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。     

Mast Cells Produce a Unique Chondroitin Sulfate Epitope

The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells.     

Isolation and characterization of proteoglycans synthesized by rat myoblasts L6J1 in culture

Proteoglycans synthesized by rat myoblasts L6J1 in culture were isolated using sorbent Q-Sepharose from culture medium, extracellular matrix (ECM), and cells. Elution of the sorbed material in a NaCl gradient separated proteoglycans from the bulk of proteins eluted at low concentration of the salt. Four fractions (fractions I-IV) were obtained for each component of the cell culture, including two proteoglycan fractions for the ECM and culture medium and one fraction for the myoblasts. Proteoglycans of the culture medium were virtually completely represented by proteoglycans of fetal calf serum. With enzymes chondroitinase ABC and heparinase III chondroitin/dermatan sulfate proteoglycans were shown to prevail in all components of the myoblast culture. The core proteins of proteoglycans were characterized by electrophoresis.     

Membrane associated proteoglycans in rat testicular peritubular cells

Confluent testicular peritubular cells derived from immature rats were used to study membrane associated proteoglycans (PG) Peripheral material (heparin releasable), membrane and intracellular material (Triton X-100 releasable) were collected, purified by anion exchange chromatography then characterized by gel filtration and by hydrophobic interaction chromatography, followed by enzymatic digestion and chemical treatment. The peripheral material was constituted of two populations of PG (K_av=0 and 0.10 on Superose 6 column), each containing both heparan sulfate proteoglycans (HSPG) and chondroitin proteoglycans (CSPG) and perhaps a hybrid PG (HSCSPG). These PG being not retained on an octyl Sepharose column they were devoided of hydrophobic properties. The integral membrane proteoglycans isolated on the basis of their hydrophobic properties represented 20% of the Triton X-100 releasable material, and were exclusively constituted of proteoheparan sulfate. There were no relationships between this membrane HSPG and the peripheral HSPG as evidenced by pulse chase experiments. The mode of intercalation of the hydrophobic HSPG in the cell membrane was studied. The majority of these macromolecules (80%) were sensitive to trypsin and only a minor proportion (20%) were sensitive to phosphatidylinositol specific phospholipase C. Thus, about 80% of the hydrophobic HSPG were intercalated in the cell membrane by a hydrophobic segment of the core protein whereas about 20% were associated with the cell membrane via a phosphatidylinositol residue covalently bound to the core protein of the PG.Abbreviations PG Proteoglycans - CSPG Chondroitin Sulfate Proteoglycans - HSPG Heparan Sulfate Proteoglycans - HSCSPG Heparan and Chondroitin Sulfate Proteoglycans - DNAse I Deoxyribonuclease I - DMEM Dulbeccos modified Eagle's medium - H/D HAM F12/DMEM - ECM Extracellular Matrix - PBS Phosphate Buffered Saline - PI Phosphatidylinositol - GPI Glycosyl Phosphatidylinositol - PI-PLC Phosphatidylinositol Specific Phospholipase C - TBS Tris Buffered Saline - STI Soybean Trypsin Inhibitor - GAG Glycosaminoglycans - HA Hyaluronic Acid     

Biological functions of proteoglycans: use of specific inhibitors of proteoglycan synthesis

The use of specific inhibitors of proteoglycan synthesis have demonstrated essential functions for these molecules. Proteoglycans do not appear to be essential for cell viability or proliferation but are necessary for stable assembly of ECM and functional cell-ECM interaction. Because of their ability to nearly completely abolish ECM assembly in cell culture systems, proteoglycan synthesis inhibitors are useful tools to examine effects of ECM on the phenotypic behavior of cells.Despite its usefulness, the use of proteoglycan synthesis inhibitors has some drawbacks. The most significant of these is the fact that synthesis of all proteoglycans is inhibited, making it difficult to assign a particular function to a specific proteoglycan type. For this reason, inhibition studies need to be done in conjunction with additional biochemical, immunological or molecular biological studies.     

Adherence of Platelets Under Flow Conditions Results in Specific Phosphorylation of Proteins at Tyrosine Residues

Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (Coll). Studies under flow conditions were performed using two different adhesive substrata: Coll and endothelial cells extracellular matrix (ECM). Coverslips coated with Coll or ECM were perfused through a parallel-plate perfusion chamber at 800s^?1 for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on Coll or on ECM were almost identical and lacked proteins p95, p80, p66, and p64. which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.     

A pH-regulated dimeric bouquet in the structure of von Willebrand factor

At the acidic pH of the trans-Golgi and Weibel-Palade bodies (WPBs), but not at the alkaline pH of secretion, the C-terminal ～1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine-knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower-like domains. N-terminal VWF domains mediate helical tubule formation in WPBs and template N-terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow-regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.     

几种哺乳类动物胸主动脉中的蛋白聚糖

本文报道了北京(As高发)、广西南宁(As低发)两地区的人、兔(“易感”As)及狗(“不易感”As)胸主动脉中三种蛋白聚糖(CSPG,DSCSPG及HSPG)的含量。结果表明:a.广西南宁样品的总PGs,HSPG及DSCSPG含量均高于北京,尤以HSPG差异显著。b.兔、狗胸主动脉PG总量均低于人的。其中HSPG,CSPG含量及百分率以及兔的DSCSPG含量亦低于人的相应PG含量,而狗的DSCSPG含量与人的类似。c.南宁人及狗的DSCSPG含量及相对百分率虽高,但其DSCSPG中DS链所占相对百分率低于北京人和兔的。以上结果提示动脉壁PG质与量的差异可能与AS发病有关。     

An erroneous glycosaminoglycan metabolism leads to corneal opacification in macular corneal dystrophy

Macular corneal dystrophy (MCD) is a rare, potentially blinding disease whose fundamental genetic defect and exact pathogenesis are yet to be elucidated. It is, however, an especially interesting pathology, which highlights how an erroneous glycosaminoglycan or proteoglycan metabolism can induce physical symptoms in a specific connective tissue. Based on immunochemical data, MCD is a heterogeneous condition, and at least two types of the disease have been identified. The cornea, cartilage, and serum from MCD type I patients all contain an unsulphated form of keratan sulphate. In contrast, these tissues contain normally sulphated keratan sulphate in MCD type II patients. A normal population of keratan sulphate proteoglycans (and chondroitin/dermatan sulphate proteoglycans) in the cornea seems to be a requirement of corneal transparency. However, a clinical diagnosis of MCD is unable to distinguish between the keratan sulphate positive and negative types of MCD. The histopathology of MCD is fairly well established, and various corneal aberrations—such as fibrillogranular and glycosaminoglycan deposits, abnormal diameter collagen, and collagen-free lacunae—result in a breakdown of the regular corneal architecture that presumably contributes to the subsequent corneal opacification.     

Thrombin Regulation of Cell Function through Protease-Activated Receptors: Implications for Therapeutic Intervention

The serine protease thrombin is well recognized as being pivotal to the maintenance of hemostasis under both normal and pathological conditions. Its cellular actions are mediated through a unique family of protease-activated receptors (PARs). These receptors represent a novel family of G protein-coupled receptors that undergo proteolytic cleavage of their amino terminus and subsequent autoactivation by a tethered peptide ligand. This paper reviews the consequences of PAR activation in thrombosis, vascular injury, inflammation, tissue injury, and within the tumor microenvironment.     

Extracellular matrix components in uterine leiomyoma and their alteration during the tumour growth

It was found that both normal human myometrium and uterine leiomyoma contain several glycosaminoglycans. In contrast to many normal and tumour tissues the amount of hyaluronic acid is very low and the proportional amount of sulphated glycosaminoglycans is distinctly higher. It is of interest that heparan sulphate is the major glycosaminoglycan component both in normal myometrium, and in leiomyoma. The amount of hyaluronic acid in myometrium and in the leiomyoma is very low. No significant change in hyaluronate content was observed during the tumour growth. In contrast to that the amount of some sulphated glycosaminoglycans (heparan sulphate, keratan sulphate, chondroitin sulphates and heparin) distinctly increased. It is suggested that some of the GAGs participate in the creation of a storage depot for biologically active molecules (growth factors, enzymes) which are thereby stabilized and protected. Hydrolytic degradation of some GAGs may result in the release of some cytokines which may promote the tumour growth and stimulate collagen biosynthesis by tumour cells.     

Connective tissue activation in guinea pig lung fibroblast cultures: Regulatory effects of glucocorticoids

Summary Earlier studies showed that guinea pig lung fibroblasts in cell culture could be “activated” by naturally occurring peptides with a resultant increase in glycolysis and glycosaminoglycan formation. Such connective tissue activation (CTA) in human cell systems (synovial, cartilage, dermal) has proved a useful tool for studying the mechanisms of inflammation and dissecting the efficacy and actions of anti-inflammatory drugs. The present study examined the consequences of treating basal and activated guinea pig lung fibroblasts with glucocorticoids. The data indicate that glucocorticoids minimally suppress glycosaminoglycan (GAG) synthesis in nonactivated cultures. Further, CTA was inhibited to only a minor degree in activated lung fibroblast cultures by steroids, and even markedly supraphysiologic concentrations of glucocorticoids were not notably inhibitory. It was of interest that thiols enhanced suppression of incremental GAG synthesis by some glucocorticoids in activated lung fibroblast cultures. This study was supported by USPHS Grant HL-19685.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

Synthesis of a novel glycosaminoglycan pentasaccharide serine having anN-acetylgalactosamine residueα-linked to the core linkage tetrasaccharide

A novel pentaosyl serine; GalNAc(1–4)GlcA(1–3)Gal(1–3)Gal(1–4)Xyl(1–3)Ser (2), a putative intermediate of chondroitin sulfate and/or heparan sulfate biosynthesis, was synthesized.