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Biomedical subjects

A Ratcliffe

Publications and source records attributed to A Ratcliffe.

At least 19 recordsLinked to original sources
PubMed1992

Antigenic properties of keratan sulfate: influence of antigen structure, monoclonal antibodies, and antibody valency.

The influence of (a) antigen structure, (b) type of monoclonal antibody, and (c) antibody bivalency on the immunochemical detection and quantification of keratan sulfate (KS) from aggrecan has been studied. Apparent KS epitope levels were determined by immunoglobulin G (IgG)-enzyme-linked immunosorbent assay (ELISA) in preparations of human aggrecan and in a defined series of lower molecular weight proteoglycan preparations generated by proteolytic and alkali treatment of aggrecan. Gel filtration chromatography showed KS epitope to be preferentially detected in the higher molecular weight fragments of the preparations. In single KS chains the epitope was detected in the chains of higher M(r). The ability of the proteoglycan to inhibit in the IgG-ELISA decreased with a reduction in proteoglycan fragment size, ranging between 6- and 260-fold, depending on the antibody used. This was considered to be a cooperative binding effect. With most antibodies, the sensitivity of the IgG-ELISA (represented by the steepness of the inhibition slope) was also reduced with smaller inhibitor sizes. The lowest limit of detectability (the amount of KS required to generate 20% inhibition) varied by up to 60-fold depending on the antibody used. The use of monovalent Fab fragments instead of the whole IgG anti-KS antibody in the ELISA showed that the bivalency of the antibody also affected the quantitation of the assay. In the Fab-ELISA the assay was found to have an increased detectability (by 9.5-fold with aggrecan as the inhibitor), and the proteoglycan fragments and aggrecan all generated parallel inhibition curves. Although the Fab-ELISA was somewhat influenced by the structural presentation of the KS, this was not apparent for small fragments and single chains. Thus the effects of cooperative binding and antibody valency could be overcome and quantitative data could be obtained for all samples, using papain-digested samples and the Fab-ELISA. Application of this assay to analysis of body fluids showed the KS-containing fragments in synovial fluid, serum, and urine were of different sizes and could be quantified.

Aggrecans↗
PubMed1992

The microstructural tensile properties and biochemical composition of the bovine distal femoral growth plate.

The tensile properties of distal femoral growth plates from 12-month-old cows were determined on uniformly prepared straight bone-growth plate-bone specimens (7 x 7 mm2 in cross-section) from predetermined anatomical sites on the physis. Each specimen was distracted to failure using a computer-controlled servo-hydraulic testing machine at a strain rate of 0.004 s-1. It was found that the exponential constitutive law, using finite deformation formulation for strain, provides an excellent description of the stress-strain behavior of all the specimens up to the point of failure. The ultimate stress and both tangent moduli (i.e., the toe region tangent modulus and the tangent modulus calculated at 75% of ultimate strain) varied with anatomical site. The anterior region was the strongest, followed by the posterior/lateral. The anterior and posterior/lateral regions were also the stiffest, whereas the posterior/medial and center regions were the weakest and most compliant. The bone-growth plate-bone specimen exhibited a low ultimate strain (13.8% +/- 6%) that did not vary significantly throughout the growth plate. This result suggests that disruption of the physis may occur in vivo even at the lower distractions currently recommended for the clinical chondrodiatasis procedure for leg lengthening. The biochemical composition of the growth plate in the anatomical regions correlated well with the tensile properties. There was a greater collagen content in the regions that were the stiffest and strongest. The gross morphology of the growth plate of the bovine distal femur is also described in this study. There is a regular pattern to the undulations of the physis at several dimensional levels. Histologic findings showed that orientation of the hypertrophic cell columns and transphyseal septa are aligned nearly parallel to the longitudinal axis of the diaphyseal shaft. This column orientation is not affected by the undulation of the primary contour of the physis, which at certain locations may be inclined as much as 60 degrees relative to the diaphyseal axis. The orientation of the hypertrophic cell columns appears to be one of the dominant microstructural features influencing the tensile behavior of the bone-growth plate-bone specimens.

Animals↗
PubMed1992

Increased release of matrix components from articular cartilage in experimental canine osteoarthritis.

The release rates of specific components of the proteoglycan aggregates (G1 domain, the chondroitin sulfate and keratan sulfate containing portion of the protein core, and link protein) of the articular cartilage of mature beagles were studied at early stages of canine experimental osteoarthritis (OA), generated by transection of the anterior cruciate ligament. Analysis of cartilage explants and synovial fluids indicates that at early stages of experimental OA, there is increased release of the proteoglycan aggregates of the articular cartilage. This involves a release from the tissue of the components of the proteoglycan that are specifically involved with aggregation together with the glycosaminoglycans of the proteoglycan. These components were detected at elevated levels in the media of explants of cartilage from the operated joint, and in the synovial fluids of the operated joints.

Animals↗
PubMed1992

Correlating magnetic resonance imaging with the biochemical content of the normal human intervertebral disc.

Magnetic resonance imaging was used to determine the T2 relaxation times of prepared proteoglycan solutions and of normal human intervertebral disc tissue from the annulus fibrosus (AF) and nucleus pulposus (NP). The collagen, proteoglycan, and water contents of the disc tissue samples were determined by biochemical assays after they were scanned. Correlations among 1/T2, collagen, proteoglycan, and water contents of the tissue samples and among 1/T2, water, and proteoglycan contents of the proteoglycan solutions were calculated. A moderate negative correlation between 1/T2 and water content was noted for the tissue samples, and a very high negative correlation was found between 1/T2 and water content for the proteoglycan solutions. The very high positive correlation between 1/T2 and proteoglycan content of the proteoglycan solutions is probably due to this negative correlation between 1/T2 and water content. There was no significant correlation between 1/T2 and proteoglycan content of the tissues. The moderate positive correlation between 1/T2 and collagen content is probably due to the high negative correlation between collagen content and water content. No significant correlation was found between the collagen and proteoglycan contents of the tissues. Thus it appears that the data confirm previous reports in the literature that the collagen of the disc tissue functions to control its water content.

Adult↗
PubMed1992

Cartilage and diarthrodial joints as paradigms for hierarchical materials and structures.

The anatomic forms of diarthrodial joints are important structural features which provide and limit the motions required for the joint. Typically, the length scale of topographic variation of anatomic forms ranges from 0.5 to 15 cm. Articular cartilage is the thin layer of hydrated soft tissue (0.5-5.0 mm thick) covering the articulating bony ends in diarthrodial joints. This tissue has a set of unique mechanical and physicochemical properties which are responsible for its load-carrying capabilities and near-frictionless qualities. The mechanical properties of articular cartilage are determined at the tissue-scale level and these properties depend on the composition of the tissue, mainly collagen and proteoglycan, and their molecular and ultrastructural organization (ultra-scale: 10(-8)-10(-6) m). Because proteoglycans possess a high density of fixed negative charges, articular cartilage exhibits a significant Donnan osmotic pressure effect. This physicochemically derived osmotic pressure is an important component of the total swelling pressure; the other component of the total swelling pressure stems from the charge-to-charge repulsive force exerted by the closely spaced (1-1.5 nm) negative charge groups along the proteoglycan molecules. Thus these interactions take place at a nano-scale level: 10(-10)-10(-9) m. Finally, cartilage biochemistry and organization are maintained by the chondrocytes which exist at a micro-scale level (10(-7)-10(-6) m). Significant mechanoelectrochemical transduction occurs within the extracellular matrix at the micro-scale level which affects and modulates cellular anabolic and catabolic activities. At present, the exact details of these transduction mechanisms are unknown. In this review, we present a summary of the hierarchical features for articular cartilage and diarthrodial joints and tables of known material properties for cartilage. Also we summarize how the multi-scale interactions in articular cartilage provide for its unique material properties and tribological characteristics.

Animals↗
PubMed1992

Changes in proteoglycan turnover in experimental canine osteoarthritic cartilage.

The metabolism of newly-synthesised and total ("resident") proteoglycans was examined in control and osteoarthritic cartilage explants obtained from an experimental model (Pond and Nuki, 1973) of canine osteoarthritis. The following findings were obtained: (i) Non-labelled proteoglycans extracted from normal cartilage with 4 M guanidine HCl showed two bands visualised by staining with toluidine blue. The electrophoretic mobilities of proteoglycans from osteoarthritic cartilage were unchanged but the relative abundance of the slower migrating band increased with time after surgery. (ii) There were qualitative differences in the proteoglycan breakdown products released into the medium of explant cultures of osteoarthritic compared with control cartilage. This was apparent for both labelled and total unlabelled proteoglycans. (iii) There were similarities in the electrophoretic mobilities of the major labelled and non-labelled proteoglycan breakdown products suggesting that total ("resident") proteoglycans and newly-formed proteoglycans were degraded by similar mechanisms. There were however some differences in the labelled and non-labelled proteoglycans, suggesting that the mechanisms of breakdown were not identical. (iv) Immunoblotting techniques showed differences in the distribution of various glycosaminoglycans in proteoglycan breakdown products from control compared with osteoarthritic cartilage explant cultures. (v) Monoclonal antibodies 7-D-4 and 3-B-3 (which recognise unusual native chondroitin sulphate epitopes) showed greatly increased expression on proteoglycans from osteoarthritic cartilage compared with controls.

Animals↗
PubMed1992

Urinary hydroxypyridinium cross-links of collagen in primary hyperparathyroidism.

Urinary concentrations of the collagen cross-links, pyridinoline (PYD) and deoxypyridinoline (DPD), were determined in 87 patients with untreated or surgically treated primary hyperparathyroidism (PHPT). Eighty-four healthy individuals, matched for age and sex, constituted the control group for the excretion of pyridinium cross-links. In addition, a subgroup of 25 patients with PHPT was followed longitudinally for up to 2 yr after successful parathyroidectomy. Mean urinary excretion of PYD (46.8 +/- 2.7 nmol/mmol creatinine) and DPD (17.6 +/- 1.3 nmol/mmol creatinine) was significantly higher in patients with untreated PHPT than in normal subjects (P less than 0.001). In the group undergoing successful parathyroidectomy, mean urinary concentrations of PYD (34 +/- 2.5) and DPD (9.4 +/- 0.8) were similar to those in normal controls and significantly lower than those in the untreated patient population (P less than 0.001). The urinary concentration of both cross-links was significantly correlated with serum levels of both alkaline phosphatase and PTH. Mean urinary concentrations of both cross-link compounds decreased significantly within 6 months in patients followed longitudinally and as early as 2 weeks after surgery in individual patients compared to presurgical baseline values. These changes preceded the reduction in serum alkaline phosphatase and hydroxyproline by approximately 6 months. The results demonstrate that urinary hydroxypyridinium cross-links of collagen are useful indices in the clinical assessment of bone involvement in PHPT.

Alkaline Phosphatase↗
PubMed1991

mShal, a subfamily of A-type K+ channel cloned from mammalian brain.

We have cloned and expressed a mouse brain cDNA, mShal, that encodes a transient, A-type K+ current. mShal, the vertebrate homolog of the Drosophila Shal gene, defines a distinct subfamily of voltage-gated K+ channels. The Shal deduced proteins are more highly conserved between mouse and Drosophila than other presently known K+ channels. mShal carries a "low-threshold" A-type current with a hyperpolarized steady-state inactivation midpoint. Marked similarity was observed between mShal and its Drosophila homolog, fShal, with regard to voltage sensitivity of activation, macroscopic inactivation, steady-state inactivation, and 4-aminopyridine sensitivity. Sequence conservation for Shal proteins is unusually high at the amino terminus, an area considered important for inactivation. Removal of conserved amino-terminal residues from mShal modifies macroscopic inactivation but the transient nature of the current is preserved. Underlying the very high conservation of mShal and fShal may be a role in the nervous system that is conserved in widely divergent species.

Amino Acid Sequence↗
PubMed1991

Early diagnosis of osteoarthrosis of the temporomandibular joint: correlation between arthroscopic diagnosis and keratan sulfate levels in the synovial fluid.

The role of osteoarthrosis (OA) and proteoglycan degradation in the pathogenesis of temporomandibular joint (TMJ) disorders has not been well established. The orthopaedic literature has demonstrated that proteoglycan degradation plays a significant role in the pathology of many joints. The purpose of this investigation was to determine if levels of immunoreactive keratan sulfate (an important component of cartilage proteoglycans) present in synovial fluid aspirates from TMJs correlated with arthroscopically demonstrated OA. Temporomandibular joint arthroscopy was performed on 25 joints in 20 patients and synovial fluid aspirates were obtained just prior to the insertion of arthroscopic cannulas. The results showed that synovial fluid aspirates from joints that arthroscopically demonstrated OA had significantly higher levels of keratan sulfate than synovial fluid aspirates from those joints that showed no evidence of OA (NON-OA). This study gives support to the theory that the pathogenesis of OA of the TMJ is similar to that of chondromalacia of other synovial joints. The combination of TMJ arthroscopy and synovial fluid analysis is an important model that can be used for investigation of the pathogenesis of TMJ disorders.

Adult↗
PubMed1991

Experimental studies on repair of large osteochondral defects at a high weight bearing area of the knee joint: a tissue engineering study.

There is a vast clinical need for the development of an animal model to study the fundamentals of healing of injured or diseased diarthrodial joints (knee, hip, shoulder, wrist, etc). Current prosthetic replacements do not offer acceptable treatment for injuries and diseases of these joints in young active individuals. New clinical treatment modalities, based on sound biologic principles, are sought for the development of repair or healing tissues engineered to have similar biomechanical properties as normal articular cartilage. In this paper we present a brief review of this need, and propose a grafting procedure which may lead to a successful animal model for studies of long term repair of major osteochondral defects. This grafting procedure uses an autologous periosteum-bone graft or an autologous-synthetic bone replacement graft. We have applied these grafts for in vivo repair of large surgically created defects in the high weight bearing area of the distal femoral condyle of mature New Zealand white rabbits. Further, an interdisciplinary study, including histochemistry, biochemistry (composition and metabolic activities), and biomechanics (biphasic properties), was performed to assess the feasibility of our animal model to generate viable repair tissues. We found our grafting procedure produced, 8 weeks postoperatively, tissues which were very similar to those found in normal articular cartilage. However, our histological studies indicate incomplete bonding between the repair tissue and the adjacent cartilage, and lack of an appropriate superficial zone at the articular surface. These deficiencies may cause long term failure of the repair tissue. Further studies must be undertaken to enhance development of a strong bond and a collagen-rich surface zone. This may require the use of growth factors (e.g., transforming growth factors beta) capable of simulating extra collagen production, or the use of serum derived tissue glue for bonding. At present, we are pursuing these studies.

Animals↗
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↗