Hepatocytes exhibit a regulatory volume decrease (RVD) during hypotonic shock, which comprises loss of intracellular K+ and Cl- accompanied by hyperpolarization of transmembrane potential (Vm) due to an increase in membrane K+ conductance, (GK). To examine hepatocyte K+ homeostasis during RVD, double-barrel, K+-selective microelectrodes were used to measure changes in steady-state intracellular K+ activity (aKi) and Vm during hyposmotic stress. Cell water volume change was evaluated by measuring changes in intracellular tetramethylammonium (TMA+). Liver slices were superfused with modified Krebs physiological salt solution. Hyposmolality (0.8×300 mosm) was created by a 50 m m step-decrease of external sucrose concentration. Hepatocyte Vm hyperpolarized by 19 mV from -27 ± 1 to -46 ± 1 mV and aKidecreased by 14% from 91 ± 4 to 78 ± 4 m m when slices were exposed to hyposmotic stress for 4-5 min. Both Vm and aKireturned to control level after restoring isosmotic solution. In paired measurements, hypotonic stress induced similar changes in Vm and aKiboth control and added ouabain (1 m m) conditions, and these values returned to their control level after the osmotic stress. In another paired measurement, hypotonic shock first induced an 18-mV increase in Vm and a 15% decrease in aKiin control condition. After loading hepatocytes with TMA+, the same hypotonic shock induced a 14-mV increase in Vm and a 14% decrease in aTMAi. This accounted for a 17% increase of intracellular water volume, which was identical to the cell water volume change obtained when aKiwas used as the marker. Nonetheless, hyposmotic stress-induced changes in Vm and aKiwere blocked partly by Ba2+ (2 m m). We conclude that (i) hepatocyte Vm increases and aKidecreases during hypotonic shock; (ii) the changes in hepatocyte Vm and aKiduring and after hypotonic shock are independent of the Na+-K+ pump; (iii) the decrease in aKiduring hypotonic stress results principally from hepatocyte swelling.

<p> Abiotic stresses such as extreme temperatures, drought and high salinity severely compromise plant productivity, and have placed selective pressure for the acquisition of traits enabling plants to adjust to and recover from these unfavorable environmental conditions. Thellungiella salsuginea is a plant that is native to highly saline and semiarid environments and exhibits an exceptional ability to tolerate abiotic stress. In this thesis, I report on laboratory and field studies aimed at identifying traits that allow Thellungiella to tolerate harsh environmental conditions. It was found that Thellungiella accumulates organic solutes in response to abiotic stress. Transcript and metabolite profiling approaches were used to identify metabolic pathways important for the accumulation of compatible organic solutes in Thellungiella in response to sub-optimal environmental conditions. The relative abundance of transcripts encoding enzymes associated with the biosynthesis of compatible organic solutes such as proline or galactinol showed stress-responsive increases in cabinet-grown material and these metabolites were accumulated in salt or drought treated plants, respectively. However, proline and galactinol were found to be of low relative abundance in leaves of field plants. In contrast, several carbohydrates including sucrose, glucose, and fructose made a greater relative contribution to the field plant profiles suggesting that carbohydrates play an important role in plant abiotic stress tolerance during growth under field conditions. The identification of stress-specific metabolic changes can be used to identify important biochemical traits underlying environmental stress tolerance in Thellungiella. This information can be used to improve the tolerance of stress -sensitive crops (including a related crucifer species, canola) that are grown in areas where persistent droughts, saline soils and early or late frosts frequently occur. </p> / Thesis / Doctor of Philosophy (PhD)

Generation and phenotypic analyses of stzA gene deletion strains of Aspergillus nidulans implies that stzA is allelic to sltA, with the encoded transcription factor regulating tolerance to cations, DNA-damaging agents and high arginine concentrations. The similar severe sensitivity of a sltA1 mutant (GO281) and stzA deletion mutants to these stresses indicated that the premature termination codon in sltA1 represents a total loss-of-function mutation. It was also verified that StzA has no regulatory role in the utilisation of carbon sources. Findings were supported by phenotypic analyses of recombinant progeny resulting from sexual crosses between sltA1 and sltA+ strains. Bioinformatic analysis of genes involved in the osmotic stress response revealed that their promoters were significantly enriched for StzA binding site motifs compared to those of the control group, indicating that StzA may regulate many of these genes that comprise the High Osmolarity Glycerol (HOG) pathway. Although this pathway is activated by fludioxonil, stzA deletants and stzA+ strains showed similar sensitivities to this fungicide. Phenotypic analyses indicate that StzA does not regulate tolerance to sources of oxidative stress, non-ionic osmotic stress or components of the Cell Wall Integrity (CWI) pathway. A. nidulans StzA appears to have no role in cellulase or xylanase expression as revealed by the results of a dinitrosalicylic acid (DNS) assay. Trichoderma reesei ace1 deletant and wild-type strains showed similar sensitivities to cations, DNA-damaging agents, arginine, neomycin, acidic and alkaline pH. These results confirm that A. nidulans StzA and T. reesei Ace1 regulate the distinct phenotypes of abiotic stress tolerance and cellulase and xylanase expression, respectively, despite these two proteins sharing 58% overall amino acid similarity. All twenty-nine StzA orthologues identified are restricted to filamentous ascomycetes of the Pezizomycotina subphylum and may therefore represent specific and novel antifungal drug targets. The C-termini of StzA proteins are highly variable in both length and sequence, with no apparent conservations in amino acids or predicted secondary structure. This region is considered most likely to influence the divergent functions of StzA proteins. Conservations of individual residues in the N-termini correspond to conserved secondary structure (alpha helices) among StzA proteins, implying shared functions for StzA proteins in this region. Regulators of two major nitrogen metabolic pathways (CpcA and AreA) may regulate stzA expression. Statistically significant putative CpcA binding sites were positionally conserved in 26 out of 29 stzA orthologue promoters, indicating an interaction between stzA and CpcA, a transcription factor that mediates the cross pathway control of amino acid biosynthesis. REALALE sequences, likely to be of retrotransposon origin, containing putative overlapping binding sites for StzA and AreA, were found in eleven stzA promoters of the Eurotiomycetes class, indicating an interaction between stzA and the global nitrogen metabolite repressor AreA. Intriguingly, REALALE-containing promoters identified across the genome of A. nidulans were significantly enriched for StzA binding site motifs when compared to a control group of genes. Hence, REALALE may have regulatory significance that extends to other A. nidulans genes.

Acute brain cell swelling (cytotoxic edema) can occur in the first minutes of stroke, presumably as a result of brain cells taking up water. In extreme hypo-osmotic situations such as excessive water-loading by patients, uptake by brain cells can expand the brain, causing seizures. But is ischemic brain cell swelling the same as hypo-osmotic swelling? Water can passively diffuse across the plasma membrane. However the presence of water channels termed aquaporins (AQP) facilitates passive water diffusion by 10-100 times. Unlike astrocytes, there is no evidence of water channels on neuronal plasma membrane. However, there is still much debate about which cells (neurons or astrocytes) swell during over-hydration or during stroke and if neurons and astrocytes can volume-regulate during osmotic stress. The purpose of this study was to examine and compare the volume responses of PyNs and magnocellular neuroendocrine cells (MNCs) to acute osmotic challenge and to OGD. We examined MNCs because they are intrinsically osmosensitive to small changes (2-3 mOsm) of plasma osmolality. We also examined if the same neurons behave similarly in brain slices or when dissociated and if they respond differently to acute osmotic stress and stroke-like stress. Our results indicate that during acute osmotic stress (±40 mOsm) half of dissociated PyNs and MNCs tended to show appropriate responses. MNCs in brain slices showed similar responses to when they were dissociated, while brain slice PyNs were less responsive than when dissociated. Exposure to OGD resulted in obvious differences between the two types of in vitro preparations. Dissociated PyNs and MNCs showed no consistency in their volume responses to 10 minutes of OGD. Dissociated neurons swelled, shrunk or were unchanged in about equal numbers. In contrast, brain slice PyNs underwent profound swelling whereas, brain slice MNCs showed minor volume decreases. We conclude that about half of our dissociated neurons were too variable and unpredictable in their osmotic volume responses to be useful for osmotic studies. They also were too resistant to stroke-like stress to be good models for ischemia. Brain slice neurons were similar in their osmotic responses to dissociated neurons but proved to have consistent and predictable responses to stroke-like stress. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2011-02-07 17:55:08.333

No description available.

The phospholipid membrane is a self-assembled, dynamic molecular system that may exist alone in association with only water, or in complex systems comprised of multiple lipid types and proteins. In this dissertation the intra- and inter-molecular forces responsible for the atomistic, molecular and collective equilibrium structure and dynamics are studied by nuclear magnetic resonance spectroscopy (NMR). The multinuclear NMR measurements and various experimental techniques are able to provide data that enable the characterization of the hierarchical spatio-temporal organization of the phospholipid membrane. The experimental and theoretical studies conducted target membrane interactions ranging from model systems composed of only water and lipids, to multiple component domain forming membranes that are in association with peripheral and trans-membrane proteins. These measurements consisit of frequency spectrum lineshapes and nuclear-spin relaxation rates obtained using 2 H NMR, 13 C NMR, 31 P NMR and 1 H NMR. The changes of these experimental observables are interpreted within a statistical thermodynamic framework that allows the membrane structure, activation energies, and correlation times of motion to be determined. The cases presented demonstrate how fundamental principles of NMR spectroscopy may be applied to a host of membranes, leading to the biophysical characterization of membrane structure and dynamics.

Abstract Salinity has considerable adverse effects on agriculture through reduction in plant growth and water use. Sodium chloride salinity has both an osmotic effect on plant water relations, and a toxic effect on cellular processes. The relative contribution of these two effects to plant growth depends on a range of factors including plant specific tolerance mechanisms, such as Na and Cl exclusion, compartmentation of ions at a whole plant and cellular level, and synthesis of organic osmotic compounds for plant osmotic adjustment. Plants growing in saline soil would also experience reduced plant available water, due to the additional osmotic effect on soil water potential. The effect of salinity on plant growth is further complicated by the interactions of environmental conditions with plant water and ion uptake. This thesis examines the osmotic and toxic effects of salinity on wheat (Triticum aestivum L.) and chickpea (Cicer arietinum L.), with particular focus on plant water availability, effects of Na and Cl toxicity, and temperature and humidity effects. While considerable research has been undertaken into the physiological response of plants to NaCl, our understanding of the capacity of plants to extract water from saline soils has remained largely theoretical. Total plant available water is largely determined by the matric potential of the soil. Presence of sodium chloride would have an additional osmotic effect, and previous theory stated that the salt tolerance of the plant determined the extent to which this osmotic potential reduced plant available water. The capacity of wheat and chickpea to extract water from saline soils was examined in a soil experiment where water stress was imposed on established plants, which were then grown until permanent wilting point (PWP) was reached. Wheat extracted to lower soil water potentials (-1.5 MPa), than chickpea (-900 kPa) in 0 NaCl treatments. Where salinity was low to moderate, plants were able to extract water to a PWP determined by the combined total of matric and osmotic potentials. Wheat extracted water to PWP in salinity treatments up to soil ECse of 5.3 dS/m, and chickpea to 2.9 dS/m. As salinity increased, toxic effects of salinity dominated, and water extraction by plants was significantly lower than that determined by total soil water potential. Solution culture experiments investigated the comparative toxic effects of Na, Cl and salt mixtures. Growth of wheat was reduced by Na toxicity, but not Cl toxicity, with Na causing a small, but significant additional reduction in growth, compared to high Cl or a salt mixture. Reductions in growth of 50% from control treatments occurred at -500 kPa for the Na treatment, and -630 kPa for the Cl and mixed salt treatments. In contrast, growth of chickpea was significantly reduced by both Na and Cl toxicity, with a large difference in growth compared to the salt mixture. Growth reductions of 50% occurred at -330 kPa for the Cl treatments, and -450 kPa for the Na treatment. A 50% growth reduction was not observed in the mixed salt treatment. Tolerance of saline conditions is reduced under stressful environmental conditions, such as high temperature and low relative humidity. Hot and dry conditions were shown to reduce the tolerance of saline conditions by both wheat and chickpea, compared to cool or humid conditions. Tissue concentrations of Na in wheat were disproportionately high in treatments with high evaporative demand, while tissue Cl was not related to evaporative demand. Tissue concentrations of Na in chickpea increased with temperature, but not relative humidity, while tissue Cl concentrations were highly correlated with evaporative demand. The relationships between NaCl salinity, plant water use, and environmental conditions were examined, allowing further development of the two phase salinity model. In particular, the transition point between the osmotic and toxic salinity effects. While the concentration of NaCl in the soil remains the primary factor, soil water status, environmental stresses and presence of other salts may dictate whether salinity be tolerated by the plant or not. The ability of the plant to extract water to PWP, as determined by total matric and osmotic potential has been identified as a useful indicator of salinity tolerance. The point at which toxicity of Na and/or Cl is observed is associated with a rapid increase in Na and Cl uptake by the shoot tissue, and a decrease in the amount of water the plant is able to extract from the soil. Within the osmotic region of salinity stress, the plant is able to extract water to PWP, but as NaCl becomes toxic the plant is unable to utilize this water.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 3 uppsatser.

>Magister Scientiae - MSc / Drought stress is a major contributor to reduced soybean crop yield and quality, this can however be mitigated by the plant’s antioxidant defence mechanisms. One group of antioxidant enzymes that are active in these defence mechanisms are glutathione peroxidases (GPXs). GPXs are antioxidant proteins which are able to reduce H2O2, a toxic reactive oxygen species which accumulates under stress conditions. This study aims at isolating the protein encoded by Glyma01g42840 and determining if it has Phospholipid hydroperoxidase glutathione peroxidase (PHGPX) and/or Thioredoxin dependent peroxidase (TRX-PX) activity as well as assaying the effect of Drought stress on the expression of this putative GPX . This will be accomplished by molecular cloning, sequencing as well as the expression of the isolated protein to assay it enzymatic activity. It was found that the enzyme encoded by Glyma01g42840 is able to use glutathione and thioredoxin as electron donors for the detoxification peroxides, however enzymatic activity is more efficient when using glutathione as an electron donor. In conclusion it was found that glyma01g42840 encodes an enzyme which is able to utilise more than one electron donor and as glutathione produces the greatest amount of enzymatic activity it can be said that glyma01g42840 encodes a GPX.

No description available.