Membrane fluidizing effects of the general anesthetic methoxyflurane elicit an acclimation response in Tetrahymena.
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Biomedical subjects
Publications and source records attributed to Y Kitajima.
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Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change being very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by membrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.
When cells of Tetrahymena pyriformis, strain NT-1, were chilled from their growth temperature of 39.5 degrees C to lower temperatures, the plasma membrane, outer alveolar, nuclear, outer mitochondrial, food vacuolar, and endoplasmic reticulum membranes each responded in a fashion quite characteristic of the membrane type. In most cases a distinctive rearrangement of intramembrane particles, as discerned by freeze-fracture electron microscopy, began abruptly at a definitive temperature. By comparing the freeze-fracture patterns of membranes in cells grown at 39.5, 27, and 15 degrees C, it was shown that the initial particle rearrangement in a given membrane always occurred at a fixed number of degrees below the growth temperature of the cell. Gradual chilling of a cell grown at constant temperature induced these membrane changes first in the outer alveolar membrane, then, in order of decreasing response to temperature, in the endoplasmic reticulum, outer mitochondrial membrane, nuclear envelope, and vacuolar membrane. The normally stable relationships between the physical properties of the several membrane types could in some cases be reversed, but only temporarily, by fatty acid supplementation or during the initial phases of acclimation to growth at a different temperature. The system provides a unique opportunity to study the effects of environmental change upon the physical properties of several functionally distinct but metabolically interrelated membranes within a single cell.
The origin and differentiation of Tetrahymena pyriformis food vacuolar membranes has been studied by freeze-fracture electron microscopy. By measuring the temperature needed to induce the onset of lipid phase separation (as inferred by the appearance of particle-free regions in replicas) and calculating the changes in average intramembrane particle distribution, a distinct modification of the vacuolar membrane could be observed from the time of its formation from disk-shaped vesicles to its maturation before egestion of its indigestible contents. Whereas the nascent vacuolar membrane first showed signs of phase separation at 9 degrees C, this temperature rose to 14 degrees C in the completed vacuole and then, after lysosomal fusion, eventually declined to 12 degrees C. The average membrane particle density on the PF face increased from 761 +/- 219 to 1,625 +/- 350 per micron 2 during membrane differentiation. Like other membranes of the cell, the vacuolar membrane underwent adaptive changes in its physical properties in cells maintained for several hours at low temperature. This exposure to low temperature caused an equal effect in vacuoles formed before, during, or after the temperature shift-down. Normal changes in the properties of the vacuolar membrane may have some bearing on its programmed sequence of fusion reactions.
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The effects of chemically different polyenes on fungal membranes (Epidermaphyton floccosum, a human pathogenic fungus, and Saccharomyces cerevisiae) and human red blood cell membranes were studied by freeze-fracture electron microscopy in order to elucidate the interaction of these antibiotics with ergosterol. Each type of neutral, small amphoteric and large amphoteric polyenes produces a distinct morphoneutral, small amphoteric and large amphoteric polyenes produces a distinct morphological effect on the fungal membranes: (1) Pit formation type. Filipin, a neutral polyene, produces 250-300 A diameter "pits" or "invagination" both in ergosterol-containing fungal plasma membranes and cholesterol-containing red blood cell ghost membranes. (2) Network particle aggregation type. The small amphoteric polyene, pimaricin, produces a network of membrane particle aggregation which encloses 1000 A diameter particle-free areas in fungal membranes. These areas are slightly elevated toward the outside of the cell. (3) Random particle aggregation type. The large amphoteric polyenes, amphotericin B and nystatin, cause a random segregation of the fungal plasma membrane and the red blood cell ghost membranes into particle-free and aggregated areas. It is concluded that these morphological differences are due to different mechanisms of polyene-sterol interactions in which the different size of the mocrolide ring in the antibiotic structure may be involved. Since all of these antibiotics, except filipin, cause no alterations on whole red blood cells detectable by negative staining and freeze-fracture electron microscopy, it is possible that they have a higher affinity to ergosterol than cholesterol in membranes.
This is a study of the molecular mechanisms employed by Tetrahymena pyriformis to change the lipid composition and thereby the fluidity of its various membranes during temperature acclimation. By quantitatively measuring the intramembrane particle aggregation using freeze-fracture electron microscopy, membrane physical properties in 39.5 degrees C grown cells shifted to 15 degrees C were found to be correlated with the degree of phospholipid fatty acid desaturation. Alteration of the phospholipid polar head group distribution from that of 39.5 degrees C-grown cells to the significantly different pattern of 15 degrees C grown cells appeared not to be of critical importance in the acclimation process. Changes in fatty acid desaturation during acclimation from high to low temperatures and vice versa were analyzed using normal cells and cells fed large amounts of polyunsaturated fatty acids. Fatty acid desaturase activity corresponded to the degree of membrane fluidity but not to the cell temperature. All evidence was compatible with the hypothesis that membrane fluidity is self-regulating, with the action of fatty acid desaturases being modulated by the physical state of their membrane environment.
Further studies on the molecular mechanisms of temperature acclimation have been carried out using the ciliate Tetrahymena pyriformis. The most prominent change in lipid metabolism during acclimation to high temperature--depression of fatty acid desaturase activity--could be simulated by supplementing the growth medium of isothermally-grown cells with polyunsaturated fatty acids. Such cells resisted the membrane-fluidizing effect of the incorporated exogenous acids by increased use of de novo synthesized saturated acids in their phospholipids. The data support the conclusions arising from earlier experiments with temperature-shifted cells (Martin, C.E., Hiramitsu, K., Kitajima, Y., Nozawa, Y., Skriver, L., and Thompson, G.A., Jr. (1976), Biochemistry 15), showing that, when membrane fluidity increased to a superoptimal level, the activity of membrane-associated fatty acid desaturases was decreased. Since the reaction is controlled by membrane fluidity, rather than temperature per se, we postulate that it is the general mechnaism employed by cells adjusting to any fluidity-modifying factor, such as cations, drugs, etc.
Experiments on temperature adaptation have been conducted using a thermotolerant clone of Tetrahymena pyriformis designated as strain NT-1. The strain was able to grow well at 39.5 and 15 degrees C and could adapt quickly when transferred from one of these temperatures to the other. Cells grown at the extreme temperatures differed markedly in their membrane lipid composition, particularly in the phospholipid polar head groups and hydrocarbon chains. The levels of fatty acid unsaturation increased at the lower temperature (e.g. 15 degrees C cells contained 31% gamma-linolenic acid vs. 25% at 39.5 degrees C) as did the content of alkyl glyceryl ether derivatives. Ethanolamine phosphoglycerides decreased by more than 10 mol % of the lipid phosphorus with the drop in temperature, the decrease being offset by a concomitant rise in 2-aminoethylphosphonolipid. These temperature-induced changes were noted in certain purified membrane preparations as well as in whole cells. Experiments with [14C]palmitic acid and sodium[14C]acetate showed that fatty acids are first incorporated into phospholipids predominantly in a saturated form. The membranes served as a reservoir of fatty acid substrate for desaturase activity. Tetrahymena pyriformis, strain NT-1, was proposed as a useful model system for studying the temperature adaptation process in eukaryotic cells.
The outer-most layer ("exo-layer") of the wall was isolated from cell walls of Epidermophyton floccosum. The pure cell walls, obtained by disruption in a Ribi cell fractionator, sonication and centrifugation, were digested with snail enzyme for 12 h. Thereafter, the exo-layer preparation was obtained as the fraction resistant to the snail enzyme. Electron microscopy showed that the exo-layer is a thin, stranded network structure 10-20 nm thick. Chemical analysis of the exo-layer showed that the main components are protein (63 percent), mannose (10 percent) and glucosamine (17 percent). Sodium dodecyl sulfate polyacrylamide gel electrophoresis has revealed that the main band is a glycoprotein containing mannose.
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PURPOSE: To investigate the prevalence of normal-tension glaucoma (NTG) and primary open-angle glaucoma (POAG) in patients with collagen diseases and determine whether an immunocompromised condition is present in a subset of glaucoma patients. METHODS: Three glaucoma specialists prospectively examined patients with collagen diseases. The diagnostic process included applanation tonometry, slit-lamp examination, gonioscopy, direct ophthalmoscopy, and automated static perimetry. Twenty-four-hour intraocular pressure monitoring was done when necessary. Using the results of a population-based survey conducted in Japan, we calculated an expected number of cases of NTG and POAG, and compared these with the actual number of cases. RESULTS: Of the 153 patients with collagen diseases examined, we found 6 patients with NTG and 2 patients with POAG. Of these 8 patients, 2 with progressive systemic sclerosis (PSS), one with NTG, and the other, POAG, had a history of being on systemic steroidal therapy. The prevalence of NTG and POAG was significantly higher in women patients having collagen diseases as compared with normal women (P = .027). CONCLUSION: Women patients with collagen diseases are highly susceptible to NTG and POAG.