Intracellular Osmotic Pressure Research Articles

Microbial diversity and biogeochemical cycling in soda lakes.

Soda lakes contain high concentrations of sodium carbonates resulting in a stable elevated pH, which provide a unique habitat to a rich diversity of haloalkaliphilic bacteria and archaea. Both cultivation-dependent and -independent methods have aided the identification of key processes and genes in the microbially mediated carbon, nitrogen, and sulfur biogeochemical cycles in soda lakes. In order to survive in this extreme environment, haloalkaliphiles have developed various bioenergetic and structural adaptations to maintain pH homeostasis and intracellular osmotic pressure. The cultivation of a handful of strains has led to the isolation of a number of extremozymes, which allow the cell to perform enzymatic reactions at these extreme conditions. These enzymes potentially contribute to biotechnological applications. In addition, microbial species active in the sulfur cycle can be used for sulfur remediation purposes. Future research should combine both innovative culture methods and state-of-the-art ‘meta-omic’ techniques to gain a comprehensive understanding of the microbes that flourish in these extreme environments and the processes they mediate. Coupling the biogeochemical C, N, and S cycles and identifying where each process takes place on a spatial and temporal scale could unravel the interspecies relationships and thereby reveal more about the ecosystem dynamics of these enigmatic extreme environments.

Calcium-mediated pore expansion and cell death following nanoelectroporation

Opening of long-lived pores in the cell membrane is the principal primary effect of intense, nanosecond pulsed electric field (nsPEF). Here we demonstrate that the evolution of pores, cell survival, the time and the mode of cell death (necrotic or apoptotic) are determined by the level of external Ca2+ after nsPEF. We also introduce a novel, minimally disruptive technique for nsEP exposure of adherent cells on indium tin oxide (ITO)-coated glass coverslips, which does not require cell detachment and enables fast exchanges of bath media. Increasing the Ca2+ level from the nominal 2–5μM to 2mM for the first 60–90min after permeabilization by 300-nsPEF increased the early (necrotic) death in U937, CHO, and BPAE cells. With nominal Ca2+, the inhibition of osmotic swelling rescued cells from the early necrosis and increased caspase 3/7 activation later on. However, the inhibition of swelling had a modest or no protective effect with 2mM Ca2+ in the medium. With the nominal Ca2+, most cells displayed gradual increase in YO-PRO-1 and propidium (Pr) uptake. With 2mM Ca2+, the initially lower Pr uptake was eventually replaced by a massive and abrupt Pr entry (necrotic death). It was accompanied by a transient acceleration of the growth of membrane blebs due to the increase of the intracellular osmotic pressure. We conclude that the high-Ca2+-dependent necrotic death in nsPEF-treated cells is effected by a delayed, sudden, and osmotically-independent pore expansion (or de novo formation of larger pores), but not by the membrane rupture.

An efficient method for the sanitary vitrification of bovine oocytes in straws

BackgroundAt present, vitrification has been widely applied to humans, mice and farm animals. To improve the efficiency of vitrification in straw, bovine oocytes were used to test a new two-step vitrification method in this study.ResultsWhen in vitro matured oocytes were exposed to 20% ethylene glycol (EG20) for 5 min and 40% ethylene glycol (EG40) for 30 s, followed by treatment with 30% glycerol (Gly30), Gly40 or Gly50, a volume expansion was observed in Gly30 and Gly40 but not Gly50. This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40 (approximately 5.6 mol/L) and Gly50 (approximately 7.0 mol/L). Since oocytes are in EG40 just for only a short period of time (30 s) and at a lower temperature (4°C), we hypothesize that the main function of this step in to induce dehydration. Based on these results, we omitted the EG40 step, before oocytes were pretreated in EG20 for 5 min, exposed to pre-cooled (4°C) Gly50, for 30 s, and then dipped into liquid nitrogen. After warming, 81.1% of the oocytes survived, and the surviving oocytes developed into cleavage stage embryos (63.5%) or blastocysts (20.0%) after parthenogenetic activation.ConclusionsThese results demonstrate that in a two-step vitrification procedure, the permeability effect in the second step is not necessary. It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.

Erg Channel Is Critical in Controlling Cell Volume during Cell Cycle in Embryonic Stem Cells

The cell cycle progression in mouse embryonic stem cells (mESCs) is controlled by ion fluxes that alter cell volume [1]. This suggests that ion fluxes might control dynamic changes in morphology over the cell cycle, such as rounding up of the cell at mitosis. However, specific channels regulating such dynamic changes and the possible interactions with actomyosin complex have not been clearly identified. Following RNAseq transcriptome analysis of cell cycle sorted mESCs, we found that expression of the K+ ion channel Erg1 peaked in G1 cell cycle phase, which was confirmed by immunostaining. Inhibition of Erg channel activity caused loss of G1 phase cells via non-apoptotic cell death. Cells first lost the ability of membrane blebbing, a typical feature of cultured embryonic stem cells. Continued Erg inhibition further increased cell volume and the cell eventually ruptured. In addition, atomic force measurements on live cells revealed a decreased cortical stiffness after treatment, suggesting alterations in actomyosin organization. When the intracellular osmotic pressure was experimentally decreased by hypertonic solution or block of K+ ion import via the Na, K-ATPase, cell viability was restored and cells acquired normal volume and blebbing activity. Our results suggest that Erg channels have a critical function in K+ ion homeostasis of mESCs over the cell cycle, and that cell death following Erg inhibition is a consequence of the inability to regulate cell volume.

Integrin-mediated Membrane Blebbing Is Dependent on Sodium-Proton Exchanger 1 and Sodium-Calcium Exchanger 1 Activity

Integrin signaling and membrane blebbing modulate cell adhesion, spreading, and migration. However, the relationship between integrin signaling and membrane blebbing is unclear. Here, we show that an integrin-ligand interaction induces both membrane blebbing and changes in membrane permeability. Sodium-proton exchanger 1 (NHE1) and sodium-calcium exchanger 1 (NCX1) are membrane proteins located on the bleb membrane. Inhibition of NHE1 disrupts membrane blebbing and decreases changes in membrane permeability. However, inhibition of NCX1 enhances cell blebbing; cells become swollen because of NHE1 induced intracellular sodium accumulation. Our study found that NHE1 induced sodium influx is a driving force for membrane bleb growth, while sodium efflux (and calcium influx) induced by NCX1 in a reverse mode results in membrane bleb retraction. Together, these findings reveal a novel function for NHE1 and NCX1 in membrane blebbing and permeability, and establish a link between membrane blebbing and integrin signaling.

Polymorphisme biochimique chez quelques halophytes autochtones du Nord Tunisien

Chemical polymorphism of some North Tunisian autochthonous halophytes. The study aims to explore and analyse osmoticums synthesized by four perennial halophytic Chenopodiaceae : Salicornia arabica, Atriplex portulacoides, Atriplex halimus and Suaeda fruticosa from Menzel Jemil, Soliman and Gammarth regions which belong to sub-humid and semi-arid bioclimatic zones. A sampling of aerial vegetative organs of these four halophytes has been made. Analyses focused on reducing sugars, starch, total proteins, soluble proteins and total lipids. Atriplex portulacoides harvested from Menzel Jemil station accumulates large amounts of starch, indicating a high photosynthetic activity. This could suggest that this species avoids drought and high salinity by an acceleration of its growth cycle and reproductive systems. Suaeda fruticosa is characterized by the highest levels in soluble sugars. This would mean that it is capable of synthesizing carbohydrate osmoticums for its osmotic adjustment. Atriplex sp. and Suaeda fruticosa have the highest levels of soluble protein indicating the possible intervention of amino acids in the regulation of intracellular osmotic pressure. This behavior was essentially observed at Soliman sampling site characterized by an upper semi-arid bioclimate and saline soils.

Through a glass darkly

Through a glass darkly

Comparative genomics study of polyhydroxyalkanoates (PHA) and ectoine relevant genes from Halomonas sp. TD01 revealed extensive horizontal gene transfer events and co-evolutionary relationships

BackgroundHalophilic bacteria have shown their significance in industrial production of polyhydroxyalkanoates (PHA) and are gaining more attention for genetic engineering modification. Yet, little information on the genomics and PHA related genes from halophilic bacteria have been disclosed so far.ResultsThe draft genome of moderately halophilic bacterium, Halomonas sp. TD01, a strain of great potential for industrial production of short-chain-length polyhydroxyalkanoates (PHA), was analyzed through computational methods to reveal the osmoregulation mechanism and the evolutionary relationship of the enzymes relevant to PHA and ectoine syntheses. Genes involved in the metabolism of PHA and osmolytes were annotated and studied in silico. Although PHA synthase, depolymerase, regulator/repressor and phasin were all involved in PHA metabolic pathways, they demonstrated different horizontal gene transfer (HGT) events between the genomes of different strains. In contrast, co-occurrence of ectoine genes in the same genome was more frequently observed, and ectoine genes were more likely under coincidental horizontal gene transfer than PHA related genes. In addition, the adjacent organization of the homologues of PHA synthase phaC1 and PHA granule binding protein phaP was conserved in the strain TD01, which was also observed in some halophiles and non-halophiles exclusively from γ-proteobacteria. In contrast to haloarchaea, the proteome of Halomonas sp. TD01 did not show obvious inclination towards acidity relative to non-halophilic Escherichia coli MG1655, which signified that Halomonas sp. TD01 preferred the accumulation of organic osmolytes to ions in order to balance the intracellular osmotic pressure with the environment.ConclusionsThe accessibility of genome information would facilitate research on the genetic engineering of halophilic bacteria including Halomonas sp. TD01.

Complete Genome Sequence of the Anaerobic, Halophilic Alkalithermophile Natranaerobius thermophilus JW/NM-WN-LF

The genome of the anaerobic halophilic alkalithermophile Natranaerobius thermophilus consists of one 3,165,557-bp chromosome and two plasmids (17,207 bp and 8,689 bp). The present study is the first to report the completely sequenced genome of an anaerobic polyextremophile and genes associated with roles in regulation of intracellular osmotic pressure, pH homeostasis, and growth at elevated temperatures.

Single-cell mechanics provides a sensitive and quantitative means for probing amyloid-β peptide and neuronal cell interactions

By using a highly sensitive technique of atomic force microscopy-based single-cell compression, the rigidity of cultured N2a and HT22 neuronal cells was measured as a function of amyloid-beta42 (Abeta42) protein treatment. Abeta42 oligomers led to significant cellular stiffening; for example, 90-360% higher force was required to reach 80% deformation for N2a cells. Disaggregated or fibrillar forms of Abeta42 showed much less change. These observations were explained by a combination of two factors: (i) incorporation of oligomer into cellular membrane, which resulted in an increase in the Young's modulus of the membrane from 0.9+/-0.4 to 1.85+/-0.75 MPa for N2a cells and from 1.73+/-0.90 to 5.5+/-1.4 MPa for HT22 cells, and (ii) an increase in intracellular osmotic pressure (e.g., from 7 to 40 Pa for N2a cells) through unregulated ion influx. These findings and measurements provide a deeper, more characteristic, and quantitative insight into interactions between cells and Abeta42 oligomers, which have been considered the prime suspect for initiating neuronal dysfunction in Alzheimer's disease.

Cell volume regulation in oocytes and early embryos: connecting physiology to successful culture media

Preimplantation embryos are particularly susceptible to in vitro developmental blocks. These could be alleviated by lowering culture medium osmolarity. Because mammalian cells regulate their volumes by adjusting intracellular osmotic pressure, cell volume regulation could be critical to early embryos. We reviewed the literature on cell volume regulation in preimplantation embryos and the effects of increased osmolarity on embryo development, focusing also on the relation with improvements in embryo culture media. Embryos failed to develop from fertilized oocytes when osmolarity is increased. This could be alleviated by decreasing osmolarity or including certain compounds such as certain amino acids. Early preimplantation mouse embryos require intracellular accumulation of glycine to provide osmotic support and thus control cell volume. The glycine-specific transporter, GLYT1, mediates osmoregulated glycine accumulation in mouse embryos and likely in human embryos. GLYT1 is activated during meiotic maturation starting at ovulation. Prior to this, oocyte size is not independently controlled but instead is determined by strong adhesion between the oocyte plasma membrane and the inner surface of the zona pellucida. Early preimplantation embryos are particularly sensitive to increased osmolarity, and require the importation of glycine to regulate their cell volumes using a mechanism unique to early embryos. Cell volume regulation first appears when ovulation is triggered, oocyte zona pellucida adhesion is released, and glycine transport is activated. The requirement for supporting these physiological functions in oocytes and embryos should be taken into account when developing and improving systems for in vitro oocyte maturation and embryo culture.

Contributions of inorganic ions, soluble carbohydrates, and multiatomic alcohols to water homeostasis in Artemisia lerchiana and A. pauciflora

Two morphological forms of wormwood Artemisia lerchiana (f. erecta and f. nutans) and A. pauciflora Web. (morphological form erecta) were grown on sand culture at a range of NaCl concentrations in the nutrient medium and then assayed for Na+, K+, and Cl− content in various organs. In addition, the content of mono-, di-, and trisaccharides and multiatomic alcohols (mannitol and glycerol); water content; and organ biomass were determined. All plants examined showed high NaCl tolerance, comparable to that of halophytes. They were able to maintain high tissue hydration under conditions of salinity-induced growth suppression. The intracellular osmotic pressure in wormwood organs was mainly determined by the presence of Na+, K+, and Cl−, as well as by mono-, di-, and trisaccharides, mannitol, and glycerol. The high content of Na+ and Cl− in wormwood organs was also observed in the absence of salinity, which implies the ability of these organs to absorb ions from diluted NaCl solutions and accumulate ions in cells of their tissues. With the increase in salinity, the content of Na+ and Cl− in roots and leaves increased to even higher levels. It is concluded that the ability of wormwood plants to absorb and accumulate inorganic ions provides for sustainable high intracellular osmotic pressure and, accordingly, low water potential under drought and salinity conditions. Growing plants under high salinity lowered the content of monosaccharides in parallel with accumulation of the trisaccharide raffinose. It is supposed that soluble carbohydrates and multiatomic alcohols are not only significant for osmoregulation but also perform a protective function in wormwood plants. The lower osmotic pressure in root cells compared to that in leaf cells of all plants examined was mainly due to the gradient distribution of K+ and Cl− between roots and leaves. The two Artemisia species and two morphological forms of A. lerchiana did not differ appreciably in the ways of water balance regulation. It is found that different morphologies of two A. lerchiana forms are unrelated to variations in intracellular osmotic and turgor pressures.

Molecular basis of transport and regulation in the Na+/betaine symporter BetP

Osmoregulated transporters sense intracellular osmotic pressure and respond to hyperosmotic stress by accumulation of osmolytes to restore normal hydration levels. Here we report the determination of the X-ray structure of a member of the family of betaine/choline/carnitine transporters, the Na(+)-coupled symporter BetP from Corynebacterium glutamicum, which is a highly effective osmoregulated uptake system for glycine betaine. Glycine betaine is bound in a tryptophan box occluded from both sides of the membrane with aromatic side chains lining the transport pathway. BetP has the same overall fold as three unrelated Na(+)-coupled symporters. Whereas these are crystallized in either the outward-facing or the inward-facing conformation, the BetP structure reveals a unique intermediate conformation in the Na(+)-coupled transport cycle. The trimeric architecture of BetP and the break in three-fold symmetry by the osmosensing C-terminal helices suggest a regulatory mechanism of Na(+)-coupled osmolyte transport to counteract osmotic stress.

Biotechnology applications of amino acids in protein purification and formulations

Amino acids are widely used in biotechnology applications. Since amino acids are natural compounds, they can be safely used in pharmaceutical applications, e.g., as a solvent additive for protein purification and as an excipient for protein formulations. At high concentrations, certain amino acids are found to raise intra-cellular osmotic pressure and adjust to the high salt concentrations of the surrounding medium. They are called "compatible solutes", since they do not affect macromolecular function. Not only are they needed to increase the osmotic pressure, they are known to increase the stability of the proteins. Sucrose, glycerol and certain amino acids were used to enhance the stability of unstable proteins after isolation from natural environments. The mechanism of the action of these protein-stabilizing amino acids is relatively well understood. On the contrary, arginine was accidentally discovered as a useful reagent for assisting in the refolding of recombinant proteins. This effect of arginine was ascribed to its ability to suppress aggregation of the proteins during refolding, thereby increasing refolding efficiency. By the same mechanism, arginine now finds much wider applications than previously anticipated in the research and development of proteins, in particular in pharmaceutical applications. For example, arginine solubilizes proteins from loose inclusion bodies, resulting in efficient production of active proteins. Arginine suppresses protein-protein interactions in solution and also non-specific adsorption to gel permeation chromatography columns. Arginine facilitates elution of bound proteins from various column resins, including Protein-A or dye affinity columns and hydrophobic interaction columns. This review covers various biotechnology applications of amino acids, in particular arginine.

Water channel proteins of adipocytes

After the first water channel protein was discovered in the red blood cell membrane in 1985, more than 200 water channel proteins have been identified in all living organisms. In mammalians there are 13 members of the water channel protein family, with two groups of members: aquaporins, which are considered as specific water channels, and aquaglyceroporins, which are permeable to water (however to varying degrees), but are also permeated by other small molecules, in particular glycerol. Adipocytes express aquaporin 1 (AQP1) and aquaglyceroporin (AQP7). Aquaporin 1 is abundant in adipocytes but its functional significance is not yet precisely known. Aquaporin 7 may prevent the acute rise in intracellular osmotic pressure due to glycerol production during lipolysis, which could damage the cell. Dysregulation of AQP7 is increasingly implicated in the pathogenesis of obesity. This is supported by studies on cultured adipocytes and on animals in which AQP7 was knocked down. Such studies are briefly reviewed, and adipopharmacology of AQP7 in obesity is outlined. Biomedical Reviews 2006; 17: 105-110.

Toluene-induced accumulation of trehalose by Pseudomonas sp. BCNU 106 through the expression of otsA and otsB homologues

The objective of this study was to investigate toluene-induced accumulation mechanism of trehalose in a toluene-tolerant bacterium Pseudomonas sp. BCNU 106. The accumulation of trehalose by a toluene-tolerant bacterium Pseudomonas sp. BCNU 106 was examined at various cultivation time by measuring the total intracellular trehalose content, trehalase activity and mRNA levels of the trehalose-biosynthetic genes. The pattern of trehalose accumulation corresponded to the mRNA expression pattern of the trehalose-biosynthetic genes with the maximum level at 12 h or 4 h of cultivation with 10% (v/v) toluene, respectively. The trehalose-biosynthetic genes were also cloned and sequenced. Furthermore, the effects of toluene addition on the intracellular osmotic pressure and pH were investigated. It was shown that homeostasis was maintained in the bacterial cells. In a toluene-tolerant bacterium Pseudomonas sp. BCNU 106, a significant amount of trehalose was accumulated through the toluene-induced expression of the trehalose-biosynthetic genes after the exposure to toluene. The accumulation of the high level of intracellular trehalose was preceded by the expression of otsA/B genes in toluene-tolerant bacteria, contributing to the elucidation of the tolerance mechanism.

TMAO and other organic osmolytes in the muscles of amphipods (Crustacea) from shallow and deep water of Lake Baikal

Concentrations of trimethylamine oxide (TMAO) and other ‘compatible’ osmolytes were analyzed in the muscle tissue of Lake Baikal amphipods (Crustacea) in relation to water depth of the freshwater Lake Baikal. Using HPLC and mass spectrometry, glycerophosphoryl choline (GPC), betaine, S-methyl-cysteine, sarcosine, and taurine were detected for the first time in freshwater amphipods. These osmolytes were frequently found in the five species studied but mixtures were too complex to be quantified. The pattern of these osmolytes did not change with respect to water depth. The TMAO concentration, however, was significantly higher in the muscle tissue of amphipods living in deep water than of those living in shallow water, which supports the hypothesis that TMAO acts as a protective osmolyte at increased hydrostatic pressure. We propose that eurybathic amphipods, exposed to raised hydrostatic pressure in the extremely deep freshwater Lake Baikal, have elevated TMAO levels to counteract the adverse effect of high pressure on protein structure. The elevated intracellular osmotic pressure is balanced by upregulating the extracellular hemolymph NaCl concentration.

Lactose-induced cell death of ?-galactosidase mutants in

The Kluyveromyces lactis lac4 mutants, lacking the beta-galactosidase gene, cannot assimilate lactose, but grow normally on many other carbon sources. However, when these carbon sources and lactose were simultaneously present in the growth media, the mutants were unable to grow. The effect of lactose was cytotoxic since the addition of lactose to an exponentially-growing culture resulted in 90% loss of viability of the lac4 cells. An osmotic stabilizing agent prevented cells killing, supporting the hypothesis that the lactose toxicity could be mainly due to intracellular osmotic pressure. Deletion of the lactose permease gene, LAC12, abolished the inhibitory effect of lactose and allowed the cell to assimilate other carbon substrates. The lac4 strains gave rise, with unusually high frequency, to spontaneous mutants tolerant to lactose (lar1 mutation: lactose resistant). These mutants were unable to take up lactose. Indeed, lar1 mutation turned out to be allelic to LAC12. The high mutability of the LAC12 locus may be an advantage for survival of K. lactis whose main habitat is lactose-containing niches.

Swelling of hepatocytes injured by oxidative stress suggests pathological changes related to macromolecular crowding

A fundamental property of living matter is the ability to establish and maintain order. Mild changes in cell volume have a role in metabolic control. Furthermore, cellular swelling is a way of living cells to react to a variety of stressors. Data from experimental pathology, biochemistry and biophysics and theoretical arguments from biology, biochemical evolution, cytology and biophysics are considered to attempt an integration of several current concepts on different subjects (intracellular compartmentation, cellular swelling, macromolecular crowding, perturbing and non-perturbing solutes). The purpose is to provide a framework for conceptualizing in modern terms the question whether cellular swelling induced by oxidative stress should be considered merely a cell adaptation balanced by antioxidant defenses and by other biochemical devices apt to preserve the intracellular environment and normal cell functioning, or whether swelling of high amplitude should be regarded as a true pathological change. The basic question dated 1982: "how crowded is the cytoplasm?" is a matter for discussion as far as swollen cells are concerned. This paper examines the liver for cellular swelling of high amplitude (about+30%) caused by iron or by thyroid hormone+iron (histological picture of "cloudy swelling") or the steatogenic poison CCl(4), also known as a source of oxyradicals, which causes an even more pronounced cellular swelling. In CCl(4)-toxic fatty liver the strong increase of tissue water is substantially masked by the parallel increase of tissue dry solids due to fat accumulation. This example of a "tissue dilution artefact" is discussed in connection with the increase of tissue water also in toxic fatty liver induced by white phosphorus and ethanol. In CCl(4)-toxic fatty liver the normal K(+)/Na(+) ratio (about 3) is substantially maintained, whereas the concentrations of the two cations ("perturbing osmolytes") in tissue water are noticeably decreased, a finding which was not further studied at the time the observations were made because biochemistry was not yet advanced enough to allow an explanation. Today, a logic hypothesis is that an increase of non-perturbing solutes such as taurine and betaine, maintains the physiological intracellular osmotic pressure and that the harmful effects of CCl(4) are limited because of the protective effects of these molecules and of molecular chaperones against damage by oxyradicals. However, as a consequence of cellular swelling, intracellular changes in ionic strength and macromolecular crowding should occur thus affecting enzyme activities. Models and techniques apt to investigate this problem experimentally are suggested.

In vitro effects of wastewater treatment plant effluent on sea bass red blood cells

Red blood cells of marine fish have been used in suitable biological assays to study the (eco)toxicity of wastewater treatment plant effluents. The aim of the present work was to draw upon their more relevant effects on cell hemolysis, ATP content, osmotic resistance and cell volume regulation. Following physico-chemical treatment, treatment plant effluents showed a residual toxicity resulting from multiple impairment of cell metabolism and structures. The earliest and most sensitive effects were related to the regulation of intracellular osmotic pressure leading to decreased cell water volume. Such effects were also observed following short-term incubation in 10-fold diluted effluent. Other damages were found following incubation in non-diluted effluent. Membrane structure was affected leading to increased osmotic resistance. Later, a decrease of the intracellular ATP level was found, followed by hemolysis. The presence of glucose in the incubation medium lessened the fall in ATP content and hemolysis in the treated cells but also in control cells.