Osmotic equilibria in fibroblasts in tissue culture measured by immersion refractometry

1958 ◽  
Vol 149 (934) ◽  
pp. 130-143 ◽  
Keyword(s):  
Water Content ◽  
Osmotic Pressure ◽  
Regression Line ◽  
Fluid Medium ◽  
Physiological Conditions ◽  
Chick Heart ◽  
Osmotic Behaviour ◽  
Osmotic Properties ◽  
Saline Medium

Volume-osmotic pressure relationships at equilibrium have been obtained in chick heart fibroblasts grown in slide-coverslip cultures in a fluid medium consisting of heparinized plasma and embryo extract. The refractive index of the fibroblast gives a direct measure of its solid concentration, and the volume is estimated as the reciprocal of concentration. The volume is found to be linearly related to the reciprocal of the osmotic pressure over a range from 130 to 587 m-osm, provided the measurements are carried out rapidly at 38°C. The isotonic water content of the cells derived from the gradient of the regression line on the basis of the simple Boyle-van’t Hoff Law was found to be less than actual water content obtained by direct refractometry, i. e. the value of Ponder’s ℛ was 0⋅94 (s. d. 0⋅04). In cultures grown in a simple saline medium and measured at 22°C the volume was related linearly to the reciprocal of the osmotic pressure only between the limits of 330 and 191 m-osm. Outside these limits the volume was greater than expected and this was attributed to alterations in the semi-permeable properties of the cell membrane. The value of Ponder’s ℛ in these cultures was 1⋅15. The importance of the quantity, ℛ, as applied to cells other than the erythrocyte, is indicated. The value, 0⋅94 (s. d. 0⋅04), obtained in fibroblasts under physiological conditions is not explicable on the basis of the probable osmotic properties in vitro of the cell proteins. The discrepancy is within the experimental error, but it may also be due to abnormal osmotic behaviour of the cell proteins resulting from some form of intermolecular structure in the cytoplasm.

Osmotic equilibria in human erythrocytes studied by immersion refractometry

1958 ◽  
Vol 148 (931) ◽  
pp. 241-256 ◽  
Keyword(s):  
Water Content ◽  
Osmotic Pressure ◽  
Regression Line ◽  
Osmotic Coefficients ◽  
Haemoglobin Concentration ◽  
Human Erythrocytes ◽  
Erythrocyte Volume ◽  
Immersion Medium ◽  
A Cell ◽  
Limits Of Error

New data are presented on volume-osmotic pressure relationships at equilibrium in human erythrocytes. The inadequacy of previous theoretical treatments of the subject is pointed out and a new theoretical approach is presented. The refractive index of the erythrocyte gives a direct measure of its solid concentration, and the volume is estimated as the reciprocal of concentration. The erythrocyte volume was found to be linearly related to the reciprocal of the osmotic pressure over a range from 300 m-osm. down to a lower limit of between 130 and 200 m-osm. (depending on the pH of the immersion medium). The gradient of the regression line was not, however, that expected from the simple Boyle-van’t Hoff law. The isotonic water content of the erythrocyte derived from the gradient on the basis of the simple Boyle-van’t Hoff Law was found to be less than the actual water content obtained by direct refractometry, i.e. the value of Ponder’s R was found to average 0.95. Below a point between 130 and 200 m-osm. it was found that the erythrocyte volume did not increase in proportion to the fall of osmotic pressure. This was attributed to incipient haemolysis. The isotonic haemoglobin concentration of erythrocytes was found to vary from 38.5% at pH 6.9 to 30.3% at pH 5.6 in the same subject. The thermodynamic basis of the Boyle-van’t Hoff law is examined. It is explained that a cell will obey the simple Boyle-van’t Hoff law only if the osmotic coefficients of all fractions of the cell solute do not change significantly within the range of intracellular concentrations produced in osmotic experiments. From the fact that the osmotic coefficient of haemoglobin changes rapidly with concentration, it is concluded that the erythrocyte will not obey the simple Boyle-van’t Hoff law; swelling in hypotonic solutions will be less than that predicted from the law. The degree of the discrepancy from the Boyle-van’t Hoff law is calculated from known osmotic coefficients of haemoglobin, and is shown to result in an expected value of Ponder’s R less than 1.0, and ranging from 0.925 to 0.975 depending on the corpuscular haemoglobin concentration. The observed values of R in different experiments are compared with the expected values and agreement is found within the limits of error of the experimental method.

Action of Lysolecithin on Blood Platelets

1963 ◽  
Vol 09 (03) ◽  
pp. 512-524 ◽  
Author(s):  
Chava Kirschmann ◽  
Sara Aloof ◽  
Andre de Vries
Keyword(s):  
Blood Platelets ◽  
Protective Action ◽  
Platelet Surface ◽  
Washed Platelet ◽  
Sufficient Concentration ◽  
Saline Medium

SummaryLysolecithin is adsorbed to washed blood platelets and, at sufficient concentration, lyses them, inhibits their clot-retracting activity and promotes their thromboplastin-generating activity. Lysolecithin adsorption to the platelet was studied by using P32-labelled lysolecithin obtained from the liver of rats injected with labelled orthophosphate. The amount of lysolecithin adsorbed to the surface of the washed platelet in saline medium is dependent on the concentration of lysolecithin in solution and reaches saturation — 5 × 10-8 jig per platelet — at a concentration of 9—10 µg per ml. Platelet lysis in saline medium begins at a lysolecithin concentration higher than 18 jig per ml. Plasma and albumin prevent adsorption of lysolecithin to the platelet and protect the platelet from damage by lysolecithin. Albumin is able to remove previously adsorbed lysolecithin from the platelet surface. The protective action of plasma explains the lack of platelet damage in blood, the plasma lecithin of which has been converted to lysolecithin by the action of Vipera palestinae venom phosphatidase, in vitro and in vivo.

scholarly journals Design and Analysis of a Continuous and Non-Invasive Multi-Wavelength Optical Sensor for Measurement of Dermal Water Content

Sensors ◽  
2021 ◽  
Vol 21 (6) ◽  
pp. 2162
Author(s):  
Mohammad Mamouei ◽  
Subhasri Chatterjee ◽  
Meysam Razban ◽  
Meha Qassem ◽  
Panayiotis A. Kyriacou
Keyword(s):  
Water Content ◽  
Optical Sensor ◽  
Broad Band ◽  
Gravimetric Analysis ◽  
Skin Damage ◽  
Biophysical Parameter ◽  
Non Invasive ◽  
Multi Wavelength ◽  
Invasive Measurement

Dermal water content is an important biophysical parameter in preserving skin integrity and preventing skin damage. Traditional electrical-based and open-chamber evaporimeters have several well-known limitations. In particular, such devices are costly, sizeable, and only provide arbitrary outputs. They also do not permit continuous and non-invasive monitoring of dermal water content, which can be beneficial for various consumer, clinical, and cosmetic purposes. We report here on the design and development of a digital multi-wavelength optical sensor that performs continuous and non-invasive measurement of dermal water content. In silico investigation on porcine skin was carried out using the Monte Carlo modeling strategy to evaluate the feasibility and characterize the sensor. Subsequently, an in vitro experiment was carried out to evaluate the performance of the sensor and benchmark its accuracy against a high-end, broad band spectrophotometer. Reference measurements were made against gravimetric analysis. The results demonstrate that the developed sensor can deliver accurate, continuous, and non-invasive measurement of skin hydration through measurement of dermal water content. Remarkably, the novel design of the sensor exceeded the performance of the high-end spectrophotometer due to the important denoising effects of temporal averaging. The authors believe, in addition to wellbeing and skin health monitoring, the designed sensor can particularly facilitate disease management in patients presenting diabetes mellitus, hypothyroidism, malnutrition, and atopic dermatitis.

scholarly journals Regulation of Mitochondria-Associated Membranes (MAMs) by NO/sGC/PKG Participates in the Control of Hepatic Insulin Response

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1319 ◽  
Author(s):  
Arthur Bassot ◽  
Marie-Agnès Chauvin ◽  
Nadia Bendridi ◽  
Jingwei Ji-Cao ◽  
Guillaume Vial ◽  
...  
Keyword(s):  
Insulin Sensitivity ◽  
Insulin Signaling ◽  
Soluble Guanylate Cyclase ◽  
Insulin Response ◽  
Cyclophilin D ◽  
Hepatic Insulin Sensitivity ◽  
Physiological Conditions ◽  
Huh7 Cells ◽  
The Impact

Under physiological conditions, nitric oxide (NO) produced by the endothelial NO synthase (eNOS) upregulates hepatic insulin sensitivity. Recently, contact sites between the endoplasmic reticulum and mitochondria named mitochondria-associated membranes (MAMs) emerged as a crucial hub for insulin signaling in the liver. As mitochondria are targets of NO, we explored whether NO regulates hepatic insulin sensitivity by targeting MAMs. In Huh7 cells, primary rat hepatocytes and mouse livers, enhancing NO concentration increased MAMs, whereas inhibiting eNOS decreased them. In vitro, those effects were prevented by inhibiting protein kinase G (PKG) and mimicked by activating soluble guanylate cyclase (sGC) and PKG. In agreement with the regulation of MAMs, increasing NO concentration improved insulin signaling, both in vitro and in vivo, while eNOS inhibition disrupted this response. Finally, inhibition of insulin signaling by wortmannin did not affect the impact of NO on MAMs, while experimental MAM disruption, using either targeted silencing of cyclophilin D or the overexpression of the organelle spacer fetal and adult testis-expressed 1 (FATE-1), significantly blunted the effects of NO on both MAMs and insulin response. Therefore, under physiological conditions, NO participates to the regulation of MAM integrity through the sGC/PKG pathway and concomitantly improves hepatic insulin sensitivity. Altogether, our data suggest that the induction of MAMs participate in the impact of NO on hepatocyte insulin response.

scholarly journals Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants

2006 ◽  
Vol 397 (3) ◽  
pp. 509-518 ◽  
Author(s):  
Andreas Fischbach ◽  
Stephan Adelt ◽  
Alexander Müller ◽  
Günter Vogel
Keyword(s):  
Dictyostelium Discoideum ◽  
Genetic Manipulation ◽  
Inositol Phosphate ◽  
Targeted Mutagenesis ◽  
Inositol Polyphosphate ◽  
Fluid Medium ◽  
Physiological Processes ◽  
Evolutionarily Conserved ◽  
Cellular Slime

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.

scholarly journals Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase

1980 ◽  
Vol 192 (1) ◽  
pp. 155-163 ◽  
Author(s):  
R Odessey
Keyword(s):  
Skeletal Muscle ◽  
Liver Mitochondria ◽  
Initial Activity ◽  
Rat Skeletal Muscle ◽  
Kidney Mitochondria ◽  
Physiological Conditions ◽  
Acid Dehydrogenase ◽  
Skeletal Muscle Mitochondria ◽  
Branched Chain

The branched chain 2-oxo acid dehydrogenase from rat skeletal muscle, heart, kidney and liver mitochondria can undergo a reversible activation-inactivation cycle in vitro. Similar results were obtained with the enzyme from kidney mitochondria of pig and cow. The dehydrogenase is markedly inhibited by ATP and the inhibition is not reversed by removing the nucleotide. The non-metabolizable ATP analogue adenosine 5′-[beta gamma-imido] triphosphate can block the effect of ATP when added with the nucleotide, but has no effect by itself, nor can it reverse the inhibition in mitochondria preincubated with ATP. These findings suggest that the branched chain 2-oxo acid dehydrogenase undergoes a stable modification that requires the splitting of the ATP gamma-phosphate group. In skeletal muscle mitochondria the rate of inhibition by ATP is decreased by oxo acid substrates and enhanced by NADH. The dehydrogenase can be reactivated 10-20 fold by incubation at pH 7.8 in a buffer containing Mg2+ and cofactors. Reactivation is blocked by NaF (25 mM). The initial activity of dehydrogenase extracted from various tissues of fed rats varies considerably. Activity is near maximal in kidney and liver whereas the dehydrogenase in heart and skeletal muscle is almost completely inactivated. These studies emphasize that comparisons of branched chain 2-oxo acid dehydrogenase activity under various physiological conditions or in different tissues must take into account its state of activation. Thus the possibility exists that the branched chain 2-oxo acid dehydrogenase may be physiologically regulated via a covalent mechanism.

Selective recruitment of arrestin-3 to clathrin coated pits upon stimulation of G protein-coupled receptors

2000 ◽  
Vol 113 (13) ◽  
pp. 2463-2470 ◽  
Author(s):  
F. Santini ◽  
R.B. Penn ◽  
A.W. Gagnon ◽  
J.L. Benovic ◽  
J.H. Keen
Keyword(s):  
G Protein ◽  
G Protein Coupled Receptors ◽  
Coated Pits ◽  
Over Expression ◽  
Physiological Conditions ◽  
A Cell ◽  
G Protein Coupled ◽  
Comparable Magnitude

Non-visual arrestins (arrestin-2 and arrestin-3) play critical roles in the desensitization and internalization of many G protein-coupled receptors. In vitro experiments have shown that both non-visual arrestins bind with high and approximately comparable affinities to activated, phosphorylated forms of receptors. They also exhibit high affinity binding, again of comparable magnitude, to clathrin. Further, agonist-promoted internalization of many receptors has been found to be stimulated by exogenous over-expression of either arrestin2 or arrestin3. The existence of multiple arrestins raises the question whether stimulated receptors are selective for a specific endogenous arrestin under more physiological conditions. Here we address this question in RBL-2H3 cells, a cell line that expresses comparable levels of endogenous arrestin-2 and arrestin-3. When (beta)(2)-adrenergic receptors are stably expressed in these cells the receptors internalize efficiently following agonist stimulation. However, by immunofluorescence microscopy we determine that only arrestin-3, but not arrestin-2, is rapidly recruited to clathrin coated pits upon receptor stimulation. Similarly, in RBL-2H3 cells that stably express physiological levels of m1AChR, the addition of carbachol selectively induces the localization of arrestin-3, but not arrestin-2, to coated pits. Thus, this work demonstrates coupling of G protein-coupled receptors to a specific non-visual arrestin in an in vivo setting.

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