Héctor Riveros-Rosas

Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans

NADPH oxidases (Nox) have been characterized as higher eukaryotic enzymes used deliberately to produce reactive oxygen species (ROS). The recent discovery of new functional members of the Nox family in plants and animals has led to the recognition of the increasing importance of ROS as signals involved in regulation of diverse cellular processes such as defence, growth and signalling. Here, we address the role of NADPH oxidase‐generated ROS in the biology of the filamentous fungus Aspergillus nidulans. We characterize the noxA gene and show that it encodes a member of a novel NADPH oxidase subfamily ubiquitous in lower eukaryotes. Deletion of noxA specifically blocks differentiation of sexual fruit bodies (cleistothecia), without affecting hyphal growth or asexual development. Accordingly, the noxA gene is induced during sexual development, peaking at the time of cleistothecia differentiation and in parallel with the hülle cell‐associated catalase peroxidase gene cpeA. This expression pattern is not dependent on transcription factors SteA and StuA, which are essential for cleistothecia formation. In contrast, noxA‐dependent premature sexual development correlates with noxA derepression in ΔsakA null mutants, connecting stress MAPK signalling to the regulated production of ROS. Using a nitroblue tetrazolium (NBT) assay to detect superoxide, we found that hülle cells and cleistothecia initials produce superoxide in a process inhibited by NADPH oxidase inhibitor DPI and markedly reduced in ΔnoxA mutants. Furthermore, using H2DCFDA, we detected that H2O2 and possibly other ROS are generated in a NoxA‐dependent fashion, mainly in the external walls from cleistothecia initials. The essential role of NoxA‐generated ROS in A. nidulans sexual differentiation and the presence of one or two noxA homologues in all analysed filamentous fungi suggest that NADPH oxidase‐generated ROS play important roles in fungal physiology and differentiation.

Mechanisms of bacterial resistance to chromium compounds

Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidusmetallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.

Bacterial transport of sulfate, molybdate, and related oxyanions.

Sulfur is an essential element for microorganisms and it can be obtained from varied compounds, sulfate being the preferred source. The first step for sulfate assimilation, sulfate uptake, has been studied in several bacterial species. This article reviews the properties of different bacterial (and archaeal) transporters for sulfate, molybdate, and related oxyanions. Sulfate uptake is carried out by sulfate permeases that belong to the SulT (CysPTWA), SulP, CysP/(PiT), and CysZ families. The oxyanions molybdate, tungstate, selenate and chromate are structurally related to sulfate. Molybdate is transported mainly by the high-affinity ModABC system and tungstate by the TupABC and WtpABC systems. CysPTWA, ModABC, TupABC, and WtpABC are homologous ATP-binding cassette (ABC)-type transporters with similar organization and properties. Uptake of selenate and chromate oxyanions occurs mainly through sulfate permeases.

Phylogenetic analysis of the chromate ion transporter (CHR) superfamily

ChrA is a membrane protein that confers resistance to the toxic ion chromate through the energy‐dependent chromate efflux from the cytoplasm. In the protein databases, ChrA is a member of the chromate ion transporter (CHR) superfamily, composed of at least several dozens of members, distributed in the three domains of life. The aim of this work was to perform a phylogenetic analysis of the CHR superfamily. An exhaustive search for ChrA homologous proteins was carried out at the National Center for Biotechnology Information database. One hundred and thirty‐five sequences were identified as members of the CHR superfamily [77 long‐chain sequences, or bidomains (LCHR), and 58 short‐chain sequences, or monodomains (SCHR)], organized mainly as tandem pairs of genes whose resultant proteins probably possess oppositely oriented membrane topology. LCHR sequences were split into amino and carboxyl domains, and the resultant domains were aligned with the SCHR proteins. A phylogenetic tree was reconstructed using four different methods, obtaining similar results. The domains were grouped into three clusters: the SCHR proteins cluster, the amino domain cluster of LCHR proteins and the carboxyl domain cluster of LCHR proteins. These results, as well as differences in the genomic context of CHR proteins, enabled the proteins to be sorted into two families (SCHR and LCHR), and 10 subfamilies. Evidence was found suggesting an ancient origin of LCHR proteins from the fusion of two SCHR protein‐encoding genes; however, some secondary events of fusion and fission may have occurred later. The separate distribution of the LCHR and SCHR proteins, differences in the genomic context in both groups and the fact that chromate transport has been demonstrated only in LCHR proteins suggest that the CHR proteins comprise two or more paralogous groups in the CHR superfamily.

Signaling the Signal, Cyclic AMP-dependent Protein Kinase Inhibition by Insulin-formed H2O2 and Reactivation by Thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H2O2 generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H2O2 added before cAMP suppressed cAMP activation of type IIβ cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H2O2 impaired activation of type IIα PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIα and catalytic α subunits. H2O2 was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic α subunit, which is active independently of cAMP activation, and (c) if the catalytic α subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H2O2 inhibition of PKA activation remained after H2O2 elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H2O2 incubation. These data strongly suggest that H2O2 promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIα or IIβ subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic α subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

Personal exposure to elements in Mexico City air

Personal exposures to various metals in airborne particulates in Mexico City were measured over a seven consecutive-day period. Subjects were divided into two groups, Group A, whose work required them to spend considerable time outdoors and in traffic (messengers, delivery men, taxi drivers, salesmen), and Group B who spent most of their time indoors (university professors, consultants, managers and research workers). Group A spent 32 +/- 8.5% of their time outdoors, while Group B spent 10.7 +/- 6.7% of their time outdoors. Group A had higher exposures to airborne lead, zinc, vanadium, manganese and chromium than did Group B. There was no difference between the groups with respect to airborne copper exposures. Overall exposures to the various airborne metals were: lead, 435 +/- 220 ng m-3; zinc, 361 +/- 253 ng m-3; vanadium, 23 +/- 12 ng m-3; manganese, < or = 30 +/- 25 ng m-3; chromium, < or = 8.5 +/- 5.5 ng m-3; and copper, < or = 45 +/- 32 ng m-3. A significant number of samples were below the analytical limits of detection for manganese and copper.

Natural alcohol exposure: is ethanol the main substrate for alcohol dehydrogenases in animals?

Alcohol dehydrogenase (ADH) activity is widely distributed in all phyla. In animals, three non-homologous NAD(P)(+)-dependent ADH protein families are reported. These arose independently throughout evolution and possess different structures and mechanisms of reaction: type I (medium-chain) ADHs are zinc-containing enzymes and comprise the most studied group in vertebrates; type II (short-chain) ADHs lack metal cofactor and have been extensively studied in Drosophila; and type III ADHs are iron-dependent/-activated enzymes that were initially identified only in microorganisms. The presence of these different ADHs in animals has been assumed to be a consequence of chronic exposure to ethanol. By far the most common natural source of ethanol is fermentation of fruit sugars by yeast, and available data support that this fruit trait evolved in concert with the characteristics of their frugivorous seed dispersers. Therefore, if the presence of ADHs in animals evolved as an adaptive response to dietary ethanol exposure, then it can be expected that the enzymogenesis of these enzymes began after the appearance of angiosperms with fleshy fruits, because substrate availability must precede enzyme selection. In this work, available evidence supporting this possibility is discussed. Phylogenetic analyses reveal that type II ADHs suffered several duplications, all of these restricted to flies (order Diptera). Induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow larval flies to exploit new restricted niches with high ethanol content. In contrast, phyletic distribution of types I and III ADHs in animals showed that these appeared before angiosperms and land plants, independently of ethanol availability. Because these enzymes are not induced by ethanol exposure and possess a high affinity and/or catalytic efficiency for non-ethanol endogenous substrates, it can be concluded that the participation of types I and III ADHs in ethanol metabolism can be considered as incidental, and not adaptive.

Dichotomic Phylogenetic Tree of the Pyruvate Kinase Family K+-DEPENDENT AND -INDEPENDENT ENZYMES

K+ dependence was assumed to be a feature of all pyruvate kinases until it was discovered that some enzymes express K+ -independent activity. Almost all the K+-independent pyruvate kinases have Lys at position 117, instead of the Glu present in the K+-dependent muscle enzyme. Mutagenesis studies show that the internal positive charge substitutes for the K+ requirement (Laughlin, L. T. & Reed, G. H. (1997) Arch. Biochem. Biophys. 348, 262–267). In this work a phylogenetic analysis of pyruvate kinase was performed to ascertain the abundance of K+ -independent activities and to explore whether the K+ activating effect is related to the evolutionary history of the enzyme. Of the 230 studied sequences, 46% have Lys at position 117, and the rest have Glu. Pyruvate kinases with Lys117 and Glu117 are separated in two clusters. All of the enzymes of the Glu117 cluster that have been characterized are K+-dependent, whereas those of the Lys117 cluster are K+-independent. Thus, there is a strict correlation between the dichotomy of the tree and the dependence of activity on K+. 77% of the pyruvate kinases that possess Lys117 have Lys113/Gln114; they also have Ile, Val, or Leu at position 120. These residues are replaced by Glu117 and Thr113/Lys114/Thr120 in 80% of K+-dependent pyruvate kinases. Structural analysis indicates that these residues are in a hinge region involved in the acquisition of the catalytic conformation of the enzyme. The route of conversion from K+-independent to K+-dependent pyruvate kinases is described. A plausible explanation of how enzymes developed K+ dependence is put forth.

Short-Chain Chromate Ion Transporter Proteins from Bacillus subtilis Confer Chromate Resistance in Escherichia coli

Tandem paired genes encoding putative short-chain monodomain protein members of the chromate ion transporter (CHR) superfamily (ywrB and ywrA) were cloned from genomic DNA of Bacillus subtilis strain 168. The transcription of the paired genes, renamed chr3N and chr3C, respectively, was shown to occur via a bicistronic mRNA generated from a promoter upstream of the chr3N gene. The chr3N and chr3C genes conferred chromate resistance when expressed in Escherichia coli strain W3110. The cloned chr3N gene alone did not confer chromate resistance on E. coli, suggesting that both chr3N and chr3C genes are required for function. E. coli cells expressing paired chr3N and chr3C genes demonstrated diminished uptake of chromate compared to that by a vector-only control strain. These results suggest that short-chain CHR proteins form heterodimer transporters which efflux chromate ions from the cytoplasm.

Hydrocarbons and Carbon Monoxide in the Atmosphere of Mexico City

Abstract In Mexico City, the use and composition of fuels determine that carbon monoxide (CO) comes mostly from mobile sources, and sulfur dioxide (SO2) from fixed and mobile sources. By simultaneously measuring hydrocarbons (HC), CO, and SO2 in the atmosphere of Mexico City, the relative amounts coming from different sources can be estimated. Assuming that some HC are emitted proportionally to CO emissions, we can establish that [HC]1= m1• [CO], where the proportionality constant ml corresponds to the ratio of emissions factor for HC and CO in mobile sources. Similarly for fuels containing sulfur, it can be assumed that [HC]2 = m2 • [SO2]. In this way, the total HC are [HC]total=[HC]0+ ml • [CO]+ m2 • [SO2], where [HC]0 corresponds mainly to other sources like solvent evaporation, gas consumption, and natural emissions. In this way, it can be estimated that in Mexico City 75% of average HC comes from mobile sources, 5% from sulfur-related sources, and 19% from natural sources and solvent evaporation. Com...