Effect of nutrient intake on intramuscular glucose metabolism during the early growth stage in cross-bred steers (Japanese Black male × Holstein female).

The objective was to investigate the impact of nutrient intake during the early growth period on the expression of glucose metabolism-related genes in skeletal muscle of cross-bred cattle. From 1.5 to 5 months of age, group H (n=7) animals were intensively fed a high-protein and low-fat milk replacer [crude protein (CP) 28%; ether extracts (EE) 18%; max: 2.0 kg, 12 l/day], and group R (n=7) animals were fed a restricted amount of normal milk replacer (CP 25%; EE 23%; max 0.5 kg, 4 l/day). From 6 to 10 months of age, group H cattle were fed a high-nutrition total mixed ration mainly prepared from grain feed, and group R cattle were fed only roughage. Blood samples were taken from each animal at three biopsy times (1.5, 5 and 10 months of age), and the blood plasma concentration of glucose and insulin was analysed. In glucose concentration, there were no significant differences; however, the concentrations of insulin were higher in group H than in group R at 5 and 10 months of age. Muscle samples were taken by biopsy from longissimus thoracis muscle (LT) at 1.5, 5 and 10 months of age. We analysed mRNA expression levels using the quantitative real-time polymerase chain reaction (PCR) assay for glucose transporters (GLUT1 and GLUT4), insulin receptor, phosphatidylinositol 3-kinase (PI-3K), protein kinase B (PKB, also known as Akt), hexokinase 1 (HK1) and tumour necrosis factor alpha (TNFα). Although no differences were detected at 1.5 and 5 months of age, at 10 months of age, GLUT1, HK1 and TNFα mRNA expression levels were significantly higher in group H than in group R. These results suggested Glut1 that affects insulin-independently mediated glucose uptake was more responsive to improved nutrition during early growth stage than GLUT4 that insulin-dependently mediated glucose uptake in LT of cattle.

Three-dimensional observation of connective tissue of bovine masseter muscle under concentrate- and roughage-fed conditions by using immunohistochemical/confocal laser-scanning microscopic methods.

We investigated changes in connective tissue components of masseter (MA) muscle in Japanese black heifers (n= 6) in concentrate- and roughage-fed groups (groups C and R, respectively). Body weight, at slaughter, of experimental heifers in group C (272.3 +/- 22.3 kg) was higher (P < 0.05) than that of group R (213.8 +/- 27.5 kg). However, muscle weight and myofiber diameter (superficial and deep layers) of MA muscle did not differ between groups C and R. In contrast, total mastication duration of group R was longer (P < 0.05) than that of group C. MA muscle of groups C and R was composed only of type I myofiber. Using immunohistochemical/confocal laser-scanning microscopy, type I collagen was observed mainly in perimysium, and type V and VI collagen were observed in perimysium and endomysium of both groups. Type IV collagen and laminin were observed only in the endomysium in both groups. However, type III collagen and fibronectin were strongly apparent in the perimysium and endomysium in group R. Connective tissue components in the perimysium of groups C and R were observed to form plate-shaped layers. On the other hand, honeycomb-shaped connective tissue components were seen in the endomysium-surrounded muscle fibers. In particular, fibronectin was strongly observed in the perimysium and endomysium in group R. These results indicate that there are different developmental changes among connective tissue components in MA muscle in response to mastication. The immunohistochemical/confocal laser-scanning microscopic method is useful to investigate the structural relationship among connective tissue components in skeletal muscle.

Demonstration of HCG on the surface of maternal lymphocytes and discrimination of T and B cells by esterase cytochemistry.

Immunological functions pregnant women in the early fetomaternal relationship were investigated. Maternal T cell population as determined by the standard method gradually decreases as plasma HCG increases from between 5 and 10 weeks of gestation. In this study, the presence of HCG on the surface of maternal lymphocytes was detected by the direct immuno-histochemical method using enzyme-labeled antibodies: (1) HRP-anti human beta-HCG rabbit Fab', (2) HRP-anti alpha-HCG rabbit Fab', (3) HRP-anti human native HCG rabbit Fab'. Further (4) HRP-normal rabbit Fab', (5) HRP-anti rabbit IgG goat F(ab')2 and (6) activated HRP were used as staining controls. In order to determine whether the maternal lymphocytes masked with HCG were T or B cells, a modified technique of esterase cytochemistry was employed. The reaction of esterase cytochemistry on rosette forming lymphocytes by SRBC exhibited the features of T cells, as the lymphocytes display a dense localized positivity made up of one to four coarse granules.

[A study on the mechanism of production and secretion of HPL (author's transl)].

Our preliminary data suggest that HPL was present in the largest quantity in chorionic villi of 16-18 weeks of pregnancy but the concentration of HPL in mother blood was maximum at 30-40 weeks of pregnancy. To clarify the discrepancy of term of those maximum values, the following experiments were done. The immuno-precipitate caused by the reaction with anti-HPL serum in the crude extract from chorionic villi incubated with MEM medium containing [3H]-leucine was subjected to SDS gel electrophoresis. Three radioactive bands were observed. Their molecular weights were 22000, 29000 and 48000, native HPL is 22000 molecular weights and other heavier molecular weights protein are surmised pre- or pro-HPL. Native HPL was few in 30 min. incubated cases but increased in over night incubated cases. Other heavier molecular weights protein changed into native HPL with the lapse of time. The concentration of HPL in mother blood is surmised to reflect the production of two pro-HPL is syncytium cells, change into free HPL and furthermore their secretion.

[A study on the immunity of pregnancy. The localization of HCG on the cell membrane of lymphocytes (author's transl)].

The study was made for women in 10 weeks and 12 weeks of pregnancy, and also for non-pregnant women and men as controls. Heparinized peripheral venous blood of them were obtained, and lymphocytes were separated by specific gravity centrifugation by Lymphoprep, to be used as samples. For enzyme reaction, at first a separated lymphocyte PBS suspension was smeared on a glass sheet, dried in air and fixed for 15 minutes by 4% PLP solution, then to make antigen-antibody reaction by direct method. After the reaction, the reaction product was fixed for 10 minutes by 2% glutaraldehyde and, with color developed by Karnovsky method. Antibodies used were (1) HRP-anti human alpha-HCG rabbit Fab', (2) HRP-anti human beta-HCG rabbit Fab' and (3) HRP-anti human native HCG rabbit Fab', and furthermore, (4) HRP-N rabbit serum and (5) HRP-anti rabbit IgG goat F(ab)2 were used as staining controls. In the reaction group of pregnant cases with alpha-HCG, beta-HCG and N-HCG antisera, the brown color of reaction products was observed on lymphocyte cell membranes to prove the localization of HCG. The existence of lymphocytes negative in the reaction and the difference in color development degrees among lymphocytes positive in the reaction were also observed, to show quantitative difference in localization. In the group of the controls and in the staining control group of pregnant cases, no reaction was observed at all.

[Immunological responses in pregnancy. The studies on the role of the uterus in gestation and effects to lymphocytes subpopulation (author's transl)].

The lymphocytes that isolated from the both peripheral venous blood (PVB) and uterine venous blood (UVB) of pregnant women with a complication of myoma were studied on the subpopulation (E-rosette and S-Ig method) and lymphoblastoid transformation to mitogens. Two methods on the E-rosette in this study were applied to make a clear correlation between masking phenomenon and humoral factors. One is to be used lymphocytes that washed five times with P.B.S., the other one cultured in RPMI 1640 with 20% FCS for 72 hours afterward. The results were obtained as follows. 1) Although T-cell of UVB displayed significantly lower than that of PVB. The count of T-cell of both PVB and UVB elevated to standard level after washing five times with P.B.S. and/or cultured for 72 hours. 2) The average counts of B-cell by the S-Ig were not detected on the difference between of PVB and UVB. 3) No differences in ratio of lymphoblastoid were recognized on the both lymphocytes of PVB and UVB to PHA and PWM too. 4) AFter five times washing, the counts of T-cell in UVB improved to standard value range. Therefore no difference of the counts in T-cell were detected between in PVB and UVB.