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Biomedical subjects

F Schroeder

Publications and source records attributed to F Schroeder.

At least 163 records · Page 9Linked to original sources
PubMed1984

Membrane anomalies in Huntington's disease fibroblasts.

Plasma membranes, microsomes, and mitochondria were isolated from paired, passage number matched, cultured human fibroblasts. The cells were obtained from skin biopsies of Huntington's disease (HD) subjects and from sex and age matched controls. All fibroblasts were cultured in identical media for three to seven passages. Enrichment of surface marker enzymes such as Na+,K+-ATPase indicated a 10-fold purification of the isolated plasma membrane. The specific activity of Na+,K+-ATPase was 62 and 82% greater in the crude homogenate and isolated plasma membrane, respectively, of HD fibroblasts than in control fibroblasts. The specific activity of plasma membrane Na+,K+-ATPase was correlated with lipid composition and with membrane structure as determined by measurement of the rotational relaxation time and limiting anisotropy of fluorescence probe molecules. Major alterations in the structure of the plasma membranes in HD fibroblasts were not noted. The rotational relaxation time and limiting anisotropy of 1,6-diphenyl-1,3,5-hexatriene and of trans-parinaric acid were not significantly different between the plasma membrane, microsomes, or mitochondria of HD versus those of control fibroblasts. trans-Parinaric acid demonstrated the coexistence of fluid and solid domains in all three subcellular membrane fractions of the normal and HD skin fibroblasts. Lastly, both trans-parinaric acid and 1,6-diphenyl-1,3,5-hexatriene displayed characteristic breakpoints in Arrhenius plots of absorbance corrected fluorescence in plasma membranes, microsomes, and mitochondria. In all cases, similar breakpoint temperatures, indicative of phase alterations, were noted near 20 degrees and 30 degrees C. These breakpoints were unaltered in HD. In summary, the data do not support the concept of major membrane structural defects in HD.

Cell Fractionation↗
PubMed1984

Membrane lipids and enzymes of cultured high- and low-metastatic B16 melanoma variants.

B16 melanoma cell variants were used to determine if the metastatic properties of these cells could be correlated to distinct plasma membrane, microsome, and mitochondrial membrane lipid compositions and membrane-bound enzyme activities in high- and low-metastatic cell variants, respectively. The high-metastatic B16-F10 melanoma cell membranes had lower cholesterol/phospholipid ratios, lower arachidonic acid content, lower polyunsaturated fatty acid content, higher phosphatidylcholine/phosphatidylethanolamine ratios, and higher succinate cytochrome c reductase activity than those of B16-F1 melanoma cell membranes. No differences in cholesterol/phospholipid ratio were noted in the mitochondria. Na+-K+-adenosinetriphosphatase activity and solubility of 5'-nucleotidase activity were also similar. The data indicate that the membrane lipid composition of B16-F10 melanoma cells is distinct from that of B16-F1 melanoma cells and may help to elucidate the molecular basis for the different metastatic properties of these cell lines in vivo.

Adenosine Triphosphatases↗
PubMed1983

Binding of [3H]flunitrazepam to the LM cell, a transformed murine fibroblast.

LM cells have a saturable, high affinity binding site for [3H]flunitrazepam with a KD of 13 nM and a Bmax of 19 pmoles/mg protein. The IC50 values for Ro 5-4864, flunitrazepam and clonazepam against [3H]flunitrazepam were 6, 23 and 2800 nM, respectively, indicating that this receptor is of the peripheral type. A decrease of 37, 26 and 26% in Bmax was associated with substituting dimethylethanolamine, monomethylethanolamine or ethanolamine, respectively, for choline in the cell culture medium. These treatments did not change either the KD of [3H]flunitrazepam binding or the IC50 values of the different benzodiazepine drugs. Metastatic cell lines of the LM cell obtained from either athymic or C3H/Hef mice exhibited alterations in the binding parameters of [3H]flunitrazepam. There was a reduction in the Bmax values of the athymic (34%) and the C3H/Hef (44%) cell lines compared to the LM cell. In both groups there was a 90% increase in the KD. In the C6 astrocytoma, the peripheral type receptor appears to regulate plasma membrane mediated synthesis of phosphatidylcholine from phosphatidylethanolamine. However, this was not observed in the LM cell. Nor did it modulate cyclic AMP metabolism as assessed by measurement of cyclic AMP levels in whole cells after drug treatment.

Animals↗
PubMed1983

Lipid domains in plasma membranes from rat liver.

The existence of fluid and solid lipid domains in isolated rat-liver plasma membranes was evaluated using the fluorescent fatty acids trans-parinaric and cis-parinaric acid as probe molecules for solid and fluid membrane areas, respectively. The fluorescence probe 1,6-diphenyl-1,3,5-hexatriene indicated that a phase transition was present in the liver plasma membrane between 18 degrees C and 30 degrees C. At intermediate temperatures, cis-parinaric acid, which partitioned approximately equally into fluid and solid lipid areas, detected two lipid domains: the mole fractions of fluid and solid lipid domains at 24 degrees C were 0.32 and 0.68 while the mole fractions of cis-parinaric acid in each domain were 0.34 and 0.66, respectively. The dissociation constant, aqueous to membrane lipid partition coefficient, and bound to free ratio for trans-parinaric acid were 7.0 +/- 0.7 microM, 4.0 +/- 0.6 x 10(6), and 83:17, respectively. The affinity of the membrane for cis-parinaric acid was twofold lower than for trans-parinaric acid. The trans-parinaric acid partitioned preferentially into solid lipid, Ksp/f = 3.30, while the cis-parinaric acid partitioned equally between fluid and solid phases Ksp/f = 0.92. Thus, the data demonstrate the coexistence of fluid and solid domains in rat liver plasma membranes.

Animals↗
PubMed1983

Calcium modulates fatty acid dynamics in rat liver plasma membranes.

Modulation of free fatty acid binding in isolated rat liver plasma membranes was evaluated using the fluorescent fatty acids trans-parinaric and cis-parinaric acid as analogues for saturated and unsaturated fatty acids, respectively. Binding of trans-parinarate but not cis-parinarate was inhibited by physiological levels of Ca2+. The effect was reversed by addition of excess EGTA. Calcium decreased the aqueous to lipid partition coefficient, Kp, of trans-parinaric acid for liver plasma membranes while increasing the Kp for cis-parinaric acid. In addition, Ca2+ also altered the fluorescence lifetime, the quantum yield, and the relative partitioning of trans-parinaric and cis-parinaric acid into fluid and solid phases. Calcium and EGTA did not affect the binding of 1,6-diphenyl-1,3,5-hexatriene. The effect of Ca2+ on the liver plasma membrane structure was to increase the rigidity of the membrane, primarily the solid domain. The fluorescence polarization of trans-parinarate, cis-parinarate, and 1,6-diphenyl-1,3,5-hexatriene at 24 degrees C in liver plasma membranes in the absence of Ca2+ was 0.295 +/- 0.008, 0.253 +/- 0.007, and 0.284 +/- 0.005, respectively. Calcium (2.4 mM) increased the polarization of these probe molecules in liver plasma membranes by 8-10%. EGTA (3.4 mM) reversed or abolished the increase in polarization. Thus, the fluorescent fatty acids trans-parinarate and cis-parinarate may be used to monitor fatty acid binding by isolated membranes, to evaluate factors such as Ca2+ which modulate fatty acid binding, and to investigate the microenvironment in which the fatty acids residue. The data suggest that Ca2+ may be an important regulator of fatty acid uptake by the liver plasma membrane, and thereby interact with intermediary metabolism of lipids at a step not involving lipolytic or synthetic enzymes.

Animals↗
PubMed1983

Lipid composition alters phagocytosis of fluorescent latex beads.

A new assay using fluoresbrite microspheres was developed to determine phagocytic rate in LM fibroblasts grown in a variety of culture conditions. Fluoresbrite beads of diameter 0.86 microns or greater were taken up by the cells at a linear rate for 60 min over a wide range of bead/cell ratios. Phagocytosis was measured as the difference in fluorescence of cells exposed to beads at 37 degrees C and at 4 degrees C, to correct for adsorbance of beads to the cells. Fluoresbrite bead phagocytosis was zero at zero time and was saturable. LM fibroblasts cultured in a serum-free, chemically-defined medium were supplemented with choline analogues or fatty acids to alter plasma membrane lipid composition. Choline analogue supplementation (N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine) altered the plasma membrane phospholipid polar head group composition and dramatically decreased the phagocytic rate as compared with choline fed cells. Supplementation with polyunsaturated and monounsaturated fatty acids, but not saturated fatty acids, increased the phagocytic rate. The phagocytic rate was correlated with the plasma membrane phospholipid fatty acid index of unsaturation.

Animals↗
PubMed1983

Measurement of phagocytosis using fluorescent latex beads.

Fluorescent monodisperse latex beads and a computer-centered spectrofluorimeter were used to devise a sensitive new assay for phagocytosis. LM fibroblasts, a transformed cell line with a high endocytic rate, were exposed to fluoresbrite beads and the following parameters were investigated: incubation time, incubation temperature and bead/cell ratio. The bead uptake was linear for 60 min over a wide range of bead/cell ratios up to 130 beads/cell. Phagocytosis was inhibited at 4 degrees C, by incubation in the presence of colchicine, and by glucose deprivation. Scanning and transmission electron microscopy were used to confirm that at 37 degrees C both bead adsorption and internalization occurred while at 4 degrees C only bead adsorption but not endocytosis occurred. Large bead sizes (0.86 and 1.72 micrometer diameter) were most useful due to higher fluorescence and higher signal to noise ratios than smaller beads (0.25 and 0.57 micrometer diameter). Beads (0.86 micrometer diameter) were taken up at a rate of 4.4 beads/cell/h at 37 degrees C when a bead/cell ratio of 70 was used. The uptake was zero when assayed at zero time. These criteria establish that fluoresbrite beads provide a useful new fluorimetric assay for phagocytosis.

Animals↗
PubMed1982

LM fibroblast plasma membrane subfractionation by affinity chromatography on con A-sepharose.

Affinity chromatography was used to determine the heterogeneity and orientation of plasma membrane vesicles isolated from LM fibroblasts subjected to Dounce homogenization. Two plasma membrane subfractions were obtained by Con A-Sepharose affinity chromatography of LM fibroblast plasma membranes prepared by Dounce homogenization. The desmosterol-phospholipid molar ratio, the phospholipid composition, and the phospholipid fatty acid composition were almost identical between the two fractions. However, the lipid to protein ratio was almost 2-fold greater in the nonadherent fraction A. The binding of fluorescein-concanavalin A was the same in both fractions indicating a right-sided-out orientation of the vesicles. Similarly and asymmetric distribution of phosphatidylethanolamine in both membrane fractions was the same. In contrast, sialic acid content, 5'-nucleotidase activity, and (Na+ + K+)-ATPase activity were 47%, 3.7-fold, and 2.5-fold greater, respectively, in the nonadherent, lipid-rich fraction A. Structural properties of the two membrane fractions determined by fluorescence polarization and arrhenius plots of trans-parinaric acid fluorescence were similar. These results indicate that concanavalin-A affinity chromatography separates two membrane fractions differing in sialic acid content, lipid content, and enzyme profile but having the same right-side-out orientation.

Animals↗
PubMed1982

Asymmetric transbilayer distribution of sterol across plasma membranes determined by fluorescence quenching of dehydroergosterol.

A new method for measurement of transbilayer distribution of sterol in plasma membranes is reported. The procedure utilized a fluorescent sterol, dehydroergosterol, and a chemical quenching agent, trinitrobenzenesulfonic acid. Dehydroergosterol was useful as a probe molecule for sterols for the following reasons, (a) Dehydroergosterol contained no bulky side chains as reporter groups. (b) Dehydroergosterol structurally resembled cholesterol and desmosterol, the primary sterol synthesized by LM fibroblasts. (c) Dehydroergosterol interacted with digitonin, filipin, and served as a substrate for cholesterol oxidase. (d) The phase transition of dipalmitoylglycerophosphocholine was completely abolished by dehydroergosterol. (e) The native sterol of LM fibroblasts, desmosterol, was completely replaced by dehydroergosterol without effect on LM cell growth, cell doubling time, plasma membrane (Na+, K+)-ATPase and 5'-nucleotidase activity, microsomal NADPH-dependent cytochrome c reductase activity, and mitochondrial succinate-dependent cytochrome c reductase activity. (f) Neither the phospholipid composition nor the sterol/phospholipid ratio of LM fibroblasts were altered by supplementation with dehydroergosterol. The trinitrophenyl group of trinitrophenylglycine or of surface membranes of LM fibroblasts or red blood cells treated with trinitrobenzenesulfonic acid was an excellent quencher of dehydroergosterol fluorescence. Fluorescence in mouse very-low-density lipoproteins, LM fibroblasts plasma membranes, red blood cell surface membranes, and in rat red blood cell membranes was quenched 95 +/- 3%, 20 +/- 2%, 75 +/- 4%, and 69 +/- 4% respectively when the quenching agent was present on only the extracellular site of the membrane. Trinitrophenyl residues effectively quenched the dehydroergosterol fluorescence in the plasma membrane of LM cells by 20% when dehydroergosterol was present from 1-85 mol/100 ml of the membrane sterol. When both sides of the plasma membrane were trinitrophenylated, greater than 95% of the dehydroergosterol fluorescence was quenched. In addition, when LM cells were cultured with dehydroergosterol, exposed latex beads, and the endocytosed particles isolated as phagosomes and treated with trinitrobenzenesulfonic acid under non-penetrating conditions, the fluorescence of the dehydroergosterol was quenched nearly 64%. From these and other results we deduced that the inner monlayer of the LM fibroblasts plasma membrane was enriched with dehydroergosterol. In contrast, the distribution of the sterol in red blood cell membranes indicated an enrichment in the outer monolayer.

Animals↗
PubMed1982

Phagosomal membrane lipids of LM fibroblasts.

Murine fibroblasts, LM cells, were cultured in suspension or monolayer in a chemically defined medium without serum and exposed to polystyrene beads. The LM cells endocytized the beads in direct proportion to the bead/cell ratio and the bead surface area. However, equal volumes of beads irrespective of size or surface area were internalized. The lipid composition of the phagosome membrane differed significantly from the parent primary membrane in having higher contents of phosphatidylcholine, phosphatidylserine, and sterol but lower contents of sphingomyelin and lysophosphatidylcholine. When phagosomes isolated from suspension-cultured LM fibroblasts were exposed to trinitrobenzene-sulfonic acid at 4 degrees C, 55 +/- 1.6% of the phagosomal membrane phosphatidylethanolamine was trinitrophenylated. The asymmetric distribution of phosphatidylethanolamine across the phagosomal membrane was not affected by the bead/cell ratio, bead diameter, or exposure time of LM fibroblasts to the beads. When cells were reacted with trinitrobenzenesulfonic acid at 4 degrees C prior to phagocytosis, the amount of trinitrophenylphosphatidylethanolamine was greater in the isolated phagosomes than in the parent primary plasma membrane. Culturing LM fibroblasts in suspension or monolayer had no effect on the asymmetric distribution of phosphatidylethanolamine across primary plasma membrane bilayers. The data are consistent with the observation that LM fibroblasts grown either in suspension or monolayer internalize polystyrene beads at selective sites in the surface membrane.

Animals↗
PubMed1982

Development of metastatic tumors in athymic (nude) mice from LM cells grown in vitro.

The capacity of LM cells to initiate tumor formation in vivo was investigated in athymic (nude) BALB/c mice. Tumor cells inoculated s.c. over the left thighs of mice formed large, grossly visible tumors within 2 weeks postinoculation. Tumor doubling times were slightly longer than doubling times of LM cells in suspension culture. Histological examination of necropsied mice revealed that tumor cells were invasive into host tissue and formed numerous bizarre mitotic figures. The tumors were classified a fibrosarcoma-like tumors. Metastasis to the lungs was observed in nude mice inoculated with a variety of tumor cells. LM cells cultured in the presence of choline analogues (N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine) had grossly altered membrane lipid compositions. However, all formed tumors in the nude mice with tumor volume doubling times similar to that of LM cells grown in choline medium. LM cells grown with 10% calf serum had doubling times about 42% shorter. LM fibroblasts injected into nude mice provide a model system for investigation of tumor biology related to metastasis formation.

Animals↗
PubMed1982

Effects of thyroid status on the structure of the very low density lipoprotein secreted by the perfused liver.

To determine whether hyperthyroidism or hypothyroidism affected the structure of the very low density lipoprotein (VLDL) secreted by the perfused rat liver, rats were pretreated with 0.9% NaCl, T3, or propylthiouracil. The livers were perfused with 0-332 mumol bovine serum albumin-oleate/h for 4 h. The structure of the secreted VLDL was determined by compositional analysis and the use of the fluorescence probe molecules diphenylhexatriene, trans-parinaric acid, and cis-parinaric acid. Compositional analysis indicated that the VLDL secreted by livers from rats pretreated with T3 had lower unsaturated to saturated fatty acid ratios than did controls, regardless of the amount of oleate infused. Fluorescence polarization of diphenylhexatriene indicated that the interior core lipids of VLDL secreted by livers from T3-treated rats were more rigid than those secreted by livers of euthyroid animals. Arrhenius plots of polarization and corrected fluorescence of diphenylhexatriene and trans-parinaric acid, but not cis-parinaric acid, revealed that characteristic temperatures were shifted 8 to 10 C higher in VLDL secreted by livers from rats pretreated with T3. Treatment of rats for 7 days with propylthiouracil under these mild conditions did not alter the structure or composition of the VLDL secreted by the perfused liver. In summary, the lipid changes in the VLDL secreted by perfused rat livers from hyperthyroid animals were consistent with these VLDL being more rigid particles than those secreted by livers from euthyroid rats, independent of the infusion rate of the FFA.

Animals↗
PubMed1982

Thyroid status alters structure of VLDL secreted by perfused rat liver.

The interaction of thyroid status and oleic acid infusion rate on the thermal behavior of the very low density lipoprotein (VLDL) secreted by isolated perfused rat liver was examined. The livers were infused at 37 degrees C with oleate at rates of 0, 83, 166, or 332 mumoles/hr for 4 hours and VLDL was isolated from the perfusate at 12 degrees C. The lipid composition of the VLDL secreted by the perfused liver from hyperthyroid animals was dramatically different from controls at all infusion rates of oleate. Significant changes in the ratio of [phospholipid + cholesterol]/[triglyceride] and in fatty composition of secreted triglycerides occurred. Differential scanning calorimetry of the intact VLDL and extracted triglycerides secreted by euthyroid rats suggested the existence of four thermotropic endothermic transitions centered at -20.5, -14.0, -3.0, and 9.0 degrees C. Both the total enthalpies and the temperatures at which the phase alterations occurred in the triglyceride fraction from VLDL secreted by livers from euthyroid rats were highly dependent on the rate of infusion of oleate. Pretreatment of the rats with triiodothyronine and infusion of 332 mumoles oleate/hr abolished in the intact VLDL the temperature transitions centered at -20.8 and at 10.5 degrees C and decreased the total enthalpy from 8.13 to 38.5 cal/gm. Livers from rats pretreated with propylthiouracil and infused with oleate at 332 mumoles/hr secreted VLDL in which only one transition centered at -5.0 degrees C remained. The total enthalpy was unaffected. At all rates of infusion of oleate, the phase behavior of the intact VLDL or triglycerides extracted from the VLDL was altered by prior treatment of the rats with triiodothyronine or propylthiouracil. The thyroid state of the rat profoundly affected the thermal properties of the VLDL secreted by the perfused liver infused with the unsaturated fatty acid oleate.

Animals↗
PubMed1982

Effects of barbiturates and ethanol on the physical properties of brain membranes.

Synaptic plasma membranes (SPM) and myelin were prepared from mouse brain and their physical properties evaluated by fluorescence probes. 1,6,-Diphenyl-1,3,5-hexatriene (DPH) was used as a probe of the membrane core and endogenous tryptophan as a probe of membrane proteins. In vitro addition of pentobarbital or ethanol decreased the fluorescence polarization of DPH in SPM. These decreases were not due to changes in fluorescence lifetime and indicate that both drugs increased the rotation mobility of the probe in the membrane core. This action was shared by other barbiturates and their potencies were correlated with their lipid solubility. In contrast to the effect on SPM, pentobarbital increased the fluorescence polarization of DPH in SPM phospholipids but had little effect on the fluorescence polarization of DPH in a total lipid extract from SPM. Pentobarbital did not affect fluorescence polarization of DPH in myelin. Analysis of the temperature dependence of pentobarbital effects indicated that the initial rigidity of the membranes was one factor determining whether the drug decreased, increased or did not change the membrane fluidity. Pentobarbital decreased the fluorescence intensity of SPM tryptophan, but did not affect the fluorescence of free tryptophan. Ethanol did not alter tryptophan fluorescence. Thus, pentobarbital, but not ethanol, produced a detectable perturbation of synaptic proteins. These results indicate that both pentobarbital and ethanol fluidized the hydrophobic core of the membrane, but pentobarbital differed from ethanol in its action on very fluid lipid domains and on membrane proteins.

Animals↗
PubMed1981

Altered phospholipid composition affects endocytosis in cultured LM fibroblasts.

The phospholipid polar head group composition of LM fibroblast membranes was altered by growing the cells in a chemically defined, serum-free medium containing choline, N,N'-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine. The cells incorporated these bases into their membrane phospholipid such that 29-40% of the total plasma membrane phospholipids contained these polar head groups. Alteration of the phospholipid composition correlated with a depression of polystyrene bead phagocytosis by 36, 55 and 85% when the cells had been supplemented with N,N'-dimethylethanolamine, N-monoethylethanolamine, or ethanolamine, respectively. Pinocytotic uptake of horseradish peroxidase was depressed 44, 39, and 32%, respectively. The phagosomal membrane phospholipid composition qualitatively resembled that of the primary plasma membrane from which it was derived. However, enrichment of phosphatidylcholine, and other quantitative differences were noted in the phagosomal membranes as compared to the parent primary plasma membrane. Approx. 50% of the phagosomal membrane's phosphatidylethanolamine was accessible to the chemical labelling reagent trinitrobenzenesulfonate at 4 degrees C. The asymmetric distribution of phosphatidylethanolamine across the phagosomal membrane did not appear to be altered by base analogues except in the case of phagosomes from cells supplemented with ethanolamine. The data were consistent with a nonrandom site for endocytosis with regard to phospholipid composition.

Animals↗