Organ-specific distribution and subcellular localisation of ascorbate peroxidase isoenzymes in potato (Solanum tuberosum L.) plants

Summary. Ascorbate peroxidase (EC 1.11.1.11), a heme-containing homodimeric protein, is a hydrogen peroxide-scavenging enzyme, playing an important role in plants in order to protect them from oxidative stress, thus adverting cellular damage. Several ascorbate peroxidase isoenzymes have been reported but the understanding of their physiological role still depends on a better knowledge of their precise localisation within plant organs. Immunocytochemistry techniques were performed in order to elucidate the peroxisomal and cytosolic ascorbate peroxidase distribution within tissues of leaves and sprouts of potato plants. The peroxisomal isoenzyme was found to have a broad distribution in sprouts, but a differential one in leaves, being restricted to the spongy parenchyma. This differential expression may be associated to the mesophyll asymmetry and the diverse physiological processes that occur in it. The cytosolic isoenzyme was not detected in leaves under the used conditions, probably because it is present in low amounts in these tissues. The results obtained in sprouts were at least curious: cytosolic ascorbate was found to be adjacent to the amyloplasts. Given these results, it is possible to state that apart from their similarity, these two isoenzymes reside in different organelles and seem to take part in different physiological processes as suggested by their organ- and tissue-specific distribution. Correspondence and reprints: Plant Functional Biology Department, Institute for Cell and Molecular Biology, University of Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Effect of the Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts against the stannous chloride oxidative damage in Escherichia coli

Stannous ion has been used in different sectors of human interest, such as in food industry and in health sciences. Much is known about stannous chloride (SnCl(2)) toxicity, although, there is no general agreement regarding its genotoxicity. Cymbopogon citratus, Maytenus ilicifolia and Baccharis genistelloides extracts have been used in popular medicine. We evaluated the influence of these crude extracts on the survival of the Escherichia coli wild type (AB 1157) strain submitted to SnCl(2) treatment. Reactive oxygen species (ROS) can be generated by a Fenton like reaction induced by SnCl(2). E. coli culture was treated simultaneously with SnCl(2) and a specific extract. Our results showed a reduction of the SnCl(2) effect on the survival of the cultures in presence of the crude extracts. The extract of M. ilicifolia showed the highest level of protection action against the SnCl(2) effect in comparison with the other extracts. This protector effect could due to the redox properties of these crude extracts. The compounds in the crude extracts could (i) chelate stannous ions, protecting them against the oxidation and avoiding the generation of ROS, (ii) be a scavenger of the ROS generated by the SnCl(2) oxidation and/or (iii) have oxidant compounds that could oxidise the stannous ions, abolishing or reducing the SnCl(2) effect.     

Isolation and characterization of a cDNA encoding a novel member of the human regenerating protein family: Reg IV

Human Reg and Reg-related genes constitute a multi-gene family belonging to the calcium (C-type) dependent lectin superfamily. Regenerating gene family members are expressed in the proximal gastrointestinal (GI) tract and ectopically at other sites in the setting of tissue injury. By high-throughput sequence analysis of a large inflammatory bowel disease library, two cDNAs have been isolated which encode a novel member of this multigene family. Based on primary sequence homology, tissue expression profiles, and shared exon-intron junction genomic organization, we assign this gene to the regenerating gene family. Specific protein structural differences suggest that the current three regenerating gene subtypes should be expanded to four. We demonstrate that Reg IV has a highly restricted tissue expression pattern, with prominent expression in the gastrointestinal tract. Reg IV mRNA expression is significantly up-regulated by mucosal injury from active Crohn's disease or ulcerative colitis.     

Less DeltamtDNA4977 than normal in various types of tumors suggests that cancer cells are essentially free of this mutation

Levels of mtDNA(4977) deletions (DeltamtDNA(4977)) have been found to be lower in tumors than in adjacent non-tumoral tissues. In 87 cancer patients, DeltamtDNA(4977) was detected by multiplex polymerase chain reaction (PCR) amplification in 43 (49%) of the tumors and in 74 (85%) of the samples of non-tumoral tissues that were adjacent to the tumors. DeltamtDNA(4977) deletions were detected in 24% of the breast tumors, 52% of the colorectal tumors, 79% of the gastric tumors, and 40% of the head and neck tumors as compared with 77, 83, 100, and 90% of the adjacent respective non-tumoral tissues at the same DNA template dilution. Based on limiting dilution PCR of 16 tumors and their adjacent non-tumoral tissues, it was found that the amount of DeltamtDNA(4977) was 10- to 100-fold lower in the tumor than in the respective control non-tumoral tissues. Real-time PCR experiments were performed to quantify the number of DeltamtDNA(4977) deletions per cell, by determining the mitochondrial-to-nuclear DNA ratio. In all of the cases of breast, colorectal, gastric, and head and neck cancer the proportion of DeltamtDNA(4977) in tumors was lower than that of the respective non-tumoral tissue. Traces of DeltamtDNA(4977) in tumors were apparently due to contamination of tumor tissue with surrounding non-tumoral tissue, as evidenced by tumor microdissection and in situ PCR techniques, suggesting that tumors are essentially free of this mutation. Although the metabolic effect of DeltamtDNA(4977) may be minimal in normal (non-tumor) tissue, in tissue under stress, such as in tumors, even low levels of DeltamtDNA(4977) deletions may be intolerable.     

Synthesis of the racemate of (Z)-exo-alpha-bergamotenal,a pheromone component of the white-spotted spined bug,Eysarcoris parvus Uhler

The racemate of (Z)-exo-alpha-bergamotenal, a sex pheromone component of the white-spotted spined bug, was synthesized from racemic exo-alpha-bergamotene by a five-step sequence involving regioselective epoxidation and (Z)-selective Wittig olefination reactions. The 1H- and 13C-NMR spectra of the synthetic sample were identical with those of the natural material.     

The mechanism of superoxide scavenging by Archaeoglobus fulgidus neelaredoxin

Neelaredoxin is a mononuclear iron protein widespread among prokaryotic anaerobes and facultative aerobes, including human pathogens. It has superoxide scavenging activity, but the exact mechanism by which this process occurs has been controversial. In this report, we present the study of the reaction of superoxide with the reduced form of neelaredoxin from the hyperthermophilic archaeon Archaeoglobus fulgidus by pulse radiolysis. This protein reduces superoxide very efficiently (k = 1.5 x 10(9) m(-1)s(-1)), and the dismutation activity is rate-limited, in steady-state conditions, by the much slower superoxide oxidation step. These data show unambiguously that the superfamily of neelaredoxin-like proteins (including desulfoferrodoxin) presents a novel type of reactivity toward superoxide, a result of particular relevance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced by the defense cells in eukaryotes. The actual in vivo functioning of these enzymes will depend strongly on the cell redox status. Further insight on the catalytic mechanism was obtained by the detection of a transient intermediate ferric species upon oxidation of neelaredoxin by superoxide, detectable by visible spectroscopy with an absorption maximum at 610 nm, blue-shifted approximately 50 nm from the absorption of the resting ferric state. The role of the iron sixth ligand, glutamate-12, in the reactivity of neelaredoxin toward superoxide was assessed by studying two site-directed mutants: E12Q and E12V.     

Stomach content analyses of Simulium perflavum Roubaud 1906 (Diptera: Simuliidae) larvae from streams in Central Amazônia, Brazil

Stomach contents of Simulium perflavum Roubaud larvae were analyzed and compared with plankton and periphyton collected in five streams, in Central Amazonia (Manaus and Presidente Figueiredo counties), in Sep./Oct.1996 (dry season) and Feb./Mar. 1997 (rainy season). A total of 1,400 last-instar larvae were dissected; the stomach contents were analyzed using different methods: fresh and after oxidation. A total of 87 taxa (algae, diatoms and rotifers) were found in the stomach contents. In each stream, qualitative samples of plankton and periphyton were collected; these were mounted between slides and cover slips. A total of 94 taxa of plankton and 54 taxa of periphyton were collected. One species of Rotifera was present in the stomach contents, plankton and periphyton. Cluster analysis based on species composition of the organisms present in the stomach contents grouped the streams into two major groups, each belonging to a different drainage area. Correlations based on presence/absence of species of microalgae in the stomach contents, plankton and periphyton indicated significant associations (p<0.05) between stomach contents and plankton and between plankton and periphyton (z test); the Sorensen coefficient and cluster analysis corroborate the same associations.