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1

Pobieranie i transport jonów azotanowych w roślinach oraz ich wpływ na architekturę korzeni

100%

Naprzał M., Janota A., Szopiński M., Sitko K., Małkowski E.

Kosmos

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2016

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vol. 65

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issue 3

411-417

PL

W odpowiedzi na zróżnicowaną dostępność azotu w glebie rośliny wytworzyły mechanizmy zwiększające efektywność pobierania tego pierwiastka, w szczególności szereg systemów transportujących NO3-. Należą do nich systemy transportowe NPF (daw. NRT1), NRT2, CLC i SLAC1. NPF (symportery) są aktywne, gdy stężenie NO3- w środowisku zewnętrznym jest większe niż 0,5 mmol · dm-3, ponadto dzięki niskiej specyficzności mogą transportować inne substraty niż jony azotanowe np. aminokwasy. Białka rodziny NRT2 (symportery) transportują wyłącznie azotany i działają, gdy zewnętrzne stężenia NO3- wynoszą poniżej 0,5 mmol · dm-3. Białka SLAC1 (kanały anionowe) zlokalizowane są w komórkach szparkowych i uczestniczą w usuwaniu jonów chlorkowych i azotanowych podczas zamykania szparek. Rodzina CLC oprócz transportu chloru wykazuje duże powinowactwo do NO3-. Jednym z przedstawicieli tych białek jest CLCa, który zlokalizowany jest w tonoplaście i bierze udział w transporcie jonów azotanowych do wakuoli w antyporcie z protonami. Po pobraniu przez komórki korzenia, jony azotanowe podlegają dystrybucji do różnych tkanek i organów rośliny, dzięki aktywności kolejnych transporterów należących do wyżej wymienionych rodzin białek. Jedną z najbardziej widocznych zmian zachodzących w odpowiedzi na zróżnicowane zasoby azotanów w podłożu jest zmiana architektury korzenia. Wysokie stężenia NO3- (50 mmol · dm-3) powodują zahamowanie rozgałęziania systemu korzeniowego. W przypadku ograniczenia dostępności azotanów (0,01 mmol · dm-3) następuje stymulacja rozwoju korzeni bocznych w kierunku obszarów gleby o wyższych zawartościach azotanów (1,0 mmol · dm-3). U A. thaliana zmiany w architekturze korzeni przebiegają bez znaczących zmian w biomasie korzeni, gdyż rozwój korzeni bocznych jest równoważony skracaniem korzeni głównych. Wyczerpywanie zasobów jonów azotanowych jest sygnałem do rozbudowania systemu korzeniowego, podczas gdy wysokie zawartości tego jonu jest odczytywane, jako sygnał do rozwoju pędów.

EN

In response to varied availability of nitrogen in the soil plants developed mechanisms to improve the efficiency of uptake of this element, in particular several NO3- transport systems. These transport systems belong to the following protein families: NPF (formerly NRT1), NRT2, CLC and SLAC1. NPF (symporters) are active when the NO3- concentration in the environment is higher than 0,5 mmol · dm-3 and due to its low specifity for nitrate ions it also may transport other substrates such as e.g. amino acids. NRT2 protein family (symporters) transports nitrates and operates only when the external NO3- concentrations are below 0,5 mmol · dm-3. SLAC1 proteins (anion channels) are located in the guard cells and are involved in the efflux of chloride and nitrate ions during the closing of stomata. CLC family apart from transporting Cl- has a high affinity for NO3-. One of the members of these proteins, CLCa, is located in the tonoplast and is involved in the transport of nitrate ions to the vacuole in antiport with protons. After uptake by root cells, nitrate ions are distributed to various tissues and organs of the plant due to activity of other transporters belonging to the above-mentioned protein families. One of the most visible changes in response to the diverse resources of nitrates is a change in the architecture of roots. High concentrations of NO3- (50 mmol · dm-3) in a substrate cause inhibition of branching of the root system. In the case of limited availability of nitrates (0,01 mmol · dm-3) stimulation of lateral root development occurs towards soil areas with higher NO3- content (1,0 mmol · dm-3). In A. thaliana modifications of roots architecture occur without significant alteration in the biomass of roots, because the development of laterals is balanced by shortening of the main root. The low content of nitrate ions is a signal to expand the root system, while a high content of this ions is the signal to develop shoots.

2

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76).

88%

Awasthi S., Sharma R., Yang Y., Singhal S. S., Pikula S., Bandorowicz-Pikula J., Singh S. V., Zimniak P., Awasthi Y. C.

Acta Biochimica Polonica

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2002

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vol. 49

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issue 4

855-867

EN

We have recently demonstrated that a previously known Ral-binding GTPase activating protein, RLIP76, can also catalyze ATP-dependent transport of various structurally unrelated xeno- and endobiotics irrespective of their net charge (Awasthi et al., 2000, Biochemistry, 39: 9327). RLIP76 is a non-ATP binding cassette (ABC) protein but it has two ATP-binding sites and shows basal ATPase activity which is stimulated in the presence of its transport substrates (allocrites) such as doxorubicin (DOX) and S-(2,4-dinitrophenyl) glutathione (DNP-SG). Proteoliposomes reconstituted with purified RLIP76 catalyze ATP-dependent, saturable transport of DOX, as well as of glutathione-conjugates including leukotrienes (LTC4) and the GSH-conjugate of 4-hydroxynonenal (GS-HNE). In erythrocytes the majority of transport activity for DOX, GS-HNE, and LTC4 is accounted for by RLIP76. Cells exposed to mild oxidative stress show a rapid and transient induction of RLIP76 resulting in an increased efflux of GS-HNE and acquire resistance to oxidative stress mediated toxicity and apoptosis. Cells transfected with RLIP76 acquire resistance to DOX through increased efflux of the drug suggesting its possible role in the mechanisms of drug-resistance. In this article, we discuss the significance of transport functions of RLIP76 highlighting its role in the defense mechanisms against oxidative injury, and modulation of signaling mechanisms.

3

Functional reconstitution of Ral-binding GTPase activating protein, RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal.

88%

Sharma R., Sharma A., Yang Y., Awasthi S., Singhal S. S., Zimniak P., Awasthi Y. C.

Acta Biochimica Polonica

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2002

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vol. 49

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issue 3

693-701

EN

Earlier studies from our laboratories have shown that RLIP76, a previously described Ral-binding GTPase activating protein (Jullien-Flores et al., 1995, J. Biol. Chem. 270: 22473), is identical with the xenobiotic transporter DNP-SG ATPase, and can catalyze ATP-dependent transport of glutathione-conjugates as well as doxorubin (Awasthi et al., 2000, Biochemistry, 39: 9327). We have now reconstituted purified bacterially expressed RLIP76 in proteoliposomes, and have studied ATP-dependent uptake of the glutathione conjugate of 4-hydroxynonenal (GS-HNE) by these vesicles. Results of these studies show that RLIP76 reconstituted in proteoliposomes catalyzes ATP-dependent transport of GS-HNE against a concentration gradient. The transport of GS-HNE is saturable with respect to ATP as well as GS-HNE with Km values of 1.4 mM and 2.5 μM, respectively. These studies demonstrate that RLIP76 mediates active transport of GS-HNE, and are consistent with our previous work showing that RLIP76-mediated efflux of GS-HNE regulates the intracellular concentration of 4-HNE and thereby affects 4-HNE mediated signaling.

4

Purification and functional reconstitution of intact ral-binding GTPase activating protein, RLIP76, in artificial liposomes.

88%

Singhal S., Singhal J., Cheng J., Pikuła S., Sharma R., Zimniak P., Awasthi Y., Awasthi S.

Acta Biochimica Polonica

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2001

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vol. 48

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issue 2

551-562

EN

We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39, 9327, 2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 μM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.

5

Transport of organic anions by multidrug resistance-associated protein in the erythrocyte.

88%

Rychlik B., Pułaski Ł., Sokal A., Soszyński M., Bartosz G.

Acta Biochimica Polonica

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2000

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vol. 47

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issue 3

763-772

EN

The active transport of oxidized glutathione and glutathione S-conjugates has been demonstrated for the first time in erythrocytes and this cell remained the main subject of research on the "glutathione S-conjugate pump" for years. Further studies identified the "glutathione S-conjugate pump" as multidrug resistance-associated protein (MRP). Even though cells overexpressing MRP and isolated MRP provide useful information on MRP structure and function, the erythrocyte remains an interesting model cell for studies of MRP1 in its natural environment, including the substrate specificity and ATPase activity of the protein.

6

Controlling introduction in the seaport transport technology systems

75%

Zinchenko S., Parkhotko A., Khlopetskaya L.

Teka Commission of Motorization and Power Industry in Agriculture

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2016

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vol. 16

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issue 3

7

Coulomb blockade in molecular quantum dots

63%

Walczak K.

Open Physics

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2006

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vol. 4

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issue 1

8-19

EN

The rate-equation approach is used to describe sequential tunneling through a molecular junction in the Coulomb blockade regime. Such device is composed of molecular quantum dot (with discrete energy levels) coupled with two metallic electrodes via potential barriers. Based on this model, we calculate nonlinear transport characteristics (conductance-voltage and current-voltage dependences) and compare them with the results obtained within a self-consistent field approach. It is shown that the shape of transport characteristics is determined by the combined effect of the electronic structure of molecular quantum dots and by the Coulomb blockade. In particular, the following phenomena are discussed in detail: the suppression of the current at higher voltages, the charging-induced rectification effect, the charging-generated changes of conductance gap and the temperature-induced as well as broadening-generated smoothing of current steps.

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Pobieranie i transport jonów azotanowych w roślinach oraz ich wpływ na architekturę korzeni

Transport functions and physiological significance of 76 kDa Ral-binding GTPase activating protein (RLIP76).

Functional reconstitution of Ral-binding GTPase activating protein, RLIP76, in proteoliposomes catalyzing ATP-dependent transport of glutathione conjugate of 4-hydroxynonenal.

Purification and functional reconstitution of intact ral-binding GTPase activating protein, RLIP76, in artificial liposomes.

Transport of organic anions by multidrug resistance-associated protein in the erythrocyte.

Controlling introduction in the seaport transport technology systems

Coulomb blockade in molecular quantum dots