Polymer Two-phase Systems Research Articles

Analysis of miRNAs in the PSMA-positive fraction of plasma nano-sized extracellular vesicles in patients with prostate cancer

Background. MicroRNAs (miRNAs) circulating in plasma are promising markers for the diagnosis of malignant tumors, including prostate cancer. However, the existing techniques used for their detection fail to ensure sufficient diagnostic accuracy. One of the possible ways to improve it is to isolate membrane nano-sized extracellular vesicles (nsEVs) secreted by prostate cells. Presumably, the analysis of miRNAs originating from this prostate-specific fraction of nsEVs more accurately reflects the process of prostate cancer development and has a greater diagnostic potential. Objective: to develop the method of miRNA isolation from the prostate-specific fraction of plasma nsEVs and to evaluate its performance characteristics.Materials and methods. Prostate-specific membrane antigen (PSMA) was used as a prostate-specific marker of nsEVs. The total population of plasma nsEVs was isolated using a two-phase polymer system. To isolate PSMA-positive (PSMA(+)) nsEVs, we used superparamagnetic particles with PSMA-binding DNA aptamer immobilized on their surface. The efficacy of PSMA(+) nsEV isolation was assessed using flow cytometry and dot-blotting. RNA from nsEVs was isolated using proteolysis; miRNA analysis was performed using reverse transcription polymerase chain reaction. Plasma samples collected from patients with prostate cancer (n = 33) and healthy donors (controls) (n = 30) were used to evaluate the diagnostic parameters of the method.Results. We developed the method of PSMA(+) nsEV isolation from plasma and estimated its performance characteristics. We found that measurement of potential miRNA markers in PSMA(+) nsEVs was more effective than its measurement in the entire nsEV population and could distinguish between patients with prostate cancer and controls.Conclusion. The new technique of PSMA(+) nsEV isolation can be used for the development of novel diagnostic methods for the diagnosis of prostate cancer.

Structural properties, antioxidant and hypoglycemic activities of polysaccharides purified from pepper leaves by high-speed counter-current chromatography

In the present study, polysaccharides with antioxidant and hypoglycemic activities were isolated from the crude extract of pepper leaves by high-speed countercurrent chromatography in one step for the first time. An aqueous polymer two-phase system comprising PEG1000-K2HPO4-KH2PO4-H2O (0.8:1.25:1.25:6.5, w/w) was used for separation. During each separation, 2.0 g of the crude sample yielded two homogeneous components of polysaccharides PLP1 (226 mg) and PLP2 (273 mg). The molecular weights of PLP1 and PLP2 were 123 kDa and 243 kDa, respectively. PLP1 primarily consisted of mannose and glucose at a molar ratio of 1:2.4, whereas PLP2 was mainly composed of galactose, glucose, and rhamnose at a molar ratio of 2.6:1:2.2 with 10.3% (w/w) sulphates. Biological activity showed that sulfated PLP2 exhibited stronger antioxidant activity in vitro and hypoglycemic activity in diabetic mice than PLP1. The results demonstrated that sulfated PLP2 possessed potential as natural hypoglycemic agents or antioxidants in food or pharmaceuticals.

Rapid large‐scale preparation of polysaccharides from jackfruit peel waste by high‐speed countercurrent chromatography and their antioxidant and hypoglycemic activities

Polysaccharides with antioxidant and hypoglycemic activities were first isolated from jackfruit (Artocarpus heterophyllus Lam.) peel through the one-step high-speed countercurrent chromatography. The separation process was completed using the polymer two-phase aqueous system constituted by PEG1000-K2 HPO4 -KH2 PO4 -H2 O (0.8:1.25:1.25:6.5, w/w). For every separation process, two main polysaccharides, namely, fraction-1 and fraction-2 (165 and 225mg, respectively) were obtained from a 2.0g crude sample. As suggested by high-performance gel permeation chromatography, jackfruit peel polysaccharides had the mean molecular weight values of 113.3 and 174.3kDa, separately. Physicochemical analysis suggested that two polysaccharides were dominant in galacturonic acid, galactose, rhamnose, arabinose, glucose, mannose, as well as fucose, which were highly esterified. Biological activity analysis showed that fraction-1 exhibited stronger antioxidant activity in vitro and hypoglycemic activity in streptozotocin-induced diabetic mice compared with fraction-2. The results suggest that polysaccharide fraction-1 may be developed as a potential functional food supplement.

Evaluation of Colon-Specific Plasma Nanovesicles as New Markers of Colorectal Cancer.

Simple SummaryHigh mortality in patients with colorectal cancer (CRC) is one of the main problems in healthcare. This is due to the use of invasive and existing noninvasive screening methods whose resources are limited. A promising alternative is a study of circulating in plasma extracellular nanovesicles (ENVs) reflecting the same composition of biomarkers with the secreted cells, forming subpopulations of tissue-specific ENVs. During the work, we have selected potential colon biomarkers from databases. The study aims to develop a quantitative method for isolating colon-specific ENVs based on the formation of immunocomplexes «beads with antibodies to specific biomarkers». We found that the amount of ENVs carrying potential colon biomarkers was higher in the patients with an IV CRC-stage compared with the healthy donors. It showed a high coefficient of diagnostic significance of these biomarkers in the CRC prognosis. These results will give an impetus to a deeper study of the ENVs as identifiers of cancer’s development.Purpose: Developing new and efficient approaches for the early diagnosis of colorectal cancer (CRC) is an important issue. Circulating extracellular nanovesicles (ENVs) present a promising class of cancer markers. Cells of well-differentiated adenocarcinomas retain the molecular characteristics of colon epithelial cells, and the ENVs secreted by these cells may have colon-specific surface markers. We hypothesize that an increase in the number of ENVs carrying colon-specific markers could serve as a diagnostic criterion for colorectal cancer. Experimental design: Potential colon-specific markers were selected based on tissue-specific expression profile and cell surface membrane localization data. Plasma was collected from CRC patients (n = 48) and healthy donors (n = 50). The total population of ENVs was isolated with a two-phase polymer system. ENVs derived from colon epithelium cells were isolated using immune-beads with antibodies to colon-specific markers prior to labelling with antibodies against exosomal tetraspanins (CD63 and CD9) and quantification by flow cytometry. Results: The number of ENVs positive for single colon cancer markers was found to be significantly higher in the plasma of CRC patients compared with healthy donors. The efficacy of detection depends on the method of ENV labelling. The diagnostic efficacy was estimated by ROC analysis (the AUC varied between 0.71 and 0.79). The multiplexed isolation of colon-derived ENVs using immune-beads decorated with antibodies against five markers allowed for a further increase in the diagnostic potency of the method (AUC = 0.82). Conclusions: ENVs derived from colon epithelium may serve as markers of differentiated CRC (adenocarcinomas). The composition of ligands used for capturing colon-derived ENVs and their method of labelling are critical for the efficacy of this proposed diagnostic approach.

P-245 Evaluation of colon-specific plasma nanovesicles as new markers of colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of cancer death worldwide, which is associated with low detection efficiency in the early stages of its development. The traditional methods of CRC screening are not effective enough. The liquid biopsy, based on the identification of extracellular nanovesicles (ENVs) in plasma, appears to be a promising diagnostic technology. Current approaches ENV-based cancer diagnostics suppose either quantification of tumor-derived ENVs or detection of cancer-associated components (e.g. miRNA) in total population of plasma ENVs. The first approach lacks specificity due to an absence of well-established vesicular membrane-associated markers of CRC. The second approach lacks sensitivity due to the extremely heterogeneous composition of plasma ENVs derived mostly from endothelium and blood cells. We hypothesized that development of a well-differentiated adenocarcinoma from colon epithelium should result in increased release of ENVs bearing markers of the intestinal differentiation. If this hypothesis is true, these vesicles might serve as markers of CRC. Plasma samples from CRC patients (n=48) and healthy donors (n.=50) were used. Design of study was confirmed by the local ethics committee, each participant signed a form of informed consent. The total population of ENVs was isolated from plasma using a two-phase polymer system and analyzed in terms of size, concentration, morphology and surface markers using NTA, Cryo-EM and on-bead flow cytometry. To isolate tissue-specific exosomes, we used streptavidin-coated super-paramagnetic particles (SPMP) and biotinylated antibodies to CLRN3, GPA33, GCNT3, PIGY, REG4, and other ten potential markers. The efficiency of the immune-sorption of such exosomes to SPMP was assessed by flow cytometry after labeling with CD63 FITC and/or CD9 PerCP-Cy5.5 antibodies. Potential markers were selected on the basis of their colon-specific expression pattern and cell surface membrane-associated localization. We selected 16 potential markers and evaluated the amount of ENVs positive for these markers in plasma of CRC patients and healthy donors. The amount of ENVs bearing CLRN3, GPA33, GCNT3, PIGY and REG4 differed between groups of patients and donors, difference was statistically significant. The value of AUC varied between 0,6 and 0,81. Multiplexed analysis allowed us to observe an increased difference between compared groups (CRC vs HD) and to achieve AUC = 0.72. The presented study is the first attempt to explore intestine-specific ENVs as a marker of cancer arising from this tissue. Our results confirmed the initial hypothesis and showed the effectiveness of the proposed approach. Further investigations are still required to identify the best markers of colon epithelium cell-derived ENVs, to better understand the link between CRC characteristics and the profile of tumor-released ENVs and to optimize techniques of quantification of the intestine-specific ENVs in plasma.

Formation and Evaluation of a Two-Phase Polymer System in Human Plasma as a Method for Extracellular Nanovesicle Isolation.

The aim of the study was to explore the polyethylene glycol–dextran two-phase polymer system formed in human plasma to isolate the exosome-enriched fraction of plasma extracellular nanovesicles (ENVs). Systematic analysis was performed to determine the optimal combination of the polymer mixture parameters (molecular mass and concentration) that resulted in phase separation. The separated phases were analyzed by nanoparticle tracking analysis and Raman spectroscopy. The isolated vesicles were characterized by atomic force microscopy and dot blotting. In conclusion, the protein and microRNA contents of the isolated ENVs were assayed by flow cytometry and by reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR), respectively. The presented results revealed the applicability of a new method for plasma ENV isolation and further analysis with a diagnostic purpose.

Directed assembly of multiplexed single chirality carbon nanotube devices

Herein, we present the fabrication of multiplexed single-walled carbon nanotube (SWCNT) devices, where selected chiralities were separately immobilized on one chip with single-tube precision. Each chirality was subsequently electrically measured individually. Specifically, (6,5) and (7,5) SWCNT species were isolated via aqueous two-phase polymer systems, after which dielectrophoresis was used to precisely control the placement of each chirality, along with a metallic species, separately on prepatterned electrodes on a single chip.

Controlling Macroscopic Phase Separation of Aqueous Two-Phase Polymer Systems in Porous Media.

In previous work, our group discovered a phenomenon in which a mixed polymer-salt or mixed micellar aqueous two-phase system (ATPS) separates into its two constituent phases as it flows within paper. While these ATPSs worked well in their respective studies to concentrate the target biomarker and improve the sensitivity of the lateral-flow immunoassay, different ATPSs can be advantageous for new applications based on factors such as biomarker partitioning or biochemical compatibility between ATPS and sample components. However, since the mechanism of phase separation in porous media is not completely understood, introducing other ATPSs to paper is an unpredictable process that relies on trial and error experiments. This is especially true for polymer-polymer ATPSs in which the characteristics of the two phases appear quite similar. Therefore, our group aimed to develop semiquantitative guidelines for choosing ATPSs that can phase separate in paper. In this work, we evaluated the Washburn equation and its parameters as a potential mathematical framework to describe the flow behavior of polymer-salt and micellar ATPSs in fiberglass paper. We compared bulk phase fluid characteristics and identified the viscosity difference between the phases as a key determinant of the potential for phase separation in paper. We then used this parameter to predict the phase separation capabilities of polyethylene glycol (PEG)-dextran ATPSs in paper and control the composition of the leading and lagging phases. We also, for the first time, successfully demonstrated the phase separation phenomenon in hydrogels, thereby extending its application and potential benefits to an alternative porous medium.

Role of PIP-Aquaporin Phosphorylation in Redox-Dependent Modulation of Osmotic Water Permeability in Plasmalemma from Roots of Pea Seedlings

Investigations were directed to study the mechanism based on redox-dependent modulation of osmotic water permeability in plasmalemma from roots of pea (Pisum sativum L.) seedlings. This mechanism may be associated with oxidation of cysteine residues in PIP-aquaporins or involves redox-dependent phosphorylation of these proteins. Plasmalemma was isolated by partitioning of microsomal membranes in the aqueous polymer two-phase system and using the roots homogenization medium additionally contained SH-reagents, dithiothreitol or diamide, and also phenylarsin oxide, an inhibitor of tyrosine protein phosphatases. Water permeability of plasmalemma was estimated by the kinetics of light scattering changes of membrane vesicles due to their osmotic shrinkage and recorded by the stopped flow method. In order to elucidate whether PIP-aquaporins contain cysteines available for oxidation by SH-reagents, isolated plasmalemma was subjected to PEGylation (reaction, based on binding of 5 kD methoxypolyethylene glycol maleimide with available membrane proteins SH-groups) and further western blot analysis was performed to reveal shift of aquaporins molecular mass by 5 kD after their immunodetection with antibodies. Relative content of PIP-aquaporin phosphorylated forms in plasmalemma was determined using fluorescent labeling proteins in gels with ProQ Diamond after membrane proteins separation by two-dimensional electrophoresis (blue native (BN/PAGE) in first dimension and denaturing (SDS/PAGE) in second dimension). It was established that conservative cysteines in molecules of PIP-aquaporins are available for SH-reagents only under denaturing conditions. This fact excludes the possibility of these cysteines participation in modulation of plasmalemma osmotic water permeability. Comparative analysis of phosphorylated PIP-aquaporins pool with different redox status of membrane proteins and osmotic water permeability coefficients and also the inhibitor analysis data showed that redox-dependent modulation of these proteins activity is due to their phosphorylation with involvement of serine-threonine protein phosphatases of PP2C type and/or thyrosine protein phosphatases.

Membrane Protein Identification in Rodent Brain Tissue Samples and Acute Brain Slices.

Acute brain slices are a sample format for electrophysiology, disease modeling, and organotypic cultures. Proteome analyses based on mass spectrometric measurements are seldom used on acute slices, although they offer high-content protein analyses and explorative approaches. In neuroscience, membrane proteins are of special interest for proteome-based analysis as they are necessary for metabolic, electrical, and signaling functions, including myelin maintenance and regeneration. A previously published protocol for the enrichment of plasma membrane proteins based on aqueous two-phase polymer systems followed by mass spectrometric protein identification was adjusted to the small sample size of single acute murine slices from newborn animals and the reproducibility of the results was analyzed. For this, plasma membrane proteins of 12 acute slice samples from six animals were enriched and analyzed by liquid chromatography-mass spectrometry. A total of 1161 proteins were identified, of which 369 were assigned to membranes. Protein abundances showed high reproducibility between samples. The plasma membrane protein separation protocol can be applied to single acute slices despite the low sample size and offers a high yield of identifiable proteins. This is not only the prerequisite for proteome analysis of organotypic slice cultures but also allows for the analysis of small-sized isolated brain regions at the proteome level.

PIP1 aquaporins, sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm3) and “heavy” (≥1.146 g/cm3) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.

Comparative proteomic analysis reveals a dynamic pollen plasma membrane protein map and the membrane landscape of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils in rice

BackgroundThe coordination of pollen tube (PT) growth, guidance and timely growth arrest and rupture mediated by PT-pistil interaction is crucial for the PT to transport sperm cells into ovules for double fertilization. The plasma membrane (PM) represents an important interface for cell–cell interaction, and PM proteins of PTs are pioneers for mediating PT integrity and interaction with pistils. Thus, understanding the mechanisms underlying these events is important for proteomics.ResultsUsing the efficient aqueous polymer two-phase system and alkali buffer treatment, we prepared high-purity PM from mature and germinated pollen of rice. We used iTRAQ quantitative proteomic methods and identified 1,121 PM-related proteins (PMrPs) (matched to 899 loci); 192 showed differential expression in the two pollen cell types, 119 increased and 73 decreased in abundance during germination. The PMrP and differentially expressed PMrP sets all showed a functional skew toward signal transduction, transporters, wall remodeling/metabolism and membrane trafficking. Their genomic loci had strong chromosome bias. We found 37 receptor-like kinases (RLKs) from 8 kinase subfamilies and 209 transporters involved in flux of diversified ions and metabolites. In combination with the rice pollen transcriptome data, we revealed that in general, the protein expression of these PMrPs disagreed with their mRNA expression, with inconsistent mRNA expression for 74% of differentially expressed PMrPs.ConclusionsThis study identified genome-wide pollen PMrPs, and provided insights into the membrane profile of receptor-like kinases and transporters important for pollen tube growth and interaction with pistils. These pollen PMrPs and their mRNAs showed discordant expression. This work provides resource and knowledge to further dissect mechanisms by which pollen or the PT controls PMrP abundance and monitors interactions and ion and metabolite exchanges with female cells in rice.

Plasma Membrane Preparation from Lilium davidii and Oryza sativa Mature and Germinated Pollen.

Pollen germination is an excellent process to study cell polarity establishment. During this process, the tip-growing pollen tube will start elongating. The plasma membrane as the selectively permeable barrier that separates the inner and outer cell environment plays crucial roles in this process. This protocol described an efficient aqueous polymer two-phase system followed by alkaline solution washing to prepare Lilium davidii or Oryza sativa plasma membrane with high purity.

Extraction of natural red colorants from the fermented broth of Penicillium purpurogenum using aqueous two-phase polymer systems.

Safety concerns related to the increasing and widespread application of synthetic coloring agents have increased the demand for natural colorants. Fungi have been employed in the production of novel and safer colorants. In order to obtain the colorants from fermented broth, suitable extraction systems must be developed. Aqueous two-phase polymer systems (ATPPS) offer a favorable chemical environment and provide a promising alternative for extracting and solubilizing these molecules. The aim of this study was to investigate the partitioning of red colorants from the fermented broth of Penicillium purpurogenum using an ATPPS composed of poly(ethylene glycol) (PEG) and sodium polyacrylate (NaPA). Red colorants partitioned preferentially to the top (PEG-rich phase). In systems composed of PEG 6,000 g/mol/NaPA 8,000 g/mol, optimum colorant partition coefficient (KC ) was obtained in the presence of NaCl 0.1 M (KC = 10.30) while the PEG 10,000 g/mol/NaPA 8,000 g/mol system in the presence of Na2 SO4 0.5 M showed the highest KC (14.78). For both polymers, the mass balance (%MB) and yield in the PEG phase (%ηTOP ) were close to 100 and 79%, respectively. The protein selectivity in all conditions evaluated ranged from 2.0-3.0, which shows a suitable separation of the red colorants and proteins present in the fermented broth. The results suggest that the partitioning of the red colorants is dependent on both the PEG molecular size and salt type. Furthermore, the results obtained support the potential application of ATPPS as the first step of a purification process to recover colorants from fermented broth of microorganisms.

DNA-Controlled Partition of Carbon Nanotubes in Polymer Aqueous Two-Phase Systems

Sorting single-wall carbon nanotubes (SWCNTs) of different chiralities is both scientifically interesting and technologically important. Recent studies have shown that polymer aqueous two-phase extraction is a very effective way to achieve nanotube sorting. However, works published to date have demonstrated only separation of surfactant-dispersed SWCNTs, and the mechanism of chirality-dependent SWCNT partition is not well understood. Here we report a systematic study of spontaneous partition of DNA-wrapped SWCNTs in several polymer aqueous two-phase systems. We show that partition of DNA-SWCNT hybrids in a given polymer two-phase system is strongly sequence-dependent and can be further modulated by salt and polymer additives. With the proper combination of DNA sequence, polymer two-phase system, and partition modulators, as many as 15 single-chirality nanotube species have been effectively purified from a synthetic mixture. As an attempt to provide a unified partition mechanism of SWCNTs dispersed by surfactants and by DNA, we present a qualitative analysis of solvation energy for SWCNT colloids in a polymer-modified aqueous phase. Our observation and analysis highlight the sensitive dependence of the hydration energy on the spatial distribution of hydrophilic functionalities.

5CN05 partitioning in an aqueous two-phase system: A new approach to the solubilization of hydrophobic drugs

Liquid–liquid extraction for the purification of molecules has been central to many advances in the pharmaceutical industry. These processes were developed based on the property that some polymer and/or micellar solutions present to separate into a concentrated phase and a diluted phase. Based on the differences in the physical and chemical environments of the two coexisting phases, and since both phases contain approximately 60–90% of water, liquid–liquid extraction provides a powerful alternative to both extract and solubilize a molecule. This paper examines the partition behavior of the synthetic drug, 2-[(3,4-dichlorine-benzylidene)-amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile (5CN05), in an aqueous two-phase polymer system (ATPPS) and also in an aqueous two-phase micellar system (ATPMS). The results showed that both systems are favorable for extraction the 5CN05 drug high partition coefficient values (K5CN05>200) and yield (Y5CN05>99.48%) in the concentrated phase were achieved with the systems. However, the ATPPS generated a partition coefficient (K5CN05) higher than the one obtained with ATPMS. The results suggest that both processes may be used for the extraction and concentration of molecules with hydrophobic characteristics, such as 5CN05. They also provide an optimal environment for the solubilization of such molecules, allowing for greater efficiency when purifying many classes of drugs.

Monitoring the native phosphorylation state of plasma membrane proteins from a single mouse cerebellum

Neuronal processing in the cerebellum involves the phosphorylation and dephosphorylation of various plasma membrane proteins such as AMPA or NMDA receptors. Despite the importance of changes in phosphorylation pattern, no global phospho-proteome analysis has yet been performed. As plasma membrane proteins are major targets of the signalling cascades, we developed a protocol to monitor their phosphorylation state starting from a single mouse cerebellum. An aqueous polymer two-phase system was used to enrich for plasma membrane proteins. Subsequently, calcium phosphate precipitation, immobilized metal affinity chromatography, and TiO2 were combined to a sequential extraction procedure prior to mass spectrometric analyses. This strategy resulted in the identification of 1501 different native phosphorylation sites in 507 different proteins. 765 (51%) of these phosphorylation sites were localized with a confidence level of 99% or higher. 41.4% of the identified proteins were allocated to the plasma membrane and about half of the phosphorylation sites have not been reported previously. A bioinformatic screen for 12 consensus sequences identified putative kinases for 642 phosphorylation sites. In summary, the protocol deployed here identified several hundred novel phosphorylation sites of cerebellar proteins. Furthermore, it provides a valuable tool to monitor the plasma membrane proteome from any small brain samples of interest under differing physiological or pathophysiological conditions.

Phosphatidic Acid Binds and Stimulates Arabidopsis Sphingosine Kinases

Phosphatidic acid (PA) and phytosphingosine-1-phosphate (phyto-S1P) have both been identified as lipid messengers mediating plant response to abscisic acid (ABA). To determine the relationship of these messengers, we investigated the direct interaction of PA with Arabidopsis sphingosine kinases (SPHKs) that phosphorylate phytosphingosine to generate phyto-S1P. Two unique SPHK cDNAs were cloned from the annotated At4g21540 locus of Arabidopsis, and the two transcripts are differentially expressed in Arabidopsis tissues. Both SPHKs are catalytically active, phosphorylating various long-chain sphingoid bases (LCBs) and are associated with the tonoplast. They both interact with PA as demonstrated by lipid-filter binding, liposome binding, and surface plasmon resonance (SPR). SPHK1 and SPHK2 exhibited strong binding to 18:1/18:1, 16:0/18:1, and 16:0/18:2 PA, but poor binding to 16:0/16:0, 8:0/8:0, 18:0/18:0, and 18:2/18:2 PA. Surface dilution kinetics analysis indicates that PA stimulates SPHK activity by increasing the specificity constant through decreasing K(m)(B). The results show that the annotated At4g21540 locus is actually comprised of two separate SPHK genes. PA binds to both SPHKs, and the interaction promotes lipid substrate binding to the catalytic site of the enzyme. The PA-SPHK interaction depends on the PA molecular species. The data suggest that these two Arabidopsis SPHKs are molecular targets of PA, and the PA stimulation of SPHK is part of the signaling networks in Arabidopsis.

Affinity of PIP-aquaporins to sterol-enriched domains in plasma membrane of the cells of etiolated pea seedlings

The hypothesis that sterol-enriched domains represent sites of preferred localization of PIP-aquaporins was tested in experiments on plasma membranes isolated from cells of etiolated pea (Pisum sativum L.) seedlings. Plasma membranes were isolated from microsomes by the partition in the aqueous two-phase polymer system and separated into vesicle fractions of different buoyant density by flotation in discontinuous OptiPrep gradient. Two types of plasma membrane preparations were used: one was treated with cold 1% Triton X-100 and the other was not. In untreated preparations, three populations of plasma membrane vesicles were obtained, while in the case of treated preparations, fractions of detergent-resistant membranes (DRM) and solubilized membrane proteins were obtained. In all membrane fractions collected after OptiPrep flotation, the amounts of proteins, sterols, and PIP-aquaporins were determined. The highest sterol content was detected in the membrane fraction with buoyant density 1.098 g/cm3 and in the DRM fraction (1.146 g/cm3). These fractions contained much more PIP-aquaporins than the other ones. Phase state of the lipid bilayer was determined by measuring generalized polarization excitation of fluorescence (GPEX) of laurdan incorporated into the membranes of different fractions. It was revealed that the lipid bilayer of the membranes with density of 1.098 g/cm3 had a higher extent of ordering than that of the fractions with density of ∼1.146 g/cm3. The results indicated that uppermost local concentrations of PIP-aquaporins were associated with tightly packed sterol-enriched domains. Moreover, upon solubilization of plasma membrane with Triton X-100, PIP-aquaporins mainly resided in DRM, thus exhibiting a high affinity to sterols.

Effect of chilling temperatures on osmotic water permeability and aquaporin activity in the plasma membranes from pea roots

The osmotic water permeability of plasma membrane vesicles was examined after isolation from the roots of 7-day-old etiolated pea ( Pisum sativum, cv. Orlovchanin) seedlings grown at optimal temperature and those exposed to 1-day chilling at 8°C in the end of the growth period. The homogenization medium for obtaining plasma membranes was supplemented with either SH-reagents or protein phosphatase inhibitors. The plasmalemma vesicles were purified from the microsome fraction by means of two-phase polymer system. The osmotic water permeability of membrane vesicles was evaluated from the rate of their osmotically induced shrinkage. The lowering of growth temperature was accompanied by the increase in osmotic water permeability of plasmalemma. These changes occurred without the corresponding increase in aquaporin content or permeability of membrane lipid matrix. The membranes from cooled seedlings were markedly depleted in the content of SH-groups. Furthermore, the treatment of membrane samples with a thiol-reducing agent, tributylphosphine did not raise the SH-group content in membranes from chilled plants, unlike such changes in membranes from warm-grown plants. When the homogenization medium contained dithiothreitol and phenylarsine oxide (an inhibitor of tyrosine protein phosphatases), the osmotic permeability of plasmalemma in preparations from warm-grown seedlings also increased. Based on these results, it is supposed that aquaporin-mediated water permeability of membranes is regulated through different pathways under optimal and adverse conditions for plant growth. Direct action of endogenous SH redox regulators on aquaporin activity is likely under optimal growth conditions, while protein phosphatase might mediate changes in aquaporin activity under unfavorable growth conditions.