High Molecular Weight Proteoglycans Research Articles

Alterations in the Structure, Composition, and Organization of Galactosaminoglycan-Containing Proteoglycans and Collagen Correspond to the Progressive Stages of Dupuytren's Disease.

Dupuytren's disease (DD) is a prevalent fibroproliferative disorder of the hand, shaped by genetic, epigenetic, and environmental influences. The extracellular matrix (ECM) is a complex assembly of diverse macromolecules. Alterations in the ECM's content, structure and organization can impact both normal physiological functions and pathological conditions. This study explored the content and organization of glycosaminoglycans, proteoglycans, and collagen in the ECM of patients at various stages of DD, assessing their potential as prognostic indicators. This research reveals, for the first time, relevant changes in the complexity of chondroitin/dermatan sulfate structures, specifically an increase of disaccharides containing iduronic acid residues covalently linked to either N-acetylgalactosamine 6-O-sulfated or N-acetylgalactosamine 4-O-sulfated, correlating with the disease's severity. Additionally, we noted an increase in versican expression, a high molecular weight proteoglycan, across stages I to IV, while decorin, a small leucine-rich proteoglycan, significantly diminishes as DD progresses, both confirmed by mRNA analysis and protein detection via confocal microscopy. Coherent anti-Stokes Raman scattering (CARS) microscopy further demonstrated that collagen fibril architecture in DD varies importantly with disease stages. Moreover, the urinary excretion of both hyaluronic and sulfated glycosaminoglycans markedly decreased among DD patients.Our findings indicate that specific proteoglycans with galactosaminoglycan chains and collagen arrangements could serve as biomarkers for DD progression. The reduction in glycosaminoglycan excretion suggests a systemic manifestation of the disease.

Glucose enhances aggrecan expression in chondrocytes via the PKCα/p38-miR141-3p signaling pathway.

Aggrecan is a high molecular weight proteoglycan that plays a critical role in cartilage structure and the function of joints, providing intervertebral disc and cartilage with the ability to resist compressive loads. Aggrecan degradation in articular cartilage is a significant event in early-stage osteoarthritis (OA). Currently, no effective treatment exists for OA other than pain relief. Dextrose (D-glucose) prolotherapy has shown promising activity in the treatment of different musculoskeletal disorders, including OA. However, little is known about the molecular mechanism of the glucose effect in OA and on the regulation of chondrogenesis. We show for the first time that glucose upregulates aggrecan expression and subsequent chondrogenesis in ATDC5 cells. Moreover, we found that glucose-induced aggrecan expression is mediated through the protein kinase Cα (PKCα)- and p38-dependent pathway. As demonstrated by microRNA (miR) and luciferase analyses, the glucose-induced PKCα/p38 signaling axis is responsible for downregulating miR141-3p which targets to the 3'untranslated region of aggrecan. In summary, we show that glucose enhances chondrogenesis by upregulating aggrecan expression via the PKCα-p38-miR141-3p signaling pathway. This result provides new insights into the mechanism of glucose on chondrogenesis.

Anti-aging effects of high molecular weight proteoglycan from salmon nasal cartilage in hairless mice

Proteoglycans comprise a family of complex macromolecules consisting of a core protein with covalently attached glycosaminoglycan (GAG) chains. The skin anti-aging effects of oral administration of proteoglycan fractions with different molecular weights from salmon nasal cartilage were investigated in a hairless mouse model of skin aging; aging was caused by repeated ultraviolet B (UVB) irradiation. Three proteoglycan fractions of different molecular weights were prepared from salmon nasal cartilage water extract by ion-exchange column chromatography and gel filtration column chromatography. Physiological and histological analysis of the skin indicated that oral administration of high molecular weight proteoglycan inhibited UVB-induced skin aging, defined as increased erythema, increased transepidermal water loss (TEWL), decreased hydration, and epidermal and dermal hypertrophies. The serum and dorsal skin inflammatory cytokine levels indicated that high molecular weight proteoglycan acts on gut immunity and improves skin by inhibiting surplus inflammatory cytokines produced by UVB irradiation. These results suggest that high molecular weight proteoglycan from salmon nasal cartilage is effective in preventing skin aging.

La thérapie cellulaire du cartilage : bases cellulaires et moléculaires

The destruction of articular cartilage represents the outcome of most inflammatory and degenerative rheumatic diseases and leads to severe disability. Articular cartilage being unable to repair spontaneously, alterations of the joint surface often results in end-stage osteoarthritis, requiring surgical intervention and total joint replacement. This makes damaged tissues repair a major challenge in our aging society. Cartilage harbors only one cell type, the chondrocyte, which synthesizes and secretes specific matrix proteins such as type II collagen and high molecular weight proteoglycans. Matrix proteins are responsible for the conservation of the chondrocyte phenotype and the maintenance of the mechanical functions of cartilage. Development of therapeutic strategies for cartilage repair should thus comprise not only the replacement of lost cartilage cells but also that of extracellular matrix with cartilage-like properties. Different protocols are under investigation. The most commonly employed materials include transplantation of autologous osteochondral tissue. More recently, cell-based therapies using autologous mature chondrocytes or pre-chondrogenic stem cells have drawn particular attention. Tissue-engineering procedures represent the actual trend in cartilage repair. This approach combines biodegradable polymeric three-dimensional matrixes and isolated prechondrogenic stem cells. The cells are seeded within the biocompatible matrix and then implanted into the joint. Numerous non-degradable and degradable polymers, which efficiently "mimic" the natural surroundings of cartilage cells, are currently under investigation.

Biochemical and morphological changes in endothelial cells in response to hypoxic interstitial edema

BackgroundA correlation between interstial pulmonary matrix disorganization and lung cellular response was recently documented in cardiogenic interstitial edema as changes in the signal-cellular transduction platforms (lipid microdomains: caveoale and lipid rafts). These findings led to hypothesize a specific "sensing" function by lung cells resulting from a perturbation in cell-matrix interaction. We reason that the cell-matrix interaction may differ between the cardiogenic and the hypoxic type of lung edema due to the observed difference in the sequential degradation of matrix proteoglycans (PGs) family. In cardiogenic edema a major fragmentation of high molecular weight PGs of the interfibrillar matrix was found, while in hypoxia the fragmentation process mostly involved the PGs of the basement membrane controlling microvascular permeability. Based on these considerations, we aim to describe potential differences in the lung cellular response to the two types of edema.MethodsWe analysed the composition of plasma membrane and of lipid microdomains in lung tissue samples from anesthetized rabbits exposed to mild hypoxia (12 % O2 for 3–5 h) causing interstitial lung edema. Lipid analysis was performed by chromatographic techniques, while protein analysis by electrophoresis and Western blotting. Lipid peroxidation was assessed on total plasma membranes by a colorimetric assay (Bioxytech LPO-586, OxisResearch). Plasma membrane fluidity was also assessed by fluorescence. Lipid microdomains were isolated by discontinuous sucrose gradient. We also performed a morphometric analysis on lung cell shape on TEM images from lung tissue specimen.ResultsAfter hypoxia, phospholipids content in plasma membranes remained unchanged while the cholesterol/phospholipids ratio increased significantly by about 9% causing a decrease in membrane fluidity. No significant increase in lipid peroxidation was detected. Analysis of lipid microdomains showed a decrease of caveolin-1 and AQP1 (markers of caveolae), and an increase in CD55 (marker of lipid rafts). Morphometry showed a significant decrease in endothelial cell volume, a marked increase in the cell surface/volume ratio and a decrease in caveolar density; epithelial cells did not show morphological changes.ConclusionThe biochemical, signaling and morphological changes observed in lung endothelial cell exposed to hypoxia are opposite to those previously described in cardiogenic edema, suggesting a differential cellular response to either type of edema.

Propagation of Human Nasal Chondrocytes in Microcarrier Spinner Culture

The aim of this study was to test the effectiveness of nasal septal chondrocytes, propagated in microcarrier spinner culture, as an alternative tissue source of chondrocytic cells for cartilage grafts for head and neck surgery and for articular cartilage repair. We harvested chondrocytes from 159 patients, ranging in age from 15 to 80 years and undergoing repair of a deviated nasal septum, and propagated the cells in a microcarrier spinner culture system. The nasal chondrocytes proliferated and produced extracellular matrix components similar to that produced by articular chondrocytes. In microcarrier spinner culture on collagen beads, chondrocyte numbers increased up to 14-fold in 2 weeks. After a month, the microcarriers seeded with nasal chondrocytes began to aggregate, producing a dense cartilage-like material. The newly synthesized extracellular matrix was rich in high molecular weight proteoglycans, and the chondrocytes expressed type II collagen and aggrecan but not type I collagen. These studies support the feasibility of engineering cartilage tissue using chondrocytes harvested from the nasal septum. Injectable and solid formulations based on this technology are being evaluated for applications in craniomaxillofacial reconstructive surgery and for plastic and orthopedic surgery practices.

Improved production by domain inversion of single-chain Fv antibody fragment against high molecular weight proteoglycan for the radioimmunotargeting of melanoma.

Melanoma is among the few cancers with rising incidence. Currently there is no effective treatment for metastatic disease, but improved detection of melanoma has the potential to benefit the management of patients with early disease. Radioimmunodection by imaging with single-chain Fv (scFv) antibody fragments is one such emerging diagnostic method. However, the amount of scFv that can be produced at a scale suitable for use in patients is limiting. We have previously shown that the bacterial expression of a scFv derived from a monoclonal antibody (MAb) specific for melanoma-associated proteoglycan can be increased by light chain shuffling. In this report we show that a further increase in expression yield can be obtained by reversing the usual V(H)-V(L) orientation of scFvs to V(L)-V(H). Such seemingly minor changes have previously been reported to have unexpected effects on the in vitro and in vivo binding properties of recombinant antibodies. Our results show that reversal of the V domain orientation of the scFv improves expression by 150% without an adverse effect on melanoma binding in vitro and tumor targeting in vivo. Therefore, our results show that alteration of V domain orientation can improve the production yield of clinically useful antibody fragments. When used in combination with other antibody engineering approaches for increased antibody production changing the domain orientation is a simple strategy to achieve significant improvements in the production of scFvs for tumor radioimmunodetection for patient studies.

Efficient in vivo targeting of malignant melanoma by single-chain Fv antibody fragments.

Single-chain antibody fragments (scFvs) have superior pharmacokinetic and biodistribution characteristics compared with larger antibody fragments when used for radioimaging of human tumours. An scFv specific for malignant melanoma (B4 scFv) was tested in a mouse xenograft model for human melanoma and compared with the monoclonal antibody (MAb) LHM2. LHM2 is specific for the high molecular weight proteoglycan melanoma-associated antigen (HMW-MAA), while B4 scFv binds a novel melanoma-specific antigen. The B4 scFv was cleared rapidly from the circulation (t(1/2)alpha = 7 min) compared with LHM2 MAb (t(1/2)alpha = 37 min). However, the B4 scFv had an unusually long t(1/2)beta (437 min compared with 384 min for LHM2 MAb). Biodistribution studies showed that B4 accumulated specifically in the tumour grafts. Although non-specific accumulation in the liver, lung, spleen and blood was lower than with LHM2, non-specific accumulation of B4 in the kidney was higher. Despite this, the overall targeting efficiency in this study, as determined by tumour:normal tissue ratios, showed that B4 was superior to whole antibody. Immunoscintigraphy images showed good correlation with the biodistribution data for B4 and LHM2. This study represents the first use of an anti-melanoma scFv in vivo.

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, alpha-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 beta-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.

Study on porcine proteoglycans relevant to structure and function of temporomandibular joint

The purpose of this study was to characterize the biochemical and immunological properties of the temporomandibular joint (TMJ). Proteoglycans (PGs) were extracted from the porcine TMJ retrodiscal tissue (RT) and superficial fibrous layer of condyle (SLC). The proportion of Glycosaminoglycans (GAGs) with TMJ disc. RT and SLC resembled that of disc and RT GAGs, but SLC GAGs were different from others. TMJ disc had a higher Dermatan sulfate/Chondroitin sulfate ratio than that of RT. and TMJ disc had stronger tensional load resistance than that of RT. Immunologically, a high molecular weight PG resembled Aggrecan and two low molecular weight PGs resembled Decorin and Biglycan. Total RNA was isolated from SLC, and RT-PCR was performed. These products were sequenced, and these sequences were compared to the sequences of porcine Aggrecan, human Decorin and Biglycan. These results suggested that TMJ had Decorin, Biglycan, and Aggrecan. The difference of PG quantity in each region seemed to be associated with the pathological conditions found on each component of TMJ.

Generation and selection of monoclonal antibodies, single-chain Fv and antibody fusion phage specific for human melanoma-associated antigens

A panel of 13 murine monoclonal antibodies (mAbs) recognizing antigens on human melanoma cells but not on melanocytes was generated. Two mAbs (LHM3 and LHM5) stained sections of melanoma but not normal tissues. mAbs LHM2 and LHM8 stained only a minority of normal tissues. The mAbs differed further in their staining patterns on melanoma cell lines HMB2, DX3 and SK23 in FACS. The mAbs recognize antigens of 34, 38, 57, 94, 190-200 and > 200 kD. One mAb each bound to each of the antigens HLA DR (LHM4) and high molecular weight proteoglycan (LHM2). The high molecular weight proteoglycan-specific mAb was used to construct a single-chain Fv (scFv) antibody fragment and an antibody fusion phage in Escherichia coli. Both the scFv and the fusion phage were shown to bind specifically to melanoma cells. A method for the selection of melanoma cell-binding phages from phage libraries is described.

Action of metalloproteinases on porcine dentin mineralization

Samples containing predentin and mineralized dentin involving the mineralized front (newly formed dentin) were prepared by scraping developing porcine teeth after odontoblastic cell debris had been removed from the predentin surfaces. An extract was obtained separately from the matrices of predentin and of the newly formed dentin with a 4 M guanidine solution before and after demineralization with acetic acid solution. Enzymography detected 56 and 61 kDa gelatinases and 25 kDa proteoglycanase as neutral metalloproteinases in both extracts and proved them to be in an active form. Approximately half of the 56 and 61 kDa gelatinases binds to collagen fibers in predentin matrix. Three high molecular weight proteoglycans (70-85 kDa, 130-180 kDa, and 290 kDa) were found in the predentin matrix, but not in the newly formed dentin. The proteoglycanases in predentin degraded 290 kDa proteoglycan, if incubated together with calcium (Ca) ions. The results of this investigation indicate that active proteoglycanases which existed in the predentin perform no substantial work in proteoglycan degradation because the Ca ions are masked in the predentin matrix by coexisting proteoglycans. When mineralization occurs, however, they can degrade the proteoglycan at the mineralization front because excess Ca ions may be supplied via odontoblastic processes.

A high molecular weight proteoglycan is differentially expressed during development of the mollusc Concholepas concholepas (Mollusca; Gastropoda; Muricidae).

Incorporation of radioactive sulfate to hatched veliger larvae of the gastropod muricid Concholepas concholepas indicated that over 87% of the sulfated macromolecules were found in the detergent insoluble fraction, rich in extracellular matrix (ECM) components. The sulfated material was solubilized with guanidine salt followed by urea dialysis and fractionated by DEAE-Sephacel chromatography. Three sulfated compounds eluting at 0.7, 1.1, and 3.0 M NaCl, called peaks I, II, and III, respectively, were obtained. The sulfated compound present in peak I was degraded by pronase or sodium alkaline treatment to a small sulfated resistant material, suggesting the presence of a proteoglycan (PG). Filtration analysis on Sephacryl S-500 and SDS-PAGE of the intact PG indicates that it has a high molecular weight (360,000 to over 1 x 10(6)). Monoclonal antibodies (mAb) against this PG were produced. The specificity of one mAb, the 6H2, was demonstrated by size chromatography and ELISA analysis. The epitope recognized by this mAb seems to be present in the core protein of the PG. Both the extent of sulfation and the presence of different sulfated species of PGs were evaluated during the development of this mollusc. A twelvefold increase in the incorporation of sulfate to PGs per milligram of protein was found in veliger larvae compared to blastula-glastula stages. This change correlated well with the differential expression of the sulfated PG present in peak I. Biochemical and immunological analysis indicate that high levels of this PG are found in veliger and trocophore larvae in comparison with blastula-gastrula and early juveniles.(ABSTRACT TRUNCATED AT 250 WORDS)

Chondroitin sulfate proteoglycan form of the Alzheimer's beta-amyloid precursor.

The Alzheimer's amyloid beta protein is derived from a family of membrane glycoproteins termed amyloid precursor proteins (APP). Here we show that APP exists as the core protein of a chondroitin sulfate (CS) proteoglycan, ranging in apparent molecular size from 140 to 250 kDa, secreted by glial cell line C6. After partial purification on ion-exchange and gel chromatography, the secreted APP proteoglycan was recognized on Western blots by several antibodies specific to different regions of APP. Chondroitinase AC or ABC treatment of our samples completely eliminated the high molecular weight proteoglycan with a concomitant increase in the APP protein. This digested product reacted with an anti-stub antibody which recognizes 4-sulfated disaccharide. Sequencing of the N terminus of the core protein of this CS proteoglycan yielded 18 residues identical to the N terminus sequence of the mature APP. Quantitative analysis showed that, in this cell line, about 90% of the secreted nexin II form of APP occurs in the proteoglycan form, suggesting that the CS chains have a role in the biological function of this protein. The close proximity of two consensus CS attachment sites to both the N terminus of the amyloid beta protein and the secretase cleavage site, suggests that the CS chains may affect the proteolysis of APP and production of the amyloid beta protein.

Glycosaminoglycan patterns in gingival proteoglycans of rat with age

Among the potential biochemical indices that are closely associated with craniofacial development are the proteoglycans. Gingival segments from the palate of 4-,6-,8-,12- and 18-week-old rats were incubated for 4 h in medium containing [ 3H]-glucosamine and [ 35S]-Na 2SO 4, and subjected to proteoglycan isolation and glycosaminoglycan analysis. Two distinct proteoglycan fractions differing in the degree of sulphation were obtained by ion-exchange chromatography. The incorporation of both labels in the undersulphated fraction increased with age; there was a pronounced decrease with age in the sulphated proteoglycan fraction. The undersulphated proteoglycans showed an age-dependent decrease in hyaluronic acid, and increase in dermatan sulphate and chondroitin 4- and 6-sulphates. Gel filtration of the sulphated proteoglycan fraction yielded high and low molecular-weight proteoglycans, the glycosaminoglycans of which were particularly rich (61–76%) in dermatan sulphate. Smaller quantities of chondroitin 4- and 6-sulphates, and heparan sulphate were also present. All glycosaminoglycans showed a decrease in content with age. The findings suggest a possible correlation between gingival proteoglycan/glycosaminoglycan patterns and development.

Studies on matrix components relevant to structure and function of the temporomandibular joint

Proteoglycans (PGs), or their component glycosaminoglycan (GAG) chains, have long been recognized as small in quantity but as significant components for many connective tissues. The temporomandibular joint (TMJ) disc and synovial fluid were rarely investigated in this regard. This research was aimed at the biochemical analysis of the composition of collagen, GAGs and PGs in the normal human and porcine TMJ discs, and of PGs in the synovial fluid. 1. The collagen type analysis by gel electrophoresis revealed that the human and porcine TMJ discs were composed only of type I collagen. 2. The human TMJ disc contained 69.9% chondroitin sulfate, 24.5% dermatan sulfate, 5.6% hyaluronic acid and a trace amount of keratan sulfate, and the porcine TMJ disc, 5.8%, 91.4%, 2.8% and a trace amount, respectively. Immunohistochemical staining of the functional parts of the TMJ disc showed a universal distribution of these GAGs. 3. PGs were extracted from the TMJ disc with 4 M guanidine chloride and separated by ion-exchange and gel-filtration chromatography. A high molecular weight PG resembling the cartilage PG and two low molecular weight PGs having a core protein of Mr 40 K were isolated. 4. PGs suggested to be derived from the matrix of other tissues were found in the abnormal human synovial fluid. From these results it was suggested that the TMJ disc was thought not to be fibrocartilage but a fibrous tissue adapted against to the compressive and tensile force.

A carbohydrate epitope defined by monoclonal antibody VC1.1 is found on N-CAM and other cell adhesion molecules

VC1.1 is a monoclonal antibody that stains the surfaces of neuronal subsets in the brain. Previous work showed that VC1.1 recognizes 3 polypeptide bands with molecular weights of 95–105 kDa, 140 kDa and 170 kDa and two high molecular weight proteoglycans with weights of approximately 680 and 650–700 kDa. The heterogeneity and molecular weight range of these bands suggested that VC1.1 might recognize a carbohydrate moiety associated with a family of cell surface molecules. It had been previously demonstrated that a separate monoclonal antibody, HNK-1 also recognized a cell surface associated epitope characterized as a sulfate- and glucuronic acid-containing N-linked carbohydrate. This epitope has been shown to be present on members of the N-CAM adhesion molecule family. In this report, we demonstrate that VC1.1 recognizes an N-linked carbohydrate group that is attached to myelin-associated glycoprotein and N-CAM. Immunocytochemical and biochemical comparisons of VC1.1 and HNK-1 staining in rat and cat brain indicate that these two antibodies probably recognize overlapping, or identical carbohydrate epitopes.

Coordinate secretion and functional synergism of T cell‐associated serine proteinase‐1 (MTSP‐1) and endoglycosidase(s) of activated T cells

Cell lysates and exocytosed soluble mediator(s) (ESM) released from CD8+ T cell lines (TCL) by receptor-triggered secretory exocytosis were tested for degradation of proteoglycans associated with in vitro produced subendothelial extracellular matrix (ECM). ESM was found to release low-molecular weight (kav 0.5-0.6) fragments from the sulfated proteoglycans in ECM. In the presence of heparin, an inhibitor for endoglycosidase activity, only high-molecular-weight products (kav 0.2) were formed. Preincubation of ESM with HD-prolylphenylalanyl-arginyl-chloromethylketone (PFR-CK) an inhibitor for the T cell-associated serine proteinase-1 (MTSP-1) totally prevented release of high- and low-molecular weight proteoglycan fragments. Furthermore, it was shown that purified MTSP-1 is able to release from ECM high-molecular weight proteoglycans and that this process is inhibitable by PFR-CK but not by heparin. Further treatment of these soluble high-molecular weight sulfated proteoglycans with ESM from TCL 1.D9 led to appearance of low-molecular weight split products (kav 0.5-0.6). This conversion was inhibitable by heparin but not by PFR-CK. These findings indicate that activated T cells contain two enzymatic activities, i.e. MTSP-1 and at least one endoglycosidase, which after receptor-triggered secretion can synergize in the degradation of sulfated proteoglycans in subendothelial ECM.

The biosynthesis of proteoglycans and interstitial collagens by bovine pericardial fibroblasts

The biosynthesis of interstitial collagens (types I and III) and proteoglycans was studied in fibroblasts isolated from the parietal layer of bovine pericardium. Confluent cultures were labeled with Na 2 35SO 4 for proteoglycans or 14C-proline for collagens. The proteoglycans synthesized by pericardial fibroblasts were purified by DEAE-Sephacel chromatography and further fractionated into three components by gelfiltration. Two minor high molecular weight proteoglycans were shown by SDS-PAGE to be resistant to chondroitinase ABC and AC, and partially degraded by nitrous acid. The major, low molecular weight proteoglycan had a core protein of 45 kDa and is considered to be a dermatan sulfate/chondroitin sulfate proteoglycan since it was resistant to nitrous acid, but digested partially by chondroitinase AC and completely by ABC. The pericardial fibroblasts synthesized predominantly type I collagen and low amounts (about 10%) of type III collagen which was detected by delayed reduction on SDS-PAGE. The data show that pericardial fibroblasts synthesize the same macromolecules that can be extracted from the intact tissue and suggest that the proteoglycan may play a structural as well as physiological role.

Effect of puromycin on metanephric differentiation: Morphological, autoradiographic and biochemical studies

Effect of aminonucleoside of puromycin (PAN) on metanephric development and proteoglycans (PGs) was investigated. Murine metanephric tissues, obtained on the thirteenth day of gestation, were exposed to PAN in a culture medium for one to seven days and processed for morphological, histochemical and immunofluorescent studies. For tissue autoradiographic and biochemical studies, kidneys were labelled with a precursor product of PGs, that is, [35S]-sulfate. A generalized decrease in the glomerular population along with swelling and deformation in the ureteric bud branches was observed. These changes were accompanied with a diminution in the total incorporated radioactivity and a reduction in the autoradiographic grains, especially over the tips of ureteric bud branches. Sepharose CL-4B chromatography revealed a major high molecular weight PG (Mr greater than 2.5 x 10(6], and a relative increase in the chondroitinase-ABC sensitive PGs. The media PGs were of relatively smaller size. Immunoprecipitation experiments with [35S]-methionine-labeled tissues and immunofluorescent studies revealed a significant decrease of PGs in metanephric tissues, while type IV collagen and laminin were relatively unaffected. Significant glomerular changes included failure in differentiation of the visceral epithelial foot processes, formation of villi and in maturation of glomerular basement membrane. The latter was seen as fragments of extracellular matrices interspersed among undifferentiated podocytes and had reduced staining with ruthenium red--a dye marker for the PGs. This deficiency of PGs was confirmed by electron microscopic autoradiography, where a reduction in the number of silver grains was observed. The fact that the PAN-induced cellular and extracellular alterations were associated with perturbances in biosynthesis of PGs, suggests that the morphogenetic regulators, that is, PGs play a vital role in various differentiation processes involved during metanephric development.