Age-related cellular copper dynamics in the fungal ageing model Podospora anserina and in ageing human fibroblasts

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

Unmasking a temperature-dependent effect of the P. anserina i-AAA protease on aging and development

Different molecular pathways involved in maintaining mitochondrial function are of fundamental importance to control cellular homeostasis. Mitochondrial i-AAA protease is part of such a surveillance system, and PaIAP is the putative ortholog in the fungal aging model Podospora anserina. Here, we investigate the role of PaIAP in aging and development. Deletion of the gene encoding PaIAP resulted in a specific phenotype. When incubated at 27°C, spore germination and fruiting body formation are not different from that of the corresponding wild-type strain. Unexpectedly, the lifespan of the deletion strain is strongly increased. In contrast, cultivation at an elevated temperature of 37°C leads to impairments in spore germination and fruiting body formation and to a reduced lifespan. The higher PaIAP abundance in wild-type strains of the fungus grown at elevated temperature and the phenotype of the deletion strain unmasks a temperature-related role of the protein. The protease appears to be part of a molecular system that has evolved to allow survival under changing temperatures, as they characteristically occur in nature

An Interdisciplinary Diagnostic Approach to Guide Therapy in C3 Glomerulopathy

Since the re-classification of membranoproliferative glomerulonephritis the new disease entity C3 glomerulopathy is diagnosed if C3 deposition is clearly dominant over immunoglobulins in immunohistochemistry or immunofluorescence. Although this new definition is more orientated at the pathophysiology as mediated by activity of the alternative complement pathway C3 glomerulopathy remains a heterogenous group of disorders. Genetic or autoimmune causes are associated in several but not in all patients with this disease. However, prognosis is poorly predictable, and clinicians cannot directly identify patients that might benefit from therapy. Moreover, therapy may range from supportive care alone, unspecific immune suppression, plasma treatment, or plasma exchange to complement inhibition. The current biopsy based diagnostic approaches sometimes combined with complement profiling are not sufficient to guide clinicians neither (i) whether to treat an individual patient, nor (ii) to choose the best therapy. With this perspective, we propose an interdisciplinary diagnostic approach, including detailed analysis of the kidney biopsy for morphological alterations and immunohistochemical staining, for genetic analyses of complement genes, complement activation patterning in plasma, and furthermore for applying novel approaches for convertase typing and complement profiling directly in renal tissue. Such a combined diagnostic approach was used here for a 42-year-old female patient with a novel mutation in the Factor H gene, C3 glomerulopathy and signs of chronic endothelial damage. We present here an approach that might in future help to guide therapy of renal diseases with relevant complement activation, especially since diverse new anti-complement agents are under clinical investigation

L' expression linguistique du doute dans les "Essais" de Montaigne (une approche polyphonique)

POITIERS-BU Droit Lettres (861942101) / SudocSudocFranceF

Examining the validity of numerical ratios in loudness fractionation

Udgivelsesdato: MAYDirect psychophysical scaling procedures presuppose that observers are able to directly relate a numerical value to the sensation magnitude experienced. This assumption is based on fundamental conditions (specified by Luce, 2002), which were evaluated experimentally. The participants' task was to adjust the loudness of a 1-kHz tone so that it reached a certain prespecified fraction of the loudness of a reference tone. The results of the first experiment suggest that the listeners were indeed able to make adjustments on a ratio scale level. It was not possible, however, to interpret the nominal fractions used in the task as "true" scientific numbers. Thus, Stevens's (1956, 1975) fundamental assumption that an observer can directly assess the sensation magnitude a stimulus elicits did not hold. In the second experiment, the possibility of establishing a specific, strictly increasing transformation function that related the overt numerals to the latent mathematical numbers was investigated. The results indicate that this was not possible for the majority of the 7 participants

The copper-binding metallothionein PaMT1 is successfully targeted into the mitochondrial matrix.

<p>(A) The expression-level in the transformants T4 and T5, both express PaMT1 directed into the matrix, is comparable to wild-type s grown under pronounced copper-stress (+250 µM CuSO₄) (upper panel). Porin was used as control for equal loading (middle panel) and absence of genomic DNA in RNA samples (lower panel). (B) <i>In situ</i> immunodetection of PaMT1-His verifies its mitochondrial localization in T4 (Bf: brightfield images) Scale bar: 10 µm. (C) Western blot analysis shows that his-tagged PaMT1 in transformant T4 is targeted to mitochondria. α-Porin: mitochondrial loading control. (D) Total copper content of mitochondria is unaffected in the matrix-PaMT1 transformant T4 (filled bar) compared to wild-type s (open bar) as measured by TXRF.</p

Analysis of senescence biomarkers in WI-38 human diploid fibroblasts (HDFs) incubated with copper sulfate (CuSO₄) at 500 µM for 16 h.

<p>(A) Senescence-associated β-galactosidase. Control (CTL): cells not exposed to CuSO₄ are considered as 100%. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. ***: p<0.001. (B) Estimation of the proliferative potential. At 48 h after the end of incubation, 10,000 cells were incubated for 24 h with 1 µCi [³H]-thymidine. Mean value±SD of three independent experiments. Statistical analysis: Student's t-test. *: 0.01</p

The abundance of mRNA of the copper-regulated genes (<i>MT2A</i>, <i>Hsp70</i> and <i>PrP</i>) is increased in WI-38 human diploid fibroblasts (HDFs) incubated with copper salts (CuSO₄ or CuCl₂) at 500 µM for 16 h.

<p>Sodium sulfate (Na₂SO₄) and sodium chloride (NaCl) were used as control salts. CTL represented the same treatment without copper or sodium salts. The results of real-time PCR are expressed as fold induction compared to controls (CTL). RPL13A, ribosomal protein L13A, was used as housekeeping gene. The presented graph is representative of two independent experiments.</p