Acute effects of intravenous pimobendan administration in dog models of chronic precapillary pulmonary hypertension.

INTRODUCTION: Pulmonary hypertension (PH) is a progressive and potentially life-threatening disease. Several drugs are used for the treatment of dogs with precapillary PH. Pimobendan is an inotropic drug with phosphodiesterase 3 inhibitory and calcium-sensitizing effects. Pimobendan administration improved right ventricular (RV) function and lowered pulmonary arterial pressure in some human patients with precapillary PH. However, the efficacy of pimobendan in dogs with precapillary PH is unknown. ANIMALS, MATERIAL, AND METHODS: An implantable port device was percutaneously placed in the cranial vena cava of five laboratory beagles. Chronic embolic precapillary PH was induced via the repeated injection of microspheres every 1-2 days. Microsphere injection was continued until systolic pulmonary arterial pressure reached 50 mmHg. Right heart catheterization and echocardiography were performed at baseline and after injections of placebo and pimobendan (0.15 mg/kg). RESULTS: Repeated injections of microspheres caused an increase in pulmonary vascular resistance, a decrease in stroke volume, RV dilation, left ventricular (LV) and RV dysfunction, and RV dyssynchrony as assessed using echocardiography. Compared with placebo, pimobendan improved LV and RV function based on the LV Tei index from 0.48 to 0.38 (p=0.002) and the RV Tei index from 0.76 to 0.61 (p=0.008), as well as the stroke volume index from 29.4 to 36.7 ml/m2 (p=0.012), respectively. CONCLUSIONS: In dog models of chronic PH, intravenous pimobendan effectively improved RV and LV function and increased stroke volume. However, pimobendan administration did not decrease pulmonary arterial pressure or produce hypotension.

The Repeatability and Left Atrial Strain Analysis Obtained via Speckle Tracking Echocardiography in healthy Dogs.

INTRODUCTION: In left atrial (LA) strain-derived two-dimensional speckle tracking echocardiography, the reference intervals in healthy dogs can provide useful information to evaluate the LA function in dogs with heart disease. ANIMALS: Six laboratory beagles and 120 privately owned dogs without cardiac diseases were recruited. MATERIALS AND METHODS: The LA strain and strain rate (SR) and echocardiographic indices were obtained in dogs who underwent standard echocardiography and offline analysis for LA strain and SR measurement by speckle tracking echocardiography. RESULTS: The intra-observer within-day variations of strain variables showed adequate repeatability (coefficient of variation <20%). The mean values of strain were 25.37 for the LA reservoir function, 11.06 for the LA conduit function, and 14.17 for the LA booster-pump function; the strain was significantly correlated with the LA fractional volume change at each phasic function. The left atrial longitudinal strain during early ventricular diastole showed moderate correlation with the peak velocity of early diastolic transmitral flow (r = 0.5560) and ratio of peak velocity of early diastolic transmitral flow to peak velocity of late transmitral flow (r = 0.5515). In multiple regression analysis, only age was significantly related to the strain/SR and volumetric change indices, indicating conduit function. CONCLUSIONS: Left atrial speckle tracking echocardiographic analysis provided useful information to assess the LA function in healthy dogs. The influencing factors on strain and SR variables including the age, body weight, and heart rate should be considered in interpretation of these parameters in a clinical setting.

Epidemiology of massive hepatocellular carcinoma in dogs: A 4-year retrospective study.

Hepatocellular carcinoma (HCC) is the most common primary liver tumour in dogs. However, the clinical features and risk factors of HCC have not been confirmed. The objective of this study was to investigate the clinical features and risk factors for canine HCC. Medical records of 44 dogs diagnosed with HCC at Hokkaido University Veterinary Teaching Hospital between 2013 and 2017 were retrospectively reviewed. All dogs evaluated at the teaching hospital during the study period were used as the reference population for breed, age, sex predispositions or possible related factors for HCC, including concurrent disorders. Clinical characteristics of HCC were determined using propensity score matching analysis. The prevalence of HCC diagnosis was 0.96%. Multivariate analysis revealed that dogs diagnosed with HCC were significantly older (odds ratio [OR], 1.20; 95% confidence intervals [CI], 1.07-1.33) than the reference population. Welsh Corgis (OR, 3.68; 95% CI, 1.56-8.67) and Beagles (OR, 4.33; 95% CI, 1.58-11.90) were significantly predisposed to HCC. Twenty-seven of 44 dogs with HCC had at least one concurrent disorder. The most common concurrent disorder was hyperadrenocorticism (n = 10), and the adjusted odds of hyperadrenocorticism in dogs with HCC were 4.13 higher than those of the reference population (95% CI, 1.95-8.76). Propensity score matching analysis revealed that thrombocytosis (n = 30/43), increased alanine aminotransferase (n = 41/44), increased alkaline phosphatase (n = 42/44), and hypercalcemia (n = 13/32) were significantly associated with HCC diagnosis. The results of this study suggest that Welsh Corgis and Beagles are breeds with a predisposition for HCC and that hyperadrenocorticism might be a potential risk factor.

Transcranial Doppler Ultrasound Examination in Dogs with Suspected Intracranial Hypertension Caused by Neurologic Diseases.

BACKGROUND: Transcranial Doppler ultrasound examination (TCD) is a rapid, noninvasive technique used to evaluate cerebral blood flow and is useful for the detection of intracranial hypertension in humans. However, the clinical usefulness of TCD in diagnosing intracranial hypertension has not been demonstrated for intracranial diseases in dogs. OBJECTIVES: To determine the association between the TCD variables and intracranial hypertension in dogs with intracranial diseases. ANIMALS: Fifty client-owned dogs with neurologic signs. METHODS: Cross-sectional study. All dogs underwent TCD of the basilar artery under isoflurane anesthesia after magnetic resonance imaging (MRI). Dogs were classified into 3 groups based on MRI findings: no structural diseases (group I), structural disease without MRI evidence of intracranial hypertension (group II), and structural disease with MRI evidence of intracranial hypertension (group III). The TCD vascular resistance variables (resistive index [RI], pulsatility index [PI], and the ratio of systolic to diastolic mean velocity [Sm/Dm]) were measured. RESULTS: Fifteen, 22, and 13 dogs were classified into groups I, II, and III, respectively. Dogs in group III had significantly higher Sm/Dm (median, 1.78; range, 1.44-2.58) than those in group I (median, 1.63; range, 1.43-1.75) and group II (median, 1.62; range, 1.27-2.10). No significant differences in RI and PI were identified among groups. CONCLUSIONS AND CLINICAL IMPORTANCE: Our findings suggest that increased Sm/Dm is associated with MRI findings of suspected intracranial hypertension in dogs with intracranial diseases and that TCD could be a useful tool to help to diagnose intracranial hypertension.

Central fibroma in the ascending ramus of the mandible. Case report.

A case of central fibroma involving the mandible in a 58 year old woman is described. There was slight swelling of the left cheek and bone-hard bulging was detected on palpation but the patient had not complained of the swelling. The lesion was removed under general anaesthesia and then examined histopathologically. There was no sign of recurrence eleven months after the operation.

Brain-derived neurotrophic factor increases the stimulation-evoked release of glutamate and the levels of exocytosis-associated proteins in cultured cortical neurons from embryonic rats.

Differentiation and survival of neurons induced by neurotrophins have been widely investigated, but little has been reported about the long-term effect of brain-derived neurotrophic factor (BDNF) on synaptic transmission. Among many steps of neurotransmission, one important step is regulated release of transmitters. Therefore, the release of glutamate and GABA from cortical neurons cultured for several days with or without BDNF was measured by an HPLC-fluorescence method. Although BDNF had little effect on the basal release of glutamate, high K(+)-evoked release was greatly increased by BDNF. BDNF also tended to increase evoked release of GABA. Recently, several proteins involved in the step of "regulated release" have been identified. Thus, the effect of BDNF on the levels of these proteins was then investigated. Neurons were cultivated with or without BDNF, collected, and electrophoresed for western blotting. BDNF increased levels of synaptotagmin, synaptobrevin, synaptophysin, and rab3A, which were known as vesicle protein. Levels of syntaxin, SNAP-25, and beta-SNAP were also increased by BDNF. In addition, the numbers of cored and clear vesicles in nerve terminals or varicosities were also increased by BDNF. These results raise the possibility that BDNF increases regulated release of neurotransmitters through the up-regulation of secretory mechanisms.

BDNF increases the expression of neuropeptide Y mRNA and promotes differentiation/maturation of neuropeptide Y-positive cultured cortical neurons from embryonic and postnatal rats.

The effects of neurotrophic factor on the expression of neuropeptide Y (NPY) mRNA and on morphology of NPY-immunoreactive neurons were investigated. Brain-derived neurotrophic factor (BDNF) increased the expression of NPY mRNA in cultured cortical neurons from both embryonic and postnatal rats. BDNF also increased the number of NPY neurons. Furthermore, multipolar neurites from NPY neurons were observed in cultures treated with BDNF, whereas only monopolar and bipolar neurites were observed in control cultures. These results suggest that BDNF not only increases the expression of NPY mRNA but also promotes the differentiation/maturation of NPY ergic neurons both in number and morphology. NPY expression was strongly increased by neurotrophin-4/5 similarly to BDNF and neurotrophin-3 evoked a slight increase. In contrast, basic fibroblast growth factor, cilliary neurotrophic factor and interferon-gamma had no effect on NPY expression.

Detection of NG,NG-dimethylarginine dimethylaminohydrolase in the nitric oxide-generating systems of rats using monoclonal antibody.

In order to elucidate the biological role of NG,NG-dimethylarginine dimethylaminohydrolase (EC 3.5.3.18), we prepared monoclonal antibodies (mAbs) against the enzyme from rat kidney and examined the distribution of the enzyme in rats. Four mAbs have been obtained by the fusion of the spleen cells from BALB/c mouse immunized with the sodium dodecyl sulfate-denatured or native enzyme and P3X63Ag8U1 myeloma cells. All the mAbs were shown to bind to the denatured enzyme, but none of them could recognize the native enzyme. The occurrence of the enzyme protein in various rat tissues and cell systems such as peritoneal neutrophils and macrophages was examined using an immunoblotting technique with one of the mAbs. The immunoblotting analyses showed that the enzyme protein is widely distributed in rats, particularly, in kidney, pancreas, liver, brain, and aorta at high concentrations. Furthermore, the enzyme protein was clearly shown to exist in peritoneal neutrophils and macrophages. Since NG-monomethylarginine and NG,NG-dimethylarginine have been suggested to be specific blockers of the systems generating nitric oxide (NO), the above findings are of great interest in connection with the regulation of the NO production in such tissues and cell systems as aorta, brain, peritoneal neutrophils, and macrophages.

Preparation and characterization of monoclonal antibodies against 4-aminobenzoate hydroxylase from Agaricus bisporus.

A monoclonal antibody (mAb, A) recognizing the FAD-binding domain of 4-aminobenzoate hydroxylase (4-aminobenzoate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating, decarboxylating), EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, had been produced (Tsuji, H., Ogawa, T., Bando, N., Kimoto, M. and Sasaoka, K. (1990) J. Biol. Chem. 265, 16064-16067). In the present study, three other mAbs (B1, B2 and B3) against the enzyme have been further prepared in order to facilitate the structural characterization of the enzyme. The three new mAbs immunoblotted the enzyme. The four mAbs, including A, were specific for different epitopes on the enzyme. B1 and B2 immunoprecipitated the apoenzyme and the immunoprecipitation was inhibited in the presence of FAD, whereas B3 failed to immunoprecipitate the apoenzyme in the absence or presence of FAD. B1 and B2 competed with FAD for the binding to the apoenzyme. These findings show that B1 and B2 recognize the FAD-binding domain of the enzyme in analogy with A. The immunoblotting analyses of the peptides obtained from the enzyme by digestion with lysyl endopeptidase (EC 3.4.21.50) provided useful knowledge as to the location of the epitopes to the mAbs on the enzyme, suggesting that the FAD-binding domain of the enzyme can be located and characterized by detailed investigations on the location of the epitopes.

Investigation of the IgE-binding proteins in soybeans by immunoblotting with the sera of the soybean-sensitive patients with atopic dermatitis.

The IgE-binding proteins in soybeans were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractionated soybean proteins probed with the sera of the patients with atopic dermatitis. About 20% of the patients examined were shown to have specific IgE antibodies to soybean proteins. At least 16 soybean proteins with molecular weights ranging from about 70,000 to 14,000 were recognized by the sera of the patients: 10 major IgE-binding components were found in the 7S-globulin fraction, and the others mainly in the 2S-globulin and whey fractions. The IgE antibodies of the patients bound most strongly and frequently to a unique protein with molecular weight of about 30,000 in the 7S-globulin fraction, which appeared to be the major allergen in soybeans and was named as Gly m Bd 30 K. The proteins in the 11S-globulin fraction were scarcely recognized by the patients' sera and assumed to be less allergenic for the patients with atopic dermatitis.

Accumulation of 3-hydroxy-L-kynurenine sulfate and ethanolamine in urine of the rat injected with 1-aminoproline.

By intraperitoneal injection of 1-amino-L-proline, a vitamin B6 antagonist, two unidentified ninhydrin-positive compounds were excreted abnormally in rat urine. One of them was definitely identified as 3-hydroxy-L-kynurenine sulfate by chromatographic and spectroscopic analyses. The other was proved to be ethanolamine. This is the first report on the abnormal excretion of ethanolamine in urine of the vitamin B6-deficient rats.

Dimethylarginine:pyruvate aminotransferase in rats. Purification, properties, and identity with alanine:glyoxylate aminotransferase 2.

Dimethylarginine:pyruvate aminotransferase, which plays a role in the metabolism of dimethylarginines, has been purified to homogeneity from rat kidney. The enzyme has a molecular weight of approximately 200,000 and an isoelectric point at about pH 6.3. The enzyme consists of four similar subunits having a molecular weight of about 50,000. The enzyme catalyzes the effective transaminations of guanidino-N methylated L-arginines (e.g. NG,NG-dimethyl-L-arginine, NG,N'G-dimethyl-L-arginine and NG-monomethyl-L-arginine) and the alpha-amino group of L-ornithine to pyruvate or glyoxylate. The enzyme was always accompanied by the known alanine:glyoxylate amino-transferase activity with the ratios of their specific activities remaining constant during the purification steps. The physicochemical and immunological properties of the purified enzyme were shown to be identical with those of the isozyme of alanine:glyoxylate aminotransferase (EC 2.6.1.44), designated as alanine:glyoxylate aminotransferase 2 (Noguchi, T. (1987) in Peroxisomes in Biology and Medicine (Fahimi, H. D., and Sies, H., eds) pp. 234-243, Springer-Verlag, Heidelberg). The distribution profiles in tissues and the negative response to glucagon treatment further supported the identity of the two enzymes. The present data show that alanine:glyoxilate aminotransferase 2 functions in dimethylarginine metabolism in vivo in rats.

Enzymatic method for selective determination of 4-aminobenzoic acid in urine.

We developed a method for the selective determination of 4-aminobenzoic acid by use of 4-aminobenzoate hydroxylase (EC 1.14.13.27) to convert 4-aminobenzoic acid to 4-hydroxyaniline. The subsequent conversion of 4-hydroxyaniline to indophenol dye was quantified colorimetrically. The method is reproducible, and the assay response is linear up to 40 mumol/L. Selectivity exceeding that of the current colorimetric assays was demonstrated for these enzymatic determinations of 4-aminobenzoic acid concentrations in urine samples from patients undergoing tests of pancreatic function with bentiromide. This method effectively minimizes interferences from drugs and diet, a problem in current colorimetric methods.

A comparison of two methods for calculating CR (time constant) during studies of arterial blood flow in rats.

Some of our earlier reports have dealt with experiments on the central caudal arteries of a series of anesthetized rats. The results of these experiments were expressed by a relationship derived from the Windkessel theory where f(t) = alpha dz(t)/dt + beta z(t). When this theory is used, the measured blood flow forms f(t) and calculated wave forms alpha dz(t)/dt + beta z(t) agree closely. In these studies, we discovered that, when blood flow adz(t)/dt + beta z(t) agree closely. In these studies, we discovered that, when blood flow decreases, CR (time constant tau, the product of the blood vessel compliance C and the peripheral resistance R) values increase and vary widely. In the present study, 1) we investigated changes in CR when blood flow increases, and, 2) the method of least squares was used in calculating the formula given above. We achieved a better conformity between measured blood flow and calculated blood flow and perceived a clearer relationship between mean blood flow and CR than when they were calculated by the old method.

A monoclonal antibody recognizing the FAD-binding site of 4-aminobenzoate hydroxylase from Agaricus bisporus.

A monoclonal antibody against 4-aminobenzoate hydroxylase (EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, has been produced by the fusion of BALB/c mouse spleen cells immunized with the denatured enzyme and P3x63Ag8U1 myeloma cells in order to locate and characterize the catalytic site of the enzyme. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated its apoenzyme. The immunoprecipitation was inhibited in the presence of FAD, and the monoclonal antibody competitively inhibited the binding of FAD to the apoenzyme. The monoclonal antibody, therefore, recognizes the FAD-binding site of 4-aminobenzoate hydroxylase. Interestingly, it was shown that the monoclonal antibody was cross-reactive with FAD-dependent enzymes such as salicylate hydroxylase (EC 1.14.13.1) and D-amino acid oxidase (EC 1.4.3.3), and that it was specific for the FAD-binding sites of these enzymes. This fact suggests that these FAD-dependent enzymes have immunologically similar structures on their FAD-binding sites.

Amebiasis in institutions for the mentally retarded in Kanagawa Prefecture, Japan.

A parasitologic survey of 620 mentally retarded patients, institutionalized in five different facilities in Kanagawa Prefecture, revealed a high incidence (12.6%) of infection with Entamoeba histolytica. A concomitant serologic survey, by the indirect fluorescent antibody test, gave a much higher incidence (26.5%). Moreover, most zymodeme patterns of the amebae isolated from infected individuals were of a pathogenic type (Zymodeme II). Our findings demonstrate that the mentally retarded in Japan, as in the United States, still are plagued by a high rate of amebic infection.

An immunological study of tricalcium phosphate supplied by three different manufacturers.

Antigenicity of tricalcium phosphate (TCP) ceramics which were supplied by two Japanese manufacturers and a manufacturer of United States was studied by means of delayed skin reactions in guinea pigs. Skin reactions were elicited 13 days after being immunized by intradermal injection of the ceramics into the dorsal flanks of guinea pigs. After 24 and 48 hr, these reactions were assessed by measuring the diameter of the erythema, the degree of hemorrhaging and its induration. Antigenicity was not detected in the TCP obtained from the Asahi Optical Co., Ltd. and the Kyocera Corporation by means of skin reactions. In contrast, TCP from Miter, Inc. (Synthograft) raised a delayed type hypersensitivity (DTH) 24 hr after antigen elicitation. The reaction appeared to be based on tuberculin type and Jones-Mote type of reactions. Arthus reactions were not observed in either the normal groups or groups immunized with TCP.

Purification and properties of a new enzyme, NG,NG-dimethylarginine dimethylaminohydrolase, from rat kidney.

A new enzyme, NG, NG-dimethylarginine dimethylaminohydrolase which plays a role in the metabolism of NG,NG-dimethyl-L-arginine, has been purified to homogeneity from rat kidney. The enzyme consists of a single polypeptide and its molecular weight is about 33,000. The isoelectric point of the enzyme is at pH 5.2. The enzyme catalyzes the hydrolytic liberation of the dimethylamino moiety of NG,NG-dimethyl-L-arginine and forms L-citrulline and dimethylamine. It is highly specific for NG,NG-dimethyl-L-arginine and NG-monomethyl-L-arginine, and the Km values for these amino acids are 0.18 and 0.36 mM, respectively. The enzyme shows the maximum activity at pH 6.5 and requires no cofactor. The activity is strongly inhibited by SH-blocking reagents (e.g. p-chloromercuribenzoate and HgCl2) and divalent metal ions (e.g. Cd2+, Cu2+, and Zn2+).