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Biomedical subjects

M Schindler

Publications and source records attributed to M Schindler.

At least 127 records · Page 7Linked to original sources
PubMed1987

A nuclear specific glycoprotein representative of a unique pattern of glycosylation.

Whole rat liver nuclei were reacted with UDP-[14C]galactose in the presence of bovine beta(1----4) galactosyltransferase. The reaction mixture was electrophoresed on a reducing sodium dodecyl sulfate-polyacrylamide gel. Autoradiograms of the gel demonstrated a major labeled broad band migrating with an apparent molecular weight of 65,000-66,000. A number of other less prominently labeled bands were also present. The labeled 65,000-66,000 band when cut from the gel and subjected to alkaline reduction while in the gel matrix exclusively yielded a 14C-labeled disaccharide that co-migrated with a [14C]Gal-GlcNAcol standard in descending paper chromatography. Treatment of this disaccharide with beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) from Aspergillus niger removed all the [14C]galactose label. Treatment of the labeled 65,000-66,000 polypeptide with Endoglycosidase F, however, did not remove the [14C]galactose label. Western transfer blots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels performed with horseradish peroxidase-labeled succinyl wheat germ agglutinin, a lectin specific for GlcNAc, on unlabeled nuclei revealed a dominant band at 63,000-64,000. Subjecting 14C-labeled nuclei to this procedure resulted in a shift of the major horseradish peroxidase-labeled succinyl wheat germ agglutinin band to 65,000-66,000. The shifted band was coincident with the [14C]galactose band as visualized on an autoradiogram. A survey of other rat tissue nuclei revealed the same spectrum of [14C]galactose acceptor proteins with a dominant 65,000-66,000 galactose-labeled band.

Animals↗
PubMed1987

Immunological evidence for gap junction polypeptide in plant cells.

A whole cell homogenate prepared from soybean (Glycine max (L.) Merr. cv. Mandarin) root cells (SB-1 cell line) was electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and transferred to nitrocellulose paper. The nitrocellulose was probed with a monospecific antibody capable of recognizing the Mr 27,000 polypeptide of rat liver gap junctions; this antibody was prepared from immune serum raised against gap junctions purified from V79 cells (Chinese lung fibroblasts). The immunoblots afforded two polypeptides migrating at Mr 29,000 and 48,000. This pattern of blotting was also observed when homogenates of soybean or poinsettia leaves excised from whole plants were probed with anti-V79 gap junction antiserum. Gap junction purification schemes, developed for rat liver (Hertzberg, E. L. (1984) J. Biol. Chem. 259, 9936-9943), were employed on soybean protoplast homogenates yielding a significant enrichment for the Mr 29,000 and 48,000 polypeptides as judged by Coomassie Blue staining and immunoblotting with anti-V79 gap junction antiserum. These immunological results provide the first reported evidence for a homologous gap junction polypeptide in plant cells.

Animals↗
PubMed1987

Enucleation of normal and transformed cells.

A quantitative analysis based on centrifugal force requirements for enucleation was developed to examine the response of a number of untransformed and transformed cell lines to cytochalasin mediated enucleation. Examination of the extent of cell enucleation as a function of centrifugal force resulted in a series of response curves demonstrating that enucleation g force requirements varied between Balb/c 3T3, Swiss 3T3, and Kirsten sarcoma virus transformed Balb/c 3T3 (3T3-K). A four times greater centrifugal force was required to reach 50% enucleation for transformed Balb/c 3T3-K when compared to Swiss 3T3. A qualitative correlation could be observed between ease of enucleation and the existence of a well-formed stress fiber network. A comparison of cytochalasin B and D suggested that cytochalasin D was far more effective in the enucleation of transformed cells. Experiments with 2-deoxyglucose and monensin provided evidence that decreasing cellular ATP levels, either directly or potentially by uncoupling ion transport from ATP generation, can decrease the efficiency of enucleation. It is suggested that the organization of the cytoskeleton is affected by the altered cellular ATP levels which can affect the centrifugal requirements of enucleation.

Actins↗
PubMed1987

Endogenous lectins from cultured cells: nuclear localization of carbohydrate-binding protein 35 in proliferating 3T3 fibroblasts.

Proliferating 3T3 mouse fibroblasts contain higher levels of the lectin carbohydrate-binding protein 35 (CBP35) than do quiescent cultures of the same cells. An immunofluorescence study was carried out with a rabbit antiserum directed against CBP35 to map the cellular fluorescence distribution in a large population of cells under different growth conditions. This cytometric analysis showed that the lectin is predominantly localized in the nucleus of the proliferating cells. In quiescent 3T3 cultures, the majority of the cells lost their nuclear staining and underwent a general decrease in the overall fluorescence intensity. Stimulation of serum-starved quiescent 3T3 cells by the addition of serum resulted in an increase in the level of CBP35. The percentage of cells showing distinct punctate intranuclear staining reached a maximum at about the same time as the onset of the first S-phase of the cell cycle. All of these results suggest that CBP35 may be a protein whose presence in the nucleus, in discrete punctate distribution, is coordinated with the proliferation state of the cell.

Animals↗
PubMed1987

Epidermal growth factor and insulin stimulate nuclear pore-mediated macromolecular transport in isolated rat liver nuclei.

Fluorescence photobleaching was used to measure the effect of epidermal growth factor (EGF), insulin, and glucagon on the nuclear transport of fluorescent-labeled dextrans across the nuclear pore complex. EGF and insulin were found to stimulate transport approximately 200%, while boiling these polypeptide growth factors greatly diminished this enhancement activity. Glucagon demonstrated no enhancement effect. The nuclear transport enhancement effects were observed at EGF and insulin concentrations that elicit the various physiological responses, e.g., nanomolar range.

Animals↗
PubMed1987

Pharmacokinetics of vancomycin in serum and tissue of patients undergoing open-heart surgery.

Penetration of vancomycin into serum, heart valves, subcutaneous tissue and muscle was determined in 33 adult patients undergoing open-heart surgery. Each patient received 15 mg/kg vancomycin as a 30-min intravenous infusion preoperatively. Within 6 h vancomycin plasma concentrations declined from 28.9 to 4.2 mg/l. Vancomycin concentrations decreased in subcutaneous tissue slowly and varied in muscle between 1.2 and 3.2 mg/kg, in subcutaneous tissue between 1.3 and 4.4 mg/kg and in heart valves between 2.3 and 4.2 mg/kg. Vancomycin concentrations in heart valves are high enough to inhibit most oxacillin-resistant Staphylococcus aureus, and coagulase-negative staphylococci causing postoperative wound infections and endocarditis.

Adipose Tissue↗
PubMed1987

Penetration of ceftazidime into heart valves and subcutaneous and muscle tissue of patients undergoing open-heart surgery.

Patients undergoing open-heart surgery were given an intravenous bolus injection of 2 g of ceftazidime as a single dose over a period of 5 min. Within 12 h, ceftazidime concentrations in serum declined from 55.8 to 3.9 mg/liter. Subcutaneous tissue concentrations of the drug decreased from 21.0 to 2.7 micrograms/g, and muscle concentrations decreased from 34.5 to 2.5 micrograms/g. Ceftazidime concentrations in cardiac valvular tissue were even higher than those in muscle or fat, declining from 37.4 to 6.3 micrograms/g within 10 h.

Adipose Tissue↗
PubMed1986

A fluorescence photobleaching assay of gap junction-mediated communication between human cells.

Gap junction-mediated communication between contiguous cells has been implicated in the regulation of cell proliferation and differentiation. This report describes a new technique to measure cell-cell communication, gap fluorescence redistribution after photobleaching, which is based on the diffusion-dependent return of 6-carboxyfluorescein-mediated fluorescence in a photobleached cell that is in contact with other fluorescently labeled cells. Fluorescence recovery rates are interpreted as dye transport across gap junctions. Results of experiments on normal human fibroblasts and human teratocarcinoma cells show that this technique can measure rapid dye transfer and detect inhibition of communication (between teratocarcinoma cells) by the tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and the pesticide dieldrin.

Cell Communication↗
PubMed1986

Lateral diffusion of phospholipids in the plasma membrane of soybean protoplasts: Evidence for membrane lipid domains.

Fluorescent lipid and phospholipid probes were incorporated at 4 degrees C into soybean protoplasts prepared from cultured soybean (SB-1) cells. Fluorescence microscopy showed that the plasma membrane as well as the nucleus were labeled. Fluorescence redistribution after photobleaching (FRAP) analysis was performed on these cells at 18 degrees C to monitor the lateral mobility of the incorporated probes. After labeling at low concentrations (40 mug/ml) of phosphatidyl-N-(4-nitrobenzo-2-oxa-1,3-diazolyl)ethanolamine (NBD-PtdEtn), a single mobile component was observed with a diffusion coefficient (D) of approximately 3 x 10(-9) cm(2)/sec. After labeling at higher probe concentrations (>/=100 mug/ml), two diffusing species were observed, with diffusion coefficients of approximately 3 x 10(-9) cm(2)/sec ("fast") and approximately 5 x 10(-10) cm(2)/sec ("slow"). Similar results were observed with fluorescent derivatives of phosphatidylcholine and fatty acids. In contrast to these results, parallel analysis of 3T3 fibroblasts, using the same probes and conditions, yielded only a single diffusion component. These results suggest that the soybean plasma membrane may contain two distinct lipid domains in terms of lipid mobility. Consistent with this idea, experiments with soybean protoplasts yielded a single diffusion component under the following conditions: (i) labeling with NBD-PtdEtn (100 mug/ml), FRAP analysis at 37 degrees C (D = 1.1 x 10(-8) cm(2)/sec); (ii) labeling with NBD-PtdEtn (100 mug/ml), FRAP analysis at 18 degrees C in the presence of 2 mM EGTA (D = 4.2 x 10(-9) cm(2)/sec); (iii) labeling with 5-(N-dodecanoyl)aminofluorescein (a short-chain lipid probe), FRAP analysis at 18 degrees C or 37 degrees C (D = 2.5 x 10(-8) cm(2)/sec). These results suggest that the plasma membrane of soybean cells may contain stable immiscible domains of fluid and gel-like lipids.

Journal Article↗
PubMed1986

Chemical factors that influence nucleocytoplasmic transport: a fluorescence photobleaching study.

The technique of fluorescence redistribution after photobleaching was used to measure the translocation rate of fluorescein-labeled dextrans across the nuclear pore complex in isolated rat liver nuclei. A transport assay system was established that could monitor the effect of biologically active molecules, e.g., ATP, GTP, cAMP on the translocation process. The results show that ATP, phosphoinositides, RNA, and insulin can enhance transport rates from 195 to 432%. It was further demonstrated that concanavalin A, but not wheat germ or soybean agglutinin, can block dextran transport completely. The effectors of dextran transport are similar to substances demonstrated to effect the efflux of RNA from isolated nuclei. A model for translocation through the nuclear pore is now presented that incorporates data from protein influx and RNA efflux experiments into a single pathway controlled by ATP.

Adenosine Triphosphate↗
PubMed1986

Nuclear actin and myosin as control elements in nucleocytoplasmic transport.

Fluorescence redistribution after photobleaching (FRAP) was used to examine the role of actin and myosin in the transport of dextrans through the nuclear pore complex. Anti-actin antibodies added to isolated rat liver nuclei significantly reduced the flux rate of fluorescently labeled 64-kD dextrans. The addition of 3 mM ATP to nuclei, which enhances the flux rate in control nuclei by approximately 250%, had no enhancement effect in the presence of either anti-actin or anti-myosin antibody. Phalloidin (10 microM) and cytochalasin D (1 micrograms/ml) individually inhibited the ATP stimulation of transport. Rabbit serum, anti-fibronectin, and anti-lamins A and C antibodies had no effect on transport. These results suggest a model for nuclear transport in which actin/myosin are involved in an ATP-dependent process that alters the effective transport rate across the nuclear pore complex.

Actins↗
PubMed1986

Monoclonal antibodies directed against protoplasts of soybean cells: analysis of the lateral mobility of plasma membrane-bound antibody MVS-1.

A monoclonal antibody (MVS-1) was used to monitor the lateral mobility of a defined component (Mr approximately 400,000) of the plasma membrane of soybean protoplasts prepared from suspension cultures of Glycine max (SB-1 cell line). The diffusion coefficient (D) of antibody MVS-1 bound to its target was determined (D = 3.2 X 10(-10) cm2/s) by fluorescence redistribution after photobleaching. Pretreatment of the protoplasts with soybean agglutinin (SBA) resulted in a 10-fold reduction of the lateral mobility of antibody MVS-1 (D = 4.1 X 10(-11) cm2/s). This lectin-induced modulation could be partially reversed by prior treatment of the protoplasts with either colchicine or cytochalasin B. When used together, these drugs completely reversed the modulation effect induced by SBA. These results have refined our previous analysis of the effect of SBA on receptor mobility to the level of a defined receptor and suggest that the binding of SBA to the plasma membrane results in alterations in the plasma membrane such that the lateral diffusion of other receptors is restricted. These effects are most likely mediated by the cytoskeletal components of the plant cell.

Antibodies, Monoclonal↗
PubMed1986

Endogenous lectins from cultured soybean cells: isolation of a protein immunologically cross-reactive with seed soybean agglutinin and analysis of its role in binding of Rhizobium japonicum.

Incubation of Rhizobium japonicum with the cultured soybean cell line SB-1, originally derived from the roots of Glycine max, resulted in specific adhesion of the bacteria to the plant cells. This binding interaction appears to be mediated via carbohydrate recognition, since galactose can inhibit the heterotypic adhesion but glucose cannot. Affinity chromatography, on a Sepharose column derivatized with N-caproyl-galactosamine, of the supernatant fraction of a SB-1 cell suspension after enzymatic removal of cell wall yielded a single polypeptide (Mr approximately 30,000) on immunoblotting analysis with rabbit antibodies directed against seed soybean agglutinin. Fluorescently labeled rabbit anti-seed soybean agglutinin also yielded specific immunofluorescent staining on the cell wall and plasma membrane of the SB-1 cells. These results suggest that one likely candidate that may mediate the recognition between the Rhizobium and the soybean cells is the endogenously produced SB-1 lectin. This notion is supported by the observation that rabbit anti-seed soybean agglutinin blocked the Rhizobium-soybean cell adhesion, whereas control antibodies did not.

Cell Adhesion↗
PubMed1985

Mobility in the mitochondrial electron transport chain.

The role of lateral diffusion in mitochondrial electron transport has been investigated by measuring the diffusion coefficients for lipid, cytochrome c, and cytochrome oxidase in membranes of giant mitoplasts from cuprizone-fed mice using the technique of fluorescence redistribution after photobleaching (FRAP). The diffusion coefficient of the phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine is dependent on the technique used to remove the outer mitochondrial membrane. A sonication technique yields mitoplasts with monophasic recovery of the lipid probe (D = 6 X 10(-9) cm2/s), while digitonin-treated mitochondria show biphasic recoveries (D1 = 5 X 10(-9) cm2/s; D2 = 1 X 10(-9) cm2/s). Digitonin appears to incorporate into mitoplasts, giving rise to decreased lipid mobility concomitant with increased rates of electron transfer from succinate to oxygen, in a manner reminiscent of the effects of cholesterol incorporation [Schneider, H., Lemasters, J. J., Hochli, M., & Hackenbrock, C. R. (1980) J. Biol. Chem. 255, 3748-3756]. FRAP measurements on tetramethylrhodamine cytochrome c modified at lysine-39 and on a mixture of active morpholinorhodamine derivatives of cytochrome c gave diffusion coefficients of (3.5-7) X 10(-10) cm2/s depending on the assay medium. With morpholinorhodamine-labeled antibodies purified on a cytochrome oxidase affinity column, the diffusion coefficient for cytochrome oxidase was determined to be 1.5 X 10(-10) cm2/s. The results are discussed in terms of a dynamic aggregate model in which an equilibrium exists between freely diffusing and associated electron-transfer components.

Animals↗
PubMed1985

Automated analysis and survival selection of anchorage-dependent cells under normal growth conditions.

An instrument is described that can automatically analyze and select for a subpopulation of anchorage-dependent cells in tissue culture. Cells that label with fluorescently tagged antibodies or demonstrate structural variations are saved from exposure to a destructive high-intensity argon laser beam. The surviving population may then be cloned. The cell selection may occur in a tissue culture plate or in a microflow incubator which is designed to maintain a constant flow of media at 37 degrees C across cells growing on a glass coverslip. This incubator sits on an inverted microscope which focuses the laser beam to a diameter as small as 1 micron. A high-speed computer-controlled two-dimensional stage moves the cells past the beam for analysis, the results of which determine the fate of each cell: whether it is to be destroyed by radiant energy or selected for survival and subsequent proliferation. Another selection strategy performed by the instrument involves growing the cells on a thin, blackened polyester film which can be cut by the argon laser beam. Cells selected for cloning are then circumscribed. The heat of cutting welds the circumscribed film to a plastic coverslip surface or tissue culture chamber bottom. Nonselected cells may be removed by pulling the unattached polyester sheet from the attachment surface. The selected cells remain on polyester film disks welded to the plastic. Selections may be done automatically under computer control or manually by operator direction of stage movements. This instrument extends the art of automated cell selection and analysis to normal cell lines that must maintain cell-substratum contact (anchorage dependence) for differentiated cell function, e.g., neurons, fibroblasts, or kidney cells.

Cell Adhesion↗