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1.
Gene ; 713: 143976, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31306715

RESUMO

Naturally evolved saline tolerant rice landraces found along the coastline of India are a valuable genomic resource to explore the complex, polygenic nature of salinity tolerance. In the present study, a set of 28 genome wide SSR markers, 11 salt responsive genic SSR markers and 8 Saltol QTL linked SSR markers were used to estimate genetic relatedness and population structure within a collection of 47 rice landraces (including a tolerant and 2 sensitive checks) originating from geographically divergent coastal regions of India. All three marker types identified substantial genetic variation among the landraces, as evident from their higher PIC values (0.53 for genomic SSRs, 0.43 for Genic SSRs and 0.59 for Saltol SSRs). The markers RM431, RM484 (Genomic SSRs), OsCAX (D), OsCAX (T) (Genic SSRs) and RM562 (Saltol SSR) were identified as good candidates to be used in breeding programs for improving salinity tolerance in rice. STRUCTURE analysis divided the landraces into five distinct populations, with classification correlating with their geographical locations. Principal coordinate and hierarchical cluster analyses (UPGMA and neighbor joining) are in close agreement with STRUCTURE results. AMOVA analysis indicated a higher magnitude of genetic differentiation within individuals of groups (58%), than among groups (42%). We also report the development and validation of a new Cleavage Amplified Polymorphic Sequence (CAPS) marker (OsHKT1;5V395) that targets a codon in the sodium transporter gene OsHKT1;5 (Saltol/SKC1 locus) that is associated with sodium transport rates in the above rice landraces. The CAPS marker was found to be present in all landraces except in IR29, Kamini, Gheus, Matla 1 and Matla 2. Significant molecular genetic diversity established among the analyzed salt tolerant rice landraces will aid in future association mapping; the CAPS marker, OsHKT1;5V395 can be used to map rice landraces for the presence of the SNP (Single Nucleotide Polymorphism) associated with increased sodium transport rates and concomitant salinity tolerance in rice.


Assuntos
Marcadores Genéticos , Variação Genética , Repetições de Microssatélites , Oryza/genética , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Sódio/metabolismo , Genótipo , Filogenia
2.
World J Microbiol Biotechnol ; 35(8): 124, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31346773

RESUMO

Candida glabrata is a haploid yeast that is considered to be an emergent pathogen since it is the second most prevalent cause of candidiasis. Contrary to most yeasts, this species carries only one plasma membrane potassium transporter named CgTrk1. We show in this work that the activity of this transporter is regulated at the posttranslational level, and thus Trk1 contributes to potassium uptake under very different external cation concentrations. In addition to its function in potassium uptake, we report a diversity of physiological effects related to this transporter. CgTRK1 contributes to proper cell size, intracellular pH and membrane-potential homeostasis when expressed in Saccharomyces cerevisiae. Moreover, lithium influx experiments performed both in C. glabrata and S. cerevisiae indicate that the salt tolerance phenotype linked to CgTrk1 can be related to a high capacity to discriminate between potassium and lithium (or sodium) during the transport process. In summary, we show that CgTRK1 exerts a diversity of pleiotropic physiological roles and we propose that the corresponding protein may be an attractive pharmacological target for the development of new antifungal drugs.


Assuntos
Candida glabrata/genética , Proteínas de Transporte de Cátions/genética , Proteínas Fúngicas/genética , Candida glabrata/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Homeostase , Concentração de Íons de Hidrogênio , Potássio/metabolismo , Sódio/metabolismo
3.
BMC Plant Biol ; 19(1): 316, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307394

RESUMO

BACKGROUND: HKT channels mediate sodium uniport or sodium and potassium symport in plants. Monocotyledons express a higher number of HKT proteins than dicotyledons, and it is only within this clade of HKT channels that cation symport mechanisms are found. The prevailing ion composition in the extracellular medium affects the transport abilities of various HKT channels by changing their selectivity or ion transport rates. How this mutual effect is achieved at the molecular level is still unknown. Here, we built a homology model of the monocotyledonous OsHKT2;2, which shows sodium and potassium symport activity. We performed molecular dynamics simulations in the presence of sodium and potassium ions to investigate the mutual effect of cation species. RESULTS: By analyzing ion-protein interactions, we identified a cation coordination site on the extracellular protein surface, which is formed by residues P71, D75, D501 and K504. Proline and the two aspartate residues coordinate cations, while K504 forms salt bridges with D75 and D501 and may be involved in the forwarding of cations towards the pore entrance. Functional validation via electrophysiological experiments confirmed the biological relevance of the predicted ion coordination site and identified K504 as a central key residue. Mutation of the cation coordinating residues affected the functionality of HKT only slightly. Additional in silico mutants and simulations of K504 supported experimental results. CONCLUSION: We identified an extracellular cation coordination site, which is involved in ion coordination and influences the conduction of OsHKT2;2. This finding proposes a new viewpoint in the discussion of how the mutual effect of variable ion species may be achieved in HKT channels.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons , Proteínas de Plantas/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/genética , Cátions/metabolismo , Clonagem Molecular , Eletrofisiologia , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Relação Estrutura-Atividade , Xenopus laevis
4.
Plant Cell Rep ; 38(9): 1151-1163, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31152194

RESUMO

KEY MESSAGE: Piriformospora indica confers salt tolerance in tomato seedlings by increasing the uptake of nutrients such as N, P and Ca, improving K+/Na+ homoeostasis by regulating the expression of NHXs, SOS1 and CNGC15 genes, maintaining water status by regulating the expression of aquaporins. Piriformospora indica, an endophytic basidiomycete, has been shown to increase the growth and improve the plants tolerance to stressful conditions, especially salinity, by establishing the arbuscular mycorrhiza-like symbiotic relationship in various plant hosts. In the present research, the effect of NaCl treatment (150 mM) and P. indica inoculation on growth, accumulation of nutrients, the transcription level of genes involved in ionic homeostasis (NHXs, SOS1 and CNGC15) and regulating water status (PIP1;2, PIP2;4, TIP1;1 and TIP2;2) in roots and leaves of tomato seedlings were investigated. The P. indica improved the uptake of N, P, Ca and K, and reduced Na accumulation, and had no significant effect on Cl accumulation in roots and leaves. The endophytic fungus also increased in K+/Na+ ratio in roots and leaves of tomato by regulating the expression of NHX isoforms and upregulating SOS1 and CNGC15 expression. Salinity stress increased the transcription of PIP2;4 gene and reduced the transcription of PIP1;2, TIP1;1 and TIP2;2 genes compared to the control treatment. However, P. indica inoculation upregulated the expression of PIP1;2 and PIP2;4 genes versus non-inoculated plants but did not have a significant effect on TIP1;1 and TIP2;2 expression. These results conclude that the positive effects of P. indica on nutrients accumulation, ionic homeostasis and water status lead to the increased salinity tolerance and the improved plant growth under NaCl treatment.


Assuntos
Basidiomycota/fisiologia , Lycopersicon esculentum/microbiologia , Nutrientes/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Água/metabolismo , Homeostase , Lycopersicon esculentum/genética , Lycopersicon esculentum/fisiologia , Salinidade , Tolerância ao Sal , Estresse Fisiológico , Simbiose
5.
Nat Commun ; 10(1): 2714, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31221956

RESUMO

The dopamine transporter is a member of the neurotransmitter:sodium symporters (NSSs), which are responsible for termination of neurotransmission through Na+-driven reuptake of neurotransmitter from the extracellular space. Experimental evidence elucidating the coordinated conformational rearrangements related to the transport mechanism has so far been limited. Here we probe the global Na+- and dopamine-induced conformational dynamics of the wild-type Drosophila melanogaster dopamine transporter using hydrogen-deuterium exchange mass spectrometry. We identify Na+- and dopamine-induced changes in specific regions of the transporter, suggesting their involvement in protein conformational transitions. Furthermore, we detect ligand-dependent slow cooperative fluctuations of helical stretches in several domains of the transporter, which could be a molecular mechanism that assists in the transporter function. Our results provide a framework for understanding the molecular mechanism underlying the function of NSSs by revealing detailed insight into the state-dependent conformational changes associated with the alternating access model of the dopamine transporter.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Dopamina/metabolismo , Proteínas de Drosophila/metabolismo , Simulação de Dinâmica Molecular , Sódio/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/isolamento & purificação , Proteínas de Drosophila/química , Proteínas de Drosophila/isolamento & purificação , Células HEK293 , Humanos , Ligantes , Espectrometria de Massas , Conformação Proteica em alfa-Hélice/fisiologia , Domínios Proteicos/fisiologia , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
6.
Plant Sci ; 285: 55-67, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203894

RESUMO

C2H2-type zinc finger proteins play important roles in plant growth, development, and abiotic stress tolerance. Here, we explored the role of the C2H2-type zinc finger protein SALT INDUCED ZINC FINGER PROTEIN1 (AtSIZ1; At3G25910) in Arabidopsis thaliana under salt stress. AtSIZ1 expression was induced by salt treatment. During the germination stage, the germination rate, germination energy, germination index, cotyledon growth rate, and root length were significantly higher in AtSIZ1 overexpression lines than in the wild type under various stress treatments, whereas these indices were significantly reduced in AtSIZ1 loss-of-function mutants. At the mature seedling stage, the overexpression lines maintained higher levels of K+, proline, and soluble sugar, lower levels of Na+ and MDA, and lower Na+/K+ ratios than the wild type. Stress-related marker genes such as SOS1, AtP5CS1, AtGSTU5, COR15A, RD29A, and RD29B were expressed at higher levels in the overexpression lines than the wild type and loss-of-function mutants under salt treatment. These results indicate that AtSIZ1 improves salt tolerance in Arabidopsis by helping plants maintain ionic homeostasis and osmotic balance.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Ligases/fisiologia , Dedos de Zinco/fisiologia , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Homeostase , Ligases/genética , Filogenia , Potássio/metabolismo , Prolina/metabolismo , Estresse Salino , Tolerância ao Sal , Sódio/metabolismo , Dedos de Zinco/genética
7.
Int J Mol Sci ; 20(9)2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035668

RESUMO

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na+,K+-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine ß1 subunit become adhesive, and those homotypic interactions amongst ß1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between ß1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the ß1 subunit of the Na+,K+-ATPase (CHO ß1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO ß1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the ß1 subunit of the Na+,K+-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO ß1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO ß1 cells. Collectively, our findings suggest that the ß1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.


Assuntos
Adesão Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ouabaína/farmacologia , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Expressão Gênica , Ligação Proteica , Subunidades Proteicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Quinases da Família src/metabolismo
8.
BMC Genomics ; 20(1): 418, 2019 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126236

RESUMO

BACKGROUND: To control the osmotic pressure in the body, physiological adjustments to salinity fluctuations require the fish to regulate body fluid homeostasis in relation to environmental change via osmoregulation. Previous studies related to osmoregulation were focused primarily on the gill; however, little is known about another organ involved in osmoregulation, the kidney. The salinity adaptation of marine fish involves complex physiological traits, metabolic pathways and molecular and gene networks in osmoregulatory organs. To further explore of the salinity adaptation of marine fish with regard to the role of the kidney, the euryhaline fish Scatophagus argus was employed in the present study. Renal expression profiles of S. argus at different salinity levels were characterized using RNA-sequencing, and an integrated approach of combining molecular tools with physiological and biochemical techniques was utilized to reveal renal osmoregulatory mechanisms in vivo and in vitro. RESULTS: S. argus renal transcriptomes from the hyposaline stress (0‰, freshwater [FW]), hypersaline stress (50‰, hypersaline water [HW]) and control groups (25‰) were compared to elucidate potential osmoregulatory mechanisms. In total, 19,012 and 36,253 differentially expressed genes (DEGs) were obtained from the FW and HW groups, respectively. Based on the functional classification of DEGs, the renal dopamine system-induced Na+ transport was demonstrated to play a fundamental role in osmoregulation. In addition, for the first time in fish, many candidate genes associated with the dopamine system were identified. Furthermore, changes in environmental salinity affected renal dopamine release/reuptake by regulating the expression of genes related to dopamine reuptake (dat and nkaα1), vesicular traffic-mediated dopamine release (pink1, lrrk2, ace and apn), DAT phosphorylation (CaMKIIα and pkcß) and internalization (akt1). The associated transcriptional regulation ensured appropriate extracellular dopamine abundance in the S. argus kidney, and fluctuations in extracellular dopamine produced a direct influence on Na+/K+-ATPase (NKA) expression and activity, which is associated with Na+ homeostasis. CONCLUSIONS: These transcriptomic data provided insight into the molecular basis of renal osmoregulation in S. argus. Significantly, the results of this study revealed the mechanism of renal dopamine system-induced Na+ transport is essential in fish osmoregulation.


Assuntos
Dopamina/metabolismo , Peixes/genética , Rim/metabolismo , Estresse Salino/genética , Sódio/metabolismo , Transcriptoma , Animais , Células Cultivadas , Peixes/metabolismo , Perfilação da Expressão Gênica , Homeostase , Transporte de Íons , Rim/enzimologia , Anotação de Sequência Molecular , Osmorregulação/genética , Potássio/metabolismo , Tolerância ao Sal , Análise de Sequência de RNA , ATPase Trocadora de Sódio-Potássio/metabolismo
9.
Plant Sci ; 283: 202-210, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128690

RESUMO

Salinity is one of the most frequent limiting conditions in pasture production for grazing livestock. Legumes, such as Lotus spp. with high forage quality and capable of adapting to different environments, improves pasture performance in restrictive areas. In order to determine potential cultivars with better forage traits, the current study assess the response to salt stress of L. tenuis, L. corniculatus and a novel L. tenuis x L. corniculatus accession. For this purpose, chlorophyll fluorescence, biomass production, ion accumulation and anthocyanins and proanthocyanidins levels have been evaluated in control and salt-treated plants PSII activity was affected by salt in L. tenuis, but not in L. corniculatus or hybrid plants. Analyzed accessions showed similar values of biomass, Na+ and K+ levels after salt treatment. Increasing Cl- concentrations were observed in all accessions. However, hybrid plants accumulate Cl- in stems at higher levels than their parental. At the same time, the levels of anthocyanins considerably increased in L. tenuis x L. corniculatus stems. Chloride and anthocyanin accumulation in stems could explain the best performance of hybrid plants after a long saline treatment. Finally, as proanthocyanidins levels were no affected by salt, L. tenuis x L. corniculatus plants maintained adequate levels to be used as ruminant feed. In conclusion, these results suggest that hybrid plants have a high potential to be used as forage on salt-affected lands. High Cl- and anthocyanins accumulation in Lotus spp. stems seems to be a trait associated to salinity tolerance, with the possibility of being used in legume breeding programs.


Assuntos
Lotus/metabolismo , Antocianinas/metabolismo , Clorofila/metabolismo , Hibridização Genética , Lotus/crescimento & desenvolvimento , Lotus/fisiologia , Melhoramento Vegetal/métodos , Potássio/metabolismo , Estresse Salino , Plantas Tolerantes a Sal , Sódio/metabolismo
10.
J Forensic Leg Med ; 65: 61-67, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31103925

RESUMO

Often, pleural effusion is noted at autopsy when the cause of death is drowning or diseases such as heart, renal and liver failure. Several studies have established a correlation between the concentrations of electrolytes in pleural effusion and the post-mortem interval (PMI) or those concentrations and drowning site. The present study aims to investigate the relationship between the amount of pleural effusion, concentrations of electrolytes and total protein in pleural effusion, by integrated interpretation using various factors such as the deceased's gender, age, cause of death, drowning site, PMI and body temperature. We included 40 cadavers (26 male, 14 female) with >20-mL pleural effusion, which were categorised into four groups as follows: freshwater drowning; brackish water drowning; seawater drowning (drowning group); and not drowning. An equation derived to assess the lung weight revealed that the drowning site affected the lung weight. An equation for the amount of pleural effusion in the drowning group for the first time revealed that the amount of pleural effusion was directly proportional to the PMI. Using an equation to assess the PMI, we could estimate the PMI within 13.0-13.2 h in cases with >20-mL pleural effusion. Despite a small number of cases in the present study, we attained exciting results from the integrated statistical analysis.


Assuntos
Afogamento/diagnóstico , Modelos Estatísticos , Derrame Pleural/metabolismo , Derrame Pleural/patologia , Mudanças Depois da Morte , Cloretos/metabolismo , Feminino , Patologia Legal/métodos , Água Doce , Humanos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Tamanho do Órgão , Potássio/metabolismo , Águas Salinas , Sódio/metabolismo
11.
Plant Physiol Biochem ; 140: 151-157, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31103797

RESUMO

Na+/H+ antiporter (NHX), responsible for counter-transport of Na+ and H+ across membranes (Na+ compartmentalization), plays a central role in plant salt-tolerance. In order to explore the Na+ compartmentalization modes and salt tolerance strategy in Chinese cabbage (Brassica rapa L. ssp. pekinensis), the seedlings of a salt-susceptible cabbage cultivar (Kuaicai 38) and a salt-tolerant cabbage cultivar (Qingmaye) were exposed to 100-400 mM NaCl for 30 days. Both of these cultivars showed a gradual decrease in fresh weight and water content and an increase in root-shoot ratio with the increasing NaCl-treatment concentration. The distribution of Na+ in these two cultivars was similar, with the green leaves showing the highest Na+ content, followed by inflated midribs, stems, and roots. The Na+ concentration in the apoplast was higher than that in the protoplast of the leaves. The expression levels of BrNHX1-1 and BrNHX1-2 in the leaves of Qingmaye were the highest among all BrNHX members, and increased after salt treatment. However, only BrNHX1-1 was expressed in Kuaicai 38. These results indicate that Na+ compartmentation into vacuoles is the major salt-adaptation strategy in Chinese cabbage. Coordinated overexpression of BrNHX1-1 and BrNHX1-2 may confer greater salt-tolerance for Chinese cabbage.


Assuntos
Brassica/metabolismo , Sódio/metabolismo , Brassica/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Caules de Planta/efeitos dos fármacos , Caules de Planta/metabolismo , Estresse Salino , Tolerância ao Sal
12.
Toxicol Lett ; 311: 27-36, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31039415

RESUMO

Methotrexate (MTX) is a derivate of folic acid, commonly used as an anchor drug for the treatment and management of malignant diseases and autoimmune disorders. However, nephrotoxicity is an important drawback of MTX therapy. Unfortunately, there are not enough studies reporting the nature of the renal failure induced by MTX. Thus, the aim of this study was to evaluate the time course of renal handling of water and electrolytes in male Wistar rats, after the exposure to a unique dose of MTX (80 mg/kg b.w.). Experiments were carried out at day 2, day 4, day 8 and day 14 after MTX administration. Several parameters of kidney function related to water and electrolytes handling were evaluated. Renal expression and urinary excretion of aquaporin-2 (AQP2) and Na-K-2Cl-cotransporter (NKCC2) were determined by Western blotting. MTX produced alterations on water handling on the second day after treatment, showing a significant increase in solute free water reabsorption which might be mediated by the increased expression of AQP2 in apical membranes. On the other hand, MTX produced alterations on electrolytes handling on the fourth day after treatment, showing a significant decrease of sodium chloride excretion, mediated at least in part, by the increase renal expression of NKCC2. These results provide valuable information to clinical practice in order to be able to find therapeutic targets that diminish adverse effects and health deterioration. Moreover, MTX treatment altered AQP2 and NKCC2 urinary excretion allowing postulating these transporters as potential biomarkers of MTX induced nephrotoxicity.


Assuntos
Aquaporina 2/metabolismo , Eletrólitos/metabolismo , Nefropatias/induzido quimicamente , Túbulos Renais/efeitos dos fármacos , Metotrexato/toxicidade , Reabsorção Renal/efeitos dos fármacos , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Água/metabolismo , Animais , Biomarcadores/metabolismo , Cloretos/metabolismo , Nefropatias/metabolismo , Túbulos Renais/metabolismo , Masculino , Potássio/metabolismo , Ratos Wistar , Sódio/metabolismo , Fatores de Tempo , Urodinâmica/efeitos dos fármacos
13.
Plant Cell Rep ; 38(8): 915-926, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31037366

RESUMO

KEY MESSAGE: Transgenic callus and roots of ice plant with altered SnRK1 function were established using Agrobacterium-mediated transformation. The role of McSnRK1 in controlling Na+ influx and Na/K ratio was demonstrated. SnRK1 kinases (SNF1-related protein kinase1) control metabolic adaptation during energy deprivation and regulate protective mechanisms against environmental stress. Yeast SNF1 activates a P-type ATPase, the Na+ exclusion pump, under glucose starvation. The involvement of plant SnRK1 in salt stress response is largely unknown. We previously identified a salt-induced McSnRK1 in the halophyte ice plant (Mesembryanthemum crystallinum). In the current study, the function of McSnRK1 in salt tolerance was analyzed in transgenic cultured cells and roots of ice plant. Ice plant callus constitutively expressed a high level of McSnRK1 and introducing the full-length McSnRK1 did not alter the Na/K ratio at 24 h after 200 mM NaCl treatment. However, interfering with McSnRK1 activity by introducing a truncate McSnRK1 to produce a dominant-negative form of McSnRK1 increased cellular Na+ accumulation and Na/K ratio. As a result, the growth of cultured cells diminished under salt treatment. Hydroponically grown ice plants with roots expressing full-length McSnRK1 had better growth and lowered Na/K ratio compared to the wild-type or vector-only plants. Roots expressing a truncate McSnRK1 had reduced growth and high Na/K ratio under 400 mM NaCl treatment. The changes in Na/K ratio in transgenic cells and whole plants demonstrated the function of SnRK1 in controlling Na+ flux and maintaining Na/K homeostasis under salinity. The Agrobacterium-mediated transformation system could be a versatile tool for functional analysis of genes involved in salt tolerance in the ice plant.


Assuntos
Mesembryanthemum/enzimologia , Mesembryanthemum/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Homeostase , Mesembryanthemum/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Potássio/metabolismo , Proteínas Serina-Treonina Quinases/genética , Plantas Tolerantes a Sal/efeitos dos fármacos , Plantas Tolerantes a Sal/genética , Sódio/metabolismo , Cloreto de Sódio/farmacologia
14.
Int J Mol Sci ; 20(7)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987285

RESUMO

Diabetes mellitus (DM) has significant effects on cardiac calcium (Ca2+) and sodium (Na⁺) regulation. Clinical studies have shown that empagliflozin (Jardiance™) has cardiovascular benefits, however the mechanisms have not been fully elucidated. This study aimed to investigate whether empagliflozin modulates cardiac electrical activity as well as Ca2+/Na⁺ homeostasis in DM cardiomyopathy. Electrocardiography, echocardiography, whole-cell patch-clamp, confocal microscopic examinations, and Western blot, were performed in the ventricular myocytes of control and streptozotocin-induced DM rats, with or without empagliflozin (10 mg/kg for 4 weeks). The results showed that the control and empagliflozin-treated DM rats had smaller left ventricular end-diastolic diameters and shorter QT intervals than the DM rats. In addition, the prolonged action potential duration in the DM rats was attenuated in the empagliflozin-treated DM rats. Moreover, the DM rats had smaller sarcoplasmic reticular Ca2+ contents, intracellular Ca2+ transients, L-type Ca2+, reverse mode Na⁺-Ca2+exchanger currents, lower protein expressions of sarcoplasmic reticulum ATPase, ryanodine receptor 2 (RyR2), but higher protein expressions of phosphorylated RyR2 at serine 2808 than the control and empagliflozin-treated DM rats. The incidence and frequency of Ca2+ sparks, cytosolic and mitochondrial reactive oxygen species, and late Na⁺ current and Na⁺/hydrogen-exchanger currents were greater in the DM rats than in the control and empagliflozin-treated DM rats. Empagliflozin significantly changed Ca2+ regulation, late Na⁺ and Na⁺/hydrogen-exchanger currents and electrophysiological characteristics in DM cardiomyopathy, which may contribute to its cardioprotective benefits in DM patients.


Assuntos
Compostos Benzidrílicos/uso terapêutico , Cálcio/metabolismo , Glucosídeos/uso terapêutico , Miocárdio/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/metabolismo , Eletrofisiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismo
15.
Plant Biol (Stuttg) ; 21(5): 825-831, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31034750

RESUMO

Accumulation of NaCl in soil causes osmotic stress in plants, and sodium (Na+ ) and chloride (Cl- ) cause ion toxicity, but also reduce the potassium (K+ ) uptake by plant roots and stimulate the K+ efflux through the cell membrane. Thus, decreased K+ /Na+ ratio in plant tissue lead us to hypothesise that elevated levels of K+ in nutrient medium enhance this ratio in plant tissue and cytosol to improve enzyme activation, osmoregulation and charge balance. In this study, wheat was cultivated at different concentrations of K+ (2.2, 4.4 or 8.8 mm) with or without salinity (1, 60 or 120 mm NaCl) and the effects on growth, root and shoot Na+ and K+ distribution and grain yield were determined. Also, the cytosolic Na+ concentration was investigated, as well as photosynthesis rate and water potential. Salinity reduced fresh weight of both shoots and roots and dry weight of roots. The grain yield was significantly reduced under Na+ stress and improved with elevated K+ fertilisation. Elevated K+ level during cultivation prevented the accumulation of Na+ into the cytosol of both shoot and root protoplasts. Wheat growth at vegetative stage was transiently reduced at the highest K+ concentration, perhaps due to plants' efforts to overcome a high solute concentration in the plant tissue, nevertheless grain yield was increased at both K+ levels. In conclusion, a moderately elevated K+ application to wheat seedlings reduces tissue as well as cytosolic Na+ concentration and enhances wheat growth and grain yield by mitigating the deleterious effects of Na+ toxicity.


Assuntos
Potássio/farmacologia , Sódio/metabolismo , Triticum/metabolismo , Pressão Osmótica , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Potássio/metabolismo , Estresse Salino , Triticum/crescimento & desenvolvimento , Triticum/fisiologia
16.
Microb Pathog ; 131: 87-97, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30951817

RESUMO

Occasionally, endophytic fungal species cognize as a hidden prospective source of plant secondary metabolites. In this study, a potent Penicillium setosum sp. nov. was explored for its detailed antibacterial action on Escherichia coli and Staphylococcus aureus through different in vitro and in silico assays. Fluorescence based viability assay determined increase in the number of dead cells in course of time with the continual exposure of extract during a 4 h period. Scanning electron micrographs reflect the distinguishable morphological changes in treated cells, namely shortening of size, bubbles, and blisters on the surface of E. coli, as well as open holes and deep craters on the surface of S. aureus, ultimately leading to rupture of cells. Significant intracellular changes in bacteria were remarkably noticed through different membrane permeabilization assays. The rate of Na+ and K+ leakage with respect to time, intracellular material and cytoplasmic ß-galactosidase release were measured spectroscopically. The results indisputably prove that membrane disruption of S. aureus cells occurs within 2 h and in E.coli occurs in between 2 and 4 h of exposure. Crude extract of P. setosum was fractioned using semi-preparative HPLC and the separated antibacterial active fraction showed antibacterial efficacy with the minimum inhibitory concentration of 8 µg/mL against both organisms. Active fraction contains four well-known plant metabolite belongs to the polyphenolic group (Leucodelphinidin, dihydroquercetin, kaempferol, and quercetin) and one polyketide (patulin) familiar as fungal metabolite, identified through high resolution LC-MS. Interaction mechanisms of identified compounds with nine important antimicrobial drug targets showed highest binding affinity by leucodelphinidin followed by dihydroquercetin > kaempferol > quercetin. This is the first instance of using leucodelphinidin and dihydroquercetin for detailed interaction study with multiple targets, and it was found that they showed more effective interaction than quercetin, which was earlier utilized for antibacterial studies.


Assuntos
Antibacterianos/farmacologia , Simulação por Computador , Simulação de Acoplamento Molecular , Penicillium/metabolismo , Antibacterianos/biossíntese , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Flavonoides/biossíntese , Flavonoides/farmacologia , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Potássio/metabolismo , Quercetina/análogos & derivados , Quercetina/biossíntese , Quercetina/farmacologia , Metabolismo Secundário , Sódio/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , beta-Galactosidase/metabolismo
17.
Pharm Res ; 36(6): 84, 2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30997560

RESUMO

PURPOSE: ß-Hydroxy-ß-methylbutyrate (HMB), a nutritional supplement, elicits anabolic activity in muscle. Here we investigated the mechanism of HMB uptake in muscle cells. METHODS: Murine muscle cells (C2C12) and human mammary epithelial cells (MCF7) were used for uptake. As HMB is a monocarboxylate, focus was on monocarboxylate transporters, monitoring interaction of HMB with H+-coupled lactate uptake, and influence of H+ directly on HMB uptake. Involvement of MCT1-4 was studied using selective inhibitors and gene silencing. Involvement of human Na+/monocarboxylate transporter SMCT1 was also assessed using Xenopus oocytes. RESULTS: H+-coupled lactate uptake was inhibited by HMB in both mammalian cells. HMB uptake was H+-coupled and inhibited by lactate. C2C12 cells expressed MCT1 and MCT4; MCF7 cells expressed MCT1-4; undifferentiated C2C12 cells expressed SMCT1. SMCT1 mediated Na+-coupled HMB transport. Inhibitors of MCT1/4, siRNA-mediated gene silencing, and expression pattern showed that MCT1-4 were responsible only for a small portion of HMB uptake in these cells. CONCLUSION: HMB uptake in C2C12 and MCF7 cells is primarily H+-coupled and inhibited by lactate, but MCT1-4 are only partly responsible for HMB uptake. SMCT1 also transports HMB, but in a Na+-coupled manner. Other, yet unidentified, transporters mediate the major portion of HMB uptake in C2C12 and MCF7 cells.


Assuntos
Suplementos Nutricionais , Transportadores de Ácidos Monocarboxílicos/metabolismo , Valeratos/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Células Epiteliais/metabolismo , Inativação Gênica , Humanos , Ácido Láctico/metabolismo , Células MCF-7 , Camundongos , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Células Musculares/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Sódio/metabolismo , Xenopus laevis
18.
Nat Commun ; 10(1): 1742, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30988359

RESUMO

Na+/H+ antiporters exchange sodium ions and protons on opposite sides of lipid membranes. The electroneutral Na+/H+ antiporter NhaP from archaea Pyrococcus abyssi (PaNhaP) is a functional homolog of the human Na+/H+ exchanger NHE1, which is an important drug target. Here we resolve the Na+ and H+ transport cycle of PaNhaP by transition-path sampling. The resulting molecular dynamics trajectories of repeated ion transport events proceed without bias force, and overcome the enormous time-scale gap between seconds-scale ion exchange and microseconds simulations. The simulations reveal a hydrophobic gate to the extracellular side that opens and closes in response to the transporter domain motion. Weakening the gate by mutagenesis makes the transporter faster, suggesting that the gate balances competing demands of fidelity and efficiency. Transition-path sampling and a committor-based reaction coordinate optimization identify the essential motions and interactions that realize conformational alternation between the two access states in transporter function.


Assuntos
Pyrococcus abyssi/metabolismo , Trocadores de Sódio-Hidrogênio/fisiologia , Simulação por Computador , Interações Hidrofóbicas e Hidrofílicas , Transporte de Íons , Modelos Moleculares , Prótons , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo
19.
mSphere ; 4(2)2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944211

RESUMO

Inorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPi and other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Pi conductance in Xenopus oocytes expressing a Saccharomyces cerevisiae or Trypanosoma brucei Na+/Pi symporter. PPi is not taken up by Xenopus oocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPi generated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type or pho91Δ yeast strains expressing TbPHO91 but not from the pho91Δ strains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing Pi from S. cerevisiae vacuoles and T. brucei acidocalcisomes.IMPORTANCE Acidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities with S. cerevisiae vacuoles in their structure and composition. Both organelles share a Na+/Pi symporter involved in Pi release to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in either Xenopus oocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.


Assuntos
Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/metabolismo , Simportadores/genética , Trypanosoma brucei brucei/metabolismo , Vacúolos/metabolismo , Animais , Potenciais da Membrana , Oócitos/metabolismo , Fosfatos/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sódio/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/genética , Trypanosoma brucei brucei/genética , Xenopus/metabolismo
20.
Aquat Toxicol ; 211: 92-104, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30954848

RESUMO

The impact of freshwater (FW) salinization on osmoregulation as well as tracheal gill morphology and function was examined in nymphs of the mayfly Hexagenia rigida following exposure to salt contaminated water (SCW, 7.25 g/l NaCl) for a 7-day period. Ionoregulatory homeostasis was perturbed in SCW exposed H. rigida nymphs as indicated by increased hemolymph Na+, K+ and Cl- levels as well as hemolymph pH and water content. Despite this, SCW did not alter gill Na+-K+-ATPase (NKA) or V-type H+-ATPase (VA) activity. In addition, NKA and VA immunolocalization in gill ionocytes did not show alterations in enzyme location or changes in ionocyte abundance. The latter observation was confirmed using scanning electron microscopy (SEM) to examine exposed tracheal gill ionocyte numbers. Ionocyte surface morphometrics also revealed that SCW did not change individual ionocyte surface area or ionocyte fractional surface area. Nevertheless, analysis of Na+ movement across the tracheal gill of mayfly nymphs using scanning ion-selective electrode technique indicated that FW nymphs acquired Na+ from surrounding water, while tracheal gills of SCW nymphs had the capacity to secrete Na+. Because Na+ secretion across the gill of SCW-exposed animals occurred in the absence of any change in (1) NKA and VA activity or (2) ionocyte numbers/surface exposure, it was reasoned that Na+ movement across the gill of SCW animals may be occurring, at least in part, through the paracellular pathway. The ultrastructure of tracheal gill septate junctions (SJs) supported this idea as they exhibited morphological alterations indicative of a leakier pathway. Data provide a first look at alterations in osmoregulatory mechanisms that allow H. rigida nymphs to tolerate sub-lethal salinization of their surroundings.


Assuntos
Ephemeroptera/efeitos dos fármacos , Água Doce/química , Brânquias/efeitos dos fármacos , Osmorregulação/efeitos dos fármacos , Cloreto de Sódio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Ephemeroptera/metabolismo , Brânquias/metabolismo , Hemolinfa/metabolismo , Salinidade , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Equilíbrio Hidroeletrolítico/efeitos dos fármacos
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