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R E Stephens

Publications and source records attributed to R E Stephens.

At least 73 records · Page 4Linked to original sources
PubMed1989

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57- and 65-kD bands present in the sensory epithelium and the cytoskeleton. It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57- and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125 A transverse stripping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.

Animals↗
PubMed1989

Retention of ciliary ninefold structure after removal of microtubules.

When axonemes of isolated gill cilia from the bay scallop Aequipecten irradians are heated at 45 degrees C for a minimum of 8 min in a 10 mM-Tris-HCl (pH 8), 1 mM-EDTA solution, nearly 80% of the tubulin is solubilized but most minor structural proteins are retained in a ninefold symmetrical configuration. This remnant consists of the junctional protofilaments, derived from outer doublet tubules, interconnected by nexin linkages, with radial spoke components still directed inwards. The remnant is of the same length as the original cilium, with the junctional protofilaments attached at the distal end to the ciliary tip and at the proximal end to the basal plate. Virtually identical fractionations can be achieved with blastula cilia isolated from both arctic and tropical sea-urchin embryos. The remnant is resistant to salt up to at least 1 M concentration, judged by the constancy of protein composition. Immunoblotting with antibodies against sea-urchin sperm flagellar tektins indicates that the tektins remain within the ciliary remnant, supporting their location within the junctional protofilament domain. The fractionation is inhibited by low pH, by magnesium or calcium ions in the millimolar range, and by monovalent ions at 10-fold higher concentrations. About a quarter of the total ciliary calmodulin is bound to the axoneme at micromolar calcium levels but most is released upon thermal fractionation. Polymerization of tubulin in the presence of the remnant results in singlet microtubules, separate from the remnant proper, suggesting that doublet formation may require coordinate co-assembly of tubulin with skeletal proteins. These observations demonstrate the existence of a fibrous skeleton in the axoneme, composed largely of ciliary tektins, nexin linkages, and other structural proteins.

Animals↗
PubMed1989

Quantal tektin synthesis and ciliary length in sea-urchin embryos.

Previous work using pulse-chase labelling of embryos from the sea-urchin Strongylocentrotus droebachiensis during ciliogenesis, regeneration or steady-state maintenance and elongation showed that a ciliary outer doublet microtubule-associated protein, originally termed component-20, was synthesized in a fixed or quantal amount. This suggested that the limited synthesis of component-20 might limit ciliary length, since the embryo has a large pool of most other ciliary components. Labelling experiments with S. purpuratus embryos now confirm quantal synthesis of component-20, while antibodies to S. purpuratus sperm flagellar tektins identify component-20 as the ciliary equivalent of the flagellar 55 x 10(3) Mr tektin, tektin A. Sequential pulse-chase labelling at various times prior to isolation of cilia proves that the high specific activity of this protein truly reflects de novo synthesis of a structurally stable protein and not rapid protein turnover. Embryos may be animalized by growth in the presence of zinc ions, resulting in cilia averaging nearly twice the normal 20 microns length. When these embryos are pulse-chase labelled during ciliary growth and elongation, labelling of tektin A is proportional to the greater ciliary length, as is the pool of labelled but unincorporated tektins and other minor proteins. Deciliated animalized and control embryos, pulse-chase labelled during their identical phases of ciliary regeneration, incorporate labelled tektin A to the same extent and have similar pools of unincorporated proteins. The correlation of enhanced tektin A synthesis with increased ciliary length and the coincidence of tektin A synthesis with ciliary elongation are observations consistent with the hypothesis that tektin A is a ciliary length-limiting structural element.

Animals↗
PubMed1989

Cytomegalovirus infection of gastrointestinal endothelium demonstrated by simultaneous nucleic acid hybridization and immunohistochemistry.

To demonstrate the degree of involvement of endothelial cells in cytomegalovirus (CMV) infection of the gastrointestinal tract we have stained sections from gastrointestinal specimens that showed inclusion bodies on hematoxylin-eosin staining. Factor VIII was first detected using a rabbit anti-factor VIII primary antibody and an alkaline phosphatase-labeled sheep anti-rabbit secondary antibody. The CMV was then visualized with a biotin-labeled CMV probe detected by a streptavidin peroxidase technique with aminoethyl carbazole as the chromogen. Factor VIII staining was a bright blue and CMV a brick red. The specimens included one small-bowel resection and four colonic resections, as well as an esophageal biopsy. The patients' diagnoses included bone marrow transplant recipient, acquired immunodeficiency syndrome, ulcerative colitis, and renal transplant recipient. Cells positive for both CMV and factor VIII ranged from 35% to 60% of positive cells in a representative section, and the relative percentages (mean +/- SE) for cell type for infected cells were: endothelial, 48.9 +/- 4.5; vascular luminal (factor VIII negative), 6.1 +/- 1.7; perivascular (factor VIII negative in vascular wall), 16.2 +/- 3.2; and other cell (non-vascular factor VIII negative), 28.9 +/- 5.1. These findings and clustering of infected cells around the vessels provide evidence that CMV infection of the gastrointestinal tract is primarily vasculitic and related to infection of endothelial cells.

Adult↗
PubMed1988

Separation of tubulin subunits by reversed-phase high-performance liquid chromatography.

When properly solubilized with trifluoroacetic acid (TFA), alpha- and beta-tubulin subunits from a variety of sources may be resolved at high yield by reversed-phase high-performance liquid chromatography (HPLC), using a Waters muBondapak C18 column and simple linear aqueous acetonitrile gradients containing TFA. The tubulin subunits are typically the most non-polar proteins present, with the beta-tubulin subunit eluting before the alpha. Column temperature above ambient improve both the resolution and the yield; less polar solvent systems do not. Tubulins not freely soluble in aqueous TFA may be solubilized in 6 M guanidine-hydrochloric acid with no change in retention time. Other columns with shorter carbon chain lengths and larger pore size produce a single, unresolved tubulin peak. Reversed-phase HPLC analysis provides an independent comparative evaluation of organelle-specific tubulins, with characteristic retention time differences observed between homologous ciliary and flagellar outer doublet tubulin subunits and also between them and their cytoplasmic counterparts.

Animals↗
PubMed1988

Neutralization of thrombin by antithrombin III in the presence of cultured human fibroblasts.

Recent evidence suggests that heparan sulfate on endothelial cell surfaces acts as a catalyst for the neutralization of thrombin by antithrombin III (AT III). Fibroblasts also produce heparan sulfate which is present on the cell surface and secreted into the extracellular matrix. We evaluated the ability of cultured human fibroblasts to catalyze the interaction between thrombin and AT III and found that heparan sulfate produced by post-confluent fibroblasts was anticoagulantly active. Furthermore, after initial binding of thrombin to cells, thrombin-heparan sulfate appeared in the fluid phase above the cells; this thrombin could be rapidly neutralized by AT III independent of the further presence of cells. These results indicate that fibroblasts do produce an anticoagulantly active species of heparan sulfate and that the normal interaction between AT III and thrombin may be driven by initial release of heparan sulfate from the cell surface by thrombin followed by AT III interaction with the soluble thrombin-heparan sulfate complex.

Antithrombin III↗
PubMed1988

EGTA induces prolonged summed depolarizations in Mytilus gill coupled ciliated epithelial cells: implications for the control of ciliary motility.

Abfrontal ciliated cells of Mytilus edulis gill beat when mechanically stimulated, a consequence of a Ca++-based generator potential and regenerative response. In contrast, the lateral ciliated epithelial cells arrest when stimulated, a consequence of a Ca++-based generator potential and a Na+/Ca++-based regenerative response. Iontophoretic injection of EGTA in abfrontal cells, followed by mechanical stimulation, results in a large, prolonged depolarization that returns to the resting level stepwise. It has been hypothesized that this phenomenon is caused by successive Ca++-dependent repolarizations in coupled cells, first in adjacent cells and then in the injected cell, in accord with relative EGTA loading. We have now demonstrated this same stepwise repolarization phenomenon in the Na+/Ca++-dependent lateral ciliated cells. In this case, each repolarization step is often preceded by a small spike. With either cell type, using two-electrode recording techniques, we can detect the stepwise repolarization in distant cells, proportionately decremented when the second (KCl) electrode is some distance from the injection (EGTA) electrode and stimulus. When force is applied between the electrodes and nearest the KCl electrode, a greater initial response is recorded from this electrode, presumably resulting from depolarization of its impaled cell, prolonged by EGTA diffusion through the intervening cell junctions. The subsequent repolarization steps are of approximately the same size, suggesting repolarization of cells between the two electrodes. These observations are consistent with the cell coupling/EGTA loading hypothesis and indicate that both cell types mediate repolarization through Ca++ and propagate ciliary beat or arrest through intracellular coupling.

Animals↗
PubMed1988

Sialyltransferase-1 in a human malignant glioma cell line. Kinetic characteristics and effect of human interferon-beta.

Sialyltransferase-1 activity was studied in cultured 12-18 human glioma cells. The apparent Km and Vmax with variable LacCer concentrations were 32 microM and 197 pmoles/mg protein/hr and with variable CMP-NeuAc concentrations were 172 microM and 877 pmoles/mg protein/hr., respectively. The pH optimum towards exogenous LacCer was 6.0 and towards endogenous acceptors was 6.2. The optimum protein:detergent ratio was 1:1. Human beta interferon (1000 units/ml medium) increased sialyltransferase-1 activity only slightly on a protein basis but increased it 47% on a per cell basis. These results demonstrate that one of the biochemical effects of beta-interferon on 12-18 human glioma cells is to stimulate ganglioside synthesis.

Cell Line↗
PubMed1988

Growth inhibition of cultured human glioma cells by beta-interferon is not dependent on changes in ganglioside composition.

This investigation tested the hypothesis that the growth inhibiting effects of human beta-interferon on cultured human glioma cells involves changes in the ganglioside composition of these cells. Four cell lines derived from human malignant gliomas (12-18, U-251 MG, I29-A, 7-24) and two lines from human fetal brain (CHI, CHII) were cultured in the presence and in the absence of human beta-interferon (HuIFN-beta), 1,000 units per ml medium for three days before harvesting. Human beta-interferon had an inhibitory effect on growth of glioma but not fetal brain cells. Total ganglioside sialic acid for all cell lines ranged between 3.5 and 13.8 micrograms/10(7) cells (0.6-3.9 micrograms/mg protein). No distinct difference in the amount of total ganglioside per cell was observed between neoplastic and non-neoplastic cells, but the latter had more ganglioside per mg total protein. All cell lines displayed different patterns of gangliosides determined by high performance thin layer chromatography, but there was no distinct difference between glioma and fetal brain cells. Human beta-interferon increased the total amount of ganglioside per cell in one fetal brain and two glioma lines, but on a protein basis in only one glioma cell line (I29-A); HuIFN-beta had only minor effects on ganglioside patterns. There was a slight shift towards a greater proportion of structurally simpler gangliosides in both fetal brain and two glioma cell lines exposed to HuIFN-beta, but the reverse occurred in glioma U-251 MG. None of these changes strongly correlated with the degree of growth inhibition due to HuIFN-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Neoplasms↗
PubMed1987

Biochemical characterization of tektins from sperm flagellar doublet microtubules.

Tektins, protein components of stable protofilaments from sea urchin sperm flagellar outer doublet microtubules (Linck, R. W., and G. L. Langevin, 1982, J. Cell Sci., 58:1-22), are separable by preparative SDS PAGE into 47-, 51-, and 55-kD equimolar components. High resolution two-dimensional tryptic peptide mapping reveals 63-67% coincidence among peptides of the 51-kD tektin chain and its 47- and 55-kD counterparts, greater than 70% coincidence between the 47- and 55-kD tektins, but little obvious similarity to either alpha- or beta-tubulin. With reverse-phase HPLC on a C18 column, using 6 M guanidine-HCl solubilization and a 0.1% trifluoroacetic acid/CH3CN gradient system (Stephens, R. E., 1984, J. Cell Biol. 90:37a [Abstr.]), the relatively less hydrophobic 51-kD tektin elutes at greater than 45% CH3CN, immediately followed by the 55-kD chain. The 47-kD tektin is substantially more hydrophobic, eluting between the two tubulins. The amino acid compositions of the tektins are very similar to each other but totally distinct from tubulin chains, being characterized by a greater than 50% higher arginine plus lysine content (in good agreement with the number of tryptic peptides) and about half the content of glycine, histidine, proline, and tyrosine. The proline content correlates well with the fact that tektin filaments have twice as much alpha-helical content as tubulin. Total hydrophobic amino acid content correlates with HPLC elution times for the tektins but not tubulins. The average amino acid composition of the tektins indicates that they resemble intermediate filament proteins, as originally postulated from structural, solubility, and electrophoretic properties. Tektins have higher cysteine and tryptophan contents than desmin and vimentin, which characteristically have only one residue of each, more closely resembling certain keratins in these amino acids.

Amino Acids↗
PubMed1987

Evidence that tubulin forms an integral membrane skeleton in molluscan gill cilia.

Controlled extraction of intact gill tissue, isolated cilia or reconstituted membrane vesicles with Nonidet P-40 at greater than 4 times the critical micelle concentration, or with octyl glucoside at the critical micelle concentration, delipidates the membrane, leaving a membrane remnant or skeleton of membrane tubulin and associated proteins. This skeleton consists of a disordered reticular protein network in reconstituted membrane vesicles and a similar but more compact sleeve in cilia of extracted tissue. The membrane skeleton is closely apposed to the axoneme and is attached to the outer doublets by fine radial bridges having a 20-24 nm longitudinal periodicity, supporting earlier observations made utilizing a lipophilic cross-linking agent. Higher concentrations of detergent solubilize the membrane tubulin-protein complex, producing 5-10 nm particulate material of low sedimentation coefficient. Dilution of an octyl glucoside solution to below the critical micelle concentration results in disappearance of the particles and reformation of the membrane, indicating that the particles are protein-detergent micelles and not denatured protein. Freeze-fracture electron microscopy reveals no comparable-sized natural particles in the ciliary membrane proper. The reticular material of the membrane skeleton contains tubulin, demonstrated on Lowicryl K4M thin sections by a rabbit polyclonal antibody to sea-urchin egg cytoplasmic tubulin, using gold-labelled secondary antibody. Minimal cross-reactivity is detected prior to Triton-delipidation, suggesting that most membrane tubulin antigenic sites are buried within the bilayer and that the tubulin is not simply adsorbed to the lipid bilayer.

Animals↗
PubMed1986

DNA excision repair in spontaneously diabetic BB Wistar rats.

BB Wistar rats develop a syndrome characterized by spontaneous diabetes mellitus as well as a wide variety of autoimmune, neoplastic, and degenerative disorders which do not occur in the outbred Wistar strain from which they were derived. This syndrome also includes elements of premature ageing (i.e., a markedly shortened lifespan and premature occurrence of diseases associated with ageing). Excision DNA repair capacity which has been reported to be directly proportional to maximal achievable life span was estimated in neonatal BB Wistar and outbred Wistar rats. Excision repair was assayed autoradiographically by determining unscheduled DNA synthesis following UV radiation of passage 3 cultured skin fibroblasts. No difference in excision repair capacity could be demonstrated between the two strains.

Animals↗
PubMed1986

Membrane tubulin.

Tubulin has been identified as a membrane component of nerve synaptosomes and myelin, plasma membranes of platelets, thyroid, and tissue culture cells, brain and liver coated vesicles, mitochondria, and in cilia but not flagella of certain molluscs. Membrane tubulin can differ from cytoplasmic forms in isoelectric point, non-polar amino acid substitutions, lack of carboxy-terminal tyrosine, carbohydrate content, and selective ability to reassociate with lipids. This tubulin may function as an attachment site for binding vesicles or plasma membranes to cytoplasmic microtubules, as a source of precursor tubulin at the growing tips of axonemes, or as a component of signal transduction in sensory cilia.

Animals↗
PubMed1986

A novel technique for viable cell determinations.

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 micrograms/ml; acridine orange, 1-5.0 micrograms/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.

Acridine Orange↗
PubMed1985

Ciliary membrane tubulin and associated proteins: a complex stable to Triton X-114 dissociation.

When either membranes from scallop gill cilia or reconstituted membranes from the same source are solubilized with Triton X-114 and the detergent is condensed by warming, no significant fraction of any major membrane protein partitions into the micellar detergent. Rather, most of the membrane lipids condense with the detergent phase, forming mixed micelles from which nearly pure lipid vesicles may be produced by adsorption of detergent with polystyrene beads. One minor membrane protein, with a molecular weight of about 20 000, is associated consistently with these vesicles. The aqueous phase contains a fairly homogeneous protein-Triton X-114 micelle sedimenting at 2.6 S in the analytical ultracentrifuge. Sucrose gradient velocity analysis in a detergent-free gradient indicates moderate size polydispersity but constant polypeptide composition throughout the sedimenting protein zone. Sucrose gradient equilibrium analysis (also in a detergent-free gradient) results in a protein-detergent complex banding at a density of 1.245 g/cm3. Sedimentation of the protein-detergent complex in the ultracentrifuge, followed by fixation and normal processing for electron microscopy, reveals a fine, reticular material consisting of 5-10-nm granules. These data are consistent with previous evidence that membrane tubulin and most other membrane proteins exist together as a discrete lipid-protein complex in molluscan gill ciliary membranes.

Animals↗
PubMed1985

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia.

Pure lateral (L) cilia may be separated from the remaining (R) cilia types of Mytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with 32P-labeled ATP at low Ca++ (10(-7) M), where L cilia beat, or in high Ca++ (2-20 microM), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5-15% gels, and autoradiographed. Neither cilia type undergoes Ca++-dependent phosphorylation of specific proteins, suggesting that neither Ca++-induced arrest in L cilia nor the Ca++ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca++-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca++-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP2). PIP2 increases at least 3-fold under Ca++-arrest conditions. Aequipecten gill lateral cilia, which require higher Ca++ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca++ levels. Phosphorylation of other lipids is Ca++-independent. In the Ca++-insensitive or activated R cilia, PIP2 levels are intermediate, increasing only marginally with increased [Ca++]. The formation of PIP2 in response to Ca++, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca++ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca++ ions, acting directly on the axoneme.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗