Thermodynamics of interaction of octyl glucoside with phosphatidylcholine vesicles: partitioning and solubilization as studied by high sensitivity titration calorimetry

The interaction of the surfactant octyl glucoside (OG) with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and soy bean phosphatidylcholine (soy bean PC) was studied using high-sensitivity titration calorimetry. We determined the partition coefficient of OG between water and lipid bilayers and the transfer enthalpy of the surfactant by addition of lipid vesicles to OG monomers or vice versa. Comparison with the micellization enthalpy of the surfactant gives information on differences in the hydrophobic environment of OG in a liquid-crystalline bilayer or a micelle. The average partition coefficient P in mole fraction units for x_e≈0.12–0.2 decreases slightly from 4152 at 27°C to 3479 at 70°C for DMPC and from 4260 to 3879 for soy bean PC, respectively. The transfer enthalpy ΔH^T of OG into lipid vesicles is positive at 27°C and negative at 70°C. Its temperature dependence is larger for the incorporation of OG into DMPC than into soy bean PC vesicles. It is concluded that OG in DMPC vesicles is better shielded from water than in soy bean PC vesicles or in micelles. Titration calorimetry was also used to determine the phase boundaries of the coexistence region of mixed vesicles and mixed micelles in the systems OG/DMPC, OG/DPPC, OG/DSPC, and OG/soy bean PC vesicles at 70°C in the liquid-crystalline phase. DMPC and soy bean PC solubilization was also studied at 27°C to investigate the effect of temperature. The effective surfactant to lipid ratios at saturation, R_e^sat, for all PCs studied are in the range between 1.33–1.72 and the ratios at complete solubilization, R_e^sol, are between 1.79–3.06. At 70°C, the R_e^sat values decrease with increasing chain length of the saturated PC. The ratios depend also slightly on temperature and the degree of unsaturation of the fatty acyl chains. For the OG/soy bean PC system, the coexistence range for mixed vesicles and mixed micelles is larger than for the corresponding PCs with saturated chains.     

Protein Hydroxylation Catalyzed by 2-Oxoglutarate-dependent Oxygenases

The post-translational hydroxylation of prolyl and lysyl residues, as catalyzed by 2-oxoglutarate (2OG)-dependent oxygenases, was first identified in collagen biosynthesis. 2OG oxygenases also catalyze prolyl and asparaginyl hydroxylation of the hypoxia-inducible factors that play important roles in the adaptive response to hypoxia. Subsequently, they have been shown to catalyze N-demethylation (via hydroxylation) of N^ϵ-methylated histone lysyl residues, as well as hydroxylation of multiple other residues. Recent work has identified roles for 2OG oxygenases in the modification of translation-associated proteins, which in some cases appears to be conserved from microorganisms through to humans. Here we give an overview of protein hydroxylation catalyzed by 2OG oxygenases, focusing on recent discoveries.     

Structure and Functional Analysis of YcfD,a Novel 2-Oxoglutarate/Fe-Dependent Oxygenase Involved in Translational Regulation in Escherichia coli

The 2-oxoglutarate (2OG)/Fe^2 +-dependent oxygenases (2OG oxygenases) are a large family of proteins that share a similar overall three-dimensional structure and catalyze a diverse array of oxidation reactions. The Jumonji C (JmjC)-domain-containing proteins represent an important subclass of the 2OG oxygenase family that typically catalyze protein hydroxylation; however, recently, other reactions have been identified, such as tRNA modification. The Escherichia coli gene, ycfD, was predicted to be a JmjC-domain-containing protein of unknown function based on primary sequence. Recently, YcfD was determined to act as a ribosomal oxygenase, hydroxylating an arginine residue on the 50S ribosomal protein L-16 (RL-16). We have determined the crystal structure of YcfD at 2.7 Å resolution, revealing that YcfD is structurally similar to known JmjC proteins and possesses the characteristic double-stranded β-helix fold or cupin domain. Separate from the cupin domain, an additional globular module termed α-helical arm mediates dimerization of YcfD. We further have shown that 2OG binds to YcfD using isothermal titration calorimetry and identified key binding residues using mutagenesis that, together with the iron location and structural similarity with other cupin family members, allowed identification of the active site. Structural homology to ribosomal assembly proteins combined with GST (glutathione S-transferase)-YcfD pull-down of a ribosomal protein and docking of RL-16 to the YcfD active site support the role of YcfD in regulation of bacterial ribosome assembly. Furthermore, overexpression of YcfD is shown to inhibit cell growth signifying a toxic effect on ribosome assembly.     

Catalytic promiscuity of rice 2-oxoglutarate/Fe(II)-dependent dioxygenases supports xenobiotic metabolism

The rice (Oryza sativa) 2-oxoglutarate (2OG)/Fe(II)-dependent dioxygenase HIS1 mediates the catalytic inactivation of five distinct β-triketone herbicides (bTHs). By assessing the effects of plant growth regulators on HIS1 enzyme function, we found that HIS1 mediates the hydroxylation of trinexapac-ethyl (TE) in the presence of Fe²⁺ and 2OG. TE blocks gibberellin biosynthesis, and we observed that its addition to culture medium induced growth retardation of rice seedlings in a concentration-dependent manner. Similar treatment with hydroxylated TE revealed that hydroxylation greatly attenuated the inhibitory effect of TE on plant growth. Forced expression of HIS1 in a rice his1 mutant also reduced its sensitivity to TE compared with that of the nontransformant. These results indicate that HIS1 metabolizes TE and thereby markedly reduces its ability to slow plant growth. Furthermore, analysis of five rice HIS1-like (HSL) proteins revealed that OsHSL2 and OsHSL4 also metabolize TE in vitro. HSLs from wheat (Triticum aestivum) and barley (Hordeum vulgare) also showed such activity. In contrast, OsHSL1, which shares the highest amino acid sequence identity with HIS1 and metabolizes the bTH tefuryltrione, did not manifest TE-metabolizing activity. Site-directed mutagenesis of OsHSL1 informed by structural models showed that substitution of three amino acids with the corresponding residues of HIS1 conferred TE-metabolizing activity similar to that of HIS1. Our results thus reveal a catalytic promiscuity of HIS1 and its related enzymes that support xenobiotic metabolism in plants.     

Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF

Background

Enterococcus faecalis has emerged as a major hospital pathogen. To explore its diversity, we sequenced E. faecalis strain OG1RF, which is commonly used for molecular manipulation and virulence studies.

Results

The 2,739,625 base pair chromosome of OG1RF was found to contain approximately 232 kilobases unique to this strain compared to V583, the only publicly available sequenced strain. Almost no mobile genetic elements were found in OG1RF. The 64 areas of divergence were classified into three categories. First, OG1RF carries 39 unique regions, including 2 CRISPR loci and a new WxL locus. Second, we found nine replacements where a sequence specific to V583 was substituted by a sequence specific to OG1RF. For example, the iol operon of OG1RF replaces a possible prophage and the vanB transposon in V583. Finally, we found 16 regions that were present in V583 but missing from OG1RF, including the proposed pathogenicity island, several probable prophages, and the cpsCDEFGHIJK capsular polysaccharide operon. OG1RF was more rapidly but less frequently lethal than V583 in the mouse peritonitis model and considerably outcompeted V583 in a murine model of urinary tract infections.

Conclusion

E. faecalis OG1RF carries a number of unique loci compared to V583, but the almost complete lack of mobile genetic elements demonstrates that this is not a defining feature of the species. Additionally, OG1RF's effects in experimental models suggest that mediators of virulence may be diverse between different E. faecalis strains and that virulence is not dependent on the presence of mobile genetic elements.     

Surface active site model for Ni2+ adsorption of the surface imprinted adsorbent

In this work, a new surface active site (SAS) adsorption equilibrium model was presented, which explicitly accounted for the H⁺ competitive adsorption with Ni²⁺ in adsorption equilibrium. Static adsorption experiments with Ni²⁺ as a model metal ion were carried out to determine the model parameters, those were, equilibrium constant for Ni²⁺ (K_a), for H⁺ (K_s), characteristic number of binding sites for Ni²⁺ (n), for H⁺ (a), and the non-imprinted factor (σ). It was found that those model parameters n and a were all constant, and that they all expressed that one active site bound two Ni²⁺ or two H⁺, while the non-imprinted factor, σ, was effected by Ni²⁺ concentration, H⁺ concentration in solution and imprinted Ni²⁺ concentration in the preparation. Simulated result was compared with experimental data of the adsorption for Ni²⁺. It was showed that this model could be well used to predict the adsorption equilibrium for Ni²⁺ on the surface imprinted adsorbent. And it was demonstrated that the efficacy of the active sites formalism could be used in describing adsorption behavior for Ni²⁺ on the surface imprinted adsorbent.     

The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases

Mutations in isocitrate dehydrogenases (IDHs) have a gain-of-function effect leading to R(−)-2-hydroxyglutarate (R-2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R- and S-2HG inhibit 2-oxoglutarate (2OG)-dependent oxygenases with varying potencies. Half-maximal inhibitory concentration (IC₅₀) values for the R-form of 2HG varied from approximately 25 μM for the histone N^ɛ-lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH-associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation.     

Spectroscopic Analyses of Manganese Ions Effects on the Conformational Changes of Inorganic Pyrophosphatase from Psychrophilic Shewanella sp. AS-11

Mn²⁺ ions influence the activity, temperature dependence, and thermostability of the psychrophilic Shewanella-PPase (Sh-PPase), and are required to function in cold environments. The functional characteristics of Sh-PPase on activation with Mn²⁺ ions are possibly related to conformational changes in the molecule. In this study, conformational changes of Sh-PPase on activation with Mn²⁺ ions were analyzed in solution by fluorescence spectroscopy analysis of intrinsic tryptophan residues, 1-anilino-8-naphthalene sulfonate fluorescence, and circular dichroism spectroscopy. For Sh-PPase, Mn²⁺ ions did not affect the flexibility of the tryptophan residues and secondary structure of the enzyme. However, the microenvironment of the tryptophan residues and surface area of Sh-PPase were more hydrophilic on activation with Mn²⁺ ions. These results indicate that activation with Mn²⁺ ions causes conformational changes around the aromatic amino acid residues and affects the hydrophobicity of the enzyme surface, which results in conformational changes. Substrate-induced conformational changes reflect that metal-free Sh-PPase in solution indicated an open structure and will be a close structure when binding substrate. In combination of our spectroscopic analyses on Sh-PPase, it can be concluded that activation with Mn²⁺ ions changes some conformation of Sh-PPase molecule in solution.     

The effect of different Fe2+ concentrations in culture media on the recycling of ground tyre rubber by Acidithiobacillus ferrooxidans YT-1

Waste rubber has posed challenging environmental and disposal problems across the world. This study focused on the microbial reclaiming of ground tyre rubber (GTR) by Acidithiobacillus ferrooxidans YT-1 cultured in media with variable Fe²⁺ concentrations. The Acidithiobacillus ferrooxidans YT-1 strain with the ability of oxidizing sulfur and reclaiming waste rubber was isolated and identified. Toxicity tests of different rubber and additives in tyre rubber compounds to microorganisms was quantitatively investigated. After desulfurization, there were many small colonies on the surface of the desulfurizated GTR (DGTR), due to surface degradation by A. ferrooxidans YT-1. The amount of small colonies increased and sulfur content decreased with the increase of Fe²⁺ concentrations in the media, implying that Fe²⁺ concentration had a great influence on the degradation ability of A. ferrooxidans YT-1. A medium with a high Fe²⁺ concentration was good for growth of A. ferrooxidans YT-1. Compared with styrene butadiene rubber (SBR)/GTR blends, the tensile strength and elongation at the break of the SBR/DGTR blends were significantly improved. The scanning electron microscope (SEM) photographs of the fracture surface further indicated a good coherency between DGTR and the SBR matrix. These results revealed that A. ferrooxidans YT-1 cultured in a medium with a high Fe²⁺ concentration could improve the reclaiming efficiency of waste rubber.     

Entamoeba histolytica: ultrastructural localization of Ca2+-dependent nucleotidases

Localization of nucleotidases dependent on Ca²⁺ was investigated cytochemically in axenically cultivated trophozoites of Entamoeba histolytica, strain HM-1:IMSS, with an electron microscope. Ca²⁺-dependent ATPase (EC 3.6.1.3) activity was found on the plasma membrane and on the inner surface of the limiting membrane of a few cytoplasmic vacuoles. Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase, and acid phosphatase (EC 3.1.3.2) activities were detected on the inner surface of the limiting membrane of most of the cytoplasmic vacuoles but not on the plasma membrane. Cytoplasmic vacuoles with these enzymatic activities seemed similar in morphological characteristics. Moreover, the reaction product formed by Ca²⁺-dependent ADPase, Ca²⁺-dependent thiamine pyrophosphatase and acid phosphatase was demonstrable on the inner surface of the limiting membrane of vacuoles containing ingested red blood cells. The reaction product formed by these enzymes was also observed on the periphery of ingested red blood cells. The findings suggest that cytoplasmic vacuoles with these enzymatic activities are lysosomal in nature, probably phagolysosomes; therefore, the enzymes appear to be at least partially associated with primary lysosomes of E. histolytica.     

In Vivo Epithelial Wound Repair Requires Mobilization of Endogenous Intracellular and Extracellular Calcium

We report that a localized intracellular and extracellular Ca²⁺ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca²⁺-sensitive protein (yellow cameleon 3.0) report that intracellular Ca²⁺ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca²⁺ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca²⁺ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca²⁺ mobilization. Indomethacin and verapamil also inhibit the luminal Ca²⁺ increase. Intracellular Ca²⁺ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca²⁺ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca²⁺ and unevenly inhibits late-phase intracellular Ca²⁺ mobilization. Both modes of Ca²⁺ chelation slow gastric repair. In plasma membrane Ca-ATPase 1^+/− mice, but not plasma membrane Ca-ATPase 4^−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca²⁺ increase. We conclude that endogenous Ca²⁺, mobilized by signaling pathways and transmembrane Ca²⁺ transport, causes increased Ca²⁺ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo.     

New Insights into Histidine Triad Proteins: Solution Structure of a Streptococcus pneumoniae PhtD Domain and Zinc Transfer to AdcAII

Zinc (Zn²⁺) homeostasis is critical for pathogen host colonization and invasion. Polyhistidine triad (Pht) proteins, located at the surface of various streptococci, have been proposed to be involved in Zn²⁺ homeostasis. The phtD gene, coding for a Zn²⁺-binding protein, is organized in an operon with adcAII coding for the extracellular part of a Zn²⁺ transporter. In the present work, we investigate the relationship between PhtD and AdcAII using biochemical and structural biology approaches. Immuno-precipitation experiments on purified membranes of Streptococcus pneumoniae (S. pneumoniae) demonstrate that native PhtD and AdcAII interact in vivo confirming our previous in vitro observations. NMR was used to demonstrate Zn²⁺ transfer from the Zn²⁺-bound form of a 137 amino acid N-terminal domain of PhtD (t-PhtD) to AdcAII. The high resolution NMR structure of t-PhtD shows that Zn²⁺ is bound in a tetrahedral site by histidines 83, 86, and 88 as well as by glutamate 63. Comparison of the NMR parameters measured for apo- and Zn²⁺-t-PhtD shows that the loss of Zn²⁺ leads to a diminished helical propensity at the C-terminus and increases the local dynamics and overall molecular volume. Structural comparison with the crystal structure of a 55-long fragment of PhtA suggests that Pht proteins are built from short repetitive units formed by three β-strands containing the conserved HxxHxH motif. Taken together, these results support a role for S. pneumoniae PhtD as a Zn²⁺ scavenger for later release to the surface transporter AdcAII, leading to Zn²⁺ uptake.     

Nucleation of Celestite and Strontianite on a Cyanobacterial S-Layer

Synechococcus strain GL24 is a unicellular cyanobacterium that was isolated from Fayetteville Green Lake, New York, a meromictic lake which has high Ca²⁺ and SO₄^2- concentrations. Epicellular mineralization of Synechococcus cells in the lake is the mechanism by which extensive calcitic bioherms (or microbial reefs) have been formed on the lake's shore and a marl sediment has been built on the lake bottom. Previous studies have shown that calcium carbonate (calcite) formation on the Synechococcus surface is dependent upon an alkaline pH, which is produced in the cellular microenvironment by the cells as their activity increases with seasonal warming of the lake water. At the circumneutral pH of bulk lake water, calcium sulfate (gypsum) is formed. In this study, we show that Synechococcus mediates a similar sulfate-to-carbonate transformation when Sr²⁺ is the major divalent cation present, forming celestite and strontianite. In experimental systems to which equimolar amounts of Ca²⁺ and Sr²⁺, Ca²⁺ or Sr²⁺ and Mg²⁺, or all three ions together were added to artificial lake water, Ca²⁺ and Sr²⁺ were incorporated equally into mineral formation to form CaSr(CO₃)₂. No Mg²⁺ -containing carbonates were formed when either or both of the other two ions were present. Mineral formation takes place on a hexagonally arranged proteinaceous template (an S-layer) which forms the outermost surface of the Synechococcus cell. Our results provide evidence that the S-layer exhibits selectivity with respect to the ions bound and subsequently incorporated into carbonate minerals and that celestite and strontianite, previously thought to be purely evaporitic minerals, can be biogenically formed.     

Nickel binding properties of Helicobacter pylori UreF,an accessory protein in the nickel-based activation of urease

Helicobacter pylori UreF (HpUreF) is involved in the insertion of Ni²⁺ in the urease active site. The recombinant protein in solution is a dimer characterized by an extensive α-helical structure and a well-folded tertiary structure. HpUreF binds two Ni²⁺ ions per dimer, with a micromolar dissociation constant, as shown by calorimetry. X-ray absorption spectroscopy indicated that the Ni²⁺ ions reside in a five-coordinate pyramidal geometry comprising exclusively N/O-donor ligands derived from the protein, including one or two histidine imidazole and carboxylate ligands. Binding of Ni²⁺ does not affect the solution properties of the protein. Mutation to alanine of His229 and/or Cys231, a pair of residues located on the protein surface that interact with H. pylori UreD, altered the affinity of the protein for Ni²⁺. This result, complemented by the findings from X-ray absorption spectroscopy, indicates that the Ni²⁺ binding site involves His229, and that Cys231 has an indirect structural role in metal binding. An in vivo assay of urease activation demonstrated that H229A HpUreF, C231A HpUreF, and H229/C231 HpUreF are significantly less competent in this process, suggesting a role for a Ni²⁺ complex with UreF in urease maturation. This hypothesis was supported by calculations revealing the presence of a tunnel that joins the Cys-Pro-His metal binding site on UreG and an opening on the UreD surface, passing through UreF close to His229 and Cys231, in the structure of the H. pylori UreDFG complex. This tunnel could be used to transfer nickel into the urease active site during apoenzyme-to-holoenzyme activation.     

Potentiation of the efficacy of murine L1210 leukemia vaccine by a novel immunostimulator 7-thia-8-oxoguanosine: Increased survival after immunization with vaccine plus 7-thia-8-oxoguanosine

Summary We have investigated the ability of a novel immunopotentiator, 7-thia-8-oxoguanosine (7T8OG) to increase the efficacy of a weakly immunogenic murine L1210 leukemia vaccine. The vaccine was prepared by irradiating L1210 leukemia cells in a cesium source with a total of 6000-R dose. DBA/2 mice were treated with 150 mg/kg 7T8OG and/or with vaccine consisting of 10⁷ irradiated cells. In combination therapy, mice first received the vaccine and then were injected with 75 mg/kg 7T8OG 2 h and 4 h after vaccination. One week after the last treatment all mice were inoculated with 10⁴ live leukemia cells intraperitoneally. Control, untreated mice (n = 66) injected with 10⁴ live leukemia cells had a mean survival time ± standard error of 10.5±0.2 days. Treating mice (n = 66) with one, two or three doses of 7T8OG administered i.p. 1 week apart did not increase survival (mean survival time = 10.7 days). Mice immunized with one, two or three doses of vaccine had 14.5±1.1, 45.4±6.2 and 68.3±10.6 days mean survival, respectively. 7T8OG-stimulated vaccination increased the survival dramatically. The best survival was noted when the mice were treated with 2× (vaccine + 7T8OG). Immunization of mice (n = 30) with this treatment regimen increased the mean survival to 156±10.0 days. Over 90% of mice that were treated this way had a cumulative survival time greater than 160 days. In contrast, only 12% of the mice immunized twice with the leukemia vaccine alone survived over 160 days. These results suggest a rationale for the use of this immuno-potentiator with various vaccines for a more effective immunization.     

Adsorption kinetics of Pb and Cd by two plant growth promoting rhizobacteria

A bench study was carried out to characterize the kinetics of two plant growth promoting rhizobacteria (PGPR) Azotobacter chroococcum and Bacillus megaterium to adsorb heavy metals from solution. Adsorption of Pb²⁺ and Cd²⁺ by bacterial cells was processed quickly with an equilibration achieved within 5 min. The adsorptions were fitted well with Freundlich and Langmuir isotherm models. The comparison of isotherm parameters indicated that A. chroococcum had a stronger capacity to bind metal ions than B. megaterium, with an average increase of 59.8% for Pb²⁺ and 75.6% for Cd²⁺, respectively. Both bacteria had a stronger affinity to Pb²⁺ than Cd²⁺ since Pb²⁺ was more easily bound with the phosphoryl groups on the cell surface than Cd²⁺. This demonstrated that the presence of bacteria in the rhizosphere may result in the reduction of mobile ions in soil solution.     

Morphological,anatomical, and ultrastructural changes (visualized through scanning electron microscopy) inducedin Triticum aestivum by Pb2+ treatment

Lead (Pb) causes severe damage to crops, ecosystems, and humans, and alters the physiology and biochemistry of various plant species. It is hypothesized that Pb-induced metabolic alterations could manifest as structural variations in the roots of plants. In light of this, the morphological, anatomical, and ultrastructural variations (through scanning electron microscopy, SEM) were studied in 4-day-old seedlings of Triticum aestivum grown under Pb stress (0, 8, 16, 40, and 80 mg Pb²⁺ l^?1; mild to highly toxic). The toxic effect was more pronounced in radicle growth than on the plumule growth. The SEM of the root of T. aestivum depicted morphological alterations and surface ultrastructural changes. Compared to intact and uniform surface cells in the control roots, cells were irregular and desiccated in Pb²⁺-treated roots. In Pb²⁺-treated roots, the number of root hairs increased manifold, showing dense growth, and these were apparently longer. Apart from the deformity in surface morphology and anatomy of the roots in response to Pb²⁺ toxicity, considerable anatomical alterations were also observed. Pb²⁺-treated root exhibited signs of injury in the form of cell distortion, particularly in the cortical cells. The endodermis and pericycle region showed loss of uniformity post Pb²⁺ exposure (at 80 mg l^?1 Pb²⁺). The cells appeared to be squeezed with greater depositions observed all over the tissue. The study concludes that Pb²⁺ treatment caused structural anomalies and induced anatomical and surface ultrastructural changes in T. aestivum.     

Moist deciduous forest identification using temporal MODIS data — A comparative study using fuzzy based classifiers

The two soft fuzzy based classifiers, Possibilistic c-Means (PCM) approach and Noise Clustering (NC) were compared for the Moist Deciduous Forest (MDF) identification from MODIS temporal data. Seven date temporal MODIS data were used to identify MDF and temporal Advanced Wide Field Sensor (AWiFS) data was used as reference data for testing. Simple Ratio (SR), Normalized Difference Vegetation Index (NDVI), Soil Adjusted Vegetation Index (SAVI) and Enhanced Vegetation Index 2 (EVI2) were used to generate the temporal spectral index datasets for both the MODIS and AWiFS. The parameter weighting exponent m for PCM and resolution parameter δ for NC were optimized. Results show that the optimized value of m for MDF is 2.1, while δ value is 3.6 × 10⁴ for temporal MODIS data. For assessment of the accuracy AWiFS datasets were also optimized using entropy approach. The optimized dataset of AWiFS was then used for accuracy assessment of the soft classified outputs from MODIS using Fuzzy ERror Matrix (FERM). It was found from this study that, for PCM classifier the highest fuzzy overall accuracy of 97.44% was obtained using the SAVI for the temporal dataset ‘Five’ consisting to one scene of ‘Full greenness’, three scenes in ‘Intermediate frequency stage of Onset of Greenness (OG) and End of Senescence (ES) activity’ and the last image pertaining corresponds to the ‘Maximum frequency stage of OG activity’ as per phenology of MDF. Similarly, for NC classifier the highest fuzzy overall accuracy of 95.19% was obtained for the EVI2 with temporal dataset ‘Five’ consisting with two scene of ‘Full greenness’, two scenes in ‘Intermediate frequency stage of OG and ES activity’ and the last one corresponds to the ‘Maximum frequency stage of OG activity’as per phenology of MDF.     

The Extracellular K+ Concentration Dependence of Outward Currents through Kir2.1 Channels Is Regulated by Extracellular Na+ and Ca2+

It has been known for more than three decades that outward Kir currents (I_K1) increase with increasing extracellular K⁺ concentration ([K⁺]_o). Although this increase in I_K1 can have significant impacts under pathophysiological cardiac conditions, where [K⁺]_o can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K⁺]_o dependence of Kir2.1-mediated outward I_K1 was due to [K⁺]_o-dependent inhibition of outward I_K1 by extracellular Na⁺ and Ca²⁺. This could be accounted for by Na⁺/Ca²⁺ inhibition of I_K1 through screening of local negative surface charges. Consistent with this, extracellular Na⁺ and Ca²⁺ reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I_K1 in a similar [K⁺]_o-dependent manner as Na⁺/Ca²⁺. Site-directed mutagenesis studies identified Asp¹¹⁴ and Glu¹⁵³ as the source of surface charges. Reducing K⁺ activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na⁺ and Ca²⁺, suggesting that in addition to exerting a surface electrostatic effect, Na⁺ and Ca²⁺ might inhibit outward I_K1 by inhibiting K⁺ activation. This study identified interactions of K⁺ with Na⁺ and Ca²⁺ that are important for the [K⁺]_o dependence of Kir2.1-mediated outward I_K1.