On the new mechanism of adaptive response

The role of changes in cell composition of population of human blood lymphocytes in the forming of an adaptive response (AR) has been studied. By micronuclei assay and cytokinetic block with cytohalasin B the frequency of mono-, bi- and multinuclear cells with micronuclei (MN) and without MN were determined in the initial population. The same parameters have been studied after exposure of the population to the adaptive (0.05 Gy), challenge (1.0 Gy) doses and to doses 0.05 + 1.0 Gy 5 hours after. 13 from 23 investigated individuals manifested the AR: the decreasing of the ratio of damaged binuclear cells to the all binuclear cells after the adaptive and challenge exposure. It was shown that the ways of an AR forming are different: in 7 of 13 individuals with AR the number of binuclear cells with MN did not decrease but the amount of binuclear undamaged cells increased. The ratio of these parameters enhances but not for the account of cells with MN decreasing. There is the linear correlation between the frequency of cells with MN and the frequency of binuclear cells in population (spontaneous, after irradiation with doses of 0.05, 1.0 and 0.05 + 1.0 Gy) with the coefficient of correlation about -1. These results show the presence of new mechanism of AR forming, which is not connect with the induction of damage repair and rather with the stimulation of cell division. In the another group of individuals the decrease in damaged cells number after irradiation with doses of 0.05 + 1.0 Gy have been observed. Probably the stimulation of repair system occurred to the moment of 1.0 Gy irradiation. Thus, the mechanism of an AR forming depends on the individual properties of organism. The work was suppoted by RFBR grant 03-04-48325a.     

Acid resistance systems required for survival of Escherichia coli O157:H7 in the bovine gastrointestinal tract and in apple cider are different

Escherichia coli O157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider. This property is thought to contribute to the low infectious dose of the organism. Three acid resistance (AR) systems are expressed in stationary-phase cells. AR system 1 is sigma(S) dependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively. In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract. Wild-type and mutant E. coli O157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves. Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider. Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH. Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells. AR system 3-deficient cells survived well in both apple cider and calves. Taken together, these results indicate that E. coli O157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered.     

Adenosine A<Subscript>2A</Subscript> receptor as a drug target for treatment of sepsis

Sepsis is a generalized infection accompanied by response of the body that manifests in a clinical and laboratory syndrome, namely, in the systemic inflammatory response syndrome (SIRS) from the organism to the infection. Although sepsis is a widespread and life-threatening disease, the assortment of drugs for its treatment is mostly limited by antibiotics. Therefore, the search for new cellular targets for drug therapy of sepsis is an urgent task of modern medicine and pharmacology. One of the most promising targets is the adenosine A_2A receptor (A_2AAR). The activation of this receptor, which is mediated by extracellular adenosine, manifests in almost all types of immune cells (lymphocytes, monocytes, macrophages, and dendritic cells) and results in reducing the severity of inflammation and reperfusion injury in various tissues. The activation of adenosine A_2A receptor inhibits the proliferation of T cells and production of proinflammatory cytokines, which contributes to the activation of the synthesis of anti-inflammatory cytokines, thereby suppressing the systemic response. For this reason, various selective A_2AAR agonists and antagonists may be considered to be drug candidates for sepsis pharmacotherapy. Nevertheless, they remain only efficient ligands and objects of pre-clinical and clinical trials. This review examines the molecular mechanisms of inflammatory response in sepsis and the structure and functions of A_2AAR and its role in the pathogenesis of sepsis, as well as examples of using agonists and antagonists of this receptor for the treatment of SIRS and sepsis.     

Chinese hamster cell variants resistant to the A chain of ricin carry altered ribosome function

Ricin, a toxic lectin from Ricinus communis, is composed of two different polypeptide chains, A and B, and the ricin A chain (RA) blocks protein synthesis. We studied cell lines resistant to cytotoxic action of RA. One low-RA-resistant cell line, AR10, isolated from Chinese hamster ovary (CHO) cells, was resistant to a low dose of RA (1 microgram/ml) and showed a 10-fold-higher resistance to RA and ricin than that of CHO. We further mutagenized AR10 to isolate high-RA-resistant cell lines AR100-6, AR100-9, and AR100-13, which were resistant to higher doses of RA and ricin (100- to 1,000-fold) than CHO was. The binding of [125I]ricin to AR10, AR100-6, AR100-9, and AR100-13 cells was decreased to about 30% of that of CHO. The internalization of [125I]ricin in AR10 cells and in the high-RA-resistant clones was the same. Polyuridylate-dependent polyphenylalanine synthesis, using S-30 extracts from either AR100-9 or AR100-13, was about 100-fold more resistant to the inhibitory action of RA than when CHO, AR10, and AR100-6 cells extracts were used. The protein synthesis with ribosomes (80S) from AR100-9 or AR100-13 was 10- to 100-fold more resistant to RA than it was with parental ribosomes when combined with the S-100 fraction of CHO cells. The polyphenylalanine synthesis assay using the ribosomes constituted from the 60S subunit of AR100-9 and the 40S subunit of CHO indicated that the resistant phenotype of AR100-9 cells is due to an alteration of the 60S ribosomal subunit.     

Acid Resistance Systems Required for Survival of Escherichia coli O157:H7 in the Bovine Gastrointestinal Tract and in Apple Cider Are Different

Escherichia coli O157:H7 is a highly acid-resistant food-borne pathogen that survives in the bovine and human gastrointestinal tracts and in acidic foods such as apple cider. This property is thought to contribute to the low infectious dose of the organism. Three acid resistance (AR) systems are expressed in stationary-phase cells. AR system 1 is σ^S dependent, while AR systems 2 and 3 are glutamate and arginine dependent, respectively. In this study, we sought to determine which AR systems are important for survival in acidic foods and which are required for survival in the bovine intestinal tract. Wild-type and mutant E. coli O157:H7 strains deficient in AR system 1, 2, or 3 were challenged with apple cider and inoculated into calves. Wild-type cells, adapted at pH 5.5 in the absence of glucose (AR system 1 induced), survived well in apple cider. Conversely, the mutant deficient in AR system 1, shown previously to survive poorly in calves, was susceptible to apple cider (pH 3.5), and this sensitivity was shown to be caused by low pH. Interestingly, the AR system 2-deficient mutant survived in apple cider at high levels, but its shedding from calves was significantly decreased compared to that of wild-type cells. AR system 3-deficient cells survived well in both apple cider and calves. Taken together, these results indicate that E. coli O157:H7 utilizes different acid resistance systems based on the type of acidic environment encountered.     

Adaptation to irradiation in vivo

The data about the increasing of radioresistance of cells and organism after the acute, chronic and prolonged irradiation in vivo were presented. The possible mechanisms of adaptation to irradiation connected with selection, stimulation of proliferative activity, forming of protecting system (protected proteins, antioxidant enzymes e.a.), activation of DNA repair and cAMP-, and Ca(2+)-dependent phosphorylation systems were observed. The conclusion about complex mechanism of radiation adaptation was drawn.     

β‐adrenergic blockade attenuates cardiac dysfunction and myofibrillar remodelling in congestive heart failure

Although β‐adrenoceptor (β‐AR) blockade is an important mode of therapy for congestive heart failure (CHF), subcellular mechanisms associated with its beneficial effects are not clear. Three weeks after inducing myocardial infarction (MI), rats were treated daily with or without 20 and 75 mg/kg atenolol, a selective β₁‐AR antagonist, or propranolol, a non‐selective β‐AR antagonist, for 5 weeks. Sham operated rats served as controls. All animals were assessed haemodynamically and echocardiographically and the left ventricle (LV) was processed for the determination of myofibrillar ATPase activity, α‐ and β‐myosin heavy chain (MHC) isoforms and gene expression as well as cardiac troponin I (cTnI) phosphorylation. Both atenolol and propranolol at 20 and 75 mg/kg doses attenuated cardiac hypertrophy and lung congestion in addition to increasing LV ejection fraction and LV systolic pressure as well as decreasing heart rate, LV end‐diastolic pressure and LV diameters in the infarcted animals. Treatment of infarcted animals with these agents also attenuated the MI‐induced depression in myofibrillar Ca²⁺‐stimulated ATPase activity and phosphorylated cTnI protein content. The MI‐induced decrease in α‐MHC and increase in β‐MHC protein content were attenuated by both atenolol and propranolol at low and high doses; however, only high dose of propranolol was effective in mitigating changes in the gene expression for α‐MHC and β‐MHC. Our results suggest that improvement of cardiac function by β‐AR blockade in CHF may be associated with attenuation of myofibrillar remodelling.     

Assessment of DNA base oxidation and glutathione level in patients with type 2 diabetes

Genetic instability resulting from the disturbances in various mechanisms of DNA-repair is the characteristic feature of cancer cells. One of the possibilities to evaluate the effectiveness of DNA-repair system is the adaptive response (AR) analysis. The AR is a phenomenon by which cells exposed to low, non-genotoxic doses of a mutagen become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. Generally, it is postulated that AR is related to a reduction of damage by the induction of free radical detoxification and/or DNA-repair systems.The existence of various DNA-repair mechanisms poses the question whether there are differences in AR induced by chemicals causing DNA-damage that requires different pathways for its repair. In this paper we present the study on the AR induced by two chemical mutagens, bleomycin (BLM) and mitomycin C (MMC), which differ in their action on DNA. BLM is a radiomimetic agent causing mainly single-strand breaks (SSB) and double-strand breaks (DSB) and, thus, inducing chromosomal aberrations (CA). MMC is a potent bifunctional mutagen acting as an alkylating agent, causing DNA cross-links and inducing sister chromatid exchanges (SCEs).The protective effect induced by low doses of tested chemicals was analysed in whole blood human lymphocytes using cytogenetic endpoints (CA for BLM and SCE for MMC, respectively) as a measure of chromosomal instability. There was a significant difference between the protective effects induced by BLM and MMC in the lymphocytes of the same group of donors. The pre-treatment with a low dose of BLM-induced almost 50% decrease in the frequency of CA induced by challenging dose (CD), while the protective effect of MMC was below 20%. The higher AR induced by BLM may be related to the repair processing of BLM-induced DNA-damages. There was also a variability in ARs among individuals, which may reflect the differences in individual DNA-repair capacity.     

Analysis of adaptive response to bleomycin and mitomycin C

Genetic instability resulting from the disturbances in various mechanisms of DNA-repair is the characteristic feature of cancer cells. One of the possibilities to evaluate the effectiveness of DNA-repair system is the adaptive response (AR) analysis. The AR is a phenomenon by which cells exposed to low, non-genotoxic doses of a mutagen become significantly resistant to a subsequent higher dose of the same or another genotoxic agent. Generally, it is postulated that AR is related to a reduction of damage by the induction of free radical detoxification and/or DNA-repair systems.The existence of various DNA-repair mechanisms poses the question whether there are differences in AR induced by chemicals causing DNA-damage that requires different pathways for its repair. In this paper we present the study on the AR induced by two chemical mutagens, bleomycin (BLM) and mitomycin C (MMC), which differ in their action on DNA. BLM is a radiomimetic agent causing mainly single-strand breaks (SSB) and double-strand breaks (DSB) and, thus, inducing chromosomal aberrations (CA). MMC is a potent bifunctional mutagen acting as an alkylating agent, causing DNA cross-links and inducing sister chromatid exchanges (SCEs).The protective effect induced by low doses of tested chemicals was analysed in whole blood human lymphocytes using cytogenetic endpoints (CA for BLM and SCE for MMC, respectively) as a measure of chromosomal instability. There was a significant difference between the protective effects induced by BLM and MMC in the lymphocytes of the same group of donors. The pre-treatment with a low dose of BLM-induced almost 50% decrease in the frequency of CA induced by challenging dose (CD), while the protective effect of MMC was below 20%. The higher AR induced by BLM may be related to the repair processing of BLM-induced DNA-damages. There was also a variability in ARs among individuals, which may reflect the differences in individual DNA-repair capacity.     

Stimulation of expression for the adenosine A2A receptor gene by hypoxia in PC12 cells. A potential role in cell protection.

The purpose of this study was to examine the regulation of adenosine A2A receptor (A2AR) gene expression during hypoxia in pheochromocytoma (PC12) cells. Northern blot analysis revealed that the A2AR mRNA level was substantially increased after a 3-h exposure to hypoxia (5% O2), which reached a peak at 12 h. Immunoblot analysis showed that the A2AR protein level was also increased during hypoxia. Inhibition of de novo protein synthesis blocked A2AR induction by hypoxia. In addition, removal of extracellular free Ca2+, chelation of intracellular free Ca2+, and pretreatment with protein kinase C inhibitors prevented A2AR induction by hypoxia. Moreover, depletion of protein kinase C activity by prolonged treatment with phorbol 12-myristate 13-acetate significantly inhibited the hypoxic induction of A2AR. A2AR antagonists led to a significant enhancement of A2AR mRNA levels during hypoxia, whereas A2AR agonists caused down-regulation of A2AR expression during hypoxia. This suggests that A2AR regulates its own expression during hypoxia by feedback mechanisms. We further found that activation of A2AR enhances cell viability during hypoxia and also inhibits vascular endothelial growth factor expression in PC12 cells. Thus, increased expression of A2AR during hypoxia might protect cells against hypoxia and may act to inhibit hypoxia-induced angiogenic activity mediated by vascular endothelial growth factor.     

Relation between the changes in the structure and synthesis of DNA in the cells of mouse leukemia L1210 induced by 1-methyl-1-nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea

The damage of DNA structure and synthesis in murine leukemia L1210 cells upon single administration in therapeutic doses of antitumour agents of N-nitrosourea type, such as 1-methyl-1-nitrosourea (MNU) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) was studied. MNU and BCNU were characterized by stronger inhibitory effects on de novo DNA synthesis compared to additional pathway of DNA synthesis in leukemia L1210 cells in vivo. Centrifugation in alkaline sucrose density gradients of L1210 cell lysates has revealed persistent single-strand breaks and alkaline-labile sites in newly replicated DNA. Parental DNA structure was more stable to damaging drug effects than that of newly replicated DNA. The results are consistent with our previous data on the differences in the mechanisms of MNU and BCNU action and the absence of complete cross resistance between the drugs.     

Differential effect of adenosine on tumor and normal cell growth: focus on the A3 adenosine receptor

Adenosine is an ubiquitous nucleoside present in all body cells. It is released from metabolically active or stressed cells and subsequently acts as a regulatory molecule through binding to specific A1, A2A, A2B and A3 cell surface receptors. The synthesis of agonists and antagonists to the adenosine receptors and their cloning enabled the exploration of their physiological functions. As nearly all cells express specific adenosine receptors, adenosine serves as a physiological regulator and acts as a cardioprotector, neuroprotector, chemoprotector, and as an immunomodulator. At the cellular level, activation of the receptors by adenosine initiates signal transduction mechanisms through G-protein associated receptors. Adenosine's unique characteristic is to differentially modulate normal and transformed cell growth, depending upon its extracellular concentration, the expression of adenosine cell surface receptors, and the physiological state of the target cell. Stimulation of cell proliferation following incubation with adenosine has been demonstrated in a variety of normal cells in the range of low micromolar concentrations, including mesangial and thymocyte cells, Swiss mouse 3T3 fibroblasts, and bone marrow cells. Induction of apoptosis in tumor or normal cells was shown at higher adenosine concentrations (>100 microM) such as in leukemia HL-60, lymphoma U-937, A431 epidermoid cells, and GH3 tumor pituitary cell lines. It was further noted that the A3 adenosine receptor (A3AR) plays a key role in the inhibitory and stimulatory growth activities of adenosine. Modulation of the A3AR was found to affect cell growth either positively or negatively depending on the concentration of the agonist, similar to the effect described for adenosine. At nanomolar concentrations, the A3AR agonists possess dual activity, i.e., antiproliferative activity toward tumor cells and stimulatory effect on bone marrow cells. In vivo, these agonists exerted anti-cancer effects, and when given in combination with chemotherapy, they enhanced the chemotherapeutic index and acted as chemoprotective agents. Taken together, activation of the A3AR, by minute concentrations of its natural ligand or synthetic agonists, may serve as a new approach for cancer therapy.     

The effects of several croton oil constituents on two types of DNA repair and cyclic nucleotide levels in mammalian cells in vitro.

The purpose of the present study was to determine the effects of two potent tumor-promoting agents on two DNA repair mechanisms and cyclic nucleotide levels in mammalian cells. Human amnion (AV3) cells were treated with low dose levels of either UV of N-acetoxy-acetylaminofluorene. Subsequently, DNA excision repair as measured by unscheduled DNA synthesis was followed in the absence or presence of non-toxic levels of either 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibenzoate (PDB), both potent tumor promoters, or phorbol, a non-promoter. Neither of these compounds inhibited DNA repair synthesis occurring in response to low doses of the carcinogenic agents. In addition, TPA did not inhibit "post-replication repair" in response to UV irradiation of growing Chinese hamster (V79-4) cells. However, both TPA and PDB did cause rapid dramatic increases in cyclic guanosine monophosphate levels in human amnion cells; phorbol had no effect. Neither of these compounds affected cyclic adenosine monophosphate levels. These results are discussed in the light of a possible mechanism of the action of tumor promoters involving "post-replication repair".     

Resistance of the human beta 1-adrenergic receptor to agonist-mediated down-regulation. Role of the C terminus in determining beta-subtype degradation

Prolonged agonist stimulation results in down-regulation of most G protein-coupled receptors. When we exposed baby hamster kidney cells stably expressing the human beta1-adrenergic receptor (beta 1AR) to agonist over a 24-h period, we instead observed an increase of approximately 30% in both beta 1AR binding activity and immune-detected receptors. In contrast, beta 2AR expressed in these cells exhibited a decrease of > or =50%. We determined that the basal turnover rates of the two subtypes were similar (t(1/2) approximately 7 h) and that agonist stimulation increased beta 2AR but not beta 1AR turnover. Blocking receptor trafficking to lysosomes with bafilomycin A1 had no effect on basal turnover of either subtype but blocked agonist-stimulated beta 2AR turnover. As beta 1AR mRNA levels increased in agonist-stimulated cells, beta 1AR up-regulation appeared to result from increased synthesis with no change in degradation. To explore the basis for the subtype differences, we expressed chimeras in which the C termini had been exchanged. Each chimera responded to persistent agonist stimulation based on the source of its C-tail; beta 1AR with a beta 2AR C-tail underwent down-regulation, and beta 2AR with a beta 1AR C-tail underwent up-regulation. The C-tails had a corresponding effect on agonist-stimulated receptor phosphorylation and internalization with the order being beta 2AR > beta 1AR with beta 2AR C-tail > beta 2AR with a beta 1AR C-tail > beta 1AR. As internalization may be a prerequisite for down-regulation, we addressed this possibility by co-expressing each subtype with arrestin-2. Although beta 1AR internalization was increased to that of beta 2AR, down-regulation still did not occur. Instead, beta 1AR accumulated inside the cells. We conclude that in unstimulated cells, both subtypes appear to be turned over by the same mechanism. Upon agonist stimulation, both subtypes are internalized, and beta 2AR but not beta 1AR undergoes lysosomal degradation, the fate of each subtype being regulated by determinants in its C-tail.     

Action of granulopoiesis-stimulating cytokines rhG-CSF, rhGM-CSF, and rmGM-CSF on murine haematopoietic progenitor cells for granulocytes and macrophages (GM-CFC)

The aim of this study was to provide new data to the knowledge of mechanisms by which recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant murine granulocyte-macrophage colony-stimulating factor (rmGM-CSF) enhance the numbers of colonies growing from hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) in the murine bone marrow. The in vitro technique for cultivating GM-CFC from normal bone marrow cells was used. For evaluation of stimulatory actions of the drugs studied, the factors themselves or sera of mice given these factors were added to the cultures. The factors or the sera were present in the cultures either as the only potentially stimulatory agents or acted jointly with a suboptimum concentration of recombinant murine interleukin-3 (rmIL-3). It was found that both rhG-CSF and rmGM-CSF stimulate the proliferation of GM-CFC by a combination of direct mechanisms (direct actions on the target cells) and indirect effects (effects mediated through the induction of other cytokines and/or growth factors in the murine organism). The rhGM-CSF exhibited somewhat weaker in vitro effects in comparison with the other two factors and only indirect effects were noted. Additional in vivo experiments documented that, in spite of differences in mechanisms of action of the individual drugs studied on murine bone marrow cells in vitro, equal in vivo doses of the factors induce quantitatively similar effects on the production of GM-CFC in vivo.     

Comparative studies of 5α-reductase inhibitors within MCF-7 human breast cancer cells

We have compared the inhibitory effects of six synthetic steroid analogs (17β-carboxy-4-androsten-3-one benzylanilide (VP-1), 17α -acetoxy-6-methylene-4-pregnene-3, 20-dione (VP-2), 6-methylene-4-pregnene-3, 20-dione (VP-3), 17β-acetoxy-6-methylene-4-androsten-3-one (VP-4), 17β -acetoxy-16, 16-dimethyl-6-methylene-4-androsten-3-one (VP-5), and 3β-hydroxy-16-methylene-5-androsten-17-one (VP-6)) upon 5α-reductase activity within MCF-7 human breast cancer cells and rat prostate. Enzyme assays were performed by quantifying the amounts of [³H]5α-androstan-3α-17β-diol and/or [³H]dihydrotestosterone formed from 40 nM [³H]testosterone within each system. Five μM concentrations of VP-2 and VP-3 inhibited prostatic 5α-reductase by 55 and 65%, respectively, whereas the other analogs showed little activity. In contrast, each of the six analogs was active against MCF-7 homogenate 5α reductase activity. VP-2 and VP-4 demonstrated approx 65 and 70% inhibitions, respectively, whereas the other four compounds inhibited enzyme activity by 40–55% in this system. These results suggest that rat prostate and MCP-7 cells contain different 5α-reductase isozymes. When these agents were examined for 5α-reductase inhibitory activity following 1 h preincubations with intact MCF-7 cultures, VP-1 and 3 demonstrated potencies similar to those in MCF-7 homogenate. The other compounds, however, were far less active under these conditions. Longer culture preincubations (16 h) were associated with substantially increased VP-6 potency, moderate increases for VP-4 and 5, but no change in VP-2 activity. Additional studies examining the abilities of these agents to bind to MCF-7 androgen receptor (AR) and progesterone receptor (PR) revealed moderate AR binding activities of VP-2, 3, and 4, and substantial PR binding for VP-2 and 3. Finally, VP-4 failed to inhibit estrogen-dependent MCF-7 PR synthesis, suggesting that it has no androgenic activity despite its ability to interact with MCF-7 AR.