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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.
Dose responses to inhalation of endotoxin, hay dust suspension and Aspergillus fumigatus extract in horses as measured by levels and activation of matrix metalloproteinase-9. Airway matrix metalloproteinases (MMPs) increase after endotoxin (LPS) exposure, but there are no reports describing dose-dependent increases or activation following exposure. Objective: To study matrix metalloproteinase-9 (MMP-9) and -2 (MMP-2) responses in bronchoalveolar lavage fluid (BALF) from heaves-susceptible and control horses following inhalation of hay dust suspension (HDS), LPS and Aspergillus fumigatus extract (AFE). Methods: Heaves-susceptible (n = 7) and control (n = 6) horses received inhalation challenges with 3 different doses of HDS and LPS. Heaves-susceptible horses (n = 6)...
Ribotyping of Burkholderia mallei isolates. In this study, the subspecies differentiation of 25 isolates of Burkholderia mallei was attempted based on their ribotype polymorphisms. The isolates were from human and equine infections that occurred at various times around the world. DNA samples from each isolate were digested separately with PstI and EcoRI enzymes and probed with an Escherichia coli-derived 18-mer rDNA sequence to identify diagnostic fragments. Seventeen distinct ribotypes were identified from the combined data obtained with the two restriction enzymes. The results demonstrate the general utility of ribotyping for the subs...
Neuronal nitric oxide synthase is heterogeneously distributed in equine myofibers and highly expressed in endurance trained horses. Mammalian skeletal muscle expresses splice variants of neuronal nitric oxide synthase (nNOS). Skeletal muscles have a metabolically heterogeneous population of myofibers, and fiber composition in equine skeletal muscle is correlated with athletic ability in endurance events. In this study, we investigated whether nNOS expression in equine skeletal muscle is related to fiber type and endurance training. Biopsy samples obtained from the gluteus medius of sedentary- (SH) and endurance-trained (TH) horses were examined for the electrophoretic mobility of myosin heavy chain (MHC) and NOS activity. ...
Expression of endothelial and inducible nitric oxide synthases is modulated in the endometrium of cyclic and early pregnant mares. The expression of the endothelial and inducible nitric oxide synthases (eNOS and iNOS, respectively) was examined in the endometrium of cyclic and pregnant mares by real-time polymerase chain reaction and immunohistology. The concentration of eNOS mRNA varied throughout the oestrous cycle, with significantly higher transcripts on Day 5 of the oestrous cycle (P 0.05). In early pregnant mares both eNOS and iNOS mRNA increased between Days 12 and 15 (P < 0.05). In cyclic mares, eNOS protein was detected immunocytochemically in endometrial epithelia, the basement membrane, the endothelial laye...
Gentamicin nephrotoxicity–a comparison of in vitro findings with in vivo experiments in equines. The aminoglycoside gentamicin is often used in equine practice. Despite its clinical use, concerns remain regarding the potential toxic side-effects, such as nephrotoxicity, in equine patients, particularly after repeated dosing. The aim of the study was to investigate first in vitro the mechanisms contributing to the renal toxicity of gentamicin and to identify sensitive biomarkers indicating proximal tubule damage. To this end, the kidney-derived cell lines LLC-PKI and MDCK were treated with gentamicin at different concentrations. Toxicity was assessed by measuring the release of gamma-gluta...
Activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5 degrees C. Sperm cell membranes are susceptible to peroxidative damage by an excess of reactive oxygen species (ROS). Antioxidative defence systems consisting of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) physiologically control the balance between ROS production and neutralization. In the present study the hypothesis was tested that lipid peroxidation occurs during storage of semen at 5 degrees C and that semen extender has positive effects on the antioxidative potential of equine semen. The aim of the study was to determine the activity of GSH-Px, SOD and CAT and the...
Protein folding in classical perspective: folding of horse cytochrome c. Proteins meet with the stipulations of Levinthal. Two test tube variants of ferrocytochrome c (ferrocyt c) whose thermodynamic stabilities are vastly different refold to the same global minimum under a given final native condition, and they do so quickly at rates that do not reflect a strong dependence on the thermodynamic driving force. The transition-state ensemble is more unfolded-like, and the folding barrier offered is energetically sizable. The experiments involve neutral- (pH 7) and alkaline ferrocyt c pH (12.7), whose aqueous stabilities are 18 (+/-0.3) and 3 (+/-0.5) kcal mol(-)(1), r...
Measurement of erythrocyte carbonic anhydrase isozymes (CA-I and CA-II) in racehorses and riding horses. Equine carbonic anhydrase isozymes (CA-I and CA-II) were purified from erythrocytes by several column chromatography. Polyclonal anti-CA-I and anti-CA-II sera were produced in rabbits. Sensitive competitive enzyme-linked immunosorbent assays (ELISA) were established to determine the developmental changes in CA-I and CA-II levels in equine erythrocytes. Concentrations of CA-I and CA-II in erythrocytes from 150 clinically normal thoroughbreds (123 racehorses and 27 riding horses) were determined by ELISA. Mean (+/- SD) concentrations of CA-I and CA-II in racehorses were 1.70 +/- 0.48 and 0.94 +/...
Cytokine induced metalloproteinase expression and activity does not correlate with focal susceptibility of articular cartilage to degeneration. To determine whether the focal susceptibility to cartilage degeneration in joints is related to a differential response to cytokine stimulation. Methods: Compare aggrecan and collagen catabolism in in-vitro models of cartilage degradation induced by retinoic acid (RA), interleukin-1 (IL-1), tumor necrosis factor alpha (TNF) and IL-1 plus oncostatin M (OSM). Glycosaminoglycan (GAG) and hydroxyproline (HyPro) quantification and Western immunoblot analyses of aggrecan and collagen degradation products were undertaken in explant cultures of normal cartilage from regions of equine joints with a kno...
A comparison of solution conformation and hydrodynamic properties of equine, porcine and rabbit serum albumin using viscometric measurements. This paper presents the results of viscosity determinations on aqueous solutions of equine, porcine and rabbit serum albumin over a wide range of concentrations and at temperatures ranging from 5 degrees C to (42-45) degrees C. The results are compared with human and bovine serum albumin previously studied. Viscosity-temperature dependence is discussed on the basis of the modified Arrhenius formula. The effective specific volume, the activation energy and entropy of viscous flow for all investigated albumins are compared. Viscosity-concentration dependence, in turn, is discussed on the basis o...
Pharmacodynamics and pharmacokinetics of nonsteroidal anti-inflammatory drugs in species of veterinary interest. This review summarises selected aspects of the pharmacokinetics (PK) and pharmacodynamics (PD) of nonsteroidal anti-inflammatory drugs (NSAIDs). It is not intended to be comprehensive, in that it covers neither minor species nor several important aspects of NSAID PD. The limited objective of the review is to summarise those aspects of NSAID PK and PD, which are important to an understanding of PK-PD integration and PK-PD modelling (the subject of the next review in this issue). The general features of NSAID PK are: usually good bioavailability from oral, intramuscular and subcutaneous administ...
Profiles of matrix metalloproteinase activity in equine tear fluid during corneal healing in 10 horses with ulcerative keratitis. Levels of tear film matrix metalloproteinases (MMPs) activity are significantly elevated in horses with ulcerative keratitis and contribute to the excessive breakdown of stromal collagen. Changes in the amount of proteolytic activity in horse tear film during corneal healing and stromal remodeling have not yet been reported, but we hypothesize they should decrease. In the present study we analyzed serial tear fluid from horses with ulcerative keratitis to identify any changes in MMP activity during corneal healing and stromal remodeling. Methods: Samples of tear fluid were obtained from both e...
Crystallization of a proteolyzed form of the horse pancreatic lipase-related protein 2: structural basis for the specific detergent requirement. Horse pancreatic lipase-related proteins PLRP1 and PLRP2 are produced by the pancreas together with pancreatic lipase (PL). Sequence-comparison analyses reveal that the three proteins possess the same two-domain organization: an N-terminal catalytic domain and a C-terminal domain, which in PL is involved in colipase binding. Nevertheless, despite the high level of sequence identity found, they exhibit distinct enzymatic properties. The intrinsic sensitivity of the peptide bond between Ser245 and Thr246 within the flap region of PLRP2 to proteolytic cleavage probably complicates PLRP2 crystalli...
Effect of frozen semen on the uterus of mares with pathological uterine changes. Pregnancy rates after frozen semen inseminations (AI), particularly in older and problem mares, are lower than after fresh semen AI. Uterine contractility and the inflammatory reaction after frozen semen insemination were studied in two groups of mares: the abnormal group comprised of 6 old barren mares categorized in biopsy category IIB or III, and the control group including 6 reproductively normal young maiden mares in biopsy category I or IIA. All 12 mares were inseminated in the first cycle with 2 mL of phosphate-buffered saline (PBS) and in their second cycle with 2 mL of frozen semen co...
Proteolytic degradation of IGF-binding protein (IGFBP)-2 in equine ovarian follicles: involvement of pregnancy-associated plasma protein-A (PAPP-A) and association with dominant but not subordinated follicles. In the ovary of mammalian species, terminal follicular growth is accompanied by a decrease in intrafollicular levels of IGF-binding protein-2 (IGFBP-2) and IGFBP-4. The decrease in IGFBP-4 levels is essentially due to an increase in proteolytic cleavage by intrafollicular pregnancy-associated plasma protein-A (PAPP-A) in growing healthy follicles. The decrease in IGFBP-2 levels is partly due to a decrease in mRNA expression by follicular cells. In addition, we have recently shown that IGFBP-2 is also proteolytically cleaved by PAPP-A in bovine and porcine growing follicles. In the present work...
Anticorrelated motions as a driving force in enzyme catalysis: the dehydrogenase reaction. Molecular dynamics and cross-correlation analysis of the horse liver alcohol dehydrogenase HLADH.NAD(+).PhCH(2)O(-) complex has established anticorrelated motions between the NAD(+)-binding domain and other portions of the enzyme. Four pairs of anticorrelated interactions are (i and ii) cofactor-binding domain: C(alpha) of V292 and the CG1 of V203 with C7 of PhCH(2)O(-); (iii) cofactor-binding domain: amide carbonyl oxygen of I318 with amide N of H67; and (iv) cofactor domain: C(alpha) of T178 with carbonyl oxygen of L141. The average distances between pairs are 9.2 A for i, 8.2 A for ii, 14.7...
The diagnostic and prognostic value of alkaline phosphatase activity in serum and peritoneal fluid from horses with acute colic. Alkaline phosphatase (ALP) is an enzyme present in intestinal mucosa, bile, bone, and renal tubule cells. We sought to assess the diagnostic and prognostic relationships of total ALP (ALPt) activity and that of intestine-derived ALP (ALPi) in serum and peritoneal fluid of 126 horses with colic. ALPt and ALPi activities were measured in both serum and peritoneal fluid by using both standard and L-phenylalanine-based buffers, respectively. Neither ALPt nor ALPi activity were useful in classifying type or severity of intestinal damage. ALPt and ALPi activities in peritoneal fluid were lowest in h...
Effect of matrix depleting agents on the expression of chondrocyte metabolism by equine chondrocytes. This study was carried out to investigate the effect of two enzymes (collagenase and chondroitinase) and two cytokines/metabolites (interleukin-1beta and retinoic acid) of known catabolic activity on the expression of cartilage metabolism/phenotype in equine articular cartilage. Articular cartilage explants from 11 horses (5-13 years old) were treated for 48 h and assayed for total sulphated glycosaminoglycan (GAG), the incorporation of 35S-sulphate, collagen degradation and mRNA expression of the proteoglycans collagen II, collagen IIA, collagen III, collagen IX, collagen X, collagen XI and g...
The apparent digestibility of phytate phosphorus and the influence of supplemental phytase in horses. Availability of phytate-bound P as influenced by supplemental phytase was studied in eight horses consuming four diets in a 4 x 4 Latin square design experiment. The treatments were a control (containing a low P level, 18.4 g/d) and three high-P diets. These diets contained P as monocalcium phosphate (MCP; 43.7 g/d), myoinositol hexakisphosphate in the form of wheat and rice bran (MIHP; 41.8 g/d), or MIHP with microbial phytase (MIHPP; 42.5 g/d). The proportions of phytate-bound P were 3, 1, 55, and 56% for the control, MCP, MIHP, and MIHPP, respectively. The MIHPP diet was supplemented with 3...
Enzyme activities in some types of peripheral leukocytes of thoroughbred race horses before and after the races. Glucose, free fatty acid (FFA), triglyceride (TG) and immunoreactive insulin (IRI) concentrations in plasma and activities of enzymes related to energy metabolism in some types of peripheral leukocytes were measured in thoroughbred race horses before and after racing. Glucose, FFA, TG and IRI concentrations and enzyme activities did not change significantly in plasma. However, the activities of cytosolic hexokinase, malate dehydrogenase (MDH), mitochondrial MDH and aspartate aminotransferase decreased significantly in leukocytes of the horses after the races. The cytosolic ratio of MDH/LDH act...
Equine laminitis: glucose deprivation and MMP activation induce dermo-epidermal separation in vitro. Acute laminitis is characterised by hoof lamellar dermal-epidermal separation at the basement membrane (BM) zone. Hoof lamellar explants cultured in vitro can also be made to separate at the basement membrane zone and investigating how this occurs may give insight into the poorly understood pathophysiology of laminitis. Objective: To investigate why glucose deprivation and metalloproteinase (MMP) activation in cultured lamellar explants leads to dermo-epidermal separation. Methods: Explants, cultured without glucose or with the MMP activator p-amino-phenol-mercuric acetate (APMA), were subject...
Evidence for vascular and enzymatic events in the pathophysiology of acute laminitis: which pathway is responsible for initiation of this process in horses? To date, there is a substantial amount of data to support the hypotheses that vascular and enzymatic changes are ongoing in experimental laminitis. Furthermore, there is substantial in vitro evidence that the enzymatic changes weaken the dermo-epidermal attachments leading to mechanical failure of the hoof-bone interface of the equine digit. However, investigators of both the vascular and enzymatic theories have, to date, been unable to substantiate the effects of these pathophysiological changes in vivo on laminar tissues of horses afflicted with experimentally induced or naturally acquired l...
Allergen challenge alters lymphocyte phosphodiesterase activity in horses with heaves. Heaves is an allergic airway disease in horses characterised by reversible airway obstruction, bronchial hyperresponsiveness and airway inflammation associated with a Th(2) response. Cyclic nucleotide-dependent signalling pathways can regulate lymphocyte function. In this study, we examined lymphocyte PDE activity comparing horses with heaves to healthy control animals. Total PDE activity and the effects of isoenzyme selective inhibitors were measured before, 5 and 24 h after the start of a 7 h allergen challenge. Allergen challenge had no effect on either total cAMP PDE activity or its inhibi...
Equine catechol estrogen 4-hydroxyequilenin is a more potent inhibitor of the variant form of catechol-O-methyltransferase. Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of t...
Characterization of the pharmacokinetic and pharmacodynamic properties of the angiotensin-converting enzyme inhibitor, enalapril, in horses. The pharmacokinetics of enalapril (0.5 mg/kg i.v.) and the pharmacodynamics of enalapril (0.5 mg/kg PO) in 5 mares were investigated. After single i.v. dosing, concentrations of enalapril and enalaprilat, its active metabolite, were measured. Two weeks later, enalapril was administered by nasogastric tube. Potassium, creatinine, blood urea nitrogen (BUN), enalapril, and enalaprilat concentrations and angiotensin converting enzyme (ACE) activity were measured in serum. In addition, heart rate, blood pressure, digital venous blood gases, and lactate were measured. Two weeks later, enalapril was ...
Expression of messenger ribonucleic acid encoding for steroidogenic acute regulatory protein and enzymes, and luteinizing hormone receptor during the spring transitional season in equine follicles. The period of spring transition, from the anovulatory to the ovulatory season, is characterized in many mares by cyclical growth and regression of large dominant follicles. These follicles produce only low concentrations of estradiol and it is thought that acquisition of steroidogenic competence by large follicles during spring transition is prerequisite in stimulating LH prior to first ovulation. In situ hybridization was used to localize and quantify expression of factors that play a key role in follicular steroidogenesis: StAR, P450scc (CYP11A1), P450c17 (CYP17), P450arom (CYP19), and LH re...
Identification and partial characterization of alpha-1,4-glucosidase activity in equine epididymal fluid. The expression of alpha-1,4-glucosidase activity was fluorometrically and electrophoretically assessed in the epididymal fluid and seminal plasma of stallions. alpha-Glucosidase specific activity in the epididymis increased significantly from the proximal caput to the cauda. Stallion epididymal glucosidase maintained activity in a wide range of pH, with two distinct peaks (around pH 4.0 and 6.0, respectively). Enzyme activities at different pH, inhibition assays with sodium dodecyl sulfate (SDS) and maltotriose (MTT, selective inhibitors of alpha-glucosidases "acidic" and "neutral" isoforms, d...
Biochemical and functional assessment of equine lymphocyte phosphodiesterases and protein kinase C. Lymphocytes play an important role in allergic inflammation and have been implicated in the pathogenesis of equine allergic skin and respiratory disease. Targeting intracellular signalling pathways in human lymphocytes has demonstrated a role for both phosphodiesterase and protein kinase C in cell activation. The aim of this study was to measure total cyclic nucleotide hydrolysing phosphodiesterase activity and to identify the phosphodiesterase and protein kinase C isoenzymes present in equine lymphocytes. The functional significance of these isoenzymes was then investigated by examining their...
Matrix metalloproteinase-2 and -9 secretion by the equine ovary during follicular growth and prior to ovulation. Profound hormonally controlled tissue remodelling occurs in the equine ovary for follicle growth and development, and also for the alteration in follicle shape directed towards the ovulation fossa, the site where ovulation occurs. The aim of this study was to examine the spatial and temporal regulation of matrix metalloproteinases (MMP)-2 and MMP-9, important enzymes in tissue remodelling, during follicle growth, and ovulation. Using gelatin substrate zymography, we measured these MMPs in follicular fluid of large anovulatory follicles collected during spring transition, early dominant follicl...
Neuropeptide distribution in the stellate ganglia of the domestic animal. We used immunohistochemical techniques to analyze the localization and distribution of the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) and the neuropeptides methionine-endephalin (M-Enk), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), calretinin (Cal), calcitonin gene-related peptide (CGRP), substance P (SP), and galanin (Gal) in the stellate ganglia of two species of domestic animal (cattle and horses). NPY, VIP and Gal immunoreactive neurons (both cell body and nerve fiber) were observed in the stellate ganglia of both animals. M-Enk and CGRP immunoreactive ne...
