The rapidly expanding CREC protein family: members,localization, function,and role in disease

Although many aspects of the physiological and pathophysiological mechanisms remain unknown, recent advances in our knowledge suggest that the CREC proteins are promising disease biomarkers or targets for therapeutic intervention in a variety of diseases. The CREC family of low affinity, Ca²⁺‐binding, multiple EF‐hand proteins are encoded by five genes, RCN1, RCN2, RCN3, SDF4, and CALU, resulting in reticulocalbin, ER Ca²⁺‐binding protein of 55 kDa (ERC‐55), reticulocalbin‐3, Ca²⁺‐binding protein of 45 kDa (Cab45), and calumenin. Alternative splicing increases the number of gene products. The proteins are localized in the cytosol, in various parts of the secretory pathway, secreted to the extracellular space or localized on the cell surface. The emerging functions appear to be highly diverse. The proteins interact with several different ligands. Rather well‐described functions are attached to calumenin with the inhibition of several proteins in the endoplasmic or sarcoplasmic reticulum membrane, the vitamin K₁ 2,3‐epoxide reductase, the γ‐carboxylase, the ryanodine receptor, and the Ca²⁺‐transporting ATPase. Other functions concern participation in the secretory process, chaperone activity, signal transduction as well as participation in a large variety of disease processes.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

Rise of intracellular Ca2+ level causes the decrease of cyclin B1 and Mos in the newt eggs at fertilization

Unfertilized eggs of the newt, Cynops pyrrhogaster, are arrested at the second meiotic metaphase, with activity of the M‐phase promoting factor (MPF) maintained at a high level. After fertilization, the eggs resume the cell cycle, and emit the second polar body. When the change in [Ca²⁺]_i in the fertilized eggs was monitored by aequorin, an early increase in [Ca²⁺]_i was observed 5–10 min after insemination and continued for about 30 sec. A late increase in [Ca²⁺]_i then occurred 10–15 min after fertilization and continued for 30–40 min. The injection of 1,2‐Bis (2 aminophenoxy) ethane‐N,N,N′,N′,‐tetraacetic acid (BAPTA) into unfertilized eggs inhibited reinitiation of the cell cycle after fertilization. Western blot analysis with antibodies against cyclin B1 or Mos indicated that both cyclin B1 and Mos were present in unfertilized eggs, but both disappeared within 30 min after fertilization. Treatment with Ca²⁺‐ionophore decreased both cyclin B1 and Mos. Chymotryptic activity in Cynops egg extracts was not significantly increased after fertilization or activation by treatment with the Ca²⁺‐ionophore. No change in [Ca²⁺]_i was observed following treatment with cycloheximide, but the amount of both cyclin B1 and Mos rapidly decreased. These results indicate that resumption of meiosis in Cynops eggs is induced by an increase in [Ca²⁺]_i at fertilization, which causes degradation of both cyclin B1 and Mos by inhibition of de novo synthesis of those proteins. Mol. Reprod. Dev. 53:341–349, 1999. © 1999 Wiley‐Liss, Inc.     

Modular architecture of Munc13/calmodulin complexes: dual regulation by Ca2+ and possible function in short‐term synaptic plasticity

Ca²⁺ signalling in neurons through calmodulin (CaM) has a prominent function in regulating synaptic vesicle trafficking, transport, and fusion. Importantly, Ca²⁺–CaM binds a conserved region in the priming proteins Munc13‐1 and ubMunc13‐2 and thus regulates synaptic neurotransmitter release in neurons in response to residual Ca²⁺ signals. We solved the structure of Ca²⁺₄–CaM in complex with the CaM‐binding domain of Munc13‐1, which features a novel 1‐5‐8‐26 CaM‐binding motif with two separated mobile structural modules, each involving a CaM domain. Photoaffinity labelling data reveal the same modular architecture in the complex with the ubMunc13‐2 isoform. The N‐module can be dissociated with EGTA to form the half‐loaded Munc13/Ca²⁺₂–CaM complex. The Ca²⁺ regulation of these Munc13 isoforms can therefore be explained by the modular nature of the Munc13/Ca²⁺–CaM interactions, where the C‐module provides a high‐affinity interaction activated at nanomolar [Ca²⁺]_i, whereas the N‐module acts as a sensor at micromolar [Ca²⁺]_i. This Ca²⁺/CaM‐binding mode of Munc13 likely constitutes a key molecular correlate of the characteristic Ca²⁺‐dependent modulation of short‐term synaptic plasticity.     

Characterization of the Antiviral Activity for Influenza Viruses M1 Zinc Finger Peptides

We sought to investigate the cellular uptake and antiviral activity for the M1 zinc finger peptides derived from influenza A and influenza B viruses in vitro. No cellular uptake was detected by fluorescent microscopy for the synthetic zinc finger peptides. When flanked to a cell permeable peptide Tp10, the zinc finger recombinant proteins were efficiently internalized by MDCK cells. However, no antiviral activity was detected against homologous or heterologous virus infections for the synthetic peptides or the Tp10-flanked recombinant proteins, regardless treated with or without Zn²⁺. Nevertheless, MDCK cell constitutively expressing the M1 zinc finger peptides in cell nuclei potently inhibited replication of homologous, but not heterologous influenza viruses. Adenoviral vector delivered M1 zinc finger peptides also exhibited potent antiviral activity against homologous viruses challenge. Transduction at 100 PFU dose of recombinant adenovirus efficiently protected 99% of the cells from 100 TCID₅₀ of different virus infections for both peptides. These results brought new insight to the antiviral researches against influenza virus infections.     

Specific ion effects on the particle size distributions of cell culture–derived influenza A virus particles within the Hofmeister series

Virus particle (VP) aggregation can have serious implications on clinical safety and efficacy of virus‐based therapeutics. Typically, VP are suspended in buffers to establish defined product properties. Salts used to achieve these properties show specific effects in chemical and biological systems in a reoccurring trend known as Hofmeister series (HS). Hofmeister series effects are ubiquitous and can affect colloidal particle systems. In this study, influences of different ions (anions: SO₄^2?, HPO₄^2?, Cl^?, Br^?, NO₃^?, I^?; cations: K⁺, Na⁺, Li⁺, Mg²⁺, Ca²⁺) on particle size distributions of cell culture‐derived influenza VP were investigated. For the experimental setup, influenza virus A/Puerto Rico/8/34 (H1N1) VP produced in adherent and suspension Madin Darby canine kidney cells were used. Inactivated and concentrated virus harvests were dialyzed against buffers containing the ions of interest, followed by differential centrifugal sedimentation to measure particle size distributions. VP from both cell lines showed no aggregation over a wide range of buffers containing different salts in concentrations ≥60 mM. However, when dialyzed to low salt or Ca²⁺ buffers, VP produced in adherent cells showed increased aggregation compared to VP produced in suspension cells. Additionally, changes in VP diameters depending on specific ion concentrations were observed that partially reflected the HS trend.     

Antiviral activity of Distictella elongata (Vahl) Urb. (Bignoniaceae), a potentially useful source of anti-dengue drugs from the state of Minas Gerais, Brazil

Aims: To investigate the in vitro antiviral activity of Distictella elongata (Vahl) Urb. ethanol extracts from leaves (LEE), fruits (FEE), stems and their main components. Methods and Results: The antiviral activity was evaluated against human herpesvirus type 1 (HSV‐1), murine encephalomyocarditis virus (EMCV), vaccinia virus Western Reserve (VACV‐WR) and dengue virus 2 (DENV‐2) by the 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) colorimetric assay. LEE presented anti‐HSV‐1 [EC₅₀ 142·8 ± 5·3 μg ml^?1; selectivity index (SI) 2·0] and anti‐DENV‐2 activity (EC₅₀ 9·8 ± 1·3 μg ml^?1; SI 1·5). The pectolinarin ( 1 ) isolated from LEE was less active against HSV‐1 and DENV‐2. A mixture of the triterpenoids ursolic, pomolic and oleanolic acids was also obtained. Ursolic and oleanolic acids have shown antiviral activity against HSV‐1. A mixture of pectolinarin ( 1 ) and acacetin‐7‐O‐rutinoside ( 2 ) was isolated from FEE and has presented anti‐DENV‐2 activity (EC₅₀ 11·1 ± 1·6 μg ml^?1; SI > 45). Besides the antiviral activity, D. elongata has disclosed antioxidant effect. Conclusions: These data shows that D. elongata has antiviral activity mainly against HSV‐1 and DENV‐2, besides antioxidant activity. These effects might be principally attributed to flavonoids isolated. Significance and Impact of the Study: Distictella elongata might be considered a promising source of anti‐dengue fever phytochemicals.     

拟南芥钙调素定点突变基因分离及其在钙不依赖钙调素结合蛋白检测中的应用

动植物系统研究表明,钙调素不仅在结合钙离子时调节多种靶酶或靶蛋白的活性,而且没有钙离子结合时,还可以通过结合钙不依赖的钙调素结合蛋白,发挥多种生物学作用．然而,目前却没有体内分析钙调素与钙不依赖钙调素结合蛋白相互作用的方法．首先,采用定点突变的方式,得到了拟南芥钙调素亚型2的多个突变基因mCaM2,随后,大肠杆菌重组表达突变蛋白的电泳迁移率及⁴⁵Ca²⁺覆盖分析表明,得到了编码失去钙结合能力的钙调素的突变基因mCaM2₁₂₃₄, mCaM2₁₂₃₄突变钙调素中所有4个钙结合EF-hand结构域中的关键氨基酸谷氨酸均突变为谷氨酰胺．在酵母双杂交体系中,作为诱饵蛋白的突变钙调素mCaM2₁₂₃₄与我们前期体外方法报道的钙不依赖性钙调素结合蛋白AtIQD26存在相互作用．这将为钙不依赖性钙调素结合蛋白提供有用的体内研究工具,有利于我们全面认识钙-钙调素-钙调素结合蛋白信号途径．     

Novel Daidzein Analogs and Their in Vitro Anti‐Influenza Activities

A series of novel isoflavonoids were synthesized based on structural modifications of daidzein, an active ingredient of traditional Chinese medicine (TCM) and evaluated for their anti‐influenza activity, in vitro, against H1N1 Tamiflu‐resistant (H1N1 TR) virus in the MDCK cell line. Among them, 4‐oxo‐4H‐1‐benzopyran‐8‐carbaldehydes 11a – 11g were most promising, and they demonstrated better activities and selectivities comparable to those the reference ribarivin, a nucleoside antiviral agent. 3‐(4‐Bromophenyl)‐7‐hydroxy‐4‐oxo‐4H‐1‐benzopyran‐8‐carboxaldehyde ( 11c ) displayed the best inhibitory activity (EC₅₀, 29.0 μM ) and selectivity index (SI>10.3). Analysis of the structure? activity relationships (SAR) indicated that both the non‐naturally‐occurring Br‐substituted B‐ring and appropriate CHO and OH groups on the A‐ring might be critical for the activity and selectivity against H1N1 TR influenza viruses.     

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca²⁺] ([Ca²⁺]_i), and also on mechanisms that are independent of [Ca²⁺]_i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca²⁺]_i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca²⁺]_i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca²⁺ sensitivity of anion and inward‐rectifying K⁺ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca²⁺]_i elevations and clamping [Ca²⁺]_i to resting levels. The absence of [Ca²⁺]_i elevations was confirmed by ratiometric [Ca²⁺]_i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca²⁺]_i elevations above the physiological resting [Ca²⁺]_i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca²⁺]_i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca²⁺]_i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca²⁺ to activate S‐type anion channels and down‐regulate inward‐rectifying K⁺ channels, providing strong evidence for a Ca²⁺ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca²⁺]_i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca²⁺]_i sensitivity of stomatal closure mechanisms.     

Recombinant Influenza Virus Carrying the Respiratory Syncytial Virus (RSV) F85-93 CTL Epitope Reduces RSV Replication in Mice

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants worldwide. Despite decades of research, there is still no registered vaccine available for this major pathogen. We investigated the protective efficacy of a recombinant influenza virus, PR8/NA-F_85–93, that carries the RSV CD8⁺ T cell epitope F_85–93 in its neuraminidase stalk. F_85–93-specific cytotoxic T lymphocytes (CTLs) were induced in mice after a single intranasal immunization with PR8/NA-F_85-93 virus, and these CTLs provided a significant reduction in the lung viral load upon a subsequent challenge with RSV. To avoid influenza-induced morbidity, we treated mice with matrix protein 2 (M2e)-specific monoclonal antibodies before PR8/NA-F_85-93 virus infection. Treatment with anti-M2e antibodies reduced the infiltration of immune cells in the lungs upon PR8/NA-F_85-93 infection, whereas the formation of inducible bronchus-associated lymphoid tissue was not affected. Moreover, this treatment prevented body weight loss yet still permitted the induction of RSV F-specific T cell responses and significantly reduced RSV replication upon challenge. These results demonstrate that it is possible to take advantage of the infection-permissive protection of M2e-specific antibodies against influenza A virus to induce heterologous CD8⁺ T cell-mediated immunity by an influenza A virus vector expressing the RSV F_85-93 epitope.     

Highly efficient antibacterial diblock copolypeptides based on lysine and phenylalanine

A series of amphiphilic diblock copolypeptides (K₃₀‐b ‐F₁₅, K₃₀‐b ‐F₃₀, and K₃₀‐b ‐F₄₅) were synthesized via N ‐carboxy‐α‐amino‐anhydride ring‐opening polymerization. The copolypeptides had excellent antibacterial efficacy to both Gram positive (S. aureus ) and Gram negative (E. coli ) bacteria. The minimum inhibitory concentrations (MICs) against E. coli and S. aureus are 8 μg mL^?1 and 2 μg mL^?1, respectively, lower than most natural and artificial antimicrobial peptides (AMPs). The morphological changes of the bacteria treated with diblock copolypeptides were investigated by transmission electron microscopy; the results proved that the diblock copolypeptides had a similar antibacterial pore‐forming mechanism to natural cationic peptides. This was confirmed by laser scanning confocal microscope images. CCK‐8 results and the MICs showed that the diblock copolypeptides have high selectivity to bacteria, which suggested that the diblock copolypeptides could be excellent candidates to replace traditional antibiotics in future.     

Identification of a novel compound targeting the nuclear export of influenza A virus nucleoprotein

Although antiviral drugs are available for the treatment of influenza infection, it is an urgent requirement to develop new antiviral drugs regarding the emergence of drug‐resistant viruses. The nucleoprotein (NP) is conserved among all influenza A viruses (IAVs) and has no cellular equivalent. Therefore, NP is an ideal target for the development of new IAV inhibitors. In this study, we identified a novel anti‐influenza compound, ZBMD‐1, from a library of 20,000 compounds using cell‐based influenza A infection assays. We found that ZBMD‐1 inhibited the replication of H1N1 and H3N2 influenza A virus strains in vitro, with an IC₅₀ ranging from 0.41–1.14 μM. Furthermore, ZBMD‐1 inhibited the polymerase activity and specifically impaired the nuclear export of NP. Further investigation indicated that ZBMD‐1 binds to the nuclear export signal 3 (NES3) domain and the dimer interface of the NP pocket. ZBMD‐1 also protected mice that were challenged with lethal doses of A/PR/8/1934 (H1N1) virus, effectively relieving lung histopathology changes, as well as strongly inhibiting the expression of pro‐inflammatory cytokines/chemokines, without inducing toxicity effects in mice. These results suggest that ZBMD‐1 is a promising anti‐influenza compound which can be further investigated as a useful strategy against IAVs in the future.     

Pharmacological characterization of a Bombyx mori α‐adrenergic‐like octopamine receptor stably expressed in a mammalian cell line

Series of agonists and antagonists were examined for their actions on a Bombyx moriα‐adrenergic‐like octopamine receptor (OAR) stably expressed in HEK‐293 cells. The rank order of potency of the agonists was clonidine>naphazoline>tolazoline in Ca²⁺ mobilization assays, and that of the antagonists was chlorpromazine>yohimbine. These findings suggest that the B. mori OAR is more closely related to the class‐1 OAR in the intact tissue than to the other classes. N′‐(4‐Chloro‐o‐tolyl)‐N‐methylformamidine (DMCDM) and 2‐(2,6‐diethylphenylimino)imidazolidine (NC‐5) elevated the intracellular calcium concentration ([Ca²⁺]_i) with EC₅₀s of 92.8 µM and 15.2 nM, respectively. DMCDM and NC‐5 led to increases in intracellular cAMP concentration ([cAMP]_i) with EC₅₀s of 234 nM and 125 nM, respectively. The difference in DMCDM potencies between the cAMP and Ca²⁺ assays might be due to “functional selectivity.” The Ca²⁺ and cAMP assay results for DMCDM suggest that the elevation of [cAMP]_i, but not that of [Ca²⁺]_i, might account for the insecticidal effect of formamidine insecticides. © 2009 Wiley Periodicals, Inc.     

Synthesis of Camphecene and Cytisine Conjugates Using Click Chemistry Methodology and Study of Their Antiviral Activity

A series of camphecene and quinolizidine alkaloid (?)‐cytisine conjugates has been obtained for the first time using ‘click’ chemistry methodology. The cytotoxicity and virus‐inhibiting activity of compounds were determined against MDCK cells and influenza virus A/Puerto Rico/8/34 (H1N1), correspondingly, in in vitro tests. Based on the results obtained, values of 50 % cytotoxic dose (CC₅₀), 50 % inhibition dose (IC₅₀) and selectivity index (SI) were determined for each compound. It has been shown that the antiviral activity is affected by the length and nature of linkers between cytisine and camphor units. Conjugate 13 ((1R,5S)‐3‐(6‐{4‐[(2‐{(E)‐[(1R,4R)‐1,7,7‐trimethylbicyclo[2.2.1]heptan‐2‐ylidene]amino}ethoxy)methyl]‐1H‐1,2,3‐triazol‐1‐yl}hexyl)‐1,2,3,4,5,6‐hexahydro‐8H‐1,5‐methanopyrido[1,2‐a][1,5]diazocin‐8‐one), which contains cytisine fragment separated from triazole ring by –C₆H₁₂– aliphatic linker, showed the highest activity at relatively low toxicity (CC₅₀=168 μmol, IC₅₀=8 μmol, SI=20). Its selectivity index appeared higher than that of reference compound, rimantadine. According to theoretical calculations, the antiviral activity of the lead compound 13 can be explained by its influence on the functioning of neuraminidase.     

Synthesis of Novel Chiral Sulfonamide‐Bearing 1,2,4‐Triazole‐3‐thione Analogs Derived from D‐ and L‐Phenylalanine Esters as Potential Anti‐Influenza Agents

Novel enantiopure 1,2,4‐trizole‐3‐thiones containing a benzensulfonamide moiety were synthesized via multistep reaction sequence starting with D‐phenylalanine methyl ester and L‐phenylalanine ethyl ester as a source of chirality. The chemical structures of all compounds were characterized by elemental analysis, UV, IR, ¹H NMR, ¹³C NMR, 2D NMR (HETCOR), and mass spectral data. All compounds were tested in vitro antiviral activity against a broad variety of DNA and RNA viruses and in vitro cytostatic activity against murine leukemia (L1210), human T‐lymphocyte (CEM) and human cervix carcinoma (HeLa) cell lines. Although enantiopure 1,2,4‐triazole‐3‐thione analogs in (R) configuration emerged as promising anti‐influenza A H1N1 subtype in Madin Darby canine kidney cell cultures (MDCK), their enantiomers exhibited no activity. Especially compounds 18a , 21a , 22a , 23a , and 24a (EC₅₀: 6.5, 6.1, 2.4, 1.6, 1.7 μM, respectively) had excellent activity against influenza A H1N1 subtype compared to the reference drug ribavirin (EC₅₀: 8.0 μM). Several compounds have been found to inhibit proliferation of L1210, CEM and HeLa cell cultures with IC₅₀ in the 12–53 μM range. Compound 5a and 27a in (R) configuration were the most active compounds (IC₅₀: 12–22 μM for 5a and IC₅₀: 19–23 μM for 27a ). Chirality 28:495–513, 2016. © 2016 Wiley Periodicals, Inc.     

Quantitative cortical synapse proteomics of a transgenic migraine mouse model with mutated CaV2.1 calcium channels

Familial hemiplegic migraine type 1 (FHM1) is caused by missense mutations in the CACNA1A gene that encodes the α1A pore‐forming subunit of Ca_V2.1 Ca²⁺ channels. Knock‐in (KI) transgenic mice expressing Ca_V2.1 Ca²⁺ channels with a human pathogenic FHM1 mutation reveal enhanced glutamatergic neurotransmission in the cortex. In this study, we employed an iTRAQ‐based LC‐LC MS/MS approach to identify differentially expressed proteins in cortical synapse proteomes of Cacna1a R192Q KI and wild‐type mice. All expression differences determined were subtle and in the range of 10–30%. Observed upregulated proteins in the mutant mice are involved in processes, such as neurite outgrowth and actin dynamics, vesicle turnover, and glutamate transporters. Our data support the view that in Cacna1a R192Q KI mice, several compensatory mechanisms counterbalancing a dysregulated glutamatergic signaling have come into effect. We propose that such adaptation mechanisms at the synapse level may play a role in the pathophysiology of FHM and possibly in the common forms of migraine.     

Calcium supply effects on wheat cultivars differing in salt resistance with special reference to leaf cytosol ion homeostasis

Salinity causes changes in cytosolic Ca²⁺, [Ca²⁺]_cyt, Na⁺, [Na⁺]_cyt and pH, pH_cyt, which induce specific reactions and signals. Reactions causing a rebalancing of the physiological homeostasis of the cytosol could result in plant resistance and growth. Two wheat cultivars, Triticum aestivum, Seds1 and Vinjett, were grown in nutrient solution for 7 days under moderate salinity (0 and 50 mM NaCl) with and without extra addition of 5 mM CaSO₄ to investigate the seedling‐ion homeostasis under salinity. In the leaf protoplasts [Ca²⁺]_cyt, [Na⁺]_cyt and pH_cyt were detected using acetoxymethyl esters of the ion‐specific dyes, Fura 2, SBFI and BCECF, respectively, and fluorescence microscopy. In addition, both cultivars were grown for 3 weeks at 0, 50 and 125 mM NaCl with, or without, extra addition of 5 mM CaSO₄ to detect overall Na⁺ and Ca²⁺ concentrations in leaves and salinity effects on dry weights. In both cultivars, salinity decreased [Ca²⁺]_cyt, while at extra Ca²⁺ supplied, [Ca²⁺]_cyt increased. The [Ca²⁺]_cyt increase was accompanied by increase in the overall Ca²⁺ concentrations in leaves and decrease in the overall Na⁺ concentration. Moreover, irrespective of Ca²⁺ treatment under salinity, the cultivars reacted in different ways; [Na⁺]_cyt significantly increased only in cv. Vinjett, while pH_cyt increased only in cv. Seds1. Even at rather high total Na⁺ concentrations, the cytosolic concentrations were kept low in both cultivars. It is discussed whether the increase of [Ca²⁺]_cyt and pH_cyt can contribute to salt tolerance and if the cytosolic changes are due to changes in overall Ca²⁺ and Na⁺ concentrations.     

Analysis of the sensing and transducing processes implicated in the stomatal responses to carbon dioxide in Commelina communis L.

It has been previously debated whether CO₂ would depolarize the guard cell plasma membrane through malate‐mediated activation of the anion channel. Moreover, it has been assessed that the CO₂ signal would be transduced via cytosolic free Ca^{2 +} . In the present study, the CO₂ sensing and transducing processes were reinvestigated in Commelina communis (L.) mainly by studying how L ‐(–)‐malic acid and Ca^{2 +} flux modulators affected different CO₂ stomatal responses. L ‐(–)‐malic acid (1 m M ) inhibited by about 50% both CO₂‐induced stomatal closing and CO₂‐triggered inhibition of the stomatal opening response to the auxin indolyl‐3‐butyric acid. Stomatal closing in response to atmospheric CO₂ was strongly inhibited by the 1,4 dihydropyridines SDZ‐202 791 R(–) (SDZ (–)) and nifedipine, and this inhibition was attenuated by the 1,4 dihydropyridines SDZ‐202 791 S( + ) and S‐(–)‐BAY K8644. Suboptimal concentrations of the slow anion channel blockers 5‐nitro‐2,3‐phenylpropyllamine benzoic acid and anthracene‐9‐carboxylic acid increased the 50% inhibition of the CO₂ closing response by the Ca^{2 +} flux modulators SDZ (–) and 1,2‐bis(o‐aminophenoxy)ethane‐N,N,N ′ N ′ ‐tetraacetic acid in a stronger manner than an additive one. Together, these results support the view that CO₂ is sensed through reducing proton extrusion. Moreover, they might suggest that the CO₂ signal is transduced through Ca^{2 +} signalling arising from depolarization‐mediated activation of a putative plasma membrane voltage‐gated L‐type Ca^{2 +} channel and for which the plasma membrane slow anion channel is a potential target.     

Cell surface display of highly pathogenic avian influenza virus hemagglutinin on the surface of Pichia pastoris cells using α‐agglutinin for production of oral vaccines

Yeast is an ideal organism to express viral antigens because yeast glycosylate proteins more similarly to mammals than bacteria. Expression of proteins in yeast is relatively fast and inexpensive. In addition to the convenience of production, for purposes of vaccination, yeast has been shown to have natural adjuvant activity making the expressed proteins more immunogenic when administered along with yeast cell wall components. Development of genetic systems to display foreign proteins on the surface of yeast via fusion to glycosylphosphatidylinositol‐anchored (GPI) proteins has further simplified the purification of recombinant proteins by not requiring harsh treatments for cellular lysis or protein purification. We have expressed the hemagglutinin protein from a highly pathogenic avian influenza (HPAI) virus [A/Egret/HK/757.2/02], subtype H5N1, on the surface of the yeast strain Pichia pastoris, as an anchored C‐terminal fusion with the Saccharomyces cerevisiae GPI‐anchored cell wall protein, α‐agglutinin. Surface expression of the hemagglutinin fusion protein was demonstrated by immunofluorescence microscopy. Functionally, the fusion protein retained hemagglutinin agglutinating activity, and oral vaccination with the yeast resulted in production of virus neutralizing antibodies. This study represents the first steps in the generation of a yeast‐based vaccine for protection against highly pathogenic strains of avian influenza. Published 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010