Effect of NO3- transport and reduction on intracellular pH: an in vivo NMR study in maize roots

The effect of NO3- uptake on cellular pH was studied in maize roots by an in vivo 31P-NMR technique. In order to separate the effects on cytoplasmic pH due to NO3- uptake from those due to NO3- reduction, tungstate was used to inhibit nitrate reductase (NR). The results confirm that in maize roots tungstate inhibited NR activity. 15N-NMR in vivo experiments demonstrated the cessation of nitrogen flux from nitrate to organic compounds. Tungstate affected neither NO3- uptake nor the levels of the main phosphorylated compounds. Slight changes in cytoplasmic pH were observed during NO3- uptake and reduction (i.e. control). By contrast, in the presence of tungstate, a consistent decrease in cytoplasmic pH occurred. The vacuolar pH did not change in any of the conditions tested. These data show that NO3- uptake is an acidifying process and suggest a possible involvement of NO3- reduction in pH homeostasis. In the presence of NO3-, a transient depolarization of transmembrane electric potential difference (Em) was observed in all the conditions analysed. However, in tungstate-treated roots, a lesser depolarization accompanied by a greater ability to recover Em was found. This was related to a higher activity of the plasma membrane (PM) H+-ATPase. When NO3- was administered as potassium salt, its uptake increased and a greater depolarization of Em took place, whilst the changes in cytoplasmic pH were remarkably reduced, according to the central role played by K+ in the control of plasma membrane activities and cell pH homeostasis. A possible involvement of cytoplasmic pH in the control of PM H+-ATPase expression during nitrate exposure is suggested.     

Employment of a noninvasive magnetic method for evaluation of gastrointestinal transit in rats

ABSTRACT: AC Biosusceptometry (ACB) was previously employed towards recording gastrointestinal motility. Our data show a reliable and successful evaluation of gastrointestinal transit of liquid and solid meals in rats, considering the methods scarcity and number of experiments needed to endorsement of drugs and medicinal plants. ACB permits real time and simultaneous experiments using the same animal, preserving the physiological conditions employing both meals with simplicity and accuracy.     

Cadmium induces acidosis in maize root cells

* Cadmium (Cd) stress increases cell metabolic demand for sulfur, reducing equivalents, and carbon skeletons, to sustain phytochelatin biosynthesis for Cd detoxification. In this condition the induction of potentially acidifying anaplerotic metabolism in root tissues may be expected. For these reasons the effects of Cd accumulation on anaplerotic metabolism, glycolysis, and cell pH control mechanisms were investigated in maize (Zea mays) roots. * The study compared root apical segments, excised from plants grown for 24 h in a nutrient solution supplemented, or not, with 10 microM CdCl(2), using physiological, biochemical and (31)P-nuclear magnetic resonance (NMR) approaches. * Cadmium exposure resulted in a significant decrease in both cytosolic and vacuolar pH of root cells and in a concomitant increase in the carbon fluxes through anaplerotic metabolism leading to malate biosynthesis, as suggested by changes in dark CO2 fixation, metabolite levels and enzyme activities along glycolysis, and mitochondrial alternative respiration capacity. This scenario was accompanied by a decrease in the net H(+) efflux from the roots, probably related to changes in plasma membrane permeability. * It is concluded that anaplerotic metabolism triggered by Cd detoxification processes might lead to an imbalance in H(+) production and consumption, and then to cell acidosis.     

SIRT1 is involved in adrenocortical cancer growth and motility

Adrenocortical cancer (ACC) is a rare tumour with unfavourable prognosis, lacking an effective treatment. This tumour is characterized by IGF-II (insulin-like growth factor II) overproduction, aromatase and ERα (oestrogen receptor alpha) up-regulation. Previous reports suggest that ERα expression can be regulated by sirt1 (sirtuin 1), a nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylases that modulates activity of several substrates involved in cellular stress, metabolism, proliferation, senescence, protein degradation and apoptosis. Nevertheless, sirt1 can act as a tumour suppressor or oncogenic protein. In this study, we found that in H295R and SW13 cell lines, sirt1 expression is inhibited by sirtinol, a potent inhibitor of sirt1 activity. In addition, sirtinol is able to decrease ACC cell proliferation, colony and spheroids formation and to activate the intrinsic apoptotic mechanism. Particularly, we observed that sirtinol interferes with E2/ERα and IGF1R (insulin growth factor 1 receptor) pathways by decreasing receptors expression. Sirt1 involvement was confirmed by using a specific sirt1 siRNA. More importantly, we observed that sirtinol can synergize with mitotane, a selective adrenolitic drug, in inhibiting adrenocortical cancer cell growth. Collectively, our data reveal an oncogenic role for sirt1 in ACC and its targeting could implement treatment options for this type of cancer.     

Comparison of the behaviour of supported homogeneous catalysts in the synthesis of dimethylcarbonate from methanol and carbon dioxide: Polystyrene-grafted tin-metallorganic species versus silesquioxanes linked Nb-methoxo species

Two kinds of supported homogeneous catalysts have been used in the synthesis of dimethylcarbonate from methanol and carbon dioxide. and [Nb(OMe)₅]₂, that are reported to be active catalysts under homogeneous conditions, have been bound to a solid support. The moiety [-BuⁿSn(OMe)₂] has been grafted on a polystyrene resin by using a phenylethylene spacer, while the [Nb(OMe)₅]₂ moiety and NbOCl₃ have been linked to a silesquioxane matrix. The grafted tin metallorganic species is still active in the synthesis of DMC from methanol and CO₂: tin is not lost during catalysis and the heterogenized catalyst can be easily recovered and reused. Conversely, the Nb-silesquioxane adduct is either a monomer or a dimer that does not bring any -OMe moiety linked to Nb and does not react with CO₂ nor with methanol, resulting to be inactive in catalysis.     

Cadmium retention in rice roots is influenced by cadmium availability, chelation and translocation

Analysis of rice plants exposed to a broad range of relatively low and environmentally realistic Cd concentrations showed that the root capacity to retain Cd ions rose from 49 to 79%, corresponding to increases in the external Cd2+ concentration in the 0.01-1 μM range. Fractioning of Cd ions retained by roots revealed that different events along the metal sequestration pathway (i.e. chelation by thiols, vacuolar compartmentalization, adsorption) contributed to Cd immobilization in the roots. However, large amounts of Cd ions (around 24% of the total amount) predictable as potentially mobile were still found in all conditions, while the amount of Cd ions loaded in the xylem seemed to have already reached saturation at 0.1 μM Cd2+, suggesting that Cd translocation may also play an indirect role in determining Cd root retention, especially at the highest external concentrations. In silico search and preliminary analyses in yeast suggest OsHMA2 as a good candidate for the control of Cd xylem loading in rice. Taken as a whole, data indicate Cd chelation, compartmentalization, adsorption and translocation processes as components of a complex 'firewall system' which acts in limiting Cd translocation from the root to the shoot and which reaches different equilibrium positions depending on Cd external concentration.     

Tungstate-Targeting of BKαβ1 Channels Tunes ERK Phosphorylation and Cell Proliferation in Human Vascular Smooth Muscle

Despite the substantial knowledge on the antidiabetic, antiobesity and antihypertensive actions of tungstate, information on its primary target/s is scarce. Tungstate activates both the ERK1/2 pathway and the vascular voltage- and Ca²⁺-dependent large-conductance BKαβ₁ potassium channel, which modulates vascular smooth muscle cell (VSMC) proliferation and function, respectively. Here, we have assessed the possible involvement of BKαβ₁ channels in the tungstate-induced ERK phosphorylation and its relevance for VSMC proliferation. Western blot analysis in HEK cell lines showed that expression of vascular BKαβ₁ channels potentiates the tungstate-induced ERK1/2 phosphorylation in a G_i/o protein-dependent manner. Tungstate activated BKαβ₁ channels upstream of G proteins as channel activation was not altered by the inhibition of G proteins with GDPβS or pertussis toxin. Moreover, analysis of G_i/o protein activation measuring the FRET among heterologously expressed G_i protein subunits suggested that tungstate-targeting of BKαβ₁ channels promotes G protein activation. Single channel recordings on VSMCs from wild-type and β₁-knockout mice indicated that the presence of the regulatory β₁ subunit was essential for the tungstate-mediated activation of BK channels in VSMCs. Moreover, the specific BK channel blocker iberiotoxin lowered tungstate-induced ERK phosphorylation by 55% and partially reverted (by 51%) the tungstate-produced reduction of platelet-derived growth factor (PDGF)-induced proliferation in human VSMCs. Our observations indicate that tungstate-targeting of BKαβ₁ channels promotes activation of PTX-sensitive G_i proteins to enhance the tungstate-induced phosphorylation of ERK, and inhibits PDGF-stimulated cell proliferation in human vascular smooth muscle.