[Analysis of gene expression pattern in peripheral blood leukocytes during experimental heat wave]. / Analiz izmeneniia ékspressii genov v leikotsitakh krovi cheloveka vo vremia ispytaniia, modeliruiushchego usloviia teplovoi volny.

The conditions of Moscow 2010 summer heat wave were simulated in an accommodation module. Six healthy men aged from 22 to 46 years stayed in the module for 30 days. Measurements of gene expression in peripheral blood leukocytes before, during and 3 day after simulated heat wave were performed using qRT-PCR. We observed a shift in the expression level of certain genes after heat exposure for a long time, and rapid return to the initial level, when volunteers leaved the accommodation module. Eight genes were chosen to form the "heat expression signature". EGR2, EGR3 were upregulated in all six volunteers, EGR1, SIRT1, CYP51A1, MAPK9, BAG5, MNDA were upregulated in 5 volunteers.

[Pharmacokinetic and pharmacodynamic synergism between neuropeptides and lithium in the neurotrophic and neuroprotective action of cerebrolysin].

OBJECTIVE: To study the synergism between neuropeptides and lithium ions. MATERIAL AND METHODS: An experimental model of stroke (chronic bilateral occlusion of the common carotid arteries in rats), neuronal culture studies, histomorphological analyses, determination of micronutrient profile of brain substrates were used. RESULTS: A complex of experimental studies revealed that the effect of cerebrolysin is influenced by the synergism between lithium ions and the neuropeptide contentof this drug. Pharmacokinetic synergism promotes the accumulation of lithium in brain tissues during cerebrolysin treatment. The existence of the pharmacokinetic synergism is evident from the potentiation of neuroprotective effects of the drug under the action of lithium ions established in the model of stroke. CONCLUSION: Lithium ions potentiate neuroprotective effects of cerebrolysin.

[Comparative transcriptome analysis of human aorta atherosclerotic lesions and peripheral blood leukocytes from essential hypertension patients].

One of the major cardiovascular risk factor which predisposes to and accelerates atherosclerosis is arterial hypertension (AH). To determine the molecular basis of the crosslink between AH and atherosclerosis for the development of new treatment strategies large-scale transcriptome analysis of the cells implicated in atherogenesis is needed. We used cDNA microarray technique for simultaneous analysis of gene expression in human abdominal aorta normal sites and atherosclerotic lesions of different histological types, as well as in peripheral blood leukocytes from patients with essential hypertension (EH) and donors. The microarray data were verified by quantitative RT-PCR (reverse transcription coupled with polymerase chain reaction) and immunohistochemical analysis. Differential expression of 40 genes has been found, among which twenty two genes demonstrated up-regulation and 18 genes demonstrated down-regulation in atherosclerotic aorta compared with normal vessel. New gene-candidates, implicated in atherogenesis, have been identified - FPRL2, CD37, CD53, RGS1, LCP1, SPI1, CTSA, EPAS1, FHL1, GEM, RHOB, SPARCL1, ITGA8, PLN, and COL14A1. These genes participate in cell migration and adhesion, phenotypic changes of smooth muscle cells, immune and inflammatory reactions, oxidative processes and extracellular matrix remodeling. We have found increased expression levels of CD53, SPI1, FPRL2, SPP1, CTSD, ACP5, LCP1, CTSA and LIPA genes in peripheral blood leukocytes from EH patients and in atherosclerotic lesions of human aorta. The majority of these genes significantly (p<0.005) positively (r>0.5) correlated with AH stage as well as with histological grading of atherosclerotic lesions.

[Gene expression analysis in myocytes of right atrial appendages in patients with atrial fibrillation using cDNA microarray technique].

Gene expression level of 2900 genes was studied by cDNA microarray in patients with atrial fibril-lation (AF) or sinus rhythm. Gene transcripts were analysed in samples of right atrial appendages from 47 patients undergoing surgery for valve repair or coronary artery bypass. Standard correlation analysis and two dimensional hierarchical clustering were used for study of differentially expressed genes in patient groups. A highly positive correlation of gene expression with AF was shown for cardiac muscle LIM domain protein (CSRP3), cardiac muscle myosin heavy chain beta isoform (MYH7), calmodulin (CALMS) and homeobox protein (PKNOXl) genes (r > 0.77, p < 0.007). In contrast, metallothionein (MT1/2), mitochondrial aldehyde dehydrogenase 2 (ALDH2), ras-related protein (RaplA) and guanine nucleotide binding protein G (GNAL) genes revealed highly negative correlation with AF (r < -0.75, p < 0.002). Alterations of gene activity were more evident at permanent as compared with paroxysmal AF. In addition, genes overexpressed in AF patients demonstrated underexpression in coronary artery disease patients (r=-0.8, p=0.0002) and conversely. Genes correlating with AF belong to different functional categories, including sarcomere organization, contraction, Ca2+ homeostasis, signaling and transcription regulation, extracellular matrix interactions and oxidative stress. Downregulation of MT1/2 and ALDH2 genes, known protectors against oxidative stress, may contribute to maintenance of oxidative stress in myocardial tissues of AF patients. The identification of novel genes - participants of pathological process in AF may open new perspective for search of therapeutic agents.

[Phospholipase A2, group IIa, as an earlier unknown immunohistological marker of cardiac myxoma].

Phospholipase A2, group IIA, gene expression has been analyzed in primary heart tumors. High expression has been demonstrated through several ways: reverse-transcriptase chain polymerase chain, Northern blotting hybridization at the RNA level and immunoblotting, immunohistochemical assay at the protein level. Human cardiac myxoma exhibits highly positive phospholipase A2, group IIA, immunophenotype (100% positive cases). The immunophenotype is unique among human primary cardiac tumors. Phospholipase A2, group IIA, can be proposed as a tissue marker for pathological examination after heart tumor resection.

Altered gene expression pattern in peripheral blood leukocytes from patients with arterial hypertension.

The role of various inflammatory mechanisms and oxidative stress in the development of atherosclerosis and arterial hypertension (AH) has been increasingly acknowledged during recent years. Hypertension per se or factors that cause hypertension along with other complications lead to infiltration of activated leukocytes in the vascular wall, where these cells contribute to the development of vascular injury by releasing cytokines, oxygen radicals, and other toxic mediators. However, molecular mechanisms underlying leukocyte activation at transcriptional level in AH are still far from being clear. To solve this problem we employed cDNA microarray technology to reveal the differences in gene expression in peripheral blood leukocytes from patients with AH compared with healthy individuals. The microarray data were verified by a semi-quantitative RT-PCR method. We found 25 genes with differential expression in leukocytes from AH patients among which 21 genes were upregulated and 4 genes were downregulated. These genes are implicated in apoptosis (CASP2, CASP4, and CASP8, p53, UBID4, NAT1, and Fte-1), inflammatory response (CAGC, CXCR4, and CX3CR1), control of MAP kinase function (PYST1, PAC1, RAF1, and RAFB1), vesicular trafficking of molecules among cellular organelles (GDI-1 and GDI-2), cell redox homeostasis (GLRX), cellular stress (HSPA8 and HSP40), and other processes. Gene expression pattern of the majority of genes was similar in AH patients independent of the disease stage and used hypotensive therapy, but was clearly different from that of normotensive subjects.

Gene expression analysis to identify mRNA markers of cardiac myxoma.

cDNA expression arrays were used to identify mRNA expression markers for cardiac myxoma. The RNA profile analysis suggests that cardiac myxoma should be considered as a stand-alone tissue rather than a pathological modification of particular normal tissue. The analysis reveals a set of genes which are highly and steadily expressed in cardiac myxomas and can serve as an mRNA expression markers of the tumour. Marker status of selected genes was confirmed by reverse transcriptase polymerase chain reaction analysis. Genes MIA (melanoma inhibitory activity) and PLA2G2A (phospholipase A2, group IIA) show the highest specificity as cardiac myxoma markers, since they have more than 10-fold higher RNA level in cardiac myxomas than in any one of 15 normal tissues tested. Among markers of myxoma at least three are participants of phospholipid metabolism: ANXA3, PLA2G2A, and phospholipid transfer protein. Tissue inhibitor of metalloproteinase 1 and secretory leucocyte protease inhibitor are inhibitors of proteases degrading extracellular matrix proteins and participating in cell proliferation regulation. MIA, SPP1, fibromodulin are modulators or participants of the interaction between extracellular matrix proteins and their cell surface receptors. SOX9 is a transcription factor required for chondrocyte differentiation. Calretenin (CALB2) is an intracellular calcium-binding protein with poorly understood function.

[PRKAR1A gene mutations in two patients with myxoma syndrome (Carney complex)]. / Mutatsii v gene reguliatornoi sub"edinitsy 1-alpha tsiklo-AMF-zavisimoi proteinkinazy A (PRKAR1A) u dvukh bol'nykh s miksomnym sindromom (kompleksom Carney).

Carney complex is an autosomic dominant disorder initially described as the association of cardiac myxomas, spotty skin pigmentation and endocrine overactivity and considered as a multiple neoplasia and lentiginosis syndrome. Mutations in the tumor suppressor gene PRKAR1A, coding for the type 1-alpha regulatory subunit of cAMP-depended protein kinase A have been previously identified in about half of the Carney complex kindreds. In this paper we report identification of the molecular defect in PRKARIA gene in two Carney complex patients. A new mutation (403delAC) located in a 3rd exon of PRKARIA gene has been observed in one case, and a previously described mutation in exon 7 (847delTC) in the second case.

Nitric oxide increases gene expression of Ca(2+)-ATPase in myocardial and skeletal muscle sarcoplasmic reticulum: physiological implications.

The aim of the study was to verify the hypothesis that NO-dependent regulation of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) gene expression can play an important role in prevention of calcium overload under the influence of detrimental factors. It was shown that 2 hours after the administration of the NO donor dinitrosyl iron complex (DNIC), the gene expression of myocardial SERCA was increased by 20% as compared to the control. In skeletal muscles, the maximum increase in SERCA expression was observed in 6 hours and amounted to 156% as compared with the initial value. Simultaneously DNIC enhanced the resistance of isolated heart and the organism as a whole to damaging effects of intracellular calcium overload induced by post-ischemic reperfusion or vigorous exercise, respectively. The results obtained confirm the existence of NO-dependent activation of SERCA expression and the important role of this mechanism in restriction of calcium overload.

[A method for obtaining the normalized cDNA libraries based on the effect of suppression of polymerase chain reaction]. / Metod polucheniia normalizovannykh bibliotek kDNK, osnovannyi na éffekte supressii polimeraznoi tsepnoi reaktsii.

A new efficient method for obtaining cDNA libraries with equal representation of all cDNA types (equalized libraries) in a single round of equalization was developed. The method is based on differences in the renaturation kinetics of double-stranded cDNAs of different genes and allow the selection of the equalized single-stranded fraction resulted from the incomplete reassociation of the total cDNA without laborious and inefficient physical separation. The equalized single-stranded fractions are selectively amplified by polymerase chain reaction (PCR). The amplification of other DNA molecules is inhibited due to PCR suppression, i.e. the suppression of amplification of the DNA molecules flanked with long interval terminal repeats in PCR with a primer corresponding to the external moiety of the repeat. The efficiency of the developed method was estimated in obtaining an equalized cDNA library based on mRNA from the activated human T lymphocyte Jurkat cell line.

Equalizing cDNA subtraction based on selective suppression of polymerase chain reaction: cloning of Jurkat cell transcripts induced by phytohemaglutinin and phorbol 12-myristate 13-acetate.

The major drawback of subtractive cDNA libraries is that the original disproportion in concentrations of different types of transcripts is preserved. This usually makes the isolation of specific rare transcripts extremely difficult. To overcome this difficulty, we propose a strategy that introduces the equalization of concentrations (normalization) of specific transcripts during the subtractive process. This makes possible obtaining both rare and highly abundant transcripts in the resulting subtracted library. This technique has been applied for isolation of transcripts activated upon induction of Jurkat cells by phytohemaglutinin and phorbol 12-myristate 13-acetate. Six novel up-regulated sequences belonging to a low-abundance class of transcripts have been obtained.

[Analysis of effectiveness of cDNA synthesis, induced using complementary primers and primers containing a noncomplementary base matrix]. / Analiz éffektivnost' sinteza kDNK, initsiirovannogo s komplementarnykh praimerov i praimerov, soderzhashchikh nekomplementarnye matritse osnovaniia.

We have studied the efficiency of DNA synthesis catalyzed by M-MLV reverse transcriptase or Thermus aquaticus DNA polymerase for primers (4-17 nucleotides long) either completely matched or possessing a single mismatched base pair at all possible positions in the primer. It has been shown that DNA synthesis efficiency depends not only on the position of mismatched base pair but on the length and primary structure of the primer. The enzyme, template, and primer concentrations determine the relative level of mismatched DNA synthesis.

[Isolation and analysis of brain-specific sequences from cDNA libraries for various segments of the human brain]. / Vydelenie i analiz mozgospetsificheskikh posledovatel'nostei iz bibliotek kDNK raznykh otedelov mozga cheloveka.

The cDNA libraries in gt10 were constructed from total poly(A)+RNA of human forebrain cortex, cerebellar cortex and medulla oblongata. We selected the clones which gave hybridization signal with brain cDNA only, or gave no signal from these libraries. Expression pattern and structure of two brain-specific clones Hfb1 from forebrain library and Hmob3 from medulla oblongata library were analyzed in detail. Hfb1 hybridized to two different transcripts (about 5 and 2 kb) from frontal cortex, but to a single (longest) from cerebellum. Hfb1 sequence includes 958 nucleotides. Comparison of Hfb1 with the Gene Bank revealed no homology with the sequences present in the Bank. At 3'-end there is poly(A) tail of 24 bases, there is the AATCAA sequence 55 nucleotides upstream which probably serves as a polyadenylation signal. However, AATCAA directs polyadenylation in vitro with very low efficiency. We found no open reading frame in the clone and this is in agreement with the data indicating that brain-specific RNAs has extremely long 3'-untranslated regions. Hmob3 was partially sequences. We compared its primary structure with the sequences from the Gene Bank and revealed no homology. Hmob3 expresses in different parts of human brain and in sceletal muscle but does not express in other tissues.