Pharmacokinetics and effects on clinical and physiological parameters following a single bolus dose of propofol in common marmosets (Callithrix jacchus)

The objectives of this study were (a) to establish a population pharmacokinetic model and (b) to investigate the clinical and physiological effects of a single bolus dose of propofol in common marmosets. In Study 1, pharmacokinetic analysis was performed in six marmosets under sevoflurane anaesthesia. 8 mg/kg of propofol was administrated at a rate of 4 mg kg^?1 min^?1. Blood samples were collected 2, 5, 15, 30, 60, 90, 120 or 180 min after starting propofol administration. Plasma concentration was measured, and population pharmacokinetic modelling was performed. A two‐compartment model was selected as the final model. The population pharmacokinetic parameters were as follows: V₁ = 1.14 L, V₂ = 77.6 L, CL₁ = 0.00182 L/min, CL₂ = 0.0461 L/min. In Study 2, clinical and physiological parameters were assessed and recorded every 2 min after 12 mg/kg of propofol was administrated at a rate of 4 mg kg^?1 min^?1. Immobilization was sustained for 5 min following propofol administration without apparent bradycardia. While combination of propofol and sevoflurane caused apnoea in Study 1, apnoea was not observed following single administration of propofol in Study 2. These data provide bases for further investigation on intravenous anaesthesia using propofol in common marmosets.     

Heterotrophic respiration causes seasonal hysteresis in soil respiration in a warm-temperate forest

To assess the effect of changes in organic litter stock on seasonal changes in heterotrophic respiration (R _H), soil respiration (R _S), and total ecosystem respiration (R _E), we measured seasonal changes in leaf litter respiration (R _LL) by the chamber method and estimated the seasonal change in total R _H using the RothC model in a warm-temperate mixed deciduous?Cevergreen forest in Japan. Both R _E and R _S had seasonal hysteresis and were higher in spring than at the same temperature during autumn. Under warm and humid conditions, the rate of decomposition of newly supplied leaf litter in one?year was high (60% loss). Consequently, R _LL and R _H were higher in spring after leaf drop, when more fresh material was available, than in autumn. In this study, 42 and 88% of the difference in R _E and R _S between spring and autumn (soil temperature 16?C18°C) could be accounted for by the difference in R _H, respectively, and 71% of the difference in R _H could be accounted for by the difference in R _LL. This study showed that seasonal changes in heterotrophic respiration (R _LL and R _H) could be a major factor in the seasonal hysteresis of R _E and R _S.     

Identification of c-kit mutations-independent neoplastic cell proliferation of canine mast cells

Gain-of-function mutations in the proto-oncogene c-kit have been considered the molecular mechanism of neoplastic proliferation of mast cells. However, the importance of c-kit gene mutations is not well evaluated in canine mast cell tumors (MCTs). In the present study, we established and characterized a mast cell line, HRMC, derived from a dog with MCT. We also examined c-kit mutations in HRMC cells and assessed an inhibitory effect of a tyrosine kinase inhibitor, STI571, on HRMC cells. HRMC cells had cytoplasmic metachromatic granules, chymase and tryptase, and expressed both KIT and FcepsilonRI on the cell surface. HRMC cells contained histamine and released beta-hexosaminidase through FcepsilonRI cross-linking and calcium ionophore stimulation. Nucleotide sequence analysis demonstrated no mutations in an open reading frame of c-kit cDNA and genomic DNA of the juxtamembrane domain of c-kit in HRMC cells. STI571 did not show any inhibitory effects on the proliferation of HRMC cells. These findings clearly demonstrated the existence of c-kit mutations-independent neoplastic canine mast cell proliferation. The growth factor-independent mast cell line established in this study might be valuable to explore novel mechanisms of c-kit mutations-independent neoplastic proliferation of mast cells in dogs.     

Comparison of the characters of the plaque-purified viruses from foot-and-mouth disease virus O/JPN/2000

At least two biotypes were observed at the 2nd passage stage after the isolation of Foot-and-mouth disease Virus (FMDV) O/JPN/2000 strain. These 2 types of viruses differed from their plaque phenotypes and were distinguishable by using a monoclonal antibody (MAb) 64G8 that was made for the FMDV O/JPN/2000 strain. One of these 2 biotypes formed small plaque (SP) and with immuno staining showed a positive reaction to MAb 64G8, while the other formed clear large plaque (LP) and did not react with MAb 64G8. The amino acid sequences of the capsid coding region (VP1-VP4) of the SP virus (SPV) and the LP virus (LPV) revealed two substitutions on the 133rd amino acid in VP2, and the 56th amino acid in VP3. These amino acid changes of SPV and LPV are Asn to Asp, Arg to His, respectively. The Arg of the 56th amino acid in VP3 that have been known as critical position of cell culture adapted virus. Only LPV showed high pathogenicity in suckling mice, and its LD(50) was calculated to be about 10(2) TCID(50)/0.1 ml. These results showed that the SPV that existed at the 2nd passage stage from isolation was a low virulence virus, which may suggest why the pathogenicity of O/JPN/2000 did not show clear symptoms in infected cattle.     

Chronic otitis externa with heat shock protein 70-positive intranuclear inclusion bodies in the ceruminous gland epithelium of a Chihuahua dog

A Chihuahua dog showed persistent itching in the right ear canal. Anti-inflammatory medicines and prednisolone were ineffective and total ear canal ablation was performed. Histological diagnosis was chronic otitis externa. Eosinophilic intranuclear inclusion bodies (Cowdry type A and full-type) were occasionally observed in the ceruminous gland epithelium. The inclusion bodies were negative for nucleic acid and ultrastructurally composed of fibrous structures (approximately 10 nm in width). Viral infection was initially suspected, but polymelase chain reaction tests did not detect the expected viral genes. Immunohistochemistry revealed that the inclusion bodies were positive for heat shock protein 70 (HSP70), suggesting that these bodies could be protein aggregates including HSP70. The etiology of this lesion has not been elucidated, but chronic inflammation may influence the cytoplasm-to-nuclear transportation of HSP70. To the best of our knowledge, this is the first report of canine chronic otitis externa with HSP70-positive intranuclear inclusion bodies.     

Antioxidative activity of heterocyclic compounds found in coffee volatiles produced by Maillard reaction

Typical heterocyclic compounds substituted with various functional groups found in Maillard reaction products were examined for antioxidant activity. Pyrroles exhibited the greatest antioxidant activity among all heterocyclic compounds tested. All pyrroles inhibited hexanal oxidation by almost 100% at a concentration of 50 microg/mL over 40 days. Addition of formyl and acetyl groups to a pyrrole ring enhanced antioxidative activity remarkably. Pyrrole-2-carboxaldehyde, 2-acetylpyrrole, 1-methyl-2-pyrrolecarboxaldehyde, and 2-acetyl-1-methylpyrrole inhibited hexanal oxidation by >80% at 10 microg/mL. Unsubstituted furan exhibited the greatest antioxidant activity among furans tested. Addition of all functional groups used in this study to furan decreased antioxidative activity. The antioxidant activity of thiophene increased with the addition of methyl and ethyl groups, but the addition of formyl or acetyl groups to thiophene decreased antioxidant activity. Thiazoles and pyrazines were ineffective antioxidants at all concentrations tested. Reaction of all heterocyclic compounds with hydrogen peroxide resulted in the formation of various oxidized products.     

Establishment of a quantitative ELISA for the measurement of allergen-specific IgE in dogs using anti-IgE antibody cross-reactive to mouse and dog IgE

As IgE plays a pivotal role in type I hypersensitivity-mediated allergic diseases, it is valuable to measure absolute quantity of serum antigen-specific IgE for clinical and research purposes. Here we describe a novel ELISA system that enables quantification of antigen-specific IgE in ng/ml in dogs. A newly developed monoclonal antibody (CRE-DM) was shown to recognize canine and mouse IgE equally in a dose dependent manner, but it did not recognize canine IgG. The reactivity of CRE-DM to canine IgE was also confirmed by an inhibition ELISA using canine IgE as an inhibitor and the maximum inhibition rate was 91.3%. In order to know whether canine IgE specific to an allergen could be quantitatively measured with an ELISA using CRE-DM, we established a quantitative ELISA that could measure canine IgE recognizing Cry j 1, one of the major allergens of Japanese cedar pollen. In this ELISA, a standard curve was created by using concentration-predetermined Cry j 1-specific monoclonal mouse IgE. According to the standard curve, the concentration of Cry j 1-specific IgE in dogs that were experimentally sensitized to Japanese cedar pollen could be calculated and determined in ng/ml. The specificity of the Cry j 1-specific IgE ELISA using CRE-DM was also confirmed by inhibition ELISA using canine IgE as an inhibitor and the inhibition rate was 97.0%. Reproducibility of the ELISA in three independent assays was determined using groups of pooled canine sera whose Cry j 1-IgE titers ranged from 155.9 to 888.2 ng/ml. Intra- and inter-assay reproducibility was determined with coefficient of variation ranging between 3.1-5.2% and 2.2-8.0%, respectively. These results demonstrated that the ELISA utilizing CRE-DM was a specific, reliable and robust new laboratory test that could quantify absolute amount of antigen-specific IgE in canine serum. The ELISA will serve as a useful tool in the clinics to evaluate the change of serum IgE titers during anti-allergic treatments as well as during seasonal fluctuation of allergen exposure.     

Isolation and characterization of a novel immunomodulatory alpha-glucan-protein complex from the mycelium of Tricholoma matsutake in basidiomycetes

Tricholoma matsutake, a high-class edible mushroom in Japan, has been reported to have excellent biological activities, but difficulty in cultivating the fruit bodies and limited bulk availability have restricted detailed studies. We have developed a method of culturing in tanks, enabling the bulk supply of the mycelia. The preparation (CM6271) exerts modulative effects on the immune competence of mice and rats. In this study, a sodium hydroxide extract of CM6271 was defatted followed by fractionation with a combination of ion exchange chromatography and gel filtration in order to identify the components involved in the expression of the activity, and a single peak fraction (MPG-1) was obtained with reversed phase chromatography. MPG-1 was a glycoprotein (sugar:protein ratio, 94.3:5.7) with a relative molecular mass of 360 kDa, and the sugar moiety contained about 90% glucose. NMR spectra and methylation analysis revealed that the alpha-1,4-linkage was the predominant glucan linkage with alpha-1,6- and alpha-1,2-linkages in the minority. The amino acid composition in the protein moiety was rich in glutamine, alanine, asparagine, leucine, glycine, valine, serine, threonine, isoleucine, and proline. MPG-1 was resistant to degradation with amylase or protease. The oral administration of MPG-1 promoted, in a dose-dependent manner, the recovery of the mouse natural killer cell activity and serum IL-12 level that had been reduced by the loading of restraint stress. The dose of MPG-1 (25 mg/kg) required for the expression of the effect decreases to 1/12 of that of CM6271 (300 mg/kg). Furthermore, MPG-1 formed a complex with TGF-beta1 in vitro, modulating the biological activity of TGF-beta1 by binding to its active form. These results indicate that the mycelium of T. matsutake contains a novel alpha-glucan-protein complex with immunomodulatory activities.     

Evaluation of urine sulfated and nonsulfated bile acids as a diagnostic test for liver disease in dogs

OBJECTIVE: To evaluate 3 methods for measuring urine bile acids (UBA) and compare their diagnostic performance with that of the serum bile acids (SBA) test and other routine screening tests in dogs with hepatic disorders. DESIGN: Prospective study. ANIMALS: 15 healthy dogs, 102 dogs with hepatic disorders, and 9 dogs with clinical signs of hepatic disorders that were found to have nonhepatic disorders. PROCEDURES: Blood and urine samples were collected from sick dogs and healthy dogs for serum biochemical analyses, and determination of concentrations of SBA and UBA. Urine samples were obtained from 15 healthy dogs to establish an upper cutoff value for UBA concentrations. The UBA were measured by use of a quantitative-linked enzymatic colorimetric method. Three analytical modifications were evaluated; 1 quantified only urine sulfated bile acids (USBA), 1 only urine nonsulfated bile acids (UNSBA), and 1 quantified both (USBA plus UNSBA). The UBA values were standardized with the urine creatinine concentration. RESULTS: The UNSBA-to-creatinine ratio and USBA plus UNSBA-to-creatinine ratio tests had the best diagnostic performance of the UBA tests; each had a substantially higher specificity, slightly higher positive predictive value, slightly lower negative predictive value, and lower sensitivity than the SBA test. These UBA-to-creatinine values were positively correlated with SBA values. The USBA-to-creatinine ratio had poor sensitivity, indicating a low rate of bile acid sulfation in dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The UBA can be measured in dogs with sufficient repeatability and accuracy for clinical application. The UNSBA-to-creatinine ratio and USBA plus UNSBA-to-creatinine ratio identified dogs with hepatic disorders nearly as well as the SBA test.     

Identification of CpG oligodeoxynucleotide sequences that induce IFN-gamma production in canine peripheral blood mononuclear cells

Oligodeoxynucleotides containing the cytosine-phosphate-guanine (CpG) motif (CpG-ODNs) have been shown to induce T(H)1 immune responses in animals. Since the sequences of CpG-ODNs that induce T(H)1 responses are considered to vary among animal species, it is necessary to identify effective CpG-ODNs in each animal. In order to identify the sequences of CpG-ODNs that induce T(H)1 responses in dogs, mRNA expression and protein production of IFN-gamma were examined in peripheral blood mononuclear cells (PBMCs) from healthy dogs treated with 11 kinds of synthetic CpG-ODNs. One of the 11 CpG-ODNs (No. 2 CpG-ODN, 5'-GGTGCATCGATGCAGGGGGG-3') was shown to significantly increase mRNA expression and protein production of IFN-gamma in canine PBMCs in a manner dependent on the sequence of the CpG motif. This CpG-ODN also enhanced the expression of IL-12 p40 mRNA in canine PBMCs, whereas expression of IL-12 p35, IL-18, and IL-4 mRNAs was not induced by this CpG-ODN. These results indicate that this CpG-ODN was able to produce IFN-gamma by induction of T(H)1-skewed immune response in dogs. CpG-ODNs may be useful for inducing prophylactic and therapeutic immunity against allergic diseases, viral infection, and tumors in dogs.