Biomedical subjects

K L Williams

Publications and source records attributed to K L Williams.

At least 37 records · Page 2Linked to original sources

PubMed2000

Multiple O-glycoforms on the spore coat protein SP96 in Dictyostelium discoideum. Fuc(alpha1-3)GlcNAc-alpha-1-P-Ser is the major modification.

A decreased level of fucosylation on certain spore coat proteins of Dictyostelium discoideum alters the permeability of the spore coat. Here the post-translational modifications of a major spore coat protein, SP96, are studied in a wild type strain (X22) and a fucosylation-defective mutant (HU2470). A novel phosphoglycan structure on SP96 of the wild type strain, consisting of Fuc(alpha1-3)GlcNAc-alpha-1-P-Ser(,) was identified by electrospray ionization mass spectrometry and NMR. It was shown using monosaccharide and gas chromatography mass spectrometry analysis that SP96 in the mutant HU2470 contained approximately 20% of wild type levels of fucose, as a result of a missing terminal fucose on the novel glycan structure. The results support previous predictions, based on inhibition studies on different fucose-deficient strains, about the nature of monoclonal antibody epitopes identified by monoclonal antibodies MUD62 and MUD166, which are known to identify O-linked glycans (Champion, A., Griffiths, K., Gooley, A. A., Gonzalez, B. Y., Gritzali, M., West, C. M., and Williams, K. L. (1995) Microbiology 141, 785-797). Quantitative studies on wild type SP96 indicated that there were approximately 60 sites with phosphodiester-linked N-acetylglucosamine-fucose disaccharide units and a further approximately 20 sites with fucose directly linked to the protein. Over 70% of the serine sites are modified, with less than 1% of these sites as phosphoserine. Threonine and tyrosine residues were not found to be modified.

Amino Acids↗

PubMed2000

A major susceptibility locus influencing plasma triglyceride concentrations is located on chromosome 15q in Mexican Americans.

Although several genetic forms of rare or syndromic hypertriglyceridemia have been reported, little is known about the specific chromosomal regions across the genome harboring susceptibility genes for common forms of hypertriglyceridemia. Therefore, we conducted a genomewide scan for susceptibility genes influencing plasma triglyceride (TG) levels in a Mexican American population. We used both phenotypic and genotypic data from 418 individuals distributed across 27 low-income, extended Mexican American families. For the analyses, TG values were log transformed (ln TG). We used a variance-components technique to conduct multipoint linkage analyses for localizing susceptibility genes that determine variation in TG levels. We used an approximately 10-15-cM map, which was made on the basis of information from 295 microsatellite markers. After accounting for the effects of sex and sex-specific age terms, we found significant evidence for linkage (LOD = 3.88) of ln TG levels to a genetic location between the markers GABRB3 and D15S165 on chromosome 15q. This putative locus explains 39.7+/-7% (P=.000012) of total phenotypic variation in ln TG levels. Suggestive evidence was found for linkage of ln TG levels to two different locations on chromosome 7, which are approximately 85 cM apart from each other. Also, there is some evidence for linkage of high-density lipoprotein cholesterol concentrations to a genetic location near one of the regions on chromosome 7. In conclusion, we found strong evidence for linkage of ln TG levels to a genetic location on chromosome 15q in a Mexican American population, which is prone to disease conditions such as type 2 diabetes and the insulin-resistance syndrome that are associated with hypertriglyceridemia. This putative locus appears to have a major influence on ln TG variation.

Adult↗

PubMed2000

Proteomics: capacity versus utility.

Until recently scientists studied genes or proteins one at a time. With improvements in technology, new tools have become available to study the complex interactions that occur in biological systems. Global studies are required to do this, and these will involve genomic and proteomic approaches. High-throughput methods are necessary in each case because the number of genes and proteins in even the simplest of organisms are immense. In the developmental phase of genomics, the emphasis was on the generation and assembly of large amounts of nucleic acid sequence data. Proteomics is currently in a phase of technological development and establishment, and demonstrating the capacity for high throughput is a major challenge. However, funding bodies (both in the public and private sector) are increasingly focused on the usefulness of this capacity. Here we review the current state of proteome research in terms of capacity and utility.

Animals↗

PubMed2000

Automatic poisson peak harvesting for high throughput protein identification.

High throughput identification of proteins by peptide mass fingerprinting requires an efficient means of picking peaks from mass spectra. Here, we report the development of a peak harvester to automatically pick monoisotopic peaks from spectra generated on matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF) mass spectrometers. The peak harvester uses advanced mathematical morphology and watershed algorithms to first process spectra to stick representations. Subsequently, Poisson modelling is applied to determine which peak in an isotopically resolved group represents the monoisotopic mass of a peptide. We illustrate the features of the peak harvester with mass spectra of standard peptides, digests of gel-separated bovine serum albumin, and with Escherictia coli proteins prepared by two-dimensional polyacrylamide gel electrophoresis. In all cases, the peak harvester proved effective in its ability to pick similar monoisotopic peaks as an experienced human operator, and also proved effective in the identification of monoisotopic masses in cases where isotopic distributions of peptides were overlapping. The peak harvester can be operated in an interactive mode, or can be completely automated and linked through to peptide mass fingerprinting protein identification tools to achieve high throughput automated protein identification.

Animals↗

PubMed2000

Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [(15)N]NH(4)Cl and [(13)C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly (13)C,(15)N-labeled protein secreted by approximately 10(10) D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional (1)H-(13)C HSQC spectrum confirms (13)C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.

Animals↗

PubMed2000

Serotype classification and characterisation of the rotavirus SA11 VP6 protein using mass spectrometry and two-dimensional gel electrophoresis.

VP6, which makes up the inner capsid of rotavirus, is the major structural protein of this virus. Whilst VP6 has been sequenced at the DNA level in several rotavirus strains, there has been less effort to characterise the protein at the amino acid level. This paper reports the use of peptide mass fingerprinting and post-source decay fragmentation studies using MALDI-TOF and electrospray ionisation mass spectrometry to identify and characterise, in detail, the VP6 protein. We show that mass spectrometric analysis of VP6 peptides successfully distinguished SA11 from other rotavirus serotypes, and identify unique peptides that can be used for serotypic differentiation. For VP6 characterisation, the ExPASy FindMod tool was used to predict post-translational modifications on the protein. Analysis of trypsin and AspN digests predicted that the N-terminal methionine of VP6 was acetylated and this was confirmed using post source decay and electrospray ionisation mass spectrometry-mass spectrometry. An asparagine residue (aa107), which is followed by a glycine residue, was shown to undergo partial deamidation to aspartic acid. VP6 has two additional asparagine-glycine sequences and, in this sequence context, asparagine is known to be particularly susceptible to deamidation. Two-dimensional gel electrophoresis revealed a complex series of VP6 isoforms with an apparent molecular mass of approximately 45,000 Da and a pI ranging from 5.25 to 5.8. This pattern could partly be explained by the potential for deamidation at several sites within the protein.

Acetylation↗

PubMed2000

Characterization of a major sporozoite surface glycoprotein of Cryptosporidum parvum.

We have identified S60, a new Cryptosporidium parvum sporozoite surface glycoprotein. S60 was cleaved into two subunits, S16 and S45, approximately 16-18 and 45-47 kDa respectively, with cleavage occurring at an SRSRR motif likely to be sensitive to trypsin in vivo. Analysis by surface biotinylation, lectins and monoclonal antibodies suggests S60 is an abundant sporozoite surface glycoprotein that is shed by migrating sporozoites. The major glycosylation on S60 was identified as single O-linked N-acetylgalactosamine sugars on threonine and serine residues. The gene encoding the S60 protein was identified and revealed a C-terminal consensus sequence for the addition of a glycosylphosphatidyl inositol anchor. Antisera raised against recombinant S60 produced in Escherichia coli reacted with the surface of sporozoites and the inner wall of excysted oocysts.

Amino Acid Motifs↗

PubMed2000

Proteomic analysis of the Escherichia coli outer membrane.

Outer membrane proteins (OMPs) of Gram-negative bacteria are key molecules that interface the cell with the environment. Traditional biochemical and genetic approaches have yielded a wealth of knowledge relating to the function of OMPs. Nonetheless, with the completion of the Escherichia coli genome sequencing project there is the opportunity to further expand our understanding of the organization, expression and function of the OMPs in this Gram-negative bacterium. In this report we describe a proteomic approach which provides a platform for parallel analysis of OMPs. We propose a rapid method for isolation of bacterial OMPs using carbonate incubation, purification and protein array by two-dimensional electrophoresis, followed by protein identification using mass spectrometry. Applying this method to examine E. coli K-12 cells grown in minimal media we identified 21 out of 26 (80%) of the predicted integral OMPs that are annotated in SWISS-PROT release 37 and predicted to separate within the range of pH 4-7 and molecular mass 10-80 kDa. Five outer membrane lipoproteins were also identified and only minor contamination by nonmembrane proteins was observed. Importantly, this research readily demonstrates that integral OMPs, commonly missing from 2D gel maps, are amenable to separation by two-dimensional electrophoresis. Two of the identified OMPs (YbiL, YeaF) were previously known only from their ORFs, and their identification confirms the cognate genes are transcribed and translated. Furthermore, we show that like the E. coli iron receptors FhuE and FhuA, the expression of YbiL is markedly increased by iron limitation, suggesting a putative role for this protein in iron transport. In an additional demonstration we show the value of parallel protein analysis to document changes in E. coli OMP expression as influenced by culture temperature.

Bacterial Outer Membrane Proteins↗

PubMed2000

Phenotypic and genetic characterization of Paecilomyces lilacinus strains with biocontrol activity against root-knot nematodes.

Efficient selection of fungi for biological control of nematodes requires a series of screening assays. Assessment of genetic diversity in the candidate species maximizes the variety of the isolates tested and permits the assignment of a particular genotype with high nematophagous potential using a rapid novel assay. Molecular analyses also facilitate separation between isolates, allowing the identification of proprietary strains and trace biocontrol strains in the environment. The resistance of propagules to UV radiation is an important factor in the survival of a biocontrol agent. We have analyzed 15 strains of the nematophagous fungus Paecilomyces lilacinus using these principles. Arbitrarily primed DNA and allozyme assays were applied to place the isolates into genetic clusters, and demonstrated that some genetically related P. lilacinus strains exhibit widespread geographic distributions. When exposed to UV radiation, some weakly nematophagous strains were generally more susceptible than effective isolates. A microtitre tray-based assay used to screen the pathogenic activity of each isolate to Meloidogyne javanica egg masses revealed that the nematophagous ability varied between 37%-100%. However, there was no clear relationship between nematophagous ability and genetic clusters. Molecular characterizations revealed sufficient diversity to allow tracking of strains released into the environment.

Animals↗

PubMed2000

Fibrogenesis. IV. Fibrosis and inflammatory bowel disease: cellular mediators and animal models.

The cellular mediators of intestinal fibrosis and the relationship between fibrosis and normal repair are not understood. Identification of the types of intestinal mesenchymal cells that produce collagen during normal healing and fibrosis is vital for elucidating the answers to these questions. Acute injury may cause normal mesenchymal cells to convert to a fibrogenic phenotype that is not maintained during normal healing but may lead to fibrosis when inappropriately sustained. Proliferation of normal or fibrogenic mesenchymal cells may lead to muscularis overgrowth associated with fibrosis. The presence of increased numbers of vimentin-positive cells within fibrotic, hypertrophied muscularis in Crohn's disease suggests that changes in mesenchymal cell phenotype and number may indeed be associated with fibrosis. Fibrosis is induced in rats by peptidoglycan polysaccharides or trinitrobenzene sulfonic acid-ethanol administration, but inducing fibrosis in mice has been technically challenging. The development of current mouse models of colitis, such as dextran sodium sulfate or trinitrobenzene sulfonic acid-ethanol administration, into models of fibrosis will allow us to use genetic manipulation to study molecular mediators of fibrosis.

Animals↗

PubMed2000

Effect of screening algorithm, parameter values and median smoothing on patient-specific risk estimates for Down's syndrome screening.

The efficiency of a screening programme for Down's syndrome is usually expressed in terms of the detection rate for a given false positive rate. Two programmes with approximately equal detection rates and false positive rates would then be regarded as being broadly equivalent. While this might be the case at a population level, we show in this paper that it may be far from true at the individual patient level. Different algorithms, or even different implementations of the same algorithm can lead to calculation of very different individual risks and can result in different decisions for individual patients. Even though two programmes might lead to the same number of referrals, they could be referring quite different women. We consider the effect of using different parameter values within an algorithm, different algorithms and alternative ways of estimating the gestational age-dependent medians of the marker values. We show that the combined effects of these factors could explain much of the range of risks reported in the UK National External Quality Assessment Scheme for Down's syndrome screening.

Adult↗

PubMed2000

A behavioral economic analysis of concurrent ethanol- and water-reinforced responding in different preference conditions.

BACKGROUND: The reinforcing properties of orally self-administered drugs have been evaluated by using choice procedures. The preference for the drug over a nondrug alternative has indicated that the drug has greater value than the nondrug alternative as a reinforcer at some drug concentrations. However, at large drug concentrations, the fluid deliveries of the drug may be equal to or less than those of the nondrug alternative, whereas the actual drug intake (milligrams per kilogram of body weight) may continue to increase. In this study, we used behavioral economics to evaluate the reinforcing strength of ethanol in conditions where baseline ethanol fluid deliveries were greater than, equal to, or less than those of the concurrently available water. METHODS: Four male rhesus monkeys were allowed access to ethanol (2%, 8%, or 32%) and water for 2 hr/day under a fixed ratio (FR) 4 reinforcement schedule. At each ethanol concentration, the FR for both fluids was gradually increased to FR 64. RESULTS: During the FR 4 schedule, the fluid deliveries of ethanol at 2%, 8%, and 32% were greater than, equal to, and less than those of water, respectively. When the FR was increased at 2% ethanol, fluid deliveries and responding decreased for both the ethanol and water. When the FR was increased at 8% ethanol, water fluid deliveries and responding decreased more rapidly than did those of ethanol. When the FR was increased at 32% ethanol, the ethanol fluid deliveries remained the same across all FRs, whereas water fluid deliveries decreased rapidly. At 8% and 32% ethanol, the responding for ethanol, relative to water, increased dramatically. CONCLUSIONS: In behavioral economic terms, demand for ethanol was more inelastic regardless of whether the ethanol or water maintained more absolute fluid deliveries at baseline FRs. Therefore, researchers should examine the reinforcing effects of ethanol in a variety of concentration and schedule conditions rather than drawing inferences regarding reinforcing effects simply based on a preference measure.

Alcohol Drinking↗

PubMed1999

Flow cytometry and RT-PCR for rotavirus detection in artificially seeded oyster meat.

A flow cytometry (FC)-based method was developed for the detection of rotavirus in oyster meat using simian rotavirus SA11 as a model. To study virus recovery, oyster meat was injected with rotavirus and the oyster extract used to infect MA104 cell monolayers. Following varying periods of infection, the cells were recovered and reacted with the monoclonal antibody M60 which is specific for the rotavirus group A serotypes 1-4 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. To evaluate the sensitivity of the method, non-inoculated oysters were processed following the same extraction protocol and, at the end, they were seeded with the same amount of virus used for oyster inoculation. This seeded oyster extract was then used to infect MA104 cells and the number of infected cells determined using the same FC procedure. A semi-nested two-step PCR for detection of rotavirus nucleic acid was undertaken to compare the sensitivity of FC with RT-PCR. Using FC, as little as 0.02 flow cytometry units (fcu) (number of infected cells counted by FC) could be detected after 72 h of cell infection. This is a very similar limit of sensitivity to that obtained with RT-PCR. Both methods are approximately 100 times more sensitive than the plaque-forming units (pfu) assay.

Animals↗

PubMed1999

High-throughput mass spectrometric discovery of protein post-translational modifications.

The availability of genome sequences, affordable mass spectrometers and high-resolution two-dimensional gels has made possible the identification of hundreds of proteins from many organisms by peptide mass fingerprinting. However, little attention has been paid to how information generated by these means can be utilised for detailed protein characterisation. Here we present an approach for the systematic characterisation of proteins using mass spectrometry and a software tool FindMod. This tool, available on the internet at http://www.expasy.ch/sprot/findmod.html , examines peptide mass fingerprinting data for mass differences between empirical and theoretical peptides. Where mass differences correspond to a post-translational modification, intelligent rules are applied to predict the amino acids in the peptide, if any, that might carry the modification. FindMod rules were constructed by examining 5153 incidences of post-translational modifications documented in the SWISS-PROT database, and for the 22 post-translational modifications currently considered (acetylation, amidation, biotinylation, C-mannosylation, deamidation, flavinylation, farnesylation, formylation, geranyl-geranylation, gamma-carboxyglutamic acids, hydroxylation, lipoylation, methylation, myristoylation, N -acyl diglyceride (tripalmitate), O-GlcNAc, palmitoylation, phosphorylation, pyridoxal phosphate, phospho-pantetheine, pyrrolidone carboxylic acid, sulphation) a total of 29 different rules were made. These consider which amino acids can carry a modification, whether the modification occurs on N-terminal, C-terminal or internal amino acids, and the type of organisms on which the modification can be found. We illustrate the utility of the approach with proteins from 2-D gels of Escherichia coli and sheep wool, where post-translational modifications predicted by FindMod were confirmed by MALDI post-source decay peptide fragmentation. As the approach is amenable to automation, it presents a potentially large-scale means of protein characterisation in proteome projects.

Acetylation↗

PubMed1999

BOLD--a biological O-linked glycan database.

Glycans can be O-linked to proteins via the hydroxyl group of serine, threonine, tyrosine, hydroxylysine or hydroxyproline. Sometimes the glycan is O-linked to the hydroxyl group via a phosphodiester bond. The core monosaccharide residue may be N-acetylgalactosamine, N-acetylglucosamine, galactose, glucose, fucose, mannose, xylose or arabinose. These O-linked glycans can remain as a monosaccharide, but often a complex structure is built up by stepwise addition of monosaccharides. Monosaccharides known to be added include galactose, N-acetylglucosamine, fucose, N-acetylneuraminic acid, N-glycolylneuraminic acid and 2-keto-3-deoxynonulosonic acid. O-linked glycans can also contain sulfate and phosphate residues. This leads to the possibility of the existence of numerous O-glycan structures. The biological O-linked database (BOLD) is a relational database that contains information on O-linked glycan structures, their biological sources (with a link to the SWISS-PROT protein database), the references in which the glycan was described (with a link to MEDLINE), and the methods used to determine the glycan structure. The database provides a valuable resource for glycobiology researchers interested in O-linked oligosaccharide structures that have been previously described on proteins from different species and tissues.

Carbohydrate Conformation↗

PubMed1999

Quadazocine decreases responding reinforced by ethanol, sucrose, and phencyclidine fluid deliveries in rhesus monkeys: comparison to naltrexone's effects.

RATIONALE: The endogenous opioid system may mediate the reinforcing effects of ethanol as well as sweet-tasting solutions. For example, opioid antagonists, such as naltrexone, reduce ethanol- and sucrose-reinforced responding in rhesus monkeys. If these effects are due to blockade of the mu-receptor, then an opioid antagonist such as quadazocine with a receptor selectivity profile similar to that of naltrexone should reduce responding at doses correlated with its mu-selectivity. OBJECTIVES: To determine whether quadazocine would reduce responding for ethanol and sucrose at mu-selective doses, and whether quadazocine and naltrexone would reduce responding for a bitter-tasting drug solution such as phencyclidine. METHODS: Rhesus monkeys were given access to ethanol, sucrose, or phencyclidine concurrently with water. Prior to the drinking sessions, quadazocine (0.032-3.2 mg/kg) or saline was injected intramuscularly. During the phencyclidine experiment, naltrexone (0.1 and 0.32 mg/kg) was also tested. RESULTS: The highest quadazocine doses (1 and 3.2 mg/kg) reduced ethanol and sucrose fluid deliveries without affecting the concurrently available water. Quadazocine reduced the fluid deliveries of both phencyclidine and water when concurrently available. Naltrexone reduced only phencyclidine fluid deliveries. CONCLUSIONS: The opioid antagonist effect on oral-reinforced responding is not selective for ethanol or sweet-tasting solutions; responding for phencyclidine was reduced as well. Quadazocine and NTX may reduce responding by blocking the mu-receptor because the relative potency of these antagonists to reduce oral self-administration was similar to their relative potency to produce withdrawal in morphine-dependent monkeys. However, water responding was low in these experiments, and thus we cannot rule out rate-dependent effects of the antagonists.

Alcohol Drinking↗

PubMed1999

Food restriction and sex differences on concurrent, oral ethanol and water reinforcers in juvenile rhesus monkeys.

The present study assessed the liability of ethanol to be established as an oral reinforcer in 24 juvenile rhesus monkeys. All of the monkeys had a prior oral self-administration history with concurrently available methadone and water. To determine if food restriction and sex differences would contribute to ethanol preference, the monkeys were divided into two groups of 12. Twelve monkeys received 30 biscuits of food each day while the other group received 15 biscuits; six monkeys were male and six were female in each food restriction group. Fluid deliveries (0.5 ml) were provided following contact responses on solenoid-operated drinking spouts. All monkeys were exposed to concurrent water on two spouts and, subsequently, ethanol was available on one spout with water in the alternate spout. Ethanol concentrations (0.25-16 g/l) were doubled weekly. Subsequently, some ethanol concentration exposures were repeated, as was the concurrent water condition. Ethanol (1-2 g/l) served to reinforce responding under most conditions except with the 30-biscuit females; where ethanol so functioned, water responding was reduced. At concentrations less than 1 g/l, ethanol and water were consumed in equal amounts. At 8-16 g/l, ethanol maintained less responding than water. Food restriction amplified ethanol preference in both males and females, although perhaps less in females. A complex set of relations exist among variables that control oral ethanol preference in rhesus monkeys.

Animals↗

PubMed1999

Linkage of type 2 diabetes mellitus and of age at onset to a genetic location on chromosome 10q in Mexican Americans.

Since little is known about chromosomal locations harboring type 2 diabetes-susceptibility genes, we conducted a genomewide scan for such genes in a Mexican American population. We used data from 27 low-income extended Mexican American pedigrees consisting of 440 individuals for whom genotypic data are available for 379 markers. We used a variance-components technique to conduct multipoint linkage analyses for two phenotypes: type 2 diabetes (a discrete trait) and age at onset of diabetes (a truncated quantitative trait). For the multipoint analyses, a subset of 295 markers was selected on the basis of optimal spacing and informativeness. We found significant evidence that a susceptibility locus near the marker D10S587 on chromosome 10q influences age at onset of diabetes (LOD score 3.75) and is also linked with type 2 diabetes itself (LOD score 2.88). This susceptibility locus explains 63.8%+/-9.9% (P=. 000016) of the total phenotypic variation in age at onset of diabetes and 65.7%+/-10.9% (P=.000135) of the total variation in liability to type 2 diabetes. Weaker evidence was found for linkage of diabetes and of age at onset to regions on chromosomes 3p, 4q, and 9p. In conclusion, our strongest evidence for linkage to both age at onset of diabetes and type 2 diabetes itself in the Mexican American population was for a region on chromosome 10q.

Age Factors↗