Category Archives: Enzymes

Activities of Glucose Metabolic Enzymes in Human Preantral Follicles: MATERIALS AND METHODS(5)

MATERIALS AND METHODS(5)

Therefore, hormones and growth factors that stimulate cellular growth may stimulate PK activity for ATP supply. PK activity was determined in 1 ml of 50 mM tri-ethanolamine buffer, pH 7.5, containing 2.25 M KCl, 0.24 M MgSO4, 6 ^M ADP, 18 U/ml lactic dehydrogenase, 1.4 ^mol NADH, and 5 ^l of follicular supernatant at 340 nm at 37°C. After recording of the background rate of NADH oxidation for 5 min without substrate, 33.3 ^l of a 45 mM phosphoenol pyruvate was added to the mixture and mixed immediately. The rate of NADH oxidation was recorded at 1-min intervals for 5 min. The rate of NADH oxidation was linear during the reaction period, and the enzyme activity was expressed as millimoles NADH oxidized per minute per milligram protein. ampicillin antibiotic

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Activities of Glucose Metabolic Enzymes in Human Preantral Follicles: MATERIALS AND METHODS(4)

The enzyme reaction was conducted at 37°C in a temperature-controlled quartz cuvette, and the absorbance of NADH was recorded at 340 nM in a Milton Roy (Rochester, NY) spectrophotometer. After recording of the background rate of NADH oxidation for 5 min without samples, 10 ^l of supernatant was added to the reaction mixture and mixed, and the rate of NADH oxidation was recorded at 1-min intervals for 5 min. The linearity of NADH oxidation throughout the recording duration indicated that the fall in absorbance was not due to substrate limitation. The enzyme activity was expressed as millimoles NADH oxidized per minute per milligram protein. antibiotics levaquin

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Activities of Glucose Metabolic Enzymes in Human Preantral Follicles: MATERIALS AND METHODS(3)

MATERIALS AND METHODS(3)

The supernatant was used to determine the activities of PFK and PK, two key regulatory enzymes of glycolysis, and the pellet was resuspended in 25 ^l of ho-mogenization buffer to determine the activity of malate dehydrogenase (MDH), an important enzyme of the Krebs cycle. All chemicals for enzyme assay were from Sigma Chemical Company (St. Louis, MO) except those that were from Boehringer-Mannheim GmbH (Mannheim, Germany). antibiotic levaquin

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Activities of Glucose Metabolic Enzymes in Human Preantral Follicles: MATERIALS AND METHODS(2)

Follicles from each ovary were cultured separately (to get n = 3) for 24 h in DMEM with ITS+ under 5% CO2 in air at 37°C in a Heraeus (Life Scientific, Inc., St. Louis, MO) incubator. Follicles were exposed to either 100 ng/ml ovine FSH (NIH-18; Bethesda, MD), 50 ng/ml ovine LH (NIH-25), 50 ng/ml recombinant murine IGF-I (Mallinckrodt, Phillipsburg, NJ), 50 ng/ml murine EGF (Mallinckrodt), or 10 ng/ml human TGFp1 (R&D Systems, Minneapolis, MN). buy cheap antibiotics

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Activities of Glucose Metabolic Enzymes in Human Preantral Follicles: MATERIALS AND METHODS(1)

MATERIALS AND METHODS(1)

Ovarian pieces were collected from women < 35 yr of age who were undergoing surgery because of clinical conditions other than ovarian pathology. Human tissue collection was approved by the Institutional Review Board. Ovary samples were rinsed with Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 100 U/ml penicillin, 10 ^g/ml streptomycin, 50 ^g/ml amphotericin B, and 0.5% BSA and dissected free of any broken antral follicle and luteal remnant. Ovarian pieces from each woman were processed separately for follicle isolation, and class 1 and 2 follicles were dissociated and collected as described previously. birth control yasmin

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Activities of Glucose Metabolic Enzymes in Human Preantral Follicles(2)

Previously we have shown that class 1 and 2 human preantral follicles grow in vitro in response to FSH and produce estradiol. Moreover, epidermal growth factor (EGF) modulation of transforming growth factor (3 (TGFp) receptor induction in human preantral follicles in vitro has been demonstrated. The presence of EGF and TGFp in the human ovary has been documented. Although the effect of EGF on ovarian glucose metabolism is unknown, EGF has been shown to transiently increase hepatic Krebs cycle and gluconeogenesis via a calcium-sensitive metabolic flux through mitochondrial 2-oxoglutarate dehydrogenase systems and inhibition of pyruvate kinase (PK).

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Activities of Glucose Metabolic Enzymes in Human Preantral Follicles(1)

Preantral Follicles(1)

Follicular development through preantral stages requires several cycles of granulosa cell division, which is an energy-dependent process. Under in vitro condition, glycolysis is the predominant source of ATP for mouse preantral follicles. Because an oxygen concentration gradient exists in ovarian follicles, glucose-handling capacity may differ in developing follicles because the layers of granulosa cells increase as follicles grow. Increased follicular glycolytic activity has been reported in the rat treated with LH, and increased lactic acid production in vitro by rat eCG-treated follicles has been observed in response to FSH. buy yasmin online

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