Bradykinin receptor gene variant and human physical performance.

Accumulating evidence suggests that athletic performance is strongly influenced by genetic variation. One such locus of influence is the gene for angiotensin-I converting enzyme (ACE), which exhibits a common variant [ACE insertion (I)/deletion (D)]. ACE can drive formation of vasoconstrictor ANG II but preferentially degrades vasodilator bradykinin. The ACE I allele is associated with higher kinin activity. A common gene variant in the kinin beta(2) receptor (B(2)R) exists: the -9 as opposed to +9 allele is associated with higher receptor mRNA expression. We tested whether this variant was associated with the efficiency of muscular contraction [delta efficiency (DE)] in 115 healthy men and women, or with running distance among 81 Olympic standard track athletes. We further sought evidence of biological interaction with ACE I/D genotype. DE was highly significantly associated with B(2)R genotype (23.84 +/- 2.41 vs. 24.25 +/- 2.81 vs. 26.05 +/- 2.26% for those of +9/+9 vs. +9/-9 vs. -9/-9 genotype; n = 25, 61, and 29, respectively; P = 0.0008 for ANOVA adjusted for sex). There was evidence for interaction with ACE I/D genotype, with individuals who were ACE II, with B(2)R -9/-9 having the highest DE at baseline. The ACE I/B(2)R -9 "high kinin receptor activity" haplotype was significantly associated with endurance (predominantly aerobic) event among elite athletes (P = 0.003). These data suggest that common genetic variation in the B(2)R is associated with efficiency of skeletal muscle contraction and with distance event of elite track athletes and that at least part of the associations of ACE and fitness phenotypes is through elevation of kinin activity.     

Ciprofibrate therapy in patients with hypertriglyceridemia and low high density lipoprotein (HDL)-cholesterol: greater reduction of non-HDL cholesterol in subjects with excess body weight (The CIPROAMLAT study)

Background

Microalbuminuria and subsequent progression to proteinuria and nephropathy is associated with increased oxidative stress, increased inflammatory cytokines and increased cardiovascular (CVD) risk. The common functional IL-6 -174G>C gene variant is also associated with elevated levels of inflammatory cytokines and CVD risk.

Methods

The aim of this study was to examine the association between the IL-6 -174G>C gene variant with plasma total antioxidant status (TAOS) in 552 subjects with type 2 diabetes in relation to urinary protein excretion.

Results

In subjects free from CVD, there was a significant interaction between urinary protein excretion (normoalbuminuria/ microalbuminuria/proteinuria) and the -174C allele (compared to -174GG) in determining plasma TAOS (p value for interaction = 0.03). In the -174C allele carriers there was a significant association between plasma TAOS and urinary protein excretion: normalbuminuria v microalbuminuria v proteinuria: 44.30% ± 11.32 vs. 39.74% ± 14.83 vs. 37.93% ± 16.42, ANOVA p = 0.025. In those with CVD, no interaction or association was observed with the -174C allele (p = 0.246).

Conclusion

The IL-6 -174G>C gene variant is associated with differences in plasma oxidative stress in response to altered protein excretion in subjects with type 2 diabetes.     

Site-specific genomic (SSG) and random domain-localized (RDL) mutagenesis in yeast

Background  

A valuable weapon in the arsenal available to yeast geneticists is the ability to introduce specific mutations into yeast genome. In particular, methods have been developed to introduce deletions into the yeast genome using PCR fragments. These methods are highly efficient because they do not require cloning in plasmids.     

Acridine orange induces translocation of phosphatidylserine to red blood cell surface

Clustering of band-3 on red blood cell (RBC) surface has been assumed to catalyze RBC phagocytosis. In studying this subject, acridine orange (AO) has commonly been employed on the assumption that it specifically induces band-3 clustering. In the present study, we show that AO strongly induces translocation of phosphatidylserine (PS) to RBC surface. Because surface PS is well known to induce RBC intercellular interaction, these findings suggest that the use of AO as a specific inducer of band-3 clustering is questionable. It is possible that band-3 clustering and PS translocation are interdependent, and this interrelationship has yet to be explored. erythrocytes; adherence; acridine orange; band-3; phosphatidylserine     

Aryl tetrahydropyridine inhibitors of farnesyltransferase: bioavailable analogues with improved cellular potency

Inhibitors of farnesyltransferase are effective against a variety of tumors in mouse models of cancer. Clinical trials to evaluate these agents in humans are ongoing. In our effort to develop new farnesyltransferase inhibitors, we have discovered bioavailable aryl tetrahydropyridines that are potent in cell culture. The design, synthesis, SAR and biological properties of these compounds will be discussed.     

Charge-charge interactions are key determinants of the pK values of ionizable groups in ribonuclease Sa (pI=3.5) and a basic variant (pI=10.2)

The pK values of the titratable groups in ribonuclease Sa (RNase Sa) (pI=3.5), and a charge-reversed variant with five carboxyl to lysine substitutions, 5K RNase Sa (pI=10.2), have been determined by NMR at 20 degrees C in 0.1M NaCl. In RNase Sa, 18 pK values and in 5K, 11 pK values were measured. The carboxyl group of Asp33, which is buried and forms three intramolecular hydrogen bonds in RNase Sa, has the lowest pK (2.4), whereas Asp79, which is also buried but does not form hydrogen bonds, has the most elevated pK (7.4). These results highlight the importance of desolvation and charge-dipole interactions in perturbing pK values of buried groups. Alkaline titration revealed that the terminal amine of RNase Sa and all eight tyrosine residues have significantly increased pK values relative to model compounds.A primary objective in this study was to investigate the influence of charge-charge interactions on the pK values by comparing results from RNase Sa with those from the 5K variant. The solution structures of the two proteins are very similar as revealed by NMR and other spectroscopic data, with only small changes at the N terminus and in the alpha-helix. Consequently, the ionizable groups will have similar environments in the two variants and desolvation and charge-dipole interactions will have comparable effects on the pK values of both. Their pK differences, therefore, are expected to be chiefly due to the different charge-charge interactions. As anticipated from its higher net charge, all measured pK values in 5K RNase are lowered relative to wild-type RNase Sa, with the largest decrease being 2.2 pH units for Glu14. The pK differences (pK(Sa)-pK(5K)) calculated using a simple model based on Coulomb's Law and a dielectric constant of 45 agree well with the experimental values. This demonstrates that the pK differences between wild-type and 5K RNase Sa are mainly due to changes in the electrostatic interactions between the ionizable groups. pK values calculated using Coulomb's Law also showed a good correlation (R=0.83) with experimental values. The more complex model based on a finite-difference solution to the Poisson-Boltzmann equation, which considers desolvation and charge-dipole interactions in addition to charge-charge interactions, was also used to calculate pK values. Surprisingly, these values are more poorly correlated (R=0.65) with the values from experiment. Taken together, the results are evidence that charge-charge interactions are the chief perturbant of the pK values of ionizable groups on the protein surface, which is where the majority of the ionizable groups are positioned in proteins.     

Effects of different beta73 amino acids on formation of 14-stranded fibers of Hb S versus double-stranded crystals of Hb C-Harlem

Hb S (alpha(2)beta(2)(6Glu-->Val)) forms polymers, while Hb C-Harlem (alpha(2)beta(2)(6Glu-->Val,73Asp-->Asn)) forms crystals upon oversaturation. Since the only difference between the two is the beta73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. Beta73 Asp in Hb S forms a hydrogen bond with beta4 Thr, while beta73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix beta6 Val acceptor sites. Two new beta73 Hb S variants (beta73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the beta73-beta4 hydrogen bond (beta73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix beta6 Val acceptor sites (beta73 Leu). beta73 His Hb S formed fibers but at a lower concentration than Hb S, while beta73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of beta73 His Hb S was (1)/(7) of that of Hb S, while the solubility of beta73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for beta73 His Hb S was 10(5)-fold shorter than that for Hb S, while that for beta73 Leu Hb S was 10(5)-fold longer in 1.0 M phosphate buffer. NMR results indicate beta73 amino acid changes induce alteration in the beta-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the beta73-beta4 hydrogen bond and/or induced changes in the heme pocket by the beta73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the beta73-beta4 hydrogen bond and enhancing the hydrophobicity of the EF helix beta6 Val acceptor sites by the beta73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals.