[Biological activities and partial characterization of guinea pig T cell-activating monokine].
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Biomedical subjects
Publications and source records attributed to S Sakamoto.
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The activities of 5-methyltetrahydrofolate (5-CH3THF) related enzymes and DNA polymerase alpha were determined in bone marrow cells obtained from patients with vitamin B12 deficient megaloblastic anemia and compared with those from healthy volunteers and patients with hemolytic anemia. 5-CH3THF homocysteine methyltransferase activity was significantly lower than that in the control subjects. 5,10-methylenetetrahydrofolate reductase activity was only slightly elevated to that in the control subjects. DNA polymerase alpha activity was significantly higher than that in the control. High deoxyuridine suppression test values in vitamin B12 deficient bone marrow cells were improved by tetrahydrofolate, but not by 5-CH3THF. These data indicate that, even though the reverse reaction catalyzed by 5,10-methylenetetrahydrofolate reductase may be operative in vitamin B12 deficiency, it is not sufficient to correct the disturbance in folate metabolism in vitamin B12 deficiency. Increased DNA polymerase alpha activity may be due to compensation for disarranged DNA synthesis.
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In medical clinics or experiments, progesterone (P) has been used for treatment of adenomatous hyperplasia or cancer of the endometrium. In the present paper, effects of P in combination with or without estradiol-17 beta (E2) on the estrogen receptor (ER) and thymidine kinase (TK) activity in immature rat uterus are described. P has been reported to interfere with the replenishment of the cytoplasmic ER of the rat uterus. In the present study, no translocation of the cytoplasmic ER to the nucleus was found after the injection of P, whereas P slightly enhanced the wet weight and the TK activity of the uterus. The TK activity increased 20 to 30 times the control level at 30 hours after the injection of E2. However, the enzyme activity induced by E2 was inhibited by P which was approximately 1,000-fold or more dose of E2. This enzyme was separated into isozymes by DEAE-cellulose column chromatography. Induction of the specific TK isozyme, which was induced by E2 in immature rat uterus and closely related to DNA synthesis, was found to be inhibited by P.
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A microassay method sensitive enough to analyze the enzyme activities in one oocyte was developed using enzymatic cycling for amplifying the reaction product to 10,000 fold. An oil-well technique was applied in the assay for achieving the reaction in the medium as small as 1.0 to 5.0 microliter. Immature Wistar rats were superovulated by PMS-hCG administration. Oocytes were collected by the puncture of the follicle and the flushing of the tube. They were freeze-dried after washing to remove cumulus cells. The dry weight was about 50ng on a quartz fiber fishpole balance. The activity of hexokinase was 1.75 +/- 0.14 picomol/oocyte/hr corresponding to one-tenth of the ovarian homogenate as control, indicating low capacity of glucose utilization in the oocyte. The activities of G6PD, LDH, and MDH were 8.41 +/- 0.34, 35.7 +/- 2.89. 11.1 +/- 2.5 picomol/oocyte/min, respectively. High activity of G6PD suggests the pentose phosphate shunt concerned with steroidogenesis is active in the oocyte. HCG increased the activities of hexokinase and MDH and decreased that of G6PD. The activity of LDH remained unchanged.
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The inhibitors of prostaglandin (PG) production (indomethacin, etc.) block ovulation. But little is known about how the inhibition of PG production effects cell function in the follicle. This study was done to clarify the morphological changes and steroidogenesis in the follicles treated with the inhibitor. Fifteen gilts treated with PMS and hCG were administered with indomethacin of 7.6 mg/kg (enough for a complete block) 24 hrs. after hCG. The morphological study of the follicles was done by serial section. The levels of estradiol-17 beta, testosterone, 17 alpha OH progesterone and progesterone were estimated by RIA in the follicular fluid, ovarian vein and peripheral blood. A histologic section of the treated follicles revealed the presence of entrapped oocytes surrounded by heavily luteinized granulosa-theca cells. No morphological changes were found in the luteal cells at any stage of the luteal phase except for retention of fluid in the central cavity of the corpus luteum. The life span of the corpus luteum (18 days) was not affected by indomethacin. No differences in steroid levels were detected in the peripheral and ovarian vein blood or in the follicular fluid at any time compared with the control. Inhibition of PG production results in the luteinized unruptured follicle without interference with steroidogenesis.
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The cyanogen bromide method was applied to the assay of vitamin B12-dependent methyltetrahydrofolate:homocysteine methyltransferase activity in normal and leukemic human hematopoietic cells. Normal peripheral lymphocytes and leukemia cells of lymphoid origin wuch as CLL and ALL, contained higher levels of enzyme activity than did normal human bone marrow cells. Normal granulocytes and leukemia cells of myeloid origin, such as CML in the chronic phase and AML, contained lower enzyme activity. Leukemia cells of CML in blast crisis showed higher mean activity than in the chronic phase of the disease.
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In order to investigate involvement of prostaglandins (PGs) in ovulation, we examined whether the antiserum to cyclooxygenase was able to inhibit superovulation in immature female rats as indomethacin did. The antiserum to cyclooxygenase was raised in rabbits using as antigen the solubilized cyclooxygenase extracted and purified from bovine seminal vesicles. The in vivo administration of the antiserum inhibited superovulation in female rats dose-dependently at doses of 0.1, 0.2 and 0.4 ml but did not affect production of progesterone. Inhibition of cyclooxygenase in the follicular cells due to the antiserum causing no effect on steroidogenesis was observed in organ cultures of the follicles excised from rats primed with PMS-hCG and the antiserum. In contrast to this, no inhibition of cyclooxygenase was detected upon adding the antiserum to the culture medium. The present study showed PG is as indispensable a factor in ovulation as are the steroids.
In order to study production of prostaglandins (PGs) by human pre-ovulatory ovarian tissue, bioconversion of 14C-arachidonic acid to PGs was examined using homogenates of human Graafian follicles and stromal tissues fully stimulated by endogenous follicle-stimulating hormone in the regular menstrual cycle. Among the radioactive metabolites extracted, PGE2 and 6-keto PGF1 alpha were purified and identified by silicic acid column-, thin layer-, reversed phase partition chromatographies and radio-gas chromatography. The follicular tissue was shown to convert 14C-arachidonic acid to PGE2 at a conversion rate of 0.34% but not to 6-keto PGF1 alpha. On the other hand, the stromal tissue was demonstrated to produce not only 14C-labeled PGE2 at a conversion of 0.09% but mainly 14C-labelled 6-keto PGF1 alpha at 0.26%.
The production of prostaglandins (PGs) E and F and 6-keto PGF1 alpha was investigated by using human decidua, amnion, and pregnant myometrium obtained before and after onset of labor, and human nonpregnant myometrium collected during luteal phase. PGs produced were measured by radioimmunoassay developed in our laboratory. The radioimmunoassay for 6-keto PGF1 alpha is specific and accurate. PGE was formed at an almost constant rate regardless of the condition under which the three tissues tested were obtained, with the amnion showing the highest activity compared with decidua and myometrium. PGF production increased remarkably during labor in the myometrium and decidua but not in the amnion. In the myometrium during labor, PGF levels were three times higher than before labor. The pregnant myometrium produced more PGs than the nonpregnant myometrium. The production of 6-keto PGI1 alpha, a stable, non-enzymatically formed derivative of PGI2, was observed to be much higher in the pregnant myometrium than in decidua, amnion, or nonpregnant myometrium. Contrary to the pattern of PGF production, 6-keto PGF1 alpha was lower during labor. The physiological significance of PG production by decidua, amnion, and myometrium in relation to parturition is discussed.