scholarly journals Absence of keratan sulphate from skeletal tissues of mouse and rat

1985 ◽  
Vol 228 (2) ◽  
pp. 443-450 ◽  
Author(s):  
G Venn ◽  
R M Mason
Keyword(s):  
Monoclonal Antibody ◽  
Degradation Products ◽  
Chondroitin Sulphate ◽  
Lumbar Disc ◽  
Heparan Sulphate ◽  
Costal Cartilage ◽  
Animal Experiments ◽  
Keratan Sulphate

The absence of keratan sulphate synthesis from skeletal tissues of young and mature mice and rats has been confirmed by (1) analysis of specific enzyme degradation products of newly synthesized glycosaminoglycans, and (2) immunohistochemistry and radioimmunoassay using a monoclonal antibody directed against keratan sulphate. Approx. 98% of the [35S]glycosaminoglycans synthesized in vivo by mouse and rat costal cartilage, and all of those of lumbar disc, are chondroitin sulphate. The remainder in costal cartilage were identified as heparan sulphate in mature rats. In contrast, [35S]glycosaminoglycans synthesized by cornea of both species comprised both chondroitin sulphate and keratan sulphate. In mice keratan sulphate accounted for 12-25% and in rats 40-50% of the total [35S]glycosaminoglycans, depending on the age of the animal. Experiments in vitro with organ culture of cartilage and cornea confirm these results. Absence of keratan sulphate from mouse costal cartilage and lumbar disc D1-proteoglycans was corroborated by inhibition radioimmunoassay with the monoclonal antibody MZ15 and by lack of staining for keratan sulphate in indirect immunofluorescence studies using the same antibody.

scholarly journals Renal glomerular proteoglycans. An investigation of their synthesis in vivo using a technique for fixation in situ

1988 ◽  
Vol 251 (2) ◽  
pp. 411-418 ◽  
Author(s):  
L A Beavan ◽  
M Davies ◽  
R M Mason
Keyword(s):  
Basement Membrane ◽  
Glomerular Basement Membrane ◽  
Membrane Fraction ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Basement Membranes ◽  
Chondroitin Sulphate Proteoglycans

Newly synthesized rat glomerular [35S]proteoglycans were labelled in vivo after injecting Na2[35S]SO4 intraperitoneally. At the end of the labelling period (7 h) the kidneys were perfused in situ with 0.01% (w/v) cetylpyridinium chloride. This fixed proteoglycans in the tissue and increased their recovery 2-3-fold during subsequent isolation of glomeruli from the renal cortex. The glomeruli were fractionated by a modified osmotic lysis and detergent extraction procedure [Meezan, Brendel, Hjelle & Carlson (1978) in The Biology and Chemistry of Basement Membranes (Kefalides, N.A., ed.), Academic Press, New York; Kanwar & Farquhar (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4493-4497] to obtain a basement membrane preparation. The proteoglycans released at each stage of the procedure were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC and HNO2 digestion and Sepharose CL-4B gel-permeation chromatography. About 85% of the [35S]proteoglycans synthesized were of the heparan sulphate variety, the remainder being chondroitin sulphate proteoglycans. Three sizes of heparan sulphate proteoglycans were identified. The largest (HS1, Kav. 0.47) accounts for 44% of the total extractable heparan sulphates. About one third of HS1 were extracted from the glomerular basement-membrane fraction with 8 M-urea and 4 M-guanidine hydrochloride but the remainder were released from the glomerulus during preparation of the fraction. The two smaller molecules (HS2, Kav. 0.56 and HS3, Kav. 0.68) accounted for 27% and 28% of the extractable heparan sulphate respectively and were not associated with the basement membrane fraction. HS1, HS2 and HS3 were also isolated from non-fixed glomeruli labelled in vivo but with much lower recovery. In glomeruli labelled in vitro, heparan sulphate accounted for only 35% of the proteoglycans, the remainder being of the chondroitin sulphate type. Proteoglycans similar to HS1, HS2 and HS3 were present in glomeruli labelled in vitro but, in addition, a large, highly charged heparan sulphate (HS1a) was extracted from the glomerular basement-membrane fraction of these glomeruli. It accounted for 6% of the total heparan sulphate.

scholarly journals Physical disruption of intervertebral disc promotes cell clustering and a degenerative phenotype

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Polly Lama ◽  
Harry Claireaux ◽  
Luke Flower ◽  
Ian J. Harding ◽  
Trish Dolan ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
Association Studies ◽  
Lumbar Disc ◽  
Animal Experiments ◽  
Herniated Disc ◽  
Cell Phenotype ◽  
Cell Clustering ◽  
Collagen Denaturation

AbstractTo test the hypothesis that physical disruption of an intervertebral disc disturbs cell-matrix binding, leading to cell clustering and increased expression of matrix degrading enzymes that contribute towards degenerative disc cell phenotype. Lumbar disc tissue was removed at surgery from 21 patients with disc herniation, 11 with disc degeneration, and 8 with adolescent scoliosis. 5 μm sections were examined with histology, and 30-µm sections by confocal microscopy. Antibodies were used against integrin α5beta1, matrix metalloproteinases (MMP) 1, MMP-3, caspase 3, and denatured collagen types I and II. Spatial associations were sought between cell clustering and various degenerative features. An additional, 11 non-herniated human discs were used to examine causality: half of each specimen was cultured in a manner that allowed free ‘unconstrained’ swelling (similar to a herniated disc in vivo), while the other half was cultured within a perspex ring that allowed ‘constrained’ swelling. Changes were monitored over 36 h using live-cell imaging. 1,9-Di-methyl methylene blue (DMMB) assay for glycosaminoglycan loss was carried out from tissue medium. Partially constrained specimens showed little swelling or cell movement in vitro. In contrast, unconstrained swelling significantly increased matrix distortion, glycosaminoglycan loss, exposure of integrin binding sites, expression of MMPs 1 and 3, and collagen denaturation. In the association studies, herniated disc specimens showed changes that resembled unconstrained swelling in vitro. In addition, they exhibited increased cell clustering, apoptosis, MMP expression, and collagen denaturation compared to ‘control’ discs. Results support our hypothesis. Further confirmation will require longitudinal animal experiments.

scholarly journals Biosynthesis and metabolism in vivo of intervertebral-disc proteoglycans in the mouse

1983 ◽  
Vol 215 (2) ◽  
pp. 217-225 ◽  
Author(s):  
G Venn ◽  
R M Mason
Keyword(s):  
Chondroitin Sulphate ◽  
Costal Cartilage ◽  
Intervertebral Discs ◽  
Hydrodynamic Size ◽  
Keratan Sulphate ◽  
Major Species ◽  
Average Size ◽  
A Minor ◽  
Turn Over

The synthesis and turnover in vivo of 35S-labelled proteoglycans in mouse cervical, thoracic and lumbar intervertebral discs, and in costal cartilage, was investigated after intraperitoneal injection of [35S]sulphate. Intervertebral discs and costal cartilage synthesize similar amounts of 35S-labelled proteoglycans per microgram of DNA. Discs and cartilage all synthesize a major proteoglycan species (approx. 85%) of large hydrodynamic size and a minor species (approx. 15%) of small size. Both proteoglycans carry chondroitin sulphate chains. Keratan sulphate was not found associated with either species. The total 35S-labelled proteoglycan pool had a metabolic half-life (t1/2) of 10-12 days in discs, and 17 days in cartilage. The extractable major and minor species turned over at similar rates. Those proteoglycans left in the tissue after 29 days turn over very slowly. Approx. 50% of the major 35S-labelled proteoglycan species formed mixed aggregates with hyaluronic acid and rat chondrosarcoma proteoglycan. The ability to form aggregates did not decrease up to 45 days after synthesis. Of the heterogeneous population of proteoglycans comprising the major species, those remaining in the tissue 9 days after synthesis were of smaller average hydrodynamic size and had shorter chondroitin sulphate side chains than the average size at the time of synthesis. With increasing time after synthesis, proteoglycans were less readily extracted from the tissue by 4.0 M-guanidinium chloride than at the time of synthesis.

scholarly journals Bioactivity of Recombinant Humanized Monoclonal Antibody Against HER2 and Its Mechanism of Action on Ovarian Cancer

2021 ◽  
Author(s):  
Jinfeng Zeng ◽  
Rui Zhang ◽  
Rui Guan
Keyword(s):  
Gene Expression ◽  
Ovarian Cancer ◽  
Monoclonal Antibody ◽  
Lactate Dehydrogenase ◽  
Transient Gene Expression ◽  
Animal Experiments ◽  
Growth Factor Receptor ◽  
Lactate Dehydrogenase Release

Abstract BACKGROUND: Human epidermal growth factor receptor 2 (HER2) protein is overexpressed on the surface of various epithelial ovarian cancer tissues, which mediates the proliferation, differentiation, metastasis and signal transduction of tumor cells and is a potential cancer therapeutic target.METHODS: In this paper, the recombinant anti-HER2 humanized IgG1 monoclonal antibody (rhHer2-mAb) was expressed in HEK293F cells by constructing mammalian cell expression vector and optimizing transfection conditions. The antibody was purified by rProtein A affinity chromatography, and its mediated antibody dependent cytotoxicity (ADCC) was identified by lactate dehydrogenase(LDH) lactate dehydrogenase release assay. The anti-tumor activity of rhHer2-mAb was evaluated in NOD/SCID mice. RESULTS: The expression of rhHer2-mAb in HEK293F cells was at the highest level (100.5 mg/L) when the ratios of DNA/PEI and light chain/heavy chain was 1:4 and 1:2, respectively. The IC50 on ADCC of antibodies against SK-OV-3, OVCAR-3 and A-2780 cells were 12.36, 5.43 and 102.90ng/ml, respectively. Animal experiments in mice showed that rhHer2-mAb could effectively inhibit the growth of SK-OV-3 tumor at the dose of 10 mg/kg.CONCLUSIONS: The recombinant monoclonal antibody was obtained by transient gene expression technology and its bioactivity was studied in vitro and in vivo , providing a novel insight for the development and production of future biotechnology-based drugs using transient gene expression technology of HEK293F.

Endothelial cell protein C receptor plays an important role in protein C activation in vivo

Blood ◽  
2001 ◽  
Vol 97 (6) ◽  
pp. 1685-1688 ◽  
Author(s):  
Fletcher B. Taylor ◽  
Glenn T. Peer ◽  
Marion S. Lockhart ◽  
Gary Ferrell ◽  
Charles T. Esmon
Keyword(s):  
Monoclonal Antibody ◽  
Endothelial Cell ◽  
Protein C ◽  
Degradation Products ◽  
Activated Protein C ◽  
Vital Signs ◽  
Cell Protein ◽  
Increased Risk

Endothelial cell protein C receptor (EPCR) augments protein C activation by the thrombin-thrombomodulin complex about 5-fold in vitro. Augmentation is EPCR concentration dependent even when the EPCR concentration is in excess of the thrombomodulin. EPCR is expressed preferentially on large blood vessel endothelium, raising questions about the importance of protein C-EPCR interaction for augmenting systemic protein C activation. In these studies, this question was addressed directly by infusing thrombin into baboons in the presence or absence of a monoclonal antibody to EPCR that blocks protein C binding. Activated protein C levels were then measured directly by capturing the enzyme on a monoclonal antibody and assaying with chromogenic substrate. Blocking protein C-EPCR interaction resulted in about an 88% decrease in circulating activated protein C levels generated in response to thrombin infusion. Leukocyte changes, fibrinogen consumption, fibrin degradation products, and vital signs were similar between the animals infused with thrombin alone and those infused with thrombin and the anti-EPCR antibody. The results indicate that EPCR plays a major role in protein C activation and suggest that defects in the EPCR gene might contribute to increased risk of thrombosis.

scholarly journals Isolation and characterization of proteoglycans synthesized by mouse osteoblastic cells in culture during the mineralization process

1990 ◽  
Vol 266 (1) ◽  
pp. 15-24 ◽  
Author(s):  
Y Takeuchi ◽  
T Matsumoto ◽  
E Ogata ◽  
Y Shishiba
Keyword(s):  
Core Protein ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Osteoblastic Cell ◽  
Guanidinium Chloride ◽  
Mineralization Process ◽  
Osteoblastic Cell Line ◽  
Isolation And Characterization

Proteoglycans in mineralized (0.5 M-EDTA/4 M-guanidinium chloride-extractable) and non-mineralized (4 M-guanidinium chloride-extractable) matrices synthesized by a mouse osteoblastic-cell line MC3T3-E1 were characterized at different phases of mineralization in vitro. Cell cultures were labelled with [35S]sulphate and either [3H]glucosamine or 3H-labelled amino acids. At the mineralization phase a large majority of proteoglycans were extracted with 4 M-guanidinium chloride (G extract), and at least five species of labelled proteoglycans were identified; dermatan sulphate proteoglycans (DSPG), apparent Mr approx. 120,000 and 70,000), heparan sulphate proteoglycans (HSPG, apparent Mr approx. 200,000 and 120,000) and DS chains with very little core protein. DSPGs weakly bound to an octyl-Sepharose CL-4B column and HSPGs bound more tightly, whereas DS chains did not bind to the column. Amounts of labelled proteoglycans extracted with 0.5 M-EDTA/4 M-guanidinium chloride (EDTA extract) were much less than those in G extract. Although the predominant species in the EDTA extract were comparable with the DS or DSPGs in the G extract, none of them bound to octyl-Sepharose CL-4B, indicating their lack of hydrophobicity. At the nonmineralizing phase a large chondroitin sulphate proteoglycan (Mr greater than 600,000) was found in the matrix in addition to the five proteoglycan species similar to those at the mineralization phase. Although DS chains at the early phase were similar in size to those at the mineralization phase, the ratio of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose to 2-acetamido-2-deoxy-3-O-(beta-D-gluculo-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose was less than that at the mineralization phase. These results agree with those of previous studies performed in vivo and suggest that alteration in the synthesis of proteoglycans is involved in the mineralization process. They also suggest that at the osteoblastic mineralization front proteoglycans undergo partial degradation and lose their hydrophobicity.

scholarly journals Mature bovine articular cartilage contains abundant aggrecan that is C-terminally truncated at Ala719-Ala720, a site which is readily cleaved by m-calpain

2004 ◽  
Vol 382 (1) ◽  
pp. 253-259 ◽  
Author(s):  
Hidefumi OSHITA ◽  
John D. SANDY ◽  
Kiichi SUZUKI ◽  
Atsushi AKAIKE ◽  
Yun BAI ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Globular Domain ◽  
Cleavage Sites ◽  
Keratan Sulphate ◽  
Bovine Articular Cartilage ◽  
The Matrix ◽  
A Site

Extracts of normal mature articular cartilage contain aggrecan molecules which bear the G1 domain (the N-terminal globular domain of aggrecan) and are C-terminally truncated by proteolysis at a number of sites. A proportion of these molecules are generated by an aggrecanase and/or matrix-metalloproteinase-mediated cleavage in the IGD (interglobular domain between the G1 and G2 domains of aggrecan). However, the proteinase(s) responsible for formation of the majority of the larger G1-G2 and glycosaminoglycan-bearing truncated species is (are) unknown. N-terminal sequencing of aggrecan core fragments generated by m-calpain digestion of bovine aggrecan has identified four novel cleavage sites: one within the CS (chondroitin sulphate)-1 domain (at one or more of the bonds Ser1229–Val1230, Ser1249–Val1250, Ser1287–Val1288, Gly1307–Val1308 and Ser1346–Val1347), two within the IGD (at bonds Ala474–Ala475 and Gly365–Gly366) and one within the KS (keratan sulphate) domain (at Ala719–Ala720). A new monoclonal antibody (SK-28) to the C-terminal neoepitope at M710VTQVGPGVA719 showed that aggrecan products generated by this cleavage are present in high abundance in mature bovine articular cartilage extracts. We conclude that m-calpain, or an unidentified proteinase with the capacity to cleave at the same site, is active during aggrecan biosynthesis/secretion by mature chondrocytes or in the matrix of mature bovine articular cartilage in vivo.

In Vitro Versus In Vivo Propagation of Monoclonal Antibodies: A Test Case for the Effectiveness of Swedish Ethical Review Committees

1997 ◽  
Vol 25 (5) ◽  
pp. 517-526
Author(s):  
Ulrika Hansson
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Antibody Production ◽  
Animal Experiments ◽  
Alternative Methods ◽  
Ethical Review ◽  
Test Case ◽  
Monoclonal Antibody Production

In descriptions of the Swedish system for the control of animal experiments, it is often implied that the system guarantees that the animals are not subjected to unnecessary suffering and that available alternative methods are used. A study was carried out to examine applications for the use of the ascites method for monoclonal antibody production during 1995 and 1996. The data show that approval was given to all the applications received, with scanty justification. Thus, public reassurance that the Swedish system prevents the unnecessary use of animals is undermined by these findings.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Comparison of High Purity Factor IX Concentrates and a Prothrombin Complex Concentrate in a Canine Model of Thrombogenicity

1991 ◽  
Vol 66 (05) ◽  
pp. 609-613 ◽  
Author(s):  
I R MacGregor ◽  
J M Ferguson ◽  
L F McLaughlin ◽  
T Burnouf ◽  
C V Prowse
Keyword(s):  
High Purity ◽  
Time Course ◽  
Prothrombin Complex Concentrate ◽  
Degradation Products ◽  
Canine Model ◽  
Factor Ix ◽  
In Vitro Tests ◽  
Albumin Infusion

SummaryA non-stasis canine model of thrombogenicity has been used to evaluate batches of high purity factor IX concentrates from 4 manufacturers and a conventional prothrombin complex concentrate (PCC). Platelets, activated partial thromboplastin time (APTT), fibrinogen, fibrin(ogen) degradation products and fibrinopeptide A (FPA) were monitored before and after infusion of concentrate. Changes in FPA were found to be the most sensitive and reproducible indicator of thrombogenicity after infusion of batches of the PCC at doses of between 60 and 180 IU/kg, with a dose related delayed increase in FPA occurring. Total FPA generated after 100-120 IU/kg of 3 batches of PCC over the 3 h time course was 9-12 times that generated after albumin infusion. In contrast the amounts of FPA generated after 200 IU/kg of the 4 high purity factor IX products were in all cases similar to albumin infusion. It was noted that some batches of high purity concentrates had short NAPTTs indicating that current in vitro tests for potential thrombogenicity may be misleading in predicting the effects of these concentrates in vivo.

Sign in / Sign up

Export Citation Format

Share Document