Coupling of Membrane Nanodomain Formation and Enhanced Electroporation near Phase Transition

Biological cells are enveloped by a heterogeneous lipid bilayer that prevents the uncontrolled exchange of substances between the cell interior and its environment. In particular, membranes act as a continuous barrier for salt and macromolecules to ensure proper physiological functions within the cell. However, it has been shown that membrane permeability strongly depends on temperature and, for phospholipid bilayers, displays a maximum at the transition between the gel and fluid phase. Here, extensive molecular dynamics simulations of dipalmitoylphosphatidylcholine bilayers were employed to characterize the membrane structure and dynamics close to phase transition, as well as its stability with respect to an external electric field. Atomistic simulations revealed the dynamic appearance and disappearance of spatially related nanometer-sized thick ordered and thin interdigitating domains in a fluid-like bilayer close to the phase transition temperature (T_m). These structures likely represent metastable precursors of the ripple phase that vanished at increased temperatures. Similarly, a two-phase bilayer with coexisting gel and fluid domains featured a thickness minimum at the interface because of splaying and interdigitating lipids. For all systems, application of an external electric field revealed a reduced bilayer stability with respect to pore formation for temperatures close to T_m. Pore formation occurred exclusively in thin interdigitating membrane nanodomains. These findings provide a link between the increased membrane permeability and the structural heterogeneity close to phase transition.     

The Balance of Fluid and Osmotic Pressures across Active Biological Membranes with Application to the Corneal Endothelium

The movement of fluid and solutes across biological membranes facilitates the transport of nutrients for living organisms and maintains the fluid and osmotic pressures in biological systems. Understanding the pressure balances across membranes is crucial for studying fluid and electrolyte homeostasis in living systems, and is an area of active research. In this study, a set of enhanced Kedem-Katchalsky (KK) equations is proposed to describe fluxes of water and solutes across biological membranes, and is applied to analyze the relationship between fluid and osmotic pressures, accounting for active transport mechanisms that propel substances against their concentration gradients and for fixed charges that alter ionic distributions in separated environments. The equilibrium analysis demonstrates that the proposed theory recovers the Donnan osmotic pressure and can predict the correct fluid pressure difference across membranes, a result which cannot be achieved by existing KK theories due to the neglect of fixed charges. The steady-state analysis on active membranes suggests a new pressure mechanism which balances the fluid pressure together with the osmotic pressure. The source of this pressure arises from active ionic fluxes and from interactions between solvent and solutes in membrane transport. We apply the proposed theory to study the transendothelial fluid pressure in the in vivo cornea, which is a crucial factor maintaining the hydration and transparency of the tissue. The results show the importance of the proposed pressure mechanism in mediating stromal fluid pressure and provide a new interpretation of the pressure modulation mechanism in the in vivo cornea.     

Toxic-Metabolite-Producing Bacteria and Fungus in an Indoor Environment

Toxic-metabolite-emitting microbes were isolated from the indoor environment of a building where the occupant was suffering serious building-related ill-health symptoms. Toxic substances soluble in methanol and inhibitory to spermatozoa at <10 μg (dry weight) ml⁻¹ were found from six bacterial isolates and one fungus. The substances from isolates of Bacillus simplex and from isolates belonging to the actinobacterial genera Streptomyces and Nocardiopsis were mitochondriotoxic. These substances dissipated the mitochondrial membrane potential (Δψ) of boar spermatozoa. The substances from the Streptomyces isolates also swelled the mitochondria. The substances from isolates of Trichoderma harzianum Rifai and Bacillus pumilus damaged the cell membrane barrier function of sperm cells.     

Clonal analysis of synovial fluid stem cells to characterize and identify stable mesenchymal stromal cell/mesenchymal progenitor cell phenotypes in a porcine model: a cell source with enhanced commitment to the chondrogenic lineage

Background aimsPrevious studies have demonstrated that porcine synovial membrane stem cells can adhere to a cartilage defect in vivo through the use of a tissue-engineered construct approach. To optimize this model, we wanted to compare effectiveness of tissue sources to determine whether porcine synovial fluid, synovial membrane, bone marrow and skin sources replicate our understanding of synovial fluid mesenchymal stromal cells or mesenchymal progenitor cells from humans both at the population level and the single-cell level. Synovial fluid clones were subsequently isolated and characterized to identify cells with a highly characterized optimal phenotype.MethodsThe chondrogenic, osteogenic and adipogenic potentials were assessed in vitro for skin, bone marrow, adipose, synovial fluid and synovial membrane–derived stem cells. Synovial fluid cells then underwent limiting dilution analysis to isolate single clonal populations. These clonal populations were assessed for proliferative and differentiation potential by use of standardized protocols.ResultsPorcine-derived cells demonstrated the same relationship between cell sources as that demonstrated previously for humans, suggesting that the pig may be an ideal preclinical animal model. Synovial fluid cells demonstrated the highest chondrogenic potential that was further characterized, demonstrating the existence of a unique clonal phenotype with enhanced chondrogenic potential.ConclusionsPorcine stem cells demonstrate characteristics similar to those in human-derived mesenchymal stromal cells from the same sources. Synovial fluid–derived stem cells contain an inherent phenotype that may be optimal for cartilage repair. This must be more fully investigated for future use in the in vivo tissue-engineered construct approach in this physiologically relevant preclinical porcine model.     

THE STRUCTURE OF THE COLLODION MEMBRANE AND ITS ELECTRICAL BEHAVIOR : IX. WATER UPTAKE AND SWELLING OF COLLODION MEMBRANES IN AQUEOUS SOLUTIONS OF ORGANIC ELECTROLYTES AND NON-ELECTROLYTES

1. Dried collodion membranes are known to swell in water and to the same limited extent also in solutions of strong inorganic electrolytes (Carr and Sollner). The present investigation shows that in solutions of organic electrolytes and non-electrolytes, the swelling of dried collodion membranes is not as uniform, but depends on the nature of the solute. 2. The solutions of typically "hydrophilic" substances, e.g., glycerine, glucose, and citric acid, swell collodion membranes only to the same extent as water and solutions of strong electrolytes. In solutions of typically carbophilic substances (e.g., butyric acid, valeric acid, isobutyl alcohol, valeramide, phenol, and m-nitrophenol) the swelling of the membranes is much stronger than in water, according to the concentration used. For the brand of collodion used the swelling in 0.5 M solution was in some cases as high as 26 per cent of the original volume, as compared to 6 to 7 per cent in water. Therefore, in these solutions the "water-wetted dried" collodion membrane is not rigid, inert, and non-swelling, but behaves as a swelling membrane. 3. The solutes which cause an increased swelling of the membranes are accumulated in the latter, the degree of accumulation being markedly parallel with the degree of their specific swelling action. 4. The anomalously high permeabilities of certain carbophilic organic solutes reported by Michaelis, Collander, and Höber find an explanation in the specific interaction of these substances with collodion. 5. The use of the collodion membrane as a model of the ideal porous membrane is restricted to those instances in which no specific interaction occurs between the solute and the collodion.     

Effect of cold plasma on the E. coli cell wall and plasma membrane

The effect of cold plasma on E. coli cells was studied. It was shown that the treatment of E. coli cells with cold plasma caused partial or total disruption of the plasma membrane integrity, which was accompanied by a release of intracellular substances into the extracellular environment. A quantitative assessment of the extent of the damage to the cell membrane showed that a loss of no more than 23.6% of intracellular substances (calculated by the proportion of the intracellular nucleotide release) is sufficient to lead to cell death. The use of media with different ionic strength levels to create osmotic shock showed that the treatment of E. coli cells with cold plasma significantly decreased the cell wall strength.     

Lipid-cell interactions: Liposome adsorption and cell-to-liposome lipid transfer are mediated by the same cell-surface sites

The competitive behavior of solid vs. fluid liposomes in liposome-to-cell adsorption and cell-to-liposome lipid transfer processes was investigated with L cells and FBT epithelial sheets. Binding, transfer and ³¹P-NMR experiments have demonstrated that: (i) solid liposomes adhere to the cell surface as integral vesicles retaining the entrapped substances; (ii) fluid liposomes are partly disintegrated at the cell surface with concomitant entry of entrapped substances into the cytoplasm, while their lipids remain on the cell surface; (iii) fluid liposomes that escape lysis dissociate from the cell, taking away cell lipid molecules. The latter process underlies the mechanism of cell-to-fluid liposome lipid transfer. In contrast, no lipid transfer occurs between the plasma membrane and solid liposomes. Cell-bound solid liposomes interfere with the transfer of cell lipids to fluid liposomes, while these in turn inhibit the binding of solid liposomes to the cell surface. Moreover, cell-induced aggregation of both fluid and solid freshly added liposomes is also inhibited by preincubation of the cells with either solid or fluid liposomes. Thus, different types of interaction of both fluid and solid liposomes with the cell are mediated by the same (or closely related) sites on the cell surface.     

Countercurrent flows,water movements and nutrient absorption in the locust midgut

Using amaranth dye as a marker solute, the movements of fluids in the gut of Schistocerca gregaria was studied, either by feeding a meal containing the dye or by injecting the dye into the haemolymph, and by comparing the distribution of amaranth with those of naturally-occurring solutes in the alimentary tract.In animals deprived of food for more than 2–4 hr, some of the fluid from the Malpighian tubules moves forward through the solid food matrix in the midgut carrying solutes into the anterior midgut and gastric caeca, where water is absorbed. After a meal the crop empties at a rate which saturates the absorptive capacity of the anterior caeca, producing a net movement of fluid down the midgut and so such a countercurrent system is not observed in animals fed ad lib., where dye introduced into the gut always moves posteriorly.A countercurrent fluid movement confers several advantages on the alimentary system which act to maximise the efficiency of nutrient absorption: the principal disadvantage of the countercurrent system is that noxious solutes, as well as nutrients, will accumulate at high concentrations near the permeable site of nutrient uptake. Thus a countercurrent flow of solutes is observed only when the insect is deprived of food and the need to conserve nutrient resources exceeds that of excretion of noxious substances. Ways in which the site of nutrient absorption may be protected from noxious solutes are discussed.The anterior caeca gradually become bloated with dark fluid as digestion proceeds; this is expelled into the midgut when a fresh meal is ingested.     

Induction of oviposition by injection of male-derived extracts in two Callosobruchus species

In some insect species, certain substances in the seminal fluid of males induce egg production and laying in females. We determined the effects of male-derived substances on female oviposition behaviour in two Callosobruchus species, C. chinensis and C. maculatus. Aqueous extracts of the accessory gland; testis; and seminal vesicle, including the ejaculatory duct, were prepared. The injection of these extracts into abdomen of females induced oviposition in both species. Oviposition was induced by the testis and seminal vesicle extracts in C. chinensis and by the accessory gland extracts in C. maculatus. The extracts were separated into three fractions by ultrafiltration: fractions I, molecular weight (MW) <3 kDa; fraction II, 3-14 kDa; and fraction III, >14 kDa. Fraction III induced oviposition in both species. These results suggest that in these two species, the substances that induce oviposition have similar MW but are present in different organs. Oviposition was induced by high-MW (>14 kDa) substances in the testis and seminal vesicle in C. chinensis, and by high-MW substances in accessory gland in C. maculatus. Here, we have discussed the relationship between oviposition and the abovementioned male-derived substances.     

OSMOSIS OF LIQUIDS. III

If only one substance S passes through a membrane, the nature of this membrane is not of importance with respect to the direction of the diffusion; this is namely determined only by the O.S.A. of the two liquids. If, however, more substances pass through a membrane, the nature of this membrane is of great importance. If n substances diffuse through a membrane, we can distinguish 2ⁿ cases, when we take into consideration only the direction in which each of these substances passes through the membrane; if we call each of these cases a D.T. (diffusion-type), 2ⁿ D.T.''s may be conceived. Now we can deduce: one of these D.T.''s is not possible, the other 2ⁿ x 1 D.T.''s are thermodynamically admissible. The composition of the two liquids determines which of the D.T.''s is not possible; the nature of the membrane determines which of the 2ⁿ x 1 other D.T.''s will occur.     

Loss of carotenoids from membranes of Pantoea sp. YR343 results in altered lipid composition and changes in membrane biophysical properties

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.     

Bioactive substances produced by marine isolates of Pseudomonas

Pseudomonas is a genus of non-fermentative gram-negative Gammaproteobacteria found both on land and in the water. Many terrestrial isolates of this genus have been studied extensively. While many produce bioactive substances, enzymes, and biosurfactants, other Pseudomonas isolates are used for biological control of plant diseases and bioremediation. In contrast, only a few marine isolates of this genus have been described that produce novel bioactive substances. The chemical structures of the bioactive substances from marine Pseudomonas are diverse, including pyroles, pseudopeptide pyrrolidinedione, phloroglucinol, phenazine, benzaldehyde, quinoline, quinolone, phenanthren, phthalate, andrimid, moiramides, zafrin and bushrin. Some of these bioactive compounds are antimicrobial agents, and dibutyl phthalate and di-(2-ethylhexyl) phthalate have been reported to be cathepsin B inhibitors. In addition to being heterogeneous in terms of their structures, the antibacterial substances produced by Pseudomonas also have diverse mechanisms of action: some affect the bacterial cell membrane, causing bacterial cell lysis, whereas others act as acetyl-CoA carboxylase and nitrous oxide synthesis inhibitors. Marine Pseudomonas spp. have been isolated from a wide range of marine environments and are a potential untapped source for medically relevant bioactive substances.     

Specific targeting of membrane nodulins to the bacteroid-enclosing compartment in soybean nodules

Rhizobium bacteroids in nodule cells are surrounded by the peribacteroid membrane (pbm), which is derived from the host plasma membrane during infection. The pbm was purified from R. japonicum 61A76-induced soybean nodules and analyzed by comparing it with the host cell plasma membrane for the presence of nodulins, nodule-specific plant proteins. Nodulins were found in pbm by reacting Western blots with a nodule-specific antiserum raised against the pbm. Peribacteroid fluid (the fluid enclosed in the pbm) was also found to contain several nodulins. The pbm nodulins were confirmed to be of plant origin by in vitro translation of poly(A)⁺ nodule mRNA followed by immunoprecipitation by the nodule-specific antiserum. Antibodies raised against a synthetic peptide corresponding to a repeated domain in nodulin-24, a pbm nodulin, and the nodule-specific pbm antiserum reacted exclusively with the pbm. The absence of pbm-nodulins in the plasma membrane suggests that the infected cells direct the intracellular transport of the pbm nodulins exclusively to this de novo synthesized subcellular compartment essential for symbiotic nitrogen fixation.     

Male Accessory Gland Protein Reduces Egg Laying in a Simultaneous Hermaphrodite

Seminal fluid is an important part of the ejaculate of internally fertilizing animals. This fluid contains substances that nourish and activate sperm for successful fertilization. Additionally, it contains components that influence female physiology to further enhance fertilization success of the sperm donor, possibly beyond the recipient''s optimum. Although evidence for such substances abounds, few studies have unraveled their identities, and focus has been exclusively on separate-sex species. We present the first detailed study into the seminal fluid composition of a hermaphrodite (Lymnaea stagnalis). Eight novel peptides and proteins were identified from the seminal-fluid-producing prostate gland and tested for effects on oviposition, hatching and consumption. The gene for the protein found to suppress egg mass production, Ovipostatin, was sequenced, thereby providing the first fully-characterized seminal fluid substance in a simultaneous hermaphrodite. Thus, seminal fluid peptides and proteins have evolved and can play a crucial role in sexual selection even when the sexes are combined.     

Ascaris suum: Free amino acids and proteins in the pseudocoelom,seminal vesicle and glandular vas deferens

The free amino acids and proteins of the seminal vesicle and pseudocoelomic fluids in the male Ascaris suum were examined and compared. The seminal fluid contained a high concentration of lysine (lysine: glutamic acid ratio of 5:1) while the pseudocoelomic fluid contained more glutamic acid than lysine with alanine, serine, glycine and proline being the most abundant free amino acids. The proteins present in the seminal fluid differed from those in the pseudocoelomic fluid in both number and molecular weight. The sperm activating substance (SAS) present in homogenates of the glandular vas deferens of male worms is nondialyzable, heat-sensitive and can be precipitated using 45% saturated ammonium sulfate. Active moieties can be recovered following passage of the ammonium sulfate precipitates through ultrafiltration membranes or by applying gland homogenates to an ion exchange column. When subjected to SDS-polyacrylamide gel electrophoresis, the active fractions revealed both low and high molecular weight substances. During attempts to purify a single activating substance, it was noted that the more heterogeneous fractions contained the highest activating capacity. Thus, no precise relationship between the biological activity and the purity of the various fractions was determined.     

Black Yeasts-Like Fungi Isolated from Dialysis Water in Hemodialysis Units

Hemodialysis in patients with chronic renal failure promotes the removal of toxic substances, water, and minerals from the body and often takes place in specialized clinics. Microbial contamination of dialysis fluid is a serious problem in therapy. One of the sources of contamination is the water used to prepare the dialysate. In Brazil, legislation regulating the microbiological quality of water for dialysis does not cover waterborne microbes such as Pseudomonas, mycobacteria, and fungi. The aim of the present study was to quantify, isolate, and identify fungi present in water systems in six hemodialysis units in Curitiba, Paraná state, Brazil. Fungi were analyzed by surface plating and membrane filtration. Isolates were identified by morphology, while the dematiaceous fungi were identified by sequencing the rDNA ITS region. It was found that 66 % of the samples presented fungi, while black fungi were present in 46 % of all samples. Twenty-eight isolates from treated water for dialysis and dialysate were identified by sequencing and were found to be Exophiala pisciphila, E. cancerae, E. equina, and Rhinocladiella similis. The presence of dematiaceous fungi may pose a risk for debilitated hospitalized patients.     

Apolipoprotein-A1 as a Damage-Associated Molecular Patterns Protein in Osteoarthritis: Ex Vivo and In Vitro Pro-Inflammatory Properties

Osteoarthritis (OA) is associated with a local inflammatory process. Dyslipidemia is known to be an underlying cause for the development of OA. Therefore, lipid and inflammatory levels were quantified ex vivo in blood and synovial fluid of OA patients (n=29) and compared to those of rheumatoid arthritis (RA) patients (n=27) or healthy volunteers (HV) (n=35). The role of apolipoprotein A-I (ApoA1) was investigated in vitro on inflammatory parameters using human joint cells isolated from cartilage and synovial membrane obtained from OA patients after joint replacement. Cells were stimulated with ApoA1 in the presence or not of serum amyloid A (SAA) protein and/or lipoproteins (LDL and HDL) at physiological concentration observed in OA synovial fluid. In our ex vivo study, ApoA1, LDL-C and total cholesterol levels were strongly correlated to each other inside the OA joint cavity whereas same levels were not or weakly correlated to their corresponding serum levels. In OA synovial fluid, ApoA1 was not as strongly correlated to HDL as observed in OA serum or in RA synovial fluid, suggesting a dissociative level between ApoA1 and HDL in OA synovial fluid. In vitro, ApoA1 induced IL-6, MMP-1 and MMP-3 expression by primary chondrocytes and fibroblast-like synoviocytes through TLR4 receptor. HDL and LDL attenuated joint inflammatory response induced by ApoA1 and SAA in a ratio dependent manner. In conclusion, a dysregulated lipidic profile in the synovial fluid of OA patients was observed and was correlated with inflammatory parameters in the OA joint cavity. Pro-inflammatory properties of ApoA1 were confirmed in vitro.     

THE MECHANISM OF THE INHIBITION OF HEMOLYSIS

The principal conclusion of this investigation is that the inhibitory effect of plasma or serum on hemolysis by saponin and lysins of the same type is similar in nature to the inhibitory effects of certain sugars and electrolytes, which again are similar to the acceleratory effects produced by indol, benzene, and other substances already studied. All these effects, both inhibitory and acceleratory, are the result of reactions between the inhibitors or accelerators and those components of the red cell membrane which are broken down by lysins. The inhibitory effect of plasma on saponin hemolysis has a number of properties in common with the inhibition produced by sugars and electrolytes and with accelerations in general. (a) The temperature coefficient is small and negative. (b) The extent of the inhibition depends on the type of red cell used in the hemolytic system. (c) The most satisfactory measure of the extent of the inhibition, the constant R, is a function of the concentration of lysin in the system, and (d) R is a linear function of the quantity of inhibitor present. It is also shown that the inhibitory effect of plasma, and serum is not entirely dependent on its protein content. The process underlying the phenomenon of lysis and its acceleration or inhibition seems to be one in which the lysin reacts with a component or components of the cell membrane in such a way as to break down its semipermeability to hemoglobin, and in which the accelerator or inhibitor also reacts with the same component in such a way as to increase or decrease the effectiveness of the lysin in producing breakdown. The membrane is considered as being an ultrastructure made up of small areas or spots of varying degrees of resistance to breakdown, the resistances being distributed according to a negatively skew type of frequency curve, and the process of lysis seems to begin with the least resistant spots breaking down first. These spots may be arranged in some regular spatial pattern, and the membrane has also to be regarded as possessing spots of varying rigidity of form. The accelerator or inhibitor changes the resistance of every reactive spot in the ultrastructure by a factor R, which suggests that acceleration and inhibition are results of some over-all effect, such as that of changing the extent to which lysin is concentrated at the surface or partitioned between the material of the membrane and the surrounding fluid. Some kind of combination between the accelerator or inhibitor and the material of the ultrastructure is presumably involved; at first the combination seems to be a loose one and partly reversible, but later some of the loose links are replaced by more permanent combinations involving the same types of bond as are broken down by the lysins themselves.     

Survey of Extreme Solvent Tolerance in Gram-Positive Cocci: Membrane Fatty Acid Changes in Staphylococcus haemolyticus Grown in Toluene

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.     

Dependence of phytochrome action in seeds on membrane organization

Germination of Amaranthus retroflexus L. seeds imbibed at 40 C is enhanced by establishing the active form of phytochrome before a reduction in temperature to <32 C. The half-time for effectiveness of the lower temperature is about 8 min at 15 C. Isolated membrane fragments of A. retroflexus seeds associated with the fluorescent probe 1,8-anilino-naphthalene sulfonate (ANS) increase in structural order as the temperature is lowered through the 32 C region. The germination response is decreased by the membrane-disruptive substances tris, octonoate, and ethanol. The results show that phytochrome activity is associated with an organized membrane. By using ANS with membrane fragments from Setaria faberi Herrm. seeds, leakage of amino acid was found to be enhanced at temperatures >32 C by a transition in the plasmalemma.