Effect of hydrogen peroxide on intracellular pH in the human atrial myocardium

The cardiac injury observed during myocardial ischemia and reperfusion has been shown to be a consequence of a complex mechanism in which the accumulation of hydrogen peroxide (H2O2) and other oxygen free radicals (OFRs), and intracellular pH (pHi) are believed to play a major role. However, the effect of H2O2 on pHi has not been well characterized in the human atrial myocardium. In the present study, we superfused hydrogen peroxide into the human atrial tissue in order to assess the effects of oxygen free radicals on the pHi, and, furthermore, to test the ability of certain potential cardioprotective agents, including scavengers of the *OH free radical (N-(mercaptopropionyl)-glycine; N-MPG) and the HOCl free radical (L-methionine), to protect against oxidative-induced pHi challenge. The human atrial tissues were obtained from patients undergoing corrective open-heart surgery. The ratiometric recordings of pHi were measured using the pH-sensitive, dual-excitation and dual-emission fluorescent dye BCECF (2', 7'-bis(carboxyethyl)-5, 6-carboxyfluorescein acetoxymethyl ester). By continuously monitoring pHi changes in human atrial myocardium, we have found, for the first time, that (a) H2O2 (30 microM-3 mM) induced a significant dose-dependent intracellular acidosis, (b) N-MPG caused a significant block on the intracellular acidosis induced by 3 mM H2O2, whereas L-methionine did not, and (c) Hoe 694, a specific Na+/H+ exchanger (NHE) inhibitor, caused a similar extents like that induced by 3 mM H2O2. Our data suggest that the effects of H2O2 are caused mainly through the generation of *OH, which is attributed to the intracellular acidosis seen in the human atrial trabecular muscle. The possible underlying mechanism for H2O2-induced acidosis is likely due to its inhibition on the activity of NHE and other acid extruders, as the pHi changes after H2O2 exposure could be detected even though the activity of NHE was completely blocked by 30 mM Hoe 694.     

Resistance of cultivated tomato to cell content-feeding herbivores is regulated by the octadecanoid-signaling pathway

The octadecanoid signaling pathway has been shown to play an important role in plant defense against various chewing insects and some pathogenic fungi. Here, we examined the interaction of a cell-content feeding arachnid herbivore, the two-spotted spider mite (Tetranychus urticae Koch), with cultivated tomato (Lycopersicon esculentum) and an isogenic mutant line (defenseless-1 [def-1]) that is deficient in the biosynthesis of the octadecanoid pathway-derived signal, jasmonic acid (JA). Spider mite feeding and fecundity on def-1 plants was significantly greater than on wild-type plants. Decreased resistance of def-1 plants was correlated with reduced JA accumulation and expression of defensive proteinase inhibitor (PI) genes, which were induced in mite-damaged wild-type leaves. Treatment of def-1 plants with methyl-JA restored resistance to spider mite feeding and reduced the fecundity of female mites. Plants expressing a 35S::prosystemin transgene that constitutively activates the octadecanoid pathway in a Def-1-dependent manner were highly resistant to attack by spider mites and western flower thrips (Frankliniella occidentalis), another cell-content feeder of economic importance. These findings indicate that activation of the octadecanoid signaling pathway promotes resistance of tomato to a broad spectrum of herbivores. The techniques of amplified fragment length polymorphism (AFLP) and bulk segregant analysis were used to map the Def-1 gene to a region on the long arm of chromosome 3 that is genetically separable from the map position of known JA biosynthetic genes. Tight linkage of Def-1 to a T-DNA insertion harboring the maize (Zea mays) Dissociation transposable element suggests a strategy for directed transposon tagging of the gene.     

Variability of Wing Size and Shape in Three Populations of a Recent Brazilian Invader, Zaprionus indianus (Diptera: Drosophilidae), from Different Habitats

Zaprionus indianus (Diptera: Drosophilidae) is an African species that was introduced in Brazil near the end of the 1990’s decade. To evaluate the adaptive potential of morphological traits in natural populations of this recently introduced species, we have investigated wing size and shape variation at Rio de Janeiro populations only two years after the first record of Z. indianus in Brazil. Significant genetic differences among populations from three distinct ecological habitats were detected. The heritability and evolvability estimates show that, even with the population bottleneck that should have occurred during the invasion event, an appreciable amount of additive genetic variation for wing size and shape was retained. Our results also indicated a greater influence of environmental variation on wing size than on wing shape. The importance of quantitative genetic variability and plasticity in the successful establishment and dispersal of Z. indianus in the Brazilian territory is then discussed.     

Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis

We compared secretion kinetics for four different fluorescent cargo proteins, each targeted to the lumen of insulin secretory vesicles. Upon stimulation, individual vesicles displayed one of four distinct patterns of fluorescence change: i), disappearance, ii), dimming, iii), transient brightening, or iv), persistent brightening. For each fusion protein, a different pattern of fluorescence change dominated. Furthermore, we demonstrated that the dominant pattern depends upon both i), the specific choice of fluorescent protein, and ii), the sequence of amino acids linking the cargo protein to the fluorescent protein. Thus, in beta-cells, experiments involving fluorescent cargo proteins for the study of exocytosis must be interpreted carefully, as design of a fluorescent cargo protein determines secretion kinetics at exocytosis.     

The "impact factor" revisited

The number of scientific journals has become so large that individuals, institutions and institutional libraries cannot completely store their physical content. In order to prioritize the choice of quality information sources, librarians and scientists are in need of reliable decision aids. The "impact factor" (IF) is the most commonly used assessment aid for deciding which journals should receive a scholarly submission or attention from research readership. It is also an often misunderstood tool. This narrative review explains how the IF is calculated, how bias is introduced into the calculation, which questions the IF can or cannot answer, and how different professional groups can benefit from IF use.     

Induced mutations in cassava using somatic embryos and the identification of mutant plants with altered starch yield and composition

A cyclic somatic embryogenic system was used to induce mutations in cassava variety PRC 60a in vitro. Globular-stage somatic embryos were selected as suitable experimental materials, and 50 Gy of -rays was determined to be the optimal dose for inducing mutations. During subsequent field trials, more than 50% of the regenerated mutant lines varied morphologically from wild-type plants. Consequently, we used this approach to induce genetic variability for obtaining novel cassava cultivars. Among the different mutant lines obtained, lines S14 and S15 showed large morphological variations. In 10-month-old S14 and S15 mutant lines, storage root yield was reduced 17-fold and 60-fold, respectively, compared to wild-type plants, while the storage roots of S15 mutant plants also exhibited an almost 50% decrease in starch content and a significant reduction (30%) in amylose content. These two features were observed throughout the different developmental stages of the storage roots in S15 plants.Abbreviations BA 6-Benzylaminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid - EMS Ethylmethanesulfonate - IBA Indole-3-butyric acid     

An increase in side chain entropy facilitates effector binding: NMR characterization of the side chain methyl group dynamics in Cdc42Hs

Cdc42Hs is a signal transduction protein that is involved in cytoskeletal growth and organization. We describe here the methyl side chain dynamics of three forms of (2)H,(13)C,(15)N-Cdc42Hs [GDP-bound (inactive), GMPPCP-bound (active), and GMPPCP/PBD46-bound (effector-bound)] from (13)C-(1)H NMR measurements of deuterium T(1) and T(1 rho) relaxation times. A wide variation in flexibility was observed throughout the protein, with methyl axis order parameters (S(2)(axis)) ranging from 0.2 to 0.4 (highly disordered) in regions near the PBD46 binding site to 0.8--1.0 (highly ordered) in some helices. The side chain dynamics of the GDP and GMPPCP forms are similar, with methyl groups on the PBD46 binding surface experiencing significantly greater mobility (lower S(2)(axis)) than those not on the binding surface. Binding of PBD46 results in a significant increase in the disorder and a corresponding increase in entropy for the majority of methyl groups. Many of the methyl groups that experience an increase in mobility are found in residues that are not part of the PBD46 binding interface. This entropy gain represents a favorable contribution to the overall entropy of effector binding and partially offsets unfavorable entropy losses such as those that occur in the backbone.     

An intronic Ikaros-binding element mediates retinoic acid suppression of the kappa opioid receptor gene, accompanied by histone deacetylation on the promoters

The mouse kappa opioid receptor (KOR) gene is constitutively expressed in mouse embryonal carcinoma P19 stem cells and suppressed by retinoic acid (RA) in cells undergoing neuronal differentiation. A negative regulatory element is located within intron 1 of the KOR gene, which contains an Ikaros (Ik)-binding site (GGGAAgGGGAT). This sequence is an Ik-1 respondive, functionally negative element as demonstrated in the context of both natural KOR and heterologous promoters. The two underlined G residues of the second half-site are critical for Ik-1 binding and Ik-mediated repression of the KOR gene. RA induces Ik-1 expression within 1 day of treatment and suppresses KOR expression between 2 and 3 days. Overexpression of Ik-1 in P19 suppresses endogenous KOR gene expression, accompanied by increased binding of Ik-1 to the Ik-binding site and chromatin histone deacetylation on KOR promoters. It is proposed that in an RA-induced P19 differentiation model, RA elevates Ik-1 expression, which recruits histone deacetylase to intron 1 of the KOR gene and silences KOR gene promoters.     

Mutational analysis of the MutH protein from Escherichia coli

Site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA-binding residues; to determine whether MutH has the same mechanism for DNA binding and catalysis as PvuII; and to localize the residues responsible for MutH stimulation by MutL. No DNA-binding residues were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. Two histidines in MutH are in a similar position as two histidines (His-84 and His-85) in PvuII that signal for DNA binding and catalysis. These MutH histidines (His-112 and His-115) were changed to alanines, but the mutant proteins had wild-type activity both in vivo and in vitro. The results indicate that the MutH signal for DNA binding and catalysis remains unknown. Instead, a lysine residue (Lys-48) was found in the first flexible loop that functions in catalysis together with the three presumed catalytic amino acids (Asp-70, Glu-77, and Lys-79). Two deletion mutations (MutHDelta224 and MutHDelta214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala-220, Leu-221, Leu-222, Ala-223, and Arg-224).     

Differential expression of two soybean apyrases, one of which is an early nodulin

Two cDNA clones were isolated from soybean (Glycine soja) by polymerase chain reaction with primers designed to conserved motifs found in apyrases (nucleotide phosphohydrolase). The two cDNAs are predicted to encode for two, distinct, apyrase proteins of approximately 50 kDa (i.e., GS50) and 52 kDa (i.e., GS52). Phylogenetic analysis indicated that GS52 is orthologous to a family of apyrases recently suggested to play a role in legume nodulation. GS50 is paralogous to this family and, therefore, likely plays a different physiological role. Consistent with this analysis, GS50 mRNA was detected in root, hypocotyls, flowers, and stems, while GS52 mRNA was found in root and flowers. Neither gene was expressed in leaves or cotyledons. Inoculation of roots with Bradyrhizobium japonicum, nitrogen-fixing symbiont of soybean, resulted in the rapid (<6 h) induction of GS52 mRNA expression. The level of GS50 mRNA expression was not affected by bacterial inoculation. Western blot (immunoblot) analysis of GS50 expression mirrored the results obtained by mRNA analysis. However, in contrast to the mRNA results, GS52 protein was found in stems. Interestingly, anti-GS52 antibody recognized a 50-kDa protein found only in nodule extracts. Treatment of roots with anti-GS52 antibody, but not anti-GS50 antibody or preimmune serum, blocked nodulation by B. japonicum. Fractionation of cellular membranes in sucrose density gradients and subsequent Western analysis of the fractions revealed that GS50 colocalized with marker enzymes for the Golgi, while GS52 colocalized with marker enzymes for the plasma membrane. Restriction fragment length polymorphism (RFLP)-based mapping placed the gs52 gene on major linkage group J of the integrated genetic map of soybean. These data suggest that GS50 is likely an endo-apyrase involved in Golgi function, while GS52 is localized on the root surface and appears to play an important role in nodulation.