Targeting effective blood pressure control with angiotensin receptor blockers

Angiotensin receptor blockers (ARBs) have become established as a major class of antihypertensive on the basis of their powerful effects on blood pressure (BP), excellent tolerability and pleiotropic end-organ-protective effects. However, individual ARBs vary in antihypertensive efficacy, which may be important to clinical outcome. Several strategies are available to ensure that BP reductions with ARBs are at least as great as that which can be achieved with other antihypertensive classes. Firstly, several newer ARBs, including irbesartan, candesartan, telmisartan and olmesartan, have been reported to provide equivalent antihypertensive efficacy to amlodipine and greater efficacy than either losartan, valsartan or both. Secondly, increases in dose may improve the antihypertensive efficacy of agents such as valsartan, although clinical studies are necessary to provide characterisation of new, higher-dose monotherapies. Thirdly, fixed dose combinations with hydrochlorothiazide (HCTZ) increase the antihypertensive effect of all ARBs. It is likely that differences in efficacy between newer and older ARBs will in some cases be sustained in combination therapy, such that the most potent ARBs and HCTZ will provide another tier of control. The future use of ARBs is likely to involve a growing emphasis on compound-specific data, with regard to the antihypertensive efficacy and pleiotropic protective actions of agents

Articles Mechanism of Neomycin and Rev Peptide Binding to the Rev Responsive Element of HIV-1 As Determined by Fluorescence and NMR Spectroscopy †

ABSTRACT: Rev is an essential HIV-1 regulatory protein that binds the Rev responsive element (RRE) within the enV gene of the HIV-1 RNA genome and is involved in transport of unspliced or partially spliced viral mRNA from the cell nucleus to the cytoplasm. Previous studies have shown that a short R-helical peptide derived from Rev , and a truncated form of the RRE sequence provide a useful in vitro system to study this interaction while still preserving the essential aspects of the native complex. We have selectively incorporated the fluorescent probe 2-aminopurine 2′-O-methylriboside (2-AP) into the RRE sequence in nonperturbing positions (A68 and U72) such that the binding of both Rev peptide and aminoglycoside ligands could be characterized directly by fluorescence methods. Rev peptide binding to the RRE-72AP variant resulted in a 2-fold fluorescence increase that provided a useful signal to monitor this binding interaction (K D ) 20 ( 7 nM). Using stopped-flow kinetic measurements, we have shown that specific Rev peptide binding occurs by a two-step process involving diffusion-controlled encounter, followed by isomerization of the RNA. Using the RRE-68AP and -72AP constructs, three classes of binding sites for the aminoglycoside neomycin were unambiguously detected. The first site is noninhibitory to Rev binding (K D ) 0.24 ( 0.040 µM), the second site inhibited Rev binding in a competitive fashion (K D ) 1.8 ( 0.8 µM), and the third much weaker site (or sites) is attributed to nonspecific binding (K D g 40 µM). Complementary NMR measurements have shown that neomycin forms both a specific binary complex with RRE and a specific ternary complex with RRE and Rev. NMR data further suggest that neomycin occupies a similar high-affinity binding site in both the binary and ternary complexes, and that this site is located in the lower stem region of RRE. Rev is an important HIV-1 1 regulatory protein that binds to part of the enV gene within the HIV-1 RNA genome, the so-called Rev responsive element (1-5). Rev is involved in the transport of unspliced and incompletely spliced viral mRNAs, which encode the structural proteins essential for viral replication, from the nucleus to the cytoplasm of the host cell. Stem-loop IIB of RRE, which contains a purinerich internal bulge, has been identified as the high-affinity Rev binding site (6, 7), and small peptides containing a 17 amino acid arginine-rich region of Rev (amino acids 34-51) have been shown to bind this stem-loop specifically (8-14). Since Rev protein binding to viral mRNA at the RRE is essential for HIV replication, acting as a crucial switch between viral latency and active viral replication, the development of new drug therapies against HIV-1 infection based on inhibitors of this essential interaction is desirable. Members of the aminoglycoside family of antibiotics have been shown to interact with a variety of RNA molecules, including the RRE IIB stem-loo

Primary Structure and Catalytic Mechanism of the Epoxide Hydrolase from Agrobacterium radiobacter AD1

The epoxide hydrolase gene from Agrobacterium radiobacter AD1, a bacterium that is able to grow on epichlorohydrin as the sole carbon source, was cloned by means of the polymerase chain reaction with two degenerate primers based on the N-terminal and C-terminal sequences of the enzyme. The epoxide hydrolase gene coded for a protein of 294 amino acids with a molecular mass of 34 kDa. An identical epoxide hydrolase gene was cloned from chromosomal DNA of the closely related strain A. radiobacter CFZ11. The recombinant epoxide hydrolase was expressed up to 40% of the total cellular protein content in Escherichia coli BL21(DE3) and the purified enzyme had a kcat of 21 s-1 with epichlorohydrin. Amino acid sequence similarity of the epoxide hydrolase with eukaryotic epoxide hydrolases, haloalkane dehalogenase from Xanthobacter autotrophicus GJ10, and bromoperoxidase A2 from Streptomyces aureofaciens indicated that it belonged to the α/β-hydrolase fold family. This conclusion was supported by secondary structure predictions and analysis of the secondary structure with circular dichroism spectroscopy. The catalytic triad residues of epoxide hydrolase are proposed to be Asp107, His275, and Asp246. Replacement of these residues to Ala/Glu, Arg/Gln, and Ala, respectively, resulted in a dramatic loss of activity for epichlorohydrin. The reaction mechanism of epoxide hydrolase proceeds via a covalently bound ester intermediate, as was shown by single turnover experiments with the His275 → Arg mutant of epoxide hydrolase in which the ester intermediate could be trapped.

Biochemical Discrimination between Selenium and Sulfur 1: A Single Residue Provides Selenium Specificity to Human Selenocysteine Lyase

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism

Fluorescent probing for RNA molecules by an unnatural base-pair system

Fluorescent labeling of nucleic acids is widely used in basic research and medical applications. We describe the efficient site-specific incorporation of a fluorescent base analog, 2-amino-6-(2-thienyl)purine (s), into RNA by transcription mediated by an unnatural base pair between s and pyrrole-2-carbaldehyde (Pa). The ribonucleoside 5′-triphosphate of s was site-specifically incorporated into RNA, by T7 RNA polymerase, opposite Pa in DNA templates. The fluorescent intensity of s in RNA molecules changes according to the structural environment. The site-specific s labeling of RNA hairpins and tRNA molecules provided characteristic fluorescent profiles, depending on the labeling sites, temperature and Mg2+ concentration. The Pa-containing DNA templates can be amplified by PCR using 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), another pairing partner of Pa. This site-specific fluorescent probing by the unnatural pair system including the s-Pa and Ds-Pa pairs provides a powerful tool for studying the dynamics of the local structural features of 3D RNA molecules and their intra- and intermolecular interactions

Biochemical Discrimination between Selenium and Sulfur 2: Mechanistic Investigation of the Selenium Specificity of Human Selenocysteine Lyase

Selenium is an essential trace element incorporated into selenoproteins as selenocysteine. Selenocysteine (Sec) lyases (SCLs) and cysteine (Cys) desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys, respectively, and generally accept both substrates. Intriguingly, human SCL (hSCL) is specific for Sec even though the only difference between Sec and Cys is a single chalcogen atom

Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

Selenocysteine (Sec)-decoding archaea and eukaryotes employ a unique route of Sec-tRNASec synthesis in which O-phosphoseryl-tRNASec kinase (PSTK) phosphorylates Ser-tRNASec to produce the O-phosphoseryl-tRNASec (Sep-tRNASec) substrate that Sep-tRNA:Sec-tRNA synthase (SepSecS) converts to Sec-tRNASec. This study presents a biochemical characterization of Methanocaldococcus jannaschii PSTK, including kinetics of Sep-tRNASec formation (Km for Ser-tRNASec of 40 nM and ATP of 2.6 mM). PSTK binds both Ser-tRNASec and tRNASec with high affinity (Kd values of 53 nM and 39 nM, respectively). The ATPase activity of PSTK may be activated via an induced fit mechanism in which binding of tRNASec specifically stimulates hydrolysis. Albeit with lower activity than ATP, PSTK utilizes GTP, CTP, UTP and dATP as phosphate-donors. Homology with related kinases allowed prediction of the ATPase active site, comprised of phosphate-binding loop (P-loop), Walker B and RxxxR motifs. Gly14, Lys17, Ser18, Asp41, Arg116 and Arg120 mutations resulted in enzymes with decreased activity highlighting the importance of these conserved motifs in PSTK catalysis both in vivo and in vitro. Phylogenetic analysis of PSTK in the context of its ‘DxTN’ kinase family shows that PSTK co-evolved precisely with SepSecS and indicates the presence of a previously unidentified PSTK in Plasmodium species

Effects of losartan vs candesartan in reducing cardiovascular events in the primary treatment of hypertension

Although angiotensin receptor blockers have different receptor binding properties no comparative studies with cardiovascular disease (CVD) end points have been performed within this class of drugs. The aim of this study was to test the hypothesis that there are blood pressure independent CVD-risk differences between losartan and candesartan treatment in patients with hypertension without known CVD. Seventy-two primary care centres in Sweden were screened for patients who had been prescribed losartan or candesartan between the years 1999 and 2007. Among the 24 943 eligible patients, 14 100 patients were diagnosed with hypertension and prescribed losartan (n=6771) or candesartan (n=7329). Patients were linked to Swedish national hospitalizations and death cause register. There was no difference in blood pressure reduction when comparing the losartan and candesartan groups during follow-up. Compared with the losartan group, the candesartan group had a lower adjusted hazard ratio for total CVD (0.86, 95% confidence interval (CI) 0.77–0.96, P=0.0062), heart failure (0.64, 95% CI 0.50–0.82, P=0.0004), cardiac arrhythmias (0.80, 95% CI 0.65–0.92, P=0.0330), and peripheral artery disease (0.61, 95% CI 0.41–0.91, P=0.0140). No difference in blood pressure reduction was observed suggesting that other mechanisms related to different pharmacological properties of the drugs may explain the divergent clinical outcomes