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.     

Effect of regular training on the myocardial and plasma concentrations of taurine andα-amino acids in thoroughbred horses

Summary Exercise induces significant changes in the free intracellular amino acid pool in skeletal muscle but little is known of whether such changes also occur in cardiac muscle. In this study the effect of regular exercise on the size and the constituents of the free amino acid pool in the hearts and in the plasma of thoroughbred horses was investigated. The total free intracellular amino acid pool in the hearts of control horses was 30.9 ± 1.2mol/g wet weight (n = 6). Glutamine but not taurine was present at the highest concentration (13.5 ± 0.9 and 7.7 ± 0.69mol/g wet weight for glutamine and taurine respectively). As for the rest of the amino acids in the pool, only glutamate and alanine were present at levels greater than 1mol/g wet weight (4.6 ± 0.25 and 1.7 ± 0.14 for glutamate and alanine respectively). The tissue to plasma ratio was highest for taurine at 155, followed by glutamate at 111, aspartate and glutamine at 37, alanine at 5.8 and ratios of less than 3 for the rest of the amino acids. The total free intracellular amino acid pool in the hearts of exercised horses was slightly but not significantly lower than control (28.1 ±1.1mol/g wet weight, n = 6). Regular exercise increased the intracellular concentration of threonine, valine, isoleucine, leucine and phenylalanine but was only significant (p < 0.05) for threonine. This work has documented the profile of taurine and protein amino acids in the heart and in the plasma of thoroughbred horses and showed that in contrast to skeletal muscle, heart muscle does not show major changes in amino acids during regular exercise.     

Genetic analysis of an F2 intercross between two strains of Japanese quail provided evidence for quantitative trait loci affecting carcass composition and internal organs

The purpose of this study was to identify genomic regions, quantitative trait loci (QTL), affecting carcass traits on chromosome 1 in an F2 population of Japanese quail. For this purpose, two white and wild strains of Japanese quail (16 birds) were crossed reciprocally and F1 generation (34 birds) was created. The F2 generation was produced by intercrossing of the F1 birds. Phenotypic data including carcass weight, internal organs and carcass parts were collected on F2 animals (422 birds). The total mapping population (472 birds) was genotyped for 8 microsatellite markers on chromosome 1. QTL analysis was performed with interval mapping method applying the line-cross model. Significant QTL were identified for breast weight at 0 (P < 0.01), 172 (P < 0.05) and 206 (P < 0.01), carcass weight at 91 (P < 0.05), carcass fatness at 0 (P < 0.01), pre-stomach weight at 206 (P < 0.01) and uropygial weight gland at 197 (P < 0.01) cM on chromosome 1. There was also evidence for imprinted QTL affecting breast weight (P < 0.01) on chromosome 1. The proportion of the F2 phenotypic variation explained by the significant additive, dominance and imprinted QTL effects ranged from 1.0 to 7.3 %, 1.2 to 3.3 % and 1.4 to 2.2 %, respectively.     

L‐leucine transport in rat heart under normal conditions and effects of a simulated hypoxia

L-leucine plays a central role in the regulation of protein metabolism in heart and has been implicated in myocardial protection, but little is known about the relationship between these phenomena and leucine transport across the cardiac sarcolemma. In this study we used sarcolemmal vesicles and ventricular myocytes isolated from rat heart to characterise L-leucine transport under normal conditions and to investigate the effect of simulated hypoxia or inhibition of protein synthesis. The Km and Vmax of leucine uptake were 5.24+/-0.65 mM and 1.43+/-1.84 nmol min(-1) mg(-1) protein in vesicles compared to 2.17+/-0.13 mM and 1.7+/-0.76 nmol min(-1) microl(-1) intracellular space in cells. Transport was not dependent on Na+ or H+ gradients. In vesicles L-leucine uptake was increased by trans-stimulation, whilst inhibition was observed with classical system L substrates including 2-aminobicyclo[2,2,1]-heptane-2-carboxylic acid (BCH) suggesting that this system mediated L-leucine transport in heart. L-Leucine uptake into isolated cardiac myocytes was inhibited after 20, 30 and 60 min of simulated hypoxia. This was not caused by reduced cell viability, although the cells underwent a rigor contracture. Inhibition of protein synthesis did not affect L-leucine transport.     

Predator–prey relationships in a middle Asian Montane steppe: Persian leopard versus urial wild sheep in Northeastern Iran

Management controversies arise when both of the prey and predator in an ecosystem are species of conservation concern. We investigated trophic interactions between the endangered Persian leopard (Panthera pardus saxicolor) and a declining mountain ungulate, urial wild sheep (Ovis vignei), on a high-altitude steppe of Iran. During two consecutive photo-trapping seasons of 1,300 nights in total, a minimum population of four adult leopards (one female and three males) was documented. Scat analysis indicated that urial wild sheep was the staple of the leopard diet with 48.44 % of total biomass consumed. Remains of domestic livestock in leopard scats were negligible yet alarming (14.53 % biomass consumed), followed by wild pigs (8.13 %) and wild goat (1.26 %). Financial costs of leopard depredation to livestock breeders during our study period were comparatively lower than livestock–leopard conflict hotspots across Iran. Using distance sampling, urial density was 15.8 individuals km^?2 (±SE 6.2), and a total biomass of 47,621.5 kg for wild ungulates in the study area was estimated. We estimated that the annual removal rate of urial by leopards during our study period was 9.4 % of the total urial population. We suggest that continuous monitoring of the leopard and prey populations to assess predation impact should be considered, particularly in areas where a single species comprises a remarkable proportion of the leopard diet. In the meantime, assessing probable conflicts with local communities is recommended as a parallel management action to ensure long-term human–leopard coexistence. Our findings will aid wildlife managers in prey-depleted arid environments of western Asia to identify susceptible wild prey populations to predation by large carnivores; hence, significantly contribute in development and implementation of effective conservation measures to mitigate management conflicts.     

Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth

The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.     

Kinetics of infected insect cell osmolysis and enhanced protein release using a modified disruption method

We have studied and characterized a cell disruption method to produce a protein extract from recombinant Baculovirus infected insect cells based on osmotic lysis. Cell lysis kinetics were measured during a 24-h incubation in lysis buffer and resulting data sets were curve fitted to a hyperbola, visually similar to the Michaelis–Menten curve, to determine the maximum concentration of released protein and the time required to reach equilibrium. Effect of parameters such as pH, ionic strength and infection phase were evaluated, and based on fittings optimal protein release conditions were obtained for total cell protein as well as the recombinant protein, HPV 16 L1. It was demonstrated that pH and the phase of infection can vastly influence the amount of release while ionic strength only effects the time required to achieve equilibrium in protein release. Osmolysis can be a mild, yet effective method to release recombinant protein with high recovery levels and hence can be used in capacities where stringent criteria regarding contamination with surfactant or non-cytoplasmic contents are observed.     

Decellularized amniotic membrane Scaffolds improve differentiation of iPSCs to functional hepatocyte-like cells

Human-induced pluripotent stem cells-derived hepatocyte-like cells (hiPSCs-HLCs) holds considerable promise for future clinical personalized therapy of liver disease. However, the low engraftment of these cells in the damaged liver microenvironment is still an obstacle for potential application. In this study, we explored the effectiveness of decellularized amniotic membrane (dAM) matrices for culturing of iPSCs and promoting their differentiation into HLCs. The DNA content assay and histological evaluation indicated that cellular and nuclear residues were efficiently eliminated and the AM extracellular matrix component was maintained during decelluarization. DAM matrices were developed as three-dimensional scaffolds and hiPSCs were seeded into these scaffolds in defined induction media. In dAM scaffolds, hiPSCs-HLCs gradually took a typical shape of hepatocytes (polygonal morphology). HiPSCs-HLCs that were cultured into dAM scaffolds showed a higher level of hepatic markers than those cultured in tissue culture plates (TCPs). Moreover, functional activities in term of albumin and urea synthesis and CYP3A activity were significantly higher in dAM scaffolds than TCPs over the same differentiation period. Thus, based on our results, dAM scaffold might have a considerable potential in liver tissue engineering, because it can improve hepatic differentiation of hiPSCs which exhibited higher level of the hepatic marker and more stable metabolic functions.