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1.
Nat Commun ; 11(1): 4791, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963229

RESUMO

The ability to absorb ingested nutrients is an essential function of all metazoans and utilizes a wide array of nutrient transporters found on the absorptive enterocytes of the small intestine. A unique population of patients has previously been identified with severe congenital malabsorptive diarrhea upon ingestion of any enteral nutrition. The intestines of these patients are macroscopically normal, but lack enteroendocrine cells (EECs), suggesting an essential role for this rare population of nutrient-sensing cells in regulating macronutrient absorption. Here, we use human and mouse models of EEC deficiency to identify an unappreciated role for the EEC hormone peptide YY in regulating ion-coupled absorption of glucose and dipeptides. We find that peptide YY is required in the small intestine to maintain normal electrophysiology in the presence of vasoactive intestinal polypeptide, a potent stimulator of ion secretion classically produced by enteric neurons. Administration of peptide YY to EEC-deficient mice restores normal electrophysiology, improves glucose and peptide absorption, diminishes diarrhea and rescues postnatal survival. These data suggest that peptide YY is a key regulator of macronutrient absorption in the small intestine and may be a viable therapeutic option to treat patients with electrolyte imbalance and nutrient malabsorption.


Assuntos
Células Enteroendócrinas/metabolismo , Absorção Intestinal/fisiologia , Transporte de Íons/fisiologia , Nutrientes/metabolismo , Animais , Enterócitos , Glucose/metabolismo , Células-Tronco Embrionárias Humanas , Humanos , Intestino Delgado , Intestinos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeo YY , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Peptídeo Intestinal Vasoativo/metabolismo , Trocador 3 de Sódio-Hidrogênio , Água/metabolismo
2.
Rev Med Suisse ; 16(701): 1450-1455, 2020 Aug 05.
Artigo em Francês | MEDLINE | ID: mdl-32833367

RESUMO

The Covid 19 pandemic remains a serious public health problem until effective drugs and/or vaccines are available. Can we explain why so many people remain asymptomatic but nevertheless highly contagious explaining the speed with which the pandemic has spread around the world? Can we explain why the acute respiratory distress syndrome (ARDS) appears late but can so quickly have a fatal outcome? In the lung, mucociliary clearance (CMC) and alveolar clearance (CA) depend on the transport of sodium through the plasma membrane of epithelial cells. This transport is mediated by a highly selective sodium channel (Epithelial Sodium Channel = ENaC) which could be a key element in the pulmonary pathophysiology of SARS-CoV-2 infection.


Assuntos
Betacoronavirus/patogenicidade , Infecções por Coronavirus/metabolismo , Modelos Biológicos , Pneumonia Viral/metabolismo , Sódio/metabolismo , Infecções por Coronavirus/virologia , Humanos , Transporte de Íons , Pandemias , Pneumonia Viral/virologia
3.
Elife ; 92020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32840481

RESUMO

In order to enter a cell, an ammonium ion must first dissociate to form an ammonia molecule and a hydrogen ion (a proton), which then pass through the cell membrane separately and recombine inside.


Assuntos
Compostos de Amônio , Nitrosomonas , Amônia , Transporte de Íons , Oxirredução
4.
Nature ; 585(7823): 129-134, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848250

RESUMO

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.


Assuntos
Simulação por Computador , Genes Sintéticos/genética , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Biologia Sintética , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Condutividade Elétrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canais Iônicos/metabolismo , Transporte de Íons , Lipossomos/metabolismo , Técnicas de Patch-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Engenharia de Proteínas , Estrutura Secundária de Proteína , Solubilidade , Água/química
5.
Aquat Toxicol ; 226: 105561, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32688145

RESUMO

In the aquatic environment, metals are present as mixtures, therefore studies on mixture toxicity are crucial to thoroughly understand their toxic effects on aquatic organisms. Common carp (Cyprinus carpio) were used to assess the effects of short-term Cu(II) and Cd(II) mixtures, using a fixed concentration of one of the metals, representing 25 % of its individual 96h-LC50 (concentration lethal for 50 % of the population) combined with a variable concentration of the other metal corresponding to 10, 25 or 50 % of its 96h-LC50, and vice versa. Our results showed a fast Cu and Cd bioaccumulation, with the percentage of increase in the order gill > liver > carcass. An inhibitory effect of Cu on Cd uptake was observed; higher Cu concentrations at fixed Cd levels resulted in a decreased accumulation of Cd. The presence of the two metal ions resulted in losses of total Na, K and Ca. Fish tried to compensate for the Na loss through the induction of the genes coding for Na+/K+-ATPase and H+-ATPase. Additionally, a counterintuitive induction of the gene encoding the high affinity copper transporter (CTR1) occurred, while a downregulation was expected to prevent further metal ion uptake. An induction of defensive mechanisms, both metal ion binding protein and anti-oxidant defences, was observed. Despite the metal accumulation and electrolyte loss, the low mortality suggest that common carp is able to cope with these metal levels, at least during a one-week exposure.


Assuntos
Bioacumulação/efeitos dos fármacos , Cádmio/toxicidade , Carpas/metabolismo , Cobre/toxicidade , Homeostase/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Cádmio/metabolismo , Carpas/genética , Cobre/metabolismo , Transportador de Cobre 1/genética , Transportador de Cobre 1/metabolismo , Eletrólitos/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Transporte de Íons , Dose Letal Mediana , Potássio/metabolismo , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Poluentes Químicos da Água/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(29): 16743-16748, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611809

RESUMO

Mammalian nervous systems, as natural ionic circuitries, stand out in environmental perception and sophisticated information transmission, relying on protein ionic channels and additional necessary structures. Prosperously emerged ionic regulated biomimetic nanochannels exhibit great potentialities in various application scenarios, especially signal transduction. Most reported direct current systems possess deficiencies in informational density and variability, which are superiorities of alternating current (AC) systems and necessities in bioinspired nervous signal transmission. Here, inspired by myelinated saltatory conduction, alternating electrostatic potential controlled nanofluidics are constructed with a noncontact application pattern and MXene nanosheets. Under time-variant external stimuli, ions confined in the interlaminar space obtain the capability of carriers for the AC ionic circuit. The transmitted information is accessible from typical sine to a frequency-modulated binary signal. This work demonstrates the potentiality of the bioinspired nervous signal transmission between electronics and ionic nanofluidics, which might push one step forward to the avenue of AC ionics.


Assuntos
Potenciais de Ação , Materiais Biomiméticos/química , Condutividade Elétrica , Microfluídica/métodos , Modelos Neurológicos , Nanoestruturas/química , Dimetilpolisiloxanos/química , Equipamentos e Provisões Elétricas , Transporte de Íons , Microfluídica/instrumentação
7.
Nature ; 583(7815): 314-318, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499654

RESUMO

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.


Assuntos
Flavobacteriaceae/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efeitos da radiação , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/efeitos da radiação , Sítios de Ligação , Cristalografia , Elétrons , Transporte de Íons , Isomerismo , Lasers , Prótons , Teoria Quântica , Retinaldeído/química , Retinaldeído/metabolismo , Bases de Schiff/química , Sódio/metabolismo , Análise Espectral , Eletricidade Estática , Fatores de Tempo
8.
Eur J Pharmacol ; 882: 173237, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32525005

RESUMO

Pirfenidone (PFD), a pyridone compound, is well recognized as an antifibrotic agent tailored for the treatment of idiopathic pulmonary fibrosis. Recently, through its anti-inflammatory and anti-oxidant effects, PFD based clinical trial has also been launched for the treatment of coronavirus disease (COVID-19). To what extent this drug can perturb membrane ion currents remains largely unknown. Herein, the exposure to PFD was observed to depress the amplitude of hyperpolarization-activated cation current (Ih) in combination with a considerable slowing in the activation time of the current in pituitary GH3 cells. In the continued presence of ivabradine or zatebradine, subsequent application of PFD decreased Ih amplitude further. The presence of PFD resulted in a leftward shift in Ih activation curve without changes in the gating charge. The addition of this compound also led to a reduction in area of voltage-dependent hysteresis evoked by long-lasting inverted triangular (downsloping and upsloping) ramp pulse. Neither the amplitude of M-type nor erg-mediated K+ current was altered by its presence. In whole-cell potential recordings, addition of PFD reduced the firing frequency, and this effect was accompanied by the depression in the amplitude of sag voltage elicited by hyperpolarizing current stimulus. Overall, this study highlights evidence that PFD is capable of perturbing specific ionic currents, revealing a potential additional impact on functional activities of different excitable cells.


Assuntos
Membrana Celular/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Piridonas/farmacologia , Animais , Betacoronavirus/metabolismo , Cátions/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Infecções por Coronavirus/virologia , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Potássio/metabolismo , Piridonas/uso terapêutico , Ratos , Sódio/metabolismo
9.
Nat Commun ; 11(1): 3024, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541684

RESUMO

The canonical mechanistic model explaining potassium channel gating is of a conformational change that alternately dilates and constricts a collar-like intracellular entrance to the pore. It is based on the premise that K+ ions maintain a complete hydration shell while passing between the transmembrane cavity and cytosol, which must be accommodated. To put the canonical model to the test, we locked the conformation of a Kir K+ channel to prevent widening of the narrow collar. Unexpectedly, conduction was unimpaired in the locked channels. In parallel, we employed all-atom molecular dynamics to simulate K+ ions moving along the conduction pathway between the lower cavity and cytosol. During simulations, the constriction did not significantly widen. Instead, transient loss of some water molecules facilitated K+ permeation through the collar. The low free energy barrier to partial dehydration in the absence of conformational change indicates Kir channels are not gated by the canonical mechanism.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potássio/metabolismo , Citosol/química , Citosol/metabolismo , Condutividade Elétrica , Impedância Elétrica , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Humanos , Transporte de Íons , Íons/química , Íons/metabolismo , Simulação de Dinâmica Molecular , Potássio/química , Conformação Proteica , Água/metabolismo
10.
Nat Commun ; 11(1): 2924, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522996

RESUMO

Biological membranes play pivotal roles in the cellular activities. Transmembrane proteins are the central molecules that conduct membrane-mediated biochemical functions such as signal transduction and substance transportation. Not only the molecular functions but also the supramolecular properties of the transmembrane proteins such as self-assembly, delocalization, orientation and signal response are essential for controlling cellular activities. Here we report anisotropic ligand responses of a synthetic multipass transmembrane ion channel. An unsymmetrical molecular structure allows for oriented insertion of the synthetic amphiphile to a bilayer by addition to a pre-formed membrane. Complexation with a ligand prompts ion transportation by forming a supramolecular channel, and removal of the ligand deactivates the transportation function. Biomimetic regulation of the synthetic channel by agonistic and antagonistic ligands is also demonstrated not only in an artificial membrane but also in a biological membrane of a living cell.


Assuntos
Transporte de Íons/fisiologia , Anisotropia , Biomimética , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência
11.
Nature ; 582(7813): 592-596, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555458

RESUMO

Proteins carry out the vast majority of functions in all biological domains, but for technological reasons their large-scale investigation has lagged behind the study of genomes. Since the first essentially complete eukaryotic proteome was reported1, advances in mass-spectrometry-based proteomics2 have enabled increasingly comprehensive identification and quantification of the human proteome3-6. However, there have been few comparisons across species7,8, in stark contrast with genomics initiatives9. Here we use an advanced proteomics workflow-in which the peptide separation step is performed by a microstructured and extremely reproducible chromatographic system-for the in-depth study of 100 taxonomically diverse organisms. With two million peptide and 340,000 stringent protein identifications obtained in a standardized manner, we double the number of proteins with solid experimental evidence known to the scientific community. The data also provide a large-scale case study for sequence-based machine learning, as we demonstrate by experimentally confirming the predicted properties of peptides from Bacteroides uniformis. Our results offer a comparative view of the functional organization of organisms across the entire evolutionary range. A remarkably high fraction of the total proteome mass in all kingdoms is dedicated to protein homeostasis and folding, highlighting the biological challenge of maintaining protein structure in all branches of life. Likewise, a universally high fraction is involved in supplying energy resources, although these pathways range from photosynthesis through iron sulfur metabolism to carbohydrate metabolism. Generally, however, proteins and proteomes are remarkably diverse between organisms, and they can readily be explored and functionally compared at www.proteomesoflife.org.


Assuntos
Classificação , Aprendizado Profundo , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma/química , Proteoma/isolamento & purificação , Proteômica/métodos , Animais , Bacteroides/química , Bacteroides/classificação , Metabolismo dos Carboidratos , Cromatografia , Glicólise , Homeostase , Transporte de Íons , Proteínas com Ferro-Enxofre/metabolismo , Oxirredução , Fotossíntese , Biossíntese de Proteínas , Dobramento de Proteína , Proteólise , Especificidade da Espécie
12.
Nucleic Acids Res ; 48(13): 7454-7467, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32520327

RESUMO

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, encoding an anion channel that conducts chloride and bicarbonate across epithelial membranes. Mutations that disrupt pre-mRNA splicing occur in >15% of CF cases. One common CFTR splicing mutation is CFTR c.3718-2477C>T (3849+10 kb C>T), which creates a new 5' splice site, resulting in splicing to a cryptic exon with a premature termination codon. Splice-switching antisense oligonucleotides (ASOs) have emerged as an effective therapeutic strategy to block aberrant splicing. We test an ASO targeting the CFTR c.3718-2477C>T mutation and show that it effectively blocks aberrant splicing in primary bronchial epithelial (hBE) cells from CF patients with the mutation. ASO treatment results in long-term improvement in CFTR activity in hBE cells, as demonstrated by a recovery of chloride secretion and apical membrane conductance. We also show that the ASO is more effective at recovering chloride secretion in our assay than ivacaftor, the potentiator treatment currently available to these patients. Our findings demonstrate the utility of ASOs in correcting CFTR expression and channel activity in a manner expected to be therapeutic in patients.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , Células Epiteliais/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Processamento de RNA , Aminofenóis/farmacologia , Brônquios/citologia , Linhagem Celular Tumoral , Células Cultivadas , Agonistas dos Canais de Cloreto/farmacologia , Cloretos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Transporte de Íons/efeitos dos fármacos , Mutação , Quinolonas/farmacologia
13.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G142-G150, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32567323

RESUMO

Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl- cotransporter-1 (KCC1) operating in parallel with K+ and Cl- channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl- in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl--dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl- exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl- cotransporter-1 (KCC1) activity.


Assuntos
Canais de Cloreto/metabolismo , Colo/fisiologia , Mucosa Intestinal/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Animais , Canais de Cloreto/genética , Cloretos/metabolismo , Feminino , Transporte de Íons , Masculino , Técnicas de Patch-Clamp , Canais de Potássio/genética , Transporte Proteico , Ratos , Ratos Sprague-Dawley
14.
PLoS Genet ; 16(6): e1008831, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555673

RESUMO

Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and brain-transcriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing, and biting/striking occurred only during intervals between mouth-locking. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pair showed a strong gene expression correlation, whereas those in D20 fighting pairs showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in a grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in brain-transcriptomic synchronization (PIBS) suggested that this synchronization provides a physiological basis for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 group found genes enriched for ion transport, synaptic function, and learning and memory. Brain-transcriptomic synchronization could be a general phenomenon and may provide a new cornerstone with which to investigate coordinating and sustaining social interactions between two interacting partners of vertebrates.


Assuntos
Comportamento Animal/fisiologia , Encéfalo/fisiologia , Peixes/fisiologia , Regulação da Expressão Gênica/fisiologia , Transcriptoma/fisiologia , Agressão , Animais , Técnicas de Observação do Comportamento , Comportamento Cooperativo , Relações Interpessoais , Transporte de Íons/fisiologia , Aprendizagem/fisiologia , Masculino , Memória/fisiologia , RNA-Seq , Gravação em Vídeo
15.
Bull Environ Contam Toxicol ; 104(6): 755-762, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32394051

RESUMO

We examined copper accumulation in the hemolymph, gills and hepatopancreas, and hemolymph osmolality, Na+ and Cl- concentrations, together with gill Na+/K+-ATPase and carbonic anhydrase activities, after dietary copper delivery (0, 100 or 500 Cu µg g-1) for 12 days in a fiddler crab, Minuca rapax. In contaminated crabs, copper concentration decreased in the hemolymph and hepatopancreas, but increased in the gills. Hemolymph osmolality and gill Na+/K+-ATPase activity increased while hemolymph [Na+] and [Cl-] and gill carbonic anhydrase activity decreased. Excretion likely accounts for the decreased hemolymph and hepatopancreas copper titers. Dietary copper clearly affected osmoregulatory ability and hemolymph Na+ and Cl- regulation in M. rapax. Gill copper accumulation decreased carbonic anhydrase activity, suggesting that dietary copper affects acid-base balance. Elevated gill Na+/K+-ATPase activity appears to compensate for the ion-regulatory disturbance. These effects of dietary copper illustrate likely impacts on semi-terrestrial species that feed on metal-contaminated sediments.


Assuntos
Bioacumulação , Braquiúros/efeitos dos fármacos , Cobre/metabolismo , Osmorregulação/efeitos dos fármacos , Poluentes Químicos da Água/metabolismo , Animais , Braquiúros/metabolismo , Cobre/análise , Exposição Dietética , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Hemolinfa/metabolismo , Hepatopâncreas/metabolismo , Transporte de Íons , Concentração Osmolar , Alimentos Marinhos , ATPase Trocadora de Sódio-Potássio/metabolismo , Poluentes Químicos da Água/análise
16.
Nat Commun ; 11(1): 2642, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457313

RESUMO

Bacteria acquire phosphate (Pi) by maintaining a periplasmic concentration below environmental levels. We recently described an extracellular Pi buffer which appears to counteract the gradient required for Pi diffusion. Here, we demonstrate that various treatments to outer membrane (OM) constituents do not affect the buffered Pi because bacteria accumulate Pi in the periplasm, from which it can be removed hypo-osmotically. The periplasmic Pi can be gradually imported into the cytoplasm by ATP-powered transport, however, the proton motive force (PMF) is not required to keep Pi in the periplasm. In contrast, the accumulation of Pi into the periplasm across the OM is PMF-dependent and can be enhanced by light energy. Because the conventional mechanism of Pi-specific transport cannot explain Pi accumulation in the periplasm we propose that periplasmic Pi anions pair with chemiosmotic cations of the PMF and millions of accumulated Pi pairs could influence the periplasmic osmolarity of marine bacteria.


Assuntos
Bactérias/metabolismo , Fosfatos/metabolismo , Alphaproteobacteria/metabolismo , Alphaproteobacteria/efeitos da radiação , Oceano Atlântico , Bactérias/efeitos da radiação , Membrana Celular/metabolismo , Transporte de Íons/efeitos da radiação , Luz , Modelos Biológicos , Concentração Osmolar , Osmose , Periplasma/metabolismo , Fitoplâncton/metabolismo , Fitoplâncton/efeitos da radiação , Prochlorococcus/metabolismo , Prochlorococcus/efeitos da radiação , Força Próton-Motriz , Água do Mar/microbiologia , Synechococcus/metabolismo , Synechococcus/efeitos da radiação
17.
Am J Respir Cell Mol Biol ; 63(3): 362-373, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32374624

RESUMO

Defective airway mucus clearance is a defining characteristic of cystic fibrosis lung disease, and improvements to current mucolytic strategies are needed. Novel approaches targeting a range of contributing mechanisms are in various stages of preclinical and clinical development. ARINA-1 is a new nebulized product comprised of ascorbic acid, glutathione, and bicarbonate. Using microoptical coherence tomography, we tested the effect of ARINA-1 on central features of mucociliary clearance in F508del/F508del primary human bronchial epithelial cells to assess its potential as a mucoactive therapy in cystic fibrosis. We found that ARINA-1 significantly augmented mucociliary transport rates, both alone and with CFTR (cystic fibrosis transmembrane conductance regulator) modulator therapy, whereas airway hydration and ciliary beating were largely unchanged compared with PBS vehicle control. Analysis of mucus reflectivity and particle-tracking microrheology indicated that ARINA-1 restores mucus clearance by principally reducing mucus layer viscosity. The combination of bicarbonate and glutathione elicited increases in mucociliary transport rate comparable to those seen with ARINA-1, indicating the importance of this interaction to the impact of ARINA-1 on mucus transport; this effect was not recapitulated with bicarbonate alone or bicarbonate combined with ascorbic acid. Assessment of CFTR chloride transport revealed an increase in CFTR-mediated chloride secretion in response to ARINA-1 in CFBE41o- cells expressing wild-type CFTR, driven by CFTR activity stimulation by ascorbate. This response was absent in CFBE41o- F508del cells treated with VX-809 and primary human bronchial epithelial cells, implicating CFTR-independent mechanisms for the effect of ARINA-1 on cystic fibrosis mucus. Together, these studies indicate that ARINA-1 is a novel potential therapy for the treatment of impaired mucus clearance in cystic fibrosis.


Assuntos
Ácido Ascórbico/farmacologia , Bicarbonatos/farmacologia , Fibrose Cística/tratamento farmacológico , Glutationa/farmacologia , Transporte de Íons/efeitos dos fármacos , Depuração Mucociliar/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos
18.
Science ; 368(6493): 893-897, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32439795

RESUMO

Synaptic vesicles accumulate neurotransmitters, enabling the quantal release by exocytosis that underlies synaptic transmission. Specific neurotransmitter transporters are responsible for this activity and therefore are essential for brain function. The vesicular glutamate transporters (VGLUTs) concentrate the principal excitatory neurotransmitter glutamate into synaptic vesicles, driven by membrane potential. However, the mechanism by which they do so remains poorly understood owing to a lack of structural information. We report the cryo-electron microscopy structure of rat VGLUT2 at 3.8-angstrom resolution and propose structure-based mechanisms for substrate recognition and allosteric activation by low pH and chloride. A potential permeation pathway for chloride intersects with the glutamate binding site. These results demonstrate how the activity of VGLUTs can be coordinated with large shifts in proton and chloride concentrations during the synaptic vesicle cycle to ensure normal synaptic transmission.


Assuntos
Canais de Cloreto/química , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Ácido Glutâmico/metabolismo , Vesículas Sinápticas/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/química , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Microscopia Crioeletrônica , Concentração de Íons de Hidrogênio , Transporte de Íons , Potenciais da Membrana , Domínios Proteicos , Ratos
19.
Invest Ophthalmol Vis Sci ; 61(3): 42, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32232342

RESUMO

Purpose: While the association between the gut microbiome and the immune system has been studied in autoimmune disorders, little is known about ocular disease. Previously we reported that IRT5, a mixture of five probiotic strains, could suppress autoimmune dry eye. In this study, we investigated the mechanism by which IRT5 performs its immunomodulatory function in a mouse model of autoimmune dry eye. Methods: NOD.B10.H2b mice were used as an autoimmune dry eye model. Either IRT5 or PBS was gavaged orally for 3 weeks, with or without 5 days of antibiotic pretreatment. The effects on clinical features, extraorbital lacrimal gland and spleen proteins, and fecal microbiota were analyzed. Results: The ocular staining score was lower, and tear secretion was higher, in the IRT5-treated groups than in the PBS-treated groups. After IRT5 treatment, the downregulated lacrimal gland proteins were enriched in the biological processes of defense response and immune system process. The relative abundances of 33 operational taxonomic units were higher, and 53 were lower, in the feces of the IRT5-treated groups than in those of the PBS-treated groups. IRT5 administration without antibiotic pretreatment also showed immunomodulatory functions with increases in the Lactobacillus helveticus group and Lactobacillus hamsteri. Additional proteomic assays revealed a decrease of proteins related to antigen-presenting processes in the CD11b+ and CD11c+ cells of spleen in the IRT5-treated groups. Conclusions: Changes in the gut microbiome after IRT5 treatment improved clinical manifestations in the autoimmune dry eye model via the downregulation of antigen-presenting processes in immune networks.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Fatores Imunológicos/metabolismo , Aparelho Lacrimal/metabolismo , Órbita/metabolismo , Probióticos/administração & dosagem , Animais , Doenças Autoimunes/metabolismo , Síndromes do Olho Seco/metabolismo , Fezes/microbiologia , Microbioma Gastrointestinal/fisiologia , Transporte de Íons/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Microbiota , Proteômica , RNA Ribossômico 16S/genética , Espectrometria de Massas em Tandem , Lágrimas/fisiologia
20.
PLoS One ; 15(4): e0231348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298338

RESUMO

Salt stress is one of the devastating factors that hampers growth and productivity of soybean. Use of Pseudomonas pseudoalcaligenes to improve salt tolerance in soybean has not been thoroughly explored yet. Therefore, we observed the response of hydroponically grown soybean plants, inoculated with halotolerant P. pseudoalcaligenes (SRM-16) and Bacillus subtilis (SRM-3) under salt stress. In vitro testing of 44 bacterial isolates revealed that four isolates showed high salt tolerance. Among them, B. subtilis and P. pseudoalcaligenes showed ACC deaminase activity, siderophore and indole acetic acid (IAA) production and were selected for the current study. We determined that 106 cells/mL of B. subtilis and P. pseudoalcaligenes was sufficient to induce tolerance in soybean against salinity stress (100 mM NaCl) in hydroponics by enhancing plant biomass, relative water content and osmolytes. Upon exposure of salinity stress, P. pseudoalcaligenes inoculated soybean plants showed tolerance by the increased activities of defense related system such as ion transport, antioxidant enzymes, proline and MDA content in shoots and roots. The Na+ concentration in the soybean plants was increased in the salt stress; while, bacterial priming significantly reduced the Na+ concentration in the salt stressed soybean plants. However, the antagonistic results were observed for K+ concentration. Additionally, soybean primed with P. pseudoalcaligenes and exposed to 100 mM NaCl showed a new protein band of 28 kDa suggesting that P. pseudoalcaligenes effectively reduced salt stress. Our results showed that salinity tolerance was more pronounced in P. pseudoalcaligenes as compared to B. subtilis. However, a detailed study at molecular level to interpret the mechanism by which P. pseudoalcaligenes alleviates salt stress in soybean plants need to be explored.


Assuntos
Bacillus subtilis/patogenicidade , Pseudomonas pseudoalcaligenes/patogenicidade , Tolerância ao Sal , Soja/metabolismo , Bacillus subtilis/metabolismo , Transporte de Íons , Malondialdeído/metabolismo , Peroxidase/genética , Peroxidase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Pseudomonas pseudoalcaligenes/metabolismo , Espécies Reativas de Oxigênio , Soja/microbiologia
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