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.
Weir J, Li H, Warren LK, Macon E, Wickens C.Evaluating impact of animal agriculture on air quality has been the focus of recent research. Ammonia (NH) volatilization occurs when undigested protein in feces and urea in urine is broken down by bacteria and enzymes. Information regarding NH emission from equine facilities is limited, and effects of CP intake on NH emissions have not been investigated. Nine mature geldings were used in a 3 × 3 replicated Latin square design study to determine effects of dietary CP on potential NH losses from feces and urine. We hypothesized feeding horses above the CP requirement would result in an increas...
Swegen A, Grupen CG, Gibb Z, Baker MA, de Ruijter-Villani M, Smith ND, Stout TAE, Aitken RJ.Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule. Embryos were recovered on day 8 after ovulation and cultured for 48 hours. Secretomes of day 9 and day 10 embryos were analyzed by LC-MS/MS and supported by analysis of blast...
Ly HGT, Parac-Vogt TN.A recent study [Angew. Chem. Int. Ed. 2015, 54, 7391-7394] has shown that horse heart myoglobin (HHM) is selectively hydrolyzed by a range of zirconium(IV)-substituted polyoxometalates (POMs) under mild conditions. In this study, the molecular interactions between the Zr-POM catalysts and HHM are investigated by using a range of complementary techniques, including circular dichroism (CD), UV/Vis spectroscopy, tryptophan fluorescence spectroscopy, and H and P NMR spectroscopy. A tryptophan fluorescence quenching study reveals that, among all examined Zr-POMs, the most reactive POM, 2:2 Zr -...
Steward KF, Robinson C, Maskell DJ, Nenci C, Waller AS.The Gram-positive bacterium subspecies () is the causative agent of strangles, among the most frequently diagnosed infectious diseases of horses worldwide. Genome analysis of strain 4047 (4047) identified a putative operon, Fim1, with similarity to the pilus loci of other Gram-positive bacteria. The Fim1 locus was present in all strains of and its close relative subspecies () that have been studied to date. In this study we provide evidence that the putative structural pilus proteins, SEQ_0936 and CNE, are produced on the cell surface during growth and infection. Although the proteins ...
Lepczyński A, Ożgo M, Dratwa-Chałupnik A, Robak P, Pyć A, Zaborski D, Herosimczyk A.The main objectives of the study were to: (1) deeply analyse the serum protein composition of Equus caballus, (2) assess the effectiveness of the high-abundant protein depletion and improve the concentration of medium- and low-abundant proteins. The analysis were performed on the blood plasma of three healthy part-Arabian mares. The implementation of two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation - time of flight mass spectrometry allowed us to establish a horse plasma proteome map. Serum proteins were resolved at pH 4 to 7, followed by 12% SDS-PAGE. As a resul...
Plapp BV, Savarimuthu BR, Ferraro DJ, Rubach JK, Brown EN, Ramaswamy S.During catalysis by liver alcohol dehydrogenase (ADH), a water bound to the catalytic zinc is replaced by the oxygen of the substrates. The mechanism might involve a pentacoordinated zinc or a double-displacement reaction with participation by a nearby glutamate residue, as suggested by studies of human ADH3, yeast ADH1, and some other tetrameric ADHs. Zinc coordination and participation of water in the enzyme mechanism were investigated by X-ray crystallography. The apoenzyme and its complex with adenosine 5'-diphosphoribose have an open protein conformation with the catalytic zinc in one pos...
Dohre SK, Kamthan A, Singh S, Alam SI, Kumar S.Glanders is a disease of horses, donkeys and mules. The causative agent Burkholderia mallei, is a biorisk group 3 pathogen and is also a biothreat agent. Simple and rapid diagnostic tool is essential for control of glanders. Using a proteomic approach and immunoblotting with equine sera, we identified 12 protein antigens that may have diagnostic potential. Various immunoreactive proteins e.g. GroEL, translation elongation factor Tu, elongation factor Ts, arginine deiminase, malate dehydrogenase, DNA directed RNA polymerase subunit alpha were identified on 2-dimentional immunoblots. One of thes...
Dube S, Chionuma H, Matoq A, Alshiekh-Nasany R, Abbott L, Poiesz BJ, Dube DK.In order to better understand the training and athletic activity of horses, we must have complete understanding of the isoform diversity of various myofibrillar protein genes like tropomyosin. Tropomyosin (TPM), a coiled-coil dimeric protein, is a component of thin filament in striated muscles. In mammals, four TPM genes (TPM1, TPM2, TPM3, and TPM4) generate a multitude of TPM isoforms via alternate splicing and/or using different promoters. Unfortunately, our knowledge of TPM isoform diversity in the horse is very limited. Hence, we undertook a comprehensive exploratory study of various TPM i...
Pinto-Bravo P, Galvão A, Rebordão MR, Amaral A, Ramilo D, Silva E, Szóstek-Mioduchowska A, Alexandre-Pires G, Roberto da Costa R, Skarzynski DJ....The oviduct plays important roles in the early reproductive process. The aim of this study was to evaluate gene transcription and protein expression of progesterone receptor (PGR), estrogen receptors 1 (ESR1) and 2 (ESR2); oxytocin receptor (OXTR); prostaglandin F2α synthase (AKR1C3), and prostaglandin E2 synthase (Ptges) in mare oviduct in different estrous cycle stages. Estradiol (E), progesterone (P), oxytocin (OXT), and tumor necrosis factor α (TNF) effect on in vitro PGE and prostaglandin F2α (PGF) secretion by equine oviduct explants or by oviductal epithelial cells (OECs) were also ...
Ashraf Kharaz Y, Zamboulis D, Sanders K, Comerford E, Clegg P, Peffers M.Exploring the tendon proteome is a challenging but important task for understanding the mechanisms of physiological/pathological processes during ageing and disease and for the development of new treatments. Several extraction methods have been utilised for tendon mass spectrometry, however different extraction methods have not been simultaneously compared. In the present study we compared protein extraction in tendon with two chaotropic agents, guanidine hydrochloride (GnHCl) and urea, a detergent, RapiGest™, and their combinations for shotgun mass spectrometry. An initial proteomic analysi...
Strauch S, Wichert B, Greef JM, Hillegeist D, Zeyner A, Liesegang A.The aim of this study was to improve an in vitro system in order to gather optimized information on the digestion of different forages in the horse's upper gastrointestinal tract. Therefore, foregut digestion of several forages was simulated in vitro (Part 1). The effect of different pH values on in vitro fructan degradation of two selected grasses (Part 2) was tested subsequently. Part 1: We hypothesized that our system produces representative results simulating digestive processes in the upper alimentary tract, but neglects microbial fermentation. In vitro digestion of six forages (grass ...
Prokhorov NS, Riccio C, Zdorovenko EL, Shneider MM, Browning C, Knirel YA, Leiman PG, Letarov AV.Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and funct...
Mendoza FJ, Serrano-Rodriguez JM, Buzon-Cuevas A, Perez-Ecija A.Alternatives to surgical castration are necessary for controlling the sexual behaviour of stallions with breeding potential in training and competition. Flutamide is a potent selective non-steroidal androgen receptor competitive antagonist that has been used in human beings as an anti-androgenic drug. In this study, the pharmacokinetics and bioavailability of flutamide and its main active metabolite, 2-hydroflutamide, were determined in seven healthy mature stallions. Single doses of flutamide (1mg/kg intravenously, 1mg/kg orally in fasted horses, 5mg/kg orally in fasted horses and 5mg/kg oral...
Pihl TH, Jacobsen S, Olsen DT, Højrup P, Grosche A, Freeman DE, Andersen PH, Houen G.OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease. ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectr...
Bowker RM, Lancaster LS, Isbell DA.OBJECTIVE To examine the equine foot for the presence of sensory receptors including Merkel cells and small lamellated Pacinian-like corpuscles (SLPCs). SAMPLE Forefeet obtained from 7 horses following euthanasia for reasons other than foot disease. PROCEDURES Disarticulated feet were cut into either sagittal sections or cross sections and immersed in neutral-buffered 4% formalin. Following fixation, samples were obtained from the midline of the dorsal aspect of the hoof wall and from the frog (cuneus ungulae) between the apex and central sulcus. The formalin-fixed, paraffin-embedded hoof wall...
Collas C, Sallé G, Dumont B, Cabaret J, Cortet J, Martin-Rosset W, Wimel L, Fleurance G.The spread of anthelmintic resistance in equine strongyle nematodes has become a major problem, advocating for the development of alternative control for strongyles. Our study consisted of both in vivo and in vitro experiments. We investigate for the first time the efficacy of a short-term consumption of tannin-rich sainfoin (Onobrychis viciifolia) or extra proteins in naturally infected horses. We used 30 horses allocated into three groups of 10 individuals that received for 18 days either (i) a tannin-rich diet with 70% DM sainfoin pellets (Sd), (ii) a protein-rich diet with 52% DM Italian r...
Maia L, de Moraes CN, Dias MC, Martinez JB, Caballol AO, Testoni G, de Queiroz CM, Peña RD, Landim-Alvarenga FC, de Oliveira E.To the best of our knowledge, this is the first study describing the proteome of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs) in a global and functional manner. The aim of this work was to analyze the proteome of previously characterized UCIM-MSCs to determine protein abundance and classify the identified proteins according to Gene Ontology (GO) terms. Protein classification analysis according to biological process, molecular function and cellular component was performed using the PANTHER (Protein ANalysis THrough Evolutionary Relationships) Classification Syst...
Warnken T, Brehm R, Feige K, Huber K.The aim of the study was to analyze key proteins of the equine insulin signaling cascade and their extent of phosphorylation in biopsies from muscle tissue (MT), liver tissue (LT), and nuchal AT, subcutaneous AT, and retroperitoneal adipose tissues. This was investigated under unstimulated (B1) and intravenously insulin stimulated (B2) conditions, which were achieved by injection of insulin (0.1 IU/kg bodyweight) and glucose (150 mg/kg bodyweight). Twelve warmblood horses aged 15 ± 6.8 yr (yr), weighing 559 ± 79 kg, and with a mean body condition score of 4.7 ± 1.5 were included in the s...
Bellefeuille M, Peters DF, Nolin M, Slusarewicz P, Telgenhoff D.Collagen cross-linking is an attractive therapeutic route aimed at supplementing natural collagen stabilisation. In this study the toxicity of the cross-linker genipin (GP) was examined in avascular (tendon) and vascular (dermis) tissue. Methods: High doses of GP were injected intratendinously into three yearling horses and evaluated at various time points up to 30 days. A second group of three yearlings were injected into the dermis and evaluated at various time points up to 1 year. Metrics used included lameness, circumferential swelling, ultrasound evaluation, microscopic morphology, collag...
Harman RM, Bihun IV, Van de Walle GR.The prevalence of cutaneous fibroproliferative disorders (CFPDs) is high and almost exclusively occurs in humans (keloids and hypertrophic scars) and horses (exuberant granulation tissue), making the horse a valuable translational model for studies on prevention and treatment of human CFPDs. CFPDs arise as a result of dysregulated wound healing characterized by persistently high levels of cytokines, such as transforming growth factor beta 1 (TGF-β1), that contribute to excessive extracellular matrix deposition, and the physical disorganization of dermal fibroblasts (DF). The mesenchymal strom...
Gastal GDA, Aguiar FLN, Alves BG, Alves KA, de Tarso SGS, Ishak GM, Cavinder CA, Feugang JM, Gastal EL.Ovarian tissue cryopreservation allows the preservation of the female fertility potential for an undetermined period. The objectives of this study were to compare the efficiency of cryoprotective agents (CPAs; dimethyl sulfoxide, DMSO; ethylene glycol, EG; and propylene glycol, PROH) using slow-freezing and vitrification methods, and evaluate the viability of cryopreserved equine ovarian tissue after 7 days of culture. Fresh and cryopreserved ovarian fragments were evaluated for preantral follicle morphology, stromal cell density, EGFR, Ki-67, Bax, and Bcl-2 protein expression, and DNA fragmen...
Braus BK, Miller I, Kummer S, Kleinwort KJH, Hirmer S, Hauck SM, McMullen RJ, Kerschbaumer M, Deeg CA.Immune mediated keratitis (IMMK) is primarily a non-ulcerative keratitis in horses causing intermittent ocular pain, eventually resulting in visual impairment. Affected horses typically respond to immunomodulatory treatment. However, the underlying cause of the disease remains enigmatic. The current study was undertaken to investigate the presence of autoantibodies in horses with immune mediated keratitis. Using 28 horses with IMMK and 27 healthy controls screening for serum autoantibodies against the corneal proteome using indirect immunofluorescence, one-dimensional (1DE) and two-dimensional...
Wang J, Wen S, Zhao R, Qi J, Liu Z, Li W, An J, Wood C, Wang Y.The conjugation of small ubiquitin-like modifier (SUMO) to the target protein, namely, SUMOylation, is involved in the regulation of many important biological events including host-pathogen interaction. Some viruses have evolved to exploit the host SUMOylation machinery to modify their own protein. Retroviral Gag protein plays critical roles in the viral life cycle. The HIV-1 p6 and the Moloney murine leukemia virus CA have been reported to be conjugated with SUMO. In this study, we report for the first time, to our knowledge, the covalent conjugation of equine infectious anemia virus (EIAV) G...
Murungi EK, Kariithi HM.The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM), a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs) have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clust...
Sedykh SE, Purvinish LV, Monogarov AS, Burkova EE, Grigor'eva AE, Bulgakov DV, Dmitrenok PS, Vlassov VV, Ryabchikova EI, Nevinsky GA.Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel fi...
Reinholt BM, Bradley JS, Jacobs RD, Ealy AD, Johnson SE.Rapid morphological and gene expression changes occur during the early formation of a mammalian blastocyst. Critical to successful retention of the blastocyst and pregnancy is a functional trophectoderm (TE) that supplies the developing embryo with paracrine factors and hormones. The contribution of TE conformational changes to gene expression was examined in equine induced trophoblast (iTr) cells. Equine iTr cells were cultured as monolayers or in suspension to form spheres. The spheres are hollow and structurally reminiscent of native equine blastocysts. Total RNA was isolated from iTr monol...
van Gennip RGP, van de Water SGP, Potgieter CA, van Rijn PA.The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essen...
Lee YS, Jung HJ, Yoon MJ.Putative markers for each specific germ cell stage can be a useful tool to study the fate and functions of these cells. Undifferentiated embryonic cell transcription factor 1 (UTF1) is a putative marker for undifferentiated spermatogonia in humans, rats and horses. The deleted in azoospermia-like (DAZL) protein is also expressed by differentiated spermatogonia and primary spermatocytes in several species. However, whether the expression patterns of these molecular markers are identical and applicable to donkeys remains to be elucidated. The objective of this study was to investigate the expres...
Bian J, Khaychuk V, Angers RC, Fernández-Borges N, Vidal E, Meyerett-Reid C, Kim S, Calvi CL, Bartz JC, Hoover EA, Agrimi U, Richt JA, Castilla J....Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrP) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrP Although disease transmission to only a subset of infecte...
Pastuszka R, Barłowska J, Litwińczuk Z.Protein content in cow milk (with over 20 proteins, and peptides may also occur as a result of enzymatic hydrolysis) ranges from 2.5% to 4.2% and is about 1.5-2 times higher than in human milk. Its most important allergens are considered to be β-lactoglobulin (absent in human milk) and αs1-casein. The most similar in composition to human milk is horse and donkey milk. It contains considerably more whey proteins (35-50%) than cow milk (about 20%), and the concentration of the most allergenic casein fraction αs1 is 1.5-2.5 g/l. In comparison, the content of αs1-casein in cow milk is about 10...
Qureshi SH, Moza B, Yadav S, Ahmad F.The denaturation of bovine and horse cytochromes-c by weak salt denaturants (LiCl and CaCl(2)) was measured at 25 degrees C by observing changes in molar absorbance at 400 nm (Delta epsilon(400)) and circular dichroism (CD) at 222 and 409 nm. Measurements of Delta epsilon(400) and mean residue ellipticity at 409 nm ([theta](409)) gave a biphasic transition for both modes of denaturation of cytochromes-c. It has been observed that the first denaturation phase, N (native) conformation X (intermediate) conformation and the second denaturation phase, X conformation D (denatured) conformation are...
Crossett B, Allen WR, Stewart F.Large quantities of an unusual 19 kDa protein (p19) are secreted into the lumen of the uterus of the mare (Equus caballus) during the oestrous cycle and early pregnancy. p19 associates strongly with the acellular capsule that surrounds the young horse conceptus and is believed to be important in maintaining pregnancy. Here we report the complete cDNA sequence encoding p19, its expression patterns in horse tissues and a Southern blot analysis of the gene in horse DNA. The predicted amino acid sequence of the p19 cDNA demonstrated a signal peptide of 18 residues and a mature protein of 162 resid...
Blommaert D, Sergeant N, Delehedde M, Donnay I, Lejeune JP, Franck T, Serteyn D.The quality of fresh or thawed sperm in stallions has been generally determined by the viability and total and progressive motility of the sperm. Today, the expression of ProAKAP4, a protein present in the flagellum of spermatozoa, appears to be an innovative and relevant functional marker to assess semen quality and male fertility. This study aims to compare the concentration of ProAKAP4 in the semen from 5 stallions frozen with two different extenders immediately after thawing (T0) and 4 h post-thawing (T4). Viability, total and progressive motility were measured in parallel. Significant di...
Chanter N, Talbot NC, Newton JR, Hewson D, Verheyen K.The M-protein genes of Streptococcus equi isolated from 17 outwardly healthy horses after 4 strangles outbreaks had ended, including a quarantined animal, were compared with those of S. equi isolates from 167 active cases of strangles across 4 countries. The healthy horses included 16 persistent S. equi carriers, at least one from each of the four outbreaks. These carriers, despite being outwardly healthy, had empyema of the guttural pouch(es), an enlargement of the equine Eustachian tube. A persistent carrier from two of these outbreaks, the quarantined animal and a healthy animal with normal...
Grund CH, Lechman ER, Issel CJ, Montelaro RC, Rushlow KE.In this study we used immune sera from equine infectious anaemia virus (EIAV)-infected horses which uniquely display broad reactivity with different lentivirus capsid proteins (CA) to characterize the cross-reactive determinants of lentivirus CA proteins. In particular, the role of the major homology region (MHR) of lentivirus CA proteins in this serological cross-reactivity was evaluated using both equine immune serum and murine monoclonal antibodies (MAbs) directed against the MHR segment of different lentiviruses. The results of our studies indicate that about 80% of sera from long-term exp...
Dobrinski I, Ignotz GG, Thomas PG, Ball BA.To test the hypotheses that the attachment of equine spermatozoa to uterine tubal (oviductal) epithelial cells (OEC) in vitro is mediated by glycoproteins, and that proteins with carbohydrate-binding properties are present in the periacrosomal plasma membrane of equine spermatozoa. Methods: 4 reproductively sound stallions, and 1 mare in estrus. Methods: In experiment 1a, fluorescent-labeled spermatozoa were cocultured with monolayers of OEC in the presence of 50 mM glucose, fructose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid, or 10 mg of fet...
Lacombe VA, Hinchcliff KW, Devor ST.To characterize insulin-sensitive glucose-transporter (GLUT-4) protein in equine tissues and determine effects of exercise and glucose administration on content of GLUT-4 protein in equine skeletal muscle. Methods: Tissue samples from 9 horses. Methods: Western blot analyses were performed on crude membrane preparations of equine tissues to characterize GLUT-4. In a crossover, randomized study, horses were strenuously exercised for 3 consecutive days and then administered 13.5% glucose or isotonic saline (0.9% NaCl; control) solution, i.v., at similar infusion rates for 12.1 hours. Samples wer...
Borzacchiello G, Mogavero S, De Vita G, Roperto S, Della Salda L, Roperto F.The equine sarcoid is the most common dermatologic neoplasm reported in horses. Bovine papillomavirus (BPV) types 1 and 2 are associated with sarcoids, in which the expression of the major transforming oncoprotein (E5) is often recorded. The transformation activity of the virus is due to the binding of the E5 to the platelet-derived growth factor beta receptor (PDGFbeta-r). In the present study, we show by Western blot in 4 sarcoid samples and 3 normal equine skin samples that the PDGFbeta-r is more phosphorylated in sarcoid tissue than in normal skin (P < .001). Furthermore, the physical i...
Boivin R, Vargas A, Lefebvre-Lavoie J, Lauzon AM, Lavoie JP.Overexpression of the (+)insert smooth muscle myosin heavy chain (SMMHC) isoform could contribute to airway bronchospasm by increasing the velocity of contraction. Whether the (+)insert isoform is present in the small airways and its expression is reversible in asthma are unknown. Objective: To determine the anatomical location and the expression kinetics of the (+)insert SMMHC isoform in airways of horses with heaves and to evaluate its modulation in response to disease status. Methods: We evaluated the (+)insert SMMHC isoform in the airways of horses with heaves during disease exacerbation a...
Bousfield GR, Ward DN.A method has been developed for the purification of equine lutropin (eLH) and equine follitropin (eFSH) from horse pituitary glands which attains high yields of both hormones in contrast to previous methods that were devoted to one or the other with inferior recovery of the hormones. Two-pass chromatography over CM-Sephadex was used to separate eLH from eFSH. Subsequent steps employing QAE (quaternary amino-ethyl)-Sephadex chromatography and gel filtration on Sephacryl S-200 produced highly purified hormone preparations. Yields of purified eLH and eFSH were 110 and 60 mg/kg of frozen pituitari...
Hoffmann C, Bazer FW, Klug J, Aupperle H, Ellenberger C, Schoon HA.Although alterations in patterns of protein secretion revealed in uterine flushings from mares suffering from endometrosis have been described, little is known about alterations at the cellular level. Hence, the aim of this study was to characterize deviations in patterns of uterine gland secretion patterns using endometrial biopsies, histochemical and newly established immunohistochemical methods. Forty-eight endometrial biopsies were obtained from mares suffering from various types of endometrosis (active and inactive, destructive and non-destructive) and degree (mild to severe) were analyze...
Zhang B, Sun C, Jin S, Cascio M, Montelaro RC.The equine lentivirus receptor 1 (ELR1), a member of the tumor necrosis factor receptor (TNFR) protein family, has been identified as a functional receptor for equine infectious anemia virus (EIAV). Toward defining the functional interactions between the EIAV SU protein (gp90) and its ELR1 receptor, we mapped the gp90 binding domain of ELR1 by a combination of binding and functional assays using the EIAV SU gp90 protein and various chimeric receptor proteins derived from exchanges between the functional ELR1 and the nonbinding homolog, mouse herpesvirus entry mediator (murine HveA). Complement...
Birch-Machin I, Rowan A, Pick J, Mumford J, Binns M.The nucleotide sequence of the nonstructural protein NS1 of the influenza virus A/equine 2/Suffolk/89 was determined and found to be 97% identical to that of A/equine 2/Miami/63. A similar level of identity was shown for the deduced NS1 amino acid sequence. The NS1 gene was expressed, in its entirety and in part, as fusion proteins with glutathione S-transferase using the pGEX-3X expression vector. Antibodies to NS1 protein were detected in serum samples from ponies experimentally infected with influenza virus, but not in animals vaccinated with whole inactivated virus or in unprimed control a...
Johnson A, Smith R, Saxne T, Hickery M, Heinegård D.Previous experiments have shown that addition of fragmented fibronectin can induce cartilage chondrolysis. In this study we investigated the fate of the collagen- and cell-binding molecules Cartilage oligomeric matrix protein (COMP) and chondroadherin. Methods: Equine articular cartilage explants were stimulated with the C-terminal and the N-terminal heparin-binding fragments of fibronectin respectively, and the conditioned media were analysed by both quantitative (ELISA) and qualitative (mass spectrometry, Western blots) methods. Results: Both COMP and chondroadherin were released in a dose-d...
Klein C, Troedsson MH, Rutllant J.The expression of 12 different aquaporin subtypes in equine endometrium was examined at the mRNA and protein level. Endometrial samples were obtained during anoestrus, oestrus, 8, and 14 days after ovulation in non-pregnant mares, and 14 days after ovulation in pregnant mares. Quantitative PCR revealed a time-dependent pattern for all aquaporin subtypes examined except for AQP10 and 12. AQP3, 5 and 7 showed highest mRNA abundance 8 days after ovulation, while AQP0 and 2 were most abundant at Day 14 of the cycle in non-pregnant mares. At 14 days of pregnancy, AQP1, 4, 8, 9 and 11 displayed ...
Segura A, Herrera M, González E, Vargas M, Solano G, Gutiérrez JM, León G.Liquid formulations of antivenom require a cold chain for their distribution and storage, especially in tropical countries characterized by high temperature and humidity (climatic zone IV). Since cold chain is often deficient in many regions, there is a need to develop novel formulations of liquid antivenoms of higher stability at room temperatures. The effect of addition of the polyols mannitol and sorbitol on the thermal stability of caprylic acid-fractionated equine whole IgG antivenoms was assessed in preparations having different concentrations of protein and phenol. Results evidenced tha...
Sabeur K, Ball BA, Corbin CJ, Conley A.Testis-specific protein kinases are important because of their potential role in spermiogenesis, sperm maturation, and sperm function. In the present study, a novel serine-threonine kinase with high identity to human serine-threonine kinase 31 (STK31) was cloned from equine testis and expression of the protein was characterized in equine testis and ejaculated spermatozoa. Five over-lapping independent clones were plaque purified after screening of a lambda ZAP cDNA expression library constructed from equine testis. Sequence analysis and alignment of all five clones showed high identity with hu...
Harman RM, Bihun IV, Van de Walle GR.The prevalence of cutaneous fibroproliferative disorders (CFPDs) is high and almost exclusively occurs in humans (keloids and hypertrophic scars) and horses (exuberant granulation tissue), making the horse a valuable translational model for studies on prevention and treatment of human CFPDs. CFPDs arise as a result of dysregulated wound healing characterized by persistently high levels of cytokines, such as transforming growth factor beta 1 (TGF-β1), that contribute to excessive extracellular matrix deposition, and the physical disorganization of dermal fibroblasts (DF). The mesenchymal strom...
Longland AC, Barfoot C, Harris PA.The aim of this study was to determine the amounts of water-soluble carbohydrate (WSC), WSC constituents and protein leached from a range of U.K. hays soaked according to common practice. Initial hay WSC content ranged from 123 to 230 g/kg dry matter (DM). Soaking the hays for up to 16 hours in water at a mean temperature of 8°C resulted in a mean loss of 27 per cent (range 6 to 54 per cent) of hay WSC. The mean percentage losses of WSC constituents were 24 per cent (range 14 to 31) for fructan, 41 per cent (range 21 to 70) for fructose, 45 per cent (range 28 to 100) for sucrose and 56 per ce...
Williams ZJ, Velez-Irizarry D, Gardner K, Valberg SJ.Myofibrillar myopathy in humans causes protein aggregation, degeneration, and weakness of skeletal muscle. In horses, myofibrillar myopathy is a late-onset disease of unknown origin characterized by poor performance, atrophy, myofibrillar disarray, and desmin aggregation in skeletal muscle. This study evaluated molecular and ultrastructural signatures of myofibrillar myopathy in Warmblood horses through gluteal muscle tandem-mass-tag quantitative proteomics (5 affected, 4 control), mRNA-sequencing (8 affected, 8 control), amalgamated gene ontology analyses, and immunofluorescent and electron m...
Ji S, Na L, Ren H, Wang Y, Wang X.Human myxovirus resistance protein 2 (huMxB) has been shown to be a determinant type I interferon (IFN)-induced host factor involved in the inhibition of human immunodeficiency virus type 1 (HIV-1) as well as many other primate lentiviruses. This blocking occurs after the reverse transcription of viral RNA and ahead of integration into the host DNA, which is closely connected to the ability of the protein to bind the viral capsid. To date, Mx2s derived from nonprimate animals have shown no capacity for HIV-1 suppression. In this study, we examined the restrictive effect of equine Mx2 (eqMx2) o...
Go YY, Wong SJ, Branscum AJ, Demarest VL, Shuck KM, Vickers ML, Zhang J, McCollum WH, Timoney PJ, Balasuriya UB.The development and validation of a microsphere immunoassay (MIA) to detect equine antibodies to the major structural proteins of equine arteritis virus (EAV) are described. The assay development process was based on the cloning and expression of genes for full-length individual major structural proteins (GP5 amino acids 1 to 255 [GP5(1-255)], M(1-162), and N(1-110)), as well as partial sequences of these structural proteins (GP5(1-116), GP5(75-112), GP5(55-98), M(88-162), and N(1-69)) that constituted putative antigenic regions. Purified recombinant viral proteins expressed in Escherichia col...
Mugnier S, Kervella M, Douet C, Canepa S, Pascal G, Deleuze S, Duchamp G, Monget P, Goudet G.Oviduct epithelial cells (OEC) co-culture promotes in vitro fertilization (IVF) in human, bovine and porcine species, but no data are available from equine species. Yet, despite numerous attempts, equine IVF rates remain low. Our first aim was to verify a beneficial effect of the OEC on equine IVF. In mammals, oviductal proteins have been shown to interact with gametes and play a role in fertilization. Thus, our second aim was to identify the proteins involved in fertilization in the horse. Results: In the first experiment, we co-incubated fresh equine spermatozoa treated with calcium ionophor...
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....
