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Biomedical subjects

E A Chernoff

Publications and source records attributed to E A Chernoff.

At least 19 recordsLinked to original sources
PubMed2001

The effects of collagen synthesis inhibitory drugs on somitogenesis and myogenin expression in cultured chick and mouse embryos.

The role of fibrillar collagen on myogenic differentiation has previously been studied in tissue culture cell lines but has not been studied in situ. We treated cultured chick and mouse embryos with collagen synthesis inhibitors to determine the role of fibrillar collagen on somitogenesis and on myogenic differentiation in vivo. Stage 12 chick embryos and 8.7 dpc mouse embryos were cultured in control medium or a range of concentrations of the collagen synthesis inhibitors ethyl-3,4-dihydroxybenzoate (EDHB) or cis-hydroxy-proline (CHP). Chick embryos were cultured for 24 h and mouse embryos were cultured for 30 h. Both collagen synthesis inhibitors produced a range of somite abnormalities including formation of fewer and irregular somites in both chick and mouse at high drug concentrations, as well as formation of double somites in EDHB-treated chick embryos. Examination of EDHB-treated mouse embryos by scanning electron microscopy demonstrated a dosage-dependent loss of fibrillar collagen and associated extracellular matrix. Expression of myogenin in EDHB-treated mouse embryos, examined by whole-mount in situ hybridization, was suppressed at higher dosage levels. This study suggests that inhibition of fibrillar collagen production and/or loss of fibrillar collagen in the developing avian and mammalian embryo results in abnormal somite formation and perturbed myogenic differentiation.

Animals↗
PubMed1997

cDNA sequence and catalytic properties of a chick embryo alcohol dehydrogenase that oxidizes retinol and 3beta,5alpha-hydroxysteroids.

This study was undertaken to identify the cytosolic 40-kDa zinc-containing alcohol dehydrogenases that oxidize all-trans-retinol and steroid alcohols in fetal tissues. Degenerate oligonucleotide primers were used to amplify by polymerase chain reaction 500-base pair fragments of alcohol dehydrogenase cDNAs from chick embryo limb buds and heart. cDNA fragments that encode an unknown putative alcohol dehydrogenase as well as the class III alcohol dehydrogenase were identified. The new cDNA hybridized with two messages of approximately 2 and 3 kilobase pairs in the adult chicken liver but not in the adult heart, muscle, testis, or brain. The corresponding complete cDNA clones with a total length of 1390 base pairs were isolated from a chicken liver lambdagt11 cDNA library. The open reading frame encoded a 375-amino acid polypeptide that exhibited 67 and 68% sequence identity with chicken class I and III alcohol dehydrogenases, respectively, and had lower identity with mammalian class II (55-58%) and IV (62%) isozymes. Expression of the new cDNA in Escherichia coli yielded an active alcohol dehydrogenase (ADH-F) with subunit molecular mass of approximately 40 kDa. The specific activity of the recombinant enzyme, calculated from active site titration of NADH binding, was 3.4 min-1 for ethanol at pH 7.4 and 25 degrees C. ADH-F was stereospecific for the 3beta,5alpha- versus 3beta,5beta-hydroxysteroids. The Km value for ethanol at pH 7.4 was 17 mM compared with 56 microM for all-trans-retinol and 31 microM for epiandrosterone. Antiserum against ADH-F recognized corresponding protein in the chicken liver homogenate. We suggest that ADH-F represents a new class of alcohol dehydrogenase, class VII, based on its primary structure and catalytic properties.

Alcohol Dehydrogenase↗
PubMed1996

Spinal cord regeneration: a phenomenon unique to urodeles?

Studies of neuronal survival and axonal regeneration in birds and mammals have made it clear that the microenvironment of the CNS is critical to the failure of CNS regeneration in these animals. This environment includes growth and trophic factors, ECM components and matrix turnover enzymes, cytokines and other immune system contributions. Urodele amphibians (salamanders and newts) can regenerate spinal cord even as adults, and environmental contributions of glial populations are a major part of the difference between urodele and higher vertebrate spinal cord regeneration. In particular, the behavior of injury-reactive ependymal cells (radial glia) is critical to the regenerative capacity of urodele spinal cord. In this review we examine what is known about cell-cell interactions between ependymal cells and neurons and between ependymal cells and other glial populations. The known contributions of ependymal cell products such as matrix metalloproteinases and trophic factors are discussed. There is evidence in the literature that an ependymal response occurs during embryonic or fetal development in birds and mammals following spinal cord transection, and this review discusses the implications of such a process for future studies of spinal cord injury.

Animals↗
PubMed1994

Growth factor modulation of injury-reactive ependymal cell proliferation and migration.

Injury-reactive ependymal cells from regenerating axolotl spinal cord can be maintained in their mesenchymal outgrowth phase in culture (O'Hara et al., 1992). To address the ability of specific growth factors in stimulating or maintaining migration and proliferation, mesenchymal ependymal cell cultures derived from injured axolotl spinal cord at 2 weeks post-lesioning were used to determine the potential effects of epidermal growth factor, platelet-derived growth factor and transforming growth factor-beta 1. In our cultures, medium containing epidermal growth factor alone or in combination with the other growth factors permitted significant migration and proliferation from ependymal explants. Platelet-derived growth factor alone was shown to have a small positive effect on ependymal cell migration and no effect on proliferation. Transforming growth factor-beta 1 alone did not support cell migration and was found to be inhibitory towards cellular proliferation. Lastly, medium containing platelet-derived growth factor and transforming growth factor-beta 1, but not epidermal growth factor, caused ependymal cell explants to break apart and migrate on the dish as cords. Migration and proliferation of injury-reactive ependymal cells was shown to be dependent on epidermal growth factor in vitro. These results suggest that epidermal growth factor may be a critical component in vivo during the initiation of ependymal migration and proliferation following transection of the axolotl spinal cord. The reorganization of cultured ependymal cells in response to the combination of platelet-derived growth factor and transforming growth factor-beta shows that ependymal organization can be modulated by growth factors. This suggests that the progressive changes observed during regeneration may be under the control of growth factors.

Ambystoma↗
PubMed1992

Reorganization of the ependyma during axolotl spinal cord regeneration: changes in intermediate filament and fibronectin expression.

Changes in intermediate filament content and extracellular matrix material showed that the injury response of ependymal cells in lesioned axolotl spinal cord involves an epithelial-to-mesenchymal transformation, and that fibrous astrocytes are excluded from the remodeling lesion site. Antibody localization was used to visualize cytokeratin-, vimentin-, and glial fibrillary acidic protein- (GFAP-) containing intermediate filaments, as well as the adhesive glycoprotein fibronectin. In normal axolotl spinal cord cytokeratins were found near the apical surface of the ependymal cells. Transmission electron microscopic examination suggested that these cytokeratins were in tonofilaments. Cytokeratin expression was lost and vimentin production was initiated in ependymal cells 2-3 weeks following spinal cord injury. There was a period of approximately 1-2 weeks when cytokeratins and vimentin were co-expressed in vivo. This co-expression was maintained in vitro by culture on a fibronectin-coated substratum. As the central canal reformed, vimentin expression was lost. Ependymal cells lacked GFAP intermediate filaments, but GFAP was present in fibrous astrocytes of the neuropil and white matter. Following injury, GFAP localization showed that fibrous astrocytes disappeared from the remodeling lesion site and reappeared only after the ependymal epithelium reformed and newly myelinated axons were found. Fibronectin expression closely followed the expression of vimentin during mesenchymal ependymal cell outgrowth. These results suggest that the ependymal cell outgrowth requires changes in cell shape followed by changes in production of extracellular matrix.

Ambystoma↗
PubMed1990

Epidermal growth factor and the onset of epithelial epidermal wound healing.

At 10 days in ovo the embryonic chick epidermis acquires the ability to spread as a cohesive epithelial sheet when wounded. A tissue culture system has been constructed that supports epidermal cell outgrowth consistent with epidermal behaviour in vivo and permits experimental manipulation of the isolated tissue with growth factors and other hormones. This culture system consists of embryonic chick epidermis isolated at days 8, 10, and 12 of development, serum-free, chemically-defined culture medium, and the inner surface of the vitelline membrane of the hen's egg as the culture substratum. At 8 days the cellular outgrowth is mesenchymal in the absence of exogenous EGF. The 8 day tissues responds to added EGF by exhibiting precocious epithelial outgrowth. The results suggest that sensitivity to EGF or EGF-like growth factors is part of the mechanism underlying the developmental onset of epidermal wound healing in skin. The epidermal origin of the outgrowth is determined by antibody staining for specific cytokeratins. The epithelial character of the outgrowth is determined by visualizing actin microfilament distribution. The normal epithelial outgrowth shows apical/basal polarization of the sheet except at the edge. From 10 days on, the isolated epidermis exhibits epithelial outgrowth from explants in culture in the absence of exogenous EGF, suggesting endogenous production of an EGF-like factor. Glucocorticoid and mineralocorticoid hormones both produce a reduced amount of epithelial outgrowth. This retardation of the early outgrowth by glucocorticoids and mineralocorticoids could result from a reduced ability of the cut edge of the epidermis to 'disorganize' and assume the unpolarized migratory form required for rapid epidermal wound healing.

Actin Cytoskeleton↗
PubMed1989

Adhesion and fusion of the extraembryonic epiblast.

A tissue culture model system has been devised to examine the attachment, expansion, and fusion of epithelial cell sheets. A normal embryonic epithelial tissue, the extraembryonic epiblast of the chick, is isolated mechanically and cultured on its natural substratum, the vitelline membrane. This persistently migratory tissue has distinct adhesive and non-adhesive regions. A serum-free chemically-defined culture medium has been formulated that permits determination of the effects of individual growth and trophic factors. Attachment of transferred epiblasts is dependent upon the presence of mineralocorticoids in the medium. This suggests that fluid transport is required for the cell sheet to make its initial attachment to the culture substratum. Expansion of the cell sheet following attachment, and the fusion of epiblasts advancing toward each other, does not require the presence of mineralocorticoid. No exogenous adhesive glycoproteins are required for attachment, expansion, or fusion. Antibody localization shows that endogenous laminin is present on the attachment surface of the specialized adhesive edge region of the extraembryonic epiblast. Following fusion of confronted epiblasts into one coherent cell sheet, the laminin disappears. Throughout these studies the adhesive and non-adhesive regions of the epiblast are identified by their characteristic distributions of actin microfilaments, localized using rhodamine-phalloidin staining.

Animals↗
PubMed1988

The role of endogenous heparan sulfate proteoglycan in adhesion and neurite outgrowth from dorsal root ganglia.

Some phases of dorsal root ganglion (DRG) substratum attachment and growth cone morphology are mediated through endogenous cell surface heparan sulfate proteoglycan. The adhesive behavior of intact embryonic chicken DRG (spinal sensory ganglia) is examined on substrata coated with fibronectin, fibronectin treated with antibody to the cell-binding site (anti-CBS), and the heparan sulfate-binding protein platelet factor four. DRG attach to fibronectin, anti-CBS-treated fibronectin, and platelet factor four. The ganglia extend an extensive halo of unfasciculated neurites on fibronectin and produce fasciculated neurite outgrowth on platelet factor four and anti-CBS antibody-treated FN. Treatment with heparinase, but not chondroitinase, abolishes adhesion to fibronectin and platelet factor four. Growth cones of DRG on fibronectin have well-spread lamellae and microspikes. On platelet factor four, and anti-CBS-treated FN, growth cones exhibit microspikes only. Isolated Schwann cells adhere equally well to fibronectin and platelet factor four, spreading more rapidly on fibronectin. Isolated DRG neurons adhere equally well on both substrata, but only 10% of the neurons extend long neurites on platelet factor four. The majority of the isolated neurons on platelet factor four exhibit persistent microspike production resembling that of the early stages of normal neurite extension. Endogenous heparan sulfate proteoglycan supports the adhesion of whole DRG, isolated DRG neurons, and Schwann cells, as well as extensive microspike activity by DRG neurons, one important part of growth cone activity.

Animals↗
PubMed1985

Cell movement and contraction in somite development.

During somite formation the segmental plate mesoderm, lying on either side of the axial organs, reorganizes into roughly spherical pairs of epithelial structures. This segmentation process includes changes in cell shape and position, cell-cell and cell-substratum adhesive properties and accumulation of extracellular matrix material which proceed down the anterior-posterior axis. Later in somite development the sclerotome region "disperses", migrating around the spinal cord where it produces the cartilage model of the vertebral column. Experimental manipulation of segmentation and sclerotome dispersal with drugs affecting microfilaments, microtubules and calcium-dependent contraction suggest that cells migrate into position, elongate, and undergo apical contraction as part of the segmentation process. This process of calcium-dependent, possibly calmodulin-mediated, contraction can be both stimulated precociously and inhibited, showing similarities with contractile morphogenetic events in epithelial organ systems such as eye and thyroid. Similar experiments with drugs affecting contractile microfilaments demonstrate that active cell movement, along with extracellular matrix production, is involved in sclerotome dispersal.

Animals↗
PubMed1984

Heparan sulfate proteoglycans of human neuroblastoma cells: affinity fractionation on columns of platelet factor-4+.

Human neuroblastoma cells (Platt) were detached from tissue culture substrata with a Ca2+ chelating agent, and then the suspended cells were extracted with a sodium dodecyl sulfate (SDS)-containing buffer to maximally solubilize their sulfate-radiolabeled proteoglycans. The majority of the high-molecular-weight material in these dissociative extracts was heparan sulfate proteoglycan, which resolves into two heterodisperse size classes upon gel filtration on columns of Sepharose CL4B. After removal of SDS from these extracts by hydrophobic chromatography on Sep-Pak C18 cartridges, extracts were further fractionated on various affinity matrices. All of the sulfate-radiolabeled material eluted as one peak from DEAE-Sephadex ion-exchange columns. In contrast, affinity fractionation on Sepharose columns derivatized with the heparan sulfate-binding protein, platelet factor-4, resolved three major and one minor subsets of these components. The nonbinding fraction contained some heparan sulfate proteoglycan and some chondroitin sulfate. The weak-binding fraction contained principally heparan sulfate proteoglycan, as well as a small amount of chondroitin sulfate proteoglycan; the gel-filtration properties of these proteoglycans before or after alkaline borohydride treatment indicated that they were small in size, containing perhaps 2 to 4 glycosaminoglycan chains. The high-affinity fraction eluted from platelet factor 4-Sepharose was composed entirely of "single-chain" heparan sulfate. A portion of the heparan sulfate proteoglycan of the original extract bound to the hydrophobic affinity matrix, octyl-Sepharose, and this hydrophobic proteoglycan partitioned into the nonbinding and weak-binding fractions of the platelet factor 4-Sepharose affinity columns. These studies reveal that the majority of the proteoglycan made by these neuronal cells in culture is of the heparan sulfate class, is small in size when compared to other characterized proteoglycans, and can be resolved into several overlapping subsets when fractionated on affinity matrices.

Calcium↗
PubMed1983

Isolation and characterization of a large, neurite-associated glycoconjugate from neuroblastoma cells.

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.

Animals↗
PubMed1982

Calcium dependence and contraction in somite formation.

The existence of a calcium-dependent contractile process in the formation of somites from segmental plate mesoderm was investigated using a Ca2+ agonist and Ca2+ and calmodulin antagonists. The contribution of cell movement and apical constriction in the segmentation process were assessed using SEM of normal and drug-treated somite and segmental plate tissue. Explants that contained segmental plates of stage 14-15 chick embryos were cultured on vitelline membranes in calcium- and magnesium-free (CMF) Hands' solution, liquid culture medium, and medium containing drugs. Ca2+ ionophore A23187 promoted the rapid completion of one new somite pair. CMF halted segmentation. The Ca2+ antagonists verapamil and papaverine reversibly inhibited segmentation. Theophylline did not inhibit segmentation, suggesting that the effects of the Ca2+ antagonists are not due to inhibition of phosphodiesterase activity. These results suggest that somitogenesis is Ca2+-dependent. Two drugs that inhibit the binding of calmodulin, chlorpromazine and trifluoperazine (TFP), halted segmentation. The inhibitory effect of TFP was reversible. The effects of TFP on somites were compared with those of cytochalasin D. The contribution of microtubules to cell shape and movement in somitogenesis was examined by incubation with nocodazole, a reversible inhibitor of tubulin polymerization. Cell elongation and somitogenesis were inhibited.

Animals↗
PubMed1981

Epithelial cell-cell and cell substrate contacts.

An epithelial sheet maintains its normal morphology by an appropriate balance between cell to cell and cell to substrate contacts which must be adjusted as proliferation occurs. We review our studies which attempts to analyze some of these problems by recombining cell and matrix tissue components, or by analyzing developmental changes in intact tissue. Epithelial and mesenchymal cells placed on tissue matrices which have been denuded of their normal tissue components behave distinctively, suggesting that this procedure could reveal inherent differences. When chick epiblast is grown on the outer surface of the vitelline membrane, the mode of expansion changes from that seen in the normal site, the vitelline membrane inner surface. Dispersed skin and corneal cells studied during subsequent aggregation show that one cell can influence desmosome formation in another. Finally, recent studies on developing chick liver indicate a correlation between the percent of lateral cell surface in the junctional domain and the proliferative rate of these cells.

Animals↗
PubMedDate not supplied

An ependymal cell culture system for the study of spinal cord regeneration.

Successful regeneration of lesioned adult spinal cord in urodele (caudate) amphibians requires the action of injury-responsive ependymal cells (ependymoglia). The epithelial-to-mesenchymal transformation of ependymal cells following transection of the salamander spinal cord and the subsequent reformation of an epithelial tube have been described previously. A complete tissue culture model system has now been devised to study mesenchymal ependymal cells, epithelial ependymal cells, and ependymal/neuronal interactions in vitro. Here, we review critical aspects of substrate and growth factor environments required to produce mesenchymal ependymal cells in culture and present the first culture system for epithelial salamander ependymal cells and central nervous system neurons suitable for cell-cell interaction studies. Critical to ependymal epithelialization in culture is the removal of epidermal growth factor and addition of thrombin. Epithelialization occurs on tissue culture plastic as well as on permeable culture substrates. This culture system can now be used to determine the initial trigger for the ependymal response. A preliminary examination of ependymal/neuronal interactions shows that coculture of mesenchymal ependymal cells and central nervous system neurons prolongs survival of the neurons.

Animals↗