Detection of Transversions and Transitions in HBG2 Cis-Elements Associated with Sickle Cell Allele in Ghanaians.

Short tandem repeats located 5' prime to the ß-globin gene, have been observed to be in linkage disequilibrium with the HbS allele, and thought to affect the severity of sickle cell disease. Here, we report on new mutants within the HBG2 region that may impact sickle cell disease. To determine the cis-acting elements microsatellites, indels and single nucleotide polymorphisms (SNPs), within the HBG2 region by sequencing, in subjects with sickle cell disease. The case-control study was located at the Center for Clinical Genetics, Sickle cell unit, Korle-Bu Teaching Hospital. A questionnaire was used for demographic data and clinical information. Hematological profile (red blood cell, white blood cell, platelet, hemoglobin and mean corpuscular volume) were assessed in 83 subjects. A set of 45 samples comprising amplified DNA on the HBG2 gene from HbSS (22), HbSC (17) and 6 controls (HbAA) were sequenced. Differences in the microsatellite region between sickle cell disease (SCD) (HbSS and HbSC) genotypes and control subjects were identified by counting and assessed by Chi-square analysis. Red blood cells, hematocrit, platelets, white blood cells and hemoglobin indices differed in genotypic groups. HbSS subjects were affirmed to have severer hemolytic anemia than HbSC subjects. Two indels (T1824 and C905) were seen in both SS and SC genotypes. Two peculiar SNPs: G:T1860 (transition) and A:G1872 transversions were found within the HBG2 gene that were significantly associated with the HbSS genotype (Fisher's exact test, p = 0.006) and HbS allele respectively (Fisher's exact test, p = 0.006). Cis-acting elements in HbSS and HbSC were different and may contribute to the phenotype seen in the disease state.

Tobacco smoking is associated with decreased semen quality.

BACKGROUND: Tobacco smoking is a public health issue and has been implicated in adverse reproductive outcomes including semen quality. Available data however provides conflicting findings. The objective of this study was to evaluate the effect of tobacco smoking on semen quality among men in Ghana. METHODS: In this study, a total of 140 subjects were recruited, comprising 95 smokers and 45 non-smokers. Smokers were further categorized into mild, moderate and heavy smokers. Semen parameters such as sperm concentration, motility, viability and normal morphology were measured according to the World Health Organisation criteria. RESULTS: The study showed that smokers had significantly lower semen volume, sperm concentration, sperm motility, total sperm count, sperm morphology, free testosterone and follicle stimulating hormone (p <0.05 respectively), compared with non-smokers. Smokers were at a higher risk of developing oligospermia, asthenozoospermia and teratozoospermia (OR = 3.1, 4.2 and, 4.7; p <0.05) than non-smokers. CONCLUSION: Results demonstrated a decline in semen quality in a dose dependent tobacco smoking manner.

Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites.

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg(1137)/Leu(1138)) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg(1137). Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg(1203). These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg(1203)/Ser(1204) bond, identical with the Arg(627)/Ser(628) site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer.

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 microM, whereas TAPI-2 was slightly less potent (IC50 18 microM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is realted to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton-X-100 and CHAPS, but not with n-octyl beta-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.

Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane.

Angiotensin-converting enzyme (ACE; EC 3.4.1.15.1) exists in both membrane-bound and soluble forms. Phase separation in Triton X-114 and a competitive e.l.i.s.a. have been employed to characterize the activity which post-translationally converts the amphipathic, membrane-bound form of ACE in pig kidney microvilli into a hydrophilic, soluble form. This secretase activity was enriched to a similar extent as other microvillar membrane proteins, was tightly membrane-associated, being resistant to extensive washing of the microvillar membranes with 0.5 M NaCl, and displayed a pH optimum of 8.4. The ACE secretase was not affected by inhibitors of serine-, thiol- or aspartic-proteases, nor by reducing agents or alpha 2-macroglobulin. The metal chelators, EDTA and 1,10-phenanthroline, inhibited the secretase activity, with, in the case of EDTA, an inhibitor concentration of 2.5 mM causing 50% inhibition. In contrast, EGTA inhibited the secretase by a maximum of 15% at a concentration of 10 mM. The inhibition of EDTA was reactivated substantially (83%) by Mg2+ ions, and partially (34% and 29%) by Zn2+ and Mn2+ ions respectively. This EDTA-sensitive secretase activity was also present in microsomal membranes prepared from pig lung and testis, and from human lung and placenta, but was absent from human kidney and human and pig intestinal brush-border membranes. The form of ACE released from the microvillar membrane by the secretase co-migrated on SDS/PAGE with ACE purified from pig plasma, thus the action and location of the secretase would be consistent with it possibly having a role in the post-translational proteolytic cleavage of membrane-bound ACE to generate the soluble form found in blood, amniotic fluid, seminal plasma and other body fluids.

Inhibition by converting enzyme inhibitors of pig kidney aminopeptidase P.

Several inhibitors of angiotensin converting enzyme were also found to inhibit aminopeptidase P, whereas inhibitors of other mammalian aminopeptidases were ineffective. Aminopeptidase P purified from pig kidney cortex was found to contain one atom of zinc per polypeptide chain, confirming its metalloenzyme nature. The concentrations of converting enzyme inhibitors required to cause 50% inhibition (I50) of aminopeptidase P were in the low micromolar range. The most potent converting enzyme inhibitors toward aminopeptidase P were the carboxylalkyl compounds, cilazaprilat, enalaprilat, and ramiprilat (I50 values of 3-12 microM). The sulfhydryl compounds captopril (I50 110 microM) and YS980 (I50 20 microM) were slightly less potent at inhibiting aminopeptidase P. In contrast, the carboxylalkyl compounds benazeprilat, lisinopril, and pentoprilat; the sulfhydryl compound rentiapril; and the phosphoryl compounds ceranopril and fosinoprilat had no inhibitory effect against aminopeptidase P. This compares with I50 values in the 1-6 nM range for these inhibitors with angiotensin converting enzyme. Inhibition of aminopeptidase P may account for some of the effects or side effects noted with the clinical use of converting enzyme inhibitors. These results may provide the basis for the design of more selective inhibitors of angiotensin converting enzyme or mixed inhibitors of aminopeptidase P and angiotensin converting enzyme, or both.