Seasonal changes in serum total thyroxine, free thyroxine, and canine thyroid-stimulating hormone in clinically healthy beagles in Hokkaido.

The purpose of this study was to evaluate seasonal influences on thyroid hormone levels of healthy outdoor dogs in Hokkaido. We surveyed serum basal total thyroxine (tT4), free thyroxine (fT4), and canine thyroid-stimulating hormone (cTSH) levels, and tT4 levels after administration of TSH for a year. Basal tT4 levels decreased in January, and increased in August and September. fT4 levels increased in January and November. No significant seasonal variation was found in cTSH. tT4 levels after administration of TSH in August and November increased. These results suggested that the thyroid gland may have been activated in November. We should take seasonal variation into consideration when thyroid function is tested.     

Oxidative damage to the membrane of canine erythrocytes with inherited high Na, K-ATPase activity.

Oxidative damage to the membrane in canine erythrocytes with inherited high Na, K-ATPase activity (HK cells) was compared with that in normal canine cells (LK cells). When 30 mM beta-acetylphenylhydrazine (APH) was applied to HK and LK cells, lipid peroxidation and hemoglobin denaturation occurred. Lipid peroxidation determined from malondialdehyde (MDA) formation was significantly lower in HK than in LK cells so far as endogenous glutathione (GSH) concentration was maintained at appropriate levels. With the depletion of GSH, MDA formation was accelerated and difference between HK and LK cells was not significant. Denatured hemoglobin bound to the membrane protein was less in HK than in LK cells. During incubation with APH, osmotic fragility increased markedly in LK cells, while HK cells showed very little change. The amounts of total lipid, total and free cholesterol, glycolipid, phospholipid and fatty acids were essentially the same in both cell types. Fatty acid compositions showed very small differences. The membrane of HK cells thus appear to have greater protection against oxidative damage induced by APH, owing to the presence of excess GSH in HK cells. The capability of HK cells to withstand oxidative damage would not be due to differences in membrane lipid compositions.     

Reduced mitotic activity and increased apoptosis of fetal sertoli cells in rat hypogonadic (hgn/hgn) testes

Sterility in male hypogonadic (hgn/hgn) rats results from congenital testicular dysplasia caused by a single recessive gene hgn on rat chromosome 10. We recently identified an insertion mutation in the Spag5/astrin gene of hgn/hgn rats that may cause defective proliferation of immature Sertoli cells in the postnatal hgn/hgn testis. Since the pathological alterations were present in the testes at birth, we examined the involvement of defective mitosis and apoptotic cell death in embryonic development of hgn/hgn testes. Testicular hypoplasia was apparent at embryonic day (ED) 18.5. Immunostaining of hgn/hgn testes at ED 21.5 with antibody to GATA-4, which is specific for fetal Sertoli cells in the seminiferous cords, showed that the significant decrease in the number of fetal Sertoli cells was accompanied by a two fold increase in their mitotic index and abnormal mitosis and apoptosis. Prior to this, we observed a decrease in the number of BrdU-labeled cells, an increase in the number of TUNEL-positive apoptotic cells, and presence of MIS-positive apoptotic cells in hgn/hgn testes on ED 17.5 and 18.5. These results suggest that the Spag5 mutation may cause a reduction in mitotic activity and an increase in apoptosis of fetal Sertoli cells in hgn/hgn testes.     

Increase in oxidized proteins in Theileria sergenti-infected erythrocyte membrane

As a part of the elucidation of the pathogenesis of anemia in Theileria sergenti infection, oxidized-erythrocyte membrane proteins (OEMPs) collected from T. sergenti-infected calves were examined. The amount of OEMPs were seen to increase with the progress of the anemia and showed a maximum value around the crisis period of the infection. The increase of OEMPs coincided with band Nos. 1, 2, 2.1, 3, 4.1, 5, 6, and 7. The majority of them was located at the Triton X-100 un-extractive phase, and was confirmed as cytoskeletal proteins. This evidence indicates the enhancement of erythrocytic oxidation, and suggests that it might be one of the aggravating factors of anemia in T. sergenti infection.     

Effect of intramammary infusion of tumour necrosis factor-alpha on milk protein composition and induction of acute-phase protein in the lactating cow

The present study was designed to evaluate the effects of tumour necrosis factor-alpha (TNF-alpha) on lactating bovine mammary functions such as milk protein secretion and the integrity of the milk-blood barrier. The effect on the induction of the systemic inflammatory response was also examined using concentrations of serum haptoglobin (Hp), a major inflammatory acute-phase protein, as an index. One hundred micrograms per mammary gland of recombinant bovine (rBo) TNF-alpha or placebo saline was individually infused into a rear mammary gland of each of four lactating cows, and milk and blood samples were collected before and 4, 8, 24, 32, 48, 96 and 168 h after infusion. In the rBoTNF-alpha-infused gland, increases of somatic cell counts were observed at 4-48 h. Although concentrations of total milk protein were not changed, compositions of milk proteins varied following rBoTNF-alpha infusion. Concentrations of caseins, alpha-lactalbumin and beta-lactoglobulin were significantly decreased at 4 and 8 h. Lactoferrin concentrations were significantly increased at 4 h. Significant infiltrations of serum albumin, immunoglobulin G1 (IgG1) and IgG2 were observed at 4 and 8 h. Elevations of the serum concentration of Hp were detected at 8-32 h, but were very small in comparison with those reported in inflammatory diseases. Changes in rectal temperature and white blood cell counts were not significant. These results show that single rBoTNF-alpha infusion into the lactating mammary gland suppresses the lactogenic function of the gland and influences the function of the milk-blood barrier, with little effect on the generalized inflammatory response.     

Development of an enzyme-linked immunosorbent assay to detect an immunomodulatory alpha-D-glucan-protein complex, MPG-1, in basidiomycete Tricholoma matsutake and related processed foods

We previously isolated a novel immunomodulatory alpha-(1,4)(1,6)(1,2)- d-glucan-protein complex (MPG-1) from mycelia of Tricholoma matsutake in basidiomycetes. In the present study, we raised a polyclonal antibody by immunizing rabbits with MPG-1 and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) system to examine the distribution of MPG-1 among edible mushrooms and related processed foods. The system detected MPG-1 quantitatively at concentrations of more than 10 ng/mL, with a coefficient of variation of less than 10% by intra-assay and interassay precision. Analysis with the system of chemically modified MPG-1 suggested that the sugar moiety was mainly involved in the detection. The system detected MPG-1 in the extracts of the fruiting bodies of T. matsutake but not in those of 34 other basidiomycete species. Moreover, a significant amount of MPG-1 was detected in the extracts of their cultured mycelia. The antigenic structure of MPG-1 was relatively stable in terms of pH and temperature. MPG-1 was detected in processed foods supplemented with T. matsutake. These results suggest that MPG-1 is distributed predominantly in T. matsutake species and that the ELISA system can detect it in processed foods supplemented with T. matsutake.     

Anti-inflammatory activities of cucurbitacin E isolated from Citrullus lanatus var. citroides: role of reactive nitrogen species and cyclooxygenase enzyme inhibition

The in vivo and in vitro mechanistic anti-inflammatory actions of cucurbitacin E (CE) (Citrullus lanatus var. citroides) were examined. The results showed that LPS/INF-γ increased NO production in RAW264.7 macrophages, whereas L-NAME and CE curtailed it. CE did not reveal any cytotoxicity on RAW264.7 and WRL-68 cells. CE inhibited both COX enzymes with more selectivity toward COX-2. Intraperitoneal injection of CE significantly suppressed carrageenan-induced rat's paw edema. ORAC and FRAP assays showed that CE is not a potent ROS scavenger. It could be concluded that CE is potentially useful in treating inflammation through the inhibition of COX and RNS but not ROS.     

Isolation and characterization of a novel immunomodulatory alpha-glucan-protein complex from the mycelium of Tricholoma matsutake in basidiomycetes

Tricholoma matsutake, a high-class edible mushroom in Japan, has been reported to have excellent biological activities, but difficulty in cultivating the fruit bodies and limited bulk availability have restricted detailed studies. We have developed a method of culturing in tanks, enabling the bulk supply of the mycelia. The preparation (CM6271) exerts modulative effects on the immune competence of mice and rats. In this study, a sodium hydroxide extract of CM6271 was defatted followed by fractionation with a combination of ion exchange chromatography and gel filtration in order to identify the components involved in the expression of the activity, and a single peak fraction (MPG-1) was obtained with reversed phase chromatography. MPG-1 was a glycoprotein (sugar:protein ratio, 94.3:5.7) with a relative molecular mass of 360 kDa, and the sugar moiety contained about 90% glucose. NMR spectra and methylation analysis revealed that the alpha-1,4-linkage was the predominant glucan linkage with alpha-1,6- and alpha-1,2-linkages in the minority. The amino acid composition in the protein moiety was rich in glutamine, alanine, asparagine, leucine, glycine, valine, serine, threonine, isoleucine, and proline. MPG-1 was resistant to degradation with amylase or protease. The oral administration of MPG-1 promoted, in a dose-dependent manner, the recovery of the mouse natural killer cell activity and serum IL-12 level that had been reduced by the loading of restraint stress. The dose of MPG-1 (25 mg/kg) required for the expression of the effect decreases to 1/12 of that of CM6271 (300 mg/kg). Furthermore, MPG-1 formed a complex with TGF-beta1 in vitro, modulating the biological activity of TGF-beta1 by binding to its active form. These results indicate that the mycelium of T. matsutake contains a novel alpha-glucan-protein complex with immunomodulatory activities.