• Animal Bioscience
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• 제34권10호
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• pp.1590-1599
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• 2021

Objective: This study investigates the expression patterns of toll-like receptors (TLRs) and intracellular mediators in horse muscle cells after exercise, and the relationship between TLRS expression in stressed horse muscle cells and immune cell migration toward them. Methods: The expression patterns of the TLRs (TLR2, TLR4, and TLR8) and downstream signaling pathway-related genes (myeloid differentiation primary response 88 [MYD88]; activating transcription factor 3 [ATF3]) are examined in horse tissues, and horse peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs) and muscles in response to exercise, using the quantitative reverse transcription-polymerase chain reaction (qPCR). Expressions of chemokine receptor genes, i.e., C-X-C motif chemokine receptor 2 (CXCR2) and C-C motif chemokine receptor 5 (CCR5), are studied in PBMCs and PMNs. A horse muscle cell line is developed by transfecting SV-T antigen into fetal muscle cells, followed by examination of muscle-specific genes. Horse muscle cells are treated with stressors, i.e., cortisol, hydrogen peroxide (H₂O₂), and heat, to mimic stress conditions in vitro, and the expression of TLR4 and TLR8 are examined in stressed muscle cells, in addition to migration activity of PBMCs toward stressed muscle cells. Results: The qPCR revealed that TLR4 message was expressed in cerebrum, cerebellum, thymus, lung, liver, kidney, and muscle, whereas TLR8 expressed in thymus, lung, and kidney, while TLR2 expressed in thymus, lung, and kidney. Expressions of TLRs, i.e., TLR4 and TLR8, and mediators, i.e., MYD88 and ATF3, were upregulated in muscle, PBMCs and PMNs in response to exercise. Expressions of CXCR2 and CCR5 were also upregulated in PBMCs and PMNs after exercise. In the muscle cell line, TLR4 and TLR8 expressions were upregulated when cells were treated with stressors such as cortisol, H₂O₂, and heat. Migration of PBMCs toward stressed muscle cells was increased by exercise and oxidative stresses, and combinations of these. Treatment with methylsulfonylmethane (MSM), an antioxidant on stressed muscle cells, reduced migration of PBMCs toward stressed muscle cells. Conclusion: In this study, we have successfully cultured horse skeletal muscle cells, isolated horse PBMCs, and established an in vitro system for studying stress-related gene expressions and function. Expression of TLR4, TLR8, CXCR2, and CCR5 in horse muscle cells was higher in response to stressors such as cortisol, H₂O₂, and heat, or combinations of these. In addition, migration of PBMCs toward muscle cells was increased when muscle cells were under stress, but inhibition of reactive oxygen species by MSM modulated migratory activity of PBMCs to stressed muscle cells. Further study is necessary to investigate the biological function(s) of the TLR gene family in horse muscle cells.

• Bulletin of the Korean Chemical Society
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• 제11권6호
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• pp.472-473
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• 1990

• 약학회지
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• 제41권6호
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• pp.756-764
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• 1997

Ile-224 in I269S mutant horse liver alcohol dehydrogenase isoenzyme S (HLADH-S) was mutated to serine by site-directed mutagenesis in order to study the role of the residue in c oenzyme binding to the enzyme. The specific activity of the I269S and I224S mutant enzyme to ethanol was increased 6-fold and all Michaelis constants($K_a,\;K_b,\;K_p,\;and\;K_q$,/TEX>) were larger than those for the wild-type and I269S enzyme. The substitution decreased the afffinity to coenzymes and increased the specific activity of the enzyme. The mutant enzyme showed the highest catalytic efficiency for octanol among the primary alcohols. But it didn`t have activities on retinoids and 5${\beta}$-cholanic acid-3-one. From these results, it was confirmed that the hydrophobic interaction of Ile-224 residue with coenzyme was related to coenzyme affinity in ADH reaction. The substitution also affected the substrate affinities to the enzyme.

• 대한의생명과학회지
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• 제8권3호
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• pp.179-184
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• 2002

We investigated the morphological changes and TUNEL reaction of apoptotic cells in the liver of D-galactosamine (20 mg/mouse) and lipopolysaccharide (5 $\mu\textrm{g}$/mouse)-treated 30 mice (BALB/c), and in additioa also of apoptotic cells in kidney and spleen. The livers and other some organs of mice at 6, 12, 24, 48 and 72 hrs after treatment were collected and were fixed with 10% neutral formalin and paraffin sections were stained with hematoxylin-eosin or terminal deoxynucleotidly transferase-mediated dUTP nick end labeling (TUNEL) method. Morphological changes in apoptotic hepatocytes were chondensation of nuclei and density of cytoplasms, then the margination and pyknosis of chromatin, the formation of half-moon- or horse-shoe- or ship-like shapes of condensed chromatin mass, lastly formation of apoptotic bodies, disappearance of nuclear envelopes, decrease of stainability, then lysis and disappearance of apoptotic bodies. TUNEL positive reactions of hepatocytes were appeared first moderate in uncondensed hepatocytes, severe in condensed hepatocytes, moderate in chromatin-marginated hepatocytes. These reactions also were appeared moderate in hepatocytes with half-moon- or horse-shoe- or ship-like pyknotic chromatin mass or apoptotic bodies, and mild or negative in hepatocytes with lysed apoptotic bodies or with disappeared nuclear envelopes. Consequently these results suggested that TUNEL positive reactions of hepatocytes appeared at more early stages than appearance of chromatin condensation and disappeared at more early stage than disappearance of histological findings of apoptosis. We also confirmed that the differentiation of apoptotic cells from normal healthy cells of Kupffer cells and vascular endothelial cells in liver, reticular cells and lymphocytes in spleen and epithelial cells of tubules and ducts in kidney was impossible in H-E preparations but was possible in TUNEL preparations.

• 대한수의학회지
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• 제47권3호
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• pp.315-319
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• 2007

Fibrinopurulent peritonitis and abdominal abscesses associated with Streptococcus (S.) equi subsp. zooepidemicus is reported in a 1-year-old female thoroughbred horse. The horse died after showing 5-day history of the cold, severe abdominal distention, abdominal breathing, hyperthermia, anorexia, and loss of intestinal sound. At necropsy, several liters of turbid yellowish green fluid were seen in the abdominal cavity. Yellowish creamy and fibrinous or bloody materials were presented on the serosa of various abdominal organs that were intensively adhered with each other. Spleen and mesenteric lymph node were remarkably enlarged. Affected lobes of the lung showed severe congestion, hemorrhage and doughy consistency. Histologically, the lung showed hemorrhagic pneumonia with diffuse congestion and edema. Severe diffuse fibrinopurulent peritonitis with Gram-positive bacterial cocci and adjacent fibrosis were showed in the serosa of various abdominal organs such as liver, spleen, stomach, and intestine. And multifocal abscess pouches were presented in the granulation tissue of abdominal viscera. S. equi subsp. zooepidemicus was isolated from the peritoneal swab, abdominal organs, and lung. Hematogenous dissemination of bacteria from hemorrhagic pneumonia is proposed as the route of infection in this case.

• 대한수의학회지
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• 제25권2호
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• pp.113-123
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• 1985

This paper dealt with the distribution of normal mast cells in the spleen, liver and lung on cattle, horses, pigs and dogs, and also degranulation of mast cells in the dogs infected with Rompun (2% Xylazine HCl). The results observed are summarized as follows. Normal mast cells were distributed in spleen, liver and lung on cattle, horse, pig and dog. Mast cells were observed in both red pulp and surroundings of white pulp of the spleen in horse, in the white pulp of the spleen in cattle, in the trabeculae of the spleen in pigs, and in white pulp and red pulp of the spleen in dogs, respectively. Mast cells were observed in the portal triad of the liver in cattle and horses, in both portal triad and interlobular connective tissues of the liver in pigs, and not only the portal triad but also walls of the sinusoids and the central veins in dogs. A large number of mast cells were observed in the interlobular septa and peribronchioles of lung on all the species in this experiment. The mast cells are more numerous in the lungs than other organs. Author considers that numbers of normal mast cells distributed in the tissue is related to the dosage of Rompun in animal. The degranulation of mast cells were observed in the subcutaneous tissues of dog intramuscularly injected with Rompun(0.5ml/times) for 4 or 5 times and subcutaneously injected with Rompun(0.3ml/times) for 4 times. In dog intradermally injected with 0.1ml of Rompun, mast cells were decreased in number at 30 minutes and markedly decreased in number at 2 hours, but more or less increased in number at 3 hours after injection. In addition, the granules of the mast cells were decreased in number at 30 minutes and marked degranulation of the mast cells were recognized at 2 hours after injections, but normal mast cells begun to appear in subcutaneous tissue with the lapse of time from 3 hours after injection. There was also observed local infiltration of neutrophils in subcutaneous tissues of dogs intradermally injected with 0.1ml of Rompun at 30 minutes. At 2 hours after injection, numerous neutrophils and a small number of eosinophils were observed in the site of injection. Conclusionally, Rompun was regarded as a factor which causes the degranulateon of mast cell and the authors considered that histamine released from the mast cells by Rompun might cause relaxation of skeletal muscle.

• Asian Pacific Journal of Cancer Prevention
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• 제15권2호
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• pp.605-610
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• 2014

Chronic liver diseases, including cancer, are characterized by inflammation and elevated serum ferritin (SF). However, the causal-relationship remains unclear. This study used primary rat hepatic stellate cells (HSC) as a model to investigate effects of physiological SF concentrations (10, 100 and 1000 pM) because HSCs play a central role in the development and progression of liver fibrosis. Physiological concentrations of SF, either horse SF or human serum, induced pro-inflammatory cytokine $IL1{\beta}$, IL6 and $TNF{\alpha}$ secretion in rat activated HSCs (all p<0.05). By contrast, treatment did not alter activation marker ${\alpha}SMA$ expression. The presence of SF markedly enhanced expression of Grp78 mRNA (p<0.01). Furthermore, transient knock down of Grp78 by endotoxin EGF-SubA abolished SF-induced $IL1{\beta}$ and $TNF{\alpha}$ secretion in activated HSCs (all p<0.05). In conclusion, our results showed that at physiological concentrations SF functions as a pro-inflammatory mediator in primary rat HSCs. We also provide a molecular basis for the action of SF and identified Grp78-associated ER stress pathways as a novel potential therapeutic target for resolution of fibrosis and possible prevention of liver cancer.

• Archives of Pharmacal Research
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• 제16권3호
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• pp.231-236
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• 1993

Gly-224 residue of yeast alcohol dehydrogenase was mutated by site-directed mufagenesis to isoleucine, which is the corresponding amino acid residue of horse liver alcohol dehydrogenase. The mutated gene on M13 vector was subcloned in YEp13 and used to transform Saccharomyces cerevisiae 302-21 #2 strain, and the expressed protein was purified. The tumover numbers of mutant enzyme for ethanol and acetaldehyde were decreased copared to wild-type enzyme. The results of product inhibition studies indicated that the reaction mechanism was changed to Iso Theorell-Chance from Ordered Bi Bi. We supposed that Gly-224 was related to the enzyme reaction mechanism.

• 약학회지
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• 제36권5호
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• pp.469-473
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• 1992

Yeast alcohol dehydrogenase (YADH) has an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the $NAD^+$ coenzyme. The acidic residue of Asp-223 (according to horse liver alcohol dehydrogenase amino acid sequence) is supposed to determine the coenzyme specificity for $NAD^+$ rather than $NADP^+$. We mutated Asp-223 to leucine and the mutant YADH was expressed in yeast and characterized for the coenzyme specificity. The turnover numbers of mutant enzyme for $NAD^+$ and ethanol were decreased 3.5- and 4.8-fold compared to wild-type enzyme, respectively. Contrastively, catalytic specificity for $NADP^+$ was increased 13-fold. As a result, the mutant YADH also employed $NADP^+$ as a coenzyme.

• 한국임상수의학회지
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• 제38권6호
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• pp.299-304
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• 2021

A 3-year-old female Connemara pony was referred to Jeju National University Equine Hospital because of weight loss and prolonged anorexia. On admission, heart and respiratory rates were slightly elevated while body temperature was within the normal range. The color of the conjunctival and oral mucosa was yellowish pink. The blood chemistry results showed that total bilirubin, aspartate aminotransferase, and gamma-glutamyl transferase levels were remarkably elevated while blood urea nitrogen was within the reference range. Transcutaneous abdominal ultrasound revealed an enlarged right lobe of the liver with prominently increased parenchymal echogenicity, reduced hepatic vessels, and rounded caudal border. The pony was tentatively diagnosed with chronic hepatitis and icterus; rest and supportive treatments were provided. Clinical signs aggravated on day 2 with hind quarter paresis and cranial nerve signs such as circling, drooping, jerking, and head pressing against walls. Recumbency and generalized ataxia (grade 5/5) were shown on day 3. Plasma ammonia concentration on day 3 was as high as 656 µmol/L. Necropsy and histopathologic examinations strongly supported a diagnosis of hepatic encephalopathy. This case of hepatic encephalopathy exhibited rapid progress from low to terminal grade within 4 days in a Connemara pony. The results provide well-established clinical and pathological data for future application.