Cloning and biological function of laminin in Hydra vulgaris.

The Cnidarian, hydra, lends itself to studies related to the role of extracellular matrix (ECM) components in development because of its high regenerative capacity and its simple structure, which is organized as an epithelial bilayer with an intervening ECM termed the mesoglea. Previous immunocytochemical and biochemical studies have established that hydra mesoglea contains many of the major matrix components (e.g., fibronectin, laminin, type IV collagen, and heparan sulfate proteoglycan) associated with the ECM of vertebrate and more complex invertebrate species. Additional studies have also established that ECM components have a critical role in hydra development as monitored during head regeneration and morphogenesis of hydra cell aggregates. In the present study a monoclonal antibody (mAb52) raised to isolated hydra mesoglea was used as a probe in additional functional studies and to screen a cDNA expression library made from poly(A)+ RNA isolated from Hydra vulgaris. Immunofluorescent analysis indicated that mAb52 was localized along the entire longitudinal axis of adult polyps in what is termed the subepithelial zones of hydra mesoglea. Cytochemical studies found these subepithelial zones to be rich in anionic sites. Previous studies have shown that mAb52 blocks hydra cell aggregate development and experiments in the current study have shown that mAb52 also blocks in vivo interstitial cell (I-cell) migration in hydra grafts. Sequence analysis of cDNA clones isolated using mAb52 indicated that the protein encoded by these clones had structural homology with mammalian and Drosophila laminin B1 chain and hybridized to a single 6.75-kb band on Northern blots of total hydra RNA. One interesting difference in hydra laminin B1 was the presence of a FTGTQ amino acid sequence in place of the vertebrate YIGSR cell binding domain. Under nonreducing conditions, polyclonal antibodies against FTGTQ bound to the same > 200-kDa band on Western blots of mesoglea as mAb52 and also immunolocalized to the subepithelial zones. Under reducing conditions, anti-FTGTQ antibodies bound to a single band with a mass of approximately 200 kDa. In addition, FTGTQ peptide inhibited adhesion of dissociated hydra cells to mesoglea and anti-FTGTQ antibodies inhibited hydra cell binding to substrates coated with mesoglea or FTGTQ peptide. Anti-FTGTQ antibodies also inhibited in vivo I-cell migration in hydra grafts. Given the early divergence of Cnidarians during evolution, these studies indicate the highly conserved nature of laminin and provide additional information regarding the critical role of ECM components during hydra development.

Extracellular matrix (mesoglea) of Hydra vulgaris III. Formation and function during morphogenesis of hydra cell aggregates.

Hydra, as a member of the phylum Cnidaria, is characterized by a body lining organized as an epithelial bilayer with an intervening extracellular matrix (ECM) termed the mesoglea. Previous studies have established that the mesoglea has components indicative of mammalian ECM such as type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan, and these components appear to play a critical role in hydra head regeneration. A remarkable feature of hydra is its ability to reorganize into its adult structure within 96 hr to 7 days from pellets formed from dissociated hydra cells. This regenerative model has been termed the hydra cell aggregate system. The present study has been designed to characterize the biogenesis of mesoglea in hydra cell aggregates and to determine its role in morphogenesis of aggregates. We find that hydra cell aggregates first form an epithelial bilayer by 12 hr of development and then subsequently develop a mesoglea. Morphogenesis of hydra structure then follows formation of the mesoglea. Immunofluorescence studies indicate that mesoglea components are first deposited between the epithelial bilayer by about 12-17 hr of pellet formation, and pulse-labeling studies indicate that the translation rate of matrix components peaks by 48-72 hr of development. Ultrastructural studies indicate that a mature mesoglea is formed by 48-96 hr of pellet formation. Drugs such as beta-aminoproprionitrile and 2,2'-dipydridyl, which interfere with the cross-linking of collagens, and p-nitrophenyl-beta-D-xylopyranoside, which interferes with the addition of GAG moieties to proteoglycan core molecules, were found to reversibly block development of hydra cell aggregates. Transmission electron microscopy studies indicate that these drugs affect the ultrastructure of the mesoglea. In addition, both polyclonal and monoclonal antibodies raised to isolated mesoglea were found to block development of hydra cell aggregates. These studies indicate that (1) mesoglea formation is rapid and precedes morphogenetic processes during aggregate development, and (2) formation of mesoglea is essential for normal morphogenesis of hydra cell aggregates.

High molecular weight secretory products of the human pancreatic ductal epithelium and the effect of secretin on their discharge.

Principal cells of the pancreatic ductal epithelium have been reported to secrete high molecular weight (HMW) glycoconjugates such as mucins into the ductal lumen. We used a human pancreatic carcinoma cell line of ductal origin (PANC-1) which has retained some of the morphological and biochemical characteristics of normal ductal principal cells as a source for isolation of HMW secretory products. The present study was designed to isolate these HMW secretory products, partially characterize them through biochemical and immunological approaches, and determine the effects of secretin on their synthesis and discharge from PANC-1 cells. Our results indicated that when PANC-1 cells are grown on collagen-coated beads in defined serum-free medium, at least three HMW secretory products could be isolated from the medium. These secretory products had a mass of approximately 200, 280, and 370 kDa based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The 200-kDa species made up proportionally less of the three in the gel-staining pattern. Polyclonal antibodies raised to the 370-kDa secretory product cross-reacted with the 280-kDa species. The 370-kDa secretory product was sulfated and wheat germ agglutinin (WGA) binding indicated that the 370-kDa species was a glycoconjugate. The 280-kDa and 200-kDa species were sulfated to a much lesser degree than the 370-kDa species and WGA binding could not be clearly demonstrated with the 280 kDa or 200 kDa species. Glycosidase and selective degradation studies, however, indicated that all three species contained glycosaminoglycan moieties. Antibodies raised to the 370-kDa secretory product localized to the epithelium of human pancreatic carcinomas but not to other cell types in this neoplastic tissue. The antibody also cross-reacted with the ductal epithelium of normal human pancreas and could be localized to centroacinar cells, the epithelium of intralobular ducts, and the epithelium of interlobular ducts. The antibody did not cross-react with goblet cells of the human small or large intestine, indicating no generalized reactivity to gastrointestinal mucins. ELISA and pulse-chase immunoprecipitation studies indicated an increase in the cellular content and synthesis of these HMW secretory products after stimulation of PANC-1 cells with 10(-8)- to 10(-11)-M secretin. We correlated secretin stimulation with the appearance of numerous membrane bound vesicles throughout the cytoplasm as monitored at the ultrastructural level. Optimal secretin concentration were in the range of 10(-10) M. The content of these vesicles was immunoreactive to the antibody raised against the 370-kDa secretory product as determined by ultrastructural immunocytochemical techniques.(ABSTRACT TRUNCATED AT 400 WORDS)

Extracellular matrix (mesoglea) of Hydra vulgaris. I. Isolation and characterization.

Hydrozoans such as Hydra vulgaris, as with all classes of Cnidaria, are characterized by having their body wall organized as an epithelial bilayer with an intervening acellular layer termed the mesoglea. The present study was undertaken to determine what extracellular matrix (ECM) components are associated with Hydra mesoglea. Using polyclonal antibodies generated from vertebrate ECM molecules, initial light and electron microscopic immunocytochemical studies indicated the presence of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin immunoreactive components in Hydra mesoglea. These immunocytochemical observations were in part supported by biochemical analyses of isolated Hydra mesoglea which indicated the presence of fibronectin and laminin based on Western blot analysis. Amino acid analysis of total mesoglea and some of its isolated components confirmed the presence of collagen molecules in mesoglea. Additional studies indicated the presence of (1) a gelatin binding protein in Hydra which was immunoreactive with antibodies raised to human plasma fibronectin and (2) a noncollagen fragment extracted from mesoglea which was immunoreactive to antibodies raised to the NC1 domain (alpha 1 subunit) of bovine glomerular basement membrane type IV collagen. These observations indicate that Hydra mesoglea is evolutionarily a primitive basement membrane that has retained some properties of interstitial ECM.

A non-mammalian in vivo model for cellular and molecular analysis of glucose-mediated thickening of basement membranes.

An increase of basement membrane thickness in renal glomeruli, blood vessels, and other tissues is a consistent pathological observation in individuals with diabetes mellitus. Although a number of pathological complications of the disease are thought to result from this structural abnormality in basement membranes, the mechanism(s) responsible for this glucose-mediated process remain unknown. The current study was designed to develop a non-mammalian in vivo epithelial/basement membrane model which would facilitate detailed analysis of the cellular and molecular processes which lead to thickening of basement membrane under hyperglycaemic conditions. The system developed utilizes the Cnidarian, Hydra vulgaris. Hydra lends itself to such studies because of (1) its simplified body structure which is composed of an epithelial bilayer with an intervening basement membrane (mesoglea) and (2) its extensive regenerative capacity which allows cell pellets (Hydra cell aggregates), formed from isolated Hydra cells, to develop into adult Hydra within 72-96 h. This process involves reformation of an epithelial bilayer and de novo biosynthesis of a basement membrane. Our studies indicate that exposure of developing Hydra cell aggregates to levels of D-glucose which mimic that observed in the human diabetic patient (15 mmol/l) induces a doubling of Hydra basement membrane thickness within 72-96 h of pellet formation. The same results were obtained using 15 mmol/l D-Ribose which is a highly efficient glycating agent. The data presented support the use of the Hydra cell aggregate system as a potentially powerful non-mammalian in vivo model to investigate the cellular and molecular mechanism(s) underlying glucose-mediated basement membrane thickening.

Comparative analysis of a human pancreatic undifferentiated cell line (MIA PaCa-2) to acinar and ductal cells.

Pancreatic ductal cell secretion has not been well characterized due to the difficulty in obtaining sufficient quantities of purified ductal cells. To determine if the MIA PaCa-2 cell line would provide a useful model for in vitro studies of pancreatic ductal cell secretion, the present study was designed to characterize these cells in greater detail. In this investigation, the human pancreatic undifferentiated cell line, MIA PaCa-2, was compared with PANC-1 cells (a human ductal cell line previously characterized), isolated rat and human ducts, acinar cells, and nonpancreatic cell lines. The results indicate that while the morphology of the MIA PaCa-2 cell line is nonpolarized and generally atypical of either ductal or acinar cells, the cell line has retained certain biochemical similarities to ductal cells. Additional morphological studies indicated (a) the presence of intermediate filaments characteristic of epithelial cells, (b) the absence of zymogen granules, and (c) an apparent basolateral plasma membrane localization of Na+, K+-ATPase. Similar to ductal cells, biochemical analyses indicated (a) the presence of Na+, K+-ATPase based on [3H]-ouabain binding assays, (b) high levels of carbonic anhydrase, (c) low levels of gamma-glutamyl transpeptidase, (d) nondetectable levels of amylase, and (e) protein composition and protein synthetic patterns comparable to PANC-1 cells. Finally, as with PANC-1 cells and isolated rat and human ducts, the major sulfated secretory product of MIA PaCa-2 cells was a protein with a molecular weight of approximately 660,000 to 1 million.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of cell surface glycoconjugates in fibroblasts from patients with cystic fibrosis.

Although the basic biochemical defect in cystic fibrosis (CF) is unknown, previous studies have indicated that errors in protein glycosylation may be involved in the pathogenesis of the disease. Utilizing human skin fibroblasts, the present study was designed to quantitatively analyze glycosylation of cell surface glycoconjugates in CF and normal cells. Cell surface glycoconjugates were analyzed using 125I-concanavilin A (Con A), 125I-WGA, and Con A-ferritin conjugates. Under our binding conditions, Con A was used as a probe for mannose residues and WGA was used as a probe for N-acetylglucosamine residues. Saturable binding of both probes was observed and appropriate sugar controls confirmed the specificity of each lectin. When compared on a DNA basis, iodinated lectin binding studies indicated that no consistent differences existed between CF and normal strains of human skin fibroblasts. Ultrastructural quantitative morphometric analysis of Con A-ferritin conjugate binding indicated that neither proteolysis of cell surface glycoconjugates or internalization of lectin probes was occurring at saturable binding concentrations. In summary, our results indicated that no consistent differences in cell surface mannose and N-acetylglucosamine residues could be detected between the normal and CF strains of human skin fibroblasts used in these studies.