No association between Angiotensin Converting Enzyme (ACE) gene variation and endurance athlete status in Kenyans

East African runners are continually successful in international distance running. The extent to which genetic factors influence this phenomenon is unknown. The insertion (I) rather than deletion (D) of a 287 bp fragment in the human angiotensin converting enzyme (ACE) gene is associated with lower circulating and tissue ACE activity and with endurance performance amongst Caucasians. To assess the association between ACE gene variation and elite endurance athlete status in an African population successful in distance running, DNA samples were obtained from 221 national Kenyan athletes (N), 70 international Kenyan athletes (I), and 85 members of the general Kenyan population (C). Blood samples were obtained from C and assayed for circulating ACE activity. ACE I/D (rs????--from NCBI SNPdb first time poly mentioned) genotype was determined, as was genotype at A22982GD (rs????--from NCBI SNPdb first time poly mentioned) which has been shown to associate more closely with ACE levels in African subjects than the I/D polymorphism. ACE I/D and A22982G genotypes explained 13 and 24% of variation in circulating ACE activity levels (P = 0.034 and <0.001 respectively). I/D genotype was not associated with elite endurance athlete status (df = 4, chi(2) = 4.1, P=0.39). In addition, genotype at 22982 was not associated with elite endurance athlete status (df = 4, chi(2) = 5.7, P = 0.23). Nor was the A allele at 22982, which is associated with lower ACE activity, more prevalent in N (0.52) or I (0.41) relative to C (0.53). We conclude that ACE I/D and A22982G polymorphisms are not strongly associated with elite endurance athlete status amongst Kenyans.     

Arg16/Gly beta2-adrenergic receptor polymorphism alters the cardiac output response to isometric exercise.

Normotensive adults homozygous for glycine (Gly) of the Arg16/Gly beta2-adrenergic-receptor polymorphism have 1) greater forearm beta2-receptor mediated vasodilation and 2) a higher heart rate (HR) response to isometric handgrip than arginine (Arg) homozygotes. To test the hypothesis that the higher HR response in Gly16 subjects serves to maintain the pressor response [increased cardiac output (CO)] in the setting of augmented peripheral vasodilation to endogenous catecholamines, we measured continuous HR (ECG), arterial pressure (Finapres), and CO (transthoracic echocardiography) during isometric, 40% submaximal handgrip to fatigue in healthy subjects homozygous for Gly (n = 30; mean age +/- SE: 30 +/- 1.2, 13 women) and Arg (n = 17, age 30 +/- 1.6, 11 women). Resting data were similar between groups. Handgrip produced similar increases in arterial pressure and venous norepinephrine and epinephrine concentrations; however, HR increased more in the Gly group (60.1 +/- 4.3% increase from baseline vs. 45.5 +/- 3.9%, P = 0.03), and this caused CO to be higher (Gly: 7.6 +/- 0.3 l/m vs. Arg: 6.5 +/- 0.3 l/m, P = 0.03), whereas the decrease in systemic vascular resistance in the Gly group did not reach significance (P = 0.09). We conclude that Gly16 homozygotes generate a higher CO to maintain the pressor response to handgrip. The influence of polymorphic variants in the beta2-adrenergic receptor gene on the cardiovascular response to sympathoexcitation may have important implications in the development of hypertension and heart failure.     

Both rotor and stator subunits are necessary for efficient binding of F1 to F0 in functionally assembled Escherichia coli ATP synthase

In F1F0-ATP synthase, the subunit b2delta complex comprises the peripheral stator bound to subunit a in F0 and to the alpha3beta3 hexamer of F1. During catalysis, ATP turnover is coupled via an elastic rotary mechanism to proton translocation. Thus, the stator has to withstand the generated rotor torque, which implies tight interactions of the stator and rotor subunits. To quantitatively characterize the contribution of the F0 subunits to the binding of F1 within the assembled holoenzyme, the isolated subunit b dimer, ab2 subcomplex, and fully assembled F0 complex were specifically labeled with tetramethylrhodamine-5-maleimide at bCys64 and functionally reconstituted into liposomes. Proteoliposomes were then titrated with increasing amounts of Cy5-maleimide-labeled F1 (at gammaCys106 and analyzed by single-molecule fluorescence resonance energy transfer. The data revealed F1 dissociation constants of 2.7 nm for the binding of F0 and 9-10 nm for both the ab2 subcomplex and subunit b dimer. This indicates that both rotor and stator components of F0 contribute to F1 binding affinity in the assembled holoenzyme. The subunit c ring plays a crucial role in the binding of F1 to F0, whereas subunit a does not contribute significantly.     

Experimental Helicobacter pylori infection induces antral-predominant, chronic active gastritis in hispid cotton rats (Sigmodon hispidus)

BACKGROUND: The hispid cotton rat has proven to be an excellent animal model for a variety of human infectious disease agents. This study was performed to evaluate the use of the cotton rat as a model of Helicobacter pylori infection. MATERIALS AND METHODS: Thirty-eight inbred cotton rats were orogastrically inoculated with a human strain of H. pylori. Twenty-eight control cotton rats were dosed with vehicle only. Animals were sacrificed at 2, 4, 12, 26, or 38 weeks after inoculation for bacterial and histologic and immunologic examinations. RESULTS: Helicobacter pylori was cultured from the glandular stomach of 89% of the infected cotton rats. The level of colonization was consistently high (approximately 10(4-6) colony-forming units/g tissue). Histologically, the spiral bacteria were demonstrated on the epithelial surface and in the foveolae of the gastric mucosa with highest numbers in the antrum. H. pylori infection was associated with antral-predominant, chronic active gastritis which progressively increased in severity over time. By week 26 of infection, moderate antral gastritis had developed with frequent involvement of the submucosa and formation of lymphocytic aggregates. Splenic T cells from infected cotton rats expressed mRNAs for interferon-gamma, interleukin-4, interleukin-6, and interleukin-10 following in vitro stimulation with H. pylori. Serum levels of H. pylori-specific immunoglobulin G were significantly elevated after 12 weeks of infection. CONCLUSIONS: The H. pylori-infected cotton rat represents a novel animal model that should prove useful for studies of H. pylori-induced chronic active gastritis and factors affecting gastric colonization by this pathogen.     

A single amino acid change in rabies virus glycoprotein increases virus spread and enhances virus pathogenicity

Several rabies virus (RV) vaccine strains containing an aspartic acid (Asp) or glutamic acid (Glu) instead of an arginine (Arg) at position 333 of the RV glycoprotein (G) are apathogenic for immunocompetent mice even after intracranial inoculation. However, we previously showed that the nonpathogenic phenotype of the highly attenuated RV strain SPBNGA, which contains a Glu at position 333 of G, is unstable when this virus is passaged in newborn mice. While the Glu(333) remained unchanged after five mouse passages, an Asn(194)-->Lys(194) mutation occurred in RV G. This mutation was associated with increased pathogenicity for adult mice. Using site-directed mutagenesis to exchange Asn(194) with Lys(194) in the G protein of SPBNGA, resulting in SPBNGA-K, we show here that this mutation is solely responsible for the increase in pathogenicity and that the Asn(194)-->Lys(194) mutation does not arise when Asn(194) is exchanged with Ser(194) (SPBNGA-S). Our data presented indicate that the increased pathogenicity of SPBNGA-K is due to increased viral spread in vivo and in vitro, faster internalization of the pathogenic virus into cells, and a shift in the pH threshold for membrane fusion. These results are consistent with the notion that the RV G protein is a major contributor to RV pathogenesis and that the more pathogenic RVs escape the host responses by a faster spread than that of less pathogenic RVs.     

Excess of de novo deleterious mutations in genes associated with glutamatergic systems in nonsyndromic intellectual disability

Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.     

Matrix metalloproteinase secretion by gastric epithelial cells is regulated by E prostaglandins and MAPKs

Because matrix metalloproteinases (MMPs) play roles in inflammatory tissue injury, we asked whether MMP secretion by gastric epithelial cells may contribute to gastric injury in response to signals involved in Helicobacter pylori-induced inflammation and/or cyclooxygenase inhibition. Tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and epidermal growth factor (EGF) stimulated gastric cell MMP-1 secretion, indicating that MMP-1 secretion occurs in inflammatory as well as non-inflammatory situations. MMP-1 secretion required activation of the MAPK Erk and subsequent protein synthesis but was down-regulated by the alternate MAPK, p38. In contrast, secretion of MMP-13 was stimulated by TNF-alpha/IL-1beta but not EGF and was Erk-independent and mediated by p38. MMP-13 secretion was more rapid (peak, 6 h) than MMP-1 (peak > or =30 h) and only partly depended on protein synthesis, suggesting initial release of a pre-existing MMP-13 pool. Therefore, MMP-1 and MMP-13 secretion are differentially regulated by MAPKs. MMP-1 secretion was regulated by E prostaglandins (PGEs) in an Erk-dependent manner. PGEs enhanced Erk activation and MMP-1 secretion in response to EGF but inhibited Erk and MMP-1 when TNF-alpha and IL-1beta were the stimuli, indicating that the effects of PGEs on gastric cell responses are context-dependent. These data show that secretion of MMPs is differentially regulated by MAPKs and suggest mechanisms through which H. pylori infection and/or cyclooxygenase inhibition may induce epithelial cell signaling to contribute to gastric ulcerogenesis.     

Growth hormone receptor is a target for presenilin-dependent gamma-secretase cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-alpha-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of gamma-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the gamma-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and gamma-secretase activities and may suggest GH-independent roles for the GHR.     

Symbiotic leghemoglobins are crucial for nitrogen fixation in legume root nodules but not for general plant growth and development

Hemoglobins are ubiquitous in nature and among the best-characterized proteins. Genetics has revealed crucial roles for human hemoglobins, but similar data are lacking for plants. Plants contain symbiotic and nonsymbiotic hemoglobins; the former are thought to be important for symbiotic nitrogen fixation (SNF). In legumes, SNF occurs in specialized organs, called nodules, which contain millions of nitrogen-fixing rhizobia, called bacteroids. The induction of nodule-specific plant genes, including those encoding symbiotic leghemoglobins (Lb), accompanies nodule development. Leghemoglobins accumulate to millimolar concentrations in the cytoplasm of infected plant cells prior to nitrogen fixation and are thought to buffer free oxygen in the nanomolar range, avoiding inactivation of oxygen-labile nitrogenase while maintaining high oxygen flux for respiration. Although widely accepted, this hypothesis has never been tested in planta. Using RNAi, we abolished symbiotic leghemoglobin synthesis in nodules of the model legume Lotus japonicus. This caused an increase in nodule free oxygen, a decrease in the ATP/ADP ratio, loss of bacterial nitrogenase protein, and absence of SNF. However, LbRNAi plants grew normally when fertilized with mineral nitrogen. These data indicate roles for leghemoglobins in oxygen transport and buffering and prove for the first time that plant hemoglobins are crucial for symbiotic nitrogen fixation.     

Synthesis, in vitro, and in vivo antibacterial activity of nocathiacin I thiol-Michael adducts

The synthesis and antibacterial activity of a series of nocathiacin I derivatives (4-20) containing polar water solubilizing groups is described. Thiol-Michael adducts containing acidic polar groups have reduced antibacterial activity whereas those with basic polar groups have retained very good antibacterial activity.