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Biomedical subjects

K Simons

Publications and source records attributed to K Simons.

At least 163 records · Page 9Linked to original sources
PubMed1987

Television retinoscopy with a slit-aperture retinoscope and a highly sensitive camera.

The low light level of the retinoscopic reflex and the small aperture required to obtain the customary shape of the reflex make it difficult to photograph the actual reflex for teaching or permanent recording. We describe the design and application of a television retinoscopy system that solves this problem. The system uses a standard streak retinoscope modified to gather more light, a highly sensitive television camera, and an image processor. The system can be used for live demonstration of retinoscopic techniques or for videotape recordings for teaching or research.

Equipment Design↗
PubMed1987

Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells.

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.

Animals↗
PubMed1986

Reconstitution of the fusogenic activity of vesicular stomatitis virus.

Enveloped virus glycoproteins exhibit membrane fusion activity. We have analysed whether the G protein of vesicular stomatitis virus, reconstituted into liposomes, is able to fuse nucleated cells in a pH-dependent fashion. Proteoliposomes produced by octylglucoside dialysis did not exhibit cell fusion activity of the G protein. However, by making use of n-dodecyl octaethylene monoether (C12E8) as the solubilizing agent and by removal of the detergent in two steps, we were able to produce fusogenic G protein liposomes. These G protein liposomes fuse to the BHK-21 cell surface at pH 5.7-6.0 with an efficiency of fusion comparable with that of the parent virus. Physical and chemical analysis revealed that the fusogenic liposomes exhibited a protein to lipid weight ratio of 0.67 and showed an average diameter of 130 nm.

Animals↗
PubMed1986

The trans Golgi network: sorting at the exit site of the Golgi complex.

The Golgi complex is a series of membrane compartments through which proteins destined for the plasma membrane, secretory vesicles, and lysosomes move sequentially. A model is proposed whereby these three different classes of proteins are sorted into different vesicles in the last Golgi compartment, the trans Golgi network. This compartment corresponds to a tubular reticulum on the trans side of the Golgi stack, previously called Golgi endoplasmic reticulum lysosomes (GERL).

Animals↗
PubMed1986

Floppy eyelid syndrome and blepharochalasis.

Floppy eyelid syndrome and blepharochalasis may represent a spectrum of one underlying disease. Two patients with floppy eyelid syndrome and one with blepharochalasis shared important clinical similarities. All three patients displayed eyelid laxity associated with papillary conjunctivitis and ocular surface abnormalities. Histopathologic findings were similar in the three cases, characterized by chronic conjunctival inflammation with normal tarsal collagen. In all three patients surgical horizontal eyelid shortening led to improvement in symptoms and findings.

Adult↗
PubMed1986

Two strains of the Madin-Darby canine kidney (MDCK) cell line have distinct glycosphingolipid compositions.

The glycosphingolipids (GSLs) of two sublines of Madin-Darby canine kidney (MDCK) cells, an epithelial cell line, were characterized by t.l.c., antibody overlay and mass spectrometry. The major characteristic which distinguishes the two MDCK cell strains is their trans-epithelial electrical resistance which is typically of the order of 3000 ohm.cm2 for strain I and 100 ohm.cm2 for strain II cells. Strain I and II cells were equally rich in glycolipids, the cellular GSL/phospholipid ratio being 0.04. However, while the phospholipid patterns were identical, the GSLs showed striking differences, and each cell strain expressed appreciable amounts of GSLs that were not found in the other strain. Both cell types possessed neutral GSLs with one, two or three carbohydrate moieties. The monoglycosylceramide accounted for 50% of the total GSLs in each strain. However, while in strain I cells over 90% of this monoglycosylceramide was monoglucosylceramide, in strain II cells over 90% consisted of monogalactosylceramide. In addition, MDCK strain II cells selectively expressed GSLs belonging to the globo series (26% of its neutral GSLs), including globoside and Forssman antigen, a globoside derivative. MDCK strain I cells, on the other hand, expressed another series of GSLs with 4-7 carbohydrate moieties characterized by the common sequence Hex-HexNAc-Hex-Hex-Cer. The presence of two fucosylated GSLs in these series was established. Both MDCK strain I and II cells contained negatively charged GSLs, the major component of which was the ganglioside GM3. MDCK strain II cells in addition expressed sulfatide, the sulfated derivative of galactosylceramide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

The function of tight junctions in maintaining differences in lipid composition between the apical and the basolateral cell surface domains of MDCK cells.

Tight junctions in epithelial cells have been postulated to act as barriers inhibiting lateral diffusion of lipids and proteins between the apical and basolateral plasma membrane domains. To study the fence function of the tight junction in more detail, we have fused liposomes containing the fluorescent phospholipid N-Rh-PE into the apical plasma membrane of MDCK cells. Liposome fusion was induced by low pH and mediated by the influenza virus hemagglutinin, which was expressed on the apical cell surface after viral infection. Redistribution of N-Rh-PE to the basolateral surface, monitored at 0 degree C by fluorescence microscopy, appeared to be dependent on the transbilayer orientation of the fluorescent lipids in the plasma membrane. Asymmetric liposomes containing over 85% of the N-Rh-PE in the external bilayer leaflet, as shown by a phospholipase A2 assay, were generated by octyl beta-D-glucoside dialysis. When these asymmetric liposomes were fused with the apical plasma membrane, fluorescent lipid did not move to the basolateral side. Symmetric liposomes which contained the marker in both leaflets were obtained by freeze-thawing asymmetric liposomes or by reverse-phase evaporation. Upon fusion of these with the apical membrane, redistribution to the basolateral membrane occurred immediately. Redistribution could be observed with asymmetric liposomes only when the tight junctions were opened by incubation in a Ca2+-free medium. During the normal experimental manipulations the tight junctions remained intact since a high trans-epithelial electrical resistance was maintained over the cell monolayer. We conclude that the tight junction acts as a diffusion barrier for the fluorescent phospholipid N-Rh-PE in the exoplasmic leaflet of the plasma membrane but not in the cytoplasmic leaflet.

Animals↗
PubMed1986

The budding mechanism of spikeless vesicular stomatitis virus particles.

Virus particles, lacking the spike G-glycoproteins, are produced during infection of Vero cells with the vesicular stomatitis virus mutant ts045 at the restrictive temperature 39.5 degrees C. At this temperature the mutated G proteins are blocked in their intracellular transport in the endoplasmic reticulum. We have studied the role of the G proteins in the formation of these spikeless virus particles. The results showed that the spikeless particles contain a full complement of membrane anchors, derived from the carboxy-terminal end of the G protein. Our observations suggest that virus particles are formed at the restrictive temperature with G protein which is later cleaved to produce spikeless particles. We suggest that this is due to a leak of G protein to the cell surface at 39.5 degrees C where budding then takes place, presumably driven by a G protein C-terminal tail--nucleocapsid interaction.

Animals↗
PubMed1986

A functional assay for proteins involved in establishing an epithelial occluding barrier: identification of a uvomorulin-like polypeptide.

A functional assay has been developed to identify cell surface proteins involved in the formation of epithelial tight junctions. Transepithelial electrical resistance was used to measure the presence of intact tight junctions in monolayers of Madin-Darby canine kidney (MDCK) cells cultured on nitrocellulose filters. The strain I MDCK cells used have a transmonolayer resistance greater than 2,000 ohm . cm2. When the monolayers were incubated at 37 degrees C without Ca2+, the intercellular junctions opened and the transmonolayer resistance dropped to the value of a bare filter, i.e., less than 40 ohm . cm2. When Ca2+ was restored, the cell junctions resealed and the resistance recovered rapidly. Polyclonal antibodies raised against intact MDCK cells inhibited the Ca2+-dependent recovery of electrical resistance when applied to monolayers that had been opened by Ca2+ removal. Cross-linking of cell surface molecules was not required because monovalent Fab' fragments also inhibited. In contrast, a variety of other antibodies that recognize specific proteins on the MDCK cell surface failed to inhibit the recovery of resistance. Monoclonal antibodies have been raised and screened for their ability to inhibit resistance recovery. One such monoclonal antibody has been obtained that stained the lateral surface of MDCK cells. This antibody, rr1, recognized a 118-kD polypeptide in MDCK cell extracts and an 81-kD fragment released from the cell surface by trypsinization in the presence of Ca2+. Sequential immunoprecipitation with antibody rr1 and a monoclonal antibody to uvomorulin showed that this polypeptide is related to uvomorulin. The role of uvomorulin-like and liver cell adhesion molecule (L-CAM)-like polypeptides in the establishment of the epithelial occluding barrier is discussed.

Animals↗
PubMed1986

Transferrin receptor polarity and recycling accuracy in "tight" and "leaky" strains of Madin-Darby canine kidney cells.

We have characterized the polarity of the transferrin receptor in the epithelial Madin-Darby canine kidney (MDCK) cell line. The receptor is present in approximately 165,000 copies per cell, migrates as a diffuse band upon SDS gel electrophoresis with Mr 90,000, displays a dissociation constant for diferritransferrin at neutral pH of approximately 2 nM, and is active in essentially all of the cells of the population. Transferrin-mediated 55Fe uptake was used to measure the polarity of active transferrin receptors in filter-grown MDCK cells. The ratio of basolateral to apical receptors was approximately 800:1 for the high resistance strain I MDCK cells (typically greater than 2,000 ohm X cm2) and approximately 300:1 for the lower resistance strain II cells (less than 350 ohm X cm2). In combination with morphometric data this shows that a difference in resistance between these two strains is not reflected in a significant difference in cell surface polarity. We used the recycling of transferrin receptor in filter-grown MDCK cells to evaluate the accuracy of the sorting of a basolateral protein during endocytosis. Monitoring the amount of apically released 125I-labeled transferrin after application of 55Fe- and 125I-labeled transferrin to the basolateral surface provided a sensitive assay of the accuracy of sorting during recycling of the receptor from endosomes to the plasma membrane. The accuracy of transferrin receptor sorting (greater than 99.88%) during a single cycle of transit between the endosome and the plasma membrane is sufficient to maintain the high level of polarity of the cell.

Animals↗
PubMed1985

Parameters affecting low-pH-mediated fusion of liposomes with the plasma membrane of cells infected with influenza virus.

Unilamellar liposomes can be fused at low pH with the plasma membrane of cells that express the hemagglutinin glycoprotein of influenza virus on their surface [van Meer, G., & Simons, K. (1983) J. Cell Biol. 97, 1365-1374]. Here, we have resolved this fusion process into two kinetically distinct steps. The first and more rapid step converts the bound liposome to a form that can no longer be released by neuraminidase. The second step is the actual membrane fusion as measured by the loss of resonance energy transfer between two liposomal fluorescent phospholipids, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dioleoylphosphatidylethanolami ne (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)dioleoylphosphatidylethanolamine (N-Rh-PE). In contrast to the first step, the rate of the second one was highly dependent on the liposomal lipid composition and the cell type used. The replacement of 50% of the phosphatidylcholine (PC) in egg PC-cholesterol liposomes by unsaturated phosphatidylethanolamine (PE) species increased the rate of fusion at least 2-fold. Of the PE-containing liposomes that were associated with Madin-Darby canine kidney (MDCK) cells after 30 s of fusion, 80% had actually fused with the plasma membrane. Fringe pattern fluorescence photobleaching experiments showed that after fusion a fraction of the cell-associated N-Rh-PE diffused laterally in the plasma membrane. Without fusion, the N-Rh-PE was completely immobile. Under optimal conditions, the mobile fractions were 65% on MDCK cells and 78% on baby hamster kidney cells. The mobility was acquired simultaneously with the dilution of the fluorescent phospholipids as measured from the loss of resonance energy transfer. The mobile fraction of N-Rh-PE on the cell surface can therefore be used as a second independent measure of actual membrane fusion. Finally, we observed that upon fusion up to 80% of the nonexchangeable liposome markers cholesterol [14C]oleate and glycerol tri[14C]oleate became accessible to cellular hydrolases. The results showed that this hydrolysis assay can also be used to monitor the second step of the fusion process.

Animals↗
PubMed1985

An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments.

The expression of viral envelope proteins on the plasma membrane domains of the epithelial cell line, MDCK, is polar. Influenza virus infection of these cells leads to expression of the viral haemagglutinin and neuraminidase glycoproteins on the apical domain of the plasma membrane while vesicular stomatitis virus (VSV) infection yields basolateral expression of the sialic acid-bearing G protein. We have exploited the ability of the influenza neuraminidase to desialate the G protein of VSV to test for contact between these proteins during their intracellular transport to separate plasma membrane domains. We were able to select for VSV-G protein expression in doubly-infected cells because VSV protein production was accelerated in cells pre-infected with influenza virus. During double infection the envelope proteins of both viruses displayed the same polar localization as during single infection but the VSG-G protein was undersialated due to the action of the influenza neuraminidase. Incubation of singly-infected cells at 20 degrees C blocked the transport of VSV-G protein to the cell surface and resulted in increased sialation of the protein over that seen at 37 degrees C. This suggests that G protein is held in contact with the sialyl transferase at this temperature. 20 degrees C incubations of doubly-infected cells also produced the undersialated G protein characteristic of interaction with the neuraminidase. We conclude that most of the newly synthesised basolaterally-directed G protein is in physical contact with the majority of the neuraminidase through the terminal steps of Golgi processing.

Animals↗
PubMed1985

Apical and basolateral endocytosis in Madin-Darby canine kidney (MDCK) cells grown on nitrocellulose filters.

Madin-Darby canine kidney (MDCK) cells (strain I) grown on 0.45 micron pore size nitrocellulose filters formed monolayers which were highly polarized and had high transepithelial electrical resistance (greater than 3000 ohm X cm2). Morphometric analysis showed that the area of the basolateral surface domain was 7.6 times larger than that of the apical. The uptake of fluid-phase markers [3H]inulin and horseradish peroxidase (HRP) was studied from the apical and the basal side of the monolayer. Uptake of [3H]inulin was biphasic and the rate during the first 40 min corresponded to a fluid phase uptake of 20.5 X 10(-8) nl/min per cell from the basolateral side, and 1.0 X 10(-8) nl/min per cell from the apical side. Electron micrographs of the monolayers after HRP uptake showed that the marker was rapidly delivered into endosome-like vesicles and into multivesicular bodies. No labelling of the Golgi complex could be observed during 2 h of uptake. Evidence was obtained for the transport of fluid phase markers across the cell. HRP and fluorescein isothiocyanate-dextran crossed the monolayers in either direction at a rate corresponding to approximately 3 X 10(-8) nl of fluid/min/cell. Adding the transcytosis rate to the rate of fluid accumulation into the cell yielded a total basolateral endocytic rate which was 6-fold greater than the apical rate. When the uptake rates were normalized for membrane area the apical and basolateral endocytic rates were about equal per unit cell surface area.

Animals↗
PubMed1985

Subunit vaccines against enveloped viruses: virosomes, micelles and other protein complexes.

The envelope proteins (the peplomers) of enveloped viruses are the components that are important for induction of protective immunity. This article reviews methods and problems of making subunit vaccines of peplomers. In the first section, the solubilization of enveloped viruses with detergent is discussed. The preparation of envelope proteins into defined different physical forms is described, i.e. monomeric and micelle forms and the reconstitution of the protein into lipid vesicles (virosomes). Finally, the preparation of a new type of complex is described (named iscom), which is highly immunogenic. In the following sections the efficacy of the different physical forms are reviewed and it is concluded that monomeric forms must be avoided since they are poorly immunogenic and they may even have a suppressive effect on the immune response. The multimeric micelles, virosomes and iscoms are all immunogenic. The iscom is an interesting new concept that can be used to produce efficient subunit vaccines.

Adjuvants, Immunologic↗
PubMed1985

Intracellular sorting and basolateral appearance of the G protein of vesicular stomatitis virus in Madin-Darby canine kidney cells.

The polarity of the surface distribution of viral glycoproteins during virus infection has been studied in the Madin-Darby canine kidney epithelial cell line on nitrocellulose filters. Using a surface radioimmunoassay on Madin-Darby canine kidney strain I cells that had been infected with vesicular stomatitis virus or with avian influenza fowl plague virus, we found that the surface G protein was 97% basolateral, whereas the fowl plague virus hemagglutinin was 88% apical. Newly synthesized, pulse-labeled vesicular stomatitis virus appeared first on the basolateral plasma membrane as measured by an immunoprecipitation assay in which the anti-G protein antibody was applied to the monolayer either from the apical or the basolateral side. Labeled G protein could be accumulated inside the cell at a late stage of transport by decreasing the temperature to 20 degrees C during the chase. Reversal to 37 degrees C led to its rapid and synchronous transport to the basolateral surface at an initial rate 61-fold greater than that of transport to the apical side. These results demonstrate that the newly synthesized G protein is transported directly to the basolateral membrane and does not pass over the apical membrane en route. Since a previous study of the surface appearance of influenza virus hemagglutinins showed that the newly synthesized hemagglutinins were inserted directly from an intracellular site into the apical membrane (Matlin, K., and K. Simons, 1984, J. Cell Biol., 99:2131-2139), we conclude that the divergence of the transport pathway for the apical and basolateral viral glycoproteins has to occur intracellularly, i.e., before reaching the cell surface.

Animals↗