Genetic polymorphisms within immunity-related candidate genes in pigs have been identified to control variations in immune functions and/or disease resistance. It has become necessary to evaluate the effects of other genetic markers of economically important traits prior to introducing them into marker-assisted selection programs. In this study, polymorphisms of porcine genes coding Interferon-induced Gunylate binding protein 1 (GBP1), GBP2, CD163, and CD169 were investigated for their association with growth and meat quality traits in a Korean native pig breed -Yorkshire inter-crossed F2 pig population (KY-F2). KY-F2 animals (n=346) have been successfully used for linkage mapping to identify quantitative loci that control meat quality, growth, and immunity traits. In our results, polymorphisms in genes GBP1 and GBP2 showed association with pig growth rate as well as meat quality traits such as crude fat, drip loss, and meat color (yellowness) in the KY-F2 population. The polymorphism in gene CD163 only showed association with crude fat, as a meat quality trait. CD169 gene was associated with pork tenderness. In conclusion, four immune-related genetic markers were validated for their association with growth and meat quality traits to gauge their potential use in a swine selection program. The results warrant further studies in other commercial pig populations.

Osteoclasts originated from hematopoietic stem cells are multi-nucleated cells that can resorb the bone matrix. Receptor activator of nuclear factor kappa-B (RANK)/RANK ligand (RANKL) signaling pathway is crucial for the differentiation and activation of osteoclasts. In this study, we investigated for the first time whether or not RANKL induced mitogen- and stress-activated kinase 1 (MSK1) phosphorylation at Ser 376. Activation of MSK1 was detected as soon as 5 min after RANKL stimulation and sparsely detected at 30 min after stimulation. RANKL-induced MSK1 phosphorylation occurred in a dose-dependent manner. MSK1 is known as a downstream signaling molecule of cAMP-dependent protein kinase (PKA). Treatment with the PKA inhibitor H89 significantly suppressed c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) induction upon RANKL stimulation. In addition, cAMP response element-binding protein (CREB) phosphorylation was extremely inhibited by H89 treatment. Mitogen-activated protein kinases (MAPKs) have been investigated for induction of MSK1 phosphorylation. Specific signaling pathway inhibitors for p38 and extracellular signal-regulated kinases (ERKs) significantly blocked RANKL-induced MSK1 activation. Finally, as a downstream effector of the p38-MSK1 pathway, c-Fos transcriptional activity was determined. RANKL-mediated elevation of c-Fos transcriptional activity was significantly suppressed by p38 inhibitor. Moreover, a dominant negative form of CREB suppressed activation of NFATc1. In conclusion, RANKL-stimulated MSK1 phosphorylation could play a role in induction of NFATc1 through CREB and c-Fos activation as a downstream molecule of p38, ERK MAPKs, and PKA. Our results support basic information for the development of osteoclast specific inhibitors.

Sucrose and alcohol are rewarding and appetitive. They are occasionally over-consumed and cause addiction. The parabrachial nuclei (PbN) are the second taste relay in the central taste pathway. The nucleus accumbens (NAcc) is an important neural substrate in the reward system. Intake of sucrose or alcohol induces dopamine release in the NAcc. Although alcohol is not classified as a taste stimulus, a substantial number of sucrose-responsive neurons in the PbN respond to stimulation by alcohol on the tongue. In the present study, we investigated whether or not application of 0.5 M sucrose, 10% ethanol (EtOH), mixture of sucrose and EtOH, and double-distilled water (DDW) to the tongue induces c-Fos-like immunoreactivity (cFLI) in the PbN and NAcc. We also examined whether or not the number of cFLI following sucrose/EtOH is comparable to the number of cFLIs following sucrose and EtOH, respectively. Male Sprague-Dwaley rat was anesthetized with a mixture of Zoletil and Rompun while stimulation solution was applied to the anterior tongue. The rat was sacrificed by perfusion, and the fixed brain was sectioned and immunostained. Data from a total of 18 animals were analyzed. The number of cFLI following stimulation with sucrose and/or EtOH was greater than that of DDW in the PbN. Numbers of cFLI following sucrose, EtOH, and sucrose/EtOH were not significantly different from each other in the PbN. The number of cFLI in response to stimulation solution was not different from that of DDW in the NAcc. The result of the present study suggests that not only sucrose but also EtOH activates some neurons in the PbN, and that some pontine neurons possibly respond to both sucrose and EtOH.

Insulin detemir is a long-acting basal insulin analogue recently introduced in veterinary medicine for treatment of canine diabetes mellitus. As there are only limited studies in dogs, long-term evaluation of insulin detemir in veterinary medicine is required. In this study, we investigated trends in12-hour blood glucose concentration during hospitalization and evaluated initial and following doses of insulin detemir for several months in six diabetic dogs. The mean levels of blood glucose over 12-hour periods were between 113.5 to 327.2 mg/dL, and the average glucose nadir was 103 mg/dL in the six dogs. The dogs were treated with a mean dosage of 0.24 U/kg of insulin detemir, but hypoglycemia was observed in four of the dogs at the first monthly follow-up. Thus, insulin doses were adjusted according to the nadir levels of glucose observed during the follow-up periods (range, 1 to 16 months). The total range of insulin doses throughout the study period was between 0.1 and 0.4 U/kg. Changes in insulin doses in each dog during the follow-up period were not variable. We suggest that insulin detemir might be not only an alternative choice against traditional insulin for patients with insulin resistance or concurrent disease but also an effective home therapy medication in canine patients with DM. This study could help inform veterinary practitioners regarding the use of insulin detemir for canine insulin-dependent DM.

Canine respiratory coronavirus (CRCoV) is commonly associated with canine kennel cough worldwide. Clinically infected dogs present coughing, sneezing, and nasal discharge. Severe infections may progress to pneumonia. Through serological surveys, CRCoV has been identified as a worldwide pathogen found in the respiratory tracts of dogs suffering from mild or severe respiratory disease. In this study, three dogs were obtained from a dog kennel. Over the previous 5 days, the dogs showed coughing, sneezing, and nasal discharge. To detect the etiologic pathogen, we performed multiplex RT-PCR (mRT-PCR) to amplify the genes encoding canine influenza virus matrix protein, canine distemper virus nucleocapsid protein, and CRCoV spike protein. Dot blotting was achieved with a CRCoV-specific probe. Nasal-secreting CRCoV was detected by the 442 bp CRCoV-positive PCR reaction in the nasal swabbing samples from dogs. Further, CRCoV-positive reactions by dot blot hybridization were detected in the nasal swabbing samples from dogs. In conclusion, we detected CRCoV in kenneled dogs with respiratory disease in Korea. Multiplex RT-PCR was able to detect successfully CRCoV infection in dogs. We suggest that mRT-PCR would be useful and effective for monitoring CRCoV infection in various kinds of dogs.

Neural crest and placodes share a number of important features, pointing to a possible common evolutionary origin. They both arise from the neural plate border, which is the boundary between the non-neural ectoderm and neural plate. The transcription factor Sox9 has been implicated in neural crest and otic placode induction in several species. To investigate the differential regulation of neural crest and otic placode induction by Sox9, a gain of function assay was performed using a hormone-inducible version of the Sox9 construct at different doses and time periods. Sox9 was expressed in both neural crest and otic placode cell populations in the same stage embryos by in situ hybridization. Using a gain of function approach, increased expression of neural crest marker (Snail2) and otic placode marker (Pax8) in Sox9-overexpressed embryos was observed. Higher dose of Sox9 reduced or eliminated both neural crest and placode cells in the embryos. Interestingly, otic placodes cells were more strongly affected as compared to neural crest cells. So, optimal dosage and timing of Sox9 expression are important for the development of the neural crest and otic placode. The development of the neural crest and otic placode are affected by Sox9 in a time- and dose-dependent manner.

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin–Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.

Antibiotics have been used to prevent disease, promote growth rate, and improve feed efficiency. However, the use of antibiotics in livestock has been restricted worldwide due to problems such as bacterial resistance. Therefore, probiotics among alternatives to antibiotics have gained attention in the livestock feed industry these days. This study was conducted to investigate the effects of dietary supplementation with probiotic 379D on safety, growth rate, and feed efficiency. In this study, bacterial strain 379D was isolated from soil and identified as a Bacillus sp. according to 16S rRNA sequence analysis. In an in vitro test, in-gel activity assay and antimicrobial susceptibility test were conducted to evaluate 379D. In an in vivo study, 379D was administered at concentrations of 0.1% and 1% to broiler chickens for 28 days. The results of in-gel activity assay and antimicrobial susceptibility test showed that strain 379D had broad spectrum antimicrobial activity. Furthermore, no adverse 379D-related effects were observed in 0.1% and 1% groups. Feed efficiency was higher in the 379D-treated groups than in the control group. In conclusion, 379D is expected to be used as a safe alternative to antibiotics in a feed supplement and will improve feed efficiency in broiler chickens.

A 10-year-old castrated male Korean shorthair cat weighing 4 kg was referred with signs of insulin-resistant diabetes mellitus based on clinical signs of polyuria, polydipsia, and polyphagia. Diagnosis of pituitary-dependent hyperadrenocorticism (PDH) was made based on results of an adrenocorticotropic hormone stimulation test and a dexamethasone screening test. In addition, plasma concentrations of insulin-like growth factor 1 (IGF-1) increased. Radiography, ultrasonography, and computed tomography (CT) revealed hepatomegaly, renomegaly, and adrenomegaly affecting both adrenal glands as well as multiple cysts in a generally enlarged pancreas. Magnetic resonance imaging (MRI) showed that the cat’s pituitary gland was enlarged. The pituitary gland had a predominantly unilateral extension to the left. The signal intensity of the pituitary gland on precontrast T1 weighted images was hypointense compared to that of soft tissue and hyperintense compared to that of cerebrospinal fluid. On T2 weighted images, the pituitary gland was predominantly hypointense with a hyperintense rim. Contrast enhancement of the pituitary gland was not evident, and a mild degree of ring-like enhancement was seen. In addition, mild peritumoral edema was present. This is the first report of a cat with suspected double adenoma of the pituitary gland on the basis of compatible clinical signs, increased serum IGF-1 concentration, PDH, CT images, and MRI findings in diabetic cats with insulin resistance.

The brachiocephalic muscle in domestic mammals is formed as a homology of the sternocleidomastoid muscle and the clavicular part of the deltoid muscle since it results from reduction of the clavicle as a clavicular intersection. The cranial insertions of the brachiocephalic muscle vary among species in domestic mammals. In the dog, the brachiocephalic muscle comprises three parts, which arise from the clavicular intersection and insert at the humerus, the dorsal cervical raphe, and the mastoid process of the temporal bone. These three parts are named the cleidobrachial muscle, the cervical part of the cleidocephalic muscle, and the mastoid part of the cleidocephalic muscle, respectively. This complexity could confuse veterinarians and complicate surgical procedures in this area. Information about the normal structure of this muscle, and any variation therein, would help to avoid such situations. During dissections of a male cross-breed dog, we found that the brachiocephalic muscle had two bellies located on the mastoid part of the cleidocephalic muscle that extended from the clavicular intersection to the wing of the atlas and the mastoid process of the temporal bone. They were innervated by the accessory nerve and the ventral branches of the second, third, and fifth cervical nerves, and they were supplied by the ascending branch of the superficial cervical artery. These bellies were considered to be a rare variation of the muscle. This is the second report of a brachiocephalic muscle variation in a dog, in which the mastoid part of the cleidocephalic muscle was made of two bellies inserted independently. Such variations should be considered during anatomical dissections and surgical procedures.

The levonorgestrel-releasing intrauterine system (LNG-IUS) is used for contraception and treatment of heavy menstrual bleeding as well as endometrial hyperplasia and early endometrial carcinoma. A 48-year-old woman visited an Internal Medicine outpatient clinic due to significantly elevated CA-125 and CA-19-9 levels in a routine health examination. She had been using LNG-IUS for 3 years. Before LNG-IUS insertion, she suffered from heavy menstrual bleeding and severe dysmenorrhea. Her endometrial sampling and ultrasonographic imaging showed no evidence of endometrial carcinoma at the time of LNS-IUS insertion. After insertion, she complained of neither abnormal uterine bleeding nor dysmenorrhea. She received a routine health check-up every year and showed results within normal range until last year. To rule out pancreatic cancer due to significantly elevated CA-19-9 levels, her physician performed positron emission tomography–computed tomography, which demonstrated increased FDG uptake in the endometrial cavity. We obtained endometrial biopsy and found endometrial carcinoma in her uterus and performed radical hysterectomy with bilteral pelvic lymphadectomy. Permanent pathology confirmed endometrial carcinoma with lymph node metastasis. She received concurrent chemoradiation therapy. We emphasize the necessity of regular follow-ups with ultrasonography and assessment of serum tumor markers for the early detection of endometrial carcinoma, although rare, in women using LNG-IUS, including those without abnormal uterine bleeding.

Glycoproteins isolated from fruit bodies and mycelial cultures of mushrooms exhibit anti-carcinogenic actions in human cancer cells and animal tumor cells by induction of apoptosis. Here, we report that isoflavone-conjugated glycoproteins (designate Gluvone), exhibit strong anti-carcinogenic effects on human breast cancer MCF-7 cells by induction of apoptosis. Gluvone with 9.4 kDa of molecular weight was isolated from submerged-liquid culture of Agaricus blazei mycelia (ABM) in soy flake-containing liquid medium. MCF-7 cells were incubated with various amounts of Gluvone (0~250 μM) for a period of 6 days. Gluvone exhibited anti-proliferative actions in a dose-dependent manner and 62% growth inhibition at 200 μM for 4 days relative to control. Hoechst 33258 staining analysis revealed that Gluvone induced formation of apoptotic bodies. Gluvone was associated with down-regulation of anti-apoptotic Bcl-2 protein expression as well as up-regulation of pro-apoptotic Bax protein expression. Gluvone treatment induced proteolytic activation of caspase-9 and caspase-3 through cytochrome c release from mitochondria to cytosol as well as concomitant degradation of poly (ADP-ribose) polymerase (PARP). In addition, Gluvone induced activation of caspase-8. Taken all together, these results indicate that the anti-proliferative effect of Gluvone is associated with induction of apoptotic cell death through the mitochondrial dysfunction pathway mediated by enhancement of Bax protein expression and suppression of Bcl-2 protein expression.

The ubiquitous Na, K-ATPase is a membrane-bound ion pump located in the plasma membrane in all animal cells and plays an essential role in a variety of cellular functions. Studies in several organisms have shown that this protein regulates different aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Na, K-ATPase is an important factor for retinal development, and combinations of the isoforms of each of its subunits are expressed in different cell types and determine its functional properties. In this study, we performed RT-PCR assay to determine temporal expression and in situ hybridization to determine spatial expression of Na, K-ATPase β2 isoform (atp1b2) in Xenopus laevis. Focusing on retinal expression to distinguish the specific expression domain, we used retinal marker genes sox4, sox11, vsx1, and pax6. Xenopus atp1b2 was expressed from late gastrulation to the tadpole stage. Using whole mount in situ hybridization, we showed that Xenopus atp1b2 was expressed broadly in the eye, the whole surface ectoderm, and gills. In situ hybridization on sections revealed detailed and specific expression in the outer nuclear layer of the retina, which consists of two major classes of photoreceptors, rods and cones, surface ectoderm, pharyngeal epithelium, and gills. These findings indicate that atp1b2 may play an important role for the development of Xenopus retina.

Toxoplasmosis is an important cause of foodborne, inflammatory, as well as congenital abnormalities. There is an urgent need for safe and effective therapies to eliminate or treat this cosmopolitan infectious disease. A medicinal herbal plant, Meliae fructus, has been used to soothe the liver and kills worms in Chinese medicine. In this study, Meliae fructus ethanol extract was examined and screened for its anti-T. gondii activity. For anti-T. gondii activity screening, in vitro study of Meliae fructus extract using tachyzoit of T. gondii RH strain-infected HeLa cells was performed. Further, in vivo anti-T. gondii study using a mouse infection model was conducted. Safety of herbal compounds was evaluated in SD rats by treatment with Meliae fructus extract for 28 days. As a result, selectivity of Meliae fructus ethanol extract was 5.85, which was higher than sulfadiazine selectivity (2.06). We also performed an in vivo study to evaluate the anti-T. gondii activity of Meliae fructus extract in a mouse model. The inhibition rate of Meliae fructus extract was as high as that of sulfadiazine. These results demonstrate that Meliae fructus can successfully cure T. gondii infection and could be a promising native herb treatment for prevention of T. gondii infection.

Characteristics and immuno-modulatory effects of Enterococcus (E.) faecium JS1-8 isolated from Kimchi were investigated for potential probiotic use. We measured their acid, bile, and heat tolerances, adhesion properties in Caco-2 cells, antimicrobial activity against pathogenic bacteria, and bacteriocin-like substance-producing activity. Immuno-modulatory effects of E. faecium JS1-8 were measured by determination of nitric oxide (NO), nuclear factor-κB (NF-κB), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) production in RAW 264.7 cells or RAW BLUE cells. JS1-8 survived at pH 2.0 for 2 hr and showed tolerance to 0.3% oxgall bile salt, and it survived after exposure for 5 min at 80°C. JS1-8 showed high antimicrobial inhibition zones to Staphylococcus aureus (460 mm), Listeria monocytogenes (310 mm), Salmonella enteritidis (280 mm), and E. coli (150 mm). Bacteriocin-like substance produced by JS1-8 showed a broad spectrum of activity against Listeria monocytogenes KCCM 40307 and Lactobacillus sake KCCM 40264. Low concentration (1 × 107 CFU/mL) of heat-killed E. faecium JS1-8 induced statistically higher production of NO than Lactobacillus rhamnosus GG (LGG), which is a well-known immuno-modulatory lactic acid bacteria. Low and high (5 × 107 CFU/mL) concentrations of JS1-8 induced statistically higher production of NF-κB than that produced by LGG. We also found that JS1-8 increased production of pro-inflammatory cytokines such as IL-1β and TNF-α in a concentration-dependent manner. As a result, E. faecium JSI-8 could be used as a useful probiotic for controlling pathogens and enhancing host immune responses.

Skin is the outermost organ and acts as a barrier between the organism and environment. Skin protects the organism from environmental insults, such as chemicals, pathogens, and UV light. Much of the protective function of skin is dependent on the epidermis, a multi-layered epithelium that is composed of various cell types such as keratinocytes and melanocytes. Keratinocytes produce protective components through a sophisticated differentiation process. Disturbance of keratinocyte differentiation is related to several skin diseases such as psoriasis and atopic dermatitis. In this study, we prepared extract of combined medicinal plants (ECMP) consisting of Taraxacum platycarpum H. Dahlstedt, Heartleaf Houttuynia, Glycyrrhiza uralensis Fischer, and root bark of Ulmus davidiana. We demonstrated that ECMP enhanced keratinocyte differentiation and barrier functionality using an in vitro cell culture system and in vivo animal test. Treatment of cultured keratinocytes with ECMP resulted in induction of keratinocyte differentiation, as evidenced by increased differentiation markers such as involucrin, loricrin, and filaggrin. In line with these results, ECMP decreased proliferation of keratinocytes cultured in vitro. ECMP applied topically to tape-stripped mouse skins accelerated reduction of transepidermal water loss (TEWL), indicating fast recovery of barrier function. Immunohistochemistry showed that ECMP increased the filaggrin level in tape-stripped mouse skins. These results suggest that ECMP may be applicable for keratinocyte differentiation-related skin diseases.

Salsola komarovi Iljin is a halophyte and herbaceous annual native to the sand dunes and beaches of Japan, northern China, Sakhalin, and Korea. The plants have been known as an ecologically important species for enhancing formation of sand dunes in Korea. The purpose of this study was to examine the anti-gastric ulcer effect of Salsola komarovi Iljin halophyte in an HCl-ethanol-induced gastritis model. SD rats (7-weeks-old) were divided into normal (I, n=10), control (II, 60% HCl-ethanol + water, n=10), 60% HCl-ethanol + Ranitidine 300 mg/kg (III, n=10), 60% HCl-ethanol + Salicornia herbacea L. 500 mg/kg (IV, n=10), 60% HCl-ethanol + 50% alcohol extract of Salsola komarovi Iljin 500 mg/kg (V, n=10), and 60% HCl-ethanol + water extract of Salsola komarovi Iljin 500 mg/kg (VI, n=10) groups. Salsola komarovi Iljin significantly suppressed gastric lesions and ulcers in the 60% HCl-ethanol-induced gastric model. Especially, 500 mg/kg of 50% alcohol extract of Salsola komarovi Iljin showed significant inhibitory effects against gastritis. Especially, 50% alcohol extract of Salsola komarovi Iljin 500 mg/kg showed a significantly inhibitory effect, which was more potent than that of 300 mg/kg of Ranitidine. In histopathological analysis of the animal model, Salsola komarovi Iljin attenuated gastric ulcer formation. Our results suggest that Salsola komarovi Iljin has inhibitory effects against gastritis and gastric ulcers and could be developed as a new anti-gastric ulcer agent.

Canine atopic dermatitis (CAD) is an allergic skin disease with characteristic clinical features associated with immunoglobulin E (IgE) antibodies. Identification of the causative allergens is the diagnostic goal, which is essential to treat and manage CAD patients. CAD is commonly associated with environmental allergens surrounding the patients. For this reason, it is important for diagnostic tests to select allergens that are related to the environment of each country and each province. There are two main allergen-specific tests, serological IgE test (SAT) and intradermal skin test (IDT). SAT did not show direct cutaneous reaction but did show serological reaction against allergens. However, SAT is simpler and more convenient than IDT in small animal practice. In this study, we selected domestically prevalent allergens for SAT, including 60 food allergens and 60 inhalant allergens, and tested eight dogs tentatively diagnosed with CAD based on Favrot’s criteria. Furthermore, IDT was performed on four dogs from the SAT group for comparison of SAT and IDT, and the results were very similar. In SAT, four types of mites (Bloomia tropicalis, Glycophagus domesticus, Euroglyphus maynei, and mite mixture 1 Korea; house dust mites), four types of molds (Botrytis cinerea, Alternaria alternata, mold fungi mixture 11, mold fungi mixture), and one type of pollen (tree pollen mix 3 Korea) induced a reaction in more than half of dogs tested. In IDT, all four dogs reacted positively to Dermatophagoides farinae, and three reacted positively to Dermatophagoides pteronyssinus and house dust. The mean agreement rate between SAT and IDT in this study was 76.3%. This is the first trial to apply local allergens for SAT in Korean veterinary medicine, and it might play an important role for diagnoses and management of animal allergic diseases.