Limited ventilation causes stress and changes in Arabidopsis morphological, physiological and molecular phenotype during in vitro growth.

A huge number of experiments in plant biology are conducted in sterile, sealed containers, providing environmental stability and full control of factors influencing the plant system. With respect to roots the in vitro growth has another benefit - the ease of conducting visual observations when grown in transparent media. Moreover, straightforward measurements of in vitro grown root systems make them a sensitive and convenient sensor of multiple stresses which may occur during experiments. In order to optimize root nematode infection tests for Arabidopsis mutants with relatively mild phenotypes, two Petri dish sealing techniques were tested (permeable medical adhesive tape and a popular non-permeable plastic film). Using standard experimental settings applied for infection tests, the root architecture, nematode infections, ion leakage, efficiency of photosynthesis, ethylene (ET) production, and CO2 accumulation were monitored in Arabidopsis thaliana Ws-0 wild-type and lsd1 (lesion stimulating disease 1) plants, which is a conditional dependent programmed cell death mutant. All tested parameters gave statistically significant differences between the analyzed sealing tapes, indicating the importance of air exchange. This factor is quite obvious but often ignored in experiments performed in Petri dishes. The results clearly indicate that stress is greater in air-tight sealed plates. These observations were supported by the great expression variation of several marker genes associated with reactive oxygen species (ROS), ET, salicylic (SA), and jasmonic acid (JA) biosynthesis and signaling in two-week-old seedlings. These results are discussed in light of the observed changes in the ET and CO2 concentration. Our results clearly indicate the importance of culture parameters for monitoring of abiotic and biotic stress responses in laboratory conditions, including accurate mutant phenotyping.

Widespread but small-scale changes in the structural and dynamic properties of vaccinia virus poly(A) polymerase upon association with its processivity factor in solution.

Vaccinia virus poly(A) polymerase (VP55) has been analyzed via hydrogen-deuterium exchange (HDX) mass spectrometry in the absence and presence of its processivity factor, VP39, to improve our understanding of the mechanism by which processivity is impressed on the polymerase. For 119 peptic peptides covering 74.1% of VP55, the extent of HDX at 900 s was interpreted in the context of parameters deduced from the VP55-VP39 X-ray crystal structure. While HDX exhibited a degree of correlation with the mean SASA of whole residues within each peptide segment, HDX was generally more active than expected from either the SASA or hydrogen bonding status of the exchangeable amide proton, indicating a significant molecular dynamics contribution to amide proton deprotection. Peptic peptides undergoing either more or less HDX than expected were distributed throughout VP55 and showed consistency between multiple overlapping peptides. VP39 had a net, marginal cooling effect on VP55, indicating a possible restriction of VP55's flexibility. VP39's cooling effect was most extensive within the central domain of VP55's three domains, while a patch within VP55's C-terminal domain showed an increased level of HDX in the presence of VP39. Langevin dynamics all-atom simulations of VP55 motions showed slower relaxation to equilibrium in the absence of VP39. At equilibrium, regions showing extremes of variation in simulated atomic fluctuation were localized within VP55's N- and C-terminal domains, and VP39 had a predominantly cooling effect on VP55. Broadly, across VP55's peptic peptides, a mild negative correlation was noted between the extent to which deuteration was more active than predicted from the structure and the amplitudes of the simulated atomic fluctuation and/or degree of disorder at equilibrium.

Assessment of dietary amino acid scarcity on growth and blood plasma proteome status of broiler chickens.

Dietary Lys needs for chicks were studied. A titration diet consisting of progressive amounts of dietary Lys from 0.95% up to 1.40% was fed to broiler chicks from 0 to 18 d of age. Optimal dietary Lys level was calculated using regression analysis. Body weight gain and feed conversion were maximized at Lys levels of 1.24% (1.10% digestible) and 1.27% (1.13% digestible) of diet, respectively. Blood samples were then collected from 2 groups: birds fed the lowest Lys level and birds fed dietary Lys nearest the determined requirement level (1.25% Lys). Plasma was analyzed for protein spectra via mass spectrometry and then classified by their functional characteristics. The number of proteins was similar between the 2 samples, but there was a tendency toward increased peptides for specific proteins in plasma from chicks fed adequate Lys levels. Furthermore, after these proteins were classified, more muscle-related proteins were found in plasma samples of birds fed Lys-adequate diets. It would appear that an individual dietary amino acid deficiency does not necessarily translate into decreasing protein synthesis proportionate to body weight, but rather significant changes may be occurring within the types of proteins undergoing anabolism. In conclusion, results herein illustrate the potential for using functional genomics in nutritionally related responses of poultry.

[Evaluation of some selected structural and functional parameters of red blood cells as oxidative stress markers in elderly people]. / Ocena wybranych parametrów strukturalnych i czynnosciowych krwinek czerwonych jako markerów stresu oksydacyjnego u osób w wieku podeszlym.

UNLABELLED: The ageing process induces age-related involutionary changes and leads to increased occurrence of many diseases. One of the most important theories of ageing and development of many pathologies is the free radical theory, which assumes that ageing process leads to lost of oxidative balance. THE AIM: of the research was to evaluate the degree of membrane lipid peroxidation, internal microviscosity, activity of membrane ATPase, both total and Na(+)K(+)-dependent, and markers of oxidative damage in erythrocyte membrane protein in elderly people. MATERIAL: The examination was performed on 35 people. The examined group (15 persons, mean age 71,3) consisted of healthy elderly people. The reference group was formed with younger healthy people (20 persons, mean age 55). RESULTS: Erythrocyte membrane lipid peroxidation was found stronger in the group of elderly people. Erythrocyte internal microviscosity was significantly higher in the elderly. The activity of ATPase, both total and Na(+)K(+)-dependent, appeared remarkably greater in the group of younger people. Stronger membrane lipid damage was observed in older age group, which may be implied by lower--SH group concentration, and higher W/S parameter value. CONCLUSION: The obtained results reveal that in elderly people the intensification of oxidative stress in the entire body occurs, which may be confirmed by structural and functional oxidative erythrocyte damage. This conclusion may be significant for pathogenesis of many diseases in this period of life.

The effect of atorvastatin on erythrocyte membranes and serum lipids in patients with type-2 hypercholesterolemia.

BACKGROUND: . The beneficial effects of statins on clinical events may involve mechanisms that modify endothelial dysfunction, plaque stability, thrombus formation, and inflammatory responses. To determine the effect of atorvastatin on blood rheology in patients with familial hypercholesterolemia (FH), we prospectively studied serum lipid concentration, red cell cholesterol content, lipid peroxidation and erythrocyte membrane fluidity. The aim of this paper was to evaluate the effects of atorvastatin therapy on the erythrocyte membrane structure and the hypolipemic efficacy in patients with FH. MATERIALS, METHODS AND SUBJECTS STUDIED:. The study involved 31 patients with FH and 20 healthy individuals as a control group. The program lasted 20 weeks. For the first 8 weeks, the patients were on a hypolipemic diet only and for the subsequent 12 weeks, alongside the diet they were given 10 mg atorvastatin per day. Laboratory tests were carried out before and after 4 weeks and 12 weeks of the pharmacological treatment. Erythrocyte membrane fluidity was determined using the spin labeled method. The peroxidation of lipids was measured in whole erythrocytes as well as in erythrocyte plasma membranes by means of the thiobarbituric acid technique. RESULTS: . Treatment with atorvastatin reduced serum total cholesterol concentration from 310+/-29 mg/dl in a basal situation to 203+/-34 mg/dl ( P<0.001) at the end of the treatment and low-density lipoprotein (LDL) cholesterol concentration from 225+/-30 mg/dl to 126+/-30 mg/dl ( P<0.001), respectively. The changes observed in the plasma lipids correlate with a significant decrease in erythrocyte membrane cholesterol, from 2.24+/-1.69 to 1.17+/-0.75 mg/mg protein ( P<0.001) after 12 weeks of treatment. The lipid peroxidation in membranes of erythrocytes was lowered from the basal value 0.171+/-0.097 to 0.100+/-0.024 mmol/mg protein ( P<0.05) after 4 weeks of treatment and to 0.057+/-0.020 mmol/mg protein ( P<0.001) after 12 weeks of treatment, and in total erythrocytes from 4.78+/-1.49 to 3.99+/-1.39 mmol/g Hb ( P<0.02) and 2.43+/-0.87 mmol/g Hb ( P<0.001), respectively. The membrane fluidity was estimated by means of parameter S at the depth of the fifth carbon atom. Atorvastatin in hypercholesterolemic erythrocytes enhances the fluidity of the superficial layer from 0.758+/-0.009 up to the values observed in the control group 0.744+/-0.009 ( P<0.001). There is no impact on the microviscosity of the hydrophobic core observed. CONCLUSION: . Our findings suggest that the atorvastatin therapy reverses the alteration of erythrocyte plasma membrane properties. It may improve blood rheology in patients with FH. This improvement in blood properties may contribute to the well-known beneficial effects of atorvastatin on cardiovascular risk in patients with severe hyperlipidemia and atherosclerotic vascular disease.

Interaction of perindoprilat with human red blood cells.

The effects of perindoprilat on the morphology and dynamic properties of human erythrocytes were studied by light microscopy, electron spin resonance spectroscopy and spectrophotometric methods. Erythrocytes were exposed to perindoprilat at 37 degrees C for 30 and 120 min. It was shown that the drug at a concentration of 0.75 microg/ml did not cause significant changes in the structure of erythrocyte membranes. Higher doses of the drug (7.5 and 75 microg/ml) induced changes in membrane fluidity in the hydrophobic core of the lipid bilayer, the conformation of membrane proteins, the number of SH groups and the activity of membrane-bound acetylcholinesterase (AChE). These modifications were accompanied by changes in the shape of erythrocytes and did not depend on time of incubation. Therefore, it is proposed that perindoprilat perturbs the lipid bilayer and disturbs the organization of the protein-lipid environment.

The biostimulatory effect of red laser irradiation on pig blood platelet function.

The molecular mechanisms of laser-induced changes in the cell structure and function are not well known. The authors examined the effects of low-power laser irradiation on unnucleated pig blood platelets. The obtained results showed that laser irradiation (1-5 J) caused in blood platelets lipid peroxidation (measured as thiobarbituric acid reactive substances) and superoxide anion generation, concomitant with the release of adenine nucleotides and proteins from platelets. The maximum platelet response to laser irradiation was observed when doses of 1.8-2 J were used. Our results indicate that red laser irradiation induces: (1) platelet secretory process and the release of substances stored in the specific granules (adenine nucleotides, proteins); and (2) lipid peroxidation partly due to stimulation of endogenous arachidonate and production of its metabolites reacting with thiobarbituric acid.

Effect of perindopril therapy on fluidity and potential of erythrocyte membrane from individuals with coronary heart disease.

Erythrocyte membrane fluidity and membrane potential were measured in patients suffering from coronary heart disease (CHD) and treated with perindopril. Membrane fluidity was determined using electron paramagnetic resonance (EPR) spectroscopy, and membrane potential was evaluated using potential-sensitive fluorescent dyes. CHD does not change membrane fluidity at the depth of the 5 carbon in the fatty acid chain of membrane phospholipids. However the hydrophobic core of the membrane is altered in CHD. For 19 CHD patients, the correlation times tau B and tau C of a spin label 16DS were higher than for controls: tau B = (1.84 +/- 0.04) x 10(-9) s and tau C = (2.54 +/- 0.04) x 10(-9) s vs. tau B = (1.62 +/- 0.06) x 10(-9) s; and tau C = (2.24 +/- 0.07) x 10(-9) s (results given as mean +/- SEM). Such results indicate the increased microviscosity in hydrophobic regions of CHD erythrocyte membranes in comparison with controls. Perindopril therapy partly abolished these changes. The membrane potential of CHD red blood cells -17.89 +/- 1.36 mV was higher than the control value -9.83 +/- 0.59 mV. Perindopril treatment shifted the membrane potential value to -13.45 +/- 0.99 mV when measured after a single dose of the drug, or even depolarized the membrane after 7 days of therapy -4.95 +/- 0.73 mV. It is concluded that the erythrocyte membrane is more rigid and hyperpolarized in CHD, and perindopril therapy partly abolishes these changes as early as 3 h after administration.

Changes in erythrocyte membranes after fractioned hyperthermia.

The structural changes in erythrocytes membranes were examined before and after the second heat shock of erythrocytes. Electrophoretic separation of protein erythrocyte membranes for cells incubated at 48.5 degrees C was different from control i.e. from erythrocytes incubated at 37 degrees C. No quantitative or qualitative changes were spotted in comparison with protein membranes isolated from the erythrocytes following single or double heat shock. Fluidity of erythrocytes membranes was determined by using spin labels, 5-doxylstearic acid and 16-doxylstearic acid. The membranes were more rigid in their hydrophobic regions after incubation of cells at 44 degrees C. It can be suggested that erythrocyte membranes play some role in thermotolerance and heat damage of enucleate cells.

Effect of hyperthermia on the internal microviscosity of erythrocytes and lymphocytes: a spin-label study.

The internal microviscosity of human erythrocytes and porcine lymphocytes was investigated, from the electron-spin resonance spectral characteristics of the nitroxyl spin probe, 4-amino-2,2,6,6-tetramethylpiperidinyloxy. Heat-induced permanent damage to cells was observed as an increase in the intracellular microviscosity of nucleate and enucleate cells. Induction of heat resistance was found for erythrocytes treated with a double heat shock.

Does thermotolerance occur in human red cells?

Osmotic fragility and autohemolysis were used as endpoints in the measurement of damage to the plasma membrane in human erythrocytes, after single or double heat treatments. Injury recorded above 46 degrees C, and the induction of thermotolerance in the enucleate cells after a primary heat treatment of 44 degrees C for 15 min, indicates that the plasma membrane plays an important role in heat injury and in thermotolerance.

Effect of hyperthermia on lymphocyte plasma membrane. 3 electrolyte spin labels permeability.

The effect of elevated temperatures on the transport of electrolyte spin labels anionic c-TEMPIR and cationic TEMPO-choline character across the porcine lymphocyte plasma membrane was investigated. Breaks in the Arrhenius plot for permeability of both spin-labels occurred at 42 to 43 degrees C. TEMPO-choline and c-TEMPIR transport are probably critical targets in hyperthermic cell killing.

Effect of hyperthermia on lymphocyte membrane. 2. Spin-labelled non-electrolyte permeability.

The effect of elevated temperatures on the permeability of non-electrolyte spin labels, hydrophilic TEMPOL and more hydrophobic TEMPO across the porcine lymphocyte membrane was investigated. In the range of 41-44 degrees C, temperature-induced changes in the permeation constant were lower for TEMPO than TEMPOL. The data obtained may suggest that the permeability of spin labels across the membrane is sensitive to changes of temperature especially above 43 degrees C.

Effect of hyperthermia on lymphocyte plasma membrane. 1. Permeability for glyceraldehyde-3-phosphate dehydrogenase substrates.

The effect of hyperthermia on the permeability of porcine lymphocyte plasma membranes for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) substrates was investigated. The permeability increased in the temperature range of 40-45 degrees C. The temperature dependence for the permeability of G3PDH substrates and for cell viability was not well correlated.

Phase transitions in spherical bilayer membranes prepared of bulk erythrocyte membrane lipids.

The phase transitions of spherical lipid bilayers built of bovine red cell lipids have been studied. The electrical and ESR measurements revealed some conformational changes in lipid bilayers taking place at 36-38 degrees C.

Hyperthermic modification of bleomycin--DNA interaction detected by electron spin resonance.

Electron spin resonance spectra of DNA labeled with each of four spin-labeling compounds have been studied to detect interaction between the antibiotic bleomycin and DNA. Only one of these labels, compound IV, resulted in a modified spectrum when bound to DNA and the latter was subjected to bleomycin. This property has been used to monitor DNA-bleomycin interactions under physiological and hyperthermic conditions. Bleomycin produced an increase in rotational correlation time of the residue bound to DNA at 37 degrees C and a significantly higher increase at 43 degrees C. Some effect was still detected with bleomycin at 37 degrees C after preheating at 43 degrees C. Parallel studies have revealed enhanced binding of 59Fe-bleomycin to DNA during and after hyperthermic treatment.

The effects of glutaraldehyde and osmium tetroxide on the erythrocyte membrane. A spin label study.

A nitroxide spin label probe technique was applied to study the interaction between glutaraldehyde or osmium tetroxide (OsO4) amd the membranes of horse erythrocytes, ghosts and liposomes prepared from erythrocyte lipids. Two major conclusions have been established: (1) Reaction of the fixation reagents with the membrane is selective. OsO4 reacts predominantly with lipids and glutaraldehyde with membrane proteins. (2) The lipid-protein interactions change after pretreatment by OsO4 or glutaraldehyde.

Ultrastructural modifications of the erythrocyte membrane in Down's syndrome.

Spin label studies of erythrocyte membranes from patients with Down's syndrome showed no differences in the rigidity of membrane lipids with respect to normal subjects and a tendency for alterations in the state of membrane proteins. Electron microscopic studies demonstrated the occurrence of ultrastructural defects in membranes from trisomics which can be due to accelerated red cell aging as similar alterations were found in the fraction of oldest (most dense) cells in normal subjects.