Different frequencies of angiotensin-converting enzyme genotypes in older hypertensive individuals.

The frequency of the D allele of an insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene has been reported to be elevated in myocardial infarction and other patients. We therefore hypothesized that death rate of DD individuals should be increased in the population as a whole and this should be evident as a decrease in DD frequency with age. This hypothesis was tested in 118 Caucasian subjects who were already at high risk of cardiovascular events by having severe, early onset, familial hypertension (HT). A group of 196 age-, sex- and body mass index-matched normotensives (NTs) was used as a control. In the NT group II, ID, and DD genotype frequencies were similar for different age groups. DD frequency was 0.42 in NTs, but in HTs was 0.28, 0.26, and 0.10 for the age groups < 50, 50-59, and > or = 60 yr, respectively. Corresponding D allele frequencies were 0.52, 0.46, and 0.40 in the respective age groups of HTs, compared with 0.61 in NTs (by chi 2-analysis, P = 0.1, 0.047, and 0.0006, respectively). In HTs aged > or = 60, DD frequency was only 14% of expected. Plasma ACE activity tracked similarly with I/D genotype in HTs (P = 0.027; n = 35) as in NTs (P = 0.0001; n = 94) and Michaelis constant was identical for DD and II. Neither blood pressure, body mass index, nor sex bore any relationship with I/D genotype. In conclusion, in a group of severely HT patients not selected for cardiac pathology, there appeared to be a marked, selective decrease, in subgroups of increasing age, in frequency of the ACE DD genotype. One possibility suggested by this data might be that DD increases risk of premature death, at least in HTs who have two HT parents.

Translation mapping with the flavivirus Kunjin: gene order and anomalies in translation of Ns5.

Kunjin (KUN) virus-infected cells were synchronized in translation by reversal of hypertonic inhibition; cells were then pulse-labelled with [35S]methionine. Electrophoretic analyses defined the sequence of incorporation of label into all the known KUN gene products shown previously to be unique and unrelated. GP44 or NS1 was inadequately labelled for analysis and was assumed to be translated sequentially after E in the polyprotein sequence. The relationship of the KUN gene order obtained by translation mapping to that proposed by Rice et al. Science (1985) 229, 726-733 based on the nucleotide sequence of yellow fever virus is as follows: KUN: 5'-C.GP20.E.GP44.P19.P10.P71. (?).P21.P98-3' YF: 5'-C.prM.E.NS1.ns2a.ns2b.NS3.ns4a.ns4b. NS5-3'. These results eliminate the ambiguities in identities of the previously hypothetical ns2a, ns2b and ns4b. Although ns4a was not positively identified, a labelled protein of Mr 12,000 to 14,000 was observed in one experiment and it mapped in the appropriate position for ns4a. Variation occurred in translation of NS5 when the hypertonic treatment of 40 min at 37 degrees C was reduced in time or in temperature.

Effect of angiotensin-converting enzyme genotype on renin-angiotensin components in hypertensives.

Plasma angiotensin-converting enzyme (ACE) tracks with the deletion allele of an insertion/deletion (I/D) polymorphism of the ACE gene. The aim of the present study was to determine plasma renin and angiotensinogen for each ACE genotype in 54 hypertensive and 96 normotensive subjects. Plasma renin was reduced by 52 +/- 15 S.E.% in DD and by 24 +/- 12% in ID hypertensives when compared to II hypertensives (P < 0.05 by one-way ANOVA), but in the normotensive subjects, renin values were similar for each genotype. Plasma angiotensinogen did not differ across ACE genotypes, but was elevated by 25 +/- 3 S.E.% in hypertensives and showed a significant correlation with blood pressure. In conclusion, although renin does not differ significantly between ACE genotypes of normotensives, a reciprocal relationship may exist between plasma renin and plasma ACE in hypertensives.

Successful competition in translation by the flavivirus Kunjin with poliovirus during co-infections in Vero cells.

Infection with poliovirus effectively inhibited the translation of Vero cell messengers (carrying type 1 or type 2 caps at the 5' end) and of influenza virus messengers (type 1 caps) in co-infections. In contrast, Kunjin virus RNA (type 1 caps) and Semliki Forest virus RNA (a togavirus, with type 0 caps) continued to be translated in the presence of co-infecting poliovirus. Translation of Kunjin virus RNA was also unaffected during co-infections with either influenza virus or Semliki Forest virus. Guanidine treatment effectively blocked poliovirus replication only, but the inhibitory effect on translation of cell messengers and influenza virus messengers was still observed, indicating that this effect was not caused by competition in translation with poliovirus messengers. It was therefore concluded that the observed inhibition was most likely caused by cleavage of the p220 subunit of the cap binding protein (CBP) complex of the cell normally required for translation of capped messengers, as reported by others. However, Kunjin virus RNA could be efficiently translated apparently in the absence of a functional CBP complex, except when its secondary structure was stabilized by hypertonic salt in the culture medium.

No effect of kinins on DNA synthesis in LNCaP prostate cancer cells.

1. Prostate has kininogenase activity and expresses members of the tissue kallikrein gene family. The present study examined the effect of exogenous and endogenous kinins on growth of LNCaP prostate adenocarcinoma cells. 2. Rate of DNA synthesis was measured by incorporation over 4 h of [3H]-thymidine into a TCA insoluble fraction of LNCaP cells that had been cultured for 24 h. 3. Increased [3H]-thymidine incorporation was seen in response to 10 nmol/L testosterone (+103 +/- 5 s.e.%), dihydrotestosterone (+113 +/- 14%) and R1881 (+64 +/- 10%) (P < or = 0.001; n = 4). 4. In contrast 0.05, 5 and 1000 nmol/L lysyl-bradykinin had no effect (15 +/- 4, 10 +/- 9 and 5 +/- 3 s.e.%, respectively; n = 7). Des-Arg9[Leu8]-bradykinin (a B1 receptor antagonist) and/or D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (a B2 receptor antagonist), 1 nmol/L, and indomethacin, 5 mumol/L, also had little or no effect. 5. In conclusion, kallidin and endogenous kinins and prostaglandins have little or no effect on DNA synthesis and therefore on the growth of LNCaP cells in comparison to the two-fold stimulation produced by androgens.

Cross-sectional analysis of Met235-->Thr variant of angiotensinogen gene in severe, familial hypertension.

A recent cross-sectional study of HTs in Salt Lake City and Paris has reported a significant association of a T704-->C (Met235-->Thr) variant in exon 2 of the angiotensinogen gene (AGT) with essential hypertension (HT). The present study used a new, direct PCR technique to detect this variant in 92 Caucasians with severe hypertension (HT) and two HT parents and 94 normotensive (NT) controls. Although frequency of the variant in HTs (0.42) was higher than in NTs (0.39), the difference was not significant (chi 2 = 0.24; P = 0.63). Plasma angiotensinogen showed a weak, nonsignificant relationship with AGT genotype in females and no genotypic relationship was apparent for blood pressure. Thus, if the Met235-->Thr variant of AGT is involved in essential HT, then its contribution may be, at best, much weaker in other HT groups.

Association analyses of NsiI RFLP of human insulin receptor gene in hypertensives.

Plasma angiotensinogen is elevated in essential hypertensives and shows a strong correlation with blood pressure. Patients with hypertension often display insulin resistance and we have found previously an association of a RsaI RFLP in intron 9 of the insulin receptor gene (INSR) with hypertension. Since insulin resistance is accompanied by hyperinsulinaemia and insulin can stimulate angiotensinogen production, we hypothesized that hypertension-associated genotypes of INSR may be associated with elevation in plasma angiotensinogen. We used PCR to detect a NsiI RFLP in exon 8 of INSR and examined its relationship with plasma angiotensinogen, as well as hypertension, in 134 Caucasian hypertensives with two hypertensive parents and in 126 normotensives. Plasma angiotensinogen tracked weakly with the major allele of the NsiI RFLP in hypertensives (p = 0.08). Moreover, the frequency of this allele was higher in lean hypertensives than in lean normotensives (p < 0.05) and in normolipidaemic hypertensives than normolipidaemic normotensives (p < 0.02). The present study thus suggests that there could be a relationship of plasma angiotensinogen with INSR genotype, and of each with hypertension.

Frequencies of variants of candidate genes in different age groups of hypertensives.

1. In severe, familial hypertension, we have reported that the proportion of patients homozygous for the deletion allele of an insertion/deletion polymorphism of the angiotensin I-converting enzyme gene is markedly decreased in older age groups, suggesting that this genotype is associated with increased risk of premature death. The aim of the present study was to examine the relationship with age, of variants of other genes that encode proteins having an influence on the cardiovascular system. 2. Genotypes of 13 different variants at 12 relevant genetic loci were determined by either Southern blotting, followed by hybridization probing, or polymerase chain reaction techniques, as appropriate, using genomic DNA extracted from blood leukocytes. Genotype numbers were then assigned to the age categories of < 50, 50-59 and > or = 60 years. 3. Polymorphisms at the atrial natriuretic factor, antithrombin III, renin, angiotensinogen, neuropeptide-Y Y1 receptor, insulin, alpha 2-adrenoceptor, beta 1-adrenoceptor, growth hormone, low density lipoprotein receptor, insulin receptor and renal kallikrein gene loci were found to display similar allele frequencies in each age group of hypertensives, as well as in normotensive controls. 4. In conclusion, we were unable to detect any difference with age for a range of variants of genes whose products have cardiovascular significance, suggesting that, like most polymorphisms, they carry no selective survival advantage or disadvantage in the hypertensive and normotensive population groups studied.

Genotypic influence on plasma dipeptidyl carboxypeptidase-1 activity in hypertensives.

1. Plasma dipeptidyl carboxypeptidase-1 (DCP1; angiotensin I-converting enzyme, kininase II; EC 3.4.15.1) tracks with the deletion allele in genotypes of a 287 bp insertion/deletion (I/D) polymorphism of its gene, DCP1, in healthy Caucasian populations. The aim of the present study was to see whether genotype has a similar influence on plasma DCP1 in hypertensives. 2. The study involved 35 Caucasian patients with severe, familial essential hypertension, who were not being treated with DCP1 inhibitors, and 94 normotensives. Genotyping for the I/D polymorphism was performed by polymerase chain reaction and plasma DCP1 activity was measured by rate of hydrolysis of both [3H]-Hip-Gly-Gly and Hip-His-Leu. 3. Plasma DCP1 activity (nmol Gly-Gly/min per mL; mean +/- s.e.m.) was 67 +/- 2, 82 +/- 4 and 91 +/- 6 in II, ID and DD hypertensives, respectively, which was similar to values of 68 +/- 4, 82 +/- 3 and 94 +/- 3 in normotensives (P = 0.0001 by one-way analysis of variance). Results for the His-Leu assay indicated similar tracking with genotype. 4. The Michaelis constant (mumol Hip-Gly-Gly/mL; mean +/- s.e.m., n = 10) for DD subjects was the same as for II subjects (10.6 +/- 1.6 vs 11.1 +/- 2.3; P = 0.86). 5. In conclusion, in severely hypertensive Caucasian subjects, plasma DCP1 activity is subject to a similar genotypic influence in hypertensives as has been reported previously in normotensives. Furthermore, the plasma DCP1 enzyme itself appears to be functionally similar for each genotype.

Association of HincII RFLP of low density lipoprotein receptor gene with obesity in essential hypertensives.

Obese (BMI > or = 26 kg/m2; n = 51) and lean (BMI < 26 kg/m2; n = 61) Caucasian patients with severe, familial essential hypertension, were compared with respect to genotype and allele frequencies of a HincII RFLP of the low density lipoprotein receptor gene (LDLR). A similar analysis was performed in obese (n = 28) and lean (n = 68) normotensives. A significant association of the C allele of the T-->C variant responsible for this RFLP was seen with obesity (chi 2 = 4.6, P = 0.029) in the hypertensive, but not in the normotensive, group (odds ratio = 3.0 for the CC genotype and 2.7 for CT). Furthermore, BMI tracked with genotypes of this allele in the hypertensives (P = 0.046). No significant genotypic relationship was apparent for plasma lipids. Significant linkage disequilibrium was, moreover, noted between the HincII RFLP and an ApaLI RFLP (chi 2 = 33, P < 0.0005) that has previously shown even stronger association with obesity (odds ratio 19.6 for cases homozygous for the susceptibility allele and 15.2 for heterozygotes). The present study therefore adds to our previous evidence implicating LDLR as a locus for obesity in patients with essential hypertension.

Influence of angiotensin converting enzyme (ACE) genotype on interpretation of diagnostic tests for serum ACE activity.

BACKGROUND: The discovery that an insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene influences the circulating concentration of ACE may have implications for the proper use of serum ACE activity measurements in screening for sarcoidosis. AIM: To determine whether the sensitivity of the serum ACE test improves if ACE genotype is taken into account. METHODS: A retrospective determination of ACE genotype and clinical diagnosis was done in 54 patients with serum ACE activity above the upper limit of the reference range for the insertion (II) genotype. ACE was measured by radioenzymatic and spectrophotometric techniques, and genotype by PCR. RESULTS: When serum ACE values determined diagnostically were related to the appropriate genotype-specific reference range, sensitivity and specificity for diagnosis of sarcoidosis were 65-70% and 58% respectively, compared to 47-57% and 77% with a reference range unsegregated for genotype. CONCLUSION: ACE genotyping may be helpful in determining the diagnostic significance of mildly elevated serum ACE activity in patients with the II and ID genotypes.