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K L Williams

Publications and source records attributed to K L Williams.

At least 109 records · Page 6Linked to original sources
PubMed1995

Comparison of 1-(1-naphthyl)ethylcarbamate derivatives of a carbohydrate bonded chiral stationary phase.

New chiral stationary bonded phases (CSPs) based on derivatized malto-oligosaccharides are reported. Chiral separations are reported for 3,5-dinitrobenzoyl-derivatized amines and amino acids as well as some 3,5-dinitrophenylcarbamoylated alcohols. These new CSPs incorporate a 1-(1-naphthyl)ethylcarbamate (NEC) moiety which introduces additional stereogenicity and provides a useful probe for investigating chiral recognition. The elution order, when known, was dependent upon the configuration of the NEC substituent; that is, the S enantiomers were retained longest on the S column and the R enantiomers were retained longest on the R column. Elution order from the RS column, when known, was the same as that observed for the S column. In most cases, retention correlated with bonded ligand concentration on the silica substrate. In general, the best enantioselectivities and resolution were obtained on the S column.

Alcohols↗
PubMed1995

Expression, purification and characterisation of secreted recombinant glycoprotein PsA in Dictyostelium discoideum.

Dictyostelium discoideum is a newly developed eukaryotic expression system which is an alternative to tissue cultures for the production of recombinant proteins requiring eukaryotic folding and post-translational modifications. The homologous glycoprotein PsA (prespore specific antigen) is a glycosyl phosphatidylinositol (GPI) anchored membrane protein from D. discoideum. A truncated form of PsA has been expressed in D. discoideum and secreted into a peptone based broth at levels of 10 mg per 1 growth medium. A simple purification protocol for recombinant PsA (rPsA) involved three steps: the concentration of the culture supernatant by ammonium sulfate precipitation, Mono Q anion-exchange chromatography, followed by size exclusion chromatography on Superdex 75. 20 mg of rPsA was purified to 98% purity from 37.1 culture supernatant. Purified rPsA was characterised. The molecular mass of the purified rPsA is 15.6 kDa, which suggests that the molecule is secreted as a monomer and contains 12% (w/w) carbohydrate. The protein sequence of rPsA proved identical to that of the predicted DNA construct. Although the recombinant form of PsA is expressed at a different developmental stage from the native molecule, the same Thr residues that are O-glycosylated in the authentic molecule are glycosylated in the recombinant protein.

Amino Acid Sequence↗
PubMed1995

Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium.

A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of 'hierarchical' analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or 'proteome' is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.

Amino Acids↗
PubMed1995

Cross-species identification of proteins separated by two-dimensional gel electrophoresis using matrix-assisted laser desorption ionisation/time-of-flight mass spectrometry and amino acid composition.

Amino acid analysis and matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry were used to identify nine of twelve proteins originally separated by two-dimensional electrophoresis and derived from an organism poorly defined at the molecular level (Spiroplasma melliferum). Two of three unidentified proteins appeared to be novel. The percentage amino acid composition and the molecular mass of peptide fragments generated by tryptic digestion were used to search the PIR/SWISS-PROT and MOWSE databases respectively. Lists of candidate proteins were independently generated and ranked from data obtained by both methods. A putative identification was allocated when a single candidate protein appeared in both lists of computer-generated rankings. Results were verified using N-terminal protein microsequencing. The combined use of amino acid composition and MALDI-TOF mass spectrometry allowed a high degree of confidence to be placed in such identifications because they were based upon homologous data sets of at least 20 parameters (16 amino acids and 4-10 tryptic digest fragments). A further two parameters, estimated M(r) and, to a lesser extent, pI, were also used to reinforce this measure of confidence. Ranking of candidate proteins by one method alone could lead to false identification. Both techniques can process large numbers of samples rapidly. In light of the increasing number of entries in both gene and protein databases, this approach is likely to become an essential first step for the characterisation of proteins, particularly across species boundaries.

Amino Acid Sequence↗
PubMed1995

Identification of macrophage activation associated proteins by two-dimensional gel electrophoresis and microsequencing.

To understand activation in monocytes and macrophages we have studied changes in protein synthesis using the human monocytoid U937 cell line and two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and protein sequencing. U937 cells that had been metabolically labeled during treatment with PMA, LPS, or IFN-gamma showed appreciable increases or decreases in synthesis of 14 proteins when analyzed by 2D PAGE. Although some 20 proteins are reported to be affected by these agents in U937 cells, none of them correspond with the 14 proteins studied here. Of the 14 observed changes, four spots (p41/65, p35/65, p26/44, p20/53) were up-regulated by PMA only, one (p16/44) by LPS only, five spots (p29/47, p26/45, p26/48, p12/47, p10/45) by both LPS and PMA, and, finally, one (p29/45) by all three agents. Two spots (p20/59 and p20/61) were down-regulated by IFN-gamma and one of these spots (p20/59) was up-regulated by LPS. Only one spot (p20/48) was up-regulated by IFN-gamma. Eleven spots with matching mobilities (both M(r) and pI) to those identified in U937 were observed on 2D PAGE gels from human culture derived macrophages. Ten spots from U937 were sequenced by Edman degradation. Two were could not identified from information contained in the available DNA and protein databases and thus represent novel proteins, whereas a further six of the proteins were N-terminally blocked. The remaining two (29/47 and 12/47, respectively) were identified from existing protein databases as translationally controlled tumor protein (TCTP) and cytokeratin. This is the first report of the presence of TCTP in hemopoietic cells and its modulation by PMA or LPS in any cell type. We believe that 2D PAGE and sequencing is a powerful approach for identifying key proteins in macrophage cellular activation.

Amino Acid Sequence↗
PubMed1995

Glycoprotein complexes interacting with cellulose in the "cell print" zones of the Dictyostelium discoideum extracellular matrix.

Cellulose is one of the commonest structural biopolymers. How cellulose is organized in extracellular matrices is a mystery. Here we investigate a model system, the extracellular matrix (ECM) of Dictyostelium discoideum which is composed of proteins and cellulose. A group of glycoproteins, the sheathins, which colocalize with cellulose in the ECM of D. discoideum are characterized. Sheathins are dimeric or trimeric forms of molecular mass 53-68 kDa, where the monomers are 12-35 kDa. The sheathin subunits are similar but not identical proteins. The sheathin family comprises sheathin 68, (68-kDa trimer); sheathin 62, (62-kDa dimer); sheathin 55, (55-kDa dimer), and sheathin 53, (53-kDa dimer). The subunits which assemble into the four sheathins represent at least three gene products: ShC, ShD, and ShE which are linked by disulphide bonds. Protein sequence analysis shows two of the sheathin genes encode products ShC and ShD with very similar amino terminal sequences. This group of D. discoideum ECM glycoproteins has homology with two other much larger ECM proteins of D. discoideum, ST430 and ST310, which are located in a more dispersed fashion in the ECM. Sheathins are tightly but non-covalently associated with the ECM, and this association requires strong denaturing conditions for disruption, e.g., SDS or 8 M urea. Sheathins form a component of the "cell prints" which are believed to have a role in cell-ECM interactions and slug cell migration.

Amino Acid Sequence↗
PubMed1995

Dictyostelium discoideum mitochondrial DNA encodes a NADH:ubiquinone oxidoreductase subunit which is nuclear encoded in other eukaryotes.

Complex I, a key component of the mitochondrial electron transport system, is thought to have evolved from at least two separate enzyme systems prior to the evolution of mitochondria from a bacterial endosymbiont, but the genes for one of the enzyme systems are thought to have subsequently been transferred to the nuclear DNA. We demonstrated that the cellular slime mold Dictyostelium discoideum retains the ancestral characteristic of having mitochondria encoding at least one gene (80-kDa subunit) that is nuclear encoded in other eukaryotes. This is consistent with the cellular slime molds of the family Dictyosteliaceae having diverged from other eukaryotes at an early stage prior to the loss of the mitochondrial gene in the lineage giving rise to plants and animals. The D. discoideum mitochondrially encoded 80-kDa subunit of complex I exhibits a twofold-higher mutation rate compared with the homologous chromosomal gene in other eukaryotes, making it the most divergent eukaryotic form of this protein.

Animals↗
PubMed1995

Glycine enhances the central depressant properties of ethanol in mice.

The interaction between ethanol and glycine in the central nervous system was investigated in male Swiss-Webster mice. The loss of the righting reflex (LORR) was used as a measure of central nervous system depression. Mice were injected with ethanol (4.0 g/kg, IP), causing an ethanol-induced LORR. Immediately after the animals regained the righting reflex from ethanol administration, they received an intracerebroventricular (ICV) injection of saline or glycine (1, 15, 25, or 50 mumol/kg) in a volume of 5 microliters. Upon ICV injection of glycine, the mice lost the righting reflex once again. This effect of glycine in the presence of ethanol occurred rapidly and in a dose-dependent manner. Glycine induced a return to the LORR of 12.6 +/- 0.7, 24.5 +/- 1.3, 32.8 +/- 2.0, and 46.8 +/- 4.5 min when doses of 1, 15, 25, and 50 mumol/kg, respectively, were injected. D-Serine (15, 25, or 50 mumol/kg), an amino acid precursor of glycine, was injected (ICV) after the animals regained the righting reflex following ethanol injection (IP). Serine caused a return to the LORR of 0.5 +/- 0.5, 6.0 +/- 1.0, and 6.5 +/- 0.9 min when doses of 15, 25, and 50 mumol/kg, respectively, were injected. Strychnine was used to attenuate the ability of glycine and serine to cause a return to the LORR in the presence of ethanol. Strychnine, a competitive antagonist of glycine, significantly reduced the ability of glycine and serine to enhance the depressant action of ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Recombinant glycoprotein production in the slime mould Dictyostelium discoideum.

Dictyostelium discoideum is a well known amoeboid organism, with unicellular and multicellular life-cycle stages, that is used for studying cell and developmental biology. With advances in gene-disruption technology and transformation of this organism, many homologous proteins have been expressed either to complement defective proteins or to study basic cell biology. Now, D. discoideum is being used to express heterologous proteins that are difficult to study in other systems, and its unique cell biology is being exploited to facilitate a wide range of protein modifications. In the past year, substantial progress has been made in expressing correctly folded forms of malarial circumsporozoite antigen and rotavirus surface glycoprotein VP7. Exciting developments have also been made in expressing human muscarinic receptors.

Animals↗
PubMed1995

From virus to vaccine: developments using the simple eukaryote, Dictyostelium discoideum.

Mass vaccination compaigns against viral diseases, both human and anim al, depend on the availability of cheap viral antigens. The eukaryote Dictyostelium discoideum has simple growth requirements and rapid growth rates and forms stable cell lines. These features, together with the possibility of secreting recombinant (glyco)proteins into a defined buffer, make the D. discoideum expression system an attractive option for producing economical recombinant subunit vaccines.

Animals↗
PubMed1995

Immunochemical, genetic and morphological comparison of fucosylation mutants of Dictyostelium discoideum.

Mutations in three loci in Dictyostelium discoideum which affect fucosylation are described. Mutations in two of these loci resulted in the simultaneous loss of two separate carbohydrate epitopes. The GA-X epitope, which was competed by L-fucose, was absent in strains carrying a modC354, modD352 or modE353 mutation. These strains exposed a new carbohydrate epitope, competed by N-acetylglucosamine, and the size of several glycoproteins was reduced. A second epitope (GA-XII) was also absent in strains carrying the modC354 or modE353 mutations, reducing the size of the glycoprotein which normally expresses it. Fucose content was reduced in the three mutants, suggesting that each mutation affected a separate step in fucosylation. The lesions did not appear to inhibit synthesis of the underlying carbohydrate, because detergent extracts of mutant vesicles were more active than normal vesicles at transferring [14C]fucose from GDP-[14C]fucose to endogenous acceptor species. The modD352 and modE353 mutant strains incorporated exogenous [3H]fucose poorly, suggesting that lesions in the modD and modE genes interfere with the biosynthesis of fucoconjugates downstream from the previously described GDP-fucose synthesis defect of the modC mutation. Intact modE353 mutant vesicles were relatively inefficient in in vitro assays, suggesting a global fucosylation defect (which is consistent with the loss of both glycoantigens, GA-X and GA-XII, in this mutant). Finally, the modC354 mutation led to delayed accumulation of slime sheath in vitro. The three genetic loci define a fucosylation pathway in D. discoideum comprising defined biochemical steps which contribute to multicellular morphogenesis in this organism.

Animals↗
PubMed1995

Expression of the rotavirus SA11 protein VP7 in the simple eukaryote Dictyostelium discoideum.

The outer capsid protein of rotavirus, VP7, is a major neutralization antigen and is considered a necessary component of any subunit vaccine developed against rotavirus infection. For this reason, significant effort has been directed towards producing recombinant VP7 that maintains the antigenic characteristics of the native molecule. Using a relatively new expression system, the simple eukaryote Dictyostelium discoideum, we have cloned the portion of simian rotavirus SA11 genome segment 9, encoding the mature VP7 protein, downstream of a native D. discoideum secretion signal sequence in a high-copy-number extrachromosomal vector. The majority of the recombinant VP7 expressed by transformants was intracellular and was detected by Western immunoblot following gel electrophoresis as two or three bands with an apparent molecular mass of 35.5 to 37.5 kDa. A small amount of VP7 having an apparent molecular mass of 37.5 kDa was secreted. Both the intracellular VP7 and the secreted VP7 were N glycosylated and sensitive to endoglycosidase H digestion. Under nonreducing electrophoresis conditions, over half the intracellular VP7 migrated as a monomer while the remainder migrated with an apparent molecular mass approximately twice that of the monomeric form. In an enzyme-linked immunosorbent assay, intracellular VP7 reacted with both nonneutralizing and neutralizing antibodies. The monoclonal antibody recognition pattern paralleled that found with VP7 expressed in either vaccinia virus or herpes simplex virus type 1 and confirms that D. discoideum-expressed VP7 is able to form the major neutralization domains present on viral VP7. Because D. discoideum cells are easy and cheap to grow, this expression system provides a valuable alternative for the large-scale production of recombinant VP7 protein.

Animals↗
PubMed1994

Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water.

Cryptosporidium and Giardia are common causes of waterborne disease. The currently used methods of detecting these organisms in water rely on filtration capture, immunofluorescence labelling, and epifluorescence microscopy. These methods are inefficient, labour intensive, and require a highly skilled microscopist. We describe an alternative technique using flocculation concentration, followed by flow cytometry with fluorescence activated cell sorting. Environmental samples were analysed, and protozoan-like particles were sorted and collected before confirmation with epifluorescence microscopy. The technique was found to be significantly more sensitive and considerably faster than the conventional methods.

Animals↗
PubMed1994

Nucleotide sequence of Ddp1, a high copy number nuclear plasmid of Dictyostelium discoideum.

The complete nucleotide sequence of Ddp1, a high copy number 13.7-kbp endogenous nuclear plasmid of Dictyostelium discoideum is presented. Previous studies have shown nine transcripts which map to five different regions of Ddp1, suggesting alternative transcription initiation sites and/or post-transcriptional processing. The sequence presented here shows five long open reading frames corresponding to previously known transcribed regions, as well as an additional reading frame, in the region of the presumed origin of replication. Two of the predicted proteins from the Ddp1 reading frames have potential leader sequences and transmembrane domains, while the rest, if translated, would encode soluble proteins. One of these has both leucine zipper and zinc finger-like motifs. Sequences upstream of the reading frames show strong similarities to chromosomal promoter elements, suggesting that transcription of at least some of the plasmid-encoded genes is regulated by factors which regulate chromosomal transcription in this organism. The region containing the presumptive origin of replication contains the promoters of the major growth-specific gene, as well as a second gene; thus, the processes of transcription and replication of Ddp1 may be intimately linked. The origin-encompassing region has a long homopurine/homopyrimidine stretch, which is similar to sequences shown in vitro to be capable of forming a triple helix structure through Hoogsteen base pairing. Possible roles for these sequences and the products of the ORFs in plasmid maintenance are discussed.

Animals↗
PubMed1994

The Dictyostelium discoideum mitochondrial genome: a primordial system using the universal code and encoding hydrophilic proteins atypical of metazoan mitochondrial DNA.

A 3,345-bp fragment of Dictyostelium discoideum mitochondrial DNA (mtDNA) has been sequenced. This fragment contained the 80-kDa subunit of complex I (NADH:ubiquinone oxidoreductase), encoding a predicted amino acid sequence of 688 residues and a molecular mass of 79,805 daltons which is nuclear encoded in other metazoa. The C-terminus of the D. discoideum complex I gene shared a 10-bp overlap with NADH:ubiquinone oxidoreductase chain 5 (ND5), while 21 bp 5' were three tRNA genes (two isoleucine and a histidine) and a further 25 bp 5' of these genes is the partial sequence (104 residues) of an unidentified open reading frame (ORF104). Both the 80-kDa subunit and the ORF104 were hydrophilic and highly charged, suggesting they are not membrane associated, unlike most mitochondrially encoded proteins in the metazoa. Sequence analysis of the 80-kDa subunit, its adjacent ND5 gene, and ORF104 indicates the universal stop codon TGA, which codes for tryptophan in nearly all nonplant mtDNA, is either unassigned or coding for a stop codon in D. discoideum. The large size of the mitochondrial genome (54 kb), the lack of intergenic sequence, and the apparent use of the universal code suggest D. discoideum mtDNA may encode many primitive genes that are nuclear encoded in higher organisms.

Amino Acid Sequence↗
PubMed1994

Characterization of a single glycosylated asparagine site on a glycopeptide using solid-phase Edman degradation.

The characterization of site-specific glycosylation is traditionally dependent on the availability of suitable proteolytic cleavage sites between each glycosylated residue, so that peptides containing individual glycosylation sites are recovered. In the case of heavily glycosylated domains such as the O-glycosylated mucins, which have no available protease sites, this approach is not possible. Here we introduce a new method to gain site-specific compositional data on the oligosaccharides attached to a single amino acid. Using a model glycopeptide from a mutant human albumin Casebrook, glycosylated PTH-Asn was recovered after sequential solid-phase Edman degradation, subjected to acid hydrolysis and the sugars were identified by high performance anion exchange chromatography with pulsed amperometric detection. The PTH-Asn(Sac) derivative was further characterized by ionspray mass spectrometry. Comparison between an endoproteinase Glu-C glycopeptide and a tryptic glycopeptide showed that the oligosaccharide attached to Asn494 was stable after at least 10 cycles of Edman degradation.

Amino Acid Sequence↗
PubMed1994

Production and secretion of recombinant proteins in Dictyostelium discoideum.

We have expressed useful amounts of three recombinant proteins in a new eukaryotic host/vector system. The cellular slime mold Dictyostelium discoideum efficiently secreted two recombinant products, a soluble form of the normally cell surface associated D. discoideum glycoprotein (PsA) and the heterologous protein glutathione-S-transferase (GST) from Schistosoma japonicum, while the enzyme beta-glucuronidase (GUS) from Escherichia coli was cell associated. Up to 20mg/l of recombinant PsA and 1mg/l of GST were obtained after purification from a standard, peptone based growth medium. The secretion signal peptide was correctly cleaved from the recombinant GST- and PsA-proteins and the expression of recombinant PsA was shown to be stable for at least one hundred generations in the absence of selection.

Amino Acid Sequence↗