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PubMed2006

Experimental study of recombinant eukaryotic expression vector of human eNOS in ECV304.

BACKGROUND AND PURPOSE: Gene transfer with recombinant non-viral vectors encoding vasodilator proteins, such as endothelial nitric oxide synthase (eNOS), maybe a preferential choice in gene therapy of artery restenosis following angioplasty, stent or anastomosis. However, the transfection rate of a non-viral vector, the harmful effects of eNOS transfection on endothelial cells (EC) and the control release of nitric oxide (NO) have been controversial. We designed the eukaryotic expression vector pcDNA3.1-eNOS to study the regulated expression of eNOS (in the presence of various chemical agents) and to evaluate the exogenous NO effect on EC proliferation in vitro. METHODS: The full-length human eNOS cDNA was inserted into the EcoRI cloning site of the pcDNA3.1 expression plasmid and the eNOS direction was tested by restriction enzyme digestion with XhoI to construct recombinant pcDNA3.1-eNOS. After co-transfection of pcDNA3.1-eNOS with pcDNA3.0-EGFP mediated by cationic liposomes into Human umbilical vein endothelial cells (ECV304), the transfection rate and the effect on ECV304 proliferation were calculated by fluorescence microscopy and flowcytometry. eNOS mRNA and protein were detected by reverse transcription-PCR (RT-PCR) and immunofluorescence, respectively. The eNOS activity, NO release and changes of the relevant cells growth curve were assessed after treating the transfected cells with four independent factors including ie Ca2+, L-arginine (L-Arg), Ethylene Diamine Tetraacetic Acid (EDTA) and N-nitro-L-arginine methylester (L-NAME). In addition, we examined the non-transfected cells status by isolated sodium nitroprusside (SNP) treatment. RESULT: eNOS cDNA was inserted into pcDNA3.1 in the proper direction. RT-PCR analysis showed that pcDNA3.1-eNOS transfected cells could express eNOS mRNA. The rate of eNOS transfection was 39.6 +/- 3.4%. Immunofluorescence staining displayed that subcellular localisation of eNOS was most prominent in plasma membrane and perinuclear regions of the cell. The eNOS activity of eNOS transfected cells had not increased significantly, whereas, in the presence of Ca2+, L-Arg, EDTA, and L-NAME, the eNOS activity was 96.98 +/- 13.47, 32.57 +/- 6.39, 11.63 +/- 3.02, 15.56 L 7.34 U/ml respectively and the NO level was 55.34 +/- 11.19, 9.43 +/- 4.51, 2.63 +/- 1.41, 3.73 +/- 1.65 mmol/L, respectively. Meanwhile, the growth curves of EC shifted. SNP also had obvious growth-inhibiting effects on the cells. Together, the ECV304 growth curve went downward in a NO concentration-dependent manner. CONCLUSION: Eukaryotic expression vector pcDNA3.1-eNOS was constructed successfully with the ability to express human eNOS mRNA and protein in EC effectively. The activity of eNOS in EC could be regulated by certain exogenous factors. Ca2+ was an important factor promoting NO release and excess NO had a cytotoxic effect on EC in vitro. Controlled release of NO in vivo and polygenic measurements might be considered in more clinical gene therapy studies.

Cell Line↗
PubMed1997

Alterations in follicular maturation associated with within-breed variation in ovulation rate in Chios sheep.

Within-breed comparisons may be helpful to identify, in a given genetic background (Chios sheep), ovarian strategies and control mechanisms associated with altered ovulation rate. High and low ewes were identified from two large groups (n = 27 and n = 33 in Exp. 1 and Exp. 2 respectively) of Chios ewes submitted to repeated laparoscopies (24 times in Exp. 1 and six times in Exp. 2). High ovulatory ewes (n = 6 and n = 7 in Exp. 1 and Exp. 2 respectively) had mean ovulation rates of 4.3 (Exp. 1) and 4.2 (Exp. 2) while low ovulatory ewes (n = 6 and n = 7 in Exp. 1 and Exp. 2 respectively) had mean ovulation rates of 2.5 (Exp. 1) and 1.9 (Exp. 2). In Exp. 1, follicular function was compared in these two groups of ewes using follicles obtained at 30 h following luteolysis in the same ewes before and after unilateral ovariectomy (ULO). In Exp. 2, circulating follicle stimulating hormone (FSH) concentrations were measured from the end of the luteal phase up to the preovulatory surge in high and low ewes. Thereafter, to demonstrate a causal link between high FSH and high ovulation rate, pituitary downregulation was achieved by a 17-day gonadotrophin releasing hormone (GnRH) agonist treatment and the ovarian response to similar amounts of exogenous gonadotrophins compared between high and low ewes. Numbers of oestrogenic (in vitro oestradiol > 250 pg ml-1 h-1) follicles on the first ovary removed (Exp. 1) were 2.16 +/- 0.5 vs. 1.33 +/- 0.17 in high and low ewes (P = 0.1). Following ULO, these numbers were 3.33 +/- 0.33 and 2.5 +/- 0.18 (P < 0.05 between high and low ewes). There were no significant differences between the first and second ovaries for any of the parameters studied. Follicles from high ovulatory ewes (n = 33) differed from those of low ovulatory ewes (n = 23) by a smaller size (P < 0.01), a reduced number of granulosa cells (P < 0.01) together with decreased oestradiol (P < 0.05) and testosterone (P < 0.01) production in vitro. However, steroid production per cell (oestradiol per granulosa cell, testosterone per thecal cell) was similar in the two groups of sheep. FSH concentrations (Exp. 2) in high ovulatory ewes were significantly higher than those of low ovulatory ewes during the late luteal phase, and the decrease in FSH concentrations was steeper (1.4 ng) during the early follicular phase for high ovulatory ewes than low ovulatory ewes. Chemical hypophysectomy achieved by a 17-day treatment with a GnRH agonist demonstrated that these high FSH concentrations may be important to generate the high ovulation rate of the 'high' ewes as ovulation rate of high and low ewes was similar following chemical hypophysectomy followed by administration of similar amounts of exogenous gonadotropins to both groups of ewes. It is concluded that, despite different genetic control of their high ovulation rate (Chios-polygenic; Booroola-major gene), alterations in follicular function and its control are very similar in high ovulatory Chios and in FecB carriers.

Analysis of Variance↗
PubMed2002

[Genetic factors in myocardial infarction--Results from a candidate gene and a genome-wide approach between beta blockers].

BACKGROUND: Coronary artery disease and myocardial infarction are the most frequent causes of death in the Western societies. Even nowadays, every second myocardial infarction is lethal and hits the patients unexpectedly without previous signs or symptoms. In order to install preventive measures most efficiently, it is necessary to have a detailed knowledge on the pathophysiology of the disease. The identification of patients who are at high risk for suffering from myocardial infarction can be done with epidemiological methods, such as the determination of "traditional" risk factors, like arterial hypertension, hypercholesterolemia, diabetes mellitus or smoking), or eventually in the future using molecular genetic testing. This is of great importance especially for asymptomatic siblings and children from myocardial infarction patients. POLYMORPHISMS: Although traditional risk factors occur frequently in families, they explain only in part the familial accumulation of coronary artery disease. Furthermore, stron genetic effects on the development of coronary artery disease and myocardial infarction have been demonstrated in several studies. These genetic effects can be examined by 1. a candidate gene approach, or 2. a systematic screening of the whole genome. In the first step, several polymorphisms (sequence variations) wee examined in several candidate genes in which a significant influence on a cardiovascular risk factor or intermediate phenotype (such as atherogeneic lipid profile or arterial hypertension) has been shown in the literature. We thus examined in a large population of patients with myocardial infarction and a sample of the general population the effects of the HindIII polymorphism in the lipoproteinlipase gene, of the -344T/C promoter polymorphism in the aldosterone synthase gene and of the 825C/T polymorphism in the gene of the beta3 subunit of the G protein gene (GNB3). In the general population, we could show an association with unfavorable lipid levels in men and in postmenopausal (but not premenopausal) women for the H2H2 genotype of the HindIII lipoproteinlipase polymorphism. However, the theoretical increase in risk for this genotype is not large enough to demonstrate a significant association with myocardial infarction in the population examined. With the promoter polymorphism in the aldosterone synthase gene, anthropometrical and echocardiographical data did not suggest that the polymorphism is a risk factor for myocardial infarction nor for left ventricular remodeling after myocardial infarction, which was observed in earlier studies. Furthermore, we could show an association with arterial hypertension in our general population sample with the polymorphism in the GNB3 gene. However, no association could be demonstrated for this polymorphism with myocardial infarction. AFFECTED SIB-PAIR APPROACH: In a systematic screening of the genome for genes that are relevant in the pathogenesis of coronary artery disease or myocardial infarction, an affected sib-pair approach was followed. 1,261 families were identified in which at least two brothers or sisters were affected with myocardial infarction or severe coronary artery disease, such as percutaneous coronary intervention or coronary after bypass grafting. In a subpopulation of 513 families and 1,407 individuals, we performed a total genome screening. The analyses using the variance component method and the SOLAR program revealed a susceptibility locus for myocardial infarction of chromosome 14q32 with a lod score of 3.89 (genome-wide p < 0.05). This locus comprises a region of about seven centi-Morgan and contains approximately 150 genes. Furthermore, a comprehensive analysis including the cardiovascular risk factors showed that 1. this myocardial infarction locus is unique and does not overlap with chromosomal loci for well-established risk factors, 2. cardiovascular risk factors, such as Lp(a), diabetes mellitus, serum lipids, or arterial hypertension have strong genetic components. CONCLUSION: These findings do not exclude a role of cardiovascular s do not exclude a role of cardiovascular risk factors or candidate genes in the pathogenesis of myocardial infarction, but rather demonstrate that risk factors may act as surrogates of specific underlying disease mechanisms. It is thus necessary to perform a comprehensive analysis of complex polygenic diseases, such as myocardial infarction, including both, established cardiovascular risk factors and genomic data.

Adrenergic beta-Antagonists↗
PubMed2004

The sudden infant death syndrome gene: does it exist?

BACKGROUND: Sudden infant death syndrome (SIDS) is in a difficult position between the legal and medical systems. In the United Kingdom, prosecutors have for years applied the simple rule that 1 unexpected death in a family is a tragedy, 2 are suspicious, and 3 are murder. However, it seems that the pendulum has now swung to the opposite extreme; mutations or polymorphisms with unclear biological significance are accepted in court as possible causes of death. This development makes research on genetic predisposing factors for SIDS increasingly important, from the standpoint of the legal protection of infants. The genetic component of sudden infant death can be divided into 2 categories, ie (1) mutations that give rise to genetic disorders that constitute the cause of death by themselves and (2) polymorphisms that might predispose infants to death in critical situations. Distinguishing between these 2 categories is essential, and cases in which a mutation causing a lethal genetic disorder is identified should be diagnosed not as SIDS but as explained death. GENETIC ALTERATIONS THAT MAY CAUSE SUDDEN INFANT DEATH: Deficiencies in fatty acid metabolism have been extensively studied in cases of SIDS, and by far the most well-investigated mutation is the A985G mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene, which is the most prevalent mutation causing MCAD deficiency. However, <1% of sudden infant death cases investigated have this mutation, and findings of biochemical profiles seen in specific fatty acid oxidation disorders in a number of such cases emphasize the importance of investigating fatty acid oxidation disorders other than MCAD deficiency. Severe acute hypoglycemia may cause sudden death among infants, but only rare novel polymorphisms have been found when key proteins involved in the regulation of blood glucose levels are investigated in cases of SIDS. The long QT syndrome (LQTS) is another inherited condition proposed as the cause of death in some cases of sudden infant death. The LQTS is caused by mutations in genes encoding cardiac ion channels, and mutations in the genes KVLQT1 and SCNA5 have been identified in cases initially diagnosed as SIDS, in addition to several polymorphisms in these 2 genes and in the HERG gene. In addition, genetic risk factors for thrombosis were investigated in a small number of SIDS cases; the study concluded that venous thrombosis is not a major cause of sudden infant death. GENE POLYMORPHISMS THAT MAY PREDISPOSE INFANTS TO SUDDEN INFANT DEATH UNDER CERTAIN CIRCUMSTANCES: Many SIDS victims have an activated immune system, which may indicate that they are vulnerable to simple infections. One reason for such vulnerability may be partial deletions of the complement component 4 gene. In cases of SIDS, an association between slight infections before death and partial deletions of the complement component 4 gene has been identified, which may indicate that this combination represents increased risk of sudden infant death. There have been a few studies investigating HLA-DR genotypes and SIDS, but no association has been demonstrated. The most common polymorphisms in the interleukin-10 (IL-10) gene promoter have been investigated in SIDS cases, and the ATA/ATA genotype has been reported to be associated with both SIDS and infectious death. The findings may indicate that, in a given situation, an infant with an unfavorable IL-10 genotype may exhibit aberrant IL-10 production, and they confirm the assumption that genes involved in the immune system are of importance with respect to sudden unexpected infant death. Another gene that has been investigated is the serotonin transporter gene, and an association between the long alleles of this gene and SIDS has been demonstrated. Serotonin influences a broad range of physiologic systems, as well as the interactions between the immune and nervous systems, and findings of decreased serotonergic binding in parts of the brainstem, together with the findings in the serotonin transporter gene, may indicate that serotonin plays a regulatory role in SIDS. It has also been speculated that inadequate thermal regulation is involved in SIDS, but investigations of genes encoding heat-shock proteins and genes encoding proteins involved in lipolysis from brown adipose tissue have not found evidence of linkages between common polymorphisms in these genes and SIDS. A number of human diseases are attributable to mutations in mitochondrial DNA (mtDNA), and there are several reasons to think that mtDNA mutations also are involved in SIDS. Both a higher substitution frequency and a different substitution pattern in the HVR-I region of mtDNA have been reported in SIDS cases, compared with control cases. A number of coding region mtDNA mutations have also been reported, but many are found only in 1 or a few SIDS cases, and, to date, no predominant mtDNA mutation has been found to be associated with SIDS. CONCLUSIONS: All mutations giving rise to metabolic disorders known to be associated with life-threatening events are possible candidates for genes involved in cases of sudden infant death, either as a cause of death or as a predisposing factor. It is necessary to distinguish between lethal mutations leading to diseases such as MCAD and LQTS, and polymorphisms (for instance, in the IL-10 gene and mtDNA) that are normal gene variants but might be suboptimal in critical situations and thus predispose infants to sudden infant death. It is unlikely that one mutation or polymorphism is the predisposing factor in all SIDS cases. However, it is likely that there are "SIDS genes" operating as a polygenic inheritance predisposing infants to sudden infant death, in combination with environmental risk factors. For genetically predisposed infants, a combination of, for instance, a slight infection, a prone sleeping position, and a warm environment may trigger a vicious circle with a death mechanism, including hyperthermia, irregular breathing, hypoxemia, and defective autoresuscitation, eventually leading to severe hypoxia, coma, and death.

Acyl-CoA Dehydrogenase↗