Oxidative Stress Induced in Sunflower Seedling Roots by Aqueous Dry Olive-Mill Residues

The contamination of soils with dry olive-mill residue can represent a serious problem as being an environmental stressor in plants. It has been demonstrated that inoculation of aqueous extract of olive oil-mill residue (ADOR) with saprobe fungi removes some phenolic compounds. In this paper we studied the effect of ADOR uninoculated or inoculated with saprobe fungi in sunflower seedling roots. The germination and root growth, O₂·^- generation, superoxide dismutase (SOD) and extracellular peroxidases (EC-POXs) activities, and the content of some metabolites involved in the tolerance of stress were tested. The roots germinated in ADOR uninoculated show a decrease in meristem size, resulting in a reduction of the root length and fresh weight, and in the number of layers forming the cortex, but did not alter the dry weight, protein and soluble amino acid content. ADOR caused the decreases in O₂·^- generation and EC-POX′s activities and protein oxidation, but enhanced SOD activity, lipid peroxidation and proline content. Fluorescence imaging showed that ADOR induced O₂·^- and H₂O₂ accumulation in the roots. The increase in SOD and the decrease in EC-POX′s activities might be involved in the enhancement of H₂O₂ content and lipid peroxidation. Control roots treated with ADOR for 10 min show an oxidative burst. Roots germinated in ADOR inoculated with saprobe fungi partially recovered normal levels of ROS, morphological characteristics and antioxidant activities. These results suggested that treatment with ADOR caused a phytotoxic effect during germination inducing an oxidative stress. The inoculation of ADOR with saprobe fungi limited the stress.     

Research on arbuscular mycorrhizae in Mexico: an historical synthesis and future prospects

This review analyzes the historical development and advances of the research on arbuscular mycorrhizal fungi (AMF) in Mexico, as well as the prospects for future research. AMF-research has been focused on studying both diversity and functionality in several ecosystems of Mexico, but mainly in the tropical dry and rainy ecosystems, and the agricultural systems. In Mexico, 95 species of AMF have been recorded, representing 41% of the known species worldwide. The functional effects of AMF colonization have been examined in approximately 10% of the known host plants, but greenhouse studies continue to dominate over those conducted under field conditions. Even though research to date has been at the organismic level, further effort is needed due to the high plant diversity in Mexico. Studies on AMF biomass under field conditions and more taxonomic determination are required based on morphological features, biochemical determinations (fatty acids) and molecular tools. In addition, ecophysiological and ecological in situ studies would help in understanding the relationships among AMF, soil fauna, nutrients, and host plants. The contribution of AMF to ecosystemic processes is a priority line of research that requires an integrated approach (inter- and multidisciplinary) in order to define the role of AM symbioses for biogeochemical models. The creation of a Mexican mycorrhizal research network has and will help to identify the main challenges. Generating similar research protocols, and sharing databases and experience will assist mycorrhizologists working under the diverse financial and ecological contexts that is to be found in Mexico and Latin America.     

α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α_1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α_1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α_1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α_1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α_1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs. heterologous).     

Detergent-activated BAX Protein Is a Monomer

BAX is a pro-apoptotic member of the BCL-2 protein family. At the onset of apoptosis, monomeric, cytoplasmic BAX is activated and translocates to the outer mitochondrial membrane, where it forms an oligomeric pore. The chemical mechanism of BAX activation is controversial, and several in vitro and in vivo methods of its activation are known. One of the most commonly used in vitro methods is activation with detergents, such as n-octyl glucoside. During BAX activation with n-octyl glucoside, it has been shown that BAX forms high molecular weight complexes that are larger than the combined molecular weight of BAX monomer and one detergent micelle. These large complexes have been ascribed to the oligomerization of BAX prior to its membrane insertion and pore formation. This is in contrast to the in vivo studies that suggest that active BAX inserts into the outer mitochondrial membrane as a monomer and then undergoes oligomerization. Here, to simultaneously determine the molecular weight and the number of BAX proteins per BAX-detergent micelle during detergent activation, we have used an approach that combines two single-molecule sensitivity technique, fluorescence correlation spectroscopy, and fluorescence-intensity distribution analysis. We have tested a range of detergents as follows: n-octyl glucoside, dodecyl maltoside, Triton X-100, Tween 20, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and cholic acid. With these detergents we observe that BAX is a monomer before, during, and after interaction with micelles. We conclude that detergent activation of BAX is not congruent with oligomerization and that in physiologic buffer conditions BAX can assume two stable monomeric conformations, one inactive and one active.BAX² is a pro-apoptotic member of the BCL-2 protein family. In a simplified apoptosis model, monomeric inactive BAX is localized in the cytoplasm of healthy nondying cells (1). During apoptosis BAX is activated and translocates to the outer mitochondrial membrane (2) where it inserts as a monomer (3), undergoes oligomerization (4), and forms a pore through which cytochrome c and other apoptotic factors are released into the cytoplasm. Once in the cytoplasm, these apoptotic factors induce the activation of the effector caspases that execute the cell death process. This mechanism, which is generally correct, requires that soluble BAX becomes integrated into the mitochondrial membrane where it forms a functional oligomeric pore capable of cytochrome c release. However, the molecular mechanism of BAX activation remains controversial (5, 6).It has been understood for some time, but frequently ignored, that activity of the BCL-2 family proteins is exhibited in cells when these proteins are associated with the hydrophobic environment of membranes. Therefore, it has always seemed that attention to the effect of hydrophobic environments on the BCL-2 family proteins would be rewarding. It has been shown that BAX can be directly activated by treatment with nonionic detergents such as n-octyl glucoside, dodecyl maltoside, and Triton X-100 (1, 7). During activation by nonionic detergents, to gain the ability to form pores in a bilayer membrane, BAX needs to undergo a major conformational transition from a globular protein with two pore-forming α-helices 5 and 6 hidden in the protein core (8) to a conformation in which these two helices are exposed and inserted into a lipid membrane (3, 5, 9). The nature of this active conformation of BAX is important for the understanding of the death decision in cells. Most proposals suggest that in a cell this activated form of BAX protein is initiated and maintained by the interactions with other proteins, such as tBID, or by BAX itself as a homo-oligomer (7, 10).Nonionic detergents have been commonly used to activate BAX for in vitro studies because they are reliably effective and simple to employ. However, little is known about the detailed molecular mechanism of BAX activation by these detergents and its comparability with in vivo activation of BAX. What is known is that concentrations of detergent above their critical micelle concentration (CMC) are necessary for BAX activation. This suggests that, to be activated, BAX needs to interact with detergent micelles instead of monomeric detergent molecules. For example, in the case of BAX activation by n-octyl glucoside, it has been shown that n-octyl glucoside concentration should be 1% (w/v) (7), which is well above the CMC for this detergent (0.6% w/v) (11). In addition, it has also been shown that above their individual CMC concentrations most BAX-activating detergents produce a change in BAX conformation that can be detected by a conformation-sensitive 6A7 antibody against BAX (1, 12, 13). In cellular experiments this feature of BAX reactivity to 6A7 antibody is commonly associated with the onset of apoptosis (14, 15). However, CHAPS does not generate the antibody-detected conformational change or the activation of BAX. The small micelle size of this detergent (10 kDa) suggests that perhaps BAX cannot adopt an activated state with this detergent. However, cholic acid with even smaller micelle size (4 kDa) can partially activate BAX (1).Many important detergent properties are associated with micelles. The formation of detergent micelles in solution is concentration-dependent beginning at the CMC. The CMC value for a detergent has practical importance because in most cases only monomers of detergent can be removed by dialysis, and therefore, it is easier to remove detergent monomers for a detergent with high CMC value than for a detergent with low CMC (11). For BAX this same consideration applies to its activation with n-octyl glucoside (CMC ∼23 mm) as compared with its activation with Triton X-100 (CMC ∼0.25 mm). The ease of dialysis is why, in most cases, OG is used to activate BAX in vitro.It has been shown by analytical gel filtration that, when incubated with n-octyl glucoside, BAX creates complexes with molecular weight larger than the combined size of a BAX monomer (21 kDa) and an n-octyl glucoside micelle (∼26 kDa) (7, 11). It has also been shown that in defined liposomes BAX pore formation requires oligomerization (16). These data combined with the knowledge that oligomerization is important for the biological function of BAX led to a hypothesis that BAX oligomerizes during its detergent activation prior to membrane insertion (7). However, it has been shown that in vivo activated BAX inserts into the outer mitochondrial membrane as a monomer (3), and to create a pore, BAX undergoes oligomerization in this membrane (4). This discrepancy between the oligomeric state of active BAX prior to its insertion into a lipid membrane in vivo (monomer) and in vitro (possibly hexamer or octamer) led us to study the oligomerization state of BAX in detergent micelles. The important issue is whether BAX activation requires protein oligomerization or whether active BAX conformation can be generated from a single protein monomer. To solve this issue we used two single-molecule sensitivity techniques: fluorescence correlation spectroscopy (FCS) (17) and fluorescence-intensity distribution analysis (FIDA) (18). Combined use of FCS and FIDA allows simultaneous determination of the apparent molecular weight and the number of fluorescently labeled BAX monomers per protein-detergent micelle. Our results are consistent with previously established results in which BAX forms high molecular weight protein-detergent micelles with n-octyl glucoside (4) and show that BAX is present as a monomer in these complexes. In addition, we determined the apparent molecular weight and the number of BAX proteins bound per protein-detergent micelles formed by BAX and micelles of five additional detergents (dodecyl maltoside, Triton X-100, Tween 20, cholic acid, and CHAPS). Our data show that BAX is a monomer before, during, and after interaction with the micelles of all tested detergents.     

Photosynthetic performance in cyanobacteria with increased sulphide tolerance: an analysis comparing wild-type and experimentally derived strains

Sulphide is proposed to have influenced the evolution of primary stages of oxygenic photosynthesis in cyanobacteria. However, sulphide is toxic to most of the species of this phylum, except for some sulphide-tolerant species showing various sulphide-resistance mechanisms. In a previous study, we found that this tolerance can be induced by environmental sulphidic conditions, in which two experimentally derived strains with an enhanced tolerance to sulphide were obtained from Microcystis aeruginosa, a sensitive species, and Oscillatoria, a sulphide-tolerant genus. We have now analysed the photosynthetic performance of the wild-type and derived strains in the presence of sulphide to shed light on the characteristics underlying the increased tolerance. We checked whether the sulphide tolerance was a result of higher PSII sulphide resistance and/or the induction of sulphide-dependent anoxygenic photosynthesis. We observed that growth, maximum quantum yield, maximum electron transport rate and photosynthetic efficiency in the presence of sulphide were less affected in the derived strains compared to their wild-type counterparts. Nevertheless, in ¹⁴C photoincoporation assays, neither Oscillatoria nor M. aeruginosa exhibited anoxygenic photosynthesis using sulphide as an electron donor. On the other hand, the content of photosynthetic pigments in the derived strains was different to that observed in the wild-type strains. Thus, an enhanced PSII sulphide resistance appears to be behind the increased sulphide tolerance displayed by the experimentally derived strains, as observed in most natural sulphide-tolerant cyanobacterial strains. However, other changes in the photosynthetic machinery cannot be excluded.

     

Cycloheximide: An adrenergic agent

Cycloheximide, a widely used inhibitor of protein synthesis, stimulates glycogenolysis, gluconeogenesis and ureogenesis in isolated rat hepatocytes. The effects of cycloheximide were compared to those of norepinephrine. Both agents, cycloheximide and norepinephrine, produced slight increases in the levels of cyclic AMP (30% increases) which were blocked by propranolol. Interestingly, it was found that the metabolic actions of norepinephrine and cycloheximide (stimulation of glycogenolysis, gluconeogenesis and ureogenesis) were only slightly diminished by the β adrenergic antagonist propranolol but abolished by the selective α₁ adrenergic antagonist prazosin. The ability of cycloheximide to inhibit protein synthesis was not affected by either prazosin or propranolol. It is concluded that the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by cycloheximide in rat hepatocytes, is an effect of the antibiotic independent of its ability to inhibit protein synthesis and that is mediated through activation of α₁ adrenoceptors. The adrenergic activity of cycloheximide should be considered when this drug is used as an inhibitor of protein synthesis.     

Probing the specificity of the subclass B3 FEZ-1 metallo-beta-lactamase by site-directed mutagenesis

The subclass B3 FEZ-1 beta-lactamase produced by Fluoribacter (Legionella) gormanii is a Zn(II)-containing enzyme that hydrolyzes the beta-lactam bond in penicillins, cephalosporins, and carbapenems. FEZ-1 has been extensively studied using kinetic, computational modeling and x-ray crystallography. In an effort to probe residues potentially involved in substrate binding and zinc binding, five site-directed mutants of FEZ-1 (H121A, Y156A, S221A, N225A, and Y228A) were prepared and characterized using metal analyses and steady state kinetics. The activity of H121A is dependent on zinc ion concentration. The H121A monozinc form is less active than the dizinc form, which exhibits an activity similar to that of the wild type enzyme. Tyr156 is not essential for binding and hydrolysis of the substrate. Substitution of residues Ser221 and Asn225 modifies the substrate profile by selectively decreasing the activity against carbapenems. The Y228A mutant is inhibited by the product formed upon hydrolysis of cephalosporins. A covalent bond between the side chain of Cys200 and the hydrolyzed cephalosporins leads to the formation of an inactive and stable complex.