scholarly journals Effect of calcipenia on proteoglycan metabolism and aggregation in normal articular cartilage in vitro

1979 ◽  
Vol 182 (2) ◽  
pp. 399-406 ◽  
Author(s):  
M J Palmoski ◽  
K D Brandt
Keyword(s):  
Articular Cartilage ◽  
Normal Adult ◽  
Leucine Incorporation ◽  
Hydrodynamic Size ◽  
Knee Cartilage ◽  
Glycosaminoglycan Synthesis ◽  
Normal Concentration ◽  
Proteoglycan Aggregates

Glycosaminoglycan synthesis in normal adult dog knee cartilage cultured in medium containing 0, 0.3 MM- and 0.9 mM-Ca2+ was 52, 67 and 78%, respectively, of that in cartilage from the same joints cultured in a normal concentration of Ca2+, i.e. 1.8 mM. Pulse-chase experiments indicated that the rate of degradiation of glycosaminoglycans in cartilage cultured in the absence of Ca2+ was similar to that of glycosaminoglycans in cartilage cultured in 1.8 mM-Ca2+. Although [35S]sulphate incorporation into glycosaminoglycans was decreased in the presence of calcipenia, [3H]leucine incorporation into protein was unaffected. The average hydrodynamic size of newly synthesized proteoglycan aggregates and purified disaggregated proteoglycans from cartilage cultured in the absence of Ca2+ was similar to that of aggregates and disaggregated proteoglycans from cartilage cultured in 1.8 mM-Ca2+.

Directing Chondrogenesis of Primary Chondrocytes by Exposure to Glucose Concentrations

2015 ◽  
Vol 24 ◽  
pp. 30-42
Author(s):  
Samuel C. Uzoechi ◽  
Kennedy O. Ejeta ◽  
Goddy C. Okoye ◽  
Gideon I. Ndubuka ◽  
Patrick Ugochukwu Agbasi ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Glucose Concentration ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Glycosaminoglycan Synthesis ◽  
Chondrogenic Medium ◽  
Hypoxic Conditions ◽  
Matrix Production ◽  
Engineered Cartilage

Since articular cartilage is avascular, both nutrient supply and metabolic waste excretion depend on diffusion. However, the major cause of the progression of articular cartilage defect is the poor inherent regenerative capacity of chondrocytes which limits the process of cartilage tissue repair. Creation of nutrient gradients in in vitro cell culture, however, can provide a clue on zonal distributions of cells and glycosaminoglycan synthesis throughout the tissue engineered cartilage. We hypothesized that glucose gradient, in combination with growth factors, could induce differences in matrix distributions for articular cartilage regeneration. Chondrocytes were harvested from bovine cartilage and expanded in monolayers. First, either p0 or p2 chondrocytes were differentiated in serum-free chondrogenic medium containing different glucose concentrations supplemented with TGFβ3/dex or IGF-1under hypoxic or normoxic conditions for 7 days in monolayer. The results indicate that cellular metabolism, cell numbers and glycosaminoglycan (GAG) content increased with increase in glucose concentration in all conditions. Aggrecan (AGC) expression consistently increased with decreasing glucose concentration in both normoxic and hypoxic conditions. COL II and COL I expressions increased with increasing glucose concentration up to 5mmol/L. The expression of COMP increased with increasing glucose concentration under hypoxic conditions and interestingly showed an opposite trend under normoxic conditions. However, comparing the chondrogenic capacity of p0 and p2 cells in the different glucose concentrations did not show differences, but the potential of p2 cells was in general lower compared to p0. Hypoxia had stimulatory effects on matrix production compared to normoxia in both passages. Therefore, supplemented glucose concentration in monolayer could induce differences in matrix production, but the chondrogenic potential remained equal. Therefore, this information could be use to a create gradients through a tissue-engineered cartilage.

scholarly journals Catabolism of Fibromodulin in Developmental Rudiment and Pathologic Articular Cartilage Demonstrate Novel Roles for MMP-13 and ADAMTS-4 in C-Terminal Processing of SLRPs

2018 ◽  
Author(s):  
Cindy Shu ◽  
Carl R Flannery ◽  
Christopher B Little ◽  
James Melrose
Keyword(s):  
Articular Cartilage ◽  
Growth Plate ◽  
Cartilage Regeneration ◽  
In Vitro Digestion ◽  
Knee Cartilage ◽  
Western Blots ◽  
Human Knee ◽  
C Terminus ◽  
Total Knee

Cartilage regeneration requires a balance of anabolic and catabolic processes. This study examined the susceptibility of fibromodulin (FMOD) and lumican (LUM) to degradation by MMP-13, ADAMTS-4 and ADAMTS-5, the three major degradative proteinases in articular cartilage in osteoarthritis (OA). Immunolocalisation of FMOD and LUM in foot sections of developmental cartilages demonstrated prominent localisations in metatarsal and phalangeal foetal rudiment cartilages and growth plate. An MMP-13 neoepitope antibody (TsYG11) demonstrated localisation of MMP-13 cleaved FMOD in the hypertrophic chondrocytes of the metatarsal growth plate. FMOD was more prominently localised in the superficial cartilage of normal and fibrillated zones in OA cartilage, TsYG11 positive FMOD was located deeper in the cartilage samples. Ab TsYG11 also identified FMOD fragmentation in Western blots of extracts of normal and fibrillated cartilage and total knee replacement OA cartilage.  The C-terminal anti-FMOD used in this study (PR-184) failed to identify FMOD fragmentation due to C terminal processing, an equivalent Ab to the C-terminus of LUM (pAb PR-353) identified 3 prominent LUM fragments in OA human knee cartilages. In-vitro digestion of human knee cartilage with MMP-13, ADAMTS-4 and ADAMTS-5 generated equivalently sized FMOD fragments of 54, 45 and 32kDa to those in blots of OA cartilage, LUM was not less susceptible to fragmention in in-vitro digestions however Ab PR-353 detected N-terminally processed LUM fragments of 39, 38 and 22 kDa in 65-80 year old OA knee cartilage. FMOD and LUM were differentially processed during in-vitro digestions with MMP-13, ADAMTS-4 and ADAMTS-5 with FMOD susceptible to degradation by MMP-13, ADAMTS-4 and to a lesser extent ADAMTS-5 however LUM was less susceptible to fragmentation. FMOD was processed by MMP-13 in metatarsal and phalangeal foetal rudiment developmental cartilages and growth plate indicating a role in skeletogenesis.

scholarly journals Catabolism of Fibromodulin in Developmental Rudiment and Pathologic Articular Cartilage Demonstrates Novel Roles for MMP-13 and ADAMTS-4 in C-terminal Processing of SLRPs

2019 ◽  
Vol 20 (3) ◽  
pp. 579 ◽  
Author(s):  
Cindy Shu ◽  
Carl Flannery ◽  
Christopher Little ◽  
James Melrose
Keyword(s):  
Articular Cartilage ◽  
Total Knee Replacement ◽  
Knee Replacement ◽  
In Vitro Digestion ◽  
Cartilage Explants ◽  
Knee Cartilage ◽  
Western Blots ◽  
Growth Plates ◽  
Total Knee

Background: Cartilage regeneration requires a balance of anabolic and catabolic processes. Aim: To examine the susceptibility of fibromodulin (FMOD) and lumican (LUM) to degradation by MMP-13, ADAMTS-4 and ADAMTS-5, the three major degradative proteinases in articular cartilage, in cartilage development and in osteoarthritis (OA). Methods: Immunolocalization of FMOD and LUM in fetal foot and adult knee cartilages using an FMOD matrix metalloprotease (MMP)-13 neoepitope antibody (TsYG11) and C-terminal anti-FMOD (PR184) and anti-LUM (PR353) antibodies. The in vitro digestion of knee cartilage with MMP-13, A Disintegrin and Metalloprotease with Thrompospondin motifs (ADAMTS)-4 and ADAMTS-5, to assess whether FMOD and LUM fragments observed in Western blots of total knee replacement specimens could be generated. Normal ovine articular cartilage explants were cultured with interleukin (IL)-1 and Oncostatin-M (OSM) ± PGE3162689, a broad spectrum MMP inhibitor, to assess FMOD, LUM and collagen degradation. Results and Discussion: FMOD and LUM were immunolocalized in metatarsal and phalangeal fetal rudiment cartilages and growth plates. Antibody TsYG11 localized MMP-13-cleaved FMOD in the hypertrophic chondrocytes of the metatarsal growth plates. FMOD was more prominently localized in the superficial cartilage of normal and fibrillated zones in OA cartilage. TsYG11-positive FMOD was located deep in the cartilage samples. Ab TsYG11 identified FMOD fragmentation in Western blots of normal and fibrillated cartilage extracts and total knee replacement cartilage. The C-terminal anti-FMOD, Ab PR-184, failed to identify FMOD fragmentation due to C-terminal processing. The C-terminal LUM, Ab PR-353, identified three LUM fragments in OA cartilages. In vitro digestion of human knee cartilage with MMP-13, ADAMTS-4 and ADAMTS-5 generated FMOD fragments of 54, 45 and 32 kDa similar to in blots of OA cartilage; LUM was less susceptible to fragmentation. Ab PR-353 detected N-terminally processed LUM fragments of 39, 38 and 22 kDa in 65–80-year-old OA knee replacement cartilage. FMOD and LUM were differentially processed in MMP-13, ADAMTS-4 and ADAMTS-5 digestions. FMOD was susceptible to degradation by MMP-13, ADAMTS-4 and to a lesser extent by ADAMTS-5; however, LUM was not. MMP-13-cleaved FMOD in metatarsal and phalangeal fetal rudiment and growth plate cartilages suggested roles in skeletogenesis and OA pathogenesis. Explant cultures of ovine cartilage stimulated with IL-1/OSM ± PGE3162689 displayed GAG loss on day 5 due to ADAMTS activity. However, by day 12, the activation of proMMPs occurred as well as the degradation of FMOD and collagen. These changes were inhibited by PGE3162689, partly explaining the FMOD fragments seen in OA and the potential therapeutic utility of PGE3162689.

CONTRIBUTION TO THE EXISTENCE OF REGULATORY MECHANISMS AT THE ADRENAL LEVEL

1972 ◽  
Vol 70 (4) ◽  
pp. 741-757
Author(s):  
Otto Linèt
Keyword(s):  
Orotic Acid ◽  
Adrenal Glands ◽  
Actinomycin D ◽  
Delay Period ◽  
Regulatory Mechanisms ◽  
Leucine Incorporation ◽  
Total Period ◽  
Free Media

ABSTRACT Rat adrenal glands atrophied by the administration of cortisol acetate in vivo were used as a model for the study of early metabolic processes occurring in vitro. Atrophied adrenals incubated in the presence of 14C-leucine incorporated subnormal quantities of this amino acid per mg of protein for the first 120 min. When the incubation lasted for a total period of 180 or 240 min a supranormal rise in the 14C-leucine incorporation was observed. Similar changes occurred with some delay with regard to corticosterone production as expressed per 100 mg of tissue. No differences in 14C-leucine incorporation were observed between the control and atrophied adrenals in vivo. Homogenates from atrophied glands incorporated 14C-leucine to a greater extent than the control homogenates. The in vitro incorporation of 14C-orotic acid into the RNA was also higher in atrophied adrenals. The in vitro use of actinomycin D, cycloheximide and amphenone indicated that corticosterone production depended on the incorporation of 14C-leucine. The addition of cortisol to the incubation media markedly decreased the enhancement of 14C-lysine incorporation into the protein of atrophied adrenals. These, as well as additional results suggest rebound phenomena: once atrophic adrenals are transferred to cortisol-free media, reparative processes begin after a delay period. Such phenomena seem to be mediated by regulatory mechanisms at the adrenal level.

EFFECT OF ACTINOMYCIN D ON TSH-INDUCED STIMULATION OF THYROIDAL PROTEIN SYNTHESIS IN THE RAT

1974 ◽  
Vol 77 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Gustav Wägar
Keyword(s):  
Protein Synthesis ◽  
Actinomycin D ◽  
The Other ◽  
Leucine Incorporation ◽  
Short Term ◽  
Other Hand ◽  
Thyroid Glands ◽  
Translational Level ◽  
Stimulation Of

ABSTRACT Whether the short-term regulation of thyroidal protein synthesis by TSH occurs at the transcriptional or the translational level was tested by measuring the effect of actinomycin D (act D) on the TSH-induced stimulation of L-14C-leucine incorporation into the thyroidal proteins of rats. TSH was injected 6 h before the rats were killed. The thyroid glands were then removed and incubated in vitro in the presence of L-14C-leucine for 2 h. The pronounced stimulation of leucine incorporation in the TSH-treated animals was depressed as compared with controls but still significant even when the animals had been pre-treated with 100 μg act D 24 and 7 h before sacrifice. On the other hand, act D strongly decreased incorporation of 3H-uridine into RNA. Short-term regulation of thyroidal protein synthesis by TSH appears to be partly but not wholly dependent on neosynthesis of RNA. Hence regulation may partly occur at the translation level of protein synthesis.

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