Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.2, #2)

Problem of proteolytic enzyme evolution by N. N. Nemova; L. A. Bondareva (115-125).
Own and literature data on putative evolution of proteolytic enzymes have been reviewed. Modern principles of peptidase classification based on evaluation of homology of more than 66 thousand gene sequences and similarity of general structural organization of almost 2.5 thousands of known enzymes are considered. The review highlights not only evolutionary interrelationships inside related peptidase families, their possible evolutionary background, but also evolutionary determined differences in certain proteolytic pathway in organisms belonging to different taxons.
Keywords: proteolytic enzymes; classification; molecular evolution

Single nucleotide polymorphisms (SNPs) are now considered as the most perspective and convenient markers for studies of genetic basis of multifactorial diseases. Fast development of technologies for exact screening of a large volume of genetic information and construction of genomic maps of SNP-markers promote the development of innovative diagnostic systems on the basis of significant SNP for an estimation of individual genetic risk of development of various diseases. This review considers the basic aspects of genetics of diabetes mellitus type 1 and 2 and an opportunity to use SNP as markers for the estimation of individual genetic predisposition to this disease.
Keywords: genetic markers; SNP; diabetes mellitus; genetic predisposition

Magnesium homeostasis: Mechanisms and inherited disorders by V. N. Zinov’eva; I. N. Iezhitsa; A. A. Spasov (133-147).
Mechanisms of magnesium homeostasis intensively studied over the last 10–15 years by means of pathophysiological and molecular genetic approaches have been considered. Impairments of magnesium homeostasis causes the development of magnesium-deficient states, which have been found in many common diseases (diabetes mellitus, cardiovascular diseases, chronic fatigue syndrome, alcoholism, psychiatric and neurologic diseases, etc.), stress condition, effects of some environmental factors as well as therapy with some drugs. Special attention is paid to familial hypomagnesemias caused by genetic defects of magnesium transport systems. The review considers clinical and biochemical characteristics of twelve familial disorders including mechanisms of their development. Deeper understanding of mechanisms of regulation of magnesium homeostasis will results in the development of new approaches in diagnostics, prophylaxis and treatment of magnesium-deficient conditions.
Keywords: magnesium; homeostasis; genes controlling magnesium transport; inherited disorders; hypomagnesemia

Inclusion membrane proteins of Chlamydiaceae by E. S. Kostryukova; V. N. Lazarev; V. M. Govorum (148-159).
Inclusion membrane proteins (Inc-proteins) belong to the family of unique chlamydial proteins. Members of this family attract attention of scientists because Inc-proteins are localized in the inclusion membrane, they have been found in all chlamydial species, expression of the most part of their genes begins during the first hours after the infection of cell culture. Biological functions of Inc-proteins remain unknown, but these proteins are suggested to play a key role in process of the development of the chlamydial infection.
Keywords: Chlamydiaceae ; Chlamydia ; inclusion membrane proteins; Inc-proteins

Age-related changes in albumin and actin of human myocardium by M. A. Kovalyova; L. I. Kovalyov; M. V. Serebryakova; S. A. Moshkovskii; S. S. Shishkin (160-165).
Proteomic technologies revealed products of 2 genes (α-actin and albumin) in a human myocardium tissue. They exist as fragments and their appearance and increased content correlated with age. The age-related variants differ from the mature forms of these proteins by the absence of N-terminal fragments of the amino acid sequences. In the chronic ischemic heart disease (CIHD), these age-related proteins were found in 50% of cases (in the age group 31–40 years), whereas in the control group such combination was detected only in 10% of the examined individuals. Subsequent studies in this field would probably reveal molecular mechanisms responsible for impairment and/or ageing of the myocardium and also of adaptation/disadaptation mechanisms in the CIHD.
Keywords: myocardium; man; proteomics; age-related changes

Changes in phospholipid composition of the erythrocyte membranes have been studied in experimental postmyocardial infarction cardiosclerosis. Erythrocyte membranes from animals with cardiosclerosis formed after experimental occlusions of coronary arteries were characterized by significant decrease of a minor phospholipid, phosphatydylinositol (by more than 40%) and the increase of the major phospholipid, phosphatydylethanolamine (by 20%). There was high content of lipid peroxidation products, malondialdehyde and conjugated dienes and the decrease in the activity of antioxidant enzymes, catalase and superoxide dismutase in blood serum of these animals. We have concluded the formation of postmyocardial infarction cardiosclerosis is accompanied by the increase of free radical reactions. This causes changes in phospholipid composition of cell membranes and the decrease of compensatory capacities of the enzymatic antioxidant system. These changes form a metabolic background, which can influence cardiac remodeling properties.
Keywords: phospholipids; lipid peroxidation; experimental postmyocardial infarction cardiosclerosis

The effect of mildronate and related substances on levels of thyroid hormones and some intermediates of lipid and carbohydrate metabolism in hyperthyroid and hypothyroid rats by E. M. Sharipova; E. V. Salna; M. Ya. Dzintare; L. Ya. Lauberte; D. V. Meirena; N. I. Sjakste; V. S. Gordjushina; I. Kalvinsh (169-175).
The effects of mildronate [3(2,2,2-trimethylhydrazinium) propionate dihydrate], γ-butyrobetaine (GBB) and their combination (neomildronate) on the level of thyroid hormones and some intermediates of basal metabolism (free fatty acids, triglycerides, glucose) in serum of laboratory rats with various dysfunctions of thyroid glands including idiopathic hyperfunction and also hypofunction induced by administration of 6-propyl-2-thiouracil (PTU) or L-carnitine administration. Intraperitoneal injections of mildronate (150 mg/kg) during 20 days to male Wistar rats with elevated level of thyroid hormones and basal metabolism normalized thyroxin level and parameters of lipid metabolism in serum. Administration of the compounds studied to rats with hypothyroidism induced by administration of PTU or L-carnitine did not influence natural recovery of the hormonal level. Possible biochemical role of these pharmacological treatments is discussed in terms of in regulation of thyroid gland function.
Keywords: mildronate and its derivatives; rat thyroid gland dysfunction; experimental models; thyroid hormones; intermediates of basal metabolism in blood serum

Entrapment of herbal extracts into biodegradable microcapsules by T. N. Borodina; L. D. Rumsh; S. M. Kunizhev; G. B. Sukhorukov; G. N. Vorozhtsov; B. M. Feldman; A. V. Rusanova; T. V. Vasil’eva; S. M. Strukova; E. A. Markvicheva (176-182).
The microcapsules with entrapped herbal water-soluble extracts of plantain Plantago major and calendula Calendula officinalis L. (PCE) were prepared by layer-by-layer (LbL)-adsorption of carrageenan and oligochitosan onto CaCO3 microparticles with their subsequent dissolving after the treatment of EDTA. Entrapment of PCE was performed by using adsorption and co-precipitation techniques. The co-precipitation provided better entrapment of PCE into the carbonate matrix compared to adsorption. In vitro release kinetics (AGJ) was studied using artificial gastric juice. Using the model of acetate ulcer in rats it has been demonstrated that PCE released from the microcapsules accelerates gastric tissue repair.
Keywords: microcapsules; herbal extracts; oligochitosan; carrageenan; pepsin; biodegradation; sustained drug release

The effect of a single dose of arecholine and atropine administration on the activities of carboxypeptidase H and phenylmethylsulfonyl fluoride-inhibited carboxypeptidase involved into the final stage of formation of biologically active neuropeptides from precursors has been studied in brain and adrenals. Changes in the enzyme activities were evident during at least 72 h after administration of these drugs to rats. These results suggest that the decrease in the activity of these enzymes may represent one of possible mechanisms involved into reduction of neuropeptide levels during inhibition and activation of cholinergic system.
Keywords: carboxypeptidase H; phenylmethylsulfonyl fluoride-inhibited carboxypeptidase; atropine; arecholine; neuropeptides; brain

Assessment of some oxidative stress parameters in patients with bronchial asthma after selenium supplementation by J. G. Voicehovska; A. Shkesters; G. A. Orlikov; A. A. Silova; N. J. Rusakova; L. T. Larmane; J. G. Karpov; A. D. Ivanov; E. Maulinsh (189-193).
The goal of this study was to investigate the effect of selenium deficit replenishment in patients with bronchial asthma (BA) on manifestations of oxidative stress and conditions of the antioxidant system (AOS). The need of correction of selenium deficit in BA-patients is determined by increased requirements in antioxidants due to chronic inflammatory process responsible for pathogenesis of BA. Latvia as well as Eastern Finland, Byelorussia, some regions of Ukraine, North-Western Russia, and New Zealand belong to the endemic areas with marked selenium deficit in soils and foodstuff. Twenty patients (7 men and 13 women) with selenium deficit and verified diagnosis of BA have been examined. In addition to basic therapy all patients received organic selenium as SelenoPRECISE (PharmaNord) 200 μg daily for 16 weeks. This caused statistically significant increase of plasma selenium from 50.94 ± 7.58 to 63.59 ± 10.87 μg/l (p < 0.001), the increase of selenium-dependent glutathione peroxidase (from 38.64 ± 10.72 U/g Hb to 58.57 ± 14.64 U/g Hb, p = 0.01). Treatment of patients with selenium also normalized the parameters characterizing oxidative stress (chemiluminescence), significantly exceeded normal values before this treatment. The use of selenium in addition to basic therapy allows to abolish or reduce oxidative stress by correcting the antioxidant system.
Keywords: bronchial asthma; inflammation; oxidative stress; selenium

Glutathione system in erythrocytes and blood plasma in strokes and dyscirculatory encephalopathy by L. S. Kolesnichenko; V. I. Kulinsky; V. V. Shprakh; V. V. Bardymov; N. V. Verlan; L. P. Gubina; G. A. Pensionerova; M. P. Sergeeva; L. M. Stanevich; G. T. Philippova (194-197).
Dyscirculatory encephalopathy and mild ischemic stroke are characterized by solitary changes in components of glutathione metabolism. In moderate and severe ischemic strokes essential changes have been found. Changes in glutathione metabolism are also expressed in hemorrhagic stroke. The clearest increase was found in activities of glutathione peroxidase and glutathione transferase and in rare cases in activities of glutathione reductase and GSH concentration. The increase of enzymes activity was not found in patients with delayed onset of treatment (more than 3 days) and also in severe cases terminated by subsequent death of patients. Glutathione system is obviously important for tolerance to cerebral ischemia.
Keywords: glutathione system; stroke; dyscirculatory encephalopathy

Ribosomal repeat in cell free DNA as a marker for cell death by N. N. Veiko; N. A. Bulycheva; O. A. Roginko; R. V. Veiko; E. S. Ershova; O. A. Kozdoba; V. A. Kuzmin; A. M. Vinogradov; A. A. Yudin; A. I. Speranskyi (198-207).
A novel method for in vivo evaluation of cell death in patients with acute and/or chronic heart diseases, accompanied by apoptosis or cell necrosis has been developed. The method is based on the analysis of cell free DNA (cfDNA) in the blood serum (plasma). It includes estimation of concentration of serum ribosomal repeat (rDNA), content of rDNA in total cfDNA, as well as factors of cfDNA elimination, such as nuclease activity and anti-DNA antibody. We have found a fivefold increase in serum cfDNA concentration and a 12-fold increase of serum rDNA concentration in patients with acute myocardial infarction compared with healthy individuals. In chronic coronary ischemia serum cfDNA concentration was nearly normal, but the content of rDNA in cfDNA was 4.8-fold higher, and the serum rDNA concentration was increased sevenfold. We hypothesize that putative reason for accumulation of rDNA within cfDNA might be attributed to the previously reported resistance of rDNA to the ds-fragmentation by serum endonucleases. In acute and chronic coronary disease serum nuclease activity and the titer of anti-DNA antibodies (which are mainly bound to the cfDNA) was substantially higher than in the healthy controls. It is suggested that release of rDNA fragments into blood not only reflects cellular death in the body but also determines the response of the organism to the disease-associated stress.
Keywords: ribosomal genes; cell free DNA; endonuclease activity; acute myocardial infarction; ischemic heart disease

Breast cancer diagnostics based on extracellular DNA and RNA circulating in blood by E. Y. Rykova; T. E. Skvortsova; A. L. Hoffmann; S. N. Tamkovich; A. V. Starikov; O. E. Bryzgunova; V. I. Permjakova; J. M. Warnecke; G. Sczakiel; V. V. Vlassov; P. P. Laktionov (208-213).
Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of healthy women and patients with fibroadenoma and breast cancer. Frequency of methylation of RASSF1A, Cyclin D2, and RARβ2 genes was detected in the extracellular DNA using methylation-specific PCR. Methylation of at least one of these genes was found in plasma of 13% patients with nonmalignant breast fibroadenoma and in 60% of breast cancer patients. Employment cell-surface bound DNA as the substrate for PCR increased the detection frequency of gene methylation up to 87% in patients with fibroadenoma and 95% in breast cancer patients. In clinically healthy women the methylation markers have not been found in extracellular DNA. GAPDH, RASSF8, Ki-67 mRNAs, and 18S rRNA copies were quantified using RT-qPCR of extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The major part of blood extracellular RNA is associated with cell surface. ROC analysis has shown that differences in concentrations 18S RNA, RASSF8, and Ki-67 mRNAs in blood plasma are highly sensitive and specific in discrimination of benign and malignant breast tumors. Thus, analysis of methylated forms of tumor suppressor genes in blood extracellular and quantification of specific extracellular RNA circulating in blood plasma may detect mammary gland tumors and discriminate malignant and benign neoplasms.
Keywords: circulating extracellular DNA; circulating extracellular RNA; methylation-specific PCR; RT-PCR; breast tumor