ABSTRACT
Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism.
ABSTRACT
Background: Hypertensive states could result from constitutive activation of mineralorticoid receptor (MR) that generates salt retention and blood pressure elevation. Moreover, microsatellite regions can be associated to the regulation of the gene expression, producing subtle pathologies. Aim: To determine the influence of microsatellite marker AGAT of the mineralocorticoid receptor gene in the plasma renin activity (PRA) and serum aldosterone (SA) levels of essential hypertensives (HT). Patients and Methods: We studied 292 HT patients and 57 normotensive (NT) controls. Blood samples were collected for PRA, SA and DNA isolation. Subjects were genotyped according to the length of the tetranucleotide AGAT repeat using polymerase chain reaction and polyacrylamide gel electrophoresis. Based on the normal distribution, we considered 13 to 15 repeats as a habitual (H) length and less than 13 or more than 15 repeats, as non-habitual (non-H). Results: We detected 8 different lengths in the AGAT repeat (allele) in both groups, ranging from 9-17 repeats, where the allele 11 was not detected in either hypertensive or normotensive groups. The allelic distribution was different in both groups (c2=37.57, 4GL, p <0.001). In hypertensive patients, the H group showed higher PRA levels (median (Q1-Q3)) than the non-H group: 1.3 (0-7-3.5) vs 1.0 (0.5-2.3) ng/mL*h, p <0.05. The SA levels did not show differences between both groups, but the SA*PRA product was higher in the H group than the no-H group: 9.3 (3.0-24.6) vs 6.5 (2.5-14.6) p <0.05. In normotensive patients, no differences were observed in PRA, SA and SA*PRA between both groups. Conclusion: These results show association between the length of the AGAT repeat with the PRA in HT, suggesting a plausible role in the control of the MR gene expression, and secondarily in the regulation of blood pressure .
Subject(s)
Female , Humans , Male , Middle Aged , Aldosterone/blood , Hypertension/genetics , Microsatellite Repeats/genetics , Receptors, Mineralocorticoid/genetics , Renin/blood , Alleles , Body Mass Index , Case-Control Studies , Genetic Markers , Genotype , Hypertension/enzymology , Polymerase Chain Reaction , Receptors, Mineralocorticoid/bloodABSTRACT
BACKGROUND: Hypertensive states could result from constitutive activation of mineralorticoid receptor (MR) that generates salt retention and blood pressure elevation. Moreover, microsatellite regions can be associated to the regulation of the gene expression, producing subtle pathologies. AIM: To determine the influence of microsatellite marker AGAT of the mineralocorticoid receptor gene in the plasma renin activity (PRA) and serum aldosterone (SA) levels of essential hypertensives (HT). PATIENTS AND METHODS: We studied 292 HT patients and 57 normotensive (NT) controls. Blood samples were collected for PRA, SA and DNA isolation. Subjects were genotyped according to the length of the tetranucleotide AGAT repeat using polymerase chain reaction and polyacrylamide gel electrophoresis. Based on the normal distribution, we considered 13 to 15 repeats as a habitual (H) length and less than 13 or more than 15 repeats, as non-habitual (non-H). RESULTS: We detected 8 different lengths in the AGAT repeat (allele) in both groups, ranging from 9-17 repeats, where the allele 11 was not detected in either hypertensive or normotensive groups. The allelic distribution was different in both groups (c2 =37.57, 4GL, p <0.001). In hypertensive patients, the H group showed higher PRA levels (median (Q1-Q3)) than the non-H group: 1.3 (0-7-3.5) vs 1.0 (0.5-2.3) ng/mL*h, p <0.05. The SA levels did not show differences between both groups, but the SA*PRA product was higher in the H group than the no-H group: 9.3 (3.0-24.6) vs 6.5 (2.5-14.6) p <0.05. In normotensive patients, no differences were observed in PRA, SA and SA*PRA between both groups. CONCLUSION: These results show association between the length of the AGAT repeat with the PRA in HT, suggesting a plausible role in the control of the MR gene expression, and secondarily in the regulation of blood pressure.
