Direct high-performance liquid chromatography determination of diastereomeric oxprenolol glucuronides

The β-blocking agent oxprenolol is used therapeutically as the racemate. In humans and animals it is metabolized i.a. to ether glucuronide diastereomers. A stereoselective HPLC assay was developed to determine directly, without hydrolysis to their parent enantiomers, the oxprenolol glucuronides in biological samples. The glucuronide standards for this direct assay are prepared by incubation of rabbit liver microsomes with RS-oxprenolol. The glucuronides obtained are purified and concentrated with solid-phase extraction, and their concentration is measured by an indirect method, i.e. HPLC assay of the oxprenolol enantiomers after enzymatic hydrolysis with β-glucuronidase. The direct assay involves separation by HPLC using a C₁₈-reversed-phase column, with UV detection at 274 nm; nalorphine is used as internal standard. On injection onto the column, without previous hydrolysis, the limit of detection is 20 ng for both glucuronides. The assay is sensitive, accurate and reproducible. The method is suitable for the assay of glucuronides in liver microsomal incubates and plasma.     

Determination of serotonin, catecholamines and their metabolites by direct injection of supernatants from chicken brain tissue homogenate using liquid chromatography with electrochemical detection

An isocratic liquid chromatographic method with electrochemical detection for the determination of

Full-size image

-3,4-dihydroxyphenylalanine, dopamine, norepinephrine, epinephrine, serotonin, and their major metabolites, 3,4-dihydroxyphenylacetic acid, 4-hydroxy-3-methoxyphenylacetic acid and 5-hydroxyindole-3-acetic acid in chicken brain tissue is described. Chickens were killed at different ages, the brains were quickly frozen and 300-μm cryostat sections were made. From these sections, two to six tissue micropunches (1 mm in diameter) were punched out from 20 different areas of the hypothalamus and homogenated in 100 μl 0.1 M perchloric acid which included 0.01% cysteine as antioxidant. Fifty-μl supernatants were injected directly onto the LC system, separated on a 3-μm Phase II ODS column (100×3.2 mm I.D.) and detected by an electrochemical detector at a potential of +0.75 V. Standard curves, recoveries, analytical precision and detection limits were investigated for each monoamine neurotransmitter and its metabolites. The method was applied to study the influence of food restriction on the concentration of monoamine neurotransmitters in different brain areas, known to be involved in feeding and reproductive behaviour of female broiler chickens. Over 1000 micropunched tissue samples from ad libitum fed and food-restricted female broiler chickens were analyzed. Our results provide a possible role for catecholamines and indolamines in the altered feeding and reproductive behaviour of the broiler chicken.     

Effect of an 18-wk weight-training program on energy expenditure and physical activity

Van Etten, Ludo M. L. A., Klaas R. Westerterp, Frans T. J. Verstappen, Bart J. B. Boon, and Wim H. M. Saris. Effect of an18-wk weight-training program on energy expenditure and physicalactivity. J. Appl. Physiol. 82(1):298-304, 1997.The purpose of this study was to examine theeffect of an 18-wk weight-training program on average daily metabolicrate (ADMR). Before the intervention and in weeks8 and 18 (T₀,T₈, andT₁₈, respectively) data on bodycomposition, sleeping metabolic rate (SMR), food intake, energy cost ofthe weight-training program(EE_ex), and nontraining physicalactivity (accelerometer) were collected in the exercise group (EXER,n = 18 males). ADMR was determined ina subgroup (EX12, n = 12) by usingdoubly labeled water. At T₀ andT₁₈, data (except ADMR) were alsocollected in a control group (Con, n = 8). Body mass did not change in EXER or Con. Fat-free mass increased only in EXER with 2.1 ± 1.2 kg, whereas fat mass decreased in EXERas well as Con (2.0 ± 1.8 and 1.4 ± 1.0 kg, respectively). Initial ADMR (12.4 ± 1.2 MJ/day) increased atT₈ (13.5 ± 1.3 MJ/day, P < 0.001) with no further increaseat T₁₈ (13.5 ± 1.9 MJ/day). SMR did not change in EXER (4.8 ± 0.5, 4.9 ± 0.5, 4.8 ± 0.5 kJ/min) or Con (4.7 ± 0.4, 4.8 ± 0.4 kJ/min). Energy intake didnot change in EXER (10.1 ± 1.8, 9.7 ± 1.8, 9.2 ± 1.9 MJ/day) or Con (10.2 ± 2.6, 9.4 ± 1.8, 10.1 ± 1.5 MJ/day)and was systematically underreported in EX12 (21 ± 14, 28 ± 18, 34 ± 14%,P < 0.001).EE_ex (0.47 ± 0.20, 0.50 ± 0.18 MJ/day) could only explain 40% of the increase in ADMR.Nontraining physical activity did not change in both groups. Inconclusion, although of modest energy cost, weight-training induces asignificant increase in ADMR.

     

Influence of age and breed on the binding of oxygen to red blood cells of bovine calves

Gustin, P., B. Detry, A. Robert, M. L. Cao, F. Lessire, C. Cambier, V. Katz, M. Ansay, A. Frans, and T. Clerbaux.Influence of age and breed on the binding of oxygen to red bloodcells of bovine calves. J. Appl.Physiol. 82(3): 784-790, 1997.The influence ofsomatic growth and genetic selection on the whole blood oxygen equilibrium curve (OEC) was measured under standard conditions indouble-muscled and dairy calves during their first 3 mo of life.Crossbreed animals were also investigated. Hemoglobin,2,3-diphosphoglycerate (DPG), Cl, andP_i concentrations were alsomeasured. The percentage of fetal hemoglobin (HbF) was determined. Theinfluence of exogenous Cl, P_i, andpH on the OEC was also assessed. ThePO₂ at 50% hemoglobin saturation(P₅₀) increased during somaticgrowth, probably because of the increase in DPG recorded indouble-muscled neonates and to the progressive disappearance of HbF inboth breeds. The oxygen exchange fraction (OEF%) was used to assessthe combined influence of the OEC shift and OEC shape changes on bloodoxygen desaturation under standard conditions, when thePO₂ decreases within a physiologicalrange. The OEF% showed an increase during the first month, then astabilization. The effects of Cl, P_i, and pH in Friesian calves weresimilar as in adult cattle. Double-muscled neonates had alower P₅₀, OEF% values, and DPG concentrations and higher hemoglobin and Cl concentrations than Friesian neonates. The P_iconcentration and the percentage of HbF were similar in both breeds.The pH and the Cl concentration had significantly less effect on theOEC in double-muscled than in Friesian calves. Crossbreed animalsexhibited intermediate parameter values, between those recorded fordouble-muscled and Friesian calves. All differences between breedsprogressively disappeared during the first month. These data show thatblood function changes markedly in calves during the first month of life and that genetic selection can alter blood function.

     

Characterization of csi52, a Cs+ resistant mutant of Arabidopsis thaliana altered in K+ transport

Plant roots accumulate potassium from a wide range of soil concentrations, utilizing at least two distinct plasma membrane uptake systems with different affinities for the cation. Although details on the structure and function of these transporters are beginning to emerge many prominent questions remain concerning how these proteins function in planta. Such questions can be addressed through the use of well-defined transport mutants. Csi52, a caesium-insensitive mutant of Arabidopsis thaliana which is defective in potassium transport, is further characterized here using conventional electrophysiology, patch-clamp and radiometric approaches to identify the nature of the potassium transport lesion. Rb⁺ uptake experiments reveal a reduced uptake in csi52 in both the high- and low-affinity uptake range. Patch-clamp analysis indicates that the activity of the predominant inward rectifying channel observed in wild-type cells is extremely low in root protoplasts isolated from csi52, whereas outward rectifying channel activity is comparable between wild-type and mutant. Rb⁺ uptake studies show that in both wild-type and csi52 the high-affinity uptake pathway is considerably less sensitive to Cs⁺ than the low-affinity pathway with K_1/2 values for Cs⁺ of around 1.3 and 0.2 mM, respectively. Furthermore, K⁺ starvation leads to a larger relative increase in high-affinity K⁺ uptake in the mutant than the wild-type. The results demonstrate the Cs⁺ sensitivity of each individual uptake pathway is comparable in wild-type and csi52 but the high-affinity pathway is less Cs⁺ sensitive (in both wild-type and csi52). Therefore, the larger shift toward high-affinity uptake in the mutant compared with the wild-type under K⁺-starvation conditions will endow the mutant with a higher degree of overall Cs⁺ resistance. The data supply evidence for the hypothesis that the csi52 mutation lies within a gene that regulates the activity of several potassium transport systems and coordinates their relative contribution to overall root K⁺ uptake.     

Enhanced stiffness of silk‐like fibers by loop formation in the corona leads to stronger gels

We study the self‐assembly of protein polymers consisting of a silk‐like block flanked by two hydrophilic blocks, with a cysteine residue attached to the C‐terminal end. The silk blocks self‐assemble to form fibers while the hydrophilic blocks form a stabilizing corona. Entanglement of the fibers leads to the formation of hydrogels. Under oxidizing conditions the cysteine residues form disulfide bridges, effectively connecting two corona chains at their ends to form a loop. We find that this leads to a significant increase in the elastic modulus of the gels. Using atomic force microscopy, we show that this stiffening is due to an increase of the persistence length of the fibers. Self‐consistent‐field calculations indicate a slight decrease of the lateral pressure in the corona upon loop formation. We argue that this small decrease in the repulsive interactions affects the stacking of the silk‐like blocks in the core, resulting in a more rigid fiber.     

Strategies to enable the adoption of animal biotechnology to sustainably improve global food safety and security

The ability to generate transgenic animals has existed for over 30 years, and from those early days many predicted that the technology would have beneficial applications in agriculture. Numerous transgenic agricultural animals now exist, however to date only one product from a transgenic animal has been approved for the food chain, due in part to cumbersome regulations. Recently, new techniques such as precision breeding have emerged, which enables the introduction of desired traits without the use of transgenes. The rapidly growing human population, environmental degradation, and concerns related to zoonotic and pandemic diseases have increased pressure on the animal agriculture sector to provide a safe, secure and sustainable food supply. There is a clear need to adopt transgenic technologies as well as new methods such as gene editing and precision breeding to meet these challenges and the rising demand for animal products. To achieve this goal, cooperation, education, and communication between multiple stakeholders—including scientists, industry, farmers, governments, trade organizations, NGOs and the public—is necessary. This report is the culmination of concepts first discussed at an OECD sponsored conference and aims to identify the main barriers to the adoption of animal biotechnology, tactics for navigating those barriers, strategies to improve public perception and trust, as well as industry engagement, and actions for governments and trade organizations including the OECD to harmonize regulations and trade agreements. Specifically, the report focuses on animal biotechnologies that are intended to improve breeding and genetics and currently are not routinely used in commercial animal agriculture. We put forward recommendations on how scientists, regulators, and trade organizations can work together to ensure that the potential benefits of animal biotechnology can be realized to meet the future needs of agriculture to feed the world.     

A Novel Triazolopyridine-Based Spleen Tyrosine Kinase Inhibitor That Arrests Joint Inflammation

Autoantibodies and the immunoreceptors to which they bind can contribute to the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). Spleen Tyrosine Kinase (Syk) is a non-receptor tyrosine kinase with a central role in immunoreceptor (FcR) signaling and immune cell functionality. Syk kinase inhibitors have activity in antibody-dependent immune cell activation assays, in preclinical models of arthritis, and have progressed into clinical trials for RA and other autoimmune diseases. Here we describe the characterization of a novel triazolopyridine-based Syk kinase inhibitor, CC-509. This compound is a potent inhibitor of purified Syk enzyme, FcR-dependent and FcR-independent signaling in primary immune cells, and basophil activation in human whole blood. CC-509 is moderately selective across the kinome and against other non-kinase enzymes or receptors. Importantly, CC-509 was optimized away from and has modest activity against cellular KDR and Jak2, kinases that when inhibited in a preclinical and clinical setting may promote hypertension and neutropenia, respectively. In addition, CC-509 is orally bioavailable and displays dose-dependent efficacy in two rodent models of immune-inflammatory disease. In passive cutaneous anaphylaxis (PCA), CC-509 significantly inhibited skin edema. Moreover, CC-509 significantly reduced paw swelling and the tissue levels of pro-inflammatory cytokines RANTES and MIP-1α in the collagen-induced arthritis (CIA) model. In summary, CC-509 is a potent, moderately selective, and efficacious inhibitor of Syk that has a differentiated profile when compared to other Syk compounds that have progressed into the clinic for RA.     

Trends in Food Habits and Their Relation to Socioeconomic Status among Nordic Adolescents 2001/2002-2009/2010

Background

In the Nordic countries, substantial policy and intervention efforts have been made to increase adolescents'' consumption of fruit and vegetables and to reduce their intake of sweets and soft drinks. Some initiatives have been formulated in a Nordic collaboration and implemented at national level. In recent years, social inequalities in food habits have been attracted particular governmental interest and several initiatives addressing the socioeconomic gradient in food habits have been highlighted. However, few internationally published studies have evaluated how trends in adolescents'' food habits develop in the context of Nordic nutrition policy, or have compared differences between the Nordic countries.

Methods

The study was based on Danish, Finnish, Norwegian and Swedish cross-sectional data from the international Health Behaviour in School-Aged Children (HBSC) study, collected via three nationally representative and comparable questionnaire surveys in 2001/2002, 2005/2006 and 2009/2010. Food habits were identified by students'' consumption of fruit, vegetables, sweets and sugar sweetened soft drink. Socioeconomic status (SES) was measured with the Family Affluence Scale (FAS). Multilevel logistic regression was used to analyze the data.

Results

Trends in fruit consumption developed differently across countries, characterized by an increase in Denmark and Norway and more stable trends in Sweden and Finland. Vegetable consumption increased particularly in Denmark and to a lesser extent in Norway, whereas Sweden and Finland displayed stable trends. Decreased trends were observed for sweet and soft drink consumption and were similar in Norway, Sweden and Finland. Sweet consumption decreased across all survey years, whereas soft drink consumption decreased between 2001/2002–2005/2006 and was stable thereafter. Denmark displayed an increase between 2001/2002–2005/2006 followed by a similar decrease between 2005/2006–2009/2010 for both sweet and soft drink consumption. Socioeconomic inequalities in fruit and vegetable consumption were observed in all countries, with no cross-country differences, and no changes over time. Small but not significant cross-country variation was identified for SES inequalities in sweet consumption. Reduced SES inequalities were observed in Sweden between 2005/2006 and 2009/2010. SES was not associated with soft drink consumption in this study population, with the exception of Denmark for the survey year 2009/2010.

Conclusion

Different trends resulted in increased country differences in food habits during the time of observations. In survey year 2009/2010, Danish students reported a higher intake of fruit and vegetable consumption than their counterparts in the other Nordic countries. Finnish students reported the lowest frequency of sweets and soft drink consumption. Despite the positive dietary trends documented in the present study, the majority of Nordic adolescents are far from meeting national dietary recommendations. Our findings underline the need for more comprehensive initiatives targeting young people''s food habits as well as a more deliberate and focused action to close gaps in social inequalities that affect food choices.     

Redox state of respiratory chain enzymes and potassium transport in silkworm mid-gut

The midgut of Hyalophora cecropia actively transports potassium from hemolymph to lumen and the energy for this process appears to be intimately linked to oxidative metabolism. In the present investigation, we monitored concurrently the rate of active transport and the redox levels of the components of the respiratory chain in the intact tissue under a variety of experimental conditions. Approximately equal concentrations of cytochromes a₃, a, c and b-557 were found. Other investigators (Pappenheimer, Jr, A. M. and Williams, C. M. (1954) J. Biol. Chem. 209, 915, Shappirio, D. G. and Williams, C. M. (1957) Proc. R. Soc. Lond. Ser. B 147, 233 and Chance, B. and Pappenheimer, Jr, A. M. (1954) J. Biol. Chem. 209, 931) have identified cytochrome b-557 with b₅ and found that it exists primarily in an extramitochondrial location.Steady-state experiments demonstrated that all these cytochromes were approximately 50% reduced while active transport proceeded at a high rate in regular cecropia Ringer containing 32 mM KCl. When the potassium concentration was reduced, the active transport decreased and all the cytochromes became more oxidized. Addition of 1 mM cyanide inhibited active transport by 90% and caused a 100% reduction of all cytochromes. Redox state and short circuit current (I_sc) kinetics measured as the tissue was made anoxic showed that all the respiratory enzymes, except cytochrome b-557, became fully reduced at a faster rate than the rate of inhibition of the I_sc. The rate of cytochrome b-557 reduction followed kinetically the I_sc.These observations are interpreted in a scheme where cytochrome b-557 (possibly b₅) branches off cytochrome c from the conventional respiratory chain, utilizing cytochrome a₃ as the terminal oxidase for both branches. Cytochrome b-557 may be involved in providing a direct link between oxidative metabolism and active transport in the midgut of the silkworm.