Search

The possibility of producing the material based on collagen and biologically active polyphenol taxifolin was explored, and the properties of the material were studied. The data on the dynamics of the release of polyphenol chemically linked to collagen are represented, and the metal-reducing activity of polyphenol released from the gel is determined. The effect of taxifolin, taxifolin glutarate and gel containing polyphenol on the production of reactive oxygen species by neutrophils stimulated with phorbol myristate acetate was examined. It was shown that polyphenol released from the gel material exerts antioxidant and metal-reducing properties, suggesting that unoxidized polyphenol linked to collagen.

Mesenchymal stromal cells (MSC) are considered to be promising tool of regenerative medicine. Migration of MSC toward damaged inflammatory site is essential for physiological tissue reparation. Therefore we studied modifications of migratory features of adipose tissue derived MSC (AT-MSC) after co-cultivation with activated monocytes derived from THP-1 cell line. As a result, we have observed an increased migration rate of AT-MSC in vitro in the absence of chemoattractant gradient as well as toward the gradient of PDGF BB (platelet-derived growth factor BB), which is well known chemoattractant for the cells of mesenchymal origin. Furthermore, the rate of directional AT-MSC migration through fibronectin was also increased. We have established that signaling from PDGFRβ which is activated through binding of integrin receptors with extracellular matrix may be possible way to stimulate cellular migration under simulated inflammatory conditions.

The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

Phorbol esters are known to modulate protein kinase C activity - enzyme involved in cell proliferation, differentiation and apoptosis regulation in skin cells. Besides it phorbol-12-myristate 13-acetate was shown possible to modulate promoter activity of TsPO - protein that is involved in steroidogenesis and cell proliferation regulation. Caspase-3 and TsPO expression was measured in squamous cell carcinoma cells after incubation with phorbol-12-myristate 13-acetate and ultraviolet radiation. Following alterations of TsPO and caspase-3 levels are explained by different mechanisms of regulation.

Regularities of biologically active lipid metabolites formation in dynamics (5, 10, 30, 60 s) by phorbol 12-miristate 13-acetate stimulation in [14C]palmitic acid have been investigated in normal and leukemia peripheral blood lymphocytes prelabeled with [14C]palmitate. In normal cells there was two-phase formation of 1,2-diacylglycerol (5, 30 s), lysophosphatidylcholine (10, 60 s), as well as free palmitic acid at 10 s of stimulation. Under the identical experimental conditions there was inhibition of investigated lipid release processes at early (5 and 10 s) stages of stimulation of leukemic lymphocytes. At later (30, 60 s) terms of these lymphocytes the activation, basically, similar to norm changes in the formation of palmitic acid-containing metabolites except free palmitic acid (the level of which raised only at 60 second of the post-stimulation) was found. Various protein kinases C are involved in the regulation of investigated lipid levels at certain stages of signal transduction both in norm, and in blast cells. Short-term (5, 10 s) activations of healthy donors lymphocytes are coupled to functioning of Ca2+-independent isoforms of protein kinase C. The inhibition of this protein kinase C in leukemic cells leads to normalization of the investigated lipid release. The data obtained suggests disorders of early membrane-bound reactions in agonist - and a protein kinase C-mediated processes of formation palmitic acid-containing lipid metabolites in the leukemic cells in comparison with the norm.

Production of reactive oxygen species (ROS) by macrophages from blood monocytes of healthy donors (MP(N)) and patients with IHD (MP(IHD)) before, during, and after their incubation with low-density lipoprotein (LDL) isolated from the blood plasma of healthy donors (LDL(N)) and patients with a high cholesterol level (LDL(H)) was estimated by the method of luminol-dependent and stimulated by opsonized zymosan (OZ) or phorbol-12-myristate-13-acetate (PMA) chemiluminescence (CL). Intrinsic luminol-dependent, and zymosan- or PMA-stimulated chemiluminescence of MP(IHD) have exceeded the same types of chemiluminescence of MP(N) by factors of 1.4, 1.8, 2.7, and 1.6, respectively (p < 0.05-0.01). The effect of zymosan on MP(N) and MP(IHD) was stronger than that of TPA by factors of 4.3 and 3.2, respectively, but manifested itself 2.5-3.0 times slower. LDL(N) and LDL(H) incubated with MP(N) during 15-60 min increased ROS production by a factor of 1.4 and 2.5 respectively, but influenced ROS production by MP(IHD) (as estimated by luminol-dependent chemiluminescence). Effects LDL(N) and LDL(H) on MP(IHD) were not detected at all. Repeated increase in zymosan-stimulated CL of MP(N) was also observed after their 15-180 min preincubation with LDL(N) and LDL(H) which followed after taking out LDL, washing MP(N) and adding Hanks' solution with opsonized zymosan. This increase was also stronger after MP(N) incubation with LDL(H) than after MP(N) incubation with LDL(N), and no increase was observed in experiments with MP(IHD). Thus, the results obtained by a chemiluminescent method showed that fresh macrophages from the blood of patients with IHD had higher ROS production than macrophages from healthy donors. LDL(N) and LDL(H) could exhibit primary and secondary (after preincubation) stimulating effect on CL in MP(N); but had no effect on MP(IHD). An analysis of macrophage chemiluminescence is a sensitive test for evaluation the degree of macrophage's stimulation and it may be effectively used for the dynamic control for treatment effectiveness in clinics.

Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 microM or forskolin at 10 microM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 microM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 microM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists-->changes in the level of calcium ions in cytoplasm-->increased protein kinase activities-->protein phosphorylation-->modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ.

Possible mechanisms of antioxidant activity of glycyrrhizinic acid (GA) were studied. GA did not exhibit antiradical properties at the range of concentrations 1-100 mM as was shown in the experiments with stable radical 1,1-Diphenyl-2-picrylhydrazyl. These data were conformed by the study of GA influence on chemiluminescence of luminol in cell-free system with hydrogen peroxide. However, GA decreased generation of reactive oxygen species by PMA-FMLF-activated neutrophils. Addition of GA did not ifluence free radical level in neurons, however, cell preincubation with GA resulted in the decrease of free radicals production and the increase of intracellular glutathione level.

We studied the ability of phorbol 12-myristate 13-acetat to prevent erythroid differentiation and apoptosis in erythroleukemic K562 cells induced by cytidine, thymidine, and guanosine. The exposure of cancer cells to combinations of phorbol 12-myrsitate 13-acetate (100 nM) nucleosides for two days led to a loss of hemoglobin production (marker of erythroid differentiation) in cells and increased expression of monocyte-macrophage lineage associated surface antigen CD14. The treatment of K562 cells with nucleosides only was accompanied by the activation of caspase-3 and caspase-9, rather than caspase-6, increased fluorescence of ethidium bromide and DAPI upon binding to DNA, and apoptosis. Intracellular activation of caspase-6, inhibition of caspase-9, a markedly decreased activity of caspase-3 and of fluorescence of DNA-binding dyes, and inhibition of apoptosis were observed when the cells were treated with phorbol 12-myeristet 13-acetate combined with nucleosides.

A vast number of studies, including the authors' own research, support the important role polymorphonuclear leukocytes (PMNL) in the development of ascite tumors. The method of luminol-dependent chemiluminescence (CL) was used to show the presence of two functionally different PMNL pools in a tumor-bearing organism: 1) "primed" PMNL, which circulate in the blood stream, and 2) "activated" PMNL, which are accumulated in the tumor zone and are capable of spontaneous CL. The purpose of the present investigation was to compare cytotoxic effects of primed and activated PMNL on tumor cells (ascite Ehrlich carcinoma (AEC), ascite Zajdel hepatoma) upon co-cultivation, as well as on normal cells of the organism, erythrocytes in vitro and in vivo. Upon stimulation with phorbol myristate acetate (PMA), PMNL effectively damaged AEC cells within the first 24 h until PMNL apoptosis occurred. Upon further co-cultivation, the tumor cells grew in number, which suggest the participation of PMNL in tumor protection. When stimulated with PMNL, pools suppressed tumor growth in vitro, since in this case the cytotoxicity was due to both reactive oxygen species and proteolytic enzymes. As it has been shown earlier by the authors, the functional potential of PMNL increases many times during tumor growth, and we suggested that not only tumor but also normal cells could be damaged. In this connection, we have studied the cytotoxic effect of primed and activated PMNL on rat erythrocytes in vitro on their co-cultivation. On stimulation with PMA, the rate of lysis of erythrocytes by primed PMNL increase many times compared to the norm. The fMLP-stimulated cytotoxity was 1.5-2.0 times higher than in the norm. Activated PMNL without stimulation are capable of producing only a partial lysis of erythrocytes (5-7 %). In order to assess the cytotoxic action of PMNL on erythrocytes in vivo, the hemoglobin content in erythrocytes and blood plasm of rats was measured in the course of tumor growth. The hemoglobin content in erythocytes during growth tumor decreased from 135 +/- 10 to 85 +/- 5 g/l, whereas in the blood plasm the hemoglobin content gradually increased by almost two times. The results enable us to suggest that one of death causes of tumor-bearing organisms may be the cytotoxic action of PMNL on normal cells of the organism caused by hyperproduction of ROS.

The action of 12-O-tetradecanoyl-13-acetate (TPA) in vitro in a wide range of concentration from 10(-3) mol/l down to ultra-low doses 10(-23) mol/l and dilution 10(-24) mol/l on the microsome membranes isolated from tumor--Ehrlich ascite carcinoma (EAC) has been studied by ESR-method using two spin probes: 5- and 16-doxyl stearates (5- and 16-DS) localized in the different regions of lipid bilayer. From the ESR spectra obtained it was calculated the following parameters: an order of the long axis 5-DS (S) related to order of the fatty acids chains in the lipid bilayer; two rotation correlation times (Tc1 and Tc2) of 16-DC to estimate a microviscosity value and structural-sensitive ones. It was found the stage changes of all these parameters (increase and decrease) as compared with control level (the membranes untreated by TPA) depending on TPA concentration into the range of 10(-3)-10(-24) mol/l; in particular, the most significant shape changes of structural-sensitive parameters have been observed at TPA doses below 10(-16) mol/l. It is concluded that tumor membranes are very sensitive to TPA action in vitro in a wide range of concentration included ultra-low doses.

The in vitro effect of sanazole (AK-2123; N-(2'-methoxyethyl)-2-[3"-nitro-1"-triazolyl]acetamide) and metronidazole (1-beta-hydroxyethyl-2-methyl-5-nitroimidazole) on phorbol-12-myristate-13-acetate (PMA)-stimulated and spontaneous (without stimulation by PMA) production of reactive oxygen species (ROS) by peritoneal and splenic murine macrophages was studied. ROS production was analyzed using fluorescent probe 2',7'-dichlorofluoresceine diacetate (DCFH-DA). An increase in the spontaneous production of ROS by macrophagal cells with therapeutic concentration of sanazole (0.6-1.25 mM) in the incubation medium was observed. At these concentrations metronidazole had no effect on spontaneous production of ROS by macrophagal cells. PMA-stimulated ROS production was inhibited by high concentrations (2.5-10 mM) of sanazole and metronidazole. The spontaneous generation of ROS by peritoneal macrophages was stimulated by sanazole at all tested concentrations (0.6-10 mM).

Using the voltage-clamp technique, a possible role of protein kinase C in regulation of Na+ transport in the skin of the frog Rana temporaria was investigated. It was shown that protein kinase C activator phorbol ester 12-myristate 13-acetate (PMA), applied to the apical surface of the skin, stimulated transepithelial Na+ transport, measured as amiloride-sensitive short-circuit current, and also increased such electrical characteristics of frog skin as the open-circuit potential and transepithelial conductance. PMA exerts a similar stimulation effect on Na+ transport across the tadpole skin. Specific inhibitors of protein kinase C, chelerythryne or H-7, almost fully prevented the PMA-induced stimulation of Na+ transport. These data support a concept that the response to PMA was indeed mediated by PKC activation. The results are compatible with the important role played by protein kinase C in regulation of transepithelial Na+ transport in the skin of R. temporaria.

The effect of modulators of protein kinase C activity on Ca2+ translocation in dark-adapted and bleached retinal rod outer segments (ROS) was studied. The activators (1,2-diacyl glycerol and phorbol-12-myristate-13-acetate) and the inhibitor (chelerythrine chloride) of protein kinase C were shown to stimulate and inhibit the ATP-dependent Ca(2+)-uptake in dark-adapted retinal ROS, correspondingly. Apparently, this action is due to the influence of protein kinase C on Ca(2+)-ATPase activity in these vesicular structures. No involvement of modulators of protein kinase C activity on ATP-dependent Ca(2+)-uptake in bleached retinal ROS was found. The influence of protein kinase C on Ca(2+)-release from retinal ROS was observed. It was shown that the activators and inhibitors of protein kinase C increased the efficiency of this process both in dark-adapted and bleached retinal ROS. The mechanisms of action of the protein kinase C activity modulators on the Ca(2+)-uptake and Ca(2+)-release in retinal ROS are discussed.

The differentiating effect of DMSO on K562 cells was studied against the background of pretreatment of the cells by the modulators of activities of protein kinase C and Ca signaling, phorbol 12-myristate 13-acetate, and ionophore A23187. The 2-hour pretreatment of K562 cells with A23187 (1 microM), rather than phorbol 12-myristate 13-acetate (0.1 microM), led to inhibition of the differentiating effect of DMSO (0.1%) during the subsequent four days of incubation. When the cells were pretreated jointly with A23187 and phorbol 12-myristate 13-acetate, the DMSO-induced erythroid differentiation was restored. Analysis of the DNA-degrading effect of the reagents used on K562 cells by fluorescent dyes under the same conditions suggests that this activity is induced in the presence of DMSO in the cells pretreated with a combination of phorbol 12-myristate 13-acetate and A23187 or without pretreatment.

The role of some components of the phospholipid metabolism in the activation of neutrophil respiratory burst and its inhibition by electromagnetic radiation (EMR) of extremely high frequencies (EHF) was studied. It was shown that EHF EMR has effect on cells with a high sensitivity to the inhibitor of phospholipase A2 4-bromophenacyl bromide. However, againsts the background of the inhibitor, the effect of EHF EMR was not observed on cells with either high or low sensitivity to the inhibitor. EHF EMR was also inefficient with cells pretreated with proadifen, an inhibitor of epoxygenase (cytochrome P-450). The results obtained suggest that the effect of EHF EMR manifests itself in cells with a high activity of phospholipase A2 and is realized with the participation of epoxygenase metabolites of arachidonic acid.

A new mRNA coding for the heparin-binding EGF-like growth factor (HB-EGF) was found in Vero cells. The corresponding cDNA had C-156 in place of T, which resulted in a loss of the NheI site and a substitution of Leu-33 with Pro in the HB-EGF precursor. The known and new forms of the precursor were accordingly termed L and P. A possible conformational change in the corresponding propeptide region were assumed to affect processing of soluble secreted HB-EGF. The L and P mRNAs are differently expressed in various cell lines and have the identical 5'-untranslated sequences. Possibly, they are transcribed from one promoter and then alternatively spliced. Stimulation of resting Vero cells with tetraphorbol ester (TPA) substantially increased production of the L form, decreased production of the P form, and did not affect expression of the total HB-EGF mRNA. This was associated with an increase in binding of the diphtheria toxin, suggesting that the L HB-EGF precursor acts as its receptor.

Membrane lymphotoxin (LT) is heterotrimer LT alpha 1 beta 2, and its production depends on two genes. Northern blotting was employed in studying their transcription in B- and T-lymphoma cell lines and in peripheral blood lymphocytes before and after induction with phorbol myristate acetate (PMA). Transcription of either gene proved similarly regulated in several cell lines and in blood lymphocytes. Activation of the LT alpha gene was associated with induction of transcription factor NF-kappa B (p50/p65) upon cell treatment with PMA. On evidence of RT-PCR, two transcripts of the LT beta gene occurred in equimolar amounts in all lymphoid cells. A product of alternative splicing contained an open reading frame coding for the cytoplasmic portion of LT beta.

Defensive conditioning in Helix is associated with activation of C/EBP family of transcription factors. The incubation of the CNS with serotonin that imitates the effects of conditioning or stimulation of adenylate cyclase with forscolin also increase the DNA-binding activity of C/EBP. The induction of protein kinase C by phorbol ester does not stimulate the formation of these transcription complexes. However, a slight increase in the cAMP-induced influence on the activation of C/EBP transcription factors was observed during a combined action of phorbol ester and serotonin. The more substantial cAMP-induced rise in DNA-binding activity of C/EBP family transcription factors was observed during increase in the intracellular Ca2+ concentration (incubation of the CNS with calcium ionophor A23187 and forscolin). Thus, the convergence of Ca2+ and cAMP-dependent regulating signals that reflect the interaction of different modal stimuli during learning can produce the intensification of formation of the C/EBP transcription complexes.

Three main effects of the amides on embryos of opistobranch molluscs, sea urchins and starfish, were revealed. First, a rather independent and clear protective action of 5-HYDROXYTRYPTAMIDES AND 3-HYDROXYTYRAMIDES against cytostatic antagonists of serotonin and dopamine, resp. Second, prevention of developmental abnormalities induced by protein kinase C activators both by hydroxytryptamides and hydroxytyramides. Third, the cytostatic effect of 3-HYDROXYTRIPTAMIDES eliminated or prevented by 5-HYDROXYTRYPTAMIDES. These effects quantitatively depended on the structure of their fatty acids part. Some functionally active regulatory substances similar to 5-HYDROXYTRYPTAMIDES AND 3-HYDROXYTYRAMIDES may exist in the early embryos.

The expression of a major stress protein Hsp70 may be induced by a great number of cytotoxic agents and also by factors known to cause process of cell proliferation or apoptosis. After induction the synthesis of Hsp70 is reduced to its basic level within first 2-6 hours after inducer application, while the high content of the protein may persist up to several days. The aim of this study was to understand the reason of such a long storage of Hsp70 in U-937 cells induced with three distinct factors. The data of immunoblotting showed that the mild heat shock at 43 degrees C for 60 min, phorbol ester and tumor necrosis factor (the two latter known to induce differentiation and apoptosis, respectively) caused Hsp70 accumulation, the protein level being high within 48 hours and then slowly declined to the basal level. According to the results of chromatography on anti-Hsp70 antibody-Sepharose gel, approximately 50% of the protein was retarded by the gel, while the other part was not recognized by immunoglobulins. Among proteins bound to the Hsp70, three polypeptides were identified as actin, p65 and c-Rel, two latter being constituents of NF-kappa B regulatory complex. The data demonstrate that besides its traditional target, i.e. newly synthesized or abnormal polypeptides, Hsp70 is able to bind mature, active proteins.

We studied the ability of inducers and inhibitors of erythroid differentiation of K562 leukemia cells, such as sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate, respectively, to modulate sensitivity of these cells to non-specific lysis (non-restricted with respect to antigens of the major histocompatibility complex) mediated by natural human or rat killer cells. Unfractionated leukocytes from human peripheral blood or rat splenocytes were used as sources of natural killers. The induction of erythroid differentiation by sodium butyrate was accompanied by a significant increase in cell sensitivity to lysis with human peripheral blood lymphocytes; incubation of K562 cells in the mixture of sodium butyrate and dimethyl sulfoxide did not change cell sensitivity to lysis by both types of effector cells. The inhibition of sodium butyrate-induced erythroid differentiation with high doses of phorbol-12-myristate-13-acetate (100 nM; incubation was in the presence of both these agents simultaneously) resulted in an increased cell sensitivity to lysis with rat splenocytes. Incubation of K562 cells in a mixture of sodium butyrate, dimethyl sulfoxide, and phorbol-12-myristate-13-acetate (100 nM) produced greater lysis by human leukocytes, as compared with incubation in the mixture of sodium butyrate and dimethyl sulfoxide.

Generation of reactive oxygen species (ROS) in A431 cells, NIH3T3 fibroblasts expressing normal epidermal growth factor (EGF) receptor, L929 fibroblasts, and in mouse peritoneal macrophages (professionally phagocytic cells) upon the effect of different activators has been studied. It has been shown that ROS formation in A431 and NIH3T3 cells upon the effect of EGF is time- and dose-dependent process. A variety of stimuli were used to stimulate macrophage ROS production. However, the effect of only phorbol ester, opsonized zymozan, peptide fMLP, and platelet activating factor led to ROS generation, whereas tumor necrosis factor alpha, interferon gamma, and lipopolysaccharide did not stimulate macrophage oxidative burst. The literature data on ROS generation in a variety of cell types are presented. ROS formed in cells acted upon certain agents are considered as the molecules participating in intracellular signaling.

Aim: To study chemiluminescence (CL) of polymorphonuclear neutrophils (PMNL) in peripheral blood of patients with acute mild and moderate dysentery caused by Shig. Flexneri in response to Re- and S-chemotype lipopolysaccharide (LPS) Sh. Flexneri and agonists fMLP, PMA and OZ., Results: PMNL population proved functionally heterogeneic, correlating with acute dysentery severity. Subgroups of PMNL varied by CL amplitude, reserve ability to additional stimulation. LPS rather modulated CL of PMNL than stimulated it. CL intensity depended on LPS structure and concentration. Mild acute dysentery was characterized by pool of primed PMNL which had higher functional reserves., Conclusion: CL analysis can be used for raising accuracy of dysentery prognosis and severity, assessment of implications of functional state of neutrophil granulocytes for support of antiendotoxin immunity in acute dysentery.

Agonists of nicotinic acetylcholine receptors (nAChR) nicotine and 1-acetyl-4-methylpiperazine do not act on the early sea urchin embryogenesis but evoke calcium shock in both oocytes and early embryos under certain conditions. Many nAChR ligands protect both oocytes and embryos against this shock. There seem to exist putative nAChR on the cell surface of the early sea urchin oocytes and early embryos. Pre-nervous acetylcholine seems to be functionally coupled via these receptors with the second messengers, endogenous activators of the protein kinase C.

Effects of substances affecting intracellular secondary messengers on the membrane currents evoked by ionophoretic application of acetylcholine (ACh currents) and on the excitatory postsynaptic currents (EPSC) evoked by single stimuli applied to preganglionic nerve fibres, were studied in neurones of the rat isolated superior cervical ganglion. Forskolin, the protein kinase A activator, and isobutyl-methyxanthine, the phosphodiesterase inhibitor, decreased the ACh currents. Neither forskolin nor isobutyl-methylxanthine affected the EPSC amplitude or the EPSC decay time constant. Phorbol ester, the protein kinase C activator, decreased the ACh current but did not affect either EPSC amplitude or the EPSC decay time constant. Thapsigargin, the intracellular calcium releaser, decreased the ACh current and the EPSC amplitude but did not affect the EPSC decay time constant. The data obtained suggest that nicotinic acetylcholine receptors (nAChRs) of ganglion neurones are not modulated through the pathways involving protein kinase A or protein kinase C. The nAChRs sensitivity to both exogenous and nerve-released acetylcholine is reduced by intracellular calcium without affecting kinetics of their ionic channels.

It was shown that vitamin E decreased the stimulating role of oxidative burst activators which influenced on Ca(2+)-dependent mechanisms (A23187, verapamil, FMLP). Jointly addition of this vitamin and blood plasma contained tocopherol-binding proteins influenced on mechanisms associated with protein kinase C. It was shown that Ca ions can also take part in tocopherol's action on blast transformation of lymphocytes.

The effect of protein kinase C activating phorbol ester, phorbol-12-myristate-13-acetate (PMA), on purinergic agonists- and thapsigargin-induced Ca2+ signals in Fura-2 loaded rat peritoneal macrophages was investigated. PMA (100 ng/ml) was shown to inhibit 200 muM ATP- or 200 microM UTP-evoked Ca2+ entry in macrophages. Protein kinase C activation by PMA also inhibits the store-dependent or "capacitative" Ca2+ influx stimulated by emptying the intracellular Ca2+ stores with endoplasmic Ca(2+)-ATPase inhibitor thapsigargin (0.5 microM). Inhibition of entry by PMA was fully prevented by protein kinase C inhibitor 50 microM H-7. These data are compatible with the important role played by protein kinase C in the control of Ca2+ entry in rat peritoneal macrophages.

In the present paper we have shown that JB6 and PDV murine skin carcinoma cells, as well as previously described sarcoma B6-4 cells, can revert to a nontumor phenotype. Revertant carcinoma clones could not grow in soft agar conditions and like sarcoma revertants acquired dependence on peptide growth factors, and exhibited a reduced expression of c-jun. Spontaneous revertants were shown to be instable. They could revert back to a transformed phenotype in 1-5 months of in vitro passaging. Being inoculated in syngeneic animals, these transformed cells show a recurrence in 2-5 months, similar to that of a dormant metastasis. Thus, dormant revertant cells are believed to be included in many tumors of different origin. So, spontaneous reversions of tumor cells may play an important role in the dormant metastatic process. The cause of these frequent spontaneous transient reversions and revertant instability appears to be of epigenetic nature. Causes and mechanisms of cell transformations and reversions remain to be clarified.

The effect of extremely high frequency electromagnetic field (mm-waves) on respiratory burst of neutrophils was studied. The peritoneal evoked neutrophils of the mice (NMRI line) were used. The production of reactive oxygen species was estimated by luminol-dependent chemiluminescence technique. Cells were irradiated by the mm-waves of 41. 95 GHz in the far field zone of the channel radiator during 20 min. Absorbed energy flux density was 150 microW/cm2. The irradiation was carried out at different concentrations of calcium ionophore A23187 and then neutrophils were stimulated by phorbol 12-myristate 13-acetate (PMA) 1 microM, activator of PKC. At irradiation of neutrophils the synergistic action of A23187 and PMA was not changed at low concentration of ionophore 10 nM-0.5 microM and was suppressed at high concentrations 0.5-10 microM. The largest inhibition of about 60% was obtained at the concentration of A23187 20 microM. The effect of mm-waves was not found under exposure in Ca(2+)-free medium for all used A23187 concentrations. We suggest that the mm-wave effect on the production of reactive oxygen species by neutrophils is determined by the influx of extracellular Ca2+ and PKC.

Direct and modulate effects of platelet activating factor (PAF), its structural analogues and ATP on primary and second processes at peritoneal neutrophils and P388D1 cells activation has been studied. The effect of compounds was evaluated on changes in Ca2+ transport and generation of reactive oxygen species. It was shown, that the synthetic analogues of MS series interact with PAF receptor, mobilize Ca2+ from thapsigargin-dependent intracellular stores and inhibit Ca2+ response on PAF in both types of cells. Unlike PAF the analogues do not induce the formations of reactive oxygen species in neutrophils and inhibit the PMA-induced respiratory burst. The activation of pyrinoreceptor of P388D1 cells by exogenous ATP does not inhibit PAF induced Ca2+ rise in cytoplasm, though partly releases Ca2+ from the same store.

The influence of low-intensity modulated electromagnetic radiation of extremely high frequencies (EHF EMR) on synergistic reaction of calcium ionophore A23187 and phorbol ester PMA in activation of the respiratory burst of the peritoneal neutrophils of mice line NMRI was investigated. The production of reactive oxygen species by the neutrophils was estimated by luminol-dependent chemiluminescence technique. The cells were irradiated in the far field zone of the channel radiator for 20 min in the presence of A23187 and then were activated by PMA after switching off the irradiation. It was shown, that continuous EHF EMR (50 microW/cm2) inhibited quasi-resonantly the synergistic reaction. The maximum effect was about 25% at carrier frequency of 41.95 GHz. Modulated radiation with carrier frequency of 41.95 GHz and modulation frequency of 1 Hz activated the synergistic reaction, but at modulation frequencies of 0.1, 16 and 50 Hz inhibited one. At fixed modulation frequency of 1 Hz the nonlinear dependence of the effect on the carrier frequency was found. The synergistic reaction was activated in the frequency range of 41.95-42.05 GHz and was inhibited at the frequencies of 41.8-41.9 GHz. The effect was observed only at raised intracellular free calcium concentration and at calcium fluxes through plasma membrane. The obtained results prove the possibility of control over cell functioning by low-intensity modulated EHF EMR, presumably, manipulating by connected systems of enzyme reactions.

We investigated the ability of gamma-interferon (IFN-gamma) and phorbol 12-myristate 13-acetate (PMA) to influence the ligand-binding of Fc-receptors for immunoglobulin G (Fc gamma R) (conjugation of IgG to fluorescein izothiocyanate--FITC) of bovine blood lymphocytes and the antibody-dependent cell-mediated cytotoxic activity (ADCC). The results demonstrate that the IFN-gamma or PMA induced augmentation of ligand-binding capacity of Fc gamma R of bovine lymphocytes, and correlated well with the enhanced capacity to lyse L cells coated with anti-L cell antibody. The number of conjugates effector--target, formed by bovine lymphocytes in the reaction of ADCC, correlates well with ligand-binding capacity of Fc gamma R. It can be suggested that IFN-gamma or PMA may control the ADCC caused by Fc gamma R regulating its expression.

The effect of thyroxine on total phospholipid, phosphatidic acid, phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate synthesis in albino rat hepatocytes, has been studied. It has been found that the hormone enhances the [14C]palmitic acid incorporation into lipids. It has been shown also that phorbol-12-myristate-13-acetate mimicks the effect of thyroxine on lipid synthesis, while exogenous sphingosine abolishes it. Sphingosine causes a significant inhibition of phosphoinositide synthesis in intact liver cells.

The activator of protein kinase C phorbol-12-myristat-13-acetate (PMA) induces maturation of the definitive intact (coated with the follicular envelopes) and defolliculated oocytes of the green toad. This effect is more pronounced in case of defolliculated oocytes. The amount of matured oocytes depends on the PMA concentration in solution. Serotonin (5-HT) inhibits or blocks maturation of the oocytes induced either by progesterone or by PMA. A possible mechanism of this effect is discussed with special reference to its role in regulation of oocyte maturation.

Effects of AML and normal mononuclear phagocyte conditioned media (CM) on the proliferation and differentiation of monoblastic human cell line U-937 have been studied. The normal mononuclear phagocyte CM inhibited proliferation and weakly stimulated differentiation of U-937 cells, whereas AML CM exerted no effect. Activation of both normal and AML macrophages with phorbol ether (TPA) was followed by enhancement of CM effect. TPA treatment corrected defects of secretory activity of AML mononuclear phagocytes. A possible role of different monokines in the regulation of proliferation and differentiation of leukaemic cells is discussed.

The effects of thyroxine, phorbol esters and sphingosine on incorporation of [14C]linoleic acid into phospholipids, triacylglycerols and cholesterol esters of albino rat liver have been studied. It has been found that phorbol-12-myristate-13-acetate and thyroxine activate the [14C]linoleic acid incorporation into liver cell phospholipids and triacylglycerols. On the other hand, methyl esters of phorbol-12-myristate-13-acetate do not influence, while mercasolyl and sphingosine decrease the fatty acid incorporation into liver glycerolipids. It has been found that sphingosine abolishes the thyroxine effect on glycerolipid activation. It has been shown also that sphingosine, thyroxine and phorbol-12-myristate-13-acetate methyl esters do not influence the cholesterol ester synthesis. However, phorbol-12-myristate-13-acetate enhances the cholesterol ester activation in liver cells twofold.

The influence of tetra phorbol diesther (TPA) on primary blast cells of patients with acute leukemia and blastic crises of chronic myelogenous leukemia and the influence of the condition medium (CM) of the primary and TPA-treated blast cells on the proliferation of HL-60 cell line has been studied. The level of interferon-alpha (IFN-alpha) in CM was tested. TPA inhibited proliferation and induced macrophage-like differentiation of primary AML blast cells and these changes were accompanied by modulation of IFN-alpha expression in CM. The effect of blast CM on proliferation of HL-60 was both inhibitory and stimulating and depended on the time of treatment and individual characteristics of patients. It has been shown that the level of IFN-alpha in CM was not correlated with antiproliferative effect of CM. The role of individual differences in capability of primary blast cells to be induced by differentiated agents and the nature of these differences are discussed.

Nedocromil sodium in concentrations of 10(-3) to 10(-9) M dose-dependently inhibits a zymosan (2 mg/ml)-induced chemiluminescence response (CLR) of whole blood leukocytes and, to a lesser extent, phorbol myrystate acetate (PMA)-induced CLR. The count of diformazan-positive granulocytes in the zymosan-induced nitroblue tetrazolium test slightly reduced in the presence of 10(5) nedocromil sodium. PMA in a dose of 10(-9) M did not induce CLR, but brought about leukocyte priming, thus sensitizing them to secondary triggering by suboptimal zymosan concentrations (0.5 mg/ml). A subsequent stimulation of leukocytes with PMA + zymosan demonstrated that nedocromil sodium (10(-7) M) inhibited mainly priming PMA rather than the zymosan-triggering step of leukocyte activation.

The roles of phospholipase A2, 5-lipoxygenase and cyclooxygenase in activation of "oxidative burst" in human neutrophils have been studied under isoosmotic (320 mOsM), hypoosmotic (200 mOsM) and hyperosmotic (420 mOsM) conditions. The oxidative burst induced by phorbol-12-myristate-13-acetate (PMA), calcium ionophore A23187 and opsonized zymosan was followed by luminol-dependent chemiluminescence (CL). There were no differences in the concentration dependences of CL inhibition at 320 mOsM and 200 mOsM in the presence of the phospholipase A2 inhibitor, p-bromphenacylbromide. Contrariwise, at 420 mOsM the sensitivity to inhibition was decreased by PMA and A23187 but increased by opsonized zymosan. A change in the medium osmolality in the presence of the 5-lipoxygenase inhibitor quercetin did not result in the modification of the concentration dependence of CL inhibition induced by any of the activators. In the presence of the cyclooxygenase inhibitor, meclofenamic acid, the "oxidative burst" induced by PMA and A23187 at 200 mOsM was manifested as a decrease of the sensitivity to inhibition, while at 420 mOsM this sensitivity was increased in comparison with that observed under isoosmotic conditions. The data obtained suggest that phospholipase A2 and cyclooxygenase play a role in the mechanism of the modulating effect of the medium osmolality on the "oxidative burst" in neutrophils, while 5-lipoxygenase is unimportant for this process.