Hesperidin protects against behavioral alterations and loss of dopaminergic neurons in 6-OHDA-lesioned mice: the role of mitochondrial dysfunction and apoptosis.

Hesperidin is a flavonoid glycoside that is frequently found in citrus fruits. Our group have demonstrated that hesperidin has neuroprotective effect in 6-hydroxydopamine (6-OHDA) model of Parkinson's disease (PD), mainly by antioxidant mechanisms. Although the pathophysiology of PD remains uncertain, a large body of evidence has demonstrated that mitochondrial dysfunction and apoptosis play a critical role in dopaminergic nigrostriatal degeneration. However, the ability of hesperidin in modulating these mechanisms has not yet been investigated. In the present study, we examined the potential of a 28-day hesperidin treatment (50 mg/kg/day, p.o.) in preventing behavioral alterations induced by 6-OHDA injection via regulating mitochondrial dysfunction, apoptosis and dopaminergic neurons in the substantia nigra pars compacta (SNpc) in C57BL/6 mice. Our results demonstrated that hesperidin treatment improved motor, olfactory and spatial memory impairments elicited by 6-OHDA injection. Moreover, hesperidin treatment attenuated the loss of dopaminergic neurons (TH+ cells) in the SNpc and the depletion of dopamine (DA) and its metabolities 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the striatum of 6-OHDA-lesioned mice. Hesperidin also protected against the inhibition of mitochondrial respiratory chain complex-I, -IV and V, the decrease of Na + -K + -ATPase activity and the increase of caspase-3 and -9 activity in the striatum. Taken together, our findings indicate that hesperidin mitigates the degeneration of dopaminergic neurons in the SNpc by preventing mitochondrial dysfunction and modulating apoptotic pathways in the striatum of 6-OHDA-treated mice, thus improving behavioral alterations. These results provide new insights on neuroprotective mechanisms of hesperidin in a relevant preclinical model of PD.

In Vitro Development and Mitochondrial Gene Expression in Brown Brocket Deer (Mazama gouazoubira) Embryos Obtained by Interspecific Somatic Cell Nuclear Transfer.

The genetic diversity of Neotropical deer is increasingly jeopardized, owing to declining population size. Thus, the formation of cryobanking of somatic cells is important for the preservation of these species using cloning. The transformation of these cells into viable embryos has been hampered by a lack of endangered species oocytes. Accordingly, the aim of this study was to produce brown brocket deer embryos by interspecific somatic cell nuclear transfer (iSCNT), using goat or cattle oocytes as cytoplasts, and to elucidate embryo mitochondrial activity by measuring the expression levels of ATP6, COX3, and ND5. Cattle embryos produced by in vitro fertilization (IVF) were used as a control. There were no differences in the development of embryos produced by traditional SCNT and iSCNT when using either the goat cytoplasts (38.4% vs. 25.0% cleaved and 40.0% vs. 50.0% morula rates, respectively) or cattle cytoplast (72.8% vs. 65.5% cleaved and 11.3% vs. 5.9% blastocyst rates, respectively). Concerning the gene expression, no significant difference was observed when goat oocytes were used as cytoplasts. However, when using cattle oocytes and 16S as a reference gene, the iSCNT upregulated COX3, when compared with SCNT group. In contrast, when GAPDH was used as a reference gene, all the evaluated genes were upregulated in the iSCNT group, when compared with the IVF group. When compared with the SCNT group, only the expression of ATP6 was statistically different. In conclusion, it was demonstrated that interspecific nuclear transfer is a potentially useful tool for conservation programs of endangered similar deer species.

Mitochondrial Pentatricopeptide Repeat Protein, EMB2794, Plays a Pivotal Role in NADH Dehydrogenase Subunit nad2 mRNA Maturation in Arabidopsis thaliana.

The Arabidopsis genome encodes >450 proteins containing the pentatricopeptide repeat (PPR) motif. The PPR proteins are classified into two groups, termed as P and P Long-Short (PLS) classes. Typically, the PLS subclass proteins are mainly involved in the RNA editing of mitochondrial and chloroplast transcripts, whereas most of the analyzed P subclass proteins have been mainly implicated in RNA metabolism, such as 5' or 3' transcript stabilization and processing, splicing and translation. Mutations of PPR genes often result in embryogenesis and altered seedling developmental defect phenotypes, but only a limited number of ppr mutants have been characterized in detail. In this report, we show that null mutations in the EMB2794 gene result in embryo arrest, due to altered splicing of nad2 transcripts in the Arabidopsis mitochondria. In angiosperms, nad2 has five exons that are transcribed individually from two mitochondrial DNA regions. Biochemical and in vivo analyses further indicate that recombinant or transgenic EMB2794 proteins bind to the nad2 pre-mRNAs in vitro as well as in vivo, suggesting a role for this protein in trans-splicing of nad2 intron 2 and possibly in the stability of the second pre-mRNA of nad2. Homozygous emb2794 lines, showing embryo-defective phenotypes, can be partially rescued by the addition of sucrose to the growth medium. Mitochondria of rescued homozygous mutant plants contain only traces of respiratory complex I, which lack the NADH-dehydrogenase activity.

Characterization of sequence variability hotspots in Cranichideae plastomes (Orchidaceae, Orchidoideae).

This study reports complete plastome sequences for six species of Neotropical Cranichideae and focuses on identification of the most variable regions (hotspots) in this group of orchids. These structure of these six plastomes is relatively conserved, exhibiting lengths ranging between 142,599 to 154,562 bp with 36.7% GC on average and exhibiting typical quadripartite arrangement (LSC, SSC and two IRs). Variation detected in the LSC/IR and SSC/IR junctions is explained by the loss of ndhF and ycf1 length variation. For the two genera of epiphytic clade in Spiranthinae, almost whole sets of the ndh-gene family were missing. Eight mutation hotspots were identified based on nucleotide diversity, sequence variability and parsimony-informative sites. Three of them (rps16-trnQ, trnT-trnL, rpl32-trnL) seem to be universal hotspots in the family, and the other five (trnG-trnR, trnR-atpA, trnP-psaJ, rpl32-infA, and rps15-ycf1) are described for the first time as orchid molecular hotspots. These regions have much more variation than all those used previously in phylogenetics of the group and offer useful plastid markers for phylogenetic, barcoding and population genetic studies. The use of whole plastomes or exclusive no-gap matrices also positioned with high support the holomycotrophic Rhizanthella among Orchidoideae plastomes in model-based analyses, showing the utility of plastomes for phylogenetic placement of this unusual genus.

miR-450a Acts as a Tumor Suppressor in Ovarian Cancer by Regulating Energy Metabolism.

Dysregulation of miRNA expression is associated with multiple diseases, including cancers, in which small RNAs can have either oncogenic or tumor suppressive functions. Here we investigated the potential tumor suppressive function of miR-450a, one of the most significantly downregulated miRNAs in ovarian cancer. RNA-seq analysis of the ovarian cancer cell line A2780 revealed that overexpression of miR-450a suppressed multiple genes involved in the epithelial-to-mesenchymal transition (EMT). Overexpression of miR-450a reduced tumor migration and invasion and increased anoikis in A2780 and SKOV-3 cell lines and reduced tumor growth in an ovarian tumor xenographic model. Combined AGO-PAR-CLIP and RNA-seq analysis identified a panel of potential miR-450a targets, of which many, including TIMMDC1, MT-ND2, ACO2, and ATP5B, regulate energetic metabolism. Following glutamine withdrawal, miR-450a overexpression decreased mitochondrial membrane potential but increased glucose uptake and viability, characteristics of less invasive ovarian cancer cell lines. In summary, we propose that miR-450a acts as a tumor suppressor in ovarian cancer cells by modulating targets associated with glutaminolysis, which leads to decreased production of lipids, amino acids, and nucleic acids, as well as inhibition of signaling pathways associated with EMT. SIGNIFICANCE: miR-450a limits the metastatic potential of ovarian cancer cells by targeting a set of mitochondrial mRNAs to reduce glycolysis and glutaminolysis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3294/F1.large.jpg.

Modulation of Mitochondrial and Epigenetic Targets by Polyphenols-rich Extract from Araucaria angustifolia in Larynx Carcinoma.

BACKGROUND: Araucaria angustifolia extract (AAE) is a polyphenol-rich extract that has gained interest as a natural anticancer agent. Recent work suggests that AAE induces oxidative damage and apoptosis through its action on decreasing complex I activity of the mitochondrial Electron Transport Chain (ETC). AIMS AND METHODS: In the present study, we aimed to further examine the specific targets by which AAE exerts proapoptotic effects in HEp-2 cancer cells. Specifically, the effect of AAE on the: 1) levels of pyruvate dehydrogenase was assessed by ELISA assay; 2) levels of mitochondrial ETC complexes, focusing on complex I at the gene transcript and protein level relevant to ROS generation was evaluated by multiplex ELISA followed by qRT-PCR and immunoblotting; 3) mitochondrial network distribution analysis was assessed by MitoTracker Red CMXRos; and 4) chemical variations on DNA was evaluated by dot-blotting in HEp-2 cells. RESULTS: Results demonstrated that AAE increased protein levels of PDH, switching energy metabolism to oxidative metabolism. Protein expression levels of complex I and III were found decreased in AAE-treated HEp-2 cells. Analyzing the subunits of complex I, changes in protein and gene transcript levels of NDUFS7 and NDUFV2 were found. Mitochondria staining after AAE incubation revealed changes in the mitochondrial network distribution. AAE was able to induce DNA hypomethylation and decreased DNA (cytosine-5)-methyltransferase 1 activity. CONCLUSION: Our data demonstrate for the first time that AAE alters expression of NDUFS7 and NDUFV2 mitochondrial subunits and induce epigenetic changes in HEp-2 cancer cells leading to a possible suppression of oncogenes.

Xenotopic expression of alternative electron transport enzymes in animal mitochondria and their impact in health and disease.

The mitochondrial respiratory chain in vertebrates and arthropods is different from that of most other eukaryotes because they lack alternative enzymes that provide electron transfer pathways additional to the oxidative phosphorylation (OXPHOS) system. However, the use of diverse experimental models, such as human cells in culture, Drosophila melanogaster and the mouse, has demonstrated that the transgenic expression of these alternative enzymes can impact positively many phenotypes associated with human mitochondrial and other cellular dysfunction, including those typically presented in complex IV deficiencies, Parkinson's, and Alzheimer's. In addition, these enzymes have recently provided extremely valuable data on how, when, and where reactive oxygen species, considered by many as "by-products" of OXPHOS, can contribute to animal longevity. It has also been shown that the expression of the alternative enzymes is thermogenic in cultured cells, causes reproductive defects in flies, and enhances the deleterious phenotype of some mitochondrial disease models. Therefore, all the reported beneficial effects must be considered with caution, as these enzymes have been proposed to be deployed in putative gene therapies to treat human diseases. Here, we present a brief review of the scientific data accumulated over the past decade that show the benefits and the risks of introducing alternative branches of the electron transport into mammalian and insect mitochondria, and we provide a perspective on the future of this research field.

The Chloroplast Genome of Utricularia reniformis Sheds Light on the Evolution of the ndh Gene Complex of Terrestrial Carnivorous Plants from the Lentibulariaceae Family.

Lentibulariaceae is the richest family of carnivorous plants spanning three genera including Pinguicula, Genlisea, and Utricularia. Utricularia is globally distributed, and, unlike Pinguicula and Genlisea, has both aquatic and terrestrial forms. In this study we present the analysis of the chloroplast (cp) genome of the terrestrial Utricularia reniformis. U. reniformis has a standard cp genome of 139,725bp, encoding a gene repertoire similar to essentially all photosynthetic organisms. However, an exclusive combination of losses and pseudogenization of the plastid NAD(P)H-dehydrogenase (ndh) gene complex were observed. Comparisons among aquatic and terrestrial forms of Pinguicula, Genlisea, and Utricularia indicate that, whereas the aquatic forms retained functional copies of the eleven ndh genes, these have been lost or truncated in terrestrial forms, suggesting that the ndh function may be dispensable in terrestrial Lentibulariaceae. Phylogenetic scenarios of the ndh gene loss and recovery among Pinguicula, Genlisea, and Utricularia to the ancestral Lentibulariaceae cladeare proposed. Interestingly, RNAseq analysis evidenced that U. reniformis cp genes are transcribed, including the truncated ndh genes, suggesting that these are not completely inactivated. In addition, potential novel RNA-editing sites were identified in at least six U. reniformis cp genes, while none were identified in the truncated ndh genes. Moreover, phylogenomic analyses support that Lentibulariaceae is monophyletic, belonging to the higher core Lamiales clade, corroborating the hypothesis that the first Utricularia lineage emerged in terrestrial habitats and then evolved to epiphytic and aquatic forms. Furthermore, several truncated cp genes were found interspersed with U. reniformis mitochondrial and nuclear genome scaffolds, indicating that as observed in other smaller plant genomes, such as Arabidopsis thaliana, and the related and carnivorous Genlisea nigrocaulis and G. hispidula, the endosymbiotic gene transfer may also shape the U. reniformis genome in a similar fashion. Overall the comparative analysis of the U. reniformis cp genome provides new insight into the ndh genes and cp genome evolution of carnivorous plants from Lentibulariaceae family.

Neuroprotective Effects of Açaí (Euterpe oleracea Mart.) against Rotenone In Vitro Exposure.

Neuropsychiatric diseases, such as bipolar disorder (BD) and schizophrenia (SCZ), have a very complex pathophysiology. Several current studies describe an association between psychiatric illness and mitochondrial dysfunction and consequent cellular modifications, including lipid, protein, and DNA damage, caused by cellular oxidative stress. Euterpe oleracea (açaí) is a powerful antioxidant fruit. Açaí is an Amazonian palm fruit primarily found in the lowlands of the Amazonian rainforest, particularly in the floodplains of the Amazon River. Given this proposed association, this study analyzed the potential in vitro neuropharmacological effect of Euterpe oleracea (açaí) extract in the modulation of mitochondrial function and oxidative metabolism. SH-SY5Y cells were treated with rotenone to induce mitochondrial complex I dysfunction and before and after we exposed the cells to açaí extract at 5 µg/mL. Treated and untreated cells were then analyzed by spectrophotometric, fluorescent, immunological, and molecular assays. The results showed that açaí extract can potentially increase protein amount and enzyme activity of mitochondrial complex I, mainly through NDUFS7 and NDUFS8 overexpression. Açaí extract was also able to decrease cell reactive oxygen species levels and lipid peroxidation. We thus suggest açaí as a potential candidate for drug development and a possible alternative BD therapy.

Phylogeography of endemic Xantus' hummingbird (Hylocharis xantusii) shows a different history of vicariance in the Baja California Peninsula.

Studies of phylogeographic patterns provide insight into the processes driving lineage divergence in a particular region. To identify the processes that caused phylogeographic breaks, it is necessary to use historical information and a set of appropriate molecular data to explain current patterns. To understand the influence of geological or ecological processes on the phylogeography of the only species of hummingbird endemic to the Baja California Peninsula, Hylocharis xantusii, mitochondrial DNA sequences of three concatenated genes (Cyt-b, COI and ND2; 2297bp in total) in 100 individuals were analyzed. The spatial analyses of genetic variation showed phylogeographic structure consisting of a north, central and south regions. According to estimated divergence times, two vicariant events are supported, permanent separation of the peninsula and formation of the Gulf of California at 5mya and temporary isolation of the southern region at the Isthmus of La Paz at 3mya. The temporal frame of genetic differentiation of intraspecific haplotypes indicates that 90% of haplotypes diverged within the last 500,000years, with a population expansion 80,000years ago. Only four haplotypes diverged ∼2.2 my and occurred in the south (Hxan_36, 38 and 45), and north (Hxan_45 and 56) regions; only haplotype 45 is shared between south and north populations. These regions also have the most recent haplotypes from 12,500 to 16,200years ago, and together with high levels of genetic diversity, we suggest two refuge areas, the Northern and Southern regions. Our results indicate that the phylogeographic pattern first results from vicariance processes, then is followed by historical and recent climate fluctuations that influenced conditions on the peninsula, and it is also related to oases distribution. This study presents the first investigation of phylogeography of the peninsular' endemic Xantus' hummingbird.

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations.

The CreBC (carbon source-responsive) two-component regulation system of Escherichia coli affects a number of functions, including intermediary carbon catabolism. The impacts of different creC mutations (a ΔcreC mutant and a mutant carrying the constitutive creC510 allele) on bacterial physiology were analyzed in glucose cultures under three oxygen availability conditions. Differences in the amounts of extracellular metabolites produced were observed in the null mutant compared to the wild-type strain and the mutant carrying creC510 and shown to be affected by oxygen availability. The ΔcreC strain secreted more formate, succinate, and acetate but less lactate under low aeration. These metabolic changes were associated with differences in AckA and LdhA activities, both of which were affected by CreC. Measurement of the NAD(P)H/NAD(P)(+) ratios showed that the creC510 strain had a more reduced intracellular redox state, while the opposite was observed for the ΔcreC mutant, particularly under intermediate oxygen availability conditions, indicating that CreC affects redox balance. The null mutant formed more succinate than the wild-type strain under both low aeration and no aeration. Overexpression of the genes encoding phosphoenolpyruvate carboxylase from E. coli and a NADH-forming formate dehydrogenase from Candida boidinii in the ΔcreC mutant further increased the yield of succinate on glucose. Interestingly, the elimination of ackA and adhE did not significantly improve the production of succinate. The diverse metabolic effects of this regulator on the central biochemical network of E. coli make it a good candidate for metabolic-engineering manipulations to enhance the formation of bioproducts, such as succinate.

Genotypic Analysis of Genes Associated with Independent Resistance and Cross-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Clinical Isolates.

Ethionamide (ETH) is an antibiotic used for the treatment of multidrug-resistant (MDR) tuberculosis (TB) (MDR-TB), and its use may be limited with the emergence of resistance in the Mycobacterium tuberculosis population. ETH resistance in M. tuberculosis is phenomenon independent or cross related when accompanied with isoniazid (INH) resistance. In most cases, resistance to INH and ETH is explained by mutations in the inhA promoter and in the following genes: katG, ethA, ethR, mshA, ndh, and inhA. We sequenced the above genes in 64 M. tuberculosis isolates (n = 57 ETH-resistant MDR-TB isolates; n = 3 ETH-susceptible MDR-TB isolates; and n = 4 fully susceptible isolates). Each isolate was tested for susceptibility to first- and second-line drugs using the agar proportion method. Mutations were observed in ETH-resistant MDR-TB isolates at the following rates: 100% in katG, 72% in ethA, 45.6% in mshA, 8.7% in ndh, and 33.3% in inhA or its promoter. Of the three ETH-susceptible MDR-TB isolates, all showed mutations in katG; one had a mutation in ethA, and another, in mshA and inhA. Finally, of the four fully susceptible isolates, two showed no detectable mutation in the studied genes, and two had mutations in mshA gene unrelated to the resistance. Mutations not previously reported were found in the ethA, mshA, katG, and ndh genes. The concordance between the phenotypic susceptibility testing to INH and ETH and the sequencing was 1 and 0.45, respectively. Among isolates exhibiting INH resistance, the high frequency of independent resistance and cross-resistance with ETH in the M. tuberculosis isolates suggests the need to confirm the susceptibility to ETH before considering it in the treatment of patients with MDR-TB.

Fumonisin B1 facilitates seizures induced by pentylenetetrazol in mice.

Fumonisin B1 (FB1) is a Fusarium spp. mycotoxin which constitutes a major public health issue because of its worldwide distribution and diversity of toxic effects.While the liver and kidney are considered the major target organs of FB1 toxicity in several species, evidence indicates that FB1 may be toxic to the brain. To further investigate the effects of FB1 on the central nervous system the present study aimed to test the hypothesis that acute FB1 exposure causes brain hyperexcitability and the potential underlying mechanisms. For these purposes, adult male C57BL/6 mice were injected with FB1 (8 mg/kg, i.p.) or its vehicle and 30 min thereafter received with a low dose of the classical convulsant pentylenetetrazol (PTZ, 30 mg/kg, i.p.) or its vehicle. After behavioral evaluation the cerebral cortex and the hippocampus were collected for analysis of Na(+),K(+)-ATPase activity, mitochondrial membrane potential (ΔΨm) and mitochondrial complex I and II activities. We found that FB1 reduced the latency for PTZ-induced myoclonic jerks and increased the number of these events. After exposure to FB1 total and α1 Na(+),K(+)-ATPase activities increased in cerebral cortex, whereas the same enzyme activities decreased in the hippocampus. Although no changes in mitochondrial complex I and II activities were found, acute exposure to FB1 increased ΔΨm in the cerebral cortex. Altogether, present results indicate that FB1 causes brain hyperexcitability in vivo, and that mitochondrial dysfunction may represent a potential underlying mechanism.

Spatio-temporal distribution of Aedes aegypti (Diptera: Culicidae) mitochondrial lineages in cities with distinct dengue incidence rates suggests complex population dynamics of the dengue vector in Colombia.

BACKGROUND: Aedes aegypti is the primary vector of the four serotypes of dengue virus (DENV1-4), Chikungunya and yellow fever virus to humans. Previous population genetic studies have revealed a particular genetic structure among the vector populations in the Americas that suggests differences in the ability to transmit DENV. In Colombia, despite its high epidemiologic importance, the genetic population structure and the phylogeographic depiction of Ae. aegypti, as well as its relationship with the epidemiologic landscapes in cities with heterogeneous incidence levels, remains unknown. We conducted a spatiotemporal analysis with the aim of determining the genetic structure and phylogeography of Colombian populations of Ae. aegypti among cities with different eco-epidemiologic characteristics with regard to DENV. METHODS/FINDINGS: Mitochondrial cytochrome oxidase C subunit 1 (COI)--NADH dehydrogenase subunit 4 (ND4) genes were sequenced and analyzed from 341 adult mosquitoes collected during 2012 and 2013 in the Colombian cities of Bello, Riohacha and Villavicencio, which exhibit low, medium and high levels of incidence of DENV, respectively. The results demonstrated a low genetic differentiation over time and a high genetic structure between the cities due to changes in the frequency of two highly supported genetic groups. The phylogeographic analyses indicated that one group (associated with West African populations) was found in all the cities throughout the sampling while the second group (associated with East African populations) was found in all the samples from Bello and in only one sampling from Riohacha. Environmental factors such as the use of chemical insecticides showed a significant correlation with decreasing genetic diversity, indicating that environmental factors affect the population structure of Ae. aegypti across time and space in these cities. CONCLUSIONS: Our results suggest that two Ae. aegypti lineages are present in Colombia; one that is widespread and related to a West African conspecific, and a second that may have been recently introduced and is related to an East African conspecific. The first lineage can be found in cities showing a high incidence of dengue fever and the use of chemical insecticides, whereas the second is present in cities showing a low incidence of dengue fever where the use of chemical insecticides is not constant. This study helps to improve our knowledge of the population structure of Ae. aegypti involved in the diversity of dengue fever epidemiology in Colombia.

Tellurite-mediated damage to the Escherichia coli NDH-dehydrogenases and terminal oxidases in aerobic conditions.

Escherichia coli exposed to tellurite shows augmented membrane lipid peroxidation and ROS content. Also, reduced thiols, protein carbonylation, [Fe-S] center dismantling, and accumulation of key metabolites occur in these bacteria. In spite of this, not much is known about tellurite effects on the E. coli electron transport chain (ETC). In this work, tellurite-mediated damage to the E. coli ETC's NADH dehydrogenases and terminal oxidases was assessed. Mutant lacking ETC components showed delayed growth, decreased oxygen consumption and increased ROS in the presence of the toxicant. Membranes from tellurite-exposed E. coli exhibited decreased oxygen consumption and dNADH/NADH dehydrogenase activity, showing an impairment of NDH-I but not of NDH-II activity. Regarding terminal oxidases, only the bo oxidase complex was affected by tellurite. When assaying NDH-I and NDH-II activity in the presence of superoxide, the NDH-I complex was preferentially damaged. The activity was partly restored in the presence of reducing agents, sulfide and Fe(2+) under anaerobic conditions, suggesting that damage affects NDH-I [4Fe-4S] centers. Finally, augmented membrane protein oxidation along with reduced oxidase activity was observed in the presence of the toxicant. Also, the increased expression of genes encoding alternative terminal oxidases probably reflects a cell's change towards anaerobic respiration when facing tellurite.

FAD binding properties of a cytosolic version of Escherichia coli NADH dehydrogenase-2.

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein. By eliminating its C-terminal region, a water soluble truncated version was obtained in our laboratory. Overall conformation of the mutant version resembles the wild-type protein. Considering these data and the fact that the mutant was obtained as an apo-protein, the truncated version is an ideal model to study the interaction between the enzyme and its cofactor. Here, the FAD binding properties of this version were characterized using far-UV circular dichroism (CD), differential scanning calorimetry (DSC), limited proteolysis, and steady-state and dynamic fluorescence spectroscopy. CD spectra, thermal unfolding and DSC profiles did not reveal any major difference in secondary structure between apo- and holo-protein. In addition, digestion site accessibility and tertiary conformation were similar for both proteins, as seen by comparable chymotryptic cleavage patterns. FAD binding to the apo-protein produced a parallel increment of both FAD fluorescence quantum yield and steady-state emission anisotropy. On the other hand, addition of FAD quenched the intrinsic fluorescence emission of the truncated protein, indicating that the flavin cofactor should be closely located to the protein Trp residues. Analysis of the steady-state and dynamic fluorescence data confirms the formation of the holo-protein with a 1:1 binding stoichiometry and an association constant KA=7.0(±0.8)×10(4)M(-1). Taken together, the FAD-protein interaction is energetically favorable and the addition of FAD is not necessary to induce the enzyme folded state. For the first time, a detailed characterization of the flavin:protein interaction was performed among alternative NADH dehydrogenases.

The effects of lead and copper on the cellular architecture and metabolism of the red alga Gracilaria domingensis.

The effect of lead and copper on apical segments of Gracilaria domingensis was examined. Over a period of 7 days, the segments were cultivated with concentrations of 5 and 10 ppm under laboratory conditions. The samples were processed for light, confocal, and electron microscopy, as well as histochemistry, to evaluate growth rates, mitochondrial activity, protein levels, chlorophyll a, phycobiliproteins, and carotenoids. After 7 days of exposure to lead and copper, growth rates were slower than control, and biomass loss was observed on copper-treated plants. Ultrastructural damage was primarily observed in the internal organization of chloroplasts and cell wall thickness. X-ray microanalysis detected lead in the cell wall, while copper was detected in both the cytoplasm and cell wall. Moreover, lead and copper exposure led to photodamage of photosynthetic pigments and, consequently, changes in photosynthesis. However, protein content and glutathione reductase activity decreased only in the copper treatments. In both treatments, decreased mitochondrial NADH dehydrogenase activity was observed. Taken together, the present study demonstrates that (1) heavy metals such as lead and copper negatively affect various morphological, physiological, and biochemical processes in G. domingensis and (2) copper is more toxic than lead in G. domingensis.

Contiguous RNA editing sites in the mitochondrial nad1 transcript of Arabidopsis thaliana are recognized by different proteins.

Pentatricopeptide repeat (PPR) proteins have been identified as site-specific factors for RNA editing in plant organelles. These proteins recognize cis-elements near the editing site. It is unclear how contiguous sites are addressed, and whether one or two factors are required. We here show the PPR MEF25 to be essential for RNA editing at the nad1-308 site in Arabidopsis mitochondria. Another editing site just one nucleotide upstream, nad1-307, is edited normally in mef25 mutant lines. This finding shows that two independent factors recognizing similar cis-elements are involved at these contiguous sites without competing with each other in vivo.

Using population genetic methods to identify the origin of an invasive population and to diagnose cryptic subspecies of Telchin licus (Lepidoptera: Castniidae).

Telchin licus, the giant sugarcane borer, is an important pest species of sugarcane in northeast Brazil. Four subspecies of Telchin licus are recognized in Brazil based on their geographic distribution and subtle differences in wing colour pattern. Some taxa are morphologically indistinguishable, and their accurate identification is key to their efficient control. Mitochondrial genes sequences (cytochrome oxidase I and subunit 6 of the nicotinamide adenine dinucleotide dehydrogenase) were applied to delimit taxonomic entities of T. licus, and to infer the origin of a newly established population in the state of São Paulo. The molecular data indicated that specimens sampled at different regions in Brazil are morphologically cryptic but genetically isolated entities, and at least three subspecies were assigned to the sampled localities. These data also suggested that the population collected from the state of São Paulo must have a common origin with populations from northeast Brazil, which corroborate the hypothesis that ornamental plants infested with larvae of T. licus might have been transported from the northeast to the southeast regions.

Modulation of mitochondrial activity by S-nitrosoglutathione reductase in Arabidopsis thaliana transgenic cell lines.

The enzyme S-nitrosoglutathione reductase (GSNOR) has an important role in the metabolism of S-nitrosothiols (SNO) and, consequently, in the modulation of nitric oxide (NO)-mediated processes. Although the mitochondrial electron transport chain is an important target of NO, the role of GSNOR in the functionality of plant mitochondria has not been addressed. Here, we measured SNO content and NO emission in Arabidopsis thaliana cell suspension cultures of wild-type (WT) and GSNOR overexpressing (GSNOR(OE)) or antisense (GSNOR(AS)) transgenic lines, grown under optimal conditions and under nutritional stress. Respiratory activity of isolated mitochondria and expression of genes encoding for mitochondrial proteins were also analyzed. Under optimal growth conditions, GSNOR(OE) had the lowest SNO and NO levels and GSNOR(AS) the highest, as expected by the GSNO-consuming activity of GSNOR. Under stress, this pattern was reversed. Analysis of oxygen uptake by isolated mitochondria showed that complex I and external NADH dehydrogenase activities were inhibited in GSNOR(OE) cells grown under nutritional stress. Moreover, GSNOR(OE) could not increase alternative oxidase (AOX) activity under nutritional stress. GSNOR(AS) showed constitutively high activity of external NADH dehydrogenase, and maintained the activity of the uncoupling protein (UCP) under stress. The alterations observed in mitochondrial protein activities were not strictly correlated to changes in gene expression, but instead seemed to be related with the SNO/NO content, suggesting a post-transcriptional regulation. Overall, our results highlight the importance of GSNOR in modulating SNO and NO homeostasis as well mitochondrial functionality, both under normal and adverse conditions in A. thaliana cells.