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.
Pazzi KA, Kraegel SA, Griffey SM, Theon AP, Madewell BR.Wild type equine p53 was amplified between exons 2 and 9 by the polymerase chain reaction using primers designed from conserved regions in other species. An 828 base pair region, corresponding to codons 25-313 of human p53, was sequenced in both directions. Human and equine amino acid sequences were 87% homologous in this region and 96% homologous in conserved domains II-V. Of eight equine cutaneous or mucocutaneous squamous cell carcinomas directly sequenced from exons 5-8, two had p53 point mutations resulting in single amino acid substitutions.
Wilson SM, Rakhit S, Murdoch R, Pediani JD, Elder HY, Baines DL, Ko WH, Wong PY.Experiments were undertaken using cultured equine sweat gland epithelial cells that express purine receptors belonging to the P2U subclass which allow the selective agonist uridine triphosphate (UTP) to increase the concentration of intracellular free Ca2+ ([Ca2+]i). Experiments using pertussis toxin (Ptx), which inactivates certain guanine-nucleotide-binding proteins (G-proteins), showed that this response consisted of Ptx-sensitive and Ptx-resistant components, and immunochemical analyses of the G-protein alpha subunits present in the cells showed that both Ptx-sensitive (alpha i1-3) and Ptx...
Gasser RB, Stevenson LA, Chilton NB, Nansen P, Bucknell DG, Beveridge I.Five species of equine strongyle belonging to the subfamily Strongylinae (Strongylus edentatus, S. equinus, S. vulgaris, Oesophagodontus robustus and Triodontophorus serratus) and 11 species belonging to the subfamily Cyathostominae (Poteriostomum imparidentatum, P. ratzii, Cylicocyclus insignis, Cc. leptostomus, Cc. nassatus, Cylicostephanus calicatus, Cs. longibursatus, Cs. goldi, Cyathostomum catinatum, Cy. labiatum and Cy. pateratum) were characterized using a polymerase chain reaction-linked restriction fragment length polymorphism technique (PCR-RFLP). Internal transcribed spacer ribosom...
Grøndahl C, Hyttel P.The nucleolus is believed to be the active site of rRNA synthesis in all eukaryotic cells. In preimplantation embryos, the embryonic genome is apparently more or less silent up to a species-specific developmental stage at which a major burst of transcription occurs. Here we report on nucleologenesis and some ultrastructural aspects of the onset of RNA synthesis in equine embryos during in vivo development. The zygotes and embryos up to blastocyst stages were surgically recovered from normally cycling mares. Mares were induced to ovulate by treatment with 3000 IU hCG and inseminated 20 and 34 h...
Wijesundera WS, Chandrasekharan NV, Karunanayake EH, Dharmasena SP.Two repetitive sequences (IpSdM and IpSdS) have been cloned and sequenced from the genome of Setaria digitata. When IpSdM (214 bp) and IpSdS (201 bp) were aligned, a high degree of homology (85%) was observed, indicating that they belong to the same family of repeats. IpSdM represents a complete repeating element while IpSdS consists of two partial repeating elements arranged in tandem. The elements are present in about 10 000 copies comprising 2.8% of the S. digitata genome. As a diagnostic probe IpSdM detects as little as 100 pg DNA of both S. digitata and S. labiato-papillosa. It can also d...
Engvall EO, Pettersson B, Persson M, Artursson K, Johansson KE.A PCR-based assay was developed for detecting DNA of granulocytic ehrlichiae in blood samples from dogs, horses, and cattle, Primers were designed from 16S rRNA sequence information to specifically amplify DNA from a newly identified Swedish Ehrlichia species. The 16S rRNA nucleotide sequence of this Swedish species differs in only two and three positions from the sequences of Ehrlichia phagocytophila and Ehrlichia equi, respectively, which were also amplified by this PCR system. For evaluation, PCR results were compared with microscopic examination of stained blood smears for the detection of...
Yang X, Chasteen ND.Electron paramagnetic resonance spectroscopy and gel permeation chromatography were employed to study the molecular diffusion of a number of small nitroxide spin probes (approximately 7-9 A diameter) into the central cavity of the iron-storage protein ferritin. Charge and polarity of these radicals play a critical role in the diffusion process. The negatively charged radical 4-carboxy-2,2,6,6-tetramethylpiperidine-N-oxyl (4-carboxy-TEMPO) does not penetrate the cavity whereas the positively charged 4-amino-TEMPO and 3-(aminomethyl)-proxyl radical and polar 4-hydroxy-TEMPO radical do. Unlike th...
Lunn DP, Schram BR, Vagnoni KE, Schobert CS, Horohov DW, Ginther OJ.Lymphokine activated killing (LAK) is an example of natural cytotoxicity, and as such is a critical means of defense against diseases such as viral infection and neoplasia. Despite this important role, the specific molecular interactions involved in LAK or other forms of natural cytotoxicity are only partially understood. In some species, cells capable of mediating natural cytotoxicity express the CD8 molecule, although no specific role has been demonstrated for CD8 in non-MHC restricted cytotoxicity. In this study the role of the EqCD8 equine homolog of CD8 in LAK cell activity was examined. ...
Johnsen , Thanh DB, Ly Q, Smestad Paulsen B, Wold JK.IgE-binding components in an extract of horse dander were analyzed, especially with regard to the glycoprotein allergens. After SDS-PAGE under reducing conditions and blotting, several of the glycoprotein IgE-binding components, including two distinct bands of 27 and 31 kDa, were detected. Together with several other bands, they were shown to bind to the lectins Sambucus nigra agglutinin (SNA) and Datura stramonium agglutinin (DSA), indicating terminal sialic acid linked alpha 2 --> 6 to galactose, and galactose linked beta 1 --> 4 to N-acetylglucosamine, respectively. Carbohydrate analy...
Bucher K, Szalai G, Marti E, Griot-Wenk ME, Lazary S, Pauli U.The tumour suppressor protein p53 enhances the genetic stability of the cell and plays a critical role in tumour suppression. Equine p53 was analysed by sequencing exons 5 to 9, a region which includes most known mutations and all the mutational hotspots in the species that have been investigated. The fragment was amplified, cloned and sequenced from genomic and complementary DNA. A comparison of the predicted amino acid sequences between the horse and other species resulted in identities between 66 per cent with the clawed frog and 92 per cent with the cat. Using the single strand conformatio...
MacLeod JN, Burton-Wurster N, Gu DN, Lust G.Fibronectin is an extracellular matrix glycoprotein encoded by a single gene. Alternative RNA splicing has been reported at three sites, ED (extra type III domain)-A, ED-B, and the variable or V region. Articular cartilage fibronectin monomers are rarely (ED-A)+, but approximately 25% are (ED-B)+. RNA gel electrophoresis and Northern blot analysis identified two (ED-B)+ and two (ED-B)- fibronectin transcripts in cartilage, each pair differing by approximately 750 bases. This difference results from a previously unreported RNA splicing pattern that eliminates not only the V region but also nucl...
Yu MF, Ewaskiewicz JI, Adda S, Bailey K, Harris V, Sosnoski D, Tomasic M, Wilson J, Kotlikoff MI.We report adenovirus-mediated gene transfer into airway smooth muscle cells in cultured cells and organ-cultured tracheal segments. Incubation of cultured rat tracheal myocytes with virus (5 x 10(8) pfu/ml) for 6 h resulted in beta-galactosidase expression in 94.8 +/- 2.5% of cells (n = 4). Following incubation of thin (less than 200 microns diameter) equine trachealis muscle segments with virus in organ culture (5 x 10(8)-5 x 10(10) pfu/ml) the average expression of the Lac Z gene was approximately 19 +/- 10% (n = 9). Expression was markedly improved, however, in segments from neonatal rats (...
Bell KS, Philp JC, Christofi N, Aw DW.Two regions in the gene coding for 16S rRNA in Rhodococcus equi were selected as species-specific primer sequences for the polymerase chain reaction (PCR). PCR using these primers was tested against 10 strains of R. equi (including the type strain) and gave positive results for all but was negative for all other tested species of Rhodococcus; representatives of the most closely related genera and a number of other bacterial species. This method could therefore be used to identify this species which can infect the lungs or other organs of horses, pigs, humans and other animals.
Salamon Z, Tollin G.Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, ...
Wutz G, Auer H, Nowotny N, Grosse B, Skern T, Kuechler E.Equine rhinoviruses (ERVs) are picornaviruses which cause a mild respiratory infection in horses. The illness resembles the common cold brought about by rhinoviruses in humans; however, the presence of a viraemia during ERV-1 infection, the occurrence of persistent infections and the physical properties are all more reminiscent of foot-and-mouth disease virus (FMDV). cDNA cloning and sequencing of the genomes of ERV-1 and ERV-2 between the poly(C) and poly(A) tracts showed that the serotypes are heterogeneous. Nevertheless, the genomic architecture of both serotypes is most similar to that of ...
Gustchina A, Kervinen J, Powell DJ, Zdanov A, Kay J, Wlodawer A.Equine infectious anemia virus (EIAV), the causative agent of infectious anemia in horses, is a member of the lentiviral family. The virus-encoded proteinase (PR) processes viral polyproteins into functional molecules during replication and it also cleaves viral nucleocapsid protein during infection. The X-ray structure of a complex of the 154G mutant of EIAV PR with the inhibitor HBY-793 was solved at 1.8 A resolution and refined to a crystallographic R-factor of 0.136. The molecule is a dimer in which the monomers are related by a crystallographic twofold axis. Although both the enzyme and t...
McDowell KJ, Little TV, Timoney PJ, Adams MH.The major proteins in stallion seminal plasma were characterised by two-dimensional polyacrylamide gel electrophoresis, and compared with the patterns of proteins in normal geldings (castrated males) and geldings supplemented with testosterone. The major proteins or groups of proteins identified according to their approximate relative molecular weight in kilodaltons (kDa) and apparent isoelectric point (pl) were: 1) 60 kDa. pl 7; 2) 23 kDa, pl 4-5; 3) 25-30 kDa, pl 5.5-6; 4) 23 kDa, pl 7-8; and 5) 15-20 kDa, pl 6-7.5. Protein groups 1 and 2 were more prominent in the seminal plasma from the st...
Maher JK, Tresnan DB, Deacon S, Hannah L, Antczak DF.Down-regulation of major histocompatibility complex (MHC) genes by trophoblast cells is considered to be a primary mechanism preventing maternal immune rejection of the fetal-placental unit in mammalian pregnancy by rendering these cells, which form the primary barrier between mother and fetus, relatively non-antigenic. In situ hybridization with probes encoding human and horse MHC class I genes was used to characterize the pattern of MHC class I mRNA expression in the various forms of horse trophoblast. Strong hybridization signals were observed in the invasive trophoblast cells of chorionic ...
Gould AR.The nucleotide sequence of the matrix protein of equine morbillivirus (EMV) was determined to be 1062 nucleotides and coded for a deduced protein of M(r) 40148 having a net charge of + 19 at neutral pH. The matrix protein gene was separated from the P and F genes by intercistronic regions of 546 and 469 nucleotides, respectively. The nucleotide sequence which coded for the F protein was 1641 nucleotides and coded for a deduced protein of 546 amino acids having an M(r) of 60,447 and a charge + 4 at neutral pH. Partial sequence information was also determined for the P/V proteins. M, P and F pro...
Butnev VY, Gotschall RR, Baker VL, Moore WT, Gout PW, Bousfield GR.Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structu...
The Journal of physiologyJune 15, 1996
Volume 493 ( Pt 3), Issue Pt 3 885-894 doi: 10.1113/jphysiol.1996.sp021431
Ko WH, Chan HC, Chew SB, Wong PY.1. The ionic mechanism involved in Ca(2+)-stimulated electrolyte transport in cultured equine sweat gland epithelial cells was studied using the short-circuit current (ISC) technique. 2. Microscopy revealed that the cultured cells grown on Millipore filters formed polarized monolayers with tight junctions. Monolayers exhibited a mean transepithelial resistance of 333.9 +/- 40.4 omega cm2. 3. Ca(2+)-mobilizing agents, A23187 (1 microM) or thapsigargin (0.01-1 microM), stimulated ISC while forskolin exerted little effect on the ISC. 4. Replacement of external Cl- by gluconate significantly reduc...
Barteri M, Gaudiano MC, Santucci R.The influence of glycerol on the structural properties of Fe(III)-horse heart myoglobin has been investigated by absorbance, CD and SR-SAXS spectroscopy. The results obtained indicate that both the tertiary and the secondary (alpha-helix) conformations of the protein are influenced by glycerol; in particular, an increase of approx. 8% in helical content was observed. Further, analysis of both the acid- and guanidine-induced denaturation transitions points to a glycerol-induced decreased stability of the tertiary structure; conversely, the alpha-helix conformation is found to be stabilized by t...
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...
Deeb RS, Peyton DH.Tin protoporphyrin IX (SnPP) is being used in the treatment of hyperbilirubinemia. We have studied the SnPP complex with equine myoglobin (EqMb) by 1H and 119Sn nuclear magnetic resonance spectroscopy (NMR) as a general model for SnPP interaction with hemoproteins. The complex formed from SnPP and EqMb, SnPP.EqMb, was found to have essentially the same porphyrin-binding pocket as EqMbCO, including the same porphyrin orientation in the major form of EqMbCO. 119Sn NMR spectroscopy has been used to demonstrate that the proximal His93F8-metal coordination is likely to be intact in SnPP.EqMb. Minor...
Peyton DH.Specific heme protons for the majority of resonances in the downfield resolved region of equine met-azido myoglobin have been assigned using solely the two-dimensional 1H NMR experiments NOESY and COSY. Metazido myoglobin provides a useful test case for the applicability of these techniques to paramagnetic proteins for the following reasons. First met-azido myoglobin is a mixed spin-state protein, with significantly shorter relaxation times and broadened lines relative to pure low-spin systems (eg., met-cyano myoglobin). Second, met-azido hemoglobin and met-azido myoglobin are important as mod...
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...
Szalai G, Bailey E, Gerber H, Lazary S.The genetic diversity at the ELA DQ beta locus was investigated using polymerase chain reaction and DNA sequencing. Based upon serological methods 16 class II homozygous animals were selected and their genomic DNA was used. A DQ beta gene from an equine cDNA library was also sequenced. Our methodology and the similarity between the genomic and the cDNA sequences suggest that the studied locus is expressed on equine lymphocytes. In the predicted amino acid sequence the most extensive variation is located at residues 56-60. The pattern of these five amino acids is strongly correlated to the sero...
D'Arcy-Moskwa E, Weston L, Noble GN, Raidal SL.Mucosal integrity may be objectively assessed by determination of the absorption of exogenous substances such as sucrose. Gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) have been reported for the accurate quantification of low concentrations of sucrose in serum. LC/MS offered the advantage of high sensitivity and mass selectivity without the need for extensive sample derivatization required for GC/MS methods. However, the high polarity and non-volatile nature of the sucrose molecule renders LC/MS techniques challenging. Previously published rep...
Guzeloglu A, Atli MO, Kurar E, Kayis SA, Handler J, Semacan A, Aslan S.The aims of the present study were to elucidate the expression profiles of leukotriene (LT) pathway mRNA transcription and to determine the possible interaction of LT and prostaglandin (PTG) pathways genes in equine endometrium during the estrous cycle and early pregnancy. Endometrial biopsies were obtained from mares on the day of ovulation (d0), at late diestrous (LD, n = 4), and after luteolysis in the estrus phase (AL, n = 4) of the cycle. Biopsies were also taken on Days 14 (P14; n = 4), 18 (P18, n = 4), and 22 (P22, n = 4) during early pregnancy that were comparable days to cyclic sampli...
Watson ED, Bae SE, Al-Zi'abi MO, Hogg CO, Armstrong DG.The insulin-like growth factors, IGF-I and -II, have been shown to play a key role in luteal function in some species. The IGF binding proteins, IGFBP-2 and -3, have been shown to inhibit binding of IGF-I and -II to bovine luteal cells and decrease progesterone production. We have recently shown that equine follicles have the genetic capacity to produce IGFBP-2, and that levels decrease in healthy preovulatory follicles. In the present study expression of mRNAs encoding IGFBP-2, as well as the rate-limiting steroidogenic enzyme, P450scc, were studied in equine corpora lutea to investigate whet...
Saari H, Tulamo RM, Konttinen YT, Sorsa T.A high performance liquid chromatography (HPLC) procedure suitable for the simultaneous determination of the molecular size and concentration of macromolecular hyaluronate and proteoglycans in synovial fluid has been developed. Irrigation of the equine tarsocrural joint with 20 ml physiological saline (PSS) caused a mild inflammation with an increase of proteoglycans in the synovial fluid over the baseline arthrocentesis control sample. Proteoglycan and hyaluronate in the synovial fluid did not interact to form hyaluronate-proteoglycan aggregates, but separated as distinct chromatographic peak...
Bartosik J, Łojek J, Długosz E, Górski P, Zygner W.Tapeworm infections in Konik Polski horses from Biebrza National Park were investigated in this study. Faecal samples were collected 10 times: in 2012 - 1 time, in 2013 - 4 times, in 2014 - 4 times and in 2015 - 1 time. In total, 162 faecal samples were collected and tested. Faecal egg counts (FECs) method was used in the study. Positive samples with cestode eggs were noted only twice - in October 2012 and December 2013 in two adult mares (9 and 11 years old). The determined prevalence was surprisingly low comparing to other studies, 4.3% in October 2013 and 28.5% in December 2013. Parasite ge...
Cappugi G, Chellini PC, Nassi P, Ramponi G.A ninhydrin-negative peptide fraction obtained from tryptic digest of carboxymethyl acylphosphatase was isolated by chromatography on a column of PA 28 Beckman resin and analysed for the amino acid composition. Degradation with carboxypeptidase B and A indicated that the sequence of this peptide was: X-Thr-Ala-Arg. The amino-terminal residue was identified as N-acetylserine by high voltage electrophoresis. It is therefore suggested that the sequence of the NH2-terminal portion of CM-acylphosphatase is N-acetyl-Ser-Thr-Ala-Arg. Digestion with carboxypeptidase A and B indicated also that the COO...
Feughelman M, Lyman DJ, Willis BK.Recent Fourier transform infrared spectroscopy (FTIR) with attenuated total reflection technique (ATR) has been applied to alpha-keratin fibers (horse-hair) extended in water both at 21 and 95 degrees C. Infrared absorption bands in the Amide 1 region indicated that at extensions to 40-50% strain in water at 21 degrees C alpha-helices had completely disappeared and parallel beta-sheets were formed [Appl. Spectrosc. 55 (2001) 552]. However, when the hair fibers were extended to the same strain at 95 degrees C in water the result was the formation of anti-parallel beta-sheets. These results sugg...
The Journal of parasitologyNovember 26, 2004
Volume 90, Issue 5 1027-1033 doi: 10.1645/0022-3395(2004)090[1027:AEOARB]2.0.CO;2
Gupta GD, Lakritz J, Saville WJ, Livingston RS, Dubey JP, Middleton JR, Marsh AE.Sarcocystis neurona is the primary parasite associated with equine protozoal myeloencephalitis (EPM). This is a commonly diagnosed neurological disorder in the Americas that infects the central nervous system of horses. Current serologic assays utilize culture-derived parasites as antigen. This method requires large numbers of parasites to be grown in culture, which is labor intensive and time consuming. Also, a culture-derived whole-parasite preparation contains conserved antigens that could cross-react with antibodies against other Sarcocystis species and members of Sarcocystidae such as Neo...
Adolf GR, Traxler E, Maurer-Fogy I.Equine interferon-beta 1 (EqIFN-beta 1) was purified from extracts of recombinant Escherichia coli by sequential chromatography on hydroxylapatite, anion-, and cation-exchangers. The resulting protein was greater than 98% pure as determined by sodium dodecylsulfate gel electrophoresis, gel permeation HPLC, and reverse-phase HPLC. Amino-terminal amino acid sequencing revealed that essentially all molecules contained an additional amino-terminal methionine. The specific antiviral activity of EqIFN-beta 1 determined on equine dermal fibroblasts challenged with vesicular stomatitis virus (VSV) was...
Lopez MJ, Robinson SO, Cooley AJ, Prichard MA, McGinnis MR.An 18-year-old mare was evaluated for an oral mass that developed after extraction of a broken incisor. Results: An ulcerated, firm, darkly pigmented, approximately 5-cm-diameter spherical mass involved the gingiva lateral and dorsal to the right first to third maxillary incisors. Osteolysis of the roots of the first and second right maxillary incisors and periosteal proliferation of the adjacent premaxilla margins were apparent on radiographs. Histologic examination of the mass revealed multiple coalescing and ramifying foci of abscess formation, each containing a well-defined, discrete, blac...
Medda R, Cara N, Floris G.Ceruloplasmin is a blue copper-containing serum glycoprotein with oxidase activity. It as been proposed that the physiological function of ceruloplasmin involves the oxidation of ferrous iron and its incorporation into apotransferrin. There are several reports demonstrating that ceruloplasmin is made up of multiple chains. Ryden has questioned the multichain structure of ceruloplasmin from human, pig, horse and rabbit sera, arguing that the dissociation observed by previous workers could be attributed to cleavage of labile bands in the protein by enzymatic contaminants present in commercial pr...
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...
