Search PubMed⌕ Search

Biomedical subjects

B M Vertel

Publications and source records attributed to B M Vertel.

34 records · Page 2Linked to original sources
PubMed1985

Immunofluorescence studies on cartilage matrix synthesis. The synthesis of link protein, chondroitin sulfate proteoglycan monomer and type II collagen.

A comparison of the synthesis and deposition of fibrous type II collagen and the constituents of chondroitin sulfate proteoglycan (CSPG) aggregates, CSPG monomer and link protein, was made for chicken sternal chondrocytes in culture, using simultaneous double immunofluorescence and lectin localization. Chondrocytes deposited only CSPG constituents--and not type II collagen--into the extracellular matrix (ECM). Intracellular precursors of CSPG monomer were localized primarily in perinuclear regions, but were observed in other cytoplasmic vesicles as well. Link protein antibodies stained the same intracellular structures, but stained the perinuclear cytoplasm less intensely. In contrast, type II procollagen was distributed in vesicles throughout the cytoplasm and was clearly absent from the distinctive, CSPG precursor-containing vesicles. Fluorescence-labelled lectins were used to further identify intracellular membrane compartments. Wheat germ agglutinin (WGA) and Ricinus lectins (which recognize carbohydrates added in the Golgi) stained the perinuclear cytoplasm, while concanavalin A (conA) (which recognizes mannose-rich oligosaccharides added co-translationally) stained vesicles throughout the rest of the cytoplasm and not the perinuclear cytoplasm. The distinctive CSPG-containing vesicles were not stained with WGA or Ricinus agglutinins. Data presented elsewhere demonstrate that the vesicles do not react with monoclonal antibodies which recognize chondroitin sulfate (CS) or keratan sulfate (KS) determinants. Thus, we conclude that the vesicles accumulate CSPG precursors which have not been modified by Golgi-mediated processes. The data indicate that matrix molecules may be segregated selectively prior to transit through the Golgi complex. The co-distribution of link protein and CSPG monomer precursors in vesicles prior to further, Golgi-mediated modification may reflect an as yet undetermined function of these vesicles in the processing or assembly of CSPG.

Animals↗
PubMed1984

Cell-free translation of messenger RNA for chondroitin sulphate proteoglycan core protein in rat cartilage.

Total RNA was extracted from the cartilage tissues rat Swarm chondrosarcoma, neonatal-rat breastplate and embryonic-chicken sterna and translated in wheat-germ cell-free reactions. The core protein of the chondroitin sulphate proteoglycan subunit was identified among translation products of rat mRNA by its apparent Mr of 330 000 and by its immunoprecipitation with specific antisera prepared against rat or chicken proteoglycan antigens. The apparent Mr of the rat proteoglycan core protein is 8000-10000 less than that of the equivalent chicken cartilage core-protein product.

Animals↗
PubMed1984

Stimulation and inhibition of myoblast differentiation by hormones.

The growth and differentiation of L6 myoblasts are subject to control by two proteins secreted by cells of the Buffalo rat liver line. The first of these, rat insulinlike growth factor-II (formerly designated multiplication stimulating activity) is a potent stimulator of myoblast proliferation and differentiation, as well as associated processes such as amino acid uptake and incorporation into protein, RNA synthesis, and thymidine incorporation into DNA. In addition, this hormone causes a significant decrease in the rate of protein degradation. All of these actions seem to be attributable to a single molecular species, although their time courses and sensitivity to the hormone differ substantially. The second protein, the differentiation inhibitor (DI), is a nonmitogenic inhibitor of all tested aspects of myoblast differentiation, including fusion and the elevation of creatine kinase. Indirect immunofluorescence experiments demonstrated that DI also blocks accumulation of myosin heavy chain and myomesin. Upon removal of DI after 72 h incubation, all of these effects were reversed and normal myotubes containing the usual complement of muscle-specific proteins were formed. Thus, this system makes it possible to achieve specific stimulation or inhibition of muscle cell differentiation by addition of purified proteins to cloned cells in serum-free medium.

Animals↗
PubMed1984

Immunofluorescence studies of chondroitin sulfate proteoglycan biosynthesis: the use of monoclonal antibodies.

Several monoclonal antibodies which recognize different antigenic determinants of chondroitin sulfate proteoglycan were used to study chondroitin sulfate proteoglycan biosynthesis in chicken chondrocyte cultures. The intracellular sites of synthesis and processing and extracellular deposition in matrix were localized by double immunofluorescence reactions. One rat monoclonal antibody, S103L , which recognizes an antigenic determinant of the core protein of the chicken cartilage chondroitin sulfate proteoglycan monomer, was used to identify both extracellular chondroitin sulfate proteoglycan and intracellular compartments containing chondroitin sulfate proteoglycan precursors. Intracellular staining with S103L was localized to perinuclear regions, and, in some chondrocytes, to a few other cytoplasmic vesicles as well. When chondrocytes were not fed for several days, intracellular chondroitin sulfate proteoglycan precursors were accumulated in larger compartments distributed throughout the cytoplasm. Polyclonal chondroitin sulfate proteoglycan antibodies displayed similar staining characteristics. In contrast, several of the monoclonal antibodies, including the rat monoclonals S11D and P100D , and the mouse monoclonals 1-B-5, 3-B-3 and 9-A-2, did not recognize native chondroitin sulfate proteoglycan, but reacted only with chondroitinase ABC-digested (and/or hyaluronidase-digested) chondroitin sulfate proteoglycan. These antibodies were particularly useful in the demonstration of the extracellular codistribution of chondroitin sulfate proteoglycan with either type II collagen or fibronectin. In other experiments, the monoclonal antibodies to chondroitin sulfate proteoglycan served to demonstrate that the perinuclear subset of intracellular compartments is uniquely involved in the addition of chondroitin sulfate oligosaccharides to the chondroitin sulfate proteoglycan core protein. Lastly, using the mouse monoclonal 5-D-4, which recognizes keratan sulfate determinants, the perinuclear region was identified as the site for keratan sulfate addition. Results suggest heterogeneity of keratan sulfate synthesis at the level of individual chondrocytes, even for cells apparently containing equivalent amounts of intracellular chondroitin sulfate proteoglycan.

Animals↗
PubMed1983

Isolation and characterization of chondrocytes and non-chondrocytes from high-density chick limb bud cell cultures.

This communication describes a replating technique for the separation of the chondrogenic and non-chondrogenic cells from stage-24 chick limb bud mesenchymal cell cultures by means of sequential digestion with collagenase. Four sub-populations of cells were obtained: The first consisted solely of non-chondrocytes and the next three were progressively enriched in chondrocytes. In addition to morphological differences, the four cell populations differed from each other in their rates of incorporation of sulfate into macromolecular material which were roughly proportional to the percentage of chondrocytes. The chondrocytes and non-chondrocytes no longer exhibited a density dependence of phenotype. In addition, the normal multilayered nodular morphology associated with cartilage development was not observed. These isolated cells have been used as starting material for detailed biochemical studies. Together, these studies indicate that the expressional program governing biosynthetic changes in chondrocytes is not controlled by the extracellular matrix.

Animals↗
PubMed1981

Extracellular matrix formation by chondrocytes in monolayer culture.

In previous studies were have reported on the secretion and extracellular deposition of type II collagen and fibronectin (Dessau et al., 1978, J. Cell Biol., 79:342-355) and chondroitin sulfate proteoglycan (CSPG) (Vertel and Dorfman, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:1261-1264) in chondrocyte cultures. This study describes a combined effort to compare sequence and pattern of secretion and deposition of all three macromolecules in the same chondrocyte culture experiment. By immunofluorescence labeling experiments, we demonstrate that type II collagen, fibronectin, and CSPG reappear on the cell surface after enzymatic release of chondrocytes from embryonic chick cartilage but develop different patterns in the pericellular matrix. When chondrocytes spread on the culture dish, CSPG is deposited in the extracellular space as an amorphous mass and fibronectin forms fine, intercellular strands, whereas type II collagen disappears from the chondrocyte surface and remains absent from the extracellular space in early cultures. Only after cells in the center of chondrocyte colonies shape reassume spherical shape does the immunofluorescence reveal type II collagen in the refractile matrix characteristic of differentiated cartilage. By immunofluorescence double staining of the newly formed cartilage matrix, we demonstrate that CSPG spreads farther out into the extracellular space that type II collagen. Fibronectin finally disappears from the cartilage matrix.

Animals↗
PubMed1979

Translation and characterization of messenger RNAs in differentiating chicken cartilage.

Total RNA, prepared from chicken limb bud cultures undergoing differentiation to cartilage, has been translated in a wheat germ cell-free protein-synthesizing system. Antibodies against chondroitin sulfate proteoglycan core protein immunoprecipitate a single component which migrates as a protein of 340,000 daltons in sodium dodecyl sulfate/polyacrylamide gels. The messenger RNA for this protein sediments at approximately 27 S in 70% formamide or aqueous sucrose gradients. The 340,000-dalton protein is present in cell-free translation products directed by RNA prepared from limb bud cultures and sternae and is absent in cell-free translation directed by RNA prepared from embryonic calvaria or liver. The level of synthesis of this protein is greatly reduced when RNA prepared from limb bud cultures inhibited from differentiation by BrdUrd is used. (Pre)pro alpha 1(I), -alpha 2(I), and -alpha 1(II) collagen bands have been identified on gels by electrophoresis of collagenase-digested or immunoprecipitated cell-free translation products directed by RNA from differentiating limb bud cultures, embryonic sternae, and embryonic calvaria.

Animals↗
PubMed1979

Simultaneous localization of type II collagen and core protein of chondroitin sulfate proteoglycan in individual chondrocytes.

In order to investigate the coordinated synthesis of matrix components by individual chondrocytes, specific antibodies to type I collagen, type II collagen, and chondroitin sulfate proteoglycan core protein were used in simultaneous double immunofluorescence reactions. Extensive accumulation of core protein surrounding chondrocytes and the intracellular accumulation of type II collagen were observed. Extracellular core protein immunofluorescence obscured the intracellular reaction product, but the extracellular immunoreactive material could be removed by digestion with purified testicular hyaluronidase prior to fixation. Subsequent to digestion, core protein and type II collagen were observed in the same chondrocytes within discrete, sometimes identical, cytoplasmic regions, thus demonstrating the simultaneous localization of these two products characteristic of differentiating cartilage.

Animals↗