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Topic:Enzymes
Enzymes are biological catalysts that facilitate biochemical reactions in horses by lowering the activation energy required for these processes. They are involved in various physiological functions, including digestion, metabolism, and cellular repair. Common enzymes in equine biology include amylase, lipase, and lactate dehydrogenase, each playing a specific role in the breakdown of nutrients and energy production. The activity and concentration of these enzymes can vary in response to different physiological and pathological conditions, serving as potential indicators in veterinary diagnostics. This page compiles peer-reviewed research studies and scholarly articles that explore the function, regulation, and clinical implications of enzymes in equine health.
Laminar xanthine oxidase, superoxide dismutase and catalase activities in the prodromal stage of black-walnut induced equine laminitis. REASONS FOR STUDY: Xanthine oxidase (XO)-dependent production of superoxide anion and hydrogen peroxide, a characteristic of ischaemia-reperfusion injury, may contribute to the development of equine laminitis. Objective: To determine the levels of XO and antioxidant enzymes (catalase, superoxide dismutase [SOD]) in the digital laminae of normal horses (CON) and horses in the developmental stage of laminitis using the black walnut extract (BWE) model. Methods: Healthy horses (n = 12) were administered BWE (BWE group, n = 6), or water (CON group, n = 6) through a nasogastric tube. At the onset o...
Effect of heat on synthesis of gelatinases and pro-inflammatory cytokines in equine tendinocytes. The aim of this study was to clarify whether matrix metalloproteinases (MMP-2 and -9: gelatinases) and pro-inflammatory cytokines [tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta] are induced by heat in tendon tissue in vitro and to test the hypothesis that heat exposure causes tendinocytes to synthesize pro-inflammatory cytokines and that synthesis of these cytokines, in turn, leads to up-regulation of synthesis of gelatinases. Isolated tendinocytes from equine superficial digital flexor tendons were cultured and all experiments were performed on cells passaged 3 or 4 times. In t...
Proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7. The positive-stranded RNA genome of the arterivirus Equine arteritis virus (order Nidovirales) encodes the partially overlapping replicase polyproteins pp1a (1727 aa) and pp1ab (3175 aa). Previously, three viral proteinases were reported to cleave these large polyproteins into 12 non-structural proteins (nsps). The chymotrypsin-like viral main proteinase residing in nsp4 is responsible for eight of these cleavages. Processing of the C-terminal half of pp1a (the nsp3-8 region) was postulated to occur following either of two alternative proteolytic pathways (the 'major' and 'minor' pathways). He...
Anti-oxidative status and semen quality during cooled storage in stallions. Activity of the anti-oxidative enzymes glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), content of thiobarbituric acid reactive substances (TBARS) and SH-groups were determined in native stallion semen (n = 8 stallions). Semen was then diluted in Kenney extender, EquiPro((R)) extender either with or without addition of N-acetyl cysteine or phosphate-buffered saline (PBS) and stored for 72 h at 5 degrees C. Correlations between initial activity of enzymes and development of semen motility and membrane integrity were calculated. Activities of GSH-Px, SOD and CAT im...
Mass spectrometry studies of demetallation of haemin by recombinant horse L chain apoferritin and its mutant (E 53,56,57,60 Q). An essential difference between eukaryotic ferritins and bacterioferritins is that the latter contain naturally, in vivo haem as Fe-protoporphyrin IX. This haem is located in a hydrophobic pocket along the 2-fold symmetry axes and is liganded by two Met 52. However, in in vivo studies, a cofactor has been isolated in horse spleen apoferritin similar to protoporphyrin IX; in in vitro experiments, it has been shown that horse spleen apoferritin is able to interact with haem. Studies of haemin (Fe(III)-PPIX) incorporation into horse spleen apoferritin have been carried out, which show that the me...
Do the matrix degrading enzymes cathepsins B and D increase following a high intensity exercise regime? It has been shown by others that levels of matrix degrading enzymes are increased in osteoarthritis (OA) and so are proposed to be involved in the aetiopathogenesis of the disease, including exercise-associated OA. Therefore we hypothesised that cathepsin B and cathepsin D were increased in cartilage samples previously shown to have early stage OA from 2-year-old Thoroughbred horses, euthanased for reasons other than this study, that had a history of 19-week high intensity exercise (n=6) compared to age and sex-matched horses with a history of low intensity exercise (n=6). Methods: Cartilage s...
The effect on creatinine kinase activity of freezing plasma from healthy foals. To assess the stability of creatine kinase (CK) activity in plasma collected from healthy foals and frozen at -20 degrees C for up to 12 weeks. Methods: Samples of venous blood drawn from 25 foals were analysed for CK activity soon after collection, and again after 1 and 12 weeks of freezing at -20 degrees C. Results: CK activity decreased (p<0.001) between Week 0 and Week 1 and between Week 0 and Week 12. Conclusions: Decreases in CK activity were statistically significant but clinically insignificant.
cDNA cloning and substrate specificity of equine tryptase, a possible mediator in equine heaves. Mast cell mediators are believed to play a central role in inflammatory lung disorders such as human allergic and occupational asthma. Equine heaves is characterized by reversible neutrophilic airway inflammation and airway obstruction, primarily due to bronchospasm and mucus hypersecretion, following exposure of susceptible horses to organic stable dusts. As such, heaves shares many similarities with human occupational dust-induced asthma and therefore it is proposed that mast cells may also be implicated in the pathogenesis of heaves. Tryptase, a mast cell-specific proteinase, can be used as...
Equine mandibular gland: in situ characterisation of sialoderivatives. Sialic acids modulate the metabolite transport across membranes and may be involved in protection against pathogenic agents. The presence of sialoderivatives in the equine mandibular gland requires further study. Objective: To biochemically visualise in situ the presence of sialoderivatives, by means of mild and strong periodate oxidation and alcoholic saponification, combined with lectin histochemistry and sialidase digestion in order to hypothesise roles for detached sialoderivatives. Methods: Mandibular glands were removed from 8 mature horses of both sexes and subjected to histochemical pr...
Hormonal status of male reproductive system: androgens and estrogens in the testis and epididymis. In vivo and in vitro approaches. The purpose of this article was to summarize our results on the role of androgens and estrogens in human, rodent and equine testes and epididymides, in both, physiological and patological conditions, obtained in the space of the Solicited Project (084/PO6/2002) financially supported by the State Committee for Scientific Research during the last three years. Testosterone produced by Leydig cells of the testes is clearly the major androgen in the circulation of men and adult males of most mammalian species. However, androgen metabolites make up a significant fraction of total circulating steroid...
The crystal structure of the Venezuelan equine encephalitis alphavirus nsP2 protease. Alphavirus replication and propagation is dependent on the protease activity of the viral nsP2 protein, which cleaves the nsP1234 polyprotein replication complex into functional components. Thus, nsP2 is an attractive target for drug discovery efforts to combat highly pathogenic alphaviruses. Unfortunately, antiviral development has been hampered by a lack of structural information for the nsP2 protease. Here, we report the crystal structure of the nsP2 protease (nsP2pro) from Venezuelan equine encephalitis alphavirus determined at 2.45 A resolution. The protease structure consists of two dist...
Quantitation of 17beta-nandrolone metabolites in boar and horse urine by gas chromatography-mass spectrometry. A method to quantify metabolites of 17beta-nandrolone (17betaN) in boar and horse urine has been optimized and validated. Metabolites excreted in free form were extracted at pH 9.5 with tert-butylmethylether. The aqueous phases were applied to Sep Pak C18 cartridges and conjugated steroids were eluted with methanol. After evaporation to dryness, either enzymatic hydrolysis with beta-glucuronidase from Escherichia coli or solvolysis with a mixture of ethylacetate:methanol:concentrated sulphuric acid were applied to the extract. Deconjugated steroids were then extracted at alkaline pH with tert-...
A specific method for measurement of equine active myeloperoxidase in biological samples and in in vitro tests. An original method called SIEFED (specific immunological extraction followed by enzymatic detection) was developed for the specific detection of the activity of equine myeloperoxidase (MPO). The method consists of the extraction of MPO from aqueous solutions by immobilized anti-MPO antibodies followed by washing (to eliminate proteins and interfering molecules) and measurement of MPO activity using a detection system containing a fluorogenic substrate, hydrogen peroxide, and nitrite as reaction enhancer. The SIEFED technique was applied to study active MPO in horse biological fluids and the ef...
IdeE, an IgG-endopeptidase of Streptococcus equi ssp. equi. Streptococcus equi ssp. equi is the causative agent of strangles, a highly contagious and serious disease in the upper respiratory tract of horses. The present study describes the characterization of IdeE, a homolog of the secreted IgG-specific protease IdeS/Mac of Streptococcus pyogenes. The activity of IdeE is compared with the activity of IdeZ, the corresponding enzyme of the closely related S. equi ssp. zooepidemicus. A study of the proteolytic activity of recombinant IdeE and IdeZ on IgG from a selection of mammals shows that only antibodies containing the substrate site of IdeS/Mac are c...
Effects of initial and extended exposure to an endophyte-infected tall fescue seed diet on faecal and urinary excretion of ergovaline and lysergic acid in mature geldings. To determine the amount of ergovaline and lysergic acid retained or excreted by geldings fed endophyte-infected seed containing known concentrations of these alkaloids, and the effects of exposure time on clinical expression of toxicosis. Methods: Mature geldings (n=10) received diets containing either endophyte-free (E-) or endophyte-infected (E+) tall fescue seed during three experimental phases. The first phase (Days -14 to -1) was an adaptation phase, to allow all horses to adapt to a diet containing E- tall fescue seed. The second (Days 0 to 3) was the initial exposure phase to E+ tall fe...
Changes in MMP-2 and -9 activity and MMP-8 reactivity after amphotericin B induced synovitis and treatment with bufexamac. The objective here was to evaluate the acute effects of induced arthritis on synovial fluid (SF) levels of matrix metalloproteinases (MMP) -2, -8 and -9 in horses. To evaluate MMP-2 and -9 activities and the effect of non-steroidal anti-inflammatory drug (NSAID) bufexamac during remission from acute arthritis. Aseptic arthritis was induced in 24 Standardbred horses using 20 mg of amphotericin B as a single intra-articular (IA) injection in the right intercarpal joint. After 1 week and 2 weeks, horses were treated intra-articularly with 10, 20, or 40 mg of bufexamac suspension or with sterile s...
Ferritin-catalyzed consumption of hydrogen peroxide by amine buffers causes the variable Fe2+ to O2 stoichiometry of iron deposition in horse spleen ferritin. Ferritin catalyzes the oxidation of Fe2+ by O2 to form a reconstituted Fe3+ oxy-hydroxide mineral core, but extensive studies have shown that the Fe2+ to O2 stoichiometry changes with experimental conditions. At Fe2+ to horse spleen ferritin (HoSF) ratios greater than 200, an upper limit of Fe2+ to O2 of 4 is typically measured, indicating O2 is reduced to 2H2O. In contrast, a lower limit of Fe2+ to O2 of approximately 2 is measured at low Fe2+ to HoSF ratios, implicating H2O2 as a product of Fe2+ deposition. Stoichiometric amounts of H2O2 have not been measured, and H2O2 is proposed to react ...
Matrix metalloproteinase-9 in laminae of black walnut extract treated horses correlates with neutrophil abundance. We sought to determine whether a correlation exists between neutrophil infiltration and tissue matrix metalloproteinase-9 (MMP-9) content in digital laminae collected during the prodromal and acute phases of laminitis in horses treated with an aqueous black walnut heartwood extract (BWE). Hoof laminar tissue was obtained at the onset of leukopenia and at the onset of clinical signs of lameness from BWE-treated horses and at equivalent times from control horses. Thin sections of laminae were screened for neutrophils by immunohistochemistry with an anti-CD13 monoclonal antibody and extracts of t...
Equine laminitis: bites by Bothrops spp cause hoof lamellar pathology in the contralateral as well as in the bitten limb. The envenoming caused by Bothrops snakebite includes local symptoms, such as pronounced edema, hemorrhage, intense pain, vesicles, blisters and myonecrosis. The principal systemic symptom consists in the alteration of blood clotting, due to fibrinogen consumption and platelet abnormalities. The horses involved in this study had this symptomatology and one of them exhibited symptoms consistent with laminitis in the bitten and in the contralateral limbs. Laminitis lesions were characterized by separation of the hoof lamellar basement membrane (BM) from basal cells of the epidermis. These results...
Pathogenesis, laboratory diagnosis, and clinical implications of erythrocyte enzyme deficiencies in dogs, cats, and horses. Deficiencies of enzymes involved in erythrocyte metabolism can have significant effects on erythrocyte function and survival. Animals with pyruvate kinase (PK) or phosphofructokinase (PFK) deficiencies have shortened erythrocyte life spans and regenerative anemia. PK-deficient dogs (but not PK-deficient cats) develop progressive myelofibrosis and osteosclerosis of bone marrow and hemochromatosis and cirrhosis of the liver. PFK-deficient dogs have sporadic episodes of hyperventilation-induced intravascular hemolysis and hemoglobinuria. Cytochrome b5 reductase (Cb5R) deficiency in dogs and cats ...
Evidence for the expression and enzymatic activity of haem oxygenase-1 in the lungs of horses. The aim of this study was to characterise the expression of haem oxygenase-1 (HO-1) in healthy lung tissue from horses and to measure its activity. Samples of lung tissue were collected from six horses euthanased for reasons other than respiratory disease. HO-1 expression and activity were detected in type II alveolar epithelial cells, macrophages and neutrophils in all the samples examined. The activity was dependent on the presence of NADPH and inhibited quantitatively by the addition of increasing concentrations of a competitive inhibitor of HO-1, tin mesoporphyrin IX.
A comparative kinetic analysis of the reactivity of plant, horse, and human respiratory cytochrome c towards cytochrome c oxidase. Two synthetic genes coding for human and Arabidopsis cytochrome c, respectively, have been designed and constructed, and the recombinant proteins have been over-expressed in Escherichia coli cells. Thus a comparative analysis of the two heme proteins, including horse cytochrome c as a reference, has been performed. In addition to their physico-chemical properties, the redox behavior of the three proteins has been analyzed by following the kinetics of both their reduction by flavin semiquinones (lumiflavin, riboflavin, and FMN) and oxidation by cytochrome c oxidase. The resulting data indicate ...
Determination of acrosin amidase activity in equine spermatozoa. Acrosin amidase activity of spermatozoa has been been associated with in vitro fertilization success in humans and has been proposed as an additional method for assessing sperm function in vitro. In this study, acrosin amidase activity was determined in equine spermatozoa by the hydrolysis of an arginine amide substrate. This assay includes a detergent to release acrosomal enzymes into a medium of basic pH to activate proacrosin to acrosin, which subsequently hydrolyses N-alpha-benzoyl-DL-arginine para-nitroanilide-HCl (BAPNA) to a chromogenic product. Spermatozoa (n = 3 ejaculates from each o...
Human chorionic gonadotropin-dependent induction of an equine aldo-keto reductase (AKR1C23) with 20alpha-hydroxysteroid dehydrogenase activity during follicular luteinization in vivo. Aldo-keto reductases (AKRs) are multifunctional enzymes capable of acting on a wide variety of substrates, including sex steroids. AKRs having 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) activity can reduce progesterone to 20alpha-hydroxy-4-pregnen-3-one (20alpha-DHP), a metabolite with lower affinity for the progesterone receptor. The objective of this study was to investigate the regulation of equine AKR1C23 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The equine AKR1C23 cDNA was cloned and shown to encode a 322 amino acid protein that is conserved (71-81...
Flow microcalorimetric study of butyrylcholinesterase kinetics and inhibition. The enzymatic hydrolysis of butyrylcholine, catalyzed by horse serum butyrylcholinesterase (EC 3.1.1.8), was studied at 37 degrees C in Tris buffer (pH 7.5) by flow microcalorimetry. A convolution procedure, using the Gamma distribution to represent the impulse response of the calorimeter, was developed to analyze the microcalorimetric curves. After correction for buffer protonation, the hydrolysis reaction was found to be slightly endothermic, with Delta H=+9.8 kJ mol(-1). Enzyme kinetics was studied with both the differential and integrated forms of the Michaelis equation with equivalent res...
Internal restriction sites: quality assurance aids in genotyping. Improvements to restriction fragment length polymorphism (RFLP)-based genotyping assays currently used for detection of mutations responsible for bovine ferrochelatase and myophosphorylase deficiencies, and equine hyperkalemic periodic paralysis (HYPP) are described. Reports of sporadic inhibition of restriction enzyme activity suggest a critical factor in RFLP-based genotyping assays should be assurance that restriction enzymes perform to specification with every sample. The RFLP genotyping assays that use either a mismatched recognition sequence in one or both of the oligonucleotides, or inc...
Screening for basic drugs in equine urine using direct-injection differential-gradient LC-LC coupled to hybrid tandem MS/MS. A rapid, selective and robust direct-injection LC/hybrid tandem MS method has been developed for simultaneous screening of more than 250 basic drugs in the supernatant of enzyme hydrolysed equine urine. Analytes, trapped using a short HLB extraction column, are refocused and separated on a Sunfire C(18) analytical column using a controlled differential gradient generated by proportional dilution of the first column's eluent with water. Independent data acquisition (IDA) was configured to trigger a sensitive enhanced product ion (EPI) scan when a multiple reaction monitoring (MRM) survey scan s...
Detection of superoxide anion generation by equine spermatozoa. To identify the generation of the superoxide anion by equine spermatozoa. Methods: Multiple ejaculates collected from 3 Thoroughbred stallions. Methods: Induced superoxide production by reduced nicotinamide adenine dinucleotides (NAD[P]H; ie, reduced nicotinamide adenine dinucleotide [NADH] and reduced nicotinamide adenine dinucleotide phosphate [NADPH]) was measured by use of a nitroblue tetrazolium (NBT) reduction assay on whole spermatozoa and a cytochrome c reduction assay on isolated membrane fractions of spermatozoa. Localization of superoxide generation was determined by use of NBT cyto...
Lack of clinical efficacy of a phosphodiesterase-4 inhibitor for treatment of heaves in horses. Phosphodiesterase-4 (PDE 4) enzyme inhibitors have been shown to have anti-inflammatory properties in various animal disease processes and therefore could be effective drugs for the treatment of equine airway diseases. The purpose of this study was to evaluate the efficacy and adverse effects of the PDE 4 inhibitor L-826,141 in horses with heaves. In a blinded parallel design, horses with heaves exposed daily to moldy hay were given a placebo for 14 days and then administered either L-826,141 (n = 6; loading dose of 1 mg/kg IV followed by 0.5 mg/kg IV q48h) or dexamethasone (n = 6; 0.04 mg/kg ...
