Acetyl-L-carnitine and alpha-lipoic acid supplementation of aged beagle dogs improves learning in two landmark discrimination tests.

Beagle dogs between 7.6 and 8.8 years of age administered a twice daily supplement of alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC) over approximately 2 months made significantly fewer errors in reaching the learning criterion on two landmark discrimination tasks compared to controls administered a methylcellulose placebo. Testing started after a 5 day wash-in. The dogs were also tested on a variable delay version of a previously acquired spatial memory task; results were not significant. The improved performance on the landmark task of dogs supplemented with LA + ALC provides evidence of the effectiveness of this supplement in improving discrimination and allocentric spatial learning. We suggest that long-term maintenance on LA and ALC may be effective in attenuating age-associated cognitive decline by slowing the rate of mitochondrial decay and cellular aging.

Alternatively spliced annexin XI transcripts encode proteins that differ near the amino-terminus.

Annexins are a family of structurally related calcium-dependent phospholipid binding proteins. We recently described a new member of this family, bovine annexin XI [1]. Two kinds of cDNAs were identified corresponding to annexin XI mRNA variants A and B, which are generated by alternative splicing of identical primary transcripts. Annexin XI isoforms are predicted to differ at the amino-terminus, suggesting that they may have distinct biological roles.

Matrix metalloproteinase digestion of aggrecan in human cartilage tumours.

Substantial experimental and clinical evidence suggests that the catabolism of extracellular matrix components is a prerequisite for invasive and metastatic behaviour of solid tumours. Chondrosarcomas are malignant cartilaginous tumours that most commonly arise in bone, and the large aggregating proteoglycan aggrecan is a major component of the extracellular matrix of these tumours. Matrix metalloproteinases (MMPs) have been implicated in tumour invasiveness. The purpose of this study was to determine whether MMPs play a role in aggrecan catabolism in cartilage tumours. In order to detect aggrecan digestion products resulting from in vivo cleavage at the MMP site, protein extracts from human articular cartilage and from various cartilage tumours were analysed by Western blot using an antibody to the FVDIPEN neoepitope generated by MMP cleavage. Examination of cartilage extracts revealed a trend of increasing aggrecan digestion at the MMP site with age. One hyaline chondrosarcoma and three osteochondromas lacked detectable aggrecan fragments with the carboxy terminal FVDIPEN neoepitope. Two osteochondromas gave weak signals. However, all chondrosarcomas with degenerating extracellular matrix or with a myxoid component exhibited strong FVDIPEN immunoreactivity. These results demonstrate that, in contrast to the benign cartilage tumour osteochondroma, human chondrosarcomas contain abundant aggrecan degradation products resulting from cleavage in vivo at the MMP site in the interglobular domain. These data support the concept that MMPs participate in the degradation of extracellular matrix in chondrosarcoma, allowing the neoplastic chondrocytes to escape local confinement, migrate, and invade neighbouring and remote tissues.

Cartilage synthesizes the serine protease inhibitor PAI-1: support for the involvement of serine proteases in cartilage remodeling.

The work described here demonstrates the synthesis by human articular cartilage of plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of the serine protease tissue plasminogen activator (tPA). We also present data demonstrating an increase in PAI-1 messenger ribonucleic acid (mRNA) in chondrocytes exposed to the cytokine interleukin-1 (IL-1). Interestingly, this elevation of steady-state mRNA levels does not appear to result in an increase in synthesis of PAI-1 protein. Northern blot analysis reveals that of the two mRNA species (3.4 kb, 2.4 kb) previously reported for PAI-1, only the larger species (3.4 kb) appears to be synthesized by chondrocytes. Our data demonstrate the IL-1-stimulated production by cartilage of tissue plasminogen activator. We also show evidence for the presence of plasminogen in cartilage. A scheme is presented indicating the probable importance of the serine proteases (tPA and plasminogen) and PAI-1 in cartilage degradation.

Induction of heat-shock protein synthesis in chondrocytes at physiological temperatures.

Induction of heat-shock protein (HSP) synthesis is demonstrated in cultured calf-chondrocytes at temperatures shown to occur in normal human cartilage during experiments subjecting intact cadaverous hip joints to the parameters of level walking. A 70,000 MW heat-shock protein (HSP-70) is synthesized by chondrocytes at temperatures above 39 degrees C, while induction of synthesis of a 110,000 MW HSP only occurs at temperatures of 45 degrees C or greater. These differences in critical temperatures for induction, and data showing differences in kinetics of induction and repression of synthesis, suggest that there are differences in the mechanism of induction of the two HSPs. The duration of HSP synthesis and inhibition of synthesis of normal cellular proteins is directly proportional to the duration and magnitude of the temperature rise. Possible relationships between these new findings and the initiation and progression of degenerative joint disease are discussed.

Evaluation of fluorescein diacetate for flow cytometric determination of cell viability in orthopaedic research.

Accurate estimation of cellular viability is important both in research and in aspects of orthopaedic clinical practice. We have been interested in the potential for flow cytometric application of fluorescein diacetate (FDA) in evaluating chondrocyte survival following cryopreservation of osteochondral allografts as well as in the assessment of sarcoma necrosis following preoperative chemotherapy. In order to evaluate the suitability of this method for cell viability assays, this study compared FDA with more traditional methodology (trypan blue, clonigenic assay, metabolic activity analysis, measurement of DNA synthesis, and histological assessment of necrosis). Both chondrocytes and sarcoma cells were exposed to various experimental injuries prior to viability analysis. Although it is evident from these experiments that FDA accurately reflects cell survival after physical injury, it underestimates the effect of chemotherapy on cell reproductive potential in vitro. However, FDA is highly correlated with histological assessment of tumor viability after chemotherapy in vivo. It is apparent that the methodology chosen for determination of viability should be appropriate for the type of experimental injury and should analyze the cell function (i.e., metabolic activity or reproductive capacity) that is appropriate for the experimental model.

Irrigating solutions for arthroscopy. A metabolic study.

UNLABELLED: In an effort to determine the optimum solution for irrigation during arthroscopic procedures, an in vitro metabolic experiment was performed in which cartilage slices were incubated with 35SO4 in various commercially available solutions, harvested at regular intervals, and assayed for incorporated radioactivity. The solutions were compared with Ham F12 medium, a complex, ionically balanced salt and amino-acid solution that is used for tissue culture. The data show that neither normal saline or phosphate-buffered saline supports metabolic activity as well as Ringer lactate or acetate, both of which approximate the values for the control, Ham F12 solution. CLINICAL RELEVANCE: Arthroscopy and arthroscopic surgical procedures have become commonplace in orthopaedic practice. Normal saline solution, commonly used in large quantities as an irrigating solution, is in fact not physiological, and we showed that it inhibits normal synthesis of proteoglycan by the chondrocytes. Ringer lactate seemed to support cartilage metabolism as well as an "ideal" tissue-culture medium (Ham F12 solution). Since Ringer solution and normal saline cost the same in our hospital, we strongly recommend that this more physiological solution be used for arthroscopic procedures.

Effect of interleukin 1 on articular cartilage from young and aged horses and comparison with metabolism of osteoarthritic cartilage.

The effect of interleukin 1 (IL-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of IL-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to IL-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metalloproteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The IL-1-induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of IL-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of interleukin 1-mediated proteoglycan degradation in bovine articular cartilage explants by addition of sodium hyaluronate.

The effect of exogenous hyaluronate on normal cartilage metabolism and interleukin-1 (IL-1)-induced cartilage matrix degradation was investigated in a bovine cartilage explant culture system. Addition of hyaluronate at a concentration of 1.5 mg/ml to cartilage culture explants consistently decreased normal proteoglycan release from the matrix to a value less than that found in control cultures. Addition of 1.5 mg of hyaluronate/ml to IL-1 stimulated cartilage culture systems reduced proteoglycan release from the matrix by 83 to 113%. The reduction in control and IL-1-stimulated proteoglycan degradation by hyaluronate had a concentration-dependent trend. Evaluation of alterations in protein (enzyme) release by IL-1-stimulated chondrocytes after introduction of hyaluronate was evaluated by use of sodium dodecyl sulfate agar gel electrophoresis of cartilage-conditioned media. The quantity or the molecular weight profile of IL-1-induced proteins did not differ after introduction of hyaluronate into the culture system. Results indicate that introduction of high molecular weight hyaluronate into cartilage culture systems results in a decrease in proteoglycan release from the matrix in control systems, as well as in cultures incubated with IL-1. Because IL-1-stimulated protein synthesis by chondrocytes remains unchanged after addition of exogenous hyaluronate, the mechanism of inhibition of matrix degradation does not appear to be interference with binding of IL-1 to chondrocytes or to be inhibition of the production of neutral metalloproteases, including stromelysin.

Growth factors and articular cartilage.

Studies of articular cartilage over the decades have demonstrated a surprisingly brisk rate of synthesis of the matrix proteins which appears to vary considerably with metabolic, physicochemical and pathological state of the tissue. It has become evident that much of this activity is directed by low molecular weight protein mediators which act at specific receptor sites. Platelet derived growth factor (PDGF) is of limited action in normal cartilage, but insulin and its analogues, insulin growth factor-I and II are powerful stimulants of DNA synthesis. Basic fibroblast growth factor stimulates both DNA and protein synthesis and works synergistically with other factors. Transforming growth factor beta potentiates the action of the mitogens and enhances and regulates proteoglycan synthesis. These actions may be of special importance in osteoarthritis and lacerative injury to cartilage.

The synthetic processes of articular cartilage.

The cells of cartilage are constantly remodeling the matrix in which they are suspended. The stimulus to initiate remodeling is probably the chondrocyte's response to physical and or chemical changes in the environment. Heat, pressure, friction, load, pH, and growth are examples of such factors, which, if altered, would have a dramatic effect on the cell's state of health. The mode of response by the chondrocyte is specific for a given stimulus. Elevated temperature, for example, switches on a set of genes, the heat shock genes, in chondrocytes. This results in the synthesis of a series of cellular protein that presumably in turn protects the cell from the injurious effects of heat. Load and pressure affect both the synthetic rate of matrix protein and the degradation rates of preexisting matrix. A number of low-molecular-weight proteins appear to be involved in anabolic and catabolic processes of cartilage. A protein recently isolated from synovium stimulates the synthesis of degradative enzymes in cartilage. This factor is probably involved in the remodeling process under normal physiologic conditions. More recently, it has been found in elevated levels under pathologic conditions such as in the synovial fluid of patients with rheumatoid and osteoarthritis. The mechanism by which this factor turns on the degradative pathway appears complex and is under investigation.

Identification of a novel mammalian annexin. cDNA cloning, sequence analysis, and ubiquitous expression of the annexin XI gene.

Annexins (or lipocortins) are a family of at least 10 structurally related calcium- and phospholipid-binding proteins. Each protein consists of a conserved core domain having four (or eight) repeats of a segment approximately 70 amino acids in length and a nonconserved, usually short, amino-terminal domain. To date, amino acid sequences for eight distinct mammalian annexins have been predicted from cDNAs. This report describes an additional member of this family, bovine annexin XI, identified by cDNA cloning and sequence analysis. The 503-amino acid deduced protein consists of a core domain of four annexin repeats and a long amino-terminal domain rich in glycine, proline, and tyrosine. This novel annexin gene is expressed in a wide variety of tissues and isolated cells in culture.

A plasminogen-related gene is expressed in cancer cells.

The breakdown of blood clots requires the fibrinolytic action of the serine proteinase plasmin, a two-chain polypeptide derived posttranslationally from its precursor zymogen, plasminogen. While investigating plasminogen gene expression in human extrahepatic tissues, a cDNA sequence was obtained which closely resembled the plasminogen cDNA, yet appeared to represent a distinct gene product. This sequence, which represents the transcript of the recently characterized plasminogen-related gene B, encodes a putative polypeptide of Mr 8800 and is expressed most prominently in malignant cancer cells.

De novo protein synthesis by human chondrosarcoma in cell and organ culture: evidence of unusually high collagen production by a neoplastic tissue.

Protein synthesis by human chondrosarcoma tissue and normal articular cartilage was studied in organ and primary monolayer cell culture systems. Protein synthesis by cell cultures was evaluated at 2.7 and 21 days after plating. When compared to normal, incorporation of 3H-proline and 35S-methionine into proteins was elevated in chondrosarcoma samples under both culture conditions. Hydroxyproline analyses of tissue hydrolysates indicated that chondrosarcoma samples contained significantly less collagen than normal articular cartilage, yet were incorporating significantly greater amounts of 3H-proline into 3H-hydroxyproline. Collagen production by cell cultures was assessed by digestion of samples with purified clostridial collagenase. Chondrosarcoma cells produced more collagenase-sensitive protein than normal cells at all intervals. SDS polyacrylamide gel analyses of all preparations showed two collagenase-sensitive proteins with apparent molecular weights of 165,000 and 175,000. Decreased synthesis of another major protein, apparent molecular weight 210-220,000 was noted in chondrosarcoma preparations in both culture systems.

Characterisation of human articular cartilage link proteins from normal and osteoarthritic cartilage.

Proteoglycan link proteins were isolated from human articular cartilage obtained from normal and osteoarthritic femoral heads and purified to homogeneity employing a method previously described by this laboratory. The link proteins were analysed for amino acid composition, molecular weight on sodium dodecyl sulphate polyacrylamide gels, and ability to stabilise proteoglycan aggregates. The results of these studies were compared with those obtained with bovine link proteins. Two link proteins were identified in the purified fraction from normal and osteoarthritic human cartilage with apparent molecular weights of 54 000 (link 1) and 48 000 (link 2). Functionally the link proteins, isolated from osteoarthritic and normal cartilage, were indistinguishable as measured by their ability to stabilise aggregate. The amino acid compositions of normal and osteoarthritic link proteins were also found to be similar to each other but significantly different from the amino acid composition reported for the bovine link proteins. The quantities of these proteins in extracts from normal and diseased tissue were similar, as was the ratio of link protein 1 to link protein 2.

Biochemical and metabolic abnormalities in normal and osteoarthritic human articular cartilage.

Incorporation of radioactive precursors into macromolecules was studied with human normal and osteoarthritic articular cartilage organ culture. Analysis of the salt extracted matrix components separated by cesium chloride buoyant density gradient centrifugation showed an increase in the specific activities of all gradient fractions prepared from the osteoarthritic cartilage. Further analysis of these fractions showed the osteoarthritic cartilage contained 5 times as much sulfate incorporated into proteoglycans, and an even greater amount of 3H-glucosamine incorporated into material sedimenting to the middle of the gradient. Greater than half of this radioactive middle fraction appears to be hyaluronate, as judged by the position it elutes from a DEAE column and its susceptibility to hyaluronidase digestion. This study supports earlier findings showing increased rates of macromolecular synthesis in osteoarthritis, and in addition, an even greater synthetic rate for hyaluronic acid is demonstrated.