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M Lyon

Publications and source records attributed to M Lyon.

At least 19 recordsLinked to original sources
PubMed1992

Molecular cloning of the major cell surface heparan sulfate proteoglycan from rat liver.

We have used an antiserum raised against a purified heparan sulfate proteoglycan (HSPG) preparation isolated from rat liver to screen a lambda gt11 expression library and have obtained overlapping cDNA clones that contain the full-length coding sequence of an HSPG core protein capable of spanning the plasma membrane. The open reading frame of the rat cDNA encodes a protein of 211 amino acids. The predicted protein sequence (23 kDa) has a high degree of homology with the published partial sequence of the human lung fibroblast HSPG, fibroglycan. The deduced protein sequence contains a 24-amino acid transmembrane domain and a 33-amino acid cytoplasmic domain, both of which are identical with the corresponding regions of human fibroglycan and are highly homologous to the human, hamster, and mouse epithelial HSPG, syndecan. The putative ectodomain, which has 85% homology to fibroglycan, contains three possible glycosaminoglycan attachment sites that may be occupied by heparan sulfate chains. The major 49-kDa core protein in the liver HSPG preparation was found to be reactive to an antibody that specifically recognizes the cytoplasmic domain of fibroglycan. We have used the full-length cDNA clone to analyze the expression of this transmembrane core protein gene in whole tissues and several epithelial and fibroblastoid cell lines. It hybridizes to three mRNA species in all cell and tissue types examined, but in liver, isolated hepatocytes, and kidney, an additional 0.8-kilobase mRNA was detected. The three common messages arise from differential use of alternative polyadenylation sites, whereas the fourth tissue-restricted RNA species represents a related gene transcript. The rat equivalent of human fibroglycan therefore appears to be the major transmembrane proteoglycan in liver, and its widespread expression in many diverse tissues and cells suggests that it plays an important role in cellular interactions.

Amino Acid Sequence↗
PubMed1992

Patterns of sulphation in heparan sulphate: polymorphism based on a common structural theme.

HS appears to be a well-organised molecule with a domain structure that is apparently unique amongst the GAG family (Gallagher, 1989). Further refinements in sequence analysis are needed to corroborate the simplified model proposed in Fig. 4. It is still not clear why evolution has favoured a structural motif of widely spaced sulphated domains. Presumably, some advantages must accrue to the organism from this design, and one idea, that we have discussed previously, is that the polysaccharide functions as a "template" for the organisation of structural proteins in the ECM and for the binding and presentation of growth factors within the matrix polymer network. The sulphated regions are likely to display considerable conformational versatility as a result of the presence of the iduronate residues, and this property may be very important for the protein-binding properties of the polysaccharides (Casu et al., 1988). Sulphation patterns within these regions could favour oligosaccharide conformations necessary for specific protein interactions. An important question in this context is why different cells express on their surfaces HS with subtle differences in sulphation pattern. Perhaps the polymorphic features of HS are involved in higher-order tissue- and organ-specific mechanisms controlling cellular recognition and morphogenesis. The consistency with which aberrant sulphation of HS is detected in malignant disease (Gallagher and Lyon, 1989) in which cellular recognition and differentiation are impaired, adds some substance to this view.

Amino Acid Sequence↗
PubMed1992

Genetic diversity among Australian and New Zealand isolates of Leptospira interrogans serovar pomona.

Restriction endonuclease analysis of 16 Australian and 4 New Zealand isolates of Leptospira interrogans serovar pomona showed that they could be divided into 3 genetic groups. Most of the isolates closely resembled the serovar kennewicki reference strain, and they all differed from the reference strain of serovar pomona. Based on these findings, it is suggested that vaccine manufacturers re-evaluate their choice of serovar pomona vaccine strain.

Animals↗
PubMed1991

Isolation and characterization of the integral glycosaminoglycan constituents of human amyloid A and monoclonal light-chain amyloid fibrils.

Amyloid fibrils were isolated by extraction in water from the livers and spleens of four patients who had died of monoclonal, light-chain (AL)-type, systemic amyloidosis and one with reactive systemic, amyloid A protein (AA)-type amyloidosis. Each fibril preparation contained 1-2% by weight of glycosaminoglycan (GAG) which was tightly associated with the fibrils and not just co-isolated from the tissues with them. After exhaustive digestion of the fibrils with papain and Pronase, the GAGs were specifically precipitated with cetylpyridinium chloride and were identified by cellulose acetate electrophoresis and selective susceptibility to specific glycosidases. All the preparations contained approximately equal amounts of heparan sulphate and dermatan sulphate. There was no evidence for the presence of chondroitin sulphate or other GAGs. Fine structural analysis by oligosaccharide mapping in gradient polyacrylamide gels, following partial digestion with specific glycosidases, showed very similar structures among the heparan sulphates and the dermatan sulphates, respectively. GAGs were also extracted by solubilizing amyloid fibrils in 4 M-guanidinium chloride followed by CsCl density-gradient ultracentrifugation. Although a minor proportion of the GAG material obtained in this way was apparently in the form of proteoglycan molecules, most of it was free GAG chains. The presence in amyloid fibrils of different types, in different organs and from different patients of particular GAG classes with similar structures supports the view that these molecules may be of pathogenic significance.

Amyloidosis↗
PubMed1991

Purification and partial characterization of the major cell-associated heparan sulphate proteoglycan of rat liver.

Heparan sulphate proteoglycans were solubilized from whole rat livers by homogenization and dissociative extraction with 4 M-guanidinium chloride containing Triton X-100 and proteinase inhibitors. The extract was subjected to trichloroacetic acid precipitation and the proteoglycan remained soluble. This was then purified to apparent homogeneity by a combination of (a) DEAE-Sephacel chromatography, (b) digestion with chondroitinase ABC followed by f.p.l.c. Mono Q ion-exchange chromatography, and (c) density-gradient centrifugation in CsCl and 4 M-guanidinium chloride. Approx. 1.5 mg of proteoglycan was obtained from 30 livers with an estimated recovery of 25%. The purified proteoglycan was eluted from Sepharose CL6B as an apparently single polydisperse population with a Kav. of 0.19 and displayed a molecular mass of greater than or equal to 200 kDa (relative to protein standards) by SDS/PAGE. Its heparan sulphate chains were eluted with a Kav. of 0.44 and have an estimated molecular mass of 25 kDa. Digestion of the proteoglycan with a combination of heparinases yielded core proteins of 77, 49 and 44 kDa. Deglycosylation using trifluoromethanesulphonic acid, though slightly decreasing the sizes, gave an identical pattern of core proteins. Electrophoretic detergent blotting demonstrated that all of the core proteins were hydrophobic and are probably integral plasma membrane molecules. The peptide maps generated by V8 proteinase digestion of the two major core proteins (77 and 49 kDa) were very similar, suggesting that these two core proteins are structurally related.

Animals↗
PubMed1991

Measuring the efficiency of an artificial lung: 1. Carbon dioxide transfer.

The performance of a hollow fibre artificial lung ('Capiox E') was analysed by measurement of the 'parallel deadspace' of the device under varying conditions in 21 patients. The efficiency with which carbon dioxide was exchanged was determined by the time available for equilibration between the blood and gas phases. When this equilibration coefficient was less than 12 seconds per litre of blood flow per litre of gas flow, there was a marked reduction in the efficiency of gas exchange. Under certain conditions, the 'counter-current' design of the device apparently permitted the clearance of carbon dioxide at a partial pressure greater than that which was found in the mixed venous blood. This anomalous behaviour may represent in vivo confirmation of the Haldane effect.

Adult↗
PubMed1990

The synthesis of proteoglycans by human T lymphocytes.

We have examined the proteoglycans produced by highly-purified cultures of human T-lymphocytes. The proteoglycans were metabolically labelled with [35S]sulphate and analysed in cellular and medium fractions using DEAE-cellulose chromatography, gel filtration and specific enzymatic and chemical degradations. The results showed that the T cells synthesized a relatively homogeneous, proteinase-resistant chondroitin 4-sulphate proteoglycan that accumulated in the culture medium during a 48 h incubation period. The cellular fraction contained a significant amount of free chondroitin sulphate chains that were not secreted into the medium. These polysaccharides were formed by intracellular degradation of proteoglycan in a chloroquine-sensitive process, indicating a requirement for an acidic environment. In contrast to chondroitin sulphate derived from proteoglycan, chondroitin sulphates synthesized on the exogenous primer, beta-D-xyloside, were mainly secreted by the cells. beta-D-Xylosides caused an 8-fold stimulation in the synthesis of chondroitin sulphate, but decreased the synthesis of proteoglycan by about 50%. These proteoglycans contained shorter chondroitin sulphate chains than their normal counterparts. The results indicate that although proteoglycans are mainly secretory components in human T-cell cultures, a specific metabolic step leads to the intracellular accumulation of free glycosaminoglycans. Separate functions are likely to be associated with the intracellular and secretory pools of chondroitin sulphate.

Cells, Cultured↗
PubMed1990

A general method for the detection and mapping of submicrogram quantities of glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure A and ammoniacal silver.

A sensitive method has been developed for the visualization of nonradiolabeled glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1-2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.

Azure Stains↗
PubMed1990

In vitro divergence of HSV-1 populations propagated in different cell lines.

To investigate how the structure of a virus population is influenced by the particular cell types in which the virus is propagated, laboratory populations of HSV-1 have been serially passaged onto a number of different cell lines, differing either in species or in tissue specificity. After a limited number of in vitro passages, several of the daughter virus populations have diverged in the expression of at least one phenotype, suggesting that different cell types have selected different variants contained in the parental virus population.

Animals↗
PubMed1990

Inhibition of HSV-1 multiplication in rat embryo fibroblasts constitutively expressing the EJ-ras oncogene.

In order to examine cellular gene involvement in HSV-1 expression, we constructed different rat embryo fibroblast cell lines immortalized by adenovirus E1A or c-myc, with or without the human EJ bladder carcinoma transforming oncogene EJ-ras. HSV-1 multiplication was strongly inhibited in cells expressing EJ-ras genes compared to immortalized control cells. Virus adsorption and penetration were not quantitatively modified, but HSV-1 DNA replication was inhibited. The expression of viral thymidine kinase (TK) activity after infection by recombinant virus with the TK coding sequence under immediate-early (IE) promoter control showed that IE gene expression is inhibited in cells expressing EJ-ras. Analysis of IE gene transcription by Northern-blot hybridization and by nuclear run-off transcription assay indicates that this inhibition takes place at the transcriptional level.

Adenovirus Early Proteins↗
PubMed1989

Fetal neural development and schizophrenia.

The conference on Fetal Neural Development and Schizophrenia which was held in Washington, DC, May 31-June 1, 1988, focused on factors of possible etiological significance in fetal development. Schizophrenia researchers joined experts in brain imaging, neuropathological, and neurochemical changes in brain development and investigators of potential genetic and neurobehavioral causes of psychosis. The combined evidence suggested dysfunction in frontal and parieto-occipital neocortex, basal ganglia, hippocampus, and amygdala. Dopamine transmission was implicated both in basal ganglia deficits and in widespread neocortical disturbances. Viral infection, or excessive stress, during the second trimester of pregnancy, as well as obstetrical complications, minor physical anomalies, and brain defects, correlated positively with incidence of adult schizophrenia. Autonomic nonresponding, birth complications, and ventricular enlargement were found to be closely related to negative symptom schizophrenia in high-risk populations. A dual factor model of schizophrenia was suggested, where genetic and environmental influences combine to produce psychosis.

Brain↗
PubMed1988

Chondroitin sulphate proteoglycan production by NK cells and T cells: effects of xylosides on proliferation and cytotoxic function.

Cultured human NK cells and T cells grown in the presence of IL-2 and phytohaemagglutinin incorporated 35S sulphate into two distinct macromolecular species. The larger molecule was identified as a chondroitin-4-sulphate proteoglycan and was present in both cell-associated and secreted material. The smaller component was identified as free glycosaminoglycan and was present only in the cell-associated material. The sulphated macromolecules synthesized by NK cells were smaller than those produced by T cells. Growth in the presence of beta-D-xyloside led to a decrease in proteoglycan production, together with an increase in the synthesis of free glycosaminoglycan. The latter molecule was found in the secreted as well as the cell-associated fraction. In all instances, growth of T cells was inhibited by xyloside in a dose-dependent fashion. However, growth of NK cells from 3/7 donors was stimulated at low concentrations of xyloside (0.25 and 0.5 mM). Growth of NK cells in xyloside had no effect on their lytic activity, and the 'NK-like' cytolytic capacity of cultured T cells was similarly unaffected. Both NK cells and T cells grown in xyloside at a concentration resulting in a 50% inhibition of intact proteoglycan synthesis did not show increased susceptibility to autolysis in the presence of NK-cell targets. These findings suggest that optimal production of the intact proteoglycan molecule may not be essential for NK-cell lytic function or protection of effector cells in vitro.

Cells, Cultured↗
PubMed1987

Identification of an extended N-acetylated sequence adjacent to the protein-linkage region of fibroblast heparan sulphate.

The distribution of N-sulphate groups within fibroblast heparan sulphate chains was investigated. The detergent-extractable heparan sulphate proteoglycan from adult human skin fibroblasts, radiolabelled with [3H]glucosamine and [35S]sulphate, was coupled to CNBr-activated Sepharose 4B. After partial depolymerization of the heparan sulphate with nitrous acid, the remaining Sepharose-bound fragments were removed by treatment with alkali. These fragments, of various sizes, but all containing an intact reducing xylose residue, were fractionated on Sephacryl S-300 and the distribution of the 3H and 35S radiolabels was analysed. A decreased degree of sulphation was observed towards the reducing termini of the chains. After complete nitrous acid hydrolysis of the Sepharose-bound proteoglycan, analysis of the proximity of N-sulphation to the reducing end revealed the existence of an extended N-acetylated sequence directly adjacent to the protein-linkage sequence. The size of this N-acetylated domain was estimated by gel filtration to be approximately eight disaccharide units. This domain appears to be highly conserved, being present in virtually all the chains derived from this proteoglycan, implying the existence of a mechanism capable of generating such a non-random sequence during the post-polymeric modification of heparan sulphate. Comparison with the corresponding situation in heparin suggests that different mechanisms regulate polymer N-sulphation in the vicinity of the protein-linkage region of these chemically related glycosaminoglycans.

Acetylation↗