Synthesis of morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) by dipeptidyl aminopeptidase IV derived from Aspergillus oryzae

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)), tetrapeptide, was synthesized using dipeptidyl aminopeptidase IV (DP IV, EC 3.4.14.5) derived from Aspergillus oryzae RIB 915 as a catalyst. Tyr-Pro-OEt was incubated with Phe-Pro-NH(2) in the presence of DP IV under various conditions of temperature, concentrations of ethylene glycol, pH, reaction time, and others. Morphiceptin was obtained at 40% yield under the optimal reaction conditions: substrate, 4 mM Tyr-Pro-OEt.HCl and 20 mM Phe-Pro-NH(2).HCl; enzyme, DP IV, 0.275 nkat; solvent, 60% ethylene glycol containing 20 mM phosphate buffer at pH 7.0; amine, 4.2 mM diisopropylamine at 4 degrees C for 24 h. Amino group protection was unnecessary for synthesis of morphiceptin by DP IV.     

Isolation and purification of a low molecular weight protein from bovine urine

A low molecular weight protein was separated from urine samples obtained from a heifer with spontaneous renal disease and from cows with CaNa2EDTA-induced renal dysfunction. The molecular weight and electrophoretic mobility of the separated protein were examined. The low molecular weight protein collected by gel filtration chromatography was further separated into two fractions by ion exchange chromatography using DEAE-cellulose. One of the two fractions, the lowest molecular weight protein showed a single band in SDS-PAGE, and its molecular weight was approximately 12,000. An antiserum against this protein formed a single precipitin line with the urine from cows with experimentally induced renal dysfunction and a heifer with spontaneous renal disease by the double immunodiffusion technique. However, the antiserum did not form any precipitin line with the concentrated urine of healthy cow and human beta 2-microglobulin. In cellulose acetate membrane electrophoresis, this protein migrated in the same position as that of serum gamma-globulin from healthy cow.     

Diurnal changes in plasma melatonin and the timing of reproductive onset in anestrous sheep fed melatonin.

Seasonally anestrous Suffolk ewes (n = 28) were randomly divided into 5 groups and treated with varying doses of melatonin as follows; Groups C (n = 4), M1 (n = 6), M2 (n = 6), M3 (n = 6), M4 (n = 6) of ewes were fed pellets containing 0, 1, 2, 3 and 4 mg melatonin, respectively, daily for 60 days from May 17 (Day 0). Following feeding of the pellets at 13.00 hr plasma levels of melatonin rapidly increased reaching the peak values within 30 min, which ranged from 92.0 to 292.7 pg/ml and were highly correlated with the dose of melatonin administered (r = 0.986, P less than 0.01). Maximum dose of melatonin (4 mg) produced an increase of plasma melatonin similar in magnitude to nocturnal peaks of endogenous secretion. The onset of ovulatory cyclicity, assessed from plasma progesterone profiles, was advanced by melatonin administration. The mean +/- SEM intervals from the commencement of melatonin treatment until the onset of ovulatory cyclicity were 53.0 +/- 5.8, 53.6 +/- 2.5, 42.0 +/- 5.6 and 44.3 +/- 4.3 days for the Groups M1, M2, M3 and M4, respectively, which were shorter (M1, P less than 0.05; M2, M3 & M4, P less than 0.01) than that for the Group C (72.5 +/- 1.4 days). The melatonin treatment also suppressed, in a dose related manner, the rise in plasma prolactin under the lengthening photoperiod. We conclude that the dose-related efficacy of melatonin could be ascribed to the difference in the diurnal profiles of circulating melatonin.     

The Effect of Diminazene Aceturate on Spienectomized Dogs with Babesia gibsoni Infection

The effect of diminazene aceturate on splenectomized and nonspienectomized dogs with Babesia gibsoni (B. gibsoni) infection was investigated. In splenectomized dogs, the fissional and multiplicational stages of B. gibsoni were observed in peripheral blood films, and hemoglobinuria was frequently observed. These findings were different from previous reports and were not changed by administration of diminazene aceturate. It is clear that the intramuscular administration of diminazene aceturate at the dose of 3 mg/kg body weight for 3 days is not effective against B. gibsoni infection in splenectomized dogs.     

Epidemiological and pathological studies on congenital diffuse hyperplastic goiter in calves.

Perinatal diseases such as weak calf, stillbirth, or abortion were found in 86 calves from about 600 cows in a farm (A Farm) in 1987 and 1988. Such perinatal diseases were decreased to 36 calves in 1989 and 1990 after an additional supply of seaweed was made to the maternal feeds. From these cases, 23 stillborn and weak calves were pathologically examined. The mean weight with standard deviation of the 18 thyroid glands was 36.3 +/- 28.6 g before feeding of the seaweed supplement, and 12 (67%) of the glands showed moderate to marked degree of diffuse hyperplastic goiter in histological criteria. On the other hand, 5 glands weighed 12.0 +/- 3.4 g and revealed no such histological lesions after the additional supply. These results suggest that the goiter had been associated with birth of weak calves. Further study was performed on 37 newborn calves affected with Akabane disease in 1986 in A Farm and the neighbouring B Farm located. Seven (54%) of 13 thyroid glands from A Farm and 1 (4%) of 24 glands from B Farm showed histologically moderate to marked goitrous lesions. There were dotted "Inugarashi", Rorippa indica, family Cruciferae in both pastures. The mean iodine contents of the orchard grasses were 87 and 121 micrograms/kg on dry basis in A and B Farms. Marginal deficiency or lower limit of iodine and possible thiocyanate content of "Inugarashi" may have been responsible for the goiter in the farms.     

High level expression of biologically active canine interferon-alpha subtype 4 using a baculovirus

In this study, a high amount of bioactive recombinant canine interferon-alpha subtype 4 (CaIFN-alpha4) was expressed in a baculovirus system. For easy purification, it was expressed as a CaIFN-alpha4 bearing histidine hexamer at the C-terminal region, designated CaIFN-alpha4His. CaIFN-alpha4His was detected in culture supernatants of insect cells infected with the recombinant virus using sodium dodecyl sulfate-polyarcylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue staining. The level of expression was very high, and approximately 1 mg of purified protein, with 5.0 x 10(7) units/mg, was obtained from 300 ml of culture supernatant. The purified product showed antiviral activity against Vesicular stomatitis virus on canine tumor cell line A72 and chicken embryo fibroblast cells.     

Genotyping of Giardia intestinalis from domestic and wild animals in Japan using glutamete dehydrogenase gene sequencing

To determine the genotypes of Giardia intestinalis from domestic and wild animals in Japan, Giardia isolates obtained from feces of 24 dogs kept in households and breeding kennels, three companion cats, five dairy calves and three wild monkeys, Macaca fuscata, were genotyped using the 177 bp sequence of the glutamete dehydrogenase gene (gdh). The genotypes were assemblages A, C, D or A/D for dog isolates, Assemblage F for cat isolates, assemblages A or E for calf isolates and assemblage B for monkey isolates. This is the first report on the genotypes of Giardia isolates from cats, calves and wild monkeys in Japan.     

Clinicopathological survey of 101 canine mammary gland tumors: differences between small-breed dogs and others

Clinicopathological features of mammary gland tumors (MGTs) in 101 dogs were evaluated retrospectively. The incidence of histological malignancy in 60 small- and 41 other-breed dogs were 25% and 58.5%, respectively. In 82 epithelial MGTs, small-sized tumors (< 3 cm) or non-invasive tumors were predominant in small breeds. In multivariate survival analysis, small breed (p=0.048) and lower stage of tumor cell invasion (p=0.006) were significantly associated with longer survival time. These results suggest that the incidence of histological or biological malignancy in MGTs is lower in small-breed dogs than in others.     

Active immunization against inhibin improves superovulatory response to exogenous FSH in cattle

The effect of active immunization against inhibin on the response to superovulatory treatment by porcine FSH (pFSH) was investigated in cattle. Japanese black cows were sc injected with 1 mg of porcine inhibin alpha-subunit fragment (1-26) conjugated with rabbit serum albumin (inhibin-immunized group; n=14) or rabbit serum albumin alone (control group; n=12) in Freund's complete adjuvant. Booster injections (half the amount of the primary injection) were given 35 and 70 days after the primary injection. All cows were superovulated three times with pFSH. Three days after each injection of the antigen, a progesterone-releasing intravaginal device (CIDR-B) was inserted vaginally into all animals and left in place for 10 days. Forty-eight hours before CIDR-B removal, all animals were sc injected with 30 mg pFSH dissolved in 40% polyvinylpyrrolidone, and im injected with 750 microg of PGF2alpha at CIDR-B removal. Cows were artificially inseminated twice during estrus, and ova or embryos were collected 7 or 8 days after estrus. The number of corpora lutea, the number of ova or embryos and the number of transferable embryos in inhibin-immunized cows (12.1+/-1.2, 11.1+/-1.3 and 6.2+/-1.0, respectively) were significantly greater than those in the controls (8.2+/-1.0, 5.7+/-1.1 and 3.1+/-0.7, respectively). These results indicate that active immunization against inhibin enhanced ovarian response to the usual superovulatory treatment in cattle. Therefore, immunization against inhibin may be a useful approach for improving the response to superovulation in cattle.