Proteins are essential macromolecules that play diverse roles in the physiology and health of horses. They are composed of amino acids and are involved in various biological processes, including tissue growth, repair, and the synthesis of enzymes and hormones. Dietary proteins are a key component of equine nutrition, influencing muscle development, immune function, and overall performance. Horses require a balanced intake of essential amino acids, which must be obtained through their diet, as they cannot be synthesized endogenously. This page compiles peer-reviewed research studies and scholarly articles that explore the types, functions, and dietary requirements of proteins in horses, as well as their impact on equine health and performance.
Sartori C, Guzzo N, Normando S, Bailoni L, Mantovani R.The present work is aimed at evaluating the behaviour of Italian Heavy Draught Horse (IHDH) foals reared in semi-covered stables and fed two isoenergetic total mixed rations with different dietary protein levels (13.2% and 10.6% of CP on dry matter). The study was prompted by the restrictions for nitrate emissions in farms of the European Nitrate Directive. One suggested solution is to reduce dietary protein while maintaining normal performance and welfare, but there is a lack of literature in studies of horses. The behaviours of 20 foals of 437±60 kg of BW, aged 379±37 days and stabled in f...
Liu ZQ, Xia J, Wang GL, Kuermanali N.Hyalomma asiaticum tick, an important ectozoic parasite causes tickle, pain, anemia, weight loss, and paralysis in its hosts, which include humans, cattle, sheep, horses, camels, and hares. The 4D8 gene can be a potential vaccine candidate antigen for H. asiaticum. In the present study, we cloned and expressed the 4D8 gene of H. asiaticum from Xinjiang Province. Primers were designed according to the H. asiaticum tick 4D8 gene sequence available in GenBank. The gene was amplified by reverse transcription-polymerase chain reaction and the fragments were subcloned into the prokaryotic expression...
Pérez-Nogués M, Encinas T, López-SanRoman J.Searching for new therapeutic options against septic arthritis in horses, this research was focused on the study of the kinetics and local side effects after the intra-articular treatment of horses with cefovecin sodium. A single dose (240 mg) of the drug (Convenia ) was administered into the radiocarpal joint of adult healthy horses (n = 6), and drug concentrations in plasma and synovial fluid were determined by high-performance liquid chromatography (HPLC). Local tolerance was also studied based on the modification of different joint physiopathological parameters (pH, cellular, and protei...
Vondran S, Venner M, Vervuert I.Feeding alfalfa hay is often recommended for its buffering components, like protein and calcium, to prevent lesions of the gastric mucosa in horses. Until now, there has been no information regarding the influence of alfalfa particle size on the gastric mucosa. The aim of this study was to investigate the effects of feeding two alfalfa preparations with different particle sizes (alfalfa chaff vs alfalfa pellets) in comparison with grass hay on the gastric mucosa in weanling horses. We hypothesized that feeding a high proportion of fine alfalfa particles would negatively impact gastric mucosa a...
Hughes KJ, Rendle DI, Higgins S, Barron R, Cowling A, Love S, Durham AE.Delays between collection and laboratory analysis of equine body fluid samples are common in practice; however, the effects of delays on the accuracy of results and diagnostic interpretation are unknown. Objective: To assess the effects of storage time and temperature combination on protein and cell parameters of equine synovial and mesothelial cavity fluids and determine whether any changes affect clinicopathological interpretation. Methods: In vitro experiment. Methods: Body fluid samples obtained from horses during diagnostic investigation were divided into 7 aliquots and total protein conc...
Gottschalk M, Metzger J, Martinsson G, Sieme H, Distl O.We performed a genome-wide association study for semen quality traits in 139 German Warmblood stallions. Stallions were genotyped using the Illumina equine SNP50 Beadchip. Traits analysed were de-regressed estimated breeding values (EBVs) for gel-free volume, sperm concentration, total number of sperm, progressive motility and the total number of progressively motile sperm. The GWAS revealed 29 SNPs on 12 different chromosomes as genome-wide significantly associated with semen quality traits. For ten genomic regions we could retrieve candidate genes influencing stallion fertility. Among the ca...
Aleksic D, Blaschke L, Mißbach S, Hänske J, Weiß W, Handler J, Zimmermann W, Cabrera-Sharp V, Read JE, de Mestre AM, O'Riordan R, Moore T....Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family that are secreted by trophoblast cells. PSGs may modulate immune, angiogenic and platelet responses during pregnancy. Until now, PSGs are only found in species that have a highly invasive (hemochorial) placentation including humans, mice and rats. Surprisingly, analyzing the CEACAM gene family of the horse, which has a non-invasive epitheliochorial placenta, with the exception of the transient endometrial cups, we identified equine CEACAM family members that seem to be rel...
Gałęcki K, Kowalska-Baron A.In this study, the influence of heavy-atom perturbation, induced by the addition of iodide ions, on the fluorescence and phosphorescence decay parameters of some single tryptophan containing serum albumins isolated from: human (HSA), equine (ESA) and leporine (LSA) has been studied. The obtained results indicated that, there exist two distinct conformations of the proteins with different exposure to the quencher. In addition, the Stern-Volmer plots indicated saturation of iodide ions in the binding region. Therefore, to determine quenching parameter, we proposed alternative quenching model and...
Choi YH, Gibbons JR, Canesin HS, Hinrichs K.Prospective studies were conducted to help define procedural factors affecting in vitro embryo production via intracytoplasmic sperm injection (ICSI) of equine oocytes. In experiment 1, use of 10% fetal bovine serum as a protein source in embryo culture medium resulted in a higher blastocyst rate than did use of a combination of 3% fetal bovine serum, 3% equine preovulatory follicular fluid, and 4% human serum substitute (37% vs. 15%, respectively, P < 0.05). In experiment 2, the effect of zinc supplementation (0, 0.5, 1, or 1.5 μg/mL) during IVM was examined. There were no significant di...
Yamanaka Y, Matsugano S, Yoshikawa Y, Orino K.Human immunoglobulin G (IgG) binding with zinc ions was examined using zinc ions immobilized on chelating Sepharose beads (Zn-beads). Human IgG bound to Zn-beads but not to Sepharose beads (control beads). Mouse, rat, bovine and equine IgGs also bound to Zn-beads, similar to human IgG. The human IgG F(c) fragment showed zinc ion-binding activity whereas the Fab fragment did not. Ethylenediaminetetraacetic acid (EDTA)-treated Zn-beads no longer bound human IgG; however, washing the beads, followed by the addition of zinc ions, restored the binding activity towards human IgG. Zn-beads saturated ...
Lee KH, Park C, Kim J, Moon C, Ahn M, Shin T.The morphological characteristics and glycoconjugate composition of the vomeronasal organ (VNO) of the horse was investigated using histological, immunohistochemical, and lectin histochemical methods. The VNO is bilaterally located at the base of the nasal septum, has a tubular structure surrounded by cartilage, and consists of sensory and non-sensory epithelia. Immunohistochemical examination showed that the vomeronasal sensory epithelium (VSE) consisted of receptor cells positive for both olfactory marker protein (OMP) and protein gene product 9.5 (PGP 9.5), supporting cells, and basal cells...
Mitchell MC, Tzelos T, Handel I, McWilliam HE, Hodgkinson JE, Nisbet AJ, Kharchenko VO, Burgess ST, Matthews JB.Cyathostomins are ubiquitous nematodes of horses. Once ingested, they can spend a substantial time as encysted larvae in the intestinal wall. The larvae can comprise up to 90% of the total burden, with up to several million worms reported in individuals. These stages can emerge in large numbers to cause life-threatening colitis. Direct methods for detection of encysted larval burdens in live horses do not exist. Previously, two antigen complexes were identified as promising markers for infection. A component of these, cyathostomin gut associated larval antigen-1 (Cy-GALA-1), was identified fol...
Walter S, Bollenbach A, Doerrbecker J, Pfaender S, Brown RJP, Vieyres G, Scott C, Foster R, Kumar A, Zitzmann N, Griffin S, Penin F, Pietschmann T....Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for the production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 using various nucle...
Smits K, Nelis H, Van Steendam K, Govaere J, Roels K, Ververs C, Leemans B, Wydooghe E, Deforce D, Van Soom A.The equine oviduct plays a pivotal role in providing the optimal microenvironment for early embryonic development, but little is known about the protein composition of the oviducal fluid in the horse. The aim of the present study was to provide a large-scale identification of proteins in equine oviducal fluid and to determine the effects of ovulation and pregnancy. Four days after ovulation, the oviducts ipsilateral and contralateral to the ovulation side were collected from five pregnant and five non-pregnant mares. Identification and relative quantification of proteins in the oviducal fluid ...
Thorpe CT, Karunaseelan KJ, Ng Chieng Hin J, Riley GP, Birch HL, Clegg PD, Screen HR.Although the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the energetic cost of locomotion. To maximise energy storage and return, energy-storing tendons need to be more extensible and elastic than tendons with a purely positional function. These properties are conferred in part by a specialisation of a specific compartment of the tendon, the interfascicular matrix, which enables sliding and recoil between adjacent fascicles. However, the composition of the interfascicular matr...
Brinkmann J, Jagannathan V, Drögemüller C, Rieder S, Leeb T, Thaller G, Tetens J.The casein genes are known to be highly variable in typical dairy species, such as cattle and goat, but the knowledge about equine casein genes is limited. Nevertheless, mare milk production and consumption is gaining importance because of its high nutritive value, use in naturopathy, and hypoallergenic properties with respect to cow milk protein allergies. In the current study, the open reading frames of the 4 casein genes CSN1S1 (αS1-casein), CSN2 (β-casein), CSN1S2 (αS2-casein), and CSN3 (κ-casein) were resequenced in 253 horses of 14 breeds. The analysis revealed 21 nonsynonymous nucle...
Kumar J, Chaudhury A, Yadav SC.Trypanosomosis (Surra) is an economically important disease caused by Trypanosoma evansi which is an extracellular parasite present in the plasma, tissues and other body fluids of a wide range of hosts including domesticated animals. Currently, serological reports are based on detection of antibodies by ELISA using whole cell lysate (WCL) antigen, which has a limitation of persistence of anti-trypanosomal antibodies after successful treatment of the disease. Moreover, it has some ethical issues also like requirement of mice for in vivo maintenance of parasite for preparing the antigen. Therefo...
Takesue M, Osaka Y, Muranaka M, Katayama Y, Ikadai H.In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings.
Kumar CS, Swamy MJ.The major protein of equine seminal plasma, HSP-1/2 exhibits chaperone-like activity and protects a variety of target proteins against thermal and chemical stress conditions. Here, we show that HSP-1/2 is able to protect enzymes such as alcohol dehydrogenase and glucose-6-phosphate dehydrogenase against H2O2 induced stress, clearly demonstrating that HSP-1/2 acts as a chaperone against oxidative stress. Further, the present studies show that HSP-1/2 also inhibits lipid (linoleic acid) peroxidation by hydroxyl radicals in vitro. These results are of great significance considering that so far l...
Lee KH, Park C, Bang H, Ahn M, Moon C, Kim S, Shin T.The olfactory mucosae of the horse were examined by using histology and lectin histochemistry to characterize the carbohydrate sugar residues therein. Histological findings revealed that olfactory epithelium (OE) consisted of both olfactory marker protein (OMP)- and protein gene product (PGP) 9.5-positive receptor cells, supporting cells and basal cells with intervening secretory ducts from Bowman's glands. Mucus histochemistry showed that Bowman's gland acini contain periodic acid-Schiff (PAS) reagent-positive neutral mucins and alcian blue pH 2.5-positive mucosubstances. Lectin histochemistr...
Kumar CS, Sivaramakrishna D, Ravi SK, Swamy MJ.Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigate...
Handing KB, Shabalin IG, Szlachta K, Majorek KA, Minor W.Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in h...
Matta C, Zhang X, Liddell S, Smith JR, Mobasheri A.There is insufficient knowledge about the chondrocyte membranome and its molecular composition. Objective: To develop a Triton X-114 based separation technique using nanoLC-MS/MS combined with shotgun proteomics to identify chondrocyte membrane proteins. Methods: Articular chondrocytes from equine metacarpophalangeal joints were separated into hydrophobic and hydrophilic fractions; trypsin-digested proteins were analysed by nanoLC-MS/MS. Results: A total of 315 proteins were identified. The phase extraction method yielded a high proportion of membrane proteins (56%) including CD276, S100-A6 an...
Thorpe CT, Peffers MJ, Simpson D, Halliwell E, Screen HR, Clegg PD.Tendon is a simple aligned fibre composite, consisting of collagen-rich fascicles surrounded by a softer interfascicular matrix (IFM). The composition and interactions between these material phases are fundamental in ensuring tissue mechanics meet functional requirements. However the IFM is poorly defined, therefore tendon structure-function relationships are incompletely understood. We hypothesised that the IFM has a more complex proteome, with faster turnover than the fascicular matrix (FM). Using laser-capture microdissection and mass spectrometry, we demonstrate that the IFM contains more ...
Macías-García B, Rocha A, González-Fernández L.Protein tyrosine phosphorylation (PY), a hallmark of sperm capacitation, is inhibited by extracellular calcium in stallion sperm. The objective of this study was to determine the presence and influence of the calcium-sensing receptor (CaSR) in this phenomenon. First, the presence of the CaSR was demonstrated in stallion sperm. We then tested its function in these gametes using its inhibitor NPS2143 or its agonist AC34356. Sperm were capacitated for 4 hr in modified Whitten's medium with 25 mM bicarbonate plus NPS2143 and 2.4 mM calcium or AC34356 alone, followed by analysis of PY. Inhibi...
Mazhitova AT, Kulmyrzaev AA.This study was carried out to determine the influence of milking season on amino acid (AA) profile and chemical composition of milk samples from Kyrgyz native breed mares under traditional pasture conditions. Milk samples were collected monthly from May to August 2014 from mares grazing at 2,200 m above sea level. The AA composition was determined by precolumn derivatization with diethyl ethoxymethylenemalonate in HPLC and 18 AA were determined in mare milk. The analytical parameters of linearity (0.01-4 μg/mL), precision of the method (0.26-4.88% relative standard deviation), derivatization ...
Sanchez-Teran AF, Bracamonte JL, Hendrick S, Burguess HJ, Duke-Novakovski T, Schott M, Hoff B, Rubio-Martínez LM.To evaluate the effect of arthroscopic lavage on systemic serum amyloid A (SAA) and SAA, total protein, nucleated cell count, and percentage of neutrophils in synovial fluid in healthy horses. Methods: Prospective experimental study. Methods: Healthy adult horses (n = 6). Methods: Middle carpal joints were randomly assigned to 1 of 2 treatments: arthrocentesis (controls) or arthroscopic lavage, with 30 day washout period between treatments. Synovial fluid and blood samples were collected at 0, 24, 48, 72, 96, and 120 hours. Measurements included systemic and synovial fluid SAA, as well as ...
McGorum BC, Scholes S, Milne EM, Eaton SL, Wishart TM, Poxton IR, Moss S, Wernery U, Davey T, Harris JB, Pirie RS.Equine grass sickness (EGS) is of unknown aetiology. Despite some evidence suggesting that it represents a toxico-infection with Clostridium botulinum types C and/or D, the effect of EGS on the functional targets of botulinum neurotoxins, namely the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, is unknown. Further, while it is commonly stated that, unlike EGS, equine botulism is not associated with autonomic and enteric neurodegeneration, this has not been definitively assessed. Objective: To determine: 1) whether botulism causes autonomic and enteric neurodeg...
Sanchez-Teran AF, Bracamonte JL, Hendrick S, Riddell L, Musil K, Hoff B, Rubio-Martínez LM.The objective of this study was to evaluate the effect of through-and-through joint lavage on systemic and synovial serum amyloid A (SAA), total protein, nucleated cell count and percentage of neutrophils in the synovial fluid of six healthy horses. A prospective experimental study was performed where one healthy tarsocrural joint of each horse was randomly assigned to receive repeated through-and-through joint lavage at 0, 48 and 96 h. Synovial fluid and blood samples were collected at 0 (baseline), 24, 48, 72, 96 and 120 h. Systemic and synovial SAA, total protein, nucleated cell count a...
Anand T, Vaid RK, Bera BCh, Singh J, Barua S, Virmani N, Rajukumar K, Yadav NK, Nagar D, Singh RK, Tripathi BN.A bacteriophage (VTCCBPA6) against a pathogenic strain of Aeromonas hydrophila was isolated from the sewage of an organized equine breeding farm. On the basis of TEM analysis, phage belonged to family Myoviridae. PCR amplification and sequence analysis of gp23 gene (encoding for major capsid protein) revealed phylogenetic resemblance to T4 like virus genus. Protein profiling by SDS-PAGE also indicated its resemblance to T4 like phage group. However, the comparison of its gp23 gene sequence with previously reported phages showed similarity with T4-like phages infecting Enterobacteriaceae instea...
Gibbs PG, Potter GD, Schelling GT, Kreider JL, Boyd CL.Mature ponies fitted with permanent ileal cannulas were used in a 3 X 3 Latin square experiment to quantify prececal, postileal and total tract digestion of hay protein. Coastal Bermuda grass (CB), containing 11.7% crude protein, and two qualities of alfalfa, containing 15.0% (low-protein alfalfa; LA) and 18.1% (high-protein alfalfa; HA) crude protein, were fed in coarsely chopped form at 2% of body weight daily. Total tract apparent digestibility of the N in HA (73.8%) was higher than that in CB (57.0%; P less than .05) and was slightly higher than that in LA (66.1%; P less than .10). Nitroge...
Morozova-Roche LA, Arico-Muendel CC, Haynie DT, Emelyanenko VI, Van Dael H, Dobson CM.Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated par...
Sironi G, Riccaboni P, Mertel L, Cammarata G, Brooks DE.The expression of p53 protein was investigated in eight formalin-fixed, paraffin-embedded conjunctival squamous cell carcinomas of five horses and one cow, dog and cat each by an immunohistochemical procedure in order to evaluate protein overexpression. Anti-human p53 protein mouse monoclonal antibodies known to be cross-reactive with p53 protein of the animal species examined were used. Positive p53 nuclear immunostaining was detected in five equine, one bovine and one feline cases. Conversely, no p53 immunostaining was found in the only canine case examined. These results demonstrate a frequ...
Bundgaard L, Åhrman E, Malmström J, Auf dem Keller U, Walters M, Jacobsen S.The objectives of this study was to establish a sensitive and reproducible method to map the cartilage and subchondral bone proteomes in quantitative terms, and mine the proteomes for proteins of particular interest in the pathogenesis of osteoarthritis (OA). The horse was used as a model animal. Protein was extracted from articular cartilage and subchondral bone samples from three horses in triplicate by pressure cycling technology or ultrasonication. Digested proteins were analysed by data independent acquisition based mass spectrometry. Data was processed using a pre-established spectral li...
Myers R, Cembran A, Fernandez-Funez P.Prion diseases are a group of neurodegenerative diseases endemic in humans and several ruminants caused by the misfolding of native prion protein (PrP) into pathological conformations. Experimental work and the mad-cow epidemic of the 1980s exposed a wide spectrum of animal susceptibility to prion diseases, including a few highly resistant animals: horses, rabbits, pigs, and dogs/canids. The variable susceptibility to disease offers a unique opportunity to uncover the mechanisms governing PrP misfolding, neurotoxicity, and transmission. Previous work indicates that PrP-intrinsic differences (s...
Kim SK, Kim S, Dai G, Zhang Y, Ahn BC, O'Callaghan DJ.The equine herpesvirus 1 (EHV-1) negative regulatory IR2 protein (IR2P), an early 1,165-amino acid (aa) truncated form of the 1487-aa immediate-early protein (IEP), lacks the trans-activation domain essential for IEP activation functions but retains domains for binding DNA, TFIIB, and TBP and the nuclear localization signal. IR2P mutants of the N-terminal region which lack either DNA-binding activity or TFIIB-binding activity were unable to down-regulate EHV-1 promoters. In EHV-1-infected cells expressing full-length IR2P, transcription and protein expression of viral regulatory IE, early EICP...
Guan F, Uboh CE, Soma LR, Birks E, Chen J, Mitchell J, You Y, Rudy J, Xu F, Li X, Mbuy G.Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS....
Graham-Thiers PM, Bowen LK.Plasma AA in horses fed either an all-hay or a hay and grain diet in a traditional format have not been investigated. Eight horses were divided into 2 groups: a hay group fed only grass hay or a hay and a grain group (HG) fed in a crossover design for two 5-wk periods. After the first period, horses were fasted overnight, followed by feeding with blood sampling every hour for 6 h. A 4-d total fecal and urine collection to evaluate N balance followed. A 10-d washout period separated the 5-wk feeding periods, during which horses switched diets. The second period was also followed by fasting, fee...
Jemmerson R, Margoliash E.Seven populations of site-specific antibodies were isolated from each of three sera of rabbits immunized against glutaraldehyde-polymerized horse cytochrome c. The antibodies were separated using an immunoadsorption scheme which employed the following cytochromes c: horse, beef, guanaco, rabbit, mouse testicular, pigeon, and the cyanogen-bromide cleaved fragment of the rabbit protein containing residues 1 to 65. The monovalent, antigen-binding fragments of the antibodies (Fab') gave 1:1 stoichiometries with native horse cytochrome c in fluorescence quenching assays. Cross-reactivities with het...
Ricci G, Del Boccio G, Pennelli A, Aceto A, Whitehead EP, Federici G.Glutathione transferase (EC 2.5.1.18) from horse erythrocytes has been purified and some molecular and kinetic properties have been investigated. It appears to be a dimeric protein composed of subunits of about 23 kDa, indistinguishable either in sodium dodecyl sulfate or in urea electrophoresis. Amino acid composition, substrate specificities, sensitivity to inhibitors, CD spectra, and immunological studies provide evidence that the horse enzyme is related to the pi class transferases. This enzyme has only two reactive thiol groups/dimer whose integrity appears to be essential for the activit...
García-Prieto A, Alonso J, Muñoz D, Marcano L, Abad Díaz de Cerio A, Fernández de Luis R, Orue I, Mathon O, Muela A, Fdez-Gubieda ML.It is generally accepted that the mineral core synthesized by ferritin-like proteins consists of a ferric oxy-hydroxide mineral similar to ferrihydrite in the case of horse spleen ferritin (HoSF) and an oxy-hydroxide-phosphate phase in plant and prokaryotic ferritins. The structure reflects a dynamic process of deposition and dissolution, influenced by different biological, chemical and physical variables. In this work we shed light on this matter by combining a structural (High Resolution Transmission Electron Microscopy (HRTEM) and Fe K-edge X-ray Absorption Spectroscopy (XAS)) and a magneti...
Radcliffe PA, Sion CJ, Wilkes FJ, Custard EJ, Beard GL, Kingsman SM, Mitrophanous KA.Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) ar...
Tözsér J, Friedman D, Weber IT, Bláha I, Oroszlan S.The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage site...
Egito AS, Miclo L, López C, Adam A, Girardet JM, Gaillard JL.The equine alpha(s1)- and beta-caseins (CN) were purified by chromatography on DEAE-cellulose and by reversed-phase HPLC. The alpha(s1)-, beta-, and kappa-CN were characterized either by monodimensional urea-PAGE or sodium dodecylsulfate (SDS)-PAGE or by bidimensional electrophoresis. Kappa-casein was characterized after electrophoresis by glycoprotein-specific staining. To identify alpha(s1)-CN without ambiguity, internal sequences were determined after trypsin or chymosin digestion of purified alpha(s1)-CN. These sequences, that could be estimated to correspond to 62% of the full protein, pr...
Ascenzi P, di Masi A, Gullotta F, Mattu M, Ciaccio C, Coletta M.Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (k(off)). Since no techniques are available to estimate k(off), we report herewith a method to determine k(off) based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k(off) value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)-cyanide) was determined at pH 9.2 and 20.0 degrees C. Mixing Mb(III)-cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)-cyanide w...
Southgate CD, Green MR.Lentivirus Tat proteins comprise a novel class of RNA-binding transcriptional activators that are essential for viral replication. In this study, we performed a series of protein fusion experiments to delineate the minimal protein domains and promoter elements required for Tat action. We show that a 15-amino-acid region of equine infectious anemia virus (EIAV) Tat protein, when fused to the GAL4 or LexA DNA binding domain, can activate transcription in appropriate promoter contexts. In the natural human immunodeficiency virus type 1 long terminal repeat, activation by Tat is dependent on multi...
Handing KB, Shabalin IG, Szlachta K, Majorek KA, Minor W.Serum albumin (SA) is the main transporter of drugs in mammalian blood plasma. Here, we report the first crystal structure of equine serum albumin (ESA) in complex with antihistamine drug cetirizine at a resolution of 2.1Å. Cetirizine is bound in two sites--a novel drug binding site (CBS1) and the fatty acid binding site 6 (CBS2). Both sites differ from those that have been proposed in multiple reports based on equilibrium dialysis and fluorescence studies for mammalian albumins as cetirizine binding sites. We show that the residues forming the binding pockets in ESA are highly conserved in h...
Waller AP, Huettner L, Kohler K, Lacombe VA.Although insulin resistance (IR) has been increasingly recognized in horses, a clear understanding of its pathophysiology is lacking. The purpose of the present study was to determine the early pathologic changes in IR horses by characterizing alterations in proteins that play key roles in innate immunological responses and inflammatory pathways, and by identifying potential links with glucose transport and insulin signaling. Visceral (VIS) and subcutaneous (SC) adipose tissue and skeletal muscle (SM) biopsies were collected from horses, which were classified as insulin-sensitive (IS) or IR ba...
Tang X, Sayre LM, Tochtrop GP.Cytochrome c is a key mitochondrial respiratory protein that is particularly susceptible to modification during oxidative stress. The nature of this susceptibility is linked to the mitochondrial membrane being rich in esterified linoleic acid, which predisposes this organelle to the formation of lipid peroxidation products such as 4-hydroxy-2-(E)-nonenal (4-HNE). To better understand the nature of cytochrome c modification by 4-HNE, we initiated an in vitro study utilizing a combination of MALDI-TOF mass spectrometry, LC-ESI-MS/MS and isotope labeling to monitor 4-HNE modification of cytochrom...
Range F, Mundhenk L, Gruber AD.The equine putative chloride channel protein eCLCA1 is thought to be critically involved in the pathogenesis of recurrent airway obstruction (RAO) via modulation of the hydration of airway mucins. A recent study revealed a strong increase of eCLCA1 messenger ribonucleic acid (mRNA) in the lungs of horses with RAO. In this study, eCLCA1 protein and mRNA expression were quantified in airway goblet cells of 9 horses affected with RAO and 9 control horses by using immunohistochemistry and laser microdissection followed by real-time quantitative reverse transcription polymerase chain reaction, resp...
Thorpe CT, McDermott BT, Goodship AE, Clegg PD, Birch HL.Advancing age is a well-known risk factor for tendon disease. Energy-storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro-damage in the extracellular matrix (ECM). Several authors suggest that age-related micro-damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and m...
Jammes M, Cassé F, Velot E, Bianchi A, Audigié F, Contentin R, Galéra P.Osteoarthritis (OA) is a widespread osteoarticular pathology characterized by progressive hyaline cartilage degradation, exposing horses to impaired well-being, premature career termination, alongside substantial financial losses for horse owners. Among the new therapeutic strategies for OA, using mesenchymal stromal cell (MSC)-derived exosomes (MSC-exos) appears to be a promising option for conveying MSC therapeutic potential, yet avoiding the limitations inherent to cell therapy. Here, we first purified and characterized exosomes from MSCs by membrane affinity capture (MAC) and size-exclusio...
Anderson JR, Johnson E, Jenkins R, Jacobsen S, Green D, Walters M, Bundgaard L, Hausmans BAC, van den Akker G, Welting TJM, Chabronova A, Kharaz YA....Extracellular vesicles (EVs) contribute to osteoarthritis pathogenesis through their release into joint tissues and synovial fluid. Synovial fluid-derived EVs have the potential to be direct biomarkers in the causal pathway of disease but also enable understanding of their role in disease progression. Utilizing a temporal model of osteoarthritis, we defined the changes in matched synovial fluid and plasma-derived EV small non-coding RNA and protein cargo using sequencing and mass spectrometry. Data exploration included time series clustering, factor analysis and gene enrichment interrogation. ...
Murray SJ, Santangelo KS, Bertone AL.To evaluate early cellular influences of bone morphogenetic protein (BMP)12 and BMP2 on equine superficial digital flexor tenocytes (SDFTNs) and equine bone marrow-derived mesenchymal stem cells (BMDMSCs). Methods: 9 adult clinically normal horses. Methods: BMDMSCs and SDFTNs were cultured in monolayer, either untreated or transduced with adenovirus encoding green fluorescent protein, adenovirus encoding BMP12, or adenovirus encoding BMP2. Cytomorphologic, cytochemical, immunocytochemical, and reverse transcriptase-quantitative PCR (RT-qPCR) analyses were performed on days 3 and 6. Genetic pro...
Singha H, Malik P, Goyal SK, Khurana SK, Mukhopadhyay C, Eshwara VK, Singh RK.To express truncated TssB protein of Burkholderia mallei and to evaluate its diagnostic efficacy for serological detection of glanders among equines. Methods: In an attempt to develop recombinant protein based enzyme-linked immunosorbent assay (ELISA), N-terminal 200 amino acid sequences of B. mallei TssB protein-a type 6 secretory effector protein--were expressed in prokaryotic expression system. Diagnostic potential of recombinant TssB protein was evaluated in indirect ELISA using a panel of glanders positive (n = 49), negative (n = 30), and field serum samples (n = 1811). Cross-reactivity o...
Schochetman G, Fine D, Arthur L, Gilden R, Heberling R.A new retravirus (SMRV) isolated from a squirrel monkey, Saimiri sciureus, has an Mg2+-dependen reverse transcriptase and a buoyant density of 1.17 g/cm3 in sucrose and 1.21 g/cm3 in cesium chloride, similar to the mouse mammary tumor virus and the Mason-Pfizer monkey virus. The polypeptide patter of SMRV as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was distinct from the reported polypeptide patterns of known retraviruses. Four major polypeptides of molecular weights 40,000, 20,000, 14,000 and 8,000 were resolved in virus propagated in human, mink, and canine cell...
Ramos-Vara JA, Frank CB, DuSold D, Miller MA.The immunoreactivity of PNL2, Melan A, and protein gene product (PGP) 9.5 was compared with that of S100 protein in 50 formalin-fixed, paraffin-embedded equine melanocytic neoplasms. PNL2, PGP 9.5, and S100 protein were detected in all 50 neoplasms; none expressed Melan A. PNL2 was not expressed in 62 nonmelanocytic tumors (equine sarcoids, schwannomas, carcinomas, sarcomas, endocrine tumors, sex-cord stromal tumors, germ cell tumors, and leukocytic tumors) or in normal tissues other than epidermis. In summary, antibody PNL2 is a sensitive marker of equine melanocytic neoplasms and is more spe...
Norcross NL, Coggins L.The spleens of horses infected with equine infectious anemia contain an antigen that is useful for a diagnostic immunodiffusion test. This antigen was extracted from the spleen by homogenization of the tissue, centrifugation, and precipitation from the supernatant fluid at 50% saturation with (NH(4))(2)SO(4). The antigen was purified by subjecting it to two cycles of electrophoresis in a continuous free-flow electrophoresis cell and finally filtering through a column of Sephadex G-200 gel. The antigen was found to be a small protein with a molecular weight of 27,500 and sedimentation coefficie...
