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Biomedical subjects

E D Hay

Publications and source records attributed to E D Hay.

At least 73 records · Page 4Linked to original sources
PubMed1978

Freeze-fracture studies of the developing cell surface. II. Particle-free membrane blisters on glutaraldehyde-fixed corneal fibroblasts are artefacts.

We describe, in sections and by freeze-fracture, four classes of intramembrane particle (IMP)-free membrane blebs or "blisters" associated with glutaraldehyde-fixed embryonic corneal fibroblasts: (a) Single blisters attached to the cell membrane; (b) free (detached) vesicles; (c) myelin figures; (d) multivesicular protrusions which resemble the "mounds" described by others on nerve growth cones. The IMP-free, membrane-bounded blisters contain no ground cytoplasm or organelles, in contrast to blebs on trypsin-isolated fibroblasts, which we show here do contain cytoplasm and IMP-rich membranes. That the IMP-free membrane blisters in embryonic corneas are artefacts of fixation is demonstrated by (a) their absence in replicas of fibroblasts frozen and fractured without prior aldehyde fixation and (b) their absence in sections of fibroblasts fixed in a combination of glutaraldehyde and osmium tetroxide. We suggest that the addition of osmium prevents postfixation movement of membrane lipids, especially the negatively charged "fluid" lipids which others have shown are capable of considerable mobility after aldehyde fixation alone. Recent literature has implicated membrane blistering in secretory processes and in growth of nerves, but before the functional significance of such IMP-free blisters is assessed, membrane mobility of the type shown here should be taken into consideration.

Cell Membrane↗
PubMed1977

Synthesis of two collagen types by embryonic chick corneal epithelium in vitro.

To better understand the mechanisms involved in matrix development, we have analyzed the collagen synthesized by embryonic corneal epithelium, the tissue known to produce the collagenous component of the primary corneal stroma. Isolated epithelia were cultured in vitro in medium containing [oH]proline and the newly synthesized, labeled collagen was extracted, fractionally salt precipitated, and analyzed by carboxymethyl-cellulose chromatography and peptide mapping after cleavage with cyanogen bromide. The data show that the cornela epithelium produces at least two different types of collagen, one similar, if not identical, to the type I molecule of skin, and a second similar, if not identical, to the type II molecule of cartilage. Type II, heretofore, had been thought to be characteristic of cartilage extracellular matrix.

Animals↗
PubMed1977

Ultrastructural and histochemical differences in cell surface properties of strain-specific and nonstrain-specific TA3 adenocarcinoma cells.

Transmission and scanning electron microscopy and histochemical and biochemical methods were used to investigate differences in cell structure and cell surface properties between the strain-specific TA3-St and nonstrain-specific TA3-Ha ascites sublines of the TA3 murine mammary adenocarcinoma. The TA3-St subline is lethal only to the syngeneic strain A mouse (the strain of origin), whereas the TA3-Ha subline is lethal even to foreign species. In contrast to the TA3-St cell surface, which has numerous folds and irregular microprojections, the TA3-Ha cell has abundant long microvilli of uniform dimensions. An extensive cell surface coat which resembles the "fuzz" coat found on microvilli of normal epithelium was present on the TA3-Ha, but not on the TA3-St cells. After routine fixation, the surface coat of the TA3-Ha cell usually appeared as a filamentous network extending 30-50 nm from the plasmalemma; occasionally, longer filamentous or rod-like structures were found extending 200-400 nm from the plasmalemma. The cell coat material was more extensive on the microvilli than on the intermicrovillous membranes. Free virus-like particles associated with TA3-Ha cells have a similar-appearing surface coat on their outer membranes. The density of surface anionic sites, determined with polycationic ferritin, was greater on the TA3-Ha than on the TA3-St cell surface, consistent with the presence at the TA3-Ha cell surface of several-fold more neuraminidase-susceptible sialic acid groups. The observed surface features of the nonstrain-specific TA3-Ha cell, in comparison to the strain-specific TA3-St cell, are consistent with the suggestion that sialic acid-rich glycoproteins at the TA3-Ha cell surface mask histocompatibility antigens and enhance the ability of malignant cells to invade foreign species.

Binding Sites↗
PubMed1977

Freeze-fracture studies of the developing cell surface. I. The plasmalemma of the corneal fibroblast.

The freeze-fracture technique was used to study changes in the corneal fibroblast cell membrane during morphogenesis in chick embryos. Fibroblasts migrate into the acellular primary corneal stroma on day 6 of embryogenesis, moving between the orthogonal layers of collagen fibrils which serve as their substratum. Morphometric analysis of the intramembrane particles (IMP) reveals their concentration on the P face to decrease from 756 to 534/mum2 from day 6 to day 14. After day 14, fibroblast migration and cell division cease and the stroma condenses due to dehydration, so that by day 18 all of the layers of fibroblasts are extremely flattened and the cornea has taken on its mature, transparent form. The cell membranes of the terminally differentiated, highly compacted fibroblasts are rich in IMP (1,300/MUM2, P face). In seeking to relate the particle increase to cell differentiation, we analyzed synthetic events taking place at this time, but no correlation, we analyzed synthetic events taking place at this time, but no correlation with 25SO4 or proline-3H incorporation was found. The event which seems best correlated with the doubling of P face particles between days 15 and 18 is the dehydration and condensation of the stroma, an event which is associated with cessation of both cell division and migration. Thyroxine stimulates premature condensation of the stroma, whereas thiouracil delays condensation, but neither of these treatments affects IMP concentration. Interestingly, IMP concentration on the filopodia of migrating fibroblasts is similar to that on the cell bodies, suggesting that the new membrane has the same composition as the pre-existing membrane. Observations are also presented on tight and gap junctions between fibroblasts and on the relation of extracellular matrix to the outer etched surface of the fibroblast plasmalemma.

Animals↗
PubMed1975

Location of procollagen in chick corneal and tendon fibroblasts with ferritin-conjugated antibodies.

Three distinct antiprocollagen preparations were characterized and used in immunocytochemical staining of chick embryo corneal and tendon cells. The several ferritin-conjugated antibody preparations permitted similar location of procollagen in the cisternae of the rough endoplasmic reticulum and in Golgi elements in both cell types. The ability to demonstrate and interpret specific ferritin staining was dependent on the extent of membrane breakage in each of those organelles, coupled with adequate retention of cell morphology. Corneal fibroblasts appeared to suffer more extensive intracellular membrane damage under controlled conditions of homogenization than tendon fibroblasts, facilitating the identification of procollagen in Golgi vacuoles of these cells. None of the labeled material appeared to by cytoplasmic in origin since ferritin was observed in the cytoplasm only in the vicinity of Golgi elements that were extensively broken. This study extends previous immunological evidence for the presence of procollagen in the Golgi complex and calls attention to the problems to be encountered in locating the antigen in small Golgi vesicles and lamellae.

Animals↗
PubMed1975

Stimulation of corneal differentiation by interaction between cell surface and extracellular matrix. I. Morphometric analysis of transfilter "induction".

The present study was undertaken to determine whether or not physical contact with the substratum is essential for the stimulatory effect of extracellular matrix (ECM) on corneal epithelial collagen synthesis. Previous studies showed that collagenous substrata stimulate isolated epithelia to produce three times as much collagen as they produce on noncollagenous substrate; killed collagenous substrata (e.g., lens capsule) are just as effective as living substrata (e.g., living lens) in promoting the production of new corneal stroma in vitro. In the experiments to be reported here, corneal epithelia were placed on one side of Nucleopore filters of different pore sizes and killed lens capsule on the other, with the expectation that contact of the reacting cells with the lens ECM should be limited by the number and size of the cell processes that can tranverse the pores. Transfilter cultures were grown for 24 h in [3H]proline-containing median and incorporation of isotope into hot trichloroacetic acid-soluble protein was used to measure corneal epithelial collagen production. Epithelial collagen synthesis increases directly as the size of the pores in the interposed filter increases and decreases as the thickness of the filter layer increases. Cell processes within Nucleopore filters were identified with the transmission electron microscope with difficulty; with the scanning electron microscope, however, the processes could easily be seen emerging from the undersurface of even 0.1-mum pore size filters. Morphometric techniques were used to show that cell surface area thus exposed to the underlying ECM is linearly correlated with enhancement of collagen synthesis. Epithelial cell processes did not pass through ultrathin (25-mum thick) 0.45-mum pore size Millipore filters nor did "induction" occur across them. The results are discussed in relation to current theories of embryonic tissue interaction.

Animals↗
PubMed1975

The behavior of fibroblasts from the developing avian cornea. Morphology and movement in situ and in vitro.

The early chick cornea is composed of an acellular collagenous stroma lined with an anterior epithelium and a posterior endothelium. At stage 27-28 of development (5 1/2 days), this stroma swells so that the cornea is 75-120 mum thick. At the same time, fibroblasts that originate from the neural crest begin to invade this stroma. Using Nomarski light microscopy, we have compared the behavior of moving cells in isolated corneas with the migratory activities of the same cells in artificial collagen lattices and on glass. In situ, fibroblasts have cyclindrical bodies from which extend several thick pseudopodia and/or finer filopodia. Movement is accompanied by activity in these cytoplasmic processes. The flat ruffling lamelli-podia that characterize these cells on glass are not seen in situ, but the general mechanism of cell movement seems to be the same as that observed in vitro: either gross contraction or recoil of the cell body (now pear shaped) into the forward cell process, or more subtle "flowing" of cytoplasm into the forward cell process without immediate loss of the trailing cell process. We filmed collisions between cells in situ and in three-dimensional collagen lattices. These fibroblasts show, in their pair-wise collisions, the classical contact inhibition of movement (CIM) exhibited in vitro even though they lack ruffled borders. On glass these cells multi-layer, showing that, while CIM affects cell movement, fibroblasts can use one another as a substratum. Postmitotic cells show CIM in moving away from each other. Interestingly, dividing cells in situ do not exhibit surface blebbing, but do extend filopodia at telophase. The role of CIM in controlling cell movement in vivo and in vitro is stressed in the discussion.

Animals↗
PubMed1974

Stimulation of extracellular matrix synthesis in the developing cornea by glycosaminoglycans.

Previously, it was demonstrated that the embryonic corneal epithelium produces the chondroitin sulfate and heparan-sulfate-like compounds and the collagen of the primary corneal stroma. Synthesis of all of these extracellular materials is greatly enhanced in vitro when isolated epithelium is grown on collagenous substrata instead of Millipore filters. We report here that chondroitin sulfate, heparin, and heparan sulfate added to the culture medium at a concentration of 200 mug/ml enhance the synthesis by the epithelium of chondroitin sulfate and heparan-sulfate-like compounds 2-fold, whether or not collagenous substrata are employed. Collagen synthesis is unaffected by adding glycosaminoglycan to the medium. Chondroitin sulfate proteoglycan (chondromucoprotein) has the same stimulatory effect as chondroitin sulfate, but dermatan sulfate and hyaluronate have no measurable effect on glycosaminoglycan production by epithelial cells. Keratan sulfate however, seems to depress glycosaminoglycan synthesis. Thus, in this system, only sulfated polyanions like those produced by the corneal epithelium have a stimulatory effect on glycosaminoglycan synthesis. The results are discussed in terms of how the tissues of the cornea (epithelium, endothelium, keratocytes) may interact by changing the composition of the stromal extracellular matrix.

Animals↗
PubMed1973

Collagen synthesis in vitro by embryonic spinal cord epithelium.

Isolated spinal cords from chick embryos (stages 12 to 15) were incubated in vitro with radioactive proline. The proteins synthesized were fractionated by coprecipitation with added carrier collagen, followed by molecularsieve and ion-exchange chromatography. A portion of the isotopically labeled proteins were found to be collagen molecules consisting only of alpha1 chains.

Amino Acids↗