PubMed Health⌕ Search

Biomedical subjects

K R Griffiths

Publications and source records attributed to K R Griffiths.

5 recordsLinked to original sources
PubMed2000

Sequencing and expression of additional xylanase genes from the hyperthermophile Thermotoga maritima FjSS3B.1.

Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.

Bacteria↗
PubMed1997

Evaluation of fluorochromes and excitation sources for immunofluorescence in water samples.

Fluorescent labelling methods for detecting microorganisms in water have limited sensitivity partly due to the natural autofluorescence from environmental particles. The aim of this study was to examine the autofluorescence of water samples to determine the optimal excitation source and fluorescent labels for minimising background autofluorescence and therefore enhancing sensitive detection of Cryptosporidium oocysts. Particles concentrated from water were examined using fluorimetry at a wide range of excitation wavelengths to determine their autofluorescent properties. Two major peaks were identified emitting at 390 to 510 nm and at 640 to 700 nm. Flow cytometry was used to define the optical properties of oocysts immunofluorescently labelled with a range of fluorochromes. Concentrated water samples were analysed using flow cytometry and the number of particles with fluorescence and light scatter properties similar to the fluorescently labelled oocysts recorded. Fluorescein isothiocyanate exited at 488 nm was the most suitable label for oocysts in untreated water with less than 70 particles having optical properties similar to labelled oocysts, detected in 10 litre concentrates. The fluorochromes CY3, phycoerythrin (PE), and tetramethylrhodamine B thioisocyanate (TRITC) excited at 542 nm were the most suitable labels for oocysts in drinking water with less than 40 particles having optical properties similar to labelled oocysts, detected in 100 litre concentrates.

Animals↗
PubMed1997

Simple and rapid measurement of Cryptosporidium excystation using flow cytometry.

In vitro excystation is commonly used to determine the viability of samples of purified Cryptosporidium parvum oocysts. Following exposure to conditions that stimulate excystation, samples are examined microscopically to determine the number of excysted oocysts. The microscopy procedure is tedious and time consuming, and difficult to apply to most oocyst samples without a purification step. A simple flow cytometric method was developed for determining the numbers of oocysts that had excysted following the in vitro excystation procedure. Differences in light-scatter properties were used to differentiate intact, partially empty and empty oocysts. By staining samples with a monoclonal antibody specific to the oocyst wall it was possible to apply the technique to unpurified oocysts from faeces. Correlation of the flow cytometric and microscopic method was statistically significant (P < 0.05), resulting in a calculated correlation coefficient of 0.994. The flow cytometry method is faster and more sensitive than the microscopy procedure, and enables analysis of large numbers of samples and of many thousands of oocysts in each sample.

Animals↗
PubMed1996

Isolation of glycosylation mutants in Dictyostelium discoideum using flow cytometry.

Fluorescence-activated cell sorting was used to isolate five spores of the soil amoeba Dictyostelium discoideum that carried new glycosylation mutations, which were produced by restriction enzyme-mediated integration (REMI)-induced gene disruption and which occurred at frequencies of around 10(-5). These mutations were identified by the loss of an O-glycosylation epitope found on surface proteins of wild type D. discoideum spores that is recognised by the monoclonal antibody MUD62. A secondary antibody conjugated to the fluorochrome fluorescein isothiocyanate identified MUD62 bound to spores. Spores lacking this epitope did not fluoresce, allowing this population to be separated. Samples were found to contain around 0.1% of viable spores that were wild type but lacked the MUD62 epitope at the time of sorting. To remove these spores from the unlabelled population, samples were labelled with monoclonal antibody MUD50, which recognises surface proteins on immature spores and proteins exposed from an inner coat layer. Double labelling with MUD50 and MUD62 reduced the unlabelled viable population to less than 0.002%, allowing the glycosylation-defective spores to be isolated. This is the first use of a selective approach to isolate nonmorphological REMI-induced mutants in D. discoideum. This study also characterises the surface properties of spore types found in mature fruiting bodies of D. discoideum.

Animals↗