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Biomedical subjects

A Ratcliffe

Publications and source records attributed to A Ratcliffe.

At least 73 records · Page 4Linked to original sources
PubMed1991

A mouse brain homolog of the Drosophila Shab K+ channel with conserved delayed-rectifier properties.

We have cloned and expressed a mouse brain K+ channel that is the homolog of the Drosophila Shab K+ channel. Mouse and Drosophila Shab K+ channels (mShab and fShab, respectively) represent an instance of K+ channels and structurally related species that are both functionally and structurally conserved; most kinetic, voltage-sensitive, and pharmacological properties are similar for the 2 channels. The greatest functional difference between the currents is recovery from inactivation, which is several times slower for mShab than for fShab currents. In addition to conserved structure, the mShab polypeptide has an unusually long nonconserved region at the carboxyl end of the protein. Truncation of 293 residues from the carboxyl end produced no noticeable change in voltage-sensitive, kinetic, or pharmacological properties. Thus, the measured functional properties of mShab are determined by the remaining 564 residues, most of which are conserved. The mShab and fShab channels are naturally occurring structural variants having substitutions in conserved portions that appear relatively neutral with respect to all measured properties except for, possibly, the rate of recovery from inactivation. The mShab current closely resembles a native delayed-rectifier-type potassium current, IK, in hippocampal neurons.

Amino Acid Sequence↗
PubMed1990

Biochemical markers of osteoarthritis.

The recent attempts to discover markers of early degenerative joint disease need to be considered from several viewpoints. 1. Our basic understanding of events in joint disease has increased considerably as a result of these studies, and the information gained will collectively contribute to future developments. 2. It should be clearly stated that at present, no marker has been found in biologic fluids that can be used for diagnosing and monitoring degenerative joint disease. 3. Current data provide optimism that the analysis of biologic fluids, perhaps by using more than one marker or fluid, will in the future offer clinically useful information allowing an earlier diagnosis and more appropriate monitoring of the disease. 4. Genetic markers will provide information on subpopulations of patients susceptible to disease development, which should allow early intervention in disease activity and progression in these patients.

Animals↗
PubMed1990

Modulation of native chondroitin sulphate structure in tissue development and in disease.

Chondroitin sulphate proteoglycans are synthesised by different tissues and cell types, and the chondroitin sulphate chains are variably sulphated. Three monoclonal antibodies 3B3, 7D4 and 6C3 that recognise different native chondroitin sulphate epitopes have been used to investigate changes in structure during embryonic tissue development in the chick and in the response of mature canine articular cartilage during experimental osteoarthritis. Strong focal expression of the epitopes was seen during development of chick bursa, which was different for the three epitopes and which changed during 5 days of development. In embryonic chick limb, although chondroitin sulphate is present throughout the cartilage, the 3B3 epitope, which is at the non-reducing terminus of chains, was only expressed on chondroitin sulphate within one region of the sub-articular cartilage. In mature canine articular cartilage the expression of this epitope on proteoglycans was very low, but when determined 3 or 6 months after induction of experimental osteoarthritis the level was greatly increased in all joints tested (23/23). The abundance of the other two native chondroitin sulphate epitopes was also increased in this experimental disease. The results show that expression of the chondroitin sulphate epitopes detected by the monoclonal antibodies changes during cellular differentiation and development and suggests that it is closely controlled by the cells synthesising chondroitin sulphate chains.

Animals↗
PubMed1989

Structural and immunological studies of keratan sulphates from mature bovine articular cartilage.

Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.

Animals↗
PubMed1989

Cartilage proteoglycan depletion in acute and chronic antigen-induced arthritis.

We examined the kinetics of proteoglycan (PG) depletion in rabbits with antigen-induced arthritis. There was a rapid loss of PG from arthritic cartilage, reaching 35-40% at day 7. Thereafter, the rate of PG depletion declined, and by day 42, the maximum loss was 55-60%. The initial loss of PG was accompanied by the appearance of large amounts of sulfated glycosaminoglycans (GAGs) in the joint fluid (measured as total sulfated GAGs by dye binding and as keratan sulfate by radioimmunoassay). However, by day 14, the levels of sulfated GAGs in arthritic joint fluid declined to control levels, even though the cartilage demonstrated a sustained depletion of PG. The cartilage PG degradation observed in antigen-induced arthritis could also be produced in normal animals by a single intraarticular injection of recombinant interleukin-1. The acute loss of cartilage PG occurred independently of neutrophil accumulation, both in the case of antigen-induced arthritis and after injection of interleukin-1.

Animals↗
PubMed1989

Asthma in pregnancy complicated by iatrogenic pulmonary oedema.

We report a unique case of near fatal acute pulmonary oedema developing with intravenous ritodrine, given in an attempt to suppress premature labour. The novel aspect of the case is that the patient had also been treated in the previous week with high dose nebulized beta-agonists for an episode of acute severe asthma, demonstrating that this idiosyncratic reaction to beta-adrenergic agents only occurs with the intravenous route of administration. The management of acute severe asthma occurring in pregnancy is discussed with a review of previous literature regarding possible mechanisms of beta 2-agonist-induced pulmonary oedema.

Acute Disease↗
PubMed1988

Increased concentrations of proteoglycan components in the synovial fluids of patients with acute but not chronic joint disease.

Synovial fluid samples (139) from 121 patients with rheumatoid arthritis, osteoarthritis, pseudogout, chronic pyrophosphate arthritis, gout, and reactive arthritis were analysed for cartilage proteoglycan components. Keratan sulphate (KS) epitope was determined by a competitive radioimmunoassay, and total sulphated glycosaminoglycans (S-GAG) were determined after papain digestion by a specific dye binding assay. Increased concentration of both KS epitope and S-GAG were found in synovial fluid from joints with acute inflammatory arthropathy (gout, pseudogout, and reactive arthritis). Analysis of consecutive samples from the same joint at different stages showed that the concentration of KS epitope or total S-GAG varied with acute inflammatory activity. In samples from patients with chronic conditions during active and inactive inflammatory phases concentrations were much lower and not distinguishable among these disease groups. The detection of raised concentration of proteoglycan components may reflect the rapid depletion or greatly increased turnover of proteoglycan in the articular cartilage during acute inflammation in the joint. This did not appear to be sustained in most patients with chronic joint diseases.

Acute Disease↗
PubMed1987

The detection of substructures within proteoglycan molecules. Electron-microscopic immuno-localization with the use of Protein A-gold.

Proteoglycan monomers from pig laryngeal cartilage were examined by electron microscopy with benzyldimethylalkylammonium chloride as the spreading agent. The proteoglycans appeared as extended molecules with a beaded structure, representing the chondroitin sulphate chains collapsed around the protein core. Often a fine filamentous tail was present at one end. Substructures within proteoglycan molecules were localized by incubation with specific antibodies followed by Protein A-gold (diameter 4 nm). After the use of an anti-(binding region) serum the Protein A-gold (typically one to three particles) bound at the extreme end of the filamentous region. A small proportion of the labelled molecules (10-15%) showed the presence of gold particles at both ends. A monoclonal antibody specific for a keratan sulphate epitope (MZ15) localized a keratan sulphate-rich region at one end of the proteoglycan, but gold particles were not observed along the extended part of the protein core. This distribution was not changed by prior chondroitin AC lyase digestion of the proteoglycan. Localization with a different monoclonal antibody to keratan sulphate (5-D-4) caused a change in the spreading behaviour of a proportion (approx. 20%) of the proteoglycan monomers that lost their beaded structure and appeared with the chondroitin sulphate chains projecting from the protein core. In these molecules the Protein A-gold localized antibody (5-D-4) along the length of the protein core whereas in those molecules with a beaded appearance it labelled only at one end. Labelling with either of the monoclonal antibodies was specific, as it was inhibited by exogenously added keratan sulphate. The differential localization achieved may reflect structural differences within the proteoglycan population involving keratan sulphate and the protein core to which it is attached. The results showed that by this technique substructures within proteoglycan molecules can be identified by Protein A-gold labelling after the use of specific monoclonal or polyclonal antibodies.

Animals↗
PubMed1987

Immunochemical studies on the synthesis and secretion of link protein and aggregating proteoglycan by chondrocytes.

Chondrocytes from pig laryngeal cartilage were maintained in culture, and the biosynthesis and secretion of link protein and proteoglycan were studied using immunochemical, biochemical and immunolocalisation techniques. In the presence of monensin there was a dose-dependent inhibition of link protein secretion which was very similar to that of aggregating proteoglycan, and suggested that they followed the same intracellular pathway during biosynthesis. In the presence of cycloheximide there was a similar dose-dependent inhibition of the secretion of both link protein and proteoglycan. Kinetics of secretion following inhibition of synthesis by cycloheximide showed that both proteins had similar intracellular pool sizes. Analysis of protein core and glycosaminoglycan biosynthesis showed that the time for synthesis and glycosylation of proteoglycan was 22 minutes, and this was quickly followed (within 6 minutes) by secretion. Intracellular electron microscopic immunolocalisation using protein A-gold showed link protein to be present in the Golgi cisternae and vesicles, and double-labelling experiments showed link protein only to be detected in vesicles that also labelled for proteoglycan protein core. When chondrocytes were maintained in monolayer culture for 10 days the rate of biosynthesis and secretion of proteoglycan increased although that of link protein remained constant. The control of their biosynthesis was thus shown to be independent. Within 4 hours of secretion a high proportion of link protein was incorporated into proteoglycan aggregates.

Animals↗
PubMed1986

Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments.

Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.

Animals↗
PubMed1986

Cartilage proteoglycans.

The structure of the protein core of the high molecular weight aggregating proteoglycan from pig laryngeal cartilage has been investigated. Mild trypsin digestion of proteoglycan aggregates released a large (Mr approximately equal to 150K) protein-rich fragment that contained the hyaluronate-binding region (Mr 66K). Rotary-shadowing electron microscopy of this preparation showed it to contain 'double globe' structures, similar to those seen with intact proteoglycans. Interaction studies and immunochemical evidence showed that one of the globular domains was the binding region. The second globular domain did not interact with hyaluronate or share any major antigenic determinants with the binding region and its function remains unknown. Further evidence from rotary shadowing also suggested that the protein core contained a third globular domain at the C-terminal end. The complete protein core sequence thus contains long folded globular protein regions, in addition to the extended regions bearing glycosaminoglycan chains. Studies of proteoglycan turnover in explants of pig articular cartilage showed that proteoglycan fragments were continuously released into the medium during culture. These included large non-aggregating proteoglycan fragments, free binding region and also link protein. Proteoglycans retained within the cartilage matrix remained intact and able to aggregate. Only in the presence of interleukin 1 was there evidence of more extensive proteolytic digestion. The results suggest normal turnover to be a conservative mechanism involving the selective cleavage of proteoglycan close to the hyaluronate-binding region. This releases the major glycosaminoglycan-bearing domain and enables it to diffuse out of the matrix. The site of the initial cleavage appears to be in the region of the N-terminal globular domains.

Animals↗
PubMed1985

The intracellular localisation of proteoglycans and their accumulation in chondrocytes treated with monensin.

Pig laryngeal chondrocytes incubated in the presence of monensin showed inhibition of [35S]sulphate incorporation and decreased secretion of proteoglycan into the culture medium, but no large decrease in protein synthesis. This lead to the intracellular accumulation of proteoglycan protein core, which was detected in immunoprecipitates of cell extracts. Using the same antiserum protein core was localised by electron microscopy with protein A-coated gold. In control chondrocytes, it was detected only in elements of the Golgi and in secretory vesicles, but following monensin treatment labelling was more intense in the Golgi and extended into the distended cisternae of the rough endoplasmic reticulum. The results suggest that monensin blocks proteoglycan protein core translocation between different elements of the Golgi and that this occurs prior to the major site of chondroitin sulphate synthesis on proteoglycan.

Animals↗
PubMed1985

Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate.

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.

Animals↗
PubMed1985

Proteoglycan biosynthesis in chondrocytes: protein A-gold localization of proteoglycan protein core and chondroitin sulfate within Golgi subcompartments.

The intracellular pathway of cartilage proteoglycan biosynthesis was investigated in isolated chondrocytes using a protein A-gold electron microscopy immunolocalization procedure. Proteoglycans contain a protein core to which chondroitin sulfate and keratan sulfate chains and oligosaccharides are added in posttranslational processing. Specific antibodies have been used in this study to determine separately the distribution of the protein core and chondroitin sulfate components. In normal chondrocytes, proteoglycan protein core was readily localized only in smooth-membraned vesicles which co-labeled with ricin, indicating them to be galactose-rich medial/trans-Golgi cisternae, whereas there was only a low level of labeling in the rough endoplasmic reticulum. Chondroitin sulfate was also localized in medial/trans-Golgi cisternae of control chondrocytes but was not detected in other cellular compartments. In cells treated with monensin (up to 1.0 microM), which strongly inhibits proteoglycan secretion (Burditt, L.J., A. Ratcliffe, P. R. Fryer, and T. Hardingham, 1985, Biochim. Biophys. Acta., 844:247-255), there was greatly increased intracellular localization of proteoglycan protein core in both ricin-positive vesicles, and in ricin-negative vesicles (derived from cis-Golgi stacks) and in the distended rough endoplasmic reticulum. Chondroitin sulfate also increased in abundance after monensin treatment, but continued to be localized only in ricin-positive vesicles. The results suggested that the synthesis of chondroitin sulfate on proteoglycan only occurs in medial/trans-Golgi cisternae as a late event in proteoglycan biosynthesis. This also suggests that glycosaminoglycan synthesis on proteoglycans takes place in a compartment in common with events in the biosynthesis of both O-linked and N-linked oligosaccharides on other secretory glycoproteins.

Animals↗
PubMed1984

The distribution of aggregating proteoglycans in articular cartilage: comparison of quantitative immunoelectron microscopy with radioimmunoassay and biochemical analysis.

Electron microscopic immunolocalization and radioimmunoassay have been used to determine the variation with depth of the hyaluronate-binding region of proteoglycan in articular cartilage. The cartilage was cut into serial sections from the articular surface to the bony margin, the proteoglycans were extracted from each section and determined by radioimmunoassay using antibodies raised against proteoglycan binding region. Proteoglycans were found to be most abundant in the middle zone and least abundant near the articular surface. Biochemical analysis for hexuronate in the same extracts showed a distribution of proteoglycan in agreement with these and other published results. The binding region antiserum was used for electron microscopic immunolocalization of proteoglycan with ultrathin sections of cartilage embedded in Lowicryl K4M resin. After digestion of the sections with chondroitinase ABC, the proteoglycans were localized using the antiserum and protein A-coated gold particles as immunolabel. The density of labeling was quantified using a Magiscan image analysis system. Throughout the depth of the cartilage matrix labeling was higher in the pericellular regions compared to the intercellular regions, and variation of the amount of immunolabel with depth was found to show a good correlation with the results from radioimmunoassay. Intracellular labeling of proteoglycans was mainly found over the Golgi region and in membrane-bound (secretory) vesicles.

Acrylic Resins↗