Expression pattern in a modified equalized kidney cDNA library of hypertensive rat.

BACKGROUND: Genes with important functions and rarely expressed would probably more easily be cloned from a modified equalized kidney cDNA library for further investigation. METHODS: A kidney cDNA library of a spontaneously hypertensive rat was synthesized by a modified equalization method. Inserts of random clones were amplified by PCR and sequenced. Sequences were compared against a nonredundant database in GenBank. The cDNA profile was compared with an expression profile of a mouse renal proximal tubule cDNA library. Seven clones were analyzed by Northern blot analysis. The cDNA ends of two novel genes were amplified by PCR, sequenced and analyzed. RESULTS: 336 cDNA clones were analyzed and grouped into 323 species of transcript with 77 species similar to previously reported genes. Northern blot analysis identified one kidney-specific, one rarely expressed and lung-specific, and another relatively testis-specific gene. Two novel genes were cloned. One was 4.1 kb in length and encoded a 390-amino acid zinc-finger protein. Another was 2.5 kb and encoded a 474-amino acid protein of unknown function. Compared with the expression profile of a mouse renal proximal tubule cDNA library, this kidney library had a lower proportion of ribosomal genes and had a greater proportion of genes for signal transduction and DNA or RNA binding. CONCLUSIONS: Rare or novel genes could be more easily isolated from this library for molecular study of hypertension and renal pathophysiology.

Cloning a novel metallophosphoesterase gene from a kidney cDNA library of hypertensive rat.

BACKGROUND AND PURPOSE: Genetic and environmental factors may contribute to the pathogenesis of essential hypertension. To facilitate genetic studies of hypertension and renal disorders, we sought to clone novel genes from a modified, equalized kidney (MEK) cDNA library of a spontaneously hypertensive rat (SHR). METHODS: A kidney cDNA library of an SHR was synthesized using the modified equalization method. Inserts of 350 random clones were amplified by polymerase chain reaction (PCR) and sequenced, of which 246 were presumably unknown after being compared against a nonredundant database in the GenBank. The cDNA ends of clone 38S were obtained by rapid amplification of cDNA ends, sequenced, and then analyzed with Translate, Prosite, Profile, SignalP, and TMpred programs. RESULTS: The full-length cDNA was 938 bp, and translated into a 182-amino acid protein. The deduced protein had a metallophosphoesterase domain, a signal peptide at its amino end, a protein kinase C phosphorylation site, and a transmembrane domain. Northern blot analysis revealed that this gene was expressed in the heart, brain, spleen, lungs, liver, skeletal muscles, kidneys and testes of Sprague-Dawley rats. A putative protein of Arabidopsis thaliana shares 62% homology with protein 38S, but the two proteins differ in terms of function and structure. CONCLUSIONS: Our results support that protein 38S is a novel membrane metallophosphoesterase, although its function in the kidneys remains to be elucidated. This study also demonstrates the feasibility of using PCR to clone novel genes from our MEK cDNA library.

Effect of acupuncture at nei-kuan on left ventricular function in patients with coronary artery disease.

Effect of acupuncture at Nei-Kuan (EH-6) on left ventricular ejection fraction (LVEF) was examined in 22 patients with angiographically proved coronary artery disease (CAD) and 22 normal subjects. Serial equilibrium radionuclide angiography was done to measure LVEF at 4 different times (at baseline, at 1 to 15 minutes, and 16 to 30 minutes during acupuncture, and immediately after acupuncture). One week later, each patient had an identical imaging protocol with acupuncture performed at a dummy point. Our results showed that in normal subjects, the mean values of LVEF did not change significantly during or after acupuncture. In contrast, in patients with CAD, the mean values of LVEF in the initial 15 minutes of acupuncture significantly increased from baseline (42.5 +/- 15.6% vs. 40.6 +/- 15.4%, p < 0.05). The increase persisted through the next 15 minutes of acupuncture and 15 minutes after acupuncture, but became insignificant at one week. Thus, acupuncture at Nei-Kuan can temporarily improve LV function in patients with CAD.

Association of the renin gene polymorphism with essential hypertension in a Chinese population.

To study the association of renin gene polymorphism with essential hypertension in the Chinese population, 86 hypertensive and 107 normotensive subjects were enrolled from an epidemiologic survey. Leukocyte DNA was extracted and digested with Hind III and Bgl I restriction enzymes. Southern hybridization was done with digoxigenin-incorporated renin gene probes generated by polymerase chain reaction. The restriction fragments were detected by anti-digoxigenin antibody and enzyme methods. Two Hind III polymorphysms of the renin gene (8.7 kb and 6.2 kb) were identified. The allele frequences were 129(75%) and 43(25%), respectively, in hypertensives; they were 139(65%) and 75(35%), respectively, in normotensives (chi2 = 4.074, p = 0.044). The genotypes of 8.7/8.7,8.7/6.2 and 6.2/6.2 were significantly different between hypertensives and normotensives, being 45(52%), 39(45%), 2(3%) and 48(45%), 43(40%), and 16(15%), respectively (chi2 = 9.002, p = 0.11). The Bgl I polymorphism did not show a difference between hypertensives and normotensives. Thus, we conclude that the renin gene Hind III polymorphysm is associated with hypertension in this Chinese population.

Molecular variant M235T of the angiotensinogen gene is associated with essential hypertension in Taiwanese.

OBJECTIVE: To examine the association of the molecular variants of the angiotensinogen (AGT) gene with essential hypertension in Taiwanese. METHODS: We conducted a case-control study concerning 151 subjects, 102 hypertensives and 49 normotensives. We created a rapid mini-sequencing method based on dye-terminator cycle sequencing to simultaneously detect the M235T and T174M variants of the AGT gene for each subject. RESULTS: The genotype and allele distribution of the M235T variant differed significantly in hypertensives and normotensives (chi 2 = 11.106, P = 0.004 and chi 2 = 6.453, P = 0.011, respectively), whereas those of the T174M variant did not differ (chi 2 = 0.004, P = 0.998 and chi 2 = 0.032, P = 0.858, respectively). The odds ratio for hypertension was 3.64 (95% confidence interval 1.56-8.49) for subjects with the C/C genotype of the M235T variant compared with other genotypes of 2.87 (95% confidence interval 1.76-4.68) for those carrying allele C versus those carrying allele T. CONCLUSION: The molecular variant M235T, but not T174M, of the AGT gene is associated significantly with essential hypertension in this Taiwanese population. The genotype C/C or allele C is a risk factor for hypertension. The underlying mechanism of this association needs to be elucidated further.

Lack of association between angiotensin-converting enzyme gene polymorphism and coronary heart disease in a Chinese population.

Insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been postulated as a risk factor for coronary heart disease. We conducted a case-control study of 271 Chinese, including 114 subjects with coronary artery disease (CAD), 42 with non-CAD and 115 apparently normal controls to examine the association of I/D polymorphism and CAD. The genotypes were identified by polymerase chain reaction and the plasma ACE activity was assayed by spectrophotometry. The allele and genotype frequencies were not different among the CAD, non-CAD and apparently normal groups (p = 0.42 and 0.63). Plasma ACE activity was not different among the three groups (p = 0.32). The D-allele and DD genotype were not more prevalent in subjects with low risk CAD (p = 0.07 and 0.16) and subjects with myocardial infarction (p = 0.79 and p = 0.35). No association was found between I/D polymorphism and severity of CAD (p = 0.42 and 0.70). In conclusion, the deletion polymorphism of the ACE gene may not be an independent risk factor in the development of CAD or myocardial infarction in this Chinese population. The unique or synergistic effect of other genes needs further study.

Lack of association of the angiotensin converting enzyme polymorphism with essential hypertension in a Chinese population.

To examine the association between insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene and essential hypertension in a Chinese population, a case-control study was conducted using 157 hypertensive and 115 normotensive subjects. The I/D polymorphism of the ACE gene was identified by polymerase chain reaction. Plasma ACE activity was determined using spectrophotometry. The difference of allele frequencies between normotensives and hypertensives was statistically significant (chi 2 = 4.467, P = .035), while the genotype distribution was not different between normotensive and hypertensive subjects (chi 2 = 3.954, P = .138). Plasma ACE activity was highest in the DD genotype, followed by the ID genotype, and the lowest in the II genotype (P = .0001 in normotensives and P = .163 in hypertensives, respectively). Thus, we conclude that the ACE gene polymorphism is not associated with essential hypertension in this Chinese population, but plasma ACE activity is genetically determined in the normotensive Chinese.

Age- and gender-dependent association of the angiotensin-converting enzyme gene with essential hypertension in a Chinese population.

A case-control study was carried out on 272 Chinese subjects over 40 years of age, including 157 hypertensives and 115 normotensives, to examine the association between angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and blood pressure (BP) status. The I/D polymorphism of the ACE gene was identified by polymerase chain reaction. As a whole group, the difference of allele frequencies between normotensives and hypertensives was statistically significant (chi 2 = 4.46, P = 0.03; D/I odds = 1.46), while there was no difference in the genotype distribution (chi 2 = 3.95, P = 0.13). In a subgroup with elderly hypertension (age > 65), the frequencies of D-allele and DD genotype significantly increased (chi 2 = 4.43, P = 0.03 and chi 2 = 4.03, P = 0.08, respectively; D/I odds = 2.28). The association and relative risk increased further in the male gender (chi 2 = 6.65, P = 0.01 and chi 2 = 7.51, P = 0.02 respectively; D/I odds = 4.57 and DD/II odds = 12.00 respectively). The D-allele increased with age in the hypertensives, while the I-allele increased with age in normotensives. Thus, we conclude that the deletion polymorphism of the ACE gene is significantly associated with male elderly hypertension, at least in this Chinese population. This observation, if proved in a larger population, may have some implications for the prevention and treatment strategy for elderly hypertension.

Effects of linolenic acid on the canine heart.

Disturbances in lipid metabolism have been observed during the early phase of acute myocardial ischemia. Accumulation of fatty acids in and around the ischemic cardiac cells has been implicated to play a role in both contractile and electrophysiological abnormalities. Linolenic acid is an essential fatty acid and constitutes the phospholipid moiety of the cell membrane. The purpose of this work was to study the effects of linolenic acid on the heart using canine preparations. A direct left atrial injection was used as the route of administration because intravenous injections of linolenic acid inevitably cause pulmonary edema. A surface lead electrocardiogram (ECG), an epicardial electrogram, femoral arterial pressure, left ventricular pressure and its time derivative (dp/dt) were recorded before and after drug administration. Various dosages of linolenic acid (1 mg, 5 mg, 10 mg, 20 mg, 30 mg and 60 mg/kg) and a control buffer solution were tested. The results showed that linolenic acid has a potent dose-dependent bradycardic and myocardial depression effect starting from a dose of 5 mg/kg (delta HR = -20.1 +/- 4.0 bpm, delta dp/dt = 364.3 +/- 66.0 mmHg, sec-1, p less than 0.01 vs. control). At a high dose of 30 mg/kg, linolenic acid induced premature ventricular complexes. Furthermore, ventricular tachycardia was observed in 5 of the 8 dogs (62.5%) receiving the high dose of 60 mg/kg. We conclude that linolenic acid has profound effects on the canine heart; at a low dose, it causes bradycardia and myocardial depression, while at a high dose, it also produces ventricular irritability.

Time course of the ventricular arrhythmias following coronary reperfusion in dogs.

The time course of ventricular arrhythmia following reperfusion was investigated in 12 dogs undergoing a 2-hour coronary artery occlusion followed by reperfusion. At days 1, 3 and 7 following coronary reperfusion, the cardiac rhythm was monitored with a 24-hour Holter electrocardiographic recorder. Six dogs with sham operations were also studied with Holter monitoring at the same time periods as a control group. This showed that, following coronary reperfusion, all 12 dogs (group A) developed frequent premature ventricular complexes (PVCs) at day 1, with a total count (beats per hour, bph) of 1,806 +/- 390 (mean +/- SD). The PVC count decreased to 298 +/- 96 at day 3 (p less than 0.001) and 0.4 +/- 0.1 at day 7 (p less than 0.001). The incidence of spontaneous sustained ventricular tachycardia (VT) at day 1 was 100%; 1 of them deteriorated to ventricular fibrillation (VF). At day 3, 9 of the remaining 11 dogs (82%) had recurrent VTs (p greater than 0.05 vs day 1) which were of shorter duration and a slower rate. One of the 11 dogs died at day 5 and the remaining 10 dogs survived to day 7, with all the VTs subsiding (p less than 0.001 vs day 1 or 3). In the control group, only isolated PVCs were observed at day 1 (7 +/- 3 bph, p less than 0.001 vs group A) and no spontaneous VT or VF was noted. We conclude that spontaneous ventricular arrhythmias were observed in all dogs with coronary reperfusion following a 2-hour coronary artery occlusion and the arrhythmias could subside in 1 week of those survived. Only rarely did VT deteriorate to VF.