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Biomedical subjects

R Walmsley

Publications and source records attributed to R Walmsley.

18 recordsLinked to original sources
PubMed2000

Application of yeast cells transformed with GFP expression constructs containing the RAD54 or RNR2 promoter as a test for the genotoxic potential of chemical substances.

Yeast strains transformed with high copy number plasmids carrying the gene encoding a green fluorescent protein optimised for yeast (yEGFP3) under the control of the RAD54 or RNR2 promoter were used to investigate the activity of potentially DNA-damaging substances. The assays were performed on 96-well microtitre plates in the presence of different concentrations of the test substances. The synthesis of GFP protein was measured through the fluorescence signal and cell growth was monitored by absorption. Here, we demonstrate that this system can be used as a biosensor to assess the genotoxic potential of drugs and other chemical substances. The use of microtitre plates will enable full automation of the system and allows the inclusion of internal reference standards in each assay.

Biosensing Techniques↗
PubMed2000

Regulation of tyrosinase expression and activity in cultured human retinal pigment epithelial cells.

The purpose of this study was to investigate the regulation of tyrosinase gene expression and activity in cultured human retinal pigment epithelial (RPE) cells. The tyrosinase promoter (Ty.prom) region (400 bp) was PCR amplified and cloned into a modified mammalian expression vector (pcDNA3.1) upstream of a firefly luciferase (Luc) cDNA and was designated 'pcDNA3.1-Ty.prom.Luc'. The plasmid was co-transfected into RPE cells with a second mammalian expression plasmid (pRL-TK) containing a herpes simplex virus thymidine kinase promoter region upstream of Renilla Luc in a protocol designated the 'dual luciferase assay' (DLA). After co-transfection, cells were treated with a range of potential melanogenic agents; basic fibroblast growth factor (bFGF), methyl methane sulphonate, alpha-melanocyte stimulating hormone, verapamil, phorbol myristate acetate, cholera toxin (CT), pigment epithelium derived factor (PEDF), and L-tyrosine. The expression of tyrosinase promoter and enzymatic activities were determined 48 hr post-transfection using the DLA and DOPA oxidase assays, respectively. Tyrosinase activity could not be detected in RPE cells with any of the treatments. Tyrosinase promoter activity was significantly up-regulated in RPE cells treated with bFGF, PEDF, verapamil, CT and tyrosine compared with control cells. In conclusion, the tyrosinase gene is not only expressed but can be regulated in response to different chemicals in cultured human RPE cells. However, it appears that RPE cells in culture lack a post-transcriptional and/or translational modification point(s), which are necessary for tyrosinase enzymic activity.

Adjuvants, Immunologic↗
PubMed2000

Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells.

PURPOSE: To optimise the high efficiency, non-viral transfer of DNA to retinal pigment epithelial (RPE) cells in vitro. METHODS: A mammalian expression vector (pcDNA3.1) containing a firefly luciferase (luc) cDNA was used to transfect RPE cells using different chemical methods; calcium phosphate, DEAE-dextran and, liposomes-based transfection techniques. Transfection was optimised for both dose and time of exposure. The efficiency of gene transfer and cytotoxicity was measured 48 hours post-transfection using luciferase and MTT assays, respectively. The percentage of transfected cells (using optimal conditions) was determined with a construct expressing a jellyfish green fluorescent protein (GFP) using flow cytometery. RESULTS: Calcium phosphate and DEAE-dextran techniques failed to transfect the vector and led to high cytotoxicity. Liposomes-based methods successfully transferred the vector to RPE cells, but the efficiency varied for different liposomes; Tfx-50 > Lipofectin > Lipofectamine > Cellfectin > DMRIE-C. No significant cytotoxicity was observed with any of the liposome treatments. Optimal transfection was achieved with Tfx-50 at a 3:1 ratio of DNA:liposome; between 12-15% of cells being transfected. CONCLUSIONS: Efficient and non-toxic transfer of functional genes into primary RPE cells in vitro can be successfuly achieved by liposomes-based techniques. Tfx-50 appears to be a promising non-viral vector for RPE gene transfer.

Aged↗
PubMed1999

Application of an in vitro model to evaluate bioadhesion of fibroblasts and epithelial cells to two different dressings.

The cellular component of a healing wound consists of many cell types and the environment in which these cells grow is important to the rate and quality of healing which can be influenced by the type of dressing used. The most commonly used dressings are traditional gauze-type dressings. In many cases these dressings may adhere to the wound surface, and subsequent removal is often traumatic, causing pain and tissue reinjury. Some modern gelling dressings have been developed to overcome this adherence problem. In order to evaluate in more detail cell-dressing interactions, an in vitro model has been developed utilising wound fibroblasts and epithelial cells. Quantitative evaluation of adherence of cells cultured with a traditional gauze or a new gelling dressing has been undertaken using radiolabel and manual counting techniques. Scanning electron microscopy has been used to visualise the cells adherent to dressings allowing evaluation of their adhesion-morphology. The results show differential attachment of cells to viscose and gelling fibres of the dressings; considerably reduced cell adhesion to the gelling fibre was evident, and it was apparent that cells adhered predominantly to the viscose component of the dressing. This model can be used to investigate and compare the adhesion of cells to different dressings and their components.

Animals↗
PubMed1999

Clinical correlation of a skin antisepsis model.

The use of pigskin as a test substrate for evaluating topical antimicrobial activity has been developed. Simulated handwashing protocols with this in vitro model in parallel with in vivo studies have been evaluated, based on an ASTM method for the clinical evaluation of a healthcare personnel handwash. Using Serratia marcescens as the test organism, similar log reductions were observed using the in vitro model when compared to in vivo efficacy. Results suggest that this model can be used as a reliable indicator of antiseptic efficacy on the skin. The use of sterilized skin simplifies the use of this model for both efficacy and skin-pathogen interaction studies.

Adolescent↗
PubMed1998

The first births and ongoing pregnancies associated with sperm cryopreservation within evacuated egg zonae.

This new procedure principally aims to avoid a second or possibly multiple surgical procedures for sperm extraction from the male partner in cases of limited amounts of sperm cells, where normal freeze-thaw protocols would fail. Patients (n = 34) diagnosed as azoospermic, extreme oligozoospermic, or oligoasthenozoospermic underwent the process of sperm cryopreservation within evacuated egg zonae. Other samples were allocated to conventional sperm freezing. Sperm samples were acquired using testicular sperm extraction (TESE), microepididymal sperm aspiration (MESA), or fresh ejaculate. Subsequently, five of these 34 couples have undergone in-vitro fertilization (IVF) and achieved normal fertilization using post-thawed spermatozoa frozen under zonae pellucidae in conjunction with intracytoplasmic sperm injection (ICSI). The average fertilization rate for the post-thaw injected spermatozoa was 65%. This is comparable with the regular fertilization rate of 65% for combined MESA and TESE using fresh spermatozoa. All patients underwent embryo transfer. The average implantation rate per embryo was 31%; nearly the same for regular MESA/TESE ICSI cycles (32%). The first pregnancy associated with this procedure concluded with the full term delivery of healthy twin girls on July 18, 1997. The remaining four thaw procedures resulted in another twin delivery, an ongoing singleton gestation, a negative pregnancy test and a biochemical pregnancy respectively.

Adult↗
PubMed1993

The nucleotide sequence of genes involved in the leucine biosynthetic pathway of Clostridium pasteurianum.

A 2.2 kb SphI/ClaI fragment of the Clostridium pasteurianum chromosome has previously been cloned and shown to complement leuB401 and leuC171 mutations in Escherichia coli. The nucleotide sequence of this fragment has been determined (2327 bp) and carries three open reading frames. The products of translation of these reading frames display significant homologies with the alpha-isopropylmalate isomerase subunit (leuD) gene of Salmonella typhimurium, the beta-isopropylmalate dehydrogenase (leuB) genes of several organisms, and the dihydroxyacid dehydrase (ilvD) gene of E. coli.

3-Isopropylmalate Dehydrogenase↗
PubMed1988

The heart of the newborn child: an anatomical study based upon transverse serial sections.

This study of the newborn child heart is based mainly on observations made on thin (0.2 mm) serial transverse sections of an entire thorax. Several features of the cardiovascular system associated with the fetal circulation are discussed. Despite considerable differences between the cardiac form in the newborn child and that in later postnatal life, the orientation of the heart in the newborn child has already attained many of its postnatal features. For example, it lies more nearly in the horizontal than the vertical plane and the atrial and ventricular septa are in line with each other at an angle of about 45 degrees to the median plane. This angulation of the septa determines that the right atrium and ventricle lie as much in front of the corresponding chambers of the left heart as they do to their right side. The cardiothoracic ratio and thoracic index are derived from a section through the thorax of the newborn child and these values are compared with those from a section through an adult man. A major purpose of this communication is to make a plea that the nomenclature given to all cardiac structures, even in the newborn child, should be consistent with the heart in situ in the anatomical position.

Adult↗