The effects of 4beta-phorbol-12-myristate-13-acetate (PMA) and 1,2-(sn)-dioctanoylglycerol (DOCG) on the phosphatidylcholine (PC) turnover (defined as degradation to diacylglycerol followed by PC resynthesis) and on the activity of PC-specific phospholipase D were investigated in placental mince incubated with various radiolabelled precursors in vitro. Experiments with [32P]phosphate indicated that 1 microM PMA and 125-250 microM DOCG were the lowest concentrations that led to maximal and selective stimulation of PC labelling. Moreover, PMA and DOCG acted along different time courses: PMA enhanced labelling after 60 min incubation, with a lag period of at least 30 min, whereas DOCG stimulated PC labelling after only 30 min with no further increase in the next 30 min. The following findings suggest that increased labelling of PC with [32P]phosphate in PMA-treated tissue reflects an increased rate of PC turnover: (1) the effects of PMA and DOCG were additive and PMA did not have any effect on the labelling of PC(DOCG) indicating that it stimulated PC labelling even if it did not activate CTP:choline cytidylyl transferase, the regulatory enzyme of PC synthesis de novo; (2) PMA did not increase the labelling of PC from [3H]glycerol or [3H]glucose ruling out a PMA-promoted availability of glycolytic and/or lipolytic intermediates for PC formation; and (3) the PMA effect was attended by an increased labelling of phosphatidic acid whereas there was no change in the labelling of lyso-PC, indicating the activation of phospholipase D. Experiments in which the transphosphatidylation reaction between [3H]myristic acid-labelled PC and ethanol was used to estimate phospholipase D activity showed 2.4-fold and 1.4-1.8-fold activations by PMA and DOCG, respectively, with no additivity noted. These results suggest that PMA stimulates PC turnover in the early human placenta via the activation of phospholipase D. Rapid metabolic conversion decreases the capacity of DOCG to accelerate PC-turnover and to activate phospholipase D. The early DOCG-induced stimulation of PC labelling with [32P]phosphate is attributed mainly to its known activating effect on CTP: choline cytidylyl transferase.

Carcinogens↗

PubMedDate not supplied

Activation and dimerization of type III nitric oxide synthase by submicromolar concentrations of tetrahydrobiopterin in microsomal preparations from human primordial placenta.

We have found in a previous study that 5-50 microM tetrahydrobiopterin (BH4) stimulated 1.2-2.5-fold Ca(2+)-dependent nitric-oxide synthase (NOS) activity in homogenates prepared from primordial human placentae. Now we report on the dramatic, about sixfold, activating effect of BH4 on this activity measured in microsomal preparations. Firstly, both in the absence and presence of BH4, arginine bound to kinetically homogeneous sites, with no significant change between the apparent KM values for arginine (3.12 +/- 1.99 microM and 2.06 +/- 1.13 microM in the absence and presence of 50 microM BH4 respectively, mean +/- s.d., n = 3). On the other hand, the Vmax values measured in different pools of placenta tissue varied between 2.5-7.55 (no BH4 added) and 13.3-58.5 (with BH4 added) pmol/min/mg protein. Secondly, the microsomal preparations responded sensitively to BH4 addition. A dose-response study indicated that as low as 79 nM final BH4 concentration stimulated NOS activity half-maximally, and 1 microM BH4 resulted in an almost maximal effect. Thirdly, immunoblot analysis combined with laser densitometric evaluation demonstrated that BH4 efficiently promoted the aggregation of microsomal NOS type III isozyme into a protein having the characteristics (electrophoretic mobility, resistance of SDS) of the dimeric form. Half-maximal dimerizing activity was reached at 148 +/- 33 nM BH4 (mean +/- s.d., n = 3), whereas 1 microM BH4 led to almost, maximal aggregation of monomers. This is the first time that BH4-induced dimerization of a NOS type III isoform has been demonstrated. Considering that human placenta predominantly expresses NOS type III isoform and BH4 concentration in this tissue is 207 +/- 87 nM, the present results strongly suggest that the dimerizing effect of BH4 is a crucial physiological mechanism for the assembly of active Ca(2+)-dependent NOS in the human primordial placenta.

Biopterins↗

PubMedDate not supplied

Leptin inhibits cortisol and corticosterone secretion in pathologic human adrenocortical cells.

Regulation of adrenal corticosteroid secretion by leptin may involve interactions at multiple levels of the hypothalamic-pituitary-adrenal axis. To investigate the possible direct effects of leptin on corticosteroid secretion of human adrenocortical adenomas, cells from adrenocortical adenomas causing primary aldosteronism (n = 1) and Cushing's syndrome (n = 1), as well as cells from nonhyperfunctioning adrenocortical adenomas (n = 5) were isolated and incubated for 2 h with human recombinant leptin (1-1000 ng/ml) in the presence and absence of adrenocorticotrop hormone (ACTH), then cortisol, corticosterone and aldosterone concentrations in incubating media were determined using radioimmunoassays. It was found that leptin effectively and dose-dependently inhibited basal and ACTH-stimulated cortisol and corticosterone secretion in the three types of human adrenocortical adenoma cells. The inhibiting effect of basal corticosterone secretion was detectable in the presence of leptin concentration as low as 1 ng/ml, with decreases of corticosterone secretion to 34+/-4%, 57+/-11% and 79+/-9% in Cushing's syndrome, primary aldosteronism, and nonhyperfunctioning adrenocortical adenoma cells, respectively. The inhibition of basal cortisol secretion in the presence of low concentration of leptin was less prominent, but 10 ng/ml leptin significantly diminished basal cortisol secretion to 81+/-9% in adrenocortical adenoma cells from Cushing's syndrome, to 68+/-6% in adenoma cells from primary aldosteronism, and to 83+/-8% in cells from nonhyperfunctioning adenomas. The inhibition of ACTH-stimulated cortisol and corticosterone secretion by leptin was similar to those found in cells without ACTH stimulation. By contrast, leptin even at 1000 ng/ml concentration exerted no clear effect on basal and ACTH-stimulated aldosterone secretion in cells from primary aldosteronism and in those nonhyperfunctioning adenoma cells in which aldosterone secretion was detectable. These results indicate that leptin is a potent inhibitor of cortisol and corticosterone secretion in human adenomatous adrenocortical cells. The inhibition of these corticosteroids by leptin may represent a potentially important interaction that exists between leptin and the hypothalamic-pituitary-adrenal axis.

Adenoma↗

PubMedDate not supplied

Sterically inhomogenous viscoelastic behavior of human saccular cerebral aneurysms.

To clarify the mechanism leading to the development and rupture of intracranial aneurysms, tensile strength and viscoelastic parameters of 22 human saccular aneurysms were investigated. Meridional and circumferential strips from the thin and the thick part of the aneurysm sack and 18 control strips from the basilar artery of 8 patients with pathologies not affecting the cerebral arterial system were studied. The length of the strips was increased in 200- microm steps, while distending force was recorded. Tensile strength and viscoelastic parameters were computed. In both directions, tensile strength of thick strips was significantly lower than that of controls. In the meridional direction, tensile strength of thin strips was significantly larger than that of thick ones (14.5 +/- 4.1 x 10(6) vs. 7.5 +/- 2.0 x 10(6) dyn/cm2, p < 0.05). In the circumferential direction, thin strips tore at lower strain values than thick ones (29 +/- 4 vs. 55 +/- 16%, p < 0.05). Viscoelastic parameters changed in parallel. In circumferential direction, values of thick and thin strips were significantly lower than those of controls. In the meridional direction, values of thin strips were significantly higher than those of the thick ones. These observations show that characteristic mechanical deterioration and steric inhomogeneities accompany the loss of smooth muscle cells and the derangement of connective tissue elements in the wall of intracranial aneurysms, which may explain certain steps in their initiation, enlargement and rupture.

Adult↗

PubMedDate not supplied

[Molecular aspects of interferon induction by adenoviruses].

The capacity of adenovirus to induce interferon in the infected cells was studied. The examined adenovirus strains of early types were grouped in 2 groups according to their capacity to induce interferon and to the sensitivity of the infecting and interferon-inducing activity to UV-irradiation. A common property of adenoviruses, potent interferon inducers, is their high sensitivity to UV-irradiation.

Adenoviruses, Human↗