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V Regnier

Publications and source records attributed to V Regnier.

15 recordsLinked to original sources
PubMed2001

Creation and characterization of temperature-sensitive CENP-C mutants in vertebrate cells.

CENP-C is an evolutionarily conserved centromere protein that is thought to be an important component in kinetochore assembly in vertebrate cells. However, the functional role of CENP-C in cell cycle progression remains unclear. To further understand CENP-C function, we developed a method incorporating the hyper-recombinogenic chicken B lymphocyte cell line DT40 to create several temperature-sensitive CENP-C mutants in DT40 cells. We found that, under restrictive conditions, one temperature-sensitive mutant, ts4-11, displayed metaphase delay and chromosome missegregation but proceeded through the cell cycle until arrest at G(1) phase. Furthermore, ts4-11 cells were transfected with a human HeLa cell cDNA library maintained in a retroviral vector, and genes that suppressed the temperature-sensitive phenotype were identified. One of these suppressor genes encodes SUMO-1, which is a ubiquitin-like protein. This finding suggests that SUMO-1 may be involved in centromere function in vertebrate cells. The novel strategy reported here will be useful and applicable to a wide range of proteins that have general cell-autonomous function in vertebrate cells.

Animals↗
PubMed2001

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells.

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.

Amino Acid Sequence↗
PubMed2000

Electroporation-mediated delivery of 3'-protected phosphodiester oligodeoxynucleotides to the skin.

The feasibility of topical delivery in the skin of 3' end modified phosphodiester oligonucleotides using electroporation was investigated. Experiments were performed in vitro, using hairless rat skin. Five pulses of (200 V, 450 ms) were applied. The 3' end modifications of the 15 mer oligonucleotide were: (1) 3'-aminohexyl, (2) biotin, with a triethyleneglycol arm, (3) methylphosphonate links between nucleotides 13, 14 and 15, and (4) 2-O-methyl nucleotides at 13, 14 and 15 positions. All the modifications were efficient to protect the oligonucleotides against degradation in the skin. Electroporation increased the topical delivery of the 3' end-modified phosphodiesters by two orders of magnitude compared to passive diffusion, without significant differences between the derivatives. Oligonucleotide concentrations in the range of 1 microm could be achieved in the viable skin. The delivery of a phosphorothioate congener was lower than phosphodiester delivery due to the interaction of phosphorothioate with the stratum corneum. Consequently, 3' end-protected phosphodiesters could be an interesting alternative to phosphorothioate oligonucleotides for topical treatment of cutaneous diseases.

Administration, Topical↗
PubMed1999

Mechanisms of a phosphorothioate oligonucleotide delivery by skin electroporation.

Skin electroporation has great potential for topical delivery of oligonucleotides. Controled therapeutic levels of an intact phosphorothioate oligonucleotide (PS) can be reached in the viable tissue of the skin. The aim of this work was to investigate the transport mechanisms of a PS in hairless rat skin by electroporation, and hence to allow optimization of oligonucleotides (ONs) topical delivery. The pulsing condition used was five exponentially-decaying pulses of 100 V and 500 ms pulse time. The main mechanism of PS transport in the skin viable tissues during pulsing was electrophoresis. The electroosmosis contribution was negligible. Electrophoresis created within minutes a reservoir of PS in the skin viable tissues, which persisted within a therapeutic range of hours. A strong PS/stratum corneum interaction occurred.

Animals↗
PubMed1998

Localization of a FITC-labeled phosphorothioate oligodeoxynucleotide in the skin after topical delivery by iontophoresis and electroporation.

PURPOSE: The aim of this study was to verify the hypothesis that the application of high voltage to the skin enhances both stratum corneum and keratinocyte permeability. Therefore, the transport of FITC labelled phosphorothioate oligonucleotides (FITC-PS) administered by passive diffusion, iontophoresis or electroporation was localized. METHODS: Fluorescent microscopy and laser scanning confocal microscopy were used to visualize the FITC-PS transport at the tissue and cell level respectively in hairless rat skin after electroporation (5 x (200 V approximately 500 ms) or iontophoresis (same amount of charges transferred). RESULTS: FITC-PS did not penetrate the viable skin by passive diffusion. Molecular transport in the skin upon electroporation or iontophoresis was localized and implied mainly hair follicles for iontophoresis. In the stratum corneum, the pathways for FITC-PS transport were more transcellular during electroporation and paracellular during iontophoresis. FITC-PS were detected in the nucleus of the keratinocytes a few minutes after pulsing. In contrast, iontophoresis did not lead to an uptake of the oligomer. CONCLUSIONS: The internalization of FITC-PS in the keratinocytes after electroporation confirms the hypothesis and suggests that electroporation, which allows both efficient topical delivery and rapid cellular uptake of the oligonucleotides, might be useful for antisense therapy of epidermal diseases.

Administration, Topical↗
PubMed1998

Parameters controlling topical delivery of oligonucleotides by electroporation.

Electroporation, using high voltage electrical pulses has been recognized as a powerful method for delivering macromolecules such as DNA and proteins in cells, or smaller molecules through the skin. Transdermal electroporation could combine targeted delivery of drugs to the skin and permeabilization of skin cells, suggesting that electroporation could be an interesting alternative for topical delivery of oligonucleotides. This work is devoted to the determination of the electroporation parameters that allow optimal delivery of oligonucleotides to the viable tissues of hairless rat skin in vitro. Phosphorothioate derivatives were preferred to the phosphodiester congeners as the former were found to be much less degraded when extracted from the tissues. Long duration (100-500 ms)--medium voltage (100-200 V)--exponentially decaying pulses appeared to be the best conditions for delivering oligonucleotides to the skin. The oligonucleotide quantity permeating the viable tissues of the skin was controlled by the selection of the electrical parameters of the pulses (voltage, pulse time and number of pulses) or by the ON concentration in the donor compartment. After delivery by electroporation, therapeutic levels of oligonucleotides were reached in the viable tissues of the skin (above 1 microM or 10 microM in intact or stripped skin respectively). Taken together, our results show that electroporation could be an interesting method for the delivery of oligonucleotides to the skin.

Administration, Cutaneous↗
PubMed1994

1994 Architectural Design awards. Windemere Nursing & Rehabilitation Center, Martha's Vineyard; Harold & Patricia Toppel Center for Life Enhancement, Miami; Westminster Place, Oakmont, Pa.

At the 11th annual Architectural Design Awards, sponsored by Contemporary Long Term Care, the following projects were singled out for their innovative designs: New nursing home with 130 beds or less: Windemere Nursing and Rehabilitation Center on Martha's Vineyard. New nursing home with more than 130 units: Harold & Patricia Toppel Center for Life Enhancement, Miami, Fla. Renovated retirement center with 150 beds or less: Westminster Place, Oakmont, Pa. No awards were given in one nursing-home and three retirement-center categories. The competition was held on March 20 in the offices of Backen Arrigoni & Ross of San Francisco. Following is an overview of the competition and a narrative on each of the three entries honored for notable innovations in design.

Aged↗