Effects of hypothalamic dopamine (DA) on salsolinol (SAL)‐induced prolactin (PRL) secretion in male goats

The aim of the present study was to clarify the effects of hypothalamic dopamine (DA) on salsolinol (SAL)‐induced prolactin (PRL) release in goats. The PRL‐releasing response to an intravenous (i.v.) injection of SAL was examined after treatment with augmentation of central DA using carbidopa (carbi) and L‐dopa in male goats under 8‐h (8 h light, 16 h dark) or 16‐h (16 h light, 8 h dark) photoperiod conditions. The carbi and L‐dopa treatments reduced basal PRL concentrations in the 16‐h photoperiod group (P < 0.05), while a reduction was not observed in the 8‐h photoperiod group. The mean basal plasma PRL concentration in the control group for the 8‐h photoperiod was lower than that for the 16‐h photoperiod (P < 0.05). SAL significantly stimulated the release of PRL promptly after the injection in both the 8‐ and 16‐h photoperiod groups (P < 0.05). PRL‐releasing responses for the 16‐h photoperiod were greater than those for the 8‐h photoperiod (P < 0.05). The carbi and L‐dopa treatments blunted SAL‐induced PRL release in both the 8‐ and 16‐h photoperiods (P < 0.05). These results indicate that hypothalamic DA blunts the SAL‐induced release of PRL in male goats, regardless of the photoperiod, which suggests that both SAL and DA are involved in regulating the secretion of PRL in goats.     

Effects of Ghrelin on growth hormone secretion from cultured adenohypophysial cells in pigs

To clarify the direct effects of Ghrelin on growth hormone (GH) release from anterior pituitary (AP) cells in pigs, GH-releasing effects of human Ghrelin (hGhrelin) and rat Ghrelin (rGhrelin) on porcine AP cells were compared with GHRH in vitro. The AP cells were obtained from 6-month-old pigs and the cells (2 x 10(5) cells per well) were incubated for 2 h with the peptides after incubating in DMEM for 3 days. hGhrelin and rGhrelin significantly stimulated GH release from the cultured cells at doses of 10(-8) and 10(-7)M (P < 0.05). The rates of increase in GH at 10(-8) and 10(-7)M of hGhrelin were 82.7 and 131.9%, while those with rGhrelin were 43.9 and 79.5%, respectively. GHRH significantly stimulated GH release from the cells at a dose as low as 10(-11)M (P < 0.05), and the response to GHRH was greater than that induced by Ghrelins. In time-course experiments, GHRH continued to increase GH concentrations in media until 120 min after incubation; however, those in media treated with hGhrelin reached a plateau 60 min after incubation, and the maximal value was approximately one third that obtained with GHRH. When hGhrelin (10(-8)M) and GHRH (10(-8)M) were added together, additive effects of both peptides on the release of GH were observed (P < 0.05). Somatostatin (SS, 10(-7)M) significantly blunted GH release induced by hGhrelin (10(-8)M) and GHRH (10(-8)M) (P < 0.05). In the presence of SS, additive effects of hGhrelin and GHRH on the release of GH were observed (P < 0.05). These results show that Ghrelin directly stimulates GH release from anterior pituitary cells in pigs; however, the GH-releasing effect is weaker than that of GHRH in vitro. The present results also show that Ghrelin interacts with GHRH and SS to in the release of GH from porcine adenohypophysial cells.     

Novel variations and loss of heterozygosity of BRCA2 identified in a dog with mammary tumors

OBJECTIVE: To establish novel polymorphic markers for analysis of loss of heterozygosity (LOH), so as to study the possible involvement of BRCA2 in mammary tumors obtained from dogs. SAMPLE POPULATION: Blood samples, mammary gland specimens, or mammary tumors from 3 tumor-bearing dogs and 10 tumor-free dogs. PROCEDURES: Nucleotide sequence analysis was performed with a DNA autosequencer. Loss of heterozygosity analysis was performed for markers established in the present study. The expression level of canine BRCA2 was quantified by real-time PCR analysis. RESULTS: 3 novel microsatellite markers with high heterozygosity rates (> 50%) were established, and the previously reported marker for canine BRCA2 gene locus was improved. These markers were used for the analysis of DNA from formalin-fixed and paraffin-embedded samples. By use of these markers, LOH in canine BRCA2 was identified as a result of recombination. In mammary tumor DNA that corresponded to the LOH-positive dog, the level of canine BRCA2 expression was decreased compared with that of nonneoplastic mammary gland tissue; the open reading frame contained 4 missense variations, 1 insertion variation, and 1 silent variation, some of which were localized to functional domains. CONCLUSIONS AND CLINICAL RELEVANCE: 3 novel polymorphic markers were developed for LOH analysis of canine BRCA2 and identified a dog with LOH with some variations in the functional domains. These markers could be useful for assessing the relevance of BRCA2 variation in mammary tumors of dogs.     

Detection of the anti-P53 antibodies in dogs with tumors

To detect the anti-P53 antibodies of dogs with tumors, a GST-recombinant canine (rc) P53 fusion protein was expressed and purified. Immunoblot analysis was performed using this GST-rcP53 fusion protein as an antigen and serum samples from dogs suffering from tumors as primary antibodies. Out of 16 serum samples obtained from various tumor cases, four samples showed reaction with GST-rcP53. In contrast, serum from other 12 dogs with tumors, four dogs with non-neoplastic diseases and two control healthy dogs (as controls) did not show any reaction with GST-rcP53 in immunoblotting. The p53 gene mutation and the P53 protein expression were examined, using the tumor tissues to explore the relationship between the existence of the GST-rcP53 bands, gene mutations of p53 and the accumulation of P53 protein. One case, which showed a clear GST-rcP53 band, had a point mutation of the p53 cDNA and showed nuclear accumulation of P53 protein. These results suggest that the anti-P53 antibodies are also produced in tumor dogs with p53 gene mutations.     

Plasma growth hormone (GH) responses after administration of the peptidergic GH secretagogue KP102 into the oral cavity, rumen, abomasum and duodenum in adult goats

The study was performed to determine whether orally administered KP102 (also known as GHRP-2) stimulates GH release in adult goats, and how the orally administered KP102 passes through the digestive tract and stimulates GH release in ruminant animals. Five mg/kg body weight (BW) of KP102 dissolved in 9 ml of saline were administered into the oral cavity, rumen, omasum and duodenum of adult goats, and GH release after administration of KP102 was examined. The GH levels were significantly elevated at 20 min after administration of KP102 into the oral cavity, and plasma concentrations of GH remained significantly elevated until 60 min (P < 0.05). The GH levels after administration of KP102 into the abomasum were variable. However, the GH level tended to increase within 30 min after administration, and were significantly higher than those of controls after 120 to 150 min (P < 0.05). The GH levels after administration of KP102 into the duodenum were significantly elevated at 40 min after administration, and plasma concentrations of GH remained significantly elevated until 140 min (P < 0.05). The administration of KP102 into the rumen failed to stimulate GH release. The GH response curves (AUC) produced after administration of KP102 into the abomasum or duodenum were 2.2-fold greater than those for after administration into the oral cavity (P < 0.05). The oral administration of 5 mg/kg BW of KP102 in the powder state, not dissolved in 9 ml of saline, failed to stimulate GH release. These results suggested that orally administered KP102 dissolved in saline transiently stimulates GH release in adult goats, and this phenomenon might be due to small amounts of the peptides entering directly into the abomasum with liquid bypassing the rumen.     

Molecular cloning of canine monoamine oxidase subtypes A (MAOA) and B (MAOB) cDNAs and their expression in the brain

The role of monoamine oxidase has been shown to be related to some behavioral changes including aggression and cognitive dysfunction. In order to demonstrate the basic expression patterns of monoamine oxidase in the canine brain, we determined the full-length nucleotide sequences of cDNA for canine monoamine oxidase type A (MAOA) and type B (MAOB) genes that were isolated from the canine brain cDNA library. Oligonucleotide primers for PCR were constructed based on the conserved sequences reported thus far for other mammalian species. The nucleotide sequences had open reading frames of 1584 and 1563 bp for MAOA and MAOB, respectively. Both of these genes showed relatively high homology with other species in both nucleotide (> 81%) and deduced amino acid (> 85%) sequences. In Northern blot analyses MAOA mRNA was expressed broadly in various parts of the canine brain, whereas MAOB mRNA was found only in specific brain regions, such as the hypothalamus, hippocampus, brain stem and olfactory bulb. These results suggest that MAOA and MAOB mRNAs have subtype-specific expression patterns in the canine brain.     

Fecal progesterone analysis by time-resolved fluoroimmunoassay (TR-FIA) for monitoring of luteal function in the sika doe (Cervus nippon centralis)

Fecal progesterone content was measured by time-resolved fluoroimmunoassay (TR-FIA) in the sika doe (Cervus nippon). The total recovery rate of fecal progesterone by twice extraction with diethylether was about 60%. The displacement curve of TR-FIA with serial doses of fecal extract (0.156-5.0 mg feces) was closely parallel to that of the reference standard. Fecal progesterone content was correlated with that of plasma (r=0.829, n=16), but the values were 100-fold higher in feces than in plasma. Fecal progesterone content periodically changed during the breeding season suggesting the estrous cycle in the doe. The fecal progesterone content was higher between the estruses, and decreased after estrus. The time between the onset of estrous signs and the lowest fecal progesterone content was 1-2 days suggesting the time required for hepatic metabolism and intestinal passage. Fecal progesterone content was also decreased around the time of vaginal discharge. The discharge took place within a few days, suggesting a short luteal phase. Not of all decreases in fecal progesterone values were preceded by estrous behavior or vaginal discharge. Fecal progesterone content was further increased in pregnancy rather than in the preceding estrous cycle and the levels were maintained up to term. These results suggest that fecal progesterone measurement is a useful tool for non-invasive analysis of luteal function in the sika doe. The TR-FIA kit, designed for the human hospital market, was shown to be successfully utilized for fecal assay in the sika doe with minor modifications.     

Identification of an cysteine-to-arginine substitution caused by a single nucleotide polymorphism in the canine monoamine oxidase B gene

Monoamine oxidase B catalytically oxidizes biogenic amines such as phenylethylamine and dopamine, and its activity is presumed to be related to particular behavioral traits. In this study, we first identified a single nucleotide polymorphism (T199C) located on the putative third exon of the canine monoamine oxidase B gene, which causes an amino acid substitution from cysteine to arginine. We then examined the allelic frequencies in five dog breeds (Golden Retriever, Labrador Retriever, Maltese, Miniature Schnauzer, and Shiba) and found significant variation among them. The present results suggest that analysis of the monoamine oxidase B polymorphism could be a useful means of elucidating the genetic background of breed-specific behavioral characteristics in dogs.     

Comparison of the 16S-23S rRNA intergenic spacer regions between Fusobacterium varium and "Fusobacterium pseudonecrophorum" strains

The 16S-23S rRNA intergenic spacer regions (ISRs) of five strains of "Fusobacterium pseudonecrophorum" which had been proposed as a new species, were compared with those of F. varium ATCC 8501T. All the strains of "F. pseudonecrophorum" exhibited of sequence similarities of 97.7% to 100% to the strain of F. varium in their 16S-23S rRNA ISR sequences. This indicates that the strains of "F. pseudonecrophorum" and the type strain of F. varium are identical at the species level.     

Differences of tensile strength distribution between mechanically high-grade and low-grade Japanese larch lumber 11: Effect of knots on tensile strength distribution

The tensile strength (TS) test results of Japanese larch (Larix kaempferi, Carriere) lumber of varying length have shown that the length effects on TS were different between high-grade (H) and low-grade (L) lumber. In this paper, we examined the effect of knots on the TS distribution by measuring the number of knots and the knot area ratio of each specimen. There were more knots in L than in H; and the knot area ratio in L distinctly increased as the length increased compared to that in H. The correlation coefficients between physical properties and TS indicated that knots were the most influencial factor for TS among several physical properties: annual ring width, distance from pith, density, dynamic Young's modulus, and knots. We attempted to estimate the length effect parameters by introducing the concept of assumed knot strength. We thought that the length effect parameters for 50th percentiles of TS could be estimated well with fitted 3P-Weibull, and that the parameters for 5th-percentiles could be estimated well with 2P-Weibull fitted to lower-tail 10% data by the likelihood method. The differences of length effect on TS between H and L should be governed by the presence of knots. The independent model based on the concept of assumed knot strength may express the TS of structural lumber of various lengths.