Molecular control of copper homeostasis in filamentous fungi: increased expression of a metallothionein gene during aging of Podospora anserina.

The lifespan of the ascomycete Podospora anserina was previously demonstrated to be significantly increased in a copper-uptake mutant, suggesting that copper is a potential stressor involved in degenerative processes. In order to determine whether changes in copper stress occur in the cells during normal aging of cultures, we cloned and characterized a gene coding for a component of the molecular machinery involved in the control of copper homeostasis. This gene, PaMt1, is a single-copy gene that encodes a metallothionein of 26 amino acids. The coding sequence of PaMt1 is interrupted by a single intron. The deduced amino acid sequence shows a high degree of sequence identity to metallothioneins of the filamentous ascomycete Neurospora crassa and the basidiomycete Agaricus bisporus, and to the N-terminal portion of mammalian metallothioneins. Levels of PaMt1 transcript increase in response to elevated amounts of copper in the growth medium and during aging of wild-type cultures. In contrast, in the long-lived mutant grisea, transcript levels first increase but then decrease again. The ability of wild-type cultures to respond to exogenous copper stress via the induction of PaMt1 transcription is not affected as they grow older.

Copper-modulated gene expression and senescence in the filamentous fungus Podospora anserina.

We have previously shown that the control of cellular copper homeostasis by the copper-modulated transcription factor GRISEA has an important impact on the phenotype and lifespan of Podospora anserina. Here we demonstrate that copper depletion leads to the induction of an alternative respiratory pathway and to an increase in lifespan. This response compensates mitochondrial dysfunctions via the expression of PaAox, a nuclear gene coding for an alternative oxidase. It resembles the retrograde response in Saccharomyces cerevisiae. In P. anserina, this pathway appears to be induced by specific impairments of the copper-dependent cytochrome c oxidase. It is not induced as the result of a general decline of mitochondrial functions during senescence. We cloned and characterized PaAox. Transcript levels are decreased when cellular copper, superoxide, and hydrogen peroxide levels are raised. Copper also controls transcript levels of PaSod2, the gene encoding the mitochondrial manganese superoxide dismutase (PaSOD2). PaSod2 is a target of transcription factor GRISEA. During the senescence of wild-type strain s, the activity of PaSOD2 decreases, whereas the activity of the cytoplasmic copper/zinc superoxide dismutase (PaSOD1) increases. Collectively, the data explain the postponed senescence of mutant grisea as a defined consequence of copper depletion, ultimately leading to a reduction of oxidative stress. Moreover, they suggest that during senescence of the wild-type strain, copper is released from mitochondria. The involved mechanism is unknown. However, it is striking that the permeability of mitochondrial membranes in animal systems changes during apoptosis and that mitochondrial proteins with an important impact on this type of cellular death are released.

[Day-care and day-treatment in psychogeriatrics. Changes in the characteristics of users in the course of a year]. / Dagverzorging en dagbehandeling in de psychogeriatrie. Veranderingen in de kenmerken van gebruikers in de loop van een jaar.

Users of day-treatment had a different need for care than users of day-care. The behaviour measured by the GIP was different on the cognitive and social scales: 100 users of day-treatment showed more disturbed behaviour than 63 users of day-care. Geronte also showed that they were in need of more help than users of day-care: at the start and also after twelve months with respect to their mental, physical and social functions as well as in home keeping. Users who left the facility within one year showed a lower level of selfcontainment at the start compared with users still visiting the facility after one year. The need profile of users leaving day-care resembled that of the users that started day-treatment. Fifteen percent of users who left day-care directly started day-treatment.

Cellular copper homeostasis, mitochondrial DNA instabilities, and lifespan control in the filamentous fungus Podospora anserina.

In the fungal aging model Podospora anserina, lifespan is controlled by mitochondrial and nuclear genetic traits. Different nuclear genes are known to affect the integrity of the mitochondrial DNA (mtDNA). One gene of this type is Grisea encoding a copper-modulated transcription factor involved in the control of cellular copper homeostasis. The characterization of a long-lived mutant with a loss-of-function mutation in this gene revealed that the last step in the pathway, homologous recombination, leading to the characteristic age-related mtDNA reorganizations is copper-dependent. In growing parts of the culture, the stabilization of the mtDNA has an important impact on the biogenesis of functional mitochondria, on their capacity to remodel damaged respiratory chains and on longevity.

Mitochondrial oxidative stress and aging in the filamentous fungus Podospora anserina.

In the filamentous fungus Podospora anserina, mitochondrial oxidative stress is a major contributor to aging. Reactive oxygen species (ROS) generated as a result of electron leakage during respiration lead to damage of components of the electron transport chain. In aging wild-type cultures, damaged proteins cannot be replaced because the mitochondrial genes encoding some of the corresponding subunits gradually become deleted from the mitochondrial DNA (mtDNA). Consequently, these defects result in an increased generation of reactive oxygen species and respiration deficits leading to cell death. Analyses of wild-type strains and of different long-lived mutants of P. anserina provide strong evidence that molecular mechanisms controlling aging processes in this fungus are complex and act at different levels. A basic mechanism (e.g., damage by ROS) appears to be overlaid by prominent instabilities of the mtDNA.

Copper-dependence of mitochondrial DNA rearrangements in Podospora anserina.

Rearrangements of the mitochondrial DNA (mtDNA) are a hallmark of senescence in wild-type strains of the ascomycete P. anserina. These rearrangements include the systematic amplification of the first intron (p1-intron) of the cytochrome oxidase subunit-I gene (CoI) as a circular DNA molecule (p1DNA). In addition, deletions and amplifications of other regions of the mtDNA occur. The molecular basis of the underlying processes is not understood in detail. A comparative analysis of the wild-type strain and of the long-lived mutant grisea, affected in the uptake of copper, revealed that mtDNA instabilities are dependent on the availability of cellular copper. In the mutant, the first steps in the corresponding pathway, including the transcription of the CoI gene, the splicing of the p1-intron and the transposition of this mobile element, are not impaired. In contrast, recombination processes between short direct repeats, as well as rearrangements between two tandem intron copies leading to the formation of p1DNA, appear to be affected. Additional copper in the growth medium rescues this molecular phenotype. We suggest that copper is a cofactor of a component of the molecular machinery leading to the characteristic age-related mtDNA rearrangements.

[Instrument for determination and planning of care]. / Een instrument ter bepaling en visualisering van zorgbehoefte.

Geronte is a French instrument to assess and visualize the need of care of elderly people. In 1995 Geronte was translated into Dutch. In order to be able to determine differences in the need of care, it was investigated whether the Dutch Geronte is able to discriminate between the users of different care-providing institutions. Five groups of fifteen elderly people in five different situations were examined: residents with predominantly psycho-geriatric impairment, residents with predominantly somatic impairment and residents of a home for the elderly, elderly people living at home with home help and elderly people living at home without any assistance. The Dutch Geronte proved to be able to reveal differences between the users of the different types of care, with the exception of residents of the home for the elderly and elderly people living at home with home help. The Dutch Geronte proved to be useful in the assessment and visualization of the need of care of elderly people.

GRISEA, a copper-modulated transcription factor from Podospora anserina involved in senescence and morphogenesis, is an ortholog of MAC1 in Saccharomyces cerevisiae.

The initial characterization of Grisea suggested that this gene codes for a transcription factor involved in the genetic control of cellular copper homeostasis in Podospora anserina. Here we demonstrate that GRISEA activates in vivo gene expression in Saccharomyces cerevisiae and is characterized by a modular organization. The DNA-binding domain was mapped to the first 168 N-terminal amino acids and the transactivation domain to the C-terminal half of the protein. Increased levels of copper in the growth medium lead to repression of the transactivation function possibly via intramolecular interactions between parts of the DNA-binding domain and the transactivation domain. The wild-type copy of Grisea was found to complement the phenotype of the mac1-1 mutant of S. cerevisiae. GRISEA is able to bind to the promoter of CTR1, a MAC1 target gene that encodes a high-affinity copper transporter. Taken together, the data reported here and in earlier investigations indicate that GRISEA is an ortholog of the yeast transcription factor MAC1 and suggest at least a partial conservation of the molecular machinery involved in the control of cellular copper homeostasis in eukaryotes. Remarkably, in P. anserina, the spectrum of phenotypes affected by this regulatory protein is much broader than that known in yeast and includes morphogenetic traits as well as lifespan and senescence.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea.

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5' upstream region of the grisea ORF. One mutation is a G --> A transition at the 5' splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

Genomic cloning and characterization of the nonoccupied allele corresponding to the integration site of human papillomavirus type 16 DNA in the cervical cancer cell line SiHa.

Human papillomavirus (HPV) type 16 DNA sequences have been found integrated into the host cell genome in a large number of cervical tumors and cell lines derived therefrom. In this study, we have cloned and analyzed the nonoccupied allele corresponding to the integration site of HPV-16 in the cervical cancer cell line SiHa. Our mapping analyses revealed an approximately 7.8-kb deletion of cellular DNA upon viral integration. Computer analysis of 2.3 kb of DNA sequences from the deleted genomic region as well as 1.0 kb of sequences upstream of the viral integration site showed no significant homology to any known human sequences. DNase I mapping experiments on native chromatin demonstrated the existence of two hypersensitive sites in both the HPV-16-containing and nonoccupied alleles located approximately 1.1 and 1.7 kb upstream of the viral integration site. This suggests that viral integration occurred close to putative regulatory sequences and that recombination with host cellular DNA was not followed by a reorganization of the chromatin structure upstream of the integration site. Nuclear run-on and RT-PCR experiments showed HPV-specific transcription spanning the E2, E4, E5, and L1/L2 open reading frames (ORFs) located upstream of the HPV-16 regulatory region (URR). Taken together, our data suggest that the cellular DNA region upstream of the HPV-16 integration site in the SiHa cell line contains regulatory elements affecting transcription of HPV-16 ORFs located upstream of the HPV-16 URR.