Effects of cannabidiol without delta-9-tetrahydrocannabinol on canine atopic dermatitis: A retrospective assessment of 8 cases.

Objective: We aimed to examine the effects of cannabidiol (CBD)-containing hemp oil without delta-9-tetrahydrocannabinol (THC) as a supplemental treatment for canine atopic dermatitis (CAD), as well as its adverse effects, and effects on concurrent drug use in dogs. Animal: In this retrospective case series, 8 dogs with CAD were diagnosed by veterinary dermatologists certified by the Japanese Society of Veterinary Dermatology. Procedure: The medical records of dogs supplemented with CBD-containing hemp oil were evaluated with respect to signalment, physical examination, plasma C-reactive protein concentrations, pharmacologic management, the CAD Extent and Severity Index (4th iteration), and the Pruritus Visual Analog Scale. Results: Overall, CBD, used as a supplement in combination with other drugs, was well-tolerated over a wide dose range and decreased the occurrence of pruritus in dogs with CAD when ingested twice a day. Conclusion: This study provides the first report of supplementation with CBD without THC that was effective in controlling pruritic behavior in dogs with CAD. Clinical relevance: Further controlled studies are required to investigate the dose range, efficacy, and safety.

Effets du cannabidiol sans delta-9-tétrahydrocannabinol sur la dermatite atopique canine : évaluation rétrospective de huit cas. Objectif: Nous avons cherché à examiner les effets de l'huile de chanvre contenant du cannabidiol (CBD) sans delta-9-tétrahydrocannabinol (THC) en tant que traitement complémentaire de la dermatite atopique canine (CAD), ainsi que ses effets indésirables et ses effets sur les médicaments concomitants utilisés chez le chien. Animal: Dans cette étude rétrospective de cas, huit chiens atteints de CAD ont été diagnostiqués par des dermatologues vétérinaires certifiés par la Société japonaise de dermatologie vétérinaire. Procédure: Les dossiers médicaux des chiens supplémentés avec de l'huile de chanvre contenant du CBD ont été évalués en ce qui concerne le signalement, l'examen physique, les concentrations plasmatiques de protéine C-réactive, la gestion pharmacologique, l'indice CAD Extent and Severity Index (4ème itération) et le Pruritus Visual Analog Scale. Résultats: Dans l'ensemble, le CBD, utilisé comme supplément en association avec d'autres médicaments, a été bien toléré sur une large gamme de doses et a diminué l'apparition de prurit chez les chiens atteints de CAD lorsqu'il est ingéré deux fois par jour. Conclusion: Cette étude fournit le premier rapport de supplémentation en CBD sans THC efficace pour contrôler le comportement prurigineux chez les chiens atteints de CAD. Pertinence clinique: D'autres études contrôlées sont nécessaires pour étudier la gamme de doses, l'efficacité et l'innocuité.(Traduit par Dr Serge Messier).

Molecular characterization of feline melanocortin 4 receptor and melanocortin 2 receptor accessory protein 2.

Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein-protein interactions, and functions. We found high sequence homology (>88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells; stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.

Genetic and Phenotypic analyses of Calcineurin A subunit in Arthroderma vanbreuseghemii.

Calcineurin is a serine/threonine protein phosphatase that consists of catalytic (calcineurin A) and regulatory (calcineurin B) subunits. The conserved protein plays important roles in various biological processes. Drug combination of fluconazole and the calcineurin inhibitor (FK506) showed synergistic effects against dermatophytes. In the current study, we identified the calcineurin A homologous gene (TmcanA) in the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes). Knockdown mutants were produced from A. vanbreuseghemii, resulting in a defection in growth properties in accordance with dose of the suppressing reagent. The TmcanA gene restored the ability of calcineurin A-deficient Cryptococcus neoformans strain to grow at elevated temperatures. Repression of TmcanA at 37°C resulted in severely stunted growth, suggesting that this protein plays a role in tolerance to elevated temperatures. In addition, TMCANA showed an interaction with high osmolarity glycerol (HOG) signalling pathway by governing the secretion of a secondary metabolite. Moreover, expression of the hydrophobin A gene (TmHF) decreased significantly under the TmcanA-repressive condition, suggesting that TMCANA is involved in its regulation. In conclusion, calcineurin A is a multifunctional gene that is involved in the regulation of several biological processes and therefore is worth being considered as a drug target for treatment of dermatophytoses.

Effectiveness of feline body mass index (fBMI) as new diagnostic tool for obesity.

Feline body mass index (fBMI), BW/PCL, length from top of patella to end of calcaneus, was developed as a new diagnostic tool for obesity in cats. To evaluate the effectiveness of fBMI for obese cats in short-term, 6 cats were induced weight gain by over-feeding with high fat diet and then they were induced weight reduction by restrict-feeding with low fat diet to measure changes in fBMI and plasma metabolite concentrations and enzyme activities. BCS 3 is correlated with fBMI 24.6-32.0, BCS 4 is correlated with fBMI 33.1-37.1 and BCS 5 is correlated with fBMI 29.9-40.3, respectively. On the correlation coefficient analysis by Pearson's method (P < 0.05), positive correlations (r = 0.403) were seen between the fBMI and plasma triglyceride (TG) levels. From these findings, fBMI seems to be more sensitive and useful indicator for obesity diagnosis in cats.

Changes in fatty acid composition in tissue and serum of obese cats fed a high fat diet.

BACKGROUND: Obesity and overweight have been frequently observed in dogs and cats in recent years as in humans. The compositions of fatty acids (FAs) in the accumulated lipids in tissues of obese animals may have important roles in the process and mechanisms related to the onset of metabolic disorders. The purpose of this study was to evaluate the effects of a high fat (HF) diet, which contained a higher proportion of saturated FAs, on FA metabolism and distribution in obese cats. Cats (N = 12) were divided into control diet group (crude fat; 16.0 %) (n = 4) or a high fat (HF) diet group (crude fat; 23.9 %) (n = 8). The HF diet contained up to 60 % of calories from fat and was rich in stearic acid. Blood samples were collected at 0, 2, 4 and 6 weeks after the feeding. Adipose and liver tissues were collected at the 6(th) week after feeding. We performed analysis of histological findings and fatty acid composition in serum and tissues. RESULTS: Body weights of the cats significantly increased in the HF group. The increased activities of hepatic enzymes and the accumulation of lipid droplets were found in hepatocytes in the HF group at the 6(th) week after feeding. In this study, the stearic acid (C18:0)-rich HF diet contained less oleic acid (C18:1n-9) and more linoleic acid (C18:2n-6) than the control. However, the composition of oleic acid in the liver was higher, and those of stearic acid and linoleic acid were lower in the HF group at the 6(th) week after feeding. The higher oleic acid:stearic acid ratio suggests an increase in the conversion from saturated FA to mono-unsaturated FAs, which may reflect the hepatic storage of FAs as a relatively harmless form. CONCLUSION: The stearic acid-rich HF diet increased hepatic lipid accumulation accompanied by the increased of hepatic oleic acid, increased serum oleic acid and activation of hepatic enzymes. These findings could be an important sign of early stages of dyslipidemia and hepatic damage. Also, the higher oleic acid:stearic acid ratio might be related to the increased activity of SCD-1, which suggests that the stearic acid-rich HF diet evoked hepatic lipogenesis in the feline liver.

Comparison of plasma lipoprotein profiles and malondialdehyde between hyperlipidemia dogs with/without treatment.

BACKGROUND: The aim of this study is to compare metabolic parameters, malondialdehyde as a lipid oxidation marker, and lipid profiles between dogs with untreated hyperlipidemia and hyperlipidemia with treatment, in order to examine the usefulness of malondialdehyde and lipid profiles as diagnostic parameters at early stages of hyperlipidemia. RESULTS: Dog samples were collected from four different veterinary clinics across Japan from March to June 2013. They were separated into three groups: control, untreated hyperlipidemia based on temporally screening, and hyperlipidemia with current anti-hyperlipidemic (statins and fibrates) treatment. Triglyceride levels of untreated hyperlipidemia dogs were significantly higher than those of control dogs. ALT levels of hyperlipidemic dogs with treatment were the highest among three groups. VLDL and LDL of both cholesterol and triglyceride of untreated hyperlipidemia dogs were the highest among three groups. HDL1 levels in triglyceride of hyperlipidemia dogs with treatment were significantly higher than those of control and untreated hyperlipidemia dog. Malondialdehyde concentrations of untreated hyperlipidemia dogs were significantly higher than those of control and hyperlipidemic dogs with treatment. CONCLUSIONS: In this study, dogs with untreated hyperlipidemia clearly showed abnormal lipid status, whereas hyperlipidemic dogs under anti-hyperlipidemia treatment showed more normal lipid status suggesting the effectiveness of the therapy. Anti-hyperlipidemics (statins and fibrates) for dogs are also effective in relieving elevated levels of lipids and lipid oxidation. Plasma lipid (triglyceride and cholesterol) profiles and malondialdehyde are useful diagnostic tools for identifying early stages of untreatment hyperlipidemia in dogs.

Age effects on plasma cholesterol and triglyceride profiles and metabolite concentrations in dogs.

BACKGROUND: In dogs, occurrence of lipid metabolism disorders such as obesity and diabetes mellitus has increased markedly in recent years. Hyperlipidemia has been regarded as a common characteristic for obese animals and hyperlipidemic condition may be associated with inflammation, oxidative stress and lipid composition changes. In this study, we investigated the changes in plasma cholesterol and triglyceride (TG) profiles and metabolite concentrations in 24 dogs (young group: 0-7 years old, n = 12, aged group: 8-13 years old, n = 12). RESULTS: Plasma adiponectin (ADN) concentrations were significantly lower in aged dogs than those in young dogs (mean ± SD, 17.2 ± 10.0 µg mL-1 vs 29.3 ± 12.5 µg mL-1, respectively; P <0.05). Although there were no significant differences statistically, aged dogs showed significantly higher plasma alpha1- acid glycoprotein (alpah1-AG) levels compared to those in young dogs. Plasma cholesterol lipoprotein and TG lipoprotein were divided into four fractions by biphasic agarose gel electrophoresis technique. The levels of the third TG-lipoprotein fraction from the positive pole (TG Fraction 3) were significantly higher in aged dogs than in young dogs (mean ± SD, 143.0 ± 109.3 mg dL-1 vs 55.2 ± 31.3 mg dL-1, respectively; P <0.05). On the correlation coefficient analysis by Peason's method, moderate positive correlations were seen between the age and TG (r = 0.446, P = 0.029), TG Fraction 3 (r = 0.516, P = 0.010), malondialdehyde (r = 0.146, P = 0.043), alpha-1 AG (r = 0.448, P = 0.028) levels, respectively. Moderate negative correlations were seen the age and total cholesterol (TC) Fraction 2 (r = -0.446, P = 0.029), glucose (r = -0.637, P = 0.001), ADN (r = -0.408, P = 0.048), respectively. CONCLUSIONS: Present data suggest biochemical characteristics of lipid metabolism disorder may be affected by aging in dogs.

Shiitake mushroom powder supplementation increase antioxidative activity in dogs.

Introduction: The prevalence of age-related diseases, including obesity (a lipid metabolism disorder), increases with the increase in a dog's lifespan. Most of age-related diseases are associated with oxidative stress by excessive production of reactive oxygen species (ROS) from impaired mitochondrial functions. Safe and effective supplements with antioxidative and anti-inflammatory activities are required to prevent obesity and associated complications. Shiitake mushroom exhibit various functions including antioxidant activity. We investigated the effect of shiitake powder supplementation in healthy dogs. Methods: Shiitake powder was supplemented at a dose of 800 mg/kg body weight/day for 4 weeks. The dose was set as 0.60-0.65 mg/kg/day of eritadenine, a hypocholesterolemic factor. Results: The body weight and body condition score of the dogs did not change after shiitake supplementation. In contrast, plasma total cholesterol concentrations decreased and superoxide dismutase activity and leukocyte sirtuin1 mRNA expression increased significantly in the dogs that received the supplement. Discussion: Oral administration of shiitake powder increased antioxidative activity. The supplement may be useful in ameliorating the signs of age-related diseases, including obesity, in dogs.

Changes in plasma metabolite concentrations and enzyme activities in aging riding horses.

In older horses, basal metabolic rate decreases, and plasma metabolite and hormone concentrations related to energy metabolism change. The occurrence of age-related diseases, which increases in old animals, may enhance inflammatory reactivity (inflammaging). Finding the appropriate treatment for inflammaging at an early stage may prevent various age-related diseases. Changes in metabolite and hormone concentrations and enzyme activities involved in energy metabolism in the plasma of clinically healthy riding horses of various ages were measured to identify biomarkers of inflammaging (persistent low-grade inflammation that occurs with aging). All horses were clinically healthy, and their body condition scores (BCSs) were 4 or 5 (9-point scale). Plasma triglyceride (TG), total cholesterol (T-Cho), blood urea nitrogen (BUN), insulin concentrations, malondialdehyde (MDA), and serum amyloid A (SAA) concentrations generally increased with age. Adiponectin concentrations, plasma superoxide dismutase (SOD), and leukocyte AMP-activated protein kinase (AMPK) activities decreased, while plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), and glutathione peroxidase (GPx) remained unchanged as horses aged. Although riding horses that partake in continuous exercise seems to be less likely to develop inflammaging, horses over 17 years of age tend to show proinflammatory signs with disordered lipid metabolism. In riding horses, SAA, in combination with other markers, may be a useful biomarker for inflammaging and dysregulated lipid metabolism in aging horses.

Antitumor effect of bevacizumab in a xenograft model of canine hemangiopericytoma.

Canine hemangiopericytoma (CHP) is characterized by frequent local recurrence and increased invasiveness. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis in tumors. The aim of the present study was to investigate the effect of a single dose of bevacizumab on a xenograft model of CHP. VEGF protein was secreted from cultured CHP cells and interacted with bevacizumab. Bevacizumab treatment suppressed tumor growth by inhibiting tumor angiogenesis, whereas no significant differences were observed in the proliferation index and apoptosis rates of treated and untreated mice. Thus, bevacizumab had antitumor effects in a xenograft model of CHP.

Change in mRNA expression of sirtuin 1 and sirtuin 3 in cats fed on high fat diet.

BACKGROUND: Mammalian sirtuins are homologs to the yeast silent information regulator 2 (Sir2), which is an NAD-dependent deacetylase. Sirtuins are comprised of 7 proteins, and each has different target proteins. Sirtuin 1 (SIRT1) plays important roles in maintaining metabolic functions and immune responses, and SIRT3 protects cells from oxidative stress-induced cell death. Both SIRT1 and SIRT3 are regulated by metabolic status and aging. Hence, SIRT1 and SIRT3 have been researched in metabolic diseases, such as type 2 diabetes mellitus (DM), fatty liver, and heart diseases. Although these diseases have been increasing, there is little information about relation between the diseases and SIRT1 and SIRT3 in cats. Therefore we cloned SIRT1 and SIRT3 cDNA, examined mRNA expression in cat tissues, and investigated the changes in SIRT1 and SIRT3 mRNA expression in peripheral blood leukocyte of cats fed on HFD for 6 weeks. RESULTS: Cat SIRT1 and SIRT3 contained a catalytic core region and showed high sequence homology with other vertebrate SIRT1 (>61.3%) and SIRT3 (>65.9%) amino acids. Real-time polymerase chain reaction analyses revealed that high expression levels were observed in the liver and skeletal muscle for SIRT1 and in the heart for SIRT3 in cats. In addition, both cat SIRT1 and SIRT3 expression levels in the pancreas were different between individuals. Cat SIRT1 mRNA expression in peripheral blood leukocytes was significantly elevated in obese cats fed on HFD (P < 0.05). CONCLUSIONS: Cat SIRT1 and SIRT3 genes are highly conserved among vertebrates, and HFD feeding may be related to SIRT1 mRNA expression mechanisms in cat peripheral blood leukocytes.

Mechanism of insulin production in canine bone marrow derived mesenchymal stem cells.

Insulin is a critical hormone in the regulation of blood glucose levels and is produced exclusively by pancreatic islet beta-cells. Insulin deficiency due to reduced pancreatic islet beta-cell number underlies the progression of diabetes mellitus, prompting efforts to develop beta-cell replacement therapies. However, precise information on beta-cell replacement and differentiation in canines is limited. In this study, we established insulin-producing cells from bone marrow derived mesenchymal stem cells transiently expressing canine pancreatic and duodenal homeobox 1 (Pdx1), beta cell transactivator 2 (Beta2) and V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) using a gene transfer technique. Real-time PCR analysis revealed an increase in insulin mRNA expression of transfected cells. And ELISA revealed that insulin protein expressed was detected in cytoplasmic fraction. Insulin immunostaining analysis was performed and observed in cytoplasmic fraction. These results suggest that co-transfection of Pdx1, Beta2 and Mafa induce insulin production in canine BMSCs. Our findings provide a clue to basic research into the mechanisms underlying insulin production in the canines.

Effect of acupuncture on the energy metabolism of dogs with intervertebral disk disease and cervical disk herniation: A pilot study.

Thirteen dogs with intervertebral disk disease (IVDD) and 3 dogs with cervical disk herniation (CVDH) were examined to determine the effects of acupuncture on energy metabolism. Acupuncture points GV14, GV20-1, BL18, BL23, BL26, GB30, and ST36 were selected for IVDD, while GV14, GV20-1, GB20, and BL23 were selected for CVDH. All dogs except no.13 did not receive medication during acupuncture treatment. Acupuncture effects were evaluated based on the IVDD/CVDH evaluation scales in Oji 2015 and Tanaka and Nakayama 2015. Blood samples were taken before and 30 min after acupuncture treatment. Pyruvate and lactate concentrations, lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) activity, the MDH/LDH ratio (M/L ratio), and LDH isozyme electrophoretic patterns served as energy metabolism markers. In IVDD/CVDH dogs that showed improvements, plasma pyruvate concentrations significantly decreased, the M/L ratio increased, and the plasma LDH isozyme pattern changed from predominantly LDH5 to predominantly LDH1. These data suggest that local redox potential is improved and energy metabolism is increased in dogs with IVDD/CVDH after acupuncture treatment. Acupuncture treatments may activate the citric acid cycle and increase ATP production, followed by improvement of the disease. Future studies with a large sample size are needed to clarify this hypothesis.

Comparison of bone marrow and adipose tissue-derived canine mesenchymal stem cells.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis. RESULTS: Samples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs. CONCLUSION: Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.

Two chicken neuromedin U receptors: characterization of primary structure, biological activity and tissue distribution.

Neuromedin U (NMU) is a bioactive peptide that is involved in a variety of physiological functions. Two of its receptors, NMUR1 and NMUR2, have been identified and characterized in mammals. In this study, we performed cDNA cloning of chicken NMUR1 and NMUR2, and characterized their primary structure, biological activity, and expression patterns in chicken tissues. The chicken NMUR1 and NMUR2 cDNAs encoded 438 and 395 amino acid sequences, respectively. Chicken NMUR1 showed 54.8%-56.5% sequence identity with human, rat, and mouse NMUR1, and NMUR2 shared 67.3%-70.1% sequence identity with mammalian orthologs. Both chicken receptors have typical characteristics of G-protein-coupled receptors with seven transmembrane domains and the D/ERY motif. An increase in intracellular Ca(2+) mobilization was observed in HEK293 cells transfected with chicken NMUR1 or NMUR2 cDNA and treated with chicken or rat NMU. Real-time PCR analysis revealed that NMUR1 mRNA was preferentially expressed in the intestinal tissues such as the duodenum, jejunum, ileum, cecum, and colon/rectum, and brain regions such as the midbrain and optic lobe, and the ovary in adult hens. NMUR2 mRNA was exclusively expressed in the brain regions such as the cerebrum and midbrain. These results indicate that NMUR1 and NMUR2 mRNAs, which encode functional receptor proteins, are expressed in chicken tissues with different distribution patterns.

Evaluation of artificial pancreas technology for continuous blood glucose monitoring in dogs.

Artificial pancreas technology, involving "closed-loop" controls with real-time blood glucose monitoring, has been increasing in reliability as its potential for clinical use and application grows. One such device, based on this technology, is the STG-22 (Nikkiso Co., Ltd., Tokyo, Japan) artificial pancreas apparatus. In order to assess the reliability and accuracy of the device for measuring blood glucose, it is important to compare its readings to those obtained using a 'gold standard' method, such as the hexokinase method. Therefore, in the present study, canine blood [glucose] measurements using the STG-22 were compared to those obtained using a previously established commercial reagent, Quickauto-Neo GLU-HK. Furthermore, two different sample types (whole blood versus plasma constituent) were compared to determine which sample type results in more accurate and optimal readings with the STG-22. Given that the STG-22 was not primarily designed for canine blood samples, results for canine blood samples were not accurate. Measurements performed by the STG-22 with whole blood were significantly lower than reference [glucose] counterparts. Alternatively, an opposite trend was observed with plasma measurements that were significantly higher. A conversion format using the following formula, Hexokinase [glucose] = STG-22 [glucose] × 1.407 + 1.532, was observed with canine samples in our study.

Molecular characterization of free fatty acid receptors FFAR2 and FFAR3 in the domestic cat.

G protein-coupled receptors 41 and 43 were identified and characterized as free fatty acid receptors (FFAR) 3 and 2, respectively. FFAR2 and FFAR3 mediate short-chain fatty acids (SCFAs) as signalling molecules. The present study aimed to give molecular characterization of FFAR2 and FFAR3 in the domestic cat. High homology with that in other mammals was revealed by cDNA cloning of cat FFAR2 FFAR3. We analyzed the tissue distribution of cat FFAR2 and FFAR3 mRNA using quantitative polymerase chain reaction. The inhibition of intracellular cAMP concentrations was observed in cells transfected with cat FFAR2 or FFAR3 and treated with SCFAs. The activation of nuclear factor of activated T cells-luciferase reporter was only observed in cat FFAR2 transfected cells but not in FFAR3. Split luciferase assay (NanoLuc Binary Technology; NanoBiT) for FFAR2 or FFAR3 and Arrestin-3/ß-arrestin-2 revealed acetate-/propionate-induced recruitment to cat FFAR2 or FFAR3 in CHO-K1 cells. Our results indicate that FFAR2 and FFAR3 are functional receptor proteins that are expressed in cat tissues and show differential distribution patterns.

A novel rapid genotyping technique for Collie eye anomaly: SYBR Green-based real-time polymerase chain reaction method applicable to blood and saliva specimens on Flinders Technology Associates filter paper.

Collie eye anomaly (CEA) is a canine inherited ocular disease that shows a wide variety of manifestations and severity of clinical lesions. Recently, a CEA-associated mutation was reported, and a DNA test that uses conventional polymerase chain reaction (PCR) has now become available. The objective of the current study was to develop a novel rapid genotyping technique by using SYBR Green-based real-time PCR for future large-scale surveys as a key part in the strategy to eradicate CEA by selective breeding. First, a SYBR Green-based real-time PCR assay for genotyping of CEA was developed and evaluated by using purified DNA samples from normal, carrier, and affected Border Collies in which genotypes had previously been determined by conventional PCR. This real-time PCR assay demonstrated appropriate amplifications in all genotypes, and the results were consistent with those of conventional PCR. Second, the availability of Flinders Technology Associates filter paper (FTA card) as DNA templates for the real-time PCR assay was evaluated by using blood and saliva specimens to determine suitability for CEA screening. DNA-containing solution prepared from a disc of blood- or saliva-spotted FTA cards was available directly as templates for the real-time PCR assay when the volume of solution was 2.5% of the PCR mixture. In conclusion, SYBR Green-based real-time PCR combined with FTA cards is a rapid genotyping technique for CEA that can markedly shorten the overall time required for genotyping as well as simplify the sample preparation. Therefore, this newly developed technique suits large-scale screening in breeding populations of Collie-related breeds.

Rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs by real-time polymerase chain reaction with TaqMan minor groove binder probes.

Real-time polymerase chain reaction (PCR) with TaqMan minor groove binder (MGB) probes was examined to establish a rapid and reliable genotyping technique for GM1 gangliosidosis in Shiba dogs. This technique was applied to DNA samples extracted from the blood, umbilical cord, or postmortem liver tissue specimens, and to DNA-containing solutions prepared from blood and saliva that had been applied to Flinders Technology Associates filter papers (FTA cards). The amplification of the targeted sequence in all the samples was sufficient to determine the genotypes of GM1 gangliosidosis. Forty-seven DNA samples that had previously been obtained from blood or tissue specimens of Shiba dogs were examined using this real-time PCR technique, and the findings were consistent with the data obtained by the earlier PCR-restriction fragment length polymorphism (RFLP) assay. In addition, the use of this new technique in combination with FTA cards for sampling could markedly shorten the time required for genotyping, as well as simplify the procedure. Furthermore, in the present study, the results of a previous epidemiological screening of 96 Shiba dogs in the Czech Republic were rechecked by this real-time PCR technique using stored crude buccal cell DNA-containing solutions directly as DNA templates. The results provided clear-cut genotyping in all the samples although the earlier PCR-RFLP assay could not determine the genotype in all cases. In conclusion, this new real-time PCR technique is a simple, rapid, and reliable choice for large-scale screening to detect an abnormal allele indicating GM1 gangliosidosis in Shiba dogs.

Effects of Age on Inflammatory Profiles and Nutrition/Energy Metabolism in Domestic Cats.

BACKGROUND: Animals tend to increase in body weight and body condition score (BCS) with aging. Serum diagnostic markers related to energy metabolism may show changes even in healthy cats with aging. MATERIALS AND METHODS: Seventy domestic cats were recruited for this study. Based upon the modified AAFP-AAHA Feline Life Stage Guidelines, animals were divided into six groups: Junior (7 months-2 years), Prime (3 -6 years), Mature (7-10 years), Senior (11-14 years), Geriatric-obese (15 years ≤) and Geriatric-thin (15 years ≤). Their body condition scores (BCS) ranged from 3/9 to 9/9. Changes in metabolites, inflammatory markers, hormone concentrations and enzyme activities related to energy metabolism were investigated in serum of 70 domestic cats of various ages. RESULTS: Serum glucose (GLU) concentrations in the Mature, Senior, and Geriatric-obese groups were significantly higher than those in the Junior group. Serum amyloid A (SAA) concentrations in the Geriatric-thin group were significantly increased compared with the Junior group. SAA concentrations in the Geriatric-obese group tended to increase although there were no statistically significant differences. In the Mature, Senior, Geriatric-obese and Geriatric-thin groups, malate dehydrogenase/lactate dehydrogenase (M/L) ratio, an energy metabolic indicator, tended to decrease compared with the Junior group. In the Senior group, triglyceride (TG) concentrations were significantly increased compared with the Junior group. In the Geriatric-obese and Geriatric-thin groups, blood urea nitrogen (BUN) concentrations were significantly increased compared with the Junior group. In the Geriatric-obese group, albumin (ALB) concentrations were decreased compared with the Junior group. CONCLUSION: Aged domestic cats tend to increase in body weight and BCS. In addition, serum GLU, TG, SAA, and BUN concentrations increased and serum ALB concentrations and M/L ratio decreased. These diagnostic markers may be useful to detect small changes related to energy metabolism with aging that may cause obesity with light inflammation in healthy cats.