Novel Proteins of the High-Affinity Nitrate Transporter Family NRT2, SaNRT2.1 and SaNRT2.5, from the Euhalophyte Suaeda altissima: Molecular Cloning and Expression Analysis.

Two genes of nitrate transporters SaNRT2.1 and SaNRT2.5, putative orthologs of high-affinity nitrate transporter genes AtNRT2.1 and AtNRT2.5 from Arabidopsis thaliana, were cloned from the euhalophyte Suaeda altissima. Phylogenetic bioinformatic analysis demonstrated that the proteins SaNRT2.1 and SaNRT2.5 exhibited higher levels of homology to the corresponding proteins from the plants of family Amaranthaceae; the similarity of amino acid sequences between proteins SaNRT2.1 and SaNRT2.5 was lower (54%). Both SaNRT2.1 and SaNRT2.5 are integral membrane proteins forming 12 transmembrane helices as predicted by topological modeling. An attempt to demonstrate nitrate transporting activity of SaNRT2.1 or SaNRT2.5 by heterologous expression of the genes in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the only yeast nitrate transporter was not successful. The expression patterns of SaNRT2.1 and SaNRT2.5 were studied in S. altissima plants that were grown in hydroponics under either low (0.5 mM) or high (15 mM) nitrate and salinity from 0 to 750 mM NaCl. The growth of the plants was strongly inhibited by low nitrogen supply while stimulated by NaCl; it peaked at 250 mM NaCl for high nitrate and at 500 mM NaCl for low nitrate. Under low nitrate supply, nitrate contents in S. altissima roots, leaves and stems were reduced but increased in leaves and stems as salinity in the medium increased. Potassium contents remained stable under salinity treatment from 250 to 750 mM NaCl. Quantitative real-time PCR demonstrated that without salinity, SaNRT2.1 was expressed in all organs, its expression was not influenced by nitrate supply, while SaNRT2.5 was expressed exclusively in roots-its expression rose about 10-fold under low nitrate. Salinity increased expression of both SaNRT2.1 and SaNRT2.5 under low nitrate. SaNRT2.1 peaked in roots at 500 mM NaCl with 15-fold increase; SaNRT2.5 peaked in roots at 500 mM NaCl with 150-fold increase. It is suggested that SaNRT2.5 ensures effective nitrate uptake by roots and functions as an essential high-affinity nitrate transporter to support growth of adult S. altissima plants under nitrogen deficiency.

Yeast Heterologous Expression Systems for the Study of Plant Membrane Proteins.

Researchers are often interested in proteins that are present in cells in small ratios compared to the total amount of proteins. These proteins include transcription factors, hormones and specific membrane proteins. However, sufficient amounts of well-purified protein preparations are required for functional and structural studies of these proteins, including the creation of artificial proteoliposomes and the growth of protein 2D and 3D crystals. This aim can be achieved by the expression of the target protein in a heterologous system. This review describes the applications of yeast heterologous expression systems in studies of plant membrane proteins. An initial brief description introduces the widely used heterologous expression systems of the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeast Pichia pastoris. S. cerevisiae is further considered a convenient model system for functional studies of heterologously expressed proteins, while P. pastoris has the advantage of using these yeast cells as factories for producing large quantities of proteins of interest. The application of both expression systems is described for functional and structural studies of membrane proteins from plants, namely, K+- and Na+-transporters, various ATPases and anion transporters, and other transport proteins.

Chloride Channel Family in the Euhalophyte Suaeda altissima (L.) Pall: Cloning of Novel Members SaCLCa2 and SaCLCc2, General Characterization of the Family.

CLC family genes, comprising anion channels and anion/H+ antiporters, are widely represented in nearly all prokaryotes and eukaryotes. CLC proteins carry out a plethora of functions at the cellular level. Here the coding sequences of the SaCLCa2 and SaCLCc2 genes, homologous to Arabidopsis thaliana CLCa and CLCc, were cloned from the euhalophyte Suaeda altissima (L.) Pall. Both the genes cloned belong to the CLC family as supported by the presence of the key conserved motifs and glutamates inherent for CLC proteins. SaCLCa2 and SaCLCc2 were heterologously expressed in Saccharomyces cerevisiae GEF1 disrupted strain, Δgef1, where GEF1 encodes the only CLC family protein, the Cl− transporter Gef1p, in undisrupted strains of yeast. The Δgef1 strain is characterized by inability to grow on YPD yeast medium containing Mn2+ ions. Expression of SaCLCa2 in Δgef1 cells growing on this medium did not rescue the growth defect phenotype of the mutant. However, a partial growth restoration occurred when the Δgef1 strain was transformed by SaCLCa2(C544T), the gene encoding protein in which proline, specific for nitrate, was replaced with serine, specific for chloride, in the selectivity filter. Unlike SaCLCa2, expression of SaCLCc2 in Δgef1 resulted in a partial growth restoration under these conditions. Analysis of SaCLCa2 and SaCLCc2 expression in the euhalophyte Suaeda altissima (L.) Pall by quantitative real-time PCR (qRT-PCR) under different growth conditions demonstrated stimulation of SaCLCa2 expression by nitrate and stimulation of SaCLCc2 expression by chloride. The results of yeast complementation assay, the presence of both the "gating" and "proton" glutamates in aa sequences of both the proteins, as well results of the gene expression in euhalophyte Suaeda altissima (L.) Pall suggest that SaCLCa2 and SaCLCc2 function as anion/H+ antiporters with nitrate and chloride specificities, respectively. The general bioinformatic overview of seven CLC genes cloned from euhalophyte Suaeda altissima is given, together with results on their expression in roots and leaves under different levels of salinity.

Whither repeat players? Litigation experience and success in court: Evidence from Russian commercial courts.

This paper tests the party capability theory of Galanter (1974) using the universe of legal entities and their 9.8 million commercial disputes in Russia in 2012-2019. We find that repetitiveness is negatively associated with litigation success in general. We explain this by selection into litigation. Entities engaged in unfair business practices are repeatedly selected into disputes as respondents becoming repeat players in spite of themselves. We argue that repetitiveness should be decoupled from party capability and urge for conceptual revision of the notion of a repeat player in court.

Could vesicular transport of Na+ and Cl- be a feature of salt tolerance in halophytes?

Background: Halophytes tolerate external salt concentrations of 200 mm and more, accumulating salt concentrations of 500 mm and more in their shoots; some, recretohalophytes, excrete salt through glands on their leaves. Ions are accumulated in central vacuoles, but the pathway taken by these ions from the outside of the roots to the vacuoles inside the cells is poorly understood. Do the ions cross membranes through ion channels and transporters or move in vesicles, or both? Vesicular transport from the plasma membrane to the vacuole would explain how halophytes avoid the toxicity of high salt concentrations on metabolism. There is also a role for vesicles in the export of ions via salt glands. Scope and Methods: We have collected data on the fluxes of sodium and chloride ions in halophytes, based on the weight of the transporting organs and on the membrane area across which the flux occurs; the latter range from 17 nmol m-2 s-1 to 4.2 µmol m-2 s-1 and values up to 1 µmol m-2 s-1 need to be consistent with whatever transport system is in operation. We have summarized the sizes and rates of turnover of vesicles in plants, where clathrin-independent vesicles are 100 nm or more in diameter and can merge with the plasma membrane at rates of 100 s-1. We gathered evidence for vesicular transport of ions in halophytes and evaluated whether vesicular transport could account for the observable fluxes. Conclusions: There is strong evidence in favour of vesicular transport in plants and circumstantial evidence in favour of the movement of ions in vesicles. Estimated rates of vesicle turnover could account for ion transport at the lower reported fluxes (around 20 nmol m-2 s-1), but the higher fluxes may require vesicles of the order of 1 µm or more in diameter. The very high fluxes reported in some salt glands might be an artefact of the way they were measured.

In vitro phosphorylation as tool for modification of silk and keratin fibrous materials.

An overview is given of the recent work on in vitro enzymatic phosphorylation of silk fibroin and human hair keratin. Opposing to many chemical "conventional" approaches, enzymatic phosphorylation is in fact a mild reaction and the treatment falls within "green chemistry" approach. Silk and keratin are not phosphorylated in vivo, but in vitro. This enzyme-driven modification is a major technological breakthrough. Harsh chemical chemicals are avoided, and mild conditions make enzymatic phosphorylation a real "green chemistry" approach. The current communication presents a novel approach stating that enzyme phosphorylation may be used as a tool to modify the surface charge of biocompatible materials such as keratin and silk.

Phosphorylated silk fibroin matrix for methotrexate release.

Silk-based matrix was produced for delivery of a model anticancer drug, methotrexate (MTX). The calculation of net charge of silk fibroin and MTX was performed to better understand the electrostatic interactions during matrix formation upon casting. Silk fibroin films were cast at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated silk fibroin. The phosphorylation content of matrix was controlled by mixing at specific ratios the phosphorylated and unphosphorylated solutions. In vitro release profiling data suggest that the observed interactions are mainly structural and not electrostatical. The release of MTX is facilitated by use of proteolytic enzymes and higher pHs. The elevated ß-sheet content and crystallinity of the acidified-cast fibroin solution seem not to favor drug retention. All the acquired data underline the prevalence of structural interactions over electrostatical interactions between methotrexate and silk fibroin.

Molecular Cloning, Expression and Transport Activity of SaNPF6.3/SaNRT1.1, a Novel Protein of the Low-Affinity Nitrate Transporter Family from the Euhalophyte Suaeda altissima (L.) Pall.

The SaNPF6.3 gene, a putative ortholog of the dual-affinity nitrate (NO3-) transporter gene AtNPF6.3/AtNRT1.1 from Arabidopsis thaliana, was cloned from the euhalophyte Suaeda altissima. The nitrate transporting activity of SaNPF6.3 was studied by heterologous expression of the gene in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the original nitrate transporter. Expression of SaNPF6.3 in Δynt1 cells rescued their ability to grow on the selective medium in the presence of nitrate and absorb nitrate from this medium. Confocal laser microscopy of the yeast cells expressing the fused protein GFP-SaNPF6.3 revealed GFP (green fluorescent protein) fluorescence localized predominantly in the cytoplasm and/or vacuoles. Apparently, in the heterologous expression system used, only a relatively small fraction of the GFP-SaNPF6.3 reached the plasma membrane of yeast cells. In S. altissima plants grown in media with either low (0.5 mM) or high (15 mM) NO3-; concentrations, SaNPF6.3 was expressed at various ontogenetic stages in different organs, with the highest expression levels in roots, pointing to an important role of SaNPF6.3 in nitrate uptake. SaNPF6.3 expression was induced in roots of nitrate-deprived plants in response to raising the nitrate concentration in the medium and was suppressed when the plants were transferred from sufficient nitrate to the lower concentration. When NaCl concentration in the nutrient solution was elevated, the SaNPF6.3 transcript abundance in the roots increased at the low nitrate concentration and decreased at the high one. We also determined nitrate and chloride concentrations in the xylem sap excreted by detached S. altissima roots as a function of their concentrations in the root medium. Based on a linear increase in Cl- concentrations in the xylem exudate as the external Cl- concentration increased and the results of SaNPF6.3 expression experiments, we hypothesize that SaNPF6.3 is involved in chloride transport along with nitrate transport in S. altissima plants.

System analysis of the fast global coronavirus disease 2019 (COVID-19) spread. Can we avoid future pandemics under global climate change?

The recent fast global spread of COVID-19 caused by a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) questions why and how the disease managed to be so effective against existing health protection measures. These measures, developed by many countries over centuries and strengthened over the last decades, proved to be ineffective against COVID-19. The sharp increase in human longevity and current transport systems in economically developing countries with the background of persisting cultural frameworks and stable local pools of high bacterial and viral mutations generated the wide gap between the established health protection systems and the new emerging diseases. SARS-CoV-2 targets human populations over the world with long incubation periods, often without symptoms, and serious outcomes. Hence, novel strategies are necessary to meet the demands of developing economic and social environments. Moreover, the ongoing climate change adds extra challenges while altering the existing system of interactions in biological populations and in human society. Climate change may lead to new sources of viral and microbial mutations, new ways of zoonotic disease transmission and to huge social and economic transformations in many countries. The present short Opinion applies a system approach linking biomedical, climate change, social and economic aspects and, accordingly, discusses the measures and more efficient means to avoid future pandemics.

Molecular Cloning and Characterization of SaCLCd, SaCLCf, and SaCLCg, Novel Proteins of the Chloride Channel Family (CLC) from the Halophyte Suaeda altissima (L.) Pall.

Coding sequences of the CLC family genes SaCLCd, SaCLCf, and SaCLCg, the putative orthologs of Arabidopsis thaliana AtCLCd, AtCLCf, and AtCLCg genes, were cloned from the euhalophyte Suaeda altissima (L.) Pall. The key conserved motifs and glutamates inherent in proteins of the CLC family were identified in SaCLCd, SaCLCf, and SaCLCg amino acid sequences. SaCLCd and SaCLCg were characterized by higher homology to eukaryotic (human) CLCs, while SaCLCf was closer to prokaryotic CLCs. Ion specificities of the SaCLC proteins were studied in complementation assays by heterologous expression of the SaCLC genes in the Saccharomyces cerevisiae GEF1 disrupted strain Δgef1. GEF1 encoded the only CLC family protein, the Cl- transporter Gef1p, in undisrupted strains of this organism. Expression of SaCLCd in Δgef1 cells restored their ability to grow on selective media. The complementation test and the presence of both the "gating" and "proton" conservative glutamates in SaCLCd amino acid sequence and serine specific for Cl- in its selectivity filter suggest that this protein operates as a Cl-/H+ antiporter. By contrast, expression of SaCLCf and SaCLCg did not complement the growth defect phenotype of Δgef1 cells. The selectivity filters of SaCLCf and SaCLCg also contained serine. However, SaCLCf included only the "gating" glutamate, while SaCLCg contained the "proton" glutamate, suggesting that SaCLCf and SaCLCg proteins act as Cl- channels. The SaCLCd, SaCLCf, and SaCLCg genes were shown to be expressed in the roots and leaves of S. altissima. In response to addition of NaCl to the growth medium, the relative transcript abundances of all three genes of S. altissima increased in the leaves but did not change significantly in the roots. The increase in expression of SaCLCd, SaCLCf, and SaCLCg in the leaves in response to increasing salinity was in line with Cl- accumulation in the leaf cells, indicating the possible participation of SaCLCd, SaCLCf, and SaCLCg proteins in Cl- sequestration in cell organelles. Generally, these results suggest the involvement of SaCLC proteins in the response of S. altissima plants to increasing salinity and possible participation in mechanisms underlying salt tolerance.

Analysis of spliceosomal proteins in Trypanosomatids reveals novel functions in mRNA processing.

In trypanosomatids, all mRNAs are processed via trans-splicing, although cis-splicing also occurs. In trans-splicing, a common small exon, the spliced leader (SL), which is derived from a small SL RNA species, is added to all mRNAs. Sm and Lsm proteins are core proteins that bind to U snRNAs and are essential for both these splicing processes. In this study, SmD3- and Lsm3-associated complexes were purified to homogeneity from Leishmania tarentolae. The purified complexes were analyzed by mass spectrometry, and 54 and 39 proteins were purified from SmD3 and Lsm complexes, respectively. Interestingly, among the proteins purified from Lsm3, no mRNA degradation factors were detected, as in Lsm complexes from other eukaryotes. The U1A complex was purified and mass spectrometry analysis identified, in addition to U1 small nuclear ribonucleoprotein (snRNP) proteins, additional co-purified proteins, including the polyadenylation factor CPSF73. Defects observed in cells silenced for U1 snRNP proteins suggest that the U1 snRNP functions exclusively in cis-splicing, although U1A also participates in polyadenylation and affects trans-splicing. The study characterized several trypanosome-specific nuclear factors involved in snRNP biogenesis, whose function was elucidated in Trypanosoma brucei. Conserved factors, such as PRP19, which functions at the heart of every cis-spliceosome, also affect SL RNA modification; GEMIN2, a protein associated with SMN (survival of motor neurons) and implicated in selective association of U snRNA with core Sm proteins in trypanosomes, is a master regulator of snRNP assembly. This study demonstrates the existence of trypanosomatid-specific splicing factors but also that conserved snRNP proteins possess trypanosome-specific functions.

Stabilizing RNA by the sonochemical formation of RNA nanospheres.

Biological macromolecules, including DNA, RNA, and proteins, have intrinsic features that make them potential building blocks for the bottom-up fabrication of nanodevices. Unlike DNA, RNA is a more versatile molecule whose range in the cell is from 21 to thousands of nucleotides and is usually folded into stem and loop structures. RNA is unique in nanoscale fabrication due to its diversity in size, function, and structure. Because gene expression analysis is becoming a clinical reality and there is a need to collect RNA in minute amounts from clinical samples, keeping the RNA intact is a growing challenge. RNA samples are notoriously difficult to handle because of their highly labile nature and tendency to degrade even under controlled RNase-free conditions and maintenance in the cold. Silencing the RNA that induces the RNA interference is viewed as the next generation of therapeutics. The stabilization and delivery of RNA to cells are the major concerns in making siRNAs usable drugs. For the first time, ultrasonic waves are shown to convert native RNA molecules to RNA nanospheres. The creation of the nanobubbles is performed by a one-step reaction. The RNA nanospheres are stable at room temperature for at least one month. Additionally, the nanospheres can be inserted into mammalian cancer cells (U2OS). This research achieves: 1) a solution to RNA storage; and 2) a way to convert RNA molecules to RNA particles. RNA nanosphere formation is a reversible process, and by using denaturing conditions, the RNA can be refolded into intact molecules.

Cloning and Characterization of Two Putative P-Type ATPases from the Marine Microalga Dunaliella maritima Similar to Plant H+-ATPases and Their Gene Expression Analysis under Conditions of Hyperosmotic Salt Shock.

The green microalga genus Dunaliella is mostly comprised of species that exhibit a wide range of salinity tolerance, including inhabitants of hyperhaline reservoirs. Na+ content in Dunaliella cells inhabiting saline environments is maintained at a fairly low level, comparable to that in the cells of freshwater organisms. However, despite a long history of studying the physiological and molecular mechanisms that ensure the ability of halotolerant Dunaliella species to survive at high concentrations of NaCl, the question of how Dunaliella cells remove excess Na+ ions entering from the environment is still debatable. For thermodynamic reasons it should be a primary active mechanism; for example, via a Na+-transporting ATPase, but the molecular identification of Na+-transporting mechanism in Dunaliella has not yet been carried out. Formerly, in the euryhaline alga D. maritima, we functionally identified Na+-transporting P-type ATPase in experiments with plasma membrane (PM) vesicles which were isolated from this alga. Here we describe the cloning of two putative P-type ATPases from D. maritima, DmHA1 and DmHA2. Phylogenetic analysis showed that both ATPases belong to the clade of proton P-type ATPases, but the similarity between DmHA1 and DmHA2 is not high. The expression of DmHA1 and DmHA2 in D. maritima cells under hyperosmotic salt shock was studied by qRT-PCR. Expression of DmHA1 gene decreases and remains at a relatively low level during the response of D. maritima cells to hyperosmotic salt shock. In contrast, expression of DmHA2 increases under hyperosmotic salt shock. This indicates that DmHA2 is important for overcoming hyperosmotic salt stress by the algal cells and as an ATPase it is likely directly involved in transport of Na+ ions. We assume that it is the DmHA2 ATPase that represents the Na+-transporting ATPase.

Seroprevalence of SARS-CoV-2 antibodies in Saint Petersburg, Russia: a population-based study.

Properly conducted serological survey can help determine infection disease true spread. This study aims to estimate the seroprevalence of SARS-CoV-2 antibodies in Saint Petersburg, Russia accounting for non-response bias. A sample of adults was recruited with random digit dialling, interviewed and invited for anti-SARS-CoV-2 antibodies. The seroprevalence was corrected with the aid of the bivariate probit model that jointly estimated individual propensity to agree to participate in the survey and seropositivity. 66,250 individuals were contacted, 6,440 adults agreed to be interviewed and blood samples were obtained from 1,038 participants between May 27 and June 26, 2020. Naïve seroprevalence corrected for test characteristics was 9.0% (7.2-10.8) by CMIA and 10.5% (8.6-12.4) by ELISA. Correction for non-response decreased estimates to 7.4% (5.7-9.2) and 9.1% (7.2-10.9) for CMIA and ELISA, respectively. The most pronounced decrease in bias-corrected seroprevalence was attributed to the history of any illnesses in the past 3 months and COVID-19 testing. Seroconversion was negatively associated with smoking status, self-reported history of allergies and changes in hand-washing habits. These results suggest that even low estimates of seroprevalence can be an overestimation. Serosurvey design should attempt to identify characteristics that are associated both with participation and seropositivity.

A Quest for Mechanisms of Plant Root Exudation Brings New Results and Models, 300 Years after Hales.

The review summarizes some of our current knowledge on the phenomenon of exudation from the cut surface of detached roots with emphasis on results that were mostly established over the last fifty years. The phenomenon is quantitatively documented in the 18th century (by Hales in 1727). By the 19th century, theories mainly ascribed exudation to the secretion of living root cells. The 20th century favored the osmometer model of root exudation. Nevertheless, growing insights into the mechanisms of water transport and new or rediscovered observations stimulated the quest for a more adequate exudation model. The historical overview shows how understanding of exudation changed with time following experimental opportunities and novel ideas from different areas of knowledge. Later theories included cytoskeleton-dependent micro-pulsations of turgor in root cells to explain the observed water exudation. Recent progress in experimental biomedicine led to detailed study of channels and transporters for ion transport via cellular membranes and to the discovery of aquaporins. These universal molecular entities have been incorporated to the more complex models of water transport via plant roots. A new set of ideas and explanations was based on cellular osmoregulation by mechanosensitive ion channels. Thermodynamic calculations predicted the possibility of water transport against osmotic forces based on co-transport of water with ions via cation-chloride cotransporters. Recent observations of rhizodermis exudation, exudation of roots without an external aqueous medium, segments cut from roots, pulses of exudation, a phase shifting of water uptake and exudation, and of effects of physiologically active compounds (like ion channel blockers, metabolic agents, and cytoskeletal agents) will likely refine our understanding of the phenomenon. So far, it seems that more than one mechanism is responsible for root pressure and root exudation, processes which are important for refilling of embolized xylem vessels. However, recent advances in ion and water transport research at the molecular level suggest potential future directions to understanding of root exudation and new models awaiting experimental testing.

Electrophysiological characterization of pathways for K(+) uptake into growing and non-growing leaf cells of barley.

Potassium is a major osmolyte used by plant cells. The accumulation rates of K(+) in cells may limit the rate of expansion. In the present study, we investigated the involvement of ion channels in K(+) uptake using patch clamp technique. Ion currents were quantified in protoplasts of the elongation and emerged blade zone of the developing leaf 3 of barley (Hordeum vulgare L.). A time-dependent inward-rectifying K(+)-selective current was observed almost exclusively in elongation zone protoplasts. The current showed characteristics typical of Shaker-type channels. Instantaneous inward current was highest in the epidermis of the emerged blade and selective for Na(+) over K(+). Selectivity disappeared, and currents decreased or remained the same, depending on tissue, in response to salt treatment. Net accumulation rates of K(+) in cells calculated from patch clamp current-voltage curves exceeded rates calculated from membrane potential and K(+) concentrations of cells measured in planta by factor 2.5-2.7 at physiological apoplastic K(+) concentrations (10-100 mm). It is concluded that under these conditions, K(+) accumulation in growing barley leaf cells is not limited by transport properties of cells. Under saline conditions, down-regulation of voltage-independent channels may reduce the capacity for growth-related K(+) accumulation.

Potassium channels in barley: cloning, functional characterization and expression analyses in relation to leaf growth and development.

It is not known how the uptake and retention of the key osmolyte K(+) in cells are mediated in growing leaf tissue. In the present study on the growing leaf 3 of barley, we have cloned the full-length coding sequence of three genes which encode putative K(+) channels (HvAKT1, HvAKT2, HvKCO1/HvTPK1), and of one gene which encodes a putative K(+) transporter (HvHAK4). The functionality of the gene products of HvAKT1 and HvAKT2 was tested through expression in Xenopus laevis oocytes. Both are inward-rectifying K(+) channels which are inhibited by Cs(+). Function of HvAKT1 in oocytes requires co-expression of a calcineurin-interacting protein kinase (AtCIPK23) and a calcineurin B-like protein (AtCBL9) from Arabidopsis, showing cross-species complementation of function. In planta, HvAKT1 is expressed primarily in roots, but is also expressed in leaf tissue. HvAKT2 is expressed particularly in leaf tissue, and HvHAK4 is expressed particularly in growing leaf tissue. Within leaves, HvAKT1 and HvAKT2 are expressed predominantly in mesophyll. Expression of genes changes little in response to low external K(+) or salinity, despite major changes in K(+) concentrations and osmolality of cells. Possible contributions of HvAKT1, HvAKT2, HvKCO1 and HvHAK4 to regulation of K(+) relations of growing barley leaf cells are discussed.

Enzymatic phosphorylation of hair keratin enhances fast adsorption of cationic moieties.

The current study describes the in vitro phosphorylation of a human hair keratin, using protein kinase for the first time. Phosphorylation of keratin was demonstrated by (31)P NMR (Nuclear Magnetic Resonance) and Diffuse Reflectance Infrared Fourier Transform (DRIFT) techniques. Phosphorylation induced a 2.5 fold increase of adsorption capacity in the first 10 min for cationic moiety like methylene blue (MB). Thorough description of MB adsorption process was performed by several isothermal models. Reconstructed fluorescent microscopy images depict distinct amounts of dye bound to the differently treated hair. The results of this work suggest that the enzymatic phosphorylation of keratins might have significant implications in hair shampooing and conditioning, where short application times of cationic components are of prime importance.

BSA/HSA ratio modulates the properties of Ca(2+)-induced cold gelation scaffolds.

An effective tissue engineering approach requires adjustment according to the target tissue to be engineered. The possibility of obtaining a protein-based formulation for the development of multivalent tunable scaffolds that can be adapted for several types of cells and tissues is explored in this work. The incremental substitution of bovine serum albumin (BSA) by human serum albumin (HSA), changing the scaffolds' hydrophilic/hydrophobic ratio, on a previously optimized scaffold formulation resulted in a set of uniform porous scaffolds with different physical properties and associated cell proliferation profile along time. There was a general trend towards an increase in hydrophilicity, swelling degree and in vitro degradation of the scaffolds with increasing replacement of BSA by HAS. The set of BSA/HSA scaffolds presented distinct values for the storage (elastic) modulus and loss factor which were similar to those described for different native tissues such as bone, cartilage, muscle, skin and neural tissue. The preferential adhesion and proliferation of skin fibroblasts on the BSA25%HSA75% and HSA100% scaffolds, as predicted by their viscoelastic properties, demonstrate that the BSA/HSA scaffold formulation is promising for the development of scaffolds that can be tuned according to the tissue to be repaired and restored.

Salinity tolerance in plants. Quantitative approach to ion transport starting from halophytes and stepping to genetic and protein engineering for manipulating ion fluxes.

Ion transport is the fundamental factor determining salinity tolerance in plants. The Review starts from differences in ion transport between salt tolerant halophytes and salt-sensitive plants with an emphasis on transport of potassium and sodium via plasma membranes. The comparison provides introductory information for increasing salinity tolerance. Effects of salt stress on ion transport properties of membranes show huge opportunities for manipulating ion fluxes. Further steps require knowledge about mechanisms of ion transport and individual genes of ion transport proteins. Initially, the Review describes methods to measure ion fluxes, the independent set of techniques ensures robust and reliable basement for quantitative approach. The Review briefly summarizes current data concerning Na(+) and K(+) concentrations in cells, refers to primary thermodynamics of ion transport and gives special attention to individual ion channels and transporters. Simplified scheme of a plant cell with known transport systems at the plasma membrane and tonoplast helps to imagine the complexity of ion transport and allows choosing specific transporters for modulating ion transport. The complexity is enhanced by the influence of cell size and cell wall on ion transport. Special attention is given to ion transporters and to potassium and sodium transport by HKT, HAK, NHX, and SOS1 proteins. Comparison between non-selective cation channels and ion transporters reveals potential importance of ion transporters and the balance between the two pathways of ion transport. Further on the Review describes in detail several successful attempts to overexpress or knockout ion transporters for changing salinity tolerance. Future perspectives are questioned with more attention given to promising candidate ion channels and transporters for altered expression. Potential direction of increasing salinity tolerance by modifying ion channels and transporters using single point mutations is discussed and questioned. An alternative approach from synthetic biology is to create new regulation networks using novel transport proteins with desired properties for transforming agricultural crops. The approach had not been widely used earlier; it leads also to theoretical and pure scientific aspects of protein chemistry, structure-function relations of membrane proteins, systems biology and physiology of stress and ion homeostasis. Summarizing, several potential ways are aimed at required increase in salinity tolerance of plants of interest.