bluuebunny/crossref_metadata_2025_split huggingface dataset:
Expression and Regulation of Glycogen Synthase in Equine Type 1 Polysaccharide Storage Myopathy
Authors: Maile C , Hingst J , Wojtaszewski J , Piercy R 
Published: June 2014

IntroductionEquine Type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense, gain‐of‐function mutation in the equine skeletal muscle glycogen synthase gene (GYS1). Affected horses have excessive glycogen storage, amylopectate‐like inclusions in their muscle and higher muscle glycogen synthase activities (GS) than controls. GS is normally allosterically‐regulated by glucose‐6‐phosphate and enzyme activity is reduced by phosphorylation. We hypothesised that the higher GS activity in PSSM1‐affected horses might be due either to 1) increased expression or reduced degradation of the mutant enzyme or 2) due to reduced phosphorylation.MethodsSemimembranosus muscle samples were biopsied from 4 homozygotes (HH), 13 heterozygotes (RH) and 12 control (RR) Belgian Draught horses. Samples were analysed for glycogen content and GS activity. Western immunoblotting was used to examine expression of total and phosphorylated forms of GS, GSK3β and GLUT4. Data was analysed using non‐parametric ANOVA statistical tests with post‐hoc Dunn's multiple comparison tests.ResultsPSSM1 affected heterozygotes and homozygotes had significantly higher muscle glycogen contents than controls (RR vs. RH P = 0.005, RR vs. HH P = 0.041) and GS activity was significantly higher in PSSM1‐affected homozygotes than in heterozygotes and control horses (P = 0.03). Although there was no difference in total GS expression between groups, there was significantly more expression of GS phosphorylated at site 2 + 2a in PSSM1‐affected horses in comparison with controls (P = 0.009).ConclusionsOur data suggest that increased GS activity in PSSM1‐affected horses is not associated with increased expression or degradation of the mutant enzyme and occurs despite increased phosphorylation of GS at site 2 + 2a. Alternative mechanisms, including aberrant ligand binding might account for the increased GS activity in PSSM1‐affected muscle.Ethical Animal ResearchMuscle biopsy samples were collected according to ethical approval from the local Institutional Animal Care and Use Committee. Explicit owner informed consent for participation in this study is not stated. Sources of funding: Petplan Charitable Trust and The Royal Veterinary College. Competing interests: none.
In Silico Three Dimensional Modelling of Mutant Glycogen Synthase Associated with Equine Type 1 Polysaccharide Storage Myopathy
Authors: Maile C , O′Reilly A , Wallace B , Piercy R 
Published: June 2014

IntroductionEquine Type 1 polysaccharide storage myopathy (PSSM1) is associated with a dominant, missense mutation in the equine glycogen synthase gene (GYS1) that increases the enzyme's activity. The amino acid sequence surrounding the PSSM1 mutation is highly conserved amongst species from yeast to horses suggesting that this region plays an important role, but its functional significance has yet to be determined.MethodsComputer modelling can predict three dimensional (3D) protein structures using related proteins as templates. Yeast glycogen synthase (GS) has high sequence homology (73%) to the equine enzyme and since the yeast enzyme's structure has been solved, we hypothesised that in silico modelling based on the yeast protein using MODELLER and SwissPDBViewer software would predict the enzyme's structure and explain the mutant enzyme's increased activity.ResultsHomology‐modelling revealed that, like the yeast enzyme, equine GS likely forms a tetramer surrounding a catalytic cleft and undergoes large structural rearrangements upon G6P binding to highly‐conserved binding sites. The enzyme's glycogen binding sites are located on the extremities of the enzyme and are also conserved in the equine protein. The PSSM1 mutation lay close to the G6P binding site, but did not confer any clear structural rearrangement on the protein's 3D configuration. The mutation was distant from the known phosphorylation sites and the glycogen binding sites.ConclusionsThe close proximity of the mutation to the G6P binding site suggests that it might influence substrate interaction or allosteric regulation. Further studies evaluating G6P ligand binding and enzyme activation are warranted and might best be made following purification of the mutant and wild type proteins.Ethical Animal ResearchNo animals were used in the study. Sources of funding: Petplan Charitable Trust and The Royal Veterinary College. Competing interests: none.
Mitochondrial Respirometry in Skeletal Muscle of Exercising Eventing Horses Run to Exhaustion
Authors: Miranda ACT , Ramos D , Chicaybam G , Almeida F , Galina A 
Published: June 2014

IntroductionMitochondria play a central role in energy metabolism during muscle contraction and in the balance of anaerobic and aerobic energy production. This study evaluated mitochondrial respirometry in skeletal muscle of eventing horses before and after an intense effort.MethodsFourteen Brazilian Sport Horses 4 to 6 years old performed an incremental velocity test on a high speed treadmill inclined to 3% and galloped at speeds up to 8 m/s until exhaustion. Biopsies from the gluteus medius muscle were obtained before and immediately after the end of the test. Mitochondrial respiration was assessed by O2K oxygraph of high resolution (Oroboros). The results were standardized by minute and per gram of wet weight of muscle and expressed as mean ± SD. Plasma lactate was measured using a spectrophotometer. Results were compared by paired t–test (P<0.05).ResultsThe maximal respiration value of mitochondria did not change in gluteus medius muscle of horses at exhaustion. However, there was an increase in stage 3 mitochondrial respiration with pyruvate/malate substrate, and also an increase of non‐mitochondrial respiration after exercise. Lactate values before and after exercise were 0.4 ± 0.1 mmol/L and 12.1 ± 4.6 mmol/L, respectively.ConclusionsThere is little change in mitochondrial respirometry in the gluteus medius muscle after intense exercise indicating predominantly anaerobic metabolism in the muscle of the eventing horses, as supported by the high plasma lactate concentrations after the test. The increase of oxidase reactions after intense exercise is related to an increase in non‐mitochondrial respiration.Ethical Animal ResearchAll procedures were approved by the Board of Ethical Research of Universidade Federal Rural do Rio de Janeiro: number 264/2012. Sources of funding: Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq; PRONEX and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro – FAPERJ. Competing interests: none.
Effect of Sedation and Exercise on Phenylalanine Pharmacokinetics in Horses
Authors: Naylor R , Smith K , Rankin D , Blake V , Atherton P , Piercy R 
Published: June 2014

IntroductionMeasurement of incorporation of stable isotope‐containing amino acids into muscle is the gold‐standard technique for measuring muscle protein synthesis (MPS), but its use in assessing MPS in horses has not been evaluated. Determination of MPS requires steady‐state tracer plasma enrichment and serial muscle biopsy. Given that MPS is often assessed in response to exercise and that muscle biopsy in the standing horse typically requires sedation, the influence of exercise and sedation on tracer kinetics was determined.MethodsThree mature Thoroughbred geldings received 0.1 mg/kg D5‐phenylalanine (D5‐Phe) IV and plasma D5‐Phe enrichment was measured over 3 hrs. Pharmacokinetic modeling determined the rate of appearance and phenylalanine pool size, from which a loading dose and infusion rate that would achieve a steady 5–10% enrichment were calculated. Subsequently, steady‐state was confirmed by measuring plasma enrichment during 6 hours of infusion after loading. Horses then received the same dose and completed a 12‐minute exercise test and were sedated with xylazine 0.4 mg/kg IV. Plasma D5‐Phe enrichment was determined every 5–15 minutes for 2 hours after both exercise and sedation.ResultsThe mean rate of phenylalanine appearance in the horse was 56 umol/kg/hr (SD 24) and mean phenylalanine pool size 41 umol/kg (SD 8.8). A loading dose of 3.3 umol/kg and an infusion rate of 4.5 umol/kg/hr D5‐phenylalanine was calculated and subsequently, confirmed to achieve steady state tracer concentration at 5.6–9.1% APE in sedentary horses. Exercise caused a transitory, and sedation a more prolonged reduction in plasma enrichment (up to 20%).ConclusionsD5‐phenylalanine is a suitable metabolic tracer in the horse as a steady‐state can be achieved, however interventions should be taken into account.Ethical Animal ResearchThe study was performed in in accordance with the local Ethics Committee approval and Home office project license (PPL 70/7523) under the UK A(SP)A Act 1986. Sources of funding: Boehringer Ingelheim. Competing interests: R Naylor is completing a PhD studentship sponsored by Boehringer Ingelheim.
Influence of Weight Loss on Skeletal Muscle Mitochondrial Function in the Mature Horse
Authors: Zambito J , Nichols C , Barnes K , Spooner H , Hollander J 
Published: June 2014

IntroductionObesity creates many metabolic issues in horses, yet stepwise alterations in mitochondrial capacity and oxidant status during weight loss have not been assessed. Skeletal muscle contains subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which respond differently to physiological stimuli, impacting tissue and systemic oxidant status. It was hypothesised that horses would display improved muscle mitochondrial subpopulation function in middle gluteal muscle, and decreases in circulating oxidant status markers during weight loss from an obese (7 to 8) to moderate (5) body condition score (BCS).Methods8 light‐type horses were studied. Mitochondrial subpopulations were isolated through differential centrifugation and all enzymatic activity through colorimetric kinematic assays. Effects of BCS reduction or percent weight loss were evaluated using SAS (mixed procedure with repeated measures).ResultsPlasma nitrate decreased in response to BCS reduction (P = 0.06) and percent weight loss (P = 0.06), whereas erythrocyte total glutathione (P = 0.06) concentration increased with decreasing BCS. Mitochondrial electron transport chain complexes I and IV displayed greater activity in SSM than IFM (P≤0.03), while all complexes in IFM had decreased activity due to both weight parameters (P≤0.01). Interactions between subpopulation complex IV activity and weight loss markers (P<0.05) were displayed. Citrate synthase activity, indicating mitochondrial number, was greater in SSM than IFM (P<0.0001) but was unaffected with weight loss. Lipid peroxidation was decreased with BCS change (P = 0.01) and weight loss (P = 0.02), displaying greater amounts in SSM than IFM (P≤0.02).ConclusionsAlterations in complex activities and lipid peroxidation suggest IFM are more affected by weight loss, with large contributions from complex IV by‐products. Mitochondrial component flexibility may contribute individually to disease development and performance of equine athletes.Ethical Animal ResearchThe study was approved by the Middle Tennessee State University Institutional Animal Care and Use Committee. Sources of funding: West Virginia University Summer Undergraduate Research Experience Program and Middle Tennessee State University. Competing interests: none.
The Use of Nutritional Supplements in Dressage and Eventing Horses
Authors: Agar C , Gemmill R , Hollands T , Freeman S 
Published: June 2014

IntroductionSelecting the appropriate nutritional supplements for performance horses is difficult, due to significant gaps in the current scientific evidence. Our hypothesis was that nutritional supplements are chosen based on owner's perception of discipline specific health and performance problems.MethodsA mixed methods (open and closed questions) online questionnaire was distributed through British Eventing and Dressage websites, to collect data on demographics of owners and their horses, supplements used, and their opinion on health and performance problems in their discipline, and in their own horses. Data was evaluated using descriptive analysis and Sign and Fisher's exact tests for quantitative data, and thematic analysis for qualitative data.Results599 responses met the inclusion criteria, of which 441 were or had competed in dressage, and 158 in eventing (unaffiliated novice to affiliated advanced levels). Participants had been riding for 26.4 (3–60) (mean (range)) years, currently competed 1.2 (0–10) horses, and used 2 (0–12) supplements in their top horse. Participants ranked the most important health and performance issues as (1) Energy and behaviour, (2) Lameness, (3) Back and muscle problems for dressage, and (1) Lameness, (2) Energy and behaviour, (3) Stamina and fitness for eventing horses. The most common reason supplements were used in individual horses were (1) Joints and mobility, (2) Behavioural problems for dressage, and (1) Electrolytes, (2) Joints and mobility for eventing.ConclusionsThere was discrepancy between participants' opinions of the main health and performance problems within their discipline, and their reasons for using supplements.Ethical Animal ResearchThe study was approved by the School of Veterinary Medicine and Science Ethics Committee at University of Nottingham. Sources of funding: School of Veterinary Medicine and Science, University of Nottingham. Competing interests: Teresa Hollands is employed by Dodson and Horrell Ltd.
Effect of Chelated Chromium and L‐carnitine on Subcutaneous Fat Deposition in the Mangalarga Marchador Breed after Training
Authors: Barcelos K , Jordão L , Ferraz G , Lana A , Rezende A 
Published: June 2014

IntroductionThe Brazilian Mangalarga Marchador breed is valued for its gait. However few studies have assessed the use of nutritional additives on this breed's performance. This study was designed to test the hypothesis that chelated trivalent chromium and L‐carnitine feed supplementation may reduce subcutaneous fat deposition after training.Methods13 mares (2.5 to 3 years old) were assigned to one of three treatments (T1, 10 mg Cr/day; T2, 10 g LCN/day; T3, control) according to a completely randomized design, with split‐plot arrangement. The diet was Panicum maximum cv. Massai and concentrate (50:50 ratio and 2.5% BWT). During 42 days the mares received supplementation and exercised for 50 min on a treadmill and 60 min on an automatic walker every other day, resting on Sundays. Subcutaneous fat was measured with ultrasound before and after training in the back‐loin(GDL), croup(GG), and base of the tail(GBC). Results were compared by Tukey test (P<0.05).ResultsBefore training, the variables were similar across the groups (P>0.05). After training, in GDL, T2 (1.76 mm) was 37.37% lower (P<0.05) than control (2.81 mm), while T1 (2.22 mm) was similar to T2 and control. There was no difference between treatments (P>0.05) in GG. In GBC, there was a 24.45% increase from pre (8.59 mm) to post‐treatment (10.69 mm) evaluations, regardless of the treatment.ConclusionsSupplementation with L‐carnitine reduces subcutaneous fat deposition in the lumbar region of Mangalarga Marchador mares after training, which may be indicative of increased use of subcutaneous fat in this region as a substrate for exercise.Ethical Animal ResearchProcedures were approved research by the University (no. 237 / 2012 protocol). Sources of funding: CNPq, CAPES, FAPEMIG, ABCCMM, Lab TORTUGA, Haras Esperança e Grande Sertão. Competing interests: none.
Effect of Diet and Road Transport on Post‐Exercise Plasma Lactate Concentration
Authors: Connysson M , Jansson A 
Published: June 2014

IntroductionHorses are usually transported before competitions. The aim of this study was to compare the lactate response in horses on two diets that were transported or not transported before exercise.MethodsSix trained and raced Standardbred trotter mares, all accustomed to transportation, were used. Two diets were fed in two periods of 29 days in a crossover design; one forage‐only diet (F) and one diet with 50% of DM intake from forage and 50% from oats (FO). The horses had slow exercise 1–3 times/week (approximately 1 h walk and slow trot [6–7 m/s]) and intensive exercise1–2 times/week (4000 m slow trot warm‐up, 2000 m at 10–11 m/s on a race track or five 500 m intervals at 9 m/s on a slope, slow trot downhill). At day 21, horses were subjected to transport for 100 kilometres before they performed an exercise test (TT) (4000 meter warm‐up [6.3–10 m/s] and 2140 m “race” [11.6–12.7 m/s]). At day 26, the horses performed a control test (CT) where they were kept in their stall before the exercise test was performed. Blood samples were collected after the finish line and 10 and 30 min post‐exercise. Plasma lactate (Lac) was analysed using a spectrophometric method. ANOVA was performed using PROC MIXED (significant difference P<0.05).ResultsThere was no difference in Lac (after finish and 10 and 30 min post‐exercise) between diets (F: 14.9 vs. FO: 17.1 mmol/l SEM: 2.1) or exercise tests (TT: 15.9 vs. CT: 16.1 mmol/l SEM 2.0).ConclusionsDiet and transport seem not to affect post‐exercise plasma lactate in experienced competition horses transported 100 km.Ethical Animal ResearchThe Umeå local ethics committee approved this study. Sources of funding: KSLA (The Royal Swedish Academy of Agriculture and Forestry). Competing interests: none.
Effect of Non‐Structural Carbohydrate, Fat and Fiber Intake on Glycogen Repletion Following Intense Exercise
Authors: Mesquita VS , Pagan JD , Valberg SJ , Waldridge BM , Whitehouse C 
Published: June 2014

IntroductionMuscle glycogen is a potentially limiting substrate for horses during intense exercise. Low‐starch, high‐fat concentrates have become popular for performance horses, but their effect on muscle glycogen usage and repletion is unclear.MethodsSix Thoroughbred horses were studied in a 3 x 3 Latin Square design to measure the effect of non‐structural carbohydrate (NSC), fat and fiber intake on glycogen repletion following intense exercise. Horses were fed isocaloric, isonitrogenous rations that supplied high (HS), medium (MS) or low (LS) NSC intake. The horses were fed 1.25% BW/d grass‐hay and 1.0% BW/d concentrate. These rations provided 45%, 36% and 18% of total digestible energy (DE) from NSC, 11%, 15% and 23% of total DE from fat, and 30%, 33% and 45% of total DE from fiber. During each 1‐month period horses were trained for three weeks on a high‐speed treadmill followed by a three‐day glycogen‐depletion period comprising multiple bouts of intense exercise. Muscle biopsies were taken before and 0, 24, 48 and 72 hours post‐depletion. A standardized exercise test (SET) was performed on day 1 of the depletion period.ResultsDuring the SET, lactate was lower in LS compared to MS or HS (P<0.05). Heart rate, plasma glucose, VO2, VCO2 were unaffected by treatment. Muscle glycogen depletion averaged 33%, 30%, and 36% in the HS, MS and LS treatments. Muscle glycogen repletion was significantly lower in the LS group 72 hours post exercise (P<0.05).ConclusionsLow NSC concentrates may not provide enough substrate for glycogen repletion following multiple bouts of intense exercise.Ethical Animal ResearchApproved by Kentucky Equine Research animal use committee. Sources of funding: Kentucky Equine Research and Cargill. Competing interests: Some of the concentrates tested are owned by the funding sources. Dr Pagan is president of Kentucky Equine Research.
The Kv1.3 potassium channel is localized to the cis‐Golgi and Kv1.6 is localized to the endoplasmic reticulum in rat astrocytes
Authors: Zhu Jing , Yan Jenny , Thornhill William B. 
Published: June 2014

The functions of voltage‐gated potassium (Kv) channels in neurons have been well defined, whereas their roles in glial cells are not fully understood. Kv1.1, Kv1.3 and Kv1.6 are endogenously expressed in C6 astrocytoma cells, but their trafficking and subcellular localization have not been well studied. In C6 cells, Kv1.1 was localized to the cell surface, Kv1.3 was predominantly localized in the cis‐Golgi, and Kv1.6 was enriched in the endoplasmic reticulum. Disruption of the Golgi stacks with brefeldin A treatment redirected Kv1.3 to the endoplasmic reticulum, further confirming that Kv1.3 was localized in the Golgi. Denaturing and reducing immunoblot analysis identified an expected Kv1.3 monomer and an unexpected Kv1.3 dimer/aggregate. These two forms had different protein half‐lives: that of the monomer form T1/2 was 5.1 h, whereas the dimer/aggregate form was stable over the 8‐h measurement period. The Kv1.3 dimer/aggregate form on immunoblots appeared to be correlated with its Golgi retention, based on examination with several cell types that expressed Kv1.3. Glycosidase treatment showed that Kv1.3 contained complex‐type N‐glycans terminated with sialic acids, suggesting that Kv1.3 had traveled to the trans‐Golgi network for sialylation before it was recycled to the cis‐Golgi for retention. Inhibition of N‐glycosylation did not affect Kv1.3 localization, indicating that N‐glycans did not play a role in its Golgi retention. Thus, Kv1.3 appears to be distributed to the cis‐Golgi membrane of rat astrocytes in a similar way as a Golgi resident protein, and this unusual distribution appears to be correlated with its SDS/2‐mercaptoethanol‐resistant dimer/aggregate forms on immunoblots.
The association of −330 interleukin‐2 gene polymorphism and HLA‐DR15 allele in Iranian patients with multiple sclerosis
Authors: Sayad A. 
Published: June 2014

SummaryThe purpose of this case–control study was to evaluate the frequencies and potential genetic susceptibility of the −330 IL2 T and G alleles and HLA‐DRB1*1501 allele in Iranian patients with multiple sclerosis (MS) compared to healthy controls. Two hundred and sixty Iranian patients with MS from medical genetics department of Sarem Women hospital were selected. Besides, 450 ethnically age‐ and sex‐matched healthy individuals without personal or family backgrounds of autoimmune disorders were enrolled as a control group. All polymorphisms were analysed using RFLP‐PCR technique. HLA‐DRB1 genotyping was carried out by HISTO TYPE SSP high‐resolution Kits according to the manufacturer's suggestions. The frequency of the T allele at the −330 IL2 polymorphism was significantly higher in patients with MS than controls (OR: 2.45, 95 CI: 1.9–3, P = 4 × 10−14). Moreover, the T/T genotype was more frequent in patients than in controls (51% vs. 30%). This study indicated that the −330 T IL2 allele and the T/T genotype were related to increased plasma concentration of IL2 and a higher risk of developing MS among Iranian patients. Carrying both the −330 T IL2 and the HLA, DRB1* 1501 alleles showed the most susceptibly effect to MS. Our data demonstrated −330 T IL2 allele provided major susceptibility to MS and HLA‐DRB1* 1501 allele had an additive effect. In addition, it seems that studies with larger sample size are required to bring about more authentic results. Our findings suggest that IL2 gene polymorphisms influence the susceptibility to MS in Iranian patients.
Two novel alleles HLA‐DRB1*11:150 and HLA‐DRB1*14:145 identified in Saudi individuals
Authors: Fakhoury H. A. , Cereb N. , Jawdat D. , Al Jumah M. , Alaskar A. S. , Hajeer A. H. 
Published: June 2014

SummaryTwo new HLA‐ DRB1 alleles were identified by sequence‐based typing method (SBT) in 1100 participants in the Saudi Stem Cell Donor Registry. HLA‐DRB1*11:150 differs from HLA‐DRB1*11:01:01G by a single C to A substitution at nucleotide position 5580 in exon 2, resulting in an amino acid change from alanine to glutamic acid at position 74. HLA‐DRB1*14:145 differs from HLA‐DRB1*14:04 by a C to G substitution at nucleotide position 5511 in exon 2, resulting in an amino acid change from threonine to arginine at position 51.