Biomedical subjects

N Ponelies

Publications and source records attributed to N Ponelies.

7 recordsLinked to original sources

PubMed2006

Regulation of the ubiquitin proteasome system in mechanically injured human skeletal muscle.

Metabolic consequences of direct muscle trauma are insufficiently defined. Their effects on the ubiquitin-proteasome pathway (UPP) of protein degradation in human skeletal muscles are as yet unknown. Thus, we investigated whether the UPP is involved in the metabolic response evoked in directly traumatized human skeletal muscles. Biopsies were obtained from contused muscles after fractures and from normal muscles during elective implant removal (control). As estimated by western blot analyses, concentrations of free ubiquitin and ubiquitin protein conjugates were similar in extracts from injured and uninjured muscles. Ubiquitin protein ligation rates were reduced after injury (1.5+/-0.2 vs. 1.0+/-0.15 fkat/microg; p=0.04). Chymotryptic-, tryptic- and caspase-like proteasome peptidase activities (total activity minus activity in the presence of proteasome inhibitors) increased significantly after trauma (p=0.04 - 0.001). Significant increases in total chymotryptic- and caspase-like activities were attributable to proteasome activation. Our results extend the possible role of the UPP in muscle wasting to direct muscle trauma. They further suggest that the effects of direct mechanical trauma are not limited to the proteasome and imply that ubiquitin protein ligase systems are also involved. Based on the potential role of the UPP in systemic diseases, it might also be a therapeutic target to influence muscle loss in critically ill blunt trauma patients, in which large proportions of muscle are exposed to direct trauma.

Adolescent↗

PubMed2000

Physical localization of single-copy sequences on pachytene chromosomes in maize (Zea mays L.) by chromosome in situ suppression hybridization.

A new approach for locating single-copy DNA sequences on pachytene chromosomes of maize (Zea mays L.) was developed. A cosmid clone with homologous sequences to a molecular marker (umc105a) linked to a quantitative trait locus (QTL) for resistance against sugarcane borer (SCB) was physically mapped by fluorescence in situ hybridization (FISH) to the short arm of chromosome 9. The marker umc105a was genetically placed in the centromeric region. To suppress signals generated by maize repetitive DNA, competitive in situ suppression (CISS) hybridization was necessary to obtain specific signals from umc105a. A centromere specific DNA probe (CentC) was used in a double-labeling technique as a reference marker. Fluorescence signals generated by umc105a cosmid and CentC were specific and highly reproducible. Thus the single-copy DNA sequence of umc105a was physically localized on the short arm of chromosome 9 near the telomere. This is the first report of physical localization of single-copy DNA sequence by CISS hybridization to a maize pachytene chromosome.

Chromosome Mapping↗

PubMed1997

Microamplification of specific chromosome sequences; an improved method for genome analysis.

An improved method was developed for microdissection and cloning of metaphase as well as pachytene chromosomes. The protocol incorporates efficient ligation of chromosomal DNA with linker adaptors, abolishment of microcloning steps and the reduction of micromanipulation. The threshold for amplifying genomic DNA template was in the range of 2-20 femtogram. The amplification products had a size distribution between 200 and 1300 bp (average 500 bp). Using pachytene chromosomes of maize the selectivity for segment-specific libraries can be increased between 10- and 20-fold. The approach described here is being applied to the fine mapping and isolation of genes conveying resistance against plant pathogens.

Chromosomes↗

PubMed1994

Laser micromanipulators for biotechnology and genome research.

The use of lasers for complete micromanipulation of metaphase chromosomes, cells and subcellular structures is reviewed. DNA probes from single microdissected chromosome segments can be prepared using Alu or Adaptor PCR. In plant biotechnology, laser microsurgery can be used to prepare non-enzymatically protoplasts from Medicago sativa. Microgravity can be simulated in the alga Chara by lifting intracellular gravity transmitting elements with the optical tweezers.

Base Sequence↗

PubMed1991

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes.

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.

Cells, Cultured↗

PubMed1989

Telomeric sequences derived from laser-microdissected polytene chromosomes.

Telomeric fragments from salivary gland squashes of Drosophila melanogaster Oregon R. were produced by a new microdissection technique, UV laser microbeam dissection. Microdissection, an essential step in microcloning procedures, is usually performed using micromanipulators and microneedles. Recently it has been shown that microdissection can be improved to very high precision if a laser coupled into a microscope is used. A laser microbeam, generated by an excimer pumped dye laser, allows chromosomes to be cut into slices of less than 0.5 micron. Here it is shown, that single copy DNA probes prepared from Drosophila chromosomes by laser microdissection and microcloning relocalize to the chromosomal regions from which they are derived. The combination of laser technique and microcloning provides an advantageous approach for rapid genetic analysis with potential for the study of genetic diseases and genome mapping.

Animals↗

PubMed1988

The nasal polyps as a tool for basic research in cystic fibrosis.

Total RNA and mRNA were prepared from cystic fibrosis (CF) and control nasal polyps and nasal epithelial cells. Genomic clones from the chromosomal region of the CF locus were screened by northern blots. A representative cDNA library from nasal polyps was cloned in the vector lambda gt10. For the construction of a physical genomic map around the CF locus single gene markers were isolated from metaphase 1:7q2qter chromosomes by laser micro-dissection and subsequent microcloning. A linkage study with the polymorphic markers met-H, met-D, and pJ3.11 was performed in 53 German CF families with at least 2 children. No significant correlation of any haplotype on the CF chromosomes with the clinical severity of the course of the disease could be observed, which provides evidence that cystic fibrosis is genetically homogeneous.

Chromosome Mapping↗