The Influence of Sodium-Free Solutions on the Membrane Potential of Frog Muscle Fibers

The membrane potential of frog sartorius muscle fibers in a Cl- and Na-free Ringer's solution when sucrose replaces NaCl is about the same as that in normal Ringer's solution. The K⁺ efflux is also about the same in the two solutions but muscles lose K and PO₄ in sucrose Ringer's solutions. The membrane potential in sucrose Ringer's solution is equal to that given by the Nernst equation for a K⁺ electrode, when corrections are made for the activity coefficients for K⁺ inside and outside the fiber. For a muscle in normal Ringer's solution, the measured membrane potential is within a few millivolts of E_K. This finding is incompatible with a 1:1 coupled Na-K pump. It is consistent with either no coupling of Na efflux to K influx, or a coupling ratio of 3 or greater.     

Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species

In this study, we investigated the possibility of using the droplet-vitrification technique for cryopreserving nodal segments of in vitro plantlets of the endangered plant species Lithodora rosmarinifolia. Among the three vitrification solutions tested, only solutions B1, containing (w/v) 50 % glycerol and 50 % sucrose, and B3, containing 40 % glycerol and 40 % sucrose, were able to induce cryotolerance in nodal explants, resulting in intermediate survival and recovery after cryopreservation. A three-step vitrification protocol, including an additional dehydration treatment with half-strength vitrification solution for 30 min before the treatment with full-strength vitrification solution, did not lead to any improvement in survival and recovery compared with the two-step protocol. The optimal protocol was the following: preculture of nodal segments in liquid medium with 0.3 M sucrose for 16 h and 0.7 M sucrose for 5 h, treatment for 20 min in loading solution containing 1.9 M glycerol + 0.5 M sucrose, dehydration with vitrification solution B1 (glycerol 50.0 %, sucrose 50.0 %, w/v) for 60 min at room temperature, rapid cooling in minute droplets of vitrification solution, and rapid rewarming by immersion of nodal segments for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 33 % recovery of cryopreserved nodal explants was achieved. Regrowth of cryopreserved samples was rapid and direct. These results indicate that long-term storage of L. rosmarinifolia by means of cryopreservation of nodal segments is possible, thereby contributing to securing the diversity of this rare and endangered plant species.     

An in vivo-in vitro alkaline DNA unwinding assay for hepatic DNA damage: comparison with the alkaline sucrose gradient centrifugation technique

The addition of glycerol, sucrose, or other diol-containing reagents to solutions of aminoacyl-tRNA (aa-tRNA) substantially increased the rate of hydrolysis of the aminoacyl ester bond. Glycerol at 4.9% (v/v) doubled the rate of deacylation for several aa-tRNAs and peptidyl-tRNAs, including fMet-tRNAMetf, while 1% (v/v) glycerol increased the deacylation rate by 20%. This effect was not caused by a nuclease contamination, and tRNA deacylated in the presence of glycerol could be fully recharged. The deacylation of aa-tRNA was accelerated by glycerol and sucrose even in the presence of EF-Tu X GTP. In addition, the extent of tRNA aminoacylation was reduced when glycerol was present at concentrations above 2% (v/v). Thus, glycerol and sucrose are not necessarily inert or neutral additions to an in vitro incubation.     

Effect of acetylcholine on ion transport in the frog skeletal muscle

1. The effect of acetylcholine (ACh) on the ion transport of frog (Rana esculenta) sartorius muscles was studied. ACh was applied in bathing solution, Na influx and K efflux were measured using 24Na and 42K isotopes. 2. Na influx of sartorius muscles was increased by 1 mmol/1 ACh 2-10 fold depending on the experimental arrangement. The increase was greater if Na influx was measured at the beginning of ACh depolarization. During ACh treatment the Na influx took about the same time course as the depolarization recorded extracellularly. This type of recording approximately reflects the depolarization proceeding on the sartorius muscle fibres. 3. The presence of 31 nmol/l tetrodotoxin (TTX) did not modify the degree of increase of Na influx. 4. Rate coefficients for K efflux were increased 2-5 fold by ACh. The maximum rate coefficients were obtained in the first minute of ACh treatment. 5. Increase in K loss evolves also in the presence of 31 nmol/l TTX. The increase in rate coefficients was found to be about 30% less than without TTX in the first minute of ACh action. 6. The results indicate that in the presence of ACh the observed increase in Na influx and K efflux is brought about mainly by changes in Na and K conductance induced by ACh at the end-plates rather than by the action potentials accompanying ACh depolarization.     

A method for one-step freezing of mouse embryos

A simple one-step method of freezing mouse embryos directly in liquid nitrogen is described. The main objective of this study was to assess post-thaw survival following predehydration in various mixtures of glycerol and sucrose. Also investigated was pretreatment with glycerol prior to dehydration and effects of embryo stage. When sucrose was held constant (0.25 M) and glycerol concentration varied (1.0-4.0 M), post-thaw survival was best (67%) in 2.0 M glycerol. Pretreatment in glycerol provided no advantage over no pretreatment. When glycerol was held constant (2.0 M) and sucrose concentration varied (0-1.0 M), optimum post-thaw survival (81%) was found in 0.5 M sucrose. Morulae survived better than blastocysts (86% vs 72%, respectively). Transfer of thawed embryos frozen by the optimum treatment (2.0 M glycerol + 0.5 M sucrose) resulted in a birthrate of 41%, compared to 54% for fresh controls. This technique could find application in freezing and thawing of livestock embryos on the farm.     

The subcellular distribution of acetylcholine, choline acetyltransferase and cholinesterases in lobster walking leg nerves

—Nerve bundles from the walking legs of the lobster have been homogenized in either isosmotic or hyperosmotic sucrose solutions and subjected to differential and discontinuous density gradient centrifugation. The resulting subcellular fractions were analysed for their concentrations of acetylcholine (ACh), choline acetyltransferase (ChAc) and cholinesterase (ChE). Enzymic characteristics were used for a biochemical identification of the subfractions. Regardless of the osmotic conditions, ACh was always found in the free form; there was no evidence of any‘bound’ester. After ultracentrifugation of homogenates in the hyperosmotic medium a pellet floating atop was formed; it consisted of membrane fragments and contained more than 30 per cent of the choline-esterhydrolysing enzymes, with a 3 to 4-fold increased specific activity. ChAc was found to be increasingly soluble if the ionic concentration was raised to that of the haemolymph body fluid of the lobster. Thus, all the components of the ACh system were present in substantial amounts but the question of their physiological function remains open.     

The effect of salt or various cryoprotective agents on frog sciatic nerves

Desheathed frog sciatic nerves were tested for membrane damage by noting changes in their conduction velocity, externally recorded action potential, and absolute refractory period. Nerves were exposed for 45–60 min depending on the treatment to 1.5 osmolar environments consisting of concentrated Ringer's solution or Ringer's solution plus glycerol, sucrose, or dimethyl sulfoxide (DMSO). The nerves were exposed to these hypertonic solutions in one of four ways: (1) Gradual exposure plus gradual de-exposure, (2) Gradual exposure (plus direct de-exposure), (3) (direct exposure plus) plus gradual de-exposure, (4) Direct exposure (plus direct de-exposure). After treatment the nerves were allowed to recover for 45–60 min depending on the type of treatment in normal Ringer's solution. The results suggest the following: (1) Concentrated salt solutions damage nerve axons due to increased ion concentration rather than increased tonicity. (2) The effect of osmotic shock on sciatic nerves is relatively minor and completely reversible. (3) Axon membranes behave as simple permeability barriers with respect to sucrose and DMSO, but they appear to behave as dynamic entities which alter their metastable states with respect to concentrated salt or concentrated glycerol in Ringer's solution. (4) Freeze-thaw damage to sciatic nerve axon membranes—and perhaps to any cell membrane—is probably due to increased ionic concentration as well as increased osmolarity.     

Effect of vinblastine sulfate, colchicine and lumicolchicine on membrane organization of normal and transformed cells

Differences in the distribution of plasma membrane intramembranous particles (PMP) have been demonstrated in normal and transformed fibroblasts using freeze fracture and electron microscopy. Transformed 3T3 cells contain randomly distributed PMP and contact-inhibited 3T3 cells have aggregated PMP when frozen in medium, glycerol, sucrose, or following stabilization in 1 % formaldehyde. To define some of the mechanisms controlling the organization of PMP in this system we have examined the effects of microtubule disruptive drugs including vinblastine sulfate and colchicine on SV3T3 cells. These drugs were observed to induce a dose- and time-dependent aggregation of PMP at concentrations between 10⁻⁹ and 10⁻⁵ M. These results suggest that modulation of PMP distribution in these cells may be influenced by an interaction of microtubules with plasma membrane components. However, the observation that lumicolchicine, a derivative of colchicine which does not disrupt microtubules, also promotes PMP aggregation, suggests that these drugs may also have a primary effect on the plasma membrane in addition to the disruption of microtubules. This is supported by the observation that reduced temperature (4 °C) which is known to disrupt microtubules fails to induce PMP aggregation in SV3T3 cells, suggesting the hypothesis that changes in the interaction of plasma membrane or plasma membrane associated constituents may control the distribution of PMP in this cell system.     

Modifications of single acetylcholine-activated channels in BC3H-1 cells. Effects of trimethyloxonium and pH

We have examined the effects of chemical modification with trimethyloxonium (TMO) and changes in external pH on the properties of acetylcholine (ACh)-activated channels in BC3H-1 cells, a clonal muscle cell line. TMO reacts covalently and specifically with carboxylic acid moieties in proteins to convert them to neutral methyl esters. In BC3H-1 cells TMO modification reduces the whole-cell response to ACh measured at negative membrane potentials by approximately 60%. G omega seal patch-clamp recordings of single ACh channel currents showed that the reduction in ACh sensitivity is due to alterations in both the current-carrying and the kinetic properties of the channels. Under all our experimental conditions, i.e., in external solutions of normal or low ionic strength, with or without external divalent cations, and at external pHs between 5.5 and 8.1, TMO treatment reduced ACh single-channel conductance to 70-90% of normal. The effects of TMO on channel kinetics were dependent on the ionic conditions. In normal ionic strength solutions containing both calcium and magnesium ions TMO modification reduced the channel average open time by approximately 25%. A similar reduction in open time was seen in calcium-free solution, but was not present when both calcium and magnesium ions were absent from the external solution. Lowering the ionic strength of the solution increased the mean open time in normal channels by about threefold, but did not affect the kinetics of modified channels. In low ionic strength solutions normal ACh channel open times were maximal at approximately pH 6.7 and decreased by three- to fourfold at both acid and alkaline pH. TMO modification removed the pH dependence of channel kinetics, and average open times were short at all pHs between 5.5 and 8.1. We suggest that TMO modifies normally titratable groups on the external surface of ACh channels that help to determine both the gating and permeability properties of ACh channels.     

Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.     

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).     

Survival of Cryptosporidium species in environments relevant to foods and beverages

AIMS: To provide data on the survival of Cryptosporidium oocysts in a range of conditions relevant to foods and beverages. METHODS AND RESULTS: Cryptosporidium parvum and C. hominis oocysts were stored in buffered media at different pH values and with various acids. In addition, neutral solutions with high salt (4.5% w/v), glycerol (20% v/v), sucrose (50% w/v) or ethanol (9 and 40% v/v) were used to determine their effects on survival. After storage periods of between 1 h and 14 days, viability was assessed using sporozoite ratio or infection of MRC-5 cell monolayers (not previously reported for culture of this organism). With all treatments, and with both assay techniques, viable oocysts were found at the end of the storage periods. However, treatments with one of the following additions: high salt, glycerol, sucrose or ethanol showed a negative and statistically significant effect on survival. Decline was noted after 1 day or even 1 h of treatment. CONCLUSIONS: MRC-5 cells are suitable for infection by C. parvum and C. hominis. Both tissue culture and sporozoite ratio gave broadly similar survival results and the greatest effects were seen with addition of components which reduced water activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has provided useful additional information to the food industry when considering the risk posed by this organism.     

Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant Klebsiella oxytoca CECT 4460 in Batch and Chemostat Cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h⁻¹, whereas with glycerol it was 0.45 h⁻¹. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y_C) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y_N) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h⁻¹ the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h⁻¹ the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

Modulation of the distribution of plasma membrane intramembranous particles in contact-inhibited and transformed cells.

The intrinsic organization of the plasma membrane differs in normal and transformed cells. With the technique of freeze fracture and electron microscopy contact inhibited 3T3 cells have been shown to contain aggregated plasma membrane intramembranous particles, while transformed cells demonstrate a uniform particle distribution. The distribution of intramembrous particles in transformed cells can be affected by colchicine or vinblastine which induces a dose- and time-dependent particle aggregation. These observations suggest that microtubules and other membrane-associated colchicine-sensitive proteins probably influence the distribution of intrinsic membrane proteins and intramembranous particles in nucleated mammalian cells. An aggregated particle distribution has been observed in 3T3 cells or colchicine-treated transformed cells frozen in media, phosphate-buffered saline or following brief exposure to glycerol, sucrose or dimethyl sulfoxide containing solutions, independent of whether specimens were rapidly frozen from 37 degrees C, room temperature or 4 degrees C incubations. Cells briefly stabilized in 1% formaldehyde yields similar patterns of particle distribution as cells rapidly frozen in media or cryoprotectants. Glutaraldehyde fixation of cells, however, appears to alter the fracturing process in these cells, as visualized by an altered fracture face appearance, decreased numbers of particles, and no particle aggregates. Differences in membrane organization between normal and transformed cells have therefore been demonstrated using a series of preparative methods and colchicine and vinblastine have been shown to modulate intramembranous particle distribution in transformed 3T3 cells.     

Potassium Exchange and Afterpotentials in Frog Sartorius Muscles Treated with Glycerol

The potassium exchange properties of glycerol-treated sartorius muscles of the frog were determined. Potassium (⁴²K) uptake, efflux, and net flux were measured in the presence of glycerol and at various times after exposure to glycerol and return to isotonic Ringer solution. Potassium uptake was not altered by the presence of glycerol but was reduced on the average 53% after glycerol treatment. Efflux transiently increased in the presence of glycerol and was reduced 37% after glycerol removal. Consequently, there was a net loss of intracellular potassium as well as a gain of sodium. In contrast to the irreversible alterations of potassium exchange induced by glycerol treatment, action potentials with normal negative afterpotentials (N.A.P.) were elicited 4–5 hr after glycerol removal. The reappearance of the N.A.P. was associated with a return of the membrane potential to normal values (90 ± 2 mv). However, the response of these muscles to reduced extracellular potassium was anomalous. In K⁺-free Ringer solution the average resting membrane potential was 74 ± 3 mv and a positive afterpotential of 11 ± 3 mv was associated with the action potential.     

Effects on sodium efflux of treating frog sartorius muscles with hypertonic glycerol solutions

Summary Efflux of sodium from frog sartorius muscles was measured during and after exposure to Ringer's fluid made hypertonic by addition of 400mm glycerol. Effects of strophanthidin, removal of external Na, and variation of external K were determined. During exposure to glycerol-containing solutions, Na efflux increased. Upon return to Ringer's fluid, Na efflux at first increased further. After the initial increase, Na efflux gradually declined; for the first two hours the efflux of Na from treated muscles was higher than that from untreated muscles. In the second hour, the strophanthidin-sensitive fractions of Na efflux were slightly increased while the strophanthidin-insensitive fractions were slightly decreased when compared with untreated muscles. The responses of Na efflux to removal of external sodium and to varying external K were comparable in both treated and untreated muscles. This shows that, at first, the membranes which remained after glycerol treatment exhibited the normal characteristics of Na extrusion. For at least eight hours after glycerol withdrawal the Na efflux from treated muscles declined relative to that of untreated muscles. The decline was largely due to reduction in strophanthidinsensitive fractions of efflux. Six to eight hours after glycerol withdrawal the Na efflux in treated muscles was less responsive to alterations in external K and Na than it was in untreated muscles. This indicates that aged glycerol-treated sartorii lost a substantial part of their capacity to actively transport sodium.     

Cryoprotectants and components induce plasmolytic responses in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) suspension cells

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.     

An antiserum specific for cholinergic synaptic vesicles from electric organ

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle- specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.     

Differential adaptation of membranes of two osmotolerant fungi,Aspergillus chevalieri and Penicillium expansum to high sucrose concentrations

Aspergillus chevalieri and Penicillium expansum were able to tolerate sucrose concentrations in the growth media up to 80% (w/v). At 50% sucrose the growth rate is approximately 1.4 and 1.2 times, respectively, higher than in the control. While at 80% sucrose it drops to 35% and 45% of the control level for both fungi. Lipids and proteins in plasma membranes increased with increasing sucrose concentrations in the growth medium. Phospholipid content in membranes of both organisms being also increased, phosphatidyl glycerol was the major detected phospholipid and represented the highest increase. The fatty acid composition of fraction enriched plasma membrane of both fungi changed when they were grown in high sucrose concentrations. Some fatty acids which had not been detected in control cultures were present and the proportions of other fatty acids changed. At 50% sucrose the unsaturation index of membranes decreased by 20-25% in both fungi, indicating that the plasma membrane is less fluid at this concentration. At 80% sucrose a similar trend was observed for P. expansum but for A. chevalieri the unsaturation index was little changed compared with the control. The fluorescence polarization values of 1,6-diphenyl 1,3,5-hexatriene (DPH) in membranes of both fungi grown at 80% sucrose increased, indicating a decrease in membrane fluidity. At 50% sucrose the increase in saturation of membrane fatty acids would tend to reduce membrane fluidity but in A. chevalieri at 80% sucrose fatty acids did not become more saturated. In this case the marked increase in sterols at this sucrose concentration may be responsible for low membrane fluidity.     

The effect of different alkali metal ions on the release of muscle potassium during glycerol treatment

Frog sartorius muscles were pretreated in 11.6 M (87%) ion-free glycerol. Then half of them were put in 5.8 M glycerol solutions containing 55 mM LiCl, NaCl, RbCl or CsCl and their pairs in ion-free 5.8 M glycerol solutions at 1:40 Vol. muscle:Vol. solution ratio. The release of potassium during exposure to the 5.8 M glycerol solution was significantly faster in the presence of Li or Na, but significantly slower in the presence of Rb than in the ion-free circumstance. The effect of Rb can be related to a cooperative phenomenon that implies existence of a structured phase of K-protein complexes. Some postulates of Ling's association-induction hypothesis are used for interpretation of two phases and their phase transitions in this type of bound-K structure in muscle. The phase transitions in this system could have an important role in the fast processes of excitation in muscle.