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M R Davey

Publications and source records attributed to M R Davey.

At least 19 recordsLinked to original sources
PubMed1997

Detection of single-copy genes in DNA from transgenic plants by nonradioactive Southern blot analysis.

Improvements to the sensitivity, speed, and reproducibility of digoxigenin (DIG)-labeled probes and chemiluminescent substrates makes these compounds increasingly popular to detect nucleic acids. High sensitivity and low background are essential in Southern blot analysis, particularly with plant DNA. This article describes a nonradioactive system to detect single-copy genes in transgenic plants. Labeling using the polymerase chain reaction (PCR) was employed to produce highly sensitive and reusable DIG-labeled probes. The background was reduced by immobilizing the DNA onto nylon filters by alkaline transfer and by minimized gel handling; the signal-to-noise ratio was improved by modification of the detection procedure.

Blotting, Southern↗
PubMed1997

Pharmacological activity of feverfew (Tanacetum parthenium (L.) Schultz-Bip.): assessment by inhibition of human polymorphonuclear leukocyte chemiluminescence in-vitro.

The bioactivity of feverfew (Tanacetum parthenium) leaf extracts has been analysed, by use of a human polymorphonuclear leukocyte (PMNL) bioassay, to assess the relative contributions of solvent extraction and parthenolide content to the biological potency of the extract. Extracts prepared in acetone-ethanol (system 1) contained significantly more parthenolide (mean +/- s.d. 1.3 +/- 0.2% dry leaf weight) than extracts in chloroform-PBS (phosphate-buffered saline; system 2; 0.1 +/- 0.04% dry leaf weight) or PBS alone (system 3; 0.5 +/- 0.1% dry leaf weight). Extract bioactivity, measured as inhibition of phorbol 12-myristate 13-acetate-induced, 5-amino-2,3-dihydro-1,4-phthalazinedione (luminol)-enhanced PMNL, chemiluminescence, followed a similar trend. Extracts inhibited phorbol 12-myristate 13-acetate-induced oxidative burst by amounts which, if solely attributable to parthenolide, indicated parthenolide concentrations for the respective solvent systems of 2.2 +/- 0.6%, 0.2 +/- 0.1% and 0.9 +/- 0.1% dry leaf weight. The mean ratio of parthenolide concentration to the parthenolide equivalent/PMNL-bioactivity value, for acetone-ethanol and PBS extracts were both 1:1.7. Parthenolide, although a key determinant of biological activity for T. parthenium leaf extracts based on the PMNL-bioassay, seems not to be the sole pharmacologically-active constituent. The identical and elevated bioactivity-parthenolide ratios for both organic and aqueons-phase leaf extracts suggest that a proportion of the other bioactive compounds have solubilities similar to that of parthenolide.

Chromatography, High Pressure Liquid↗
PubMed1997

Haemoglobin-enhanced mitotic division in cultured protoplasts.

Protoplasts from cell suspensions of albino Petunia hybrida cv. Comanche were cultured for 9 days in nutrient medium containing Erythrogen, a purified bovine haemoglobin solution (supplied at 10% w/v) at 1:50-1:500 (v/v). In some assessments, the non-ionic surfactant Pluronic F-68 (Poloxamer 188), was also added to the culture medium at 0.01-1.0% (w/v). Erythrogen at 1:50 (v/v) increased the mean initial protoplast plating efficiency (IPE; 18.5 +/- 0.8%, n = 5 throughout) by 64% (P < 0.001) above that of controls (11.3 +/- 0.4%). Supplementation of medium with 1:50 (v:v) Erythrogen and 0.01% (w/v) Pluronic F-68, increased the mean IPE (24.4 +/- 1.4%) by 92% (P < 0.001) over control (12.7 +/- 1.1%). Similar results were obtained for mesophyll protoplasts of Passiflora suberosa, with 1:50 and 1:100 (v/v) Erythrogen increasing the mean IPEs to 87% and 93% respectively, over controls. This beneficial and synergistic effect of Erythrogen with Pluronic F-68, on mitotic division of cultured Petunia and Passiflora protoplasts, should also facilitate the culture of isolated protoplasts and cells of other, agronomically-important, species.

Animals↗
PubMed1997

Changes in cell biochemistry in response to culture of protoplasts with oxygenated perfluorocarbon.

Superoxide dismutase (superoxide oxidoreductase; EC 1.15.1.1; SOD) was measured in enzymatically isolated protoplasts of Salpiglossis sinuata following culture in aqueous nutrient medium overlaying oxygen-gassed perfluorodecalin (Flutec PP6; BNFL Fluorochemicals, UK). SOD was extracted from harvested, lysed protoplast-derived cells after 1, 3, 7 and 14 days of culture and assayed spectrophotometrically. Protoplasts cultured with oxygenated PFC (+/- s.e.m, n = 5) showed significant increases in mean SOD activity to 4.2 +/- 0.1 U after 1 day (P < 0.05) and 9.3 +/- 0.7 U after 3 days (P < 0.01), with a fall in mean SOD after 7 days (5.1 +/- 0.9 U), similar to control. The decrease in SOD after 7 days correlated closely with a progressive fall in pO2 in the PFC phase over the same period. In contrast, control protoplasts (medium alone) or protoplasts cultured in medium overlaying non-oxygenated PFC showed no significant changes in mean SOD activity over the 14-day culture assessment period.

Cells, Cultured↗
PubMed1997

Perfluorochemicals and cell biotechnology.

Perfluorochemical (PFC) liquids have properties, especially high gas solubility, which make these compounds useful in medicine and biotechnology. PFCs are being employed to facilitate respiratory gas supply to both prokaryotic and eukaryotic cells and, in some systems, to improve biomass production and yields of commercially-important cellular products. Animal (including human) and plant cells have also been cultured at the interface between PFC liquids and aqueous culture medium, while fluorocarbon polymers have been employed as gaspermeable membranes in eukaryotic cell cultures. This paper presents an overview of the applications and beneficial effects of PFCs in microbial, animal and plant culture systems. PFCs have been compared with other physical and chemical options for manipulating respiratory gas supply to cultured cells. PFC-facilitated improvements in cell culture technology will have increasingly important biotechnological implications.

Animals↗
PubMed1997

Beneficial effects of oxygenated fluorocarbon on the in vitro culture of protoplasts and cell electrofusion products.

Electrofused Passiflora protoplasts (P. edulis, P. giberti) were plated in KPR medium overlaying oxygen-gassed perfluorodecalin (Flutec PP6). Oxygenated PFC significantly (P < 0.05) enhanced protoplast division, as reflected by an increase in mean plating efficiency of up to 62% (P < 0.05) over 14 days. After 21 days of culture, the liquid phase containing dividing protoplast-derived cells, was removed from the PFC surface and overlaid onto MS-based agar medium for callus proliferation. Forty days later, protoplast-derived calli were transferred to one of two regeneration protocols, previously determined using unfused parental protoplasts. Calli derived from electrofusion-treated protoplasts exhibited organogenesis or somatic embryogenesis, depending on the regeneration procedure. The regeneration efficiency after 121 days for protoplasts initially cultured with oxygenated PFC was over 2-fold greater (P < 0.01) than control. These results indicate that oxygenated PFC can enhance growth and regeneration of protoplasts and their fusion products.

Cell Fusion↗
PubMed1996

Preservation of viable biological samples for experiments in space laboratories.

Standard viable preservation methods for biological samples using low temperatures have been investigated concerning their storage capabilities under higher temperature levels than usual. For a representative set of organism classes (plants, mammalian cells, arthropods and aquatic invertebrates), the minimum appropriate storage conditions have been identified by screening storage temperatures at -196 degrees, -80 degrees, -20 degrees, +4 degrees, +20 degrees/25 degrees C for periods from 2 days to 4 weeks. For storage below 0 degree C, as a typical cryopreservative, dimethylsulfoxide (DMSO) was used. For some samples, the addition of trehalose (as cryopreservative) and the use of a nitrogen atmosphere were investigated. After storage, the material was tested for vitality. The findings demonstrated that acceptable preservation can be achieved under higher storage temperatures than are typically applied. Small, dense cultured plant cells survive for 21 d when moderately cooled (+4 degrees to -20 degrees C); addition of trehalose enhances viability at -20 degrees C. For mammalian cells, the results show that human lymphocytes can be preserved for 3 d at 25 degrees C, 7 d at 4 degrees C and 28 d at -80 degrees C. Friend leukaemia virus transformed cells can be stored for 3 d at 25 degrees C, 14 d at 4 degrees C and 28 d at -80 degrees C. Hybridoma cells can be kept 7 d at 4 degrees C and 28 d at -20 degrees C or -80 degrees C. Model arthropod systems are well preserved for 2 weeks if maintained at lower temperatures that vary depending on the species and/or stage of development; e.g., 12 degrees C for Drosophila imagoes and 4-6 degrees C for Artemia nauplii. For aquatic invertebrates such as sea urchins, embryonic and larval stages can be preserved for several weeks at +6 degrees C, whereas sperm and eggs can best be stored at + 4 degrees C for up to 5 d at maximum. These results enhance the range of feasible space experiments with biological systems. Moreover, for typical terrestrial preservation methods, considerable modification potential is identified.

Animals↗
PubMed1995

Enhanced protoplast growth at the interface between oxygenated fluorocarbon liquid and aqueous culture medium supplemented with pluronic F-68.

Protoplasts isolated from cell suspensions of albino Petunia hybrida were grown for 10 d at the interface between aqueous culture medium and oxygenated perfluorodecalin. Protoplasts synthesised new cell walls and divided normally at the fluorocarbon/culture medium interface, the mean division frequency of protoplasts being increased by 37% (P < 0.05) in this system when the perfluorodecalin was saturated with oxygen prior to use. The mean division frequency of protoplasts was further increased to a maximum of 52% above control (P < 0.01) when the medium overlaying the oxygenated perfluorodecalin was supplemented with 0.01% (w/v) of the co-polymer surfactant, Pluronic F-68. These results demonstrate a beneficial and synergistic effect of supplementing protoplast culture systems with oxygenated perfluorodecalin and Pluronic F-68.

Cell Division↗
PubMed1994

Transformation of Solanum dulcamara protoplasts and regeneration of transgenic plants.

We have achieved successful transformation of Solanum dulcamara protoplasts by direct DNA uptake and regeneration of transgenic plants. The plasmids pDW2 carrying CAT gene and pCaMVNEO carrying NPTII gene were used. The electroporation voltage was 1500 V, which gave a field strength of 1500 V/cm with a time decay constant of 59.4 sec. The concentration of plasmid was 20 micrograms/2 x 10(6) protoplasts. Under these conditions, a very high transformation efficiency was obtained, with relative transformation frequency being up to 12.4% and absolute transformation frequency 2.4 x 10(-4). The activity of NPTII was detected in 75% of the kanamycin resistant calli and all of the plants regenerated from resistant calli. Southern blot analysis showed that the DNA sequence of NPTII gene derived from pCaMVNEO plasmid existed in the transformed plants of S. dulcamara.

Blotting, Southern↗
PubMed1994

Equipment for the large-scale electromanipulation of plant protoplasts.

Electric fields are now used extensively for the genetic manipulation of plant cells through protoplast fusion and direct gene uptake. The cost of commercially available electrofusion and electroporation equipment remains prohibitive for many laboratories. This paper describes an electronic apparatus, suitable for the large-scale electrofusion and electroporation of plant protoplasts that is compatible in both function and cost with commercially available equipment.

Biomedical Engineering↗
PubMed1992

Quantification and comparison of chloramphenicol acetyltransferase activity in transformed plant protoplasts using high-performance liquid chromatography- and radioisotope-based assays.

Rice and petunia leaf and cell suspension protoplasts were transformed by electroporation in the presence of pDW2. This plasmid contains a chloramphenicol acetyltransferase (CAT) coding region under the control of a promoter constructed from sequences of the cauliflower mosaic virus genome. We have compared two different approaches to measuring CAT activity in this system, namely high-performance liquid chromatography (HPLC) and a radioisotope-based method. Our results show that both techniques have a similar detection limit of 10 mU CAT and (with an activity greater than 10 mU CAT) the standard error for measuring known amounts of CAT activity was less than +/- 12% for both assays. The HPLC technique, however, has a greater linear response range of 10-600 mU CAT than the radioisotope method, which has a range of 10-400 mU CAT. The HPLC assay also requires a shorter assay time. As a result of this work we believe that HPLC is a viable alternative to the radioisotope-based assay described.

Cells, Cultured↗
PubMed1991

Design and use of synthetic oligonucleotide probes in the cloning of delta-endotoxin genes from Bacillus thuringiensis.

A detailed protocol is described for the design and use of synthetic oligonucleotide probes for screening DNA libraries from Bacillus thuringiensis var. kurstaki (strain HD191) for copies of the gene (tox) encoding the insecticidal delta-endotoxin. Two homologous tox genes were identified in this organism; one of these was located on a 75-kb plasmid and the other on a second large plasmid or the bacterial chromosome. A tox gene was isolated as a 6.5-kb HindIII fragment of B. thuringiensis plasmid DNA.

Bacillus thuringiensis↗
PubMed1991

Surface exposure of O1 serotype lipopolysaccharide in Klebsiella pneumoniae strains expressing different K antigens.

Surface exposure of the O1 serotype lipopolysaccharide in encapsulated Klebsiella pneumoniae strains belonging to different serotypes was examined by using the O1 antigen-specific bacteriophages FC3-1 and FC3-2 in conjunction with immunogold electron microscopy and enzyme immunoassays with specific antisera. Despite the presence of the capsular polysaccharide, the O1 antigen was exposed at the cell surface in strains producing K2, K7, K8, K12, K19, K21, K22, K34, K35, K42, K45, K55, K57, K62, K66, K69, and K70 capsular polysaccharides. However, in strains producing K1, K10, and K16 capsular polysaccharides, the O1 antigen was masked by the K antigen. These results suggest that, since the O1 antigen is surface exposed in many different strains of K. pneumoniae with different capsular serotypes and is also able to immunoprotect, its potential as a useful vaccine component should not be overlooked.

Animals↗