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.
Ing NH, Laughlin AM, Varner DD, Welsh TH, Forrest DW, Blanchard TL, Johnson L.In the testis of the 1.5-year-old horse, spermatogenesis initiates locally in grossly light, central areas that contrast with grossly dark, peripheral areas that are as yet inactive in spermatogenesis. Gene expression was compared between "light" and "dark" tissues of 1.5-year-old horse testes to identify mechanisms important to the initiation of spermatogenesis. Microarrays containing human cDNAs were used to assess expression levels of 9132 genes simultaneously in matched pairs of dark and light testis tissues from 3 prepubertal colts. In all 3 analyses, dysferlin (DYS), down-regulated in ov...
Wu D, Murakami K, Liu N, Konishi M, Muneta Y, Inumaru S, Kokuho T, Sentsui H.The equine interleukin-18 (IL-18) cDNA that contains the coding sequence was cloned and a recombinant baculovirus, named AcEIL-18, was constructed. The recombinant protein of the equine IL-18 was expressed by AcEIL-18 and its expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Insect cells infected with AcEIL-18 secreted a precursor IL-18 with 24 kilo dalton (kDa) into the culture supernatant. Western blot analysis showed that mature equine IL-18 about 18 kDa was also confirmed without co-expression of caspase-1. Culture supern...
Cêtre-Sossah C, Baldet T, Delécolle JC, Mathieu B, Perrin A, Grillet C, Albina E.Bluetongue (BT) and African Horse Sickness (AHS) are infectious arthropod-borne viral diseases affecting ruminants and horses, respectively. Culicoides imicola Kieffer, 1913, a biting midge, is the principal vector of these livestock diseases in Africa and Europe. Recently bluetongue disease has re-emerged in the Mediterranean Basin and has had a devastating effect on the sheep industry in Italy and on the islands of Sicily, Sardinia, Corsica and the Balearics, but fortunately, has not penetrated onto mainland France and Spain. To survey for the presence of C. imicola, an extensive light-trap ...
Dai CM, Zhang XY, Zhang RR, Shao YM, Shen RX.To develop a novel vaccine candidate of Equine infectious anemia virus(EIAV). Methods: env genes of EIAV Chinese donkey leukocyte attenuated strain (EIAV DLV) and its parental virus strain (EIAV LN) were expressed using the BAC-To-BAC system, and Env proteins were confirmed by SDS-PAGE and Western blot. BALB/c mice were immunized with recombinant vaccinia viruses containing env genes of EIAV alone or boosted with Env proteins expressed by recombinant baculovirus. Both protective humoral and cellular immune responses were detected. Results: Recombinant baculovirus could express complete Env pro...
Dell'Aquila ME, Caillaud M, Maritato F, Martoriati A, Gérard N, Aiudi G, Minoia P, Goudet G.The aim of this study was to investigate cumulus expansion, nuclear maturation and expression of connexin 43, cyclooxygenase-2 and FSH receptor transcripts in equine cumuli oophori during in vivo and in vitro maturation in the presence of equine FSH (eFSH) and precursors for hyaluronic acid synthesis. Equine cumulus-oocyte complexes (COC) were cultured in a control defined medium supplemented with eFSH (0 to 5 micrograms/ml), Fetal Calf Serum (FCS), precursors for hyaluronic acid synthesis or glutamine according to the experiments. After in vitro maturation, the cumulus expansion rate was incr...
Spier SJ, Leutenegger CM, Carroll SP, Loye JE, Pusterla JB, Carpenter TE, Mihalyi JE, Madigan JE.To develop and use a sensitive molecular assay for detecting the phospholipase D (PLD) exotoxin gene of Corynebacterium pseudotuberculosis in an attempt to identify insect vectors that may be important in transmission of clinical disease in horses. Methods: 2,621 flies of various species. Methods: A real-time polymerase chain reaction (PCR)-based fluorogenic 5' nuclease (TaqMan) system (ie, TaqMan PCR assay) was developed for the detection of the PLD gene in insects. Flies were collected monthly (May to November 2002) from 5 farms in northern California where C. pseudotuberculosis infection in...
Raudsepp T, Santani A, Wallner B, Kata SR, Ren C, Zhang HB, Womack JE, Skow LC, Chowdhary BP.We herein report a detailed physical map of the horse Y chromosome. The euchromatic region of the chromosome comprises approximately 15 megabases (Mb) of the total 45- to 50-Mb size and lies in the distal one-third of the long arm, where the pseudoautosomal region (PAR) is located terminally. The rest of the chromosome is predominantly heterochromatic. Because of the unusual organization of the chromosome (common to all mammalian Y chromosomes), a number of approaches were used to crossvalidate the results. Analysis of the 5,000-rad horse x hamster radiation hybrid panel produced a map spannin...
Chopineau M, Martinat N, Gibrat JF, Galet C, Lecompte F, Foulon-Gauze F, Pourchet C, Guillou F, Combarnous Y.To identify amino-acids in the alpha-subunit important for expression of heterospecific FSH activity of horse (e) LH/choriogonadotropin (CG) (eLH) and donkey (dk) LH/CG (dkLH) (FSH/LH ratio ten times higher for eLH than for dkLH); this FSH activity absolutely requires an equid (donkey or horse) alpha-subunit combined with an equid beta-LH subunit. Methods: Chimeric alpha-subunits possessing the first 63 amino-acids of the porcine (p) and the last 33 amino-acids of the donkey alpha-subunit (alphap-dk) and the inverse (alphadk-p) were constructed. Porcine-specific amino-acids were introduced by ...
Paulis M, Moralli D, Bensi M, De Carli L, Raimondi E.Tigger elements are human DNA transposons homologous to the pogo element of Drosophila melanogaster. They contain an open reading frame for a transposase very similar to the major mammalian centromere protein CENP-B. We found in the horse genome a DNA element ( Ecatig3) sharing 88% homology with human Tigger3. The presence of Tigger elements in the horse genome confirms previous data that date these elements before the divergence between Perissodactyla and Primates (80-90 Myr ago). Copy number evaluation indicates that the horse element is much more abundant than its human counterpart. Souther...
Chikuni K, Muroya S, Nakajima I.Nucleotide sequences which included the full coding region for three types of myosin heavy chain (MyHC) isoforms were determined from equine skeletal muscles. The deduced amino acid sequences were 1937, 1938, and 1935 residues for the MyHC-2a, -2x, and -slow, respectively. No MyHC-2b isoform was amplified from the equine muscle cDNA except for one pseudogene fragment. One nucleotide was inserted in the coding region of the equine pseudogene product, a minute amount of which was expressed in the skeletal muscle. The 596 bp sequence of the equine MyHC pseudogene was categorized into the MyHC-2b ...
Oleksiewicz MB, Snijder EJ, Normann P.A putative zinc finger (ZF) domain in the Equine arteritis virus (EAV) nsp1 protein was described recently to be required for viral transcription. The nsp1 ZF (50 aa) was expressed on the surface of M13KE gIII phage, fused to the N terminus of the phage pIII protein. To evaluate the functionality of the ZF domain, a binding assay was developed, based on the use of immobilized Ni(2+) ions (Ni-NTA). Phages displaying ZF bound significantly better to Ni-NTA than did phages displaying negative-control peptides, which also contained metal-coordinating residues. Also, binding of ZF-displaying phages...
Kyaw-Tanner M, Pollitt CC.The dysadhesion and destruction of lamellar basement membrane of laminitis may be due to increased lamellar metalloproteinase activity. Characterising lamellar metalloproteinase-2 (MMP-2) and locating it in lamellar tissues may help determine if laminitis pathology is associated with increased MMP-2 transcription. Objective: To clone and sequence the cDNA encoding lamellar MMP-2, develop antibody and in situ hybridisation probes to locate lamellar MMP-2 and quantitate MMP-2 transcription in normal and laminitis tissue. Methods: Total RNA was isolated, fragmented by RT-PCR, cloned into vector a...
Muscatello G, Browning GF.Selective agar media have been used for many years to facilitate the isolation of Rhodococcus equi from environmental and clinical samples. However, characterisation of R. equi still requires the use of immunochemical or polymerase chain reaction (PCR) analysis to differentiate between virulent and avirulent isolates. Here, we describe a novel method to detect and differentiate between R. equi isolates using colony blotting and DNA hybridization. Radiolabelled PCR product derived from the R. equi rrnA gene and specific hybridization conditions enabled differentiation of colonies of R. equi fro...
Davis EG, Sang Y, Blecha F.beta-Defensins are cysteine-rich endogenously produced antimicrobial peptides that play an important role in innate immune defense. Although, previous investigations have identified beta-defensins in several mammalian species, no reports have identified equine beta-defensins. Using a strategy of database searching for expressed sequence tags (EST) we identified putative expression of equine beta-defensins in hepatic tissue. Based on this information, sequence specific primers were designed for the equine gene enabling the identification of the full-length cDNA sequence of equine beta-defensin-...
Li Y, Yao J, Chang M, Nikolic D, Yu L, Yager JD, Mesecar AD, van Breemen RB, Bolton JL.Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of t...
Studdert MJ, Azuolas JK, Vasey JR, Hall RA, Ficorilli N, Huang JA.To develop and validate specific, sensitive and rapid diagnostic tests using RT-PCR for the detection of Ross River virus (RRV), Kunjin virus (KV) and Murray Valley encephalitis virus (MVEV) infections in horses. Methods: Primer sets based on nucleotide sequence encoding the envelope glycoprotein E2 of RRV and on the nonstructural protein 5 (NS5) of KV and MVEV were designed and used in single round PCRs to test for the respective viruses in infected cell cultures and, in the case of RRV, in samples of horse blood and synovial fluid. Results: The primer pairs designed for each of the three vir...
Wallner B, Piumi F, Brem G, Müller M, Achmann R.Y chromosome polymorphisms such as microsatellites or single nucleotide polymorphisms represent a paternal counterpart to mitochondrial DNA (mtDNA) for evolutionary and phylogeographic studies. The use of Y chromosome haplotyping in natural populations of species other than humans is still hindered by the lack of sequence information necessary for polymorphism screening. Here we used representational difference analysis (RDA) followed by a screen of a bacterial artificial chromosome (BAC) library for repetitive sequences to obtain polymorphic Y-chromosomal markers. The procedure was performed ...
Poloumienko A.A growing body of evidence suggests the involvement of sex chromosome genes in mammalian development. We report the cloning and characterization of the complete coding regions of the bovine Y chromosome ZFY and X chromosome ZFX genes, and partial coding regions of porcine and equine ZFX and ZFY genes. Bovine ZFY and ZFX are highly similar to each other and to ZFX and ZFY from other species. While bovine and human ZFY proteins are both 801 amino acids long, bovine ZFX is 5 amino acids shorter than human ZFX. Like in humans, both bovine ZFY and ZFX contain 13 zinc finger motifs and belong to the...
Balasuriya UB, Dobbe JC, Heidner HW, Smalley VL, Navarrette A, Snijder EJ, MacLachlan NJ.We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 pro...
Collins MD, Lundström T, Welinder-Olsson C, Hansson I, Wattle O, Hudson RA, Falsen E.Phenotypic and phylogenetic studies were performed on four unidentified Gram-positive staining, catalase-negative, alpha-hemolytic Streptococcus-like organisms recovered from the teeth of horses. SDS PAGE analysis of whole-cell proteins and comparative 16S rRNA gene sequencing demonstrated the four strains were highly related to each other but that they did not correspond to any recognised species of the genus Streptococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed the unidentified organisms form a hitherto unknown sub-line within the Streptococcus genus, displaying a clos...
Di Bernardo G, Galderisi U, Del Gaudio S, D'Aniello A, Lanave C, De Robertis MT, Cascino A, Cipollaro M.DNA extracted from the skeletons of five equids discovered in a Pompeii stable and of a horse found in Herculaneum was investigated. Amino acid racemization level was consistent with the presence of DNA. Post-mortem base modifications were excluded by sequencing a 146 bp fragment of the 16S rRNA mitochondrial gene. Sequencing of a 370 bp fragment of mitochondrial (mt)DNA control region allowed the construction of a phylogenetic tree that, along with sequencing of nuclear genes (epsilon globin, gamma interferon, and p53) fragments, gave us the possibility to address some questions puzzling arch...
Gasser RB, Hung GC, Chilton NB, Beveridge I.Infections of equids with parasitic nematodes of the order Strongylida (subfamilies Strongylinae and Cyathostominae) are of major veterinary importance. In last decades, the widespread use of drugs against these parasites has led to problems of resistance within the Cyathostominae, and to an increase in their prevalence and intensity of infection. Novel control strategies, based on improved knowledge of parasite biology and epidemiology, have thus become important. However, there are substantial limitations in the understanding of fundamental biological and systematic aspects of these parasite...
Dias AJ, Maia MS, Retamal CA, López ML.The expression of alpha-1,4-glucosidase activity was fluorometrically and electrophoretically assessed in the epididymal fluid and seminal plasma of stallions. alpha-Glucosidase specific activity in the epididymis increased significantly from the proximal caput to the cauda. Stallion epididymal glucosidase maintained activity in a wide range of pH, with two distinct peaks (around pH 4.0 and 6.0, respectively). Enzyme activities at different pH, inhibition assays with sodium dodecyl sulfate (SDS) and maltotriose (MTT, selective inhibitors of alpha-glucosidases "acidic" and "neutral" isoforms, d...
Terry RB, Archer S, Brooks S, Bernoco D, Bailey E.A single autosomal dominant locus, leopard complex (LP) controls the presence of appaloosa pigmentation patterns in the horse. The causative gene for LP is unknown. This study was undertaken to map LP in the horse. Two paternal half sib families segregating for the LP locus and including a total of 47 offspring were used to perform a genome scan which localized LP to horse chromosome 1 (ECA1). LP was linked to ASB08 (LOD = 9.99 at Theta = 0.02) and AHT21 (LOD = 5.03 at Theta = 0.14). To refine the map position of LP, eight microsatellite markers on ECA1 (UM041, LEX77, 1CA41, TKY374, COR046, 1C...
Lai AC, Rogers KM, Glaser A, Tudor L, Chambers T.Phylogenetic and antigenic analyses indicate that recent circulating equine-2 influenza viruses in the United States have been alternating between two genetic and antigenic distinct lineages since 1996. The evolution rates for these two lineages, the Kentucky and the Florida lineage, are very similar. For the earlier isolates in the Kentucky lineage, there are multiple and sequential nonsynonymous substitutions at antigenic sites B and D. However, there are no changes at any of these antigenic sites for KY98 and OK00. In the Florida lineage, except for NY99 with one amino acid substitution at ...
Perryman LE.Severe combined immunodeficiency (SCID) is an inherited disorder of humans, mice, horses, and dogs, in which affected individuals are incapable of generating antigen-specific immune responses. It occurs when lymphocyte precursors fail to differentiate into mature lymphocytes because of mutations within recombinase-activating genes 1 and 2 or within the genes encoding deoxyribonucleic acid (DNA)-dependent protein kinase (DNA-PK). It also occurs when differentiated lymphocytes are incapable of completing signal transduction pathways because of defects in cell surface receptors for interleukins (...
Hines R, Sorensen BR, Shea MA, Maury W.Binding of the transcription factor PU.1 to its DNA binding motif regulates the expression of a number of B-cell- and myeloid-specific genes. The long terminal repeat (LTR) of macrophage-tropic strains of equine infectious anemia virus (EIAV) contains three PU.1 binding sites, namely an invariant promoter-proximal site as well as two upstream sites. We have previously shown that these sites are important for EIAV LTR activity in primary macrophages (W. Maury, J. Virol. 68:6270-6279, 1994). Since the sequences present in these three binding motifs are not identical, we sought to determine the r...
Breathnach CC, Rudersdorf R, Lunn DP.Recombinant modified vaccinia Ankara (MVA) vectors expressing equine influenza virus genes were constructed and evaluated for use in equine vaccination. Two strains of recombinant MVA, expressing either hemagglutinin (HA) or nucleoprotein (NP) genes were constructed. Each influenza virus gene was cloned from A/equine/Kentucky/1/81 (Eq/Ky) into an MVA construction plasmid, and was introduced to the deletion III locus of the wild type MVA genome by homologous recombination. Recombinant viruses were plaque purified, and antigen expression was confirmed by immunostaining. Two ponies were primed by...
Riley SC, Thomassen R, Bae SE, Leask R, Pedersen HG, Watson ED.Profound hormonally controlled tissue remodelling occurs in the equine ovary for follicle growth and development, and also for the alteration in follicle shape directed towards the ovulation fossa, the site where ovulation occurs. The aim of this study was to examine the spatial and temporal regulation of matrix metalloproteinases (MMP)-2 and MMP-9, important enzymes in tissue remodelling, during follicle growth, and ovulation. Using gelatin substrate zymography, we measured these MMPs in follicular fluid of large anovulatory follicles collected during spring transition, early dominant follicl...
Gasa S, Makita A.Gangliosides were isolated from equine kidney and spleen, and their carbohydrate and lipid moieties were characterized. Among the long-chain bases, considerable proportions of trihydroxy bases (42.3 to 61.2% of the total bases), in which phytosphingosine was predominant were found in all the ganglioside classes. The other major base was sphingosine. Among the constituent fatty acids, long-chain acids (with a carbon number of more than 20), comprised approximately half the total acids, with some alpha-hydroxy and mono-unsaturated acids. By means of sequential hydrolysis with glycosidases couple...
Fachal MV, Furlan M, Clark R, Card CE, Chedrese PJ.The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in...
Clegg PD, Coughlan AR, Carter SD.Matrix metalloproteinases (MMPs) are the main enzymes involved in connective tissue turnover. Regulation of MMPs is achieved by controlling production, activation of the pro-enzymes together with the presence of inhibitors, such as, tissue inhibitors of metalloproteinases (TIMPS). The presence of TIMPs in equine synovial fluid was assessed by the ability of the fluid to inhibit equine MMP-9 activity using a gelatin degradation ELISA. The cellular source of the TIMPs was determined using culture supernatants of resident articular cells (chondrocytes and synovial fibroblasts) and invading inflam...
Andrews RK, Booth WJ, Bendall LJ, Berndt MC.von Willebrand Factor (vWF) is a multifunctional glycoprotein in plasma and vascular subendothelial matrix which plays a major role in cellular adhesion. vWFdependent adhesion of platelets to the subendothelium at high shear rates involves a specific platelet membrane receptor, the glycoprotein (GP) Ib-IX complex. We have previously purified a 39/34-kiloDalton (kDa) dispase fragment of vWF (Leu-480/Val-481 to Gly-718) and demonstrated that this fragment contains the binding site for the GP Ib-IX complex [Andrews R K, et al. Biochemistry 1989; 28: 8326-83361. vWF also mediates agglutination of ...
Narváez SÁ, Fernández I, Patel NV, Sánchez S. is an important veterinary pathogen that takes the lives of many foals every year. With the emergence and spread of MDR to current antimicrobial treatment, new tools that can provide a fast and accurate diagnosis of the disease and antimicrobial resistance profile are needed. Here, we have developed and analytically validated a multiplex qPCR for the simultaneous detection of and related macrolide resistance genes in equine respiratory samples. The three sets of oligos designed in this study to identify housekeeping gene and macrolide resistance genes (46) and (51) showed high analytic se...
Bulone V, Rademaker GJ, Pergantis S, Krogstad-Johnsen T, Smestad-Paulsen B, Thomas-Oates J.Separation of horse dander allergens using two-dimensional PAGE resulted in the identification of 16 proteins that react with allergic patient sera. A sensitive method has been developed for analysing the structures of the glycan chains of individual glycoprotein allergens transferred to blots following two-dimensional PAGE, and has allowed the structural identification of the glycan chains of the most abundant isoforms of Equ c 1, a glycosylated horse dander major allergen. The method involves separation of the allergens by two-dimensional PAGE, transfer to polyvinylidene difluoride membranes...
Pereira GR, Lorenzo PL, Carneiro GF, Bilodeau-Goeseels S, Kastelic J, Liu IK.The objectives of this study were firstly to determine whether the stimulatory function of equine growth hormone (eGH) on equine oocyte maturation in vitro is mediated via cyclic adenosine monophosphate (cAMP); and secondly if the addition of eGH in vitro influences oocyte nuclear maturation and if this effect is removed when GH inhibitors are added to the culture. Cumulus-oocyte complexes (COCs) were recovered from follicles <25 mm in diameter and randomly allocated as follows: (i) control (no additives); and (ii) 400 ng/ml of eGH. A specific inhibitor against cyclic AMP-dependent protein ...
Watts EJ, Rose MT.Horses are more prone to complications in the wound healing process than other species, and problems such as chronic inflammation, delayed epithelialization, poor wound contraction, and exuberant granulation tissue are commonly seen, particularly in wounds on the distal limbs. In comparison, wounds of the oral mucosa heal rapidly in a scarless fashion with a high degree of wound contraction. The effect of platelet-derived growth factor BB (PDGF), insulin-like growth factor (IGF)-1, and transforming growth factor beta1 (TGFbeta1) on the contraction of a fibroblast-populated collagen matrix (FPC...
Suzuki Y.All types of the hemagglutinin(HA) of human, pig, horse and aq. bird influenza A viruses, recognize sialyl lacto-series type I and II sugar chains(Sialic acid(SA) alpha 2-3(6)Gal beta 1-3(4) GlcNAc beta 1-) in glycoproteins and glycolipids in the target cells as common receptor molecules. Avian and equine influenza viruses preferentially binds the terminal sialic acid alpha 2-3Gal(SA2-3Gal) linkage, while human influenza viruses preferentially bind the SA2-6Gal linkage. SA distribution in animal species influence influenza virus host range. Swine trachea has both receptors for avian influenza ...
Wagner B, Siebenkotten G, Radbruch A, Leibold W.IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Cvarepsilon gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Cvarepsilon alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cvarepsilon(a) and Cvarepsilon(b). Sequence analysis of both, Cvarepsilon(a) and Cvarepsilon(b) cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences hav...
Desantis S, Acone F, Corriero A, Deflorio M, Zubani D, Ventriglia G, Palmieri G, De Metrio G.The distribution of sialic acid residues as well as other glycosidic sugars has been investigated in the horse oviductal isthmus during anoestrus, oestrus and pregnancy by means of lectin and pre-lectin methods. Ciliated cells and non-ciliated (secretory) cells exhibited different lectin binding profiles that were found to change during the investigated stages. Ciliated cells did not show any reactivity in the basal cytoplasm, while the supra-nuclear cytoplasm displayed a few of oligosaccharides with terminal and internal alphamannose (Man) and/or alphaglucose (Glc) during oestrus and pregnanc...
Barr AR, Duance VC, Wotton SF, Waterman AE, Holt PE.Cyanogen bromide was used to solubilise and specifically fragment purified equine Type I and II collagen and equine articular surface repair tissue. The resultant peptides were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and quantified by densitometric scanning. Measurement of the relative amounts of the peptides alpha 2(I) CB3, 5 and alpha 1(II)CB10 provided an accurate method of establishing the ratio of Type I to Type II collagen in mixtures of purified equine collagens. The method was sensitive to 6% Type II collagen when the band areas were corrected for peptid...
BMC research notesApril 16, 2013
Volume 6 152 doi: 10.1186/1756-0500-6-152
Christensen MB, Sørensen JC, Jacobsen S, Kjelgaard-Hansen M.Serum amyloid A (SAA) is useful as a diagnostic marker of systemic inflammation in horses, but only heterologous assays based on non-equine calibration and standardization are available for measurements of equine SAA. More accurate measurements could be obtained using purified species-specific SAA in native conformation for assay calibration and standardization. Further knowledge about the biochemical properties of SAA would facilitate a future production of native species-specific calibration material Therefore, the aim of the study was an investigation of the solubility and potentials for pu...
Santucci R, Ascoli F, La Mar GN, Pandey RK, Smith KM.The reconstitution kinetics of horse heart myoglobin, as met-cyano derivative, with two synthetic hemins in which the 6- or the 7-propionate is replaced by a methyl group, has been investigated by circular dichroism, in order to gain information on the heme re-orientation process following the heme insertion into the globin pocket. The results obtained confirm that the preferred heme orientation places the sole propionate into the position occupied by the 6-propionate in the crystal structure, supporting the importance of the salt bridge occurring between this propionate and the basic CD3 resi...
Cristoni S, Bernardi LR, Biunno I, Guidugli F.A new approach, based on the use of atmospheric pressure chemical ionization ion trap mass spectrometry (APCI-ITMS), but without a corona discharge, was investigated for application to creating and monitoring protein ions. It must be emphasized that APCI is not usually used in protein analysis. In order to verify the applicability of the proposed method to the analysis of proteins, two standard proteins (horse cytochrome c and horse myoglobin) were analyzed. A mixture of the two proteins was also analyzed showing that this novel approach, based on the use of APCI, can be used in the analysis o...
Livengood JL, Lanka S, Maddox C, Tewari D.Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is an important equine pathogen. Strangles is a highly contagious disease and a commercial modified live vaccine (MLV) is used for protection, which although effective, may also result in clinical signs of the disease. A rapid means to differentiate between the MLV and wild-type infection is crucial for quarantine release and limiting the disease spread. This study describes the use of a pyrosequencing assay targeting a single nucleotide deletion upstream of the SzPSe gene to distinguish between the wild-type and v...
Sakagami M, Hirota K, Awata T, Yasue H.We have molecularly cloned portions of equine satellite-type DNA and investigated the organization of the DNA sequence of the cloned segments. Sequence analysis and dot-blot analysis, using the cloned sequence (ES200) as a probe, indicate that the satellite-type DNA sequence consists mainly of 221-bp tandem repeats and represents 3.7-11% of the equine genome. Southern blot analysis further shows that (1) no sequences homologous to ES200 exist in the human, swine, and bovine genomes and that (2) the fragment pattern of the satellite-type DNA produced by ApaI cleavage shows a slight difference a...
Cook SJ, Li G, Zheng Y, Willand ZA, Issel CJ, Cook RF.Although the equine lentivirus (equine infectious anemia virus [EIAV]) poses a major threat to equid populations throughout most regions of the world, detailed knowledge concerning its molecular epidemiology is still in its infancy. Such information is important because the few studies conducted to date suggest there is extensive genetic variation between viral isolates that if confirmed has significant implications for future vaccine design and development of newer diagnostic procedures. Here, we avoid potential assembly artifacts inherent in composite sequencing techniques by using long-rang...
Kumar CS, Sivaramakrishna D, Ravi SK, Swamy MJ.Seminal fibronectin type-II (Fn-II) proteins interact with choline phospholipids present on the sperm plasma membrane and play a crucial role in sperm capacitation. Crystal structure of phosphorylcholine (PrC) complex of PDC-109, the major bovine Fn-II protein, together with fluorescence spectroscopic studies has shown that tryptophan residues are crucial for its specific interaction with choline phospholipids. In the present study, the heterogeneity and microenvironment of tryptophan residues in HSP-1/2, a major protein of horse seminal plasma (which is homologous to PDC-109) were investigate...
Bhuyan AK, Rao DK, Prabhu NP.Proteins meet with the stipulations of Levinthal. Two test tube variants of ferrocytochrome c (ferrocyt c) whose thermodynamic stabilities are vastly different refold to the same global minimum under a given final native condition, and they do so quickly at rates that do not reflect a strong dependence on the thermodynamic driving force. The transition-state ensemble is more unfolded-like, and the folding barrier offered is energetically sizable. The experiments involve neutral- (pH 7) and alkaline ferrocyt c pH (12.7), whose aqueous stabilities are 18 (+/-0.3) and 3 (+/-0.5) kcal mol(-)(1), r...
Amaral A, Fernandes C, Szóstek-Mioduchowska A, Lukasik K, Rebordão MR, Pinto-Bravo P, Skarzynski DJ, Ferreira-Dias G.Cathepsin G (CAT) is a protease released by neutrophils when forming neutrophil extracellular traps that was already associated with inducing type I collagen (COL1) in equine endometrium in vitro. Endometrosis is a fibrotic condition mainly characterized by COL1 deposition in the equine endometrium. The objective was to evaluate if noscapine (an alkaloid for cough treatment with anti-neoplastic and anti-fibrotic properties) would reduce transcription (evaluated by qPCR) and COL1 protein relative abundance (evaluated by western blot) induced by CAT in equine endometrial explants from follicula...
Stanley SM, Poljak A.Mass spectrometric analyses of the recombinant proteins in Eprex and Aranesp were undertaken with the goal of producing reference mass spectra and evaluating strategies to improve its applicability as a method for equine and canine doping control of these substances. A simple, low chemical noise deglycosylation reaction removed microheterogeneity due to post-translational carbohydrate attachment and both proteins were detectable using MALDI-TOF-MS. Deglycosylated human erythropoietin (hEPO) was also detected using HPLC-ESI-MS. This is the first time that spectra of deglycosylated Eprex and Ara...
Rose BV, Cabrera-Sharp V, Firth MJ, Barrelet FE, Bate S, Cameron IJ, Crabtree JR, Crowhurst J, McGladdery AJ, Neal H, Pynn J, Pynn OD, Smith C....Early pregnancy loss occurs in 6-10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14-65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in Amnio...
Scacchia M, Molini U, Marruchella G, Maseke A, Bortone G, Cosseddu GM, Monacoo F, Savini G, Pini A.African horse sickness (AHS) is a vector‑borne viral disease of equids, endemic in Sub‑Saharan Africa. This article reports the clinic‑pathological and laboratory findings observed in the framework of passive surveillance during the AHS outbreaks which occurred in Namibia between 2006 and 2013. This study was conducted in the framework of the collaboration among the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise (Teramo, Italy), the Namibian Ministry of Agriculture Water and Forestry, and the Namibian National Veterinary Association. A total of 92 horses were investigate...
Desantis S, Ventriglia G, Zizza S, Guaricci AC, Losurdo M, Zarrilli A, Albrizio M.The presence of the mu-opioid receptor (MOR) was investigated in the mare oviduct during oestrus and anoestrus, by means of immunoblotting and immunohistochemistry. Immunoblotting analysis showed that the MOR protein is expressed as 65, 50 and 30 kDa forms in the infundibulum and ampulla both in oestrus and anoestrus, while the 30 kDa form is absent in the isthmus. Moreover, different levels of expression were observed along the ampulla in the two periods examined. Immunohistochemistry revealed MOR in the mucosal epithelium, stromal cells, myocytes and blood vessels. Ciliated cells expressed M...
Bousfield GR, Ward DN.After dissociating equine gonadotropins as a function of time at pH 3, we examined them by radioligand assay and sodium dodecyl sulfate polyacrylamide gel electrophoresis under nondissociating conditions (low, 0.1% SDS). Equine follicle-stimulating hormone (FSH) rapidly lost its receptor-binding activity, and low SDS-polyacrylamide gels demonstrated dissociation into subunits. Maximum dissociation occurred after 20-30 min of pH 3 incubation. Equine luteinizing hormone (LH), however, retained most biologic activity and was largely intact after 72 h of pH 3 incubation. Dose-response curves of ac...
Ryan PL, Klonisch T, Yamashiro S, Renaud RL, Wasnidge C, Porter DG.Immunoreactive, chromatographic and molecular techniques were used to study the expression of relaxin in mare ovaries at different stages of the oestrous cycle. Relaxin in follicular fluid ranged from 1.6 to 2.5, from 1.4 to 5.2, from 1.2 to 6.7 and from 1.0 to 3.5 ng ml-1 in small ( 2 3 4 cm) follicles, respectively, and total content of fluid relaxin per follicle increased (P < 0.05) with follicular size. When subjected to reverse phase HPLC analysis, follicular fluid yielded absorbance profiles corresponding closely to those of purified relaxin, and immunoreactive peaks in follicular f...
Tachibana Y, Sakurai T, Bai H, Shiota K, Nambo Y, Nagaoka K, Imakawa K.Extensive studies have been conducted to characterize the unique phenomena of equine pregnancy. Most studies have focused on embryo transmigration when the embryo is covered with a mucin-like glycoprotein capsule and on the characterization of the chorionic girdle and chorionic gonadotropin (CG) secretion. However, the events preceding and following capsule disappearance have not been well studied. In this study, the mRNA expression in conceptus membranes at days 19, 21, and 25 (day 0 = day of ovulation) was analyzed by RNA-seq (SOLiD3), and transcript levels on these three days and day 13...
Moloney E, Kavanagh KS, Buckley TC, Cooney JC.Streptococcus equi ssp. equi is the causative agent of 'Strangles' in horses. This is a debilitating condition leading to economic loss, yard closures and cancellation of equestrian events. There are multiple genotypes of S. equi ssp. equi which can cause disease, but to date there has been no systematic study of strains which are prevalent in Ireland. This study identified and classified Streptococcus equi ssp. equi strains isolated from within the Irish equine industry. Results: Two hundred veterinary isolates were subjected to SLST (single locus sequence typing) based on an internal sequenc...
Bayless RL, Sheats MK, Jones SL.Neutrophils play a major role in many equine conditions, including equine asthma, laminitis, and intestinal ischemia and reperfusion injury, and therefore represent an attractive target for innovative therapeutic approaches. Novel strategies for reducing neutrophilic inflammation include modulation of neutrophil functions and lifespan. Withaferin A (WFA) is a phytochemical with well-established and anti-inflammatory properties, but its direct effects on neutrophils are largely unknown. We hypothesized that WFA would inhibit adhesion, migration, and respiratory burst by equine neutrophils and...
