Structural and Functional Features of Ca2+ Transport Systems in Liver Mitochondria of Rats with Experimental Hyperthyroidism.

We analyzed structural and functional features of the main mitochondrial Ca2+-transporting systems, mitochondrial Ca2+ uniporter complex (MCUC) and Ca2+-dependent cyclosporin A-sensitive mitochondrial permeability transition pore (MPT pore), in rats with hyperthyroid state. It was found that, the rate of Ca2+ accumulation by rat liver mitochondria in this pathology increases by 1.3 times, which can be associated with higher level of the channel-forming subunit of the uniporter MCU and lower content of dominant-negative subunit of this complex MCUb. At the same time, the level of the regulatory subunit MICU1 remained unchanged. It was shown that calcium retention capacity of liver mitochondria in rats with experimental hyperthyroidism decreased by 2 times in comparison with the control, which attested to reduced resistance of liver mitochondria of hyperthyroid rats to induction of the MPT pore. The observed changes are consistent with the data on increased amount of cyclophilin D, a mitochondrial matrix peptidyl-prolyl isomerase that is known to modulate the MPT pore opening and expression of the Ppif gene that encodes mitochondrial cyclophilin D in rats with experimental hyperthyroidism.

[A role for calreticulin in functioning of mitochondrial ATP-dependent potassium channel].

Homology of the amino acid sequence of the mitochondrial potassium-transporting protein (MW 57kDa), having the properties of a channel subunit of the mitochondrial ATP-dependent potassium channel, and calreticulin (MW 55kDa) was detected by MALDI-TOF-TOF analysis method. Inhibitory analysis of ATP-dependent potassium transport in mitochondria with polyclonal antibodies to calreticulin was carried out. A dose-dependent inhibition of potassium transport in mitochondria by these antibodies was shown. The maximum value of inhibition was 55-60%. Based on these data it is hypothesized that at least two types of ATP-sensitive potassium channels are localized in mitochdndrial membrane. It is expected that the type of mitochondrial ATP-dependent potassium channel, which includes homologous calreticulin protein is localized mainly at mitochondrial and reticulum membrane contact sites.

Oxidative phosphorylation and ion transport in the mitochondria of two strains of rats varying in their resistance to stress and hypoxia.

The role of mitochondria in the inherited or ontogenetically acquired reactions of organism to stress is not studied enough. In the present work, we examined the functional state of the coupled respiratory chain, potassium and calcium transport and rate of hydrogen peroxide production on two rat lines: August and Wistar-which possess different resistance to emotional stress and hypoxia. It was established that the respiration rate and efficiency of oxidative phosphorylation were higher in August rats than in Wistar ones. In August rats, the rate of potassium transport and ATP-dependent mitochondrial swelling as well as the concentration of the ion in the mitochondrial matrix were almost twice as higher comparatively to those parameters in Wistar rats. The rate of H2O2 production was found to be decreased in the mitochondria of August rats. It was also demonstrated that the two rat lines differed by their resistance to the opening of the palmitate/Ca(2+)-induced pore and by their ability to retain calcium within mitochondria. The paper discusses the involvement of the mitochondrial ATP-dependent potassium channel in the adaptation of animals to adverse effects.

[Isolation and purification of human blood plasma proteins able to form potassium channels in artificial bilayer lipid membrane].

Protein fraction able to induce K(+)-selective transport across bilayer lipid membrane was isolated from human blood plasma with the use of the detergent and proteolytic enzyme-free method developed at our laboratory. After addition of the studied sample to the artificial membrane in the presence of 100 mM KCl, a discrete current change was observed. No channel activity was recorded in the presence of calcium and sodium ions. Channel forming activity of fraction was observed only in the presence of K+. Using a threefold gradient of KCl in the presence of studied proteins the potassium-selective potential balanced by voltage of -29 mV was registered. This value is very close to the theoretical Nernst potential in this case. This means that the examined ion channel is cation-selective. According to data obtained with MS-MALDI-TOF/TOF and database NCBI three protein components were identified in isolated researched sample.

[Antioxidant enzymes in erythrocytes from hypertension patients receiving lisinopril monotherapy or combined lisinopril plus simvastatin therapy].

Statins and angiotensin-converting enzyme (ACE) inhibitors have beneficial impact on the serum cholesterol and blood pressure. It is supposed that statins and ACE inhibitors may modify the antioxidative status of erythrocytes. The study objective was to compare the effects of two treatments, lisinopril alone vs lisinopril plus simvastatin, on erythrocyte antioxidant enzyme activities. The study involved 32 patients with arterial hypertension, the initial serum total cholesterol, LDL-cholesterol and triglycerides within the normal range. Patients of two groups, each of 16 subjects, were treated with lisinopril (10 mg/day) or with lisinopril (10 mg/day) plus simvastatin (20 mg/day). Before and after 3 and 6 months of follow-up therapy, activities of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), glutathione reductase (GLR) in purified erythrocytes were determined. In all patients, significantly higher catalase activity (by 79.3-106.5%, p < 0.0001) and significantly lower GPx activity (by 20.7-30.6%, p < 0.001) were observed after therapy as compared to the baselines. Just the same results were obtained in both groups (lisinopril and lisinopril + simvastatin), after both periods (3 and 6 month) of treatments. SOD activity was increased only in the lisinopril group and only after 6 months (p = 0.0345). No changes of GLR reductase activity were seen under all conditions indicated. Thus, the lisinopril monotherapy and combined lisinopril plus simvastatin therapy exhibit specific, pronounced and equipotent effects on antioxidant enzymes in human erythrocytes. Administration of lisinopril or lisinopril plus simvastatin may protect erythrocytes and other tissues from oxidative damage.

[The drug hypoxen--a new inhibitor of mitochondrial respiration and mitochondrial dehydrogenases].

The effect of hypoxen on the oxygen consumption and activity of dehydrogenases in rat liver mitochondria has been studied. The addition of hypoxen to mitochondria caused a speed reduction of phosphorylating and uncoupling respiration. The minimal effective concentration of hypoxen was 15 microg/ml with succinate, 60 microg/ml with pyruvate or palmitoylcarnitine, and 120 microg/ml with glutamate as the substrates. The activities of malate, glutamate, and succinate dehydrogenases in mitochondria were significantly decreased by the effect of hypoxen.

[Role of glycolysis and antioxidant enzymes in the toxicity of amyloid beta peptide Abeta25-35 to erythrocytes].

The role of glycolysis and antioxidant enzymes in amyloid beta peptide Abeta(25-35) toxicity to human and rat erythrocytes was studied. The erythrotoxicity of Abeta(25-35) was shown to increase two- to fourfold both in the absence of glucose in the incubation medium and upon the addition of sodium fluoride, an enolase inhibitor. Potassium cyanide, a Cu,Zn-superoxide dismutase inhibitor, abolishes the toxic effect of Abeta(25-35) to erythrocytes, whereas mercaptosuccinate, a glutathione peroxidase inhibitor, and ouabain, a Na+,K+-ATPase inhibitor, promote it. Sodium azide, a catalase inhibitor, did not affect the cell lysis under the action of Abeta(25-35) . The results support the hypothesis that H2O2, Cu,Zn superoxide dismutase, and glutathione peroxidase are involved in the toxicity mechanism rather than superoxide radical. Glycolysis and Na+,K+-ATPase play a substantial protective role. Fullerene C(60) nanoparticles are toxic to erythrocytes of both types; their toxicity is not related to enhanced oxidative stress and the mechanism of toxicity differs from that of Abeta(25-35) .

[Toxic effect of Abeta25-35 and fullerene C60 on erythrocytes].

Using light microscopy and spectrophotometry, it has been shown that amyloid beta-peptide Abeta25-35 and water-soluble fullerene C60 cause lysis of human and rat erythrocytes. Both fullerene C60 and Abeta25-35 partly inhibited the activities of membrane-associated phosphofructokinase and plasmalemmal lactate dehydrogenase in erythrocytes.

Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes.

Mitochondrial diseases are not uncommon, and may result from mutations in both nuclear and mitochondrial DNA (mtDNA). At present, only palliative therapies are available for these disorders, and interest in the development of efficient treatment protocols is high. Here, we demonstrate that in cells heteroplasmic for the T8993G mutation, which is a cause for the NARP and MILS syndromes, infection with an adenovirus, which encodes the mitochondrially targeted R.XmaI restriction endonuclease, leads to selective destruction of mutant mtDNA. This destruction proceeds in a time- and dose-dependent manner and results in cells with significantly increased rates of oxygen consumption and ATP production. The delivery of R.XmaI to mitochondria is accompanied by improvement in the ability to utilize galactose as the sole carbon source, which is a surrogate indicator of the proficiency of oxidative phosphorylation. Concurrently, the rate of lactic acid production by these cells, which is a marker of mitochondrial dysfunction, decreases. We further demonstrate that levels of phosphorylated P53 and gammaH2ax proteins, markers of nuclear DNA damage, do not change in response to infection with recombinant adenovirus indicating the absence of nuclear DNA damage and the relative safety of the technique. Finally, some advantages and limitations of the proposed approach are discussed.

Studies on ammocytes: development, metabolic characteristics, and detoxication of ammonium.

The possibility of reducing ammonium concentration in the blood of mice with hyperammonemia with ammocytes (erythrocytes loaded with glutamate synthase) and the metabolic characteristics of these cells were studied. Injection of ammocytes into the blood stream of animals with hyperammonemia led to reduction of the blood ammonium concentration within the first 30-120 min and this activity of ammocytes was retained for at least 2 days. Endogenous phosphofructokinase, glucose-6-phosphate dehydrogenase, hexokinase, lactate dehydrogenase, pyruvate kinase, and Na(+),K(+)-ATPase in ammocytes remained at the levels of catalytic activities characteristic of intact erythrocytes. Hence, ammocytes are functionally active cells and can be used as a protective system in pathological hyperammonemia, while the method can be regarded as a new technology for medicine and veterinary.

[Antioxidant enzymes, hydrogen peroxide metabolism, and respiration in rat heart during experimental hyperammonemia].

The effects of toxic ammonia doses on H2O2 metabolism, energy metabolism, and antioxidant enzyme activities in rat heart were studied. Ammonium acetate administration to animals proved to increase total superoxide dismutase (SOD), catalase, and glutathione peroxidase activities in the heart cytoplasmic fraction as well as Mn-SOD, catalase, and glutathione reductase in heart mitochondria. Conversely, ammonia inhibited the same activities in the brain, liver, and erythrocytes. Hyperammonemia had no effect on the levels of ATP, ADP and total adenine nucleotides in the heart but decreased them in the brain. Ammonia impaired oxidative phosphorylation and increased the rate of H202 production in heart and brain mitochondria. The ammonia concentration inhibiting antioxidant enzymes in the liver and brain can be insufficient for such effect in the heart.

[Antioxidant enzymes of the rat liver, brain, heart and erythrocytes in ammonia intoxication].

Injection of large amounts of ammonium salts leads to rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of toxic dose of ammonium acetate on the activities of antioxidant enzymes in rat liver, brain, heart and erythrocytes. Acute ammonia intoxication resulted in rapid (within 11 min) decrease in superoxide dismutase, catalase and glutathione peroxidase activities in liver and brain mitochondria and cytosol and in erythrocytes, but in increase of these enzyme activities in heart. Diminished activities of the antioxidant enzymes in liver, brain and erythrocytes suggest that the systemic oxidative stress takes place, whereas their elevated activities in heart can be the adaptive reaction to oxidative stress in hyperammoniemia.

Antioxidant status of erythrocytes after acupuncture treatment.

A course of acupuncture therapy in patients with locomotor, peripheral nervous system, gynecological, and bronchopulmonary diseases led to complete or partial normalization of nonspecifically changed MDA content, catalase and glutathione peroxidase activities in erythrocytes. SOD activity increased after therapy and did not differ from the control. Acupuncture stabilized disordered LPO processes and improved the antioxidant status of erythrocytes.

[Calcium and ammonia stimulate monoamine oxidase A activity in brain mitochondria]. / Kal'tsii i ammiak stimuliruiut aktivnost' monoaminoksidazy A v mitokhondriiakh mozga.

The effect of ammonia and calcium on the activity of monoamine oxidase (MAO) was studied. The enzyme activity in non-synaptic brain mitochondria isolated from rats administered with ammonium acetate was assayed by release of H2O2 using spectrophotometry. The effect of calcium on MAO was assayed directly after addition of Ca2+ to the non-synaptic mitochondria isolated from the forebrain of the control rats. Both ammonium acetate injection in vivo and Ca2+ addition in vitro stimulated the activity of MAO A but not that of MAO B in mitochondria. This is the first evidence that ammonia and Ca2+ regulate MAO A in non-synaptic mitochondria in the forebrain and can contribute to oxidative stress in the neurons via MAO A activation.

Preparation and handling of brain mitochondria useful to study uptake and release of calcium.

There is increasing evidence for a critical role of mitochondria in calcium homeostasis and neuronal death in excitotoxicity. In spite of much work during the last two decades, the kinetic parameters of Ca(2+) transport in brain mitochondria remain controversial. Analysis of the literature data suggests that these contradictions can be due to differences in the methodology used to prepare or to incubate brain mitochondria. In the present communication, the whole protocol for preparation of non-synaptic rat forebrain mitochondria is described. This report shows that this preparation is well coupled and essentially free of non-mitochondrial contaminants. The mitochondria obtained are useful to study Ca(2+) uptake and release. Both Na(+)-independent, Na(+)-dependent and spontaneous Ca(2+) release may be studied with this preparation. This system is also useful in studies on the role of mitochondria and other intracellular Ca(2+) stores in disturbance of Ca(2+) homeostasis and delayed cell death under excitotoxic conditions.

[Role of parotitis virus in the development of insulin-dependent diabetes mellitus]. / O roli virusa parotita v razvitii insulinzavisimogo sakharnogo diabeta.

Viral infection is one of the factors provoking the development of insulin-dependent diabetes mellitus (IDDM). Epidemic parotitis more frequently than other infections is connected with IDDM manifestations, but the results of examinations of convalescents after epidemic parotitis and of patients with newly diagnosed IDDM are contradictory. Parotitis viruses are believed to injure pancreatic beta-cells and trigger on the autoimmune process in carriers of certain antigens. In none of the 200 cases of IDDM manifestation a relationship with epidemic parotitis was established and none of the 268 convalescents after epidemic parotitis developed IDDM. Screening for antibodies to epidemic parotitis virus carried out in 61 patients with newly detected IDDM revealed low antibody titers in the majority (83.3%); HLA-B8 antigen was detected in 23% of these examinees. In the group of subjects with persistent high antibody titers and their diagnostic increment or reduction HLA-B8 antigen was found in only 12.5%. These results do not permit a conclusion on the relationship between IDDM manifestation and parotitis infection.

[Immune structure of the pediatric population in relation to the mumps virus and the morbidity of serous meningitis of mumps etiology in Sverdlovsk]. / Immunostruktura detskogo naseleniia k virusu épidemicheskogo parotitai zabolevaemost' seroznym meningitom parotitnoi étiologiii v Sverdlovske.

The work presents the results of the longterm study of regularities in the formation of herd immunity to mumps virus in children in Sverdlovsk with due regard to vaccination carried out in this city and morbidity level in serous meningitis of mumps etiology. The inverse correlation between the number of seropositive persons in the total child population and the level of morbidity in the nervous form of mumps has been established. No differences in the size of the immune stratum and the intensity of immunity to mumps virus among boys and girls have been revealed. Vaccination against mumps, carried out at the period of a natural drop in mumps morbidity, has produced no essential effect on the level of herd immunity, though it has led to a considerable decrease in the incidence of serous meningitis of mumps etiology.

[Gastroenteritis of a nonbacterial nature in Sverdlovsk children]. / Gastroénterity nebakterial'noi prirody u detei Sverdlovska.

The analysis of the results of the clinico-epidemiological and laboratory examination of children with clinical manifestations of acute gastroenteritis, admitted to specialized hospitals in Sverdlovsk during 1982-1983, showed that morbidity rate in Sverdlovsk was determined mainly by cases registered among children during the first 2 years of their life. Morbidity rate among children aged up to 1 year was 3-6 times higher than that among children of other age groups. Cases of gastroenteritis among children not belonging to organized groups were observed 3.9 times more frequently than in organized groups of children. Gastroenteritis of viral etiology constituted 50% of all nonbacterial enteric infections in young children, the course of gastroenteritis being more favorable in children aged up to 3 months than in older children. 54% of gastroenteritis cases in children aged up to 1 year and 71% of such cases in children aged 1-2 years proved to be of rotavirus etiology.

[Characteristics of the dynamics of herd immunity to the poliovirus and of the quality of the vaccination carried out against poliomyelitis]. / Kharakteristika dinamiki kollektivnogo immuniteta k poliovurusu i kachestva provodimoi protiv poiliomielita vaktsinatsii.

The aim of this work was to study the state of immunity in children immunized with liquid poliomyelitis vaccine. During 1976-1981 the intensity of immunity to poliomyelitis in 653 children aged 1-3 years was studied. The study showed that at the period when liquid poliomyelitis vaccine was used the share immune to polioviruses, types 1 and 2, constituted 89.4% and 98.0%, respectively, as compared with 67.0% and 68.0% at the period when sugar-coated live dried poliomyelitis vaccine was administered. Changes in such epidemiological characteristics, as the percentage of persons with antibodies to poliovirus, were particularly essential in the age group of 1-2 years. In 1979 persons with antibodies to type 1 poliovirus constituted 94.0% as compared with 44.0% in 1976, while the percentage of persons found to be seronegative in respect of antibodies to polioviruses, types 1, 2 and 3, decreased 5-7 times. The quality of immunization carried out at the period, when liquid poliomyelitis vaccine was used, was shown to influence the size of the immune share, while having no effect on the geometric mean of the titer of antibodies to polioviruses.