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.
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...
Heim A, Passos LM, Ribeiro MF, Costa-Júnior LM, Bastos CV, Cabral DD, Hirzmann J, Pfister K.Blood samples were collected from 487 adult horses, including 83 pregnant mares, at a slaughterhouse located in Araguari, Minas Gerais State, Brazil. For each blood sample, the packed cell volume (PCV) was determined, and Giemsa-stained smears were microscopically examined for the presence of hemoparasites. The plasma was examined by the indirect fluorescent antibody test for detection of antibodies against Babesia caballi and Theileria equi. In addition, DNA was extracted and analyzed by a multiplex real-time polymerase chain reaction (PCR), specific for B. caballi and T. equi. Products of PC...
Van de Walle GR, May ML, Sukhumavasi W, von Einem J, Osterrieder N.Glycoprotein G (gG) of alphaherpesviruses has been described to function as a viral chemokine-binding protein (vCKBP). More recently, mutant viruses devoid of gG have been shown to result in increased virulence, but it remained unclear whether the potential of gG to serve as a vCKBP is responsible for this observation. In this study, we used equine herpesvirus type 1 (EHV-1) as a model to study the pathophysiological importance of vCKBP activity. First, in vitro chemotaxis assays studying migration of immune cells, an important function of chemokines, were established. In such assays, supernat...
Allen CA, Payne SL, Harville M, Cohen N, Russell KE.The study of the equine immune system and inflammatory responses, by measuring cytokine expression, can provide important insight into disease pathogenesis in the horse. A set of quantitative real-time polymerase chain reaction (QPCR) assays for the equine cytokines IL-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha were validated using QPCR primers and probes which were generated for the equine IL-1alpha, IL-1beta, IL-6, IL-8, TNF-alpha and 18S genes. Amplification efficiency, intra-assay and inter-assay variation were determined using 10-fold dilutions of plasmid for each gene. Under these condit...
Sun WC, Moore JN, Hurley DJ, Vandenplas ML, Linden JM, Murray TF.To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes. Methods: Neutrophils isolated from 8 healthy horses. Methods: Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A(1), A(2A), and A(3) receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrop...
Endo A, Okada S, Morita H.Diversity and compositions of the Lactobacillus, Streptococcus, and Bifidobacterium group in the feces of six healthy, actively racing horses (Thoroughbreds) were analyzed by using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR with primer sets specific for each group. PCR-DGGE analysis of the feces showed that Lactobacillus equi, Lactobacillus johnsonii, a phylogenetic relative of Lactobacillus salivarius, a phylogenetic relative of Lactobacillus gastricus, and Weissella confusa were predominant in almost all of the feces tested, and Streptococcus bovis/Streptococcus equ...
Strüder-Kypke MC, Kornilova OA, Lynn DH.Ciliates of the subclass Trichostomatia inhabit the fermentative regions of the digestive tract of herbivores. Most available small subunit ribosomal RNA (SSrRNA) gene sequences of trichostomes are from species isolated from the rumen of cattle or sheep and from marsupials. No ciliate species endosymbiotic in horses has yet been analyzed. We have sequenced the SSrRNA genes of five ciliate species, isolated from the cecum and colon of four Yakut horses: Cycloposthium edentatum, Cycloposthium ishikawai, Tripalmaria dogieli, Cochliatoxum periachtum, and Paraisotricha colpoidea. Based on their mor...
Herszberg B, Mata X, Giulotto E, Decaunes P, Piras FM, Chowdhary BP, Chaffaux S, Guérin G.Glycogen debranching enzyme (AGL) is a multifunctional enzyme acting in the glycogen degradation pathway. In humans, the AGL activity deficiency causes a type III glycogen storage disease (Cori-Forbes disease). One particularity of AGL gene expression lies in the multiple alternative splicing in its 5' region. The AGL gene was localized on ECA5q14-q15. The sequence of the equine cDNA was determined to be 7.5 kb in length with an open reading frame of 4602 bp. The gene is 69 kb long and contains 35 exons. The equine AGL gene has an ubiquitous expression and presents five tissue-dependent cDNA v...
Musilova P, Kubickova S, Zrnova E, Horin P, Vahala J, Rubes J.Using laser microdissection we prepared a set of horse chromosome arm-specific probes. Most of the probes were generated from horse chromosomes, some of them were derived from Equus zebra hartmannae. The set of probes were hybridized onto E. grevyi chromosomes in order to establish a genome-wide chromosomal correspondence between this zebra and horse. The use of arm-specific probes provided us with more information on the mutual arrangement of the genomes than we could obtain by means of whole-chromosome paints generated by flow sorting, even if we used reciprocal painting with probe sets from...
King SS, Jones KL, Mullenix BA, Heath DT.Dopamine (DA) blockade during anestrus or early spring transition can facilitate ovarian recrudescence and advance the timing of the first ovulation of the season. Some laboratories have reported variable results using DA antagonists to stimulate follicular growth during the mid-portion of the anestrual period. Differences in DA antagonist efficacy may be due to the FSH secretory status of the anestrous mare and the presence or absence of functional ovarian FSH receptors. We hypothesize that direct ovarian dopaminergic input can affect follicular growth through regulation of FSH receptor (FSHr...
Xu S, Luosang J, Hua S, He J, Ciren A, Wang W, Tong X, Liang Y, Wang J, Zheng X.To investigate genetic mechanisms of high altitude adaptations of animals living in the Tibetan Plateau, three mitochondrial genomes (mt-genome) of Tibetan horses living in Naqu (4,500 m) of Tibetan, Zhongdian (3,300 m) and Deqin (3,100 m) of Yunnan province were sequenced. The structures and lengths of these three mt-genomes are similar to the Cheju horse, which is related to Tibetan horses, but little shorter than the Swedish horse. The pair-wise identity of these three horses on nucleotide level is more than 99.3%. When the gene encoding the mitochondrial protein of Tibetan horses was analy...
Sharp EL, Farrell HE, Borchers K, Holmes EC, Davis-Poynter NJ.Equid herpesvirus 2 (EHV-2), in common with other members of the subfamily Gammaherpesvirinae, encodes homologues of cellular seven-transmembrane receptors (7TMR), namely open reading frames (ORFs) E1, 74 and E6, which each show some similarity to cellular chemokine receptors. Whereas ORF74 and E6 are members of gammaherpesvirus-conserved 7TMR gene families, E1 is currently unique to EHV-2. To investigate their genetic variability, EHV-2 7TMRs from a panel of equine gammaherpesvirus isolates were sequenced. A region of gB was sequenced to provide comparative sequence data. Phylogenetic analysi...
Golomidova A, Kulikov E, Isaeva A, Manykin A, Letarov A.The diversity of coliphages and indigenous coliform strains (ICSs) simultaneously present in horse feces was investigated by culture-based and molecular methods. The richness of coliforms (as estimated by the Chao1 method) is about 1,000 individual ICSs distinguishable by genomic fingerprinting present in a single sample of feces. This unexpectedly high value indicates that some factor limits the competition of coliform bacteria in the horse gut microbial system. In contrast, the diversity of phages active against any selected ICS is generally limited to one to three viral genotypes present in...
Nielsen MK, Peterson DS, Monrad J, Thamsborg SM, Olsen SN, Kaplan RM.Strongylus vulgaris is an important strongyle nematode with high pathogenic potential infecting horses world-wide. Several decades of intensive anthelmintic use has virtually eliminated clinical disease caused by S. vulgaris, but has also caused high levels of anthelmintic resistance in equine small strongyle (cyathostomin) nematodes. Recommendations aimed at limiting the development of anthelmintic resistance by reducing treatment intensity raises a simultaneous demand for reliable and accurate diagnostic tools for detecting important parasitic pathogens. Presently, the only means available t...
Casini A, Mastrobuoni G, Terenghi M, Gabbiani C, Monzani E, Moneti G, Casella L, Messori L.The interactions with protein targets of the ruthenium(III) complex imidazolium trans-[tetrachloro(dimethyl sulfoxide)(imidazole)ruthenate(III)], NAMI-A, an effective anticancer and antimetastatic agent now in clinical trials, deserve great attention as they are believed to be at the basis of the mechanism of action of this innovative molecule. Here, we report on the reactions of NAMI-A with two well-known model proteins, namely, hen egg white lysozyme and horse heart cytochrome c; these reactions were investigated by a variety of physicochemical methods, including optical spectroscopy, (1)H N...
Iwatani S, Zendo T, Yoneyama F, Nakayama J, Sonomoto K.A novel bacteriocin, lacticin Z, produced by Lactococcus lactis QU 14 isolated from a horse's intestinal tract was identified. Lacticin Z was purified through a three step procedure comprised of hydrophobic-interaction, cation-exchange chromatography, and reverse-phase HPLC. ESI-TOF MS determined the molecular mass of lacticin Z to be 5,968.9 Da. The primary structure of lacticin Z was found to consist of 53 amino acid residues without any leader sequence or signal peptide. Lacticin Z showed homology to lacticin Q from L. lactis QU 5, aureocin A53 from Staphylococcus aureus A53, and mutacin BH...
Zhang J, Miszczak F, Pronost S, Fortier C, Balasuriya UB, Zientara S, Fortier G, Timoney PJ.Genetic variation and phylogenetic relationships among 22 French isolates of equine arteritis virus (EAV) obtained over four breeding seasons (2001-2004) were determined by sequencing open reading frames (ORFs) 2a-7. The ORFs 2a-7 of 22 isolates differed from the prototype virulent Bucyrus strain of EAV by between 14 (99.5% identity) and 328 (88.7% identity) nucleotides, and differed from each other by between 0 (100% identity) and 346 (88.1% identity) nucleotides, confirming genetic diversity among EAV strains circulating in France. Phylogenetic analysis based on the partial ORF5 sequences (n...
Osborne RL, Coggins MK, Walla M, Dawson JH.The heme-containing respiratory protein, myoglobin (Mb), best known for oxygen storage, can exhibit peroxidase-like activity under conditions of oxidative stress. Under such circumstances, the initially formed ferric state can react with H2O2 (or other peroxides) to generate a long-lived ferryl [Fe(IV)=O] Compound II (Cpd II) heme intermediate that is capable of oxidizing a variety of biomolecules. In this study, the ability of Mb Cpd II to catalyze the oxidation of carcinogenic halophenols is demonstrated. Specifically, 2,4,6-trichlorophenol (TCP) is converted to 2,6-dichloro-1,4-benzoquinone...
Sabeur K, Ball BA.Reactive oxygen species (ROS) play an important role in normal sperm function, and spermatozoa possess specific mechanisms for ROS generation via an NAD(P)H-dependent oxidase. The aim of this study was to identify the presence of an NADPH oxidase 5 (NOX5) in equine testis and spermatozoa. The mRNA of NOX5 was expressed in equine testis as detected by northern blot probed with human NOX5 cDNA and by RT-PCR. Immunoblotting with affinity purified alpha-NOX5 revealed one major protein in equine testis and other tissues. Immunolocalization of NOX5 showed labeling over the rostral sperm head with so...
Quinn BA, Hayes MA, Waelchli RO, Kennedy MW, Betteridge KJ.During the third week of pregnancy, the equine conceptus is enclosed within a capsule, the glycan composition of which changes at around day 16 (ovulation = day 0) when the conceptus becomes immobilized (fixed) in the uterine lumen. Our objective was to characterize the process of fixation by identifying changes in major capsule-associated proteins. Individual equine conceptuses (n = 55) were collected transcervically by uterine lavage between days 13.5 and 26.5. Major proteins extracted from capsules were compared with those in fluids from the uterus and yolk sac by SDS-PAGE. Until day 14, a ...
Bruhn O, Regenhard P, Michalek M, Paul S, Gelhaus C, Jung S, Thaller G, Podschun R, Leippe M, Grötzinger J, Kalm E.Defensins are a predominant class of antimicrobial peptides, which act as endogenous antibiotics. Defensins are classified into three distinct sub-families: theta-, beta-, and alpha-defensins. Synthesis of alpha-defensin has been confirmed only in primates and glires to date and is presumably unique for a few tissues, including neutrophils and Paneth cells of the small intestine. Antimicrobial activities of these peptides were shown against a wide variety of microbes including bacteria, fungi, viruses and protozoan parasites. In the present study, we report the characterization of the equine a...
Nomura M, Hosaka Y, Kasashima Y, Ueda H, Takehana K, Kuwano A, Arai K.The DNA microarray analysis for matrix metalloproteinase (MMP)-related mRNA expression in equine superficial digital flexor tendinitis indicated that mRNA level of MMP-13 was apparently up-regulated in the tendinitis as compared to normal tendon. In situ hybridization also revealed that fibroblastic cells proliferated in the granulation tissue generated in the tendinitis actively expressed MMP-13 mRNA. On the other hand, in normal tendon, a few fibroblastic cells and vascular components lied in the endotenon barely expressed its mRNA, but other cellular components in the tendon bundle were not...
Boehm AK, Seth M, Mayr KG, Fortier LA.Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1beta (IL-1beta) signaling in chondrocytes. Methods: Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain rea...
Allsopp MT, Lewis BD, Penzhorn BL.The intra-erythrocytic parasite Theileria equi is one of two tick-transmitted causative agents of equine piroplasmosis. Piroplasms of T. equi can be transmitted across the equine placenta and once a horse is infected, it appears to remain a lifelong carrier, since anti-theilerial drugs suppress but do not eliminate the parasite. Carrier mares may transmit the organism to their offspring and this may result in abortion or neonatal piroplasmosis, but observations by some researchers suggest that foals may be born as carriers yet remain apparently healthy. Using a T. equi-specific oligonucleotide...
Njiru ZK, Constantine CC.To investigate the population genetic structure of Trypanosoma evansi from domesticated animals, we have analysed 112 stocks from camels, buffaloes, cattle and horses using the tandemly repeated coding sequence (MORF2) and minisatellite markers 292 and cysteine-rich acidic integral membrane protein (CRAM). We recorded a total of six alleles at the MORF2 locus, seven at 292 and 12 at the CRAM loci. Nei's genetic distance showed reduced allelic diversity between buffaloes and cattle stocks (1.2) as compared to the diversity between camels and buffaloes (3.75) and camels and cattle stock (1.69). ...
Ramery E, Closset R, Bureau F, Art T, Lekeux P.Environmental causes of heaves are well described, but the molecular mechanisms of the disease remain unclear. Previous studies have highlighted the implications of variations in gene expression, most using reverse transcription polymerase chain reaction (RT-PCR). This well-known technique limits the number of genes that can be studied in a single assay. Microarray appears to be a valuable tool to by-pass this limitation, but so far there has been no equine-specific microarray available on the market. The present study was performed to determine whether a human microarray could be used to stud...
Eckert RE, Neuder LE, Bell JL, Trujillo JC, Jones SL.The goal of this study was to define the role for p38 mitogen-activated kinase (MAPK) in the signaling mechanism regulating pro-inflammatory cyclooxygenase (COX) gene expression in lipopolysaccharide (LPS)-activated equine leukocytes for the purposes of identifying novel targets for anti-inflammatory therapy in endotoxemic horses. The p38 MAPK has been shown to positively regulate inflammatory gene expression in human leukocytes and can be activated by a variety of stimuli including LPS, TNF-alpha, and IL-1. Activation-associated phosphorylated p38 MAPK has been implicated in the up-regulation...
Yasui M, Suzuki N, Liu X, Okamoto Y, Kim SY, Laxmi YR, Shibutani S.4-Hydroxyequilenin (4-OHEN)-dC is a major, potentially mutagenic DNA adduct induced by equine estrogens used for hormone replacement therapy. To study the miscoding property of 4-OHEN-dC and the involvement of Y-family human DNA polymerases (pols) eta, kappa and iota in that process, we incorporated 4-OHEN-dC into oligodeoxynucleotides and used them as templates in primer extension reactions catalyzed by pol eta, kappa and iota. Pol eta inserted dAMP opposite 4-OHEN-dC, accompanied by lesser amounts of dCMP and dTMP incorporation and base deletion. Pol kappa promoted base deletions as well as ...
Gold JR, Perkins GA, Erb HN, Ainsworth DM.Septicemia initiates the production of pro-inflammatory (interleukin [IL] 1-beta [IL-1beta], interferon-gamma [IFN-gamma], IL-6), and anti-inflammatory (IL-4) cytokines. The transcription of some of these proteins (IL-8, IL-6) is linked to endotoxin-induced activation of the toll-like receptor 4 (TLR4) on peripheral blood mononuclear cells (PBMC). Objective: Septic foals fail to increase gene expression of IFN-gamma. Nonsurviving septic foals exhibit distinctive cytokine profiles. Methods: Twenty-one septic and 20 healthy neonatal foals. Methods: Using real-time polymerase chain reaction, gene...
Silva CJD, Lima KPB, Monteiro JFDCLS, Silva AKSFD, Silva FJD, Pereira AMS, Hernandes VP, Silva EDD, Silva CSAGE, Brandão Filho SP, Brito MEF.American cutaneous leishmaniasis is a commonly neglected, vector-borne tropical parasitic disease that is a major public health concern in Brazil. Leishmania (Viannia) braziliensis is the main species associated with the disease. Accurate diagnosis is based on epidemiological surveillance, clinical assessment, and laboratory testing. Leishmania (V.) braziliensis has been detected in several wild and synanthropic mammals. Their epidemiological role has not been entirely elucidated. This study aimed to assess potential L. braziliensis infections in asymptomatic domestic animals, by molecular and...
Nikbakhsh M, Varkoohi S, Seyedabadi HR.Kurdish horse is one of the most valuable horse genetic resources in the Middle East. To assess the genetic diversity of Kurdish horses, Mitochondrial DNA D-loop hyper-variable region1 (HVR1) was sequenced in 29 non-related Kurdish horses which were sampled from diverse geographic regions of Iran. Total DNA was extracted from the collected blood samples by modified salting out method. The HVR1 was amplified by PCR and then sequenced using ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit. Consequently, the sequences were trimmed to 294 bp using BIOEDIT to become comparable wit...
Journal of proteomicsAugust 5, 2020
Volume 228 103927 doi: 10.1016/j.jprot.2020.103927
Chiaradia E, Pepe M, Sassi P, Mohren R, Orvietani PL, Paolantoni M, Tognoloni A, Sforna M, Eveque M, Tombolesi N, Cillero-Pastor B.Osteochondrosis is a developmental orthopedic disease affecting growing cartilage in young horses. In this study we compared the proteomes of equine chondrocytes obtained from healthy and osteochondrotic cartilage using a label-free mass spectrometry approach. Quantitative changes of some proteins selected for their involvement in different functional pathways highlighted by the bioinformatics analysis, were validated by western blotting, while biochemical alterations of extracellular matrix were confirmed via Raman spectroscopy analysis. In total 1637 proteins were identified, of which 59 wer...
Nilsson K, Larsson PO.Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants w...
Bailly-Chouriberry L, Baudoin F, Cormant F, Glavieux Y, Loup B, Garcia P, Popot MA, Bonnaire Y.The improvement of doping control is an ongoing race. Techniques to fight doping are usually based on the direct detection of drugs or their metabolites by analytical methods such as chromatography hyphenated to mass spectrometry after ad hoc sample preparation. Nowadays, omic methods constitute an attractive development and advances have been achieved particularly by application of molecular biology tools for detection of anabolic androgenic steroids (AAS), erythropoiesis-stimulating agent (ESA), or to control human growth hormone misuses. These interesting results across different animal spe...
Silva Serra AC, Júnior EC, Cruz JF, Lobo PS, Júnior ET, Bandeira RS, Bezerra DA, Mascarenhas JD, Santos Guerra SF, Soares LS. To perform a molecular analysis of rotavirus A (RVA) G3P[6] strains detected in 2012 and 2017 in the Amazon region of Brazil. Eighteen RVA G3P[6] strains were collected from children aged under 10 years hospitalized with acute gastroenteritis, and partial sequencing of each segment genome was performed using Sanger sequencing. Phylogenetic analysis showed that all G3P[6] strains had a DS-1-like genotype constellation. Two strains had the highest nucleotide identities with equine-like G3P[6]/G3P[8] genotypes. Several amino acid alterations in VP4 and VP7 neutralizing epitopes of equine-l...
Albornoz A, Morales N, Uberti B, Henriquez C, Burgos RA, Alarcon P, Moran G.Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its me...
Wagner B, Greiser-Wilke I, Antczak DF.Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse ( Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5' end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding ...
Dobrinski I, Ignotz GG, Fagnan MS, Yudin SI, Ball BA.The periacrosomal plasma membrane of spermatozoa is involved in sperm binding to oviductal epithelial cells and to the zona pellucida. A protein of 68-70 kD molecular mass was purified biochemically from the isolated periacrosomal plasma membrane of equine spermatozoa as a possible receptor for adhesion of spermatozoa to oviductal epithelial cells. A polyclonal antibody raised in rabbits against the purified equine sperm membrane protein recognized the 70 kD and an antigenically related to 32 kD protein in preparations of isolated periacrosomal sperm plasma membrane and in detergent extracted ...
Derochette S, Serteyn D, Mouithys-Mickalad A, Ceusters J, Deby-Dupont G, Neven P, Franck T.Excessive neutrophil stimulation and reactive oxygen species (ROS) production are involved in numerous human or horse pathologies. The modulation of the neutrophil NADPH oxidase (NOX) has a great therapeutic potential since this enzyme produces superoxide anion whose most of the other ROS derive. The measurement of NOX activity by cell-free systems is often used to test potential inhibitors of the enzyme. A major drawback of this technique is the possible interferences between inhibitors and the probe, ferricytochrome c, used to measure the activity. We designed the "EquiNox2", a new pharmacol...
equine sarcoids are the most frequent skin tumors in equidae worldwide. It is well known that delta bovine papillomaviruses are their causative agents. We have recently shown the presence in equine sarcoids of abnormal vessel structures, which could cause a hypoxic condition. The aim of this study was to analyze the expression of hypoxia-inducible factor-1 alpha (HIF-1α) in a subset of BPV positive equine sarcoids and explore the relationship with vascular endothelial growth factor (VEGF) expression. Results: 80% of equine sarcoids showed strong cytoplasmic staining in >60% of neoplastic f...
Ababneh MM, Troedsson MH, Michelson JR, Seguin BE.Equine conceptuses are thought to produce antiluteolytic factors that inhibit endometrial PGF2alpha and, thus, prevent luteolysis in pregnant mares. The aim of the present study was to characterize partially the chemical nature of the prostaglandin inhibitory factor (PIF) produced by equine conceptuses in vitro. Embryos were collected from pregnant mares 13 +/- 0.5 days after ovulation and were cultured for 24 h. Harvested equine conceptus conditioned media (CCM) were assayed for antiluteolytic activity by determining the inhibition of endometrial PGF2alpha synthesis in vitro. Significant anti...
Haouz A, Glandieres JM, Zentz C, Pin S, Ramstein J, Tauc P, Brochon JC, Alpert B.The effects of the solvent conditions (buffer pH 9, 8, or 7 or buffer pH 6.5 alone or mixed with 3.2% ethanol or 6.2% formamide) on the protein dynamics of horse apomyoglobin were investigated through tryptophan fluorescence quenching, spectra, and decay properties. Raising the pH (which induces discontinuous protein conformation changes) increases the structural fluctuations inside the hydrophobic A, G, and H helix core. Mixed solutions containing either 3.2% ethanol or 6.2% formamide (which redistribute water molecules on the protein surface) produce protein dynamics changes in the vicinity ...
Iyer LK, Qasba PK.Alpha-lactalbumins (LAs) and c-type lysozymes (LYZs) are two classes of proteins which have a 35-40% sequence homology and share a common three dimensional fold but perform different functions. Lysozymes bind and cleave the glycosidic bond linkage in sugars, where as, alpha-lactalbumin does not bind sugar but participates in the synthesis of lactose. Alpha-lactalbumin is a metallo-protein and binds calcium, where as, only a few of the LYZs bind calcium. These proteins consist of two domains, an alpha-helical and a beta-strand domain, separated by a cleft. Calcium is bound at a loop situated at...
Gautam A, Dubey JP, Saville WJ, Howe DK.Sarcocystis neurona is a two-host coccidian parasite whose complex life cycle progresses through multiple developmental stages differing at morphological and molecular levels. The S. neurona merozoite surface is covered by multiple, related glycosylphosphatidylinositol-linked proteins, which are orthologous to the surface antigen (SAG)/SAG1-related sequence (SRS) gene family of Toxoplasma gondii. Expression of the SAG/SRS proteins in T. gondii and another related parasite Neospora caninum is life-cycle stage specific and seems necessary for parasite transmission and persistence of infection. I...
McMonagle EL, Taylor S, van Zuilekom H, Sanders L, Scholtes N, Keanie LJ, Hopkins CA, Logan NA, Bain D, Argyle DJ, Onions DE, Schijns VE, Nicolson L.Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p...
Rice NR, Coggins L.In the endogenous reverse transcriptase reaction, equine infectious anemia virus is able to synthesize complementary DNA (cDNA) of 8,000 nucleotides in high yield. After 2 h in 50 muM dNTP, about 2.8 mug of cDNA per mg of protein is produced, almost 30% of which is long cDNA. The system thus compares favorably with the other two well-characterized endogenous reaction systems, Moloney murine leukemia virus and avian sarcoma virus. Elongation rates of 100 to 150 nucleotides per min have been observed; these rates are comparable to those seen with purified avian myeloblastosis virus reverse trans...
Semik-Gurgul E, Ząbek T, Fornal A, Wnuk M, Pawlina-Tyszko K, Gurgul A, Klukowska-Rötzler J, Koch C, Mählmann K, Bugno-Poniewierska M.Genetic and epigenetic alterations in the equine sarcoid, a locally invasive skin tumour of equids, are still poorly characterized. Numerous studies have provided reliable evidence for the relationship between the development of cancer and the loss of function of a number of tumour suppressor genes. In the present study, we assessed methylation levels in the promoter region of SFN, S100A14 and POU2F3 genes in sarcoid samples to clarify whether DNA methylation may be associated with previously identified changes in the expression level of these genes during the course of tumour progression. Usi...
Mendes MA, Chies JM, de Oliveira Dias AC, Filho SA, Palma MS.Most commercial recombinant proteins used as molecular biology tools, as well as many academically made preparations, are generally maintained in the presence of high glycerol concentrations after purification to maintain their biological activity. The present study shows that larger proteins containing high concentrations of glycerol are not amenable to analysis using conventional electrospray ionization mass spectrometry (ESI-MS) interfaces. In this investigation the presence of 25% (v/v) glycerol suppressed the signals of Taq DNA polymerase molecules, while 1% (v/v) glycerol suppressed the ...
Green MK, Johnston MV, Larsen BS.To elucidate the role of high mass accuracy in mass spectrometric peptide mapping and database searching, selected proteins were subjected to tryptic digestion and the resulting mixtures were analyzed by electrospray ionization on a 7 Tesla Fourier transform mass spectrometer with a mass accuracy of 1 ppm. Two extreme cases were examined in detail: equine apomyoglobin, which digested easily and gave very few spurious masses, and bovine alpha-lactalbumin, which under the conditions used, gave many spurious masses. The effectiveness of accurate mass measurements in minimizing false protein match...
Abraham G, Kottke C, Ammer H, Dhein S, Ungemach FR.Muscarinic receptors are considered to be of comparable clinical importance in chronic obstructive pulmonary disease (COPD) in equines and in humans. At present, data are scarce on the expression and distribution of probable subtypes of these receptors and their signalling pathways in airway segments, including lung parenchyma and bronchial and tracheal epithelium with the underlying smooth muscle in horses. Specific [N-methyl-3H]scopolamine chloride ([3H]NMS) binding to all three tissues was saturable and of high affinity, with KD values ranging between 1.6+/-0.7 and 1.9+/-0.3 nmol/L. [3H]NMS...
Marsico TV, Valente RS, Annes K, Oliveira AM, Silva MV, Sudano MJ.A wide-ranging review study regarding the molecular characterization of the first cell lineages of the developmental embryo is lacking, especially for the primary events during earliest differentiation which leads to the determination of cellular fate. Here, a systematic review and meta-analysis were conducted according to PRISMA guidelines. MEDLINE-PubMed was searched based on an established search strategy through April 2021. Thirty-six studies fulfilling the inclusion criteria were subjected to qualitative and quantitative analysis. Among the studies, 50 % (18/36) used mice as an animal mod...
Thevis M, Opfermann G, Krug O, Schänzer W.Isotope-dilution mass spectrometry has been employed successfully in numerous fields of analytical chemistry enabling the establishment of fast and reliable procedures. In equine sports, xanthine derivatives such as caffeine and theobromine are prohibited, and doping control laboratories analyze horse urine specimens regarding these illicit performance-enhancing drugs. Theobromine has to exceed a threshold level of 2 microg/mL, hence a robust and reliable quantitation is required. Stably deuterated theobromine and caffeine were synthesized by the reaction of xanthine or theobromine with iodome...
Studdert MJ, Fitzpatrick DR, Horner GW, Westbury HA, Gleeson LJ.Representative strains of EHV isolated from an aborted foetus and from a horse with rhinopneumonitis in New Zealand had restriction endonuclease DNA fingerprints typical of those usually associated with these syndromes elsewhere and now designated EHV1 and 4 respectively. EHV1 was isolated from the brain and spinal cord of a 4-year-old gelding that died of myeloencephalitis. A mare on the same farm, at about the same time as the gelding developed myeloencephalitis, aborted and EHV1 was isolated from the tissues of the aborted foetus. Restriction endonuclease DNA fingerprints of the viruses iso...
Matsumura Y, Li J, Ikeguchi M, Kihara H.Acidic buffer conditions are known to stabilize helix-rich states of even those proteins with a predominantly beta-sheet native secondary structure. Here we investigated whether such states also exist under alkaline buffer conditions. The guanidine hydrochloride (GuHCl)-induced unfolding transition and kinetic refolding of equine beta-lactoglobulin (ELG) by GuHCl-jump were investigated at pH 8.7 by far-ultraviolet circular dichroism. We found that an equilibrium intermediate appeared in 45% ethylene glycol (EGOH) buffer with 1.5 M GuHCl. The intermediate is rich in non-native alpha-helix, whic...
Miksovská J, Day JH, Larsen RW.Carbon monoxide binding to myoglobin was characterized using the photothermal beam deflection method. The volume and enthalpy changes coupled to CO dissociation were found to be 9.3+/-0.8 mL x mol(-1) and 7.4+/-2.8 kcal x mol(-1), respectively. The corresponding values observed for CO rebinding have the same magnitude but opposite sign: Delta V=-8.6+/-0.9 mL x mol(-1) and Delta H=-5.8+/-2.9 kcal x mol(-1). Ligand rebinding occurs as a single conformational step with a rate constant of 5 x 10(5) M(-1) s(-1) and with activation enthalpy of 7.1+/-0.8 kcal x mol(-1) and activation entropy of -22.4...
Piquette GN, Kenney RM, Sertich PL, Yamoto M, Hsueh AJ.The association of equine granulosa-theca cell tumors with atrophied contralateral ovaries and abnormal estrous cycles suggests that these tumors produce hormones that affect pituitary gonadotropin production. Because inhibin, a heterodimer protein secreted by granulosa cells, decreases FSH production, we examined the presence of inhibin alpha- and beta A-subunits and their mRNAs in ovarian tumors obtained from three mares. These tumors contained neoplastic cords and nodules, multiple fluid-filled cysts, and a predominance of neoplastic granulosa cells. Reduced proteins from tumor-conditioned ...
Kakoi H, Kijima-Suda I, Gawahara H, Kinoshita K, Tozaki T, Hirota K, Yoshizawa M.To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26-plex single-nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot(™) technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26-plex assay showed a probability of identity of 5.80 × 10(-11). Compared to the conventional short tan...
Cheevers WP, Fatemi-Nainie S, Anderson LW.A retrovirus is spontaneously released into the culture medium of the equine sarcoid-derived MC-1 cell line. The MC-1 virus did not exhibit in vitro transforming activity or replication when tested on equine fibroblasts or a variety of other mammalian cell cultures. Complementary DNA, synthesized using detergent-activated MC-1 virus RNA-dependent DNA polymerase, detected homologous sequences in the DNA of an established equine dermal cell line and in the DNA of primary equine dermal fibroblasts. Iododeoxyuridine or azacytidine induced a replication-deficient endogenous retrovirus in the normal...
Takeda S, Ohta M, Ebina S, Nagayama K.Horse L-ferritin cDNA was cloned from horse liver, and the base sequence was determined. The L-ferritin was expressed using pTZ18U encoding lac promoter, and found to possess an additional 8-amino acid sequence at the N-terminus as compared with commercially obtained horse spleen (natural) ferritin. It was determined that there was Pro at position 94 in both the recombinant and natural L-ferritin, although it was previously reported that Leu was in this position in the natural species. Transmission electron microscopy showed that this recombinant ferritin formed a 24-mer shell.
