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Biomedical subjects

R C Murray

Publications and source records attributed to R C Murray.

At least 19 recordsLinked to original sources
PubMed2006

Do the matrix degrading enzymes cathepsins B and D increase following a high intensity exercise regime?

OBJECTIVE: It has been shown by others that levels of matrix degrading enzymes are increased in osteoarthritis (OA) and so are proposed to be involved in the aetiopathogenesis of the disease, including exercise-associated OA. Therefore we hypothesised that cathepsin B and cathepsin D were increased in cartilage samples previously shown to have early stage OA from 2-year-old Thoroughbred horses, euthanased for reasons other than this study, that had a history of 19-week high intensity exercise (n=6) compared to age and sex-matched horses with a history of low intensity exercise (n=6). METHODS: Cartilage samples were used from four specific sites within the carpal joints. Standard immunolocalisation protocols and blind counting of positive and negative cells within the articular surface, mid-zone and deep zone (DZ) were used to test our hypothesis. RESULTS: A high intensity exercise regime did not significantly alter the number of chondrocytes positive for cathepsin B, whereas a significant decrease was found for cathepsin D in the DZ, indicating that these enzymes are regulated differently by mechanical loading. Furthermore, cathepsin D varied according to the topographical location within the joint, reflecting biomechanical differences experienced during a high compared to a low intensity exercise regime. CONCLUSION: This study disproves our hypothesis that cathepsins B and D are increased following a high intensity exercise regime unlike that reported for other matrix enzymes.

Animals↗
PubMed2005

Is there a characteristic distal tarsal subchondral bone plate thickness pattern in horses with no history of hindlimb lameness?

REASONS FOR PERFORMING STUDY: Osteoarthritis (OA) is a common cause of distal tarsal pain, but disease development is poorly understood. Awareness of normal tarsal structure and function is important in order to understand the pathogenesis of OA. Thickening of the subchondral bone (SCB) plate has been related to the development of OA, but SCB plate patterns in the equine tarsus have not been documented. HYPOTHESES: There is a repeatable pattern of SCB thickness across the distal tarsal joints, and specifically that thickness would be greatest dorsally and laterally. METHODS: Twenty cadaver tarsi were collected from mature horses that had undertaken low-level exercise only with no history of hindlimb lameness. Magnetic resonance images were acquired using a high-resolution sagittal 3-dimensional T1-weighted spoiled gradient echo sequence. Subchondral bone thickness was measured on sagittal images at dorsal and plantar locations on the proximal and distal aspects of the central (CT) and third (T3) tarsal bones and proximal aspect of the third metatarsal bone (Mt3). RESULTS: On the proximal aspect of CT, medial and lateral SCB thickness were significantly greater than midline. On the distal aspect of CT and T3 and proximal Mt3, lateral SCB thickness was significantly greater than medial and midline sites. Dorsal SCB thickness was greatest on the proximal and distal aspects of CT and proximal Mt3. Subchondral bone accounted for a greater proportion of CT and T3 on the dorsal aspect than the plantar. CONCLUSIONS: There is a repeatable pattern of SCB thickness in the distal tarsal bones of horses with no history of hindlimb lameness. This reflects the pattern of loading across the joints. POTENTIAL RELEVANCE: This study provides evidence of a consistent osteochondral pattern in the equine tarsus for reference in identification of osteoarticular pathologies.

Animals↗
PubMed2002

The effect of intra-articular methylprednisolone acetate and exercise on equine carpal subchondral and cancellous bone microhardness.

Dorsal carpal osteochondral injury is a major cause of lameness in horses undergoing high intensity training. Intra-articular corticosteroid treatment is used commonly to manage exercise-associated articular pain, but its use remains highly controversial in the equine athlete. This project, therefore, aimed to compare the mechanical properties of intra-articular MPA and diluent-treated middle carpal subchondral and cancellous bone in horses undergoing a short-term treadmill exercise programme. It was hypothesised that subchondral and cancellous bone mechanical properties are influenced by intra-articular administration of methylprednisolone acetate (MPA). Eight 2-year-old female horses had MPA or diluent administered into contralateral middle carpal joints at 14 day intervals, for a total of 4 treatments per horse. Horses underwent a standard treadmill exercise protocol until euthanasia (Day 70). Standard sites were located on the dorsal aspect of third, radial and intermediate carpal bones. Osteochondral samples from each test site were divided into subchondral bone and cancellous bone portions. These were dried, resin-embedded and gold-coated. Microhardness measurements were obtained at each test site. No significant effect of intra-articular treatment was detected. At each site, cancellous bone trabecular struts had an 18-19% higher microhardness value than the overlying subchondral bone. These findings indicate that intra-articular administration of MPA at this dose has no effect on subchondral or cancellous bone adaptation to short-term exercise and, therefore, on the propensity of carpal bones to injury. Further investigation into the calcified cartilage layer, effect of different corticosteroid preparations and diffusion of medication are required.

Animals↗
PubMed2001

Subchondral bone thickness, hardness and remodelling are influenced by short-term exercise in a site-specific manner.

It was hypothesised that subchondral bone thickness, hardness and remodelling are influenced by exercise intensity, and by location within a joint. Dorsal carpal osteochondral injury is a major cause of lameness in horses undergoing high intensity training. This project aimed to determine the subchondral bone thickness, formation, resorption and hardness at sites with high and low incidence of pathology in 2 year-old horses undergoing 19 weeks high intensity treadmill training or low intensity exercise, and to compare these factors between exercise groups. Dorsal and palmar test sites were identified on radial, intermediate and third carpal articular surfaces after euthanasia. Adjacent osteochondral samples from each test site underwent histomorphometric analysis (for subchondral bone thickness, osteoid perimeter, osteoid seam width, eroded cavity area and eroded cement line surface length) and microhardness testing. Bone from horses undergoing high intensity training was thicker with a greater osteoid perimeter, and at individual sites had a smaller osteoid seam width and eroded cavity. Exercise-related differences were most marked at dorsal locations. Maximal differences in bone formation indices were observed at dorsal radial and medial third carpal locations. Overall subchondral bone from dorsal sites was thicker with a greater osteoid perimeter. Subchondral bone from dorsal sites was approximately 35% harder than bone from palmar sites. These results show topographical variations in subchondral bone structure, formation, resorption and material properties and a site-specific response to exercise. The maximal response to exercise was at high load sites with a clinical predisposition to injury. These findings indicate that the combined effect of exercise and local load variations within a joint may lead to maximal adaptive responses or overload of these responses at sites predisposed to injury.

Animals↗
PubMed2001

Biochemical composition of equine carpal articular cartilage is influenced by short-term exercise in a site-specific manner.

UNLABELLED: It was hypothesized that cartilage macro-molecular characteristics are influenced by exercise intensity and by location within a joint. OBJECTIVE: To determine the macromolecular characteristics of carpal articular cartilage at common and uncommon sites of pathology in horses undergoing high or low intensity exercise, and to compare this composition between exercise groups. DESIGN: Twelve horses (19.3+/-0.9 years) were assigned to exercise groups. Each group underwent 19 weeks high-intensity treadmill training (N=6) or low-intensity exercise (N=6). Dorsal and palmar test sites were identified on radial, intermediate and third carpal articular surfaces after euthanasia. Cartilage was collected from each site, freeze-dried and assessed for water content. Hydroxyproline, glycosaminoglycan and DNA analyses were performed on cartilage from each test site. Adjacent cartilage underwent histological preparation and assessment for chondrocyte numerical density at each site and proteoglycan distribution through the depth of cartilage. RESULTS: Dorsal cartilage had a higher collagen content, DNA content, and chondrocyte numerical density, but lower glycosaminoglycan content than palmar cartilage. Cartilage from horses undergoing high-intensity training had a significantly higher glycosaminoglycan content than cartilage from horses undergoing low-intensity exercise, with maximal difference being observed in cartilage from dorsal radial and dorsal intermediate carpal articular surfaces. Overall no effect of exercise on collagen was observed, but at sites predisposed to clinical lesions cartilage from horses undergoing high-intensity training contained significantly less collagen than from horses undergoing low-intensity exercise. Distribution of proteoglycan was non-uniform in 52% of the sections examined, with superficial loss of toluidine blue staining primarily at dorsal sites and in the high-intensity exercise group. CONCLUSIONS: These results indicate that topographical and exercise related differences exist in carpal cartilage composition, and that the effect of exercise on overall composition and distribution within the cartilage was maximal at sites predisposed to clinical lesions. These findings could indicate that the combined effect of exercise and local load variations within a joint may lead to a risk of exceeding the physiologic threshold at high load sites that are predisposed to clinical injury.

Animals↗
PubMed2001

The distribution of cartilage oligomeric matrix protein (COMP) in equine carpal articular cartilage and its variation with exercise and cartilage deterioration.

Based on previous studies where tendons receiving the most load have been shown to have the highest levels of cartilage oligomeric matrix protein (COMP), we hypothesized that COMP distribution in articular cartilage may be influenced by mechanical loading. This investigation aimed (a) to describe the pattern of COMP immunoreactivity in middle carpal joint cartilage of two-year-old Thoroughbred horses; (b) to determine topographical variations; (c) to compare high (group 1) and low (group 2) intensity training and (d) to describe COMP immunoreactivity at sites with early osteoarthritis. Group 1 (n =6) underwent a 19 week high-intensity treadmill training programme and group 2 (n =6) were given daily walking until euthanasia. Dorsal and palmar sites on radial and third carpal articular surfaces were prepared. Immunohistochemistry was performed with polyclonal rabbit anti-equine COMP antiserum using a biotin-streptavidin/peroxidase method. Results showed: (a) intracellular immunoreactivity was present in all cartilage zones, but the distribution of COMP staining within the matrix varied between cartilage zones; (b) differences in distribution between sites were not observed, but total COMP levels in exercised horses (n =2) did vary between sites with dorsal sites containing less COMP than palmar sites on the radial, intermediate and third carpal lateral facet; (c) group 1 cartilage showed marked interterritorial distribution in the deep layer compared to group 2 where staining was more generalized throughout the matrix and (d) fibrillated cartilage showed increased local immunoreactivity in the matrix. These findings demonstrate zonal variations in equine COMP distribution which may be influenced by loading.

Animals↗
PubMed2000

Mutations in the reduced-folate carrier affect protein localization and stability.

The reduced-folate-carrier (rfc) gene has been shown to be functionally important for reduced-folate transport in mammalian cells. In the present paper we describe the identification of alterations in both alleles of the rfc gene in a mutant Chinese-hamster ovary cell line deficient in methotrexate transport. One allele of the rfc gene contains a point mutation resulting in a Gly(345)-->Arg substitution in the predicted amino acid sequence. In this case, a protein of similar size to the wild-type protein is produced, although it remains as an immature, core-glycosylated, form. The second allele contains a point mutation in the last base of intron 5 that results in the utilization of a cryptic splice site leading to a seven-base deletion in the mRNA. The use of an alternate splice site changes the reading frame to yield a truncated protein with 68 different C-terminal amino acids as compared with the wild-type. Both of these altered gene products were monitored by fusion with green fluorescent protein and found to be non-functional with an increased rate of turnover. The protein with the point mutation is trapped in the endoplasmic reticulum with subsequent degradation, whereas the product of the splice mutation is not membrane-associated and is partially degraded. Thus mutations in both alleles of the rfc gene in this resistant cell line account for the loss of reduced-folate transport. The observations made regarding the degradation of these mutant gene products also provide support for putative checkpoints in the endoplasmic reticulum.

Alleles↗
PubMed2000

A fragment of the Neurogenin1 gene confers regulated expression of a reporter gene in vitro and in vivo.

The basic helix-loop-helix transcription factor neurogenin1 is required for proper nervous system development in vertebrates. It is expressed in neuronal precursors during embryonic development and is thought to play a role in specifying neuronal fate. To investigate the regulation of neurogenin1 expression, the transcriptional start site of the gene was identified and a 2.7-kb fragment ending in the first exon was shown to possess basal promoter activity. This 2.7-kb fragment was able to promote expression of reporter genes in P19 cells under conditions in which expression of endogenous neurogenin1 was induced. Importantly, the 2.7-kb fragment was able to drive expression of a lacZ reporter gene in transgenic mice in most tissues in which neurogenin1 is normally expressed, including those peripheral ganglia that fail to develop in neurogenin1 "knockout" mice. These findings identify a regulatory region containing elements responsible for appropriate expression of a gene with a crucial role in generating the vertebrate nervous system.

Animals↗
PubMed2000

Opposing effects of bone morphogenetic proteins on neuron production and survival in the olfactory receptor neuron lineage.

In olfactory epithelium (OE) cultures, bone morphogenetic proteins (BMPs) can strongly inhibit neurogenesis. Here we provide evidence that BMPs also promote, and indeed are required, for OE neurogenesis. Addition of the BMP antagonist noggin inhibited neurogenesis in OE-stromal cell co-cultures. Bmp2, Bmp4 and Bmp7 were expressed by OE stroma, and low concentrations of BMP4 (below the threshold for inhibition of neurogenesis) stimulated neurogenesis; BMP7 did not exhibit a stimulatory effect at any concentration tested. Stromal cell conditioned medium also stimulated neurogenesis; part of this effect was due to the presence within it of a noggin-binding factor or factors. Studies of the pro-neurogenic effect of BMP4 indicated that it did not increase progenitor cell proliferation, but rather promoted survival of newly generated olfactory receptor neurons. These findings indicate that BMPs exert both positive and negative effects on neurogenesis, depending on ligand identity, ligand concentration and the particular cell in the lineage that is responding. In addition, they reveal the presence of a factor or factors, produced by OE stroma, that can synergize with BMP4 to stimulate OE neurogenesis.

Animals↗
PubMed2000

Equine carpal articular cartilage fibronectin distribution associated with training, joint location and cartilage deterioration.

Processes involved in equine carpal osteochondral injury have not been established. In other species, fibronectin appears important in chondrocyte-matrix interactions, and levels are increased in osteoarthritis. This investigation aimed to (a) describe fibronectin immunoreactivity in the middle carpal joint of 2-year-old Thoroughbreds, (b) determine topographical variations, (c) compare strenuously trained (Group 1) or gently exercised horses (Group 2) and (d) describe sites with early osteoarthritis. Group 1 (n = 6) underwent a 19 week high intensity treadmill training programme. Group 2 (n = 6) underwent 40 min walking until euthanasia. Dorsal and palmar sites on radial, intermediate and third carpal articular surfaces were prepared. Immunohistochemistry was performed using a biotin-streptavidin/peroxidase method. Cross-reactivity of rabbit antihuman fibronectin antiserum with equine fibronectin was confirmed using Western blotting. Results showed: (a) fibronectin was present primarily in pericellular and interterritorial matrix locations, (b) dorsal sites had zonal immunoreactivity compared to palmar sites, (c) Group 1 dorsal radial carpal cartilage had increased superficial staining compared to Group 2 and (d) fibrillated cartilage showed increased intracellular and local matrical immunoreactivity (superficial zone). These findings suggest topographical and exercise-related variations in fibronectin distribution, and indicate equine fibronectin is localised at sites of cartilage degeneration and released into the matrix by chondrocytes in the local area.

Animals↗
PubMed1999

Exercise affects the mechanical properties and histological appearance of equine articular cartilage.

Dorsal carpal osteochondral injury is a major cause of reduced performance in horses undergoing high-intensity training. It was hypothesised that the mechanical behaviour and histology of cartilage are influenced by the intensity of exercise and by location within a joint. Relationships between histology and mechanical behaviour were identified in 2-year-old horses undergoing 19 weeks of high-intensity treadmill training or low-intensity exercise and then compared between groups. Dorsal and palmar test sites were identified on radial, intermediate, and third carpal articular surfaces after euthanasia. The mechanical properties of cartilage were determined with an automated creep indentation apparatus as previously described for equine cartilage. Cartilage morphology was assessed with use of sections stained with haematoxylin and eosin and toluidine blue. Dorsal cartilage was less permeable, thinner, and had a loss of chondrocyte alignment compared with palmar cartilage. Cartilage from strenuously trained horses showed more fibrillation and chondrocyte clusters than did cartilage from gently exercised animals. Dorsal radial carpal cartilage and third carpal cartilage of strenuously trained animals were significantly less stiff than that from gently exercised animals, and the former had reduced superficial toluidine blue staining compared with that from the gently exercised group. These results indicate that topographical and exercise-related differences exist in the morphology and mechanical properties of carpal cartilage and suggest that strenuous training may lead to deterioration of cartilage at sites with a high clinical incidence of lesions.

Animals↗
PubMed1999

An MRI study of the effect of treadmill training on bone morphology of the central and third tarsal bones of young thoroughbred horses.

Training results in marked modelling of the subchondral bone of the carpus, but the effect of training on the subchondral bone of the distal tarsal joints is unknown. The aim of this study was to determine whether training influenced modelling of the third and central tarsal bones in Thoroughbred horses. Twelve untrained Thoroughbred horses were divided into 2 groups. Group 1 underwent a 19 week progressive training regimen on a high speed treadmill. Group 2 were walked for 40 min daily. Images of left tarsi were obtained by magnetic resonance imaging (MRI) with a 0.5 Tesla superconducting magnet using a spin echo sequence. Sagittal and oblique sagittal slices were made perpendicular to the articular surfaces of the intertarsal joints and were analysed using image analysis software to measure the proportion of dense subchondral bone in the dorsal facet of each bone. Mean +/- s.d. percentage area of dense subchondral bone in the dorsal facet of the central tarsal bone in Group 1 was 47 +/- 8 medially, 46 +/- 4 sagitally and 50 +/- 11 dorsolaterally, whereas in Group 2 it was 39 +/- 16 medially, 43 +/- 8 sagitally and 53 +/- 7 dorsolaterally. For the third tarsal bone mean +/- s.d. percentage area of dense subchondral bone in Group 1 was 32 +/- 10 medially, 39 +/- 11 sagitally and 44 +/- 8 dorsolaterally, whereas in Group 2 it was 28 +/- 8 medially, 37 +/- 6 sagitally and 41 +/- 9 dorsolaterally. There was no significant difference in percentage area of dense subchondral bone between the trained and untrained horses. An effect of treadmill training of Thoroughbred horses on modelling of the central and third tarsal bones could not be demonstrated.

Animals↗
PubMed1999

The effect of training on the calcified zone of equine middle carpal articular cartilage.

Carpal osteochondral injury is frequently observed in strenuously trained horses. It is clear that the integrity of articular cartilage and subchondral bone are intimately related, although there is controversy about which component is the most important. Calcified cartilage provides the mechanical link between soft hyaline cartilage and stiff subchondral bone so it is essential to understand the tissue's response to exercise. Middle carpal calcified and uncalcified (hyaline) cartilage thickness was investigated in horses undergoing high- and low-intensity exercise. Twelve untrained horses (18-21 months) were paired, and randomly assigned to an exercise group. Group 1 underwent 19 weeks progressive high-intensity training on a high-speed treadmill. Group 2 underwent walking exercise only. Osteochondral specimens were obtained from 8 test sites per carpus immediately after euthanasia. Histomorphometric measurements of total cartilage, hyaline layer, and calcified zone thickness were obtained from decalcified and undecalcified samples. Mean +/- s.d. (micron) calcified cartilage thickness for dorsal cartilage from Group 1 was 271 +/- 73 and from Group 2 was 163 +/- 49; for palmar cartilage from Group 1 was 195 +/- 42 and Group 2 was 150 +/- 52. Group 1 horses had significantly thicker total cartilage (P < 0.0001) and calcified zone (P < 0.0001) than Group 2, but there was no difference in hyaline layer. Within each group all dorsal sites had thicker calcified cartilage (P < 0.003) than palmar sites, but no difference in hyaline or total cartilage. These findings indicate that high-intensity exercise leads to greater calcified zone depth without alteration in hyaline cartilage thickness, and that this response is maximal at sites that withstand high, intermittent loads. Increasing calcified cartilage thickness with exercise may maintain the articular surface stiffness gradient in the face of alterations in hyaline cartilage and/or subchondral bone stiffness.

Analysis of Variance↗
PubMed1998

The effects of intra-articular methylprednisolone and exercise on the mechanical properties of articular cartilage in the horse.

INTRODUCTION: Intra-articular corticosteroids are widely used as anti-inflammatory agents for symptomatic management of arthritis, but their administration with concurrent exercise remains controversial. Biochemical and morphologic analysis of treated cartilage has revealed conflicting results, but previous biomechanical assessment has not been undertaken. OBJECTIVE: To compare the biomechanical properties of intra-articular methylprednisolone acetate (MPA) and diluent treated cartilage in treadmill exercised horses. METHODS: Eight 2-year-old female horses had MPA or diluent administered into contralateral middle carpal joints at 14 day intervals for a total of four treatments per horse. Horses underwent a standard treadmill exercise protocol until euthanasia (day 70). Standard sites were tested on the third, radial and intermediate carpal bones using an automated indentation apparatus to obtain the creep and recovery behavior of the articular cartilage. Using previously validated biphasic creep indentation methodology, aggregate modulus, Poisson's ratio, permeability, shear modulus, thickness, creep and recovery equilibrium times and percent recovery were obtained at each site. Results were analyzed using ANOVA and multiple comparisons of the means (P < 0.05). RESULTS: Cartilage intrinsic material properties and thickness demonstrated significant differences between MPA and diluent treated joints. Diluent treated cartilage had a 97% increase in compressive stiffness modulus (P = 0.0001), was 121% more permeable (P = 0.0001), had 88% increase in shear modulus (P = 0.0001), and was 24% thicker (P = 0.0001) than MPA treated articular cartilage. CONCLUSIONS: The findings indicate that repetitive intra-articular administration of MPA to exercising horses alters the mechanical integrity of articular cartilage, which could lead to early cartilage degeneration.

Analysis of Variance↗
PubMed1996

Structural organization of the reduced folate carrier gene in Chinese hamster ovary cells.

The reduced folate carrier gene (rfc) encodes a putative protein that is involved in the intracellular accumulation of folates. In this report, we describe the organization of the rfc gene from Chinese hamster ovary cells. The hamster rfc gene contains 7 exons and 6 introns, which span 15.3 kilobases. It codes for two alternatively spliced messenger RNAs, one that contains all 7 exons and one that lacks exon 2 but contains the remaining 6 exons. The transcriptional start of the gene has been mapped to six sites approximately 200 base pairs upstream of the putative ATG initiation codon. The promoter region has no TATA box-like sequence but contains a consensus Sp1 binding site. This is the first report of the genomic structure of the reduced folate carrier gene from any species.

Alternative Splicing↗
PubMed1994

Isolation of a hamster cDNA clone coding for a function involved in methotrexate uptake.

A clone has been isolated from a Chinese hamster ovary cell cDNA expression library that complements mutant cells defective in the uptake of the folate analogue methotrexate. When transfected with this clone, the mutant cells regain the ability to bind and transport the drug and, as a consequence, become sensitive to its cytotoxic action. The clone is 2314 base pairs long and has an open reading frame of 1557 base pairs that codes for a putative protein of 58 kDa. This novel putative protein has a high content of hydrophobic residues and has a large part of its predicted secondary structure in the form of beta-sheets. In the wild-type cell line and in one of the mutant cell lines, this clone detects an mRNA of 2.5 kilobases, while in another mutant cell line, this message is absent. The data are consistent with this clone encoding either the reduced folate transporter or an auxiliary function that interacts with this transporter. This is the first report of a cDNA coding for a function, other than folate-binding protein, that is involved in the transport of methotrexate.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1994

Primary pulmonary germ cell tumor with blastomatous differentiation.

We describe the clinical and pathologic findings of a patient with mixed blastoma-germ cell malignancy primary in the lung. Serum alpha-fetoprotein levels were elevated at presentation, and normalized with anti-germ cell chemotherapy. The resection specimen contained massively necrotic germ cell tumor with viable mature neural tissue, plus viable biphasic blastoma with stromal bone and skeletal muscle differentiation. It is not clear whether the germ cell component represents unusual differentiation of a somatic cell line or whether the blastoma component represents an unusual pattern of teratomatous differentiation.

Cystadenoma↗