Effect of outer sphere anions on the structure and color of nitrosylpentaamminechromium complex

Three kinds of crystalline compounds containing the nitrosylpentaamminechromium complexes [Cr(NO)(NH₃)₅]²⁺(A) were obtained: chloride ACl₂ (red-orange), chloride perchlorate ACl(ClO₄) (brown), and perchlorate A(ClO₄)₂ (green). The cause of the color change of the complex A with the change of outer sphere anions was sought using X-ray structural data of ACl₂, ACl(ClO₄), and A(ClO₄)₂. Crystal data: ACl₂, orthorhombic, space group Cmcm, a=10.0236 (9) Å, b=9.098 (3) Å, c=10.357(1) Å, V=944.5 (5) ų, Z=4; ACl(ClO₄), tetragonal, space group P4/nmm, a=7.6986 (8) Å, c=9.9566(8) Å, V=590.1 (1) Å^3,Z=2; A(ClO₄)₂, orthorhombic, space group Pnma, a=15.760 (2) Å, b=11.480(2) Å, c=7.920 (2) Å, V=1432.9 (4) ų, Z=4. The complex cation in ACl₂ has a distorted octahedral structure with a linear CrNO moiety. The short CrN (nitrosyl) distance of 1.692 (7) Å indicates the presence of multiple bonding between the chromium atom and the nitrogen atom in the nitrosyl group. The interatomic distances and angles within the complex cations hardly change with the change of the counter anions, while the distances between the complex cations in each crystal increase in the order ACl₂<ACl(ClO₄)<A(ClO₄)₂. The bulky perchlorate anions seems to separate the complex cations, while smaller chloride anions are not large enough to separate them. The distance (3.213(5) Å) between O(NO) and N(NH₃ in the adjacent complex cation) is rather short in the crystal of ACl₂, and there are six hydrogen bonds, where the NO group is surrounded by four NH₃ ligands. The distance (4.002(5) Å) between O(NO) and N(NH₃) is much longer in the crystal of A(ClO₄)₂, indicating the presence of no hydrogen bonding. In the crystal of ACl(ClO₄) the distance (3.452(4) Å) between O(NO) and N(NH₃) is in between those of ACl₂ and A(ClO₄)₂. The presence of hydrogen bonding between O(NO) and N(NH₃ in the adjacent complex cation) seems to cause the color change with the change of outer sphere anions.     

CO2 provides an intermediate link in the red light response of guard cells

Guard cells in intact leafs display light-induced membrane potential changes, which alter the direction of K+-transport across the plasma membrane (Roelfsema et al., 2001). A beam of blue light, but not red light, directed at the impaled guard cell triggers this response, while both light qualities induce opening of stomata. To gain insight into this apparent contradiction, we explored the possible interaction between red light and CO2. Guard cells in the intact plant were impaled with double-barrelled electrodes and illuminated with red light. Cells that were hyperpolarized in CO2-free air, depolarized after a switch to air with 700 micro l l(-1) CO2, in a reversible manner. As a result, K+-fluxes across the plasma membrane changed direction, to favour K+ extrusion and stomatal closure in the presence of CO2. Concurrent with the depolarization, an inward current across the plasma membrane appeared, most likely due to activation of anion channels. Guard cell responses to CO2 could be recorded in darkness as well as in red light. However, in darkness some cells spontaneously depolarized, these cells hyperpolarized again in red light. Here, red light was projected on a large area of the leaf and decreased the intracellular CO2 concentration by about 250 micro l l(-1), as measured with a miniature CO2 sensor placed in the substomatal cavity. We conclude, that in intact leaves the red light response of guard cells is mediated through a decrease of the intercellular CO2 concentration.     

Transport of cadmium across the apical membrane of epithelial cell lines

Cadmium (Cd) uptake and secretion across the apical membrane of epithelial cells was studied using LLC-PK1 cells cultured on Petri dishes and permeable membranes, respectively. Cd accumulation in cells from the apical medium was decreased by low temperature and metabolic inhibitors. A saturable tendency was observed between initial Cd accumulation and increased concentrations of Cd in the apical medium at 37 degrees C, but not at 4 degrees C. Co-incubation with ZnCl2 or CuCl2 competitively decreased Cd accumulation at 37 degrees C. A decrease in the pH of the apical medium markedly decreased Cd accumulation. Pretreatment of cells with an inorganic anion-exchange inhibitor significantly decreased Cd uptake at pH 7.4 in the presence of bicarbonate, but only marginally in its absence. A decrease in the pH of the apical medium increased the secretory (basolateral-to-apical) transport of Cd, with a concomitant decrease in the cellular accumulation of Cd. Co-incubation with Cd and tetraethylammonium, a typical substrate of the organic cation transporter, decreased Cd transport, with a concomitant increase in cellular Cd accumulation. The uptake and secretion of Cd across the apical membrane appear to be partly mediated via an inorganic anion exchanger and a H+ antiport of the organic cation transport system, respectively. Therefore, a decrease in pH of the apical medium markedly decreases Cd accumulation, possibly as a result of not only the decrease in Cd uptake via an inorganic anion exchanger, but also the increase in Cd secretion via the Cd2+/H+ antiport. Further evidence of the antiport was obtained from experiments using brush border membrane vesicles isolated from rat kidney and small intestine. In addition, passive diffusion of Cd appears to be decreased by low temperature and a decrease in pH.     

Tetrahydrobiopterin improves vascular endothelial function in ovariectomized rats

The goal of the present study is to investigate the role of tetrahydrobiopterin (BH4) in the vascular response in ovariectomized rats. Rats were randomly assigned to two groups: (1) sham group: sham-operated female rats, and (2) Ovx group: rats were ovariectomized. Our results have shown that the plasma 17 beta-estradiol levels in the Ovx group at the end of the experiment were significantly lower than in the sham group. Vasoreactivity assessed with intact aortic rings indicated that the phenylephrine-induced vasocontractile response to aortic rings from the Ovx group was greater than that of the sham group. In contrast, the vasodilator responses to acetylcholine and L-arginine (L-Arg) in the sham group were significantly greater than in the Ovx group. Differences in vasoreactivity in denuded aorta between the two groups were not noted. Moreover, exogenous BH4 significantly restored L-Arg-induced vasodilator responses in the Ovx group. However, this improvement effect was not found in the sham group. In addition, there were significant increases in superoxide anion production in aortic tissue and significant decreases in plasma nitric oxide levels in the Ovx group. Furthermore, BH4 contents in the aorta in the Ovx group were significantly decreased compared with the sham group. In conclusion, the present study demonstrates that the impairment of vascular reactivity was found in the ovariectomized rats. The possible mechanism of this defect may have resulted from the deficiency of available BH4. Thus, this study may provide a novel therapeutic strategy for the treatment of postmenopausal cardiovascular disorders.     

Change of Cu,Zn-superoxide dismutase activity of guinea pig lung in experimental asthma

Correlation between the level of reactive oxygen species (ROS) generated by airway inflammatory cells and superoxide dismutase (SOD) activity of pulmonary tissue during an asthma attach was investigated in a guinea pig model of allergic asthma. In addition, the influence of SOD inhibition by diethyldithiocarbamate (DDC, Cu-chelating agent) on the airway was investigated in terms of pulmonary function during an asthma attach. Relative to controls, the capacity of bronchoalveolar lavage fluid (BAL) cells to release ROS was significantly increased in guinea pigs sensitized with ovalbumin (OA) as the antigen, and significantly increased in guinea pigs with an asthma attack provoked by the inhalation of OA. SOD activity was increased significantly in the antigen-sensitized group. The asthma provocation group showed a tendency for increase in total SOD activity, compared with the sensitization group, whose increase was dependent on the increase in copper, zinc-SOD (Cu, Zn-SOD) activity. Pretreatment with DDC increased the severity and duration of the asthma attack. These results were indicated that Cu, Zn-SOD was closely involved in the asthma process, particularly in the scavenging of oxygen radicals secreted from BAL cells.     

Cloning of the pig renal organic anion transporter 1 (pOAT1)

A pig kidney cDNA library was screened for the porcine ortholog of the multispecific organic anion transporter 1 (pOAT1). Several positive clones were isolated resulting in two alternatively spliced cDNA clones of pOAT1 (pOAT1 and pOAT1A). pOAT1-cDNAs consist of 2126 or 1895 base pairs (EMBL Acc. No. AJ308234 and AJ308235) encoding 547 or 533 amino acid residue proteins with 89, 87, 83 and 81% homology to the human, rabbit, rat, and mouse OAT1, respectively. Heterologous expression of pOAT1 in Xenopus laevis oocytes revealed an apparent K(m) for [3H]PAH of 3.75 +/- 1.6 microM. [3H]PAH uptake mediated by pOAT1 was abolished by 0.5 mM glutarate or 1 mM probenecid. Functional characterization of pOAT1A did not show any affinity for [3H]PAH. In summary, we cloned two alternative splice variants of the pig ortholog of organic anion transporter 1. One splice form (pOAT1) showed typical functional characteristics of organic anion transporter 1, whereas the second form appears not to transport PAH.     

Orientation selection in photosynthetic PS I multilayers: structural investigation of the charge separated state P700A1 by high-field/high-frequency time-resolved EPR at 3.4 T/95 GHz

The radical-pair state of the primary electron donor and the secondary electron acceptor (P₇₀₀⁺A₁⁻) of the photosynthetic reaction center (RC) photosystem I (PS I) of Synechocystis PCC 6803 was studied by time-resolved electron paramagnetic resonance (TREPR) at high field/high frequency (3.4 T/95 GHz) using orientation selection in multilayers. The goal of the present article is to work out the basis for future studies, in which the improved resolution of such multilayers may be used to detect mutation-induced structural changes of PS I in membrane preparations. This approach is particularly interesting for systems that cannot be prepared as single crystals. However, in order to use such multilayers for structural investigations of protein complexes, it is necessary to know their orientation distribution. PS I was chosen as a test example because the wild type was recently crystallized and its X-ray structure determined to 2.5 Å resolution [Nature 411 (2001) 909]. On the basis of our experimental results we determined the orientation distribution. Furthermore, a simulation model for the general case in which the orientation distribution is not axially symmetric about the C₂ symmetry axis of the RC is developed and discussed. Spectra simulations show that changes in the TREPR spectra of PS I are much more significant for these oriented multilayers than for disordered samples. In this way the use of oriented multilayers, in conjunction with multifrequency TREPR measurements on oriented as well as on disordered samples, is a promising approach for studies of structural changes of PS I systems that are induced by point mutations.     

Cell death in human acute myelogenous leukemic cells induced by pyrrolidinedithiocarbamate

Pyrrolidinedithiocarbamate (PDTC) is a metal chelating compound, which exerts both pro-apoptotic effect and pro-oxidant activity on many cells. Our objective was to investigate whether PDTC was able to interfere with apoptotic process in leukemic and normal bone marrow CD34⁺ cells. Since hematopoietic growth factors stimulate growth and differentiation and prevent apoptosis, we therefore studied the effect of growth factors pretreatment, such as interleukin-3 and granulocyte-macrophage colony stimulating factor, in human myeloid CD34⁺ cells to evaluate whether they protect the cells from the apoptotic action of PDTC.We revealed that PDTC exerted an apoptotic effect in leukemic CD34⁺ cells. This effect was dependent on the ability of this compound to generate the oxidation of cellular glutathion to its disulphide and consequently to induce an intracellular oxidative stress. Hematopoietic growth factors did not protect cells from apoptosis induced by previous treatment with PDTC. The ability of PDTC to induce apoptosis was restricted to acute myelogenous leukaemia CD34⁺ cells, since normal CD34⁺ cells were insensitive to the pro-oxidant effect of PDTC.These findings imply that normal cells are equipped with mechanisms by which they respond differently to PDTC effects with respect to leukemic cells.     

Membrane oxidation assay--a novel lipoxygenase-based evaluation of membrane oxidizability

A membrane oxidation assay is presented which uses isolated erythrocyte membranes ("ghosts") and lipoxygenase as a selective catalyst for the transfer of oxygen to cis - cis -1,4-pentadiene-moieties. The latter are, for instance, present in linoleic and arachidonic acids, both of which are integral parts of membranes. These non-conjugated double bonds represent energetically favorable sites for oxidative attack and therefore, may be rearranged and partially consumed during oxidative stress. Consequently, the measurement of oxygen consumption in the course of the lipoxygenase-mediated oxidation provides a tool for the quick and reliable determination of such double bonds. Significant inter-individual differences have been noted in 11 subjects, which also correlate to the total radical antioxidant parameter (TRAP) values obtained. This assay will be helpful in the assessment of oxidizable structures in erythrocyte membranes that may be diminished as a consequence of oxidative damage suffered by an individual. In conclusion, a simple and rapid assay for the assessment of the oxidizability of erythrocyte membranes is presented complementing the TRAP assay for plasma antioxidative status.