Osmotically Active Research Articles

Effect of trophic, osmotically active and solidifying components of nutrient medium on the direct embryognesis in spring barley anther culture in vitro

Aim. Ambryoidogenesis, or somatic embryogenesis, is known to be the most efficient mode of plant regeneration in plant cell, tissue and organ culture. The investigation was aimed to elucidate effects of trophic and osmogenic components of inductive medium on the frequency of direct embryoidogenesis in spring barley anther culture in vitro and to determine mechanism of morphogenesis improvement coursed by chemically modified starch D-5aM used as a gelling agent instead of agar. Methods. Anthers of DH-line with a high androgenetic capacity were inoculated on inductive media containing N6 macro-, MS micronutrients, organic supplements, maltose or mannitol (0.3 M) and solidified with agar or chemically modified starch. Results. A positive effect of combination of high maltose content and chemically modified starch on the induction and regeneration processes in spring barley anther culture in vitro was confirmed. It was also shown that mannitol didn’t keep any growth or development processes going in barley anther and embryo culture, but at the same time this substance had no toxic effect. Conclusions. In order to achieve a high frequency of induction in spring barley anther culture, it is necessary to use medium containing maltose – a low weight component both with trophic and with osmotic activity. Keywords: Hordeum vulgare L., anther culture in vitro, mannitol, maltose, embryo formation, plant regeneration.

Effect of cooling rate and cryoprotectant concentration on intracellular ice formation of small abalone (Haliotis diversicolor) eggs

The intracellular ice formation (IIF) behavior of Haliotis diversicolor (small abalone) eggs is investigated in this study, in relation to controlling the cooling rate and the concentration of dimethyl sulfoxide (DMSO). The IIF phenomena are monitored under a self-developed thermoelectric cooling (TEC) cryomicroscope system which can achieve accurate temperature control without the use of liquid nitrogen. The accuracy of the isothermal and ramp control is within ±0.5°C. The IIF results indicate that the IIF of small abalone eggs is well suppressed at cooling rates of 1.5, 3, 7 and 12°C/min with 2.0, 2.5, 3.0 and 4.0M DMSO in sea water. As 2.0M DMSO in sea water is the minimum concentration that has sufficient IIF suppression, it is selected as the suspension solution for the cryopreservation of small abalone eggs in order to consider the solution’s toxicity effect. Moreover, IIF characteristics of the cumulative probability of IIF temperature distribution are shown to be well fitted by the Weibull probabilistic distribution. According to our IIF results and the Weibull distribution parameters, we conclude that cooling at 1.5°C/min from 20 to −50°C with 2.0M DMSO in sea water is more feasible than other combinations of cooling rates and DMSO concentrations in our experiments. Applying this protocol and observing the subsequent osmotic activity, 48.8% of small abalone eggs are osmotically active after thawing. In addition, the higher the cooling rate, the less chance of osmotically active eggs. A separate fertility test experiment, with a cryopreservation protocol of 1.5°C/min cooling rate and 2.0M DMSO in sea water, achieves a hatching rate of 23.7%. This study is the first to characterize the IIF behavior of small abalone eggs in regard to the cooling rate and the DMSO concentration. The Weibull probabilistic model fitting in this study is an approach that can be applied by other researchers for effective cryopreservation variability estimation and analysis.

Osmotic behaviour of polyhedral non-ionic surfactant vesicles (niosomes).

In addition to common spherical non-ionic surfactant vesicles (niosomes), disc-like, tubular, and polyhedral niosomes have also been reported. The permeability and osmotic activity of niosomes are important in determining their use as controlled-release drug-delivery systems. These properties have been compared for polyhedral niosomes prepared by hydrating a mixture of a hexadecyl diglycerol ether (C16G2), a poly(24)oxyethylene cholesteryl ether (Solulan C24), 91:9 or 98:2, and conventional spherical niosomes prepared from the same surfactants but with cholesterol. When subjected to osmotic gradients, polyhedral niosomes, the membranes of which are in the gel phase, swell and shrink less than their spherical counterparts and they are more permeable to the hydrophilic solute 5(6)-carboxyfluorescein. In 2 M NaCl the rate of release of carboxyfluorescein from polyhedral niosomes (both containing 9% Solulan C24) into either a hypotonic (water) or an isotonic medium (2 M NaCl) was low. This contrasted with similarly loaded spherical niosomes and polyhedral niosomes containing 2% Solulan C24, from which release was high in hypotonic media (e.g. water) but less in an isotonic medium (2 M NaCl). For both polyhedral and spherical niosomes encapsulating carboxyfluorescein (pKa = 6.4), release rates were higher at pH 8 than at pH 5. Polyhedral niosomes are thus, in general, less osmotically active than spherical niosomes because of their rigid but highly permeable membranes. The unusual polyhedral membrane impermeability to carboxyfluorescein co-entrapped with salt in hypotonic media is a function of Solulan C24 content, and is possibly a result of salting out of the polyoxyethylene chains; this is, therefore, a property that might be manipulated in the design of a drug-delivery system.

Importance of the Body Water Management for Winter Cold Survival of the European Corn Borer Ostrinia Nubilalis Hübn. (Lepidoptera: Pyralidae)

ABSTRACTWinter diapause, a common strategy of many insect species occupying temperate regions, is usually closely related to and coincides with their cold hardiness. Freezing of body fluids represents one of the major obstacles for sub-zero temperatures survival and thus the body water management is an important part of cold hardiness. In this study, we examined some cryobiological parameters, as well as content of glycerol and trehalose in non-diapausing and freeze tolerant diapausing larvae of the European corn borer, Ostrinia nubilalis. Diapausing larvae were divided into two experimental groups—a group exposed to field temperatures (which were in average above 0°C) and a group exposed to -8°C for ten days. Contents of the total body water, osmotically active (OA) and inactive (OI), as well as the supercooling point (SCP) of hemolymph and fat body, were measured by differential scanning calorimetry (DSC). The content of glycerol and trehalose was analysed by gas chromatography. Compared to diapausing groups, non -diapausing larvae had higher SCP, lower content of trehalose and glycerol in both tissues. The content of total and OA water in both tissues of diapausing larvae had changed with low temperatures exposure. At -8°C, the amount of total and OA body water was decreased in hemolymph and increased in fat body while the content of OI water was slightly increased in hemolymph but remained unchanged in fat body. Mean SCPs of both tissues were significantly different—for hemolymph it was around -21°C, which was almost two times lower than for fat body (-10°C). However, the SCPs of fat body and hemolymph had not significantly changed after the exposure to low temperature. The content of glycerol and trehalose was far greater in hemolymph than in fat body for all groups, which is in accordance with the difference between the SCPs of these tissues. Furthermore, exposure of diapausing larvae to sub-zero temperatures (-8°C) had simultaneously provoked an increase in glycerol/trehalose concentration in hemolymph and the decrease in fat body. These adjustments of water and cryoprotectors distribution are an important part of cold hardiness mechanisms.

Improved long term stability of aqueous ethylcellulose film coatings: Importance of the type of drug and starter core

Instability during long term storage due to further gradual coalescence of the film remains one of the major challenges when using aqueous polymer dispersions for controlled release coatings. It has recently been shown that the addition of small amounts of poly(vinyl acetate)–poly(ethylene glycol)-graft-copolymer (PVA–PEG-graft-copolymer) to aqueous ethylcellulose dispersion provides long term stable drug release patterns even upon open storage under stress conditions in the case of theophylline matrix cores. However, the transferability of this approach to other types of drugs and starter cores exhibiting different osmotic activity is yet unknown. The aim of this study was to evaluate whether this novel approach is also applicable to freely water-soluble drugs and osmotically active sugar starter cores. Importantly, long term stable drug release profiles from coated diltiazem HCl-layered sugar cores could be achieved even upon open storage for 1 year under stress conditions (40°C and 75% relative humidity). However, to provide desired drug release profiles the amount of added PVA–PEG-graft-copolymer must be adjusted. A minimal critical content of 10% (w/w) of this hydrophilic additive was identified, under which further polymer particle coalescence upon long term storage under stress conditions cannot be excluded. Potentially too rapid drug release can effectively be slowed down by increasing the coating level. Thus, adapting the polymer blend ratio and coating thickness desired and long term stable drug release profiles (even under stress conditions and open storage) can be provided for very different types of drugs and starter cores by the addition of small amounts of PVA–PEG-graft-copolymer to aqueous ethylcellulose dispersion.

Water Transporting Properties of Hepatocyte Basolateral and Canalicular Plasma Membrane Domains

Previous work from our laboratory supports an important role for aquaporins (AQPs), a family of water channel proteins, in bile secretion by hepatocytes. To further define the pathways and molecular mechanisms for water movement across hepatocytes, we directly assessed osmotic water permeability (Pf) and activation energy (Ea) in highly purified, rat hepatocytes basolateral membrane vesicles (BLMV) and canalicular membrane (CMV) vesicles by measuring scattered light intensity using stopped-flow spectrophotometry. The time course of scattered light for BLMV and CMV fit well to a single-exponential function. In BLMV, Pf was 108 +/- 4 mum.s-1 (25 degrees C) with an Ea of 7.7 kcal/mol; in CMV, Pf was 86 +/- 5 mum.s-1 (25 degrees C) with an Ea of 8.0 kcal/mol. The AQP blocker, dimethyl sulfoxide, significantly inhibited the Pf of both basolateral (81 +/- 4 mum.s-1; -25%) and canalicular (59 +/- 4 mum.s-1; -30%) membrane vesicles. When CMV were isolated from hepatocytes treated with dibutyryl cAMP, a double-exponential fit was needed, implying two functionally different vesicle populations; one population had Pf and Ea values similar to those of CMV from untreated hepatocytes, but the other population had a very high Pf (655 +/- 135 mum.s-1, 25 degrees C) and very low Ea (2.8 kcal/mol). Dimethyl sulfoxide completely inhibited the high Pf value in this second vesicle population. In contrast, Pf and Ea of BLMV were unaltered by cAMP treatment of hepatocytes. Our results are consistent with the presence of both lipid- and AQP-mediated pathways for basolateral and canalicular water movement across the hepatocyte plasma membrane barrier. Our data also suggest that the hepatocyte canalicular membrane domain is rate-limiting for transcellular water transport and that this domain becomes more permeable to water when hepatocytes are exposed to a choleretic agonist, presumably by insertion of AQP molecules. These data suggest a molecular mechanism for the efficient coupling of osmotically active solutes and water transport during canalicular bile formation.

Steady-state volumes and metabolism-independent osmotic adaptation in mammalian erythrocytes.

Erythrocytes of various mammalian species -- including human -- maintain osmotic balance with the blood plasma (osmotic activity 270-310 mosmol). However, their intracellular levels of osmotically active ions (potassium, sodium, chloride, and hydrogencarbonate), water content and osmotic resistance deviate significantly. In the present report we study the relationship among intracellular water, potassium and sodium levels of the erythrocytes of various mammalian species and in the developing calf. In addition, the osmotic resistance, K(+) (Rb(+)) uptake and the DPH fluorescence anisotropy of various erythrocytes and erythrocyte ghost membranes were correlated. The results show no statistically significant relationship between erythrocyte water content and [K(+)+Na(+)] levels or K(+)/Na(+) ratios. The reversal of erythrocyte K(+)/Na(+) ratios coincides with the decrease of steady-state ATP levels in the developing calf. The mobility of lipids within the hydrophobic inner layer of the plasma membrane relates closely to passive K(+) (Rb(+)) uptake, and plays a significant role in regulatory volume changes.

The State of Water in Biological Systems

This paper addresses the issue of how the aqueous cytoplasm is organized on a macroscopic scale. Mitochondria were used as the experimental model, and a unique experimental approach was used to probe the properties of water in the mitochondrial matrix. The results demonstrate aqueous phase separation into two distinct phases with different osmotic activity and different solute partition coefficients. The larger phase, designated "normal water," is osmotically active and behaves in every respect like a bulk, dilute salt solution. The smaller phase, designated "abnormal water," is osmotically inactive and comprises the water of hydration of matrix proteins. It is, nevertheless, solvent water, with highly selective partition coefficients, and behaves like a Lewis base.

A mechanistic study of the release of osmotic agents from polymeric monoliths

The mechanisms governing the release of osmotically active agents from hydrophobic polymeric monoliths are outlined. The release kinetics are loading dependent. Below the percolation threshold the bulk of the loaded excipient is released with zero order kinetics. Above the percolation threshold, diffusional and osmotic release mechanisms occur simultaneously resulting in a release profile that appears to be diffusionally controlled but whose slope increases with excipient osmotic activity. Current models explaining the observed zero order release rate are reviewed and their limitations outlined. A model was developed to overcome these limitations. The main parameters affecting release were determined to be the osmotic activity, saturation concentration and density of the incorporated agent, the tensile strength, elastic modulus and hydraulic permeability of the polymer and the fraction of particles released by dissolution and diffusion. The model was correlated against data from experiments and the literature and provided an excellent fit.

Studies for the osmotic parameter of liposomes

By using the former equation (8), we modified the equation which can show the dissimilar osmotic behavior of liposome with composition change. The slope of the new equation was presented as the ratio of osmotically active volume (V act ) to the total volume (V total =V act +V dead ; V dead is osmotically inactive volume) of liposomes. We defined it as a Z-value, which can elucidate the dissimilarity of the osmotic activity of multilamellar liposomes with the change of phospholipid composition and the differences of physicochemical properties of liposomes. Z-value was applied for studying the physico-chemical properties of liposomal membrane. The factor that affects on the Z-value was not the lipid concentration of liposome stock dispersion but the lipid composition of liposomal membrane. As the content of dicetylphosphate, the negative charged phospholipid, was increased, the osmotic activity, represented by Z-value, of multilamellar liposome was decreased. Under the hypertonic conditions (shrinking region), Z-value steadily increased and reached a maximum at 10 mole percent cholesterol with increasing the cholesterol content.

Permeability of rod outer segment disk membranes as probed by ficoll density gradient centrifugation and by turbidimetry

Osmotic volume changes of isolated disks prepared from bovine rod outer segments were investigated utilizing the capacity of osmotically intact disks to float on 2·5% ( w v ) Ficoll under hypotonic conditions. The amount of floating disks decreases steadily and approximately linearly when the osmolarity is increased stepwise with an osmotically active solute. The slope of the decrease becomes smaller for partially permeable substances and no decrease is observed for fully permeable solutes. All ions assayed by this method as well as sucrose were osmotically fully active to disk membranes. The buffer molecules Tris and imidazole partially penetrated the disk membranes at pH 7, whereas the zwitterionic buffer Hepes was impermeable. Ethanol showed absolutely no osmotic activity to disk membranes and, therefore, can easily penetrate these membranes. Concurrent turbidimetric measurements confirmed these results. Upon prolonged exposure of disk membranes to 5% ( w v ) Ficoll, a considerable modification of the osmotic properties of disk membranes is observed, indicating a significant interaction of 5% ( w v ) Ficoll with disk membranes. No influence of 2·5% ( w v ) Ficoll on the osmotic properties of disk membranes was detected. The restrictions of the use of Ficoll for disk membrane preparative purposes will be discussed.

Osmotic activity of dimethyl sulfoxide in the renal distal tubule

Dimethyl Sulfoxide (DMSO) is a dipolar organic solvent that has received widespread publicity as a possible topical analgesic [II and has also been used with increasing frequency as a solvent to dissolve hydrophobic drugs prior to their use in biological systems [2, 31. Because it penetrates biological membranes rapidly, some investigators have stated that DMSO is not osmotically active in the kidney [2, 41. However, we are not aware of any systematic investigation of the effects of DMSO on water transport by kidney tubules. Recently, in beginning a study of the effects of a rather insoluble diuretic on electrolyte transport in the renal distal tubule, we used DMSO to dissolve the drug in aqueous perfusion solutions. In these experiments, we observed changes in net volume fluxes that depended on the concentration of DMSO in the perfusion solutions. Because it would be important to know whether DMSO can exert osmotic effects when it is used as a vehicle in experimental studies, and because of the continued use of intravenous DMSO in a variety of clinical settings, the present experiments were conducted to determine whether DMSO is osmotically active and whether it affects volume fluxes in the distal tubule of the rat. Methods. Experiments were performed on 200 to 340 g Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, Indiana) that were allowed free access to commercial rat chow (Ralston Purina, St. Louis, Missouri) and tap water up to the time of the experiment. The rats were anesthetized by intraperitoneal injection of 100 to 110 mg/kg body weight thiobutabarbital (mactin, Byk-Gulden, Konstanz, Federal Repubic of Germany). A salt solution (140 mrvi NaCI, 4 mM KCI) was infused at 10 mi/hr/kg of body wt. The left kidney was exposed through a flank incision and was prepared for micropuncture as described previously [5]. When two distal segments belonging to one nephron were found at the kidney surface, a pipette filled with an artificial solution and connected to a microperfusion pump was placed in the upstream segment. The last surface proximal segment was blocked with castor oil and a complete timed collection of fluid from the downstream segment of the distal tubule was accomplished using a pipette filled with stained (Sudan Black) mineral oil as described previously [5, 61. It was frequently possible subsequently to insert a second collection pipette at the same collection site and, after removing the first perfusion pipette, to insert a pipette filled with a different perfusion solution. Two collections were obtained from the same nephron segment in this manner in 48 cases. Latex casts were made of 14 nephrons. The mean length of the perfused segments was 1.3 0.05 mm and the mean length

Studies on Water in Barnacle Muscle Fibres I. The Dry Weight Components of Fresh Fibres

ABSTRACT The dry weight components comprise 25 % of the total weight of fresh muscle fibres of the giant barnacle Balanus nubilis. Half of the solids are water insoluble; 6 % of this material is fat, and the rest is mostly myofilament protein. Less than 3 % of the dry weight consists of inorganic ions, and these account for only a quarter of the osmotic activity in the fibre water. Seventy per cent of the osmotic activity is due to nitrogenous solutes which comprise 18% of the dry weight. Glycine predominates, followed by TMAO + betaine, proline, glutamine + asparagine, arginine, and taurine. About 13% of the dry weight is soluble protein and not more than 10 % is an unidentified soluble substance of high molecular weight. Thus, Balanus conforms with other marine invertebrates in the high content of osmotically active nitrogenous solutes in its myoplasm.

Electrolyte and non-electrolyte distribution in the Ehrlich ascites tumor cells during the cell cycle.

In a previous study, evidence was presented for changes in the state of water and osmotically active solutes during the cell cycle. Total water was constant at 82% (w/w), while the fraction of water that was osmotically active decreased from a maximum during S to a minimum at mitosis. Total Na+, K+, and C1- in milliequivalents per liter of cell water remained constant. Therefore, electrolytes are sequestered in the osmotically inactive water. Evidence is now presented that Na+ exists primarily as one compartment, with a second, slower compartment appearing during S and disappearing during G2. Na+ is completely exchangeable during the entire cell cycle. The distribution of other penetrating solutes was also investigated. When placed in hyperosmotic ethylene glycol solutions, cells first shrink, then swell to their original volumes. 14C-ethylene glycol distributes in 89% of cell water throughout the cell cycle. However, 14C-urea distributes in anywhere from 86-100% of the cell water, depending on the stage in the cell cycle. Both solutes are at chemical equilibrium in water in which they are distributed, but they differ in their effects on cell volume. The final volume at which cells equilibrate in urea varies with the concentration of urea in the environment and with time into the cell cycle. Results suggest a loss of osmotically active particles or decreased osmotic activity of urea.

Some physical and chemical properties of the lettuce seed endosperm

Lettuce seeds (Lactuca sativa) are shown to be encased in an osmotically active cellulose membrane derived from the endosperm. The osmotic activity of this membrane is maintained in seeds treated with strong acid (4 M HCl), strong base (4.4 M NaOH), organic solvents, and protein solvents, indicating that there is no contribution of lipid, protein, or living cells to the osmotic properties of the membrane. Only cellulose solvents such as xanthate and cellulase were effective in disrupting the membrane.When dry seeds are treated with 50% solutions of methanol, ethanol, or acetone or with 10 mM HgCl2, they become osmotically distended seeds (ods) as a result of the accumulation of soluble molecules in the fluid layer (EEF) between the membrane and embryo. This process is temperature dependent and appears to require enzymatic activity.The molecular exclusion limit of the ods membrane was measured by osmotic equilibration techniques and was found to be about 300 daltons. This then explains previous observations on the impermeability of lettuce seeds to substances such as actinomycin D (mol. wt. 1255.5) during germination. Molecules of molecular weight greater than about 300 daltons will be excluded until the endospermal barrier is ruptured at about 10–12 h after germination onset.

Permeability and integrity properties of lecithin-sphingomyelin liposomes

The properties of multibilayered liposomes formed from mixtures of sphingomyelin and phosphatidylcholine in varying mole ratio (all containing one mole dicetylphosphate per 10 moles of phospholipids) have been studied. The principal findings are: (1) Over the range 0 to 1 mole fraction sphingomyelin the liposomes exhibit multibilayer structure as visualized by electron microscopy using negative staining. (2) The two phospholipids differ in their interaction with dicetylphosphate in a bilayer structure. In mixtures of the two the effect of sphingomyelin is dominant. (3) The ability of sphingomyelin to form osmotically active liposomes depends on its fatty acids' composition. (4) Liposomes of all mole fractions of sphingomyelin are osmotically active if the C 24 : 1 fatty acid content of sphingomyelin exceeds 10% of the total acyl residues. The degree of osmotic activity, however, depends upon the molar ratio between the two phospholipids. The highest initial rate of water permeability was found for lecithin liposomes. The maximal change of volume by osmotic gradients was obtained for liposomes composed of 1 : 1 lecithin to sphingomyelin (mole ratio). (5) Permeability to glucose increased with increasing lecithin mole fraction. (6) Liposomes composed of 1 : 1 lecithin to sphingomyelin have the largest aqueous volume per mole of phospholipid as measured by glucose trapping. (7) The osmotic fragility of liposomes made of sphingomyelin is higher than for those made of lecithin but the highest osmotic fragility was obtained for liposomes containing lecithin and sphingomyelin in 1 : 1 molar ratio. (8) When the temperature is abruptly lowered to about 2°C, liposomes formed from phosphatidylcholine release about 20% of trapped glucose during a transient increase in permeability. Liposomes containing 0.5 mole fraction sphingomyelin release about 30% of the trapped glucose under these conditions. Liposomes composed of sphingomyelin alone do not exhibit this phenomenon.

Model connective tissue systems. A physical study of gelatin gels containing proteoglycans.

AbstractThe swelling behavior of gelatin gels containing proteoglycans (sulphated proteoglycans from bovine intervertebral discs and a hyaluronate proteoglycan from bovine synovial fluid) when immersed in osmotically active solutions of dextran have been measured. The presence of the proteoglycans markedly affects the internal osmotic contribution to the swelling pressure of the gel. These internal osmotic pressures are considerably in excess of the sum of the osmotic activities of the individual components. This behavior is understood in terms of an entropic interaction between the gelatin and the proteoglycan molecules. By use of the “dilute solution” treatment of Flory, the osmotic pressure excesses are related to the volumes and hence dimensions of the interact acting species. A comparison of these values with those calculated by other means shows good agreement. The osmotic behavior of the complex gels can be understood on a mechanistic basis, if we regard the gelatin and sulphated proteoglycans as spheres and the hyaluronate proteoglycan as a rod.

The osmotic effects of electron microscope fixatives.

The reflecting cells on the scales of sprat and herring contain ordered arrays of guanine crystals. The spacing of the crystals within these cells determines the wave bands of the light which they reflect, hence volume changes in the reflecting cells can be observed as color changes directly. This property of the scales is used to show that (a) fixation with osmium tetroxide solutions destroys osmotic activity; (b) fixation with aldehyde solutions does not destroy osmotic activity and does not cause volume changes if the aldehydes are made up in salt or sucrose solutions whose osmolarities, discounting the aldehyde, are about 60% of those to which the cells are in equilibrium in life, and (c) after aldehyde fixation the cells are osmotically active but come to a given volume in salt and sucrose solutions of concentrations only 60% of those which give their volume before fixation. Various possible mechanisms underlying the change of osmotic equilibrium caused by aldehyde fixation are discussed.

Action of Triton X-100 on chloroplast membranes. Mechanisms of structural and functional disruption.

Addition of Triton X-100 to chloroplast suspensions to a final concentration of 100-200 microM causes an approximate tripling of chloroplast volume and complete inhibition of light-induced conformational changes, light-dependent hydrogen ion transport, and photophosphorylation. Electron microscopic studies show that chloroplasts treated in this manner manifest extensive swelling in the form of vesicles within their inner membrane structure. Triton was adsorbed to chloroplast membranes in a manner suggesting a partition between the membrane phase and the suspending medium, rather than a strong, irreversible binding. This adsorption results in the production of pores through which ions may freely pass, and it is suggested that the inhibition of conformational changes, hydrogen ion transport, and photophosphorylation by Triton is due to an inability of treated chloroplast membranes to maintain a light-dependent pH gradient. The observed swelling is due to water influx in response to a fixed, osmotically active species within the chloroplasts, after ionic equilibrium has occurred. This is supported by the fact that chloroplasts will shrink upon Triton addition if a nonpenetrating, osmotically active material such as dextran or polyvinylpyrrolidone is present externally in sufficient concentration (>0.1 mM) to offset the osmotic activity of the internal species.

THE CONTRACTILE VACUOLE AND THE ADJUSTMENT TO CHANGING CONCENTRATION IN FRESH WATER AMOEBAE

1. Amoeba lacerata, a fresl water amoeba, is able to adjust and live in any concentration of the salts of sea water up to 125 per cent.2. Contractile vacuoles are formed in all concentrations and are separate from food vacuoles.3. Food is engulfed with little or no water and small protoplasmic vacuoles coalesce with it. When digestion is completed, the food residues are eliminated with little or no fluid.4. The major part of fluid elimination occurs by way of the contractile vacuoles.5. Contractile vacuoles grow in size by coalescence and osmotic swelling.6. The rate of fluid elimination is affected by a number of factors, but under constant optimal conditions it varies inversely with the concentration of the medium. It is suggested that the rate is proportional to the rate of catabolism.7. The osmotic pressure of the protoplasm varies in the same direction as that of the outside medium. The protoplasm of completely adjusted amoebae is very nearly equal to that of the medium, the osmotic difference being less than that of 5 per cent sea water (0.13 atmosphere).8. Contractile vacuoles have been shown to contain osmotically active substances; they shrink in hypertonic and swell in hypotonic solutions regardless of whether remain in the cell or have been removed from the cell.9. The volume of the amoeba is only temporarily dependent on the concentration of the medium. After adjustment, the volume shows no dependence upon concentration of the medium. If the amoeba is placed in a hypertonic solution the cell shrinks and then swells to its original volume within three hours time. Conversely, if placed in a hypotonic solution, it swells and then shrinks to its original volume.10. The cell membrane is either permeable to substances in the medium especially when adjusting to a change in the concentration of the medium, or is capable of modifying its internal osmotic activity in some other way.11. The contractile vacuoles are obviously excretory and also osmoregulatory organelles since they remove waste substances which would otherwise cause increases in the osmotic pressure in the cell.