Sulfonamides as selective oestrogen receptor β agonists

A series of p-hydroxybenzenesulphonamides ERβ receptor agonists were discovered and several compounds identified had excellent selectivity over the related ERα receptor. One of these, compound 11, had an interesting binding conformation determined by X-ray and represents an excellent starting point in the quest for further selective ERβ agonists.     

The discovery of novel 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamides as vasopressin V1A receptor antagonists

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V_1A receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V_1A antagonists.     

IL28B, HLA-C, and KIR variants additively predict response to therapy in chronic hepatitis C virus infection in a European Cohort: a cross-sectional study

Background

To date, drug response genes have not proved as useful in clinical practice as was anticipated at the start of the genomic era. An exception is in the treatment of chronic hepatitis C virus (HCV) genotype 1 infection with pegylated interferon-alpha and ribavirin (PegIFN/R). Viral clearance is achieved in 40%–50% of patients. Interleukin 28B (IL28B) genotype predicts treatment-induced and spontaneous clearance. To improve the predictive value of this genotype, we studied the combined effect of variants of IL28B with human leukocyte antigen C (HLA-C), and its ligands the killer immunoglobulin-like receptors (KIR), which have previously been implicated in HCV viral control.

Methods and Findings

We genotyped chronic hepatitis C (CHC) genotype 1 patients with PegIFN/R treatment-induced clearance (n = 417) and treatment failure (n = 493), and 234 individuals with spontaneous clearance, for HLA-C C1 versus C2, presence of inhibitory and activating KIR genes, and two IL28B SNPs, rs8099917 and rs12979860. All individuals were Europeans or of European descent. IL28B SNP rs8099917 “G” was associated with absence of treatment-induced clearance (odds ratio [OR] 2.19, p = 1.27×10⁻⁸, 1.67–2.88) and absence of spontaneous clearance (OR 3.83, p = 1.71×10⁻¹⁴, 2.67–5.48) of HCV, as was rs12979860, with slightly lower ORs. The HLA-C C2C2 genotype was also over-represented in patients who failed treatment (OR 1.52, p = 0.024, 1.05–2.20), but was not associated with spontaneous clearance. Prediction of treatment failure improved from 66% with IL28B to 80% using both genes in this cohort (OR 3.78, p = 8.83×10⁻⁶, 2.03–7.04). There was evidence that KIR2DL3 and KIR2DS2 carriage also altered HCV treatment response in combination with HLA-C and IL28B.

Conclusions

Genotyping for IL28B, HLA-C, and KIR genes improves prediction of HCV treatment response. These findings support a role for natural killer (NK) cell activation in PegIFN/R treatment-induced clearance, partially mediated by IL28B. Please see later in the article for the Editors'' Summary     

Can Dietary Nitrates Enhance the Efficiency of Mitochondria?

A decline in mitochondrial function occurs in many conditions. A report in this issue of Cell Metabolism (Larsen et al., 2011) shows that dietary inorganic nitrates enhance muscle mitochondrial efficiency. It is an attractive hypothesis that dietary changes enhance energy efficiency, but its potential application depends on long-term studies investigating net benefits versus adverse effects.     

A Molecular Switch Governs the Interaction between the Human Complement Protease C1s and Its Substrate,Complement C4

The complement system is an ancient innate immune defense pathway that plays a front line role in eliminating microbial pathogens. Recognition of foreign targets by antibodies drives sequential activation of two serine proteases, C1r and C1s, which reside within the complement Component 1 (C1) complex. Active C1s propagates the immune response through its ability to bind and cleave the effector molecule complement Component 4 (C4). Currently, the precise structural and biochemical basis for the control of the interaction between C1s and C4 is unclear. Here, using surface plasmon resonance, we show that the transition of the C1s zymogen to the active form is essential for C1s binding to C4. To understand this, we determined the crystal structure of a zymogen C1s construct (comprising two complement control protein (CCP) domains and the serine protease (SP) domain). These data reveal that two loops (492–499 and 573–580) in the zymogen serine protease domain adopt a conformation that would be predicted to sterically abrogate C4 binding. The transition from zymogen to active C1s repositions both loops such that they would be able to interact with sulfotyrosine residues on C4. The structure also shows the junction of the CCP1 and CCP2 domains of C1s for the first time, yielding valuable information about the exosite for C4 binding located at this position. Together, these data provide a structural explanation for the control of the interaction with C1s and C4 and, furthermore, point to alternative strategies for developing therapeutic approaches for controlling activation of the complement cascade.     

Examining the link between personality and laterality in a feral guppy Poecilia reticulata population

This study examined whether variation in the strength and direction of lateralization in a detour task was linked with variation in three common personality measurements: boldness, activity and sociability, in a population of wild guppies Poecilia reticulata. Additionally, the aim was to determine whether any consistent correlations between these behavioural traits, known as behavioural syndromes, were present in the study population. The results revealed that all three personality traits were highly repeatable over time in both sexes. Evidence of a complex syndrome in the form of a correlation between boldness, sociability and activity was found; however, this relationship was only present in males. Males that were more active in a familiar environment emerged more quickly from shelter into a novel environment and were more social. In general, male P. reticulata were bold, active and antisocial compared to females, with these differences probably a reflection of opposing life‐history strategies. Only a weak link between the strength of cerebral lateralisation and personality was discovered and this was mediated by sex.     

Involvement of Arabidopsis ACYL-COENZYME A DESATURASE-LIKE2 (At2g31360) in the Biosynthesis of the Very-Long-Chain Monounsaturated Fatty Acid Components of Membrane Lipids

The Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A (CoA) desaturase-like (ADS) gene family contains nine genes encoding fatty acid desaturase-like proteins. The biological function of only one member of the family, fatty acid desaturase5 (AtADS3/FAD5, At3g15850), is known, and this gene encodes the plastidic palmitoyl-monogalactosyldiacylglycerol Δ7 desaturase. We cloned seven members of the gene family that are predicted not to have a chloroplast transit peptide and expressed them in the yeast Saccharomyces cerevisiae. All seven have previously undescribed desaturase activity on very-long-chain fatty acid (VLCFA) substrates and exhibit diverse regiospecificity, catalyzing introduction of double bonds relative to the methyl end of the molecule (n-x) at n-6 (AtADS4, At1g06350), n-7 (AtADS1.3, At1g06100 and AtADS4.2, At1g06360), n-9 (AtADS1, At1g06080 and AtADS2, At2g31360) or Δ9 (relative to the carboxyl end of the molecule) positions (AtADS1.2, At1g06090 and AtADS1.4, At1g06120). Through forward and reverse genetics it was shown that AtADS2 is involved in the synthesis of the 24:1(n-9) and 26:1(n-9) components (X:Y, where X is chain length and Y is number of double bonds) of seed lipids, sphingolipids, and the membrane phospholipids phosphatidylserine, and phosphatidylethanolamine. Plants deficient in AtADS2 expression showed no obvious phenotype when grown under normal growing conditions, but showed an almost complete loss of phosphatidylethanolamine(42:4), phosphatidylserine(42:4), dihydroxy-monohexosylceramide(42:2)-2, trihydroxy-monohexosylceramide(42:2)-3, and trihydroxy-glycosylinositolphosphoceramide(42:2)-3, lipid species that contain the VLCFA 24:1(n-9), and trihydroxy-glycosylinositolphosphoceramide(44:2)-3, a lipid containing 26:1(n-9). Acyl-CoA profiling of these plants revealed a major reduction in 24:1-CoA and a small reduction in 26:1-CoA. Overexpression of AtADS2 resulted in a substantial increase in the percentage of glycerolipid and sphingolipids species containing 24:1 and a dramatic increase in the percentage of very-long-chain monounsaturated fatty acids in the acyl-CoA pool. Plants deficient in AtADS1 expression had reduced levels of 26:1(n-9) in seed lipids, but no significant changes in leaf phospholipids or sphingolipids were observed. These findings indicate that the 24-carbon and 26-carbon monounsaturated VLCFAs of Arabidopsis result primarily from VLCFA desaturation, rather than by elongation of long chain monounsaturated fatty acids.The ADS (for acyl-CoA desaturase-like) gene family of Arabidopsis (Arabidopsis thaliana) encodes a group of nine proteins with homology to the Δ9 acyl-lipid desaturases of cyanobacteria, the Δ9 acyl-CoA desaturases of yeast (Saccharomyces cerevisiae) and mammals (Fukuchi-Mizutani et al., 1998; Heilmann et al., 2004b) and the membrane-bound desaturases of insects (Knipple et al., 2002). Eight of these genes are located in three clusters on chromosomes I and III. The remaining gene, designated AtADS2 (At2g31360), is present on chromosome II. With the exception of Arabidopsis fatty acid desaturase5 (AtADS3/FAD5, At3g15850), which encodes the plastidic palmitoyl-monogalactosyldiacylglycerol Δ7 desaturase (Heilmann et al., 2004b), the biological role of these enzymes in Arabidopsis is currently unknown. AtADS3/FAD5 and a second closely linked homolog designated AtADS3.2 (At3g15870), are the only members of the gene family predicted to encode proteins with a plastid transit peptide.The first study, to our knowledge, to report evidence of desaturase activity associated with an Arabidopsis ADS, AtADS1 (At1g06080), described the heterologous expression of the gene in Brassica juncea. Seeds from transformed plants contained decreased levels of saturated fatty acids and a slight increase in oleic acid content (Yao et al., 2003). Although the evidence was indirect, the results suggested that AtADS1 may encode a Δ9 desaturase. More detailed studies involving in vivo expression of AtADS1, AtADS2, and AtADS3 (without the plastid transit peptide) in yeast (Saccharomyces cerevisiae) have shown that all three enzymes can catalyze the Δ9 or Δ7 desaturation of palmitic (16:0) and stearic (18:0) acids (X:Y, where X is chain length and Y is number of double bonds), with regiospecificity being partly influenced by fatty acid substrate (Heilmann et al., 2004a). In this work, the substrate for desaturation was suggested to be a glycerolipid rather than acyl-CoA. The bifunctionality of these enzymes was further demonstrated by expression in Arabidopsis. When AtADS3 was expressed as the full-length form including the plastid transit peptide, or when AtADS1 and AtADS2 were retargeted to the plastid by the addition of a plastid transit peptide, 16:1Δ7 became the predominant monounsaturated 16-carbon (C16) fatty acid. The Arabidopsis plants used in the study were fab1/fae1 (for fatty acid elongase1) double mutant lines lacking the activity of KASII (for 3-ketoacyl-acyl-carrier protein synthase; FAB1, At1g74960) and the FAE1 condensing enzyme (At4g34520), and consequently exhibiting higher than normal levels of 16:0 and low very-long-chain unsaturated fatty acid content in the seed lipids.Homologs of the Arabidopsis ADS enzymes have been identified in other plant species, but their catalytic activity and acyl-substrates are not well characterized. Heterologous expression of a complementary DNA (cDNA) encoding an ADS-like protein from white spruce (Picea glauca) gave evidence of Δ9 activity when expressed in yeast (Marillia et al., 2002). The lipid substrate of this desaturase was not determined and the cDNA appeared to encode an enzyme with a plastid transit peptide. The Δ5 desaturase catalyzing the synthesis of 20:1Δ5 in the seeds of Limnanthes alba is also an ADS homolog (Cahoon et al., 2000). The substrate for this reaction is thought to be the 20:0-CoA thioester (Pollard and Stumpf, 1980; Moreau et al., 1981). Δ5 desaturase activity on fatty acids with chain length longer than 18 carbons (very-long-chain fatty acids [VLCFAs]) has also been demonstrated from two ADS homologs isolated from Anemone leveillei (Sayanova et al., 2007). Indirect evidence suggesting that both enzymes utilized acyl-CoA substrates was presented based on characterization of acyl-CoA pools in developing seeds of transgenic Arabidopsis expressing the A. leveillei desaturases.In addition to functioning in the synthesis of chloroplast lipids (AtADS3/FAD5) and VLCFAs of certain seed oils, ADS proteins have been suggested to play a role in petal senescence in roses (Rosa spp.; Fukuchi-Mizutani et al., 1995) and the expression of Arabidopsis AtADS1 and AtADS2 appears to be regulated in response to changes in temperature (Fukuchi-Mizutani et al., 1998; Byun et al., 2009). A potential role in drought tolerance has also been suggested for a member of the gene family (At1g06100; Allen et al., 2012). This group of plant enzymes therefore appears to contain members showing a diversity of lipid substrate utilization, desaturation regiospecificity and biological function that merits further investigation.Only four members of the Arabidopsis ADS gene family have documented nomenclature. Based on the existing literature we propose a systematic nomenclature of the ADS gene family based on their chromosomal location and grouping (VLCFA substrates. Forward and reverse genetics have revealed a role for AtADS2 in the production of very-long-chain monounsaturated fatty acids (VLCMUFAs) in seed lipids and in membrane phospholipids and sphingolipids.

Table I.

The Arabidopsis ADS gene family

Molecular regulation of CCN2 in the intervertebral disc: Lessons learned from other connective tissues

Connective tissue growth factor (CCN2/CTGF) plays an important role in extracellular matrix synthesis, especially in skeletal tissues such as cartilage, bone, and the intervertebral disc. As a result there is a growing interest in examining the function and regulation of this important molecule in the disc. This review discusses the regulation of CCN2 by TGF-β and hypoxia, two critical determinants that characterize the disc microenvironment, and discusses known functions of CCN2 in the disc. The almost ubiquitous regulation of CCN2 by TGF-β, including that seen in the disc, emphasizes the importance of the TGF-β–CCN2 relationship, especially in terms of extracellular matrix synthesis. Likewise, the unique cross-talk between CCN2 and HIF-1 in the disc highlights the tissue and niche specific mode of regulation. Taken together the current literature supports an anabolic role for CCN2 in the disc and its involvement in the maintenance of tissue homeostasis during both health and disease. Further studies of CCN2 in this tissue may reveal valuable targets for the biological therapy of disc degeneration.     

X-ray diffraction measurements of the extensibility of actin and myosin filaments in contracting muscle.

We have used a small angle scattering system assembled on the high flux multipole wiggler beam line at CHESS (Cornell) to make very accurate spacing measurements of certain meridional and layer-line reflections from contracting muscles. During isometric contraction, the actin 27.3 A reflection increases in spacing from its resting value by approximately 0.3%, and other actin reflections, including the 59 and 51 A off-meridional reflections, show corresponding changes in spacing. When tension is augmented or diminished by applying moderate speed length changes to a contracting muscle, changes in spacing in the range of 0.19-0.24% (when scaled to full isometric tension) can be seen. The larger difference between the resting and isometric spacings suggests either nonlinearity at low tension levels or the presence of a component related to activation itself. Myosin filaments also show similar increases in axial period during slow stretch, in addition to the well known larger change associated with activation. An actin spacing change of 0.25-0.3% can also be measured during a 2 ms time frame immediately after a quick release, showing that the elastic behavior is rapid. These observations of filament extensions totaling 2-3 nm per half-sarcomere may necessitate some significant revision of the interpretation of a number of mechanical experiments in muscle, in which it has usually been assumed that virtually all of the elasticity resides in the cross-bridges.     

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin β-subunit by disruption of disulfide bonds

The human chorionic gonadotropin β-subunit (hCGβ) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGβ produced in Chinese hamster ovary (CHO) cells: wild-type hCGβ (βWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of βWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled βWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGβ molecule. Abbreviations: hCGβ, human chorionic gonadotropin β-subunit; βWT, wild type hCGβ; CHO, Chinese hamster ovary; Endo-H, endoglycosidase H; Endo-F, endoglycosidase F This revised version was published online in November 2006 with corrections to the Cover Date.