Coexistence of Wolbachia with Buchnera aphidicola and a secondary symbiont in the aphid Cinara cedri.

Intracellular symbiosis is very common in the insect world. For the aphid Cinara cedri, we have identified by electron microscopy three symbiotic bacteria that can be characterized by their different sizes, morphologies, and electrodensities. PCR amplification and sequencing of the 16S ribosomal DNA (rDNA) genes showed that, in addition to harboring Buchnera aphidicola, the primary endosymbiont of aphids, C. cedri harbors a secondary symbiont (S symbiont) that was previously found to be associated with aphids (PASS, or R type) and an alpha-proteobacterium that belongs to the Wolbachia genus. Using in situ hybridization with specific bacterial probes designed for symbiont 16S rDNA sequences, we have shown that Wolbachia was represented by only a few minute bacteria surrounding the S symbionts. Moreover, the observed B. aphidicola and the S symbionts had similar sizes and were housed in separate specific bacterial cells, the bacteriocytes. Interestingly, in contrast to the case for all aphids examined thus far, the S symbionts were shown to occupy a similarly sized or even larger bacteriocyte space than B. aphidicola. These findings, along with the facts that C. cedri harbors the B. aphidicola strain with the smallest bacterial genome and that the S symbionts infect all Cinara spp. analyzed so far, suggest the possibility of bacterial replacement in these species

Comparative analysis of two genomic regions among four strains of Buchnera aphidicola, primary endosymbiont of aphids.

Preliminary analysis of two selected genomic regions of Buchnera aphidicola BCc, the primary endosymbiont of the cedar aphid Cinara cedri, has revealed a number of interesting features when compared with the corresponding homologous regions of the three B. aphidicola genomes previously sequenced, that are associated with different aphid species. Both regions exhibit a significant reduction in length and gene number in B. aphidicola BCc, as it could be expected since it possess the smallest bacterial genome. However, the observed genome reduction is not even in both regions, as it appears to be dependent on the nature of their gene content. The region fpr-trxA, that contains mainly metabolic genes, has lost almost half of its genes (45.6%) and has reduced 52.9% its length. The reductive process in the region rrl-aroK, that contains mainly ribosomal protein genes, is less dramatic, since it has lost 9.3% of genes and has reduced 15.5% of its length. Length reduction is mainly due to the loss of protein-coding genes, not to the shortening of ORFs or intergenic regions. In both regions, G+C content is about 4% lower in BCc than in the other B. aphidicola strains. However, when only conserved genes and intergenic regions of the four B. aphidicola strains are compared, the G+C reduction is higher in the fpr-trxA region

Plasmids in the aphid endosymbiont Buchnera aphidicola with the smallest genomes. A puzzling evolutionary story.

Buchnera aphidicola, the primary endosymbiont of aphids, has undergone important genomic and biochemical changes as an adaptation to intracellular life. The most important structural changes include a drastic genome reduction and the amplification of genes encoding key enzymes for the biosynthesis of amino acids by their translocation to plasmids. Molecular characterization through different aphid subfamilies has revealed that the genes involved in leucine and tryptophan biosynthesis show a variable fate, since they can be located on plasmids or on the chromosome in different lineages. This versatility contrasts with the genomic stasis found in three distantly related B. aphidicola strains already sequenced. We present the analysis of three B. aphidicola strains (BTg, BCt and BCc) belonging to aphids from different tribes of the subfamily Lachninae, that was estimated to harbour the bacteria with the smallest genomes. The presence of both leucine and tryptophan plasmids in BTg, a chimerical leucine-tryptophan plasmid in BCt, and only a leucine plasmid in BCc, indicates the existence of many recombination events in a recA minus bacterium. In addition, these B. aphidicola plasmids are the simplest described in this species, indicating that plasmids are also involved in the genome shrinkage process

Smooth Muscle-Generated Methylglyoxal Impairs Endothelial Cell-Mediated Vasodilatation of Cerebral Microvessels in Type 1 Diabetic Rats

Background and Purpose Endothelial cell-mediated vasodilatation of cerebral arterioles is impaired in individuals with Type 1 diabetes (T1D). This defect compromises haemodynamics and can lead to hypoxia, microbleeds, inflammation and exaggerated ischaemia-reperfusion injuries. The molecular causes for dysregulation of cerebral microvascular endothelial cells (cECs) in T1D remains poorly defined. This study tests the hypothesis that cECs dysregulation in T1D is triggered by increased generation of the mitochondrial toxin, methylglyoxal, by smooth muscle cells in cerebral arterioles (cSMCs). Experimental Approach Endothelial cell-mediated vasodilatation, vascular transcytosis inflammation, hypoxia and ischaemia-reperfusion injury were assessed in brains of male Sprague-Dawley rats with streptozotocin-induced diabetes and compared with those in diabetic rats with increased expression of methylglyoxal-degrading enzyme glyoxalase-I (Glo-I) in cSMCs. Key Results After 7–8 weeks of T1D, endothelial cell-mediated vasodilatation of cerebral arterioles was impaired. Microvascular leakage, gliosis, macrophage/neutrophil infiltration, NF-κB activity and TNF-α levels were increased, and density of perfused microvessels was reduced. Transient occlusion of a mid-cerebral artery exacerbated ischaemia-reperfusion injury. In cSMCs, Glo-I protein was decreased, and the methylglyoxal-synthesizing enzyme, vascular adhesion protein 1 (VAP-1) and methylglyoxal were increased. Restoring Glo-I protein in cSMCs of diabetic rats to control levels via gene transfer, blunted VAP-1 and methylglyoxal increases, cECs dysfunction, microvascular leakage, inflammation, ischaemia-reperfusion injury and increased microvessel perfusion. Conclusions and Implications Methylglyoxal generated by cSMCs induced cECs dysfunction, inflammation, hypoxia and exaggerated ischaemia-reperfusion injury in diabetic rats. Lowering methylglyoxal produced by cSMCs may be a viable therapeutic strategy to preserve cECs function and blunt deleterious downstream consequences in T1D

Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation

Advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs) have a pathogenetic role in the development and progression of different oxidative-based diseases including diabetes, atherosclerosis, and neurological disorders. AGEs and ALEs represent a quite complex class of compounds that are formed by different mechanisms, by heterogeneous precursors and that can be formed either exogenously or endogenously. There is a wide interest in AGEs and ALEs involving different aspects of research which are essentially focused on set-up and application of analytical strategies (1) to identify, characterize, and quantify AGEs and ALEs in different pathophysiological conditions ; (2) to elucidate the molecular basis of their biological effects ; and (3) to discover compounds able to inhibit AGEs/ALEs damaging effects not only as biological tools aimed at validating AGEs/ALEs as drug target, but also as promising drugs. All the above-mentioned research stages require a clear picture of the chemical formation of AGEs/ALEs but this is not simple, due to the complex and heterogeneous pathways, involving different precursors and mechanisms. In view of this intricate scenario, the aim of the present review is to group the main AGEs and ALEs and to describe, for each of them, the precursors and mechanisms of formation

Structure-function studies of the Parkinsonism-linked protein DJ-1

DJ-1 is a multifunctional protein linked to familial Parkinson’s disease. DJ-1 has been suggested to exert its cytoprotective function, in part, by acting as a copper carrier that can sequester the reactive metal and/or provide the copper cofactor for the activation of the Cu-Zn superoxide dismutase (SOD1). Using absorption spectroscopy and mass spectrometry, we found that DJ-1 binds one Cu(I) ion per DJ-1 homodimer. The structure of Cu(I)-bound DJ-1 reveals a new biscysteinate metal binding motif formed by juxtaposed Cys-53 at the homodimer interface. We calculated a subfemtomolar dissociation constant (Kd = 6.41 x 10-16 M) for Cu(I) that supports the physiological intracellular retention of the metal. Cu(I)-bound DJ-1 was not capable of interacting and activating SOD1 in vitro. We posit that DJ-1 sequester copper to protect against metal-induced cytotoxicity. Our results illuminate the molecular basis on how disease-linked mutations that impairs homodimerisation could disrupt the metal binding site. In the second part of this dissertation, we sought to determine the impact of a Parkinsonism-linked A107P mutation on DJ-1 structure and glyoxalase activity. The A107P variant abrogates the ability of DJ-1 to protect against glyoxal-induced cytotoxicity and carboxymethyllysine protein modification. A crystal structure of DJ-1 C106S variant with glycerol and sulphate bound in the active site suggests that Ala-107 is critical for the stabilization of the transition state of the nucleophilic addition step. In our hands, the protein levels of DJ-1 A107P mutant in SH-SH5Y cells were ostensibly similar to the wild-type level but reduced levels were found in HEK 293E and MEF cells. Using CD and NMR spectroscopy, we found that the structural defect caused by the mutation extends beyond the active site. The A107P mutation resulted in a remarkable misfolding of the protein providing a basis for the reduced intracellular protein level and the abrogation of enzymatic activity

Modulation of the glyoxalase system in the aging model Podospora anserina : effects on growth and lifespan

The eukaryotic glyoxalase system consists of two enzymatic components, glyoxalase I (lactoylglutathionelyase) and glyoxalase II (hydroxyacylglutathione hydrolase). These enzymes are dedicated to the removal of toxic alpha-oxoaldehydes like methylglyoxal (MG). MG is formed as a by-product of glycolysis and MG toxicity results from its damaging capability leading to modifications of proteins, lipids and nucleic acids. An efficient removal of MG appears to be essential to ensure cellular functionality and viability. Here we study the effects of the genetic modulation of genes encoding the components of the glyoxalase system in the filamentous ascomycete and aging model Podospora anserina. Overexpression of PaGlo1 leads to a lifespan reduction on glucose rich medium, probably due to depletion of reduced glutathione. Deletion of PaGlo1 leads to hypersensitivity against MG added to the growth medium. A beneficial effect on lifespan is observed when both PaGlo1 and PaGlo2 are overexpressed and the corresponding strains are grown on media containing increased glucose concentrations. Notably, the double mutant has a ‘healthy’ phenotype without physiological impairments. Moreover, PaGlo1/PaGlo2_OEx strains are not long-lived on media containing standard glucose concentrations suggesting a tight correlation between the efficiency and capacity to remove MG within the cell, the level of available glucose and lifespan. Overall, our results identify the up-regulation of both components of the glyoxalase system as an effective intervention to increase lifespan in P. anserina. Key words: Podospora anserina, aging, lifespan, glycation, glucose, methylglyoxal, advanced glycation end product

The critical role of methylglyoxal and glyoxalase 1 in diabetic nephropathy

The discovery of increased formation of methylglyoxal (MG) by cell metabolism in high glucose concentration in vitro suggested possible relevance to diabetes and diabetes complications (1,2). MG is the precursor of quantitatively important advanced glycation end products (AGEs) of protein and DNA- and MG-derived AGEs increase in experimental and clinical diabetes (3,4). Increased MG and its metabolism by glyoxalase 1 (Glo1) was linked to clinical microvascular complications (nephropathy, retinopathy, and neuropathy) (5). Current clinical treatment decreasing MG and MG-derived AGEs, such as insulin lispro (6,7), has some clinical benefit in diabetic nephropathy (8), although the decrease in MG-derived AGE exposure is minor—∼17% (7). Greater benefits may be achieved with specific and effective anti-MG targeted therapy. An outstanding research problem is to gain unequivocal evidence that MG glycation is a key mediator of vascular complications and, if possible, provide some pointers as to how MG glycation could be effectively countered. In this issue, the study by Giacco et al. (9) provides key evidence by a functional genomic approach manipulating expression of Glo1 to increase or decrease endogenous MG glycation. The outcomes show that development of experimental diabetic nephropathy is driven by increased levels of MG glycation and increasing renal expression of Glo1 prevents this. Recent research has shown Glo1 expression may be increased by small molecule inducers (10). Taken together, these findings suggest that prevention and treatment of diabetic nephropathy and possibly other complications of diabetes may be improved by development of Glo1 inducers

Spectroscopic Studies on \u3cem\u3eArabidopsis\u3c/em\u3e ETHE1, a Glyoxalase II-like Protein

ETHE1 (ethylmalonic encephalopathy protein 1) is a β-lactamase fold-containing protein that is essential for the survival of a range of organisms. In spite of the apparent importance of this enzyme, very little is known about its function or biochemical properties. In this study Arabidopsis ETHE1 was over-expressed and purified and shown to bind tightly to 1.2 ± 0.2 equivalents of iron. 1H NMR and EPR studies demonstrate that the predominant oxidation state of Fe in ETHE1 is Fe(II), and NMR studies confirm that two histidines are bound to Fe(II). EPR studies show that there is no antiferromagnetically coupled Fe(III)Fe(II) center in ETHE1. Gel filtration studies reveal that ETHE1 is a dimer in solution, which is consistent with previous crystallographic studies. Although very similar in terms of amino acid sequence to glyoxalase II, ETHE1 exhibits no thioester hydrolase activity, and activity screening assays reveal that ETHE1 exhibits low level esterase activity. Taken together, ETHE1 is a novel, mononuclear Fe(II)-containing member of the β-lactamase fold superfamily

Ferricytochrome c Directly Oxidizes Aminoacetone to Methylglyoxal, a Catabolite Accumulated in Carbonyl Stress

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH4+ ion, and H2O2 coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. the participation of O-2(center dot-) and HO center dot radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5'-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E-0 values = -0.51 and -1.0 V) to ferricytochrome c (E-0 = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH4+ ion. in the presence of oxygen, aminoacetone enoyl and O-2(center dot-) radicals propagate aminoacetone oxidation to methylglyoxal and H2O2. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INCT Processos Redox em Biomedicina (Brazil)Univ São Paulo, Dept Bioquim, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim & Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, São Paulo, BrazilUniv São Paulo, Dept Fis & Informat, São Paulo, BrazilUniv Fed ABC, Ctr Ciencias Nat & Humanas, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim & Biol Mol, São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, São Paulo, BrazilWeb of Scienc