Category Archives: Plasminogen

Prolactin Regulation of Tissue: DISCUSSION(4)

The PA and PAI complex formation subsequently leads to the cleavage and inactivation of both tPA and PAI-I activity. Any change in tPA and PAI-I activity in the cells or in the cell-conditioned media is therefore determined by the relative level of both of these molecules. flovent inhaler

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Prolactin Regulation of Tissue: DISCUSSION(3)

DISCUSSION(3)

These results suggest that the effects of PRL on ovarian cells may largely depend on the differentiation of PRL receptors. Using a eCG-primed mouse granulosa cell culture model, we have demonstrated that PRL significantly enhances FSH- and LH-induced progesterone production, while it inhibits aromatase activity. In vivo experiments also showed that PRL inhibits hCG-induced ovulation in mice by decreasing ovarian PA activity and serum estrogen concentrations. Both FSH and LH have been demonstrated to induce tPA mRNA and activity in cultured granulosa cells. buy ortho tri-cyclen

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Prolactin Regulation of Tissue: DISCUSSION(2)

During GnRH-induced ovulation in hypophysec-tomized rats, both PAI-I mRNA and activity were also increased in a time-dependent fashion. These data suggest that the action of PRL on PAI-I gene expression in granulosa cells might be through a protein kinase C-dependent pathway. Buy Asthma Inhalers Online

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Prolactin Regulation of Tissue: DISCUSSION(1)

DISCUSSION(1)

In the present study, we have demonstrated that PRL stimulates PAI-I gene expression in a time- and dose-dependent manner in eCG-primed rat granulosa cells in vitro. This stimulation of PAI-I expression is dramatically enhanced by the presence of LH. PRL is also capable of inhibiting LH-induced tPA expression. The mechanism by which PRL with the gonadotropin synergistically super-in-duces PAI-I, while inhibiting tPA expression, in the eCG-primed granulosa cells is unknown. It has been demonstrated that a low dose of FSH through activation of adenylate cyclase induces PAI-I production in granulosa cells. ventolin inhaler

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Prolactin Regulation of Tissue: RESULTS(3)

Time- and Dose-Dependent Inhibition of LH-Induced tPA mRNA and Activity Levels by PRL in Cultured Granulosa Cells

To examine whether PRL inhibits LH-induced tPA gene expression in cultured granulosa cells, 2 X 106 cells obtained from eCG-primed ovaries were preincubated for 6 h in the presence of 2% calf serum, and then further incubated in serum-free medium for the indicated times. As shown in Figure 4, LH alone dramatically stimulated tPA mRNA levels in a time-dependent manner. Cotreatment with PRL time-dependently decreased the LH-induced tPA mRNA levels. Significant inhibition could be observed at 3-, 6-, and 12-h culture, with 36.4%, 51.4%, and 66.7% decreases, respectively, of the LH-induced tPA mRNA levels.

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Prolactin Regulation of Tissue: RESULTS(2)

RESULTS(2)

After 24-h incubation, an obvious synergistic increase in PAI-I activity could be observed in the culture of LH with PRL (data not shown).

PRL Dose-Dependent Stimulation of the Synthesis of PAI-I mRNA and Activity Levels in Cultured Granulosa Cells

To estimate the dose-dependent effect of PRL on PAI-I mRNA levels in the culture, granulosa cells (2 X 106 cells/ dish) were preincubated for 6 h in medium containing 2% calf serum, and then incubated for 24 h in serum-free medium. As shown in Figure 2, addition of various doses of PRL in the presence or absence of LH stimulated PAI-I mRNA induction in a dose-dependent manner. The presence of LH resulted in a synergistic induction of PAI-I mRNA levels. buy diabetes drugs

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Prolactin Regulation of Tissue: RESULTS(1)

Time-Dependent Effect of PRL on PAI-I mRNA and Activity in eCG-Primed Granulosa Cells

To examine whether PRL stimulates PAI-I expression in cultured granulosa cells, 2 X 106 cells obtained from eCG-treated ovaries were preincubated for 6 h in the presence of 2% calf serum, and then incubated in fresh serum-free medium for the indicated times with or without LH (100 ng/ml) and PRL (1000 ng/ml) alone or in combination. The conditioned media were collected for PAI-I assay, and the cells were frozen in liquid nitrogen for mRNA preparation. As shown in Figure 1, LH and PRL alone slightly induced PAI-I mRNA production in a time-dependent manner. After 48-h incubation, 1.6- and 4.5-fold increases in the mRNA production were observed in the presence of LH and PRL, respectively, as compared with the control. birth control pills

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Prolactin Regulation of Tissue: MATERIALS AND METHODS(7)

MATERIALS AND METHODS(7)

The glass slides were air-dried and fixed in 4% paraformaldehyde in PBS. After fixation, the slides were digested with proteinase K for 10 min at 37°C, rinsed in PBS, and fixed again in 4% paraformaldehyde for 5 min. After being washed in PBS for 5 min, the slides were dehydrated in a series of ethanol and air-dried. They were then prehybridized in 50% formamide/double-strength SSC for 2 h at room temperature. Then the slides were hybridized with dig-labeled antisense cRNA probes in hybridization solution. Hybridization was performed in a humidified box for 20 h at 48°C. The slides were washed in double-strength, then single-strength, and then 0.1-strength SSC, each twice for 15 min at 40°C. The hybridized probes were detected using an alkaline phosphatase-coupled anti-dig Fab fragment. The color reaction was developed by incubation with NBT and BCIP in color development buffer. The reactions were terminated by immersing the slides in buffer I.

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Prolactin Regulation of Tissue: MATERIALS AND METHODS(6)

Nylon filters were cross-linked by UV light in a Strata-linker (Stratagene, La Jolla, CA) and prehybridized at 62°C for 2 h in 50% formamide, 5-strength saline sodium citrate (SSC; single-strength SSC IS 0.15 M sodium chloride, 0.015 M sodium citrate), 8-strength Denhardt’s solution (1.6 mg/ml Ficoll, 1.6 mg/ml polyvinylpyrrolidone, 1.6 mg/ ml BSA), 0.1% SDS, 10 mM EDTA, 25 mM Tris-HCl (pH 7.0), 250 mg/ml heat-denatured herring sperm DNA, and 250 mg/ml yeast tRNA. The hybridization was carried out in the same solution containing 2.5 X 106 cpm/ml of each probe for 16 h at 64°C. The filters were washed in doublestrength SSC, 0.1% SDS twice for 15 min at room temperature and then washed twice in 0.01-strength SSC, 0.1% SDS for 40 min at 66°C. Hybridizations using the p-actin probe was performed at 42°C as described. After hybridization, the filters were exposed to autoradiographic films or analyzed by Phosphorimager (Molecular Dynamics, Sunnyvale, CA). buy levaquin online

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Prolactin Regulation of Tissue: MATERIALS AND METHODS(5)

MATERIALS AND METHODS(5)Synthesis of RNA and DNA Probes

A rat PAI-I cDNA cloned into a pBluescript vector and a 400-basepair (bp) £coRI fragment from rat tPA cDNA clone lambda 15 was subcloned into a pGEM-1 vector. Both vectors were linearized by HindIII and used as templates for probe synthesis using an in vitro transcription system (Promega-Biotech). A 400-bp tPA probe complementary to the 5′ region of rat tPA mRNA and a 376-bp PAI-I probe complementary to the 3′-untranslated region of PAI-I mRNA were obtained using the T7 and T3 promoters, respectively. The 32P-labeled antisense RNA probes were tested for specificity by hybridization to ovarian total RNA fractionated by formaldehyde agarose gel electrophoresis and blotted to nylon filters. The probes were found to be specific for tPA and PAI-I mRNA since they hybridized only to specific mRNA species with the size corresponding to tPA and PAI-I mRNA. A 250-nucleotide single-stranded p-actin DNA probe was prepared by primer extension as described. buy ampicillin

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