Lifespan and reproduction in brain-specific miR-29-knockdown mouse.

The microRNA miR-29 is widely distributed and highly expressed in adult mouse brain during the mouse's lifetime. We recently created conditional mutant mice whose miR-29 was brain-specifically knocked down through overexpression of an antisense RNA transgene against miR-29. To explore a role for brain miR-29 in maximizing organismal fitness, we assessed somatic growth, reproduction, and lifespan in the miR-29-knockdown (KD) mice and their wild-type (WT) littermates. The KD mice were developmentally indistinguishable from WT mice with respect to gross morphology and physical activity. Fertility testing revealed that KD males were subfertile, whereas KD females were hyperfertile, only in terms of reproductive success, when compared to their gender-matched WT correspondents. Another phenotypic difference between KD and WT animals appeared in their lifespan data; KD males displayed an overall increasing tendency in post-reproductive survival relative to WT males. In contrast, KD females were prone to shorter lifespans than WT females. These results clarify that brain-targeted miR-29 knockdown affects both lifespan and reproduction in a gender-dependent manner, and moreover that the reciprocal responsiveness to the miR-29 knockdown between these two phenotypes in both genders closely follow life-course models based on the classical trade-off prediction wherein elaborate early-life energetic investment in reproduction entails accelerated late-life declines in survival, and vice versa. Thus, this study identified miR-29 as the first mammalian miRNA that is directly implicated in the lifetime trade-off between the two major fitness components, lifespan and reproduction.

Possible role of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in growth promotion of Arabidopsis seedlings by low levels of selenium.

We explored functional significance of selenium (Se) in Arabidopsis physiology. Se at very low concentrations in cultivation exerted a considerable positive effect on Arabidopsis growth with no indication of oxidative stress, whereas Se at higher concentrations significantly suppressed the growth and brought serious oxidative damage. Respiration, ATP levels, and the activity of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NAD-GAPDH) were enhanced in Arabidopsis grown in the medium containing 1.0 µM Se. Addition of an inhibitor of glutathione (GSH) synthesis to the medium abolished both of the Se-dependent growth promotion and NAD-GAPDH up-regulation. Assay of NAD-GAPDH purified from seedlings subjected to Se interventions raised the possibility of a direct connection between the activity of this enzyme and Arabidopsis growth. These results reveal that trace amounts of Se accelerate Arabidopsis growth, and suggest that this pro-growth effect of Se arises enhancing mitochondrial performance in a GSH-dependent manner, in which NAD-GAPDH may serve as a key regulator.

Post-translational activation of non-selenium glutathione peroxidase of Chlamydomonas reinhardtii by specific incorporation of selenium.

Glutathione peroxidase (GPX) plays a pivotal role in the protection of cells against oxidative damage. The green alga Chlamydomonas reinhardtii expresses both selenocysteine-containing GPX and the non-selenium GPX homolog (GPXH). We previously reported that supplementation of selenium to algal culture induces GPXH to exhibit GPX activity. Here we investigated the incorporation of selenium into GPXH and its causal relationship with the upregulation of the enzymatic activity. GPXH was purified from algal cells grown with selenium and proteolytically digested into four fragments. Selenium content analysis for these proteolytic fragments confirmed that GPXH-incorporated selenium is predominantly enriched in a fragment that carries the putative catalytic residue Cys-38. We next constructed three kinds of engineered GPXH proteins by substituting Ser for one of three Cys residues in native GPXH, Cys-38, -66, and -84, using a bacterial overexpression system, resulting in Cys38Ser, Cys66Ser, and Cys84Ser derivatives, respectively. Of these, the Cys66Ser and Cys84Ser derivatives exhibited the same level of selenium-dependent GPX activity as the normal recombinant GPXH, whereas the Cys38Ser mutant GPXH not only lost its activity completely but also demonstrated severely impaired incorporation of selenium. These findings strongly suggest that selenium is post-translationally assimilated into the Cys-38 of the GPXH protein, thereby enhancing its enzymatic activity.

The combination of strong immunohistochemical mtTFA expression and a high survivin index predicts a shorter disease-specific survival in pancreatic ductal adenocarcinoma.

Mitochondrial transcription factor A (mtTFA) plays a crucial role in both the transcription and maintenance of mitochondrial DNA. A high expression of mtTFA has been demonstrated in several solid tumors, and is closely associated with cancer cell survival/apoptosis and growth. However, its expression pattern in pancreatic ductal adenocarcinoma (PAC) remains to be elucidated. Additionally, our groups have recently revealed that a subset of apoptosis-related genes is strongly regulated by mtTFA, and that two putative mtTFA binding sites are present in the promoter region of the survivin gene, which is a member of the inhibitor-of-apoptosis protein family. We therefore investigated the correlation of the immunohistochemical mtTFA expression and the survivin index with various clinicopathological variables and the prognosis, using 70 paraffin-embedded tumor samples from patients with surgically-resected PAC. The mtTFA expression or survivin index was considered to be strong or high when ≥30% or 10% of the PAC cells showed positive staining, respectively. Strong mtTFA expression and/or a high survivin index was revealed to have a significant relationship to a pathologically high tumor grading and advanced tumor stage. Moreover, mtTFA showed significantly high co-expression with survivin. Univariate and multivariate analyses demonstrated that both the strong mtTFA expression and high survivin index groups had significantly shorter survival rates, especially within the first two years postoperatively. The combination of strong mtTFA expression and a high survivin index may predict a poor prognosis in patients with PAC, and these new biomarkers might offer useful information for the early clinical management.

The combination of a nuclear HMGB1-positive and HMGB2-negative expression is potentially associated with a shortened survival in patients with pancreatic ductal adenocarcinoma.

High-mobility group box (HMGB) proteins are ubiquitous, abundant nuclear non-histone chromosomal proteins that play a critical role in binding to distorted DNA structures and subsequently regulating DNA transcription, replication, repair, and recombination. Both HMGB1 and HMGB2 exhibit a high expression in several human cancers and are closely associated with tumor progression and a poor prognosis. However, the expression patterns of these molecules in pancreatic ductal adenocarcinoma (PDAC) remain to be elucidated. As most cases of postoperative relapse of PDAC occur within the first 2 years, the clinical significance of accurate biomarkers is needed. Therefore, we investigated the correlation between the immunohistochemical HMGB1 and HMGB2 expression and the clinicopathological characteristics and prognosis using 62 paraffin-embedded tumor samples obtained from patients with surgically resected PDAC. The HMGB1/2 expression was considered to be positive when 10 % or more of the cancer cells showed positive nuclear, not merely cytoplasmic, staining. Consequently, the expression of HMGB1/2 was observed in 54 (87.1 %) and 31 (50.0 %) patients, respectively. Unexpectedly, a positive HMGB1 expression was found to have a significantly close relationship with a negative HMGB2 expression. The univariate and multivariate analyses demonstrated that the patients with a HMGB1+ and HMGB2- status had markedly lower disease-specific survival rates, especially within the first 2 years postoperatively, whereas those with a HMGB1+ status alone did not. Therefore, the combination of a HMGB1+ and HMGB2- expression potentially predicts a poor prognosis in patients with PDAC, and these new biomarkers may be useful parameters for clinical management in the early postoperative phase.

Comparison of three Chlamydomonas strains which show distinctive oxidative stress tolerance.

Methyl viologen (MV) causes severe oxidative stress by generating superoxide in the photosystem. The marine Chlamydomonas strain W80 is highly tolerant to MV (inhibitory concentration 50% [IC50]=110 µM), and another marine Chlamydomonas strain HS5 shows also relatively a high tolerance (IC50=12 µM). These two marine strains and a freshwater Chlamydomonas reinhardtii, which is highly sensitive to MV (IC50=0.03 µM), were compared with respect to their reactive oxygen species (ROS) eliminating enzymes (superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase), intracellular free amino acids, and antioxidant activities of the cell extracts. The marked difference between the marine Chlamydomonas strains and C. reinhardtii is the much higher (more than 5 fold) ascorbate peroxidase (APX) activity in the marine strains. The marine strains also kept the high APX activities (more than 100% of non-stressed condition) under the MV stressed condition, while the APX activity in C. reinhardtii was significantly decreased (36% of non-stressed condition) under the stressed condition, indicating that APX activity potentially contributes to the oxidative stress tolerance in Chlamydomonas. In addition, the levels of intracellular free proline, which is supposed to ameliorate oxidative stress, were several tens of times higher in the marine Chlamydomonas strains than in C. reinhardtii.

The contribution of Arabidopsis homologs of L-gulono-1,4-lactone oxidase to the biosynthesis of ascorbic acid.

To clarify the involvement of seven Arabidopsis homologs of rat L-gulono-1,4-lactone (L-GulL) oxidase, AtGulLOs, in the biosynthesis of L-ascorbic acid (AsA), transgenic tobacco cells overexpressing the various AtGulLOs were generated. Under treatment with L-GulL, the levels of total AsA in three transgenic tobacco cell lines, overexpressing AtGulLO2, 3, or 5, were significantly increased as compared with those in control cells.

Hydroperoxide reduction by thioredoxin-specific glutathione peroxidase isoenzymes of Arabidopsis thaliana.

Arabidopsis thaliana contains eight glutathione peroxidase (GPX) homologs (AtGPX1-8). Four mature GPX isoenzymes with different subcellular distributions, AtGPX1, -2, -5 and -6, were overexpressed in Escherichia coli and characterized. Interestingly, these recombinant proteins were able to reduce H2O2, cumene hydroperoxide, phosphatidylcholine and linoleic acid hydroperoxides using thioredoxin but not glutathione or NADPH as an electron donor. The reduction activities of the recombinant proteins with H2O2 were 2-7 times higher than those with cumene hydroperoxide. Km values for thioredoxin and H2O2 were 2.2-4.0 and 14.0-25.4 microM, respectively. These finding suggest that GPX isoenzymes may function to detoxify H2O2 and organic hydroperoxides using thioredoxin in vivo and may also be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP balance.

Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate.

We have studied the enzymological properties of L-galactose dehydrogenase (l-GalDH), a key enzyme in the biosynthetic pathway of l-ascorbate (AsA) in plants. L-GalDH was purified approximately 560-fold from spinach leaves. The enzyme was a homodimer with a subunit mass of 36 kDa. We also cloned the full-length cDNA of spinach L-GalDH, which contained an open reading frame encoding 322 amino acid residues with a calculated molecular mass of 35,261 Da. The deduced amino acid sequence of the cDNA showed 82, 79 and 75% homology to L-GalDH from kiwifruit, apple and Arabidopsis, respectively. Recombinant enzyme expressed from the cDNA in Escherichia coli showed L-GalDH activity. Southern blot analysis revealed that the spinach L-GalDH gene occurs in a single copy. Northern blot analysis suggests that L-GalDH is expressed in different organs of spinach. The purified native L-GalDH showed high specificity for L-galactose with a Km of 116.2+/-3.2 microM. Interestingly, spinach L-GalDH exhibited reversible inhibition by AsA, the end-product of the biosynthetic pathway. The inhibition kinetics indicated a linear-competitive inhibition with a Ki of 133.2+/-7.2 microM, suggesting feedback regulation in AsA synthesis in the plant.

Induction and functional analysis of two reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione peroxidase-like proteins in Synechocystis PCC 6803 during the progression of oxidative stress.

Synechocystis PCC 6803 contains two types of glutathione peroxidase-like proteins (GPX-1 and GPX-2) that utilize NADPH but not reduced glutathione and unsaturated fatty acid hydroperoxides or alkyl hydroperoxides. The steady-state transcript level of gpx-1 gradually increased under oxidative stress conditions imposed by high light intensity, high salinity, or application of methylviologen or t-butyl hydroperoxide in the wild-type and GPX-2 knock-out mutant (gpx-2Delta) cells. To examine the ability of GPX-1, GPX-2, and thioredoxin peroxidase to scavenge lipid hydroperoxide in vivo, we measured the photosynthetic evolution of O(2) and the level of lipid peroxidation in the wild-type and each type of mutant cell after the application of t-butyl hydroperoxide or H(2)O(2). The data reported here indicate that GPX-1 and GPX-2 are essential for the removal of lipid hydroperoxides under normal and stress conditions, leading to the protection of membrane integrity.

Enhancement of stress tolerance in transgenic tobacco plants overexpressing Chlamydomonas glutathione peroxidase in chloroplasts or cytosol.

To evaluate the physiological potential of the defense system against hydroperoxidation of membrane-lipid components caused by environmental stresses in higher plants, we generated transgenic tobacco plants expressing a glutathione peroxidase (GPX)-like protein in the cytosol (TcGPX) or chloroplasts (TpGPX). The activities toward alpha-linolenic acid hydroperoxide in TcGPX and TpGPX plants were 47.5-75.3 and 32.7-42.1 nM min(-1) mg(-1) protein, respectively, while no activity was detected in wild-type plants. The transgenic plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV: 50 microM) under moderate light intensity (200 micro E m(-2) sec(-1)), chilling stress under high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)), or salt stress (250 mM NaCl). Under these stresses, the lipid hydroperoxidation (the production of malondialdehyde (MDA)) of the leaves of TcGPX and TpGPX plants was clearly suppressed compared with that of wild-type plants. Furthermore, the capacity of the photosynthetic and antioxidative systems in the transgenic plants remained higher than those of wild-type plants under chilling or salt stress. These results clearly indicate that a high level of GPX-like protein in tobacco plants functions to remove unsaturated fatty acid hydroperoxides generated in cellular membranes under stress conditions, leading to the maintenance of membrane integrity and increased tolerance to oxidative stress caused by various stress conditions.

Molecular characterization of glutathione peroxidase-like protein in halotolerant Chlamydomonas sp. W80.

A cDNA clone encoding a glutathione peroxidase (GPX)-like protein was isolated from the cDNA library from halotolerant Chlamydomonas W80 (C. W80) by a simple screening method based on the bacterial expression system. The cDNA clone contained an open reading frame encoding a mature protein of 163 amino acids with a calculated molecular mass of 18 267 Da. No potential signal peptide was found. The deduced amino acid sequence of the cDNA showed 40-63% and 37-46% homology to those of GPX-like proteins from higher plants and mammalian GPXs, respectively. The C. W80 GPX-like protein contained a normal cysteine residue instead of a selenocysteine at the catalytic site. However, it contained amino acid residues (glutamine and tryptophan) that are involved in three protein loops and are important for the catalytic activity in the mammalian GPX. Interestingly, the native and recombinant GPX-like proteins showed activities towards unsaturated fatty acid hydroperoxides, but not towards either H2O2 or phospholipid hydroperoxide. Transformed E. coli cells expressing the C. W80 GPX-like protein showed enhanced tolerance to 5% NaCl or 0.2 mM paraquat treatments. Accession number: The nucleotide sequence data reported have been submitted to the DDBJ, EMBL, and GenBank nucleotide sequence databases with the following accession number AB009083.

Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress.

To evaluate the physiological importance of thylakoid membrane-bound ascorbate peroxidase (tAPX) in the active oxygen species-scavenging system of chloroplasts, the level of tAPX in tobacco plants was altered by expression of the tAPX cDNA in both sense and antisense orientation. The tobacco plants transformed with constructs of antisense tAPXs from spinach and tobacco could not be obtained, suggesting that the suppression of tAPX in higher plants had a severe effect on the growth even under normal conditions. In contrast, the transgenic tobacco plants (TpTAP-12) overexpressing tAPX, which had approximately 37-fold higher activity than that of the wild-type plants, were generated. The TpTAP-12 plants showed increased tolerance to oxidative stress caused by application of methylviologen (MV, 50 microm) under light intensity (300 and 1600 microE m(-2) sec(-1)) and by chilling stress with high light intensity (4 degrees C, 1000 microE m(-2) sec(-1)). At 24 h after the MV treatment under illumination at 300 microE m-2 sec-1, destruction of chlorophyll was observed in the wild-type plants, but not in the TpTAP-12 plants. The activities of thiol-modulated enzymes in the Calvin cycle, the level and redox status of ascorbate (AsA), and the activity of tAPX in the wild-type plants significantly decreased, while those in the TpTAP-12 plants were hardly changed. These observations suggest that tAPX is a limiting factor of antioxidative systems under photo-oxidative stress in chloroplasts, and that the enhanced activity of tAPX functions to maintain the AsA content and the redox status of AsA under stress conditions.

Regulation and function of ascorbate peroxidase isoenzymes.

Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.

Crystallization and preliminary X-ray diffraction analysis of chloroplastic ascorbate peroxidase of tobacco plants.

The stromal ascorbate peroxidase of tobacco plants was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 37.2, b = 76.8, c = 98.8 A. The calculated V(M) value based on a monomer in the asymmetric unit is 2.2 A(3)Da(-1). A data set was successfully collected to 1.6 A resolution from a frozen crystal using synchrotron radiation of wavelength 1.0 A at KEK-PF, Japan.