Peroxynitrite oxidizes erythrocyte membrane band 3 protein and diminishes its anion transport capacity

We describe an altered membrane band 3 protein-mediated anion transport in erythrocytes exposed to peroxynitrite, and relate the loss of anion transport to cell damage and to band 3 oxidative modifications. We found that peroxynitrite down-regulate anion transport in a dose dependent relation (100–300 μmoles/l). Hemoglobin oxidation was found at all peroxynitrite concentrations studied. A dose-dependent band 3 protein crosslinking and tyrosine nitration were also observed. Band 3 protein modifications were concomitant with a decrease in transport activity. ( ? )-Epicatechin avoids band 3 protein nitration but barely affects its transport capacity, suggesting that both processes are unrelated. N-acetyl cysteine partially reverted the loss of band 3 transport capacity. It is concluded that peroxynitrite promotes a decrease in anion transport that is partially due to the reversible oxidation of band 3 cysteine residues. Additionally, band 3 tyrosine nitration seems not to be relevant for the loss of its anion transport capacity.     

Increased dosage of Ink4/Arf protects against glucose intolerance and insulin resistance associated with aging

Recent genome‐wide association studies have linked type‐2 diabetes mellitus to a genomic region in chromosome 9p21 near the Ink4/Arf locus, which encodes tumor suppressors that are up‐regulated in a variety of mammalian organs during aging. However, it is unclear whether the susceptibility to type‐2 diabetes is associated with altered expression of the Ink4/Arf locus. In the present study, we investigated the role of Ink4/Arf in age‐dependent alterations of insulin and glucose homeostasis using Super‐Ink4/Arf mice which bear an extra copy of the entire Ink4/Arf locus. We find that, in contrast to age‐matched wild‐type controls, Super‐Ink4/Arf mice do not develop glucose intolerance with aging. Insulin tolerance tests demonstrated increased insulin sensitivity in Super‐Ink4/Arf compared with wild‐type mice, which was accompanied by higher activation of the insulin receptor substrate (IRS)‐PI3K‐AKT pathway in liver, skeletal muscle and heart. Glucose uptake studies in Super‐Ink4/Arf mice showed a tendency toward increased ¹⁸F‐fluorodeoxyglucose uptake in skeletal muscle compared with wild‐type mice (P = 0.079). Furthermore, a positive correlation between glucose uptake and baseline glucose levels was observed in Super‐Ink4/Arf mice (P < 0.008) but not in wild‐type mice. Our studies reveal a protective role of the Ink4/Arf locus against the development of age‐dependent insulin resistance and glucose intolerance.     

Vibrational Study of a Nucleoside Analogue with Antiviral Activity, 5-Chloro-2′-deoxyuridine,CDU

Abstract

The experimental FTIR and FT-Raman spectra of 5-chloro-2′-deoxyuridine have been assigned on the basis of normal coordinate analyses, in the light of observed and calculated wavenumbers and isotopic shifts. The results indicate that virtually all normal modes of IDU involve some degree of vibrational coupling between the chlorouracil base and the deoxyribose moiety.     

New Arabidopsis thaliana Cytochrome c Partners: A Look Into the Elusive Role of Cytochrome c in Programmed Cell Death in Plants

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.Programmed cell death (PCD)¹ is a fundamental event for the development of multicellular organisms and the homeostasis of their tissues. It is an evolutionarily conserved mechanism present in organisms ranging from yeast to mammals (1–3).In mammals, cytochrome c (Cc) and dATP bind to apoptosis protease-activating factor-1 (Apaf-1) in the cytoplasm, a process leading to the formation of the Apaf-1/caspase-9 complex known as apoptosome. This apoptosome subsequently activates caspases-3 and -7 (4, 5). In other organisms, such as Caenorhabditis elegans or Drosophila melanogaster, however, Cc is not essential for the assembly and activation of the apoptosome (6) despite the presence of proteins homologous to Apaf-1—cell death abnormality-4 (CED-4) in C. elegans and Drosophila Apaf-1-related killer (Dark) in D. melanogaster—which have been found to be essential for caspase cascade activation. Furthermore, other organisms such as Arabidopsis thaliana lack Apaf-1 (7). In fact, only highly distant caspase homologues (metacaspases) (8, 9), serine proteases (saspases) (10), phytaspases (11) and VEIDases (12–14) with caspase-like activity have been detected in plants; however, their targets remain veiled and whether they are activated by Cc remains unclear.Intriguingly, the release of Cc from mitochondria into the cytoplasm during the onset of PCD is an evolutionarily conserved event found in organisms ranging from yeast (15) and plants (16) to flies (17), and mammals (18). However, understanding of the roles of this phenomenon in different species can be said to be uneven at best. In fact, the release of Cc from mitochondria has thus far been considered a random event in all organisms, save mammals. Thus, the participation of Cc in the onset and progression of PCD needs to be further elucidated.Even in the case of mammals, the role(s) of Cc in the cytoplasm during PCD remain(s) controversial. Recently, new putative functions of Cc, going beyond the already-established apoptosome assembly process, have been proposed in the nucleus (19, 20) and the endoplasmic reticulum (21–23). Neither these newly proposed functions nor other arising functions, such as oxidative stress (24), are as yet fully understood. This current state of affairs demands deeper exploration of the additional roles played by Cc in nonmammalian species.In this study, putative novel Cc-partners involved in plant PCD were identified. For this identification, a proteomic approach was employed based on affinity chromatography and using Cc as bait. The Cc-interacting proteins were identified using nano-liquid chromatography tandem mass spectrometry (NanoLC-MS/MS). These Cc-partners were then further confirmed in vivo through bimolecular fluorescence complementation (BiFC) in A. thaliana protoplasts and human HEK293T cells, as a heterologous system. Finally, the Cc-GLY2, Cc-NRP1 and Cc-TCL interactions were corroborated in vitro using surface plasmon resonance (SPR).These results indicate that Cc is able to interact with targets in the plant cell cytoplasm during PCD. Moreover, they provide new ways of understanding why Cc release is an evolutionarily well-conserved event, and allow us to propose Cc as a signaling messenger, which somehow controls different essential events during PCD.     

Reproductive strategy of the invasive Oxalis pes-caprae: distribution patterns of floral morphs, ploidy levels and sexual reproduction

Oxalis pes-caprae, a tristylous flowering plant native to South Africa, is described in the western Mediterranean basin as an asexual—only 5x short-styled morph (5x S-morph) invasive weed losing all mating partners after introduction. The objective of this study was to reassess the patterns of floral morph and cytotype distribution and the sexual reproduction ability in this invaded range. For that, floral morph and cytotype composition were evaluated in 39 populations of O. pes-caprae in a methodical sampling. The reproductive success of natural populations was assessed as fruit and seed production and seed germination for all floral morphs and cytotypes detected. Self- and morph-incompatibility were also studied with controlled hand pollinations. A remarkable diversity in floral morph and cytotype composition was observed. Furthermore, we observed successful sexual reproduction in several localities across the surveyed area. The S-morph is still dominant in this invaded area, and although it was mostly 5x, an additional cytotype (4x) was also recorded. Records of both a mid-styled morph (M-morph) and an area with trimorphic populations of this species are reported here for the first time in the invasive range of the Mediterranean basin. The long-styled morph appears to occur randomly across the surveyed area, while the M-morph is concentrated mainly in Estremadura province (Portugal), where a breakdown in the incompatibility system was observed. These distribution patterns may result from events of sexual reproduction after incompatibility breakdown and/or from multiple introduction events from the native area. The ability to reproduce sexually, undetected so far, may have important impacts in the population dynamics and major consequences for the adaptation and selection potential of O. pes-caprae in this invaded area.     

Effect of invader removal: pollinators stay but some native plants miss their new friend

Removal of invasive species often benefits biological diversity allowing ecosystems’ recovery. However, it is important to assess the functional roles that invaders may have established in their new areas to avoid unexpected results from species elimination. Invasive animal-pollinated plants may affect the plant–pollination interactions by changing pollinator availability and/or behaviour in the community. Thus, removal of an invasive plant may have important effects on pollinator community that may then be reflected positive or negatively on the reproductive success of native plants. The objective of this study was to assess the effect of removing Oxalis pes-caprae, an invasive weed widely spread in the Mediterranean basin, on plant–pollinator interactions and on the reproductive success of co-flowering native plants. For this, a disturbed area in central Portugal, where this species is highly abundant, was selected. Visitation rates, natural pollen loads, pollen tube growth and natural fruit set of native plants were compared in the presence of O. pes-caprae and after manual removal of their flowers. Our results showed a highly resilient pollination network but also revealed some facilitative effects of O. pes-caprae on the reproductive success of co-flowering native plants. Reproductive success of the native plants seems to depend not only on the number and diversity of floral visitors, but also on their efficiency as pollinators. The information provided on the effects of invasive species on the sexual reproductive success of natives is essential for adequate management of invaded areas.     

Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions

In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4–7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively.     

Asymmetric reduction of (4S)-(+)-carvone catalyzed by baker's yeast: A green method for monitoring the conversion based on liquid–liquid–liquid microextraction with polypropylene hollow fiber membranes

The α,β-unsaturated carbonyl compound (4S)-(+)-carvone was selectively reduced to (1R,2R,4S)-iso-dihydrocarveol using baker's yeasts. The conversion of the bioreduction reaction was monitored using a green hollow-fiber liquid–liquid–liquid microextraction (HF-LLLME) technique. Several parameters which may affect the bioreduction of (4S)-(+)-carvone, such as temperature, time, substrate/enzyme ratio, pH and buffer concentration, were evaluated. The effect of some additives, such as trehalose, DMSO and the ionic liquid [BMIm][PF₆], was also studied. The (1R,2R,4S)-iso-dihydrocarveol was recovered with 52.7% conversion and diastereoisomeric excess >99% after 48 h of reaction at 40 °C in an aqueous monophasic system, with 0.1 mol L^?1 buffer concentration (pH 7.5) and a substrate/yeast cell mass ratio of 8.0 mg g^?1. The HF-LLLME microextraction technique allowed the optimization of the reaction with a reduction of over 99.5% in relation to the use of organic solvents.