Emotional Stress Induces Adaptive Response in Rat Lymphocytes to Subsequent Ionizing Radiation Exposure.

We studied the molecular mechanisms of cross-adaptation to ionizing radiation (1 Gy) of lymphocytes isolated from rats subjected to emotional stress. The effects of chronic (CES; various types of stress exposure) and acute (AES; forced swimming) emotional stress in rats on indicators of oxidative stress, cell death, and levels of NRF2 and NOX4 proteins involved in the development of the adaptive response were analyzed in isolated lymphocytes. It was found that stress induced an adaptive response in rat lymphocytes and triggered processes similar to the adaptive response induced by low doses of ionizing radiation: an increase in the level of oxidized DNA and cell death, as well as an increase in the content of NOX4 and NRF2 proteins. In animals subjected to emotional stress, suppressed DNA oxidation in response to irradiation, reduced levels of protective factor NRF2, as well as lymphocyte death were observed.

[Antioxidant status of blood plasma of acutely psychotic patients and its correlation with Nrf2 activation]. / Antioksidantnyi status plazmy krovi patsientov s ostrym psikhozom i ego svyaz' s aktivatsiei transkriptsionnogo faktora Nrf2.

OBJECTIVE: To study the correlation between the blood plasma antioxidant profile and the transcriptional activity of the Nrf2 gene in acute psychosis in patients with schizophrenia and alcoholism. MATERIAL AND METHODS: The study included 40 patients with the first episode of the paranoid form of schizophrenia, 33 patients with schizophrenic psychosis who had previously received therapy, 22 patients with first-time acute alcohol psychosis, and 25 healthy volunteers. The level of Nrf2 in peripheral blood mononuclear cells was estimated by flow cytometry, and the antioxidant profile of blood plasma was estimated with chemiluminometry. RESULTS: The total and «thiol¼ antioxidant capacity were reduced in patients with initially diagnosed schizophrenic psychosis and alcoholic psychosis. In patients after treatment, the total antioxidant capacity was higher compared to previously untreated patients. The level of Nrf2 protein in mononuclear cells in patients with the first psychotic episode was significantly lower than in patients with alcoholism and lower than in the control group. In patients with alcoholic psychosis, Nrf2 level was correlated with both the total antioxidant capacity due to uric acid and the «thiol¼ antioxidant capacity; in patients with psychosis in schizophrenia, Nrf2 level was correlated only with the «thiol¼ antioxidant capacity. CONCLUSIONS: The correlation between the total and «thiol¼ antioxidant capacity and the level of Nrf2 in mononuclear cells of patients with alcohol delirium indicates the undamaged state of the regulation. The absence of a correlation between the total antioxidant capacity and the level of Nrf2 in patients with schizophrenia indicates a disturbance of the activation of the Nrf2 pathway due to, possibly, a part associated with the participation of uric acid.

Accumulation of Circulating Cell-Free CpG-Enriched Ribosomal DNA Fragments on the Background of High Endonuclease Activity of Blood Plasma in Schizophrenic Patients.

INTRODUCTION: Schizophrenia (SZ) increases the level of cell death, leading to an increase in the concentration of circulating cell-free DNA (cfDNA). Ribosomal DNA (rDNA) contains many unmethylated CpG motifs that stimulate TLR9-MyD88-NF-κB signaling and the synthesis of proinflammatory cytokines. The number of rDNA copies in the genomes of SZ patients is increased; therefore, we expect that the concentration of cell-free rDNA in the plasma of the SZ patients also increases. This may be one of the explanations of the proinflammatory cytokine increase that is often observed in SZ. The major research question is what is the rDNA copy number in cfDNA (cf-rDNA CN) and its putative role in schizophrenia? Materials and Methods. We determined cfDNA concentration (RNase A/proteinase K/solvent extraction; fluorescent dye PicoGreen) and endonuclease activity (NA) of blood plasma (radial diffusion method) in the untreated male SZ group (N = 100) and in the male healthy control group (HC) (N = 96). Blood leukocyte DNA and cfDNA rDNA CN were determined with nonradioactive quantitative hybridization techniques. Plasma concentration of cf-rDNA was calculated. RESULTS: In the subjects from the SZ group, the mean cfDNA plasma concentration was twofold higher and NA of the plasma was fourfold higher than those in the healthy controls. rDNA CN in the blood leukocyte genome and in the cfDNA samples in the SZ group was significantly higher than that in the HC group. cf-rDNA concentration was threefold higher in the SZ group. CONCLUSION: Despite the abnormally high endonuclease activity in the blood plasma of SZ patients, the circulating cfDNA concentration is increased. Fragments of cf-rDNA accumulate in the blood plasma of SZ patients. Potentially, SZ patients' cfDNA should be a strong stimulating factor for the TLR9-MyD88-NF-κB signaling pathway.

Oxidized Cell-Free DNA Role in the Antioxidant Defense Mechanisms under Stress.

The present study focuses on the investigation of the oxidized cell-free DNA (cfDNA) properties in several experimental models, including cultured cerebellum cells, peripheral blood lymphocytes (PBL), plasma, and hippocampus under an acute and chronic unpredictable stress model in rats. Firstly, our study shows that Spectrum Green fluorescence-labeled oxidized cfDNA fragments were transferred into the cytoplasm of 80% of the cerebellum culture cells; meanwhile, the nonoxidized cfDNA fragments do not pass into the cells. Oxidized cfDNA stimulates the antioxidant mechanisms and induction of transcription factor NRF2 expression, followed by an activation of NRF2 signaling pathway genes-rise of Nrf2 and Hmox1 gene expression and consequently NRF2 protein synthesis. Secondly, we showed that stress increases plasma cfDNA concentration in rats corresponding with the duration of the stress exposure. At the same time, our study did not reveal any significant changes of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) level in PBL of rats under acute or chronic stress, probably due to the significantly increased Nrf2 expression, that we found in such conditions. 8-oxodG is one of the most reliable markers of DNA oxidation. We also found an increased level of 8-oxodG in the hippocampal homogenates and hippocampal dentate gyrus in rats subjected to acute and chronic stress. Taken together, our data shows that oxidized cfDNA may play a significant role in systemic and neuronal physiological mechanisms of stress and adaptation.

[Heterochronies in the Formation of the Nervous and Digestive Systems in Early Postlarval Development of Opistobranch Mollusks: Organization of Basic Functional Systems of the Arctic Dorid Cadlina laevis].

For the first time using laser confocal microscopy and histochemical and immunocytochemical methods (detection of F-actine, catecholamines, acetylcholintransferase, substances of P and FM RFamide) in combination with classical histological methods and electron microscopy of total preparations, the general structure and regularities of formation of the main organs and the nervous, muscular, and digestive systems in early postlarval development (2 to 4 months) in the opistobranch mollusk Cadlina laevis were studied. Heterochronies manifested in positive allometry of the sensory organs, ganglia of the central nervous system, and the pharyngeal region of the digestive system in relation to general body sizes in juvenile individuals compared to adult animals were detected.

[Evolutionary history of Metazoa, ancestral status of the bilateria clonal reproduction, and semicolonial origin of the mollusca].

Evolutionary history of any metazoan group is a history of the entire ontogenetic cycles instead of separate stages and genes only. Ontogeny in the most objective way links two key components of the biological systematics: historically-independent characters attribution and phylogeny itself. A general theory encompassing "static" traditional taxonomy and dynamic evolutionary process, based on the ontogenetic transformation of the organisms' shape is suggested here to term as ontogenetic systematics. As an important practical implication of the ontogenetic systematics, a new model of the bilaterian metazoans evolution is suggested. The new model considers asexual clonal reproduction as a central feature of the ancestral ontogenetic cycles of basal Bilateria. The new scenario resolves several notable contradictions, e.g. morphological, ontogenetic and molecular similarities of Pogonophora, Vestimentifera, Phoronida simultaneously to protostomian Spiralia (Lophotrochozoa) and Deuterostomia. The suggested model implies individuation (possibly multiple) of ancestral semicolonial sedentary group as a major factor of the basal Bilateria diversification. In the late Ediacaran and early Cambrian thus existed ancestral bilaterian group that shared characters of both Spiralia and Deuterostomia and possessed polyp-shape body and cephalic secretory shield (like in modern Pterobranchia and Vestimentifera), that later on reduced in various lines. This ancestral taxon in rank of supraphylum is suggested to term as Carmaphora (shield-bearers). Presence of the enigmatic sedentary fossil of the genus Cloudina with vestimentiferan-like tubes and evident clonal reproduction already in the late Ediacaran, and most recent found of an unquestionable pterobranch already in the early Cambrian support the new model of Bilateria evolution.

[Ontogeny, systematics, and phylogenetics: space of future synthesis and a new model of the evolution of bilateria].

Ontogeny is considered as a process that allows combining two key components of biological systematics in an objective way: historically independent indicative attribution and phylogeny. It is proposed to designate the general theory that unifies the "statistical" traditional taxonomy and the dynamic evolutionary process on the basis ofontogenetic transformation of shapes of organisms as the ontogenetic systematics. One of the important practical applications is a new model of the evolution of bilaterally symmetric animals, which supposes an ancestral status of clonal asexual reproduction and its multiple reduction in a variety of Bilateria lines.

[Chemically modified specific immunoglobulins for legionellosis laboratory diagnostics].

On an example of the rabbit antilegionella immunoglobulins (antLegIgRb) and monospecific human serum (antLegSerHum) the influence of a protein with different acylation level on specific and nonspecific activity of the above immunobiopreparations was investigated. It was determined, that 5 % level of chemical modification provides the highest level of augmentation (2.0-2.7 times) of specific and nonspecific activity of antLegIgRb and antLegSerHum. Acylated forms of immunoglobulins and sera retain the beneficial biological properties (high level of specific activity, ability to be labelled with fluorochromes, resistance at long-term storage) and can be used for producing diagnostic immunobiological drugs.

Antiproliferative properties of chemically modified recombinant IFN-alpha2b.

The antiproliferative activity of aconitylated (AIFN) and succinylated (SIFN) derivatives of recombinant interferon- alpha2b (IFN-alpha2b) was examined. Acylation of IFN-alpha2b was performed by succinic and cis-aconitic anhydrides. Antiproliferative properties of AIFN and SIFN were studied in vitro on the CaOv cell line, highly sensitive to IFN, and on the SW-480 cell line, with low sensitivity to IFN-alpha2b. Acylation of one lysine in the IFN-alpha2b molecule with cis-aconitic or succinic anhydride resulted in a 3-3.5-fold increase of its antiproliferative activity on CaOv cells. The highest antiproliferative activity of acylated IFN-alpha2b on SW-480 cells was observed for both AIFNs and SIFNs with three modified lysine residues. In conclusion, aconitylated and succinylated IFNs may be useful antiproliferative agents for cancer treatment.

[Effect of succinylation of Klebsiella 45 bacteriophage proteins on specific lytic activity].

Effect of different levels of virions protein acylation on lytic activity, parameters of interaction with sensitive bacterial cells and other biological properties of phages has been studied in experiments on Klebsiella 45 bacteriophage. It was shown that at 5-, 10- and 20% levels of the phage sample acylation the specific lytic activity increased 90, 12 and 8 times, respectively, adsorbtion velocity constant increased 9, 8 and 8 times, respectively, and latent period decreases by 20% in all cases of the development of productive infection caused by succinylated phages. Such chemical modification did not change morphology, antigenic structure of phase virions, did not take essential effect on the spectrum of lytic activity and average yield of infectious phage corpuscles per a sensitive bacterial cell.

[The use of liposomal form of phenylimide of cis-aconitic acid in herpes therapy]. / Primenenie fenilimida tsisakonitovoi kisloty v liposomal'noi forme dlia terapii gerpesa.

The results of study of the antiviral activity and pharmacokinetics of phenylimide of cis-aconitic acid (PCAA) is presented. The 20% increase of the antiviral activity of PCAA incorporated into liposomes in comparison with the antiviral activity of the pure substance was shown. Liposomes with PCAA were tropic to lymphocytes and macrophages with maximum fluorescence being observed in the spleen, while empty liposomes were accumulated mainly in the liver. After the treatment with liposomal PCAA the symptoms of herpetic meningoencephalitis became less severe with 100% survival of the experimental animals. In the control group of rabbits 50% of the animals died, and in the surviving animals blindness or paralysis developed.

Biochemical nature of high-molecular-weight prolactin of human serum: identification of a prolactin-binding protein.

The biochemical nature of a high-molecular-weight immunoreactive prolactin (HMW-irPRL) prepared by gel filtration of women's sera with predominance of this hormone form was studied. Immunochemical characteristics of HMW-irPRL are different from those of 23 kD prolactin (23 kD-PRL). A protein which specifically and reversibly binds to human [125I]PRL is isolated from the pooled fractions of HMW-irPRL by affinity chromatography on prolactin-Sepharose. According to gel filtration, the binding protein (BP) has molecular weight about 150 kD, and it reversibly binds to protein A immobilized on Sepharose. Analysis of BP by SDS-PAGE resulted in two major protein bands, of 65-70 and approximately 150 kD. Both the bands, when transferred to nitrocellulose, interacted with [125I]protein A. Binding of highly purified human pituitary prolactin to the BP significantly decreased the immunoreactivity of the hormone. The molecular weight of BP and its interaction with protein A and recognition by poly- and monoclonal antibodies against human (but not guinea pig) IgG indicate that BP may be an immunoglobulin. Thus, our data demonstrate that HMW-irPRL is formed by the binding of 23 kD-PRL to a specific serum protein which is probably an anti-prolactin IgG.

[Molecular forms of human growth hormone and prolactin, secreted by cultured pituitary tumor cells]. / Molekuliarnye formy gormona rosta i prolaktina cheloveka, sekretiruemye kul'turami kletok opukholei gipofiza.

Functionally active cultures of human pituitary adenoma cells producing excessive amounts of the growth hormone (somatotropinomas), of prolactin (prolactinomas) or of the both hormones (mixed type adenomas) have been prepared and their secreted molecular forms studied. SDS-PAAG electrophoresis combined with immunoblotting making use of poly- and monoclonal antibodies revealed that the growth hormone and prolactin are secreted by adenoma cells in several molecular forms typical of normal human pituitary. The major form secreted by the growth hormone is 22K; the minor forms are 20K (the product of alternative splicing of pre-mRNA) and the split-off two-chain form 25K. The major form secreted by prolactin is 23K; the minor form is glycosylated 25K. No significant differences in the ratios of molecular forms of the hormones were found either under basal conditions of culturing or under the influence of the pituitary function regulators, somatostatin and thyroliberin. At the same time, the data obtained suggest that pituitary adenoma cells can secrete some amount of "abnormal" molecular forms of the hormones, e.g., immature products of postribosomal processing or large-sized immunoreactive fragments. Hence, pituitary adenoma cell cultures are an effective tool in biochemical and physiological studies of molecular forms of the human growth hormone and prolactin and of their secretion.

[An electrophoretic analysis of human glycoprotein hormones and their subunits]. / Elektroforeticheskii analiz glikoproteinovykh gormonov cheloveka i ikh sub''edinits.

Stability of heterodimers of human glycoprotein hormones with gonadotropic and thyrotropic activities in sodium dodecylsulfate (SDS) under non-reducing conditions at low temperature permits to resolve the native molecules of these hormones in SDS-PAG and to distinguish from their dissociated subunits by electrophoretical mobility. The analysis of dimers and alpha-, beta-subunits in one polyacrylamide gel allows to detect certain human glycoprotein hormones and to study some of their physico-chemical properties. Using two polyclonal antisera against human LH and FSH by the Western blot immunoassay it was shown that heterodimers as well as alpha and beta subunits after SDS-PAGE retain antigenic activity of native hormones. The method gave possibility to characterize the specificity of the given sera to different glycoprotein hormones.

Lung is the target organ for a monoclonal antibody to angiotensin-converting enzyme.

125I-labeled mouse monoclonal antibody (MoAb) to human angiotensin-converting enzyme (ACE), termed 9B9 and cross-reacting with rat and monkey ACE, when injected into the circulation, accumulates in the lung in up to 10 to 20 greater concentrations than in other organs and blood. That 111In-labeled MoAb 9B9 also accumulates in the lungs of both rats and monkeys very selectively, was clearly revealed by gamma-scintigraphy. Unlike polyclonal anti-ACE antibodies that induce an immunodependent lethal reaction when administered intravenously, MoAb 9B9 was well tolerated by rats even at very high doses (up to 300 mg/kg/body weight). At the same time, the administration of this antibody (which does not inhibit the catalytic activity of ACE) resulted in both a 3-fold decrease of the lung ACE activity and an increase in the activity of serum ACE. The highly organ-specific, nondamaging accumulation of the MoAb 9B9 makes it a promising vector for targeted drug delivery to the lung, for modeling of lung pathology, and for gamma-scintigraphic visualization of the lung vascular bed. We also suggest that MoAb 9B9 accumulation in the lung may serve as a highly sensitive marker of lung vessel damage upon various lung pathology.

Long-term serial cultivation of human vascular endothelial cells.

Long-term mass culture of human large vessel endothelial cells (EC) from aorta, umbilical cord vein, and pulmonary artery was undertaken and the cells characterized. The cells possessed all the major features of EC, proliferated rapidly [population doubling (PD) time 17-42 hours] and survived 40-60 PD. The cells were cultured under different conditions, and the following combination chosen as optimal for routine culturing: Medium 199, supplemented with 20% human serum, 4 mM glutamine, 200 micrograms ml-1 EC growth factor from human brain, 100 micrograms ml-1 heparin. The cells were grown on gelatin-coated or fibronectin-coated substrate. The results of the experiments with large vessel EC cultivation were used to develop a system for the culture of capillary EC from human lung and kidney. Mass-scale culture of large vessel and capillary EC is valuable in the investigation of EC function and for clinical purposes.

Radioimmunoimaging of lung vessels: an approach using indium-111-labeled monoclonal antibody to angiotensin-converting enzyme.

A murine monoclonal antibody against human angiotensin-converting enzyme was radiolabeled with 111In via diethylenetriaminepentaacetic acid without substantial loss of antigen-binding capacity. This monoclonal antibody designated 9B9 cross-reacted with rat and monkey angiotensin-converting enzyme. Indium-111-labeled 9B9 selectively accumulated 10-20 times greater in the lung than in blood or other organs following intravenous administration in rats. Kinetics of lung accumulation and blood clearance were studied for 111In-9B9-antibody and compared to that of 125I-labeled 9B9 in rat. Highly specific accumulation of 111In-9B9-antibody in the lung of Macaca Rhesus monkeys after intravenous injection was monitored by gamma-imaging. Images of 111In-labeled antibody 9B9 biodistribution in monkey lung noticeably differ from the images of biodistribution of 99mTc-labeled albumin microspheres. This difference may provide information concerning the state of the endothelium of lung capillaries, which is different from the blood flow characteristics determined with routine microsphere technique.

In vivo administration of glucose oxidase conjugated with monoclonal antibodies to angiotensin-converting enzyme. The tissue distribution, blood clearance, and targeting into rat lungs.

A conjugate between glucose oxidase (GO) and monoclonal antibody to human angiotensin-converting enzyme (ACE) cross-reacting with rat ACE (MoAb9b9) has been prepared by oxidation of the cardohydrate moiety of the enzyme with sodium periodate. The conjugate (GO-MoAb9b9) thus obtained retained both antigen-binding capacity and enzymatic activity. The fate of the conjugate in vivo after intravenous injection was studied using conjugates containing radiolabeled enzyme. GO-MoAb9b9 was specifically accumulated in rat lungs upon in vivo administration, as compared with free enzyme and nonimmune IgG-conjugated glucose oxidase. The specificity of the conjugate accumulation expressed as the localization ratio (the ratio between radioactivity of gram tissue to that of blood) (Loc. Ratio) reached a value up to 50 on the second day after injection, in contrast to native enzyme and to IgG-conjugated enzyme (Loc. Ratio was less than 0.5 for both preparations). The Loc. Ratio of GO-MoAb9b9 was even higher than that of the original antibody MoAb9b9 and was equal to 20, which is probably explained by an extremely rapid blood clearance of the conjugate from the circulation. The administration of excess free MoAb9b9 dramatically inhibited the conjugate targeting in the lung without any effect on liver uptake. At doses ranging from 10 to 1,000 micrograms/rat, the conjugate was accumulated in the lung without saturation of the antigen determinants of the target. At minimal doses, the efficiency of targeting achieved 5 to 7% of the conjugate injected. With elevation of the dose, the efficiency of targeting decreased to 2.5% of the injected dose (1 mg of GO-MoAb9b9 per rat). A sixfold greater accumulation of unmodified radiolabeled MoAb9b9 compared with the GO-MoAb9b9 conjugate in rat lung has been observed, though the kinetics of desorption from the target organ was similar for both the antibody and the conjugate. In the bloodstream, the conjugate persisted for at least 5 days without binding to blood cells; all circulating radioactivity was associated with proteins. A considerable part of the conjugate (to 50%) circulated as a tight antibody-enzyme complex for several days. The conjugate retained its antigen-binding capacity for at least 24 h; during this period, its enzymatic activity decreased by less than 40%. The results obtained provide the experimental ground for further attempts to apply glucose oxidase conjugates for local modulation of inflammation and elimination of the target cells.

Blood clearance of radiolabeled antibody: enhancement by lactosamination and treatment with biotin-avidin or anti-mouse IgG antibodies.

Methods of rapid blood clearance of 111In-labeled mouse monoclonal antibody 9B9 against angiotensin-converting enzyme were studied. Indium-111-9B9 is specifically accumulated in rat lung, but its blood clearance is relatively slow and target-to-blood radioactivity ratio/g tissue (localization ratio) increases from 11 to 30 only 48 hr postinjection. Injection of second (anti-mouse immunoglobulin) antibodies results in slight (1.8-fold) increase of 9B9 localization ratio. Chemical modification of 9B9 aminogroups with lactose results in enhanced liver uptake and rapid blood clearance of antibody. Blood radioactivity level decreases tenfold, and as a result localization ratio increases threefold (up to 38 in 30 min). Injection of avidin following the injection of biotinylated 9B9 results in rapid clearance of blood radioactivity with increased uptake in liver and spleen. Lung uptake is not changed. Localization ratio increases fivefold over the avidin-untreated animal value. Implications of these approaches for various applications in immunoimaging are discussed.