Isolation and analysis of genes mainly expressed in adult mouse heart using subtractive hybridization cDNA library.

Subtractive hybridization cDNA library (SHL) is one of the powerful approaches for isolating differentially expressed genes. Using this technique between mouse heart and skeletal muscle (skm) tissues, we aimed to construct a cDNA-library that was specific to heart tissue and to identify the potential candidate genes that might be responsible for the development of cardiac diseases or related pathophysiological conditions. In the first step of the study, we created a cDNA-library between mouse heart and skm tissues. The homologies of the randomly selected 215 clones were analyzed and then classified by function. A total of 146 genes were analyzed for their expression profiles in the heart and skm tissues in published mouse microarray dataset. In the second step, we analyzed the expression patterns of the selected genes by Northern blot and RNA in situ hybridization (RISH). In Northern blot analyses, the expression levels of Myl3, Myl2, Mfn2, Dcn, Pdlim4, mt-Co3, mt-Co1, Atpase6 and Tsc22d1 genes were higher in heart than skm. For first time with this study, expression patterns of Pdlim4 and Tsc22d1 genes in mouse heart and skm were shown by RISH. In the last step, 43 genes in this library were identified to have relationships mostly with cardiac diseases and/or related phenotypes. This is the first study reporting differentially expressed genes in healthy mouse heart using SHL technique. This study confirms our hypothesis that tissue-specific genes are most likely to have a disease association, if they possess mutations.

Niemann-Pick type C fibroblasts have a distinct microRNA profile related to lipid metabolism and certain cellular components.

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression at post-transcriptional level. Dysregulation of miRNA expression may lead to severe pathophysiologies in human cells. Niemann-Pick type C (NPC) disease is a complex lipid storage disease characterized by late endosomal-lysosomal accumulation of multiple lipid molecules. Our aim was to characterize the miRNA profile in NPC fibroblasts as they may play an active role in the NPC disease associated changes in the cellular physiology. To investigate the miRNA expression, total RNAs were isolated from cultured human NPC fibroblasts and healthy fibroblasts and then, TaqMan Low-Density Array system containing 365 mature human miRNAs was used. Expression differences between the healthy and NPC cells were detected according to the relative quantification values. Target genes were predicted by using three different algorithms and classified regarding NPC related biological processes and cellular components. We found that three miRNAs, miR-196a, miR-196b and miR-296 were up-regulated (>3.5-fold increase, p<0.05) whereas 38 miRNAs were significantly down-regulated in NPC cells (>3.5-fold decrease, p<0.05). Among these non-coding RNAs, miR-98 was the most down-regulated (-33.3-fold) miRNA and miR-143, the lipid biosynthesis associated miRNA, had a 20-fold decreased expression in the NPC cells. Additionally, gene ontology analyses of the target genes suggested a distinct role for each miRNA. Our results show that NPC fibroblasts have an altered miRNA expression profile and certain miRNAs have importance in disease pathogenesis as well as the therapeutic capacity to correct lipid related pathophysiologies in the NPC cells.

ADAM-9, ADAM-15, and ADAM-17 are upregulated in macrophages in advanced human atherosclerotic plaques in aorta and carotid and femoral arteries--Tampere vascular study.

BACKGROUND AND AIMS: The expression of disintegrin and metalloprotease ADAM-9, ADAM-15, and ADAM-17 has been associated with cell-cell, cell-platelet, and cell-matrix interactions and inflammation. They are possibly implicated in the pathophysiology of atherosclerosis. METHODS AND RESULTS: Whole-genome expression array and quantitative real-time polymerase chain reaction (PCR) analysis confirmed that ADAM-9, ADAM-15, and ADAM-17 are upregulated in advanced human atherosclerotic lesions in samples from carotid, aortic, and femoral territories compared to samples from internal thoracic artery (ITA) free of atherosclerotic plaques. Western analysis indicated that the majority of these ADAMs were in the catalytically active form. ADAM-9, ADAM-15, and ADAM-17-expressing cells were shown to co-localize with CD68-positive cells of monocytic origin in the atherosclerotic plaques using immunohistochemistry and double-staining immunofluorescence analysis. Co-localization was demonstrated in all vascular territories. In the carotid territory, cells expressing the ADAMs co-distributed also with smooth muscle cells and, in femoral territory, with CD31-positive endothelial cells, indicating that the ADAM expression pattern depends on vascular bed territory. CONCLUSIONS: Present findings provide strong evidence for the involvement of catalytically active ADAM-9, ADAM-15, and ADAM-17 in advanced atherosclerosis, most notably associated with cells of monocytic origin.

CETP TaqIB polymorphism in Turkish adults: association with dyslipidemia and metabolic syndrome.

OBJECTIVE: In this study, our aim was to investigate the association of cholesterol ester transfer protein (CETP) TaqIB polymorphism with the likelihood of metabolic syndrome (MetS). METHODS: Study was designed as a cross-sectional analysis of the Turkish Adult Risk Factor follow-up study. Randomly selected sample of 1585 persons were included in the analyses. Genomic DNAs were isolated and the genotyping was performed using TaqMan system. ANOVA, Chi-square, univariate analyses and logistic regression models were used to investigate the association of genotypes with clinical and biochemical measurements. RESULTS: The frequencies of the B1B1, B1B2 and the B2B2 genotypes were 33.3%, 46.4% and 20.3%, respectively. The B2B2 genotype was associated with elevated high-density lipoprotein -cholesterol (HDL-C) levels (p<0.0001). After adjusting for sex and age B2B2 individuals had 15.9% higher HDL-C levels than B1B1 individuals. Furthermore, the likelihood of dyslipidemia was lower in the presence of the B2B2 genotype (30.9% non-dyslipidemic vs. 69.1% dyslipidemic, p=0.001) when compared to the other genotypes. Moreover, in a logistic regression model comprising age and environmental factors, B1 allele carriers showed higher odds ratios of 1.49 (OR=1.49, 95% CI; 1.03-2.14, p=0.032) for MetS only in females. CONCLUSIONS: These results suggest that B1 allele is associated with MetS in adult females. However, TaqIB polymorphism appears not associated with the components of MetS other than atherogenic dyslipidemia in adult Turkish population.

Effect of leukaemia inhibitory factor on long-term sperm motility and survival.

Leukaemia inhibitory factor (LIF) is expressed at high constitutive levels in the human Fallopian tubal epithelium. In this study, the effect of human recombinant LIF on sperm motility and survival in vitro was investigated. Human spermatozoa were incubated in sperm washing medium that contained various concentrations of LIF at 37 degrees C and under 5% of CO(2) in air for up to 48 h. Sperm motion characteristics were measured using a sperm motility analyser. Sperm survival was determined by the hypo-osmotic swelling test. The effect of LIF on sperm motility was concentration-dependent and maximal effect was observed at a concentration of 5 ng/ml. Sperm motility was significantly higher after 24 h exposure to LIF compared with control (P < 0.001). Sperm survival was also prolonged in a concentration-dependent manner. LIF significantly enhanced sperm survival at higher concentrations (10 ng/ml) and the result was significant after 48 h exposure (P < 0.05). LIF increased long-term sperm motility and survival in vitro.