Hormonal influences on in vitro [35S]-sulfate uptake by gill arches from the goldfish (Carassius auratus L.)

A bioassay for insulin-like growth factor (IGF), based on the in vitro incorporation of [³⁵S]-sulfate into gill arch tissue was used to study the hormonal regulation of proteoglycan synthesis in the goldfish (Carassius auratus). [³⁵S]-sulfate incorporation into gill arch tissue was found to be time-dependent with maximal uptake occurring by 48h, suggesting that proteoglycan synthesis in this tissue was maintained for at least 48h in vitro. The addition of human recombinant IGF-I (IGF-I) to the incubation medium was found to significantly stimulate [³⁵S]-sulfate uptake into the gill arches, whereas bovine growth hormone (GH) was without effect. Porcine insulin was also stimulatory, but results indicate that the effects of porcine insulin and IGF-I may be mediated by a single receptor system. Finally, arches from hypophysectomized fish were significantly less responsive to IGF-I than were arches from sham-operated fish. Furthermore, administration of ovine GH in vivo appeared to increase subsequent responsiveness in vitro. Together, these results provide evidence that the growth-promoting actions of GH in the goldfish may be mediated, at least in part, by a peptide related in structure to mammalian IGF-I.     

Interaction between myostatin and extracellular matrix components

Myostatin, a member of the TGF-β superfamily, is a negative regulator of skeletal muscle mass. We have recently demonstrated that decorin binds to myostatin in vitro , and that immobilized decorin within the collagen matrix prevents myostatin-mediated inhibition of myoblast proliferation. However, little is known about other ECM molecules that bind to myostatin and modulate its activity. Thus, in the present study, we investigated the interaction of several other ECM molecules with myostatin. We here show that fibromodulin, fibronectin and laminin bind to myostatin in the presence of Zn²⁺ with a dissociation constant ( K_D ) of 10⁻¹⁰∼10⁻⁸ mol/L. Fibromodulin shows the highest affinity for myostatin among them. These results suggest that these ECM molecules may modulate myostatin activity like decorin does.     

Isolation and partial characterization of a novel keratan sulfate proteoglycan from metamorphosing bonefish (Albula) larvae

Proteoglycans (PGs) were isolated from leptocephalous larvae of the bonefish (Albula sp.), which were in the early stages of metamorphosis, using both associative and dissociative conditions in the presence of protease inhibitors. The procedure was rapid and resulted in an extraction efficiency of 75% (associative) and 85–90% (dissociative). The majority of co-extracted protein could be effectively separated from the PGs by utilizing either Sepharose CL-2B or CL-6B gel chromatography. Sepharose CL-2B chromatography of extracted PGs after treatment with bacterial keratan sulfate-endo--galactosidase (keratanase) showed that most of the high molecular weight (M _r) carbohydrate was degraded. Free keratan sulfate (KS) chains were prepared from whole-larva extracts (which also contain small amounts of chondroitin sulfate) by both chondroitinase ABC treatment and ethanol fractionation. Sepharose CL-6B chromatography under dissociative conditions showed that larval KS chains were much larger (M _r55,000) than those from cornea. These chains tended to aggregate when chromatographed under associative conditions. Larval KS was degraded by keratanase and resistant to chondroitinase, ABC and testicular hyaluronidase. Differences were also noted in the oligosaccharides produced by keratanase treatment of the two preparations. However, biochemical composition of larval and corneal KS was similar.Abbreviations KS keratan sulfate - CS chondroitin sulfate - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - EDTA ethylenediaminetetraacetic acid - PG proteoglycan - GAG glycosaminoglycan - SDS sodium dodecyl sulfate - PAGE polyacrylamide-gel electrophoresis - TLC thin-layer chromatography - V _o void volume - V _t total volume - M _r molecular weight - U units     

Expression of NG2 proteoglycan in the degenerated intervertebral disc in dachshunds

The pathogenesis of intervertebral disc (IVD) degeneration is not fully understood. The biomolecular signaling pathways involved in the IVD degeneration require further investigation. The aim of this study was to investigate the expression of NG2 proteoglycan in the degenerated IVD. IVD samples were obtained from 16 Dachshunds that were confirmed to have IVD herniation and subsequently underwent hemilaminectomy. The samples were subjected to histological and immunohistochemical (IHC) examinations. IHC revealed positive results for the expression of NG2 proteoglycan in all examined samples. The results showed the expression of NG2 proteoglycan by the degenerated IVDs.     

Influence of exercise and joint topography on depth‐related spatial distribution of proteoglycan and collagen content in immature equine articular cartilage

Reasons for performing study: There is ample evidence on topographical heterogeneity of the principal biochemical components of articular cartilage over the surface of the joint and the influence of loading thereon, but no information on depth‐related zonal variation in horses. Objectives: To study depth‐related zonal variation in proteoglycan (PG) and collagen content in equine articular cartilage. Methods: Two techniques (safranin‐O densitometry and Fourier transform infrared spectroscopy) were applied to sections of articular cartilage from the proximal phalangeal bone of the metacarpophalangeal joint of 18‐month‐old Thoroughbreds that had been raised at pasture from age 0–18 months without (PASTEX) and with (CONDEX) additional exercise. Two sites were investigated: site 1 at the joint margin that is unloaded at rest or at slow gaits, but subjected to high‐intensity loading during athletic activity; and site 2, a continuously, but less intensively, loaded site in the centre of the joint. Results: Proteoglycan values increased from the surface to the deep layers of the cartilage, collagen content showed a reverse pattern. PG content was significantly higher at site 2 in both PASTEX and CONDEX animals without an effect of exercise. In the PASTEX animals collagen content was significantly higher at site 1, but in the CONDEX group the situation was reversed, due to a significant exercise effect on site 1, leading to a reduced collagen content. Conclusions: Collagen and PG content gradients agree with findings in other species. The observations on PG levels suggest that the exercise level was not strenuous. The collagen results in the PASTEX group confirmed earlier findings, the lower levels at site 1 in the CONDEX group being possibly due to an advancement of the physiological maturation process of collagen remodelling. Potential relevance: This study confirms earlier observations that even moderate variations in exercise level in early age may have significant effects on the collagen network of articular cartilage.     

The role of intramuscular connective tissue in meat texture

The structure, composition and amount of intramuscular connective tissue (IMCT) vary tremendously between muscles, species and breeds, and certainly contribute to meat texture. With animal growth, collagen crosslinks become more stable, and the structural integrity of IMCT increases. These changes increase the mechanical properties of IMCT, contributing to the toughening of meat. Intramuscular fat deposits, mainly in the perimysium between muscle fiber bundles, result in marbling. This causes the remodeling of IMCT structures and reduces the mechanical strength of IMCT, contributing to the tenderization of beef. The IMCT has been thought to be rather immutable compared to myofibrils during postmortem ageing of meat. However, recent studies have shown the disintegration of IMCT during postmortem ageing of meat and its relationship to tenderization of raw meat, although its contribution to cooked meat is still controversial. Given the large influence of IMCT on meat texture, further elucidations of molecular mechanisms which change the structural integrity of IMCT during chronological ageing of animals and postmortem ageing of meat are needed.     

The diversity of structure and function of heparin sulfate proteoglycans via modification of some relative enzymes

The cytokine-receptor- heparin sulfate functional complex combined by cytokines, cytokine receptors, and heparin sulfate chains formed by concatenation of heparin sulfate proteoglycans (HSPG), an important component of extracellular matrix and modified by some relative enzymes, can regulate the density of cytokine receptors and their intracellular signal transduction. This article focused on the regulatory function of this complex. Many morphological abnormalities and diseases occur when the complex is dysfunctional.     

HAPLN1 induces resistance to MTX in human colorectal cancer HT-29 cells though IKK/p65 pathway

AIM:To investigate the changes of hyaluronan and proteoglycan link protein 1 (HAPLN1) expression before and after resistance to methotrexate (MTX) in human colorectal cancer HT-29 cells and its effect on this drug resistance, and to explore the molecular mechanism in the process. METHODS:The drug-resistant HT-29/MTX cells were established by stepwise exposure of the cells to MTX, and then the HT-29/MTX cells were stably transfected with specific shRNA interference plasmid vectors targeting HAPLN1 and multidrug resistance-associated protein 2 (MRP2). The mRNA expression levels of HAPLN1 and MRP2 were measured by RT-PCR. CCK-8 assay was used to detect the viability of HT-29/MTX cells. The apoptosis rate was analyzed by flow cytometry. The protein levels of HAPLN1, MRP2, IκB kinase (IKK) α/β, p-IKKα/β (Ser176/Ser177), p65 and p-p65 (Ser536) were determined by Western blot. RESULTS:The HT-29/MTX cells had significantly higher mRNA and protein levels of HAPLN1 and MRP2 than HT-29 cells (P<0.05) with resistant factor of 463.756. HAPLN1 and MRP2 gene silencing significantly increased the cytotoxicity and apoptosis of HT-29/MTX cells induced by MTX (P<0.05). The IC₅₀ value was decreased from 15.304 μmol/L to 6.119 μmol/L and 7.801 μmol/L, respectively, and their reversal folds were 2.501 and 1.962, respectively. Silencing of HAPLN1 and IKK inhibitor IKK16 inhibited the phosphorylation of IKKα/β and p65 (P<0.05), and down-regulated the protein level of MRP2 in the HT-29/MTX cells (P<0.05). However, IKK16 did not affect the protein level of HAPLN1 in the HT-29/MTX cells.CONCLUSION:Knock-down of HAPLN1 gene expression reverses the resistance to MTX in human colorectal cancer HT-29/MTX cells possibly by blocking the IKK/p65 signaling pathway and thus down-regulating the expression of MRP2.