Yu. N. Korystov

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A2, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A2 can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.

The effect of inhibitors of arachidonic acid metabolism on proliferation and death of tumor cells

The effect of inhibitors of arachidonic acid metabolism on proliferation and death of tumor P‐388 cells in a broad concentration range was studied. Cell proliferation was estimated by the metaphase frequency and the proportion of cells in S phase; cell death was determined from lysis, staining of cells with trypan blue, nuclear damage, percentage of cells with subdiploid DNA and the type of DNA fragmentation. It was shown that low concentrations of phospholipase A2 and lipoxygenase inhibitors stimulated the proliferation of P‐388 cells. At higher concentrations, the inhibitors suppressed cell proliferation by blocking the G1‐S transition and induced cell death of the apoptosis type. Indomethacin, an inhibitor of cyclooxygenase, did not initiate cell death, nor did it affect the proliferation of P‐388 cells at concentrations of up to 10 μM.

Opposite Effects of Low Oligomycin Concentrations on the Apoptosis of Normal and Tumor Cells

Oligomycin is widely used in laboratory studies as an ATPase inhibitor. It binds to F 0 F 1 ATPase and inhibits the mitochondrial proton pump [1], which results in respiration suppression and a decrease in the cell ATP content. Oligomycin concentrations completely inhibiting respiration vary from 80 to 300 nmol/l for different cell types [2]. A decrease in ATP concentration has been previously assumed to be the only cause of the toxic effect of oligomycin on cells. Recent data suggest, however, that some oligomycin effects do not depend on ATP. For example, the inhibition of Baxinduced apoptosis does not depend on ATP [1]. It is supposed [1] that, in this case, a blockage of mitochondrial pore opening accounts for the oligomycin effect.

[Apoptosis in P388 leukemia cells induced by specific inhibitors of 5- and 12-lipoxygenase and the product of cyclooxygenase, prostaglandin E2].

We studied the effect of specific inhibitors of 5- and 12-lipoxygenases as well as the product of cyclooxygenase activity, prostaglandin E2 , on proliferation and death of P388 leukemia cells. Inhibition of 5- and 12-lipoxygenases in the cells inhibits proliferation and induces apoptosis. The concentrations of baicalein, an inhibitor of 12-lipoxygenase, and AA861, an inhibitor of 5-lipoxygenase, causing a 50% death rate (LC50) proved to be the same, 50 μM. Excessive prostaglandin also inhibited proliferation of the cells and induced apoptosis. The LC50 for prostaglandin E2 was 4 μM. The obtained data suggest that apoptosis in P388 cells after lipoxygenase inhibition can be induced by both deficiency of lipoxygenase products and excess of prostaglandins in the cell.

Taxifolin and Fucoidin Abolish the Irradiation-Induced Increase in the Production of Reactive Oxygen Species in Rat Aorta

We studied changes in ROS content in the aorta of Wistar rats at early terms after irradiation in doses equal to single fraction used in tumor radiotherapy and the effects of taxifolin and fucoidin, blockers of leukocyte adhesion to endothelium, on ROS content. Male rats were exposed to X-rays (200 kW) in doses of 1-7.5 Gy. ROS production in aorta segments was measured in 1-48 h after irradiation by dichlorodihydrofluorescein oxidation. The content of ROS in the aorta of rats exposed to radiation in doses of 1-2.5 Gy increased in 1-24 h after irradiation, the peak ROS content was found in 2 h after irradiation. Taxifolin (100 μg/kg dihydroquercetin once a day with drinking water) and fucoidin (10 mg/kg, i.v.) abolished ROS accumulation. The content of ROS in rat aorta increased in 1-24 h after irradiation in doses used for tumor radiotherapy and this increase can be determined by leukocyte adhesion to the endothelium.

Ionizing Radiation Enhances Activity of Angiotensin-Converting Enzyme in Rat Aorta

We analyzed changes in angiotensin-converting enzyme activity in rat aorta at the early terms after irradiation in doses equal to one fraction dose used in tumor radiotherapy. Male Wistar rats were exposed to whole body or local (chest) X-ray irradiation (200 kV, 1-7.5 Gy). The activity of the enzyme in aorta segments was measured in 1-48 h after irradiation by hydrolysis of hippuryl-histidine-leucine. Activity of angiotensin-converting enzyme in rat aorta was increased 1-24 h after whole body irradiation in a dose of 2.5 Gy with a peak in 2 h after exposure. After local exposure, enzyme activity also increased in 2 h, but returned to the control level in 24 h. In 2 h after whole-body irradiation in doses >2.5 Gy, the increase in enzyme activity was less pronounced and after exposure to 7.5 Gy, it did not differ from the control. During local exposure, the effect did not decrease with increasing the irradiation dose. The fraction of blood monocytes adherent to plastic in rats subjected to whole body irradiation decreases with increasing the dose. In rats subjected to local irradiation in a dose of 7.5 Gy, monocyte adhesion to plastic did not differ from the control. These data suggest that the increase in activity of angiotensin-converting enzyme in the aorta after irradiation is determined by monocyte adhesion to the endothelium; the decrease in this effects with increasing the dose can be explained by radiation damage of monocytes.

Lipoxygenase Inhibitors Nordihydroguaiaretic Acid and Fungus Lecanicillum lecanii Extract Induce Death of Lymphoid Leukemia Cells

We studied the effect of lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and fungus Lecanicillum lecanii extract on lymphatic leukemia P388 cells. The cells grown in the abdominal cavity of DBA2 mice for 7 days were transferred into a nutrient medium. The effect of lipoxygenase inhibitors was evaluated by changes in cell number, trypan blue staining, nucleus damage, and changes in cell distribution by DNA content after 22-h incubation. NDGA and fungus extract induced apoptotic death of lymphatic leukemia cells, which was seen from nucleus damage and reduced DNA content in cells. IC50 for NDGA and fungus extract was 0.66 and 5.5 μg/ml, respectively.

The Hexapeptide Immunofan Inhibits the MRP-dependent Multidrug Resistance of Tumor Cells

The phenomenon of resistance to a broad range of drugs is widely known for many biological objects: bacteria, protists, fungi, and mammalian cells. The fact that plasma membrane contains transport proteins capable of using the energy of ATP for exporting various compounds out of cells against the concentration gradient is one of the main factors of multidrug resistance (MDR) [1]. The transport of various compounds out of cells causes a substantial decrease in the efficacy of remedies used in therapy of bacterial, parasitic, and fungal diseases, as well as malignant neoplasms. MDR makes it difficult or even impossible to treat a broad spectrum of diseases. Therefore, it is necessary to find effective inhibitors of MDR. Transport proteins of two types are mainly responsible for MDR in mammalian cells: multidrug resistance associated protein (MRP) and P-glycoprotein (P-gp) [1]. Both MRP and P-gp have been found in tumors of various origin: lymphomas, sarcomas, carcinomas, and neuroblastomas [1]. A broad range of antitumor drugs are substrates of transport proteins of the two types: anthracyclines, Etoposide, Vincristine, Taxol, etc. [1]. However, in contrast to P-gp, MRP mediates the transport of various compounds out of cells in a modified form, namely, as conjugates with gluthatione or glucuronic acid [1].

[Analysis of 15-lipoxygenase activity in irradiated thymocytes].

We studied the effect of γ-irradiation on 15-lipoxygenase activity in rat thymocytes. The enzyme activity was determined as the rate of linoleic acid oxidation by the protein fraction isolated from the control and irradiated thymocytes under standard conditions. We demonstrate lipoxygenase activation immediately after irradiation of thymocytes. High lipoxygenase activity is observed in the cells for no more than an hour after irradiation. No high lipoxygenase activity later indicates that its synthesis is not directly induced by irradiation. Irradiation-induced generation of lipid peroxides can be the factor of lipoxygenase activation.

Social Stress in Groups of Mice: Methods for Recording Conflicts and Their Consequences

When animals intensely use a territory, they form groups whose members are linked with one another by domination–subordination relationships [1]. This system of relationships is generally called hierarchical. The formation of the group and changes in its structure are determined by aggressive interactions that establish or change the structure of domination–subordination. This is accompanied by social stress determined by the mutual interactions of the animals in the group. Social stress causes various diseases and can even lead to death of the animals [1, 2]. The hierarchical organization of the groups is characteristic not only of nonhuman animals, but also of humans. In humans, social stress is considered one of the main causes of neurological disorders [3], circulatory diseases [4], tumors [5, 6], and other diseases [7]. The necessity of experimental study of social stress, its effects, and measures to prevent or mitigate its consequences is obvious.