Histochemical localization of oxidized glutathione-catalysing enzymes in human term placenta

Summary This paper reports a new cytochemical affinity technique for detecting oxidized-glutathione-binding enzymes by light microscopy and for carrying out fine structural analyses by means of oxidized glutathione labelled with colloidal gold. Albumin-colloidal gold particles were prepared. Oxidized glutathione, added to the solution, replaced albumin on the surface of the gold, thus forming a new histochemical reagent. In human placenta, oxidized-glutathione-catalysing activity was detected on the brush border of the placental villi, over the foldings of endothelial membranes, and in the ground substance of connective tissue in the villous core. Every process of the cell membrane demonstrated enhanced oxidized-glutathione-catalysing activity. This new reagent is a unique example of a low-molecular-weight compound labelled with colloidal gold, and it permits direct measurement of oxidized-glutathione-binding activity in tissue sections.     

Short term culture of human midterm and term placenta: parameters of hormonogenesis

Monolayer cultures of human midterm and term placentae have been established following trypsin dispersion of placental minces. Maintenance of endocrine function was monitored by the concentrations of specific hormones in the culture media. At either gestational age the cultures 1) secret estradiol-17beta(1) and estrone (in a ratio of about 1:20) and aromatize 3H- or 14C-dehydroepiandrosterone sulfate and 14C-androstenedione, estrogen production being markedly enhanced by addition of dehydroepiandrosterone (10(-6)7) to the culture medium; 2) metabolize 3H-pregnenolone to progesterone and 14C-cortisol to cortisone; and 3) produce increasing amounts of chorionic gonadotropin and decreasing amounts of placental lactogen during the first week in culture. It is proposed that the model is highly suited to the study of factors affecting hormonogenesis by the human placenta whether they be of maternal or of fetal origin.     

Dexamethasone inhibition of cyclooxygenase expression in bovine term placenta.

Since both prostaglandin (PG) F2 alpha and corticosteroids are elevated in mammals before the onset of parturition, we studied the effect of the synthetic corticosteroid dexamethasone on PGF2 alpha accumulation and cyclooxygenase (prostaglandin synthase, PGS) expression in the bovine fetal placenta. Cultures were prepared from cotyledons at different stages of gestation. The effect of dexamethasone on PGF2 alpha accumulation and PGS expression was determined by radioimmunoassay and [35S]methionine metabolic labeling followed by immunoprecipitation with specific anti-cyclooxygenase antibodies, respectively. Data demonstrate that in fetal placental cells at term, both PGF2 alpha accumulation and cyclooxygenase expression are significantly inhibited after 18 hours of dexamethasone treatment (150 nM). In contrast, neither first nor second trimester cells were sensitive to dexamethasone treatment. Dexamethasone inhibition of PGF2 alpha synthesis in fetal cells at term was abolished in the presence of RNA or protein synthesis inhibitors (actinomycin D or puromycin, 10 micrograms/ml each). Neither progesterone nor 17 beta-estradiol accumulation were affected by dexamethasone treatment at any stage of gestation. Data suggest that corticosteroids play a role in parturition through PGF2 alpha synthesis regulation by fetal placental cells. Since abnormalities during parturition e.g. retained placenta, are common following dexamethasone induction of labor in cows, we postulate that the local inhibition of PGF2 alpha accumulation by cotyledon cells after corticosteroid administration, may be involved in placental retention.     

Prostaglandin E2 9-keto reductase from bovine term placenta.

The aim of the following study was to determine the activity and physico-chemical properties of prostaglandin E2 9-keto reductase from bovine placenta. Placental tissues obtained immediately after parturition were subjected to purification procedure consisting of homogenization, affinity chromatography, gel filtration and allowed to electrophoresis. The activity of enzyme was measured spectrophotometrically. The purification procedures receive 135-fold purified enzyme preparate of the molecular weight of 45 kDa with the following kinetic values: Michaelis constant for PGE2, 117 microM and max velocity 183 pmol/min. The activity of enzyme was also detected with 20 alpha-hydroxypregn-4en-3-one and with 9,10-phenanthrenquinone (Michaelis constant 22 microM and 6 microM, respectively). The determination of physico-chemical properties of prostaglandin E2 9-keto reductase, performed for the first time in bovine placenta, should aid the understanding of the metabolism of prostaglandins and their biological importance in physiological and pathological conditions in cattle.     

Tolerance of human corneal endothelium to glycerol

As an initial step in the development of a method for corneal cryopreservation by vitrification, we attempted to establish the maximum concentration of glycerol to which human corneal endothelium could be exposed at 4 degrees C for 15 min without damage. Damage was defined as an increase in mean endothelial cell size or the inability to maintain corneal thickness for 1 week after exposure to glycerol. Using a system for long-term corneal perfusion, we perfused 24 paired human corneas with glycerol at 4 degrees C. The concentration of glycerol increased at a rate of 20% (w/v) (2.2 M) per hour until the desired maximum concentration was reached for that cornea, stabilized for 15 min, and then decreased at the same rate. The corneas were then perfused at 37 degrees C with Dulbecco's medium at a rate of 5 microliters/min under 18 mm Hg intracameral pressure for 7 days with daily measurements of corneal thickness. Endothelial morphology was examined by specular microscopy and by scanning electron microscopy. After 7 days of perfusion at 37 degrees C, there was a statistically significant direct relationship between the maximum concentration of glycerol to which the experimental eyes had been exposed and the increase in mean endothelial cell size. The mean endothelial cell size increased in corneas exposed to glycerol concentrations of 40, 50, and 60% (w/v), but did not differ significantly from baseline measurements in the corneas exposed to 30% glycerol or less. Thus, there was no detectable damage to human corneas exposed to 30% (w/v) (3.3 M) glycerol in this system. Tolerance of higher concentrations may be achieved by changes in the rates of addition and removal of glycerol or in the composition of the perfusate.     

Effect of alpha-tocopherol and ascorbic acid on bovine in vitro fertilization

The aim of this work was to study the effect of alpha-tocopherol (vitamin E) and ascorbic acid on the in vitro fertilization process. Frozen bovine semen was prepared using extenders with and without addition of vitamin E. Samples were capacitated with heparin in the fertilization medium. In vitro matured oocytes were inseminated with spermatozoa frozen with and without vitamin E and, after thawing, fertilized in TALP medium (control) and in TALP medium with vitamin E (1 mg/ml), with ascorbic acid (5 mM) and with vitamin E plus ascorbic acid. Gametes were incubated in the respective fertilization medium for 48 h; those frozen without vitamin E yielded 75, 76, 69 and 49% of fertilized oocytes in the control, vitamin E, ascorbic acid and vitamin E plus ascorbic acid media, respectively. The last value was significantly different (P < 0.01). In bovine sperm frozen with vitamin E, fertilization rates were 74, 50, 47 and 34%, respectively for the 4 groups. Values observed for the different supplements were significantly different inter se (P < 0.01), except between the media with vitamin E and with ascorbic acid. These results indicate that preserved antioxidant capacity of vitamin E impairs the success of the in vitro fertilization process.     

Subcellular distribution of isocitrate dehydrogenase in early and term human placenta.

The activities of NAD-specific and NADP-specific isocitrate dehydrogenases were measured in early and term human placenta. In both tissues the activity of NADP-specific isocitrate dehydrogenase was severalfold higher than that of the NAD-dependent enzyme. Subcellular distribution of these two enzymes in the placental tissue was estimated. About 60% of the total NADP-specific isocitrate dehydrogenase activity was found in the mitochondrial fraction and about 40% in the cytosol fraction. Insignificant amounts of the total activity were bound to the microsomal fraction. The whole of the NAD-specific isocitrate dehydrogenase activity was localized in the mitochondrial fraction. The total mitochondrial NADP-specific isocitrate dehydrogenase activity in both early and term placenta was also estimated from the mitochondrial specific activity of this enzyme and the amount of mitochondrial protein in wet tissue, calculated from the activities of citrate synthase or cytochrome c oxidase assayed in the isolated mitochondrial fraction and in the tissue of early and term human placenta.     

Differential effects of magnesium on the hydrolysis of ADP and ATP in human term placenta. Effect of substrates and potassium

This study evaluates the effect of Mg2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K+, and different oxidizable substrates. Mg2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K+, succinate and 1mM Mg2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM     

Proteomic and redox‐proteomic evaluation of homogentisic acid and ascorbic acid effects on human articular chondrocytes

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues up to the deposition of melanin‐like pigments (ochronosis). Since little is known on the effects of HGA and its metabolites on articular cells, we carried out a proteomic and redox‐proteomic analysis to investigate how HGA and ascorbic acid (ASC) affect the human chondrocytic protein repertoire. We settled up an in vitro model using a human chondrocytic cell line to evaluate the effects of 0.33 mM HGA, alone or combined with ASC. We found that HGA and ASC significantly affect the levels of proteins with specific functions in protein folding, cell organization and, notably, stress response and cell defense. Increased protein carbonyls levels were found either in HGA or ASC treated cells, and evidences produced in this paper support the hypothesis that HGA‐induced stress might be mediated by protein oxidation. Our finding can lay the basis towards the settling up of more sophisticated models to study AKU and ochronosis. J. Cell. Biochem. 111: 922–932, 2010. © 2010 Wiley‐Liss, Inc.