Regioselective preparation of 5-hydroxypropranolol and 4'-hydroxydiclofenac with a fungal peroxygenase

An extracellular peroxygenase of Agrocybe aegerita catalyzed the H(2)O(2)-dependent hydroxylation of the multi-function beta-adrenergic blocker propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) and the non-steroidal anti-inflammatory drug diclofenac (2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid) to give the human drug metabolites 5-hydroxypropranolol (5-OHP) and 4'-hydroxydiclofenac (4'-OHD). The reactions proceeded regioselectively with high isomeric purity and gave the desired 5-OHP and 4'-OHD in yields up to 20% and 65%, respectively. (18)O-labeling experiments showed that the phenolic hydroxyl groups in 5-OHP and 4'-OHD originated from H(2)O(2), which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites.

Circulating transforming growth factor-beta in Marfan syndrome

BACKGROUND: Marfan syndrome (MFS) is caused by mutations in the fibrillin-1 gene and dysregulation of transforming growth factor-beta (TGF-beta). Recent evidence suggests that losartan, an angiotensin II type 1 blocker that blunts TGF-beta activation, may be an effective treatment for MFS. We hypothesized that dysregulation of TGF-beta might be mirrored in circulating TGF-beta concentrations. METHODS AND RESULTS: Serum obtained from MFS mutant mice (Fbn1(C1039G/+)) treated with losartan was analyzed for circulating TGF-beta1 concentrations and compared with those from placebo-treated and wild-type mice. Aortic root size was measured by echocardiography. Data were validated in patients with MFS and healthy individuals. In mice, circulating total TGF-beta1 concentrations increased with age and were elevated in older untreated Fbn1(C1039G/+) mice compared with wild-type mice (P=0.01; n=16; mean+/-SEM, 115+/-8 ng/mL versus n=17; mean+/-SEM, 92+/-4 ng/mL). Losartan-treated Fbn1(C1039G/+) mice had lower total TGF-beta1 concentrations compared with age-matched Fbn1(C1039G/+) mice treated with placebo (P=0.01; n=18; 90+/-5 ng/mL), and circulating total TGF-beta1 levels were indistinguishable from those of age-matched wild-type mice (P=0.8). Correlation was observed between circulating TGF-beta1 levels and aortic root diameters in Fbn1(C1039G/+) and wild-type mice (P=0.002). In humans, circulating total TGF-beta1 concentrations were elevated in patients with MFS compared with control individuals (P<0.0001; n=53; 15+/-1.7 ng/mL versus n=74; 2.5+/-0.4 ng/mL). MFS patients treated with losartan (n=55) or beta-blocker (n=80) showed significantly lower total TGF-beta1 concentrations compared with untreated MFS patients (P< or =0.05). CONCLUSIONS: Circulating TGF-beta1 concentrations are elevated in MFS and decrease after administration of losartan, beta-blocker therapy, or both and therefore might serve as a prognostic and therapeutic marker in MFS.

Cathepsin D specifically cleaves the chemokines macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, and SLC that are expressed in human breast cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895–1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.

Base pairing and miscoding properties of 1,N(6)-ethenoadenine- and 3,N(4)-ethenocytosine-containing RNA oligonucleotides

Two RNA phosphoramidites containing the bases 1,N(6)-ethenoadenine (εA) and 3,N(4)-ethenocytosine (εC) were synthesized. These building blocks were incorporated into two 12-mer oligoribonucleotides for evaluation of the base pairing properties of these base lesions by UV melting curve (Tm) and circular dichroism measurements. The Tm data of the resulting duplexes with the etheno modifications opposing all natural bases showed a substantial destabilization compared to the corresponding natural duplexes, confirming their inability to form base pairs. The coding properties of these lesions were further investigated by introducing them into 31-mer oligonucleotides and assessing their ability to serve as templates in primer extension reactions with HIV, AMV, and MMLV reverse transcriptases (RT). Primer extension reactions showed complete arrest of the incorporation process using MMLV RT and AMV RT, while HIV RT preferentially incorporates dAMP opposite εA and dAMP as well as dTMP opposite εC. The properties of these RNA lesions are discussed in the context of its putative biological role.

Copy number variation of the Beta-defensin genes in europeans: no supporting evidence for association with lung function, chronic obstructive pulmonary disease or asthma

Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02-1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72-1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.

Distinct roles of estrogen receptors alpha and beta mediating acute vasodilation of epicardial coronary arteries.

This study investigated the contribution of estrogen receptors (ERs) alpha and beta for epicardial coronary artery function, vascular NO bioactivity, and superoxide (O(2)(-)) formation. Porcine coronary rings were suspended in organ chambers and precontracted with prostaglandin F(2alpha) to determine direct effects of the selective ER agonists 4,4',4''-(4-propyl-[(1)H]pyrazole-1,3,5-triyl)tris-phenol (PPT) or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or the nonselective ER agonist 17beta-estradiol. Indirect effects on contractility to U46619 and relaxation to bradykinin were assessed and effects on NO, nitrite, and O(2)(-) formation were measured in cultured cells. Within 5 minutes, selective ERalpha activation by PPT, but not 17beta-estradiol or the ERbeta agonist DPN, caused rapid, NO-dependent, and endothelium-dependent relaxation (49+/-5%; P<0.001 versus ethanol). PPT also caused sustained endothelium- and NO-independent vasodilation similar to 17beta-estradiol after 60 minutes (72+/-3%; P<0.001 versus ethanol). DPN induced endothelium-dependent NO-independent relaxation via endothelium-dependent hyperpolarization (40+/-4%; P<0.01 versus ethanol). 17beta-Estradiol and PPT, but not DPN, attenuated the responses to U46619 and bradykinin. All of the ER agonists increased NO and nitrite formation in vascular endothelial but not smooth muscle cells and attenuated vascular smooth muscle cell O(2)(-) formation (P<0.001). ERalpha activation had the most potent effects on both nitrite formation and inhibiting O(2)(-) (P<0.05). These data demonstrate novel and differential mechanisms by which ERalpha and ERbeta activation control coronary artery vasoreactivity in males and females and regulate vascular NO and O(2)(-) formation. The findings indicate that coronary vascular effects of sex hormones differ with regard to affinity to ERalpha and ERbeta, which will contribute to beneficial and adverse effects of hormone replacement therapy.

The cannabinoid CB2 receptor-selective phytocannabinoid beta-caryophyllene exerts analgesic effects in mouse models of inflammatory and neuropathic pain

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB2). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB2 is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB2 receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB2 agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions.

Effects of beta-alanine supplementation and interval training on physiological determinants of severe exercise performance.

INTRODUCTION We aimed to manipulate physiological determinants of severe exercise performance. We hypothesized that (1) beta-alanine supplementation would increase intramuscular carnosine and buffering capacity and dampen acidosis during severe cycling, (2) that high-intensity interval training (HIT) would enhance aerobic energy contribution during severe cycling, and (3) that HIT preceded by beta-alanine supplementation would have greater benefits. METHODS Sixteen active men performed incremental cycling tests and 90-s severe (110 % peak power) cycling tests at three time points: before and after oral supplementation with either beta-alanine or placebo, and after an 11-days HIT block (9 sessions, 4 × 4 min), which followed supplementation. Carnosine was assessed via MR spectroscopy. Energy contribution during 90-s severe cycling was estimated from the O2 deficit. Biopsies from m. vastus lateralis were taken before and after the test. RESULTS Beta-alanine increased leg muscle carnosine (32 ± 13 %, d = 3.1). Buffering capacity and incremental cycling were unaffected, but during 90-s severe cycling, beta-alanine increased aerobic energy contribution (1.4 ± 1.3 %, d = 0.5), concurrent with reduced O2 deficit (-5.0 ± 5.0 %, d = 0.6) and muscle lactate accumulation (-23 ± 30 %, d = 0.9), while having no effect on pH. Beta-alanine also enhanced motivation and perceived state during the HIT block. There were no between-group differences in adaptations to the training block, namely increased buffering capacity (+7.9 ± 11.9 %, p = 0.04, d = 0.6, n = 14) and glycogen storage (+30 ± 47 %, p = 0.04, d = 0.5, n = 16). CONCLUSIONS Beta-alanine did not affect buffering considerably, but has beneficial effects on severe exercise metabolism as well as psychological parameters during intense training phases.

Determination of the Weak Axial Vector Coupling λ=g_{A}/g_{V} from a Measurement of the β-Asymmetry Parameter A in Neutron Beta Decay

We report on a new measurement of the neutron beta-asymmetry parameter A with the instrument \perkeo. Main advancements are the high neutron polarization of P=99.7(1) from a novel arrangement of super mirror polarizers and reduced background from improvements in beam line and shielding. Leading corrections were thus reduced by a factor of 4, pushing them below the level of statistical error and resulting in a significant reduction of systematic uncertainty compared to our previous experiments. From the result A0=−0.11996(58), we derive the ratio of the axial-vector to the vector coupling constant λ=gA/gV=−1.2767(16)

Search for squarks and gluinos with the ATLAS detector in final states with jets and missing transverse momentum using 4.7 fb⁻¹ of sqrts=7 TeV proton-pro collision data

A search for squarks and gluinos in final states containing jets, missing transverse momentum and no high-p(T) electrons or muons is presented. The data represent the complete sample recorded in 2011 by the ATLAS experiment in 7 TeV proton-proton collisions at the Large Hadron Collider, with a total integrated luminosity of 4.7 fb(-1). No excess above the Standard Model background expectation is observed. Gluino masses below 860 GeV and squark masses below 1320 GeV are excluded at the 95% confidence level in simplified models containing only squarks of the first two generations, a gluino octet and a massless neutralino, for squark or gluino masses below 2 TeV, respectively. Squarks and gluinos with equal masses below 1410 GeV are excluded. In minimal supergravity/constrained minimal supersymmetric Standard Model models with tan beta = 10, A(0) = 0 and mu > 0, squarks and gluinos of equal mass are excluded for masses below 1360 GeV. Constraints are also placed on the parameter space of supersymmetric models with compressed spectra. These limits considerably extend the region of supersymmetric parameter space excluded by previous measurements with the ATLAS detector.

Genomic cloning and expression of three murine UDP-galactose: beta-N-acetylglucosamine beta1,3-galactosyltransferase genes.

Based on the detection of expressed sequence tags that are similar to known galactosyltransferase sequences, we have isolated three novel UDP-galactose:beta-N-acetylglucosamine beta1, 3-galactosyltransferase (beta3GalT) genes from a mouse genomic library. The three genes, named beta3GalT-I, -II, and -III, encode type II transmembrane proteins of 326, 422, and 331 amino acids, respectively. The three proteins constitute a distinct subfamily as they do not share any sequence identity with other eucaryotic galactosyltransferases. Also, the entire protein-coding region of the three beta3GalT genes was contained in a single exon, which contrasts with the genomic organization of the beta1,4- and alpha1, 3-galactosyltransferase genes. The three beta3GalT genes were mainly expressed in brain tissue. The expression of the full-length murine genes as recombinant baculoviruses in insect cells revealed that the beta3GalT enzymes share the same acceptor specificity for beta-linked GlcNAc, although they differ in their Km for this acceptor and the donor UDP-Gal. The identification of beta3GalT genes emphasizes the structural diversity present in the galactosyltransferase gene family.

Beta-alanine supplementation improves jumping power and affects severe-intensity performance in professional alpine skiers.

INTRODUCTION Supplementation with beta-alanine may have positive effects on severe-intensity, intermittent, and isometric strength-endurance performance. These could be advantageous for competitive alpine skiers, whose races last 45 to 150 s, require metabolic power above the aerobic maximum, and involve isometric muscle work. Further, beta-alanine supplementation affects the muscle force-frequency relationship, which could influence explosiveness. We explored the effects of beta-alanine on explosive jump performance, severe exercise energy metabolism, and severe-intensity ski-like performance. METHODS Nine male elite alpine skiers consumed 4.8 g/d beta-alanine or placebo for 5 weeks in a double-blind fashion. Before and after, they performed countermovement jumps (CMJ), a 90-s cycling bout at 110% VO2max (CLT), and a maximal 90-s box jump test (BJ90). RESULTS Beta-alanine improved maximal (+7 ± 3%, d = 0.9) and mean CMJ power (+7 ± 2%, d = 0.7), tended to reduce oxygen deficit (-3 ± 8%, p = .06) and lactate accumulation (-12 ± 31%) and enhance aerobic energy contribution (+1.3 ± 2.9%, p = .07) in the CLT, and improved performance in the last third of BJ90 (+7 ± 4%, p = .02). These effects were not observed with placebo. CONCLUSIONS Beta-alanine supplementation improved explosive and repeated jump performance in elite alpine skiers. Enhanced muscle contractility could possibly explain improved explosive and repeated jump performance. Increased aerobic energy production could possibly help explain repeated jump performance as well.