Detection of chromogranin A in the adrenal gland extracts of different animal species by an enzyme-linked immunosorbent assay using Thomsen-Friedenreich antigen-specific Amaranthus caudatus lectin.

The reactivity of different lectins with crude chromogranin A (CgA) obtained from different animals, namely, cow, horse, dog, pig, and dolphin, was examined to identify lectin(s) that would be useful as coating reagent(s) in a sandwich enzyme-linked immunosorbent assay (ELISA). Of the different lectins studied, the Amaranthus caudatus lectin (ACA), which is specific for the Thomsen-Friedenreich (T)-antigen (Galß1-3GalNAc), was found to react with the CgA from different animals by western blotting. Purified rabbit anti-bovine CgA antibody was also found to cross-react with the crude CgA preparations. On the basis of these findings, a sandwich ELISA was developed with ACA as the coating reagent and anti-bovine CgA antibody as the probing antibody. Using this method, concentration-dependent curves ranging from 0.003 µg/mL to 25 µg/mL and from 0.02 µg/mL to 25 µg/mL were obtained for bovine CgA and canine CgA, respectively. Similarly, concentration-dependent curves were obtained for the equine, swine, and dolphin crude CgA extracts. Thus, ACA is concluded to be a valuable reagent for CgA detection in crude extracts from different animal species, and for CgA isolation/purification.

Expression patterns of the BRCA1 splicing variants in canine normal tissues and mammary gland tumors.

Human BRCA1 is familial breast cancer susceptibility gene. Recently, decreased BRCA1 mRNA and protein expression has been identified in sporadic breast tumors. In the reported human BRCA1 splicing variants, delta11b lacks the majority of exon11 and is suspected to have a distinct function in normal tissues. The splicing variants display a variety of expression pattern in breast cancer samples. Although mammary gland tumor is important disease in dog, there are few reports for BRCA1 in the canine tumors. In this study, we examined the relative amounts of BRCA1 splicing variants mRNA in canine normal and mammary tumor samples by RT-PCR to investigate whether there is the altered expression of variant mRNAs in the canine tumor as reported in human. The exon11b-defecting RT-PCR products were observed in all the normal tissues examined and the nucleotide sequence was quite similar to that of human BRCA1 delta11b. In some tumor samples, we did not detect the products targeted for exon10-13 and exon14-15, while these products were observed in all the normal samples examined. Especially, the relative amounts of the exon11-defecting products were remarkably decreased in most of the tumors (11/16).

Relationship between transportation stress and polymorphonuclear cell functions of bottlenose dolphins, Tursiops truncatus.

Dolphins in a captive environment are exposed to various kinds of stresses. Handling and transportation are stressful events for terrestrial mammals, and such stress may affect immune system function and increase susceptibility to infectious diseases. The same phenomenon could occur in dolphins, however, few studies have reported this in dolphins. The objective of this study was to evaluate the relationship between stress and polymorphonuclear (PMN) cell function of dolphins during transportation. Four bottlenose dolphins (Tursiops truncatus) were transported for 6 hr by truck. Serum cortisol levels, leukograms, phagocytosis, and superoxide production of PMN cells were evaluated during handling and transportation compared to resting values. The mean serum cortisol level was significantly increased during handling and transportation (p<0.05) when compared with the resting values. White blood cell (WBC) counts, eosinophil counts, phagocytosis, and superoxide production of PMN cells during handling and transportation stages decreased significantly in comparison with the resting stage (p<0.05). The concentration of serum cortisol was significantly correlated with the results of the WBC counts, eosinophil counts, superoxide production, and phagocytosis (p<0.01, p<0.05, p<0.05, and p<0.001, respectively). The present results indicate that handling and transportation are stressful events for dolphins and could affect their PMN cell functions, thereby leading to the impairment of the immune system.

Measurement of plasma chromogranin A concentrations for assessment of stress responses in dogs with insulin-induced hypoglycemia.

OBJECTIVE: To determine whether cross-reactivity exists between canine chromogranin A (CgA) and anti-human CgA antibody and investigate the usefulness of plasma CgA concentration measurements as an index of acute stress responses in dogs. ANIMALS: 12 healthy Beagles. PROCEDURE: Canine CgA was extracted and purified from canine adrenal glands of cadaver dogs for studying cross-reactivity with anti-human CgA antibody. Western blotting with anti-human CgA antibody was performed. Blood samples were collected from dogs at 0, 10, 20, 30, 40, 60, 120, and 180 minutes after IV administration of saline (0.9% NaCl) solution or insulin. Canine plasma CgA concentrations were determined by use of a CgA ELISA kit with rabbit antiserum against the carboxy-terminal fragment of human CgA. Plasma cortisol and catecholamine (ie, norepinephrine and epinephrine) concentrations were measured by use of an ELISA and a high-performance liquid chromatography method, respectively. RESULTS: Purified canine CgA was specifically detected by use of western blot analysis and an ELISA with anti-human CgA antibody. An increase in plasma CgA concentrations was observed in insulin-induced hypoglycemic dogs. Changes in plasma CgA concentration were correlated with changes in plasma cortisol or catecholamine concentrations of hypoglycemic dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Use of the CgA ELISA kit for determination of human plasma CgA concentrations is applicable to the measurement of canine plasma CgA concentrations. Canine plasma CgA concentrations, along with measurements of plasma cortisol and catecholamine concentrations, correctly reflect insulin-induced hypoglycemic stressed conditions in dogs. Measurement of canine plasma CgA concentrations may provide a useful index for evaluation of an acute stress response.

Renal effects of medetomidine in isoflurane-anesthetized dogs with special reference to its diuretic action.

Renal effects of the selective alpha(2)-adrenoceptor agonist, medetomidine, were investigated in anesthetized dogs. Animals were administered medetomidine 20 and 40 microg/kg intravenously (IV) and 80 mug/kg intramuscularly (IM) or 1 ml of saline IV. Urine and blood samples were collected before and at 30, 60, 90 and 120 min following medetomidine injection. Mean arterial blood pressure (MABP), renal blood flow (RBF), glomerular filtration rate (GFR), urine volume (U(v)), urine osmolality (U(osm)), free water clearance (C(H2O)), fractional clearance of sodium (F(Na)), plasma osmolality (P(osm)), plasma glucose levels and plasma antidiuretic hormone (ADH) concentrations were measured. The results showed that IV administration of medetomidine initially increased MABP 5-15 min followed by long-lasting decrease. The initial hypertension was not observed after IM administration, which was accompanied by a more profound hypotensive effects. RBF, GFR, U(v), C(H2O) increased after IV injection and decreased after IM. Medetomidine increased FNa and Posm and decreased U(osm). Plasma glucose levels initially increased and subsequently decreased. Plasma ADH concentration was decreased by IV injection but increased by IM administration. Our data imply that: 1) IV administration of medetomidine at dose rates of 20 and 40 microg/kg results in profound diuresis up to 2 hr; 2) Suppression of ADH release from the CNS is one of the mechanisms of medetomidine-induced diuresis although it may not be the principal one.

Enzyme-linked immunosorbent assay for detection of canine chromogranin A by use of immunological cross-reactivity of rabbit anti-bovine chromogranin A antibody.

Bovine and canine chromogranin A were extracted and purified from each specie's adrenal glands. Isolated bovine 70 kDa protein showed 100% identity to bovine CgA reported previously, whereas isolated canine 68 kDa protein showed 83.3% identity to bovine CgA by the NH(2)-terminal amino acid sequence analysis. Rabbit antibody to purified bovine protein (CgA) was found to immunologically cross-reacted with purified canine protein (CgA). In sandwich ELISA with anti-bovine CgA, concentration-dependent curves were obtained ranging from 0.3 to 20 mug/ml for canine CgA. From these findings, sandwich ELISA with anti-bovine CgA is found to be useful to determine the concentration of canine CgA.

A primary hepatic plasma cell tumor in a dog.

An 8-year-old female Shetland sheep dog had hyperproteinemia with a monoclonal gammopathy and a solid mass on the liver, which was histologically diagnosed as a plasma cell tumor. After the treatment of surgery and chemotherapy, serum protein level reduced to the normal range and the gammopathy was disappeared. These findings indicate the plasma cell tumor developed primarily from the liver.