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In previous investigations an impact of cellular copper homeostasis on ageing of the ascomycete Podospora anserina has been demonstrated. Here we provide new data indicating that mitochondria play a major role in this process. Determination of copper in the cytosolic fraction using total reflection X-ray fluorescence spectroscopy analysis and eGfp reporter gene studies indicate an age-related increase of cytosolic copper levels. We show that components of the mitochondrial matrix (i.e. eGFP targeted to mitochondria) become released from the organelle during ageing. Decreasing the accessibility of mitochondrial copper in P. anserina via targeting a copper metallothionein to the mitochondrial matrix was found to result in a switch from a copper-dependent cytochrome-c oxidase to a copper-independent alternative oxidase type of respiration and results in lifespan extension. In addition, we demonstrate that increased copper concentrations in the culture medium lead to the appearance of senescence biomarkers in human diploid fibroblasts (HDFs). Significantly, expression of copper-regulated genes is induced during in vitro ageing in medium devoid of excess copper suggesting that cytosolic copper levels also increase during senescence of HDFs. These data suggest that the identified molecular pathway of age-dependent copper dynamics may not be restricted to P. anserina but may be conserved from lower eukaryotes to humans.
Potentiation of glycine-gated NR1/NR3A NMDA receptors relieves Ca2+-dependent outward rectification
(2010)
Glycine has diverse functions within the mammalian central nervous system. It inhibits postsynaptic neurons via strychnine-sensitive glycine receptors (GlyRs) and enhances neuronal excitation through co-activation of N-methyl-D-aspartate (NMDA) receptors. Classical Ca2+-permeable NMDA receptors are composed of glycine-binding NR1 and glutamate-binding NR2 subunits, and hence require both glutamate and glycine for efficient activation. In contrast, recombinant receptors composed of NR1 and the glycine binding NR3A and/or NR3B subunits lack glutamate binding sites and can be activated by glycine alone. Therefore these receptors are also named “excitatory glycine receptors”. Co-application of antagonists of the NR1 glycine-binding site or of the divalent cation Zn2+ markedly enhances the glycine responses of these receptors. To gain further insight into the properties of these glycine-gated NMDA receptors, we investigated their current-voltage (I–V) dependence. Whole-cell current-voltage relations of glycine currents recorded from NR1/NR3B and NR1/NR3A/NR3B expressing oocytes were found to be linear under our recording conditions. In contrast, NR1/NR3A receptors displayed a strong outwardly rectifying I–V relation. Interestingly, the voltage-dependent inward current block was abolished in the presence of NR1 antagonists, Zn2+ or a combination of both. Further analysis revealed that Ca2+ (1.8 mM) present in our recording solutions was responsible for the voltage-dependent inhibition of ion flux through NR1/NR3A receptors. Since physiological concentrations of the divalent cation Mg2+ did not affect the I–V dependence, our data suggest that relief of the voltage-dependent Ca2+ block of NR1/NR3A receptors by Zn2+ may be important for the regulation of excitatory glycinergic transmission, according to the Mg2+-block of conventional NR1/NR2 NMDA receptors. Keywords: NMDA receptor, excitatory glycine receptor, voltage block, NR3 subunit, supralinear potentiation, Zn2+, NR1 antagonist, ligand-binding domain
The editorial board of Aging reviews research papers published in 2009,which they believe have or will have a significant impact on aging research.Among many others, the topics include genes that accelerate aging or incontrast promote longevity in model organisms, DNA damage responsesand telomeres, molecular mechanisms of life span extension by calorierestriction and pharmacologic interventions into aging. The emergingmessage in 2009 is that aging is not random but determined by agenetically-regulated longevity network and can be decelerated bothgenetically and pharmacologically.
Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ~4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.
We solved the crystal structure of a novel type of c-ring isolated from Bacillus pseudofirmus OF4 at 2.5 Å, revealing a cylinder with a tridecameric stoichiometry, a central pore, and an overall shape that is distinct from those reported thus far. Within the groove of two neighboring c-subunits, the conserved glutamate of the outer helix shares the proton with a bound water molecule which itself is coordinated by three other amino acids of outer helices. Although none of the inner helices contributes to ion binding and the glutamate has no other hydrogen bonding partner than the water oxygen, the site remains in a stable, ion-locked conformation that represents the functional state present at the c-ring/membrane interface during rotation. This structure reveals a new, third type of ion coordination in ATP synthases. It appears in the ion binding site of an alkaliphile in which it represents a finely tuned adaptation of the proton affinity during the reaction cycle. Formal Correction: This article has been formally corrected to address the following errors. 1. The images for Figures S2 and S3 were incorrectly switched. The image that appears as Figure S2 should be Figure S3, and the image that appears as Figure S3 should be Figure S2. The figure legends appear in the correct order. Please view the correct... (read formal correction) 2. The images for Figures S2 and S3 were incorrectly switched. The image that appears as Figure S2 should be Figure S3, and the image that appears as Figure S3 should be Figure S2. The figure legends appear in the correct order. Please view the correct... (read formal correction)
Background: Falciparum Malaria, an infectious disease caused by the apicomplexan parasite Plasmodium falciparum, is among the leading causes of death and morbidity attributable to infectious diseases worldwide. In Gabon, Central Africa, one out of four inpatients have severe malarial anemia (SMA), a life-threatening complication if left untreated. Emerging drug resistant parasites might aggravate the situation. This case control study investigates biomarkers of enhanced hemolysis in hospitalized children with either SMA or mild malaria (MM). Methods and Findings: Ninety-one children were included, thereof 39 SMA patients. Strict inclusion criteria were chosen to exclude other causes of anemia. At diagnosis, erythrophagocytosis (a direct marker for extravascular hemolysis, EVH) was enhanced in SMA compared to MM patients (5.0 arbitrary units (AU) (interquartile range (IR): 2.2–9.6) vs. 2.1 AU (IR: 1.3–3.9), p<0.01). Furthermore, indirect markers for EVH, (i.e. serum neopterin levels, spleen size enlargement and monocyte pigment) were significantly increased in SMA patients. Markers for erythrocyte ageing, such as CD35 (complement receptor 1), CD55 (decay acceleration factor) and phosphatidylserine exposure (annexin-V-binding) were investigated by flow cytometry. In SMA patients, levels of CD35 and CD55 on the red blood cell surface were decreased and erythrocyte removal markers were increased when compared to MM or reconvalescent patients. Additionally, intravascular hemolysis (IVH) was quantified using several indirect markers (LDH, alpha-HBDH, haptoglobin and hemopexin), which all showed elevated IVH in SMA. The presence of both IVH and EVH predicted the need for blood transfusion during antimalarial treatment (odds ratio 61.5, 95% confidence interval (CI): 8.9–427). Interestingly, this subpopulation is characterized by a significantly lowered reticulocyte production index (RPI, p<0.05). Conclusions: Our results show the multifactorial pathophysiology of SMA, whereby EVH and IVH play a particularly important role. We propose a model where removal of infected and non-infected erythrocytes of all ages (including reticulocytes) by EVH and IVH is a main mechanism of SMA. Further studies are underway to investigate the mechanism and extent of reticulocyte removal to identify possible interventions to reduce the risk of SMA development.
Proteins can be acetylated at the alpha-amino group of the N-terminal amino acid (methionine or the penultimate amino acid after methionine removal) or at the epsilon-amino group of internal lysines. In eukaryotes the majority of proteins are N-terminally acetylated, while this is extremely rare in bacteria. A variety of studies about N-terminal acetylation in archaea have been reported recently, and it was revealed that a considerable fraction of proteins is N-terminally acetylated in haloarchaea and Sulfolobus, while this does not seem to apply for methanogenic archaea. Many eukaryotic proteins are modified by differential internal acetylation, which is important for a variety of processes. Until very recently, only two bacterial proteins were known to be acetylation targets, but now 125 acetylation sites are known for E. coli. Knowledge about internal acetylation in archaea is extremely limited; only two target proteins are known, only one of which--Alba--was used to study differential acetylation. However, indications accumulate that the degree of internal acetylation of archaeal proteins might be underestimated, and differential acetylation has been shown to be essential for the viability of haloarchaea. Focused proteomic approaches are needed to get an overview of the extent of internal protein acetylation in archaea.
Background: Current conventional vaccination approaches do not induce potent CD8 T-cell responses for fighting mostly variable viral diseases such as influenza, avian influenza viruses or HIV. Following our recent study on vaccine penetration by targeting of vaccine to human hair follicular ducts surrounded by Langerhans cells, we tested in the first randomized Phase-Ia trial based on hair follicle penetration (namely transcutaneous route) the induction of virus-specific CD8 T cell responses. Methods and Findings: We chose the inactivated influenza vaccine – a conventional licensed tetanus/influenza (TETAGRIP®) vaccine – to compare the safety and immunogenicity of transcutaneous (TC) versus IM immunization in two randomized controlled, multi-center Phase I trials including 24 healthy-volunteers and 12 HIV-infected patients. Vaccination was performed by application of inactivated influenza vaccine according to a standard protocol allowing the opening of the hair duct for the TC route or needle-injection for the IM route. We demonstrated that the safety of the two routes was similar. We showed the superiority of TC application, but not the IM route, to induce a significant increase in influenza-specific CD8 cytokine-producing cells in healthy-volunteers and in HIV-infected patients. However, these routes did not differ significantly for the induction of influenza-specific CD4 responses, and neutralizing antibodies were induced only by the IM route. The CD8 cell response is thus the major immune response observed after TC vaccination. Conclusions: This Phase Ia clinical trial (Manon05) testing an anti-influenza vaccine demonstrated that vaccines designed for antibody induction by the IM route, generate vaccine-specific CD8 T cells when administered transcutaneously. These results underline the necessity of adapting vaccination strategies to control complex infectious diseases when CD8 cellular responses are crucial. Our work opens up a key area for the development of preventive and therapeutic vaccines for diseases in which CD8 cells play a crucial role.