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1.
Chemosphere ; 239: 124822, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31726527

RESUMO

Arsenic (As) methylation is regarded as an efficient strategy for As contamination remediation by As volatilization. However, most microorganisms display low As volatilization efficiency, which is possibly linked to As efflux transporters competing for cytoplasmic As(III) as a substrate. Here, we developed two types of As biosensors in Escherichia coli to compare the As efflux rate of three efflux transporters and to further investigate the correlation between As efflux rates and As volatilization. The engineered As-sensitive E. coli AW3110 expressing arsBRP, acr3RP or arsBEC displayed a higher As resistance compared to the control. The fluorescence intensity was in a linear correlation in the range of 0-2.0 µmol/L of As(III). The intracellular As(III) concentration was negatively related to As efflux activity of As efflux transporter, which was consistent with the As resistance assays. Moreover, arsM derived from R. palustris CGA009 was subsequently introduced to construct an E. coli AW3110 co-expressing arsB/acr3 and arsM, which exhibited higher As(III) resistance, lower fluorescence intensity and intracellular As concentration compared to the engineered E. coli AW3110 expressing only arsB/acr3. The As volatilization efficiency was negatively related to As efflux activity of efflux transporters, the recombinants without arsB/acr3 displayed the highest rate of As volatilization. This study provided new insights into parameters affecting As volatilization with As efflux being the main limiting factor for As methylation and subsequent volatilization in many microorganisms.


Assuntos
Arsênico/metabolismo , Arsenitos/metabolismo , Escherichia coli/metabolismo , Bombas de Íon/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Técnicas Biossensoriais , Catálise , Escherichia coli/genética , Bombas de Íon/genética , Proteínas de Membrana Transportadoras/genética , Metilação , Volatilização
2.
Biochem Soc Trans ; 47(5): 1247-1257, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31671180

RESUMO

P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.


Assuntos
Bombas de Íon/química , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/química , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Catálise , Humanos , Hidrólise , Bombas de Íon/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo
3.
Cell Mol Life Sci ; 76(19): 3783-3800, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31165202

RESUMO

In the male reproductive tract, ionic equilibrium is essential to maintain normal spermatozoa production and, hence, the reproductive potential. Among the several ions, HCO3- and H+ have a central role, mainly due to their role on pH homeostasis. In the male reproductive tract, the major players in pH regulation and homeodynamics are carbonic anhydrases (CAs), HCO3- membrane transporters (solute carrier 4-SLC4 and solute carrier 26-SLC26 family transporters), Na+-H+ exchangers (NHEs), monocarboxylate transporters (MCTs) and voltage-gated proton channels (Hv1). CAs and these membrane transporters are widely distributed throughout the male reproductive tract, where they play essential roles in the ionic balance of tubular fluids. CAs are the enzymes responsible for the production of HCO3- which is then transported by membrane transporters to ensure the maturation, storage, and capacitation of the spermatozoa. The transport of H+ is carried out by NHEs, Hv1, and MCTs and is essential for the electrochemical balance and for the maintenance of the pH within the physiological limits along the male reproductive tract. Alterations in HCO3- production and transport of ions have been associated with some male reproductive dysfunctions. Herein, we present an up-to-date review on the distribution and role of the main intervenient on pH homeodynamics in the fluids throughout the male reproductive tract. In addition, we discuss their relevance for the establishment of the male reproductive potential.


Assuntos
Genitália Masculina/metabolismo , Concentração de Íons de Hidrogênio , Animais , Bicarbonatos/metabolismo , Anidrases Carbônicas/metabolismo , Fertilidade , Genitália Masculina/química , Homeostase , Humanos , Canais Iônicos/metabolismo , Bombas de Íon/metabolismo , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
4.
Int J Mol Sci ; 20(10)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137773

RESUMO

Ion channels and transporters play essential roles in excitable cells including cardiac, skeletal and smooth muscle cells, neurons, and endocrine cells. In pancreatic beta-cells, for example, potassium KATP channels link the metabolic signals generated inside the cell to changes in the beta-cell membrane potential, and ultimately regulate insulin secretion. Mutations in the genes encoding some ion transporter and channel proteins lead to disorders of glucose homeostasis (hyperinsulinaemic hypoglycaemia and different forms of diabetes mellitus). Pancreatic KATP, Non-KATP, and some calcium channelopathies and MCT1 transporter defects can lead to various forms of hyperinsulinaemic hypoglycaemia (HH). Mutations in the genes encoding the pancreatic KATP channels can also lead to different types of diabetes (including neonatal diabetes mellitus (NDM) and Maturity Onset Diabetes of the Young, MODY), and defects in the solute carrier family 2 member 2 (SLC2A2) leads to diabetes mellitus as part of the Fanconi-Bickel syndrome. Variants or polymorphisms in some ion channel genes and transporters have been reported in association with type 2 diabetes mellitus.


Assuntos
Canalopatias/metabolismo , Transtornos do Metabolismo de Glucose/metabolismo , Canais Iônicos/metabolismo , Bombas de Íon/metabolismo , Animais , Canalopatias/genética , Transtornos do Metabolismo de Glucose/genética , Humanos , Canais Iônicos/genética , Bombas de Íon/genética
5.
Int J Mol Sci ; 20(7)2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30925682

RESUMO

Aluminum (Al) toxicity is one of the major constraints to agricultural production in acid soils. Molecular mechanisms of coping with Al toxicity have now been investigated in a range of plant species. Two main mechanisms of Al tolerance in plants are Al exclusion from the roots and the ability to tolerate Al in the roots. This review focuses on the recent discovery of novel genes and mechanisms that confer Al tolerance in plants and summarizes our understanding of the physiological, genetic, and molecular basis for plant Al tolerance. We hope this review will provide a theoretical basis for the genetic improvement of Al tolerance in plants.


Assuntos
Alumínio/metabolismo , Alumínio/toxicidade , Raízes de Plantas/metabolismo , Plantas/metabolismo , Adaptação Fisiológica , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Bombas de Íon/genética , Bombas de Íon/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Micorrizas/genética , Micorrizas/metabolismo , Micorrizas/fisiologia , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Plantas/genética
6.
Physiol Biochem Zool ; 91(6): 1148-1171, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30334669

RESUMO

Pupfishes (genus Cyprinodon) evolved some of the broadest salinity tolerances of teleost fishes, with some taxa surviving in conditions from freshwater to nearly 160 ppt. In this study, we examined transcriptional dynamics of ion transporters and aquaporins in the gill of the desert Amargosa pupfish (Cyprinodon nevadensis amargosae) during rapid salinity change. Pupfish acclimated to 7.5 ppt were exposed to freshwater (0.3 ppt), seawater (35 ppt), or hypersaline (55 ppt) conditions over 4 h and sampled at these salinities over 14 d. Plasma osmolality and Cl- concentration became elevated 8 h after the start of exposure to 35 or 55 ppt but returned to baseline levels after 14 d. Osmolality recovery was paralleled by increased gill Na+/K+-ATPase activity and higher relative levels of messenger RNAs (mRNAs) encoding cystic fibrosis transmembrane conductance regulator (cftr) and Na+/K+/2Cl- cotransporter-1 (nkcc1). Transcripts encoding one Na+-HCO3- cotransporter-1 isoform (nbce1.1) also increased in the gills at higher salinities, while a second isoform (nbce1.2) increased expression in freshwater. Pupfish in freshwater also had lower osmolality and elevated gill mRNAs for Na+/H+ exchanger isoform-2a (nhe2a) and V-type H+-ATPase within 8 h, followed by increases in Na+/H+ exchanger-3 (nhe3), carbonic anhydrase 2 (ca2), and aquaporin-3 (aqp3) within 1 d. Gill mRNAs for Na+/Cl- cotransporter-2 (ncc2) also were elevated 14 d after exposure to 0.3 ppt. These results offer insights into how coordinated transcriptional responses for ion transporters in the gill facilitate reestablishment of osmotic homeostasis after changes in environmental salinity and provide evidence that the teleost gill expresses two Na+-HCO3- cotransporter-1 isoforms with different roles in freshwater and seawater acclimation.


Assuntos
Aclimatação/genética , Aquaporinas/genética , Proteínas de Peixes/genética , Expressão Gênica , Bombas de Íon/genética , Peixes Listrados/fisiologia , Salinidade , Animais , Aquaporinas/metabolismo , Feminino , Proteínas de Peixes/metabolismo , Água Doce , Brânquias , Bombas de Íon/metabolismo , Peixes Listrados/genética , Masculino , Água do Mar
7.
Cell Death Dis ; 9(10): 984, 2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30250248

RESUMO

Glioblastoma (GBM) is characterized by highly aggressive growth and invasive behavior. Due to the highly lethal nature of GBM, new therapies are urgently needed and repositioning of existing drugs is a promising approach. We have previously shown the activity of Proscillaridin A (ProA), a cardiac glycoside inhibitor of the Na(+)/K(+) ATPase (NKA) pump, against proliferation and migration of GBM cell lines. ProA inhibited tumor growth in vivo and increased mice survival after orthotopic grafting of GBM cells. This study aims to decipher the mechanism of action of ProA in GBM tumor and stem-like cells. ProA displayed cytotoxic activity on tumor and stem-like cells grown in 2D and 3D culture, but not on healthy cells as astrocytes or oligodendrocytes. Even at sub-cytotoxic concentration, ProA impaired cell migration and disturbed EB1 accumulation at microtubule (MT) plus-ends and MT dynamics instability. ProA activates GSK3ß downstream of NKA inhibition, leading to EB1 phosphorylation on S155 and T166, EB1 comet length shortening and MT dynamics alteration, and finally inhibition of cell migration and cytotoxicity. Similar results were observed with digoxin. Therefore, we disclosed here a novel pathway by which ProA and digoxin modulate MT-governed functions in GBM tumor and stem-like cells. Altogether, our results support ProA and digoxin as potent candidates for drug repositioning in GBM.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Microtúbulos/metabolismo , Proscilaridina/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Astrócitos/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Bombas de Íon/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação/efeitos dos fármacos , Polimerização/efeitos dos fármacos , Tubulina (Proteína)/metabolismo
8.
Chem Rev ; 118(21): 10710-10747, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29985590

RESUMO

Synthetic photoswitches have been known for many years, but their usefulness in biology, pharmacology, and medicine has only recently been systematically explored. Over the past decade photopharmacology has grown into a vibrant field. As the photophysical, pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biological targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), soluble proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, we provide an overview of photopharmacology using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. We discuss the scope and limitations of this approach to study biological function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.


Assuntos
Compostos Azo/química , Derivados de Benzeno/química , Farmacologia , Processos Fotoquímicos , Animais , Transporte Biológico Ativo , Reagentes para Ligações Cruzadas/química , Citoesqueleto/metabolismo , Enzimas/metabolismo , Humanos , Canais Iônicos/metabolismo , Bombas de Íon/metabolismo , Ligantes , Luz , Optogenética , Biossíntese de Proteínas/efeitos da radiação
9.
Curr Opin Nephrol Hypertens ; 27(4): 305-313, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29847376

RESUMO

PURPOSE OF REVIEW: Uric acid homeostasis in the body is mediated by a number of SLC and ABC transporters in the kidney and intestine, including several multispecific 'drug' transporters (e.g., OAT1, OAT3, and ABCG2). Optimization of uric acid levels can be viewed as a 'systems biology' problem. Here, we consider uric acid transporters from a systems physiology perspective using the framework of the 'Remote Sensing and Signaling Hypothesis.' This hypothesis explains how SLC and ABC 'drug' and other transporters mediate interorgan and interorganismal communication (e.g., gut microbiome and host) via small molecules (e.g., metabolites, antioxidants signaling molecules) through transporters expressed in tissues lining body fluid compartments (e.g., blood, urine, cerebrospinal fluid). RECENT FINDINGS: The list of uric acid transporters includes: SLC2A9, ABCG2, URAT1 (SLC22A12), OAT1 (SLC22A6), OAT3 (SLC22A8), OAT4 (SLC22A11), OAT10 (SLC22A13), NPT1 (SLC17A1), NPT4 (SLC17A3), MRP2 (ABCC2), MRP4 (ABCC4). Normally, SLC2A9, - along with URAT1, OAT1 and OAT3, - appear to be the main transporters regulating renal urate handling, while ABCG2 appears to regulate intestinal transport. In chronic kidney disease (CKD), intestinal ABCG2 becomes much more important, suggesting remote organ communication between the injured kidney and the intestine. SUMMARY: The remote sensing and signaling hypothesis provides a useful systems-level framework for understanding the complex interplay of uric acid transporters expressed in different tissues involved in optimizing uric acid levels under normal and diseased (e.g., CKD, gut microflora dysbiosis) conditions.


Assuntos
Transporte Biológico , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Ácido Úrico/metabolismo , Animais , Humanos , Mucosa Intestinal/metabolismo , Bombas de Íon/genética , Bombas de Íon/metabolismo , Rim/metabolismo , Transdução de Sinais , Biologia de Sistemas
10.
Biochim Biophys Acta Mol Cell Res ; 1865(7): 995-1001, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29694915

RESUMO

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL deamidation. The conservation of such histidines implies that human Bcl-xL is in essence "designed" to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.


Assuntos
Histidina/química , Bombas de Íon/metabolismo , Proteína bcl-X/química , Proteína bcl-X/metabolismo , Células 3T3 , Animais , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Desaminação , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteína bcl-X/genética
11.
J Am Chem Soc ; 140(11): 4085-4091, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29489362

RESUMO

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.


Assuntos
Bacteriorodopsinas/química , Bombas de Íon/química , Luz , Ressonância Magnética Nuclear Biomolecular , Bacteriorodopsinas/isolamento & purificação , Bacteriorodopsinas/metabolismo , Halobacterium salinarum/química , Halobacterium salinarum/citologia , Halobacterium salinarum/metabolismo , Bombas de Íon/metabolismo
12.
Nutrients ; 10(3)2018 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-29510506

RESUMO

BACKGROUND: Beneficial effects of Resveratrol (RSV) have been demonstrated, including effects on transporters and channels. However, little is known about how RSV influences intestinal transport. The aim of this study was to further characterize the effects of RSV on intestinal transport and the respective mechanisms. METHODS: Porcine jejunum and ileum were incubated with RSV (300 µM, 30 min) in Ussing chambers (functional studies) and tissue bathes (detection of protein expression, phosphorylation, association with detergent resistant membranes (DRMs)). RESULTS: RSV reduced alanine and glucose-induced short circuit currents (ΔIsc) and influenced forskolin-induced ΔIsc. The phosphorylation of sodium-glucose-linked transporter 1 (SGLT1), AMP-activated protein kinase (AMPK), protein kinase A substrates (PKA-S) and liver kinase B1 (LKB1) increased but a causative relation to the inhibitory effects could not directly be established. The DRM association of SGLT1, peptide transporter 1 (PEPT1) and (phosphorylated) Na⁺/H⁺-exchanger 3 (NHE3) did not change. CONCLUSION: RSV influences the intestinal transport of glucose, alanine and chloride and is likely to affect other transport processes. As the effects of protein kinase activation vary between the intestinal localizations, it would appear that increasing cyclic adenosine monophosphate (cAMP) levels are part of the mechanism. Nonetheless, the physiological responses depend on cell type-specific structures.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Alanina/metabolismo , Membrana Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glucose/metabolismo , Íleo/efeitos dos fármacos , Bombas de Íon/metabolismo , Jejuno/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Estilbenos/farmacologia , Animais , Membrana Celular/enzimologia , Cloretos/metabolismo , AMP Cíclico/metabolismo , Íleo/enzimologia , Técnicas In Vitro , Jejuno/enzimologia , Potenciais da Membrana , Transportador 1 de Peptídeos/metabolismo , Fosforilação , Resveratrol , Transportador 1 de Glucose-Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Sus scrofa
13.
J Nutr Biochem ; 56: 193-204, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29587242

RESUMO

Anthocyanins (ANT) are polyphenolic flavonoids with antioxidant and neuroprotective properties. This study evaluated the effect of ANT treatment on cognitive performance and neurochemical parameters in an experimental model of sporadic dementia of Alzheimer's type (SDAT). Adult male rats were divided into four groups: control (1 ml/kg saline, once daily, by gavage), ANT (200 mg/kg, once daily, by gavage), streptozotocin (STZ, 3 mg/kg) and STZ plus ANT. STZ was administered via bilateral intracerebroventricular (ICV) injection (5 µl). ANT were administered after ICV injection for 25 days. Cognitive deficits (short-term memory and spatial memory), oxidative stress parameters, and acetylcholinesterase (AChE) and Na+-K+-ATPase activity in the cerebral cortex and hippocampus were evaluated. ANT treatment protected against the worsening of memory in STZ-induced SDAT. STZ promoted an increase in AChE and Na+-K+-ATPase total and isoform activity in both structures; ANT restored this change. STZ administration induced an increase in lipid peroxidation and decrease in the level of antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), in the cerebral cortex; ANT significantly attenuated these effects. In the hippocampus, an increase in reactive oxygen species (ROS), nitrite and lipid peroxidation levels, and SOD activity and a decrease in CAT and GPx activity were seen after STZ injection. ANT protected against the changes in ROS and antioxidant enzyme levels. In conclusion, the present study showed that treatment with ANT attenuated memory deficits, protected against oxidative damage in the brain, and restored AChE and ion pump activity in an STZ-induced SDAT in rats.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Antocianinas/farmacologia , Bombas de Íon/metabolismo , Transtornos da Memória/tratamento farmacológico , Estresse Oxidativo , Acetilcolinesterase/metabolismo , Doença de Alzheimer/induzido quimicamente , Animais , Antioxidantes/uso terapêutico , Encéfalo/metabolismo , Catalase/metabolismo , Córtex Cerebral/metabolismo , Cognição , Modelos Animais de Doenças , Glutationa Peroxidase/metabolismo , Hipocampo/metabolismo , Infusões Intraventriculares , Peroxidação de Lipídeos , Masculino , Aprendizagem em Labirinto , Memória/efeitos dos fármacos , Transtornos da Memória/induzido quimicamente , Fármacos Neuroprotetores/uso terapêutico , Ratos , Ratos Wistar , Estreptozocina/efeitos adversos , Compostos de Sulfidrila , Superóxido Dismutase/metabolismo
14.
Microbes Environ ; 33(1): 89-97, 2018 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-29553064

RESUMO

Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl--pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl- pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl--transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl--pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR.


Assuntos
Cloretos/metabolismo , Cianobactérias/genética , Halorrodopsinas/genética , Bombas de Íon/genética , Água do Mar/microbiologia , Archaea , Cianobactérias/classificação , Cianobactérias/metabolismo , Escherichia coli/genética , Evolução Molecular , Transferência Genética Horizontal , Genoma Bacteriano , Halorrodopsinas/metabolismo , Bombas de Íon/metabolismo , Luz , Filogenia , Rodopsina/genética
15.
Arch Oral Biol ; 90: 19-26, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29524788

RESUMO

OBJECTIVE: Homeostasis of intracellular pH (pHi) plays vital roles in many cell functions, such as proliferation, apoptosis, differentiation and metastasis. Thus far, Na+-H+ exchanger (NHE), Na+-HCO3- co-transporter (NBC), Cl-/HCO3- exchanger (AE) and Cl-/OH- exchanger (CHE) have been identified to co-regulate pHi homeostasis. However, functional and biological pHi-regulators in human dental pulp stem cells (hDPSCs) have yet to be identified. DESIGN: Microspectrofluorimetry technique with pH-sensitive fluorescent dye, BCECF, was used to detect pHi changes. NH4Cl and Na+-acetate pre-pulse were used to induce intracellular acidosis and alkalosis, respectively. Isoforms of pHi-regulators were detected by Western blot technique. RESULTS: The resting pHi was no significant difference between that in HEPES-buffered (nominal HCO3--free) solution or CO2/HCO3-buffered system (7.42 and 7.46, respectively). The pHi recovery following the induced-intracellular acidosis was blocked completely by removing [Na+]o, while only slowed (-63%) by adding HOE694 (a NHE1 specific inhibitor) in HEPES-buffered solution. The pHi recovery was inhibited entirely by removing [Na+]o, while adding HOE 694 pulse DIDS (an anion-transporter inhibitor) only slowed (-55%) the acid extrusion. Both in HEPES-buffered and CO2/HCO3-buffered system solution, the pHi recovery after induced-intracellular alkalosis was entirely blocked by removing [Cl-]o. Western blot analysis showed the isoforms of pHi regulators, including NHE1/2, NBCe1/n1, AE1/2/3/4 and CHE in the hDPSCs. CONCLUSIONS: We demonstrate for the first time that resting pHi is significantly higher than 7.2 and meditates functionally by two Na+-dependent acid extruders (NHE and NBC), two Cl--dependent acid loaders (CHE and AE) and one Na+-independent acid extruder(s) in hDPSCs. These findings provide novel insight for basic and clinical treatment of dentistry.


Assuntos
Equilíbrio Ácido-Base/fisiologia , Citoplasma/metabolismo , Polpa Dentária/metabolismo , Homeostase/fisiologia , Células-Tronco/metabolismo , Desequilíbrio Ácido-Base , Ácidos/farmacologia , Cloreto de Amônio , Antiporters/metabolismo , Apoptose , Tampões (Química) , Diferenciação Celular , Proliferação de Células , Citoplasma/efeitos dos fármacos , Guanidinas/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Bombas de Íon/efeitos dos fármacos , Bombas de Íon/metabolismo , Metástase Neoplásica , Isoformas de Proteínas , Sódio/farmacologia , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Células-Tronco/efeitos dos fármacos , Sulfonas/farmacologia
16.
Bioorg Med Chem ; 26(4): 833-844, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29373270

RESUMO

Novel reversed isoniazid (RINH) agents were synthesized by covalently linking isoniazid with various efflux pump inhibitor (EPI) cores and their structural motifs. These RINH agents were then evaluated for anti-mycobacterial activity against sensitive, isoniazid mono-resistant and MDR clinical isolates of M. tuberculosis and a selected number of compounds were also tested ex vivo for intracellular activity as well as in the ethidium bromide (EB) assay for efflux pump inhibition efficacy. The potency of some compounds against various strains of M. tuberculosis (4a-c, 7 and 8; H37Rv-MIC99 ≤1.25 µM, R5401-MIC99 ≤2.5 µM, X_61-MIC99 ≤5 µM) demonstrated the potential of the reversed anti-TB agent strategy towards the development of novel anti-mycobacterial agents to address the rapidly growing issue of resistance. Further, macrophage activity with >90% inhibition by 1a-c and 3b (MIC90 ≤13.42 µM) and inhibition of EB efflux demonstrated by these compounds are encouraging.


Assuntos
Antituberculosos/síntese química , Desenho de Fármacos , Isoniazida/química , Animais , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Células CHO , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Humanos , Bombas de Íon/antagonistas & inibidores , Bombas de Íon/metabolismo , Isoniazida/síntese química , Isoniazida/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-Atividade
17.
Phys Chem Chem Phys ; 20(5): 3165-3171, 2018 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-28975940

RESUMO

Light-driven H+, Na+ and Cl- pumps have been found in eubacteria, which convert light energy into a transmembrane electrochemical potential. A recent mutation study revealed asymmetric functional conversion between the two pumps, where successful functional conversions are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Although this fact suggests that the essential structural mechanism of an ancestral function is retained even after gaining a new function, questions regarding the essential structural mechanism remain unanswered. Light-induced difference FTIR spectroscopy was used to monitor the presence of strongly hydrogen-bonded water molecules for all eubacterial H+, Na+ and Cl- pumps, including a functionally converted mutant. This fact suggests that the strongly hydrogen-bonded water molecules are maintained for these new functions during evolution, which could be the reason for successful functional conversion from Na+ to H+, and from Cl- to H+ pumps. This also explains the successful conversion of the Cl- to the H+ pump only for eubacteria, but not for archaea. It is concluded that water-containing hydrogen-bonding networks constitute one of the essential structural mechanisms in eubacterial light-driven ion pumps.


Assuntos
Proteínas de Bactérias/metabolismo , Bombas de Íon/metabolismo , Luz , Água/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cloretos/metabolismo , Temperatura Baixa , Cristalografia por Raios X , Ligação de Hidrogênio , Bombas de Íon/química , Bombas de Íon/genética , Transporte de Íons/efeitos da radiação , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Sódio/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier , Água/química
18.
Physiol Rep ; 5(19)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29038365

RESUMO

It was evaluated whether upper-body compared to lower-body musculature exhibits a different phenotype in relation to capacity for handling reactive oxygen species (ROS), H+, La-, Na+, K+ and also whether it differs in adaptive potential to exercise training. Eighty-three sedentary premenopausal women aged 45 ± 6 years (mean ± SD) were randomized into a high-intensity intermittent swimming group (HIS, n = 21), a moderate-intensity swimming group (MOS, n = 21), a soccer group (SOC, n = 21), or a control group (CON, n = 20). Intervention groups completed three weekly training sessions for 15 weeks, and pre- and postintervention biopsies were obtained from deltoideus and vastus lateralis muscle. Before training, monocarboxylate transporter 4 (MCT4), Na+/K+ pump α2, and superoxide dismutase 2 (SOD2) expressions were lower (P < 0.05) in m deltoideus than in m vastus lateralis, whereas deltoid had higher (P < 0.05) Na+/H+ exchanger 1 (NHE1) expression. As a result of training, Na+/K+ pump α2 isoform expression was elevated only in deltoideus muscle, while upregulation (P < 0.05) of the α1 and ß1 subunits, phospholemman (FXYD1), NHE1, and superoxide dismutase 1 expression occurred exclusively in vastus lateralis muscle. The increased (P < 0.05) expression of MCT4 and SOD2 in deltoid muscle after HIS and vastus lateralis muscle after SOC were similar. In conclusion, arm musculature displays lower basal ROS, La-, K+ handling capability but higher Na+-dependent H+ extrusion capacity than leg musculature. Training-induced changes in the ion-transporting and antioxidant proteins clearly differed between muscle groups.


Assuntos
Adaptação Fisiológica , Músculo Deltoide/metabolismo , Treinamento Intervalado de Alta Intensidade , Bombas de Íon/metabolismo , Músculo Quadríceps/metabolismo , Superóxido Dismutase/metabolismo , Adulto , Braço/fisiologia , Músculo Deltoide/fisiologia , Feminino , Humanos , Bombas de Íon/genética , Perna (Membro)/fisiologia , Pessoa de Meia-Idade , Músculo Quadríceps/fisiologia , Superóxido Dismutase/genética
19.
Biotechnol J ; 12(9)2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28731528

RESUMO

Biologically fixation of CO2 has great potential as a significant carbon source for biosynthesis, which is also a major way to reduce CO2 accumulation in atmosphere. Phosphoenolpyruvate (PEP) carboxylation is the key step of anaerobic succinate production in Escherichia coli. In this reaction, one mole CO2 is assimilated with PEP to form oxaloacetate by PEP carboxykinase (PCK). The preferred substrate of PCK is CO2 , which is very limited in cytoplasm. In this study, the carbon concentration mechanism (CCM) of cyanobacteria was introduced into Escherichia coli to enhance the intracellular inorganic carbon concentration for improving carboxylation velocity. Overexpression of the bicarbonate transporter (BT) or carbonic anhydrase (CA) gene from Synechococcus sp. PCC7002 led to a 22 or 35% increase in succinate titer at 36 h, respectively. The carboxylation rate of PCK increased from 2.46 to 3.92 µmol min-1 mg-1 protein by overexpression of the CA gene. In addition, co-overexpression of BT and CA genes had a synergetic effect, leading to a 44% increase in succinate titer at 36 h. This work is the first attempt to increase carbon fixation involved in microbial biosynthesis by engineering a biological CO2 delivery system, which provides new direction and strategies for improving industrial fermentations based on biological CO2 assimilation pathways.


Assuntos
Dióxido de Carbono/metabolismo , Escherichia coli/metabolismo , Engenharia Metabólica/métodos , Ácido Succínico/metabolismo , Synechococcus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Escherichia coli/genética , Bombas de Íon/genética , Bombas de Íon/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácido Succínico/análise , Synechococcus/enzimologia
20.
J Phys Chem B ; 121(33): 7899-7906, 2017 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-28745057

RESUMO

Biological molecular machines perform the work of supporting life at the smallest of scales, including the work of shuttling ions across cell boundaries and against chemical gradients. Systems of artificial channels at the nanoscale can likewise control ionic concentration by way of ionic current rectification, species selectivity, and voltage gating mechanisms. Here, we theoretically show that a voltage-gated, ion species-selective, and rectifying ion channel can be built using the components of a biological water channel aquaporin. Through all-atom molecular dynamics simulations, we show that the ionic conductance of a truncated aquaporin channel nonlinearly increases with the bias magnitude, depends on the channel's orientation, and is highly cation specific but only for one polarity of the transmembrane bias. Further, we show that such an unusually complex response of the channel to transmembrane bias arises from mechanical motion of a positively charged gate that blocks cation transport. By combining two truncated aquaporins, we demonstrate a molecular system that pumps ions against their chemical gradients when subject to an alternating transmembrane bias. Our work sets the stage for future biomimicry efforts directed toward reproducing the function of biological ion pumps using synthetic components.


Assuntos
Bombas de Íon/química , Simulação de Dinâmica Molecular , Nanoestruturas/química , Água/química , Bombas de Íon/metabolismo , Água/metabolismo
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