Leisure activities of adolescents-associations with demographic characteristics, well-being and parental leisure engagement.

Background: The present study aimed to investigate associations between leisure activities and well-being, behavioral difficulties, and parental leisure time engagement.

Methods: A total of 959 adolescents aged 10 to 18 years were included in the present study. We assessed their leisure activities (e.

Open the Pores: Electroporation for the Transformation of Trichoderma reesei.

During the electroporation of T. reesei, linearized exogenous DNA is absorbed into swollen conidia by an electrical impulse. The advantage of this method is that it is less time-consuming, less expensive, and easier to perform than the classical protoplast transformation while at the same time having a comparable efficiency.

Deletion of the small GTPase in provokes hyperbranching and impacts growth and cellulase production.

Background: is widely known for its enormous protein secretion capacity and as an industrially relevant producer of cellulases and hemicellulases. Over the last decades, rational strain engineering was applied to further enhance homologous and heterologous enzyme yields. The introduction of hyperbranching is believed to increase protein secretion, since most exocytosis is located at the hyphal apical tip.

The Promoter Toolbox for Recombinant Gene Expression in .

The ascomycete is one of the main fungal producers of cellulases and xylanases based on its high production capacity. Its enzymes are applied in food, feed, and textile industry or in lignocellulose hydrolysis in biofuel and biorefinery industry. Over the last years, the demand to expand the molecular toolbox for to facilitate genetic engineering and improve the production of heterologous proteins grew.

Highly active promoters and native secretion signals for protein production during extremely low growth rates in Aspergillus niger.

Background: The filamentous ascomycete Aspergillus niger is used in many industrial processes for the production of enzymes and organic acids by batch and fed-batch cultivation. An alternative technique is continuous cultivation, which promises improved yield and optimized pipeline efficiency.

Results: In this work, we have used perfusion (retentostat) cultivation to validate two promoters that are suitable for A.

Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in Aspergillus.

In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is activated in a titratable manner by addition of the tetracycline derivative doxycycline. The complementary Tet-off system utilises the tetracycline-controlled transactivator (tTA) component to quantitatively reduce gene expression. In this study, we utilised a synthetic biological approach to engineer highly optimised Tet-off conditional expression systems in Aspergillus niger and Aspergillus fumigatus.

Engineering of for the production of secondary metabolites.

Background: Filamentous fungi can each produce dozens of secondary metabolites which are attractive as therapeutics, drugs, antimicrobials, flavour compounds and other high-value chemicals. Furthermore, they can be used as an expression system for eukaryotic proteins. Application of most fungal secondary metabolites is, however, so far hampered by the lack of suitable fermentation protocols for the producing strain and/or by low product titers.

Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeast.

Oxidative protein folding can exceed the cellular secretion machinery, inducing the unfolded protein response (UPR). Sustained endoplasmic reticulum (ER) stress leads to cell stress and disease, as described for Alzheimer, Parkinson, and diabetes mellitus, among others. It is currently assumed that the redox state of the ER is optimally balanced for formation of disulfide bonds using glutathione as the main redox buffer and that UPR causes a reduction of this organelle.

Fungal gene expression on demand: an inducible, tunable, and metabolism-independent expression system for Aspergillus niger.

Filamentous fungi are the cause of serious human and plant diseases but are also exploited in biotechnology as production platforms. Comparative genomics has documented their genetic diversity, and functional genomics and systems biology approaches are under way to understand the functions and interaction of fungal genes and proteins. In these approaches, gene functions are usually inferred from deletion or overexpression mutants.

Cellular organelle transport and positioning by plasma streaming.

Our analysis of known data reveals that translocations of passively movable cellular organelles from tiny granules up to large cell nuclei can be ascribed to transport by streaming cytoplasm. The various behaviours, such as velocity changes during more or less interrupted movements, forth and back shuttling and particle rotation result from different types of plasma circulation. Fast movements over long distances, as observed in the large characean internodial cells occur in strong streams generated by myosin in bundles of actin filaments in the direction of the barbed filament ends.

Force generation by cellular motors.

Cell motility processes in non-muscle cells depend on the activity of motor proteins that bind to either microtubules or actin filaments. From presently available data it must be concluded that the driving force is generated by transient interaction of the respective motors with microtubules or actin filaments which then activates the binding and hydrolysis of ATP. This reaction results in an abrupt discharge of the motor molecule, the direction of which is determined by the spatial orientation of its binding to the helical and polar vehicle.

Functional aspects of the nuclear matrix.

A model is proposed of the way in which the unwinding of the chromosomal DNA loops is controlled during DNA replication. It is based on the observation of a permanent binding of replication origins to the nuclear matrix and of a transient attachment of replicating DNA regions to sites in the immediate neighbourhood. DNA unwinding is controlled while the replicating loops are reeled through the replication binding sites.

Control of eukaryotic DNA replication at the chromosomal level.

A hypothesis for the control of eukaryotic DNA replication at the chromosomal level is proposed. The specific regulatory problem arises from the subdivision of the genome into thousands of individually replicating units, each of which must be duplicated a single time during S-phase. The hypothesis is based on the finding of direct repeats at replication origins.

Direct repeats at nuclear matrix-associated DNA regions and their putative control function in the replicating eukaryotic genome.

Short DNA regions, known to contain replication origins, were isolated from 2 M NaCl resistant nuclear structures of Physarum polycephalum after predigestion with DNase. Regions of 100 bp average length were cloned and sequenced. About 25% of the clones contained direct repeats of 12 to 16 bp and variable base sequences, that have been shown to possess the potential of playing a crucial role in the control of DNA replication.

Induction of DNA replication in mammalian G1 nuclei by a heat-inactivible component from permeable S phase cells; an assay system.

The in vitro initiation of DNA replication was studied in permeable mammalian cells by a newly developed procedure. Pairs of monolayer cultures, one synchronized in G1 and the other in S phase, were incubated in a sandwich with assay solution, containing Triton X-100 for permeabilization and [3H]TTP as a tracer. After 1.

Nuclear matrix and chromosome scaffold preparations of in vitro cultured bovine liver cells have two proteins in common.

Nuclear matrices and chromosome scaffolds of in vitro cultured bovine liver cells were prepared under conditions that preserve the specific binding of the DNA. Protein compositions were analysed by electrophoresis and peptide mapping. Two slightly acidic polypeptides of apparent molecular masses 47 and 53 kDa were present in nuclear matrix as well as chromosome scaffold preparations.

Evidence for the persistence of a decondensed chromosome scaffold in the interphase nucleus.

Nuclei of in vitro cultured bovine liver cells, deprived of the membranes by Triton X-100, were treated with 2 M-NaCl and DNase. Changes in ultrastructure and protein composition were studied at successive steps during treatment. Electron micrographs of nuclei treated with 2 M-NaCl showed a peripheral lamina and an internal system of randomly coiled filaments embedded in a mass of DNA fibres.

Evidence for contamination of origin DNA isolated after an in vivo treatment of mammalian cells with 4,5',8-trimethylpsoralen.

We have studied the effect of in vivo treatment with trioxsalen on DNA replication in mammalian cells. In vitro cultured bovine liver cells were exposed to two or four cycles of treatment with 45 microM trioxsalen followed by irradiation with long-wave ultraviolet light. Thymidine incorporation was reduced by about 95% during the first hour after a double treatment.

Chromatin degradation in isolated nuclei of normal and transformed baby hamster kidney cells.

In a transformed cell line, derived from baby hamster kidney cells by treatment with ethylnitrosourea, degradation of DNA in isolated nuclei by endogenous nuclease was studied. Compared to the nontransformed cell line, the nuclear DNA of the transformed cells was found to be degraded to a much greater extent. This was reflected by a markedly lower proportion of DNA attached to the nuclear protein matrix in the transformed compared to the nontransformed cells.

Protein composition of the chromosomal scaffold and interphase nuclear matrix.

Residual protein structures were prepared from isolated chromosomes and interphase nuclei of in vitro cultured bovine liver cells and the protein compositions were analysed. Chromosomes with minimal cytoplasmic contamination were obtained by a simple procedure using a pH 8 isolation medium containing Triton X-100 and polyamines, and residual protein-DNA complexes were prepared by extraction with 2 M NaCl. Residual protein structures were also obtained by digesting isolated chromosomes with staphylococcal nuclease.

Newly-initiated DNA isolated from Physarum in early S phase consists of nascent-nascent duplexes.

We have studied the nature of newly initiated DNA released during DNA isolation at the beginning of S phase of Physarum polycephalum. The released DNA was separated from the bulk DNA by sedimentation through sucrose gradients. Gentle shearing strongly enhanced the release of newly initiated DNA.

In vitro DNA replication in association with the nuclear matrix of permeable mammalian cells.

DNA replication has been studied in in vitro cultured bovine liver cells permeabilized in 0.02% Triton X-100. The Km for TTP was 20 microM.

Attachment of origins of replication to the nuclear matrix and the chromosomal scaffold.

We have investigated the attachment of DNA to the nuclear matrix and chromosomal scaffold in synchronized bovine liver cells. Label incorporated at the onset of the S phase remained preferentially associated with the matrix during the subsequent G1 phase and with a residual protein structure from dehistonized chromosomes during mitosis. On the other hand label incorporated during mid or late S phase was about equally distributed over the DNA molecule after a chase into the G1 phase.

Organization of DNA replication in Physarum polycephalum. Attachment of origins of replicons and replication forks to the nuclear matrix.

We have investigated the attachment of the DNA to the nuclear matrix during the division cycle of the plasmodial slime mold Physarum polycephalum. The DNA of plasmodia was pulse labelled at different times during the S phase and the label distribution was studied by graded DNase digestion of the matrix-DNA complexes prepared from nuclei isolated by extraction with 2 M NaCl. Pulse labelled DNA was preferentially recovered from the matrix bound residual DNA at any time of the S phase.