Encapsulation of macromolecules by lipid vesicles under simulated prebiotic conditions

Summary Phospholipid vesicles (liposomes) were subjected to dehydration-hydration cycles in the presence of 6-carboxyfluorescein or salmon sperm DNA. We found that the vesicles fused into multilamellar structures during dehydration with solutes trapped between the lamellae. Upon rehydration the lamellae swelled and formed large vesicular structures containing solute. This model can be used to study encapsulation of macromolecules by lipid membranes to form protocellular structures under prebiotic conditions.     

Anhydrobiosis: Inside yeast cells

Under natural conditions yeast cells as well as other microorganisms are regularly subjected to the influence of severe drought, which leads to their serious dehydration. The dry seasons are then changed by rains and there is a restoration of normal water potential inside the cells. To survive such seasonal changes a lot of vegetative microbial cells, which belong to various genera and species, may be able to enter into a state of anhydrobiosis, in which their metabolism is temporarily and reversibly suspended or delayed. This evolutionarily developed adaptation to extreme conditions of the environment is widely used for practical goals – for conservation of microorganisms in collections, for maintenance and long storage of different important strain-producers and for other various biotechnological purposes. This current review presents the most important data obtained mainly in the studies of the structural and functional changes in yeast cells during dehydration. It describes the changes of the main organelles of eukaryotic cells and their role in cell survival in a dry state. The review provides information regarding the role of water in the structure and functions of biological macromolecules and membranes. Some important intracellular protective reactions of eukaryotic organisms, which were revealed in these studies and may have more general importance, are also discussed. The results of the studies of yeast anhydrobiosis summarized in the review show the possibilities of improving the conservation and long-term storage of various microorganisms and of increasing the quality of industrially produced dry microbial preparations.     

Effect of water on the structure of bacteriorhodopsin and photochemical processes in purple membranes

Visible and infrared spectra of bacteriorhodopsin films under different humidities at room and low temperatures are investigated. On dehydration of purple membranes at room temperatures an additional chromophore state with the absorption band at 506 nm is revealed. The photocycle of purple membranes in the dry state is devoid of the 550 nm intermediate and involves the long-lived intermediate at 412 nm. As water is removed, the 550 nm intermediate becomes undetectable. The analysis of the infrared spectra shows that dehydration does not affect the ordering of the main network of the interpeptide hydrogen bonds which stabilizes the -helical conformation (slightly distorted in the initial humid dark- and light-adapted state); light adaptation (cis-trans isomerization) of bacteriorhodopsin results in an increase of sorbed water in purple membranes. Dehydration of purple membranes decreases the reaction rate of cis-trans isomerization.     

Dielectric relaxation of water and water-plasticized biomolecules in relation to cellular water organization, cytoplasmic viscosity, and desiccation tolerance in recalcitrant seed tissues

To understand the relationship between the organization of cellular water, molecular interactions, and desiccation tolerance, dielectric behaviors of water and water-plasticized biomolecules in red oak (Quercus rubra) seeds were studied during dehydration. The thermally stimulated current study showed three dielectric dispersions: (a) the relaxation of loosely-bound water and small polar groups, (b) the relaxation of tightly-bound water, carbohydrate chains, large polar groups of macromolecules, and (c) the "freezing in" of molecular mobility (glassy state). Seven discrete hydration levels (water contents of 1.40, 0.55, 0.41, 0.31, 0.21, 0.13, and 0.08 g/g dry weight, corresponding to -1.5, -8, -11, -14, -24, -74, and -195 MPa, respectively) were identified according to the changes in thermodynamic and dielectric properties of water and water-plasticized biomolecules during dehydration. The implications of intracellular water organization for desiccation tolerance were discussed. Cytoplasmic viscosity increased exponentially at water content < 0.40 g/g dry weight, which was correlated with the great relaxation slowdown of water-plasticized biomolecules, supporting a role for viscosity in metabolic shutdown during dehydration.     

Novel fluorescent core-shell nanocontainers for cell membrane transport

The synthesis and characterization of novel core-shell macromolecules consisting of a fluorescent perylene-3,4,9,10-tetracarboxdiimide chromophore in the center surrounded by a hydrophobic polyphenylene shell as a first and a flexible hydrophilic polymer shell as a second layer was presented. Following this strategy, several macromolecules bearing varying polymer chain lengths, different polymer shell densities, and increasing numbers of positive and negative charges were achieved. Because all of these macromolecules reveal a good water solubility, their ability to cross cellular membranes was investigated. In this way, a qualitative relationship between the molecular architecture of these macromolecules and the biological response was established.     

Evaluation of ultrafiltration membranes with biological macromolecules

Eighteen ultrafiltration membranes ranging in molecular weight cutoff ratings from 500 to 300,000 were tested with water, 0.5M NaCl solution, and, in some cases, with macromolecules and urea in a 3-in. stirred filter cell. Approximately half of the membranes showed a significant decrease in filtration rate during the first 24-hr period. The steady-state rates were less than the manufacturers' rating for about two thirds of the membranes, the discrepancy being greater for the membranes with high molecular weight cutoffs. The filtration rates were linearly dependent on applied pressure over the range at least as great as 15 to 55 psig. The rate decreased as the concentration of macromolecules such as transfer ribonucleic acid (tRNA) increased; the rate for a concentration of 3 mg tRNA/per ml was one-fourth of that observed when no tRNA was present. Some increase in rate (～33 to 50%) was obtained by increasing the stirring speed from 100 rpm to 1000 rpm. The membranes were effective for desalting and concentration of macromolecules but not for separation of large molecules from each other, such as tRNA from bovine serum albumin. Easily denatured molecules such as catalase were not deactivated by filtration at 4°C.     

Role of trehalose in the spores of Streptomyces

Abstract Dormant spores of Streptomyces antibioticus contain large amounts of trehalose (11–12% of dry weight) and can be subjected to a dehydration treatment without a significant loss of viability. Loss of dehydration resistance coincided with a decrease in the trehalose level of the spores, under different conditions of incubation. The viability of dehydration-sensitive cells was enhanced by the presence of exogenous trehalose during dehydration. The morphology and functional activity of isolated membranes of S. antibioticus can be retained when dehydrated in the presence of trehalose. It is suggested that, in dormant spores of S. antibioticus , trehalose may serve to protect cellular components during dehydration by acting as a substitute for water.     

Freezing, drying, and/or vitrification of membrane- solute-water systems.

Membranes are often damaged by freezing and/or dehydration, and this damage may be reduced by solutes. In many cases, these phenomena can be explained by the physical behavior of membrane-solute-water systems. Both solutes and membranes reduce the freezing temperature of water, although their effects are not simply additive. The dehydration of membranes induces large mechanical stresses in the membranes. These stresses produce a range of physical deformations and changes in the phase behavior. These membrane stresses and strains are in general reduced by osmotic effects and possibly other effects of solutes-provided of course that the solutes can approach the membrane in question. Membrane stresses may also be affected by vitrification where this occurs between membranes. Many of the differences among the effects of different solutes can be explained by the differences in the crystallization, vitrification, volumetric, partitioning, and permeability properties of the solutes.     

Dehydration-inducible changes in expression of two aquaporins in the sleeping chironomid, Polypedilum vanderplanki

Aquaporin, AQP, is a channel protein that allows water to permeate across cell membranes. Larvae of the sleeping chironomid, Polypedilum vanderplanki, can withstand complete dehydration by entering anhydrobiosis, a state of suspended animation; however, the mechanism by which water flows out of the larval body during dehydration is still unclear. We isolated two cDNAs (PvAqp1 and PvAqp2) encoding water-selective aquaporins from the chironomid. When expressed in Xenopus oocytes, PvAQP1 and PvAQP2 facilitated permeation of water but not glycerol. Northern blots and in situ hybridization showed that expression of PvAqp1 was dehydration-inducible and ubiquitous whereas that of PvAqp2 was dehydration-repressive and fat body-specific. These data suggest distinct roles for these aquaporins in P. vanderplanki, i.e., PvAqp2 controls water homeostasis of fat body during normal conditions and PvAqp1 is involved in the removal of water during induction of anhydrobiosis.     

The alpha-helix to beta-sheet transition in poly(L-lysine): effects of anesthetics and high pressure.

Poly(L-lysine) exists in a random-coil formation at a low pH, alpha-helix at a pH above 10.6, and transforms into beta-sheet when the alpha-helix polylysine is heated. Each conformation is clearly distinguishable in the amide-I band of the infrared spectrum. The thermotropic alpha-to-beta transition was studied by using differential scanning calorimetry. At pH 10.6, the transition temperature was 43.5 degrees C and the transition enthalpy was 170 cal/mol residue. At pH 11.85, the measurements were 36.7 degrees C and 910 cal/mol residue, respectively. Volatile anesthetics (chloroform, halothane, isoflurane and enflurane) partially transformed alpha-helix polylysine into beta-sheet. The transformation was reversed by the application of hydrostatic pressure in the range of 100-350 atm. Apparently, the alpha-to-beta transition was induced by anesthetics through partial dehydration of the peptide side-chains (beta-sheet surface is less hydrated than alpha-helix). High pressure reversed this process by re-hydrating the peptide. Because the membrane spanning domains of channel and receptor proteins are predominantly in the alpha-helix conformation, anesthetics may suppress the activity of excitable cells by transforming them into a less than optimal structure for electrogenic ion transport and neurotransmission. Proteins and lipid membranes maintain their structural integrity by interaction with water. That which attenuates the interaction will destabilize the structure. These data suggest that anesthetics alter macromolecular conformations essentially by a solvent effect, thereby destroying the solvation water shell surrounding macromolecules.     

Glycosaminoglycans of the plasma membranes of renal tubule and liver cells

Glycosaminoglycans were isolated from plasma membranes of hepatic and renal tubule cells of guinea pig. Plasmalemma of renal tubule cells contained more total glycosaminoglycans, hyaluronic acid, chondroitin-4 sulfates and chondroitin-6 sulfates, and less dermatan sulfates and heparin sulfates than liver plasma membranes. These glycocalyx components, owing to their polyanionic properties, may have a role in the transport of water, ions, and macromolecules across the cell membrane.     

ROLE OF EDTA AND METALS IN MITOCHONDRIAL CONTRACTION

Studies comparing the state of hydration and dehydration of rat liver mitochondria to their content of ATP, Ca, and fatty acid, along with the rate of ATP hydrolysis, as well as microscopic appearance of mitochondria, have led to the following generalizations: 1. The competition between cationic translocations and water translocation for the available chemical energy (ATP) determines under many circumstances the water content of mitochondria. 2. Swelling of mitochondria by electron transport substrates is an example of the activation of the cationic translocations at the expense of water translocation. 3. Electron micrographic studies are interpreted to indicate that EDTA alone can cause condensation and dehydration of the mitochondrial matrix. However, both EDTA and substrate are necessary to remove appreciable quantities of water from mitochondrial intramembranous spaces. 4. Since the data in the accompanying report indicated that EDTA, in the absence of energy, decreased the permeability of mitochondrial membranes, it appears likely that ballooning of intramembranous spaces, following addition of EDTA, represents trapping of water between two semipermeable membranes following dehydration of mitochondrial matrix.     

Chitosan-silica complex membranes from sulfonic acid functionalized silica nanoparticles for pervaporation dehydration of ethanol-water solutions

Nanosized silica particles with sulfonic acid groups (ST-GPE-S) were utilized as a cross-linker for chitosan to form a chitosan-silica complex membranes, which were applied to pervaporation dehydration of ethanol-water solutions. ST-GPE-S was obtained from reacting nanoscale silica particles with glycidyl phenyl ether, and subsequent sulfonation onto the attached phenyl groups. The chemical structure of the functionalized silica was characterized with FTIR, (1)H NMR, and energy-dispersive X-ray. Homogeneous dispersion of the silica particles in chitosan was observed with electronic microscopies, and the membranes obtained were considered as nanocomposites. The silica nanoparticles in the membranes served as spacers for polymer chains to provide extra space for water permeation, so as to bring high permeation rates to the complex membranes. With addition of 5 parts per hundred of functionalized silica into chitosan, the resulting membrane exhibited a separation factor of 919 and permeation flux of 410 g/(m(2) h) in pervaporation dehydration of 90 wt % ethanol aqueous solution at 70 degrees C.     

Aquaporins of the PIP2 class are required for efficient anther dehiscence in tobacco

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues. Before flower anthesis, anther and pollen dehydration takes place before the release of mature pollen at dehiscence. Aquaporins represent a class of proteins that mediates the movement of water over cellular membranes. Aquaporins of the plasmamembrane PIP2 family are expressed in tobacco (Nicotiana tabacum) anthers and may therefore be involved in the movement of water in this organ. To gain more insight into the role these proteins may play in this process, we have analyzed their localization using immunolocalizations and generated plants displaying RNA interference of PIP2 aquaporins. Our results indicate that PIP2 protein expression is modulated during anther development. Furthermore, in tobacco PIP2 RNA interference plants, anther dehydration was slower, and dehiscence occurred later when compared with control plants. Together, our results suggest that aquaporins of the PIP2 class are required for efficient anther dehydration prior to dehiscence.     

脱水素研究进展

脱水素(dehydrin)是植物体内的一种LEA蛋白,能够在植物胚胎发育后期以及逆境下大量表达,广泛存在于植物界。它是具有高度热稳定性的亲水性蛋白,有三类非常保守的区域,即K,Y和S片段。依据这三类片段的组成情况,可将脱水素分为5个基本类别。脱水素可通过多种转运方式定位于植物细胞的不同部位,以行使其功能。其基因的表达存在依赖ABA和不依赖ABA两种途径,并且受到多种环境因素的影响,能稳定细胞膜和许多大分子的结构以避免脱水对细胞造成的伤害。近年来,脱水素的结构和组成、在细胞中的定位及转运、基因的表达与调控、功能与作用机理等方面的研究已取得了很大的进展。     

Prediction of functions for two LEA proteins from mung bean

LEA (late embryogenesis abundant) proteins are associated with tolerance to water stress resulting from desiccation and cold shock. Although various functions have been proposed to LEA proteins, their precise role is not fully defined. In silico analysis of the amino acid sequence of two LEA proteins (early methionine-labeled Vigna, EMV) from the tropical legume crop, Vigna radiata identified a 20 residues motif 'GGQTRKQQLGSEGYHEMGRK' characteristic to group 1 LEA proteins. Structural analyses hypothesize these proteins to function like DNA/RNA binding proteins in protecting macromolecules/ membrane stabilization at the time of dehydration process.     

Isolated water in desiccated microorganisms

The presence of isolated mobile water in dehydrated eukaryotic microorganisms established earlier by NMR has been confirmed by direct chemical registration of this water in the yeast Cryptococcus albidus. This water constitutes several per cent of the dry biomass weight. Apparently, its preservation should be attributed to changes in the permeability of intracellular membranes upon dehydration. The water is released by the cells when they are heated to 150--200 degrees C and the cellular structures containing water are destroyed.     

植物复苏性状的分子机理研究进展

复苏性状是某些生物应对水分剧烈变化恶劣环境的一种特殊适应能力,在植物界广泛分布于苔藓和蕨类低等植物,一些高等植物也具有这种性状。复苏植物可以在损失体内95％以上的水分后,遇水而复苏,以此度过环境恶劣的时段。复苏性状的分子机制一直让人们着迷,但对其认知还十分有限。近年来的研究表明,一些小分子代谢物和特殊蛋白的大量积累在复苏植物脱水过程中对生物膜和大分子结构起保护作用;复苏性状中的信号转导与基因调控可能包括ABA在内的一系列信号分子和途径。随着“组学”技术的发展,更宽广角度的研究将会极大的促进人们对复苏性状的认知。对于复苏性状的深入研究,可能为农作物和蔬菜的改良提供一个全新的方向,具有很大的潜在应用价值。     

脱水在聚乙二醇诱导膜融合中的作用

随着各种诱导膜融合的因子相继发现,人们建立了各种膜融合的模型.我们通过对聚乙二醇PEG诱导脂质体融合的分析,认为膜融合的关键在于脱去膜表面的结合水,而其它作用诸如膜脂缺陷.膜脂分相以及脂多型性等尽管是不同膜体系中直接观察到的膜融合形式,都是膜脱去结合水带来的必然结果.膜表面结合水的排除是前因,本文着重讨论脱水及脱水后膜脂结构的一系列变化.     

Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae

Summary This paper describes the characteristics of the structural and functional organization of cellular membranes rehydrated after dehydration of the yeast Saccharomyces cerevisiae. It was noted that dehydration and subsequent rehydration of yeast cells causes a considerable increase of cytoplasmic membrane permeability. Addition of CaCl₂, glucose and polyethyleneglycol to the rehydration medium caused a decrease in cell permeability, assessed as the losses of potassium ions, nucleotides, as well as the total losses of intracellular compounds. KCl had a positive effect only at concentrations above 10%. Yeast cells, dried to residual moisture lower than 20%, showed a decrease in membrane permeability as temperatures of the rehydration medium increased up to 38°–43°C. Upon reactivation of viable dehydrated cells in a nutrient medium, a reparation of the structural damages of various intracellular membranes takes place. It was established that at cell dehydration to residual moistures of 8%–12% all the free and a part of bound water is evaporated from cells.