments in cultured primary rat proximal tubule epithelial cells labelled with fluorescein phalloidin

When proximal tubule epithelial cells are exposed to HgCl₂, cytoplasmic blebs are formed. These represent on early, potentially reversible response to injury. These blebs are accompanied by reorganization of cytoskeletal proteins, and pre-sumably by alternations in cytoskeletal-plasma membrane interactions. Ca²⁺-activated proteinases, such as calpain, are known to affect cytoskeletal proteins and to be involved in diverse cellular processes. However, the role of calpains in cytotoxicity d due to HgCl₂ is unknown. To determine the relationship between Factin, calpain, and HgCl₂ toxicity, cells were stained with fluorescein phalloidin before and after treatment with HgCl₂. Cells were grown on coverslips and exposed to HgCl₂ (10 or 25 M) in the presence or absence of the calpain inhibitor, leupeptin. Untreated cells were flat, polygonal, and contained many fluorescent-stained cables of actin filaments. Generally, cells exposed to HgCl₂ became pleomorphic and contracted as the blebs formed. These cells showed fewer actin cables and fluorescence was seen mostly as either compact areas of dense stain or as peripheral rings. In many cells, actin cables and filaments were completely absent. Disappearance of F-actin was initially seen by 2 min after exposure to HgCl₂. Thus, disruption of the actin cytoskeleton and blebbing were found to be early events in HgCl₂ toxicity. When leupeptin was used with HgCl₂ treatment, the actin staining appeared similar to that of untreated cells. These findings clearly illustrate that HgCl₂ injury to proximal tubule epithelial cells causes rearrangement and alteration of F-actin which may involve the activation of calpain.Abbreviations HgCl₂ mercuric chloride - PTE proximal tubule epithelium - [Ca²⁺]_i cytosolic ionized calcium - [Ca²⁺]_e extracellular calcium - PBS phosphate buffered saline Supported by Navy N00014-88-K-0427 & NIH DK15440. This is contribution No. 2905 from the Cellular Pathobiology Laboratory.     

The Composition of Jerusalem Artichoke (Helianthus tuberosus L.) Spirits Obtained from Fermentation with Bacteria and Yeasts

The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains 3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains Bc16a and D₂ and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K. fragilis. The highest ethanol yield was achieved when Z. mobilis 3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94 % of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by‐products compared to the distillates resulting from the single species process. Ester production of 0.30–2.93 g/L, responsible for the aromatic quality of the spirits, was noticed when K. fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04 g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z. mobilis, were lower than those achieved for yeasts.