[Variations of some physiological and biochemical indices in the population of red-backed vole (Clethrionomys rutilus)]. / Izmenchivost' nekotorykh fiziologo-biokhimicheskikh pokazatelei fluktuiruiushchei populiatsii krasnoi polevki (Clethrionomys rutilus).

We studied the changes in the population density of the red-backed vole (Clethrionomys rutilus Pall.) on the northern coast of the Sea of Okhotsk (60 degrees N) during three population cycles (1980-1990). A total of 3111 animals were studied, including 1006 alive voles. For evaluation of the population stress and food deficit, a complex of biochemical (blood level of glucose and liver levels of glycogen and lipids) and physiological (blood content of leucocytes, spleen weight, etc.) indices was used. At a high population density, the influence of stress continues during the reproductive period, after its termination, and, supposedly, until the beginning of a new reproductive period, while at a low population density, the consequences of stress were expressed only in the second half of the reproductive period. The signs of starvation were recorded only in some semiadult voles in a high density population. It has been confirmed that stress acts as a mechanism involved in regulation of the population density of small rodents.

[Specificity of morphological changes and DNA degradation in the Ehrlich ascite carcinoma cells exposed to various damaging agents]. / Spetsifika strukturnykh izmenenil i degradatsii DNK v kletkakh astsitnol kartsinomy erlikha, podvergnutykh razlichnym povrezhdaiushchim vozdelstviiam.

Using the Ehrlich ascite carcinoma cells, exposed to oxidative stress, heat shock, cytochalasin B, vinblastine, Triton X-100 and energy starvation, morphological changes, DNA degradation, and form of cell death were investigated. A rather clear specificity of cell morphology, characteristic of apoptosis, was revealed for the most of treatments, including membrane blebs, chromatin condensation and apoptotic body formation. Karyorhexis was not common for the examined cells. After energy starvation, oxidative stress, heat shock and Triton X-100 treatment, the apoptotic cells were seen to undergo secondary necrosis rather soon; this was not the case after cytochalasin B or vinblastine treatments. In cells exposed to heat shock, cellular pyknosis was frequently developed. Such cells did not undergo secondary necrosis for a long time. The DNA degradation in cells occurred in chaotic manner (without ladder pattern). The fraction of degraded DNA corresponded to that of cells undergoing secondary necrosis.

Resistance of Ehrlich tumor cells to apoptosis can be due to accumulation of heat shock proteins.

Previously we have found that stationary Ehrlich ascites carcinoma (EAC) cells in vivo accumulated heat shock proteins (HSPs) and became resistant to necrotic death induced by prolonged energy deprivation of hyperthermia. Here we report that apoptotic death induced by nutrient starvation, transient ATP depletion, heat shock and a microtubule-disrupting drug, vinblastine, was also suppressed in stationary EAC cells comparing with exponential cells. When exponential (sensitive) cells were subjected to short-term heating with recovery to accumulate inducible form of HSP70, they also became resistant to all of the employed apoptosis-inducing exposures, and an inhibitor of cytosolic protein synthesis, cycloheximide, prevented acquisition of the resistance. It is suggested that in vivo accumulation of HSPs in stationary tumor cells can be endogenous protective device against apoptotic death induced by starvation or some anticancer treatments.

Adaptation of Ehrlich ascites carcinoma cells to energy deprivation in vivo can be associated with heat shock protein accumulation.

Tumor adaptation to chronic energy starvation in vivo was studied on Ehrlich ascites carcinoma (EAC) cells. EAC cells were isolated from mice and incubated in a glucose-free medium containing blocators of mitochondrial ATP generation (rotenone, 2,4-dinitrophenol, or oligomycin). ATP level in the treated cells decreased to 3-4% of the initial during 30 min of the incubation. The aggregation of cytoskeletal proteins, blebbing, and necrotic death within 2-3 h were observed in ATP-depleted EAC which were isolated and treated in the exponential phase of growth (5 days after inoculation), whereas stationary EAC (8 days after inoculation) were considerably more resistant to ATP depletion, and actin aggregation as well as bleb formation were suppressed in these cells despite the ATP loss. In contrast to the exponentially growing cells, thermotolerance and unexpected expression of inducible HSP68 and HSP27 as well as an elevated level of HSP90 were found in stationary EAC. Since the stationary cells had decreased content of ATP, ATP/ADP ratio, and energy charge, we suggest that this energy dysbalance may be conducive to HSP induction within the ascites tumor in vivo, and, at the same time, EAC cells with elevated content of HSPs acquire resistance to chronic energy starvation occurring in late stages of the tumor growth.

[Lack of serum causes apoptosis of thymocytes not requiring protein synthesis and ATP generation]. / Lishenie syvorotki vyzyvaet apoptoz timotsitov, ne trebuiushchii sinteaz belka i generatsii ATP.

Incubation of rat thymocytes in serum-free media was found to result in their apoptotic death characterized by internucleosomal DNA fragmentation, nuclear pyknosis and subsequent irreversible plasma membrane damage. As in the case of glucocorticoid (hydrocortisone)-induced apoptosis, DNA fragmentation under serum withdrawal was suppressed by endonuclease inhibitors (Zn2+ and spermine). At the same time, protein synthesis inhibitors (cycloheximide and puromycin) failed to block the apoptosis induced by serum withdrawal but inhibited the hydrocortisone-induced apoptosis. Various inhibitors of oxidative phosphorylation (uncoupler, rotenone, oligomycin), causing sharp decrease in cellular ATP did not suppress DNA fragmentation, whereas thymocyte plasma membrane damage accelerated under their effect. The results obtained indicate that intact thymocytes contain all the components of the apoptotic system; however, in the absence of apoptotic stimuli (e.g., hydrocortisone) the system is blocked by some growth factors of serum origin. Serum withdrawal is sufficient by itself to induce apoptosis and does not require the synthesis of special proteins.

[Fragmentation of Ehrlich ascites carcinoma DNA during influences causing aggregation of cytoskeletal proteins]. / Fragmentatsiia DNK astsitnoi kartsinomy Erlikha pri vozdeistviiakh, vyzyvaiushchikh agregatsiiu belkov tsitoskleta.

Upon exposures of Ehrlich ascites carcinoma cells to heat shock (44 degrees, 1 hr), oxidative stress or energy deprivation, their DNA undergoes fragmentation (35-45% after 5 hrs of incubation) which is considered as a hallmark of apoptosis. Prior to DNA fragmentation the cells exhibited blebbing (55-90% after 1 hr), thus being suggestive of cytoskeletal damage and a 1.5-2-fold increase in the Triton-insoluble protein concentration (protein aggregation) after 3 hrs. Rapid cell death (75% after 4 hrs) occurred only under oxidative stress. Electrophoresis of the Triton-insoluble protein fraction revealed that the common feature of all stress exposures used in this study was a dramatic increase in the aggregation of cytoskeletal proteins--actin and the 57 kDa protein. No dependence of DNA fragmentation on intracellular Ca2+ increase was found. Both DNA fragmentation and protein aggregation were suppressed by glucose, whereas Zn2+, an endonuclease inhibitor, suppressed only DNA fragmentation without any effect on protein aggregation. It is suggested that cytoskeletal damage may trigger tumor cell apoptosis.

[Damage to and interphase death of Ehrlich ascites carcinoma tumor cells at different growth stages during energy starvation and heat shock]. / Povrezhdenie i interfaznaia gibel' opukholevykh kletok astsitnoi kartsinomy Erlikha, nakhodiashchikhsia na raznykh stadiiakh rosta, pri énergeticheskom golodanii i teplovom shoke.

The reaction of the Ehrlich ascite carcinoma cells, being at different phases of their growth, to the energy deprivation (rotenone in glucose-free medium) and heat shock (HS) was investigated. The criteria of this reaction were interphase death (according to Trypan blue staining) and structural changes (appearance of big blebs). It was found that proliferating cells (from log phase), judging from the two criteria, were more sensitive to a separate action of both energy deprivation and HS, than the resting ones (from stationary phase). Under combined actions (energy deprivation plus HS), when cell damage is much accelerated, the difference in their sensitivity was revealed only in relation to structural damages. Under the action of starvation and HS, changes in ATP content in the cells of both ages were similar; the reaction of cells to both the agents, after removing the calcium from medium (by chelator), was not changed. It means that specificity of the reaction of cells being at different growth phases to the damaging agents is not determined by disturbance in their energetic and calcium homeostases. In the proliferating cells, the cytoskeletal protein aggregation under energetic deprivation proceeded faster than that in the resting ones. It is proposed that the reaction of cells being on different growth phases depends on the stability of cytoskeletal proteins and on the content of stress proteins.

Association of blebbing with assembly of cytoskeletal proteins in ATP-depleted EL-4 ascites tumour cells.

ATP depletion in EL-4 ascites tumour cells rapidly induced the changes in cell morphology (blebbing), cytoskeletal protein assembly and finally resulted in cell death. After 1 hr of incubation with 2 microM rotenone (inhibitor of respiration) in glucose-free medium, when ATP level was 4% of the initial level, there were increases in triton-insoluble actin and vinculin levels (2.5-fold and 2.8-fold, respectively) and 44% of cells showed blebs; such treatment damaged cells irreversibly. Ca2+ removal did not diminish the effect of ATP depletion on cytoskeleton, blebbing and cell death, although the elevation of free intracellular Ca2+ in rotenone-treated cells was prevented. The role of ATP in maintaining cytoskeleton and cell shape is discussed.

[Change in energy metabolism of ascites cancer cells with a decrease in pH]. / Izmenenie énergeticheskogo obmena astsitnykh opukholevykh kletok pri snizhenii pH.

In Hank's balanced salt solution EL-4 ascites thymoma cells possessed endogenous respiration which was sufficient for the maintenance of their ATP level: pH decrease down to 6.0 had no effect either on endogenous respiration or the ATP level. Glucose had no influence on the respiration of EL-4 cells but inhibited that of Ehrlich ascites carcinoma (EAC) cells by 40% (Crabtree effect); respiration of the both cell lines was strongly (4-fold) inhibited after simultaneous addition of glucose, lactate and pH decrease. EL-4 cells had no endogenous glycolysis; EAC cells showed a low level of glycolysis only after pH decrease. Glucose addition led to activation of glycolysis (both inhibited 2-fold after a decrease of pH down to 6.0. The respiration inhibition at pH 7.3 and 6.0 caused no decrease of ATP depletion when glucose was present in the medium; this result may be due to suppression of ATP consumption. Incubation of EL-4 cells under respiration and glycolysis deficiency conditions resulted in a sharp ATP depletion; pH decrease delayed this depletion.

[Increasing the concentration of free calcium in thymocytes during gamma-irradiation may induce their death]. / Povyshenie kontsentratsii svobodnogo kal'tsiia v timotsitakh pri gamma-obluchenii mozhet byt' induktorom ikh gibeli.

Free Ca2+ concentration in thymocytes increased 0.5-1.5h after gamma-irradiation (10 Gy) as was measured by Quin-2AM fluorescent probe. Cycloheximide, a protein synthesis inhibitor, suppressed Ca2+ increase and inhibited radiation-induced thymocyte death. EL-4 thymoma cells did not exhibit any changes in free Ca2+ concentration and interphase death after gamma-irradiation. It is believed that the radiation-induced increase of free Ca2+ concentration in thymocytes may induce their death.

Oxidative stress, disturbance of energy balance, and death of ascites tumour cells under menadione (vitamin K3) action.

Resting ascites tumour cells (Ehrlich and EL-4 thymoma) treated with menadione (50 microM) died (up to 80% cell death over 2 h) without dividing (i.e. interphase). Glucose (25 mM) added to the cell suspensions partially protected these cells from menadione action. During incubation of the cells with menadione, the rates of free oxidation and lipid peroxidation were elevated, cellular ATP and nonprotein SH-group levels were much decreased, and [Ca2+]i was moderately increased. From a comparison of these effects and cell survival rates with those seen with rotenone, KCN (both inhibitors of oxidative phosphorylation), and H2O2 (an inducer of oxidative stress), it is concluded that ATP depletion is the main factor leading to the death of cells treated with menadione. The level of cellular ATP dropped to less than 10% of its initial value after 1 h incubation with menadione and may have resulted in irreversible damage to cytoskeletal structures, bleb formation, and changes in plasma membrane permeability that are incompatible with cell viability.

[The relationship of damage to and death of ascitic tumor cells during starvation to the ATP and free calcium content in the cells]. / Zavisimost' povrezhdeniia i gibeli astsitnykh opukholevykh kletok pri golodanii ot soderzhaniia v kletkakh ATF i svobodnogo kal'tsiia.

Ascite tumor cells EL-4 were incubated in conditions of energy starvation (Hanks salt solution with rothenone and without glucose) at 37 degrees C for 3 hours. Under these conditions, some structural cell damages appeared within the first hours: enlarging and flattening of the cells, blebbing, vacuolization of the cytoplasm, nuclear chromatin condensation. Later on, a share of cells with obvious damage decreased, whereas that of the cells stained with trypan blue (dead cells) much increased (up to 90% after a 3 hour incubation). The cellular ATP decreased abruptly (up to 10% of the control) during the first 10 minutes of starvation. Free Ca2+ concentration increased within 1 hour of incubation more than two-fold. The conditions promoting Ca2+ influx (ionophore A23187 + Ca2+ in medium) accelerated the damage and cell death. However, the increase in free Ca2+ concentration did not trigger any damage in the energy-starved cells, since in the Ca2(+)-depleted medium (no increase in free Ca2(+)-concentration) the development of damages was not prevented. The damage initiation was irreversible: the addition of glucose to cell suspensions after 0.5-1 hour of their incubation in energy-starved condition did not prevent the development of damage, while ATP content in these cells was much increased.

[DNA degradation and changes in the permeability and form of Ehrlich ascites tumor cells when incubated in an anaerobic medium without glucose]. / Degradatsiia DNK, izmenenie pronitsaemosti i formy kletok astsitnoi opukholi Erlikha pri inkubatsii ikh v anaérobnoi srede bez gliukozy.

After a 3-hour incubation of the Ehrlich ascite tumor cells in buffered Hanks solution, without glucose and oxygen, the extensive cell injuries were observed. The time-course of appearance of these injuries was as follows: cell blebbing, staining of the cells with trypan blue, and then their staining with ethidium bromide. The DNA degradation registered with fluorometric method coincided in time with cell staining with trypan blue. All injuries (except DNA degradation) were delayed at pH 6.0 compared with those at pH 7.3. Glucose added to the cell suspension greatly protected the cells from these injuries, although DNA degradation at pH 6.0 in these conditions was a little higher than that at pH 7.3.

On the energy fuel in voles during their starvation.

Starved voles derive energy predominantly from endogenous carbohydrates, unlike small laboratorial rodents which use free fatty acids as fuel during their fasting. This seems the reason why deep hypoglycemia is developed in fasting voles. The rate of gluconeogenesis in starved voles is rather high while oxidation of carbohydrates in that state is more rapid than their formation. Utilization of fat in fasted voles is relatively small. Voles can die after starvation still having a lot of fats in their bodies. Oxygen consumption in starved voles decreases to a small extent.

Some physiological and biochemical features of starvation and refeeding in small wild rodents (Microtinae).

Blood glucose and leucocytes and liver glycogen and lipids were investigated in Cl. rutilus and Cl. rufocanus fasted for 0, 6, 12, 18 and 22 hr. It was observed: 1. In both species blood glucose content drops (1.3-1.5 times) by 6 hr, slowly rises by 12 hr and then progressively and strongly declines up to the end of starvation (36% and 27% of control for Cl. rutilus and Cl. rufocanus respectively). 2. Liver glycogen was depleted by 6 hr of fasting while lipids accumulate in liver during starvation in a large quantity (3.4-3.6 times at the end). 3. White blood cells content in fasted animals decreases. At one end of starvation it equals 23% and 16% of control for Cl. rutilus and Cl. rufocanus, respectively. 4. Hypoglycemia in Microtinae during starvation is pronounced compared with that in Muridae. Leucopenia and accumulation of lot of lipids in liver are new phenomena for fasting. 5. After refeeding hyperglycemia develops, the liver accumulates large quantities of glycogen. Recovery of all indexes slow. Complete normalization does not occur by 16 hr of refeeding.

[Metabolic and hematologic changes in the northern red-backed vole Clethrionomys rutilus and the large-toothed red-backed vole Clethrionomys rufocanus during short-term starvation and refeeding]. / Metabolicheskie i gematologicheskie sdvigi u krasnoi Clethrionomys rutilus i krasno-seroi Clethrionomys rufocanus polevok pri neprodolzhitel'nom golodanii i vozobnovlenii pitaniia.

The effect of starvation has been investigated on the content of glucose and lipids in the blood plasma, on the level of leucocytes in the peripheral blood and on the concentration of total lipids and glycogen in the liver. It was found that in both species of the voles, starvation within 20-22 hours sharply decreases glucose content and increases lipid content of the blood plasma. The liver glycogen becomes almost completely exhausted. These changes are more significant than those observed under similar conditions in laboratory rodents (albino mice and rats). During starvation of the voles, the level of leucocytes drastically decreases, whereas lipid content of the liver significantly increased. These changes are absent in laboratory rodents. The decrease in the content of glucose and in the level of leucocytes in the blood is more significant in C. rufocanus than in C, rutilis. On the contrary, accumulation of the lipids in the liver is more intense in the latter. The recovery of the investigated indices after refeeding is very slow. Possible significance of these re-arrangements for temperature and immune homeostases, as well as for regulation of other functions in the organism, is discussed.

Some features of the reaction of intracellular bacteriophages T1 to UV irradiation.

The reaction of complexes pf phage T1-cells of E. coli B or E. coli Bs-1 to UV irradiation was investigated. The complexes were irradiated at various stage of infection, and their survival, extent of Hcr and Phr, were evaluated. It was found that the UV resistance of phage DNA in the second half of the latent period fluctuates. Hcr after UV exposure at these stages of infection operates in a small volume. The ability of intracellular phage to photoreactivate when cells of E. coli B were infected is constant after irradiation at many stages of infection, except the early ones. In the complexes of phage T1-bacteria of E. coli Bs-1 this ability declines while infection is promoted. The daughter phage particles released from UV irradiated complexes undergo Phr and Hcr only after irradiation at the late stages of infection. This was not the cases when complexes of phage-bacteria were irradiated during the first half of the latent period. A possible tole of UV-damaged phage DNA in propagation of infection and in maturation of phage particles is discussed.

Effect of chloramphenicol and cyanide on the increase in UV resistance of intracellular bacteriophage T1.

The effects of chloramphenicol and cyanide on the increase in UV resistance of intracellular phage T1 infecting cells of E. coli B or E. coli Bs-1 were investigated. The inhibitiors were added to the cells 3 min prior to infection and to the complexes of phage-bacteria 3.5 and 6.5 min after adsorption of phage by the cells. The data obtained are not in agreement with the suggestion that increase in UV resistance of intracellular phage is mainly due to the accumulation of phage DNA inside the host cells. It is suggested that a very important role in this resistance is played by the interaction of phage DNA with the cell membranes.