Molecular biology in horses involves the study of molecular processes and genetic mechanisms that underpin equine physiology and health. This field encompasses the analysis of DNA, RNA, proteins, and other biomolecules to understand gene expression, genetic variation, and cellular functions in horses. Techniques such as genomic sequencing, gene expression profiling, and molecular diagnostics are employed to explore topics like hereditary diseases, performance traits, and immune responses in equines. This page assembles peer-reviewed research studies and scholarly articles that investigate the molecular biology of horses, focusing on genetic research, molecular techniques, and their applications in equine science.
Calvete JJ, Mann K, Schäfer W, Sanz L, Reinert M, Nessau S, Raida M, Töpfer-Petersen E.We report the complete amino acid sequence of HSP-1, a major protein isolated from stallion seminal plasma or acid extracts of ejaculated spermatozoa. The protein consists of 121 amino acids organized in two types of homologous repeats arranged in the pattern AA'BB'. Each of the 13-15-residue A-type repeats contains two O-linked oligosaccharide chains. The B-type repeats span 44-47 amino acids each, are not glycosylated, and have the consensus pattern of the gelatin-binding fibronectin type-II module. This domain also occurs in the major bovine seminal plasma heparin-binding proteins PDC-109 (...
Hurwitz A, Finci-Yeheskel Z, Dushnik M, Milwidsky A, Shimonovitz S, Yagel S, Adashi EY, Mayer M.This study examines the effects of interleukin-1 (IL-1) on plasminogen activator (PA) activity and prostaglandin (PG) E production in pregnant mare serum gonadotropin (PMSG)-primed granulosa cells and the potential involvement of PGE in the regulation of ovarian plasminogen activation. Methods: Granulosa cells were obtained from PMSG-primed rat (27-day-old) ovaries and cultured in serum-free conditions for 48 hours in the absence or presence of IL-1 beta (10 ng/mL) with and without transforming growth factor-beta 1 (10 ng/mL). Cellular PA activity was measured through the conversion of plasmin...
Marti E, Szalai G, Bucher K, Dobbelaere D, Gerber H, Lazary S.In order to isolate a part of the immunoglobulin E (IgE) heavy chain cDNA of the horse, primers have been designed based upon well conserved sequences in humans, sheep and rats. The PCR resulted in a 500 bp fragment which hybridised with a human IgE constant region probe. The fragment was cloned and sequenced and its derived protein sequence compared with the corresponding sequences in humans, sheep and mice. Most amino acids common to these three species are also shared by the horse.
Moulay S, Zientara S, Sailleau C, Cruciere C.In order to develop a non-radioactive dot-blot hybridization assay, for the detection of African-horse sickness virus (AHSV), genome segment 7 from 9 serotypes was amplified by RT-PCR. The resulting PCR products were denatured, immobilized on nylon membranes and then hybridized to a non-radioactive digoxigenin-labelled probe. This probe (265 bp in length) was generated by nested-PCR using genome segment 7 of AHSV, serotype 4 as a template. The dot-blot was visualized by chemiluminescence. Positives were obtained from the PCR products amplified from all 9 AHSV serotypes, but not from any other ...
Ishida N, Oyunsuren T, Mashima S, Mukoyama H, Saitou N.The noncoding region between tRNAPro and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA D-loop region. This variable region (ca. 270 bp) of four species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data were compared with our recently published Thoroughbred horse mitochondrial DNA sequences. The evolutionary rate of this region among the four species of Equus was estimated to be 2-4 x 10(-8) per site per year. Phylogenetic trees of Equus species demo...
Guo Y, Wang M, Zheng GS, Li WK, Kawaoka Y, Webster RG.In May 1993, a severe epidemic of respiratory disease began in horses in Inner Mongolia and spread throughout horses in China. The disease affected mules and donkeys as well as horses but did not spread to other species, including humans. The severity of the disease raised the question of whether the outbreak might have been caused by the new avian-like influenza viruses detected in horses in China in 1989 or by current variants ofA/equine/Miami/1/63 (H3N8) (equine-2) or by a reassortant between these viruses. Antigenic and sequence analysis established that all gene segments of the influenza ...
Chirnside ED, de Vries AA, Mumford JA, Rottier PJ.Complementary DNAs encoding ORFs 2 to 7 equine arteritis virus (EAV) have been cloned into the expression vector pGEX to produce glutathione-S-transferase fusion proteins. Recombinant proteins were affinity purified and screened in ELISA with equine sera to identify immunoreactive polypeptides. The large envelope glycoprotein (GL) was identified as the most reactive to EAV-positive equine sera and an immuno-dominant epitope was mapped between amino acids 55 and 98 by subcloning and expression. A fusion protein covering this region and a GL-specific synthetic peptide (residues 75 through 97) in...
Ochoa M, Bárcena J, de la Luna S, Melero JA, Douglas AR, Nieto A, Ortín J, Skehel JJ, Portela A.Characterization of the epitopes recognized by 21 monoclonal antibodies (MAbs) specific for the influenza A virus PA (13 MAbs) and PB2 (8 MAbs) polypeptides (Bárcena et al. (1994) J. Virol. 68, 6900-6909) raised against denatured polypeptides produced in E. coli is described. MAbs were characterized by: (1) competitive binding ELISAs; (2) mapping of the protein regions that specify their binding sites; and (3) analyses of their ability to recognize the corresponding viral protein in a number of viral isolates. Five and three non-overlapping antigenic areas were defined by the anti-PA and anti...
Chopineau M, Stewart F, Allen WR.The coding regions of the horse (Equus caballus) and donkey (E. asinus) luteinizing hormone (LH) beta-subunit transcripts were cloned from pituitary gland RNA, in order to investigate their relationships to the corresponding equine chorionic gonadotropin (CG) beta-subunits and to further understand the unusual receptor-binding properties of equine LH and CG. The horse and donkey LH beta-subunit sequences were very similar (97% identity at the nucleotide (nt) level; 93% at the amino acid (aa) level), confirming their very close evolutionary linkage and also indicating that the C-terminal extens...
Dillon AM, Heath MF.The effects of the protein tyrosine kinase inhibitors tyrphostins B42 and B46 on equine platelet function and protein tyrosine phosphorylation (PTP) were assessed. Tyrphostins B42 and B46 (both at 100 microM concentration) produced significant inhibition of thrombin-stimulated equine platelet aggregation. The effect of tyrphostin B46 was also time-dependent. The same concentration of these inhibitors produced very little or no inhibition of platelet-activating factor (PAF)-induced aggregation. The effects of tyrphostins B42 and B46 on thrombin- and PAF-stimulated PTP were generally similar, al...
Steagall WK, Robek MD, Perry ST, Fuller FJ, Payne SL.The retrovirus equine infectious anemia virus (EIAV) encodes a dUTPase situated between reverse transcriptase and integrase. We have described the inability of EIAV with a 270-bp dUTPase deletion, delta DU EIAV, to replicate to wild-type (WT) levels in equine macrophages (D. S. Threadgill, W. K. Steagall, M. T. Flaherty, F. J. Fuller, S. T. Perry, K. E. Rushlow, S. F. J. LeGrice, and S. L. Payne, J. Virol. 67, 2592-2600, 1993). Here we describe the construction of a second dUTPase-deficient virus (DUD71E) containing a single amino acid substitution in dUTPase. delta DU and DUD71E replicate to ...
Wagner B, Radbruch A, Richards C, Leibold W.In order to define equine immunoglobulins (Igs) and to produce monoclonal reference Igs we fused equine peripheral blood mononuclear cells with X63-Ag8.653 non Ig producing murine myeloma cells. A total of 29 equine Ig producing equi-murine heterohybridomas were obtained, of which ten expressed equine Ig for more than 3 months. One of these heterohybridoma lines produced monoclonal IgM, an equine isotype which has not been available in monoclonal form before. Four lines secreted equine IgG of two distinct Ig heavy chain types as assessed by the molecular weight (MW), while the remaining five l...
Dudhia J, Platt D.Investigation of the structure of the equine articular cartilage link protein (LP) from individuals ranging in age from 1 to 15 years identified 3 distinct isoforms having molecular weights of 46,000, 43,000 and 41,000. The relative amounts of each of the 3 isoforms altered with age. The largest form did not change with age; however, amounts of the Mr 43,000 and 41,000 forms increased with increasing age. The results suggested that an accumulation, in the extracellular matrix of cartilage, of these 2 smaller products may have arisen from proteolytic cleavage. The complete amino acid sequence o...
Foygel K, Spector S, Chatterjee S, Kahn PC.Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be -136 +/- 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded + 100 +/- 6 mL/mol. MG to N was broken into two steps, N to NClx at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClx = N interacting with an unknown number, X, of chloride ions), and MG to NClx by jumping MG at pH 2 in 0.5 M KCl to pH7 at the same salt concentration. The d...
Pead S, Durrant E, Webb B, Larsen C, Heaton D, Johnson J, Watt GD.The binding of Cd2+, Zn2+, Cu2+, Ni2+, Co2+, Mn2+, and Mg2+ to apo, holo, reconstituted horse spleen ferritin (HoSF), and native holo HoSF with phosphate removed was measured by gel-exclusion chromatography. Three classes of strong binding interactions (Kd < 10(-7) M) with apo HoSF at pH 7.5 were found for the various M2+ studied: high stoichiometric binding (30-54 M2+/HoSF) for Cd2+, Zn2+, Cu2+, with two protons released per metal bound; intermediate binding (16 M2+/HoSF) for Ni2+ and Co2+, with one proton released per metal bound; and low levels of binding (2-12 M2+/HoSF) for Mn2+, Mg2+, and...
Telford EA, Watson MS, Aird HC, Perry J, Davison AJ.The complete DNA sequence of equine herpesvirus 2 (EHV-2) strain 86/67 was determined. The genome is 184,427 bp in size and has a base composition of 57.5% G + C. Unusually for a herpesvirus, about a third of the sequence distributed in several large blocks appears not to encode proteins. The 79 open reading frames that were identified as probably polypeptide-coding are predicted to encode 77 distinct proteins. Amino acid sequence comparisons confirmed that EHV-2 is a gamma-herpesvirus that is genetically collinear with herpesvirus saimiri (HVS; a gamma 2-herpesvirus) and Epstein-Barr virus (E...
Gargiulo AM, Pedini V, Ceccarelli P, Lorvik S.In the present work, gustatory glands (von Ebner's glands) of the horse tongue were examined by means of five peroxidase-conjugated lectins (PNA, DBA, SBA, UEA I, WGA), with and without prior sialidase digestion, in order to investigate the presence and distribution of carbohydrate residues in secretory cells and duct cells. The most intense staining of secretory cells was observed with PNA after pre-treatment with neuraminidase. This indicates that the terminal trisaccharide sequence sialic acid- (alpha 2-->3, 6) galactosyl (beta 1-->3) N-acetylgalactosamine is the most frequent oligosa...
Klonisch T, Ryan PL, Yamashiro S, Porter DG.To determine the site of relaxin gene expression in equine placentae, a set of degenerate oligonucleotide primers was made according to the published amino acid sequence of the A- and B-chain of equine relaxin (eRXN). Total cellular RNA (tcRNA) from equine placentae at about 120 and 300 days of pregnancy was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) with use of these primers. A single amplification product of approximately 430 bp was detected in each case by agarose gel electrophoresis. The PCR product was ligated into Bluescript plasmid and sequenced to confirm the...
Ellis SA, Martin AJ, Holmes EC, Morrison WI.Nineteen horse MHC class I specificities have been serologically identified previously at a single locus (ELA-A), and two other specificities appear to be coded at other loci. Biochemical studies indicate that there are at least two expressed loci. In order to establish the number of transcribed horse MHC class I genes, we made a cDNA library from a heterozygous animal (ELA-A3/A7), and screened for positive clones using a bovine class I probe. More than 200 class I clones were isolated in this way, and so far seven unique full length sequences have been identified. All of the sequences are pre...
Marklund S, Chaudhary R, Marklund L, Sandberg K, Andersson L.The hypervariable D-loop region of mitochondrial DNA (mtDNA) was amplified with the polymerase chain reaction using total horse DNA samples. Analysis of single strand conformation polymorphism (SSCP) of denatured amplification products was carried out by native polyacrylamide (8%) gel electrophoresis followed by silver staining. As many as 15 distinct SSCP variants were revealed when screening a total of 78 maternally unrelated horses representing five different breeds. All breeds showed a high degree of polymorphism and the estimated probability (PImt) that two maternally unrelated individual...
Yoshida S, Yamanouchi K, Hasegawa T, Ikeda A, Suzuki M, Chang KT, Matsuyama S, Nishihara M, Takahashi M.cDNAs encoding equine inhibin/activin beta A subunit were isolated from an equine follicle cDNA library and characterized. Using primers based on the rat inhibin/activin beta A subunit cDNA sequence, a RT-PCR was performed to generate the probe for screening. Four positive clones were isolated. Analysis of the nucleotide sequence of these clones revealed that two pairs of identical clones were present, Eq-beta A-1 (0.9 kb) and Eq-beta A-2 (1.5 kb). Eq-beta A-2 clone contained a complete open reading frame encoding 426 amino acids. The deduced amino acid sequence of equine inhibin/activin beta ...
Cannon SC, Hayward LJ, Beech J, Brown RH.1. Equine hyperkalemic periodic paralysis (E-HPP) is a dominantly inherited disorder of muscle that causes recurrent episodes of stiffness (myotonia) and weakness in association with elevated serum K+. Affected horses carry a mutant allele of the skeletal muscle isoform of the Na channel alpha-subunit. To understand how this mutation may cause the disease phenotype, the functional defect in Na channel behavior was defined physiologically by recording unitary currents from cell-attached patches on normal and affected equine myotubes. 2. The presence of the mutation was confirmed in our cell lin...
Lichtenstein DL, Rushlow KE, Cook RF, Raabe ML, Swardson CJ, Kociba GJ, Issel CJ, Montelaro RC.As an important enzyme in DNA synthesis, dUTPase is present in a wide variety of organisms and viruses and has been identified as a component of the equine infectious anemia virus (EIAV) pol gene. The role of EIAV dUTPase, designated DU, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of EIAV. A deletion mutant that was deficient in dUTPase activity was constructed, and its replication kinetics was examined in fetal equine kidney (FEK) cells and primary equine bone marrow macrophage (EBMM) cells. In FEK cells, which are permissive...
Zhao Y, Holden VR, Smith RH, O'Callaghan DJ.The UL3 protein of equine herpesvirus 1 (EHV-1) KyA strain is a homolog of the ICP27 alpha regulatory protein of herpes simplex virus type 1 (HSV-1) and the ORF 4 protein of varicella-zoster virus. To characterize the regulatory function of the UL3 gene product, a UL3 gene expression vector (pSVUL3) and a vector expressing a truncated version of the UL3 gene (pSVUL3P) were generated. These effector plasmids, in combination with an EHV-1 immediate-early (IE) gene expression vector (pSVIE) and chimeric EHV-1 promoter-chloramphenicol acetyltransferase (CAT) reporter constructs, were used in trans...
Pérez MD, Calvo M.beta-Lactoglobulin is the major whey protein in the milk of ruminants and some nonruminants, such as pigs and horses. Although beta-lactoglobulin was first isolated 60 yr ago, no function has been definitely ascribed to beta-lactoglobulin. Recent x-ray crystallographic studies have advanced knowledge of the structure of beta-lactoglobulin, which is homologous with that of retinol-binding protein and lipocalycins; the function of these proteins seems to be participation in the transport of small hydrophobic substances. By analogy, this protein has been suggested as having a role as a transporte...
Hartmann J, Lindau M.Exocytosis begins with formation of a small fusion pore which then expands allowing rapid release of granular contents. We studied the influence of cytoplasmic free Ca2+ ([Ca2+]i) on the conductance of the initial pore and on the dynamics of subsequent expansion in horse eosinophils using the patch clamp technique. The mean initial conductance is approximately 200 pS independent of [Ca2+]i. This value is close to that previously found in beige mouse mast cells. The pore subsequently expands by 18 nS/s at [Ca2+]i < 10 nM, by 40 nS/s at [Ca2+]i = 1.5 microM and by 90 nS/s at [Ca2+]i = 10 micr...
Fan F, Plapp BV.The contributions of isoleucine residues 224 and 269 of horse liver alcohol dehydrogenase to binding of the adenine moiety of NAD and to catalysis were studied by replacing Ile-224 with glycine (I224G) and Ile-269 with serine (I269S). The kinetic mechanisms of wild-type and both mutated liver enzymes were ordered. Affinities for several adenosine derivatives were decreased 5-50-fold by both substitutions. The I269S mutation differentially destabilized binding of the complete coenzyme, as affinities for NAD+ and NADH were decreased about 60-fold with the I224G enzyme and 350-fold for the I269S ...
Eizema K, van der Wal DE, van den Burg MM, de Jonge HW, Everts ME.During early postnatal development, the myosin heavy chain (MyHC) expression pattern in equine gluteus medius muscle shows adaptation to movement and load,resulting in a decrease in the number of fast MyHC fibers and an increase in the number of slow MyHC fibers. In the present study we correlated the expression of MyHC isoforms to the expression of sarcoplasmic(endo)reticulum Ca2+-ATPase 1 and 2a (SERCA), phospholamban (PLB), calcineurin A (CnA), and calcineurin B (CnB). Gluteus medius muscle biopsies were taken at 0, 2, 4, and 48 weeks and analyzed using immunofluorescence. Both SERCA isofor...
da Silva CMB, Cano FEM, Gaitskell-Phillips G, Vega FJP.Flow cytometry is a powerful tool for the analysis of cell samples formed of multipopulations, such as spermatozoa. In recent years, multiparametric cytometers have evolved, allowing the study of different cellular characteristics, such as protein expression, DNA analysis, or mitochondrial activity. Whether using traditional fluorescent dyes or fluorophore-conjugated antibodies, each cell or cellular component is individually stained, the sample is analyzed at high velocities, and then is displayed and interpreted in a dot-plot. We hereby describe the procedure to perform a multiparametric flo...
Echigoya Y, Sato T, Itou T, Endo H, Sakai T.To elucidate the molecular properties of the equine glycolytic enzymes equine M(1) (eM(1)) and M(2) (eM(2))-pyruvate kinase (PK), mRNAs were isolated from thoroughbred horse skeletal muscle and hair roots, respectively. The full-length eM(1) and eM(2)-PK cDNAs consist of 2,320 and 2,376 bp, respectively, containing a 1596 bp open reading frame. The cDNAs were mapped to equine chromosome 1, and the equine pyruvate kinase M (PKM) gene consists of twelve exons. Exon 9 of eM(1)-PK and exon 10 of eM(2)-PK were further investigated in five equine species. Out of 55 amino acids encoded by exon 9 in e...
Zhang B, Wilson PE, Watt GD.Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react ...
Marchant EA, Semevolos SA.The marginal transitional zone is peripherally located within the diarthrodial joint, and represents the interface of articular cartilage, periosteum, and the fibrous joint capsule. The purpose of this study is to characterize the protein expression of matrix and molecular regulators in the marginal transitional zone of foals having osteochondrosis (OC) compared to normal foals. Several families of proteins with known roles in cartilage and bone development are investigated, including matrix molecules, Wnt signaling, apoptotic factors and paracrine cell signaling molecules. Our results demonst...
Porter RM, Akers RM, Howard RD, Forsten-Williams K.Interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I), which have opposing effects on matrix metabolism within articular cartilage, are thought to play prominent roles in the pathogenesis of osteoarthritis. To better understand the link between these anabolic (IGF-I) and catabolic (IL-1) stimuli, we examined exogenous IL-1 regulation of the IGF-I signaling system of articular chondrocytes (ACs). Equine ACs from non-arthritic stifle joints were expanded in monolayer culture, encapsulated for 10 days in alginate beads, and stimulated as high-density monolayers with recombinant equine IL-...
Alim S, Cheppali SK, Laitaoja M, Talluri TR, Jänis J, Swamy MJ.Fibronectin type-II (FnII) family proteins are the major proteins in many mammalian species including bull, horse and pig. In the present study, a major FnII protein has been identified and isolated from donkey (Equus hemionus) seminal plasma, which we refer to as Donkey Seminal Plasma protein-1 (DSP-1). The amino acid sequence determined by mass spectrometry and computational modeling studies revealed that DSP-1 is homologous to other mammalian seminal plasma proteins, including bovine PDC-109 (also known as BSP-A1/A2) and equine HSP-1/2. High-resolution LC-MS analysis indicated that the prot...
Mata X, Vaiman A, Ducasse A, Diribarne M, Schibler L, Guérin G.Gene characterization is an important feature for genome annotation and more particularly for candidate genes that could be selected in domestic species. Associations between an alpha-actinin-3 gene polymorphism and muscle performance were reported in humans involving a nonsense mutation (R577X) and in mice after inactivation of the gene. Here, we characterized the equine alpha-actinin-3 (ACTN3) gene by sequencing and transcript analysis. The cDNA was determined to be 3.47 kb in length with an open reading frame of 2709 bp expectedly encoding a protein 902 amino acids long. The ACTN3 gene is 1...
Duan H, Gao JF, Su X, Fu X, Yue DM, Gao Y, Qiu JH, Wang CR.Triodontophorus spp. parasitizes the large intestine of equine, causing strongylid diseases. The present study assessed genetic variation in five gene regions within and between Triodontophorus brevicauda and Triodontophorus nipponicus from Heilongjiang Province and the Inner Mongolia Autonomous region. The five gene markers were three mitochondrial (mt) genes, cytochrome c oxidase subunit I (cox1), NADH dehydrogenase subunit 5 (nad5), cytochrome b (cytb); and two ribosomal RNA genes, the internal transcribed spacer 1 (ITS1) and the internal transcribed spacer 2 (ITS2). Partial (p) sequences o...
Ma J, Zhang Z, Yao Q, Su C, Yin X, Wang X.Rev, an important accessory protein of equine infectious anaemia virus (EIAV), induces the nuclear export of incompletely spliced viral mRNAs. Rev is translated from the tat-rev mRNA through leaky scanning of the tat CUG. In this study, the function of the Kozak sequence at the beginning of the rev ORF was investigated. Deletion or attenuation of the Kozak sequence resulted in expression of an N-terminal 11 aa-truncated Rev in addition to WT Rev. Truncated Rev displayed weaker promotion of Gag expression and processing than WT Rev. Furthermore, EIAV rescued from an infectious molecular clon...
Kinoshita Y, Kakoi H, Ishige T, Yamanaka T, Niwa H, Uchida-Fujii E, Nukada T, Ueno T.Taylorella equigenitalis causes contagious equine metritis. Here we compared seven nucleic acid amplification tests for T. equigenitalis to select a rapid and reliable diagnostic method. The 95% detection limits of each assay varied greatly: real-time PCR had the lowest detection limit (0.77 fg/reaction); those of some of the conventional PCRs (cPCRs) were >100 fg/reaction. In experimentally infected samples, real-time PCR and semi-nested PCR showed the highest positive numbers (33 out of 42 samples), but two of the cPCRs detected only 2 and 7 positive results. Our results indicate that the us...
Potempa J.The proteolytic activity of the elastase-like proteinase from granules of horse blood leukocytes is retained on a column of Cibacron Blue-Sepharose and can be eluted with 0.5 M KSCN. During this procedure its mol. wt. is reduced from 49000 to 30000 and isoelectric point is shifted towards higher pH. The inactive protein not adsorbed on Cibacron Blue-Sepharose is strongly acidic and shows a mol. wt. of 20000. Upon mixing this protein with the modified enzyme the native proteinase is reconstituted as shown by polyacrylamide gel electrophoresis at pH 8.3 and isoelectric focusing in a sucrose grad...
Hesford F, Lazary S, Curty-Hänni K, Gerber H.A number of horse alloantisera were characterized biochemically as being directed against MHC class I or class II antigens by immunoprecipitation of the corresponding antigens from lysates of biosynthetically radioactively labelled lymphocytes and determination of their molecular weights by SDS-PAGE and fluorography. Sera recognizing A2 and A3 specificities precipitated antigens of 44,000 Daltons molecular weight (class I heavy chain), whereas sera with specificities W13, W22 and W23 precipitated antigens corresponding to class II dimers (30,000 and 32,000 Daltons). Comparison with antigens pr...
Mugnier S, Boittin S, Douet C, Monget P, Magistrini M, Goudet G.In human and rodents, sperm-zona pellucida binding is mediated by a sperm surface Galactosyltransferase that recognizes N-Acetylglucosamine residues on a glycoprotein ZPC. In large domestic mammals, the role of these molecules remains unclear: in bovine, they are involved in sperm-zona pellucida binding, whereas in porcine, they are not necessary. Our aim was to clarify the role of Galactosyltransferase and N-Acetylglucosamine residues in sperm-zona pellucida binding in ungulates. For this purpose, we analyzed the mechanism of sperm-zona pellucida interaction in a third ungulate: the horse, si...
Montelaro RC, West M, Ivey M.Representative glycoproteins including fetuin, protein A, ovalbumin, alpha 1 acid glycoprotein, and the major glycoprotein of equine infectious anemia virus were labelled with 125I by the chloramine-T or Bolton-Hunter procedure and their binding to immobilized Con A or lentil lectin compared to untreated samples of each glycoprotein. Glycoprotein modification was no greater than one substituted residue per protein molecule. Yet the radioiodinated glycoproteins typically displayed only 0-50% of the lectin binding observed with untreated samples. These results indicate that lectin glycoprotein b...
Arosio P, Gatti G, Bolognesi M.Various precipitating agents were examined in order to crystallize horse heart and spleen ferritins. Cadmium sulfate induced the crystallization of the spleen ferritin, while 2-methyl-2,4-pentanediol and poly(ethylene glycol) only induced that of the heart ferritin. Isoelectric focusing analysis showed that the crystals grown from cadmium sulfate contained only the more acidic isoferritins, and those grown from methyl pentanediol only the less acidic isoferritins. Heart ferritin crystallizes in a cubic space group, as previously reported for spleen ferritin crystals grown from cadmium sulfate....
Warme PK, Scheraga HA.The reported X-ray structure of horse-heart ferricytochrome c has been refined by conformational energy calculations, using a three-stage computational procedure. In stage I, the atomic positions are adjusted to conform to idealized bond lengths and bond angles characteristic of small amino acid derivatives, while yet remaining as close as possible to the X-ray coordinates. In stage II, atomic overlaps are eliminated by adjusting the backbone and side-chain dihedral angles to minimize the nonbonded energy, hydrogen-bonded energy, and rotational energy contributions. In the final stage of refin...
Gurusinghe S, Hilbert B, Trope G, Wang L, Bandara N, Strappe P.Immortalization of chondrocytes enables long term in vitro culture; however, the chondrogenic capacity of transformed cells varies, thus highlighting the need to develop a proliferative and tuneable chondrocyte cell line where hypertrophic differentiation can be controlled. In this study the SV40 large T antigen and human telomerase reverse transcriptase were employed to immortalize pooled equine chondrocytes through lentiviral vector mediated transduction either singly or on combination. Transformed chondrocytes proliferated stably over multiple passages, but resulted in significantly lower e...
García-Rubio I, Fittipaldi M, Trandafir F, Van Doorslaer S.In this work, we show the extreme power of multifrequency HYSCORE (hyperfine sublevel correlation spectroscopy) techniques to unravel the hyperfine interactions of the electron spin with the remote nuclei in the heme site of high-spin ferric heme proteins. Horse heart aquo-metmyoglobin was used as a model system to demonstrate the power of these techniques. Experimental evidence was collected and assigned to protons of the proximal histidine ligand, to the mesoprotons of the heme ligand, and to two different protons of the distal water ligand. The latter difference relates to the stabilization...
Allen GP, Cohen JC, Randall CC, O'Callaghan DJ.The replication of equine herpesvirus type 1 (EHV-1) and type 3 (EHV-3) was unimpeded in three different cell types-equine epithelial cells, equine fibroblasts, and mouse fibroblasts-which had been blocked in their capacity to synthesize host DNA by 2.5 mM hydroxyurea (HU) or 2 mM thymidine (TdR). The rate of DNA synthesis in uninfected or equine herpesvirus-infected cells in the presence of HU or TdR was measured by pulse-labeling cell samples with a labeled DNA precursor at different times after infection. DNA synthesis in uninfected cultures was completely inhibited by both compounds. Howev...
Balkman CE, Nixon AJ.To clone and determine molecular structure of equine stromelysin 1 (matrix metalloproteinase 3) and examine stromelysin expression in articular cartilage. SAMPLES AND PROCEDURE: Total RNA was harvested from equine arthritic cartilage specimens and was used for reverse transcription and polymerase chain reaction amplification to develop overlapping complementary DNA (cDNA) clones. Four cDNA sequences were ligated into plasmid (pGEM3Z) constructs and subcloned into bacterial expression vectors, and sequence was determined by automated dye terminator sequencing. Stromelysin mRNA expression was as...
Kerfelec B, Foglizzo E, Bonicel J, Bougis PE, Chapus C.The complete sequence of the horse pancreatic lipase was elucidated by combining polypeptide chain and cDNA sequencing. Among the structural features of horse lipase, it is worth mentioning that Lys373 is not conserved. This residue, which is present in human, porcine and canine lipases, has been assumed to be involved in p-nitrophenyl acetate hydrolysis by pancreatic lipases. Kinetic investigation of the p-nitrophenyl acetate hydrolysis by the various pancreatic lipases and by the C-terminal domain (336-449) of human lipase reveals that this hydrolysis is the result of the superimposition of ...
Traversa D.The recognition that the health and welfare of some humans are improved through contact and relationships with animals is now established. Two commonly recognized assistance animals are dogs and horses. Both provide therapeutic benefits to humans with some physical and mental illnesses and both assist people with disabilities. Moreover, the public and scientific attention to the health and conservation of many animal breeds is also increasing worldwide. In the past few years, two potentially life-threatening nematode infections that can induce tumours or tumour-like masses in canids and equids...
Thougaard AV, Jaliashvili I, Christiansen M.The glycoprotein tetranectin (TN) found in human serum is a 90-kDa homotrimeric C-type lectin binding Ca2+, heparin and plasminogen kringle 4. TN is suggested as being implicated in tissue remodelling. The antigenic reactivity of putative TN was examined in serum from 14 different animal species using three sandwich enzyme immunoassays for human TN. Crab-eating macaque serum showed the strongest reaction, followed by horse and cat. Serum from cow, goat, pig, mouse and chicken reacted weakly, while dog, trout, and the amphibian and the reptile species did not react. The TN-like protein from mac...
Lorenzo PL, Liu IK, Illera JC, Picazo RA, Carneiro GF, Illera MJ, Conley AJ, Enders AC, Illera M.Epidermal growth factor (EGF) has been reported to promote different functions in mammalian ovaries, including oocyte maturation. The aim of the present study was to establish: that EGF influences oocyte maturation in ovine and equine, that a tyrosine kinase-dependent intracellular mechanism mediates EGF effect and, that EGF-R receptor is detectable in ovarian follicles by immunohistochemistry methods. Selected ovine and equine oocytes were aspirated from 2-5 mm (ovine) or 25 mm (equine) follicles and cultured in TCM 199 for 22 (ovine) or 36 hours (equine). They are then subjected to culture w...
Park HB, Marklund S, Jeon JT, Mickelson JR, Valberg SJ, Sandberg K, Andersson L.The PRKAG3 gene encodes a muscle-specific isoform of the regulatory gamma subunit of AMP-activated protein kinase (AMPK). A major part of the coding PRKAG3 sequence was isolated from horse muscle cDNA using reverse-transcriptase (RT)-PCR analysis. Horse-specific primers were used to amplify genomic fragments containing 12 exons. Comparative sequence analysis of horse, pig, mouse, human, Fugu, and zebrafish was performed to establish the exon/intron organization of horse PRKAG3 and to study the homology among different isoforms of AMPK gamma genes in vertebrates. The results showed conclusively...
Hristov P, Yordanov G, Vladov V, Neov B, Palova N, Radoslavov G.It is well known that horse breeding in Bulgaria is a cultural heritage in Bulgaria, dating from prehistoric and historic times. Until now, molecular data on Bulgarian horses from the plain regions of the country were not available. Therefore, for the first time, we have collected genetic information about some modern horse breeds from the plain regions in Bulgaria. A total of 50 horses originating from different families from two different breeds were investigated: the first one was the Pleven horse (n = 11, breeding in the Danubian Plain), and the second one was the East Bulgarian horse bre...
Tulamo RM, Raekallio M, Taylor P, Johnson CB, Salonen M.Both morphine and physiologic saline injected intra-articularly into healthy equine tarsocrural joints induced a release of large molecular size proteoglycan (PG) subunits into the synovial fluid (SF) analysed 24 h postinjection. High-performance liquid chromatography (HPLC) with a size-exclusion column was used to assess the high molecular weight proteoglycans in equine synovial fluid (SF). The PG peaks of SF samples eluated separately from SF hyaluronate and other molecular components of the SF in the HPLC chromatographies indicating no interaction between hyaluronate and PG in the SF. Indiv...
Song X, Jiang H, Lv P, Cui K, Liu Q, Yin S, Liu H, Li Z.Oocyte in vitro maturation is necessary for the study and application of animal-assisted reproduction technology in animal reproduction and breeding. The comprehensive transcriptional profile of equine oocyte maturated in vitro has not been fully mined yet, which makes many key transcriptional events still unidentified. Here, Smart-seq2 was performed to analyse the gene expression pattern and the underlying regulatory mechanism of horse germinal vesicle (GV) and in vitro metaphase II (MII) oocytes. The results showed that 6402 genes (2640 up-regulated and 3762 down-regulated in MII samples com...
O'Rourke BA, Dennis JA, Healy PJ.Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or inc...
