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One of the urgent problems of gene therapy is the search for effective transfection methods. Synthetic cationic peptides (CPs) are considered to be one of the most promising approaches for intracellular transport of oligonucleotides. Almost unlimited possibilities of the architectural design of CPs (linear and cyclic structures with a variation of chirality as well as dendrimers) make CPs an effective tunable carrier in this field. Cationic peptide dendrimers (PDs), as a relatively new direction, have significant advantages as gene delivery vehicles by virtue of non-natural ε-amide bonds that significantly increase their resistance to proteolysis. Moreover they also possess much lower cytotoxicity than linear peptides, which is crucial for the potential clinical application of CPs. In a further development of oligonucleotide delivery systems, we have synthesized a collection of 14 CPs, including linear peptides, lipopeptides and PDs. Their activity was evaluated by transfection of 293T cells with plasmids containing reporter genes encoding luciferase or a green fluorescent protein. The obtained results demonstrated that the greatest activity was exhibited by PDs, particularly LTP, an arginine-rich peptide dendrimer, which possesses low cytotoxic and hemolytic activity. The peptide exhibited high cell-penetrating activity, confirmed by fast dissipation of the membrane potential of cells probed by dis-C3-(5). The quantitative analysis of labelled LTP in tissue samples of mice revealed that the Cy5-LTP/siRNA complexes have a reasonable tropism to lung tissues.

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Symbiosomes are organelle-like compartments responsible for nitrogen fixation in infected nodule cells of legumes, which are formed as a result of symbiotic association of soil bacteria rhizobia with certain plant root cells. They are virtually the only source of reduced nitrogen in the Earth's biosphere, and consequently, are of great importance. It has been proven that the functioning of symbiosomes depends to a large extent on the transport of various metabolites and ions - most likely including Ca2+ - across the symbiosome membrane (SM). Although it has been well established that this cation is involved in the regulation of a broad spectrum of processes in cells of living organisms, its role in the functioning of symbiosomes remains obscure. This is despite available data indicating both its transport through the SM and accumulation within these compartments. This review summarises the results obtained in the course of studies on the given aspects of calcium behaviour in symbiosomes, and on this basis gives a possible explanation of the proper functional role in them of Ca2+.

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Histidine plays a crucial role in nickel (Ni) translocation in Ni-hyperaccumulating plants. Here, we investigated its role in zinc (Zn) translocation in four accessions of the Zn hyperaccumulator, Noccaea caerulescens, using the related non-hyperaccumulator, Thlaspi arvense, as a reference. We compared the effects of exogenous histidine supply on Zn xylem loading, and of Zn-histidine complex formation on Zn uptake in energized tonoplast vesicles. The Zn distribution patterns over root tissues were also compared. Exogenous histidine supply enhanced Zn xylem loading in all the N. caerulescens accessions, but decreased it in T. arvense. Zn distribution patterns over root tissues were similar, apart from the accumulation in cortical and endodermal cells, which was much lower in N. caerulescens than in T. arvense. Zn uptake in energized tonoplast vesicles was inhibited significantly in N. caerulescens, but not affected significantly in T. arvense, when Zn was supplied in combination with histidine in a 1:2 molar ratio. Histidine-mediated Zn xylem loading seems to be a species-wide character in N. caerulescens. It may well have evolved as a component trait of the hyperaccumulation machinery for Zn, rather than for Ni.

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Ca(2+)-ATPase in the peribacteroid membrane (PBM) of symbiosomes isolated from Vicia faba root nodules was characterized in terms of its hydrolytic and transport activities. Both activities were found to be pH-dependent and exhibit pH optimum at pH 7.0. Translocation of Ca(2+) through the PBM by the Ca(2+)-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP > ITP ≅ GTP ≅ UTP ≅ CTP, the K m of the enzyme for MgATP being about 100 µM. Ca-dependent ITP-hydrolytic activity of symbiosomes was investigated in the presence of the Ca-EGTA buffer system and showed the affinity of PBM Ca(2+)-ATPase for Ca(2+) of about 0.1 µM. The transport activity of Ca(2+)-ATPase was inhibited by erythrosin B as well as orthovanadate, but markedly stimulated by calmodulin from bovine brain. These results allowed us to conclude that this enzyme belongs to IIB-type Ca(2+)-ATPases which are present in other plant membranes.

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The hypothesis that the primary Na+-pump, Na+-ATPase, functions in the plasma membrane (PM) of halotolerant microalga Dunaliella maritima was tested using membrane preparations from this organism enriched with the PM vesicles. The pH profile of ATP hydrolysis catalyzed by the PM fractions exhibited a broad optimum between pH 6 and 9. Hydrolysis in the alkaline range was specifically stimulated by Na+ ions. Maximal sodium dependent ATP hydrolysis was observed at pH 7.5-8.0. On the assumption that the ATP-hydrolysis at alkaline pH values is related to a Na+-ATPase activity, we investigated two ATP-dependent processes, sodium uptake by the PM vesicles and generation of electric potential difference (Deltapsi) across the vesicle membrane. PM vesicles from D. maritima were found to be able to accumulate 22Na+ upon ATP addition, with an optimum at pH 7.5-8.0. The ATP-dependent Na+ accumulation was stimulated by the permeant NO3- anion and the protonophore CCCP, and inhibited by orthovanadate. The sodium accumulation was accompanied by pronounced Deltapsi generation across the vesicle membrane. The data obtained indicate that a primary Na+ pump, an electrogenic Na+-ATPase of the P-type, functions in the PM of marine microalga D. maritima.

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Our previous investigations have established that Na+ translocation across the Tetraselmis viridis plasma membrane (PM) mediated by the primary ATP-driven Na+-pump, Na+-ATPase, is accompanied by H+ counter-transport [Y.V. Balnokin et al. (1999) FEBS Lett 462:402-406]. The hypothesis that the Na+-ATPase of T. viridis operates as an Na+/H+ exchanger is tested in the present work. The study of Na+ and H+ transport in PM vesicles isolated from T. viridis demonstrated that the membrane-permeant anion NO3- caused (i) an increase in ATP-driven Na+ uptake by the vesicles, (ii) an increase in (Na(+)+ATP)-dependent vesicle lumen alkalization resulting from H+ efflux out of the vesicles and (iii) dissipation of electrical potential, deltapsi, generated across the vesicle membrane by the Na+-ATPase. The (Na(+)+ATP)-dependent lumen alkalization was not significantly affected by valinomycin, addition of which in the presence of K+ abolished deltapsi at the vesicle membrane. The fact that the Na+-ATPase-mediated alkalization of the vesicle lumen is sustained in the absence of the transmembrane deltapsi is consistent with a primary role of the Na+-ATPase in driving H+ outside the vesicles. The findings allowed us to conclude that the Na+-ATPase of T. viridis directly performs an exchange of Na+ for H+. Since the Na+-ATPase generates electric potential across the vesicle membrane, the transport stoichiometry is mNa+/nH+, where m> n.

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Cells of the cyanobacterium Synechocystis sp. PCC 6803 are equipped with a mechanosensitive ion channel MscL that is located in their plasma membrane. However, the exact function of the channel in this freshwater cyanobacterium is unknown. This study shows that cells of Synechocystis are capable of releasing Ca(2+) in response to depolarization of the plasma membrane by the K(+) ionophore valinomycin in the presence of K(+) or by tetraphenylphosphonium (TPP(+)). A fluorescent dye, diS-C(3)-(5), sensitive to membrane potential and the metallochromic Ca(2+) indicator arsenazo III were used to follow the plasma membrane depolarization and the Ca(2+) release, respectively. The Ca(2+) release from wild-type cells was temperature-dependent and it was strongly inhibited by the Ca(2+) channel blocker verapamil and by the mechanosensitive channel blocker amiloride. In MscL-deficient cells, Ca(2+) release was about 50 % of that from the wild-type cells. The mutant cells had lost temperature sensitivity of Ca(2+) release completely. However, verapamil and amiloride inhibited Ca(2+) release from these cells in same manner as in the wild-type cells. This suggests the existence of additional Ca(2+) transporters in Synechocystis, probably of a mechanosensitive nature. Evidence for the putative presence of intracellular Ca(2+) stores in the cells was obtained by following the increase in fluorescence intensity of the Ca(2+) indicator chlortetracycline. These results suggest that the MscL of Synechocystis might operate as a verapamil/amiloride-sensitive outward Ca(2+) channel that is involved in the plasma-membrane depolarization-induced Ca(2+) release from the cells under temperature stress conditions.

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