Chronic Peripheral Administration of Kappa-Opioid Receptor Antagonist Advances Puberty Onset Associated with Acceleration of Pulsatile Luteinizing Hormone Secretion in Female Rats

Puberty in mammals is timed by an increase in gonadotropin-releasing hormone (GnRH) secretion. Previous studies have shown involvement of the two neuropeptides, kisspeptin and neurokinin B (NKB), in controlling puberty onset. Little is known about the role of the other key neuropeptide, dynorphin, in controlling puberty onset, although these three neuropeptides colocalize in the arcuate kisspeptin neurons. The arcuate kisspeptin neuron, which is also referred to as the KNDy neuron, has recently been considered to play a role as an intrinsic source of the GnRH pulse generator. The present study aimed to determine if attenuation of inhibitory dynorphin-kappa-opioid receptor (KOR) signaling triggers the initiation of puberty in normal developing female rats. The present study also determined if stimulatory NKB-neurokinin 3 receptor (NK3R) signaling advances puberty onset. Female Wistar-Imamichi rats were weaned and intraperitoneally implanted with osmotic minipumps filled with nor-binaltorphimine (nor-BNI), a KOR antagonist, or senktide, a NK3R agonist, at 20 days of age. Fourteen days of intraperitoneal infusion of nor-BNI or senktide advanced puberty onset, manifested as vaginal opening and the first vaginal estrus in female rats. Frequent blood sampling showed that nor-BNI significantly increased luteinizing hormone (LH) pulse frequency at 29 days of age compared with vehicle-treated controls. Senktide tended to increase this frequency, but its effect was not statistically significant. The present results suggest that the inhibitory input of dynorphin-KOR signaling plays a role in the prepubertal restraint of GnRH/LH secretion in normal developing female rats and that attenuation of dynorphin-KOR signaling and increase in NKB-NK3R signaling trigger the onset of puberty in female rats.     

Diversity and population structure of black soybean landraces originating from Tanba and neighboring regions

Black soybean landraces that had been cultivated in Tanba region and the neighboring regions and conserved black soybean landraces, including those from other regions in Japan, were used in this study. The polymorphisms of 78 SSR markers in nuclear DNA and 6 SSRs in chloroplast DNA were analyzed in the black soybean landrace populations. The result of phylogenic analysis revealed that the black soybeans can be classified into six clades. The landraces originating from Tanba region were classed into first and second clades, and two chloroplast genotypes were found in the population of black soybeans from the Tanba region. Genotype A chloroplast was predominantly identified in major populations of the Tanba, while genotype B was widely distributed in the black soybean population. Population structure analysis in the Japanese black soybean accessions inferred there are six groups. The black soybean landrace from the Tanba region was classified into three groups, mainly corresponding to the distance-based phylogenic results. The two groups were probably derived from different ancestors with Type A and B chloroplast genomes, respectively, whereas the other group showed both types of chloroplast genome. The admixture situations suggested that the landraces in the main group have been widely cultivated in Tanba region, while the landraces that belong to other groups were cultivated in localized area. Several phenotypes were compared among genotype groups, dividing into two sub-groups: founder sub-group and admixed sub-group. Phenotypic differences were observed between founder landraces in group 1 and group 3. On the other hand, landraces in admixture landraces in group 1 and group 2 segregated for several traits, while founder landraces in group 1 were stabled for each trait. These observations suggest that gene flow events have occurred between different founder landraces.     

Association between preoperative characteristics and risk of anaesthesia-related death in dogs in small-animal referral hospitals in Japan

Objective

To explore the major risk factors linking preoperative characteristics and anaesthesia-related death in dogs in referral hospitals in Japan.

A case of congenital hyperostosis in a newborn piglet

One female newborn piglet showed prominent thickening of both forelimbs and died soon after birth. Histopathologically, thin and woven trabeculae of bone was extending out from the edge of cortical bone in the affected forelimbs, and diagnosed as congenital hyperostosis. The extent of radially proliferated trabeculae was most prominent in radioulna. Many round- to spindle-shaped cells were observed in periosteum, which were considered to be osteoblasts. Around the periosteum, the mesenchymal proliferation was extensive with abundant mucus, and cartilaginous metaplastic changes were observed mainly around the radioulna and humerus. Dilatation of vessels with fibrin deposition in vessel walls was often observed, which were considered to reflect the localized circulatory disturbance.     

Development of high-grade B-cell lymphoma concurrent with T-cell chronic lymphocytic leukemia in a dog

Second malignancies are frequent complications in human patients with chronic lymphocytic leukemia (CLL). However, the clinical details and outcome of this phenomenon were unclear in their canine counterparts. Here, we report a dog with high-grade lymphoma concurrent with T-cell CLL. A 10-year-old male golden retriever presented with lymphadenopathies. The lymph nodes contained large-sized lymphocytes, raising suspicion of high-grade lymphoma. Meanwhile, small lymphocytic lymphocytosis in the peripheral blood was consistent with CLL. Interestingly, molecular biological analyses revealed that CLL cells were of the T-cell type, whereas lymphoma cells were of the B-cell type. Chemotherapy using the L-VCA short protocol was effective for 155 days, but the dog died on day 194 after diagnosis, despite rescue therapies.     

Ehrlichia canis infection in two dogs that emigrated from endemic areas

Two dogs, emigrated from Zambia and China to Japan, were diagnosed with Ehrlichia canis infection. Both cases had thrombocytopenia, non-regenerative anemia, and hypergloblinemia with polyclonal gammopathy. Case 1 had ataxia of the hind limbs. Severe meningitis was revealed by magnetic resonance imaging examination. Intracytoplasmic inclusions were observed in mononuclear cells of cerebrospinal fluid. Case 2 had a history of bilateral epistaxis, and severe pancytopenia was noticed in complete blood count. Diagnosis was finally achieved by nested polymerase chain reaction and sequence analysis. Thus, even in non-endemic areas, E. canis infection should be included in the differential diagnosis of clinically ill dogs that emigrated from endemic areas.     

Simultaneous estimation of the pH of rumen and reticulum fluids of cows using a radio-transmission pH-measurement system

Circadian pH changes in the fluid of the rumen (bottom and middle) and reticulum were assessed simultaneously using wireless and wired radio-transmission pH-measurement systems in cows fed a control diet (C diet) or rumen-acidosis-inducing diet (RAI diet). The pH in the three sites decreased following the morning and evening feedings. In cows fed the C diet, the bottom-rumen and reticular pH reverted to the basal level by the next morning, while the middle-rumen pH did not recover completely, suggesting that active fermentation occurred in the middle of the rumen. The mean pH at 1 hr intervals was higher in the reticulum than at the bottom and in the middle of the rumen. The relatively stable reticular pH may result from dilution due to salivation. In cows fed the RAI diet, the bottom-rumen pH fell to approximately 5.2 after the evening feeding, but returned to the basal level by the next morning. In contrast, the middle-rumen pH did not return to the basal level (6.5) within 24 hr, presumably owing to continuous, vigorous fermentation. There were positive correlations between the pH at the bottom and in the middle of the rumen and at the bottom of the rumen and in the reticulum. These findings indicate that our radio-transmission pH-measurement system may be suitable tool for simultaneous measurement of pH in the rumen and reticulum fluid.     

Fourth ventricular alloxan injection suppresses pulsatile luteinizing hormone release in female rats

Previous studies have suggested the presence of a glucose-sensing mechanism in the hindbrain that appears to regulate reproductive function as well as feeding behavior. The ependymocytes lining the ventricular wall of the hindbrain showed immunoreactivities to pancreatic glucokinase (GK), a key enzyme for glucose sensing in pancreatic B cells. Our goal in the present study was to test whether the GK-immunopositive ependymocytes in the wall of the fourth cerebroventricle (4V) play a role in regulating gonadal activity. Our approach was to determine the effect of injecting alloxan, a GK inhibitor, into the 4V on pulsatile luteinizing hormone (LH) secretion. Estrogen-primed ovariectomized rats received an injection of alloxan (10 or 20 microg/animal) into the 4V and blood samples were collected every 6 min for 3 h for measurement of blood LH, corticosterone and glucose levels. Pulsatile LH secretion was suppressed after alloxan injection and all pulse parameters were significantly (P<0.05) inhibited by 20 microg alloxan. Plasma corticosterone levels were increased significantly (P<0.05) by 20 microg alloxan, suggesting that LH pulse suppression by alloxan may be at least partly mediated by activation of the hypothalamo-pituitary-adrenal axis. The present results suggest that acute suppression of GK activity in the hindbrain inhibits pulsatile LH secretion in female rats, and supports the idea that GK-immunopositive ependymocytes may sense glucose levels in the cerebrospinal fluid and play a role in regulation of LH secretion.     

Absorption and tissue distribution of an immunomodulatory alpha-D-glucan after oral administration of Tricholoma matsutake

Alpha-D-glucan (MPG-1) separated from Tricholoma matsutake (CM6271) has been reported to show immunomodulatory activities. In this study, the plasma concentration and tissue distribution of MPG-1 after CM6271 oral administration were investigated as part of the action mechanism analysis. When CM6271 was orally administered in a single dose to mice, MPG-1 was absorbed via the intestinal tract, appeared in plasma after 16 h, was gradually excreted from the blood, and fell to background level after 48 h. The time course analysis of MPG-1 in plasma showed the following pharmacokinetic parameters of MPG-1: tmax = 24 h; Cmax = 161.1 ng/mL; AUC(0-infinity) = 2559.7 ng x h/mL. Moreover, MPG-1 was confirmed to localize in Peyer's patches, mesenteric lymph nodes (MLN), and the spleen and to promote IL-12 p70 production and NK cell activity. These results suggest that MPG-1 stimulated the intestinal immune system through Peyer's patches; moreover, it was taken into the blood and stimulated the systemic immune system.     

Induction of dendritic cell-mediated immune responses against canine malignant melanoma cells

To establish the basis for the use of dendritic cells (DC) in the treatment of canine melanoma, dogs were vaccinated using autologous DC pulsed with canine melanoma CMM2 cell lysate in the presence of keyhole limpet haemocyanin (KLH) in vitro (CMM2-KLH-DC), and the induction of immune responses against CMM2 cells in vivo was examined using the delayed-type hypersensitivity (DTH) skin test. The DTH responses against CMM2 cells and KLH were observed in dogs vaccinated with CMM2-KLH-DC, while the responses against KLH but not CMM2 cells were detected with DC pulsed with KLH alone (KLH-DC). Recruitment of CD8 and CD4 T cells was detected in the positively responding sites, suggested that vaccination with CMM2-KLH-DC efficiently elicits T cell-mediated immunity against CMM-2 cells in vivo. These findings demonstrate the potential utility of DC-based tumour vaccination in the treatment of canine malignant melanoma.