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Electron-microscopic and electrophoretic studies of bovine femoral-head cartilage proteoglycan fractions.

ABSTRACT: Proteoglycans (A1D1) extracted from bovine femoral-head cartilage were examined by electron microscopy using benzyldimethylammonium chloride as a spreading agent. The preparation contained a mixture of particles, some with a 'beaded' structure and a contiguous filamentous 'tail' at one end and others which appeared as round 'blobs', some of which also had filamentous tails. Previous electron-microscopic studies of proteoglycan monomers have indicated that their length distributions were apparently unimodal, a finding that contrasted with agarose/polyacrylamide-gel-electrophoresis results, which generally indicated two bands. In the present study proteoglycans isolated from the slowly migrating electrophoretic band were shown to be predominantly the larger molecules of beaded appearance, whereas the rapidly migrating proteoglycans were predominantly molecules with the 'blob-like' appearance. Gel-filtration, isopycnic-density-gradient-centrifugation and rate-zonal-centrifugation techniques were evaluated as means of proteoglycan fractionation by electron microscopy and agarose-gel electrophoresis. Rate-zonal centrifugation in mixed-salt gradients of caesium chloride/4 M-guanidinium chloride yielded the most effective fractionation.

SUBMITTER: Thornton DJ

PROVIDER: S-EPMC1147373 | biostudies-other | 1986 Nov

REPOSITORIES: biostudies-other

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Project description:1. Proteoglycans were extracted from bovine nasal cartilage with 2.0M-CaC2 or with 0.15M-KCl followed by 2.0M-CaC2.. Proteoglycan fractions were prepared from the extracts by density-gradient centrifugation in CsCl under 'associative' and 'dissociative' conditions. 2. The heterogeneity of the proteoglycan fractions was investigated by large-pore-gel electrophoresis. It was concluded that extracts made with 2.0M-CaCl2 or sequential 2.0M-CaCl2 contain two major species of proteoglycan 'subunit' of different hydrodynamic size, together with proteoglycan aggregates. Both 'subunits' have mobilities that are greater than those of proteoglycans obtained from pig articular cartilage McDevitt & Muir (1971) Anal. Biochem. 44, 612-622] and are therefore probably smaller in size than the latter. 3. Proteoglycan fractions isolated from cartilage extracted lith 0.15M-KCl separated into two main components on large-pore-gel electrophoresis with mobilities greater than those of proteoglycans extracted with 2.0M-CaCl2. Proteoglycans extracted at low ionic strength from bovine nasal cartilage are of similar hydrodynamic size to those extracted from pig articular cartilage under the same conditions [McDevitt & Muir (1971) Anal. Biochem. 44, 612-622]. 4. The role of endogenous proteolytic enzymes in producing proteoglycan heterogeneity, particularly in low-ionic-strength cartilage extracts is discussed. 5. Hyaluronic acid and 'link proteins' were present in the proteoglycan fraction separated from KCl extracts as well as in the fraction separated from CaCl2 extracts. Hyaluronic acid can only be identified in proteoglycan fractions by large-pore-gel electrophoresis after proteolysis and further purification of the fraction. 6. Collagen was extracted by both salt solutions and was tentatively identified as type II. Small amounts of collagen appear to be associated with the proteoglycan-aggregate fraction from the high-ionic-strength extract but not with the corresponding fraction from the KCl extract.

Project description:(1) Background: Since the large-scale poultry industry has been established, femoral head necrosis (FHN) has always been a major leg disease in fast-growing broilers worldwide. Previous research suggested that cartilage homeostasis could be taken into consideration in the cause of FHN, but the evidence is insufficient. (2) Methods: One-day-old broiler chickens were randomly divided into three groups, 16 broilers per group. The birds in group L were injected intramuscularly with methylprednisolone (MP) twice a week for four weeks (12.5 mg·kg-1). The birds in group H were injected intramuscularly with MP (20 mg·kg-1·d-1) for 7 d (impulse treatment). The birds in group C were treated with sterile saline as a control group. Broilers were sacrificed at 42 and 56 d. Blood samples were collected from the jugular vein for ELISA and biochemical analysis. Bone samples, including femur, tibia, and humerus, were collected for histopathological analysis, bone parameters detection, and real-time quantitative PCR detection. (3) Results: The FHN broilers in group L and H both showed lower body weight (BW) and reduced bone parameters. In addition, the MP treatment resulted in reduced extracellular matrix (ECM) anabolism and enhanced ECM catabolism. Meanwhile, the autophagy and apoptosis of chondrocytes were enhanced, which led to the destruction of cartilage homeostasis. Moreover, the impulse MP injection increased the portion of birds with severer FHN, whereas the MP injection over a long period caused a more evident change in serum cytokine concentrations and bone metabolism indicators. (4) Conclusions: The imbalance of cartilage homeostasis may play a critical role in the development of FHN in broilers. FHN broilers induced by MP showed a more pronounced production of catabolic factors and suppressed the anabolic factors, which might activate the genes of the WNT signal pathway and hypoxia-inducible factors (HIFs), and then upregulate the transcription expression of ECM to restore homeostasis.

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Electron-microscopic and electrophoretic studies of bovine femoral-head cartilage proteoglycan fractions.

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