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Topic:Molecular biology
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.
The open reading frame 3 of equine arteritis virus encodes an immunogenic glycosylated, integral membrane protein. Open reading frame 3 (ORF 3) of equine arteritis virus (EAV) is predicted to encode a glycosylated membrane protein (GP3) that is uncharacterized. ORF 3 of the American Type Culture Collection strain of EAV was in vitro transcribed and the encoded GP3 protein was in vitro translated with and without canine microsomal membranes. The GP3 protein was approximately 17 kDa after in vitro translation without canine microsomal membranes whereas the glycosylated form, after translation with microsomal membranes, was a diffuse band of 36-42 kDa, indicating that the GP3 protein is extensively glycosylat...
Structure of oxalate-substituted diferric mare lactoferrin at 2.7 A resolution. Lactoferrin binds two Fe(3+) and two CO(2-)(3) ions with high affinity. It can also bind other metal ions and anions. In order to determine the perturbations in the environments of the binding sites in the N and C lobes and elsewhere in the protein, the crystal structure of oxalate-substituted diferric mare lactoferrin has been determined at 2.7 A resolution. The final model has a crystallographic R factor of 21.3% for all data in the resolution range 17.0-2.7 A. The substitution of an oxalate anion does not perturb the overall structure of the protein, but produces several significant changes...
MR 20492 and MR 20494: two indolizinone derivatives that strongly inhibit human aromatase. In this study, we describe the synthesis of a new family of indolizinone derivatives designed to fit an extrahydrophobic pocket within the active site of aromatase and to strongly inhibit human aromatase. This could help improve the specificity of the inhibitors. Equine aromatase, very well characterized biochemically, is used as a comparative model. Indeed, in a previous comparison between both human and equine aromatases, we described the importance of the interaction between the inhibitor and this pocket for the indane derivative MR 20814. MR 20492 and MR 20494 are more potent inhibitors of...
Cloning and expression of a 48-kilodalton Babesia caballi merozoite rhoptry protein and potential use of the recombinant antigen in an enzyme-linked immunosorbent assay. A cDNA expression library prepared from Babesia caballi merozoite mRNA was screened with a monoclonal antibody BC11D against the rhoptry protein of B. caballi merozoite. A cDNA encoding a 48-kDa protein of B. caballi was cloned and designated BC48. The complete nucleotide sequence of the BC48 gene had 1,828 bp and was shown to contain no intron. Southern blotting analysis indicated that the BC48 gene contained more than two copies in the B. caballi genome. Computer analysis suggested that this sequence contained an open reading frame of 1,374 bp with a coding capacity of approximately 52 kDa. ...
The molecular genetics of red and green color vision in mammals. To elucidate the molecular mechanisms of red-green color vision in mammals, we have cloned and sequenced the red and green opsin cDNAs of cat (Felis catus), horse (Equus caballus), gray squirrel (Sciurus carolinensis), white-tailed deer (Odocoileus virginianus), and guinea pig (Cavia porcellus). These opsins were expressed in COS1 cells and reconstituted with 11-cis-retinal. The purified visual pigments of the cat, horse, squirrel, deer, and guinea pig have lambdamax values at 553, 545, 532, 531, and 516 nm, respectively, which are precise to within +/-1 nm. We also regenerated the "true" red ...
Age-related changes to the molecular and cellular components of equine flexor tendons. Specific tendons show a high incidence of partial central core rupture which is preceded by degeneration. In the performance horse, the superficial digital flexor tendon (SDFT) is most often affected. We have described previously the molecular changes that are associated with degeneration in the central core region of the equine SDFT. The pathophysiological mechanism leading to change in synthetic activity of central zone cells in degenerated tendons is not known. In this study, we test the hypothesis that ageing results in matrix composition changes within the central zone of the SDFT. Extrac...
Modulation of cytokine response of pneumonic foals by virulent Rhodococcus equi. The ability of Rhodococcus equi to induce pneumonia in foals depends on the presence of an 85- to 90-kb plasmid. In this study, we evaluated whether plasmid-encoded products mediate virulence by modulating the cytokine response of foals. Foals infected intrabronchially with a virulence plasmid-containing strain of R. equi had similar gamma interferon (IFN-gamma) and interleukin-12 (IL-12) p35 but significantly higher IL-1beta, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-alpha) mRNA expression in lung tissue compared to foals infected with the plasmid-cured derivative. IFN-gamma mRNA...
The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase. The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine ...
Pyrimidine nucleotide-evoked inhibition of cyclic AMP accumulation in equine epithelial cells. Uridine triphosphate (UTP) evoked inhibition of adrenaline-evoked cAMP accumulation in cultured equine epithelial cells (EC50, 1.8 +/- 0.2 microM) and this effect was mimicked by 5-Br-UTP (EC50, 6.6 +/- 1.8 microM) and uridine diphosphate (UDP; EC50, 96 +/- 26 microM). This inhibitory action of UTP was abolished by pre-treating cells with pertussis toxin (10 ng ml-1, 24 h). UTP (EC50, 2.3 +/- 0.3 microM) and 5-Br-UTP (EC50, 29.4 +/- 9.4 microM) also increased intracellular free calcium ([Ca2+]i) whilst UDP did not; the two effects are thus differentially sensitive to these pyrimidine nucleotid...
Evolutionary history of MHC class I genes in the mammalian order Perissodactyla. We carried out an analysis of partial sequences from expressed major histocompatibility complex (MHC) class I genes isolated from a range of equid species and more distantly related members of the mammalian order Perissodactyla. Phylogenetic analysis revealed a minimum of six groups, five of which contained genes and alleles that are found in equid species and one group specific to the rhinoceros. Four of the groups contained only one, or very few sequences, indicating the presence of relatively nonpolymorphic loci, while another group contained the majority of the equid sequences identified. ...
Novel cathelicidins in horse leukocytes(1). Cathelicidins are precursors of defense peptides of the innate immunity and are widespread in mammals. Their structure comprises a conserved prepropiece and an antimicrobial domain that is structurally varied both intra- and inter-species. We investigated the complexity of the cathelicidin family in horse by a reverse transcription-PCR-based cloning strategy of myeloid mRNA and by Southern and Western analyses. Three novel cathelicidin sequences were deduced from bone marrow mRNA and designated equine cathelicidins eCATH-1, eCATH-2 and eCATH-3. Putative antimicrobial domains of 26, 27 and 40 r...
Latency-associated transcripts of equine herpesvirus type 4 in trigeminal ganglia of naturally infected horses. Equine herpesvirus type 4 (EHV-4) is a major respiratory pathogen of horses. Unlike most other members of the Alphaherpesvirinae, EHV-4 was regarded as non-neurotropic. Here, neural and lymphoid tissues of 17 horses have been analysed post-mortem. EHV-4 DNA was detected in 11 cases (65%) by PCR, exclusively in the trigeminal ganglia. In order to define the transcriptional activity, RNA preparations of 10 EHV-4 DNA-positive ganglia were investigated by nested RT-PCR. EHV-4-specific transcripts derived from genes 63 [herpes simplex virus type 1 (HSV-1) ICPO gene homologue] and 64 (HSV-1 ICP4 gen...
Dual regulation of promoter II- and promoter 1f-derived cytochrome P450 aromatase transcripts in equine granulosa cells during human chorionic gonadotropin-induced ovulation: a novel model for the study of aromatase promoter switching. Estradiol biosynthesis is a key biochemical trait of developing follicles. To study its regulation in equine follicles, the objectives of this study were to clone and determine the structure of equine cytochrome P450 aromatase (P450AROM), and characterize the regulation of P450AROM and P450 17alpha-hydroxylase/C17-20 lyase (P45017alpha) messenger RNAs (mRNAs) in vivo in equine preovulatory follicles isolated during hCG-induced ovulation. Two distinct P450AROM complementary DNAs (cDNAs) were isolated from an equine preovulatory follicle cDNA library. One clone was 2682 bp in length and included...
Matrix metalloproteinase-2 and -9 are activated in joint diseases. A study was performed to identify the activation status of the gelatinase MMPs, MMP-2 and -9, in both normal and diseased equine articular tissues. In addition, the production and activation status of equine MMP-2 and -9 by equine articular cells and tissues in response to increasing IL-1beta concentrations was assessed. The study was performed to test the hypothesis that activation of MMPs is a fundamental step in the pathogenesis of joint diseases; and that this activation is mediated by the cytokine IL-1. Using purified equine MMP-2 and -9, the molecular weights of the zymogen and activated...
Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers. A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus specie...
CD26 and adenosine deaminase interaction: its role in the fusion between horse membrane vesicles and spermatozoa. Membrane vesicles of horse seminal plasma present at their surface a highly specific serine-type protease, dipeptidyl peptidase IV/CD26, a surface antigen known to characterize human prostasomes. Horse sperm cells expressed at their surface A(1) adenosine receptors (A(1)AR) and ecto-adenosine deaminase (ecto-ADA), both detected by immunoblot analysis, whereas CD26 was visualized at the equatorial segment by immunofluorescence microscopy. In addition to CD26, horse membrane vesicles showed ecto-ADA. The fusion process between horse sperm cells and vesicles was evidenced by confocal microscopy, ...
Species-specific amplification by PCR of ribosomal DNA from some equine strongyles. The first and second internal transcribed spacer sequences of 28 morphologically-defined species of horse strongyle were characterized, and specific oligonucleotide primers were designed for some species based on the nucleotide differences. Utilizing these primers, a PCR approach was developed for the specific amplification of ribosomal DNA of Strongylus vulgaris, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicostephanus longibursatus or Cylicostephanus goldi. The method allowed the species-specific amplification of parasite DNA derived from faecal samples and/or copro-cultures, demonstra...
Molecular detection of Babesia equi and Babesia caballi in horse blood by PCR amplification of part of the 16S rRNA gene. Babesia equi and Babesia caballi are tick-borne haemoparasites that may cause babesiosis of Equidae. In southern Europe B. equi is enzootic and infections may occur asymptomatically and more frequently than those due to B. caballi. Complement fixation test (CFT) is the official serological test for the diagnosis of equine babesiosis, but it has low sensitivity during early and latent stages of the disease. With the aim of developing more sensitive and rapid direct diagnostic alternatives, PCR systems that amplified DNA targets of 664 or 659 bp regions of the 16S rRNA genes were designed and de...
Identification of Ruminococcus flavefaciens as the predominant cellulolytic bacterial species of the equine cecum. Detection and quantification of cellulolytic bacteria with oligonucleotide probes showed that Ruminococcus flavefaciens was the predominant species in the pony and donkey cecum. Fibrobacter succinogenes and Ruminococcus albus were present at low levels. Four isolates, morphologically resembling R. flavefaciens, differed from ruminal strains by their carbohydrate utilization and their end products of cellobiose fermentation.
A PCR based method for the identification of equine influenza virus from clinical samples. In this paper we describe the development of a nested RT-PCR assay for the rapid diagnosis and characterisation of influenza virus directly from clinical specimens. Viral RNA is extracted from nasal swabs by the guanidine thiocyanate extraction method, and subsequently reverse transcribed. The complementary DNA is then used as template in a nested PCR reaction. Primers designed for use in this assay are specific for three templates; (1) the nucleoprotein (NP) gene, (2) the haemagglutinin gene of the H7N7 equine influenza virus (A1), and (3) the haemagglutinin gene of the H3N8 equine influenza ...
Phylogenetic relationships of Cheju horses to other horse breeds as determined by mtDNA D-loop sequence polymorphism. Historical records suggest that horses inhabiting the island of Cheju in Korea are descendants of Mongolian horses introduced in 1276. Other studies, however, suggest that horses may have been present on the island prior to the Mongolian introduction. To determine the origin of the Cheju horses we used a phylogenetic analysis of sequences of the mitochondrial DNA (mtDNA) D-loop region, including tRNA Pro and parts of tRNA thr and tRNA Phe sequences (1102-bp excluding the tandem repeat region). Maximum parsimony and neighbor-joining trees were constructed using sequences determined for seven Ch...
Independent nucleation and heterogeneous assembly of structure during folding of equine lysozyme. The refolding of equine lysozyme from guanidinium chloride has been studied using hydrogen exchange pulse labelling in conjunction with NMR spectroscopy and stopped flow optical methods. The stopped flow optical experiments indicate that extensive hydrophobic collapse occurs rapidly after the initiation of refolding. Pulse labelling experiments monitoring nearly 50 sites within the protein have enabled the subsequent formation of native-like structure to be followed in considerable detail. They reveal that an intermediate having persistent structure within three of the four helices of the alph...
Three-dimensional structure of mare diferric lactoferrin at 2.6 A resolution. Lactoferrin is a monomeric glycoprotein with a molecular mass of approximately 80 kDa. The three-dimensional structure of mare diferric lactoferrin (mlf) has been determined at 2.6 A resolution. The protein crystallizes in the space group P 212121with a=85.2 A, b=99.5 A, c=103.1 A with a solvent content of 55 % (v/v). The structure was solved by the molecular replacement method using human diferric lactoferrin as the model. The structure has been refined using XPLOR to a final R -factor of 0.194 for all data in the 15-2.6 A resolution range. The amino acid sequence of mlf was determined using ...
Detection of equine herpesvirus types 2 and 5 (EHV-2 and EHV-5) in Przewalski’s wild horses. In blood samples of seven captive equid species from four German zoos EHV-1 specific antibodies were detected in 76% and EHV-4 specific antibodies in 73% of the 55 animals, whereas 93% were tested positive for EHV-2 and EHV-5, respectively. In only one blood sample from a Przewalski's wild horse EHV-4 DNA was amplified by PCR. From seven Przewalski's wild horses EHV-2, and from another one EHV-5 was isolated by cocultivation. The identity of the virus isolates was verified by PCR and restriction enzyme digestion.
Oxytocin-neurophysin I mRNA abundance in equine uterine endometrium. A positive-feedback loop between luteal oxytocin and uterine prostaglandin F2 alpha (PGF) is a major signal for luteolysis in ruminants. Likewise, uterine PGF causes luteolysis in mares, but the involvement of oxytocin in this process is unclear. We wanted: 1) to determine if the oxytocin-neurophysin I (OT-NP I) gene is transcribed into mRNA in the endometrium of mares; and, if so, 2) to analyze relative changes in abundance of endometrial OT-NP I mRNA throughout the estrous cycle and during early stages of pregnancy. Endometrial biopsies were obtained from nonbred mares during estrus, and 5, ...
