Localization of ferredoxin isoproteins in mesophyll and bundle sheath cells in maize leaf

Four ferredoxin isoproteins were identified in the C₄ plant Zea mays L. by analysis of extracts from leaves, mesocotyls, and roots of the young seedlings. The relative amounts of the isoproteins isolated from the photosynthetic and nonphotosynthetic organs were different. All the isoproteins were present in the leaves of green and etiolated plants, whereas two out of the four isoproteins were not detected in the roots or in the mesocotyls. During the greening of etiolated seedlings, the level of the two isoproteins unique to the leaf increased markedly. Analysis of the cellular and subcellular distribution of the two major leaf isoproteins showed that one isoprotein was present in the chloroplasts of both mesophyll and bundle sheath cells, whereas the other was only found in the chloroplasts of bundle sheath cells. This is the first report of the cell-specific expression of ferredoxin isoproteins in the leaves of a C₄ plant.     

Degradation of type IX collagen by matrix metalloproteinase 3 (stromelysin) from human rheumatoid synovial cells

The degradation of type IX collagen, a minor collagen in cartilage, was examined by treatment with three different types of matrix metalloproteinases (MMPs) purified from the culture medium of rheumatoid synovial cells. Neither MMP-1 (collagenase) nor MMP-2 (so-called 'gelatinase') could digest type IX collagen, but MMP-3 (stromelysin) readily degraded it into smaller fragments. This suggests that MMP-3 may be responsible for the pathological degradation and/or normal turnover of type IX collagen.     

Changes in proteoglycan composition during development of rat skin. The occurrence in fetal skin of a chondroitin sulfate proteoglycan with high turnover rate

Extraction of the skin of newborn rat yielded two populations of galactosaminoglycan-containing proteoglycan: a Mr = 111,000-200,000 dermatan sulfate proteoglycan (DS-PG) with a Mr congruent to 55,000 core glycoprotein and a Mr congruent to 10(6) chondroitin sulfate proteoglycan (CS-PGs) composed of two subpopulations with different size core-glycoproteins (Mr congruent to 480,000 and 520,000). Tryptic peptide mapping of chondroitinase-treated DS-PG and CS-PGs indicated that the peptide patterns observed with the two core molecules from CS-PGs were identical with each other but distinct from the peptide pattern of the DS-PG core molecule. It is likely therefore that the two forms of CS-PGs are derived from the same gene product by post-translational modification or partial degradation, but DS-PG is derived from a distinct gene product. Comparison of the concentration (hexuronate/DNA) of the proteoglycans in newborn and fetal rat skin showed an age-related change in proteoglycan composition; at 4 days before birth, the uronic acid proportions, DS-PG:CS-PGs, were about 14:1 and during the next 4 days, DS-PG increased 2.2-fold whereas CS-PGs decreased 4-fold. On a per DNA basis, the rate of [3H]serine incorporation into CS-PGs was 2.5 times the rate for DS-PG at 4 days before birth but decreased by 95% during the next 4 days. The rate for DS-PG also decreased but to a much lesser extent, so that by 2 days before birth, it began to exceed the rate for CS-PGs. The striking change in the concentration and labeling rate of CS-PGs can be interpreted either as a decrease of CS-PGs synthesis, or as an increase of CS-PGs breakdown, or both, a process which might be involved in the transition of extracellular matrix from a fetal type to a newborn or adult type.     

Isolation of two forms of basement membrane proteoglycans

Sequential extractions of the basement membrane producing Engelbreth-Holm-Swarm tumor yielded heparan sulfate proteoglycans with different size core proteins, but the same size heparan sulfate side chains. Saline, a nondenaturing solvent, extracted a small high density proteoglycan with a heterodisperse core protein of Mr = 95,000-130,000 whereas subsequent extraction with 7 M urea, a denaturing solvent, removed a large, low density proteoglycan with a Mr = 350,000-400,000 protein core. The denaturing conditions required for extraction of the large proteoglycan suggest that it interacts strongly with other basement membrane components. Antibodies to these proteoglycans cross-react with both proteoglycans, but the large proteoglycan has additional antigenic sites not present on the small proteoglycan. These proteoglycans may be derived from the same or similar gene products.     

The core molecule from type H proteoglycan. Release of mannose-containing oligosaccharides by digestion with N-oligosaccharide glycopeptidase.

Chick-embryo cartilage contains a unique set of proteoglycans. Type H proteoglycan (PG-H) is the most abundant, constituting over 90% of the total cartilage hexuronate. We previously showed that treatment of PG-H with chondroitinase ACII and keratanase yields a protein-enriched core molecule [PG(-CS,KS)] with enzymically modified linkage oligosaccharides of the chondroitin sulphate and keratan sulphate chains. We report here that further treatment of PG(-CS,KS) with pepsin and N-oligosaccharide glycopeptidase (almond glycopeptidase) released four distinct types of mannose-containing oligosaccharide. Two of them were shown to be: (Formula: see text). Of the mannose-containing glycopeptides formed by pepsin digestion, about 40% (as mannose) were resistant to N-oligosaccharide glycopeptidase. Since the resistant fraction was enriched in keratan sulphate remnants, it is suggest that the mannose-containing oligosaccharides in this fraction represent those located in a keratan sulphate-enriched region of PG-H.     

Isolation and characterization of a third proteoglycan (PG-Lt) from chick embryo cartilage which contains disulfide-bonded collagenous polypeptide

Chick embryo epiphyseal cartilage has been shown to contain three different proteoglycan species (PG-H, PG-Lb, and PG-Lt). This report is concerned with the purification and characterization of the third proteoglycan, PG-Lt. The proteoglycan can be separated from the other two by virtue of its low buoyant density in a CsCl density gradient and further purified by consecutive ion exchange and gel chromatography. The final preparation is composed of PG-Lt monomer and PG-Lt oligomer. The amino acid composition of PG-Lt is quite different from that of PG-H and PG-Lb and rather resembles that of collagens with respect to high content of glycine and high degrees of hydroxylation of proline and lysine. PG-Lt monomer is composed of disulfide-bonded subunits of Mr congruent to 120,000 and 190,000 as demonstrated by its gel electrophoretic behavior after reduction with 2-mercaptoethanol. The latter, but not the former, contains dermatan sulfate chains with glucuronic acid/iduronic acid residues and yields a protein-enriched core molecule of Mr congruent to 100,000 after digestion with chondroitinase ABC. Both of the protein subunits are completely digestible with bacterial collagenase. Immunofluorescence microscopic examination of cartilage tissues, using an antibody against PG-Lt, shows that this proteoglycan exists in both the cartilage matrix and perichondrial noncartilagenous region. When chondrocytes are plated onto tissue culture dishes, the antibody stains strands found on the cell surfaces and in the intercellular space of substrate-attached cell layers, suggesting that PG-Lt mediates cell-to-cell and cell-to-substrate contacts.     

Aortic endothelial cells synthesize a large chondroitin sulphate proteoglycan capable of binding to hyaluronate.

Confluent cultures of mouse aortic endothelial (END-D) were incubated with either [35S]methionine or 35SO4 2-, and the radiolabelled proteoglycans in media and cell layers were analysed for their hyaluronate-binding activity. The proteoglycan subfraction which bound to hyaluronate accounted for about 18% (media) and 10% (cell layers) of the total 35S radioactivity of each proteoglycan fraction. The bound proteoglycan molecules could be dissociated from the aggregates either by digestion with hyaluronate lyase or by treatment with hyaluronate decasaccharides. Digestion of [methionine-35S]proteoglycans with chondroitinase and/or heparitinase, followed by SDS/polyacrylamide-gel electrophoresis, indicated that the medium and cell layer contain at least three chondroitin sulphate proteoglycans, one dermatan sulphate proteoglycan, and two heparan sulphate proteoglycans which differ from one another in the size of core molecules. Among these, only the hydrodynamically large chondroitin sulphate species with an Mr 550,000 core molecule was shown to bind to hyaluronate. A very similar chondroitin sulphate proteoglycan capable of binding to hyaluronate was also found in cultures of calf pulmonary arterial endothelial cells (A.T.C.C. CCL 209). These observations, together with the known effects of hyaluronate on various cellular activities, suggest the existence of possible specialized functions of this proteoglycan subspecies in cellular processes characteristic of vascular development and diseases.     

Complex carbohydrates in cartilaginous and other tissues of cartilage matrix deficiency (cmd/cmd) mice as studied by light microscopic histochemical methods

Summary Mice homozygous for the autosomal recessive gene, cartilage matrix deficiency (cmd/cmd) are characterized by disproportionate dwarfism and cleft palate and are suffered from genetic failure in biosynthesis of cartilagetype proteoglycans. To investigate histochemical aspects of the defects in cartilaginous and other tissues of the mutant mice, complex carbohydrates in the tissues have been studied by methods of light microscopy. As a result of the present investigations, the intercellular matrix of cartilaginous tissues from the mutant was shown to exhibit weaker positive reactions for sulfated and acidic complex carbohydrates, as compared with the corresponding tissues from the control. In certain muscular and nervous tissues from the mutant, the reactions for sulfated complex carbohydrates tended to be weaker in intensity than those in the control. Furthermore, the dermal tissues of the mutant showed apparent mastocytosis. All these results indicate that there exist accessory effects in non-cartilaginous tissues in addition to the defects of cartilaginous tissues in the mutant mice.     

Induction of diabetes by PolyI:C and anti-RT6.1 antibody treatment in DR-BB rats.

To determine whether environmental factors could affect the incidence of diabetes in RT6.1+ lymphocytes-depleted diabetes resistant (DR) BB rats, we tested polyinosinic-polycytidylic acid (Poly I:C), as an immune activator, in conjunction with anti-RT6.1 antibody in DR-BB rats which were bred in a specific pathogen free (SPF) condition. Diabetes was induced by the combined administration of poly I:C and anti-RT6.1 antibody. The use of poly I:C or anti-RT6.1 antibody alone did not cause diabetes. These results suggest that RT6.1+ T lymphocytes regulate autoimmune diabetes and that non-specific immune activation caused by environmental factors plays a key role in inducing diabetes in DR-BB rats.