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 characterisation of equine interleukin-1. Equine interleukin-1 has been produced from peripheral blood monocytes by stimulation with E. coli lipopolysaccharide. Sephacryl S200 gel filtration revealed a molecular weight of 17-18 kD. Chromatofocusing of the 17-18 kD peak identified four active fractions. Two major peaks were detected at pH 6.7 and pH 7, with smaller peaks at pH 6.3 and pH 5.9. The pI 7 molecule is probably the equine form of IL-1 beta.
An improved method for the study of equine haptoglobin heterogeneity. Equine serum haptoglobin was separated by polyacrylamide gel isoelectric focusing and visualized by protein staining or Western blotting. Conventional protein staining revealed up to three bands in the pI range 4.17 to 4.44. The blotting technique, however, showed an anodal group of 8 to 10 bands with a pI range of 4.11 to 4.52 and a cathodal group of 4 to 6 bands with a range of 4.55 to 5.14. The blotting method revealed that equine haptoglobin migrates outside the prealbumin area, in contrast to previous reports.
Conformational comparison in the growth hormone family. 1. The method of Kubota et al. [Biochim. biophys. Acta 701, 242-252 (1982)] was applied to several members of the growth hormone family in order to examine their conformational homology. 2. The method neither detects differences between rat, cow, sheep, horse and alpaca hormones, nor between monkey and human hormones. 3. Lack of homology between primate and non-primate growth hormones was found in segments 42-49 and 184-191. The first fragment could be linked to species-specificity.
Molecular forms of gastrin in antral mucosa of the horse. The predominant form of gastrin in the antral mucosa of the stomach of virtually all species previously examined is the 17 amino acid peptide little gastrin (G17). This report describes the occurrence in equine antral mucosa of an immunoreactive form of gastrin with elution properties on Sephadex G-50 superfine similar to human unsulfated big gastrin (G34-I). This putative equine big gastrin was a major component of the gastrin immunoreactivity present. A second peak of activity in equine antral mucosa eluted in an identical manner to human little gastrin (hG17-I). Inhibition curves of equine ...
Equus przewalskii plasma protease inhibitor (Pi) system. A detailed biochemical characterization of four of the five previously described alleles of the plasma protease inhibitor (Pi) system of Equus przewalskii was performed using both one- and two-dimensional electrophoretic techniques. The proteins have been characterized in terms of isoelectric point, relative molecular mass, inhibitory activity to bovine trypsin and chymotrypsin, immunochemical cross-reactivity, terminal sialic acid content and enzyme:inhibitor complex formation and the oxidation sensitivity of this interaction. Using these functional criteria, only three loci (Spi 1, 2 and 3) ...
Comparison of heparan sulfate proteoglycans from equine and human glomerular basement membranes. 1. Proteoglycans extracted from human and equine glomerular basement membranes (GBM) were purified by ion-exchange chromatography and gel filtration. 2. The glycoconjugates had an apparent molecular mass of 200-400 kDa and consisted of 75% protein and 25% glycosaminoglycan. Glycosidase and HNO2 treatment and the amino sugar and sulfate composition of both proteoglycan preparations identified heparan sulfate (HS) as the predominant saccharide chain. 3. Hydrolysis with trifluoromethanesulfonic acid yielded comparable core proteins with molecular masses of ca 160 and 120 kDa. 4. The HS chains had...
The carbohydrate side chains of the major plasma serpins of horse and wallaby: analyses of enzymatic and chemically treated (including ‘Smith degradation’) protein blots by lectin binding. The carbohydrate side chains of the major plasma serpins of the horse and wallaby have been characterized by lectin analyses of protein blots from two-dimensional gels using the major human plasma serpin, alpha 1-protease inhibitor, as a control. Eight lectins were used in the characterization in conjunction with enzymatic deglycosylation of complex and high mannose side chains, chemical desialylation and defucosylation, and one round of 'Smith degradation', all being performed on the nitrocellulose blots. Assuming a standard complex side chain structure, the results of the 21 lectin/treatment...
Functional and morphological stasis during molecular evolution. The evolutionary distance between two sets of proteins was estimated using the techniques of Miyata and Yasunaga (1980) and Kimura (1980). Human beta 2-microglobulin was compared with the homologous murine molecule, while human and equine alpha-globin were similarly treated. It was found that a large amount of molecular evolution has occurred in beta 2-microglobulin since its divergence from the common ancestor of mice and humans. Kimura's estimate of evolutionary distance, K, is 0.353, while those of Miyata and Yasunaga are KS = 0.708 and KA = 0.171. The respective values for human and equine...
High-performance liquid chromatography/tandem mass spectrometry: its use for the identification of stanozolol and its major metabolites in human and equine urine. A screening procedure for the anabolic steroid stanozolol in human and equine urine was developed based on enzymatic hydrolysis, liquid-liquid extraction and reversed-phase liquid chromatography combined on-line with tandem mass spectrometry. The column effluent was introduced into the atmospheric pressure ionization source of a triple-quadrupole mass spectrometer via a heated pneumatic nebulizer liquid chromatograph/mass spectrometer interface. Abundant protonated molecular ions were generated by corona discharge ionization. Confirmation of stanozolol and several of its hydroxylated and dihyd...
Structure and expression of the equine infectious anemia virus transcriptional trans-activator (tat). Equine infectious anemia virus (EIAV) encodes a tat gene which is closely related to the trans-activators encoded by the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat message is composed of three exons; the first two encode tat and the third may encode rev.. Interestingly, EIAV tat translation is initiated at a non-AUG codon in the first exon of the message, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, ...
Rapid determination of methandrostenolone in equine urine by isotope dilution liquid chromatography-tandem mass spectrometry. Urine samples were spiked with [17-methyl-2H3]methandrostenolone as internal standard and extracted with a mixture of dichloromethane and cyclohexane. The organic phase was concentrated and injected onto a short octyl-silica column (30 mm x 4.6 mm I.D.) for separation of methandrostenolone and 17-epimethandrostenolone. The effluent from the column was connected to a Sciex TAGA 6000E triple quadrupole mass spectrometer equipped with an atmospheric pressure ion source for sampling of ions generated by a heated pneumatic nebulizer with corona discharge ionization. This ion source produced abundan...
Primary structure of horse serotransferrin glycans. Demonstration that heterogeneity is related to the number of glycans and to the presence of N-acetylneuraminic acid and N-acetyl-4-O-acetylneuraminic acid. Three serotransferrin variants Tf 2a, Tf 4b and Tf 5b were isolated in an homogeneous form from a preparation of homozygous horse serotransferrin Tf 0. On the basis of the results concerning molecular mass determination and the carbohydrate analysis, it is concluded that the serotransferrin variant Tf 2a contains only one glycan while variants Tf 4b and Tf 5b contain two glycans. The structure of all of the glycans has been established by combining methylation analysis, mass spectrometry and 400-MHz 1H-NMR spectroscopy. From the obtained results, it appears that the two glycans of Tf 5b varian...
Transbilayer movement of phosphatidylserine in nonhuman erythrocytes: evidence that the aminophospholipid transporter is a ubiquitous membrane protein. A 31-32-kDa integral membrane protein has been previously identified in erythrocytes as the protein most likely to be responsible for the transbilayer movement of phosphatidylserine (PS) [Connor & Schroit (1988) Biochemistry 27, 848-851]. Using similar techniques, we have identified analogous proteins of identical molecular weights in bovine, equine, ovine, porcine, canine, caprine, and rhesus red blood cells. Similar to human red blood cells, all of the mammalian cells were able to specifically transport an exogenously supplied fluorescent PS analogue from their outer-to-inner membrane le...
Concentration and degree of polymerization of hyaluronate in equine synovial fluid. In addition to its well-known physicochemical properties, hyaluronate (HA) has recently been shown to have important biological and pathophysiologic regulatory effects on granulocytes, monocytes, fibroblasts, and endothelial cells, as well as on the healing of wounds and various joint disorders. Many of these effects depend on or are reflected in the concentration and degree of polymerization of HA. Therefore, high-performance liquid chromatography with size-exclusion column was used to characterize the concentration and degree of polymerization of HA in equine synovial fluid (SF). The mean (+...
Histochemical and molecular determination of fiber types in chemically skinned single equine skeletal muscle fibers. Until now, there has been no reliable method for histochemical determination of fiber types of single skinned muscle fibers. The major problem arises from the fact that most histochemical techniques use cross-sections of a large group of fibers and compare a given fiber with those surrounding it. This is not possible with a single skinned fiber which has been separated from a bundle to be used for mechanical analysis. A further problem is that the skinning procedure itself may alter the staining pattern. We have developed a procedure by which multiple cross-sections of single skinned fibers ca...
Androgen and 19-norandrogen aromatization by equine and human placental microsomes. The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone oc...
Comparison of the binding of Ca2+ and Mn2+ to bovine alpha-lactalbumin and equine lysozyme. The enthalpy change of the binding of Ca2+ and Mn2+ to equine lysozyme was measured at 25 degrees C and pH 7.5 by batch microcalorimetry: delta H degrees Ca2+ = -76 +/- 5 kJ mol-1, delta H degrees Mn2+ = -21 +/- 10 kJ mol-1. Binding constants, log KCa2+ = 6.5 +/- 0.2 and log KMn2+ = 4.1 +/- 0.5, were calculated from the calorimetric data. Therefore, delta S degrees Ca2+ = -131 +/- 20 JK-1 mol-1 and delta S degrees Mn2+ = 8 +/- 44 JK-1 mol-1. Removal of Ca2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformatio...
Molecular confirmation of an abortigenic strain of equine herpesvirus 1 (subtype 1) in a pregnant mare study. Four pregnant mares were inoculated intranasally and/or intravenously with equine herpesvirus 1 (EHV-1), subtype 1 during the third trimester of gestation. One mare aborted on postinfection day 15, one mare delivered a sick, weak full term foal, and two mares delivered healthy, full term foals. EHV-1, subtype 1 was isolated from several tissues of the aborted fetus and from the thymus of the sick foal. DNA restriction endonuclease patterns of the recovered EHV-1 viruses were identical to those of the EHV-1 challenge strain, documenting the origin of the abortigenic viruses.
Difference in content ratio of components among horse serum transferrin variants. Each of five genetic variants of horse serum transferrin (Tf), D, F, H, O, and R, was separated into two bands by polyacrylamide gel isoelectric focusing (PAGIEF). The more acidic band, termed component a, was more abundant than the other one, termed component b, in all variants. Components a and b of TFO variant were immunologically indistinguishable from each other by double immunodiffusion test. Determination of the content ratio of component a to component b in each variant revealed that the variants were classified into two groups: one group (D, F, and H) had a relatively high ratio withi...
Isolation and characterization of equine microvascular endothelial cells in vitro. The use of cultured tissue has not yet become widespread in research involving equine disease, and this may be attributable in part to the scarcity of published reports concerning tissue culture methods for this species. We report here the isolation of equine microvascular endothelium (EMVE) from fresh omental tissue of horses and ponies. Fresh donor tissue was minced, subjected to collagenase digestion, and filtered. Cells were layered on 5% bovine serum albumin for gravity sedimentation, the bottom layer was collected, and the cells were plated onto fibronectin-coated flasks. Medium consiste...
Regulation of ovarian function by catecholestrogens: current concepts. Development of the ovarian follicle(s) destined for ovulation appears to be a process in which antral follicles undergo a recruitment, selection and subsequent dominance phase. Several intraovarian or autocrine/paracrine regulatory mechanisms have been evoked to explain these processes. One of these potential autocrine/paracrine regulators is a catecholestrogen, 2-hydroxy-estradiol (2-OH-E2). Evidence implicating 2-OH-E2 as an autocrine/paracrine regulator of follicular function is reviewed. Studies have shown 2-OH-E2 to be present in nanomolar concentrations in fluid of human and equine folli...
Comparison of the use of mass spectrometry and methylene unit values in the determination of the stereochemistry of estranediol, the major urinary metabolite of 19-nortestosterone in the horse. The stereochemistry of an isomer of 5-estrane-3,17 alpha-diol, the major metabolite of 19-nortestosterone in horse urine has been established by the use of methylene unit (MU) values. The empirical MU values of the bis-trimethylsilyl (TMS) derivatives of the eight available isomers of 5-androstane-3,17-diol and four isomers of 5-estrane-3,17 beta-diol were determined by capillary gas chromatography using three different columns. From this data the theoretical MU values for the bis-TMS derivatives of the four 5-estrane-3,17 alpha-diol isomers were predicted. Comparison of the experimentally det...
Electron microscopy of gold-labeled human and equine chromosomes. We present an immunochemical technique for the detection of 5-bromo-2'-deoxyuridine (BrdU) incorporated discontinuously into the chromosomal DNA. A monoclonal anti-BrdU antibody and a protein A-gold complex were used to produce chromosome banding of human and equine chromosomes, specific for electron microscopy (EM). Well-defined bands, symmetry of sister chromatids, concordance between homologues, and band patterns similar to those observed by light microscopy facilitate chromosome identification and karyotyping. From prophase to late metaphase, chromosomes condense and bands appear to fuse. ...
DNA sequence and comparative analyses of the equine herpesvirus type 1 immediate early gene. The immediate early (IE) proteins of herpesviruses are important regulatory factors which control the expression of genes at the transcriptional level. We report the DNA sequence of the immediate early gene of the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This sequence is shown to be extremely rich in guanine and cytosine, resulting in a highly biased codon usage. The IE gene region possesses 38 open reading frames (ORFs) greater than 300 bp in length, 11 of which have coding regions of at least 100 amino acids (aa) following potential translation initiator codons. The largest ORF co...
DNA restriction analysis of equine adenovirus serotype I. The three equine adenovirus strains isolated in different locations showed a similar cleavage pattern with HindIII and the DNA homology among the strains was confirmed by Southern blot hybridization. The three strains revealed differences in cleavage patterns with BamHI, EcoRI and PstI, suggesting the presence of DNA polymorphisms among equine adenoviruses.
Genetic heterogeneity within Echinococcus granulosus: isolates from different hosts and geographical areas characterized with DNA probes. A segment of the ribosomal RNA gene of Schistosoma mansoni and a DNA fragment specific to Echinococcus granulosus, cloned in plasmids, have been used as DNA probes to assess the extent of genetic variability within E. granulosus and some distinct strains have been identified. The DNA analysis, involving restriction endonuclease digestion and Southern blot hybridization with the probes, did not demonstrate any significant genetic variation within the U.K. horse/dog or sheep/dog strains but confirmed the distinctiveness of the two strains shown in previous studies. The sheep/dog strain was shown...
Identification of carbohydrate-binding proteins in mammalian spermatozoa (human, bull, boar, ram, stallion and hamster) using [125I]fucoidin and [125I]neoglycoprotein probes. A protein-carbohydrate recognition system is thought to be involved in the early stages of fertilization in mammals. In this investigation carbohydrate-binding proteins have been identified in extracts of human, bull, boar, ram, stallion and hamster spermatozoa using [125I]fucoidin and [125I]neoglycoproteins (BSA-fucose and BSA-mannose) to probe Western blots. Results show that proacrosin is the major protein species recognized in extracts of human, bull, boar and ram spermatozoa. In hamster and stallion spermatozoa, carbohydrate-binding activity was associated with several low molecular weigh...
The primary structure of equine serum amyloid A (SAA) protein. The complete amino acid sequence of equine serum amyloid A (SAA) was elucidated. The protein consists of 110 amino acid residues and contains an 8-amino acid residue insertion tentatively located between positions 69 and 70, as compared with human SAA. Microheterogeneities were detected at positions 16, 44, and 59, compatible with the existence of more than one SAA gene in the horse. This corresponds to the situation in man and mouse. Pronounced homology with SAA from man and several animal species was observed, thus confirming the conserved structure of this acute phase reactant and apoprotei...
Mössbauer spectroscopic study of the initial stages of iron-core formation in horse spleen apoferritin: evidence for both isolated Fe(III) atoms and oxo-bridged Fe(III) dimers as early intermediates. Ferritin stores iron within a hollow protein shell as a polynuclear Fe(III) hydrous oxide core. Although iron uptake into ferritin has been studied previously, the early stages in the creation of the core need to be clarified. These are dealt with in this paper by using Mössbauer spectroscopy, a technique that enables several types of Fe(II) and Fe(III) to be distinguished. Systematic Mössbauer studies were performed on samples prepared by adding 57Fe(II) atoms to apoferritin as a function of pH (5.6-7.0), n [the number of Fe/molecule (4-480)], and tf (the time the samples were held at room ...