Single X-ray irradiation modulates proteoglycan expression in brain tissue: investigation using mouse model.

Radiotherapy is an integral part of glioblastoma treatment affecting both cancer cells and tumour microenvironment, where proteoglycans (PGs) are key extracellular components. However, the molecular effects of radiotherapy on PGs expression and functional activity in brain tissue are poorly understood. Here, we aimed to study the short-term effects of X-ray irradiation on PGs expression in normal brain tissue in mouse model in vivo. Two-month-old male CBL/6Bl mice (n = 54) were used in this study, animals' brains were irradiated using either research synchrotron VEPP-4 or clinical linear accelerator ElektaAxesse. Control (n = 18) and irradiated (n = 36) brain tissues were analysed at 24 h, 48 h and 72 h after irradiation. Morphology of the cortex and hippocampus was accessed by H&E staining, and expression of PGs (syndecan-1, glypican-1, HSPG2/perlecan, versican, brevican, neurocan, NG2/CSPG4, CD44, decorin, biglycan) was determined by RT-PCR. Single irradiation of mouse brain with a 7 Gy dose did not affect tissue morphology and mRNA levels of most highly-expressed PGs decorin and neurocan, although resulted in significant downregulation of brevican (3-10-fold) and NG2/CSPG4 (8-9-fold) expression both in cerebral cortex and subcortex. Research synchrotron and clinical linear accelerators demonstrated minor variability in their effects. Single X-ray irradiation with a 7 Gy dose does not significantly affect the mouse brain tissue morphology but selectively decreases expression levels of some PGs. The downregulation of brevican and NG2/CSPG4 but not decorin and neurocan reflects alteration of extracellular matrix in irradiated brain tissue, which might contribute to the formation of a permissive microenvironment for glioblastoma relapse development.

Expression of the progenitor marker NG2/CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma.

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O(6)-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling.

Transcriptional regulation of proteoglycans and glycosaminoglycan chain-synthesizing glycosyltransferases by UV irradiation in cultured human dermal fibroblasts.

Various kinds of glycosaminoglycans (GAGs) and proteoglycans (PGs) have been known to be involved in structural and space-filling functions, as well as many physiological regulations in skin. To investigate ultraviolet (UV) radiation-mediated regulation of GAGs and PGs in cultured human dermal fibroblasts, transcriptional changes of many types of PGs and GAG chain-synthesizing enzymes at 18 hr after 75 mJ/cm(2) of UV irradiation were examined using quantitative real-time polymerase chain reaction methods. Hyaluronic acid synthase (HAS)-1, -2, and -3 and hyaluronidase-2 mRNA expressions were significantly increased by UV irradiation. Expressions of lumican, fibromodulin, osteoglycin, syndecan-2, perlecan, agrin, versican, decorin, and biglycan were significantly decreased by UV irradiation, while syndecan-1 was increased. Expressions of GAG chain-synthesizing glycosyltransferases, xylosyltransferase-1, ß1,3-glucuronyltransferase-1, ß1,4-galactosyltransferase-2, -4, exostosin-1, chondroitin polymerizing factor, and chondroitin sulfate synthase-3 were significantly reduced, whereas those of ß1,3-galactosyltransferase-6, ß1,4-galactosyltransferase-3, -7, ß-1,3-N-acetylglucosaminyltran sferase-2, and -7 were increased by UV irradiation. Heparanase-1 mRNA expression was increased, but that of heparanase-2 was reduced by UV irradiation. Time-course investigation of representative genes showed consistent results. In conclusion, UV irradiation may increase hyaluronic acid production through HAS induction, and decrease other GAG productions through downregulation of PG core proteins and GAG chain-synthesizing glycosyltransferases in cultured human dermal fibroblasts.

Chondroprotective effects of pulsed electromagnetic fields on human cartilage explants.

This study investigated the effects of pulsed electromagnetic fields (PEMFs) on proteoglycan (PG) metabolism of human articular cartilage explants from patients with osteoarthritis (OA). Human cartilage explants, recovered from lateral and medial femoral condyles, were classified according to the International Cartilage Repair Society (ICRS) and graded based on Outerbridge scores. Explants cultured in the absence and presence of IL-1ß were treated with PEMF (1.5 mT, 75 Hz) or IGF-I alone or in combination for 1 and 7 days. PG synthesis and release were determined. Results showed that explants derived from lateral and medial condyles scored OA grades I and III, respectively. In OA grade I explants, after 7 days exposure, PEMF and IGF-I significantly increased (35) S-sulfate incorporation 49% and 53%, respectively, compared to control, and counteracted the inhibitory effect of IL 1ß (0.01 ng/ml). The combined exposure to PEMF and IGF-I was additive in all conditions. Similar results were obtained in OA grade III cartilage explants. In conclusion, PEMF and IGF-I augment cartilage explant anabolic activities, increase PG synthesis, and counteract the catabolic activity of IL-1ß in OA grades I and III. We hypothesize that both IGF-I and PEMF have chondroprotective effects on human articular cartilage, particularly in early stages of OA.

Ultraviolet B and A irradiation induces fibromodulin expression in human fibroblasts in vitro.

Ultraviolet (UV) radiation affects the extracellular matrix (ECM) of the human skin. The small leucine-rich repeat protein fibromodulin interacts with type I and II collagen fibrils, thereby affecting ECM assembly. The aim of this study was to evaluate whether short wave UV (UVB) or long wave UV (UVA) irradiation influences fibromodulin expression. Exponentially growing human fibroblasts (IMR-90 cells) were exposed to increasing doses of UVB (2.5-60 mJ/cm(2)) or UVA (0.5-10 J/cm(2)). After UV irradiation fibromodulin, p21 and GADD45 levels were evaluated as well as cell viability, reactive oxygen species formation (ROS) and DNA damage. We found that fibromodulin expression: (i) increased after UVB and UVA irradiation; (ii) was 10-fold higher after UVA (10 J/cm(2)) versus 5-fold with UVB (10 mJ/cm(2)); (iii) correlated with reactive oxygen species formation, particularly after UVA; and (iv) was linked to the DNA damage binding protein (DDB1) translocation in the nucleus, particularly after UVB. These results further suggest that the UV-induced fibromodulin increase could counteract the UV-induced connective tissue damage, promoting the assembly of new collagen fibrils.

Proteoglycan synthesis in bovine articular cartilage explants exposed to different low-frequency low-energy pulsed electromagnetic fields.

OBJECTIVE: To investigate the role of pulsed electromagnetic field (PEMF) exposure parameters (exposure length, magnetic field peak amplitude, pulse frequency) in the regulation of proteoglycan (PG) synthesis of bovine articular cartilage explants. METHODS: Bovine articular cartilage explants were exposed to a PEMF (75 Hz; 2 mT) for different time periods: 1, 4, 9, 24 h. Then, cartilage explants were exposed for 24 h to PEMFs of different magnetic field peak amplitudes (0.5, 1, 1.5, 2 mT) and different frequencies (2, 37, 75, 110 Hz). PG synthesis of control and exposed explants was determined by Na2-35SO4 incorporation. RESULTS: PEMF exposure significantly increased PG synthesis ranging from 12% at 4 h to 17% at 24 h of exposure. At all the magnetic field peak amplitude values, a significant PG synthesis increase was measured in PEMF-exposed explants compared to controls, with maximal effect at 1.5 mT. No effect of pulse frequency was observed on PG synthesis stimulation. CONCLUSIONS: The results of this study show the range of exposure length, PEMF amplitude, pulse frequency which can stimulate cartilage PG synthesis, and suggest optimal exposure parameters which may be useful for cartilage repair in in vivo experiments and clinical application.

The effect of irradiation and hyperbaric oxygenation (HBO) on extracellular matrix of the condylar cartilage after mandibular distraction osteogenesis in the rabbit.

The effects of irradiation and hyperbaric oxygenation (HBO) on the extracellular matrix of condylar cartilage after mandibular distraction were evaluated. Unilateral distraction was performed on 19 rabbits. Five study groups were included: control, low- and high-dose irradiation, and low- and high-dose irradiation groups with HBO. Additionally, four temporomandibular joints (TMJ) were used as control material. The high-dose irradiated animals were given in the TMJ 22.4 Gy/4 fractions irradiation (equivalent to 50 Gy/25 fractions). Low-dose irradiation group received a 2.2 Gy dosage. Two groups were also given preoperatively HBO 18 x 2.5ATA x 90 min. After a two-week distraction period (14 mm lengthening) and four-week consolidation period the TMJs were removed. Proteoglycan (PG) distribution of the extracellular matrix was evaluated using safranin O staining and collagen I and II using immunohistochemistry. The organization of fibrillar network was studied by polarized light microscopy. On the operated side of the control group and on the unoperated side in all, except for high-dose irradiated group, PG distribution and fibrillar network were normal appearing. In the irradiated groups, with or without HBO, the cartilaginous layer was partially or totally devoid of PG and the network structure was severely damaged. In conclusion, irradiation in conjunction with the pressure applied by distraction causes severe damage to extracellular matrix of condylar cartilage.

Fibromodulin gene transcription is induced by ultraviolet irradiation, and its regulation is impaired in senescent human fibroblasts.

Cells undergoing replicative senescence display an altered pattern of gene expression. Senescent fibroblasts show significant changes in the expression of mRNAs encoding extracellular matrix-remodeling proteins; among these mRNAs, the mRNA encoding fibromodulin is highly decreased in these cells. To understand the molecular basis of this phenomenon, we explored the regulatory mechanisms of the human fibromodulin gene. We found that fibromodulin gene promoter contains a cis-element, crucial for its basal expression, that forms a DNA-protein complex when exposed to nuclear extracts from exponentially growing human fibroblasts and not to extracts from cells undergoing senescence by repeated in vitro passages or by mild oxidative stress. The purification of this complex showed that it contains the damage-specific DNA-binding protein DDB-1. The latter is known to be induced by UV irradiation; therefore we checked whether fibromodulin gene promoter is regulated upon the exposure of the cells to UV rays. The results showed that, in exponentially growing fibroblasts, the promoter efficiency is increased by UV irradiation and the DDB-1-containing complex is robustly enriched in cells exposed to UV light. Accordingly, in these experimental conditions the endogenous fibromodulin mRNA accumulates to very high levels. On the contrary, senescent cells did not show any activation of the fibromodulin gene promoter, any induction of the DDB-1-containing complex, or any accumulation of fibromodulin mRNA. These phenomena are accompanied in senescent cells by a decrease of the UV-damaged DNA binding activity.

Exogenous modulation of TGF-beta(1) influences TGF-betaR-III-associated vascularization during wound healing in irradiated tissue.

BACKGROUND AND PURPOSE: Following preoperative radiotherapy prior to ablative surgery of squamous epithelial cell carcinomas of the head and neck region, wound-healing disorders occur. Previous experimental studies showed altered expression of transforming growth factor-(TGF-)beta isoforms following surgery in irradiated graft beds. Altered levels of TGF-beta(1) are reported to promote fibrosis and to suppress vascularization during wound healing, whereas expression of TGF-beta receptor-III (TGF-betaR-III) is associated with vascularization. The aim of the study was to analyze the influence of anti-TGF-beta(1) treatment on TGF-betaR-III-associated vascularization in the transition area between irradiated graft bed and graft. MATERIAL AND METHODS: Wistar rats (male, weight 300-500 g) underwent preoperative irradiation of the head and neck region with 40 Gy (four fractions of 10 Gy each; n = 16 animals). A free myocutaneous gracilis flap taken from the groin was then transplanted to the neck in all rats. The time interval between operation and transplantation was 4 weeks. Eight animals received 1 micro g anti-TGF-beta(1) into the graft bed by intradermal injection on days 1-7 after surgery. On days 3, 7, 14, 28, 56, and 120, skin samples were taken from the transition area between transplant and graft bed and from the graft bed itself. Immunohistochemistry was performed using the ABC-POX method to analyze the TGF-betaR-III and E-selectin expression. Histomorphometry was performed to analyze the percentage and the area of positively stained vessels. RESULTS: A significantly higher expression of TGF-betaR-III was seen in the irradiated and anti-TGF-beta(1)-treated graft bed in comparison to the group receiving preoperative irradiation followed by transplantation alone. The percentage of TGF-betaR-III positively staining capillaries from the total amount of capillaries in the anti-TGF-beta(1)-treated graft bed was higher than in the group irradiated only. The total area of capillaries was also higher in the TGF-beta(1)-treated group. CONCLUSION: Neutralizing of TGF-beta(1) activity in irradiated tissue undergoing surgery leads to a higher expression of TGF-betaR-III and increased vascularization. TGF-betaR-III seems to be associated with newly formed blood vessels during neovascularization in wound healing.

Effects of electromagnetic fields on proteoglycan metabolism of bovine articular cartilage explants.

Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of interleukin-1beta (IL-1beta) in the culture medium. Proteoglycan synthesis and the residual PG tissue content resulted significantly higher in EMF-exposed explants than in controls, whereas no effect was observed on PG release and nitric oxide (NO) production. IL-1beta induced both a reduction in PG synthesis and an increase in PG release, related to a strong stimulation of NO production, which resulted in a net loss of tissue PG content. In IL-1beta-treated explants, EMF increased PG synthesis, whereas in spite of a slight stimulation of NO production EMF did not modify PG release. This resulted in the residual PG tissue content being maintained at the control level. In both experimental conditions, the effects of EMF were associated with an increase in lactate production. The results of our study show that EMFs are able to promote anabolic activities and PG synthesis in bovine articular cartilage explants. This effect also is maintained in the presence of IL-1beta, thus counteracting the catabolic activity of the cytokine. Altogether, these data suggest that EMF exposure exerts a chondroprotective effect on articular cartilage in vitro.

Search for genes responsible for UV susceptibility of human cells: involvement of syndecan-1 in UV resistance.

UVr-1 UV-resistant cells were established from UV-sensitive human RSa cells. We looked for genes expressed differentially between UVr-1 and RSa cells using PCR-based mRNA differential display to elucidate the molecular mechanisms underlying UV resistance. The transcription levels of syndecan-1 mRNA were increased in UVr-1 cells compared with those of RSa cells. Syndecan-1 is a transmembrane heparan sulfate proteoglycan and associates with cell adhesion and the cytoskeleton. Flow cytometric analysis using anti-syndecan-1 monoclonal antibody revealed that syndecan-1 was more abundant in UVr-1 cells than in RSa cells. The MTT method revealed that UVr-1 cells treated with the antibody showed higher sensitivity to UV cell killing than mock-treated cells. Studies using antisense oligonucleotides for syndecan-1 showed that antisense-treated UVr-1 cells became sensitive to UV cell killing. Thus, syndecan-1 might be involved in UV resistance in UVr-1 cells.

Pulsed electromagnetic fields influence hyaline cartilage extracellular matrix composition without affecting molecular structure.

Pulsed electromagnetic fields (PEMF) influence the extracellular matrix metabolism of a diverse range of skeletal tissues. This study focuses upon the effect of PEMF on the composition and molecular structure of cartilage proteoglycans. Sixteen-day-old embryonic chick sterna were explanted to culture and exposed to a PEMF for 3 h/day for 48 h. PEMF treatment did not affect the DNA content of explants but stimulated elevation of glycosaminoglycan content in the explant and conserved the tissue's histological integrity. The glycosaminoglycans in sterna exposed to PEMF were indistinguishable from those in controls in their composition of chondroitin sulfate resulting from chondroitinase ABC digestion. Specific examination with [35S]-sulfate labels showed that PEMF treatment significantly suppressed both the degradation of pre-existing glycosaminoglycans biosynthetically labeled in ovo and the synthesis of new [35S]-sulfated glycosaminoglycans. The average size and aggregating ability of pre-existing and newly synthesized [35S]-sulfated proteoglycans extracted with 4 M guanidinium chloride from PEMF-treated cartilage explants were identical to controls. The chain length and degree of sulfation of [35S]-sulfated glycosaminoglycans also were identical in control and PEMF-treated cultures. PEMF treatment also reduced the amount of both unlabeled glycosaminoglycans and labeled pre-existing and newly synthesized [35S]-sulfated glycosaminoglycans recovered from the nutrient media. [35S]-Sulfated proteoglycans released to the media of both control and PEMF-treated cultures were mostly degradation products although their glycosaminoglycan chain size was unchanged. These results demonstrate that exposure of embryonic chick cartilage explants to PEMF for 3 h/day maintains a balanced proteoglycan composition by down-regulating its turnover without affecting either molecular structure or function.

In situ fluorescence spectroscopic studies on bovine cornea.

The cornea is a transparent ocular tissue and its transparency is thought to be a result of intramolecular interactions and the supramolecular organization of its protein constituents. We have studied the intrinsic fluorescence properties of intact bovine corneas and compared these with that of the opaque sclera. It was observed that with increasing excitation wavelengths the emission maxima shifted toward the red edge exhibiting the phenomenon of red edge excitation shift, which is indicative of immobilization of the constituent fluorophores. The magnitude of the shift increased after photodamage by irradiation at 295 nm. Many of the spectral characteristics of the cornea are shown to be due to its proteoglycans, which show surprisingly significant red edge excitation shift in solution.

Effects of ionizing radiation on the size distribution of proteoglycan aggregates synthesized by chondrocytes in agarose.

Effects of ionizing radiation on the structure and size-distribution of newly synthesized proteoglycan aggregates are studied in-vitro. Chondrocytes, isolated from embryonic chick sterna, are cultured for 7 days in a tri-dimensional agarose system. Single radiation doses of 10, 20 and 50 Gy are given before starting the culture. Digestion of the artificial agarose matrix liberates the newly synthesized proteoglycans. Spreading with cytochrome C allows electron microscopic investigations of the individual, newly synthesized molecules. The structure of aggregates synthesized by control and irradiated chondrocytes is comparable. However, radiation causes alterations in the size-distributions of the aggregate-populations. For the control cultures, an average aggregate contains 27 aggrecans per aggregate. 34 pro mille of the molecules contain more than 100 aggrecans per aggregate. Irradiation with 10 Gy doesn't cause alterations. With radiation doses of 20 and 50 Gy, an average molecule contains 20 aggrecans. Only about 9 pro mille of the aggregates contain more than 100 aggrecans. Stimulation of lysosomal activity after irradiation could explain the observed alterations.

Alterations of proteoglycans in ultraviolet-irradiated skin.

The effect of UVB exposure on the distribution and synthesis of dermal proteoglycans was measured in the skin of hairless mice. Two groups of mice were included: one was irradiated for 10 weeks; the other was kept as control. After intraperitoneal injection of sodium 35-S-sulfate, punch biopsies were taken for histology and proteoglycans were extracted from the remaining skin with 4 M guanidinium chloride, containing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (0.5%, weight per volume). Following proteolytic digestion, the glycosaminoglycan constituents were isolated and analyzed by quantitative cellulose acetate electrophoresis and enzymatic digestibility. Under the influence of UVB radiation, newly synthesized proteoglycans measured by 35SO4 uptake increased as much as 60%. In addition, the irradiated skin had a higher average content of proteoglycan than had control skin (4981 micrograms vs 4134 micrograms/g dry weight). This could be ascribed to an increase in heparin (1400 vs 533 micrograms/g dry weight) and heparan sulfate (472 vs 367 micrograms/g dry weight), whereas no change in the concentration of hyaluronic acid (1243 vs 1372 micrograms/g dry weight) and dermatan sulfate (1866 vs 1863 micrograms/g dry weight) was observed. The irradiated animals also exhibited a marked increase in the synthesis of heparan sulfate and heparin (62% and 71%, respectively). These results demonstrate that chronic doses of UVB altered proteoglycan metabolism through both quantitative and qualitative changes.

The irradiation effect on the initial mechanical properties of meniscal grafts.

In this study, the effect of sterilization was investigated on the mechanical properties of rabbit's meniscal grafts. The processes of freezing and irradiation were carried out and their effects upon the elastic and viscoelastic properties measured using a tensile testing machine and an indentation test. It was found that gamma irradiation, at dosages commonly used for sterilizing grafts, had a significant adverse influence on the elastic and viscous response of the grafts. Freezing led also to a small significant decrease in elastic stiffness but no change in failure strength. It is concluded that irradiation sterilization is unsuitable for clinical use and other techniques should be used whenever possible.

Cell-cell matrix interactions in induced lung injury. I. The effects of X-irradiation on basal laminar proteoglycans.

The lungs of male LAF1/J mice were locally irradiated with graded doses of radiation ranging from 5 to 13 Gy. The animals were sacrificed at 1 hr, 1 day, 1 week, 4 weeks, and 12 weeks postirradiation (PI), fixed with Ruthenium Red/Triton X-100 for demonstration of basal laminar anionic sites, and processed for electron microscopy. Sham-irradiated (0 Gy, 0 time) and untreated control groups were also processed. Sections of lungs were examined ultrastructurally and changes in both alveolar and capillary basal laminar anionic sites quantitated. A marked decrease in the number of basal laminar anionic sites was noted 1 hr PI in both alveolar and capillary basal laminae at all dose levels. The decline continued to 1 week for doses of 13 Gy and more gradually to 4 weeks following doses of 5 and 9 Gy, when the number of sites began to increase. By 12 weeks animals receiving 13 Gy were approaching normal levels while those receiving 5 or 9 Gy remained subnormal. The potential effects of the loss of proteoglycans with radiation on lung basal laminar permeability and cell-extracellular matrix interactions are discussed.

Ultrastructure of hydrated proteoglycans using a pulsed plasma source.

During the past 10 years, attempts have been made to examine hydrated biological specimens by using wet chambers (at ambient temperature) [1-3] or cold stages (at -30 degrees C and below) during electron microscopic examination. Obtaining sufficient contrast of unstained hydrated biological samples has proven a considerable problem using both of these methods. Many fragile biological specimens, when examined hydrated, frozen or dried, are severely damaged by the electron beam and cannot be imaged by conventional scanning or transmission electron microscopy. In order to increase specimen contrast and eliminate electron beam induced trauma to the specimen, we have developed a wet-cell [4], which when used in concert with a pulsed plasma soft X-ray source, provides high contrast contact replicas of totally hydrated, unstained biological specimens. Although it has been postulated that hydrated unstained samples can be imaged by soft X-ray contact microscopy [5-7], to date there has been little success due to cell movement or degradation of the wet sample during the long exposure period necessary for an adequate imaging dose [8]. With the pulsed plasma source described in this study we have been able to use exposure times of approximately 40-60 ns while maintaining the sample in its hydrated state at atmospheric pressure. The resultant contact replicas exhibit good contrast and better than 30 nm spatial resolution when examined by conventional scanning electron microscopy.

Imaging unstained proteoglycan aggregates by soft x-ray contact microscopy.

Soft X-ray contact microscopy is a relatively new form of ultrastructural imaging, having better than 6 nm resolution and being uniquely well suited for the examination of fragile, unstained biological specimens. The biological specimen placed on a layer of photoresist and exposed to soft X-rays (1-10 nm lambda) of a specific wavelength or broad band. After X-ray exposure, the specimen is removed from the photoresist and the latter chemically developed. When the developed replica is examined by high resolution scanning electron microscopy, the fine structure of the original biological specimen is faithfully reproduced. Since the soft X-ray replica is initially formed due to the differential absorption of the incident X-rays by the biological specimen, the resultant contact replica also reveals information about the elemental composition of the sample. This paper presents our application of this new technique for the study of the proteoglycans, the complex polyanionic macromolecules comprising the gel phase in the matrix of mammalian cartilage.

Extracellular matrix metabolism by chondrocytes. III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture.

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-beta-D-xyloside, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.