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Topic:Biochemistry
The study of biochemistry in horses encompasses the chemical processes and substances that occur within equine organisms. This field investigates the molecular interactions and pathways that are fundamental to horse physiology, including metabolism, enzyme activity, and genetic expression. Key areas of interest include the examination of metabolic disorders, nutrient absorption, and the biochemical basis of muscle function and energy production. Researchers utilize biochemical analysis to understand health and disease mechanisms in horses, contributing to the development of diagnostic tools and therapeutic strategies. This page gathers peer-reviewed studies and scholarly articles that explore various biochemical processes and their implications for equine health and performance.
High expression in adult horse of PLRP2 displaying a low phospholipase activity. The physiological role of the two lipase-related proteins, PLRP1 and PLRP2, still remains obscure although some propositions have been made concerning PLRP2. In this paper, we report the presence of high amounts of PLRP2 in adult horse pancreas whereas no PLRP1 could be detected. As well, a non-parallel expression of PLRP2 and PLRP1 is observed in adult cat and dog, since no PLRP2 could be detected in these two species. In adult ox, neither PLRP2 nor PLRP1 could be found. These findings are in favor of a different regulation of the expression of the genes encoding pancreatic lipase and the rel...
Oxidative monensin metabolism and cytochrome P450 3A content and functions in liver microsomes from horses, pigs, broiler chicks, cattle and rats. The oxidative metabolism of monensin, an ionophore antibiotic extensively used in veterinary practice as a coccidiostat and a growth promoter, was studied in hepatic microsomal preparations from horses, pigs, broiler chicks, cattle and rats. As assayed by the measurement of the amount of the released formaldehyde, the rate of monensin O-demethylation was nearly of the same order of magnitude in all species, but total monensin metabolism, which was estimated by measuring the rate of substrate disappearance by a high-performance liquid chromatography (HPLC) method, was highest in cattle, interme...
Cultivation and characterisation of primary and subcultured equine keratinocytes. We describe the establishment and characterisation of equine keratinocyte cultures with maintenance of a high proliferative capacity up to the second passage. Improved attachment and growth were obtained by seeding primary cells on equine feeder layers. Subcultured keratinocytes showed optimal growth when seeded on collagen type I. The proliferation rate of cells on this substrate exceeded that seen for cells seeded on equine feeder layers. By immunohistochemistry, epithelial origin and state of differentiation of the equine keratinocytes were determined. They expressed keratin and desmoplakin...
Growth hormone response to a novel growth hormone-releasing tripeptide in horses: interaction with gonadotropin-releasing hormone, thyrotropin-releasing hormone, and sulpiride. A series of experiments was performed to determine the factor(s) responsible for an apparent inhibition of GH secretion in mares administered the GH secretagogue EP51389 in combination with GnRH, thyrotropin-releasing hormone (TRH), and sulpiride. Experiment 1 tested the repeatability of the original observation: 10 mares received EP51389 at 10 microg/kg BW; five received TRH (10 microg/kg BW), GnRH (1 microg/kg BW), and sulpiride (100 microg/kg BW) immediately before EP51389, and five received saline. The mixture of TRH, GnRH, and sulpiride reduced (P = 0.0034) the GH response to EP51389, con...
A cytochrome c mutant with high electron transfer and antioxidant activities but devoid of apoptogenic effect. A cytochrome c mutant lacking apoptogenic function but competent in electron transfer and antioxidant activities has been constructed. To this end, mutant species of horse and yeast cytochromes c with substitutions in the N-terminal alpha-helix or position 72 were obtained. It was found that yeast cytochrome c was much less effective than the horse protein in activating respiration of rat liver mitoplasts deficient in endogenous cytochrome c as well as in inhibition of H(2)O(2) production by the initial segment of the respiratory chain of intact rat heart mitochondria. The major role in the di...
Characterization of the H- and L-subunit ratios of ferritins by sodium dodecyl sulfate-capillary gel electrophoresis. Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was used to characterize the H- and L-subunit ratios of several mammalian ferritins and one bacterioferritin. Traditionally, SDS-PAGE has been used to characterize the H- and L-subunit ratios in ferritin; however, this technique is relatively slow and requires staining, destaining, and scanning before the data can be processed. In addition, the H- and L-subunits of ferritin are fairly close in molecular weight (approximately 21,000 and approximately 20,000, respectively) and are often difficult to resolve in SDS-PAGE slab gels. In ...
Surface immunolocalisation of HPr in the equine pathogen Streptococcus equi. We have investigated the surface localisation of the phosphotransferase system protein HPr in the equine pathogen Streptococcus equi subsp. equi using immunogold localisation and transmission electron microscopy. Like the LppC acid phosphatase lipoprotein, a reference surface antigen, the S. equi HPR could be clearly detected on the surfaces of intact cells. This study is consistent with previous reports that some streptococcal HPr is cell surface associated and suggests that the extracytoplasmic mobilisation and transfer of phosphate groups by streptococci warrant further investigation.
Detection of lipid peroxidation in equine spermatozoa based upon the lipophilic fluorescent dye C1l-BODIPY581/591. The lipophilic fluorescent probe, 4,4-difluoro-5-(4-phenyl-1 ,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591) was used to evaluate changes in lipid peroxidation in equine spermatozoa during both short-term exposure to ferrous sulfate and sodium ascorbate in the presence of cumene hydroperoxide as well as during storage of spermatozoa at 5 degrees C for 48 hours. Peroxidation of C11-BODIPY581/591 was accompanied by a shift in fluorescence from red to green, and the relative amount of nonoxidized probe was determined as the ratio of red:(red + green) fluorescenc...
Detection of fenspiride and identification of in vivo metabolites in horse body fluids by capillary gas chromatography-mass spectrometry: administration, biotransformation and urinary excretion after a single oral dose. Studies related to the in vivo biotransforrmation and urinary excretion of fenspiride hydrochloride in the horse are described. After oral administration, the drug is metabolised by both phase I functionalisation and phase II conjugation pathways. Following enzymatic deconjugation, fenspiride and its phase I metabolites were isolated from post-administration biofluids using bonded co-polymeric mixed mode solid-phase extraction cartridges to isolate the basic compounds. Following trimethylsilylation (TMS), the parent drug and metabolites were identified by capillary gas chromatography-mass spec...
Prostaglandin E(2) and F(2 alpha) production by equine conceptuses and concentrations in conceptus fluids and uterine flushings recovered from early pregnant and dioestrous mares. A growing equine conceptus must suppress the cyclical release of PGF(2 alpha) from the endometrium to effect maternal recognition of its presence in the uterus. Paradoxically, the conceptus itself secretes PGF(2 alpha), together with other prostaglandins. In this study, the PGF(2 alpha) and PGE(2) content of, and production in vitro by, day 10-32 equine conceptuses were measured and the influence of pregnancy on the concentrations of these prostaglandins in the uterine lumen was examined. In vitro, the release of both prostaglandins per mg conceptus tissue was very high on day 10 after ovulati...
Equine infectious anemia virus and the ubiquitin-proteasome system. Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9(Gag) proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecip...
Prostaglandin E2 and reactive oxygen metabolite damage in the cecum in a pony model of acute colitis. The objective of this project was to determine early tissue biochemical events associated with increased colonic secretion during the acute stage of castor-oil-induced colitis by measuring cecal mucosal and submucosal malondialdehyde (MDA) and prostaglandin E2 (PGE2), levels in ponies. Intestinal tissue (inflamed or healthy) samples were obtained from 4 age- and sex-matched Shetland ponies. Biochemical methods were used to determine MDA and PGE2 levels in intestinal tissue samples from inflamed and healthy equine intestine. Inflamed tissue MDA and PGE2 levels increased with time after castor o...
Purification and quantification of lactoferrin in equine seminal plasma. Lactoferrin with a molecular mass of 80 kDa was purified from equine seminal plasma by heparin-Agarose affinity chromatography and Sephacryl S-200 gel filtration. Purified lactoferrin was found to be highly homogeneous on the bases of its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of the monospecificity of rabbit antibodies to the purified protein in immunoblotting of seminal plasma proteins. A sandwich enzyme-linked immunosorbent assay was developed for quantifying lactoferrin in equine seminal plasma. Seminal plasma lactoferrin concentrations ...
Effect of dietary medium chain triacylglycerols on plasma triacylglycerol levels in horses. The hypothesis tested was that the feeding of medium chain triacylglycerols (MCT) to horses would raise the level of plasma triacylglycerols by increasing the availability of glucose as lipogenic substrate, implying that the MCT effect would be greater with glucose in the diet instead of cellulose. A Latin square experiment was carried out with 4 horses and 4 dietary treatments. The experimental periods lasted 21 d. Blood samples were taken 16 h after feeding. The diets consisted of hay and experimental concentrates, differing in fat source (MCT or soybean oil) and carbohydrate source (corn st...
Phenotypic expression of equine articular chondrocytes grown in three-dimensional cultures supplemented with supraphysiologic concentrations of insulin-like growth factor-1. To assess the effects of supraphysiologic concentrations of insulin-like growth factor-1 (IGF-1) on morphologic and phenotypic responses of chondrocytes. Methods: Articular cartilage obtained from 2 young horses. Methods: Chondrocytes were suspended in fibrin cultures and supplemented with 25, 12.5, or 0 mg of IGF-1/ml of fibrin. Chondrocyte morphology and phenotypic expression were assessed histologically, using H&E and Alcian blue stains, immunoreaction to collagen type I and II, and in situ hybridization. Proteoglycan content, synthesis, and monomer size were analyzed. The DNA content w...
Changes in follicular fluid steroids, insulin-like growth factors (IGF) and IGF-binding protein concentration, and proteolytic activity during equine follicular development. The objective of the present study was to evaluate changes in equine follicular fluid insulin-like growth factor binding protein (IGFBP) proteolytic activity as well as steroid, IGF, and IGFBP concentrations during follicular development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6 to 15 mm; n = 54), medium (16 to 25 mm; n = 23), or large (> 25 mm; n = 15), and follicular fluid was collected. Estradiol and androstenedione levels in follicular fluid were greater (P < 0.05), an...
Urea as a measure of dilution of equine synovial fluid. This paper tests the hypothesis that serum and synovial urea concentrations are similar and that urea concentration can be used as an accurate marker for synovial fluid dilution in normal equine joints. Serum and synovial fluid urea concentrations were compared in 42 horses and were equivalent for individual horses (P<0.0001). Mean +/- s.e. serum concentration was 6.1+/-0.552 mmol/l and synovial concentration 6.0+/-0.459 mmol/l. The normal range for synovial urea concentration was determined as 2.5-7.7 mmol/l. The synovial urea concentration from different synovial structures in individual hor...
Metabolism of methandrostenolone in the horse: a gas chromatographic-mass spectrometric investigation of phase I and phase II metabolism. The phase I and phase II metabolism of the anabolic steroid methandrostenolone was investigated following oral administration to a standardbred gelding. In the phase I study, metabolites were isolated from the urine by solid-phase extraction, deconjugated by acid catalysed methanolysis and converted to their O-methyloxime trimethylsilyl derivatives. GC-MS analysis indicated the major metabolic processes to be sequential reduction of the A-ring and hydroxylation at C6 and C16. In the phase II study, unconjugated, beta-glucuronidated and sulfated metabolites were fractionated and deconjugated us...
Glycogen branching enzyme deficiency in quarter horse foals. Seven related Quarter Horse foals that died by 7 weeks of age were examined for glycogen branching enzyme (GBE) deficiency. Clinical signs varied from stillbirth, transient flexural limb deformities, seizures, and respiratory or cardiac failure to persistent recumbency. Leukopenia (5 of 5 foals) as well as high serum creatine kinase (CK; 5 of 5), aspartate transaminase (AST; 4 of 4), and gamma glutamyl transferase (GGT; 5 of 5) activities were present in most foals, and intermittent hypoglycemia was present in 2 foals. Gross postmortem lesions were minor, except for pulmonary edema in 2 foals....
Thermolysin activates equine lamellar hoof matrix metalloproteinases. Cultured equine lamellar hoof explants secrete the pro-enzymes matrix metalloproteinase-2 (MMP-2, 72 kDa) and MMP-2 (92 kDa). Untreated explants remained intact when tested on a calibrated force transducer, but when treated with an MMP activator, developed "in-vitro laminitis", separating at the dermal-epidermal junction. Explants treated with the bacterial protease thermolysin separated dose-dependently; this was accompanied by activation of both MMP-2 and -9. Thermolysin-mediated MP activation did not occur in a cell-free system and was not inhibited by the addition of the MMP inhibitor and ...
Mammalian lipocalin-type prostaglandin D2 synthase in the fluids of the male genital tract: putative biochemical and physiological functions. Prostaglandin D2 synthase (PGDS) is a major epididymal secretory protein in several species. We quantified PGDS in ram and bull semen using a specific antiserum. Strong variations in PGDS concentration existed between animals. In the bull, the highest concentrations were found preferentially in animals with normal or high fertility, as was previously suggested. However, low concentrations were found in males with all ranges of fertility, suggesting that the function of PGDS either is not necessary for male fertility or can be assumed by other proteins when its concentration is low. In the ram ...
Rapid intrachain binding of histidine-26 and histidine-33 to heme in unfolded ferrocytochrome C. Time-resolved spectroscopic studies of unfolded horse iron(II) cytochrome c have suggested that the imidazole side chains of His26 and His33 bind transiently to the heme iron on microsecond time scales, after photodissociation of a carbon monoxide ligand from the heme. Our studies of four variants of cytochrome c (horse wild type, horse H33N, horse H33N/H26Q, and tuna wild type), unfolded in guanidine hydrochloride at pH 6.5, demonstrate that these side chains are responsible for the observed microsecond spectral changes. As His33 and then His26 are eliminated from the horse wild-type sequence...
Structure of equine infectious anemia virus matrix protein. The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric...
IGF -I plasma concentrations in non-treated horses and horses administered with methionyl equine somatotropin. Insulin-like growth factor-I (IGF -I) is likely to be an indicator of somatotropin (ST) administration in the horse. To investigate the different ways ST administration may be detected, the following aspects of IGF -I concentrations in plasma were studied: (i) the daily variation; (ii) variation following a treadmill test; (iii) concentrations at rest and after exercise; and (iv) concentrations in plasma from two young horses and two adults treated with methionyl equine somatotropin (e ST). In the population of horses at rest, IGF -I mean concentration (SEM) was 261 (104) ng ml(-1). In post ra...
Synovial fluid studies in navicular disease. The purpose of this study was to investigate biochemical changes in synovial fluid in navicular disease, and to establish if synovial fluid from the distal interphalangeal joint (DIP) could be used diagnostically to assess alterations in the synovial fluid of the navicular bursa. Cartilage oligomeric matrix protein (COMP), total glycosaminoglycans (GAG), hyaluronan (HA), metalloproteinases 2 and -9 (MMP-2 and MMP-9) and total protein (TP) levels were determined in synovial fluids obtained from 18 navicular bursae and 35 DIP -joints from animals suffering from navicular disease, and the same sy...
Identification and characterization of Streptococcus agalactiae isolated from horses. Seven group B streptococcal cultures isolated from three horses reacted with group B-specific antiserum, were CAMP positive, pigmented and showed the typical biochemical properties of Streptococcus agalactiae. The identification could be confirmed by PCR amplification of the 16S rRNA gene and a subsequent RsaI restriction pattern typical for S. agalactiae. In addition, the isolates were identified by amplification of species specific parts of the 16S rRNA gene, the 16S-23S rRNA intergenic spacer region and by amplification of the CAMP-factor (cfb) gene. Six isolates could be classified as sero...
In vitro microelectrode study of the electrical properties of smooth muscle in equine ileum. Intracellular microelectrode recordings were made from smooth muscle cells in cross-sectional preparations of equine ileum, superfused in vitro. Membrane potential oscillations and spike potentials were recorded in all preparations, but recordings were made more readily from cells in the longitudinal muscle layer than from cells in the circular layer. The mean (se) resting membrane potential (RMP) of smooth muscle cells in the longitudinal muscle layer was -51.9 (1.2) mV, and the membrane potential oscillations in this layer had a mean amplitude of 4.8 (0.4) mV, a frequency of 9.0 (0.1) cycles...
[Eight polymorphic blood protein systems in Arab horses from Turkey]. Analysis of the blood protein system was used to study the genetic composition of Arabian horses. Biochemical markers of eight polymorphic loci (Tf, Al, Es, AlB, Gc, Hb, PGD, and PGM) were electrophoretically identified in blood samples. A total of 43 phenotypes were identified for these polymorphic systems. The Tf, Hb, and Es loci appeared to be more polymorphic than the other loci studied. Statistically significant differences between the observed and expected genotypic frequencies were found for the PGD and PGM loci (P < 0.05 and P < 0.01, respectively). Individual allele frequencies,...
The C-terminal portion of the fibrinogen-binding protein of Streptococcus equi subsp. equi contains extensive alpha-helical coiled-coil structure and contributes to thermal stability. The major cell wall-associated protein of the equine pathogen Streptococcus equi subsp. equi is a fibrinogen-binding protein (FgBP) which binds horse fibrinogen and equine IgG-Fc avidly through residues located in the N-terminal half and central regions of the molecule, respectively. The molecule is a major virulence factor for the organism and displays protective potential. In the present study, we use circular dichroism spectroscopy to investigate the secondary structure of the protein and show through the analysis of a panel of recombinant FgBP truncates that the C-terminal portion of FgBP ...
Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems. 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...
