Skin Fibroblasts as the Object for Clinical Diagnosis of Parkinson's Disease in Persons of Different Ages.

We compared the expression of Aß42 peptide, τ-protein, and α-synuclein in the substantia nigra and skin fibroblasts of elderly and senile patients with Parkinson's disease and subjects without neuropathology. Expression of markers in the studied tissues was assessed by immunohistochemical and immunocytochemical methods. The expression of Aß42 peptide, τ-protein, and α-synuclein in the substantia nigra of elderly and senile patients with Parkinson's disease was higher by 11-31 times than in subjects without neuropathology. In skin fibroblasts of patients with Parkinson's disease, the expression of Aß42 peptide and α-synuclein was 3-14 times higher than in subjects without neuropathology, and expression of τ-protein did not significantly differ in the studied groups. Thus, immunocytochemical analysis of the expression Aß42 peptide and α-synuclein in skin fibroblasts can be a simple method of early diagnosis of Parkinson's disease in elderly persons.

[The personalized medicine and organization of geriatric care of population].

The article presents advisability of applying approaches of personalized medicine to solve problems of modern gerontology and geriatrics. The actuality of issue is conditioned by population aging due to rapid increasing of both percentage and absolute number of elderly people in total population of most countries. The ideology of personalized medicine or P4 medicine is targeted to developing individual preventive and therapeutic activities for specific personality. The fundamental basis of developing personalized medicine is ensured by biomarkers, specific for every patient and certain disease. The elderly people health is characterized by increased susceptibility to diseases, polymorbidity and heterogeneity in health status. The solution of actual problems in gerontology and geriatrics improvement of basic principles of health care is needed with an emphasis on prognostication, prevention and personalized approach applied to elderly patients with the purpose of ameliorating their quality of life. The development and promotion of complex inter-disciplinary programs of personalized treatment of elderly patients is required.

[Neurochemical markers of neurodegeneration in the early diagnosis of Alzheimer's disease, vascular and mixed dementia].

A study of 27 patients with dismnestic variant of moderate cognitive impairment (MCI) and 20 patients with neurodynamic-dysregulatory variant was carried out. The more intensive atrophic process supported by neuroimaging data and changed levels of neurodegeneration biomarkers - phosphorylated tau-protein 181 and beta-amyloid 42 - were found in patients with dismnestic MCI variant. The follow-up study showed that the large number of patients with progressive cognitive disorders associated with conversion to dementia was observed in this group. In conclusion, the determination of the neurochemical markers in the cerebrospinal fluid allows to detect the neurodegenerative process at early stages of the disease and assists in detection of cases with increased risk of Alzheimer's disease.

[Cell death and specifics of its regulation in tumour cells].

Disorders of apoptosis regulation leading to development oftumours associated with expression of one of the main links in the regulation of programmed death of cells (proteins of the Bcl-2 family) were studied.

[Peculiarities of regulation of tumour cell apoptosis].

Studies of expression of BCL-2 gene family revealed abnormal regulation of biosynthesis of apoptosis-related proteins in tumour cells involved in the development and progress of neoplastic diseases. Expression of BCL-2 proteins in highly differentiated gastric and colonic tumours was roughly 1.2 times lower than in the normal cells. A 3.5-fold rise in BCL-2 expression was documented in a group of moderately differentiated adenocarcinomas and undifferentiated tumours. The most striking (9-fold) difference between BCL expression in untransformed and atypical cells was recorded in moderately differentiated metastatic colonic adenocarcinoma. Immunohistochemical studies of BAX expression in untransformed and atypical gastric and colonic cells demonstrated activation of apoptosis-inducing mechanisms in the pathologically changed tissue. Investigations with the use of SSCP showed that 20% of the patients had BAX gene mutations affecting codons 38 to 41 of exone 3. This region was found to contain octadeoxyguanosine G8 sequence. The mutations were caused by deletion of G7 or insertion of G9.

[Inactivation and sensitization of the tumor cells by the gene Bax transfection].

After the transfection of the gene Bax into the cultured tumor cells of human ovary adenocarcinoma SKOV3 and uterus carcinoma HeLa in vitro the high sensitivity of the cells SKOV3 to the protein Bax produced after the gene Bax transfection was found. The sensitivity of the cells HeLa to the gene Bax transfection was much smaller. The hyperexpression of gene Bax and hypersensitivity to doxorubicin were seen in HeLa cells received as a result of the gene Bax transfection and subsequent selection. All cells of the line SKOV3 with the increased expression of the transfected gene Bax died. In the cell line SKOV3 the mutation in a gene Bax was found which has a genotype G7/G9 against a native type of a gene Bax--G8/G8. It was concluded that the found in the exone 3 of the gene Bax mutation G7/G9 in cells SKOV3 results in an inactivation of proapoptotic activity of the protein Bax.

[The role of Fas/FasL system in induction of hepatocyte apoptosis in chronic viral hepatitides]. / Rol' sistemy Fas/FasL v induktsii apoptoza gepatotsitov pri khronicheskikh virusnykh gepatitakh.

The aim of the study was assessment of hepatocyte apoptosis depending on expression of Fas and FasL proteins by various liver cells in patients with chronic viral hepatitis B (CVHB) or chronic viral hepatitis C (CVHC). The symptoms of hepatocyte apoptosis were observed in 3 of 12 patients with CVHB and in 9 of 14 patients with CVHC, the proportion of apoptotic cells being 12-65%. Hepatocytes of healthy people and patients with hepatitis B or C express Fas protein in the cytoplasm diffusely, as granules or on cell membrane. In health, hepatocytes do not express FasL, but in CVH they do. The highest apoptosis was observed in Fas protein location as granules in cytoplasm or in their preferable location on the cell membrane. The severity of hepatocyte apoptosis in CVH directly correlated with FasL expression by the cells of the lymphoid-histiocytic infiltrate in the liver and inversely correlated with FasL expression by hepatocytes. Thus, a great part of hepatocytes in CVH are killed by the virus; Fas/FasL interaction is leading in damage to hepatocytes in CVH.

[Molecular mechanisms of apoptosis pathology]. / Molekuliarnye osnovy patologii apoptoza.

Apoptosis--a programmed cell death--is a general biological mechanism responsible for maintenance of a permanent cell number. Impairment of apoptosis regulation entails disorders in homeostasis and various diseases. This explains biological and medical importance of apoptosis. Molecular grounds of apoptosis regulation, physiological regulation depending on the cell type, examples of apoptosis disregulation leading to definite diseases, approaches to treatment of some diseases basing on knowledge of apoptosis regulation are provided.

Vasorelaxant and antiplatelet activity of 4,7-dimethyl-1,2, 5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide: role of soluble guanylate cyclase, nitric oxide and thiols.

1. Certain heterocyclic N-oxides are vasodilators and inhibitors of platelet aggregation. The pharmacological activity of the furoxan derivative condensed with pyridazine di-N-oxide 4,7-dimethyl-1,2, 5-oxadiazolo[3,4-d]pyridazine 1,5,6-trioxide (FPTO) and the corresponding furazan (FPDO) was studied. 2. FPTO reacted with thiols generating nitrite (NO), S-nitrosoglutathione and hydroxylamine (nitroxyl) and converted oxyHb to metHb. FPDO did not generate detectable amounts of NO-like species but reacted with thiols and oxyHb. 3. FPTO and FPDO haem-dependently stimulated the activity of soluble guanylate cyclase (sGC) and this stimulation was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and by 0.1 mM dithiothreitol. 4. FPTO relaxed noradrenaline-precontracted aortic rings and its concentration-response curve was biphasic (pIC(50)=9. 03+/-0.13 and 5.85+/-0.06). FPDO was significantly less potent vasodilator (pIC(50)=5.19+/-0.14). The vasorelaxant activity of FPTO and FPDO was inhibited by ODQ. oxyHb significantly inhibited only FPTO-dependent relaxation. 5. FPTO and FPDO were equipotent inhibitors of ADP-induced platelet aggregation (IC(50)=0.63+/-0.15 and 0.49+/-0. 05 microM, respectively). The antiplatelet activity of FPTO (but not FPDO) was partially suppressed by oxyHb. The antiaggregatory effects of FPTO and FPDO were only partially blocked by sGC inhibitors. 6. FPTO and FPDO (10 - 20 microM) significantly increased cyclic GMP levels in aortic rings and platelets and this increase was blocked by ODQ. 7. Thus, FPTO can generate NO and, like FPDO, reacts with thiols and haem. The vasorelaxant activity of FPTO and FPDO is sGC-dependent and a predominant role is played by NO at FPTO concentrations below 1 microM. On the contrary, inhibition of platelet aggregation is only partially related to sGC activation.

[Inhibition of ADP-induced platelet aggregation by guanidine thiols--a novel class of guanylate cyclase activators and NO-synthase substrates]. / Tormozhenie ADP-indutsirovannoi agregatsii trombotsitov guanidinotiolami--nvym klassom aktivatorov guanilattsiklazy i substratov NO-sintazy.

Antiaggregatory properties of guanidine thiol derivatives and their effect on human platelet guanylate cyclase activity were studied. The molecules of guanidine thiols contain guanidine and thiol groups which are the donor and acceptor of nitric oxide (NO), respectively. Three synthetic guanidine thiol derivatives were studied including mercaptoethylguanidine (MEG), mercaptoethylguanidine disulfide (MEG-disulfide), and mercaptoethylguanidine methylated at SH-group (S-methylmercaptoethylguanidine (S-methyl MEG)). All compounds are the substrates of NO-synthase and activators of human platelet guanylate cyclase. The stimulatory effects of MEG and MEG-disulfide on guanylate cyclase activity were 2- and 4-fold higher, respectively, than the effect of L-arginine. Stimulation of the enzyme by S-methyl MEG is similar to L-arginine. Antiaggregatory properties of these compounds correspond to the extent of guanylate cyclase activation. Extent of guanylate cyclase activation (S-methyl MEG < MEG < MEG-disulfide) significantly correlates with inhibition of ADP-induced platelet aggregation and with acceleration of spontaneous disaggregation of platelets. The mechanism of directed enhancement of antiaggregatory properties of the compounds can depend on their chemical structure and extent of guanylate cyclase activation.

[A new activator of soluble guanylate cyclase generated by nitric oxide and having antihypertensive and anti-aggregation properties]. / Novyi aktivator rastvorimoi guanilattsiklazy, generiruiushchii oksid azota i obladaiushchii antigipertenzivnym i antiagregatsionnym svoistvami.

The effects of aminoethylisothiuronium bromide known as a radioprotector on the activity of human platelet soluble guanylate cyclase and on ADP-induced aggregation of human platelets have been studied. It has been shown that in Tris-buffer and at definite pH values aminoethylisothiuronium bromide is converted into mercaptoethylguanidine as a result of a transguanidine rearrangement. The latter contains in its molecule both guanidine and SH-groups which appear to be the donor and acceptor of nitric oxide, respectively. It was found that after its rearrangement into mercaptoethylguanidine, aminoethylisothiuronium bromide activates human platelet soluble guanylate cyclase, inhibits ADP-induced aggregation of human platelets and accelerates their spontaneous disaggregation. The stimulating effect of aminoethylisothiuronium bromide depends on the effectiveness of its transguanidine rearrangement into mercaptoethylguanidine. A molecular mechanism of the hypotensive side effect of aminoethylisothiuronium bromide is proposed.

[The preliminary results of a study of the role of endogenous nitric oxide in pregnant women with chronic glomerulonephritis, hypertension and puerperal eclampsia]. / Predvaritel'nye rezul'taty izucheniia roli éndogennogo oksida azota u beremennykh s khronicheskim glomerulonefritom, gipertonicheskoi bolezn'iu, rodovoi éklampsiei.

NO-synthase activity and formation of ERP/NO were evaluated in 19 pregnant women by high activity of platelet guanylate-cyclase (GC) and presence of z-arginine. The examinees were divided into 4 groups: 3 women with chronic glomerulonephritis (CGN), 9 patients with essential hypertension (EH), 3 nonpregnant women of reproductive age with EH, 3 healthy pregnant women. One patient had puerperal eclampsia. Z-arginine activation failed to change GC activity in healthy pregnant women, reduced it in pregnant and nonpregnant women with EH, increased GC activity 1.6-fold in a patient with puerperal eclampsia and in CGN pregnant females. It is inferred that changes in NO-dependent synthesis of cGMP manifested more distinctly in a patient with puerperal eclampsia.

Guanidine thiol--a new activator of soluble guanylate cyclase with antihypertensive and antiaggregatory properties.

Effects of aminoethylisothiuronium bromide (AET), known as radioprotector, on human platelet soluble guanylate cyclase and on ADP-induced human platelets aggregation were studied. It was shown that AET - in Tris buffer and at certain pH values - is converted, via transguanidine rearrangement, to mercaptoethylguanidine. The latter contains in its molecule both the guanidine and SH groups which act as donor and acceptor of nitric oxide (NO), respectively. It was demonstrated that AET, after its rearrangement to mercaptoethylguanidine, is able to activate human platelet soluble guanylate cyclase, as well as to inhibit ADP-induced human stimulatory effect of AET is dependent on the effectiveness of its transguanidine rearrangement to mercaptoethylguanidine. The molecular mechanism of the hypotensive by - effect of AET is proposed.

[Endothelial relaxation factor in the development of diabetic nephropathy]. / Endotelial'nyi faktor relaksatsii v razvitii diabeticheskoi nefropatii.

Progression of diabetic nephropathy is now associated with intrarenal hemodynamic disorders (renal hyperperfusion, hyperfiltration, intraglomerular hypertension). The cause of these disorders is unclear. It is supposed that the relaxation factor which is produced by the vascular endothelium (endothelial relaxation factor-ERF) and an endogenous nitrogen oxide (NO) can cause the above intrarenal hemodynamic alterations in diabetes mellitus. The production of ERF/NO in 35 patients with insulin-dependent diabetes mellitus who had varying severities of diabetic nephropathies were examined. These included the following groups: 1) patients without diabetic nephropathy (n = 9); 2) those with incipient diabetes mellitus (n = 12), 3) those with severe diabetes mellitus (n = 14). From groups 1 and 2, 5 patients with hyperfiltration were identified, their glomerular filtration rate were more than 140 ml/ml. The ability of the cells to produce ERF/NO was indirectly estimated, by determining the levels of human platelet guanylate cyclase in the presence of L-arginine, a NO precursor, the accumulation of cGMP in the cells and plasma. When L-arginine was present, the activity of guanylate cyclase was virtually unchanged in Group 1, but it was substantially increased in Groups 2 and 3, by reaching its peak in patients with hyperfiltration (Group 4). The platelet and plasma levels of cGMP corresponded to the enhancement of guanylate cyclase activity in the presence of L-arginine and increased as diabetic nephropathy progressed. Thus, it is suggested that there is ERF/NO hyperproduction in patients at a high risk for diabetic nephropathy (those having hyperfiltration). ERF/NO is likely to promote the dilation of glomerular arterioles, which results in the development of hyperfiltration and intraglomerular hypertension, causing diabetic nephropathy progression.

[Inhibition of human platelet aggregation by a new class of soluble guanylate cyclase inhibitor, generating nitric oxide]. / Ingibirovanie agregatsii trombotsitov cheloveka novym klassom aktivatorov rastvorimoi guanilattsiklazy, generiruiushchikh oksid azota.

Diacetidine-di-N-oxide derivatives have been found to be capable of generating nitric oxide (NO) non-enzymatically, via an entirely new mechanism--NO splitting at physiological pH. The effects of the synthesized compounds on the human platelet soluble guanylate cyclase activity and ADP-induced human platelet aggregation have been investigated. Four out of seven derivatives tested exhibited a distinct correlation between the intensity of platelet guanylate cyclase activation, inhibition of platelet aggregation and acceleration of their disaggregation. The ability of the compounds to be decomposed under the given experimental conditions with NO formation and the observed correlation between the amount of the NO formed and the intensity of guanylate cyclase activation suggest that the NO-dependent mechanism of guanylate cyclase activation and the intraplatelet cGMP accumulation are responsible for the antiaggregating/disaggregating properties of the compounds used. The data obtained suggest that 1.2-diacetidine-di-N-oxide derivatives may be regarded as antiaggregating agents of a new class.

Inhibition of ADP-induced human platelet aggregation by a new class of soluble guanylate cyclase activators capable of nitric oxide generation.

Diazetidine-di-N-oxide derivatives have been found capable of the nonenzymatic generation of nitric oxide by a principally new mechanism of nitric oxide splitting at physiological pH values. The effect of the synthesized compounds on human platelet soluble guanylate cyclase activity and ADP-induced human platelets aggregation were studied. Four of 7 derivatives studied exhibited a distinct correlation between the intensity of platelet guanylate cyclase activation, inhibition of platelets aggregation and acceleration of their disaggregation. The NO-dependent mechanism of guanylate cyclase activation and intraplatelet cGMP accumulation are suggested to be responsible for antiaggregatory/disaggregatory properties of the compounds used. Data presented allow us to regard 1,2-diazetidine-di-N-oxide derivatives as antiaggregatory agents of a new class.

Increase in activating ability of human platelet guanylate cyclase during aggregation.

The dynamics of changes in the stimulation of human platelet guanylate cyclase by some activators in aggregating platelets was studied. It was shown that ADP-induced aggregation of human platelets (donors) is accompanied by the enhancement of the intensity of guanylate cyclase activation by sodium nitroprusside, L-arginine, protoporphyrin IX and arachidonic acid and also by the increase in cGMP content. Immediately after the induction of aggregation the intensity of guanylate cyclase activation and cGMP content begin to increase. The rise reaches its maxima within several minutes, then followed by a fall to the initial level. The peaks of the enhanced capacity for guanylate cyclase activation by the above compounds coincide in time and intensity. On the basis of the proposed hypothetical scheme of cGMP action as a regulator of platelet aggregation a possible mechanism of enhancing the capacity of guanylate cyclase to be stimulated by various activators in aggregating platelets is suggested.

Increase in reactivity of human platelet guanylate cyclase during aggregation potentiates the disaggregating capacity of sodium nitroprusside.

1. Basal and stimulated guanylate cyclase activity during ADP-induced human platelet aggregation in comparison with the actions of sodium nitroprusside (SNP) on platelets was investigated. 2. Sodium nitroprusside exhibited both ex vivo and in vitro antiplatelet effects, as assessed by inhibition of subsequent ADP-induced aggregation in platelet-rich plasma. A strong correlation between decrease in aggregation and increase in platelet guanylate cyclase activity in the presence of SNP was obtained. 3. When SNP was administered after the induction of aggregation, it caused acceleration of disaggregation (in reversible aggregation) and produced disaggregation (under conditions of otherwise irreversible aggregation) which was time-dependent. 4. Platelet aggregation was accompanied by a transient increase in platelet cyclic GMP content and guanylate cyclase activation by the nitric oxide (NO) donor SNP. Changes in guanylate cyclase activity were haem-associated and probably reflected saturation of enzyme by haem. 5. Maximal SNP disaggregating effect coincided with peak guanylate cyclase responsiveness to SNP. 6. The present investigation provides evidence that increased responsiveness of platelet guanylate cyclase to NO during aggregation facilitates disaggregation in the presence of SNP. Thus, availability of NO (endogenous or exogenous) at sites of incipient platelet aggregation in vivo may play a pivotal role regarding limitation of this process.

[Changes in the guanylate cyclase activity of human thrombocytes during ADP-inducible aggregation]. / Izmeneniia v aktivnosti guanilattsiklazy trombotsitov cheloveka pri ADF-indutsiruemoi agregatsii.

Activity of guanylate cyclase (GC) and its capacity for sodium nitroprusside (SNP) activation were determined in platelets with different state of aggregation. The development of ADP-induced reversible aggregation was accompanied by a decrease in the basal GC activity and by an increase in the SNP activation of GC. It was shown that elevation of GC sensitivity to SNP during the aggregation might be due to the decrease in the state of enzyme blood deficiency. Preincubation of platelets with SNP before ADP adding markedly diminished or even prevented aggregation, depending on SNP concentration. GC parameters in platelets with prevented aggregation were just the same as in control. It is suggested that the regulatory role of cGMP system in platelet aggregation may be seen in the increase in GC sensitivity to endogenous activator, presumably to NO.