Selenium supplementation on plasma glutathione peroxidase activity in patients with end-stage chronic renal failure

Patients with chronic renal failure (CRF) usually have a lower than healthy level of selenium (Se) in whole blood and plasma. Plasma glutathione peroxidase (GSH-Px) is synthesized mostly in the kidney. In CRF patients, activity of this enzyme is significantly reduced and its reduction increases with the progress of the disease. The aim of the study was to evaluate the effect of Se supplementation to CRF patients at various stages of the disease on Se concentration in blood components and on plasma GSH-Px activity. The study group comprised 53 CRF patients at various stages of the disease supplemented with Se (200 μg/d for 3 mo as Se-enriched yeast, containing about 70% l-selenomethionine [SeMet]). The control group consisted of 20 healthy subjects. The Se concentration in blood components was measured spectrofluorometrically with 2,3-diaminonaphthalene as a complexing reagent. GSH-Px activity in red cell hemolysates and plasma was assayed by the coupled method with tert-butyl hydroperoxide as a substrate. The Se concentration in whole blood and plasma of CRF patients is significantly lower as compared with healthy subjects, but similar at all stages of the disease. In the patients’ plasma, total protein and albumin levels are also significantly lower than in healthy subjects. Plasma GSH-Px activity in patients is extremely low, and contrary to Se concentration, it decreases linearly with the increasing stage of the illness. Se-supplied patients show an increased Se concentration in all blood components and at all disease stages, whereas plasma GSH-Px activity is enhanced only at the incipient stage of the disease. Se supply has no effect on plasma GSH-Px activity in uremic patients at the end stage of the disease. Total plasma protein and albumin levels did not change after Se supplementation. Our data seem to show that in patients with CRF lower total protein and albumin levels in plasma may be the chief cause of the low blood and plasma Se concentrations. GSH-Px activity decreases along with the kidney impairment. At the end stage of the disease, Se supplementation in the form of Se-enriched yeast has no effect on the increase in plasma GSH-Px activity.     

Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton

Modulation of the cytoskeletal architecture was shown to regulate the expression of CTGF (connective tissue growth factor, CCN2). The microtubule disrupting agents nocodazole and colchicine strongly up-regulated CTGF expression, which was prevented upon stabilization of the microtubules by paclitaxel. As a consequence of microtubule disruption, RhoA was activated and the actin stress fibers were stabilized. Both effects were related to CTGF induction. Overexpression of constitutively active RhoA induced CTGF synthesis. Interference with RhoA signaling by simvastatin, toxinB, C3 toxin, and Y27632 prevented up-regulation of CTGF. Likewise, direct disintegration of the actin cytoskeleton by latrunculin B interfered with nocodazole-mediated up-regulation of CTGF expression. Disassembly of actin fibers by cytochalasin D, however, unexpectedly increased CTGF expression indicating that the content of F-actin per se was not the major determinant for CTGF gene expression. Given the fact that cytochalasin D sequesters G-actin, a decrease in G-actin increased CTGF, while increased levels of G-actin corresponded to reduced CTGF expression. These data link alterations in the microtubule and actin cytoskeleton to the expression of CTGF and provide a molecular basis for the observation that CTGF is up-regulated in cells exposed to mechanical stress.     

Nitrate reduction and nitrogen fixation in symbiotic association Rhizobium-legumes

The inhibitory effect of nitrate on nitrogenase activity in root nodules of legume plants has been known for a long time. The major factor inducing changes in nitrogenase activity is the concentration of free oxygen inside nodules. Oxygen availability in the infected zone of nodule is limited, among others, by the gas diffusion resistance in nodule cortex. The presence of nitrate may cause changes in the resistance to O2 diffusion. The aim of this paper is to review literature data concerning the effect of nitrate on the symbiotic association between rhizobia and legume plants, with special emphasis on nitrogenase activity. Recent advances indicate that symbiotic associations of Rhizobium strains characterized by a high nitrate reductase activity are less susceptible to inhibition by nitrate. A thesis may be put forward that dissimilatory nitrate reduction, catalyzed by bacteroid nitrate reductase, significantly facilitates the symbiotic function of bacteroids.     

Metabolic and ultrastructural responses of lupine embryo axes to sugar starvation

Embryo axes isolated from germinating lupine seeds were cultivated in vitro for 24-96 h over media containing either 60 mmol/L sucrose or no sucrose. Ultrastructural studies showed that large vacuoles were accumulating in a central region of primary parenchyma cells in sucrose starved lupine embryo axes, whereas cytoplasm along with organelles were forced to a periphery of the cells. We suggest that the autolysis of cytoplasmic proteins contributes to the accumulation of the vacuoles and this suggestion is consistent with the results of the characterisation of protein content. The level of cytosolic proteins was reduced by 50% and the activity of cytosolic marker enzyme, PEP carboxylase, was reduced by 46% in starved embryos as compared to control. The mitochondria from starved tissues were not degraded. The level of mitochondrial proteins was reduced by only 10% and the activity of mitochondrial NAD-isocitrate dehydrogenase decreased by 8% as a result of starvation. As demonstrated by the results of Percoll density gradient centrifugation, sucrose starvation caused an increase of 49% in many of the higher density mitochondria fractions, whereas many of the lower density mitochondria fractions were decreased by 33%. The samples of mitochondria from starved embryo axes were determined to have higher respiration activity in the presence of glutamate and malate as compared to control samples. EPR-based analyses of free radicals showed the presence of free radicals with a signal at g = 2.0060 in embryo axes. The level of the radical was two times higher in sucrose-starved embryo axes than in control (the level of this radical increased in senescing plant tissues as well). The results of EPR-based quantitation of Mn2+ ions revealed that the level was a few times higher in starved material than in control. Starved embryo axes, however, do possess a number of adaptive mechanisms protecting them from oxidative damage. Densitometric analyses of gels revealed an increase in the activity of SOD in sugar-starved embryos, whereas CAT and POX activities were lower in axes grown without sucrose as compared to control. Superoxide dismutase, catalase and peroxidase zymogram analyses showed that synthesis of new isoforms was not induced by sugar starvation. An accumulation of phytoferritin was found in plastids of sucrose starved embryos. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.     

Effect of Seed Freezing and Storage Conditions on Plasma Membrane Properties of Norway Spruce (Piceo abies Karst.) Seedlings

Norway spruce (Picea abies Karst.) seeds were frozen and stored for 15 months at + 3, ? 25, ? 75 or ? 196°C. After storage, seeds were germinated for 9?14 days to determine viability and plasma membrane protein composition, H⁺-ATPase activity and fluidity. The results indicate no significant differences in viability of seed 14 days after germination. Biochemical analyses revealed increased plasma membrane fluidity in 9-day-old Norway spruce seedlings raised from seeds pretreated at ? 75 °C. and changes in the temperature profile of membrane fluidity in seedlings after pre-treatment of seeds at ? 25 °C. On the other hand, the same treatments did not result in changes in plasma membrane protein content, protein composition or ATPase activity. There was also no difference in plasma membrane H⁺-ATPase activity assayed in the presence of different ATP hydrolysis inhibitors. Based on the presented results, and other experimental data, we suggest that during early seedling growth, adaptation of seeds to ? 25 and ? 75°C freezing and/or storage temperature results in stability of the plasma membrane protein function and composition and increased fluidity or changes in the temperature-dependent fluidity profile of these membranes.     

Metabolism of amino acids in germinating yellow lupin seeds. II. Pathway of conversion of aspartate to alanine during the imbibition

The incorporation of ¹⁴C-aspartate during the imbibition of yellow lupin seeds resulted in the production of ¹⁴C-alanine and ¹⁴CO₂. On the basis of tracer and enzymatic assays, conducted in vitro on the extract obtained from lupin seeds, it is postulated that aspartate can be converted to oxaloacetate, then, by phosphoenolopyruvate and pyruvate to alanine. This pathway can be catalyzed by the following enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, pyruvate kinase and alanine aminotransferase.