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K L Williams

Publications and source records attributed to K L Williams.

At least 73 records · Page 4Linked to original sources
PubMed1998

Development of a rapid and sensitive quantitative assay for rotavirus based on flow cytometry.

A very sensitive and accurate flow cytometry (FC) based method have developed to quantitate rotavirus infection in MA104 cells. Confluent cell monolayers were infected with serial dilutions of rotavirus SA11. After infection, the cells were recovered with the aid of trypsin and then reacted with monoclonal antibody M60 (specific for the rotavirus outer capsid protein, VP7), followed by a second antibody (anti-mouse IgG-FITC). A FACScan FC was used to estimate the number of infected cells, as well as the level of infection. Viral infection was optimised by varying the concentration of trypsin used in the maintenance medium. The FC method enables many cells to be screened quickly for infectivity, and can detect low levels of virus. This method can be adapted to monitor the presence of other viruses in clinical and environmental samples without the need for prolonged periods of adaptation to growth in tissue culture.

Animals↗
PubMed1998

NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility.

The specificities of the UDP-GalNAc:polypeptide Nacetylgalactosaminyltransferases which link the carbohydrate GalNAc to the side-chain of certain serine and threonine residues in mucin type glycoproteins, are presently unknown. The specificity seems to be modulated by sequence context, secondary structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. Charged residues were disfavoured at position -1 and +3. A jury of artificial neural networks was trained to recognize the sequence context and surface accessibility of 299 known and verified mucin type O-glycosylation sites extracted from O-GLYCBASE. The cross-validated NetOglyc network system correctly found 83% of the glycosylated and 90% of the non-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predictions of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based on the amino acid sequence. The server addresses are http://www.cbs.dtu.dk/services/NetOGlyc/ and netOglyc@cbs.dtu.dk.

Algorithms↗
PubMed1998

The use of a ribosomal RNA targeted oligonucleotide probe for fluorescent labelling of viable Cryptosporidium parvum oocysts.

A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescent labelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employs a fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence in the 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resulted in fluorescence of sporozoites within oocysts that were capable of excystation, while oocysts that were dead prior to fixation did not fluoresce. Correlation of the FISH method with viability as measured by in vitro excystation was statistically highly significant, with a calculated correlation coefficient of 0.998. Examination of sequence data for Cryptosporidium spp. other than C. parvum suggests that the Cry1 probe is C. parvum-specific. In addition, 19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1 probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresce after hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was not bright enough to enable detection of oocysts in environmental water concentrates containing autofluorescent algae and mineral particles. However, in combination with immunofluorescence staining, FISH enabled species-specific detection and viability determination of C. parvum oocysts in water samples.

Animals↗
PubMed1998

Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization.

Oligonucleotide probes specific to Cryptosporidium parvum (CRY1) were conjugated with a range of fluorochromes. The fluorescence after in situ hybridization (FISH) labelling of oocysts and controls was assessed. The objective was to determine the most suitable conjugate for FISH labelling, followed by analysis with a 488 nm laser flow cytometer. The most promising candidate was fluorescein isothiocyanate but only when linked to the CRY1 probe via an 18-carbon spacer arm consisting of six ethylene glycol moieties. The use of the spacer increased fluorescent signals fivefold compared with an equivalent probe in which the FITC was linked directly to the 5'-amino group of the DNA.

Animals↗
PubMed1998

Oral ethanol-reinforced responding in rhesus monkeys: effects of opioid antagonists selective for the mu-, kappa-, or delta-receptor.

To determine the mechanism by which naltrexone (NTX) reduces oral ethanol-reinforced responding, opioid antagonists that show mu-, kappa-, or delta-selectivity were evaluated. Rhesus monkeys (n = 6) were given opportunities to respond and receive ethanol (1% or 2%) or water during daily 3-hr drinking sessions. Before some drinking sessions, the monkeys received intramuscular injections of saline or the following drugs: the mu-selective irreversible antagonist clocinnamox (CCAM), the kappa-selective long-lasting antagonist nor-binaltorphimine (nor-BNI), or the delta-selective antagonist naltrindole. Also, NTX was administered along with either CCAM or nor-BNI. When given alone, CCAM (0.1 mg/kg) had no effect on ethanol-reinforced responding. When NTX (0.32 mg/kg) was given with CCAM, responding maintained by ethanol was decreased. Nor-BNI (3 mg/kg) reduced ethanol-reinforced responding only on the day of injection. On subsequent days, when other studies report continued kappa-antagonism, responding maintained by ethanol returned to control levels. Also, NTX (0.32 mg/kg), administered in the presence of nor-BNI, was still able to reduce ethanol-reinforced responding. Naltrindole failed to alter responding maintained by ethanol. Because selective antagonism at the mu-, kappa-, or delta-receptor did not reduce ethanol-reinforced responding, NTX's ability to reduce ethanol consumption may not be mediated by these previously characterized opioid receptors. NTX may exert its effects through an uncharacterized opioid binding site or through a nonopioid mechanism.

Alcohol Drinking↗
PubMed1998

Reversion from type 2 diabetes to nondiabetic status. Influence of the 1997 American Diabetes Association criteria.

OBJECTIVE: To determine the incidence and the rate of reversion of type 2 diabetes to a nondiabetic status in the 7- to 8-year follow-up of the San Antonio Heart Study, and to determine the influence of the recent 1997 American Diabetes Association (ADA) criteria for diabetes on these rates. Individuals who revert have been problematic for those developing criteria for the diagnosis of type 2 diabetes. Few studies have addressed this issue using 1979 National Diabetes Data Group/1980 World Health Organization (WHO) criteria. RESEARCH DESIGN AND METHODS: We studied 3,682 Mexican-American and non-Hispanic white men and nonpregnant women who completed both the baseline and follow-up examination of the San Antonio Heart Study. Incidence and reversion rates were calculated using both the 1980 WHO and the 1997 ADA criteria. Risk factors for reversion were identified, and the best fitting model using multiple logistic regression was determined using both the 1980 WHO and the 1997 ADA criteria. RESULTS: Using the 1997 ADA criteria, the age-adjusted incidences of type 2 diabetes for Mexican-American men and women were 10.8 and 12.2%, respectively. For non-Hispanic white men and women, the age-adjusted incidence rates were 5.5 and 5.1%, respectively. Similar age-adjusted incidences were recorded using the 1980 WHO criteria. The reversion rate for individuals with type 2 diabetes was 11.5% using the 1980 WHO criteria and 12.5% using the 1997 ADA criteria. These rates were not significantly different. Numerous risk factors for reversion were identified. The best fitting model, after controlling for age, sex, and ethnicity, included baseline 2-h glucose level, baseline HDL cholesterol, and previous diagnosis of diabetes. The models were the same for both the 1980 WHO and the 1997 ADA criteria. CONCLUSIONS: There was no significant difference in the incidence or the reversion rates for diabetic subjects using either 1980 WHO or 1997 ADA criteria. In addition, the risk factors for reversion were very similar using either set of criteria. The revision of the ADA criteria did not have a significant influence on reversion in this study.

Adult↗
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

Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum.

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we described the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution.

Amino Acid Sequence↗
PubMed1997

Cross-species protein identification using amino acid composition, peptide mass fingerprinting, isoelectric point and molecular mass: a theoretical evaluation.

Proteins can be identified by rapid techniques that do not involve Edman degradation sequencing. These approaches entail the matching of amino acid compositions or tryptic peptide masses of proteins against databases, often in conjunction with estimated protein molecular weight and isoelectric point. As genome sequencing projects progress, proteins from poorly molecularly defined organisms will increasingly be identified by cross-species comparison to proteins from well-defined organisms. To investigate the application of rapid techniques for cross-species protein identification, a total of 65 theoretical cross-species comparisons involving 21 proteins (nine human and 12 E. coli) were undertaken. The degree of conservation of amino acid composition, tryptic peptides, protein isoelectric point and mass was established. Protein amino acid composition was well conserved across species boundaries, whilst tryptic peptides were poorly conserved. The molecular weight of proteins was generally well conserved, but protein isoelectric point was not. These results suggest that cross-species protein identification by rapid techniques will be done best by protein amino acid composition and protein molecular weight.

Amino Acids↗
PubMed1997

An in vivo approach for the identification of acceptor sites for O-glycosyltransferases: motifs for the addition of O-GlcNAc in Dictyostelium discoideum.

To identify and analyze acceptor sequences for O-glycosylation, we have developed an in vivo system expressing short peptides as glutathione S-transferase fusion proteins in the eukaryotic host Dictyostelium discoideum. Using this approach, we show that a short peptide motif (PTVTPT), present in the D. discoideum cell-surface glycoprotein PsA, is sufficient as a signal for O-glycosylation, even when fused to a heterologous protein. Monosaccharide analysis and solid-phase protein sequencing showed that the modification is a single N-acetylglucosamine attached to threonine residues. This was further confirmed by electrospray-mass spectrometry. The O-linked glycosylation of both this peptide and authentic PsA presents the modB-dependent carbohydrate-specific epitope identified by the monoclonal antibody MUD50. Substitution of threonine by serine residues in this peptide also yields a glycosylated fusion protein which is modified with single N-acetylglucosamine residues, but not all of the serines are glycosylated.

Acetylglucosamine↗
PubMed1997

A role for Edman degradation in proteome studies.

Advances in protein database design and the software used to access the sequence data has led to progress in using protein attributes such as amino acid composition and peptide masses to identify proteins separated by two-dimensional electrophoresis. However, Edman degradation remains the principal technique for protein identification and it presents a significant bottleneck in the progress towards rapid protein identification. Simple modifications to the sequencing hardware, which automate the delivery of protein spots into the sequencer, and parallel sequencing of the protein spots represent a significant advance in the use of Edman degradation to rapidly generate the powerful protein attribute, an N-terminal sequence tag.

Amino Acid Sequence↗
PubMed1997

Identification of wallaby milk whey proteins separated by two-dimensional electrophoresis, using amino acid analysis and sequence tagging.

Micropreparative two-dimensional polyacrylamide gel electrophoresis has been used to separate milk whey proteins from the Tammar wallaby (Macropus eugenii). We have used a combination of amino acid analysis and N-terminal sequence tagging as a rapid and sensitive method to identify the major whey proteins. Using these techniques, we confidently identified alpha-lactalbumin and late lactation protein. While these are the only two M. eugenii whey proteins with a corresponding SWISS-PROT entry, we demonstrate that by using amino acid analysis and matching across species boundaries, we can identify previously unsequenced conserved wallaby whey proteins including beta-lactoglobulin and serum albumin.

Amino Acid Sequence↗
PubMed1997

Establishment of the human reflex tear two-dimensional polyacrylamide gel electrophoresis reference map: new proteins of potential diagnostic value.

To understand the changes in protein expression associated with various physiological states as well as the development of pathological eye disease, we have begun to map the protein components of normal human reflex tears. An analytical reference map of normal human reflex tears was created using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with pH 3.5-10 immobilized pH gradients (IPGs). Micropreparatively loaded gels were transferred to polyvinylidene difluoride (PVDF) and analysed by a combination of N-terminal sequence tagging and amino acid compositional analysis. Thirty spots were sequence tagged, resulting in identification of six different proteins (lipocalin, lysozyme, lactotransferrin, zinc-alpha-2 glycoprotein, cystatin S, cystatin SN) that matched to entries in the SWISS-PROT database. A group of N-terminally blocked proteins was clearly identified from SWISS-PROT by amino acid analysis, isoelectric point (pI) and molecular weight (Mr). A number of highly expressed protein components remain unidentified despite being subjected to amino acid analysis and Edman sequencing. A majority of the abundant proteins showed varying degrees of charge heterogeneity attributed to post-translational processing such as glycosylation and N-terminal truncation. We have identified a previously undescribed protein that we have named lacryglobin. This protein displays strong homology with mammaglobin, a protein overexpressed in breast cancer. The discovery of this homologue in tears offers the potential for disease diagnosis by screening tear fluid proteins.

Databases, Factual↗
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↗
PubMed1997

Comparison of liquid and supercritical fluid chromatography for the separation of enantiomers on chiral stationary phases.

Comparisons of liquid (LC) and supercritical fluid chromatography (SFC) were conducted using commercially available chiral stationary phases (CSPs) bearing three different types of chiral selectors. Chiral compounds of pharmaceutical and agricultural interest were used to probe advantages of limitations of SFC relative to LC for enantiomeric separations. Column equilibrium and parameter optimization were generally accomplished more rapidly in SFC than in LC. Although improved resolution was often observed in SFC, analysis times were not always lower in SFC than in LC. In some instances, SFC provided separation capabilities not readily accessible in LC.

Chromatography, Liquid↗
PubMed1997

A simple method for evaluating Cryptosporidium-specific antibodies used in monitoring environmental water samples.

A simple method is described for the evaluation and quality control of Cryptosporidium-specific antibodies used in monitoring environmental water samples. Purified oocysts were fluorescently labelled with a test antibody at the appropriate concentration. Labelled oocysts were analysed using flow cytometry and a region was defined on a bivariate dotplot of fluorescence versus light scatter that enclosed all oocysts. Concentrates of environmental water samples that did not contain oocysts were then incubated with the test antibody and analysed using flow cytometry. The number of particles that appeared in the region defined for oocysts was recorded and was a measure of non-specific binding. The technique provides a simple, rapid and quantitative tool for both evaluating the binding specificity of test antibodies and optimizing sample staining conditions.

Animals↗