Moderately Elevated Temperature Eliminates Resistance of Rice Plants with Enhanced Expression of Glutathione Reductase to Intensive Photooxidative Stress

We developed transgenic rice plants (Oryza sativa L. cv. Daeribbyeo) overproducing cytosolic glutathione reductase (GR) using a GR gene from Brassica campestris and studied their response to photo-oxidative stress in the presence of methyl viologen (MV, 10 and 50 M concentrations) under room (25 °C) and moderately elevated (35 °C) temperature by analysis of chlorophyll (Chl) a fluorescence parameters (F_V/F_M, q_N, and q_P) and of Chl content. Elevated temperature enhanced and accelerated the photo-oxidative damage to photosynthetic apparatus expressed mainly by a fast decrease of q_N. Higher temperature supported the protective reaction in transformed rice plants for lower MV concentration (10 M) and eliminated the enhanced tolerance of photosystem 2 photochemistry to photooxidative stress for higher (50 M) MV concentration. Different mechanisms and temperature dependence of oxidative and protective reactions explain the results.     

Nucleophilic reaction by carbonic anhydrase model zinc compound: characterization of intermediates for CO2 hydration and phosphoester hydrolysis

The partially hydrophilic and hydrophobic tripodal ligands, tris(hydroxy-2-benzimidazolylmethyl)amine L1h and tris(2-benzimidazolyl)amine L1 were used for the preparation of biomimetic complex of carbonic anhydrase. The CO(2) hydration using [L1hZn(OH)]ClO(4).1.5H(2)O provided the zinc-bound and free HCO(3)(-)s, which were formed by nucleophilic attack of Zn-OH toward CO(2) in dimethyl sulfoxide (DMSO). The phenolic OH in L1h can recognize water molecules through hydrogen bonds to facilitate the collection of the water molecules around a biomimetic zinc compound; the molecular structure of [L1hZn(OH)](+) was revealed. The packing diagram has demonstrated the all the water molecules are hydrogen bonded to each phenolic OH. The nucleophilic attack of zinc-bound OH(-) to substrate is used to catalyze the CO(2) hydration and phosphoester hydrolysis. The carbonic anhydrase model compound [L1Zn(OH(2))](2+) was applied for the hydrolysis of phosphoesters, parathion and bis(p-nitrophenyl)phosphate (BNPP(-)). The low reactivity of [L1Zn(OH)](+) for parathion hydrolysis is attributed to the stability of the intermediate [L1Zn(OP(S)(OEt)(2))](+). Since the structures of the intermediates [L1Zn(OH(2))](BNPP)(2) (1) and [L1Zn(OP(S)(OEt)(2))]ClO(4) (2) formed on the way of hydrolysis are too stable to realize the catalytic cycle and are not active for hydrolysis, carbonic anhydrase model compound [L1Zn(OH(2))](2+) was not suitable for phosphoester hydrolysis; the zinc model compound surrounded by three benzimidazolyl groups is used to have the steric hindrance for bulky substrate, such as parathion and BNPP(-).     

High affinity for farnesyltransferase and alternative prenylation contribute individually to K-Ras4B resistance to farnesyltransferase inhibitors

Farnesyltransferase inhibitors (FTIs) block Ras farnesylation, subcellular localization and activity, and inhibit the growth of Ras-transformed cells. Although FTIs are ineffective against K-Ras4B, the Ras isoform most commonly mutated in human cancers, they can inhibit the growth of tumors containing oncogenic K-Ras4B, implicating other farnesylated proteins or suggesting distinct functions for farnesylated and for geranylgeranylated K-Ras, which is generated when farnesyltransferase is inhibited. In addition to bypassing FTI blockade through geranylgeranylation, K-Ras4B resistance to FTIs may also result from its higher affinity for farnesyltransferase. Using chimeric Ras proteins containing all combinations of Ras background, CAAX motif, and K-Ras polybasic domain, we show that either a polybasic domain or an alternatively prenylated CAAX renders Ras prenylation, Ras-induced Elk-1 activation, and anchorage-independent cell growth FTI-resistant. The polybasic domain alone increases the affinity of Ras for farnesyltransferase, implying independent roles for each K-Ras4B sequence element in FTI resistance. Using microarray analysis and colony formation assays, we confirm that K-Ras function is independent of the identity of the prenyl group and, therefore, that FTI inhibition of K-Ras transformed cells is likely to be independent of K-Ras inhibition. Our results imply that relevant FTI targets will lack both polybasic and potentially geranylgeranylated methionine-CAAX motifs.     

Cultivation and characterization of planarian neuronal cells isolated by fluorescence activated cell sorting (FACS)

Studies using molecular markers have revealed that planarians possess a highly organized brain. Here we separated brain neurons from dissociated planarian head cells by fluorescence activated cell sorting (FACS), and characterized them by single cell PCR analysis and cell culture. Dissociated cells were labeled with three different fluorescent dyes, Hoechst 33258, Merocyanine 540, and Propidium Iodide (PI), and fractioned by FACS. Interestingly, we have succeeded in identifying a cell fraction specific to the head, which we have named the head-abundant cell fraction (HAC). Most of the HAC expressed neuron-specific genes and proteins. When they were cultured in vitro, they showed an ability to extend neurites on several types of extracellular matrices (ECMs), and, depending on the ECM type used, presented a high level of plasticity in morphology and gene expression.     

Kinetic study of catalytic CO(2) hydration by water-soluble model compound of carbonic anhydrase and anion inhibition effect on CO(2) hydration

A kinetic study of CO(2) hydration was carried out using the water-soluble zinc model complex with water-soluble nitrilotris(2-benzimidazolylmethyl-6-sulfonate) L1S, [L1SZn(OH(2))](-), mimicking the active site of carbonic anhydrase, in the presence and absence of anion inhibitors NCS(-) and Cl(-). The obtained rate constants k(cat) for CO(2) hydration were 5.9x10(2), 1. 7x10(3), and 3.1x10(3) M(-1) s(-1) at 5, 10, and 15 degrees C, respectively: the k(cat)=ca. 10(4) M(-1) s(-1) extrapolated towards 25 degrees C has been the largest among the reported k(cat) using zinc model complexes for carbonic anhydrase. It was also revealed that NCS(-), Cl(-) and acetazolamide play a role of inhibitors by the decrease of k(cat): 7x10(2) and 2x10(3) M(-1) s(-1) for NCS(-) and Cl(-) at 15 degrees C, respectively. The sequence of their magnitudes in k(cat) is Cl(-) approximately acetazolamide>NCS(-), where the sequence Cl(-)>NCS(-) is confirmed for native carbonic anhydrase. The difference of k(cat) or k(obs) between NCS(-) and Cl(-) resulted from that between the stability constants K(st)=2x10(3) for [L1SZn(NCS)](2-) and 1x10(2) M(-1) for [L1SZnCl](2-) in D(2)O: for water-insoluble tris(2-benzimidazolylmethyl)amine L1, K(st)=1.8x10(4) for [L1Zn(NCS)](2-) and 1.5x10(3) M(-1) for [L1ZnCl](2-)in CD(3)CN/D(2)O (50% v/v). The crystal structure of anion-binding zinc model complexes [L1Zn(OH(2))](0.5)[L1ZnCl](0.5) (ClO(4))(1.5) 1(0.5)2(0.5)(ClO(4))(1.5) was revealed by X-ray crystallography. The geometry around Zn(2+) in 1 and 2 was tetrahedrally coordinated by three benzimidazolyl nitrogen atoms and one oxygen atom of H(2)O, or Cl(-).     

Blazeispirols B, C, E and F, des-A-ergostane-type compounds, from the cultured mycelia of the fungus Agaricus blazei

Four new des-A-ergostane derivatives including blazeispirols B, C, E and F were isolated from the cultured mycelia of fungus Agaricus blazei Murill and were established to be (20S, 22R, 23R, 24S)-14beta,22: 22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9,11-tetraen-23-ol; (20S, 22S, 23R, 24S)-14beta,22: 22,25-diepoxy-5-methoxy-des-A-ergosta-5,7,9-trien-23-ol; (20S, 22S, 23R, 24S)-14beta, 22: 22, 25-diepoxy-5-methoxy-des-A-ergosta-5,7,9,11-tetraene-19,23-diol and (20S, 22S, 23R, 24S)-14beta,22: 22,25-diepoxy-des-A-ergosta-5,7,9-triene-5,23-diol by comparison of extensive 1D and 2D NMR spectral data with that of blazeispirol A.     

Postischemic Reperfusion Induces α-Fodrin Proteolysis by m-Calpain in the Synaptosome and Nucleus in Rat Brain

Abstract: A membrane cytoskeletal protein, fodrin, is a substrate for a Ca²⁺-dependent protease, calpain. It remains unknown whether μ-calpain or m-calpain is involved in the proteolysis of either α- or β-fodrin and in what subcellular localization during ischemia and reperfusion of the brain. To address these issues, we examined the distribution of fodrin and calpain and the activities of calpain and calpastatin (endogenous calpain inhibitor) in the same subcellular fractions. Rat forebrain was subjected to ischemia by a combination of occlusion of both carotid arteries and systemic hypotension, whereas reperfusion was induced by releasing the occlusion. Immunoblotting, activity measurement, and casein zymography did not detect the presence of μ-calpain or a significant change of m-calpain level after ischemia or reperfusion. However, casein zymography revealed a unique Ca²⁺-dependent protease that was eluted with both 0.18 and 0.40 M NaCl from a DEAE-cellulose column. α- and β-fodrins and m-calpain were found to be rich in the synaptosomal, nuclear, and cytosolic subfractions by immunoblotting analysis. Reperfusion (60 min) following ischemia (30 min) induced selective proteolysis of α-fodrin, which was inhibited by a calpain inhibitor, acetylleucylleucylnorleucinal (400 µ M , 1 ml, i.v.). The μ-calpain-specific fragment of β-fodrin was not generated during ischemia-reperfusion, supporting the possibility of the involvement of m-calpain rather than μ-calpain in the α-fodrin proteolysis.     

Phylogeny and divergence time estimation of the genus Didymodon (Pottiaceae) based on nuclear and chloroplast markers

Didymodon Hedw., with approximately 140 species in the family Pottiaceae, is distributed nearly throughout the world, with the greatest diversity and important ecological functions in drought lands and alpine ecosystems. Several studies involving morphology, molecular systematics, and macro-systematic analysis have addressed the infrageneric classification of Didymodon, but controversy over the position of the infrageneric and species classification remains due to its high degree of morphological variation in micro-habitats and strong sensitivity to climate change at regional and global scale. To date, only a few phylogenetic studies have been conducted with an incomplete number of Didymodon species; further, there is no study published regarding the divergence time of Didymodon. Consequently, we conducted a comprehensive phylogenetic analysis of Didymodon species, sampling a total of 107 species, based on one nuclear (ITS) and five chloroplast DNA. Moreover, divergence time analysis was conducted to infer the age of origin and divergence of Didymodon species. Our results presented the largest scale phylogenetic relationship of Didymodon to date and resolved the phylogenetic status of some controversial taxa and the new species. The divergence time estimation showed that Didymodon species originated around the early Cretaceous, and the diversification was concentrated in the Cretaceous and Eocene. Paleoclimate and environmental change have a direct impact on the origin and divergence of Didymodon species by shaping their morphology, resource availability and ecological niche. Our study will help understand species origin and speciation of Didymodon as well as reflecting species adaptability and experience to historical events.     

Studies on the fungal flora in the lung of human necropsy cases. A critical survey in connection with the pathogenesis of opportunistic fungus infections

An ecological study was carried out on the fungal flora in the human lung of 159 autopsy cases. Fungi were isolated from 129 cases (81.1%). Filamentous fungi consisting of 918 strains were isolated from 113 cases, and the dominant genera were Aspergillus and Penicillium. Yeasts were isolated from 58 cases, and the dominant genus appeared to be Candida.With morbid anatomical study, the incidence of the fungus isolation was examined from various points of view. It was concluded that even a healthy respiratory parenchyma of the human lung cannot be assumed as aseptic. However, only a few numbers of a few genera of air-born fungi were isolated.Special stress was laid on the exposure of the respiratory parenchyma of the human lung to air-born fungi in connection with the pathogenesis of opportunistic fungus infections.