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Biomedical subjects

G Weeks

Publications and source records attributed to G Weeks.

At least 73 records · Page 4Linked to original sources
PubMed1984

A plasma membrane Mg2+-ATPase in the cellular slime mold Dictyostelium discoideum.

Evidence is presented for the existence of a plasma membrane ATPase in Dictyostelium discoideum. The enzyme is dependent on Mg+2, and is insensitive to both azide and oligomycin. It is, however, sensitive to diethylstilbestrol, dicyclohexylcarbodiimide, vanadate, and thimerosal. Monovalent cations (Na+, K+, and choline) have no effect on enzyme activity, but high concentrations of Ca+2 are highly inhibitory. Vegetative cells express the highest amount of enzyme activity; the activity decreases three- to fourfold during the early stages of differentiation, and then remains constant during the latter stages.

Adenosine Triphosphatases↗
PubMed1984

The spore cell induction activity of conditioned media and subcellular fractions of Dictyostelium discoideum.

While certain sporagenous mutants of Dictyostelium discoideum do not display a density dependence for spore cell formation under in vitro differentiating conditions, other sporagenous mutants (HM18, HM18-2 and FR17) do exhibit density dependence. In addition, pre-spore cell formation in the wild-type strain V12M2 is density-dependent. Spore cell formation in HM18 and HM18-2 is stimulated at low cell densities by a membrane fraction from pseudoplasmodial cells. Stimulation is also effected by extracts of these membranes or conditioned media from starving cells and these preparations also stimulate pre-spore cell formation in V12M2. The active factor in conditioned media is of low molecular weight, heat-stable and insensitive to treatment by pronase and glycosidase; the factor is not extractable by hexane. The available evidence suggests that the active factor in conditioned media and in membrane extracts is the same molecule and, although ammonia also stimulates spore cell formation under these conditions, the active factor is not ammonia. More activity is produced extracellularly by cells in shake suspension than by cells in monolayers, and some strains produce more activity than others.

Culture Media↗
PubMed1983

The effect of ammonia on stalk cell formation in submerged monolayers of Dictyostelium discoideum.

It has been shown that ammonia inhibits stalk cell formation in monolayers of V12M2, and it was suggested that this inhibition was due to an antagonism of the differentiation-inducing factor (DIF) (Gross, J.D. et al., Nature, 303, 244-246, 1983). However, the results presented here indicate that ammonia inhibition is independent of DIF concentration, and that it occurs well in advance of the period of DIF requirement. Ammonia completely inhibits DIF accumulation and inhibits stalk cell differentiation, but there is no inhibition of prespore cell formation. These results imply the existence of an early ammonia-sensitive event that influences terminal cell type differentiation.

Ammonia↗
PubMed1983

Inhibition of Dictyostelium discoideum differentiation in monolayers in vitro by endogenous and exogenous lectins.

Spore-cell differentiation in monolayers in vitro of two sporagenous mutants of Dictyostelium discoideum, HM18 and HM15, is markedly inhibited by relatively low concentrations of the exogenous lectins, Concanavalin A (ConA) and wheat germ agglutinin (WGA) and by somewhat higher concentrations of the endogenous lectin, discoidin. The selective inhibition of spore cell formation by ConA occurs toward the end of the differentiation process, as shown by studies involving the addition or removal of ConA at various times during the period of differentiation. In addition, ConA has no deleterious effect on the expression of the pre-spore cell specific enzyme, UDP-galactosyl transferase. It is likely, therefore, that the maturation of pre-spore cells into spore cells is the most sensitive locus of lectin inhibition. At higher concentrations of WGA and ConA, both stalk- and spore-cell formation are inhibited, but increased concentrations of discoidin do not have this effect.

Carrier Proteins↗
PubMed1982

Extraction of membrane factors that inhibit aggregation in Dictyostelium discoideum.

Plasma membrane preparations partially inhibit the reaggregation of dissociated aggregation-component cells of Dictyostelium discoideum, and membranes from aggregation-competent cells are more inhibitory than membranes from vegetative cells. Inhibitory activity can be extracted from the membranes of aggregation-competent cells with the detergent octyl glucoside or with EDTA, although the inhibition is never more than partial. Extracts of vegetative membranes have relatively little inhibitory activity and the amount of inhibitory activity that can be extracted increases as cells become aggregation-competent. The inhibitory activity is sensitive to Pronase, but relatively stable to heat. Inhibitory activity can also be extracted from intact aggregation-competent cells by 20 mM-EDTA or 0.25% octyl glucoside.

Agglutination↗
PubMed1980

Reversible heat activation of alkaline phosphatase of dictyostelium discoideum and its developmental implication.

The activities of some enzymes increase during the development of the cellular slime mould, Dictyostelium discoideum. Because optimal specific activity is attained by eachenzyme at a specific developmental stage, development can be followed as a function of enzyme activity. The activity of one of these enzymes, alkaline phosphatase, increases markedly during the late stages of development, making it a potentially useful marker for terminal differentiation. It has been suggested that this increase in activity is due to de novo enzyme synthesis because the increase in activity does not occur in the presence of inhibitors of RNA and protein synthesis. However, we present evidence here of reversible heat activation of membrane-bound alkaline phosphatase which suggests that the increase in alkaline phosphatase activity may be due to an unmasking of pre-existing enzyme by a novel membrane regulatory mechanism.

Alkaline Phosphatase↗
PubMed1980

The fluidity of plasma membranes of Dictyostelium discoideum. The effects of polyunsaturated fatty acid incorporation assessed by fluorescence depolarization and electron paramagnetic resonance.

Two probe techniques, fluorescence depolarization (using diphenylhexatriene) and electron paramagnetic resonance (using 5-doxyl stearic acid), have been used to assess the fluidity of the purified plasma membranes of Dictyostelium discoideum. Both techniques indicate that a large incorporation of polyunsaturated fatty acids into the plasma membranes does not significantly change membrane fluidity In addition, phosphatidylcholines isolated from cells grown on both polyunsaturated fatty acid-supplemented and unsupplemented media exhibit similar mobilities of an incorporated spin probe when dispersed in aqueous solution. This result suggests that the enrichment of a membrane already high in content of fatty acyl chains containing two double bonds with those containing three or more double bonds does not markedly change fluidity.

Cell Membrane↗
PubMed1980

Quantitative study of the fluidity of Escherichia coli membranes using deuterium magnetic resonance.

Specifically deuterated palmitic acid was incorporated into the membrane phospholipids of the L51 strain of Escherichia coli. The cytoplasmic and outer membranes were separated by using standard techniques and studied by deuterium nuclear magnetic resonance between 0 and 40 degrees C. Distinctive liquid-crystalline and gel spectra were observed to coexist over a wide temperature range. The relative intensities of these spectra provided a direct measure of the fraction of the deuterium-labeled phospholipids in the fluid state as a function of temperature. Above 37 degrees C, the amount of immobilized or gel-phase phospholipid is estimated to be less than 3% of the total phospholipid. The gel to liquid-crystalline transition region for the outer membrane was shifted upwards by approximately 7 degrees C relative to that of the cytoplasmic membrane, in agreement with previous studies [Davis, J. H., Nichol, C. P., Weeks, G., & Bloom, M. (1979) Biochemistry 18, 2103]. The orientational order in the fluid phase of both membranes decreased gradually with increasing temperature and was greater in the outer membrane than in the cytoplasmic membrane. The orientational order of the gel-phase component was the same for both membranes, within an experimental uncertainty of 10%, and was independent of temperature from 0 to 30 degrees C for the outer membrane and from 10 to 30 degrees C for the cytoplasmic membrane.

Cell Membrane↗
PubMed1980

The lipid composition and membrane fluidity of Dictyostelium discoideum plasma membranes at various stages during differentiation.

There are only minor changes in the amounts of the major lipid constituents of Dictyostelium discoideum plasma membranes during the early stages in the differentiation of this organism. By the time cells reach the pseudoplasmodial stage of development there are small increases in the amounts of phosphatidylinositol, phosphatidylglycerol, and lysophosphatidylethanolamine, and small decreases in the amounts of phosphatidylethanolamine and its plasmalogen form. There is also a slight decrease in the total amount of sterol in the plasma membrane during the transition from aggregation to pseudoplasmodium formation. However, no significant change in membrane fluidity as determined by electron paramagnetic resonance (EPR) accompanies these minor changes in lipid composition. It can be concluded that the establishment of cell-cell interaction in D. discoideum does not involve gross changes in plasma membrane fluidity or lipid composition. It was found that the plasmalogen form of phosphatidylethanolamine is a major phospholipid constituent in D. discoideum, and that this species is somewhat enriched in the plasma membrane.-Weeks, G., and F. G. Herring. The lipid composition and membrane fluidity of Dictyostelium discoideum plasma membranes at various stages during differentiation.

Cell Membrane↗
PubMed1979

Study of the cytoplasmic and outer membranes of Escherichia coli by deuterium magnetic resonance.

The cytoplasmic and outer membranes of Escherichia coli were studied between 0 and 40 degrees C by deuterium magnetic resonance quadrupolar echo spectroscopy. The L51 strain of E. coli was used to incorporate perdeuterated palmitic acid into the membrane phospholipids. The cytoplasmic and outer membranes were separated using standard techniques. The spectrum of each membrane preparation was dominated at high temperatures (greater than or equal to 37 degrees C) by the characteristic liquid-crystalline plateau previously observed for perdeuterated palmitate chains in model phospholipid membranes. At low temperatures, the shape and width of the spectrum were characteristic of the gel phase. The relative intensities of the liquid-crystalline and gel features varied systematically with temperature. A quantitative analysis of the acyl chain orientational order was carried out by using the method of moments. The orientational order at each temperature was greater in the outer membrane sample than in that of the cytoplasmic membrane, indicating that the liquid-crystalline-gel transition region in the outer membrane is shifted to higher temperatures than that of the cytoplasmic membrane by about 7 degrees C. It is clear from the results that most of the phospholipid molecules participate in the phase transition.

Cell Membrane↗
PubMed1979

Analysis of Dictyostelium discoideum plasma membrane fluidity by electron spin resonance.

Dictyostelium discoideum grown axenically in media containing polyunsaturated fatty acids exhibited normal growth rates but impaired differentiation (Weeks, G. (1976) Biochim. Biophys. Acta 450, 21--32). Since cell-cell contact is vital for differentiation but unnecessary for growth we have examined the isolated plasma membranes of these cells. The lipids of the plasma membranes of cells grown in the presence of polyunsaturated fatty acids contain considerable quantities of these acids, but the total phospholipid and sterol contents of the plasma membrane are close to normal. Electron spin resonance studies using 5-doxyl-stearic acid as the spin probe reveal two things. Firstly, there are no detectable characteristic transition temperatures in the plasma membranes of D. discoideum. Secondly, the plasma membranes of cell grown in the presence of polyunsaturated fatty acids have essentially the same fluidity as that of the control cells. The possible significance of this result to impaired cell-cell interaction is discussed.

Cell Membrane↗
PubMed1979

An analysis of the protein, glycoprotein and monosaccharide composition of Dictyostelium discoideum plasma membranes during development.

Qualitative and quantitative changes in the protein and glycoprotein components of the plasma membrane of the cellular slime mould Dictyostelium discoideum have been detected by analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoretic patterns. The amounts of proteins of subunit molecular weight 220 000, 91 000, 63 000, 59 000, 56 000 increased during the acquisition of aggregation competence, while proteins of subunit molecular weight 82 000 and 22 000 decreased. The amounts of glycoproteins with apparent subunit molecular weights 285 000, 150 000, 137 000, 100 000, 53 000, 50 500 and 30 500 increased during differentiation while a 125 000 dalton component decreased dramatically in amount. The neutral and amino sugar composition of the plasma membrane was also analyzed and found to remain essentially unchanged during the first 12 h of differentiation. The major sugars were mannose, fucose, and glucosamine; galactose and galactosamine were also present, but in lower amounts.

Carbohydrates↗
PubMed1979

Bilayer rigidity of the erythrocyte membrane2H-NMR of a perdeuterated palmitic acid probe.

Perdeuterated palmitic acid was intercalated into the human erythrocyte membrane and its motion studied by dueterium nuclear magnetic resonance (2H-NMR). From analysis of temperature dependent changes in the 2H-NMR spectra and from an analysis of derived moments we conclude that the acyl chains of the erythrocyte lipids do not exhibit a detectable phase transition.

Deuterium↗
PubMed1979

Detection of erythrocyte membrane structural abnormalities in lecithin: cholesterol acyltransferase deficiency using a spin label approach.

The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCAT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different from that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physiocochemical state of the membrane. An investigation of the spectral lineshapes below 10 degrees C allowed a distinction to be made at the membrane level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of elec-ron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.

Electron Spin Resonance Spectroscopy↗
PubMed1978

Fatty acyl chain order in lecithin model membranes determined from proton magnetic resonance.

Proton magnetic resonance (1H NMR) has been used to compare the local orientational order of acyl chains in phospholipid bilayers of multilamellar and small sonicated vesicular membranes of dipalmitoyllecithin (DPL) at 50 degrees C and egg yolk lecithin (EYL) at 31 degrees C. The orientational order of the multilamellar systems was characterized using deuterium magnetic resonance order parameters and 1H NMR second moments. 1H NMR line shapes in the vesicle samples were calculated using vesicle size distributions, determined directly using electron microscopy, and a theory of motional narrowing, which takes into account the symmetry properties of the bilayer systems. The predicted non-Lorentzian line shapes and widths were found to be in good agreement with experimental results, indicating that the local orientational order (called "packing" by many workers) in the bilayers of small vesicles and in multilamellar membranes is substantially the same. This results was found to be true not only for the largest 1H NMR line associated with the nonterminal methylene protons but also for the resolved 1H NMR lines due to the alpha-CH2 and the terminal CH3 positions on the acyl chain. Analysis of the vesicle 1H NMR spectra of EYL taken with different medium viscosities yielded a value of approximately 4 X 10(-8) cm2 s-1 for the lateral diffusion constant of the phospholipid molecules at 31 degrees C.

Fatty Acids↗