Growth and senescence of Medicago truncatula cultured cells are associated with characteristic mitochondrial morphology

Here mitochondrial morphology and dynamics were investigated in Medicago truncatula cell-suspension cultures during growth and senescence. Cell biology techniques were used to measure cell growth and death in culture. Mitochondrial morphology was investigated in vivo using a membrane potential sensor probe coupled with confocal microscopy. Expression of a senescence-associated gene (MtSAG) was evaluated in different cell-growth phases. Mitochondria appeared as numerous, punctuate organelles in cells at the beginning of the subculture cycle, while interconnected networks were observed in actively growing cells. In senescent cells, giant mitochondria were associated with dying cells. The release of cytochrome c from mitochondria was detected in different growth phases of cultured cells. Studies on plant cell cultures allowed us to identify physiological and molecular markers of senescence and cell death, and to associate distinct mitochondrial morphology with cells under different physiological conditions.     

Molecular mechanisms of nucleoside recycling in the brain

A major role of plasma membrane bound ectonucleotidases is the modulation of ATP, ADP, adenosine (the purinergic agonists), UTP, and UDP (the pyrimidinergic agonists) availability in the extracellular space at their respective receptors. We have recently shown that an ATP driven uridine-UTP cycle is operative in the brain, based on the strictly compartmentalized processes of uridine salvage to UTP and uridine generation from UTP, in which uptaken uridine is anabolized to UTP in the cytosol, and converted back to uridine in the extracellular space by the action of ectonucleotidases (Ipata et al. Int J Biochem Cell Biol 2010;42:932-7). In this paper we show that a similar cytidine-CTP cycle exists in rat brain. Since (i) brain relies on imported preformed nucleosides for the synthesis of nucleotides, RNA, nuclear and mitochondrial DNA, coenzymes, pyrimidine sugar- and lipid-conjugates and (ii) no specific pyrimidinergic receptors have been identified for cytidine and their nucleotides, our results, taken together with previous studies on the intra- and extracellular metabolic network of ATP, GTP, UTP, and their nucleosides in the brain (Barsotti and Ipata. Int J Biochem Cell Biol 2004;36:2214-25; Balestri et al. Neurochem Int 2007;50:517-23), strongly suggest that, apart from the modulation of ligand availability, ectonucleotidases may serve the process of local nucleoside recycling in the brain.     

Natural-product diversity of the New Caledonian marine ecosystem compared to other ecosystems: a pharmacologically oriented view

In comparison with other ecosystems, biodiversity and natural-product diversity of the New Caledonian marine ecosystem, comprising lagoons, barrier reefs, and deep waters in seamount regions, are described here phylogenetically with the aid of molecular drawings and tabulation of data. Admittedly, since the inception of these studies in 1977, the comparison is biased by selection of New Caledonian organisms on the basis of positive pharmacologically oriented bioassays. However, we show that these and other distortions must be accepted to draw any comparison on a regional basis, which, nonetheless, turn out to be useful for the progress of knowledge, particularly in directing future explorations of biodiversity in the search for new pharmacologically active metabolites.     

Effects of the regulatory ligands calcium and GTP on the thermal stability of tissue transglutaminase

Tissue transglutaminase undergoes thermal inactivation with first-order kinetics at moderate temperatures, in a process which is affected in opposite way by the regulatory ligands calcium and GTP, which stabilize different conformations. We have explored the processes of inactivation and of unfolding of transglutaminase and the effects of ligands thereon, combining approaches of differential scanning calorimetry (DSC) and of thermal analysis coupled to fluorescence spectroscopy and small angle scattering. At low temperature (38-45°C), calcium promotes and GTP protects from inactivation, which occurs without detectable disruption of the protein structure but only local perturbations at the active site. Only at higher temperatures (52-56°C), the protein structure undergoes major rearrangements with alterations in the interactions between the N- and C-terminal domain pairs. Experiments by DSC and fluorescence spectroscopy clearly indicate reinforced and weakened interactions of the domains in the presence of GTP and of calcium, and different patterns of unfolding. Small angle scattering experiments confirm different pathways of unfolding, with attainment of limiting values of gyration radius of 52, 60 and 90?? in the absence of ligands and in the presence of GTP and calcium. Data by X-rays scattering indicate that ligands influence retention of a relatively compact structure in the protein even after denaturation at 70°C. These results suggest that the complex regulation of the enzyme by ligands involves both short- and long-range effects which might be relevant for understanding the turnover of the protein in vivo.     

Drug-induced expansion and differentiation of V gamma 9V delta 2 T cells in vivo: the role of exogenous IL-2

Human Vgamma9Vdelta2 T cells recognize nonpeptidic Ags generated by the 1-deoxy-d-xylulose 5-phosphate (many eubacteria, algae, plants, and Apicomplexa) and mevalonate (eukaryotes, archaebacteria, and certain eubacteria) pathways of isoprenoid synthesis. The potent Vgamma9Vdelta2 T cell reactivity 1) against certain cancer cells or 2) induced by infectious agents indicates that therapeutic augmentations of Vgamma9Vdelta2 T cell activities may be clinically beneficial. The functional characteristics of Vgamma9Vdelta2 T cells from Macaca fascicularis (cynomolgus monkey) are very similar to those from Homo sapiens. We have found that the i.v. administration of nitrogen-containing bisphosphonate or pyrophosphomonoester drugs into cynomolgus monkeys combined with s.c. low-dose (6 x 10(5) U/animal) IL-2 induces a large pool of CD27+ and CD27- effector/memory T cells in the peripheral blood of treated animals. The administration of these drugs in the absence of IL-2 is substantially less effective, indicating the importance of additional exogenous costimuli. Shortly after the costimulatory IL-2 treatment, only gammadelta (but not alphabeta) T cells expressed the CD69 activation marker, indicating that Vgamma9Vdelta2 T lymphocytes are more responsive to low-dose IL-2 than alphabeta T cells. Up to 100-fold increases in the numbers of peripheral blood Vgamma9Vdelta2 T cells were observed in animals receiving the gammadelta stimulatory drug plus IL-2. Moreover, the expanded Vgamma9Vdelta2 T cells were potent Th1 effectors capable of releasing large amounts of IFN-gamma. These results may be relevant for designing novel (or modifying current) immunotherapeutic trials with nitrogen-containing bisphosphonate or pyrophosphomonoester drugs.     

Delayed luminescence: a novel technique to obtain new insights into water structure

Fully understanding the structure of water is a crucial point in biophysics because this liquid is essential in the operation of the engines of life. Many of its amazing anomalies seem to be tailored to support biological processes and, during about a century, several models have been developed to describe the water structuring. In particular, a theory assumes that water is a mixture of domains constituted by two distinct and inter-converting structural species, the low-density water (LDW) and the high-density water (HDW). According to this theory, by using some particular solutes or changing the water temperature, it should be possible to modify the equilibrium between the two species, changing in this way the water behavior in specific biological processes, as in governing the shape and stability of the structures of proteins. In this work, we assess the possibility of obtaining information on the structures induced in water by specific salts or by temperature by measuring the delayed luminescence (DL) of some salt solutions and of water in the super-cooled regime. Previous works have demonstrated that the delayed luminescence of a system is correlated with its dynamic ordered structures. The results show significant DL signals only when the formation of LDW domains is expected. The measurement reveals a similar activation energy for the domains both in aqueous salt solutions and super-cooled water. It is worth noting that the time trend of DL signals suggests the existence of structures unusually long-lasting in time, up to the microsecond range.