人正常软骨和软骨肉瘤蛋白多糖的比较研究

取三例一般型一级的软骨肉瘤及不同年令正常人股骨头关节软骨,经4mol/L盐酸胍提取和氯化铯平衡等密度梯度离心,分析蛋白多糖组份的化学组成,并用Sepharose CL-2B层析分析蛋白多糖分子大小。初步结果,两例软骨肉瘤蛋白多糖的化学组成和分子大小与新生儿接近,另一例软骨肉瘤蛋白多糖的化学组成和分子大小与成年人相似。此结果说明软骨肉瘤生物学行为的多变性及其蛋白多糖的异质性。     

Polyamine-dependent alterations in the structure of microfilaments,golgi apparatus,endoplasmic reticulum,and proteoglycan synthesis in BHK cells

The activity of ornithine decarboxylase, the key enzyme in the synthesis of polyamines, is essential for proliferation and differentiation of all living cells. Two inhibitors of ornithine decarboxylase, α-difluoromethylornithine (DFMO) and 1-aminooxy-3-aminopropane (APA), caused swelling of endoplasmic reticulum (ER) and medial and trans Golgi cisternae, and the disappearance of stress fibers, as visualized by staining with fluorescent concanavalin A (ConA), C6-NBD-ceramide or wheat germ agglutinin (WGA), and phalloidin, respectively. In contrast, the pattern of microtubules, stained with a β-tubulin antibody, was not affected. Rough ER seemed to be especially affected in polyamine deprivation forming whorls and involutions, which were observed by transmission electron microscopy. Since ER and Golgi apparatus are vital parts of the glycosylation and secretory machinery of the cell, we tested the ability of these structurally altered cell organelles to synthesize proteoglycans using [³H]glucosamine and [³⁵S]sulfate as precursors. The total incorporation rate into proteoglycans and hyaluronan was not reduced in polyamine-deprived cells, suggesting that the total glycosylation capacity of cells was not affected. However, the synthesis of a high molecular weight proteoglycan containing chondroitin and keratan sulfate was completely inhibited. The remodeling of cytoskeleton and rough endoplasmic reticulum in polyamine deprivation may perturb the synthesis and secretion of the components of membrane skeleton and of the extracellular matrix, e.g., proteoglycans. Rough ER and cytoskeleton may be the targets where polyamines affect cell proliferation and differentiation. J. Cell Biochem. 66:165-174, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of Glycos-aminoglycans Associated with Rauscher Murine Leukemia Virions

Host-derived sulfated components that copurify and are physically associated with the envelope of Rauscher murine leukemia virions grown in JLS-V9 cells were characterized by digestion with chondroitinase ABC and chondroitinase AC II, as well as nitrous acid degradation. A dermatan-sulfate-chondroitin-sulfate copolymer and heparin or heparan sulfate were shown to be associated with the virions. Competitive binding studies indicated a specificity of the virions for association with heparan sulfate. The physiological importance of the association is discussed.     

Reduced Sulfation of Chondroitin Sulfate but Not Heparan Sulfate in Kidneys of Diabetic db/db Mice

Heparan sulfate proteoglycans are hypothesized to contribute to the filtration barrier in kidney glomeruli and the glycocalyx of endothelial cells. To investigate potential changes in proteoglycans in diabetic kidney, we isolated glycosaminoglycans from kidney cortex from healthy db/+ and diabetic db/db mice. Disaccharide analysis of chondroitin sulfate revealed a significant decrease in the 4-O-sulfated disaccharides (D0a4) from 65% to 40%, whereas 6-O-sulfated disaccharides (D0a6) were reduced from 11% to 6%, with a corresponding increase in unsulfated disaccharides. In contrast, no structural differences were observed in heparan sulfate. Furthermore, no difference was found in the molar amount of glycosaminoglycans, or in the ratio of hyaluronan/heparan sulfate/chondroitin sulfate. Immunohistochemical staining for the heparan sulfate proteoglycan perlecan was similar in both types of material but reduced staining of 4-O-sulfated chondroitin and dermatan was observed in kidney sections from diabetic mice. In support of this, using qRT-PCR, a 53.5% decrease in the expression level of Chst-11 (chondroitin 4-O sulfotransferase) was demonstrated in diabetic kidney. These results suggest that changes in the sulfation of chondroitin need to be addressed in future studies on proteoglycans and kidney function in diabetes.     

Application of a 22L scale membrane bioreactor and cross-flow ultrafiltration to obtain purified chondroitin

Recently, the possibility of producing fructosylated chondroitin from the capsular polysaccharide of Escherichia coli O5:K4:H4, in fed‐batch and microfiltration experiments was assessed on a 2 L bioreactor. In this work, a first scale‐up step was set on a 22 L membrane reactor with modified baffles to insert ad hoc designed microfiltration modules permanently inside the bioreactor vessel. Moreover, the downstream polysaccharide purification process, recently established on the A¨?KTA cross‐flow instrument, was translated to a UNIFLUX‐10, a tangential flow filtration system suitable for prepilot scale. In particular, the microfiltered permeates obtained throughout the fermentation, and the supernatant recovered from the centrifuged broth at the end of the process, were treated as two separate samples in the following ultrafiltration procedure, and the differences in the two streams and how these affected the ultrafiltration/diafiltration process performance were analysed. The total amount of K4 capsular polysaccharide was about 85% in the broth and 15% in the microfiltered permeates. However, the downstream treatment was more efficient when applied to the latter. The major contaminant, the lipopolysaccharide, could easily be separated by a mild hydrolysis that also results in the elimination of the unwanted fructosyl residue, which is linked to the C‐3 of glucuronic acid residues. The tangential ultrafiltration/diafiltration protocols developed in a previous work were effectively scaled‐up, and therefore in this research proof of principle was established for the biotechnological production of chondroitin from the wild‐type strain E. coli O5:K4:H4. The complete downstream procedure yielded about 80% chondroitin with 90% purity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1012–1018, 2012     

Determination of unsaturated glycosaminoglycan disaccharides by spectrophotometry on thin-layer chromatographic plates

Chondroitin sulfate isomers and hyaluronate, digested with chondroitinase AC II and separated as unsaturated disaccharides by thin-layer chromatography on cellulose, were quantitated with scanning spectrophotometry using reflectance measurement at 232 nm. The sensitivity of the assay was increased by one order of magnitude as compared to previous modifications of the disaccharide analysis on cellulose. The method allowed quantitation of 0.2–20 μg of glycosaminoglycan uronic acid. Since the elution of the disaccharides from the plate could be omitted, the speed and reproducibility of the assay was enhanced.     

Active synthesis of glycosaminoglycans by liver parenchymal cells in primary culture

Rat liver parenchymal cells were evaluated after 2 days of primary culture for their ability to synthesize and accumulate heparan sulfate as the major component and low-sulfated chondroitin sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid as the minor ones. The newly synthesized glycosaminoglycans secreted into the medium were different from those remaining with and/or on the cell layer. Low-sulfated chondroitin 4-sulfate, a major glycosaminoglycan in blood, was synthesized in the order of 320 μg/liver per day, more than 90% of which was secreted into the medium within 16 h and 40% of the glycan secreted was degraded during that time. On the other hand, heparan sulfate, the major glycosaminoglycan synthesized by the parenchymal cells, was mainly distributed in the cell layer. After 8 days of culture, the synthesis of glycosaminoglycans by the cells increased markedly, especially dermatan sulfate, chondroitin sulfate and hyaluronic acid.     

MMPs are less efficient than ADAMTS5 in cleaving aggrecan core protein

Aggrecan degradation in articular cartilage occurs predominantly through proteolysis and has been attributed to the action of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) families. Both families of enzymes cleave aggrecan at specific sites within the aggrecan core protein. One cleavage site within the interglobular domain (IGD), between Glu^373-374Ala and five additional sites in the chondroitin sulfate-2 (CS-2) region of aggrecan were characterized as “aggrecanase” (ADAMTS) cleavage sites, while cleavage between Ser^341-342Phe within the IGD of bovine aggrecan is attributed to MMP action. The objective of this study was to assess the cleavage efficiency of MMPs relative to ADAMTS and their contribution to aggrecan proteolysis in vitro. The analysis of aggrecan IGD degradation in bovine articular cartilage explants treated with catabolic cytokines over a 19-day period showed that MMP-mediated degradation of aggrecan within the IGD can only be observed following day 12 of culture. This delay is associated with the lack of activation of proMMPs during the first 12 days of culture. Analysis of MMP1, 2, 3, 7, 8, 9, 12, 13 and ADAMTS5 efficiencies at cleaving within the aggrecan IGD and CS-2 region in vitro was carried out by the digestion of bovine aggrecan with the various enzymes and Western blot analysis using aggrecan anti-G1 and anti-G3 antibodies. Of these MMPs, MMP12 was the most efficient at cleaving within the aggrecan IGD. In addition to cleavage in the IGD, MMP, 3, 7, 8 and 12 were also able to degrade the aggrecan CS-2 region. MMP3 and MMP12 were able to degrade aggrecan at the very C-terminus of the CS-2 region, cleaving the Glu^2047-2048Ala bond which was previously shown to be cleaved by ADAMTS5. However, in comparison to ADAMTS5, MMP3 was about 100 times and 10 times less efficient at cleaving within the aggrecan IGD and CS-2 regions, respectively. Collectively, our results showed that the delayed activation of proMMPs and the relatively low cleavage efficiency of MMPs can explain the minor contribution of these enzymes to aggrecan catabolism in vivo. This study also uncovered a potential role for MMPs in the C-terminal truncation of aggrecan.     

Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene

Aims: Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. Methods and Results: Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in the rough variants of the same strain. Temperature significantly increased the adhesion of rhizoid variants up to 20°C. Modification of bacterial surface suggested that adhesion molecules contain both carbohydrates and proteins. LPS did not differ between the variants of the same strain. Conclusions: The results suggest that in Fl. columnare both rhizoid colony morphology and high chondroitinase activity are needed for virulence and that temperature may promote the adhesion of the virulent variants to surfaces at fish farms. Significance and Impact of the Study: New information is produced on the virulence mechanisms of Fl. columnare and the reasons behind the survival of the bacterium at fish farms.