[ABCG1 transporter gene expression in peripheral blood mononuclear cells of patients with atherosclerosis].

Accumulation of cholesterol in arterial wall macrophages is a main hallmark of atherosclerosis. The ABCG1 transporter mediates cholesterol efflux to high density lipoproteins (HDL) and plays an important role in macrophage foam cell formation. The goal of our study was to investigate the potential role of ABCG1 in atherosclerosis development in humans. ABCG1 gene expression has been examined in leukocytes, monocytes and monocyte-derived macrophages of patients with atherosclerosis and in the control group. Real time PCR and Western blotting were used to determine ABCG1 mRNA and ABCG1 protein levels. Monocyte ABCG1 mRNA level was inversely correlated with the rate of artery occlusion (r = -0.45, P = 0.016). Patients with 100% artery occlusions had decreased monocyte ABCG1 mRNA levels compared to patients who had smaller plaques and controls (P < 0.05). ABCG1 mRNA (P < 0.001) and ABCG1 protein (P < 0.05) levels in macrophages of patients with coronary artery stenosis were significantly reduced compared to the control group. No significant correlation between the ABCG1 gene expression in mononuclear cells and HDL cholesterol concentration has been found. Our study suggests that decrease in the ABCG1 gene expression in macrophages is associated with atherosclerosis.

[Investigation of neuroprotective activity of apolipoprotein E peptide mimetic Cog1410 in transgenic lines of Drosophila melanogaster].

The neuroprotective activity of apolipoprotein E (apoE) peptide mimetic Cog1410, containing amino acid sequence of the receptor-binding domain apoE, has been investigated in transgenic lines of Drosophila melanogaster expressing human APP and beta-secretase. Expression of two transgenes caused neuropathological processes attributed to Alzheimer's disease: neurodegeneration, cognitive abnormality and amyloid deposits formation in brain. It was shown that Cog 1410 reduces neurodegeneration in brain of transgenic flies and improves cognitive functions (odor recognition). These data suggest that Cog1410 is a potential neuroprotector that can be used in AD treatment.

[ABCA1 mRNA and protein levels in M-CSF-activated macrophages from patients with arterial stenosis].

ABCA1 transporter is one of the key factors defining the level of antiatherogenic HDL in plasma. It is actively involved in the removal of cholesterol from peripheral tissues by reverse cholesterol transport. However, the influence of the level of ABCA1 mRNA and the level of ABCA1 protein 1 in macrophages in atherosclerosis remains unexplored. Using real time PCR we determined ABCA1 mRNA level in macrophages cultured for 5 days with macrophage colony-stimulating factor (M-CSF). ABCA1 mRNA levels in macrophages from patients with arterial stenosis were increased when compared with the control group, P = 0.04. According to a Western blot analysis ABCA1 protein level in macrophages from patients was significantly lower than in the control group, P = 0.01. Our results suggest that the level of ABCA1 mRNA and level of ABCA1 protein in macrophages may be important factors in the development of atherosclerosis.

[Morphological and functional abnormalities in neuromuscular junctions of Drosophila melanogaster induced by the expression of human APP gene].

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the loss of neurocortical and hippocampal synapses that precedes amyloidosis and neurodegeneration and closely correlates with memory impairment. Mutations in the amyloid precursor protein (APP) cause familial AD and result in the increased production of amyloid-beta-protein (Abeta). To gain insights into synaptic effects of APP, we expressed APP, mutant form APP-Swedish and BACE in the motor neurons of fly larvae. We have shown that targeted expression of APP (APP-Swedish) in Drosophila larval motor neurons causes significant morphological and functional changes in neuromuscular junctions (NMJs): a dramatic increase in the number of synaptic buttons and changes in exocytosis as revealed by incorporation of the styryl dye FM4-64. Analysis of the number and distribution of mitochondria showed that motor neurons overexpressing APP (APP-Swedish) had a significant reduction of functional mitochondria in the presynaptic terminal. Significant synaptic abnormalities were observed for APP (APP-Swedish) and human beta-secretase (BACE) resulting in secretion of amyloid beta protein (Abeta). We suggest that APP participates in regulation of synaptic functions and its elevated expression leads to synaptic pathology independently from neurotoxic effects of Abeta.

[Apoptosis of peripheral blood lymphocytes in patients with LRRK2-assoctated Parkinson's disease].

Mutations in the Leucine Reach Repeat Kinase 2 (LRRK2) gene are the most frequent cause of familial Parkinson's disease (PD). Although the precise physiological and pathological role of LRRK2 is unclear, a direct link between mutant LRRK2 and apoptosis has been suggested. Using flow cytometric analysis (PI+Annexin V(FITC)) we showed increased spontaneous apoptosis of peripheral blood lymphocytes in patients with LRRK.2-associated PD compared to controls after 24 (P < 0.016) and 48 (P < 0.031 ) h of incubation (5 % CO2, 37 degrees C). We found the increased FAS mRNA level in peripheral blood lymphocytes of patients with LRRK2-associated PD compared to controls (P < 0.05) and to sporadic PD (sPD) (P < 0.002). Significant difference in FAS expression between patients with LRRK2-associated PD and controls remained after three years and was detected after 1 and 24 h during lymphocyte incubation (P < 0.03 and 0.05, respectively). Increased spontaneous lymphocytes apoptosis along to increased FAS expression in patients with LRRK2-associated PD suggest that LRRK2 mutations may lead to the activation of extrinsic apoptotic way.

[ABCA1 gene expression in peripheral blood lymphocytes and macrophages in patients with atherosclerosis].

ABCA1 transporter is known to play important role in the cholesterol transport from peripheral tissues. However its contribution in atherosclerosis development remains not completely understood. Using Real Time PCR, a significant reduction of ABCA1 mRNA level in leukocytes of patients with atherosclerosis was determined when compared with controls. Mean ABCA1 expression levels in leukocytes for the group of patients and for the control group are 0.57 +/- 0.28 and 0.93 +/- 0.14 (p = 0.02). At the same time we detected a significant increase of ABCA1 mRNA level in macrophages of patients when compared with controls. Mean ABCA1 expression levels in macrophages for the group of patients and for the control group are 1.32 +/- 0.10 and 0.90 +/- 0.14 (p = 0.014). In summary, we suggest that expression level of ABCA1 gene may contribute to the development of atherosclerosis.

[Potential role of presenilin 1 in regulation of synaptic function].

One of the earliest neuropathological symptoms of Alzheimer's disease is the loss of synapses, which preceed the formation of amyloidosis and neurodegeneration. Although most cases of early-onset familial Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene, the functions of PS1 and its role in synaptic disfunction are not yet completely understood. In this paper we analysed of the intracellular and extracellular distribution of PS1 in the cultures of mouse cortical embryonic neurons. We found that PS1 is concentrated on the surface of the growth cone and at neurite contact sites. PS1 was also found in synapses where it is co-localized with synaptophysin. Independent evidense of involvement of PS1 in synaptic function we obtained by transfection of neurons with GFP-PS1 cDNA. GFP was colocalized with synaptophysin in transfected cultures. GFP-immunoprecepitates from transfected neurons contained processed N-cadherin. This result presents an additional proof of involvment PS1 in synapse formation. To evaluate the role of PS1 inactivation in the synaptic functions, we compare synaptic density in neuronal cell cultures from PS1 knockout mice PS1 (-/-) and wild type mice PS1 (+/+). Our results clearly show that PS1 (-/-) displayed a low number of morphological synapses in comparing with wild type culture PS1 (+/+). In summary, our results indicate a role of PS1 in synaptic function.

[Familial Alzheimer's disease mutations in the presenilin 1 gene reduce cell-cell adhesion in transfected fibroblasts].

Experimental evidence has been obtained that mutations in the presenilin 1 (PS1) gene in familial Alzheimer's disease can lead to the disturbance of cell adhesion in model cell cultures. It was shown that, in L fibroblasts of mice with stable expression of GFP-PS1 cDNA containing G209V or E319G mutations, cell-cell interactions and the accumulation of GFP-PS1 cDNA in intercellular contacts are disturbed. Similar results were obtained in transfected human epithelial Hep2 cells. It is assumed that mutations in familial Alzheimer's disease lead to the disturbance of the functions of presenelin 1 in cell adhesion.

[Modern genetic approaches to the search for targets for medicinal preparations].

In spite of a vast number of drug preparations used in medicine, advances in treating most socially important human diseases remain modest. Historically, many drugs were developed without clear understanding of the mechanisms of their action and were aimed only at correcting symptoms of the disease. Identification of molecular targets in pharmacological screening of new pharmaceuticals plays a key role not only in defining the strategy of the treatment, but also in understanding the general development of the disease. Sequencing of the genomes of various organisms, human in particular, and the development of new modern techniques of research have created the prerequisites for targeted screening for genes that are potentially interesting for designing new drugs.

[Genetic variants of platelet ADP receptor P2Y12 associated with changed platelet functional activity and development of cardiovascular diseases].

The key role in platelet aggregation is played by the platelet ADP receptor P2Y12, which is the target for antiaggregant drugs, clopidogrel and ticlopidine. At present, only sporadic data on genetic variants of platelet ADP receptor P2Y12 are available from literature, and their association with thromboembolic and cardiovascular diseases still remains obscure. Analysis of the group of subjects with high platelet reactivity resulted in identification of two nucleotide substitutions, C18T and G36T, in the coding region of the P2Y12 gene. The frequency of the P2Y12 T1 8 allele was higher in control group than in the group of patients survived from myocardial infarction at the age under 45 years (39% versus 28%, respectively, P = 0.04). Moreover, in the T18 carriers, platelet aggregation activity was lower than in the carriers of the wild-type genotype (0.84 +/- 0.05% versus 1.01 +/- 0.08%, respectively, P = 0.03). In the group of patients with early myocardial infarctions, a tendency towards the increased frequency of 16T allele in comparison with control group (20 and 12%, respectively, P = 0.07) was observed. The rate of ADP-induced platelet aggregation in the carriers of 16T allele from the control group was somewhat higher than in the subjects with the GG36 genotype (1.31 +/- 0.16% versus 1.12 +/- 0.06%, respectively, P = 0.07). The nucleotide substitutions identified were in absolute disequilibrium, i.e., allele T18 conformed to allele G36. On the contrary, allele C18 conformed to allele T36. Haplotype T18G36 was found to be responsible for the decreased risk of myocardial infarction and decreased platelet reactivity. It is suggested that polymorphisms of the P2Y12 gene identified can be used for determination of the risk group for myocardial infarction in the young males.

[Protein transduction domain peptide mediates delivery to the brain via the blood-brain barrier in Drosophila].

Protein transduction domain (PTD)-peptides greatly facilitate the delivery of high molecular weight macromolecules across the blood-brain barrier (BBB). This BBB-transport function is highly desirable and helps to enable the development of new therapeutics for treatment of brain disorders. However, the drug discovery process is limited by the generation of a simple and reliable BBB model that is amenable to testing of large number of samples and simultaneously, reproduces the physiological and functional characteristics of the human BBB. To address these challenges, we have studied whether the PTD-peptide penetratin, derived from a Drosophila Antennapedia homeodomain protein, is capable of crossing the BBB in Drosophila while carrying a cargo into the fly brain. An initial in vivo experiment in Drosophila showed that abdominal injection of biotin-tagged penetratin permeated the BBB. The same effect was observed for biotin-tagged penetratin fused with apoE mimetic peptide with demonstrated anti-inflammatory and neuroprotective activities.

[Studying pathogenesis of Alzheimer's disease in a Drosophila melanogaster model: human APP overexpression in the brain of transgenic flies leads to deficit of the synaptic protein synaptotagmin].

Alzheimer's disease (AD) is a progressive neurodegenerative disease whose main pathomorphological sign is synapse degeneration in the cortex and hippocampus. Abnormal synaptogenesis precedes amyloidosis and neurodegeneration and correlates with memory impairment during the early clinical phase. Mutations in the amyloid precursor protein (APP) gene cause familial AD and enhance the secretion of amyloid-beta-protein (Abeta). However, it remains unclear in what way APP and Abeta are involved in synaptic disorder in the absence of visible amyloid structures. In this study, the role of the human APP gene in synaptogenesis in transgenic lines of Drosophila melanogaster whose nerve cells express the human APP695 isoform, truncated APPs, and the presynaptic marker synaptotagmin driving the sequence of the green fluorescent protein. The expression of APP and its truncated forms caused a decrease in the synaptotagmin content of antennal lobes and mushroom lobes of the D. melanogaster brain, as well as neurodegeneration that progressed with age. The results suggest that that abnormal synaptogenesis and neurodegeneration occur in the Drosophila brain in the absence of Abeta. It is assumed that impaired cellular functions of APP and secretion of Abeta independently contribute to the pathogenesis of AD.

[Degeneration of growth cones in a culture of embryonic neurons of mouse with presenilin 1 gene knockout].

A comparative study of growth cone morphology in cultured embryonic neurons derived from wild type PS 1(+/+) and knockout PS 1(-/-) mice has been performed. Growth cones from wild type PS 1(+/+) mice were well spread and usually formed radially continuous and regular lamellar extensions with numerous filopodia. In contrast, most growth cones from knockout PS 1(-/-) mice exhibited small lamellar extensions, short filopodia, and pure adhesion. A significant amount of growth cones from knockout PS 1(-/-) mice collapsed after 3-4 days in culture. It was suggested that PS 1 plays an important role in growth cone structure by stabilizing the integrity of the cytoskeleton. The growth cone collapse may be the main reason for abnormal neuronal migration and impaired synaptic function in PS 1(-/-) mice.

[Genetic risk factors for development of myocardial infarction in young men living in North-West region of Russia].

With the aim to detect genetic factors of risk of development of early myocardial infarction (MI) we studied 29 allele variants of 19 genes in 206 men who had survived MI in the age before 45 years and in 195 men of similar age without cardiovascular diseases. All subjects were inhabitants of North-West region of Russia. The following factors were associated with history of myocardial infarction: genotype RR191 of paraoxonase-1 (PON1) gene (RR 2.8 [95% CI: 1.24 - 6.30]), P1A2 allele of glycoprotein (GP) IIIa subunit of platelet fibrinogen receptor GPIIb/IIIa (RR 1.8 [95% CI: 1.11 - 2.93]), and Met145 allele of GPIbalpha platelet von Willebrand factor receptor gene. Genotype CC ( - 108) PON1 was associated with lowered risk of MI development (RR 0.6 [95% CI: 0.40 - 0.91]). During 7 years of follow-up 30 men from MI group died of recurrent acute coronary syndromes. In the group of those who died we noted increased prevalence of P1A2 GPIIIa allele compared with those who survived (p < 0.03). The results allow to suggest that contribute to development of MI in young men factors associated with elevation of functional state of platelets and levels of oxidized lipids in blood plasma.

Participation of IIb-IIIa glycoprotein in spontaneous platelet aggregation.

In some patients with stable and unstable angina pectoris and in some donors without clinical manifestations of cardiovascular diseases and other pathologies, spontaneous platelet aggregation was completely suppressed by glycoprotein IIb-IIIa antagonists blocking the interaction of this glycoprotein with fibrinogen. Antibodies inhibiting binding of glycoprotein Ib with von Willebrand factor had no effect on the level and rate of spontaneous platelet aggregation. In the donor group, the level of spontaneous aggregation was almost 1.5-fold higher in persons with a certain genetic polymorphism (Leu-->Pro substitution in position 33 of glycoprotein IIIa). The level of spontaneous aggregation correlated with the amount of glycoprotein IIb-IIIa on the platelet surface (r = 0.41).

[Expression of presenilin 1 on the cell surface in motile polarized cells].

Most cases of familial early-onset Alzheimer's disease are caused by mutations in the presenilin 1 (PS1) gene. However, the cellular functions of PS1 are not yet completely understood. We showed that endogenous PS1 and the adhesion protein CD44 are redistributed on the surface of cell projections (lamellipodia) in polarized T- lymphocytes (Jurkat cells) after the adhesion to a collagen matrix. This effect was not observed for another surface protein of T lymphocytes, which is not involved in cell adhesion processes, the T cell receptor. In primary cultures of mouse cortical neurons, PS1 was concentrated at the surface of extended growth cones and at the sites of neurite contacts. The concentration of PS1 at the surface of cellular structures that promote cell motility and cell contacts suggests an important role of PSI in cell adhesion in motile polarized cells.

Role of the C-terminal fragment of human transthyretin in abnormal fibrillogenesis.

Polypeptide chain fragments of recombinant transthyretin (TTR) with leucine-55 substituted by proline (L55P), which are involved in abnormal fibrillogenesis of this protein, were studied. No fibrils were produced in purified preparations of TTR(L55P) under the optimum conditions for fibrillogenesis but in absence of protease inhibitors. The ability of TTR for fibrillogenesis was lost because of a limited proteolysis resulting in detachment of the TTR polypeptide chain C-terminal fragment of approximately 18 amino acid residues in length. This proteolysis seemed to occur with involvement of a bacterial serine endopeptidase sohB (EC 3.4.21), which was identified in TTR preparations by the MALDI-TOF method. The presence of the C-terminal fragment of the TTR polypeptide chain seems to be crucial for production of abnormal fibrils.

[Molecular heterogeneity of familial hypercholesterolemia in the St. Petersburg population]. / Molekuliarnaia geterogennost' semeinoi giperkholesterinemii v populiatsii zhitelei Sankt-Peterburga.

Inheritance of Taq I, BstE II, and Nco I restriction fragment length polymorphisms (RFLP) in three families from St. Petersburg with familial hypercholesterolemia (FH) was studied. In two of these families, polymorphic markers of the low density lipoprotein receptor (LDLR) gene cosegregated with the disease. This data confirmed FH diagnosis based on the analysis of blood plasma lipid levels. Three different RFLP haplotypes were associated with the disease, suggesting the presence of at least three point mutations in the LDLR gene in the population studied, i.e., suggesting molecular heterogeneity of FH in the St. Petersburg population.

[A multiexon deletion in the human low density lipoprotein receptor gene as a reason for familial hypercholesterolemia]. / Mul'tiékzonnaia deletsiia v gene retseptora lipoproteinov nizkoi plotnosti cheloveka kak prichina semeinoi giperkholesterinemii.

Inheritance of Taq I, BstE II, and Nco I restriction fragment length polymorphisms (RFLP) in three families from St. Petersburg with familial hypercholesterolemia (FH) was studied. In two of these families, polymorphic markers of the low density lipoprotein receptor (LDLR) gene cosegregated with the disease. This data confirmed FH diagnosis based on the analysis of blood plasma lipid levels. Three different RFLP haplotypes were associated with the disease, suggesting the presence of at least three point mutations in the LDLR gene in the population studied, i.e., suggesting molecular heterogeneity of FH in the St. Petersburg population.