Category: Ascorbic Acid

DISCUSSION(2)

Vitamin C did not directly deactivate MMP-2 but had an effect on MMP-2 genomic expression as revealed by the Western blots, Northern blots, and run-on assays. Down-regulation of MMP-2 gene expression by vitamin C is exerted at physiologic concentrations if we consider the concentrations in amniotic fluid a good indicator of what is occurring inside the chorioam-nion. A nonspecific effect of vitamin C on gene expression, mediated by changes in the cellular redox potential, may be overruled by the lack of effect of equimolar amounts of glutathione and the parallel observations of no change in p-actin gene expression and increase in type I collagen gene expression.

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Type I, type III, and type IV collagens are the main structural constituents of chorioamniotic membranes, and they are responsible for the strength and flexibility of the membranes. In this study, it was demonstrated that vitamin C, in addition to its well-known role in collagen biosynthesis, modulates the degradation of this protein. The regulatory effect of vitamin C is exerted on the 72-kDa type IV collagenase or MMP-2 gene expression. MMP-2 is the only MMP secreted by WISH cells under experimental conditions. This peculiarity makes WISH cells a good model for study of MMP synthesis and secretion in chorioamnion during gestation. The unique presence of MMP-2 in am-niotic fluid and fetal membranes during midgestation suggests its role in extracellular matrix remodeling in cho-rioamnion. MMP-2 degrades type IV collagen, which is found in association with basal membranes but in chorioamnion is very widespread in a network. buy prednisone

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RESULTS(2)

Equimolar amounts of glutathione did not affect the basal expression of MMP-2 (Fig. 1B). According to semiquantitative den-sitometric analysis and taking the vitamin C-free cell media as 100%, residual activity was approximately 60% in the 29 ^g/ml dose (range from 53% to 72%, n = 8) and 18% in the 200 ^g/ml vitamin C dose (range from 15% to 30%, n = 8). ANOVA on ranks showed differences between all treatments (p < 0.0001). Ascorbic acid did not have a direct effect on enzymatic activity as demonstrated by the gel-substrate assay of purified human proenzyme 72-kDa type IV collagenase in the presence of equivalent amounts of ascorbic acid (Fig. 1C). buy flovent inhaler

Western blotting of all the assayed media revealed the presence of a 71-kDa molecular mass band detected with the antibody directed against MMP-2. The relative intensity of this band decreased to 72% with 29 ^g/ml (range from 70% to 78%, n = 8) and to 55.6% with 200 ^g/ml of vitamin C (range from 45.0% to 65.1%, n = 5) compared to that for the cells incubated in absence of vitamin C (Fig. 2A). ANOVA on ranks revealed significant differences between all groups (p < 0.001).

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Media from WISH cells incubated in the absence of vitamin C showed a gelatinase-specific activity (mean ± SD, n = 8) of 124.3 ± 4.9 ^g degraded gelatin/^g protein. Gelatinolytic-specific activity decreased progressively with 10 ^g of vitamin C to 82.1 ± 9.7, with 29 ^g to 63.0 ± 7.8, with 50 ^g to 52.6 ± 9.6, with 100 ^g to 47.8 ± 6.1, and with 200 ^g to 37.2 ± 6.1. All concentrations of vitamin C significantly (ANOVA, p < 0.01) diminished the gelatinolytic activity considering the absence of vitamin C as the basal production. A 4-h incubation in the presence of vitamin C was enough to obtain a maximum effect (data not shown). Concentration of intracellular vitamin C during this time was assessed. Most of the ascorbic acid (86%, range from 83.0 to 88.3, n = 8) remained in the extracellular space under experimental conditions. buy asthma inhalers

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MATERIALS AND METHODS(3)Western Blotting

Ten micrograms of protein from conditioned media per lane was applied to a 10% SDS-polyacrylamide gel under nonreducing conditions. Protein electrotransference to Im-mobilon-P membranes (Millipore, Medford, MA) using a semi-dry system was performed according to Towbin et al.. Membrane was developed with monospecific polyclonal antibodies against 72-kDa type IV collagenase (MMP-2), generously donated by Dr. William Stetler-Ste-venson from National Institute of Cancer (NIH, Bethesda, MD). Primary antibodies were detected with the ABC method (Vector Labs., Burlingame, CA).

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Vitamin C Quantitation

Ascorbic acid was quantitated at different times (0-4 h) to ensure that vitamin C was present under experimental conditions. Medium and cells were precipitated with 0.35 M perchloric acid, and their filtrates were processed for HPLC. Chromatography was carried out as suggested by Lee et al.. Intracellular ascorbic acid was normalized to 50 ^g DNA. Total DNA per well was quantitated according to Burton.

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MATERIALS AND METHODS(1)

Cell Culture

WISH cells, a line of human amnion cells (American Type Culture Collection, Rockville, MD; ATCC CCL5) were cultured in 25-cm2 flasks. Cells were grown in Dul-becco’s Modified Eagle’s medium, supplemented with 10% fetal calf serum (Gibco BRL, Gaithersburg, MD), 25 mM Hepes, 110 mg/L sodium pyruvate, and 20 mM L-gluta-mine, in a 95% air:5% CO2 incubator at 37°C with humid atmosphere. flovent inhaler

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