The impact of dehydration rate on the production and cellular location of reactive oxygen species in an aquatic moss

Background and Aims

The aquatic moss Fontinalis antipyretica requires a slow rate of dehydration to survive a desiccation event. The present work examined whether differences in the dehydration rate resulted in corresponding differences in the production of reactive oxygen species (ROS) and therefore in the amount of cell damage.

Methods

Intracellular ROS production by the aquatic moss was assessed with confocal laser microscopy and the ROS-specific chemical probe 2,7-dichlorodihydrofluorescein diacetate. The production of hydrogen peroxide was also quantified and its cellular location was assessed.

Key Results

The rehydration of slowly dried cells was associated with lower ROS production, thereby reducing the amount of cellular damage and increasing cell survival. A high oxygen consumption burst accompanied the initial stages of rehydration, perhaps due to the burst of ROS production.

Conclusions

A slow dehydration rate may induce cell protection mechanisms that serve to limit ROS production and reduce the oxidative burst, decreasing the number of damaged and dead cells due upon rehydration.     

The impact of reactive oxygen species and genetic mitochondrial mutations in Parkinson's disease

The exact pathogenesis of Parkinson's disease (PD) is still unknown and proper mechanisms that correspond to the disease remain unidentified. It is understood that PD is age-related; as age increases, the chance of onset responds accordingly. Although there are no current means of curing PD, the understanding of reactive oxygen species (ROS) provides significant insight to possible treatments. Complex I deficiencies of the respiratory chain account for the majority of unfavorable neural apoptosis generation in PD. Dopaminergic neurons are severely damaged as a result of the deficiency. Symptoms such as inhibited cognitive ability and loss of smooth motor function are the results of such impairment. The genetic mutations of Parkinson's related proteins such as PINK1 and LRRK2 contribute to mitochondrial dysfunction which precedes ROS formation. Various pathways are inhibited by these mutations, and inevitably causing neural cell damage. Antioxidants are known to negate the damaging effects of free radical overexpression. This paper expands on the specific impact of mitochondrial genetic change and production of free radicals as well as its correlation to the neurodegeneration in Parkinson's disease.     

Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.2) with several substrates by monitoring the heat released during the reaction. The method is based on the proportionality between the reaction rate and the thermal power (heat/time) generated. Microcalorimetric assays carried out at different temperatures provided the dependence of the catalytic rate constant on temperature. We show that ITC assay is a nondestructive method that allows the determination of the catalytic rate constant (kcat), Michaelis constant (KM), activation energy and activation Gibbs energy, enthalpy and entropy of this reaction.     

The cold denatured state is compact but expands at low temperatures: hydrodynamic properties of the cold denatured state of the C-terminal domain of L9

A point mutation of a small globular protein, the C-terminal domain of L9 destabilizes the protein and leads to observable cold-denaturation at temperatures above zero. The cold denatured state is in slow exchange with the native state on the NMR time scale, and this allows the hydrodynamic properties of the cold unfolded state and the native state to be measured under identical conditions using pulsed-field gradient NMR diffusion measurements. This provides the first experimental measurement of the hydrodynamic properties of a cold unfolded protein and its folded form under identical conditions. Hydrodynamic radii of the cold-induced unfolded states were measured for a set of temperatures ranging from 2 °C to 25 °C at pD 6.6 in the absence of denaturant. The cold unfolded state is compact compared to the urea or acid unfolded state and a trend of increasing radii of hydration is observed as the temperature is lowered. These observations are confirmed by experiments on the same protein at pD 8.0, where it is more stable, in the presence of a modest concentration of urea. The expansion of the cold-denatured state at lower temperatures is consistent with the temperature dependence of hydrophobic interactions.     

Bimodal actions of reactive oxygen species in the differentiation and bone-resorbing functions of osteoclasts

In order to demonstrate that cellular redox status undergoes decreased reduction during osteoclast differentiation and further decreased reduction during osteoclastic bone resorption, we analyzed gamma-glutamylcysteinyl synthetase activity, a glutathione synthesis rate-limiting enzyme, and total glutathione and thiol groups. Moderate and severe redox shifts towards a more oxidizing environment induced gradual increases and decreases in osteoclastogenesis. Moreover, while severe glutathione depletion inhibited bone resorption, moderate glutathione repletion enhanced bone resorption. In summary, our observations suggest that there is a threshold for redox status, representing biphasic patterns in osteoclast differentiation and function.     

Effects of N-acylethanolamines on the respiratory chain and production of reactive oxygen species in heart mitochondria

Long-chain N-acylethanolamines (NAEs) have been found to uncouple oxidative phosphorylation and to inhibit uncoupled respiration of rat heart mitochondria [Wasilewski, M., Wieckowski, M.R., Dymkowska, D. and Wojtczak, L. (2004) Biochim. Biophys. Acta 1657, 151-163]. The aim of the present work was to investigate in more detail the mechanism of the inhibitory effects of NAEs on the respiratory chain. In connection with this, we also investigated a possible action of NAEs on the generation of reactive oxygen species (ROS) by respiring rat heart mitochondria. It was found that unsaturated NAEs, N-oleoylethanolamine (N-Ole) and, to a greater extent, N-arachidonoylethanolamine (N-Ara), inhibited predominantly complex I of the respiratory chain, with a much weaker effect on complexes II and III, and no effect on complex IV. Saturated N-palmitoylethanolamine had a much smaller effect compared to unsaturated NAEs. N-Ara and N-Ole were found to decrease ROS formation, apparently due to their uncoupling action. However, under specific conditions, N-Ara slightly but significantly stimulated ROS generation in uncoupled conditions, probably due to its inhibitory effect on complex I. These results may contribute to our better understanding of physiological roles of NAEs in protection against ischemia and in induction of programmed cell death.     

Zinc induces distinct changes in the metabolism of reactive oxygen and nitrogen species (ROS and RNS) in the roots of two Brassica species with different sensitivity to zinc stress

Background and Aims Zinc (Zn) is an essential micronutrient naturally present in soils, but anthropogenic activities can lead to accumulation in the environment and resulting damage to plants. Heavy metals such as Zn can induce oxidative stress and the generation of reactive oxygen and nitrogen species (ROS and RNS), which can reduce growth and yield in crop plants. This study assesses the interplay of these two families of molecules in order to evaluate the responses in roots of two Brassica species under high concentrations of Zn.Methods Nine-day-old hydroponically grown Brassica juncea (Indian mustard) and B. napus (oilseed rape) seedlings were treated with ZnSO₄ (0, 50, 150 and 300 µm) for 7 d. Stress intensity was assessed through analyses of cell wall damage and cell viability. Biochemical and cellular techniques were used to measure key components of the metabolism of ROS and RNS including lipid peroxidation, enzymatic antioxidants, protein nitration and content of superoxide radical (O2·−), nitric oxide (NO) and peroxynitrite (ONOO⁻).Key Results Analysis of morphological root damage and alterations of microelement homeostasis indicate that B. juncea is more tolerant to Zn stress than B. napus. ROS and RNS parameters suggest that the oxidative components are predominant compared with the nitrosative components in the root system of both species.Conclusions The results indicate a clear relationship between ROS and RNS metabolism as a mechanism of response against stress caused by an excess of Zn. The oxidative stress components seem to be more dominant than the elements of the nitrosative stress in the root system of these two Brassica species.     

Unusual binding of Grb2 protein to a bivalent polyproline-ligand immobilized on a SPR sensor: Intermolecular bivalent binding

The Grb2 adapter protein is involved in the activation of the Ras signaling pathway. It recruits the Sos protein by binding of its two SH3 domains to Sos polyproline sequences. We observed that the binding of Grb2 to a bivalent ligand, containing two Sos-derived polyproline-sequences immobilized on a SPR sensor, shows unusual kinetic behavior. SPR-kinetic analysis and supporting data from other techniques show major contributions of an intermolecular bivalent binding mode. Each of the two Grb2 SH3 domains binds to one polyproline-sequence of two different ligand molecules, facilitating binding of a second Grb2 molecule to the two remaining free polyproline binding sites. A molecular model based on the X-ray structure of the Grb2 dimer shows that Grb2 is flexible enough to allow this binding mode. The results fit with a role of Grb2 in protein aggregation, achieving specificity by multivalent interactions, despite the relatively low affinity of single SH3 interactions.     

The genus Galumna in Nepalese oribatid mite fauna,with notes on systematic placement of some species (Acari,Oribatida, Galumnidae)

The oribatid mite genus Galumna (Oribatida, Galumnidae) is recorded for the first time in Nepal. A new species, Galumna tetraporosasp. n., is described from soil of secondary mixed broadleaved forest. It is most similar morphologically to G. tokyoensis Aoki, 1966 and G. valida Aoki, 1994, however, it differs from both by the absence of interlamellar setae and the presence of two pairs of notogastral porose areas Aa. Galumna granalata Aoki, 1984 is redescribed on the basis of specimens from Nepal. Galumna floridae (Jacot, 1929) and G. hexagona Balogh, 1960 are transferred to the genus Notogalumna; G. mauritii Mahunka, 1978 is transferred to the genus Dimidiogalumna.     

Computational modeling of cell adhesion and movement using a continuum-kinetics approach

Adhesion of leukocytes to substrate involves the coupling of disparate length and timescales between molecular mechanics and macroscopic transport, and existing models of cell adhesion do not use full cellular information. To address these challenges, a multiscale computational approach for studying the adhesion of a cell on a substrate is developed and assessed. The cellular level model consists of a continuum representation of the field equations and a moving boundary tracking capability to allow the cell to change its shape continuously. At the receptor-ligand level, a bond molecule is mechanically represented by a spring. Communication between the macro/micro- and nanoscale models is facilitated interactively during the computation. The computational model is assessed using an adherent cell, rolling and deforming along the vessel wall under imposed shear flows. Using this approach, we first confirm existing numerical and experimental results. In this study, the intracellular viscosity and interfacial tension are found to directly affect the rolling of a cell. Our results also show that the presence of a nucleus increases the bond lifetime, and decreases the cell rolling velocity. Furthermore, it is found that a cell with a larger diameter rolls faster, and decreases the bond lifetime. This study shows that cell rheological properties have significant effects on the adhesion process contrary to what has been hypothesized in most literature.     

The impact of cold on photosynthesis in genotypes of Coffea spp.--photosystem sensitivity, photoprotective mechanisms and gene expression

Environmental constraints disturb plant metabolism and are often associated with photosynthetic impairments and yield reductions. Among them, low positive temperatures are of up most importance in tropical plant species, namely in Coffea spp. in which some acclimation ability has been reported. To further explain cold tolerance, the impacts on photosynthetic functioning and the expression of photosynthetic-related genes were analyzed. The experiments were carried out along a period of slow cold imposition (to allow acclimation), after chilling (4 °C) exposure and in the following rewarming period, using 1.5-year-old coffee seedlings of 5 genotypes with different cold sensitivity: Coffea canephora cv. Apoatã, Coffea arabica cv. Catuaí, Coffea dewevrei and 2 hybrids, Icatu (C. arabica × C. canephora) and Piatã (C. dewevrei × C. arabica). All genotypes suffered a significant leaf area loss only after chilling exposure, with Icatu showing the lowest impact, a first indication of a higher cold tolerance, contrasting with Apoatã and C. dewevrei. During cold exposure, net photosynthesis and Chl a fluorescence parameters were strongly affected in all genotypes, but stomatal limitations were not detected. However, the extent of mesophyll limitation, reflecting regulatory mechanisms and/or damage, was genotype dependent. Overnight retention of zeaxanthin was common to Coffea genotypes, but the accumulation of photoprotective pigments was highest in Icatu. That down-regulated photochemical events but efficiently protected the photosynthetic structures, as shown, e.g., by the lowest impacts on A_max and PSI activity and the strongest reinforcement of PSII activity, the latter possibly reflecting the presence of a photoprotective cycle around PSII in Icatu (and Catuaí). Concomitant to these protection mechanisms, Icatu was the sole genotype to present simultaneous upregulation of caCP22, caPI and caCytf, related to, respectively, PSII, PSI and to the complex Cytb₆/f, which could promote better repair ability, contributing to the maintenance of efficient thylakoid functioning. We conclude that Icatu showed the best acclimation ability among the studied genotypes, mostly due to a better upregulation of photoprotection and repair mechanisms. We confirmed the presence of important variability in Coffea spp. that could be exploited in breeding programs, which should be assisted by useful markers of cold tolerance, namely the upregulation of antioxidative molecules, the expression of selected genes and PSI sensitivity.     

Production of reactive oxygen species by photosystem II

Photosysthetic cleavage of water molecules to molecular oxygen is a crucial process for all aerobic life on the Earth. Light-driven oxidation of water occurs in photosystem II (PSII) — a pigment-protein complex embedded in the thylakoid membrane of plants, algae and cyanobacteria. Electron transport across the thylakoid membrane terminated by NADPH and ATP formation is inadvertently coupled with the formation of reactive oxygen species (ROS). Reactive oxygen species are mainly produced by photosystem I; however, under certain circumstances, PSII contributes to the overall formation of ROS in the thylakoid membrane. Under limitation of electron transport reaction between both photosystems, photoreduction of molecular oxygen by the reducing side of PSII generates a superoxide anion radical, its dismutation to hydrogen peroxide and the subsequent formation of a hydroxyl radical terminates the overall process of ROS formation on the PSII electron acceptor side. On the PSII electron donor side, partial or complete inhibition of enzymatic activity of the water-splitting manganese complex is coupled with incomplete oxidation of water to hydrogen peroxide. The review points out the mechanistic aspects in the production of ROS on both the electron acceptor and electron donor side of PSII.     

Dual role of FMN in flavodoxin function: Electron transfer cofactor and modulation of the protein-protein interaction surface

Flavodoxin (Fld) replaces Ferredoxin (Fd) as electron carrier from Photosystem I (PSI) to Ferredoxin-NADP⁺ reductase (FNR). A number of Anabaena Fld (AnFld) variants with replacements at the interaction surface with FNR and PSI indicated that neither polar nor hydrophobic residues resulted critical for the interactions, particularly with FNR. This suggests that the solvent exposed benzenoid surface of the Fld FMN cofactor might contribute to it. FMN has been replaced with analogues in which its 7- and/or 8-methyl groups have been replaced by chlorine and/or hydrogen. The oxidised Fld variants accept electrons from reduced FNR more efficiently than Fld, as expected from their less negative midpoint potential. However, processes with PSI (including reduction of Fld semiquinone by PSI, described here for the first time) are impeded at the steps that involve complex re-arrangement and electron transfer (ET). The groups introduced, particularly chlorine, have an electron withdrawal effect on the pyrazine and pyrimidine rings of FMN. These changes are reflected in the magnitude and orientation of the molecular dipole moment of the variants, both factors appearing critical for the re-arrangement of the finely tuned PSI:Fld complex. Processes with FNR are also slightly modulated. Despite the displacements observed, the negative end of the dipole moment points towards the surface that contains the FMN, still allowing formation of complexes competent for efficient ET. This agrees with several alternative binding modes in the FNR:Fld interaction. In conclusion, the FMN in Fld not only contributes to the redox process, but also to attain the competent interaction of Fld with FNR and PSI.     

Enzymatic oxidation of manganese ions catalysed by laccase

The principal possibility of enzymatic oxidation of manganese ions by fungal Trametes hirsuta laccase in the presence of oxalate and tartrate ions, whereas not for plant Rhus vernicifera laccase, was demonstrated. Detailed kinetic studies of the oxidation of different enzyme substrates along with oxygen reduction by the enzymes show that in air-saturated solutions the rate of oxygen reduction by the T2/T3 cluster of laccases is fast enough not to be a readily noticeable contribution to the overall turnover rate. Indeed, the limiting step of the oxidation of high-redox potential compounds, such as chelated manganese ions, is the electron transfer from the electron donor to the T1 site of the fungal laccase.     

In vitro metabolism of zolmitriptan in rat cytochromes induced with beta-naphthoflavone and the interaction between six drugs and zolmitriptan

Zolmitriptan is a novel and highly selective 5-HT(1B/1D) receptor agonist used as an acute oral treatment for migraine. There are few reports regarding the in vitro metabolism of zolmitriptan. Previous studies indicated zolmitriptan was metabolized via CYP1A2 in human hepatic microsomes. In order to study the enzyme kinetics and drug interaction, the metabolism of zolmitriptan and possible drug-drug interactions were investigated in rat hepatic microsomes induced with different inducers. An active metabolite, N-demethylzolmitriptan, was detected and another minor, inactive metabolite that was reported in human hepatic microsomes was not detected in this study. The enzyme kinetics for the formation of N-demethylzolmitriptan from zolmitriptan in rat liver microsomes pretreated with BNF were 96+/-22 microM (K(m)), 11+/-3 pmol min(-1)mg protein(-1) (V(max)), and 0.12+/-0.02 microl min(-1)mg protein(-1) (CL(int)). Fluvoxamine and diphenytriazol inhibited zolmitriptan N-demethylase activity catalyzed by CYP1A2 (K(i)=3.8+/-0.3 and 3.2+/-0.1 microM, respectively). Diazepam and propranolol elicited a slight inhibitory effect on the metabolism of zolmitriptan (K(i)=70+/-11 and 90+/-18 microM, respectively). Cimetidine and moclobemide produced no significant effect on the metabolism of zolmitriptan. Fluvoxamine yielded a k(inactivation) value of 0.16 min(-1), and K(i) of 57 microM. The results suggest that rat hepatic microsomes are a reasonable model to study the metabolism of zolmitriptan, although there is a difference in the amount of minor, inactive metabolites between human hepatic microsomes and rat liver microsomes. The results of the inhibition experiments provided information for the interactions between zolmitriptan and drugs co-administrated in clinic, and it is helpful to explain the drug-drug interactions of clinical relevance on enzyme level. This study aso demonstrated that fluvoxamine may be a mechanism-based inactivator of CYP1A2.     

Photoelectrochemical control of the balance between cyclic- and linear electron transport in photosystem I. Algorithm for P700 induction kinetics

Redox transients of chlorophyll P700, monitored as absorbance changes ΔA₈₁₀, were measured during and after exclusive PSI excitation with far-red (FR) light in pea (Pisum sativum, cv. Premium) leaves under various pre-excitation conditions. Prolonged adaptation in the dark terminated by a short PSII + PSI− exciting light pulse guarantees pre-conditions in which the initial photochemical events in PSI RCs are carried out by cyclic electron transfer (CET). Pre-excitation with one or more 10 s FR pulses creates conditions for induction of linear electron transport (LET). These converse conditions give rise to totally different, but reproducible responses of P700⁻ oxidation. System analyses of these responses were made based on quantitative solutions of the rate equations dictated by the associated reaction scheme for each of the relevant conditions. These provide the mathematical elements of the P700 induction algorithm (PIA) with which the distinguishable components of the P700⁺ response can be resolved and interpreted. It enables amongst others the interpretation and understanding of the characteristic kinetic profile of the P700⁺ response in intact leaves upon 10 s illumination with far-red light under the promotive condition for CET. The system analysis provides evidence that this unique kinetic pattern with a non-responsive delay followed by a steep S-shaped signal increase is caused by a photoelectrochemically controlled suppression of the electron transport from Fd to the PQ-reducing Q_r site of the cytb₆f complex in the cyclic pathway. The photoelectrochemical control is exerted by the PSI-powered proton pump associated with CET. It shows strong similarities with the photoelectrochemical control of LET at the acceptor side of PSII which is reflected by release of photoelectrochemical quenching of chlorophyll a fluorescence.     

Effects of above- and below-ground competition from shrubs on photosynthesis, transpiration and growth in Quercus robur L. seedlings

For a tree seedling to successfully establish in dense shrubbery, it must maintain function under heterogeneous resource availability. We evaluated leaf-level acclimation in photosynthetic capacity, seedling-level transpiration, and seedling morphology and growth to gain an understanding of the effects of above- and below-ground competition on Quercus robur seedlings. Experimental seedlings were established in a typical southern Swedish shrub community where they received 1 of 4 competition levels (above-ground, below-ground, above- and below-ground, or no competition), and leaf-level responses were examined between two growth flushes. Two years after establishment, first-flush leaves from seedlings receiving above-ground competition showed a maximum rate of photosynthesis (A_max) 40% lower than those of control seedlings. With the development of a second flush above the shrub canopy, A_max of these seedlings increased to levels equivalent to those of seedlings free of light competition. Shrubby competition reduced oak seedling transpiration such that seedlings exposed to above- and below-ground competition showed rates 43% lower than seedlings that were not exposed to competition. The impaired physiological function of oak seedlings growing amid competition ultimately led to a 60-74% reduction in leaf area, 29-36% reduction in basal diameter, and a 38-78% reduction in total biomass accumulation, but root to shoot ratio was not affected. Our findings also indicate that above-ground competition reduced A_max, transpiration and biomass accumulation more so than below-ground competition. Nevertheless, oak seedlings exhibited the ability to develop subsequent growth flushes with leaves that had an A_max acclimated to utilize increased light availability. Our findings highlight the importance of flush-level acclimation under conditions of heterogeneous resource availability, and the capacity of oak seedlings to initiate a positive response to moderate competition in a shrub community.     

The surface structure of a complex substrate revealed by enzyme kinetics and Freundlich constants for α-amylase interaction with the surface of starch

Background

Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates.

Methods

For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution–solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR).

Results

Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and Δ_gelH was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid–liquid interface and slowest to granules possessing ordered crystalline surfaces.

Conclusions

Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods.

General Significance

Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.     

The family Ctenobelbidae (Acari,Oribatida), with description of a new species and discussion on systematic placement and taxonomic status of the genus Berndamerus Mahunka, 1977

The oribatid mite genus Berndamerus Mahunka, 1977 is transferred into the family Ctenobelbidae as the subgenus Ctenobelba (Berndamerus) Mahunka, 1977, stat. n. from the family Amerobelbidae. The known species of Berndamerus combined: C. (B.) bicostata (Berlese, 1910), comb. n., C. (B.) eremuloides (Berlese, 1910), comb. n., C. (B.) hellenica (Mahunka, 1977), comb. n. A new species, Ctenobelba (Berndamerus) bugiamapensis sp. n., is described from soil, Bu Gia Map National Park, southern Vietnam. It differs from the other species of the subgenus by the heterotrichy of notogastral setae, presence of adanal neotrichy and localization of adanal lyrifissures. Ctenobelbidae is recorded in Vietnam for the first time. A new diagnosis of the family Ctenobelbidae and the identification keys to the known subgenera of the genus Ctenobelba and species of the subgenus Ctenobelba (Berndamerus) are provided.