Rotational dynamics of erythrocyte spectrin.

The rotational diffusion of erythrocyte spectrin has been measured using time-resolved phosphorescence anisotropy. The anisotropy of the spectrin dimer decays to zero with a time constant of 3 microseconds at 21 degrees C. The results are compared with the correlation times predicted for the anisotropy decay of an equivalent sphere and rigid rod. The data indicate that the ribbon-like spectrin molecule possesses considerable torsional and segmental flexibility. These motions are restricted, but not abolished, when spectrin is reconstituted into cross-linked cytoskeletal protein networks, or bound to spectrin-actin depleted erythrocyte membrane vesicles.

T helper lymphocyte depression in early human pregnancy.

Peripheral blood lymphocyte subclasses were determined in 60 women with normal pregnancies, 20 from each trimester, and in 20 controls using automated flow cytofluorimetry. The cells were stained with the monoclonal antibodies OKT3, OKT4 and OKT8 to stain total T cells, T helper and T suppressor-cytotoxic lymphocytes, respectively. A polyvalent rabbit anti-human Ig serum was used to stain B lymphocytes. Absolute numbers of T lymphocytes were significantly reduced in both the first and second trimesters. This was due to a significant decrease in T helper lymphocytes and a smaller, statistically not significant, reduction in the number of T suppressor lymphocytes. There was no significant change in lymphocyte subclasses during the third trimester. Total lymphocyte numbers were normal throughout pregnancy.

Induction of heat, freezing and salt tolerance by heat and salt shock in Saccharomyces cerevisiae.

Stress tolerance of Saccharomyces cerevisiae was examined after exposure to heat and salt shock in the presence or absence of the protein synthesis inhibitor cycloheximide. Cells heat-shocked (37 degrees C for 45 min) in the absence of cycloheximide demonstrated increased tolerance of heat, freezing and salt stress. For cells heat-shocked in the presence of cycloheximide, heat and salt tolerance could still be induced, although at lower levels, while induction of freezing tolerance was completely inhibited. These results indicated that while heat shock proteins (hsps) may contribute to induced heat and salt tolerance they are not essential, although induction of freezing tolerance appears to require protein synthesis. Exposure of cells to salt shock (300 mM NaCl for 45 min) induced stress protein synthesis and the accumulation of glycerol, responses analogous to induction of hsp synthesis and trehalose accumulation in cells exposed to heat shock. Cells salt-shocked in the absence of cycloheximide showed a similar pattern of induced stress tolerance as with heat, with increased tolerance of heat, salt and freezing. Cells salt-shocked in the presence of cycloheximide continued to show induced heat and salt tolerance, but freezing tolerance could not be induced. These results lend support to the hypothesis that hsp synthesis is not essential for induced tolerance of some forms of stress and that accumulated solutes such as trehalose or glycerol may contribute to induced stress tolerance.

Stress tolerance and membrane lipid unsaturation in Saccharomyces cerevisiae grown aerobically or anaerobically.

Saccharomyces cerevisiae cells grown either aerobically or anaerobically were tested for tolerance to a brief heat stress (52 degrees C, 5 min) or oxidative stress (20 mM H2O2, 15 min). Tolerance was related to growth phase, in that stationary phase cells were intrinsically more resistant to heat or oxidative stress than exponential phase cells. A mild heat shock (37 degrees C, 30 min) induced thermotolerance and oxidative tolerance in both aerobic and anaerobic cells. However, prior exposure to a low concentration of H2O2 (0.1 mM, 60 min) induced protection against the lethal concentration of H2O2 but not against the lethal temperature. Sensitivity to both heat and oxidative stress was dependent on membrane lipid composition. In the case of anaerobic cells, the most stress resistant had membranes enriched in saturated fatty acids, followed in order by cells enriched in oleic and linolenic acids. Aerobic cells with membranes enriched in palmitoleic and oleic acids showed the highest resistance to stress under all conditions. In both aerobic and anaerobic cells, a mild heat shock or oxidative shock induced markedly increased levels of thiobarbituric acid reactive substance (TBARS), indicative of malondialdehyde formation and lipid damage. Anaerobic cells with membranes enriched in linolenic acid had the highest TBARS, followed by cells enriched in oleic acid, with cells enriched in saturated fatty acids showing the lowest TBARS. The results suggest that heat and oxidative stress may share a common mechanism of damage through induction of oxygen-derived free radicals, resulting in membrane lipid damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryoprotection of yeast by alcohols during rapid freezing.

We have investigated the cryoprotective effect of alcohols on Saccharomyces cerevisiae and other yeast under various freezing conditions. For S. cerevisiae, at a cooling rate of 3 degrees C min-1 methanol and ethanol acted as cryosensitizers. However, at a cooling rate of 200 degrees C min-1, both methanol and ethanol proved superior to all other cryoprotectants tested, including glycerol, dimethyl sulfoxide, lactose, trehalose, polyethylene glycol, and polyvinyl pyrrolidone. Propan-2-ol also demonstrated a small but measurable effect although propan-1-ol and butan-1-ol demonstrated no cryoprotective effect. A minimum cooling rate of 25 degrees C min-1 to elicit the cryoprotective effect of ethanol was necessary; below this rate it acted as a cryosensitizer. At cooling rates up to 650 degrees C min-1 substantial cryoprotective effect was still evident. Although the effect of ethanol was variable for other yeast genera tested, ethanol acted positively for all strains of S. cerevisiae. We hypothesize that the cryoprotective effect of alcohols during rapid cooling is a result of their ability to induce increased membrane permeability, allowing rapid water equilibration during extracellular freezing and avoidance of intracellular ice crystal formation.

Role of growth phase and ethanol in freeze-thaw stress resistance of Saccharomyces cerevisiae.

The freeze-thaw tolerance of Saccharomyces cerevisiae was examined throughout growth in aerobic batch culture. Minimum tolerance to rapid freezing (immersion in liquid nitrogen; cooling rate, approximately 200 degrees C min-1) was associated with respirofermentative (exponential) growth on glucose. However, maximum tolerance occurred not during the stationary phase but during active respiratory growth on ethanol accumulated during respirofermentative growth on glucose. The peak in tolerance occurred several hours after entry into the respiratory growth phase and did not correspond to a transient accumulation of trehalose which occurred at the point of glucose exhaustion. Substitution of ethanol with other carbon sources which permit high levels of respiration (acetate and galactose) also induced high freeze-thaw tolerance, and the peak did not occur in cells shifted directly from fermentative growth to starvation conditions or in two respiratorily incompetent mutants. These results imply a direct link with respiration, rather than exhaustion of glucose. The role of ethanol as a cryoprotectant per se was also investigated, and under conditions of rapid freezing (cooling rate, approximately 200 degrees C min-1), ethanol demonstrated a significant cryoprotective effect. Under the same freezing conditions, glycerol had little effect at high concentrations and acted as a cryosensitizer at low concentrations. Conversely, under slow-freezing conditions (step freezing at -20, -70, and then -196 degrees C; initial cooling rate, approximately 3 degrees C min-1), glycerol acted as a cryoprotectant while ethanol lost this ability. Ethanol may thus have two effects on the cryotolerance of baker's yeast, as a respirable carbon source and as a cryoprotectant under rapid-freezing conditions.

The need for consistent nomenclature and assessment of growth phases in diauxic cultures of Saccharomyces cerevisiae.

The need for consistent nomenclature and accurate assessment of late growth phases in diauxic yeast cultures is highlighted by the substantial variation of stress tolerance in Saccharomyces cerevisiae after the exhaustion of the initial fermentable carbon source. At present, a wide variety of assessment methods and confused terminology exists in the literature, leading to difficulties in the interpretation and comparison of published results. A method based on the depletion of ethanol accumulated during the respiro-fermentative growth phase is suggested as suitable for assessing subsequent growth phases and reporting results. Consistent application of nomenclature for growth phases is recommended to assist the interpretation of published experimental results. It is suggested that the phases of growth in diauxic batch culture should be referred to using the terms (1) initial lag phase, (2) respiro-fermentative phase, (3) diauxic lag phase, (4) respiratory phase, (5) stationary phase, and (6) death phase.

Lymphocyte subclasses in pregnancy induced hypertension.

Peripheral blood lymphocyte subclasses were studied by flow cytofluorimetry and monoclonal antibodies in 21 women with Pregnancy Induced Hypertension (PIH), 20 healthy women in their third trimester of pregnancy and in 20 nulliparous, nonpregnant women. The cells were stained with the monoclonal antibodies OKT3, OKT4 and OKT8 to define total T cells, T helper cells (Th) and T suppressor-cytotoxic cells (Ts/c) respectively. B lymphocytes were defined by their surface immunoglobulin. Absolute numbers of total T cells and Ts/c cells were significantly decreased (p less than 0.05) in patients with PIH compared to either control group. The proportion of B lymphocytes was significantly (p less than 0.01) increased and absolute numbers were marginally increased. These findings reflect an immune disturbance which may be of prime importance in the pathogenesis of this disease.

Stress co-tolerance and trehalose content in baking strains of Saccharomyces cerevisiae.

Fourteen wild-type baking strains of Saccharomyces cerevisiae were grown in batch culture to true stationary phase (exogenous carbon source exhausted) and tested for their trehalose content and their tolerance to heat (52 degrees C for 4.5 min), ethanol (20% v/v for 30 min), H2O2 (0.3 M for 60 min), rapid freezing (-196 degrees C for 20 min, cooling rate 200 degrees C min-1), slow freezing (-20 degrees C for 24 h, cooling rate 3 degrees C min(-1)), salt (growth in 1.5 M NaCl agar) or acetic acid (growth in 0.4% w/v acetic acid agar) stresses. Stress tolerance among the strains was highly variable and up to 1000-fold differences existed between strains for some types of stress. Compared with previously published reports, all strains were tolerant to H2O2 stress. Correlation analysis of stress tolerance results demonstrated relationships between tolerance to H2O2 and tolerance to all stresses except ethanol. This may imply that oxidative processes are associated with a wide variety of cellular stresses and also indicate that the general robustness associated with industrial yeast may be a result of their oxidative stress tolerance. In addition, H2O2 tolerance might be a suitable marker for the general assessment of stress tolerance in yeast strains. Trehalose content failed to correlate with tolerance to any stress except acetic acid. This may indicate that the contribution of trehalose to tolerance to other stresses is either small or inconsistent and that trehalose may not be used as a general predictor of stress tolerance in true stationary phase yeast.

Altered blood lymphocyte subclasses in patients with ulcerative colitis.

Peripheral blood lymphocytes were studied by flow cytofluorimetry and monoclonal antibody techniques in 107 patients with ulcerative colitis and in 20 healthy controls of similar ages. Total T, T helper (TH), and T cytotoxic/suppressor (TC/S) lymphocytes were defined by the monoclonal antibodies OKT3, OKT4 and OKT8, respectively, while B lymphocytes were defined by surface immunoglobulin. Patients had a significantly (P less than 0.05) lower proportion of TC/S lymphocytes than the controls, and patients with quiescent disease had a reduced proportion of B lymphocytes compared to controls and those with active disease. Patients with marked mucosal dysplasia had a significantly (P less than 0.025) lower proportion of TH lymphocytes and a higher (P less than 0.01) proportion of B lymphocytes than those without dysplasia. There were no significant associations between lymphocyte levels and any other clinicopathological features assessed.