Intracellular Ionic Variations in the Apoptotic Death of L Cells by Inhibitors of Cell Cycle Progression

Treatment with VP-16 (1-50 μM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light seattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca²⁺ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K⁺ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD⁺ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD⁺-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD⁺ triggered Golgi disassembly by BFA. This effect of NAD⁺ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the Golgi disassembling activity of BFA, and suggest that the ADP-ribosylated substrates are components of the machinery controlling the structure of the Golgi apparatus.     

Time linkages between pollination onsets of different taxa over an 11-year period in Perugia,Central Italy

Times of pollination of different taxa in the atmosphere of Perugia (Central Italy) over an 11-year period (1982–1992) were recorded and analysed by means of a 7-day volumetric Hirst-type pollen trap. For some taxa, the pollination period varied from year to year from a chronological and/or quantitative point of view. Several taxa showed a linkage in their starting dates of pollination. Knowledge of this kind of linkage allows us to build a forecasting model.     

Combined effects of vasoactive intestinal peptide and dopamine on adenylate cyclase in prolactin-secreting cells

VIP stimulates adenylate cyclase activity of male and female rat anterior pituitaries and human prolactinomas, while dopamine inhibits the enzyme activity of female rat pituitaries and prolactinomas. A dopamine inhibited cyclase can be detected also in male rats provided the enzyme activity is increased by VIP. The analysis of the dose-response curves for one agent (VIP or dopamine) in the absence or in the presence of the other indicates that the two agents exhibit a different pattern of interaction in the different systems. In fact, in female rat pituitaries and in human prolactinomas, the curves for dopamine±VIP and for VIP±dopamine were parallel, indicating that the two agents exherted their effects independently from one another. On the contrary, in male rat pituitaries, the curves were definitively non parallel, that is, the inhibitory effect of dopamine was greatly amplified by VIP. In no case was the apparent affinity (EC₅₀) of one agent modified by the presence of the other. It is concluded that two different modes of interaction between stimulatory and inhibitory neurohormones might exist at the level of adenylate cyclase from anterior pituitary cells.     

Interaction of α-cyano[14C]cinnamate with the mitochondrial pyruvate translocator

The binding of α-cyanocinnamate to rat-heart mitochondrial membrane was investigated using α-cyano[¹⁴C]cinnamate. The binding was correlated to the inhibition of pyruvate transport. The results obtained demonstrate that both these functions reach saturation at the same titre of the inhibitor. Quantitative parameters of α-cyano[¹⁴C]cinnamate binding have been determined. The binding can be prevented by pyruvate and other substrates of the carrier but not by acetate. Pyruvate decreases the affinity of α-cyanocinnamate binding, leaving the maximum number of binding unchanged. It is concluded that rat-heart mitochondria contain a specific site at which α-cyanocinnamate binds which is directly involved in the inhibition of pyruvate transport.     

Effects of GTP on binding of (3H) glucagon to receptors in rat hepatic plasma membranes.

In this study, we report the preparation of [3H]glucagon and its characteristics of binding to receptors in the rat liver plasma membrane. Binding of the labeled hormone is optimal at pH 7.0. In the absence of GTP, [3H]glucagon binding to receptors is slow and the time of equilibration is inversely proportional to the hormone concentration. In the presence of GTP, equilibrium is reached within 30 s regardless of hormone levels, and the kinetics of binding are in accord with the kinetics of activation of adenylate cyclase by native glucagon in the presence of the nucleotide. Equilibrium binding measurements indicate that, in the absence of GTP, the binding isotherm is sigmoidal with an apparent Kd of 2 nM. The addition of GTP results in a complex binding isotherm with about 90% of the binding sites having a considerably lower apparent dissociation constant (greater than 10 nM) and a small population of sites having high affinity for the hormone. The binding properties of [3H]glucagon are compared with those of 125I-glucagon, and the implications of the actions of GTP on glucagon binding are discussed in relation to the overall regulation of adenylate cyclase by hormone and the nucleotide.     

Dihydroteugin,a neo-clerodane diterpenoid from Teucrium chamaedrys

From the aerial part of Teucrium chamaedrys a new neo-clerodane diterpenoid, dihydroteugin, has been isolated, besides the previously known diterpenoids teucrin A and teugin. The structure of dihydroteugin, (12S)-15,16-epoxy-2β,6β-dihydroxy-neo-cleroda-13(16), 14-diene-18,19:20,12-diolide, was established by chemical and spectroscopic means and by partial synthesis from teugin.