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.
Singh UK, Patel R.In vitro refolding of denatured protein and the influence of the alkyl chain on the refolding of a protein were tested using long chain imidazolium chloride salts, 1-methyl-3-octylimidazolium chloride [Cmim][Cl], and 1-decyl-3-methylimidazolium chloride [Cmim][Cl]. The horse heart cytochrome c (h-cyt c) was denatured by urea and guanidinium hydrochloride (GdnHCl), as well as by base-induced denaturation at pH 13, to provide a broad overview of the overall refolding behavior. The variation in the alkyl chain of the ionic liquids (ILs) showed a profound effect on the refolding of denatured h-cyt...
Steger K, Balhorn R.Protamines are nuclear proteins which are specifically expressed in haploid male germ cells. Their replacement of histones and binding to DNA is followed by chromatin hypercondensation that protects DNA from negative influences by environmental factors. Mammalian sperm contain two types of protamines: PRM1 and PRM2. While the proportion of the two protamines is highly variable between different species, abnormal ratios within a species are known to be associated with male subfertility. Therefore, it is more than likely that correct protamine expression represents a kind of chromatin checkpoint...
Vanniasinkam T, Barton MD, Das TP, Heuzenroeder MW.This chapter describes a strategy for mapping linear B-cell epitopes of proteins using synthetic biotinylated peptides in an ELISA.A set of overlapping peptides were designed based upon a known amino acid sequence of the target protein, VapA (Virulence-associated Protein A) of the bacterium Rhodococcus equi, an important pulmonary pathogen in foals. The peptides synthesized as biotinylated peptides were coated directly onto micro titer plates which had been pre-coated with NeutrAvidin™ and used to screen sera from foals confirmed to have R. equi disease. A linear B-cell epitope was identifie...
Read JE, Cabrera-Sharp V, Kitscha P, Cartwright JE, King PJ, Fowkes RC, de Mestre AM.Equine chorionic gonadotrophin (eCG) is a placental glycoprotein critical for early equine pregnancy and used therapeutically in a number of species to support reproductive activity. The factors in trophoblast that transcriptionally regulate eCGβ-subunit (), the gene which confers the hormones specificity for the receptor, are not known. The aim of this study was to determine if glial cells missing 1 regulates promoter activity. Here, studies of the proximal promoter identified four binding sites for glial cells missing 1 (GCM1) and western blot analysis confirmed GCM1 was expressed in equi...
Galvão A, Wolodko K, Rebordão MR, Skarzynski D, Ferreira-Dias G.In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin-like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast-like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid-, and Late-CL (p < 0.05). Subsequently, using an in vitro model with Mid-CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone...
Smits K, Willems S, Van Steendam K, Van De Velde M, De Lange V, Ververs C, Roels K, Govaere J, Van Nieuwerburgh F, Peelman L, Deforce D, Van Soom A.During maternal recognition of pregnancy (MRP), a conceptus-derived signal leads to the persistence of the corpus luteum and the maintenance of gestation. In the horse, the nature of this signal remains to be elucidated. Several studies have focused on the changes in gene expression during MRP, but little information exists at the protein level. The aim of this study was to identify the proteins at the embryo-maternal interface around signalling of MRP in the horse (day 13) by means of mass spectrometry. A distinct influence of pregnancy was established, with 119 proteins differentially expres...
Fedorka CE, Scoggin KE, Boakari YL, Hoppe NE, Squires EL, Ball BA, Troedsson MHT.The deposition of semen into the uterus of the horse induces a transient innate immune response that lasts 24-36 h in the normal mare. There exists a subset of mares that are unable to resolve this inflammation in a timely manner, and are classified as susceptible to the disease of persistent breeding-induced endometritis (PBIE). Lactoferrin is a protein of interest as a potential therapeutic for this persistent inflammation due to its anti-inflammatory and bactericidal properties. The addition of human recombinant lactoferrin (hrLF) to the insemination dose was previously shown to suppress ...
Loux SC, Ball BA.Placental inflammation (placentitis) is one of the leading causes of late-term abortion in mares. Although prognosis is good assuming early diagnosis and treatment, diagnostics are limited. To better characterize the disease and identify potential biomarkers, we analyzed the proteome of fetal fluids (amniotic and allantoic) in both control mares (n = 5) and mares with experimentally-induced placentitis (n = 5) using LTQ-Orbitrap mass-spectrometry. Placentitis was induced via trans-cervical inoculation of Streptococcus equi ssp. zooepidemicus. In total, 130 proteins were identified in e...
Luciani M, Di Febo T, Orsini M, Krasteva I, Cattaneo A, Podaliri Vulpiani M, Di Pancrazio C, Bachi A, Tittarelli M.The diagnosis of dourine can be difficult because the clinical signs of this disease in horses are similar to those of surra, caused by . Moreover, and are closely related and, so far, they cannot be distinguished using serological tests. In a previous work, the protein pattern recognized by antibodies from dourine-infected horses and the humoral immune response kinetics were investigated by immunoblotting assay; a total of 20 sera from naturally and experimentally infected horses and from healthy animals were tested. Immunoblotting analysis showed that antibodies from infected horses speci...
DeBoer ML, Martinson KM, Pampusch MS, Hansen AM, Wells SM, Ward C, Hathaway M.Leucine has been shown to stimulate the mammalian/mechanistic target of rapamycin (mTOR) signaling pathway which plays numerous key regulatory roles in cell growth, survival, and metabolism including protein synthesis in a number of species. However, previous work with equine satellite cells has suggested distinct species differences in regards to physiological effects and the magnitude of responses to growth factors and regulators. Because there is limited research available regarding the role of leucine in regulating equine skeletal muscle protein synthesis, the objective of this study was t...
Sudheer Kumar C, Swamy MJ.The major protein of horse seminal plasma, HSP-1/2, exhibits membranolytic and chaperone-like activities and plays a crucial role in regulating sperm capacitation. L-Carnitine is a small polar molecule present in high concentrations in mammalian seminal plasma. The present results demonstrate that L-carnitine binds to HSP-1/2 and increases its thermal stability, enhances cooperativity of its chemical unfolding and decreases both chaperone-like and membranolytic activities of this protein. The HSP-1/2-L-carnitine complex exhibits anti-oxidative behaviour by inhibiting the production of hydroxyl...
Carmalt JL, Mortazavi S, McOnie RC, Allen AL, Unniappan S.Equine pituitary pars intermedia dysfunction (PPID) is characterized by hyperplasia of the pars intermedia (PI) melanotrophs of the pituitary gland (PG), and increased production of proopiomelanocortin (POMC). POMC is cleaved by prohormone convertase 1 (PC1) to produce adrenocorticotropic hormone (ACTH), and further processing of ACTH by PC2 to produce alpha-melanocyte stimulating hormone (α-MSH) and corticotropin-like intermediate peptide (CLIP). High plasma ACTH concentrations in horses with PPID might be related to reduced conversion of ACTH to α-MSH by PCs. The hypothesis of this study w...
Reisbig NA, Hussein HA, Pinnell E, Bertone AL.OBJECTIVE To create a bioactive synovium scaffold by infusing decellularized synovial-derived extracellular matrix (synECM) with synovial-derived mesenchymal stem cells (synMSCs). SAMPLE Synovium from the femoropatellar and medial femorotibial joints of equine cadavers. PROCEDURES The synMSCs were cultured in monolayer and not treated or cotransduced to enhance expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. The synECM was decellularized with 0.1% peracetic acid and then seeded with synMSCs (0.5 × 10 cells/0.5 mL) by use of a 30% serum gradient. Sam...
Okada A, Suganuma S, Badr Y, Omatsu T, Mizutani T, Ohya K, Fukushi H.VP22 is a major tegument protein of equine herpesvirus type 1 (EHV-1). In the present study, we examined functions of VP22 in EHV-1 replication by viral protein expression analyses in cells infected with the VP22-deficient virus. The expressions of several viral proteins in the cells infected with the VP22-deficient virus were lower than those in the cells infected with the parent virus. One of the weakly expressed proteins was identified as ICP4, which is a major regulatory protein encoded by an immediate early gene of EHV-1. A real-time PCR analysis showed that the mRNA expression of ICP4 wa...
Shibany KA, Tötemeyer S, Pratt SL, Paine SW.1. Scaling factor values for the in vitro-in vivo extrapolation of hepatic metabolic clearance for xenobiotics have not yet been determined in horses. Scaling factors were determined by comparing the total protein and or cytochrome (CYP) P450 content in microsomes and cryopreserved hepatocytes against the content in the liver. 2. Microsomal protein per gram of liver (MPPGL) and hepatocellularity number per gram of liver (HPGL) using CYP P450 content method ranged 41-73 mg/gram of liver (mean= 57 mg/gram of liver, n = 39) and 146-320 × 10 cells/g of liver (mean = 227× 10 c...
Tesena P, Korchunjit W, Taylor J, Wongtawan T.Gastric tissue biopsy and gene expression analysis are important tools for disease diagnosis and study of the physiology of the equine stomach. However, RNA extraction from gastric biopsy samples is a complex procedure because the samples contain low quantities of RNA and are contaminated with mucous protein and bacterial flora. The objectives of these studies were to compare the performance of RNA extraction methods and to investigate the sensitivity of commercial qPCR master mixes for gene expression analysis of gastric biopsy samples. Three commercial RNA extraction methods (TRIzol, GENEzol...
Ketchum CC, Larsen CD, McNeil A, Meyer-Ficca ML, Meyer RG.Chromatin remodeling during spermatogenesis culminates in the exchange of nucleosomes for transition proteins and protamines as an important part of spermatid development to give rise to healthy sperm. Comparative immunofluorescence analyses of equine and murine testis histological sections were used to characterize nucleoprotein exchange in the stallion. Histone H4 hyperacetylation is considered a key event of histone removal during the nucleoprotein transition to a protamine-based sperm chromatin structure. In the stallion, but not the mouse, H4 was already highly acetylated in lysine residu...
Compton JR, Mickey MJ, Hu X, Marugan JJ, Legler PM.The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated antiviral drug target. Clan CN proteases contain a cysteine protease domain that is intimately packed with an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. Within a cleft formed at the interface of these two domains, the peptide substrate is thought to bind. The nucleophilic cysteine can be found within a conserved motif, NVCWAK, which differs from that of papain (CGSCWAFS). Mutation of the motif residue, N475, to alanine unexpectedly produced a self-inhibited s...
Romagnoli N, Zannoni A, Bernardini C, Gobbetti T, Bombardi C, Rambaldi AM, Morini M, Dondi F, Rinnovati R, Lambertini C, Spadari A.Proteinase-activated receptor 2 (PAR) is a G-protein-coupled receptor for trypsin and mast cell tryptase; it is highly expressed at the intestinal level with multiple functions, such as epithelial permeability and intestinal motility. The aim of the study was to evaluate the distribution and expression of proteinase-activated receptor 2 in the small intestine during herniation through epiploic foramen. In this prospective clinical study, eight horses admitted for colic and which underwent exploratory laparotomy were considered. During surgery, the jejunum or the ileum was sampled by enterectom...
Rossi HS, Koho NM, Ilves M, Rajamäki MM, Mykkänen AK.OBJECTIVE To examine whether expression of extracellular matrix metalloproteinase inducer (EMMPRIN) can be detected in equine lungs and whether it correlates with matrix metalloproteinase (MMP)-2 and -9 expression in bronchoalveolar lavage fluid (BALF) of horses with chronic inflammation of the lungs (ie, lower airway inflammation [LAI]). ANIMALS 29 horses with signs of chronic respiratory tract disease, which were classified as the LAI (n = 17) and LAI with respiratory distress (RDLAI [12]) groups, and 15 control horses. PROCEDURES BALF, tracheal aspirate, and blood samples were obtained, and...
Bonilla-Correal S, Noto F, Garcia-Bonavila E, Rodríguez-Gil JE, Yeste M, Miro J.Aquaporins (AQPs) are transmembrane proteins found in all cells and are responsible for the transport of water and small solutes. While these proteins have been found in the spermatozoa of humans, rodents, pigs and cattle, where not only do they play a role for the regulation of sperm volume but are also related with the sperm resilience to withstand freeze-thawing procedures, their presence in stallion sperm is yet to be reported. Therefore, the objectives of this work were as follows: (i) to determine whether AQP3, AQP7 and AQP11 are present in stallion sperm and (ii) to investigate whether ...
Yuan M, Breitkopf SB, Asara JM.Horse urine is easily collected and contains molecules readily measurable using mass spectrometry that can be used as biomarkers representative of health, disease or drug tampering. This study aimed at analyzing microliter levels of horse urine to purify, identify and quantify proteins, polar metabolites and non-polar lipids. Urine from a healthy 12 year old quarter horse mare on a diet of grass hay and vitamin/mineral supplements with limited pasture access was collected for serial-omics characterization. The urine was treated with methyl tert-butyl ether (MTBE) and methanol to partition into...
Martin EM, Messenger KM, Sheats MK, Jones SL.Pro-inflammatory cytokines including tumor necrosis factor α (TNFα), IL-1β, IL-6, and IL-8 are potent immune mediators that exacerbate multiple equine diseases such as sepsis and laminitis. Unfortunately, safe and effective cytokine-targeting therapies are lacking in horses; therefore, novel mechanisms of inhibiting cytokine production are critically needed. One potential mechanism for inhibiting cytokine synthesis is elevation of intracellular cyclic AMP (cAMP). In human leukocytes, intracellular cAMP production is induced by activation of E-prostanoid (EP) receptors 2 and 4. These recepto...
Hall SE, Upton RMO, McLaughlin EA, Sutherland JM.The mare ovary is unique in its anatomical structure; however, the signalling pathways responsible for physiological processes, such as follicular activation, remain uncharacterised. This provided us with the impetus to explore whether signalling molecules from important folliculogenesis pathways, phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and Janus kinase/signal transducer and activator of transcription (JAK/STAT), are conserved in the mare ovary. Messenger RNA expression of six genes important in follicle development was measured using quantitative polymerase chain reaction and pr...
Chen W, Zhang Y, Li X, Chen H, Sun J, Feng F.We took advantage of gasotransmitter HS as a chemical reaction-based trigger for controlled release of doxorubicin which is precoordinated by copper ions and enclosed in horse spleen apoferritin. The nanocomposite is stable at physiological pH and temperature before HS activation. The drug release process avoids disassembly of protein shells and is controllable by the strong affinity of sulfide with copper ions. The in vitro cytotoxicity assay indicates the antitumor effect of doxorubicin toward tumor cells could be achievable by HS activation.
Usuga A, Rojano BA, Restrepo G.Contribution of seminal plasma proteins to semen freezability has been reported in several species, suggesting these proteins as genetic markers. The aim of this study was to evaluate the relationship between cysteine-rich secretory protein-3 (CRISP-3) and some of its single-nucleotide polymorphisms (SNPs) with post-thawing semen quality in stallions. DNA was obtained from 100 stallions, regions of interest were amplified by polymerase chain reaction and sequenced. Evaluated SNPs within the equine CRISP-3 gene were CRISP3c.+199A>G (SNP1), CRISP3c.+566C>A (SNP2), CRISP3c.+622G>A (SNP3)...
Godinho MSC, Thorpe CT, Greenwald SE, Screen HRC.Tendon is composed of fascicles bound together by the interfascicular matrix (IFM). Energy storing tendons are more elastic and extensible than positional tendons; behaviour provided by specialisation of the IFM to enable repeated interfascicular sliding and recoil. With ageing, the IFM becomes stiffer and less fatigue resistant, potentially explaining why older tendons become more injury-prone. Recent data indicates enrichment of elastin within the IFM, but this has yet to be quantified. We hypothesised that elastin is more prevalent in energy storing than positional tendons, and is mainly lo...
Balmer P, Bauer A, Pujar S, McGarvey KM, Welle M, Galichet A, Müller EJ, Pruitt KD, Leeb T, Jagannathan V.Keratins represent a large protein family with essential structural and functional roles in epithelial cells of skin, hair follicles, and other organs. During evolution the genes encoding keratins have undergone multiple rounds of duplication and humans have two clusters with a total of 55 functional keratin genes in their genomes. Due to the high similarity between different keratin paralogs and species-specific differences in gene content, the currently available keratin gene annotation in species with draft genome assemblies such as dog and horse is still imperfect. We compared the National...
Khan SH, Islam A, Hassan MI, Sharma S, Singh TP, Ahmad F.A sequence alignment of horse cytochrome c (cyt c) with all known cyts c shows that Leu at position 94 is conserved, except in 14 species which have either Val or Ile at this position. It is also known that Leu94 of the mammalian cyt c plays an important role in folding and stability. The important question here is as to what will happen in terms of folding and stability if Leu94 of the mammalian cyt c is substituted by Val or Ile. To answer this question, we introduced natural substitutes of Leu94 by Val and Ile in horse cyt c. The purified L94V and L94I mutants under native condition (pH 6.0...
Bone reportsAugust 18, 2017
Volume 7 98-107 doi: 10.1016/j.bonr.2017.08.002
Ayodele BA, Mirams M, Pagel CN, Mackie EJ.Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (, , , , , , , , , , and ) were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas and were expressed at lower levels in the hypertrophic zone than in the reserve zone. , which encodes vacuolar H ATPase (V-ATP...
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...
Hamada D, Kuroda Y, Kataoka M, Aimoto S, Yoshimura T, Goto Y.One unique aspect of cytochrome c folding concerns the involvement of the covalently attached heme group and its axial ligands. To elucidate the role of the ligands in stabilizing the native and molten globule states, we studied the conformational and thermodynamic features of the iron-free derivative of horse cyctochrome c (porphyrin-cytochrome c). At neutral pH, far-UV circular dichroism suggested that porphyrin-cytochrome c has native-like alpha-helices, whereas near-UV CD suggested that the side-chains are flexible. Its stability against heat or denaturants was much less than that of the i...
Lindsay AM, Zhang M, Mitchell Z, Holden MTG, Waller AS, Sutcliffe IC, Black GW.Streptococcus equi causes equine 'strangles'. Hyaluronate lyases, which degrade connective tissue hyaluronan and chondroitins, are thought to facilitate streptococcal invasion of the host. However, prophage-encoded hyaluronate lyases are hyaluronan-specific and are thought to be primarily involved in the degradation of the hyaluronan capsule of streptococci during bacteriophage infection. To understand the role of prophage-encoded hyaluronate lyases further, we have biochemically characterized such a hyaluronate lyase, SEQ2045 from S. equi, and have shown that it is produced during equine infe...
Taddei N, Stefani M, Magherini F, Chiti F, Modesti A, Raugei G, Ramponi G.Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were...
dos Santos MC, D'Império Lima MR, Furtado GC, Colletto GM, Kipnis TL, Dias da Silva W.Pooled horse plasma containing antibodies against Crotalus durissus terrificus whole venom were digested with pepsin at an enzyme-substrate ratio of 8:1, pH 3.1, for 40 min and the F(ab')2M fragments purified by adding 8.7% caprylic acid (pH 5.0). For comparison, F(ab')2B purified by precipitation with ammonium sulphate and uncleaved IgG purified with caprylic acid were also prepared. Fab' fragments were obtained by reduction and alkylation of F(ab')2B. The anti-whole C.d. terrificus venom titers, determined by Dot-Blot were 12,800 (IgG), 6400 [F(ab')2B], 4800 [F(ab')2M] and 3200 (Fab'B). Immu...
Muhonen S, Connysson M, Lindberg JE, Julliand V, Bertilsson J, Jansson A.The objective of this study was to investigate the effect of CP intake from 2 grass silage-only diets, differing in CP concentration, fed at similar DMI on the equine colon ecosystem after an abrupt feed change between the diets. Four adult right ventral colon-fistulated geldings were fed one silage-only diet high in CP (HP, 873 g of CP/d) and one diet providing recommended intakes (RP, 615 g of CP/d). An adaptation period of 15 d on either the HP or the RP diet was followed by 2 experimental periods when the diets were fed for 22 d each in a crossover design. Colon samples were taken before a...
Waller AP, Lindinger MI.Carbohydrate (CHO) stored in the form of skeletal muscle glycogen is the main energy source for glycolytic and oxidative ATP production during vigorous exercise in mammals. In man, horse and dog both short-term high intensity and prolonged submaximal exercise deplete muscle glycogen. In horses, however, muscle glycogen synthesis is 2-3-fold slower than in man and rat, even when a diet high in soluble CHO is fed. There appear to be significant differences in CHO and glycogen metabolism between horses and other mammals, and it is becoming increasingly clear that many conclusions drawn from human...
Van Dael H, Haezebrouck P, Morozova L, Arico-Muendel C, Dobson CM.Despite their homologous structure, c-type lysozymes and alpha-lactalbumins have been found to differ profoundly in their unfolding behavior, in that the alpha-lactalbumins readily enter a partially unfolded collapsed state (the "molten globule"), whereas lysozymes unfold cooperatively to a highly unfolded state. The calcium-binding property of lysozyme from equine milk provides an evolutionary link between the two families of proteins. We demonstrate here that equine lysozyme undergoes a two-stage unfolding transition upon heating or in the presence of guanidine hydrochloride that is highly d...
Dordas-Perpinyà M, Yanez-Ortiz I, Sergeant N, Mevel V, Bruyas JF, Catalán J, Delehedde M, Briand-Amirat L, Miró J.ProAKAP4 is the precursor of AKAP4 (A-kinase Anchor protein 4), the main structural protein of the fibrous sheath of sperm. The amount of proAKAP4 reflects the ability of spermatozoa to maintain the flagellum activity and functionality up to the site of fertilization and is positively correlated with progressive motility in several mammalian species. The aim of this study was to investigate the relationship between proAKAP4 concentration with horse sperm motility descriptors and spermatic motile subpopulations. For this purpose, a total of 48 ejaculates from 13 different stallions were analyze...
McPartlin LA, Littell J, Mark E, Nelson JL, Travis AJ, Bedford-Guaus SJ.Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating mediu...
Restrepo G, Rojano B, Usuga A.Cysteine-rich secretory protein-3 (CRISP-3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP-3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP-3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP-3 was ident...
Guina T, Oliver DB.We cloned the gene encoding a membrane-interactive protein of Borrelia burgdorferi by means of its haemolytic activity in Escherichia coli. The haemolytic activity was erythrocyte-species specific, with progressively decreasing activity for erythrocytes from horse, sheep, and rabbit, respectively. Genetic analysis of the haemolytic determinant revealed two borrelia haemolysin genes, blyA and blyB, that are part of a predicted four-gene operon which is present in multiple copies on the 30 kb circular plasmid(s) of B. burgdorferi B31. blyA encodes a predicted alpha-helical 7.4 kDa protein with a...
Kim YC, Won SY, Do K, Jeong BH.Prion diseases, a protein misfolded disorder (PMD) caused by misfolded prion protein (PrP), present in a wide variety of hosts, ranging from ungulates to humans. To date, prion infections have not been reported in horses, which are well-known as prion disease-resistant animals. Several studies have attempted to identify distinctive features in the prion protein of horses compared to prion disease-susceptible animals, without the study on polymorphisms of the horse prion protein gene (PRNP). Since single nucleotide polymorphisms (SNPs) of PRNP in prion disease-susceptible animals are major susc...
Della Malva A, De Palo P, Lorenzo JM, Maggiolino A, Albenzio M, Marino R.The aim of this study was to evaluate the effect of post-mortem aging on tenderness development and proteolysis of myofibrillar proteins in 3 different horse muscles. Warner-Bratzler shear force decreased during aging in all muscles, showing the lowest values in longissimus lumborum (LL) in all sampling points. Myofibril fragmentation index significantly increased in LL and semimembranosus (SM) muscles throughout aging time whereas in semitendinosus (ST) it increased after 14 days of aging. Proteomics analysis revealed the major content of intact myofibrillar proteins with high molecular wei...
Coleman SJ, Zeng Z, Hestand MS, Liu J, Macleod JN.Sequencing of equine mRNA (RNA-seq) identified 428 putative transcripts which do not map to any previously annotated or predicted horse genes. Most of these encode the equine homologs of known protein-coding genes described in other species, yet the potential exists to identify novel and perhaps equine-specific gene structures. A set of 36 transcripts were prioritized for further study by filtering for levels of expression (depth of RNA-seq read coverage), distance from annotated features in the equine genome, the number of putative exons, and patterns of gene expression between tissues. From ...
Yang F, Richardson DW.The tendon is highly prone to injury, overuse, or age-related degeneration in both humans and horses. Natural healing of injured tendon is poor, and cell-based therapeutic treatment is still a significant clinical challenge. In this study, we extensively investigated the expression of tenogenic genes in equine bone marrow mesenchymal stem cells (BMSCs) and tenocyte-derived induced pluripotent stem cells (teno-iPSCs) stimulated by growth factors (TGF-3 and BMP12) combined with ectopic expression of tenogenic transcription factor MKX or cyclic uniaxial mechanical stretch. Western blotting reveal...
Degroote RL, Hauck SM, Amann B, Hirmer S, Ueffing M, Deeg CA.Equine recurrent uveitis is a spontaneous, lymphocyte-driven autoimmune disease. It affects horses worldwide and presents with painful remitting-relapsing inflammatory attacks of inner eye structures eventually leading to blindness. Since lymphocytes are the key players in equine recurrent uveitis, we were interested in potential changes of their protein repertoire which may be involved in disease pathogenesis. To create a reference for differential proteome analysis, we first unraveled the equine lymphocyte proteome by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis ...
Moczygemba C, Guidry J, Wittung-Stafshede P.Native myoglobin (Mb) consists of two populations which differ in the orientation of the heme by 180 degrees rotation (as verified by nuclear magnetic resonance) but have identical absorption spectra and equilibrium-thermodynamic stability. Here, we report that these two fractions of native oxidized Mb (from horse) both unfold and refold (chemical denaturant, pH 7, 20 degrees C) in two parallel kinetic reactions with rate constants differing 10-fold. In accord, the oxidized heme remains coordinated to unfolded horse Mb in up to 4 M guanidine hydrochloride (pH 7, 20 degrees C).
Evenberg A, Meyer H, Gaastra W, Verheij HM, De Haas GH.The complete amino acid sequence of phosphlipase A2 (EC 3.1.1.4) from horse pancreas was determined. The protein controls of a single polypeptide chain of 125 amino acids and has a molecular weight of 13,927. The chain is crosslinked by seven disulfide bridges. The sequence was determined by automated Edman degradation of the intact protein and several of the large peptide fragments. Smaller peptides were analyzed by manual Edman degradation. Fragmentation of the peptide chain was accomplished by enzymatic digestion with trypsin, chymotrypsin, and thermolysin. The final overlap was found by di...
Wagner B, Robeson J, McCracken M, Wattrang E, Antczak DF.Recombinant cytokines are valuable tools for functional studies and candidates for vaccine additives or therapeutic use in various diseases. They can also be used to generate specific antibodies to analyze the roles of different cytokines during immune responses. We generated a mammalian expression system for recombinant cytokines using the equine IgG1 heavy chain constant region as a tag for detection and purification of the expressed cytokine, demonstrated here using equine interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL4) and transforming growth factor-beta1 (TGF-beta1...
Caballero AR, Lottenberg R, Johnston KH.Streptokinases secreted by nonhuman isolates of group C streptococci (Streptococcus equi, S. equisimilis, and S. zooepidemicus) have been shown to bind to different mammalian plasminogens but exhibit preferential plasminogen activity. The streptokinase genes from S. equisimilis strains which activated either equine or porcine plasminogen were cloned, sequenced, and expressed in Escherichia coli. The streptokinase secreted by the equine isolate had little similarity to any known streptokinases secreted by either human or porcine isolates. The streptokinase secreted by the porcine isolate had li...
Chagas JR, Hirata IY, Juliano MA, Xiong W, Wang C, Chao J, Juliano L, Prado ES.The present studies demonstrate the importance of subsite interactions in determining the cleavage specificities of kallikrein gene family proteinases. The effect of substrate amino acid residues in positions P3-P'3 on the catalytic efficiency of tissue kallikreins (rat, pig, and horse) and T-kininogenase was studied using peptidyl-pNA and intramolecularly quenched fluorogenic peptides as substrates. Kinetic analyses show the different effects of D-amino acid residues at P3, Pro at P'2, and Arg at either P'1 or P'3 on the hydrolysis of substrates by tissue kallikreins from rat and from horse o...
Albrecht RA, Kim SK, O'Callaghan DJ.The equine herpesvirus 1 (EHV-1) EICP27 protein cooperates with either the immediate-early (IE) or the EICP0 protein to synergistically trans-activate viral promoters. GST-pulldown and co-immunoprecipitation assays revealed that the EICP27 protein's cooperation with the IE or the EICP0 protein involves its physical interaction with these viral proteins. In the case of the IE-EICP27 protein interaction, IE residues 424 to 826 and EICP27 residues 41 to 206 harbor the interactive domains. Electrophoretic mobility shift assays (EMSA) suggested that the EICP27 protein is not a sequence-specific DNA...
X-ray structures of homopolymeric human L-ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa-nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (μ -oxo)tris[(μ -glutamato-κO:κO')](glutamato-κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the l...
Bauminger ER, Harrison PM, Nowik I, Treffry A.Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room ...
Baumann U, Bode W, Huber R, Travis J, Potempa J.The crystal structure of active-site cleaved equine leucocyte elastase inhibitor, a member of the serpin superfamily, has been solved and refined to a crystallographic R-factor of 17.6% at 1.95 A resolution. Despite being an intracellular inhibitor with rather low sequence homology of 30% to human alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor, the three-dimensional structures are very similar, with deviations only at the sites of insertions and few mobile secondary structure elements. The better resolution in comparison with the structures of other cleaved serpins allows a more pre...
Thoburn R, Hurvitz AI, Kunkel HG.Various mammalian species contain an anionic serum protein that reacts specifically with native DNA. It is considerably less reactive with single-strand DNA and does not react with monodeoxyribonucleotides, homopolyribonucleotides, or duplexes of homopolyribonucleotides. Synthetic dA.dT was an effective inhibitor of the reaction with native DNA, while Micrococcus luteus DNA and dG.dC were not inhibitory. This protein was encountered in the course of studies on DNA antibodies. Although it reacted with red cells coated with DNA and gave agar precipitation bands, it was clearly distinct from DNA ...
Morozova-Roche LA, Jones JA, Noppe W, Dobson CM.The refolding of equine lysozyme from guanidinium chloride has been studied using hydrogen exchange pulse labelling in conjunction with NMR spectroscopy and stopped flow optical methods. The stopped flow optical experiments indicate that extensive hydrophobic collapse occurs rapidly after the initiation of refolding. Pulse labelling experiments monitoring nearly 50 sites within the protein have enabled the subsequent formation of native-like structure to be followed in considerable detail. They reveal that an intermediate having persistent structure within three of the four helices of the alph...
Côté O, Lillie BN, Hayes MA, Clark ME, van den Bosch L, Katavolos P, Viel L, Bienzle D.Secretoglobin 1A1 (SCGB 1A1), also called Clara cell secretory protein, is the most abundantly secreted protein of the airway. The SCGB1A1 gene has been characterized in mammals as a single copy in the genome. However, analysis of the equine genome suggested that horses might have multiple SCGB1A1 gene copies. Non-ciliated lung epithelial cells produce SCGB 1A1 during inhalation of noxious substances to counter airway inflammation. Airway fluid and lung tissue of horses with recurrent airway obstruction (RAO), a chronic inflammatory lung disease affecting mature horses similar to environmental...
