chondroitin-sulfates and copper-phthalocyanine

Recently, gene-targeted strains of mice with null mutations for specific proteoglycans (PGs) have been used for investigations of the functional role of these molecules. In the present study, the corneal stroma of the mouse was examined to provide some baseline PG morphologies in this species.. Monoclonal antibodies to specific glycosaminoglycan (GAG) chain sulfation patterns were used to characterize PG composition in corneal extracts by SDS-PAGE and Western blot analysis and to identify their tissue distribution by immunofluorescence microscopy. PGs were also visualized by transmission electron microscopy after contrast enhancement with cationic dye fixation.. Western blot analysis of pooled corneal extracts and immunofluorescence of tissue sections identified 4-sulfated, but not 6-sulfated, chondroitin sulfate/dermatan sulfate (CS/DS). Keratan sulfate (KS) was present only as a low-sulfated moiety. Electron microscopic histochemistry disclosed a complex array of corneal PGs present as (1) fine filaments radiating from collagen fibrils, and (2) elongate, straplike structures, running either along the fibril axis or weaving across the primary fibril orientation. These large structures were digested by chondroitinase ABC, but not by keratanase.. KS in the mouse is predominantly undersulfated and generates an immunostaining pattern that differs from that observed in corneas of other mammalian species thus far investigated. The mouse cornea resembles other mammalian corneas in the presence of filamentous arrays of small, collagen-associated stromal PGs visualized by cationic dye staining. However, large dye-positive structures with a CS/DS component are also present and appear to be unique to the cornea of this species.

Topics: Animals; Blotting, Western; Chondroitin Sulfates; Coloring Agents; Corneal Stroma; Dermatan Sulfate; Electrophoresis, Polyacrylamide Gel; Indoles; Keratan Sulfate; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microscopy, Fluorescence; Organometallic Compounds; Proteoglycans; Staining and Labeling

To determine whether aging vitreous collagen fibrils undergo ultrastructural changes that might underlie vitreous liquefaction and posterior vitreous detachment.. Vitreous collagen fibrils from 21 human subjects (age range, 3-89 years) and from bovine eyes were isolated on electron microscopy grids. Cupromeronic blue labeling in the presence of 0.3 M MgCl(2) and immunogold labeling for collagen types II and IX were analyzed by transmission electron microscopy.. Aging was associated with marked changes on the surface of human vitreous collagen fibrils, including an exponential loss of type IX collagen along with its chondroitin sulfate side-chains (half-life, 11 years) and a fourfold increase in the exposure of type II collagen.. Despite being a minor component of vitreous collagen fibrils, type IX collagen, probably by virtue of its chondroitin sulfate side-chains, shields type II collagen from exposure on the fibril surface. With aging, this shielding diminishes, resulting in the surface exposure of "sticky" type II collagen and thus predisposing the vitreous collagen fibrils to fusion. These changes could underlie vitreous liquefaction and weakening of vitreoretinal adhesion.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Aging; Animals; Blotting, Western; Cattle; Child; Child, Preschool; Chondroitin Sulfates; Collagen Type II; Collagen Type IX; Humans; Immunohistochemistry; Indoles; Male; Microscopy, Immunoelectron; Middle Aged; Organometallic Compounds; Vitreous Body

Supramolecular organization involving a polyanionic glycan in a bacterial capsule (hyaluronan, HA, in streptococcal capsules) is revealed, for the first time, by electron-histochemical methodology previously used to elucidate ultrastructure in extracellular matrix. Capsular HA filaments thereby revealed closely resemble aligned linear structures demonstrated by similar technology in HA solution. These parallel arrays, spontaneously formed, are based on HA tertiary structures (beta sheet-like) which are stabilized by hydrophobic and hydrogen bonds. HA tertiary structures in aqueous solutions resist shear stress as shown by rheo-NMR. Thus, supramolecular HA wrapping covering many cells probably stabilizes chains of bacteria. Streptococci possibly templated the ordered structures since eukaryotic B6 cell HA did not produce similarly organized envelopes. Supramolecular organization in streptococcal and pneumococcal capsules are compared. Their glycans are very similar but the potential for HA-like tertiary structures is not present in the pneumococcal type 3 polysaccharide and chains of cells are not formed to the same extent by pneumococci. We suggest that the streptococcal capsule exemplifies a simple extracellular matrix analogous to those in animal connective tissues, which contain glycans (chondroitin, keratan, and dermochondan sulfates) of the HA family, capable of undergoing aggregation to similar tertiary structures.

Topics: Bacterial Capsules; Carbohydrate Conformation; Chondroitin Sulfates; Hyaluronic Acid; Hyaluronoglucosaminidase; Indicators and Reagents; Indoles; Organometallic Compounds; Polysaccharides; Streptococcus

We investigated histo-chemically the composition and distribution of proteoglycans in the trabecular tissue of eyes with neovascular glaucoma. Cupromeronic blue in combination with a series of enzyme digestions and nitrous acid treatment were used. The spaces between the trabecular beams were lined by a single layer of vascular endothelium and were filled with red blood cells. A basal lamina and microfibrils were detected just beneath the newly formed vascular endothelial cells. Chondroitin-sulfate- and dermatan-sulfate-type proteoglycans were present in association with collagen fibrils in the extracellular matrix. Heparan-sulfate-type proteoglycans were present in association with the basal lamina of both the vascular endothelial cells and the trabecular cells. It is unlikely that these abnormalities in the type or distribution of proteoglycans in the trabecular meshwork have a major role in the pathogenesis of glaucoma.

Topics: Adult; Basement Membrane; Chondroitin Sulfates; Chondroitinases and Chondroitin Lyases; Collagen; Coloring Agents; Copper; Dermatan Sulfate; Extracellular Matrix; Glaucoma, Neovascular; Heparitin Sulfate; Humans; Indoles; Male; Middle Aged; Organometallic Compounds; Trabecular Meshwork

Glycosaminoglycans (GAGs) are essential components of the extracellular matrix contributing to the mechanical properties of connective tissues as well as to cell recognition and growth regulation. The ultrastructural localization of GAGs in porcine lung was studied by means of the dye Cupromeronic Blue in the presence of 0.3 M MgCl2 according to Scott's critical electrolyte concentration technique. GAGs were observed in locations described as follows. Pleura: Dermatan sulphate (DS) and chondroitin sulphate (CS) attached in the region of the d-band of collagen fibrils, interconnecting the fibrils; heparan sulphate (HS) at the surface of elastic fibers and in the basement membrane of the mesothelium and blood vessels. Bronchial cartilage: Abundant amounts of GAGs were observed in three zones: pericellular, in the intercellular matrix and at the perichondrial collagen. By enzyme digestion a superficial cartilage layer with predominantly CS could be distinguished from a deep zone with CS and keratan sulphate. The structure of the large aggregating cartilage proteoglycan was confirmed in situ. Airway epithelium: HS at the whole surface of cilia and microvilli and in the basement membrane of the epithelial cells. Alveolar wall: CS/DS at collagen fibrils, HS at the surface of elastic fibers and in the basement membranes of epithelium and endothelium.

Topics: Animals; Basement Membrane; Bronchi; Cartilage; Chondroitin Sulfates; Collagen; Coloring Agents; Connective Tissue; Dermatan Sulfate; Epithelium; Extracellular Matrix; Glycosaminoglycans; Heparitin Sulfate; Indoles; Lung; Microscopy, Electron; Organometallic Compounds; Pleura; Pulmonary Alveoli; Pulmonary Circulation; Swine

Human gingiva was stained with cupromeronic blue according to Scott's critical electrolyte concentration technique in order to localize glycosaminoglycans (GAG) in the electron microscope. Identification was performed by digestion with chondroitinase AC, ABC and heparinase. The GAG were localized in three compartments of the connective tissue: the supra-alveolar fiber apparatus, the loose connective tissue and the basement membranes. In the supra-alveolar fiber apparatus, consisting mainly of densely packed parallel collagen fibrils, dermatan sulfate GAG are regularly attached to the d-band of the collagen fibrils. The precipitates (6-7 nm in diameter) aggregate to thicker precipitates (up to 16 nm), thus possibly providing stability to the fiber system. In the loose connective tissue with sparse collagen fibrils dermatan and chondroitin sulfate GAG form very large precipitates (up to 30 nm in diameter and 400 nm length) which interconnect the few collagen fibrils. The basement membranes of the epithelium and capillary endothelium contain heparan sulfate GAG as fine precipitates (4-6 nm in diameter) which form a meshwork. These findings are consistent with the Scott model (1) for the interactions among glycans and glycans and collagen fibrils in connective tissues.

Topics: Basement Membrane; Chondroitin Sulfates; Collagen; Coloring Agents; Connective Tissue; Dermatan Sulfate; Endothelium, Vascular; Gingiva; Glycosaminoglycans; Histocytochemistry; Humans; Indoles; Organometallic Compounds; Protein Structure, Tertiary; Tissue Distribution

The structural organization of integral and associated components of the ciliary zonule is still not fully understood. The present study is to localize and characterize the proteoglycans associated with the ciliary zonule of the rat eye by Cuprolinic blue (CB) staining and immunocytochemistry. After CB staining, the proteoglycans appeared as electron dense elongated rodlets and were localized with the zonular fibers. They were seen lying on the periphery of the zonular fibers or along the length of the individual fibrils. Most of the CB rodlets had a size of 60-170 nm long (average 130 nm) and 25 nm wide. Smaller CB rodlets measuring 25-60 nm long (average 45 nm) and 12 nm wide were sometimes found associated with the individual zonular fibrils. The CB rodlets were removed after chondroitinase ABC or chondroitinase AC treatment, but were resistant to heparitinase, nitrous acid, keratanase or Streptomyces hyaluronidase digestions. The ciliary zonule was also immunostained with three monoclonal antibodies: 2-B-6 specific for chondroitin 4-sulfate, 3-B-3 for chondroitin 6-sulfate and 1-B-5 for unsulfated chondroitin, using indirect immunoperoxidase or immuno-colloidal gold methods. The zonular fibers were immunoperoxidase stained and immunogold labeled by 2-B-6, but were not reactive to 3-B-3 and 1-B-5. The results demonstrate that chondroitin sulfate proteoglycan is associated with the ciliary zonule of the rat eye.

Topics: Animals; Chondroitin Sulfates; Ciliary Body; Enzymes; Eye; Histocytochemistry; Immunoenzyme Techniques; Immunohistochemistry; Indicators and Reagents; Indoles; Magnesium Chloride; Male; Microscopy, Electron; Nitrous Acid; Organometallic Compounds; Proteoglycans; Rats; Rats, Sprague-Dawley

The time course of development of extracellular matrix (ECM) in experimentally induced fibrosis (thioacetamide administration followed for 12 weeks or bile duct ligation for 8 weeks) in adult rats was examined by light and electron microscopy, using Alcian blue or Cupromeronic blue staining for sulphated proteoglycans (PGs) in critical electrolyte concentration techniques. Proteodermatan sulphate (PDS) was regularly observed at the gap zone of the collagen fibrils. Morphometry of uranyl acetate-stained collagen fibrils, polarity of their banding patterns (a-e), statistics of d/e band occupancies by PDS, and lengths and thicknesses of PG filaments were quantified. Biochemical analyses showed that the ECM components collagen, hyaluronan, chondroitin and dermatan sulphates increased by 5-10 fold, roughly in parallel, as did heparan sulphate and DNA. Water and lipid contents also increased sharply. Thioacetamide treatment was much slower than bile duct ligation in producing fibrotic changes of equal severity. Sulphation of anionic glycosaminoglycans (AGAGs) decreased with increasing severity of fibrosis. Biochemical and ultrastructural methods correlated well. The large increase in dermatan sulphate was quantitatively as expected, given that it is collagen fibril surface-associated, and there was an increase of collagen content together with a decrease in fibril thicknesses. The increase in DNA reflected the marked increase in cell numbers in fibrotic livers. The chemical morphology of the new connective tissue closely resembled that in e.g. developing young tendon, in that fibrils were thinner, and AGAG levels were higher. The collagen fibrils were often disarranged, rather than ordered and parallel as in normal ECM. No other indication of abnormality in the new ECM was obtained.

Topics: Alcian Blue; Animals; Chondroitin Sulfates; Collagen; Connective Tissue; Dermatan Sulfate; DNA; Extracellular Matrix; Female; Hyaluronic Acid; Indoles; Liver Cirrhosis, Experimental; Male; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Rats; Rats, Sprague-Dawley; Staining and Labeling

We evaluated histochemically the distribution of proteoglycans in the trabecular tissue of goniodysgenetic (developmental) glaucoma. Nine trabecular tissue specimens obtained at trabeculectomy from seven patients with goniodysgenetic glaucoma were stained with either cuprolinic blue or cupromeronic blue in combination with a series of enzyme and nitrous acid treatments. Within the extracellular matrix of the trabecular meshwork, many cupromeronic blue- or cuprolinic blue-positive filaments were observed in association with collagen fibrils, basal lamina, and basal lamina-like material. The extracellular matrices of elastin-like fibers, fine fibrillar materials, and fine granular materials were free from any reaction products. The enzyme and nitrous acid treatments disclosed that the reaction products associated with collagen fibrils represented both chondroitin sulfate and dermatan sulfate types, while those with basal lamina and basal lamina-like material represented heparan sulfate-type proteoglycans. Extensive accumulations of basal lamina-like material contained a great deal of heparan sulfate-type proteoglycans in the thick subcanalicular tissue of goniodysgenetic glaucoma. These results indicate that the class and distribution of proteoglycans in the goniodsygenetic trabecular tissues are virtually the same as that in the normal tissues. However, the large accumulation of basal lamina-like material with heparan sulfate-type proteoglycans can be one of the causes of the intraocular pressure increase in goniodysgenetic glaucoma.

Topics: Adolescent; Adult; Anterior Eye Segment; Child; Child, Preschool; Chondroitin Sulfates; Dermatan Sulfate; Glaucoma; Heparitin Sulfate; Humans; Indicators and Reagents; Indoles; Infant; Male; Organometallic Compounds; Trabecular Meshwork; Trabeculectomy

Sulfated proteoglycans in the lamina cribrosa of the optic nerve head from individuals aged 2 months, 18 months, and 23, 35, 44, 55, 67, 74, and 88 years were studied by electron microscopy after cuprolinic blue dye binding. Within the cores of the laminar plates, cuprolinic blue-positive chondroitin/dermatan sulfate proteoglycan filaments of different sizes were found associated with collagen fibers. In addition, small punctate and filamentous structures that represented heparan sulfate proteoglycan molecules were associated with the basal laminae of astrocytes and blood vessels. In the eyes of older individuals, the chondroitin/dermatan sulfate and heparan sulfate proteoglycan filaments were found to be shorter than those in younger persons. A mild decline with aging in the diameter of the filaments was also noted. Our findings illustrate the age-related changes in the proteoglycans in the human lamina cribrosa, which may help explain why the optic nerve head is more susceptible to damage with aging.

Topics: Adult; Aged; Aged, 80 and over; Aging; Chondroitin Sulfates; Coloring Agents; Dermatan Sulfate; Extracellular Matrix; Heparitin Sulfate; Histocytochemistry; Humans; Indoles; Infant; Middle Aged; Optic Disk; Organometallic Compounds

The proteoglycans (PGs) in the guinea pig seminal vesicle were demonstrated ultrastructurally by both cuprolinic blue (CB) and ruthenium red (RR) staining. The PGs appeared as electron-dense granules with RR, but were filamentous following CB staining using the critical electrolyte concentration method. Three major types of PGs (T1, T2, T3) have been described according to their different locations and sizes. T1 filaments were short and were found mostly on both sides of the lamina densa of the basal lamina of the glandular epithelium (40-60 nm long) and also on the basal laminae of smooth muscle cells and capillary endothelial cells (20-30 nm long). In the epithelial basal lamina they were regularly spaced at an interval of 40-60 nm. T1 filaments in the lamina densa were smaller and more randomly distributed. Cytochemical characterisation of these PGs by various GAG degrading enzymes showed that T1 PGs are rich in heparan sulphate. T2 filaments were 30-40 nm long and closely associated with the collagen fibrils. They were arranged perpendicular to the long axis of collagen fibrils, also at intervals of about 60 nm. T2 filaments were removed by chondroitinase (Ch)-ABC, Ch-ABC plus Streptomyces (S)-hyaluronidase and pronase, but resistant to nitrous acid, heparitinase, heparinase, neuraminidase and S-hyaluronidase. These show that T2 filaments are rich in dermatan sulphate. T3 filaments (60-100 nm) were widely distributed in the stroma at sites such as the interstitial spaces of the lamina propria, the reticular layer below the basal lamina, around individual collagen fibrils or bundles of such fibres, and on the cell surfaces of fibroblasts. The T3 filaments were removed by Ch-ABC, Ch-AC and pronase but were resistant to heparitinase, heparinase, S-hyaluronidase, neuraminidase and nitrous acid. They are therefore rich in chondroitin sulphate.

Topics: Animals; Basement Membrane; Chondroitin Sulfates; Dermatan Sulfate; Epithelium; Guinea Pigs; Heparitin Sulfate; Histocytochemistry; Indoles; Male; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Ruthenium Red; Seminal Vesicles; Staining and Labeling

The sulfated proteoglycans in the normal human lamina cribrosa were studied by electron microscopy after cuprolinic blue dye binding. Within the cores of the laminar plates, three types of cuprolinic blue-positive proteoglycan filaments with different sizes were associated with collagen fibers. These filaments, which were partially sensitive to chondroitinase AC and chondroitinase B, were completely removed by chondroitinase ABC and were identified as chondroitin/dermatan sulfate proteoglycans. In addition, small punctate and filamentous structures that stained with cuprolinic blue were associated with the basal laminae of astrocytes and blood vessels. Enzyme chondroitinase ABC had no effect, but heparinase digested all of these basement membrane-associated structures, indicating that they represented heparan sulfate proteoglycan molecules. Keratanase did not affect any of the cuprolinic blue-positive materials. This investigation illustrates the ultrastructural distribution and morphology of proteoglycans in the human lamina cribrosa and provides baseline information for future studies regarding the roles of proteoglycan molecules in diseases such as glaucoma.

Topics: Aged; Basement Membrane; Chondroitin Lyases; Chondroitin Sulfates; Coloring Agents; Copper; Dermatan Sulfate; Female; Glycoside Hydrolases; Heparitin Sulfate; Histocytochemistry; Humans; Indicators and Reagents; Indoles; Male; Middle Aged; Optic Disk; Organometallic Compounds

The distribution of sulfated proteoglycans in Bruch's membrane of the human eye was evaluated histochemically using Cupromeronic Blue in combination with specific enzyme digestions and nitrous acid treatment. Five distinct categories of filament-shaped profiles were present following staining with this dye. Type 1 (90 +/- 13 nm long and 7 +/- 1 nm in diameter) (mean +/- S.D.) and type 2 (43 +/- 7 nm long and 5 +/- 1 nm in diameter) filaments were associated with collagen fibrils in the inner and outer collagenous zones. Type 3 profiles (70 +/- 18 nm long and 8 +/- 1 nm in diameter) were present in two locations--along the cortical border of the central elastic zone and within the basal infoldings of the pigment epithelium. Type 4 (60 +/- 11 nm long and 6 +/- 1 nm in diameter) and type 5 (200 +/- 100 nm long and 100 +/- 50 nm in diameter) filaments were associated with the basal laminae of the retinal pigment epithelium and choriocapillaris. Chondroitinase AC treatment eliminated the staining of type 1 filaments. Chondroitinase ABC treatment eliminated the staining of both type 1 and type 2 filaments. Nitrous acid eliminated the staining of type 4 and type 5 filaments. Incubations with keratanase or hyaluronidase did not alter the staining of any filament type. Type 3 filaments were resistant to all enzyme digestions and nitrous acid treatment. These results are consistent with an interpretation that Bruch's membrane contains chondroitin sulfate, dermatan sulfate and heparan sulfate-type proteoglycans. Proteoglycans containing chondroitin sulfate (type 1) and dermatan sulfate (type 2) are associated uniquely with collagen fibrils. Heparan sulfate type proteoglycans (types 4 and 5) are associated with the basal lamina of the pigment epithelium and choriocapillaris. The identity of type 3 profiles, which were resistant to all enzyme and nitrous acid digestions employed, could not be established at this time.

Topics: Adolescent; Adult; Aged; Basement Membrane; Chondroitin Sulfates; Chondroitinases and Chondroitin Lyases; Choroid; Coloring Agents; Dermatan Sulfate; Female; Heparitin Sulfate; Humans; Indoles; Male; Microscopy, Electron; Middle Aged; Nitrous Acid; Organometallic Compounds

Ultrastructural localization of proteoglycans (PGs) in 1-week- to 2-year-old scar was determined by staining with cuprolinic blue dye (CBD) after specific enzymatic digestion of keratan sulfate (KS) glycosaminoglycans (GAGs) or chondroitin sulfate glycosaminoglycans (CSs). High critical electrolyte conditions were maintained for CBD-staining, specific for high-sulfated GAGs. Although KS was detected in the 1-week-old wound, no CBD-stained KS was seen in the anterior stroma adjacent to the wound. The CS was present throughout the 1-week-old wound and adjacent stroma, and PGs were biosynthetically 35SO4-labeled in normal stroma. Subsequently, radioactivity from labeled PGs in normal stroma adjacent to the wound moved into scar tissue during healing. Marked sensitivity of PGs to Chondroitinase ABC indicated an abundance of CS in 2-week-old scars. Punctate CBD-staining and immunohistochemical evidence suggested chemically altered KS is present in the 2-week-old anterior scar. The pattern of CBD-staining in 1- and 2-week scars, after chondroitinase treatment, suggested KS in the younger scar is similar to adult high-sulfated GAG, whereas KS in the 2-week scar contains primarily newly synthesized low-sulfated KS. The latter is consistent with previous immunochemical and biochemical analyses. Cytochemical and immunohistochemical evidence indicated that KS is not present in the 2-week-old posterior scar. By the week 8 of healing, CBD-stained KS was present throughout most of the scar, except along the posterior margin, consistent with earlier stages of healing. The CBD-stained structures in the first 8 weeks of healing were reminiscent of stained GAGs in normal developing cornea. This fetal-like CBD-staining pattern seen in scar, however, changed to that of the normal adult by the 2nd year of healing. The significance of these observations relate to our contention that healing adult cornea recapitulates some ontogenetic events of the normal cornea, and that the nonuniform distribution and chemical properties of GAGs in scar tissue are a function of the movement of existing proteoglycans and de novo synthesis of altered macromolecules.

Topics: Animals; Antibodies, Monoclonal; Chondroitin Sulfates; Cicatrix; Cornea; Glycosaminoglycans; Immunohistochemistry; Indoles; Keratan Sulfate; Organometallic Compounds; Proteoglycans; Rabbits; Wound Healing

Proteoglycans stained specifically with cuprolinic blue have been visualized in electron micrographs of bovine arterial tissue. Three differently sized proteoglycan-cuprolinic blue precipitates, designated as types I, II, and III, could be detected in the extracellular matrix. The precipitates could be distinguished by their length, width, area, topographical distribution, and their characteristic association with other matrix components. By taking into account the available biochemical data and the individual susceptibilities of the precipitates towards specific glycosaminoglycan-degrading enzymes, each type of proteoglycan-cuprolinic blue precipitate could be attributed to a proteoglycan population containing dermatan sulfate, chondroitin sulfate, or heparan sulfate as its main glycosaminoglycan component.

Topics: Animals; Arteries; Cattle; Chemical Precipitation; Chondroitin Sulfates; Collagen; Dermatan Sulfate; Elastin; Extracellular Matrix; Heparitin Sulfate; Indoles; Microscopy, Electron; Organometallic Compounds; Proteoglycans; Staining and Labeling