Structural and Functional Analysis of a β₂-Adrenergic Receptor Complex with GRK5

Komolov, Konstantin E.,Du, Yang,Duc, Nguyen Minh,Betz, Robin M.,Rodrigues, Joã,o P.G.L.M.,Leib, Ryan D.,Patra, Dhabaleswar,Skiniotis, Georgios,Adams, Christopher M.,Dror, Ron O.,Chung, Ka Young Cell Press 2017 Cell Vol. No.

<P><B>Summary</B></P> <P>The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β<SUB>2</SUB>-adrenergic receptor (β<SUB>2</SUB>AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the β<SUB>2</SUB>AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs.</P> <P><B>PaperClip</B></P> <P>Display Omitted</P> <P><B>Highlights</B></P> <P> <UL> <LI> GRK5-β<SUB>2</SUB>AR binding is enhanced by receptor and kinase ligands and acidic lipids </LI> <LI> GRK5 binding to the β<SUB>2</SUB>AR involves a multi-site interaction </LI> <LI> Receptor binding triggers substantial conformational changes in GRK5 </LI> <LI> RH/catalytic domain separation in GRK5 is essential for receptor phosphorylation </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Ingestive Behavior of Heifers Supplemented with Glycerin in Substitution of Corn on Brachiaria brizantha Pasture

Facuri, L.M.A.M.,Silva, Roberio Rodrigues,da Silva, F.F.,de Carvalho, G.G.P.,Sampaio, C.B.,Mendes, F.B.L.,Lisboa, M.M.,Barroso, D.S.,Carvalho, V.M.,Pereira, M.M.S. Asian Australasian Association of Animal Productio 2014 Animal Bioscience Vol.27 No.11

The objective was to evaluate the ingestive behavior of crossbred heifers finished on a Brachiaria brizantha cv. Marandu pasture receiving four levels of glycerin in their supplementation. Thirty-six crossbred heifers with average initial weight of $264.83{\pm}3.83kg$ and 20 months of age were distributed into a completely randomized design with four treatments and nine replications: control (0%), 4.82%, 10.12%, and 15.56% glycerin in the dry matter. The grazing time reduced linearly (p<0.05), whereas the time spent on activities like rumination, idleness, trough and total chewing time were quadratically affected (p<0.05). Bite rate and number of bites/day were quadratically influenced (p<0.05). The number of bites/swallowed cud and the number of bites/minute, however, increased linearly (p<0.05). Although the time spent on each cud and number of chews per cud were not affected (p>0.05).The number of rumination periods reduced linearly (p<0.05), whereas the number of grazing, idle and trough periods, and the times per grazing, idle, rumination and trough periods were quadratically affected (p<0.05). The feed and rumination efficiencies of the dry matter, non-fibrous carbohydrates, pasture dry matter and concentrate were quadratically affected (p>0.05) whereas the feed efficiency of neutral detergent fiber reduced linearly (p<0.05). Addition of glycerin in substitution of corn in supplements for animals managed on pastures does not influenced feed intake, but reduces the grazing time and increases the idle time. The supplementation also improves feed and rumination efficiencies.

Phase II study of S-1 combined with oxaliplatin as therapy for patients with metastatic biliary tract cancer: influence of the <i>CYP2A6</i> polymorphism on pharmacokinetics and clinical activity

Kim, K-p,Jang, G,Hong, Y S,Lim, H-S,Bae, K-s,Kim, H-S,Lee, S S,Shin, J-G,Lee, J-L,Ryu, M-H,Chang, H-M,Kang, Y-K,Kim, T W Nature Publishing Group 2011 The British journal of cancer Vol.104 No.4

<P><B>Background:</B></P><P>Advanced biliary cancer is often treated with fluoropyrimidine-based chemotherapy. In this study, we evaluated the efficacy and tolerability of a combination of S-1, an oral fluoropyrimidine prodrug, and oxaliplatin in patients with metastatic biliary cancer.</P><P><B>Methods:</B></P><P>Patients with histologically confirmed metastatic biliary cancer and no history of radiotherapy or chemotherapy were enrolled. Oxaliplatin was administered intravenously (130 mg m<SUP>−2</SUP>), followed by 14-day administration of oral S-1 (40 mg m<SUP>−2</SUP> twice daily) with a subsequent 7-day rest period every 21 days. Pharmacokinetic analysis of S-1 was performed at cycle 1. Patients were genotyped for <I>CYP2A6</I> polymorphisms (<SUP>*</SUP>1, <SUP>*</SUP>4, <SUP>*</SUP>7, <SUP>*</SUP>9 or <SUP>*</SUP>10), and pharmacokinetic and clinical parameters compared according to the <I>CYP2A6</I> genotype.</P><P><B>Results:</B></P><P>In total, 49 patients were evaluated, who received a median of four cycles. The overall response rate was 24.5%. Median progression-free and overall survival was 3.7 and 8.7 months, respectively. The most common haematological grade 3 out of 4 toxicity was neutropenia (14%), while non-hematological grade 3 out of 4 toxicities included anorexia (14%), nausea (12%), asthenia (10%), vomiting (10%), and diarrhoea (4%). Biotransformation of S-1 (AUC<SUB>0−24 h</SUB> of 5-fluorouracil/AUC<SUB>0−24 h</SUB> of tegafur) was 1.85-fold higher for the <I>*1/*1</I> group than for the other groups (90% confidence interval 1.37–2.49). Diarrhoea (<I>P</I>=0.0740), neutropenia (<I>P</I>=0.396), and clinical efficacy (response rate, <I>P</I>=0.583; PFS, <I>P</I>=0.916) were not significantly associated with <I>CYP2A6</I> genotype, despite differences in 5-FU exposure.</P><P><B>Conclusion:</B></P><P>The combination of S-1 and oxaliplatin appears to be active and well tolerated in patients with metastatic biliary cancer, and thus is feasible as a therapeutic modality. <I>CYP2A6</I> genotypes are associated with differences in the biotransformation of S-1. However, the impact of the <I>CYP2A6</I> polymorphism on variations in clinical efficacy or toxicity requires further evaluation.</P>

High polarity analytes in food - enrofloxacin and sulfadiazine in bovine tissue (CCQM-K141)

Windust, Anthony,McRae, Garnet,Meija, Juris,Mester, Zoltá,n,Melanson, Jeremy E,Croft, Meg,Johnston, Lesley,Murby, John,Rego, Eliane C P do,Violante, Fernando G M,Fernandes, Jane L N,Wollinger, W BUREAU INTERNATIONAL DES POIDS ET MESURES 2019 METROLOGIA -BERLIN- Vol.56 No.-

<P></P> <P>Within the Organic Analysis Working Group (OAWG) of the Comité Consultatif pour la Quantitè de Matiére (CCQM), key comparison CCQM-K141 and associated pilot study CCQM-P178 were coordinated by the National Research Council Canada (NRC). This comparison was a Track A key comparison that formed part of the OAWG 10-year strategic plan. The comparison demonstrated capabilities for measuring high-polarity analytes in a high-fat and high-protein matrix. The measurand chosen as the model system was enrofloxacin and sulfadiazine in bovine tissue. Thirteen National Metrology Institutes or Designated Institutes participated in the CCQM-K141, while two National Metrology Institutes participated in CCQM-P178.</P> <P>The bovine muscle tissue study material was derived from a single live animal that was administered with chemical based pharmaceutical agents prior to processing. Therefore, the study material was naturally incurred, providing a true test of extraction procedures relative to more commonly encountered spiked materials. NRC confirmed excellent homogeneity and stability of the material prior to shipping. Three 10 g bottles of freeze dried powdered muscle tissue were supplied. NRC also provided isotopically labelled solutions of the two measurands, <SUP>13</SUP>C<SUB>6</SUB>-sulfadiazine and enrofloxacin-d<SUB>5</SUB> (HI Salt), to those interested in using isotope dilution mass spectrometry (IDMS) methodologies. Procurement and purity assignment with appropriate metrological traceability of native calibrants were the responsibility of individual participants. The study required extraction, clean-up, analytical separation, and selective detection of the analytes.</P> <P>The level of agreement was reasonable given the measurands and matrix were new for most laboratories. The KCRV values and their uncertainties at the 95% confidence level of 57.81 ± 2.57 μg/kg for enrofloxacin and 2285 ± 68 μg/kg for sulfadiazine were calculated using the DSL means. While one participant's value was voluntarily excluded from the KCRV calculations for enrofloxacin, all other participants demonstrated equivalence for both measurands.</P> <P>Significant effort was undertaken post-study to identify the major sources of variability between results. In particular, the various extraction conditions used by participants were investigated thoroughly. While there appeared to be a correlation between highly acidic conditions and higher recovery, this was not definitive and could not be confirmed. The form of standards employed (i.e. free base vs salts) and potential differential solubility between forms was also a suspected source of variability. Biases could also have been introduced with the choice of solvents used for standard preparation, with some solvents better able to minimize adsorption of the analytes to glass surfaces.</P> <P>KEY WORDS FOR SEARCH</P> <P>enrofloxacin, sulfadiazine, bovine tissue, incurred, key comparison</P> <P></P> <H2>Main text</H2> <P> To reach the main text of this paper, click on <A HREF='https://www.bipm.org/utils/common/pdf/final_reports/QM/K141/CCQM-K141.pdf'>Final Report</A>. Note that this text is that which appears in Appendix B of the BIPM key comparison database <A HREF='http://kcdb.bipm.org/'>kcdb.bipm.org/</A>.</P> <P>The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).</P>

Report of the CCQM-K97: measurement of arsenobetaine standard solution and arsenobetaine content in fish tissue (tunafish)

Ma, L D,Wang, J,WEI, C,Kuroiwa, T,Narukawa, T,Ito, N,HIOKI, A,CHIBA, K,Yim, Y H,Lee, K S,Lim, Y R,Turk, G C,Davis, C W,Mester, Z,Yang, L,McCooeye, M,Maxwell, P,Cankur, O,Tokman, N,Coskun, F G BUREAU INTERNATIONAL DES POIDS ET MESURES 2017 METROLOGIA -BERLIN- Vol.54 No.-

<P></P> <P>The CCQM-K97 key comparison was organized by the inorganic analysis working group (IAWG) of CCQM as a follow-up to completed pilot study CCQM-P96 and P96.1 to test the abilities of the national metrology institutes to accurately quantitate the mass fraction of arsenobetaine (AsB) in standard solution and in fish tissue. A pilot study CCQM-P133 was parallelized with this key comparison. National Institute of Metrology (NIM), China and National Metrology Institute of Japan (NMIJ) acted as the coordinating laboratories.</P> <P>Six NMIs participated in CCQM-K97 and two institutes participated in CCQM-P133, and all of them submitted the results. Some NMIs submitted more than one results by different methods. The results were in excellent agreement with each other, and obviously better than those of previous P96 and P96.1. Therefore the calibrant which each NMI used was comparable. It shows that the capabilities of some of the participants have been improved after the previous pilot studies.</P> <H2>Main text</H2> <P> To reach the main text of this paper, click on <A HREF='http://www.bipm.org/utils/common/pdf/final_reports/QM/K97/CCQM-K97.pdf'>Final Report</A>. Note that this text is that which appears in Appendix B of the BIPM key comparison database <A HREF='http://kcdb.bipm.org/'>kcdb.bipm.org/</A>.</P> <P>The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).</P>

Comparison of Methodologies to Quantify Phytate Phosphorus in Diets Containing Phytase and Excreta from Broilers

de P. Naves, L.,Rodrigues, P.B.,Bertechini, A.G.,Correa, A.D.,de Oliveira, D.H.,de Oliveira, E.C.,Duarte, W.F.,da Cunha, M.R.R. Asian Australasian Association of Animal Productio 2014 Animal Bioscience Vol.27 No.7

The use of a suitable methodology to quantify the phytate phosphorus ($P_{phy}$) content in both the feed and the excreta from broilers is required to enable accurate calculation of the catalytic efficiency of the phytase supplemented in the feed. This study was conducted to compare 2 analytical methodologies (colorimetry and also high-performance liquid chromatography with a refractive index detector) in order to calculate the phytase efficiency by utilizing the results from the methodology that was shown to be the most appropriate. One hundred and twenty broilers were distributed in a $(4+1){\times}2$ factorial arrangement, corresponding to 4 diets that were equally deficient in P supplemented with increasing levels of phytase (0, 750, 1,500, and 2,250 units of phytase activity - FTU - per kg of feed) plus 1 positive control diet without phytase, supplied to male and female birds. The result indicated that the colorimetric methodology with an extraction ratio of 1:20 (mass of sample in g:volume of the solvent extractor in mL) was shown to be the most adequate. There was no interaction between the phytase level and the sex of the broilers (p>0.05). Males consumed 12% more $P_{phy}$ than did females (p<0.01), but the sex of the broilers did not affect (p>0.05) the excretion and retention coefficient of $P_{phy}$. The increase in the phytase level of the diet reduced (linear, p<0.01) the $P_{phy}$ excretion. The greatest $P_{phy}$ retention was estimated at 87.85% when the diet contained 1,950 FTU/kg (p<0.01), indicating that it is possible to reduce the inorganic P in the formulation at an amount equivalent to 87.85% of the $P_{phy}$ content present in the feed, which, in this research, corresponds to a decrease in 2.86 g of P/kg of the feed.

Intramuscular Administration of Zinc Metallothionein to Preslaughter Stressed Pigs Improves Anti-oxidative Status and Pork Quality

Li, L.L.,Hou, Z.P.,Yin, Y.L.,Liu, Y.H.,Hou, D.X.,Zhang, B.,Wu, G.Y.,Kim, S.W.,Fan, M.Z.,Yang, C.B.,Kong, X.F.,Tang, Z.R.,Peng, H.Z.,Deng, D.,Deng, Z.Y.,Xie, M.Y.,Xiong, H.,Kang, P.,Wang, S.X. Asian Australasian Association of Animal Productio 2007 Animal Bioscience Vol.20 No.5

This study was conducted to determine the effects of exogenous zinc-metallothionein (Zn-MT) on anti-oxidative function and pork quality. After feeding a corn-soybean meal-based diet for two weeks, 48 pigs ($Duroc{\times}Landrace{\times}Chinese\;Black Pig$) were assigned randomly to four groups. Pigs in Group 1 were maintained under non-stress conditions, whereas pigs in Groups 2, 3 and 4 were aggressively handled for 25 min to produce stress. Pigs in Groups 1, 2, 3, and 4 received intramuscular administration of saline (control group; CON), 0 (negative control group; NCON), 0.8 (low dose group; LOW), and 1.6 (high dose group; HIGH) mg rabbit liver Zn-MT per kg body weight, respectively. Pigs were slaughtered at 3 and 6 h post-injection. Zn-MT treatment increased (p<0.05) the activities of superoxide dismutase (SOD) and glutathione-peroxidase (GSH-PX) while decreasing the concentration of malondialdehyde (MDA) in liver. These responses were greater (p<0.05) at 6 h than at 3 h post Zn-MT injection. Zn-MT treatment increased (p<0.05) hepatic SOD mRNA levels in a time and dose-dependent manner and decreased (p<0.05) serum glutamate-pyruvate transaminase and lactate dehydrogenase activities (indicators of tissue integrity). Zn-MT administration decreased (p<0.05) lactate concentration and increased (p<0.05) pH and water-holding capacity in the longissimus thorasis meat. Collectively, our results indicate that intramuscular administration of Zn-MT to pre-slaughter stressed pigs improved tissue anti-oxidative ability and meat quality.

Reproductive Response of Ewes Fed with Taiwan Grass Hay (Pennisetum purpureum Schum.) Supplemented with Duckweed (Lemna sp. and Spirodela sp.)

Zetina-Cordoba, P.,Ortega-Cerrilla, M.E.,Torres-Esqueda, M.T. Sanchez,Herrera-Haro, J.G.,Ortega-Jimenez, E.,Reta-Mendiola, J.L.,Vilaboa-Arroniz, J. Asian Australasian Association of Animal Productio 2012 Animal Bioscience Vol.25 No.8

The effect of duckweed (DW) supplementation was evaluated on dry matter intake (DMI), presence and duration of estrus, percentage of ewes repeating estrus and pregnancy rate, as well as the concentration of progesterone ($P_4$) in multiparous crossbred ewes from Pelibuey, Dorper, and Katahdin breeds, fed with Taiwan grass hay (TWH). Eighteen ewes with $39.7{\pm}4kg$ mean body weight, kept in individual pens, were randomly assigned to one of the following treatments: $T_1$: TWH, $T_2$: TWH plus 200 g DW, $T_3$: TWH plus 300 g DW. The ewes were synchronized with 40 mg fluorogestone acetate (FGA) and 400 UI equine chorionic gonadotropin (eCG). Data were analyzed as a completely randomized design using the GLM procedure. DW supplementation had no effect on dry matter intake (p>0.05); however, a slight decrease of TWH intake was observed as DW supplementation increased. No differences (p>0.05) were found in the beginning of estrus, percentage of ewes presenting it, its duration, or pregnancy rate. There were no differences (p>0.05) on $P_4$ concentration among treatments, or $treatment{\times}period$ interaction (p>0.05). However the period was significant (p<0.01), since the $P_4$ levels increased as time increased after the removal of the FGA device and eCG application.

Nitrogen Metabolism in Lactating Goats Fed with Diets Containing Different Protein Sources

Santos, A.B.,Pereira, M.L.A.,Silva, H.G.O.,Pedreira, M.S.,Carvalho, G.G.P.,Ribeiro, L.S.O.,Almeida, P.J.P.,Pereira, T.C.J.,Moreira, J.V. Asian Australasian Association of Animal Productio 2014 Animal Bioscience Vol.27 No.5

This study aimed to evaluate urea excretion, nitrogen balance and microbial protein synthesis in lactating goats fed with diets containing different protein sources in the concentrate (soybean meal, cottonseed meal, aerial part of cassava hay and leucaena hay). Four Alpine goats whose mean body weight was $42.6{\pm}6.1kg$ at the beginning of the experiment, a mean lactation period of $94.0{\pm}9.0days$ and a production of $1.7{\pm}0.4kg$ of milk were distributed in a $4{\times}4$ Latin square with four periods of 15 days. Diets were formulated to be isonitrogenous, containing 103.0 g/kg of CP, 400 g/kg of Tifton 85 hay and 600 g/kg of concentrate. Diet containing cottonseed meal provided (p<0.05) increased excretion of urea and urea nitrogen in the urine (g/d and mg/kg of BW) when compared with leucaena hay. The diets affected the concentrations of urea nitrogen in plasma (p<0.05) and excretion of urea nitrogen in milk, being that soybean meal and cottonseed meal showed (p<0.05) higher than the average aerial part of the cassava hay. The use of diets with cottonseed meal as protein source in the concentrate in feeding of lactating goats provides greater nitrogen excretion in urine and negative nitrogen balance, while the concentrate with leucaena hay as a source of protein, provides greater ruminal microbial protein synthesis.

Characterization of Nutritional Value for Twenty-one Pork Muscles

Kim, J.H.,Seong, P.N.,Cho, S.H.,Park, B.Y.,Hah, K.H.,Yu, L. H.,Lim, D.G.,Hwang, I.H.,Kim, D.H.,Lee, J.M.,Ahn, C.N. Asian Australasian Association of Animal Productio 2008 Animal Bioscience Vol.21 No.1

A study was conducted to evaluate nutritional value for twenty-one pork muscles. Ten market-weight crossbred pigs (five gilts and five barrows) were used for evaluating proximate chemical composition, cholesterol, total iron, calorie and fatty acid contents. As preliminary analysis revealed no noticeable sex effect, pooled data from both sexes were used for the final analysis. M. rectus femoris had the highest moisture content, while m. latissimus dorsi was lowest in moisture content (p<0.05). Protein content was highest for m. longissimus dorsi and lowest for m. supraspinatus (p<0.05). The tensor fasciae and latissimus dorsi muscles contained the highest intramuscular fat (p<0.05), while rectus femoris, adductor and vastus lateralis were lowest in intramuscular fat content. When simple correlations between chemical values were computed for the pooled dataset from all muscles, intramuscular fat had significant (p<0.05) negative linear relationships with moisture (r = -0.85) and protein (r = -0.51) contents. Calorie levels were not significantly affected by fat content, while rectus femoris and latissimus dorsi muscles showed lowest and highest calorie contents, respectively (p<0.05). Polyunsaturated fatty acid content was highest (p<0.05) for both m. adductor and m. rectus femoris, while it was lowest for m. longissimus dorsi. Collectively, the current study identified a large amount of variation in nutritional characteristics between pork muscles, and the data can be used for the development of muscle-specific strategies to improve eating quality of meats and meat products.