Characterization of the residues in helix 8 of the human beta1-adrenergic receptor that are involved in coupling the receptor to G proteins

Several key amino acids within amphipathic helix 8 of the human beta1-adrenergic receptor (beta1-AR) were mutagenized to characterize their role in signaling by G protein-coupled receptors. Mutagenesis of phenylalanine at position 383 in the hydrophobic interface to histidine (F383H) prevented the biosynthesis of the receptor, indicating that the orientation of helix 8 is important for receptor biosynthesis. Mutagenesis of aspartic acid at position 382 in the hydrophilic interface to leucine (D382L) reduced the binding and uncoupled the receptor from G protein activation. Mutagenesis of the basic arginine residue at position 384 to glutamine (R384Q) or to glutamic acid (R384E) increased basal and agonist-stimulated adenylyl cyclase activities. R384Q and R384E displayed features associated with constitutively active receptors because inverse agonists markedly reduced their elevated basal adenylyl cyclase activities. Isoproterenol increased the phosphorylation and promoted the desensitization of the Gly389 or Arg389 allelic variants of the wild type beta1-AR but failed to produce these effects in R384Q and R384E, because these receptors were maximally phosphorylated and desensitized under basal conditions. In contrast to the membranous distribution of the wild type beta1-AR, R384Q and R384E were localized mostly within intracellular punctate structures. Inverse agonists restored the membranous distribution of R384Q and R384E, indicating that they recycled normally when their constitutive internalization was blocked by inverse agonists. These data combined with computer modeling of the putative three-dimensional organization of helix 8 indicated that the amphipathic character of helix 8 and side chain projections of Asp382 and Arg384 within the hydrophilic interface might serve as a tethering site for the G protein.     

Myocardial taurine,development and vulnerability to ischemia

Summary. Depleting intracellular taurine in heart cells improves their resistance to ischemia and reperfusion injury. The aim of this work was to see whether physiologically low levels of endogenous taurine also reflect a reduced vulnerability of the myocardium to cardiac insults. The myocardial concentration of taurine was measured during different stages of development and compared with vulnerability to ischemia and reperfusion injury in the rat and in pediatric patients undergoing cardiac surgery.Rat hearts with relatively lower levels of taurine were significantly more resistant to an ischemic inult and there was a strong negative correlation between taurine content and recovery. Childrens hearts had significantly lower taurine levels compared to infants hearts which was consistent with their known increased resistance to an ischemic cardioplegic insult (Imura et al., 2001). This work shows that the changes in the concentration of myocardial taurine during development correlate with vulnerability to ischemia where low myocardial taurine is associated with improved recovery upon reperfusion.     

Parental genome composition and genetic classifications of derivatives from intergeneric crosses of Festuca mairei and Lolium perenne

Intergeneric hybridization between Festuca and Lolium has been a long-term goal of forage and turfgrass breeders to generate improved cultivars by combining stress tolerance of Festuca and rapid establishment of Lolium. However, wide-distance hybridizations usually result in the wild genome being eliminated from the hybrid due to incomplete chromosome pairing and crossovers. In this study, random amplified polymorphic DNA (RAPD) and simple sequence repeat (SSR) markers were used to detect the parental genome composition of F₁ hybrids and backcross, generated from crosses between Festuca mairei St. Yves (Fm) and Lolium perenne L. (Lp). Each of the hybrids exhibited integration of Fm and Lp genomes with varying levels of Fm/Lp genome ratios. However, cluster and principle component analyses of the progeny consistently revealed four groups depending on the amount of genome introgression from both parents. The parental genome composition and classifications of intergeneric progeny would be useful for breeding material selection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Associations between Cognition,Gender and Monocyte Activation among HIV Infected Individuals in Nigeria

The potential role of gender in the occurrence of HIV-related neurocognitive impairment (NCI) and associations with markers of HIV-related immune activity has not been previously examined. In this study 149 antiretroviral-naïve seropositive subjects in Nigeria (SP, 92 women and 57 men) and 58 seronegative (SN, 38 women and 20 men) were administered neuropsychological testing that assessed 7 ability domains. From the neuropsychological test scores was calculated a global deficit score (GDS), a measure of overall NCI. Percentages of circulating monocytes and plasma HIV RNA, soluble CD163 and soluble CD14 levels were also assessed. HIV SP women were found to be younger, more educated and had higher CD4+ T cell counts and borderline higher viral load measures than SP men. On the neuropsychological testing, SP women were more impaired in speed of information processing and verbal fluency and had a higher mean GDS than SN women. Compared to SP men, SP women were also more impaired in speed of information processing and verbal fluency as well as on tests of learning and memory. Numbers of circulating monocytes and plasma sCD14 and sCD163 levels were significantly higher for all SP versus all SN individuals and were also higher for SP women and for SP men versus their SN counterparts. Among SP women, soluble CD14 levels were slightly higher than for SP men, and SP women had higher viral load measurements and were more likely to have detectable virus than SP men. Higher sCD14 levels among SP women correlated with more severe global impairment, and higher viral load measurements correlated with higher monocyte numbers and sCD14 and sCD14 levels, associations that were not observed for SP men. These studies suggest that the risk of developing NCI differ for HIV infected women and men in Nigeria and, for women, may be linked to effects from higher plasma levels of HIV driving activation of circulating monocytes.     

In vitro propagation of the medicinal plant Ziziphora tenuior L. and evaluation of its antioxidant activity

Ziziphora tenuior L. (Lamiaceae) is an aromatic herb used for its medicinal values against fungi, bacteria. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Z. tenuior, and to compare the antioxidant activity between in vitro propagated and wild plants.The explants were sterilized and cultured on MS medium containing different concentrations of growth regulators naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA) with 0.5 mg/L of kinetin (Kin) callus formation was 70.2% after 45 days of incubation in dark on medium supplemented with 1.5 mg/L of NAA. After one month of callus culture on medium supplemented with 2 mg/L BA the shoot number was 5.12 and for the multiplication stage. The shoot number was 4.21 and length was 6.17 cm on medium supplemented with 1 mg/L Kin + 0.1 mg/L NAA.DPPH• reagent was used to test the antioxidant activity. The aqueous and methanol extracts of in vitro plants which were treated with 1.5 and 1 mg/L of kin plus 0.1 mg/L of NAA showed a strong DPPH• scavenging activity where IC₅₀ was 0.307 and 0.369 mg/ml, respectively, while the IC₅₀ of aqueous and methanol extracts of wild plants was 0.516 and 9.229 mg/ml, respectively. Our results suggested that plant growth regulators and in vitro culture conditions increased the antioxidant activity.     

Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long-distance transport of phosphate. The effects of two hormones, gibberellic acid (GA₃) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (³²P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA³ treatment (10⁻⁴ M) of the tuber as well as of the leaves led to reduced transport of ³²P into the tuber. By contrast, treatment of the tuber with ABA (10⁻⁴M) did not change the ³²P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.     

Effects of storage,mucilage presence,photoperiod, thermoperiod and salinity on germination of Farsetia aegyptia Turra (Brassicaceae) seeds: implications for restoration and seed banks in Arabian Desert

Seed viability and germination are key factors in the success of restoration efforts, especially when stored seeds are used. However, the effect of seed storage on germination of most of the native Arabian species is not well documented. We investigated the effect of storage time and role of the seed mucilage in regulating germination, dormancy, salinity tolerance and consequential survival strategy of F. aegyptia in an unpredictable arid desert setting. Effect of light and temperature during germination was studied under two photoperiods and two thermoperiods using intact and de-mucilaged seeds. Presence of mucilage and thermoperiod did not affect the germination. However, seed collection year and photoperiod had a highly significant effect on the germination. Increasing salinity levels decreased the germination of F. aegyptia but ungerminated seeds were able to germinate when salinity stress was alleviated. Seed storage at room temperature enhances the germination percentage, indicating that F. aegyptia seeds have physiological dormancy and it can be alleviated by after-ripening at dry storage. In addition, F. aegyptia seeds show ability to germinate at lower salinity concentration and remain viable even at higher saline conditions, indicating their adaptability to cope with such harsh environmental conditions.     

Metabolic derangement and cardiac injury early after reperfusion following intermittent cross-clamp fibrillation in patients undergoing coronary artery bypass graft surgery using conventional or miniaturized cardiopulmonary bypass

Reactive oxygen species (ROS)-induced oxidative stress increases in skeletal muscle with aging and decreases the viability of implanted cells. Type 1 insulin-like growth factor (IGF-1) promotes the survival of skeletal muscle cells under oxidative stress. It is unknown whether IGF-1 protects muscle-derived stem cells (MDSCs) from oxidative stress. In this study, we genetically engineered rat MDSCs to overexpress IGF-1 and determined cell viability, apoptosis, and VEGF secretion under oxidative stress. Overexpression of IGF-1 prevented MDSCs from H₂O₂-induced caspase-dependent apoptotic cell death by upregulating the PI3K/AKT pathway, accompanied with an increase of NF-κB, p-NF-κB, Bcl-2, and VEGF, as well as a decrease of Bax. In contrast, pre-administration of picropodophyllinb, wortmannin, 1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate), or pyrrolidine-dithiocarbamate, specific inhibitors of IGF-1R, PI3K, AKT, and NF-κB, respectively, followed by treatment with H₂O₂, resulted in cell death of MDSCs. Our data indicated that IGF-1 suppresses apoptosis and enhances the paracrine function of MDSCs under oxidative stress via enhancing IGF-1R/PI3K/AKT signaling. Thus, IGF-1 gene-modified MDSCs present a potential application in the treatment of muscle wasting, such as urethra intrinsic sphincter deficiency.     

Dual role of the beta2-adrenergic receptor C terminus for the binding of beta-arrestin and receptor internalization

Homologous desensitization of beta2-adrenergic and other G-protein-coupled receptors is a two-step process. After phosphorylation of agonist-occupied receptors by G-protein-coupled receptor kinases, they bind beta-arrestins, which triggers desensitization and internalization of the receptors. Because it is not known which regions of the receptor are recognized by beta-arrestins, we have investigated beta-arrestin interaction and internalization of a set of mutants of the human beta2-adrenergic receptor. Mutation of the four serine/threonine residues between residues 355 and 364 led to the loss of agonist-induced receptor-beta-arrestin2 interaction as revealed by fluorescence resonance energy transfer (FRET), translocation of beta-arrestin2 to the plasma membrane, and receptor internalization. Mutation of all seven serine/threonine residues distal to residue 381 did not affect agonist-induced receptor internalization and beta-arrestin2 translocation. A beta2-adrenergic receptor truncated distal to residue 381 interacted normally with beta-arrestin2, whereas its ability to internalize in an agonist-dependent manner was compromised. A similar impairment of internalization was observed when only the last eight residues of the C terminus were deleted. Our experiments show that the C terminus distal to residue 381 does not affect the initial interaction between receptor and beta-arrestin, but its last eight amino acids facilitate receptor internalization in concert with beta-arrestin2.     

In vitro screening of Fe2+‐chelating effect by a Fenton's reaction–luminol chemiluminescence system

In vitro screening of a Fe²⁺‐chelating effect using a Fenton's reaction–luminol chemiluminescence (CL) system is described. The luminescence between the reactive oxygen species generated by the Fenton's reaction and luminol was decreased on capturing Fe²⁺ using a chelator. The proposed method can prevent the consumption of expensive seed compounds (drug discovery candidates) owing to the high sensitivity of CL detection. Therefore, the assay could be performed using small volumes of sample solution (150 μL) at micromolar concentrations. After optimization of the screening conditions, the efficacies of conventional chelators such as ethylenediaminetetraacetic acid (EDTA), diethylentriaminepentaacetic acid (DETAPAC), deferoxamine, deferiprone and 1,10‐phenanthroline were examined. EC₅₀ values for these compounds (except 1,10‐phenanthroline) were in the range 3.20 ± 0.87 to 9.57 ± 0.64 μM (n = 3). Rapid measurement of the Fe²⁺‐chelating effect with an assay run time of a few minutes could be achieved using the proposed method. In addition, the specificity of the method was discussed. Copyright © 2014 John Wiley & Sons, Ltd.