Comparative analysis of quality and nutritional traits from Lonicera caerulea (Honeyberry) cultivars and other berries grown in Scotland

Lonicera caeruleabrix is a perennial shrub native to North America, Europe and Asia. It produces dark blue berries known as honeyberries or haskap berries which are produced commercially in several territories including Canada, Japan, Russia and Poland. Plants are suited to UK environments, but it is yet to be widely commercially developed in the UK. In the present work, quality and nutritional traits of six honeyberry cultivars grown in Scotland were compared with other commonly grown berry fruits (strawberry, raspberry, blueberry, blackberry, blackcurrant) to aid the identification of environmentally stable, high-quality honeyberry cultivars suitable for UK cultivation. Differences were observed in fruit quality variables (soluble solids, titratable acidity and Brix/acid ratios) between honeyberry cultivars. Three of six cultivars examined exhibited notable variation in soluble solids dependent on harvest year with ‘Aurora’ and ‘Strawberry Sensation’ having consistently high ^oBrix values. Titratable acidity exhibited cultivar differences and there was limited variation over harvest years. ‘Aurora’ exhibited consistently high ^oBrix/titratable acidity ratio reflected by high glucose and fructose content. Honeyberry fruit had good nutritional profile relative to other soft fruits with higher polyphenol and anthocyanin content than strawberry, blueberry, blackberry and raspberry, manifested in greater antioxidant capacity. The major anthocyanins in aqueous honeyberry fruit extracts were cyanidin, pelargonidin and peonidin glycosides. These findings indicate that L. caerulea represents a crop suitable for UK cultivation capable of producing high quality fruit with a valuable nutritional profile relative to other soft fruits. Cultivars exhibit significant differences in fruit quality and nutritional profile as well as harvest consistency and growers should consider this when establishing new plantations.     

Dynamic Changes of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript> Regulatory T Cells in NOD.H-2<Superscript>h4</Superscript> Mice with Iodine-Induced Autoimmune Thyroiditis

Iodine is an essential trace element for thyroid hormone synthesis and metabolism, either low or high intake may lead to thyroid disease, but the pathogenetic mechanisms by which iodine interacts with the thyroid autoimmune are poorly understood. We investigated the dynamic changes of CD4⁺CD25⁺ regulatory T cells in NOD.H-2^h4 mice with iodine-induced autoimmune thyroiditis (AIT), and explore potential immune mechanism of AIT induced by iodine. NOD.H-2^h4 mice were randomly divided into two groups, and received plain water or water containing 0.005% sodium iodide. Eight weeks after iodine provision, the incidences of thyroiditis, relative weights of thyroids, and serum thyroglobulin antibody titers in the iodine-supplied groups were significantly increased compared to the control groups (p < 0.05). The AIT mice had fewer CD4⁺CD25⁺Foxp3⁺ T cells and reduced Foxp3 mRNA expression in splenocytes compared with the controls (p < 0.01), and maintained relatively low levels during the development of thyroiditis. The changes described above aggravated gradually with the extension of iodine treatment. These data suggest that CD4⁺CD25⁺ regulatory T cells may be involved in the pathogenesis and development of AIT induced by iodine.     

A <Emphasis Type="Italic">FRUITFULL-like</Emphasis> gene is associated with genetic variation for fruit flesh firmness in apple (<Emphasis Type="Italic">Malus domestica</Emphasis> Borkh.)

The FRUITFULL (FUL) and SHATTERPROOF (SHP) genes are involved in regulating fruit development and dehiscence in Arabidopsis. We tested the hypothesis that this class of genes are also involved in regulating the development of fleshy fruits, by exploring genetic and phenotypic variation within the apple (Malus domestica) gene pool. We isolated and characterised the genomic sequences of two candidate orthologous FUL-like genes, MdMADS2.1 and MdMADS2.2. These were mapped using the reference population ‘Prima x Fiesta’ to loci on Malus linkage groups LG14 and LG06, respectively. An additional MADS-box gene, MdMADS14, shares high amino acid identity with the Arabidopsis SHATTERPROOF1/2 genes and was mapped to Malus linkage group LG09. Association analysis between quantitative fruit flesh firmness estimates of ‘Prima x Fiesta’ progeny and the MdMADS2.1, MdMADS2.2 and MdMADS14 loci was carried out using a mixed model analysis of variance. This revealed a significant association (P < 0.01) between MdMADS2.1 and fruit flesh firmness. Further evidence for the association between MdMADS2.1 and fruit flesh firmness was obtained using a case–control population-based genetic association approach. For this, a polymorphic repeat, (AT)_n, in the 3′ UTR of MdMADS2.1 was used as a locus-specific marker to screen 168 apple accessions for which historical assessments of fruit texture attributes were available. This analysis revealed a significant association between the MdMADS2.1 and fruit flesh firmness at both allelic (χ ² = 34, df = 9, P < 0.001) and genotypic (χ ² = 57, df = 32, P < 0.01) levels.     

Computational study of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter from <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Sodium proton antiporters are ubiquitous membrane proteins that catalyze the exchange of Na⁺ for protons throughout the biological world. The Escherichia coli NhaA is the archetypal Na⁺/H⁺ antiporter and is absolutely essential for survival in high salt concentrations under alkaline conditions. Its crystal structure, accompanied by extensive molecular dynamics simulations, have provided an atomically detailed model of its mechanism. In this study, we utilized a combination of computational methodologies in order to construct a structural model for the Na⁺/H⁺ antiporter from the gram-negative bacterium Vibrio parahaemolyticus. We explored its overall architecture by computational means and validated its stability and robustness. This protein belongs to a novel group of NhaA proteins that transports not only Na⁺ and Li⁺ as substrate ions, but K⁺ as well, and was also found to miss a β-hairpin segment prevalent in other homologs of the Bacteria domain. We propose, for the first time, a structure of a prototype model of a β-hairpin-less NhaA that is selective to K⁺. Better understanding of the Vibrio parahaemolyticus NhaA structure-function may assist in studies on ion transport, pH regulation and designing selective blockers.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Superscript>137</Superscript>Cs, <Superscript>40</Superscript>K, <Superscript>238</Superscript>Pu, <Superscript>239+240</Superscript>Pu and <Superscript>90</Superscript>Sr in biological samples from King George Island (Southern Shetlands) in Antarctica

There are few data reported on radionuclide contamination in Antarctica. The aim of this paper is to report ¹³⁷Cs, ⁹⁰Sr and ^238,239+240Pu and ⁴⁰K activity concentrations measured in biological samples collected from King George Island (Southern Shetlands, Antarctica), mostly during 2001–2002. The samples included: bones, eggshells and feathers of penguin Pygoscelis papua, bones and feathers of petrel Daption capense, bones and fur of seal Mirounga leonina, algae Himantothallus grandifolius, Desmarestia anceps and Cystosphaera jacquinotii, fish Notothenia corriceps, sea invertebrates Amphipoda, shells of limpet Nacella concina, lichen Usnea aurantiaco-atra, vascular plants Deschampsia antarctica and Colobanthus quitensis, fungi Omphalina pyxidata, moss Sanionia uncinata and soil. The results show a large variation in some activity concentrations. Samples from the marine environment had lower contamination levels than those from terrestrial ecosystems. The highest activity concentrations for all radionuclides were found in lichen and, to a lesser extent, in mosses, probably because lichens take up atmospheric pollutants and retain them. The only significant correlation (except for that expected between ²³⁸Pu and ^239+240Pu) was noted for moss and lichen samples between plutonium and ⁹⁰Sr. A tendency to a slow decrease with time seems to be occurring. Analyses of the activity ratios show varying fractionation between various radionuclides in different organisms. Algae were relatively more highly contaminated with plutonium and radiostrontium, and depleted with radiocesium. Feathers had the lowest plutonium concentrations. Radiostrontium and, to a lesser extent, Pu accumulated in bones. The present low intensity of fallout in Antarctic has a lower ²³⁸Pu/^239+240Pu activity ratio than that expected for global fallout.     

Dynamics of <Superscript>18</Superscript>O Incorporation from H <Subscript>2</Subscript><Superscript>18</Superscript>O into Soil Microbial DNA

Heavy water (H₂¹⁸O) has been used to label DNA of soil microorganisms in stable isotope probing experiments, yet no measurements have been reported for the ¹⁸O content of DNA from soil incubated with heavy water. Here we present the first measurements of atom% ¹⁸O for DNA extracted from soil incubated with the addition of H₂¹⁸O. Four experiments were conducted to test how the atom% ¹⁸O of DNA, extracted from Ponderosa Pine forest soil incubated with heavy water, was affected by the following variables: (1) time, (2) nutrients, (3) soil moisture, and (4) atom% ¹⁸O of added H₂O. In the time series experiment, the atom% ¹⁸O of DNA increased linearly (R ² = 0.994, p < 0.01) over the first 72 h of incubation. In the nutrient addition experiment, there was a positive correlation (R ² = 0.991, p = 0.006) between the log₁₀ of the amount of tryptic soy broth, a complex nutrient broth, added to soil and the log₁₀ of the atom% ¹⁸O of DNA. For the experiment where soil moisture was manipulated, the atom% ¹⁸O of DNA increased with higher soil moisture until soil moisture reached 30%, above which ¹⁸O enrichment of DNA declined as soils became more saturated. When the atom% ¹⁸O for H₂O added was varied, there was a positive linear relationship between the atom% ¹⁸O of the added water and the atom% ¹⁸O of the DNA. Results indicate that quantification of ¹⁸O incorporated into DNA from H₂¹⁸O has potential to be used as a proxy for microbial growth in soil.     

<Superscript>15</Superscript>N and <Superscript>13</Superscript>C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated,selectively [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C]-labeled proteins

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and ¹⁵N, methyl labeled samples in H₂O. The experiments benefit from a combination of selective T ₁ relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear ¹⁵N,¹³C-edited, or with diagonal-free ¹³C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.     

CD133<Superscript>+</Superscript>CD44<Superscript>+</Superscript> subgroups may be human small intestinal stem cells

The identification and separation of small intestinal epithelial stem cells are still on the preliminary stage. In this study, we planned to utilize immunohistochemistry, fluorescence-activated cell sorting (FACS) and RT-PCR to investigate the possibility of CD133 and CD44 as markers of human small intestinal epithelial stem cells. The expressions of CD133, CD44 and Lgr5 were studied by immunohistochemistry. Four subgroups of CD133⁺CD44⁺, CD133⁺CD44⁻, CD133⁻CD44⁺, CD133⁻CD44⁻ were sorted out through FACS and the expression level of Lgr5 gene was measured by RT-PCR and polyacrylamide gel electropheresis (PAGE) with sliver stained. Ten cases of samples were available for analyzing. By immunohistochemical staining, few cells with positive expressions of CD133, CD44 and Lgr5 were distributed in the bottom of crypts with the expression locations somewhat overlapped. The average percentage of CD133⁺CD44⁺ cells was 0.0580 ± 0.0403%, while the corresponding contents of CD133⁺CD44⁻ cells, CD133⁻CD44⁺ cells and CD133⁻CD44⁻ cells were 0.4000 ± 0.1225%, 0.7000 ± 0.2646% and 76.5600 ± 3.5529% respectively. Ten times of positive expressions of Lgr5 were detected in the CD133⁺CD44⁺ groups, while 9/10, 8/10 and 4/10 times for CD133⁺CD44⁻, CD133⁻CD44⁺ and CD133⁻CD44⁻ subgroups respectively. With the help of Quantityone 4.62 software, the densities of corresponding place to Lgr5 and reference gene were obtained. The density ratios of corresponding place to Lgr5 to reference gene were significant difference between subgroups (P < 0.001). By means of LSD method, the density ratios in CD133⁺CD44⁺ subgroups had statistical differences from the other subgroups (P < 0.05). We concluded CD133⁺CD44⁺ cells may be human small intestinal epithelial stem cells, which need further researches to confirm.     

Salt tolerance of barley: Relations between expression of isoforms of vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter and <Superscript>22</Superscript>Na<Superscript>+</Superscript> accumulation

Two barley cultivars (Hordeum vulgare L., cvs. Elo and Belogorskii) differing in salt tolerance were used to study ²²Na⁺ uptake, expression of three isoforms of the Na⁺/H⁺ antiporter HvNHX1-3, and the cellular localization of these isoforms in the elongation zone of seedling roots. During short (1 h) incubation, seedling roots of both cultivars accumulated approximately equal quantities of ²²Na⁺. However, after 24-h incubation the content of ²²Na⁺ in roots of a salt-tolerant variety Elo was 40% lower than in roots of the susceptible variety Belogorskii. The content of ²²Na⁺ accumulated in shoots of cv. Elo after 24-h incubation was 6.5 times lower than in shoots of cv. Belogorskii and it was 4 times lower after the salt stress treatment. The cytochemical examination revealed that three proteins HvNHX1-3 are co-localized in the same cells of almost all root tissues; these proteins were present in the tonoplast and prevacuolar vesicles. Western blot analysis of HvNHX1-3 has shown that the content of isoforms in vacuolar membranes increased in response to salt stress in seedling roots and shoots of both cultivars, although the increase was more pronounced in the tolerant cultivar. The content of HvNHX1 in the seedlings increased in parallel with the enhanced expression of HvNHX1, whereas the increase in HvNHX2 and HvNHX3 protein content was accompanied by only slight changes in expression of respective genes. The results provide evidence that salt tolerance of barley depends on plant ability to restrict Na⁺ transport from the root to the shoot and relies on regulatory pathways of HvNHX1-3 expression in roots and shoots during salt stress.     

Chilling Injury,Physicochemical Properties,and Antioxidant Enzyme Activities of Red Pitahaya (<i>Hylocereus polyrhizus</i>) Fruits under Cold Storage Stress

Low-temperature storage is extensively used to optimize the postharvest life of various fresh fruits. However, red pitahaya (Hylocereus polyrhizus) fruits are sensitive to chilling injury (CI), which leads to the limitation of low-temperature storage. In this study, red pitahaya fruits were stored at 2, 4, 6, 8, and 10°C, respectively, for 27 days to determine the appropriate storage temperature. During the storage of red pitahaya fruits, storage at 8°C was more effective in suppressing decay and maintaining quality than other low temperatures. Low-temperature (2, 4, and 6°C) storage decreased weight loss (WL) and maintained higher content of titratable acidity (TA), soluble sugars (SS), and total phenolics (TP) but different degrees of CI were detected. No CI was observed at 8°C and 10°C. Red pitahay as stored at 8 and 10°C were associated with better color evaluation, lower electrolyte leakage (EL), respiration rate, and lipoxygenase (LOX) activity, and higher fruit firmness, superoxide dismutase (SOD) activity, and catalase (CAT) activity. However, higher storage temperature (10°C) resulted in higher metabolic activity leading to lower quality and antioxidant capacities compared with 8°C. Therefore, our results demonstrated that red pitahaya stored at 8°C exhibited a protective effect on fruit quality and resisted CI development during storage.     

<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume     

Modulation of the reaction cycle of the Na<Superscript>+</Superscript>:Ca<Superscript>2+</Superscript>, K<Superscript>+</Superscript> exchanger

Ca²⁺ concentration in retinal photoreceptor rod outer segment (OS) strongly affects the generator potential kinetics and the receptor light adaptation. The response to intense light stimuli delivered in the dark produce potential changes exceeding 40 mV: since the Ca²⁺ extrusion in the OS is entirely controlled by the Na⁺:Ca²⁺, K⁺ exchanger, it is important to assess how the exchanger ion transport rate is affected by the voltage and, in general, by intracellular factors. It is indeed known that the cardiac Na⁺:Ca²⁺ exchanger is regulated by Mg-ATP via a still unknown metabolic pathway. In the present work, the Na⁺:Ca²⁺, K⁺ exchanger regulation was investigated in isolated OS, recorded in whole-cell configuration, using ionic conditions that activated maximally the exchanger in both forward and reverse mode. In all species examined (amphibia: Rana esculenta and Ambystoma mexicanum; reptilia: Gecko gecko), the forward (reverse) exchange current increased about linearly for negative (positive) voltages and exhibited outward (inward) rectification for positive (negative) voltages. Since hyperpolarisation increases Ca²⁺ extrusion rate, the recovery of the dark level of Ca²⁺ (and, in turn, of the generator potential) after intense light stimuli results accelerated. Mg-ATP increased the size of forward and reverse exchange current by a factor of ∼2.3 and ∼2.6, respectively, without modifying their voltage dependence. This indicates that Mg-ATP regulates the number of active exchanger sites and/or the exchanger turnover number, although via an unknown mechanism. Proceedings of the XVIII Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), Palermo, Sicily, September 2006.     

<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignments for pyrazinoic acid binding domain of ribosomal protein S1 from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Ribosomal protein S1 of Mycobacterium tuberculosis (MtRpsA) binds to ribosome and mRNA, and plays significant role in the regulation of translation initiation, conventional protein synthesis and transfer-messenger RNA (tmRNA) mediated trans-translation. It has been identified as the target of pyrazinoic acid (POA), a bactericidal moiety from hydrolysis of pyrazinamide, which is a mainstay of combination therapy for tuberculosis. POA prevented the interactions between the C-terminal S1 domain of MtRpsA (residues 280–368, MtRpsA^CTD_S1) and tmRNA; so that POA can inhibit the trans-translation, which is a key component of multiple quality control pathways in bacteria. However, the details of molecular mechanism and dynamic characteristics for MtRpsA^CTD_S1 interactions with POA, tmRNA or mRNA are still unclear. Here we present the ¹H, ¹⁵N, ¹³C resonance assignments of MtRpsA^CTD_S1 as well as the secondary structure information based on backbone chemical shifts, which lay foundation for further solution structure determination, dynamic properties characterization and interactions investigation between MtRpsA^CTD_S1 and tmRNA, RNA or POA.     

Molecular characterization and functional analysis of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene (<Emphasis Type="Italic">HcNHX1</Emphasis>) from <Emphasis Type="Italic">Halostachys caspica</Emphasis>

According to sequences of several vacuolar Na⁺/H⁺ antiporter genes from Xinjiang halophytic plants, a new vacuolar Na⁺/H⁺ antiporter gene (HcNHX1) from the halophyte Halostachys caspica was obtained by RACE and RT-PCR using primers corresponding to conserved regions of the coding sequences. The obtained HcNHX1 cDNA was 1,983 bp and contained a 1,656 bp open reading frame encoding a deduced protein of 551 amino acid residues. The deduced amino acid sequence showed high identity with other NHX1 we have cloned previously from halophyte in Xinjiang desert area. The phylogenetic analysis showed that HcNHX1 formed a clade with NHX homologs of Chenopodiaceae. Expression profiles under salt treatment and ABA induction were investigated, and the results revealed that expression of HcNHX1 was induced by NaCl and ABA. To compare the degree of salt tolerance, we over-expressed HcNHX1 in Arabidopsis. Two transgenic lines grew more vigorously than the wild type (WT) under salt stress. The analysis of ion contents indicated that under salt stress, the transgenic plants compartmentalized more Na⁺ in the leaves compared with wild-type plants. Together, these results suggest that the products of the novel gene HcNHX1 from halophyte Halostachys caspica is a functional tonoplast Na⁺/H⁺ antiporter.     

Over-expression of a vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene improves salt tolerance in an upland rice

To develop a salt-tolerant upland rice cultivar (Oryza sativa L.), OsNHX1, a vacuolar-type Na⁺/H⁺ antiporter gene from rice was transferred into the genome of an upland rice cultivar (IRAT109), using an Agrobacterium-mediated method. Seven independent transgenic calli lines were identified by polymerase chain reaction (PCR) analysis. These 35S::OsNHX1 transgenic plants displayed a little accelerated growth during seedling stage but showed delayed flowering time and a slight growth retardation phenotype during late vegetative stage, suggesting that the OsNHX1 has a novel function in plant development. Northern and western blot analyses showed that the expression levels of OsNHX1 mRNA and protein in the leaves of three independent transgenic plant lines were significantly higher than in the leaves of wild type (WT) plants. T₂ generation plants exhibited increased salt tolerance, showing delayed appearance and development of damage or death caused by salt stress, as well as improved recovery upon removal from this condition. Several physiological traits, such as increased Na⁺ content, and decreased osmotic potential in transgenic plants grown in high saline concentrations, further indicated that the transgenic plants had enhanced salt tolerance. Our results suggest the potential use of these transgenic plants for further agricultural applications in saline soil.     

The control of CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>Foxp3<Superscript>+</Superscript>regulatory T cell survival

   

CD4⁺CD25⁺Foxp3⁺ regulatory T (T_reg) cells are believed to play an important role in suppressing autoimmunity and maintaining peripheral tolerance. How their survival is regulated in the periphery is less clear. Here we show that T_reg cells express receptors for gamma chain cytokines and are dependent on an exogenous supply of these cytokines to overcome cytokine withdrawal apoptosis in vitro. This result was validated in vivo by the accumulation of T_reg cells in Bim^-/- and Bcl-2 tg mice which have arrested cytokine deprivation apoptosis. We also found that CD25 and Foxp3 expression were down-regulated in the absence of these cytokines. CD25⁺ cells from Scurfy mice do not depend on cytokines for survival demonstrating that Foxp3 increases their dependence on cytokines by suppressing cytokine production in T_reg cells. Our study reveals that the survival of T_reg cells is strictly dependent on cytokines and cytokine producing cells because they do not produce cytokines. Our study thus, demonstrates that different gamma chain cytokines regulate T_reg homeostasis in the periphery by differentially regulating survival and proliferation. These findings may shed light on ways to manipulate T_reg cells that could be utilized for their therapeutic applications.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.