Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modifi... more Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. Primary cultures of MMEC were incubated in the presence of 3H-palmitate for 4 h. A cytoskeletal (CS) fraction was prepared by treatment of the cells with 1.5M KCl and 1% Triton X-100. The residual material, consisting primarily of keratin and actin filaments was exhaustively (10-20 rounds, including sonications) extracted with chloroform/methanol to remove non-covalently bound labeled lipids. The CS protein was then acid-hydrolyzed and the chloroform-soluble products subjected to thin layer chromatography (TLC). Two-thirds of the covalently bound radiolabel appeared as a very hydrophobic peak on a TLC system optimized for separation of neutral lipids. Ten percent separated into 4-5 peaks on a polar lipid TLC system. A small amount of label was traced to fatty acid-like components. Autoradiography of two-dimensional gels indicated that all the CS proteins resolvable by Coomassie blue staining were also radiolabeled. The results are discussed in terms of CS-lipid-membrane interactions.
The neutral lipid content of cells and cilia of Paramecium tetraurelia was determined as a functi... more The neutral lipid content of cells and cilia of Paramecium tetraurelia was determined as a function of growth stage and of growth medium composition. The major sterol(s) of deciliated cells and of cilia were the sterol provided in the growth medium (e.g., stigmasterol) and its 7-dehydro derivative. Body sterol esters and triglycerides accumulated during exponential cell growth and became depleted during stationary phase. Isolated cilia contained neither sterol esters nor triglycerides. The ratio of phospholipid to free sterol in cilia was constant (approximately 5:1) throughout exponential growth in axenic or bacterized medium, but the relative proportions of free sterol and 7-dehydrosterol varied with growth phase and with growth medium composition. Cholesterol did not support the growth of Paramecium, but was taken up rapidly into cells and cilia as the free sterol and its 7-dehydro derivative. The fatty acids of sterol esters and of triglycerides were similar to those of membrane phospholipids.
Injections of cyclic AMP (cAMP) and 8-Br-cAMP into Paramecium and external application of isobuty... more Injections of cyclic AMP (cAMP) and 8-Br-cAMP into Paramecium and external application of isobutylmethylxanthine (IBMX), an inhibitor of cAMP breakdown, to these cells increased the frequency of ciliary beating and hyperpolarized the membrane potential. When the membrane potential was held equal to the resting potential under voltage clamp, the same experimental conditions which serve to increase intracellular cAMP did not raise the ciliary frequency. We conclude that cAMP is presumably not the direct mediator of the hyperpolarization-induced ciliary activation, although it may be associated with this motor response.
The purpose of the present study was to examine the effect of beta-sitosterol, the main dietary p... more The purpose of the present study was to examine the effect of beta-sitosterol, the main dietary phytosterol on the growth of HT-29 cells, a human colon cancer cell line. In addition, the incorporation of this phytosterol into cellular membranes and how this might influence the lipid composition of the membranes were investigated. Tumor cells were grown in DMEM containing 10% FBS and supplemented with sterols (cholesterol or beta-sitosterol) at final concentrations up to 16 microM. The sterols were supplied to the media in the form of sterol cyclodextrin complexes. The cyclodextrin used was 2-hydroxypropyl-beta-cyclodextrin. The sterol to cyclodextrin molar ratio was maintained at 1:300. The study indicated that 8 and 16 microM beta-sitosterol were effective at cel growth inhibition as compared to cholesterol or to the control (no sterol supplementation). After supplementation with 16 microM beta-sitosterol for 9 days, cell growth was only one-third that of cells supplemented with equimolar concentration of cholesterol. No effect was observed on total membrane phospholipid concentration. At 16 microM beta-sitosterol supplementation, membrane cholesterol was reduced by 26%. Cholesterol supplementation resulted in a significant increase in the cholesterol/phospholipid ratio compared to either beta-sitosterol supplemented cells or controls. There was a 50% reduction in membrane sphingomyelin (SM) of cells grown in 16 microM beta-sitosterol. Additional changes were observed in the fatty acid composition of minor phospholipids of beta-sitosterol supplemented cells, such as SM, phosphatidylserine (PS), and phosphatidylinositol (PI). Only in the case of PI, was there an effect of these fatty acid changes on the unsaturation index, beta-sitosterol incorporation resulted in an increase in the U.I. It is possible that the observed growth inhibition by beta-sitosterol may be mediated through the influence of signal transduction pathways that involve membrane phospholipids.
Comparative Biochemistry and Physiology Part A: Physiology, 1989
1. 1. The amplitude of the isolated voltage dependent inward calcium current of axenically grown ... more 1. 1. The amplitude of the isolated voltage dependent inward calcium current of axenically grown Paramecium was greater in cells grown with sitosterol than those grown in stigmasterol supplemented medium. ... 2. 2. The duration of high K + stimulated backward swimming, a bioassay for ...
Biochimica Et Biophysica Acta - Biomembranes, Feb 1, 1983
... a Department of Biochemistry, College of Agricultural and Life Sciences, University of Wiscon... more ... a Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706 USA. Received 29 July 1982. Available online 30 January 2003. Abstract. Transfer ...
Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral Physiology, Nov 1, 1994
Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptati... more Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptation and cellular sensory integration. While millimolar amounts of many attractants hyperpolarize and cause faster forward swimming, oxidants are repellents that depolarize and cause backward swimming at micromolar concentrations. The non-permeant oxidants cytochrome c, nitro blue tetrazolium and ferricyanide are repellents with half maximal concentrations of 0.4 microM, 2.2 microM and 100 microM respectively. In vivo reductase activities follow the same order of potencies. The concentration dependence of the cytochrome c reductase activity is well correlated with cytochrome c-induced depolarizations. This suggests that plasma membrane reduction of external cytochrome c is electrogenic, causing membrane depolarization and chemorepulsion. The reductase activity also appears to be voltage dependent. Depolarization by either K+, Na+, Ca++ or Mg++ correlates with inhibition of both in vivo reductase activities and cytochrome c-induced membrane potential changes. These responses were also seen in deciliated cells, showing that the body plasma membrane is sufficient for the response. Both chloroquine and diphenyleneiodonium inhibited reductase activities but only at unusually high concentrations. This activity showed no pH dependence in the physiological range. We propose that a plasma membrane bound NA-DPH-dependent reductase controls oxidant-induced depolarizations and consequent chemorepulsion.
Micromolar concentrations of adenosine triphosphate (ATP) and its non-hydrolyzable analog β- γ -m... more Micromolar concentrations of adenosine triphosphate (ATP) and its non-hydrolyzable analog β- γ -methylene ATP are both effective depolarizing chemorepellents in Tetrahymena thermophila. Chemorepellent behavior consists of repeated bouts of backward swimming (avoidance reactions) that can easily be quantified to provide a convenient bioassay for purinergic reception studies. Chemosensory adaptation occurs following prolonged exposure (10 min) to the repellents, and cells regain normal swimming behavior. Adaptation is specific since cells that are behaviorally adapted to either ATP or β- γ -methylene ATP still retain full responsiveness to the chemorepellents GTP and lysozyme. However, cross adaptation occurs between ATP and β- γ -methylene ATP, suggesting that they involve the same receptor. Behavioral sensitivity to both ATP and β- γ -methylene ATP is increased by the addition of Na+, but addition of either Ca2+ or Mg2+ dramatically decreases the response to ATP. These ionic effects...
Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modifi... more Cytoskeletal proteins obtained from mouse mammary epithelial cells (MMEC) were found to be modified by covalent attachment of lipids. Primary cultures of MMEC were incubated in the presence of 3H-palmitate for 4 h. A cytoskeletal (CS) fraction was prepared by treatment of the cells with 1.5M KCl and 1% Triton X-100. The residual material, consisting primarily of keratin and actin filaments was exhaustively (10-20 rounds, including sonications) extracted with chloroform/methanol to remove non-covalently bound labeled lipids. The CS protein was then acid-hydrolyzed and the chloroform-soluble products subjected to thin layer chromatography (TLC). Two-thirds of the covalently bound radiolabel appeared as a very hydrophobic peak on a TLC system optimized for separation of neutral lipids. Ten percent separated into 4-5 peaks on a polar lipid TLC system. A small amount of label was traced to fatty acid-like components. Autoradiography of two-dimensional gels indicated that all the CS proteins resolvable by Coomassie blue staining were also radiolabeled. The results are discussed in terms of CS-lipid-membrane interactions.
The neutral lipid content of cells and cilia of Paramecium tetraurelia was determined as a functi... more The neutral lipid content of cells and cilia of Paramecium tetraurelia was determined as a function of growth stage and of growth medium composition. The major sterol(s) of deciliated cells and of cilia were the sterol provided in the growth medium (e.g., stigmasterol) and its 7-dehydro derivative. Body sterol esters and triglycerides accumulated during exponential cell growth and became depleted during stationary phase. Isolated cilia contained neither sterol esters nor triglycerides. The ratio of phospholipid to free sterol in cilia was constant (approximately 5:1) throughout exponential growth in axenic or bacterized medium, but the relative proportions of free sterol and 7-dehydrosterol varied with growth phase and with growth medium composition. Cholesterol did not support the growth of Paramecium, but was taken up rapidly into cells and cilia as the free sterol and its 7-dehydro derivative. The fatty acids of sterol esters and of triglycerides were similar to those of membrane phospholipids.
Injections of cyclic AMP (cAMP) and 8-Br-cAMP into Paramecium and external application of isobuty... more Injections of cyclic AMP (cAMP) and 8-Br-cAMP into Paramecium and external application of isobutylmethylxanthine (IBMX), an inhibitor of cAMP breakdown, to these cells increased the frequency of ciliary beating and hyperpolarized the membrane potential. When the membrane potential was held equal to the resting potential under voltage clamp, the same experimental conditions which serve to increase intracellular cAMP did not raise the ciliary frequency. We conclude that cAMP is presumably not the direct mediator of the hyperpolarization-induced ciliary activation, although it may be associated with this motor response.
The purpose of the present study was to examine the effect of beta-sitosterol, the main dietary p... more The purpose of the present study was to examine the effect of beta-sitosterol, the main dietary phytosterol on the growth of HT-29 cells, a human colon cancer cell line. In addition, the incorporation of this phytosterol into cellular membranes and how this might influence the lipid composition of the membranes were investigated. Tumor cells were grown in DMEM containing 10% FBS and supplemented with sterols (cholesterol or beta-sitosterol) at final concentrations up to 16 microM. The sterols were supplied to the media in the form of sterol cyclodextrin complexes. The cyclodextrin used was 2-hydroxypropyl-beta-cyclodextrin. The sterol to cyclodextrin molar ratio was maintained at 1:300. The study indicated that 8 and 16 microM beta-sitosterol were effective at cel growth inhibition as compared to cholesterol or to the control (no sterol supplementation). After supplementation with 16 microM beta-sitosterol for 9 days, cell growth was only one-third that of cells supplemented with equimolar concentration of cholesterol. No effect was observed on total membrane phospholipid concentration. At 16 microM beta-sitosterol supplementation, membrane cholesterol was reduced by 26%. Cholesterol supplementation resulted in a significant increase in the cholesterol/phospholipid ratio compared to either beta-sitosterol supplemented cells or controls. There was a 50% reduction in membrane sphingomyelin (SM) of cells grown in 16 microM beta-sitosterol. Additional changes were observed in the fatty acid composition of minor phospholipids of beta-sitosterol supplemented cells, such as SM, phosphatidylserine (PS), and phosphatidylinositol (PI). Only in the case of PI, was there an effect of these fatty acid changes on the unsaturation index, beta-sitosterol incorporation resulted in an increase in the U.I. It is possible that the observed growth inhibition by beta-sitosterol may be mediated through the influence of signal transduction pathways that involve membrane phospholipids.
Comparative Biochemistry and Physiology Part A: Physiology, 1989
1. 1. The amplitude of the isolated voltage dependent inward calcium current of axenically grown ... more 1. 1. The amplitude of the isolated voltage dependent inward calcium current of axenically grown Paramecium was greater in cells grown with sitosterol than those grown in stigmasterol supplemented medium. ... 2. 2. The duration of high K + stimulated backward swimming, a bioassay for ...
Biochimica Et Biophysica Acta - Biomembranes, Feb 1, 1983
... a Department of Biochemistry, College of Agricultural and Life Sciences, University of Wiscon... more ... a Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, 420 Henry Mall, Madison, WI 53706 USA. Received 29 July 1982. Available online 30 January 2003. Abstract. Transfer ...
Journal of Comparative Physiology A-neuroethology Sensory Neural and Behavioral Physiology, Nov 1, 1994
Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptati... more Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptation and cellular sensory integration. While millimolar amounts of many attractants hyperpolarize and cause faster forward swimming, oxidants are repellents that depolarize and cause backward swimming at micromolar concentrations. The non-permeant oxidants cytochrome c, nitro blue tetrazolium and ferricyanide are repellents with half maximal concentrations of 0.4 microM, 2.2 microM and 100 microM respectively. In vivo reductase activities follow the same order of potencies. The concentration dependence of the cytochrome c reductase activity is well correlated with cytochrome c-induced depolarizations. This suggests that plasma membrane reduction of external cytochrome c is electrogenic, causing membrane depolarization and chemorepulsion. The reductase activity also appears to be voltage dependent. Depolarization by either K+, Na+, Ca++ or Mg++ correlates with inhibition of both in vivo reductase activities and cytochrome c-induced membrane potential changes. These responses were also seen in deciliated cells, showing that the body plasma membrane is sufficient for the response. Both chloroquine and diphenyleneiodonium inhibited reductase activities but only at unusually high concentrations. This activity showed no pH dependence in the physiological range. We propose that a plasma membrane bound NA-DPH-dependent reductase controls oxidant-induced depolarizations and consequent chemorepulsion.
Micromolar concentrations of adenosine triphosphate (ATP) and its non-hydrolyzable analog β- γ -m... more Micromolar concentrations of adenosine triphosphate (ATP) and its non-hydrolyzable analog β- γ -methylene ATP are both effective depolarizing chemorepellents in Tetrahymena thermophila. Chemorepellent behavior consists of repeated bouts of backward swimming (avoidance reactions) that can easily be quantified to provide a convenient bioassay for purinergic reception studies. Chemosensory adaptation occurs following prolonged exposure (10 min) to the repellents, and cells regain normal swimming behavior. Adaptation is specific since cells that are behaviorally adapted to either ATP or β- γ -methylene ATP still retain full responsiveness to the chemorepellents GTP and lysozyme. However, cross adaptation occurs between ATP and β- γ -methylene ATP, suggesting that they involve the same receptor. Behavioral sensitivity to both ATP and β- γ -methylene ATP is increased by the addition of Na+, but addition of either Ca2+ or Mg2+ dramatically decreases the response to ATP. These ionic effects...
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